[CANCER RESEARCH 47, 241-250. January 1. 1987]

Tissue Distribution, Immunochemical Characterization, and Biosynthesis of 47D10,a Tumor-associated Surface Glycoprotein

May-Kin Ho,1 Kenneth P. Kato, Paul J. Durda, James H. Murray, Hubert Wolfe, Harvey Rabin, and

Walter P. CarneyBiomedicai Products Department, E. I. DuPont de Nemours & Co., North Billerica, Massachusetts 01862 [M.-K. H., K. P. K., P. J. D., J. H. M., H. R., W. P. CJ, andDepartment of Pathology, New England Medical Center, Boston, Massachusetts 02111 [H. W.]

ABSTRACT

This report describes a new monoclonal antibody (MAli) designated47D10 wh¡i-hwas produced by immunizing mice against a human lung

adenocarcinoma line, A549. The MAb 47D10 reacts with a surfaceantigen found in 95% of adenocarcinomas of the pancreas as well as onhigh percentages of adenocarcinomas from colon, breast, lung, and bileduct. The antigen was not detected in normal pancreas, in pancreatitis,or in a variety of normal tissues with the exception of colon and maturegranulocytes. Lymphocytes and erythrocytes were also negative. Thebinding of 47D10 to tumor cells was unaffected by treatment of cells withneuraminidase.

Immunoprecipitation followed by polyacrylamide gel electrophoresisshowed that 47D10 MAb recognized a group of glycoproteins ranging inmolecular weight from 67,000-98,000 on AS49 lung carcinoma cells.Pulse-chase labeling showed two precursor proteins with molecularweights of 69,000 and 67,000 which were processed to the larger poh -peptides in 1.5 h. At least part of the carbohydrates associated with the47D10 antigen was asparagine linked because the antigen was sensitiveto endoglycosidases, and tunicamycin inhibited the biosynthesis of 47D10antigen. The 47D10 antigen was expressed on the cell surface because itcould be detected on live A549 cells by enzyme-linked immunosorbantassays as well as by immunofluorescent staining. Furthermore, 47D10antigens on tumor cell lines and granulocytes were vectorially labeledwith 125I.The antigen found on granulocytes showed a higher molecularweight of 150,000-180,000, which was digested by endoglycosidase F topolypeptides with molecular weights ranging from 23,000-27,000. Incontrast, the degradation product of the A549 antigen was a \1,39,000polypeptide after treatment with endoglycosidase F. The immunochemi-cal characteristics of 47D10 antigen suggest that it is distinct from otherantigens associated with pancreatic tumors, such as carcinoembryonicantigen, 19-9, and Du-PAN-2. By virtue of its broad range of tumor cellreactivity and low activity on normal cells, the 47D10 MAb may representan important immunological reagent for differential diagnosis, especiallyof pancreatic carcinoma.

INTRODUCTION

There is, at present, a need to improve upon existing meansfor diagnosis and therapy of cancer. Because of their versatilityof application and large repertoire of potential specificities,much effort has gone into the development of immunologicalreagents, primarily MAbs,2 for this purpose. Some of these

MAbs have been used for classification, diagnosis, and treatment of neoplasms (reviewed Ref. l). To obtain MAb againsttumor-associated antigens on epithelial tumors, we immunizedmice with a human lung adenocarcinoma line, A549. One of

Received 4/25/86; revised 8/19/86; accepted 9/11/86.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, at Biomedicai Products

Department, E.I. DuPont de Nemours and Co., North Billerica, MA 01862.2The abbreviations used are: MAb, monoclonal antibody(ies); CEA, carci

noembryonic antigen; ELISA, enzyme-linked immunosorbent assay; endo H,endoglycosidase H; endo F, endoglycosidase F; PCS, fetal calf serum; BSA,bovine serum albumin; 2-ME, 2-mercaptoethanol; DMEM, Dulbecco's minimumessential medium; NCA, nonspecific cross-reacting antigen; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline (0.01 M N:i.,l'()4 0.14M NaCl, pH 7.3); PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecylsulfate.

the MAbs generated was designated as 47D10. In this report,we describe the distribution, biosynthesis, and structural characteristics of the 47D10 antigen. Of particular interest is thereactivity of the 47D10 MAb with epithelial cells of pancreaticadenocarcinoma and cystadenocarcinoma, but not with ductalelements, acinar, and islets in regenerative, or inflammatorypancreas. The 47D10 MAb cross reacts with granulocytes.However, the granulocyte antigen has a higher molecular weightthan the 47D10 antigen on A549 cells. Furthermore, the degradation product of the granulocyte antigen is smaller than thatof the A549 antigen. The distribution and immunochemicalcharacteristics of 47D10 antigen suggest that it is distinct fromother antigens associated with pancreatic carcinoma, such asCEA (2), 19-9 (3), Du-Pan-1, Du-Pan-2, and Du-Pan-3 (4).Therefore, the 47D10 MAb could be used as a new immunoh-istochemical reagent for differentiating neoplastic from regenerative proliferation of the pancreas.

MATERIALS AND METHODSCell Lines and Antibodies. MCF-7 and MDA MB231 were obtained

from EG & G Mason Research Institute, Breast Cancer Human CellCulture Bank, Worcester, MA. Embryonic lung fìbroblastsWI38 andHEL were purchased from Flow Laboratories, McLean, VA. Lungcarcinoma line A549 (5) and human foreskin fibroblast HSF wereobtained from the American Type Culture Collection, Rockville, MD.The remaining cell lines were obtained in collaboration with the late J.Fogh, Sloan-Kettering Cancer Institute, Rye, NY. All cell lines weremaintained in DMEM supplemented with 10% FCS and 1% L-gluta-mine. MAb 19-9 was generously supplied as a crude hybrid supernatantfluid by Dr. Z. Steplewski, Wistar Institute (Philadelphia, PA). MAbto CEA was purchased from Hybritech (La Jolla, CA; Catalogue No.1022).

Hybridonta Production. Ten 6-8-week-old BALB/c x C57BL/6 F,mice were inoculated i.p. on days I, 30, 60, and 90 with IO7 viableA549 cells. Three days prior to fusion, mice were boosted i.p. with IO7

viable cells. Fusion of splenocytes with P3X63 Ag8.653 mouse myeloma cells was performed as described (6). Approximately 2-3 weeksafter fusion, the hybrid culture supernatants were screened in a fixed-cell ELISA.

Purification of Antibodies and Coupling to Sepharose. The hybridomassecreting 47D10 antibodies was cloned twice in soft agar and inoculatedinto pristane-primed BALB/c x C57BL/6 mice for asdics production.The ascites fluids were precipitated with ammonium sulfate (pH 8.0),redissolved in 50 HIMTris-HCl, and dialyzed against the same. Theantibodies were then purified by fast protein liquid chromatography.Antibodies, 10-20 mg, in 50 mM Tris-HCl, pH 8.0, were loaded ontoa Mono Q column (Pharmacia, Piscataway, NJ) and eluted with 0-500mM NaCl in the same buffer. The peaks at 170-190 mM NaCl werepooled and analyzed for specific antibodies in a cellular ELISA. Antibodies were conjugated to Sepharose 4B using the procedure of Cua-trecasas (7).

Fixed Cell ELISA. Cell monolayers were prepared by dispensing 5x IO4cells/well onto 96-well microtiter plates. After incubation overnight at 37°C,the cells were fixed with methanol, washed with PBS

(pH 7.2), and blocked with PBS-1 % BSA. Fifty ¿tlof hybrid supernatantfluids were added per well and incubated for 1 h at 37°C.The plates

were washed three times with PBS, and 50 ^1of horseradish peroxidase-conjugated sheep F(ab')2 anti-mouse immunoglobulin (DuPont-NEN,

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DISTRIBUTION AND CHARACTERIZATION OF 47D10 GLYCOPROTEIN

Boston, MA) were added per well. After incubation at 37°Cfor 1 h, the

plates were washed three times with PBS, and the enzyme was detectedby the addition of 100 n\ of o-phenylenediamine and 0.15% hydrogenperoxide. All antibodies were diluted in PBS-1% BSA. In some experiments, the methanol-fixed cells were treated with neuraminidase(Cooper Biomédical,Malvern, PA; 1 unit/ml in 0. l M sodium acetatebuffer, pH 5.0) for 16 h at 37*C before being used in ELISA. Control

cells were treated with acetate buffer only.Immunohistochemical Analysis. Immunoperoxidase studies used the

avidin-biotin complex system (Vector Laboratories, Burlingame, CA)as described previously (8). Human tissues were formalin fixed beforebeing embedded in paraffin. 47D10 and the class-matched, negativecontrol MOPC 21 (Litton Bionetics, Rockville, MD) were used at 0.5jig/slide.

Immunofluorescent Staining. For one-color immunofluorescence ofcell lines, 2 x IO6cells were incubated with 1 ^g of 47D10 or MOPC21 mouse myeloma (Litton Bionetics) in 100 ¿tlof Hanks' balanced salt

solution (Gibco, Grand Island, NY) supplemented with 2% PCS and0.1% sodium azide for 30 min. The cells were then washed three timesin this medium and 100 v\ of fluorescein-conjugated sheep F(ab')2 anti-

mouse immunoglobulin (DuPont-NEN; diluted 1:100) were added.After another incubation of 30 min, the cells were washed three times,resuspended in PBS-1% formalin, and analyzed by an EPICS V flowcytometer (Coulter, Hialeah, FL). All incubations were at 4°C.

For two-color immunofluorescence of peripheral blood cells, buffycoat cells were depleted of erythrocytes by lysis in 10 volumes of 17mM Tris-HCl-144 HIM ammonium chloride, pH 7.2. The leukocyteswere first stained with 2 Mg/ml of mouse MAb to granulocytes, T-cells,B-cells, or monocytes (DuPont-NEN) as described above. Then 10 tig/ml of normal mouse IgG were added to block unoccupied sites of thefluorescein-conjugated sheep F(ab')2 anti-mouse immunoglobulin. The

cells were then stained with biotinylated 47D10 for 1 h, washed insaline, and incubated with avidin-rhodamine. Cells were fixed andanalyzed as above. Biotinylated 47D10 were prepared as described (8).Briefly, 0.25 ml containing 1 mg of activated W-hydroxysuccinimidebiotin (Sigma) in dimethyl sulfoxide was mixed with 2 ml of 47D10 at1 mg/ml of 0.1 M NaHCO3, pH 9.O. The mixture was incubated atroom temperature for 4 h and dialyzed against PBS.

Radiolabeling of Cells and Immunoprecipitation. For biosyntheticlabeling with [35S]methionine, 1.5 x 10' cells were labeled with 400 ¿iCi[35S]methionine (DuPont-NEN) for 6 h as described (9). Radiolabeledcells were solubilized with ice-cold 10 mM Tris-HCl-140 mM NaCl-1%Triton X-100-0.5% deoxycholate-0.1 % SDS-1 mM EDTA-1 mMphen-

ylmethylsulfonyl fluoride, pH 7.5. All lysates were centrifuged at 8000x g for 15 min and the supernatants were precleared with a mixture ofSepharose conjugated with normal mouse immunoglobulin and mock-conjugated Sepharose before use. In some experiments, cells werelabeled in the presence of 0.1-8.1 ¿ig/mlof tunicamycin (BoehringerMannheim, Indianapolis, IN). Tunicamycin was dissolved in 25 mMsodium hydroxide to 1 mg/ml and various amounts of tunicamycinwere added to the cells 2 h before the addition of [35S]methionine. Forpulse-chase labeling, cells were preincubated with methionine-freeDMEM for 30 min before the addition of 400 nC\ [35S]methionine.

After a pulse of 1 to 30 min, cells were either solubilized as above orchased with 5 ml of DMEM supplemented with 10% PCS at 37°Cfor

various times before solubilization. Similar procedures were used forlabeling with tritiated leucine, lysine, or valine (DuPont-NEN) exceptthat DMEM, depleted of the corresponding amino acid, was used asthe labeling medium. For each 1.5 x 10* cells, 200 tiC\ tritiated amino

acid in 2 ml media were used. For labeling of carbohydrates, cells werepreincubated with RPMI with 80 g/liter glucose (low-sugar media)supplemented with 5% each of dialyzed and undialyzed PCS for 1 h.Then 200 ßCiof tritiated mannose, glucosamine, or fucose in 2 ml low-sugar media were added per dish. The cells were incubated at 37°Cfor

24 h before washing and solubilization. Immunoprecipitations usingMAb coupled to Sepharose 4B were performed as described (9).

Treatment with Endoglycosidases. Digestions with endo H (MilesScientific, Naperville, IL) were performed as described (10). Sampleswere digested with 0.025 or 0.5 unit/ml of enzyme for 18 h at 37°C.

For treatment with endo F, a modification of a procedure described by

Kanellopoulos et al. (11) was used. Immune precipitates were elutedfrom the Sepharose by boiling in 20 /tl of 100 mM Tris-HCl, pH 7.4,containing 1% SDS and 1% 2-mercaptoethanol, followed by additionof 20 M!of endo F (DuPont-NEN) to give a final enzyme concentrationof 0.15 or 10 units/ml. The mixture was incubated for 18 h at 37°C.

Proteins digested with endo H or endo F were precipitated with 5volumes of ice-cold ethanol. The precipitates were dried under nitrogenand redissolved in reducing sample buffer for analysis by SDS-PAGE.

RESULTS

Reactivity of 47D10 with Cell Lines. MAb 47D10 was derivedby fusion of spleen cells from mice immunized against the lungadenocarcinoma line, A549. Initial screening by fixed-cellELISA revealed that 47D10 was strongly reactive with methanol-fixed A549 cells, but not with normal fibroblasts, HSF. Atitration of 47D10 on these two lines is shown in Fig. 1. Half-maximal binding to A549 cells was achieved using 0.05 ¿ig/mlof 47D10 MAb. Isotyping indicated that 47D10 was an IgGlMAb.

To study the distribution of 47D10 on lines derived fromother tumors, 47D10 was evaluated on a panel of cell lines(Table 1). The 47D10 MAb was reactive with lung carcinomalines A549 and SW900; breast carcinoma lines ZR.75-1, BT20,and MCF-7; colon carcinoma line LS174T; melanoma linesG361 and Mel-1; as well as pancreatic tumor lines RWP-1 andRWP-2. Other lines derived from breast, lung, and adrenalcortex tumors were negative. No reactivity was observed withIgGl control antibody, MOPC 21 (not shown).

47D10 Antigen is Surface Associated. To establish whether47D10 was a cell surface antigen, the antibody was tested forbinding to live A549, SW900, or HSF cells using the fluorescence-activated cell sorter. Data in Fig. 2 showed that 47D10bound 77.3% of the A549 cells, 74.2% SW900 cells, but only2% of HSF cells. The binding of HSF cells with 47D10 wasequivalent to MOPC 21 control. Binding of A549 and SW900was also observed when viable adherent cells were used inimmunofluorescence and in ELISA (data not shown). Thesestudies showed that the 47D10 MAb recognized a determinanton the cell surface.

Immunoperoxidase Staining of Formalin-fixed Tissues. Todetermine its utility in immunohistochemistry, 47D10 wastested for binding to tissue sections using an avidin-biotinimmunoperoxidase system. Initially, A549 cells were cytocen-trifuged onto glass slides, air dried, and stained with 47D10either before or after fixation with formalin. No decrease in

i.o 10

Concentration of 47D10 Antibody /¿g/ml

Fig. 1. Titration of 47D10 MAb on A549 lung tumor cells and HSF normalfibroblasts. Binding of indicated concentrations of 47D10 MAb to met hanoi-fixed AS49 or HSF cells was determined by ELISA as described in "Materialsand Methods." Binding by equivalent concentrations of MOPC 21, the control

IgG antibody, was less than 0.2 absorbance unit.

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DISTRIBUTION AND CHARACTERIZATION OF 47D10 GLYCOPROTEIN

Table 1 Reactivity of47DIO with tumor cell lines as determined by fixed cellELISA

Binding of 2 Mgof 47D10 to methanol-fixed cells was determined in a cellularELISA as described in "Materials and Methods." Binding by 2 fig of control

IgGl antibody, MOPC 21, was less than 0.2 absorbante unit on all cell lines.

Origin of celllineLung

carcinomaBreast

carcinomaColon

carcinomaDesignationA549SW900SK-Lu-19812SW1573SW1271Calu

1SK-MES-1SW161Calu

6SHP-77ZR.75-1BT20MCF-7T47DMDA.MB.231WiDrLS174TAbsorbante"1.21.40.20.10000.10.1001.00.90.5001.41.5

Adrenal cortexSWI3Melanoma

G361Mel-1Pancreatic

carcinoma RWP-1RWP-2Normal

fibroblasts WI38HSF00.7

1.01.8

1.90

0"Net absorbance units at 488 nm.

47D10 reactivity was observed after fixation. Formalin-fixed,paraffin-embedded A549 cell pellets were also strongly reactivewith 47D10 (2-3+), but not with MOPC 21 (data not shown).Therefore, the reactivity of 47D10 was evaluated on a varietyof formalin-fixed, paraffin-embedded neoplastic (Table 2) andnonneoplastic (Table 3) human tissues. The 47D10 MAb recognized adenocarcinoma arising from pancreas, bile duct, colon, breast, lung, bile duct, ovary, and prostate. Nonadenoma-tous cancers were not reactive. Staining of the malignant ductalepithelial elements by MAb 47D10 was observed with 35 of 37pancreatic adenocarcinomas (Table 2). The percentage of positive cells in each case ranged between 30 and 95%. The majorityof cases were 2-3+ in intensity with a few cases showing theless intense 1+ reactivity. Staining was mostly limited to thecytoplasm (Fig. 3). MAb 47D10 was also reactive with 2 of 4papillary cystadenocarcinomas, as well as 3 of 3 mucinouscystadenocarcinomas and 2 of 7 periampullary adenocarcinomas of pancreatic origin. None of the 11 cases of islet celltumors was reactive with 47D10. Staining by 47D10 was seenin both primary and metastatic foci. There was no correlationbetween 47D10 expression and the grade of differentiation ofthe tumors. MAb 47D10 was unreactive with regenerativeductal epithelial cells seen in chronic pancreatitis (7 cases)(Table 2). Normal pancreatic ductal acinar and islet cell constituents also failed to stain with 47D10 (Fig. 3). In sections oflymph nodes infiltrated with metastatic pancreatic adenocarcinoma, only the tumor cells, but not the surrounding lymphoidelements, were stained.

Six cases of bile duct adenocarcinoma were tested and werefound to be strongly reactive (3+) with 47D10 MAb. Nine of11 colon adenocarcinomas (1-3+) and 3 of 4 villous adenomas(3+) were stained by 47D10 (Table 2). There was weak stainingof some (3 of 11 cases) normal colon tissues (Table 3). In thecase of breast tumors, 4 of 7 cases of infiltrating ductal carci-

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A549 77.3%

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SW900 74.2%

HSF 2.0%

FLUORESCENCE INTENSITY (LOG,0)

Fig. 2. Immunofluorescent flow cytometric analysis of 47D10 antigens onA549, SW900, and HSF cells. Cells were first labeled with 47DIO ( ) orMOPC 21 control ( ) antibody (1 ¿ig/2x 10' cells), followed by fluorescein-conjugated goat anti-mouse immunoglobulin. The labeled cells were analyzed byEPICS V flow cytometer. Data are plotted as cell number versus log greenfluorescence. The percentages of cells stained by 47D10 above the MOPC 21background are indicated.

noma showed weak to moderate staining, whereas tissues fromfibrocystic diseases and fibroadenomas were not reactive. Ad-enocarcinomas of lung were positive in 5 of 8 cases (2-3+).Squamous cell lung carcinoma, oat cell lung carcinoma, chronicpneumonitis tissues, and normal lung were negative.

In addition to normal colon, breast, and pancreas, severalother nonneoplastic tissues were also tested. Normal liver,breast, and kidney were negative for 47D10. However, 11 of 17specimens of spleen were reactive. The positive cells in thespleen were polymorphonuclear leukocytes by morphology. Thelymphoid and erythroid elements were negative for 47D10.

Cross-Reactivity of 47D10 with Granulocytes. Since immu-noperoxidase results suggested that 47D10 was reactive withgranulocytes in the spleen, we utilized flow cytometry to definethe lymphoid cells which were reactive with 47D10. Dualstaining of peripheral blood leukocytes with 47D10 and 1G10(12), a MAb against granulocytes, indicated that 47D10 reactedstrongly with granulocytes (Fig. 4). Parallel two-color immu-nofluorescent studies involving 47D10 and M Ab against otherleukocyte antigens showed that 47D10 was not present on T-cells, B-cells, and monocytes (not shown). In addition, peripheral blood leukocytes from eight normal donors were examinedby one-color immunofluorescence. All eight samples were positive with 19-31% of the cells stained by 47D10 (not shown).This suggests that the 47D10 antigen on granulocytes is notpolymorphic. In other experiments, the reactivity of 47D10with erythrocytes was examined by agglutination and indirectimmunofluorescence. Red cells from A, B, and O blood groups

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DISTRIBUTION AND CHARACTERIZATION OF 47D10 GLYCOPROTEIN

47D10

1G10

1G10 + 47D10

Fig. 4. Two-color immunofluorescent staining of peripheral blood leukocytewith 1G10 antigranulocyte or 47D10 MAb. Peripheral blood leukocytes werelabeled with IG10 (top right), biotin-conjugated 47D10 (bottom left), or bothMAb (bottom right) as described in "Materials and Methods." Data are plotted

as cell number (vertical) versus green fluorescence (1G10 expression) versus redfluorescence (47D10 expression).

Antibody Dilution (fold)Fig. 5. Effect of neuraminidase treatment of methanol-fixed SW900 cells on

the binding of MAb 47D10 and 19-9. Methanol-fixed SW900 cells were treatedwith neuraminidase (I unit/ml in acetate buffer) (•,A) or acetate buffer only (O,A). Binding of indicated concentrations of MAb 47D10 (•,O) and 19-9 (A, A)on the treated cells was determined by ELISA. Points, averages of duplicates aftersubtraction of background (0.073 ±0.01 absorbance unit); bars, SD.

reproducibly titratable and exquisitely sensitive to neuraminidase. These results are consistent with previous findings showing that 19-9 antigen is a monosialoganglioside and that sialicacid residues are involved in 19-9 antibody binding (3).

Biochemical Characterization of 47D10 Antigen. To identifythe 47D10 antigen, immunoprecipitations were performed.A549 cells, the immunogen, were vectorially labeled with 125Ior metabolically labeled with [35S]methionine. The radiolabeled

cells were detergent solubilized and incubated with 47D10 MAbconjugated to Sepharose 4B. The 47D10 antigen from bothI25I- and [35S]methionine-labeled cells appeared as a diffuseband with a molecular weight of 67,000-98,000 when analyzedby SDS-PAGE (Fig. 6). Therefore, the 47D10 polypeptideswere synthesized by the A549 tumor cells and expressed on thecell surface. Migration of the [35S]methionine-labeled antigenin the presence of 2-mercaptoethanol was similar to that observed in the presence of iodoacetamide (Fig. 6). The similarmobility of the antigen under reducing and nonreducing conditions suggests the absence of intrachain or interchain disulfidebonds. Immunoperoxidase studies on tissues showed that47D10 antigen was strongly expressed by pancreatic carcinoma.To determine whether pancreatic tumor lines also express the47D10 antigen, PANC-1, MIA, RWP-1, and RWP-2 were

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t- [35s] Met —'Fig. 6. SDS-PAGE of immunoprecipitates from '"!- or ("Sjmethionine-

labeled A549 cells. A549 cells were labeled with 125Ior [35S]tnethionine, detergentsolubilized, and immunoprecipitated with 47D10 conjugated to Sepharose CL-4B. The immunoprecipitates were treated with reduced [+5% 2-mercaptoethanol(ME)] or nonreduced [+ 50 min iodoacetamide (IAA)} sample buffer at 100°Candsubjected to SDS-5-15% PAGE. Controls were immunoprecipitated with MOPC21 conjugated to Sepharose CL-4B. The molecular weight standards were myosin,phosphorylase />'.BSA, ovalbumin, carbonic anhydrase, and cytochrome c.

labeled with [35S]methionine and used for immunoprecipitation.RWP-1 and RWP-2, but not PANC-1 or MIA, were found tosynthesize 47D10 antigens with molecular weights similar tothat found in A549 cells (not shown).

The diffuse appearance of the 47D10 antigen in SDS-PAGEsuggests that it may be glycosylated. To explore the nature ofthis antigen, we metabolically labeled A549 cells with tritiatedfucose, mannose, and glucosamine. As seen in Fig. 7, all threesugars were incorporated into the 47D10 polypeptides. Migration of the sugar-labeled antigen was similar to that of ["Sinici h¡<mine-labeled antigen. These results indicate that 47D10antigen is a glycoprotein. To study the carbohydrates of the47D10 antigen, the effect of tunicamycin on its biosynthesiswas examined. Tunicamycin blocks the formation of jY-acetyl-glucosaminylpyrophosphoryldolichol, thereby inhibiting N-linked glycosylation (reviewed in Ref. 13). When used at 0.1Mg/ml, tunicamycin partially inhibited the formation of the47D10 antigen (Fig. 8). Inhibition was complete at greater than0.3 ng tunicamycin per ml. Nonspecific polypeptides with molecular weights of 33,000, 35,000, and 44,000 were observed intunicamycin-treated cells after immunoprecipitation with47D10 as well as MOPC 21. There was an increase in intensity

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DISTRIBUTION AND CHARACTERIZATION OF 47D10 GLYCOPROTEIN

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Fig. 8. Effect of tunicamycin on immunoprecipitation of 47D10 antigen from¡"Sjmethionine-labeled A549 cells. A549 cells were labeled with ["Sjmethionine

in the presence or absence of tunicamycin, detergent solubilized, and used forimmunoprecipitation with 47D10 MAb or MOPC 21 control antibody. Theconcentrations of tunicamycin used were: Lane 1, 8.1 *ig/ml; Lane 2, 2.7 (ig/ml;Lane 3, 0.9 Mg/ml; Lane 4, 0.3 Mg/ml; Lane 5, 0.1 nu ml: Lane S, none. Lanes 6and 7 were treated with buffers in which equivalents of 8.1 and 2.7 ^g oftunicamycin per ml were dissolved, respectively.

-30

47DIO ControlFig. 7. SDS-PAGE of immunoprecipitates of A549 cells labeled with [35S]-

methionine. tritiated fucose. mannose. or glucosamine. Detergent lysates of A549cells labeled with ["SJmethionine, [3H]fucose, [3H]mannose, or [3H]glucosamine

were immunoprecipitated with 47D10 or MOPC 21 control antibodies conjugatedwith Sepharose CL-4B. The immunoprecipitates were reduced in sample buffercontaining 5% 2-mercaptoethanol and subjected to SDS-PAGE and fluorography.

of a MT 82,000 polypeptide with increasing concentrations oftunicamycin. This polypeptide might not be related to 47D10because smaller amounts of a MT 82,000 band were also seenin the MOPC 21 immunoprecipitates. In parallel experiments,the biosynthesis of 89E5, a keratin-like nonglycosylated antigenwith molecular weights of 53,000 and 45,000, was not inhibitedby 0.1-8.1 Mgtunicamycin per ml.'

Biosynthesis of 47D10 Antigen. Biosynthesis of the 47D10antigen was studied by pulse-chase labeling. Two polypeptideswith molecular weights of 67,000 and 69,000 were immunoprecipitated from A549 cells labeled for 1 min with [35S]methionine

(Fig. 9). The intensity of these polypeptides increased when thetime of pulse was increased to 3, 5, and 10 min. When the cellslabeled for 10 min were chased for 0.25, 0.5, and 1 h, theamounts of these precursor polypeptides decreased. Concomi-tantly, there was an increase in polypeptides of higher molecularweight. Similar observations were made when cells were labeled

for 30 min followed by chase periods of 0.5, 1.5, and 4.5 h.Essentially all the M, 67,000 and 69,000 precursors were processed to higher molecular weight forms by 1.5 h of chase.Therefore, the higher molecular weight forms of the 47D10antigen were derived from intermediates with molecular weightsof 67,000 and 69,000.

To study the carbohydrates on the intermediates as well asthe mature 47D10 antigen, immunoprecipitates from pulse-chase-labeled A549 cells were treated with endoglycosidases.Carbohydrates with high mannose-type side chains have beenshown to be sensitive to endo H and endo F. In contrast, themore mature, complex carbohydrate side chains are susceptibleonly to endo F ( 14, 15). As seen in Fig. 10/4, the M, 67,000 and69,000 intermediates obtained by pulsing A549 cells for 0.5 h(Lane 1) were digested by endo H, 0.025 unit/ml, to M, 44,000,41,000, and 39,000 polypeptides. At 0.5 unit/ml, ofendo H,the predominant degradation product was M, 39,000. A M,47,000 band was observed in all immunoprecipitates with47D10 but the same polypeptide was found in control precipitates with MOPC 21 (see controls for Figs. 5, 7, and 8).4 After

a chase period of 0.5 h, some of the precursors were processedto higher molecular weight forms (Lane 2). Digestion with endoH, 0.025 unit/ml, resulted in a downward shift in the averagemolecular weight of the larger antigens which was accompaniedby the appearance of two polypeptides with molecular weightsof 41,000 and 39,000. At higher concentration (0.5 unit/ml) ofendo H, only the M, 39,000 polypeptide was seen. Decreasingamounts of the M, 46,000 and 39,000 polypeptides were foundwith digestion of antigens obtained after increasing times ofchase. This suggested that the higher molecular weight forms

' K. Kalo, unpublished data. * Unpublished results.

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DISTRIBUTION AND CHARACTERIZATION OF 47D10 GLYCOPROTEIN

A. 47D10 B. Control

Pulse (min) I 3 5 I 10 II 30—1360

Chose(tus) O O O O .255 I 2 24 0 .5 1545 0

I 3 5 I 10 II—30—1360

000 0 .25.5 I 2 24 0 .5 1.54.5 0Mr«IO3

Fig. 9. Biosynthesis of 47D10 antigen byA549 cells. A549 cells were labeled with [3!S]

methionine and chased for the indicatedlengths of time. Cell lysates were immunopre-cipitated with Sepharose conjugated with47D10 MAb or MOPC 21 control antibody.Immunoprecipitates were analyzed by 5-15%reduced SDS-PAGE.

Precursor- ..- .. •47D10

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-30

-I2

Fig. 10. Effects of endoglycosidases on47D10 antigens from A549 cells. A549 cellswere pulse-labeled with ["Sjmethionine for 0.5h (Lane 1) or 6 h (Lane 5). Cells labeled for0.5 h were chased for 0.5 (Lane 2), 1.5 (Lane3). or 4.5 h (Lane 4). Cell lysates were immu-noprecipitated with 47D10 MAb conjugatedto Sepharose CL-4B and treated with indicatedconcentrations of endo H or endo F as described in "Materials and Methods" beforeanalysis by reduced SDS-PAGE.

A. EndoH

i None—ii—0.5U/mHi-O.OZ5U/ml-i

123451234512345

B. EndoF

—None—11—10U/ml—it-0.!5U/ml-t

23451234512345

Precursor-i ...I ' III"1

••..

«.

Mr«IO3

-97

-69

. -46

-30

of the antigen was relatively resistant to endo H (Lanes 3 and for binding to 47D10 MAb, attempts were made to reimmu-4). The mature antigen obtained after 6 h (Lane 5) was onlyslightly digested by endo H.

The effect of treatment with endo F is shown in Fig. 105.The M, 67,000 and 69,000 intermediates obtained after a 30-

min pulse were partially digested by endo F, 0.15 unit/ml, tolower molecular weight forms ranging from M, 49,000 to72,000. Increasing the concentration of the enzyme to 10 units/ml resulted in a polypeptide of M, 39,000 (Lane 1). The highermolecular weight forms of the antigen obtained after chaseperiods of 0.5, 1.5, and 4.5 h (Lanes 2 and 4) were also reducedto A/, 39,000 by enzyme 10 units/ml. However, the intensity ofthe M, 39,000 polypeptide decreased with increasing times ofchase. With the antigen obtained after 6 h of labeling, a broadband with an average molecular weight of 48,000 was obtainedin addition to the M, 39,000 degradation product. With endoF, 8 units/ml, M, 45,000 intermediate product was also observed. In parallel experiments, human IgA2 was treated withendo H and endo F at concentrations used for treating 47D10antigens. No digestion of the protease-sensitive immunoglobu-

lin was observed (not shown). Therefore, digestion of the 47D10antigens was not due to contaminating proteases in the enzymepreparations. To determine whether carbohydrates are required

noprecipitate the polypeptides resulted from endo F treatment.However, none of these polypeptides could be reimmunoprecip-itated.

47D10 Antigen on Granulocytes. To study the 47D10 antigenon granulocytes, these cells were surface labeled with 125Ianddetergent-solubilized for immunoprecipitations. The 47D10 antigen on granulocytes appeared as a less diffuse band than thosefrom A549 cells (Fig. 11). Furthermore, the granulocyte antigens showed a higher molecular weight of 150,000-180,000.No antigen was detected when an isotype-matched control,MOPC 21, was used in these studies (not shown). To comparethe antigen from granulocytes with that from A549 tumor cells,I25l-labeled 47D10 from both cell types were treated with endo

F. Digestion with endo F, 0.15 unit/ml, led to a slight decreasein the molecular weight of the antigens from both A549 cellsand granulocytes. At 10 units endo F per ml, the 47D10antigens from A549 cells were digested to a broad band at M,48,000 and a polypeptide of M, 39,000. In contrast, the degradation products from granulocytes showed a lower molecularweight of 23,000-27,000 (Fig. 12).

Comparison of 47D10 with Anti-CEA MAb. The associationof 47D10 with carcinoma cells and granulocytes is reminiscent

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DISTRIBUTION AND CHARACTERIZATION OF 47D10 GLYCOPROTEIN

O

O

MrxlO*

-200

-97

-69

-46

-30

Fig. 11. SDS-PAGE of 47D10 antigen from '"I-labeled A549 cells and

granulocytes. AS49 cells and human peripheral blood granulocytes were labeledwith '-"'I. detergent solubilized. and immunoprecipitated with 47D10 MAb con

jugated to Sepharose CL-4B. The immunoprecipitates were eluted in reducedsample buffer and analyzed by SDS 5-15% PAGE.

of the expression of another tumor-associated glycoprotein,CEA. The molecular weight of CEA, 180,000, is higher thanthat of 47D10 antigens. However, CEA-related antigens withmolecular weight of 160,000, 90,000, 50,000, and 40,000 havebeen described (16, 17). To examine the relationship of 47D10MAb and anti-CEA, ELISA was performed on two clones ofSW900, a human lung adenocarcinoma line. We have foundpreviously that the two clones, SW900-1 and SW900-2, expressdifferent levels of CEA.5 Table 4 shows that SW900-1 expressedat least 4 times more CEA than SW900-2 when determined bybinding of an anti-CEA MAb (Hybritech), 0.16-10 /xg/ml.However, the binding of 47D10 and 89E5, a MAb to keratin-like polypeptides,5 was similar for the two cell clones.

DISCUSSION

In this report, we described the distribution and the natureof a glycoprotein defined by the 47D10 MAb. In fixed cell

Gronulocytesi 2 3

BA5491 2 3

Mr

200-

97-

69-

46-

30-

Fig. 12. Effect ofendo F on 47D10 antigens from '"I-labeled A549 cells andhuman peripheral blood granulocytes. Lysates of '"I-labeled human granulocytes(.•I)and AS49 cells (B) were immunoprecipitated with 47D10 conjugated toSepharose < I 411. The immunoprecipitates were treated with (Lane I) buffercontrol with 10 units equivalent of endo F per ml; (Lane 2) endo F, 10 unit/ml;and (Lane 3) endo F, 0.1 S unit/ml; before analysis by reduced SDS-PAGE.

Table 4 Binding of47D10 and anti-CEA to melhanol-fixed SW900 cellsIndicated concentration of 47D10, Anti-CEA (Hybritech), 89E5, and MOPC

21 were used in cellular ELISA as described in "Materials and Methods." All

four MAb are of the IgG 1 subclass.

MAb47D10Anti-CEA89E5MOPC

21MAb

concentration (tm

ml)102.50.630.16102.50.630.16102.50.630.1610Absorbance"SW900-11.00.91.01.00.90.80.80.61.11.11.10.80.1SW900-20.80.90.90.80.20.10.20.11.11.11.11.10.1

* Ho. M K , unpublished observations.

" Net absorbance units at 488 nm.

ELISA, 47D10 reacted with cell lines derived from tumors ofthe breast, lung, colon, and pancreas. Similarly, immunoper-oxidase staining of tumor tissue sections showed the expression

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DISTRIBUTION AND CHARACTERIZATION OF 47DIO GLYCOPROTEIN

of 47D10 by pancreatic adenocarcinoma and tumors of thebreast, lung, and colon. Of particular interest was the strongassociation of 47D10 with pancreatic adenocarcinoma andcystadenocarcinoma, but not chronic pancreatitis or normalpancreas. The 47D10 M Ab cross-reacts with granulocytes, butnot with lymphocytes, monocytes, or erythrocytes.

The 47D10 antigen is expressed on the cell surface as evidentfrom the binding of 47D10 M Ab to live A549 and SW900 cellsby immunofluorescence and in ELISA. Furthermore, the47D10 antigen was vectorially labeled by U5I. Immunoprecipi-tations and SDS-PAGE showed that the molecular weight ofthe 47D10 antigen ranged from 67,000-98,000 (average,83,000). Incorporation of methionine, glucosamine, mannose,and fucose into the antigen suggested that it was a glycoproteinsynthesized by the cells. At least part of the oligosaccharide on47D10 was linked to asparagine (jY-linked) because the synthesis of the mature antigen was inhibited by tunicamycin. Pulse-chase labeling studies showed that the heterogeneous group of47D10 polypeptides with molecular weights ranging from67,000-98,000 were derived from two intermediates, M, 67,000and 69,000. These intermediates were processed in approximately 1.5 h to the mature forms. Endo H treatment reducedthe intermediates to lower molecular weight forms, M, 44,000,41,000, and 39,000. The mature form of the antigen wasrelatively resistant to endo H. In contrast, both the intermediates and mature antigen were degraded by endo F to polypeptides with molecular weights of 48,000 and 39,000. Therefore,the 47D10 antigen was synthesized from a precursor with amolecular weight of at least 39,000 which was glycosylated toform the M, 67,000 and 69,000 intermediates with high man-nose-type carbohydrates. These intermediates were in turn processed in 1.5 h to form the mature antigen with mostly complexoligosaccharide side chains. Because of the heterogeneity andhigh degree of glycosylation, it is difficult to accurately determine the molecular weight of the 47D10 antigen by SDS-PAGE. Assuming an average molecular weight of 83,000 forthe mature antigen and 39,000 for the precursor, up to 50% ofthe antigen could be carbohydrate by weight. Two observationssuggested that the precursor forms of the antigen were notreadily recognized by 47D10 M Ab: (a) the lower molecularweight forms of the antigen (those below M, 67,000 and 69,000)seen after endoglycosidase treatment were not detected in im-munoprecipitates of pulse-labeled cells or tunicamycin-treatedcells; (b) we failed to reimmunoprecipitate the endo F-digestedpolypeptides. The failure of the precursors to bind 47D10 M Absuggested that carbohydrates were part of the antigenic determinant or that conformation of the antibody-binding site wasaltered in the absence of carbohydrates. The carbohydratesinvolved are probably not sialic acid residues because the binding of 47D10 to SW900 cells was not affected by treatment ofthe cells with neuraminidase. Herlyn et al. (18) found that thecarbohydrate structure defined by the 19-9 M Ab is present onglycolipids as well as on glycomucins. It is not clear whetherthe 47D10 M Ab will react with glycolipids from tumor cells.

Previously, a number of MAbs have been shown to be associated with pancreatic adenocarcinoma. However, only a few ofthe antigens recognized by these MAbs were biochemicallycharacterized. Comparison of these antigens with the 47D10antigen suggests that the latter is distinct from previouslydefined antigens. The C54-0 MAb developed by Schmiegel etal. (19) recognizes a broad band of M, 120,000. However, incontrast to 47 D10, C54-0 was reactive with normal pancreaticducts but not with lung cancer tissues. The DU-PAN-2 antigenis a high molecular weight mucin which is not labeled by

radioactive amino acids whereas the 47D10 antigen is labeledby [35S]methionine and 125Iat tyrosine residues (20). AnotherMAb highly reactive with pancreatic tumors, 19-9, defines amonosialoganglioside on tumors of the gastrointestinal tract.Unlike 47D10, the 19-9 antigen was sensitive to neuraminidase(3, 21). The molecular weight of 47D10 (67,000-98,000) alsodistinguished it from antigens defined by MAb DU-PAN-1, Mr30,000-35,000 (4); DU-PAN-3, M, 70,000-75,000 (4); C1-N3,MT 135,000 and 105,000 (19); and AR2-20, M, 190,000 (22).

Therefore, 47D10 seems to be distinct from the other MAbagainst pancreatic tumors that have been described previously.Additional proof of the uniqueness of the 47DIO antigen willrequire cross-inhibition studies and sequential immunoprecipi-

tations.The cross-reactivity with granulocytes raises the possibility

that 47D10 may be against CEA. This is unlikely because: (a)binding studies on two SW900 clones show that they expresseddifferent levels of CEA but similar amounts of 47D10; (b) CEAin tumor tissues shows a molecular weight of 180,000, whichis much higher than the M, 83,000 calculated for 47D10 antigenon tumor cell lines; (c) CEA cannot be readily labeled by[35SJmethionine because it contains extremely low or undetect-

able levels of this amino acid (23, 24). In contrast, the 47D10antigen is well-labeled by [35S]methionine. The CEA-relatedantigens present on normal tissues, including polymorphonu-clear leukocytes, are broadly termed NCA. The various formsof NCA have molecular weights of 160,000, 90,000, 50,000,and 40,000, which are lower than that of CEA in carcinomatissues or cell lines (16, 17). Blaszcyk et al. (16) used an MAbC4-20-32 to identify a M, 50,000 NCA from human spleen,which contained large numbers of granulocytes. Muraro et al.(17) found a M, 90,000 NCA in spleen by immunoblotting withBl.l, a MAb to CEA. The higher molecular weight (150,000-180,000) of the 47D10 antigen on granulocytes suggests that itis distinct from the NCA defined by C420-32 and Bl.l. However, the possibility that 47D10 might recognize other forms ofNCA cannot be ruled out.

The 47D10 antigen on granulocytes has a different molecularweight than that on A549 cells. One approach to compareantigens from the two cell types is by comparing the intermediates in pulse-chase labeling experiments. However, we failedto identify the intermediates from granulocytes, probably because these cells did not actively synthesize surface antigens.Another approach for comparing antigens is by treatment withendoglycosidases. Endo F digestion of the 125I-labeled immu-noprecipitates resulted in a diffuse band at A/r 23,000-27,000,which was smaller than the polypeptides derived by similartreatment of I25l-labeled A549 antigens. One possible cause for

the difference in molecular weight of the polypeptides obtainedafter endo F digestion could be incomplete digestion of theA549 antigens. This was unlikely because the same M, 39,000polypeptide was seen even after digestion with higher concentrations ofendo F. Another possibility could be that the 47D10antigen from A549 cells had similar carbohydrate structuresbut different protein backbones than the antigen from granulocytes. Labeling of the antigens followed by peptide mappingmight shed light on the latter notion. If the two antigens dopossess different proteins, then it might be possible to raisesecond-generation MAb against purified 47D10 antigens fromA549 cells and select MAb reactive with tumor cells, but notwith granulocytes.

In view of its strong tumor association, 47D10 MAb may beuseful as an in vitro diagnostic agent, especially for pancreaticadenocarcinoma. For immunohistochemical staining of tumor

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DISTRIBUTION AND CHARACTERIZATION OF 47D10 GLYCOPROTEIN

tissues, the cross-reactivity of granulocytes should not pose aproblem because these cells are morphologically distinct fromtumor cells. However, the potential of 47D10 as an in vivoreagent will depend on the significance of granulocyte-bindingin vivo and the feasibility of producing second-generation MAbthat do not react with granulocytes.

ACKNOWLEDGMENTS

We are grateful to David Dombkowski for expert technical assistancein flow cytometry.

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1987;47:241-250. Cancer Res   May-Kin Ho, Kenneth P. Kato, Paul J. Durda, et al.   GlycoproteinBiosynthesis of 47D10, a Tumor-associated Surface Tissue Distribution, Immunochemical Characterization, and

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