membrane-associated glycoprotein (gploo) identified on ... · the 5e8 antibody and the m, 160,000...

[CANCER RESEARCH 46, 6446-6451, December 1986]

Membrane-associated Glycoprotein (gploO) Identified on Human Lung Tumors bya Monoclonal Antibody1

Samuel Zylstra, Fang-An Chen, Swapan K. Ghosh, Elizabeth A. Repasky, Urna Rao, Hiroshi Takita, andRichard B. Bankert2

Departments of Molecular Immunology Â¡S.Z., F-A. C., S. K. G., E. A. R., K. B. B.J, Pathology Â¡U.K.J, and Thoracic Surgery [H. T.], Roswelt Park Memorial Institute,New York State Department of Health, Buffalo, New York 14263 and T and B Bioclone, Inc., Â¡E.A.R.,H. T., R.B.B.], Buffalo, New York Â¡4212

ABSTRACT

A monoclonal antibody (5E8) has been used to identify and structurallycharacterize a previously unreported macromolecule present on the surface of human lung tumors. This antibody was derived from a hybridclone that was produced using spleen cells of mice immunized with asurgically excised squamous cell carcinoma. Using immunofluorescence,the 5E8 antibody was observed to stain many different human lung tumorcell lines and surgically excised human lung tumors including squamouscell carcinomas, adenocarcinomas, alveolar carcinomas, and a portion ofthe large cell tumors tested. With few exceptions, notably the basal layerof the skin, little or no detectable staining of 5E8 to normal humantissues (lung, brain, kidney, heart, stomach, breast, erythrocytes, orlymphocytes) was observed. The 5E8 antibody was used to immunopre-cipitate detergent lysates of biosynthetically labeled or surface radioiodi-nated lung tumors. Analysis of the immunoprecipitates by sodium dodi-evi

sulfate gel electrophoresis revealed a major band and a faster migratingsecond minor band. The molecular weights of these two proteins wereestimated to be 160,000 and 120,000, respectively. The addition of areducing agent to the gels did not alter the migration pattern of theimmunoprecipitated macromolecules. The removal of a terminal carbohydrate, sialic acid, did not restrict the binding of 5E8 to the tumor-associated antigen. However, labeling studies using galactose oxidaseand tritiated borohydride revealed the presence of galactose on theimmunoprecipitated protein. This major V/r 160,000 glycoprotein thatwas identified on two different human lung tumor cell lines was alsofound on a human large cell tumor tissue obtained by surgical biopsy.The 5E8 antibody and the M, 160,000 glycoprotein that it recognizesrepresent two very useful components with which to test several newantibody-mediated drug delivery systems in the treatment of human lungtumors. The tumor-associated glycoprotein also represents a potentialanal) u- for a diagnostic or prognostic immunoassay for lung cancer.

INTRODUCTION

Several investigators have produced monoclonal antibodiesspecific for human lung tumor-associated antigens and haveidentified glycoproteins with molecular weights ranging from40,000 to 149,000 (1-5). The immunogens used for all of thesestudies were cultured human tumor cell lines. A possibilityexists that, in using cultured cell lines (both as the immunogenand for the initial screening test), one may overlook potentiallyimportant tumor-associated molecules that are present on freshtumors, but are decreased in their expression during cell culture.In a previous study, we established a hybridoma production andscreening protocol in which human lung tumor tissues obtainedfrom surgery (squamous cell and adenocarcinoma) were usedas the immunogen and target cells in the screening assay (6).Using a monoclonal antibody, 5E8, obtained with this approach, we report here the identification of a new lung tumor-

Received5/20/86;revised8/29/86;accepted9/2/86.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.

1This work was supported in part by grants from the NIH, CA33462 andContract N43-CM-57779.

7To whom requests for reprints should be addressed.

associated glycoprotein (gploO3) that is membrane associated

and is present on a large percentage of primary human lungcancers. In addition to characterizing this new lung tumor-associated antigen, we report the reactivity pattern of the 5E8monoclonal antibody with 5 histologically distinct lung tumortypes, 11 non-lung tumor biopsies, and a large panel of normalhuman lung tissues. Based upon its size, its location on the cellsurface, its expression on a large percentage of primary lungtumor types, and its limited expression on normal human tissue,gploO represents a viable target for the immunospecific deliveryof cytotoxic drugs.

MATERIALS AND METHODS

Monoclonal Antibodies. Monoclonal antibodies specific for the gpl60on human lung tumors were obtained from the hybridoma clone 5E8.This hybridoma was produced by the fusion of the drug-resistantmyeloma X63-Ag8.653 with spleen cells from a BALB/c mouse immunized with a poorly differentiated squamous cell carcinoma of thelung obtained by surgical biopsy. The initial specificity of the 5E8monoclonal antibody was established using a very sensitive immuno-cytoadherence assay (6). The 5E8 antibody was isotyped by Ouchterlonyanalysis and by a radioimmunoassay as a -,, heavy chain and * lightchain molecule. A control murine monoclonal antibody (2C3) specificfor the hapten phthalate was also isotyped as a 71, Kmolecule. Anothercontrol monoclonal antibody 5C7 was prepared by the fusion of X63-Ag8.653 with spleen cells from mice immunized with another humanlung tumor (adenocarcinoma) obtained by surgical biopsy. The specificity of 5C7 antibody was reported previously (6).

Monoclonal antibodies were precipitated by the addition of onevolume of a saturated solution of ammonium sulfate to an equal volumeof serum or ascites fluid from mice bearing the hybridoma, 5E8, 5C7,or 2C3. The 2C3 antibody was isolated by affinity chromatographyusing an affinity column containing phthalate coupled to Sepharose 4B(7). The 7-globulin precipitates containing monoclonal antibodies 5E8and 5C7 were dissolved in borate-buffered saline, pH 8, and afterextensive dialysis the mouse immunoglobulin was bound to an affinitycolumn containing rabbit anti-mouse -,, specific antibodies coupled toSepharose 4B (8). The mouse immunoglobulin was eluted from thecolumn with 0.2 M glycine-HCl, pH 2.8.

Human Tumor Cell Lines and Tissues. Human lung cancer cell linesused in this study include PC-3, an adenocarcinoma (9), PC-9, a poorlydifferentiated adenocarcinoma (10), PC 10, a squamous cell carcinoma,and A549, an alveolar cell tumor (10). The non-lung tumor cell linesthat were used include MCF-7, an adenocarcinoma of breast (11), HT-29, an adenocarcinoma of the colon (12), PANC-C and P17, eachadenocarcinomas of pancreas, and HeLa, a squamous carcinoma of thecervix.

Other human tissues including lung tumors, non-lung tumors, andnormal human tissues were obtained by surgical biopsy of patients fromthe RPMI clinic.

Radiolabeling of Cells and Immunoprecipitation. Cell surface labelingwith I251and biosynthetic labeling with [35S]methionine were performed

as previously described (13, 14). The labeled cells were washed andthen lysed in phosphate-buffered saline containing 1% NP-40, 0.5 M

3The abbreviations used are: gploO, M, 160,000 glycoprotein; SDS, sodiumdodecyl sulfate; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis;PBS, phosphate-buffered saline.

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LUNG TUMOR-ASSOCIATED ANTIGEN

Fig. 1. Staining of PC-9 cells by fluorescence microscopy. A, staining of PC-9 cells by monoclonal antibody 5E8. The staining pattern reveals numerous punctatesites of fluorescence at the surface of most cells. The region of membrane above the periphery of the nucleus is shown in focus. In addition, the antibody showspreferential accumulation at membrane surfaces involved in the formation of ruffles at the periphery of the cell (arrow). B, staining of PC-9 cells (a poorly differentiatedadenocarcinoma cell line) with normal mouse serum yielding a negative staining reaction.

EDTA, and 1 HIMphenylmethylsulfonyl fluoride. Following centrifuga-tion, the soluble material from the cell lysate was immunoprecipitatedaccording to the following protocol. Twenty p\ of either 5E8, 5C7, ornormal mouse serum (at a concentration of 0.8 to 1.0 mg/ml) wereadded to 500 n\ of the radiolabeled cell lysates. After a 12- to 14-hincubation at 4"( '. 10 /il of rabbit anti-mouse immunoglobulin anti-serum (2 mg/ml) were added and incubated for 15 min at 4Â°C.Precip

itation of the immune complex was accomplished by the addition of100 n\ of 10% suspension of Staphylococcus aureus microorganisms.As indicated in "Results," some samples were precleared with normal

mouse serum prior to immunoprecipitation with monoclonal antibody.This was accomplished by adding normal mouse serum to the radiolabeled lysate, followed by rabbit anti-mouse immunoglobulin and 5.aureus. The resultant supernatant was then immunoprecipitated by theaddition of 5E8 antibody, the rabbit anti-mouse immunoglobulin, andS. aureus as indicated above.

SDS-PAGE Analysis. One-dimensional SDS-PAGE analysis andautography/fluorography were performed as previously described (14,15). The molecular weight standards were l4C-labeled proteins andincluded cytochrome c (M, 12,300), /3-lactoglobulin (M, 18,400), a-chymotrypsinogen (M, 25,700), ovalbumin (M, 43,000), bovine serumalbumin (M, 68,000), phosphorylase ft (M, 92,500), and myosin H-chain (M, 200,000).

Neuraminidase Treatment and Carbohydrate Labeling of Lung TumorCell Lines. PC-9 and A549 cells were suspended in 1 ml of 0.1 MNaHPO4 buffer, pH 6, at a concentration of 5 x IO7cells/ml. The cellsuspensions were incubated at 37Â°Cfor l h with 50 units of neuramin-

idase (Vibriocholerae from GIBCO, Grand Island, NY). The labelingof cell surface carbohydrates was accomplished as previously reported(16). Neuraminidase-treated cells were incubated with 15 ng of galactose oxidase (Sigma Co., St. Louis, MO) at 37Â°Cfor 2.5 h. The cells

were then pelleted and resuspended several times to remove the galactose oxidase.

The washed cells were next incubated with 50 n\ of NaB3H4 (Amher-

sham; 13.7 Ci/mmol) for 45 min at room temperature. Subsequent tothis incubation, 1 nn of \aH'lI., was added to complete the reduction.

The cells were then washed and lysed with 0.5% NP-40. The labeledlysates were immunoprecipitated and electrophoresed as outlined abovefor the '"I- and [35S]methionine-labeled cell lysates.

Cells, Immunization of Mice, Production of Hybridomas, ScreeningAssay, and Isotype Determinations. The protocol using human lungtumor tissue obtained from surgery for both immunization and screening is as previously described (6). The cells used for immunization werefrom a 60-yr-old male operated on for a poorly differentiated squamouscell carcinoma of left lung. The initial screening assay was an inuminocytoadherence assay (6). Clones secreting antibody binding to thepatient's tumor but not to the Epstein Barr virus-transformed B-cells

of the patient were selected for further study.Purification of Monoclonal Human Lung Specific Antibody 5E8. After

repeated cloning by a limiting dilution technique (17), the selectedhybrid cells were cultivated in vivo by injecting them i.p. into pristane(2,6,10',4-tetramethylpentadecane)-treated BALB/c mice. Lung tu

mor-specific antibodies were purified from the serum and ascites fluidfrom mice bearing the hybridoma lines. The immunoglobulin fractionswere isolated from pooled sera and ascites by precipitation with 25%sodium sulfate which was subsequently reprecipitated with 18 and 14%sodium sulfate. The precipitate dissolved in borate buffer (pH 8) wasdialyzed extensively to remove sodium sulfate and passed throughaffinity columns containing rabbit a-mouse-coupled Sepharose 4B (8).The lung specific antibody 5E8 was eluted from the affinity columnwith 0.2 M glycine-HCI, pH 2.7, and subsequently neutralized to pH7.4.

Immunofluorescence Studies. Human lung and non-lung tumor cellswere cultured in 10% RPMI-1640 medium and grown on glass cover-slips in Petri dishes. After several days, coverslips were rinsed inDulbecco's modified Eagle's medium and fixed in cold acetone for 10

to 15 min. Normal and neoplastic adult tissues were obtained at thetime of surgery or autopsy. Tissues were immediately frozen in isopen-

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tane and stored at â€”70Â°C.Cryostat sections (5 to 6 Mm in thickness)

were melted onto glass coverslips, air dried for 1 h, fixed in cold acetonefor 10 min, and washed in PBS. The coverslips were then incubatedwith 5E8 antibody, a nonrelated monoclonal of the same isotype (2C3)or normal mouse serum at various concentrations ranging from 5 to SOMg/ml for a period of 45 min at room temperature. The slides wererinsed 3 times with PBS and incubated with a 1:40 dilution of fluores-cein isothiocyanate-conjugated goat anti-mouse IgG for 30 min at roomtemperature. The sections were then rinsed 3 times with PBS andmounted onto slides with elvanol.

Fluorescence was visualized using a Zeiss Photomicroscope IIequipped for epifluorescence. Photography on test and control slideswas performed using ASA 400 Kodak Tri-X film for constant time

intervals of 3 min.

RESULTS

Reactivity of Monoclonal Antibody 5E8 for Human LungTumor Cell Lines, Surgically Excised Lung and Non-Lung Tumors, and Normal Human Tissue Determined by Immunofluo-rescence. The pattern of antigen distribution was examinedusing 5E8 antibody on various cells grown in culture and onfrozen sections of human tissue. The cells grown in culture gavethe clearest view of this distribution pattern due to the flatnessand large size of the cells. Shown in Fig. \A is the typicalpattern observed in all positive lines examined. Staining isconcentrated into numerous intensely stained surface aggregates approximately 0.2 to 0.5 nM in diameter. This punctatepattern appears to be most concentrated near areas of membrane ruffles and near the region above the nucleus.

The reactivity of the 5E8 antibody with four histologicallydistinct human lung tumor cell lines and five non-lung tumor

cell lines was determined by indirect immunofluorescence. Allfour of the lung tumor lines exhibited a distinctly positiveimmunofluorescent staining with 5E8 compared to the negligible staining observed with normal mouse serum (Table 1). Acontrol hapten-specific monoclonal antibody (having the samelight and heavy chain isotype as 5E8) produced only backgroundimmunofluorescence equal to or less than that observed withnormal mouse serum.

Of the five non-lung tumor cell lines examined only one, anadenocarcinoma of the colon, exhibited an immunofluorescence

Table 1 Reactivity ofSES with established cell lines determined byimmunofluorescence

No. Cell line HistolÃ³gica! type

Immunofluorescence"

5E8 2C3

LungTumor1234PC-10PC-3PC-9A549SquamouscellAdenocarcinomaPoorly

differentiatedadenocarcinomaAlveolar

cell+

â€”+++

-

Non-lungtumor56789MCF-7HT

29PANC-1P17HELABreast/adenocarcinomaâ€”Colon/adenocarcinomaÂ±Pancreas/adenocarci-nomaPancreas/adenocarci-

â€”nomaCervix/squamous

â€”â€”â€”---

" Indirect immunofluorescent staining was accomplished by incubation of the

cells with either normal mouse serum or one of two monoclonal antibodies (5E8,the lung tumor-specific antibody, or 2C3, a hapten-specific antibody with thesame isotype, yÂ¡,*, as 5E8). Following treatment with the first reagent the washedcells were stained with a fluorescein-labeled goat anti-mouse immunoglobulinreagent. Positive (+) indicates an intense staining pattern relative to the negligiblefluorescence observed with normal mouse serum. Negative (â€”)indicates fluorescence that was equal to or less than that observed with normal mouse serum.

with 5E8 that was greater than that which was observed witheither normal mouse serum or the control monoclonal antibody(Table 1).

The indirect immunofluorescence assay was next extended tofrozen sections of lung tumor as well as non-lung tumor tissuesobtained by surgical biopsy. Nine of the 13 lung tumor biopsiesexamined were positive by immunofluorescence with the 5E8antibody (Table 2). The 13 individual biopsies included 5 histologically distinct lung tumor types. Of the 11 non-lung tumorbiopsies tested only one (a squamous cell carcinoma of theesophagus) was positive with the 5E8 antibody. Immunofluorescent staining of frozen sections of lung tumor biopsiesexhibited staining patterns resembling those observed with cultured lung tumor cell lines. The tumor cells display a densearray of punctate sites of antigen concentration on the cellsurface, while surrounding normal cells are negative with respect to staining with the 5E8 antibody. No staining of any ofthe biopsies tested was observed with the control isotyped-matched hapten-specific monoclonal antibody 2C3.

In addition to the neoplastic cell lines and biopsy tissues, the5E8 antibody was tested by indirect immunofluorescence todetermine its reactivity with normal human tissue. Most of thehuman tissues tested (including erythrocytes, lymphocytes, tes-tis, cardiac muscle, connective tissue, esophagus, pancreas,mammary gland, lung, pancreas, blood vessels, brain, kidney,and breast) were negative by immunofluorescence with the 5E8antibody. These same tissues were also found to be negativeaccording to immunoperoxidase staining. The basal layer of theskin, the luminal surface of the epithelium of the stomach andcolon, and restricted areas of the lung (primarily the terminalbronchial epithelium) all exhibited some degree of reactivityabove background with the 5E8 antibody according to theimmunoperoxidase staining.

The reactivity pattern of 5E8 that has been established byimmunofluorescence has been confirmed using an immunohis-

Table 2 Reactivity of SES with lung and non-lung tumor tissue determined byimmunofluorescence

Immuno-fluoresence"

TissueLung

tumorSquamous cell carcinomaAdenocarcinomaLarge cell carcinomaAlveolar cell carcinomaMesotheliomaNo.

5E84

+2 +4* +

212C3-

Total 13 9/13 0/-13

Non-lung tumorsOvarian/anaplastic mucinous 2 â€”Colon/adenocarcinomaNeck/anaplasticEsophagus/squamousSubmucosae glandLiver/hepatocellularShoulder/small cellSarcomaPancreatic/adenocarcinoma

Total 11 1/10 0/10" Indirect immunofluorescent staining was accomplished by incubation of

frozen tissue sections with either normal mouse serum or one of two monoclonalantibodies (5E8, the lung tumor-specific antibody, or 2C3, a hapten-specificantibody with the same isotype, -.,. K,as 5E8). Following treatment with the firstreagent the washed sections were stained with a fluorescein-labeled goat anti-mouse immunoglobulin reagent. Positive ( - ) indicates an intense staining patternrelative to the negligible fluorescence observed with normal mouse serum. Negative (â€”)indicates immunofluorescence that was equal to or less than that observedwith normal mouse serum.

'Three of the four tumors tested were positive for immunofluorescence.

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tochemical stain and an immunocytoadherence assay (6). Inthis assay the binding of 5E8 antibody to cells in suspension isdetected by indicator sheep RBC which have rabbit anti-mouse

immunoglobulin covalently linked to their surface. The percentage of tumor target cells with adherent rabbit anti-mousecoupled sheep RBC (i.e., perentage of rosettes) is determined.The tumor cell lines and tumor biopsies that are positive forgploO by immunofluorescence are also positive for 5E8 binding(i.e., 70 to 95% rosettes) by immunocytoadherence. Cell lines,tumor biopsies, and normal tissues (i.e., lung, lymphocytes, anderythrocytes) that are negative for gploO according to theimmunofluorescence assay are also negative (i.e., <2% rosettes)in the immunocytoadherence assay.

Immunoprecipitation of V/, 160,000 Protein from Surface-iodinated Lung Tumor Cell Lines. In view of the frequency ofthe antigen recognized by 5E8 on such a wide variety of differentlung tumor types and its limited expression on normal humantissue, this cell surface marker represents a biologically important tumor-associated macromolecule that is potentially usefulboth diagnostically and therapeutically. In order to establishthe relationship of this antigen with other lung tumor-associated molecules, it was necessary to characterize the 5E8-definedantigen biochemically and structurally. The surfaces of twodifferent 5E8-positive lung tumor cell lines, A549 (an alveolarcell carcinoma) and PC-9 (a poorly differentiated adenocarci-noma), were radioiodinated. Detergent lysates of the labeledcells were immunoprecipitated with the 5E8 monoclonal antibody, and the immunoprecipitates were subjected to SDS-PAGE analysis either in an unreduced state or following reduction with 2-mercaptoethanol. The arrows in Fig. 2 reveal themajor band of radioactivity (Fig. 2, A and B, Lane b) that is notpresent in the control lanes for both the A549 and PC-9 tumorcell lines. The molecular weight of the immunoprecipitatedmacromolecule (both for A549 and PC-9) was estimated to be

200 Kd

116 Kd92 Kd

66 Kd

45 Kd

abed

tâ€¢

abc

200 Kd

116 Kd92 Kd

66 Kd

45 Kd

Â«*.Fig. 2. SDS-PAGE analysis of immunoprecipitates of surface radiolabeled

(I2'I) lung tumor cell lines A549. an alveolar cell tumor (A), and PC9, a poorly

differentiated adenocarcinoma (B). Immunoprecipitates of the radiolabeled celllysates were formed by incubation with either normal mouse serum (A, Lane a,and B, Lane a), the monoclonal anti-lung tumor antibody 5E8 (A, Lane h, and B,iMne b). or a control monoclonal antibody 5C7 (A, Lane d, and B, Lane c). In A,Lane c, the immunoprecipitate was formed by treatment of precleared radiolabeledcell lysate with 5E8 (see "Materials and Methods" for details). The molecularweight standards used here and in all of the subsequent gels were myosin M-chain(M, 200.000). /i-galactosidase (M, 116.000). phosphorylase b (M, 92,000), bovineserum albumin (M, 66.000). and ovalbumin (M, 4S.OOO)supplied by Bio-RadLaboratories. Richmond. CA.

160,000 based upon its migration pattern in the gel. Themigration pattern of the tumor-associated protein derived fromA549 and PC-9 was not altered by reduction wtih 2-mercapto

ethanol (Fig. 3, A and B, Lanes b and c). In these studies andin subsequent studies with biosynthetically labeled cells, controlprecipitates using normal mouse serum and another lung tumor-specific monoclonal antibody (5C7) were run on the samegel with the 5E8 precipitates. While some lower molecularweight proteins were nonspecifically precipitated by both thenormal mouse serum and the control monoclonal antibodies(Fig. 2A, Lanes a and d; Fig. IB, Lanes a and c; Fig. 3, A and

abc abc

200 Kd

116 Kd92 Kd

66 Kd

45 Kd

200 Kd

(16 Kd92 Kd

Fig. 3. SDS-PAGE analysis of immunoprecipitates of surface radiolabeled(I25I) lung tumor cell lines A549. an alveolar cell tumor (A), and PC9. a poorlydifferentiated adenocarcinoma (B). All of the samples were reduced with 2-mercaptoethanol prior to electrophoresis. The immunoprecipilates of the radio-labeled cell lysates were formed either by incubation with normal mouse serum(Lanes a) or with 5E8 monoclonal antibody (Lanes b). Immunoprecipitates runin Lanes c were formed by treatment of precleared radiolabeled cell lysates with5E8 (see "Materials and Methods" for details).

200 Kd

116 Kd

92 Kd

66 Kd

45 Kd

a b

Fig. 4. SDS-PAGE analysis of immunoprecipitates of surface radiolabeled(125I) lung tumor obtained by surgical biopsy. The tumor was identified by

histopathology as a large cell tumor. Radiolabeled cell lysates were formed byincubation with normal mouse serum (Lane a) or the SE8 monoclonal antibody(Lane b). See "Materials and Methods" for details.

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B, Lane a), none of the control sera or antibodies precipitatedthe A/r 160,000 protein. Moreover, the nonspecific immuno-precipitation could be eliminated by preclearing the cell lysates(i.e., treatment of lysates with normal mouse serum, rabbit anti-mouse immunoglobulin, and Protein A) prior to immunopre-cipitation with the 5E8 antibody (see Figs. 2A and 3A, Lane c).In addition to the major specific band immunoprecipitated bythe 5E8 antibody, a faster migrating minor band precipitatedby the 5E8 antibody was often observed (Fig. 3B, Lane c). Themolecular weight of this second component which may represent a proteolytic fragment of the larger protein was estimatedto be 120,000 (see below). Based upon these studies, we conclude that the 5E8 antibody recognizes antigenic determinantsof A549 and PC-9 that are expressed by a common protein.

Immunoprecipitation of Biosynthetically Labeled Lung TumorCell Lines. In order to establish that the M, 160,000 proteinimmunoprecipitated by 5E8 was produced by the tumor itselfand not simply a protein in the tissue culture fluid that wasnonspecifically adsorbed to the cell surface of the tumor cells,a similar immunoprecipitation and SDS-PAGE analysis wereperformed using biosynthetically labeled tumor cells. Detergentcell lysates of biosynthetically labeled A549 and PC-9 that wereimmunoprecipitated with the 5E8 monoclonal antibody revealed a single band in SDS-PAGE corresponding to a proteinwith an apparent molecular weight of 160,000. From these datait was concluded that the M, 160,000 molecule recognized bythe 5E8 antibody was a membrane-associated structural proteinsynthesized by both A549 and PC-9 lung tumor cell lines.

Carbohydrate Association with M, 160,000 Protein. Neura-minidase treatment of either A549 or PC9 lung tumor cells didnot alter the ability of the 5E8 antibody to immunoprecipitatethe tumor-associated antigen (indicating that the 5E8 antibodywas not specific for sialic acid residues). But, it remained to bedetermined whether or not the antigen contained any carbohydrate. In order to pursue this question further, neuraminidase-treated A549 or PC9 tumor cells were incubated with galactoseoxidase and subsequently incubated with tritiated borohydrideto label the sugars on the cell surface. The labeled cells werelysed and immunoprecipitated with the 5E8 antibody. Analysisof these precipitates in SDS-PAGE revealed a radiolabeled M,160,000 band in the gels for both the alveolar and adenocarci-noma cell lines. The second minor band with an apparentmolecular weight of 120,000 was again noted. Normal mouseserum or a control monoclonal antibody (2C3) did not immunoprecipitate either of these labeled proteins. The data indicatethat the M, 160,000 protein is glycosylated and confirm that itexists on the cell surface of both A549 and PC-9 cell lines.Following this observation, the lung tumor-associated antigen

was termed gpl60.Presence of gpl60 on Surgically Excised Lung Tumors. Hav

ing identified and partially characterized the 5E8 specific antigenic moiety associated with the lung tumor cell lines, it was ofconsiderable interest to us to know whether or not the 5E8antibody was binding to the same or to a similar glycoproteinon fresh lung tumor biopsy tissues. The first available lungtumor that was obtained from a patient in our clinic was a largecell carcinoma. This patient's tumor was determined to express

the 5E8 specific antigen according to immunocytoadherenceand immunofluorescence. The cells of this tumor were surfacelabeled with '"I, and the cell lysates were subjected to immunoprecipitation and SDS-PAGE analysis according to the sameprotocol that was used for the lung tumor cell lines. Two specificbands with molecular weight estimates of 160,000 and 120,000were resolved in the gel (see arrow and arrowhead in Fig. 4,

Lane b). The relative increase in the lower molecular weightcomponent observed here, compared to that which was observedin the cell lines, is likely related to the fact that the tumor tissuewas frozen and thawed prior to the surface labeling procedure,thus increasing the probability of proteolytic fragment formation due to endogenous protease activity. A control monoclonalantibody (2C3) did not immunoprecipitate either of these twoproteins (data not shown). We conclude that the 5E8 antibodyrecognizes the same or a very similar molecule on histologicallydistinct lung tumors and that this gpl60 is present on lungtumor cell lines propagated /// vitro as well as lung tumorsobtained by surgical biopsy.

DISCUSSION

We describe here that the reactivity of a monoclonal antibody5E8 is directed towards an antigenic determinant containedwithin a M, 160,000 non-disulfide-bonded glycoprotein. Thismolecule has been identified as a plasma membrane-associatedglycoprotein that is found on both human lung tumor cell linesand on surgically excised fresh lung tumor tissue.

In view of the molecular weight of the gp 160 and the reactivity pattern described here, we conclude that we have identifieda new and potentially very useful tumor-associated antigen ofhuman lung tumors. While it is clear that there is little or nodetectable binding of the 5E8 antibody to normal lung tissue itis still possible that the gpl60 is present in such low concentrations or on so few cells that it is not possible to detect themolecule in our assay.

gpl60 can be added to the list of human lung tumor-associated antigens that have been previously identified (1-5). Thecharacteristics of this new antigen suggest an excellent potentialfor immunotherapeutic application to human lung cancer. Thisis one of the largest cell surface antigens reported, and it isexpressed by a large percentage of primary lung tumor types.Its very limited expression in normal human tissues and thelack of any observable molecular heterogeneity make this asuitable target for one of several immunospecific targetingprotocols (18). We are currently attempting to exploit gpl60as a tumor target for immunospecific drug delivery. Our preliminary work indicates that the 5E8 monoclonal antibody can becovalently coupled either directly to cytotoxic drugs or to thelipid bilayer of large unilamellar drug-containing liposomeswithout loss of the antibody's ability to bind to gpl60 on the

tumor cell surface (19). The 5E8 antibody has also been usedin a diagnostic immunoassay which detects the gpl60 antigenor the presence of antibodies specific for gpl60 in the bodyfluids of cancer patients or in the serum of immunodeficientmice bearing human lung tumors.4

Much is yet to be determined regarding possible functionalsignificance of gpl60 with respect to its association with celltransformation. It is of interest that this molecule is very similarin molecular weight (20), cell distribution (21), and biochemicalcharacteristics (22) to the epidermal growth factor receptor. Amore complete comparison between gpl60, the epidermalgrowth factor receptor, or possibly oncogene products willrequire primary amino acid sequence or nucleotide sequencedata. Such efforts are currently under way.

ACKNOWLEDGMENTS

The authors wish to thank Donna Ovak and Cheryl Zuber forassistance in the preparation of this manuscript. We would like to

' F-A. Chen, S-C. Luo, S. Reddi, and R. B. Banker!, unpublished observation.

6450




acknowledge the significant contribution of Dr. Shigetoyo Saji whohelped us cultivate the SES hybridoma clone.

REFERENCES

1. Varki, N. M., Reisfield, R. A., and Walker. L. E. Antigens associated withhuman lung adenocarcinoma defined by monoclonal antibodies. Cancer Res.,44:681-687. 1984.

2. Mazauric, T., Mitchell, K. F., Letchworth, G. J., Ill, and Koprowski, H.Monoclonal antibody-defined human lung cell surface protein antigens.Cancer Res., 42:150-154, 1982.

3. Brown, D. T., and Moore, M. Monoclonal antibodies against two humanlung carcinoma cell lines. Br. J. Cancer, 46: 794-801, 1982.

4. Mulshine, J. L., Cuttitta, G., Bibro, M., el al. Monoclonal antibodies thatdistinguish non-small cell lung cancer from small cell lung cancer. J. Imumnoi., 131:497-502, 1983.

5. Radosevich, J. A., Ma, Y., Lee, I., Salwen, H. R., Gould, V. E., and Rosen,S. T. Monoclonal antibody 44-3A6 as a probe for a novel antigen found onhuman lung carcinomas with glandular differentiation. Cancer Res., 45:5808-5812, 1985.

6. Saji, S., Zylstra, S., Schepart, B. S., Ghosh, S. K., Jou, Y-H., Takita, H., andBankert, R. B. Monoclonal antibodies specific for two different histologicaltypes of human lung carcinoma. Hybridoma, 3:119-129, 1984.

7. Bloor. A. G.. Jou, Y-H., Hoeplinger, S., Gartner, J. E., Mayers. G. L., andBankert. R. B. Hapten-specific B cell repertoire probed by hybridoma technology: selection and characterization of representative clonotypes from theantibody-forming cell pool. J. Immunol., 128: 1443-1449, 1982.

8. Mayers. G. L., and Bankert. R. B. Immunochemistry of monoclonal antibodies. Transplant. Proc., 12: 413-416, 1980.

9. Tsuji. K., Hayata, Y., Oko, T., Sato. M., Omtani, T., Shimasato, Y., andKoide. T. Studies in ectopie hormone-producing tumors with tissue culturetechniques. Saishin-lgaku (Jpn.), 28: 1861-1869, 1973.

10. Lieber, M., Smith, G., Sakal, A., Nelson-Rees, W., and Todaro. G. Acontinuous tumor-cell line from a human lung carcinoma with properties of

type II alveolar epithelial cells. Int. J. Cancer, 17: 62-70, 1976.11. Soule, D. M., Vazquez, J., Long, A., Albert, S., and Brennan, M. A human

cell line from a pleural effusion derived from a breast carcinoma. J. Nati.Cancer Inst., 51: 1409-1416, 1973.

12. Fogh, J., and Trempe, G. New human tumor cell lines, in: J. Fogh (ed.).Human Tumor Cells in Vitro, pp. 115-159. New York: Plenum Press, 1975.

13. Kubo, R. T, and Pelanne, M. L. Tunicamycin inhibits the expression ofmembrane IgM in the human lymphoblastoid cell line Daudi. Mol. Immunol.,20:67-76, 1983.

14. KlÃ¶ppel,T. M., Kubo, R. T., Cain, P. S., Brown, C. P., Colon, S. M.,Kearney, J. F., and Grey, H. M. Structural analysis of the Â»i-chainsynthesizedby fetal liver hybridomas. J. Immunol.. 126: 1346-1350, 1981.

15. Laemmli, U. K. Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.

16. Gahmberg, C. G., and Hakomori, S-I. External labelling of cell surfacegalactose and galactosamine glycolipid and glycoprotein of human erythro-cytes. J. Biol. Chem., 248:4311-4317. 1973.

17. Bankert, R. B., Mazzaferro, D.. and Mayers, G. L. Hybridomas producinghemolytic plaques used to study the relationship between monoclonal antibody affiiiii) and the efficiency of plaque inhibition with increasing concentrations of antigen. Hybridoma. 7:47-58, 1981.

18. Bankert, R. B. Development and use of monoclonal antibodies in the treatment of cancer. Cancer Drug Delivery. /: 251-267, 1984.

19. Yokota, S., Mayhew, E., Chen. F. A., and Bankert, R. B. Immunospccifictargeting of drug-containing liposomes to a murine B-cell line and humanlung tumor (abstract). In: Sixth International Congress of Immunology, p.551. Toronto, Ont., 1986.

20. Carpenter, G. The biochemistry and physiology of the receptor-kinase forepidermal growth factor. Mol. Cell. Endocrinol., 31:1-19, 1983.

21. Hendler, F. J., and Ozanne, B. W. Human squamous cell lung cancersexpress increased epidermal growth factor receptors. J. Clin. Invest., 74:647-651, 1984.

22. Chen. F. A., Repasky, E. A., Zylstra. S.. Ghosh, S. K., Rao. K.. Takita, H.,and Bankert. R. B. Glycoprotein associated with human lung tumors that issimilar to but distinct from EGF receptor (abstract). Sixth InternationalCongress of Immunology, p. 518. Toronto, Ont., 1986.

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1986;46:6446-6451. Cancer Res Samuel Zylstra, Fang-An Chen, Swapan K. Ghosh, et al. Human Lung Tumors by a Monoclonal AntibodyMembrane-associated Glycoprotein (gp160) Identified on

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