[CANCER RESEARCH 47, 4835-4841, September 15, 1987]

Cross-Reactivity of Murine Anti-Human High Molecular Weight-MelanomaAssociated Antigen Monoclonal Antibodies with Guinea Pig Melanoma Cells1

Shuen-Kuei Liao, James W. Smith, Pak C. Kwong, Pier G. Natali, Mikihiro Kusama, Carl V. Hamby, andSoldano Ferrone2

Departments of Pathology and Pediatrics [S-K. L, J. W. S., P. C. KJ, McMaster University, Hamilton, Ontario L8N 32.5, Canada; Biotnerapeutics, Inc. fS-K. L.J,Franklin, Tennessee 3 7064; Immunology Laboratory [P. G. N.], Regina Elena Cancer Institute, Rome, Italy; and Department of Microbiology and Immunology [M. K.,C. y. H., S. F.], New York Medical College, Valhalla, New York 10595

ABSTRACT

To identify melanoma associated antigens (MAAs) shared by humanand guinea pig melanoma cells, a battery of murine monoclonal antibodies(MoAl>s) to human MAA and an antiserum to S100 protein were testedwith four newly established guinea pig melanoma cell lines. Only themonoclonal antibodies 149.53 and 225.28 which recognize distinct determinants of the human high molecular weight-MAA (HMW-MAA) reacted with all four guinea pig melanoma cell lines. To compare thebinding site of MoAbs 149.53 and 225.28 with guinea pig and humanmelanoma cells, inhibition binding experiments were performed withantiidiotypic monoclonal antibodies which completely inhibit the bindingof MoAbs 149.53 and 225.28 to human melanoma cells. The binding ofM«Ah149.53 to guinea pig melanoma cells was partially inhibited byantiidiotypic MoAbs MF9-10 and MK1-180 which recognize distinctprivate idiotopes within the antigen combining site of MoAb 149.53. Onthe other hand the binding of MoAb 225.28 to guinea pig melanoma cellswas completely inhibited by antiidiotypic MoAbs MF11-30 and I M-12which recognize distinct private idiotopes within the antigen combiningsite of MoAb 225.28. These results suggest that the determinant recognized by MoAb 149.53 on guinea pig melanoma cells is similar but notidentical to that recognized on human melanoma cells, while the determinants recognized by MoAb 225.28 on the two types of cells do notdisplay any detectable differences under the experimental conditionstested. The target structure on the guinea pig melanoma cells identifiedby MoAbs 149.53 and 225.28 is a M, 280,000 molecule which has thesame apparent molecular weight as one of the two subunits of the HMW-MAA synthesized by human melanoma cells. Sequential immunoprecip-itation experiments with guinea pig melanoma cells showed that thedeterminant recognized by MoAb 149.53 is expressed on a subpopulationof the molecules recognized by MoAb 225.28. Immunohistochemicalstaining with MoAb 225.28 of a variety of different tissues from normaladult guinea pigs showed that the corresponding antigenic determinant isdetectable only in basal cells of epidermis and hair follicles of skin. SIIMIprotein, which is a cytoplasmic constituent of normal human melanocytes,benign nevi, and malignant melanocytes, was also detected in the cytoplasm of the four cultured guinea pig melanoma cells lines. The resultsof the present investigation may lead to a better understanding of thephylogenetic evolution of the human HMW-MAA and suggest thatguinea pig melanoma may serve as a useful animal model for immuno-biological studies and carcinogen-induced tumorigenesis investigations.

INTRODUCTION

Melanoma is similar in human and other animal species inseveral aspects of its biological and clinical behavior; amongthem is the appearance of MAAs3 on melanocytes which have

Received 10/27/86; revised 4/7/87; accepted 6/19/87.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 by grants from the Medical Research Council of

Canada (MA 5429), the National Institute of Canada (Project 400, type 3), theJ. P. Bickell Foundation, Toronto, Canada, the Consiglio Nazionale delle Ricerche Progetto Finalizzato Oncologia (Grant 860069044), the AssociazioneItaliana per la Ricerca sul Cancro, and NIH Grants CA37959 and CA39559.

2To whom requests for reprints should be addressed.3The abbreviations used are: MAA, melanoma associated antigen; ELISA,

enzyme linked immunosorbent assay; HM W, high molecular weight; III, indirectimmunofluorescence; Kd, kilodaltons; MoAb, monoclonal antibody; PBS, phosphate buffered saline (10 HIMsodium phosphate-150 HIMNaCI, pH 7.2); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

undergone malignant transformation (for review, see Refs. 1and 2). While early studies with conventional antisera did notdetect any cross-reactivity between human and murine MAAs(3, 4), recently a murine monoclonal antibody raised againstsyngeneic melanoma B16 cells has been shown to cross-reactwith cultured human melanoma cells (S) and with surgicallyremoved melanoma lesions (6). The latter findings raise thepossibility that monoclonal antibodies to human MAAs maycross-react with xenogeneic melanoma cells. Therefore in thisstudy we have tested a panel of anti-human MAA monoclonalantibodies and a rabbit anti-Si00 protein antiserum with fourmelanoma cell lines that we have established from cutaneousand visceral tumor lesions induced by 7,12-dimethylbenzan-thracene in albino guinea pigs (7).

MATERIALS AND METHODS

Cells, Tissues, and Purified Bovine S100 Protein. The melanoma celllines GP-1, GP-5, GP-6, and GP-8 were established from primary andmetastatic tumor lesions which developed in Hartley Strain albinoguinea pigs (Bio-Breeding Laboratories of Canada, Ltd., Ottawa, Canada) following 7,12-dimethylbenzanthracene treatment on their skins(7). The cell lines have been grown in vitro for more than 28 passages,and each cell line has exhibited relatively stable morphology and growthcharacteristics (Table 1). Cells were shown by electron microscopy tocontain melanosomes and/or premelanosomes.4 The cells were maintained in Eagle's minimum essential medium containing 10% fetal calfserum at 37°Cin an incubator with a humidified atmosphere of 5%

CO., in air. The guinea pig hepatoma cell lines LI (8) and LIO (9) wereobtained from Dr. B. Zbar of the National Cancer Institute in Frederick,MD. The human melanoma cell lines CaCL 73-36, CaCL 78-1, Colo38, NKI-302, and SK Mel 93, the human carcinoma cell lines HCT-8R and KB, and the human lymphoid cell line MANN were maintainedin RPMI 1640 medium containing 10% fetal calf serum and antibiotics(100 units/ml penicillin and 100^g/ml streptomycin). Tissues obtainedfrom normal adult guinea pigs were frozen sectioned for immunofluorescence staining and were homogenized for absorption analysis. Purified bovine SI00 protein was prepared and kindly supplied by Dr. A.Marks, Banting and Best Department of Medical Research, Universityof Toronto, Toronto, Canada.

Monoclonal Antibodies and Conventional Antisera. The sources andreferences of anti-human MAA monoclonal antibodies, of anti-HLAmonoclonal antibodies, and of anti-bovine SI 00 antiserum are listed inTable 2. The antiidiotypic MoAbs MF9-10, MK1-180, and MK1-104to MoAb 149.53 and the antiidiotypic MoAbs MF11-30, TK1-F2, andTK1-G3 to MoAb 225.28 are secreted by hybridomas constructed withsplenocytes from BALB/c mice immunized with the MoAbs 149.53and 225.28, respectively. The latter two antibodies are directed todistinct and spatially distant epitopes of the human HMW-MAA.MoAbs MF9-10 and MK1-180 recognize private idiotopes within theantigen combining site of MoAb 149.53, while MoAb MK1-104 recognizes a private idiotope which does not inhibit the binding of MoAb149.53 to human melanoma cells. MoAbs MF11-30 and TK1-F2recognize private idiotopes within the antigen combining site of MoAb225.28, while MoAb TK1-G3 recognizes a private idiotope which doesnot inhibit the binding of MoAb 225.28 to human melanoma cells. The

4A. Pawlowski and S-K. Liao, unpublished observations.

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CROSS-REACTING HUMAN AND GUINEA PIG MAA

Table 1 Morphology and growth characteristics of guinea pig melanoma cell lines

CelllineGP-1GP-5

GP-6GP-8Animal

no.2620

2020Tumor

site fromwhich cell line was

derivedSkinInguinal

lymph nodeLiverSkinPassage

no.during which

study wasconducted20

to2526

to 2923 to2627

to 30MorphologyMixed

polygonal andepithelial cells

Polygonal cellsMixed polygonal and

epithelial cellsPolygonal cellsIn

vitroDoubling

time(h)11863

10247Saturation

density(cells/cm2)

xIO"13.125.31

4.019.04

Table 2 Characteristics of antigens immunoprecipitated from human melanoma cells by antibodies tested for cross-reactivity with guinea pig melanoma cells

AntibodySource designation Isotype Specificity and antigenic structure Ref.

Murine monoclonal antibodiesBecton-Dickinson

Brown, J. P.Cheung, N. K.Ferrone, S.

Gani, M.Houghton, A.

Liao. S-K.

Schlom. J.Taniguchi, M.

Rabbit antiserumCochran, A.

L243

96.53F8*

149.53225.28345.134376.96763.74902.5NAMB/IQ5/13

W6/32

1176.2Ir

A010A092R-247.51140.240140.72225.27C10Bl.l'

Anti-Si 00

IgG2a Monomorphic determinant of HLA 10Class II antigens

IgG2a M, 97,000 glycoprotein (p97°) 11IgG3 <.,,. ganglioside 12IgGl HMW-MAA proteoglycan 13IgG2a HMW-MAA proteoglycan 14IgG2a M, 115,000 protein 15IgG2a M, 100,000 proteoglycan 16IgGl HMW-MAA proteoglycan 13IgGI HMW-MAA proteoglycan 17IgGl ft-microglobulin 18IgG2a Monomorphic determinant of HLA 19

Class II antigenslgG2a Monomorphic determinant of HLA 20

Class I antigensM, 44,000/50,000 glycoprotein

IgGl M-3, undefined structure 21IgGl M-2, undefined structure 21IgG3 GD3ganglioside 22IgGl M, 42,000 protein 23IgG2a M, 87,000 glycoprotein (identical to p97) 24-26IgGl M, 95,000/150,000 glycoprotein 27IgG2a M, 87,000 glycoprotein (identical to p97) 28IgG2a M, 95.000/150,000 glycoprotein' 29

IgM M, 31,000 glycoprotein 5

SIOO 30" p97, a protein with a molecular weight of 97,000.* MoAb 3F8 was raised against human neuroblastoma cells and cross-reacts with human melanoma and sarcoma cells.' MoAb 1176.21 was raised against carcinoembryonic antigen and cross-reacts with human melanoma cells (S-K. Liao, unpublished results).'' Mu \li Bl.l was raised against human breast carcinoma cell membranes and immunoprecipitates a M, 180.000 glycoprotein from some of the human carcinoma

cell lines tested.* MoAb Bl.l immunoprecipitates a M, 95,000/150,000 glycoprotein from cultured human melanoma cells (S-K. Liao, unpublished results).

\l'i \l> M2590 was raised against murine BI6 melanoma cells and cross-reacts with human melanoma cells.

production and characterization of these six antiidiotypic monoclonalantibodies will be described in detail elsewhere.5

Horseradish peroxidase-conjugated affinity-purified goat anti-mouseIgG + IgM + IgA antibodies and fluorescein isothiocyanate conjugatedrabbit anti-mouse Ig antibodies were purchased from JacksonImmunoResearch Laboratories, Inc., West Grove, PA and from CooperBiomedicai, Inc., Malvern, PA. respectively.

Monoclonal antibodies were purified from ascites by caprylic acidprecipitation (31). Purified antibodies were labeled with '"I utilizing

the chloramine-T method (32).Serological Assays. The mixed hemadsorption assay (33), the double

determinant immunoassay (13), the IIFassay (34), and the absorptionassay (25) were performed as previously described. The ELISA wasperformed in 96-well U-bottomed polyvinyl chloride plates (Becton-Dickinson, Oxnard, CA) precoated with 0.5% (w/v) non-fat dry milkdissolved in PBS. The latter will be referred to as PBS/dry milk buffer.After flipping out the PBS/dry milk buffer, antibody containing super-natants (50 ¿tl/well)were added to the plates and incubated for l h at4°Cwith washed target cells (1 x 10s cells/well) suspended in PBS/dry

milk buffer. Unbound antibodies were removed by centrifuging plates

at 700 x g for 3 min, flipping out the buffer, and resuspending cells infresh PBS/dry milk buffer. After three washes with PBS/dry milkbuffer, 100 n\ of horseradish peroxidase-conjugated goat anti-mouseIgG + IgM + IgA antibodies diluted 1:2000 in PBS/dry milk bufferwere added to each well. Plates were incubated for l h at 4°Cfollowed

by three washes with PBS/dry milk and a final wash with PBS. Freshlyprepared substrate solution (0.05% o-phenylenediamine and 0.0075%hydrogen peroxide in Mcllvaine's buffer, pH 6.0) was added to each

test well (200 ¿il/weli),and the absorbance of each test well was read at405 nm on a Titertek Multiskan plate reader (Flow Laboratories,McLean, VA) after 30 min of incubation.

Competitive Binding Assay with Antiidiotypic Monoclonal Antibodies.Competition binding assays with antiidiotypic monoclonal antibodieswere performed by incubating dilutions of purified antiidiotypic monoclonal antibodies for 2 h at 4°Cwith '"I-labeled anti-HMW-MAAmonoclonal antibodies (2 x IO5cpm/well) in duplicate wells of 96-wellU-bottomed plates. Target cells (2 x 10' cells/well) were added to theplates and incubated for 2 h at 4°C.Following five washes with PBS/

dry milk buffer, radioactivity bound to target cells was measured witha gamma counter. The percentage of binding inhibition was calculatedby the formula

* M. Kusama. T. Kageshita, J. W. Kim. and S. Ferrone. Antiidiotypic monoclonal antibodies to murine anti-human high molecular weight-melanoma associated antigen monoclonal antibodies, to be submitted for publication.

4836

cpm bound with antiidiotypic monoclonal antibodycpm bound without antidiotypic monoclonal antibody

100

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Radiolabeling, Immunoprecipitation, and SDS-PAGE. Labeling ofcells with 12SI,biosynthetic labeling of cells with [35S]methionine. indirect immunoprecipitation of antigens from Nonidet P-40 cell lysates,and one-dimensional SDS-PAGE were carried out as previously described (14).

RESULTS

Serological Reactivity of Anti-Human MAA and Anti-HLAMonoclonal Antibodies with Guinea Pig Melanoma Cells. Eighteen monoclonal antibodies to human membrane bound MAA,two anti-HLA Class I monoclonal antibodies, and two anti-HLA Class II monoclonal antibodies were tested in the mixedhemadsorption assay with the guinea pig melanoma cell linesGP-1, GP-5, GP-6, and GP-8. Only MoAbs 149.53 and 225.28reacted with guinea pig melanoma cells (Table 3). The titers ofMoAbs 149.53 and 225.28 with the four guinea pig melanomacell lines ranged from 1:160 to 1:20,480 compared to titersranging from 1:1,280 to 1:20.480 with the human melanomacell lines CaCL 73-36, CaCL 78-1, and NKI-302. In agreementwith previous results (14) MoAbs 149.53 and 225.28 did notreact with the two human carcinoma cell lines tested. Resultsfrom the ELISA confirmed the serological cross-reactivity ofMoAbs 149.53 and 225.28 with guinea pig melanoma cells anddetected no reactivity of guinea pig melanoma cells with theanti-HLA Class I MoAb W6/32, with the anti-human 02-microglobulin MoAb NAMB-1, and with the anti-HLA ClassII MoAb Q5/13 (Table 4).

Characterization with Antiidiotypic Monoclonal Antibodies ofthe Cross-Reactivity of Anti-Human HMW-MAA MoAbs149.53 and 225.28 with Guinea Pig Melanoma Cells. To furthercharacterize the cross-reactivity of MoAbs 149.53 and 225.28with guinea pig melanoma cells, competitive blocking assayswere performed to compare the extent to which preincubationof antiidiotypic monoclonal antibodies with MoAbs 149.53 and225.28 could inhibit their binding with guinea pig and humanmelanoma cells (Fig. 1). The binding of MoAb 149.53 to guineapig melanoma GP-5 cells was partially inhibited by amounts ofMoAbs MF9-10 and MK1-180 higher than those which completely inhibited the binding of MoAb 149.53 to human melanoma cells Colo 38 (2 to 3 log difference). On the other handthe binding of MoAb 149.53 to cultured guinea pig and humanmelanoma cells was not affected by the antiidiotypic MoAbMK1-104 which recognizes an idiotope outside of the combining site. The reactivity of MoAb 225.28 with the guinea pig

Table 3 Differential reactivity of cultured guinea pig melanoma cells withmonoclonal antibodies to distinct determinants of human HMW-MAA

Monoclonal antibody"

Table 4 Reactivity ofanli-MAA, anti-HLA Class I and anti-HL4 Class IImonoclonal antibodies with guinea pig and human cultured melanoma cells

CelllineGP-IGP-5GP-6GP-8LILIOSpeciesHistotypeGuinea

pigMelanomaGuineapigMelanomaGuineapigMelanomaGuineapigMelanomaGuinea

pigHepatomaGuineapig Hepatoma149.53

225.28 763.74902.510,240*20.480 <5<1010,240

5.120 <5<10160320 <5NT*5.120

640 <5<10NT

<5 NTNTNT<5 NT NT

CaCL 73-36 Human MelanomaCaCL 78-1 Human MelanomaColo 38 Human MelanomaNKI-302 Human Melanoma

20,480 1,280 10,240 20,48020,480 10,240 5.120 20,48010,240 20,480 20,480 10.240

NT 10,240 NT NT

HCT-8RKB

HumanHuman

Colon carcinomaOral carcinoma

<5NT

<5 NT<5 NT

<5NT

' MoAbs 149.53, 225.28. 763.74, and 902.5 recognize distinct determinantsof the human HMW-MAA.

* Reciprocal of highest antibody dilution with positive reaction in MHA.c NT, not tested.

Guinea pig melanomacells GP-5

Human melanomacells Colo 38

MoAb Specificity

Radiobindingassay* (cpm

ELISA x 10-')

Radiobindingassay* (cpm

ELISA x 10-')

149.53*225.28763.74902.5345.134376.96W6/32NAMB-1Q5/I3Ag8HMW-MAAHMW-MAAHMW-MAAHMW-MAA115KMAA100KMAAHLA

Class1antigensiJi-roicro-globulinHLA

ClassIIantigensNot

known0.94C0.900.180.160.180.280.380.320.140.204.513.11.21.3ND'NDN

DNDN

n1.40.900.980.780.680.740.760.881.000.940.2012.815.423.513.2N

DNDNDNDN

D1.6

" Target cells were incubated with I2*l-labeled monoclonal antibody.* MoAbs 149.53. 225.28. 763.74, and 902.5 recognize distinct determinants

of human HMW-MAA.' Absorbance at 405 nm. Results were scored positive when absorbance was at

least 0.4.'NO. not done.

IOC

i•950

100 100 001 100 100 001Antiidiotypic Monoclonal Antibody (^q/ml)

Fig. 1. Inhibition of anti-human HMW-MAA MoAbs 149.53 and 225.28binding to human and guinea pig melanoma cells by antiidiotypic monoclonalantibodies. A, '"I-labeled MoAb 149.53 (2 x 10*cpm/well) was preincubated for2 h at 4'C with purified antiidiotypic MoAb MF9-10 (O. •)and MoAb MK1-

180 (A, A) to idiotopes within the antigen combining site of MoAbs 149.53 andMK l UM (D. •)to an idiotope which does not inhibit the binding of MoAb149.53 to human melanoma cells. The mixture was then incubated for 2 h at 4"Cwith guinea pig GP-5 melanoma cells (•.A, •)and human Colo 38 melanomacells (O. A. D) at a concentration of 2 x 10s cells/well. Cells were washed, bound

radioactivity was measured with a gamma counter, and the percentage of bindinginhibition was calculated. B. '"I-labeled MoAb 225.28 was preincubated withantiidiotypic MoAb MF11-30 (O, •)and MK1-F2 (A. A) to idiotopes within theantigen combining site of MoAbs 225.28 and TKI-G3 (D. •)to an idiotope whichdoes not inhibit the binding of MoAb 225.28 to human melanoma cells. Thebinding of MoAb 225.28 to guinea pig GP-5 (•.A, •)and human Colo 38melanoma cells (O, A, D) was assayed as described above.

melanoma cells GP-5 was inhibited by MoAbs MF11-30 andTK1-F2, both of which recognize idiotopes within the antigencombining site of MoAb 225.28. The inhibition was dose dependent, and the inhibition curves were similar to those obtained when cultured human melanoma cells Colo 38 were usedas targets. The antiidiotypic MoAb TK1-G3 which recognizesan idiotope outside of the combining site inhibited the reactivityof MoAb 225.28 with neither guinea pig nor human melanomacells.

Level of HMW-MAA Homologue in the Lysate and SpentMedium from Guinea Pig Melanoma Cells. The double determinant immunoassay was performed with the lysate and spentmedium of the guinea pig melanoma cells GP-5 to determinewhether the determinants recognized by MoAbs 149.53 and225.28 are expressed on the same molecule and to quantitatethe level of cellular and shed antigens cross-reacting with theanti-human HMW-MAA MoAbs 149.53 and 225.28 (Fig. 2).Antigen could be detected in the lysate but not in the spentmedium of guinea pig melanoma cells GP-5. On the other hand

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15

10

1000 100 10 I

Protein Concentration ofCell Lysote (tig/ml)

IO'1 IO'2 IO"3 IO"4

Dilution of Culture Supernatant

Fig. 2. Detection with the double determinant immunoassay of the HMW-MAA in lysates (I) and spent medium I«)of human Colo 38 melanoma cells(positive control, O). human MANN B-lymphoid cells (negative control. D), andguinea pig GP-5 melanoma cells (•).Cell lysates and spent medium wereincubated for 24 h at 4*C in plates previously coated with anti-human HMW-MAA MoAb 149.53. After washing, plates were incubated with '"I-labeled anti-human HMW-MAA MoAb 225.28 (2 X 10* cpm/well) for 4 h at 4'C. washed,

and counted with a gamma counter.

the HMW-MAA was detected in both the lysate and the spentmedium of cultured human melanoma cells Colo 38. The levelof antigen detected in the lysate of a given number of guineapig melanoma cells was markedly lower than that found in thesame number of human melanoma cells.

Immunochemical Analysis of Cross-Reactivity of Guinea PigMelanoma Cells with Anti-Human HMW-MAA MonoclonalAntibodies. To define the molecular basis of the serologicalcross-reactivity of MoAbs 149.53 and 225.28 with guinea pigmelanoma cells, antigens were immunoprecipitated from radio-labeled guinea pig melanoma cells and analyzed by SDS-PAGE.One component with an apparent Mr of 280,000 was immunoprecipitated by MoAbs 225.28 and 149.53 from guinea pigmelanoma cells, while the two characteristic subunits of theHMW-MAA with the apparent M, of 28,000 and greater thanA/r 440,000 were immunoprecipitated from cultured humanmelanoma cells (Fig. 3). It is noteworthy that the intensity ofthe component immunoprecipitated by MoAb 149.53 fromguinea pig melanoma cells is less than that immunoprecipitatedby MoAb 225.28 (Fig. 4). Sequential immunoprecipitationexperiments showed that MoAb 149.53 recognized only a sub-population^) of the MT280,000 molecules recognized by MoAb225.28, since the latter immunoprecipitated molecules from theguinea pig melanoma cell lysate following immunodepletionwith MoAb 149.53. On the other hand no specific componentwas detected in the immunoprecipitate with MoAb 149.53 fromthe guinea pig melanoma cell lysate which had been ¡mimmo-depleted with MoAb 225.28 (Fig. 5).

Distribution of MoAb 225.28 Defined Antigen in Normal AdultGuinea Pig Tissues and in Guinea Pig Hepatoma Cell Lines. Todetermine the extent of cross-reactivity of MoAb 225.28 withnormal guinea pig tissues, we tested the antibody with 17normal adult guinea pig tissues in III and absorption assays.The basal epidermis and hair follicles in guinea pig skin werestained by MoAb 225.28 in the IIP assay. On the other hand,no reactivity was detected with brain, eye, salivary glands, heart,lung, liver, kidney, small and large gastrointestinal tracts, pancreas, endometrium. urinary bladder, testis, uterus, skin, nucleated bone marrow cells, and peripheral blood lymphocytes.

To determine if the expression of the epitope defined by

200Kd

974Kd

i

Õmabcde

Fig. 3. SDS-PAGE analysis of antigen(s) immunoprecipitated by anti-humanHMW-MAA MoAb 225.28 from ["Sjmethionine-labeled guinea pig melanomacell lines GP-1 (lane a), GP-6 (lane c), GP-8 (lanes d and e). and human melanomacell line CaCL 78-1 (laneb). Cells were metabolically labeled with ("SJmethioninefor 18 h and solubilized with NP40. Following preclearing with normal mouseserum, antigens were immunoprecipitated with MoAb 225.28 (lanes a to d) orwith normal murine IgG (lane e). Immunoprecipitates were analyzed on a 7.5 to15% polyacrylamide gradient gel under reducing conditions. The molecular weightmarkers [mefA>7-'4CJmethyIated myosin (M, 200,000) and phosphorylase B (M,

97,000) are shown in lane m. Large arrowhead, a M, 280,000 component; smallarrowhead, a M, >440,000 component. Kd, expressed as M,.

280 Kd-

B C D E F GFig. 4. SDS-PAGE analysis of antigen(s) immunoprecipitated by anti-human

HMW-MAA MoAbs 149.53 and 225.28 from the lysate and spent medium ofradiolabeled guinea pig melanoma cell line GP-5. Cells were labeled with '"I,solubilized with NP40, and immunoprecipitated with MoAb 149.53 (lane A),murine IgG (lane B), and MoAb 225.28 (lanes C and D). Immunoprecipitateswere analyzed on a 7.5 to 15% polyacrylamide gradient gel under reducingconditions. X-ray films were exposed for 5 (lanes A to C), and 2 (lane D) days.Spent medium was harvested from a culture of [35S]methionine-labeled culturedguinea pig melanoma cells, immunoprecipitated with MoAbs 149.53 (lane E) and225.28 (lane F) and murine IgG (lane G). Immunoprecipitates were analyzed ona 7.5 to 15% polyacrylamide gradient gel under reducing conditions. Kd, expressedas A/,.

MoAb 225.28 was a general phenomenon related to the malignant transformation of guinea pig cells, the guinea pig hepa-toma cell lines LI and LIO were tested for reactivity with MoAb225.28 in the ELISA and IIP assay. No reaction was detectedin either assay.

Expression and Synthesis of S100 by Cultured Guinea Pig4838

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ABCDEF _A

280 Kd-

Fig. 5. SDS-PAGE analysis of components immunoprecipitated by anti-human HMW-MAA MoAbs 149.53 and 225.28 from '"I-labeled guinea pig melanoma cell line GP-5 following reciprocal immunodepletions. Cells were labeledwith '"I, solubilized with NP40, and immunodepleted with MoAbs 225.28 (lanesA to C) and 149.53 (lanes D to /•').Immunodepleted cell extracts were immunoprecipitated with MoAbs 225.28 (lanes A and /') and 149.53 (lanes B and /-.')and

murine IgG (lanes C and F). Immunoprecipitates were analyzed on a 7.5 to 15%polyacrylamide gradient gel under reducing conditions. Kd, expressed as V,

Table 5 Indirect immunofluorescence staining with rabbit anti-SWO antiserum ofcultured guinea pig melanoma cells

CelllineGP-1

GP-5GP-6GP-8LILIOCaCL

78-1CaCL 73-36NKI302HCT-8R

KBSpeciesGuinea

pigGuinea pigGuinea pigGuineapigGuinea

pigGuineapigHuman

HumanHumanHuman

HumanHistotypeMelanoma

MelanomaMelanomaMelanomaHepatoma

HepatomaMelanoma

MelanomaMelanomaColon

carcinomaOral carcinomaReactivity+++•

+++++++-++++++

++-

" Relative intensity of immunofluorescence staining.

Melanoma Cells. Since normal and neoplastic cells of humanmelanocytic lineage have been shown to express SIOO proteinin the cytoplasm (35-37), the four cultured guinea pig melanoma cell lines were tested with a rabbit anti-Si00 antiserum

in IIP. Three human melanoma, two human carcinoma, andtwo guinea pig hepatoma cell lines were included as positiveand negative controls. As summarized in Table 5, the fourguinea pig melanoma cell lines as well as the three humanmelanoma cell lines reacted with the rabbit anti-Si00 antiserum

strongly. On the other hand the guinea pig hepatoma cell linesLI and LIO and the human carcinoma cell lines HCT-8R and

KB were not stained.SDS-PAGE analysis under reducing conditions showed that

a component with an estimated M, of 9500 was precipitated bythe anti-Si00 antiserum from each of the four guinea pig

melanoma cell lines (Fig. 6, A and B) and from the humanmelanoma cell lines CaCL 78-1 and NKI-302 (results notshown). The position of the molecules precipitated from theguinea pig melanoma cell lines corresponded to that of thepurified bovine SIOO protein (Fig. 6, B and Q.

abed200 Kd -

974 Kd

69Kd — Î

53 Kd46 Kd —^

22.5 Kd>

abc

• - <200Kd

i;--97.4Kd

<69Kd

•53Kd<46Kd

•22.5Kd

-S100-S100

Fig. 6. SDS-PAGE analysis of components immunoprecipitated by a rabbitanti-Si00 protein antiserum from [35S]methionine-labeled guinea pig melanomacell lines and of puntici! bovine S100 protein. Guinea pig melanoma cells GP-I(.-(.lanes b and </).GP-S (A, lane c), GP-6 (B, lane a), and GP-8 (I!, lane ft) werelabeled with ("Sjmethionine, solubilized with NP40, and immunoprecipitatedwith rabbit anti-Si 00 protein antiserum (I. lanes b and <: It. lanes a and b) andnormal rabbit serum if, lane d). Immunoprecipitates were analyzed on a 7.5 to15% polyacrylamide gradient gel under reducing conditions. The molecular weightmarkers [meiAy/-MC]methylated myosin (M, 200,000), phosphorylase B (M,97,400), bovine serum albumin (M, 69,000), -y-globulin subunits (M, 53,000 and22,500), and ovalbumin (M, 46.000) are shown in lanes a and c of A and li.respectively. Highly purified bovine SIOO protein (C, lane b) was electrophoresedon a 7.5 to 15% polyacrylamide gradient gel under reducing conditions andstained with Coomassie brilliant blue. The unlabeled molecular weight markersphosphorylase B IA/, 97,400), bovine serum albumin (M, 46,000), ovalbumin (M,69,000), carbonic anhydrase (M, 30,000), trypsin inhibitor (M, 20,000), and a-lactalbumin (M, 14,400) are shown in lanes a and c of C. Kd, expressed as M,.

DISCUSSION

Serological and immunochemical studies have shown for thefirst time cross-reactivity of the anti-human HMW-MAAMoAbs 149.53 and 225.28 with guinea pig melanoma cells.Inhibition experiments with antiidiotypic monoclonal antibodies suggest that the determinant recognized by MoAb 149.53on guinea melanoma cells is similar but not identical to thatrecognized on human melanoma cells. On the other hand, thedeterminants recognized by MoAb 225.28 on both types of cellsdo not display detectable differences under the conditions ofthe assay system. In view of current applications of MoAb225.28 and antiidiotypic monoclonal antibodies to developimmunodiagnostic and immunotherapeutic approaches to malignant melanoma (38-41), the cross-reactivity of MoAb 225.28with guinea pig melanoma cell lines and the restricted distribution of the corresponding determinant in normal adult guineapig tissues suggest that this animal model may be useful forimmunobiological studies of melanoma.

The molecular profile of the antigen immunoprecipitated byanti-human HMW-MAA MoAbs 149.53 and 225.28 fromguinea pig melanoma cells is different from that synthesized byhuman melanoma cells, since the antigen synthesized by guineapig melanoma cells consists of only one M, 280,000 subunit,while the HMW-MAA synthesized by human melanoma cellsconsists of two subunits of M, 280,000 and >440,000 (14, 42-44). It is of interest that the antigen immunoprecipitated byanti-HMW-MAA monoclonal antibodies from hybrids derivedfrom the fusion of human and mouse neuroblastoma cellsconsists mainly of the lower molecular weight component (45).Since biosynthetic studies performed by Bumol and Reisfeld(44) and by Kantor et al. (46) using human melanoma cells

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have shown that the higher molecular weight subunit (M,>440,000) of the HMW-MAA derives from the smaller one(M, 280,000) through the addition of carbohydrates, in guineapig melanoma cells the maturation of this molecule may bearrested at the stage of M, 280,000. On the other hand it isunlikely, although it cannot be excluded, that the M, 280,000molecule expressed by guinea pig melanoma cells and the M,280,000 molecule expressed by human melanoma cells arestructurally different but share some epitopes.

The antigenic profile of the human HMW-MAA and itsguinea pig counterpart are different since they share only 2 ofthe 4 determinants recognized by the anti-HMW-MAA monoclonal antibodies tested in the present study and none of the 6additional determinants recognized by a panel of 21 anti-humanHMW-MAA monoclonal antibodies.6 It remains to be deter

mined whether the lack of reactivity of guinea pig melanomacells with most of the anti-human HMW-MAA monoclonal

antibodies reflects the lack of synthesis or of cell surface expression of the corresponding determinants. Furthermore, althoughthe human HMW-MAA and its guinea pig counterpart are bothheterogeneous in the expression of the determinants recognizedby monoclonal antibodies (13, 17, 47, 48), the degree of heterogeneity is different on the two types of antigens. While thelarge majority of HMW-MAA molecular species synthesizedby human melanoma cells express the determinants recognizedby MoAbs 149.53 and 225.28 (13), a much higher number ofmolecules synthesized by guinea pig melanoma cells expressthe determinant defined by MoAb 225.28, but lack that definedby MoAb 149.53.

S100 refers to a heterogeneous population of low molecularweight acidic proteins which bind calcium (49, 50). S100 wasinitially considered a nervous system-specific antigen localizedin glial cells and Schwann cells (51-53). However, it has alsobeen detected in adipose tissues (54, 55), thymocytes (56), andin normal, benign, and malignant melanocytes (34-36, 57). Itis of interest that guinea pig melanoma cells also express andsynthesize S100 protein which has an apparent molecularweight similar to that of S100 proteins isolated from humanmelanocytes and melanoma cells and from bovine brain tissues.Aside from the presence of melanosomes and/or premelano-somes in these cells, the S100 and M, 280,000 moleculesprovide useful additional markers to characterize guinea pigmelanoma cells.

The lack of reactivity of guinea pig melanoma cells withMoAbs R-24 and M2590 is noteworthy in view of their cross-reactivity with xenogeneic melanoma cells. The anti-disialogan-glioside GD3 MoAb R-24 elicited with human melanoma cellsin FI hybrids of BALB/c x C57BL/6 mice cross-reacts withfish melanoma cells.7 MoAb M2590 elicited with murine melanoma cells B16 in C57BL/6 mice cross-reacts with hamsterand human melanoma cells (5, 6). In view of the differentialreactivity of guinea pig melanoma cells with monoclonal antibodies recognizing distinct determinants of the human HMW-MAA, we do not know whether their lack of reactivity withMoAbs R-24 and M2590 reflects the lack of correspondingdeterminants or of corresponding antigenic structures. If thelatter is the case, malignant transformation of melanocytes isassociated with an increased expression of different antigenicstructures in different animal species. Correlation of thesefindings with the biological properties of melanoma cells indifferent animal species may contribute to our understanding

6C. V. Hamby, unpublished results.7 F. Anders, personal communication.

of the functional role of MAA defined by monoclonal antibodies.

In view of the expression of HLA Class II antigens oncultured melanoma cells (58, 59), the lack of reactivity of theanti-HLA Class II monoclonal antibodies with guinea pig melanoma cells is noteworthy, especially since MoAb Q5/13 hasbeen shown to cross-react with guinea pig Class II histocom-patibility antigens (60). This finding in conjunction with thelack of expression of Class II histocompatibility antigens bymurine melanoma cells B168 suggests that the appearance of

HLA Class II antigens associated with malignant transformation of melanocytes is restricted to the human species. Characterization of the mechanism(s) underlying this phenomenonand comparison of the immune response to MAA in humansand other animal species may provide useful information aboutthe pathogenesis of melanoma and the role of HLA Class IIantigens in the interaction of melanoma cells with the host's

immune system.

ACKNOWLEDGMENTS

The authors wish to acknowledge the expert technical assistance ofAnna Fenyes, Patricia Chmilewski, and M. Rita Nicotra and wish tothank Harriett V. Harrison, Edwina L. Jones, and Deborah O. Kimbofor excellent secretarial assistance. Appreciation is also expressed tothe investigators named in the text who kindly supplied various antibodies and cell lines.

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1987;47:4835-4841. Cancer Res   Shuen-Kuei Liao, James W. Smith, Pak C. Kwong, et al.   with Guinea Pig Melanoma CellsWeight-Melanoma Associated Antigen Monoclonal Antibodies Cross-Reactivity of Murine Anti-Human High Molecular

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