suppression of spontaneous melanoma metastasis in … · suppression of spontaneous melanoma...

1991;51:2193-2198.Cancer Res Barbara M. Mueller, Cynthia A. Romerdahl, Jeffrey M. Trent, et al.

ReceptorMice with an Antibody to the Epidermal Growth Factor Suppression of Spontaneous Melanoma Metastasis in Scid

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(CANCER RESEARCH 51. 2193-2198. April 15, 1991]

Suppression of Spontaneous Melanoma Metastasis in Seid Mice with an Antibody

to the Epidermal Growth Factor ReceptorBarbara M. Mueller, Cynthia A. Romerdahl,1 Jeffrey M. Trent, and Ralph A. Reisfeld2

Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037 [B. M. M., C. A. R., R. A. R.J, and Departments of Radiation Oncology andHuman Genetics, University of Michigan, Ann Arbor, MI 48109 [J. M. T.]

ABSTRACT

The human melanoma cell line M24met metastasizes spontaneouslyfrom s.c. tumors to multiple distant sites in mice with severe combinedimmunodeficiency. Metastasis to lymph nodes and lungs is found in 100%of the animals. M24met has an undifferentiated phenotype and extracopies of the short arm of chromosome 7. This cell line expresses theepidermal growth factor receptor, and 425.3, a monoclonal antibody tothe epidermal growth factor receptor, binds to 291,000 receptor moleculesper M24met cell with a KDof 2.3 x 10"'Â°M. This antibody has no effect

on the proliferation of M24met cells under tissue culture conditions anddoes not mediate effector cell or complement-dependent cytotoxicity ofthese cells in vitro. However, treatment of established s.c. M24mettumors in mice with severe combined immunodeficiency with monoclonalantibody 425.3 specifically suppresses spontaneous metastasis of thesetumors. Total doses of 4, 2, and 1 mg antibody per mouse decrease thenumber and size of melanoma mÃ©tastasesand prolong the life span oftreated animals. Treatment with 4 mg of the F(ab'>2 fragment of mono

clonal antibody 425.3 does not influence M24met melanoma metastasis,implying a significant contribution of the lv portion to the antimetastaticeffect of this antibody.

INTRODUCTION

Tumor xenografts in athymic nude mice are well-establishedanimal models for the study of human cancers. Several humanmelanoma cell lines were recently reported to spontaneouslymetastasize in athymic nude mice (1-4), providing models forthis fatal event in clinical disease. They are useful in the studyof mechanisms of metastasis, as well as in the preclinicalevaluation of new therapies for human melanoma. The latterrequires a model that produces mÃ©tastasesof significant numberand size in a high percentage of animals inoculated with theprimary tumor. We describe here a novel, very aggressive modelof spontaneous melanoma metastasis in seid3 mice based on

the human cell line, M24met. This model differs from thoseabove, as mÃ©tastasesform in 100% of the animals within only5-6 wk from the time of the initial tumor cell inoculum.

The metastatic cell line M24met strongly expresses the EGFreceptor. The expression of this receptor by melanoma cell linesis closely related to that of phenotypic differentiation markers.Thus, less-differentiated melanomas have an epithelioid morphology, are nonpigmented, and are EGF receptor-positive (5).The EGF receptor seems to be expressed selectively on humanmelanoma cell lines that contain an extra copy of chromosome7 (6). We find that the M24 and M24met cells contain additional copies of the short arm of chromosome 7. Melanomacell lines isolated from clinical metastasis can grow in tissue

Received 9/11/90; accepted 2/6/91.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.

1Present address: BASF Research, 195 Albany Street, Cambridge, MA 02139.2To whom requests for reprints should be addressed, at Department of

Immunology, Research Institute of Scripps Clinic, 10666 North Torrey PinesRoad, La Jolla, CA 92037.

3The abbreviations used are: seid, severe combined immunodeficiency; mAb,monoclonal antibody; EGF, epidermal growth factor; FBS, fetal bovine serum;ADCC, antibody-dependent cellular cytotoxicity; ADMC, antibody-dependentmacrophage cytotoxicity.

culture independent from exogenous growth factors, but a highpercentage of these cells is growth-responsive to EGF and othergrowth factors (7, 8). In melanoma biopsies, the presence ofEGF receptor correlates with malignancy, exhibiting the highest expression on advanced metastatic lesions (9).

mAbs to the EGF receptor were reported to suppress thegrowth of solid tumors implanted in the skin of nude mice (10,11). Here, we describe the effects of anti-EGF receptor antibodytherapy on spontaneous and aggressively metastasizing humanmelanoma M24met tumors in seid mice.

MATERIALS AND METHODS

Animals. Athymic nude mice (nu/nu) were purchased from theNational Cancer Institute (Bethesda, MD). C.B-17 seid/seid mice wereobtained from the rodent breeding colony of the Research Institute ofScripps Clinic. Six- to 8-wk-old female mice were used for metastasisexperiments. Mice were housed under specific pathogen-free conditionsand were handled in a laminar flow air cabinet. Experiments wereperformed according to the guidelines set down in the NIH Guide forthe Care and Use of Laboratory Animals.

Cell Lines. The human melanoma cell line, M24, was derived froma biopsy of a lymph node metastasis and found to be tumorigenic innude mice (12). This cell line was provided to us by Dr. D. L. Morton(UCLA, Los Angeles, CA). A total of IO6M24 cells (passage 30) wereinjected s.c. into the flank of 6-wk-old nude mice. These animals (3/3)grew large s.c. tumors and were found with multiple metastatic foci intheir lymph nodes when sacrificed 8 wk after the injection of the tumorcells. A cell line was established from an invaded lymph node of a nudemouse and designated M24met.

M24 and M24met cells were grown in RPMI 1640 tissue culturemedium and supplemented with 10% fetal bovine serum (FBS), 2 mMglutamine, and 50 Mg/ml gentamycin sulfate. The cells were detachedfrom tissue culture plastic with 0.5 mM EDTA, 0.15 M NaCl, 0.02 M4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, washed once, andused as a single-cell suspension.

M21, a subclone derived in this laboratory from the human melanoma cell line UCLA-SO-M21, originally provided by Dr. D. L. Morton (UCLA, Los Angeles, CA), was grown in RPMI 1640 tissue culturemedium supplemented with 10% FBS.

Chromosome Analysis. Exponentially growing cultures of M24 andM24met were harvested for karyotypic analysis, slides were prepared,and G-banding was performed, as previously described (13). A minimum of 30 cells per cell line was analyzed, with results expressedaccording to ISCN recommendations (14).

Monoclonal Antibodies and Fragments. Murine mAbs were used totest for the expression of melanoma-associated cell surface antigens.Hybridoma cells producing mAb 425.3 (IgG2a) were provided by Dr.H. Koprowski (The Wistar Institute, Philadelphia, PA). Hybridomasproducing mAb 96.5 and mAb B3/25 were made available by Dr. I.HellstrÃ¶m(Oncogene Corp., Seattle, WA) and Dr. I. Trowbridge (SalkInstitute, La Jolla, CA), respectively. Antibodies were purified fromhybridoma supernatant fluids by affinity chromatography on proteinA-Sepharose. mAb H36 (IgG2a) directed against influenza hemagglu-tinin was kindly provided by Dr. W. Gerhard (The Wistar Institute,Philadelphia, PA) and used as a control antibody.

F(ab')2 fragments of mAb 425.3 were produced by peptic digestion.

Five mg pepsin (Sigma, St. Louis, MO) was incubated with 100 mgmAb 425.3 in 0.13 M acetate and 0.1 M NaCl, pH 4.0, for 4 h at 37Â°C.

Undigested antibody was removed by absorption to protein A-Sepha-

2193




SUPPRESSION OF MELANOMA METASTASIS BY ANTI-EGF-R mAb

rose. The digestion of 80 mg purified 425.3 yielded 50 mg F(ab')2fragment. The binding activity of the F(ab')3 of 425.3 to M24met cells

was verified in a direct binding assay.Indirect Immunofluorescence. Melanoma cells (IO6) were incubated

for l h at 4Â°Cwith 10 pg mAb. The cells were washed 3 times in

phosphate-buffered saline containing 1% FBS and 0.02% NaNj andwere stained for l h at 4Â°Cwith goat antimouse IgG conjugated to

fluorescein isothiocyanate (Boehringer, Indianapolis, IN). The cellswere again washed 3 times, and IO4cells were analyzed in a FACS 440

flow cytometer (Becton Dickinson, Mountain View, CA).Saturation Binding. For direct binding assays, 425.3 was iodinated

by the lodo-Gen method. Antibody (500 //g) was incubated for 25 minon ice in a polystyrene tube coated with 100 //g lodo-Gen (PierceChemical Co., Rockford, IL) with 1.0 mCi Na'25I (Amersham, Arlington Heights, IL). Nonincorporated 125Iwas removed by gel filtrationon Sephadex G25 (PD10 columns; Pharmacia Fine Chemicals, Pisca-taway, NJ). The specific activity was 1.7 nCi/ng antibody.

M24met cells (2 x 10") were suspended in ice-cold RPMI 1640

medium containing 1% bovine serum albumin and 1.25 to 400 ng of'"I-labeled antibody. Nonspecific binding was determined in the presence of a 200-fold excess of unlabeled antibody and was found to be10-15% of the total bound radiolabel. After 2 h incubation at 4Â°C,the

tumor cells were washed in ice-cold RPMI 1640 with 1% bovine serumalbumin. Cell-bound radiolabel in duplicate samples was determined ina 7-scintillation counter, and the data were analyzed by the method ofScatchard (15).

Cell Proliferation Assay. M24 and M24met cells were suspended inRPMI 1640 supplemented with 1% Nutridoma-HU (Boehringer, Indianapolis, IN), and aliquots of 5 x 10' cells in 100 p\ medium were

plated into each well of a 96-well tissue culture plate. Human recombinant EGF (a kind gift of Creative BioMolecules, Hopkinton, MA)and/or mAb 425.3 were added in serial dilutions to triplicate wellscovering a range from 16-0.25 n.Mfor EGF and 100-0.4 n.Mfor 425.3.The cells were cultured for 4 days at 37Â°Cand then received anotherdose of EGF and antibody on day 3. On day 4, 1 Â¿<Ciof ['HJthymidine

(ICN, Irvine, CA) was added to each well, the plates were incubatedovernight, and cells were harvested onto glass fiber filters. The filterswere placed in Ecolume scintillation solution (ICN, Irvine, CA) andwere counted in a liquid scintillation counter. ['H]Thymidine incorpo

ration as percentage of untreated controls was used to quantify cellproliferation.

Antibody-mediated Cytotoxicity. The ability of mAb 425.3 to mediatecomplement-dependent cytotoxicity and antibody-dependent cellularcytotoxicity (ADCC) was measured, as described previously (16).Briefly, tumor cells were labeled with Na5'Cr and incubated with 10-

0.001 Mg mAb/ml. Sera from either normal volunteers or seid micewere the source of complement in a 2-h "Cr-release assay, and human

peripheral blood mononuclear cells or seid mouse spleen cells servedas effector cells in a 4-h 5ICr-release assay.

The ability of mAb 425.3 to mediate antibody-dependent macrophage cytotoxicity (ADMC) was tested, as described (17). Briefly,female seid mice were injected i.p. with 2 ml thioglycollate medium(Difco, Detroit, MI), and 4 days later peritoneal exÃºdate cells wereharvested as effector cells. Tumor cells were labeled with ['Hjthymidine

overnight and incubated with murine effector cells at a ratio of 1:50and 1:100 in the presence of 50-0.5 /jg mAb/ml. Three days later,adherent cells and supernatant fluids were harvested separately and['HJthymidine release was measured to evaluate ADMC.

Spontaneous Metastasis in Mice. M24met cells (2 x IO6), in 100 n\phosphate-buffered saline, were injected s.c. into the flank of seid ornude mice. At the times indicated, mice were given i.p. injections ofeither mAb in 150 /Â¿Isaline or only 150 jul saline as a control. Tumorsizes were measured with calipers, and tumor volumes were calculatedby using the formula (a2 x Â¿>)/2,with a being the width and h the length

of the tumor. Subcutaneous M24met tumors routinely reach a size of700-900 mm1 within 14-18 days postinjection. At that time, mice were

anesthetized by injecting 2.5 mg ketamine i.p. and tumors were excisedunder aseptic conditions. Typically, 22 days after this procedure orwhen animals were moribund, they were sacrificed and examined formetastasis. The difference between the extent of metastasis of controland experimental groups was determined using the nonparametric

Wilcox rank sum test. Lungs were fixed in Bouin fixative and examinedunder a low magnification microscope for tumor foci on the surface ofthe lungs. Enlarged lymph nodes and ovaries were excised, weighed,and fixed. Sections from lungs, lymph nodes, and ovaries were stainedwith hematoxylin/eosin and examined histologically.

RESULTS

Characterization of M24 and M24met Cells. M24 andM24met are nonpigmented, human melanoma cell lines. Theygrow adherent to tissue culture plastic with an irregular morphology, revealing both spindle-shaped and epithelioid cells.

Chromosome banding of both the M24 and the M24met linesdemonstrates multiple clonal karyotypic abnormalities. M24has a hypertetraploid chromosome number (range, 54-127)with 56% of cells indicating 117-122 chromosomes/cell. Establishment of the M24met line has selected for hyperdiploid cells(range, 41-74) with 68% of cells containing 53-57 chromosomes/cell. The karyotype of M24 cells is complex, demonstrating several identifiable clonal structural chromosome rearrangements including: i(lq), t(5;ll)(ql3;q21), i(6p),del(6)(ql4:), del(7)(q32:), t(9;14)(qll;pl3), t(10;10)(pl3;qll)+ 1-8 unidentified marker (Umar). Fig. 1 demonstrates a G-banded karyotype from the M24met subline. The subline sharesnumerous markers in common with the parental M24 subline(Fig. IA), confirming its origin from a common progenitor.However, three markers from the parent were lost in theM24met cell (Fig. IB), and one structural alteration is foundto be unique to the M24met cell: t(l I;14)(pl4;ql3).

M24 and M24met cells were tested for the expression ofhuman melanoma-associated antigens by indirect immunoflu-orescence (Table 1). Both cell lines stain positively for mela-notransferrin (p97). They also express the transferrin receptorand the EGF receptor, both receptors being present on a higherpercentage of M24met than M24 cells. Fig. 2 shows a flowcytometric analysis of M24 and M24met cells stained withmAb 425.3 directed against the EGF receptor. An early passageof M24 cells consists of an EGF receptor-negative and an EGFreceptor-positive subpopulation; however, all M24met cells areEGF receptor positive. M24 and M24met cells tested essentially negative for melanoma-associated proteoglycan (mAb9.2.27), the melanoma-associated vitronectin receptor av/33(mAb LM609), and the disialogangliosides GD2 (mAb 14.G2a)and Grx, (mAb MB3.6). After many passages on tissue cultureplastic, however, the percentage of G^-positive M24 andM24met cells can increase quite dramatically.

A direct binding assay with 125I-labeled mAb 425.3 was used

to determine the number of EGF receptor molecules per cell.Under saturation conditions, 291,000 Â±33,000 molecules werefound to bind per M24met cell (n = 4) with a A"Dof 2.3 x 10~'Â°

M when the binding curves were analyzed by the method ofScatchard (Fig. 3). F(ab')? fragments of mAb 425.3 also boundwith a Ko of 2.3 x IO'10 M to M24met cells.

Effect of Anti-EGF Receptor mAb 425.3 on M24 and M24metCells in Vitro. The effect of EGF and anti-EGF receptor antibodies on the growth of M24 and M24met cells was examinedunder serum-free conditions. Both cell lines grew well underserum-free conditions, i.e., independently of exogenous growthfactors. At all concentrations tested, EGF alone and in combination with 425.3 leads to a small increase in [-'Hjthymidine

incorporation of these cells (10% over untreated controls),whereas mAb 425.3 alone has no effect on the proliferation ofthese cells.

The ability of mAb 425.3 to mediate in vitro antibody-dependent cytotoxicity was tested in the presence of human and

2194





piiiliiiilliiiiB

) v,Â¡(imâ€¢iiiiiiiiiiiiiiA

it iiMl "IH W Â«l)W â€¢'?â€¢''Â»

3

6 9 10 11

Ã‰ÃŒII iÂ»13 14 15

ti il16 17 18

Â«1iil1920At

Ã¯1'Der

1 6q-Â«

l7q-tl9;*>)I

Ã¨Ã¬*limar21jtl10;?lB1tl1;?lttl5;11l*22i(6p)

Fig. 1. G-banded karyotypc of the M24met cell line. Cross-hatching illustrates the occurrence as a clonal marker of a der(l) chromosome in the M24met sublinenot found in this cell. A, examples of structurally altered clonal abnormalities from the M24 parental cell line shared in common with the M24met subline; B, clonalchromosome alterations unique to the M24 parental cell line which were lost in the M24met subline.

Table 1 Expression of melanoma-associated antigens on M24 and M24met cells

Antigen mAB Reference M24 M24met

Melanotransferrin(p97)EGFreceptorTransferrin

receptorMelanoma-associatedproteoglycanVitronectin

receptor(Â«A)GDIGo396.5425.3B3/259.2.27LM60914.G2aMB3.61819202122232488.0

Â±10.668.2Â±18.519.5Â±3.30.15

Â±0.10.2

Â±0.252.3

Â±1.90.3Â±0.296.7

Â±3.497.0Â±2.725.5Â±2.90.8Â±0.65.2

Â±4.94.1

Â±4.00.9Â±0.3

Â°Percentage of cells that stained positive in flow cytometric analyses of indirect

immunofluorescence with the specific mAb listed. Values represent means andstandard deviations of three independent experiments.

murine complement, human peripheral blood mononuclearcells, seid mouse spleen cells, and seid mouse macrophages.M24met cells could not be lysed by mAb 425.3 in complement-

dependent cytotoxicity, ADCC, or ADMC assays. On the otherhand, the positive control M21 melanoma cells, when incubatedwith the anti-GD2 antibody 14.G2a, were effectively lysed byall effector mechanisms mentioned above, except with seidmouse complement.

Spontaneous Metastasis of \124niel. M24met metastasizesspontaneously from s.c. tumors to multiple distant sites. Thisis observed in a model system where the s.c. tumor is completelyexcised and rarely grows back (5% of the animals) at the primarysite. MÃ©tastasesto lymph nodes and lungs are found to occurin 80-90% of the nude mice and in 100% of the seid mice.Lymph nodes ipsilateral to the primary tumor are enlarged inall animals with mÃ©tastases;in approximately 20% of theanimals, however, contralateral lymph nodes are affected. Enlarged lymph nodes can reach weights up to 4 g. On the surfaceof the lungs, one can often count up to 500 metastatic foci. Insome of the animals, however, the lungs are covered with tumorand distinct foci cannot be counted. Melanoma mÃ©tastasestothe ovaries are observed in 20-25% of the mice. Table 2

summarizes the pattern of metastasis of M24met cells and theweight of enlarged lymph nodes in nude and in seid mice.

HistolÃ³gica! sections of the lungs demonstrate nests andnodules of metastatic tumor in alveolar spaces. In some animals, >50% of the lung is replaced by tumor and the remainderof the lung shows atelectasis. HistolÃ³gica! sections of enlargedlymph nodes demonstrate sheets of malignant polygonal cells.The center of very large lymph nodes is often necrotic.

2195




SIPPRKSSION OF MELANOMA METASTASIS BY ANTI-EGF-R mAb

300-TT

Fig. 2. Flow cytometric analysis of indirectimmunofluorescence after staining M24 andM24met cells with mAb 425.3 and goat anti-mouse IgG antibodies conjugated to fluores-cein isothiocyanate (solid line) or only with thesecondary antiserum (broken line).

03~E

CDC_5

FluorescenceIntensity

0.06

0.05

LL

0

0.03

0.02

0.010 0.2 04 0.6 0.8 10 1 2 1.4 1.6 1.8 20

Input Antibody |nM)

4 6 8 10

Bound Antibody (pM)

12

Fig. 3. Saturation binding and Scatchard plot analysis for '"I-labeled mAb

425.3 on M24met cells. B/F, bound versus free.

Table 2 Spontaneous metastasis ofM24met melanomaMice received s.c. injections of 2 X 10* M24met cells in the flank. Primary

tumors were removed when they had reached a size of 500-800 mm1, and animals

were sacrificed 22 days later. Data shown for nude and seid mice are compiledfrom 3 and 4 independent experiments, respectively.

Athymic nudemiceOrganIALNÂ°IILNCALNCILNOvariesLungsAny

siteNo.

micewithapparentmÃ©tastases8/1912/192/194/194/197/1983Â«Weightorno.490*(90-1400)1.400(150-4320)20,15050(10-740)57r(2->500)Seid

miceNo.

micewithapparentmÃ©tastases22/2311/232/234/236/2323/23100%Weightorno.1.950(450-4120)740(250-2400)80.24090(40-370)>500r(150->500)

" IALN, ipsilateral axillary lymph node; CALN. contralateral axillary lymphnode; IILN, ipsilateral inguinal lymph node; CILN. contralateral inguinal lymphnode.

* Weight of enlarged lymph nodes (mg); median and range are shown.' Number of metastatic foci on the lungs of affected mice: median and range

are shown.

Suppression of Metastasis with Anti-EGF Receptor Antibody.Treatment of established s.c. M24met tumors in seid mice withmAb 425.3 specifically suppresses spontaneous metastasis ofthese tumors. In an initial experiment, groups of 10 mice eachwere given injections of either 1 mg 425.3 or an isotype-matchedcontrol mAb H36 6 days after tumor cell inoculation, when thetumor volumes were 30-60 mm', and also on days 10, 14, and

16. Subcutaneous tumors showed essentially identical growthrates whether the animals had been given a total injected doseof 4 mg mAb 425.3, control antibody, or saline. The primarytumors were excised on day 16, when they reached volumes of

500-800 mm3. Injection of mAb 425.3 markedly decreased the

number and size of melanoma mÃ©tastases,whereas the nonspecific antibody H36 showed no effect (Table 3).

The experiment was repeated under the same conditions, andthe injection of 4 mg mAb 425.3 was shown to prolong the lifespan of treated animals (Fig. 4). In this case, all but one of thecontrol mice had died 54 days after surgical removal of theprimary tumor. On this day, all mice were sacrificed and noevidence of melanoma metastasis was found in 3 of the 7 micetreated with mAb 425.3, whereas 4 of these animals had mÃ©tastases to one or more sites.

The effect of smaller doses of antibody on melanoma mÃ©tastases was also studied, and essentially identical results wereobtained with total doses of 4, 2, and 1 mg of 425.3 antibody,respectively. Table 4 provides a summary of three independentexperiments. A total dose of 4 mg F(ab')2 fragment of mAb

425.3 was administered in daily injections from day 6 to day16 to compensate for the more rapid blood clearance of suchantibody fragments; however, it had no effect on the growth ofthe s.c. M24met tumors. After the primary tumors were excisedon day 16, it was evident that treatment with F(ab')2 of mAb

425.3 had no effect on M24met melanoma metastasis in seidmice (Table 4).

DISCUSSION

In this report, we describe a model of spontaneous metastasisfor human melanoma in immunodeficient seid mice and demonstrate significant suppression of metastasis using an antibodyto the EGF receptor. The metastatic process involves distinctphases, including the release of tumor cells from the primarylesion into blood or lymph vessels, the survival of tumor cellsin the circulation, and the ability of tumor cells to arrest indistant sites and to invade the extracellular matrix (25, 26).Spontaneous metastasis models are particularly useful becausethey are thought to emulate the major events of the metastasiscascade. Our seid mouse model, based on the cell line M24met,has several distinct practical advantages over other spontaneousmetastatic models reported thus far (1-4). This model formsspontaneous mÃ©tastasesin 100% of the animals inoculated withprimary tumor and requires only 5-6 wk from the time of theinitial tumor cell inoculation to the establishment of fulminantmÃ©tastases.Others have observed that the seid mouse is a betterhost than the nude mouse for some, but not all s.c. humantumor xenografts (27, 28) and for the disseminated growth ofB-cell tumors (29). We describe here M24met, a very aggressivehuman melanoma cell line, which metastasizes more reproduc-ibly in seid than in nude mice.

M24 and M24met cells express some typical melanoma-

2196





Table 3 Specific suppression ofM24mel metastasis with anti-EGF receptor mAbM24met cells (2 x 10') were injected s.c. into the (lank of seid mice. On day 16, the primary tumors were excised. The animals were sacrificed 22 days later, and

melanoma mÃ©tastaseswere evaluated.

Weight or no.

425.3 (anti-EGF receptor) H36 (isotype control)

IALNIILNCALNCILNLungs

5/9*(60,310.460,580,4360)c

1/9(470)0/90/98/9(3,4,6,26,65,130,>200.>500)Â¿

IO/10*(600,730,940,950,1300,1390,1420.1540,1810,2250)

4/10(40,390,480,1630)1/10(10)1/10(290)10/10 (all animals more than 500 foci)

0.003

0.0004Â°Probability of no difference between the groups as determined by Wilcoxon rank sum test.* Number positive for metastasis of number that received i.p. injections of 1 mg mAb 425.3 or mAb H36 on days 6, 10, 14, and 16 after s.c. injections of M24met

cells.' Weight of invaded lymph nodes.**Number of metastatic foci detected on the lungs of each animal.

<^

0 10 20 30 40 50 60

Days After Removal of the Primary Lesion

Fig. 4. Survival of seid mice with M24met melanoma mÃ©tastases.One mgmAb 425.3 (solid line) or saline (broken line) was injected i.p. on days 7, 10, 13,and 16 after 2 x 10' M24met cells were injected s.c. into seid mice. On day 16,

the primary tumors were excised and no further treatment was applied.

associated antigens, such as melanotransferrin and growth factor receptors. Other antigens, like the melanoma-associatedproteoglycan and GDz, as well as GDj disialogangliosides thatare usually found on the vast majority of melanoma cell linesand biopsies (30), are missing from M24 and M24met cells.Karyotype analysis showed M24 and M24met to have additional copies of the short arm of chromosome 7. The karyotypeand the phenotype of M24 and M24met cells, including theexpression of the EGF receptor, are consistent with that ofrelatively undifferentiated, malignant melanoma cells (5, 6).

We report here on the treatment of established s.c. melanomatumors with mAb 425.3 directed against the EGF receptor.Although this treatment fails to influence the growth of theprimary tumor during the 10 days of antibody administration,it drastically decreases the number and size of mÃ©tastasesandprolongs the survival of treated animals when compared tountreated controls. This is noteworthy, as others have shownthat the size of the primary tumor at the time of its removalcan determine the incidence of spontaneous metastasis (31).Our data suggest that M24met cells that are in circulation or

have formed micrometastases in lymph nodes or lungs areeliminated by the anti-EGF receptor antibody.

We are aware of only one other report in the literature wheresuppression of spontaneous metastasis is achieved by mAb (2).In this regard, Iliopoulos et al. described the inhibition ofmelanoma metastasis using two different isotypes of mAb tothe disialoganglioside antigens GD2/GD> These investigatorsobserved inhibition of metastasis in two different situations,which both contrast with our observations. First, anti-GD2/GD3mAb (IgG2a) suppresses metastasis only when the growth ofthe primary tumor is also decreased. Second, anti-GD2/GD3mAb (IgGl), which fails to suppress growth of the primarytumors, can suppress metastasis only when injected concomi-tantly with the initial s.c. tumor inoculum (2).

Masui et al. reported that two antibodies directed against theEGF receptor, 225 (IgGl) and 528 (IgG2a), compete with EGFfor receptor binding and inhibit proliferation of the humanepidermoid carcinoma cell line A431 in culture, which expresses2.6 x 10" EGF receptor molecules/cell (10). The same investi

gators also observed that mAb 528, but not mAb 225, activatesmacrophage- and complement-dependent lysis of A431 cells invitro (32). However, irrespective of whether these two anti-EGFreceptor mAbs are able to mediate cytolysis in vitro, they areboth able to suppress the growth of s.c. A431 grafts in nudemice. From these observations, Masui et al. concluded thatblocking the binding of EGF to its receptor, as well as theimmune mechanisms of the host animal, is involved in theantitumor effect of anti-EGF receptor mAb on A431 cells (10).

Rodeck et al., when applying a total dose of 2 mg mAb 425.3directed against the EGF receptor, demonstrated suppressionof s.c. A431 tumors, as well as of tumors formed by the humancolorectal carcinoma cell line SW948 in nude mice. Monoclonalantibody 425.3 binds to a different epitope of the EGF receptorthan mAb 225 and 528, but also inhibits the growth of A431cells in vitro by blocking EGF binding and/or by inducing downregulation of the EGF receptor, and mAb 425.3 also mediatesmonocyte- and lymphocyte-dependent cytotoxicity in vitro (I I).

Aboud-Pirak et al. used mAb 108.4, also directed against thehuman EGF receptor, against s.c. tumors and experimental

Table 4 Suppression ofM24met spontaneous metastasis

Treatment:Totaldose:No.

animals with no evidence ofmetastasisNo.animals with metastasis to 1siteNo.animals with metastasis to more than 1siteNo.animals with enlargedIALNMedian

weight and range of IALN(mg)No.

animals with metastasis to thelungsMedianno. and range of foci/lungsSaline0/141/1413/1413/142100(650-4120)13/14>500(150->500)425.3

4mg3/167/166/166/16500(60-4360)10/1625(1->500)425.32mg3/61/62/62/6240.7001/63425.31mg0/52/53/55/5160(20-680)3/525(15-250)F(ab')2of425.34mg0/90/99/99/92040(210-4020)9/9>500(40->500)

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SUPPRESSION OF MELANOMA METASTASIS BY ANT1-EGF-R mAb

lung mÃ©tastasesof the epidermoid carcinoma cell line KB innude mice (33). They achieved growth inhibition of the s.c.tumors and a reduction of experimental metastasis by treatmentwith both mAb 108.4 (IgG2a) and its F(ab')2 fragment.

In our hands, mAb 425.3 specifically suppresses spontaneousmetastasis of M24met in seid mice after the injection of totaldoses of 4, 2, and 1 mg mAb, respectively. A 6-fold molarexcess of a F(ab')2 fragment of 425.3 showed no effect on either

number or size of spontaneous melanoma mÃ©tastases.Thisfinding implies a significant contribution of the Fc portion ofmAb 425.3 to its antimetastatic effect. We were unable todemonstrate effector cell- or complement-mediated lysis ofM24met cells in vitro even though 425.3, an antibody of IgG2aisotype, mediates both ADCC and ADMC against A431 epidermoid carcinoma cells (11). However, M24met melanomacells that are resistant to cell-mediated lysis in vitro are apparently sensitive to macrophages and/or natural killer cells in seidmice. Such discrepancies between in vitro and in vivo resultsmay be explained by effector cell-mediated mechanisms oftumor cell killing other than ADCC or ADMC (34, 35).

In conclusion, we describe here a novel spontaneous metastasis model for human melanoma, and we demonstrate suppression but not complete prevention of metastasis by treatmentwith anti-EGF receptor mAb 425.3. This effective spontaneousmetastasis model provides a challenging preclinical system fordeveloping improved immunothÃ©rapies for human melanoma.

ACKNOWLEDGMENTS

The authors wish to thank the researchers mentioned in "Materialsand Methods" for generously providing cell lines and reagents. The

authors also extend thanks to Dr. Candece Gladson for advice with thehistopathology; Sue Knowles, Dorothy Markowitz, and David Vilettfor technical assistance; and Lynne Kottel for preparing this manuscript. This work was supported by grants from the National Institutesof Health, CA42508 (RAR) and CA41183 (JMT), as well as CA29476(JMT). This is the Research Institute of Scripps Clinic Manuscript#6529-IMM.

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