(CANCER RESEARCH54, 2448-2455, May I. 19941

ABSTRACT

The M0v18 (71,ic) and M0v19 (72a,ic) murine monoclonal antibodies(MAbs) recognize different epitopes on the human folate binding receptor

which is overexpressed on 90% of nonmucinous epithelial ovarian tumors.A chimeric murine-human (human yl,.c) version of both antibodies wasconstructed and expressed. The genes encoding the munne heavy andlight chain variable regions of the MOviS and M0v19 MAbs were clonedfrom the parental hybridomas, fused with genes encoding the humanheavy (yl) and light (ic) chain constant regions, respectively, and ex

pressed in the SP2/O murine myeloma cell line. Using human peripheralblood mononuclear cells as effector cells and conditions that provide for

maximum lysis (effector target = 50:1, saturating antibody concentration), the murine MOviS MAb (IgGi) mediated variable levels of specificcytolysis of the target ovarian cancer cell line IGROV1. In contrast, thechimeric M0v18 MAb mediated higher and more consistent lysis even at

a 10—100-foldlower antibody concentration. The munne M0v19 MAb(IgG2a) mediated specific lysis of IGROV1 cells, and the chimeric versionof this antibody mediated an amount of lysis at least equal to thatmediated by its murine counterparL A comparison of the ED@ valuesobtained for the murine M0v19 and chimeric M0v19 antibodies indicatesthat the chimeric M0v19 MAb was 3 to 10 times more potent than themurine M0v19 antibody. In addition, the EDso values obtained for thechimeric M0v18 and chimeric M0v19 MAbs were similar, indicating thatthese MAin are equally potent. The level of maximal lysis obtained wasdependent on the number of target molecules/cell; the same high level oflysis mediated by cMOvl8, M0v19, and cMOvl9 was observed with bothIGROV1 and OvCA432 target cells. However, only low levels oflysis wereobtained when the SW626 cell line, which expresses 1 x 1O@folate bindingprotein sites/cell, was used as a target. An equimolar mixture of thechimenc MOviS and M0v19 MAbs was no more effective in the mediation of lysis than an equivalent amount of either chimeric MAb alone.These data suggest that the folate binding receptor is expressed onIGROV1 cells at a density sufficient to provide for optimal levels ofantibody-mediated lysis using a single chimeric antibody directed at thefolate binding receptor.

INTRODUCTION

In recent years, significant advances in the systemic treatment ofpatients with ovarian carcinoma using combination chemotherapyhave reduced the short-term mortality caused by this disease (1).Despite these advances, the long-term prognosis has remained poorfor the two-thirds or more of patients presenting with extrapelvicdisease. In the majority of these patients, residual tumor cells becomerefractory to chemotherapeutic agents and eventually repopulate theperitoneal cavity, resulting in clinical relapse and death (2). Consequently, a search for alternative approaches to systemic therapy forovarian cancer has continued.

Received 8/9/93; accepted 3/1/94.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Research supported in part by Associazione Italians Ricerca Cancro and CNR-ACRO

(Committee of National Research-of the Cancer Research Oncologic).2 To whom requests for reprints should be addressed, at Apollon, Inc., One Great

Valley Parkway, Malvern, PA 19355.3 Present address: Apollon, Inc., One Great Valley Parkway, Malvern, PA 19355.

MAbs4 directed at ovarian tumor cell surface antigens have beenused in clinical trials to target radioisotopes (3, 4) to ovarian tumorswith some minimal responses. It has also been reported that MAbsdirected against tumor-associated antigens mediate lysis of tumorcells by effector immunocytes, such as natural killer cells and activated macrophages, that possess cell surface receptors for the Fcportion of antibodies (5, 6). Hybridoma B cell lines synthesizing 2murine MAbs (M0v18:yl,K; MOv19:y2a,K) that recognize noncross-reactive epitopes on the same ovarian tumor-associated antigenhave been established (7). This antigen has been identified as thehuman FBP (8, 9). These human FBP epitopes are expressed on thecell surface of the majority of both malignant and benign nonmucinous epithelial ovarian tumor cells and are limited to adult oviductepithehium and kidney proximal and distal tubules among normal cells(7, 10). The antigenic determinants recognized by MOv18 andMOv19 are present on the cell surface of ovarian cancer cells in a highcopy number (approximately 1 X 106 molecules/cell) (7). Therefore,these MAbs might be useful targeting agents or mediators of ADCC

or complement-dependent cytotoxicity for the treatment of epithehialovarian malignant tumors.

The administration of murine monoclonal antibodies to humansfrequently elicits an anti-murine immunoglobuhin response (11—15),resulting in measurable levels of FIAMA. The presence of HAMAmay result in the generation of toxic immune complexes and, furthermore, HAMA blocking the antigen combining site or Fc region of anantibody could reduce the ability of a potentially therapeutic antibodyto bind to target tumor cells or effector cells (12). In addition, murineIgG1 antibodies do not bind to human Fc receptors and are incapableof mediating cytolysis via cellular mechanisms. Replacement of themurine heavy and light chain constant regions with their humancounterparts can be accomplished using recombinant DNA techniques

(16—18),yielding a chimeric mouse-human monoclonal antibody thatshould be less immunogenic. Additionally, construction of the chimeric antibody gene with an appropriate human heavy chain geneallows for selection of an isotype with maximum biological effectorpotential.

Here we describe the construction and expression of chimeric,mouse-human MOv18 and MOv19 MAbs. The genes encoding the VLand VH regions of each antibody were cloned and fused with thehuman Ck gene and the human Cyl gene, respectively. The fusiongenes encoding the chimeric heavy and light chains of MOv18 orM0v19 were introduced into the SP2/O non-producing murine myeloma cell line by electroporation. The resulting transfectants produced immunofunctional chimeric antibodies that could be comparedwith their murine counterparts. The immunoreactivity of each chimeric antibody (cMOvl8 and cMOvl9) was similar to that of theircorresponding murine antibody, and both mediated specific cellularlysis of ovarian carcinoma cell lines.

4 The abbreviations used are: MAbs, monoclonal antibodies; FCS, fetal calf serum;

HAMA, human anti-mouse antibody; V@,L-chain variable region; VH, H-chain variableregion; PBMC, peripheral blood mononuclear cells; ADCC, antibody-dependent cellularcytotoxicity; FBP, folate binding protein; ELISA, enzyme-linked immunosorbent assay;PBS, phosphate-buffered saline; BSA, bovine serum albumin; c, chimeric.

2448

Chimeric Murine-Human Antibodies Directed against Folate Binding Receptor AreEfficient Mediators of Ovarian Carcinoma Cell Killing'

Leslie R. Coney,2 Delia Mezzanzanica, David Sanborn,3 Patrizia Casalini, Maria I. Colnaghi, and

Vincent R. Zurawski, Jr.3From Centocor, Malvern, Pennsylvania 19355 [L. R. C., D. S., V. R. Z., Jr.]; Istituto Nazionale Per Lo Studio E La Cura Dei Tumori, via Venezian 1, Milan, Italy [D. M., P. C.,M. I. C.]; and the Department of Obstetrics and Gynecology, Harvard Medical School, Boston, Massachusetts 02114 [V. R. Z., Jr.]

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OVARIAN CARCINOMA: SPECIFIC CHIMERIC ANTIBODIES MEDIATE ADCC

MATERIALS AND METhODS

Antibody Purification. The munne MOv18 antibody was purified from

murine ascites by protein A-Sepharose (Pharmacia, Piscataway, NJ) chromatography. Ascitic fluid was applied to the column in 3 MNaC1,1.5 Mglycine,pH 8.9. Antibody was eluted with 0.1 Msodium citrate, pH 5.5. The antibodywas further purified by Mono 0 (Pharmacia) chromatography. The murine

M0v19 antibody was also purified from murine ascites by protein A-Sepharose chromatography. Ascitic fluid was applied in 0.1 MTris, pH 8.0, at 10mg/ml, and antibody was eluted with 0.1 M citrate, pH 3.5. The chimericM0v18 and M0v19 antibodies were purified from spent tissue culture supernatants harvested from cultures seeded with 1.0 X 10@cells/ml and allowed togrow for 7 days. After the cells were pelleted and discarded, the supernatantwas adjusted to pH 8.5 with 0.1 volume lOX protein A running buffer (0.2 MTris, pH 8.5, 0.01 MEDTA, 1.4 MNaCI, 1.0% NaN3)and applied to a proteinA-Sepharose column equilibrated with 1x protein A running buffer (0.02 MTris, pH 8.5, 0.001 M EDTA, 0.14 M NaC1, 0.1% NaN3). Antibodies were

eluted with a pH gradient from 6.5 to 3.5 in 0.1 MNa citrate. The absorbanceat 280 mu of the antibody samples was measured with a Beckman DU-50spectrophotometer to quantitate antibody concentration.

Antibody Labeling. The murine M0v18 or MOv19 antibodies used as

tracers in the solid phase inhibition radioimmunoassays were labeled with ‘@I.Vials containing 2 or 4 mCi of Bolton-Hunter reagent (Amersham, ArlingtonHeights, IL) were used to label the M0v18 antibody, and M0v19 was labeledusing iodobeads (Pierce Chemical Company, Rockford, IL). The specificactivity of the labeled M0v18 samples ranged from 4 to 19 @.tCi/p@g,while thatof the M0v19 samples was approximately 6—13pCi/gig. ‘@I-labeledMOv18or -MOv19 was diluted to a final concentration of approximately 1 x 106cpm/ml in tracer buffer consisting of PBS, 1% BSA.

The murine and chimeric antibodies used for the cell-binding assays wereradiolabeled by lactoperoxidase-catalyzed iodination (19). The specific activ

ities obtained ranged from 7 to 13 @tCi/@g.DNA Probes and Expression Vectors. The @Kprobe is a 1.5-kb MstII

fragment which contains 1.1 kilobases of the murine JK-CKintron and the J4through iS coding regions (20). The @Hprobe is a 2.0-kilobase EcoRl-BamHIfragment which contains the murine J3 and J4 coding regions and part of theJHCH intron (21). All probes were labeled with 5'-[a-32PJdC1'P by random

priming reactions using a kit purchased from Boehringer Mannheim (Indianapolis, IN).

The L-chain expression vector, which contains the human Ckexon, and theH-chain expression vector, which carries the human heavy chain yl constantregion exons, have been described (16, 17). The light and heavy chainexpression vectors carry the 0418 resistance gene (neo) and the mycophenolicacid resistance gene (gpt), respectively. The neo gene confers resistance to theantibiotic 0418 and the gpt gene allows selection in medium containinghypoxanthine, mycophenolic acid, and xanthine.

Murine Cell Lines and Cell CUltUre. The MOv18 and MOvI9 hybridoma(7) cell lines and the NS1 fusion partner cell line (T1B 18; American TypeCulture Collection, Rockville, MD) were grown in Iscove's modified Dulbecco's medium with 10% fetal calf serum, 1 m@isodium pyruvate, and 2 mr@iL-glutamine. The carcinoma cell lines IGROV1 (22), 0VCA432, and SW626were kindly provided by Dr. J. Bénard(Institute Gustave Roussy, Villejuif,France), Dr. R. Knapp (Dana Farber Cancer Institute, Boston, MA), andSloan-Kettering Cancer Center (New York, NY), respectively. These cell lineswere grown in RPM! 1640 with 10% fetal calf serum, 1 mMsodium pyruvate,2 mt@tL-glutamine. The nonproducing myeloma cell line SP2/0-AG14 (CRL

1581; American Type Culture Collection) was used for transfection. This cellline and the transfectants derived from it were grown in Iscove's modifiedDulbecco's medium supplemented with 10% fetal calf serum, 1 mMsodiumpyruvate, 2 mML-glutamine.All media, sodium pyruvate, and L-glutaminewaspurchased from Hazleton Biologics, Inc. (Lenexa, KS), and the fetal calf serumfrom HyClone (Logan, UT). All cells were grown in a humidified atmospherewith 5% CO2 at 37°C.

Bacterial Cultures. Phage particles were plated on LE392, and Escherichia coil HB1O1 was used for all plasmid constructions and plasmid DNApreparations (23).

Isolation of Murine MOviS and M0v19 VKGenes. The MOv18andMOv19 VL genes were isolated from recombinant libraries constructed inbacteriophage AEMBL3 (Stratagene, La Jolla, CA). Total genomic DNA was

partially digested with Sau3A and fractionated by centrifugation through a10—40%sucrose density gradient. Restriction fragments ranging in size from9 to 20 kilobases were ligated with AEMBL3predigestedwith BamHI andEcoRI (Stratagene).This ligation was packaged with Gigapack Gold packaging extract (Stratagene), plated on a LE392 bacterial lawn at a density ofapproximately 25,000 plaque forming units/150 X 15-mm Petri plate, and

screened with the @Kprobe. Positive clones identified on duplicate filters wereplaque-purified and phage DNA was prepared.

To identify expressed variable region clones, phage insert DNA was punfled, labeled with 5'-[a-32PJdCTP, and hybridized to a Northern blot of RNAextracted from the original MOv18 or MOv19 hybnidoma and from the NS1myeloma fusion partner. Inserts resulting in a positive Northern hybridizationsignal with RNA from the hybnidoma but not with the NS1 RNA were ligatedinto the HindIII site of the L-chain expression vector.

Isolation of Murine MOviS and M0v19 V@Genes. The M0v18 andM0v19 VH genes were isolated from recombinant libraries constructed inbacteriophage AgtlO (Stratagene). Southern blot analysis of EcoRI-digestedM0v18 hybnidoma DNA indicated that 3 EcoRl fragments, ranging in sizefrom approximately 4.4 to 6.3 kilobases, were homologous with the @Hprobe

(data not shown). A similar analysis of DNA isolated from the M0v19

hybridoma indicated that a single 6.0-kilobase EcoRI fragment was similarlyhomologous with the @Hprobe (data not shown). A range of EcoRI fragmentssize-selected to include the fragments homologous to the J,1 probe was ligatedto AgtlO vector digested with EcoRl and packaged with Gigapack Goldpackaging extract (Stratagene). The libraries were plated at a density ofapproximately 25,000 plaque forming units/iSO x 15-mm Petri plate andscreened by plaque hybridization with the @Hprobe as detailed above. Positive

clones identified on duplicate filters were plaque purified, and phage DNA wasthen prepared. Phage insert DNA was purified and hybridized to a Northern

blot of RNA extracted from the original MOv18 or M0v19 hybnidoma andfrom the NS1 myeloma fusion partner to identify expressed variable regionclones. Inserts resulting in a positive Northern hybridization signal with thehybnidoma RNA but not with the NS1 RNA were ligated into the EcoRI siteof the H-chain expression vector.

Introduction of DNA into Murine Cells by Electroporation. PlasmidDNA was purified by centnifugation sequentially through 2 cesium chloride

gradients. Purified DNA was then introduced into SP2/0 cells by electropora

tion. The SP2/O cells were seeded at a density of 2—3x 10@cells/mI andallowed to grow overnight; 1—2x i0@cells were harvested by centnifugationand resuspended in 0.8 ml Hanks' balanced salts without Mg and Ca (Hazieton). A mixture containing 30 @.tgof the heavy chain expression vector and 30i.Lgof the light chain expression vector in 100 @lof glass distilled water wasmixed with the SP2/0 cells in a 0.4-cm electrode gap gene pulser cuvette(Bio-Rad, Richmond, CA). The mixture was pulsed at 200 V in a Bio-RadGene Pulser electroporation apparatus and diluted to 1 x 10@cells/mi. This cellsuspension was plated in 96-well tissue culture clusters with flat bottomed

wells (Costar, Cambridge, MA) at a density of I x iO@cells/well. Drugselection was added to a final concentration of 0.25 p@g/mlmycophenolic acid,

1.25 @glmlhypoxanthine,25 @tglmlxanthine,and 0.5 or 0.25 mg/miG4l8after 48 h. Colonies resistant to the drug selection were scored, screened for

antibody production, and expanded after 1—2weeks.

Quantitation of Antibody Production by EUSA Tissue culture supernatants were screened for chimeric antibody production with an enzymeimmunoassay. AffiniPure goat anti-human IgG Fc fragment (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) served as the capture antibody,and alkaline phosphatase conjugated Affinipure goat anti-human IgG (heavyand light) was used to detect bound chimeric antibody. A standard curve was

used to generate supennatant concentrations.

Assay of Antibody Immunoreactivity. The M0v18 and M0v19 MAbsrecognize different epitopes on the same antigen (7) and, therefore, it waspossible to use a double determinant, solid phase radioimmunoassay to determine the immunoreactivities of chimenic M0v18 and chimeric M0v19. Ingeneral, the assay was designed with either M0v18 or M0v19 adsorbed tosolid phase beads. The solid phase beads were incubated simultaneously witha source of FBP and the appropriate radiolabeled MAb (M0v18 or M0v19).

Inhibition of binding of the radiolabeled trace MAb to antigen bound toimmobilized MAb was tested by inclusion of various dilutions of unlabeledmunine or chimeric antibody. The antigen source was spent tissue culturesupernatant generated by culture of IGROV1 cells. The FBP antigen recog

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ov@itia@NCARCINOMA:SPECIFICCHIMERICANTIBODIESMEDIATEADCC

nized by M0v18 and M0v19 is shed by IGROV1 cells into the tissue culturemedium. A gold standard antigen source that allowed near-maximum bindingof the radiolabeled second antibody to the coated beads was generated.

Specifically, the assay was performed as follows. To test the immunoreactivity of chimeric M0v18, M0v19 antibody-coated beads were prepared by

passive adsorption as described (9). The antibody-coated beads were incubatedfor 18 h at room temperature with 1.0 to 2.0 x 1O@counts of 1@I-labeled

M0v18 tracer in 100 @.tland 50 pi of the antigen standard solution, and 50 @.dof tissue culture supematant (negative control) or dilutions of the purifiedchimenic or murine M0v18 antibody in PBS, 1% BSA. The beads werewashed 3 times with distilled water and counted in an LKB y-counter. Thenumber of counts bound in each experimental sample was used to calculate apercent of counts bound in a control sample that did not contain competingantibody (0% inhibition). This number was subtracted from 100% to determine

percent inhibition.The assay used to test the immunoreactivity of chimenicM0v19 was similar

to that described above, however, in this case, the solid phase beads werecoated with MOv18. These beads were incubated with approximately 1.0 X

@ counts of the ‘@I-labeledM0v19 tracer in 100 @.dand 50 pi of standard

solution, and 50 pi of tissue culture supernatant (negative control) or dilutionsof purified chimeric or munine M0v19 antibody in PBS, 1% BSA. Calculations of percent inhibition were determined as described above.

Cross-Competition Experiments. Cross-competition experiments between the murine and chimenic M0v18 and M0v19 MAbs were performedwith the IGROV1 cell line. Cells were seeded in 96-well culture plates

(Costar, Cambridge, MA) and grown until a monolayer was obtained. Thecells were then fixed with 0.1% glutaraldehyde. Fixed cell monolayers

were incubated for 3 h at 37°Cwith 50 ,.d of various triplicate dilutions ofunlabeled murine or chimeric MAbs (starting from 150 pg/mi) in the

presence of a fixed amount of ‘25I-labeledmurine MAbs (5 X iO@cpm/50p1). The number of counts bound in each experimental sample was used tocalculate a percent of counts bound in a control sample that did not containcompeting antibody (0% inhibition). This number was subtracted from

100% to determine percent inhibition.Cell-binding Assays. Ovarian carcinoma cells cultured in TiSO flasks

were allowed to reach approximately 100% confluence and harvested bytreatment with 0.25% trypsin-EDTA. After trypsinization, IGROV1 cells wereleft for 3 h at room temperature in RPMI 1640 containing 5% FCS. After 2washes with RPM!, 1% FCS, 3 x i0@cells/well were plated in U-bottomed96-well polyvinylchlonide plates (Dynatec, Chatilly, VA). Cell pellets wereincubated with dilutions of the labeled antibody for 3 h at 4°Cwith gentleshaking. After centnifugation, the pellets were washed 3 times with RPM!, 1%FCS, and the bound radioactivity was determined after cutting the wells off theplate using a CRYSTALL II y-counter (Packard). The nonspecific bindingsubtracted from the total binding was evaluated by incubating the cells with the

same dilution of labeled antibodies in the presence of a i0O@ excess ofcorresponding cold antibody. The binding data were evaluated on a desktopcompact computer with a program that calculated the molarity of boundantibody, the ratio of bound/free ligand molecules, the significance of thecurve as r and p. and the intercepts giving the values of apparent K..@,,and thenumber of sites per cell. All reported data showed a significant regressionanalysis (P < 0.05 and r@> 0.7).

Effector Cells. HumanPBMCwere isolatedfromthebloodof leukapheresed healthy donors by Ficoll-Paque density gradient centrifugation. PBMCwere maintained in RPM! 1640 supplemented with 0.6 mg/ml glutamine, 0.1mg/mi penicillin, 0.27 mg/mi streptomycin, 5% fetal calf serum, and cultured

at 37°C,5% CO2 for 1—3days before performing the cytotoxic assay.Cell Lysis Assay. A standard4-h 51Cr-releaseassay was carriedout with

ovarian carcinoma cells in U-bottomed 96-well plates (24); 1—3X 106 cellswere labeled with 100—309 @Ci51Cr (Amersham), in the presence of 100 @lFCS, for 1 h at 37°C.After 2 washeswith culturemedium,the cells (5 x103/well)were incubated in triplicate with different concentrations of effectorcells and antibodies. The spontaneous release, evaluated by incubating thelabeled cells with medium alone, was <10% of maximum release determinedby using 2% Triton X-100 detergent. Percent specific lysis was calculated by:

100 X experimental cpm— spontaneous release

RESULTS

Isolation and Sequence Analysis of the MOviS and M0v19 VKand VH(@e'i@PhageclonescontainingantibodyL-chainvariableregion genes were isolated from genomic AEMBL3 libraries by hybridization with the @kprobe. Seven and 14 positive plaques wereobtained by screening 8.0 X 10@and 7.0 X 10@plaques from theMOv18 and MOv19 libraries, respectively. The purified insert fromeach plaque was hybridized to a Northern blot as described in “Materials and Methods.― Only one clone isolated from each of thegenomic libraries constructed with the MOv18 and MOv19 hybridoma genomic DNA produced a positive hybridization signal with thecorresponding hybridoma RNA but not with the fusion partner RNA(data not shown). The MOv18 VK clone was isolated as a 3.5-kilobaseHindIII-SalI fragment and the MOv19 VK clone was isolated as5.0-kilobase Hindu fragment. Each fragment was inserted into theHindu! site of the light chain vector for expression.

Size-selected AgtlO libraries constructed with EcoRI fragmentsgenerated from MOv18 and MOv19 hybridoma genomic DNA werescreened with the @Hprobe to identify clones containing antibodyH-chain variable regions; 4.0 X 10@and 7.0 X i0@ plaques werescreened to obtain 14 and 11 positive phages from the MOv18 andMOv19 libraries, respectively. The purified inserts from these cloneswere hybridized with a Northern blot as described in “MaterialsandMethods.―Only one clone isolated from MOv18 and MOv19 produced a positive hybridization signal with the hybridoma RNA but notwith the fusion partner RNA (data not shown). The M0v18 andMOv19 H-chain variable region genes were present on 4.3- and6.0-kilobase EcoRI fragments, respectively. These fragments wereinserted into the EcoRI site of the heavy chain vector for expression.

Expression of the cMOvl8 and cMOvl9 Antibodies. Anequimolar mixture of the H-chain and L-chain expression vectorscontaining the heavy and light chain variable region genes for eitherthe MOv18 or MOv19 antibodies was introduced into SP2/0 cells byelectroporation. Drug-resistant clones were tested for chimeric immunoglobulin production by ELISA 2 weeks after drug selection wasinitiated. The clones producing the highest level of antibody wereexpanded and retested for antibody production by ELISA. The transfectants producing the highest level of antibody were grown as described in “Materialsand Methods,―and antibody was purified fromspent tissue culture supernatant by protein A chromatography. Thecell line used to produce the chimeric MOv19 MAb yielded 94 @glmlof purified cMOvl9, and the cell line used to produce the chimericMOv18 MAb yielded 34 .tglml of purified cMOvl8.

Comparisonof Murineand ChimericAntibodyImmunoreactivities. The MOv18 and MOv19 antibodies recognize differentepitopes on the same antigen; therefore, they can be used in simultaneous double-determinant sandwich competition assays. ChimericM0v18 or MOv19 and the murine counterparts of these antibodieswere used to inhibit binding of the radioiodinated murine MOv18 orMOv19 antibodies. The munne and chimeric forms of both theMOv18 and MOv19 antibodies were similarly capable of inhibitingbinding of the corresponding radiolabeled murine antibody to theantigen (Fig. 1, A and B). In addition, the relative ability of eachantibody to bind IGROV1 ovarian carcinoma cells was assessed withcell binding assays. The K...@,,for each antibody was determined byScatchard analysis of the cell binding data. The murine and chimericforms of both the MOv18 and MOv19 antibodies had similar affinitybinding constants when tested with IGROV1 cells. The K...5,,for themurine and chimeric MOv18 MAbs was 2.2 X 108 liters/mol and2.7 X 108 liters/mol, respectively (Fig. 2A). The K.@ for the murineand chimeric MOv19 MAbs was 1.2 X 108 liters/mol and 1.3 X 108liters/mol, respectively (Fig. 2B).total release —spontaneous release

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0.04

50:1 100:1

OVARIAN CARCINOMA: SPECIFIC CHIMERIC ANTIBODIES MEDIATE ADCC

A. 0.12 0.12

NLu 0.10Lu

@0.08

@0.06

:@0

0.10

0.08

0.06

0.04

0.02 Murlna M0v18 0.02 Chimerlc M0v181 I I I I I

0.1 02 0.30

B.

00.12

0.10

0.08

0.06

0.04

0

@_I I I I

0.102

LU 0.10

@0.08

@0.06

@ 0.040m 0.02N.

MurineM0v19 0.02 ChlmericM0v19L I I I I I I I I

0 02 0.4 0.6 0 02 0.4

BOUND(nM)Fig. 2. Analysis of antibody affinity. The ability of each antibody to bind IGROV1

ovarian carcinoma cells was tested by cell binding assays. The K,@,,for each antibody wasdetermined by Scatchard plot analysis of the cell binding data. A, Scatchard plot analysisofmurine MOvl8 and chimeric M0v18 MAb binding to IGROV1 cells. B, Scatchard plotanalysis of murine MOvI9 and chimeric M0v19 MAb binding to IGROV1 cells.

AFig. 1. Assay of antibody immunoreactivity. Binding of ‘@I-labeledmurine M0v18 or

M0v19antibodywascompetitivelyinhibitedbythechimericandmurineversionsofeachantibody using a solid phase inhibition radioimmunoassay. Results are presented as themean of triplicate sample values and verticalbars indicate SD.A, mean percent inhibitionof lssI@labeledM0v18.B, meanpercentinhibitionof ‘@I-labeledMOv19.0, murineM0v18 and MOvI9 antibody. S. chimeric M0v18 and MOv19 antibody.

Assay of the MOv18 and MOv19 Antibodies in Antibodydependent Cellular Cytotoxicity. The ability of the murine andchimeric M0v18 and M0v19 antibodies to mediate the lysis ofcultured ovarian carcinoma cells by human PBMC was tested. 51Crlabeled IGROV1 ovarian carcinoma cells were incubated with either1.0 or 0.1 p.g/ml of antibody and human PBMC at effector:targetratiosof12:1,25:1,50:1,and100:1.

The results in Fig. 3A indicate that the cMOvl8 antibody mediatedcytolysis more efficiently than did the murine MOv18 antibody at anantibody concentration of 1 @tWml.The increased lysis with cMOvl8was observed at all effector:target ratios tested. The MOv19 murineantibody mediated some specific lysis of IGROV1 cells, but thechimeric antibody was a more efficient mediator. Chimeric MOv19was twice as effective for the mediation of specific lysis of carcinomacells at all effector:target ratios. Maximum total lysis was achieved forboth M0v18 and M0v19 chimeric antibodies at an effector:targetratioof50:1.

Both chimeric antibodies were also efficient mediators of cytolysiswhen tested in ADCC assays with an antibody concentration of 0.1p@g/ml.As shown in Fig. 3B, the cMOvl8 antibody mediated 6 to 20times more specific lysis than did the murine antibody at this antibodyconcentration depending on the effector:target ratio used, and thecMOvl9 antibody was 2.5- to 5-fold more efficient than the murineantibody. Maximum total lysis was achieved for both chimeric antibodies at an effector:target ratio between 50:1 and 100:1. The maxi

4C

20

CO

‘.4 °12:i 2b:1

B

40

20

0@ 12:1 25:1 50:1 100:1EFFECTOR:TARGET RATIO

Fig. 3. Lysis of 51Cr-labeledovarian carcinoma cell line IGROV1 mediated by murineor chimeric antibodies and human PBMC at various effector:target ratios over a 4-hperiod. Results are presented as the mean of triplicate sample values, and vertical barsindicate the SD. Specific 51Crrelease obtained with effector cells alone was 6.3 ±0.3 foran effector:target ratio of 100:1; 4.0 ±1.3 for an effector:target ratio of 50:1; 2.0 ±0.6for an effector:target ratio of 25.1; and 2.6 ±0.8 for an effector:target ratio of 12: 1. A,specific 51Cr release obtained with 1 @.&g/mlantibody. B, specific 51Cr release obtainedwith 0.1 @sg/m1antibody. 0, murine M0v18 antibody; L@,chimeric M0v18 antibody;•,murine M0v19 antibody; A, chimeric M0v19 antibody; 0, control chimeric antibody; 0,no antibody.

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ANTIBODYCONCENTRATION(pig/mI)

c@)

Co

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Ovariantarget cellNo.

of bFBP sites

(X 1O@)Maximum

lysisc(above PBMC alone)

% gLg/mldED50

(ng/ml)IGROV1

Murine M0v18Chimeric M0v18Murine MOv19Chimeric M0v1910034.0

±8.7 0.250.0 ±7.2 0.249.4 ±2.9 241.0 ±6.8 22

<0.22

<0.2OVCA

432MurineMOv18Chimeric M0v18MunineMOv19Chimeric MOv19200

227.4 ±1.8 265.5 ±7.3 258.0 ±4.3 220

303SW626

Murine MOv18Chimeric M0v18Murine M0v19Chimeric M0v1910

27.7 ±2.2 26.6 ±2.9 2

16.2 ±23 2—

<0.20.203

Table 1 ADCC mediated by murine and chinsericantibodies°PBMCMaximum

lysisED5OC

(ng/ml)%b@g/mlHealthy

donor 1Murine M0v18Chimeric M0v1813.2

±1.347.6 ±5.82.00.0252Murine

M0v19Chimeric MOv1924.5

±2.759.1 ±3.80.220 0.074103Healthy

donor 2Murine MOvI8Chimeric MOv1816.0

±1.457 ±7.02.0 0.031——Murine

MOv19Chimeric MOv1949

±3.054 ±2.10.031 0.031230.250Healthy

donor 3Murine M0v18Chimeric MOvl820.0

±2.858.0 ±2.02.0 0.125—0.6Murine

MOv19Chimeric MOv1940

±3.062 ±6.00.031 0.0313 1

OVARIAN CARCINOMA: SPECIFIC CH1MERICANTIBODIES MEDIATE ADCC

mal lysis obtained with 1 and 0.1 @WmIantibody was very similar.This indicates that the 0.1-@Wml antibody concentration is saturating

in this system and that a 10-fold increase in antibody does not resultin a significant increase in the lysis of target cells.

To further evaluate dependence of lysis on antibody concentration,cytolysis experiments were performed at several antibody concentrations at an effector:target ratio of 50: 1. Three separate experiments

were performed, each with PBMC isolated from the blood of differentdonors. Data from the 3 experiments are summarized in Table 1. Theresults in Table 1 indicate that cMOvl8 was consistently a moreeffective mediator of ADCC than murine MOv18. When maximumlysis obtained with each antibody was compared, the maximum lysisobserved in each experiment with cMOvl8 was similar (specific lysis:47.6, 57, and 58%) and achieved at similar antibody concentrations(0.025, 0.031, and 0.125 @Wml).However, the maximum specificlysis observed with murine MOv18 was lower (13.2, 16.0, and 20.0)and was obtained at the highest antibody concentration (2 @iWml)ineach case.

The maximum lysis results obtained with cMOvl9 and MOv19were less consistent. Chimeric MOv19 was 2.4-fold more efficientthan MOv19 in the mediation of ADCC when PBMC from donor 1were used as effector cells. In this experiment, the lower specific lysisobtained with MOv19 was achieved with a higher antibody concentration (0.220 @Wml)than that at which the highest levels of specificlysis were obtained with cMOvl9 (0.074 @g/ml). In separate exper

iments, chimeric M0v19 was only a slightly more efficient mediatorof ADCC than MOv19 when the maximum lysis obtained with theeffector cells from donors 2 and 3 were compared. In both of theseexperiments, maximal lysis was achieved at the same antibody concentration with the murine and chimeric antibodies (0.031 p@g/ml).Acomparison of the ED50 values obtained for murine MOv19 andchimeric MOv19 antibodies indicates that the chimeric MOv19 MAbwas 3 to 10 times more potent than the murine M0v19. In addition,the ED50 values obtained for the chimeric M0v18 and chimericMOv19 MAbs were similar, indicating that these MAbs are equallypotent mediators of tumor cell lysis.

Effect of Target Cell Antigen Density on ADCC of Carcinoma

Cells. To determine whether the results obtained with the IGROV1

Table 2 ADCC mediated by murine and chimeric antibodies:influence of antigen expressiona

a A standard 4-h 51Cr-release assay was carried out with different tumor cells and

human PBMC from the same healthy donor at an effector:target ratio of 100:1 asdescribed in “Materialsand Methods.―Specific 51Crrelease obtained with effector cellsalone was 29.1 ±6.4 for IGROV1, 203 ±7.9 for 0VCA432, and 22.3 ±2.8 for SW 626.

b Determined by Scatchard analysis of the cell binding data as described in “Materials

and Methods.―CMean ±SD of triplicatevalues.d Concentration of MAb producing half-maximal lysis.

e not evaluable.

target cell line were generally representative, the ability of the chimenc and murine versions of MOv18 and M0v19 to lyse othercarcinoma target cells was tested. The results obtained when theIGROV1, 0VCA432, and 5W626 cell lines were used as target cellsare summarized in Table 2; these cell lines express different amountsof the FBP target molecule. When maximum lysis obtained with eachantibody was compared, it was apparent that chimeric MOv18, murineM0v19, and chimeric M0v19 were equally capable of lysing all 3target cell lines. Murine MOv18 was less lytically active whenIGROV1 cells were used as a target and not active at all withOVCA432 and SW626 target cells. The level of maximal lysisachieved was dependent on the number of target molecules/cell; thesame high level of lysis mediated by cMOV18, murine MOV19, andcMOV19 was observed with both IGROV1 and OVCA432 targetcells. A comparison of the ED50 values obtained with each antibodyusing IGROV1 and 0VCA432 target cells indicates that the chimericantibodies were more potent than the murine antibodies. However,only low levels of lysis were obtained with each antibody when usedto lyse the SW626 cell line which expressed only 1 X 10' FBPsites/cell. Similar results were obtained in a separate experiment withPBMC from a different healthy donor (data not shown).

Effect of Chimeric MOv1S and MOv19 in Combination onADCC ofIGROV Cells. The M0v18 and M0v19 antibodies recognize different epitopes on the same antigen (7). Therefore, bothantibodies together could be used to determine whether local antibodydensity on target cells affected specific cellular lysis. A mixturecontaining a 1:1 ratio of cMOvl8 and cMOvl9 was tested for itsability to mediate ADCC with PBMC and 51Cr-labeled IGROV1 cellsand compared with the amount of ADCC mediated by an equalamount of either cMOvl8 or cMOvl9 alone (Fig. 4). The combinationof cMOvl8 and cMOvl9 mediated an equal amount of cellular lysisas the same amount of either antibody alone at all concentrations,including antibody concentrations at and above target cell bindingsaturation (determined by cell binding assays, data not shown). Inaddition, cross-competition cell binding assays demonstrated thatbinding ofthe MOv18 MAb to FBP-positive cells was not inhibited inthe presence of either the murine or chimeric M0v19 antibodies (Fig.

a A standard 4-h 51Cr-release assay was carried out with IGROV1 tumor cells and

human PBMC at an effector:target ratio of 50:1 as described in “Materialsand Methods.―Specific 51Cr release obtained with effector cells alone was 8.4 ±0.1, 18.1 ±9.7, and25 ±93 for healthy donors numbered 1, 2, and 3, respectively.

M@ ±SDof triplicatesamplevalues.CConcentration of MAb producing half-maximal lysis.d not evaluable.

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@60

Cl)

C'.)Li'.()

Cl) 20

ov,@m@CARCINOMA:SPECIFICCHIMERICANTIBODIESMEDIATEADCC

human yl constant region resulted in a chimeric antibody havingequivalent or superior ability to mediate ADCC. This is in keepingwith the findings of Steplewski et a!. (27) which indicated that themurine 17—lAMAb (y2a,.c) exhibited a capacity equal to that ofchimeric 17—lA(-yl,.c) to perform ADCC. In each case, the minimaldifference in the ability of the chimeric (cyl,K) antibodies and themurine (my2a,K) antibodies to mediate ADCC probably is due to thesimilar affinity of the human @y1and murine -y2a constant regions forFcyRl (29), a major effector molecule for ADCC (30, 31). The causeof the decreased relative ability of the murine y2a M0v19 MAb tomediate ADCC in some experiments is not clear.

Replacement of the murine yl constant region of the MOv18antibody with the human yl constant region greatly enhanced theability of the resulting chiineric antibody to mediate ADCC. Thisenhanced ADCC activity of the chimeric antibody is most likely dueto the high affmity of the human yl constant region for FcyRl relativeto the affinity of the murine yl isotype which fails to interact with thiseffector population (29). The inability of the murine M0v18 antibodyto mediate ADCC substantiates the findings of others which indicatethat murine antibodies of the ‘ylisotype are relatively inefficientmediators of ADCC (32, 33).

In an attempt to augment the amount of specific lysis mediated bycMOvl8 and cMOvl9, we tested the amount of lysis observed with anequimolar mixture of the 2 antibodies and the same amount of eitherantibody alone. At saturating concentrations of antibody, it is possiblethat more antibody would be bound per cell in the sample containingthe mixture of antibodies because cMOvl8 and cMOvl9 recognizedifferent epitopes on the same antigen. However, no augmentation ofspecific lysis with the antibody mixture was observed, although crosscompetition binding studies indicated that binding of one antibody isnot inhibited in the presence of the other antibody. Consistent withthis finding, Herlyn et a!. (34) demonstrated that the use of a mixtureof 2 murine IgG2a monoclonal antibodies directed at differentepitopes of the same tumor-associated antigen did not result in increased antibody-dependent macrophage cytotoxicity with mousemacrophages as effector cells. In contrast, exposure of the same tumorcell targets to mixtures of antibodies which recognize different tumorassociated antigens resulted in increased antibody-dependent macro

40-

0

Fig. 4. ADCC activity mediated by an equimolar mixture of the M0v18 and MOv19chimeric antibodies and by an equal amount of each chimeric antibody alone. Lysis of51Cr-labeled ovarian carcinoma tissue culture cells mediated by human PBMC at aneffector:target ratio of 50:1 over a 4-h period. Results are presented as the mean oftriplicate sample values and the vertical bars indicate the SD. 0, chimeric MOv19antibody; •,chimeric M0v18 antibody; A, an equimolar mixture of chimeric M0v18antibody and chimeric MOv19 antibody.

5A). Similarly, the presence of the munine or chimeric MOv18 antibodies did not inhibit binding of the M0v19 antibody (Fig. SB).

DISCUSSION

The murine MOv18 and M0v19 monoclonal antibodies were raisedagainst an ovarian carcinoma cell membrane preparation and bound toa cell surface antigen expressed on 90% of nonmucinous ovariancarcinomas tested by immunofluorescence and immunoperoxidasestaining (7, 25). We have recently identified the antigen recognized bythe M0v18 and MOv19 MAbs as human folate binding receptor (9).Here we describe the production and characterization of chimericversions of both antibodies which lyse target ovarian carcinoma cellsefficiently when human peripheral blood mononuclear cells are usedas effector cells. The immunoreactivities of the chimeric and murineversions of both M0v18 and MOv19 are virtually identical. Thisconfirms the findings of other groups which indicate that the effectorcapacity of an antibody may be altered without changing the bindingspecificity or affinity of the variable region (16, 18). In this way, itwill hopefully be possible to decrease the immunogenicity of theantibody molecule without altering its ability to bind target cells. Inaddition, the stability of the antibody molecule may be increased ashas been shown for the chimeric version of the 17—lAantibody (26).Both increased half-life and decreased immunogenicity will be extremely important in clinical applications; decreased immunogenicitywill allow administration of multiple antibody doses, and increasedhalf-life may result in increased antibody localization at the tumorsite. In addition to decreasing immunogenicity, the substitution of themurine constant region with a human constant region of choice allowsthe construction of an antibody molecule with increased effectorcapacity.

It has previously been published that the human IgG1 isotype issuperior to the human IgG2, IgG3, and IgG4 isotypes in its ability tomediate ADCC of human tumor cell lines using human effector cells(27, 28) and we, therefore, substituted the constant region of a murine

.y1 and @y2aantibody with the human yl constant region. Replacementof the murine ‘y2aconstant region of the MOv19 antibody with the

0.1W @1@ 10 10@ 1000

ANTIBODYCONCENTRATION(pg/mi)

0I-'.

Fig. 5. Cross-competition cell binding assays. The binding of @‘l-labeledmurineM0v18 or M0v19 antibody to IGROV1 cells was tested in the presence of increasingconcentrations of the murine or chimeric M0v18 and M0v19 MAbs. A, percent inhibitionof WI-labeled MOv18 binding; B, percent inhibition of ‘@I-labeIedMOvl9 binding. 0,murine MOV18 antibody; •,chimeric MOvI8 antibody; 0, murine MOv 19 antibody; S.chimenc M0v19 antibody.

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(,.ig/mI)

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OVARIAN CARCINOMA: SPECIFIC CHIMERIC ANTIBODIES MEDIATE ADCC

phage cytotoxicity. Ralph and Nakoinz (35) also showed that a cornbination of antibodies recognizing the same antigen did not mediateincreased ADCC, although a mixture of antibodies that recognizeddifferent tumor-associated antigens did boost ADCC. However, cornpetitive binding assays of the antibodies that recognized the sameantigen were not performed to determine whether the antibodies in themixture recognized the same or different epitopes. In another study,Fogler et a!. (36) demonstrated that mixtures of MAbs containingsaturating amounts of 2 antibodies that recognize different epitopes onthe same tumor-associated antigen mediated more cellular lysis than asaturating amount of either antibody alone. Together these resultsimplicate antibody density as a parameter important for ADCC.

The reason for the variable effect of a set of antibodies whichrecognize different epitopes on the same antigen is not clear andindicates that the effect is specific for a given set of antibodies. Whenno augmentation is observed, it is possible that binding of one antibody inhibits binding of the other. It is also possible that incubation ofthe target cells with saturating amounts of both antibodies may increase the antibody density, however steric hindrance may preventincreased interaction between the antibodies and effector cells. Sterichindrance may also be responsible for the lack of ADCC enhancementobserved with the mixture of the cMOvl8 and cMOvl9 antibodies.On the other hand, these antibodies may mediate ADCC so efficientlythat it is possible to attain a maximum specific lysis with one antibodyalone under the assay conditions described here. The efficient targetcell lysis caused by cMOvl8 and cMOvl9 alone may simply be afunction of the MOv18 antigen density on IGROV1 cells. The FBPantigen is expressed at approximately 1.0 X 106 antigen sites/cell bythe IGROV1 target cell line. Similar high levels of lyric activity wereobserved when either the IGROV1 or the OvCA432 cell lines wereused as target cells for ADCC mediated by cMOvl8 or cMOvl9,although the FBP antigen was expressed 5-fold more by the IGROV1cells than by the OvCA432 cells. These results may indicate that athreshold number of antigen molecules per target cell is necessary formaximal ADCC under the conditions used here. Above that thresholdlevel, increased antibody bound/cell may not result in increasedcellular lysis.

A number of variables including antibody density on the target andeffector cells, FcyRI availability, and physical interaction betweeneffector and target cells influence the occurrence of maximum specificlysis. Rather than the number of armed FcyRI molecules/cell or thenumber of antibody molecules/target cell being the limiting factor, theamount of lysis may be limited by interaction between target andeffector cells. Experiments performed to determine the effect ofantibody concentration on specific target cell lysis indicate that aprozone effect was observed at high antibody concentrations (data notshown). At the highest antibody concentration tested in these 51Crrelease assays, 2 ,@g/ml, 1.6 X 1012 antibody molecules were present.The average number of binding sites per IGROV1 target cell was4.3 X iO@ and, therefore, the total number of binding sites per well

(S X i0@cells/well) was 2.2 X iOu. Therefore, antibody was in excessat the higher antibody concentrations tested, and optimum interactionbetween effector and target cells actually occured at an antibodyconcentration lower than that at which saturation was observed. Theseresults indicate that doubling the number of antibody molecules/targetcell with a saturating concentration of the equimolar cMOV18/cMOV19 mixture would not increase the amount of specific lysisobserved because of limitations imposed by target:effector cell interaction. These observations may be important for the selection ofoptimum antibody dosing regimens for use in therapy.

Identification of the antigen recognized by the MOv18 and MOv19MAbs as human folate binding receptor indicates that this molecule ishighly expressed on a majority of ovarian carcinomas and provides a

suitable target for therapy using the cMOvl8 and cMOvl9 MAbs.Folate binding receptor is known to internalize 5-methyltetrahydrofolate, and it will be interesting to determine the fate of the MOv18and MOv19 antibodies after binding to the surface of tissue culturecells adapted for growth in physiological concentrations of folate (37,38). Internalization studies previously indicated that the MOv18/19antigen is stable on the surface of ovarian carcinoma cells grown instandard tissue culture media, consistent with its status as a glycosylphosphatidylinositol labeled protein (39, 40). The fate of antibodiesafter binding to the target cell is important when considering therapeutic applications. The data presented here indicate that a chimericversion of either the MOv18 or MOv19 antibody will provide for themaximum mediation of cell lysis attainable when using human folatereceptor as a target antigen. The recent confirmation that this antigenis highly overexpressed on certain tumor types, especially epithelial

ovarian tumors (25), supports the testing of both these antibodies inthe clinic as therapeutic agents for ovarian carcinoma.

ACKNOWLEDGMENTS

We wouldlike to acknowledgeMonicaSinquettfor her help in preparingthis manuscript, and Stephanie Branche and Daniel V. Zurawski for excellenttechnical assistance. In addition, we thank Dr. John Ghrayeb and Dr. David

Knightfor valuablediscussionsand reagents,and Dr. SilvanaCanevari,Dr.Jeffrey Krieger, and Dr. David Woo for a critical review of the manuscript.

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1994;54:2448-2455. Cancer Res   Leslie R. Coney, Delia Mezzanzanica, David Sanborn, et al.   Cell KillingBinding Receptor Are Efficient Mediators of Ovarian Carcinoma Chimeric Murine-Human Antibodies Directed against Folate

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