expression of the df3-p epitope in human ovarian …...treated with aqunmount (lernen labs,...

Vol. 1, 565-571, May 1995 Clinical Cancer Research 565

Expression of the DF3-P Epitope in Human Ovarian Carcinomas

Keiko Ichige, Lucien Perey, Charles A. Vogel,

Franz Buchegger, and Donald Kufe’

Division of Cancer Pharmacology, Dana-Farber Cancer Institute,

Harvard Medical School, Boston, Massachusetts 02115 [K. I., D. K.]

and Centre Pluridisciplinaine d’Oneologie [L. P.], Division of Nuclear

Medicine [C. A. V., F. B.], Centre Hospitalien, Universitaine Vaudois,

CH-101 1 Lausanne, Switzerland

ABSTRACT

Recent studies have described the generation of a mAb,designated DF3-P, which reacts with underglycosylated pre-

cursors of the DF3IMUC1 mucin-like glycoprotein. Thepresent work demonstrates that the epitope recognized by

mAb DF3-P is expressed by cell lines derived from humanepithelial ovarian carcinomas and not a teratocarcinoma.Indirect immunofluorescence assays of single-cell suspen-sions support expression of the DF3-P epitope on the surfaceof ovarian carcinomas. Immunofluorescence studies onchamber slides further demonstrate that the mAb DF3-P-reactive cells are present in clusters. We also demonstrate

that ‘251-labeled mAb DF3-P selectively localizes to humanovarian carcinoma xenografts in athymic mice. The percent-age ofinjected 125! dose/g tissue ranged between 10 and 17%

for implanted CAOV-3 and OVCAR-3 tumors. Finally, theresults of immunoperoxidase staining studies demonstrate

that the DF3-P epitope is detectable in formalin-fixed sec-

tions of ovarian tumors and that mAb DF3-P exhibits littleif any reactivity with normal surrounding tissues. Selectiveexpression of the DF3-P epitope may be useful as a target for

radioimaging or immunotherapeutic approaches to ovarian

cancer.

INTRODUCTION

Several mAbs have demonstrated varying patterns of im-

munological reactivity with ovarian cancers. mAb 0C125 reacts

with both serous and endometnioid adenocnrcinomns, but lacks

reactivity with mucinous ndenocarcinomas (1). Conversely,

mAbs OCA and OCAA react with mucinous adenocarcinomas

and not senous on endometniod ndenocarcinomas (2). Other

studies have demonstrated that mAb DF3 reacts with an antigen

present in serous, endometnioid, mucinous, clean cell, and un-

differentiated epithelial ovarian carcinomas (3). Similar findings

were obtained for the related F36/22 antigen which is detectable

in 100% of human ovarian carcinomas, regardless of histology

(4). The demonstration that DF3 antigen circulates at elevated

levels in patients with ovarian cancer as compared to normal

Received 11/11/94; accepted 2/17/95.1 To whom requests for reprints should be addressed, at Dana-FarberCancer Institute, 44 Binney Street, Boston, MA 02115.

2 The abbreviations used are: ATCC, American Type Culture Collec-

tion; FBS, fetal bovine serum; eIgG, control IgG; % ID/g, percentage of

the injected dose per gram tissue.

women (5, 6) may be related to cell surface shedding on lysis of

tumor cells. In this regard, DF3 antigen is detectable at high

levels on the surface of ovarian carcinomas and is shed into the

supernatant of ovarian cancer cell lines (3).

DF3 antigen (also designated episinlin and MUC1) is a

member of a family of high molecular weight glycoproteins

which are aberrantly expressed in carcinomas (7). Sequence

analysis of eDNA clones coding for the core protein revealed

highly conserved (G + C)-rich 60-base pain tandem repeats

(8-10). These repents code for the epitopes identified by mAb

DF3 and other mAbs directed against the intact glycoproteins on

the deglycosylated protein core (9-12). The availability of

eDNA clones coding for the tandem repents has provided an

opportunity to prepare mAbs against recombinant peptide. In-

deed, recent studies have described the isolation of one such

antibody, designated DF3-P, which reacts with an epitope sim-

ilan to that identified for mAb DF3 (13). However, in contrast to

mAb DF3, mAb DF3-P binds to the tandem repeat protein cone

and exhibits little if any binding to the mature glycoprotein (13).

The present studies have examined mAb DF3-P reactivity

with human ovarian carcinomas. The results demonstrate that

mAb DF3-P reacts with precursor forms of the mature glyco-

protein in ovarian carcinoma cell lines. Other findings by mdi-

rect immunofluorescence demonstrate that the DF3-P epitope is

expressed on the surface of ovarian carcinoma cells. The ability

to specifically localize ‘25I-labeled mAb DF3-P to ovarian can-

cinomn xenografts in nude mice and detection of the DF3-P

epitope in tumor specimens suggest that this antibody may be

useful in nadioimaging of patients with these tumors.

MATERIALS AND METHODS

Cell Culture. CAOV-3 cells obtained from the ATCC2

(Rockville, MD) were grown in DMEM with 10% fetal bovine

serum (FBS) and 4.5 g/liter glucose. The OV-D line was grown

in DMEM with 10% FBS, 1% L-glutnmine, 1% nonessential

amino acids, and 1% sodium pyruvate (14). OVCAR-3 (ATCC)

was grown in RPMI 1640 medium with 10% FBS and 10 p.g/ml

insulin. SK-OV-3 cells (ATCC) were grown in McCoy’s Sn

medium with 15% FBS. The PA-i cell line (ovarian terntocnr-

cinoma) was grown in Eagle’s minimal essential medium with

10% FBS and 1% nonessential amino acids (15).

Indirect Immunofluorescence Analysis. Cultured cellswere harvested with a rubber policeman and passed through a

21-gauge needle. The single-cell suspension was then washed

three times with 1% BSA in PBS. Cells were incubated in

blocking buffer (3% BSA/0.002% sodium azide/PBS) at 4#{176}Cfor

30 mm and then with mAb DF3-P, mAb DF3, on an isotype-

identical control antibody (IgG2�, UPC 10; IgG1, MOPC 21;

Sigma Chemical Co., St. Louis, MO) for 1 h. After washing with

1% BSA/PBS, cells were reacted with FITC-conjugnted goat

anti-mouse IgG (F-2012; Sigma) at 4#{176}Cfor 1 h, washed, and

incubated with S p.g/ml propidium iodide. Fluorescence was

determined for 106 stained viable cells using a BDIS Consort

flow cytometen.

Research. on July 17, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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A. MAb DF3-P B. MAb DF3

C,, (f)

e� I I

>0< Oi.-Oio Ii<>> �<000 (00.

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e� I

� ?<>> �000(0 0.

46-

566 Expression of the DF3-P Epitope

Fig. 1 Immunoblot analysis of human ovarian carcinoma cell lines

with mAbs DF3-P and DF3. Extracts of the indicated cells (10�) were

subjected to electrophoresis in 3-10% SDS-polyacrylamide gels, trans-

ferred to nitrocellulose filters, and monitored for reactivity with: A, mAb

DF3-P; B, mAb DF3.

Cell Surface Assay on Chamber Slides. Cells (1-2 X

10�) were plated on plastic chamber slides (Lab-Tek; Nunc, Inc.,

Naperville, IL) for at least 24 h, incubated in blocking buffer at

4#{176}Cfor 1 h, and then reacted with mAb DF3-P, mAb DF3, on

isotype-identical control antibodies for another 1 h. After expo-

sure to FITC-conjugated gont anti-mouse antibody, cells were

washed and then fixed in 10% formalinfPBS. The slides were

treated with Aqunmount (Lernen Labs, Pittsburgh, PA) and then

analyzed by fluorescent microscopy.

ELISA. CAOV-3 cells (2 X 10�) were sonicated and the

extract was incubated to dryness at 37#{176}Cin individual wells of

a 96-well assay plate (Falcon 391 1). After washing with PBS,

the wells were incubated with 5% horse serum/PBS for 1 h and

then with various concentrations of mAb DF3-P for 2 h at 37#{176}C.

Excess mAb DF3-P was removed by washing and the plates

were incubated with horseradish penoxidase-conjugated goat

anti-mouse immunoglobulin (Boehningen Mnnnheim, Indiannp-

ohs, IN) for 1 h. 0-phenylene diamine (Sigma) in 0.02 M citrate

buffer (pH 5.3) was used as substrate, and development was

monitored by absonbance at 492 nm.

Radiolabeling of Antibodies. mAb DF3-P and a eIgG

produced by P3 x 63Ag8 mouse myeloma (16) were purified

from aseites by precipitation in 45% ammonium sulfate at 4#{176}C

(17). The antibodies were labeled by the Iodogen method using

50 p.Ci (1.8 MBq) 1251 (DF3-P) on 50 p.Ci ‘�‘I (eIgG). Immu-

noneactivity of nadiolabeled mAb DF3-P as determined by bind-

ing assays with CAOV-3 cells was 41%. Tniehlonoaeetic acid

MAb DF3-P MAb DF3

� L� � � CAOV�3

1[L�& ��JOV�D

� �I�s�1 � ��OVCAR-3

I �L1 PA�1100 101 102 io� 10#{176}101 102 iO�

Fluorescence Intensity

Fig. 2 Cell surface expression of the DF3-P and DF3 epitopes on

human ovarian carcinoma cells. The indicated human cell lines were

incubated with mAb DF3-P or mAb DF3 (_) and isotype-identical

control antibodies (-). Reactivity was determined by flow cytom-

etry.

precipitation demonstrated 96 ± 4% (mean ± SD) protein-

bound radioactivity for different preparations.

Nude Mouse Tumor Models. The CAOV-3 and

OVCAR-3 tumors were serially transplanted (50 mm3 of

minced tumor) into the night flank of 7-9-week-old male Swiss

homozygous nu/nu mice kept in aseptic conditions. Mice ne-

eeived 6 Gy whole-body irradiation using a cesium source 24 h

before transplantation. Three animals from an identical passage

were used for each experiment. Thyroid glands were blocked for

uptake of free iodine by addition of 5% Lugol iodine solution

into the drinking water (0.4 mi/liter) from 3 days before the

injection of nadiolnbeled antibodies until the end of the expeni-

ments.

Biodistribution Studies. Within 3-4 weeks after trans-

plantation, groups of three animals beaning CAOV-3 on

OVCAR-3 tumors weighing between 0. 1 and 0.4 g were coin-

jeeted into the tail vein with nndiolnbeled antibodies (18). The

injected dose pen mouse was 4 p.g each of mAb DF3-P and eIgG

nadiolabeled with 1.6-1.8 p.Ci 1251 on ‘�‘i respectively. As a

further control, a group of three mice bearing CAOV-3 tumors

was given injections of ‘25I-labeled mAb DF3-P in combination

with 4 �.i.g isotype-matched IgG2� (UPC 10; Sigma) labeled

with 1311 At 48 on 96 h after injection, mice were killed by CO2

inhalation; about 0.5 ml blood was taken from the vena enva, the



A. B.

C.

�#{149}#{149}:�.:�‘

�

Clinical Cancer Research 567

MAb DF3-P MAb DF3

‘�

I :

-IfD #{149} � � :

�. .�t �p

‘�

CAOV-3

OVCAR--3

Fig. 3 Immunofluorescence staining patterns with mAbs DF3-P and DF3. CAOV-3 (A and B) and OVCAR-3 (C and D) cells were grown on

chamber slides to 50-70% confluence, reacted with mAbs DF3-P or DF-3, and then a FITC-conjugated second antibody. The cells were then fixed

in formalin and visualized for fluorescence staining.

tumor, and various organs; and the carcasses were dissected, and

weighed, and the radioactivity for both iodine isotopes was

measured in a dual-channel gamma scintillation counter. The

data were connected for the overlap (14%) of 1311 -y radiation

into the 1251 channel and for the physical half-life of both iodine

isotopes. From the differential radioactivity analysis, we deter-

mined the concentration of mAb DF3-P and eIgG and expressed

the data as a percentage of the total injected dose of radioactivity

pen g tissue (% ID/g).

Immunoperoxidase Staining. Four-p.m sections of

formalin-fixed panaffm-embedded tissue were incubated at 60#{176}C

for 1-2 h, incubated with 3% H2O1/methanol for 20 mm, and then

blocked with 8% horse serum/PBS for 20 mm. Incubation with

primary antibodies (mAb DF3-P, 1 p.g/ml; niAb DF3, 0.5 p.g/ml;

and mouse IgG�,,, 1 p.g/ml) was performed overnight at 4#{176}C.

Antibody reactivity was assessed using an avidin-biotin-penoxidase

staining technique using biotinylated antimouse IgG (BA-2000;

Vector Laboratories, Burlingame, CA) and peroxidase-conjugated

streptavidin (P397; DAKO, Canpinterin, CA).

RESULTS

Reactivity of mAb DF3-P with Human Ovarian Carci-

noma Cell Lines. Ovarian carcinoma cell lines were studied

by immunoblotting for expression of the DF3-P epitope. mAb

DF3-P reacted with extracts of OVCAR-3, OV-D, CAOV-3,

and SK-OV-3 cells (Fig. 1A). In contrast, there was no detect-

able reactivity with similar preparations of the PA-I tenatocan-

cinoma cells (Fig. 1A). The apparent molecular weight of the

mAb DF3-P reactive species varied among the cell lines. For

example, mAb DF3-P reactivity was predominant against Mr

170,000-250,000 proteins (Fig. 1A). In contrast, mAb DF3

identified higher molecular weight species in each of the mAb

DF3-P-positive lines (Fig. 1B). These findings were in concert

with the binding of mAb DF3-P to cone proteins or partially

glycosylated species.

Previous work has demonstrated that the DF3 epitope is

detectable on the surface of human ovarian carcinoma cell lines

and in the culture supernatants (3). While these findings are

consistent with the properties of a shed antigen, less is known

about cell surface expression of the immature precursors of this

glycoprotein. Consequently, we compared flow cytometry pat-

terns using mAbs DF3-P and DF3. mAb DF3-P reacted with the

intact CAOV-3, OV-D, and OVCAR-3 cells (Fig. 2). CAOV-3

cells also exhibited reactivity with mAb DF3-P and the pattern

was similar to that obtained with mAb DF3 (Fig. 2). When using

OV-D and OVCAR-3 cells, reactivity with mAb DF3 was

greater than that obtained with mAb DF3-P (Fig. 2). In contrast

to these findings, there was no detectable reactivity of either



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mAb DF3-P on mAb DF3 with PA-i tenatocaneinoma cells (Fig.

2).

In order to further identify cell surface patterns of DF3-P

and DF3 expression, immunofluoneseenee staining was pen-

formed on cells growing as monolayens on chamber slides. In

these studies, cells at over 50-70% confluence were incubated

with mAb DF3-P, mAb DF3, on isotope-identical control anti-

bodies and then exposed to a FITC-conjugnted second antibody.

After fixation, the cells were visualized for flounescence stain-

ing. mAb DF3-P-neactive CAOV-3 cells were detectable in

clusters (Fig. 3). A similar pattern was observed with mAb DF3

(Fig. 3). Comparable findings were obtained with OVCAR-3

cells, although the intensity of staining with mAb DF3 was

somewhat greaten than that obtained with mAb DF3-P (Fig. 3).

The specificity of these findings was supported by the absence

of reactivity with isotype-identical control antibodies (data not

shown).

Affinity of mAb DF3-P Binding. Two approaches were

used to estimate the Kd of mAb DF3-P binding. Intact CAOV-3

cells were incubated with varying concentrations of mAb DF3-P

and binding was determined by indirect immunofluoneseence.

Using this approach, the concentration of antibody that resulted

in half-maximal binding was 46 p.g (Kd = 1.6 X 10_6 M; Fig.

4A). In other studies, membrane preparations of CAOV-3 cells

were incubated with mAb DF3-P and binding was determined

by ELISA. Half-maximal binding using this method was found

at 6.25 �ig mAb DF3-P (Kd 4.2 X i07 M; Fig. 4B).

Biodistribution of Radiolabeled mAb DF3-P. Biodis-tnibution of mAb DF3-P and cIgG was assessed by measuring

radioactivity concentrations, expressed as % ID/g, in the tumor,

blood, and various organs at different times after injection. In

six mice beaning CAOV-3 tumors that were analyzed 48 h after

injection, nndiolabeled mAb DF3-P uptake in tumor reached

14.6 ± 4.4% (mean ± SD) ID/g (Fig. 5A). Radioactivity in the

other organs was low for both mAb DF3-P and cIgG. The

tumon:blood ratio with mAb DF3-P was 1.95, while this ratio

was 0.43 for cIgG. In three other mice beaning CAOV-3 tumors,

mAb DF3-P biodistnibution at 48 h was similar with 13.2 ±

5.9% ID/g tumor. Uptake of coinjected control mouse IgG2� in

these mice (4.5 ± 0.8% ID/g tumor) was comparable to that

obtained with the P3 X 63Ag8 myelomn protein with the

exception of somewhat higher uptake in the spleen. At 96 h after

injection, mAb DF3-P uptake in CAOV-3 tumor was similar to

that at 48 h, while it was lower in normal tissues and blood

(tumon:blood ratio, 2.5 for mAb DF3-P and 0.4 for cIgG; Fig.

SB).

In three mice beaning OVCAR-3 tumors at 48 h after

injection, tumor radioactivity was 16.7 ± 4.9% ID/g for mAb

DF3-P and relatively low in normal organs (Fig. 6). Localization

in tumor and normal organs was also low for cIgG. The tumor:

blood ratio of mAb DF3-P was 1.4 and that for cIgG was 0.4. In

another group of three mice beaning OVCAR-3 tumors of a later

passage (fourth; Fig. 6 representing mice beaning tumors of the

second passage), specific localization of mAb DF3-P at 48 h

was lower; 10.2 ± 4.4% ID/g tumor, giving a tumor:blood ratio

of 0.9 (data not shown).

Reactivity of mAb DF3-P with Ovarian Tumors. For-malin-fixed paraffin-embedded sections of malignant and be-

nign ovarian tumors were evaluated for reactivity with mAbs

300 A.

200

100

.01 .1 1 10 100

MAb DF3-P (�ig)

Fig. 4 Determination of mAb DF3-P binding affinity. A, indirect im-

munofluoreseence. CAOV-3 cells (2.5 X 10�/200 �i.l) were incubatedwith the indicated amounts of mAb DF3-P in 2% BSA and then a

FITC-conjugated second antibody. Reactivity was determined by flowcytometry. Half-maximal binding detected at 46 �.tg. B, ELISA. Ex-tracts of CAOV-3 cells (2 X 10�/100 p.1) adsorbed to plates were

incubated with mAb DF3-P and then a peroxidase-conjugated second

antibody. Development was monitored at 492 nm. Half-maximal bind-

ing was found at 6.25 pg.

DF3-P and DF3 using an immunopenoxidnse staining technique.

Staining of a senous cystndenoeancinoma of the ovary with mAb

DF3-P was detectable throughout the cytoplasm and cell mem-

branes (Fig. 7A). Reactivity of this tumor with mAb DF3 dem-

onstnnted less intense staining with a pattern that was predom-

inantly membrane in location (Fig. 7C). There was little if any

staining with either antibody in normal surrounding tissue.

There was also no detectable staining of a serous adenoma of the

ovary using mAb DF3-P (Fig. 7B). However, reactivity of this

benign tumor was apparent with mAb DF3 (Fig. 7D).

Eleven malignant tumors of the ovary that included senous

cystadenocancinomas, mucinous cystndenocancinomas, endo-

metnioid carcinomas, and clear cell carcinomas were evaluated

for mAb DF3-P and DF3 reactivity on the basis of percentage of

stained cells (Table 1). Six of the 1 1 tumors demonstrated

positive staining of 30% on more of the malignant cells with

mAb DF3-P. In contrast, mAb DF3-P staining of nine benign

tumors was less intense with none exhibiting 30% or more

positive cells (Table 1). As demonstrated previously (3), mAb



�- L� � �) � 0) a��E > a) � a, U)�

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Fig. 5 Biodistnibution of radiolabeled mAb DF3-P in nude mice bear-

ing CAOV-3 tumors. Six mice with CAOV-3 tumor xenografts were

injected with ‘25I-labeled mAb DF3-P (U) and ‘311-labeled cIgG (a).The results (mean ± SD) are expressed as the percentage of injecteddose per g of tissue in tumor, blood, and the indicated organs at 48 (A)

and 96 (B) h after injection. S, small; L, large.

DF3 reacted with the ovarian carcinomas and demonstrated

relatively strong staining of the benign tumors (Table 1).

DISCUSSION

mAb DF3-P was generated against a recombinant fusion

protein containing 3-galnetosidase and 309 base pains of tandem

repeat sequences from the DF3 gene (13). This mAb exhibited

reactivity with immature precursors of the DF3 antigen and not

the secreted form. These findings contrast those obtained with

mAb DF3, which reacts predominantly with the mature glyco-

protein (13). Mapping studies have demonstrated that the DF3

and DF3-P epitopes are located at a TRPAPGS domain in the

20-amino acid tandem repent. The DF3 epitope includes thneo-

nine at position 7 and serine at position 13. Glycosylation of

these amino acids appears to contribute to the affinity of mAb

DF3 binding. Moreover, glycosylation of the threonine and/on

senine residues appears to inhibit reactivity of mAb DF3-P

through stenic hindrance on confonmational changes. Thus, gly-

cosylation at 7Thr on ‘3Ser probably accounts for the different

patterns of reactivity obtained with mAbs DF3 and DF3-P. This

difference in antibody binding was supported by immunoper-

oxidase staining of human breast tissues. mAb DF3 reacts with

the apical borders of normal secretory mammary epithelial cells

10

5

15

10

a,Cl)Cl)

I-

E

0a)0.a,U)0

aV

C.)�0)C

Fig. 6 Localization of radiolabeled mAb DF3-P in OVCAR-3 tumors.

Three mice bearing OVCAR-3 tumors were given injections of 125!..

labeled mAb DF3-P (U) and ‘311-labeled eIgG (�). The results (mean± SD) are expressed as the percentage of injected dose per g tissue in

tumor, blood, and the indicated organs at 48 h after injection. S. small;

L, large.

and the cytosol of malignant breast cells (19). While mAb

DF3-P also exhibited reactivity with breast carcinomas, there

was little if any binding of this antibody to normal mammary

epithelium (13).

The previous finding that mAb DF3 reacts with human

ovarian carcinomas (3) prompted the present studies of DF3-P

expression in these tumors. The results with ovarian carcinoma

cell lines demonstrate that the mAb DF3-P epitope is detectable

in lower molecular weight precursors and not the mature gly-

coproteins. Other studies have shown that the protein core is

processed in the endoplasmic reticulum and that extensive 0-

glyeosylation is primarily responsible for the increase in molee-

ulnn weight of the mature glycoprotein (20). Precursors with

incomplete glycosylation patterns are routed to the cell mem-

bnane, but undergo internalization and further addition of gly-

cans before secretion (21). The finding that the DF3-P epitope is

detectable on the surface of ovarian cancer cells and not in

antigen secreted into the culture supernntant (data not shown)

supports similar mechanisms of rerouting. Other work has dem-

onstrated that the mature glycoprotein circulates at elevated

levels in the plasma of patients with ovarian cancer (5). The

finding that circulating DF3-P-positive proteins are undetectable

in these patients by immunoblot analysis (data not shown)

would support, at least in part, the finding that undenglycosy-

lated forms are not subject to secretion.

The finding that mAb DF3-P reactivity is detectable on the

surface of ovarian carcinoma cells suggested that this epitope

might serve as a target for localization of tumor with nadiola-

beled antibody. A Kd of 10_6 to 5 x i07 for mAb DF3-P

binding to intact cells and cell membranes further supported this

possibility. Radioimaging trials for the detection of human ovan-

inn carcinomas have been performed with limited success using

nndiolnbeled mAbs generated against human milk fat globule

membranes (22, 23), placental alkaline phosphatnse (24, 25),

and other tumors (26). Consequently, we asked whether mAb



A.

C. D.


B..1

MAb DF3-P

MAb DF3

Fig. 7 Immunopenoxidase staining of ovarian tumors. Fonmalin-fixed tissue sections of a human serous cystadenocarcinoma of the ovary (A and C)

and a benign serous adenoma (B and D) were stained with mAb DF3-P on mAb DF3 by an avidin-biotin-penoxidase method.

Table 1 Reactivity of mAbs DF 3-P and DF3 with human ovarian tumor tissues

mAb Tumors

% of Stained cells”

Total- + + + + + +

DF3-P

DF3

MalignantBenignMalignantBenign

1 (9.1%)4 (44.4%)

1 (9.1%)1 (11.1%)

4 (36.4%) 2 (18.2%)5 (55.6%) 00 4 (36.4%)4 (44.4%) 3 (33.3%)

4 (36.4%)06 (54.5%)

1 (11.1%)

119

119

(,-, 0-10%; +, 11-30%; ++, 31-50%; +++, 51-100%.

DF3-P might be useful for the radiodetection of these tumors.

Distribution studies were therefore performed with ‘251-lnbeled

mAb DF3-P in nude mice bearing either CAOV-3 on OVCAR-3

tumors. mAb DF3-P preferentially accumulated in both tumors

as compared to multiple other organs and the blood pool. More-

oven, coinjection of 131I-lnbeled cIgG demonstrated selective

tumor localization with mAb DF3-P in the same animal and

therefore under conditions of identical biological parameters.

The finding of selective uptake of nadiolabeled mAb DF3-P in

ovarian carcinoma xenogrnfts, as well as the absence of detect-

able circulating DF3-P antigen in patients (data not shown),

indicates that mAb DF3-P may be useful in the rndioimmuno-

detection of this disease.

Expression of the DF3-P epitope in ovarian carcinoma

specimens was also detectable by immunoperoxidase staining

studies. We had previously shown that mAb DF3 reacts with

carcinomas of the ovary (3). The present results extend those

findings by demonstrating expression of the DF3-P epitope in

ovarian cancers and to a lessen extent in benign tumors. More-

oven, there was little if any staining of normal surrounding

tissue. Heterogeneity of DF3-P expression was apparent in both

the ovarian carcinoma specimens and in the cell lines where

clusters of positive cells were primarily responsible for mAb

DF3-P reactivity. While the basis for these findings are not

clear, heterogeneity of DF3-P expression may be relevant in

approaches that involve active specific immunotherapy. In this

context, expression of the protein core in an underglycosylated

state appears to be responsible for the induction of immune

responses.

Cytotoxic T cells from tumor-draining lymph nodes of

patients with breast cancer recognize epitopes on the protein

core that are not expressed in the mature glycoprotein (27).

Furthermore, antibodies generated by an Epstein-Barr virus

immortalized B cells from a patient with ovarian cancer have

demonstrated reactivity with the tandem repent peptide (28).

These findings have suggested that accessibility of certain




protein core sequences, such as the DF3-P epitope, might

serve as a target for active immunotherapy. Indeed, recent

studies have demonstrated that immunization of mice with

vaccinia virus recombinants expressing a cell-associated

form of this glycoprotein protects against tumor challenge

(29). Thus, expression of the DF3-P epitope in ovarian cnn-

cinomas may be useful for actively inducing immunity

against these tumors.

REFERENCES

1. Kabawat, S. E., Bast, R. C., Welch, W. R., Knapp, R. C., and Colvin,R. B. Immunopathologic characterization of a monoclonal antibody thatrecognizes common surface antigens of human ovarian tumors on se-

nous, endometrioid and clean cell types. Am. J. Clin. Pathol., 79:

98-104, 1983.

2. Bhattachanya, M., Chattenjee, S. K., Barlow, J. J., and Fuji, H.

Monoelonal antibodies recognizing tumor-associated antigen of humanovarian mucinous cystadenocancinomas. Cancer Res., 42: 1650-1654,

1982.

3. Friedman, E. L., Hayes, D. G., and Kufe, D. W. Reactivity of

monoclonal antibody DF3 with a high molecular weight antigen ex-pressed in human ovarian carcinomas. Cancer Res., 46: 5189-5194,

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1995;1:565-571. Clin Cancer Res K Ichige, L Perey, C A Vogel, et al. Expression of the DF3-P epitope in human ovarian carcinomas.

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