Monoclonal antibodies to ovarian, cervical and uterine human cancers and method of diagnosis

Mouse monoclonal antibodies to several cell antigens of human ovarian, cervical and endometrial carcinomas have been produced and characterized. The distribution of the antigens was determined by mixed hemagglutination assays on 153 normal and malignant cell cultures of various types, and by immunoperoxidase staining of frozen sections of 27 normal adult and 24 fetal tissues. five monoclonal antibodies representative of five classes of mAb raised to restricted ovarian, cervical and endometrial cells were tested extensively producing mAb reactive with cancer but not normal cells. One such mAb, MF116 was readily detected in the spent culture medium of metabolically radiolabeled cells. These antibodies, reacting with relatively restricted cell surface antigens, are useful in the analysis of epithelial cell differentiation, in cancer diagnosis and therapy and in tissue typing of normal or abnormal cells.

This invention relates to a method for the production of monoclonal 
antibodies (mAbs) to restrictive antigenic human cell components 
especially in human ovarian and endometrial tissues. Such mAbs have use in 
cancer diagnosis and therapy, as well as other cell disorders. 
BACKGROUND 
Conventional antisera, produced by immunizing animals with tumor cells or 
other antigens, contain a myriad of different antibodies differing in 
their specificity and properties. In 1975 Kohler and Milstein (Nature, 
256:495) introduced a procedure which leads to the production of 
quantities of antibodies of precise and reproducible specificity. The 
Kohler-Milstein procedure involves the fusion of spleen cells (from an 
immunized animal) with an immortal myeloma cell line. By antibody testing 
of the fused cells (hybridomas), clones of the hybridomas are selected 
that produce antibody of the desired specificity. Each clone continues to 
produce only that one antibody, monoclonal antibody (mAb). As hybridoma 
cells can be cultured indefinitely (or stored frozen in liquid nitrogen), 
a constant, adequate supply of antibody with uniform characteristics is 
assured. 
Antibodies are proteins that have the ability to combined with and 
recognize other molecules, known as antigens. Monoclonal antibodies are no 
different from other antibodies except that they are very uniform in their 
properties and recognize only one antigen or a portion of an antigen known 
as a determinant. 
In the case of cells, the determinant recognized is an antigen on or in the 
cell which reacts with the antibody. It is through these cell antigens 
that a particular antibody recognizes, i.e. reacts with, a particular kind 
of cell. Thus the cell antigens are markers by which the cell is 
identified. 
These antigenic markers may be used to observe the normal process of cell 
differentiation and to locate abnormalities within a given cell system. 
The process of differentiation is accompanied by changes in the cell 
surface antigenic phenotype, and antigens that distinguish cells belonging 
to distinct differentiation lineages or distinguish cells at different 
phases in the same differentiation lineage may be observed if the correct 
antibody is available. 
The preparation of hybridoma cell lines can be successful or not depending 
on such experimental factors as nature of the innoculant, cell growth 
conditions, hybridization conditions etc. Thus it is not always possible 
to predict successful hybridoma preparation of one cell line although 
success may have been achieved with another cell line. But it is often 
true that selected mAb may be representative of a class of mAb raised by a 
particular immunogen. Members of that class share similar characteristics, 
reacting with the same cell antigen. Thus the invention includes hybridoma 
cell lines and mAb with like or similar characteristics. 
Progress in defining cell surface antigens is of great importance in 
differentiation and disease as markers for normal and diseased cells, 
thereby furthering diagnosis and treatment. Thus work on melanocytes was 
made possible by the recently discovered technique of culturing 
melanocytes from normal skin (Eisinger, et al., Proc. Nat'l. Acad. Sci. 
U.S.A., 79 2018 (March 1982). This method provides a renewable source of 
proliferating cells for the analysis of melanocyte differentiation 
antigens. Likewise, a large number of cell lines derived from melanomas 
have now been established and these have facilitated the analysis of 
melanoma surface antigens. The advent of mAbs has greatly accelerated 
knowledge about the surface antigens of malignant melanoma, cell markers 
on both melanomas and melanocytes have been identified. A panel of typing 
monoclonal antibodies has been selected which recognizes differentiation 
antigen characteristics at each stage of development in both melanocytes 
and melanomas. These differentiation antigens may be used to classify 
melanocytes and melanomas and to group them into characteristic sub-sets. 
[Dippold et al. Proc. Nat'l. Acad. Sci. U.S.A. 77, 6114 (1980) and 
Houghton, et al, J. Exp. Med. 156, 1755 (1982)]. Immunoassay of 
melanocytes and melanoma cells within sub-sets is thus made possible. 
Initial recognition of differentiation antigens came about through analysis 
of surface antigens of T-cell leukemias of the mouse and the description 
of the TL, Thy-1, and Lyt series of antigens. (Old, Lloyd J., Cancer 
Research, 41, 361-375, February 1981) The analysis of these T-cell 
differentiation antigens was greatly simplified by the availability of 
normal T cells and B cells of mouse and man. (See U.S. Pat. Nos. 
4,361,549-559; 4,364,932-37 and 4,363,799 concerning mAb to Human T-cell 
antigens). 
The existence of human leukemia specific antigens has been suggested by 
studies using heterologous antibodies developed by immunization with human 
leukemic cells [Greaves, M. F. et al. Clin. Immunol. and Immunopathol 
4:67, (1975); Minowada, J., et al. J. Nat'l. Cancer Insti. 60:1269, 
(1978); Tanigaki, N., et al. J. Immunol. 123:2906, (1979)] or by using 
autologous antisera obtained from patients with leukemia [Garret, T. J., 
et al., Proc. Nat'l. Acad. Sci. U.S.A. 74:4587, (1977); Naito, K., et al., 
Proc. Nat'l. Acad. Sci. U.S.A., 80: 2341, (1983)]. The common acute 
lymphoblastic leukemia antigen (CALLA) which is present on leukemia cells 
from many patients with non-T, non-B, acute lymphoblastic leukemia 
(N-ALL), some chronic myelocytic leukemias (CML) in blast crisis and a few 
acute T-lymphoblastic leukemias (T-ALL) was originally described using 
conventional rabbit heteroantisera [Greaves, M. F. et al. Supra ]. 
By the autologous typing technique [Garret, T. J., et al. Supra; Naito, K., 
et al. Supra 1983; Old, L. J. Cancer Res. 41:361, (1981)], antibodies 
uniquely reacting with ALL cells were found in sera obtained from patients 
with ALL, and seemed to recognize very similar antigens to CALLA (Garret, 
T. J., et al. Supra; Naito, K., et al. Supra). Another leukemia associated 
antigen detected by heterologous antisera is the human thymus leukemia 
(TL)-like antigen, which is present on thymocytes as well as leukemia 
cells (Tanigaki, N. et al. Supra). This antigen, is therefore, a normal 
differentiation antigen which is composed of a heavy chain (MW 
44,000-49,000) and light chain (MW 12,000-14,000) similar to the class I 
HLA antigens (Tanigaki, N., et al. Supra). These investigations have, 
however, been hampered by the need for vigorous absorptions with normal 
tissues as well as the relatively small quantity and low titer of the 
antisera. 
In vitro production of monoclonal antibodies by the technique of Kohler and 
Milstein, Supra has provided a better system for the identification and 
detection of leukemia specific antigens. A panel of monoclonal antibodies 
detecting cell surface antigens of human peripheral blood lymphocytes and 
their precursor cells have been investigated in detail [Reinherz, E. L., 
et al. Proc. Nat'l. Acad. Sci. U.S.A. 77:1588, (1980)]. While monoclonal 
antibodies detecting antigens characteristic for different lymphocyte 
lineages can be used for classification of human lymphocytic leukemia 
[Schroff, R. W., et al. Blood 59:207, (1982)], such antibodies have only 
limited therapeutic applications. Monoclonal antibodies detecting human 
leukemia associated antigens have also been produced. These include 
several antibodies detecting the human equivalents of the murine TL 
antigens. One TL-like antigen is recognized by NA134 [McMichael, A. J., et 
al. Eur. J. Immunol. 9:205, (1979)], OKT6 (Reinherz, E. L., et al. Supra) 
and Leu 6 (R. Evans, personal communication). A second TL-like antigen is 
recognized by M241 (Knowles, R. W., et al. Eur. J. Immunol. 12:676, 1982). 
Monoclonal antibodies with specificities for common acute lymphoblastic 
leukemia antigens J-5 (Ritz, J., et al. Nature 283:583, 1980), NL-1 and 
NL-22 (Ueda, R., et al. Proc. Nat'l. Acad. Sci. U.S.A. 79:4386, 1982) have 
also been produced. Recently, Deng, C-T, et al. Lancet. i:10, 1982) 
reported a complement fixing monoclonal antibody (CALLA-2) which reacts 
with most cultured human T-ALL cell lines and also reacts with most fresh 
T-ALL cells. 
Mouse monoclonal antibodies to human tumor cell surface antigens have been 
produced in many laboratories (Lloyd, K. O. (1983) In: Basic and Clinical 
Tumor Immunology, Vol. 1 (R. B. Herberman, Ed.), Nijhoff, The Hague (in 
press)). The intention of these studies often has been to identify 
tumor-associated antigens that could be useful in tumor therapy or 
diagnosis. An inherent difficulty in this approach is the diversity of 
antigens on the cell surface. Although it has been possible to identify 
some antigens with a very restricted distribution, antibodies to antigens 
that elicit very weak immune responses may be missed due to their 
scarcity. These restricted antigens may be quite difficult to identify. 
Also, immunization with a complex mixture of antigens such as tumor cells 
may suppress the antibody response to relatively less immunogenic 
molecules, in a manner resembling antigenic competition (Taussig, M. J. 
(1973). Curr. Top. Micro. Immuno. 60:125). Thus production of mAb to 
restricted cell sites is an especially difficult task. The present 
invention provide cancer diagnosis and therapy and overcome problems 
heretofor encountered in the prior art with respect to ovarian and 
endometrial human cell antigens. 
A number of ovarian tumor antigens have been detected using xenogeneic 
polyclonal sera (reviewed in Lloyd, K. O. (1982) Serono Symposium No. 46 
(M. I. Colnagki, G. L. Buraggi and M. Ghrone, Eds.) Academic press. N.Y. 
pp. 205-211) but none are related to the antigens of the invention. Other 
laboratories have also described monoclonal antibodies to human ovarian 
carcinoma different from those of the invention. Bhattacharya et al. 
(Bhattacharya, M., et al. (1982) Cancer Res., 42:1650-1654) produced an 
antibody to a saline-extracted antigen detected only in mucinous cyst 
adenocarcinomas of the ovary and in fetal intestine. Serous cyst 
adenocarcinomas, the most common ovarian carcinoma, did not contain this 
antigen. Bast et al. produced an antibody (OC 125) reactive with an 
antigen present on 6/6 ovarian carcinoma cell lines and one melanoma of 14 
non-ovarian cell lines tested. This antibody reacted with sections of 
12/20 ovarian carcinomas and was nonreactive with 12 non-ovarian 
carcinomas and with most normal tissues, including normal adult and fetal 
ovary. Weak reactivity was observed with adult fallopian tube, endometrium 
and endocervix (Bast, R. C., et al. (1981) J. Clin. Invest. 68:1331-1336; 
Kabawat S. E., et al (1983) Amer. J. Clin. Pathol., 79:98-104). 
SUMMARY 
Monoclonal antibody representative of five separate classes of mAb to 
ovarian and uterine cancers are described. The antigenic profile of each 
of these mAbs is presented with both serological and tissue reactivity 
studies in cancer and normal cell lines and tissue sections. These mAbs 
form a panel useful for the diagnosis and therapy of cancers of the 
ovarian and uterine system.

DESCRIPTION 
The techniques described below result in the isolation of mAb of several 
classes; representative mAbs from each of these classes are described and 
characterized. These techniques can be used to isolate other mAbs from 
these classes. Thus substantially similar or functionally equivalent 
monoclonal antibodies having substantially the same characteristics and 
properties can be produced in accordance with the procedures of the 
invention. The mAb examples described herein are for illustrative purposes 
only and are not meant to limit the invention in any way. 
Target cells 
Cell lines used are listed in Table I. Preparation of cultures of normal 
human fibroblasts, kidney epithelial cells and melanocytes have been 
described (Carey, T. E., et al. (1976) Proc. Nat'l. Acad. Sci., U.S.A., 
73:3278-3282; Ueda, R., et al. (1979) J. Exp. Med., 150:564-579; Eisinger, 
M., et al. (1982) Proc. Nat'l. Acad. Sci., U.S.A., 79:2018-2022). Adherent 
cells were maintained in Eagle's minimum essential medium (GIBCO, Grand 
Island, NY) supplemented with 2.5% fetal calf serum, 5% newborn calf 
serum, 100 U/ml penicillin and 1 mg/ml streptomycin. Nonadherent cells 
were cultured in RPMI 1640 medium supplemented similarly except with 7.5% 
fetal calf serum. Cultures were regularly tested for mycoplasma and 
contaminated cultures discarded. 
Normal blood mononuclear cells were obtained by centrifuging heparinized 
blood onto a layer of Ficoll-Paque (Pharmacia, Piscataway, NJ). Total 
blood leukocytes were obtained by collecting the buffy coat after 
centrifugation for 10 min at 600 g in 100 microliter capillary tubes. 
The origins of cells and tissues is as follows: Drs. Charles Welander, 
Sloan-Kettering Institute, New York, N.Y. (SK-OV-6 and SK-UT-1), Jorgen 
Fogh, Sloan-Kettering Institute, New York, N.Y. (SK-OV-3 and SW626), G. 
Roos, University of Umea at Umea, Sweden (A7 and A10), George Moore, 
Denver General Hospital, Denver, Colo. (COLO 316) and R. S. Freedman 
University of Texas, at Houston (2774), Dr. Virginia Pierce for clinical 
specimens, the Human Cancer Serology group for cell lines and tissue 
specimens. 
Production of Mouse Monoclonal Antibodies 
BALB/c or (BALB/c.times.C57BL/6)F.sub.1 mice were immunized with the 
ovarian carcinoma cell lines SK-OV-3, SW626 or 2774, or the endometrial 
carcinoma cell line SK-UT-1. Intraperitoneal injections of approximately 
100 microliters of packed cells were given 2-5 times at intervals of two 
weeks. Three days after the last injection, the fusion of immune spleen 
cells with mouse myeloma MOPC-21 NS/1 cells was performed as described 
(Dippold, W. G., (1980) Proc. Nat'l. Acad. Sci., U.S.A., 77:6114-6118). 
Initially, cells were plated in 480 wells (Costar 3524, 24 well plates) 
Hybridoma cultures were subcloned at least two times by limiting dilution 
in 96 well plates on a feeder layer of normal mouse spleen cells. Culture 
supernatants were monitored for antibody activity by the anti-mouse Ig MHA 
(mixed hemagglutination assay) method on a panel of cultured cells 
consisting of the immunizing cell line and other types of human tumor 
cells. Cloned hybridoma cells were injected subcutaneously into nu/nu 
mice. Sera from mice with progressively growing tumors were collected and 
used for serological and biochemical characterization. Antibody subclass 
was determined by double diffusion in agar with anti-Ig heavy 
chain-specific reagents (Bionetics, Kensington, MD). 
Serological Procedures 
For adherent target cells, 200-500 trypsinized cells were plated in 10 
microliters in wells of Terasaki plates (Falcon microtest plates 3034) and 
allowed to adhere overnight. Nonadherent target cells were attached to the 
wells by pretreating the wells with concanavalin A (con A, grade IV, Sigma 
Chemicals, St. Louis, MO) (Mattes, M. J., et al. (1983) J. Immunol. 
Metho., 61:145-150). The mixed hemagglutination (MHA) assay, using rabbit 
anti-mouse Ig, has been described (Ueda, R., et al. (1979) J. Exp. Med., 
150:564-579). The CrCl.sub.3 conjugation procedure has been described 
(Koo, G. C., et al. (1978) J. Immunol. Meth., 23:197-201), except that 
undiluted rabbit anti-mouse IgG (DAKO, Accurate Chemicals, Westbury, NY) 
or the IgG fraction of goat anti-mouse IgM (Cappel Laboratories, 
Cochranville, PA), at 4.0 mg/ml, was used instead of Protein A. Monoclonal 
sera were titrated starting at 10.sup.-3. To confirm the specificity of 
antibodies, absorption tests were performed with the immunizing cell line 
and three melanomas (SK-MEL-28, SK-MEL-37 and MeWo), three astrocytomas 
(SK-MG-1, SK-MG-3 and U373 MG), three carcinomas (SK-BR-3, SK-LC-6 and 
Scaber), one T cell leukemia (MOLT-4), one B cell leukemia (Raji) and 
human erythrocytes. Absorption procedures have been described (Carey, T. 
E., et al. (1976) Supra. 
To test heat stability of antigens, cells were heated 5 min. at 100.degree. 
C. before performing absorption tests. To test the hydrophobic nature of 
antigens, cell pellets were extracted with 20 volumes of chloroform 
methanol, 2:1. Solubilized material was dried and resuspended with 
sonication in Dulbecco's phosphate-buffered saline (GIBCO), 0.5% bovine 
albumin (fraction V, Sigma Chemicals), to a volume equal to the original 
packed cell volume. This suspension was assayed for inhibitory activity of 
the appropriate antibody. 
Immunoperoxidase staining of sections employed 5 micrometer cryostat 
sections. Air-dried sections were fixed for 10 min at room temperature 
with 2.0% buffered formaldehyde (Farr, A. G., et al. (1981) J. Immunol. 
Meth., 47:129-144). A triple sandwich was used routinely which consisted 
of monoclonal antibody (nu/nu mouse serum at 1/500), biotinylated horse 
anti-mouse Ig, and complexes of avidin and biotinylated horseradish 
peroxidase (Vectastain reagents, Vector Laboratories, Burlingame, CA), 
following procedures recommended by the manufacturer. For particular 
tissues that had excessive background with this procedure, namely the 
kidney, liver and pancreas, a double sandwich was used which comprised 
monoclonal sera at 1/200 and peroxidase-conjugated anti-mouse Ig (DAKO 
P161) at 1/50. To ensure that fixation did not destroy the antigen 
investigated, each antibody was first tested on sections of tissue culture 
cells frozen in 10% dimethylsulfoxide at 50% (packed cell volume/volume). 
All antibodies tested were positive in this assay, when the immunizing 
cell line was used as the target. 
Immunofluorescent staining of blood leukocytes in suspension was performed 
as described (Mattes, M. J., et al. (1979) J. Immunol., 123: 2851-2860) 
using fluorescein-conjugated goat anti-mouse Ig (Cappel Laboratories) at 
1/40, and monoclonal sera at 1/50. Lymphocytes and granulocytes were 
distinguished by morphology. 
Immunoprecipitation Procedures 
Each antibody was tested for its ability to precipitate an antigen from 
detergent-solubilized extracts of the immunizing cell after labeling by 
three methods: metabolic incorporation of [.sup.3 H] glucosamine (Ogata, 
S-I, et al. (1981) Proc. Nat'l. Acad. Sci., U.S.A. 78:770-774), metabolic 
incorporation of [.sup.35 S]methionine (Dippold, W. G., et al. (1980) 
Proc. Nat'l. Acad. Sci., U.S.A., 77:6114-6118), or chloramine T .sup.125 I 
labeling of solubilized cell membranes (Cairncross, J. G., et al, (1982) 
Proc. Nat'l. Acad. Sci., U.S.A., 79:5641-5645). NP40 solubilization of 
labeled cells and con A-Sepharose fractionation of labeled extracts, used 
in some experiments, have been described (Dippold, W. G., et al. (1980), 
Supra; Ogata, S-I, et al, (1981), Supra, Cairncross, J. G., et al. (1982), 
Supra), as have immunoprecipitation procedures for .sup.125 I-labeled 
samples, using Staphylococcus aureus (Cairncross, J. G., et al. Supra 
(1982)). 
Aliquots of 2.times.10.sup.6 [.sup.35 S] cpm from unfractionated cell 
extracts were handled similarly except that preclearing was omitted. For 
the con A eluate fraction of [.sup.35 S]-labeled extracts and for [.sup.3 
H]-labeled extracts, aliquots of 2.times.10.sup.5 cpm and different 
washing buffers (Lloyd, K. O., et al. (1981) J. Immunol., 126:2408-2413) 
were used. Precipitated molecules were extracted with 60 microliter 0.01 M 
Tris HCl pH 7.2, 2.0% NaDodSO.sub.4 (sodium dodecylsulfate), 12.0 mg/ml 
dithiothreitol (DTT), 15% (wt/vol) sucrose, 0.01% pyronin Y by heating 5 
min at 100.degree. C., and analyzed by polyacrylamide gel electrophoresis 
(PAGE) (Dippold, W. G., et al. (1980) Supra; Laemmli, U. K., (1970) Nature 
227:680-685), using 9% gels. For 2-dimensional electrophoresis 
(isoelectric focusing followed by NaDodSO.sub.4 -electrophoresis), immune 
precipitates were extracted and handled as described (Ogata, S-I, et al. 
(1981) Supra; O'Farrell, P. H., et al. in Method in Cell Biology 
(Prescott, D. M., Eds.) (1977) Academic Press, New York, Vol. 16, pp. 
407-420)). For unreduced samples, DTT was omitted and 14.0 mg/ml 
iodacetamide was added to samples. 
Selection of Hybridomas 
Monoclonal antibodies (mAb) MD144, MF61, MF116, ME195 and ME46 were 
obtained after immunization with ovarian carcinoma cell line 2774, and 
mAbs MH55 and MH94 were obtained after immunization with endometrial 
carcinoma cell line SK-UT-1. The heavy chain subclasses of the seven 
antibodies are: MD144, gamma-sub one; MH55, mu; MF61, mu; MF116, gamma-sub 
two A; ME46, gamma-sub one; ME195, gamma-sub one; MH94, gamma-sub one. 
These monoclonal antibodies were initially selected for cloning on the 
basis of reactivity with the immunizing cell line and lack of reactivity 
with three melanomas and three astrocytomas. These antibodies were 
produced from a total of 12 fusions from mice immunized with human ovarian 
and uterine cancer cell lines. A total of 430 of the supernatants 
contained antibodies reacting with the immunizing line. Twelve cultures 
were selected for subcloning, and ten hybridomas were propagated 
successfully. The properties of seven antibodies are discussed in this 
publication and two other antibodies, which detected more wide-spread 
antigens, are described elsewhere (Mattes, M. J., et al. Hybridoma (1983) 
2:523. None of these mAbs reacted with glycoproteins carrying A, B, H, 
Le.sup.a, Le.sup.b, X, Y or I blood group structures. 
TABLE I 
__________________________________________________________________________ 
Reactivity of mouse monoclonal antibodies with 
cultured human cells and cell lines 
CELLS mAb MD144 
mAb MF61 
mAb MF116 
mAb MH94 
__________________________________________________________________________ 
Ovarian carcinomas 
2774, SK-OV-6, SW 626, SK-OV-3 
2 0 0 0.sup.1 
2 0 0 0 2 0 0 0 0 2 2 0 
SK-OV-4, Colo 316, A7, A10 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SW 626 0 0 0 0 
Endometrial carcinoma 
SK-UT-1 0 0 2 2 
Renal carcinoma 
SK-RC-1, -2, -7, -8 0 0 0 0 2 0 0 0 2 2 2 2 0 0 0 0 
SK-RC-9, -10, -12, -17 
0 0 0 0 2 2 2 2 2 0 0 0 0 
SK-RC-35, Caki-2, SK-RC-4, -6 
0 0 0 0 2 2 0 0 0 0 0 0 0 0 0 0 
SK-RC-16, -28, Caki-1 
0 0 0 0 0 0 0 0 0 0 0 0 
Bladder carcinomas 
253J, SCABER, RT4, VM-CUB-1 
0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 
VM-CUB-2, 5637, 639-V, J82 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
486-P, JCCSUP 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
Colon carcinomas 
SK-CO-10, SW403, SW480, SK-CO-1 
0 0 0 0 0 0 0 0 0 0 0 0 2 2 1 0 
SW620, SW1222, HT-29 0 0 0 0 0 0 0 0 0 0 0 0 
Breast carcinomas 
CAMA, SK-BR-3, -5, BT-20 
0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 
BT-474, MCF-7, AlAb, ZR-75-1 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
DA-MB-361, MDA-MG-231 
0 0 0 0 0 0 0 0 
Lung carcinomas 
SK-LC-9, -15, -1, -2 0 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 
SK-LC-3, -4, -5, -6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SK-LC-7, -8, -10, -13 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SK-LC-14, -16, Calu-1, SK-LC-LL 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SL-LC-12 0 0 0 0 
Cervical carcinoma 
ME 180 0 0 0 1 
Pancreatic carcinomas 
CAPAN-1, -2, ASPC-1 0 0 0 0 0 0 0 0 0 0 2 0 
Neuroblastomas 
LA-N-15, SK-N-sSH, -MC, LA-N-Is 
0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 
SH-EP1, SK-N-BE(2) 0 0 0 0 0 0 0 0 
Melanomas 
SK-MEL-13, -19, -23, -26 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SK-MEL-28, -29, -31, -37 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SK-MEL-75, -93-2, -93-3, -127 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SK-MEL-130, -153, MeWo 
0 0 0 0 0 0 0 0 0 0 0 0 
Astrocytomas 
SK-MG-1, -2, -3, -5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SK-MG-6, -8, -11, -14 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SK-MG-15, U138MG, U251MG, U373MG 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
Other carcinomas 
SK-HEP-1 (heptoma), G cc-SV 
0 0 0 0 0 0 0 0 
T-cell lymphomas and leukemias 
MOLT4, CCRF-HSB-2, CCRF-CEM, 45 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
Peer, P12/Ichikawa, HPB-ALL 
0 0 0 0 0 0 0 0 0 0 0 0 
B-cell lymphomas and leukemias 
SK-LY-16, -18, Daudi, Ball-1 
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 
SK-DLH-2, ARA-10, Raji 
0 0 0 0 0 0 0 0 0 0 0 0 
Null-cell leukemias 
NALM-1, -16 0 0 0 0 0 0 0 0 
Myeloid leukemias 
HL-60, K562, KG-1 0 0 0 0 0 0 0 0 0 0 0 0 
Myelomas 
SK-MY-1, LICR-Lon-HMy-2 
0 0 0 0 0 0 0 0 0 0 0 0 
Monocytic leukemia 
U937 0 0 0 0 
Normal cells 
Fibroblasts (6), melanocytes 
0 0 0 0 0 0 0 0 
Kidney epithelia, 1, 2 
0 0 0 0 2 1 0 0 
Non-human cells 
Vero, CHO 0 0 0 0 0 0 0 0 
__________________________________________________________________________ 
.sup.1 The symbols listed under the antibodies refer to the titer against 
the cell line in the corresponding position in the left hand side of the 
Table. The titer of the antibody was defined as the highest dilution 
producing at least 50% rosetting in the MHA assay. Symbols are: 2 = a 
range of 1 .times. 10.sup.-3 - 1 .times. 10.sup.-6 ; 1 = positive reactio 
but with less than 50% rosetting at 10.sup.-3 dilution of antibody; 0 = n 
reactivity at antibody dilution of 10.sup. -3. 
TABLE II 
__________________________________________________________________________ 
Summary of reactivities of monoclonal antibodies 
ON TISSUE ON NORMAL ADULT 
ON NORMAL FETAL 
ANTIBODY 
CULTURE CELLS.sup.1 
TISSUE SECTIONS.sup.2 
TISSUE SECTIONS.sup.3 
__________________________________________________________________________ 
MD144 Ovarian carcinomas 
Negative N.T..sup.4 
(1/8) 
MH55 Ovarian (3/8) and 
Negative N.T. 
uterine (1/1) 
carcinomas 
MF61 Ovarian (1/8) and 
Uterine epithelial 
Uterine epithelial 
renal (6/16) 
cells and thyroid 
cells positive 
carcinomas colloid positive 
MH94 Ovarian (2/8), 
Pancreas, ureter, 
Stomach, intestine, 
uterine (1/1), 
breast, prostate, 
pancreas, ureter, 
colon (3/7), breast 
cervix, urinary 
urinary bladder, 
(1/10), lung (2/18) 
bladder epithelial 
uterus and cervix 
and cervical (1/1) 
cells, sweat and 
epithelial cells 
carcinomas sebaceous glands 
positive 
of skin positive 
MF116 Normal kidney 
Negative Negative 
epithelial cells; 
ovarian (1/8), 
uterine (1/1), renal 
(6/16), bladder 
(1/10), carcinomas 
and neuroblastomas 
(1/6). 
__________________________________________________________________________ 
.sup.1 Antibodies were tested on 153 cell lines of various types (Table 
I). All tests except those listed were negative. 
.sup.2 Antibodies were tested on sections of 27 normal adult tissues: 
lung, heart, liver, spleen, gall bladder, esophagus, stomach, small 
intestine, colon, pancreas, kidney, ureter, urinary bladder, adrenal, 
thyroid, breast, prostate, testes, ovary, fallopian tube, uterus, cervix, 
placenta, skin, brain, lymph nodes and muscle. Tissues not listed were 
negative. 
.sup.3 Antibodies were tested on 24 normal fetal tissues: lung, heart, 
spleen, thymus, liver, gall bladder, esophagus, stomach, small intestine, 
colon, pancreas, kidney, ureter, urinary bladder, adrenal, testes, ovary, 
fallopian tube, uterus, cervix, skin, brain lymph nodes and muscle. 
Tissues not listed were negative. 
.sup.4 N.T.: not tested. 
MD144 
Ab MD144 reacted with only a single ovarian carcinoma cell line, 2774, with 
a titer of 10.sup.4 by immune rosetting; all 152 other cell lines tested 
were negative (Table I). The antigen was not detected in sections of the 
normal tissues (Table II). Absorption experiments also did not detect the 
antigen on any cell type except 2774; in this assay, 1-3 microliters of 
packed cells was required for nearly complete absorption. The antigen was 
not destroyed by heating at 100.degree. C. for 5 min, and it was present 
in the chloroform:methanol extract of 2774 cells. In immunoprecipitation 
experiments using cell extracts labeled with [.sup.3 H]glucosamine but not 
with [.sup.35 S]methionine, counts were precipitated which migrated at the 
dye front in both 9% and 12.5% acrylamide gels. These properties all 
strongly suggest that the antigen is a lipid. 
MH55 
Ab MH55 is an IgM antibody which reacts weakly with 4/8 ovarian carcinomas 
(2774, SK-OV-6, A10 and A7) and 1/1 uterine carcinoma (SK-UT-1) with a 
titer of 10.sup.-3 or lower; all 148 other cell types tested were 
negative. Varying the temperature of incubation with antibody, the density 
of the target cells, and the time interval between target cell plating and 
testing did not improve the titer or the consistency. We therefore have 
not done absorption experiments. Ab MH55 did not react with any tissue 
sections examined but it did react with sections of frozen pellets of A10 
ovarian carcinoma cells. 
MF61 
Antibody MF61 reacted with 1/8 ovarian carcinoma and 6/16 renal carcinoma 
cell lines (Table I); the other 146 cell types tested were negative. 
Absorption experiments revealed no additional positive cell types. Blood 
leukocytes were negative by immunofluorescence. Absorption tests were 
unusually sensitive, in that 0.1 microliters of packed cells absorbed 
nearly completely; negative absorption under our standard conditions 
therefore indicates at least a 300-fold lower expression of antigen than 
on the immunizing cell line. MF61 antigen, like the MD144 antigen, was 
heat-stable and soluble in chloroform:methanol. The chloroform:methanol 
extracts were as active as intact cells in absorption experiments. Also, 
antibody MF61 immunoprecipitated counts from [.sup.3 H]glucosamine-labeled 
cell extracts that migrated at the dye front in both 9% and 12.5% 
acrylamide gels (FIG. 1). It is believed that this antigen is a lipid. 
In tissue sections, antibody MF61 reacted with two normal tissues: 
glandular epithelial cells of the adult and fetal uterus and the 
noncellular follicles of the thyroid. It also reacted with the follicles 
of a pig thyroid. 
MF116 
Three antibodies were obtained from two fusions which react with the same 
antigen, of which the prototype antibody is MF116. The other two 
antibodies, ME46 and ME195, are IgG.sub.1, while antibody MF116 is 
IgG.sub.2a. Antibody MF116 reacted with 1/8 ovarian carcinoma, 1/1 
endometrial carcinoma, 6/16 renal carcinoma, 1/10 bladder carcinoma and 
1/6 neuroblastoma cell lines. It also reacted with 2/2 normal kidney 
epithelial cell cultures (Table I). The other 141 cell types tested were 
negative. Absorption experiments revealed no additional positive cells; 10 
microliters of packed cells was required for nearly complete absorption; 
this result is consistent with a low expression of antigen on the cell 
surface. Blood leukocytes were negative by immunofluorescence. By 
immunoperoxidase, MF116 was not detected in any normal tissues examined, 
including normal kidney, ovary and uterus. 
MF116 antigen was immunoprecipitated from [.sup.3 H]glucosamine or [.sup.35 
S]methionine labeled spent medium from ovarian carcinoma 2774. No antigen 
was detected in solubilized cell extracts labeled with [.sup.3 
H]glucosamine, [.sup.35 S]methionine or .sup.125 I. This antigen is 
preferentially shed or secreted in the medium, although it must be present 
on the cell surface since it is detected in rosetting assays. The 
molecular weight is 105,000, as estimated by PAGE. If the antigen was not 
reduced, it migrated slightly faster, indicating some intrachain disulfide 
bonds. The isoelectric point was determined to be less than pH 4.0, since 
the antigen migrated at or off the acidic end of the isoelectric focusing 
gel. This antigen bound to concanavalin A-Sepharose and was eluted with 
methyl-alpha-D-mannoside. The antigen was destroyed by heating at 
100.degree. C., as determined in absorption experiments. MF116 was not 
detected by immunoprecipitation in the spent medium of two other cell 
lines (SK-UT-1 and SK-RC-1) that were positive by rosetting. 
MH94 
MH94 antigen was detected on various carcinoma cell lines, being detected 
on 2/8 ovarian carcinoma, 1/1 endometrial carcinoma, 3/7 colon carcinoma, 
1/10 breast carcinoma, 2/18 lung carcinoma, 1/1 cervical carcinoma and 1/3 
pancreatic carcinoma cell lines (Table I). All 142 other cell types tested 
were negative. Absorption experiments did not reveal additional positive 
cells; 3 microliters packed SK-UT-1 cells gave nearly complete absorption. 
Blood leukocytes were negative by immunofluorescence. By immunoperoxidase, 
MH94 was detected in the acinar and duct lining cells of the pancreas, the 
epithelial cells of the ureter, breast, pancreas, cervix and urinary 
bladder and the sweat and sebaceous glands of the skin. It was also found 
in fetal stomach, intestine, pancreas, ureter, urinary bladder, 
endometrium and endocervix. 
The MH94 antigen was not destroyed by heating to 100.degree. C., but was 
not detected in a chloroform:methanol extract of cells. It was not 
precipitated under any conditions tested, which included labeling two cell 
lines with three isotopes. 
These studies describe five specific mAbs detecting highly restricted 
antigens that are of considerable interest for the analysis of ovarian and 
uterine tumors (Table II). More broadly reactive antibodies derived from 
the same fusions, recognizing glycoprotein antigens, were described 
previously (Mattes, M. J., et al. Hybridoma (1983) 2:523. These antibodies 
generally had higher titers than the ones described here, perhaps 
reflecting the characteristics of the antigens recognized as discussed 
below. 
Both MD144 and MF61 antigens have properties of lipids or hydrophobic 
proteins. A substantial fraction of very restricted mouse monoclonal 
antibodies, produced in several laboratories, have recognized glycolipids 
(Pukel, C. S., et al. (1982) J. Exp. Med., 155:1133-1147; Nudelman, E., et 
al. (1982) J. Biol. Chem., 257:12752-12756; Magnani, J. L., et al. (1981) 
Science, 212:55-56), a result which was quite unexpected on the basis of 
previous work using whole xenoantisera to human tumor cells. Resistance to 
heating at 100.degree. C., which is one of their characteristics, could be 
a property of lipids, carbohydrate determinants on glycoproteins or of 
exceptional protein determinants. MD144 and MF61 antigens are soluble in 
chloroform methanol, but this does not distinguish between lipids and 
hydrophobic proteins (Audubert, F., et al. (1979) Biochem. Biophys. Res. 
Comm., 91:416-426). Likewise, the immunoprecipitation by mAbs MD144 and 
MF61 of counts running at the dye front in acrylamide gel electrophoresis, 
after labeling with [.sup.3 H]glucosamine, is true of glycolipids as well 
as small glycoproteins, which would have similar properties. Also, we have 
evidence that some hydrophobic interactions are not completely disrupted 
in the presence of the detergents used to solubilize cells, so glycolipids 
might be co-precipitated by antibodies to hydrophobic proteins or to a 
nonglycosylated lipid. 
MD144 antigen which is found only a single ovarian cancer cell line, is 
unique. This component is believed to be: a rare human allele, or a rarely 
expressed gene product, or a mutant form of a normal cell component. 
Although unique antigens have been demonstrated on chemically induced 
animal tumors (Baldwin, R. W. (1973) Adv. Cancer Res., 18:1-75) and on 
human tumors (Old, L. J. (1981) Cancer Res., 41:361-375), present data 
indicate that these antigens are proteins or glycoproteins (DuBois, G. C., 
et al. (1982) Proc. Nat'l. Acad. Sci., U.S.A. 79:7669-7673; Carey, T. E., 
et al. (1979) Proc. Nat'l. Acad. Sci., U.S.A. 76:2898-2902; Real, F. X., 
et al. (1983) Proc. Amer. Assoc. Cancer Res., 24:233) and therefore differ 
in this respect from MD144. MF61 has an unusual distribution in normal 
tissue, being present in the noncellular follicles of the thyroid and in 
uterine glandular epithelial cells. The dominant antigen of the thyroid 
colloid is thyroglobulin, but antibody MF61 was not reactive with human 
thyroglobulin. A second colloid antigen has been described (Balfour, B. 
M., et al. (1961) Brit. J. Exp. Pathol, 42:307-316), but has not been 
characterized biochemically. On tumor cells, MF61 is very restricted in 
its distribution, being detected only one ovarian carcinoma line and six 
renal carcinoma cell lines. 
MH94 was detected on a small fraction of carcinoma cell lines tested, 
including carcinomas of the ovary, uterus, colon, breast, lung, cervix and 
pancreas. The fact that this antigen was detected on only 1/10 breast 
carcinomas and 2/20 lung carcinomas indicates the importance of testing 
many cell lines of each tumor type in determining the distribution of an 
antigen. In frozen sections, MH94 was detected in secretory epithelial 
cells of many normal tissues. 
On tissue culture cells, MF116 was found on normal kidney cells as well as 
on some carcinomas of the ovary, uterus, kidney, bladder and on one 
neuroblastoma. The most frequent tumor type that was positive was renal 
carcinoma, for which 6/16 cell lines were positive. MF116 was not detected 
in sections of any normal tissue. The presence of MF116 on normal kidney 
cells in tissue culture and its absence from frozen sections of normal 
kidney cannot presently be explained, but might suggest that antigen 
expression is increased in rapidly proliferating cells. MF116 is secreted 
or shed into the medium by at least some tissue culture cells, and, in 
fact, is more readily detected by immunoprecipitation using spent medium 
than with solubilized cell extracts, thus serving as a basis for human 
cancer diagnosis in this system. 
Both MF116 and MF61 show patterns of distribution which seem to be related 
to the embryological origin of the tissues. Thus, these antigens were 
detected on tumor cell lines of the ovary, uterus, kidney and bladder but 
not on cell lines from lung, colon, breast and pancreatic tumors. The 
former tumors are all from mesoderm-derived epithelia whereas the latter 
are endodermal or ectodermal in origin. The presence of the antigens in 
frozen sections of fresh tumor specimens of various types is currently 
being examined with the immunoperoxidase procedure. It is believed that 
MF116, MF61 and MH94, but not MD144, are expressed on a proportion of 
ovarian carcinomas. 
The five antibodies described were selected from a large number of 
hybridoma antibodies produced to ovarian and endometrial cancer cell 
lines. Each mAb described is representative of a mAb class with similar 
characteristics. One problem in attempting to produce antibodies to 
restricted antigens of epithelial tumors is a tendency to produce many 
antibodies to common, strongly antigenic components. Another factor is 
that only a small fraction of ovarian carcinomas can be grown and 
maintained in tissue culture. This invention overcomes the problems. 
Removing strong antigens from a solubilized cell extract, by the use of 
immunoadsorbents, before immunization is also possible (Mattes, Hybridoma 
2:523, 1983). 
Diagnosis of cancer by the present invention comprises contacting a tissue 
containing ovarian and/or endometrial and/or cervical cells with the mAbs 
recognizing such cell antigens, preferably monoclonal antibodies to one or 
more cell antigens of the ovarian and/or endometrial and/or cervical 
antigenic system, and observing the immunoserological or 
immunopathological antigenic reaction between said monoclonal antibody and 
said antigen. In a preferred embodiment of the invention, the tissue 
sample or specimen or part thereof to be contacted is ovarian, cervical or 
endometrial tissue or cells or parts thereof and the antigenic reaction of 
the contacted tissue is observed by well known techniques such as 
immunofluorescence, ELISA, radioactive mAb, rosette formation with sheep 
or human red blood cells linked to Protein A or to anti-immunoglobulin, 
direct absorption and the like. In the case of shed antigens, body fluids 
and/or excretions or secretions can be tested in this manner. 
In another preferred embodiment of the invention unknown human Cell 
specimens are analyzed for mAb reaction with each member of the cell panel 
using cell sorters for flow cytometry. Thus, the number of cells reacting 
with fluorescent mAb can be counted. The other well-known observation 
techniques can be employed to count the number of cells expressing the mAb 
antigen. In another embodiment of the present invention, the tissue to be 
assayed is first excised and is then either freshly, or after being frozen 
or embedded in paraffin by methods well-known in the art, contacted with 
the monoclonal antibodies of the invention. Observation of the reaction is 
as before. 
In another preferred embodiment of the present invention, the tissue to be 
assayed comprises the intact body of an individual or whole portion 
thereof. The antibody, tagged with a radioactive or other energy-producing 
element, is administered to the individual, and the whole body or part 
thereof is scanned externally for localization of radioactivity at the 
site of cancerous cervical, endometrial or ovarian cells. 
The present invention also makes possible the treatment of ovarian, 
cervical, or endometrial tumors in a patient wherein the monoclonal 
antibody recognizing the cell antigen of cancerous ovarian or endometrial 
cells, preferably the cell differentiation antigen, is administered to the 
patient in an amount effective to inhibit the growth or proliferation of 
cancer cells. In a preferred embodiment of this method, the antibody is 
tagged with a potentially tissue destructive agent which causes 
destruction of the cancer cells. 
Examples of tissue destructive agents comprise chemotoxic agents, 
chemotherapeutic agents including vaccines, radionuclides, toxins, 
complement activators clotting activators and the like. 
The invention also enables tissue typing using mAb-tissue immune reactions. 
The above examples are for illustrative purposes only and are not meant to 
limit the scope of the invention. 
The hybridoma cell lines producing the monoclonal antibodies of the same 
designation described above (MF116, MH94, MD144, MH55, MF61, ME46 and 
ME195) are on deposit and available at Memorial Sloan-Kettering Institute 
for Cancer Research, 1275 York Ave., New York, N.Y. 10021. 
The hybridoma cell lines producing the monoclonal antibodies of the same 
designation as described above MF116, MH94, MD144, MH55 and MF61 have been 
deposited with the American Type Culture Collection (ATCC), 12301 Parklawn 
Drive, Rockville, Md. 20852 a recognized depository on Oct. 28, 1983 and 
have been given ATCC accession numbers of HB8409 for hybridoma cell line 
MD144 producing mAb MD144, HB8411 for hybridoma cell line MF116 producing 
mAb MF116, HB8412 for hybridoma cell line producing MH55 mAb MH55, HB8410 
for hybridoma cell line MF61 producing mAb MF61 and HB8413 for hybridoma 
cell line MH94 producing mAb MH94. 
The hybridoma cell lines producing the monoclonal antibodies of the same 
designation described above ME46 and ME195 have been deposited with the 
ATCC on Nov. 16, 1983 and have been given ATCC accession numbers of HB8431 
for hybridoma cell line ME195 producing mAb ME195 and HB8430 for hybridoma 
cell line ME46 producing mAb ME46.