CA1315220C - Preparation of monoclonal antibodies capable of reacting with human breast carcinoma cells - Google Patents

Abstract

The object of the present invention is the production of monoclonal antibodies (MCAs) that react with a high degree of selectivity to human breast cancer cells for a potential use in tumor diagnosis,prognosis and therapy.
MCAs are produced by fusing Ns-l myeloma cells to spleen cells from BALB/c mice hyperimmunized with the well-characterized human breast carcinoma cell line BT-20.
Hybridoma are selected on the basis of their preferential binding in an ELISA assay to human mammary tumor cells versus normal breast cells. Ammonium sulphate precipitation and ion-exchange chromatography allow the isolation of partially purified material that is further used as a possible tool to distinguish breast carcinoma cells from normal cells of the same histogenesis. According to the invention, three different techniques are used: ELISA, dot-immunobinding, and indirect immunofluorescence, to further characterize the antigen recognized by each of the MCAs produced.

Description

This invention relates to monoclonal antibodies capable of reacting with human carcinoma cells and to a process of producing these monoclonal antibodies. __ The advent of the hydridoma technology(l) for the - production of large amounts of high titered monoclonal anti-bodies ~MCAs) with a unique specificity for the antigen has greatly improved the possibility of probing the cell-surface antigenic complex of tumor cells and spurred efforts to generate tumor-specific antibodies. Investigators have used a variety of approaches for producing MCAs that recognize various antigenic structures on malignant and/or normal breast tissues.( 1 ) In the present specification, the Applicant describes the use of hybridoma technology to generate a panel of hybridoma-secreting monoclonal antibodies that react with a high degree of selectivity to human breast cancer cells.
The potential of these MCAs to probe surface protein differ-ences between normal and malignant breast cancer cells will also be decribed. BT-20 breast carcinoma cell line was used as a source of antigens because it was derived from a poorly differentiated tumor(l9) and has been well characterized and - -~ ~ extensively studied.(20 26) More importantly for the present invention, these cells were very tumorigenic when implanted in the nude athymic mice, producing nodules with the same mor-phologic characteristics as the original tumor.(21) These cells seem to have kept the properties of breast carcinoma.
In U.S. Patent No. 4,522,918 there are described monoclonal antibodies which demonstrate a reactivity with human breast cancer. However, these antibodies do not react with 100~ certainty with the result that tests that can be made with these antibodies cannot be totally reliable.
It is the object of the present invention to provide methods of production, selection and characterization of monoclonal antibodies capable of reacting with a large panel r of cultured human breast carcinoma cells~and biopsies.

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This invention relates to a process for producing monoclonal antibodies capable of reacting with human breast carcinoma cells, which comprises:
a) providing a breast cancer cell line of known aggressiveness;
b) culturing confluent monolayers of the breast cancer cell line in serum-free culture medlum, overnight;
c) harvesting the cultured cell line mechanically, such as with a rubber policeman and in the absence of trypsin;
d) immunizing mice with the harvested cell line obtained in (c);
- e) fusing spleenocytes of mice immunized as in (d) with myeloma cell line to give hybridoma cultures;
f) selecting hybridoma culture supernatants that contain antibodies reacting in an ELISA assay with BT-20 versus HsL-100 used here as a discriminatory cell line of . normal human breast origin; and g) isolating stable hybridoma producing monoclonal antibodies by cloning.
In accordance with a preferred embodiment of the invention, the breast cancer cell line has been selected from a group of breast cancer cell lines consisting of BT-20, BT-483, BT-549, CaMa, ZR-75-30, ZR-75-1, MCF-7, SKBR 3 and T-47D, the most preferred being BT-20.
- In accordance with another preferred embodiment step (d) is carried out by hyper immunizing sALB/c mice.
In accordance with another preferred embodiment of the invention, spleenocytes of mice are fused with the myeloma cell line, NS-l.
In accordance with yet another embodiment of the invention isolated stable hybridoma producing monoclonal antibodies are separated into different groups based on several criteria. The following grouping are evident monoclonal anti-bodies which:
: 1. recognize a protein moiety;

2. correspond to an antigen which is localized in the cytoplasm;

3. react with substantially 100% of breast cancer biopsies of primary and metastatic origin in cryostat sections, 4. do not react or react very weakly with normal breast tissue;
and/or 1. recognize a glycoprotein moiety, 2. correspond to an antigen which is localized at cell surface;
3. react with a limited number of primary and metastatic breast cancer biopsies; and , - 4. also recognize normal tissues of breast and non-breast origin.
The invention is illustrated by means of the -- , annexed drawings, in which:
i~ FIGURE l represents the SDS-PAGE electrophoresis of purified monoclonal antibodies;
FIGURE 2 represents curves showing the quantitative binding of lF10B4, lFlOG2 and 3C6F9 to BT-20 and HBL-100 cell lines; and FIGURE 3 is a photomicrograph of four immuno-fluorescence staining of acetone-fixed cultured cell lines and primary breast carcinomas.
MATERIAL AND METHODS
CULTURED HUMAN CELL LINES: Nine well established human ~ ~ry tumor cell lines (26 to 32) were maintained as -~

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continuous cultured and repeatedly tested to ensure the absence of any mycoplasma infection. Most of these cell lines were either purchased from the American Type Culture Collection or kindly obtained from EG & G Mason Research Institute (Rockville, Maryland). Characteristics of each of these cell lines is given in Table 1. Additional cell lines used included cultures of apparently normal m~mm~ry cells, HBL-100, an epitheiial cell line derived from human milk sample (33), and two human fetal foreskin cells lines, SW-480 and CC-95. All cell lines were routinely maintained in RPMI-1640 medium (Grand Island Biological Co.; Burlingtony Ontario, Canada) supplemented with 0.2 mM L-glutamine, 20 mM hepes buffer, lOYo heat-inactivated fetal calf serum and antibiotics (penicillin 100 IU/ml, streptomycin 10 ~ug/ml and fungizone 10 ,ug/ml). Single cell suspensions of each monolayer culture were obtained by treatment with trypsin-EDTA in Hank's balanced salt solution.

Cells were maintained at 37C in a humidified atmosphere of 95% air and 5% CO2. Tests for mycoplasma contamination - using agar culture were performed and only negative cell lines were further used.
HYBRIDOMA METHODOLOGY:
(a) Hyperimmunization and Fusion: Female BALB/c mice were hyperimmunized by 3 subcutaneous injections of 10 BT-20 cells emulsified in complete Freund's adjuvant. For all the immunizations BT-20 cells were cultured in serum-fr-ee RPMI
medium for 24 hours before harvesting and were detached from the culture vessel by means of a rubber policeman. Rise in serum antibody titers in immunized mice was monitored weekly and animals showing high response to BT-20 by the ELISA
(Enzyme-Linked Immunosorbant Assay) technlque were selected ; for fusion. At least 1 month after the last immunization, .

1 ~ 1 5220 mice were boosted i.p. with 107 BT-20 cells. Three days later spleenocytes were fused with the non-immunoglobulin producing myeloma cell line NS-l (P3-NSl/l-Ag4-1) (34), using 50% polyethylene glycol as fusing agent.
(b) Antibody-screening assay: As already mentioned an ELISA technique was used for antibody screening in either culture supernatants, purified ascitic fluid or in sera of immunized mice. In preparation for this test, cells (BT-20 and HBL-100) were grown until confluence in sterile tissue culture multiwell plates (96 flat bottom wells, Linbro) using complete RPMI medium. Confluent monolayers were then incubated overnight at 37C in serum-free RPMI-medium and fixed with a solution of 0.1% glutaraldehyde in PBS (5 min.
at room temperature). This was followed by 3 washes in PBS
pH 7.4 and blocking of non-specific sites by an incubation at - 37C (200 yl/well) with a blocking solution. This solution TM
contained 20 ml of citrated milk (100 g of Crino milk powder ~ with 12.3 g of citric acid), 1 g of polyvinyl-pyrolidone (PVP-- ~ 40) and O.S g of BSA ~bovine serum albumin). To this was added 75 ml of Tris-HCl buffer, the volume was completed to 100 ml and the pH adjusted to 7.5. The blocking solution was always filtered on nylon membrane paper before its use.

After blocking the non-specific sites, cells were rinced twice in PBS then 100 ~ul of antibodies (at different dilutions) were added to each well and incubated at 37C for 90 min. Cells were then rinced 3 times in PBS and 100 ~1 of f __ horseradish peroxidase-labeled goat anti-mouse IgG (Miles ~~~ Lab.) diluted 1/75 in dilution buffer (0.5 g of PVP-40 and 0.5 g of BSA, pH 7.5) was added and incubated for 90 min. at 37C. ~ ;

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After 3 washes in PBS, lOO,ul of the substrate (2,2' azinodi-(3-ethylbenzthiazoline sulfonate and oxygen peroxide, v:v) was added and incubated for 10 min at 37C and for 20 min at room temperature, in the dark. The reaction was stopped by the addition of 50 ~uliwell of a solution of lOYo sodium dodecyl-sulfate. The optical density was read in a spectrophotometer at 405 nm wavelength (Titerteck Multiskan photometer, Flow Lab.).
(c) Selection and cloninq of hybridomas: Following fusion with ~S-l cells approximately 96% (311) of the seeded wells showed rapid growth within two weeks. Only 128 (40%) secreted antibodies that bound to BT-20 cells in the ELISA
assay, of these 21 (16%) reacted weakly or failed to react with the discriminatory cell line B L-100. After 3 to 5 primary screens, hybridoma cultured were cloned and recloned twice using the limiting dilution technique in the presence of irradiated 3T3 mouse fibroblasts as a feeder layer. Six of these clones were further characterized (Table 2) and injected into pristane-primed BALB/c mice (2,6,10,14-tetramethyl-pentadecane, 96%) for the production of ascitic fluid.
(d) Characterization and purification of M~A~: The heavy and light chain composition of each MCAs was determined using Ouchterlony gels first against affinity-purified goat-antiserum specific for each of the murine subclass (IgGl, IgG2a, IgG2b, IgG3, IgM and IgA) and then to monos~ecific goat-antimouse immunoglobulin isotypes (Cappel Lab., Cochranville, PA). Quantitation of MCAs in culture super-natants of the cloned hybridomas was performed by a standard radioimmunoassay using goat anti-mouse IgG (Miles Lab.).
~ 30 Mouse ascitic fluids were clarified by centrifugation (2000 -- rpm for 30 min at 4C) and the immunoglobulin fraction purified ;

1 3 t 5220 by three sequential Am~onium sulfate precipitation. In this technique, the ~onium sulfate (50/O) solution was added drop by drop to the ascitic fluid and the mixture kept under constant agitation. The mixture was then transferred to 50 ~1 conical tubes and placed for at least 16 hours at 4C. The precipitate was recovered by centrifugation for 20 min at 4C.
This procedure was repeated twice with the precipitate, but in the second and third precipitation incubation was for only - 2 hours at 4C. The final precipitate was dissolved in a O.Ol M Carbonate buffer pH 8.3 and dialysed for 16 hours at , 4C against the same buffer~ After dialysis the precipi-tate was diluted in Carbonate buffer so as to dissolve completely the precipitate. The solution was filtered first on a Watman no 1 (Canlab) filter paper then on a millipore membrane 0.2 ~m pores under vacuum (filter unit, type LS
Nalgene, 0.2 ym, Nalge), aliquoted into sterile tubes (1 mg/ml) and fro~en at -70C until use. The purity of each MCAs was confirmed by SDS-PAGE electrophoresis (Fig. 1) under reduced conditions (2-mercaptoethanol). Lanes 1 and 2 show lFlOB4 and lFlOG2(yl) while lane 3 is 3C6F9(y2a).
IMMUNOFLUORESCENCE STUDIES ON CULTURED CELL LINES:
In preparation for indirect immunofluorescence, freshly trypsinized cells obtained during the logarithymic growth phase of a culture, were seeded onto pretreated tissue culture slides and incubated overnight in complete medium. Cells - were first washed, then either fixed with cold acetone at 4C
for 10 min, or immiediately stored at -70C until used. For these studies, slides were incubated with hybridoma super-natants for 20 min at room temperature, then for 30 min with fluoresceinated goat anti-mouse Ig (Cappel Lab., Cockranville, PA) diluted 1/30 in PBS. After two washes for 10 min in _ 7 _ PBS, slides were mounted with 50% glycerol in PBS and were examined with an epifluorescent microscope. The appropriate working dilutions of these three MCAs, as tested on acetone-fixed BT-20 and HBL-100 cells, were established at 1/50 for lFlOB4 and lFlOG2 (20 ~g/ml PBS) and 1/20 for 3C6F9 (50 ~g/ml).
These dilutions were selected because they gave a strong reactivity on BT-20 cells in the presence of a clear back-ground.
IMMUNOHISTOCHEMICAL REACTIVITY ON HUMAN TISSUES:
Specimens obtained either directly from the operating room or from fresh autopsies were snap frozen in liquid nitrogen and transferred at -70C until used. The specimens included, (a) 30 primary breast tumors, (b) 7 metastatic m~m~ry cancers (5 lymph nodes and 2 liver metastasis), (c) 7 benign breast tissues including 5 normal m~mm~ry tissues, one fibrodenoma and one sclerosing adenosis; and (d) 6 normal tissues of non breast origin. Acetone-fixed cryostat sections of each specimen were studied using an indirect immunofluorescence staining. Tissue sections were incubated for 30 min at room temperature with 250,ul of the diluted MCAs (same concentrations as determined on BT-20 cells); after extensive washing with PBS, they were incubated for 30 min with 100 ~ul of fluoresceinated rabbit anti-mouse IgG (Cappel Lab., Cockranville, PA) diluted 1/30 in Pss. Fixed sT-20 and HBL-100 monolayers were always used as control slides.
These sections were examined with an epi-illumination fluorescent microscope. In all instances, an adjacent section of the frozen tissue was stained with hematoxylin-eosin and microscopic findings were correlated both with theimmunofluorescence studies and the pathoIogical diagnosis ~, ,,, performed on paraffin-embedded sections of these cases.
RESULTS
- QUANTITATIVE COMPARISON OF ANTIBODY REACTIVITY ON
BT-20 AND HBL-100: Isotype analysis of these MCAs showed that antibodies lFlOB4 and lFlOG2 were both of the yl subclass, while 3C6F9 represented a y2a immunoglobulin (Table 2). All three MCAs produced high titers of IgG in hybridoma super-natants fluid. To reach a more quantitative comparison of their affinity to the target antigens, purified ascitic fluid 10 were used at a concentration of 1 mg/ml of proteins and the effect of antibody dilution on the differential reactivity :
to BT-20 versus HBL-100 was determined (Fig. 2). Using an ELISA assay, we could demonstrate two different patterns of reactivity; lFlOB4 and lFlOG2 showing a high degree of selectivity bound strongly to BT-20 (up to 10 2 dilution for lFlOB4 and 10 3 dilution for lFlOG2 and weakly (10 1 dilution) or not at all with HBL-100 (Fig. 2). On the other hand at low dilutions, 3C6F9 reacted with both HBL-100 and BT-20, however, at 10 3 dilution reactivity was still very strong with BT-20 - 20 and was completely lost with HBL-100. We could also note that even at 10 dilution 3C6F9 reacted very strongly with BT-20 (Fig. 2). These results were further confirmed on acetone-fixed cell monolayers by a standard avidin-biotin peroxydase technique using commercially available reagents (Vectastain, Trade Mark of Cederlane Lab., Ontario Canada) and the three - MCAs at the same dilutions.

.

IMMUNOFLUORESCENCE STUDIES ON CULTURED NORMAL AND
- MALIGNANT CELL LINES. With reference to Fig. 3, there is illustrated the immunofluorescence staining of acetone-fixed cultured cell lines and primary breast carcinomas (a) HBL-100 incubated with 3C6F9, (b) BT-20 cells incubated with lFlOG2, (c) primary c-arcinoma incubated with 3C6F9 and (d) primary breast carcinoma incubated with lFlOG2. The different pattern of reactivity demonstrated by these two MCAs will be noted;
IFlOG2 shows a granular cytoplasmic reactivity often associated with a perimuclear accentuation of staining while 3C6F9 shows a fine linear positivity at the periphery of the cells. The enlargement for (a), (b), (c) and (d) is as follows: (a), (b) and (d) x 1000, c x 400. To localize the antigenic structures defined by lFlOB4, lFlOG2 and 3C6F9, indirect immunocyto-~ chemical staining of acetone-fixed and unfixed m~mm~ry ~~ epithelial cells was performed (Table 3). For both lFlOB4 and lFlOG2, a strong cytoplasmic staining was observed on acetone-fixed BT-20 cells, while unfixed preparations of the same breast cancer cell line were negative. The appearance of the cytoplasmic staining was coarsely granular and was uniformly distributed throughout the cytoplasm with some accentuation around the perinuclear membrane (Fig. 3b). A
similar pattern was noted for the nine human breast cancer cell lines studied. However, as illustrated in Table 3, the intensity of the staining varied from one cell line to another and there was no correlation between the intensity of the fluorescence staining and any of the different ,, ,~
characteristics of these cell lines i.e. tumor origin, tumorigenicity in aminals, hormonal receptors content and growth in soft agar (Table 1). In contrast to the positivity of malignant breast cancer cell lines, lFlOB4 and lFlOG2 showed no staining with the three normal cell lines examined (Table 3).
Results obtained with 3C6F9 were substantially different: this monoclonal antibody recognized both acetone-fixed and unfixed preparations of normal and malignant cell lines. A positive reaction was detected with all the cell lines examined (Table 3) regardless of their origin, the intensity of the reaction varied however, from one cell line to another and was weaker with the normal cell lines. This fluorescence was localized, in all instances, at the periphery of the cells and was uniformly distributed among the cells of a given preparation (Fig. 3a). These data indicate that the antigenic determinant recognized by 3C6F9 is associated with the plasma membrane of the cells and is expressed on both normal and malignant cultured epithelial cell lines.
REACTIVITY WITH HUMAN MAMMARY TUMORS: To determine if antigens recognized by these 3 MCAs are also present in breast tumors of human origin, tissue sections of mammary tumors were stained by indirect immunofluorescence. Two groups of m~mm~ry tumors were examined, the first consisted of 30 primary infiltrating ductal or lobular carcinomas, the second included 7 metastatic breast cancers (lymph nodes or liver metastases). As reported in Table 4, l.F10B4 and lFlOG2 exhibited the capacity to react with a wide spectrum of breast carcinomas of primary and metastatic origin. lFlOs4 recognized 97% of the primary tumors tested while lFlOG2 recognized 100%
of both infiltrating ductal or lobular m~mm~ry carcinomas.
Similar results were obtained for metastatic breast tumors (Table 4) i.e.; whatever the site of the metastasis, both MCAs ~ ~;
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recognized lO~/o of all the metastatic breast cancers examined. In 67% ( 20 out of 30) of the primary tumors studied, a clearly cytoplasmic pattern of reactivity could be identified (Fig. 3d). This type of staining was identical to that seen with m~mm~ry epithelial cell lines (~ig. 3b). In the r~m~in;ng tumors (10 out of 30) the reactivity was mainly localized at the periphery of the cells usually in a homo-geneous, discontinuous pattern.
On the other hand, 3C6F9 reacted strongly with 2~/o of the primary breast cancers examined and weakly with 4~/0 of them, illustrating a more limited spectrum of recognition of primary brea~t cancers. This feature was further accen-tuated when metastatic tumors were studied, only 4~/0 of these metastatic tumors stained weakly with 3C6F9 (Table 4). In all the positive cases, a linear reactivity localized at the cell .
membrane was observed with 3C6F9 (Fig. 3c). This pattern of staining was also similar to that noted on cultured m~ ry epithelial cell lines (Fig. 3a).
- REACTIVITY ON NORMAL BREAST AND NON BREAST TISSUES:
The reactivity of these 3 MCAs was also evaluated on cryostat sections of 7 benign breast tissues ( 5 normal - - ry tissues, one fibroadenoma and one sclerosing adenosis), and 6 normal - . tissues of non breast origin. With lFlOB4 and lFlOG2, immunofluorescence was negative in glanaular structures in - -ry lobules but few focal reactivities were sometimes found in ducts. In these areas a weak staining of the luminal pole of the epithelial cells lining the ducts was observed.
With 3C6F9 however, a peripherally located reactivity of variable intensity was noted in mammarY glands and ducts.
On normal tissues of other origin, lFlOB4 and lFlOG2 showed by fluorescence a very weak reactivity-in some ~l 2_- , r~~

renal tubular epithelial cells, in few keratinocytes and epithelial cells of sebaceous glands ln the skin and a moderate staining in biliary ducts of the liver. All mesenchymal structures, including smooth and striated muscle tissues, lymph nodes and connective tissues were negative. These data suggest that the antigenic detel ;n~nts recognized by lFlOB4 and lFlOG2 are not restricted to malignant - -ry cells but are probably present in very small amounts in few normal epithelial cells of other tissues. On the other hand, with 3C6F9 a strong reactivity was detected on all medial smooth muscle cells in blood vessels, whereas a moderate reactivity was found in biliary ducts. Moreover, a weak positivity was found focally on the basement membrane of the skin and kidneys specimens studies. Therefore, 3C6F9 recognizes an epitope that is present in the cell membrane of a large spectrum of normal epithelial cells of different origin.
An overall ~x ination of the data presented herein suggests that MCAs described can be divided into two different groups on the basis of their differential reactivities to human tumor specimens and cultured cell lines of either malignant or normal origin. Two of the MCAs namely lFlOB4 and lFlOG2, showing a wide spectrum of reactivity to human breast carcinomas, recognize cytoplasmic structures in primary and metastatic - -ry tumors but fail to react with normal cultured cell lines and react weakly or locally with biopsies of benign breast tumors and normal tissues of breast and non breast origin. On the other hand, 3C6F9 recognizes a surface membrane det~ inAnt expressed on both normal and neoplastic mammary epithelial cells as well as on medial smooth muscle cells in blood vessels.

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- ~ It is interesting to note that all three MCAs reacted with - ry tumor cell lines without discrimination.
On the other hand, while lFlOB4 and lFlOG2 show (even at low dilutions) little reactivity to ~BL-100, 3C6F9 demonstrate a comparatively stronger reactivity to the three normal cell lines studied (Table 3).
Cells in culture may change their behavior and become unrepresentative of their in vivo counterparts in expression of certain tumor-associated antigens, particularly those associated with differentiation. In order to have a more valid assessment of the antigens recognized by those three MCAs, their binding to frozen sections of mammary tumors has been examined using indirect immunofluorescence techniques.
~~ It could be demonstrated that 3C6F9 react with 630/o (19 out of 30) of the primary breast cancers examined and with only 43/0 (3 out of 7) of the metastatic breast tumors. This monoclonal antibody identifies an epitope localized at the periphery of the cells and, as revealed by examination of unfixed and acetone-fixed cultured cell lines, the antigenic determinant seems to be associated with the plasma membrane of both normal and malignant cells.
On the other hand, lFlOB4 and lFlOG2 recognize a wide spectrum of m~mm~ry tumors staining almost all of the 30 primary and 7 metastatic breast cancers examined, except for one primary tumor which was negative with lFlOB4. These two MCAs recognize structures localized in the cytoplasm of these neoplastic cells, they do not recognize (or very weakly) epithelial structures of normal breast tissues (7 biopsies) and non m~mm~ry tissues (6 biopsies).

This wide range of reactivity of lFlOB4 and lFlOG2 for carcinoma of the breast, their different pattern of Z@ .~

staining within a single tumor, between different tumors of the same histological type and between the various types is very interesting. The fact that they recognize cytoplasmic determinants, which are usually better preserved in conven-tional formalin-fixed paraffin-embedded tissues, suggested, that these two MCAs may have diagnostic, prognostic and therapeutic applications. Thus MCAs used singly or in combination could be used in:
1. Radioimmunoassay for the development of diagnostic kits for the detection of cir-culating antigens, aiding in the diagnosis, follow up and the prognosis of breast cancer patients.
2. In vivo localization of micrometastasis at the draining lymph nodes or at distant location (bone, lung, brain) when labeled with radioactive isotopes.
3. Treatment of primary and/or metastatic breast lesions using MCAs either alone or coupled to toxins, drugs or radioactive isotopes.

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l6 INITIAL SCREENING AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES
IN THE SUPERNATANTS OF CLONED HYBRIDOMAS

Antibody Immunoglobulin ELISAC
Designation Class and Concentration BT-20 HBL-100 subclass (ug/ml) 3C6F9 IgG2ak 81.2 +++ +
3C6H6 IgGlk 88.0 + o 3C6D7 IgGlk 19.6 + o lFlOB4 IgGlk 126.0 ++ 0 ~1 lFlOHl IgGlk 108.9 +++ +
lFlOG2 IgGlk 135.7 ++ +

__ a. Initial screenings and characterization of cloned hybridomas was performed using culture supernatant diluted 1:10 in PBS. r~
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c. Optical density (405 nm) was graded a~ follows: (O) = 0.2 or lower, (+) = 0.4 - 02: (++) = 0.6 - 0.4, and (+++) = 0.8 or higher.

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Claims (4)

1. Two monoclonal antibodies which:
a. specifically bind with a 250 Kd molecular weight cytoplasmic antigen of BT-20 breast carcinoma cells;
b. are of the IgG1 isotype;
c. specifically bind to 100% of 9 acetone-fixed breast cancer lines tested;
d. do not bind to acetone-fixed HBL-100, a normal breast epithelial cell line tested;
e. do not bind to 2 acetone-fixed normal colon cell lines tested;
f. one specifically binds to approximately 97%
(IF10-G2) and the other to 100% (IF10-B4) of cryostat sections of 30 primary breast cancer biopsies tested;
g. specifically bind to approximately 100% of cryostat section of the 7 metastatic biopsies of breast cancer lesions tested.

2. A monoclonal antibody according to claim 1, wherein said antibody is selected from the group consisting of: IF10-B4, IF10-G2.

3. A monoclonal antibody which:
a. specifically binds with a 39 Kd molecular weight membrane glycoprotein antigen of BT-20 breast carcinoma cells;

b. is of the IgG2a isotype;
c. specifically binds to 100% of 9 acetone-fixed and non-fixed breast cancer lines tested;

d. specifically binds to acetone-fixed HBL-100, a normal breast epithelial cell line tested;
e. specifically binds to 100% of 2 acetone-fixed and non-fixed normal colon cell lines tested;
f. specifically binds to 100% of 8 biopsies from resting normal breast tissues tested;
g. specifically binds to approximately 66% (3 fibroadenoma) to 87.5% (8 fibrocystic diseases) of cryostat sections of the 11 benign mammary lesions tested;
h. specifically binds to approximately 72% of cryostat sections of 39 breast cancer biopsies tested and 67% of the metastatic lesions of breast cancer biopsies tested;
i. does not bind with most of 28 biopsies from normal extra-mammary tissues tested with the exception for the weak staining of glandular epithelium of the esophagus and larynx, alveolar wall cells of lung and uterine myometrium and strong staining of the smooth muscle cells and basement membrane lining of all of the epithelia tested;
j. does not bind to most of 34 biopsies of the extra-mammary neoplasia tested with the exception for the weak staining of ovarian (3 biopsies), pancreatic (1 biopsy) kidney (1 biopsy) and appendix (1 biopsy) and strong staining of hepatic (4 biopsies) carcinoma tested.

4. A monoclonal antibody according to claim 3, wherein said antibody is 3C6-F9.