Monoclonal antibodies demonstrating a reactivity with human breast
cancer are produced. The hybridoma cultures secreting immunoglobins
are produced by hydridoma technology. Splenic lymphocytes of mice,
immunized with membrane-enriched fractions of metastatic human mammary
carcinoma tissue are fused with the NS-1 non-immunoglobulin-secreting
murine myeloma cell line. Screening of immunoglobulin reactivities
and double cloning of cultures yielded 11 monoclonal antibodies
that demonstrated activities with the surface of human mammary tumor
cells and not with the surface of apparently normal human tissues.
These monoclonal antibodies aid in the diagnosis, prognosis and
treatment of human breast cancer.
1. Antibodies from hybridoma cultures produced by the steps of
(1) taking splenic lymphocytes of mice previously immunized with
membrane-enriched fractions of immunoglobulin-depleted cancer cells;
(2) fusing the lymphocytes with a myeloma cell line, and
(3) culturing the hybridoma cell line in an in vitro culture medium
or in vivo therefor to produce antibodies, which antibodies are
selected from one member of the group consisting B6.2, B14.2, B39.1,
F64.5,B25.2, B84.1, B38.1, B50.4 and B50.1 and which
(a) react and bind with extracts from human metastatic mammary
carcinoma cells from involved livers but not with liver cell extracts;
(b) react and bind with at least one of the mammary carcinoma cell
lines, BT-20, MCF-7, ZR-75-1, but not with lung, vulva epidermoid
or oral epidermoid and not with rhabdomyosarcoma, fibrosarcoma and
(c) do not react with normal cell derived from breast, skin, lung,
bone marrow, kidney, spleen and uterus.
2. Antibodies selected from one member of the group consisting
of B6.2, B25.2, B72.3, F25.2, B38.1, and B50.4.
3. Antibodies selected from one member of the group consisting
of B6.2, B25.2, B38.1, F25.2, B72.3 and B50.4, which bind human
mammary carcinoma lines designated BT-20, MCF-7 and ZR-75-1.
4. A mouse myeloma cell line selected from the group consisting
of ATCC #HB8106, #HB8107, #HB8108, #HB8109, #HB8110, and #HB8111.
5. A method of detecting a small number of mammary cancer cells
in micro-lesions in thin tissue sections or body fluids by applying
monoclonal antibodies selected from the group consisting of monoclonal
antibodies designated B6.2, B25.2, B72.3, F25.2, B38.1, and B50.4
to the tissue, adding quantity of goat (anti-mouse Ig) antibody
conjugated with peroxidase and fixing with diaminobenzidene and
peroxide and staining with hemotoxylin, and after fixing, examining
for reddish brown colored cells indicative of mammary cancer cells.
6. In an assay to detect mammary carcinoma cells involving contacting
a sample with an antibody under conditions which allow the formation
of an antibody antigen complex and measurement of the complex formation,
the improvement comprises using monoclonal antibodies selected from
the group consisting of B6.2, B25.2, B72.3, F25.2, B38.1, and B50.4.
PRIOR ART STATEMENT
Hybridoma technology was developed by Kohler and Milstein [Kohler,
G. and Milstein, C. (1975) Nature (London), 256, 494-497].
The technique is also set out in detail in the following excerpt
from a basic immunology textbook, Herzenberg and Milstein, Handbook
of Exerimental Immunology, ed. Weir (Blackwell Scientific, London),
1979, pp. 25.1-25.7.
Assaying for antibody in solid-phase RIA is described by the inventor
in Colcher, Horan Hand, Teramoto, Wunderlich, and Schlom, Cancer
Research, 41, 1451-1459 (1981).
Background art by the inventors is in Colcher, Horan Hand, Nuti,
and Schlom, J. Proc. Natl. Acad. Sci., May 1981, Vol. 78, No. 5,
Patents relating to other tumors produced by hybridoma technology
are U.S. Pat. Nos. 4,172,124 and 4,196,265.
These eleven monoclonal antibodies aid in the diagnosis, prognosis,
and treatment of human breast cancer. These antibodies are activated
only by tumor cells from human mammary cells and not by apparently
normal human tissues.
STATEMENT OF DEPOSIT
The following hybridoma cell lines were deposited in the ATCC with
the following accession numbers: B6.2, ATCC #HB8106; B25.2, #HB8107;
B72.3, #HB8108; F25.2, #HB8109; B38.1, #HB8110; and B50.4, #HB8111.
SUMMARY OF THE INVENTION
Mice were immunized with membrane-enriched fractions of human metastatic
mammary carcinoma cells from either of two involved livers (designated
Met 1 and Met 2). Spleens of immunized mice were fused with NS-1
myeloma cells to generate 4250 primary hybridoma cultures. Supernatant
fluids from these cultures were screened in solid phase RIAs for
the presence of immunoglobulin reactive with extracts of metastatic
mammary tumor cells from involved livers and not reactive with similar
extracts of apparently normal liver. Whereas many cultures demonstrated
immunoglobulin reactive with all test antigens, 370 cultures contained
immunoglobulin reactive only with the metastatic carcinoma cell
extracts. Following passage and double cloning of these cultures
by endpoint dilution, the monoclonal immunoglobulins from eleven
hybridoma cell lines were chosen for further study. The isotypes
of all eleven antibodies were determined; ten were IgG of various
subclasses and one was an IgM.
DETAILS OF THE INVENTION
FIG. 1 shows tissue sections and the detection of binding of monoclonal
antibodies to human mammary adenocarcinoma cells using the immunoperoxidose
MATERIALS AND METHODS
Cell Extracts. Cell extracts were prepared from breast tumor metastases
to the liver from two patients as well as from apparently normal
liver. Tissues were minced finely and homogenized for 2 to 3 min.
at 4.degree. C. in 10 mM Tris-HCl, (pH 7.2), 0.2 mM CaCl.sub.2 (10
gm/100 ml). The homogenate was then subjected to nitrogen cavitation
using a cell disruption bomb (Parr Instrument Co., Moline, IL) for
5 min. at 1000 lg/in.sup.2 and then clarified at 1000.times.g for
5 min. Cell membrane enriched fractions were prepared from this
extract by centrifugation of the 1000.times.g supernatant in a discontinuous
20%/40% (w/w/, 10 ml of each) sucrose gradient in 10 mM Tris-HCl,
(pH 7.2), containing 2 mM CaCl.sub.2 for 16 hr at 130,000.times.g.
The material obtained from the 20-40% interface was diluted and
pelleted at 130,000.times.g for 60 min (SW27 rotor, Beckman). The
pellet was resuspended in PBS buffer and sonicated at 4.degree.
C. for 1 min. at 15 sec. intervals (Branson Sonifier, setting 7).
The sonicate was centrifuged at 10,000.times.g for 10 min. and the
supernatant was used for immunizations. The balance of the cell
extract was repeatedly passed through columns of Protein A-Sepharose
(Pharmacie) to remove cell associated Ig that would interfere with
the solid phase radioimmunoassays. Protein concentrations were determined
by the Lowry method (Lowry, O. H., et al, J. Biol. Chem., 193, 265-275,
Immunizations. Four-week old C57BL/6, BABL/c and C3HfC57BL mice
were immunized by intraperitoneal inoculation with 100 .mu.g of
membrane-enriched fractions of either of two breast tumor metastases
to the liver (termed Met 1 and Met 2) mixed with an equal volume
of complete Freund's adjuvant. Fourteen and 28 days later, mice
were boosted with an intraperitoneal inoculation of 100 .mu.g of
the immunogen mixed with incomplete Freund's adjuvant. After an
additional 14 days, 10 .mu.g immunogen was administered intravenously.
Spleens were removed for cell fusion 3 to 4 days later. NS-1 Cells.
The preferred embodiment of this procedure requires HL clones. H
and L are the heavy and light chains contributed by the antibody
producing parental cell. In the past, HL clones were derived from
MOPC-21 through the X63-Ag8 myeloma cell line (which secretes an
IgG1 myeloma protein). X63-Ag8 also secretes heavy and light chains
of G and K. Thus, the hybrid produces a variety of tetrameric molecules
with different H, L, G, and K combinations. Only some of these molecules
bind to the antigen and even fewer are detectable by a complement
Fortunately, it has not been difficult to obtain variants of the
selected hybrids which have lost the ability to make G and K. By
subcloning HLGK lines, HLK clones can be detected and by subcloning
these, HL variant clones can be found. The frequency of variants
differs from clone to clone but is often in the order of 1-3 percent
for each step.
Screening for HL clones can be done in favorable cases simply by
haemolytic or cytolytic activity assays, since HL and HLK have greater
activity than HLGK. A more general method of screening for HL clones
is chain analysis of secreted immunoglobulins (Ig) using acrylamide
gels. A convenient method is to intrinsically label the secreted
Ig with .sup.14 C-lysine (300 .mu.c/.mu.mol/l) in microculture wells
are cultured. The cells are centrifuged and the supernatants saved
for direct analysis by isoelectric focusing (IEF) or electrophoresis
followed by autoradiography after fixing and washing. The focusing
positions in IEF analysis for G and K are known and H and L can
often be identified as bands with different isoelectric points.
This process is complex and time consuming.
To simplify and speed up production of HL clones, rather than using
the MOPC-21 variant producing HLGK, a MOPC-21 variant called NS-1
is used. NS-1 does not make G; its initial hybrids are HLK, thus
obviating the above procedure as it relates to G.
Hybridoma Methodology. Somatic cell hybrids were prepared using
the method of Herzenberg with some modifications. Single cell suspensions
of spleen cells from immunized mice were made by passing the tissue
through a No. 3 mesh stainless steel screen (B. Fenenco Co., Inc.,
Norcester, Mass.). Spleen cells and NS-1 cells were washed in RPMI-1640,
containing 2 mM glutamine, 1 mM sodium pyruvate, 50 units/ml penicillin,
50 .mu.g/ml streptomycin, mixed at a 4:1 ratio, and fused with 50%
polyethylene glycol (M.W. 1500) (BDH Chemical Ltd., Poole, England).
After fusion, individual wells of 96-well microtiter plates (Costar,
Cambridge, Mass.) were seeded with 1.times.10.sup.6 total cells
(0.1 ml) of the cell suspension. Fused cells were then selected
for growth using RPMI medium as described above containing 15% heat-inactivated
fetal calf serum.
Cloning of hybridoma cell lines was performed by limiting dilution.
Twenty-four wells of a 96-well microtiter plate (Costar, Cambridge,
Mass.) were seeded with one of the following concentrations of hybridoma
cells: 10 cells/well, 5 cells/well, 1 cell/well, or 0.5 cell/well.
Mouse thymocytes, derived from the thymus glands of four-week old
C57BL/6 mice, were added to each well as feeder cells at a concentration
of 10.sup.6 cells/well. Wells seeded at the concentration that eventually
resulted in growth of cells in one out of two wells and containing
a single colony of hybridoma cells were chosen for further propagation.
All hybridoma cell lines were cloned twice.
Solid Phase Radioimmunoassays. All hybridoma supernatant fluids
were assayed for antibody in solid phase RIA usng cell extracts
from both breast metastases and normal liver. Fifty microliters
of the cell extracts (5 .mu.g) was added to each well of 96-well
microtiter polyvinyl plates and allowed to dry. To minimize non-speclfic
protein absorption, microtiter wells were treated with 100 .mu.l
of 5% bovine serum albumin (BSA) in phosphate buffered saline containing
calcium and magnesium (PBS) and incubated for 1 hr. This and all
subsequent incubations were at 37.degree. C. The BSA was removed
and 50 .mu.l of hybridoma supernatant fluid was added. After a 1
hr incubation, the unbound Ig was removed by washing the plates
with 1% BSA in PBS. The wells were incubated with 50 .mu.l of a
1:1000 dilution of o rabbit anti-mouse IgG F(ab').sub.2 in PBS containing
1% BSA. After 1 hr, the wells were washed three times with 1% BSA
in PBS and 50,000 cpm of [ .sup.125 I]-labeled Protein A (IPA) in
25 .mu.l was added to each well. The plates were incubated for an
additional hour and the unbound IPA was removed by extensive washing
with 1% BSA in PBS. The plates were then subjected to autoradiography
using Kodak XR film and Dupont Lighting-Plus intensifying screens.
The films were developed after 16 hr at -70.degree. C. The bound
IPA was also detected by cutting the individual wells from the plate
and measuring the radioactivity in a gamma counter.
Immunoassays for Antibodies to CEA and Ferritin. Solid phase radioimmunoassays
for the detection of antibodies to carcinoembryonic antigen (CEA)
and ferritin were run as described above with minor modifications.
CEA and a mouse monoclonal antibody to CEA was provided by Dr. Hans
Hansen (Hoffman-LaRoche, Inc., Nutley, N.J.). Thirty nanograms of
CEA was applied to each well of the microtiter plate and allowed
to dry overnight. The monoclonal antibody to CEA was used as a control
with approximately 5000 cpm bound using 1.5 ng of antibody. The
assay for ferritin was run using 2 ng of ferritin and rabbit anti-ferritin
(both obtained from Calbiochem-Behring, LaJolla, Calif.). The assay
was run as described above, but no second antibody was used on the
rabbit anti-ferritin which bound approximately 3500 cpm of IPA at
a 1:1000 dilution. A monoclonal antibody to a determinant common
to all human HLA, A, B, and C antigens (W6/32) was purchased from
Accurate Chemical and Scientific Corp. (Hicksville, N.Y.).
Cells. The BALB/c myeloma cell line, P3-NS1-1-Ag4-1 [NS-1 (see
above)], was obtained from Dr. K. J. Kim, NIH, Bethesda, Md. All
human cells lines used were obtained from either the American Type
Culture Collection, the Breast Cancer Task Force Cell Bank (NCI),
the Naval Biosciences Laboratory (Oakland, Calif.), or the Cell
Repository of Meloy Laboratories (Springfield, Va.). Cell cultures
were maintained as recommended by each laboratory, respectively.
Cell lines were tested for species of origin by isoenzyme analyses,
karyotyping, and immunofluorescence analyses and were demonstrated
to be of proper species identity. These tests were performed by
Dr. W. Peterson, Child Research Center of Michigan, via the Biological
Carcinogenesis Branch. Cell lines were tested for the presence of
Mycoplasma species and were negative.
Live Cell Immunoassay. Subconfluent established cell lines were
detached from 75 cm.sup.2 tissue culture flasks using 0.1% trypsin
containing 0.5 mM EDTA (ethylene diamine tetraacetic acid). Cells
were then seeded into 96-well, flat bottom tissue culture plates
(Limbro Scientific, Inc., Hamden, Conn.), using the appropriate
growth medium at 5.times.10.sup.4 cells per well and incubated at
37.degree. C. for 18-24 hrs in a humidified incubator containing
5% CO.sub.2. (All additional incubations were performed using identical
conditions). The growth medium was then aspirated and 100 .mu.l
of RPMI-1640 containing 10% (w/v) bovine seru albumin (BSA) and
0.08% (w/v) sodium azide were added to each well. After a 30-min.
incubation, the fluid was aspirated and the plates were washed with
RPMI-1640 with 1% (w/v) BSA and 0.08% (w/v) sodium azide (Wash Buffer).
The Wash Buffer was then removed and the plates were inverted to
remove excess fluid. All additional washes were performed in this
manner. Fifty microliters of Wash Buffer containing 5% (v/v) normal
goat serum (Antibodies, Inc., Davis, Calif.) was then added to each
well and the incubation was continued for 30 min. The plates were
rinsed with Wash Buffer and 50 .mu.l of monoclonal antibody were
added. The plates were incubated for 1 hr and then rinsed twice
with Wash Buffer. Fifty microliters of a 1:1000 dilution of rabbit
anti-mouse F(ab').sub.2 were then added to each well. The remainder
of the assay is as described for the solid phase RIA. At the end
of the assay, wells were examined for the presence of cells; more
than 95% of the cells were present. Autoradiography was performed
as described above. To determine cpm bound, 50 .mu.l of 2 N NaOH
were then added to each well. Cotton swabs were then used to absorb
the fluid from each well and then were counted in a gamma counter.
Background (the average of the cpm obtained with Wash Buffer and
NS-1 supernatant fluid) was subtracted from the cpm obtained when
monoclonal antibody was used as primary antibody.
Immunoperoxidase Studies. Five micron sections of formalin fixed
or frozen sections of tissue on slides were used. Fixed tissues
were deparaffinized in xylene and rinsed in absolute ethanol. A
10 min. incubation with 0.3% H.sub.2 O.sub.2 in methanol was used
to block any endogenous peroxidase activity. After rinsing in PBS,
the slides were incubated with a 1:20 dilution of normal swine serum
(NSS) for 15 min. This incubation and all subsequent incubations
were carried out at room temperature. The NSS was removed and 0.1
ml monoclonal antibody or normal mouse sera (at an equal or greater
immunoglobulin concentration as a control) was placed on the tissue
sections and the slides were incubated for 30 min. The antibody
was removed and the slides were rinsed for 15 min. in PBS. The slides
were then incubated for 30 min. at each step with first a 1:10,000
dilution of goat anti-murine IgG F(ab').sub.2 followed by a 1:200
dilution of swine antigoat and then 1:2000 goat peroxidase anti-peroxidase
reagent (Cappel Laboratories, Cochranville, Pa.). The sections were
then rinsed in PBS for 15 min. and reacted with 0.06% diaminobenzidine
and 0.01% H.sub.2 O.sub.2 for 5 min. The sections were rinsed in
PBS, counterstained with hematoxylin for 30 sec., dehydrated in
ethanol, rinsed in xylene and mounted.
Isotyping of Monoclonal Antibodies. Radial immunodiffusion plates
(Meloy Laboratories, Springfield, Va.) containing monospecific goat
antisera to the murine isotypes were used to determine the isotype
and to quantitate the immunoglobulin. Isotypes were also determined
using rabbit antisera (Litton Bionetics, Rockville, Md.) to specific
murine isotypes in solid phase RIAs.
Results: The primary screen for monoclonal antibodies reactive
with human mammary carcinoma cells was a solid phase RIA employing
cell extracts of two breast tumor metastases (Met 1 and Met 2) and
apparently normal human liver as test antigens. The eleven monoclonal
antibodies could immediately be divided into three major groups
based on their differential reactivity to Met 1 versus Met 2. Nine
of the eleven monoclonals were reactive with both metastases. See
TABLE 1 __________________________________________________________________________
Reactivity of Monoclonal Antibodies in Solid Phase RIAs Live Cells.sup.b
Cell Extracts.sup.a Mammary Carcinoma Mcl Ab Isotype Met 1.sup.c
Met 2 Liver BT-20 MCF-7 ZR-75-1 Carc..sup.d Sarc..sup.e Normal.sup.f
B6.2 IgG.sub.1 +++ ++ NEG ++ +++ ++ NEG NEG NEG B14.2 IgG.sub.1
+++ ++ NEG + ++ + NEG NEG NEG B39.1 IgG.sub.1 +++ ++ NEG ++ ++ ++
NEG NEG NEG F64.5 IgG.sub.2a +++ ++ NEG ++ ++ + NEG NEG NEG F25.2
IgG.sub.1 +++ ++ NEG + + + NEG NEG NEG B84.1 IgG.sub.1 +++ ++ NEG
+ + + NEG NEG NEG B38.1 IgG.sub.1 + + NEG +++ ++ +++ + NEG NEG B50.4
IgG.sub.1 ++ + NEG NEG + NEG NEG NEG NEG B50.1 IgG.sub.1 ++ + NEG
NEG + NEG NEG NEG NEG B25.2 IgM NEG +++ NEG NEG NEG NEG NEG NEG
NEG B72.3 IgG.sub.1 +++ NEG NEG NEG NEG NEG NEG NEG NEG W6/32 IgG.sub.2a
NEG NEG NEG + NEG +++ ++ ++ B139 IgG.sub.1 +++ +++ ++ ++ ++ + ++
+++ ++ __________________________________________________________________________
.sup.a Solid phase radioimmunoassays were performed as described.
NEG, <500 cpm; +, 500-2000 cpm ++, 2001-5000 cpm; +++, >5000
cpm. .sup.b The live cell immunoassay was performed on human cells
as described. NEG, <300 cpm; +, 300-1000 cpm; ++, 1001-2000 cpm;
+++, >2000 cpm. .sup.c Mets 1 and 2 are extracts from human metastatic
mammary carcinoma cells from involved livers. .sup.d The carcinoma
cells used were lung (A549), vulva epidermoid (A431) and oral epidermoid
(KB). .sup.e Rhabdomyosarcoma (A204), fibrosarcoma (HT1080), and
melanoma (A375). .sup.f Human cells lines were derived from apparently
normal breast (Hs0584Bst), skin (Detroit 550), embryonic skin (Detroit
551), fetal lung (WI38 and MRC5), fetal testis (Hs0181Tes), fetal
hymus (Hs0208Th), fetal bone marrow (Hs0074Bm), embryonic kidney
(Flow 4000), fetal spleen (Hs0203Sp), and uterus (Hs0769Ut).
The following groupings are evident.
______________________________________ Monoclonal Cell extracts
Antibody Isotype Met 1 Met 2 ______________________________________
B6.2 IgG1 +++ ++ B14.2 IgG1 +++ ++ B39.1 IgG1 +++ ++ F64.5 IgG2a
+++ ++ B84.1 IgG1 +++ ++ B38.1 IgG1 + + B50.4 IgG1 ++ + B50.1 IgG1
++ + ______________________________________
The above antibodies bind both metastases and all three mammary
tumor cell lines (MCF-7, BT-20, ZR75-1). B6.2. binds to a 90,000
______________________________________ B25.2 IgM Neg +++ ______________________________________
B25.2 binds only Metastase 2.
______________________________________ B72.3 IgG1 +++ Neg ______________________________________
B72.3 binds only Metastase 1. B72.3 reacts with a complex of proteins
ranging from 220,000 to 400,000 d.
F25.2 has a similar range of reactivity as Group 1 but binds a
220,000 d protein.
B38.1 bound to BT-20, MCF-7, ZR-75-1, A 549 lung carcinoma line,
A 431 vulva epidermoid carcinoma line and KB oral epidermoid carcinoma
line (not to carcinoma and metanoma cell lines tested). Antibody
B38.1 thus appears to possess a "pancarcinoma" pattern
of binding activity. B38.1 binds a 70,000 d protein.
______________________________________ BT-20 MCF-7 2R-75-1 ______________________________________
B50.4 Neg + Neg B50.1 Neg + Neg ______________________________________
B50.4 and B50.1 preferentially bind to the MCF-7 versus the BT-20
cell line. B50.4 binds a 90,000 d protein.
None of the 11 monoclonal antibodies bound to any of the following
cell lines derived from apparently normal human tissues; breat,
uterus, skin, embryonic skin and kidney, and fetal lung, testis,
thymus, bone marrow and spleen. Control monoclonals W6/32 and B129,
however, did bind all of these cells.
All eleven antibodies were negative when tested against similar
extracts from normal human liver, the A204 rhabdomyosarcoma cell
line, the HBL100 cell line derived from cultures of human milk,
the Mm5mt/c.sub.1 mouse mammary tumor cell line, the C3H10T.sub.1/2
mouse fibroblast cell line, the CrFK feline kidney cell line, and
disrupted mouse mammary tumor virus and mouse leukemia virus (Table
1). Two monoclonal antibodies were used as controls in all these
studies: (a) W6/32, an anti-human histocompatibility antigen and
(b) B139, which was generated against a human breast tumor metastasis,
but which showed reactivity to all human cells tested.
The solid phase RIA using cell extracts of metastatic breast tumor
cells proved quite sensitive for the detection of test antigen.
The assays employed 5 .mu.g of tissue extract, but titration experiments
showed that as little as 0.3 .mu.g of tissue extract could be used.
FIG. 1 graphically illustrates the staining.
FIG. 1. Detection of binding of monoclonal antibodies to human
mammary adenocarcinoma cells using the immunoperoxidase technique.
Tissue sections in panel A through E are from the same area of an
infiltrating duct carcinoma. Panel F is an involved axillary lymph
node of a patient with an infiltrating duct carcinoma. Panels a,
b, and c are tissues stained with monoclonal B50.4. Panels d, e,
f are tissues stained with monoclonal B6.2. Panels a-e: Note staining
of mammary tumor cells (T), and absence of staining of apparently
normal mammary epithelium (N) or stroma. Note the granular staining
observed in panels a-c, as compared to the more diffuse staining
in panels d and f. Panel f: Note the staining of mammary carcinoma
cells (T) and the absence of staining of lymphocytes (L). Panel
a is 80.times.; b is 330.times.: c is 860.times., d is 330.times.;
e is 540.times.; f is 330.times..
To further define specificity and range of reactivity of each of
the eleven monoclonal antibodies, the immunoperoxidase technique
on tissue sections was employed. As seen in Table 2, all the monoclonals
reacted with mammary carcinoma cells of primary mammary carcinomas
(both infiltrating ductal and lobular). The percentage of primary
mammary tumors that were reactive varied for the different monoclonals,
ranging from 74% (23/31) and 80% (8/10) using monoclonals B6.2 and
B38.1, respectively, to 22% (2/9) for monoclonal B50.4. In many
of the positive primary and metastatic mammary carcinomas, not all
tumor cells stained. In certain tumor masses, moreover, heterogeneity
of tumor cell staining was observed in different areas of a tumor,
and even within a given area (See FIG. 1b). A high degree of selective
reactivity with mammary tumor cells, and not with apparently normal
mammary epithelium, stroma, blood vessels, or lymphocytes of the
breast was observed with all eleven monoclonal antibodies. This
is exemplified with monoclonals B50.4 and B6.2 in FIG. 1a-e. A dark
reddish-brown stain (the result of the immunoperoxidase reaction
with the diaminobenzidine substrate) was observed only on mammary
carcinoma cells, whereas only the light blue hematoxylin counterstain
was observed on adjacent normal mammary epithelium, stroma, and
lymphocytes. Occasionally, a few of the apparently "normal"
mammary epithelial cells immediately adjacent to mammary carcinoma
cell populations did stain weakly (Table 2). However, apparently
normal mammary epithelial cells in distal areas of these same breasts
(i.e., where no tumor was present) or from breasts of apparently
normal patients were always negative (Table 2). This faint staining
of some of the "normal" mammary epithelium immediately
adjacent to tumor cells may therefore be the manifestation of antigens
shed by adjacent tumor cells, or may represent a "preneoplastic"
population expressing the antigen being detected. The staining patterns
of mammary carcinoma cells varied among the different monoclonals.
This is exemplified in FIG. 1, where it can be seen that monoclonal
B50.4 is reactive with mammary carcinoma cells displaying a dense
focal staining (FIG. 1a-c). Monoclonal B6.2, on the other hand,
reacts with alternate sections of the exact mammary carcinoma displaying
a more diffuse pattern. The monoclonal antibodies could also be
distinguished from one another on the basis of which mammary tumors
they reacted with. For example, monoclonals B72.3 and B6.2 both
reacted with infiltrating ductal mammary carcinoma 10970, but only
monoclonal B6.2 reacted with mammary tumor 2657, while conversely,
only monoclonal B72.3 reacted with mammary tumor 9388. To ensure
against artifacts or differences due to sampling of tissues, these
experiments were carried out by reacting monoclonals with alternate
tissue sections. Frozen sections of primary mammary tumors were
also tested with some of the monoclonals; as expected from the surface
binding experiments to live cells in culture (Table 1), the frozen
sections revealed membrane staining.
Experiments were then carried out to determine if the eleven monoclonals
could detect mammary carcinoma cell populations at distal sites;
i.e., in metastases. Since the monoclonals were all generated using
metastatic mammary carcinoma cells as antigen, it was not unexpected
that the monoclonals all reacted, but with different degrees, to
various metastases. Perhaps the most efficient of the monoclonals
for this purpose was B6.2, which reacted with metastatic mammary
carcinoma cells in lymph nodes of 6 of 7 patients but did not react
with uninvolved nodes of 8 patients (Table 2). As exemplified in
FIG. 1f, none of the monoclonals reacted with normal lymphocytes
or stroma from any involved or uninvolved nodes. The eleven monoclonals
also varied in their ability to detect metastatic mammary carcinoma
lesions in distal sites such as liver, uterus, and bone (Table 2).
All eleven monoclonals were negative for reactivity with apparently
normal tissues of the following organs: spleens, bone marrow, thyroid,
colon, lung, liver, bladder, tonsils, stomach, prostate, and salivary
glands (Table 2).
Staining was seen with subpopulations of polymorphonuclear leukocytes
with some of the monoclonals. Flourescent activated cell sorter
analyses of spleens and bone marrows for various individuals, however,
revealed no surface binding of these cells with these same monoclonals.
Immunoperoxidase staining of tissue sections also revealed differences
among monoclonals with respect to both the population of cells stained
and the staining pattern. None of the monoclonals are reactive with
CEA, ferritin, the mouse mammary tumor virus (MMTV) or murine mammary
tumor cells infected with MMTV.
Studies have been undertaken to determine if radiolabeled monoclonal
antibodies could be used to detect human mammary tumors growing
in athymic mice. Monoclonal B6.2, chosen as the prototype, was labeled
with .sup.125 I using the iodogen technique. Sixty microcuries (1.times.10.sup.8
cpm) of radioactive B6.2 immunoglobulin, Fab' fragments, or F(ab)'.sub.2
fragments of B6.2 were then injected intravenously into athymic
mice bearing either mammary tumors derived from injections of MCF-7
mammary tumor cells, or the Clouser transplantable human mammary
tumor. At 8, 16, 24, 48, 72, and 96 hours post inoculation of radioactive
antibody, mice were first scanned with a gamma imager, then sacrificed,
and their blood and organs monitored for uptake of radioactive antibody.
The ratio of cpm of isotope per gram of tissue for the tumor was
from 3:1 to 12:1 versus the blood or normal tissues of tumor bearing
animals. This is shown in FIG. 2a where the tumor (arrow) is clearly
seen to take up more radioactive antibody than other tissues. When
the threshold of the cpm recognized by the gamma camera is raised
(FIG. 2b), the tumor clearly is the only site that scores positive.
When this same radioactive antibody was injected into an athymic
mouse bearing a human melanoma, there was no preferential uptake
of isotope by the tumor. Alternatively, when a radioactive nonspecific
immunoglobulin (MOPC-21) was injected into mice bearing transplanted
human mammary tumors, there was no preferential uptake of isotope
by the tumor. These studies demonstrate the utility of these antibodies
in detecting human mammary tumor lesions in an in vivo system.
The eleven monoclonal antibodies (designated B6.2, B14.2, B39.1,
F64.5, F25.2, B84.1, B38.1, B50.4, B50.1, B25.2, and B72.3) may
be useful in five major areas in the management of human breast
cancer. They are:
1. The diagnosis of primary and metastatic breast tumor lesions
by the assay of human blood samples or other body fluids for reactivity
with any one or a combination of the eleven monoclonal antibodies
2. The in-situ detection (via gamma scanning) of primary or metastatic
breast tumor lesions by the coupling of one or a combination of
the eleven monoclonal antibodies described with radioactive compounds.
3. The treatment of primary or metastatic breast cancer using one
or a combination of these eleven antibodies either alone or coupled
to toxic drugs, compounds, or radioactive isotopes.
4. The use of one or a combination of the eleven monoclonal antibodies
described in the staining (via the immunoperoxidase technique) of
populations of tumor cells in thin tissue sections obtained from
primary or metastatic breast tumor lesions. This reactivity may
serve as a prognostic indicator of the degree of malignancy of those
5. The detection of micro-lesions containing only a few tumor cells
(in thin tissue sections or body fluids obtained from biopsy of
patients suspected of or known to have breast cancer) that would
not ordinarily be detected by conventional staining techniques.
This would be accomplished with one or a combination of the eleven
monoclonal antibodies described using the immunoperoxidase technique
The range of reactivity of the monoclonal antibodies generated
and described above allows for the detection of mammary cancer cells,
treatments utilizing antibodies, and monitoring of conventional
The immunoperoxidase technique is useful in the detection of micro-lesions
containing only a few tumor cells in thin tissue sections or body
fluids obtained from biopsy of patients suspected of, or known to
have, breast cancer.
The monoclonal antibodies can be used singly or mixed. The immunoperoxidase
technique is as described above.