Murine monoclonal antibodies, or fragments thereof, that bind selectively
to human breast cancer cells, are IgGs or IgMs, and when conjugated
to ricin A chain, exhibit a TCID 50% against at least one of MCF-7,
CAMA-1, SKBR-3, or BT-20 cells of less than about 10 nM. Methods
for diagnosing, monitoring, and treating human breast cancer with
the antibodies or immunotoxins made therefrom are described.
What is claimed is:
1. A monoclonal antibody that binds specifically to a monomeric
210 kD protein present in cancerous breast tissue.
2. A monoclonal antibody as described in claim 1, wherein said
antibody is selected from the group consisting of 454C11 (ATTC No.
HB8484), 452F2 (ATTC No. HB10811), 520C9 (ATTC No. HB8696), and
741F8 (ATTC No. HB10807).
3. A monoclonal antibody as described in claim 2, wherein said
antibody has an association constant of about 4.8.times.10.sup.7.
4. A monoclonal antibody as described in claim 2, wherein said
antibody has an association constant of about 8.2.times.10.sup.6.
This invention is in the fields of immunology and cancer diagnosis
and therapy. More particularly, it concerns murine monoclonal anti-human
breast cancer antibodies, hybridomas that produce those antibodies,
immunochemicals made from those antibodies, and diagnostic and therapeutic
methods that use those immunochemicals.
2. Background Art
Since the mid-1970's, there have been numerous reports of murine
monoclonal antibodies that interact with human breast cancer associated
antigens. In these reported studies, mice were immunized and boosted
with human milk fat globule proteins, breast cancer cell lines or
breast cancer membrane extracts. Immune splenocytes were fused with
mouse myeloma cells and hybridomas were selected based on some specificity
of the culture media for breast or breast cancer antigens. Taylor-Papaddimitriou,
J. et al., Int. J. Cancer (1981) 28:17-21; Yuan, D., et al., JNCI
(1982) 68:719-728; Ciocca, D. R., et al., Cancer Res. (1982) 42:4256-4258.
The normal tissue activities of these prior antibodies are different
than the normal tissue reactivities of the antibodies of the present
Numerous prior workers have suggested or reported linking cytotoxic
agents or antibodies to make "immunotoxins". Recent interest
has centered on immunotoxins of monoclonal antibodies conjugated
to the enzymatically active portions (A chains) of toxins of bacterial
or plant origin via heterobifunctional agents. Neveill, D. M. and
Youle, R. J. Immunol. Rev. (1982) 62:75-91; Ross, W. C. J., et al.,
European J. Biochem, (1980) 104; Vitteta, E. S., et al., Immunol.
Rev. (9182) 62:158-183; Raso, V.,, et al., Cancer Res. (1982) 42:457-464;
Trowbridge, I. W. and Domingo, D. L., Nature(Cond) (1981) 294:171-173.
DISCLOSURE OF THE INVENTION
A principal aspect of the invention concerns murine monoclonal
(a) bind selectively to human breast cancer cells;
(b) are IgGs or IgMs;
(c) when conjugated to rich A chain exhibit a TCID 50% of less
than about 10 mM against at least one of MCF-7, CAMA-1, SKBR-3,
or BT-20 cells.
Preferred embodiments of these antibodies are those designated
260F9, 113F1, 2G3, 280D11, 266B2, 33F8, 245E7, 454C11, 317G5, 520C9,
and 369F10, and functional equivalents thereof.
The murine x murine hybridomas that produce the above described
antibodies and progeny of those hybridomas are other aspects of
Another aspect of the invention relates to immunotoxins that are
(a) the above described monoclonal antibodies, and
(b) a cytotoxic moiety.
Another aspect of the invention concerns labeled derivatives of
the above described monoclonal antibodies that are labeled with
a detectable label that permits the derivatives to be used in diagnosing
or monitoring human breast cancer.
Another aspect of the invention concerns a method of killing human
breast cancer cells by contacting the cells with a cytocidally effective
amount of one or more of the above described immunotoxins.
Other aspects of the invention are direct and indirect immunoassays
for determining whether a human cell is a breast cancer cell. These
assays involve incubating the cells with the monoclonal antibodies
or labeled derivatives thereof. When the labeled derivatives are
used, the presence of labeled binary immune complexes on the cells
is read directly. When unlabeled antibody is used the cells are
further incubated with a labeled antibody against monoclonal antibody
and the presence of labeled ternary immune complexes on the cells
MODES FOR CARRYING OUT THE INVENTION
As used herein, the term "monoclonal antibody" means
an antibody composition having a homogeneous antibody population.
It is not intended to be limited as regards to the source of the
antibody or the manner in which it is made.
As used herein with respect to the exemplified murine monoclonal
anti-human breast cancer antibodies, the term "functional equivalent"
means a monoclonal antibody, fragments thereof, or any molecule
having the antigen binding site of the monoclonal antibody that:
(a) crossbacks an exemplified monoclonal antibody; (b) binds selectively
to human breast cancer cells; (c) has a G or M isotope; (d) binds
to the same antigen as determined by immunoprecipitation or sandwich
immunoassay; and (e) when conjugated to ricin A chain, exhibits
a TCID 50% against at least one of MCF-7, CAMA-1, SKBR-3, or BT-20
cells of less than about 10 mM.
Antibody fragments include the Fab, Fab', and F(ab').sub.2 regions,
or derivatives or combinations thereof. Fab, Fab', and F(ab').sub.2
regions of an immunoglobin may be generated by enzymatic digestion
of the monoclonal antibodies using techniques well known to those
skilled in the art. Fab fragments may be generated by digesting
the monoclonal antibody with papain and contacting the digest with
a reducing agent to reductively cleave disulfide bonds. Fab' fragments
may be obtained by digesting the antibody with pepsin and reductive
cleavage of the fragment so produce with a reducing agent. In the
absence of reductive cleavage, enzymatic digestion of the monoclonal
with pepsin produces F(ab').sub.2 fragments.
It will further be appreciated that encompassed within the definition
of monoclonal antibody is single chain antibody that can be generated
as described in U.S. Pat. No. 4,704,692, as well as hybrid antibodies
described in U.S. patent application Ser. No. 474,893 and by Munroe,
(1984) Nature 312:597; Morrison, S. L. (1985) Science 229:1202 and
Oi, et al. (1986) Biotechniques 4:214. Particularly useful hybrid
antibodies are "humanized" antibodies made as described
in European Patent Application No. 302,620.These publications are
hereby incorporated by reference.
As used herein with regard to the monoclonal antibody-producing
hybridomas of the invention, the term "progeny" is intended
to include all derivatives, issue, and offspring of the parent hybridoma
that produce the monoclonal anti-human breast cancer antibody produced
by the parent, regardless of generation of karyotypic identify.
Monoclonal Antibody Production
The antibody-producing fusion partners that are used to make the
hybridomas of this invention are generated by immunizing mice with
live human breast cancer cells or membrane extracts made therefrom.
The mice are inoculated intraperitoneally with an immunogenic amount
of the cells or extract and then boosted with similar amounts of
the immunogen. Spleens are collected from the immunized mice a few
days after the final boost and a cell suspension is prepared therefrom
for use in the fusion.
Hybridomas are prepared from the spenocytes and a murine tumor
partner using the general somatic cell hybridization technique of
Kohler, B. and Milstein, C., Nature (1975) 256:495-497 as modified
by Buck, D. W., et al., In Vitro (1982) 18:377-381. Available murine
myeloma lines, such as those from the Salk Institute, Cell Distribution
Center, San Diego, Calif., USA, may be used in the hybridization.
Basically, the technique involves fusing the tumor cells and splenocytes
using a fusogen such a polyethylene glycol, or by electrical means
well known to those skilled in the art. After the fusion the cells
are separated from the fusion medium and grown in a selective growth
medium, such as HAT medium, to eliminate unhybridized parent cells.
The hybridomas are expanded, if desired, and supernatants are assayed
for antihuman breast cancer activity by conventional immunoassay
procedures (e.g., radioimmunoassay, enzynme immunoassay, or fluoroescence
immunoassay) using the immunizing agent (breast cancer cells or
membrane extract) as antigen. Positive clones are characterized
further to determine whether they meet the criteria of the invention
Hybridomas that produce such antibodies may be grown in vitro or
in vivo using known procedures. The monoclonal antibodies may be
isolated from the culture media or body fluids, as the case may
be, by conventional immunoglobulin purification procedures such
as ammonium sulfate precipitation, gel electrophoresis, dialysis,
chromatography, and ultrafiltration, if desired.
Monoclonal Antibody Selection/Characterization
The important characteristics of the monoclonal antibodies are
(1) their immunoglobulin class, (2) their selectivity for human
breast cancer cells and the range of human breast cancer cells to
which they bind and (3) their usefulness in making effective anti-human
breast cancer immunotoxins.
The selectivity and range of a given antibody is determined by
testing it against panels of (1) human breast cancer tissues and
cells and (2) normal human tissues or cells of breast or other origin.
In selecting the claimed antibodies approximately twenty-two thousand
growing hybridoma cultures were initially screened against the immunizing
breast tumor membranes, a fibroblast cell line and a breast tumor
frozen section. Clones that reacted with the neoplastic materials
but not the normal materials were identified in this initial screen
and chosen for isotyping and additional screening for selectivity
and range. The additional screening involved: sixteen normal tissue
sections, five normal blood cell types, eleven nonbreast neoplasm
sections, twenty-one breast cancer sections and fourteen breast
cancer cell lines. Antibodies were deemed to bind selectively to
breast cancer if they bound strongly to less than about 1/3 of the
normal tissues and blood cell types. One hundred twenty-seven antibodies
were purified and tested on the additional screen.
Antibodies exhibiting acceptable selectivity and range were conjugated
to ricin A chain using N-succinimidyl-3-(2-pyridyldithio)propionate
(SPDP) or iminothiolane (IT) as a coupling agent. The conjugates
were tested against MCF-7, CAMA-1, SKBR-3, and BT-20 cells in a
24-hour tissue culture assay. Sixteen of the antibodies exhibited
acceptable immunotoxin activity (TCID 50% of less than 10 nM) against
at least one of these breast tumor lines. Seven of the sixteen were
found to recognize the same 210,000 dalton antigen, with six of
the seven probably recognizing the same epitope but differing in
Further details of the characterization of these antibodies are
provided in the examples below.
The immunochemical derivatives of the monoclonal antibodies of
this invention that are of prime importance are immunotoxins (conjugates
of the antibody and a cytotoxic moiety) and labeled (e.g., radiolabeled,
enzymelabeled, or fluorochrome-labeled) derivatives in which the
label provides a means for identifying immune complexes that include
the labeled antibody.
The cytotoxic moiety of the immunotoxins may be a cytotoxic drug
or an enzymatically active toxin of bacterial or plant origin, or
an enzymatically active fragment ("A chain") of such a
toxin. Enzymatically active toxins and fragments thereof are preferred
and are exemplified by diptheria A chain, nonbinding active fragments
of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolacca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, and enomycin. Ricin A chain, nonbinding active fragments
of diptheria toxin, abrin A chain, and PAPII are preferred. Conjugates
of the monoclonal antibody and such cytotoxic moieties may be made
using a variety of bifunctional protein coupling agents. Examples
of such reagents are SPDP, IT, bifunctional derivatives of imidoesters
such as dimethyl adipimidate. HCl, active esters such as disuccinimidyl
suberate, aldehydes such as glutaraldehyde, bis-azido compounds
such as bis(p-azidobenzoyl) hxanediamine, bis-diazonium derivaties
such as bis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates
such as tolylene 2,6-diisocyanate, and bis-active fluorine compounds
such as 1,5-difluoro-2,4-dinitrobenzene.
When used to kill human breast cancer cells in vitro for diagnostic
purposes, the conjugates will typically be added to the cell culture
medium at a concentration of at least about 10 nM. The formulation
and mode of administration for in vitro use are not critical. Aqueous
formulations that are compatible with the culture or perfusion medium
will normally be used. Cytotoxicity may be ready by conventional
techniques to determine the presence or degree of breast cancer.
When used in vivo for therapy, the immunotoxins are administered
to the patient in therapeutically effective amounts (i.e., amounts
that eliminate or reduce the patient's tumor burden). They will
normally be administered parenterally, preferably intravenously,
although other routes may be employed and even preferred depending
on the nature and site of the tumor (i.e., intratumoral administration).
The dose and dosage regimen will depend upon the nature of the cancer
(primary or metastatic) and is population, the characteristics of
the particular immunotoxin, e.g., it therapeutic index, the patient,
and the patient's history. The amount of immunotoxin administered
will typically be in the range of about 0.1 to about 10 mg/kg of
For parenteral administration the immunotoxins will be formulated
in a unit dosage injectable form (solution, suspension, emulsion)
in associated with a pharmaceutically acceptable parenteral vehicle.
Such vehicles are inherently nontoxic and nontherapeutic. Examples
of such vehicles are water, saline, Ringer's solution, dextrose
solution, and 5% human serum albumin. Nonaqueous vehicles such as
fixed oils and ethyl oleate may also be used. Liposomes may be used
as carriers. The vehicles may contain minor amounts of additives
such as substances that enhance isotonicity and chemical stability,
e.g., buffers and preservatives. The immunotoxins will typically
be formulated in such vehicles at concentrations of about 1mg/ml
to 10 mg/ml.
Cytotoxic radiopharmaceuticals for treating breast cancer may be
made by conjugating high linear energy transfer (LET) emitting isotopes
(e.g., Y, Pr) to the antibodies. The term "cytotoxic moiety"
as used herein as intended to include such isotopes.
The labels that are used in making labeled versions of the antibodies
include moieties that may be detected directly, such as fluorochromes
and radiolabeled, as well as moieties, such as enzymes, that must
be reacted or derivatized to be detected. Examples of such labels
are .sup.32 P, .sup.125 I, .sup.3 H, .sup.14 C, fluorescein and
its derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
luciferia, 2,3-dihydrophthalazinediones, horseradish peroxidase,
alkaline phosphatase lysozyme, and glucose-6-phosphate dehydrogenase.
The antibodies may be tagged with such labels by known methods.
For instance, coupling agents such as aldehydes, carbodiimides,
dimaleimide, imidates, succinimides, bis-diazotized benzidine and
the like may be used to tag the antibodies with the above-described
fluorescent, chemiluminescent, and enzyme labels.
The antibodies and labeled antibodies may be used in a variety
of immunoimaging or immunoassay procedures to detect the presence
of breast cancer in a patient or monitor the status of such cancer
in a patient already diagnosed to have it. When used to monitor
the status of a caner a quantitative immunoassay procedure may be
used. Such monitoring assays are carried out periodically and the
results compared to determine whether the patient's tumor burden
has increased or decreased. Common assay techniques that may be
used include direct and indirect assays. Direct assays involve incubating
a tissue sample or cells from the patient with a labeled antibody.
If the sample includes breast cancer cells, the labeled antibody
will bind to those cells. After washing the tissue or cells to remove
unbound labeled antibody, the tissue sample is read for the presence
of labeled immune complexes. In indirect assays the tissue or cell
sample is incubated with unlabeled monoclonal antibody. The sample
is then treated with a labeled second antibody (or antibody fragment)
against the monoclonal antibody (e.g., a labeled antimurine antibody
or fragment), washed, and read for the presence of labeled ternary
For diagnostic use the antibodies will typically be distributed
in kit form. These kits will typically comprise: the antibody in
labeled or unlabeled form in suitable containers, reagents for the
incubations and washings, a labeled antimurine antibody if the kit
is for an indirect assay, and substrates or derivatizing agents
depending on the nature of the label. Human breast cancer antigen
controls and instructions may also be included.
The following examples provide a detailed description of the preparation,
characterization, and use of representative monoclonal antibodies
of this invention. These examples are not intended to limit the
invention in any manner.
Fresh postsurgical human breast cancer tissue and a variety of
normal tissues were used to prepare membrane extracts by Polytron
homogenization and discontinuous suscrose gradient centrifugation.
Human breast cancer cell lines were obtained from the Breast Caner
Task Force, the American Type Culture Collection (ATCC), and from
Dr. Jorgen Fogh at Memorial Sloan Kettering. The cells were maintained
and passaged as recommended by the Breast Cancer Task Force, the
ATCC and Dr. Fogh. For immunizations, either membrane extract containing
100 .mu.g of protein (Lowry assay) or ten million live breast cancer
cells were inoculated intraperitoneally into five week old Balb/c
mice. The mice were boosted identically twice at monthly intervals.
Three days after the 1st boost, the spleens were removed for cell
Hybridoma supernatant was assayed for reactive antibody in either
a solid phase enzyme-linked immunosorbent assay (ELISA) with the
immunizing breast cancer membrane extract or an indirect immunofluorescence
assay with the immunizing breast cancer cell line. For the solid
phase membrane ELISA, 40 .mu.l of 0.1 mg/ml breast cancer membrane
protein were placed in polyvinyl chloride (PVC) microtiter wells
(Dynatech, Inc.) for 12 hours at 4.degree. C. The extract was aspirated
and the wells washed with phosphate buffered saline (PBS) containing
1% bovine serum albumin (BSA). The wells were the incubated with
45 .mu.l of a 1:10 dilution of hybridoma supernatant. The diluent
was RPMI 1640 media with 25 mM Hepes, 10% bovine serum, and 0.1%
sodium azide. After 30 minutes at room temperature, the wells were
again washed and incubated 45 minutes at 37.degree. C. with a 1:200
dilution of peroxidase conjugated goat anti-mouse IgG (Zymed, Inc.).
The diluent was PBS. The wells were then washed with PBS and reacted
with 200 .mu. l of 2,2-azino-di(3-ethylbenzthiazoline supphonic
acid) in 0. 1sodium citrate buffer pH 4.2 for 30 minutes at room
temperature. Optical density was measured at 405 mm on a MicroElisa
Reader (Dynatech, Inc.). For each experiment a positive control,
anti-beta 2 microglobulin at 5 .mu.g/ml (Becton Dickinson, Inc.),
was reacted with normal human kidney membrane. This gave an optical
density of 1.0.+-.0.1 (standard deviation). The background was 0.+-.0.1
optical density units (O.D.) using media without mouse monoclonal
antibody. Wells that gave a reaction on the breast cancer membrane
extract of greater than 0.7 O.D. were saved. For the indirect immunofluorescence
cell line assay was placed one hundred thousand breast cancer cells
of the immunizing cell line overnight with appropriate media in
each chamber of a set of eight chambered slides (Lab-tek, Inc.).
Similarly, one hundred thousand fibroblast cells from cell line
CC05 were incubated overnight in chambered slide wells. The cells
were washed with PBS containing 1% BSA (Miles, Inc.). The wells,
both breast cancer and fibroblast, were incubated 30 minutes at
4.degree. C. with a 1:50 dilution of fluorescein isothiocyanate
(FITC)-conjugated goat F(ab').sub.2 anti-mouse Ig (Zymed, Inc.).
The cells were washed three times, fixed in 1.5% formaldehyde in
PBS for five minutes, chambers removed and rinsed in PBS. The slides
were then mounted in Aguamount (Lenex Lab) and examined with a Laborlus
12 fluorescence microscope (Leitz, Inc.) Hybridoma wells showing
strong fluorescent binding to breast cancer cells but no fluorescent
binding to fibroblasts were saved. Five thousand one hundred fifty-six
hybridoma wells revealed breast cancer reactivity in the initial
Supernatants from the 5156 positive wells were then tested in solid
phase ELISA with eight normal tissue membrane extracts (liver, lung,
colon, stomach, kidney, tonsil, spleen and pancrease). Any well
supernatant giving an ELISA O.D. greater than 0.3 was discarded.
One thousand one hundred one of the supernatants were found to be
unreactive with the normal tissue extracts.
The 1101 hybridoma supernatants were tested on frozen sections
of human breast carcinoma tissues. Six micron sections were attached
to slide, fixed 10 minutes in acetone at 4.degree. C., dried 10
minutes at room temperature, washed with PBS, blocked with horse
serum and incubated 20 minutes at room temperature with 200 .mu.l
neat hybridoma supernatant. The slides were washed with PBS, and
finally incubated 20 minutes at 37.degree. C. with a 1:50 dilution
of peroxidase conjugated rabbit anti-mouse Ig (Tago, Inc.), washed
again with PBS, and finally incubated 7.5 minutes at 37.degree.
C. with 0.5 mg/ml diaminobenzidine (Sigma, Inc.) in 0.05 M Tris
buffer pH 7.2 containing 0.01% hydrogen peroxide (Fisher, Inc.).
The slides were stained with hematoxylin, dehydrated and mounted
in Permount (Fisher, Inc.). One hundred twenty-four wells yielded
breast cancer selective binding and were cloned.
Purification and Class Determination
Immunoglobulin class and subclass of the monoclonal breast cancer
selective antibodies were determined. Antibodies were also internally
labeled by growing 2-3.times.10.sub.6 hybridoma cells for four hours
in methionine-free medium containing 0.2 .mu.Ci .sup.35 S methionine
(New England Nuclear). .sup.35 S-labeled antibodies were immunoprecipitated
with fixed staphylococcus A cells (IgSorb, The Enzyme Center) or
with IgSorb pre-coated with rabbit anti-mouse immunoglobulin (Zymed),
and the imnmunoprecipitates were analyzed by SDS-PAGE to determine
antibody light and heavy chain mobility, lack of extra chains, and
the ability of each antibody to bind staphylococcal protein A.
The antibodies were expanded in vivo. Balb/c or F1 (C578/6.times.Balb/c)
mice were primed with 0.5 ml pristine intraperitoneally (ip) and
after 10-14 days inoculated with one million log phase hybridoma
cells in PBS. Ascites fluid was stored at -70.degree. C. and thawed
and filtered through a 0.8 micron Millex filter unit before further
IgG antibodies that bound staphylooccal protein A were purified
by affinity chromatography on protein A-sepharose (Pharmacia) with
pH step gradient elution. IgG antibodies that did not bind protein
A were precipitated by addition of ammonium sulfate to 40% saturation
at 0.degree. C. The precipitates were redissolved in PBS, dialysed
to 20 mM Tris pH 7.2 and chromatographed on a 1.6.times.50 cm column
of diethylaminoethyl cellulose (DEAE) Affi-Gel Blue (BioRad) eluting
with a 1.5 liter (0-600 mM NaCl gradient at 4.degree. C. at a flow
rate of 1 ml/min. In each case, column fractions were monitored
by SDS-PAGE and the purest antibody fractions were pooled, concentrated
to 1-3 mg/ml, dialysed to PBS/0.02% NaN.sub.3, and stored at 40.degree.
IgM antibodies were purified by gel filtration on a 2.6.times.40
cm column of Sephacryl S-300 (Pharmacia) eluting with PBS/0.01%
sodium azide at room temperature at a flow rate of 1 ml/min.
In order to evaluate their selectivity for breast cancer, the purified
antibodies were tested by immunoperoxidase section staining on sections
of sixteen normal tissues, and by immunofluorescent cell sorting
on five blood cell types. Immunoperoxidase staining was performed
as above except that known dilutions of purified antibodies in PBS
in the range of 1-40 .mu.g/ml were used instead of hybridoma supernatants.
The pure antibodies were first titrated to find the minimal concentration
giving strong immunoperoxidase staining on breast cancer sections
and then used at that concentration for the normal tissue tests.
Peripheral blood cells (platelets, lymphocytes, red blood cells,
granulocytes, and monocytes) were prepared by centrifugation using
Mono-Poly Resolving Medium (Flow Laboratories), Inc.). The cells
were reacted with antibody at the optimal concentration determined
above for 30 minutes at 4.degree. C., washed, reacted with a 1:50
dilution of FITC-conjugated goat anti-mouse Ig (Tago, Inc.) for
30 minutes at 4.degree. C., washed again and examined in an EPICS
cell sorter (Coulter Electronics, Inc.). The buffer and diluents
were PBS with 1% gelatin and 0.02% sodium azide. The EPICS V was
equipped with a 76 micron nozzle and a one watt argon ion laser
at 488 nm. An 80 mm confocal lens was used on the optical rail assembly
for focusing. Other filters used were a 515 nm interference filter
and a 515 mm absorbance filter (for scattered laser light) and a
neutral density 1.5 filter (Melles Griot) for forward angle light
scatter. Contour plots of log fluorescein fluorescence versus forward
angle light scatter were used for sample analysis.