A purified and isolated DNA sequence and the encoded mammary-specific
secreted protein, mammaglobin, are disclosed. Also disclosed are
methods for detecting breast cancer based upon the overexpression
and secretion of mammaglobin by breast cancer cells. The methods
detect and/or quantitate the presence of mammaglobin or the mRNA
encoding mammaglobin. Immunotherapy-based methods for treating a
breast cancer patient with a mammaglobin-expressing tumor are also
disclosed. The methods involve using mammaglobin antigens to induce
a humoral and/or cell-mediated immune response against the tumor.
What is claimed is:
1. A method for detecting the presence of breast cancer in a patient
which comprises detecting the presence of mammaglobin polypeptide
in a sample from the patient by reacting the mammaglobin polypeptide
with a purified antibody and detecting a binding of the mammaglobin
polypeptide with the antibody, wherein the antibody is specific
for a mammaglobin epitope comprising at least five amino acids of
SEQ ID NO:14 and wherein an elevated expression of said mammaglobin
polypeptide above the expression level in a normal sample indicates
the presence of breast cancer.
2. The method of claim 1, wherein said mammaglobin polypeptide
comprises SEQ ID NO:17.
3. The method of claim 2 wherein the purified antibody is a polyclonal
4. The method of claim 2 wherein the purified antibody is a monoclonal
5. The method of claim 1 wherein the sample comprises breast tumor
6. A kit for detecting the presence of breast cancer cells in a
sample which comprises a purified antibody packaged in a container,
wherein said antibody is specific for a mammaglobin epitope comprising
at least five amino acids of SEQ ID NO:14 and wherein said antibody
reacts with a mammaglobin polypeptide comprising SEQ ID NO:17.
7. The method of claim 1 wherein the sample is breast tissue.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention relates generally to the field of breast cancer
pathogenesis and, more particularly, to a cDNA sequence and encoded
mammary-specific protein for use in detecting and treating breast
(2) Description of the Related Art
Breast cancer is one of the most common and potentially lethal
of cancers. Although early diagnosis and treatment can reduce morbidity
and mortality related to the disease, the positive predictive value
of mammography has been estimated to be only about 25% (Hall et
al., N Engl J Med 327:319-328, 1992). It would, therefore, be desirable
to have a means for detecting the cancer earlier than the cancer
can be detected using mammography and a genetic or biochemical marker
might be able to provide such means to complement and increase the
predictive value of mammography. (Hayes, Hematol Oncol Clin N Am
The development of breast cancer is accompanied by a number of
genetic changes (For review see Porter-Jordan, Hematol Oncol Clin
N Am 8:73, 1994). Such changes include gross chromosomal alterations
and loss of genetic markers (Devilee et al, Biochim Biophys Acta
1198:113, 1994; Callahan et al, J Cell Biochem Suppl 17:167, 1993).
The progression of breast neoplasia has also been shown to result
in qualitative and quantitative changes in expression of previously
identified genes that encode growth factors and their receptors
(Zajchowski et al., Cancer Res 48:7041, 1988), structural proteins
(Trask et al., Proc Natl Acad Sci 87:2319, 1990), second messenger
proteins (Ohuchi et al., Cancer Res 26:2511, 1986), and transcription
factors (Harris, Adv Cancer Res 59:69:1992). These changes in gene
expression could potentially form the basis for developing a breast
cancer marker, although the precise role of these gene changes in
the pathogenesis of breast carcinoma in patient biopsy samples is
not well understood.
In addition to providing a genetic or biochemical marker for breast
cancer for early detection of the disease, it would also be desirable
to have a tumor marker that might provide an estimation of prognosis,
a means for selection and evaluation of therapy and a means for
the targeting of therapy. Although a number of tissue markers have
been identified, none are sufficiently sensitive or tumor specific
to be ideally suited for diagnosis or for screening the general
population. (Id.) Thus, there remains a continuing need for a breast
cancer marker such as a gene along with its expressed protein that
can be used to specifically and selectively identify the appearance
and pathogenic development of breast cancer in a patient, and that
can be used in tumor-specific immunotherapy.
Using a modified differential display polymerase chain reaction
technique to isolate differentially expressed sequence tags from
mammary carcinoma, several sequence fragments were isolated that
were uniquely expressed in neoplastic mammary epithelial tissue
as compared to normal tissue controls (Watson and Fleming, Cancer
Res 54:4598-4602, 1994). The discovery of one of these sequence
tags identified as DEST002 has led to the discovery and isolation
of the novel full length cDNA and encoded protein now referenced
as mammaglobin. The cDNA and protein are both new.
SUMMARY OF THE INVENTION
Briefly, therefore, the present invention is directed to the identification
of novel genes whose expression is increased in breast cancer and
to the isolating of cDNA's from the mRNA's of these genes. Accordingly,
applicants have succeeded in discovering a novel cDNA and the encoded
mammary-specific secretory protein, mammaglobin. The cDNA is in
purified and isolated form and comprises a nucleotide sequence identified
as SEQ ID NO:15 and the encoded protein, mammaglobin, is in purified
and isolated form and has an amino acid sequence identified as SEQ
In a small-scale study described in U.S. Pat. No. 5,668,267, mammaglobin
mRNA was overexpressed in 27% of stage I primary breast cancer tumors.
The present application describes a larger survey of primary breast
tumors of multiple grades and histological types in which mammaglobin
protein was detected in about 80% of the tumors examined. These
data suggest that dysregulation of the mammaglobin gene occurs early
and frequently in breast cancer. The discovery of mammaglobin and
its cDNA, therefore, provide the basis for the development of novel
methods and compositions for the detection and treatment of breast
neoplastic disease in humans and other mammals.
Thus, the present invention is directed to novel methods for detecting
the presence of breast neoplasia cells in a sample. In one embodiment,
a polynucleotide probe is used to detect the presence of mammaglobin
mRNA in the sample. The method comprises the steps of: (a) contacting
mRNA in the sample with a polynucleotide probe which specifically
hybridizes to a mammaglobin mRNA comprising SEQ ID NO:15 or an allelic
variant thereof, and (b) detecting a hybridization complex between
the probe and the sample mRNA.
Another aspect of the present invention provides a kit for detecting
the presence of breast neoplasia cells in a sample by hybridization.
The kit comprises a polynucleotide probe which specifically hybridizes
to a mammaglobin mRNA comprising SEQ ID NO:15 or an allelic variant
thereof packaged in a container.
In another embodiment of the present invention, mammaglobin expression
in a sample is determined by detecting the presence of cDNA that
is reverse transcribed from mammaglobin mRNA in the sample. The
method comprises the steps of: (a) producing a cDNA encoding mammaglobin
from mRNA using the reverse transcription method in a sample obtained
from a patient, (b) providing two primers for the polymerase chain
reaction method which comprise oligomers that flank or lie within
the cDNA encoding mammaglobin, and (c) amplifying the cDNA encoding
mammaglobin by the polymerase chain reaction method. Preferably,
the two primers have nucleotide sequences encoding SEQ ID NO:4 and
SEQ ID NO:16.
Another embodiment to the present invention provides a kit for
detecting the presence of breast neoplasia cells in a sample by
the polymerase chain reaction. The kit comprises two primers for
the polymerase chain reaction method which comprise oligomers that
flank or lie within a cDNA encoding mammaglobin packaged in a container.
Preferably, the two primers have nucleotide sequences comprising
SEQ ID NO:4 and SEQ ID NO:16.
In another embodiment of the present invention, the presence of
mammaglobin protein expressed by a tumor cell is detected in a sample
using specific antibodies to the mammaglobin protein. The specific
antibodies can be polyclonal or monoclonal antibodies.
The invention is also directed to novel compositions and methods
for treating breast neoplastic disease using mammaglobin antigens
capable of inducing an antibody-mediated and/or a cell-mediated,
i.e., through activated T cells, immune response against a mammaglobin-expressing
One embodiment of a composition according to the invention comprises
a mammaglobin B cell antigen capable of activating mammaglobin-specific
B cells. The B cell antigen comprises a mammaglobin-specific B cell
epitope and a T.sub.H epitope, or determinant, recognized by T helper
In another embodiment, the mammaglobin antigen is a mammaglobin
T.sub.C cell antigen recognized by mammaglobin-specific cytotoxic
T lymphocytes which comprises a T.sub.C cell epitope and a binding
site, or agretope, for a MHC class I molecule.
Yet another embodiment of a composition according to the invention
comprises B cell and T.sub.C cell antigens.
Methods for treating a patient with a mammaglobin-expressing tumor
include adoptive immunotherapy, which comprises ex vivo stimulation
with a mammaglobin antigen of mammaglobin-specific lymphocytes isolated
from the patient and subsequent administration of the activated
lymphocytes to the patient, and in vivo stimulation of an anti-mammaglobin
immune response, which comprises administering to the patient a
vaccine comprising a mammaglobin antigen.
Among the several advantages found to be achieved by the present
invention, therefore, may be noted the provision of a nucleotide
sequence and encoded amino acid sequence that can serve as markers
for breast cancer cells; the provision of methods for early detection
of the presence of breast neoplasia cells; the provision of means
for detecting breast cancer that can complement mammography and
increase the predictive value; the provision of methods that can
provide an estimation of prognosis; the provision of markers that
will allow the targeting of therapy; and the provision of compositions
for stimulating a cellular and humoral immune response against the
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the strategy used to isolate the full length
mammaglobin cDNA including the Rapid amplification of cDNA Ends
(RACE) Polymerase Chain Reaction (PCR) technique and subsequent
subcloning into vectors pGEM7Z and pCEV27.
FIG. 2 illustrates the human cDNA sequence (SEQ ID NO:1), (nucleotides
numbered above) and the amino acid sequence of the encoded mammary-specific
protein, mammaglobin (SEQ ID NO:2)(amino acids numbered below),
the solid bar indicating the 403 bp fragment (SEQ ID NO:5) isolated
by the RACE PCR method and the open bar indicating the 206 bp DEST002
sequence (SEQ ID NO:6);
FIG. 3 illustrates the amino acid sequence of the mammary-specific
protein, mammaglobin (hMAM), (SEQ ID NO:2) compared to rat prostatic
steroid binding protein subunit C3 (rPSC3)(SEQ ID NO:7) and human
clara cell 10 kD protein (hCC10)(SEQ ID NO:8) with identities marked
by bold letters and double lines and structurally similar amino
acids marked by single lines;
FIG. 4 illustrates (FIG. 4A) the Northern blot analysis of hybridization
of the human cDNA sequence encoding the mammary-specific protein,
mammaglobin (hMAM), to mRNA expressed by tissues from breast neoplasia,
normal breast and other adult tissues and (FIG. 4B) the analysis
of RT/PCR amplified samples of tissues from breast neoplasia, normal
breast and other adult tissues;
FIG. 5 illustrates the translation of the mammary-specific cDNA
sequence in an in vitro rabbit reticulocyte lysate assay system;
FIG. 6 illustrates Northern blot hybridization with the cDNA encoding
mammaglobin showing detection of mRNA in tumor 2410, in tumors from
three of eight other patients (shown in bold), and to a lesser extent,
in normal breast tissue (shown in italics), and comparing in two
cases (the four lanes on the right) mammaglobin mRNA expression
in tumor tissue and patient matched normal tissue;
FIG. 7 illustrates the Western blot analysis using polyclonal antibody
to the mammaglobin C-terminus (SEQ ID NO:14) of the conditioned
medium (S) and cell lysate (C) from MDA-MB-415 breast tumor cells
in the absence (-) and presence (+) of the immunizing peptide showing
detection of the precursor and secreted forms of mammaglobin protein
in the cell medium and cell lysate, respectively;
FIG. 8 illustrates the Western blot analysis using the anti-mammaglobin
polyclonal antibody of the conditioned medium (S) and cell lysate
(C) from MDA-MB-415 breast tumor cells grown in the absence (-)
and presence (+) of tunicamycin, which blocks glycosylation, showing
the lack of detectable mammaglobin protein in the lysate or medium
of cells in which N-linked glycosylation is inhibited;
FIG. 9 illustrates the Western blot analysis of cell lysates from
human breast tumor cells showing detection of the precursor mammaglobin
protein using the anti-mammaglobin polyclonal antibody and goat
anti-rabbit antibody visualized by enzyme-linked chemiluminescence;
FIG. 10 illustrates the Western blot analysis using the anti-mammaglobin
polyclonal antibody of fluid secretions from human breast during
pregnancy and postpartum showing detection of the secreted mammaglobin
protein in proliferating mammary gland;
FIG. 11A illustrates in color a paraffin-fixed section of breast
cancer cells from a patient specimen immunohistochemically stained
using the anti-mammaglobin polyclonal antibody and goat anti-rabbit
antibody tagged with horseradish peroxidase and DAB as substrate
showing a brown staining of cells expressing the mammaglobin protein;
FIG. 11B illustrates in black and white a paraffin-fixed section
of breast cancer cell from a patient specimen immunohistochemically
stained using the anti-mammaglobin polyclonal antibody and goat
anti-rabbit antibody tagged with horseradish peroxidase and DAB
as substrate wherein the brown staining of cells expressing the
mammaglobin protein is indicated;
FIG. 12 illustrates mammaglobin immunoreactivity in tissue sections
from the same specimens of (FIGS. 12A, 12B) pure ductal carcinoma
in situ (DCIS), (FIGS. 12C, 12D) well differentiated ductal carcinoma,
and (FIGS. 12E, 12F) poorly differentiated ductal carcinoma which
were immunohistochemically stained with (FIGS. 12A, 12C, 12E) mammaglobin
antiserum or (FIGS. 12B, 12D, 12F) pre-immune antiserum;
FIG. 13 shows a photograph of a northern blot of RNA from lymph
node specimens containing histologically documented metastases from
primary breast tumors (lanes 1-20, 27), squamous cell tumors of
the head and neck (lanes 21, 23, 24), endometrial cancer (lane 22),
adenocarcinoma of the colon (lane 25), billiary carcinoma (land
26), and adenocarcinoma of the lung (lane 28) and, as negative controls,
lymph node biopsies from patients without any known malignancy (lanes
29-33) and as a positive control, a specimen from normal breast
tissue (lane 34), and hybridized with the mammaglobin cDNA (top
panels) or a keratin cDNA (bottom panels); and.
FIG. 14 shows a photograph of a Southern blot of RT-PCR products
of RNA from patients with metastatic breast cancer (CA) or normal
donors (NL) obtained from peripheral stem cell collections and probed
with the mammaglobin cDNA probe.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the present invention is based upon the identification
and sequencing of the cDNA identified as SEQ ID NO:1 which encodes
a mammary-specific secretory protein, mammaglobin, identified by
SEQ ID NO:2 (FIG. 2). As described below, the full length mamaglobin
cDNA was isolated starting from tumor cell mRNA that was reverse
transcribed, amplified using the technique of PCR and subcloned
into expression vectors. In addition, the protein, mammaglobin,
encoded by the cDNA was identified and characterized.
Using the anonymous sequence tag previously designated DEST002,
it was demonstrated that the corresponding gene product, which was
up until now unknown but herein identified as mammaglobin, is particularly
abundant in the breast cancer tumor cell line MDA-MB-415. To isolate
the full length mammaglobin cDNA, the mRNA was reverse transcribed
from this cell line and cloned using the RACE PCR technique (Edwards
et al. Nucleic Acids Research 19:5227-32, 1991). This technique
is based upon the strategy of ligation of single-stranded oligodeoxyribonucleotide
to the 3' end of single-stranded cDNA. The method by which the mammaglobin
cDNA was isolated is represented schematically in FIG. 1.
The full length 503 bp cDNA sequence (SEQ ID NO:1) was deduced
from the sequence information obtained from the 403 bp fragment
(SEQ ID NO:5) (FIG. 2) isolated by this technique along with sequence
information previously obtained from the corresponding DEST sequence
(DEST002, SEQ ID NO:6) (FIG. 2) in our earlier study (Watson and
Fleming, supra). Within the 503 bp cDNA is a 279 bp open reading
frame (SEQ ID NO:15) which encodes a polypeptide of 93 amino acids
(SEQ ID NO:2) (FIG. 2) and predicted molecular mass of 10.5 kD.
The initial methionine of this open reading frame is within a near-perfect
Kozak consensus sequence (Kozak, Cell 22:7-8, 1980). The 60 bp upstream
of this sequence contain no other in-frame methionines or translational
stops. The 3' untranslated sequence of the cDNA constitutes 163
bp and contains a polyadenylation signal, AATAAA, 12 bp upstream
of the priming site of the original DEST002 sequence. These data
indicate that the full length mammaglobin cDNA has been isolated.
The first 19 residues of the encoded polypetide predict a hydrophobic
peptide signal sequence and residues 53-55 and 68-70 are consensus
N-linked glycosylation sites, indicating that mammaglobin is a secreted
A search for DNA sequences similar to the mammaglobin cDNA sequence
in Genbank using the BLAST algorithm (Benson et al., Nucl Acid Res
21:2963-2965, 1993; Altschul et al, J Mol Biol 215:403-410, 1990),
identified no obvious DNA sequence homologies. Thus, mammaglobin
cDNA is believed to be a novel, heretofore unknown DNA sequence.
A search of other polypeptides for sequences related to mammaglobin
revealed an amino acid sequence homology between mammaglobin and
other polypeptides. Mammaglobin exhibited 42% amino acid identity
(58% including conservative substitutions) with rat prostatic steroid
binding protein (prostatein) subunit C3 (rPSC3) (FIG. 3) (SEQ ID
NO:7). Rat prostatic steroid binding protein is a major secretory
protein in the rat ventral prostate consisting of a tetrameric protein
composed of two different dimeric subunits; C3/C1 and C3/C2 (Parker
et al., Ann N Y Acad Sci 438:115-124; Parker et al., J Steroid Biochem
20:67-71,1984). The C1, C2, and C3 genes all encode approximately
6 kD secretory proteins and are thought to have arisen from gene
duplication, but while the C1 and C2 genes show strong homology
to each other, they are much less similar to the C3 gene. Correspondingly,
mamaglobin shows no sequence homology with the C1 or C2 proteins.
As noted above, prostatic steroid binding protein (prostatein)
is the major secretory protein in the rat ventral prostate and its
expression is regulated by androgenic steroids (Parker et al, Ann
N Y Acad Sci 438:115-24, 1984; Parker et al, J Steroid Biochem 20:67-71,
1984). Another protein, human estramustin-binding protein (hEMBP)
has been reported to be expressed in human prostate, human breast
cancer and human malignant melanoma. (Bjork et al, Cancer Res 42:1935-1942,
1982; Bjork et al, Anticancer Res 11:1173-82, 1991). Human estramustin-binding
protein is immunochemically similar to rat estramustin-binding protein,
which has been postulated to be identical to rat steroid-binding
protein, prostatein. As noted above, the amino acid sequence of
mammaglobin exhibited 42% amino acid identity and 58% homology including
conservative substitutions with the C3 subunit of prostatein. Thus
it is possible that mammaglobin could be in some way related to
hEMBP. However, while both prostatein and hEMBP are detected in
the prostate gland, mammaglobin mRNA is completely absent in this
tissue. Hence, mammaglobin is neither the same protein nor a subunit
of HEMBP and, furthermore, the sequence of hEMBP has not been determined
so that it is not known whether there is even any similarity of
mammaglobin with some fragment or subunit of HEMBP.
Although recent reports have demonstrated the rPSC3 promoter fused
to SV40 T antigen produces both prostatic and mammary carcinomas
in transgenic mice (Maroulakou et al., Proc Nat Acad Sci U.S. 91:11236-11240,
1994; Sandmoller et al, Oncogene 9:2805-2815, 1994), the true biological
function of this protein is unknown. Furthermore, notwithstanding
the hypothesized relationship of rat prostatic steroid binding protein
to human EMBP, no human polypeptide or human gene corresponding
to rPSC3 has been identified. Thus, mammaglobin and the cDNA encoding
mammaglobin represent novel sequences heretofore unknown.
Using manual alignment with other sequences that had less significant
BLAST scores with both mammaglobin and rPSC3 protein sequences,
we identified other homologies with human clara cell 10 kD protein
(hCC10) (SEQ ID NO:8) (Peri et al, J Clin Invest 92:2099-2109, 1993)
(FIG. 3) and, in addition, with rabbit and mouse uteroglobin proteins
(Miele et al., Endocrine Rev 8:474-90, 1987; Cato and Beato, Anticancer
Res 5:65-72, 1985; Miele et al., J Endocrinol Invest 17:679-692,
1994). These homologies, depending on species, were 26% identity
or 40% including conservative substitutions. In particular, a number
of amino acids were perfectly conserved among all proteins, including
Cys-3 and Cys-69 which are known to play a role in disulfide bond
formation between uteroglobin subunits (see below). These homologies
suggest that mammaglobin is a novel member of a small family of
proteins that are secreted by epithelial cells (Miele et al, 1994,
The hCC10 gene is the human homologue of rabbit and mouse uteroglobin
genes (Peri et al, J Clin Invest 92:2099-2109, 1993). Uteroglobin
was originally characterized as a secretory protein in rabbit uterus,
but has since been found in other epithelial organs including lung,
breast and prostate. Unlike rat prostatein, uteroglobin is a homodimeric
protein coupled by two disulfide linkages at the conserved residues
Cys-2 and Cys-69 (Miele et al, 1994, supra). Although uteroglobin
gene transcription is regulated by steroid hormones, the ability
of the protein itself to bind progesterone or other steroid hormones
is controversial and again, the true biological function of this
protein is unknown (Miele et al., 1994, supra).
Mammaglobin expression is restricted to the mammary gland. This
is in contrast to the observation that rPSC3 is expressed in rat
ventral prostate (Parker et al., Ann N Y Acad Sci 438:115-1124,
1984), and the expression of hCC10/uteroglobin in numerous tissues
including lung, uterus, prostate, and breast (Miele et al., 1987,
supra; Cato and Beato, supra; Miele et al., 1994 supra). Because
of the sequence homology between mammaglobin and these proteins,
we determined the pattern of tissue specific expression.
The 500 bp mammaglobin mRNA was easily detected in tumor specimen
2410 (the tissue from which this original sequence tag was isolated)
and to a much less extent in normal human breast tissue (FIG. 4A).
Mammaglobin mRNA could not be detected in the immortalized breast
epithelial cell line B5-589. Expression of mammaglobin was also
undetectable in human uterus and lung, two sites of uteroglobin
Amplification using RT/PCR detected mammaglobin mRNA in both tumor
2410 and normal breast tissue, but not in 15 other tissues surveyed,
including tissues that normally express rPSC3 and uteroglobin (lung,
uterus, prostate), hormonally responsive and steroidogenic tissues
(ovary, testis, placenta), and other secretory epithelial organs
(colon) (FIG. 4B). Therefore, the expression of mammaglobin mRNA
is relatively specific for mammary tissue.
To unequivocally demonstrate the breast-specific nature of mammaglobin
expression, the full-length mammaglobin cDNA was used to probe for
mammaglobin mRNA in a more extensive and quantitative panel of polyA-selected
mRNAs from pooled populations of adult and fetal human tissues as
described below in Example 2A. Mammaglobin mRNA expression was completely
absent in other closesly related apocrine glands such as the salivary
gland and was also undetectable in peripheral leukocytes, lymph
node, and bone marrow. Apart from the mammary gland, mammaglobin
mRNA was not detected in any other of the 43 adult or seven fetal
tissues surveyed. This confirms that mammaglobin gene expression
is likely to be a very specific marker for breast cancer.
Based on the studies in this report, mammaglobin is a relatively
mammary-specific protein. Two other genes known to be overexpressed
in breast carcinoma are erb-B and cyclin D (Jardines et al, Pathobiology
61:268-282, 1994; Keyomars and Pardee, Proc Nat Acad Sci U.S. 90:1112-1116,
1993). Unlike the overexpression of erb-B or cyclin D, the overexpression
of mammaglobin may reflect a more specific alteration of the mammary
epithelial cell rather than a general increased growth potential
or mitotic rate. As such, appearance of mammaglobin gene dysregulation
may have more specific import for the therapeutic vulnerability
or clinical course of a tumor.
As reported in U.S. Pat. No. 5,668,267, overexpression of mammaglobin
mRNA was detected by northern blot hybridization in the 2410 tumor
specimen as well as 4 of 15 (27%) stage 1 primary breast carcinomas
of differing histological types. This percentage is comparable to
the prevalence of other genetic alterations such as erb-B amplification
and p53 mutation (Slamon et al. Sci 244:707-712, 1989; Thor et al,
J Nat'l Cancer Inst 84:845-855, 1992). Furthermore, because we have
restricted our analysis to stage I tumors, overexpression of mammaglobin
would actually be more prevalent than any other genetic alteration
reported in this subgroup of tumors (Alllerd et al, J Nat'l Cancer
Inst 85:200-206, 1993). These data suggest that overexpression of
mammaglobin is not unique to a single tumor specimen and is, in
fact, relatively frequent among primary breast tumors. Furthermore,
the fact that all tumors examined were stage I suggests that this
dysregulation occurs relatively early in the progression of breast
Mammaglobin expression could not be detected in normal lymph nodes
or peripheral lymphocytes at the level of sensitivity afforded by
a single step RT/PCR assay. However, as reported below, mammaglobin
mRNA was detected in over 40% of lymph nodes and in 60% of peripheral
blood stem cell collections (PBSCs) from patients with metastatic
breast cancer. These results indicate that analysis of mammaglobin
transcripts in peripheral lymph nodes and blood stem cells may be
useful for detecting occult breast cancer metastases, as has been
suggested for other epithelial specific genes (Schoenfeld et al.,
Cancer Res 54:2986-2990, 1994).
To demonstrate that the mammaglobin cDNA encoded a translatable
protein, the cDNA clone was used in an in vitro translation assay.
FIG. 5 shows the protein product from a rabbit reticulocyte lysate
programmed with the mammaglobin cDNA. An approximately 6 kD protein
is generated using the mammaglobin cDNA. The apparent molecular
weight is smaller than that predicted from conceptual translation
of the open reading frame, but this finding is commonly observed
with rabbit and human uteroglobin translation products as well.
To examine the prevalence of mammaglobin protein expression in
breast cancer, rabbit polyclonal, anti-mammaglobin antibody was
generated against a peptide corresponding to the C-terminal mammaglobin
protein sequence and used to examine 100 primary breast tumors of
multiple grades and histological types for mammaglobin protein expression
by immunohistochemical analysis. As summarized in Table 1 below,
over 80% of these tumors were strongly immunopositive for mammaglobin
protein and staining was independent of tumor grade. This result
indicates that detection of mammaglobin protein in clinical samples
can be used as a sensitive and specific marker for primary breast
Because Applicants believe mammaglobin is a secreted protein, its
presence would be expected to be detectable in sera from patients
whose tumors overexpress this gene product. As such, mammaglobin
is likely to be as clinically useful as prostate specific antigen
(PSA) and other solid tumor markers for managing patients with breast
cancer (Tumor markers in diagnostic pathology, Clin Lab Med 10:1-250,
The identification of mammaglobin as a breast cancer marker provides
the basis for another aspect of the present invention, which involves
methods for detecting the presence of breast cancer in a patient.
The term "detection" as used herein in the context of
detection of breast neoplastic disease is intended to be a comprising
aspect of the determining of the presence of breast cancer in a
patient, the distinguishing of breast cancer from other diseases,
the estimation of prognosis in terms of probable outcome of the
disease and prospect for recovery, the monitoring of the disease
status or the recurrence of the disease, the determining of a preferred
therapeutic regimen for the patient and the targeting of antitumor
One method for detecting breast cancer comprises hybridizing a
polynucleotide to mRNA from breast neoplasia cells. The polynucleotide
comprises the complement of SEQ ID NO:15 or a derivative of the
complement of SEQ ID NO:15. As used herein, a polynucleotide includes
DNA and/or RNA and thus the nucleotide sequences recited in the
Sequence Listing as DNA sequences also include the identical RNA
sequences with uracil substituted for thymine residues. A derivative
of a nucleotide sequence has sufficient sequence identity to the
sequence from which it is derived to specifically hybridize to mammaglobin
mRNA from breast neoplasia cells under the same stringency conditions
that the sequence from which it is derived specifically hybridizes
to the mammaglobin mRNA from breast neoplasia cells. The derived
nucleotide sequence is not necessarily physically derived from the
nucleotide sequence, but may be generated in any manner including
for example, chemical synthesis or DNA replication or reverse transcription
or transcription. Preferred derivative nucleotide sequences include
fragments of the complement of SEQ ID NO:15 and sequences identical
to the complement of an allelic variant of the mammaglobin coding
sequence set forth in SEQ ID NO:15.
To detect the presence of mRNA encoding mammaglobin in a detection
system for breast cancer, a sample is obtained from a patient. The
sample can be a tissue biopsy sample or a sample of blood, plasma,
serum or the like. The sample may be treated to extract the nucleic
acids contained therein. Preferably, the resulting nucleic acid
from the sample is subjected to gel electrophoresis or other size
Detection involves contacting the nucleic acids and in particular
the mRNA of the sample with the polynucleotide probe to form hybridization
duplexes. The term "probe" refers to a structure comprised
of a polynucleotide which forms a hybrid structure with a target
sequence, due to complementarily of probe sequence with a sequence
in the target region.
Detection of the resulting duplex can be accomplished by any known
methodology. The hybridization duplex is usually accomplished by
the use of labeled probes. Alternatively, the probe may be unlabeled,
but may be detectable by specific binding with a ligand which is
labeled, either directly or indirectly. Suitable labels and methods
for labeling probes and ligands are known in the art, and include,
for example, radioactive labels which may be incorporated by known
methods (e.g., nick translation or kinasing), biotin, fluorescent
groups, chemiluminescent groups (e.g., dioxetanes, particularly
triggered dioxetanes), enzymes, antibodies, and the like.
When probing with a mammaloglobin cDNA, high stringency conditions
can be used in order to prevent false positives. When using a probe
containing a sequence derived from the complement of the mammaglobin
coding sequence, less stringent conditions can be used. The stringency
of hybridization is determined by a number of factors during hybridization
and during the washing procedure, including temperature, ionic strength,
length of time and concentration of formamide. These factors are
outlined in, for example, Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 2d ed., 1989).
In order to increase the sensitivity of detection of mammaglobin
mRNA in a sample, the technique of reverse transcription/polymerization
chain reaction (RT/PCR) can be used to amplify cDNA transcribed
from mRNA encoding mammaglobin. The method of RT/PCR is well known
in the art (for example, see Watson and Fleming, supra). Oligonucleotides
useful as the amplification primers comprise SEQ ID NO:3 and SEQ
ID NO:4. Preferably, the mammaglobin forward amplification primer
consists of 5'-AGCACTGCTACGCAGGCTCT-3' (SEQ ID NO:16) and the mammaglobin
reverse amplification primer consists of 5'-ATAAGAAAGAGAAGGTGTGG-3'
(SEQ ID NO:4).
Alternatively, a mammaglobin target sequence in the reverse transcribed
cDNA can be amplified and detected using any other known methodology
such as ligase chain reaction methods, including gap LCR (G-LCR)
and other variations, or self-sustained sequence replication (3SR)
and its various modifications. In addition, the mammaglobin mRNA
can be detected directly by asymmetric gap LCR (AG-LCR). See, e.g.,
Leckie et al., "Infectious Disease Testing by Ligase Chain
Reaction" in Molecular Biology and Biotechnology, R. A. Myers,
ed., pp. 463-466, VCH Publishers, 1995.
In another embodiment of the present invention, the mammaglobin
cDNA sequence or derivative thereof can be used to characterize
any alteration of the mammaglobin gene (i.e. gene rearrangement,
gene amplification, or gene deletion) in a specimen from a breast-cancer
patient. This provides a method whereby patient specimens or samples,
which do not contain intact mRNA, can still be examined for changes
in gene structure.
In one application of this technique, the mammaglobin cDNA sequence
or derivative thereof is hybridized to patient genomic DNA that
has been isolated from a patient's tumor, normal tissue, or lymphocytes
and digested with one or more restriction endonucleases. Using the
Southern blot protocol, which is well known in the art, this assay
determines whether a patient or a patient's breast tumor has a mammaglobin
gene, which was deleted, rearranged, or amplified. Detection of
these changes can then provide important information useful for
predicting prognosis and for patient management.
In a second application of this technique, one or more pairs of
oligonucleotide primers based on the mammaglobin cDNA sequence and
complement thereof could be used in the polymerase chain reaction
to amplify segments of the mammaglobin gene from a patient sample.
Analysis of the resulting PCR products indicate whether a particular
segment of the mammaglobin gene is deleted or rearranged. Such information
is useful for prognosis and patient management.
Another method for detecting breast cancer comprises detecting
the presence of the precursor and/or secreted forms of the mammaglobin
polypeptide in a sample obtained from a patient. Any method known
in the art for detecting proteins can be used. Such methods include,
but are not limited to immunodiffusion, immunoelectrophoresis, immunochemical
methods, binder-ligand assays, immunohistochemical techniques, agglutination
and complement assays. (for example see Basic and Clinical Immunology,
Sites and Terr, eds., Appleton & Lange, Norwalk, Conn. pp 217-262,
1991). Preferred are binder-ligand immunoassay methods including
reacting antibodies with an epitope or epitopes of mammaglobin and
competitively displacing a labeled mammaglobin polypeptide or derivative
As used herein, the term "mammaglobin polypeptide" embraces
naturally occurring mammaglobin, including nonglycosylated and glycosylated
precursor forms and the glycosylated secreted form, derivatives
and fragments thereof. By naturally occurring is meant a polypeptide
that can be isolated from a source in nature, e.g., from normal
and/or diseased organisms, and that has not been intentionally modified
by man. Naturally-occurring mammaglobin polypeptides identified
herein include a precursor form which comprises SEQ ID NO:2 and
a secreted form comprising SEQ ID NO:17 (amino acids 20-93 of SEQ
ID NO:2). Allelic variants of SEQ ID NO:2 are also intended to be
included in the scope of naturally-occurring mammaglobin polypeptides.
A derivative of a mammaglobin polypeptide is intended to refer
to polypeptides which are comprised of a segment of at least 10
amino acids that has substantial identity to a portion of naturally
occurring mammaglobin. The segment having substantial identity is
preferably at least about 20 amino acids, more preferably at least
about 50 amino acids, and most preferably at least about 75 amino
acids. Two polypeptides have substantial identity when upon optimal
alignment by sequence alignment programs such as BLAST using default
settings for gap penalties and other parameters, they share at least
80% sequence identity, preferably at least 90% sequence identity,
more preferably at least 95% sequence identity, most preferably
99% sequence identity. Preferably, residue positions which are not
identical differ by conservative amino acid substitutions.
Conservative amino acid substitutions refer to the interchangeability
of residues having similar side chains. For example, a group of
amino acids having aliphatic side chains is glycine, alanine, valine,
leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl
side chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine, tyrosine,
and tryptophan; a group of amino acids having basic side chains
is lysine, arginine, and histidine; and a group of amino acids having
sulfur-containing side chains is cysteine and methionine. Preferred
conservative amino acid substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
A derivative mammaglobin polypeptide will preferably cross-react
with an anti-mammaglobin antibody, monoclonal or polyclonal, which
is specific for naturally occurring mammaglobin or fragments thereof.
As used herein the terms "fragment" and "peptide"
refer to a mammaglobin polypeptide having an amino acid sequence
identical to the amino acid sequence deduced from a full-length
mammaglobin cDNA (e.g., SEQ ID NO:1), but that has an amino-terminal
and/or carboxy-terminal deletion. Typically, mammaglobin fragments
or peptides are at least 3 amino acids long. Preferably a mammaglobin
fragment or peptide is at least 6 amino acid residues in length,
more preferably about 12 amino acid residues in length, even more
preferably about 25 amino acid residues in length, and most preferably
50 amino acid residues or greater.
Numerous competitive and non-competitive protein binding immunoassays
are well known in the art. Antibodies employed in such assays may
be unlabeled, for example as used in agglutination tests, or labeled
for use in a wide variety of assay methods. Labels that can be used
include radionuclides, enzymes, fluorescers, chemiluminescers, enzyme
substrates or co-factors, enzyme inhibitors, particles, dyes and
the like for use in radioimmunoassay (RIA), enzyme immunoassays,
e.g., enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassays
and the like.
Polyclonal or monoclonal antibodies to a mammaglobin polypeptide
comprising a B cell epitope can be made for use in immunoassays
by any of a number of methods known in the art. As used herein,
the term "B cell epitope" refers to an antigenic determinant
of a mammaglobin polypeptide. The B cell epitope could comprise
3 amino acids in a spatial conformation which is unique to the epitope.
Generally, a B cell epitope consists of at least 5 such amino acids.
Methods of determining the spatial conformation of amino acids are
known in the art, and include, for example, x-ray crystallography
and 2 dimensional nuclear magnetic resonance.
One approach for preparing antibodies to a protein is the selection
and preparation of an amino acid sequence of all or part of the
protein, chemically synthesizing the sequence and injecting it into
an appropriate animal, usually a rabbit or a mouse.
Methods for preparation of a mammaglobin polypeptide include, but
are not limited to chemical synthesis, recombinant DNA techniques
or isolation from biological samples. Chemical synthesis of a peptide
comprising an epitope can be performed, for example, by the classical
Merrifeld method of solid phase peptide synthesis (Merrifeld, J
Am Chem Soc 85:2149, 1963) or the FMOC strategy on a Rapid Automated
Multiple Peptide Synthesis system (DuPont Company, Wilmington, Del.)
(Caprino and Han, J Org Chem 37:3404, 1972).
Polyclonal antibodies can be prepared by immunizing rabbits by
injecting antigen into the popliteal lymph nodes followed by subsequent
boosts at two week intervals with intraperitoneal injection of antigen.
The animals are bled and sera assayed against purified mammaglobin
protein, usually by ELISA. Monoclonal antibodies can be prepared
after the method of Milstein and Kohler by fusing splenocytes from
immunized mice with continuously replicating tumor cells such as
myeloma or lymphoma cells. (Milstein and Kohler Nature 256:495-497,
1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical
Techniques 73:1-46, Langone and Banatis eds., Academic Press, 1981).
The hybridoma cells so formed are then cloned by limiting dilution
methods and supernates assayed for antibody production by ELISA
Thus prepared polyclonal or monoclonal antibodies to mammaglobin
may be used to isolate and purify precursor and secreted forms of
mammaglobin from cells expressing mammaglobin. For example, as shown
below, a polyclonal antibody generated against the 16 C-terminal
amino acids predicted from mammaglobin cDNA (Glu-Val-Phe-Met-Gln-Leu-Ile-Tyr-Asp-Ser-Ser-Leu-Cys-Asp-Leu-Phe,
SEQ ID NO:14) binds to precursor and secreted forms of mammaglobin,
as well as to mammaglobin that has been synthesized in an in vitro
translation system. Isolation of mammaglobin using an anti-mammaglobin
antibody may be accomplished using procedures well-known in the
art, such as affinity chromatography.
The unique ability of antibodies to recognize and specifically
bind to target antigens expressed by a tumor cell provides an approach
for the treatment of cancer. (For review see LoBuglio and Saleh,
Am J Med Sci 304:214-224, 1992; Bagshawe, Adv Pharmacol 24:99-121,
1993). Thus, another aspect of the present invention provides for
a method for preventing the onset and treating breast cancer in
an animal based upon the use of antibodies to mammaglobin, which
has been discovered to be overexpressed by breast cancer cells.
Specific antibodies to mammaglobin, either polyclonal or monoclonal,
are produced by any method known in the art. For example, murine
or human monoclonal antibodies can be produced by hybridoma technology.
Alternatively, mammaglobin, or an immunologically active derivative
or fragment thereof, or an anti-idiotypic antibody, or fragment
thereof, can be administered to an animal to elicit B cell production
of antibodies capable of recognizing the mammaglobin-expressing
The antibodies so produced or fragments thereof are labeled with
one or more oncolytic substances such as radionuclides, toxins,
or cytotoxic drugs and administered to a patient suspected of having
breast cancer. The binding of the labeled antibody to the mammaglobin
being overexpressed by the breast cancer cell will cause the death
of the cancer cell.
Any of a variety of oncolytic substances known in the art can be
used to produce such labeled antibodies. For example, immunotoxins
can be made by coupling plant and bacterial toxins to antibodies.
Such toxins include, for example, ricin, diphtheria toxin and Pseudomonas
exotoxin A. Drug-antibody conjugates can also be made in which chemotherapeutic
agents are linked to the antibody. Chemotherapeutic agents suitable
for such use include, for example, tomoxifen, doxorubicin, methotrexate,
chlorambucil, Vinca alkaloids, and mitomycin. In addition, radioimmunoconjugates
can be made in which a radionuclide is stably linked to the antibody.
Radionuclides suitable for making radioimmunoconjugates include,
for example, .beta.-emmitters such as .sup.131 I, .sup.188 Re, .sup.186
Re, .sup.67 Cu, .sup.90 Y and .sup.47 Sc; .alpha.-emitters such
as .sup.211 At, .sup.212 Bi and .sup.212 Pb; auger electron emitters
such as 125I and .sup.77 Br; and fissionable nuclides such as .sup.10
The finding that a significant percentage of breast tumors express
mammaglobin protein is the basis for another aspect of the invention,
which involves the activation of mammaglobin-specific B and/or T
cell lymphocytes (T.sub.C) with mammaglobin antigens. Accordingly,
the invention provides mammaglobin B cell antigens and T.sub.C cell
antigens; vaccines comprising at least one B cell mammaglobin antigen
and/or at least one T.sub.C mammaglobin antigen for inducing antibody-
and/or cell-mediated immune responses against mammaglobin-expressing
tumors, and methods for treating a breast cancer patient with a
mammaglobin-expressing tumor. One method according to the invention
comprises administering to the patient activated mammaglobin-specific
lymphocytes. Another method comprises administering to the patient
a mammaglobin-specific vaccine.
As used herein, "mammaglobin antigen" includes naturally
occurring mammaglobin polypeptides, derivatives, and fragments thereof
which contain a B cell or T.sub.C cell epitope recognized by mammaglobin-specific
B cells or T.sub.C cells.
A mammaglobin B cell antigen comprises a mammaglobin-specific B
cell epitope and a T.sub.H cell epitope. The term "B-cell epitope"
refers to any antigen, hapten, epitope or antigenic determinant
which is recognized by anti-mammaglobin immunoglobulin receptors
on B cells and is capable of eliciting the production of antibodies
with appropriate help from T.sub.H cells when administered to an
animal. The B cell epitope comprises an amino acid sequence of at
least 4 amino acids. Preferably, the B cell epitope is between at
least 6 and 25 amino acids in length and more preferably is between
about 15 and 22 amino acids in length. The comprising amino acid
sequence of the B cell epitope may be identical or substantially
identical to a continuous amino acid sequence in a fragment of naturally
occurring mammaglobin. Alternatively, the comprising amino acid
sequence of a B cell epitope is identical to or substantially identical
to a discontinuous amino acid sequence representing an assembled
topographic determinant of mammaglobin.
The term "T.sub.H cell epitope" refers to any antigenic
determinant recognized by T helper cells through association with
MHC class II molecules. The activation of T helper cells induces
differentiation of resting mammaglobin-specific B cells into higher
affinity IgG-secreting cells, i.e, induces a secondary antibody
response. The preparation and use of immunogenic peptides containing
B and T.sub.H cell determinants to produce higher titres of specific
antibody-producing B cells through T cell help is known in the art,
see, e.g., Cheronis et la., U.S. Pat. No. 5,573,916, Denton, et
la., Cancer Letters 70:143-150 (1993), Borras-Cuesta et al., Eur.
J Immunol. 17, 1213-1215 (1987), and Good et al., Science 235:1059-1062
(1987). The T.sub.H cell epitope may comprise an amino acid sequence
from mammaglobin or a heterologous protein. For example, Denton
et al. describe the induction of antibody responses to mucins, which
are complex glycoproteins expressed in secretory epithelia and associated
with breast and other carcinomas, in mice immunized with a synthetic
peptide containing a B cell determinant region from the core of
MUC-1 mucin linked to sequence 111-120 of influenza haemagglutinin
A/X-3 1, a known helper T cell-determinant. The T.sub.H cell epitope
comprises an amino acid sequence of between about 6 to about 20
amino acid residues, preferably between about 8 residues and 18
residues, even more preferably between 9 residues and 15 residues.
A mammaglobin T.sub.C cell antigen comprises a T.sub.C cell epitope
and a MHC class I agretope. The term "T.sub.C cell epitope"
means any antigen, epitope or antigenic determinant which is recognized
by mammaglobin-specific T.sub.C cells when presented by a MHC class
I molecule on the surface of an antigen presenting cell. The term
"MHC class I agretope" refers to any amino acid sequence
recognized by a MHC class I molecule that allows the mammaglobin
antigen to be presented to a mammaglobin-specific T.sub.C cell by
the MHC class I molecule on an antigen presenting cell (APC). The
T.sub.C cell epitope and MHC class I agretope are contained within
an amino acid sequence of between about 6 to about 11 amino acids
that is identical or substantially identical to the amino acid sequence
of a fragment of naturally occurring mammaglobin. Preferably, the
sequence is 8 or 9 amino acids in length.
Methods for identifying B and T.sub.C cell epitopes for a protein
antigen are known in the art. For example, the capacity of isolated
mammaglobin-specific B cells or mammaglobin-specific T.sub.C cells
to respond to overlapping synthetic peptides spanning secreted mammaglobin
may be determined using standard immunobiology techniques. Those
peptides identified as antigenic may then be modified one or a few
amino acids at a time to optimize their ability to stimulate mammaglobin-specific
B or T cells.
B cell epitopes can also be mapped using commercially available
epitope mapping kits which involve the screening of random peptides
bound at the C terminus to polyethylene multipin supports, e.g.,
Cambridge Research Biochemicals.
Alternatively, the predicted mammaglobin amino acid sequence may
be searched for sequences that conform to known binding motifs of
MHC class I or MHC class II molecules. See e.g, Hill et al., Nature
360:434 (1992), Pamer et al., Nature 360:852 (1992) and Hammer et
al., J. Exp. Med. 176:1007 (1992), and Falk et al., Nature 351:290-296
(1991). For example, antigenic peptides recognizable by breast tumor-specific
CTLs may be identified by searching the mammaglobin amino acid sequence
for HLA-A2-binding peptides as described by Peoples et al., Proc.
Natl. Acad. Sci. 92:432-436 (1995). The choice of HLA-A2 as the
antigen presenting molecule is appropriate where the patient expresses
HLA-A2 (approximately 50% of Caucasians express HLA-A2). The predicted
HLA-A2 binding peptides can be synthesized and tested for antigenicity
by loading the synthetic peptides onto the T2 cell line, a human
T-cell/B-cell fusion product containing a defect in antigen presentation
such that HLA-A2 molecules on the surface of T2 cells can be effectively
loaded with exogenous HLA-A2 binding peptides (Henderson, et al,
Science 255:1264-1266 (1992)). A standard cytotoxicity assay is
then carried out which comprises incubating the peptide-loaded T2
cells with breast-specific CTLs derived from tumor infiltrating
lymphocytes (TILs) isolated from a mammaglobin-expressing breast
tumor, e.g., see Peoples et al., pages 432-433 and Toso et al.,
Cancer Research 56:16-20 (1996).
Antigenic mammaglobin peptides containing T.sub.C cell epitopes
may also be identified by acid-eluting endogenous peptides presented
by HLA class I molecules on the tumor cell surface. (See, e.g.,
Peoples et al., supra, p. 433). The eluted peptides may be separated
by any number of techniques known in the art, including HPLC fractionation.
The different peptide fractions are loaded onto T2 cells and the
loaded T2 cells are incubated with breast-tumor specific CTLs to
determine which peptides are recognized by the CTLs using standard
One use of a mammaglobin antigen according to the invention is
in adoptive immunotherapy. This therapy involves in vitro activation
and expansion by a mammaglobin antigen of anti-mammaglobin antibody-producing
B cells and/or mammaglobin-specific cytotoxic T lymphocytes (CTLs)
isolated from a patient with a mammaglobin-expressing tumor. The
method may also be practiced with a composition comprising both
mammaglobin B cell and T.sub.C cell antigens. The activated lymphocytes
are then introduced back into the patient for adoptive immunotherapy.
A mammaglobin antigen according to the invention is also useful
as a component of a mammaglobin-specific vaccine. The vaccine comprises
an immunogenically-stimulatory amount of a mammaglobin antigen.
As used herein, an immunostimulatory amount refers to that amount
of antigen that is able to stimulate the desired immune response
in the recipient for the amelioration, or treatment, of breast cancer.
This amount may be determined empirically by standard procedures,
well known to those of ordinary skill in the art, without undue
The antigen may be provided in any one of a number of vaccine formulations
which are designed to induce the desired type of immune response,
e.g., antibody and/or cell mediated. Such formulations are known
in the art. See, e.g., A. Lanzavecchia, Science 260:937-944 (1993)
and U.S. Pat. No. 5,585,103 to Raychandhuri. Examples of vaccine
formulations used to stimulate immune responses include pharmaceutically
acceptable adjuvants such as aluminum salts; emulsions of squalene
or squalane and muramyl dipeptide; liposomes; and a composition
comprising a stabilizing detergent, a micelle-forming agent, and
a biodegradable and biocompatible oil (Raychandhuri, supra).
A mammaglobin-specific vaccine may also comprise a carrier cell
loaded with a mammaglobin antigen. Preferably, the carrier cell
is prepared from autologous professional antigen presenting cells
(APC) such as macrophages, dendritic cells, or activated B or T
lymphocytes. See e.g., Lanzavecchia, supra, p. 937. Professional
APCs express a ligand, B7, that binds to CD28 or CTLA4 on T cells
to deliver an antigen- nonspecific costimulatory signal known as
Signal 2 which prevents T cell anergy or inactivation. Thus, the
vaccine may also comprise interleukin-2 or another costimulatory
signal to counteract anergy induction. (Lanzavecchia, supra, p.
Another formulation of a mammaglobin-specific vaccine comprises
a recombinant vector containing a nucleotide sequence encoding for
expression a mammaglobin antigen. The use of infectious agents to
stimulate cytotoxic T lymphocytes is known in the art. (Raychaudhuri,
supra.) Chimeric vectors have been described using vaccinia, polio,
adeno- and retro-viruses, as well as bacteria such as Listeria and
BCG. For example, a canarypox virus vector, ALVAC, has been shown
to elicit strong cellular immune responses against encoded heterologous
gene products (Taylor et al, Virology 187:321-328 (1991)). In addition,
a recombinant ALVAC expressing the MZ2-E human melanoma rejection
antigen encoded by the MAGE-1 gene is able to stimulate in vitro
MAGE-1 CTL activities in a TIL population derived from a breast
tumor expressing MAGE-1 mRNA (Toso et al, supra). In another approach
described in U.S. Pat. No. 5593972 to Weiner et al, a recombinant
expression vector encoding an antigen of an immunogenic protein
to be targeted is directly administered to an individual either
in vivo, e.g., to muscle cells, or to the cells of an individual
ex vivo along with an agent that facilitates uptake of the DNA into
Those skilled in the art may readily determine how to formulate
a vaccine suitable for achieving the desired immune response. For
example, for inducing in vivo production of anti-mammaglobin antibodies,
a mammaglobin-specific vaccine comprises at least one mammaglobin
B cell antigen comprising a B cell epitope and a T.sub.H cell epitope.
The T.sub.H cell epitope is preferably matched with the appropriate
MHC class II haplotype of the intended vaccine recipient. Alternatively,
a T.sub.H cell epitope could be used that is known to be recognized
universally by humans regardless of HLA type such as the "universal"
T cell epitope from tetanus toxoid (Panina-Bordignon et al. Eur.
J. Immunol. 19:2237 (1989). Preferably, the vaccine comprises a
plurality of mammaglobin B cell antigens with T.sub.H epitopes recognized
by MHC Class II molecules of different HLA types.
Another embodiment of a mammaglobin-specific vaccine induces a
cell-mediated response and comprises at least one mammaglobin T.sub.C
antigen capable of activating mammaglobin-specific T.sub.C cells.
Preferably, the vaccine comprises several T.sub.C cell antigens.
A mammaglobin-specific vaccine may also be formulated to induce
both antibody and cell-mediated responses. This embodiment comprises
both mammaglobin B cell and T.sub.C cell antigens.
A patient with a mammaglobin-expressing tumor may be treated by
administering to the patient an immunostimulatory amount of a mammaglobin-specific
vaccine according to the present invention. Administration of the
vaccine may be by any known or standard technique. These include,
but are not limited to intravenous, intraperitoneal, intramuscular,
subcutaneous, or intramammary injection.
Preferred embodiments of the invention are described in the following
examples. Other embodiments within the scope of the claims herein
will be apparent to one skilled in the art from consideration of
the specification or practice of the invention as disclosed herein.
It is intended that the specification, together with the examples,
be considered exemplary only, with the scope and spirit of the invention
being indicated by the claims which follow the examples.
In the examples below, cell lines were obtained from American Type
Culture Collection and grown in Dulbecco's minimal essential medium
supplemented with 10% fetal calf serum. Tissue biopsy specimens
were obtained from the Human Cooperative Tissue Network (LiVolsi
et al, Cancer 71:1391-1394, 1993).
This example illustrates the isolation of mammaglobin cDNA.
Total cellular RNA from the cell line MDA-MB415 was isolated using
the standard guanidinium isothiocyanate method. (Belyavsky et al,
supra). This RNA was used in the RACE PCR procedure employing the
Amplifinder kit (Clonetech) and following the manufacturer's protocol.
The synthesis of first strand cDNA was performed in a standard
reaction containing 1 .mu.g RNA, 10 .mu.M specific mammaglobin primer
D2R (5'-ATA AGA AAG AGA AGG TGT GG-3')(SEQ ID NO:4), 4 .mu.l of
5.times.RT buffer (250 mM TrisCl pH8.3, 375 mM Kcl, 15 mM MgCl.sub.2),
2 .mu.l of 100 mM DTT, 1 .mu.l of 10 mM dNTPs and 200 units of Superscript.TM.
II reverse transcriptase (Gibco/BRL) in a reaction volume 20 .mu.l.
The reaction proceeded for 1 hour at 45.degree. C. and was terminated
by incubating at 95.degree. C. for 5 minutes. RNA was hydrolyzed
with 400 .mu.M NaOH at 65.degree. C. for 30 minutes and neutralized
with 400 .mu.M acetic acid.
The reaction mixture was then added to 3 volumes of 6M NaI and
10 .mu.l of treated glass beads. Beads were washed three times with
80% EtOH and nucleic acid was eluted from the beads in 45 .mu.l
of water. Nucleic acid was then precipitated and resuspended in
10 .mu.l of water. The purified first strand cDNA was ligated to
the manufacturer's provided anchor oligonucleotide (SEQ ID NO:9,
5'-CAC GAA TTC ACT ATC GAT TCT GGA ACC TTC AGA GG-3'), using T4
RNA ligase at 27.degree. for 20 hours. One tenth of a ligation reaction
was used for PCR amplification in a 50 .mu.l reaction containing
1 .mu.M manufacturer's anchor primer (SEQ ID NO:10, 5'-CTG GTT CGG
CCC ACC TCT GAA GGT TCC AGA ATC GAT AG-3.mu.), 1 .mu.M mammaglobin
specific primer D2Rb (SEQ ID NO:11, 5'-AAT CCG TAG TTG GTT TCT CAC
C-3'), 200 .mu.M dNTPs, 5 units of Vent.TM. DNA polymerase, and
1.times.polymerase buffer (10 mM Kcl, 20 mM TrisCl, 10 mM (NH.sub.4).sub.2
SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton X-100). The reaction was
incubated at 94.degree. for 2 minutes and then 94.degree. for 45
seconds, 50.degree. for 1 minute, and 72.degree. for 90 seconds
for a total of 40 times.
The two downstream mammaglobin-specific nested oligonucleotides
were D2R (SEQ ID NO:4) and D2Rb (SEQ ID NO:11). An upstream mammaglobin-specific
control oligonucleotide was also used as per the manufacturer's
recommendations, D2F (5'-CTT TCT GCA AGA CCT TTG GC-3')(SEQ ID NO:12).
All PCR amplifications were performed with Vent DNA polymerase (New
England Biolabs). The amplified RACE product was digested with EcoRI
and ligated into the EcoRI and SmaI sites of the plasmid vector
pGEM7Z (Promega, Madison, Wis.).
All sequencing was performed using the Taq DNA polymerase thermal
cycle sequencing kit as per the manufacture's protocol (Promega).
Briefly the procedure used is as follows.
Ten pmol of sequence specific oligonucleotide was end labeled with
10 pmol of .sup.32 P-.gamma. ATP (3,000 Ci/mmol and 10 mCi/ml) using
T4 polynucleotide kinase in a 10 .mu.l reaction for 30 minutes at
37.degree. C. A polymerization reaction containing 100 ng of plasmid
template, 1.5 pmol of labeled sequencing primer, and 5 units of
sequencing grade Taq polymerase was created in 17 .mu.l of the manufacturer's
provided sequencing buffer. This reaction was aliquoted to a set
of four reaction tubes containing manufacturer's provided mix of
deoxynucleotides and either dideoxy-A, C, G, or T. The set of four
tubes were incubated at 95.degree. C. for 2 minutes and then, 94.degree.
C. for 45 seconds, 45.degree. C. for 30 seconds, and 72.degree.
C. for 1 minute for 30 times. After reactions were completed, 3
.mu.l of 80% formamide/bromphenol blue dye was added to each tube.
Samples were heated to 70.degree. C. for 2 minutes and loaded on
a 6% acrylamide/7.5 M urea sequencing gel and run for 2-4 hours
and 60 W constant power. The gel was dried and then exposed to Kodak
XAR5 Xray film for 2 to 24 hours.
The sequence thus obtained was a 403 bp fragment (SEQ ID NO:5)
as shown in FIG. 2, solid bar. In earlier work the DEST002 Tag sequence
was isolated (Watson and Fleming, supra). This sequence was a 206
bp fragment (SEQ ID NO:6) as shown in FIG. 2, open bar. Combining
the information from these two sequences allowed the full-length
503 bp cDNA of mammaglobin to be deduced. (FIG. 2).
This example demonstrates that mammaglobin expression is restricted
to mammary gland tumor cells and, to a lesser extent, normal mammary
Total cellular RNA samples were isolated using the standard guanidinium
isothiocyanate method and treated with RNase-free DNase (Promega).
For RT/PCR analysis, 1 .mu.g of indicated total RNA was reverse
transcribed with oligo dT.sub.21 (SEQ ID NO:13) and Superscript
II reverse transcriptase (Gibco/BRL) according to the manufacture's
Two hundred ng of oligo dT.sub.21 (SEQ ID NO:13) and 1 pg of total
RNA were incubated at 65.degree. C. for 5 minutes in a 10 .mu.l
volume. Sample was chilled on ice and added to it were 4 .mu.l of
5.times.RT buffer (250 mM TrisCl pH8.3, 375 mM Kcl, 15 mM MgCl.sub.2),
2 .mu.l of 100 mM DTT, 1 .mu.l of 10 mM dNTPs and 200 units of Superscript.TM.
II reverse transcriptase (Gibco/BRL). The reaction proceeded for
1 hour at 45.degree. C. and was terminated by incubating at 95.degree.
C. for 5 minutes.
One tenth of each RT reaction was subject to PCR analysis using
the mammaglobin specific primers D2R (5'-ATA AGA AAG AGA AGG TGT
GG-3') (SEQ ID NO:4) and d2102 (5'-CAG CGG CTT CCT TGA TCC TTG-3')(SEQ
ID NO:3) and standard reaction conditions for 40 cycles at 94.degree..times.30
sec./55.degree..times.1 min./72.degree..times.1 min.
For Northern analysis, 20 .mu.g of total RNA was analyzed as previously
described (Watson and Fleming, supra) using the full length mammaglobin
cDNA probe. Integrity and equal loading of each RNA sample was assessed
by ethidium bromide staining.
As shown in FIG. 4A, the 500 bp mammaglobin message is easily detected
in tumor specimen 2410 (the tissue from which this original DEST
was isolated) and to a much lesser extent in normal human breast
tissue but not in the immortalized breast epithelial cell line B5-589,
or in human lung, placenta, uterus and ovary (FIG. 4A). Following
amplification using RT/PCR analysis, mammaglobin expression was
still not detected in 15 tissues surveyed (FIG. 4B). Detection of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) message (FIG. 4B)
and EGF receptor message (data not shown) in these reactions demonstrated
that absence of expression was not due to degraded RNA or other
trivial explanations. Thus the expression of mammaglobin mRNA is
relatively specific for mammary tissue.
This example confirms the breast specificity of mammaglobin expression
in normal tissues.
The mammaglobin cDNA probe used in Example 2 was hybridized to
a commercially prepared dot-blot of normalized, poly-A enriched
RNAs derived from pooled populations of normal human tissues (Master
Blot.TM., Clonetech, Palo Alto, Calif.). The dot-blot contained
RNA from the following human tissues: whole brain, amygdala, caudate
nucleus, cerebelum, cerebral cortex, frontal lobe, hippocamus, medulla
oblongata, occipital lobe, putamen, substantia nigra, temporal lobe,
thalamus, nucleus accumbeus, spinal cord, heart, aorta, skeletal
muscle, colon, bladder, uterus, prostate, stomach, testis, ovary,
pancreas, pituitary gland, adrenal gland, thyroid gland, salivary
gland, mammary gland, kidney, liver, small intestine, spleen, thymus,
peripheral leukocyte, lymph node, bone marrow, appendix, lung, trachea,
placenta, fetal brain, fetal heart, fetal kidney, fetal liver, fetal
spleen, fetal thymus, and fetal lung.
The mammaglobin cDNA probe was radiolabled with .sup.32 P-.alpha.-dCTP
(10 mCi/ml; >3000 Ci/mmol) and the Rediprime labeling kit (Amersham,
Arlington Heights, Ill.) following the supplier's protocol. The
dot blot was hybridized with 1.times.10.sup.6 CPM/ml of the mammaglobin
cDNA probe at 60.degree. C. for 16 hours using Rapid-Hyb hybridization
buffer (Amersham, Arlington Heights, Ill.). Filters were then washed
twice at room temperature for 15 min. in 2.times.SSC/0.1% SDS, and
washed twice again at 60.degree. C. for one hour in 0.2.times.SSC/0.1%
SDS. Washed filters were exposed to XAR5 film (Eastman-Kodak, Rochester,
N.Y.) with phosphor enhancing screens for 72 hours.
Mammaglobin mRNA was only detected in RNA from the mammary gland
(data not shown). The lack of detectable mammaglobin mRNA in normal
salivary gland and prostate indicates that mammaglobin expression
is not associated with the apocrine phenotype as opposed to the
apocrine cell specificity of GCDP15, which has been used as a breast-specific
marker. Wick et al., Hum Pathol 20:281-287, 1989; Raab et al., Am
J Clin Pathol 100:27-35, 1993. These results suggest potential advantages
for using mammaglobin gene expression as a very specific marker
for breast cancer.
This example demonstrates that the mammaglobin cDNA encodes a translatable
nucleotide sequence which results in a protein product of appropriately
predicted molecular mass. In vitro translations were performed using
the TNT.TM. rabbit reticulocyte translation kit with T7 RNA polymerase
(Promega) and .sup.35 S-Methionine (>1000 Ci/mmol; 10 mCi/ml,
Amersham) according to the manufacturer's protocol.
To 25 .mu.l of TNT.TM. rabbit reticulocyte lystae was added 2 .mu.l
of manufacturer's prepared reaction buffer, T7 RNA polymerase, 20
.mu.M amino acid mixture minus methionine, 40 .mu.Ci.sup.35 S-methionine
(1,000 Ci/mmol and 10 mCi/ml), 40 units ribonuclease inhibitor,
1 .mu.g of mammaglobin/pGEM7 plasmid, and sufficient DEPC treated
water to create a final reaction volume of 50 .mu.l. This reaction
was incubated at 30.degree. C. for 60 minutes. 5 .mu.l of this reaction
was removed into 20 .mu.l of SDS gel buffer, boiled for 2 minutes,
and loaded on a 17.5% SDS-polyacrylamide gel.
Rabbit reticulocyte lysate programmed with mammaglobin cDNA produced
a 6 kD protein while that programmed with no cDNA did not produce
any protein product.
This example illustrates the prevalence of overexpression of mammaglobin
in primary breast carcinoma.
To determine the frequency of mammaglobin overexpression in breast
carcinomas, we examined a panel of fifteen, stage I primary breast
carcinomas of differing histological types using Northern blot hybridization
with the mammaglobin cDNA probe. Patient-matched normal breast tissues
samples were also compared in tissues from two patients (FIG. 6).
The 500 bp mammaglobin mRNA was detected in normal breast tissue
and tumor 2410 and in three other tumors, two of which when tested
demonstrated little or no expression in patient-matched normal tissue
(BO15 v. BO16; B022 v. B023) (FIG. 6). In all, 4 of 15 (27%) of
tumors examined overexpressed mammaglobin mRNA.
These data indicate that overexpression of mammaglobin is not unique
to a single tumor specimen and is, in fact, relatively frequent
among primary breast tumors. Furthermore, the fact that all tumors
examined were stage I suggests that this dysregulation occurs relatively
early in the progression of breast neoplasia.
The following example illustrates the detection of the mammaglobin
protein using an anti-mammaglobin polyclonal antibody.
The anti-mammaglobin polyclonal antibody was prepared by coupling
a peptide corresponding to the 16 C-terminal amino acids predicted
from mammaglobin cDNA (Glu-Val-Phe-Met-Gln-Leu-Ile-Tyr-Asp-Ser-Ser-Leu-Cys-Asp-Leu-Phe,
SEQ ID NO:14) to Keyhole Lymphet Hemocyanin and injecting into rabbits
with Freund's adjuvant. The inoculated rabbits were boosted at three
week intervals and on week 12, the rabbits were bled and the sera
was assayed for its ability to detect mammaglobin in serum-free
conditioned medium from cultures of the breast tumor cell lines
MDA-MB-415 and MCF-7. MDA-MB-415 had been identified earlier as
a cell line that overexpresses the mammaglobin message and MCF-7
had been identified as a cell line that produces no detectable mammaglobin
The conditioned media was harvested from a 24 hr. culture and resolved
on a 12% SDS acrylamide gel under reducing conditions (i.e., the
sample was boiled in buffer containing dithiothreitol (DTT) and
2-mercaptoethanol (BME) to reduce disulfide bonds), blotted onto
a Nytran filter, and analyzed by standard Western blot protocols
using the above described antibody to the C-terminal peptide as
the primary antibody in this assay. After primary antibody binding,
the blot was washed and secondary antibody (goat anti-rabbit) was
added. Mammaglobin-antibody complexes were visualized by enzyme-linked
chemiluminescence (ECL Western Blotting Detecting Reagent, Amersham,
Arlington Heights, Ill.).
The anti-mammaglobin polyclonal antibody detected a band with an
apparent molecular weight of approximately 21 kD in the conditioned
media of the MDA-MB-415 cell culture (data not shown). No bands
were detected in the conditioned medium of the MCF-7 cell culture
(data not shown). Thus, consistent with the mRNA data, MDA-MB-415
cells secrete mammaglobin protein but MCF-7 cells do not. The apparent
molecular weight of the mammaglobin secreted into the MDA-MB-415
culture media is greater than the 10.5 kDa molecular weight calculated
from the predicted amino acid sequence of SEQ ID NO:2. Since almost
all secreted proteins are glycosylated, the cytosol of MDA-MB-415
cells was analyzed with the anti-mammaglobin polyclonal antibody
to see if any precursor forms of secreted mammaglobin could be detected.
MDA-MG-415 cells were grown for 24 hours in serum-free media, the
culture media was collected, spun, and the resulting supernatant
was collected. The attached cells were washed with phosphate buffered
saline (PBS) and lysed with 1.times.Laemmli sample buffer (2% SDS,
10% glycerol, 100 mM DTT, 60 mM Tris, pH 6.8, 0.001% Bromophenol
Blue). The lysis mixture was boiled for 5 minutes and then spun
at 10,000 g for 5 minutes to pellet the cell debris. The cell lysate
was transferred to a new tube and used for Western blot analysis
as described below.
The culture supernatant and cell lysate were run on a 12% SDS acrylamide
gel under reducing conditions (i.e., samples boiled in buffer containing
DTT and BME) and blotted onto a PVDF membrane using standard techniques.
The blot was probed with the polyclonal antibody to the C-terminal
peptide in the presence and absence of the competing peptide used
to generate the antibody.
Visualization of mammaglobin-antibody complexes were as discussed
above. As seen in FIG. 7, in the absence of competing peptide (-),
the conditioned media (S) has the 21 kD band representative of the
secreted mammaglobin protein. The cell lysate (C) showed a prominent
band at approximately 14 kD, and several higher molecular weight
bands, including one at approximately 21 kD. When the Western blot
is performed in the presence of the competing peptide (+), the secreted
form and intracellular forms of mammaglobin are not visualized,
indicating that these proteins contain the peptide to which the
antibody was synthesized.
The 14 kD band detected only in the cell lysate likely represents
a precursor, or unprocessed, form of mammaglobin. Since the predicted
amino acid sequence for mammaglobin has the consensus N-glycosylation
site, Asn-X-Thr, located at residues 53-55 and at residues 68-70
of SEQ ID NO:2, the observed, secreted 21 kD form likely represents
some glycosylated form of the protein.
This hypothesis was tested by culturing MDA-MB-415 cells in the
presence and absence of tunicamycin, a drug that blocks N-linked
glycosylation of eukaryotic proteins. Tunicamycin was added to one
of two identical cultures at 1 ug/ml and both cultures were incubated
overnight for more hours. The culture media and cell lysate from
the treated and control cultures were prepared and analyzed by Western
blot analysis as described above.
As shown in FIG. 8, media from cultures (S) treated with tunicamycin
(+) lack detectable levels of secreted mammaglobin, suggesting that
secreted mammaglobin is glycosylated. Surprisingly, the cell cytosol
form of mammaglobin (14 kD) was also not detectable in lysates of
MDA-MB-415 cells treated with tunicamycin (far right lane). We hypothesize
that blocking early glycosylation events with tunicamycin leads
to instability and degradation of precursor forms of mammaglobin,
thus explaining the lack of detectable 14 kD protein in the cytosol
of tunicamycin-treated cells.
The polyclonal antibody to the C-terminal peptide of mammaglobin
has also detected the 14 kDa precursor form of mammaglobin in cell
lysates from primary human breast tumor specimens. As seen in FIG.
9, the precursor form of mammaglobin is present in tumor specimen
B023, but is undetectable in a normal breast tissue sample from
the same patient (BO22). Interestingly, some tumor samples that
express the mammaglobin transcript (i.e., 087R, 014, 75A and 2410)
do not contain detectable levels of mammaglobin protein as assayed
by Western blot analysis. One hypothesis consistent with these data
is that mammaglobin expression is differentially regulated at the
levels of transcription and translation and that this differential
regulation is determined the developmental stage of the tumor.
The anti-mammaglobin polyclonal antibody has also been used to
look for secreted mammaglobin in breast secretions from proliferating
mammary gland. Colostrum or mature milk fluid (500 ul samples) was
collected by manual expression from a pregnant woman during the
first and third trimester, at birth, and at day 3, 14, and 21 post-partum.
The samples were diluted with an equal volume of 2.times.laemmli
sample buffer (4% SDS, 20% glycerol, 200 mM DTT, 120 mM Tris, pH
6.8, 0.002% Bromophenol Blue). The diluted samples were boiled for
5 min. and then spun at 10,000 g for 5 min. at 4.degree. C. to pellet
cell debris. The denatured samples were transferred to a new tube
and stored at -20.degree. C. prior to Western blot analysis as described
As shown in FIG. 10, the antibody detected the 21 kD secreted mammaglobin
in breast secretions sampled during pregnancy, a period of high
proliferation of breast epithelial cells. However, at the onset
of lactation, a stage of breast epithelial differentiation, mammaglobin
levels decreased significantly by 3 days post partum and was no
longer observed at 14 days post-partum. These results indicate that
secreted mammaglobin is associated with proliferating breast epithelial
cells, an observation consistent with the detection of secreted
mammaglobin in human breast cancer.
Reactivity with the antibody to the manunaglobin peptide has also
been shown for breast tumor cells by immunohistochemical staining
of paraffin-fixed sections of a breast cancer patient specimen (FIG.
11). The immunohistochemical staining was performed using the antibody
to the mammaglobin peptide as the primary antibody and then detecting
the mammaglobin-antibody complex using goat anti-rabbit antibody
tagged with horseradish peroxidase and 3,3' diamino benzene tetrahydrochloride
(DAB) as substrate. Cells expressing the mammaglobin protein showed
a brown staining.
From the results, it is believed that the mammaglobin protein is
synthesized as a precursor protein and post-translational modifications
such as N-linked glycosylation increase its apparent molecular weight
prior to secretion; that the stability of precursor forms of mammoglobin
is dependent on N-linked glycoslyation and that mammaglobin protein
is secreted by proliferating breast tumor cells.
This example illustrates detection of mammaglobin protein in primary
breast tumors by immunohistochemical analysis using the anti-mammaglobin
rabbit polyclonal antibody described in Example 5.
One hundred archived breast tumor specimens were chosen at random
from the Vanderbilt University Department of Pathology and the Washington
University Cancer Center Tumor Repository. Formalin-fixed, paraffin-embedded
tissues were cut at 5 .mu.m, mounted on charged slides, and dried.
For immunohistochemical analysis, slides were deparaffinized and
rehydrated in graded solutions of ethanol and distilled water. Tissue
sections were preincubated with normal goat serum (Vector Laboratories,
Burlingame, Calif.) at a 1:100 dilution in 3% bovine serum albumin
(BSA)/phosphate buffered saline (PBS) and then with the anti-mammaglobin
rabbit polyclonal antibody at a 1:1000 dilution for 1 hour at room
temperature. After several rinses in PBS, sections were incubated
in a solution of normal goat serum (1:1000), 3% BSA, and 6 .mu.g/ml
of biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame,
Calif.) in PBS for 1 hour. The secondary antibody solution was rinsed
four times in PBS and tissues were then incubated with a 1:1000
dilution of streptavidin peroxidase (Boehringer Mannheim, Indianapolis,
Ind.) also in a solution of 3% BSA/PBS. After a 30 minute incubation,
slides were again rinsed four times in PBS and exposed to chromagen
solution containing 1 mg/ml 3,3'-diaminobenzidine tetrahydrochloride
(Dako, Carpinteria, Calif.) and 0.02% hydrogen peroxide for 3 minutes.
Slides were rinsed briefly in deionized water, counterstained with
Harris' hemotoxylin, and mounted under coverslips.
For negative controls, tissue sections were processed identically
except a 1:500 dilution of pre-immune rabbit serum was substituted
for the anti-mammaglobin antiserum. Alternatively, for peptide competition
experiments, mammaglobin antiserum was first incubated with the
16 residue mammaglobin peptide at a concentration of 100 .mu.g/ml
in 3% BSA/PBS for 1 hour at room temperature and then applied to
Immunopositivity was scored as 0: no staining, 1: weak and sporadic
staining in less than 50% of tumor cells, 2: weak staining in greater
than 50% of tumor cells, 3: strong, diffuse cytoplasmic staining
in less than 50% of tumor cells, 4: strong, diffuse cytoplasmic
staining in greater than 50% of tumor cells. Only sections scoring
3 or 4 were considered mammaglobin "positive". The scoring
results are shown in Table I below and representative staining patterns
for three tumor types are shown in FIG. 12.
TABLE 1 Mammaglobin immunoreactive staining in human breast cancer
Staining Score.sup.2 Tumor Type.sup.1 4 3 2 1 0 Total Lobular 0
1 1 0 1 3 (33%) (33%) (33%) Well 11 0 1 0 2 14 Differentiated (78%)
(7%) (14%) Moderately 28 5 3 2 4 42 Differentiated (67%) (12%) (7%)
(5%) (10%) Poorly 24 9 2 0 3 38 Differentiated (63%) (24%) (5%)
(8%) DCIS 1 2 0 0 0 3 (33%) (67%) Total 64 17 7 2 10 100 .sup.1
Except for lobular and ductal carcinoma in situ (DCIS), all speciments
are invasive cancers. .sup.2 The degree of immunopositive staining
of breast tumor tissue sections with anti-mammaglobin antibodiy
was rated as 0: No staining, 1: Weak staining in <50% of tumor
cells, 2: Weak staining in >50% of tumor cells, 3: Strong, cytoplasmic
staining in <50% of tumor cells, 4: Strong, cytoplasmic staining
in >50% of tumor cells. For purposes of discussion, mammaglobin
`positive` tumors are those that scored 3 or 4.
Overall, 80% of ductal carcinomas examined demonstrated strong
global or focal cell staining for mammaglobin protein. Interestingly,
staining was equally frequent among well differentiated (78%), moderately
differentiated (67%), and poorly differentiated (63%) tumors (Table
1, FIG. 12). Strong staining was also seen in 3/3 cases of pure
ductal carcinoma in situ (DCIS) (FIG. 12A). The cellular staining
pattern of mammaglobin was predominantly diffuse and cytoplasmic,
although some cells also demonstrated localized staining adjacent
to the nucleus. In normal breast tissue, mammaglobin staining was
observed only in rare epithelial cells within small ducts and lobules.
However, as has been observed with other secretory proteins, increased
expression of mammaglobin coincided with features of apocrine metaplasia.
In benign breast tissue with metaplastic apocrine epithelium, mammaglobin
immunoreactivity was present both within the epithelium and in the
apocrine cyst fluid. The specificity of these patterns of positive
staining were documented by the lack of signal from identical specimens
incubated with either pre-immune rabbit serum (FIGS. 12B, 12D, 12F)
or anti-mammaglobin antiserum preincubated with competing C-terminal
peptide (data not shown).
However, mammaglobin expression was not detected in other apocrine
tissues such as the normal prostate and salivary gland. Furthermore,
breast tumor cells with both apocrine and non-apocrine features
express mammaglobin with roughly equal frequency and intensity.
There is also apparently no correlation between mammaglobin expression
and tumor grade. Therefore, it is believed that mammaglobin expression
is a marker of a unique breast tumor phenotype and may be useful
in conjunction with other established markers to further define
breast tumors at the molecular level.
The results described in Examples 5 and 6 indicate that detection
of a mammaglobin protein will be applicable in cancer diagnostics
using the mammaglobin protein as a breast tumor marker, in assessing
breast tumor relapse, in monitoring autologous bone marrow/stem
cell transplants for contaminating tumor cells, and in targeting
breast tumor cells for therapeutic intervention via antibody-mediated
complexes. A purified and isolated mammaglobin polypeptide is useful
for generating antibodies against breast tumors and in the development
of other tumor-specific immunotherapy regimens.