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 composition comprising a mammaglobin fragment, wherein said
mammaglobin fragment consists of a sequence of SEQ ID NO:2, mammaglobin,
which binds to an antibody specific for SEQ ID NO:2, mammaglobin.
2. The composition of claim 1, wherein said mammaglobin fragment
is loaded onto a carrier cell.
3. The composition of claim 1, further comprising a pharmaceutically
4. The composition of claim 1, wherein said mammaglobin fragment
is recognized by a mixture of B cells and T.sub.c cells which recognize
SEQ ID NO:2, mammaglobin.
5. The composition of claim 4, wherein said mammaglobin fragment
is recognized by B cells and/or T.sub.c cells specific for a naturally
occurring, secreted mammaglobin polypeptide which is glycosylated
and consists of amino acids 20-93 of SEQ ID NO:2.
6. The composition of claim 5, wherein the mammaglobin fragment
is recognized by B cells and/or T.sub.c cells specific for a naturally
occurring, secreted mammaglobin polypeptide which is glycosylated
and consists of SEQ ID NO:2.
7. The composition of claim 1, wherein the mammaglobin fragment
consists of at least 16 contiguous amino acids of SEQ ID NO:2.
8. The composition of claim 7, wherein the mammaglobin fragment
consists of at least 25 contiguous amino acids of SEQ ID NO:2.
9. The composition of claim 8, wherein the mammaglobin fragment
is glycosylated and consists of amino acids 20-93 of SEQ ID NO:2.
10. The composition of claim 9, wherein the mammaglobin fragment
is glycosylated and consist of SEQ ID NO:2.
11. The composition of claim 4, wherein the mammaglobin fragment
induces in vitro activation and expansion of B cells that are specific
for SEQ ID NO:2, wherein the B cells are isolated from a breast
12. The composition of claim 4, wherein the mammaglobin fragment
induces in vitro activation and expansion of T.sub.c cells that
are specific for SEQ ID NO:2, wherein the T.sub.c cells are isolated
from a breast cancer sample.
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
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 which is incorporated by reference).
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 8:485, 1994 which is incorporated
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 which is incorporated by reference). 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 which are incorporated by
reference). 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 which is
incorporated by reference), structural proteins (Trask et al., Proc
Natl Acad Sci 87:2319, 1990 which is incorporated by reference),
second messenger proteins (Ohuchi et al., Cancer Res 26:2511, 1986
which is incorporated by reference), and transcription factors (Harris,
Adv Cancer Res 59:69:1992 which is incorporated by reference). 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 which is incorporated by reference). 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 has a nucleotide sequence identified
as SEQ ID NO:1 and the encoded protein, mammaglobin, is in purified
and isolated form and has an amino acid sequence identified as SEQ
Mammaglobin is overexpressed in 27% of stage I primary breast cancer
tumors. This suggests 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 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,
cDNA encoding mammaglobin or a derivative of said cDNA is used to
detect the presence of mammaglobin mRNA in a sample. The method
comprises the steps of: (a) providing a polynucleotide containing
a nucleotide sequence having the sequence of SEQ ID NO:1 or a derivative
thereof, (b) incubating the nucleotide sequence with the sample
under conditions in which the sequence can hybridize with mRNA from
breast neoplasia cells, and (c) detecting the existence of a DNA-RNA
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 containing a nucleotide sequence
having the sequence of SEQ ID NO:1 or a derivative 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. The two primers
have nucleotide sequences comprising SEQ ID NO:3 and SEQ ID NO:4.
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.
The two primers have nucleotide sequences comprising SEQ ID NO:3
and SEQ ID NO:4.
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 of 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 (A) 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 (B) 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.
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 mammaglobin
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 which is incorporated
by reference). 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 which encodes a polypeptide of 93 amino acids and predicted
molecular mass of 10.5 kD (SEQ ID NO:2) (FIG. 2). The initial methionine
of this open reading frame is within a near-perfect Kozak consensus
sequence (Kozak, Cell 22:7-8, 1980 which is incorporated by reference).
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 glycoprotein.
A search for DNA sequences similar to the mammaglobin cDNA sequence
in Genbank using the BLAST algorithm (Benson et al., Nucl Acid Res21:2963-2965,
1993; Altschul et al, J Mol Biol 215:403-410, 1990 which are incorporated
by reference), identified no obvious DNA sequence homologies. Thus,
mammaglobin cDNA is believed to be a novel, heretofore unknown DNA
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/C.sub.1 and C3/C2
(Parker et al., Ann N Y Acad Sci 438:115-124; Parker et al., J Steroid
Biochem 20:67-71, 1984 which are incorporated by reference). 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, mammaglobin 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 which are incorporated by reference). 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 which are incorporated by reference). 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 which are incorporated
by reference), 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
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
which is incorporated by reference) (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 which are incorporated
by reference). 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 which is incorporated
by reference). 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.
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 which is incorporated by reference). 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
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. This suggests that analysis of mammaglobin
transcripts in peripheral lymph nodes may be useful for detecting
occult breast cancer metastases, as has been suggested for other
epithelial specific genes (Schoenfeld et al., Cancer Res 54:2986-90
which is incorporated by reference).
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.
Although we detected overexpression of mammaglobin RNA in one tumor
specimen (i.e. 2410), it was not clear at what frequency this overexpression
is seen in other breast carcinomas. We therefore examined a panel
of fifteen, stage I primary breast carcinomas of differing histological
types by Northern blot hybridization with the mammaglobin cDNA probe.
Because of potential variability in expression due to environment
influences (e.g. patient hormonal status), we also sought to compare
tumor specimens directly with patient-matched normal breast tissues
samples, although this was not possible in many cases. As shown
in FIG. 6, the 500 bp mammaglobin mRNA was again detected in normal
breast tissue and tumor 2410. Mammaglobin was also detected in three
other tumors, two of which demonstrated little or no expression
in patient-matched normal tissue (BO15 and BO22). In all, 4 of 15
(27%) of tumors examined overexpressed mammaglobin mRNA. 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 neoplasia.
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,
1990 which is incorporated by reference).
We determined the prevalence of mammaglobin as a tumor marker in
the general population of breast cancer tumors by examining the
expression of mammaglobin in several primary breast carcinomas.
Although the number of specimens examined in this study was small,
27% of tumors evaluated overexpressed mammaglobin mRNA. 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 which are
incorporated by reference). 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 which is incorporated by reference).
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
A method for detecting breast cancer comprises hybridizing a polynucleotide
to mRNA from breast neoplasia cells. The polynucleotide comprises
SEQ ID NO:1 or a derivative of SEQ ID NO:1. A derivative of a nucleotide
sequence means that the derived nucleotide sequence is substantially
the same as the sequence from which it is derived in that the derived
nucleotide sequence has sufficient sequence complementarity to the
sequence from which it is derived to hybridize to mRNA from breast
neoplasia cells under the same stringency conditions that the sequence
from which it is derived hybridizes to the 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.
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. The resulting nucleic acid from the sample
is subjected to gel electrophoresis or other size separation techniques.
Detection involves contacting the nucleic acids and in particular
the mRNA of the sample with a DNA sequence serving as a probe to
form hybrid 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 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 using the cDNA encoding mammaloglobin or a derivative thereof
as a probe, high stringency conditions can be used in order to prevent
false positives. When using sequences derived from mammaglobin,
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).
The RT/PCR method can be performed as follows. Total cellular RNA
is isolated by, for example, the standard guanidium isothiocyanate
method and the total RNA is reverse transcribed. The reverse transcription
method involves synthesis of DNA on a template of RNA using a reverse
transcriptase enzyme and a 3' end primer. Typically, the primer
contains an oligo(dT) sequence. The cDNA thus produced is then amplified
using the PCR method and mammaglobin specific primers. (Belyavsky
et al, Nucl Acid Res 17:2919-2932, 1989, Krug and Berger, Methods
in Enzymology, Academic Press. N.Y., Vol. 152, pp. 316-325, 1987
which are incorporated by reference)
The polymerase chain reaction method is performed using two oligonucleotide
primers that are complementary to the two flanking regions of the
DNA segment to be amplified. The upstream and down stream primers
are typically from 20 to 30 base pairs in length and hybridize to
the flanking regions for replication of the nucleotide sequence.
The primers are selected to be substantially complementary to the
strand of cDNA being amplified. Therefore, the primers need not
reflect the exact sequence of the template, but must be sufficiently
complementary to selectively hybridize with the strand being amplified.
Polymerization from the primers is catalyzed by a DNA-polymerase
in the presence of deoxynucleotide triphosphates or nucleotide analogs
to produce double-stranded DNA molecules. The double strands are
then separated by any denaturing method including physical, chemical
or enzymatic. Commonly, the method of physical denaturation is used
involving heating the nucleic acid, typically to temperatures from
about 80.degree. C. to 105.degree. C. for times ranging from about
1 to 10 minutes. The process is repeated for the desired number
Following amplification, the PCR product is then detected by ethidium
bromide staining (Sambrook, et al., 1989, supra).
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 or
derivative 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 which is incorporated by reference). 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 thereof.
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
A derivative of mammaglobin 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, they share at least 80 percent
sequence identity, preferably at least 95 percent sequence identity,
more preferably at least 95 percent sequence identity, most preferably
99 percent 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) or derivative thereof, 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 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. A B cell epitope could comprise 3
amino acids in a spacial 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 which is incorporated by reference) 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 which is incorporated by reference).
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 which are incorporated by reference).
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 which are incorporated by reference). 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 .sup.125 I and .sup.77 Br; and fissionable nuclides such
as .sup.10 B.
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.C 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), each of which is incorporated herein by reference. 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-31, 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), each of which is herein incorporated by reference. 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), which is incorporated herein by reference.
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)
incorporated herein by reference). 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), herein incorporated by reference.
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, animmunostimulatory 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, each of which is incorporated
herein by reference. 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), incorporated
herein by reference). 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. 5,593,972
to Weiner et al (herein incorporated by reference), 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), herein incorporated by reference). Preferably,
the vaccine comprises a plurality of mammaglobin B cell antigens
with T.sub.H epitopes recognized by MHC Class H1 molecules of different
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 which is incorporated by reference).
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 RCC AGA ATC GAT AG-3'), 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 .mu.g 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 nM 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
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 demonstrates that the mammaglobin cDNA encodes a translatable
nucleotide sequence which results in 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 35S-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; BO22 v. BO23) (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.