Compounds and methods for the treatment and diagnosis of breast
cancer are provided. The inventive compounds include polypeptides
containing at least a portion of a breast tumor antigen. Vaccines
and pharmaceutical compositions for immunotherapy of breast cancer
comprising such polypeptides, or polynucleotides encoding such polypeptides,
are provided, together with polynucleotides for preparing the inventive
polypeptides. The inventive polypeptides may be used to generate
antibodies useful for the diagnosis and monitoring of breast cancer.
What is claimed is:
1. An isolated polypeptide comprising SEQ ID NO: 176.
2. An isolated polypeptide comprising the amino acid sequence encoded
by SEQ ID NO: 175.
3. A composition comprising the polypepticle of any one of claims
1 and 2 and a physiologically acceptable carrier.
4. A composition comprising the polypeptide of any one of claims
1 and 2 and a non-specific immune response enhancer.
5. The composition of claim 4 wherein the non-specific immune response
enhancer is an adjuvant.
6. A fuision protein comprising at least one polypeptide according
to any one of claims 1 and 2.
7. A composition comprising a fusion protein according to claim
6 and a physiologically acceptable carrier.
8. A composition comprising a fusion protein according to claim
6 and a non-specific immune response enhancer.
9. The composition of claim 6 wherein the non-specific immune response
enhancer is an adjuvant.
The present invention relates generally to compositions and methods
for the treatment of breast cancer. The invention is more particularly
related to polypeptides comprising at least a portion of a protein
that is preferentially expressed in breast tumor tissue and to polynucleotides
encoding such polypeptides. Such polypeptides and polynucleotides
may be used in vaccines and pharmaceutical compositions for treatment
of breast cancer.
BACKGROUND OF THE INVENTION
Breast cancer is a significant health problem for women in the
United States and throughout the world. Although advances have -been
made in detection and treatment of the disease, breast cancer remains
the second leading cause of cancer-related deaths in women, affecting
more than 180,000 women in the United States each year. For women
in North America, the life-time odds of getting. breast cancer are
one in eight.
No vaccine or other universally successful method for the prevention
or treatment of breast cancer is currently available. Management
of the disease currently relies on a combination of early diagnosis
(through routine breast screening procedures) and aggressive treatment,
which may include one or more of a variety of treatments such as
surgery, radiotherapy, chemotherapy and hormone therapy. The course
of treatment for a particular breast cancer is often selected based
on a variety of prognostic parameters, including an analysis of
specific tumor markers. See, e.g., Porter-Jordan and Lippman, Breast
Cancer 8:73-100 (1994). However, the use of established markers
often leads to a result that is difficult to interpret, and the
high mortality observed in breast cancer patients indicates that
improvements are needed in the treatment, diagnosis and prevention
of the disease.
Accordingly, there is a need in the art for improved methods for
the treatment and diagnosis of breast cancer. The present invention
fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
The present invention provides compounds and methods for the treatment
and diagnosis of breast cancer. In one aspect, isolated polypeptides
are provided comprising at least an immunogenic portion of a breast
tumor antigen or a variant thereof, wherein the antigen comprises
an amino acid sequence encoded by a polynucleotide having a sequence
selected from the group consisting of: (a) nucleotide sequences
recited in SEQ ID NO: 1-61, 63-175, 178 and 180; (b) complements
of said nucleotide sequences; and (c) sequences that hybridize to
a sequence of (a) or (b) under moderately stringent conditions.
In specific embodiments, the inventive polypeptides comprise an
amino acid sequence selected from the group consisting of SEQ ID
NO: 62, 176, 179 and 181.
In related aspects, isolated polynucleotides encoding the above
polypeptides are provided. In specific embodiments, such polynucleotides
comprise a sequence selected from the group consisting of sequences
provided in SEQ ID NO: 1-61, 63-175, 178 and 180. The present invention
further provides expression vectors comprising the above polynucleotides,
together with host cells transformed or transfected with such expression
vectors. In preferred embodiments, the host cells are selected from
the group consisting of E. coli, yeast and mammalian cells.
In another aspect, the present invention provides fusion proteins
comprising a first and a second inventive polypeptide or, alternatively,
an inventive polypeptide and a known breast tumor antigen.
The present invention also provides pharmaceutical compositions
comprising at least one of the above polypeptides, or a polynucleotide
encoding such a polypeptide, and a physiologically acceptable carrier,
together with vaccines comprising at least one such polypeptide
or polynucleotide in combination with a non-specific immune response
enhancer. Pharmaceutical compositions and vaccines comprising one
or more of the above fusion proteins are also provided.
In yet another aspect, methods are provided for inhibiting the
development of breast cancer in a patient, comprising administering
an effective amount of at least one of the above pharmaceutical
compositions and/or vaccines.
The polypeptides disclosed herein may be usefully employed in the
diagnosis and monitoring of breast cancer. In one aspect of the
present invention, methods are provided for detecting breast cancer
in a patient, comprising: (a) contacting a biological sample obtained
from a patient with a binding agent that is capable of binding to
one of the above polypeptides; and (b) detecting in the sample a
protein or polypeptide that binds to the binding agent. In preferred
embodiments, the binding agent is an antibody, most preferably a
In related aspects, methods are provided for monitoring the progression
of breast cancer in a; patient, comprising: (a) contacting a biological
sample obtained from a patient with a binding agent that is capable
of binding to one of the above polypeptides; (b) determining in
the sample an amount of a protein or polypeptide that binds to the
binding agent; (c) repeating steps (a) and (b); and comparing the
amounts of polypeptide detected in steps (b) and (c).
Within related aspects, the present invention provides antibodies,
preferably monoclonal antibodies, that bind to the inventive polypeptides,
as well as diagnostic kits comprising such antibodies, and methods
of using such antibodies to inhibit the development of breast cancer.
The present invention further provides methods for detecting breast
cancer comprising: (a) obtaining a biological sample from a patient;
(b) contacting the sample with a first and a second oligonucleotide
primer in a polymerase chain reaction, at least one of the oligonucleotide
primers being specific for a polynucleotide that encodes one of
the above polypeptides; and (c) detecting in the sample a DNA sequence
that amplifies in the presence of the first and second oligonucleotide
primers. In a preferred embodiment, at least one of the oligonucleotide
primers comprises at least about 10 contiguous nucleotides of a
polynucleotide comprising a sequence selected from the group consisting
of SEQ ID NO: 1-61, 63-175, 178 and 180.
In a further aspect, the present invention provides a method for
detecting breast cancer in a patient comprising: (a) obtaining a
biological sample from the patient; (b) contacting the sample with
an oligonucleotide probe specific for a polynucleotide that encodes
one of the above polypeptides; and (c) detecting in the sample a
DNA sequence that hybridizes to the oligonucleotide probe. Preferably,
the oligonucleotide probe comprises at least about 15 contiguous
nucleotides of a polynucleotide comprising a sequence selected from
the group consisting of SEQ ID NO: 1-61, 63-175, 178 and 180.
In related aspects, diagnostic kits comprising the above oligonucleotide
probes or primers are provided.
These and other aspects of the present invention will become apparent
upon reference to the following detailed description. All references
disclosed herein are hereby incorporated by reference in their entirety
as if each was incorporated individually.
BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE IDENTIFIERS
FIG. 1 shows the results of a Northern blot of the clone SYN18C6
(SEQ ID NO: 40).
DETAILED DESCRIPTION OF THE INVENTION
As noted above, the present invention is generally directed to
compositions and methods for the treatment and diagnosis of breast
cancer. The inventive compositions are generally isolated polypeptides
that comprise at least a portion of a breast tumor antigen. Also
included within the present invention are molecules (such as an
antibody or fragment thereof) that bind to the inventive polypeptides.
Such molecules are referred to herein as "binding agents."
In particular, the subject invention discloses polypeptides comprising
at least a portion of a human breast tumor antigen, or a variant
thereof, wherein the breast tumor antigen includes an amino acid
sequence encoded by a polynucleotide including a sequence selected
from the group consisting of: nucleotide sequences recited in SEQ
ID NO: 1-61, 63-175, 178 and 180, the complements of said nucleotide
sequences, and variants thereof. As used herein, the term "polypeptide"
encompasses amino acid chains of any length, including full length
proteins, wherein the amino acid residues are linked by covalent
peptide bonds. Thus, a polypeptide comprising a portion of one of
the above breast antigens may consist entirely of the portion, or
the portion may be present within a larger polypeptide that contains
additional sequences. The additional sequences may be derived from
the native protein or may be heterologous, and such sequences may
be immunoreactive and/or antigenic.
As used herein, an "immunogenic portion" of a human breast
tumor antigen is a portion that is capable of eliciting an immune
response in a patient inflicted with breast cancer and as such binds
to antibodies present within sera from a breast cancer patient.
Such immunogenic portions generally comprise at least about 5 amino
acid residues, more preferably at least about 10, and most preferably
at least about 20 amino acid residues. Immunogenic portions of the
proteins described herein may be identified in antibody binding
assays. Such assays may generally be performed using any of a variety
of means known to those of ordinary skill in the art, as described,
for example, in Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988. For
example, a polypeptide may be immobilized on a solid support (as
described below) and contacted with patient sera to allow binding
of antibodies within the sera to the immobilized polypeptide. Unbound
sera may then be removed and bound antibodies detected using, for
example, .sup.125 -labeled Protein A. Alternatively, a polypeptide
may be used to generate monoclonal and polyclonal antibodies for
use in detection of the polypeptide in blood or other fluids of
breast cancer patients. Methods for preparing and identifying immunogenic
portions of antigens of known sequence are well known in the art
and include those summarized in Paul, Fundamental Immunology, 3.sup.rd
ed., Raven Press, 1993, pp. 243-247.
The term "polynucleotide(s)," as used herein, means a
single or double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases and includes DNA and corresponding RNA molecules, including
HnRNA and mRNA molecules, both sense and anti-sense strands, and
comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly
or partially synthesized polynucleotides. An HnRNA molecule contains
introns and corresponds to a polynucleotide in a generally one-to-one
manner. An mRNA molecule corresponds to an HnRNA and polynucleotide
from which the introns have been excised. A polynucleotide may consist
of an entire gene, or any portion thereof. Operable anti-sense polynucleotides
may comprise a fragment of the corresponding polynucleotide, and
the definition of "polynucleotide" therefore includes
all such operable anti-sense fragments.
The compositions and methods of the present invention also encompass
variants of the above polypeptides and polynucleotides. Such variants
include, but are not limited to, naturally occurring allelic variants
of the inventive sequences.
A polypeptide "variant," as used herein, is a polypeptide
that differs from the recited polypeptide only in conservative substitutions
and/or modifications, such that the antigenic properties of the
polypeptide are retained. In a preferred embodiment, variant polypeptides
differ from an identified sequence by substitution, deletion or
addition of five amino acids or fewer. Such variants may generally
be identified by modifying one of the above polypeptide sequences,
and evaluating the antigenic properties of the modified polypeptide
using, for example, the representative procedures described herein.
Polypeptide variants preferably exhibit at least about 70%, more
preferably at least about 90% and most preferably at least about
95% identity (determined as described below) to the identified polypeptides.
As used herein, a "conservative substitution" is one
in which an amino acid is substituted for another amino acid that
has similar properties, such that one skilled in the art of peptide
chemistry would expect the secondary structure and hydropathic nature
of the polypeptide to be substantially unchanged. In general, the
following groups of amino acids represent conservative changes:
(1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr,
thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5)
phe, tyr, trp, his.
Variants may also, or alternatively, contain other modifications,
including the deletion or addition of amino acids that have minimal
influence on the antigenic properties, secondary structure and hydropathic
nature of the polypeptide. For example, a polypeptide may be conjugated
to a signal (or leader) sequence at the N-terminal end of the protein
which co-translationally or post-translationally directs transfer
of the protein. The polypeptide may also be conjugated to a linker
or other sequence for ease of synthesis, purification or identification
of the polypeptide (e.g., poly-His), or to enhance binding of the
polypeptide to a solid support. For example, a polypeptide may be
conjugated to an immunoglobulin Fc region.
A nucleotide "variant" is a sequence that differs from
the recited nucleotide sequence in having one or more nucleotide
deletions, substitutions or additions. Such modifications may be
readily introduced using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis as taught, for
example, by Adelman et al. (DNA, 2:183, 1983). Nucleotide variants
may be naturally occurring allelic variants, or non-naturally occurring
variants. Variant nucleotide sequences preferably exhibit at least
about 70%, more preferably at least about 80% and most preferably
at least about 90% identity (determined as described below) to the
The breast tumor antigens provided by the present invention include
variants that are encoded by DNA sequences which are substantially
homologous to one or more of the DNA sequences specifically recited
herein. "Substantial homology," as used herein, refers
to DNA sequences that are capable of hybridizing under moderately.
stringent conditions. Suitable moderately stringent conditions include
prewashing in a solution of 5.times.SSC; 0.5% SDS, 1.0 mM EDTA (pH
8:0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC, overnight
or, in the event of cross-species homology, at 45.degree. C. with
0.5.times.SSC; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC containing
0.1% SDS. Such hybridizing DNA sequences are also within the scope
of this invention, as. are nucleotide sequences that, due to code
degeneracy, encode an immunogenic polypeptide that is encoded by
a hybridizing DNA sequence.
Two nucleotide or polypeptide sequences are said to be "identical"
if the sequence of nucleotides or amino acid residues in the two
sequences is the same when aligned for maximum correspondence as
described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A "comparison
window" as used herein, refers to a segment of at least about
20 contiguous positions, usually 30 to about 75, 40 to about 50,
in which a sequence may be compared to a reference sequence of the
same number of contiguous positions after the two sequences are
Optimal alignment of sequences for comparison may be conducted
using the Megalign program in the Lasergene suite of bioinformatics
software (DNASTAR, Inc., Madison, Wis.), using default parameters.
This program embodies several alignment schemes described in the
following references: Dayhoff, M. O. (1978) A model of evolutionary
change in proteins--Matrices for detecting distant relationships.
In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure,
National Biomedical Resarch Foundaiton, Washington D.C. Vol. 5,
Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment
and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic
Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M.
(1989) Fast and sensitive multiple sequence alignments on a microcomputer
CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) Optimal alignments
in linear space CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor
11:105; Santou, N. Nes, M. (1987) The neighbor joining method. A
new method for reconstructing phylogenetic trees Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy--the
Principles and Practice of Numerical Taxonomy, Freeman Press, San
Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Rapid
similarity searches of nucleic acid and protein data banks Proc.
Natl. Acad, Sci. USA 80:726-730.
Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a window
of comparison of at least 20 positions, wherein the portion of the
polynucleotide sequence in the comparison window may comprise additions
or deletions (i.e. gaps) of 20 percent or less, usually 5 to 15
percent, or 10 to 12 percent, as compared to the reference sequences
(which does not comprise additions or deletions) for optimal alignment
of the two sequences. The percentage is calculated by determining
the number of positions at which the identical nucleic acid bases
or amino acid residue occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the reference sequence (i.e. the
window size) and multiplying the results by 100 to yield the percentage
of sequence identity.
The breast tumor antigens of the present invention, and polynucleotides
encoding such antigens, may be isolated from breast tumor tissue
using any of a variety of methods well known in the art. DNA sequences
corresponding to a gene (or a portion thereof) encoding one of the
inventive breast tumor antigens may be isolated from a breast tumor
cDNA library using a subtraction technique as described in detail
below. Examples of such DNA sequences are provided in SEQ ID NO:
1-61, 63-175, 178 and 180. Partial DNA sequences thus obtained may
be used to design oligonucleotide primers for the amplification
of full-length DNA sequences in a polymerase chain reaction (PCR),
using techniques well known in the art (see, for example, Mullis
et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich
ed., PCR Technology, Stockton Press, N.Y., 1989). Once a DNA sequence
encoding a polypeptide is obtained, any of the above modifications
may be readily introduced using standard mutagenesis techniques,
such as oligonucleotide-directed site-specific mutagenesis as taught,
for example, by Adelman et al. (DNA, 2:183,1983).
The breast tumor polypeptides disclosed herein may also be generated
by synthetic or recombinant means. Synthetic polypeptides having
fewer than about 100 amino acids, and generally fewer than about
50 amino acids, may be generated using techniques well known to
those of ordinary skill in the art. For example, such polypeptides
may be synthesized using any of the commercially available solid-phase
techniques, such as the Merrifield solid-phase synthesis method,
where amino acids are sequentially added to a growing amino acid
chain (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149-2146,
1963). Equipment for automated synthesis of polypeptides is commercially
available from suppliers such as Perkin Elmer/Applied BioSystems
Division (Foster City, Calif.), and may be operated according to
the manufacturer's instructions.
Alternatively, any of the above polypeptides may be produced recombinantly
by inserting a DNA sequence that encodes the polypeptide into an
expression vector and expressing the protein in an appropriate host.
Any of a variety of expression vectors known to those of ordinary
skill in the art may be employed to express recombinant polypeptides
of this invention. Expression may be achieved in any appropriate
host cell that has been transformed or transfected with an expression
vector containing a polynucleotide that encodes a recombinant polypeptide.
Suitable host cells include prokaryotes, yeast and higher eukaryotic
cells. Preferably, the host cells employed are E. coli, yeast or
a mammalian cell line, such as CHO cells. The DNA sequences expressed
in this manner may encode naturally occurring polypeptides, portions
of naturally occurring polypeptides, or other variants thereof.
In general, regardless of the method of preparation, the polypeptides
disclosed herein are prepared in an isolated, substantially pure,
form (i.e., the polypeptides are homogenous as determined by amino
acid composition and primary sequence analysis). Preferably, the
polypeptides are at least about 90% pure, more preferably at least
about 95% pure and most preferably at least about 99% pure. In certain
preferred embodiments, described in more detail below, the substantially
pure polypeptides are incorporated into pharmaceutical compositions
or vaccines for use in one or more of the methods disclosed herein.
In a related aspect, the present invention provides fusion proteins
comprising a first and a second inventive polypeptide or, alternatively,
a polypeptide of the present invention and a known breast tumor
antigen, together with variants of such fusion proteins.
A DNA sequence encoding a fusion protein of the present invention
is constructed using known recombinant DNA techniques to assemble
separate DNA sequences encoding the first and second polypeptides
into an appropriate expression vector. The 3' end of a DNA sequence
encoding the first polypeptide is ligated, with or without a peptide
linker, to the 5' end of a DNA sequence encoding the second polypeptide
so that the reading frames of the sequences are in phase to permit
mRNA translation of the two DNA sequences into a single fusion protein
that retains the biological activity of both the first and the second
A peptide linker sequence may be employed to separate the first
and the second polypeptides by a distance sufficient to ensure that
each polypeptide folds into its secondary and tertiary structures.
Such a peptide linker sequence is incorporated into the fusion protein
using standard techniques well known in the art. Suitable peptide
linker sequences may be chosen based on the following factors: (1)
their ability to adopt a flexible extended conformation; (2) their
inability to adopt a secondary structure that could interact with
functional epitopes on the first and second polypeptides; and (3)
the lack of hydrophobic or charged residues that might react with
the polypeptide functional epitopes. Preferred peptide linker sequences
contain Gly, Asn and Ser residues. Other near neutral amino acids,
such as Thr and Ala may also be used in the linker sequence. Amino
acid sequences which may be usefully employed as linkers include
those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et
al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No.
4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may be
from 1 to about 50 amino acids in length. PIeptide sequences are
not required when the first and second polypeptides have non-essential
N-terminal amino acid regions that can be used to separate the functional
domains and prevent steric interference.
The ligated DNA sequences are operably linked to suitable transcriptional
or translational regulatory elements. The regulatory elements responsible
for expression of DNA are located only 5' to the DNA sequence encoding
the first polypeptides. Similarly, stop codons require to end translation
and transcription termination signals are only present 3' to the
DNA sequence encoding the second polypeptide.
Fusion proteins are also provided that comprise a polypeptide of
the present invention together with an unrelated immunogenic protein.
Preferably the immunogenic protein is capable of eliciting a recall
response. Examples of such proteins include tetanus, tuberculosis
and hepatitis proteins (see, for example, Stoute et al. New Engl.
J. Med., 336:86-91 (1997)).
Polypeptides of the present invention that comprise an immunogenic
portion of a breast tumor antigen may generally be used for immunotherapy
of breast cancer, wherein the polypeptide stimulates the patient's
own immune response to breast tumor cells. The present invention
thus provides methods for using one or more of the immunoreactive
polypeptides encoded by a polynucleotide comprising a sequence of
SEQ ID NO: 1-61, 63-175, 178 and 180 (or fusion proteins comprising
one or more such polypeptides and/or DNA encoding such polypeptides)
for immunotherapy of breast cancer in a patient. As used herein,
a "patient" refers to any warm-blooded animal., preferably
a human. A patient may be afflicted with a disease, or may be free
of detectable disease. Accordingly, the above immunoreactive polypeptides
(or fusion proteins or polynucleotides encoding such polypeptides)
may be used to treat breast cancer or to inhibit the development
of breast cancer. The polypeptides may be administered either prior
to or following surgical removal of primary tumors and/or treatment
by administration of radiotherapy and conventional chemotherapeutic
In these aspects, the polypeptide or fusion protein is generally
present within a pharmaceutical composition and/or a vaccine. Pharmaceutical
compositions may comprise one or more polypeptides, each of which
may contain one or more of the inventive sequences (or variants
thereof), and a physiologically acceptable carrier. The vaccines
may comprise one or more such polypeptides and a non-specific immune
response enhancer, wherein the non-specific immune response enhancer
is capable of eliciting or enhancing an immune response to an exogenous
antigen. Examples of non-specific-immune response enhancers include
adjuvants, biodegradable microspheres (e.g., polylactic galactide)
and liposomes (into which the polypeptide is incorporated). Pharmaceutical
compositions and vaccines may also contain other epitopes of breast
tumor antigens, either incorporated into a combination polypeptide
(i.e., a single polypeptide that contains multiple epitopes) or
present within a separate polypeptide.
Alternatively, a pharmaceutical composition or vaccine may contain
DNA encoding one or more of the above polypeptides, such that the
polypeptide is generated in situ. In such pharmaceutical compositions
and vaccines, the DNA may be present within any of a variety of
delivery systems known to those of ordinary skill in the art, including
nucleic acid expression systems, bacteria and viral expression systems.
Appropriate nucleic acid expression systems contain the necessary
DNA sequences for expression in the patient (such as a suitable
promoter). Bacterial delivery systems involve the administration
of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses
an epitope of a breast tumor cell antigen on its cell surface. In
a preferred embodiment, the DNA may be introduced using a viral
expression system (e.g., vaccinia or other pox virus, retrovirus,
or adenovirus), which may involve the use of a non-pathogenic (defective),
replication competent virus. Suitable systems are disclosed, for
example, in Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner et.
al., Ann. N.Y. Acad Sci. 569:86-103, 1989; Flexner et al., Vaccine
8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487;
WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242;
WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et
al., Science 252:431-434, 1991; Kolls et al., PNAS 91:215-219, 1994;
Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzman et al., Circulation
88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993.
Techniques for incorporating DNA into such expression systems are
well known to those of ordinary skill in the art. The DNA may also
be "naked," as described, for example, in published PCT
application WO 90/11092, and Ulmer et al., Science 259:1745-1749,
1993, reviewed by Cohen, Science 259:1691-1692, 1993. The uptake
of naked DNA may be increased by coating the DNA onto biodegradable
beads, which are efficiently transported into the cells.
Routes and frequency of administration, as well as dosage, will
vary from individual to individual and may parallel those currently
being used in immunotherapy of other diseases. In general, the pharmaceutical
compositions and vaccines may be administered by injection (e.g.,
intracutaneous, intramuscular, intravenous or subcutaneous), intranasally
(e.g., by aspiration) or orally. Between 1 and 10 doses may be administered
over a 3-24 week period. Preferably, 4 doses are administered, at
an interval of 3 months, and booster administrations may be given
periodically thereafter. Alternate protocols may be appropriate
for individual patients. A suitable dose is an amount of polypeptide
or DNA that is effective to raise an immune response (cellular and/or
humoral) against breast tumor cells in a treated patient. A suitable
immune response is at least 10-50% above the basal (i.e., untreated)
level. In general, the amount of polypeptide present in a dose (or
produced in situ by the DNA in a dose) ranges from about 1 pg to
about 100 mg per kg of host, typically from about 10 pg to about
1 mg, and preferably from about 100 pg to about 1 .mu.g. Suitable
dose sizes will vary with the size of the patient, but will typically
range from about 0.01 mL to about 5 mL.
While any suitable carrier known to those of ordinary skill in
the art may be employed in the pharmaceutical compositions of this
invention, the type of carrier will vary depending on the mode of
administration. For parenteral administration, such as subcutaneous
injection, the carrier preferably comprises water, saline, alcohol,
a lipid, a wax and/or a buffer. For oral administration, any of
the above carriers or a solid carrier, such as mannitol, lactose,
starch, magnesium stearate, sodium saccharine, talcum, cellulose,
glucose, sucrose, and/or magnesium carbonate, may be employed. Biodegradable
microspheres (e.g., polylactic glycolide) may also be employed as
carriers for the pharmaceutical compositions of this invention.
Suitable biodegradable microspheres are disclosed, for example,
in U.S. Pat. Nos. 4,897,268 and 5,075,109.
Any of a variety of non-specific immune response enhancers may
be employed in the vaccines of this invention. For example, an adjuvant
may be included. Most adjuvants contain a substance designed to
protect the antigen from rapid catabolism, such as aluminum hydroxide
or mineral oil, and a nonspecific stimulator of immune response,
such as lipid A, Bordella pertussis or Mycobacterium tuberculosis.
Such adjuvants are commercially available as, for example, Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,
Mich.) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.).
Polypeptides disclosed herein may also be employed in adoptive
immunotherapy for the treatment of cancer. Adoptive immunotherapy
may be broadly classified into either active or passive immunotherapy.
In active immunotherapy, treatment relies on the in vivo stimulation
of the endogenous host immune system to react against tumors with
the administration of immune response-modifying agents (for example,
tumor vaccines, bacterial adjuvants, and/or cytokines).
In passive immunotherapy, treatment involves the delivery of biologic
reagents with established tumor-immune reactivity (such as effector
cells or antibodies) that can directly or indirectly mediate antitumor
effects and does not necessarily depend on an intact host immune
system. Examples of effector cells include T lymphocytes (for example,
CD8+ cytotoxic T-lymphocyte, CD4+ T-helper, gamma/delta T lymphocytes,
tumor-infiltrating lymphocytes), killer cells (such as Natural Killer
cells, lymphokine-activated killer cells), B cells, or antigen presenting
cells (such as dendritic cells and macrophages) expressing the disclosed
antigens. The polypeptides disclosed herein may also be used to
generate antibodies or anti-idiotypic antibodies (as in U.S. Pat.
No. 4,918,164), for passive immunotherapy.
The predominant method of procuring adequate numbers of T-cells
for adoptive immunotherapy is to grow immune F-cells in vitro. Culture
conditions for expanding single antigen-specific T-cells to several
billion in number with retention of antigen recognition in vivo
are well known in the art. These in vitro culture conditions typically
utilize intermittent stimulation with antigen, often in the presence
of cytokines, such as IL-2, and non-dividing feeder cells. As noted
above, the immunoreactive polypeptides described herein may be used
to rapidly expand antigen-specific T cell cultures in order to generate
sufficient number of cells for immunotherapy. In particular, antigen-presenting
cells, such as dendritic, macrophage, monocyte, fibroblast or B-cells,
may be pulsed with immunoreactive polypeptides or polynucleotide
sequence(s) may be introduced into antigen presenting cells, using
standard techniques well known in the art. For example, antigen
presenting cells may be transfected or transduced with a polynucleotide
sequence, wherein said sequence contains a promoter region appropriate
for inducing expression, and can be expressed as part of a recombinant
virus or other expression system. Several viral vectors may be used
to transduce an antigen presenting cell, including pox virus, vaccinia
virus, and adenovirus. Antigen presenting cells may be transfected
with polynucleotide sequences disclosed herein by a variety of means,
including gene-gun technology, lipid-mediated delivery, electroporation,
osmotic shock, and particulate delivery mechanisms, resulting in
efficient and acceptable expression levels as determined by one
of ordinary skill in the art. For cultured T-cells to be effective
in therapy, the cultured T-cells must be able to grow and distribute
widely and to survive long term in vivo. Studies have demonstrated
that cultured T-cells can be induced to grow in vivo and to survive
long term in substantial numbers by repeated stimulation with antigen
supplemented with IL-2 (see, for example, Cheever et al. Ibid).
The polypeptides disclosed herein may also be employed to generate
and/or isolate tumor-reactive T-cells, which can then be administered
to the patient. In one technique, antigen-specific T-cell lines
may be generated by in vivo immunization with short peptides corresponding
to immunogenic portions of the disclosed polypeptides. The resulting
antigen specific CD8+ CTL clones may be isolated from the patient,
expanded using standard tissue culture techniques, and returned
to the patient.
Alternatively, peptides corresponding to immunogenic portions of
the polypeptides may be employed to generate tumor reactive T cell
subsets by selective in vitro stimulation and expansion of autologous
T cells to provide antigen-specific T cells which may be subsequently
transferred to the patient as described, for example, by Chang et
al. (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996). Cells of the
immune system, such as T cells, may be isolated from the peripheral
blood of a patient, using a commercially available cell separation
system. The separated cells are stimulated with one or more of the
immunoreactive polypeptides contained within a delivery vehicle,
such as a microsphere, to provide antigen-specific T cells. The
population of tumor antigen-specific T cells is then expanded using
standard techniques and the cells are administered back to the patient.
In other embodiments, T-cell and/or antibody receptors specific
for the polypeptides disclosed herein can be cloned, expanded, and
transferred into other vectors or effector cells for use in adoptive
immunotherapy. In particular, T cells may be transfected with the
appropriate genes to express the variable domains from tumor specific
monoclonal antibodies as the extracellular recognition elements
and joined to the T cell receptor signaling chains, resulting in
T cell activation, specific lysis, and cytokine release. This enables
the T cell to redirect its specificity in an MHC-independent manner.
See for example, Eshhar, Z., Cancer Immunol Immunother, 45(3-4):131-6,
1997 and Hwu, P., et al, Cancer Res, 55(15):3369-73, 1995. Another
embodiment may include the transfection of tumor antigen specific
alpha and beta T cell receptor chains into alternate T cells, as
in Cole, D J, et al, Cancer Res, 55(4):748-52, 1995.
In further embodiments, syngeneic or autologous dendritic cells
may be pulsed with peptides corresponding to at least an immunogenic
portion of a polypeptide disclosed herein. The resulting antigen-specific
dendritic cells may either be transferred into a patient, or employed
to stimulate T cells to provide antigen-specific T cells which may,
in turn, be administered to a patient. The use of peptide-pulsed
dendritic cells to generate antigen-specific T cells and the subsequent
use of such antigen-specific T cells to eradicate tumors in a murine
model has been demonstrated by Cheever et al. ("Therapy With
Cultured T Cells: Principles Revisited," Immunological Reviews,
157:177, 1997). Additionally vectors expressing the disclosed polynucleotides
may be introduced into stem cells taken from the patient and clonally
propagated in vitro for autologous transplant back into the same
In one specific embodiment, cells of the immune system, such as
T cells, may be isolated from the peripheral blood of a patient,
using a commercially available cell separation system, such as CellPro
Incorporated's (Bothell, Wash.) CEPRATE.TM. system (see U.S. Pat.
No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116
and WO 92/07243). The separated cells are stimulated with one or
more of the immunoreactive polypeptides contained within a delivery
vehicle, such as a microsphere, to provide antigen-specific T cells.
The population of tumor antigen-specific T cells is then expanded
using standard techniques and the cells are administered back to
Additionally vectors expressing the disclosed polynucleotides may
be introduced into stem cells taken from the patient and clonally
propagated in vitro for autologous transplant back into the same
Polypeptides of the present invention may also, or alternatively,
be used to generate binding agents, such as antibodies or fragments
thereof, that are capable of detecting metastatic human breast tumors.
Binding agents of the present invention may generally be prepared
using methods known to those of ordinary skill in the art, including
the representative procedures described herein. Binding agents are
capable of differentiating between patients with and without breast
cancer, using the representative assays described herein. In other
words, antibodies or other binding agents raised against a breast
tumor antigen, or a suitable portion thereof, will generate a signal
indicating the presence of primary or metastatic breast cancer in
at least about 20% of patients afflicted with the disease, and will
generate a negative signal indicating the absence of the disease
in at least about 90% of individuals without primary or metastatic
breast cancer. Suitable portions of such breast tumor antigens are
portions that are able to generate a binding agent that indicates
the presence of primary or metastatic breast cancer in substantially
all (i.e., at least about 80%, and preferably at least about 90%)
of the patients for which breast cancer would be indicated using
the full length antigen, and that indicate the absence of breast
cancer in substantially all of those samples that would be negative
when tested with full length antigen. The representative assays
described below, such as the two-antibody sandwich assay, may generally
be employed for evaluating the ability of a binding agent to detect
metastatic human breast tumors.
The ability of a polypeptide prepared as described herein to generate
antibodies capable of detecting primary or metastatic human breast
tumors may generally be evaluated by raising one or more antibodies
against the polypeptide (using, for example, a representative method
described herein) and determining the ability of such antibodies
to detect such tumors in patients. This determination may be made
by assaying biological samples from patients with and without primary
or metastatic breast cancer for the presence of a polypeptide that
binds to the generated antibodies. Such test assays may be performed,
for example, using a representative procedure described below. Polypeptides
that generate antibodies capable of detecting at least 20% of primary
or metastatic breast tumors by such procedures are considered to
be useful in assays for detecting primary or metastatic human breast
tumors. Polypeptide specific antibodies may be used alone or in
combination to improve sensitivity.
Polypeptides capable of detecting primary or metastatic human breast
tumors may be used as markers for diagnosing breast cancer or for
monitoring disease progression in patients. In one embodiment, breast
cancer in a patient may be diagnosed by evaluating a biological
sample obtained from the patient for the level of one or more of
the above polypeptides; relative to a predetermined cut-off value.
As used-herein, suitable "biological samples" include
blood, sera and urine.
The level of one or more of the above polypeptides may be evaluated
using any binding agent specific for the polypeptide(s). A "binding
agent," in the context of this invention, is any agent (such
as a compound or a cell) that binds to a polypeptide as described
above. As used herein, "binding" refers to a noncovalent
association between two separate molecules (each of which may be
free (i.e., in solution) or present on the surface of a cell or
a solid support), such that a "complex" is formed. Such
a complex may be free or immobilized (either covalently or noncovalently)
on a support material. The ability to bind may generally be evaluated
by determining a binding constant for the formation of the complex.
The binding constant is the value obtained when the concentration
of the complex is divided by the product of the component concentrations.
In general, two compounds are said to "bind" in the context
of the present invention when the binding constant for complex formation
exceeds about 10.sup.3 L/mol. The binding constant may be determined
using methods well known to those of ordinary skill in the art.
Any agent that satisfies the above requirements may be a binding
agent. For example, a binding agent may be a ribosome with or without
a peptide component, an RNA molecule or a peptide. In a preferred
embodiment, the binding partner is an antibody, or a fragment thereof.
Such antibodies may be polyclonal, or monoclonal. In addition, the
antibodies may be single chain, chimeric, CDR-grafted or humanized.
Antibodies may be prepared by the methods described herein and by
other methods well known to those of skill in the art.
There are a variety of assay formats known to those of ordinary
skill in the art for using a binding partner to detect polypeptide
markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment,
the assay involves the use of binding partner immobilized on a solid
support to bind to and remove the polypeptide from the remainder
of the sample. The bound polypeptide may then be detected using
a second binding partner that contains a reporter group. Suitable
second binding partners include antibodies that bind to the binding
partner/polypeptide complex. Alternatively, a competitive assay
may be utilized, in which a polypeptide is labeled with a reporter
group and allowed to bind to the immobilized binding partner after
incubation of the binding partner with the sample. The extent to
which components of the sample inhibit the binding of the labeled
polypeptide to the binding partner is indicative of the reactivity
of the sample with the immobilized binding partner.
The solid support may be any material known to those of ordinary
skill in the art to which the antigen may be attached. For example,
the solid support may be a test well in a microtiter plate or a
nitrocellulose or other suitable membrane. Alternatively, the support
may be a bead or disc, such as glass, fiberglass, latex or a plastic
material such as polystyrene or polyvinylchloride. The support may
also be a magnetic particle or a fiber optic sensor, such as those
disclosed, for example, in U.S. Pat. No. 5,359,681. The binding
agent may be immobilized on the solid support using a variety of
techniques known to those of skill in the art, which are amply described
in the patent and scientific literature. In the context of the present
invention, the term "immobilization" refers to both noncovalent
association, such as adsorption, and covalent attachment (which
may be a direct linkage between the antigen and functional groups
on the support or may be a linkage by way of a cross-linking agent).
Immobilization by adsorption to a well in a microtiter plate or
to a membrane is preferred. In such cases, adsorption may be achieved
by contacting the binding agent, in a suitable buffer, with the
solid support for a suitable amount of time. The contact time varies
with temperature, but is typically between about 1 hour and about
1 day. In general, contacting a well of a plastic microtiter plate
(such as polystyrene or polyvinylchloride) with an amount of binding
agent ranging from about 10 ng to about 10 .mu.g, and preferably
about 100 ng to about 1 .mu.g, is sufficient to immobilize an adequate
amount of binding agent.
Covalent attachment of binding agent to a solid support may generally
be achieved by first reacting the support with a bifunctional reagent
that will react with both the support and a functional group, such
as a hydroxyl or amino group, on the binding agent. For exarnple,
the binding agent may be covalently attached to supports having
an appropriate polymer coating using benzoquinone or by condensation
of an aldehyde group on the support with an amine and an active
hydrogen on the binding partner (see, e.g., Pierce Immunotechnology
Catalog and Handbook, 1991, at A12-A13).
In certain embodiments, the assay is a two-antibody sandwich assay.
This assay may be performed by first contacting an antibody that
has been immobilized on a solid support, commonly the well of a
microtiter plate, with the sample, such that polypeptides within
the sample are allowed to bind to the immobilized antibody. Unbound
sample is then removed from the immobilized polypeptide-antibody
complexes and a second antibody (containing a reporter group) capable
of binding to a different site on the polypeptide is added. The
amount of second antibody that remains bound to the solid support
is then determined using a method appropriate for the specific reporter
More specifically, once the antibody is immobilized on the support
as described above, the remaining protein binding sites on the support
are typically blocked. Any suitable blocking agent known to those
of ordinary skill in the art, such as bovine serum albumin or TWEEN
20.TM. (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody
is then incubated with the sample, and polypeptide is allowed to
bind to the antibody. The sample may be diluted with a suitable
diluent, such as phosphate-buffered saline (PBS) prior to incubation.
In general, an appropriate contact time (i.e., incubation time)
is that period of time that is sufficient to detect the presence
of polypeptide within a sample obtained from an individual with
breast cancer. Preferably, the contact time is sufficient to achieve
a level of binding that is at least about 95% of that achieved at
equilibrium between bound and unbound polypeptide. Those of ordinary
skill in the art will recognize that the time necessary to achieve
equilibrium may be readily determined by assaying the level of binding
that occurs over a period of time. At room temperature, an incubation
time of about 30 minutes is generally sufficient.
Unbound sample may then be removed by washing the solid support
with an appropriate buffer, such as PBS containing 0.1% Tween 20.TM..
The second antibody, which contains a reporter group, may then be
added to the solid support. Preferred reporter groups include enzymes
(such as horseradish peroxidase), substrates, cofactors, inhibitors,
dyes, radionuclides, luminescent groups, fluorescent groups and
biotin. The conjugation of antibody to reporter group may be achieved
using. standard methods known to those of ordinary skill in the
The second antibody is then incubated with the immobilized antibody-polypeptide
complex for an amount of time sufficient to detect the bound polypeptide.
An appropriate amount of time may generally be determined by assaying
the level of binding that occurs over a period of time. Unbound
second antibody is then removed and bound second antibody is detected
using the reporter group. The method employed for detecting the
reporter group depends upon the nature of the reporter group. For
radioactive groups, scintillation counting or autoradiographic methods
are generally appropriate. Spectroscopic methods may be used to
detect dyes, luminescent groups and fluorescent groups. Biotin may
be detected using avidin, coupled to a different reporter group
(commonly a radioactive or fluorescent group or an enzyme). Enzyme
reporter groups may generally be detected by the addition of substrate
(generally for a specific period of time), followed by spectroscopic
or other analysis of the reaction products.
To determine the presence or absence of breast cancer, the signal
detected from the reporter group that remains bound to the solid
support is generally compared to a signal that corresponds to a
predetermined cut-off value. In one preferred embodiment, the cut-off
value is the average mean signal obtained when the immobilized antibody
is incubated with samples from patients without breast cancer. In
general, a sample generating a signal that is three standard deviations
above the predetermined cut-off value is considered positive for
breast cancer. In an alternate preferred embodiment, the cut-off
value is determined using a Receiver Operator Curve, according to
the method of Sackett et al., Clinical Epidemiology: A Basic Science
for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly,
in this embodiment, the cut-off value may be determined from a plot
of pairs of true positive rates (i.e., sensitivity) and false positive
rates (100%-specificity) that correspond to each possible cut-off
value for the diagnostic test result. The cut-off value on the plot
that is the closest to the upper left-hand corner (i.e., the value
that encloses the largest area) is the most accurate cut-off value,
and a sample generating a signal that is higher than the cut-off
value determined by this method may be considered positive. Alternatively,
the cut-off value may be shifted to the left along the plot, to
minimize the false positive rate, or to the right, to minimize the
false negative rate. In general, a sample generating a signal that
is higher than the cut-off value determined by this method is considered
positive for breast cancer.
In a related embodiment, the assay is performed in a flow-through
or strip test format, wherein the antibody is immobilized on a membrane,
such as nitrocellulose. In the flow-through test, polypeptides within
the sample bind to the immobilized antibody as the sample passes
through the membrane. A second, labeled antibody then binds to the
antibody-polypeptide complex as a solution containing the second
antibody flows through the membrane. The detection of bound second
antibody may then be performed as described above. In the strip
test format, one end of the membrane to which antibody is bound
is immersed in a solution containing the sample. The sample migrates
along the membrane through a region containing second antibody and
to the area of immobilized antibody. Concentration of second antibody
at the area of immobilized antibody indicates the presence of breast
cancer. Typically, the concentration of second antibody at that
site generates a pattern, such as a line, that can be read visually.
The absence of such a pattern indicates a negative result. In general,
the amount of antibody immobilized on the membrane is selected to
generate a visually discernible pattern when the biological sample
contains a level of polypeptide that would be sufficient to generate
a positive signal in the two-antibody sandwich assay, in the format
discussed above. Preferably, the amount of antibody immobilized
on the membrane ranges from about 25 ng to about 1 .mu.g, and more
preferably from about 50 ng to about 500 ng. Such tests can typically
be performed with a very small amount of biological sample.
Of course, numerous other assay protocols exist that are suitable
for use with the antigens or antibodies of the present invention.
The above descriptions are intended to be exemplary only.
In another embodiment, the above polypeptides may be used as markers
for the progression of breast cancer. In this embodiment,. assays
as described above for the diagnosis of breast cancer may be performed
over time, and the change in the level of reactive polypeptide(s)
evaluated. For example, the assays may be performed every 24-72
hours for a period of 6 months to 1 year, and thereafter performed
as needed. In general, breast cancer is progressing in those patients
in whom the level of polypeptide detected by the binding agent increases
over time. In contrast, breast cancer is not progressing when the
level of reactive polypeptide either remains constant or decreases
Antibodies for use in the above methods may be prepared by any
of a variety of techniques known to those of ordinary skill in the
art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen
comprising the antigenic polypeptide is initially injected into
any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep
and goats). In this step, the polypeptides of this invention may
serve as the immunogen without .modification. Alternatively, particularly
for relatively short polypeptides, a superior immune response may
be elicited if the polypeptide is joined to a carrier protein, such
as bovine serum albumin or keyhole limpet hemocyanin. The immunogen
is injected into the animal host, preferably according to a predetermined
schedule incorporating one or more booster immunizations, and the
animals are bled periodically. Polyclonal antibodies specific for
the polypeptide may then be purified from such antisera by, for
example, affinity chromatography using the polypeptide coupled to
a suitable solid support.
Monoclonal antibodies specific for the antigenic polypeptide of
interest may be prepared, for example, using the technique of Kohler
and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements
thereto. Briefly, these methods involve the preparation of immortal
cell lines capable of producing antibodies having the desired specificity
(i.e., reactivity with the polypeptide of interest). Such cell lines
may be produced, for example, from spleen cells obtained from an
animal immunized as described above. The spleen cells are then immortalized
by, for example, fusion with a myeloma cell fusion partner, preferably
one that is syngeneic with the immunized animal. A variety of fusion
techniques may be employed. For example, the spleen cells and myeloma
cells may be combined with a nonionic detergent for a few minutes
and then plated at low density on a selective medium that supports
the growth of hybrid cells, but not myeloma cells. A preferred selection
technique uses HAT (hypoxanthine, aminopterin, thymidine) selection.
After a sufficient time, usually about 1 to 2 weeks, colonies of
hybrids are observed. Single colonies are selected and tested for
binding activity against the polypeptide. Hybridomas having high
reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of
growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the hybridoma
cell line into the peritoneal cavity of a suitable vertebrate host,
such as a mouse. Monoclonal antibodies may then be harvested from
the ascites fluid or the blood. Contaminants may be removed from
the antibodies by conventional techniques, such as chromatography,
gel filtration, precipitation, and extraction. The polypeptides
of this invention may be used in the purification process in, for
example, an affinity chromatography step.
Monoclonal antibodies of the present invention may also be used
as therapeutic reagents, to diminish or eliminate breast tumors.
The antibodies may be used on their own (for instance, to inhibit
metastases) or coupled to one or more therapeutic agents. Suitable
agents in this regard include radionuclides, differentiation inducers,
drugs, toxins, and derivatives thereof. Preferred radionuclides
include .sup.90 Y, .sup.123 I, .sup.125 I, .sup.131 I, .sup.186
Re, .sup.188 Re, .sup.211 At, and .sup.212 Bi. Preferred drugs include
methotrexate, and pyrimidine and purine analogs. Preferred differentiation
inducers include phorbol esters and butyric acid. Preferred toxins
include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas
exotoxin, Shigella toxin, and pokeweed antiviral protein.
A therapeutic agent may be coupled (e.g., covalently bonded) to
a suitable monoclonal antibody either directly or indirectly (e.g.,
via a linker group). A direct reaction between an agent and an antibody
is possible when each possesses a substituent capable of reacting
with the other. For example, a nucleophilic group, such as an amino
or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing
group, such as an anhydride or an acid halide, or with an alkyl
group containing a good leaving group (e.g., a halide) on the other.
Alternatively, it may be desirable to couple a therapeutic agent
and an antibody via a linker group. A linker group can function
as a spacer to distance an antibody from an agent in order to avoid
interference with binding capabilities. A linker group can also
serve to increase the chemical reactivity of a substituent on an
agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
It will be evident to those skilled in the art that a variety of
bifunctional or polyfunctional reagents, both homo- and hetero-functional
(such as those described in the catalog of the Pierce Chemical Co.,
Rockford, Ill.), may be employed as the linker group. Coupling may
be effected, for example, through amino groups, carboxyl groups,
sulflhydryl groups or oxidized carbohydrate residues. There are
numerous references describing such methodology, e.g., U.S. Pat.
No. 4,671,958, to Rodwell et al.
Where a therapeutic agent is more potent when free from the antibody
portion of the immunoconjugates of the present invention, it may
be desirable to use a linker group which is cleavable during or
upon internalization into a cell. A number of different cleavable
linker groups have been described. The mechanisms for the intracellular
release of an agent from these linker groups include cleavage by
reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710, to
Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat.
No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino
acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.),
by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958,
to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat.
No. 4,569,789, to Blattler et al.).
It may be desirable to couple more than one agent to an antibody.
In one embodiment, multiple molecules of an agent are coupled to
one antibody-molecule. In another embodiment, more than one type
of agent may be coupled to one antibody. Regardless of the particular
embodiment, immunoconjugates with, more than one agent may be prepared
in a variety of ways. For example, more than one agent may be coupled
directly to an antibody molecule, or linkers which provide multiple
sites for attachment can be used. Alternatively, a carrier can be
A carrier may bear the agents in a variety of ways, including covalent
bonding either directly or via a linker group. Suitable carriers
include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234,
to Kato et al.), peptides and polysaccharides such as aminodextran
(e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carrier may also
bear an agent by noncovalent bonding or by encapsulation, such as
within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088).
Carriers specific for radionuclide agents include radiohalogenated
small molecules and chelating compounds. For example, U.S. Pat.
No. 4,735,792 discloses representative radiohalogenated small molecules
and their synthesis. A radionuclide chelate may be formed from chelating
compounds that include those containing nitrogen and sulfur atoms
as the donor atoms for binding the metal, or metal oxide, radionuclide.
For example, U.S. Pat. No. 4,673,562, to Davison et al. discloses
representative chelating compounds and their synthesis.
A variety of routes of administration for the antibodies and immunoconjugates
may be used. Typically, administration will be intravenous, intramuscular,
subcutaneous or in the bed of a resected tumor. It will be evident
that the precise dose of the antibody/immunoconjugate will vary
depending upon the antibody used, the antigen density on the tumor,
and the rate of clearance of the antibody.
Diagnostic reagents of the present invention may also comprise
DNA sequences encoding one or more of the above polypeptides, or
one or more portions thereof. For example, at least two oligonucleotide
primers may be employed in a polymerase chain reaction (PCR) based
assay to amplify breast tumor-specific cDNA derived from a biological
sample, wherein at least one of the oligonucleotide primers is specific
for a polynucleotide encoding a breast tumor protein of the present
invention. The presence of the amplified cDNA is then detected using
techniques well known in the art, such as gel electrophoresis. Similarly,
oligonucleotide probes specific for a polynucleotide encoding a
breast tumor protein of the present invention may be used in a hybridiatioin
assay to detect the presence of an inventive polypeptide in a biological
As used herein, the term "oligonucleotide primer/probe specific
for a polynucleotide" means an oligonucleotide sequence that
has at least about 60%, preferably at least about 75% and more preferably
at least about 90%, identity to the polynucleotide in question.
Oligonucleotide primers and/or probes which may be usefully employed
in the inventive diagnostic methods preferably have at least about
10-40 nucleotides. In a preferred embodiment, the oligonucleotide
primers comprise at least about 10 contiguous nucleotides of a polynucleotide
comprising a sequence selected from SEQ ID NOS: 1-61, 63-175, 178
and 180. Preferably, oligonucleotide probes for use in the inventive
diagnostic methods comprise at least about 15 contiguous oligonucleotides
of a polynucleotide comprising a sequence provided in SEQ ID NOS:
1-61, 63-175, 178 and 180. Techniques for both PCR based assays
and hybridization assays are well known in the art (see, for example,
Mullis et al. Ibid; Ehrlich, Ibid). Primers or probes may thus be
used to detect breast tumor-specific sequences in biological samples,
including blood, urine and/or breast tumor tissue.
The following Examples are offered by way of illustration and not
by way of limitation.
Isolation and Characterization of Breast Tumor Polypeptides
This Example describes the isolation of breast tumor polypeptides
from a breast tumor cDNA library.
A cDNA subtraction library containing cDNA from breast tumor subtracted
with normal breast cDNA was constructed as follows. Total .RNA was
extracted from primary tissues using Trizol reagent (Gibco BRL Life
Technologies, Gaithersburg, Md.) as described by the manufacturer.
The polyA+RNA was purified using an oligo(dT) cellulose column according
to standard protocols. First strand cDNA was synthesized using the
primer supplied in a Clontech PCR-Select cDNA Subtraction Kit (Clontech,
Palo Alto, Calif.). The driver DNA consisted of cDNAs from two normal
breast tissues with the tester cDNA being from three primary breast
tumors. Double-stranded cDNA was synthesized for both tester and
driver, and digested with a combination of endonucleases (MluI,
MscI, PvuII, SalI and StuI) which recognize six base pairs DNA.
This modification increased the average cDNA size dramatically compared
with cDNAs generated according to Clontech's protocol. The digested
tester cDNAs were ligated to two different adaptors and the subtraction
was performed according to Clontech's protocol. The subtracted cDNAs
were subjected to two rounds of PCR amplification, following the
manufacturer's protocol. The resulting PCR products were subcloned
into the TA cloning vector, pCRII (Invitrogen, San Diego, Calif.)
and transformed into ElectroMax E. coli DH10B cells (Gibco BRL Life,
Technologies) by electroporation. DNA was isolated from independent
clones and sequenced using a Perkin Elmer/Applied Biosystems Division
(Foster City, Calif.) Automated Sequencer Model 373A.
Sixty-three distinct cDNA clones were found in the subtracted breast
tumor-specific cDNA library. The determined one strand (5' or 3')
cDNA sequences for the clones are provided in SEQ ID NOS: 1-61,
72 and 73, respectively. Comparison of these cDNA sequences with
known sequences in the gene bank using the EMBL and GenBank databases
(Release 97) revealed no significant homologies to the sequences
provided in SEQ ID NOS: 14, 21, 22, 27, 29, 30, 32, 38, 44, 45,
53, 72 and 73. The sequences of SEQ ID NOS: 1, 3, 16, 17, 34, 48,
57, 60 and 61 were found to represent known human genes, The sequences
of SEQ ID NOS: 2, 4, 23, 39 and 50 were found to show some similarity
to previously identified non-human genes. The remaining clones (SEQ
ID NOS: 5-13, 15, 18-20, 24-26, 28. 31, 33, 35-37, 40-43, 46, 47,
49, 51, 52, 54-56, 58 and 59) were found to show at least some degree
of homology to previously identified expressed sequence tags (ESTs).
To determine mRNA expression levels of the isolated cDNA clones,
cDNA clones from the breast subtraction described above were randomly
picked and colony PCR amplified. Their mRNA expression levels in
breast tumor, normal breast and various other normal tissues were
determined using microarray technology (Synteni, Palo Alto, Calif.).
Briefly, the PCR amplification products were arrayed onto slides
in an array format, with each product occupying a unique location
in the array. mRNA was extracted from the tissue sample to be tested,
reverse transcribed, and fluorescent-labeled cDNA probes were generated.
The microarrays were probed with the labeled cDNA probes, the slides
scanned and fluorescence intensity was measured. Data was analyzed
using Synteni provided GEMTOOLS Software. Of the seventeen cDNA
clones examined, those of SEQ ID NOS: 40, 46, 59 and 73 were found
to be over-expressed in breast tumor and expressed at low levels
in all normal tissues tested (breast, PBMC, colon, fetal tissue,
salivary gland, bone marrow, lung, pancreas, large intestine, spinal
cord, adrenal gland, kidney, pancreas, liver, stomach, skeletal
muscle, heart, small intestine, skin, brain and human mammary epithelial
cells). The clones of SEQ ID NOS: 41 and 48 were found to be over-expressed
in breast tumor and expressed at low levels in all other tissues
tested, with the exception of bone marrow. The clone of SEQ ID NO:
42 was found to be over-expressed in breast tumor and expressed
at low levels in all other tissues tested except bone marrow and
spinal cord. The clone of SEQ ID NO: 43 was found to be over-expressed
in breast tumor and expressed at low levels in all other tissues
tested with the exception of spinal cord, heart and small intestine.
The clone of SEQ ID NO: 51 was found to be over-expressed in breast
tumor and expressed at low levels in all other tissues tested with
the exception of large intestine. The clone of SEQ ID NO: 54 was
found to be over-expressed in breast tumor and expressed at low
levels in all other tissues tested with the exception of PBMC, stomach
and small intestine. The clone of SEQ ID NO: 56 was found to be
over-expressed in breast tumor and expressed at low levels in all
other tissues tested with the exception of large and small intestine,
human mammary epithelia cells and SCID mouse-passaged breast tumor.
The clone of SEQ ID NO: 60 was found to be over-expressed in breast
tumor and expressed at low levels in all other tissues tested with
the exception of spinal cord and heart. The clone of SEQ ID NO:
61 was found to be over-expressed in breast tumor and expressed
at low levels in all other tissues tested with the exception of
small intestine. The clone of SEQ ID NO: 72 was found to be over-expressed
in breast tumor and expressed at low levels in all other tissues
tested with the exception of colon and salivary gland.
The results of a Northern blot analysis of the clone SYN18C6 (SEQ
ID NO: 40) are shown in FIG. 1. A predicted protein sequence encoded
by SYN18C6 is provided in SEQ ID NO: 62.
Additional cDNA clones that are over-expressed in breast tumor
tissue were isolated from breast cDNA subtraction libraries as follows.
Breast subtraction libraries were prepared, as described above,
by PCR-based subtraction employing pools of breast tumor cDNA as
the tester and pools of either normal breast cDNA or cDNA from other
normal tissues as the driver. cDNA clones from breast subtraction
were randomly picked and colony PCR amplified and their mRNA expression
levels in breast tumor, normal breast and various other normal tissues
were determined using the microarray technology described above.
Twenty-four distinct cDNA clones were found to be over-expressed
in breast tumor and expressed at low levels in all normal tissues
tested (breast, brain, liver, pancreas, lung, salivary gland, stomach,
colon, kidney, bone marrow, skeletal muscle, PBMC, heart, small
intestine, adrenal gland, spinal cord, large intestine and skin).
The determined partial cDNA sequences for these clones are provided
in SEQ ID NOS: 63-87. Comparison of the sequences of SEQ ID NOS:
74-87 with those in the gene bank as described above, revealed homology
to previously identified human genes. No significant homologies
were found to the sequences of SEQ ID NOS: 63-73.
Three DNA isoforms for the clone B726P (partial sequence provided
in SEQ ID NO: 71) were isolated as follows. A radioactive probe
was synthesized from B726P by excising B726P DNA from a pT7Blue
vector (Novagen) by a BamHI/XbaI restriction digest and using the
resulting DNA as the template in a single-stranded PCR in the presence
of [.alpha.-32P]dCTP. The sequence of the primer employed for this
PCR is provided in SEQ ID NO: 177. The resulting radioactive probe
was used to probe a directional cDNA library and a random-primed
cDNA library made using RNA isolated from breast tumors. Eighty-five
clones were identified, excised, purified and sequenced. Of these
85 clones, three were found to each contain a significant open reading
frame. The determined cDNA sequence of the isoform B726P-20 is provided
in SEQ ID NO: 175, with the corresponding predicted amino acid sequence
being provided in SEQ ID NO: 176. The determined cDNA sequence of
the isoform B726P-74 is provided in SEQ ID NO: 178, with the corresponding
predicted amino acid sequence being provided in SEQ ID NO: 179.
The determined cDNA sequence of the isoform B726P-79 is provided
in SEQ ID NO: 180, with the corresponding predicted amino acid sequence
being provided in SEQ ID NO: 181.