Described herein are methods that can be used for diagnosis and
prognosis of breast cancer. Also described herein are methods that
can be used to screen candidate bioactive agents for the ability
to modulate breast cancer. Additionally, methods and molecular targets
(genes and their products) for therapeutic intervention in breast
cancer are described.
1. A method of diagnosing breast cancer in a human individual comprising:
a) determining the expression of a gene encoding an amino acid sequence
of SEQ ID NO: 5 in a first breast tissue sample obtained from a
human individual; and b) comparing the expression of said gene in
the first breast tissue sample to expression of said gene in a normal
breast tissue sample; whereby the overexpression of said gene in
the first breast tissue sample indicates breast cancer in said human
2. The method of claim 1, wherein said normal breast tissue sample
is obtained from said human individual.
3. The method of claim 1, wherein said normal breast tissue sample
is obtained from a second individual.
4. The method of claim 1, wherein said gene comprises a nucleic
acid sequence of SEQ ID NO: 4.
5. The method of claim 1, wherein said expression is measured using
a labeled nucleic acid probe.
6. The method of claim 1, wherein said expression is measured utilizing
a biochip comprising the sequence of SEQ ID NO: 4.
FIELD OF THE INVENTION
The invention relates to the identification of expression profiles
and the nucleic acids involved in breast cancer, and to the use
of such expression profiles and nucleic acids in diagnosis and prognosis
of breast cancer. The invention further relates to methods for identifying
and using candidate agents and/or targets which modulate breast
BACKGROUND OF THE INVENTION
Breast cancer is a significant cancer in Western populations. It
develops as the result of a pathologic transformation of normal
breast epithelium to an invasive cancer. There have been a number
of recently characterized genetic alterations that have been implicated
in breast cancer. However, there is a need to identify all of the
genetic alterations involved in the development of breast cancer.
Imaging of breast cancer for diagnosis has been problematic and
limited. In addition, dissemination of tumor cells (metastases)
to locoregional lymph nodes is an important prognostic factor: five
year survival rates drop from 80 percent in patients with no lymph
node metastases to 45 to 50 percent in those patients who do have
lymph node metastases. A recent report showed that micrometastases
can be detected from lymph nodes using reverse tansdptase-PCR methods
based on the presence of mRNA for carcinoembryonic antigen, which
has previously been shown to be present in the vast majority of
breast cancers but not in normal tissues. Liefers et al. New England
J. of Med. 339(4):223 (1998).
Thus, methods that can be used for diagnosis and prognosis of breast
cancer would be desirable. While academia and industry has made
an effort to identify novel sequences, there has not been an equal
effort exerted to identify the function of the novel sequences.
For example, databases show the sequence for accession number U41060,
which has been suggested to encode a protein that is inducible by
estrodiol and other simulation of estrogen receptors (i.e., LIV-1)
in a particular cell line (MCF-7) (el- Tanani et al, J. Stemid Biochem.
Mol. Biol. 60(5-6):269-276 (1997; (el- Tanani et al, Mol. Cell.
Endocrinol. 124(1-2):71-77 (1996); (el- Tanani et al, Mol. Cell.
Endrinol 121(1):29-35 (1996)), but there is no data correlating
this sequence with a function, much less a role in a disease state.
Accordingly, provided herein are methods that can be used in diagnosis
and prognosis of breast cancer. Further provided are methods that
can be used to screen candidate bioactive agents for the ability
to modulate breast cancer. Additionally, provided herein are molecular
targets for therapeutic intervention in breast and other cancers.
SUMMARY OF THE INVENTION
The present invention provides methods for screening for compositions
which modulate breast cancer. In one aspect, a method of screening
drug candidates comprises providing a cell that expresses an expression
profile gene or fragments thereof. Preferred embodiments of the
expression profile gene as described herein include the sequence
comprising BCR4 or a fragment thereof. The method further includes
adding a drug candidate to the cell and determining the effect of
the drug candidate on the expression of the expression profile gene.
In one embodiment, the method of screening drug candidates includes
comparing the level of expression in the absence of the drug candidate
to the level of expression in the presence of the drug candidate,
wherein the concentration of the drug candidate can vary when present,
and wherein the comparison can occur after addition or removal of
the drug candidate. In a preferred embodiment, the cell expresses
at least two expression profile genes The profile genes may show
an increase or decrease.
Also provided herein is a method of screening for a bioactive agent
capable of binding to a breast cancer modulating protein (BCMP)
or a fragment thereof, the method comprising combining the BCMP
or fragment thereof and a candidate bioactive agent, and determining
the binding of the candidate agent to the BCMP or fragment thereof.
In a preferred embodiment, the BCMP Is BCR4.
Further provided herein is a method for screening for a bioactive
agent capable of modulating the bioactvity of a BCMP or a fragment
thereof. In one embodiment, the method comprises combining the BCMP
or fragment thereof and a candidate bioactive agent, and determining
the effect of the candidate agent on the bioactivity of the BCMP
or the fragment thereof. In a preferred embodiment, the BCMP is
Also provided herein is a method of evaluating the effect of a
candidate breast cancer drug comprising administering the drug to
a transgenic animal expressing or over-expressing a BCMP or a fragment
thereof, or an animal lacking a BCMP for example as a result of
a gene knockout. In a preferred embodiment, the BCMP is BCR4.
Additionally, provided herein is a method of evaluating the effect
of a candidate breast cancer drug comprising administering the drug
to a patient and removing a cell sample from the patient. The expression
profile of the cell is then determined. This method may further
comprise comparing the expression profile to an expression profile
of a healthy individual.
Furthermore, a method of diagnosing breast cancer is provided.
The method comprises determining the expression of a gene which
encodes BCR4 or a fragment thereof in a first tissue type of a first
individual, and comparing this to the expression of the gene from
a second unaffected individual. A difference in the expression indicates
that the first individual has breast cancer.
In another aspect, the present invention provides an antibody which
specifically binds to BCR4, or a fragment thereof. Preferably the
antibody is a monoclonal antibody. The antibody can be a fragment
of an antibody such as a single stranded antibody as further described
herein, or can be conjugated to another molecule. In one embodiment,
the antibody is a humanized antibody.
In one embodiment a method for screening for a bioactive agent
capable of interfering with the binding of BCR4 or a fragment thereof
and an antibody which binds to said BCR4 or fragment thereof is
provided. In a preferred embodiment, the method comprises combining
BCR4 or a fragment thereof, a candidate bioactive agent and an antibody
which binds to said BCR4 or fragment thereof. The method further
includes determining the binding of said BCR4 or fragment thereof
and said antibody. Wherein there is a change in binding, an agent
is identified as an interfering agent. The interfering agent can
be an agonist or an antagonist. Preferably, the antibody as well
as the agent inhibits breast cancer.
In one aspect of the invention, a method for inhibiting the activity
of a breast cancer modulating protein are provided. The method comprises
binding an inhibitor to the protein. In a preferred embodiment,
the protein Is BCR4.
In another aspect, the invention provides a method for neutralizing
the effect of a breast cancer modulating protein. The method comprises
contacting an agent specific for the protein with the protein in
an amount sufficient to effect neutralization. In a preferred embodiment,
the protein is BCR4.
In a further aspect, a method for treating or inhibiting breast
cancer is provided. In one embodiment, the method comprises administering
to a cell a composition comprising an antibody to BCR4 or a fragment
thereof. In one embodiment, the antibody is conjugated to a therapeutic
moiety. Such therapeutic moieties include a cytotoxic agent and
a radioisotope. The method can be performed in vitro or in vivo,
preferably in vivo to an individual. In a preferred embodiment the
method of inhibiting breast cancer is provided to an individual
with such cancer.
As described herein, methods of treating or inhibiting breast cancer
can be performed by administering an inhibitor of BCR4 activity
to a cell or individual. In one embodiment, a BCR4 inhibitor is
an antisense molecule to a nucleic acid encoding BCR4.
Moreover, provided herein is a biochip comprising a nucleic acid
segment which encodes BCR4, or a fragment thereof, wherein the biochip
comprises fewer than 1000 nucleic acid probes. Preferably at least
two nucleic acid segments are included.
Also provided herein are methods of eliciting an immune response
in an individual. In one embodiment a method provided herein comprises
administering to an individual a composition comprising BCR4 or
a fragment thereof. In another aspect, said composition comprises
a nucleic acid comprising a sequence encoding BCR4 or a fragment
Further provided herein are compositions capable of eliciting an
immune response in an individual in one embodiment, a composition
provided herein comprises BCR4 or a fragment thereof and a pharmaceutically
acceptable carrier. In another embodiment, said composition comprises
a nucleic acid comprising a sequence encoding BCR4 or a fragment
thereof and a pharmaceutically acceptable carrier.
Yet further provided herein are novel nucleic acid and amino acid
compounds, and compositions thereof.
Other aspects of the invention will become apparent to the skilled
artisan by the following description of the invention.
DETAILED DESCRIPTION OF THE FIGURES
FIG. 1 shows an embodiment of a nucleic acid (mRNA) which includes
a sequence which encodes a breast cancer protein provided herein,
BCR4 (SEQ ID NO:1). The start (ATG) and stop (TAG) codons are underlined.
This sequence is similar to the published sequence for human LIV-1,
however the present sequence includes an additional 18 base sequence
(boxed GATCATCCACTCTCACCAT; SEQ ID NO:2) not found in the published
sequence for LIV-1. Also, BCR4 contains two additional thymine residues,
indicated at the ends of the boxed sequence TTTCCATATTTGAACATAAAATCGTGT
(SEQ ID NO:3) which are not found in the published sequence for
Preferred sequences of the present invention comprise the 18 base
sequence indicated above or a fragment thereof and/or a sequence
including one or both of the thymine residues indicated above.
FIG. 2 shows an embodiment of an open reading frame of a nucleic
acid encoding BCR4 (SEQ ID NO:4), wherein the start (ATG) and stop
(TAG) codons are underlined. Sequences distinguishing BCR4 from
the published sequence for human LIV-1 are boxed as in FIG. 1.
FIG. 3 shows an embodiment of an amino acid sequence of BCR4 (SEQ
ID NO:5). The signal peptide is underlined and putative transmembrane
domains are shaded. The amino acid sequence is similar to the published
sequence for human LIV-1, but differs by the sequences indicated
with boxes. The sequence HDHHSH (SEQ ID NO:6) results from the additional
18 base sequence of the mRNA. The sequence at the carboxy terminus
differs from the [published sequence for LIV-1 polypeptide, due
to a shift in the reading frame resulting from the two additional
thymine residues of BCR4 not found in the published LIV-1 nucleic
Preferred polypeptides of the present invention comprise one or
both of the boxed sequences of FIG. 3, or a fragment thereof.
In a preferred embodiment, a soluble form of BCR4 is provided wherein
the signal peptide is deleted or preferably naturally cleaved, and
the transmembrane domains are deleted, inactivated, or BCR4 is truncated
to exclude the transmembrane domains.
FIG. 4 shows the relative amount of expression of BCR4 in various
samples of breast cancer tissue (dark bars) and many normal tissue
types (fight bars).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel methods for diagnosis and
prognosis evaluation for breast cancer, as well as methods for screening
for compositions which modulate breast cancer and compositions which
bind to modulators of breast cancer. In one aspect, the expression
levels of genes are determined in different patient samples for
which either diagnosis or prognosis information is desired, to provide
expression profiles. An expression profile of a particular sample
is essentially a "fingerprint" of the state of the sample;
while two states may have any particular gene similarly expressed,
the evaluation of a number of genes simultaneously allows the generation
of a gene expression profile that is unique to the state of the
cell. That is, normal tissue may be distinguished from breast cancer
tissue, and within breast cancer tissue, different prognosis states
(good or poor long term survival prospects, for example) may be
determined. By comparing expression profiles of breast cancer tissue
in different states, information regarding which genes are important
(including both up- and down-regulation of genes) in each of these
states is obtained. The identification of sequences that are differentially
expressed in breast cancer tissue versus normal breast tissue, as
well as differential expression resulting in different prognostic
outcomes, allows the use of this information in a number of ways.
For example, the evaluation of a particular treatment regime may
be evaluated: does a chemotherapeutic drug act to improve the longterm
prognosis in a particular patient. Similarly, diagnosis may be done
or confirmed by comparing patient samples with the known expression
profiles. Furthermore, these gene expression profiles (or individual
genes) allow screening of drug candidates with an eye to mimicking
or altering a particular expression profile; for example, screening
can be done for drugs that suppress the breast cancer expression
profile or convert a poor prognosis profile to a better prognosis
profile. This may be done by making biochips comprising sets of
the important breast cancer genes, which can then be used in these
screens. These methods can also be done on the protein basis; that
is, protein expression levels of the breast cancer proteins can
be evaluated for diagnostic and prognostic purposes or to screen
candidate agents. In addition, the breast cancer nucleic acid sequences
can be administered for gene therapy purposes, including the administration
of antisense nucleic acids, or the breast cancer proteins (including
antibodies and other modulators thereof) administered as therapeutic
Thus the present invention provides nucleic acid and protein sequences
that are differentially expressed in breast cancer when compared
to normal tissue. The differentially expressed sequences provided
herein are termed "breast cancer sequences". As outlined
below, breast cancer sequences include those that are upregulated
(i.e. expressed at a higher level) in breast cancer, as well as
those that are down-regulated (i.e. expressed at a lower level)
in breast cancer. In a preferred embodiment, the breast cancer sequences
are from humans; however, as will be appreciated by those in the
art, breast cancer sequences from other organisms may be useful
in animal models of disease and drug evaluation; thus, other breast
cancer sequences are provided, from vertebrates, including mammals,
including rodents (rats, mice, hamsters, guinea pigs, etc.), primates,
farm animals (including sheep, goats, pigs, cows, horses, etc).
Breast cancer sequences from other organisms may be obtained using
the techniques outlined below.
In a preferred embodiment, the breast cancer sequences are those
of nucleic acids encoding BCR4 or fragments thereof. Preferably,
the breast cancer sequences are those depicted in FIG. 1 or FIG.
2, or fragments thereof. Preferably, the breast cancer sequences
encode a protein having the amino acid sequence depicted in FIG.
3, or a fragment thereof. In a preferred embodiment, BCR4 has the
sequence of human LIV-1 protein.
Breast cancer sequences can include both nucleic acid and amino
acid sequences. In a preferred embodiment, the breast cancer sequences
are recombinant nucleic acids. By the term "recombinant nucleic
acid" herein is meant nucleic acid, originally formed in vitro,
in general, by the manipulation of nucleic acid by polymerases and
endonucleases, in a form not normally found in nature. Thus an isolated
nucleic acid, in a linear form, or an expression vector formed in
vitro by ligating DNA molecules that are not normally joined, are
both considered recombinant for the purposes of this invention.
It is understood that once a recombinant nucleic acid is made and
reintroduced into a host cell or organism, it will replicate non-recombinantly,
i.e. using the in vivo cellular machinery of the host cell rather
than in vitro manipulations; however, such nucleic acids, once produced
recombinantly, although subsequently replicated non-recombinantly,
are still considered recombinant for the purposes of the invention.
Similarly, a "recombinant protein" is a protein made
using recombinant techniques, i.e. through the expression of a recombinant
nucleic acid as depicted above. A recombinant protein is distinguished
from naturally occurring protein by at least one or more characteristics.
For example, the protein may be isolated or purified away from some
or all of the proteins and compounds with which it is normally associated
in its wild type host, and thus may be substantially pure. For example,
an isolated protein is unaccompanied by at least some of the material
with which it is normally associated in its natural state, preferably
constituting at least about 0.5%, more preferably at least about
5% by weight of the total protein in a given sample. A substantially
pure protein comprises at least about 75% by weight of the total
protein, with at least about 80% being preferred, and at least about
90% being particularly preferred. The definition includes the production
of a breast cancer protein from one organism in a different organism
or host cell. Alternatively, the protein may be made at a significantly
higher concentration than is normally seen, through the use of an
inducible promoter or high expression promoter, such that the protein
is made at increased concentration levels. Alternatively, the protein
may be in a form not normally found in nature, as in the addition
of an epitope tag or amino acid substitutions, insertions and deletions,
as discussed below.
In a preferred embodiment, the breast cancer sequences are nucleic
acids. As will be appreciated by those in the art and is more fully
outlined below, breast cancer sequences are useful in a variety
of applications, including diagnostic applications, which will detect
naturally occurring nucleic acids, as well as screening applications;
for example, biochips comprising nucleic acid probes to the breast
cancer sequences can be generated. In the broadest sense, then,
by "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked
together. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramidate (Beaucage et
al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579
(1977); Letsinger et al., Nuci. Acids Res. 14:3487 (1986); Sawai
et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc.
110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)),
phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991);
and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J.
Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages
(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,
Oxford University Press), and peptide nucleic acid backbones and
linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et
al., Chem. Int Ed. Engl. 31:1008 (1992): Nielsen, Nature, 365:566
(1993): Carlsson et al., Nature 380:207 (1996), all of which are
incorporated by reference). Other analog nucleic acids include those
with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA
92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684,
5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem.
Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988); Letsinger at al., Nucleoside & Nudeotide
13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate
Modifications in Antisense Research", Ed. Y. S. Sanghui and
P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem.
Lett. 4:395 (1994): Jeffs et al., J. Biomolecular NMR 34:17 (1994);
Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including
those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters
6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications
in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook.
Nucleic acids containing one or more carbocyclic sugars are also
included within one definition of nucleic acids (see Jenkins et
al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs
are described in Rawls, C & E News Jun. 2, 1997 page 35. All
of these references are hereby expressly incorporated by reference.
These modifications of the ribose-phosphate backbone may be done
for a variety of reasons, for example to increase the stability
and half-life of such molecules in physiological environments or
as probes on a biochip.
As will be appreciated by those in the art, all of these nucleic
acid analogs may find use in the present invention. In addition,
mixtures of naturally occurring nucleic acids and analogs can be
made; alternatively, mixtures of different nucleic acid analogs,
and mixtures of naturally occurring nucleic acids and analogs may
Particularly preferred are peptide nucleic acids (PNA) which includes
peptide nucleic acid analogs. These backbones are substantially
non-ionic under neutral conditions, in contrast to the highly charged
phosphodiester backbone of naturally occurring nucleic acids. This
results in two advantages. First, the PNA backbone exhibits improved
hybridization kinetics. PNAs have larger changes in the melting
temperature (Tm) for mismatched versus perfectly matched basepairs.
DNA and RNA typically exhibit a 2-4.degree. C. drop in Tm for an
internal mismatch. With the non-ionic PNA backbone, the drop is
closer to 7-9.degree. C. Similarly, due to their non-ionic nature,
hybridization of the bases attached to these backbones is relatively
insensitive to salt concentration. In addition, PNAs are not degraded
by cellular enzymes, and thus can be more stable.
The nucleic acids may be single stranded or double stranded, as
specified, or contain portions of both double stranded or single
stranded sequence. As will be appreciated by those in the art, the
depiction of a single strand ("Watson") also defines the
sequence of the other strand ("Crick"); thus the sequences
described herein also includes the complement of the sequence. The
nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid,
where the nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine,
isocytosine, isoguanine, etc. As used herein, the term "nucleoside"
includes nucleotides and nucleoside and nucleotide analogs, and
modified nucleosides such as amino modified nucleosides. In addition,
"nucleoside" includes non-naturally occurring analog structures.
Thus for example the individual units of a peptide nucleic acid,
each containing a base, are referred to herein as a nucleoside.
A breast cancer sequence can be initially identified by substantial
nucleic acid and/or amino acid sequence homology to the breast cancer
sequences outlined herein. Such homology can be based upon the overall
nucleic acid or amino acid sequence, and is generally determined
as outlined below, using either homology programs or hybridization
The breast cancer sequences of the invention can be identified
as follows. Samples of normal and tumor tissue are applied to biochips
comprising nucleic acid probes. The samples are first microdissected,
if applicable, and treated as is know in the art for the preparation
of mRNA. Suitable biochips are commercially available, for example
from Affymetrix. Gene expression profiles as described herein are
generated, and the data analyzed.
In a preferred embodiment, the genes showing changes in expression
as between normal and disease states are compared to genes expressed
in other normal tissues, including, but not limited to lung, heart,
brain, liver, breast, kidney, muscle, prostate, small intestine,
large intestine, spleen, bone, and placenta. In a preferred embodiment,
those genes identified during the breast cancer screen that are
expressed in any significant amount in other tissues are removed
from the profile, although in some embodiments, this is not necessary.
That is, when screening for drugs, it is preferable that the target
be disease specific, to minimize possible side effects.
In a preferred embodiment, breast cancer sequences are those that
are unregulated in breast cancer; that is, the expression of these
genes is higher in breast carcinoma as compared to normal breast
tissue. "Up-regulation" as used herein means at least
about a 50% increase, preferably a two fold change, more preferably
at least about a three fold change, with at least about five-fold
or higher being preferred. All accession numbers herein are for
the GenBank sequence database and the sequences of the accession
numbers are hereby expressly incorporated by reference. GenBank
is known in the art, see, e.g., Benson, DA, et al., Nucleic Acids
Research 26:1-7 (1998). In addition, these genes were found to be
expressed in a limited amount or not at all in bone marrow, heart,
brain, lung, liver, kidney, muscle, pancreas, prostate, colon, skin,
testes, stomach, small intestine and spleen.
In another embodiment, breast cancer sequences are those that are
down-regulated in breast cancer; that is, the expression of these
genes is lower in, for example, breast carcinoma as compared to
normal breast tissue. "Down-regulation" as used herein
means at least about a two-fold change, preferably at least about
a three fold change, with at least about five-fold or higher being
Breast cancer proteins of the present invention may be classified
as secreted proteins, transmembrane proteins or intracellular proteins.
In a preferred embodiment the breast cancer protein is an intracellular
protein. Intracellular proteins may be found in the cytoplasm and/or
in the nucleus. Intracellular proteins are involved in all aspects
of cellular function and replication (including, for example, signaling
pathways); aberrant expression of such proteins results in unregulated
or disregulated cellular processes. For example, many intracellular
proteins have enzymatic activity such as protein kinase activity,
protein phosphatase activity, protease activity, nucleotide cyclase
activity, polymerase activity and the like. Intracellular proteins
also serve as docking proteins that are involved in organizing complexes
of proteins, or targeting proteins to various subcellular localizations,
and are involved in maintaining the structural integrity of organelles.
An increasingly appreciated concept in characterizing intracellular
proteins is the presence in the proteins of one or more motifs for
which defined functions have been attributed. In addition to the
highly conserved sequences found in the enzymatic domain of proteins,
highly conserved sequences have been identified in proteins that
are involved in protein-protein interaction. For example, Src-homology-2
(SH2) domains bind tyrosine-phosphorytated targets in a sequence
dependent manner. PTB domains, which are distinct from SH2 domains,
also bind tyrosine phosphorylated targets. SH3 domains bind to proline-rich
targets. In addition, PH domains, tetratricopeptide repeats and
WD domains to name only a few, have been shown to mediate protein-protein
interactions. Some of these may also be involved in binding to phospholipids
or other second messengers. As will be appreciated by one of ordinary
skill in the art, these motifs can be identified on the basis of
primary sequence; thus, an analysis of the sequence of proteins
may provide insight into both the enzymatic potential of the molecule
and/or molecules with which the protein may associate.
In a preferred embodiment, the breast cancer sequences are transmembrane
proteins. Transmembrane proteins are molecules that span the phospholipid
bilayer of a cell. They may have an intracellular domain, an extracellular
domain, or both. The intracellular domains of such proteins may
have a number of functions including those already described for
intracellular proteins. For example, the intracellular domain may
have enzymatic activity and/or may serve as a binding site for additional
proteins. Frequently the intracellular domain of transmembrane proteins
serves both roles. For example certain receptor tyrosine kinases
have both protein kinase activity and SH2 domeains. In addition,
autophosphorylation of tyrosines on the receptor molecule itself,
creates binding sites for additional SH2 domain containing proteins.
Transmembrane proteins may contain from one to many transmembrane
domains. For example, receptor tyrosine kinases, certain cytokine
receptors, receptor guanylyl cyclases and receptor serine/threonine
protein kinases contain a single transmembrane domain. However,
various other proteins including channels and adenylyl cyclases
contain numerous transmembrane domains. Many important cell surface
receptors are classified as "seven transmembrane domain"
proteins, as they contain 7 membrane spanning regions. Important
transmembrane protein receptors include, but are not limited to
insulin receptor, insulin-like growth factor receptor, human growth
hormone receptor, glucose transporters, transferrin receptor, epidermal
growth factor receptor, low density lipoprotein receptor, epidermal
growth factor receptor, leptin receptor, interleukin receptors,
e.g. IL-1 receptor, IL-2 receptor, etc.
Characteristics of transmembrane domains include approximately
20 consecutive hydrophobic amino acids that may be followed by charged
amino acids. Therefore, upon analysis of the amino acid sequence
of a particular protein, the localization and number of transmembrane
domains within the protein may be predicted.
The extracellular domains of transmembrane proteins are diverse;
however, conserved motifs are found repeatedly among various extracellular
domains. Conserved structure and/or functions have been ascribed
to different extracellular motifs. For example, cytokine receptors
are characterized by a cluster of cysteines and a WSXWS (SEQ ID
NO:7) (W=tryptophan, S=serine, X=any amino acid) motif. Immunoglobulin-like
domains are highly conserved. Mucin-like domains may be involved
in cell adhesion and leucine-rich repeats participate in protein-protein
Many extracellular domains are involved in binding to other molecules.
In one aspect, extracellular domains are receptors. Factors that
bind the receptor domain include circulating ligands, which may
be peptides, proteins, or small molecules such as adenosine and
the like. For example, growth factors such as EGF, FGF and PDGF
are circulating growth factors that bind to their cognate receptors
to initiate a variety of cellular responses. Other factors include
cytokines, mitogenic factors, neurotrophic factors and the like.
Extracellular domains also bind to cell-associated molecules. In
this respect, they mediate cell-cell interactions. Cell-associated
ligands can be tethered to the cell for example via a glycosylphosphatidylinositol
(GPI) anchor, or may themselves be transmembrane proteins. Extracellular
domains also associate with the extracellular matrix and contribute
to the maintenance of the cell structure.
Breast cancer proteins that are transmembrane are particularly
preferred in the present invention as they are good targets for
immunotherapeutics, as are described herein. In addition, as outlined
below, transmembrane proteins can be also useful in imaging modalities.
In a preferred embodiment, BCR4 is a transmembrane protein.
It will also be appreciated by those in the art that a transmembrane
protein can be made soluble by removing transmembrane sequences,
for example through recombinant methods. Furthermore, transmembrane
proteins that have been made soluble can be made to be secreted
through recombinant means by adding an appropriate signal sequence.
In a preferred embodiment, the breast cancer proteins are secreted
proteins; the secretion of which can be either constitutive or regulated.
These proteins have a signal peptide or signal sequence that targets
the molecule to the secretory pathway. Secreted proteins are involved
in numerous physiological events; by virtue of their circulating
nature, they serve to transmit signals to various other cell types.
The secreted protein may function in an autocrine manner (acting
on the cell that secreted the factor), a paracrine manner (acting
on cells in close proximity to the cell that secreted the factor)
or an endocrine manner (acting on cells at a distance). Thus secreted
molecules find use in modulating or altering numerous aspects of
physiology. Breast cancer proteins that are secreted proteins are
particularly preferred in the present invention as they serve as
good targets for diagnostic markers, for example for blood tests.
A breast cancer sequence is initially identified by substantial
nucleic acid and/or amino acid sequence homology to the breast cancer
sequences outlined herein. Such homology can be based upon the overall
nucleic acid or amino acid sequence, and is generally determined
as outlined below, using either homology programs or hybridization
As used herein, a nucleic acid is a "breast cancer nucleic
acid" if the overall homology of the nucleic acid sequence
to the nucleic acid sequences encoding the amino acid sequences
of the figures is preferably greater than about 75% more preferably
greater than about 80%, even more preferably greater than about
85% and most preferably greater than 90%. In some embodiments the
homology will be as high as about 93 to 95 or 98%. Homology in this
context means sequence similarity or identity, with identity being
preferred. A preferred comparison for homology purposes is to compare
the sequence containing sequencing errors to the correct sequence.
This homology will be determined using standard techniques known
in the art, including, but not limited to, the local homology algorithm
of Smith & Waterman. Adv. Appl. Math. 2:482 (1981), by the homology
alignment algorithm of Needleman & Wunsch, J. Mol. Biool. 48:443
(1970), by the search for similarity method of Pearson & Upman,
PNAS USA 85:2444 (1988), by computerized implementations of these
algorithms (GAP. BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Drive, Madison,
Wis.), the Best Fit sequence program described by Devereux et al.,
Nucl. Acid Res. 12:387-395 (1984), preferably using the default
settings, or by inspection.
In a preferred embodiment, the sequences which are used to determine
sequence identity or similarity are selected from the sequences
set forth in the figures, preferably those shown in FIGS. 1 and
2 and fragments thereof. In one embodiment the sequences utilized
herein are those set forth in the figures. In another embodiment,
the sequences are naturally occurring allelic variants of the sequences
set forth in the figures. In another embodiment, the sequences are
sequence variants as further described herein.
One example of a useful algorithm is PILEUP. PILEUP creates a multiple
sequence alignment from a group of related sequences using progressive,
pairwise alignments. It can also plot a tree showing the clustering
relationships used to create the alignment PILEUP uses a simplification
of the progressive alignment method of Feng & Doofittie, J.
Mol. Evol. 35:351-360 (1987); the method is similar to that described
by Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters
including a default gap weight of 3.00, a default gap length weight
of 0.10. and weighted end gaps.
Another example of a useful algorithm is the BLAST algorithm, described
in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin
et al., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST
program is the WU-BLAST-2 program which was obtained from Altschul
et al., Methods in Enzymology, 266: 460-480 (1996). WU-BLAST-2 uses
several search parameters, most of which are set to the default
values. The adjustable parameters are set with the following values:
overlap span=1, overlap fraction=0.125, word threshold (T)=11. The
HSP S and HSP S2 parameters are dynamic values and are established
by the program itself depending upon the composition of the particular
sequence and composition of the particular database against which
the sequence of interest is being searched; however, the values
may be adjusted to increase sensitivity. A % amino acid sequence
identity value is determined by the number of matching identical
residues divided by the total number of residues of the "longer"
sequence in the aligned region. The "longer" sequence
is the one having the most actual residues in the aligned region
(gaps introduced by WU-Blast-2 to maximize the alignment score are
Thus, "percent (%) nucleic acid sequence identity" is
defined as the percentage of nucleotide residues in a candidate
sequence that are identical with the nucleotide residues of FIG.
1 or FIG. 2. A preferred method utilizes the BLASTN module of WU-BLAST-2
set to the default parameters, with overlap span and overlap fraction
set to 1 and 0.125, respectively.
The alignment may include the introduction of gaps in the sequences
to be aligned. In addition, for sequences which contain either more
or fewer nucleosides than those of the figures, it is understood
that the percentage of homology will be determined based on the
number of homologous nucleosides in relation to the total number
of nucleosides. Thus, for example, homology of sequences shorter
than those of the sequences identified herein and as discussed below,
will be determined using the number of nucleosides in the shorter
In one embodiment, the nucleic acid homology is determined through
hybridization studies. Thus, for example, nucleic acids which hybridize
under high stringency to the nucleic acid sequences which encode
the peptides identified in the figures, or their complements, are
considered a breast cancer sequence. High stringency conditions
are known in the art; see for example Maniats et al., Molecular
Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols
in Molecular Biology, ed. Ausubel, et al., both of which are hereby
incorporated by reference. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen, Techniques
in Biochemistry and Molecular Biology--Hybridization with Nucleic
Acid Probes, "Overview of principles of hybridization and the
strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength pH. The Tm is the temperature (under defined ionic
strength, pH and nucleic acid concentration) at which 50% of the
probes complementary to the target hybridize to the target sequence
at equilibrium (as the target sequences are present in excess, at
Tm, 50% of the probes are occupied at equilibrium). Stringent conditions
will be those in which the salt concentration is less than about
1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 30.degree. C. for short probes (e.g. 10 to 50 nucleotides)
and at least about 60.degree. C. for long probes (e.g. greater than
50 nucleotides). Stringent conditions may also be achieved with
the addition of destabilizing agents such as formamide.
In another embodiment, less stringent hybridization conditions
are used; for example, moderate or low stringency conditions may
be used, as are known in the art; see Maniatis and Ausubel, supra,
and. Tijssen, supra.
In addition, the breast cancer nucleic acid sequences of the invention
are fragments of larger genes, i.e. they are nucleic acid segments.
"Genes" in this context includes coding regions, non-coding
regions, and mixtures of coding and non-coding regions. Accordingly,
as will be appreciated by those in the art, using the sequences
provided herein, additional sequences of the breast cancer genes
can be obtained, using techniques well known in the art for cloning
either longer sequences or the full length sequences; see Maniatis
et al., and Ausubel, et al., supra, hereby expressly incorporated
Once the breast cancer nucleic acid is identified, it can be cloned
and, if necessary, its constituent parts recombined to form the
entire breast cancer nucleic acid. Once isolated from its natural
source, e.g., contained within a plasmid or other vector or excised
therefrom as a linear nucleic acid segment, the recombinant breast
cancer nucleic acid can be further-used as a probe to identify and
isolate other breast cancer nucleic acids, for example additional
coding regions. It can also be used as a "precursor" nucleic
acid to make modified or variant breast cancer nucleic acids and
The breast cancer nucleic acids of the present invention are used
in several ways. In a first embodiment, nucleic acid probes to the
breast cancer nucleic acids are made and attached to biochips to
be used in screening and diagnostic methods, as outlined below,
or for administration, for example for gene therapy and/or antisense
applications. Alternatively, the breast cancer nucleic acids that
include coding regions of breast cancer proteins can be put into
expression vectors for the expression of breast cancer proteins,
again either for screening purposes or for administration to a patient
In a preferred embodiment, nucleic acid probes to breast cancer
nucleic acids (both the nucleic acid sequences encoding peptides
outlined in the figures and/or the complements thereof) are made.
The nucleic acid probes attached to the biochip are designed to
be substantially complementary to the breast cancer nucleic acids,
i.e. the target sequence (either the target sequence of the sample
or to other probe sequences, for example in sandwich assays), such
that hybridization of the target sequence and the probes of the
present invention occurs. As outlined below, this complementarity
need not be perfect; there may be any number of base pair mismatches
which will interfere with hybridization between the target sequence
and the single stranded nucleic acids of the present invention.
However, if the number of mutations is so great that no hybridization
can occur under even the least stringent of hybridization conditions,
the sequence is not a complementary target sequence. Thus, by "substantially
complementary" herein is meant that the probes are sufficiently
complementary to the target sequences to hybridize under normal
reaction conditions, particularly high stringency conditions, as
A nucleic acid probe is generally single stranded but can be partially
single and partially double stranded. The strandedness of the probe
is dictated by the structure, composition, and properties of the
target sequence. In general, the nucleic acid probes range from
about 8 to about 100 bases long, with from about 10 to about 80
bases being preferred, and from about 30 to about 50 bases being
particularly preferred. That is, generally whole genes are not used.
In some embodiments, much longer nucleic acids can be used, up to
hundreds of bases.
In a preferred embodiment, more than one probe per sequence is
used, with either overlapping probes or probes to different sections
of the target being used. That is, two, three, four or more probes,
with three being preferred, are used to build in a redundancy for
a particular target. The probes can be overlapping (i.e. have some
sequence in common), or separate.
As will be appreciated by those in the art, nucleic acids can be
attached or immobilized to a solid support in a wide variety of
ways. By immobilized and grammatical equivalents herein is meant
the association or binding between the nucleic acid probe and the
solid support is sufficient to be stable under the conditions of
binding, washing, analysis, and removal as outlined below. The binding
can be covalent or noncovalent By "noncovalent binding"
and grammatical equivalents herein is meant one or more of either
electrostatic, hydrophilic, and hydrophobic interactions. Included
in non-covalent binding is the covalent attachment of a molecule,
such as, streptavidin to the support and the non-covalent binding
of the biotinylated probe to the streptavidin. By "covalent
binding" and grammatical equivalents herein is meant that the
two moieties, the solid support and the probe, are attached by at
least one bond, including sigma bonds, pi bonds and coordination
bonds. Covalent bonds can be formed directly between the probe and
the solid support or can be formed by a cross linker or by inclusion
of a specific reactive group on either the solid support or the
probe or both molecules. Immobilization may also involve a combination
of covalent and non-covalent interactions.
In general, the probes are attached to the biochip in a wide variety
of ways, as will be appreciated by those in the art. As described
herein, the nucleic acids can either be synthesized first, with
to subsequent attachment to the biochip, or can be directly synthesized
on the biochip.
The biochip comprises a suitable solid substrate. By substrate
or "solid support" or other grammatical equivalents herein
is meant any material that can be modified to contain discrete individual
sites appropriate for the attachment or association of the nucleic
acid probes and is amenable to at least one detection method. As
will be appreciated by those in the art, the number of possible
substrates are very large, and include, but are not limited to,
glass and modified or functionalized glass, plastics (Including
acrylics, polystyrene and copolymers of styrene and other materials,
polypropylene, polyethylene, polybutylene, polyurethanes, Teflon,
etc.), polysaccharides, nylon or nitrocellulose, resins, silica
or silicabased materials including silicon and modified silicon,
carbon, metals, inorganic glasses, plastics, etc. In general, the
substrates allow optical detection and do not appreciably fluorescese.
A preferred substrate is described in copending application entitled
Reusable Low Fluorescent Plastic Biochip filed Mar. 15, 1999, herein
incorporated by reference in its entirety.
Generally the substrate is planar, although as will be appreciated
by those in the art, other configurations of substrates may be used
as well. For example, the probes may be placed on the inside surface
of a tube, for flow-through sample analysis to minimize sample volume.
Similarly, the substrate may be flexible, such as a flexible foam,
including closed cell foams made of particular plastics.
In a preferred embodiment, the surface of the biochip and the probe
may be derivatized with chemical functional groups for subsequent
attachment of the two. Thus, for example, the biochip is derivatized
with a chemical functional group including, but not limited to,
amino groups, carboxy groups, oxo groups and thiol groups, with
amino groups being particularly preferred. Using these functional
groups, the probes can be attached using functional groups on the
probes. For example, nucleic acids containing amino groups can be
attached to surfaces comprising amino groups, for example using
linkers as are known in the art; for example, homo- or hetero-bifunctional
linkers as are well known (see 1994 Pierce Chemical Company catalog,
technical section on cross-linkers, pages 155-200, incorporated
herein by reference). In addition, in some cases, additional linkers,
such as alkyl groups (including substituted and heteroalkyl groups)
may be used.
In this embodiment, the oligonucleotides are synthesized as is
known in the art, and then attached to the surface of the solid
support As will be appreciated by those skilled in the art, either
the 5' or 3' terminus may be attached to the solid support, or attachment
may be via an internal nucleoside.
In an additional embodiment, the immobilization to the solid support
may be very strong, yet non-covalent. For example, biotinylated
oligonucleotides can be made, which bind to surfaces covalently
coated with streptavidin, resulting in attachment.
Alternatively, the oligonucleotides may be synthesized on the surface,
as is known in the art. For example, photoactivation techniques
utilizing photopolymerization compounds and techniques are used.
In a preferred embodiment, the nucleic acids can be synthesized
in situ, using well known photolithographic techniques, such as
those described in WO 95/25116; WO 95135505; U.S. Pat. Nos. 5,700,637
and 5,445,934; and references cited within, all of which are expressly
incorporated by reference; these methods of attachment form the
basis of the Affimetrix GENECHIP.RTM. (DNA microarray chip) technology.
In a preferred embodiment, breast cancer nucleic acids encoding
breast cancer proteins are used to make a variety of expression
vectors to express breast cancer proteins which can then be used
in screening assays, as described below. The expression vectors
may be either self-replicating extrachromosomal vectors or vectors
which integrate into a host genome. Generally, these expression
vectors include transcriptional and translational regulatory nucleic
acid operably linked to the nucleic acid encoding the breast cancer
protein. The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are suitable
for prokaryotes, for example, include a promoter, optionally an
operator sequence, and a ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into
a functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably linked
to DNA for a polypeptide if it is expressed as a preprotein that
participates in the secretion of the polypeptide; a promoter or
enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome binding site is operably
linked to a coding sequence if it is positioned so as to facilitate
translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However, enhancers
do not have to be contiguous. Linking is accomplished by ligation
at convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in accordance
with conventional practice. The transcriptional and translational
regulatory nucleic acid will generally be appropriate to the host
cell used to express the breast cancer protein; for example, transcriptional
and translational regulatory nucleic acid sequences from Bacillus
are preferably used to express the breast cancer protein in Bacillus.
Numerous types of appropriate expression vectors, and suitable regulatory
sequences are known in the art for a variety of host cells.
In general, the transcriptional and translational regulatory sequences
may include, but are not limited to, promoter sequences, ribosomal
binding sites, transcriptional start and stop sequences, translational
start and stop sequences, and enhancer or activator sequences. In
a preferred embodiment, the regulatory sequences include a promoter
and transcriptional start and stop sequences.
Promoter sequences encode either constitutive or inducible promoters.
The promoters may be either naturally occurring promoters or hybrid
promoters. Hybrid promoters, which combine elements of more than
one promoter, are also known in the art, and are useful in the present
In addition, the expression vector may comprise additional elements.
For example, the expression vector may have two replication systems,
thus allowing it to be maintained in two organisms, for example
in mammalian or insect cells for expression and in a procaryotic
host for cloning and amplification. Furthermore, for integrating
expression vectors, the expression vector contains at least one
sequence homologous to the host cell genome, and preferably two
homologous sequences which flank the expression construct. The integrating
vector may be directed to a specific locus in the host cell by selecting
the appropriate homologous sequence for inclusion in the vector.
Constructs for integrating vectors are well known in the art.
In addition, in a preferred embodiment, the expression vector contains
a selectable marker gene to allow the selection of transformed host
cells. Selection genes are well known in the art and will vary with
the host cell used.
The breast cancer proteins of the present invention are produced
by culturing a host cell transformed with an expression vector containing
nucleic acid encoding a breast cancer protein, under the appropriate
conditions to induce or cause expression of the breast cancer protein.
The conditions appropriate for breast cancer protein expression
will vary with the choice of the expression vector and the host
cell, and will be easily ascertained by one skilled in the art through
routine experimentation. For example, the use of constitutive promoters
in the expression vector will require optimizing the growth and
proliferation of the host cell, while the use of an inducible promoter
requires the appropriate growth conditions for induction. In addition,
in some embodiments, the timing of the harvest is important. For
example, the baculoviral systems used in insect cell expression
are lytic viruses, and thus harvest time selection can be crucial
for product yield.
Appropriate host cells include yeast, bacteria, archaebacteria,
fungi, and insect and animal cells, including mammalian cells. Of
particular interest are Drosophila melangaster cells, Saccharomyces
cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells,
C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, THP1
cell line (a macrophage cell line) and human cells and cell lines.
In a preferred embodiment, the breast cancer proteins are expressed
in mammalian cells. Mammalian expression systems are also known
in the art, and include retroviral systems. A preferred expression
vector system is a retroviral vector system such as is generally
described in PCT/US97/01019 and PCT/US97/01048, both of which are
hereby expressly incorporated by reference. Of particular use as
mammalian promoters are the promoters from mammalian viral genes,
since the viral genes are often highly expressed and have a broad
host range. Examples include the SV40 early promoter, mouse mammary
tumor virus LTR promoter, adenovinis major late promoter, herpes
simplex virus promoter, and the CMV promoter. Typically, transcription
termination and polyadenylation sequences recognized by mammalian
cells are regulatory regions located 3' to the translation stop
codon and thus, together with the promoter elements, flank the coding
sequence. Examples of transcription terminator and polyadenylation
signals include those derived form SV40.
The methods of introducing exogenous nucleic acid into mammalian
hosts, as well as other hosts, is well known in the art, and will
vary with the host cell used. Techniques include dextran-mediated
transfection, calcium phosphate precipitation, polybrene mediated
transfection, protoplast fusion, electroporation, viral infection,
encapsulation of the polynucleotide(s) in liposomes, and direct
microinjection of the DNA into nuclei.
In a preferred embodiment, breast cancer proteins are expressed
in bacterial systems. Bacterial expression systems are well known
in the art. Promoters from bacteriophage may also be used and are
known in the art. In addition, synthetic promoters and hybrid promoters
are also useful; for example, the tac promoter is a hybrid of the
trp and lac promoter sequences. Furthermore, a bacterial promoter
can include naturally occurring promoters of non-bacterial origin
that have the ability to bind bacterial RNA polymerase and initiate
transcription. In addition to a functioning promoter sequence, an
efficient ribosome binding site is desirable. The expression vector
may also include a signal peptide sequence that provides for secretion
of the breast cancer protein in bacteria. The protein is either
secreted into the growth media (gram-positive bacteria) or into
the periplasmic space, located between the inner and outer membrane
of the cell (gram-negative bacteria). The bacterial expression vector
may also include a selectable marker gene to allow for the selection
of bacterial strains that have been transformed. Suitable selection
genes include genes which render the bacteria resistant to drugs
such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin
and tetracycline. Selectable markers also include biosynthetic genes,
such as those in the histidine, tryptophan and leucine biosynthetic
pathways. These components are assembled into expression vectors.
Expression vectors for bacteria are well known in the art and include
vectors for Bacillus subtilis, E. coli, Streptococcus cremoris,
and Streptococcus lividans, among others. The bacterial expression
vectors are transformed into bacterial host cells using techniques
well known in the art, such as calcium chloride treatment, electroporation,
In one embodiment, breast cancer proteins are produced in insect
cell. Expression vectors for the transformation of insect cells,
and in particular, baculovirus-based expression vectors, are well
known in the art.
In a preferred embodiment, breast cancer protein is produced in
yeast cells. Yeast expression systems are well known in the art,
and include expression vectors for Saccharomyces cerevisiae, Candida
albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis
and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces
pombe, and Yarrowia lipolytica.
The breast cancer protein may also be made as a fusion protein,
using techniques well known in the art. Thus, for example, for the
creation of monoclonal antibodies, if the desired epitope is small,
the breast cancer protein may be fused to a carrier protein to form
an immunogen. Alternatively, the breast cancer protein may be made
as a fusion protein to increase expression, or for other reasons.
For example, when the breast cancer protein is a breast cancer peptide,
the nucleic acid encoding the peptide may be linked to other nucleic
acid for expression purposes.
In one embodiment, the breast cancer nucleic acids, proteins and
antibodies of the invention are labeled. By "labeled"
herein is meant that a compound has at least one element, isotope
or chemical compound attached to enable the detection of the compound.
In general, labels fall into three classes: a) isotopic labels,
which may be radioactive or heavy isotopes; b) immune labels, which
may be antibodies or antigens; and c) colored or fluorescent dyes.
The labels may be incorporated into the breast cancer nucleic acids,
proteins and antibodies at any position. For example, the label
should be capable of producing, either directly or indirectly, a
detectable signal. The detectable moiety may be a radioisotope,
such as .sup.3 H, .sup.14 C, .sup.32 P, .sup.35 S or .sup.125 I
a fluorescent or chemiluminescent compound, such as fluorescein
isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase or horseradish peroxidase. Any method
known in the art for conjugating the antibody to the label may be
employed, including those methods described by Hunter et al., Nature,
144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain
et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem,
and Cytochem., 30:407 (1982).
Accordingly, the present invention also provides breast cancer
protein sequences. A breast cancer protein of the present invention
may be identified in several ways. "Protein" in this sense
includes proteins, polypeptides, and peptides. As will be appreciated
by those in the art, the nucleic acid sequences of the invention
can be used to generate protein sequences. There are a variety of
ways to do this, including cloning the entire gene and verifying
its frame and amino acid sequence, or by comparing it to known sequences
to search for homology to pride a frame, assuming the breast cancer
protein has homology to some protein in the database being used.
Generally, the nucleic acid sequences are input into a program that
will search all three frames for homology. This is done in a preferred
embodiment using the following NCBI Advanced BLAST parameters. The
program is blastx or blastn. The database is nr. The input data
is as "Sequence in FASTA format". The organism list is
"none". The "expect"is 10; the filter is default.
The "descriptions" is 500, the "alignments"
is 500, and the "alignment view" is pairwise. The "Query
Genetic Codes" is standard (1). The matrix is BLOSUM62; gap
existence cost is 11, per residue gap cost is 1; and the lambda
ratio is 0.85 default. This results in the generation of a putative
Also included within one embodiment of breast cancer proteins are
amino acid variants of the naturally occurring sequences, as determined
herein. Preferably, the variants are preferably greater than about
75% homologous to the wild-type sequence, more preferably greater
than about 80%, even more preferably greater than about 85% and
most preferably greater than 90%. In some embodiments the homology
will be as high as about 93 to 95 or 98%. As for nucleic acids,
homology in this context means sequence similarity or identity,
with identity being preferred. This homology will be determined
using standard techniques known in the art as are outlined above
for the nucleic acid homologies.
Breast cancer proteins of the present invention may be shorter
or longer than the wild type amino acid sequences. Thus, in a preferred
embodiment, included within the definition of breast cancer proteins
are portions or fragments of the wild type sequences, herein. In
addition, as outlined above, the breast cancer nucleic acids of
the invention may be used to obtain additional coding regions, and
thus additional protein sequence, using techniques known in the
In a preferred embodiment, the breast cancer proteins are derivative
or variant breast cancer proteins as compared to the wild-type sequence.
That is, as outlined more fully below, the derivative breast cancer
peptide will contain at least one amino acid substitution, deletion
or insertion, with amino acid substitutions being particularly preferred.
The amino acid substitution, insertion or deletion may occur at
any residue within the breast cancer peptide.
Also included in an embodiment of breast cancer proteins of the
present invention are amino acid sequence variants. These variants
fall into one or more of three classes: substitutional, insertional
or deletional variants. These variants ordinarily are prepared by
site specific mutagenesis of nucleotides in the DNA encoding the
breast cancer protein, using cassette or PCR mutagenesis or other
techniques well known in the art, to produce DNA encoding the variant,
and thereafter expressing the DNA in recombinant cell culture as
outlined above. However, variant breast cancer protein fragments
having up to about 100-150 residues may be prepared by in vitro
synthesis using established techniques. Amino acid sequence variants
are characterized by the predetermined nature of the variation,
a feature that sets them apart from naturally occurring allelic
or interspecies variation of the breast cancer protein amino acid
sequence. The variants typically exhibit the same qualitative biological
activity as the naturally occurring analogue, although variants
can also be selected which have modified characteristics as will
be more fully outlined below.
While the site or region for introducing an amino acid sequence
variation is predetermined, the mutation per se need not be predetermined.
For example, in order to optimize the performance of a mutation
at a given site, random mutagenesis may be conducted at the target
codon or region and the expressed breast cancer variants screened
for the optimal combination of desired activity. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example, M13 primer mutagenesis
and PCR mutagenesis. Screening of the mutants is done using assays
of breast cancer protein activities.
Amino acid substitutions are typically of single residues; insertions
usually will be on the order of from about 1 to 20 amino acids,
although considerably larger insertions may be tolerated. Deletions
range from about 1 to about 20 residues, although in some cases
deletions may be much larger.
Substitutions, deletions, insertions or any combination thereof
may be used to arrive at a final derivative. Generally these changes
are done on a few amino acids to minimize the alteration of the
molecule. However, larger changes may be tolerated in certain circumstances.
When small alterations in the characteristics of the breast cancer
protein are desired, substitutions are generally made in accordance
with the following chart:
CHART I Original Exemplary Residue Substitutions Ala Ser Arg Lys
Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln
Ile, Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met,
Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
Substantial changes in function or immunological identity are made
by selecting substitutions that are less conservative than those
shown in Chart 1. For example, substitutions may be made which more
significantly affect the structure of the polypeptide backbone in
the area of the alteration, for example the alpha-helical or beta-sheet
structure; the charge or hydrophobicity of the molecule at the target
site; or the bulk of the side chain. The substitutions which in
general are expected to produce the greatest changes in the polypeptide's
properties are those in which (a) a hydrophilic residue, e.g. seryl
or threonyl is substituted for (or by) a hydrophobic residue, e.g.
leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine
or proline is substituted for (or by) any other residue; (c) a residue
having an electropositive side chain, e.g. lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g. glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine,
is substituted for (or by) one not having a side chain, e.g. glycine.
The variants typically exhibit the same qualitative biological
activity and will elicit the same immune response as the naturally
occurring analogue, although variants also are selected to modify
the characteristics of the breast cancer proteins as needed. Alternatively,
the variant may be designed such that the biological activity of
the breast cancer protein is altered. For example, glycosylation
sites may be altered or removed.
Covalent modifications of breast cancer polypeptides are included
within the scope of this invention. One type of covalent modification
includes reacting targeted amino acid residues of a breast cancer
polypeptide with an organic derivatizing agent that is capable of
reacting with selected side chains or the N-or C-terminal residues
of a breast cancer polypeptide. Derivatization with bifunctional
agents is useful, for instance, for crosslinking breast cancer to
a water-insoluble support matrix or surface for use in the method
for purifying anti-breast cancer antibodies or screening assays,
as is more fully described below. Commonly used crosslinking agents
include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic
acid, homobifunctional imidoesters, including disuccimidyl esters
such as 3,3'-dithiobis(succinimidyl-propionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl
residues to the corresponding glutamyl and asparyl residues, respectively,
hydroxylation of proline and lysine, phosphorylation of hydroxy
groups of seryl, threonyl or tyrosyl residues, methylation the .alpha.-aminogroups
of lysine, arginine, and histidine side chains [T. E. Creighton,
Proteins: Structure and Molecular Properties, W. H. Freeman &
Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal
amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the breast cancer polypeptide
included within the scope of this invention comprises altering the
native glycosylation pattern of the polypeptide. "Altering
the native glycosylation pattern" is intended for purposes
herein to mean deleting one or more carbohydrate moieties found
in native sequence breast cancer polypeptide, and/or adding one
or more glycosylation sites that are not present in the native sequence
breast cancer polypeptide.
Addition of glycosylation sites to breast cancer polypeptides may
be accomplished by altering the amino acid sequence thereof. The
alteration may be made, for example, by the addition of, or substitution
by, one or more serine or threonine residues to the native sequence
breast cancer polypeptide (for O-linked glycosylation sites). The
breast cancer amino acid sequence may optionally be altered through
changes at the DNA level, particularly by mutating the DNA encoding
the breast cancer polypeptide at preselected bases such that codons
are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties
on the breast cancer polypeptide is by chemical or enzymatic coupling
of glycosides to the polypeptide. Such methods are described in
the art, e.g., in WO 87105330 published Sep. 11, 1987, and in Aplin
and Wriston, breast cancer Crit Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the breast cancer polypeptide
may be accomplished chemically or enzymatically or by mutational
substitution of codons encoding for amino acid residues that serve
as targets for glycosylation. Chemical deglycosylation techniques
are known in the art and described, for instance, by Hakimuddin,
et al. Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al.,
Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate
moieties on polypeptides can be achieved by the use of a variety
of endo- and exo-glycosidases as described by Thotakura et al.,
Meth. Enzymol., 138:350 (1987).
Another type of covalent modification of breast cancer protein
comprises linking the breast cancer polypeptide to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
Breast cancer polypeptides of the present invention may also be
modified in a way to form chimeric molecules comprising a breast
cancer polypeptide fused to another, heterologous polypeptide or
amino acid sequence. In one embodiment, such a chimeric molecule
comprises a fusion of a breast cancer polypeptide with a tag polypeptide
which provides an epitope to which an anti-tag antibody can selectively
bind. The epitope tag is generally placed at the amino- or carboxy-terminus
of the breast cancer polypeptide. The presence of such epitope-tagged
forms of a breast cancer polypeptide can be detected using an antibody
against the tag polypeptide. Also, provision of the epitope tag
enables the breast cancer polypeptide to be readily purified by
affinity purification using an anti-tag antibody or another type
of affinity matrix that binds to the epitope tag. In an alternative
embodiment, the chimeric molecule may comprise a fusion of a breast
cancer polypeptide with an immunoglobulin or a particular region
of an immunoglobulin. For a bivalent form of the chimeric molecule,
such a fusion could be to the Fc region of an IgG molecule.
Various tag polypeptides and their respective antibodies are well
known in the art. Examples include poly-histidine (poly-his) or
poly-histidineglycine (poly-his-gly) tags; the flu HA tag polypeptide
and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165
(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies
thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616
(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and
its antibody [Paborsky et al., Protein Engineering, 3(6):547-553
(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et
al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide
[Marlin et al., Science, 255:192-194 (1992)]; tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]: and the
T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)].
Also included with the definition of breast cancer protein in one
embodiment are other breast cancer proteins of the breast cancer
family, and breast cancer proteins from other organisms, which are
cloned and expressed as outlined below. Thus, probe or degenerate
polymerase chain reaction (PCR) primer sequences may be used to
find other related breast cancer proteins from humans or to other
organisms. As will be appreciated by those in the art, particularly
useful probe and/or PCR primer sequences include the unique areas
of the breast cancer nucleic acid sequence. As is generally known
in the art, preferred PCR primers are from about 15 to about 35
nucleotides in length, with from about 20 to about 30 being preferred,
and may contain inosine as needed. The conditions for the PCR reaction
are well known in the art.
In addition, as is outlined herein, breast cancer proteins can
be made that are longer than those depicted in the figures, for
example, by the elucidation of additional sequences, the addition
of epitope or purification tags, the addition of other fusion sequences,
Breast cancer proteins may also be identified as being encoded
by breast cancer nucleic acids. Thus, breast cancer proteins are
encoded by nucleic acids that will hybridize to the sequences of
the sequence listings, or their complements, as outlined herein.
In a preferred embodiment, when the breast cancer protein is to
be used to generate antibodies, for example for immunotherapy, the
breast cancer protein should share at least one epitope or determinant
with the full length protein. By "epitope" or "determinant"
herein is meant a portion of a protein which will generate and/or
bind an antibody or T-cell receptor in the context of MHC. Thus,
in most instances, antibodies made to a smaller breast cancer protein
will be able to bind to the full length protein. In a preferred
embodiment, the epitope is unique; that is, antibodies generated
to a unique epitope show little or no cross-reactivity.
In one embodiment, the term "antibody" includes antibody
fragments, as are known in the art, including Fab, Fab.sub.2, single
chain antibodies (Fv for example), chimeric antibodies, etc., either
produced by the modification of whole antibodies or those synthesized
de novo using recombinant DNA technologies.
Methods of preparing polyclonal antibodies are known to the skilled
artisan. Polyclonal antibodies can be raised in a mammal, for example,
by one or more injections of an immunizing agent and, if desired,
an adjuvant. Typically, the immunizing agent and/or adjuvant will
be injected in the mammal by multiple subcutaneous or intraperitoneal
injections. The immunizing agent may include the BCR4 or fragment
thereof or a fusion protein thereof. It may be useful to conjugate
the immunizing agent to a protein known to be immunogenic in the
mammal being immunized. Examples of such immunogenic proteins include
but are not limited to keyhole limpet hemocyanin, serum albumin,
bovine thyroglobulin, and soybean trypsin inhibitor. Examples of
adjuvants which may be employed include Freund's complete adjuvant
and MPL-TDM adjuvant (monophosphryl Lipid A, synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by
one skilled in the art without undue experimentation.
The antibodies may, alternatively, be monoclonal antibodies. Monoclonal
antibodies may be prepared using hybridoma methods, such as those
described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma
method, a mouse, hamster, or other appropriate host animal, is typically
immunized with an immunizing agent to elicit lymphocytes that produce
or are capable of producing anti-bodies, that will specifically
bind to the immunizing agent. Alternatively, the lymphocytes may
be immunized in vitro. The immunizing agent will typically include
the BCR4 polypeptide or fragment thereof or a fusion protein thereof.
Generally, either peripheral blood lymphocytes ("PBLs")
are used if cells of human origin are desired, or spleen cells or
lymph node cells are used if non-human mammalian sources are desired.
The lymphocytes are then fused with an immortalized cell fine using
a suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice,
Academic Press, (1988) pp. 59-103]. Immortalized cell lines are
usually transformed mammalian cells, particularly myeloma cells
of rodent, bovine and human origin. Usually, rat or mouse myeloma
cell lines are employed. The hybridoma cells may be cultured in
a suitable culture medium that preferably contains one or more substances
that inhibit the growth or survival of the unfused, immortalized
cells. For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("H-AT medium"), which substances prevent
the growth of HGPRT-deficient cells.
In one embodiment, the antibodies are bispecfic antibodies. Bispecific
antibodies are monoclonal, preferably human or humanized, antibodies
that have binding specificities for at least two different antigens.
In the present case, one of the binding specificities is for the
BCR4 or a fragment thereof, the other one is for any other antigen,
and preferably for a cell-surface protein or receptor or receptor
subunit, preferably one that is tumor specific.
In a preferred embodiment, the antibodies to breast cancer are
capable of reducing or eliminating the biological function of breast
cancer, as is described below. That is, the addition of anti-breast
cancer antibodies (either polyclonal or preferably monoclonal) to
breast cancer (or cells containing breast cancer) may reduce or
eliminate the breast cancer activity. Generally, at least a 25%
decrease in activity is preferred, with at least about 50% being
particularly preferred and about a 95-100% decrease being especially
In a preferred embodiment the antibodies to the breast cancer proteins
are humanized antibodies. Humanized forms of non-human (e.g., murine)
antibodies are chimeric molecules of immunoglobulins, immunoglobulin
chains or fragments thereof (such as Fv, Fab. Fab', F(ab').sub.2
or other antigen-binding subsequences of antibodies) which contain
minimal sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues form a complementary determining region (CDR) of
the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by corresponding
nonhuman residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the imported
CDR or framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions correspond
to those of a non-human immunoglobulin and all or substantially
all of the FR regions are those of a human immunoglobulin consensus
sequence. The humanized antibody optimally also will comprise at
least a portion of an immunoglobulin constant region (Fc), typically
that of a human immunoglobulin [Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta,
Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the
art. Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as import residues,
which are typically taken from an import variable domain. Humanization
can be essentially performed following the method of Winter and
co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, :1534-1536
(1988)], by substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such humanized antibodies
are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially
less than an intact human variable domain has been substituted by
the corresponding sequence from a nonhuman species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by residues
from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques
known in the art, including phage display libraries [Hoogenboom
and Winter, J. Mol Biol., 27:381 (1991); Marks et al., J. Mol. Biol.,
222:581 (1991)]. The techniques of Cole et al. and Boemer et al.
are also available for the preparation of human monoclonal antibodies
(Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(1):86-95
(1991)]. Similarly, human antibodies can be made by introducing
of human immunoglobulin loci into transgenic animals, e.g., mice
in which the endogenous immunoglobulin genes have been partially
or completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody repertoire.
This approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,681,016, and in the
following scientific publications: Marks et al., Bio/Technology
10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison,
Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology
14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996);
Lonberg and Huszar, Intern. Rev. Immunol, 13 65-93 (1995).
By immunotherapy is meant treatment of breast cancer with an antibody
raised against breast cancer proteins. As used herein, immunotherapy
can be passive or active. Passive immunotherapy as defined herein
is the passive transfer of antibody to a recipient (patient). Active
immunization is the induction of antibody and/or T-cell responses
in a recipient (patient). Induction of an immune response is the
result of providing the recipient with an antigen to which antibodies
are raised. As appreciated by one of ordinary skill in the art,
the antigen may be provided by injecting a polypeptide against which
antibodies are desired to be raised into a recipient, or contacting
the recipient with a nucleic acid capable of expressing the antigen
and under conditions for expression of the antigen.
In a preferred embodiment the breast cancer proteins against which
antibodies are raised are secreted proteins as described above.
Without being bound by theory, antibodies used for treatment, bind
and prevent the secreted protein from binding to its receptor, thereby
inactivating the secreted breast cancer protein.
In another preferred embodiment, the breast cancer protein to which
antibodies are raised is a transmembrane protein. Without being
bound by theory, antibodies used for treatment, bind the extracellular
domain of the breast cancer protein and prevent it from binding
to other proteins, such as circulating ligands or cell-associated
molecules. The antibody may cause down-regulation of the transmembrane
breast cancer protein. As will be appreciated by one of ordinary
skill in the art, the antibody may be a competitive, non-competitive
or uncompetitive inhibitor of protein binding to the extracellular
domain of the breast cancer protein. The antibody is also an antagonist
of the breast cancer protein. Further, the antibody prevents activation
of the transmembrane breast cancer protein. In one aspect, when
the antibody prevents the binding of other molecules to the breast
cancer protein, the antibody prevents growth of the cell. The antibody
also sensitizes the cell to cytotoxic agents, including, but not
limited to TNF.alpha., TNF-.beta., IL-1. INF-.gamma. and IL-2, or
chemotherapeutic agents including 5FU, vinblastine, actinomycin
D, cisplatin, methotrexate, and the like. In some instances the
antibody belongs to a sub-type that activates serum complement when
complexed with the transmembrane protein thereby mediating cytotoxicity.
Thus, breast cancer is treated by administering to a patient antibodies
directed against the transmembrane breast cancer protein.
In another preferred embodiment, the antibody is conjugated to
a therapeutic moiety. In one aspect the therapeutic moiety is a
small molecule that modulates the activity of the breast cancer
protein. In another aspect the therapeutic moiety modulates the
activity of molecules associated with or in close proximity to the
breast cancer protein. The therapeutic moiety may inhibit enzymatic
activity such as protease or protein kinase activity associated
with breast cancer.
In a preferred embodiment, the therapeutic moiety may also be a
cytotoxic agent in this method, targeting the cytotoxic agent to
tumor tissue or cells, results in a reduction in the number of afflicted
cells, thereby reducing symptoms associated with breast cancer.
Cytotoxic agents are numerous and varied and include, but are not
limited to, cytotoxic drugs or toxins or active fragments of such
toxins. Suitable toxins and their corresponding fragments include
diphtheria A chain, exotoxin A chain. ricin A chain, abrin A chain,
curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents
also include radiochemicals made by conjugating radioisotopes to
antibodies raised against breast cancer proteins, or binding of
a radionuclide to a chelating agent that has been covalently attached
to the antibody. Targeting the therapeutic moiety to transmembrane
breast cancer proteins not only serves to increase the local concentration
of therapeutic moiety in the breast cancer afflicted area, but also
serves to reduce deleterious side effects that may be associated
with the therapeutic moiety.
In another preferred embodiment, the PC protein against which the
antibodies are raised is an intracellular protein. In this case,
the antibody may be conjugated to a protein which facilitates entry
into the cell. In one case, the antibody enters the cell by endocytosis.
In another embodiment, a nucleic acid encoding the antibody is administered
to the individual or cell. Moreover, wherein the PC protein can
be targeted within a cell, i.e., the nucleus, an antibody thereto
contains a signal for that target localization, i.e., a nuclear
The breast cancer antibodies of the invention specifically bind
to breast cancer proteins. By "specifically bind" herein
is meant that the antibodies bind to the protein with a binding
constant in the range of at least 10.sup.-4 -10.sup.-8 M.sup.-1,
with a preferred range being 10.sup.-7 -10.sup.-9 M.sup.-1.
In a preferred embodiment, the breast cancer protein is purified
or isolated after expression. Breast cancer proteins may be isolated
or purified in a variety of ways known to those skilled in the art
depending on what other components are present in the sample. Standard
purification methods include electrophoretic, molecular, immunological
and chromatographic techniques, including ion exchange, hydrophobic,
affinity, and reverse-phase HPLC chromatography, and chromatofocusing.
For example, the breast cancer protein may be purified using a standard
anti-breast cancer antibody column. Ultrafiltration and diafiltration
techniques, in conjunction with protein concentration, are also
useful. For general guidance in suitable purification techniques,
see Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982).
The degree of purification necessary will vary depending on the
use of the breast cancer protein. In some instances no purification
will be necessary.
Once expressed and purified if necessary, the breast cancer proteins
and nucleic acids are useful in a number of applications.
In one aspect, the expression levels of genes are determined for
different cellular states in the breast cancer phenotype; that is,
the expression levels of genes in normal breast tissue and in breast
cancer tissue (and in some cases, for varying severities of breast
cancer that relate to prognosis, as outlined below) are evaluated
to provide expression profiles. An expression profile of a particular
cell state or point of development is essentially a "fingerprint"
of the state; while two states may have any particular gene similarly
expressed, the evaluation of a number of genes simultaneously allows
the generation of a gene expression profile that is unique to the
state of the cell. By comparing expression profiles of cells in
different states, information regarding which genes are important
(including both up- and down-regulation of genes) in each of these
states is obtained. Then, diagnosis may be done or confirmed: does
tissue from a particular patient have the gene expression profile
of normal or breast cancer tissue.
"Differential expression," or equivalents as used herein
to both qualitative as well as quantitative differences in the genes'
temporal and/or cellular expression patterns within and among the
cells. Thus, a breast cancer gene can qualitatively have its expression
altered, including an activation or inactivation, in, for example,
normal versus breast cancer tissue. That is, genes may be turned
on or turned off in a particular state, relative to another state.
As is apparent to the skilled artisan, any comparison of two or
more states can be made. Such a qualitatively regulated gene will
exhibit an expression pattern within a state or cell type which
is detectable by standard techniques in one such state or cell type,
but is not detectable in both. Alternatively, the determination
is quantitative in that expression is increased or decreased; that
is, the expression of the gene is either upregulated, resulting
in an increased amount of transcript, or downregulated, resulting
in a decreased amount of transcript. The degree to which expression
differs need only be large enough to quantify via standard characterization
techniques as outlined below, such as by use of Affymetrix GENECHIP.RTM.
(DNA microarray chip) expression arrays, Lockhart, Nature Biotechnology,
14:1675-1680 (1996), hereby expressly incorporated by reference.
Other techniques include, but are not limited to, quantitative reverse
transcriptase PCR, Northern analysis and RNase protection. As outlined
above, preferably the change in expression (i.e. upregulation or
downregulation) is at least about 50%, more preferably at least
about 100%, more preferably at least about 150%, more preferably,
at least about 200%, with from 300 to at least 1000% being especially
As will be appreciated by those in the art, this may be done by
evaluation at either the gene transcript, or the protein level;
that is, the amount of gene expression may be monitored using nucleic
acid probes to the DNA or RNA equivalent of the gene transcript,
and the quantification of gene expression levels, or, alternatively,
the final gene product itself (protein) can be monitored, for example
through the use of antibodies to the breast cancer protein and standard
immunoassays (ELISAs,e tc.) or other techniques, including mass
spectroscopy assays, 2D gel lectophoresis assays, etc. Thus, the
proteins corresponding to breast cancer genes, i.e. those identified
as being important in a breast cancer phenotype, can be evaluated
in a breast cancer diagnostic test.
In a preferred embodiment, gene expression monitoring is done and
a number of genes, i.e. an expression profile, is monitored simultaneously,
although multiple protein expression monitoring can be done as well.
Similarly, these assays may be done on an individual basis as well.
In this embodiment, the breast cancer nucleic acid probes are attached
to biochips as outlined herein for the detection and quantification
of breast cancer sequences in a particular cell. The assays are
further described below in the example.
In a preferred embodiment nucleic acids encoding the breast cancer
protein are detected. Although DNA or RNA encoding the breast cancer
protein may be detected, of particular interest are methods wherein
the mRNA encoding a breast cancer protein is detected. The presence
of mRNA in a sample is an indication that the breast cancer gene
has been transcribed to form the mRNA, and suggests that the protein
is expressed. Probes to detect the mRNA can be any nucleotide/deoxynucleotide
probe that is complementary to and base pairs with the mRNA and
includes but is not limited to oligonucleotides, cDNA or RNA. Probes
also should contain a detectable label, as defined herein. In one
method the mRNA is detected after immobilizing the nucleic acid
to be examined on a solid support such as nylon membranes and hybridizing
the probe with the sample. Following washing to remove the non-specifically
bound probe, the label is detected. In another method detection
of the mRNA is performed in situ. In this method permeabilized cells
or tissue samples are contacted with a detectably labeled nucleic
acid probe for sufficient time to allow the probe to hybridize with
the target mRNA. Following washing to remove the nonspecifically
bound probe, the label is detected. For example a digoxygenin labeled
riboprobe (RNA probe) that is complementary to the mRNA encoding
a breast cancer protein is detected by binding the digoxygenin with
an anti-digoxygenin secondary antibody and developed with nitro
blue tetrazolium and 5bromo4chloro-3indoyl phosphate.
In a preferred embodiment, any of the three classes of proteins
as described herein (secreted, transmembrane or intracellular proteins)
are used in diagnostic assays. The breast cancer proteins, antibodies,
nucleic acids, modified proteins and cells containing breast cancer
sequences are used in diagnostic assays. This can be done on an
individual gene or corresponding polypeptide level. In a preferred
embodiment, the expression profiles are used, preferably in conjunction
with high throughput screening techniques to allow monitoring for
expression profile genes and/or corresponding polypeptides.
As described and defined herein, breast cancer proteins, including
intracellular, transmembrane or secreted proteins, find use as markers
of breast cancer. Detection of these proteins in putative breast
cancer tissue of patients allows for a determination or diagnosis
of breast cancer. Numerous methods known to those of ordinary skill
in the art find use in detecting breast cancer. In one embodiment,
antibodies are used to detect breast cancer proteins. A preferred
method separates proteins from a sample or patient by electrophoresis
on a gel (typically a denaturing and reducing protein gel, but may
be any other type of gel including isoelectric focusing gels and
the like). Following separation of proteins, the breast cancer protein
is detected by immunoblotting with antibodies raised against the
breast cancer protein. Methods of immunoblotting are well known
to those of ordinary skill in the art.
In another preferred method, antibodies to the breast cancer protein
find use in in situ imaging techniques. In this method cells are
contacted with from one to many antibodies to the breast cancer
protein(s). Following washing to remove non-specific antibody binding,
the presence of the antibody or antibodies is detected. In one embodiment
the antibody is detected by incubating with a secondary antibody
that contains a detectable label. In another method the primary
antibody to the breast cancer protein(s) contains a detectable label.
In another preferred embodiment each one of multiple primary antibodies
contains a distinct and detectable label. This method finds particular
use in simultaneous screening for a plurality of breast cancer proteins.
As will be appreciated by one of ordinary skill in the art, numerous
other histological imaging techniques are useful in the invention.
In a preferred embodiment the label is detected in a fluorometer
which has the ability to detect and distinguish emissions of different
wavelengths. In addition, a fluorescence activated cell sorter (FACS)
can be used in the method.
In another preferred embodiment, antibodies find use in diagnosing
breast cancer from blood samples. As previously described, certain
breast cancer proteins are secreted/circulating molecules. Blood
samples, therefore, are useful as samples to be probed or tested
for the presence of secreted breast cancer proteins. Antibodies
can be used to detect the breast cancer by any of the previously
described immunoassay techniques including ELISA, immunoblotting
(Western blotting), immunoprecipitation, BIACORE technology and
the like, as will be appreciated by one of ordinary skill in the
In a preferred embodiment, in situ hybridizabon of labeled breast
cancer nucleic acid probes to tissue arrays is done. For example,
arrays of tissue samples, including breast cancer tissue and/or
normal tissue, are made. In situ hybridization as is known in the
art can then be done.
It is understood that when comparing the fingerprints between an
individual and a standard, the skilled artisan can make a diagnosis
as well as a prognosis. It Is further understood that the genes
which Indicate the diagnosis may differ from those which indicate
In a preferred embodiment, the breast cancer proteins, antibodies,
nucleic acids, modified proteins and cells containing breast cancer
sequences are used in prognosis assays. As above, gene expression
profiles can be generated that correlate to breast cancer severity,
in terms of long term prognosis. Again, this may be done on either
a protein or gene level, with the use of genes being preferred.
As above, the breast cancer probes are attached to biochips for
the detection and quantification of breast cancer sequences in a
tissue or patient. The assays proceed as outlined for diagnosis.
In a preferred embodiment, any of the three classes of proteins
as described herein are used in drug screening assays. The breast
cancer proteins, antibodies, nucleic acids, modified proteins and
cells containing breast cancer sequences are used in drug screening
assays or by evaluating the effect of drug candidates on a "gene
expression profile" or expression profile of polypeptides.
In a preferred embodiment, the expression profiles are used, preferably
in conjunction with high throughput screening techniques to allow
monitoring for expression profile genes after treatment with a candidate
agent, Ziokamik, et al., Science 279, 84-8 (1998), Held, 1996 #69.
In a preferred embodiment, the breast cancer proteins, antibodies,
nucleic acids, modified proteins and cells containing the native
or modified breast cancer proteins are used in screening assays.
That is, the present invention provides novel methods for screening
for compositions which modulate the breast cancer phenotype. As
above, this can be done on an individual gene level or by evaluating
the effect of drug candidates on a "gene expression profile".
In a preferred embodiment, the expression profiles are used, preferably
in conjunction with high throughput screening techniques to allow
monitoring for expression profile genes after treatment with a candidate
agent, see Ziokamik, supra.
Having identified the breast cancer genes herein, a variety of
assays may be executed. In a preferred embodiment, assays may be
run on an individual gene or protein level. That is, having identified
a particular gene as up regulated in breast cancer, candidate bioactive
agents may be screened to modulate this gene's response; preferably
to down regulate the gene, although in some circumstances to up
regulate the gene. "Modulation" thus includes both an
increase and a decrease in gene expression. The preferred amount
of modulation will depend on the original change of the gene expression
in normal versus tumor tissue, with changes of at least 10%, preferably
50%, more preferably 100-300%, and in some embodiments 300-1000%
or greater. Thus, if a gene exhibits a 4 fold increase in tumor
compared to normal tissue, a decrease of about four fold is desired;
a 10 fold decrease in tumor compared to normal tissue gives a 10
fold increase in expression for a candidate agent is desired.
As will be appreciated by those in the art, this may be done by
evaluation at either the gene or the protein level; that is, the
amount of gene expression may be monitored using nucleic acid probes
and the quantification of gene expression levels, or, alternatively,
the gene product itself can be monitored, for example through the
use of antibodies to the breast cancer protein and standard immunoassays.
In a preferred embodiment, gene expression monitoring is done and
a number of genes, i.e., an expression profile, is monitored simultaneously,
although multiple protein expression monitoring can be done as well.
In this embodiment, the breast cancer nucleic acid probes are attached
to biochips as outlined herein for the detection and quantification
of breast cancer sequences in a particular cell. The assays are
further described below.
Generally, in a preferred embodiment, a candidate bioactive agent
is added to the cells prior to analysis. Moreover, screens are provided
to identify a candidate bioactive agent which modulates breast cancer,
modulates breast cancer proteins, binds to a breast cancer protein,
or interferes between the binding of a breast cancer protein and
The term "candidate bioactive agent" or "drug candidate"
or grammatical equivalents as used herein describes any molecule,
e.g., protein, oligopeptide, small organic molecule, polysaccharide,
polynucleotide, etc., to be tested for bioactive agents that are
capable of directly or indirectly altering the breast cancer phenotype
or the expression of a breast cancer sequence, including both nucleic
acid sequences and protein sequences. In preferred embodiments,
the bioactive agents modulate the expression profiles, or expression
profile nucleic acids or proteins provided herein. In a particularly
preferred embodiment, the candidate agent suppresses a breast cancer
phenotype, for example to a normal breast tissue fingerprint. Similarly,
the candidate agent preferably suppresses a severe breast cancer
phenotype. Generally a plurality of assay mixtures are run in parallel
with different agent concentrations to obtain a differential response
to the various concentrations. Typically, one of these concentrations
serves as a negative control, i.e., at zero concentration or below
the level of detection.
In one aspect, a candidate agent will neutralize the effect of
a CRC protein. By "neutralize" is meant that activity
of a protein is either inhibited or counter acted against so as
to have substantially no effect on a cell.
Candidate agents encompass numerous chemical classes, though typically
they are organic molecules, preferably small organic compounds having
a molecular weight of more than 100 and less than about 2,500 daltons.
Preferred small molecules are less than 2000, or less than 1500
or less than 1000 or less than 500 D. Candidate agents comprise
functional groups necessary for structural interaction with proteins,
particularly hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, preferably at least
two of the functional chemical groups. The candidate agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among biomolecules
including peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations thereof.
Particularly preferred are peptides.
Candidate agents are obtained from a wide variety of sources including
libraries of synthetic or natural compounds. For example, numerous
means are available for random and directed synthesis of a wide
variety of organic compounds and biomolecules, including expression
of randomized oligonucleotides. Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant and animal extracts
are available or readily produced. Additionally, natural or synthetically
produced libraries and compounds are readily modified through conventional
chemical, physical and biochemical means. Known pharmacological
agents may be subjected to directed or random chemical modifications,
such as acylation, alkylation, esterification, amidification to
produce structural analogs.
In a preferred embodiment, the candidate bioactive agents are proteins.
By "protein" herein is meant at least two covalently attached
amino acids, which includes proteins, polypeptides, oligopeptides
and peptides. The protein may be made up of naturally occurring
amino acids and peptide bonds, or synthetic peptidomimetic structures.
Thus "amino acid", or "peptide residue", as
used herein means both naturally occurring and synthetic amino acids.
For example, homophenylalanine, citrulline and noreleucine are considered
amino acids for the purposes of the invention. "Amino acids"
also includes amino acid residues such as proline and hydroxyproline.
The side chains may be in either the (R) or the (S) configuration.
In the preferred embodiment, the amino acids are in the (S) or L-configuration.
If non-naturally occurring side chains are used, non-amino acid
substituents may be used, for example to prevent or retard in vivo
In a preferred embodiment, the candidate bioactive agents are naturally
occurring proteins or fragments of naturally occurring proteins.
Thus, for example, cellular extracts containing proteins, or random
or directed digests of proteinaceous cellular extracts, may be used.
In this way libraries of procaryotic and eucaryotic proteins may
be made for screening in the methods of the invention. Particularly
preferred in this embodiment are libraries of bacterial, fungal,
viral, and mammalian proteins, with the latter being preferred,
and human proteins being especially preferred.
In a preferred embodiment, the candidate bioactive agents are peptides
of from about 5 to about 30 amino acids, with from about 5 to about
20 amino acids being preferred, and from about 7 to about 15 being
particularly preferred. The peptides may be digests of naturally
occurring proteins as is outlined above, random peptides, or "biased"
random peptides. By "randomized" or grammatical equivalents
herein is meant that each nucleic acid and peptide consists of essentially
random nucleotides and amino acids, respectively. Since generally
these random peptides (or nucleic acids, discussed below) are chemically
synthesized, they may incorporate any nucleotide or amino acid at
any position. The synthetic process can be designed to generate
randomized proteins or nucleic acids, to allow the formation of
all or most of the possible combinations over the length of the
sequence, thus forming a library of randomized candidate bioactive
In one embodiment, the library is fully randomized, with no sequence
preferences or constants at any position. In a preferred embodiment,
the library is biased. That is, some positions within the sequence
are either held constant or are selected from a limited number of
possibilities. For example, in a preferred embodiment the nucleotides
or amino acid residues are randomized within a defined class, for
example, of hydrophobic amino acids, hydrophilic residues, sterically
biased (either small or large) residues, towards the creation of
nucleic acid binding domains, the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or histidines
for phosphorylation sites, etc., or to purines, etc.
In a preferred embodiment, the candidate bioactive agents are nucleic
acids, as defined above.
As described above generally for proteins, nucleic acid candidate
bioactive agents may be naturally occurring nucleic acids, random
nucleic acids, or "biased" random nucleic acids. For example,
digests of procaryotic or eucaryotic genomes may be used as is outlined
above for proteins.
In a preferred embodiment, the candidate bioactive agents are organic
chemical moieties, a wide variety of which are available in the
After the candidate agent has been added and the cells allowed
to incubate for some period of time, the sample containing the target
sequences to be analyzed is added to the biochip. If required, the
target sequence is prepared using known techniques. For example,
the sample may be treated to lyse the cells, using known lysis buffers,
electroporation. etc., with purification and/or amplification such
as PCR occurring as needed, as will be appreciated by those in the
art. For example, an in vitro transcription with labels covalently
attached to the nucleosides is done. Generally, the nucleic acids
are labeled with biotn-FITC or PE, or with cy3 or cy5.
In a preferred embodiment, the target sequence is labeled with,
for example, a fluorescent, a chemiluminescent, a chemical, or a
radioactive signal, to provide a means of detecting the target sequence's
specific binding to a probe. The label also can be an enzyme, such
as, alkaline phosphatase or horseradish peroxidase, which when provided
with an appropriate substrate produces a product that can be detected.
Alternatively, the label can be a labeled compound or small molecule,
such as an enzyme inhibitor, that binds but is not catalyzed or
altered by the enzyme. The label also can be a moiety or compound,
such as, an epitope tag or biotin which specifically binds to streptavidin.
For the example of biotin, the streptavidin is labeled as described
above, thereby, providing a detectable signal for the bound target
sequence. As known in the art unbound labeled streptavidin is removed
prior to analysis.
As will be appreciated by those in the art, these assays can be
direct hybridization assays or can comprise "sandwich assays",
which include the use of multiple probes, as is generally outlined
in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584,
5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352,
5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are
hereby incorporated by reference. In this embodiment, in general,
the target nucleic acid is prepared as outlined above, and then
added to the biochip comprising a plurality of nucleic acid probes,
under conditions that allow the formation of a hybridization complex.
A variety of hybridization conditions may be used in the present
invention, including high, moderate and low stringency conditions
as outlined above. The assays are generally run under stringency
conditions which allows formation of the label probe hybridization
complex only in the presence of target. Stringency can be controlled
by altering a stop parameter that is a thermodynamic variable, including,
but not limited to, temperature, formamide concentration, salt concentration,
chaotropic salt concentration pH, organic solvent concentration,
These parameters may also be used to control non-specific binding,
as is generally outlined in U.S. Pat. No. 5,681,697. Thus it may
be desirable to perform certain steps at higher stringency conditions
to reduce non-specific binding.
The reactions outlined herein may be accomplished in a variety
of ways, as will be appreciated by those in the art. Components
of the reaction may be added simultaneously, or sequentially, in
any order, with preferred embodiments outlined below. In addition,
the reaction may include a variety of other reagents may be included
in the assays. These include reagents like salts, buffers, neutral
proteins, e.g, albumin, detergents, etc which may be used to facilitate
optimal hybridization and detection, and/or reduce non-specific
or background interactions. Also reagents that otherwise improve
the efficiency of the assay, such as protease inhibitors, nuclease
inhibitors, anti-microbial agents, etc., may be used, depending
on the sample preparation methods and purity of the target
Once the assay is run, the data is analyzed to determine the expression
levels, and changes in expression levels as between states, of individual
genes, forming a gene expression profile.
The screens are done to identify drugs or bioactive agents that
modulate the breast cancer phenotype. Specifically, there are several
types of screens that can be run. A preferred embodiment is in the
screening of candidate agents that can induce or suppress a particular
expression profile, thus preferably generating the associated phenotype.
That is, candidate agents that can mimic or produce an expression
profile in breast cancer similar to the expression profile of normal
breast tissue is expected to result in a suppression of the breast
cancer phenotype. Thus, in this embodiment, mimicking an expression
profile, or changing one profile to another, is the goal.
In a preferred embodiment, as for the diagnosis and prognosis applications,
having identified the breast cancer genes important in any one state,
screens can be run to alter the expression of the genes individually.
That is, screening for modulation of regulation of expression of
a single gene can be done: that is, rather than try to mimic all
or part of an expression profile, screening for regulation of individual
genes can be done. Thus, for example, particularly in the case of
target genes whose presence or absence is unique between two states,
screening is done for modulators of the target gene expression.
In a preferred embodiment, screening is done to alter the biological
function of the expression product of the breast cancer gene. Again,
having identified the importance of a gene in a particular state,
screening for agents that bind and/or modulate the biological activity
of the gene product can be run as is more fully outlined below.
Thus, screening of candidate agents that modulate the breast cancer
phenotype either at the gene expression level or the protein level
can be done.
In addition screens can be done for novel genes that are induced
in response to a candidate agent. After identifying a candidate
agent based upon its ability to suppress a breast cancer expression
pattern leading to a normal expression pattern, or modulate a single
breast cancer gene expression profile so as to mimic the expression
of the gene from normal tissue, a screen as described above can
be performed to identify genes that are specifically modulated in
response to the agent. Comparing expression profiles between normal
tissue and agent treated breast cancer tissue reveals genes that
are not expressed in normal breast tissue or breast cancer tissue,
but are expressed in agent treated tissue. These agent specific
sequences can be identified and used by any of the methods described
herein for breast cancer genes or proteins. In particular these
sequences and the proteins they encode find use in marking or identifying
agent treated cells. In addition, antibodies can be raised against
the agent induced proteins and used to target novel therapeutics
to the treated breast cancer tissue sample.
Thus, in one embodiment, a candidate agent is administered to a
population of breast cancer cells, that thus has an associated breast
cancer expression profile. By "administration" or "contacting"
herein is meant that the candidate agent is added to the cells in
such a manner as to allow the agent to act upon the cell, whether
by uptake and intracellular action, or by action at the cell surface.
In some embodiments, nucleic acid encoding a proteinaceous candidate
agent (i.e. a peptide) may be put into a viral construct such as
a retroviral construct and added to the cell, such that expression
of the peptide agent is accomplished; see PCT US97/01019, hereby
expressly incorporated by reference.
Once the candidate agent has been administered to the cells, the
cells can be washed if desired and are allowed to incubate under
preferably physiological conditions for some period of time. The
cells are then harvested and a new gene expression profile is generated,
as outlined herein.
Thus, for example, breast cancer tissue may be screened for agents
that reduce or suppress the breast cancer phenotype. A change in
at least one gene of the expression profile indicates that the agent
has an effect on breast cancer activity. By defining such a signature
for the breast cancer phenotype, screens for new drugs that alter
the phenotype can be devised. With this approach, the drug target
need not be known and need not be represented in the original expression
screening platform, nor does the level of transcript for the target
protein need to change.
In a preferred embodiment, as outlined above, screens may be done
on individual genes and gene products (proteins). That is, having
identified a particular breast cancer gene as important in a particular
state, screening of modulators of either the expression of the gene
or the gene product itself can be done. The gene products of breast
cancer genes are sometimes referred to herein as "breast cancer
proteins" or "breast cancer modulating proteins"
or "BCMP". Additionally, "modulator" and "modulating"
proteins are sometimes used interchangeably herein. In one embodiment,
the breast cancer protein is termed BCR4. BCR4 sequences can be
identified as described herein for breast cancer sequences. In one
embodiment, BCR4 protein sequences are as depicted in FIG. 3. The
breast cancer protein may be a fragment, or alternatively, be the
full length protein to the fragment shown herein. Preferably, the
breast cancer protein is a fragment. In a preferred embodiment,
the amino acid sequence which is used to determine sequence identity
or similarity is that depicted in FIG. 3. In another embodiment,
the sequences are naturally occurring allelic variants of a protein
having the sequence depicted in FIG. 3. In another embodiment, the
sequences are sequence variants as further described herein.
Preferably, the breast cancer protein is a fragment of approximately
14 to 24 amino acids long. More preferably the fragment is a soluble
fragment. Preferably, the fragment includes a non-transmembrane
region. In a preferred embodiment, the fragment has an N-terminal
Cys to aid in solubility. In one embodiment, the c-terminus of the
fragment is kept as a free acid and the n-terminus is a free amine
to aid in coupling, i.e., to cysteine. Preferably, the fragment
of approximately 14 to 24 amino acids long. More preferably the
fragment is a soluble fragment. In another embodiment, a BCR4 fragment
has at least one BCR4 bioactivity as defined below.
In one embodiment the breast cancer proteins are conjugated to
an immunogenic agent as discussed herein. In one embodiment the
breast cancer protein is conjugated to BSA.
Thus, in a preferred embodiment, screening for modulators of expression
of specific genes can be done. This will be done as outlined above,
but in general the expression of only one or a few genes are evaluated.
In a preferred embodiment, screens are designed to first find candidate
agents that can bind to breast cancer proteins, and then these agents
may be used in assays that evaluate the ability of the candidate
agent to modulate breast cancer activity. Thus, as will be appreciated
by those in the art, there are a number of different assays which
may be run; binding assays and activity assays.
In a preferred embodiment, binding assays are done. In general,
purified or isolated gene product is used; that is, the gene products
of one or more breast cancer nucleic acids are made. In general,
this is done as is known in the art. For example, antibodies are
generated to the protein gene products, and standard immunoassays
are run to determine the amount of protein present. Alternatively,
cells comprising the breast cancer proteins can be used in the assays.
Thus, in a preferred embodiment, the methods comprise combining
a breast cancer protein and a candidate bioactive agent, and determining
the binding of the candidate agent to the breast cancer protein.
Preferred embodiments utilize the human breast cancer protein, although
other mammalian proteins may also be used, for example for the development
of animal models of human disease. In some embodiments, as outlined
herein, variant or derivative breast cancer proteins may be used.
Generally, in a preferred embodiment of the methods herein, the
breast cancer protein or the candidate agent is non-diffusably bound
to an insoluble support having isolated sample receiving areas (e.g.
a microtiter plate, an array, etc.). It is understood that alternatively,
soluble assays known in the art may be performed. The insoluble
supports may be made of any composition to which the compositions
can be bound, is readily separated from soluble material, and is
otherwise compatible with the overall method of screening. The surface
of such supports may be solid or porous and of any convenient shape.
Examples of suitable insoluble supports include microtiter plates,
arrays, membranes and beads. These are typically made of glass,
plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose,
TEFLON.RTM. (synthetic resinous flurorine-containing polymers),
etc. Microtiter plates and arrays are especially convenient because
a large number of assays can be carried out simultaneously, using
small amounts of reagents and samples. The particular manner of
binding of the composition is not crucial so long as it is compatible
with the reagents and overall methods of the invention, maintains
the activity of the composition and is nondiffusable. Preferred
methods of binding include the use of antibodies (which do not sterically
block either the ligand binding site or activation sequence when
the protein is bound to the support), direct binding to "sticky"
or ionic supports, chemical crosslinking, the synthesis of the protein
or agent on the surface, etc. Following binding of the protein or
agent, excess unbound material is removed by washing. The sample
receiving areas may then be blocked through incubation with bovine
serum albumin (BSA), casein or other innocuous protein or other
In a preferred embodiment, the breast cancer protein is bound to
the support, and a candidate bioactive agent is added to the assay.
Alternatively, the candidate agent is bound to the support and the
breast cancer protein is added. Novel binding agents include specific
antibodies, non-natural binding agents identified in screens of
chemical libraries, peptide analogs, etc. Of particular interest
are screening assays for agents that have a low toxicity for human
cells. A wide variety of assays may be used for this purpose, including
labeled in vitro protein-protein binding assays, electrophoretic
mobility shift assays, immunoassays for protein binding, functional
assays (phosphorylation assays, etc.) and the like.
The determination of the binding of the candidate bioactive agent
to the breast cancer protein may be done in a number of ways. In
a preferred embodiment, the candidate bioactive agent is labelled,
and binding determined directly. For example, this may be done by
attaching all or a portion of the breast cancer protein to a solid
support, adding a labelled candidate agent (for example a fluorescent
label), washing off excess reagent, and determining whether the
label is present on the solid support. Various blocking and washing
steps may be utilized as is known in the art.
By "labeled" herein is meant that the compound is either
directly or indirectly labeled with a label which provides a detectable
signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles
such as magnetic particles, chemiluminesers, or specific binding
molecules, etc. Specific binding molecules include pairs, such as
biotin and streptavidin, digoxin and antidigoxin etc. For the specific
binding members, the complementary member would normally be labeled
with a molecule which provides for detection, in accordance with
known procedures, as outlined above. The label can directly or indirectly
provide a detectable signal.
In some embodiments, only one of the components is labeled. For
example, the proteins (or proteinaceous candidate agents) may be
labeled at tyrosine positions using .sup.125 I, or with fluomphores.
Alternatively, more than one component may be labeled with different
labels; using .sup.125 I for the proteins, for example, and a fluorophor
for the candidate agents.
In a preferred embodiment, the binding of the candidate bioactive
agent is determined through the use of competitive binding assays.
In this embodiment, the competitor is a binding moiety known to
bind to the target molecule (i.e. breast cancer), such as an antibody,
peptide, binding partner, ligand, etc. Under certain circumstances,
there may be competitive binding as between the bioactive agent
and the binding moiety, with the binding moiety displacing the bioactive
In one embodiment, the candidate bioactive agent is labeled. Either
the candidate bioactive agent, or the competitor, or both, is added
first to the protein for a time sufficient to allow binding, if
present. Incubations may be performed at any temperature which facilitates
optimal activity, typically between 4 and 40.degree. C. Incubation
periods are selected for optimum activity, but may also be optimized
to facilitate rapid high through put screening. Typically between
0.1 and 1 hour will be sufficient. Excess reagent is generally removed
or washed away. The second component is then added, and the presence
or absence of the labeled component is followed, to indicate binding.
In a preferred embodiment, the competitor is added first, followed
by the candidate bioactive agent. Displacement of the competitor
is an indication that the candidate bioactive agent is binding to
the breast cancer protein and thus is capable of binding to, and
potentially modulating, the activity of the breast cancer protein.
In this embodiment, either component can be labeled. Thus, for example,
if the competitor is labeled, the presence of label in the wash
solution indicates displacement by the agent. Alternatively, if
the candidate bioactive agent is labeled, the presence of the label
on the support indicates displacement.
In an alternative embodiment, the candidate bioactive agent is
added first, with incubation and washing, followed by the competitor.
The absence of binding by the competitor may indicate that the bioactive
agent is bound to the breast cancer protein with a higher affinity.
Thus, if the candidate bioactive agent is labeled, the presence
of the label on the support coupled with a lack of competitor binding,
may indicate that the candidate agent is capable of binding to the
breast cancer protein.
In a preferred embodiment, the methods comprise differential screening
to identity bioactive agents that are capable of modulating the
activity of the breast cancer proteins. In this embodiment, the
methods comprise combining a breast cancer protein and a competitor
in a first sample. A second sample comprises a candidate bioactive
agent, a breast cancer protein and a competitor. The binding of
the competitor is determined for both samples, and a change, or
difference in binding between the two samples indicates the presence
of an agent capable of binding to the breast cancer protein and
potentially modulating its activity. That is, if the binding of
the competitor is different in the second sample relative to the
first sample, the agent is capable of binding to the breast cancer
Alternatively, a preferred embodiment utilizes differential screening
to identify drug candidates that bind to the native breast cancer
protein, but cannot bind to modified breast cancer proteins. The
structure of the breast cancer protein may be modeled, and used
in rational drug design to synthesize agents that interact with
that site. Drug candidates that affect breast cancer bioactivity
are also identified by screening drugs for the ability to either
enhance or reduce the activity of the protein.
Positive controls and negative controls may be used in the assays.
Preferably all control and test samples are performed in at least
triplicate to obtain statistically significant results. Incubation
of all samples is for a time sufficient for the binding of the agent
to the protein. Following incubation, all samples are washed free
of non-specifically bound material and the amount of bound, generally
labeled agent determined. For example, where a radiolabel is employed,
the samples may be counted in a scintillation counter to determine
the amount of bound compound.
A variety of other reagents may be included in the screening assays.
These include reagents like salts, neutral proteins, e.g. albumin,
detergents, etc which may be used to facilitate optimal protein-protein
binding and/or reduce non-specific or background interactions. Also
reagents that otherwise improve the efficiency of the assay, such
as protease inhibitors, nuclease inhibitors, anti-microbial agents,
etc., may be used. The mixture of components may be added in any
order that provides for the requisite binding.
Screening for agents that modulate the activity of breast cancer
proteins may also be done. In a preferred embodiment, methods for
screening for a bioactive agent capable of modulating the activity
of breast cancer proteins comprise the steps of adding a candidate
bioactive agent to a sample of breast cancer proteins, as above,
and determining an alteration in the biological activity of breast
cancer proteins. "Modulating the activity" of breast cancer
includes an increase in activity, a decrease in activity, or a change
in the type or kind of activity present. Thus, in this embodiment,
the candidate agent should both bind to breast cancer proteins (although
this may not be necessary), and alter its biological or biochemical
activity as defined herein. The methods include both in vitro screening
methods, as are generally outlined above, and in vivo screening
of cells for alterations in the presence, distribution, activity
or amount of breast cancer proteins.
Thus, in this embodiment, the methods comprise combining a breast
cancer sample and a candidate bioactive agent, and evaluating the
effect on breast cancer activity. By "breast cancer activity"
or grammatical equivalents herein is meant at least one of breast
cancer's biological activities, including, but not limited to, cell
division, preferably in breast tissue, cell proliferation, tumor
growth, and transformation of cells. In one embodiment, breast cancer
activity includes activation of BCR4 or a substrate thereof by BCR4.
An inhibitor of breast cancer activity is an agent which inhibits
any one or more breast cancer activities.
In a preferred embodiment, the activity of the breast cancer protein
is increased; in another preferred embodiment, the activity of the
breast cancer protein is decreased. Thus, bioactive agents that
are antagonists are preferred in some embodiments, and bioactive
agents that are agonists may be preferred in other embodiments.
In a preferred embodiment, the invention provides methods for screening
for bioactive agents capable of modulating the activity of a breast
cancer protein. The methods comprise adding a candidate bioactive
agent, as defined above, to a cell comprising breast cancer proteins.
Preferred cell types include almost any cell. The cells contain
a recombinant nucleic acid that encodes a breast cancer protein.
In a preferred embodiment, a library of candidate agents are tested
on a plurality of calls.
In one aspect, the assays are evaluated in the presence or absence
or previous or subsequent exposure of physiological signals, for
example hormones, antibodies, peptides, antigens, cytokines, growth
factors, action potentials, pharmacological agents including chemotherapeutics,
radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
In another example, the determinations are determined at different
stages of the cell cycle process.
In this way. bioactive agents are identified. Compounds with pharmacological
activity are able to enhance or interfere with the activity of the
breast cancer protein. In one embodiment, "breast cancer protein
activity" as used herein includes at least one of the following:
breast cancer activity, binding to BCR4, activation of BCR4 or activation
of substrates of BCR4 by BCR4. An inhibitor of BCR4 inhibits at
least one of BCR4's bioactivities.
In one embodiment, a method of inhibiting breast cancer cell division
is provided. The method comprises administration of a breast cancer
In another embodiment, a method of inhibiting breast tumor growth
is provided. The method comprises administration of a breast cancer
inhibitor. In a preferred embodiment, the inhibitor is an inhibitor
In a further embodiment, methods of treating cells or individuals
with breast cancer are provided. The method comprises administration
of a breast cancer inhibitor. In a preferred embodiment, the inhibitor
is an inhibitor of BCR4.
In one embodiment, a breast cancer inhibitor is an antibody as
discussed above. In another embodiment, the breast cancer inhibitor
is an antisense molecule. Antisense molecules as used herein include
antisense or sense oligonucleotides comprising a singe-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target mRNA (sense) or DNA (antisense) sequences for breast cancer
molecules. A preferred antisense molecule is for BCR4 or for a ligand
or activator thereof. Antisense or sense oligonucleotides, according
to the present invention, comprise a fragment generally at least
about 14 nucleotides, preferably from about 14 to 30 nucleotides.
The ability to derive an antisense or a sense oligonucleotide, based
upon a cDNA sequence encoding a given protein is described in, for
example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der
Krol et al. (BioTechniques 8:958, 1988).
Antisense molecules may be introduced into a cell containing the
target nucleotide sequence by formation of a conjugate with a ligand
binding molecule, as described in WO 91/04753. Suitable ligand binding
molecules include, but are not limited to, cell surface receptors,
growth factors, other cytokines, or other ligands that bind to cell
surface receptors. Preferably, conjugation of the ligand binding
molecule does not substantially interfere with the ability of the
ligand binding molecule to bind to its corresponding molecule or
receptor, or block entry of the sense or antisense oligonucleotide
or its conjugated version into the cell. Alternatively, a sense
or an antisense oligonucleotide may be introduced into a cell containing
the target nucleic acid sequence by formation of an oligonucleotide-lipid
complex, as described in WO 90110448. it is understood that the
use of antisense molecules or knock out and knock in models may
also be used in screening assays as discussed above, in addition
to methods of treatment.
The compounds having the desired pharmacological activity may be
administered in a physiologically acceptable carrier to a host,
as previously described. The agents may be administered in a variety
of ways, orally, parenterally e.g., subcutaneously, intraperitoneally,
intravascularly, etc. Depending upon the manner of introduction,
the compounds may be formulated in a variety of ways. The concentration
of therapeutically active compound in the formulation may vary from
about 0.1-100 wt. %. The agents may be administered alone or in
combination with other treatments, i.e., radiation.
The pharmaceutical compositions can be prepared in various forms,
such as granules, tablets, pills, suppositories, capsules, suspensions,
salves, lotions and the like. Pharmaceutical grade organic or inorganic
carriers and/or diluents suitable for oral and topical use can be
used to make up compositions containing the therapeutically-active
compounds. Diluents known to the art include aqueous media, vegetable
and animal oils and fats. Stabilizing agents, wetting and emulsifying
agents, salts for varying the osmotic pressure or buffers for securing
an adequate pH value, and skin penetration enhancers can be used
as auxiliary agents.
Without being bound by theory, it appears that the various breast
cancer sequences are important in breast cancer. Accordingly, disorders
based on mutant or variant breast cancer genes may be determined.
In one embodiment, the invention provides methods for identifying
cells containing variant breast cancer genes comprising determining
all or part of the sequence of at least one endogeneous breast cancer
gene in a cell. As will be appreciated by those in the art, this
may be done using any number of sequencing techniques. In a preferred
embodiment, the invention provides methods of identifying the breast
cancer genotype of an individual comprising determining all or part
of the sequence of at least one breast cancer gene of the individual.
This is generally done in at least one tissue of the individual,
and may include the evaluation of a number of tissues or different
samples of the same tissue. The method may include comparing the
sequence of the sequenced gene to a known gene, i.e. a wild-type
The sequence of all or part of the breast cancer gene can then
be compared to the sequence of a known breast cancer gene to determine
if any differences exist. This can be done using any number of known
homology programs, such as Bestfit, etc. In a preferred embodiment,
the presence of a difference in the sequence between the breast
cancer gene of the patient and the known breast cancer gene is indicative
of a disease state or a propensity for a disease state, as outlined
In a preferred embodiment, the breast cancer genes are used as
probes to determine the number of copies of the breast cancer gene
in the genome.
In another preferred embodiment breast cancer genes are used as
probed to determine the chromosomal localization of the breast cancer
genes. Information such as chromosomal localization finds use in
providing a diagnosis or prognosis in particular when chromosomal
abnormalities such as translocations, and the like are identified
in breast cancer gene loci.
Thus, in one embodiment, methods of modulating breast cancer in
cells or organisms are provided. In one embodiment, the methods
comprise administering to a cell an antibody that reduces or eliminates
the biological activity of an endogenous breast cancer protein.
Alternatively, the methods comprise administering to a cell or organism
a recombinant nucleic acid encoding a breast cancer protein. As
will be appreciated by those in the art, this may be accomplished
in any number of ways. In a preferred embodiment, for example when
the breast cancer sequence is down-regulated in breast cancer, the
activity of the breast cancer gene is increased by increasing the
amount in the cell, for example by overexpressing the endogenous
protein or by administering a gene encoding the sequence, using
known genetherapy techniques, for example. In a preferred embodiment,
the gene therapy techniques include the incorporation of the exogenous
gene using enhanced homologous recombination (EHR), for example
as described in PCT/US93/03868, hereby incorporated by reference
in its entirety. Alternatively, for example when the breast cancer
sequence is up-regulated in breast cancer, the activity of the endogeneous
gene is decreased, for example by the administration of an inhibitor
of breast cancer, such as an antisense nucleic acid.
In one embodiment, the breast cancer proteins of the present invention
may be used to generate polyclonal and monoclonal antibodies to
breast cancer proteins, which are useful as described herein. Similarly,
the breast cancer proteins can be coupled, using standard technology,
to affinity chromatography columns. These columns may then be used
to purify breast cancer antibodies. In a preferred embodiment, the
antibodies are generated to epitopes unique to a breast cancer protein;
that is, the antibodies show little or no cross-reactivity to other
proteins. These antibodies find use in a number of applications.
For example, the breast cancer antibodies may be coupled to standard
affinity chromatography columns and used to purity breast cancer
proteins. The antibodies may also be used as blocking polypeptides,
as outlined above, since they will specifically bind to the breast
In one embodiment, a therapeutically effective dose of a breast
cancer or modulator thereof is administered to a patient. By "therapeutically
effective dose" herein is meant a dose that produces the effects
for which it is administered. The exact dose will depend on the
purpose of the treatment, and will be ascertainable by one skilled
in the art using known techniques. As is known in the art, adjustments
for degradation, systemic versus localized delivery, and rate of
new protease synthesis, as well as the age, body weight, general
health, sex, diet time of administration, drug interaction and the
severity of the condition may be necessary, and will be ascertainable
with routine experimentation by those skilled in the art
A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals, and
organisms. Thus the methods are applicable to both human therapy
and veterinary applications. In the preferred embodiment the patient
is a mammal, and in the most preferred embodiment the patent is
The administration of the breast cancer proteins and modulators
of the present invention can be done in a variety of ways as discussed
above, including, but not limited to, orally, subcutaneously, intravenously,
intranasally, transdermally, intraperitoneally, intramuscularly,
intrapulmonary, vaginally, rectally, or intraocularty. In some instances,
for example, in the treatment of wounds and inflammation, the breast
cancer proteins and modulators may be directly applied as a solution
The pharmaceutical compositions of the present invention comprise
a breast cancer protein in a form suitable for administration to
a patient. In the preferred embodiment, the pharmaceutical compositions
are in a water soluble form, such as being present as pharmaceutically
acceptable salts, which is meant to include both acid and base addition
salts. "Pharmaceutically acceptable acid addition salt"
refers to those salts that retain the biological effectiveness of
the free bases and that are not biologically or otherwise undesirable,
formed with inorganic acids such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid and the like,
and organic acids such as acetic acid, propionic acid, glycolic
acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid.
p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically
acceptable base addition salts" include those derived from
inorganic bases such as sodium, potassium, lithium, ammonium, calcium,
magnesium, iron, zinc, copper, manganese, aluminum salts and the
like. Particularly preferred are the ammonium, potassium, sodium,
calcium, and magnesium salts. Salts derived from pharmaceutically
acceptable organic non-toxic bases include salts of primary, secondary,
and tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or more of
the following: carrier proteins such as serum albumin; buffers;
fillers such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol. Additives are well known
in the art, and are used in a variety of formulations.
In a preferred embodiment, breast cancer proteins and modulators
are administered as therapeutic agents, and can be formulated as
outlined above. Similarly, breast cancer genes (including both the
full-length sequence, partial sequences, or regulatory sequences
of the breast cancer coding regions) can be administered in gene
therapy applications, as is known in the art. These breast cancer
genes can include antisense applications, either as gene therapy
(i.e. for incorporation into the genome) or as antisense compositions,
as will be appreciated by those in the art.
In a preferred embodiment, breast cancer genes are administered
as DNA vaccines, either single genes or combinations of breast cancer
genes. Naked DNA vaccines are generally known in the art. Brower,
Nature Biotechnology, 16:1304-1305 (1998).
In one embodiment, breast cancer genes of the present invention
are used as DNA vaccines. Methods for the use of genes as DNA vaccines
are well known to one of ordinary skill in the art, and include
placing a breast cancer gene or portion of a breast cancer gene
under the control of a promoter for expression in a patient with
breast cancer. The breast cancer gene used for DNA vaccines can
encode full-length breast cancer proteins, but more preferably encodes
portions of the breast cancer proteins including peptides derived
from the breast cancer protein. In a preferred embodiment a patient
is immunized with a DNA vaccine comprising a plurality of nucleotide
sequences derived from a breast cancer gene. Similarly, it is possible
to immunize a patient with a plurality of breast cancer genes or
portions thereof as defined herein. Without being bound by theory,
expression of the polypeptide encoded by the DNA vaccine, cytotoxic
T-cells, helper T-cells and antibodies are induced which recognize
and destroy or eliminate cells expressing breast cancer proteins.
In a preferred embodiment, the DNA vaccines include a gene encoding
an adjuvant molecule with the DNA vaccine. Such adjuvant molecules
include cytokines that increase the immunogenic response to the
breast cancer polypeptide encoded by the DNA vaccine. Additional
or alternative adjuvants are known to those of ordinary skill in
the art and find use in the invention.
In another preferred embodiment breast cancer genes find use in
generating animal models of breast cancer. For example, as is appreciated
by one of ordinary skill in the art, when the breast cancer gene
identified is repressed or diminished in breast cancer tissue, gene
therapy technology wherein antisense RNA directed to the breast
cancer gene will also diminish or repress expression of the gene.
An animal generated as such serves as an animal model of breast
cancer that finds use in screening bioactive drug candidates. Similarly,
gene knockout technology, for example as a result of homologous
recombination with an appropriate gene targeting vector, will result
in the absence of the breast cancer protein. When desired, tissue-specific
expression or knockout of the breast cancer protein may be necessary.
It is also possible that the breast cancer protein is overexpressed
in breast cancer. As such, transgenic animals can be generated that
overexpress the breast cancer protein. Depending on the desired
expression level, promoters of various strengths can be employed
to express the transgene. Also, the number of copies of the integrated
transgene can be determined and compared for a determination of
the expression level of the transgene. Animals generated by such
methods find use as animal models of breast cancer and are additionally
useful in screening for bioactive molecules to treat disorders related
to the breast cancer protein.
It is understood that the examples described herein in no way serve
to limit the true scope of this invention, but rather are presented
for illustrative purposes. All references and sequences of accession
numbers cited herein are incorporated by reference in their entirety.