Compositions and methods for the detection and therapy of breast
cancer are disclosed. The compounds provided include nucleotide
sequences that are preferentially expressed in breast tumor tissue,
as well as polypeptides encoded by such nucleotide sequences. Vaccines
and pharmaceutical compositions comprising such compounds are also
provided and may be used, for example, for the prevention and treatment
of breast cancer. The polypeptides may also be used for the production
of antibodies, which are useful for diagnosing and monitoring the
progression of breast cancer in a patient.
What is claimed is:
1. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:299, wherein said polypeptide is expressed in breast tumor
2. An isolated polypeptide that is at least 90% identical to the
amino acid sequence of SEQ ID NO:299, wherein said polypeptide is
expressed in breast tumor tissue.
3. An isolated polypeptide according to claim 2, wherein said polypeptide
is at least 95% identical to the amino acid sequence of SEQ ID NO:299.
4. An isolated polypeptide comprising the amino acid sequence encoded
by the polynucleotide sequence of SEQ ID NO:292.
5. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:299.
6. An isolated polypeptide according to any one of claims 1-5,
in combination with a physiologically acceptable carrier.
7. An isolated polypeptide according to any one of claims 1-5,
in combination with an immunostimulant.
8. An isolated polypeptide according to claim 7, wherein said immunostimulant
is an adjuvant.
9. An isolated polypeptide according to claim 7, wherein said immunostimulant
induces a predominantly Type I response.
The present invention relates generally to the detection and therapy
of breast cancer. The invention is more specifically related to
nucleotide sequences that are preferentially expressed in breast
tumor tissue and to polypeptides encoded by such nucleotide sequences.
The nucleotide sequences and polypeptides may be used in vaccines
and pharmaceutical compositions for the prevention and treatment
of breast cancer. The polypeptides may also be used for the production
of compounds, such as antibodies, useful for diagnosing and monitoring
the progression of breast cancer in a patient.
BACKGROUND OF THE INVENTION
Breast cancer is a significant health problem for women in the
United States and throughout the world. Although advances have been
made in detection and treatment of the disease, breast cancer remains
the second leading cause of cancer-related deaths in women, affecting
more than 180,000 women in the United States each year. For women
in North America, the life-time odds of getting breast cancer are
now one in eight.
No vaccine or other universally successful method for the prevention
or treatment of breast cancer is currently available. Management
of the disease currently relies on a combination of early diagnosis
(through routine breast screening procedures) and aggressive treatment,
which may include one or more of a variety of treatments such as
surgery, radiotherapy, chemotherapy and hormone therapy. The course
of treatment for a particular breast cancer is often selected based
on a variety of prognostic parameters, including an analysis of
specific-tumor markers. See, e.g., Porter-Jordan and Lippman, Breast
Cancer 8:73-100 (1994). However, the use of established markers
often leads to a result that is difficult to interpret, and the
high mortality observed in breast cancer patients indicates that
improvements are needed in the treatment, diagnosis and prevention
of the disease.
Accordingly, there is a need in the art for improved methods for
therapy and diagnosis of breast cancer. The present invention fulfills
these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the subject invention provides compositions and
methods for the diagnosis and therapy of breast cancer. In one aspect,
isolated polynucleotides are provided, comprising (a) a nucleotide
sequence preferentially expressed in breast cancer tissue, relative
to normal tissue; (b) a variant of such a sequence, as defined below;
or (c) a nucleotide sequence encoding an epitope of a polypeptide
encoded by at least one of the above sequences. In one embodiment,
the isolated polynucleotide comprises a human endogenous retroviral
sequence recited in SEQ ID NO: 1. In other embodiments, the isolated
polynucleotide comprises a sequence recited in any one of SEQ ID
NO: 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198,
200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245, 247,
250, 251, 253, 255, 257-266, 268, 269, 271-273, 275, 276, 278, 280,
281, 284, 288, 291-298, 301-303, 307, 313, 314, 316 and 317.
In related embodiments, the isolated polynucleotide encodes an
epitope of polypeptide, wherein the polypeptide is encoded by a
nucleotide sequence that: (a) hybridizes to a sequence recited in
any one of SEQ ID NO: 1, 3-26, 28-77, 142, 143, 46-152, 154-166,
168-176, 178-192, 194-198, 200-204, 206, 207, 209-214, 216, 218,
219, 221-240, 243-245, 247, 250, 251, 253, 255, 257-266, 268, 269,
271-273, 275, 276, 278, 280, 281, 284, 288, 291-298, 301-303, 307,
313, 314, 316 and 317 under stringent conditions; and (b) is at
least 80% identical to a sequence recited in any one of SEQ ID NO:
1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198,
200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245, 247,
250, 251, 253, 255, 257-266, 268, 269, 271-273, 275, 276, 278, 280,
281, 284, 288, 291-298, 301-303, 307, 313, 314, 316 and 317.
In another embodiment, the present invention provides an isolated
polynucleotide encoding an epitope of a polypeptide, the polypeptide
being encoded by: (a) a nucleotide sequence transcribed from the
sequence of SEQ ID NO: 141; or (b) a variant of said nucleotide
sequence that contains one or more nucleotide substitutions, deletions,
insertions and/or modifications at no more than 20% of the nucleotide
positions, such that the antigenic and/or immunogenic properties
of the polypeptide encoded by the nucleotide sequence are retained.
Isolated DNA and RNA molecules comprising a nucleotide sequence
complementary to a polynucleotide as described above are also provided.
In related aspects, the present invention provides recombinant
expression vectors comprising a polynucleotide as described above
and host cells transformed or transfected with such expression vectors.
In further aspects, polypeptides comprising an amino acid sequence
encoded by a polynucleotide as described above, and monoclonal antibodies
that bind to such polypeptides are provided. In certain embodiments,
the inventive polypeptides comprise an amino acid sequence selected
from the group consisting of SEQ ID NO: 299, 300, 304-306, 308 and
315, and variants thereof as defined below.
In yet another aspect, methods are provided for determining the
presence of breast cancer in a patient. In one embodiment, the method
comprises detecting, within a biological sample, a polypeptide as
described above. In another embodiment, the method comprises detecting,
within a biological sample, an RNA molecule encoding a polypeptide
as described above. In yet another embodiment, the method comprises
(a) intradermally injecting a patient with a polypeptide as described
above; and (b) detecting an immune response on the patient's skin
and therefrom detecting the presence of breast cancer in the patient.
In further embodiments, the present invention provides methods for
determining the presence of breast cancer in a patient as described
above wherein the polypeptide is encoded by a nucleotide sequence
selected from the group consisting of SEQ ID NO: 78-86, 144, 145,
153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246,
248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287,
289, 290 and sequences that hybridize thereto under stringent conditions.
In a related aspect, diagnostic kits useful in the determination
of breast cancer are provided. The diagnostic kits generally comprise
either one or more monoclonal antibodies as described above, or
one or more monoclonal antibodies that bind to a polypeptide encoded
by a nucleotide sequence selected from the group consisting of sequences
provided in SEQ ID NO: 78-86, 144, 145, 153, 167, 177, 193, 199,
205, 208, 215, 217, 220, 241, 242 and 246, 248, 249, 252, 256, 267,
270, 274, 277, 279, 282, 283, 285-287, 289, 290 and a detection
Diagnostic kits are also provided that comprise a first polymerase
chain reaction primer and a second polymerase chain reaction primer,
at least one of the primers being specific for a polynucleotide
described herein. In one embodiment, at least one of the primers
comprises at least about 10 contiguous nucleotides of a polynucleotide
as described above, or a polynucleotide encoding a polypeptide encoded
by a sequence selected from the group consisting of SEQ ID NO: 78-86,
144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241,
242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283,
285-287, 289 and 290.
Within another related aspect, the diagnostic kit comprises at
least one oligonucleotide probe, the probe being specific for a
polynucleotide described herein. In one embodiment, the probe comprises
at least about 15 contiguous nucleotides of a polynucleotide as
described above, or a polynucleotide selected from the group consisting
of SEQ ID NO: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208,
215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274,
277, 279, 282, 283, 285-287, 289 and 290.
In another related aspect, the present invention provides methods
for monitoring the progression of breast cancer in a patient. In
one embodiment, the method comprises: (a) detecting an amount, in
a biological sample, of a polypeptide as described above at a first
point in time; (b) repeating step (a) at a subsequent point in time;
and (c) comparing the amounts of polypeptide detected in steps (a)
and (b), and therefrom monitoring the progression of breast cancer
in the patient. In another embodiment, the method comprises (a)
detecting an amount, within a biological sample, of an RNA molecule
encoding a polypeptide as described above at a first point in time;
(b) repeating step (a) at a subsequent point in time; and (c) comparing
the amounts of RNA molecules detected in steps (a) and (b), and
therefrom monitoring the progression of breast cancer in the patient.
In yet other embodiments, the present invention provides methods
for monitoring the progression of breast cancer in a patient as
described above wherein the polypeptide is encoded by a nucleotide
sequence selected from the group consisting of SEQ ID NO: 78-86,
144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241,
242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283,
285-287, 289, 290 and sequences that hybridize thereto under stringent
In still other aspects, pharmaceutical compositions, which comprise
a polypeptide as described above in combination with a physiologically
acceptable carrier, and vaccines, which comprise a polypeptide as
described above in combination with an immunostimulant or adjuvant,
are provided. In yet other aspects, the present invention provides
pharmaceutical compositions and vaccines comprising a polypeptide
encoded by a nucleotide sequence selected from the group consisting
of SEQ ID NO: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208,
215, 217, 220, 241, 242 and 246, 248, 249, 252, 256, 267, 270, 274,
277, 279, 282, 283, 285-287, 289, 290 and sequences that hybridize
thereto under stringent conditions.
In related aspects, the present invention provides methods for
inhibiting the development of breast cancer in a patient, comprising
administering to a patient a pharmaceutical composition or vaccine
as described above.
These and other aspects of the present invention will become apparent
upon reference to the following detailed description and attached
drawings. All references disclosed herein are hereby incorporated
by reference in their entirety as if each was incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the differential display PCR products, separated by
gel electrophoresis, obtained from cDNA prepared from normal breast
tissue (lanes 1 and 2) and from cDNA prepared from breast tumor
tissue from the same patient (lanes 3 and 4). The arrow indicates
the band corresponding to B18Ag1.
FIG. 2 is a northern blot comparing the level of B18Ag1 mRNA in
breast tumor tissue (lane 1) with the level in normal breast tissue.
FIG. 3 shows the level of B18Ag1 mRNA in breast tumor tissue compared
to that in various normal and non-breast tumor tissues as determined
by RNase protection assays.
FIG. 4 is a genomic clone map showing the location of additional
retroviral sequences obtained from ends of XbaI restriction digests
(provided in SEQ ID NO:3-SEQ ID NO:10) relative to B18 Ag1.
FIGS. 5A and 5B show the sequencing strategy, genomic organization
and predicted open reading frame for the retroviral element containing
FIG. 6 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B18Ag1.
FIG. 7 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B17Ag1.
FIG. 8 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B17Ag2.
FIG. 9 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B13Ag2a.
FIG. 10 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B13Ag1b.
FIG. 11 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B13Ag1a.
FIG. 12 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B11Ag1.
FIG. 13 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B3CA3c.
FIG. 14 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B9CG1.
FIG. 15 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B9CG3.
FIG. 16 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B2CA2.
FIG. 17 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B3CA1.
FIG. 18 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B3CA2.
FIG. 19 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B3CA3.
FIG. 20 shows the nucleotide sequence of the representative breast
tumor-specific cDNA B4CA1.
FIG. 21A depicts RT-PCR analysis of breast tumor genes in breast
tumor tissues (lanes 1-8) and normal breast tissues (lanes 9-13)
and H.sub.2 O (lane 14).
FIG. 21B depicts RT-PCR analysis of breast tumor genes in prostate
tumors (lane 1, 2), colon tumors (lane 3), lung tumor (lane 4),
normal prostate (lane 5), normal colon (lane 6), normal kidney (lane
7), normal liver (lane 8), normal lung (lane 9), normal ovary (lanes
10, 18), normal pancreases (lanes 11, 12), normal skeletal muscle
(lane 13), normal skin (lane 14), normal stomach (lane 15), normal
testes (lane 16), normal small intestine (lane 17), HBL-100 (lane
19), MCF-12A (lane 20), breast tumors (lanes 21-23), H.sub.2 O (lane
24), and colon tumor (lane 25).
FIG. 22 shows the recognition of a B11Ag1 peptide (referred to
as B11-8) by an anti-B11-8 CTL line.
FIG. 23 shows the recognition of a cell line transduced with the
antigen B11Ag1 by the B11-8 specific clone A1.
FIG. 24 shows recognition of a lung adenocarcinoma line (LT-140-22)
and a breast adenocarcinoma line (CAMA-1) by the B11-8 specific
DETAILED DESCRIPTION OF THE INVENTION
As noted above, the present invention is generally directed to
compositions and methods for the diagnosis, monitoring and therapy
of breast cancer. The compositions described herein include polypeptides,
polynucleotides and antibodies. Polypeptides of the present invention
generally comprise at least a portion of a protein that is expressed
at a greater level in human breast tumor tissue than in normal breast
tissue (i.e., the level of RNA encoding the polypeptide is at least
2-fold higher in tumor tissue). Such polypeptides are referred to
herein as breast tumor-specific polypeptides, and cDNA molecules
encoding such polypeptides are referred to as breast tumor-specific
cDNAs. Polynucleotides of the subject invention generally comprise
a DNA or RNA sequence that encodes all or a portion of a polypeptide
as described above, or that is complementary to such a sequence.
Antibodies are generally immune system proteins, or fragments thereof,
that are capable of binding to a portion of a polypeptide as described
above. Antibodies can be produced by cell culture techniques, including
the generation of monoclonal antibodies as described herein, or
via transfection of antibody genes into suitable bacterial or mammalian
cell hosts, in order to allow for the production of recombinant
Polypeptides within the scope of this invention include, but are
not limited to, polypeptides (and epitopes thereof) encoded by a
human endogenous retroviral sequence, such as the sequence designated
B18Ag1 (FIG. 5 and SEQ ID NO: 1). Also within the scope of the present
invention are polypeptides encoded by other sequences within the
retroviral genome containing B18Ag1 (SEQ ID NO: 141). Such sequences
include, but are not limited to, the sequences recited in SEQ ID
NO:3-SEQ ID NO:10. B18Ag1 has homology to the gag p30 gene of the
endogenous human retroviral element S71, as described in Werner
et al., Virology 174:225-238 (1990) and also shows homology to about
thirty other retroviral gag genes. As discussed in more detail below,
the present invention also includes a number of additional breast
tumor-specific polypeptides, such as those encoded by the nucleotide
sequences recited in SEQ ID NO: 11-26, 28-77, 142, 143, 146-152,
154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214,
216, 218, 219, 221-240, 243-245, 247, 250, 251, 253, 255, 257-266,
268, 269, 271-273, 275, 276, 278, 280, 281, 284, 288, 291-298, 301-303,
307, 313, 314, 316 and 317.
As used herein, the term "polypeptide" encompasses amino
acid chains of any length, including full length proteins containing
the sequences recited herein. A polypeptide comprising an epitope
of a protein containing a sequence as described herein may consist
entirely of the epitope, or may contain additional sequences. The
additional sequences may be derived from the native protein or may
be heterologous, and such sequences may (but need not) possess immunogenic
or antigenic properties.
An "epitope," as used herein is a portion of a polypeptide
that is recognized (i.e., specifically bound) by a B-cell and/or
T-cell surface antigen receptor. Epitopes may generally be identified
using well known techniques, such as those summarized in Paul, Fundamental
Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references
cited therein. Such techniques include screening polypeptides derived
from the native polypeptide for the ability to react with antigen-specific
antisera and/or T-cell lines or clones. An epitope of a polypeptide
is a portion that reacts with such antisera and/or T-cells at a
level that is similar to the reactivity of the full length polypeptide
(e.g., in an ELISA and/or T-cell reactivity assay). Such screens
may generally be performed using methods well known to those of
ordinary skill in the art, such as those described in Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988. B-cell and T-cell epitopes may also be predicted via computer
analysis. Polypeptides comprising an epitope of a polypeptide that
is preferentially expressed in a tumor tissue (with or without additional
amino acid sequence) are within the scope of the present invention.
The term "polynucleotide(s)," as used herein, means a
single or double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases and includes DNA and corresponding RNA molecules, including
HnRNA and mRNA molecules, both sense and anti-sense strands, and
comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly
or partially synthesized polynucleotides. An HnRNA molecule contains
introns and corresponds to a DNA molecule in a generally one-to-one
manner. An mRNA molecule corresponds to an HnRNA and DNA molecule
from which the introns have been excised. A polynucleotide may consist
of an entire gene, or any portion thereof. Operable anti-sense polynucleotides
may comprise a fragment of the corresponding polynucleotide, and
the definition of "polynucleotide" therefore includes
all such operable anti-sense fragments.
The compositions and methods of the present invention also encompass
variants of the above polypeptides and polynucleotides.
A polypeptide "variant," as used herein, is a polypeptide
that differs from the recited polypeptide only in conservative substitutions
and/or modifications, such that the antigenic properties of the
polypeptide are retained. In a preferred embodiment, variant polypeptides
differ from an identified sequence by substitution, deletion or
addition of five amino acids or fewer. Such variants may generally
be identified by modifying one of the above polypeptide sequences,
and evaluating the antigenic properties of the modified polypeptide
using, for example, the representative procedures described herein.
Polypeptide variants preferably exhibit at least about 70%, more
preferably at least about 90% and most preferably at least about
95% identity (determined as described below) to the identified polypeptides.
As used herein, a "conservative substitution" is one
in which an amino acid is substituted for another amino acid that
has similar properties, such that one skilled in the art of peptide
chemistry would expect the secondary structure and hydropathic nature
of the polypeptide to be substantially unchanged. In general, the
following groups of amino acids represent conservative changes:
(I) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr,
thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5)
phe, tyr, trp, his.
Variants may also, or alternatively, contain other modifications,
including the deletion or addition of amino acids that have minimal
influence on the antigenic properties, secondary structure and hydropathic
nature of the polypeptide. For example, a polypeptide may be conjugated
to a signal (or leader) sequence at the N-terminal end of the protein
which co-translationally or post-translationally directs transfer
of the protein. The polypeptide may also be conjugated to a linker
or other sequence for ease of synthesis, purification or identification
of the polypeptide (e.g., poly-His), or to enhance binding of the
polypeptide to a solid support. For example, a polypeptide may be
conjugated to an immunoglobulin Fc region.
A nucleotide "variant" is a sequence that differs from
the recited nucleotide sequence in having one or more nucleotide
deletions, substitutions or additions. Such modifications may be
readily introduced using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis as taught, for
example, by Adelman et al. (DNA, 2:183, 1983). Nucleotide variants
may be naturally occurring allelic variants, or non-naturally occurring
variants. Variant nucleotide sequences preferably exhibit at least
about 70%, more preferably at least about 80% and most preferably
at least about 90% identity (determined as described below) to the
The breast tumor antigens provided by the present invention include
variants that are encoded by DNA sequences which are substantially
homologous to one or more of the DNA sequences specifically recited
herein. "Substantial homology," as used herein, refers
to DNA sequences that are capable of hybridizing under moderately
stringent conditions. Suitable moderately stringent conditions include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC, overnight
or, in the event of cross-species homology, at 45.degree. C. with
0.5.times.SSC; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC containing
0.1% SDS. Such hybridizing DNA sequences are also within the scope
of this invention, as are nucleotide sequences that, due to code
degeneracy, encode an immunogenic polypeptide that is encoded by
a hybridizing DNA sequence.
Two nucleotide or polypeptide sequences are said to be "identical"
if the sequence of nucleotides or amino acid residues in the two
sequences is the same when aligned for maximum correspondence as
described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A "comparison
window" as used herein, refers to a segment of at least about
20 contiguous positions, usually 30 to about 75, 40 to about 50,
in which a sequence may be compared to a reference sequence of the
same number of contiguous positions after the two sequences are
Optimal alignment of sequences for comparison may be conducted
using the Megalign program in the Lasergene suite of bioinformatics
software (DNASTAR, Inc., Madison, Wis.), using default parameters.
This program embodies several alignment schemes described in the
following references: Dayhoff, M. O. (1978) A model of evolutionary
change in proteins--Matrices for detecting distant relationships.
In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure,
National Biomedical Resarch Foundaiton, Washington D.C. Vol. 5,
Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment
and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic
Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M.
(1989) Fast and sensitive multiple sequence alignments on a microcomputer
CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) Optimal alignments
in linear space CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor
11:105; Santou, N. Nes, M. (1987) The neighbor joining method. A
new method for reconstructing phylogenetic trees Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy--the
Principles and Practice of Numerical Taxonomy, Freeman Press, San
Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Rapid
similarity searches of nucleic acid and protein data banks Proc.
Natl. Acad., Sci. USA 80:726-730.
Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a window
of comparison of at least 20 positions, wherein the portion of the
polynucleotide sequence in the comparison window may comprise additions
or deletions (i.e. gaps) of 20 percent or less, usually 5 to 15
percent, or 10 to 12 percent, as compared to the reference sequences
(which does not comprise additions or deletions) for optimal alignment
of the two sequences. The percentage is calculated by determining
the number of positions at which the identical nucleic acid bases
or amino acid residue occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the reference sequence (i.e. the
window size) and multiplying the results by 100 to yield the percentage
of sequence identity. In general, polynucleotides encoding all or
a portion of the polypeptides described herein may be prepared using
any of several techniques. For example, cDNA molecules encoding
such polypeptides may be cloned on the basis of the breast tumor-specific
expression of the corresponding mRNAs, using differential display
PCR. This technique compares the amplified products from RNA template
prepared from normal and breast tumor tissue. cDNA may be prepared
by reverse transcription of RNA using a (dT).sub.12 AG primer. Following
amplification of the cDNA using a random primer, a band corresponding
to an amplified product specific to the tumor RNA may be cut out
from a silver stained gel and subcloned into a suitable vector (e.g.,
the T-vector, Novagen, Madison, Wis.). Polynucleotides encoding
all or a portion of the breast tumor-specific polypeptides disclosed
herein may be amplified from cDNA prepared as described above using
the random primers shown in SEQ ID NO.:87-125.
Alternatively, a polynucleotide encoding a polypeptide as described
herein (or a portion thereof) may be amplified from human genomic
DNA, or from breast tumor cDNA, via polymerase chain reaction. For
this approach, B18Ag1 sequence-specific primers may be designed
based on the sequence provided in SEQ ID NO:1, and may be purchased
or synthesized. One suitable primer pair for amplification from
breast tumor cDNA is (5' ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID
NO: 126) and (5' CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO:127).
An amplified portion of B18Ag1 may then be used to isolate the full
length gene from a human genomic DNA library or from a breast tumor
cDNA library, using well known techniques, such as those described
in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold
Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (1989). Other
sequences within the retroviral genome of which B18Ag1 is a part
may be similarly prepared by screening human genomic libraries using
B18Ag1-specific sequences as probes. Nucleotides translated into
protein from the retroviral genome shown in SEQ ID NO: 141 may then
be determined by cloning the corresponding cDNAs, predicting the
open reading frames and cloning the appropriate cDNAs into a vector
containing a viral promoter, such as T7. The resulting constructs
can be employed in a translation reaction, using techniques known
to those of skill in the art, to identify nucleotide sequences which
result in expressed protein. Similarly, primers specific for the
remaining breast tumor-specific polypeptides described herein may
be designed based on the nucleotide sequences provided in SEQ ID
NO:11-86, 142-298, 301-303, 307, 313, 314, 316 and 317.
Recombinant polypeptides encoded by the DNA sequences described
above may be readily prepared from the DNA sequences. For example,
supernatants from suitable host/vector systems which secrete recombinant
protein or polypeptide into culture media may be first concentrated
using a commercially available filter. Following concentration,
the concentrate may be applied to a suitable purification matrix
such as an affinity matrix or an ion exchange resin. Finally, one
or more reverse phase HPLC steps can be employed to further purify
a recombinant polypeptide.
In general, any of a variety of expression vectors known to those
of ordinary skill in the art may be employed to express recombinant
polypeptides of this invention. Expression may be achieved in any
appropriate host cell that has been transformed or transfected with
an expression vector containing a polynucleotide that encodes a
recombinant polypeptide. Suitable host cells include prokaryotes,
yeast and higher eukaryotic cells. Preferably, the host cells employed
are E. coli, yeast or a mammalian cell line such as COS or CHO.
Such techniques may also be used to prepare polypeptides comprising
epitopes or variants of the native polypeptides. For example, variants
of a native polypeptide may generally be prepared using standard
mutagenesis techniques, such as oligonucleotide-directed site-specific
mutagenesis, and sections of the DNA sequence may be removed to
permit preparation of truncated polypeptides. Portions and other
variants having fewer than about 100 amino acids, and generally
fewer than about 50 amino acids, may also be generated by synthetic
means, using techniques well known to those of ordinary skill in
the art. For example, such polypeptides may be synthesized using
any of the commercially available solid-phase techniques, such as
the Merrifield solid-phase synthesis method, where amino acids are
sequentially added to a growing amino acid chain. See Merrifield,
J. Am. Chem. Soc. 85:2149-2146 (1963). Equipment for automated synthesis
of polypeptides is commercially available from suppliers such as
Perkin Elmer/Applied BioSystems Division, Foster City, Calif., and
may be operated according to the manufacturer's instructions.
In specific embodiments, polypeptides of the present invention
encompass amino acid sequences encoded by a polynucleotide having
a sequence recited in any one of SEQ ID NO:1, 3-26, 28-77, 142,
143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206,
207, 209-214, 216, 218, 219, 221-240, 243-245, 247, 250, 251, 253,
255, 257-266, 268, 269, 271-273, 275, 276, 278, 280, 281, 284, 288,
291-298, 301-303, 307, 313, 314, 316 and 317, and variants of such
polypeptides. Polypeptides within the scope of the present invention
also include polypeptides (and epitopes thereof) encoded by DNA
sequences that hybridize to a sequence recited in any one of SEQ
ID NO: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192,
194-198, 200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245,
247, 250, 251, 253, 255, 257-266, 268, 269, 271-273, 275, 276, 278,
280, 281, 284, 288, 291-298, 301-303, 307, 313, 314, 316 and 317
under stringent conditions, wherein the DNA sequences are at least
80% identical in overall sequence to a recited sequence and wherein
RNA corresponding to the nucleotide sequence is expressed at a greater
level in human breast tumor tissue than in normal breast tissue.
As used herein, "stringent conditions" refers to prewashing
in a solution of 6.times.SSC, 0.2% SDS; hybridizing at 65.degree.
C., 6.times.SSC, 0.2% SDS overnight; followed by two washes of 30
minutes each in 1.times.SSC, 0.1% SDS at 65.degree. C. and two washes
of 30 minutes each in 0.2.times.SSC, 0.1% SDS at 65.degree. C. Polynucleotides
according to the present invention include molecules that encode
any of the above polypeptides.
In another aspect of the present invention, antibodies are provided.
Such antibodies may be prepared by any of a variety of techniques
known to those of ordinary skill in the art. See, e.g., Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988. In one such technique, an immunogen comprising the polypeptide
is initially injected into any of a wide variety of mammals (e.g.,
mice, rats, rabbits, sheep or goats). In this step, the polypeptides
of this invention may serve as the immunogen without modification.
Alternatively, particularly for relatively short polypeptides, a
superior immune response may be elicited if the polypeptide is joined
to a carrier protein, such as bovine serum albumin or keyhole limpet
hemocyanin. The immunogen is injected into the animal host, preferably
according to a predetermined schedule incorporating one or more
booster immunizations, and the animals are bled periodically. Polyclonal
antibodies specific for the polypeptide may then be purified from
such antisera by, for example, affinity chromatography using the
polypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for the antigenic polypeptide of
interest may be prepared, for example, using the technique of Kohler
and Milstein, Eur. J. Immunol. 6:511-519 (1976), and improvements
thereto. Briefly, these methods involve the preparation of immortal
cell lines capable of producing antibodies having the desired specificity
(i.e., reactivity with the polypeptide of interest). Such cell lines
may be produced, for example, from spleen cells obtained from an
animal immunized as described above. The spleen cells are then immortalized
by, for example, fusion with a myeloma cell fusion partner, preferably
one that is syngeneic with the immunized animal. A variety of fusion
techniques may be employed. For example, the spleen cells and myeloma
cells may be combined with a nonionic detergent for a few minutes
and then plated at low density on a selective medium that supports
the growth of hybrid cells, but not myeloma cells. A preferred selection
technique uses HAT (hypoxanthine, aminopterin, thymidine) selection.
After a sufficient time, usually about 1 to 2 weeks, colonies of
hybrids are observed. Single colonies are selected and their culture
supernatants tested for binding activity against the polypeptide.
Hybridomas having high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of
growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the hybridoma
cell line into the peritoneal cavity of a suitable vertebrate host,
such as a mouse. Monoclonal antibodies may then be harvested from
the ascites fluid or the blood. Contaminants may be removed from
the antibodies by conventional techniques, such as chromatography,
gel filtration, precipitation, and extraction. The polypeptides
of this invention may be used in the purification process in, for
example, an affinity chromatography step.
Antibodies may be used, for example, in methods for detecting breast
cancer in a patient. Such methods involve using an antibody to detect
the presence or absence of a breast tumor-specific polypeptide as
described herein in a suitable biological sample. As used herein,
suitable biological samples include tumor or normal tissue biopsy,
mastectomy, blood, lymph node, serum or urine samples, or other
tissue, homogenate, or extract thereof obtained from a patient.
There are a variety of assay formats known to those of ordinary
skill in the art for using an antibody to detect polypeptide markers
in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1988. For example, the assay
may be performed in a Western blot format, wherein a protein preparation
from the biological sample is submitted to gel electrophoresis,
transferred to a suitable membrane and allowed to react with the
antibody. The presence of the antibody on the membrane may then
be detected using a suitable detection reagent, as described below.
In another embodiment, the assay involves the use of antibody,
immobilized on a solid support to bind to the polypeptide and remove
it from the remainder of the sample. The bound polypeptide may then
be detected using a second antibody or reagent that contains a reporter
group. Alternatively, a competitive assay may be utilized, in which
a polypeptide is labeled with a reporter group and allowed to bind
to the immobilized antibody after incubation of the antibody with
the sample. The extent to which components of the sample inhibit
the binding of the labeled polypeptide to the antibody is indicative
of the reactivity of the sample with the immobilized antibody, and
as a result, indicative of the concentration of polypeptide in the
The solid support may be any material known to those of ordinary
skill in the art to which the antibody may be attached. For example,
the solid support may be a test well in a microtiter plate or a
nitrocellulose filter or other suitable membrane. Alternatively,
the support may be a bead or disc, such as glass, fiberglass, latex
or a plastic material such as polystyrene or polyvinylchloride.
The support may also be a magnetic particle or a fiber optic sensor,
such as those disclosed, for example, in U.S. Pat. No. 5,359,681.
The antibody may be immobilized on the solid support using a variety
of techniques known to those in the art, which are amply described
in the patent and scientific literature. In the context of the present
invention, the term "immobilization" refers to both noncovalent
association, such as adsorption, and covalent attachment (which
may be a direct linkage between the antigen and functional groups
on the support or may be a linkage by way of a cross-linking agent).
Immobilization by adsorption to a well in a microtiter plate or
to a membrane is preferred. In such cases, adsorption may be achieved
by contacting the antibody, in a suitable buffer, with the solid
support for a suitable amount of time. The contact time varies with
temperature, but is typically between about 1 hour and 1 day. In
general, contacting a well of a plastic microtiter plate (such as
polystyrene or polyvinylchloride) with an amount of antibody ranging
from about 10 ng to about 1 .mu.g, and preferably about 100-200
ng, is sufficient to immobilize an adequate amount of polypeptide.
Covalent attachment of antibody to a solid support may also generally
be achieved by first reacting the support with a bifunctional reagent
that will react with both the support and a functional group, such
as a hydroxyl or amino group, on the antibody. For example, the
antibody may be covalently attached to supports having an appropriate
polymer coating using benzoquinone or by condensation of an aldehyde
group on the support with an amine and an active hydrogen on the
binding partner (see, e.g., Pierce Immunotechnology Catalog and
Handbook (1991) at A12-A13).
In certain embodiments, the assay for detection of polypeptide
in a sample is a two-antibody sandwich assay. This assay may be
performed by first contacting an antibody that has been immobilized
on a solid support, commonly the well of a microtiter plate, with
the biological sample, such that the polypeptide within the sample
are allowed to bind to the immobilized antibody. Unbound sample
is then removed from the immobilized polypeptide-antibody complexes
and a second antibody (containing a reporter group) capable of binding
to a different site on the polypeptide is added. The amount of second
antibody that remains bound to the solid support is then determined
using a method appropriate for the specific reporter group.
More specifically, once the antibody is immobilized on the support
as described above, the remaining protein binding sites on the support
are typically blocked. Any suitable blocking agent known to those
of ordinary skill in the art, such as bovine serum albumin or Tween
20.TM. (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody
is then incubated with the sample, and polypeptide is allowed to
bind to the antibody. The sample may be diluted with a suitable
diluent, such as phosphate-buffered saline (PBS) prior to incubation.
In general, an appropriate contact time (i.e., incubation time)
is that period of time that is sufficient to detect the presence
of polypeptide within a sample obtained from an individual with
breast cancer. Preferably, the contact time is sufficient to achieve
a level of binding that is at least 95% of that achieved at equilibrium
between bound and unbound polypeptide. Those of ordinary skill in
the art will recognize that the time necessary to achieve equilibrium
may be readily determined by assaying the level of binding that
occurs over a period of time. At room temperature, an incubation
time of about 30 minutes is generally sufficient.
Unbound sample may then be removed by washing the solid support
with an appropriate buffer, such as PBS containing 0.1% Tween 20.TM..
The second antibody, which contains a reporter group, may then be
added to the solid support. Preferred reporter groups include enzymes
(such as horseradish peroxidase), substrates, cofactors, inhibitors,
dyes, radionuclides, luminescent groups, fluorescent groups and
biotin. The conjugation of antibody to reporter group may be achieved
using standard methods known to those of ordinary skill in the art.
The second antibody is then incubated with the immobilized antibody-polypeptide
complex for an amount of time sufficient to detect the bound polypeptide.
An appropriate amount of time may generally be determined by assaying
the level of binding that occurs over a period of time. Unbound
second antibody is then removed and bound second antibody is detected
using the reporter group. The method employed for detecting the
reporter group depends upon the nature of the reporter group. For
radioactive groups, scintillation counting or autoradiographic methods
are generally appropriate. Spectroscopic methods may be used to
detect dyes, luminescent groups and fluorescent groups. Biotin may
be detected using avidin, coupled to a different reporter group
(commonly a radioactive or fluorescent group or an enzyme). Enzyme
reporter groups may generally be detected by the addition of substrate
(generally for a specific period of time), followed by spectroscopic
or other analysis of the reaction products.
To determine the presence or absence of breast cancer, the signal
detected from the reporter group that remains bound to the solid
support is generally compared to a signal that corresponds to a
predetermined cut-off value established from non-tumor tissue. In
one preferred embodiment, the cut-off value is the average mean
signal obtained when the immobilized antibody is incubated with
samples from patients without breast cancer. In general, a sample
generating a signal that is three standard deviations above the
predetermined cut-off value may be considered positive for breast
cancer. In an alternate preferred embodiment, the cut-off value
is determined using a Receiver Operator Curve, according to the
method of Sackett et al., Clinical Epidemiology: A Basic Sciencefor
Clinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly,
in this embodiment, the cut-off value may be determined from a plot
of pairs of true positive rates (i.e., sensitivity) and false positive
rates (100%-specificity) that correspond to each possible cut-off
value for the diagnostic test result. The cut-off value on the plot
that is the closest to the upper left-hand corner (i.e., the value
that encloses the largest area) is the most accurate cut-off value,
and a sample generating a signal that is higher than the cut-off
value determined by this method may be considered positive. Alternatively,
the cut-off value may be shifted to the left along the plot, to
minimize the false positive rate, or to the right, to minimize the
false negative rate. In general, a sample generating a signal that
is higher than the cut-off value determined by this method is considered
positive for breast cancer.
In a related embodiment, the assay is performed in a flow-through
or strip test format, wherein the antibody is immobilized on a membrane,
such as nitrocellulose. In the flow-through test, the polypeptide
within the sample bind to the immobilized antibody as the sample
passes through the membrane. A second, labeled antibody then binds
to the antibody-polypeptide complex as a solution containing the
second antibody flows through the membrane. The detection of bound
second antibody may then be performed as described above. In the
strip test format, one end of the membrane to which antibody is
bound is immersed in a solution containing the sample. The sample
migrates along the membrane through a region containing second antibody
and to the area of immobilized antibody. Concentration of second
antibody at the area of immobilized antibody indicates the presence
of breast cancer. Typically, the concentration of second antibody
at that site generates a pattern, such as a line, that can be read
visually. The absence of such a pattern indicates a negative result.
In general, the amount of antibody immobilized on the membrane is
selected to generate a visually discernible pattern when the biological
sample contains a level of polypeptide that would be sufficient
to generate a positive signal in the two-antibody sandwich assay,
in the format discussed above. Preferably, the amount of antibody
immobilized on the membrane ranges from about 25 ng to about 1 .mu.g,
and more preferably from about 50 ng to about 1 .mu.g. Such tests
can typically be performed with a very small amount of biological
The presence or absence of breast cancer in a patient may also
be determined by evaluating the level of mRNA encoding a breast
tumor-specific polypeptide as described herein within the biological
sample (e.g., a biopsy, mastectomy and/or blood sample from a patient)
relative to a predetermined cut-off value. Such an evaluation may
be achieved using any of a variety of methods known to those of
ordinary skill in the art such as, for example, in situ hybridization
and amplification by polymerase chain reaction.
For example, polymerase chain reaction may be used to amplify sequences
from cDNA prepared from RNA that is isolated from one of the above
biological samples. Sequence-specific primers for use in such amplification
may be designed based on the sequences provided in any one of SEQ
ID NO: 1, 11-86, 142-298. 301-303, 307, 313, 314, 316 and 317, and
may be purchased or synthesized. In the case of B18Ag1, as noted
herein, one suitable primer pair is B18Ag1-2 (5' ATG GCT ATT TTC
GGG GGC TGA CA) (SEQ ID NO:126) and B18Ag1-3 (5' CCG GTA TCT CCT
CGT GGG TAT T) (SEQ ID NO:127). The PCR reaction products may then
be separated by gel electrophoresis and visualized according to
methods well known to those of ordinary skill in the art. Amplification
is typically performed on samples obtained from matched pairs of
tissue (tumor and non-tumor tissue from the same individual) or
from unmatched pairs of tissue (tumor and non-tumor tissue from
different individuals). The amplification reaction is preferably
performed on several dilutions of cDNA spanning two orders of magnitude.
A two-fold or greater increase in expression in several dilutions
of the tumor sample as compared to the same dilution of the non-tumor
sample is considered positive.
As used herein, the term "primer/probe specific for a polynucleotide"
means an oligonucleotide sequence that has at least about 80% identity,
preferably at least about 90% and more preferably at least about
95%, identity to the polynucleotide in question, or an oligonucleotide
sequence that is anti-sense to a sequence that has at least about
80% identity, preferably at least about 90% and more preferably
at least about 95%, identity to the polynucleotide in question.
Primers and/or probes which may be usefully employed in the inventive
diagnostic methods preferably have at least about 10-40 nucleotides.
In a preferred embodiment, the polymerase chain reaction primers
comprise at least about 10 contiguous nucleotides of a polynucleotide
that encodes one of the polypeptides disclosed herein or that is
anti-sense to a sequence that encodes one of the polypeptides disclosed
herein. Preferably, oligonucleotide probes for use in the inventive
diagnostic methods comprise at least about 15 contiguous oligonucleotides
of a polynucleotide that encodes one of the polypeptides disclosed
herein or that is anti-sense to a sequence that encodes one of the
polypeptides disclosed herein. Techniques for both PCR based assays
and in situ hybridization assays are well known in the art.
Conventional RT-PCR protocols using agarose and ethidium bromide
staining, while important in defining gene specificity, do not lend
themselves to diagnostic kit development because of the time and
effort required in making them quantitative (i.e., construction
of saturation and/or titration curves), and their sample throughput.
This problem is overcome by the development of procedures such as
real time RT-PCR which allows for assays to be performed in single
tubes, and in turn can be modified for use in 96 well plate formats.
Instrumentation to perform such methodologies are available from
Perkin Elmer/Applied Biosystems Division. Alternatively, other high
throughput assays using labeled probes (e.g., digoxygenin) in combination
with labeled (e.g., enzyme fluorescent, radioactive) antibodies
to such probes can also be used in the development of 96 well plate
In yet another method for determining the presence or absence of
breast cancer in a patient, one or more of the breast tumor-specific
polypeptides described may be used in a skin test. As used herein,
a "skin test" is any assay performed directly on a patient
in which a delayed-type hypersensitivity (DTH) reaction (such as
swelling, reddening or dermatitis) is measured following intradermal
injection of one or more polypeptides as described above. Such injection
may be achieved using any suitable device sufficient to contact
the polypeptide or polypeptides with dermal cells of the patient,
such as a tuberculin syringe or 1 mL syringe. Preferably, the reaction
is measured at least 48 hours after injection, more preferably 48-72
The DTH reaction is a cell-mediated immune response, which is greater
in patients that have been exposed previously to a test antigen
(i.e., an immunogenic portion of a polypeptide employed, or a variant
thereof). The response may measured visually, using a ruler. In
general, a response that is greater than about 0.5 cm in diameter,
preferably greater than about 5.0 cm in diameter, is a positive
response, indicative of breast cancer.
The breast tumor-specific polypeptides described herein are preferably
formulated, for use in a skin test, as pharmaceutical compositions
containing at least one polypeptide and a physiologically acceptable
carrier, such as water, saline, alcohol, or a buffer. Such compositions
typically contain one or more of the above polypeptides in an amount
ranging from about 1 .mu.g to 100 .mu.g, preferably from about 10
.mu.g to 50 .mu.g in a volume of 0.1 mL. Preferably, the carrier
employed in such pharmaceutical compositions is a saline solution
with appropriate preservatives, such as phenol and/or Tween 80.TM..
In other aspects of the present invention, the progression and/or
response to treatment of a breast cancer may be monitored by performing
any of the above assays over a period of time, and evaluating the
change in the level of the response (i.e., the amount of polypeptide
or mRNA detected or, in the case of a skin test, the extent of the
immune response detected). For example, the assays may be performed
every month to every other month for a period of 1 to 2 years. In
general, breast cancer is progressing in those patients in whom
the level of the response increases over time. In contrast, breast
cancer is not progressing when the signal detected either remains
constant or decreases with time.
In further aspects of the present invention, the compounds described
herein may be used for the immunotherapy of breast cancer. In these
aspects, the compounds (which may be polypeptides, antibodies or
polynucleotides) are preferably incorporated into pharmaceutical
compositions or vaccines. Pharmaceutical compositions comprise one
or more such compounds and a physiologically acceptable carrier.
Vaccines may comprise one or more such compounds in combination
with an immunostimulant, such as an adjuvant or a liposome (into
which the compound is incorporated). An immunostimulant may be any
substance that enhances or potentiates an immune response (antibody
and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants
include adjuvants, biodegradable microspheres (e.g., polylactic
galactide) and liposomes (into which the compound is incorporated;
see e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccine preparation
is generally described in, for example, M. F. Powell and M. J. Newman,
eds., "Vaccine Design (the subunit and adjuvant approach),"
Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines
within the scope of the present invention may also contain other
compounds, which may be biologically active or inactive. For example,
one or more immunogenic portions of other tumor antigens may be
present, either incorporated into a fusion polypeptide or as a separate
compound, within the composition or vaccine.
Alternatively, a vaccine may contain DNA encoding one or more of
the polypeptides as described above, such that the polypeptide is
generated in situ. In such vaccines, the DNA may be present within
any of a variety of delivery systems known to those of ordinary
skill in the art, including nucleic acid expression systems, bacteria
and viral expression systems. Appropriate nucleic acid expression
systems contain the necessary DNA sequences for expression in the
patient (such as a suitable promoter and terminating signal). Bacterial
delivery systems involve the administration of a bacterium (such
as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion
of the polypeptide on its cell surface. In a preferred embodiment,
the DNA may be introduced using a viral expression system (e.g.,
vaccinia or other pox virus, retrovirus, or adenovirus), which may
involve the use of a non-pathogenic (defective), replication competent
virus. Techniques for incorporating DNA into such expression systems
are well known to those of ordinary skill in the art. The DNA may
also be "naked," as described, for example, in Ulmer et
al., Science 259:1745-1749 (1993), and reviewed by Cohen, Science
259:1691-1692 (1993). The uptake of naked DNA may be increased by
coating the DNA onto biodegradable beads, which are efficiently
transported into the cells.
While any suitable carrier known to those of ordinary skill in
the art may be employed in the pharmaceutical compositions of this
invention, the type of carrier will vary depending on the mode of
administration. For parenteral administration, such as subcutaneous
injection, the carrier preferably comprises water, saline, alcohol,
a fat, a wax or a buffer. For oral administration, any of the above
carriers or a solid carrier, such as mannitol, lactose, starch,
magnesium stearate, sodium saccharine, talcum, cellulose, glucose,
sucrose, and magnesium carbonate, may be employed. Biodegradable
microspheres (e.g., polylactate polyglycolate) may also be employed
as carriers for the pharmaceutical compositions of this invention.
Any of a variety of immunostimulants may be employed in the vaccines
of this invention. For example, an adjuvant may be included. Most
adjuvants contain a substance designed to protect the antigen from
rapid catabolism, such as aluminum hydroxide or mineral oil, and
a stimulator of immune responses, such as lipid A, Bortadella pertussis
or Mycobacterium tuberculosis derived proteins. Suitable adjuvants
are commercially available as, for example, Freund's Incomplete
Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.);
Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2
(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as
aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium,
iron or zinc; an insoluble suspension of acylated tyrosine; acylated
sugars; cationically or anionically derivatized polysaccharides;
polyphosphazenes; biodegradable microspheres; monophosphoryl lipid
A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or
-12, may also be used as adjuvants.
Within the vaccines provided herein, the adjuvant composition is
preferably designed to induce an immune response predominantly of
the Th1 type. High levels of Th1-type cytokines (e.g., IFN-.gamma.,
TNF.alpha., IL-2 and IL-12) tend to favor the induction of cell
mediated immune responses to an administered antigen. In contrast,
high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10)
tend to favor the induction of humoral immune responses. Following
application of a vaccine as provided herein, a patient will support
an immune response that includes Th1- and Th2-type responses. Within
a preferred embodiment, in which a response is predominantly Th1-type,
the level of Th1-type cytokines will increase to a greater extent
than the level of Th2-type cytokines. The levels of these cytokines
may be readily assessed using standard assays. For a review of the
families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol.
Preferred adjuvants for use in eliciting a predominantly Th 1-type
response include, for example, a combination of monophosphoryl lipid
A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), together
with an aluminum salt. MPL adjuvants are available from Corixa Corporation
(Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034
and 4,912,094). CpG-containing oligonucleotides (in which the CpG
dinucleotide is unmethylated) also induce a predominantly Th1 response.
Such oligonucleotides are well known and are described, for example,
in WO 96/02555 and WO 99/33488. Immunostimulatory DNA sequences
are also described, for example, by Sato et al., Science 273:352,
1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila
Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone
or in combination with other adjuvants. For example, an enhanced
system involves the combination of a monophosphoryl lipid A and
saponin derivative, such as the combination of QS21 and 3D-MPL as
described in WO 94/00153, or a less reactogenic composition where
the QS21 is quenched with cholesterol, as described in WO 96/33739.
Other preferred formulations comprise an oil-in-water emulsion and
tocopherol. A particularly potent adjuvant formulation involving
QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described
in WO 95/17210.
Other preferred adjuvants include Montanide ISA 720 (Seppic, France),
SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron),
the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available
from SmithKline Beecham, Rixensart, Belgium), Detox (Ribi ImmunoChem
Research Inc., Hamilton, Mont.), RC-529 (Ribi ImmunoChem Research
Inc., Hamilton, Mont.) and Aminoalkyl glucosaminide 4-phosphates
Any vaccine provided herein may be prepared using well known methods
that result in a combination of antigen, immunostimulant and a suitable
carrier or excipient. The compositions described herein may be administered
as part of a sustained release formulation (i.e., a formulation
such as a capsule, sponge or gel (composed of polysaccharides, for
example) that effects a slow release of compound following administration).
Such formulations may generally be prepared using well known technology
(see, e.g., Coombes et al., Vaccine 14:1429-1438, 1996) and administered
by, for example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release formulations
may contain a polypeptide, polynucleotide or antibody dispersed
in a carrier matrix and/or contained within a reservoir surrounded
by a rate controlling membrane.
Carriers for use within such formulations are biocompatible, and
may also be biodegradable; preferably the formulation provides a
relatively constant level of active component release. Such carriers
include microparticles of poly(lactide-co-glycolide), as well as
polyacrylate, latex, starch, cellulose and dextran. Other delayed-release
carriers include supramolecular biovectors, which comprise a non-liquid
hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide)
and, optionally, an external layer comprising an amphiphilic compound,
such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT
applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount
of active compound contained within a sustained release formulation
depends upon the site of implantation, the rate and expected duration
of release and the nature of the condition to be treated or prevented.
Any of a variety of delivery vehicles may be employed within pharmaceutical
compositions and vaccines to facilitate production of an antigen-specific
immune response that targets tumor cells. Delivery vehicles include
antigen presenting cells (APCs), such as dendritic cells, macropliages,
B cells, monocytes and other cells that may be engineered to be
efficient APCs. Such cells may, but need not, be genetically modified
to increase the capacity for presenting the antigen, to improve
activation and/or maintenance of the T cell response, to have anti-tumor
effects per se and/or to be immunologically compatible with the
receiver (i.e., matched HLA haplotype). APCs may generally be isolated
from any of a variety of biological fluids and organs, including
tumor and peritumoral tissues, and may be autologous, allogeneic,
syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic
cells or progenitors thereof as antigen-presenting cells. Dendritic
cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251,
1998) and have been shown to be effective as a physiological adjuvant
for eliciting prophylactic or therapeutic antitumor immunity (see
Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). In general,
dendritic cells may be identified based on their typical shape (stellate
in situ, with marked cytoplasmic processes (dendrites) visible in
vitro), their ability to take up, process and present antigens with
high efficiency and their ability to activate naive T cell responses.
Dendritic cells may, of course, be engineered to express specific
cell-surface receptors or ligands that are not commonly found on
dendritic cells in vivo or ex vivo, and such modified dendritic
cells are contemplated by the present invention. As an alternative
to dendritic cells, secreted vesicles antigen-loaded dendritic cells
(called exosomes) may be used within a vaccine (see Zitvogel et
al., Nature Med. 4:594-600, 1998).
Dendritic cells and progenitors may be obtained from peripheral
blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating
cells, lymph nodes, spleen, skin, umbilical cord blood or any other
suitable tissue or fluid. For example, dendritic cells may be differentiated
ex vivo by adding a combination of cytokines such as GM-CSF, IL-4,
IL-13 and/or TNF.alpha. to cultures of monocytes harvested from
peripheral blood. Alternatively, CD34 positive cells harvested from
peripheral blood, umbilical cord blood or bone marrow may be differentiated
into dendritic cells by adding to the culture medium combinations
of GM-CSF, IL-3, TNF.alpha., CD40 ligand, LPS, flt3 ligand and/or
other compound(s) that induce differentiation, maturation and proliferation
of dendritic cells.
Dendritic cells are conveniently categorized as "immature"
and "mature" cells, which allows a simple way to discriminate
between two well characterized phenotypes. However, this nomenclature
should not be construed to exclude all possible intermediate stages
of differentiation. Immature dendritic cells are characterized as
APC with a high capacity for antigen uptake and processing, which
correlates with the high expression of Fcy receptor and mannose
receptor. The mature phenotype is typically characterized by a lower
expression of these markers, but a high expression of cell surface
molecules responsible for T cell activation such as class 1 and
class 11 MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory
molecules (e.g., CD40, CD80, CD86 and 4-I BB).
APCs may generally be transfected with a polynucleotide encoding
a polypeptide of the present invention (or portion or other variant
thereof) such that the polypeptide, or an immunogenic portion thereof,
is expressed on the cell surface. Such transfection may take place
ex vivo, and a composition or vaccine comprising such transfected
cells may then be used for therapeutic purposes, as described herein.
Alternatively, a gene delivery vehicle that targets a dendritic
or other antigen presenting cell may be administered to a patient,
resulting in transfection that occurs in vivo. In vivo and ex vivo
transfection of dendritic cells, for example, may generally be performed
using any methods known in the art, such as those described in WO
97/24447, or the gene gun approach described by Mahvi et al., Immunology
and cell Biology 75:456-460, 1997. Antigen loading of dendritic
cells may be achieved by incubating dendritic cells or progenitor
cells with the polypeptide, DNA (naked or within a plasmid vector)
or RNA; or with antigen-expressing recombinant bacterium or viruses
(e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior
to loading, the polypeptide may be covalently conjugated to an immunological
partner that provides T cell help (e.g., a carrier molecule). Alternatively,
a dendritic cell may be pulsed with a non-conjugated immunological
partner, separately or in the presence of the polypeptide.
Vaccines and pharmaceutical compositions may be presented in unit-dose
or multi-dose containers, such as sealed ampoules or vials. Such
containers are preferably hermetically sealed to preserve sterility
of the formulation until use. In general, formulations may be stored
as suspensions, solutions or emulsions in oily or aqueous vehicles.
Alternatively, a vaccine or pharmaceutical composition may be stored
in a freeze-dried condition requiring only the addition of a sterile
liquid carrier immediately prior to use.
The above pharmaceutical compositions and vaccines may be used,
for example, for the therapy of breast cancer in a patient. As used
herein, a "patient" refers to any warm-blooded animal,
preferably a human. A patient may or may not be afflicted with breast
cancer. Accordingly, the above pharmaceutical compositions and vaccines
may be used to prevent the development of breast cancer or to treat
a patient afflicted with breast cancer. In a preferred embodiment,
the compounds are administered either prior to or following surgical
removal of primary tumors and/or treatment by administration of
radiotherapy and conventional chemotherapeutic drugs. To prevent
or slow the development of breast cancer, a pharmaceutical composition
or vaccine comprising one or more polypeptides as described herein
may be administered to a patient. Alternatively, naked DNA or plasmid
or viral vector encoding the polypeptide may be administered. For
treating a patient with breast cancer, the pharmaceutical composition
or vaccine may comprise one or more polypeptides, antibodies or
polynucleotides complementary to DNA encoding a polypeptide as described
herein (e.g., antisense RNA or antisense deoxyribonucleotide oligonucleotides).
Routes and frequency of administration, as well as dosage, will
vary from individual to individual. In general, the pharmaceutical
compositions and vaccines may be administered by injection (e.g.,
intracutaneous, intramuscular, intravenous or subcutaneous), intranasally
(e.g., by aspiration) or orally. Between 1 and 10 doses may be administered
for a 52-week period. Preferably, 6 doses are administered, at intervals
of 1 month, and booster vaccinations may be given periodically thereafter.
Alternate protocols may be appropriate for individual patients.
A suitable dose is an amount of a compound that, when administered
as described above, is capable of promoting an anti-tumor immune
response. Such response can be monitored by measuring the anti-tumor
antibodies in a patient or by vaccine-dependent generation of cytolytic
effector cells capable of killing the patient's tumor cells in vitro.
Such vaccines should also be capable of causing an immune response
that leads to an improved clinical outcome (e.g., more frequent
remissions, complete or partial or longer disease-free survival)
in vaccinated patients as compared to non-vaccinated patients. In
general, for pharmaceutical compositions and vaccines comprising
one or more polypeptides, the amount of each polypeptide present
in a dose ranges from about 100 .mu.g to 5 mg. Suitable dose sizes
will vary with the size of the patient, but will typically range
from about 0.1 mL to about 5 mL.
Polypeptides disclosed herein may also be employed in adoptive
immunotherapy for the treatment of cancer. Adoptive immunotherapy
may be broadly classified into either active or passive immunotherapy.
In active immunotherapy, treatment relies on the in vivo stimulation
of the endogenous host immune system to react against tumors with
the administration of immune response-modifying agents (for example,
tumor vaccines, bacterial adjuvants, and/or cytokines).
In passive immunotherapy, treatment involves the delivery of biologic
reagents with established tumor-immune reactivity (such as effector
cells or antibodies) that can directly or indirectly mediate antitumor
effects and does not necessarily depend on an intact host immune
system. Examples of effector cells include T lymphocytes (for example,
CD8+ cytotoxic T-lymphocyte, CD4+ T-helper, tumor-infiltrating lymphocytes),
killer cells (Natural Killer cells, lymphokine-activated killer
cells), B cells, or antigen presenting cells (such as dendritic
cells and macrophages) expressing the disclosed antigens. The polypeptides
disclosed herein may also be used to generate antibodies or anti-idiotypic
antibodies (as in U.S. Pat. No. 4,918,164), for passive immunotherapy.
The predominant method of procuring adequate numbers of T-cells
for adoptive immunotherapy is to grow immune T-cells in vitro. Culture
conditions for expanding single antigen-specific T-cells to several
billion in number with retention of antigen recognition in vivo
are well known in the art. These in vitro culture conditions typically
utilize intermittent stimulation with antigen, often in the presence
of cytokines, such as IL-2, and non-dividing feeder cells. As noted
above, the immunoreactive polypeptides described herein may be used
to rapidly expand antigen-specific T cell cultures in order to generate
sufficient number of cells for immunotherapy. In particular, antigen-presenting
cells, such as dendritic, macrophage or B-cells, may be pulsed with
immunoreactive polypeptides or transfected with a polynucleotide
sequence(s), using standard techniques well known in the art. For
cultured T-cells to be effective in therapy, the cultured T-cclls
must bc able to grow and distribute widely and to survive long term
in vivo. Studies have demonstrated that cultured T-cells can be
induced to grow in vivo and to survive long term in substantial
numbers by repeated stimulation with antigen supplemented with IL-2
(see, for example, Cheever et al. Ibid).
The polypeptides disclosed herein may also be employed to generate
and/or isolate tumor-reactive T-cells, which can then be administered
to the patient. In one technique, antigen-specific T-cell lines
may be generated by in vivo immunization with short peptides corresponding
to immunogenic portions of the disclosed polypeptides. The resulting
antigen specific CD8+ CTL clones may be isolated from the patient,
expanded using standard tissue culture techniques, and returned
to the patient.
Alternatively, peptides corresponding to immunogenic portions of
the polypeptides may be employed to generate tumor reactive T cell
subsets by selective in vitro stimulation and expansion of autologous
T cells to provide antigen-specific T cells which may be subsequently
transferred to the patient as described, for example, by Chang et
al. (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996).
In another embodiment, syngeneic or autologous dendritic cells
may be pulsed with peptides corresponding to at least an immunogenic
portion of a polypeptide disclosed herein. The resulting antigen-specific
dendritic cells may either be transferred into a patient, or employed
to stimulate T cells to provide antigen-specific T cells which may,
in turn, be administered to a patient. The use of peptide-pulsed
dendritic cells to generate antigen-specific T cells and the subsequent
use of such antigen-specific T cells to eradicate tumors in a murine
model has been demonstrated by Cheever et al. ("Therapy With
Cultured T Cells: Principles Revisited," Immunological Reviews,
Additionally vectors expressing the disclosed polynucleotides may
be introduced into stem cells taken from the patient and clonally
propagated in vitro for autologous transplant back into the same
patient. In one embodiment, cells of the immune system, such as
T cells, may be isolated from the peripheral blood of a patient,
using a commercially available cell separation system, such as CellPro
Incorporated's (Bothell, Wash.) CEPRATE.TM. system (see U.S. Pat.
No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116
and WO 92/07243). The separated cells are stimulated with one or
more of the immunoreactive polypeptides contained within a delivery
vehicle, such as a microsphere, to provide antigen-specific T cells.
The population of tumor antigen-specific T cells is then expanded
using standard techniques and the cells are administered back to
The following Examples are offered by way of illustration and not
by way of limitation.
Preparation of Breast Tumor-Specific cDNAs Using Differential Display
This Example illustrates the preparation of cDNA molecules encoding
breast tumor-specific polypeptides using a differential display
A. Preparation of B18Ag1 cDNA and Characterization of mRNA Expression
Tissue samples were prepared from breast tumor and normal tissue
of a patient with breast cancer that was confirmed by pathology
after removal from the patient. Normal RNA and tumor RNA was extracted
from the samples and mRNA was isolated and converted into cDNA using
a (dT).sub.12 AG (SEQ ID NO:130) anchored 3' primer. Differential
display PCR was then executed using a randomly chosen primer (CTTCAACCTC)
(SEQ ID NO:103). Amplification conditions were standard buffer containing
1.5 mM MgCl.sub.2, 20 pmol of primer, 500 pmol dNTP, and 1 unit
of Taq DNA polymerase (Perkin-Elmer, Branchburg, N.J.). Forty cycles
of amplification were performed using 94.degree. C. denaturation
for 30 seconds, 42.degree. C. annealing for 1 minute, and 72.degree.
C extension for 30 seconds. An RNA fingerprint containing 76 amplified
products was obtained. Although the RNA fingerprint of breast tumor
tissue was over 98% identical to that of the normal breast tissue,
a band was repeatedly observed to be specific to the RNA fingerprint
pattern of the tumor. This band was cut out of a silver stained
gel, subcloned into the T-vector (Novagen, Madison, Wis.) and sequenced.
The sequence of the cDNA, referred to as B18Ag1, is provided in
SEQ ID NO:1. A database search of GENBANK and EMBL revealed that
the B 18Ag1 fragment initially cloned is 77% identical to the endogenous
human retroviral Element S71, which is a truncated retroviral element
homologous to the Simian Sarcoma Virus (SSV). S71 contains an incomplete
gag gene, a portion of the pol gene and an LTR-like structure at
the 3' terminus (see Werner et al., Virology 174:225-238 (1990)).
B18Ag1 is also 64% identical to SSV in the region corresponding
to the P30 (gag) locus. B18Ag1 contains three separate and incomplete
reading frames covering a region which shares considerable homology
to a wide variety of gag proteins of retroviruses which infect mammals.
In addition, the homology to S71 is not just within the gag gene,
but spans several kb of sequence including an LTR.
B18Ag1-specific PCR primers were synthesized using computer analysis
guidelines. RT-PCR amplification (94.degree. C., 30 seconds; 60.degree.
C..fwdarw.42.degree. C., 30 seconds; 72.degree. C., 30 seconds for
40 cycles) confirmed that B18Ag1 represents an actual mRNA sequence
present at relatively high levels in the patient's breast tumor
tissue. The primers used in amplification were B18Ag1-1 (CTG CCT
GAG CCA CAA ATG) (SEQ ID NO:128) and B18Ag1-4 (CCG GAG GAG GAA GCT
AGA GGA ATA) (SEQ ID NO:129) at a 3.5 mM magnesium concentration
and a pH of 8.5, and B18Ag1-2 (ATG GCT ATT TTC GGG GCC TGA CA) (SEQ
ID NO:126) and B18Ag1-3 (CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID
NO:127) at 2 mM magnesium at pH 9.5. The same experiments showed
exceedingly low to nonexistent levels of expression in this patient's
normal breast tissue (see FIG. 1). RT-PCR experiments were then
used to show that B 18Ag1 mRNA is present in nine other breast tumor
samples (from Brazilian and American patients) but absent in, or
at exceedingly low levels in, the normal breast tissue corresponding
to each cancer patient. RT-PCR analysis has also shown that the
B18Ag1 transcript is not present in various normal tissues (including
lymph node, myocardium and liver) and present at relatively low
levels in PBMC and lung tissue. The presence of B18Ag1 mRNA in breast
tumor samples, and its absence from normal breast tissue, has been
confirmed by Northern blot analysis, as shown in FIG. 2.
The differential expression of B18Ag1 in breast tumor tissue was
also confirmed by RNase protection assays. FIG. 3 shows the level
of B18Ag1 mRNA in various tissue types as determined in four different
RNase protection assays. Lanes 1-12 represent various normal breast
tissue samples, lanes 13-25 represent various breast tumor samples;
lanes 26-27 represent normal prostate samples; lanes 28-29 represent
prostate tumor samples; lanes 30-32 represent colon tumor samples;
lane 33 represents normal aorta; lane 34 represents normal small
intestine; lane 35 represents normal skin, lane 36 represents normal
lymph node; lane 37 represents normal ovary; lane 38 represents
normal liver; lane 39 represents normal skeletal muscle; lane 40
represents a first normal stomach sample, lane 41 represents a second
normal stomach sample; lane 42 represents a normal lung; lane 43
represents normal kidney; and lane 44 represents normal pancreas.
Interexperimental comparison was facilitated by including a positive
control RNA of known .epsilon.-actin message abundance in each assay
and normalizing the results of the different assays with respect
to this positive control.
RT-PCR and Southern Blot analysis has shown the B18Ag1 locus to
be present in human genomic DNA as a single copy endogenous retroviral
element. A genomic clone of approximately 12-18 kb was isolated
using the initial B18Ag1 sequence as a probe. Four additional subclones
were also isolated by XbaI digestion. Additional retroviral sequences
obtained from the ends of the XbaI digests of these clones (located
as shown in FIG. 4) are shown as SEQ ID NO:3-SEQ ID NO:10, where
SEQ ID NO:3 shows the location of the sequence labeled 10 in FIG.
4, SEQ ID NO:4 shows the location of the sequence labeled 11-29,
SEQ ID NO:5 shows the location of the sequence labeled 3, SEQ ID
NO:6 shows the location of the sequence labeled 6, SEQ ID NO:7 shows
the location of the sequence labeled 12, SEQ ID NO:8 shows the location
of the sequence labeled 13, SEQ ID NO:9 shows the location of the
sequence labeled 14 and SEQ ID NO: 0 shows the location of the sequence
Subsequent studies demonstrated that the 12-18 kb genomic clone
contains a retroviral element of about 7.75 kb, as shown in FIGS.
5A and 5B. The sequence of this retroviral element is shown in SEQ
ID NO: 141. The numbered line at the top of FIG. 5A represents the
sense strand sequence of the retroviral genomic clone. The box below
this line shows the position of selected restriction sites. The
arrows depict the different overlapping clones used to sequence
the retroviral element. The direction of the arrow shows whether
the single-pass subclone sequence corresponded to the sense or anti-sense
strand. FIG. 5B is a schematic diagram of the retroviral element
containing B18Ag1 depicting the organization of viral genes within
the element. The open boxes correspond to predicted reading frames,
starting with a methionine, found throughout the element. Each of
the six likely reading frames is shown, as indicated to the left
of the boxes, with frames 1-3 corresponding to those found on the
Using the cDNA of SEQ ID NO:1 as a probe, a longer cDNA was obtained
(SEQ ID NO:227) which contains minor nucleotide differences (less
than 1%) compared to the genomic sequence shown in SEQ ID NO:141.
B. Preparation of cDNA Molecules Encoding Other Breast Tumor-Specific
Normal RNA and tumor RNA was prepared and mRNA was isolated and
converted into cDNA using a (dT).sub.12 AG anchored 3' primer, as
described above. Differential display PCR was then executed using
the randomly chosen primers of SEQ ID NO: 87-125. Amplification
conditions were as noted above, and bands observed to be specific
to the RNA fingerprint pattern of the tumor were cut out of a silver
stained gel, subcloned into either the T-vector (Novagen, Madison,
Wis.) or the pCRII vector (Invitrogen, San Diego, Calif.) and sequenced.
The sequences are provided in SEQ ID NO:11-SEQ ID NO:86. Of the
79 sequences isolated, 67 were found to be novel (SEQ ID NO:11-26
and 28-77) (see also FIGS. 6-20).
An extended DNA sequence (SEQ ID NO: 290) for the antigen B15Ag1
(originally identified partial sequence provided in SEQ ID NO: 27)
was obtained in further studies. Comparison of the sequence of SEQ
ID NO: 290 with those in the gene bank as described above, revealed
homology to the known human .beta.-A activin gene. Further studies
led to the isolation of the full-length cDNA sequence for the antigen
B21GT2 (also referred to as B311D; originally identified partial
cDNA sequence provided in SEQ ID NO: 56). The full-length sequence
is provided in SEQ ID NO: 307, with the corresponding amino acid
sequence being provided in SEQ ID NO: 308. Further studies led to
the isolation of a splice variant of B311D. The B311D clone of SEQ
ID NO: 316 was sequenced and a XhoI/NotI fragment from this clone
was gel purified and 32P-cDTP labeled by random priming for use
as a probe for further screening to obtain additional B311D gene
sequence. Two fractions of a human breast tumor cDNA bacterial library
were screened using standard techniques. One of the clones isolated
in this manner yielded additional sequence which includes a poly
A+ tail. The determined cDNA sequence of this clone (referred to
as B311D_BT1.sub.-- 1A) is provided in SEQ ID NO: 317. The sequences
of SEQ ID NO: 316 and 317 were found to share identity over a 464
bp region, with the sequences diverging near the poly A+ sequence
of SEQ ID NO: 317.
Subsequent studies identified an additional 146 sequences (SEQ
ID NOS:142-289), of which 115 appeared to be novel (SEQ ID NOS:142,
143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206,
207, 209-214, 216, 218, 219, 221-240, 243-245, 247, 250, 251, 253,
255, 257-266, 268, 269, 271-273, 275, 276, 278, 280, 281, 284, 288
and 291). To the best of the inventors' knowledge none of the previously
identified sequences have heretofore been shown to be expressed
at a greater level in human breast tumor tissue than in normal breast
In further studies, several different splice forms of the antigen
B11Ag1 (also referred to as B305D) were isolated, with each of the
various splice forms containing slightly different versions of the
B11Ag1 coding frame. Splice junction sequences define individual
exons which, in various patterns and arrangements, make up the various
splice forms. Primers were designed to examine the expression pattern
of each of the exons using RT-PCR as described below. Each exon
was found to show the same expression pattern as the original B11Ag1
clone, with expression being breast tumor-, normal prostate- and
normal testis-specific. The determined cDNA sequences for the isolated
protein coding exons are provided in SEQ ID NO: 292-298, respectively.
The predicted amino acid sequences corresponding to the sequences
of SEQ ID NO: 292 and 298 are provided in SEQ ID NO: 299 and 300.
Additional studies using rapid amplification of cDNA ends (RACE),
a 5' specific primer to one of the splice forms of B11Ag1 provided
above and a breast adenocarcinoma, led to the isolation of three
additional, related, splice forms referred to as isoforms B11C-15,
B11C-8 and B11C-9,16. The determined cDNA sequences for these isoforms
are provided in SEQ ID NO: 301-303, with the corresponding predicted
amino acid sequences being provided in SEQ ID NO: 304-306.
In subsequent studies on B305D isoform A (cDNA sequence provided
in SEQ ID NO: 292), the cDNA sequence (provided in SEQ ID NO: 313)
was foul contain an additional guanine residue at position 884,
leading to a frameshift in the open reading frame. The determined
DNA sequence of this ORF is provided in SEQ ID NO: 314. This frameshift
generates a protein sequence (provided in SEQ ID NO: 315) of 293
amino acids that contains the C-terminal domain common to the other
isoforms of B305D but that differs in the N-terminal region.
Preparation of B18AG1 DNA from Human Genomic DNA
This Example illustrates the preparation of B18Ag1 DNA by amplification
from human genomic DNA.
B18Ag1 DNA may be prepared from 250 ng human genomic DNA using
20 pmol of B18Ag1 specific primers, 500 pmol dNTPS and 1 unit of
Taq DNA polymerase (Perkin Elmer, Branchburg, N.J.) using the following
amplification parameters: 94.degree. C. for 30 seconds denaturing,
30 seconds 60.degree. C. to 42.degree. C. touchdown annealing in
2.degree. C. increments every two cycles and 72.degree. C. extension
for 30 seconds. The last increment (a 42.degree. C. annealing temperature)
should cycle 25 times. Primers were selected using computer analysis.
Primers synthesized were B18Ag1-1, B18Ag1-2, B11Ag1-3, and B18Ag1-4.
Primer pairs that may be used are 1+3, 1+4, 2+3, and 2+4.
Following gel electrophoresis, the band corresponding to B18Ag1
DNA may be excised and cloned into a suitable vector.
Preparation of B 18AG1 DNA from Breast Tumor cDNA
This Example illustrates the preparation of B18Ag1 DNA by amplification
from human breast tumor cDNA.
First strand cDNA is synthesized from RNA prepared from human breast
tumor tissue in a reaction mixture containing 500 ng poly A+ RNA,
200 pmol of the primer (T).sub.12 AG(i.e., TTT TTT TTT TTT AG) (SEQ
ID NO: 130), IX first strand reverse transcriptase buffer, 6.7 mM
DTT, 500 mmol dNTPs, and 1 unit AMV or MMLV reverse transcriptase
(from any supplier, such as Gibco-BRL (Grand Island, N.Y.)) in a
final volume of 30 .mu.l. After first strand synthesis, the cDNA
is diluted approximately 25 fold and 1 .mu.l is used for amplification
as described in Example 2. While some primer pairs can result in
a heterogeneous population of transcripts, the primers B18Ag1-2
(5'ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO: 126) and B18Ag1-3
(5'CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO: 127) yield a single
151 bp amplification product.
Identification of B-Cell and T-Cell Epitopes or B 18AG1
This Example illustrates the identification of B18Ag1 epitopes.
The B18 .mu.l sequence can be screened using a variety of computer
algorithms. To determine B-cell epitopes, the sequence can be screened
for hydrophobicity and hydrophilicity values using the method of
Hopp, Prog. Clin. Biol. Res. 172B:367-77 (1985) or, alternatively,
Cease et al., J. Exp. Med. 164:1779-84 (1986) or Spouge et al.,
J. Immunol. 138:204-12 (1987). Additional Class II MHC (antibody
or B-cell) epitopes can be predicted using programs such as AMPHI
(e.g., Margalit et al., J. Immunol. 138:2213 (1987)) or the methods
of Rothbard and Taylor (e.g., EMBO J. 7:93 (1988)).
Once peptides (15-20 amino acids long) are identified using these
techniques, individual peptides can be synthesized using automated
peptide synthesis equipment (available from manufacturers such as
Perkin Elmer/Applied Biosystems Division, Foster City, Calif.) and
techniques such as Merrifield synthesis. Following synthesis, the
peptides can used to screen sera harvested from either normal or
breast cancer patients to determine whether patients with breast
cancer possess antibodies reactive with the peptides. Presence of
such antibodies in breast cancer patient would confirm the immunogenicity
of the specific B-cell epitope in question. The peptides can also
be tested for their ability to generate a serologic or humoral immune
in animals (mice, rats, rabbits, chimps etc.) following immunization
in vivo. Generation of a peptide-specific antiserum following such
immunization further confirms the immunogenicity of the specific
B-cell epitope in question.