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Cancer Patent Abstract
Compounds and methods for diagnosing prostate cancer are provided.
The inventive compounds include polypeptides containing at least
a portion of a prostate tumor protein. The inventive polypeptides
may be used to generate antibodies useful for the diagnosis and
monitoring of prostate cancer. Nucleic acid sequences for preparing
probes, primers, and polypeptides are also provided.
Cancer Patent Claims
The invention claimed is:
1. A method for detecting the presence of prostate cancer in a
patient, comprising the steps of: (a) detecting in a biological
sample the level of expression of a mRNA encoding a prostate tumor
protein, wherein the prostate tumor protein comprises an amino acid
sequence encoded by SEQ ID NO: 107; and (b) comparing the level
of expression detected in the biological sample to a predetermined
cut-off value, and thereby detecting the presence or absence of
prostate cancer, wherein an increase in the level of expression
in the biological sample compared to a non-cancerous sample is indicative
of the presence of prostate cancer.
2. The method of claim 1, wherein step (a) comprises an amplification
reaction.
3. The method of claim 2, wherein the amplification reaction is
a reverse transcription polymerase chain reaction.
4. The method of claim 2, wherein the amplification reaction is
a transcription-mediated amplification reaction.
5. The method of claim 1, wherein the biological sample is blood,
sera, urine, biopsies or prostate secretions.
Cancer Patent Description
TECHNICAL FIELD
The present invention relates generally to cancer diagnosis and
monitoring. The invention is more specifically related to polypeptides
comprising at least a portion of a prostate tumor protein, and to
polynucleotides encoding such polypeptides. Such polypeptides and
polynucleotides may be used to generate compounds for the diagnosis
and monitoring of prostate cancer.
BACKGROUND OF THE INVENTION
Prostate cancer is the most common form of cancer among males,
with an estimated incidence of 30% in men over the age of 50. Overwhelming
clinical evidence shows that human prostate cancer has the propensity
to metastasize to bone, and the disease appears to progress inevitably
from androgen dependent to androgen refractory status, leading to
increased patient mortality. This prevalent disease is currently
the second leading cause of cancer death among men in the U.S.
In spite of considerable research into therapies for the disease,
prostate cancer remains difficult to treat. Commonly, treatment
is based on surgery and/or radiation therapy, but these methods
are ineffective in a significant percentage of cases. Two previously
identified prostate specific proteins--prostate specific antigen
(PSA) and prostatic acid phosphatase (PAP)--have limited therapeutic
and diagnostic potential. For example, PSA levels do not always
correlate well with the presence of prostate cancer, being positive
in a percentage of non-prostate cancer cases, including benign prostatic
hyperplasia (BPH). Furthermore, PSA measurements correlate with
prostate volume, and do not indicate the level of metastasis.
In order to improve cancer treatment and survival, it would be
beneficial to identify prostate tumor proteins that permit an earlier
or more accurate diagnosis. In addition, further antigens are needed
to facilitate the selection of a course of treatment and monitoring
of patients. The present invention fulfills these needs and further
provides other related advantages.
SUMMARY OF THE INVENTION
The present invention provides methods for diagnosis and monitoring
of prostate cancer, together with kits for use in such methods.
Polypeptides are disclosed which comprise at least an immunogenic
portion of a prostate tumor protein or a variant thereof that differs
in one or more substitutions, deletions, additions and/or insertions
such that the ability of the variant to react with antigen-specific
antisera is not substantially diminished. Within certain embodiments,
the prostate tumor protein comprises an amino acid sequence encoded
by a DNA molecule having a sequence selected from the group consisting
of nucleotide sequences recited in SEQ ID NOs: 2 3, 5 107, 109 11,
115 171, 173 175, 177, 179 228, 229 305, 307 326, 328, 330, 332
335 and complements of such polynucleotides. Polynucleotides that
encode all or a portion of a prostate tumor protein are also provided.
Such polypeptides, polynucleotides, and compounds that bind to the
polypeptides, may be used in the diagnosis and monitoring of cancer,
such as prostate cancer.
In one specific aspect of the present invention, methods are provided
for determining the presence or absence of prostate cancer in a
patient, comprising: (a) contacting a biological sample obtained
from a patient with a binding agent that is capable of binding to
one of the above polypeptides; and (b) detecting in the sample an
amount of polypeptide that binds to the binding agent, relative
to a predetermined cut-off value, and therefrom determining the
presence or absence of a cancer in the patient. In preferred embodiments,
the binding agent is an antibody, most preferably a monoclonal antibody.
In related aspects, methods are provided for monitoring the progression
of prostate cancer in a patient, comprising: (a) contacting a biological
sample obtained from a patient with a binding agent that is capable
of binding to one of the above polypeptides; (b) determining in
the sample an amount of a protein or polypeptide that binds to the
binding agent; (c) repeating steps (a) and (b); and comparing the
amounts of polypeptide detected in steps (b) and (c).
Within related aspects, the present invention provides antibodies,
preferably monoclonal antibodies, that bind to a polypeptide as
described above, as well as diagnostic kits comprising such antibodies,
and methods of using such antibodies to inhibit the development
of prostate cancer.
The present invention further provides methods for determining
the presence or absence of prostate cancer in a patient, comprising
the steps of: (a) contacting a biological sample obtained from a
patient with an oligonucleotide that hybridizes to a polynucleotide
that encodes a prostate tumor protein, wherein the prostate tumor
protein comprises an amino acid sequence that is encoded by a polynucleotide
sequence selected from the group consisting of: (i) polynucleotides
recited in any one of SEQ ID NOs:2 3, 5 107, 109 11, 115 171, 173
175, 177, 179 228, 229 305, 307 326, 328, 330, and 332 335; and
(ii) complements of the foregoing polynucleotides; and (b) detecting
in the sample a level of a polynucleotide that hybridizes to the
oligonucleotide, relative to a predetermined cut-off value, and
therefrom determining the presence or absence of prostate cancer
in the patient. Within certain embodiments, the amount of mRNA is
detected via polymerase chain reaction using, for example, at least
one oligonucleotide primer that hybridizes to a polynucleotide that
encodes a polypeptide as recited above, or a complement of such
a polynucleotide. Within other embodiments, the amount of mRNA is
detected using a hybridization technique, employing an oligonucleotide
probe that hybridizes to a polynucleotide that encodes a polypeptide
as recited above, or a complement of such a polynucleotide. In a
preferred embodiment, at least one of the oligonucleotide primers
comprises at least about 10 contiguous nucleotides of a DNA molecule
having a partial sequence selected from the group consisting of
SEQ ID NOs:2 3, 5 107, 109 11, 115 171, 173 175, 177, 179 228, 229
305, 307 326, 328, 330, and 332 335.
In related aspects, methods are provided for monitoring the progression
of prostate cancer in a patient, comprising the steps of: (a) contacting
a biological sample obtained from a patient with an oligonucleotide
that hybridizes to a polynucleotide that encodes a prostate tumor
protein, wherein the antigen comprises an amino acid sequence that
is encoded by a polynucleotide sequence selected from the group
consisting of: (i) polynucleotides recited in any one of SEQ ID
NOs:2 3, 5 107, 109 11, 115 171, 173 175, 177, 179 228, 229 305,
307 326, 328, 330, and 332 335; and (ii) complements of the foregoing
polynucleotides; (b) detecting in the sample an amount of a polynucleotide
that hybridizes to the oligonucleotide; (c) repeating steps (a)
and (b) using a biological sample obtained from the patient at a
subsequent point in time; and (d) comparing the amount of polynucleotide
detected in step (c) with the amount detected in step (b) and therefrom
monitoring the progression of prostate cancer in the patient.
In related aspects, diagnostic kits comprising the above oligonucleotide
probes or primers are provided.
These and other aspects of the present invention will become apparent
upon reference to the following detailed description 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 is a graph illustrating the ability of T cells to kill fibroblasts
expressing the representative prostate tumor polypeptide P502S,
as compared to control fibroblasts. The % lysis is shown at a series
of effector:target ratios, as indicated.
FIGS. 2A and 2B are graphs illustrating the ability of T cells
to recognize cells expressing the representative prostate tumor
polypeptide P502S. In each case, the number of .gamma.-interferon
spots is shown for different numbers of responders. In FIG. 2A,
data is presented for fibroblasts pulsed with the P2S-12 peptide,
as compared to fibroblasts pulsed with a control E75 peptide. In
FIG. 2B, data is presented for fibroblasts expressing P506, as compared
to fibroblasts expressing HER-2/neu.
FIG. 3 represents a peptide competition binding assay showing that
the P1S#10 peptide, derived from P501S, binds HLA-A2. Peptide P1S#10
inhibits HLA-A2 restricted presentation of fluM58 peptide to CTL
clone D150M58 in TNF release bioassay. D150M58 CTL is specific for
the HLA-A2 binding influenza matrix peptide fluM58.
FIG. 4 is a graph illustrating the ability of T cell lines generated
from P1S#10 immunized mice to specifically lyse P1S#10-pulsed Jurkat
A2Kb targets and P501 S-transduced Jurkat A2Kb targets, as compared
to EGFP-transduced Jurkat A2Kb. The per cent lysis is shown as a
series of effector to target ratios, as indicated.
FIGS. 5 illustrates the ability of a T cell clone to recognize
and specifically lyse Jurkat A2Kb cells expressing the representative
prostate tumor polypeptide P501S, thereby demonstrating that the
P1S#10 peptide may be a naturally processed epitope of the P501S
polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
As noted above, the present invention is generally directed to
compounds and methods for the diagnosis and monitoring of prostate
cancer. The compositions described herein may include one or more
prostate tumor polypeptides, nucleic acid sequences encoding such
polypeptides, binding agents such as antibodies that bind to a polypeptide
and/or immune system cells (e.g., T cells). Prostate tumor polypeptides
of the present invention generally comprise at least a portion of
a prostate tumor protein or a variant thereof, such that the therapeutic,
antigenic and/or immunogenic properties of the polypeptide are not
substantially diminished relative to the native prostate tumor protein.
A "prostate tumor protein" is a protein that is overexpressed
(i.e., mRNA and/or protein is present at a level that is at least
two fold higher) in prostate tumor tissue, relative to normal prostate
tissue and/or relative to other tissues (e.g., brain, heart, kidney,
liver, lung, pancreas, ovary, placenta, skeletal muscle, spleen
and/or thymus). Nucleic acid sequences of the subject invention
generally comprise a DNA or RNA sequence that encodes all or a portion
of such a polypeptide, or that is complementary to such a sequence.
Antibodies are generally immune system proteins, or antigen-binding
fragments thereof, that are capable of binding to a portion of a
polypeptide as described above. T cells that may be employed within
such compositions are generally T cells that are specific for a
polypeptide as described above.
The present invention is based on the discovery of previously unknown
human prostate tumor proteins. Partial sequences of polynucleotides
encoding specific prostate tumor proteins (or complementary to such
coding sequences) are provided in SEQ ID NOs:2 3, 5 107, 109 111,
115 171, 173 175, 177, 179 228, 229 305, 307 326, 328, 330, and
332 335.
Prostrate Tumor Polynucleotides
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.
Any polynucleotide that encodes a prostate tumor protein or a portion
or other variant thereof as described herein is encompassed by the
present invention. Preferred polynucleotides comprise at least 10
consecutive nucleotides, and preferably at least 30 consecutive
nucleotides, that encode a portion of a prostate tumor protein.
More preferably, a polynucleotide encodes an immunogenic portion
of a prostate tumor protein. Polynucleotides complementary to any
such sequences are also encompassed by the present invention.
Polynucleotides may comprise a native sequence (i.e., an endogenous
sequence that encodes a prostate tumor protein or a portion thereof)
or may comprise a variant of such a sequence. Polynucleotide variants
may contain one or more substitutions, additions, deletions and/or
insertions such that the therapeutic, antigenic and/or immunogenic
properties are not substantially diminished, relative to a native
prostate tumor protein. 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). Preferably, the antigenicity or immunogenicity
of a polypeptide variant is not substantially diminished. The effect
on the immunogenicity of the encoded polypeptide may generally be
assessed as described herein. Variants preferably exhibit at least
about 70% identity, more preferably at least about 80% identity
and most preferably at least about 90% identity to a polynucleotide
sequence that encodes a native prostate tumor protein or a portion
thereof. The percent identity may be readily determined by comparing
sequences using computer algorithms well known to those of ordinary
skill in the art, such as Megalign, using default parameters. Certain
variants are substantially homologous to a native gene, or a portion
or complement thereof. Such polynucleotide variants are capable
of hybridizing under moderately stringent conditions to a naturally
occurring DNA sequence encoding a native prostate tumor protein
(or a complementary sequence). 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; 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).
It will be appreciated by those of ordinary skill in the art that,
as a result of the degeneracy of the genetic code, there are many
nucleotide sequences that encode a polypeptide as described herein.
Some of these polynucleotides bear minimal homology to the nucleotide
sequence of any native gene. Nonetheless, polynucleotides that vary
due to differences in codon usage are specifically contemplated
by the present invention.
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, more preferably
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 optimally aligned.
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 Research Foundation, 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.
Also included in the scope of the present invention are alleles
of the genes encoding the nucleotide sequences recited herein. As
used herein, an "allele" or "allellic sequence"
is an alternative form of the gene which may result from at least
one mutation in the nucleic acid sequence. Alleles may result in
altered mRNAs or polypeptides whose structure or function may or
may not be altered. Any given gene may have none, one, or many allelic
forms. Common mutational changes which give rise to alleles are
generally ascribed to natural deletions, additions, or substitutions
of nucleotides. Each of these types of changes may occur alone or
in combination with the others, one or more times in a given sequence.
Polynucleotides may be prepared using any of a variety of techniques.
For example, a polynucleotide may be identified, as described in
more detail below, using a PCR-based subtraction protocol. Alternatively,
polypeptides may be amplified via polymerase chain reaction (PCR)
from cDNA prepared from prostate tumor cells. For this approach,
sequence-specific primers may be designed based on the sequences
provided herein, and may be purchased or synthesized.
An amplified portion may be used to isolate a full length gene
from a suitable library (e.g., a prostate tumor cDNA library) using
well known techniques. Within such techniques, a library (cDNA or
genomic) is screened using one or more polynucleotide probes or
primers suitable for amplification. Preferably, a library is size-selected
to include larger molecules. Random primed libraries may also be
preferred for identifying 5' and upstream regions of genes. Genomic
libraries are preferred for obtaining introns and extending 5' sequences.
For hybridization techniques, a partial sequence may be labeled
(e.g., by nick-translation or end-labeling with .sup.32P) using
well known techniques. A bacterial or bacteriophage library is then
screened by hybridizing filters containing denatured bacterial colonies
(or lawns containing phage plaques) with the labeled probe (see
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing
colonies or plaques are selected and expanded, and the DNA is isolated
for further analysis. cDNA clones may be analyzed to determine the
amount of additional sequence by, for example, PCR using a primer
from the partial sequence and a primer from the vector. Restriction
maps and partial sequences may be generated to identify one or more
overlapping clones. The complete sequence may then be determined
using standard techniques, which may involve generating a series
of deletion clones. The resulting overlapping sequences are then
assembled into a single contiguous sequence. A full length cDNA
molecule can be generated by ligating suitable fragments, using
well known techniques.
Alternatively, there are numerous amplification techniques for
obtaining a full length coding sequence from a partial cDNA sequence.
Within such techniques, amplification is generally performed via
PCR. Any of a variety of commercially available kits may be used
to perform the amplification step. Primers may be designed using
techniques well known in the art (see, for example, Mullis et al.,
Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; Erlich ed.,
PCR Technology, Stockton Press, N.Y., 1989), and software well known
in the art may also be employed. Primers are preferably 22 30 nucleotides
in length, have a GC content of at least 50% and anneal to the target
sequence at temperatures of about 68.degree. C. to 72.degree. C.
The amplified region may be sequenced as described above, and overlapping
sequences assembled into a contiguous sequence.
One such amplification technique is inverse PCR (see Triglia et
al., Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes
to generate a fragment in the known region of the gene. The fragment
is then circularized by intramolecular ligation and used as a template
for PCR with divergent primers derived from the known region. Within
an alternative approach, sequences adjacent to a partial sequence
may be retrieved by amplification with a primer to a linker sequence
and a primer specific to a known region. The amplified sequences
are typically subjected to a second round of amplification with
the same linker primer and a second primer specific to the known
region. A variation on this procedure, which employs two primers
that initiate extension in opposite directions from the known sequence,
is described in WO 96/38591. Additional techniques include capture
PCR (Lagerstrom et al., PCR Methods Applic. 1:111 19, 1991) and
walking PCR (Parker et al., Nucl. Acids. Res. 19:3055 60, 1991).
Transcription-Mediated Amplification, or TMA is another method that
may be utilized for the amplification of DNA, rRNA, or mRNA, as
described in Patent No. PCT/US91/03184. This autocatalytic and isothermic
non-PCR based method utilizes two primers and two enzymes: RNA polymerase
and reverse transcriptase. One primer contains a promoter sequence
for RNA polymerase. In the first amplification, the promoter-primer
hybridizes to the target rRNA at a defined site. Reverse transcriptase
creates a DNA copy of the target rRNA by extension from the 3' end
of the promoter-primer. The RNA in the resulting complex is degraded
and a second primer binds to the DNA copy. A new strand of DNA is
synthesized from the end of the primer by reverse transcriptase
creating double stranded DNA. RNA polymerase recognizes the promoter
sequence in the DNA template and initiates transcription. Each of
the newly synthesized RNA amplicons re-enters the TMA process and
serves as a template for a new round of replication leading to the
expotential expansion of the RNA amplicon. Other methods employing
amplification may also be employed to obtain a full length cDNA
sequence.
In certain instances, it is possible to obtain a full length cDNA
sequence by analysis of sequences provided in an expressed sequence
tag (EST) database, such as that available from GenBank. Searches
for overlapping ESTs may generally be performed using well known
programs (e.g., NCBI BLAST searches), and such ESTs may be used
to generate a contiguous full length sequence.
Certain nucleic acid sequences of cDNA molecules encoding portions
of prostate tumor proteins are provided in SEQ ID NOS: 1 107, 109
111, 115 171, 173 175, 177, 179 228, 229 305, 307 326, 328, 330,
and 332 335. The polynucleotides recited herein, as well as full
length polynucleotides comprising such sequences, other portions
of such full length polynucleotides, and sequences complementary
to all or a portion of such full length molecules, are specifically
encompassed by the present invention.
Polynucleotide variants may generally be prepared by any method
known in the art, including chemical synthesis by, for example,
solid phase phosphoramidite chemical synthesis. Modifications in
a polynucleotide sequence may also be introduced using standard
mutagenesis techniques, such as oligonucleotide-directed site-specific
mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively,
RNA molecules may be generated by in vitro or in vivo transcription
of DNA sequences encoding a prostate tumor protein, or portion thereof,
provided that the DNA is incorporated into a vector with a suitable
RNA polymerase promoter (such as T7 or SP6). Certain portions may
be used to prepare an encoded polypeptide, as described herein.
In addition, or alternatively, a portion may be administered to
a patient such that the encoded polypeptide is generated in vivo.
A portion of a sequence complementary to a coding sequence (i.e.,
an antisense polynucleotide) may also be used as a probe or to modulate
gene expression. cDNA constructs that can be transcribed into antisense
RNA may also be introduced into cells of tissues to facilitate the
production of antisense RNA. An antisense polynucleotide may be
used, as described herein, to inhibit expression of a prostate tumor
protein. Antisense technology can be used to control gene expression
through triple-helix formation, which compromises the ability of
the double helix to open sufficiently for the binding of polymerases,
transcription factors or regulatory molecules (see Gee et al., In
Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing
Co. (Mt. Kisco, N.Y.; 1994)). Alternatively, an antisense molecule
may be designed to hybridize with a control region of a gene (e.g.,
promoter, enhancer or transcription initiation site), and block
transcription of the gene; or to block translation by inhibiting
binding of a transcript to ribosomes.
A portion of a coding sequence or a complementary sequence may
also be designed as a probe or primer to detect gene expression.
Probes may be labeled by a variety of reporter groups, such as radionuclides
and enzymes, and are preferably at least 10 nucleotides in length,
more preferably at least 20 nucleotides in length and still more
preferably at least 30 nucleotides in length. Primers, as noted
above, are preferably 22 30 nucleotides in length.
Any polynucleotide may be further modified to increase stability
in vivo. Possible modifications include, but are not limited to,
the addition of flanking sequences at the 5' and/or 3' ends; the
use of phosphorothioate or 2' O-methyl rather than phosphodiesterase
linkages in the backbone; and/or the inclusion of nontraditional
bases such as inosine, queosine and wybutosine, as well as acetyl-
methyl-, thio- and other modified forms of adenine, cytidine, guanine,
thymine and uridine.
Nucleotide sequences as described herein may be joined to a variety
of other nucleotide sequences using established recombinant DNA
techniques. For example, a polynucleotide may be cloned into any
of a variety of cloning vectors, including plasmids, phagemids,
lambda phage derivatives and cosmids. Vectors of particular interest
include expression vectors, replication vectors, probe generation
vectors and sequencing vectors. In general, a vector will contain
an origin of replication functional in at least one organism, convenient
restriction endonuclease sites and one or more selectable markers.
Other elements will depend upon the desired use, and will be apparent
to those of ordinary skill in the art.
Within certain embodiments, polynucleotides may be formulated so
as to permit entry into a cell of a mammal, and expression therein.
Such formulations are particularly useful for therapeutic purposes,
as described below. Those of ordinary skill in the art will appreciate
that there are many ways to achieve expression of a polynucleotide
in a target cell, and any suitable method may be employed. For example,
a polynucleotide may be incorporated into a viral vector such as,
but not limited to, adenovirus, adeno-associated virus, retrovirus,
or vaccinia or other pox virus (e.g., avian pox virus). Techniques
for incorporating polynucleotides into such vectors are well known
to those of ordinary skill in the art. A retroviral vector may additionally
transfer or incorporate a gene for a selectable marker (to aid in
the identification or selection of transduced cells) and/or a targeting
moiety, such as a gene that encodes a ligand for a receptor on a
specific target cell, to render the vector target specific. Targeting
may also be accomplished using an antibody, by methods known to
those of ordinary skill in the art.
Other formulations for therapeutic purposes include colloidal dispersion
systems, such as macromolecule complexes, nanocapsules, microspheres,
beads, and lipid-based systems including oil-in-water emulsions,
micelles, mixed micelles, and liposomes. A preferred colloidal system
for use as a delivery vehicle in vitro and in vivo is a liposome
(i.e., an artificial membrane vesicle). The preparation and use
of such systems is well known in the art.
Prostrate Tumor Polypeptides
Within the context of the present invention, polypeptides may comprise
at least a portion of a prostate tumor protein or a variant thereof,
as described herein. As noted above, a "prostate tumor protein"
is a protein that is overexpressed by prostate tumor cells, relative
to normal prostate cells and/or other tissues such as brain, heart,
kidney, liver, lung, pancreas, ovary, placenta, skeletal muscle,
spleen and/or thymus. Such polypeptides should comprise a portion
of a prostate tumor protein such that the therapeutic, antigenic
and/or immunogenic properties of the polypeptide are not substantially
diminished, relative to the full length protein. Within certain
preferred embodiments, a polypeptide comprises an immunogenic portion
of a native prostate tumor protein (i.e., the immunogenic properties
of the polypeptide are not substantially diminished). As used herein,
the term "polypeptide" encompasses amino acid chains of
any length, including full length proteins, wherein the amino acid
residues are linked by covalent peptide bonds. In addition to a
portion of a prostate tumor protein, additional sequences derived
from the native protein and/or heterologous sequences may be present,
and such sequences may (but need not) possess further immunogenic
or antigenic properties.
An "immunogenic portion," as used herein is a portion
of an antigen that is recognized (i.e., specifically bound) by a
B-cell and/or T-cell surface antigen receptor. Such immunogenic
portions generally comprise at least 5 amino acid residues, more
preferably at least 10, and still more preferably at least 20 amino
acid residues of a prostate tumor protein or a variant thereof.
Immunogenic portions of prostate tumor proteins provided herein
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 for the ability to react with antigen-specific
antibodies, antisera and/or T-cell lines or clones. As used herein,
antisera and antibodies are "antigen-specific" if they
specifically bind to an antigen (i.e., they react with the antigen
in an ELISA or other immunoassay, and do not react detectably with
unrelated proteins). Such antisera and antibodies may be prepared
as described herein, and using well known techniques. An immunogenic
portion of a native prostate tumor protein is a portion that reacts
with such antisera and/or T-cells at a level that is not substantially
less than the reactivity of the full length polypeptide (e.g., in
an ELISA and/or T-cell reactivity assay). Such immunogenic portions
may react within such assays at a level that is similar to or greater
than the reactivity of the full length polypeptide. Alternatively,
an immunogenic portion may react within such assays at a level that
is diminished by less than 50%, and preferably less than 20%, relative
to the full length polypeptide. 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. For example, a polypeptide
may be immobilized on a solid support and contacted with patient
sera to allow binding of antibodies within the sera to the immobilized
polypeptide. Unbound sera may then be removed and bound antibodies
detected using, for example, .sup.125I-labeled Protein A.
As noted above, a polypeptide may comprise a variant of a native
prostate tumor protein. A polypeptide "variant," as used
herein, is a polypeptide that differs from a native prostate tumor
protein in one or more substitutions, deletions, additions and/or
insertions, such that the therapeutic, antigenic and/or immunogenic
properties are not substantially diminished. Preferably, the immunogenic
properties are not substantially diminished. In other words, the
ability of a variant to react with antigen-specific antisera may
be enhanced or unchanged, relative to the native antigen, or may
be diminished by less than 50%, and preferably less than 20%, relative
to the native antigen. Polypeptide variants preferably exhibit at
least about 70%, more preferably at least about 90% and most preferably
at least about 95% identity to polypeptides encoded by polynucleotides
specifically recited herein. Identity may be determined by comparing
sequences using computer algorithms well known to those of skill
in the art, such as Megalign, using default parameters. For prostate
tumor polypeptides with immunoreactive properties, variants may
generally be identified by modifying one of the above polypeptide
sequences and evaluating the reactivity of the modified polypeptide
with antigen-specific antibodies or antisera as described herein.
For prostate tumor polypeptides useful for the generation of diagnostic
binding agents, a variant may be identified by evaluating a modified
polypeptide for the ability to generate antibodies that detect the
presence or absence of prostate cancer. Such modified sequences
may be prepared and tested using, for example, the representative
procedures described herein.
Preferably, a variant contains conservative substitutions. 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. Amino acid substitutions may generally be made on the
basis of similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and valine;
glycine and alanine; asparagine and glutamine; and serine, threonine,
phenylalanine and tyrosine. Other groups of amino acids that may
represent conservative changes include: (1) ala, pro, gly, glu,
asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu,
met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A
variant may also, or alternatively, contain nonconservative changes.
Variants containing substitutions may also (or alternatively) be
modified by, for example, the deletion or addition of amino acids
that have minimal influence on the immunogenicity, secondary structure
and hydropathic nature of the polypeptide.
As noted above, polypeptides may comprise 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.
Polypeptides may be prepared using any of a variety of well known
techniques. Recombinant polypeptides encoded by polynucleotide sequences
as described above may be readily prepared from the polynucleotide
sequences using any of a variety of expression vectors known to
those of ordinary skill in the art. Expression may be achieved in
any appropriate host cell that has been transformed or transfected
with an expression vector containing a polynucleotide molecule that
encodes a recombinant polypeptide. Suitable host cells include prokaryotes,
yeast and higher eukaryotic cells. Preferably, the host cells employed
are E. coli, yeast or a mammalian cell line, such as CHO cells.
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.
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 Applied BioSystems, Inc. (Foster City, Calif.),
and may be operated according to the manufacturer's instructions.
Within certain specific embodiments, a polypeptide may be a fusion
protein that comprises multiple polypeptides as described herein,
or that comprises one polypeptide as described herein and a known
prostate tumor antigen, or a variant of such an antigen. A fusion
protein generally comprises at least one of the above immunogenic
portions and one or more additional immunogenic prostate tumor sequences,
which are joined via a peptide linkage into a single amino acid
chain. The sequences may be joined directly (i.e., with no intervening
amino acids) or may be joined by way of a linked sequence (e.g.,
Gly-Cys-Gly) that does not significantly diminish the immunogenic
properties of the component polypeptides.
Fusion proteins may generally be prepared using standard techniques.
For example, a fusion protein may be prepared recombinantly. Briefly,
DNA sequences encoding the polypeptide components may be assembled
separately, and ligated into an appropriate expression vector. The
3' end of the DNA sequence encoding one polypeptide component is
ligated, with or without a peptide linker, to the 5' end of a DNA
sequence encoding the second polypeptide component so that the reading
frames of the sequences are in phase. This permits translation into
a single fusion protein that retains the biological activity of
both component polypeptides.
A peptide linker sequence may be employed to separate the first
and the second polypeptide components by a distance sufficient to
ensure that each polypeptide folds into its secondary and tertiary
structures. Such a peptide linker sequence may be incorporated into
the fusion protein using standard techniques well known in the art.
Suitable peptide linker sequences may be chosen based on the following
factors: (1) their ability to adopt a flexible extended conformation;
(2) their inability to adopt a secondary structure that could interact
with functional epitopes on the first and second polypeptides; and
(3) the lack of hydrophobic or charged residues that might react
with the polypeptide functional epitopes. Preferred peptide linker
sequences contain Gly, Asn and Ser residues. Other near neutral
amino acids, such as Thr and Ala may also be used in the linker
sequence. Amino acid sequences which may be usefully employed as
linkers include those disclosed in Maratea et al., Gene 40:39 46,
1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262, 1986;
U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker
sequence may generally be from 1 to about 50 amino acids in length.
Linker sequences are not required when the first and second polypeptides
have non-essential N-terminal amino acid regions that can be used
to separate the functional domains and prevent steric interference.
The ligated polynucleotide sequences are operably linked to suitable
transcriptional or translational regulatory elements. The regulatory
elements responsible for expression of polynucleotide are located
only 5' to the polynucleotide sequence encoding the first polypeptides.
Similarly, stop codons required to end translation and transcription
termination signals are only present 3' to the polynucleotide sequence
encoding the second polypeptide.
Fusion proteins are also provided that comprise a polypeptide of
the present invention together with an unrelated immunogenic protein.
Preferably the immunogenic protein is capable of eliciting a recall
response. Examples of such proteins include tetanus, tuberculosis
and hepatitis proteins (see, for example, Stoute et al. New Engl.
J. Med., 336:86 91, 1997).
In general, polypeptides (including fusion proteins) and polynucleotides
as described herein are isolated. An "isolated" polypeptide
or polynucleotide is one that is removed from its original environment.
For example, a naturally-occurring protein is isolated if it is
separated from some or all of the coexisting materials in the natural
system. Preferably, such polypeptides are at least about 90% pure,
more preferably at least about 95% pure and most preferably at least
about 99% pure. A polynucleotide is considered to be isolated if,
for example, it is cloned into a vector that is not a part of the
natural environment.
Binding Agents
The present invention further provides agents, such as antibodies
and antigen-binding fragments thereof, that specifically bind to
a prostate tumor protein. As used herein, an agent is said to "specifically
bind" to a prostate tumor protein if it reacts at a detectable
level (within, for example, an ELISA) with a prostate tumor protein,
and does not react detectably with unrelated proteins under similar
conditions. As used herein, "binding" refers to a noncovalent
association between two separate molecules such that a "complex"
is formed. The ability to bind may be evaluated by, for example,
determining a binding constant for the formation of the complex.
The binding constant is the value obtained when the concentration
of the complex is divided by the product of the component concentrations.
In general, two compounds are said to "bind," in the context
of the present invention, when the binding constant for complex
formation exceeds about 10.sup.3 L/mol. The binding constant may
be determined using methods well known in the art.
Binding agents are further capable of detecting metastatic prostate
tumors and differentiating between patients with and without prostate
cancer, using a representative assay provided herein. In other words,
antibodies or other binding agents that bind to a prostate tumor
protein will generate a signal indicating the presence of prostate
cancer in at least about 20% of patients with the disease, and will
generate a negative signal indicating the absence of the disease
in at least about 90% of individuals without the cancer. To determine
whether a binding agent satisfies this requirement, biological samples
(e.g., blood, blood-associated tumor cells, sera, urine, biopsies
and/or prostate secretions) from patients with and without prostate
cancer (as determined using standard clinical tests) may be assayed
as described herein for the presence of polypeptides or polynucleotides
that bind to the binding agent. It will be apparent that a statistically
significant number of samples with and without the disease should
be assayed. Each binding agent should satisfy the above criteria;
however, those of ordinary skill in the art will recognize that
binding agents may be used in combination to improve sensitivity.
If an immunogenic portion is employed, the resulting antibody should
indicate the presence of prostate cancer in substantially all (i.e.,
at least 80%, and preferably at least 90%) of the patients for which
prostate cancer would be indicated using an antibody raised against
the full length antigen. The antibody should also indicate the absence
of prostate cancer in substantially all of those samples that would
be negative when tested with an antibody raised against the full
length antigen. The representative assays provided herein, such
as the two-antibody sandwich assay, may generally be employed for
evaluating the ability of an antibody to detect prostate cancer.
Binding agents may be further linked to a reporter group, to facilitate
diagnostic assays. Suitable reporter groups will be apparent to
those of ordinary skill in the art, and 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.
Any agent that satisfies the above requirements may be a binding
agent. For example, a binding agent may be a ribosome, with or without
a peptide component, an RNA molecule or a polypeptide. In a preferred
embodiment, a binding agent is an antibody or an antigen-binding
fragment thereof. Such antibodies may be polyclonal or monoclonal.
In addition, the antibodies may be single chain, chimeric, CDR-grafted
or humanized.
Antibodies may be prepared by 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 general, 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
antibodies. In one 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 within,
for example, an affinity chromatography step.
Within certain embodiments, the use of antigen-binding fragments
of antibodies may be preferred. Such fragments include Fab fragments,
which may be prepared using standard techniques. Briefly, immunoglobulins
may be purified from rabbit serum by affinity chromatography on
Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain
to yield Fab and Fc fragments. The Fab and Fc fragments may be separated
by affinity chromatography on protein A bead columns.
Monoclonal antibodies of the present invention may be coupled to
one or more therapeutic agents. Suitable agents in this regard include
radionuclides, differentiation inducers, drugs, toxins, and derivatives
thereof. Preferred radionuclides include .sup.90Y, .sup.123I, .sup.125I,
.sup.131I, .sup.186Re, .sup.188Re, .sup.211At, and .sup.212Bi. Preferred
drugs include methotrexate, and pyrimidine and purine analogs. Preferred
differentiation inducers include phorbol esters and butyric acid.
Preferred toxins include ricin, abrin, diptheria toxin, cholera
toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed
antiviral protein.
A therapeutic agent may be coupled (e.g., covalently bonded) to
a suitable monoclonal antibody either directly or indirectly (e.g.,
via a linker group). A direct reaction between an agent and an antibody
is possible when each possesses a substituent capable of reacting
with the other. For example, a nucleophilic group, such as an amino
or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing
group, such as an anhydride or an acid halide, or with an alkyl
group containing a good leaving group (e.g., a halide) on the other.
Alternatively, it may be desirable to couple a therapeutic agent
and an antibody via a linker group. A linker group can function
as a spacer to distance an antibody from an agent in order to avoid
interference with binding capabilities. A linker group can also
serve to increase the chemical reactivity of a substituent on an
agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
It will be evident to those skilled in the art that a variety of
bifunctional or polyfunctional reagents, both homo- and hetero-functional
(such as those described in the catalog of the Pierce Chemical Co.,
Rockford, Ill.), may be employed as the linker group. Coupling may
be effected, for example, through amino groups, carboxyl groups,
sulfhydryl groups or oxidized carbohydrate residues. There are numerous
references describing such methodology, e.g., U.S. Pat. No. 4,671,958,
to Rodwell et al.
Where a therapeutic agent is more potent when free from the antibody
portion of the immunoconjugates of the present invention, it may
be desirable to use a linker group which is cleavable during or
upon internalization into a cell. A number of different cleavable
linker groups have been described. The mechanisms for the intracellular
release of an agent from these linker groups include cleavage by
reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710, to
Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat.
No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino
acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.),
by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958,
to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat.
No. 4,569,789, to Blattler et al.).
It may be desirable to couple more than one agent to an antibody.
In one embodiment, multiple molecules of an agent are coupled to
one antibody molecule. In another embodiment, more than one type
of agent may be coupled to one antibody. Regardless of the particular
embodiment, immunoconjugates with more than one agent may be prepared
in a variety of ways. For example, more than one agent may be coupled
directly to an antibody molecule, or linkers which provide multiple
sites for attachment can be used. Alternatively, a carrier can be
used.
A carrier may bear the agents in a variety of ways, including covalent
bonding either directly or via a linker group. Suitable carriers
include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234,
to Kato et al.), peptides and polysaccharides such as aminodextran
(e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carrier may also
bear an agent by noncovalent bonding or by encapsulation, such as
within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088).
Carriers specific for radionuclide agents include radiohalogenated
small molecules and chelating compounds. For example, U.S. Pat.
No. 4,735,792 discloses representative radiohalogenated small molecules
and their synthesis. A radionuclide chelate may be formed from chelating
compounds that include those containing nitrogen and sulfur atoms
as the donor atoms for binding the metal, or metal oxide, radionuclide.
U.S. Pat. No. 4,673,562, to Davison et al. discloses representative
chelating compounds and their synthesis.
A variety of routes of administration for the antibodies and immunoconjugates
may be used. Typically, administration is intravenous, intramuscular,
subcutaneous or in the bed of a resected tumor. It will be evident
that the precise dose of the antibody/immunoconjugate will vary
depending upon the antibody used, the antigen density on the tumor,
and the rate of clearance of the antibody.
Pharmaceutical Compositions and Vaccines
Within certain aspects, polypeptides, polynucleotides and/or binding
agents may be 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 and a non-specific immune response enhancer.
A non-specific immune response enhancer may be any substance that
enhances an immune response to an exogenous antigen. Examples of
non-specific immune response enhancers include adjuvants, biodegradable
microspheres (e.g., polylactic galactide) and liposomes (into which
the compound is incorporated). 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.
A pharmaceutical composition or vaccine may contain polynucleotides
encoding one or more of the polypeptides as described above, such
that the polypeptide is generated in situ. As noted above, the polynucleotides
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 polynucleotide 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 polynucleotides may be introduced using
a viral expression system (e.g., vaccinia or other pox virus, retrovirus,
or adenovirus), which may involve the use of a non-pathogenic (defective),
replication competent virus. Suitable systems are disclosed, for
example, in Fisher-Hoch et al., PNAS 86:317 321, 1989; Flexner et
al., Ann. N.Y. Acad. Sci. 569:86 103, 1989; Flexner et al., Vaccine
8:17 21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487;
WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242;
WO 91/02805; Berkner, Biotechniques 6:616 627, 1988; Rosenfeld et
al., Science 252:431 434, 1991; Kolls et al., PNAS 91:215 219, 1994;
Kass-Eisler et al., PNAS 90:11498 11502, 1993; Guzman et al., Circulation
88:2838 2848, 1993; and Guzman et al., Cir. Res. 73:1202 1207, 1993.
Techniques for incorporating polynucleotides into such expression
systems are well known to those of ordinary skill in the art. The
polynucleotides 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 polynucleotides
may be increased by coating the polynucleotides 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. Compositions of the present invention may be formulated
for any appropriate manner of administration including, for example,
topical, oral, nasal, intravenous, intracranial, intraperitoneal,
subcutaneous or intramuscular 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. Suitable biodegradable microspheres are disclosed,
for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
Such compositions may also comprise buffers (e.g., neutral buffered
saline or phosphate buffered saline), carbohydrates (e.g., glucose,
mannose, sucrose or dextrans), mannitol, proteins, polypeptides
or amino acids such as glycine, antioxidants, chelating agents such
as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or
preservatives. Alternatively, compositions of the present invention
may be formulated as a lyophilizate. Compounds may also be encapsulated
within liposomes using well known technology.
Any of a variety of non-specific immune response enhancers may
be employed in the vaccines of this invention. For example, an adjuvant
may be included. Most adjuvants contain a substance designed to
protect the antigen from rapid catabolism, such as aluminum hydroxide
or mineral oil, and a 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.),
alum, 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.
The compositions described herein may be administered as part of
a sustained release formulation (i.e., a formulation such as a capsule
or sponge that effects a slow release of compound following administration).
Such formulations may generally be prepared using well known technology
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. 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.
Cancer Therapy
In further aspects of the present invention, the pharmaceutical
compositions and vaccines described herein may be used for immunotherapy
of cancer, such as prostate cancer, in a patient. Polypeptides for
use within such compositions and vaccines generally comprise an
immunogenic portion of a prostate tumor protein, or a variant thereof.
Such polypeptides may stimulate the patient's own immune response
to prostate tumor cells. Alternatively, a pharmaceutical composition
or vaccine may comprise one or more fusion proteins comprising one
or more such polypeptides and/or polynucleotides encoding such one
or more such polypeptides. Monoclonal antibodies of the present
invention may also be used as therapeutic reagents, to diminish
or eliminate prostate tumors. The antibodies may be used on their
own (for instance, to inhibit metastases) or coupled to one or more
therapeutic agents, as described above.
Within such methods, pharmaceutical compositions and vaccines are
typically administered to a patient. As used herein, a "patient"
refers to any warm-blooded animal, preferably a human. A patient
may be afflicted with a disease, or may be free of detectable disease.
Accordingly, the above pharmaceutical compositions and vaccines
may be used to prevent the development of prostate cancer or to
treat a patient afflicted with prostate cancer. Prostate cancer
may be diagnosed using criteria generally accepted in the art. Pharmaceutical
compositions and vaccines may be administered either prior to or
following surgical removal of primary tumors and/or treatment such
as administration of radiotherapy or conventional chemotherapeutic
drugs.
Routes and frequency of administration, as well as dosage, will
vary from individual to individual, and may parallel those currently
being used in immunotherapy of other diseases. In general, the pharmaceutical
compositions and vaccines may be administered by injection (e.g.,
intracutaneous, intramuscular, intravenous or subcutaneous), intranasally
(e.g., by aspiration) or orally. Preferably, between 1 and 10 doses
may be administered over a 3 24 week period. Preferably, 4 doses
are administered, at an interval of 3 months, and booster administrations
may be given periodically thereafter. Alternate protocols may be
appropriate for individual patients. A suitable dose is an amount
of polypeptide or polynucleotide that is effective to raise an immune
response (cellular and/or humoral) against prostate tumor cells
in a treated patient. A suitable immune response is at least 10
50% above the basal (i.e., untreated) level. 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, the amount of polypeptide
present in a dose (or produced in situ by the polynucleotides molecule
in a dose) ranges from about 1 pg to about 100 mg per kg of host,
typically from about 10 pg to about 1 mg, and preferably from about
100 pg to about 1 .mu.g. Suitable dose sizes will vary with the
size of the patient, but will typically range from about 0.01 mL
to about 5 mL. A variety of routes of administration for the antibodies
and immunoconjugates may be used. Typically, administration will
be intravenous, intramuscular, subcutaneous or in the bed of a resected
tumor. It will be evident that the precise dose of the antibody/immunoconjugate
will vary depending upon the antibody used, the antigen density
on the tumor, and the rate of clearance of the antibody.
Polypeptides disclosed herein may also be employed in adoptive
immunotherapy for the treatment of cancer. Adoptive immunotherapy
may be broadly classified into either active or passive immunotherapy.
In active immunotherapy, treatment relies on the in vivo stimulation
of the endogenous host immune system to react against tumors with
the administration of immune response-modifying agents (for example,
tumor vaccines, bacterial adjuvants, and/or cytokines).
In passive immunotherapy, treatment involves the delivery of biologic
reagents with established tumor-immune reactivity (such as effector
cells or antibodies) that can directly or indirectly mediate antitumor
effects and does not necessarily depend on an intact host immune
system. Examples of effector cells include T lymphocytes (for example,
CD8+ cytotoxic T-lymphocyte, CD4+ T-helper, gamma/delta T lymphocytes,
tumor-infiltrating lymphocytes), killer cells (such as Natural Killer
cells, lymphokine-activated killer cells), B cells, or antigen presenting
cells (such as dendritic cells and macrophages) expressing the disclosed
antigens. The polypeptides disclosed herein may also be used to
generate antibodies or anti-idiotypic antibodies (as in U.S. Pat.
No. 4,918,164), for passive immunotherapy.
The predominant method of procuring adequate numbers of T-cells
for adoptive immunotherapy is to grow immune 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, monocyte, fibroblast, or B-cells,
may be pulsed with immunoreactive polypeptides, or polynucleotide
sequence(s) may be introduced into antigen presenting cells, using
a variety of standard techniques well known in the art. For example,
antigen presenting cells may be transfected or transduced with a
polynucleotide sequence, wherein said sequence contains a promoter
region appropriate for increasing expression, and can be expressed
as part of a recombinant virus or other expression system. Several
viral vectors may be used to transduce an antigen presenting cell,
including pox virus, vaccinia virus, and adenovirus; also, antigen
presenting cells may be transfected with polynucleotide sequences
disclosed herein by a variety of means, including gene-gun technology,
lipid-mediated delivery, electroporation, osmotic shock, and particlate
delivery mechanisms, resulting in efficient and acceptable expression
levels as determined by one of ordinary skill in the art. For cultured
T-cells to be effective in therapy, the cultured T-cells must be
able to grow and distribute widely and to survive long term in vivo.
Studies have demonstrated that cultured T-cells can be induced to
grow in vivo and to survive long term in substantial numbers by
repeated stimulation with antigen supplemented with IL-2 (see, for
example, Cheever, M., et al, "Therapy With Cultured T Cells:
Principles Revisited," Immunological Reviews, 157:177, 1997).
The polypeptides disclosed herein may also be employed to generate
and/or isolate tumor-reactive T-cells, which can then be administered
to the patient. In one technique, antigen-specific T-cell lines
may be generated by in vivo immunization with short peptides corresponding
to immunogenic portions of the disclosed polypeptides. The resulting
antigen specific CD8+ CTL clones may be isolated from the patient,
expanded using standard tissue culture techniques, and returned
to the patient.
Alternatively, peptides corresponding to immunogenic portions of
the polypeptides may be employed to generate tumor reactive T cell
subsets by selective in vitro stimulation and expansion of autologous
T cells to provide antigen-specific T cells which may be subsequently
transferred to the patient as described, for example, by Chang et
al, (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996). Cells of the
immune system, such as T cells, may be isolated from the peripheral
blood of a patient, using a commercially available cell separation
system, 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 patient.
In other embodiments, T-cell and/or antibody receptors specific
for the polypeptides disclosed herein can be cloned, expanded, and
transferred into other vectors or effector cells for use in adoptive
immunotherapy. In particular, T cells may be transfected with the
appropriate genes to express the variable domains from tumor specific
monoclonal antibodies as the extracellular recognition elements
and joined to the T cell receptor signaling chains, resulting in
T cell activation, specific lysis, and cytokine release. This enables
the T cell to redirect its specificity in an MHC-independent manner.
See for example, Eshhar, Z., Cancer Immunol Immunother, 45(3 4):131
6, 1997 and Hwu, P., et al, Cancer Res, 55(15):3369 73, 1995. Another
embodiment may include the transfection of tumor antigen specific
alpha and beta T cell receptor chains into alternate T cells, as
in Cole, D J, et al, Cancer Res, 55(4):748 52, 1995.
In a further 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, Immunological Reviews,
15 7:177, 1997).
Additionally, vectors expressing the disclosed polynucleotides
may be introduced into stem cells taken from the patient and clonally
propagated in vitro for autologous transplant back into the same
patient.
In general, an appropriate dosage and treatment regimen provides
the active compound(s) in an amount sufficient to provide therapeutic
and/or prophylactic benefit. Such a response can be monitored by
establishing an improved clinical outcome (e.g., more frequent remissions,
complete or partial, or longer disease-free survival) in treated
patients as compared to non-treated patients. Increases in preexisting
immune responses to a prostate tumor protein generally correlate
with an improved clinical outcome. Such immune responses may generally
be evaluated using standard proliferation, cytotoxicity or cytokine
assays, which may be performed using samples obtained from a patient
before and after treatment.
Methods for Detecting Cancer
In general, a cancer may be detected in a patient based on the
presence of one or more prostate tumor proteins and/or polynucleotides
encoding such proteins in a biological sample obtained from the
patient. In other words, such proteins may be used as markers to
indicate the presence or absence of prostate cancer. The binding
agents provided herein generally permit detection of the level of
protein that binds to the agent in the biological sample. Alternatively,
polynucleotide primers and probes may be used to detect the level
of mRNA encoding an antigen, which is also indicative of the presence
or absence of prostate cancer.
There are a variety of assay formats known to those of ordinary
skill in the art for using a binding agent to detect polypeptide
markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence
or absence of a cancer in a patient may be determined by (a) contacting
a biological sample obtained from a patient with a binding agent;
(b) detecting in the sample a level of polypeptide that binds to
the binding agent; and (c) comparing the level of polypeptide with
a predetermined cut-off value.
In a preferred embodiment, the assay involves the use of binding
agent immobilized on a solid support to bind to and remove the polypeptide
from the remainder of the sample. The bound polypeptide may then
be detected using a detection reagent that contains a reporter group
and specifically binds to the binding agent/polypeptide complex.
Such detection reagents may comprise, for example, a binding agent
that specifically binds to the polypeptide or an antibody or other
agent that specifically binds to the binding agent, such as an anti-immunoglobulin,
protein G, protein A or a lectin. Alternatively, a competitive assay
may be utilized, in which a polypeptide is labeled with a reporter
group and allowed to bind to the immobilized binding agent after
incubation of the binding agent with the sample. The extent to which
components of the sample inhibit the binding of the labeled polypeptide
to the binding agent is indicative of the reactivity of the sample
with the immobilized binding agent. Suitable polypeptides for use
within such assays include full length prostate tumor proteins and
portions thereof to which the binding agent binds, as described
above.
The solid support may be any material known to those of ordinary
skill in the art to which the antigen may be attached. For example,
the solid support may be a test well in a microtiter plate or a
nitrocellulose or other suitable membrane. Alternatively, the support
may be a bead or disc, such as glass, fiberglass, latex or a plastic
material such as polystyrene or polyvinylchloride. The support may
also be a magnetic particle or a fiber optic sensor, such as those
disclosed, for example, in U.S. Pat. No. 5,359,681. The binding
agent may be immobilized on the solid support using a variety of
techniques known to those of skill in the art, which are amply described
in the patent and scientific literature. In the context of the present
invention, the term "immobilization" refers to both noncovalent
association, such as adsorption, and covalent attachment (which
may be a direct linkage between the antigen and functional groups
on the support or may be a linkage by way of a cross-linking agent).
Immobilization by adsorption to a well in a microtiter plate or
to a membrane is preferred. In such cases, adsorption may be achieved
by contacting the binding agent, in a suitable buffer, with the
solid support for a suitable amount of time. The contact time varies
with temperature, but is typically between about 1 hour and about
1 day. In general, contacting a well of a plastic microtiter plate
(such as polystyrene or polyvinylchloride) with an amount of binding
agent ranging from about 10 ng to about 10 .mu.g, and preferably
about 100 ng to about 1 .mu.g, is sufficient to immobilize an adequate
amount of binding agent.
Covalent attachment of binding agent to a solid support may generally
be achieved by first reacting the support with a bifunctional reagent
that will react with both the support and a functional group, such
as a hydroxyl or amino group, on the binding agent. For example,
the binding agent may be covalently attached to supports having
an appropriate polymer coating using benzoquinone or by condensation
of an aldehyde group on the support with an amine and an active
hydrogen on the binding partner (see, e.g., Pierce Immunotechnology
Catalog and Handbook, 1991, at A12 A13).
In certain embodiments, the assay is a two-antibody sandwich assay.
This assay may be performed by first contacting an antibody that
has been immobilized on a solid support, commonly the well of a
microtiter plate, with the sample, such that polypeptides within
the sample are allowed to bind to the immobilized antibody. Unbound
sample is then removed from the immobilized polypeptide-antibody
complexes and a detection reagent (preferably a second antibody
capable of binding to a different site on the polypeptide) containing
a reporter group is added. The amount of detection reagent 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 a period of time that is sufficient to detect the presence of
polypeptide within a sample obtained from an individual with prostate
cancer. Preferably, the contact time is sufficient to achieve a
level of binding that is at least about 95% of that achieved at
equilibrium between bound and unbound polypeptide. Those of ordinary
skill in the art will recognize that the time necessary to achieve
equilibrium may be readily determined by assaying the level of binding
that occurs over a period of time. At room temperature, an incubation
time of about 30 minutes is generally sufficient.
Unbound sample may then be removed by washing the solid support
with an appropriate buffer, such as PBS containing 0.1% Tween 20.TM..
The second antibody, which contains a reporter group, may then be
added to the solid support. Preferred reporter groups include those
groups recited above.
The detection reagent 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
detection reagent is then removed and bound detection reagent 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 prostate cancer, the signal
detected from the reporter group that remains bound to the solid
support is generally compared to a signal that corresponds to a
predetermined cut-off value. In one preferred embodiment, the cut-off
value for the detection of prostate cancer is the average mean signal
obtained when the immobilized antibody is incubated with samples
from patients without prostate cancer. In general, a sample generating
a signal that is three standard deviations above the predetermined
cut-off value is considered positive for prostate cancer. In an
alternate preferred embodiment, the cut-off value is determined
using a Receiver Operator Curve, according to the method of Sackett
et al., Clinical Epidemiology. A Basic Science for Clinical Medicine,
Little Brown and Co., 1985, p. 106 7. Briefly, in this embodiment,
the cut-off value may be determined from a plot of pairs of true
positive rates (i.e., sensitivity) and false positive rates (100%-specificity)
that correspond to each possible cut-off value for the diagnostic
test result. The cut-off value on the plot that is the closest to
the upper left-hand corner (i.e., the value that encloses the largest
area) is the most accurate cut-off value, and a sample generating
a signal that is higher than the cut-off value determined by this
method may be considered positive. Alternatively, the cut-off value
may be shifted to the left along the plot, to minimize the false
positive rate, or to the right, to minimize the false negative rate.
In general, a sample generating a signal that is higher than the
cut-off value determined by this method is considered positive for
prostate cancer.
In a related embodiment, the assay is performed in a flow-through
or strip test format, wherein the binding agent is immobilized on
a membrane, such as nitrocellulose. In the flow-through test, polypeptides
within the sample bind to the immobilized binding agent as the sample
passes through the membrane. A second, labeled binding agent then
binds to the binding agent-polypeptide complex as a solution containing
the second binding agent flows through the membrane. The detection
of bound second binding agent may then be performed as described
above. In the strip test format, one end of the membrane to which
binding agent is bound is immersed in a solution containing the
sample. The sample migrates along the membrane through a region
containing second binding agent and to the area of immobilized binding
agent. Concentration of second binding agent at the area of immobilized
antibody indicates the presence of prostate cancer. Typically, the
concentration of second binding agent 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 binding
agent 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.
Preferred binding agents for use in such assays are antibodies and
antigen-binding fragments thereof. Preferably, the amount of antibody
immobilized on the membrane ranges from about 25 ng to about 1 .mu.g,
and more preferably from about 50 ng to about 500 ng. Such tests
can typically be performed with a very small amount of biological
sample.
Of course, numerous other assay protocols exist that are suitable
for use with the antigens or binding agents of the present invention.
The above descriptions are intended to be exemplary only.
In another embodiment, the above polypeptides may be used as markers
for the progression of prostate cancer. In this embodiment, assays
as described above for the diagnosis of prostate cancer may be performed
over time, and the change in the level of reactive polypeptide(s)
evaluated. For example, the assays may be performed every 24 72
hours for a period of 6 months to 1 year, and thereafter performed
as needed. In general, prostate cancer is progressing in those patients
in whom the level of polypeptide detected by the binding agent increases
over time. In contrast, the cancer is not progressing when the level
of reactive polypeptide either remains constant or decreases with
time.
As noted above, prostate cancer may also, or alternatively, be
detected based on the level of mRNA encoding a prostate tumor protein
in a biological sample. For example, at least two oligonucleotide
primers may be employed in a polymerase chain reaction (PCR) based
assay to amplify a portion of a prostate tumor protein cDNA derived
from a biological sample, wherein at least one of the oligonucleotide
primers is specific for (i.e., hybridizes to) a polynucleotide encoding
the prostate tumor protein. The amplified cDNA is then separated
and detected using techniques well known in the art, such as gel
electrophoresis. Similarly, oligonucleotide probes that specifically
hybridize to a polynucleotide encoding a prostate tumor protein
may be used in a hybridization assay to detect the presence of polynucleotide
encoding the antigen in a biological sample.
To permit hybridization under assay conditions, oligonucleotide
primers and probes should comprise an oligonucleotide sequence that
has at least about 60%, preferably at least about 75% and more preferably
at least about 90%, identity to a portion of a polynucleotide encoding
a prostate tumor protein that is at least 10 nucleotides, and preferably
at least 20 nucleotides, in length. Oligonucleotide primers and/or
probes which may be usefully employed in the diagnostic methods
described herein preferably are at least 10 40 nucleotides in length.
In a preferred embodiment, the oligonucleotide primers comprise
at least 10 contiguous nucleotides, more preferably at least 15
contiguous nucleotides, of a DNA molecule recited herein. Techniques
for both PCR based assays and hybridization assays are well known
in the art (see, for example, Mullis et al., Cold Spring Harbor
Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, Stockton
Press, N.Y., 1989).
One preferred assay employs RT-PCR, in which PCR is applied in
conjunction with reverse transcription. Typically, RNA is extracted
from a sample tissue and is reverse transcribed to produce cDNA
molecules. PCR amplification using at least one specific primer
generates a cDNA molecule, which may be separated and visualized
using, for example, gel electrophoresis. Amplification may be performed
on samples obtained from biological samples taken from a test patient
and an individual who is not afflicted with prostate cancer. The
amplification reaction may be performed on several dilutions of
cDNA spanning two orders of magnitude. A two-fold or greater increase
in expression in several dilutions of the test patient sample as
compared to the same dilutions of the non-cancerous sample is typically
considered positive.
Certain in vivo diagnostic assays may be performed directly on
a tumor. One such assay involves contacting tumor cells with a binding
agent. The bound binding agent may then be detected directly or
indirectly via a reporter group. Such binding agents may also be
used in histological applications. Alternatively, polynucleotide
probes may be used within such applications.
As noted above, to improve sensitivity, multiple prostate tumor
protein markers may be assayed within a given sample. It will be
apparent that binding agents specific for different antigens provided
herein may be combined within a single assay. Further, multiple
primers or probes may be used concurrently. The selection of antigen
markers may be based on routine experiments to determine combinations
that results in optimal sensitivity. In addition, or alternatively,
assays for antigens provided herein may be combined with assays
for other known tumor antigens.
Diagnostic Kits
The present invention further provides kits for use within any
of the above diagnostic methods. Such kits typically comprise two
or more components necessary for performing a diagnostic assay.
Components may be compounds, reagents, containers and/or equipment.
For example, one container within a kit may contain a monoclonal
antibody or fragment thereof that specifically binds to a prostate
tumor protein. Such antibodies or fragments may be provided attached
to a support material, as described above. One or more additional
containers may enclose elements, such as reagents or buffers, to
be used in the assay. Such kits may also, or alternatively, contain
a detection reagent as described above that contains a reporter
group suitable for direct or indirect detection of antibody binding.
Alternatively, a kit may be designed to detect the level of mRNA
encoding a prostate tumor protein in a biological sample. Such kits
generally comprise at least one oligonucleotide probe or primer,
as described above, that hybridizes to a polynucleotide encoding
a prostate tumor protein. Such an oligonucleotide may be used, for
example, within a PCR or hybridization assay. Additional components
that may be present within such kits include a second oligonucleotide
and/or a diagnostic reagent or container to facilitate the detection
of a polynucleotide encoding a prostate tumor protein.
The following Examples are offered by way of illustration and not
by way of limitation.
EXAMPLES
Example 1
Isolation and Characterization of Prostrate Tumor Polypeptides
This Example describes the isolation of certain prostate tumor
polypeptides from a prostate tumor cDNA library.
A human prostate tumor cDNA expression library was constructed
from prostate tumor poly A.sup.+ RNA using a Superscript Plasmid
System for cDNA Synthesis and Plasmid Cloning kit (BRL Life Technologies,
Gaithersburg, Md. 20897) following the manufacturer's protocol.
Specifically, prostate tumor tissues were homogenized with polytron
(Kinematica, Switzerland) and total RNA was extracted using Trizol
reagent (BRL Life Technologies) as directed by the manufacturer.
The poly A.sup.+ RNA was then purified using a Qiagen oligotex spin
column mRNA purification kit (Qiagen, Santa Clarita, Calif. 91355)
according to the manufacturer's protocol. First-strand cDNA was
synthesized using the NotI/Oligo-dT18 primer. Double-stranded cDNA
was synthesized, ligated with EcoRI/BAXI adaptors (Invitrogen, San
Diego, Calif.) and digested with NotI. Following size fractionation
with Chroma Spin-1000 columns (Clontech, Palo Alto, Calif.), the
cDNA was ligated into the EcoRI/NotI site of pCDNA3.1 (Invitrogen)
and transformed into ElectroMax E. coli DH10B cells (BRL Life Technologies)
by electroporation.
Using the same procedure, a normal human pancreas cDNA expression
library was prepared from a pool of six tissue specimens (Clontech).
The cDNA libraries were characterized by determining the number
of independent colonies, the percentage of clones that carried insert,
the average insert size and by sequence analysis. The prostate tumor
library contained 1.64.times.10.sup.7 independent colonies, with
70% of clones having an insert and the average insert size being
1745 base pairs. The normal pancreas cDNA library contained 3.3.times.10.sup.6
independent colonies, with 69% of clones having inserts and the
average insert size being 1120 base pairs. For both libraries, sequence
analysis showed that the majority of clones had a full length cDNA
sequence and were synthesized from mRNA, with minimal rRNA and mitochondrial
DNA contamination.
cDNA library subtraction was performed using the above prostate
tumor and normal pancreas cDNA libraries, as described by Hara et
al. (Blood, 84:189 199, 1994) with some modifications. Specifically,
a prostate tumor-specific subtracted cDNA library was generated
as follows. Normal pancreas cDNA library (70 .mu.g) was digested
with EcoRI, NotI, and SfuI, followed by a filling-in reaction with
DNA polymerase Klenow fragment. After phenol-chloroform extraction
and ethanol precipitation, the DNA was dissolved in 100 .mu.l of
H.sub.2O, heat-denatured and mixed with 100 .mu.l (100 .mu.g) of
Photoprobe biotin (Vector Laboratories, Burlingame, Calif.). As
recommended by the manufacturer, the resulting mixture was irradiated
with a 270 W sunlamp on ice for 20 minutes. Additional Photoprobe
biotin (50 .mu.l) was added and the biotinylation reaction was repeated.
After extraction with butanol five times, the DNA was ethanol-precipitated
and dissolved in 23 .mu.l H.sub.2O to form the driver DNA.
To form the tracer DNA, 10 .mu.g prostate tumor cDNA library was
digested with BamHI and XhoI, phenol chloroform extracted and passed
through Chroma spin-400 columns (Clontech). Following ethanol precipitation,
the tracer DNA was dissolved in 5 .mu.l H.sub.2O. Tracer DNA was
mixed with 15 .mu.l driver DNA and 20 .mu.l of 2.times.hybridization
buffer (1.5 M NaCl/10 mM EDTA/50 mM HEPES pH 7.5/0.2% sodium dodecyl
sulfate), overlaid with mineral oil, and heat-denatured completely.
The sample was immediately transferred into a 68.degree. C. water
bath and incubated for 20 hours (long hybridization [LH]). The reaction
mixture was then subjected to a streptavidin treatment followed
by phenol/chloroform extraction. This process was repeated three
more times. Subtracted DNA was precipitated, dissolved in 12 .mu.l
H.sub.2O, mixed with 8 .mu.l driver DNA and 20 .mu.l of 2.times.hybridization
buffer, and subjected to a hybridization at 68.degree. C. for 2
hours (short hybridization [SH]). After removal of biotinylated
double-stranded DNA, subtracted cDNA was ligated into BamHI/XhoI
site of chloramphenicol resistant pBCSK.sup.+ (Stratagene, La Jolla,
Calif. 92037) and transformed into ElectroMax E. coli DH10B cells
by electroporation to generate a prostate tumor specific subtracted
cDNA library (prostate subtraction 1).
To analyze the subtracted cDNA library, plasmid DNA was prepared
from 100 independent clones, randomly picked from the subtracted
prostate tumor specific library and grouped based on insert size.
Representative cDNA clones were further characterized by DNA sequencing
with a Perkin Elmer/Applied Biosystems Division Automated Sequencer
Model 373A (Foster City, Calif.). Six cDNA clones, hereinafter referred
to as F-13, F-12, F1-16, H1-1, H1-9 and H1-4, were shown to be abundant
in the subtracted prostate-specific cDNA library. The determined
3' and 5' cDNA sequences for F1-12 are provided in SEQ ID NO: 2
and 3, respectively, with determined 3' cDNA sequences for F1-13,
F1-16, H1-1, H1-9 and H1-4 being provided in SEQ ID NO: 1 and 4
7, respectively.
The cDNA sequences for the isolated clones were compared to known
sequences in the gene bank using the EMBL and GenBank databases
(release 96). Four of the prostate tumor cDNA clones, F1-13, F1-16,
H1-1, and H1-4, were determined to encode the following previously
identified proteins: prostate specific antigen (PSA), human glandular
kallikrein, human tumor expression enhanced gene, and mitochondria
cytochrome C oxidase subunit II. H1-9 was found to be identical
to a previously identified human autonomously replicating sequence.
No significant homologies to the cDNA sequence for F1-12 were found.
Subsequent studies led to the isolation of a full-length cDNA sequence
for F1-12. This sequence is provided in SEQ ID NO: 107, with the
corresponding predicted amino acid sequence being provided in SEQ
ID NO: 108.
To clone less abundant prostate tumor specific genes, cDNA library
subtraction was performed by subtracting the prostate tumor cDNA
library described above with the normal pancreas cDNA library and
with the three most abundant genes in the previously subtracted
prostate tumor specific cDNA library: human glandular kallikrein,
prostate specific antigen (PSA), and mitochondria cytochrome C oxidase
subunit II. Specifically, 1 .mu.g each of human glandular kallikrein,
PSA and mitochondria cytochrome C oxidase subunit II cDNAs in pCDNA3.1
were added to the driver DNA and subtraction was performed as described
above to provide a second subtracted cDNA library hereinafter referred
to as the "subtracted prostate tumor specific cDNA library
with spike".
Twenty-two cDNA clones were isolated from the subtracted prostate
tumor specific cDNA library with spike. The determined 3' and 5'
cDNA sequences for the clones referred to as J1-17, L1-12, N1-1862,
J1-13, J1-19, J1-25, J1-24, K1-58, K1-63, L1-4 and L1-14 are provided
in SEQ ID NOS: 8 9, 10 11, 12 13, 14 15, 16 17, 18 19, 20 21, 22
23, 24 25, 26 27 and 28 29, respectively. The determined 3' cDNA
sequences for the clones referred to as J1-12, J1-16, J1-21, K1-48,
K1-55, L1-2, L1-6, N1-1858, N1-1860, N1-1861, N1-1864 are provided
in SEQ ID NOS: 30 40, respectively. Comparison of these sequences
with those in the gene bank as described above, revealed no significant
homologies to three of the five most abundant DNA species, (J1-17,
L1-12 and N1-1862; SEQ ID NOS: 8 9, 10 11 and 12 13, respectively).
Of the remaining two most abundant species, one (J1-12; SEQ ID NO:30)
was found to be identical to the previously identified human pulmonary
surfactant-associated protein, and the other (K1-48; SEQ ID NO:33)
was determined to have some homology to R. norvegicus mRNA for 2-arylpropionyl-CoA
epimerase. Of the 17 less abundant cDNA clones isolated from the
subtracted prostate tumor specific cDNA library with spike, four
(J1-16, K1-55, L1-6 and N1-1864; SEQ ID NOS:31, 34, 36 and 40, respectively)
were found to be identical to previously identified sequences, two
(J1-21 and N1-1860; SEQ ID NOS: 32 and 38, respectively) were found
to show some homology to non-human sequences, and two (L1-2 and
N1-1861; SEQ ID NOS: 35 and 39, respectively) were found to show
some homology to known human sequences. No significant homologies
were found to the polypeptides J1-13, J1-19, J1-24, J1-25, K1-58,
K1-63, L1-4, L1-14 (SEQ ID NOS: 14 15, 16 17, 20 21, 18 19, 22 23,
24 25, 26 27, 28 29, respectively).
Subsequent studies led to the isolation of full length cDNA sequences
for J1-17, L1-12 and N1-1862 (SEQ ID NOS: 109 111, respectively).
The corresponding predicted amino acid sequences are provided in
SEQ ID NOS: 112 114. L1-12 is also referred to as P501 S.
In a further experiment, four additional clones were identified
by subtracting a prostate tumor cDNA library with normal prostate
cDNA prepared from a pool of three normal prostate poly A+ RNA (prostate
subtraction 2). The determined cDNA sequences for these clones,
hereinafter referred to as U1-3064, U1 3065, V1-3692 and 1A-3905,
are provided in SEQ ID NO: 69 72, respectively. Comparison of the
determined sequences with those in the gene bank revealed no significant
homologies to U1-3065.
A second subtraction with spike (prostate subtraction spike 2)
was performed by subtracting a prostate tumor specific cDNA library
with spike with normal pancreas cDNA library and further spiked
with PSA, J1-17, pulmonary surfactant-associated protein, mitochondrial
DNA, cytochrome c oxidase subunit II, N1-1862, autonomously replicating
sequence, L1-12 and tumor expression enhanced gene. Four additional
clones, hereinafter referred to as V1-3686, R1-2330, 1B-3976 and
V1-3679, were isolated. The determined cDNA sequences for these
clones are provided in SEQ ID NO:73 76, respectively. Comparison
of these sequences with those in the gene bank revealed no significant
homologies to V1-3686 and R1-2330.
Further analysis of the three prostate subtractions described above
(prostate subtraction 2, subtracted prostate tumor specific cDNA
library with spike, and prostate subtraction spike 2) resulted in
the identification of sixteen additional clones, referred to as
1G-4736, 1G-4738, 1G-4741, 1G-4744, 1G |