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Cancer Patent Abstract
A gene (designated 161P2F10B) and its encoded protein are described
wherein 161P2F10B exhibits tissue specific expression in normal
adult tissue, it is aberrantly expressed in the cancers listed in
Table I. Consequently, 161P2F10B provides a diagnostic, prognostic,
prophylactic and/or therapeutic target for cancer. The 161P2F10B
gene or fragment thereof, or its encoded protein or a fragment thereof,
can be used to elicit a humoral or cellular immune response.
Cancer Patent Claims
The invention claimed is:
1. A purified polypeptide comprising the amino acid sequence of
SEQ ID NO: 745.
2. The polypeptide of claim 1, further comprising a fusion protein
partner.
3. A composition comprising a polypeptide, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO: 745.
4. The composition of claim 3, further comprising a pharmaceutically
acceptable excipient.
5. The composition of claim 4, further comprising an adjuvant.
6. The composition of claim 3, wherein the polypeptide is present
in a concentration from at least about 2% to as much as 20% of the
composition by weight.
Cancer Patent Description
SUBMISSION ON COMPACT DISC
The content of the following submission on compact discs is incorporated
herein by reference in its entirety: A compact disc copy of the
Sequence Listing (CRF) (file name: 511582006203, date recorded:
Feb. 23, 2007, size: 169,984 bytes); a duplicate compact disc copy
of the Sequence Listing (COPY 1) (file name: 511582006203, date
recorded: Feb. 23, 2007, size: 169,984 bytes); and a duplicate compact
disc copy of the Sequence Listing (COPY 2) (file name: 511582006203,
date recorded: Feb. 23, 2007, size: 169,984 bytes).
FIELD OF THE INVENTION
The invention described herein relates to a gene and its encoded
protein, termed 161P2F10B, expressed in certain cancers, and to
diagnostic and therapeutic methods and compositions useful in the
management of cancers that express 161P2F10B.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of human death next to coronary
disease. Worldwide, millions of people die from cancer every year.
In the United States alone, as reported by the American Cancer Society,
cancer causes the death of well over a half-million people annually,
with over 1.2 million new cases diagnosed per year. While deaths
from heart disease have been declining significantly, those resulting
from cancer generally are on the rise. In the early part of the
next century, cancer is predicted to become the leading cause of
death.
Worldwide, several cancers stand out as the leading killers. In
particular, carcinomas of the lung, prostate, breast, colon, pancreas,
and ovary represent the primary causes of cancer death. These and
virtually all other carcinomas share a common lethal feature. With
very few exceptions, metastatic disease from a carcinoma is fatal.
Moreover, even for those cancer patients who initially survive their
primary cancers, common experience has shown that their lives are
dramatically altered. Many cancer patients experience strong anxieties
driven by the awareness of the potential for recurrence or treatment
failure. Many cancer patients experience physical debilitations
following treatment. Furthermore, many cancer patients experience
a recurrence.
Worldwide, prostate cancer is the fourth most prevalent cancer
in men. In North America and Northern Europe, it is by far the most
common cancer in males and is the second leading cause of cancer
death in men. In the United States alone, well over 30,000 men die
annually of this disease--second only to lung cancer. Despite the
magnitude of these figures, there is still no effective treatment
for metastatic prostate cancer. Surgical prostatectomy, radiation
therapy, hormone ablation therapy, surgical castration and chemotherapy
continue to be the main treatment modalities. Unfortunately, these
treatments are ineffective for many and are often associated with
undesirable consequences.
On the diagnostic front, the lack of a prostate tumor marker that
can accurately detect early-stage, localized tumors remains a significant
limitation in the diagnosis and management of this disease. Although
the serum prostate specific antigen (PSA) assay has been a very
useful tool, however its specificity and general utility is widely
regarded as lacking in several important respects.
Progress in identifying additional specific markers for prostate
cancer has been improved by the generation of prostate cancer xenografts
that can recapitulate different stages of the disease in mice. The
LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer
xenografts that have survived passage in severe combined immune
deficient (SCID) mice and have exhibited the capacity to mimic the
transition from androgen dependence to androgen independence (Klein
et al., 1997, Nat. Med. 3:402). More recently identified prostate
cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad.
Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto
et al., Clin Cancer Res 1996 Sep. 2 (9): 1445-51), STEAP (Hubert,
et al., Proc Natl Acad Sci USA. 1999 Dec. 7; 96(25): 14523-8) and
prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl.
Acad. Sci. USA 95: 1735).
While previously identified markers such as PSA, PSM, PCTA and
PSCA have facilitated efforts to diagnose and treat prostate cancer,
there is need for the identification of additional markers and therapeutic
targets for prostate and related cancers in order to further improve
diagnosis and therapy.
Renal cell carcinoma (RCC) accounts for approximately 3 percent
of adult malignancies. Once adenomas reach a diameter of 2 to 3
cm, malignant potential exists. In the adult, the two principal
malignant renal tumors are renal cell adenocarcinoma and transitional
cell carcinoma of the renal pelvis or ureter. The incidence of renal
cell adenocarcinoma is estimated at more than 29,000 cases in the
United States, and more than 11,600 patients died of this disease
in 1998. Transitional cell carcinoma is less frequent, with an incidence
of approximately 500 cases per year in the United States.
Surgery has been the primary therapy for renal cell adenocarcinoma
for many decades. Until recently, metastatic disease has been refractory
to any systemic therapy. With recent developments in systemic therapies,
particularly immunotherapies, metastatic renal cell carcinoma may
be approached aggressively in appropriate patients with a possibility
of durable responses. Nevertheless, there is a remaining need for
effective therapies for these patients.
Of all new cases of cancer in the United States, bladder cancer
represents approximately 5 percent in men (fifth most common neoplasm)
and 3 percent in women (eighth most common neoplasm). The incidence
is increasing slowly, concurrent with an increasing older population.
In 1998, there was an estimated 54,500 cases, including 39,500 in
men and 15,000 in women. The age-adjusted incidence in the United
States is 32 per 100,000 for men and 8 per 100,000 in women. The
historic male/female ratio of 3:1 may be decreasing related to smoking
patterns in women. There were an estimated 11,000 deaths from bladder
cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer
incidence and mortality strongly increase with age and will be an
increasing problem as the population becomes more elderly.
Most bladder cancers recur in the bladder. Bladder cancer is managed
with a combination of transurethral resection of the bladder (TUR)
and intravesical chemotherapy or immunotherapy. The multifocal and
recurrent nature of bladder cancer points out the limitations of
TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical
cystectomy and urinary diversion is the most effective means to
eliminate the cancer but carry an undeniable impact on urinary and
sexual function. There continues to be a significant need for treatment
modalities that are beneficial for bladder cancer patients.
An estimated 130,200 cases of colorectal cancer occurred in 2000
in the United States, including 93,800 cases of colon cancer and
36,400 of rectal cancer. Colorectal cancers are the third most common
cancers in men and women. Incidence rates declined significantly
during 1992-1996 (-2.1% per year). Research suggests that these
declines have been due to increased screening and polyp removal,
preventing progression of polyps to invasive cancers. There were
an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from
rectal cancer) in 2000, accounting for about 11% of all U.S. cancer
deaths.
At present, surgery is the most common form of therapy for colorectal
cancer, and for cancers that have not spread, it is frequently curative.
Chemotherapy, or chemotherapy plus radiation, is given before or
after surgery to most patients whose cancer has deeply perforated
the bowel wall or has spread to the lymph nodes. A permanent colostomy
(creation of an abdominal opening for elimination of body wastes)
is occasionally needed for colon cancer and is infrequently required
for rectal cancer. There continues to be a need for effective diagnostic
and treatment modalities for colorectal cancer.
There were an estimated 164,100 new cases of lung and bronchial
cancer in 2000, accounting for 14% of all U.S. cancer diagnoses.
The incidence rate of lung and bronchial cancer is declining significantly
in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996.
In the 1990s, the rate of increase among women began to slow. In
1996, the incidence rate in women was 42.3 per 100,000.
Lung and bronchial cancer caused an estimated 156,900 deaths in
2000, accounting for 28% of all cancer deaths. During 1992-1996,
mortality from lung cancer declined significantly among men (-1.7%
per year) while rates for women were still significantly increasing
(0.9% per year). Since 1987, more women have died each year of lung
cancer than breast cancer, which, for over 40 years, was the major
cause of cancer death in women. Decreasing lung cancer incidence
and mortality rates most likely resulted from decreased smoking
rates over the previous 30 years; however, decreasing smoking patterns
among women lag behind those of men. Of concern, although the declines
in adult tobacco use have slowed, tobacco use in youth is increasing
again.
Treatment options for lung and bronchial cancer are determined
by the type and stage of the cancer and include surgery, radiation
therapy, and chemotherapy. For many localized cancers, surgery is
usually the treatment of choice. Because the disease has usually
spread by the time it is discovered, radiation therapy and chemotherapy
are often needed in combination with surgery. Chemotherapy alone
or combined with radiation is the treatment of choice for small
cell lung cancer; on this regimen, a large percentage of patients
experience remission, which in some cases is long lasting. There
is however, an ongoing need for effective treatment and diagnostic
approaches for lung and bronchial cancers.
An estimated 182,800 new invasive cases of breast cancer were expected
to occur among women in the United States during 2000. Additionally,
about 1,400 new cases of breast cancer were expected to be diagnosed
in men in 2000. After increasing about 4% per year in the 1980s,
breast cancer incidence rates in women have leveled off in the 1990s
to about 110.6 cases per 100,000.
In the U.S. alone, there were an estimated 41,200 deaths (40,800
women, 400 men) in 2000 due to breast cancer. Breast cancer ranks
second among cancer deaths in women. According to the most recent
data, mortality rates declined significantly during 1992-1996 with
the largest decreases in younger women, both white and black. These
decreases were probably the result of earlier detection and improved
treatment.
Taking into account the medical circumstances and the patient's
preferences, treatment of breast cancer may involve lumpectomy (local
removal of the tumor) and removal of the lymph nodes under the arm;
mastectomy (surgical removal of the breast) and removal of the lymph
nodes under the arm; radiation therapy; chemotherapy; or hormone
therapy. Often, two or more methods are used in combination. Numerous
studies have shown that, for early stage disease, long-term survival
rates after lumpectomy plus radiotherapy are similar to survival
rates after modified radical mastectomy. Significant advances in
reconstruction techniques provide several options for breast reconstruction
after mastectomy. Recently, such reconstruction has been done at
the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate
amounts of surrounding normal breast tissue may prevent the local
recurrence of the DCIS. Radiation to the breast and/or tamoxifen
may reduce the chance of DCIS occurring in the remaining breast
tissue. This is important because DCIS, if left untreated, may develop
into invasive breast cancer. Nevertheless, there are serious side
effects or sequelae to these treatments. There is, therefore, a
need for efficacious breast cancer treatments.
There were an estimated 23,100 new cases of ovarian cancer in the
United States in 2000. It accounts for 4% of all cancers among women
and ranks second among gynecologic cancers. During 1992-1996, ovarian
cancer incidence rates were significantly declining. Consequent
to ovarian cancer, there were an estimated 14,000 deaths in 2000.
Ovarian cancer causes more deaths than any other cancer of the female
reproductive system.
Surgery, radiation therapy, and chemotherapy are treatment options
for ovarian cancer. Surgery usually includes the removal of one
or both ovaries, the fallopian tubes (salpingo-oophorectomy), and
the uterus (hysterectomy). In some very early tumors, only the involved
ovary will be removed, especially in young women who wish to have
children. In advanced disease, an attempt is made to remove all
intra-abdominal disease to enhance the effect of chemotherapy. There
continues to be an important need for effective treatment options
for ovarian cancer.
There were an estimated 28,300 new cases of pancreatic cancer in
the United States in 2000. Over the past 20 years, rates of pancreatic
cancer have declined in men. Rates among women have remained approximately
constant but may be beginning to decline. Pancreatic cancer caused
an estimated 28,200 deaths in 2000 in the United States. Over the
past 20 years, there has been a slight but significant decrease
in mortality rates among men (about -0.9% per year) while rates
have increased slightly among women.
Surgery, radiation therapy, and chemotherapy are treatment options
for pancreatic cancer. These treatment options can extend survival
and/or relieve symptoms in many patients but are not likely to produce
a cure for most. There is a significant need for additional therapeutic
and diagnostic options for pancreatic cancer.
As will be discussed in detail below, the gene and corresponding
protein referred to as 161P2F10B is identical to ENPP3 phosphodiesterase
(also called CD203c or PD-1beta). ENPP3 is an ecto-enzyme belonging
to a family of ectonucleotide phosphodiesterases and pyrophosphatases.
ENPP3 is a phosphodiesterase I ecto-enzyme. It is expressed in normal
prostate and uterus, as well as on basophils and mast cells. Expression
on the hematopoietic cells is upregulated in presence of allergen
or by cross-linking with IgE (Buring et al., 1999, Blood 94: 2343).
Members of the ENPP family possess ATPase and ATP pyrophosphatase
activities. They hydrolyze extracellular nucleotides, nucleoside
phosphates, and NAD. They are involved in extracellular nucleotide
metabolism, nucleotide signaling, and recycling of extracellular
nucleotides. They are also involved in cell-cell and cell-matrix
interactions. ENPP enzymes differ in their substrate specificity
and tissue distribution. ENPP enzymes also play a role in recycling
extracellular nucleotides. It has been demonstrated that ENNP1 allows
activated T-cells to use NAD+ from dying cells as a source of adenosine.
ENPP3 expressed in the intestine may also be involved in the hydrolysis
of nucleotides derived from food (Byrd et al 1985, Scott et al.
1997).
SUMMARY OF THE INVENTION
The present invention relates to a gene, designated 161P2F10B,
that has now been found to be over-expressed in the cancer(s) listed
in Table I. Northern blot expression analysis of 161P2F10B gene
expression in normal tissues shows a restricted expression pattern
in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2,
and FIG. 3) sequences of 161P2F10B are provided. The tissue-related
profile of 161P2F10B in normal adult tissues, combined with the
over-expression observed in the tumors listed in Table I, shows
that 161P2F10B is aberrantly over-expressed in at least some cancers,
and thus serves as a useful diagnostic, prophylactic, prognostic,
and/or therapeutic target for cancers of the tissue(s) such as those
listed in Table I.
The invention provides polynucleotides corresponding or complementary
to all or part of the 161P2F10B genes, mRNAs, and/or coding sequences,
preferably in isolated form, including polynucleotides encoding
161P2F10B-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more
than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55,
60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino
acids of a 161P2F10B-related protein, as well as the peptides/proteins
themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides
or oligonucleotides complementary or having at least a 90% homology
to the 161P2F10B genes or mRNA sequences or parts thereof, and polynucleotides
or oligonucleotides that hybridize to the 161P2F10B genes, mRNAs,
or to 161P2F10B-encoding polynucleotides. Also provided are means
for isolating cDNAs and the genes encoding 161P2F10B. Recombinant
DNA molecules containing 161P2F10B polynucleotides, cells transformed
or transduced with such molecules, and host-vector systems for the
expression of 161P2F10B gene products are also provided. The invention
further provides antibodies that bind to 161P2F10B proteins and
polypeptide fragments thereof, including polyclonal and monoclonal
antibodies, murine and other mammalian antibodies, chimeric antibodies,
humanized and fully human antibodies, and antibodies labeled with
a detectable marker or therapeutic agent. In certain embodiments
there is a proviso that the entire nucleic acid sequence of FIG.
2 is not encoded and/or the entire amino acid sequence of FIG. 2
is not prepared. In certain embodiments, the entire nucleic acid
sequence of FIG. 2 is encoded and/or the entire amino acid sequence
of FIG. 2 is prepared, either of which are in respective human unit
dose forms.
The invention further provides methods for detecting the presence
and status of 161P2F10B polynucleotides and proteins in various
biological samples, as well as methods for identifying cells that
express 161P2F10B. A typical embodiment of this invention provides
methods for monitoring 161P2F10B gene products in a tissue or hematology
sample having or suspected of having some form of growth dysregulation
such as cancer.
The invention further provides various immunogenic or therapeutic
compositions and strategies for treating cancers that express 161P2F10B
such as cancers of tissues listed in Table I, including therapies
aimed at inhibiting the transcription, translation, processing or
function of 161P2F10B as well as cancer vaccines. In one aspect,
the invention provides compositions, and methods comprising them,
for treating a cancer that expresses 161P2F10B in a human subject
wherein the composition comprises a carrier suitable for human use
and a human unit dose of one or more than one agent that inhibits
the production or function of 161P2F10B. Preferably, the carrier
is a uniquely human carrier. In another aspect of the invention,
the agent is a moiety that is immunoreactive with 161P2F10B protein.
Non-limiting examples of such moieties include, but are not limited
to, antibodies (such as single chain, monoclonal, polyclonal, humanized,
chimeric, or human antibodies), functional equivalents thereof (whether
naturally occurring or synthetic), and combinations thereof. The
antibodies can be conjugated to a diagnostic or therapeutic moiety.
In another aspect, the agent is a small molecule as defined herein.
In another aspect, the agent comprises one or more than one peptide
which comprises a cytotoxic T lymphocyte (CTL) epitope that binds
an HLA class I molecule in a human to elicit a CTL response to 161P2F10B
and/or one or more than one peptide which comprises a helper T lymphocyte
(HTL) epitope which binds an HLA class II molecule in a human to
elicit an HTL response. The peptides of the invention may be on
the same or on one or more separate polypeptide molecules. In a
further aspect of the invention, the agent comprises one or more
than one nucleic acid molecule that expresses one or more than one
of the CTL or HTL response stimulating peptides as described above.
In yet another aspect of the invention, the one or more than one
nucleic acid molecule may express a moiety that is immunologically
reactive with 161P2F10B as described above. The one or more than
one nucleic acid molecule may also be, or encodes, a molecule that
inhibits production of 161P2F10B. Non-limiting examples of such
molecules include, but are not limited to, those complementary to
a nucleotide sequence essential for production of 161P2F10B (e.g.
antisense sequences or molecules that form a triple helix with a
nucleotide double helix essential for 161P2F10B production) or a
ribozyme effective to lyse 161P2F10B mRNA.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. The 161P2F10B SSH sequence.
FIG. 2. The cDNA and amino acid sequence of 161P2F10B (FIG. 2A),
and the nucleic acid and amino acid sequence of 161P2F10B variant
1 (FIG. 2B). The codon for the start methionine is underlined. The
open reading frame for each extends from nucleic acid 44 to 2671
including the stop codon
FIG. 3. Amino acid sequence of 161P2F10B (FIG. 3A) and the amino
acid sequence of 161P2F10B variant 1 (FIG. 3B). Each protein has
875 amino acids.
FIG. 4. FIG. 4A provides the amino acid alignment of 161P2F10B
with ENPP3; FIG. 4B provides an amino acid alignment of 161P2F10B
with 161P2F10B variant 1; FIG. 4C provides the Alignment of 161P2F10B
and SNP variant 2 carrying a T to P mutation at position 874. The
consensus seciuence is the same as SEQ ID NO: 750, from residues
1 to 383.
FIG. 5. Hydrophilicity amino acid profile of 161P2F10B determined
by computer algorithm sequence analysis using the method of Hopp
and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci.
U.S.A. 78:3824-3828) accessed on the Protscale website through the
ExPasy molecular biology server.
FIG. 6. Hydropathicity amino acid profile of 161P2F10B determined
by computer algorithm sequence analysis using the method of Kyte
and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132)
accessed on the ProtScale website through the ExPasy molecular biology
server.
FIG. 7. Percent accessible residues amino acid profile of 161P2F10B
determined by computer algorithm sequence analysis using the method
of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale
website through the ExPasy molecular biology server.
FIG. 8. Average flexibility amino acid profile of 161P2F10B determined
by computer algorithm sequence analysis using the method of Bhaskaran
and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J.
Pept. Protein Res. 32:242-255) accessed on the ProtScale website
through the ExPasy molecular biology server.
FIG. 9. Beta-turn amino acid profile of 161P2F10B determined by
computer algorithm sequence analysis using the method of Deleage
and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294)
accessed on the ProtScale website through the ExPasy molecular biology
server.
FIG. 10. Expression of 161P2F10B by RT-PCR. First strand cDNA was
prepared from vital pool 1 (VP1: liver, lung and kidney), vital
pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool
(LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate
cancer pool, kidney cancer pool, colon cancer pool, lung cancer
pool, ovary cancer pool, breast cancer pool, metastasis cancer pool,
pancreas cancer pool, and prostate cancer metastasis to lymph node
from two different patients. Normalization was performed by PCR
using primers to actin and GAPDH. Semi-quantitative PCR, using primers
to 161P2F10B, was performed at 26 and 30 cycles of amplification.
Strong expression of 161P2F10B was observed in kidney cancer pool.
Expression was also detected in VP1, prostate cancer xenograft pool,
prostate cancer pool and colon cancer pool. Low expression was observed
in VP2, lung cancer pool, ovary cancer pool, breast cancer pool,
metastasis pool, pancreas cancer pool, and in the two different
prostate cancer metastasis to lymph node.
FIG. 11. Expression of 161P2F10B in normal human tissues. Two multiple
tissue Northern blots, with 2 mg of mRNA/lane, were probed with
the 161P2F10B sequence. Size standards in kilobases (kb) are indicated
on the side. The results show expression of two 161P2F10B transcripts
comigrating at approximately 4.4 kb, in kidney, prostate and colon,
and to lower levels, in thymus.
FIG. 12. Expression of 161P2F10B in kidney cancer xenografts. RNA
was extracted from normal kidney (N), prostate cancer xenografts,
LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI, and two kidney cancer
xenografts (Ki Xeno-1 and Ki Xeno-2). A Northern blot with 10 mg
of total RNA/lane was probed with the 161P2F10B sequence. Size standards
in kilobases (kb) are indicated on the side. The results showed
expression of 161P2F10B in both kidney xenografts, LAPC-4AI, LAPC-9AI,
but not in normal kidney or the tested cell lines.
FIG. 13. Expression of 161P2F10B in patient kidney cancer specimens
and in normal tissues. RNA was extracted from a pool of three kidney
cancers, as well as from normal prostate (NP), normal bladder (NB),
normal kidney (NK), and normal colon (NC). A Northern blot with
10 mg of total RNA/lane was probed with the 161P2F10B sequence.
Size standards in kilobases (kb) are indicated on the side. The
results showed expression of 161P2F10B in the kidney cancer pool
but not in the normal tissues tested.
FIG. 14. Expression of 161P2F10B in kidney cancer patient specimens.
RNA was extracted from kidney cancer cell lines (CL), normal kidney
(N), kidney tumors (T), and matched normal adjacent tissue (NAT)
isolated from kidney cancer patients. Northern blots with 10 mg
of total RNA/lane were probed with the 161P2F10B sequence. Size
standards in kilobases (kb) are indicated on the side. The results
showed expression of 161P2F10B in all four clear cell carcinoma
kidney tumors, but not in papillary carcinoma nor in normal kidney
tissues.
FIG. 15. Expression of 161P2F10B in kidney cancer metastasis specimens
and in normal tissues. RNA was extracted from kidney cancer metastasis
to lung, kidney cancer metastasis to lymph node, normal bladder
(NB), normal kidney (NK), and normal lung (NL), normal breast (NBr),
normal ovary (NO), and normal pancreas (NPa). Northern blots with
10 mg of total RNA/lane were probed with the 161P2F10B sequence.
Size standards in kilobases (kb) are indicated on the side. The
results showed expression of 161P2F10B in the two kidney cancer
metastasis tested. Weak expression was detected in normal kidney
and normal breast but not in other normal tissues. The ethidium-bromide
staining of the gel showed equivalent loading of the RNA samples.
FIG. 16: Detection of 161P2F10B protein by immunohistochemistry
in kidney cancer patient specimens. Renal clear cell carcinoma tissue
and its matched normal adjacent tissue as well as its metastatic
cancer to lymph node were obtained from a kidney cancer patient.
Frozen tissues were cut into 4 micron sections and fixed in acetone
for 10 minutes. The sections were then incubated with PE-labeled
mouse monoclonal anti-ENPP3 antibody (Coulter-Immunotech, Marseilles,
France) for 3 hours (FIG. 16 panels A-F), or isotype control antibody
(FIG. 16 panels G-I). The slides were washed three times in buffer,
and either analyzed by fluorescence microscopy (FIG. 16 panels A,
B and C), or further incubated with DAKO EnVision+.TM. peroxidase-conjugated
goat anti-mouse secondary antibody (DAKO Corporation, Carpenteria,
Calif.) for 1 hour (FIG. 16 panels D, E, and F). The sections were
then washed in buffer, developed using the DAB kit (SIGMA Chemicals),
counterstained using hematoxylin, and analyzed by bright field microscopy
(FIG. 16 panels D, E and F). The results showed strong expression
of 161P2F10B in the renal carcinoma patient tissue (FIG. 16 panels
A and D) and the kidney cancer metastasis to lymph node tissue (FIG.
16 panels C and F), but weakly in normal kidney (FIG. 16 B and E).
The expression was detected mostly around the cell membrane indicating
that 161P2F10B is membrane associated in kidney cancer tissues.
The weak expression detected in normal kidney was localized to the
kidney tubules. The sections stained with the isotype control antibody
were negative showing the specificity of the anti-ENPP3 antibody
(FIG. 16 panels G-I).
FIG. 17: Expression of 161P2F10B protein on the cell surface of
renal cell carcinoma xenografts. Renal cell carcinoma xenograft
tissues (FIG. 17 A) and renal cell carcinoma metastasis to lymph
node xenograft tissues (FIG. 17 B) were harvested from animals and
dispersed into single cell suspension. The cells were stained using
the commercially available antibody 97A6 specific for ENPP3 protein
(also called anti-CD203c) (Immunotech, Marseilles, France). They
were then washed in PBS and analyzed by flow cytometry. The results
showed strong expression of 161P2F10B in both renal cell carcinoma
xenograft (FIG. 17 A) as well as renal cancer metastasis xenograft
(FIG. 17 B). These data demonstrate that 161P2F10B is expressed
on the cell surface of the kidney cancer and kidney cancer metastasis
xenograft cells.
FIG. 18. Detection of 161P2F10B protein by immunohistochemistry
in human cancer xenograft tissues. Renal cell carcinoma (FIG. 18
panels A, D, G), renal cell carcinoma metastasis to lymph node (FIG.
18 panels B, E, H), and prostate cancer LAPC-4AI (FIG. 18 panels
C, F, I) xenografts were grown in SCID mice. Xenograft tissues were
harvested, 4 micron thick frozen sections were cut and fixed in
acetone for 10 minutes. The sections were then incubated with PE-labeled
mouse monoclonal anti-ENPP3 antibody (Immunotech, Marseilles, France)
for 3 hours (FIG. 18 panels A-F), or isotype control antibody (FIG.
18 panels G-I). The slides were washed three times in buffer, and
either analyzed by fluorescence microscopy (FIG. 18 panels A-C),
or further incubated with DAKO EnVision+.TM. peroxidase-conjugated
goat anti-mouse secondary antibody (DAKO Corporation, Carpenteria,
Calif.) for 1 hour (FIG. 18 panels D-I). The sections were then
washed in buffer, developed using the DAB kit (SIGMA Chemicals),
counterstained using hematoxylin, and analyzed by bright field microscopy
(FIG. 18 panels C-F). The results showed strong expression of 161P2F10B
in the renal cell carcinoma xenograft tissue (FIG. 18 panels A and
D), in the kidney cancer metastasis to lymph node (FIG. 18 panels
B and E) as well as in the LAPC4AI prostate xenograft (C and F)
but not in the negative isotype control sections (FIG. 18 panels
G, H, I). The expression was detected mostly around the cell membrane
indicating that 161P2F10B is membrane-associated.
FIG. 19. The secondary structure of 161P2F10B, namely the predicted
presence and location of alpha helices, extended strands, and random
coils, is predicted from the primary amino acid sequence using the
HNN--Hierarchical Neural Network method (Guermeur, 1997), accessed
from the ExPasy molecular biology server. The analysis indicates
that 161P2F10B is composed 31.31% alpha helix, 11.31% extended strand,
and 57.37% random coil (FIG. 19A). Shown graphically in FIG. 19
panels B and C are the results of analysis using the TMpred (FIG.
19B) and TMHMM (FIG. 19C) prediction programs depicting the location
of the transmembrane domain.
FIG. 20. Expression of 161P2F10B in Human Patient Cancers by Western
Blot. Cell lysates from kidney cancer tissues (KiCa), kidney cancer
metastasis to lymph node (KiCa Met), as well as normal kidney (NK)
were subjected to Western analysis using an anti-161P2F10B mouse
monoclonal antibody. Briefly, tissues (.about.25 .mu.g total protein)
were solubilized in SDS-PAGE sample buffer and separated on a 10-20%
SDS-PAGE gel and transferred to nitrocellulose. Blots were blocked
in Tris-buffered saline (TBS)+3% non-fat milk and then probed with
purified anti-161P2F10B antibody in TBS+0.15% Tween-20+1% milk.
Blots were then washed and incubated with a 1:4,000 dilution of
anti-mouse IgG-HRP conjugated secondary antibody. Following washing,
anti-161P2F10B immunoreactive bands were developed and visualized
by enhanced chemiluminescence and exposure to autoradiographic film.
The specific anti-161P2F10B immunoreactive bands represent a monomeric
form of the 161P2F10B protein, which runs at approximately 130 kDa.
These results demonstrate that 161P2F10B may be useful as a diagnostic
and therapeutic target for kidney cancers, metastatic cancers and
potentially other human cancers.
FIG. 21. Expression of 161P2F10B in Human Xenograft Tissues by
Western Blot. Cell lysates from kidney cancer xenograft (KiCa Xeno),
kidney cancer metastasis to lymph node xenograft (Met Xeno), as
well as normal kidney (NK) were subjected to Western analysis using
an anti-161P2F10B mouse monoclonal antibody. Briefly, tissues (.about.25
.mu.g total protein) were solubilized in SDS-PAGE sample buffer
and separated on a 10-20% SDS-PAGE gel and transferred to nitrocellulose.
Blots were blocked in Tris-buffered saline (TBS)+3% non-fat milk
and then probed with purified anti-161P2F10B antibody in TBS+0.15%
Tween-20+1% milk. Blots were then washed and incubated with a 1:4,000
dilution of anti-mouse IgG-HRP conjugated secondary antibody. Following
washing, anti-161P2F10B immunoreactive bands were developed and
visualized by enhanced chemiluminescence and exposure to autoradiographic
film. The specific anti-161P2F10B immunoreactive bands represent
a monomeric form of the 161P2F10B protein, which runs at approximately
130 kDa, and a multimer of approximately 260 kDa. These results
demonstrate that the human cancer xenograft mouse models can be
used to study the diagnostic and therapeutic effects of 161P2F10B.
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions II.) 161P2F10B Polynucleotides II.A.) Uses of 161P2F10B
Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities II.A.2.)
Antisense Embodiments II.A.3.) Primers and Primer Pairs II.A.4.)
Isolation of 161P2F10B-Encoding Nucleic Acid Molecules II.A.5.)
Recombinant Nucleic Acid Molecules and Host-Vector Systems III.)
161P2F10B-related Proteins III.A.) Motif-bearing Protein Embodiments
III.B.) Expression of 161P2F10B-related Proteins III.C.) Modifications
of 161P2F10B-related Proteins III.D.) Uses of 161P2F10B-related
Proteins IV.) 161P2F10B Antibodies V.) 161P2F10B Cellular Immune
Responses VI.) 161P2F10B Transgenic Animals VII.) Methods for the
Detection of 161P2F10B VIII.) Methods for Monitoring the Status
of 161P2F10B-related Genes and Their Products IX.) Identification
of Molecules That Interact With 161P2F10B X.) Therapeutic Methods
and Compositions X.A.) Anti-Cancer Vaccines X.B.) 161P2F10B as a
Target for Antibody-Based Therapy X.C.) 161P2F10B as a Target for
Cellular Immune Responses X.C.1. Minigene Vaccines X.C.2. Combinations
of CTL Peptides with Helper Peptides X.C.3. Combinations of CTL
Peptides with T Cell Priming Agents X.C.4. Vaccine Compositions
Comprising DC Pulsed with CTL and/or HTL Peptides X.D.) Adoptive
Immunotherapy X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes XI.) Diagnostic and Prognostic Embodiments
of 161P2F10B. XII.) Inhibition of 161P2F10B Protein Function XII.A.)
Inhibition of 161P2F10B With Intracellular Antibodies XII.B.) Inhibition
of 161P2F10B with Recombinant Proteins XII.C.) Inhibition of 161P2F10B
Transcription or Translation XII.D.) General Considerations for
Therapeutic Strategies XIII.) KITS
I.) Definitions
Unless otherwise defined, all terms of art, notations and other
scientific terms or terminology used herein are intended to have
the meanings commonly understood by those of skill in the art to
which this invention pertains. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not
necessarily be construed to represent a substantial difference over
what is generally understood in the art. Many of the techniques
and procedures described or referenced herein are well understood
and commonly employed using conventional methodology by those skilled
in the art, such as, for example, the widely utilized molecular
cloning methodologies described in Sambrook et al., Molecular Cloning:
A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory
Press, Cold Spring. Harbor, N.Y. As appropriate, procedures involving
the use of commercially available kits and reagents are generally
carried out in accordance with manufacturer defined protocols and/or
parameters unless otherwise noted.
The terms "advanced prostate cancer", "locally advanced
prostate cancer", "advanced disease" and "locally
advanced disease" mean prostate cancers that have extended
through the prostate capsule, and are meant to include stage C disease
under the American Urological Association (AUA) system, stage C1-C2
disease under the Whitmore-Jewett system, and stage T3-T4 and N+
disease under the TNM (tumor, node, metastasis) system. In general,
surgery is not recommended for patients with locally advanced disease,
and these patients have substantially less favorable outcomes compared
to patients having clinically localized (organ-confined) prostate
cancer. Locally advanced disease is clinically identified by palpable
evidence of induration beyond the lateral border of the prostate,
or asymmetry or induration above the prostate base. Locally advanced
prostate cancer is presently diagnosed pathologically following
radical prostatectomy if the tumor invades or penetrates the prostatic
capsule, extends into the surgical margin, or invades the seminal
vesicles.
"Altering the native glycosylation pattern" is intended
for purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 161P2F10B (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence 161P2F10B. In
addition, the phrase includes qualitative changes in the glycosylation
of the native proteins, involving a change in the nature and proportions
of the various carbohydrate moieties present.
The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 161P2F10B-related protein). For example an analog
of the 161P2F10B protein can be specifically bound by an antibody
or T cell that specifically binds to 161P2F10B.
The term "antibody" is used in the broadest sense. Therefore
an "antibody" can be naturally occurring or man-made such
as monoclonal antibodies produced by conventional hybridoma technology.
Anti-161P2F10B antibodies comprise monoclonal and polyclonal antibodies
as well as fragments containing the antigen-binding domain and/or
one or more complementarity determining regions of these antibodies.
An "antibody fragment" is defined as at least a portion
of the variable region of the immunoglobulin molecule that binds
to its target, i.e., the antigen-binding region. In one embodiment
it specifically covers single anti-161P2F10B antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-161P2F10B antibody compositions with polyepitopic specificity.
The term "codon optimized sequences" refers to nucleotide
sequences that have been optimized for a particular host species
by replacing any codons having a usage frequency of less than about
20%. Nucleotide sequences that have been optimized for expression
in a given host species by elimination of spurious polyadenylation
sequences, elimination of exon/intron splicing signals, elimination
of transposon-like repeats and/or optimization of GC content in
addition to codon optimization are referred to herein as an "expression
enhanced sequences."
The term "cytotoxic agent" refers to a substance that
inhibits or prevents the expression activity of cells, function
of cells and/or causes destruction of cells. The term is intended
to include radioactive isotopes chemotherapeutic agents, and toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments and/or
variants thereof. Examples of cytotoxic agents include, but are
not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain,
doxorubicin, daunorubicin, TAXOL, ethidium bromide, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin
dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)
A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin,
mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin,
calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid
and other chemotherapeutic agents, as well as radioisotopes such
as Ar.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, p.sup.32 and radioactive isotopes of Lu.
Antibodies may also be conjugated to an anti-cancer pro-drug activating
enzyme capable of converting the pro-drug to its active form.
The term "homolog" refers to a molecule which exhibits
homology to another molecule, by for example, having sequences of
chemical residues that are the same or similar at corresponding
positions.
"Human Leukocyte Antigen" or "HLA" is a human
class I or class II Major Histocompatibility Complex (MHC) protein
(see, e.g., Stites, et al., IMMUNOLOGY, 8.sup.TH ED., Lange Publishing,
Los Altos, Calif. (1994).
The terms "hybridize", "hybridizing", "hybridizes"
and the like, used in the context of polynucleotides, are meant
to refer to conventional hybridization conditions, preferably such
as hybridization in 50% formamide/6.times.SSC/0.1% SDS/100 .mu.g/ml
ssDNA, in which temperatures for hybridization are above 37 degrees
C. and temperatures for washing in 0.1.times.SSC/0.1% SDS are above
55 degrees C.
The phrases "isolated" or "biologically pure"
refer to material which is substantially or essentially free from
components which normally accompany the material as it is found
in its native state. Thus, isolated peptides in accordance with
the invention preferably do not contain materials normally associated
with the peptides in their in situ environment. For example, a polynucleotide
is said to be "isolated" when it is substantially separated
from contaminant polynucleotides that correspond or are complementary
to genes other than the 161P2F10B gene or that encode polypeptides
other than 161P2F10B gene product or fragments thereof. A skilled
artisan can readily employ nucleic acid isolation procedures to
obtain an isolated 161P2F10B polynucleotide. A protein is said to
be "isolated," for example, when physical, mechanical
or chemical methods are employed to remove the 161P2F10B protein
from cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated 161P2F10B protein. Alternatively,
an isolated protein can be prepared by chemical means.
The term "mammal" refers to any organism classified as
a mammal, including mice, rats, rabbits, dogs, cats, cows, horses
and humans. In one embodiment of the invention, the mammal is a
mouse. In another embodiment of the invention, the mammal is a human.
The terms "metastatic prostate cancer" and "metastatic
disease" mean prostate cancers that have spread to regional
lymph nodes or to distant sites, and are meant to include stage
D disease under the AUA system and stage TxNxM+ under the TNM system.
As is the case with locally advanced prostate cancer, surgery is
generally not indicated for patients with metastatic disease, and
hormonal (androgen ablation) therapy is a preferred treatment modality.
Patients with metastatic prostate cancer eventually develop an androgen-refractory
state within 12 to 18 months of treatment initiation. Approximately
half of these androgen-refractory patients die within 6 months after
developing that status. The most common site for prostate cancer
metastasis is bone. Prostate cancer bone metastases are often osteoblastic
rather than osteolytic (i.e., resulting in net bone formation).
Bone metastases are found most frequently in the spine, followed
by the femur, pelvis, rib cage, skull and humerus. Other common
sites for metastasis include lymph nodes, lung, liver and brain.
Metastatic prostate cancer is typically diagnosed by open or laparoscopic
pelvic lymphadenectomy, whole body radionuclide scans, skeletal
radiography, and/or bone lesion biopsy.
The term "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the antibodies comprising the population are identical except
for possible naturally occurring mutations that are present in minor
amounts.
A "motif", as in biological motif of an 161P2F10B-related
protein, refers to any pattern of amino acids forming part of the
primary sequence of a protein, that is associated with a particular
function (e.g. protein-protein interaction, protein-DNA interaction,
etc) or modification (e.g. that is phosphorylated, glycosylated
or amidated), or localization (e.g. secretory sequence, nuclear
localization sequence, etc.) or a sequence that is correlated with
being immunogenic, either humorally or cellularly. A motif can be
either contiguous or capable of being aligned to certain positions
that are generally correlated with a certain function or property.
In the context of HLA motifs, "motif" refers to the pattern
of residues in a peptide of defined length, usually a peptide of
from about 8 to about 13 amino acids for a class I HLA motif and
from about 6 to about 25 amino acids for a class II HLA motif, which
is recognized by a particular HLA molecule. Peptide motifs for HLA
binding are typically different for each protein encoded by each
human HLA allele and differ in the pattern of the primary and secondary
anchor residues.
A "pharmaceutical excipient" comprises a material such
as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservative, and the like.
"Pharmaceutically acceptable" refers to a non-toxic,
inert, and/or composition that is physiologically compatible with
humans or other mammals.
The term "polynucleotide" means a polymeric form of nucleotides
of at least 10 bases or base pairs in length, either ribonucleotides
or deoxynucleotides or a modified form of either type of nucleotide,
and is meant to include single and double stranded forms of DNA
and/or RNA. In the art, this term if often used interchangeably
with "oligonucleotide". A polynucleotide can comprise
a nucleotide sequence disclosed herein wherein thymidine (T) can
also be uracil (U); this definition pertains to the differences
between the chemical structures of DNA and RNA, in particular the
observation that one of the four major bases in RNA is uracil (U)
instead of thymidine (T).
The term "polypeptide" means a polymer of at least about
4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard
three letter or single letter designations for amino acids are used.
In the art, this term is often used interchangeably with "peptide"
or "protein".
An HLA "primary anchor residue" is an amino acid at a
specific position along a peptide sequence which is understood to
provide a contact point between the immunogenic peptide and the
HLA molecule. One to three, usually two, primary anchor residues
within a peptide of defined length generally defines a "motif"
for an immunogenic peptide. These residues are understood to fit
in close contact with peptide binding groove of an HLA molecule,
with their side chains buried in specific pockets of the binding
groove. In one embodiment, for example, the primary anchor residues
for an HLA class I molecule are located at position 2 (from the
amino terminal position) and at the carboxyl terminal position of
a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with
the invention. In another embodiment, for example, the primary anchor
residues of a peptide that will bind an HLA class II molecule are
spaced relative to each other, rather than to the termini of a peptide,
where the peptide is generally of at least 9 amino acids in length.
The primary anchor positions for each motif and supermotif are set
forth in Table IV. For example, analog peptides can be created by
altering the presence or absence of particular residues in the primary
and/or secondary anchor positions shown in Table IV. Such analogs
are used to modulate the binding affinity and/or population coverage
of a peptide comprising a particular HLA motif or supermotif.
A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
Non-limiting examples of small molecules include compounds that
bind or interact with 161P2F10B, ligands including hormones, neuropeptides,
chemokines, odorants, phospholipids, and functional equivalents
thereof that bind and preferably inhibit 161P2F10B protein function.
Such non-limiting small molecules preferably have a molecular weight
of less than about 10 kDa, more preferably below about 9, about
8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments,
small molecules physically associate with, or bind, 161P2F10B protein;
are not found in naturally occurring metabolic pathways; and/or
are more soluble in aqueous than non-aqueous solutions.
"Stringency" of hybridization reactions is readily determinable
by one of ordinary skill in the art, and generally is an empirical
calculation dependent upon probe length, washing temperature, and
salt concentration. In general, longer probes require higher temperatures
for proper annealing, while shorter probes need lower temperatures.
Hybridization generally depends on the ability of denatured nucleic
acid sequences to reanneal when complementary strands are present
in an environment below their melting temperature. The higher the
degree of desired homology between the probe and hybridizable sequence,
the higher the relative temperature that can be used. As a result,
it follows that higher relative temperatures would tend to make
the reaction conditions more stringent, while lower temperatures
less so. For additional details and explanation of stringency of
hybridization reactions, see Ausubel et al., Current Protocols in
Molecular Biology, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions",
as defined herein, are identified by, but not limited to, those
that: (1) employ low ionic strength and high temperature for washing,
for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1%
sodium dodecyl sulfate at 50.degree. C.; (2) employ during hybridization
a denaturing agent, such as formamide, for example, 50% (v/v) formamide
with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times. Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree.
C. in 0.2.times.SSC (sodium chloride/sodium. citrate) and 50% formamide
at 55.degree. C., followed by a high-stringency wash consisting
of 0.1.times.SSC containing EDTA at 55.degree. C. "Moderately
stringent conditions" are described by, but not limited to,
those in Sambrook et al., Molecular Cloning: A Laboratory Manual,
New York: Cold Spring Harbor Press, 1989, and include the use of
washing solution and hybridization conditions (e.g., temperature,
ionic strength and % SDS) less stringent than those described above.
An example of moderately stringent conditions is overnight incubation
at 37.degree. C. in a solution comprising: 20% formamide, 5.times.SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20
mg/mL denatured sheared salmon sperm DNA, followed by washing the
filters in 1.times.SSC at about 37-50.degree. C. The skilled artisan
will recognize how to adjust the temperature, ionic strength, etc.
as necessary to accommodate factors such as probe length and the
like.
An HLA "supermotif" is a peptide binding specificity
shared by HLA molecules encoded by two or more HLA alleles.
As used herein "to treat" or "therapeutic"
and grammatically related terms, refer to any improvement of any
consequence of disease, such as prolonged survival, less morbidity,
and/or a lessening of side effects which are the byproducts of an
alternative therapeutic modality; full eradication of disease is
not required.
A "transgenic animal" (e.g., a mouse or rat) is an animal
having cells that contain a transgene, which transgene was introduced
into the animal or an ancestor of the animal at a prenatal, e.g.,
an embryonic stage. A "transgene" is a DNA that is integrated
into the genome of a cell from which a transgenic animal develops.
As used herein, an HLA or cellular immune response "vaccine"
is a composition that contains or encodes one or more peptides of
the invention. There are numerous embodiments of such vaccines,
such as a cocktail of one or more individual peptides; one or more
peptides of the invention comprised by a polyepitopic peptide; or
nucleic acids that encode such individual peptides or polypeptides,
e.g., a minigene that encodes a polyepitopic peptide. The "one
or more peptides" can include any whole unit integer from 1-150
or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,
110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides
of the invention. The peptides or polypeptides can optionally be
modified, such as by lipidation, addition of targeting or other
sequences. HLA class I peptides of the invention can be admixed
with, or linked to, HLA class II peptides, to facilitate activation
of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines
can also comprise peptide-pulsed antigen presenting cells, e.g.,
dendritic cells.
The term "variant" refers to a molecule that exhibits
a variation from a described type or norm, such as a protein that
has one or more different amino acid residues in the corresponding
position(s) of a specifically described protein (e.g. the 161P2F10B
protein shown in FIG. 2 or FIG. 3). An analog is an example of a
variant protein.
The 161P2F10B-related proteins of the invention include those specifically
identified herein, as well as allelic variants, conservative substitution
variants, analogs and homologs that can be isolated/generated and
characterized without undue experimentation following the methods
outlined herein or readily available in the art. Fusion proteins
that combine parts of different 161P2F10B proteins or fragments
thereof, as well as fusion proteins of a 161P2F10B protein and a
heterologous polypeptide are also included. Such 161P2F10B proteins
are collectively referred to as the 161P2F10B-related proteins,
the proteins of the invention, or 161P2F10B. The term "161P2F10B-related
protein" refers to a polypeptide fragment or an 161P2F10B protein
sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at
least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or
more than 100 amino acids.
II.) 161P2F10B Polynucleotides
One aspect of the invention provides polynucleotides corresponding
or complementary to all or part of an 161P2F10B gene, mRNA, and/or
coding sequence, preferably in isolated form, including polynucleotides
encoding an 161P2F10B-related protein and fragments thereof, DNA,
RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides
complementary to an 161P2F10B gene or mRNA sequence or a part thereof,
and polynucleotides or oligonucleotides that hybridize to an 161P2F10B
gene, mRNA, or to an 161P2F10B encoding polynucleotide (collectively,
"161P2F10B polynucleotides"). In all instances when referred
to in this section, T can also be U in FIG. 2.
Embodiments of a 161P2F10B polynucleotide include: a 161P2F10B
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of 161P2F10B as shown in FIG. 2 wherein T is U; at least
10 contiguous nucleotides of a polynucleotide having the sequence
as shown in FIG. 2; or, at least 10 contiguous nucleotides of a
polynucleotide having the sequence as shown in FIG. 2 where T is
U. For example, embodiments of 161P2F10B nucleotides comprise, without
limitation: (a) a polynucleotide comprising or consisting of the
sequence as shown in FIG. 2, wherein T can also be U; (b) a polynucleotide
comprising or consisting of the sequence as shown in FIG. 2, from
nucleotide residue number 44 through nucleotide residue number 2671,
wherein T can also be U; (c) a polynucleotide that encodes an 161P2F10B-related
protein that is at least 90% homologous to the entire amino acid
sequence shown in FIG. 2; (d) a polynucleotide that encodes an 161P2F10B-related
protein that is at least 90% identical to the entire amino acid
sequence shown in FIG. 2; (e) a polynucleotide that encodes at least
one peptide set forth in Tables V-XVIII; (f) a polynucleotide that
encodes a peptide region of at least 5 amino acids of FIG. 3 in
any whole number increment up to 875 that includes an amino acid
position having a value greater than 0.5 in the Hydrophilicity profile
of FIG. 5; (g) a polynucleotide that encodes a peptide region of
at least 5 amino acids of FIG. 3 in any whole number increment up
to 875 that includes an amino acid position having a value less
than 0.5 in the Hydropathicity profile of FIG. 6; (h) a polynucleotide
that encodes a peptide region of at least 5 amino acids of FIG.
3 in any whole number increment up to 875 that includes an amino
acid position having a value greater than 0.5 in the Percent Accessible
Residues profile of FIG. 7; (i) a polynucleotide that encodes a
peptide region of at least 5 amino acids of FIG. 3 in any whole
number increment up to 875 that includes an amino acid position
having a value greater than 0.5 in the Average Flexibility profile
on FIG. 8; (j) a polynucleotide that encodes a peptide region of
at least 5 amino acids of FIG. 3 in any whole number increment up
to 875 that includes an amino acid position having a value greater
than 0.5 in the Beta-turn profile of FIG. 9; (k) a polynucleotide
that is fully complementary to a polynucleotide of any one of (a)-(j);
(l) a polynucleotide that selectively hybridizes under stringent
conditions to a polynucleotide of (a)-(k); (m) a peptide that is
encoded by any of (a)-(j); and, (n) a polynucleotide of any of (a)-(l)
or peptide of (m) together with a pharmaceutical excipient and/or
in a human unit dose form.
As used herein, a range is understood to specifically disclose
all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 161P2F10B
polynucleotides that encode specific portions of the 161P2F10B mRNA
sequence (and those which are complementary to such sequences) such
as those that encode the protein and fragments thereof, for example
of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,
220, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 900, 825,
850, or 875 contiguous amino acids.
For example, representative embodiments of the invention disclosed
herein include: polynucleotides and their encoded peptides themselves
encoding about amino acid 1 to about amino acid 10 of the 161P2F10B
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 10 to about amino acid 20 of the 161P2F10B protein shown
in FIG. 2, or FIG. 3, polynucleotides encoding about amino acid
20 to about amino acid 30 of the 161P2F10B protein shown in FIG.
2 or FIG. 3, polynucleotides encoding about amino acid 30 to about
amino acid 40 of the 161P2F10B protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 40 to about amino acid
50 of the 161P2F10B protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 50 to about amino acid 60 of the 161P2F10B
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 60 to about amino acid 70 of the 161P2F10B protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70
to about amino acid 80 of the 161P2F10B protein shown in FIG. 2
or FIG. 3, polynucleotides encoding about amino acid 80 to about
amino acid 90 of the 161P2F10B protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 90 to about amino acid
100 of the 161P2F10B protein shown in FIG. 2 or FIG. 3, in increments
of about 10 amino acids, ending at the carboxyl terminal amino acid
set forth in FIG. 2 or FIG. 3. Accordingly polynucleotides encoding
portions of the amino acid sequence (of about 10 amino acids), of
amino acids 100 through the carboxyl terminal amino acid of the
161P2F10B protein are embodiments of the invention. Wherein it is
understood that each particular amino acid position discloses that
position plus or minus five amino acid residues.
Polynucleotides encoding relatively long portions of the 161P2F10B
protein are also within the scope of the invention. For example,
polynucleotides encoding from about amino acid 1 (or 20 or 30 or
40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the
161P2F10B00 protein shown in FIG. 2 or FIG. 3 can be generated by
a variety of techniques well known in the art. These polynucleotide
fragments can include any portion of the 161P2F10B sequence as shown
in FIG. 2 or FIG. 3.
Additional illustrative embodiments of the invention disclosed
herein include 161P2F10B polynucleotide fragments encoding one or
more of the biological motifs contained within the 161P2F10B protein
sequence, including one or more of the motif-bearing subsequences
of the 161P2F10B protein set forth in Tables V-XVIII. In another
embodiment, typical polynucleotide fragments of the invention encode
one or more of the regions of 161P2F10B that exhibit homology to
a known molecule. In another embodiment of the invention, typical
polynucleotide fragments can encode one or more of the 161P2F10B
N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation
sites, casein kinase II phosphorylation sites or N-myristoylation
site and amidation sites.
II.A.) Uses of 161P2F10B Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of
different specific uses. The human 161P2F10B gene maps to the chromosomal
location set forth in Example 3. For example, because the 161P2F10B
gene maps to this chromosome, polynucleotides that encode different
regions of the 161P2F10B protein are used to characterize cytogenetic
abnormalities of this chromosomal locale, such as abnormalities
that are identified as being associated with various cancers. In
certain genes, a variety of chromosomal abnormalities including
rearrangements have been identified as frequent cytogenetic abnormalities
in a number of different cancers (see e.g. Krajinovic et al., Mutat.
Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86 (10): 3905-3914
(1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus,
polynucleotides encoding specific regions of the 161P2F10B protein
provide new tools that can be used to delineate, with greater precision
than previously possible, cytogenetic abnormalities in the chromosomal
region that encodes 161P2F10B that may contribute to the malignant
phenotype. In this context, these polynucleotides satisfy a need
in the art for expanding the sensitivity of chromosomal screening
in order to identify more subtle and less common chromosomal abnormalities
(see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057
(1994)).
Furthermore, as 161P2F10B was shown to be highly expressed in prostate
and other cancers, 161P2F10B polynucleotides are used in methods
assessing the status of 161P2F10B gene products in normal versus
cancerous tissues. Typically, polynucleotides that encode specific
regions of the 161P2F10B protein are used to assess the presence
of perturbations (such as deletions, insertions, point mutations,
or alterations resulting in a loss of an antigen etc.) in specific
regions of the 161P2F10B gene, such as such regions containing one
or more motifs. Exemplary assays include both RT-PCR assays as well
as single-strand conformation polymorphism (SSCP) analysis (see,
e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both
of which utilize polynucleotides encoding specific regions of a
protein to examine these regions within the protein.
II.A.2.) Antisense Embodiments
Other specifically contemplated nucleic acid related embodiments
of the invention disclosed herein are genomic DNA, cDNAs, ribozymes,
and antisense molecules, as well as nucleic acid molecules based
on an alternative backbone, or including alternative bases, whether
derived from natural sources or synthesized, and include molecules
capable of inhibiting the RNA or protein expression of 161P2F10B.
For example, antisense molecules can be RNAs or other molecules,
including peptide nucleic acids (PNAs) or non-nucleic acid molecules
such as phosphorothioate derivatives, that specifically bind DNA
or RNA in a base pair-dependent manner. A skilled artisan can readily
obtain these classes of nucleic acid molecules using the 161P2F10B
polynucleotides and polynucleotide sequences disclosed herein.
Antisense technology entails the administration of exogenous oligonucleotides
that bind to a target polynucleotide located within the cells. The
term "antisense" refers to the fact that such oligonucleotides
are complementary to their intracellular targets, e.g., 161P2F10B.
See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors
of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988).
The 161P2F10B antisense oligonucleotides of the present invention
include derivatives such as S-oligonucleotides (phosphorothioate
derivatives or S-oligos, see, Jack Cohen, supra), which exhibit
enhanced cancer cell growth inhibitory action. S-oligos (nucleoside
phosphorothioates) are isoelectronic analogs of an oligonucleotide
(O-oligo) in which a nonbridging oxygen atom of the phosphate group
is replaced by a sulfur atom. The S-oligos of the present invention
can be prepared by treatment of the corresponding O-oligos with
3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer
reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990);
and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).
Additional 161P2F10B antisense oligonucleotides of the present invention
include morpholino antisense oligonucleotides known in the art (see,
e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug
Development 6: 169-175).
The 161P2F10B antisense oligonucleotides of the present invention
typically can be RNA or DNA that is complementary to and stably
hybridizes with the first 100 5' codons or last 100 3' codons of
the 161P2F10B genomic sequence or the corresponding mRNA. Absolute
complementarity is not required, although high degrees of complementarity
are preferred. Use of an oligonucleotide complementary to this region
allows for the selective hybridization to 161P2F10B mRNA and not
to mRNA specifying other regulatory subunits of protein kinase.
In one embodiment, 161P2F10B antisense oligonucleotides of the present
invention are 15 to 30-mer fragments of the antisense DNA molecule
that have a sequence that hybridizes to 161P2F10B mRNA. Optionally,
161P2F10B antisense oligonucleotide is a 30-mer oligonucleotide
that is complementary to a region in the first 10 5' codons or last
10 3' codons of 161P2F10B. Alternatively, the antisense molecules
are modified to employ ribozymes in the inhibition of 161P2F10B
expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends
Genet 12: 510-515 (1996).
II.A.3.) Primers and Primer Pairs
Further specific embodiments of this nucleotides of the invention
include primers and primer pairs, which allow the specific amplification
of polynucleotides of the invention or of any specific parts thereof,
and probes that selectively or specifically hybridize to nucleic
acid molecules of the invention or to any part thereof. Probes can
be labeled with a detectable marker, such as, for example, a radioisotope,
fluorescent compound, bioluminescent compound, a chemiluminescent
compound, metal chelator or enzyme. Such probes and primers are
used to detect the presence of a 161P2F10B polynucleotide in a sample
and as a means for detecting a cell expressing a 161P2F10B protein.
Examples of such probes include polypeptides comprising all or
part of the human 161P2F10B cDNA sequence shown in FIG. 2. Examples
of primer pairs capable of specifically amplifying 161P2F10B mRNAs
are also described in the Examples. As will be understood by the
skilled artisan, a great many different primers and probes can be
prepared based on the sequences provided herein and used effectively
to amplify and/or detect a 161P2F10B mRNA.
The 161P2F10B polynucleotides of the invention are useful for a
variety of purposes, including but not limited to their use as probes
and primers for the amplification and/or detection of the 161P2F10B
gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis
and/or prognosis of prostate cancer and other cancers; as coding
sequences capable of directing the expression of 161P2F10B polypeptides;
as tools for modulating or inhibiting the expression of the 161P2F10B
gene(s) and/or translation of the 161P2F10B transcript(s); and as
therapeutic agents.
The present invention includes the use of any probe as described
herein to identify and isolate a 161P2F10B or 161P2F10B related
nucleic acid sequence from a naturally occurring source, such as
humans or other mammals, as well as the isolated nucleic acid sequence
per se, which would comprise all or most of the sequences found
in the probe used.
II.A.4.) Isolation of 161P2F10B-Encoding Nucleic Acid Molecules
The 161P2F10B cDNA sequences described herein enable the isolation
of other polynucleotides encoding 161P2F10B gene product(s), as
well as the isolation of polynucleotides encoding 161P2F10B gene
product homologs, alternatively spliced isoforms, allelic variants,
and mutant forms of the 161P2F10B gene product as well as polynucleotides
that encode analogs of 161P2F10B-related proteins. Various molecular
cloning methods that can be employed to isolate full length cDNAs
encoding an 161P2F10B gene are well known (see, for example, Sambrook,
J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold
Spring Harbor Press, New York, 1989; Current Protocols in Molecular
Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example,
lambda phage cloning methodologies can be conveniently employed,
using commercially available cloning systems (e.g., Lambda ZAP Express,
Stratagene). Phage clones containing 161P2F10B gene cDNAs can be
identified by probing with a labeled 161P2F10B cDNA or a fragment
thereof. For example, in one embodiment, the 161P2F10B cDNA (FIG.
2) or a portion thereof can be synthesized and used as a probe to
retrieve overlapping and full-length cDNAs corresponding to a 161P2F10B
gene. The 161P2F10B gene itself can be isolated by screening genomic
DNA libraries, bacterial artificial chromosome libraries (BACs),
yeast artificial chromosome libraries (YACs), and the like, with
161P2F10B DNA probes or primers.
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
The invention also provides recombinant DNA or RNA molecules containing
an 161P2F10B polynucleotide, a fragment, analog or homologue thereof,
including but not limited to phages, plasmids, phagemids, cosmids,
YACs, BACs, as well as various viral and non-viral vectors well
known in the art, and cells transformed or transfected with such
recombinant DNA or RNA molecules. Methods for generating such molecules
are well known (see, for example, Sambrook et al, 1989, supra).
The invention further provides a host-vector system comprising
a recombinant DNA molecule containing a 161P2F10B polynucleotide,
fragment, analog or homologue thereof within a suitable prokaryotic
or eukaryotic host cell. Examples of suitable eukaryotic host cells
include a yeast cell, a plant cell, or an animal cell, such as a
mammalian cell or an insect cell (e.g., a baculovirus-infectible
cell such as an Sf9 or HighFive cell). Examples of suitable mammalian
cells include various prostate cancer cell lines such as DU145 and
TsuPrl, other transfectable or transducible prostate cancer cell
lines, primary cells (PrEC), as well as a number of mammalian cells
routinely used for the expression of recombinant proteins (e.g.,
COS, CHO, 293, 293T cells). More particularly, a polynucleotide
comprising the coding sequence of 161P2F10B or a fragment, analog
or homolog thereof can be used to generate 161P2F10B proteins or
fragments thereof using any number of host-vector systems routinely
used and widely known in the art.
A wide range of host-vector systems suitable for the expression
of 161P2F10B proteins or fragments thereof are available, see for
example, Sambrook et al., 1989, supra; Current Protocols in Molecular
Biology, 1995, supra). Preferred vectors for mammalian expression
include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen)
and the retroviral vector pSR.alpha.tkneo (Muller et al., 1991,
MCB 11:1785). Using these expression vectors, 161P2F10B can be expressed
in several prostate cancer and non-prostate cell lines, including
for example 293, 293T, rat-1, NIH 3T3 and TsuPrl. The host-vector
systems of the invention are useful for the production of a 161P2F10B
protein or fragment thereof. Such host-vector systems can be employed
to study the functional properties of 161P2F10B and 161P2F10B mutations
or analogs.
Recombinant human 161P2F10B protein or an analog or homolog or
fragment thereof can be produced by mammalian cells transfected
with a construct encoding a 161P2F10B-related nucleotide. For example,
293T cells can be transfected with an expression plasmid encoding
161P2F10B or fragment, analog or homolog thereof, the 161P2F10B
or related protein is expressed in the 293T cells, and the recombinant
161P2F10B protein is isolated using standard purification methods
(e.g., affinity purification using anti-161P2F10B antibodies). In
another embodiment, a 161P2F10B coding sequence is subcloned into
the retroviral vector pSR.alpha.MSVtkneo and used to infect various
mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in
order to establish 161P2F10B expressing cell lines. Various other
expression systems well known in the art can also be employed. Expression
constructs encoding a leader peptide joined in frame to the 161P2F10B
coding sequence can be used for the generation of a secreted form
of recombinant 161P2F10B protein.
As discussed herein, redundancy in the genetic code permits variation
in 161P2F10B gene sequences. In particular, it is known in the art
that specific host species often have specific codon preferences,
and thus one can adapt the disclosed sequence as preferred for a
desired host. For example, preferred analog codon sequences typically
have rare codons (i.e., codons having a usage frequency of less
than about 20% in known sequences of the desired host) replaced
with higher frequency codons. Codon preferences for a specific species
are calculated, for example, by utilizing codon usage tables available
on the INTERNET.
Additional sequence modifications are known to enhance protein
expression in a cellular host. These include elimination of sequences
encoding spurious polyadenylation signals, exon/intron splice site
signals, transposon-like repeats, and/or other such well-characterized
sequences that are deleterious to gene expression. The GC content
of the sequence is adjusted to levels average for a given cellular
host, as calculated by reference to known genes expressed in the
host cell. Where possible, the sequence is modified to avoid predicted
hairpin secondary mRNA structures. Other useful modifications include
the addition of a translational initiation consensus sequence at
the start of the open reading frame, as described in Kozak, Mol.
Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that
the general rule that eukaryotic ribosomes initiate translation
exclusively at the 5' proximal AUG codon is abrogated only under
rare conditions (see, e.g., Kozak PNAS 92 (7): 2662-2666, (1995)
and Kozak NAR 15(20): 8125-8148 (1987)).
III.) 161P2F10B-related Proteins
Another aspect of the present invention provides 161P2F10B-related
proteins. Specific embodiments of 161P2F10B proteins comprise a
polypeptide having all or part of the amino acid sequence of human
161P2F10B as shown in FIG. 2 or FIG. 3. Alternatively, embodiments
of 161P2F10B proteins comprise variant, homolog or analog polypeptides
that have alterations in the amino acid sequence of 161P2F10B shown
in FIG. 2 or FIG. 3.
In general, naturally occurring allelic variants of human 161P2F10B
share a high degree of structural identity and homology (e.g., 90%
or more homology). Typically, allelic variants of the 161P2F10B
protein contain conservative amino acid substitutions within the
161P2F10B sequences described herein or contain a substitution of
an amino acid from a corresponding position in a homologue of 161P2F10B.
One class of 161P2F10B allelic variants are proteins that share
a high degree of homology with at least a small region of a particular
161P2F10B amino acid sequence, but further contain a radical departure
from the sequence, such as a non-conservative substitution, truncation,
insertion or frame shift. In comparisons of protein sequences, the
terms, similarity, identity, and homology each have a distinct meaning
as appreciated in the field of genetics. Moreover, orthology and
paralogy can be important concepts describing the relationship of
members of a given protein family in one organism to the members
of the same family in other organisms.
Amino acid abbreviations are provided in Table II. Conservative
amino acid substitutions can frequently be made in a protein without
altering either the conformation or the function of the protein.
Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15 conservative substitutions. Such changes
include substituting any of isoleucine (I), valine (V), and leucine
(L) for any other of these hydrophobic amino acids; aspartic acid
(D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine
(N) and vice versa; and serine (S) for threonine (T) and vice versa.
Other substitutions can also be considered conservative, depending
on the environment of the particular amino acid and its role in
the three-dimensional structure of the protein. For example, glycine
(G) and alanine (A) can frequently be interchangeable, as can alanine
(A) and valine (V). Methionine (M), which is relatively hydrophobic,
can frequently be interchanged with leucine and isoleucine, and
sometimes with valine. Lysine (K) and arginine (R) are frequently
interchangeable in locations in which the significant feature of
the amino acid residue is its charge and the differing pK's of these
two amino acid residues are not significant. Still other changes
can be considered "conservative" in particular environments
(see, e.g. Table III herein; pages 13-15 "Biochemistry"
2.sup.nd ED. Lubert Stryer ed (Stanford University); Henikoff et
al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995
May 19; 270(20):11882-6).
Embodiments of the invention disclosed herein include a wide variety
of art-accepted variants or analogs of 161P2F10B proteins such as
polypeptides having amino acid insertions, deletions and substitutions.
161P2F10B variants can be made using methods known in the art such
as site-directed mutagenesis, alanine scanning, and PCR mutagenesis.
Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331
(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette
mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection
mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415
(1986)) or other known techniques can be performed on the cloned
DNA to produce the 161P2F10B variant DNA.
Scanning amino acid analysis can also be employed to identify one
or more amino acids along a contiguous sequence that is involved
in a specific biological activity such as a protein-protein interaction.
Among the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine,
and cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant. Alanine is also typically preferred because it is
the most common amino acid. Further, it is frequently found in both
buried and exposed positions (Creighton, The Proteins, (W.H. Freeman
& Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine
substitution does not yield adequate amounts of variant, an isosteric
amino acid can be used.
As defined herein, 161P2F10B variants, analogs or homologs, have
the distinguishing attribute of having at least one epitope that
is "cross reactive" with a 161P2F10B protein having the
amino acid sequence of SEQ ID NO: 703. As used in this sentence,
"cross reactive" means that an antibody or T cell that
specifically binds to an 161P2F10B variant also specifically binds
to the 161P2F10B protein having the amino acid sequence of SEQ ID
NO: 703. A polypeptide ceases to be a variant of the protein shown
in SEQ ID NO: 703 when it no longer contains any epitope capable
of being recognized by an antibody or T cell that specifically binds
to the 161P2F10B protein. Those skilled in the art understand that
antibodies that recognize proteins bind to epitopes of varying size,
and a grouping of the order of about four or five amino acids, contiguous
or not, is regarded as a typical number of amino acids in a minimal
epitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955;
Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al.,
J Immunol (1985) 135(4):2598-608.
Another class of 161P2F10B-related protein variants share 70%,
75%, 80%, 85% or 90% or more similarity with the amino acid sequence
of FIG. 2 or a fragment thereof. Another specific class of 161P2F10B
protein variants or analogs comprise one or more of the 161P2F10B
biological motifs described herein or presently known in the art.
Thus, encompassed by the present invention are analogs of 161P2F10B
fragments (nucleic or amino acid) that have altered functional (e.g.
immunogenic) properties relative to the starting fragment. It is
to be appreciated that motifs now or which become part of the art
are to be applied to the nucleic or amino acid sequences of FIG.
2 or FIG. 3.
As discussed herein, embodiments of the claimed invention include
polypeptides containing less than the full amino acid sequence of
the 161P2F10B protein shown in FIG. 2 or FIG. 3. For example, representative
embodiments of the invention comprise peptides/proteins having any
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino
acids of the 161P2F10B protein shown in FIG. 2 or FIG. 3.
Moreover, representative embodiments of the invention disclosed
herein include polypeptides consisting of about amino acid 1 to
about amino acid 10 of the 161P2F10B protein shown in FIG. 2 or
FIG. 3, polypeptides consisting of about amino acid 10 to about
amino acid 20 of the 161P2F10B protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 20 to about amino acid
30 of the 161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 30 to about amino acid 40 of the
161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 40 to about amino acid 50 of the 161P2F10B protein
shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino
acid 50 to about amino acid 60 of the 161P2F10B protein shown in
FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60
to about amino acid 70 of the 161P2F10B protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 70 to about
amino acid 80 of the 161P2F10B protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 80 to about amino acid
90 of the 161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 90 to about amino acid 100 of the
161P2F10B protein shown in FIG. 2 or FIG. 3, etc. throughout the
entirety of the 161P2F10B amino acid sequence. Moreover, polypeptides
consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about
amino acid 20, (or 130, or 140 or 150 etc.) of the 161P2F10B protein
shown in FIG. 2 or FIG. 3 are embodiments of the invention. It is
to be appreciated that the starting and stopping positions in this
paragraph refer to the specified position as well as that position
plus or minus 5 residues.
161P2F10B-related proteins are generated using standard peptide
synthesis technology or using chemical cleavage methods well known
in the art. Alternatively, recombinant methods can be used to generate
nucleic acid molecules that encode a 161P2F10B-related protein.
In one embodiment, nucleic acid molecules provide a means to generate
defined fragments of the 161P2F10B protein (or variants, homologs
or analogs thereof).
III.A.) Motif-bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed
herein include 161P2F10B polypeptides comprising the amino acid
residues of one or more of the biological motifs contained within
the 161P2F10B polypeptide sequence set forth in FIG. 2 or FIG. 3.
Various motifs are known in the art, and a protein can be evaluated
for the presence of such motifs by a number of publicly available
Internet sites (see, e.g., Epimatrix.TM. and Epimer.TM., Brown University;
and BIMAS.
Motif bearing subsequences of the 161P2F10B protein are set forth
and identified in Table XIX.
Table XX sets forth several frequently occurring motifs based on
pfam searches. The columns of Table XX list (1) motif name abbreviation,
(2) percent identity found amongst the different member of the motif
family, (3) motif name or description and (4) most common function;
location information is included if the motif is relevant for location.
Polypeptides comprising one or more of the 161P2F10B motifs discussed
above are useful in elucidating the specific characteristics of
a malignant phenotype in view of the observation that the 161P2F10B
motifs discussed above are associated with growth dysregulation
and because 161P2F10B is overexpressed in certain cancers (See,
e.g., Table I). Casein kinase II, cAMP and camp-dependent protein
kinase, and Protein Kinase C, for example, are enzymes known to
be associated with the development of the malignant phenotype (see
e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et
al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic
Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene
18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309
(1998)). Moreover, both glycosylation and myristoylation are protein
modifications also associated with cancer and cancer progression
(see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999);
Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is
another protein modification also associated with cancer and cancer
progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.
(13): 169-175 (1992)).
In another embodiment, proteins of the invention comprise one or
more of the immunoreactive epitopes identified in accordance with
art-accepted methods, such as the peptides set forth in Tables V-XVIII.
CTL epitopes can be determined using specific algorithms to identify
peptides within an 161P2F10B protein that are capable of optimally
binding to specified HLA alleles (e.g., Table IV; Epimatrix.TM.
and Epimer.TM., Brown University; and BIMAS). Moreover, processes
for identifying peptides that have sufficient binding affinity for
HLA molecules and which are correlated with being immunogenic epitopes,
are well known in the art, and are carried out without undue experimentation.
In addition, processes for identifying peptides that are immunogenic
epitopes, are well known in the art, and are carried out without
undue experimentation either in vitro or in vivo.
Also known in the art are principles for creating analogs of such
epitopes in order to modulate immunogenicity. For example, one begins
with an epitope that bears a CTL or HTL motif (see, e.g., the HLA
Class I and HLA Class II motifs/supermotifs of Table IV). The epitope
is analoged by substituting out an amino acid at one of the specified
positions, and replacing it with another amino acid specified for
that position. For example, one can substitute out a deleterious
residue in favor of any other residue, such as a preferred residue
as defined in Table IV; substitute a less-preferred residue with
a preferred residue as defined in Table IV; or substitute an originally-occurring
preferred residue with another preferred residue as defined in Table
IV. Substitutions can occur at primary anchor positions or at other
positions in a peptide; see, e.g., Table IV.
A variety of references reflect the art regarding the identification
and generation of epitopes in a protein of interest as well as analogs
thereof. See, for example, WO 9733602 to Chesnut et al.; Sette,
Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol.
2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1):
12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney
et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature
351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker
et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.
152:163-75 (1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta
et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al., J.
Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID:
7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8):
2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander
et al., Immunol. Res. 1998 18(2): 79-92.
Related embodiments of the inventions include polypeptides comprising
combinations of the different motifs set forth in Table XIX, and/or,
one or more of the predicted CTL epitopes of Table V through Table
XVIII, and/or, one or more of the T cell binding motifs known in
the art. Preferred embodiments contain no insertions, deletions
or substitutions either within the motifs or the intervening sequences
of the polypeptides. In addition, embodiments which include a number
of either N-terminal and/or C-terminal amino acid residues on either
side of these motifs may be desirable (to, for example, include
a greater portion of the polypeptide architecture in which the motif
is located). Typically the number of N-terminal and/or C-terminal
amino acid residues on either side of a motif is between about 1
to about 100 amino acid residues, preferably 5 to about 50 amino
acid residues.
161P2F10B-related proteins are embodied in many forms, preferably
in isolated form. A purified 161P2F10B protein molecule will be
substantially free of other proteins or molecules that impair the
binding of 161P2F10B to antibody, T cell or other ligand. The nature
and degree of isolation and purification will depend on the intended
use. Embodiments of a 161P2F10B-related proteins include purified
161P2F10B-related proteins and functional, soluble 161P2F10B-related
proteins. In one embodiment, a functional, soluble 161P2F10B protein
or fragment thereof retains the ability to be bound by antibody,
T cell or other ligand.
The invention also provides 161P2F10B proteins comprising biologically
active fragments of the 161P2F10B amino acid sequence shown in FIG.
2 or FIG. 3. Such proteins exhibit properties of the 161P2F10B protein,
such as the ability to elicit the generation of antibodies that
specifically bind an epitope associated with the 161P2F10B protein;
to be bound by such antibodies; to elicit the activation of HTL
or CTL; and/or, to be recognized by HTL or CTL.
161P2F10B-related polypeptides that contain particularly interesting
structures can be predicted and/or identified using various analytical
techniques well known in the art, including, for example, the methods
of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz
or Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments
that contain such structures are particularly useful in generating
subunit-specific anti-161P2F10B antibodies, or T cells or in identifying
cellular factors that bind to 161P2F10B.
CTL epitopes can be determined using specific algorithms to identify
peptides within an 161P2F10B protein that are capable of optimally
binding to specified HLA alleles (e.g., by using the SYFPEITHI site;
the listings in Table IV(A)-(E); Epimatrix.TM. and Epimem.TM., Brown
University; and BIMAS. Illustrating this, peptide epitopes from
161P2F10B that are presented in the context of human MHC class I
molecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables
V-XVIII). Specifically, the complete amino acid sequence of the
161P2F10B protein was entered into the HLA Peptide Motif Search
algorithm found in the Bioinformatics and Molecular Analysis Section
(BIMAS) web site listed above. The HLA peptide motif search algorithm
was developed by Dr. Ken Parker based on binding of specific peptide
sequences in the groove of HLA Class I molecules, in particular
HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et
al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7
(1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm
allows location and ranking of 8-mer, 9-mer, and 10-mer peptides
from a complete protein sequence for predicted binding to HLA-A2
as well as numerous other HLA Class I molecules. Many HLA class
I binding peptides are 8-, 9-, 10 or 11-mers. For example, for class
I HLA-A2, the epitopes preferably contain a leucine (L) or methionine
(M) at position 2 and a valine (V) or leucine (L) at the C-terminus
(see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected
results of 161P2F10B predicted binding peptides are shown in Tables
V-XVIII herein. In Tables V-XVIII, the top 50 ranking candidates,
9-mers and 10-mers, for each family member are shown along with
their location, the amino acid sequence of each specific peptide,
and an estimated binding score. The binding score corresponds to
the estimated half time of dissociation of complexes containing
the peptide at 37.degree. C. at pH 6.5. Peptides with the highest
binding score are predicted to be the most tightly bound to HLA
Class I on the cell surface for the greatest period of time and
thus represent the best immunogenic targets for T-cell recognition.
Actual binding of peptides to an HLA allele can be evaluated by
stabilization of HLA expression on the antigen-processing defective
cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and
Peshwa et al., Prostate 36:129-38 (1998)). Immunogenicity of specific
peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic
T lymphocytes (CTL) in the presence of antigen presenting cells
such as dendritic cells.
It is to be appreciated that every epitope predicted by the BIMAS
site, Epimer.TM. and Epimatrix.TM. sites, or specified by the HLA
class I or class II motifs available in the art or which become
part of the art such as set forth in Table IV are to be "applied"
to the 161P2F10B protein. As used in this context "applied"
means that the 161P2F10B protein is evaluated, e.g., visually or
by computer-based patterns finding methods, as appreciated by those
of skill in the relevant art. Every subsequence of the 161P2F10B
of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I
motif, or a subsequence of 9 or more amino acid residues that bear
an HLA Class II motif are within the scope of the invention.
III.B.) Expression of 161P2F10B-related Proteins
In an embodiment described in the examples that follow, 161P2F10B
can be conveniently expressed in cells (such as 293T cells) transfected
with a commercially available expression vector such as a CMV-driven
expression vector encoding 161P2F10B with a C-terminal 6.times.His
and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation,
Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal
that can be used to facilitate the production of a secreted 161P2F10B
protein in transfected cells. The secreted HIS-tagged 161P2F10B
in the culture media can be purified, e.g., using a nickel column
using standard techniques.
III.C.) Modifications of 161P2F10B-related Proteins
Modifications of 161P2F10B-related proteins such as covalent modifications
are included within the scope of this invention. One type of covalent
modification includes reacting targeted amino acid residues of a
161P2F10B polypeptide with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or C-terminal
residues of the 161P2F10B. Another type of covalent modification
of the 161P2F10B polypeptide included within the scope of this invention
comprises altering the native glycosylation pattern of a protein
of the invention. Another type of covalent modification of 161P2F10B
comprises linking the 161P2F10B polypeptide to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The 161P2F10B-related proteins of the present invention can also
be modified to form a chimeric molecule comprising 161P2F10B fused
to another, heterologous polypeptide or amino acid sequence. Such
a chimeric molecule can be synthesized chemically or recombinantly.
A chimeric molecule can have a protein of the invention fused to
another tumor-associated antigen or fragment thereof. Alternatively,
a protein in accordance with the invention can comprise a fusion
of fragments of the 161P2F10B sequence (amino or nucleic acid) such
that a molecule is created that is not, through its length, directly
homologous to the amino or nucleic acid sequences shown in FIG.
2 or FIG. 3. Such a chimeric molecule can comprise multiples of
the same subsequence of 161P2F10B. A chimeric molecule can comprise
a fusion of a 161P2F10B-related protein with a polyhistidine epitope
tag, which provides an epitope to which immobilized nickel can selectively
bind, with cytokines or with growth factors. The epitope tag is
generally placed at the amino- or carboxyl-terminus of the 161P2F10B.
In an alternative embodiment, the chimeric molecule can comprise
a fusion of a 161P2F10B-related protein with an immunoglobulin or
a particular region of an immunoglobulin. For a bivalent form of
the chimeric molecule (also referred to as an "immunoadhesin"),
such a fusion could be to the Fc region of an IgG molecule. The
Ig fusions preferably include the substitution of a soluble (transmembrane
domain deleted or inactivated) form of a 161P2F10B polypeptide in
place of at least one variable region within an Ig molecule. In
a preferred embodiment, the immunoglobulin fusion includes the hinge,
CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule.
For the production of immunoglobulin fusions see, e.g., U.S. Pat.
No. 5,428,130 issued Jun. 27, 1995.
III.D.) Uses of 161P2F10B-related Proteins
The proteins of the invention have a number of different specific
uses. As 161P2F10B is highly expressed in prostate and other cancers,
161P2F10B-related proteins are used in methods that assess the status
of 161P2F10B |