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1
NUCLEIC ACIDS AND CORRESPONDING PROTEINS ENTITLED 109P1 D4
USEFUL IN TREATMENT AND DETECTION OF CANCER
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
FIELD OF THE INVENTION
The invention described herein relates to genes and their encoded proteins,
termed 109P1 D4 and variants thereof,
expressed in certain cancers, and to diagnostic and therapeutic methods and
compositions useful in the management of
cancers that express 109P1 D4.
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.
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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 at
al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers
include PCTA-1 (Su eta!., 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 U S A. 1999 Dec 7; 96(25): 14523-
8) and prostate stem cell antigen (PSCA)
(Reiter at aL, 1998, Proc. NatI. 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 eight 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
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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.
3
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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.
SUMMARY OF THE INVENTION
The present invention relates to a gene, designated 109P1 D4, that has now
been found to be over-expressed in
the cancer(s) listed in Table I. Northern blot expression analysis of 109P1D4
gene expression in normal tissues shows a
restricted expression pattern in adult tissues. The nucleotide (Figure 2) and
amino acid (Figure 2, and Figure 3) sequences
of 109P1 D4 are provided. The tissue-related profile of 109P1 D4 in normal
adult tissues, combined with the over-expression
observed in the tissues listed in Table I, shows that 109P1 D4 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 109P1 D4 genes,
mRNAs, and/or coding sequences, preferably in isolated form, including
polynucleotides encoding 109P1 D4-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
109P1 D4-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 109P1 D4 genes or mRNA
sequences or parts thereof, and polynucleotides or oligonucleotides that
hybridize to the 109P1 D4 genes, mRNAs, or to
109P1 D4-encoding polynucleotides. Also provided are means for isolating cDNAs
and the genes encoding 109P1 D4.
Recombinant DNA molecules containing 109P1 D4 polynucleotides, cells
transformed or transduced with such molecules, and
host-vector systems for the expression of 109P1 D4 gene products are also
provided. The invention further provides antibodies
that bind to 109P1 D4 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
Figure 2 is not encoded and/or the entire amino acid sequence of Figure 2 is
not prepared. In certain embodiments, the
entire nucleic acid sequence of Figure 2 is encoded and/or the entire amino
acid sequence of Figure 2 is prepared, either of
which are in respective human unit dose forms.
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The invention further provides methods for detecting the presence and status
of 109P1 D4 polynucleotides and
proteins in various biological samples, as well as methods for identifying
cells that express 109P1 D4. A typical embodiment of this
invention provides methods for monitoring 109P1 D4 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 109P1 D4 such as cancers of tissues listed in Table 1, including
therapies aimed at inhibiting the transcription,
translation, processing or function of 109P1 D4 as well as cancer vaccines. In
one aspect, the invention provides
compositions, and methods comprising them, for treating a cancer that
expresses 109P1 D4 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 109P1 D4. Preferably, the carrier is a uniquely
human carrier. In another aspect of the
invention, the agent is a moiety that is immunoreactive with 109P1 D4 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 109P1 D4 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 109P1 D4 as
described above. The one or more than one nucleic acid molecule may also be,
or encodes, a molecule that inhibits
production of 109P1 D4. Non-limiting examples of such molecules include, but
are not limited to, those complementary to a
nucleotide sequence essential for production of 109P1 D4 (e.g. antisense
sequences or molecules that form a triple helix with
a nucleotide double helix essential for 109P1 D4 production) or a ribozyme
effective to lyse 109PI D4 mRNA.
Note that to determine the starting position of any peptide set forth in
Tables VIII-XXI and XXII to XLIX (collectively
HLA Peptide Tables) respective to its parental protein, e.g., variant 1,
variant 2, etc., reference is made to three factors: the
particular variant, the length of the peptide in an HLA Peptide Table, and the
Search Peptides in Table VII. Generally, a
unique Search Peptide is used to obtain HLA peptides of a particular for a
particular variant. The position of each Search
Peptide relative to its respective parent molecule is listed in Table VII.
Accordingly, if a Search Peptide begins at position
"X", one must add the value "X -1" to each position in Tables VIII-XXI and
XXII to XLIX to obtain the actual position of the
HLA peptides in their parental molecule. For example, if a particular Search
Peptide begins at position 150 of its parental
molecule, one must add 150 -1, i.e., 149 to each HLA peptide amino acid
position to calculate the position of that amino acid
in the parent molecule.
One embodiment of the invention comprises an HLA peptide, that occurs at least
twice in Tables VIII-XXI and XXII
to XLIX collectively, or an oligonucleotide that encodes the HLA peptide.
Another embodiment of the invention comprises an
HLA peptide that occurs at least once in Tables VIII-XXI and at least once in
tables XXII to XLIX, or an oligonucleotide that
encodes the HLA peptide.
Another embodiment of the invention is antibody epitopes, which comprise a
peptide regions, or an oligonucleotide
encoding the peptide region, that has one two, three, four, or five of the
following characteristics:
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i) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole number increment up
to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or greater than 0.5,
0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity
profile of Figure 5;
ii) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole number increment up
to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or less than 0.5, 0.4,
0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile
of Figure 6;
iii) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole number increment up
to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or greater than 0.5,
0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible
Residues profile of Figure 7;
iv) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole number increment up
to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or greater than 0.5,
0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility
profile of Figure 8; or
v) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole number increment up
to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or greater than 0.5,
0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile
of Figure 9.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. The 109P1 D4 SSH sequence of 192 nucleotides.
Figure 2. A) The cDNA and amino acid sequence of 109P1 D4 variant 1 (also
called "109P1 D4 v.1" or "109P1 D4
variant 11") is shown in Figure 2A, The start methionine is underlined. The
open reading frame extends from nucleic acid
846-3911 including the stop codon.
B) The cDNA and amino acid sequence of 109P1 D4 variant 2 (also called "109P1
D4 v.2") is shown in Figure 2B.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 503-3667 including the
stop codon.
C) The cDNA and amino acid sequence of 109P1 D4 variant 3 (also called "109P1
D4 v.3") is shown in Figure 2C.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 846-4889 including the
stop codon.
D) The cDNA and amino acid sequence of 109P1 D4 variant 4 (also called "109P1
D4 v.4") is shown in Figure 2D.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 846-4859 including the
stop codon.
E) The cDNA and amino acid sequence of 109P1 D4 variant 5 (also called "109P1
D4 v.5") is shown in Figure 2E.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 846-4778 including the
stop codon.
F) The cDNA and amino acid sequence of 109P1 D4 variant 6 (also called "109P1
D4 v.6") is shown in Figure 2F.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 614-3727 including the
stop codon.
G) The cDNA and amino acid sequence of 109P1 D4 variant 7 (also called "109P1
D4 v.7") is shown in Figure 2G.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 735-3881 including the
stop codon.
H) The cDNA and amino acid sequence of 109P1 D4 variant 8 (also called "109P1
D4 v.8") is shown in Figure 2H.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 735-4757 including the
stop codon.
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I) The cDNA and amino acid sequence of 109P1 D4 variant 9 (also called "109P1
D4 v.9") is shown in Figure 21.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 514-3627 including the
stop codon.
J) 109P1 D4 v.1, v.2 and v.3 SNP variants. Though these SNP variants are shown
separately, they can also
occur in any combinations and in any of the transcript variants listed above.
K) I09P1 D4 v.6, v.7 and v.8 SNP variants. Though these SNP variants are shown
separately, they can also occur in ar
combinations and in any of the transcript variants listed above.
Figure 3.
A) The amino acid sequence of 109P1 D4 v.1 is shown in Figure 3A; it has 1021
amino acids.
B) The amino acid sequence of 109P1 D4 v.2 is shown in Figure 3B; it has 1054
amino acids.
C) The amino acid sequence of 109P1 D4 v.3 is shown in Figure 3C; it has 1347
amino acids.
D) The amino acid sequence of 109P1 D4 v.4 is shown in Figure 3D; it has 1337
amino acids.
E) The amino acid sequence of 109P1 D4 v.5 is shown in Figure 3E; it has 1310
amino acids.
F) The amino acid sequence of 109P1 D4 v.6 is shown in Figure 3F; it has 1037
amino acids.
G) The amino acid sequence of 109P1 D4 v.7 is shown in Figure 3G; it has 1048
amino acids.
H) The amino acid sequence of 109P1 D4 v.8 is shown in Figure 3H; it has 1340
amino acids.
I) The amino acid sequence of 109P1 D4 v.9 is shown in Figure 31; it has 1037
amino acids.
As used herein, a reference to 109P1 D4 includes all variants thereof,
including those shown in Figures 2, 3, 10, 11,
and 12 unless the context clearly indicates otherwise.
Figure 4. Alignment of 109P1 D4 v.1 Protein with protocadherin-11.
Figure 5. Hydrophilicity amino acid profile of 109P1 D4 v.1-v.9 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 located on the World Wide Web at
(expasy.ch/cgi-bin/protscale.pl) through the ExPasy
molecular biology server.
Figure 6. Hydropathicity amino acid profile of 109P1 D4 v.1-v.9 determined by
computer algorithm sequence
analysis using the method of Kyle and Doolittle (Kyle J., Doolittle R.F.,
1982. J. Mol. Biol. 157:105-132) accessed on the
ProtScale website located on the World Wide Web at (.expasy.ch/cgi-
bin/protscale.pl) through the ExPasy molecular biology
server.
Figure 7. Percent accessible residues amino acid profile of 109P1 D4 v.1-v.9
determined by computer algorithm
sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-
492) accessed on the ProtScale website
located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the
ExPasy molecular biology server.
Figure 8. Average flexibility amino acid profile of 109P1 D4 v.1-v.9
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 located on the World Wide
Web at (.expasy.ch/cgi-bin/protscale.pl)
through the ExPasy molecular biology server.
Figure 9. Beta-turn amino acid profile of 109P1 D4 v.1-v.9 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 located on the World Wide Web at (.expasy.ch/cgi-
bin/protscale.pl) through the ExPasy molecular biology
server.
Figure 10. Structure of transcript variants of 109P1 D4. Variants 109P1 D4 v.2
through v.9 were transcript
variants of M. Variant v.2 shared middle portion of v.1 sequence (the 3'
portion of exon I and 5' portion of exon 2). Variant
CA 02522994 2010-03-25
8
v.6 was similar to v.2 but added an extra exon between exons I and 2 of v.2.
V.3 shared exon I and 5' portion of exon 2 with
v.1 with five additional exons downstream. Compared with v.3, v.4 deleted exon
4 of v.3 while v.5 deleted exons 3 and 4 of
v.3. Variant v.5 lacked exons 3 and 4. This gene (called PCD11) is located in
sex chromosomes X and Y. Ends of exons in
the transcripts are marked above the boxes. Potential exons of this gene are
shown in order as on the human genome. Poly
A tails and single nucleotide differences are not shown in the figure. Lengths
of introns and exons are not proportional.
Figure 11. Schematic alignment of protein variants of 109P1D4. Variants 109P1
D4 v.2 through v.9 were
proteins translated from the corresponding transcript variants. All these
protein variants shared a common portion of the
sequence, i.e., 3-1011 of v.1, except for a few amino acids different in this
segment resulted from SNP in the transcripts.
Variant v.6 and v.9 were the same except for two amino acids at 906 and 1001.
Variant v.8 was almost the same as v.5,
except for the N-terminal end, and a 2-aa deletion at 1117-8. Single amino
acid difference was not shown. Numbers in
parentheses corresponded to positions in variant v.3.
Figure 12. Intentionally Omitted.
Figure 13. Figures 13(a)"(i): Secondary structure and transmembrane domains
prediction for 109P1 D4 protein
variants 1-9(v.1 -(SEQ ID NO: 31); v.2 - (SEQ iD NO: 32); v.3 - (SEQ iD NO:
33); v.4 - (SEQ ID NO: 34); v.5 - (SEQ ID
NO: 35); v.6 - (SEQ ID NO: 36); v.7 - (SEQ ID NO: 37); v.8 - (SEQ ID NO: 38);
v.9 - (SEQ ID NO: 39)). The secondary
structures of 109P1 D4 protein variants were predicted using the HNN -
Hierarchical Neural Network method (NPS@:
Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150
Combet C., Blanchet C., Geourjon C. and
Deleage G. ), accessed from the ExPasy molecular biology
server located on the World Wide Web. This method predicts the presence and
location of alpha
helices, extended strands, and random coils from the primary protein sequence.
The percent of the protein variant in a given
secondary structure is also listed. Figures 13(J)-(R) top panels: Schematic
representation of the probability of existence of
transmembrane regions of 109P1 D4 variants based on the TMpred algorithm of
Hofmann and Stoffel which utilizes TMBASE
(K. Hofmann, W. Stoffel. TMBASE - A database of membrane spanning protein
segments Biol. Chem. Hoppe-Seyler
374:166, 1993). Figures 13(J)-(R) bottom panels: Schematic representation of
the probability of the existence of
transmembrane regions of 109P1 D4 variants based on the TMHMM algorithm of
Sonnhammer, von Heijne, and Krogh (Erik
L.L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model
for predicting transmembrane helices in
protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for
Molecular Biology, p 175-182 Ed J. Glasgow, T.
Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, CA:
AAAI Press, 1998). The TMpred and TMHMM
algorithms are accessed from the ExPasy molecular biology server located on
the World Wide Web.
Figure 14. Expression of 109P1 D4 in Lymphoma Cancer Patient Specimens. RNA
was extracted from
peripheral blood lymphocytes, cord blood isolated from normal individuals, and
from lymphoma patient cancer specimens.
Northern blots with 10pg of total RNA were probed with the 109P1 D4 sequence.
Size standards in kilobases are on the
side. Results show expression of 109P1 D4 in lymphoma patient specimens but
not in the normal blood cells tested.
Figure 15. Expression of 109P1D4 by RT-PCR. First strand cDNA was prepared
from vital pool I (liver, lung
and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer pool,
bladder cancer pool, kidney cancer pool,
colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool,
cancer metastasis pool, and pancreas cancer
pool. Normalization was performed by PCR using primers to actin and GAPDH.
Semi-quantitative PCR, using primers to
109P1 D4, was performed at 30 cycles of amplification. Results show strong
expression of 109P1 D4 in all cancer pools
tested. Very low expression was detected in the vital pools.
Figure 16. Expression of 109P1D4 in normal tissues. Two multiple tissue
northern blots (Clontech), both with 2
pg of mRNMane, were probed with the 109P1D4 SSH fragment. Size standards In
kilobases (kb) are indicated on the side.
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Results show expression of approximately 10 kb 109P1D4 transcript in ovary.
Weak expression was also detected in
placenta and brain, but not in the other normal tissues tested.
Figure 17. Expression of 109P1 D4 in human cancer cell lines. RNA was
extracted from a number of human
prostate and bone cancer cell lines. Northern blots with 10 lag of total
RNA/lane were probed with the 109P1 D4 SSH
fragment. Size standards in kilobases (kb) are indicated on the side. Results
show expression of 109P1 D4 in LAPC-9AD,
LAPC-9AI, LNCaP prostate cancer cell lines, and in the bone cancer cell lines,
SK-ES-1 and RD-ES.
Figure 18. Figure 18A: 109P1 D4 Expression in Human Normal Tissues. An cDNA
dot blot containing 76
different samples from human tissues was analyzed using a 109P1 D4 SSH probe.
Expression was only detected in multiple
areas of the brain, placenta, ovary, and fetal brain, amongst all tissues
tested. Figure 1813: Expression of 109P1 D4 in
patient cancer specimens. Expression of 109PI D4 was assayed in a panel of
human cancers (T) and their respective
matched normal tissues (N) on RNA dot blots. Upregulated expression of 109P1
D4 in tumors compared to normal tissues
was observed in uterus, lung and stomach. The expression detected in normal
adjacent tissues (isolated from diseased
tissues) but not in normal tissues (isolated from healthy donors) may indicate
that these tissues are not fully normal and that
109P1 D4 may be expressed in early stage tumors.
Figure 19. 109P1 D4 Expression in Lung Cancer Patient Specimens. RNA was
extracted from normal lung,
prostate cancer xenograft LAPC-9AD, bone cancer cell line RD-ES, and lung
cancer patient tumors. Northern blots with 10
lag of total RNA were probed with 109P1 D4. Size standards in kilobases are on
the side. Results show strong expression of
109P1 D4 in lung tumor tissues as well as the RD-ES cell line, but not in
normal lung.
Figure 20. Expression of soluble secreted Tag5109P1 D4 in 293T cells. 293T
cells were transfected with either
an empty vector or with the Tags secretion vector encoding the extracellular
domain (ECD; amino acids 24-812) of 109P1 D4
variant I fused to a Myc/His epitope Tag. 2 days later, cells and media
harvested and analyzed for expression of the
recombinant Tag5109P1 D4 protein by SDS-PAGE followed by anti-His epitope tag
Western blotting. An arrow indicates the
immunoreactive band corresponding to the 109P1D4 ECD present in the media and
the lysate from Tag5109P1D4
transfected cells.
Figure 21. Expression of 109PI D4 protein in 293T cells. 293T cells were
transfected with either an empty vector
or with pCDNA3.1 vector encoding the full length cDNA of 109P1 D4 variant I
fused to a Myc/His epitope Tag. 2 days later,
cells were harvested and analyzed for expression of I09P1 D4 variant I protein
by SDS-PAGE followed by anti-His epitope
tag Western blotting. An arrow indicates the immunoreactive band corresponding
to the full length 109P1 D4 variant I
protein expressed in cells transfected with the 109P1 D4 vector but not in
control cells.
Figure 22. Tyrosine phosphorylation of 109P1 D4 after pervanadate treatment.
293T cells were transfected with
the neomycin resistance gene alone or with 109P1 D4 in pSRp vector. Twenty
four hours after transfection, the cells were
either left in 10% serum or grown in 0.1 % serum overnight. The cells were
then left untreated or were treated with 200 pM
pervanadate (1:1 mixture of Na3VO4 and H202) for 30 minutes. The cells were
lysed in Triton X-100, and the 109P1 D4
protein was immunoprecipitated with anti-His monoclonal antibody. The
immunoprecipitates were run on SDS-PAGE and
then Western blotted with either anti-phosphotyrosine (upper panel) or anti-
His (lower panel). The 109P1 D4 protein is
phosphorylated on tyrosine in response to pervanadate treatment, and a large
amount of the protein moves to the insoluble
fraction following pervanadate-induced activation.
Figure 23. Effect of 109P1 D4 RNAi on cell proliferation. LNCaP cells were
transfected with Lipofectamine 2000
alone or with siRNA oligonucleotides. The siRNA oligonucleotides included a
negative control, Luc4, specific for Luciferase,
a positive control, Eg5, specific for the mitotic spindle protein Eg5, or
three siRNAs specific for the 109P1 D4 protein,
109P1 D4.a,109P1 D4.c and 109P1 D4.d at 20 nM concentration. Twenty four hours
after transfection, the cells were pulsed
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with 3H-thymidine and incorporation was measured after 72 hours. All three
siRNAs to 109P1 D4 inhibited the proliferation of
LNCaP cells, indicating that 109P1 D4 expression is important for the cell
growth pathway of these cancer cells.
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
1.) Definitions
II.) 109P1 D4 Polynucleotidez
II.A.) Uses of 109P1 D4 Polynucleotides
II.A.I.) Monitoring of Genetic Abnormalitiez
II.A.2.) Antisense Embodiments
II.A.3.) Primers and Primer Pairs
II.A.4.) Isolation of 109P1D4-Encoding Nucleic Acid Molecules
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
III.) 109P1 D4-related Proteins
III.A.) Motif-bearing Protein Embodiments
III.B.) Expression of 109P1D4-related Proteins
III.C.) Modifications of I09P1 D4-related Proteins
III.D.) Uses of 109P1 D4-related Proteins
IV.) 109P1 D4 Antibodies
V.) 109P1 D4 Cellular Immune Responses
VI.) 109P1D4 Transgenic Animals
VII.) Methods for the Detection of I09P1 D4
Vill.) Methods for Monitoring the Status of 109P1 D4-related Genes and Their
Products
IX.) Identification of Molecules That Interact With 109P1D4
X.) Therapeutic Methods and Compositions
X.A.) Anti-Cancer Vaccines
X.B.) 109P1 D4 as a Target for Antibody-Based Therapy
X.C.) 109P1D4 as a Target for Cellular Immune Responses
X.C.I. 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
Xl.) Diagnostic and Prognostic Embodiments of 109P1 D4.
XII.) Inhibition of 109P1D4 Protein Function
XII.A.) Inhibition of 109PID4 With Intracellular Antibodies
XII.B.) Inhibition of 109P1D4 with Recombinant Proteins
XII.C.) Inhibition of 109P1 D4 Transcription or Translation
XII.D.) General Considerations for Therapeutic Strategies
XIII.) Identification, Characterization and Use of Modulators of 109P1D4
XIV.) KITSIArticles of Manufacture
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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
andlor 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 at 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 109P1 D4 (either by removing
the underlying glycosy)ation 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 109P1 D4. 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 109P1 D4-related protein). For example, an analog of
a 109P1 D4 protein can be specifically bound by an
antibody or T cell that specifically binds to 109P1 D4.
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-109P1 D4 antibodies comprise monoclonal
and polyclonal antibodies as well as fragments containing the antigen-binding
domain andlor 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-I09P1 D4 antibodies and
clones thereof (including agonist, antagonist and neutralizing antibodies) and
anti-I09P1 D4 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
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optimization are referred to herein as an "expression enhanced sequences."
A "combinatorial library" is a collection of diverse chemical compounds
generated by either chemical synthesis or
biological synthesis by combining a number of chemical "building blocks" such
as reagents. For example, a linear
combinatorial chemical library, such as a polypeptide (e.g., mutein) library,
is formed by combining a set of chemical building
blocks called amino acids in every possible way for a given compound length
(i.e., the number of amino acids in a
polypeptide compound). Numerous chemical compounds are synthesized through
such combinatorial mixing of chemical
building blocks (Gallop et al., J. Med. Chem. 37(9):1233-1251 (1994)).
Preparation and screening of combinatorial libraries is well known to those of
skill in the art. Such combinatorial
chemical libraries include, but are not limited to, peptide libraries (see,
e.g., U.S. Patent No. 5,010,175, Furka, Pept. Prot.
Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids
(PCT Publication No WO 91/19735), encoded
peptides (PCT Publication WO 93/20242), random bio- oligomers (PCT Publication
WO 92/00091), benzodiazepines (U.S.
Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and
dipeptides (Hobbs et al., Proc. Nat. Acad. Sci.
USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer.
Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer.
Chem. Soc. 114:9217-9218 (1992)),
analogous organic syntheses of small compound libraries (Chen et al., J. Amer.
Chem. Soc. 116:2661 (1994)),
oligocarbarnates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl
phosphonates (Campbell et al., J. Org. Chem.
59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994),
nucleic acid libraries (see, e.g., Stratagene,
Corp.), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083),
antibody libraries (see, e.g., Vaughn et al., Nature
Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate
libraries (see, e.g., Liang et al., Science
274:1520-1522 (1996), and U.S. Patent No. 5,593,853), and small organic
molecule libraries (see, e.g., benzodiazepines,
Baum, C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588;
thiazolidinones and metathiazanones, U.S.
Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Patent No.
5,506, 337; benzodiazepines, U.S. Patent No. 5,288,514; and the like).
Devices for the preparation of combinatorial libraries are commercially
available (see, e.g., 357 NIPS, 390 NIPS,
Advanced Chem Tech, Louisville KY; Symphony, Rainin, Woburn, MA; 433A, Applied
Biosystems, Foster City, CA; 9050,
Plus, Millipore, Bedford, NIA). A number of well-known robotic systems have
also been developed for solution phase
chemistries. These systems include automated workstations such as the
automated synthesis apparatus developed by
Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems
utilizing robotic arms (Zymate H, Zymark
Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.),
which mimic the manual synthetic operations
performed by a chemist. Any of the above devices are suitable for use with the
present invention. The nature and
implementation of modifications to these devices (if any) so that they can
operate as discussed herein will be apparent to
persons skilled in the relevant art. In addition, numerous combinatorial
libraries are themselves commercially available (see,
e.g., ComGenex, Princeton, NJ; Asinex, Moscow, RU; Tripos, Inc., St. Louis,
MO; ChemStar, Ltd, Moscow, RU; 3D
Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; etc.).
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 auristatins, auromycins,
maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin,
duocarmycins, dolostatins, doxorubicin, daunorubicin,
taxol, cisplatin, cc1065, 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,
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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 At211,1131,1125 Y90, Re186
Re188, Sm153, Bi212or213 P32 and radioactive isotopes of Lu including Lu177.
Antibodies may also be conjugated to an anti-
cancer pro-drug activating enzyme capable of converting the pro-drug to its
active form.
The "gene product" is sometimes referred to herein as a protein or mRNA. For
example, a "gene product of the
invention" is sometimes referred to herein as a "cancer amino acid sequence",
"cancer protein", "protein of a cancer listed in
Table I", a "cancer mRNA", "mRNA of a cancer listed in Table I", etc. In one
embodiment, the cancer protein is encoded by a
nucleic acid of Figure 2. The cancer protein can be a fragment, or
alternatively, be the full-length protein to the fragment
encoded by the nucleic acids of Figure 2. In one embodiment, a cancer amino
acid sequence is used to determine
sequence identity or similarity. In another embodiment, the sequences are
naturally occurring allelic variants of a protein
encoded by a nucleic acid of Figure 2. In another embodiment, the sequences
are sequence variants as further described
herein.
"High throughput screening" assays for the presence, absence, quantification,
or other properties of particular
nucleic acids or protein products are well known to those of skill in the art.
Similarly, binding assays and reporter gene
assays are similarly well known. Thus, e.g., U.S. Patent No. 5,559,410
discloses high throughput screening methods for
proteins; U.S. Patent No. 5,585,639 discloses high throughput screening
methods for nucleic acid binding (i.e., in arrays);
while U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput
methods of screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially available
(see, e.g., Amersham Biosciences,
Piscataway, NJ; Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor,
OH; Beckman Instruments, Inc. Fullerton,
CA; Precision Systems, Inc., Natick, MA; etc.). These systems typically
automate entire procedures, including all sample
and reagent pipetting, liquid dispensing, timed incubations, and final
readings of the microplate in detector(s) appropriate for
the assay. These configurable systems provide high throughput and rapid start
up as well as a high degree of flexibility and
customization. The manufacturers of such systems provide detailed protocols
for various high throughput systems. Thus,
e.g., Zymark Corp. provides technical bulletins describing screening systems
for detecting the modulation of gene
transcription, ligand binding, and the like.
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, 8TH ED., Lange Publishing, Los
Altos, CA (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/6XSSC/0.1 % SDS/1 00
g/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and
temperatures for washing in
0.1 XSSC/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 109P1 D4 genes or that
encode polypeptides other than 109P1 D4 gene
product or fragments thereof. A skilled artisan can readily employ nucleic
acid isolation procedures to obtain an isolated 109P1 D4
polynucleotide. A protein is said to be "isolated," for example, when
physical, mechanical or chemical methods are employed to
remove the 109P1 D4 proteins from cellular constituents that are normally
associated with the protein. A skilled artisan can readily
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employ standard purification methods to obtain an isolated 109P1 D4 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 "modulator" or "test compound" or "drug candidate" or grammatical
equivalents as used herein describe
any molecule, e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, etc., to be tested for the
capacity to directly or indirectly alter the cancer phenotype or the
expression of a cancer sequence, e.g., a nucleic acid or
protein sequences, or effects of cancer sequences (e.g., signaling, gene
expression, protein interaction, etc.) In one aspect,
a modulator will neutralize the effect of a cancer protein of the invention.
By "neutralize" is meant that an activity of a protein
is inhibited or blocked, along with the consequent effect on the cell. In
another aspect, a modulator will neutralize the effect
of a gene, and its corresponding protein, of the invention by normalizing
levels of said protein. In preferred embodiments,
modulators alter expression profiles, or expression profile nucleic acids or
proteins provided herein, or downstream effector
pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g.
to a normal tissue fingerprint. In another
embodiment, a modulator induced a cancer phenotype. Generally, a plurality of
assay mixtures is run in parallel with
different agent concentrations to obtain a differential response to the
various concentrations. Typically, one of these
concentrations serves as a negative control, i.e., at zero concentration or
below the level of detection.
Modulators, drug candidates or test compounds encompass numerous chemical
classes, though typically they are
organic molecules, preferably small organic compounds having a molecular
weight of more than 100 and less than about
2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500
or less than 1000 or less than 500 D.
Candidate agents comprise functional groups necessary for structural
interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl, hydroxyl or
carboxyl group, preferably at least two of the functional
chemical groups. The candidate agents often comprise cyclical carbon or
heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above functional
groups. Modulators also comprise biomolecules
such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines,
derivatives, structural analogs or combinations
thereof. Particularly preferred are peptides. One class of modulators are
peptides, for example of from about five to about
35 amino acids, with from about five to about 20 amino acids being preferred,
and from about 7 to about 15 being particularly
preferred. Preferably, the cancer modulatory protein is soluble, includes a
non-transmembrane region, and/or, has an N-
terminal Cys to aid in solubility. In one embodiment, the C-terminus of the
fragment is kept as a free acid and the N-terminus
is a free amine to aid in coupling, i.e., to cysteine. In one embodiment, a
cancer protein of the invention is conjugated to an
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immunogenic agent as discussed herein. In one embodiment, the cancer protein
is conjugated to BSA. The peptides of the
invention, e.g., of preferred lengths, can be linked to each other or to other
amino acids to create a longer peptide/protein.
The modulatory peptides can be digests of naturally occurring proteins as is
outlined above, random peptides, or "biased"
random peptides. In a preferred embodiment, peptide/protein-based modulators
are antibodies, and fragments thereof, as
defined herein.
Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents
can be naturally occurring nucleic
acids, random nucleic acids, or "biased" random nucleic acids. For example,
digests of prokaryotic or eukaryotic genomes
can be used in an approach analogous to that outlined above for proteins.
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 a 109P1 D4-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), as shown for example in Figure
2, 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. Alternatively, in
another embodiment, the primary anchor
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residues of a peptide binds 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.
"Radioisotopes" include, but are not limited to the following (non-limiting
exemplary uses are also set forth):
Examples of Medical Isotopes:
Isotope Description of use
Actinium-225 See Thorium-229 (Th-229)
(AC-225)
Actinium-227 Parent of Radium-223 (Ra-223) which is an alpha emitter used to
treat metastases in the skeleton
(AC-227) resulting from cancer (i.e., breast and prostate cancers), and cancer
radioimmunotherapy
Bismuth-212 See Thorium-228 (Th-228)
(Bi-212)
Bismuth-213 See Thorium-229 (Th-229)
(Bi-213)
Cadmium-109 Cancer detection
(Cd-109)
Cobalt-60 Radiation source for radiotherapy of cancer, for food irradiators,
and for sterilization of medical
(Co-60) supplies
Copper-64 A positron emitter used for cancer therapy and SPECT imaging
(Cu-64)
Copper-67 Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic
studies (i.e., breast and
(Cu-67) colon cancers, and lymphoma}
Dysprosium-166 Cancer radioimmunotherapy
(Dy-166)
Erbium-169 Rheumatoid arthritis treatment, particularly for the small joints
associated with fingers and toes
(Er-169)
Europium-152 Radiation source for food irradiation and for sterilization of
medical supplies
(Eu-152)
Europium-154 Radiation source for food irradiation and for sterilization of
medical supplies
(Eu-154)
Gadolinium-153 Osteoporosis detection and nuclear medical quality assurance
devices
(Gd-153)
Gold-198 Implant and intracavity therapy of ovarian, prostate, and brain
cancers
(Au-198)
Holmium-166 Multiple myeloma treatment in targeted skeletal therapy, cancer
radioimmunotherapy, bone
(Ho-166) marrow ablation, and rheumatoid arthritis treatment
Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer
treatment, radiolabeling,
Iodine-125 tumor imaging, mapping of receptors in the brain, interstitial
radiation therapy, brachytherapy for
(1-125) treatment of prostate cancer, determination of glomerular filtration
rate (GFR), determination of
plasma volume, detection of deep vein thrombosis of the legs
Iodine-131 Thyroid function evaluation, thyroid disease detection, treatment
of thyroid cancer as well as other
(1-131) non-malignant thyroid diseases (i.e., Graves disease, goiters, and
hyperthyroidism), treatment of
leukemia, lymphoma, and other forms of cancer (e.g., breast cancer) using
radioimmunotherapy
Iridium-192 Brachytherapy, brain and spinal cord tumor treatment, treatment of
blocked arteries (i.e.,
(Ir-192) arteriosclerosis and restenosis), and implants for breast and
prostate tumors
Lutetium-177 Cancer radioimmunotherapy and treatment of blocked arteries
(i.e., arteriosclerosis and
(Lu-177) restenosis)
Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, liver,
lungs, heart, and
Molybdenum-99 other organs. Currently, Tc-99m is the most widely used
radioisotope used for diagnostic imaging
(MO-99) of various cancers and diseases involving the brain, heart, liver,
lungs; also used in detection of
deep vein thrombosis of the legs
Osmium-194 Cancer radioimmunotherapy
(Os-194)
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Palladium-103 17
(Pd-103) Prostate cancer treatment
Platinum-195m Studies on biodistribution and metabolism of cisplatin, a
chemotherapeutic drug
(Pt-195m)
Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone
cancer
Phosphorus-32 diagnosis/treatment; colon, pancreatic, and liver cancer
treatment; radiolabeling nucleic acids for
(P-32) in vitro research, diagnosis of superficial tumors, treatment of
blocked arteries (i.e.,
arteriosclerosis and restenosis), and intracavity therapy
Phosphorus-33 Leukemia treatment, bone disease diagnosis/treatment,
radiolabeling, and treatment of blocked
(P-33) arteries (i.e., arteriosclerosis and restenosis)
Radium-223 See Actinium-227 (Ac-227)
(Ra-223)
Rhenium-186 Bone cancer pain relief, rheumatoid arthritis treatment, and
diagnosis and treatment of lymphoma
(Re-186) and bone, breast, colon, and liver cancers using radioimmunotherapy
Rhenium-188 Cancer diagnosis and treatment using radioimmunotherapy, bone
cancer pain relief, treatment of
(Re-188) rheumatoid arthritis, and treatment of prostate cancer
Rhodium-105 Cancer radioimmunotherapy
(Rh-105)
Samarium-145 Ocular cancer treatment
(Sm-145)
Samarium-153 Cancer radioimmunotherapy and bone cancer pain relief
(Sm-153)
Scandium-47 Cancer radioimmunotherapy and bone cancer pain relief
(Sc-47)
Selenium-75 Radiotracer used in brain studies, imaging of adrenal cortex by
gamma-scintigraphy, lateral
(Se-75) locations of steroid secreting tumors, pancreatic scanning, detection
of hyperactive parathyroid
glands, measure rate of bile acid loss from the endogenous pool
Strontium-85- Bone cancer detection and brain scans
(Sr-85),
Strontium-89 Bone cancer pain relief, multiple myeloma treatment, and
osteoblastic therapy
(Sr-89)
Technetium-99m See Molybdenum-99 (Mo-99)
(Tc-99m)
Thorium-228 Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in
cancer radioimmunotherapy
(Th-228)
Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-
213) which are alpha
(Th-229) emitters used in cancer radioimmunotherapy
Thulium-170 Gamma source for blood irradiators, energy source for implanted
medical devices
(Tm-170)
Tin-117m Cancer immunotherapy and bone cancer pain relief
(Sn-117m)
Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for cancer
diagnostics/treatment, bone cancer
(W-1 88) pain relief, rheumatoid arthritis treatment, and treatment of blocked
arteries (i.e., arteriosclerosis
and restenosis)
Xenon-127 Neuroimaging of brain disorders, high resolution SPECT studies,
pulmonary function tests, and
(Xe-127) cerebral blood flow studies
Ytterbium-175 Cancer radioimmunotherapy
(Yb-175)
Yttrium-90 Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver
cancer treatment
(Y-90)
Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90) which is used for
cancer radioimmunotherapy (i.e.,
(Y-91) lymphoma, breast, colon, kidney, lung, ovarian, prostate, pancreatic,
and inoperable liver
cancers)
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By "randomized" or grammatical equivalents as herein applied to nucleic acids
and proteins is meant that each
nucleic acid and peptide consists of essentially random nucleotides and amino
acids, respectively. These random peptides
(or nucleic acids, discussed herein) can incorporate any nucleotide or amino
acid at any position. The synthetic process can
be designed to generate randomized proteins or nucleic acids, to allow the
formation of all or most of the possible
combinations over the length of the sequence, thus forming a library of
randomized candidate bioactive proteinaceous
agents.
In one embodiment, a library is "fully randomized," with no sequence
preferences or constants at any position. In
another embodiment, the library is a "biased random" library. That is, some
positions within the sequence either are held
constant, or are selected from a limited number of possibilities. For example,
the nucleotides or amino acid residues are
randomized within a defined class, e.g., of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or
large) residues, towards the creation of nucleic acid binding domains, the
creation of cysteines, for cross-linking, prolines for
SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation
sites, etc., or to purines, etc.
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 109P1 D4, ligands including
hormones, neuropeptides, chemokines, odorants, phospholipids, and functional
equivalents thereof that bind and preferably
inhibit 109P1 D4 protein function. Such non-limiting small molecules
preferably have a molecular weight of less than about
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, 109P1 D4 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/0.001 5 M sodium
citrate/0.1 % sodium dodecyl sulfate at 50 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 oC; or (3) employ 50% formamide, 5 x
SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),
0.1 % sodium pyrophosphate, 5 x
Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1 % SDS, and 10%
dextran sulfate at 42 oC, with washes at
42 C in 0.2 x SSC (sodium chloride/sodium. citrate) and 50% formamide at 55
oC, followed by a high-stringency wash
consisting of 0.1 x SSC containing EDTA at 55 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
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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 370C in a solution
comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5 x
Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared
salmon sperm DNA, followed by washing the
filters in 1 x SSC at about 37-50 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.
Overall phenotypic frequencies of HLA-supertypes in different ethnic
populations are set forth in Table IV (F). The non-
limiting constituents of various supetypes are as follows:
A3: A*0201, A*0202, A*0203, A*0204, A* 0205, A*0206, A*6802, A*6901, A*0207
A3: A3, All, A31, A*3301, A*6801, A*0301, A*1101, A*3101
B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*6701,
B*7801, B*0702, B*5101, B*5602
B44: B*3701, B*4402, B*44036B*60 (B*4001), B61 (B*4006)
AF A*0102, A*2604, A*3601, A*4301, A*8001
A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003
B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901, B*3902,
B*3903-04, B*4801-02, B*7301,
B*2701-08
B58: B*1516, B*1517, B*5701, B*5702, B58
B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)
Calculated population coverage afforded by different HLA-supertype
combinations are set forth in Table IV (G).
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
109P1 D4 protein shown in Figure 2 or Figure 3. An analog is an example of a
variant protein. Splice isoforms and single
nucleotides polymorphisms (SNPs) are further examples of variants.
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The "109P1 D4-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 109P1 D4 proteins or fragments thereof, as well as fusion proteins
of a 109P1 D4 protein and a heterologous polypeptide
are also included. Such 109P1 D4 proteins are collectively referred to as the
109P1 D4-related proteins, the proteins of the
invention, or 109P1 D4. The term "109P1 D4-related protein" refers to a
polypeptide fragment or a 109P1 D4 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, 105, 110, 115, 120, 125, 130, 135,
140, 145, 150, 155, 160, 165, 170, 175, 180, 185,
190,195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,
550, 575, or 576 or more amino acids.
II.) 109P1 4 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or
complementary to all or part of a 109P1 D4
gene, mRNA, and/or coding sequence, preferably in isolated form, including
polynucleotides encoding a 109P1 D4-related
protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related
molecules, polynucleotides or oligonucleotides
complementary to a 109P1 D4 gene or mRNA sequence or a part thereof, and
polynucleotides or oligonucleotides that
hybridize to a 109P1 D4 gene, mRNA, or to a 109P1 D4 encoding polynucleotide
(collectively, "109P1 D4 polynucleotides"). In
all instances when referred to in this section, T can also be U in Figure 2.
Embodiments of a 109P1 D4 polynucleotide include: a 109P1 D4 polynucleotide
having the sequence shown in
Figure 2, the nucleotide sequence of 109P1 D4 as shown in Figure 2 wherein T
is U; at least 10 contiguous nucleotides of a
polynucleotide having the sequence as shown in Figure 2; or, at least 10
contiguous nucleotides of a polynucleotide having
the sequence as shown in Figure 2 where T is U. For example, embodiments of
109P1 D4 nucleotides comprise, without
limitation:
(I) a polynucleotide comprising, consisting essentially of, or consisting of a
sequence as shown in Figure 2,
wherein T can also be U;
(II) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as shown in Figure
2A, from nucleotide residue number 846 through nucleotide residue number 3911,
including the stop codon,
wherein T can also be U;
(III) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as shown in Figure
2B, from nucleotide residue number 503 through nucleotide residue number 3667,
including the stop codon,
wherein T can also be U;
(IV) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as shown in Figure
2C, from nucleotide residue number 846 through nucleotide residue number 4889,
including the a stop codon,
wherein T can also be U;
(V) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as shown in Figure
2D, from nucleotide residue number 846 through nucleotide residue number 4859,
including the stop codon,
wherein T can also be U;
(VI) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as shown in Figure
2E, from nucleotide residue number 846 through nucleotide residue number 4778,
including the stop codon,
wherein T can also be U;
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(VII) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as shown in Figure
2F, from nucleotide residue number 614 through nucleotide residue number 3727,
including the stop codon,
wherein T can also be U;
(VIII) a polynucleotide comprising, consisting essentially of, or consisting
of the sequence as shown in Figure
2G, from nucleotide residue number 735 through nucleotide residue number 3881,
including the stop codon,
wherein T can also be U;
(IX) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as shown in Figure
2H, from nucleotide residue number 735 through nucleotide residue number 4757,
including the stop codon,
wherein T can also be U;
(X) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as shown in Figure
21, from nucleotide residue number 514 through nucleotide residue number 3627,
including the stop codon,
wherein T can also be U;
(XI) a polynucleotide that encodes a 109P1 D4-related protein that is at least
90, 91, 92, 93, 94, 95, 96, 97,
98, 99 or 100% homologous to an entire amino acid sequence shown in Figure 2A-
I;
(XII) a polynucleotide that encodes a 109P1 D4-related protein that is at
least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99 or 100% identical to an entire amino acid sequence shown in Figure 2A-
I;
(XIII) a polynucleotide that encodes at least one peptide set forth in Tables
VIII-XXI and XXII-XLIX;
(XIV) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figures 3A in any
whole number increment up to 1021 that includes at least 1, 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 amino acid
position(s) having a value greater than
0.5 in the Hydrophilicity profile of Figure 5;
(XV) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3A in any whole
number increment up to 1021 that includes 1, 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 amino acid position(s)
having a value less than 0.5 in the
Hydropathicity profile of Figure 6;
(XVI) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3A in any whole
number increment up to 1021 that includes 1, 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 amino acid position(s)
having a value greater than 0.5 in the
Percent Accessible Residues profile of Figure 7;
(XVII) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3A in any whole
number increment up to 1021 that includes 1, 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 amino acid position(s)
having a value greater than 0.5 in the
Average Flexibility profile of Figure 8;
(XVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8,
9, 10, 11, 12,13, 14,15, 16, 17,
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18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of Figure 3A in any
whole number increment up to 1021 that includes 1, 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 amino acid
position(s) having a value greater than 0.5 in
the Beta-turn profile of Figure 9;
(XIX) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3B, 3C, and/or
3D in any whole number increment up to 1054, 1347, and/or 1337 respectively
that includes 1, 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 amino acid
position(s) having a value greater than 0.5 in the Hydrophilicity profile of
Figure 5;
(XX) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3B, 3C, and/or
3D in any whole number increment up to 1054, 1347, and/or 1337 respectively
that includes 1, 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 amino acid
position(s) having a value less than 0.5 in the Hydropathicity profile of
Figure 6;
(XXI) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3B, 3C, and or
3D in any whole number increment up to 1054, 1347, and/or 1337 respectively
that includes 1, 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 amino acid
position(s) having a value greater than 0.5 in the Percent Accessible Residues
profile of Figure 7;
(XXII) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3B, 3C, and/or
3D in any whole number increment up to 1054, 1347, and/or 1337 respectively
that includes 1, 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 amino acid
position(s) having a value greater than 0.5 in the Average Flexibility profile
of Figure 8;
(XXIII) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3B, 3C, and/or
3D in any whole number increment up to 1054, 1347, and/or 1337 respectively
that includes 1, 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 amino acid
position(s) having a value greater than 0.5 in the Beta-turn profile of Figure
9;
(XXIV) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3E, 3F, 3G, 3H
and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or
1037 respectively that includes 1,
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 amino acid position(s) having a value greater than 0.5 in the
Hydrophilicity profile of Figure 5;
(XXV) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3E, 3F, 3G, 3H
and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or
1037 respectively that includes 1,
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 amino acid position(s) having a value less than 0.5 in the Hydropathicity
profile of Figure 6;
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(XXVI) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3E, 3F, 3G, 3H
and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or
1037 respectively that includes 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15,16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34,
35 amino acid position(s) having a value greater than 0.5 in the Percent
Accessible Residues profile of Figure 7;
(XXVII) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3E, 3F, 3G, 3H
and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or
1037 respectively that includes 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34,
35 amino acid position(s) having a value greater than 0.5 in the Average
Flexibility profile of Figure 8;
(XXVIII) a polynucleotide that encodes a peptide region of at least 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 amino acids
of a peptide of Figure 3E, 3F, 3G, 3H,
and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or
1037 respectively that includes 1,
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 amino acid position(s) having a value greater than 0.5 in the Beta-turn
profile of Figure 9;
(XXIX) a polynucleotide that is fully complementary to a polynucleotide of any
one of (I)-(XXVIII);
(XXX) a polynucleotide that is fully complementary to a polynucleotide of any
one of (I)-(XXIX);
(XXXI) a peptide that is encoded by any of (I) to (XXX); and;
(XXXII) a composition comprising a polynucleotide of any of (I)-(XXX) or
peptide of (XXXI) together with a
pharmaceutical excipient and/or in a human unit dose form;
(XXXIII) a method of using a polynucleotide of any (I)-(XXX) or peptide of
(XXXI) or a composition of (XXXII) in a
method to modulate a cell expressing 109P1 D4;
(XXXIV) a method of using a polynucleotide of any (I)-(XXX) or peptide of
(XXXI) or a composition of (XXXII) in a
method to diagnose, prophylax, prognose, or treat an individual who bears a
cell expressing 109P1 D4;
(XXXV) a method of using a polynucleotide of any (I)-(XXX) or peptide of
(XXXI) or a composition of (XXXII) in a
method to diagnose, prophylax, prognose, or treat an individual who bears a
cell expressing 109P1 D4, said cell
from a cancer of a tissue listed in Table I;
(XXXVI) a method of using a polynucleotide of any (I)-(XXX) or peptide of
(XXXI) or a composition of (XXXII) in a
method to diagnose, prophylax, prognose, or treat a a cancer;
(XXXVII) a method of using a polynucleotide of any (1)-(XXX) or peptide of
(XXXI) or a composition of (XXXII) in a
method to diagnose, prophylax, prognose, or treat a a cancer of a tissue
listed in Table 1; and;
(XXXVIII) a method of using a polynucleotide of any (I)-(XXX) or peptide of
(XXXI) or a composition of (XXXII) in a
method to identify or characterize a modulator of a cell expressing 109P1 D4.
As used herein, a range is understood to disclose specifically all whole unit
positions thereof.
Typical embodiments of the invention disclosed herein include 109P1 D4
polynucleotides that encode specific
portions of 109P1 D4 mRNA sequences (and those which are complementary to such
sequences) such as those that encode
the proteins and/or fragments thereof, for example: .
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24
(a) 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,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,
600, 625, 650, 675, 700, 725, 750, 775, 800,
825, 850, 875, 900, 925, 950, 975, 1000, 1010, 1020, and 1021 or more
contiguous amino acids of 109P1 D4 variant 1; the
maximal lengths relevant for other variants are: variant 2, 1054 amino acids;
variant 3, 1347 amino acids, variant 4, 1337
amino acids, variant 5,1310 amino acids, variant 6; 1047 amino acids, variant
7; 1048 amino acids, variant 8; 1340 amino
acids and variant 9; 1037 amoni 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 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino
acid 20 of the 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 20 to about amino
acid 30 of the 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino
acid 40 of the 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino
acid 50 of the 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino
acid 70 of the 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino
acid 80 of the 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino
acid 90 of the 109P1 D4 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 90 to about amino
acid 100 of the 109P1 D4 protein shown in
Figure 2 or Figure 3, in increments of about 10 amino acids, ending at the
carboxyl terminal amino acid set forth in Figure 2
or Figure 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 109P1 D4 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 a 109P1 D4 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 109P1 D4 protein "or variant" shown in Figure 2 or Figure 3
can be generated by a variety of techniques well
known in the art. These polynucleotide fragments can include any portion of
the 109PI D4 sequence as shown in Figure 2.
Additional illustrative embodiments of the invention disclosed herein include
109P1 D4 polynucleotide fragments
encoding one or more of the biological motifs contained within a 109P1 D4
protein or variant" sequence, including one or
more of the motif-bearing subsequences of a 109P1 D4 protein "or variant" set
forth in Tables VIII-XXI and XXII-XLIX. In
another embodiment, typical polynucleotide fragments of the invention encode
one or more of the regions of 109P1 D4
protein or variant that exhibit homology to a known molecule. In another
embodiment of the invention, typical polynucleotide
fragments can encode one or more of the 109P1 D4 protein or variant N-
glycosylation sites, cAMP and cGMP-dependent
protein kinase phosphorylation sites, casein kinase II phosphorylation sites
or N-myristoylation site and amidation sites.
Note that to determine the starting position of any peptide set forth in
Tables VIII-XXI and Tables XXII to XLIX
(collectively HLA Peptide Tables) respective to its parental protein, e.g.,
variant 1, variant 2, etc., reference is made to three
factors: the particular variant, the length of the peptide in an HLA Peptide
Table, and the Search Peptides listed in Table VII.
Generally, a unique Search Peptide is used to obtain HLA peptides for a
particular variant. The position of each Search
Peptide relative to its respective parent molecule is listed in Table VII.
Accordingly, if a Search Peptide begins at position
"X", one must add the value "X minus 1" to each position in Tables VIII-XXI
and Tables XXII-IL to obtain the actual position of
the HLA peptides in their parental molecule. For example if a particular
Search Peptide begins at position 150 of its parental
molecule, one must add 150 - 1, i.e., 149 to each HLA peptide amino acid
position to calculate the position of that amino acid
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in the parent molecule.
II.A.) Uses of 109P1 D4 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different
specific uses. The human 109P1 D4
gene maps to the chromosomal location set forth in the Example entitled
"Chromosomal Mapping of 109P1 D4." For
example, because the 109P1 D4 gene maps to this chromosome, polynucleotides
that encode different regions of the
109P1 D4 proteins 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 109P1 D4 proteins provide new
tools that can be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the
chromosomal region that encodes 109P1 D4 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 109P1 D4 was shown to be highly expressed in prostate and
other cancers, 109P1 D4
polynucleotides are used in methods assessing the status of 109P1 D4 gene
products in normal versus cancerous tissues.
Typically, polynucleotides that encode specific regions of the 109P1 D4
proteins 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 109P1 D4 gene, such as 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 109P1 D4. 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
109P1 D4 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.,109P1 D4. See for example, Jack Cohen,
Oligodeoxynucleotides, Antisense Inhibitors of Gene
Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 109P1 D4
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 (0-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 0-oligos with 3H-1,2-
benzodithiol-3-one-1,I-dioxide, which is a sulfur transfer reagent. See, e.g.,
lyer, R. P. et al., J. Org. Chem. 55:4693-4698
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26
(1990); and Iyer, R. P. eta!., J. Am. Chem. Soc. 112:1253-1254 (1990).
Additional 109P1 D4 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 109P1 D4 antisense oligonucleotides of the present invention typically can
be RNA or DNA that is
complementary to and stably hybridizes with the first 100 5' colons or last
100 3' codons of a 109P1 D4 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 109P1 D4 mRNA
and not to mRNA specifying other regulatory subunits of protein kinase. In one
embodiment, 109P1 D4 antisense
oligonucleotides of the present invention are 15 to 30-mer fragments of the
antisense DNA molecule that have a sequence
that hybridizes to 109P1 D4 mRNA. Optionally, 109P1 D4 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
109P1 D4. Alternatively, the antisense molecules
are modified to employ ribozymes in the inhibition of 109P1 D4 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 these 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
109P1 D4 polynucleotide in a sample and as a means for detecting a cell
expressing a 109P1 D4 protein.
Examples of such probes include polypeptides comprising all or part of the
human 109P1 D4 cDNA sequence shown in
Figure 2. Examples of primer pairs capable of specifically amplifying 109P1 D4
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 109P1 D4 mRNA.
The 109P1 D4 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 109P1
D4 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 109P1 D4 polypeptides; as tools for modulating or inhibiting
the expression of the 109P1 D4 gene(s) and/or
translation of the 109P1 D4 transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to
identify and isolate a 109P1 D4 or 109P1 D4
related nucleic acid sequence from a naturally occurring source, such as
humans or other mammals, as well as the isolated
nucleic acid sequence perse, which would comprise all or most of the sequences
found in the probe used.
II.A.4.) Isolation of 109P1 D4-Encoding Nucleic Acid Molecules
The 109P1 D4 cDNA sequences described herein enable the isolation of other
polynucleotides encoding 109P1 D4 gene
product(s), as well as the isolation of polynucleotides encoding 109P1 D4 gene
product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of a 109P1 D4 gene product as well as
polynucleotides that encode analogs of 109P1 D4-related
proteins. Various molecular cloning methods that can be employed to isolate
full length cDNAs encoding a 109P1 D4 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 109P1 D4 gene cDNAs can be
identified by probing with a labeled 109P1 D4
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27
cDNA or a fragment thereof. For example, in one embodiment, a 109P1 D4 cDNA
(e.g., Figure 2) or a portion thereof can be
synthesized and used as a probe to retrieve overlapping and full-length cDNAs
corresponding to a 109P1 D4 gene. A 109P1 D4
gene itself can be isolated by screening genomic DNA libraries, bacterial
artificial chromosome libraries (BACs), yeast artificial
chromosome libraries (YACs), and the like, with 109P1 D4 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 a
109P1 D4 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 a!.,1989, supra).
The invention further provides a host-vector system comprising a recombinant
DNA molecule containing a 109P1 D4
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 TsuPr1, 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 109P1 D4 or a fragment, analog
or homolog thereof can be used to generate 109P1 D4 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 109P1 D4
proteins or fragments thereof are available,
see for example, Sambrook at a1.,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
pSRatkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors,
109P1 D4 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 109P1 D4 protein
or fragment thereof. Such host-vector systems
can be employed to study the functional properties of 109P1 D4 and 109P1 D4
mutations or analogs.
Recombinant human 109P1 D4 protein or an analog or homolog or fragment thereof
can be produced by
mammalian cells transfected with a construct encoding a 109P1 D4-related
nucleotide. For example, 293T cells can be
transfected with an expression plasmid encoding 109P1 D4 or fragment, analog
or homolog thereof, a 109P1 D4-related
protein is expressed in the 293T cells, and the recombinant 109P1 D4 protein
is isolated using standard purification methods
(e.g., affinity purification using anti-109P1 D4 antibodies). In another
embodiment, a 109P1 D4 coding sequence is subcloned
into the retroviral vector pSRaMSVtkneo and used to infect various mammalian
cell lines, such as NIH 3T3, TsuPr1, 293 and
rat-1 in order to establish 109P1 D4 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 a 109P1 D4 coding sequence can be used
for the generation of a secreted form of recombinant 109PI D4 protein.
As discussed herein, redundancy in the genetic code permits variation in 109P1
D4 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 colons. Codon preferences for a specific species are calculated, for
example, by utilizing codon usage tables
available on the INTERNET such as at URL dna.affrc.go.jp/-nakamura/codon.html.
Additional sequence modifications are known to enhance protein expression in a
cellular host. These include
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28
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, M l. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that
the general rule that eukaryotic ribosomes
initiate translation exclusively at the 5' proximal AUG colon 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. 109P1 D4-related Proteins
Another aspect of the present invention provides 109P1 D4-related proteins.
Specific embodiments of 109P1 D4
proteins comprise a polypeptide having all or part of the amino acid sequence
of human 109P1 D4 as shown in Figure 2 or
Figure 3. Alternatively, embodiments of 109P1 D4 proteins comprise variant,
homolog or analog polypeptides that have
alterations in the amino acid sequence of 109P1 D4 shown in Figure 2 or Figure
3.
Embodiments of a 109P1 D4 polypeptide include: a 109P1 D4 polypeptide having a
sequence shown in Figure 2, a
peptide sequence of a 109P1 D4 as shown in Figure 2 wherein T is U; at least
10 contiguous nucleotides of a polypeptide
having the sequence as shown in Figure 2; or, at least 10 contiguous peptides
of a polypeptide having the sequence as
shown in Figure 2 where T is U. For example, embodiments of 109P1 D4 peptides
comprise, without limitation:
(I) a protein comprising, consisting essentially of, or consisting of an amino
acid sequence as shown in
Figure 2A-I or Figure 3A-l;
(II) a 109P1 D4-related protein that is at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99 or 100% homologous to
an entire amino acid sequence shown in Figure 2A-I or 3A-I;
(III) a 109P1 D4-related protein that is at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99 or 100% identical to an
entire amino acid sequence shown in Figure 2A-I or 3A-l; %
(IV) a protein that comprises at least one peptide set forth in Tables VIII to
XLIX, optionally with a proviso
that it is not an entire protein of Figure 2;
(V) a protein that comprises at least one peptide set forth in Tables VIII-
XXI, collectively, which peptide is
also set forth in Tables XXII to XLIX, collectively, optionally with a proviso
that it is not an entire protein of Figure 2;
(VI) a protein that comprises at least two peptides selected from the peptides
set forth in Tables VII I-XLIX,
optionally with a proviso that it is not an entire protein of Figure 2;
(VII) a protein that comprises at least two peptides selected from the
peptides set forth in Tables VIII to XLIX
collectively, with a proviso that the protein is not a contiguous sequence
from an amino acid sequence of Figure 2;
(VIII) a protein that comprises at least one peptide selected from the
peptides set forth in Tables VIII-XXI; and
at least one peptide selected from the peptides set forth in Tables XXII to
XLIX, with a proviso that the protein is
not a contiguous sequence from an amino acid sequence of Figure 2;
(IX) a polypeptide comprising at least 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 amino acids of a protein of Figure
3A, 3B, 3C, 3D and/or 3E in any whole
number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively that
includes at least 1, 2, 3, 4, 5,
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29
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 amino
acid position(s) having a value greater than 0.5 in the Hydrophilicity profile
of Figure 5;
(X) a polypeptide comprising at least 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 amino acids of a protein of Figure
3A, 3B, 3C, 3D, and/or 3E, in any
whole number increment up to 1021, and/or 1310 respectively respectively that
includes at least
at least 1, 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 amino acid position(s) having a value less than 0.5 in the
Hydropathicity profile of Figure 6;
(XI) a polypeptide comprising at least 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 amino acids of a protein of Figure
3A, 3B, 3C, 3D, and/or 3E, in any whole
number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively
respectively that includes at least at
least 1, 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 amino acid position(s) having a value greater than 0.5 in the
Percent Accessible Residues profile of
Figure 7;
(XII) a polypeptide comprising at least 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 amino acids of a protein of Figure
3A, 3B, 3C, 3D, and/or 3E, in any
whole number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively
respectively that includes at least
at least 1, 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 amino acid position(s) having a value greater than 0.5 in the
Average Flexibility profile of Figure 8;
(XIII) a polypeptide comprising at least 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, amino acids of a protein of Figure 3A,
3B, 3C, 3D, and 3E in any whole
number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively
respectively that includes at least at
least 1, 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 amino acid position(s) having a value greater than 0.5 in the Beta-
turn profile of Figure 9;
(XIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 14, 18, 19, 20, 21,
22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure
3F, 3G, 3H, and/or 31, in any whole
number increment up to 1037, 1048, 1340, and/or 1037 respectively that
includes at least 1, 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 amino acid
position(s) having a value greater than 0.5 in the Hydrophilicity profile of
Figure 5;
(XV) a polypeptide comprising at least 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 amino acids of a protein of Figure
3F, 3G, 3H, and/or 31 in any whole
number increment up to 1037, 1048, 1340, and/or 1037 respectively that
includes at least at least 1, 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 amino acid
position(s) having a value less than 0.5 in the Hydropathicity profile of
Figure 6;
(XVI) a polypeptide comprising at least 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 amino acids of a protein of Figure
3F, 3G, 3H, and/or 31 in any whole
number increment up to 1037, 1048, 1340, and/or 1037 respectively that
includes at least at least 1, 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 amino acid
position(s) having a value greater than 0.5 in the Percent Accessible Residues
profile of Figure 7;
(XVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
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25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure
3F, 3G, 3H, and/or 31 in any whole
number increment up to 1037, 1048, 1340, and/or 1037 respectively that
includes at least at least 1, 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 amino acid
position(s) having a value greater than 0.5 in the Average Flexibility profile
of Figure 8;
(XVIII) a polypeptide comprising at least 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, amino acids of a protein of Figure 3F,
3G, 3H, and/or 31 in any whole number
increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at
least at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35 amino acid
position(s) having a value greater than 0.5 in the Beta-turn profile of Figure
9;
(XIX) a peptide that occurs at least twice in Tables VIII-XXI and XXII to
XLIX, collectively;
(XX) a peptide that occurs at least three times in Tables VIII-XXI and XXII to
XLIX, collectively;
(XXI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to
XLIX, collectively;
(XXII) a peptide that occurs at least five times in Tables VIII-XXI and XXII
to XLIX, collectively;
(XXIII) a peptide that occurs at least once in Tables VIII-XXI, and at least
once in tables XXII to XLIX;
(XXIV) a peptide that occurs at least once in Tables VIII-XXI, and at least
twice in tables XXII to XLIX;
(XXV) a peptide that occurs at least twice in Tables VIII-XXI, and at least
once in tables XXII to XLIX;
(XXVI) a peptide that occurs at least twice in Tables Vill-M, and at least
twice in tables XXII to XLIX;
(XXVII) a peptide which comprises one two, three, four, or five of the
following characteristics, or an
oligonucleotide encoding such peptide:
1) a region of at least 5 amino acids of a particular peptide of Figure 3, in
any whole number increment
up to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or
greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the
Hydrophilicity profile of Figure 5;
ii) a region of at least 5 amino acids of a particular peptide of Figure 3, in
any whole number increment
up to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or less
than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the
Hydropathicity profile of Figure 6;
iii) a region of at least 5 amino acids of a particular peptide of Figure 3,
in any whole number increment
up to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or
greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the
Percent Accessible Residues profile of
Figure 7;
iv) a region of at least 5 amino acids of a particular peptide of Figure 3, in
any whole number increment
up to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or
greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the
Average Flexibility profile of Figure 8; or,
v) a region of at least 5 amino acids of a particular peptide of Figure 3, in
any whole number increment
up to the full length of that protein in Figure 3, that includes an amino acid
position having a value equal to or
greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the
Beta-turn profile of Figure 9;;
(XXVIII) a composition comprising a peptide of (I)-(XXVII) or an antibody or
binding region thereof together with a
pharmaceutical excipient and/or in a human unit dose form.
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(XXIX) a method of using a peptide of (I)-(XXVII), or an antibody or binding
region thereof or a composition of
(XXVIII) in a method to modulate a cell expressing 109P1 D4,;
(XXX) a method of using a peptide of (I)-(XXVII) or an antibody or binding
region thereof or a composition of
(XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual
who bears a cell expressing
109P1D4;
(XXXI) a method of using a peptide of (I)-(XXVII) or an antibody or binding
region thereof or a composition
(XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual
who bears a cell expressing
109P1D4, said cell from a cancer of a tissue listed in Table I;
(XXXII) a method of using a peptide of (I)-(XXVII) or an antibody or binding
region thereof or a composition of
(XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
(XXXIII) a method of using a peptide of (I)-(XXVII) or an antibody or binding
region thereof or a composition of
(XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of
a tissue listed in Table I; and;
(XXXIV) a method of using a a peptide of (I)-(XXVII) or an antibody or binding
region thereof or a composition
(XXVIII) in a method to identify or characterize a modulator of a cell
expressing 109P1 D4
As used herein, a range is understood to specifically disclose all whole unit
positions thereof.
Typical embodiments of the invention disclosed herein include 109P1D4
polynucleotides that encode specific
portions of 109P1D4 mRNA sequences (and those which are complementary to such
sequences) such as those that encode
the proteins and/or fragments thereof, for example:
(a) 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,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,
600, 625, 650, 675, 700, 725, 750, 775, 800,
825, 850, 875, 900, 925, 950, 975, 1000, 1010, 1020, and 1021 or more
contiguous amino acids of 109P1 D4 variant 1; the
maximal lengths relevant for other variants are: variant 2, 1054 amino acids;
variant 3, 1347 amino acids, variant 4, 1337
amino acids, variant 5, 1310 amino acids, variant 6; 1037 amino acids, variant
7; 1048 amino acids, variant 8; 1340 amino
acids, and variant 9; 1037 amino acids. .
In general, naturally occurring allelic variants of human 109P1 D4 share a
high degree of structural identity and
homology (e.g., 90% or more homology). Typically, allelic variants of a 109P1
D4 protein contain conservative amino acid
substitutions within the 109P1 D4 sequences described herein or contain a
substitution of an amino acid from a corresponding
position in a homologue of 109P1 D4. One class of 109P1 D4 allelic variants
are proteins that share a high degree of homology
with at least a small region of a particular 109P1 D4 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
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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" 2nd 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
109P1D4 proteins such as polypeptides having amino acid insertions, deletions
and substitutions. 109P1D4 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 at al., Philos. Trans. R.
Soc. London SerA, 317:415 (1986)) or other known techniques can be performed
on the cloned DNA to produce the
109P1 D4 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, 109P1 D4 variants, analogs or homologs, have the
distinguishing attribute of having at least one
epitope that is "cross reactive" with a 109P1D4 protein having an amino acid
sequence of Figure 3. As used in this
sentence, "cross reactive" means that an antibody or T cell that specifically
binds to a 109P1 D4 variant also specifically binds
to a 109P1 D4 protein having an amino acid sequence set forth in Figure 3. A
polypeptide ceases to be a variant of a protein
shown in Figure 3, when it no longer contains any epitope capable of being
recognized by an antibody or T cell that
specifically binds to the starting 109P1 D4 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.
Other classes of 109P1 D4-related protein variants share 70%, 75%, 80%, 85% or
90% or more similarity with an
amino acid sequence of Figure 3, or a fragment thereof. Another specific class
of 109P1 D4 protein variants or analogs
comprises one or more of the 109P1 D4 biological motifs described herein or
presently known in the art. Thus, encompassed
by the present invention are analogs of 109P1 D4 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 Figure 2 or
Figure 3.
As discussed herein, embodiments of the claimed invention include polypeptides
containing less than the full
amino acid sequence of a 109P1 D4 protein shown in Figure 2 or Figure 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 a
CA 02522994 2010-03-25
33
109P1 D4 protein shown in Figure 2 or Figure 3.
Moreover, representative embodiments of the invention disclosed herein include
polypeptides consisting of about
amino acid I to about amino acid 10 of a 109P1 D4 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 10 to about amino acid 20 of a 109P1D4 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 20 to about amino acid 30 of a 109P1 D4 protein shown in Figure 2
or Figure 3, polypeptides consisting of about
amino add 30 to about amino acid 40 of a 109P1 D4 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 40 to about amino acid 50 of a 109P1 D4 protein shown in Figure 2
or Figure 3, polypeptides consisting of about
amino acid 50 to about amino acid 60 of a 109P1 D4 protein shown in Figure 2
or Figure 3, polypeptides consisting of about
amino acid 60 to about amino acid 70 of a 109P1D4 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 70 to about amino acid 80 of a 109P1D4 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 80 to about amino acid 90 of a 109P1D4 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 90 to about amino acid 100 of a 109P1 D4 protein shown in Figure 2
or Figure 3, etc. throughout the entirety of a
109P1 D4 amino acid sequence. Moreover, polypeptides consisting of about amino
acid I (or 20 or 30 or 40 etc.) to about
amino acid 20, (or 130, or 140 or 150 etc.) of a 109P1D4 protein shown in
Figure 2 or Figure 3 are embodiments of the
invention. lt.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.
109P1 D4-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
109P1 D4-related protein. In one embodiment, nucleic acid molecules provide a
means to generate defined fragments of a
109P1 D4 protein (or variants, homologs or analogs thereof).
III.A.) Motif-bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include
109P1D4 polypeptides comprising the
amino acid residues of one or more of the biological motifs contained within a
109P1 D4 polypeptide sequence set forth in
Figure 2 or Figure 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., EpimatrixTM and
EpimerTM, Brown University and BIMAS).
Motif bearing subsequences of all 109P1 D4 variant proteins are set forth and
identified in Tables Vlll-XXI and XXII-
XLIX.
Table V sets forth several frequently occurring motifs based on pfam searches.
The columns of Table V 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 109P1 D4 motifs discussed above are
useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation that the
109P1 D4 motifs discussed above are associated
with growth dysregulation and because 109PID4 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 Adds 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
CA 02522994 2010-03-25
34
myristoylation are protein modifications also associated with cancer and
cancer progression (see e.g. Dennis at at, Biochem.
Biophys. Acta 1473(1):21-34 (1999); Raju of 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 at 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 VIII-XXI and XXiI-XLIX. CTL epitopes can
be determined using specific algorithms to identify peptides within a 109P1 D4
protein that are capable of optimally binding to
specified HLA alleles (e.g., Table IV; EpimabixTm and Epimer1 ", Brown
University ).
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 li
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, on the basis of residues defined in Table
IV, one can substitute out a deleterious residue in favor of any other
residue, such as a preferred residue; substitute a less-
preferred residue with a preferred residue; or substitute an originally-
occurring preferred residue with another preferred
residue. 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 97/33602 to Chesnut et al.;
Sette, Immunogenetics 1999 50(3-4): 201-
212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum.
immunol.1997 58(1):12-20; Kondo et al.,
Immunogenetics 1997 45(4): 249-258; Sidney et at, J. Immunol. 1996 157(8):
3480-90; and Falk et al., Nature 351: 290-6
(1991); Hunt et at, Science 255:1261-3 (1992); Parker et al., J. Immunol.
149:3580-7 (1992); Parker at at, J. Immunol.
152:163-75 (1994)); Kast et a/., 1994 152(8): 3904-12; Borras-Cuesta et al.,
Hum. Immunol. 2000 61(3): 266-278; Alexander
et al., J. Immunol. 2000164(3);164(3):1625-1633; Alexander et al, PMID:
7895164, UI: 95202582; O'Sullivan et al., J.
Immunol. 1991147(8): 2663-2669; Alexander et al., Immunity 19941(9): 751-761
and Alexander et at, Immunol. Res. 1998
18(2): 79-92.
Related embodiments of the invention include polypeptides comprising
combinations of the different motifs set
forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables
VIII-XXI and XXII-XLIX, and/or, one or more of
the predicted HTL epitopes of Tables XLVI-XLiX, 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 within 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.
109P1 D4-related proteins are embodied in many forms, preferably in isolated
form. A purified 109P1 D4 protein
molecule will be substantially free of other proteins or molecules that impair
the binding of 109P1 D4 to antibody, T cell or
other ligand. The nature and degree of isolation and purification will depend
on the intended use. Embodiments of a 109P1 D4-
related proteins include purified 109P1D4-related proteins and functional,
soluble 109P1D4-related proteins. In one
embodiment, a functional, soluble 109P1 D4 protein or fragment thereof retains
the ability to be bound by antibody, T cell or
CA 02522994 2010-03-25
other ligand.
The invention also provides 109P1 D4 proteins comprising biologically active
fragments of a 109P1 D4 amino acid
sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of
the starting 109P1 D4 protein, such as the ability
to elicit the generation of antibodies that specifically bind an epitope
associated with the starting 109P.1 D4 protein; to be
bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to
be recognized by HTL or CTL that also specifically
bind to the starting protein.
109P1 D4-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, Gamier-Robson, Kyle-
Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on
immunogenicity. Fragments that contain such
structures are particularly useful in generating subunit-specific anti-109P1
D4 antibodies or T cells or in identifying cellular factors
that bind to 109P1 D4. For example, hydrophilicity profiles can be generated,
and immunogenic peptide fragments identified,
using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci.
U.S.A. 78:3824-3828. Hydropathicity profiles
can be generated, and immunogenic peptide fragments identified, using the
method of Kyle, J. and Doolittle, R.F., 1982, J.
Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be
generated, and immunogenic peptide fragments
identified, using the method of Janin J., 1979, Nature 277:491-492. Average
Flexibility profiles can be generated, and
immunogenic peptide fragments identified, using the method of Bhaskaran R.,
Ponnuswamy P.K., 1988, Int. J. Pept. Protein
Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide
fragments identified, using the method of
Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.
CTL epitopes can be determined using specific algorithms to identify peptides
within a 109P1 D4 protein that are
capable of optimally binding to specified HIA alleles (e.g., by using the
SYFPEITHI site at World Wide Web; EpimatrixTM
and EpimerTM; Brown University and BIMAS).,
Illustrating this, peptide epitopes from 109P1 D4 that
are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2,
A3, Al 1, A24, B7 and B35 were predicted
(see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid
sequence of the 109P1D4 protein and relevant
portions of other variants, i.e., for HLA Class I predictions 9 flanking
residues on either side of a point mutation or exon
juction, and for HLA Class II predictions 14 flanking residues on either side
of a point mutation or exon junction
corresponding to that variant, were entered into the HLA Peptide Motif Search
algorithm found in the Bioinfbrmatics and
Molecular Analysis Section (BIMAS) web site ; in addition to the site
SYFPEITHI.
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 eta/., Nature 351: 290-6 (1991);
Hunt et al., Science 255:1261-3 (1992); Parker of aL, J. Immunol. 149:3580-7
(1992); Parker et aL, J. lmmunol.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 109P1D4 predicted binding peptides are shown in Tables
VIII-XXI and XXII-XLIX herein. In Tables VIII-
XXI and XXII-XLVII, selected 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. In Tables XLVI-XLIX, selected
candidates, 15-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
CA 02522994 2010-03-25
36
complexes containing the peptide at 370C 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 at., Prostate 30:73-8
(1997) and Peshwa at a!., Prostate 36:129-38
(1998)). imrnunogenicity 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,
EpimerTM and EpimatrixTM 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 (or
determined using World Wide Web site ) are to be "applied"
to a 109P1 D4 protein in accordance with the invention. As used in this
context "applied" means that a 109P1 D4 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 a 109P1D4 protein 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.
1II.B.) Expression of 109P1D4-related Proteins
In an embodiment described in the examples that follow, 109P1 D4 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
109P1 D4 with a C-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen or
Tag5, GenHunter Corporation, Nashville
TN). The Tag5 vector provides an I9GK secretion signal that can be used to
facilitate the production of a secreted 109P1 D4
protein in transfected cells. The secreted HIS-tagged 109P1D4 in the culture
media can be purified, e.g., using a nickel
column using standard techniques.
III.C.) Modifications of 109P1D4-related Proteins
Modifications of 109P1D4-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 109P1D4 polypeptide with
an organic derivatizing agent that is capable of reacting with selected side
chains or the N- or C- terminal residues of a
109P1 D4 protein. Another type of covalent modification of a 109P1D4
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
109P1D4 comprises linking a 109P1 D4 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. Patent Nos. 4,640,835; 4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The 109P1D4-related proteins of the present invention can also be modified to
form a chimeric molecule
comprising 109P1 D4 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 a 109P1D4 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 Figure 2
or Figure 3. Such a chimeric molecule can
comprise multiples of the same subsequence of 109P1D4. A chimeric molecule can
comprise a fusion of a 109P1D4-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 a 109P1 D4
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37
protein. In an alternative embodiment, the chimeric molecule can comprise a
fusion of a 109P1 D4-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 109P1 D4 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, CHI, CH2 and CH3 regions of an IgGI molecule. For the
production of immunoglobulin fusions see,
e.g., U.S. Patent No. 5,428,130 issued June 27, 1995.
III.D.) Uses of 109P1D4-related Proteins
The proteins of the invention have a number of different specific uses. As
109P1 D4 is highly expressed in prostate
and other cancers, 109P1 D4-related proteins are used in methods that assess
the status of 109P1 D4 gene products in
normal versus cancerous tissues, thereby elucidating the malignant phenotype.
Typically, polypeptides from specific regions
of a 109P1 D4 protein are used to assess the presence of perturbations (such
as deletions, insertions, point mutations etc.) in
those regions (such as regions containing one or more motifs). Exemplary
assays utilize antibodies or T cells targeting
109P1 D4-related proteins comprising the amino acid residues of one or more of
the biological motifs contained within a
109P1 D4 polypeptide sequence in order to evaluate the characteristics of this
region in normal versus cancerous tissues or
to elicit an immune response to the epitope. Alternatively, 109P1 D4-related
proteins that contain the amino acid residues of
one or more of the biological motifs in a 109P1 D4 protein are used to screen
for factors that interact with that region of
109P1D4.
109P1D4 protein fragments/subsequences are particularly useful in generating
and characterizing domain-specific
antibodies (e.g., antibodies recognizing an extracellular or intracellular
epitope of a 109P1 D4 protein), for identifying agents or
cellular factors that bind to 109P1 D4 or a particular structural domain
thereof, and in various therapeutic and diagnostic contexts,
including but not limited to diagnostic assays, cancer vaccines and methods of
preparing such vaccines.
Proteins encoded by the 109P1 D4 genes, or by analogs, homologs or fragments
thereof, have a variety of uses,
including but not limited to generating antibodies and in methods for
identifying ligands and other agents and cellular
constituents that bind to a 109P1 D4 gene product. Antibodies raised against a
109P1 D4 protein or fragment thereof are useful
in diagnostic and prognostic assays, and imaging methodologies in the
management of human cancers characterized by
expression of 109P1 D4 protein, such as those listed in Table I. Such
antibodies can be expressed intracellularly and used in
methods of treating patients with such cancers. 109P1 D4-related nucleic acids
or proteins are also used in generating HTL
or CTL responses.
Various immunological assays useful for the detection of 109P1 D4 proteins are
used, including but not limited to various
types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-
linked immunofluorescent assays (ELIFA),
immunocytochemical methods, and the like. Antibodies can be labeled and used
as immunological imaging reagents capable of
detecting 109P1 D4-expressing cells (e.g., in radioscintigraphic imaging
methods). 109P1 D4 proteins are also particularly useful in
generating cancer vaccines, as further described herein.
IV.) 109P1 D4 Antibodies
Another aspect of the invention provides antibodies that bind to 109P1 D4-
related proteins. Preferred antibodies
specifically bind to a 109P1 D4-related protein and do not bind (or bind
weakly) to peptides or proteins that are not 109P1 D4-
related proteins under physiological conditions. In this context, examples of
physiological conditions include: 1) phosphate
buffered saline; 2) Tris-buffered saline containing 25mM Tris and 150 mM NaCl;
or normal saline (0.9% NaCI); 4) animal serum
CA 02522994 2005-10-20
WO 2004/098515 PCT/US2004/013568
38
such as human serum; or, 5) a combination of any of 1) through 4); these
reactions preferably taking place at pH 7.5, alternatively
in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5;
also, these reactions taking place at a temperature
between 4 C to 37 C. For example, antibodies that bind 109P1 D4 can bind 109P1
D4-related proteins such as the homologs or
analogs thereof.
109P1 D4 antibodies of the invention are particularly useful in cancer (see,
e.g., Table I) diagnostic and prognostic
assays, and imaging methodologies. Similarly, such antibodies are useful in
the treatment, diagnosis, and/or prognosis of
other cancers, to the extent 109P1 D4 is also expressed or overexpressed in
these other cancers. Moreover, intracellularly
expressed antibodies (e.g., single chain antibodies) are therapeutically
useful in treating cancers in which the expression of
109PI D4 is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the
detection and quantification of 109PI D4 and
mutant 109P1 D4-related proteins. Such assays can comprise one or more 109P1
D4 antibodies capable of recognizing and
binding a 109PI D4-related protein, as appropriate. These assays are performed
within various immunological assay formats well
known in the art, including but not limited to various types of
radioimmunoassays, enzyme-linked immunosorbent assays (ELISA),
enzyme-linked immunofluorescent assays (ELIFA), and the like.
Immunological non-antibody assays of the invention also comprise T cell
immunogenicity assays (inhibitory or
stimulatory) as well as major histocompatibility complex (MHC) binding assays.
In addition, immunological imaging methods capable of detecting prostate
cancer and other cancers expressing
109P1 D4 are also provided by the invention, including but not limited to
radioscintigraphic imaging methods using labeled
109P1 D4 antibodies. Such assays are clinically useful in the detection,
monitoring, and prognosis of 109P1 D4 expressing
cancers such as prostate cancer.
109P1 D4 antibodies are also used in methods for purifying a 109P1 D4-related
protein and for isolating 109P1 D4
homologues and related molecules. For example, a method of purifying a 109P1
D4-related protein comprises incubating a
109P1 D4 antibody, which has been coupled to a solid matrix, with a lysate or
other solution containing a 109P1 D4-related protein
under conditions that permit the 109P1 D4 antibody to bind to the 109P1 D4-
related protein; washing the solid matrix to eliminate
impurities; and eluting the 109P1 D4-related protein from the coupled
antibody. Other uses of 109P1 D4 antibodies in
accordance with the invention include generating anti-idiotypic antibodies
that mimic a 109P1 D4 protein.
Various methods for the preparation of antibodies are well known in the art.
For example, antibodies can be prepared
by immunizing a suitable mammalian host using a 109P1 D4-related protein,
peptide, or fragment, in isolated or
immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds.,
Harlow, and Lane (1988); Harlow, Antibodies, Cold
Spring Harbor Press, NY (1989)). In addition, fusion proteins of 109PI D4 can
also be used, such as a 109P1 D4 GST-fusion
protein. In a particular embodiment, a GST fusion protein comprising all or
most of the amino acid sequence of Figure 2 or Figure
3 is produced, then used as an immunogen to generate appropriate antibodies.
In another embodiment, a 109P1 D4-related
protein is synthesized and used as an immunogen.
In addition, naked DNA immunization techniques known in the art are used (with
or without purified 109P1 D4-related
protein or 109P1 D4 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly of
al., 1997, Ann. Rev. Immunol.15: 617-648).
The amino acid sequence of a 109P1 D4 protein as shown in Figure 2 or Figure 3
can be analyzed to select specific
regions of the 109PI D4 protein for generating antibodies. For example,
hydrophobicity and hydrophilicity analyses of a 109P1 D4
amino acid sequence are used to identify hydrophilic regions in the 109P1 D4
structure. Regions of a 109P1 D4 protein that show
immunogenic structure, as well as other regions and domains, can readily be
identified using various other methods known in the
art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-
Schultz or Jameson-Wolf analysis. Hydrophilicity
CA 02522994 2005-10-20
WO 2004/098515 PCT/US2004/013568
39
profiles can be generated using the method of Hopp, T.P. and Woods, K.R.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-
3828. Hydropathicity profiles can be generated using the method of Kyte, J.
and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-
132. Percent (%) Accessible Residues profiles can be generated using the
method of Janin J., 1979, Nature 277:491-492.
Average Flexibility profiles can be generated using the method of Bhaskaran
R., Ponnuswamy P.K., 1988, Int. J. Pept.
Protein Res. 32:242-255. Beta-turn profiles can be generated using the method
of Deleage, G., Roux B., 1987, Protein
Engineering 1:289-294. Thus, each region identified by any of these programs
or methods is within the scope of the present
invention. Methods for the generation of 109P1 D4 antibodies are further
illustrated by way of the examples provided herein.
Methods for preparing a protein or polypeptide for use as an immunogen are
well known in the art. Also well known in the art are
methods for preparing immunogenic conjugates of a protein with a carrier, such
as BSA, KLH or other carrier protein. In some
circumstances, direct conjugation using, for example, carbodiimide reagents
are used; in other instances linking reagents such as
those supplied by Pierce Chemical Co., Rockford, IL, are effective.
Administration of a 109P1 D4 immunogen is often conducted
by injection over a suitable time period and with use of a suitable adjuvant,
as is understood in the art. During the immunization
schedule, titers of antibodies can be taken to determine adequacy of antibody
formation.
109P1 D4 monoclonal antibodies can be produced by various means well known in
the art. For example, immortalized
cell lines that secrete a desired monoclonal antibody are prepared using the
standard hybridoma technology of Kohler and
Milstein or modifications that immortalize antibody-producing B cells, as is
generally known. Immortalized cell lines that secrete
the desired antibodies are screened by immunoassay in which the antigen is a
109P1D4-related protein. When the appropriate
immortalized cell culture is identified, the cells can be expanded and
antibodies produced either from in vitro cultures or from
ascites fluid.
The antibodies or fragments of the invention can also be produced, by
recombinant means. Regions that bind
specifically to the desired regions of a 109P1 D4 protein can also be produced
in the context of chimeric or complementarity-
determining region (CDR) grafted antibodies of multiple species origin.
Humanized or human 109P1 D4 antibodies can also be
produced, and are preferred for use in therapeutic contexts. Methods for
humanizing murine and other non-human antibodies, by
substituting one or more of the non-human antibody CDRs for corresponding
human antibody sequences, are well known (see for
example, Jones et al., 1986, Nature 321: 522-525; Riechmann et at, 1988,
Nature 332: 323-327; Verhoeyen et al., 1988, Science
239: 1534-1536). See also, Carter at a1,1993, Proc. Natl. Acad. Sci. USA 89:
4285 and Sims et al., 1993, J. Immunol. 151: 2296.
Methods for producing fully human monoclonal antibodies include phage display
and transgenic methods (for review,
see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 109P1
D4 monoclonal antibodies can be generated
using cloning technologies employing large human Ig gene combinatorial
libraries (i.e., phage display) (Griffiths and Hoogenboom,
Building an in vitro immune system: human antibodies from phage display
libraries. In: Protein Engineering of Antibody Molecules
for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.),
Nottingham Academic, pp 45-64 (1993); Burton and Barbas,
Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human
109P1 D4 monoclonal antibodies can also be
produced using transgenic mice engineered to contain human immunoglobulin gene
loci as described in PCT Patent Application
W098/24893, Kucherlapati and Jakobovits at al., published December 3, 1997
(see also, Jakobovits, 1998, Exp. Opin. Invest.
Drugs 7(4): 607-614; U.S. patents 6,162,963 issued 19 December 2000; 6,150,584
issued 12 November 2000; and, 6,114598
issued 5 September 2000). This method avoids the in vitro manipulation
required with phage display technology and efficiently
produces high affinity authentic human antibodies.
Reactivity of 109P1 D4 antibodies with a 109P1 D4-related protein can be
established by a number of well known
means, including Western blot, immunoprecipitation, ELISA, and FACS analyses
using, as appropriate, 109P1 D4-related
proteins,109PI D4-expressing cells or extracts thereof. A 109P1 D4 antibody or
fragment thereof can be labeled with a
detectable marker or conjugated to a second molecule. Suitable detectable
markers include, but are not limited to, a
CA 02522994 2010-03-25
radioisotope, a fluorescent compound, a bioluminescent compound,
chemiluminescent compound, a metal chelator or an
enzyme. Further, bi-specific antibodies specific for two or more 109P1 D4
epitopes are generated using methods generally
known in the art. Homodimeric antibodies can also be generated by cross-
linking techniques known in the art (e.g., Wolff et
at., Cancer Res. 53: 2560-2565).
V.) 109P1D4 Cellular Immune Responses
The mechanism by which T cells recognize antigens has been delineated.
Efficacious peptide epitope vaccine
compositions of the invention induce a therapeutic or prophylactic immune
responses in very broad segments of the world-
wide population. For an understanding of the value and efficacy of
compositions of the invention that induce cellular immune
responses, a brief review of immunology-related technology is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand
recognized by HLA-restricted T cells
(Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359,
1985; Townsend, A. and Bodmer, H., Annu. Rev.
Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol.11:403,1993). Through
the study of single amino acid
substituted antigen analogs and the sequencing of endogenously bound,
naturally processed peptides, critical residues that
correspond to motifs required for specific binding to HLA antigen molecules
have been identified and are set forth in Table IV
(see also, e.g., Southwood, et al., J. lmmunol.160:3363,1998; Rammensee, et
a!., Immunogenetics 41:178, 1995;
Rammensee et a1., SYFPEITHI, access via World Wide Web ); Sette, A.
and Sidney, J. Curr. Opin. lmmunol. 10:478, 1998; Engelhard, V. H., Cur: Opin.
Immunol. 6:13,1994; Sette, A. and Grey, H.
M., Curr. Opin. lmmunol. 4:79,1992; Sinigaglia, F. and Hammer, J. Cuff, Biol.
6:52,1994; Ruppert et al., Cell 74:929-937,
1993; Kondo et al., J. lmmunol. 155:4307-4312,1995; Sidney et al., J. lmmunol
157:3480-3490,1996; Sidney et aL, Human
Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 Nov;
50(3-4):201-12, Review).
Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have
revealed pockets within the peptide
binding cleft/groove of HLA molecules which accommodate, in an allele-specific
mode, residues borne by peptide ligands;
these residues in turn determine the HLA binding capacity of the peptides in
which they are present. (See, e.g., Madden,
D.R. Annu. Rev. Immunol. 13:587,1995; Smith, et al., Immunity 4:203, 1996;
Fremont et aL, Immunity 8:305,1998; Stern et
al., Structure 2:245, 1994; Jones, E.Y. Curr. Opin. Immunol. 9:75, 1997;
Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C.
et al., Proc. Nall. Acad. Sci. USA 90:8053,1993; Guo, H. C. et al., Nature
360:364,1992; Silver, M. L. et al., Nature 360:367,
1992; Matsumura, M. et al., Science 257:927, 1992; Madden et aL, Cell 70:1035,
1992; Fremont, D. H. et al., Science
257:919,1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol.
219:277,1991.)
Accordingly, the definition of class I and class II allele-specific HLA
binding motifs, or class I or class II supermotifs
allows identification of regions within a protein that are correlated with
binding to particular HLA antigen(s).
Thus, by a process of HLA motif identification, candidates for epitope-based
vaccines have been identified; such
candidates can be further evaluated by HLA-peptide binding assays to determine
binding affinity and/or the time period of
association of the epitope and its corresponding HLA molecule. Additional
confirmatory work can be performed to select,
amongst these vaccine candidates, epitopes with preferred characteristics in
terms of population coverage, and/or
immunogenicity.
Various strategies can be utilized to evaluate cellular immunogenicity,
including:
1) Evaluation of primary T cell cultures from normal individuals (see, e.g.,
Wentworth, P. A. et al., Mol. lmmunol.
32:603, 1995; Cells, E. et aL, Proc. Nat!. Acad. Sc!. USA 91:2105, 1994; Tsai,
V. eta!., J. lmmunol. 158:1796, 1997;
Kawashima, I. et aL, Human lmmunol 59:1, 1998). This procedure involves the
stimulation of peripheral blood lymphocytes
(PBL) from normal subjects with a test peptide in the presence of antigen
presenting cells in vitro over a period of several
CA 02522994 2005-10-20
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41
weeks. T cells specific for the peptide become activated during this time and
are detected using, e.g., a lymphokine- or
51 Cr-release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J.
Immunol. 26197,1996; Wentworth, P.
A. et at, Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol.
159:4753, 1997). For example, in such methods
peptides in incomplete Freund's adjuvant are administered subcutaneously to
HLA transgenic mice. Several weeks following
immunization, splenocytes are removed and cultured in vitro in the presence of
test peptide for approximately one week.
Peptide-specific T cells are detected using, e.g., a 51 Cr-release assay
involving peptide sensitized target cells and target
cells expressing endogenously generated antigen.
3) Demonstration of recall T cell responses from immune individuals who have
been either effectively vaccinated
and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp.
Med. 181:1047, 1995; Doolan, D. L. et al.,
Immunity 7:97, 1997; Berton!, R. et at, J. Clin. Invest, 100:503, 1997;
Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997;
Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall
responses are detected by culturing PBL from subjects
that have been exposed to the antigen due to disease and thus have generated
an immune response "naturally", or from
patients who were vaccinated against the antigen. PBL from subjects are
cultured in vitro for 1-2 weeks in the presence of
test peptide plus antigen presenting cells (APC) to allow activation of
"memory" T cells, as compared to "naive" T cells. At
the end of the culture period, T cell activity is detected using assays
including 51 Cr release involving peptide-sensitized
targets, T cell proliferation, or lymphokine release.
VI.) 109P1 D4 Transgenic Animals
Nucleic acids that encode a 109P1 D4-related protein can also be used to
generate either transgenic animals or
"knock out" animals that, in turn, are useful in the development and screening
of therapeutically useful reagents. In
accordance with established techniques, cDNA encoding 109P1 D4 can be used to
clone genomic DNA that encodes
109PI D4. The cloned genomic sequences can then be used to generate transgenic
animals containing cells that express
DNA that encode 109P1 D4. Methods for generating transgenic animals,
particularly animals such as mice or rats, have
become conventional in the art and are described, for example, in U.S. Patent
Nos. 4,736,866 issued 12 April 1988, and
4,870,009 issued 26 September 1989. Typically, particular cells would be
targeted for 109P1 D4 transgene incorporation
with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 109P1 D4 can be
used to examine the effect of
increased expression of DNA that encodes 109P1 D4. Such animals can be used as
tester animals for reagents thought to
confer protection from, for example, pathological conditions associated with
its overexpression. In accordance with this
aspect of the invention, an animal is treated with a reagent and a reduced
incidence of a pathological condition, compared to
untreated animals that bear the transgene, would indicate a potential
therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of 109P1 D4 can be used to construct a
109P1 D4 "knock out" animal that
has a defective or altered gene encoding 109P1 D4 as a result of homologous
recombination between the endogenous gene
encoding 109P1 D4 and altered genomic DNA encoding 109P1 D4 introduced into an
embryonic cell of the animal. For
example, cDNA that encodes 109P1 D4 can be used to clone genomic DNA encoding
109P1 D4 in accordance with
established techniques. A portion of the genomic DNA encoding 109P1 D4 can be
deleted or replaced with another gene,
such as a gene encoding a selectable marker that can be used to monitor
integration. Typically, several kilobases of
unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector
(see, e.g., Thomas and Capecchi, Cell, 51:503
(1987) for a description of homologous recombination vectors). The vector is
introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA
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42
are selected (see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells
are then injected into a blastocyst of an animal
(e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in
Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A
chimeric embryo can then be implanted into a
suitable pseudopregnant female foster animal, and the embryo brought to term
to create a "knock out" animal. Progeny
harboring the homologously recombined DNA in their germ cells can be
identified by standard techniques and used to breed
animals in which all cells of the animal contain the homologously recombined
DNA. Knock out animals can be characterized,
for example, for their ability to defend against certain pathological
conditions or for their development of pathological
conditions due to absence of a 109P1 D4 polypeptide.
VII.) Methods for the Detection of 109P1 D4
Another aspect of the present invention relates to methods for detecting 109P1
D4 polynucleotides and 109P1 D4-
related proteins, as well as methods for identifying a cell that expresses
109P1 D4. The expression profile of 109P1 D4 makes it
a diagnostic marker for metastasized disease. Accordingly, the status of 109P1
D4 gene products provides information useful
for predicting a variety of factors including susceptibility to advanced stage
disease, rate of progression, and/or tumor
aggressiveness. As discussed in detail herein, the status of 109P1 D4 gene
products in patient samples can be analyzed by a
variety protocols that are well known in the art including immunohistochemical
analysis, the variety of Northern blotting techniques
including in situ hybridization, RT-PCR analysis (for example on laser capture
micro-dissected samples), Western blot analysis
and tissue array analysis.
More particularly, the invention provides assays for the detection of 109P1 D4
polynucleotides in a biological sample,
such as serum, bone, prostate, and other tissues, urine, semen, cell
preparations, and the like. Detectable 109P1 D4
polynucleotides include, for example, a 109P1 D4 gene or fragment thereof,
109P1 D4 mRNA, alternative splice variant 109P1 D4
mRNAs, and recombinant DNA or RNA molecules that contain a 109P1 D4
polynucleotide. A number of methods for amplifying
and/or detecting the presence of 109PI D4 polynucleotides are well known in
the art and can be employed in the practice of this
aspect of the invention.
In one embodiment, a method for detecting a 109P1 D4 mRNA in a biological
sample comprises producing cDNA from
the sample by reverse transcription using at least one primer; amplifying the
cDNA so produced using a 109P1 D4
polynucleotides as sense and antisense primers to amplify 109P1 D4 cDNAs
therein; and detecting the presence of the
amplified 109P1D4 cDNA. Optionally, the sequence of the amplified 109P1 D4
cDNA can be determined.
In another embodiment, a method of detecting a 109P1 D4 gene in a biological
sample comprises first isolating
genomic DNA from the sample; amplifying the isolated genomic DNA using 109P1
D4 polynucleotides as sense and
antisense primers; and detecting the presence of the amplified 109P1 D4 gene.
Any number of appropriate sense and
antisense probe combinations can be designed from a 109P1 D4 nucleotide
sequence (see, e.g., Figure 2) and used for this
purpose.
The invention also provides assays for detecting the presence of a 109P1 D4
protein in a tissue or other biological
sample such as serum, semen, bone, prostate, urine, cell preparations, and the
like. Methods for detecting a 109P1 D4-related
protein are also well known and include, for example, immunoprecipitation,
immunohistochemical analysis, Western blot analysis,
molecular binding assays, ELISA, ELIFA and the like. For example, a method of
detecting the presence of a 109P1 D4-related
protein in a biological sample comprises first contacting the sample with a
109P1 D4 antibody, a 109P1 D4-reactive fragment
thereof, or a recombinant protein containing an antigen-binding region of a
109P1 D4 antibody; and then detecting the
binding of 109P1D4-related protein in the sample.
Methods for identifying a cell that expresses 109PI D4 are also within the
scope of the invention. In one embodiment,
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43
an assay for identifying a cell that expresses a 109P1 D4 gene comprises
detecting the presence of 109P1 D4 mRNA in the cell.
Methods for the detection of particular mRNAs in cells are well known and
include, for example, hybridization assays using
complementary DNA probes (such as in situ hybridization using labeled 109P1 D4
riboprobes, Northern blot and related
techniques) and various nucleic acid amplification assays (such as RT-PCR
using complementary primers specific for 109P1 D4,
and other amplification type detection methods, such as, for example, branched
DNA, SISBA, TMA and the like). Alternatively, an
assay for identifying a cell that expresses a 109P1 D4 gene comprises
detecting the presence of 109P1 D4-related protein in the
cell or secreted by the cell. Various methods for the detection of proteins
are well known in the art and are employed for the
detection of 109P1 D4-related proteins and cells that express 109P1 D4-related
proteins.
109P1 D4 expression analysis is also useful as a tool for identifying and
evaluating agents that modulate 109P1 D4 gene
expression. For example, 109P1 D4 expression is significantly upregulated in
prostate cancer, and is expressed in cancers of
the tissues listed in Table I. Identification of a molecule or biological
agent that inhibits 109P1 D4 expression or over-
expression in cancer cells is of therapeutic value. For example, such an agent
can be identified by using a screen that
quantifies 109P1 D4 expression by RT-PCR, nucleic acid hybridization or
antibody binding.
VIII.) Methods for Monitoring the Status of 109P1 D4-related Genes and Their
Products
Oncogenesis is known to be a multistep process where cellular growth becomes
progressively dysregulated and
cells progress from a normal physiological state to precancerous and then
cancerous states (see, e.g., Alers et al., Lab
Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32
(1995)). In this context, examining a biological
sample for evidence of dysregulated cell growth (such as aberrant 109P1 D4
expression in cancers) allows for early detection
of such aberrant physiology, before a pathologic state such as cancer has
progressed to a stage that therapeutic options are
more limited and or the prognosis is worse. In such examinations, the status
of 109P1 D4 in a biological sample of interest
can be compared, for example, to the status of 109P1 D4 in a corresponding
normal sample (e.g. a sample from that
individual or alternatively another individual that is not affected by a
pathology). An alteration in the status of 109P1 D4 in the
biological sample (as compared to the normal sample) provides evidence of
dysregulated cellular growth. In addition to
using a biological sample that is not affected by a pathology as a normal
sample, one can also use a predetermined
normative value such as a predetermined normal level of mRNA expression (see,
e.g., Grever et al., J. Comp. Neurol. 1996
Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,501) to compare 109P1D4 status
in a sample.
The term "status" in this context is used according to its art accepted
meaning and refers to the condition or state of a
gene and its products. Typically, skilled artisans use a number of parameters
to evaluate the condition or state of a gene and its
products. These include, but are not limited to the location of expressed gene
products (including the location of 109P1 D4
expressing cells) as well as the level, and biological activity of expressed
gene products (such as 109P1 D4 mRNA,
polynucleotides and polypeptides). Typically, an alteration in the status of
109P1 D4 comprises a change in the location of
109PI D4 and/or 109P1 D4 expressing cells and/or an increase in 109P1 D4 mRNA
and/or protein expression.
109P1 D4 status in a sample can be analyzed by a number of means well known in
the art, including without limitation,
immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser
capture micro-dissected samples, Western blot
analysis, and tissue array analysis. Typical protocols for evaluating the
status of a 109P1 D4 gene and gene products are found,
for example in Ausubel et al. eds., 1995, Current Protocols In Molecular
Biology, Units 2 (Northern Blotting), 4 (Southern
Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of
109P1 D4 in a biological sample is evaluated by
various methods utilized by skilled artisans including, but not limited to
genomic Southern analysis (to examine, for example
perturbations in a 109P1 D4 gene), Northern analysis and/or PCR analysis of
109P1 D4 mRNA (to examine, for example
alterations in the polynucleotide sequences or expression levels of 109P1 D4
mRNAs), and, Western and/or
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44
immunohistochemical analysis (to examine, for example alterations in
polypeptide sequences, alterations in polypeptide
localization within a sample, alterations in expression levels of 109P1 D4
proteins and/or associations of 109P1 D4 proteins
with polypeptide binding partners). Detectable 109P1 D4 polynucleotides
include, for example, a 109P1 D4 gene or fragment
thereof, 109P1 D4 mRNA, alternative splice variants,109PI D4 mRNAs, and
recombinant DNA or RNA molecules containing a
109P1 D4 polynucleofide.
The expression profile of 109P1 D4 makes it a diagnostic marker for local
and/or metastasized disease, and
provides information on the growth or oncogenic potential of a biological
sample. In particular, the status of 109P1 D4 provides
information useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The
invention provides methods and assays for determining 109P1 D4 status and
diagnosing cancers that express 109P1 D4, such as
cancers of the tissues listed in Table I. For example, because 109P1 D4 mRNA
is so highly expressed in prostate and other
cancers relative to normal prostate tissue, assays that evaluate the levels of
109PI D4 mRNA transcripts or proteins in a biological
sample can be used to diagnose a disease associated with 109P1 D4
dysregulation, and can provide prognostic information useful
in defining appropriate therapeutic options.
The expression status of 109P1 D4 provides information including the presence,
stage and location of dysplastic,
precancerous and cancerous cells, predicting susceptibility to various stages
of disease, and/or for gauging tumor
aggressiveness. Moreover, the expression profile makes it useful as an imaging
reagent for metastasized disease.
Consequently, an aspect of the invention is directed to the various molecular
prognostic and diagnostic methods for examining the
status of 109P1 D4 in biological samples such as those from individuals
suffering from, or suspected of suffering from a
pathology characterized by dysregulated cellular growth, such as cancer.
As described above, the status of 109P1 D4 in a biological sample can be
examined by a number of well-known
procedures in the art. For example, the status of 109P1 D4 in a biological
sample taken from a specific location in the body
can be examined by evaluating the sample for the presence or absence of 109P1
D4 expressing cells (e.g. those that express
109PI D4 mRNAs or proteins). This examination can provide evidence of
dysregulated cellular growth, for example, when
109P1D4-expressing cells are found in a biological sample that does not
normally contain such cells (such as a lymph node),
because such alterations in the status of 109P1 D4 in a biological sample are
often associated with dysregulated cellular
growth. Specifically, one indicator of dysregulated cellular growth is the
metastases of cancer cells from an organ of origin
(such as the prostate) to a different area of the body (such as a lymph node).
In this context, evidence of dysregulated
cellular growth is important for example because occult lymph node metastases
can be detected in a substantial proportion
of patients with prostate cancer, and such metastases are associated with
known predictors of disease progression (see,
e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg.
Oncol.18(1):17-28 (2000) and Freeman et aL, J
Urol 1995 Aug 154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring 109PI D4 gene
products by determining the status of
109P1 D4 gene products expressed by cells from an individual suspected of
having a disease associated with dysregulated
cell growth (such as hyperplasia or cancer) and then comparing the status so
determined to the status of 109P1 D4 gene
products in a corresponding normal sample. The presence of aberrant 109PI D4
gene products in the test sample relative to
the normal sample provides an indication of the presence of dysregulated cell
growth within the cells of the individual.
In another aspect, the invention provides assays useful in determining the
presence of cancer in an individual,
comprising detecting a significant increase in 109P1 D4 mRNA or protein
expression in a test cell or tissue sample relative to
expression levels in the corresponding normal cell or tissue. The presence of
109P1 D4 mRNA can, for example, be
evaluated in tissues including but not limited to those listed in Table I. The
presence of significant 109P1 D4 expression in
any of these tissues is useful to indicate the emergence, presence and/or
severity of a cancer, since the corresponding
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normal tissues do not express 109P1 D4 mRNA or express it at lower levels.
In a related embodiment, 109P1 D4 status is determined at the protein level
rather than at the nucleic acid level. For
example, such a method comprises determining the level of 109P1 D4 protein
expressed by cells in a test tissue sample and
comparing the level so determined to the level of 109P1 D4 expressed in a
corresponding normal sample. In one embodiment,
the presence of 109P1 D4 protein is evaluated, for example, using
immunohistochemical methods. 109P1 D4 antibodies or
binding partners capable of detecting 109P1 D4 protein expression are used in
a variety of assay formats well known in the art for
this purpose.
In a further embodiment, one can evaluate the status of 109P1 D4 nucleotide
and amino acid sequences in a biological
sample in order to identify perturbations in the structure of these molecules.
These perturbations can include insertions, deletions,
substitutions and the like. Such evaluations are useful because perturbations
in the nucleotide and amino acid sequences are
observed in a large number of proteins associated with a growth dysregulated
phenotype (see, e.g., Marrogi et al., 1999, J.
Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of
109P1 D4 may be indicative of the presence or
promotion of a tumor. Such assays therefore have diagnostic and predictive
value where a mutation in 109P1 D4 indicates a
potential loss of function or increase in tumor growth.
A wide variety of assays for observing perturbations in nucleotide and amino
acid sequences are well known in the art.
For example, the size and structure of nucleic acid or amino acid sequences of
109P1 D4 gene products are observed by the
Northern, Southern, Western, PCR and DNA sequencing protocols discussed
herein. In addition, other methods for observing
perturbations in nucleotide and amino acid sequences such as single strand
conformation polymorphism analysis are well known
in the art (see, e.g., U.S. Patent Nos. 5,382,510 issued 7 September 1999, and
5,952,170 issued 17 January 1995).
Additionally, one can examine the methylation status of a 109P1 D4 gene in a
biological sample. Aberrant
demethylation and/or hypermethylation of CpG islands in gene 5' regulatory
regions frequently occurs in immortalized and
transformed cells, and can result in altered expression of various genes. For
example, promoter hypermethylation of the pi-class
glutathione S-transferase (a protein expressed in normal prostate but not
expressed in >90% of prostate carcinomas)
appears to permanently silence transcription of this gene and is the most
frequently detected genomic alteration in prostate
carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In
addition, this alteration is present in at least 70%
of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et
al., Cancer Epidemiol. Biomarkers Prev., 1998,
7:531-536). In another example, expression of the LAGE-I tumor specific gene
(which is not expressed in normal prostate
but is expressed in 25-50% of prostate cancers) is induced by deoxy-
azacytidine in lymphoblastoid cells, suggesting that
tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer
76(6): 903-908 (1998)). A variety of assays for
examining methylation status of a gene are well known in the art. For example,
one can utilize, in Southern hybridization
approaches, methylation-sensitive restriction enzymes that cannot cleave
sequences that contain methylated CpG sites to assess
the methylation status of CpG islands. In addition, MSP (methylation specific
PCR) can rapidly profile the methylation status of all
the CpG sites present in a CpG island of a given gene. This procedure involves
initial modification of DNA by sodium bisulfite
(which will convert all unmethylated cytosines to uracil) followed by
amplification using primers specific for methylated versus
unmethylated DNA, Protocols involving methylation interference can also be
found for example in Current Protocols In Molecular
Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
Gene amplification is an additional method for assessing the status of 109P1
D4. Gene amplification is measured
in a sample directly, for example, by conventional Southern blotting or
Northern blotting to quantitate the transcription of
mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting
(DNA analysis), or in situ hybridization, using
an appropriately labeled probe, based on the sequences provided herein.
Alternatively, antibodies are employed that
recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA
hybrid duplexes or DNA-protein
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duplexes. The antibodies in turn are labeled and the assay carried out where
the duplex is bound to a surface, so that upon
the formation of duplex on the surface, the presence of antibody bound to the
duplex can be detected.
Biopsied tissue or peripheral blood can be conveniently assayed for the
presence of cancer cells using for example,
Northern, dot blot or RT-PCR analysis to detect 109P1 D4 expression. The
presence of RT-PCR amplifiable 109P1 D4 mRNA
provides an indication of the presence of cancer. RT-PCR assays are well known
in the art. RT-PCR detection assays for tumor
cells in peripheral blood are currently being evaluated for use in the
diagnosis and management of a number of human solid
tumors. In the prostate cancer field, these include RT-PCR assays for the
detection of cells expressing PSA and PSM (Verkaik et
al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol.
13:1195-2000; Heston et aL, 1995, Clin. Chem. 41:1687-
1688).
A further aspect of the invention is an assessment of the susceptibility that
an individual has for developing cancer. In
one embodiment, a method for predicting susceptibility to cancer comprises
detecting 109P1 D4 mRNA or 109P1 D4 protein in a
tissue sample, its presence indicating susceptibility to cancer, wherein the
degree of 109P1 D4 mRNA expression correlates to the
degree of susceptibility. In a specific embodiment, the presence of 109P1 D4
in prostate or other tissue is examined, with the
presence of 109P1 D4 in the sample providing an indication of prostate cancer
susceptibility (or the emergence or existence of a
prostate tumor). Similarly, one can evaluate the integrity 109P1 D4 nucleotide
and amino acid sequences in a biological sample, in
order to identify perturbations in the structure of these molecules such as
insertions, deletions, substitutions and the like. The
presence of one or more perturbations in 109P1 D4 gene products in the sample
is an indication of cancer susceptibility (or the
emergence or existence of a tumor).
The invention also comprises methods for gauging tumor aggressiveness. In one
embodiment, a method for gauging
aggressiveness of a tumor comprises determining the level of 109P1 D4 mRNA or
109P1 D4 protein expressed by tumor cells,
comparing the level so determined to the level of 109P1 D4 mRNA or 109P1 D4
protein expressed in a corresponding normal
tissue taken from the same individual or a normal tissue reference sample,
wherein the degree of 109P1 D4 mRNA or 109P1 D4
protein expression in the tumor sample relative to the normal sample indicates
the degree of aggressiveness. In a specific
embodiment, aggressiveness of a tumor is evaluated by determining the extent
to which 109P1 D4 is expressed in the tumor cells,
with higher expression levels indicating more aggressive tumors. Another
embodiment is the evaluation of the integrity of
109P1 D4 nucleotide and amino acid sequences in a biological sample, in order
to identify perturbations in the structure of these
molecules such as insertions, deletions, substitutions and the like. The
presence of one or more perturbations indicates more
aggressive tumors.
Another embodiment of the invention is directed to methods for observing the
progression of a malignancy in an
individual over time. In one embodiment, methods for observing the progression
of a malignancy in an individual over time
comprise determining the level of 109P1 D4 mRNA or 109P1 D4 protein expressed
by cells in a sample of the tumor, comparing
the level so determined to the level of 109P1 D4 mRNA or 109P1 D4 protein
expressed in an equivalent tissue sample taken from
the same individual at a different time, wherein the degree of 109PI D4 mRNA
or 109P1 D4 protein expression in the tumor
sample over time provides information on the progression of the cancer. In a
specific embodiment, the progression of a cancer is
evaluated by determining 109PI D4 expression in the tumor cells over time,
where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity 109P1 D4
nucleotide and amino acid sequences in a biological
sample in order to identify perturbations in the structure of these molecules
such as insertions, deletions, substitutions and the like,
where the presence of one or more perturbations indicates a progression of the
cancer.
The above diagnostic approaches can be combined with any one of a wide variety
of prognostic and diagnostic
protocols known in the art. For example, another embodiment of the invention
is directed to methods for observing a coincidence
between the expression of 109P1 D4 gene and 109P1 D4 gene products (or
perturbations in 109P1 D4 gene and 109P1 D4 gene
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products) and a factor that is associated with malignancy, as a means for
diagnosing and prognosticating the status of a tissue
sample. A wide variety of factors associated with malignancy can be utilized,
such as the expression of genes associated with
malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as
well as gross cytological observations (see, e.g.,
Bocking eta!., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum.
Pathol. 26(2):223-9; Thorson eta!., 1998, Mod.
Pathol. 11 (6):543-51; Baisden eta!., 1999, Am. J. Surg. Pathol. 23(8):918-
24). Methods for observing a coincidence between
the expression of 109P1 D4 gene and 109P1 D4 gene products (or perturbations
in 109P1 D4 gene and 109P1 D4 gene products)
and another factor that is associated with malignancy are useful, for example,
because the presence of a set of specific factors
that coincide with disease provides information crucial for diagnosing and
prognosticating the status of a tissue sample.
In one embodiment, methods for observing a coincidence between the expression
of 109P1 D4 gene and 109P1 D4
gene products (or perturbations in 109P1 D4 gene and 109P1 D4 gene products)
and another factor associated with malignancy
entails detecting the overexpression of 109P1 D4 mRNA or protein in a tissue
sample, detecting the overexpression of PSA mRNA
or protein in a tissue sample (or PSCA or PSM expression), and observing a
coincidence of 109P1 D4 mRNA or protein and PSA
mRNA or protein overexpression (or PSCA or PSM expression). In a specific
embodiment, the expression of 109P1 D4 and PSA
mRNA in prostate tissue is examined, where the coincidence of 109P1 D4 and PSA
mRNA overexpression in the sample indicates
the existence of prostate cancer, prostate cancer susceptibility or the
emergence or status of a prostate tumor.
Methods for detecting and quantifying the expression of 109P1 D4 mRNA or
protein are described herein, and standard
nucleic acid and protein detection and quantification technologies are well
known in the art. Standard methods for the detection
and quantification of 109P1 D4 mRNA include in situ hybridization using
labeled 109P1 D4 riboprobes, Northern blot and related
techniques using 109P1 D4 polynucleotide probes, RT-PCR analysis using primers
specific for 109P1 D4, and other amplification
type detection methods, such as, for example, branched DNA, SISBA, TMA and the
like. In a specific embodiment, semi-
quantitative RT-PCR is used to detect and quantify 109P1 D4 mRNA expression.
Any number of primers capable of amplifying
109P1 D4 can be used for this purpose, including but not limited to the
various primer sets specifically described herein. In a
specific embodiment, polyclonal or monoclonal antibodies specifically reactive
with the wild-type 109P1 D4 protein can be used in
an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules That Interact With I09P1 D4
The 109P1 D4 protein and nucleic acid sequences disclosed herein allow a
skilled artisan to identify proteins, small
molecules and other agents that interact with 109P1 D4, as well as pathways
activated by 109P1 D4 via any one of a variety
of art accepted protocols. For example, one can utilize one of the so-called
interaction trap systems (also referred to as the
"two-hybrid assay"). In such systems, molecules interact and reconstitute a
transcription factor which directs expression of a
reporter gene, whereupon the expression of the reporter gene is assayed. Other
systems identify protein-protein interactions
in vivo through reconstitution of a eukaryotic transcriptional activator, see,
e.g., U.S. Patent Nos. 5,955,280 issued 21
September 1999, 5,925,523 issued 20 July 1999, 5,846,722 issued 8 December
1998 and 6,004,746 issued 21 December
1999. Algorithms are also available in the art for genome-based predictions of
protein function (see, e.g., Marcotte, et al.,
Nature 402: 4 November 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that
interact with 109PI D4 protein sequences.
In such methods, peptides that bind to 109P1 D4 are identified by screening
libraries that encode a random or controlled
collection of amino acids. Peptides encoded by the libraries are expressed as
fusion proteins of bacteriophage coat proteins,
the bacteriophage particles are then screened against the 109P1 D4 protein(s).
Accordingly, peptides having a wide variety of uses, such as therapeutic,
prognostic or diagnostic reagents, are
thus identified without any prior information on the structure of the expected
ligand or receptor molecule. Typical peptide
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48
libraries and screening methods that can be used to identify molecules that
interact with 109P1 D4 protein sequences are
disclosed for example in U.S. Patent Nos. 5,723,286 issued 3 March 1998 and
5,733,731 issued 31 March 1998.
Alternatively, cell lines that express 109P1 D4 are used to identify protein-
protein interactions mediated by
109P1 D4. Such interactions can be examined using immunoprecipitation
techniques (see, e.g., Hamilton B.J., et al.
Biochem. Biophys. Res. Commun. 1999, 261:646-51). 109P1D4 protein can be
immunoprecipitated from 109P1D4-
expressing cell lines using anti-I 09P1 D4 antibodies. Alternatively,
antibodies against His-tag can be used in a cell line
engineered to express fusions of 109P1 D4 and a His-tag (vectors mentioned
above). The immunoprecipitated complex can
be examined for protein association by procedures such as Western blotting,
35S-methionine labeling of proteins, protein
microsequencing, silver staining and two-dimensional gel electrophoresis.
Small molecules and ligands that interact with 109P1 D4 can be identified
through related embodiments of such
screening assays. For example, small molecules can be identified that
interfere with protein function, including molecules
that interfere with 109P1 D4's ability to mediate phosphorylation and de-
phosphorylation, interaction with DNA or RNA
molecules as an indication of regulation of cell cycles, second messenger
signaling or tumorigenesis. Similarly, small
molecules that modulate 109P1 D4-related ion channel, protein pump, or cell
communication functions are identified and
used to treat patients that have a cancer that expresses 109P1 D4 (see, e.g.,
Hille, B., Ionic Channels of Excitable
Membranes 2nd Ed., Sinauer Assoc., Sunderland, MA, 1992). Moreover, ligands
that regulate 109P1 D4 function can be
identified based on their ability to bind 109P1 D4 and activate a reporter
construct. Typical methods are discussed for
example in U.S. Patent No. 5,928,868 issued 27 July 1999, and include methods
for forming hybrid ligands in which at least
one ligand is a small molecule. In an illustrative embodiment, cells
engineered to express a fusion protein of 109P1 D4 and a
DNA-binding protein are used to co-express a fusion protein of a hybrid
ligand/small molecule and a cDNA library
transcriptional activator protein. The cells further contain a reporter gene,
the expression of which is conditioned on the
proximity of the first and second fusion proteins to each other, an event that
occurs only if the hybrid ligand binds to target
sites on both hybrid proteins. Those cells that express the reporter gene are
selected and the unknown small molecule or
the unknown ligand is identified. This method provides a means of identifying
modulators, which activate or inhibit 109P1 D4.
An embodiment of this invention comprises a method of screening for a molecule
that interacts with a 109P1 D4
amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of
contacting a population of molecules with a
109P1 D4 amino acid sequence, allowing the population of molecules and the
109P1 D4 amino acid sequence to interact
under conditions that facilitate an interaction, determining the presence of a
molecule that interacts with the 109P1 D4 amino
acid sequence, and then separating molecules that do not interact with the
109P1 D4 amino acid sequence from molecules
that do. In a specific embodiment, the method further comprises purifying,
characterizing and identifying a molecule that
interacts with the 109P1 D4 amino acid sequence. The identified molecule can
be used to modulate a function performed by
109P1 D4. In a preferred embodiment, the 109P1 D4 amino acid sequence is
contacted with a library of peptides.
NJ Therapeutic Methods and Compositions
The identification of 109P1 D4 as a protein that is normally expressed in a
restricted set of tissues, but which is also
expressed in cancers such as those listed in Table I, opens a number of
therapeutic approaches to the treatment of such
cancers.
Of note, targeted antitumor therapies have been useful even when the targeted
protein is expressed on normal
tissues, even vital normal organ tissues. A vital organ is one that is
necessary to sustain life, such as the heart or colon. A
non-vital organ is one that can be removed whereupon the individual is still
able to survive. Examples of non-vital organs are
ovary, breast, and prostate.
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49
For example, Herceptin is an FDA approved pharmaceutical that has as its
active ingredient an antibody which is
immunoreactive with the protein variously known as HER2, HER2Ineu, and erb-b-
2. It is marketed by Genentech and has
been a commercially successful antitumor agent. Herceptin sales reached almost
$400 million in 2002. Herceptin is a
treatment for HER2 positive metastatic breast cancer. However, the expression
of HER2 is not limited to such tumors. The
same protein is expressed in a number of normal tissues. In particular, it is
known that HER2/neu is present in normal
kidney and heart, thus these tissues are present in all human recipients of
Herceptin. The presence of HER2/neu in normal
kidney is also confirmed by Latif, Z., et al., E.J. U. International (2002)
89:5-9. As shown in this article (which evaluated
whether renal cell carcinoma should be a preferred indication for anti-HER2
antibodies such as Herceptin) both protein and
mRNA are produced in benign renal tissues. Notably, HER2/neu protein was
strongly overexpressed in benign renal tissue.
Despite the fact that HER2/neu is expressed in such vital tissues as heart and
kidney, Herceptin is a very useful, FDA
approved, and commercially successful drug. The effect of Herceptin on cardiac
tissue, i.e., "cardiotoxicity," has merely
been a side effect to treatment. When patients were treated with Herceptin
alone, significant cardiotoxicity occurred in a very
low percentage of patients.
Of particular note, although kidney tissue is indicated to exhibit normal
expression, possibly even higher
expression than cardiac tissue, kidney has no appreciable Herceptin side
effect whatsoever. Moreover, of the diverse array
of normal tissues in which HER2 is expressed, there is very little occurrence
of any side effect. Only cardiac tissue has
manifested any appreciable side effect at all. A tissue such as kidney, where
HER2/neu expression is especially notable,
has not been the basis for any side effect.
Furthermore, favorable therapeutic effects have been found for antitumor
therapies that target epidermal growth
factor receptor (EGFR). EGFR is also expressed in numerous normal tissues.
There have been very limited side effects in
normal tissues following use of anti-EGFR therapeutics.
Thus, expression of a target protein in normal tissue, even vital normal
tissue, does not defeat the utility of a
targeting agent for the protein as a therapeutic for certain tumors in which
the protein is also overexpressed.
Accordingly, therapeutic approaches that inhibit the activity of a 109P1 D4
protein are useful for patients suffering
from a cancer that expresses 109P1 D4. These therapeutic approaches generally
fall into two classes. One class comprises
various methods for inhibiting the binding or association of a 109P1 D4
protein with its binding partner or with other proteins.
Another class comprises a variety of methods for inhibiting the transcription
of a 109P1 D4 gene or translation of 109P1 D4
mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 109P1 D4-related protein
or 109P1 D4-related nucleic acid. In
view of the expression of 109P1 D4, cancer vaccines prevent and/or treat 109P1
D4-expressing cancers with minimal or no effects
on non-target tissues. The use of a tumor antigen in a vaccine that generates
humoral and/or cell-mediated immune responses
as anti-cancer therapy is well known in the art and has been employed in
prostate cancer using human PSMA and rodent PAP
immunogens (Hodge eta/,1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J.
Immunol. 159:3113-3117).
Such methods can be readily practiced by employing a 109P1 D4-related protein,
or a 109P1 D4-encoding nucleic
acid molecule and recombinant vectors capable of expressing and presenting the
109P1 D4 immunogen (which typically
comprises a number of antibody or T cell epitopes). Skilled artisans
understand that a wide variety of vaccine systems for
delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et
al., Ann Med 1999 Feb 31(1):66-78; Maruyama
et al., Cancer Immunol Immunother 2000 Jun 49(3):123-32) Briefly, such methods
of generating an immune response (e.g.
humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the
mammal's immune system to an
CA 02522994 2010-03-25
immunoreactive epitope (e.g. an epitope present in a 109P1 D4 protein shown in
Figure 3 or analog or homolog thereof) so
that the mammal generates an immune response that is specific for that epitope
(e.g. generates antibodies that specifically
recognize that epitope). Ina preferred method, a 109P1 D4 immunogen contains a
biological motif, see e.g., Tables VIII-XXI
and XXII-XLIX, or a peptide of a size range from 109P1 D4 indicated in Figure
5, Figure 6, Figure 7, Figure 8, and Figure 9.
The entire 109P1D4 protein, immunogenic regions or epitopes thereof can be
combined and delivered by various
means. Such vaccine compositions can include, for example, lipopeptides
(e.g.,Vitiello, A. at at, J. Clin. Invest. 95:341,
1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide)
("PLG") microspheres (see, e.g., Eldridge, at al,
Molec. Immunol. 28:287-294, 1991: Alonso at at, Vaccine 12:299-306, 1994;
Jones eta!., Vaccine 13:675-681, 1995),
peptide compositions contained in immune stimulating complexes (ISCOMS) (see,
e.g., Takahashi at al., Nature 344:873-
875,1990; Hu at al., Clin Exp Immunot.113:235-243,1998), multiple antigen
peptide systems (MAPS) (see e.g., Tam, J. P.,
Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413,1988; Tam, J.P., J. Immunol.
Methods 196:17-32,1996), peptides formulated as
multivalent peptides; peptides for use in ballistic delivery systems,
typically crystallized peptides, viral delivery vectors
(Perkus, M. E. at al., in: Concepts in vaccine development, Kaufmann, S. H.
E., ed., p. 379, 1996; Chakrabarti, S. eta!.,
Nature 320:535,1986; Hu, S. L. at al., Nature 320:537,1986; Kleny, M. P. of
al., AIDS Bio/Technology 4:790,1986; Top, F.
H. at al., J. Infect. Dis.124:148,1971; Chanda, P. K. at al., Virology
175:535,1990), particles of viral or synthetic origin (e.g.,
Kofler, N. at al., J. Immunol. Methods. 192:25,1996; Eldridge, J. H. at al.,
Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. at at,
Nature Med. 7:649,1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid,
L. A. Annu. Rev. Immunol 4:369,1986;
Gupta, R. K. at at, Vaccine 11:293,1993), liposomes (Reddy, R. at al., J.
Immunol. 148:1585,1992; Rock, K. L., Immunol.
Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. at al.,
Science 259:1745,1993; Robinson, H. L.,
Hunt, L. A., and Webster, R. G., Vaccine 11:957,1993; Shiver, J. W. at al.,
In: Concepts in vaccine development, Kaufmann,
S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.
Immunol 12:923, 1994 and Eldridge, J. H. at al.,
Sam. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known
as receptor mediated targeting, such as
those of Avant Immunotherapeutics, inc. (Needham, Massachusetts) may also be
used.
In patients with 109P1 D4-associated cancer, the vaccine compositions of the
invention can also be used in
conjunction with other treatments used for cancer, e.g., surgery,
chemotherapy, drug therapies, radiation therapies, etc.
including use in combination with immune adjuvants such as IL-2, IL-12, GM-
CSF, and the like.
Cellular Vaccines:
CTL epitopes can be determined using specific algorithms to identify peptides
within 109P1 D4 protein that bind
corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown
University; and BIMAS).
In a preferred embodiment, a 109P1 D4 immunogen contains one or more amino
acid sequences identified using techniques
well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-
XLIX or a peptide of 8, 9, 10 or 11 amino
acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table
IV (D), or Table IV (E)) and/or a peptide of at
least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g.,
Table IV (B) or Table IV (C)). As is appreciated in
the art, the HLA Class I binding groove is essentially closed ended so that
peptides of only a particular size range can fit into
the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11
amino acids long. In contrast, the HLA Class II
binding groove is essentially open ended; therefore a peptide of about 9 or
more amino acids can be bound by an HLA Class
It molecule. Due to the binding groove differences between HLA Class I and iI,
HLA Class I motifs are length specific, i.e.,
position two of a Class I motif is the second amino add in an amino to
carboxyl direction of the peptide. The amino acid
positions in a Class II motif are relative only to each other, not the overall
peptide, i.e., additional amino acids can be
attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA
Class 11 epitopes are often 9,10,11,12,13,
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51
14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer
than 25 amino acids.
Antibody-based Vaccines
A wide variety of methods for generating an immune response in a mammal are
known in the art (for example as
the first step in the generation of hybridomas). Methods of generating an
immune response in a mammal comprise exposing
the mammal's immune system to an immunogenic epitope on a protein (e.g. a
109P1 D4 protein) so that an immune
response is generated. A typical embodiment consists of a method for
generating an immune response to 109P1 D4 in a
host, by contacting the host with a sufficient amount of at least one 109P1 D4
B cell or cytotoxic T-cell epitope or analog
thereof; and at least one periodic interval thereafter re-contacting the host
with the 109P1 D4 B cell or cytotoxic T-cell epitope
or analog thereof. A specific embodiment consists of a method of generating an
immune response against a 109P1 D4-
related protein or a man-made multiepitopic peptide comprising: administering
109P1 D4 immunogen (e.g. a 109P1 D4
protein or a peptide fragment thereof, a 109P1 D4 fusion protein or analog
etc.) in a vaccine preparation to a human or
another mammal. Typically, such vaccine preparations further contain a
suitable adjuvant (see, e.g., U.S. Patent No.
6,146,635) or a universal helper epitope such as a PADRETM peptide (Epimmune
Inc., San Diego, CA; see, e.g., Alexander
et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity
1994 1(9): 751-761 and Alexander et aL,
Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating
an immune response in an individual
against a 109P1 D4 immunogen by: administering in vivo to muscle or skin of
the individual's body a DNA molecule that
comprises a DNA sequence that encodes a 109P1 D4 immunogen, the DNA sequence
operatively linked to regulatory
sequences which control the expression of the DNA sequence; wherein the DNA
molecule is taken up by cells, the DNA
sequence is expressed in the cells and an immune response is generated against
the immunogen (see, e.g., U.S. Patent No.
5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids;
saponins; lectins; estrogenic compounds;
hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered.
In addition, an antiidiotypic antibody can be
administered that mimics 109P1 D4, in order to generate a response to the
target antigen.
Nucleic Acid Vaccines:
Vaccine compositions of the invention include nucleic acid-mediated
modalities. DNA or RNA that encode
protein(s) of the invention can be administered to a patient. Genetic
immunization methods can be employed to generate
prophylactic or therapeutic humoral and cellular immune responses directed
against cancer cells expressing 109P1 D4.
Constructs comprising DNA encoding a 109P1 D4-related protein/immunogen and
appropriate regulatory sequences can be
injected directly into muscle or skin of an individual, such that the cells of
the muscle or skin take-up the construct and
express the encoded 109PI D4 protein/immunogen. Alternatively, a vaccine
comprises a 109P1 D4-related protein.
Expression of the 109P1 D4-related protein immunogen results in the generation
of prophylactic or therapeutic humoral and
cellular immunity against cells that bear a 109P1 D4 protein. Various
prophylactic and therapeutic genetic immunization
techniques known in the art can be used (for review, see information and
references published at Internet address
genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff
et. al., Science 247:1465 (1990) as well as
U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524;
5,679,647; WO 98/04720. Examples of DNA-
based delivery technologies include "naked DNA", facilitated (bupivicaine,
polymers, peptide-mediated) delivery, cationic lipid
complexes, and particle-mediated ("gene gun") or pressure-mediated delivery
(see, e.g., U.S. Patent No. 5,922,687).
For therapeutic or prophylactic immunization purposes, proteins of the
invention can be expressed via viral or
bacterial vectors. Various viral gene delivery systems that can be used in the
practice of the invention include, but are not limited
to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-
associated virus, lentivirus, and sindbis virus (see, e.g.,
Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer
Inst. 87:982-990 (1995)). Non-viral delivery systems
can also be employed by introducing naked DNA encoding a 109P1 D4-related
protein into the patient (e.g., intramuscularly or
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intradermally) to induce an anti-tumor response.
Vaccinia virus is used, for example, as a vector to express nucleotide
sequences that encode the peptides of the
invention. Upon introduction into a host, the recombinant vaccinia virus
expresses the protein immunogenic peptide, and
thereby elicits a host immune response, Vaccinia vectors and methods useful in
immunization protocols are described in,
e.g., U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette
Guerin). BCG vectors are described in Stover et
at, Nature 351:456-460 (1991). A wide variety of other vectors useful for
therapeutic administration or immunization of the
peptides of the invention, e.g. adeno and adeno-associated virus vectors,
retroviral vectors, Salmonella typhi vectors,
detoxified anthrax toxin vectors, and the like, will be apparent to those
skilled in the art from the description herein.
Thus, gene delivery systems are used to deliver a 109P1 D4-related nucleic
acid molecule. In one embodiment, the full-
length human 109P1 D4 cDNA is employed. In another embodiment, I 09P1 D4
nucleic acid molecules encoding specific cytotoxic
T lymphocyte (CTL) and/or antibody epitopes are employed.
Ex Vivo Vaccines
Various ex vivo strategies can also be employed to generate an immune
response. One approach involves the use of
antigen presenting cells (APCs) such as dendritic cells (DC) to present 109P1
D4 antigen to a patient's immune system. Dendritic
cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and
are thus highly specialized antigen presenting cells.
In prostate cancer, autologous dendritic cells pulsed with peptides of the
prostate-specific membrane antigen (PSMA) are
being used in a Phase I clinical trial to stimulate prostate cancer patients'
immune systems (Tjoa eta!., 1996, Prostate 28:65-
69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be
used to present 109P1 D4 peptides to T cells in
the context of MHC class I or II molecules. In one embodiment, autologous
dendritic cells are pulsed with 109P1 D4 peptides
capable of binding to MHC class I and/or class II molecules. In another
embodiment, dendritic cells are pulsed with the
complete 109P1 D4 protein. Yet another embodiment involves engineering the
overexpression of a 109P1 D4 gene in
dendritic cells using various implementing vectors known in the art, such as
adenoviru6 (Arthur et al., 1997, Cancer Gene
Ther. 4:17-25), retrovirus (Henderson et at, 1996, Cancer Res. 56:3763-3770),
Ientivirus, adeno-associated virus, DNA
transfection (Ribas et at, 1997, Cancer Res. 57:2865-2869), or tumor-derived
RNA transfection (Ashley et al., 1997, J. Exp.
Med. 186:1177-1182). Cells that express 109P1 D4 can also be engineered to
express immune modulators, such as GM-
CSF, and used as immunizing agents.
X.B.) 109P1 D4 as a Target for Antibody-based Therapy
109P1 D4 is an attractive target for antibody-based therapeutic strategies. A
number of antibody strategies are
known in the art for targeting both extracellular and intracellular molecules
(see, e.g., complement and ADCC mediated
killing as well as the use of intrabodies). Because 109P1 D4 is expressed by
cancer cells of various lineages relative to
corresponding normal cells, systemic administration of 109P1 D4-immunoreactive
compositions are prepared that exhibit
excellent sensitivity without toxic, non-specific and/or non-target effects
caused by binding of the immunoreactive
composition to non-target organs and tissues. Antibodies specifically reactive
with domains of 109P1 D4 are useful to treat
109P1 D4-expressing cancers systemically, either as conjugates with a toxin or
therapeutic agent, or as naked antibodies
capable of inhibiting cell proliferation or function.
109P1 D4 antibodies can be introduced into a patient such~that the antibody
binds to 109PI D4 and modulates a
function, such as an interaction with a binding partner, and consequently
mediates destruction of the tumor cells and/or
inhibits the growth of the tumor cells. Mechanisms by which such antibodies
exert a therapeutic effect can include
complement-mediated cytolysis, antibody-dependent cellular cytotoxicity,
modulation of the physiological function of
109P1 D4, inhibition of ligand binding or signal transduction pathways,
modulation of tumor cell differentiation, alteration of
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tumor angiogenesis factor profiles, and/or apoptosis.
Those skilled in the art understand that antibodies can be used to
specifically target and bind immunogenic
molecules such as an immunogenic region of a 109P1 D4 sequence shown in Figure
2 or Figure 3. In addition, skilled
artisans understand that it is routine to conjugate antibodies to cytotoxic
agents (see, e.g., Sievers at at. Blood 93:11 3678-
3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are delivered
directly to cells, such as by conjugating them
to antibodies specific for a molecule expressed by that cell (e.g.109P1 D4),
the cytotoxic agent will exert its known biological
effect (i.e. cytotoxicity) on those cells.
A wide variety of compositions and methods for using antibody-cytotoxic agent
conjugates to kill cells are known in
the art. In the context of cancers, typical methods entail administering to an
animal having a tumor a biologically effective
amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent
linked to a targeting agent (e.g. an anti-
109P1 D4 antibody) that binds to a marker (e.g. 109P1 D4) expressed,
accessible to binding or localized on the cell surfaces.
A typical embodiment is a method of delivering a cytotoxic and/or therapeutic
agent to a cell expressing 109P1 D4,
comprising conjugating the cytotoxic agent to an antibody that
immunospecifically binds to a 109P1 D4 epitope, and,
exposing the cell to the antibody-agent conjugate. Another illustrative
embodiment is a method of treating an individual
suspected of suffering from metastasized cancer, comprising a step of
administering parenterally to said individual a
pharmaceutical composition comprising a therapeutically effective amount of an
antibody conjugated to a cytotoxic and/or
therapeutic agent.
Cancer immunotherapy using anti-109P1 D4 antibodies can be done in accordance
with various approaches that
have been successfully employed in the treatment of other types of cancer,
including but not limited to colon cancer (Arlen et
a!.,1998, Crit. Rev. Immunol.18:133-138), multiple myeloma (Ozaki et at.,
1997, Blood 90:3179-3186, Tsunenari et at,
1997, Blood 90:2437-2444), gastric cancer (Kasprzyk eta!., 1992, Cancer Res.
52:2771-2776), B-cell lymphoma (Funakoshi
et aL,1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong
et a/., 1996, Leuk. Res. 20:581-589),
colorectal cancer (Moun eta!., 1994, Cancer Res. 54:6160-6166; Velders et at,
1995, Cancer Res. 55:4398-4403), and
breast cancer (Shepard at a!.,1991, J. Clin. Immunol.11:117-127). Some
therapeutic approaches involve conjugation of
naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or
1131 to anti-CD20 antibodies (e.g., ZevalinTM, IDEC
Pharmaceuticals Corp. or BexxarTM, Coulter Pharmaceuticals), while others
involve co-administration of antibodies and other
therapeutic agents, such as HerceptinTM (trastuzumab) with paclitaxel
(Genentech, Inc.). The antibodies can be conjugated
to a therapeutic agent. To treat prostate cancer, for example, 109P1 D4
antibodies can be administered in conjunction with
radiation, chemotherapy or hormone ablation. Also, antibodies can be
conjugated to a toxin such as calicheamicin (e.g.,
MylotargTM, Wyeth-Ayerst, Madison, NJ, a recombinant humanized IgG4 kappa
antibody conjugated to antitumor antibiotic
calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug,
TAP, platform, ImmunoGen, Cambridge,
MA, also see e.g., US Patent 5,416,064).
Although 109P1 D4 antibody therapy is useful for all stages of cancer,
antibody therapy can be particularly
appropriate in advanced or metastatic cancers. Treatment with the antibody
therapy of the invention is indicated for patients
who have received one or more rounds of chemotherapy. Alternatively, antibody
therapy of the invention is combined with a
chemotherapeutic or radiation regimen for patients who have not received
chemotherapeutic treatment. Additionally,
antibody therapy can enable the use of reduced dosages of concomitant
chemotherapy, particularly for patients who do not
tolerate the toxicity of the chemotherapeutic agent very well. Fan et al.
(Cancer Res. 53:4637-4642, 1993), Prewett et al.
(International J. of Onco. 9:217-224, 1996), and Hancock at al. (Cancer Res.
51:4575-4580,1991) describe the use of
various antibodies together with chemotherapeutic agents.
Although 109P1 D4 antibody therapy is useful for all stages of cancer,
antibody therapy can be particularly
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appropriate in advanced or metastatic cancers. Treatment with the antibody
therapy of the invention is indicated for patients
who have received one or more rounds of chemotherapy. Alternatively, antibody
therapy of the invention is combined with a
chemotherapeutic or radiation regimen for patients who have not received
chemotherapeutic treatment. Additionally,
antibody therapy can enable the use of reduced dosages of concomitant
chemotherapy, particularly for patients who do not
tolerate the toxicity of the chemotherapeutic agent very well.
Cancer patients can be evaluated for the presence and level of 109PI D4
expression, preferably using
immunohistochemical assessments of tumor tissue, quantitative 109P1 D4
imaging, or other techniques that reliably indicate
the presence and degree of 109P1 D4 expression. Immunohistochemical analysis
of tumor biopsies or surgical specimens is
preferred for this purpose. Methods for immunohistochemical analysis of tumor
tissues are well known in the art.
Anti-I 09P1 D4 monoclonal antibodies that treat prostate and other cancers
include those that initiate a potent
immune response against the tumor or those that are directly cytotoxic. In
this regard, anti-109P1 D4 monoclonal antibodies
(mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-
dependent cell cytotoxicity (ADCC)
mechanisms, both of which require an intact Fc portion of the immunoglobulin
molecule for interaction with effector cell Fc
receptor sites on complement proteins. In addition, anti-I09P1 D4 mAbs that
exert a direct biological effect on tumor growth
are useful to treat cancers that express 109PI D4. Mechanisms by which
directly cytotoxic mAbs act include: inhibition of cell
growth, modulation of cellular differentiation, modulation of tumor
angiogenesis factor profiles, and the induction of apoptosis.
The mechanism(s) by which a particular anti-109P1 D4 mAb exerts an anti-tumor
effect is evaluated using any number of in
vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated
cell lysis, and so forth, as is generally
known in the art.
In some patients, the use of murine or other non-human monoclonal antibodies,
or human/mouse chimeric mAbs
can induce moderate to strong immune responses against the non-human antibody.
This can result in clearance of the
antibody from circulation and reduced efficacy. In the most severe cases, such
an immune response can lead to the
extensive formation of immune complexes which, potentially, can cause renal
failure. Accordingly, preferred monoclonal
antibodies used in the therapeutic methods of the invention are those that are
either fully human or humanized and that bind
specifically to the target 109P1 D4 antigen with high affinity but exhibit low
or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single
anti-I09P1 D4 mAbs as well as
combinations, or cocktails, of different mAbs. Such mAb cocktails can have
certain advantages inasmuch as they contain
mAbs that target different epitopes, exploit different effector mechanisms or
combine directly cytotoxic mAbs with mAbs that
rely on immune effector functionality. Such mAbs in combination can exhibit
synergistic therapeutic effects. In addition, anti-
109P1 D4 mAbs can be administered concomitantly with other therapeutic
modalities, including but not limited to various
chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-
CSF), surgery or radiation. The anti-
109P1 D4 mAbs are administered in their "naked" or unconjugated form, or can
have a therapeutic agent(s) conjugated to
them.
Anti-109P1 D4 antibody formulations are administered via any route capable of
delivering the antibodies to a tumor
cell. Routes of administration include, but are not limited to, intravenous,
intraperitoneal, intramuscular, intratumor,
intradermal, and the like. Treatment generally involves repeated
administration of the anti-I09P1 D4 antibody preparation,
via an acceptable route of administration such as intravenous injection (IV),
typically at a dose in the range of about 0.1, .2,
.3, .4, .5, .6, .7, .8, .9., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25
mglkg body weight. In general, doses in the range of 10-1000
mg mAb per week are effective and well tolerated.
Based on clinical experience with the HerceptinTM mAb in the treatment of
metastatic breast cancer, an initial
loading dose of approximately 4 mg/kg patient body weight IV, followed by
weekly doses of about 2 mg/kg IV of the anti-
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109P1 D4 mAb preparation represents an acceptable dosing regimen. Preferably,
the initial loading dose is administered as
a 90-minute or longer infusion. The periodic maintenance dose is administered
as a 30 minute or longer infusion, provided
the initial dose was well tolerated. As appreciated by those of skill in the
art, various factors can influence the ideal dose
regimen in a particular case. Such factors include, for example, the binding
affinity and half life of the Ab or mAbs used, the
degree of 109P1 D4 expression in the patient, the extent of circulating shed
109P1 D4 antigen, the desired steady-state
antibody concentration level, frequency of treatment, and the influence of
chemotherapeutic or other agents used in
combination with the treatment method of the invention, as well as the health
status of a particular patient.
Optionally, patients should be evaluated for the levels of 109P1 D4 in a given
sample (e.g. the levels of circulating
109P1 D4 antigen and/or 109P1 D4 expressing cells) in order to assist in the
determination of the most effective dosing
regimen, etc. Such evaluations are also used for monitoring purposes
throughout therapy, and are useful to gauge
therapeutic success in combination with the evaluation of other parameters
(for example, urine cytology and/or ImmunoCyt
levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate
cancer therapy).
Anti-idiotypic anti-I09P1 D4 antibodies can also be used in anti-cancer
therapy as a vaccine for inducing an
immune response to cells expressing a 109P1 D4-related protein. In particular,
the generation of anti-idiotypic antibodies is
well known in the art; this methodology can readily be adapted to generate
anti-idiotypic anti-I09P1 D4 antibodies that mimic
an epitope on a 109P1 D4-related protein (see, for example, Wagner et al.,
1997, Hybridoma 16: 33-40; Foon et at., 1995, J.
Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. lmmunother.
43:65-76). Such an anti-idiotypic antibody can
be used in cancer vaccine strategies.
X.C.) 109P1 D4 as a Target for Cellular Immune Responses
Vaccines and methods of preparing vaccines that contain an immunogenically
effective amount of one or more
HLA-binding peptides as described herein are further embodiments of the
invention. Furthermore, vaccines in accordance
with the invention encompass compositions of one or more of the claimed
peptides. A peptide can be present in a vaccine
individually. Alternatively, the peptide can exist as a homopolymer comprising
multiple copies of the same peptide, or as a
heteropolymer of various peptides. Polymers have the advantage of increased
immunological reaction and, where different
peptide epitopes are used to make up the polymer, the additional ability to
induce antibodies and/or CTLs that react with
different antigenic determinants of the pathogenic organism or tumor-related
peptide targeted for an immune response. The
composition can be a naturally occurring region of an antigen or can be
prepared, e.g., recombinantly or by chemical
synthesis.
Carriers that can be used with vaccines of the invention are well known in the
art, and include, e.g., thyroglobulin,
albumins such as human serum albumin, tetanus toxoid, polyamino acids such as
poly L-lysine, poly L-glutamic acid,
influenza, hepatitis B virus core protein, and the like. The vaccines can
contain a physiologically tolerable (i.e., acceptable)
diluent such as water, or saline, preferably phosphate buffered saline. The
vaccines also typically include an adjuvant.
Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum
hydroxide, or alum are examples of
materials well known in the art. Additionally, as disclosed herein, CTL
responses can be primed by conjugating peptides of
the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl- serine
(P3CSS). Moreover, an adjuvant such as a
synthetic cytosine-phosphorothiolated-guanine-containing (CpG)
oligonucleotides has been found to increase CTL
responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-
547 (2000))
Upon immunization with a peptide composition in accordance with the invention,
via injection, aerosol, oral,
transdermal, transmucosal, intrapleural, intrathecal, or other suitable
routes, the immune system of the host responds to the
vaccine by producing large amounts of CTLs and/or HTLs specific for the
desired antigen. Consequently, the host becomes
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56
at least partially immune to later development of cells that express or
overexpress 109P1 D4 antigen, or derives at least
some therapeutic benefit when the antigen was tumor-associated.
In some embodiments, it may be desirable to combine the class I peptide
components with components that
induce or facilitate neutralizing antibody and or helper T cell responses
directed to the target antigen. A preferred
embodiment of such a composition comprises class I and class II epitopes in
accordance with the invention. An alternative
embodiment of such a composition comprises a class I and/or class II epitope
in accordance with the invention, along with a
cross reactive HTL epitope such as PADRETM (Epimmune, San Diego, CA) molecule
(described e.g., in U.S. Patent Number
5,736,142).
A vaccine of the invention can also include antigen-presenting cells (APC),
such as dendritic cells (DC), as a
vehicle to present peptides of the invention. Vaccine compositions can be
created in vitro, following dendritic cell
mobilization and harvesting, whereby loading of dendritic cells occurs in
vitro. For example, dendritic cells are transfected,
e.g., with a minigene in accordance with the invention, or are pulsed with
peptides. The dendritic cell can then be
administered to a patient to elicit immune responses in vivo. Vaccine
compositions, either DNA- or peptide-based, can also
be administered in vivo in combination with dendritic cell mobilization
whereby loading of dendritic cells occurs in vivo.
Preferably, the following principles are utilized when selecting an array of
epitopes for inclusion in a polyepitopic
composition for use in a vaccine, or for selecting discrete epitopes to be
included in a vaccine and/or to be encoded by
nucleic acids such as a minigene. It is preferred that each of the following
principles be balanced in order to make the
selection. The multiple epitopes to be incorporated in a given vaccine
composition may be, but need not be, contiguous in
sequence in the native antigen from which the epitopes are derived.
1.) Epitopes are selected which, upon administration, mimic immune responses
that have been observed to
be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes
that come from at least one tumor associated
antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4
epitopes are selected from at least one TAA (see,
e.g., Rosenberg et at., Science 278:1447-1450). Epitopes from one TAA may be
used in combination with epitopes from one
or more additional TAAs to produce a vaccine that targets tumors with varying
expression patterns of frequently-expressed
TAAs.
2.) Epitopes are selected that have the requisite binding affinity established
to be correlated with
immunogenicity: for HLA Class I an IC5o of 500 nM or less, often 200 nM or
less; and for Class II an IC5o of 1000 nM or less.
3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-
specific motif-bearing peptides, are
selected to give broad population coverage. For example, it is preferable to
have at least 80% population coverage. A
Monte Carlo analysis, a statistical evaluation known in the art, can be
employed to assess the breadth, or redundancy of,
population coverage.
4.) When selecting epitopes from cancer-related antigens it is often useful to
select analogs because the
patient may have developed tolerance to the native epitope.
5.) Of particular relevance are epitopes referred to as "nested epitopes."
Nested epitopes occur where at
least two epitopes overlap in a given peptide sequence. A nested peptide
sequence can comprise B cell, HLA class I and/or
HLA class II epitopes. When providing nested epitopes, a general objective is
to provide the greatest number of epitopes per
sequence. Thus, an aspect is to avoid providing a peptide that is any longer
than the amino terminus of the amino terminal
epitope and the carboxyl terminus of the carboxyl terminal epitope in the
peptide. When providing a multi-epitopic sequence,
such as a sequence comprising nested epitopes, it is generally important to
screen the sequence in order to insure that it
does not have pathological or other deleterious biological properties.
6.) If a polyepitopic protein is created, or when creating a minigene, an
objective is to generate the smallest
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57
peptide that encompasses the epitopes of interest. This principle is similar,
if not the same as that employed when selecting
a peptide comprising nested epitopes. However, with an artificial polyepitopic
peptide, the size minimization objective is
balanced against the need to integrate any spacer sequences between epitopes
in the polyepitopic protein. Spacer amino
acid residues can, for example, be introduced to avoid junctional epitopes (an
epitope recognized by the immune system, not
present in the target antigen, and only created by the man-made juxtaposition
of epitopes), or to facilitate cleavage between
epitopes and thereby enhance epitope presentation. Junctional epitopes are
generally to be avoided because the recipient
may generate an immune response to that non-native epitope. Of particular
concern is a junctional epitope that is a
"dominant epitope." A dominant epitope may lead to such a zealous response
that immune responses to other epitopes are
diminished or suppressed.
7.) Where the sequences of multiple variants of the same target protein are
present, potential peptide
epitopes can also be selected on the basis of their conservancy. For example,
a criterion for conservancy may define that
the entire sequence of an HLA class I binding peptide or the entire 9-mer core
of a class II binding peptide be conserved in a
designated percentage of the sequences evaluated for a specific protein
antigen.
X.C.I. Minigene Vaccines
A number of different approaches are available which allow simultaneous
delivery of multiple epitopes. Nucleic
acids encoding the peptides of the invention are a particularly useful
embodiment of the invention. Epitopes for inclusion in a
minigene are preferably selected according to the guidelines set forth in the
previous section. A preferred means of
administering nucleic acids encoding the peptides of the invention uses
minigene constructs encoding a peptide comprising
one or multiple epitopes of the invention.
The use of multi-epitope minigenes is described below and in, ishioka et a1.,
J. Immunol.162:3915-3925, 1999; An,
L. and Whitton, J. L., J. Virol. 71:2292,1997; Thomson, S. A. et al., J.
ImmunoL 157:822, 1996; Whitton, J. L. et al., J. Virol.
67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-
epitope DNA plasmid encoding supermotif-
and/or motif-bearing epitopes derived 109P1 D4, the PADRE universal helper T
cell epitope or multiple HTL epitopes from
109P1 D4 (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic
reticulum-translocating signal sequence can be
engineered. A vaccine may also comprise epitopes that are derived from other
TAAs.
The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic
mice to evaluate the magnitude of
CTL induction responses against the epitopes tested. Further, the
immunogenicity of DNA-encoded epitopes in vivo can be
correlated with the in vitro responses of specific CTL lines against target
cells transfected with the DNA plasmid. Thus, these
experiments can show that the minigene serves to both: 1.) generate a CTL
response and 2.) that the induced CTLs
recognized cells expressing the encoded epitopes.
For example, to create a DNA sequence encoding the selected epitopes
(minigene) for expression in human cells,
the amino acid sequences of the epitopes may be reverse translated. A human
codon usage table can be used to guide the
codon choice for each amino acid. These epitope-encoding DNA sequences may be
directly adjoined, so that when
translated, a continuous polypeptide sequence is created. To optimize
expression and/or immunogenicity, additional
elements can be incorporated into the minigene design. Examples of amino acid
sequences that can be reverse translated
and included in the minigene sequence include: HLA class I epitopes, HLA class
Ii epitopes, antibody epitopes, a
ubiquitination signal sequence, and/or an endoplasmic reticulum targeting
signal. In addition, HLA presentation of CTL and
HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or
naturally-occurring flanking sequences adjacent
to the CTL or HTL epitopes; these larger peptides comprising the epitope(s)
are within the scope of the invention.
The minigene sequence may be converted to DNA by assembling oligonucleotides
that encode the plus and minus
strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may
be synthesized, phosphorylated, purified
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58
and annealed under appropriate conditions using well known techniques. The
ends of the oligonucleotides can be joined, for
example, using T4 DNA ligase. This synthetic minigene, encoding the epitope
polypeptide, can then be cloned into a desired
expression vector.
Standard regulatory sequences well known to those of skill in the art are
preferably included in the vector to ensure
expression in the target cells. Several vector elements are desirable: a
promoter with a down-stream cloning site for
minigene insertion; a polyadenylation signal for efficient transcription
termination; an E. coli origin of replication; and an E.
coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous
promoters can be used for this purpose, e.g., the
human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Patent Nos. 5,580,859
and 5,589,466 for other suitable promoter
sequences.
Additional vector modifications may be desired to optimize minigene expression
and immunogenicity. In some
cases, introns are required for efficient gene expression, and one or more
synthetic or naturally-occurring introns could be
incorporated into the transcribed region of the minigene. The inclusion of
mRNA stabilization sequences and sequences for
replication in mammalian cells may also be considered for increasing minigene
expression.
Once an expression vector is selected, the minigene is cloned into the
polylinker region downstream of the
promoter. This plasmid is transformed into an appropriate E. coli strain, and
DNA is prepared using standard techniques.
The orientation and DNA sequence of the minigene, as well as all other
elements included in the vector, are confirmed using
restriction mapping and DNA sequence analysis. Bacterial cells harboring the
correct plasmid can be stored as a master cell
bank and a working cell bank.
In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role
in the immunogenicity of DNA
vaccines. These sequences may be included in the vector, outside the minigene
coding sequence, if desired to enhance
immunogenicity.
In some embodiments, a bi-cistronic expression vector which allows production
of both the minigene-encoded
epitopes and a second protein (included to enhance or decrease immunogenicity)
can be used. Examples of proteins or
polypeptides that could beneficially enhance the immune response if co-
expressed include cytokines (e.g., IL-2, IL-12, GM-
CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or
for HTL responses, pan-DR binding proteins
(PADRETM, Epimmune, San Diego, CA). Helper (HTL) epitopes can be joined to
intracellular targeting signals and
expressed separately from expressed CTL epitopes; this allows direction of the
HTL epitopes to a cell compartment different
than that of the CTL epitopes. If required, this could facilitate more
efficient entry of HTL epitopes into the HLA class II
pathway, thereby improving HTL induction. In contrast to HTL or CTL induction,
specifically decreasing the immune
response by co-expression of immunosuppressive molecules (e.g. TGF-(3) may be
beneficial in certain diseases.
Therapeutic quantities of plasmid DNA can be produced for example, by
fermentation in E. coli, followed by
purification. Aliquots from the working cell bank are used to inoculate growth
medium, and grown to saturation in shaker
flasks or a bioreactor according to well-known techniques. Plasmid DNA can be
purified using standard bioseparation
technologies such as solid phase anion-exchange resins supplied by QIAGEN,
Inc. (Valencia, California). If required,
supercoiled DNA can be isolated from the open circular and linear forms using
gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of
formulations. The simplest of these is
reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS).
This approach, known as "naked DNA," is
currently being used for intramuscular (IM) administration in clinical trials.
To maximize the immunotherapeutic effects of
minigene DNA vaccines, an alternative method for formulating purified plasmid
DNA may be desirable. A variety of methods
have been described, and new techniques may become available. Cationic lipids,
glycolipids, and fusogenic liposomes can
also be used in the formulation (see, e.g., as described by WO 93/24640;
Mannino & Gould-Fogerite, BioTechniques 6(7):
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59
682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, et aL, Proc.
Nat'l Acad. Sci. USA 84:7413 (1987). In
addition, peptides and compounds referred to collectively as protective,
interactive, non-condensing compounds (PINC)
could also be complexed to purified plasmid DNA to influence variables such as
stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional assay for expression and
HLA class I presentation of
minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into
a mammalian cell line that is suitable as
a target for standard CTL chromium release assays. The transfection method
used will be dependent on the final
formulation. Electroporation can be used for "naked" DNA, whereas cationic
lipids allow direct in vitro transfection. A
plasmid expressing green fluorescent protein (GFP) can be co-transfected to
allow enrichment of transfected cells using
fluorescence activated cell sorting (FACS). These cells are then chromium-51
(51Cr) labeled and used as target cells for
epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates
both production of, and HLA presentation of,
minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in
an analogous manner using assays to
assess HTL activity.
In vivo immunogenicity is a second approach for functional testing of minigene
DNA formulations. Transgenic mice
expressing appropriate human HLA proteins are immunized with the DNA product.
The dose and route of administration are
formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for
lipid-complexed DNA). Twenty-one days after
immunization, splenocytes are harvested and restimulated for one week in the
presence of peptides encoding each epitope
being tested. Thereafter, for CTL effector cells, assays are conducted for
cytolysis of peptide-loaded, 51Cr-labeled target
cells using standard techniques. Lysis of target cells that were sensitized by
HLA loaded with peptide epitopes,
corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function
for in vivo induction of CTLs.
Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous
manner.
Alternatively, the nucleic acids can be administered using ballistic delivery
as described, for instance, in U.S.
Patent No. 5,204,253. Using this technique, particles comprised solely of DNA
are administered. In a further alternative
embodiment, DNA can be adhered to particles, such as gold particles.
Minigenes can also be delivered using other bacterial or viral delivery
systems well known in the art, e.g., an
expression construct encoding epitopes of the invention can be incorporated
into a viral vector such as vaccinia.
X.C.2. Combinations of CTL Peptides with Helper Peptides
Vaccine compositions comprising CTL peptides of the invention can be modified,
e.g., analoged, to provide desired
attributes, such as improved serum half life, broadened population coverage or
enhanced immunogenicity.
For instance, the ability of a peptide to induce CTL activity can be enhanced
by linking the peptide to a sequence
which contains at least one epitope that is capable of inducing a T helper
cell response. Although a CTL peptide can be
directly linked to a T helper peptide, often CTL epitope/HTL epitope
conjugates are linked by a spacer molecule. The spacer
is typically comprised of relatively small, neutral molecules, such as amino
acids or amino acid mimetics, which are
substantially uncharged under physiological conditions. The spacers are
typically selected from, e.g., Ala, Gly, or other
neutral spacers of nonpolar amino acids or neutral polar amino acids. It will
be understood that the optionally present spacer
need not be comprised of the same residues and thus may be a hetero- or homo-
oligomer. When present, the spacer will
usually be at least one or two residues, more usually three to six residues
and sometimes 10 or more residues. The CTL
peptide epitope can be linked to the T helper peptide epitope either directly
or via a spacer either at the amino or carboxy
terminus of the CTL peptide. The amino terminus of either the immunogenic
peptide or the T helper peptide may be
acylated.
In certain embodiments, the T helper peptide is one that is recognized by T
helper cells present in a majority of a
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genetically diverse population. This can be accomplished by selecting peptides
that bind to many, most, or all of the HLA
class 11 molecules. Examples of such amino acid bind many HLA Class lI
molecules include sequences from antigens such
as tetanus toxoid at positions 830-843 QYIKANSKFIGITE; (SEQ ID NO: 40),
Plasmodium falciparum circumsporozoite (CS)
protein at positions 378-398 DIEKKIAKMEKASSVFNWNS; (SEQ ID NO: 41), and
Streptococcus 18kD protein at positions
116-131 GAVDSILGGVATYGAA; (SEQ ID NO: 42). Other examples include peptides
bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
Alternatively, it is possible to prepare synthetic peptides capable of
stimulating T helper lymphocytes, in a loosely
HLA-restricted fashion, using amino acid sequences not found in nature (see,
e.g., PCT publication WO 95/07707). These
synthetic compounds called Pan-DR-binding epitopes (e.g., PADRETM, Epimmune,
Inc., San Diego, CA) are designed, most
preferably, to bind most HLA-DR (human HLA class II) molecules. For instance,
a pan-DR-binding epitope peptide having
the formula: xKXVAAWTLKAAx (SEQ ID NO: 43), where 'Xis either
cyclohexylalanine, phenylalanine, or tyrosine, and a is
either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles,
and to stimulate the response of T helper
lymphocytes from most individuals, regardless of their HLA type. An
alternative of a pan-DR binding epitope comprises all
"L" natural amino acids and can be provided in the form of nucleic acids that
encode the epitope.
HTL peptide epitopes can also be modified to alter their biological
properties. For example, they can be modified
to include D-amino acids to increase their resistance to proteases and thus
extend their serum half life, or they can be
conjugated to other molecules such as lipids, proteins, carbohydrates, and the
like to increase their biological activity. For
example, a T helper peptide can be conjugated to one or more palmitic acid
chains at either the amino or carboxyl termini.
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
In some embodiments it may be desirable to include in the pharmaceutical
compositions of the invention at least
one component which primes B lymphocytes or T lymphocytes. Lipids have been
identified as agents capable of priming
CTL in vivo. For example, palmitic acid residues can be attached to the c-and
a- amino groups of a lysine residue and then
linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-
Ser, or the like, to an immunogenic peptide.
The lipidated peptide can then be administered either directly in a micelle or
particle, incorporated into a liposome, or
emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred
embodiment, a particularly effective
immunogenic composition comprises palmitic acid attached to c- and a- amino
groups of Lys, which is attached via linkage,
e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
As another example of lipid priming of CTL responses, E. coli lipoproteins,
such as tripalmitoyl-S-
glycerylcysteinlyseryl- serine (P3CSS) can be used to prime virus specific CTL
when covalently attached to an appropriate
peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the
invention can be coupled to P3CSS, for example,
and the lipopeptide administered to an individual to prime specifically an
immune response to the target antigen. Moreover,
because the induction of neutralizing antibodies can also be primed with P3CSS-
conjugated epitopes, two such compositions
can be combined to more effectively elicit both humoral and cell-mediated
responses.
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
An embodiment of a vaccine composition in accordance with the invention
comprises ex vivo administration of a
cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from
the patient's blood. A pharmaceutical to
facilitate harvesting of DC can be used, such as ProgenipoietinTM (Pharmacia-
Monsanto, St. Louis, MO) or GM-CSF/IL-4.
After pulsing the DC with peptides and prior to reinfusion into patients, the
DC are washed to remove unbound peptides. In
this embodiment, a vaccine comprises peptide-pulsed DCs which present the
pulsed peptide epitopes complexed with HLA
molecules on their surfaces.
The DC can be pulsed ex vivo with a cocktail of peptides, some of which
stimulate CTL responses to 109P1 D4.
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Optionally, a helper T cell (HTL) peptide, such as a natural or artificial
loosely restricted HLA Class II peptide, can be
included to facilitate the CTL response. Thus, a vaccine in accordance with
the invention is used to treat a cancer which
expresses or overexpresses 109P1 D4.
X.D. Adoptive Immunotherapy
Antigenic 109P1 D4-related peptides are used to elicit a CTL and/or HTL
response ex vivo, as well. The resulting
CTL or HTL cells, can be used to treat tumors in patients that do not respond
to other conventional forms of therapy, or will
not respond to a therapeutic vaccine peptide or nucleic acid in accordance
with the invention. Ex vivo CTL or HTL
responses to a particular antigen are induced by incubating in tissue culture
the patient's, or genetically compatible, CTL or
HTL precursor cells together with a source of antigen-presenting cells (APC),
such as dendritic cells, and the appropriate
immunogenic peptide. After an appropriate incubation time (typically about 7-
28 days), in which the precursor cells are
activated and expanded into effector cells, the cells are infused back into
the patient, where they will destroy (CTL) or
facilitate destruction (HTL) of their specific target cell (e.g., a tumor
cell). Transfected dendritic cells may also be used as
antigen presenting cells.
X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes
Pharmaceutical and vaccine compositions of the invention are typically used to
treat and/or prevent a cancer that
expresses or overexpresses 109P1 D4. In therapeutic applications, peptide
and/or nucleic acid compositions are
administered to a patient in an amount sufficient to elicit an effective B
cell, CTL and/or HTL response to the antigen and to
cure or at least partially arrest or slow symptoms and/or complications. An
amount adequate to accomplish this is defined as
"therapeutically effective dose." Amounts effective for this use will depend
on, e.g., the particular composition administered,
the manner of administration, the stage and severity of the disease being
treated, the weight and general state of health of
the patient, and the judgment of the prescribing physician.
For pharmaceutical compositions, the immunogenic peptides of the invention, or
DNA encoding them, are
generally administered to an individual already bearing a tumor that expresses
109P1 D4. The peptides or DNA encoding
them can be administered individually or as fusions of one or more peptide
sequences. Patients can be treated with the
immunogenic peptides separately or in conjunction with other treatments, such
as surgery, as appropriate.
For therapeutic use, administration should generally begin at the first
diagnosis of 109P1 D4-associated cancer.
This is followed by boosting doses until at least symptoms are substantially
abated and for a period thereafter. The
embodiment of the vaccine composition (i.e., including, but not limited to
embodiments such as peptide cocktails,
polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic
cells) delivered to the patient may vary
according to the stage of the disease or the patient's health status. For
example, in a patient with a tumor that expresses
109P1 D4, a vaccine comprising 109P1 D4-specific CTL may be more efficacious
in killing tumor cells in patient with
advanced disease than alternative embodiments.
It is generally important to provide an amount of the peptide epitope
delivered by a mode of administration
sufficient to stimulate effectively a cytotoxic T cell response; compositions
which stimulate helper T cell responses can also
be given in accordance with this embodiment of the invention.
The dosage for an initial therapeutic immunization generally occurs in a unit
dosage range where the lower value is
about 1, 5, 50, 500, or 1,000 pg and the higher value is about 10,000; 20,000;
30,000; or 50,000 pg. Dosage values for a
human typically range from about 500 pg to about 50,000 pg per 70 kilogram
patient. Boosting dosages of between about
1.0 g to about 50,000 g of peptide pursuant to a boosting regimen over weeks
to months may be administered depending
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upon the patient's response and condition as determined by measuring the
specific activity of CTL and HTL obtained from
the patient's blood. Administration should continue until at least clinical
symptoms or laboratory tests indicate that the
neoplasia, has been eliminated or reduced and for a period thereafter. The
dosages, routes of administration, and dose
schedules are adjusted in accordance with methodologies known in the art.
In certain embodiments, the peptides and compositions of the present invention
are employed in serious disease
states, that is, life-threatening or potentially life threatening situations.
In such cases, as a result of the minimal amounts of
extraneous substances and the relative nontoxic nature of the peptides in
preferred compositions of the invention, it is
possible and may be felt desirable by the treating physician to administer
substantial excesses of these peptide compositions
relative to these stated dosage amounts.
The vaccine compositions of the invention can also be used purely as
prophylactic agents. Generally the dosage
for an initial prophylactic immunization generally occurs in a unit dosage
range where the lower value is about 1, 5, 50, 500,
or 1000 pg and the higher value is about 10,000; 20,000; 30,000; or 50,000 pg.
Dosage values for a human typically range
from about 500 pg to about 50,000 pg per 70 kilogram patient. This is followed
by boosting dosages of between about 1.0
g to about 50,000 g of peptide administered at defined intervals from about
four weeks to six months after the initial
administration of vaccine. The immunogenicity of the vaccine can be assessed
by measuring the specific activity of CTL and
HTL obtained from a sample of the patient's blood.
The pharmaceutical compositions for therapeutic treatment are intended for
parenteral, topical, oral, nasal,
intrathecal, or local (e.g. as a cream or topical ointment) administration.
Preferably, the pharmaceutical compositions are
administered parentally, e.g., intravenously, subcutaneously, intradermally,
or intramuscularly. Thus, the invention provides
compositions for parenteral administration which comprise a solution of the
immunogenic peptides dissolved or suspended in
an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8%
saline, 0.3% glycine, hyaluronic acid
and the like. These compositions may be sterilized by conventional, well-known
sterilization techniques, or may be sterile
filtered. The resulting aqueous solutions may be packaged for use as is, or
lyophilized, the lyophilized preparation being
combined with a sterile solution prior to administration.
The compositions may contain pharmaceutically acceptable auxiliary substances
as required to approximate
physiological conditions, such as pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservatives,
and the like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, etc.
The concentration of peptides of the invention in the pharmaceutical
formulations can vary widely, i.e., from less
than about 0.1 %, usually at or at least about 2% to as much as 20% to 50% or
more by weight, and will be selected primarily
by fluid volumes, viscosities, etc., in accordance with the particular mode of
administration selected.
A human unit dose form of a composition is typically included in a
pharmaceutical composition that comprises a
human unit dose of an acceptable carrier, in one embodiment an aqueous
carrier, and is administered in a volume/quantity
that is known by those of skill in the art to be used for administration of
such compositions to humans (see, e.g., Remington's
Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing
Co., Easton, Pennsylvania, 1985). For example
a peptide dose for initial immunization can be from about 1 to about 50,000
g, generally 100-5,000 lag, for a 70 kg patient.
For example, for nucleic acids an initial immunization may be performed using
an expression vector in the form of naked
nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at
multiple sites. The nucleic acid (0.1 to 1000 g)
can also be administered using a gene gun. Following an incubation period of 3-
4 weeks, a booster dose is then
administered. The booster can be recombinant fowlpox virus administered at a
dose of 5-107 to 5x1 09 pfu.
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For antibodies, a treatment generally involves repeated administration of the
anti-109P1D4 antibody preparation,
via an acceptable route of administration such as intravenous injection (IV),
typically at a dose in the range of about 0.1 to
about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb
per week are effective and well tolerated.
Moreover, an initial loading dose of approximately 4 mg/kg patient body weight
IV, followed by weekly doses of about 2
mg/kg IV of the anti-109P1 D4 mAb preparation represents an acceptable dosing
regimen. As appreciated by those of skill
in the art, various factors can influence the ideal dose in a particular case.
Such factors include, for example, half life of a
composition, the binding affinity of an Ab, the immunogenicity of a substance,
the degree of 109P1 D4 expression in the
patient, the extent of circulating shed 109P1 D4 antigen, the desired steady-
state concentration level, frequency of treatment,
and the influence of chemotherapeutic or other agents used in combination with
the treatment method of the invention, as
well as the health status of a particular patient. Non-limiting preferred
human unit doses are, for example, 500pg -1 mg, 1 mg
- 50mg, 50mg -100mg,100mg - 200mg, 200mg - 300mg, 400mg - 500mg, 500mg -
600mg, 600mg - 700mg, 700mg -
800mg, 800mg - 900mg, 900mg -1 g, or 1 mg - 700mg. In certain embodiments, the
dose is in a range of 2-5 mg/kg body
weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5mg, 1, 2, 3, 4, 5,
6, 7, 8, 9,10mg/kg body weight followed, e.g., in
two, three or four weeks by weekly doses; 0.5 -10mg/kg body weight, e.g.,
followed in two, three or four weeks by weekly
doses; 225, 250, 275, 300, 325, 350, 375, 400mg m2 of body area weekly; 1-
600mg m2 of body area weekly; 225-400mg m2
of body area weekly; these does can be followed by weekly doses for 2, 3, 4,
5, 6, 7, 8, 9, 19, 11, 12 or more weeks.
In one embodiment, human unit dose forms of polynucleotides comprise a
suitable dosage range or effective
amount that provides any therapeutic effect. As appreciated by one of ordinary
skill in the art a therapeutic effect depends
on a number of factors, including the sequence of the polynucleotide,
molecular weight of the polynucleotide and route of
administration. Dosages are generally selected by the physician or other
health care professional in accordance with a
variety of parameters known in the art, such as severity of symptoms, history
of the patient and the like. Generally, for a
polynucleotide of about 20 bases, a dosage range may be selected from, for
example, an independently selected lower limit
such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,
100, 200, 300, 400 or 500 mg/kg up to an
independently selected upper limit, greater than the lower limit, of about 60,
80, 100, 200, 300, 400, 500, 750, 1000, 1500,
2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a
dose may be about any of the following:
0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, Ito 500
mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1
to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to
1000 mg/kg, 500 to 5000 mg/kg, or 500 to
10,000 mg/kg. Generally, parenteral routes of administration may require
higher doses of polynucleotide compared to more
direct application to the nucleotide to diseased tissue, as do polynucleotides
of increasing length.
In one embodiment, human unit dose forms of T-cells comprise a suitable dosage
range or effective amount that
provides any therapeutic effect. As appreciated by one of ordinary skill in
the art, a therapeutic effect depends on a number
of factors. Dosages are generally selected by the physician or other health
care professional in accordance with a variety of
parameters known in the art, such as severity of symptoms, history of the
patient and the like. A dose may be about 104
cells to about 106 cells, about 106 cells to about 108 cells, about 108 to
about 1011 cells, or about 108 to about 5 x 1010 cells.
A dose may also about 106 cells/m2 to about 1010 cells/m2, or about 106
cells/m2 to about 108 cells/M2.
Proteins(s) of the invention, and/or nucleic acids encoding the protein(s),
can also be administered via liposomes,
which may also serve to: 1) target the proteins(s) to a particular tissue,
such as lymphoid tissue; 2) to target selectively to
diseases cells; or, 3) to increase the half-life of the peptide composition.
Liposomes include emulsions, foams, micelles,
insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar
layers and the like. In these preparations, the
peptide to be delivered is incorporated as part of a liposome, alone or in
conjunction with a molecule which binds to a
receptor prevalent among lymphoid cells, such as monoclonal antibodies which
bind to the CD45 antigen, or with other
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therapeutic or immunogenic compositions. Thus, liposomes either filled or
decorated with a desired peptide of the invention
can be directed to the site of lymphoid cells, where the liposomes then
deliver the peptide compositions. Liposomes for use
in accordance with the invention are formed from standard vesicle-forming
lipids, which generally include neutral and
negatively charged phospholipids and a sterol, such as cholesterol. The
selection of lipids is generally guided by
consideration of, e.g., liposome size, acid lability and stability of the
liposomes in the blood stream. A variety of methods are
available for preparing liposomes, as described in, e.g., Szoka, et al., Ann.
Rev. Biophys, Bioeng. 9:467 (1980), and U.S.
Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
For targeting cells of the immune system, a ligand to be incorporated into the
liposome can include, e.g.,
antibodies or fragments thereof specific for cell surface determinants of the
desired immune system cells. A liposome
suspension containing a peptide may be administered intravenously, locally,
topically, etc. in a dose which varies according
to, inter alia, the manner of administration, the peptide being delivered, and
the stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used which
include, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, talcum, cellulose, glucose,
sucrose, magnesium carbonate, and the like. For oral administration, a
pharmaceutically acceptable nontoxic composition is
formed by incorporating any of the normally employed excipients, such as those
carriers previously listed, and generally 10-
95% of active ingredient, that is, one or more peptides of the invention, and
more preferably at a concentration of 25%-75%.
For aerosol administration, immunogenic peptides are preferably supplied in
finely divided form along with a
surfactant and propellant. Typical percentages of peptides are about 0.01 %-
20% by weight, preferably about 1%-10%. The
surfactant must, of course, be nontoxic, and preferably soluble in the
propellant. Representative of such agents are the
esters or partial esters of fatty acids containing from about 6 to 22 carbon
atoms, such as caproic, octanoic, lauric, palmitic,
stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic
polyhydric alcohol or its cyclic anhydride. Mixed esters,
such as mixed or natural glycerides may be employed. The surfactant may
constitute about 0.1 %-20% by weight of the
composition, preferably about 0.25-5%. The balance of the composition is
ordinarily propellant. A carrier can also be
included, as desired, as with, e.g., lecithin for intranasal delivery.
XI.) Diagnostic and Prognostic Embodiments of 109P1D4.
As disclosed herein, 109P1 D4 polynucleotides, polypeptides, reactive
cytotoxic T cells (CTL), reactive helper T
cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic,
prognostic and therapeutic assays that
examine conditions associated with dysregulated cell growth such as cancer, in
particular the cancers listed in Table I (see,
e.g., both its specific pattern of tissue expression as well as its
overexpression in certain cancers as described for example in
the Example entitled "Expression analysis of 109P1 D4 in normal tissues, and
patient specimens").
I09P1D4 can be analogized to a prostate associated antigen PSA, the archetypal
marker that has been used by
medical practitioners for years to identify and monitor the presence of
prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2):
503-5120 (2000); Polascik et al., J. Urol. Aug; 162(2):293-306 (1999) and
Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-
1640(1999)). A variety of other diagnostic markers are also used in similar
contexts including p53 and K-ras (see, e.g.,
Tulchinsky et al., Int J Mol Med 1999 Jul 4(1):99-102 and Minimoto et al.,
Cancer Detect Prev 2000;24(1):1-12). Therefore,
this disclosure of 109P1 D4 polynucleotides and polypeptides (as well as 109P1
D4 polynucleotide probes and anti-109P1 D4
antibodies used to identify the presence of these molecules) and their
properties allows skilled artisans to utilize these
molecules in methods that are analogous to those used, for example, in a
variety of diagnostic assays directed to examining
conditions associated with cancer.
Typical embodiments of diagnostic methods which utilize the 109P1 D4
polynucleotides, polypeptides, reactive T
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cells and antibodies are analogous to those methods from well-established
diagnostic assays, which employ, e.g., PSA
polynucleotides, polypeptides, reactive T cells and antibodies. For example,
just as PSA polynucleotides are used as probes
(for example in Northern analysis, see, e.g., Sharief et aL, Biochem. Mol.
Biol. Int. 33(3):567-74(1994)) and primers (for
example in PCR analysis, see, e.g., Okegawa of aL, J. Urol. 163(4): 1189-1190
(2000)) to observe the presence and/or the
level of PSA mRNAs in methods of monitoring PSA overexpression or the
metastasis of prostate cancers, the 109P1 D4
polynucleotides described herein can be utilized in the same way to detect
109P1 D4 overexpression or the metastasis of
prostate and other cancers expressing this gene. Alternatively, just as PSA
polypeptides are used to generate antibodies
specific for PSA which can then be used to observe the presence and/or the
level of PSA proteins in methods to monitor
PSA protein overexpression (see, e.g., Stephan of al., Urology 55(4):560-3
(2000)) or the metastasis of prostate cells (see,
e.g., Alanen eta!., Pathol. Res. Pract. 192(3):233-7 (1996)), the 109P1 D4
polypeptides described herein can be utilized to
generate antibodies for use in detecting 109P1 D4 overexpression or the
metastasis of prostate cells and cells of other
cancers expressing this gene.
Specifically, because metastases involves the movement of cancer cells from an
organ of origin (such as the lung
or prostate gland etc.) to a different area of the body (such as a lymph
node), assays which examine a biological sample for
the presence of cells expressing 109P1 D4 polynucleotides and/or polypeptides
can be used to provide evidence of
metastasis. For example, when a biological sample from tissue that does not
normally contain 109P1 D4-expressing cells
(lymph node) is found to contain 109P1 D4-expressing cells such as the 109P1
D4 expression seen in LAPC4 and LAPC9,
xenografts isolated from lymph node and bone metastasis, respectively, this
finding is indicative of metastasis.
Alternatively 109P1 D4 polynucleotides and/or polypeptides can be used to
provide evidence of cancer, for
example, when cells in a biological sample that do not normally express 109P1
D4 or express 109P1 D4 at a different level are
found to express 109P1D4 or have an increased expression of 109P1D4 (see,
e.g., the 109P1D4 expression in the cancers
listed in Table I and in patient samples etc. shown in the accompanying
Figures). In such assays, artisans may further wish
to generate supplementary evidence of metastasis by testing the biological
sample for the presence of a second tissue
restricted marker (in addition to 109P1 D4) such as PSA, PSCA etc. (see, e.g.,
Alanen eta!., Pathol. Res. Pract. 192(3): 233-
237 (1996)).
The use of immunohistochemistry to identify the presence of a 109P1 D4
polypeptide within a tissue section can
indicate an altered state of certain cells within that tissue. It is well
understood in the art that the ability of an antibody to
localize to a polypeptide that is expressed in cancer cells is a way of
diagnosing presence of disease, disease stage,
progression and/or tumor aggressiveness. Such an antibody can also detect an
altered distribution of the polypeptide within
the cancer cells, as compared to corresponding non-malignant tissue.
The 109P1 D4 polypeptide and immunogenic compositions are also useful in view
of the phenomena of altered
subcellular protein localization in disease states. Alteration of cells from
normal to diseased state causes changes in cellular
morphology and is often associated with changes in subcellular protein
localization/distribution. For example, cell membrane
proteins that are expressed in a polarized manner in normal cells can be
altered in disease, resulting in distribution of the
protein in a non-polar manner over the whole cell surface.
The phenomenon of altered subcellular protein localization in a disease state
has been demonstrated with MUCI
and Her2 protein expression by use of immunohistochemical means. Normal
epithelial cells have a typical apical distribution
of MUCI, in addition to some supranuclear localization of the glycoprotein,
whereas malignant lesions often demonstrate an
apolar staining pattern (Diaz et at, The Breast Journal, 7; 40-45 (2001);
Zhang of a!, Clinical Cancer Research, 4; 2669-2676
(1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45:1547-
1557 (1997)). In addition, normal breast
epithelium is either negative for Her2 protein or exhibits only a basolateral
distribution whereas malignant cells can express
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66
the protein over the whole cell surface (De Potter, et al, International
Journal of Cancer, 44; 969-974 (1989): McCormick, et
al, 117; 935-943 (2002)). Alternatively, distribution of the protein may be
altered from a surface only localization to include
diffuse cytoplasmic expression in the diseased state. Such an example can be
seen with MUCI (Diaz, et al, The Breast
Journal, 7: 40-45 (2001)).
Alteration in the localization/distribution of a protein in the cell, as
detected by immunohistochemical methods, can
also provide valuable information concerning the favorability of certain
treatment modalities. This last point is illustrated by a
situation where a protein may be intracellular in normal tissue, but cell
surface in malignant cells; the cell surface location
makes the cells favorably amenable to antibody-based diagnostic and treatment
regimens. When such an alteration of
protein localization occurs for 109P1 D4, the 109P1 D4 protein and immune
responses related thereto are very useful.
Accordingly, the ability to determine whether alteration of subcellular
protein localization occurred for 24P4C12 make the
109P1 D4 protein and immune responses related thereto very useful. Use of the
109P1 D4 compositions allows those skilled
in the art to make important diagnostic and therapeutic decisions.
Immunohistochemical reagents specific to 109P1 D4 are also useful to detect
metastases of tumors expressing 109P1 D4
when the polypeptide appears in tissues where 109P1 D4 is not normally
produced.
Thus, 109P1 D4 polypeptides and antibodies resulting from immune responses
thereto are useful in a variety of
important contexts such as diagnostic, prognostic, preventative and/or
therapeutic purposes known to those skilled in the art.
Just as PSA polynucleotide fragments and polynucleotide variants are employed
by skilled artisans for use in
methods of monitoring PSA, 109P1 D4 polynucleotide fragments and
polynucleotide variants are used in an analogous
manner. In particular, typical PSA polynucleotides used in methods of
monitoring PSA are probes or primers which consist
of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR
amplify a PSA polynucleotide must include
less than the whole PSA sequence to function in the polymerase chain reaction.
In the context of such PCR reactions,
skilled artisans generally create a variety of different polynucleotide
fragments that can be used as primers in order to amplify
different portions of a polynucleotide of interest or to optimize
amplification reactions (see, e.g., Caetano-Anolles, G.
Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol.
Biol. 98:121-154 (1998)). An additional
illustration of the use of such fragments is provided in the Example entitled
"Expression analysis of 109P1 D4 in normal
tissues, and patient specimens," where a 109P1 D4 polynucleotide fragment is
used as a probe to show the expression of
109P1D4 RNAs in cancer cells. In addition, variant polynucleotide sequences
are typically used as primers and probes for
the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al,
Fetal Diagn. Ther. 1996 Nov-Dec
11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2,
Frederick M. Ausubel et al. eds., 1995)).
Polynucleotide fragments and variants are useful in this context where they
are capable of binding to a target polynucleotide
sequence (e.g., a 109P1 D4 polynucleotide shown in Figure 2 or variant
thereof) under conditions of high stringency.
Furthermore, PSA polypeptides which contain an epitope that can be recognized
by an antibody or T cell that
specifically binds to that epitope are used in methods of monitoring PSA.
109P1 D4 polypeptide fragments and polypeptide
analogs or variants can also be used in an analogous manner. This practice of
using polypeptide fragments or polypeptide
variants to generate antibodies (such as anti-PSA antibodies or T cells) is
typical in the art with a wide variety of systems
such as fusion proteins being used by practitioners (see, e.g., Current
Protocols In Molecular Biology, Volume 2, Unit 16,
Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s)
functions to provide the architecture with which an
antibody or T cell is reactive. Typically, skilled artisans create a variety
of different polypeptide fragments that can be used in
order to generate immune responses specific for different portions of a
polypeptide of interest (see, e.g., U.S. Patent No.
5,840,501 and U.S. Patent No. 5,939,533). For example it may be preferable to
utilize a polypeptide comprising one of the
109P1 D4 biological motifs discussed herein or a motif-bearing subsequence
which is readily identified by one of skill in the
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art based on motifs available in the art. Polypeptide fragments, variants or
analogs are typically useful in this context as long
as they comprise an epitope capable of generating an antibody or T cell
specific for a target polypeptide sequence (e.g. a
109P1D4 polypeptide shown in Figure 3).
As shown herein, the 109P1 D4 polynucleotides and polypeptides (as well as the
109P1 D4 polynucleotide probes
and anti-I 09P1 D4 antibodies or T cells used to identify the presence of
these molecules) exhibit specific properties that
make them useful in diagnosing cancers such as those listed in Table I.
Diagnostic assays that measure the presence of
109P1 D4 gene products, in order to evaluate the presence or onset of a
disease condition described herein, such as
prostate cancer, are used to identify patients for preventive measures or
further monitoring, as has been done so
successfully with PSA. Moreover, these materials satisfy a need in the art for
molecules having similar or complementary
characteristics to PSA in situations where, for example, a definite diagnosis
of metastasis of prostatic origin cannot be made
on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res.
Pract. 192(3): 233-237 (1996)), and consequently,
materials such as 109P1D4 polynucleotides and polypeptides (as well as the
109P1D4 polynucleotide probes and anti-
109P1 D4 antibodies used to identify the presence of these molecules) need to
be employed to confirm a metastases of
prostatic origin.
Finally, in addition to their use in diagnostic assays, the 109P1 D4
polynucleotides disclosed herein have a number
of other utilities such as their use in the identification of oncogenetic
associated chromosomal abnormalities in the
chromosomal region to which the 109P1 D4 gene maps (see the Example entitled
"Chromosomal Mapping of 109P1 D4"
below). Moreover, in addition to their use in diagnostic assays, the 109P1 D4-
related proteins and polynucleotides disclosed
herein have other utilities such as their use in the forensic analysis of
tissues of unknown origin (see, e.g., Takahama K
Forensic Sci Int 1996 Jun 28;80(1-2): 63-9).
Additionally, 109P1 D4-related proteins or polynucleotides of the invention
can be used to treat a pathologic
condition characterized by the over-expression of 109P1 D4. For example, the
amino acid or nucleic acid sequence of Figure
2 or Figure 3, or fragments of either, can be used to generate an immune
response to a 109P1 D4 antigen. Antibodies or
other molecules that react with 109P1 D4 can be used to modulate the function
of this molecule, and thereby provide a
therapeutic benefit.
XII.) Inhibition of 109P1 D4 Protein Function
The invention includes various methods and compositions for inhibiting the
binding of 109P1 D4 to its binding
partner or its association with other protein(s) as well as methods for
inhibiting 109P1 D4 function.
XII.A.) Inhibition of 109P1 D4 With Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies
that specifically bind to 109P1 D4 are
introduced into 109P1 D4 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-
109P1 D4 antibody is expressed intracellularly, binds to 109P1 D4 protein, and
thereby inhibits its function. Methods for
engineering such intracellular single chain antibodies are well known. Such
intracellular antibodies, also known as
"intrabodies", are specifically targeted to a particular compartment within
the cell, providing control over where the inhibitory
activity of the treatment is focused. This technology has been successfully
applied in the art (for review, see Richardson and
Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually
eliminate the expression of otherwise abundant
cell surface receptors (see, e.g., Richardson et aL.,1995, Proc. Natl. Acad.
Sci. USA 92: 3137-3141; Beerli et aL, 1994, J.
Biol. Chem. 289: 23931-23936; Deshane et aL,1994, Gene Ther. 1: 332-337).
Single chain antibodies comprise the variable domains of the heavy and light
chain joined by a flexible linker
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polypeptide, and are expressed as a single polypeptide. Optionally, single
chain antibodies are expressed as a single chain
variable region fragment joined to the light chain constant region. Well-known
intracellular trafficking signals are engineered
into recombinant polynucleotide vectors encoding such single chain antibodies
in order to target precisely the intrabody to
the desired intracellular compartment. For example, intrabodies targeted to
the endoplasmic reticulum (ER) are engineered
to incorporate a leader peptide and, optionally, a C-terminal ER retention
signal, such as the KDEL amino acid motif.
Intrabodies intended to exert activity in the nucleus are engineered to
include a nuclear localization signal. Lipid moieties are
joined to intrabodies in order to tether the intrabody to the cytosolic side
of the plasma membrane. Intrabodies can also be
targeted to exert function in the cytosol. For example, cytosolic intrabodies
are used to sequester factors within the cytosol,
thereby preventing them from being transported to their natural cellular
destination.
In one embodiment, intrabodies are used to capture 109P1 D4 in the nucleus,
thereby preventing its activity within
the nucleus. Nuclear targeting signals are engineered into such 109P1 D4
intrabodies in order to achieve the desired
targeting. Such 109P1 D4 intrabodies are designed to bind specifically to a
particular 109P1 D4 domain. In another
embodiment, cytosolic intrabodies that specifically bind to a 109P1 D4 protein
are used to prevent 109P1 D4 from gaining
access to the nucleus, thereby preventing it from exerting any biological
activity within the nucleus (e.g., preventing 109P1 D4
from forming transcription complexes with other factors).
In order to specifically direct the expression of such intrabodies to
particular cells, the transcription of the intrabody
is placed under the regulatory control of an appropriate tumor-specific
promoter and/or enhancer. In order to target intrabody
expression specifically to prostate, for example, the PSA promoter and/or
promoter/enhancer can be utilized (See, for
example, U.S. Patent No. 5,919,652 issued 6 July 1999).
XII.B.) Inhibition of 109P1 D4 with Recombinant Proteins
In another approach, recombinant molecules bind to 109P1 D4 and thereby
inhibit 109P1 D4 function. For example,
these recombinant molecules prevent or inhibit 109P1 D4 from accessing/binding
to its binding partner(s) or associating with
other protein(s). Such recombinant molecules can, for example, contain the
reactive part(s) of a 109P1 D4 specific antibody
molecule. In a particular embodiment, the 109P1 D4 binding domain of a 109P1
D4 binding partner is engineered into a dimeric
fusion protein, whereby the fusion protein comprises two 109P1 D4 ligand
binding domains linked to the Fe portion of a human
IgG, such as human IgG1. Such IgG portion can contain, for example, the CH2
and CH3 domains and the hinge region, but not
the CHI domain. Such dimeric fusion proteins are administered in soluble form
to patients suffering from a cancer associated with
the expression of 109PI D4, whereby the dimeric fusion protein specifically
binds to 109PI D4 and blocks 109P1 D4 interaction
with a binding partner. Such dimeric fusion proteins are further combined into
multimeric proteins using known antibody linking
technologies.
XII.C.) Inhibition of 109P1 D4 Transcription or Translation
The present invention also comprises various methods and compositions for
inhibiting the transcription of the
109P1 D4 gene. Similarly, the invention also provides methods and compositions
for inhibiting the translation of 109P1 D4
mRNA into protein.
In one approach, a method of inhibiting the transcription of the 109P1 D4 gene
comprises contacting the 109P1 D4
gene with a 109PI D4 antisense polynucleotide. In another approach, a method
of inhibiting 109P1 D4 mRNA translation
comprises contacting a 109P1 D4 mRNA with an antisense polynucleotide. In
another approach, a 109P1 D4 specific
ribozyme is used to cleave a 109P1 D4 message, thereby inhibiting translation.
Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the 109P1 D4 gene,
such as 109P1 D4 promoter and/or enhancer
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elements. Similarly, proteins capable of inhibiting a 109P1 D4 gene
transcription factor are used to inhibit 109P1 D4 mRNA
transcription. The various polynucleotides and compositions useful in the
aforementioned methods have been described
above. The use of antisense and ribozyme molecules to inhibit transcription
and translation is well known in the art.
Other factors that inhibit the transcription of 109P1 D4 by interfering with
109P1 D4 transcriptional activation are
also useful to treat cancers expressing 109P1D4. Similarly, factors that
interfere with 109PID4 processing are useful to treat
cancers that express 109P1 D4. Cancer treatment methods utilizing such factors
are also within the scope of the invention.
XII.D.) General Considerations for Therapeutic Strategies
Gene transfer and gene therapy technologies can be used to deliver therapeutic
polynucleotide molecules to tumor cells
synthesizing 109P1D4 (i.e., antisense, ribozyme, polynucleotides encoding
intrabodies and other 109P1D4 inhibitory molecules).
A number of gene therapy approaches are known in the art. Recombinant vectors
encoding 109P1 D4 antisense polynucleotides,
ribozymes, factors capable of interfering with 109P1 D4 transcription, and so
forth, can be delivered to target tumor cells using
such gene therapy approaches.
The above therapeutic approaches can be combined with any one of a wide
variety of surgical, chemotherapy or
radiation therapy regimens. The therapeutic approaches of the invention can
enable the use of reduced dosages of
chemotherapy (or other therapies) and/or less frequent administration, an
advantage for all patients and particularly for those that
do not tolerate the toxicity of the chemotherapeutic agent well.
The anti-tumor activity of a particular composition (e.g., antisense,
ribozyme, intrabody), or a combination of such
compositions, can be evaluated using various in vitro and in vivo assay
systems. In vitro assays that evaluate therapeutic activity
include cell growth assays, soft agar assays and other assays indicative of
tumor promoting activity, binding assays capable of
determining the extent to which a therapeutic composition will inhibit the
binding of 109P1 D4 to a binding partner, etc.
In vivo, the effect of a 109P1 D4 therapeutic composition can be evaluated in
a suitable animal model. For example,
xenogenic prostate cancer models can be used, wherein human prostate cancer
explants or passaged xenograft tissues are
introduced into immune compromised animals, such as nude or SCID mice (Klein
et at., 1997, Nature Medicine 3: 402-408). For
example, PCT Patent Application W098/16628 and U.S. Patent 6,107,540 describe
various xenograft models of human
prostate cancer capable of recapitulating the development of primary tumors,
micrometastasis, and the formation of
osteoblastic metastases characteristic of late stage disease. Efficacy can be
predicted using assays that measure inhibition
of tumor formation, tumor regression or metastasis, and the like.
In vivo assays that evaluate the promotion of apoptosis are useful in
evaluating therapeutic compositions. In one
embodiment, xenografts from tumor bearing mice treated with the therapeutic
composition can be examined for the presence
of apoptotic foci and compared to untreated control xenograft-bearing mice.
The extent to which apoptotic foci are found in
the tumors of the treated mice provides an indication of the therapeutic
efficacy of the composition.
The therapeutic compositions used in the practice of the foregoing methods can
be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery method.
Suitable carriers include any material that when
combined with the therapeutic composition retains the anti-tumor function of
the therapeutic composition and is generally
non-reactive with the patient's immune system. Examples include, but are not
limited to, any of a number of standard
pharmaceutical carriers such as sterile phosphate buffered saline solutions,
bacteriostatic water, and the like (see, generally,
Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).
Therapeutic formulations can be solubilized and administered via any route
capable of delivering the therapeutic
composition to the tumor site. Potentially effective routes of administration
include, but are not limited to, intravenous,
parenteral, intraperitoneal, intramuscular, intratumor, intradermal,
intraorgan, orthotopic, and the like. A preferred
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formulation for intravenous injection comprises the therapeutic composition in
a solution of preserved bacteriostatic water,
sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene
bags containing 0.9% sterile Sodium Chloride
for Injection, USP. Therapeutic protein preparations can be lyophilized and
stored as sterile powders, preferably under
vacuum, and then reconstituted in bacteriostatic water (containing for
example, benzyl alcohol preservative) or in sterile
water prior to injection.
Dosages and administration protocols for the treatment of cancers using the
foregoing methods will vary with the
method and the target cancer, and will generally depend on a number of other
factors appreciated in the art.
XIII.) Identification, Characterization and Use of Modulators of 109P1 D4
Methods to Identify and Use Modulators
In one embodiment, screening is performed to identify modulators that induce
or suppress a particular expression
profile, suppress or induce specific pathways, preferably generating the
associated phenotype thereby. In another
embodiment, having identified differentially expressed genes important in a
particular state; screens are performed to identify
modulators that alter expression of individual genes, either increase or
decrease. In another embodiment, screening is
performed to identify modulators that alter a biological function of the
expression product of a differentially expressed gene.
Again, having identified the importance of a gene in a particular state,
screens are performed to identify agents that bind
and/or modulate the biological activity of the gene product.
In addition, screens are done for genes that are induced in response to a
candidate agent. After identifying a
modulator (one that suppresses a cancer expression pattern leading to a normal
expression pattern, or a modulator of a
cancer gene that leads to expression of the gene as in normal tissue) a screen
is performed to identify genes that are
specifically modulated in response to the agent. Comparing expression profiles
between normal tissue and agent-treated
cancer tissue reveals genes that are not expressed in normal tissue or cancer
tissue, but are expressed in agent treated
tissue, and vice versa. These agent-specific sequences are identified and used
by methods described herein for cancer
genes or proteins. In particular these sequences and the proteins they encode
are used in marking or identifying agent-
treated cells. In addition, antibodies are raised against the agent-induced
proteins and used to target novel therapeutics to
the treated cancer tissue sample.
Modulator-related Identification and Screening Assays:
Gene Expression-related Assays
Proteins, nucleic acids, and antibodies of the invention are used in screening
assays. The cancer-associated
proteins, antibodies, nucleic acids, modified proteins and cells containing
these sequences are used in screening assays,
such as evaluating the effect of drug candidates on a "gene expression
profile," expression profile of polypeptides or
alteration of biological function. In one embodiment, the expression profiles
are used, preferably in conjunction with high
throughput screening techniques to allow monitoring for expression profile
genes after treatment with a candidate agent
(e.g., Davis, GF, et al, J Biol Screen 7:69 (2002); Zlokarnik, et al., Science
279:84-8 (1998); Heid, Genome Res 6:986-
94,1996).
The cancer proteins, antibodies, nucleic acids, modified proteins and cells
containing the native or modified cancer
proteins or genes are used in screening assays. That is, the present invention
comprises methods for screening for
compositions which modulate the cancer phenotype or a physiological function
of a cancer protein of the invention. This is
done on a gene itself or by evaluating the effect of drug candidates on a
"gene expression profile" or biological function. In
one embodiment, expression profiles are used, preferably in conjunction with
high throughput screening techniques to allow
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monitoring after treatment with a candidate agent, see Zlokamik, supra.
A variety of assays are executed directed to the genes and proteins of the
invention. Assays are run on an
individual nucleic acid or protein level. That is, having identified a
particular gene as up regulated in cancer, test compounds
are screened for the ability to modulate gene expression or for binding to the
cancer protein of the invention. "Modulation" in
this context includes an increase or a decrease in gene expression. The
preferred amount of modulation will depend on the
original change of the gene expression in normal versus tissue undergoing
cancer, with changes of at least 10%, preferably
50%, more preferably 100-300%, and in some embodiments 300-1000% or greater.
Thus, if a gene exhibits a 4-fold
increase in cancer tissue compared to normal tissue, a decrease of about four-
fold is often desired; similarly, a 10-fold
decrease in cancer tissue compared to normal tissue a target value of a 10-
fold increase in expression by the test compound
is often desired. Modulators that exacerbate the type of gene expression seen
in cancer are also useful, e.g., as an
upregulated target in further analyses.
The amount of gene expression is monitored using nucleic acid probes and the
quantification of gene expression
levels, or, alternatively, a gene product itself is monitored, e.g., through
the use of antibodies to the cancer protein and
standard immunoassays. Proteomics and separation techniques also allow for
quantification of expression.
Expression Monitoring to Identify Compounds that Modify Gene Expression
In one embodiment, gene expression monitoring, i.e., an expression profile, is
monitored simultaneously for a
number of entities. Such profiles will typically involve one or more of the
genes of Figure 2. In this embodiment, e.g., cancer
nucleic acid probes are attached to biochips to detect and quantify cancer
sequences in a particular cell. Alternatively, PCR
can be used. Thus, a series, e.g., wells of a microtiter plate, can be used
with dispensed primers in desired wells. A PCR
reaction can then be performed and analyzed for each well.
Expression monitoring is performed to identify compounds that modify the
expression of one or more cancer-
associated sequences, e.g., a polynucleotide sequence set out in Figure 2.
Generally, a test modulator is added to the cells
prior to analysis. Moreover, screens are also provided to identify agents that
modulate cancer, modulate cancer proteins of
the invention, bind to a cancer protein of the invention, or interfere with
the binding of a cancer protein of the invention and
an antibody or other binding partner.
In one embodiment, high throughput screening methods involve providing a
library containing a large number of
potential therapeutic compounds (candidate compounds). Such "combinatorial
chemical libraries" are then screened in one
or more assays to identify those library members (particular chemical species
or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds," as compounds for
screening, or as therapeutics.
In certain embodiments, combinatorial libraries of potential modulators are
screened for an ability to bind to a
cancer polypeptide or to modulate activity. Conventionally, new chemical
entities with useful properties are generated by
identifying a chemical compound (called a "lead compound") with some desirable
property or activity, e.g., inhibiting activity,
creating variants of the lead compound, and evaluating the property and
activity of those variant compounds. Often, high
throughput screening (HTS) methods are employed for such an analysis.
As noted above, gene expression monitoring is conveniently used to test
candidate modulators (e.g., protein,
nucleic acid or small molecule). After the candidate agent has been added and
the cells allowed to incubate for a period, the
sample containing a target sequence to be analyzed is, e.g., added to a
biochip.
If required, the target sequence is prepared using known techniques. For
example, a sample is treated to lyse the
cells, using known lysis buffers, electroporation, etc., with purification
and/or amplification such as PCR performed as
appropriate. For example, an in vitro transcription with labels covalently
attached to the nucleotides is performed. Generally,
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the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
The target sequence can be labeled with, e.g., a fluorescent, a
chemiluminescent, a chemical, or a radioactive
signal, to provide a means of detecting the target sequence's specific binding
to a probe. The label also can be an enzyme,
such as alkaline phosphatase or horseradish peroxidase, which when provided
with an appropriate substrate produces a
product that is detected. Alternatively, the label is a labeled compound or
small molecule, such as an enzyme inhibitor, that
binds but is not catalyzed or altered by the enzyme. The label also can be a
moiety or compound, such as, an epitope tag or
biotin which specifically binds to streptavidin. For the example of biotin,
the streptavidin is labeled as described above,
thereby, providing a detectable signal for the bound target sequence. Unbound
labeled streptavidin is typically removed prior
to analysis.
As will be appreciated by those in the art, these assays can be direct
hybridization assays or can comprise
"sandwich assays", which include the use of multiple probes, as is generally
outlined in U.S. Patent Nos. 5, 681,702;
5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670;
5,591,584; 5,624,802; 5,635,352; 5,594,118;
5,359,100; 5,124, 246; and 5,681,697. In this embodiment, in general, the
target nucleic acid is prepared as outlined above,
and then added to the biochip comprising a plurality of nucleic acid probes,
under conditions that allow the formation of a
hybridization complex.
A variety of hybridization conditions are used in the present invention,
including high, moderate and low stringency
conditions as outlined above. The assays are generally run under stringency
conditions which allow formation of the label
probe hybridization complex only in the presence of target. Stringency can be
controlled by altering a step parameter that is
a thermodynamic variable, including, but not limited to, temperature,
formamide concentration, salt concentration, chaotropic
salt concentration pH, organic solvent concentration, etc. These parameters
may also be used to control non-specific
binding, as is generally outlined in U.S. Patent No. 5,681,697. Thus, it can
be desirable to perform certain steps at higher
stringency conditions to reduce non-specific binding.
The reactions outlined herein can be accomplished in a variety of ways.
Components of the reaction can be added
simultaneously, or sequentially, in different orders, with preferred
embodiments outlined below. In addition, the reaction may
include a variety of other reagents. These include salts, buffers, neutral
proteins, e.g. albumin, detergents, etc. which can be
used to facilitate optimal hybridization and detection, and/or reduce
nonspecific or background interactions. Reagents that
otherwise improve the efficiency of the assay, such as protease inhibitors,
nuclease inhibitors, anti-microbial agents, etc.,
may also be used as appropriate, depending on the sample preparation methods
and purity of the target. The assay data
are analyzed to determine the expression levels of individual genes, and
changes in expression levels as between states,
forming a gene expression profile.
Biological Activity-related Assays
The invention provides methods identify or screen for a compound that
modulates the activity of a cancer-related
gene or protein of the invention. The methods comprise adding a test compound,
as defined above, to a cell comprising a
cancer protein of the invention. The cells contain a recombinant nucleic acid
that encodes a cancer protein of the invention.
In another embodiment, a library of candidate agents is tested on a plurality
of cells.
In one aspect, the assays are evaluated in the presence or absence or previous
or subsequent exposure of
physiological signals, e.g. hormones, antibodies, peptides, antigens,
cytokines, growth factors, action potentials,
pharmacological agents including chemotherapeutics, radiation, carcinogenics,
or other cells (i.e., cell-cell contacts). In
another example, the determinations are made at different stages of the cell
cycle process. In this way, compounds that
modulate genes or proteins of the invention are identified. Compounds with
pharmacological activity are able to enhance or
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interfere with the activity of the cancer protein of the invention. Once
identified, similar structures are evaluated to identify
critical structural features of the compound.
In one embodiment, a method of modulating (e.g., inhibiting) cancer cell
division is provided; the method
comprises administration of a cancer modulator. In another embodiment, a
method of modulating (e.g., inhibiting) cancer is
provided; the method comprises administration of a cancer modulator. In a
further embodiment, methods of treating cells or
individuals with cancer are provided; the method comprises administration of a
cancer modulator.
In one embodiment, a method for modulating the status of a cell that expresses
a gene of the invention is provided.
As used herein status comprises such art-accepted parameters such as growth,
proliferation, survival, function, apoptosis,
senescence, location, enzymatic activity, signal transduction, etc. of a cell.
In one embodiment, a cancer inhibitor is an
antibody as discussed above. In another embodiment, the cancer inhibitor is an
antisense molecule. A variety of cell
growth, proliferation, and metastasis assays are known to those of skill in
the art, as described herein.
High Throughput Screening to Identify Modulators
The assays to identify suitable modulators are amenable to high throughput
screening. Preferred assays thus
detect enhancement or inhibition of cancer gene transcription, inhibition or
enhancement of polypeptide expression, and
inhibition or enhancement of polypeptide activity.
In one embodiment, modulators evaluated in high throughput screening methods
are proteins, often naturally
occurring proteins or fragments of naturally occurring proteins. Thus, e.g.,
cellular extracts containing proteins, or random or
directed digests of proteinaceous cellular extracts, are used. In this way,
libraries of proteins are made for screening in the
methods of the invention. Particularly preferred in this embodiment are
libraries of bacterial, fungal, viral, and mammalian
proteins, with the latter being preferred, and human proteins being especially
preferred. Particularly useful test compound
will be directed to the class of proteins to which the target belongs, e.g.,
substrates for enzymes, or ligands and receptors.
Use of Soft Agar Growth and Colony Formation to Identify and Characterize
Modulators
Normal cells require a solid substrate to attach and grow. When cells are
transformed, they lose this phenotype
and grow detached from the substrate. For example, transformed cells can grow
in stirred suspension culture or suspended
in semi-solid media, such as semi-solid or soft agar. The transformed cells,
when transfected with tumor suppressor genes,
can regenerate normal phenotype and once again require a solid substrate to
attach to and grow. Soft agar growth or colony
formation in assays are used to identify modulators of cancer sequences, which
when expressed in host cells, inhibit
abnormal cellular proliferation and transformation. A modulator reduces or
eliminates the host cells' ability to grow
suspended in solid or semisolid media, such as agar.
Techniques for soft agar growth or colony formation in suspension assays are
described in Freshney, Culture of
Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the
methods section of Garkavtsev et al. (1996), supra.
Evaluation of Contact Inhibition and Growth Density Limitation to Identify and
Characterize Modulators
Normal cells typically grow in a flat and organized pattern in cell culture
until they touch other cells. When the cells
touch one another, they are contact inhibited and stop growing. Transformed
cells, however, are not contact inhibited and
continue to grow to high densities in disorganized foci. Thus, transformed
cells grow to a higher saturation density than
corresponding normal cells. This is detected morphologically by the formation
of a disoriented monolayer of cells or cells in
foci. Alternatively, labeling index with (3H)-thymidine at saturation density
is used to measure density limitation of growth,
similarly an MTT or Alamar blue assay will reveal proliferation capacity of
cells and the the ability of modulators to affect
same. See Freshney (1994), supra. Transformed cells, when transfected with
tumor suppressor genes, can regenerate a
normal phenotype and become contact inhibited and would grow to a lower
density.
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In this assay, labeling index with 3H)-thymidine at saturation density is a
preferred method of measuring density
limitation of growth. Transformed host cells are transfected with a cancer-
associated sequence and are grown for 24 hours
at saturation density in non-limiting medium conditions. The percentage of
cells labeling with (3H)-thymidine is determined
by incorporated cpm.
Contact independent growth is used to identify modulators of cancer sequences,
which had led to abnormal
cellular proliferation and transformation. A modulator reduces or eliminates
contact independent growth, and returns the
cells to a normal phenotype.
Evaluation of Growth Factor or Serum Dependence to Identify and Characterize
Modulators
Transformed cells have lower serum dependence than their normal counterparts
(see, e.g., Temin, J. NatI. Cancer
Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970));
Freshney, supra. This is in part due to release of
various growth factors by the transformed cells. The degree of growth factor
or serum dependence of transformed host cells
can be compared with that of control. For example, growth factor or serum
dependence of a cell is monitored in methods to
identify and characterize compounds that modulate cancer-associated sequences
of the invention.
Use of Tumor-specific Marker Levels to Identify and Characterize Modulators
Tumor cells release an increased amount of certain factors (hereinafter "tumor
specific markers") than their normal
counterparts. For example, plasminogen activator (PA) is released from human
glioma at a higher level than from normal
brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and
Potential Interference with Tumor Growth, in
Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly,
Tumor Angiogenesis Factor (TAF) is released
at a higher level in tumor cells than their normal counterparts. See, e.g.,
Folkman, Angiogenesis and Cancer, Sam Cancer
Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, Bet
al).
Various techniques which measure the release of these factors are described in
Freshney (1994), supra. Also,
see, Unkless et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers,
J. Biol. Chem. 251:5694-5702 (1976); Whur et
al., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor
Vascularization, and Potential Interference with Tumor
Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985);
Freshney, Anticancer Res. 5:111-130 (1985).
For example, tumor specific marker levels are monitored in methods to identify
and characterize compounds that modulate
cancer-associated sequences of the invention.
Invasiveness into Matrigel to Identify and Characterize Modulators
The degree of invasiveness into Matrigel or an extracellular matrix
constituent can be used as an assay to identify
and characterize compounds that modulate cancer associated sequences. Tumor
cells exhibit a positive correlation
between malignancy and invasiveness of cells into Matrigel or some other
extracellular matrix constituent. In this assay,
tumorigenic cells are typically used as host cells. Expression of a tumor
suppressor gene in these host cells would decrease
invasiveness of the host cells. Techniques described in Cancer Res. 1999;
59:6010; Freshney (1994), supra, can be used.
Briefly, the level of invasion of host cells is measured by using filters
coated with Matrigel or some other extracellular matrix
constituent. Penetration into the gel, or through to the distal side of the
filter, is rated as invasiveness, and rated
histologically by number of cells and distance moved, or by prelabeling the
cells with 1251 and counting the radioactivity on
the distal side of the filter or bottom of the dish. See, e.g., Freshney
(1984), supra.
Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
Effects of cancer-associated sequences on cell growth are tested in transgenic
or immune-suppressed organisms.
Transgenic organisms are prepared in a variety of art-accepted ways. For
example, knock-out transgenic organisms, e.g.,
mammals such as mice, are made, in which a cancer gene is disrupted or in
which a cancer gene is inserted. Knock-out
transgenic mice are made by insertion of a marker gene or other heterologous
gene into the endogenous cancer gene site in
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the mouse genome via homologous recombination. Such mice can also be made by
substituting the endogenous cancer
gene with a mutated version of the cancer gene, or by mutating the endogenous
cancer gene, e.g., by exposure to
carcinogens.
To prepare transgenic chimeric animals, e.g., mice, a DNA construct is
introduced into the nuclei of embryonic
stem cells. Cells containing the newly engineered genetic lesion are injected
into a host mouse embryo, which is re-
implanted into a recipient female. Some of these embryos develop into chimeric
mice that possess germ cells some of which
are derived from the mutant cell line. Therefore, by breeding the chimeric
mice it is possible to obtain a new line of mice
containing the introduced genetic lesion (see, e.g., Capecchi et al., Science
244:1288 (1989)). Chimeric mice can be derived
according to US Patent 6,365,797, issued 2 April 2002; US Patent 6,107,540
issued 22 August 2000; Hogan et al.,
Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor
Laboratory (1988) and Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press,
Washington, D.C., (1987).
Alternatively, various immune-suppressed or immune-deficient host animals can
be used. For example, a
genetically athymic "nude" mouse (see, e.g., Giovanella et al., J. Nati.
Cancer Inst. 52:921 (1974)), a SCID mouse, a
thymectornized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br.
J. Cancer 38:263 (1978); Selby et al., Br. J.
Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells
(typically about 106 cells) injected into isogenic
hosts produce invasive tumors in a high proportion of cases, while normal
cells of similar origin will not. In hosts which
developed invasive tumors, cells expressing cancer-associated sequences are
injected subcutaneously or orthotopically.
Mice are then separated into groups, including control groups and treated
experimental groups) e.g. treated with a
modulator). After a suitable length of time, preferably 4-8 weeks, tumor
growth is measured (e.g., by volume or by its two
largest dimensions, or weight) and compared to the control. Tumors that have
statistically significant reduction (using, e.g.,
Student's T test) are said to have inhibited growth.
In Vitro Assays to Identify and Characterize Modulators
Assays to identify compounds with modulating activity can be performed in
vitro. For example, a cancer
polypeptide is first contacted with a potential modulator and incubated for a
suitable amount of time, e.g., from 0.5 to 48
hours. In one embodiment, the cancer polypeptide levels are determined in
vitro by measuring the level of protein or mRNA.
The level of protein is measured using immunoassays such as Western blotting,
ELISA and the like with an antibody that
selectively binds to the cancer polypeptide or a fragment thereof. For
measurement of mRNA, amplification, e.g., using
PCR, LCR, or hybridization assays, e. g., Northern hybridization, RNAse
protection, dot blotting, are preferred. The level of
protein or mRNA is detected using directly or indirectly labeled detection
agents, e.g., fluorescently or radioactively labeled
nucleic acids, radioactively or enzymatically labeled antibodies, and the
like, as described herein.
Alternatively, a reporter gene system can be devised using a cancer protein
promoter operably linked to a reporter
gene such as luciferase, green fluorescent protein, CAT, or P-gal. The
reporter construct is typically transfected into a cell.
After treatment with a potential modulator, the amount of reporter gene
transcription, translation, or activity is measured
according to standard techniques known to those of skill in the art (Davis GF,
supra; Gonzalez, J. & Negulescu, P. Curr.
Opin. Biotechnol. 1998: 9:624).
As outlined above, in vitro screens are done on individual genes and gene
products. That is, having identified a
particular differentially expressed gene as important in a particular state,
screening of modulators of the expression of the
gene or the gene product itself is performed.
In one embodiment, screening for modulators of expression of specific gene(s)
is performed. Typically, the
expression of only one or a few genes is evaluated. In another embodiment,
screens are designed to first find compounds
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that bind to differentially expressed proteins. These compounds are then
evaluated for the ability to modulate differentially
expressed activity. Moreover, once initial candidate compounds are identified,
variants can be further screened to better
evaluate structure activity relationships.
Binding Assays to Identify and Characterize Modulators
In binding assays in accordance with the invention, a purified or isolated
gene product of the invention is generally
used. For example, antibodies are generated to a protein of the invention, and
immunoassays are run to determine the
amount and/or location of protein. Alternatively, cells comprising the cancer
proteins are used in the assays.
Thus, the methods comprise combining a cancer protein of the invention and a
candidate compound such as a
ligand, and determining the binding of the compound to the cancer protein of
the invention. Preferred embodiments utilize
the human cancer protein; animal models of human disease of can also be
developed and used. Also, other analogous
mammalian proteins also can be used as appreciated by those of skill in the
art. Moreover, in some embodiments variant or
derivative cancer proteins are used.
Generally, the cancer protein of the invention, or the ligand, is non-
diffusibly bound to an insoluble support. The
support can, e.g., be one having isolated sample receiving areas (a microtiter
plate, an array, etc.). The insoluble supports
can be made of any composition to which the compositions can be bound, is
readily separated from soluble material, and is
otherwise compatible with the overall method of screening. The surface of such
supports can be solid or porous and of any
convenient shape.
Examples of suitable insoluble supports include microtiter plates, arrays,
membranes and beads. These are
typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon,
nitrocellulose, or TeflonTM, etc. Microtiter plates
and arrays are especially convenient because a large number of assays can be
carried out simultaneously, using small
amounts of reagents and samples. The particular manner of binding of the
composition to the support is not crucial so long
as it is compatible with the reagents and overall methods of the invention,
maintains the activity of the composition and is
nondiffusable. Preferred methods of binding include the use of antibodies
which do not sterically block either the ligand
binding site or activation sequence when attaching the protein to the support,
direct binding to "sticky" or ionic supports,
chemical crosslinking, the synthesis of the protein or agent on the surface,
etc. Following binding of the protein or
ligand/binding agent to the support, excess unbound material is removed by
washing. The sample receiving areas may then
be blocked through incubation with bovine serum albumin (BSA), casein or other
innocuous protein or other moiety.
Once a cancer protein of the invention is bound to the support, and a test
compound is added to the assay.
Alternatively, the candidate binding agent is bound to the support and the
cancer protein of the invention is then added.
Binding agents include specific antibodies, non-natural binding agents
identified in screens of chemical libraries, peptide
analogs, etc.
Of particular interest are assays to identify agents that have a low toxicity
for human cells. A wide variety of
assays can be used for this purpose, including proliferation assays, cAMP
assays, labeled in vitro protein-protein binding
assays, electrophoretic mobility shift assays, immunoassays for protein
binding, functional assays (phosphorylation assays,
etc.) and the like.
A determination of binding of the test compound (ligand, binding agent,
modulator, etc.) to a cancer protein of the
invention can be done in a number of ways. The test compound can be labeled,
and binding determined directly, e.g., by
attaching all or a portion of the cancer protein of the invention to a solid
support, adding a labeled candidate compound (e.g.,
a fluorescent label), washing off excess reagent, and determining whether the
label is present on the solid support. Various
blocking and washing steps can be utilized as appropriate.
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In certain embodiments, only one of the components is labeled, e.g., a protein
of the invention or ligands labeled.
Alternatively, more than one component is labeled with different labels, e.g.,
1125, for the proteins and a fluorophor for the
compound. Proximity reagents, e.g., quenching or energy transfer reagents are
also useful.
Competitive Binding Identify and Characterize Modulators
In one embodiment, the binding of the "test compound" is determined by
competitive binding assay with a
"competitor." The competitor is a binding moiety that binds to the target
molecule (e.g., a cancer protein of the invention).
Competitors include compounds such as antibodies, peptides, binding partners,
ligands, etc. Under certain circumstances,
the competitive binding between the test compound and the competitor displaces
the test compound. In one embodiment,
the test compound is labeled. Either the test compound, the competitor, or
both, is added to the protein for a time sufficient
to allow binding. Incubations are performed at a temperature that facilitates
optimal activity, typically between four and 40 C.
Incubation periods are typically optimized, e.g., to facilitate rapid high
throughput screening; typically between zero and one
hour will be sufficient. Excess reagent is generally removed or washed away.
The second component is then added, and
the presence or absence of the labeled component is followed, to indicate
binding.
In one embodiment, the competitor is added first, followed by the test
compound. Displacement of the competitor
is an indication that the test compound is binding to the cancer protein and
thus is capable of binding to, and potentially
modulating, the activity of the cancer protein. In this embodiment, either
component can be labeled. Thus, e.g., if the
competitor is labeled, the presence of label in the post-test compound wash
solution indicates displacement by the test
compound. Alternatively, if the test compound is labeled, the presence of the
label on the support indicates displacement.
In an alternative embodiment, the test compound is added first, with
incubation and washing, followed by the
competitor. The absence of binding by the competitor indicates that the test
compound binds to the cancer protein with
higher affinity than the competitor. Thus, if the test compound is labeled,
the presence of the label on the support, coupled
with a lack of competitor binding, indicates that the test compound binds to
and thus potentially modulates the cancer protein
of the invention.
Accordingly, the competitive binding methods comprise differential screening
to identity agents that are capable of
modulating the activity of the cancer proteins of the invention. In this
embodiment, the methods comprise combining a
cancer protein and a competitor in a first sample. A second sample comprises a
test compound, the cancer protein, and a
competitor. The binding of the competitor is determined for both samples, and
a change, or difference in binding between
the two samples indicates the presence of an agent capable of binding to the
cancer protein and potentially modulating its
activity. That is, if the binding of the competitor is different in the second
sample relative to the first sample, the agent is
capable of binding to the cancer protein.
Alternatively, differential screening is used to identify drug candidates that
bind to the native cancer protein, but
cannot bind to modified cancer proteins. For example the structure of the
cancer protein is modeled and used in rational
drug design to synthesize agents that interact with that site, agents which
generally do not bind to site-modified proteins.
Moreover, such drug candidates that affect the activity of a native cancer
protein are also identified by screening drugs for
the ability to either enhance or reduce the activity of such proteins.
Positive controls and negative controls can be used in the assays. Preferably
control and test samples are
performed in at least triplicate to obtain statistically significant results.
Incubation of all samples occurs for a time sufficient to
allow for the binding of the agent to the protein. Following incubation,
samples are washed free of non-specifically bound
material and the amount of bound, generally labeled agent determined. For
example, where a radiolabel is employed, the
samples can be counted in a scintillation counter to determine the amount of
bound compound.
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A variety of other reagents can be included in the screening assays. These
include reagents like salts, neutral
proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal
protein-protein binding and/or reduce non-specific
or background interactions. Also reagents that otherwise improve the
efficiency of the assay, such as protease inhibitors,
nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of
components is added in an order that provides
for the requisite binding.
Use of Polynucleotides to Down-regulate or Inhibit a Protein of the Invention.
Polynucleotide modulators of cancer can be introduced into a cell containing
the target nucleotide sequence by
formation of a conjugate with a ligand-binding molecule, as described in WO
91/04753. Suitable ligand-binding molecules
include, but are not limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface
receptors. Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand
binding molecule to bind to its corresponding molecule or receptor, or block
entry of the sense or antisense oligonucleotide
or its conjugated version into the cell. Alternatively, a polynucleotide
modulator of cancer can be introduced into a cell
containing the target nucleic acid sequence, e.g., by formation of a
polynucleotide-lipid complex, as described in WO
90/10448. It is understood that the use of antisense molecules or knock out
and knock in models may also be used in
screening assays as discussed above, in addition to methods of treatment.
Inhibitory and Antisense Nucleotides
In certain embodiments, the activity of a cancer-associated protein is down-
regulated, or entirely inhibited, by the
use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e.,
a nucleic acid complementary to, and which
can preferably hybridize specifically to, a coding mRNA nucleic acid sequence,
e.g., a cancer protein of the invention,
mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the
mRNA reduces the translation and/or
stability of the mRNA.
In the context of this invention, antisense polynucleotides can comprise
naturally occurring nucleotides, or
synthetic species formed from naturally occurring subunits or their close
homologs. Antisense polynucleotides may also
have altered sugar moieties or inter-sugar linkages. Exemplary among these are
the phosphorothioate and other sulfur
containing species which are known for use in the art. Analogs are comprised
by this invention so long as they function
effectively to hybridize with nucleotides of the invention. See, e.g., Isis
Pharmaceuticals, Carlsbad, CA; Sequitor, Inc.,
Natick, MA.
Such antisense polynucleotides can readily be synthesized using recombinant
means, or can be synthesized in
vitro. Equipment for such synthesis is sold by several vendors, including
Applied Biosystems. The preparation of other
oligonucleotides such as phosphorothioates and alkylated derivatives is also
well known to those of skill in the art.
Antisense molecules as used herein include antisense or sense
oligonucleotides. Sense oligonucleotides can,
e.g., be employed to block transcription by binding to the anti-sense strand.
The antisense and sense oligonucleotide
comprise a single stranded nucleic acid sequence (either RNA or DNA) capable
of binding to target mRNA (sense) or DNA
(antisense) sequences for cancer molecules. Antisense or sense
oligonucleotides, according to the present invention,
comprise a fragment generally at least about 12 nucleotides, preferably from
about 12 to 30 nucleotides. The ability to derive
an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a
given protein is described in, e.g., Stein
&Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958
(1988)).
Ribozymes
In addition to antisense polynucleotides, ribozymes can be used to target and
inhibit transcription of cancer-
associated nucleotide sequences. A ribozyme is an RNA molecule that
catalytically cleaves other RNA molecules. Different
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kinds of ribozymes have been described, including group I ribozymes,
hammerhead ribozymes, hairpin ribozymes, RNase P,
and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25:
289-317 (1994) for a general review of the
properties of different ribozymes).
The general features of hairpin ribozymes are described, e.g., in Hampel et
al., Nuci. Acids Res. 18:299-304
(1990); European Patent Publication No. 0360257; U.S. Patent No. 5,254,678.
Methods of preparing are well known to
those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl.
Acad. Sci. USA 90:6340-6344 (1993); Yamada et
al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci.
USA 92:699- 703 (1995); Leavitt et al., Human
Gene Therapy 5:1151-120 (1994); and Yamada et al., Virology 205:121-126
(1994)).
Use of Modulators in Phenotypic Screening
In one embodiment, a test compound is administered to a population of cancer
cells, which have an associated
cancer expression profile. By "administration" or "contacting" herein is meant
that the modulator is added to the cells in such
a manner as to allow the modulator to act upon the cell, whether by uptake and
intracellular action, or by action at the cell
surface. In some embodiments, a nucleic acid encoding a proteinaceous agent
(i.e., a peptide) is put into a viral construct
such as an adenoviral or retroviral construct, and added to the cell, such
that expression of the peptide agent is
accomplished, e.g., PCT US97101019. Regulatable gene therapy systems can also
be used. Once the modulator has been
administered to the cells, the cells are washed if desired and are allowed to
incubate under preferably physiological
conditions for some period. The cells are then harvested and a new gene
expression profile is generated. Thus, e.g.,
cancer tissue is screened for agents that modulate, e.g., induce or suppress,
the cancer phenotype. A change in at least
one gene, preferably many, of the expression profile indicates that the agent
has an effect on cancer activity. Similarly,
altering a biological function or a signaling pathway is indicative of
modulator activity. By defining such a signature for the
cancer phenotype, screens for new drugs that alter the phenotype are devised.
With this approach, the drug target need not
be known and need not be represented in the original gene/protein expression
screening platform, nor does the level of
transcript for the target protein need to change. The modulator inhibiting
function will serve as a surrogate marker
As outlined above, screens are done to assess genes or gene products. That is,
having identified a particular
differentially expressed gene as important in a particular state, screening of
modulators of either the expression of the gene
or the gene product itself is performed.
Use of Modulators to Affect Peptides of the Invention
Measurements of cancer polypeptide activity, or of the cancer phenotype are
performed using a variety of assays.
For example, the effects of modulators upon the function of a cancer
polypeptide(s) are measured by examining parameters
described above. A physiological change that affects activity is used to
assess the influence of a test compound on the
polypeptides of this invention. When the functional outcomes are determined
using intact cells or animals, a variety of
effects can be assesses such as, in the case of a cancer associated with solid
tumors, tumor growth, tumor metastasis,
neovascularization, hormone release, transcriptional changes to both known and
uncharacterized genetic markers (e.g., by
Northern blots), changes in cell metabolism such as cell growth or pH changes,
and changes in intracellular second
messengers such as cGNIP.
Methods of Identifying Characterizing Cancer-associated Sequences
Expression of various gene sequences is correlated with cancer. Accordingly,
disorders based on mutant or
variant cancer genes are determined. In one embodiment, the invention provides
methods for identifying cells containing
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variant cancer genes, e.g., determining the presence of, all or part, the
sequence of at least one endogenous cancer gene in
a cell. This is accomplished using any number of sequencing techniques. The
invention comprises methods of identifying
the cancer genotype of an individual, e.g., determining all or part of the
sequence of at least one gene of the invention in the
individual. This is generally done in at least one tissue of the individual,
e.g., a tissue set forth in Table I, and may include
the evaluation of a number of tissues or different samples of the same tissue.
The method may include comparing the
sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene
to determine the presence of family
members, homologies, mutations or variants. The sequence of all or part of the
gene can then be compared to the
sequence of a known cancer gene to determine if any differences exist. This is
done using any number of known homology
programs, such as BLAST, Bestfit, etc. The presence of a difference in the
sequence between the cancer gene of the
patient and the known cancer gene correlates with a disease state or a
propensity for a disease state, as outlined herein.
In a preferred embodiment, the cancer genes are used as probes to determine
the number of copies of the cancer
gene in the genome. The cancer genes are used as probes to determine the
chromosomal localization of the cancer genes.
Information such as chromosomal localization finds use in providing a
diagnosis or prognosis in particular when
chromosomal abnormalities such as translocations, and the like are identified
in the cancer gene locus.
XIV.) Kits/Articles of Manufacture
For use in the laboratory, prognostic, prophylactic, diagnostic and
therapeutic applications described herein, kits
are within the scope of the invention. Such kits can comprise a carrier,
package, or container that is compartmentalized to
receive one or more containers such as vials, tubes, and the like, each of the
container(s) comprising one of the separate
elements to be used in the method, along with a label or insert comprising
instructions for use, such as a use described
herein. For example, the container(s) can comprise a probe that is or can be
detectably labeled. Such probe can be an
antibody or polynucleotide specific for a protein or a gene or message of the
invention, respectively. Where the method
utilizes nucleic acid hybridization to detect the target nucleic acid, the kit
can also have containers containing nucleotide(s)
for amplification of the target nucleic acid sequence. Kits can comprise a
container comprising a reporter, such as a biotin-
binding protein, such as avidin or streptavidin, bound to a reporter molecule,
such as an enzymatic, fluorescent, or
radioisotope label; such a reporter can be used with, e.g., a nucleic acid or
antibody. The kit can include all or part of the
amino acid sequences in Figure 2 or Figure 3 or analogs thereof, or a nucleic
acid molecule that encodes such amino acid
sequences.
The kit of the invention will typically comprise the container described above
and one or more other containers
associated therewith that comprise materials desirable from a commercial and
user standpoint, including buffers, diluents, filters,
needles, syringes; carrier, package, container, vial and/or tube labels
listing contents and/or instructions for use, and package
inserts with instructions for use.
A label can be present on or with the container to indicate that the
composition is used for a specific therapy or non-
therapeutic application, such as a prognostic, prophylactic, diagnostic or
laboratory application, and can also indicate directions for
either in vivo or in vitro use, such as those described herein. Directions and
or other information can also be included on an
insert(s) or label(s) which is included with or on the kit. The label can be
on or associated with the container. A label a can be
on a container when letters, numbers or other characters forming the label are
molded or etched into the container itself; a
label can be associated with a container when it is present within a
receptacle or carrier that also holds the container, e.g., as
a package insert. The label can indicate that the composition is used for
diagnosing, treating, prophylaxing or prognosing a
condition, such as a neoplasia of a tissue set forth in Table 1.
The terms "kit" and "article of manufacture" can be used as synonyms.
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In another embodiment of the invention, an article(s) of manufacture
containing compositions, such as amino acid
sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s),
e.g., materials useful for the diagnosis,
prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those
set forth in Table I is provided. The article of
manufacture typically comprises at least one container and at least one label.
Suitable containers include, for example,
bottles, vials, syringes, and test tubes. The containers can be formed from a
variety of materials such as glass, metal or
plastic. The container can hold amino acid sequence(s), small molecule(s),
nucleic acid sequence(s), cell population(s)
and/or antibody(s). In one embodiment, the container holds a polynucleotide
for use in examining the mRNA expression
profile of a cell, together with reagents used for this purpose. In another
embodiment a container comprises an antibody,
binding fragment thereof or specific binding protein for use in evaluating
protein expression of109P1 D4 in cells and tissues,
or for relevant laboratory, prognostic, diagnostic, prophylactic and
therapeutic purposes; indications and/or directions for
such uses can be included on or with such container, as can reagents and other
compositions or tools used for these
purposes. In another embodiment, a container comprises materials for eliciting
a cellular or humoral immune response,
together with associated indications and/or directions. In another embodiment,
a container comprises materials for adoptive
immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL),
together with associated indications and/or
directions; reagents and other compositions or tools used for such purpose can
also be included.
The container can alternatively hold a composition that is effective for
treating, diagnosis, prognosing or
prophylaxing a condition and can have a sterile access port (for example the
container can be an intravenous solution bag or
a vial having a stopper pierceable by a hypodermic injection needle). The
active agents in the composition can be an
antibody capable of specifically binding 109P1 D4 and modulating the function
of I 09P1 D4.
The article of manufacture can further comprise a second container comprising
a pharmaceutically-acceptable
buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose
solution. It can further include other materials
desirable from a commercial and user standpoint, including other buffers,
diluents, filters, stirrers, needles, syringes, and/or
package inserts with indications and/or instructions for use.
EXAMPLES:
Various aspects of the invention are further described and illustrated by way
of the several examples that follow,
none of which is intended to limit the scope of the invention.
Example 1: SSH-Generated Isolation of cDNA Fragment of the 109P1 D4 Gene
To isolate genes that are over-expressed in prostate cancer we used the
Suppression Subtractive Hybridization (SSH)
procedure using cDNA derived from prostate cancer tissues. The 109P1 D4 SSH
cDNA sequence was from an experiment where
cDNA derived from LNCaP cells that was androgen-deprived (by growing in the
presence of charcoal-stripped serum) was
subtracted from cDNA derived from LNCaP cells that were stimulated with
mibolerone for 9 hours.
Materials and Methods
Human Tissues:
The patient cancer and normal tissues were purchased from different sources
such as the NDRI (Philadelphia, PA).
mRNA for some normal tissues were purchased from different companies such as
Clontech, Palo Alto, CA.
RNA Isolation:
Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL)
using 10 ml/ g tissue to isolate total RNA.
Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and
Midi kits. Total and mRNA were quantified by
spectrophotometric analysis (O.D. 2601280 nm) and analyzed by gel
electrophoresis.
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Oligonucleotides:
The following HPLC purified oligonucleotides were used.
DPNCDN (cDNA synthesis primer):
5'TTTTGATCAAGCTT3o3' (SEQ ID NO: 44)
Adaptor 1:
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 45)
3'GG000GTCCTAG5' (SEQ ID NO: 46)
Adaptor 2:
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 47)
3'CGGCTCCTAG5' (SEQ ID NO: 48)
PCR primer 1:
5'CTAATACGACTCACTATA000C3' (SEQ ID NO: 49)
Nested primer (NP)1:
5'TCGAGCGGCCG000GGGCAGGA3' (SEQ ID NO: 50)
Nested primer (NP)2:
5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 51)
Suppression Subtractive Hybridization:
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs
corresponding to genes that may be
differentially expressed in prostate cancer. The SSH reaction utilized cDNA
from LNCaP prostate cancer cells.
The 109P1D4 SSH sequence was derived from cDNA subtraction of LNCaP stimulated
with mibolerone minus LNCaP in
the absence of androgen. The SSH DNA sequence (Figure 1) was identified.
The cDNA derived from androgen-deprived LNCaP cells was used as the source of
the "driver" cDNA, while the cDNA
from androgen-stimulated LNCaP cells was used as the source of the "tester"
cDNA. Double stranded cDNAs corresponding to
tester and driver cDNAs were synthesized from 2 g of poly(A)+ RNA isolated
from the relevant xenograft tissue, as described
above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 pg of
oligonucleotide DPNCDN as primer. First- and second-
strand synthesis were carried out as described in the Kit's user manual
protocol (CLONTECH Protocol No. PT1117-1, Catalog No.
K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37oC.
Digested cDNA was extracted with phenol/chloroform
(1:1) and ethanol precipitated.
Tester cDNA was generated by diluting I tl of Dpn II digested cDNA from the
relevant tissue source (see above) (400
ng) in 5 l of water. The diluted cDNA (2 I,160 ng) was then ligated to 2 l
of Adaptor I and Adaptor 2 (10 M), in separate
ligation reactions, in a total volume of 10 pl at 16oC overnight, using 400 pl
of T4 DNA ligase (CLONTECH). Ligation was
terminated with 1 l of 0.2 M EDTA and heating at 720C for 5 min.
The first hybridization was performed by adding 1.5 l (600 ng) of driver cDNA
to each of two tubes containing 1.5 I (20
ng) Adaptor 1- and Adaptor 2- ligated tester cDNA. In a final volume of 4 pl,
the samples were overlaid with mineral oil, denatured
in an MJ Research thermal cycler at 980C for 1.5 minutes, and then were
allowed to hybridize for 8 hrs at 68oC. The two
hybridizations were then mixed together with an additional I l of fresh
denatured driver cDNA and were allowed to hybridize
overnight at 68oC. The second hybridization was then diluted in 200 l of 20
mM Hepes, pH 8.3, 50 mM NaCI, 0.2 mM EDTA,
heated at 700C for 7 min. and stored at -20 C.
PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from
SSH:
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To amplify gene fragments resulting from SSH reactions, two PCR amplifications
were performed. In the primary PCR
reaction 1 Al of the diluted final hybridization mix was added to I Al of PCR
primer 1 (10 AM), 0.5 Al dNTP mix (10 AM), 2.5 Al 10
x reaction buffer (CLONTECH) and 0.5 l 50 x Advantage cDNA polymerase Mix
(CLONTECH) in a final volume of 25 l. PCR I
was conducted using the following conditions: 75 C for 5 min., 94 C for 25
sec., then 27 cycles of 94 C for 10 sec, 66 C for 30 sec,
72 C for 1.5 min. Five separate primary PCR reactions were performed for each
experiment. The products were pooled and
diluted 1:10 with water. For the secondary PCR reaction,1 RI from the pooled
and diluted primary PCR reaction was added to the
same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 AM)
were used instead of PCR primer 1. PCR 2 was
performed using 10-12 cycles of 940C for 10 sec, 68 C for 30 sec, and 72 C for
1.5 minutes. The PCR products were analyzed
using 2% agarose gel electrophoresis.
The PCR products were inserted into pCR2.1 using the T/A vector cloning kit
(Invitrogen). Transformed E. coli were
subjected to blue/white and ampicillin selection. White colonies were picked
and arrayed into 96 well plates and were grown in
liquid culture overnight. To identify inserts, PCR amplification was performed
on 1 p1 of bacterial culture using the conditions of
PCRI and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose
gel electrophoresis.
Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA
was prepared, sequenced, and subjected
to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP
databases.
RT-PCR Expression Analysis:
First strand cDNAs can be generated from 1 g of mRNA with oligo (dT)12-18
priming using the Gibco-BRL Superscript
Preamplification system. The manufacturer's protocol was used which included
an incubation for 50 min at 42 C with reverse
transcriptase followed by RNAse H treatment at 37 C for 20 min. After
completing the reaction, the volume can be increased to
200 p.1 with water prior to normalization. First strand cDNAs from 16
different normal human tissues can be obtained from
Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by
using the primers
5'ATATCGCCGCGCTCGTCGTCGACAA3' (SEQ ID NO: 52) and 5'AGCCACACGCAGCTCATTGTAGAAGG
3' (SEQ ID NO: 53)
to amplify (3-actin. First strand cDNAs (5 Al) were amplified in a total
volume of 50 l containing 0.4 AM primers, 0.2 AM each
dNTPs, 1X PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgC12, 50 mM KCI,
pH8.3) and IX Klentaq DNA polymerase
(Clontech). Five l of the PCR reaction can be removed at 18, 20, and 22
cycles and used for agarose gel electrophoresis. PCR
was performed using an MJ Research thermal cycler under the following
conditions: Initial denaturation can be at 94 C for 15 sec,
followed by a 18, 20, and 22 cycles of 94 C for 15, 65 C for 2 min, 72 C for 5
sec. A final extension at 72 C was carried out for 2
min. After agarose gel electrophoresis, the band intensities of the 283 base
pair j3-actin bands from multiple tissues were
compared by visual inspection. Dilution factors for the first strand cDNAs
were calculated to result in equal R-actin band intensities
in all tissues after 22 cycles of PCR. Three rounds of normalization can be
required to achieve equal band intensities in all tissues
after 22 cycles of PCR.
To determine expression levels of the 109P1 D4 gene, 5 Al of normalized first
strand cDNA were analyzed by PCR using
26, and 30 cycles of amplification. Semi-quantitative expression analysis can
be achieved by comparing the PCR products at cycle
numbers that give light band intensities. The primers used for RT-PCR were
designed using the 109P1 D4 SSH sequence and are
listed below:
109P1 D4.1
5'- TGGTCTTTCAGGTAATTGCTGTTG - 3' (SEQ ID NO: 54)
109P1 D4.2
5'- CTCCATCAATGTTATGTTGCCTGT - 3' (SEQ ID NO: 55)
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A typical RT-PCR expression analysis is shown in Figure 15.
Example 2: Isolation of Full Length 109P1D4 encoding DNA
The 109P1 D4 SSH sequence of 192 bp (Figure 1).exhibited homology to
protocadherin 11 (PCDH1 1), a cell adhesion
molecule related to the calcium dependent cadherins. The human cDNA sequence
encodes a 1021 amino acid protein with an N-
terminal leader sequence and a Iransmembrane domain. 109P1 D4 v.1 of 4603bp
was cloned from human prostate cancer
xenograft LAPC-9AD cDNA library, revealing an ORF of 1021 amino acids (Figure
2 and Figure 3). Other variants (Transcript and
SNP) of 109P1 D4 were also identified and these are listed sequentially in
Figure 2 and Figure 3.
Example 3: Chromosomal Mapping of 109P1D4
Chromosomal localization can implicate genes in disease pathogenesis. Several
chromosome mapping approaches are
available including fluorescent in situ hybridization (FISH), human/hamster
radiation hybrid (RH) panels (Walter et al., 1994;
Nature Genetics 7:22; Research Genetics, Huntsville Al), human-rodent somatic
cell hybrid panels such as is available from the
Coriell Institute (Camden, New Jersey), and genomic viewers utilizing BLAST
homologies to sequenced and mapped genomic
clones (NCBI, Bethesda, Maryland).
109P1 D4 maps to chromosome Xq21.3 using 109P1 D4 sequence and the NCBI BLAST
tool: located on the World Wide
Web. 109P1D4 was also identified on chromosome
Yp11.2, a region of 99% identity to Xq21.
Example 4: Expression Analysis of 109P1 D4 in Normal Tissues and Patient
Specimens
Expression analysis by RT-PCR and Northern analysis demonstrated that normal
tissue expression of a gene of
Figure 2 is restricted predominantly to the tissues set forth in Table I.
Therapeutic applications for a gene of Figure 2 include use as a small
molecule therapy and/or a vaccine (T cell or
antibody) target. Diagnostic applications for a gene of Figure 2 include use
as a diagnostic marker for local and/or
metastasized disease. The restricted expression of a gene of Figure 2 in
normal tissues makes it useful as a tumor target for
diagnosis and therapy. Expression analysis of a gene of Figure 2 provides
information useful for predicting susceptibility to
advanced stage disease, rate of progression, and/or tumor aggressiveness.
Expression status of a gene of Figure 2 in
patient samples, tissue arrays and/or cell lines may be analyzed by: (i)
immunohistochemical analysis; (ii) in situ
hybridization; (iii) RT-PCR analysis on laser capture micro-dissected samples;
(iv) Western blot analysis; and (v) Northern
analysis.
RT-PCR analysis and Northern blotting were used to evaluate gene expression in
a selection of normal and
cancerous urological tissues. The results are summarized in Figures 15-19.
Figure 14 shows expression of 109P1D4 in lymphoma cancer patient specimens.
RNA was extracted from
peripheral blood lymphocytes, cord blood isolated from normal individuals, and
from lymphoma patient cancer specimens.
Northern blots with 1 Opg of total RNA were probed with the 109P1 D4 sequence.
Size standards in kilobases are on the
side. Results show expression of 109P1 D4 in lymphoma patient specimens but
not in the normal blood cells tested.
Figure 15 shows expression of 109P1 D4 by RT-PCR. First strand cDNA was
prepared from vital pool 1(liver, lung
and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer pool,
bladder cancer pool, kidney cancer pool,
colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool,
cancer metastasis -pool, and pancreas cancer
pool. Normalization was performed by PCR using primers to actin and GAPDH.
Semi-quantitative PCR, using primers to
109P1 D4, was performed at 30 cycles of amplification. Results show strong
expression of 109P1 D4 in all cancer pools
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tested. Very low expression was detected in the vital pools.
Figure 16 shows expression of 109P1 D4 in normal tissues. Two multiple tissue
northern blots (Clontech), both
with 2 pg of mRNA/lane, were probed with the 109P1D4 SSH fragment. Size
standards in kilobases (kb) are indicated on
the side. Results show expression of approximately 10 kb 109P1 D4 transcript
in ovary. Weak expression was also detected
in placenta and brain, but not in the other normal tissues tested.
Figure 17 shows expression of 109P1 D4 in human cancer cell lines. RNA was
extracted from a number of human
prostate and bone cancer cell lines. Northern blots with 10 pg of total
RNA/lane were probed with the 109P1 D4 SSH
fragment. Size standards in kilobases (kb) are indicated on the side. Results
show expression of 109P1 D4 in LAPC-9AD,
LAPC-9AI, LNCaP prostate cancer cell lines, and in the bone cancer cell lines,
SK-ES-1 and RD-ES.
Extensive expression of 109P1 D4 in normal tissues is shown in Figure 18A. A
cDNA dot blot containing 76
different samples from human tissues was analyzed using a 109P1 D4 SSH probe.
Expression was only detected in multiple
areas of the brain, placenta, ovary, and fetal brain, amongst all tissues
tested.
Figure 188 shows expression of 109PID4 in patient cancer specimens. Expression
of 109P1D4 was assayed in a
panel of human cancers (T) and their respective matched normal tissues (N) on
RNA dot blots. Upregulated expression of
109P1 D4 in tumors compared to normal tissues was observed in uterus, lung and
stomach. The expression detected in
normal adjacent tissues (isolated from diseased tissues) but not in normal
tissues (isolated from healthy donors) may
indicate that these tissues are not fully normal and that 109P1 D4 may be
expressed in early stage tumors.
Figure 19 shows 109P1 D4 expression in lung cancer patient specimens. RNA was
extracted from normal lung,
prostate cancer xenograft LAPC-9AD, bone cancer cell line RD-ES, and lung
cancer patient tumors. Northern blots with 10
pg of total RNA were probed with 109P1 D4. Size standards in kilobases are on
the side. Results show strong expression of
109PI D4 in lung tumor tissues as well as the RD-ES cell line, but not in
normal lung.
The restricted expression of 109PI D4 in normal tissues and the expression
detected in cancer patient specimens
suggest that 109P1 D4 is a potential therapeutic target and a diagnostic
marker for human cancers.
Example 5: Splice Variants of 109P1 D4
Transcript variants are variants of mature mRNA from the same gene which arise
by alternative transcription or
alternative splicing. Alternative transcripts are transcripts from the same
gene but start transcription at different points.
Splice variants are mRNA variants spliced differently from the same
transcript. In eukaryotes, when a multi-exon gene is
transcribed from genomic DNA, the initial RNA is spliced to produce functional
mRNA, which has only exons and is used for
translation into an amino acid sequence. Accordingly, a given gene can have
zero to many alternative transcripts and each
transcript can have zero to many splice variants. Each transcript variant has
a unique exon makeup, and can have different
coding and/or non-coding (5' or 3' end) portions, from the original
transcript. Transcript variants can code for similar or
different proteins with the same or a similar function or can encode proteins
with different functions, and can be expressed in
the same tissue at the same time, or in different tissues at the same time, or
in the same tissue at different times, or in
different tissues at different times. Proteins encoded by transcript variants
can have similar or different cellular or
extracellular localizations, e.g., secreted versus intracellular.
Transcript variants are identified by a variety of art-accepted methods. For
example, alternative transcripts and
splice variants are identified by full-length cloning experiment, or by use of
full-length transcript and EST sequences. First,
all human ESTs were grouped into clusters which show direct or indirect
identity with each other. Second, EST's in the same
cluster were further grouped into sub-clusters and assembled into a consensus
sequence. The original gene sequence is
compared to the consensus sequence(s) or other full-length sequences. Each
consensus sequence is a potential splice
CA 02522994 2010-03-25
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variant for that gene. Even when a variant is identified that is not a full-
length clone, that portion of the variant is very useful
for antigen generation and for further cloning of the full-length splice
variant, using techniques known in the art
Moreover, computer programs are available in the art that identify transcript
variants based on genomic
sequences. Genomic-based transcript variant identification programs include
FgenesH (A. Salamov and V. Solovyev, "Ab
initio gene finding in Drosophila genomic DNA," Genome Research. 2000
April;10(4):516-22); Grail
and GenScan. For a general discussion
of splice variant identification protocols see., e.g., Southan, C., A genomic
perspective on human proteases, FEBS Lett
2001 Jun 8; 498(2-3):214-8; de Souza, S.J., of al., Identification of human
chromosome 22 transcribed sequences with ORF
expressed sequence tags, Proc. Nag Acad Sci U S A. 2000 Nov 7; 97(23):12690-3.
To further confirm the parameters of a transcript variant, a variety of
techniques are available in the art, such as
full-length cloning, proteomic validation, PCR-based validation, and 5' RACE
validation, etc. (see e.g., Proteomic Validation:
Brennan, S.O,, et al., Albumin banks peninsula: a new termination variant
characterized by electrospray mass spectrometry,
Biochem Biophys Acta. 1999 Aug 17;1433(1-2):321-6; Ferranti P, et at,
Differential splicing of pre-messenger RNA produces
multiple forms of mature caprine atpha(s1)-casein, Eur J Biochem.1997 Oct
1;249(1):1-7. For PCR-based Validation:
Wellmann S, et aL, Specific reverse transcription-PCR quantification of
vascular endothelial growth factor (VEGF) splice
variants by LightCycler technology, Clin Chem. 2001 Apr,,47(4):65460; Jia,
H.P., of al., Discovery of new human beta-
defensins using a genomics-based approach, Gene. 2001 Jan 24; 263(1-2):211-8.
For PCR-based and 5' RACE Validation:
Brigle, KE., of al., Organization of the murine reduced folate carrier gene
and identification of variant splice forms, Biochem
Biophys Acta. 1997 Aug 7; 1353(2): 191-8).
It is known in the art that genomic regions are modulated in cancers. When the
genomic region to which a gene
maps is modulated in a particular cancer, the alternative transcripts or
splice variants of the gene are modulated as well.
Disclosed herein is that 109P1 D4 has a particular expression profile related
to cancer. Alternative transcripts and splice
variants of 109P1D4 may also be involved in cancers in the same or different
tissues, thus serving as tumor-associated
markerslantigens.
Using the full-length gene and EST sequences, 8 transcript variants were
identified, designated as 109P1D4 v.2,
v.3, v.4, v.5, v.6, v.7, v.8 and v.9. The boundaries of the exon in the
original transcript, 109P1 D4 v.1, were shown in Table LI.
Compared with 109P1 D4 v.1, transcript variant 109P1 04 v.3 has spliced out
2069-2395 from variant 109P1 D4 v.1, as shown
in Figure 12. Variant 109P1D4 v.4 spliced out 1162-2096 of variant 109P1D4
v.1. Variant 109P1 D4 v.5 added one exon to
the 5' and extended 2 bp to the 5' end and 288 bp to the 3' end of variant
109P1 D4 v.1. Theoretically, each different
combination of exons in spatial order, e.g. exon 1 of v.5 and exons 1 and 2 of
v.3 or v.4, is a potential splice variant.
Tables LII through LV are set forth on a variant by-variant basis. Tables
Lll(a)-(h) show nucleotide sequence of
the transcript variants. Tables LIII(a)-(h) show the alignment of the
transcript variants with nucleic acid sequence of
109P1 D4 v.1. Tables LIV(a)-(h) lay out amino acid translation of the
transcript variants for the identified reading frame
orientation. Tables LV(a)-(h) displays alignments of the amino acid sequence
encoded by the splice variants with that of
109P1D4 v.1.
Example 6: Single Nucleotide Polymorphisms of 109P1 D4
A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a
nucleotide sequence at a specific
location. At any given point of the genome, there are four possible nucleotide
base pairs: All, C/G, G/C and T/A Genotype
refers to the specific base pair sequence of one or more locations in the
genome of an individual. Haplotype refers to the
base pair sequence of more than one location on the same DNA molecule (or the
same chromosome in higher organisms),
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often in the context of one gene or in the context of several tightly linked
genes. SNP that occurs on a cDNA is called cSNP.
This cSNP may change amino acids of the protein encoded by the gene and thus
change the functions of the protein. Some
SNP cause inherited diseases; others contribute to quantitative variations in
phenotype and reactions to environmental
factors including diet and drugs among individuals. Therefore, SNP and/or
combinations of alleles (called haplotypes) have
many applications, including diagnosis of inherited diseases, determination of
drug reactions and dosage, identification of
genes responsible for diseases, and analysis of the genetic relationship
between individuals (P. Nowotny, J. M. Kwon and A.
M. Goate, " SNP analysis to dissect human traits," Curr. Opin. Neurobiol. 2001
Oct; 11(5):637-641; M. Pirmohamed and B.
K. Park, "Genetic susceptibility to adverse drug reactions," Trends Pharmacol,
Sci. 2001 Jun; 22(6):298-305; J. H. Riley, C.
J. Allan, E. Lai and A. Roses, The use of single nucleotide polymorphisms in
the isolation of common disease genes,"
Pharmacogenomics. 2000 Feb; 1(1):39-47; R. Judson, J. C. Stephens and A.
Windemuth, The predictive power of
haplotypes in clinical response," Pharmacogenomics. 2000 Feb; 1(1):15-26).
SNP are identified by a variety of art-accepted methods (P. Bean, "The
promising voyage of SNP target discovery,"
Am. Clin. Lab. 2001 Oct-Nov; 20(9):18-20; K. M. Weiss, "In search of human
variation," Genome Res. 1998 Jul; 8(7):691-
697; M. M. She, "Enabling large-scale pharmacogenetic studies by high-
throughput mutation detection and genotyping
technologies," Clin. Chem. 2001 Feb; 47(2):164-172). For example, SNP can be
identified by sequencing DNA fragments
that show polymorphism by gel-based methods such as restriction fragment
length polymorphism (RFLP) and denaturing
gradient gel electrophoresis (DGGE). They can also be discovered by direct
sequencing of DNA samples pooled from
different individuals or by comparing sequences from different DNA samples.
With the rapid accumulation of sequence data
in public and private databases, one can discover SNP by comparing sequences
using computer programs (Z. Gu, L. Hillier
and P. Y. Kwok, "Single nucleotide polymorphism hunting in cyberspace," Hum.
Mutat. 1998; 12(4):221-225). SNP can be
verified and genotype or haplotype of an individual can be determined by a
variety of methods including direct sequencing
and high throughput microarrays (P. Y. Kwok, "Methods for genotyping single
nucleotide polymorphisms," Annu. Rev.
Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J.
Mathis, B. Erwin, P. Grass, B. Hines and A.
Duesterhoeft, "High-throughput SNP genotyping with the Masscode system," Mol.
Diagn. 2000 Dec; 5(4):329-340).
Using the methods described above, SNP were identified in the original
transcript, 109P4D4 v.1, and its variants
(see Figure 2J and Figure 2K). These alleles of the SNP, though shown
separately here, can occur in different combinations
(haplotypes) and in any one of the transcript variants (such as 109P4D4 v.4 or
v.5) that contains the site of the SNP.
Transcript variants v.4 and v.5 contained those SNP in the exons shared with
variant v.3, and transcript variant v.9 contained
all the SNP occurred in variant v.6 (see Figure 10).
Example 7: Production of Recombinant 109P1 D4 in Prokaryotic Systems
To express recombinant 109P1 D4 and 109P1 D4 variants in prokaryotic cells,
the full or partial length 109P1 D4.
and 109P1 D4 variant cDNA sequences are cloned into any one of a variety of
expression vectors known in the art. One or
more of the following regions of 109P1 D4 variants are expressed: the full
length sequence presented in Figures 2 and 3, or
any 8, 9, 10, 11, 12, 13, 14,15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30 or more contiguous amino acids
from 109P1D4, variants, or analogs thereof.
A. In vitro transcription and translation constructs:
pCRII: To generate 109P1 D4 sense and anti-sense RNA probes for RNA in situ
investigations, pCRIl constructs
(Invitrogen, Carlsbad CA) are generated encoding either all or fragments of
the 109P1 D4 cDNA. The pCRII vector has Sp6
and T7 promoters flanking the insert to drive the transcription of 109P1 D4
RNA for use as probes in RNA in situ hybridization
experiments. These probes are used to analyze the cell and tissue expression
of 109P1 D4 at the RNA level. Transcribed
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109P1 D4 RNA representing the cDNA amino acid coding region of the 109P1 D4
gene is used in in vitro translation systems
such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, WI)
to synthesize 109P1D4 protein.
B. Bacterial Constructs:
pGEX Constructs: To generate recombinant 109P1 D4 proteins in bacteria that
are fused to the Glutathione S-
transferase (GST) protein, all or parts of the 109P1 D4 cDNA protein coding
sequence are cloned into the pGEX family of
GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, NJ). These
constructs allow controlled expression of
recombinant 109P1 D4 protein sequences with GST fused at the amino-terminus
and a six histidine epitope (6X His) at the
carboxyl-terminus. The GST and 6X His tags permit purification of the
recombinant fusion protein from induced bacteria with
the appropriate affinity matrix and allow recognition of the fusion protein
with anti-GST and anti-His antibodies. The 6X His
tag is generated by adding 6 histidine colons to the cloning primer at the 3'
end, e.g., of the open reading frame (ORF). A
proteolytic cleavage site, such as the PreScissionTM recognition site in pGEX-
6P-1, may be employed such that it permits
cleavage of the GST tag from 109P1 D4-related protein. The ampicillin
resistance gene and pBR322 origin permits selection
and maintenance of the pGEX plasmids in E. coll.
pMAL Constructs: To generate, in bacteria, recombinant 109P1 D4 proteins that
are fused to maltose-binding
protein (MBP), all or parts of the 109P1 D4 cDNA protein coding sequence are
fused to the MBP gene by cloning into the
pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, MA). These
constructs allow controlled expression of
recombinant 109P1 D4 protein sequences with MBP fused at the amino-terminus
and a 6X His epitope tag at the carboxyl-
terminus. The MBP and 6X His tags permit purification of the recombinant
protein from induced bacteria with the appropriate
affinity matrix and allow recognition of the fusion protein with anti-MBP and
anti-His antibodies. The 6X His epitope tag is
generated by adding 6 histidine codons to the 3' cloning primer. A Factor Xa
recognition site permits cleavage of the pMAL
tag from 109P1 D4. The pMAL-c2X and pMAL-p2X vectors are optimized to express
the recombinant protein in the
cytoplasm or periplasm respectively. Periplasm expression enhances folding of
proteins with disulfide bonds. In one
embodiment, amino acids 24-419 of 109P1 D4 variant I was cloned into the pMAL-
c2X vector and was used to express the
fusion protein.
pET Constructs: To express 109PI D4 in bacterial cells, all or parts of the
109P1 D4 cDNA protein coding
sequence are cloned into the pET family of vectors (Novagen, Madison, WI).
These vectors allow tightly controlled
expression of recombinant 109P1 D4 protein in bacteria with and without fusion
to proteins that enhance solubility, such as
NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag TM that
aid purification and detection of the
recombinant protein. For example, constructs are made utilizing pET NusA
fusion system 43.1 such that regions of the
109P1D4 protein are expressed as amino-terminal fusions to NusA. In 2
embodiments, amino acids 24-419 and 24-815
were cloned into pET43.1 vector and used to express the fusion protein.
C. Yeast Constructs:
pESC Constructs: To express 109P1 D4 in the yeast species Saccharomyces
cerevisiae for generation of
recombinant protein and functional studies, all or parts of the 109P1 D4 cDNA
protein coding sequence are cloned into the
pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3,
TRPI, LEU2, and URA3 (Stratagene, La
Jolla, CA). These vectors allow controlled expression from the same plasmid of
up to 2 different genes or cloned sequences
containing either FIagTM or Myc epitope tags in the same yeast cell. This
system is useful to confirm protein-protein
interactions of 109P1 D4. In addition, expression in yeast yields similar post-
translational modifications, such as
glycosylations and phosphorylations, that are found when expressed in
eukaryotic cells,
pESP Constructs: To express 109P1 D4 in the yeast species Saccharomyces pombe,
all or parts of the 109P1 D4
cDNA protein coding sequence are cloned into the pESP family of vectors. These
vectors allow controlled high level of
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expression of a 109P1 D4 protein sequence that is fused at either the amino
terminus or at the carboxyl terminus to GST
which aids purification of the recombinant protein. A FlagTM epitope tag
allows detection of the recombinant protein with anti-
FlagTM antibody.
Example 8: Production of Recombinant 109P1 D4 in Higher Euka otic Systems
A. Mammalizn Constructs:
To express recombinant 109P1 D4 in eukaryotic cells, the full or partial
length 109P1 D4 cDNA sequences were
cloned into any one of a variety of expression vectors known in the art. One
or more of the following regions of 109P1 D4
were expressed in these constructs, amino acids I to 1021 or any 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 109P1 D4 v.1;
amino acids I to 1054, 1 to 1347, 1 to 1337, 1
to 1310,1 to 1037, 1 to 1048,1 to 1340 of v.2, v.3, v.4, v.5, v.6, v.7, and
v.8 respectively; or any 8, 9, 10,11, 12,13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous
amino acids from 109P1 D4 variants, or analogs
thereof.
The constructs can be transfected into any one of a wide variety of mammalian
cells such as 293T cells.
Transfected 293T cell lysates can be probed with the anti-109P1D4 polyclonal
serum, described herein.
pcDNA4IHisMax Constructs: To express 109P1 D4 in mammalian cells, a 109PI D4
ORF, or portions thereof, of
109PID4 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA).
Protein expression is driven from the
cytomegalovirus (CMV) promoter and the SPI6 translational enhancer. The
recombinant protein has XpressTM and six
histidine (6X His) epitopes fused to the amino-terminus. The pcDNA4/HisMax
vector also contains the bovine growth
hormone (BGH) polyadenylation signal and transcription termination sequence to
enhance mRNA stability along with the
SV40 origin for episomal replication and simple vector rescue in cell lines
expressing the large T antigen. The Zeocin
resistance gene allows for selection of mammalian cells expressing the protein
and the ampicillin resistance gene and CoIEI
origin permits selection and maintenance of the plasmid in E. coll.
pcDNA3.11MycHis Constructs: To express 109P1 D4 in mammalian cells, a 109P1 D4
ORF, or portions thereof,
of 109PID4 with a consensus Kozak translation initiation site was cloned into
pcDNA3.1/MycHis Version A (Invitrogen,
Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV)
promoter. The recombinant proteins have the
myc epitope and 6X His epitope fused to the carboxyl-terminus. The
pcDNA3.1/MycHis vector also contains the bovine
growth hormone (BGH) polyadenylation signal and transcription termination
sequence to enhance mRNA stability, along with
the SV40 origin for episomal replication and simple vector rescue in cell
lines expressing the large T antigen. The Neomycin
resistance gene can be used, as it allows for selection of mammalian cells
expressing the protein and the ampicillin
resistance gene and ColEl origin permits selection and maintenance of the
plasmid in E. coll.
The complete ORF of 109PID4 v.1 was cloned into the pcDNA3.1/MycHis construct
to generate
109P I D4. pcDNA3.1 /MycHis.
pcDNA3.1ICT,GFP=TOPO Construct: To express 109PID4 in mammalian cells and to
allow detection of the
recombinant proteins using fluorescence, a 109P1 D4 ORF, or portions thereof,
with a consensus Kozak translation initiation
site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression
is driven from the cytomegalovirus
(CMV) promoter. The recombinant proteins have the Green Fluorescent Protein
(GFP) fused to the carboxyl-terminus
facilitating non-invasive, in vivo detection and cell biology studies. The
pcDNA3.1 CT-GFP-TOPO vector also contains the
bovine growth hormone (BGH) polyadenylation signal and transcription
termination sequence to enhance mRNA stability
along with the SV40 origin for episomal replication and simple vector rescue
in cell lines expressing the large T antigen. The
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Neomycin resistance gene allows for selection of mammalian cells that express
the protein, and the ampicillin resistance
gene and CoIE1 origin permits selection and maintenance of the plasmid in E.
coll. Additional constructs with an amino-
terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire
length of a 109P1D4 protein.
PAPtag: A 109P1 D4 ORF, or portions thereof, is cloned into pAPtag-5
(GenHunter Corp. Nashville, TN). This
construct generates an alkaline phosphatase fusion at the carboxyl-terminus of
a 109P1 D4 protein while fusing the IgGK
signal sequence to the amino-terminus. Constructs are also generated in which
alkaline phosphatase with an amino-
terminal IgGK signal sequence is fused to the amino-terminus of a 109P1 D4
protein. The resulting recombinant 109P1 D4
proteins are optimized for secretion into the media of transfected mammalian
cells and can be used to identify proteins such
as ligands or receptors that interact with 109P1 D4 proteins. Protein
expression is driven from the CMV promoter and the
recombinant proteins also contain myc and 6X His epitopes fused at the
carboxyl-terminus that facilitates detection and
purification. The Zeocin resistance gene present in the vector allows for
selection of mammalian cells expressing the
recombinant protein and the ampicillin resistance gene permits selection of
the plasmid in E. coll.
Tp ag5: A 109P1 D4 ORF, or portions thereof, were cloned into pTag-5. This
vector is similar to pAPtag but
without the alkaline phosphatase fusion. This construct generated 109P1 D4
protein with an amino-terminal IgGK signal
sequence and myc and 6X His epitope tags at the carboxyl-terminus that
facilitate detection and affinity purification. The
resulting recombinant 109P1 D4 protein was optimized for secretion into the
media of transfected mammalian cells, and was
used as immunogen or ligand to identify proteins such as ligands or receptors
that interact with the 109P1 D4 proteins.
Protein expression is driven from the CMV promoter. The Zeocin resistance gene
present in the vector allows for selection
of mammalian cells expressing the protein, and the ampicillin resistance gene
permits selection of the plasmid in E. coil.
PsecFc: A 109P1 D4 ORF, or portions thereof, is also cloned into psecFc. The
psecFc vector was assembled by
cloning the human immunoglobulin GI (IgG) Fc (hinge, CH2, CH3 regions) into
pSecTag2 (Invitrogen, California). This
construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 109PI D4
proteins, while fusing the IgGK signal
sequence to N-terminus. 109P1 D4 fusions utilizing the murine IgG1 Fe region
are also used. The resulting recombinant
109P1 D4 proteins are optimized for secretion into the media of transfected
mammalian cells, and can be used as
immunogens or to identify proteins such as ligands or receptors that interact
with 109P1 D4 protein. Protein expression is
driven from the CMV promoter. The hygromycin resistance gene present in the
vector allows for selection of mammalian
cells that express the recombinant protein, and the ampicillin resistance gene
permits selection of the plasmid in E. coff
pSRa Constructs: To generate mammalian cell lines that express 109P1 D4
constitutively,109PI D4 ORF, or
portions thereof, were cloned into pSRa constructs. Amphotropic and ecotropic
retroviruses were generated by transfection
of pSRa constructs into the 293T-10A1 packaging line or co-transfection of
pSRa and a helper plasmid (containing deleted
packaging sequences) into the 293 cells, respectively. The retrovirus is used
to infect a variety of mammalian cell lines,
resulting in the integration of the cloned gene, 109P1 D4, into the host cell-
lines. Protein expression is driven from a long
terminal repeat (LTR). The Neomycin resistance gene present in the vector
allows for selection of mammalian cells that
express the protein, and the ampicillin resistance gene and ColEl origin
permit selection and maintenance of the plasmid in
E. co/i. The retroviral vectors can thereafter be used for infection and
generation of various cell lines using, for example,
PC3, NIH 3T3, IsuPr1, 293 or rat-1 cells.
Additional pSRa constructs are made that fuse an epitope tag such as the
FLAGTM tag to the carboxyl-terminus of
109P1 D4 sequences to allow detection using anti-Flag antibodies. For example,
the FLAGTM sequence 5' GAT TAC AAG
GAT GAC GAC GAT AAG 3' (SEQ ID NO: 56) is added to cloning primer at the 3'
end of the ORF. Additional pSRa
constructs are made to produce both amino-terminal and carboxyl-terminal GFP
and myc/6X His fusion proteins of the full-
length 109P1 D4 proteins.
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Additional Viral Vectors: Additional constructs are made for viral-mediated
delivery and expression of 109P1D4.
High virus titer leading to high level expression of 109P1 D4 is achieved in
viral delivery systems such as adenovirat vectors
and herpes amplicon vectors. A 109P1 D4 coding sequence or fragments thereof
are amplified by PCR and subcloned into
the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are
performed according to the manufacturer's
instructions to generate adenoviral vectors. Alternatively,109P1D4 coding
sequences or fragments thereof are cloned into
the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors
are thereafter used for infection of various
cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
Regulated Expression Systems: To control expression of 109P1 D4 in mammalian
cells, coding sequences of
109P1 D4, or portions thereof, are cloned into regulated mammalian expression
systems such as the T-Rex System
(Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated
Ecdysone System (Stratagene). These systems
allow the study of the temporal and concentration dependent effects of
recombinant 109P1 04. These vectors are thereafter
used to control expression of 109P1D4 in various cell lines such as PC3, NIH
3T3, 293 or rat -1 cells.
B. Baculovirus Expression Systems
To generate recombinant 109P1 D4 proteins in a baculovirus expression system,
109P1 D4 ORF, or portions
thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5
(Invitrogen), which provides a His-tag at the N-terminus.
Specifically, pBlueBac-109P1 D4 is co-transfected with helper plasmid pBac-N-
Blue (Invitrogen) into SF9 (Spodopfera
frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen
instruction manual for details). Baculovirus is
then collected from cell supernatant and purified by plaque assay.
Recombinant 109P1 D4 protein is then generated by infection of HighFive insect
cells (Invitrogen) with purified
baculovirus. Recombinant 109P1D4 protein can be detected using anti-109P1D4 or
anti-His-tag antibody. 109P1D4 protein
can be purified and used in various cell-based assays or as immunogen to
generate polyclonal and monoclonal antibodies
specific for 109P1 D4.
Example 9: Antigenicity Profiles and Secondary Structure
Figure(s) 5A-!, Figure 6A-1, Figure 7A-1, Figure 8A-I, and Figure 9A-l depict
graphically five amino acid profiles of
109P1 D4 variants I through 9, each assessment available by accessing the
ProtScale website located on the World Wide
Web on the ExPasy molecular biology server.
These profiles: Figure 5, Hydrophilicity, (Hopp T.P., Woods K.R., 1981. Proc.
Natl. Acad. Sol. U.S.A. 78:3824-
3828); Figure 6, Hydropathicity, (Kyle J., Doolittle R.F., 1982. J. Mol. Biol.
157:105-132); Figure 7, Percentage Accessible
Residues (Janin J., 1979 Nature 277:491-492); Figure 8, Average Flexibility,
(Bhaskaran R., and Ponnuswamy P.K., 1988.
Int. J. Pept. Protein Res. 32:242-255); Figure 9, Beta-turn (Deleage, G., Roux
B. 1987 Protein Engineering 1:289-294); and
optionally others available in the art, such as on the ProtScale website, were
used to identify antigenic regions of each of the
109P1 D4 variant proteins. Each of the above amino acid profiles of 109P1 D4
variants were generated using the following
ProtScale parameters for analysis: 1) A window size of 9,2) 100% weight of the
window edges compared to the window
center, and, 3) amino acid profile values normalized to lie between 0 and 1.
Hydrophilicity (Figure 5), Hydropathicity (Figure 6) and Percentage Accessible
Residues (Figure 7) profiles were
used to determine stretches of hydrophilic amino acids (i.e., values greater
than 0.5 on the Hydrophilicity and Percentage
Accessible Residues profile, and values less than 0.5 on the Hydropathicity
profile). Such regions are likely to be exposed to
the aqueous environment, be present on the surface of the protein, and thus
available for immune recognition, such as by
antibodies.
Average Flexibility (Figure 8) and Beta-turn (Figure 9) profiles determine
stretches of amino acids (i.e., values
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greater than 0.5 on the Beta-turn profile and the Average Flexibility profile)
that are not constrained in secondary structures
such as beta sheets and alpha helices. Such regions are also more likely to be
exposed on the protein and thus accessible
to immune recognition, such as by antibodies.
Antigenic sequences of the 109P1 D4 variant proteins indicated, e.g., by the
profiles set forth in Figure 5, Figure 6,
Figure 7, Figure 8, and/or Figure 9 are used to prepare immunogens, either
peptides or nucleic acids that encode them, to
generate therapeutic and diagnostic anti-109P1D4 antibodies. The immunogen can
be any 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 or more than 50
contiguous amino acids, or the corresponding
nucleic acids that encode them, from the 109P1D4 protein variants listed in
Figures 2 and 3. In particular, peptide
immunogens of the invention can comprise, a peptide region of at least 5 amino
acids of Figures 2 and 3 in any whole
number increment that includes an amino acid position having a value greater
than 0.5 in the Hydrophilicity profiles of Figure
5; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole
number increment that includes an amino acid
position having a value less than 0.5 In the Hydropathicity profile of Figure
6; a peptide region of at least 5 amino acids of
Figures 2 and 3 in any whole number increment that includes an amino acid
position having a value greater than 0.5 in the
Percent Accessible Residues profiles of Figure 7; a peptide region of at least
5 amino acids of Figures 2 and 3 In any whole
number increment that includes an amino acid. position having a value greater
than 0.5 in the Average Flexibility profiles on
Figure 8; and, a peptide region of at least 5 amino acids of Figures 2 and 3
in any whole number increment that includes an
amino acid position having a value greater than 0.5 in the Beta-turn profile
of Figures 9. Peptide immunogens of the
invention can also comprise nucleic acids that encode any of the forgoing.
All immunogens of the invention, peptide or nucleic acid, can be embodied in
human unit dose form, or comprised
by a composition that includes a pharmaceutical excipient compatible with
human physiology.
The secondary structure of 109P1 D4 protein variants, namely the' predicted
presence and location of alpha helices,
extended strands, and random coils, are predicted from the primary amino acid
sequence using the HNN - Hierarchical
Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March
Vol. 25, No 3 [2911:147-150 Combet
C., Blanchet C., Geourjon C. and Deleage G. ), accessed from
the ExPasy molecular biology server located on the World Wide Web . This
analysis for protein
variants I through 9 are shown in Figure 13A through 131 respectively. The
percent of structure for each variant comprised
of alpha helix, extended strand, and random coil is also indicated.
Analysis for the potential presence of transmembrane domains in 109P1 D4
variant proteins was carried out using
a variety of transmembrane prediction algorithms accessed from the ExPasy
molecular biology server located on the World
Wide Web at (www.expasy.ch/tools/). Shown graphically in figures 13J-R are the
results of analyses using the TMpred
program (top. panels) and the TMHMM program (bottom panels) of 109P1 D4
protein variants I through 9 respectively.
Analyses of the variants using other structural prediction programs are
summarized in Table VI and Table L.
Example 10: Generation of 109P1D4 Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more
Injections of an immunizing agent
and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant
will be injected in the mammal by multiple
subcutaneous or intraperitoneal injections. In addition to immunizing with a
full length 109P1 D4 protein variant, computer
algorithms are employed in design of immunogens that, based on amino acid
sequence analysis contain characteristics of
being antigenic and available for recognition by the immune system of the
immunized host (see the Example entitled
"Antigenicity Profiles and Secondary Structure"). Such regions would be
predicted to be hydrophilic, flexible, in beta-turn
conformations, and be exposed on the surface of the protein (see, e.g., Figure
5, Figure 6, Figure 7, Figure 8, or Figure 9 for
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amino acid profiles that indicate such regions of 109P1D4 protein variant 1).
For example, recombinant bacterial fusion proteins or peptides containing
hydrophilic, flexible, beta-turn regions of
109P1 D4 protein variants are used as antigens to generate polyclonal
antibodies in New Zealand White rabbits or
monoclonal antibodies as described in the example entitled "Generation of
109P1 D4 Monoclonal Antibodies (mAbs)". For
example, in 109P1D4 variant 1, such regions include, but are not limited to,
amino acids 22-39, amino acids 67-108, amino
acids 200-232, amino acids 454-499, amino acids 525-537, amino acids 640-660,
amino acids 834-880, and amino acids
929-942. It is useful to conjugate the immunizing agent to a protein known to
be immunogenic in the mammal being
immunized. Examples of such immunogenic proteins include, but are not limited
to, keyhole limpet hemocyanin (KLH),
serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In 2
embodiments, peptides encoding amino acids 77-90
and amino acids 929-942 of 109P1 D4 variant I were synthesized, conjugated to
KLH, and used to immunize separate
rabbits. Alternatively the immunizing agent may include all or portions of the
109P1 D4 variant proteins, analogs or fusion
proteins thereof. For example, the 109P1 D4 variant 1 amino acid sequence can
be fused using recombinant DNA
techniques to any one of a variety of fusion protein partners that are well
known in the art, such as glutathione-S-transferase
(GST) and HIS tagged fusion proteins. In I embodiment, amino acids 24-419 of
109P1D4 variant 1 was fused to NUSa
using recombinant techniques and the pET43.1 expression vector, expressed,
purified and used to immunize a rabbit. Such
fusion proteins are purified from induced bacteria using the appropriate
affinity matrix.
Other recombinant bacterial fusion proteins that may be employed include
maltose binding protein, LacZ,
thioredoxin, NusA, or an immunoglobulin constant region (see the section
entitled "Production of 109P1D4 in Prokaryotic
Systems" and Current Protocols In Molecular Biology, Volume 2, Unit 16,
Frederick M. Ausubul et al. eds., 1995; Linsley,
P.S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, J.(1991)
J.Exp. Med. 174, 561-566).
In addition to bacterial derived fusion proteins, mammalian expressed protein
antigens are also used. These
antigens are expressed from mammalian expression vectors such as the Tag5 and
Fc-fusion vectors (see the section
entitled "Production of Recombinant 109P1D4 in Eukaryotic Systems"), and
retain post-translational modifications such as
glycosylations found in native protein. In one embodiment, amino acids 24-812
of 109P1 D4 variant 1 was cloned into the
Tag5 mammalian secretion vector, and expressed in 293T cells (See Figure 20).
The recombinant protein is purified by
metal chelate chromatography from tissue culture supernatants of 293T cells
stably expressing the recombinant vector. The
purified Tag5109P1D4 protein is then used as immunogen.
During the immunization protocol, it is useful to mix or emulsify the antigen
in adjuvants that enhance the immune
response of the host animal. Examples of adjuvants include, but are not
limited to, complete Freund's adjuvant (CFA) and
MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
In a typical protocol, rabbits are initially immunized subcutaneously with up
to 200 g, typically 100-200 g, of
fusion protein or peptide conjugated to KLH mixed in complete Freund's
adjuvant (CFA). Rabbits are then injected
subcutaneously every two weeks with up to 200 g, typically 100-200 g, of the
immunogen in incomplete Freund's adjuvant
(IFA). Test bleeds are taken approximately 7-10 days following each
immunization and used to monitor the titer of the
antiserum by ELISA.
To test reactivity and specificity of immune serum, such as the rabbit serum
derived from immunization with the
NUSa-fusion of 109P1 D4 variant 1 protein, the full-length 109P1 D4 variant I
cDNA is cloned into pCDNA 3.1 myc-his
expression vector (Invitrogen, see the Example entitled "Production of
Recombinant 109P1 D4 in Eukaryotic Systems"). After
transfection of the constructs into 293T cells, cell lysates are probed with
the anti-109P1 D4 serum to determine specific
reactivity to denatured 109P1 D4 protein using the Western blot technique.
Probing with anti-His antibody serves as a
positive control for expression of 109P1D4 in the transfected cells (See
Figure 21). In addition, the immune serum is tested
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by fluorescence microscopy, flow cytometry and immunoprecipitation against
293T and other recombinant 109P1 D4-
expressing cells to determine specific recognition of native protein. Western
blot, immunoprecipitation, fluorescent
microscopy, and flow cytometric techniques using cells that endogenously
express 109P1 D4 are also carried out to test
reactivity and specificity.
Anti-serum from rabbits immunized with 109P1 D4 variant fusion proteins, such
as GST and MBP fusion proteins,
are purified by depletion of antibodies reactive to the fusion partner
sequence by passage over an affinity column containing
the fusion partner either alone or in the context of an irrelevant fusion
protein. For example, antiserum derived from a NUSa-
109P1 D4 variant 1 fusion protein is first purified by passage over a column
of MBP protein covalently coupled to AffiGel
matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by
passage over a column composed of a NUSa-
109P1 D4 fusion protein covalently coupled to Affigel matrix. The serum is
then further purified by protein G affinity
chromatography to isolate the IgG fraction, Sera from other His-tagged
antigens and peptide immunized rabbits as well as
fusion partner depleted sera are affinity purified by passage over a column
matrix composed of the original protein
immunogen or free peptide.
Example 11: Generation of 109P1D4 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 109P1 D4 variants comprise those that
react with epitopes specific for
each variant protein or specific to sequences in common between the variants
that would disrupt or modulate the biological
function of the 109P1 D4 variants, for example those that would disrupt the
interaction with ligands and binding partners.
Immunogens for generation of such mAbs include those designed to encode or
contain the entire 109P1 D4 protein variant
sequence, regions predicted to contain functional motifs, and regions of the
109P1D4 protein variants predicted to be
antigenic from computer analysis of the amino acid sequence (see, e.g., Figure
5, Figure 6, Figure 7, Figure 8, or Figure 9,
and the Example entitled "Antigenicity Profiles and Secondary Structure").
Immunogens include peptides, recombinant
bacterial proteins, and mammalian expressed Tag 5 proteins and human and
murine IgG FC fusion proteins. In addition,
cells engineered to express high levels of a respective 109P1 D4 variant, such
as 293T-109P1 D4 variant I or 300.19-
109P1 D4 variant 1 murine Pre-B cells, are used to immunize mice.
To generate mAbs to a 109P1 D4 variant, mice are first immunized
intraperitoneally (IP) with, typically, 10-50 g of
protein immunogen or 107 109P1 D4-expressing cells mixed in complete Freund's
adjuvant. Mice are then subsequently
immunized IP every 2-4 weeks with, typically, 10-50 g of protein immunogen or
107 cells mixed in incomplete Freund's
adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In
addition to the above protein and cell-based
immunization strategies, a DNA-based immunization protocol is employed in
which a mammalian expression vector
encoding a 109P1 D4 variant sequence is used to immunize mice by direct
injection of the plasmid DNA. For example,
amino acids 24-812 of 109P1 D4 of variant I is cloned into the Tags mammalian
secretion vector and the recombinant vector
will then be used as immunogen. In another example the same amino acids are
cloned into an Fc-fusion secretion vector in
which the 109P1 D4 variant I sequence is fused at the amino-terminus to an IgK
leader sequence and at the carboxyl-
terminus to the coding sequence of the human or murine IgG Fc region. This
recombinant vector is then used as
immunogen. The plasmid immunization protocols are used in combination with
purified proteins expressed from the same
vector and with cells expressing the respective 109P1D4 variant.
Alternatively, mice may be immunized directly into their footpads. In this
case, 10-50 pg of protein immunogen or
107 254P1 D613-expressing cells are injected sub-cutaneously into the footpad
of each hind leg. The first immunization is
given with Titermax (SigmaTM) as an adjuvant and subsequent injections are
given with Alum-gel in conjunction with CpG
oligonucleotide sequences with the exception of the final injection which is
given with PBS. Injections are given twice weekly
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(every three to four days) for a period of 4 weeks and mice are sacrificed 3-4
days after the final injection, at which point
lymph nodes immediately draining from the footpad are harvested and the B-
cells are collected for use as antibody
producing fusion partners.
During the immunization protocol, test bleeds are taken 7-10 days following an
injection to monitor titer and
specificity of the immune response. Once appropriate reactivity and
specificity is obtained as determined by ELISA, Western
blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric
analyses, fusion and hybridoma generation is
then carried out with established procedures well known in the art (see, e.g.,
Harlow and Lane, 1988).
In one embodiment for generating 109P1 D4 monoclonal antibodies, a Tag5
antigen of variant 1 encoding amino
acids 14-812 is expressed in 293T cells and purified from conditioned media.
Balb C mice are initially immunized
intraperitoneally with 25 lag of the Tag5109P1 D4 variant 1 protein mixed in
complete Freund's adjuvant. Mice are
subsequently immunized every two weeks with 25 lag of the antigen mixed in
incomplete Freund's adjuvant for a total of
three immunizations. ELISA using the Tag5 antigen determines the titer of
serum from immunized mice. Reactivity and
specificity of serum to full length 109P1 D4 variant I protein is monitored by
Western blotting, immunoprecipitation and flow
cytometry using 293T cells transfected with an expression vector encoding the
109P1 D4 variant 1 cDNA (see e.g., the
Example entitled "Production of Recombinant 109P1D4 in Higher Eukaryotic
Systems" and Figure 21). Other recombinant
109P1 D4 variant 1-expressing cells or cells endogenously expressing 109P1 D4
variant I are also used. Mice showing the
strongest reactivity are rested and given a final injection of antigen in PBS
and then sacrificed four days later. The spleens
of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using
standard procedures (Harlow and Lane, 1988).
Supernatants from HAT selected growth wells are screened by ELISA, Western
blot, immunoprecipitation, fluorescent
microscopy, and flow cytometry to identify I 09P1 D4 specific antibody-
producing clones. .
To generate monoclonal antibodies that are specific for a 109P1 D4 variant
protein, immunogens are designed to
encode sequences unique for each variant. In one embodiment, an antigenic
peptide composed of amino acids 1-29 of
109P1 D4 variant 2 is coupled to KLH to derive monoclonal antibodies specific
to 109P1 D4 variant 2. In another
embodiment, an antigenic peptide comprised of amino acids 1-23 of 109P1 D4
variant 6 is coupled to KLH and used as
immunogen to derive varaiant6 specific MAbs. In another example, a GST-fusion
protein encoding amino acids 1001-1347
of variant 3 is used as immunogen to generate antibodies that would recognize
variants 3, 4, 5, and 8, and distinguish them
from variants 1, 2, 6, 7and 9. Hybridoma supernatants are then screened on the
respective antigen and then further
screened on cells expressing the specific variant and cross-screened on cells
expressing the other variants to derive variant-
specific monoclonal antibodies.
The binding affinity of 109P1 D4 variant specific monoclonal antibodies are
determined using standard
technologies. Affinity measurements quantify the strength of antibody to
epitope binding and are used to help define which
109P1 D4 variant monoclonal antibodies preferred for diagnostic or therapeutic
use, as appreciated by one of skill in the art.
The BlAcore system (Uppsala, Sweden) is a preferred method for determining
binding affinity. The BlAcore system uses
surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1;
Morton and Myszka, 1998, Methods in
Enzymology 295: 268) to monitor biomolecular interactions in real time.
BlAcore analysis conveniently generates
association rate constants, dissociation rate constants, equilibrium
dissociation constants, and affinity constants.
Alternatively, equilibrium binding analysis of MAbs on 109P1 D4-expressing
cells can be used to determine affinity.
Example 12: HLA Class I and Class II Binding says
HLA class I and class II binding assays using purified HLA molecules are
performed in accordance with disclosed
protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al.,
Current Protocols in Immunology 18.3.1
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(1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol.
Immunol. 31:813 (1994)). Briefly, purified MHC
molecules (5 to 500 nM) are incubated with various unlabeled peptide
inhibitors and 1-10 nM 1251-radiolabeled probe
peptides as described. Following incubation, MHC-peptide complexes are
separated from free peptide by gel filtration and
the fraction of peptide bound is determined. Typically, in preliminary
experiments, each MHC preparation is titered in the
presence of fixed amounts of radiolabeled peptides to determine the
concentration of HLA molecules necessary to bind 10-
20% of the total radioactivity. All subsequent inhibition and direct binding
assays are performed using these HLA
concentrations.
Since under these conditions [label]<[HLA] and IC5o4HLA], the measured IC50
values are reasonable
approximations of the true KD values. Peptide inhibitors are typically tested
at concentrations ranging from 120 glml to 1.2
ng/ml, and are tested in two to four completely independent experiments. To
allow comparison of the data obtained in
different experiments, a relative binding figure is calculated for each
peptide by dividing the IC5o of a positive control for
inhibition by the IC5o for each tested peptide (typically unlabeled versions
of the radiolabeled probe peptide). For database
purposes, and inter-experiment comparisons, relative binding values are
compiled. These values can subsequently be
converted back into IC5o nM values by dividing the IC5o nM of the positive
controls for inhibition by the relative binding of the
peptide of interest. This method of data compilation is accurate and
consistent for comparing peptides that have been tested
on different days, or with different lots of purified MHC.
Binding assays as outlined above may be used to analyze HLA supermotif and/or
HLA motif-bearing peptides (see
Table IV).
Example 13: Identification of HLA Supermotif- and Motif-Bearing CTL Candidate
Epitopes
HLA vaccine compositions of the invention can include multiple epitopes. The
multiple epitopes can comprise
multiple HLA supermotifs or motifs to achieve broad population coverage. This
example illustrates the identification and
confirmation of supermotif- and motif-bearing epitopes for the inclusion in
such a vaccine composition. Calculation of
population coverage is performed using the strategy described below.
Computer searches and algorithms for identification of supermotif and/or motif-
bearing epitopes
The searches performed to identify the motif-bearing peptide sequences in the
Example entitled "Antigenicity
Profiles" and Tables VIII-XXI and XXII-XLIX employ the protein sequence data
from the gene product of 109P1 D4 set forth in
Figures 2 and 3, the specific search peptides used to generate the tables are
listed in Table VII.
Computer searches for epitopes bearing HLA Class I or Class 11 supermotifs or
motifs are performed as follows.
All translated 109P1 D4 protein sequences are analyzed using a text string
search software program to identify potential
peptide sequences containing appropriate HLA binding motifs; such programs are
readily produced in accordance with
information in the art in view of known motif/supermotif disclosures.
Furthermore, such calculations can be made mentally.
Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial
algorithms to predict their capacity
to bind to specific HLA-Class I or Class II molecules. These polynomial
algorithms account for the impact of different amino
acids at different positions, and are essentially based on the premise that
the overall affinity (or AG) of peptide-HLA molecule
interactions can be approximated as a linear polynomial function of the type:
"AG"=a1;xa21xa3;...... xan;
where a;; is a coefficient which represents the effect of the presence of a
given amino acid (I) at a given position (i)
along the sequence of a peptide of n amino acids. The crucial assumption of
this method is that the effects at each position
are essentially independent of each other (i.e., independent binding of
individual side-chains). When residue j occurs at
position i in the peptide, it is assumed to contribute a constant amount j; to
the free energy of binding of the peptide
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irrespective of the sequence of the rest of the peptide.
The method of derivation of specific algorithm coefficients has been described
in Gulukota at al., J. Mol. Biol.
267:1258-126, 1997; (see also Sidney at al., Human Immunol. 45:79-93, 1996;
and Southwood et al., J. ImmunoL 160:3363-
3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the
geometric mean of the average relative binding
(ARB) of all peptides carrying j is calculated relative to the remainder of
the group, and used as the estimate of j;. For Class
II peptides, if multiple alignments are possible, only the highest scoring
alignment is utilized, following an iterative procedure.
To calculate an algorithm score of a given peptide in a test set, the ARB
values corresponding to the sequence of the peptide
are multiplied. If this product exceeds a chosen threshold, the peptide is
predicted to bind. Appropriate thresholds are
chosen as a function of the degree of stringency of prediction desired.
Selection of HLA-A2 supertype cross-reactive peptides
Protein sequences from 109P1D4 are scanned utilizing motif identification
software, to identify 8-, 9- 10- and 11-
mer sequences containing the HLA-A2-supermotif main anchor specificity.
Typically, these sequences are then scored using
the protocol described above and the peptides corresponding to the positive-
scoring sequences are synthesized and tested
for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201
is considered a prototype A2 supertype
molecule).
These peptides are then tested for the capacity to bind to additional A2-
supertype molecules (A*0202, A*0203,
A*0206, and A*6802). Peptides that bind to at least three of the five A2-
supertype alleles tested are typically deemed A2-
supertype cross-reactive binders. Preferred peptides bind at an affinity equal
to or less than 500 nM to three or more HLA-
A2 supertype molecules.
Selection of HLA-A3 supermotif-bearing epitopes
The 109P1 D4 protein sequence(s) scanned above is also examined for the
presence of peptides with the HLA-A3-
supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-
bearing sequences are then synthesized and
tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules
encoded by the two most prevalent A3-
supertype alleles. The peptides that bind at least one of the two alleles with
binding affinities of <_500 nM, often <_ 200 nM,
are then tested for binding cross-reactivity to the other common A3-supertype
alleles (e.g., A*3101, A*3301, and A*6801) to
identify those that can bind at least three of the five HLA-A3-supertype
molecules tested.
Selection of HLA-B7 supermotif bearing epitopes
The 109P1 D4 protein(s) scanned above is also analyzed for the presence of 8-,
9- 10-, or 11-mer peptides with the
HLA-B7-supermotif. Corresponding peptides are synthesized and tested for
binding to HLA-B*0702, the molecule encoded
by the most common B7-supertype allele (i.e., the prototype B7 supertype
allele). Peptides binding B*0702 with IC5o of <_500
nM are identified using standard methods. These peptides are then tested for
binding to other common B7-supertype
molecules (e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of
binding to three or more of the five B7-
supertype alleles tested are thereby identified.
Selection of Al and A24 motif-bearing epitopes
To further increase population coverage, HLA-A1 and -A24 epitopes can also be
incorporated into vaccine
compositions. An analysis of the 109P1D4 protein can also be performed to
identify HLA-A1- and A24-motif-containing
sequences.
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High affinity and/or cross-reactive binding epitopes that bear other motif
and/or supermotifs are identified using
analogous methodology.
Example 14: Confirmation of Immunogenicity
Cross-reactive candidate CTL A2-supermotif-bearing peptides that are
identified as described herein are selected
to confirm in vitro immunogenicity. Confirmation is performed using the
following methodology:
Target Cell Lines for Cellular Screening:
The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the
HLA-A, -B, -C null mutant human B-
lymphoblastoid cell line 721.221, is used as the peptide-loaded target to
measure activity of HLA-A2.1-restricted CTL. This
cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium
pyruvate, nonessential amino acids and 10%
(vlv) heat inactivated FCS. Cells that express an antigen of interest, or
transfectants comprising the gene encoding the
antigen of interest, can be used as target cells to confirm the ability of
peptide-specific CTLs to recognize endogenous
antigen.
Primary CTL Induction Cultures:
Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 g/ml
DNAse, washed twice and
resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-
essential amino acids, sodium pyruvate, L-
glutamine and penicillin/streptomycin). The monocytes are purified by plating
10 x 106 PBMC/well in a 6-well plate. After 2
hours at 37 C, the non-adherent cells are removed by gently shaking the plates
and aspirating the supernatants. The wells
are washed a total of three times with 3 ml RPMI to remove most of the non-
adherent and loosely adherent cells. Three ml of
complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then
added to each well. TNFu is added to
the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures
on day 7.
Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive
selection with Dynal immunomagnetic
beads (Dynabeads M-450) and the detacha-bead reagent. Typically about 200-
250x106 PBMC are processed to obtain
24x106 CD8} T-cells (enough for a 48-well plate culture). Briefly, the PBMCs
are thawed in RPMI with 30pg/ml DNAse,
washed once with PBS containing 1% human AB serum and resuspended in PBS/1 %
AB serum at a concentration of
20x106cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added
to the cells (140p1 beads/20x106 cells)
and incubated for 1 hour at 4 C with continuous mixing. The beads and cells
are washed 4x with PBS/AB serum to remove
the nonadherent cells and resuspended at 100x106 cells/ml (based on the
original cell number) in PBS/AB serum containing
100pI/ml detacha-bead reagent and 30 pg/ml DNAse. The mixture is incubated
for 1 hour at room temperature with
continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the
CD8+ T-cells. The DC are collected
and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA,
counted and pulsed with 40pg/ml of
peptide at a cell concentration of 1-2x106/ml in the presence of 3pg/ml 132-
microglobulin for 4 hours at 20 C. The DC are
then irradiated (4,200 rads), washed I time with medium and counted again.
Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1x105
cells/ml) are co-cultured with 0.25m1 of
CD8+ T-cells (at 2x106 cell/ml) in each well of a 48-well plate in the
presence of 10 ng/ml of IL-7. Recombinant human IL-10
is added the next day at a final concentration of 10 nglml and rhuman IL-2 is
added 48 hours later at 10 IU/ml.
Restimulation of the induction cultures with peptide pulsed adherent cells:
Seven and fourteen days after the
primary induction, the cells are restimulated with peptide-pulsed adherent
cells. The PBMCs are thawed and washed twice
with RPMI and DNAse. The cells are resuspended at 5x106 cellslml and
irradiated at -4200 rads. The PBMCs are plated at
2x106 in 0.5 ml complete medium per well and incubated for 2 hours at 37 C.
The plates are washed twice with RPMI by
tapping the plate gently to remove the nonadherent cells and the adherent
cells pulsed with 1Opg/ml of peptide in the
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presence of 3 pg/ml 92 microglobulin in 0.25ml RPMI/5%AB per well for 2 hours
at 37 C. Peptide solution from each well is
aspirated and the wells are washed once with RPMI. Most of the media is
aspirated from the induction cultures (CD8+ cells)
and brought to 0.5 ml with fresh media. The cells are then transferred to the
wells containing the peptide-pulsed adherent
cells. Twenty four hours later recombinant human IL-10 is added at a final
concentration of 10 ng/ml and recombinant
human IL2 is added the next day and again 2-3 days later at 501U/ml (Tsai et
al., Critical Reviews in Immunology
18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL
activity in a 51Cr release assay. In some
experiments the cultures are assayed for peptide-specific recognition in the
in situ IFNy ELISA at the time of the second
restimulation followed by assay of endogenous recognition 7 days later. After
expansion, activity is measured in both assays
for a side-by-side comparison.
Measurement of CTL lytic activity by 51Cr release.
Seven days after the second restimulation, cytotoxicity is determined in a
standard (5 hr) 51Cr release assay by
assaying individual wells at a single E:T. Peptide-pulsed targets are prepared
by incubating the cells with 10pg/ml peptide
overnight at 37 C.
Adherent target cells are removed from culture flasks with trypsin-EDTA.
Target cells are labeled with 200pCi of
51Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 37 C. Labeled
target cells are resuspended at 106 per ml and
diluted 1:10 with K562 cells at a concentration of 3.3x106/ml (an NK-sensitive
erythroblastoma cell line used to reduce non-
specific lysis). Target cells (100 pl) and effectors (100p1) are plated in 96
well round-bottom plates and incubated for 5 hours
at 37 C. At that time, 100 pl of supernatant are collected from each well and
percent lysis is determined according to the
formula:
[(cpm of the test sample- cpm of the spontaneous 51Cr release sample)/(cpm of
the maximal 51Cr release sample-
cpm of the spontaneous 51Cr release sample)] x 100.
Maximum and spontaneous release are determined by incubating the labeled
targets with 1% Triton X-100 and
media alone, respectively. A positive culture is defined as one in which the
specific lysis (sample- background) is 10% or
higher in the case of individual wells and is 15% or more at the two highest
E:T ratios when expanded cultures are assayed.
In situ Measurement of Human IFNy Production as an Indicator of Peptide-
specific and Endogenous Recognition
Immulon 2 plates are coated with mouse anti-human IFNy monoclonal antibody (4
g/ml 0.1M NaHCO3, pH8.2)
overnight at 4 C. The plates are washed with Cat+, Mg2+-free PBS/0.05% Tween
20 and blocked with PBS/10% FCS for two
hours, after which the CTLs (100 l/well) and targets (100 l/well) are added
to each well, leaving empty wells for the
standards and blanks (which received media only). The target cells, either
peptide-pulsed or endogenous targets, are used
at a concentration of 1 x106 cells/ml. The plates are incubated for 48 hours
at 37 C with 5% C02.
Recombinant human IFN-gamma is added to the standard wells starting at 400 pg
or 1200pg/100 microliter/well
and the plate incubated for two hours at 37 C. The plates are washed and 100
l of biotinylated mouse anti-human IFN-
gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are
added and incubated for 2 hours at
room temperature. After washing again, 100 microliter HRP-streptavidin
(1:4000) are added and the plates incubated for
one hour at room temperature. The plates are then washed 6x with wash buffer,
100 microliter/well developing solution
(TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The
reaction is stopped with 50 microliter/well IM
H3P04 and read at OD450. A culture is considered positive if it measured at
least 50 pg of IFN-gamma/well above
background and is twice the background level of expression.
CTL Expansion.
Those cultures that demonstrate specific lytic activity against peptide-pulsed
targets and/or tumor targets are
expanded over a two week period with anti-CD3. Briefly, 5x104 CD8+ cells are
added to a T25 flask containing the following:
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1x106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2x105
irradiated (8,000 rad) EBV- transformed cells per
ml, and OKT3 (anti-CD3) at 30ng per ml in RPMI-1640 containing 10% (v/v) human
AB serum, non-essential amino acids,
sodium pyruvate, 25pM 2-mercaptoethanol, L-glutamine and
penicillin/streptomycin. Recombinant human IL2 is added 24
hours later at a final concentration of 2001U/ml and every three days
thereafter with fresh media at 501U1ml. The cells are
split if the cell concentration exceeds 1x106/ml and the cultures are assayed
between days 13 and 15 at E:T ratios of 30, 10,
3 and 1:1 in the 51Cr release assay or at 1x106/ml in the in situ IFNy assay
using the same targets as before the expansion.
Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures
that demonstrate specific lytic
activity against peptide and endogenous targets are selected and 5x104
CD8+cells are added to a T25 flask containing the
following: 1x106 autologous PBMC per ml which have been peptide-pulsed with 10
g/ml peptide for two hours at 37 C and
irradiated (4,200 rad); 2x105 irradiated (8,000 rad) EBV-transformed cells per
ml RPMI-1640 containing 10%(v/v) human AB
serum, non-essential AA, sodium pyruvate, 25mM 2-ME, L-glutamine and
gentamicin.
Immunogenicity of A2 supermotif-bearing peptides
A2-supermotif cross-reactive binding peptides are tested in the cellular assay
for the ability to induce peptide-
specific CTL in normal individuals. In this analysis, a peptide is typically
considered to be an epitope if it induces peptide-
specific CTLs in at least individuals, and preferably, also recognizes the
endogenously expressed peptide.
Immunogenicity can also be confirmed using PBMCs isolated from patients
bearing a tumor that expresses
109P1D4. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-
pulsed monocytes and assayed for the
ability to recognize peptide-pulsed target cells as well as transfected cells
endogenously expressing the antigen.
Evaluation of A*03/A1 I immunogenicity
HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated
for immunogenicity using
methodology analogous for that used to evaluate the immunogenicity of the HLA-
A2 supermotif peptides.
Evaluation of B7 immunogenicity
Immunogenicity screening of the B7-supertype cross-reactive binding peptides
identified as set forth herein are
confirmed in a manner analogous to the confirmation of A2-and A3-supermotif-
bearing peptides.
Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc. are also
confirmed using similar
methodology
Example 15: Implementation of the Extended Supermotif to Improve the Binding
Capacity of Native Epitopes
Creating Analogs
HLA motifs and supermotifs (comprising primary and/or secondary residues) are
useful in the identification and
preparation of highly cross-reactive native peptides, as demonstrated herein.
Moreover, the definition of HLA motifs and
supermotifs also allows one to engineer highly cross-reactive epitopes by
identifying residues within a native peptide
sequence which can be analoged to confer upon the peptide certain
characteristics, e.g. greater cross-reactivity within the
group of HLA molecules that comprise a supertype, and/or greater binding
affinity for some or all of those HLA molecules.
Examples of analoging peptides to exhibit modulated binding affinity are set
forth in this example.
Analoging at Primary Anchor Residues
Peptide engineering strategies are implemented to further increase the cross-
reactivity of the epitopes. For
example, the main anchors of A2-supermotif-bearing peptides are altered, for
example, to introduce a preferred L, 1, V, or M
at position 2, and I or V at the C-terminus.
To analyze the cross-reactivity of the analog peptides, each engineered analog
is initially tested for binding to the
prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is
maintained, for A2-supertype cross-reactivity.
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Alternatively, a peptide is confirmed as binding one or all supertype members
and then analoged to modulate
binding affinity to any one (or more) of the supertype members to add
population coverage.
The selection of analogs for immunogenicity in a cellular screening analysis
is typically further restricted by the
capacity of the parent wild type (WT) peptide to bind at least weakly, i.e.,
bind at an IC5o of 5000nM or less, to three of more
A2 supertype alleles. The rationale for this requirement is that the WT
peptides must be present endogenously in sufficient
quantity to be biologically relevant. Analoged peptides have been shown to
have increased immunogenicity and cross-
reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst et
al., J. Immunol. 157:2539, 1996; and Pogue of al.,
Proc. Nafl. Acad. Sci. USA 92:8166, 1995).
In the cellular screening of these peptide analogs, it is important to confirm
that analog-specific CTLs are also able
to recognize the wild-type peptide and, when possible, target cells that
endogenously express the epitope.
Analoging of HLA-A3 and B7-supermotif-bearing peptides
Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies
similar to those employed in
analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to
3/5 of the A3-supertype molecules are
engineered at primary anchor residues to possess a preferred residue (V, S, M,
or A) at position 2.
The analog peptides are then tested for the ability to bind A*03 and A*11
(prototype A3 supertype alleles). Those
peptides that demonstrate < 500 nM binding capacity are then confirmed as
having A3-supertype cross-reactivity.
Similarly to the A2- and A3- motif bearing peptides, peptides binding 3 or
more 67-supertype alleles can be
improved, where possible, to achieve increased cross-reactive binding or
greater binding affinity or binding half life. B7
supermotif-bearing peptides are, for example, engineered to possess a
preferred residue (V, I, L, or F) at the C-terminal
primary anchor position, as demonstrated by Sidney eta!. (J. Immunol. 157:3480-
3490, 1996).
Analoging at primary anchor residues of other motif and/or supermotif-bearing
epitopes is performed in a like
manner.
The analog peptides are then be confirmed for immunogenicity, typically in a
cellular screening assay. Again, it is
generally important to demonstrate that analog-specific CTLs are also able to
recognize the wild-type peptide and, when
possible, targets that endogenously express the epitope.
Analoging at Secondary Anchor Residues
Moreover, HLA supermotifs are of value in engineering highly cross-reactive
peptides and/or peptides that bind
HLA molecules with increased affinity by identifying particular residues at
secondary anchor positions that are associated
with such properties. For example, the binding capacity of a B7 supermotif-
bearing peptide with an F residue at position I is
analyzed. The peptide is then analoged to, for example, substitute L for F at
position 1. The analoged peptide is evaluated
for increased binding affinity, binding half life and/or increased cross-
reactivity. Such a procedure identifies analoged
peptides with enhanced properties.
Engineered analogs with sufficiently improved binding capacity or cross-
reactivity can also be tested for
immunogenicity in HLA-B7-transgenic mice, following for example, IFA
immunization or lipopeptide immunization. Analoged
peptides are additionally tested for the ability to stimulate a recall
response using PBMC from patients with 109P1 D4-
expressing tumors.
Other analoging strategies
Another form of peptide analoging, unrelated to anchor positions, involves the
substitution of a cysteine with C-
amino butyric acid. Due to its chemical nature, cysteine has the propensity to
form disulfide bridges and sufficiently alter the
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peptide structurally so as to reduce binding capacity. Substitution of a-amino
butyric acid for cysteine not only alleviates this
problem, but has been shown to improve binding and crossbinding capabilities
in some instances (see, e.g., the review by
Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John
Wiley & Sons, England, 1999).
Thus, by the use of single amino acid substitutions, the binding properties
and/or cross-reactivity of peptide ligands
for HLA supertype molecules can be modulated.
Example 16: Identification and confirmation of 109P1D4-derived sequences with
HLA-DR binding motifs
Peptide epitopes bearing an HLA class II supermotif or motif are identified
and confirmed as outlined below using
methodology similar to that described for HLA Class I peptides.
Selection of HLA-DR-supermotif bearing epitopes.
To identify 109PI D4-derived, HLA class II HTL epitopes, a 109P1 D4 antigen is
analyzed for the presence of
sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer
sequences are selected comprising a DR-
supermotif, comprising a 9-mer core, and three-residue N- and C-terminal
flanking regions (15 amino acids total).
Protocols for predicting peptide binding to DR molecules have been developed
(Southwood et al., J. Immunol.
160:3363-3373, 1998). These protocols, specific for individual DR molecules,
allow the scoring, and ranking, of 9-mer core
regions. Each protocol not only scores peptide sequences for the presence of
DR-supermotif primary anchors (i.e., at
position 1 and position 6) within a 9-mer core, but additionally evaluates
sequences for the presence of secondary anchors.
Using allele-specific selection tables (see, e.g., Southwood et al., ibid.),
it has been found that these protocols efficiently
select peptide sequences with a high probability of binding a particular DR
molecule. Additionally, it has been found that
performing these protocols in tandem, specifically those for DRI, DR4w4, and
DR7, can efficiently select DR cross-reactive
peptides.
The 109P1 D4-derived peptides identified above are tested for their binding
capacity for various common HLA-DR
molecules. All peptides are initially tested for binding to the DR molecules
in the primary panel: DR1, DR4w4, and DR7.
Peptides binding at least two of these three DR molecules are then tested for
binding to DR2w2 (31, DR2w2 (32, DR6w19,
and DR9 molecules in secondary assays. Finally, peptides binding at least two
of the four secondary panel DR molecules,
and thus cumulatively at least four of seven different DR molecules, are
screened for binding to DR4w1 5, DR5w11, and
DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten
DR molecules comprising the primary,
secondary, and tertiary screening assays are considered cross-reactive DR
binders. 109P1 D4-derived peptides found to
bind common HLA-DR alleles are of particular interest.
Selection of DR3 motif peptides
Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and
Hispanic populations, DR3 binding
capacity is a relevant criterion in the selection of HTL epitopes. Thus,
peptides shown to be candidates may also be
assayed for their DR3 binding capacity. However, in view of the binding
specificity of the DR3 motif, peptides binding only to
DR3 can also be considered as candidates for inclusion in a vaccine
formulation.
To efficiently identify peptides that bind DR3, target 109P1 D4 antigens are
analyzed for sequences carrying one of
the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol.
152:5742-5748, 1994). The corresponding
peptides are then synthesized and confirmed as having the ability to bind DR3
with an affinity of 1 M or better, i.e., less than
I M. Peptides are found that meet this binding criterion and qualify as HLA
class II high affinity binders.
DR3 binding epitopes identified in this manner are included in vaccine
compositions with DR supermotif-bearing
peptide epitopes.
Similarly to the case of HLA class I motif-bearing peptides, the class 11
motif-bearing peptides are analoged to
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improve affinity or cross-reactivity. For example, aspartic acid at position 4
of the 9-mer core sequence is an optimal residue
for DR3 binding, and substitution for that residue often improves DR 3
binding.
Example 17: Immunogenicity of 109P1 D4-derived HTL epitopes
This example determines immunogenic DR supermotif- and DR3 motif-bearing
epitopes among those identified
using the methodology set forth herein.
Immunogenicity of HTL epitopes are confirmed in a manner analogous to the
determination of immunogenicity of
CTL epitopes, by assessing the ability to stimulate HTL responses and/or by
using appropriate transgenic mouse models.
Immunogenicity is determined by screening for: 1.) in vitro primary induction
using normal PBMC or 2.) recall responses from
patients who have 109PlD4-expressing tumors.
Example 18: Calculation of phenotypic frequencies of HLA-supertypes in various
ethnic backgrounds to determine
breadth of population coverage
This example illustrates the assessment of the breadth of population coverage
of a vaccine composition comprised
of multiple epitopes comprising multiple supermotifs and/or motifs.
In order to analyze population coverage, gene frequencies of HLA alleles are
determined. Gene frequencies for
each HLA allele are calculated from antigen or allele frequencies utilizing
the binomial distribution formulae gf=l-(SQRT(l-
af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain
overall phenotypic frequencies, cumulative gene
frequencies are calculated, and the cumulative antigen frequencies derived by
the use of the inverse formula [af=l-(l-Cgf)2].
Where frequency data is not available at the level of DNA typing,
correspondence to the serologically defined
antigen frequencies is assumed. To obtain total potential supertype population
coverage no linkage disequilibrium is
assumed, and only alleles confirmed to belong to each of the supertypes are
included (minimal estimates). Estimates of total
potential coverage achieved by inter-loci combinations are made by adding to
the A coverage the proportion of the non-A
covered population that could be expected to be covered by the B alleles
considered (e.g., total=A+B*(l-A)). Confirmed
members of the A3-like supertype are A3, All, A31, A*3301, and A*6801.
Although the A3-like supertype may also include
A34, A66, and A*7401, these alleles were not included in overall frequency
calculations. Likewise, confirmed members of
the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205,
A*0206, A*0207, A*6802, and A*6901. Finally,
the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301,
B*5401, B*5501-2, B*5601, B*6701, and B*7801
(potentially also B*1401, B*3504-06, B*4201, and B*5602).
Population coverage achieved by combining the A2-, A3- and B7-supertypes is
approximately 86% in five major
ethnic groups. Coverage may be extended by including peptides bearing the Al
and A24 motifs. On average, Al is present
in 12% and A24 in 29% of the population across five different major ethnic
groups (Caucasian, North American Black,
Chinese, Japanese, and Hispanic). Together, these alleles are represented with
an average frequency of 39% in these
same ethnic populations. The total coverage across the major ethnicities when
Al and A24 are combined with the coverage
of the A2-, A3- and B7-supertype alleles is >95%, see, e.g., Table IV (G). An
analogous approach can be used to estimate
population coverage achieved with combinations of class II motif-bearing
epitopes.
Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.
100:503, 1997; Doolan et al., Immunity 7:97,
1997; and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that highly
cross-reactive binding peptides are almost
always recognized as epitopes. The use of highly cross-reactive binding
peptides is an important selection criterion in
identifying candidate epitopes for inclusion in a vaccine that is immunogenic
in a diverse population.
With a sufficient number of epitopes (as disclosed herein and from the art),
an average population coverage is
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predicted to be greater than 95% in each of five major ethnic populations. The
game theory Monte Carlo simulation analysis,
which is known in the art (see e.g., Osborne, M.J. and Rubinstein, A. "A
course in game theory" MIT Press, 1994), can be
used to estimate what percentage of the individuals in a population comprised
of the Caucasian, North American Black,
Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine
epitopes described herein. A preferred
percentage is 90%. A more preferred percentage is 95%.
Example 19: CTL Recognition Of Endogenously Processed Antigens After Priming
This example confirms that CTL induced by native or analoged peptide epitopes
identified and selected as
described herein recognize endogenously synthesized, i.e., native antigens.
Effector cells isolated from transgenic mice that are immunized with peptide
epitopes, for example HLA-A2
supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated
stimulator cells. Six days later, effector cells are
assayed for cytotoxicity and the cell lines that contain peptide-specific
cytotoxic activity are further re-stimulated. An
additional six days later, these cell lines are tested for cytotoxic activity
on 51Cr labeled Jurkat-A2.1/Kb target cells in the
absence or presence of peptide, and also tested on 51Cr labeled target cells
bearing the endogenously synthesized antigen,
i.e. cells that are stably transfected with 109P1 D4 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with
peptide epitope recognize
endogenously synthesized 109P1 D4 antigen. The choice of transgenic mouse
model to be used for such an analysis
depends upon the epitope(s) that are being evaluated. In addition to HLA-
A*0201/Kb transgenic mice, several other
transgenic mouse models including mice with human All, which may also be used
to evaluate A3 epitopes, and B7 alleles
have been characterized and others (e.g., transgenic mice for HLA-Al and A24)
are being developed. HLA-DR1 and HLA-
DR3 mouse models have also been developed, which may be used to evaluate HTL
epitopes.
Example 20: Activity Of CTL-HTL Conjugated Epitopes In Transgenic Mice
This example illustrates the induction of CTLs and HTLs in transgenic mice, by
use of a 109P1 D4-derived CTL and
HTL peptide vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient
with a 109P1 D4-expressing tumor. The peptide composition can comprise
multiple CTL and/or HTL epitopes. The epitopes
are identified using methodology as described herein. This example also
illustrates that enhanced immunogenicity can be
achieved by inclusion of one or more HTL epitopes in a CTL vaccine
composition; such a peptide composition can comprise
an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that
binds to multiple HLA family members at an
affinity of 500 nM or less, or analogs of that epitope. The peptides may be
lipidated, if desired.
Immunization procedures: Immunization of transgenic mice is performed as
described (Alexander et al., J.
Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic
for the human HLA A2.1 allele and are
used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-
bearing epitopes, and are primed
subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete
Freund's Adjuvant, or if the peptide composition is a
lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is
a polypeptide, in PBS or Incomplete Freund's
Adjuvant. Seven days after priming, splenocytes obtained from these animals
are restimulated with syngenic irradiated LPS-
activated lymphoblasts coated with peptide.
Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat
cells transfected with the HLA-A2.1/Kb
chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)
In vitro CTL activation: One week after priming, spleen cells (30x106
cells/flask) are co-cultured at 37 C with
syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10x106
cells/flask) in 10 ml of culture medium/T25 flask.
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After six days, effector cells are harvested and assayed for cytotoxic
activity.
Assay for cytotoxic activity: Target cells (1.0 to 1.5x106) are incubated at
37 C in the presence of 200 pl of 51Cr.
After 60 minutes, cells are washed three times and resuspended in R10 medium.
Peptide is added where required at a
concentration of I pg/ml. For the assay, 104 51Cr-labeled target cells are
added to different concentrations of effector cells
(final volume of 200 pl) in U-bottom 96-well plates. After a six hour
incubation period at 37 C, a 0.1 ml aliquot of
supernatant is removed from each well and radioactivity is determined in a
Micromedic automatic gamma counter. The
percent specific lysis is determined by the formula: percent specific release
= 100 x (experimental release - spontaneous
release)/(maximum release - spontaneous release). To facilitate comparison
between separate CTL assays run under the
same conditions, % 51Cr release data is expressed as lytic units/106 cells.
One lytic unit is arbitrarily defined as the number
of effector cells required to achieve 30% lysis of 10,000 target cells in a
six hour 51Cr release assay. To obtain specific lytic
units/106, the lytic units/106 obtained in the absence of peptide is
subtracted from the lytic units/106 obtained in the presence
of peptide. For example, if 30% 51Cr release is obtained at the effector (E):
target (T) ratio of 50:1 (i.e., 5x105 effector cells
for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5x104 effector
cells for 10,000 targets) in the presence of peptide,
the specific lytic units would be: [(1/50,000)-(1/500,000)] x 106 =18 LU.
The results are analyzed to assess the magnitude of the CTL responses of
animals injected with the immunogenic
CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the
CTL response achieved using, for
example, CTL epitopes as outlined above in the Example entitled "Confirmation
of Immunogenicity." Analyses similar to this
may be performed to confirm the immunogenicity of peptide conjugates
containing multiple CTL epitopes and/or multiple HTL
epitopes. In accordance with these procedures, it is found that a CTL response
is induced, and concomitantly that an HTL
response is induced upon administration of such compositions.
Example 21: Selection of CTL and HTL epitopes for inclusion in a 109P1134-
specific vaccine.
This example illustrates a procedure for selecting peptide epitopes for
vaccine compositions of the invention. The
peptides in the composition can be in the form of a nucleic acid sequence,
either single or one or more sequences (i.e.,
minigene) that encodes peptide(s), or can be single and/or polyepitopic
peptides.
The following principles are utilized when selecting a plurality of epitopes
for inclusion in a vaccine composition.
Each of the following principles is balanced in order to make the selection.
Epitopes are selected which, upon administration, mimic immune responses that
are correlated with 109P1 D4
clearance. The number of epitopes used depends on observations of patients who
spontaneously clear 109P1 D4. For
example, if it has been observed that patients who spontaneously clear 109P1
D4-expressing cells generate an immune
response to at least three (3) epitopes from 109P1 D4 antigen, then at least
three epitopes should be included for HLA class
1. A similar rationale is used to determine HLA class II epitopes.
Epitopes are often selected that have a binding affinity of an IC5o of 500 nM
or less for an HLA class I molecule, or
for class II, an IC5o of 1000 nM or less; or HLA Class I peptides with high
binding scores from the BIMAS web site, at URL
bimas.dcrt.nih,govl.
In order to achieve broad coverage of the vaccine through out a diverse
population, sufficient supermotif bearing
peptides, or a sufficient array of allele-specific motif bearing peptides, are
selected to give broad population coverage. In
one embodiment, epitopes are selected to provide at least 80% population
coverage. A Monte Carlo analysis, a statistical
evaluation known in the art, can be employed to assess breadth, or redundancy,
of population coverage.
When creating polyepitopic compositions, or a minigene that encodes same, it
is typically desirable to generate the
smallest peptide possible that encompasses the epitopes of interest. The
principles employed are similar, if not the same, as
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those employed when selecting a peptide comprising nested epitopes. For
example, a protein sequence for the vaccine
composition is selected because it has maximal number of epitopes contained
within the sequence, i.e., it has a high
concentration of epitopes. Epitopes may be nested or overlapping (i.e., frame
shifted relative to one another). For example,
with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be
present in a 10 amino acid peptide. Each
epitope can be exposed and bound by an HLA molecule upon administration of
such a peptide. A multi-epitopic, peptide can
be generated synthetically, recombinantly, or via cleavage from the native
source. Alternatively, an analog can be made of
this native sequence, whereby one or more of the epitopes comprise
substitutions that alter the cross-reactivity and/or
binding affinity properties of the polyepitopic peptide. Such a vaccine
composition is administered for therapeutic or
prophylactic purposes. This embodiment provides for the possibility that an as
yet undiscovered aspect of immune system
processing will apply to the native nested sequence and thereby facilitate the
production of therapeutic or prophylactic
immune response-inducing vaccine compositions. Additionally such an embodiment
provides for the possibility of motif-
bearing epitopes for an HLA makeup that is presently unknown. Furthermore,
this embodiment (absent the creating of any
analogs) directs the immune response to multiple peptide sequences that are
actually present in 109P1 D4, thus avoiding the
need to evaluate any junctional epitopes. Lastly, the embodiment provides an
economy of scale when producing nucleic
acid vaccine compositions. Related to this embodiment, computer programs can
be derived in accordance with principles in
the art, which identify in a target sequence, the greatest number of epitopes
per sequence length.
A vaccine composition comprised of selected peptides, when administered, is
safe, efficacious, and elicits an
immune response similar in magnitude to an immune response that controls or
clears cells that bear or overexpress
109P1 D4.
Example 22: Construction of "Minigene" Multi-Epitope DNA Plasmids
This example discusses the construction of a minigene expression plasmid.
Minigene plasmids may, of course,
contain various configurations of B cell, CTL and/or HTL epitopes or epitope
analogs as described herein.
A minigene expression plasmid typically includes multiple CTL and HTL peptide
epitopes. In the present example,
HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-
bearing peptide epitopes are used in
conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class
I supermotif or motif-bearing peptide
epitopes derived 109P1 D4, are selected such that multiple supermotifs/motifs
are represented to ensure broad population
coverage. Similarly, HLA class II epitopes are selected from 109P1 D4 to
provide broad population coverage, i.e. both HLA
DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are
selected for inclusion in the minigene
construct. The selected CTL and HTL epitopes are then incorporated into a
minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL
epitopes to the endoplasmic reticulum.
For example, the Ii protein may be fused to one or more HTL epitopes as
described in the art, wherein the CLIP sequence of
the Ii protein is removed and replaced with an HLA class II epitope sequence
so that HLA class II epitope is directed to the
endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction of a minigene-
bearing expression plasmid. Other
expression vectors that may be used for minigene compositions are available
and known to those of skill in the art.
The minigene DNA plasmid of this example contains a consensus Kozak sequence
and a consensus murine
kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes
selected in accordance with principles disclosed
herein. The sequence encodes an open reading frame fused to the Myc and His
antibody epitope tag coded for by the
pcDNA 3.1 Myc-His vector.
Overlapping oligonucleotides that can, for example, average about 70
nucleotides in length with 15 nucleotide
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overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the
selected peptide epitopes as well as
appropriate linker nucleotides, Kozak sequence, and signal sequence. The final
multiepitope minigene is assembled by
extending the overlapping oligonucleotides in three sets of reactions using
PCR. A Perkin/Elmer 9600 PCR machine is used
and a total of 30 cycles are performed using the following conditions: 95 C
for 15 sec, annealing temperature (5 below the
lowest calculated Tm of each primer pair) for 30 sec, and 72 C for I min.
For example, a minigene is prepared as follows. For a first PCR reaction, 5 g
of each of two oligonucleotides are
annealed and extended: In an example using eight oligonucleotides, i.e., four
pairs of primers, oligonucleotides 1+2, 3+4,
5+6, and 7+8 are combined in 100 I reactions containing Pfu polymerase buffer
(1x=10 mM KCL,10 mM (NH4)2SO4, 20
mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1 % Triton X-100, 100 g/ml BSA),
0.25 mM each dNTP, and 2.5 U of Pfu
polymerase. The full-length dimer products are gel-purified, and two reactions
containing the product of 1+2 and 3+4, and
the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles.
Half of the two reactions are then mixed, and
cycles of annealing and extension carried out before flanking primers are
added to amplify the full length product. The full-
length product is gel-purified and cloned into pCR-blunt (Invitrogen) and
individual clones are screened by sequencing.
Example 23: The Plasmid Construct and the Degree to Which It Induces
Immunogenicity.
The degree to which a plasmid construct, for example a plasmid constructed in
accordance with the previous
Example, is able to induce immunogenicity is confirmed in vitro by determining
epitope presentation by APC following
transduction or transfection of the APC with an epitope-expressing nucleic
acid construct. Such a study determines
"antigenicity" and allows the use of human APC. The assay determines the
ability of the epitope to be presented by the APC
in a context that is recognized by a T cell by quantifying the density of
epitope-HLA class I complexes on the cell surface.
Quantitation can be performed by directly measuring the amount of peptide
eluted from the APC (see, e.g., Sijts et al., J.
Immunol.156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the
number of peptide-HLA class I complexes
can be estimated by measuring the amount of lysis or lymphokine release
induced by diseased or transfected target cells,
and then determining the concentration of peptide necessary to obtain
equivalent levels of lysis or lymphokine release (see,
e.g., Kageyama et al., J. Immunol. 154:567-576,1995).
Alternatively, immunogenicity is confirmed through in vivo injections into
mice and subsequent in vitro assessment
of CTL and HTL activity, which are analyzed using cytotoxicity and
proliferation assays, respectively, as detailed e.g., in
Alexander et al., Immunity 1:751-761, 1994.
For example, to confirm the capacity of a DNA minigene construct containing at
least one HLA-A2 supermotif
peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for example, are
immunized intramuscularly with 100 g of
naked cDNA. As a means of comparing the level of CTLs induced by cDNA
immunization, a control group of animals is also
immunized with an actual peptide composition that comprises multiple epitopes
synthesized as a single polypeptide as they
would be encoded by the minigene.
Splenocytes from immunized animals are stimulated twice with each of the
respective compositions (peptide
epitopes encoded in the minigene or the polyepitopic peptide), then assayed
for peptide-specific cytotoxic activity in a 51Cr
release assay. The results indicate the magnitude of the CTL response directed
against the A2-restricted epitope, thus
indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic
vaccine.
It is, therefore, found that the minigene elicits immune responses directed
toward the HLA-A2 supermotif peptide
epitopes as does the polyepitopic peptide vaccine. A similar analysis is also
performed using other HLA-A3 and HLA-B7
transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or
supermotif epitopes, whereby it is also
found that the minigene elicits appropriate immune responses directed toward
the provided epitopes.
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To confirm the capacity of a class II epitope-encoding minigene to induce HTLs
in vivo, DR transgenic mice, or for
those epitopes that cross react with the appropriate mouse MHC molecule, I-Ab-
restricted mice, for example, are immunized
intramuscularly with 100 g of plasmid DNA. As a means of comparing the level
of HTLs induced by DNA immunization, a
group of control animals is also immunized with an actual peptide composition
emulsified in complete Freund's adjuvant.
CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals
and stimulated with each of the respective
compositions (peptides encoded in the minigene). The HTL response is measured
using a 3H-thymidine incorporation
proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994).
The results indicate the magnitude of the HTL
response, thus demonstrating the in vivo immunogenicity of the minigene.
DNA minigenes, constructed as described in the previous Example, can also be
confirmed as a vaccine in
combination with a boosting agent using a prime boost protocol. The boosting
agent can consist of recombinant protein
(e.g., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:5299-
S309,1998) or recombinant vaccinia, for
example, expressing a minigene or DNA encoding the complete protein of
interest (see, e.g., Hanke et al., Vaccine 16:439-
445,1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke
and McMichael, Immunol. Letters 66:177-
181, 1999; and Robinson et al., Nature Med. 5:526-34,1999).
For example, the efficacy of the DNA minigene used in a prime boost protocol
is initially evaluated in transgenic
mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 g of
a DNA minigene encoding the
immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide.
After an incubation period (ranging from 3-
9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia
virus expressing the same sequence
encoded by the DNA minigene. Control mice are immunized with 100 g of DNA or
recombinant vaccinia without the
minigene sequence, or with DNA encoding the minigene, but without the vaccinia
boost. After an additional incubation
period of two weeks, splenocytes from the mice are immediately assayed for
peptide-specific activity in an ELISPOT assay.
Additionally, splenocytes are stimulated in vitro with the A2-restricted
peptide epitopes encoded in the minigene and
recombinant vaccinia, then assayed for peptide-specific activity in an alpha,
beta and/or gamma IFN ELISA.
It is found that the minigene utilized in a prime-boost protocol elicits
greater immune responses toward the HLA-A2
supermotif peptides than with DNA alone. Such an analysis can also be
performed using HLA-Al I or HLA-B7 transgenic
mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif
epitopes. The use of prime boost
protocols in humans is described below in the Example entitled "Induction of
CTL Responses Using a Prime Boost Protocol."
Example 24: Peptide Compositions for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 109P1D4
expression in persons who are at
risk for tumors that bear this antigen. For example, a polyepitopic peptide
epitope composition (or a nucleic acid comprising
the same) containing multiple CTL and HTL epitopes such as those selected in
the above Examples, which are also selected
to target greater than 80% of the population, is administered to individuals
at risk for a 109P1 D4-associated tumor.
For example, a peptide-based composition is provided as a single polypeptide
that encompasses multiple
epitopes. The vaccine is typically administered in a physiological solution
that comprises an adjuvant, such as Incomplete
Freunds Adjuvant. The dose of peptide for the initial immunization is from
about 1 to about 50,000 g, generally 100-5,000
g, for a 70 kg patient. The initial administration of vaccine is followed by
booster dosages at 4 weeks followed by
evaluation of the magnitude of the immune response in the patient, by
techniques that determine the presence of epitope-
specific CTL populations in a PBMC sample. Additional booster doses are
administered as required. The composition is
found to be both safe and efficacious as a prophylaxis against 109P1D4-
associated disease.
Alternatively, a composition typically comprising transfecting agents is used
for the administration of a nucleic acid-
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based vaccine in accordance with methodologies known in the art and disclosed
herein.
Example 25: Polyepitopic Vaccine Compositions Derived from Native 109P1 D4
Sequences
A native 109P1 D4 polyprotein sequence is analyzed, preferably using computer
algorithms defined for each class I
and/or class II supermotif or motif, to identify "relatively short" regions of
the polyprotein that comprise multiple epitopes. The
"relatively short" regions are preferably less in length than an entire native
antigen. This relatively short sequence that
contains multiple distinct or overlapping, "nested" epitopes can be used to
generate a minigene construct. The construct is
engineered to express the peptide, which corresponds to the native protein
sequence. The "relatively short" peptide is
generally less than 250 amino acids in length, often less than 100 amino acids
in length, preferably less than 75 amino acids
in length, and more preferably less than 50 amino acids in length. The protein
sequence of the vaccine composition is
selected because it has maximal number of epitopes contained within the
sequence, i.e., it has a high concentration of
epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e.,
frame shifted relative to one another). For
example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope
can be present in a 10 amino acid peptide.
Such a vaccine composition is administered for therapeutic or prophylactic
purposes.
The vaccine composition will include, for example, multiple CTL epitopes from
109P1 D4 antigen and at least one
HTL epitope. This polyepitopic native sequence is administered either as a
peptide or as a nucleic acid sequence which
encodes the peptide. Alternatively, an analog can be made of this native
sequence, whereby one or more of the epitopes
comprise substitutions that alter the cross-reactivity and/or binding affinity
properties of the polyepitopic peptide.
The embodiment of this example provides for the possibility that an as yet
undiscovered aspect of immune system
processing will apply to the native nested sequence and thereby facilitate the
production of therapeutic or prophylactic
immune response-inducing vaccine compositions. Additionally, such an
embodiment provides for the possibility of motif-
bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore,
this embodiment (excluding an analoged
embodiment) directs the immune response to multiple peptide sequences that are
actually present in native 109P1 D4, thus
avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment
provides an economy of scale when
producing peptide or nucleic acid vaccine compositions.
Related to this embodiment, computer programs are available in the art which
can be used to identify in a target
sequence, the greatest number of epitopes per sequence length.
Example 26: Polyepitopic Vaccine Compositions from Multiple Antigens
The I09P1 D4 peptide epitopes of the present invention are used in conjunction
with epitopes from other target
tumor-associated antigens, to create a vaccine composition that is useful for
the prevention or treatment of cancer that
expresses 109P1 D4 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide
that incorporates multiple epitopes from 109P1 D4 as well as tumor-associated
antigens that are often expressed with a
target cancer associated with 109P1 D4 expression, or can be administered as a
composition comprising a cocktail of one or
more discrete epitopes. Alternatively, the vaccine can be administered as a
minigene construct or as dendritic cells which
have been loaded with the peptide epitopes in vitro.
Example 27: Use of peptides to evaluate an immune response
Peptides of the invention may be used to analyze an immune response for the
presence of specific antibodies,
CTL or HTL directed to 109P1 D4. Such an analysis can be performed in a manner
described by Ogg et al., Science
279:2103-2106, 1998. In this Example, peptides in accordance with the
invention are used as a reagent for diagnostic or
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prognostic purposes, not as an immunogen.
In this example highly sensitive human leukocyte antigen tetrameric complexes
("tetramers") are used for a cross-
sectional analysis of, for example, 109P1 D4 HLA-A*0201-specific CTL
frequencies from HLA A*0201 -positive individuals at
different stages of disease or following immunization comprising a 109P1 D4
peptide containing an A*0201 motif. Tetrameric
complexes are synthesized as described (Musey et al., N. Engl. J. Med.
337:1267,1997). Briefly, purified HLA heavy chain
(A*0201 in this example) and 02-microglobulin are synthesized by means of a
prokaryotic expression system. The heavy
chain is modified by deletion of the transmembrane-cytosolic tail and COOH-
terminal addition of a sequence containing a
BirA enzymatic biotinylation site. The heavy chain, 132-microglobulin, and
peptide are refolded by dilution. The 45-kD
refolded product is isolated by fast protein liquid chromatography and then
biotinylated by BirA in the presence of biotin
(Sigma, St. Louis, Missouri), adenosine 5' triphosphate and magnesium.
Streptavidin-phycoerythrin conjugate is added in a
1:4 molar ratio, and the tetrameric product is concentrated to I mg/ml. The
resulting product is referred to as tetramer-
phycoerythrin,
For the analysis of patient blood samples, approximately one million PBMCs are
centrifuged at 300g for 5 minutes
and resuspended in 50 l of cold phosphate-buffered saline. Tri-color analysis
is performed with the tetramer-phycoerythrin,
along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with
tetramer and antibodies on ice for 30 to 60 min
and then washed twice before formaldehyde fixation. Gates are applied to
contain >99.98% of control samples. Controls for
the tetramers include both A*0201-negative individuals and A*0201-positive non-
diseased donors. The percentage of cells
stained with the tetramer is then determined by flow cytometry. The results
indicate the number of cells in the PBMC sample
that contain epitope-restricted CTLs, thereby readily indicating the extent of
immune response to the 109P1 D4 epitope, and
thus the status of exposure to 109P1 D4, or exposure to a vaccine that elicits
a protective or therapeutic response.
Example 28: Use of Peptide Epitopes to Evaluate Recall Responses
The peptide epitopes of the invention are used as reagents to evaluate T cell
responses, such as acute or recall
responses, in patients. Such an analysis may be performed on patients who have
recovered from I09P1D4-associated
disease or who have been vaccinated with a 109P1 D4 vaccine.
For example, the class I restricted CTL response of persons who have been
vaccinated may be analyzed. The
vaccine may be any 109P1 D4 vaccine. PBMC are collected from vaccinated
individuals and HLA typed. Appropriate
peptide epitopes of the invention that, optimally, bear supermotifs to provide
cross-reactivity with multiple HLA supertype
family members, are then used for analysis of samples derived from individuals
who bear that HLA type.
PBMC from vaccinated individuals are separated on Ficoll-Histopaque density
gradients (Sigma Chemical Co., St.
Louis, MO), washed three times in HBSS (GIBCO Laboratories), resuspended in
RPMI-1640 (GIBCO Laboratories)
supplemented with L-glutamine (2mM), penicillin (50U/ml), streptomycin (50
lag/ml), and Hepes (10mM) containing 10%
heat-inactivated human AB serum (complete RPMI) and plated using microculture
formats. A synthetic peptide comprising
an epitope of the invention is added at 10 g/ml to each well and HBV core 128-
140 epitope is added at 1 g/ml to each well
as a source of T cell help during the first week of stimulation.
In the microculture format, 4 x 105 PBMC are stimulated with peptide in 8
replicate cultures in 96-well round bottom
plate in 100 pd/well of complete RPMI. On days 3 and 10, 100 pI of complete
RPMI and 20 U/ml final concentration of rIL-2
are added to each well. On day 7 the cultures are transferred into a 96-well
flat-bottom plate and restimulated with peptide,
rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are
tested for cytotoxic activity on day 14. A
positive CTL response requires two or more of the eight replicate cultures to
display greater than 10% specific 51Cr release,
based on comparison with non-diseased control subjects as previously described
(Rehermann, of at, Nature Med.
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2:1104,1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and
Rehermann et at. J. Clin. Invest. 98:1432-
1440,1996).
Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are
either purchased from the
American Society for Histocompatibility and Immunogenetics (ASHI, Boston, MA)
or established from the pool of patients as
described (Guilhot, et al. J. Virol, 66:2670-2678, 1992).
Cytotoxicity assays are performed in the following manner. Target cells
consist of either allogeneic HLA-matched
or autologous EBV-transformed B lymphoblastoid cell line that are incubated
overnight with the synthetic peptide epitope of
the invention at 10 M, and labeled with 100 Ci of 51Cr (Amersham Corp.,
Arlington Heights, IL) for 1 hour after which they
are washed four times with HBSS.
Cytolytic activity is determined in a standard 4-h, split well 51Cr release
assay using U-bottomed 96 well plates
containing 3,000 targets/well. Stimulated PBMC are tested at effector/target
(E/T) ratios of 20-50:1 on day 14. Percent
cytotoxicity is determined from the formula: 100 x [(experimental release-
spontaneous release)lmaximum release-
spontaneous release)]. Maximum release is determined by lysis of targets by
detergent (2% Triton X-100; Sigma Chemical
Co., St. Louis, MO). Spontaneous release is <25% of maximum release for all
experiments.
The results of such an analysis indicate the extent to which HLA-restricted
CTL populations have been stimulated
by previous exposure to 109P1 D4 or a 109P1 D4 vaccine.
Similarly, Class II restricted HTL responses may also be analyzed. Purified
PBMC are cultured in a 96-well flat
bottom plate at a density of 1.5x105 cells/well and are stimulated with 10
g/ml synthetic peptide of the invention, whole
109P1 D4 antigen, or PHA. Cells are routinely plated in replicates of 4-6
wells for each condition. After seven days of
culture, the medium is removed and replaced with fresh medium containing
IOU/ml IL-2. Two days later,1 Ci 3H-thymidine
is added to each well and incubation is continued for an additional 18 hours.
Cellular DNA is then harvested on glass fiber
mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell
proliferation is calculated as the ratio of 3H-
thymidine incorporation in the presence of antigen divided by the 3H-thymidine
incorporation in the absence of antigen.
Example 29: Induction Of Specific CTL Response In Humans
A human clinical trial for an immunogenic composition comprising CTL and HTL
epitopes of the invention is set up
as an IND Phase I, dose escalation study and carried out as a randomized,
double-blind, placebo-controlled trial. Such a
trial is designed, for example, as follows:
A total of about 27 individuals are enrolled and divided into 3 groups:
Group I: 3 subjects are injected with placebo and 6 subjects are injected with
5 g of peptide composition;
Group Il: 3 subjects are injected with placebo and 6 subjects are injected
with 50 g peptide composition;
Group III: 3 subjects are injected with placebo and 6 subjects are injected
with 500 g of peptide composition.
After 4 weeks following the first injection, all subjects receive a booster
inoculation at the same dosage.
The endpoints measured in this study relate to the safety and tolerability of
the peptide composition as well as its
immunogenicity. Cellular immune responses to the peptide composition are an
index of the intrinsic activity of this the
peptide composition, and can therefore be viewed as a measure of biological
efficacy. The following summarize the clinical
and laboratory data that relate to safety and efficacy endpoints.
Safety: The incidence of adverse events is monitored in the placebo and drug
treatment group and assessed in
terms of degree and reversibility.
Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects
are bled before and after injection.
Peripheral blood mononuclear cells are isolated from fresh heparinized blood
by Ficoll-Hypaque density gradient
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centrifugation, aliquoted in freezing media and stored frozen. Samples are
assayed for CTL and HTL activity.
The vaccine is found to be both safe and efficacious.
Example 30: Phase II Trials In Patients Expressing 109P1 D4
Phase II trials are performed to study the effect of administering the CTL-HTL
peptide compositions to patients
having cancer that expresses 109P1 D4. The main objectives of the trial are to
determine an effective dose and regimen for
inducing CTLs in cancer patients that express 109P1 D4, to establish the
safety of inducing a CTL and HTL response in
these patients, and to see to what extent activation of CTLs improves the
clinical picture of these patients, as manifested,
e.g., by the reduction and/or shrinking of lesions. Such a study is designed,
for example, as follows:
The studies are performed in multiple centers. The trial design is an open-
label, uncontrolled, dose escalation
protocol wherein the peptide composition is administered as a single dose
followed six weeks later by a single booster shot
of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection.
Drug-associated adverse effects (severity
and reversibility) are recorded.
There are three patient groupings. The first group is injected with 50
micrograms of the peptide composition and
the second and third groups with 500 and 5,000 micrograms of peptide
composition, respectively. The patients within each
group range in age from 21-65 and represent diverse ethnic backgrounds. All of
them have a tumor that expresses
109P1 D4.
Clinical manifestations or antigen-specific T-cell responses are monitored to
assess the effects of administering the
peptide compositions. The vaccine composition is found to be both safe and
efficacious in the treatment of 109P1 D4-
associated disease.
Example 31: Induction of CTL Responses Using a Prime Boost Protocol
A prime boost protocol similar in its underlying principle to that used to
confirm the efficacy of a DNA vaccine in
transgenic mice, such as described above in the Example entitled "The Plasmid
Construct and the Degree to Which It
Induces Immunogenicity," can also be used for the administration of the
vaccine to humans. Such a vaccine regimen can
include an initial administration of, for example, naked DNA followed by a
boost using recombinant virus encoding the
vaccine, or recombinant protein/polypeptide or a peptide mixture administered
in an adjuvant.
For example, the initial immunization may be performed using an expression
vector, such as that constructed in
the Example entitled "Construction of "Minigene" Multi-Epitope DNA Plasmids"
in the form of naked nucleic acid administered
IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic
acid (0.1 to 1000 g) can also be administered
using a gene gun. Following an incubation period of 3-4 weeks, a booster dose
is then administered. The booster can be
recombinant fowlpox virus administered at a dose of 5-107 to 5x109 pfu. An
alternative recombinant virus, such as an MVA,
canarypox, adenovirus, or adeno-associated virus, can also be used for the
booster, or the polyepitopic protein or a mixture
of the peptides can be administered. For evaluation of vaccine efficacy,
patient blood samples are obtained before
immunization as well as at intervals following administration of the initial
vaccine and booster doses of the vaccine.
Peripheral blood mononuclear cells are isolated from fresh heparinized blood
by Ficoll-Hypaque density gradient
centrifugation, aliquoted in freezing media and stored frozen. Samples are
assayed for CTL and HTL activity.
Analysis of the results indicates that a magnitude of response sufficient to
achieve a therapeutic or protective
immunity against 109P1 D4 is generated.
Example 32: Administration of Vaccine Compositions Using Dendritic Cells (DC)
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Vaccines comprising peptide epitopes of the invention can be administered
using APCs, or "professional" APCs
such as DC. In this example, peptide-pulsed DC are administered to a patient
to stimulate a CTL response in vivo. In this
method, dendritic cells are isolated, expanded, and pulsed with a vaccine
comprising peptide CTL and HTL epitopes of the
invention. The dendritic cells are infused back into the patient to elicit CTL
and HTL responses in vivo. The induced CTL
and HTL then destroy or facilitate destruction, respectively, of the target
cells that bear the 109P1 D4 protein from which the
epitopes in the vaccine are derived.
For example, a cocktail of epitope-comprising peptides is administered ex vivo
to PBMC, or isolated DC therefrom.
A pharmaceutical to facilitate harvesting of DC can be used, such as
ProgenipoietinTM (Monsanto, St. Louis, MO) or GM-
CSFIIL-4. After pulsing the DC with peptides, and prior to reinfusion into
patients, the DC are washed to remove unbound
peptides.
As appreciated clinically, and readily determined by one of skill based on
clinical outcomes, the number of DC
reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998;
Nature Med. 2:52, 1996 and Prostate 32:272, 1997).
Although 2-50 x 106 DC per patient are typically administered, larger number
of DC, such as 107 or 108 can also be provided.
Such cell populations typically contain between 50-90% DC.
In some embodiments, peptide-loaded PBMC are injected into patients without
purification of the DC. For
example, PBMC generated after treatment with an agent such as ProgenipoietinTM
are injected into patients without
purification of the DC. The total number of PBMC that are administered often
ranges from 108 to 1010. Generally, the cell
doses injected into patients is based on the percentage of DC in the blood of
each patient, as determined, for example, by
immunofluorescence analysis with specific anti-DC antibodies. Thus, for
example, if ProgenipoietinTM mobilizes 2% DC in
the peripheral blood of a given patient, and that patient is to receive 5 x
106 DC, then the patient will be injected with a total
of 2.5 x 108 peptide-loaded PBMC. The percent DC mobilized by an agent such as
ProgenipoietinTM is typically estimated to
be between 2-10%, but can vary as appreciated by one of skill in the art.
Ex vivo activation of CTL/HTL responses
Alternatively, ex vivo CTL or HTL responses to 109P1 D4 antigens can be
induced by incubating, in tissue culture,
the patient's, or genetically compatible, CTL or HTL precursor cells together
with a source of APC, such as DC, and
immunogenic peptides. After an appropriate incubation time (typically about 7-
28 days), in which the precursor cells are
activated and expanded into effector cells, the cells are infused into the
patient, where they will destroy (CTL) or facilitate
destruction (HTL) of their specific target cells, i.e., tumor cells.
Example 33: An Alternative Method of Identifying and Confirming Motif-Bearing
Peptides
Another method of identifying and confirming motif-bearing peptides is to
elute them from cells bearing defined
MHC molecules. For example, EBV transformed B cell lines used for tissue
typing have been extensively characterized to
determine which HLA molecules they express. In certain cases these cells
express only a single type of HLA molecule.
These cells can be transfected with nucleic acids that express the antigen of
interest, e.g. 109P1 D4. Peptides produced by
endogenous antigen processing of peptides produced as a result of transfection
will then bind to HLA molecules within the
cell and be transported and displayed on the cell's surface. Peptides are then
eluted from the HLA molecules by exposure to
mild acid conditions and their amino acid sequence determined, e.g., by mass
spectral analysis (e.g., Kubo et al., J.
Immunol. 152:3913, 1994). Because the majority of peptides that bind a
particular HLA molecule are motif-bearing, this is an
alternative modality for obtaining the motif-bearing peptides correlated with
the particular HLA molecule expressed on the
cell.
Alternatively, cell lines that do not express endogenous HLA molecules can be
transfected with an expression
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construct encoding a single HLA allele. These cells can then be used as
described, i.e., they can then be transfected with
nucleic acids that encode 109P1 D4 to isolate peptides corresponding to 109P1
D4 that have been presented on the cell
surface. Peptides obtained from such an analysis will bear motif(s) that
correspond to binding to the single HLA allele that is
expressed in the cell.
As appreciated by one in the art, one can perform a similar analysis on a cell
bearing more than one HLA allele
and subsequently determine peptides specific for each HLA allele expressed.
Moreover, one of skill would also recognize
that means other than transfection, such as loading with a protein antigen,
can be used to provide a source of antigen to the
cell.
Example 34: ComplementaryPoivnucleotides
Sequences complementary to the 109P1 D4-encoding sequences, or any parts
thereof, are used to detect,
decrease, or inhibit expression of naturally occurring 109P1 D4. Although use
of oligonucleotides comprising from about 15
to 30 base pairs is described, essentially the same procedure is used with
smaller or with larger sequence fragments.
Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software
(National Biosciences) and the coding sequence
of 109P1 D4. To inhibit transcription, a complementary oligonucleotide is
designed from the most unique 5' sequence and
used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary oligonucleotide is
designed to prevent ribosomal binding to a 109P1 D4-encoding transcript.
Example 35: Purification of Naturally-occurring or Recombinant 109P1 D4 Using
109P1 D4-Specific Antibodies
Naturally occurring or recombinant 109P1 D4 is substantially purified by
immunoaffinity chromatography using
antibodies specific for 109P1 D4. An immunoaffinity column is constructed by
covalently coupling anti-I09P1 D4 antibody to
an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham
Pharmacia Biotech). After the
coupling, the resin is blocked and washed according to the manufacturer's
instructions.
Media containing 109P1 D4 are passed over the immunoaffinity column, and the
column is washed under
conditions that allow the preferential absorbance of 109P1 D4 (e.g., high
ionic strength buffers in the presence of detergent).
The column is eluted under conditions that disrupt antibody/109PI D4 binding
(e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is
collected.
Example 36: Identification of Molecules Which Interact with 109P1 D4
109P1 D4, or biologically active fragments thereof, are labeled with 121 1
Bolton-Hunter reagent. (See, e.g., Bolton
et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in
the wells of a multi-well plate are incubated
with the labeled 109P1 D4, washed, and any wells with labeled 109P1 D4 complex
are assayed. Data obtained using
different concentrations of 109P1 D4 are used to calculate values for the
number, affinity, and association of 109PI D4 with
the candidate molecules.
Example 37: In Vivo Assay for 109P1 D4 Tumor Growth Promotion
The effect of a 109P1 D4 protein on tumor cell growth is evaluated in vivo by
gene overexpression in tumor-bearing
mice. For example, SCID mice are injected subcutaneously on each flank with I
x 106 of either PC3, DU145 or 3T3 cells
containing tkNeo empty vector or a nucleic acid sequence of the invention. At
least two strategies can be used: (1)
Constitutive expression under regulation of a promoter such as a constitutive
promoter obtained from the genomes of viruses
such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989),
adenovirus (such as Adenovirus 2), bovine
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papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-
B virus and Simian Virus 40 (SV40), or from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, provided such promoters are
compatible with the host cell systems, and (2) Regulated expression under
control of an inducible vector system, such as
ecdysone, tet, etc., provided such promoters are compatible with the host cell
systems. Tumor volume is then monitored at
the appearance of palpable tumors and followed over time to determine if the
cells expressing a gene of the invention grow
at a faster rate and whether tumors of a 109P1 D4 protein-expressing cells
demonstrate characteristics of altered
aggressiveness (e.g. enhanced metastasis, vascularization, reduced
responsiveness to chemotherapeutic drugs).
Additionally, mice can be implanted with I x 105 of the same cells
orthotopically to determine if a protein of the
invention has an effect on local growth in the prostate or on the ability of
the cells to metastasize, specifically to lungs, lymph
nodes, and bone marrow.
The assay is also useful to determine the inhibitory effect of candidate
therapeutic compositions, such as for
example, 109P1 D4 protein-related intrabodies, I09P1 D4 gene-related antisense
molecules and ribozymes.
Example 38: 109P1 D4 Monoclonal Antibody-mediated Inhibition of Tumors In Vivo
The significant expression of 109P1 D4 proteins in the cancer tissues of Table
I and its restrictive expression in
normal tissues, together with its expected cell surface expression, makes
109P1 D4 proteins excellent targets for antibody
therapy. Similarly, 109PI D4 proteins are a target for T cell-based
immunotherapy. Thus, for 109P1 D4 genes expressed,
e.g., in prostate cancer, the therapeutic efficacy of anti-109P1 D4 protein
mAbs in human prostate cancer xenograft mouse
models is evaluated by using androgen-independent LAPC-4 and LAPC-9 xenografts
(Craft, N., eta/.,. Cancer Res, 1999.
59(19): p. 5030-6) and the androgen independent recombinant cell line PC3-of
109P1 D4 (see, e.g., Kaighn, M.E., et al.,
Invest Urol,1979.17(1): p. 16-23); analogous models are used for other
cancers.
Antibody efficacy on tumor growth and metastasis formation is studied, e.g.,
in a mouse orthotopic prostate cancer
xenograft models and mouse kidney xenograft models. The antibodies can be
unconjugated, as discussed in this Example,
or can be conjugated to a therapeutic modality, as appreciated in the art.
Anti-109P1 D4 protein mAbs inhibit formation of
both the androgen-dependent LAPC-9 and androgen-independent PC3-109P1 D4
protein tumor xenografts. Anti-109P1 D4
protein mAbs also retard the growth of established orthotopic tumors and
prolonged survival of tumor-bearing mice. These
results indicate the utility of anti-I09PI D4 protein mAbs in the treatment of
local and advanced stages of prostate cancer.
(See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 or World Wide Web URL
ww.pnas.org/cgi/doi/l 0.1073/pnas.051624698).
Administration of the anti-I09P1 D4 protein mAbs lead to retardation of
established orthotopic tumor growth and
inhibition of metastasis to distant sites, resulting in a significant
prolongation in the survival of tumor-bearing mice. These
studies indicate that proteins of the invention are attractive targets for
immunotherapy and demonstrate the therapeutic
potential of anti-109P1 D4 protein mAbs for the treatment of local and
metastatic cancer. This example demonstrates that
unconjugated 109P1 D4 protein-related monoclonal antibodies are effective to
inhibit the growth of human prostate tumor
xenografts and human kidney xenografts grown in SCID mice; accordingly a
combination of such efficacious monoclonal
antibodies is also effective.
Tumor inhibition using multiple unconjugated mAbs
Materials and Methods
109P1D4 Protein-related Monoclonal Antibodies:
Monoclonal antibodies are raised against proteins of the invention as
described in the Example entitled
"Generation of 109P1D4 Monoclonal Antibodies". The antibodies are
characterized by ELISA, Western blot, FACS, and
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immunoprecipitation for their capacity to bind to the respective protein of
the invention. Epitope mapping data for, e.g., the
anti-109P1 D4 protein mAbs, as determined by ELISA and Western analysis,
indicate that the antibodies recognize epitopes
on the respective 109P1 D4 protein. Immunohistochemical analysis of prostate
cancer tissues and cells with these
antibodies is performed.
The monoclonal antibodies are purified from ascites or hybridoma tissue
culture supernatants by Protein-G
Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored
at -20 C. Protein determinations are
performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic
monoclonal antibody or a cocktail comprising a
mixture of individual monoclonal antibodies is prepared and used for the
treatment of mice receiving subcutaneous or
orthotopic injections of LAPC-9 prostate tumor xenografts.
Cancer X enoo rafts and Cell Lines
The LAPC-9 xenograft, which expresses a wild-type androgen receptor and
produces prostate-specific antigen
(PSA), is passaged in 6- to 8-week-old male ICR-severe combined
immunodeficient (SCID) mice (Taconic Farms) by s.c.
trocar implant (Craft, N., et al., supra). The prostate carcinoma cell line
PC3 (American Type Culture Collection) is
maintained in RPMI supplemented with L-glutamine and 10% FBS.
Recombinant PC3 and 3T3- cell populations expressing a protein of the
invention are generated by retroviral gene
transfer as described in Hubert, R.S., et al., STEAP: a prostate-specific cell-
surface antigen highly expressed in human
prostate tumors. Proc Nall Acad Sol U S A, 1999. 96(25): p. 14523-8. Anti-
protein of the invention staining is detected by
using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology
Associates) followed by analysis on a Coulter
Epics-XL flow cytometer.
Xenograft Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of I x 10 6 LAPC-9, PC3,
recombinant PC3-protein of the
invention, 3T3 or recombinant 3T3-protein of the invention cells mixed at a
1:1 dilution with Matrigel (Collaborative Research)
in the right flank of male SCID mice. To test antibody efficacy on tumor
formation, i.p. antibody injections are started on the
same day as tumor-cell injections. As a control, mice are injected with either
purified mouse IgG (ICN) or PBS; or a purified
monoclonal antibody that recognizes an irrelevant antigen not expressed in
human cells. In preliminary studies, no
difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are
determined by vernier caliper
measurements, and the tumor volume is calculated as length x width x height.
Mice with s.c. tumors greater than 1.5 cm in
diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit
(Anogen, Mississauga, Ontario). Circulating
levels of, e.g., anti-109P1 D4 protein mAbs are determined by a capture ELISA
kit (Bethyl Laboratories, Montgomery, TX).
(See, e.g., Saffran, D., et al., PNAS 10:1073-1078 ).
Orthotopic injections are performed under anesthesia by using ketamineh
ylazine. For prostate orthotopic studies,
an incision is made through the abdominal muscles to expose the bladder and
seminal vesicles, which then are delivered
through the incision to expose the dorsal prostate. IAPC-9 or PC3 cells (5 x
105) mixed with Matrigel are injected into each
dorsal lobe in a 10-pi volume. To monitor tumor growth, mice are bled on a
weekly basis for determination of PSA levels.
The mice are segregated into groups for the appropriate treatments, with anti-
protein of the invention or control mAbs being
injected i.p.
Anti-109P1D4 Protein mAbs Inhibit Growth of Respective 109P1D4 Protein-
Expressing Xenograft-Cancer Tumors
The effect of anti-109P1 D4 protein mAbs on tumor formation is tested by using
LAPC-9 and recombinant PC3-
protein of the invention orthotopic models. As compared with the s.c. tumor
model, the orthotopic model, which requires
injection of tumor cells directly in the mouse prostate or kidney,
respectively, results In a local tumor growth, development of
metastasis in distal sites, deterioration of mouse health, and subsequent
death (Saffran, D., et al., PNAS supra; Fu, X., et al.,
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Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1):
p. 4-8). The features make the orthotopic
model more representative of human disease progression and allowed us to
follow the therapeutic effect of mAbs on
clinically relevant end points.
Accordingly, tumor cells are injected into the mouse prostate or kidney, and 2
days later, the mice are segregated
into two groups and treated with either: a) 200-500pg, of anti-109P1 D4
protein Ab, orb) PBS three times per week for two to
five weeks.
A major advantage of the orthotopic prostate-cancer model is the ability to
study the development of metastases.
Formation of metastasis in mice bearing established orthotopic tumors is
studied by IHC analysis on lung sections using an
antibody against a prostate-specific cell-surface protein STEAP expressed at
high levels in LAPC-9 xenografts (Hubert, R.S.,
et al., Proc Natl Acad Sci U S A, 1999. 96(25): p. 14523-8).
Mice bearing established orthotopic LAPC-9 or recombinant PC3-109PI D4 protein
tumors are administered
1000pg injections of either anti-109PI D4 protein mAbs or PBS over a 4-week
period. Mice in both groups are allowed to
establish a high tumor burden (PSA levels greater than 300 ng/ml for IAPC-9),
to ensure a high frequency of metastasis
formation in mouse lungs. Mice then are killed and their prostate and lungs
are analyzed for the presence of tumor cells by
IHC analysis.
These studies demonstrate a broad anti-tumor efficacy of anti-109P1 D4 protein
antibodies on initiation and
progression of prostate cancer in xenograft mouse models. Anti-109P1 D4
protein antibodies inhibit tumor formation of both
androgen-dependent and androgen-independent tumors, retard the growth of
already established tumors, and prolong the
survival of treated mice. Moreover, anti-I09P1 D4 protein mAbs demonstrate a
dramatic inhibitory effect on the spread of
local prostate tumor to distal sites, even in the presence of a large tumor
burden. Thus, anti-109P1 D4 protein mAbs are
efficacious on major clinically relevant end points (tumor growth),
prolongation of survival, and health.
Example 39: Therapeutic and Diagnostic use of Anti-109P1D4 Antibodies in
Humans.
Anti-I 09P1 D4 monoclonal antibodies are safely and effectively used for
diagnostic, prophylactic, prognostic and/or
therapeutic purposes in humans. Western blot and immunohistochemical analysis
of cancer tissues and cancer xenografts
with anti-109P1 D4 mAb show strong extensive staining in carcinoma but
significantly lower or undetectable levels in normal
tissues. Detection of 109P1 D4 in carcinoma and in metastatic disease
demonstrates the usefulness of the mAb as a
diagnostic and/or prognostic indicator. Anti-109PI D4 antibodies are therefore
used in diagnostic applications such as
immunohistochemistry of kidney biopsy specimens to detect cancer from suspect
patients.
As determined by flow cytometry, anti-109P1D4 mAb specifically binds to
carcinoma cells. Thus, anti-1091304
antibodies are used in diagnostic whole body imaging applications, such as
radioimmunoscintigraphy and
radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res
20(2A):925-948 (2000)) for the detection of localized
and metastatic cancers that exhibit expression of 109P1 D4. Shedding or
release of an extracellular domain of 109PI D4 into
the extracellular milieu, such as that seen for alkaline phosphodiesterase BI
0 (Meerson, N. R., Hepatology 27:563-568
(1998)), allows diagnostic detection of 109P1 D4 by anti-I09P1 D4 antibodies
in serum and/or urine samples from suspect
patients.
Anti-I09P1 D4 antibodies that specifically bind 109P1 D4 are used in
therapeutic applications for the treatment of
cancers that express 109PI D4. Anti-109PI D4 antibodies are used as an
unconjugated modality and as conjugated form in
which the antibodies are attached to one of various therapeutic or imaging
modalities well known in the art, such as a
prodrugs, enzymes or radioisotopes, In preclinical studies, unconjugated and
conjugated anti-109PI D4 antibodies are
tested for efficacy of tumor prevention and growth inhibition in the SCID
mouse cancer xenograft models, e.g., kidney cancer
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models AGS-K3 and AGS-K6, (see, e.g., the Example entitled "109P1D4 Monoclonal
Antibody-mediated Inhibition of
Bladder and Lung Tumors In Vivo"). Either conjugated and unconjugated anti-
109P1 D4 antibodies are used as a therapeutic
modality in human clinical trials either alone or in combination with other
treatments as described in following Examples.
Example 40: Human Clinical Trials for the Treatment and Diagnosis of Human
Carcinomas through use of Human
Anti-109P1 D4 Antibodies In vivo
Antibodies are used in accordance with the present invention which recognize
an epitope on 109P1 D4, and are
used in the treatment of certain tumors such as those listed in Table I. Based
upon a number of factors, including 109P1 D4
expression levels, tumors such as those listed in Table I are presently
preferred indications. In connection with each of these
indications, three clinical approaches are successfully pursued.
I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-
109P1D4 antibodies in
combination with a chemotherapeutic or antineoplastic agent and/or radiation
therapy. Primary cancer targets, such as
those listed in Table I, are treated under standard protocols by the addition
anti-I09P1 D4 antibodies to standard first and
second line therapy. Protocol designs address effectiveness as assessed by
reduction in tumor mass as well as the ability to
reduce usual doses of standard chemotherapy. These dosage reductions allow
additional and/or prolonged therapy by
reducing dose-related toxicity of the chemotherapeutic agent. Anti-109P1 D4
antibodies are utilized in several adjunctive
clinical trials in combination with the chemotherapeutic or antineoplastic
agents adriamycin (advanced prostrate carcinoma),
cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer),
and doxorubicin (preclinical).
II.) Monotherapy: In connection with the use of the anti-109P1 D4 antibodies
in monotherapy of tumors, the
antibodies are administered to patients without a chemotherapeutic or
antineoplastic agent. In one embodiment,
monotherapy is conducted clinically in end stage cancer patients with
extensive metastatic disease. Patients show some
disease stabilization. Trials demonstrate an effect in refractory patients
with cancerous tumors.
III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium
(1131, Y90) to anti-109P1 D4
antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or
imaging agent. In such a role, the labeled
antibodies localize to both solid tumors, as well as, metastatic lesions of
cells expressing 109PI D4. In connection with the
use of the anti-I09P1 D4 antibodies as imaging agents, the antibodies are used
as an adjunct to surgical treatment of solid
tumors, as both a pre-surgical screen as well as a post-operative follow-up to
determine what tumor remains and/or returns.
In one embodiment, a (111 In)-109P1 D4 antibody is used as an imaging agent in
a Phase I human clinical trial in patients
having a carcinoma that expresses 109P1 D4 (by analogy see, e.g., Divgi et al.
J. Natl. Cancer Inst. 83:97-104 (1991)).
Patients are followed with standard anterior and posterior gamma camera. The
results indicate that primary lesions and
metastatic lesions are identified.
Dose and Route of Administration
As appreciated by those of ordinary skill in the art, dosing considerations
can be determined through comparison
with the analogous products that are in the clinic. Thus, anti-I09P1 D4
antibodies can be administered with doses in the
range of 5 to 400 mg/m 2, with the lower doses used, e.g., in connection with
safety studies. The affinity of anti-I09P1 D4
antibodies relative to the affinity of a known antibody for its target is one
parameter used by those of skill in the art for
determining analogous dose regimens. Further, anti-109P1 D4 antibodies that
are fully human antibodies, as compared to
the chimeric antibody, have slower clearance; accordingly, dosing in patients
with such fully human anti-109P1 D4 antibodies
can be lower, perhaps in the range of 50 to 300 mg/m2, and still remain
efficacious. Dosing in mg/m2, as opposed to the
conventional measurement of dose in mg/kg, is a measurement based on surface
area and is a convenient dosing
measurement that is designed to include patients of all sizes from infants to
adults.
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Three distinct delivery approaches are useful for delivery of anti-109P1 D4
antibodies. Conventional intravenous
delivery is one standard delivery technique for many tumors. However, in
connection with tumors in the peritoneal cavity,
such as tumors of the ovaries, biliary duct, other ducts, and the like,
intraperitoneal administration may prove favorable for
obtaining high dose of antibody at the tumor and to also minimize antibody
clearance. In a similar manner, certain solid
tumors possess vasculature that is appropriate for regional perfusion.
Regional perfusion allows for a high dose of antibody
at the site of a tumor and minimizes short term clearance of the antibody.
Clinical Development Plan (CDP)
Overview: The CDP follows and develops treatments of anti-109PID4 antibodies
in connection with adjunctive
therapy, monotherapy, and as an imaging agent. Trials initially demonstrate
safety and thereafter confirm efficacy in repeat
doses. Trails are open label comparing standard chemotherapy with standard
therapy plus anti-109PI D4 antibodies. As will
be appreciated, one criteria that can be utilized in connection with
enrollment of patients is 109P1 D4 expression levels in
their tumors as determined by biopsy.
As with any protein or antibody infusion-based therapeutic, safety concerns
are related primarily to (1) cytokine
release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the
development of an immunogenic response to the material
(i.e., development of human antibodies by the patient to the antibody
therapeutic, or HAHA response); and, (iii) toxicity to
normal cells that express 109P1 D4. Standard tests and follow-up are utilized
to monitor each of these safety concerns.
Anti-109P1D4 antibodies are found to be safe upon human administration.
Example 41: Human Clinical Trial Adjunctive Therapy with Human Anti-109P1D4
Antibody and Chemotherapeutic
Agent
A phase I human clinical trial is initiated to assess the safety of six
intravenous doses of a human anti-109PI D4
antibody in connection with the treatment of a solid tumor, e.g., a cancer of
a tissue listed in Table I. In the study, the safety
of single doses of anti-109PI D4 antibodies when utilized as an adjunctive
therapy to an antineoplastic or chemotherapeutic
agent as defined herein, such as, without limitation: cisplatin, topotecan,
doxorubicin, adriamycin, taxol, or the like, is
assessed. The trial design includes delivery of six single doses of an anti-I
09P1 D4 antibody with dosage of antibody
escalating from approximately about 25 mg/m 2to about 275 mg/m lover the
course of the treatment in accordance with the
following schedule:
Day 0 Day 7 Day 14 Day 21 Day 28 Day 35
mAb Dose 25 75 125 175 225 275
mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2
Chemotherapy + + + + + +
(standard dose)
Patients are closely followed for one-week following each administration of
antibody and chemotherapy. In
particular, patients are assessed for the safety concerns mentioned above: (I)
cytokine release syndrome, i.e., hypotension,
fever, shaking, chills; (ii) the development of an immunogenic response to the
material (i.e., development of human
antibodies by the patient to the human antibody therapeutic, or HAHA
response); and, (iii) toxicity to normal cells that
express 109P1 D4. Standard tests and follow-up are utilized to monitor each of
these safety concerns. Patients are also
assessed for clinical outcome, and particularly reduction in tumor mass as
evidenced by MRI or other imaging.
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The anti-I 09P1 D4 antibodies are demonstrated to be safe and efficacious,
Phase II trials confirm the efficacy and
refine optimum dosing.
Example 42: Human Clinical Trial: Monotherapy with Human Anti 109P1D4 Antibody
Anti-109P1D4 antibodies are safe in connection with the above-discussed
adjunctive trial, a Phase II human
clinical trial confirms the efficacy and optimum dosing for monotherapy. Such
trial is accomplished, and entails the same
safety and outcome analyses, to the above-described adjunctive trial with the
exception being that patients do not receive
chemotherapy concurrently with the receipt of doses of anti-I 09P1 D4
antibodies.
Example 43: Human Clinical Trial: Diagnostic Imaging with Anti-109P1 D4
Antibody
Once again, as the adjunctive therapy discussed above is safe within the
safety criteria discussed above, a human
clinical trial is conducted concerning the use of anti-109P1 D4 antibodies as
a diagnostic imaging agent. The protocol is
designed in a substantially similar manner to those described in the art, such
as in Divgi et al J. Natl. Cancer inst. 83:97-104
(1991). The antibodies are found to be both safe and efficacious when used as
a diagnostic modality.
Example 44:109P1D4 Functional Assays
1. Phosphorylation of 109P1D4 on tyrosine residues
One hallmark of the cancer cell phenotype is the active signal transduction of
surface bound receptor molecules,
such as the EGF receptor, through tyrosine phosphorylation of their
cytoplasmic domains and their subsequent interaction
with cytosolic signaling molecules. To address the possibility that 109P1 D4
is phosphorylated on its cytoplamsic tyrosine
residues, 293T cells were transfected with the 109P1 D4 gene in an expression
plasmid such that the 109P1 D4 gene was
fused with a Myc/His tag, and were then stimulated with pervanadate (a 1:1
mixture of Na3VO4 and H202). After
solubilization of the cells in Triton X-100, the 109P1 D4 protein was
immunoprecipitated with anti-His polyclonal antibody
(pAb), subjected to SDS-PAGE and Western blotted with anti-phosphotyrosine.
Equivalent immunoprecipitates were
Western blotted with anti-His antibody. In Figure 22, 109P1D4 exhibits
tyrosine phosphorylation only upon cell treatment
with pervanadate and not without treatment. This suggests that pervanadate,
which inhibits intracellular protein tyrosine
phosphatases (PTPs), allows the accumulation of phosphotyrosine (tyrosine
kinase activity) on 109P1 D4. Further, a large
amount of the 109P1 D4 protein is sequestered into the insoluble fraction upon
pervanadate activation, suggesting its
association with cytoskeletal components. Similar effects of partial
insolubility in Triton X-100 have been observed for
cadherins, proteins that are related to protocadherins based on homology of
their extracellular domains. Cadherins are
known to interact with cytoskeletal proteins including actin, which are not
readily soluble in the detergent conditions used in
this study. Together, these data indicate that 109P1 D4 is a surface receptor
with the capacity to be phosphorylated on
tyrosine and to bind to signaling molecules that possess SH2 or PTB binding
domains, including but not limited to,
phospholipase-Cyl, Grb2, Shc, Crk, PI-3-kinase p85 subunit, rasGAP, Src-family
kinases and abl-family kinases. Such
interactions are important for downstream signaling through 109P1 D4, leading
to changes in adhesion, proliferation,
migration or elaboration of secreted factors. In addition, 109P1 D4 protein
interacts with cytoskeletal components such as
actin that facilitates its cell adhesion functions. These phenotypes are
enhanced in 109P1 D4 expressing tumor cells and
contribute to their increased capacity to metastasize and grow in vivo.
Thus, when 109P1 D4 plays a role in cell signaling and phosphorylation, it is
used as a target for diagnostic,
prognostic, preventative and/or therapeutic purposes.
Example 45: 109P1D4 RNA Interference (RNA)
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RNA interference (RNAi) technology is implemented to a variety of cell assays
relevant to oncology. RNAi is a
post-transcriptional gene silencing mechanism activated by double-stranded RNA
(dsRNA). RNAi induces specific mRNA
degradation leading to changes in protein expression and subsequently in gene
function. In mammalian cells, these dsRNAs
called short interfering RNA (siRNA) have the correct composition to activate
the RNAi pathway targeting for degradation,
specifically some mRNAs. See, Elbashir S.M., et. al., Duplexes of 21-
nucleotide RNAs Mediate RNA interference in
Cultured Mammalian Cells, Nature 411(6836):494-8 (2001). Thus, RNAi technology
is used successfully in mammalian cells
to silence targeted genes.
Loss of cell proliferation control is a hallmark of cancerous cells; thus,
assessing the role of 109P1 D4 in cell
survival/proliferation assays is relevant. Accordingly, RNAi was used to
investigate the function of the 109P1 D4 antigen. To
generate siRNA for 109P1 D4, algorithms were used that predict
oligonucleotides that exhibit the critical molecular
parameters (G:C content, melting temperature, etc.) and have the ability to
significantly reduce the expression levels of the
109P1 D4 protein when introduced into cells. Accordingly, three targeted
sequences for the 109P1 D4 siRNA are: 5'
AAGAGGATACTGGTGAGATCT 3' (SEQ ID NO: 57)(oligo 109P1 D4.a), 5'
AAGAGCAATGGTGCTGGTAAA 3' (SEQ ID
NO: 58)(oligo 109P1 D4.c), and 5' AACACCAGAAGGAGACAAGAT 3' (SEQ ID NO:
59)(oligo 109P1 D4.d). In accordance
with this Example, 109P1 D4 siRNA compositions are used that comprise siRNA
(double stranded, short interfering RNA) that
correspond to the nucleic acid ORF sequence of the 109P1 D4 protein or
subsequences thereof. Thus, siRNA
subsequences are used in this manner are generally 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 or more than 35 contiguous RNA
nucleotides in length. These siRNA sequences
are complementary and non-complementary to at least a portion of the mRNA
coding sequence. In a preferred embodiment,
the subsequences are 19-25 nucleotides in length, most preferably 21-23
nucleotides in length. In preferred embodiments,
these siRNA achieve knockdown of 109P1 D4 antigen in cells expressing the
protein and have functional effects as described
below.
The selected siRNAs (109P1 D4.a,109P1 D4.c,109P1 D4.d oligos) were tested in
LNCaP cells in the 3H-thymidine
incorporation assay (measures cellular proliferation). Moreover, the
oligonucleotides achieved knockdown of 109P1D4
antigen in cells expressing the protein and had functional effects as
described below using the following protocols.
Mammalian siRNA transfections: The day before siRNA transfection, the
different cell lines were plated in
media (RPMI 1640 with 10% FBS w/o antibiotics) at 2x103 cells/well in 80 p (96
well plate format) for the proliferation assay.
In parallel with the 109PI D4 specific siRNA oligo, the following sequences
were included in every experiment as controls: a)
Mock transfected cells with Lipofectamine 2000 (Invitrogen) Carlsbad, CA) and
annealing buffer (no siRNA); b) Luciferase-4
specific siRNA (targeted sequence: 5'-AAGGGACGAAGACGAACACUUCTT-3') (SEQ ID NO:
60); and, c) Eg5 specific
siRNA (targeted sequence: 5'-AACTGAAGACCTGAAGACAATAA-3') (SEQ ID NO: 61).
SiRNAs were used at 10nM and
pg/ml Lipofectamine 2000 final concentration.
The procedure was as follows: The siRNAs were first diluted in OPTIMEM (serum-
free transfection media,
Invitrogen) at 0.1 pM (10-fold concentrated) and incubated 5-10 min RT.
Lipofectamine 2000 was diluted at 10 pg/ml (10-
fold concentrated) for the total number transfections and incubated 5-10
minutes at room temperature (RT). Appropriate
amounts of diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 with
diluted 10-fold concentrated siRNA and
incubated at RT for 20-30" (5-fold concentrated transfection solution). 20 pis
of the 5-fold concentrated transfection
solutions were added to the respective samples and incubated at 37 C for 96
hours before analysis.
3H-Thymidine incorporation assay: The proliferation assay is a 3H-thymidine
incorporation method for determining
the proliferation of viable cells by uptake and incorporation of label into
DNA.
The procedure was as follows: Cells growing in log phase are trypsinized,
washed, counted and plated in 96-well
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plates at 1000-4000 cells/well in 10% FBS. After 4-8 hrs, the media is
replaced. The cells are incubated for 24-72 hrs,
pulsed with 3H-Thy at 1.5 pCi/mI for 14 hrs, harvested onto a filtermat and
counted in scintillation cocktail on a Microbeta
trilux or other counter.
In order to address the function of 109P1 D4 in cells, 109P1 D4 was silenced
by transfecting the endogenously
expressing 109P1 D4 cell line (LNCaP) with the 109P1 D4 specific siRNAs (109P1
D4.a,109P1 D4.c, and 109P1 D4.d) along
with negative siRNA controls (Luc4, targeted sequence not represented in the
human genome), a positive siRNA control
(targeting Eg5) and no siRNA oligo (LF2K) (Figure 23). The results indicated
that when these cells are treated with siRNA
specifically targeting the 109P1 D4 mRNA, the resulting "109P1 D4 deficient
cells" showed diminished cell proliferation as
measured by this assay (e.g., see oligo 109P1 D4.a treated cells).
These data indicate that 109P1 D4 plays an important role in the proliferation
of cancer cells and that the lack of
109P1 D4 clearly decreases the survival potential of these cells. It is to be
noted that 109P1 D4 is constitutively expressed in
many tumor cell lines. 109P1 D4 serves a role in malignancy; its expression is
a primary indicator of disease, where such
disease is often characterized by high rates of uncontrolled cell
proliferation and diminished apoptosis. Correlating cellular
phenotype with gene knockdown following RNAi treatments is important, and
allows one to draw valid conclusions and rule
out toxicity or other non-specific effects of these reagents. To this end,
assays to measure the levels of expression of both
protein and mRNA for the target after RNAi treatments are important, including
Western blotting, FACS staining with
antibody, immunoprecipitation, Northern blotting or RT-PCR (Taqman or standard
methods). Any phenotypic effect of the
siRNAs in these assays should be correlated with the protein and/or mRNA
knockdown levels in the same cell lines.
109P1 D4 protein is reduced after treatment with siRNA oligos described above
(e.g., 109P1 D4.a, etc.)
A method to analyze 109P1 D4 related cell proliferation is the measurement of
DNA synthesis as a marker for
proliferation. Labeled DNA precursors (i.e. 3H-Thymidine) are used and their
incorporation to DNA is quantified.
Incorporation of the labeled precursor into DNA is directly proportional to
the amount of cell division occurring in the culture.
Another method used to measure cell proliferation is performing clonogenic
assays. In these assays, a defined number of
cells are plated onto the appropriate matrix and the number of colonies formed
after a period of growth following siRNA
treatment is counted.
In 109P1 D4 cancer target validation, complementing the cell
survival/proliferation analysis with apoptosis and cell
cycle profiling studies are considered. The biochemical hallmark of the
apoptotic process is genomic DNA fragmentation, an
irreversible event that commits the cell to die. A method to observe
fragmented DNA in cells is the immunological detection
of histone-complexed DNA fragments by an immunoassay (i.e. cell death
detection ELISA) which measures the enrichment
of histone-complexed DNA fragments (mono- and oligo-nucleosomes) in the
cytoplasm of apoptotic cells. This assay does
not require pre-labeling of the cells and can detect DNA degradation in cells
that do not proliferate in vitro (i.e. freshly
isolated tumor cells).
The most important effector molecules for triggering apoptotic cell death are
caspases. Caspases are proteases
that when activated cleave numerous substrates at the carboxy-terminal site of
an aspartate residue mediating very early
stages of apoptosis upon activation. All caspases are synthesized as pro-
enzymes and activation involves cleavage at
aspartate residues. In particular, caspase 3 seems to play a central role in
the initiation of cellular events of apoptosis.
Assays for determination of caspase 3 activation detect early events of
apoptosis. Following RNAi treatments, Western blot
detection of active caspase 3 presence or proteolytic cleavage of products
(i.e. PARP) found in apoptotic cells further
support an active induction of apoptosis. Because the cellular mechanisms that
result in apoptosis are complex, each has its
advantages and limitations. Consideration of other criteria/endpoints such as
cellular morphology, chromatin condensation,
membrane blebbing, apoptotic bodies help to further support cell death as
apoptotic. Since not all the gene targets that
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regulate cell growth are anti-apoptotic, the DNA content of permeabilized
cells is measured to obtain the profile of DNA
content or cell cycle profile. Nuclei of apoptotic cells contain less DNA due
to the leaking out to the cytoplasm (sub-G1
population). In addition, the use of DNA stains (i.e., propidium iodide) also
differentiate between the different phases of the
cell cycle in the cell population due to the presence of different quantities
of DNA in GO/G1, S and G2/M. In these studies the
subpopulations can be quantified.
For the 109P1 D4 gene, RNAi studies facilitate the understanding of the
contribution of the gene product in cancer
pathways. Such active RNAi molecules have use in identifying assays to screen
for mAbs that are active anti-tumor
therapeutics. Further, siRNA are administered as therapeutics to cancer
patients for reducing the malignant growth of
several cancer types, including those listed in Table I. When 109P1D4 plays a
role in cell survival, cell proliferation,
tumorigenesis, or apoptosis, it is used as a target for diagnostic,
prognostic, preventative and/or therapeutic purposes.
Throughout this application, various website data content, publications,
patent applications and patents are
referenced. (Websites are referenced by their Uniform Resource Locator, or
URL, addresses on the World Wide Web.)
The present invention is not to be limited in scope by the embodiments
disclosed herein, which are intended as
single illustrations of individual aspects of the invention, and any that are
functionally equivalent are within the scope of the
invention. Various modifications to the models and methods of the invention,
in addition to those described herein, will
become apparent to those skilled in the art from the foregoing description and
teachings, and are similarly intended to fall
within the scope of the invention. Such modifications or other embodiments can
be practiced without departing from the true
scope and spirit of the invention.
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TABLES:
TABLE I: Tissues that Express 109P1 D4 when malignant:
Prostate
Bladder
Kidney
Colon
Lymphoma
Lung
Pancreas
Ovary
Breast
Uterus
Stomach
Rectum
Cervix
Lymph Node
Bone
TABLE 11: Amino Acid Abbreviations
SINGLE LETTER THREE LETTER FULL NAME
F Phe phenylalanine
L Leu leucine
S Ser serine
Y Tyr tyrosine
C Cys cysteine
W Trp t to han
P Pro proline
H His histidine
Q Gin glutamine
R Arg arginine
I lie isoleucine
M Met methionine
T Thr threonine
N Asn as ara ine
K Lys lysine
V Val valine
A Ala alanine
D Asp aspartic acid
E Glu glutamic acid
G Gly glycine
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TABLE III: Amino Acid Substitution Matrix
Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix
(block substitution matrix). The
higher the value, the more likely a substitution is found in related, natural
proteins. (See world wide web URL
ikp.unibe.ch/manual/blosum62.htm) )
A C D E F G H I K L M N P Q R S T V W Y.
4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A
9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C
6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D
-3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2 E
6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3 F
6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G
8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2 H
4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 1
5 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K
4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L
5 -2 -2 0 -1 -1 -1 1 -1 -1 M
6 -2 0 0 1 0 -3 -4 -2 N
7 -1 -2 -1 -1 -2 -4 -3 P
5 1 0 -1 -2 -2 -1 Q
5 -1 -1 -3 -3 -2 R
4 1 -2 -3 -2 S
5 0 -2 -2 T
4 -3 -1 V
11 2 W
7 Y
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TABLE IV:
HLA Class 1111 Motifs/Supermotifs
TABLE IV (A): HLA Class I Supermotifs/Motifs
SUPERMOTIF POSITION POSITION POSITION
2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary
Anchor)
Al TILVMS FWY
A2 LIVMATQ IVMATL
A3 VSMATLI RK
A24 YFWIVLMT FIYWLM
B7 P VILFMWYA
B27 RHK FYLWMIVA
B44 ED FWYLIMVA
B58 ATS FWYLIMMA
B62 QLIVMP FWYMIVLA
MOTIFS
Al TSM Y
Al DEAS Y
A2.1 LMVQIAT VLIMAT
A3 LMVISATFCGD KYRHFA
Al 1 VTMLISAGNCDF KRYH
A24 YFWM FLIW
A*3101 MVTALI S RK
A*3301 MVALFIST RK
A*6801 AVTMSL1 RK
B*0702 P LMFWYAIV
B*3501 P LMFWYIVA
B51 P LIVFWYAM
B*5301 P IMFWYALV
B*5401 P ATIVLMFWY
Bolded residues are preferred, italicized residues are less preferred: A
peptide is considered motif-bearing if it has primary
anchors at each primary anchor position for a motif or supermotif as specified
in the above table.
TABLE IV (B): HLA Class II Supermotif
1 6 9
W, F, Y, V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y
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TABLE IV (C): HLA Class II Motifs
MOTIFS 1 anchor 1 2 3 4 5 1 anchor 6 7 8 9
DR4 preferred FMYLIVW M T I VSTCPALIM MH MH
deleterious w R WDE
DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM
deleterious C CH FD CWD GDE D
DR7 preferred MFLIVWY M W A IVMSACTPL M IV
deleterious C G GRD N G
DR3 MOTIFS 1 anchor 1 2 3 1 anchor 4 5 1 anchor 6
Motif a preferred LIVMFY D
Motif b preferred LIVMFAY DNQEST KRH
DR Supermotif MFLIVWY VMSTACPLI
Italicized residues indicate less preferred or "tolerated" residues
TABLE IV (D): HLA Class I Supermotifs
POSITION: 1 2 3 4 5 6 7 8 C-terminus
SUPER-
MOTIFS
Al 1 Anchor 1 Anchor
TILVMS FWY
A2 1 Anchor 1 Anchor
LIVMATQ LIVMAT,
A3 Preferred 1 0 Anchor YFW YFW YFW P 1 Anchor
VSMATLI (4/5) (3/5) (4/5) (4/5) RK
deleterious DE (3/5); DE
P (515) (4/5)
A24 1 Anchor 1 Anchor
YFWIVLMT FIYWLM
B7 Preferred FWY (5/5) J' Anchor FWY FWY I 'Anchor
LIVM (3/5) P (4/5) (3/5) VILFMWYA
deleterious DE (3/5); DE G QN DE
P(5/5); (3/5) (4/5) (415) (4/5)
G(415);
A(315);
QN(3/5)
B27 1 Anchor 1 Anchor
RHK FYLWMIVA
B44 1 Anchor 1 Anchor
ED FWYLIMVA
B58 10 Anchor 1 Anchor
ATS FWYLIVMA
B62 10 Anchor 1 Anchor
QLIVMP FWYMIVLA
Italicized residues indicate less preferred or "tolerated" residues
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TABLE IV (E): HLA Class I Motifs
POSITION 1 2 3 4 5 6 7 8 9 C-
terminus
or
C-terminus
Al preferred GFYW l Anchor DEA YFW P DEQN YFW l Anchor
9-mer STM y
deleterious DE RHKLIVMP A G A
Al preferred GRHK ASTCLIVM J 'Anchor GSTC ASTC LIVM DE I 'Anchor
9-mer DEAS Y
deleterious A RHKDEPYFW DE PQN RHK PG GP
Al preferred YFW l Anchor DEAQN A YFWQN PASTC GDE P 1 Anchor
10- STM Y
mer
deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A
Al preferred YFW STCLIVM 1 *Anchor A YFW PG G YFW I 'Anchor
10- DEAS Y
mer
deleterious RHK RHKDEPYFW P G PRHK QN
A2.1 preferred YFW I 'Anchor YFW STC YFW A P l *Anchor
9-mer LMIVQAT VLIMAT
deleterious DEP DERKH RKH DERKH
POSITION:1 2 3 4 5 6 7 8 9 C-
Terminus
A2.1 preferred AYFW J 'Anchor LVIM G G FYWL l 'Anchor
10- LMIVQAT VIM VLIMAT
mer
deleterious DEP DE RKHA P RKH DERKHRKH
A3 preferred RHK I 'Anchor YFW PRHKYF A YFW P I 'Anchor
LMVISATFCGD W KYRHFA
deleterious DEP DE
All preferred A I 'Anchor YFW YFW A YFW YFW P l *Anchor
VTLMISAGNCD KRYH
F
deleterious DEP A G
A24 preferred YFWRHK I 'Anchor STC YFW YFW 1 Anchor
9-mer YFWM FLIW
deleterious DEG DE G QNP DERHKG AQN
A24 Preferred l *Anchor P YFWP P l *Anchor
10- YFWM FLIW
mer
Deleterious GDE QN RHK DE A QN DEA
A3101 Preferred RHK 1 *Anchor YFW P YFW YFW AP l 'Anchor
MVTALIS RK
Deleterious DEP DE ADE DE DE DE
A3301 Preferred I 'Anchor YFW AYFW I 'Anchor
MVALFIST RK
Deleterious GP DE
A6801 Preferred YFWSTC J 'Anchor YFWLIV YFW P 1 Anchor
AVTMSLI M RK
deleterious GP DEG RHK A
B0702Preferred RHKFWY l Anchor RHK RHK RHK RHK PA l Anchor
P LMFWYAI
V
deleterious DEQNP DEP DE DE GDE QN DE
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POSITION 1 2 3 4 5 6 7 8 9 C-
terminus
or
C-terminus
Al preferred GFYW J *Anchor DEA YFW P DEQN YFW 1 *Anchor
9-mer STM y
deleterious DE RHKLIVMP A G A
Al preferred GRHK ASTCLIVM 1 Anchor GSTC ASTC LIVM DE 1 Anchor
9-mer DEAS Y
deleterious A RHKDEPYFW DE PQN RHK PG GP
63501 Preferred FWYLIVM I 'Anchor FWY FWY 1 Anchor
P LMFWYIV
A
deleterious AGP G G
B51 Preferred LIVMFWY J 'Anchor FWY STC FWY G FWY I 'Anchor
P LIVFWYA
M
deleterious AGPDER DE G DEQN GDE
HKSTC
B5301 preferred LIVMFWY 1 Anchor FWY STC FWY LIVMFWYFWY 1 Anchor
P IMFWYAL
V
deleterious AGPQN G RHKQN DE
B5401 preferred FWY 1 *Anchor FWYLIVM LIVM ALIVM FWYA 1 Anchor
P P ATIVLMF
WY
deleterious GPQNDE GDESTC RHKDE DE QNDGE DE
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TABLE IV (F):
Summa of HLA-supertypes
Overall phenotypic frequencies of HLA-su ert es in different ethnic
populations
Specificity Phenotypic frequency
Sue ePosition 2 C-Terminus Caucasian N.A. Black Ja aneseChineseHispanic
Average
B7 P ILMVFWY43.2 55.1 57.1 43.0 49.3 49.5
3 ILMVST RK 37.5 2.1 15.8 52.7 13.1 4.2
2 ILMVT ILMVT 5.8 39.0 12.4 15.9 13.0 2.2
24 F WIVLMT Fl (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0
B44 E (D) FWYLIMV 3.0 21.2 12.9 39.1 39.0 37.0
1 I (LVMS) FWY 7.1 16.1 21.8 14.7 26.3 25.2
B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4
B62 QL IVMP FWY MI 12.6 1.8 36.5 25.4 11.1 18.1
B58 TS FWY LI 10.0 25.1 11.6 9.0 5.9 10.3
TABLE IV (G):
Calculated population coverage afforded by different HLA-su ert a combinations
HLA-supertypes Phenotypic frequency
Caucasian N.A Blacks -Japanese Chinese Hispanic Average
83.0 86.1 87.5 88.4 86.3 86.2
2, A3 and B7 99.5 98.1 100.0 99.5 99.4 99.3
2, A3, B7, A24, B44 99,9 99.6 100.0 99.8 99.9 99.8
and Al
2, A3, B7, A24,
B44, Al, B27, B62,
and B 58
Motifs indicate the residues defining supertype specificites. The motifs
incorporate residues determined on the basis of
published data to be recognized by multiple alleles within the supertype.
Residues within brackets are additional residues
also predicted to be tolerated by multiple alleles within the supertype.
able V: Frequently Occurring Motifs
Name avrg. % Description Potential Function
identity
Nucleic acid-binding protein functions as
transcription factor, nuclear location
zf-C2H2 34% Zinc finger, C2H2 type probable
Cytochrome b(N- membrane bound oxidase, generate
c tochrome_b_N 68% terminal /b6/ etB su eroxide
domains are one hundred amino acids
long and include a conserved
19% Immunoglobulin domain intradomain disulfide bond.
tandem repeats of about 40 residues,
each containing a Trp-Asp motif.
Function in signal transduction and
WD40 18% WD domain, G-beta repeat protein interaction
may function in targeting signaling
PDZ 23% PDZ domain molecules to sub-membranous sites
LRR 28% Leucine Rich Repeat short sequence motifs involved in
protein-protein interactions
onserved catalytic core common to
both serine/threonine and tyrosine
protein kinases containing an ATP
Pkinase 23% Protein kinase domain binding site and a catalytic site
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pleckstrin homology involved in
intracellular signaling or as constituents
PH 16% PH domain of the cytoskeleton
30-40 amino-acid long found in the
extracellular domain of membrane-
EGF 34% EGF-like domain bound proteins or in secreted proteins
Reverse transcriptase
(RNA-dependent DNA
Rvt 9% of merase
Cytoplasmic protein, associates integral
nk 25% nk repeat membrane proteins to the cytoskeleton
NADH- membrane associated. Involved in
Ubiquinone/plastoquinone proton translocation across the
Oxidored_ 1 32% (complex 1), various chains membrane
calcium-binding domain, consists of a12
residue loop flanked on both sides by a
Efhand 24% EF hand 12 residue alpha-helical domain
Retroviral aspartyl spartyl or acid proteases, centered on
Rvp 79% protease a catalytic as art l residue
extracellular structural proteins involved
in formation of connective tissue. The
Collagen triple helix repeat sequence consists of the G-X-Y and the
Collagen 2% (20 copies) of a tide chains forms a triple helix.
Located in the extracellular ligand-
binding region of receptors and is about
200 amino acid residues long with two
pairs of cysteines involved in disulfide
Fn3 20% Fibronectin type III domain bonds
seven hydrophobic transmembrane
regions, with the N-terminus located
7 transmembrane receptor extracellularly while the C-terminus is
7tm 1 19% (rhodopsin family) cytoplasmic. Signal through G proteins
Table VI: Post-translational modifications of 109P1D4
0-glycosylation sites
231 S
238 S
240 T
266 T
346 T
467 T
551 T
552 S
555 T
595 T
652 S
654 S
660 T
790 T
795 T
798 T
804 S
808 S
923 T
927 T
954 T
979 S
982 S
983 S
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985 S
986 S
990 S
999 T
1000 T
1006 S
1017 S
1020 T
Spring phosphorylation sites
50 DLNLSLIPN (SEQ ID NO: 62)
147 VINISIPEN (SEQ ID NO: 63)
152 IPENSAINS (SEQ ID NO: 64)
238 ILQVSVTDT (SEQ ID NO: 65)
257 EIEVSIPEN (SEQ ID NO: 66)
428 LDYESTKEY (SEQ ID NO: 67)
480 PENNSPGIQ (SEQ ID NO: 68)
489 LTKVSAMDA (SEQ ID NO: 69)
495 MDADSGPNA (SEQ ID NO: 70)
559 TVFVSIIDQ (SEQ ID NO: 71)
567 QNDNSPVFT (SEQ ID NO: 72)
608 AVTLSILDE (SEQ ID NO: 73)
630 RPNISFDRE (SEQ ID NO: 74)
638 EKQESYTFY (SEQ ID NO: 75)
652 GGRVSRSSS (SEQ ID NO: 76)
654 RVSRSSSAK (SEQ ID NO: 77)
655 VSRSSSAKV (SEQ ID NO: 78)
656 SRSSSAKVT (SEQ ID NO: 79)
714 EVRYSIVGG (SEQ ID NO: 80)
789 LVRKSTEAP (SEQ ID NO: 81)
805 ADVSSPTSD (SEQ ID NO: 82)
808 SSPTSDYVK (SEQ ID NO: 83)
852 NKQNSEWAT (SEQ ID NO: 84)
877 KKKHSPKNL (SEQ ID NO: 85)
898 DDVDSDGNR (SEQ ID NO: 86)
932 FKPDSPDLA (SEQ ID NO: 87)
941 RHYKSASPQ (SEQ ID NO: 88)
943 YKSASPQPA (SEQ ID NO: 89)
982 ISKCSSSSS (SEQ ID NO: 90)
983 SKCSSSSSD (SEQ ID NO: 91)
984 KCSSSSSDP (SEQ ID NO: 92)
985 CSSSSSDPY (SEQ ID NO: 93)
990 SDPYSVSDC (SEQ ID NO: 94)
1006 EVPVSVHTR (SEQ ID NO: 95)
Threonine phosphorylation sites
29 EKNYTIREE (SEQ ID NO: 96)
81 IEEDTGEIF (SEQ ID NO: 97)
192 DVIETPEGD (SEQ ID NO: 98)
252 VFKETEIEV (SEQ ID NO: 99)
310 TGLITIKEP (SEQ ID NO: 100)
320 DREETPNHK (SEQ ID NO: 101)
551 VPPLTSNVT (SEQ ID NO: 102)
790 VRKSTEAPV (SEQ ID NO: 103)
856 SEWATPNPE (SEQ ID NO: 104)
924 NWVTTPTTF (SEQ ID NO: 105)
927 TTPTTFKPD (SEQ ID NO: 106)
999 GYPVTTFEV (SEQ ID NO: 107)
1000 YPVTTFEVP (SEQ ID NO: 108)
Tyrosine phosphorylation sites
67 FKLVYKTGD (SEQ ID NO: 109)
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158 INSKYTLPA (SEQ ID NO: 110)
215 EKDTYVMKV (SEQ ID NO: 111)
359 IDIRYIVNP (SEQ ID NO: 112)
423 ETAAYLDYE (SEQ ID NO: 113)
426 AYLDYESTK (SEQ ID NO: 114)
432 STKEYAIKL (SEQ ID NO: 115)
536 KEDKYLFTI (SEQ ID NO: 116)
599 TDPDYGDNS (SEQ ID NO: 117)
642 SYTFYVKAE (SEQ ID NO: 118)
682 SNCSYELVL (SEQ ID NO: 119)
713 AEVRYSIVG (SEQ ID NO: 120)
810 PTSDYVKIL (SEQ ID NO: 121)
919 TMGKYNWVT (SEQ ID NO: 122)
989 SSDPYSVSD (SEQ ID NO: 123)
996 SDCGYPVTT (SEQ ID NO: 124)
Table VII:
Search Peptides
109P1 D4 v.1- 9-mers, 10-mers and 15-mers (SEQ ID NO: 125)
MDLLSGTYIF AVLLACVVFH SGAQEKNYTI REEMPENVLI GDLLKDLNLS LIPNKSLTTA 60
MQFKLVYKTG DVPLIRIEED TGEIFTTGAR IDREKLCAGI PRDEHCFYEV EVAILPDEIF 120
RLVKIRFLIE DINDNAPLFP ATVINISIPE NSAINSKYTL PAAVDPDVGI NGVQNYELIK 180
SQNIFGLDVI ETPEGDKMPQ LIVQKELDRE EKDTYVMKVK VEDGGFPQRS STAILQVSVT 240
DTNDNHPVFK ETEIEVSIPE NAPVGTSVTQ LHATDADIGE NAKIHFSFSN LVSNIARRLF 300
HLNATTGLIT IKEPLDREET PNHKLLVLAS DGGLMPARAM VLVNVTDVND NVPSIDIRYI 360
VNPVNDTVVL SENIPLNTKI ALITVTDKDA DHNGRVTCFT DHEIPFRLRP VFSNQFLLET 420
AAYLDYESTK EYAIKLLAAD AGKPPLNQSA MLFIKVKDEN DNAPVFTQSF VTVSIPENNS 480
PGIQLTKVSA MDADSGPNAK INYLLGPDAP PEFSLDCRTG MLTVVKKLDR EKEDKYLFTI 540
LAKDNGVPPL TSNVTVFVSI IDQNDNSPVF THNEYNFYVP ENLPRHGTVG LITVTDPDYG 600
DNSAVTLSIL DENDDFTIDS QTGVIRPNIS FDREKQESYT FYVKAEDGGR VSRSSSAKVT 660
INVVDVNDNK PVFIVPPSNC SYELVLPSTN PGTVVFQVIA VDNDTGMNAE VRYSIVGGNT 720
RDLFAIDQET GNITLMEKCD VTDLGLHRVL VKANDLGQPD SLFSVVIVNL FVNESVTNAT 780
LINELVRKST EAPVTPNTEI ADVSSPTSDY VKILVAAVAG TITVVVVIFI TAVVRCRQAP 840
HLKAAQKNKQ NSEWATPNPE NRQMIMMKKK KKKKKHSPKN LLLNFVTIEE TKADDVDSDG 900
NRVTLDLPID LEEQTMGKYN WVTTPTTFKP DSPDLARHYK SASPQPAFQI QPETPLNSKH 960
HIIQELPLDN TFVACDSISK CSSSSSDPYS VSDCGYPVTT FEVPVSVHTR PVGIQVSNTT 1020
F 1021
109P1 D4 v.2 (both ends diff from v.1)
N' terminal
9-mers as -30 to 8
MRTERQWVLIQIFQVLCGLIQQTVTSVPGMDLLSGTY (SEQ ID NO: 126)
10-mers as -30 to 9
MRTERQWVLIQIFQVLCGLIQQTVTSVPGMDLLSGTYI (SEQ ID NO: 127)
15-mers as -30 to 14
MRTERQWVLIQIFQVLCGLIQQTVTSVPGMDLLSGTYIFAVLL (SEQ ID NO: 128)
109P1 D4 v.2
C' Terminal
9 mers: as 1004 to 1025
PVSVHTRPTDSRTSTIEICSEI (SEQ ID NO: 129)
mers: as 1003 to 1025
VPVSVHTRPTDSRTSTIEICSEI (SEQ ID NO: 130)
mers: as 997 to 1025
VTTFEVPVSVHTRPTDSRTSTIEICSEI (SEQ ID NO: 131)
109P1 D4 v.3
9 mers: as 1004 to 1347 (SEQ ID NO: 132)
PVSVHTRPPMKEVVRSCTPMKESTTMEIWIHPQPQRKSEGKVAGKSQRRVTFHLPEGSQESSSDG
GLGDHDAGSLTSTSHGLPLGYPQEEYFDRATPSNRTEGDGNSDPESTFIPGLKKAAEITVQPTVE
EASDNCTQECLIYGHSDACWMPASLDHSSSSQAQASALCHSPPLSQASTQHHSPRVTQTIALCHS
PPVTQTIALCHSPPPIQVSALHHSPPLVQATALHHSPPSAQASALCYSPPLAQAAAISHSSPLPQ
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VIALHRSQAQSSVSLQQGWVQGADGLCSVDQGVQGSATSQFYTMSERLHPSDDSIKVIPLTTFTP
RQQARPSRGDSPMEEHPL
mers: as 1003 to 1347 (SEQ ID NO: 133)
VPVSVHTRPPMKEVVRSCTPMKESTTMEIWIHPQPQRKSEGKVAGKSQRRVTFHLPEGSQESSSD
GGLGDHDAGSLTSTSHGLPLGYPQEEYFDRATPSNRTEGDGNSDPESTFIPGLKKAAEITVQPTV
EEASDNCTQECLIYGHSDACWMPASLDHSSSSQAQASALCHSPPLSQASTQHHSPRVTQTIALCH
SPPVTQTIALCHSPPPIQVSALHHSPPLVQATALHHSPPSAQASALCYSPPLAQAAAISHSSPLP
QVIALHRSQAQSSVSLQQGWVQGADGLCSVDQGVQGSATSQFYTMSERLHPSDDSIKVIPLTTFT
PRQQARPSRGDSPMEEHPL
mers: as 998 to 1347 (SEQ ID NO: 134)
VTTFEVPVSV HTRPPMKEVV RSCTPMKEST TMEIWIHPQP QRKSEGKVAG KSQRRVTFHL
PEGSQESSSD GGLGDHDAGS LTSTSHGLPL GYPQEEYFDR ATPSNRTEGD GNSDPESTFI
PGLKKAAEIT VQPTVEEASD NCTQECLIYG HSDACWMPAS LDHSSSSQAQ ASALCHSPPL
SQASTQHHSP RVTQTIALCH SPPVTQTIAL CHSPPPIQVS ALHHSPPLVQ ATALHHSPPS
AQASALCYSP PLAQAAAISH SSPLPQVIAL HRSQAQSSVS LQQGWVQGAD GLCSVDQGVQ
GSATSQFYTM SERLHPSDDS IKVIPLTTFT PRQQARPSRG DSPMEEHPL
109P1 D4 v.4 (deleting 10 aa, 1039-1048, from v.1)
9-mers as 1031-1056 (deleting 10 aa,1039-1048, from v.1)
IWIHPQPQSQRRVTFH (SEQ ID NO: 135)
10-mers as 1030- 1057 (deleting 10 aa, 1039-1048, from v.1)
EIWIHPQPQSQRRVTFHL (SEQ ID NO: 136)
15-mers as 1025- 1062 (deleting 10 aa, 1039-1048, from v.1)
ESTTMEIWIHPQPQSQRRVTFHLPEGSQ (SEQ ID NO: 137)
109P1 D4 v.5 (deleting 37 aa, 1012-1048, from v.1)
9-mers as 1004-1056 (deleting 37 aa,1012-1048, from v.1)
PVSVHTRPSQRRVTFH (SEQ ID NO: 138)
10-mers as 1003-1057 (deleting 37 aa, 1012-1048, from v.1)
VPVSVHTRPSQRRVTFHL (SEQ ID NO: 139)
15-mers as 998-1062 (deleting 37 aa, 1012-1048, from v.1)
VTTFEVPVSVHTRPSQRRVTFHLPEGSQ (SEQ ID NO: 140)
109P1 D4 v.6 (both ends diff from v.1)
N' terminal
9-mers: as -23 to 10 (excluding I and 2)
MTVGFNSDISSVVRVNTTNCHKCLLSGTYIF (SEQ ID NO: 141)
10-mers: as -23 to 11 (excluding I and 2)
MTVGFNSDISSVVRVNTTNCHKCLLSGTYIFA (SEQ ID NO: 142)
15-mers: as -23 to 17 (excluding 1 and 2)
MTVGFNSDISSVVRVNTTNCHKCLLSGTYIFAVLLVC (SEQ ID NO: 143)
109P1 D4 v.6
C' terminal
9-mers: as 1004-1016
PVSVHTRPTDSRT (SEQ ID NO: 144)
10-mers: as 1003-1016
VPVSVHTRPTDSRT (SEQ ID NO: 145)
15-mers: as 998-1016
VTTFEVPVSVHTRPTDSRT (SEQ ID NO: 146)
109P1 D4 v.7 (N-terminal 21 as diff from those in v.6)
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N' terminal
9-mers as -21 to 10 (excluding 1 and 2)
MFRVGFLIISSSSSLSPLLLVSVVRVNTT (SEQ ID NO: 147)
1 0-mers as -21 to 11 (excluding 1 and 2)
MFRVGFLIISSSSSLSPLLLVSVVRVNTTN (SEQ ID NO: 148)
15-mers as -21 to 16 (excluding 1 and 2)
MFRVGFLIISSSSSLSPLLLVSVVRVNTTNCHKCL (SEQ ID NO: 149)
109P1 D4 v.8
9-mers as 1099-1126 (excluding 1117 and 1118)
TFIPGLKKEITVQPTV (SEQ ID NO: 150)
10-mers as 1098-1127 (excluding 1117 and 1118)
STFIPGLKKEITVQPTVE (SEQ ID NO: 151)
15-mers as 1093-1131 (excluding 1117 and 1118)
NSDPESTFIPGLKKEITVQPTVEEASDN (SEQ ID NO: 152)
109P1D4 v.1, v.2 and v.3 SNP variants
Al 5V
9-mers
TYIFAVLLVCVVFHSGA (SEQ ID NO: 153)
10-mers
GTYIFAVLLVCVVFHSGAQ (SEQ ID NO: 154)
15-mers
MDLLSGTYIFAVLLVCVVFHSGAQEKNYT (SEQ ID NO: 155)
109P1D4 v.1, v.2 and v.3 SNP variants
M341
9-mers
KNYTIREEIPENVLIGD (SEQ ID NO: 156)
10-mers
EKNYTIREEIPENVLIGDL (SEQ ID NO: 157)
15-mers
HSGAQEKNYTIREEIPENVLIGDLLKDLN (SEQ ID NO: 158)
109P1D4 v.1, v.2 and v.3 SNP variants
M341 and D42N
9-mers
KNYTIREEIPENVLIGN (SEQ ID NO: 159)
10-mers
EKNYTIREEIPENVLIGNL (SEQ ID NO: 160)
15-mers
HSGAQEKNYTIREEIPENVLIGNLLKDLN (SEQ ID NO: 161)
109P1D4 v.1, v.2 and v.3 SNP variants
D42N
9-mers
MPENVLIGNLLKDLNLS (SEQ ID NO: 162)
10-mers
EMPENVLIGNLLKDLNLSL (SEQ ID NO: 163)
15-mers
YTIREEMPENVLIGNLLKDLNLSLIPNKS (SEQ ID NO: 164)
109P1D4 v.1, v.2 and v.3 SNP variants
D42N and M341
9-mers
IPENVLIGNLLKDLNLS (SEQ ID NO: 165)
10-mers
EIPENVLIGNLLKDLNLSL (SEQ ID NO: 166)
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15-mers
YTIREEIPENVLIGNLLKDLNLSLIPNKS (SEQ ID NO: 167)
109P1 D4 v.1, v.2 and v.3 SNP variants
A60T
9-mers
IPNKSLTTTMQFKLVYK (SEQ ID NO: 168)
10-mers
LIPNKSLTTTMQFKLVYKT (SEQ ID NO: 169)
15-mers
DLNLSLIPNKSLTTTMQFKLVYKTGDVPLI (SEQ ID NO: 170)
109P1D4 v.1, v.2 and v.3 SNP variants
1154V
9-mers
ISIPENSAVNSKYTLPA (SEQ ID NO: 171)
10-mers
NISIPENSAVNSKYTLPAA (SEQ ID NO: 172)
15-mers
PATVINISIPENSAVNSKYTLPAAVDPDV (SEQ ID NO: 173)
109P1 D4 v.1, v.2 and v.3 SNP variants
V2921
9-mers
IHFSFSNLISNIARRLF (SEQ ID NO: 174)
10-mers
KIHFSFSNLISNIARRLFH (SEQ ID NO: 175)
15-mers
IGENAKIHFSFSNLISNIARRLFHLNATT (SEQ ID NO: 176)
109P1 D4 v.1, v.2 and v.3 SNP variants
T420N
9-mers
FSNQFLLENAAYLDYES (SEQ ID NO: 177)
10-mers
VFSNQFLLENAAYLDYEST (SEQ ID NO: 178)
15-mers
FRLRPVFSNQFLLENAAYLDYESTKEYAI (SEQ ID NO: 179)
109P1 D4 v.1, v.2 and v.3 SNP variants
T486M
9-mers
NNSPGIQLMKVSAMDAD (SEQ ID NO: 180)
10-mers
ENNSPGIQLMKVSAMDADS (SEQ ID NO: 181)
15-mers
TVSIPENNSPGIQLMKVSAMDADSGPNAK (SEQ ID NO: 182)
109P1 D4 v.1, v.2 and v.3 SNP variants
T486M and M491T
9-mers
NNSPGIQLMKVSATDAD (SEQ ID NO: 183)
10-mers
ENNSPGIQLMKVSATDADS (SEQ ID NO: 184)
15-mers
TVSIPENNSPGIQLMKVSATDADSGPNAK (SEQ ID NO: 185)
109P1 D4 v.1, v.2 and v.3 SNP variants
T486M and M491T and K500E
15-mers
TVSIPENNSPGIQLMKVSATDADSGPNAE (SEQ ID NO: 186)
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109P1D4 v.1, v.2 and v.3 SNP variants
T486M and K500E
15-mers
TVSIPENNSPGIQLMKVSAMDADSGPNAE (SEQ ID NO: 187)
109P1D4 v.1, v.2 and v.3 SNP variants
M491T
9-mers
IQLTKVSATDADSGPNA (SEQ ID NO: 188)
10-mers
GIQLTKVSATDADSGPNAI< (SEQ ID NO: 189)
15-mers
ENNSPGIQLTKVSATDADSGPNAKINYLL (SEQ ID NO: 190)
109P1D4 v.1, v.2 and v.3 SNP variants
M491T and T486M
9-mers
IQLNKVSATDADSGPNA (SEQ ID NO: 191)
10-mers
GIQLNKVSATDADSGPNAK (SEQ ID NO: 192)
15-mers
ENNSPGIQLNKVSATDADSGPNAKINYLL (SEQ ID NO: 193)
109P1D4 v.1, v.2 and v.3 SNP variants
M491T and T486M and K500E
10-mers
GIQLNKVSATDADSGPNAE (SEQ ID NO: 194)
15-mers
ENNSPGIQLNKVSATDADSGPNAEINYLL (SEQ ID NO: 195)
109P1 D4 v.1, v.2 and v.3 SNP variants
M491 T and K500E
15-mers
ENNSPGIQLTKVSATDADSGPNAEINYLL (SEQ ID NO: 196)
109P1 D4 v.1, v.2 and v.3 SNP variants
K500E
9-mers
DADSGPNAEINYLLGPD (SEQ ID NO: 197)
10-mers
MDADSGPNAEINYLLGPDA (SEQ ID NO: 198)
15-mers
TKVSAMDADSGPNAEINYLLGPDAPPEFS (SEQ ID NO: 199)
109P1 D4 v.1, v.2 and v.3 SNP variants
K500E and M491T
1 0-mers
TDADSGPNAEINYLLGPDA (SEQ ID NO: 200)
15-mers
TKVSATDADSGPNAEINYLLGPDAPPEFS (SEQ ID NO: 201)
109P1D4 v.1, v.2 and v.3 SNP variants
K500E and M491T and T486M
15-mers
MKVSATDADSGPNAEINYLLGPDAPPEFS (SEQ ID NO: 202)
109P1 D4 v.1, v.2 and v.3 SNP variants
K500E and T486M
15-mers
MKVSAMDADSGPNAEINYLLGPDAPPEFS (SEQ ID NO: 203)
109P1 D4 v.1, v.2 and v.3 SNP variants
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C517R
9-mers
APPEFSLDRRTGMLTVV (SEQ ID NO: 204)
10-mers
DAPPEFSLDRRTGMLTVVK (SEQ ID NO: 205)
15-mers
INYLLGPDAPPEFSLDRRTGMLTVVKKLDRE (SEQ ID NO: 206)
109P1D4 v.1, v.2 and v.3 SNP variants
N576K
9-mers
PVFTHNEYKFYVPENLP (SEQ ID NO: 207)
1 0-mers
SPVFTHNEYKFYVPENLPR (SEQ ID NO: 208)
15-mers
DQNDNSPVFTHNEYKFYVPENLPRHGTVG (SEQ ID NO: 209)
109P1 D4 v.1, v.2 and v.3 SNP variants
S678Y
9-mers
KPVFIVPPYNCSYELVLPS (SEQ ID NO: 210)
10-mers
NKPVFIVPPYNCSYELVLPST (SEQ ID NO: 211)
15-mers
VDVNDNKPVFIVPPYNCSYELVLPSTNPG (SEQ ID NO: 212)
109P1D4 v.1, v.2 and v.3 SNP variants
S678Y and C680Y
9-mers
KPVFIVPPYNYSYELVLPS (SEQ ID NO: 213)
10-mers
NKPVFIVPPYNYSYELVLPST (SEQ ID NO: 214)
15-mers
VDVNDNKPVFIVPPYNYSYELVLPSTNPG (SEQ ID NO: 215)
109P1 D4 v.1, v.2 and v.3 SNP variants
C680Y
9-mers
VFIVPPSNYSYELVLPS (SEQ ID NO: 216)
10-mers
PVFIVPPSNYSYELVLPST (SEQ ID NO: 217)
15-mers
VNDNKPVFIVPPSNYSYELVLPSTNPGTV (SEQ ID NO: 218)
109P1D4 v.1, v.2 and v.3 SNP variants
C680Y and S678Y
9-mers
VFIVPPYNYSYELVLPS (SEQ ID NO: 219)
10-mers
PVFIVPPYNYSYELVLPST (SEQ ID NO: 220)
15-mers
VNDNKPVFIVPPYNYSYELVLPSTNPGTV (SEQ ID NO: 221)
109P1 D4 v.1, v.2 and v.3 SNP variants
T7901
9-mers
INELVRKSIEAPVTPNT (SEQ ID NO: 222)
1 0-mers
LINELVRKSIEAPVTPNTE (SEQ ID NO: 223)
15-mers
VTNATLINELVRKSIEAPVTPNTEIADVS (SEQ ID NO: 224)
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109P1D4 v.1, v.2 and v.3 SNP variants
K846M
9-mers
HLKAAQKNMQNSEWATP (SEQ ID NO: 225)
10-mers
PHLKAAQKNMQNSEWATPN (SEQ ID NO: 226)
1 5-mers
RCRQAPHLKAAQKNMQNSEWATPNPENRQ (SEQ ID NO: 227)
109P1D4 v.1, v.2 and v.3 SNP variants
F855V
9-mers
SPKNLLLNVVTIEETKA (SEQ ID NO: 228)
10-mers
HSPKNLLLNVVTIEETKAD (SEQ ID NO: 229)
15-mers
KKKKKHSPKNLLLNVVTIEETKADDVDSD (SEQ ID NO: 230)
109P1D4 v.1, v.2 and v.3 SNP variants
S958L
9-mers
IQPETPLNLKHHIIQEL (SEQ ID NO: 231)
10-mers
QIQPETPLNLKHHIIQELP (SEQ ID NO: 232)
15-mers
PQPAFQIQPETPLNLKHHIIQELPLDNTF (SEQ ID NO: 233)
109P1D4 v.1, v.2 and v.3 SNP variants
K980N
9-mers
FVACDSISNCSSSSSDP (SEQ ID NO: 234)
10-mers
TFVACDSISNCSSSSSDPY (SEQ ID NO: 235)
15-mers
LPLDNTFVACDSISNCSSSSSDPYSVSDC (SEQ ID NO: 236)
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Tables VIII - XXI:
à Table VIII -109P1 D4v. 1 Al Table VilI 109PI D4v 1 Al- Table VIII -109P1
D4v. 1 Al-
9-mers 9 mers 9-mers
.. .........._ ...... _..........
Each peptide is a portion of Each peptide is a portion of Each peptide is a
portion of
SEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each start
position is specified, the position is specified, the position is specified,
the
length of peptide is 9 amino length of peptide is 9 amino length of peptide is
9 amino
acids, and the end position for! acids, and the end position for acids, and
the end position fort
each peptide is the start each peptide is the start each peptide is the start
position plus eight position plus eight position plus eighth .__..J
Start Subse Subsequenceore Start Subse uence Score Start Subse uence; Score
1 DLEEQTMGK 90.000 ( 522 LTVVKKLDR #l 1.250
895 DVDSDGNRV' 0.500
399 FTDHEIPFR 25 00 85 j FTTGARIDR 1.250 700 AVDNDTGMN, 0.500
189' VIETPEGDK 18.000 779 ! ATLINELVR 1.250 ! 8 DADHNGRVT 0.500 ,
94 (VTDPDYGDN W12 500 192 TPEGDKMPQ; 1.125 1 802 jDVSS 0.500
( 278 IGENAKIHF 11.250 858 NPENRQMIM 1.125 645 AEDGGRVSR 0.500
275 DADIGENAK 10 000 148 IPENSAINS_ 1.125 740 DVTDLGLHR 0500
492 DADSGPNAK 10 000 591 LITVTDPDY 1 000 f 617 TIDSQTGVI 0.500
370 11~LSENIPLNT 6 750 37 NVLIGDLLK ( 1 00072 A(DQETGNi0 5001
_
IF9291, KPD D 16 250 172 GVQNYELIK ! 1 000 304 -ATTGLITIK 0.500
688 STNPG
TVVF ~5 000 800'JIADVSSPTS 1000 241iDTNDNHPVF 1, 0 500
1674 [ IVPPSNCSY 5 00 438 ~AADAGKPPL 1 000 ~4I[SLDCRTGML 0 500
163 (~AVDPDVGIN 5 0972 FVACDSISK 1 000 9741 j ACDSISKC 0,500
113 AILPDEIFR 5 000 518 RTGMLTWK F000 116 PDEIFRLVK ( 0 45
0
IF
242 TNDNHPVFI, j 9 8547, WATPNPENR 00 77 iEEDTGEIF 0.450
220l<VEDGGFP764.500 527 rKLDRE EEL
1.000 45 IPENNSPG(- 0.45
797' , NTEIADVSS '[:4.50O [64 ! KAEDGGRVS 0.900 258 IPENAPVGT 0450
,i 951 ;F-QPETPLNSK' _ 4 500 76 i RIEEDTGEI j 0.900 109 } ^EVVAILPD 0.450
807 TSDYVKILV 3 750 204 QKELDREEK O 900 011 DHEIPFRLR ! 0.450
'[329 ASDGGLMPA -3.750 1708 NAEVRYSIV 0.900 435 KLLAADAGK 0.400
9 TAMQFKLVY j 2.500 316 DREETPNHK, 0900 780 TLINELVRK .400
738 KCDVTDLGL 2.500 128 LIEDINDNA 0,900 256 VSIPENAPV 0.300
354 SIDIRYIVN
E2
1F .500 931 DSPDLARHY 0.750 940 KSASPQPAF 0.300
351 NVPSIDIRY 2500 2 HSGAQEKNY 0.750 851 NSEWATPNP 0.270
932 SPDLARHYK 2.500 981 CSSSSSDPY 0.750 744: LGLHRVLVK 0.250
911 ; _LEEQTMGKY 2,250 . i55 KSLTTAMQF [P.750 11794- DTGMNAEVR; 0.250
789 EAPVTPNw 2250 351 KQESYTFYV -0.675 666 VNDNKPVFI 0.250-
253 ! EIEVSIPEN 11.800 727 DQETGNITL 0.675 38 DKDADHNGR 0.250
F8971 DSDGNRVTL 1.500 9 TGDVPLI I 0.625 s 350 DNVPS(DIR ( 1 0 250
'6 N ._.....__ T9~
985 SSDPYSVSD ' 1.500 495 , SGPNAKINY , 0.625 ! 90 RIDREKLCA 0.250
991, VSDCGYPVT 1 500 804 SSPTSDYVK I 0.600
68 KTGDVPLIR 1.250 221 " VEDGGFPQR 0.500 Table IX-109P1D4v.1-
1~-741 ! VTDLGLHRV 1.250 201 LIVQKELDR _2.500 Al 10 mers
273 ATDADIGEN 1.250 609; ILDENDDFT 0.500
J
570 FTHNEYNFY 1.250 , 892 11KADDVDSDG 0 500
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.... .... _...... _-_. ..................._....
Each peptide is a portion of Table IX-109P1D4v.1- Table IX 109P1D4v.1-
SEQ ID NO: 3; each start Al IL 10-mers Al 10 mers
position is specified, the
length of peptide is 10 amino Each peptide is a portion of Each peptide is a
portion of
acids, and the end position for; SEQ ID NO: 3; each start SEQ ID NO: 3; each
start
position is specified, the position is specified, the
each peptide is the start
length of peptide is 10 amino length of peptide is 10 amino
P. plus nine.
acids, and the end position for acids, and the end position for.,
Pos, Subsequence Score each peptide is the start each peptide is the start
J189f LLETaAYLDY 225 000 position plus nine. position plus nine.
682 DLEE TMGKY' 45 000 Pos Subsequence Score Pos Subsequence ' Score
266, DSGPnAKINY 37 500 47 3 DADIgENAKI 1.000 83 j IKEPIDREET 0.450
1290 RTGMITVVKK J 1.000 , 544! I VNESvTNATL 0.450
142 LSENiPLNTK 27 000 _ !
[ HIK _
195= YLDYeSTKEY 25 000 551( ATLInELVRK 1.000 6105F QAPHIKAAQK 0.400
416' KAEDgGRVSRi 18 000 , 13 DTNDnHPVFK 1 1.000 3 DSPDIARHYK 0 300
101; ASDGgLMPAR' 15 000: 161; DADHnGRVTC 1.000 28 VSIPeNAPVG 0.300
tKADDV F 0.900 220; QSAMIFIKVK 0.300
366` VTDPdY S 12 500 65_9j TIEE
25~ EIEVsIPENA 0900 665'. ADDVdSDGNR 0.25
389! TIDS~~gTGVIR 4 10 000
757 SSDPySVSDC, 7 500 229, KDENdNAPVFI 0.900 2181 LNQSaMLFIK 0.250
'~ _
122 DNVPsIDIRY 6 250 (3381 NSPVfTHNEY 0.750 474 DNDTgMNAEV 0.250
171 FTDHeIPFRL 6 250 60 FSNLVSNIAR 0.750 701! KPDSpDLARH 0 20
5751 VSSPtSDYVK 6 000 # 278, GPDApPEFSL 10 625 530`. QPDS 0 250
407? KQES TFYVK 5 400 3351 QNDNsPVFTH 0.625 676 j TLDLpIDLEE 0.250
1445 FIVPpSNCSY 5000 120= VNDNvPSIDI 0.625233DNAPvFQSF 0.250
1561 STEApVTPNT' 00 231 END Na PVFTQt 0 .625 704 SPDLa !H (sS 0.250
I 438 VNDNkPVFIV 3 0.625 15691 NTEIaDVSSP .225
480, NAEVrYSIVG 4.500 - Y ..
1F 30 } IPENaPVGTS
579 TSDYvKILVA 3 750: X80 LITIkEPLDR 0 500-
381E ILDEnDDFT. 2 500 293 MLTVvRKLDR 0.500 ! 3 3 EKEDkYLFTI 0.225
2 AVDNdTGMNA 2 500 105! GLMFaRAMVL 0.500 Ã 2471 iPENnSPGIQ 1 0.225
129] KLDReKEDKY 2 500. ,7213 QIQPeTPLNS 0.500 351 j VPENIPRHGT ( 0.225
:1 286.~LDCrTGMLT ? 500 280 DAPPeFSLDC 10 500 723 QPETpLNSKH 0.225
1171VTD-VnDNVP 2 500 592 GTITvVVVIF 0.500- 2011 TKEYaIKLLA 0.225
m W 169 TCFTdHEIPF 0.500 50 IGENaKIHFS 0.225
250 NNSPgIQLTK 2 500 ~F~L___ .[
'150111 ETGNiTLMEK 2.500 49 DiGEnAKIHF 0 500 175 EIPFrLRPVF 0 200
1~4 6 DTGMnAEVRYI 2 500 460 STNPgTVVFQ 0 500 193 AAYLdYESTK I Q.20
2~76s LLGPdAPPEF T2 000: 435 WDVnDNKPV 0 500 598 WIFiTAVVR (_0 200
763; VSDCgYPVTT 11 1500 746 ACDSiSK-0111. 500E 456 LVLPsTNPGT 0.200 J
735IQELpLDNTF 1.350 1664 KADDvDSDGNI 0.50q
nISFDR 0. Table X-109P1D4v.1-
396 V RP
513TDLgLHRVL _1250
451 A9E IGENA 1250 332 IIDQnDNSPV 1 0 500 j A0201-9 mers
r -=~ - Each peptide is a portion of
11 I VTDTnDNHPV 1.250 12624 AMDAdSGPNA 500 SEQ ID NO: 3; each start
6301 NPENrQMIMM 1.125 510 KCDVtDLGLH E 0.500 lepo osition is specified,
9 amino
~fDVDSdGNRVT,[ 0.506-
23 ETEleVSIPE~ v1 125 al_...667
I- acids, and the end position ford
210 AADAgKPPLN 1.000 497 AIDQeTGNIT 0.500 each peptide is the start
(264 _DADSgPNAKI 1000 7131[SASPgPAFQI 0.500 position plus eight.
17 -21,KCSSsSSIDPY 0.5 Po n~nc~_ F- ~L
-00
3621 GLITvTDPDY 1.000 I11--5--50~~V1-NALiNEVR 10.500
356 FLLETAAYL 8 98.910
515 DLGLhRVLVK 1 000 L_ ,LT
1-54 _ l-PDEIFRL 1986 272
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142
........ . ....... . . . . . . . . . . .. .. . . _ ...... Table X-109P1 D4v. 1
Table X-109P1 D4v.1- LTable X-109PI D4v.1-
A0201 9 mers A0201-9-mers A0201-9-mers
Each peptide is a portion of Each peptide is a portion of Each peptide is a
portion of
SEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each start
position is specified, the position is specified, the position is specified,
the
length of peptide is 9 amino length of peptide is 9 amino length of peptide is
9 amino
acids, and the end position fort acids, and the end position ford acids, and
the end position ford
each peptide is the start each peptide is the start each peptide is the start
position plus eight. position plus eight. position plus eighty
Pos Se uence Score Pose-Se uence Score Pos Se uence I Score
697 GQPDSLFS 385 691 685! GLHRVLVKAj 1.426 8251 FVTIEETKA 2.000
----- - - _- 7091 NLFVNESVTI 11.305 j ; 906, LPLDNTFVA 1.989
2 ARAM 257.342 , FSNQFLLET 16
1~
[1LMQFKLVYK 10.931 ...._ .^ X3-54- NQFLLETAA 1.864
I R60 GMLTVVKKLt 131296 29g YIVNPVNDT 10.841
II~
765 WVIFITAV 90.423""" = 859 YNWVTTPT [1.85 T 1274I LMPARAMV 10.754 ~ 280
MVLVNVTD 88.043 ------- PARAMVL ,
- 247 GLITIKEPL 10.468 2751 V i 1.775
820 NLLLNFVTI s 73.343 l 2101, QLHATDADI E6.433 = ~( 1436 GPNAKINYL 1 764
61 RLVKIRFLI 60 510 8g FQIQPETPL 963 266 LVLASDGGL 1 528
1549 ILDENDDFT 55 992 ~mm_.. EP- 37
: 490 LTSNVTVFV 9.032 681 VTDLGLHRV
[1 575. KQESYTFYV_ 50389 843{`{ VTLID PIDL 7.652 .; 819 KNLLLNFVT 1498
598: KVTINVVDV, 48.991 423j IQLTKVSAM 7.287 386 LNQSAMLFI 1.465
234' NIARRLFHL 39184 j~68-8-1 RVLVKANDL 6.916 WVVIFITA 1.404
479;TILAKD NGV5. 385 764~~"..
- 5THNEYNFY j 6.317 708j VNLFVNESV 1.399-;
704 SWIVNLFV 3 472 V 309 VLSENIPLN F 1.195
KLVYKTGDV 31 66 = 486 GVPPLTSNV 6 086 , 6 YNF 1
I .
.w
QTMGKYNW; 673; ITLMEKCDV 6 076
854 V 29 ,487 - - 322, LITVTDKDA 1.161
630 NPGTVVFQ 6,057 F77
RQA
PHLKA
29137 V 1.159
753 ILVAAVAGT 291,37 557 AVAGTITVV 5.739 224 IHFSFSNLV 1 154
905; ELPLDNTFV 28.690 6831 DLGLHRVLVj 5.216 454 SLDCRTGM j 1.111
238 RLFHLNATT 27.572_; 300 IVNPVNDTV 5.069j'
12 SQNIFGLDV 26.797 766 VVIFITAW 4.242. w913jFACDSISKC 1106
]930 SVSDC24.952 472 KYLFT 3.789 267 VLASDGGL .098
I 75 APLFPATV` 3.671 --
r
TLMEKCDV _ 370 KEYAIKLLA 1.082
674 T 22 711 763 TVWVIFIT~ 66
" 116 YELIKSQNI 3.j53 j 407 TQSFVTVSI 1 058
123; KIHFSFSNL; 19.533
493 NVTVFVSII 3.271 1169; RSSTAILQVS _1.044
711 FVNESVTNA 18.856 I~ 735` TPNTEIADV 1 044 j
556 FTIDSQTGV, 18 19 ! 67 D FLIEDINDN 3.233
2 ' 6 ITVVVVIFI 6 1I420IISPGIQLTKVI~ 1.044
TMG li.._. F 3 11..w
855 T 16.550 190 1KETEIEVSI 2.911 1171 1 STAILQVSV 1 0.966
1939; TTFEVPVSVj~ 14 65 403 APVFTQSFV 2497 756 [AAVAGTITV 0.966
2.263 1264; KLLVLASDG 0 965
633, TVVFQVIAV 13.997 FSLDCRTG
.....
M 4531366PYESTKEYAI 0.933
625 PSTNPGT: 12 668. -
F 750[VKILVAAV jr2.254 .. I 946 SVHTRPVGI 0.913
284] NVTDVNDN 12226~
7431 VSSPTSDYVI 2.080 658 GNTRDLFAI 0.908
308; WLSENIPL 11,757 61 621 DLFAIDQET 2,068 0IF
VFSNQFLL 0 882 .j
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143
..... .............. ;_....... ..
Table X- 109P1 D4v.1- j Table Xl -109P1 D4v1 A0201- Table XI -109P1 D4v.1-
A0201-
A0201 9-mers10 mers 10 mers
Each peptide is a portion of Each peptide is a portion of SEQ Each peptide is
a portion of SEQ
SEQ ID NO: 3; each start ID NO: 3; each start position is ID NO: 3; each start
position is
position is specified, the specified, the length of peptide is specified, the
length of peptide is
length of peptide is 9 amino 10 amino acids, and the end 10 amino acids, and
the end
acids, and the end position for position for each peptide is the position for
each peptide is the
each peptide is the start start position plus nine. start position plus nine.
position plus eight Pos Subsequence Scores Pos Subsequence Score
Pos; Sequence Score x-7667 VIFItAWRC 9 882 j x-33-9 FTDHeIPFRL 3166
314 I-PL-NTK~IAL~ -q.877 L - -- 672 NITLmEKCDV 9.563 454 ~SLDCrTGMLT 2.981
..._~_ _ __ ....... _ 855 TMGKyNWVTT :.9.149 1 1-. 313 NIPLnTKIAL 2 937
Table XI -109PID4v 1 A0201-
10-mers 173 j _AILQvSVTDT 81720 109 G1NGvQNYEL 2937
....._ W - _. 123 NlFGIDVIET 820 385 PLNQsAMLF! 2.903 `'
Each peptide is a portion of SEQI ..
ID NO: 3; each start position is 934 CGYPvTTFEV 8.427 226 FSFSnLVSNI 2.666
specified, the length of peptide is '4891 i PLTSnVTVFVV 8.416 757 j AVAGtI
2.495
amino acids, and the s NT ....
88
position for each peptide is the 902 IIQEIPLDNT 8.049 370 KEYAiKLLAA 2.48
start position plus nine. 936 ~YPVTtFEVPV 7.936 440 KINY{LGPDA 2,391=
Pos Subseque Scorer 145 KELDrEEKDT_ 7.693 118 LIKSgNIFGL 2.331
_Lej 274_ LMPArAMVLV 196.407 646 GMNAeVRYSI 7.535 291 NVPSiDIRYI 2.310
54 1 LPDelFRL 184.215, 721 LINEIVRKST ! 7142 753 ILA VAaVAGTI s 2306
( 701 SLFSvVIVN 181.794 :500 IIDQnDNSPV 6.503 I 632 GTVVfQVIAV 2 222
549LDEnDFT 168.7031 590~RVSRsSSAKV 6.086 929 l YSVSdCGYPV , 2.088
153 AILPdEIFRL 44.9$1 629 LTNPGtVVFQV : 6.057 37T j LAADaGKPPL i 2.068
510 FTHNeYNFYV 141.751 120 ( KSQNiFGLDV 6038 77 PLFPaTVINI 1.953
223 ; KIHFsFSNLV 127.193; 414 SIPEnNSPGI 5.881 647 ; MNAEvRYSIV 1.946
279
_KDV
jj_ AMVLv 115 5341 402 NAPVfTQSFV 5.313 842 ~, RVTLdLPIDL 1869
76 WVViFI A ~0 423 707 I IVNLfVNESV . 5.069 307 , TVVLsENIPL 1 869
99 TLPAaVDPDV 1 69.5521 321 ALITvTDKDA 4 968 233 SNIArRLFHL 1.860
301 VLSEnIPLNT 1.940 424 QLTKvSAMDA 4.968 1316 LNTKiALITV 1175
67 FLlEdINDNA 45 911 KTGDvPLIRI .782 ; 435 SGPNaKINYL 1.764
548
1 -
SILDeNDDFT 41.891 1265 LLVLaSDGGL~ 4.721 606 VNDNkPVFIV 1.689
J
273 GLMPaRAMVL 32 407 912 FVACdSISKC 4.599 272 GGLMPARAMV 1.680
52 KILVaAVAGT 30.519 478 FTILaKDNGV 4 444 89 KNLLINFVTI _1.676
904 QELPIDNTFV 127.521 1853 EQTMgKYNWV 4 363 ; 930 ~SVSDcGYPVT 1 644
697 GQPDsLFSVV
22.523 680 ~DlGLHRV 4.304 938 ~VTTFeVPVSV 1 642
I
299 YIVNpVNDTV j 21.556 301 NLVSnIARRL -4.272 755 VAAVaGTITV 1 642
522: NLPRhGTVG 21362 75 ~VWIfITAW _; _4.242 0 LPLDnTFVAC 1.589
761 , TITVvVVIFI 18.147 " 300 11 IVNPVNDTVV ` 4 242 422 GIQLtKVSAM ^ 1.571
625 VLPStNPGTV 15 371 197 I SIPEnAPVGT ,4.201 1 ;0758 }~ VAGTiTWW 1.549
822 LLNFvTIEET 4.277 , 603. VVDVnDNKPV ! 4.138 104 1 VDPDvGINGV j 1.549
387 NQSAmLF 13.398j
F-6 41 LVLPsTNPGT 4.101 605 DVNDnKPVFI 1 544
711 FVNEsVTNAT 1 12.298 209 TQLHaTDADi 3.914 620 ` CSYEIVLPST 1.468
703 FSVV1VNLFV 11487 675 LMEKcDVTDL 3_861430 AMDAdSGPNA 1.435
696 LGQPdSLFSV 10.296 i 734 VTPNtEIADV ! 3.777 85 NIS1pENSAI 1.435
i '{ LVYKtGDVPL 10169 636 FQVIaVDNDT 11
3 476Ã
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..................... _.......... ...... .._..._e._.....................
....._._............,........._.... ............. _................
_._..._.......__....
....__................._..__..........._........__.._......_.... _............
.._..__....... _......_...._.d..... ............... _......... _
Table XII -109P1 D4v.1- Table XII -109P1 D4v.1- Table XII -109P1 D4v.1-
A3-9-mers ~ A3-9-mers A3-9-mers
Each peptide is a portion of SEQ Each peptide is a portion of SEQ Each peptide
is a portion of SEQ
ID NO: 3; each start position is ID NO: 3; each start position is ID NO: 3;
each start position is
specified, the length of peptide is 91 specified, the length of peptide is 91
specified, the length of peptide is 9
amino acids, and the end position amino acids, and the end position amino
acids, and the end position
for each peptide is the start for each peptide is the start for each peptide
is the start
position plus eight. position plus eight. position plus eight.
_Pos Subsequen Score Pos Subsequences Score; Pos Subsequence j Score
137 KMPQLIVQI4 90 000 7611 TITVWV!F 0.900 764 VVVVIFITA- j 0 270
375 KLLAADAGK 90.000 320 1 IALITVTDK 0.900, 234 NIARRLFHL 0.270
467 KLDREKD 60000 117 ELIKSQNIF 0 900: 475 KYLFTILAK~ 0.270
720 TLINELVRK 45000 58 EIFRLVKIR 0 900: 64 KIRFLIEDI 0.270
1112 GVQNYELIK _ 36000 701 , SLFSVVIVN - 0 900 680 DVTDLGLHR 0.240
850 DLEEQTMGK 18.000 389 SAMLFIKVK 0 675 ` 476: YLFTILA KD 0.225
805 IMMKKKKKK 15.000 802 RQMIMMKKKj R.675_
674 TLMEKCDVT 1 0.225
803 QMIMM KKKK 1.5.000 760 GTITVVVV. I 0 608 + 662 DLFAIDQET 0 225
781 HLKAAQKNK 10.000 719_+ ATLINELVR 0 600 872 SPDLARHYK1 0.200
806 1 MMKKKKKKK 10.".00 ' 210 3 QLHATDADI 0 600: 775 RCRQAPHLK 10.200
I ------~
_230 NLVSNIARR j 9.000 614 IVPPSNCSY 0 600 510 1_ FTHNEYNFY { , 0.2200
460 ~~ GMLTVVKKL 6.075 48 PLTSNNVTVF 0 600 464 VVKKLDREK 10 200
02 tlNVVDVNDNK 506 P53 1 GIQVSNTT F I 0,6001: 779 r APHLKAAQK P0.200_
61 RLVKIRFLI
j_ I 1_ 4.050 1 7, 39 GIPRDEHCF r0 600 j 684 {f LGLHRVL K .180
27 GLITIKEPL 1 4.50 462 -IF LTVVKKLDR 0 600 454_3 SLDCRTGML 0.180
i 912 FVACDSISK 4.000 25 FTTGARIDR 0 600. 158 KVKVEDGGF 0.180 ;
861-3 WVTTPTTFK 3.000. 249 ! ITIKEPLDR 0.600:
633 TVVFQVIAV 0180 '
820 NLLLNFVTI 2.700 NVTVFVSll 0540 769 FITAVVRC 0.180
1LPDEIFRL 1 2.700 223 KIHFSFSNL 0 540: 598 ~KVTINVVDV 0.180
11 -
6 V I R P N I S F 2.700 -576 ESYTFYVK 0 540 742 DVSSPTSDY 0.180
I 87 NQSAMLFIK 2.700 09 i NLFVNESVT 0.500 241 HLNATTGLI 0.980
i 244 ATTGLITIK 2.250 238 RLFHLNATT 0 500 308 VVLSENIPL 0.180
767 VIFITAVVR 2.000 419 NSPGIQLTK 0.450 575 I KQESYTFYV 0.162
590 RVSRSSSAK 000 ( 753 ILVAAVAGT 0.45
9j, F
391 MLFIKVKD E 0.150
KTGDVPLIR 1.800 891QPETP 0.450 910 NTFVACDSI 0.150
53 _ L AILPDEIFR 1.800 762 ITVWVIFh õ0.405 6281 STNPGTWF 0.150
804 MIMMK 1.500 õ 531 LITVTDPDY 0.400
273 GLMPA HMV 1 350 385 PLQSAMLF 1[,0.400 1 Table XIII -109P1 D4v.1 A3-
356 FLLETAAYL 1--35-0 869 i KPDSPDLAR 0 360 10-mers
{ - ~.f - - - Each peptide is a portion of SEQ
68 GLHRVLVKA 1.350 I F942 EVPVSVHTR 0.360
ID NO: 3; each start position is
141 LIVQKELDR _I 1L.200 ! 744 SSPTSDYVK-10.3001
specified, the length of peptide isl
1 NVPSIDIRY 339 FTDHEIPFR 0.300 10 amino acids, and the end
291 - 1.200
AR_ -=--= position for each peptide is the
274 , LMPAMVL (1,.200 174 ILQVSVTDT; 0.300
start position plus nine.
458 RTGMLTVVK -T.000} 548 SILDENDDF 0,300:: Pos' Subsequence Score
6 5 DLGQPDSLF 0.900 683
368 STKEYAIKL 0 270 IDLGLhRVLVK 36.000
2 VIETPEGDK 0.900 { 821 LLLNFVTIE 0.270
- y al _ L _ 1[ 319 KIAL~TVTDK 18.000
855 TMGKYNWVT 0:900.
F .. ..õ.^ 4-.. KLVYKTG .V N .n 1
I .. _ ..............._ 530 GLITvTDPDY 18.000
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........... ..._.............. ...
..... .... _.,..._........._ ...... ......
.......__............................................ ...._ ...........
..._..._ ..._._..._....... ........... ......................._.............
.,.,.. ............ _........ ... _ ..
Table XIII -109P1 D4v.1-A3- Table XIII -109P1 D4v.1 A3 Table XIII -109P1 D4v.1-
A3-
~ 10-mers _ 10-mers 10-mers
Each peptide is a portion of SEQ Each peptide is a portion of SEQ Each peptide
is a portion of SEQ
ID NO: 3; each start position is ID NO: 3; each start position is ID NO: 3;
each start position is
specified, the length of peptide is, specified, the length of peptide is'`
specified, the length of peptide is
amino acids, and the end 10 amino acids, and the end 10 amino acids, and the
end
position for each peptide is the position for each peptide is the 1 position
for each peptide is the
start position plus nine. start position plus nine. start position plus nine.
jPosSubsequence Score Pos ISubsequence Scored Pos Subsequence Score
575 KQESyTFYVI(I 16.200 53 ' AILPdEIFRL .608 337 t TCFTdHEIPF 0 200
803 CQMIMmKKKKK 15.000 128 DVIEtPEGDK 0.608 430 AMDAdSGPNA .200
805 IMMKkKKI 15.000 766 Il WIFiTAWR 0.600 778 QAPHIKAAQK 0 200 j
140 QLIVgKELDR 12000 522 NLPRhGTVGL 0.600 454 SLDCrTGMLT _0.200
467 KLDReKEDKY 12.000 354 NQFLIETAAY 0.600 386 LNQSaMLFII< 0.180
806 MMKKkKKKKK 10.000 761 TITVvWIFI 0.540 ( 418 NNSPgIQLTK 0.180 I
347 RLR_PVFSNQ'11.900 09 VLSEnIPLNT 0 450 111 NGVQnYELIK 0.180
~646~ GMNAeVRYSI 8. X80-2 RQMImMKKKK 0 450 905 t ELPLdNTFVA .0180 J
[ _ 2 T 3 1 1 123 NIFGIDVIET 6.450 217 DIGEnAKIHF 0180
461 MLTVvKKLDR T8.000 743 ~VSSPtSDYVK F-0.4507 367 1 TWLsENIPL 0180
357 LLETaAYLDY 8.00 753 lLVAaVAGTI 0.405 385 ~PLNQsAMLFI 0180_
701 ~SLFSvVIVNL 1 6.750 8 KTGDvPLIRI 6495 ! 61 RLVKiRFLIE 0 980
160 KVEDgGFPQR^3~ 3.600 ~6 7 TIDSgTGVIR 0 400 223 KIHFsFSNLV 180
361 AAYLdYESTK 3.000 [424 QLTKvSAMDA 0.400 422GIQLtKVSAM 0.180
LGPdAPPEF i 3.000 107 DVGInGVQNY 0.360 866 TTFKpDSPDL 0.150
444 L
Fj-
458 ( RTGMITWKK 3.000 93~ TTFEvPVSVH 0.338 822J LLNFvTIEET 0.150
391
549 ILDEnDDFTI 2.700 88 CIPENsAINSK 0.300 MLFIkVKDEN 0.150
77 I~PLFPaTVINI 2.700 243 "I NATTgLITIK 0.300 26 TTGArIDREK 0.150
564IVIRPnISFDR .70 6551 IVGGnTRDLF 0.300 [321 ALITvTDKDA , 0150
719 ATLInELVRK 2.250 823 _LNFVtIEETK 0 300 339 FTDHeIPFRL 13
890 IQPEtPLNSK 2,025 16~RIEEdTGEIF 0.300 230 NLVSnIARRL 0.135
760 l GTITvWVIF 2.025LVYKtGDVPL _0.30 0 356 ," FLLEtAAYLD 0.135
363 YLDYeSTKEY 2.000 274 LMPArAMVLV 0.300 764 1L:~FITAV 0.135
675 LMEKcDVTDL 1.800 767 VIFItAVVRC 0.300
55 LPDEIFRLVK 1.800 181 DTNDnHPVFK 0.300 ` Table XIV-109P1 D4v.1-A1101-
~ Table
804 MIMMkKKKKK 1.500 463 I TWKkLDREK 3001, 39 GIPRdEHCFY 1.200 99 TLPAaVDPDV
0.300 Each peptide is a portion of
._... SEQ ID NO: 3; each start
I
146 ELDReEKDTY 1 200 508 PVFThNEYNF 0 300 position is specified, the length
NiTLME of peptide is 9 amino acids, and
0.270
6-9 ETGN~TLME K ! 0.900 X7-6-3 TWVvIFITA
iX6I - - - t - the end position for each
613 FIVPpSNCSY 0.900 137 KMPQIIVQKE 0 270 peptide is the start position plus;
58 I EIFRIVKIRFW 0 900 632 GTVVfQVIAV 0.2270 eight.
`" ub seuence Score
143 , VQKEIDREEK 0.900 265 , LLVLaSDGGL 0_270 .I Pos S q
279 AMVLvNVTDV 0.900 820 NLLLnFVTIIE 0.270 j 112 GVQNYELIK 12.0001
109 i GINGvQNYEL ' 0.810 11811 LIKSgNIFGL 0.270 590 ] RVSRSSSAK 6.000-
850 DLEEgTMGKY 0 810 310 ILSENiPLNTK 0 225 i 2 .FVACDSISK 4.000
248 LITIkEPLDR 0.800 388 QSAMIFIKVK0.225 475 KYLFTILAK 3.600
67 FLIEdINDNA 0 75 241 HLNAtTGLIT 0.200 458 RTGMLTWK 3 000 -
CA 02522994 2005-10-20
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146
............... ...... .... ...... _.... ... ._...... ......._..........
Table AV-109P1 D4v.1-A1101- Table XIV-109P1 1-A`1101-1 I Table XIV 109PI D4v 1
A1101-
9-mers 9-mers 9 mers
Each peptide is a portion of Each peptide is a portion of Each peptide is a
portion of
SEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each start
position is specified, the length position is specified, the length position
is specified, the length
of peptide is 9 amino acids, and of peptide is 9 amino acids, and' of peptide
is 9 amino acids, and
the end position for each the end position for each '. the end position for
each i
peptide is the start position plus. peptide is the start position plus:.
peptide is the start position plus!:
eight. eight.
Pos' Subsequence Score Pos Subsequence Score .` Pos Subsequence Score
1602 NWDVNDNK 3 000 464 WKKLDREK 10.200 459 TGMLTWKK 0.04.0
861 1 WVTTPTTFK 2 000 563 GVIRPNISF_ 0.180 ! 1935 GYPVTTFEV 0.036
387 j F N_QS_AM_LFIK , 1 800 767 VIFITAVVR 0160 152 KDTYVMKVI< 0.030
j~375 KLLAADAGK 1800 576 QESYTFYVI< 0.120 ! 843 VTLDLPIDL_j 0.030
1 802 (RQMIMMKKK 1.800 230 NLVSNIARR .120 766_j WIFITAW 0.030
37 KMPQLIVQK 1 200 942 i EVPVSVHTR 0120_ ; 280 MVLVNVTDV , T 0.030_:
46 KLDREKEDK j 1 200: j 6881 RVLVKANDL ; 0.090 266 LVLASDGG 0.030
01 KTGDVPLIR 1.200 1811 KKKKKHSPK 0.060 762 ITVVV FI 0.030 }
244 ATTGLITIK 1.000 w 684 LGLHRVLVK ILO. 0 765 EWYOTAV _0. 0
462 LTVVKKLDR 0.600 I311 SENIPLNTK q .96O 229 SNLVSNIAR 0.024
` 720 TLINELVRK~1 0.600 598 KVTINWDV 0 060 4 ' 58 EIFRLVKIR ' 0.024
249~j ITIKEPL R 0 600 _ 3 215 DADIGENAK t [013i 0 ~j _RIDREKLCA F_ 0 024
775: RCRQAPHLK 0.600 764 VVVVIFITA 0.060_ j 273 GLMPARAMV 0024
719 ATLINELVR 0 600 644 DTGMNAEVR 0.060 800 : ENRQINIMMK 0.024 J
362 R AYLDYESTK 0.600 704 SVVIVNLFV 0060 1939] TTFEVPVSV 0.020
25 FTTGARIDR 0.400, 486 GVPPLTSNV 0.060 61 IVPPSNCSY 0 020
80 IMMKKKKKK _0.400 1432 jj: DADSGPNAK 0.060 324 TVTDKDADH 0 020 1
804 MI_MMKKKK lf_0 400 W395 KVKDENDNA' 0.060 ( 754 LVAAVAGTI 0A20
82 YVKAEDGGR 0.400 633 TVVFQVIAV 0.060 368 j STKEYAIKL 0.020
129 1 VIETPEGDK 0.400 205 GTSVTQLHA .060 1 73 WRCRQAPH 0 020
320 IALITVTDK F 0300 158 KVKVED F 0 060 946 j SVHTRPVGI~ .020
803 QMIMMKKKK 0.300 308 { VVLSENIPL .j0 00 (757 1 AVAGTITVV 0020
824 NFVTIEETK 0 300 I 61 j RLVKIRFLI . 0 054 750 YVKILVAAV 0.020
680 DVTDLGLHR 10.240 697 GQPDSLFSV 0.054
QESYTFYV 0.054 Table XV 109P1 D4v 1 IF[
` 869 KPDSPDLAR 0 240 575 K
F
A1101-10 mers
53 AILPDEIFR 0.240 22 GEIFTTGAR 0.054
Each peptide is a portion of SEW
! 850 DLEEQTMGK 0 240 760 GTITVVVVI ~0 045. ID NO: 3; each start position is
141 LIVQKELDR 0.240E 632 GTWFQVIA 0 045: specified, the length of peptide is
._.m__.._ 10 amino acids and the end
517 R FYVPENLPR 0 240 930 SVSDCGYPV 0.040
position for each peptide is the
389 ~SAMLFIKVK 0.266 801 NRQMIMMKK 0 040 start position plus nine.
781 HLKAAQKNK 0.200 i 744 1 SSPTSDYVK 0.040 Pos Subsequence E score
`~8-72-z; SPDLARHYK ?('0200 1670 I~GNITLMEK 0.040 i 575 KQESyTFY vK1 3.600
806 . MMKKKKKKK I 0.200 419 1 NSPGIQLTK 0.040 458 RTGMITWKK .3.000
779 APHLKAAQK ? 0.200 182 TNDNHPVFK 0.040 _j 802 1 RQMImMKKKK ..1 800
lnELVRK i 1500
891 QPETPLNSK 0.200 l 291 NVPSIDIRY 0.040 719 ATL
339 FTDHEIPFR 0.200 794 i WATPNPENR 0.040 1319 KIALiTVTDK1200
CA 02522994 2005-10-20
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147
........ _..... ......._ ........................ ........................ _---
........ ...... ..-..... .......__.................,...................
Table XV -109P1 D4v.1- Table XV -109P1 D4v.1 Table XV -109PI D4v.1-
A1101-10-mers A1101-10-mers A1101-10-mers
Each peptide is a portion of SEQ Each peptide is a portion of SEQ Each peptide
is a portion of SEQ
ID NO: 3; each start position is ID NO: 3; each start position is ID NO: 3;
each start position is
specified, the length of peptide is specified, the length of peptide is
specified, the length of peptide is
amino acids, and the end 10 amino acids, and the end 10 amino acids, and the
end
position for each peptide is the position for each peptide is the position for
each peptide is the
start position plus nine. start position plus nine, start position plus nine.
Pos Subsequences Score o Subsequence Score Posh SubsequenceScore
1 0 KVE
DgGFPQRT 1200 [ _55 TIDSgTGVIR 1 0.080 3 757 AVAGtlTVW 0 020
4 128 DVIEt_ GDK = 0.900 418 _N-F-6. 8lVNPvNDTW 0 020
766 }WIFiTAWR 0.600 823 LNFVtIEETK 0.080 774 VRCRgAPHLK 0.020
669 ETGNiTLMEK 0 600 33 REKLcAGIPR 0.072 I IF- 707 IVNLfVNESV 0.020
911; TFVAcDSISK 0.600 1566 RPNIsFDREK 0 060 750 --.-- -AAVA_ 0.02
-r F- 143 VQKEIDREEK 0.600 111 NGVQnYELIK 0.060 255 ; LDREeTPNHK 0.020
1 890 lQPEtPLNSK 0.600 _849 1DLEeQTMGK 0.060 866 TTFKpDSPDL 0.020
804 MIMMkKKKKK 1-0...49.0- k 601 INWdVNDNK _ 0..060 207 SVTQIHATDA 0 020
1805 ll MK KKK_~ 0 400 810 ; KKKKkKHSPK 0.060 939 TTFEvPVSVH 0.020
361 AAYLdYESTK 0 400 366 ( YESTkEYAIK 0 060 Ã 457 CRTGmLTWK 0 020
55JLPDEiFRLVK 0.400 KTGDVPLIRI~ 0.060 725 , LVRKsTEAPV_ 0 020
181 ; DTNDnHPVFK 0.300
335 RVTCfTDHEI 0 060 682 YVKAeDGGRV 0 020
_ ..
463 , TVVKkLDRE{f~~ 0 300 307 TWLsENIPL 560 ( 655; IVGGnTRDLF 0'.020
F803~ QMIMmKKKKK mm0.300 763 _TWVvIFITA 0.060 773 1VVRCrQAPHL I 0 020
1564 _VIRPnISFDR ,240 9_0 RVSRsSSAKV6 0.060 LSENiLNTK_ 0.0 0,
140 i QLIVgKELDR : 0~240~ 273 , GLMPaRAMVL 0.048 530 I GLITvTDPD 0.018
652 RYSIvGGNTR 0.240 760 GTITvVVVIF 0.045 446 GPDApPEFSL0.018 p
-831[q LGLhRVLVK 0.240 640 AVDNdTGMNA! 0.040 697 GQPDs FL SVVE 0.018 I
778 ` QAPHIKAAQK'~0.200 449 APPEfSLDCR 0.040 153 AILPdEIFRL 0.018
88 IPENsAINSK E 0.200 j _ LVYKtGDVPL 0.040 941 j EVPvS HTR 0.018
243 NATT LITIK ' 0.200 743 VSSPtSDYVK 0.040 556 j FTIDsQTGVI 0.015
t 80 MKKkKKKKK, 200 338 CFTDhEIPFR 0,040
49 j REEKdTYVMK 0.180 374 IKLLaADAGK , 0.030 Table XVI-109P1 D4v.1 A24 -
461 MLTVVKKLDR .160 1 M860 NWVTtPTTFK 0.030 -.. __.._ 9-mers
"" _ Each peptide is a portion of
516 NFYVpENLPR .160 764 WWiFITAV 030 SEQ ID N0: 3; each start
248 LITIkEPLDR 160 339 FTDHeIPFRL 0.030 position is specified, the length
386 LNQSaMLFIK 0.120 772 AVVRcRQAPH 0.030 of peptide is 9 amino acids, and,
the end position for each
581 FYVKaEDGGR. 0120 266 FLY LAsGGLM'0.030
L _.. .., , peptide is the start position plus I
842 [kV-TL-q_LPIDL_ 0.120 v50 FTHNeYNFYV 0.030 eight.
52VAILpDEIFR 0.120 765 ' VVVIfITAVV ' 0.030 PosSubsequence Score
584~EDgGRVSR 0.120 1 349 ' RPVFsNQFLL 0.027 47 FYEVEVAIL 300.000
2 TTGArIDREK_ 0.100 109 GINGvQNYEL 0.024 VYKTGDVPL 200.000 ',
589 F GRVSrSSSAK 0.090 646 GMNAeVRYSI 0 024 702 LFSWIVNL 28 000_
466 KKLDrEKEDK 0.090 0 ENRQmIMMKK 0.024 867 TFKPDSPDL 24A00.
632 j GTVVfQVIAV 0.090 474 DKYLffILAK 0 024 858 I~NWVTTPT 21 000
7 1 8 NATLiNELVR I 0.080 431 J MDADsGPNAK _0.020 _ 34 RPVFSNQFU 14.400 ,
24 IFTTgARIDR _a 0 080 214 _I TDADiGENAK 0.020 688 RVLVKANDL 14 400 J
CA 02522994 2005-10-20
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148
............ .....................__ ................ _..... .................
.... ......_,._......
..._................__...._......._..._.,............._..............
Table XVI-109P1 D4v.1-A24 - Table XVI-109P1 D4v.1-A24 Table XVI-109P1 D4v 1
A24 -
9-mers 9-mers 9-mers
-~
Each peptide is a portion of Each peptide is a portion of Each peptide is a
portion of
SEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each start
position is specified, the length position is specified, the length position
is specified, the length
of peptide is 9 amino acids, and
of peptide is 9 amino acids, and of peptide is 9 amino acids, and' I
the end position for each the end position for each the end position for each
11 peptide is the start position plus peptide is the start position plus
peptide is the start position plus:
eight._ eight. _ .._..._ < _ _ eight
Pos. I Subsequences Score Pos E Subsequence; Score_ Pos Subsequence, Score
59 IFRLVKIRF 14 000 368 STKEYAIKL 5.280 492 SNVTVFVSI _ _2.520
703 FSWVNLF 5.040 E 64 KIRFFLLIIEEDDII 2.400
652 ; RRYSI-VG-GN-T 14.000
5.0 } 4 IPFRI LRPVF 2.400
8 YCFTI HEIPF 1 371 EYAIKLLAA ' F-I
1621 FYELVLPST 1 500 E 110 INGVQNYEL 4.400_' 817 SPKNLLLNF _ 2.400
749 DYVKILVAA 10 00 28 GARIDREKL 4.400 312 ENIPLNTKI 2.376
115 NYELIKSQN 10.500 61 RLVKIRFLI _' 4.200 E j 601 F GTITVWVI 2.100
509 VFTHNEYNF 10 000 x-378-i A_ADAGKPPL 4.000 762 ITVWVIFI 2.100
3 KIHF 9 1837 DSD 4 DLGQPDSLF 2
880 ! KSASPQPAF 4.000 656 VGGNTRDLFi 2.000
4 460 GMLTWKKL 9.240
843 VTLDLPIDL 8 640 655 r IVGGNTRDL 4.000 933 DCGYPVTTF 2.000
46 CFYEVEVAIõ 8400 539 YGDNSAVTLI 4,000 ' 593' RSSSAKVTI 2.000 ,
00
839 DGNRVTLDLj 8 400 234 NIARRLFHL s 4 000 86 ISIPENSAI 1.8
247 ~GLITIKEPL 1 _ 8.400 618 SNCSYELVL' 4 000 306 DTVVLSENI 1.800
935 GYPVTTFEV 250 j 542 NSAVTLSIL 4.000 1 28_7 j
454 DVNDNVPSI , 1.800
...-^ wy j .00 102 AAVDPDVGI 1.800'
514 EYNFYVPEN? ET 25O SLDCRTGML 4
678 KCDVTDLGLj 8000 158 KVKDGGFI 4.000 820 NEED 1.800
NVRYSI 1.680
78_ LFPATVINI 7 500 [`5233 EE LPRHGTVGL 4.000 647 M AE
365 DYESTKEYA' 7 500 _16 RIEEDTGEI L3 ,960 86 HPVFKETEIw 1.650
{ 436GPNAKINYL 7.200 45 LGPDAPPEF, 3.960 73 APVTPNTEI 1.
54 1LPDEIFRL 7 200 v. 502 QNDNSPVF! 3.6_00 l j _111 j NGVQNYELI[ 1.500 ~s
356 FLLETAAYL 7.200 548 SILDENDDF~ 3.600 - 166 FPQR_Lj 1.500
17 TNATLINEL 6 336 1 17 ELIKSQNIF ! 3.600
667 DQETGNITL 6 000 605 I DVNDNKPVFI 3 600 Table XVII 109P1 D4v 1-
RAMVL 6.000 402 NAPVFTQSF L 3.600 A24 -10-mers
~~~ _T
Each peptide is a portion of SEQ
417 ENNSPGIQL 6 000 181 DTNDNHPVFj 3.600 ID NO: 3; each start position is
-~~i 71 DINDNAPLF 3 0
E specified, the length of peptide is
314: IPLNTKIAL 6.000
302 NPVNDTVVL ~-6 000000 - f 628 ~STTN-PGT~WF 3.600 10 amino acids, and the
end
position for each peptide is the
s 308 ? WLSENIPL 6.00 860 NWVTTPTTF, 3 000 j start position plus nine
9 SAINSKYTL 6000 f 39 GIPRDEHCT 3.000 Pos, Subs Score
f 538 j DYGDNSAVT, 0
r52 1VAILPDEIF _3;000 514' EYNFyVPENL 420.000
60TPNHKLLVL 6.000 55633 i GVIRPNISF 3.000 538 , DYGDnSAVTL,
2 Y240 000
L._ .
88 FQIQPETPL 6.000 232 VSNIA RRLF 3A00 P7 ! NYELiKSQNI ( 90 000
227: SFSNLVSNI 11 6.000 218 ' IGENAKIHF_I 3.000 365 DYEStKE A 75.000 266
LVLASDGGL 1 6.000 953 GIQVSNTTF , 3A00 KTgDVPLI 50.000
231 LVSNIARRL _5.600 1220 1 ENAKIHFSF 2.80 $87 AFQIgPETPL 30.000
515 YNFYVPENL 5.600_1 761 TITVWVIF 2.800 355 f QFLLeTAAYL I X000
CA 02522994 2005-10-20
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149
.............. .................... _.... ........__............ ..... .
........... .................._
Table XVII 109P1 D4v.1- Table XVII -109P1 D4v.1- Table XVII 109P1 D4v.1-
A24 -10-mers A24 -10-mers A24 10-mers
Each peptide is a portion of SEQ Each peptide'is a portion of SEQ Each peptide
is a portion of
ID NO: 3; each start position is ID NO: 3; each start position is ID NO: 3;
each start position is
specified, the length of peptide is specified, the length of peptide is
specified, the length of peptide is
amino acids, and the end 10 amino acids, and the end 10 amino acids, and the
end
position for each peptide is the position for each peptide is the position for
each peptide is the
start position plus nine. start position plus nine. start position plus nine.
Posh Subsequence, Score Posi Subse uence Score Pos Subse uence Score
k 46 CFYEVEVAIL 24.000 259 ETPNhKLLVL ` 6.000 45 KYLFtILAKD 2.310
239LFHLnATTGL 20.000 1-3-2~ TPEGdKMPQL` 6.000 33 RVTCffDHEI 200
59 IFRLVKIRFL 20.000 654 SIVGgNTRDL ,6.0 0 796 TPNPeNRQMIw 2.160
298 RYIVnPVNDL1C 18.000 347 RLRPvFSNQF 5.760 646 GMNAeVRYS 2.100
7021 LFSVvIVNLF 16 800 701 SLFSvVIVNL 55600 406 j FTQSfVTVSI 200
858 KYNWVTTPTT 15.000 339 FTDHeIPFRL 5.600 753 3 ILVAaVA 2.100
349; RPVFsNQFLL' 12000 81 LAKDnGVPPL 4.800 630 NPGTvVFQ 2.016
33 KPPLnQSAML+ 12 000 377 LAADaGKPPL 4 800 655 IVGGnTRDLF 2 000
842 RVTLdLPIDL 9.600 i 681 VTDLgLHRVL 4.800 337 TCF7T _ llPF 2 000_ 1
716 VTNAtLINEL 9 504 368 STKEyAIKLL 4 800 51 EVAIIPDEIF 2.000
59 TGMLtVVKKL 9 240 27 TGARiDREKL 4.400 231 LVSNiARRLF 2.000
138 NMPQLiVQKEL' ES
9 240 367 TKeYAIKL 4400 , 859 YNWVtTPTTF, 2,0 00
621 SYELvLPS N 9 000 ) 903 IQELpLDNTF4.320 556 f FTIDsQTGV [ 1.800
749 DYVKiLVAAV 9.000 760FGTITvWVIF 4.200 605 DVNDnKPVFI _ 1.800
1246 TGLItIKEPL 8.400 773 VVRCrQAPHL 4000 ! , 664 FAIDgETGNI 1 800
L 0. ~ 6
` 0 NLVS 8 91 NSAI 4 66 RFLIeDIN _ 1.800
1
I436 GPNAMNYLL 1 8.400 866 TTFKpDSPDL _ 4.000 414; SIPEnNSPGI ;1.800_
165 GFPQrSSTAI) _ 7.500 118 LIKSgNIFGL 4.000 731; EAPVtPNTEI 1.650
897 NSKHhIIQEL 7 392 _ ; 693 ANDLgQPDSL` 4.00 744 SSPTsDYVKI 1.650 1
16 RIEEdTGEIF 7.200 446 GDApPEFSL; 4.000
53 AILPdEIFRL jC 7.200 1541_1_p NSAvTLSIL 4.000 Table XVIIl9 09 ID4v.1-B7
435 SGPNaKINYL 7.200 1 LVYKtGDVPL 4.00_0
1 Each peptide is a portion of
273 GLMPaRAMVL' 7.200 _ 745. SPTSdYVKIL 4.000 SEQ ID N0: 3; each start
453; FSLDcRTGMLI 7.200 38 ', AGIPrDEHCF 3.600 position is specified, the
.615; VPPSnCSYEL 6.600 816 HSPKnLLLNF 3 6O0 length of peptide is 9 amino
acids, and the end position fort
109 GINGvQNYEL 6.600 819 KNLLINFVTI .600 each peptide is the start
313 NIPLnTKIAL 6000 343 EIPFrLRPVF 3.600 ` positionplus eight.
_878 HYKSaSPQPA 6.000 57 _S ILdENDDF 3.000 Pos` Subsequence Score
7-2- VN A L 0 952 VG TF 3 = LPR 80 0
0. 7qqq
'522 22 NLPRhGTVGLj 6.000 562~ TGVlrPNlSF 3.000 8 GARI DREKL ~ 18
307 TVVLsENIPL 1 6.000 401 DNAPvFTQSF 2.880 3491 RPVFSNQFL F 80000
265' LLVLaSDGGL 6.000 58 EIFRIVKIRF F 2.800 314 IPLNTKIAL 80.000
1661 FPQRsSTAIL 6,000 444 LLGPdAPPEF 2 640 436, GPNAKINYL 80.000 1
1675 LMEKcDVTDL 1 6.000 Ã 491 TSNVtVFVSI 2.520 2601 TPNHKLLVL 80.000
12021 APVGtSVTQL 6 000 14 EFSLdCRTGM 2.500 P39 NPVNDTVVL 80.000
2331 SNIArRLFHL 6. tj217 DIGEnAKIHF 2.400 j 732 APVTPNTEI 36.000
301 VNPVnDTWL 6 000 KTGDvPLIRI 2 400 76 i APLFPATVI 36 000
CA 02522994 2005-10-20
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150
. ............................ ...... .__.........................
......__................... _
Table XVIII -109P1 D4v.1-B7 Table XVIII -109P1 D4v.1-B7 Table XVIII -109P1
D4v.1-B7
9-mers 9-mers it 9-mers
Each peptide is a portion of Each peptide is a portion of Each peptide is a
portion of
SEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each start
position is specified, the position is specified, the position is specified,
the
length of peptide is 9 amino length of peptide is 9 amino length of peptide is
9 amino
acids, and the end position for; acids, and the end position for, acids, and
the end position for
each peptide is the start each peptide is the start each peptide is the start
position plus eight. position plus eight position plus eight.
Pos Subse uence Score Subsequence Score Pesos Subsequence Score
q I. I........ ......_. ......__..; I[ q.__
796; TPNPENRQM 20.000 i 626, LPSTNPGTV 4.000 539 YGDNSAVTL F1.200
655 IVGGNTRDL 20.000 !,843 VTLDLPIDL 4.000 i 667 DQETGNITL 11 200
688: RVLVKANDL 20.000 542 NSAVTLSIL 'F4.000 454' SLDCRTGML 1.200
3081 WLSENIPL 20000 234 NIARRLF L 4.000 55 LPDEIFRLV 11200
231CVSNIARRL ' 20.000 40 IPRDEHCFYW 4.000 284(NVTDVNDNV 1.000
383 1KPPLNQSAM 20.000 100 LPAAVDPDV 4.000 633~TWFQVIAV 1.000
266 LVLASDGGL 20.000 515 YNFYVPENL 4.000 80 MVLVNVTDV 1.000 _
_ 92 ; SAINSKYTL 12.000 717 TNATLINEL 4.000 750 YVKILVAAV 1.000
4 APVFTQSFV 12.000 247 GLITIK 4.000 766' VVIFITAW _1.000
378' AADAGKPPL 10.800 110 INGVQNYEL 4.000 930 i SVSDCGYPV 1 000
166' FPQRSSTAI 8 000 757 AVAGTITVV 3.000 46 GVPPLTSNV 3 1 000
745; SPTSDYVKI 8 000 639 i IAVDNDTGM 3.000 75 VVVIFITAV 1.000
~~ _
7, IVNPVNDTV 1 000 y
384w PPLNQSAML 8.000 41, IPENNSPGI 2.400
186; HPVFKETEI 8.000 203VGTSVTQL '.g.'9 00 1598! KVTINVVDV 1.000
292 VPSIDIRYI 8.000 1906? LPLDNTFVA 2.000 ? 423 IQLTKVSAM ! 1.000
894; TPLNSKHHI 8 000 946, SVHTRPVGI 2.000 26 1 VLASDGGLM 1.000
616 PPSNCSYEL 8.000 296k DIRYIVNPV~ 2.000 76: ', SVVI 1000
888; FQIQPETPL 6000 287 DVNDNVPSI 2.000 273GLMPARAMV 0.900
1449; APPEFSLDC 6.000 350 PVFSNQFLL 2.000 278 RAMVLVNyT 0.900
417; ENNSPGIQL 6.000 .E 754 LVAAVAGTI 2.000,õ_,,,
1798 NPENRQMIM 6.000 456 DCRTGMLTV 2.000 Table XIX-109PID4v.1-B7
~ 10-mers
102 AAVDPDVGI ` I ~- -. --
~i 5 400 493 NVTVFVSII 2 000
Each peptide is a portion of
735; TPNTEIADV 4 000 487 VPPLTSNVT F2-7 SEQ ID NO: 3; each start
839; DGNRVTLDL ! 4 000 51 EVAILPDEI 2.000 position is specified, the
630; NPGTVVFQV 4 000 948 HTRPVGIQV 2 000 length of peptide is 10 amino
l acids, and the end position for
27; MPARAMVLV 1_4.000_ 1 847 LPIDLEEQT ; 2000 each peptide is the start
4601 GMLTVVKKL 4 000 591, VSRSSSAKV 2 000 position plus nines
274' LMPARAMVL 4.0001 882 ASPQPAFQI 1.800 Subsequence Score
618' SNCSYELVL 4.000 756 AAVAGTITV~ 1.800 PniI
223j( KIHFSFSNL 4.000837~~DSDGNRVTL _1.800 202 APVGtSVTQL 240.00 111 368127
STKEYAIKL 4.000 I 1r
GGLMPARAM 1.500~.._W___
i 167; PQRSSTAIL 4.000 453, FSLDCRTGM 1.500 F773 VVRCrQAPHL~ 20000
54 ILPDEIFRL 4.000 678 KCDVTDLGL .200 615, VPPSnCSYELI 80.000
4201 SPGIQLTKV 4.000 243 NATTGLITI _1.200 GPNAl~INYLL 80.000
64 j KIRFLIEDI 4.000 105I DPID GINGV l 1.200 349( RPVFsNQFLL 80.000
356 FLLETAAYL 4.000 ! 698 QPDSLFSW 1.200 523j _ LPRHgTVGLI 80.000_
CA 02522994 2005-10-20
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.... ....... _.._ .............._..................
........_._..__..._........._........ .......... .............
._.............................._........__.... ..... _..... ...............
._....- ........ _..................._..
Table XIX-109P1 D4v.1-B7 Table XX -109P1 D4v.1- Table XX-109P1 D4v.1-
10-mers ~- B3501w 9-mers B3501-9-mers
Each peptide is a portion of Each peptide is a portion of Each peptide is a
portion of
SEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each start
position is specified, the position is specified, the position is specified,
the
length of peptide is 10 amino length of peptide is 9 amino length of peptide
is 9 amino
acids, and the end position for? acids, and the end position for 1 acids, and
the end position fort
each peptide is the start Leach peptide is the start each peptide is the start
position plus nine. position plus eight. _position plus eight.
I ;Posy Subsequence Score Pos Subsequence Score
Pos -_ - --_ __ -l
Subsequence Score 344 IPFRLRPVF 20000 Itg 548 ILDENDDF -__3,
000
138' MPQUVQKEL 80.000 28 GAR~IDREK13.500 52 VAILPDEIF 3.000
383; KPPLnQSAML 80w000 745' _SPT~ SDYVKI 12.000 26 VLASI GGLM: 3.000_
t
166; FPQRsSTAIL = "80 000' 798 NPENRQMIM 12.00 86 ISIPENSAI 3.000
745 i SPTSdYVKIL 80.000 2g2 VPSIDIRYI 12 000 415 IPENNSPG) = 2.400
446 , GPDApPEFSL 36.000 639 IAVDNDTGM 12 i 64 KIRFLIEDI 2.400
132 TPEGdKMPQL 24.000 t Itt _
8412 RVTL~ 2 $-~ KSASPQPAF 10.000 55 , LPDEIFRLV 2.400
91 CSSSSSDPY - 10.0000 12.400"_._.
307 TWLsENIPL 20 000 ` 158; KVKVEDGGFrt 9.000 1291 NVPSIDIRY ' 2.000 j
LPIDIEEQTM 20.00 894 TPLNSKHHI H 8.000 j 223 kLKNLL 2.000
i LVYKtGDVPL 20 000 732; APVTPNTEI w 8.000 ' 742 DVSSPTSDY 2 000 i
481; LAKDnGVPPLJ 12.00 76 APLFPATVI J-1-000-1 7 aFDINDNAPLF -2.0-005
3[ AILPdEIFRL 2.000 18, HPVFKETEI 000 356, FLLETAAYL 2 000
377',: LAADaGKPPL 12 000 166] FQRSSTA_ 8.000 41 VTLDLPIDL 2.000
459 TGMLtVVKKL 12.000 735 TPNTEIADV 6.000 487 VPPLTSNVT 00
---- - -- 368 F STKEYAIKL 6 000 614 IVPPSNCSY 2.000
Table XX 109P1 D4v 1 VSNIARRLF 5 000 435` SGPNAKINY 2.000
t232[tl-tt ~t.l
B3501 9 mers
272 ~GGLMPARAM _2.000
703' .. FSVVIVNLj 5.000
Each _
peptide
ID NO: 3; each start SEQ 1542I NSAVTLSIL H60 882, 1ASPQPAFQI 2.000
position is specified, the 906 LPLDNTFVA 4.00 616 PPSNCSYEL 2.000
length of peptide is 9 amino 714
4.000 4 ESVTNATLI 2.000
acids, and the end position fors 630; [N) F
each peptide is the start 626 LPSTNPGTV 4 000 1169; RSSTAILQV 2.000
position plus eight. i
610 KPVFIVPPS 4 000 384 PPLNQSAML , 2 000
Pos'; Subsequence Score
593 RSSSAKVTI 1 4.000 502- DQNDNSPVF _2000
1 3 6
P E R 00 ; 420 SPGIQLTKV 4 000 531 f LITVTDPDY 2 000
3-'KPPLNQSA 80.000
- 449 APPEFSLDC 1 4.000_ 423 IQLTKVSAM 1 T
:523 LPRHGTVGL 60.000 847 LPIDLEEQT . 4.000 645=ITGMNAEVRY 2 000
817 SPKNLLLNF ' 60.000] ' 100 LPAAVDPDV i 4 000 605 [DVNDNKPVF r2 000
796 TPNPENRQM 60.000 i 0
950 RPVGIQVSN -4A0 445 LGPDAPPEF , 2 000
507'. SPVFTHNEY :F40.9067!
r403 APVFTQSFV 4.000 ~~. _ 864 TPTT FKPDS
~ 2,000m
3491 RPVFSNQFL I 40.000
275 MPARAMVLV 4.000 688 RVLVKANDL 2.000
02 NPVNDTVVL
871 DSP 30 000 ([54 ILPDEIFRL_ 3.000 79 FPATVINIS 2.000
, 20.000 tIt
92 SAINSKYTL 3.000 108 VGINGVQNY 2.000
314I IPLNTKIAL 20,000 t t
; 3.000 181; DTNDNHPVF 2.000
5103 FTHNEYNFY ~
260 TPNHKLLVM 2
20.000 0.000 -- 90 f ENSAI NSKY 2.000-
1
453( FSLDCRTG _ 59 VSRSSSAKV i 3000
~ 402 NAPVFTQSF 3.000 147 LDREEKDTY 1.800
436 GPNAKINYL 20 000 s
CA 02522994 2005-10-20
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152
.._.........
.................._..........__....................................._._...
_.._...... ........ _..V...... .....
....._..,.__......_.........._.............
Table XX-109P1 D4v.1- Table XXI -109P1 D4v.1- Table XXI -109P1 D4v.1-
B3501-9-mers 83501-10-mers B3501-10-mers
Each peptide is a portion of Each peptide is a portion of Each peptide is a
portion of
SEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each start
position is specified, the position is specified, the position is specified,
the
length of peptide is 9 amino length of peptide is 9 amino length of peptide is
9 amino
acids, and the end position for acids, and the end position acids, and the end
position
each peptide is the start for each peptide is the start for each peptide is
the start
position plus eight_ position plus eighty position plus eight.
F_
Pos Subsequence Score 2 9431 VPVSvHTRPV 14.000! A~PVGt'S, L ........
Ji REKEDKYLF 1.800 _ ....,. {610 KPVFiVPPSN .000''
I _..
395; KVKDENDNA, 1.800 745 SPTSdYVKIL 2 95Q' RPVGiQVSNT 4 000',
596 SAKVTINVV 1.800 , 20.00 1487 VPPLtSNVTV 14.000
166
j[ 0
37õ DSDGNRVTL 1500 11
FPQRsSTAIL ~ 626 LPSTnPGTVV;4.000
95' NSKYTLPAA 1.500 E615t 20.00' 06 LPLDnTFVAC4000
VPPSnCSYEL
?3 0
SSSSDPYSV1.500 ;
3 664(
9 FAIDgETGNI ! 3.600
i 308 WLSENIPL 1.500 481 LAKDnGVPPL 18000 744 SSPTsDYVKI 3.000E
918, ISKCSSSSS 1.500 15.00 354 NQFLIETAAY 3.000
39 GIPRDEHCF 500 897KHhIIQEL 118 LIKSgNIFGL 3.000
196 VSIPENAPV 1.500 _ ~26--6! LVLAsDGGLM 3.000
571; FDREKQESY 1 200 798 NPENrQMIMM 1 1 000 771 VVRCrQAPHL 3.000i
468 LDREKED 1.200 12.00 39 GIPRdEHCFY 3 0
817 SPKNILLNF~=- ...... 698E QPDSLFSVV 1.200 V 95 NSKYtLPAAV 3.0 0'
10.00 ...-.......___..._.....
243; NATTGLITI -9.200 453 FSLDcRTGML 795 ATPNpENRQM 3.0001
105 DPDVGINGV 1.200 698 QPDSIFSVVI 2.400
10.00
434; DSGPnAKINY
p 885QPAFgIQPET~ 2.000'
11-
Table XXI -109P1 D4v. 1 0.00 _226 FSFSnl- I 2.000
83501-10-mers 506 1 NSPVfTHNEY
842 RVTLdLPIDL 2000
Each peptide is a portion of
SEQ ID N0: 3; each start " 796 TPNPeNRQMI 18.0001 ? 638; VIAVdNDTGM 2.0001
position is specified, the = 791 FPATvINISI 8.000 120 KSQNiFGGL V 2000]
length of peptide is 9 amino 314 IPLNtKIALI 8.000 - 1
,., 12 VPLlrlEEDT , 2.000
acids, and the end position - === -~ -- -
for each peptide is the start 630 NPGTvVFQVI 8 000 613 FIVPpSNCSY 2.000
position plus eight 894 TPLNsKHHII 18 000 76 APLFpATVIN 2,000
Pos Subsequence 1Sceor 547 LSILdENDDF 7.500 420 SPGlgLTKVS 2.000
- -- - I 368 STKEyAIKLL 6.000 384 PPLNgSAMLF 2 000
8471 LPIDIEEQTM 1000; 446 GPDApPEFSL 6.000 945 _VSVHtRPVG~ 2.000,!
377 { LAADaGKPPL 6.000
- 530 GLITvTDPDY 2000
383 KPPLnQSAML 40 OOi ; .
1 0 147 RLRPvFSNQF 6.000 F 290iCDNVPsIDIRY j 2.000Ã
40 00; 32 TPEGdKMPQL 6.0004 507; SPVFtH N 0
927! DPYSvSDCGY 0 253 EPLDrEETPN 6.000,
I -~ 22 j f GIQLtKVSAM - .000:
!140.00! 816 I,HSPKnLLLNF 344
115.000i
IPFRI P~-
-1 1 5.000VFS~ 2.000
349 RPVFsNQFLL 275 MPARa
o 91 NSAInSKYTL
MVLVN 2.000
523; LPRHgTVGLI 2 0001 [367 ESTKeYAIKL [5.060; 72 KSTEaPVTPN 2.000;
MR 1936 1 YPVTtFEVPV; 4.000+ 302 I ~!PVNdTVVLS 2.000,
[43.61 GPNAIdNYLL
0 ; 2921 VPSItlIRYIV 4 000 644 1 DTGMnAEVRY I20001
[41 MPQLiVQKEL 920 KCSSsSSDPY 4 000 L.
2.0
0
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153
._......._..... ..... _........ .... _ ............._..... _.............
__.:.
......_._._ ..................__......_.._........ _......_...................
............__... _...._.......................
.
Table XXI -109P1 109P1DTable IX -109PI D4v.1- Table IX -109P1 D4v.1-
83501-10-mers Al-10-mers Al-10-mers
Each peptide is a portion of Each peptide is a portion of Each peptide is a
portion of
SEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each start
position is specified, the position is specified, the length position is
specified, the length
length of peptide is 9 amino of peptide is 10 amino acids, of peptide is 10
amino acids,
acids, and the end position and the end position for each and the end position
for each
I for each peptide is the start peptide is the start position plus peptide is
the start position plus
position plus eight. nine. nine.
107; DVGInGVQNY 2 000 [77777 00_ 467 KLDReKEDKY ! 2 500'
779 APHLkAAQKN 2 000 [5]0 DLEEgTMGKY 45 00 640 AVDNdTGMNA j 2 500
260 TPNHkLLVLA 2 000 _ _ - o 669 ETGNiTLMEK 2.500
735 TPNTe1ADVS 2 000 434 LDSGPnAKINY 37'5 1 TGEIffTGAR 2 0
271. DGGLmPARAM 2 000; F
31]0 .0014411 LLGPdAPPEF 2.000
TSNVtVFVSI LSENiPLNTK 931= VSDCgYPVTT 1.500,
403 APVFtQSFVT 2 000, - 25.00 ' 903 IQELpLDNTF 1350
488; PPLTsNVTVF 2.000 363 YLDYeSTKEY 0 213 ATDAdIGENA 1.250
732 j APVTpNTEIA 2.000: 103: AVDPdVGING t25.00 179 VTDTnDNHPV 1 250 j
536' DPDYgDNSAV 1.800: 681? . VTDLgLHRVL
1 1,250,
F KTGDVPLIRI 1.600 160 1]LKVEDgGFPQRF 8.00 798 NPENrQMIMM 1.125
569 ISFDrEKQES 1 500 8.00 ` v191 ETEIeVSIPE 1.125
'.584 KAEDgGRVSR
53 , AILPdEIFRL 1 500 _ 0 1811 DTNDnHPVFK 1 000
307 TWLsENIPL 1 00 -269 ASDGgLMPAR 1500 37; AADAgKPPLN E 1.000
8 AQKNkQNSEW 1 500; 32 DADSgPNAKI 1000
92; SSSSsDPYSV 1.50; 55 I [iiK 12.50 " 215 DADIgENAKI 1.000
301; VNPVnDTVVL 1 500; 534 -== VTDPdYGDNS I-- 1261~GLDViETPEG 1.000
0
12.50 683, DLGLhRVLVK 'E
591 VSRSsSAKVT 1500' =
1 000
38 AGIPrDEHCF 1 500 (1 p 458 RTGMITVVKK 1`000
27 j TGARiDREKL 1 5001 557 1[' TIDSgTGVIR 0
,. _... __ 719 ATLI ELVRK 1.000
866 TTFKpDSPDL 1.500; 16 RIEEdTGEIF 1-9.000 150 GLITVTDPDY 00
840; GNRVtLDLPI 1.200; 925 SSDPySVSDC 7.500'j 329` DADHnGRVTC
_ 1.000
40 IPRDeHCFYE 1200a f 3391 FTDHeIPFRL j 6 5 397, KDENdNAPVF 0 900
692 KANDIGQPDS i 1.200; P-9911 DNVPslDlRY 6.250 827 TIEEtKADDV 0 900
16~ 1E IF 1.200; 743] VSSPtSDYVK 6.000 129 VIETpEGDKM 0 900
F - 00 575 KQESyTFYVK 5.400 j 193 EIEVsIPENA 0.900
573 REKQeSYTFY 1 2001 613,L FIVPpSNCSY 5.000 506; NSPVffHNEY 0.750
- _. _ ., 729 STEApVTPNT 4.500 22. FSNLvSNIAR IF
750
Table IX- 109P1 D4v.1- 648 NAEVrYSIVG ' 4.500 288 r(~ VNDNVPSIDI
, ...._ ~~ ;~i _.. ,_- ._._. __...__.I x=625
Al 10-mers
_.. - i 88 F EWINSK 4.500 606 VNDNkPVFIV 0.625
Each peptide is a portion of (747, TSDYvKILVA 3.750 399' ENDNaPVFTQ 0 62
SEQ ID NO: 3; each start - ,F MFR _. _- _: F -1 .
position is specified, the length ; 418; NNSPglQLTK 2.500 ,F721 INDNaPLFPA 0
.625
F
of peptide is 10 amino acids, 146; ELDReEKDTY , 2.500 503,TQNDNsPVFTH [0.6
and the end position for each
peptide is the start position plus 644 DTGMnAEVRY 2.500 or
GPDApPEFSL O.K.
549
ILDEnDDFTI 2.5001
914`
1 nine. 454
SL LT ACDSiSKCSS ~' 0.500
678 KCDVtDLGLH0 500
Subsequence i Scoe 2.500
LIL I 5j L VTDVnDNVPS 2.500~
718 NATLiNELVR_ 0.500 11
' 357; LLETaLDY 225 0
AY
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154
...........
........... __......._ .............._...-.._ ............. ...............
........._..._._........... ......
Table IX -109P1 D4v.1- Each peptide is a portion of Table V111 -109P1 D4v.2-
Al SEQ ID NO: 5; each start N' terminal - A1-9-mers
1 = position is specified, the length Each tide is a portion of
Each peptide is a portion of pap p
SEQ ID NO: 3; each start of peptide is 9 amino acids, and SEQ ID NO: 5; each
start
the end position for each position is specified, the length
position is specified, the length p of peptide is 10 amino acids, peptide is
the start position plus of peptide is 9 amino acids, and
eight. the end position for each
and the end position for each --- ---- -- p
peptide is the start position plus Pos Subsequence Score peptide is the start
position plus
nine. 1 i RTSTIEICS 0.125 _eight
2171 DIGEnAI<IHF 0,500' PTDSRTSTI 0.125 ` 16 .I LCGLIQQTV " 0 010
53 AILPdEIFRL 0.500 14 STIEICSEI~ 0.025 f 10 f IQIFQVLCG 0.007
l3 FQVLCGLIQ 0.007
628 STNPgTWFQ 0.500 E J E HTRPT_SRT 0.025 ;I--- I - -
1248] LITIkEPLDR 0.500 3 SVHTRPTDS 0.010 f L 21 QQTVTSVPG 0.003 i
151' EKDTYVMKVK 0.500 10 ~~ DSRTSTIEI 1 0.008 i _, l~NLIQIFQ 0.003
,... f 4 ERQWVLIQI 0.003
;.__
430 AMDAdSGPNA 0.500. VSVHTRPTD 0.003
832f KADDvDSDGN 0 500 RPTDS S 0.003 7 ',IGLIQQTVT 0.003
273 GLMPaRAMVL 500 13 TSTIEICSE 0.002 5 RQWVLIQIF 0.002 f
889I QIQPeTPLNS 57- 500 PVSVHTRPT 0001 23 TVTSVPGMD 0.001
564, VIRPnISFDR 0.500 VHTRPTDSR 0;001 MRTERQWVL 0.001
461 MLTVvKKLDR 0.500 - I 12 IFQVLCGLI 0 001
~ _ , .~. SRTSTIEIC .001
337( F - . 5 Q 00_ TRPTDSRT_S 0 001 TERQWVLIQ 0 000
500 -IIDQnDNSPV 0.500 s g TDSRTSTIE 0 000 28.w PGMDLLSGT MOO
448( DAPPeFSLDC 0.500 20 I66TVTSV~0.000
.,_._..
14W QLIV KELDR 0.500 S _ e
q __....-, ~...._ Table VIII 109P1D4-v.2- ~ ...,_ ...._._.~...._........-
....~..._..
VGInGVQNY 0.500 N' terminal - Al 9-mers Table VIII -109P1 D4v.3
107 D ..__lnGVQN Al 9 mers
VAILpDEIFR 0.500 Each peptide is a portion of 1 - -- 9-. ---- - -
~ SEQ ID NO: 5; each start Each peptide is a portion of SEQ
760 jj-- GTITvWVIF 0.500: position is specified, the length ID NO: 7; each
start position is
920 KCSSsSSDPY 0.500 of peptide is 9 amino acids, and specified, the length of
peptide is
the end position for each 9 amino acids, and the end
81 I SASPgPAFQI _ 0.500 peptide is the start position plus
position for each peptide is the
1603 WDVnDNKPV 0 500 eight. start position plus eight.
26 TTGArIDREK 0.50 Pos Subsequence Score Pos Subsequence Score
835DVDSdGNRVT 0 500 29 GMDLLSGTY 12.500 37 I KSEGKVAGK 4 000
665;, AIDQeTGNIT Ng , RTERQWVLI 0 450 106; NSDPESTFI 7.500
132 TPEGdKMPQL 0 450, TSVPGMDLL 0.150 78- TSHGLPLGY 3.750
21; IKEPIDREET 0.450 ' 24 VTSVPGMDL 0.125 145 HSDACW A 3.750
712 VNESvTNATL F6.45T-111 26 f SVPGMDLLS 0 050 1111 STFIPGLKK 2.500
778I QAPHIKAAQK 0 .40.0 14 QVLCGLIQQ 0050 135 NCTQECLIY _ 2.500
196, VSIPeNAPVG ; 0 300 22 QTVTSVPGM E 0 050 234 SAQA m) T 2.500
~~
388' QSAMIFIKVK F6 -736W 7 WVLIQIFQV 0.050 29 1 WIHPQPQRK 2.
871 DSPDIARHYK 0 300 ;
DPESTFIPG 1 1 125
_ _ 18 I~GLIQQTVTS 0.020 8 D
86ISIPeNSAIN 0.300 g ~LIQIFQVLC 0.020 1281 TVEEASDNC 9.966
872" SPDLaRHYKS 0.250 ] 27 VPGMDLLSG 10.013 I 120 AAEITVQPT I 0.900
833~ADDVdSDGNR 0.250 19 LIQQTVTSV 0.010_ 132 f ASDNCTQEC .750
VLIQIFQV 0.010 1' 162 SSDGGLGDH 0.750
0
IL Table VIII -109P1D4v.2 11 QIFQVLCG 0.1 288 I1[SVDQGVQGSLQ9J
nal Al-9-mers
C' Termi
-- - ' T-_ 15 VLCGLIQQT 0.010 f 154j SLDHSSSSQ _ 0 500
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155
.............. _..-...... ......._.. _.........._............
..._...._.............._ ........... ..._ ................_........
...............
Table VIII -109P1 D4v.3 Table VIII -109P1 D4v.3 Table VIII -109P1 D4v.3
Al-9-mers A1-9-mers A1-9-mers - --- -j
Each peptide is a portion of SEQ Each peptide is a portion of SEQ Each peptide
is a portion of SEQ
ID NO: 7; each start position is ID NO: 7; each start position is ID NO: 7;
each start position is
specified, the length of peptide is" specified, the length of peptide is
specified, the length of peptide is
9 amino acids, and the end j 9 amino acids, and the end 9 amino acids, and the
end
position for each peptide is the position for each peptide is the position for
each peptide is the
start position plus eight. start position plus eight. start position plus
eighty
Pos Subsequence Score Pos CSubsequence Score Pos ` Subsequen a Score
TMEIWIHPQ 0.45 0 222wLVQ4TALHNO 0,050 214 SALHHSPPL 0 020
SVHTRPPMK 0400 7 F STSHGLPL 0.050 ' 190` IALCHSPPV ;L- 0.020
110 ESTFIPGLK 0.300 } 240 LCYSPPLAQ 0.050 238 SALCYSPPL 0.020 1137 TQECLIYGH
0.270 ! 68 LCHSPPLS0 050 ! 49 RVTFHLPEG 0 020
84 LGYPQEEYF _ M 0250- RPPMKEWR 0 050 122 TALHHSPPS 0.020
20 MKESTTMEI 0.225 ! 8 HGLPLGYPQ~;~0.050 274 SLQQGWVQG 0.020
54 LPEGSQESS 0 225 178 STQHHSPR 0.0 192 LCHSPPVTQ ! 0.020
100 RTEGDGNSD 0.225 246 LAQAAAISH , 0 050 s 204 LCHSPPPIQ 0 020 1
254 ! HSSPLPQVI 0150 162 QAQASALCH F F-7 T90 66 GLGDHDAGS 0 020
230 HSPPSAQAS 0.150 322TFTPRQQAR~ 0.050 185 RVTQTIALC j 0.020
147 DACWMPASL -0.0
218 HSPPLVQAT 0150 83 PLGYPQEEY 0.050
1f77. ASTQHHSPR 0150 ! 282 GADGLCSVD 0 050
i[94 S E ~--" -TQTI T m E - . 207 SPPPIQVSA 1x050 Table VIII -109P1D4v.4
1PPVT 0150 _..___ __~ _ _..__._ __.._ _ ...... _...
206; HSPPPIQVS ,0.150 TO { MKEWRSCT 0.045 Al 9 mers
~ ---s Each peptide is a portion of SEQ
170 HSPPLSQAS.0150 88 ~EEYFDRAT04 ID N0: 9; each start position is
0.045 specified, the length of peptide is 9
242; SPPLAQAA; 0.150 F29- F F-
58 SQESSSSDGG ; 13 I [V-- C VRSCTPMK I 0.040 amino acids, and the end position
F-. 1 -- for each peptide is the start
186 VTQTIALCH 0125 287,1CSVDQGVQG 0.030 osition plus eight. 13 CTQECLIYG
0.1125 157 HSSSSQAQA t _0.030 Pos Subse uence `~i Score.
67 LGDHDAGSL 0.125 25 , S 0.
SPLPQVIA 030 HPQPQSQ 0.250'
294 QGSATSQFY 0.125 159 SSSQAQASA W
0.030 IHPQPQSQ 0.100
256; SPLPQVAL
I 0125 2VSVHTRPPM - 0.030 3 IHPQPQSQR 0 005
i
86 YPQEEYFDR 0.125 30 ( MSERLHPSD 0.027 pQSQRRVTF 0.003
69 4 DHDAGSLTS 0.125 31 IPLTTFTPR , 0.025 6 QPQSQRRVT 003
1 1 9 8 1 VTQTIALCH 0.125 27 ATSQFYTMS .025 8
QS RRVTFH 0.0021
QSQRRVTFH 2581 LPQVALHR_i 0.125 149 CWMPASLDH 0.025 IHPQPQS 1100011
333; RGDSPMEEH 0.125 JT HTRPPMKEV J 0.025 5 pQpQS-^" QRRV 0 0001
16 SCTPMKEST 0100 105 GNSDPESTF ! 0 025 -
316; KVIPLTTFT - 0100 95 ATPSNRTEG 0.025 S ; Table IX-109PID4v 4
307 RLHPSDDSI 0.100 205, CHSPP1 I01 0.025 Al-10-mers
l 124' TVQPTVEEA .100 1 a2 STTMEIWIH 0.025 Each peptide is a portion of SEQ
L J F. ~- ID N0: 9; each start position is
41 KVAGKSQRR 0.100 17 ,FCTPMKESTT~ 0.025 specified, the length of peptide is
310, PSDDSIKVI 075 3201 LTTFTPRQQ 0 025 10 amino acids, and the end
F7-61 TSTSHGLPL 0.075 r321iTTFTPR QA Ã 0.025 position for each peptide is the
1 (
start tar
sposition tion plus nine.
22 ESTTMEIWI 0 075 50 VTFHLPEGS F_0.025
- - f Pos Subse uence Score
296 GSATSQFYT 0 075 215 ALHHSPPLV 0020 1- i q QSR
1.000
252 _ISHSSPLPQ 0 075 ] 167; ALCHSPPLS 0 020 7 QPQS RRVTF 0
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156
........... .........__...._._............_._.................
_..............._..-... ......... ......... ---- _............ ._.....
._........ _............_ _........._._..._..._.. ..............
......__._....... ..._..._ ....._..._....._.....__......................
...__.._.._.._...
Table IX - 109P1 D4v.4 Table XI -109P1D4v.4 Table XIV-109P1D4v.4
Al-10-mers A0201-10-mers ,01101-9-mers
Each peptide is a portion of SEQ Each peptide is a portion of SEQ Each peptide
is a portion of SEQ
ID NO: 9; each start position is ID NO: 9; each start position is ID NO: 9;
each start position is
specified, the length of peptide is specified, the length of peptide is
specified, the length of peptide is 9
amino acids, and the end 10 amino acids, and the end amino acids, and the end
position
position for each peptide is the position for each peptide is the for each
peptide is the start
start position plus nine. start position plus nine. position plus eight.
Pos Subsequence I Score s Pos Subsequence Score Pos Subsequencey Score
HPQPgSQRRV 0.025 QPQSgRRVTF 0.0004 a y HPQPQ Q 0.040
QSQRrVTFHL 0.008 IHPQpQSQRR 1 0.000 IHPQPQSQR 0.004
IHPQpQSQRR ,0,005
... IWIHpQPQSQ 0 000 PQSQRRVTF f 0.001
FT7i
1 , EIWIhPQPQS 0.002 { WIHPQPQSQ 0.000
IWIHpQPQSQ j Or001 Table Xll -109PI D4v4 8
QSQRRVTFH 0 00
A3 9 mers '"-
PQPQsQRRVT 0.000 - '- -- - PQPQSQRRV' 000
Each peptide is a portion of SEQ
P qQ RVTFH 0,000 ID N0: 9; each start position is ~- _ IWIHPQPQS 0 000
specified, the length of peptide is 9 _ QPQSQRRVT 0.000
F Table X-109P1D4v.4 amino acids, and the end position
A0201-9-mers for each peptide is the start" ""
- - - - - Table XV-109P1D4v.4
Each peptide is a portion of SEQ position plus eight.
,01101-10 mers
ID NO: 9; each start position is Pos 1Subsequence ! Score;
specified, the length of is 9 Each peptide is a portion of SEQ
peptide EHPQPQSQRR 4 0.060:
ID NO: 9; each start position is
amino acids, and the end position
for each peptide is the start 3 IHPQPQSQR 0 006 specified, the length of
peptide is
4 10 amino acids, and the end
position plus eight. qIQ RRVTF 0.006;
position for each peptide is the
Pos Subsequence 4 Score2 ~WIHPQPQSQ 0 003? start position plus nine.
F PQPQSQRRV 0.031 QSQRRVTFH i r p., Pos
jl- Subsequence Score;
WIHPQPQSQ 0.009 6
QPQSQRRVT 'FP-90 0; 3 i WIHPgPQSQR .080
2:1 _
QSQRRVTFH 0 006j ~- IWIHPQPQS [ 0 000 IHPQpQSQRR 0.004
QPQSQRRVT 0 004 5 ,-- PQPQSQRRV 0 000' f~ QPQSgRRVTF 0 002;
PQSQRRVTF 0.000 M PQSQrRVTFH 0.001
IHPQPQSQR 0.000 , Table XIII -109P1 D4v.4 I -- QSQRrVTFHL 0.001
~- __ ,03-10-mers } ..
-
. ' I....IW(HPQPQS 0000_.. -.....- .._ _ =._ _.._..f EIWIhPQPQS 0000;
HPQPQSQRR 0 000 Each peptide is a portion of SEQ
5 HPQPgSQRRV 0.000
( ID N0: 9; each start position is
specified, the length of peptide is IWIHpQPQSQ 110.000;---
Table XI -109P1 D4v.4 10 amino acids, and the end PQPQsQRRVT 0.000;
,00201-10-mers position for each peptide is the
start position plus nine
Each peptide is a portion of SEQ
i Pos Subsequence Score Table XVI -109P1 D4v.4
ID NO: 9; each start position is C . r.,,, .... A24-9-mers
specified, the length of peptide is } 3 WIHPqPQSQR 0.900
10 amino acids, and the end Each peptide is a portion of SEQ
position for each peptide is the 7 QPQSgR 0.020 ID NO: 9; each start position
is
start osition lus nine. 9 specified, the length of e tide is 9
p p_. QSQRrVTFHL- f 0.013 9 p p
I Pos Subsequence Scored Jf. 1,_, f; EIWIhPQPQS 0.009 I ammo acids, and the
end position
I for each peptide is the start
QSQRrVTFHL 0 809, 4 -1HPQpQSQRR !0 004 tposition plus eight
0.009 f TT EIW hPQPQS Ã 0.006; ( o"~4SQrRVTFH 0.002 Pos SubsequenceScore,
. 5 HPQPgSQRRV 0.000 -7 _ PQSQRRVTF 0.200
E-L aHPQPgSQRRV 0.003 s PQPQsQRRVT 0000 QPQSQRRVT 0.150
i
i=8 11 PQSQrRVTFH ( 0.002 jt õ2 I IWIHpQPQSQ , 0.000 L 1 IWIHPQPQS 0.1501 E-TI-
PugsQRRVT ` 0.001õ 4 HPQPQSQRR 0 022
CA 02522994 2005-10-20
WO 2004/098515 PCT/US2004/013568
157
.............._V._.._........... ....... m.... __
Table XVI 109P1 D4v.4 Table XXI -109PI D4v 4
A24 9 mers Table XIX-109P1 MA B3501-10-mers
B7-10-mers
Each peptide is a portion of SEQ ' ...........
.....____.............._........... ___................... .__....._ Each
peptide is a portion of SEQ
ID NO: 9; each start position is Each peptide is a portion of SEQ ID NO: 9;
each start position is
specified, the length of peptide is 9 ID NO: 9; each start position is
specified, the length of peptide is
amino acids, and the end position specified, the length of peptide is 9 amino
acids, and the end
for each peptide is the start 10 amino acids, and the end position for each
peptide is the
position plus eight. position for each peptide is the
tart position plus eight.
s
start position plus nne.
Pos _,I Subsequence Score; ---- Pos Subsequence ,Score
Pos Subsequence Score HPQPgSQR V 4 000
QSQRRVTFH LL ? 0.0152 ;-.-9 QSQRrVTFHL 4;000 EIWIhP PQS 0.100
PQPQSQRRV 0.0 15
WIHPQPQSQ ' .014; I J I HPQPgSQRRV 4.000
PQPQsQRRVT 0010
F J
IHPQPQSQR 0 002 1 QPQSgRRVTF 0 600_ 3 WIHPgPQSQR 0.010
EIWIhPQPQS 0 03 PQSQrRVTFH 0.001
Table XVII -109P1 D4v 4 WIHPgPQSQR rn015 L i 2 IWIH
pQPQSQ .001
A24-10 mers[~ QPQsQRRVT 0.0154 iHPQpQSQRR 0.001
Each peptide is a portion of SEQ PQSQrRVTFH 0.001
ID NO: 9; each start position is
IWIHpQPQSQ 0.001 Table V111-109M4.5
specified, the length of peptide is
amino acids, and the end IHPQpQSQRR 0^001 _.A1 9-mers
position for each peptide is the Each peptide is a portion of SEQ
start position plus nine. ID NO: 11; each start position is
Table XX -109P1 D4v.4
PosSubsequence Score specified, the length of peptide is
B3501 9 mers s
9 amino acids, and the end 8 . 4 0 0
F-1 i õ Each peptide is a portion of SEQ position for each peptide is the
7 QPQS RRVTF 3.000 ID NO: 9; each start position is start position plus eight.
specified, the length of peptide is 9i ---
5 HPQPgSQRRV 0180', Pos Subse uence Score,
amino acids, and the end position
1 EIWIhPQPQS 0100 for each peptide is the start 3 SVHTRPSQR 0100
2 IWIH QPQSQ 0.018 position plus eight
RPSQRRVTF ~0 0-50
IF,
6 ' PQPQsQRRVT 0.015 IÃ._ Pos Subsequence Score E V _S . 2 VHTRPSQ mom"
_ ..._ ._._....~ 6 QPQSQRRVT 2 000 I....._._-.__.
WIHPgPQSQR_ 0.012 .- - .--- i
5 HTRPSQRRV 0.00251
_._ iHPQpQSQRR 0.002 C 4 HPQPQSQRR 0 200 PVSVHTRPS 0.001:
DD 1, PQSQrRVTFH ._' 0 001 7._ PQSQRRVTF 0.100; VHTRPSQRR 0..001
L 8 . QSQRRVTFH 0.050'7
õ-` TRPSQRRVT 0 001
TTable XVIII -109P1 D4v.4 E75 5 ! LPQ-PQ J0.9201
EEC SQRRVTFH K no
B7-9-mers IWIHPQPQS 0 010;
Each is a portion of SEQ
peptide 2 mmWIHPQPQSQ R 0 010 Table IX-109P1 D4v.5
ID NO: 9; each start position is ~- -; - , Al-10 mers
specified, the length of peptide is 9 I,.._:? IHPQPQSQR 0 001;
amino acids, and the end position Each peptide is a portion of SEQ
for each peptide is the start Table XXI - 109P1 D4v.4 ID NO: 11; each start
position is
position plus eight. B3501-10-mers specified, the length of peptide is
F-
10 amino acids, and the end
Pos Subsequence ; Score Each is a portion of SEQ' ?
peptide position for each peptide is the
QPQSQRRVT ; 3.000 ID NO: 9; each start position is start position plus,nine
specified, the length of peptide is
4 HPQPQSQRR 0.200 9 amino acids, and the end ' Pos Subsequence Score I
[ DI PQPQSQRRV 0.020 I position for each peptide is the 3 I VSVHtRPSQR 0.150
QSQRRVTFH 0.010 start position plus eight. 8- 4 SVHTrPSQRR 0.,100
Pos Subsequence
WIHPQPQSQ 0 010 ! 20.00 6 HTRPSQRRV 0.025
PQSQRRVTF ; 0.003 7 QPQS RRVTF -
q ... w _ 7 TRPSgRRVTF 0.010
IWIHPQPQS 0.003
F g QSQRrVTFHL 5.00
IHPQPQSQR 0.002 1 _!~.___I VPVSvHTRPS 0.003.._
F7E 0
CA 02522994 2005-10-20
WO 2004/098515 PCT/US2004/013568
158
...........,_ .............__.... _...... -....... ._.....
..._.................... ......... ._............._, ....._...........
....._......_......_.......... _...._...._........ ..............
Table XV-109P1 D4v.5
Table IX-109P1D4v.5 Table Xl-109P1D4v.5
Al-10-mers _ A0201-10-mers A1101-10-mers
Each peptide is a portion of SEQ Each peptide is a portion of SEQ Each peptide
is a portion of SEQ
ID NO: 11; each start position is ID NO: 11; each start position is ID NO: 11;
each start position is
specified, the length of peptide is specified, the length of peptide is 1
specified, the length of peptide is
amino acids, and the end 10 amino acids, and the end 10 amino acids, and the
end
position for each peptide is the position for each peptide is the position for
each peptide is the
start position plus nine. start position plus nine. } start position plus
nine.
s PEs Subsequence Score 11fos Subsequence_ ' Score Posi Subsequence Score
8 RPSQrRVTFH 0.003. _ HTRPSQRRVT~ 0 000' SVHTrPSQRR 0.400
2 [ PVSVhTRPS 0.002TRPSgRRVTF ! 0 000 VSVHtRPSQR .006
w_ _ .._._..._ _..__ _,.. ~. I.. I _.RPSQrRVTFH.
50o- Table X11-1 09PI D4v.5 TRPS
-,,.gRRVTF 0.000
QRR' A3-9-mars
S
FVHTRp
5 F-O000 PVSVhTRPSQ Oa000 w
Each peptide is a portion of SEQ
HTRPsQRRVT 0.000
ID NO: 11; each start position is
Table X-109P1D4v.5 specified, the length of peptide is 9 PSQRrVTFHL 0000
amino acids, and the end position
' 0.000
for each peptide is the start ..I .[ VPVSvHTRPS 000
Each peptide is a portion of SEQ position plus eight VHTRpSQRRV 0.000
ID NO: 11; each start position is }
Pos Subsequence score; ;_...___ _ ... _ _. ..___
specified, the length of peptide is 9I = Table XVI-109P1 D4v 5
amino acids, and the end position .SVHTRPSQR 0 400
for each peptide is the start A24-9-mars
7 [ RPSQRRVTF 0.0201 position plus eight. Each peptide is a portion of SEQ ID
Pos ubsequence Score E .. -_w. _VHTRPSQRR 1 0 006 N0:11; each start position
is
SVHTRPSQR 0.001 HTRPSQRRV f 0 002 specified, the length of peptide is 9
amino acids, and the end position for
PSQRRVTFH 0.000
i each peptide is the start position plus
5 HTRPSQRRV 0.000
VSVHTRPSQ 000: eight
RPSQRRVTF 0.000!
F PVSVHTRPS 0 000 Pos Subsequence Score
.... VSVHTRPSQ 0.0 0 _.~ _ _... q...
6 i TRPSQRRVT ! 0 00 7 RPSQRRVTF 4.000
PSQRRVTFH 0.000 P __.....~__...
5 4 HTRPSQRRV 0120
TRPSQRRVT IE...~... .... _._..._ C
~ Table XIV-109P1 D4v.5 TRPSQRRVT 0.015
PVSVHTRPS 0.000 A1101-9-mars - ~- -=
. _ _. _ ,..._ _.... _ .._ ! 2 VSVHTRPSQ 0 015
4 VHTRPSQRR 000
._ __ Each peptide is a portion of SEQ~j PVSVHTRPS _ 0 010 j
ID NO: 11; each start position is
Table XI-109P1D4v.5 specified, the length of peptide is 9 ~ 3 - SVHTRPSQR^
0.010 1
amino acids, and the end position
{ A0201-10-mars PSQRRVTFH 0 002
___._ __._ . .__ __ _ ~_.= for each peptide is the start
~---!-.-_-.....~.
0.001
VHTRPS
Each
peptide is a portion of SEQ 4 position plus eight. L.. ....... . _Q.RR ..
._,..... ... I
I ID NO: 11; each start position is q --- ~-
Pos I Subse uence Score
specified, the length of peptide is ( _ Table XVII-109P1 D4v.5
10 amino acids, and the end 4 SVHTrPSQRR C0 600; 9P__.._4
i~ A24 10 mars
position for each peptide is the -
3 i _ VSVHtRPSQR (F9-673-6 start position plus nine. Each peptide is a portion
of SEQ
8 !1
RPSQrRVTFH I 0 006
PosSubsequence Score ID N0:11; each start position is
C ~PSQRrVTFHL 0.018 ;t -...'. _. l.. TRPSgRRVTF 0.002 sp10 aminoo acids tend
the end is
---- - 9 PSQRrVTFHL 0 001; I
5 VHTRpSQRRV 0.016 position for each peptide is the
HTRPsQRRVT 0001 `: start position plus nine.
RPSQrRVTFH 0.006 -- _
2 PVSVhTRPSQ (O.000i Pos Subse uence Score;
4 SVHTrPSQRR 0.001 q .. _ _ _..+
9 PSQRrVTFHL 0.840
1 VPVSvHTRPS 0 000:
1F
E-7E, VPVSvHTRPS 0.000 ~5~ ",HTRPSQRRV 0.300;
VSVHtRPSQR ` 0.000
t 2 PVSVhTRPSQ 0.000. VPVSvHTRPS 0.150;
HTRPsQ 0.120!
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