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CA 02503346 2009-10-08
NUCLEIC ACID AND CORRESPONDING PROTEIN ENTITLED 24P4C12
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 a gene and its encoded protein,
termed 24P4C12, expressed in certain
cancers, and to diagnostic and therapeutic methods and compositions useful in
the management of cancers that express
24P4C12.
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 fives are dramatically
altered. Many cancer patients experience strong anxieties driven by the
awareness of the potential for recurrence or
treatment failure. Many cancer patients experience physical debilitations
following treatment. Furthermore, many cancer
patients experience a recurrence.
Worldwide, prostate cancer is the fourth most prevalent cancer in men. In
North America and Northern Europe, it
is by far the most common cancer in males and is the second leading cause of
cancer death in men. in the United States
alone, well over 30,000 men die annually of this disease - second only to lung
cancer. Despite the magnitude of these
figures, there is still no effective treatment for metastatic prostate cancer.
Surgical prostatectomy, radiation therapy,
hormone ablation therapy, surgical castration and chemotherapy continue to be
the main treatment modalities.
Unfortunately, these treatments are ineffective for many and are often
associated with undesirable consequences.
On the diagnostic front, the lack of a prostate tumor marker that can
accurately detect early-stage, localized tumors
remains a significant limitation in the diagnosis and management of this
disease. Although the serum prostate specific
antigen (PSA) assay has been a very useful tool, however its specificity and
general utility is widely regarded as lacking in
several important respects.
Progress in identifying additional specific markers for prostate cancer has
been improved by the generation of
prostate cancer xenografts that can recapitulate different stages of the
disease in mice. The LAPC ()os 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 of
al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers
include PCTA-1 (Su etal., 1996, Proc. Nati.
Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et
al., Clin Cancer Res 1996 Sep 2 (9): 1445--
51), STEAP (Hubert, etal., Proc Nati Aced Sci U S A. 1999 Dec 7; 96(25): 14523-
8) and prostate stem cell antigen (PSCA)
(Reiter etal., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
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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
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.
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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 cif DCIS
occurring in the remaining breast tissue. This is important because DCIS, if
left untreated, may develop into invasive breast
cancer. Nevertheless, there are serious side effects or sequelae to these
treatments. There is, therefore, a need for
efficacious breast cancer treatments.
There were an estimated 23,100 new cases of ovarian cancer in the United
States in 2000. It accounts for 4% of
all cancers among women and ranks second among gynecologic cancers. During
1992-1996, ovarian cancer incidence
rates were significantly declining. Consequent to ovarian cancer, there were
an estimated 14,000 deaths in 2000. Ovarian
cancer causes more deaths than any other cancer of the female reproductive
system.
Surgery, radiation therapy, and chemotherapy are treatment options for ovarian
cancer. Surgery usually includes
the removal of one or both ovaries, the fallopian tubes (salpingo-
oophorectomy), and the uterus (hysterectomy). In some
very early tumors, only the involved ovary will be removed, especially in
young women who wish to have children. In
advanced disease, an attempt is made to remove all intra-abdominal disease to
enhance the effect of chemotherapy. There
continues to be an important need for effective treatment options for ovarian
cancer.
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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 24P4C12, that has now been
found to be over-expressed in
the cancer(s) listed in Table I. Northern blot expression analysis of 24P4C12
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 24P4C12 are provided. The tissue-related profile of 24P4C12 in normal adult
tissues, combined with the over-expression
Observed in the tissues listed in Table I, shows that 24P4C12 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 Tablet.
The invention provides polynucleotides corresponding or complementary to all
or part of the 24P4C12 genes,
mRNAs, and/or coding sequences, preferably in isolated form, including
polynucleotides encoding 24P4C12-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 adds; 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
24P4C12-related protein, as well as the peptides/proteins themselves; DNA,
RNA, DNAIRNA hybrids, and related molecules,
polynucleotides or oligonucleotides complementary or having at least a 90%
homology to the 24P4C12 genes or mRNA
sequences or parts thereof, and polynucleotides or ofigonucleotides that
hybridize to the 24P4C12 genes, mRNAs, or to
24P4C12-encoding polynucleotides. Also provided are means for isolating cDNAs
and the genes encoding 24P4C12.
Recombinant DNA molecules containing 24P4C12 polynucleotides, cells
transformed or transduced with such molecules, and
host-vector systems for the expression of 24P4C12 gene products are also
provided. The invention further provides antibodies
that bind to 24P4C12 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.
The invention further provides methods for detecting the presence and status
of 24P4C12 polynucleotides and
proteins in various biological samples, as well as methods for identifying
cells that express 24P4C12. A typical embodiment of this
invention provides methods for monitoring 24P4C12 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 24P4C12 such as cancers of tissues listed in Table I, including
therapies aimed at inhibiting the transcription,
translation, processing or function of 24P4C12 as well as cancer vaccines. In
one aspect, the invention provides
compositions, and methods comprising them, for treating a cancer that
expresses 24P4C12 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
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the production or function of 24P4C12. Preferably, the carrier is a uniquely
human carrier. In another aspect of the
invention, the agent is a moiety that is immunoreactive with 24P4C12 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 1-ILA class I molecule in a human to elicit a CTL
response to 24P4C12 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 pepfides 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 24P4C12 as
described above. The one or more than one nucleic acid molecule may also be,
or encodes, a molecule that inhibits
production of 24P4C12. Non-limiting examples of such molecules include, but
are not limited to, those complementary to a
nucleotide sequence essential for production of 24P4C12 (e.g. antisense
sequences or molecules that form a triple helix with
a nucleotide double helix essential for 24P4C12 production) or a ribozyme
effective to lyse 24P4C12 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 V111-)0(1 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 )001
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 HtA 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:
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;
CA 02503346 2009-10-08
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.
DESRIPTION OF THE FIGURES
= -
Figure I. The 24P4C12 SSH sequence of 160 nucleotides.
Figure 2. A) The cDNA (SEQ ID NO.: 2) and amino acid sequence (SEQ ID NO.:3)
of 24P4C12 variant 1 (also
called "24P4C12 v. 1" or "24P4C12 variant 1") is shown in Figure 2A. The start
methionine is underlined. The open reading
frame extends from nucleic acid 6-2133 including the stop codon.
B) The cDNA (SEQ ID NO.: 4) and amino acid sequence (SEQ ID NO.: 5) of 24P4C12
variant 2 (also called
"24P4C12v.2") is shown in Figure 2B. The codon for the start methionine is
underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
C) The cDNA (SEQ ID NO.: 6) and amino acid sequence (SEQ ID NO.: 7) of 24P4C12
variant 3 (also called
"24-P4C12 v. 3") is shown in Figure 2C. The codon for the start methionine is
underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
D) The cDNA (SEQ ID NO.: 8) and amino acid sequence (SEQ ID NO.: 9) of 24P4C12
variant 4 (also called
"24P4C12 v. 4") is shown in Figure 2D. The codon for the start methionine is
underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
E) The cDNA (SEQ ID NO.: 10) and amino acid sequence (SEQ ID NO.: 11) of
24P4C12 variant 5 (also called
"24P4C12v.5") is shown in Figure 2E. The codon for the start methionine is
underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
F) The cDNA (SEQ ID NO.: 12) and amino acid sequence (SEQ ID -NO.: 13) of
24134C12 variant 6 (also called
"24P4C12 v.6") is shown in Figure 2F. The codon for the start methionine is
underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
0) The cDNA (SEQ ID NO.: 14) and amino acid sequence (SEQ ID NO.: 15) of
24P4C12 variant 7 (also called
"24P4C12 v.7") is shown in Figure 2G. The codon for the start methionine is
underlined. The open reading frame extends
from nucleic acid 6-1802 including the stop codon.
H) The cDNA (SEQ ID NO.: 16) and amino acid sequence (SEQ ID NO.: 17) of
24P4C12 variant 8 (also called
"24P4C12 v.8") is shown in Figure 2H. The codon for the start methionine is
underlined. The open reading frame extends
from nucleic acid 6-2174 including the stop codon.
I) The cDNA (SEQ ID NO.: 18) and amino acid sequence (SEQ ID NO.: 19) of
24P4C12 variant 9 (also called
"24P4C12v.9") is shown in Figure 21. The codon for the start methionine is
underlined. The open reading frame extends
from nucleic acid 6-2144 including the stop codon.
Figure 3.
A) Amino acid sequence of 24P4C12 v.1 (SEQ ID NO.: 20) is shown in Figure 3A;
it has 710 amino acids.
[3) The amino acid sequence of 24P4C12 v.3 (SEQ ID NO.: 21) is shown in Figure
313; it has 710 amino acids.
C) The amino acid sequence of 24P4C12 v.5 (SEQ ID NO.: 22) is shown in Figure
3C: it has 710 amino acids.
D) The amino acid sequence of 24P4C12v.6 (SEQ ID NO.: 23) is shown in Figure
3D; it has 710 amino acids.
E) The amino acid sequence of 24P4C12 v.7 (SEQ ID NO.: 24) is shown in Figure
3E; it has 598 amino acids.
F) The amino acid sequence of 24P4C12 v.8 (SEQ ID NO.: 25) is shown in Figure
3F; it has 722 amino acids.
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G) The amino acid sequence of 24P4C12 v.9 (SEQ ID NO.: 26) is shown in Figure
30; it has 712 amino acids. As
used herein, a reference to 24P4C12 includes all variants thereof; including
those shown in Figures 2,3, 10, and 11, unless
the context clearly indicates otherwise.
Figure 4. Alignment or 24P4C12 (SEQ ID NO.: 27) with human choline transporter-
like protein 4 (CTL4)
(gill 4249468)(SEQ ID NO.: 28).
Figure 5. Hydrophilicity amino acid profile of 24P4C12 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
through the ExPasy
molecular biology server.
Figure 6. Hydropathicity amino acid profile of 24P4C12 determined by computer
algorithm sequence analysis
using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J.
N4ol. Biol. 157: 105-132) accessed on the ProtScale
website located on the World Wide Web
through the ExPasy molecular biology server.
Figure 7. Percent accessible residues amino acid profile of 24P4C12 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 through the ExPasy molecular biology
server.
Figure 8. Average flexibility amino acid profile of 24P4C12 determined by
computer algorithm sequence analysis
using the method of Bhaskaran and Ponnuswamy (Bhaskaran R. , and Ponnuswamy P.
K. , 1988. Int..I. Pept. Protein Res.
37:242-255) accessed on the ProtScale website located on the Worid Wide Web at
(. expasy. ch/cgi-binlprotscale. pl)
through the ExPasy molecular biology server.
Figure 9. Beta-turn amino acid profile of 24P4C12 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 through the ExPasy molecular
biology server.
Figure 10. Schematic alignment of SNP variants of 24P4C12. Variants 24P4C12
v.2 through v.6 are variants with
single nucleotide differences. Though these SNP variants are shown separately,
they could also occur in any cOnibillatitinS
and in any transcript variants that contains the base pairs. Numbers
correspond to those of 24P4C12 v.1. Bled box shows
the same sequence as 24P4C12 v.1. SNPs are indicated above the box.
Figure 11. Schematic alignment of protein variants of 24P4C12. Protein
variants correspond to nucleotide
variants. Nucleotide variants 24P4C12 v.2, v.4 in Figure 10 code for the same
protein as 24P4C12 v.1. Nucleotide variants
24P4012 v.7, v.8 and v.9 are splice variants of v.1, as shown in Figure 12.
Single amino acid differences were indicated
above the boxes. Black boxes represent the same sequence as 24P4C12 v.1.
Numbers underneath the box correspond to
24P4C12 v.1.
= Figure 12. Exon compositions of transcript variants of 24P4C12. Variant
24P4C12 v.7, v.8 and v.9 are transcript
variants of 24P4C12 v.1. Variant 24P4C12 v.7 does not have exons 10 and 11 of
variant 24P4C12 v.1. Variant 24P4C12 v.8
extended 36 bp at the 3' end of exon 20 of variant 24P4C12 v.1. Variant
24P4C12 v.9 had a longer exon 12 and shorter
exon 13 as compared to variant 24P4C12 v.1. Numbers in Q" underneath the boxes
correspond to those of 24P4C12 v.1.
Lengths of introns and exons are not proportional.
Figure 13. Secondary structure and transmembrane domains prediction for
24P4C12 protein variant 1 (SEQ ID
NO: 112). k The secondary structure of 24P4C12 protein variant 1 was predicted
using the HNN - Hierarchical Neural
Network method accessed from the
ExPasy molecular biology server. 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 in a given
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secondary structure is also listed, 6: Schematic representation of the
probability of existence of transmembrane regions and
orientation of 24P4C12 variant 1 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). C: Schematic representation of the probability of the existence of
transmembrane regions and the extracellular and
intracellular orientation of 24P4C12 variant 1 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:
AAA' Press, 1998). The TMpred and TMHMM
algorithms are accessed from the ExPasy molecular biology server
(http://www.expasy.ch/tools/).
Figure 14. 24P4C12 Expression by RT-PCR. First strand cDNA was generated from
vital pool 1 (kidney, liver and
lung), vital pool 2 (colon, pancreas and stomach), a pool of prostate cancer
xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD
and LAPC-9A1), prostate cancer pool, bladder cancer pool, kidney cancer pool,
colon cancer pool, ovary cancer pool, breast
cancer pool, and cancer metastasis pool. Normalization was performed by PCR
using primers to actin. Semi-quantitative
PCR, using primers to 24P4C12, was performed at 26 and 30 cycles of
amplification. Results show strong expression of
24P4C12 in prostate cancer pool and ovary cancer pool. Expression was also
detected in prostate cancer xenografts,
bladder cancer pool, kidney cancer pool, colon cancer pool, breast cancer
pool, cancer metastasis pool, vital pool 1, and vital
pool 2.
Figure 15. Expression of 24P4C12 in normal tissues. Two multiple tissue
northern blots (Clontech) both with 2 ug
of mRNA/lane were probed with the 24P4C12 sequence. Size standards in
kilobases (kb) are indicated on the side. Results
show expression of 24P4C12 in prostate, kidney and colon. Lower expression is
detected in pancreas, lung and placenta
amongst all 16 normal tissues tested.
Figure 16. Expression of 24P4C12 in Prostate Cancer Xenografts and Cell Lines.
RNA was extracted from a
panel of cell lines and prostate cancer xenografts (PrEC, LAPC-4AD, LAPC-4A1,
LAPC-9AD, LAPC-9AI, LNCaP, PC-3,
DU145, TsuPr, and LAPC-4CL). Northern blot with 10 ug of total RNA/lane was
probed with 24P4C12 SSH sequence. Size
standards in kilobases (kb) are indicated on the side. The 24P4C12 transcript
was detected in LAPC-4AD, LAPC-4A1, LAPC-
9AD, LAPC-9A1, LNCaP, and LAPC-4 CL.
Figure 17. Expression of 24P4C12 in Patient Cancer Specimens and Normal
Tissues. RNA was extracted from a
pool of prostate cancer specimens, bladder cancer specimens, colon cancer
specimens, ovary cancer specimens, breast
cancer specimens and cancer metastasis specimens, as well as from normal
prostate (NP), normal bladder (NB), normal
kidney (NK), and normal colon (NC). Northern blot with 10 pg of total RNA/lane
was probed with 24P4C12 SSH sequence.
Size standards in kilobases (kb) are indicated on the side. Strong expression
of 24P4C12 transcript was detected in the
patient cancer pool specimens, and in normal prostate but not in the other
normal tissues tested.
Figure 18. Expression of 24P4C12 in Prostate Cancer Patient Specimens. RNA was
extracted from normal
prostate (N), prostate cancer patient tumors (T) and their matched normal
adjacent tissues (Nat). Northern blots with 10 ug
of total RNA were probed with the 24P4C12 SSH fragment. Size standards in
kilobases are on the side. Results show
expression of 24P4C12 in normal prostate and all prostate patient tumors
tested.
Figure 19. Expression of 24P4C12 in Colon Cancer Patient Specimens. RNA was
extracted from colon cancer
cell lines (CL: Cob 205, LoVo, and SK-00-), normal colon (N), colonicancer
patient tumors (T) and their matched normal
adjacent tissues (Nat). Northern blots with 10 ug of total RNA were probed
with the 24P4C12 SSH fragment. Size
standards in kilobases are on the side. Results show expression of 24P4C12 in
normal colon and all colon patient tumors
tested. Expression was detected in the cell lines Cob 205 and SK-CO-, but not
in LoVo.
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Figure 20. Expression of 24P4C12 in Lung Cancer Patient Specimens. RNA was
extracted from lung cancer cell
lines (CL: CALU-1, A427, NCI-H82, NCI-H146), normal lung (N), lung cancer
patient tumors (T) and their matched normal
adjacent tissues (Nat). Northern blots with 10 ug of total RNA were probed
with the 24P4C12 SSH fragment. Size
standards in kilobases are on the side. Results show expression of 24P4C12 in
lung patient tumors tested, but not in normal
lung. Expression was also detected in CALU-1, but not in the other cell lines
A427, NCI-H82, and NCI-H146.
Figure 21. Expression of 24P4C12 in breast and stomach human cancer specimens.
Expression of 24P4C12 was
assayed in a panel of human stomach and breast cancers (T) and their
respective matched normal tissues (N) on RNA dot
blots. 24P4C12 expression was seen in both stomach and breast cancers. 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 24P4C12 may be expressed in early stage
tumors.
Figure 22. 24P4C12 Expression in a large panel of Patient Cancer Specimens.
First strand cDNA was prepared
from a panel of ovary patient cancer specimens (A), uterus patient cancer
specimens (B), prostate cancer specimens (C),
bladder cancer patient specimens (D), lung cancer patient specimens (E),
pancreas cancer patient specimens (F), colon
cancer specimens (G), and kidney cancer specimens (H). Normalization was
performed by PCR using primers to actin.
Semi-quantitative PCR, using primers to 24P4C12, was performed at 26 and 30
cycles of amplification. Samples were run
on an agarose gel, and PCR products were quantitated using the Alphalmager
software. Expression was recorded as
absent, low, medium or strong. Results show expression of 24P4C12 in the
majority of patient cancer specimens tested,
73.3% of ovary patient cancer specimens, 83.3% of uterus patient cancer
specimens, 95.0% of prostate cancer specimens,
61.1% of bladder cancer patient specimens, 80.6% of lung cancer patient
specimens, 87.5% of pancreas cancer patient
specimens, 87.5% of colon cancer specimens, 68.4% of of clear cell renal
carcinoma, 100% of papillary renal cell carcinoma.
Figure 23. 24P4C12 expression in transduced cells. PC3 prostate cancer cells,
NIH-3T3 mouse cells and 300.19
mouse cells were transduced with 24P4C12 .pSRa retroviral vector. Cells were
selected in neomycin for the generation of
stable cell lines. RNA was extracted following selection in neomycin. Northern
blots with 10 ug of total RNA were probed
with the 24P4C12 SSH fragment. Results show strong expression of 24P4C12 in
24P4C12.pSRa transduced PC3, 3T3 and
300.19 cells, but not in the control cells transduced with the parental pSRa
construct.
Figure 24. Expression of 24P4C12 in 2931 cells. 293T cell were transiently
transfected with either pCDNA3.1
Myc-His tagged expression vector, the pSR[lexpression vector each encoding the
24P4C12 variant 1 cDNA or a control neo
vector. Cells were harvested 2 days later and analyzed by Westem blot with
anti-24P4C12 pAb (A) or by Flow cytometry (B)
on fixed and permeabilized 2931 cells with either the anti-24P4C12 pAb or anti-
His pAb followed by a PE-conjugated anti-
rabbit IgG secondary Ab. Shown is expression of the monomeric and aggregated
forms of 24P4C12 by Western blot and a
fluorescent shift of 24P4C12-293T cells compared to control neo cells when
stained with the anti-24P4C12 and anti-His pAbs
which are directed to the intracellular NH3 and COOH termini, respectively.
Figure 25. Expression and detection of 24P4C12 in stably transduced PC3 cells.
PC3 cells were infected with
retrovirus encoding the 24P4C12 variant 1 cDNA and stably transduced cells
were derived by G418 selection. Cells were
then analyzed by Western blot (A) or immunohistochemistry (B) with anti-
24P4C12 pAb. Shown with an arrow on the
Western blot is expression of a ¨94 kD band representing 24P4C12 expressed in
PC3-24P4C12 cells but not in control neo
cells. Immunohistochemical analysis shows specific staining of 24P4C12-PC3
cells and not PC3-neo cells which is
competed away competitor peptide to which the pAb was derived.
Figure 26. Expression of recombinant 24P4C12 antigens in 2931 cells. 2931
cells were transiently transfected
with Tag5 His-tagged expression vectors encoding either amino acids 59-227 or
319-453 of 24P4C12 variant 1 or a control
vector. 2 days later supernatants were collected and cells harvested and
lysed. Supematants and lysates were then
subjected to Western blot analysis using an anti-His pAb. Shown is expression
of the recombinant Tag5 59-227 protein in
9
CA 02503346 2009-10-08
both the supernatant and lysate and the Tag5 319-453 protein in the cell
lysate. These proteins are purified and used as
antigens for generation of 24P4C12-specific antibodies.
Figure 27. Monoclonal antibodies detect 24P4C12 protein expression in 293T
cells by flow cytometry. 293T cells
were transfected with either pCDNA 3.1 His-tagged expression vector for
24P4C12 or a control neo vector and harvested 2
days later. Cells were fixed, permeabilized, and stained with a 1:2 dilution
of supernatants of the indicated hybridomas
generated from mice immunized with 300.19-24P4C12 cells or with anti-His pAb.
Cells were then stained with a PE-
conjugated secondary Ab and analyzed by flow cytometry. Shown is a fluorescent
shift of 293T-24P4C12 cells but not
control neo cells demonstrating specific recognition of 24P4C12 protein by the
hybridoma supernatants.
Figure 28. Shows expression of 24P4C12 Enhances Proliferation. PC3 and 3T3
were grown overnight in low MS.
Cells were then incubated in low or 10% FBS as indicated. Proliferation was
measured by Alamar Blue. Experiments were
performed in triplicate.
Figure 29. Detection of 24P4C12 protein by immunohistochemistry in prostate
cancer patient specimens. Prostate
adenocarcinoma tissue and its matched normal adjacent tissue were obtained
from prostate cancer patients. The results showed
strong expression of 241'4;12 in the tumor cells and normal epithelium of the
prostate cancer patients'tissue (panels (A) low
grade prostate adenocarcinoma, (B) high grade prostate adenocarcinoma_ (C)
normal tissue adjacent to tumor). The expression
was detected mostly around the cell membrane indicating that 24P4C12 is
membrane associated in prostate tissues.
Figure 30. Detection of 24P4C12 protein by immunohistochemistry in various
cancer patient specimens. Tissue
was obtained from patients with colon adenocarcinoma, breast ductal carcinoma,
lung adenocarcinoma, bladder transitional
cell carcinoma, renal clear cell carcinoma and pancreatic adenocarcinoma. The
results showed expression of 24P4C12 in
the tumor cells of the cancer patients' tissue (panel (A) colon
adenocarcinoma, (B) lung adenocarcinoma, (C) breast ductal
carcinoma, (0) bladder transitional carcinoma, (E) renal clear cell carcinoma,
(F) pancreatic adenocarcinoma).
Figure 31. Shows 24P4C12 Enhances Tumor Growth in SC1D Mice. 1 x 106 PC3-
24P4C12 cells were mixed with
Matrigel and injected on the right and left subcutaneous flanks of 4 male SCID
mice per group. Each data point represents
mean tumor volume (n=8).
Figure 32. Shows 24P4C12 Enhances Tumor Growth in SCID Mice. 1 x 106 3T3-
24P4C12 cells were mixed with
Matrigel and injected on the right subcutaneous flanks of 7 male SCID mice per
group. Each data point represents mean
tumor volume (n=6).
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections =
I.) Definitions
II.) 24P4C12 Polynucleotides
II.A.) Uses of 24P4C12 Polynucleotides
Il.A.1.) Monitoring of Genetic Abnormalities
II.A.2.) Antisense Embodiments
II.A.3.) Primers and Primer Pairs
II.A.4.) Isolation of 24P4C12-Encoding Nucleic Acid Molecules
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
III.) 24P4C12-related Proteins
ILA.) Motif-bearing Protein Embodiments
III.B.) Expression of 24P4C12-related Proteins
Modifications of 24P4C12-related Proteins
III.D.) Uses of 24P4C12-related Proteins
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IV.) 24P4C12 Antibodies
V.) 24P4C12 Cellular Immune Responses
VI.) 24P4C12 Transgenic Animals
VII.) Methods for the Detection of 24P4C12
VIII.) Methods for Monitoring the Status of 24P4C12-related Genes and Their
Products
IX.) Identification of Molecules That Interact With 24P4C12
X.) Therapeutic Methods and Compositions
X.A.) Anti-Cancer Vaccines
X.B.) 24P4C12 as a Target for Antibody-Based Therapy
X.C.) 24P4C12 as a Target for Cellular Immune Responses
X.C.1. Minigene Vaccines
X.C.2. Combinations of CTL Peptides with Helper Peptides
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL andlor HTL Peptides
X.D.) Adoptive Immunotherapy
X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes
XL) Diagnostic and Prognostic Embodiments of 24P4C12.
XII.) Inhibition of 24P4C12 Protein Function
XII.A.) Inhibition of 24P4C12 With Intracellular Antibodies
XII.B.) Inhibition of 24P4C12 with Recombinant Proteins
XII.C.) Inhibition of 24P4C12 Transcription or Translation
XII.D.) General Considerations for Therapeutic Strategies
XIII.) Identification, Characterization and Use of Modulators of 24P4C12
XIV.) KITSIArticles of Manufacture
I.) Definitions:
Unless otherwise defined, all terms of art, notations and other scientific
terms or terminology used herein are
intended to have the meanings commonly understood by those of skill in the art
to which this invention pertains. In some
cases, terms with commonly understood meanings are defined herein for clarity
and/or for ready reference, and the inclusion
of such definitions herein should not necessarily be construed to represent a
substantial difference over what is generally
understood in the art. Many of the techniques and procedures described or
referenced herein are well understood and
commonly employed using conventional methodology by those skilled in the art,
such as, for example, the widely utilized
molecular cloning methodologies described in Sambrook etal., 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 Cl -
C2 disease under the Whitmore-Jewett
system, and stage 13 - 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
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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 24P4C12 (either by removing the
underlying glycosylation site or by deleting
the glycosylation by chemical and/or enzymatic means), and/or adding one or
more glycosylation sites that are not present in
the native sequence 24P4C12. 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 24P4C12-related protein). For example, an analog of a
24P4C12 protein can be specifically bound by an
antibody or T cell that specifically binds to 24P4C12.
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-24P4C12 antibodies comprise monoclonal
and polydonal antibodies as well as fragments containing the antigen-binding
domain and/or one or more complementarity
determining regions of these antibodies.
An "antibody fragment" is defined as at least a portion of the variable region
of the immunoglobulin molecule that
binds to its target, i.e., the antigen-binding region. In one embodiment it
specifically covers single anti-24P4C12 antibodies and
clones thereof (including agonist, antagonist and neutralizing antibodies) and
anti-24P4C12 antibody compositions with
polyepitopic specificity.
The term "codon optimized sequences" refers to nucleotide sequences that have
been optimized for a particular
host species by replacing any codons having a usage frequency of less than
about 20%. Nucleotide sequences that have
been optimized for expression in a given host species by elimination of
spurious polyadenylation sequences, elimination of
exon/intron splicing signals, elimination of transposon-like repeats and/or
optimization of GC content in addition to codon
optimization are referred to herein as an "expression enhanced sequences."
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)), nonpepfidal
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)),
oligocarbamates (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
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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,
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 Atm, 1131, 1125, ro, Reim,
Rein, Sm153, Bi212 or 213, p32 and radioactive isotopes of Lu including Lum.
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
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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, etal., 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/100
pg/m1 ssDNA, in which temperatures for hybridization are above 37 degrees C
and temperatures for washing in
0.1XSSC/0.1% SOS 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 polynudeotide is said to be "isolated" when it is substantially
separated from contaminant polynudeotides that
correspond or are complementary to genes other than the 24P4C12 genes or that
encode polypeptides other than 24P4C12 gene
product or fragments thereof. A skilled artisan can readily employ nucleic
acid isolation procedures to obtain an isolated 24P4C12
polynudeotide. A protein is said to be "isolated," for example, when physical,
mechanical or chemical methods are employed to
remove the 24P4C12 proteins from cellular constituents that are normally
associated with the protein. A skilled artisan can readily
employ standard purification methods to obtain an isolated 24P4C12 protein.
Alternatively, an isolated protein can be prepared by
chemical means.
The term "mammal" refers to any organism classified as a mammal, induding
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'Iocally 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
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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 adds 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
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 24P4C12-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.
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A "pharmaceutical excipienr 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 witroligonucleotide". 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
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
superrnotif 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
(AC-225)
See Thorium-229 (Th-229)
Actinium-227
(AC-227)
Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat
metastases in the skeleton resulting
from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy
Bismuth-212
(Bi-212)
See Thorium-228 (Th-228)
Bismuth-213
(Bi-213)
See Thorium-229 (Th-229)
Cadmium-109
(Cd-109)
Cancer detection
=
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Cobalt-60
(Co-60)
Radiation source for radiotherapy of cancer, for food irradiators, and for
sterilization of medical supplies
Copper-64
(Cu-64)
A positron emitter used for cancer therapy and SPECT imaging
Copper-67
(Cu-67)
Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic studies
(i.e., breast and colon
cancers, and lymphoma)
Dysprosium-166
(Dy-166)
Cancer radioimmunotherapy
Erbium-169
(Er-169)
Rheumatoid arthritis treatment, particularly for the small joints associated
with fingers and toes
Europium-152
(Eu-152)
Radiation source for food irradiation and for sterilization of medical
supplies
Europium-154
(Eu-154)
Radiation source for food irradiation and for sterilization of medical
supplies
Gadolinium-153
(Gd-153)
Osteoporosis detection and nuclear medical quality assurance devices
Gold-198
(Au-198)
Implant and intracavity therapy of ovarian, prostate, and brain cancers
Holmium-166
(Ho-166)
Multiple myeloma treatment in targeted skeletal therapy, cancer
radioimmunotherapy, bone marrow ablation,
and rheumatoid arthritis treatment
Iodine-125
(1-125)
Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer
treatment, radiolabeling, tumor
imaging, mapping of receptors in the brain, interstitial radiation therapy,
brachytherapy for treatment of
prostate cancer, determination of glomerular filtration rate (GFR),
determination of plasma volume, detection
of deep vein thrombosis of the legs
Iodine-131
(I-131)
Thyroid function evaluation, thyroid disease detection, treatment of thyroid
cancer as well as other 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
(Ir-192)
Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked
arteries (i.e., arteriosclerosis and
restenosis), and implants for breast and prostate tumors
Lutetium-177
(Lu-177)
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Cancer radioimmunotherapy and treatment of blocked arteries (i.e.,
arteriosclerosis and restenosis)
Molybdenum-99
(Mo-99)
Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, liver,
lungs, heart, and other organs.
Currently, Tc-99m is the most widely used radioisotope used for diagnostic
imaging of various cancers and
diseases involving the brain, heart, liver, lungs; also used in detection of
deep vein thrombosis of the legs ,
Osmium-194
(0s-194)
Cancer radioimmunotherapy
Palladium-103
(Pd-103)
Prostate cancer treatment
Platinum-195m
(Pt-195m)
Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic
drug
Phosphorus-32
(P-32)
Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone
cancer diagnosis/treatment; colon,
pancreatic, and liver cancer treatment; radiolabeling nucleic acids for in
vitro research, diagnosis of superficial
tumors, treatment of blocked arteries (i.e., arteriosclerosis and restenosis),
and intracavity therapy
Phosphorus-33
(P-33)
Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and
treatment of blocked arteries (i.e.,
arteriosclerosis and restenosis)
Radium-223
(Ra-223)
See Actinium-227 (Ac-227)
Rhenium-186
(Re-186)
Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and
treatment of lymphoma and bone,
breast, colon, and liver cancers using radioimmunotherapy
Rhenium-188
(Re-188)
Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain
relief, treatment of rheumatoid
arthritis, and treatment of prostate cancer
Rhodium-105
(Rh-105)
Cancer radioimmunotherapy
Samarium-145
(Sm-145)
Ocular cancer treatment
=
Samarium-153
(Sm-153)
Cancer radioimmunotherapy and bone cancer pain relief
Scandium-47
(Sc-47)
Cancer radioimmunotherapy and bone cancer pain relief
Selenium-75
(Se-75)
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Radiotracer used in brain studies, imaging of adrenal cortex by gamma-
scintigraphy, lateral locations of
steroid secreting tumors, pancreatic scanning, detection of hyperactive
parathyroid glands, measure rate of
bile acid loss from the endogenous pool
Strontium-85
(Sr-85)
Bone cancer detection and brain scans
Strontium-89
(Sr-89)
Bone cancer pain relief, multiple myeloma treatment, and osteoblastic therapy
Technetium-99m
(Tc-99m)
See Molybdenum-99 (Mo-99)
Thorium-228
(Th-228)
Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer
radioimmunotherapy
Thorium-229
(Th-229)
Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) which
are alpha emitters used in
cancer radioimmunotherapy
Thulium-170
( Tm-170)
Gamma source for blood irradiators, energy source for implanted medical
devices
Tin-117m
(Sn-117m)
Cancer immunotherapy and bone cancer pain relief
Tungsten-188
(W-188)
Parent for Rhenium-188 (Re-188) which is used for cancer
diagnostics/treatment, bone cancer pain relief,
rheumatoid arthritis treatment, and treatment of blocked arteries (i.e.,
arteriosclerosis and restenosis)
Xenon-127
(Xe-127)
Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary
function tests, and cerebral blood
flow studies
Ytterbium-175
(Yb-175)
Cancer radioimmunotherapy
Yttrium-90
. (Y-90)
Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer
treatment
Yttrium-91
(Y-91)
A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer
radioimmunotherapy (i.e., 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 vitt .
Non-limiting examples of small molecules include compounds that bind or
interact with 24P4C12, ligands including
hormones, neuropeptides, chemokines, odorants, phospholipids, and functional
equivalents thereof that bind and preferably
inhibit 24P4C12 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, 24P4C12 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
etal., Current Protocols in Molecular Biology,
Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, are
identified by, but not limited to, those
that: (1) employ low ionic strength and high temperature for washing, for
example 0.015 M sodium chloride/0.0015 M sodium
citrate/0.1% sodium dodecyl sulfate at 50 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% Fico11/0.1%
polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate
at 42 C; or (3) employ 50% formamide, 5 x
SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),
0.1% sodium pyrophosphate, 5 x
=Denhardt's solution, son icated salmon sperm DNA (50 jig/m1), 0.1% SDS, and
10% dextran sulfate at 42 C, with washes at
42 C in 0.2 x SSC (sodium chloride/sodium, citrate) and 50% formamide at 55 C,
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
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
ovemight incubation at 37 C in a solution
comprising: 20% formamide, 5 x SSC (150 mM NaC1, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5 x
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Denhardrs 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 "supermotir 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:
A2: 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: 67, 6*3501-03, 6*51, B*5301, B*5401, B*5501, 6*5502, B*5601, 8*6701,
B*7801, B*0702, B*5101, B*5602
B44: B*3701, 8*4402, 8*4403, B*60 (B*4001), B61 (B*4006)
A1: 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, 6*3901, B*3902,
B*3903-04, 6*4801-02, B*7301,
B*2701-08
B58: B*1516, B*1517, B*5701, B*5702, B58
862: B*4601, B52, B*1501 (862), B*1502 (1375), B*1513 (877)
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 polypep tides 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
ll 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
24P4C12 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.
The "24P4C12-related proteins" of the invention indude 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 24P4C12 proteins or fragments thereof, as well as fusion proteins of
a 24P4C12 protein and a heterologous polypeplide
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are also induded. Such 24P4C12 proteins are collectively referred to as the
24P4C12-related proteins, the proteins of the
invention, or 24P4C12. The term '24P4C12-related protein" refers to a
polypeptide fragment or a 24P4C12 protein sequence of 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2Z 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, 600, 625, 650, or 664 or more
amino acids.
II.) 24P4C12 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or
complementary to all or part of a 24P4C12
gene, mRNA, and/or coding sequence, preferably in isolated form, including
polynucleotides encoding a 24P4C12-related
protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related
molecules, polynucleotides or oligonucleotides
complementary to a 24P4C12 gene or mRNA sequence or a part thereof, and
polynucleotides or oligonucleotides that
hybridize to a 24P4C12 gene, mRNA, or to a 24P4C12 encoding polynucleotide
(collectively, "24P4C12 polynucleotides"). In
all instances when referred to in this section, T can also be U in Figure 2.
Embodiments of a 24P4C12 polynucleotide indude: a 24P4C12 polynucleotide
having the sequence shown in
Figure 2, the nucleotide sequence of 24P4C12 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
24P4C12 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 6 through nucleotide residue number 2138,
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 6 through nucleotide residue number 2138,
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 6 through nucleotide residue number 2138,
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 6 through nucleotide residue number 2138,
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 6 through nucleotide residue number 2138,
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 6 through nucleotide residue number 2138,
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 6 through nucleotide residue number 1802,
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 6 through nucleotide residue number 2174,
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 nudeotide residue number 6 through nucleotide residue number 2144,
including the stop codon, wherein T
can also be U;
(XI) a polynucleotide that encodes a 24P4C12-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 24P4C12-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 polynudeotide 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 Figure 3A-D in any
whole number increment up to 710 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-D in any
whole number increment up to 710 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-D in any
whole number increment up to 710 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;
(VII) 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-D in any
whole number increment up to 710 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
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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, 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-D in any
whole number increment up to 710 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 3E in any whole
number increment up to 598 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 3E in any whole
number increment up to 598 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 3E in any whole
number increment up to 598 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 3E in any whole
number increment up to 598 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 3E in any whole
number increment up to 598 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 3F in any whole
number increment up (0 722 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;
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(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 3F in any whole
number increment up (0 722 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;
(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 3F in any whole
number increment up to 722 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;
(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 3F in any whole
number increment up to 722 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;
(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 3F in any whole
number increment up to 722 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 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 3G in any whole
number increment up to 712 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;
(XO(X) 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 3G in any whole
number increment up to 712 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;
(XXXI) 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 3G in any whole
number increment up to 712 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;
(XXM) a polynudeotide 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 3G in any whole
=
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number increment up to 712 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;
(XXXII!) 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 3G in any whole
number increment up to 712 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
(XXXIV) a polynucleotide that is fully complementary to a polynucleotide of
any one of (1)-(00011).
(XXXV) a peptide that is encoded by any of (I) to (XXXIII); and
(XXXVI) a composition comprising a polynucleotide of any of (1)-(X)CIV) or
peptide of (XXXV) together with a
pharmaceutical excipient and/or in a human unit dose form.
(XXXVII) a method of using a polynucleotide of any (I)-( XXXIV) or peptide of
(XXXV) or a composition of (XXXVI)
in a method to modulate a cell expressing 24P4C12,
(XXXVIII) a method of using a polynucleotide of any (I)-( XXXIV) or peptide of
(XXXV) or a composition of (X)OVI)
in a method to diagnose, prophylax, prognose, or treat an individual who bears
a cell expressing 24P4C12
(XXXIX) a method of using a polynucleotide of any (1)-( XXXIV) or peptide of
(XXXV) or a composition of (XXXVI)
in a method to diagnose, prophylax, prognose, or treat an individual who bears
a cell expressing 24P4C12, said
cell from a cancer of a tissue listed in Table I;
(XL) a method of using a polynucleotide of any (I)-(XXXIV) or peptide of
(XXXV) or a composition of (XXXVI)
in a method to diagnose, prophylax, prognose, or treat a a cancer;
(XLI) a method of using a polynucleotide of any (I)-(XXXIV) or peptide of
(XXXV) or a composition of (000/I)
in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue
listed in Table I; and,
(XL11) a method of using a polynucleotide of any (I)-(XXXIV) or peptide of
(XXXV) or a composition of 00(X/1)
in a method to identify or characterize a modulator of a cell expressing
24P4C12.
As used herein, a range is understood to disdose specifically all whole unit
positions thereof.
Typical embodiments of the invention disclosed herein include 24P4C12
polynudeotides that encode specific
portions of 24P4C12 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, 710 or more contiguous
amino acids of 24P4C12 variant 1; the maximal lengths relevant for other
variants are: variant 3, 710 amino acids; variant 5,
710 amino acids, variant 6, 710 amino acids, variant 7, 598 amino acids,
variant 8,722 amino acids, and variant 9, 712
amino acids.
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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 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino
acid 20 of the 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 20 to about amino
acid 30 of the 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino
acid 40 of the 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino
acid 50 of the 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino
acid 70 of the 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino
acid 80 of the 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino
acid 90 of the 24P4C12 protein shown in Figure
2 or Figure 3, polynucleotides encoding about amino acid 90 to about amino
acid 100 of the 24P4C12 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 24P4C12 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 24P4C12 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 24P4C12 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 24P4C12 sequence as shown in Figure 2.
Additional illustrative embodiments of the invention disclosed herein include
24P4C12 polynucleotide fragments
encoding one or more of the biological motifs contained within a 24P4C12
protein "or variant" sequence, including one or
more of the motif-bearing subsequences of a 24P4C12 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 24P4C12
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 24P4C12 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 )(X11 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
LVII. 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 in the parent molecule.
II.A.1 Uses of 24P4C12 Polvnucleotides
IAA.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different
specific uses. The human 24P4C12
gene maps to the chromosomal location set forth in the Example entitled
"Chromosomal Mapping of 24P4C12." For
example, because the 24P4C12 gene maps to this chromosome, polynucleotides
that encode different regions of the
24P4C12 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
=
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rearrangements have been identified as frequent cytogenetic abnormalities in
inumber of different cancers (see e.g.
Krajinovic etal., Mutat. Res. 382(3-4): 81-83 (1998); Johansson etal., 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 24P4C12 proteins provide new
tools that can be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the
chromosomal region that encodes 24P4C12 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 etal., Am. J.
Obstet. Gyneco1171(4): 1055-1057
(1994)).
Furthermore, as 24P4C12 was shown to be highly expressed in bladder and other
cancers, 24P4C12
polynucleotides are used in methods assessing the status of 24P4C12 gene
products in normal versus cancerous tissues.
Typically, polynucleotides that encode specific regions of the 24P4C12
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 24P4C12 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 at at, 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 add 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 24P4C12. 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
24P4C12 polynucleotides and polynudeotide 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., 24P4C12. See for example, Jack Cohen,
Oligodeomucleotides, Antisense Inhibitors of Gene
Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 24P4C12 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-
benzodithio1-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g.,
lyer, R. P. etal., J. 'Org. Chem. 55:4693-4698
(1990); and lyer, R. P. et aL, J. Am. Chem. Soc. 112:1253-1254 (1990).
Additional 24P4C12 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 24P4C12 antisense oligonucleotides of the present invention typically can
be RNA or DNA that is
complementary to and stably hybridizes with the first 1005' codons or last 100
3' codons of a 24P4C12 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 24P4C12 mRNA
and not to mRNA specifying other regulatory subunits of protein kinase. In one
embodiment, 24P4C12 antisense
oligonucleotides of the present invention are 15 to 30-mer fragments of the
antisense DNA molecule that have a sequence
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that hybridizes to 24P4C12 mRNA. Optionally, 24P4C12 antisense oligonucleotide
is a 30-mer oligonucleotide that is
complementary to a region in the first 105' codons or last 103' codons of
24P4C12. Alternatively, the antisense molecules
are modified to employ ribozymes in the inhibition of 24P4C12 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
24P4C12 polynudeotide in a sample and as a means for detecting a cell
expressing a 24P4C12 protein.
Examples of such probes include polypeptides comprising all or part of the
human 24P4C12 cDNA sequence shown in
Figure 2. Examples of primer pairs capable of specifically amplifying 24P4C12
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 24P4C12 mRNA.
The 24P4C12 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
24P4C12 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 24P4C12 polypeptides; as tools for modulating or inhibiting
the expression of the 24P4C12 gene(s) and/or
translation of the 24P4C12 transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to
identify and isolate a 24P4C12 or 24P4C12
related nucleic acid sequence from a naturally occurring source, such as
humans or other mammals, as well as the isolated
nucleic acid sequence per se, which would comprise all or most of the
sequences found in the probe used.
II.A.4.) Isolation of 24P4C12-Encoding Nucleic Acid Molecules
The 24P4C12 cDNA sequences described herein enable the isolation of other
polynucleotides encoding 24P4C12 gene
product(s), as well as the isolation of polynucleotides encoding 24P4C12 gene
product homologs, alternatively spliced isoforrns,
allelic variants, and mutant forms of a 24P4C12 gene product as well as
polynudeotides that encode analogs of 24P4C12-related
proteins. Various molecular cloning methods that can be employed to isolate
full length cDNAs encoding a 24P4C12 gene are
well known (see, for example, Sambrook, J. et at, Molecular Cloning: A
Laboratory Manual, 2d edition, Cold Spring Harbor Press,
New York, 1989; Current Protocols in Molecular Biology. Ausubel et at, 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 24P4C12 gene cDNAs can be
identified by probing with a labeled 24P4C12
cDNA or a fragment thereof. For example, in one embodiment, a 24P4C12 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 24P4C12 gene. A 24P4C12
gene itself can be isolated by screening genomic DNA libraries, bacterial
artificial chromosome libraries (BACs), yeast artificial
chromosome libraries (YACs), and the like, with 24P4C12 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
24P4C12 polynudeotide, a fragment,
analog or homologue thereof, induding 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
iransfected with such recombinant DNA or RNA
molecules. Methods for generating such molecules are well known (see, for
example, Sambrook et at, 1989, supra).
29
CA 02503346 2009-10-08
The invention further provides a host-vector system comprising a recombinant
DNA molecule containing a 24P4C12
polynudeotide, fragment, analog or homologue thereof within a suitable
prokaryotic or eukaryolic 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, 2931 cells). More particularly, a polynucleotide comprising the
coding sequence of 24P4C12 or a fragment, analog
or homolog thereof can be used to generate 24P4C12 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 24P4C12
proteins or fragments thereof are available,
see for example, Sambrook etal., 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,
24P4C12 can be expressed in several
prostate cancer and non-prostate cell lines, including for example 293, 2931,
rat-1, NH 313 and TsuPr1. The host-vector
systems of the invention are useful for the production of a 24P4C12 protein or
fragment thereof. Such host-vector systems
can be employed to study the functional properties of 24P4C12 and 24P4C12
mutations or analogs.
Recombinant human 24P4C12 protein or an analog or homolog or fragment thereof
can be produced by
mammalian cells transfected with a construct encoding a 24P4C12-related
nudeotide. For example, 293T cells can be
transfeded with an expression plasmid encoding 24P4C12 or fragment, analog or
homolog thereof, a 24P4C12-related
protein is expressed in the 293T cells, and the recombinant 24P4C12 protein is
isolated using standard purification methods
(e.g., affinity purification using anti-24P4C12 antibodies). In another
embodiment, a 24P4C12 coding sequence is subcloned
into the retroviral vector pSRaMSVtkneo and used to infect various mammalian
cell lines, such as NIH 313, TsuPrl, 293 and
rat-1 in order to establish 24P4C12 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 24P4C12 coding sequence can be used
for the generation of a secreted form of recombinant 24P4C12 protein.
As discussed herein, redundancy in the genetic code permits variation in
24P4C12 gene sequences. In particular,
it is known in the art that specific host species often have specific codon
preferences, and thus one can adapt the disclosed
sequence as preferred for a desired host. For example, preferred analog codon
sequences typically have rare codons (i.e.,
codons having a usage frequency of less than about 20% in known sequences of
the desired host) replaced with higher
frequency codons. Codon preferences for a specific species aricalculated, for
example, by utilizing codon usage tables
available on the INTERNET.
Additional sequence modifications are known to enhance protein expression in a
cellular host. These include
elimination of sequences encoding spurious polyadenylation signals,
exonfintron splice site signals, transposon-like repeats,
andlor other such well-characterized sequences that are deleterious to gene
expression. The GC content of the sequence is
adjusted to levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell.
Where possible, the sequence is modified to avoid predicted hairpin secondary
mRNA structures. Other useful modifications
include the addition of a translational initiation consensus sequence at the
start of the open reading frame, as described in
Kozak, MoL Cell Bid., 9:5073-5080 (1989). Skilled artisans understand that the
general rule that eukaryofic ribosomes
initiate translation exclusively at the 5' proximal AUG codon is abrogated
only under rare conditions (see, 'e.g., Kozak PNAS
92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148(1987)).
24P4C12-related Proteins
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Another aspect of the present invenfion provides 24P4C12-related proteins.
Specific embodiments of 24P4C12
proteins comprise a polypeptide having all or part of the amino acid sequence
of human 24P4C12 as shown in Figure 2 or
Figure 3. Alternatively, embodiments of 24P4C12 proteins comprise variant,
homolog or analog polypeptides that have
alterations in the amino acid sequence of 24P4C12 shown in Figure 2 or Figure
3.
Embodiments of a 24P4C12 polypeptide include: a 24P4C12 polypeptide having a
sequence shown in Figure 2, a
peptide sequence of a 24P4C12 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 24P4C12 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-G;
(II) a 24P4C12-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;
(III) a 24P4C12-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-G;
(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
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 VIII-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 add 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, 38, 3C, 3D, 3E, 3F, or 3G in any
whole number increment up to 710, 710, 710, 710, 598, 722, or 712 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;
(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, 38, 3C, 3D, 3E, 3F, or 3G in any
whole number increment up to 710, 710, 710, 710, 598, 722, or 712
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 less than 0.5 in the Hydropathicity profile of
Figure 6;
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(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, 3E, 3F, or 3G in any
whole number increment up to 710, 710, 710, 710, 598, 722, or 712
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 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, 3E, 3F, or 3G in any
whole number increment up to 710, 710, 710, 710, 598, 722, or 712
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 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, 35 amino acids of a protein of Figure
3A, 3B, 3C, 3D, 3E, 3F, or 3G in any
whole number increment up to 710, 710, 710, 710, 598, 722, or 712
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 Beta-turn profile of
Figure 9;
(XIV) a peptide that occurs at least twice in Tables VIII-XXI and XXII to
XLIX, collectively;
(XV) a peptide that occurs at least three times in Tables VIII-XXI and XXII
to XLIX, collectively;
(XVI) a peptide that occurs at least four times in Tables VIII-XXI and XXII
to XLIX, collectively;
(XVII) a peptide that occurs at least five times in Tables VIII-XXI and
XXII to XLIX, collectively;
(XVIII) a peptide that occurs at least once in Tables VIII-XXI, and at
least once in tables XXII to XLIX;
(XIX) a peptide that occurs at least once in Tables VIII-XXI, and at least
twice in tables XXII to XLIX;
(XX) a peptide that occurs at least twice in Tables VIII-XXI, and at least
once in tables XXII to XLIX;
(XXI) a peptide that occurs at least twice in Tables VIII-XXI, and at least
twice in tables XXII to XLIX;
(0(11) a peptide which comprises one two, three, four, or five of the
following characteristics, or an
oligonudeotide encoding such peptide:
i) 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 add
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 add
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,
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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;
(XXIII) a composition comprising a peptide of (l)-(XXII) or an antibody or
binding region thereof together with a
pharmaceutical excipient and/or in a human unit dose form.
(XXIV) a method of using a peptide of XXXII), or an antibody or binding
region thereof or a composition of
(XXIII) in a method to modulate a cell expressing 24P4C12,
(XXV) a method of using a peptide of (I)-(XXII) or an antibody or binding
region thereof or a composition of
(XXIII) in a method to diagnose, prophylax, prognose, or treat an individual
who bears a cell expressing 24P4C12
(XXVI) a method of using a peptide of (I)-(XXII) or an antibody or binding
region thereof or a composition (XXIII)
in a method to diagnose, prophylax, prognose, or treat an individual who bears
a cell expressing 24P4C12, said cell from a
cancer of a tissue listed in Table I;
(XXVII) a method of using a peptide of (I)-(XXII) or an antibody or binding
region thereof or a composition of
(XXIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
(XXVIII) a method of using a peptide of (I)-(XXII) or an antibody or binding
region thereof or a composition of
(XXIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a
tissue listed in Table I; and,
(XXIX) a method of using a a peptide of (I)-(XXII) or an antibody or binding
region thereof or a composition
(XXIII) in a method to identify or characterize a modulator of a cell
expressing 24P4C12.
As used herein, a range is understood to specifically disclose all whole unit
positions thereof.
Typical embodiments of the invention disclosed herein include 24P4C12
polynucleotides that encode specific
portions of 24P4C12 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, 710 or more contiguous
amino acids of 24P4C12 variant 1; the maximal lengths relevant for other
variants are: variant 3, 710 amino acids; variant 5,
710 amino acids, variant 6, 710, variant 7, 598 amino acids, variant 8, 722
antino acids, and variant 9, 712 amino acids..
In general, naturally occurring allelic variants of human 24P4C12 share a high
degree of structural identity and
homology (e.g., 90% or more homology). Typically, allelic variants of a
24P4C12 protein contain conservative amino acid
substitutions within the 24P4C12 sequences described herein or contain a
substitution of an amino acid from a corresponding
position in a homologue of 24P4C12. One class of 24P4C12 allelic variants are
proteins that share a high degree of homology
with at least a small region of a particular 24P4C12 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.
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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 (0) for glutamic acid (E) and vice
versa; glutamine (CI) for asparagine (N) and vice versa; and serine (S) for
threonine (T) and vice versa. Other substitutions
can also be considered conservative, depending on the environment of the
particular amino acid and its role in the three-
dimensional structure of the protein. For example, glycine (G) and alanine (A)
can frequently be interchangeable, as can
alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic,
can frequently be interchanged with leucine and
isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are
frequently interchangeable in locations in which the
significant feature of the amino acid residue is its charge and the differing
pK's of these two amino acid residues are not
significant. Still other changes can be considered 'conservative" in
particular environments (see, e.g. Table Ill herein; pages
13-15 "Biochemistry' 2nd ED. Lubert Stryer ed (Stanford University); Henikoff
etal., PNAS 1992 Vol 89 10915-10919; Lei et
aL, J Blot 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
24P4C12 proteins such as polypeptides having amino acid insertions, deletions
and substitutions. 24P4C12 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
etal., NucL Acids Res., 10:6487 (1987)),
cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction
selection mutagenesis (Wells et at, Philos. Trans. R.
Soc. London SerA, 317:415 (1986)) or other known techniques can be performed
on the cloned DNA to produce the
24P4C12 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, 24P4C12 variants, analogs or honnologs, have the
distinguishing attribute of having at least one
epitope that is "cross reactive" with a 24P4C12 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 24P4C12 variant also specifically binds
to a 24P4C12 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 24P4C12 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
at al., Mol Immunol (1989) 26(9):865-73; Schwartz et aL, J Immunol (1985)
135(4):2598-608.
Other classes of 24P4C12-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 24P4C12 protein variants or analogs
comprises one or more of the 24P4C12 biological motifs described herein or
presently known in the art. Thus, encompassed
by the present invention are analogs of 24P4C12 fragments (nucleic or amino
acid) that have altered functional (e.g.
=
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CA 02503346 2009-10-08
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 24P4C12 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
24P4C12 protein shown in Figure 2 or Figure 3.
Moreover, representative embodiments of the invention disclosed herein include
polypeptides consisting of about
amino acid 1 to about amino acid 10 of a 24P4C12 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 10 to about amino-acid 20 of a 24P4C12 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 20 to about amino acid 30 of a 24P4C12 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 30 to about amino acid 40 of a 24P4012 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 40 to about amino acid 50 of a 24P4C12 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 50 to about amino acid 60 of a 24P4C12 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 60 to about amino acid 70 of a 24P4C12 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid iota about amino acid 80 of a 24P4C12 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 80 to about amino acid 90 of a 24P4C12 protein shown in Figure 2 or
Figure 3, polypeptides consisting of about
amino acid 9010 about amino acid 100 of a 24P4C12 protein shown in Figure 2 or
Figure 3, etc. throughout the entirety of a
24P4C12 amino acid sequence. Moreover, polypeptides consisting of about amino
acid 1 (or 20 or 30 or 40 etc.) to about
amino acid 20, (or 130, or 140 or 150 etc.) of a 24P4C12 protein shown in
Figure 2 or Figure 3 are embodiments of the
invention. It is to be appreciated that the starting and stopping positions in
this paragraph refer to the specified position as
well as that position plus or minus 5 residues.
24P4C12-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 nudeic acid molecules that encode a
24P4C12-related protein. In one embodiment, nucleic acid molecules provide a
means to generate defined fragments of a
24P4C12 protein (or variants, homologs or analogs thereof).
III.A.) Motif-bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include
24P4C12 polypeptides comprising the
amino acid residues of one or more of the biological motifs contained within a
24P4C12 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.
Motif bearing subsequences of all 24P4C12 variant proteins are set forth and
identified in Tables VIII-XXI and XXII-
XLIX.
Table V sets forth several frequently occurring motifs based on pfam searches
(see URL address plammusiedu/).
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 24P4C12 motifs discussed above are
useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation that the
24P4C12 motifs discussed above are associated
with growth dysregulation and because 24P4C12 is overexpressed in certain
cancers (See, e.g., Table I). Casein kinase II,
=
CA 02503346 2009-10-08
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 at al., Lab Invest.,
78(2): 165-174(1998); Gaiddon ef al.,
Endocrinology 136(10): 4331-4338 (1995); Halt et at, Nucleic Acids Research
24(6): 1119-1126 (1996); Peterziel etal.,
Oncogene 18(46): 6322-6329(1999) and O'Brian, Oncol. Rep. 5(2): 305-309
(1998)). Moreover, both glycosylation and
myristoylation are protein modifications also associated with cancer and
cancer progression (see e.g. Dennis et al., Biochem.
Biophys. Acta 1473(1):21-34 (1999); Raju at 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 24P4C12
protein that are capable of optimally binding to
specified HLA alleles.
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 land HLA Class II
motifs/supermotifs of Table IV). The epitope is analoged by substituting out
an amino acid at one of the specified positions,
and replacing it with another amino acid specified for that position. For
example, on the basis of residues defined in Table
IV, one can substitute out a deleterious residue irt 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 etal.;
Sette, lmmunogenetics 1999 50(3-4): 201-
212; Sette at al., J. Immunol. 2001 166(2): 1389-1397; Sidney etal., Hum.
Immunol. 1997 58(1): 12-20; Kondo etal.,
Immunogenetics 1997 45(4): 249-258; Sidney etal., J. Immunol. 1996 157(8):
3480-90; and Falk etal., Nature 351: 290-6
(1991); Hunt etal., Science 255:1261-3(1992); Parker etal., J. Immunol.
149:3580-7 (1992); Parker etal., J. Immunol.
152:163-75 (1994)); Kast etal., 1994 152(8): 3904-12; Borras-Cuesta eta!,,
Hum. Immunol. 2000 61(3): 266-278; Alexander
etal., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander etal., PMID:
7895164, Ul: 95202582; O'Sullivan et al., J.
Immunol. 1991 147(8): 2663-2669; Alexander etal., Immunity 1994 1(9): 751-761
and Alexander et al., 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-)0(1 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.
24P4C12-related proteins are embodied in many forms, preferably in isolated
form. A purified 24P4C12 protein
molecule will be substantially free of other proteins or molecules that impair
the binding of 24P4C12 to antibody, T cell or
36
CA 02503346 2009-10-08
other ligand. The nature and degree of isolation and purification will depend
on the intended use. Embodiments of a 24P4C12-
related proteins include purified 24P4C12-related proteins and functional,
soluble 24P4C12-related proteins. In one
embodiment, a functional, soluble 24P4C12 protein or fragment thereof retains
the ability to be bound by antibody, T cell or
other ligand.
The invention also provides 24P4C12 proteins comprising biologically active
fragments of a 24P4C12 amino acid
sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of
the starting 24P4C12 protein, such as the ability
to elicit the generation of antibodies that specifically bind an epitope
associated with the starting 24P4C12 protein; to be
bound by such antibodies; to elicit the activation of HTL or CTL; and/at, to
be recognized by HTL or CTL that also specifically
bind to the starling protein.
24P4C12-related polypeptides that contain particularly interesting structures
can be predicted andlor identified using
various analytical techniques well known in the art, induding, for example,
the methods of Chou-Fasman, Gamier-Robson, Kyle-
Doolittle, asenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on
immunogenicity. Fragments that contain such
structures are particularly useful in generating subunit-specific anti-24P4C12
antibodies or T cells or in identifying cellular factors
that bind to 24P4C12. 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 Kyte, 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. 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 24P4C12 protein that are
capable of optimally binding to specified HLA alleles.
Illustrating this, peptide epitopes from 24P4C12 that
are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2,
A3, All, A24, B7 and 835 were predicted
(see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid
sequence of the 24P4C12 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 Bioinformatics and
Molecular Analysis Section (BIMAS) web site,
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 FiLA-A2 (see,
e.g., Falk etal., Nature 351: 290-6 (1991);
Hunt of al., Science 255:1261-3(1992); Parker etal., J. immunol. 149:3580-7
(1992); Parker etal., J. Immunol. 152:163-75
(1994)). This algorithm allows location and ranking of 8-mer, 9-met, 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 etal., J. Immunol. 149:3580-7 (1992)),
Selected results of 24P4C12 predicted binding peptides are shown in Tables
VIII-)01 and XXI1-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
37
CA 02503346 2009-10-08
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
complexes containing the peptide at 37 C at pH 6.5. Peptides with the highest
binding score are predicted to be the most
tightly bound to HLA Class I on the cell surface for the greatest period of
time and thus represent the best immunogenic
targets for T-cell recognition.
=
Actual binding of peptides to an HLA allele can be evaluated by stabilization
of HA expression on the antigen-
processing defective cell line T2 (see, e.g., Xue et at, Prostate 30:73-8
(1997) and Peshwa at aL, Prostate 36:129-38
(1998)). Immunogenicity of specific peptides can be evaluated in vitro by
stimulation of CD8+ cytotoxic T lymphocytes (CTL)
in the presence of antigen presenting cells such as dendntic cells.
It is to be appreciated that every epitope predicted by the BIMAS site,
Epimerm4 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
are to be 'applied'
to a 24P4C12 protein in accordance with the invention. As used in this context
"applied" means that a 24P4C12 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 24P4C12 protein of 8, 9, 10, or 11 amino acid residues
that bears an HLA Class 1 motif, or a
subsequence of 9 or more amino acid residues that bear an HLA Class 11 motif
are within the scope of the invention.
111.B.) Expression of 24P4C12-related Proteins
In an embodiment described in the examples that follow, 24P4C12 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
24P4C12 with a C-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, invitrogen or
Tag5, GenHunter Corporation, Nashville
TN). The Tag5 vector provides an IgGK secretion signal that can be used to
facilitate the production of a secreted 24P4C12
protein in transfected cells. The secreted HIS-tagged 24P4C12 in the culture
media can be purified, e.g., using a nickel
column using standard techniques.
III.C.) Modifications of 24P4C12-related Proteins
Modifications of 24P4C12-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 24P4C12 polypeptide with
an organic derivatizing agent that is capable of reacting with selected side
chains or the N- or C- terminal residues of a
24P4C12 protein. Another type of covalent modification of a 24P4C12
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
24P4C12 comprises linking a 24P4C12 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;
4,301,14.4; 4,670,417; 4,791,192 or 4,179,337.
The 24P4C12-related proteins of the present invention can also be modified to
form a chimeric molecule
comprising 24P4C12 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 24P4C12 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 24P4C12. A chimeric molecule can
comprise a fusion of a 24P4C12-related
protein with a polyhistidine epitope tag, which provides an epitope to which
immobilized nickel can selectively bind, with
38
CA 02503346 2005-04-21
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PCT/US2002/038264
cytokines or with growth factors. The epitope tag is generally placed at the
amino- or carboxyl- terminus of a 24P4C12
protein. In an alternative embodiment, the chimeric molecule can comprise a
fusion of a 24P4C12-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 24P4C12 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 lgGl 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 24P4C12-related Proteins
The proteins of the invention have a number of different specific uses. As
24P4C12 is highly expressed in prostate
and other cancers, 24P4C12-related proteins are used in methods that assess
the status of 24P4C12 gene products in
normal versus cancerous tissues, thereby elucidating the malignant phenotype.
Typically, polypeptides from specific regions
of a 24P4C12 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
24P4C12-related proteins comprising the amino acid residues of one or more of
the biological motifs contained within a
24P4C12 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, 24P4C12-related
proteins that contain the amino acid residues of
one or more of the biological motifs in a 24P4C12 protein are used to screen
for factors that interact with that region of
24P4C12.
24P4C12 protein fragments/subsequences are particularly useful in generating
and characterizing domain-specific
antibodies (e.g., antibodies recognizing an extracellular or intracellular
epitope of a 24P4C12 protein), for identifying agents or
cellular factors that bind to 24P4C12 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 24P4C12 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 24P4C12 gene product. Antibodies raised against a
24P4C12 protein or fragment thereof are useful
in diagnostic and prognostic assays, and imaging methodologies in the
management of human cancers characterized by
expression of 24P4C12 protein, such as those listed in Table I. Such
antibodies can be expressed intracellularly and used in
methods of treating patients with such cancers. 24P4C12-related nucleic acids
or proteins are also used in generating HTL
or CTL responses.
Various immunological assays useful for the detection of 24P4C12 proteins are
used, induding but not limited to various
types of radioimmunoassays, enzyme-linked immunosorbent assays (EISA), enzyme-
linked immunofluorescent assays (ELIFA),
immunocytochemical methods, and the like. Antibodies can be labeled and used
as immunological imaging reagents capable of
detecting 24P4C12-expressing cells (e.g., in radioscintigraphic imaging
methods). 24P4C12 proteins are also particularly useful in
generating cancer vaccines, as further described herein.
IV.) 24P4C12 Antibodies
Another aspect of the invention provides antibodies that bind to 24P4C12-
related proteins. Preferred antibodies
specifically bind to a 24P4C12-related protein and do not bind (or bind
weakly) to peptides or proteins that are not 24P4C12-
related proteins. For example, antibodies that bind 24P4C12 can bind 24P4C12-
related proteins such as the homologs or
analogs thereof.
39
CA 02503346 2005-04-21
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PCT/US2002/038264
24P4C12 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 24P4C12 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
24P4C12 is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the
detection and quantification of 24P4C12 and
mutant 24P4C12-related proteins. Such assays can comprise one or more 24P4C12
antibodies capable of recognizing and
binding a 24P4C12-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 immunosoibent 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
24P4C12 are also provided by the invention, induding but not limited to
radioscintigraphic imaging methods using labeled
24P4C12 antibodies. Such assays are dinically useful in the detection,
monitoring, and prognosis of 24P4C12 expressing
cancers such as prostate cancer.
24P4C12 antibodies are also used in methods for purifying a 24P4C12-related
protein and for isolating 24P4C12
homologues and related molecules. For example, a method of purifying a 24P4C12-
related protein comprises incubating a
24P4C12 antibody, which has been coupled to a solid matrix, with a lysate or
other solution containing a 24P4C12-related protein
under conditions that permit the 24P4C12 antibody to bind to the 24P4C12-
related protein; washing the solid matrix to eliminate
impurities; and eluting the 24P4C12-related protein from the coupled antibody.
Other uses of 24P4C12 antibodies in
accordance with the invention include generating anti-idiotypic antibodies
that mimic a 24P4C12 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 24P4C12-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 24P4C12 can
also be used, such as a 24P4C12 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 24P4C12-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 24P4C12-related
protein or 24P4C12 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et
al., 1997, Ann. Rev. Immunol. 15: 617-648).
The amino acid sequence of a 24P4C12 protein as shown in Figure 2 or Figure 3
can be analyzed to select specific
regions of the 24P4C12 protein for generating antibodies. For example,
hydrophobicity and hydrophilicity analyses of a 24P4C12
amino acid sequence are used to identify hydrophilic regions in the 24P4C12
structure. Regions of a 24P4C12 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, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-
Schultz or Jameson-Wolf analysis. Hydrophilicity
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
CA 02503346 2005-04-21
WO 2004/050828
PCT/US2002/038264
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 24P4C12 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, KU-I 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 24P4C12 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.
24P4C12 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
24P4C12-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 24P4C12 protein can also be produced
in the context of chimeric or complementarity-
determining region (CDR) grafted antibodies of multiple species origin.
Humanized or human 24P4C12 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 etal., 1988,
Nature 332: 323-327; Verhoeyen et at, 1988, Science
239: 1534-1536). See also, Carter etal., 1993, Proc. Natl. Acad. Sci. USA 89:
4285 and Sims etal., 1993, J. Immunol. 151: 2296.
, Methods for producing fully human monoclonal antibodies indude phage
display and transgenic methods (for review,
see Vaughan etal., 1998, Nature Biotechnology 16: 535-539). Fully human
24P4C12 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
24P4C12 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 et at, 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 24P4C12 antibodies with a 24P4C12-related protein can be
established by a number of well known
means, including Western blot, immunoprecipitation, ELISA, and FACS analyses
using, as appropriate, 24P4C12-related
proteins, 24P4C12-expressing cells or extracts thereof. A 24P4C12 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
radioisotope, a fluorescent compound, a bioluminescent compound,
chemiluminescent compound, a metal chelator or an
enzyme. Further, bi-specific antibodies specific for two or more 24P4C12
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.) 24P4C12 Cellular Immune Responses
41
CA 02503346 2009-10-08
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 1-ILA molecule and a peptidic antigen acts as the ligand
recognized by HLA-restricted T cells
(Buus, S. etal., Cell 47:1071, 1986; Babbitt, B. P. etal., Nature 317:359,
1985; Townsend, A. and Bodmer, H., Annu. Rev.
immune!. 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, etal., J. Immune!. 160:3363, 1998; Rammensee,
etal., Immunogenetics 41:178, 1995;
Rammensee el al., SYFPEITHI
); Sette, A.
and Sidney, J. Curr. Opin. Immune!. 10:478, 1998; Engelhard, V. H., Curr.
Opin. ImmunoL 6:13, 1994; Sette, A. and Grey, H.
M., Curr. Opin. Immune). 4:79,1992; Sinigaglia, F. and Hammer, J. Curr. Biol.
6:52, 1994; Ruppert etal., Cell 74:929-937,
1993; Kondo etal., J. Immunot 155:4307-4312, 1995; Sidney et al., J. Immunol
157:3480-3490, 1996; Sidney et al., Human
Immunoi 45:79-93, 1996; Sette, A. and Sidney, J. lmmunogenetics 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 (igands;
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. lmmunol. 13:587, 1995; Smith, etal., Immunity 4:203, 1996;
Fremont etal., Immunity 8:305, 1998; Stern et
al., Structure 2:245, 1994; Jones, E.Y. CWT. Opin. ImmunoL 9:75, 1997; Brown,
J. H. etal., Nature 364:33, 1993; Guo, H. C.
eta)., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. etal., Nature
360:364, 1992; Silver, M. L etal., Nature 360:367,
1992; Matsumura, M. etal., Science 257:927, 1992; Madden et al., Cell 70:1035,
1992; Fremont, D. H. etal., 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 supermobfs
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 at., MoL frilmuno(.
32:603, 1995; Cells, E. at al., Proc. Natl. Acad. Sci. USA 91:2105, 1994;
Tsai, V. etal., J. ImmunoL 158:1796, 1997;
Kawashima, I. etal., Human Immunol. 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
weeks. T cells specific for the peptide become activated during this time and
are detected using, e.g., a lymphokine- or
51Cr-release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J.
Immune). 26:97, 1996; Wentworth, P.
A. el al., Int. Immunot 8:651, 1996; Alexander, J. etal., 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.
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Peptide-specific T cells are detected using, e.g., a 51Cr-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
andlor from chronically ill patients (see, e.g., Rehermann, B. etal., J. Exp.
Med. 181:1047, 1995; Doolan, D. L. etal.,
Immunity 7:97, 1997; Bertoni, R. etal., J. Clin. Invest. 100:503, 1997;
Threlkeld, S. C. etal., J. Immunot 159:1648, 1997;
Diepolder, H. M. eta)., 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 51Cr release involving peptide-sensitized
targets, T cell proliferation, or lymphokine release.
VI.) 24P4C12 Transonic Animals
Nucleic acids that encode a 24P4C12-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 24P4C12 can be used to
clone genomic DNA that encodes
24P4C12. The cloned genomic sequences can then be used to generate transgenic
animals containing cells that express
DNA that encode 24P4C12. 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 24P4C12 transgene incorporation
with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 24P4C12 can be
used to examine the effect of
increased expression of DNA that encodes 24P4C12. 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.
Altematively, non-human homologues of 24P4C12 can be used to construct a
24P4C12 'knock out" animal that
has a defective or altered gene encoding 24P4C12 as a result of homologous
recombination between the endogenous gene
encoding 24P4C12 and altered genomic DNA encoding 24P4C12 introduced into an
embryonic cell of the animal. For
example, .cDNA that encodes 24P4C12 can be used to clone genomic DNA encoding
24P4C12 in accordance with
established techniques. A portion of the genomic DNA encoding 24P4C12 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) fora 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
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,
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for example, for their ability to defend against certain pathological
conditions or for their development of pathological
conditions due to absence of a 24P4C12 polypeptide.
VII.) Methods for the Detection of 24P4C12
Another aspect of the present invention relates to methods for detecting
24P4C12 polynudeotides and 24P4C12-
related proteins, as well as methods for identifying a cell that expresses
24P4C12. The expression profile of 24P4C12 makes it
a diagnostic marker for metastasized disease. Accordingly, the status of
24P4C12 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 24P4C12 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 24P4C12
polynucleotides in a biological sample,
such as serum, bone, prostate, and other tissues, urine, semen, cell
preparations, and the like. Detectable 24P4C12
polynucleotides include, for example, a 24P4C12 gene or fragment thereof,
24P4C12 mRNA, alternative splice variant 24P4C12
mRNAs, and recombinant DNA or RNA molecules that contain a 24P4C12
polynucleotide. A number of methods for amplifying
and/or detecting the presence of 24P4C12 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 24P4C12 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 24P4C12
polynucleotides as sense and antisense primers to amplify 24P4C12 cDNAs
therein; and detecting the presence of the
amplified 24P4C12 cDNA. Optionally, the sequence of the amplified 24P4C12 cDNA
can be determined.
In another embodiment, a method of detecting a 24P4C12 gene in a biological
sample comprises first isolating
genomic DNA from the sample; amplifying the isolated genomic DNA using 24P4C12
polynucleotides as sense and
antisense primers; and detecting the presence of the amplified 24P4C12 gene.
Any number of appropriate sense and
antisense probe combinations can be designed from a 24P4C12 nucleotide
sequence (see, e.g., Figure 2) and used for this
purpose.
The invention also provides assays for detecting the presence of a 24P4C12
protein in a tissue or other biological
sample such as serum, semen, bone, prostate, urine, cell preparations, and the
like. Methods for detecting a 24P4C12-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 24P4C12-related
protein in a biological sample comprises first contacting the sample with a
24P4C12 antibody, a 24P4C12-reactive fragment
thereof, or a recombinant protein containing an antigen-binding region of a
24P4C12 antibody; and then detecting the
binding of 24P4C12-related protein in the sample.
Methods for identifying a cell that expresses 24P4C12 are also within the
scope of the invention. In one embodiment,
an assay for identifying a cell that expresses a 24P4C12 gene comprises
detecting the presence of 24P4C12 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 24P4C12
riboprobes, Northern blot and related
techniques) and various nucleic acid amplification assays (such as RT-PCR
using complementary primers specific for 24P4C12,
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 24P4C12 gene comprises detecting
the presence of 24P4C12-related protein in the
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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 24P4C12-related proteins and cells that express 24P4C12-related
proteins.
24P4C12 expression analysis is also useful as a tool for identifying and
evaluating agents that modulate 24P4C12 gene
expression. For example, 24P4C12 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 24P4C12 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 24P4C12 expression by RT-PCR, nucleic acid hybridization or
antibody binding.
VIII.) Methods for Monitoring the Status of 24P4C12-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 etal., Lab
Invest. 77(5): 437-438(1997) and Isaacs etal., Cancer Surv. 23: 19-32 (1995)).
In this context, examining a biological
sample for evidence of dysregulated cell growth (such as aberrant 24P4C12
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 24P4C12 in a biological sample of interest
can be compared, for example, to the status of 24P4C12 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 24P4C12 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., Greyer et at, J. Comp. Neurol. 1996
= Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,501) to compare 24P4C12
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 24P4C12
expressing cells) as well as the level, and biological activity of expressed
gene products (such as 24P4C12 mRNA,
polynucleotides and polypeptides). Typically, an alteration in the status of
24P4C12 comprises a change in the location of
24P4C12 and/or 24P4C12 expressing cells and/or an increase in 24P4C12 mRNA
and/or protein expression.
24P4C12 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 24P4C12 gene and gene products are found,
for example in Ausubel etal. eds., 1995, Current Protocols In Molecular
Biology, Units 2 (Northern Blotting), 4 (Southern
Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of
24P4C12 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 24P4C12 gene), Northern analysis and/or PCR analysis of
24P4C12 mRNA (to examine, for example
alterations in the polynucleotide sequences or expression levels of 24P4C12
mRNAs), and, Western and/or
immunohistochemical analysis (to examine, for example alterations in
polypeptide sequences, alterations in polypeptide
localization within a sample, alterations in expression levels of 24P4C12
proteins and/or associations of 24P4C12 proteins
with polypeptide binding partners). Detectable 24P4C12 polynucleotides
include, for example, a 24P4C12 gene or fragment
thereof, 24P4C12 mRNA, alternative splice variants, 24P4C12 mRNAs, and
recombinant DNA or RNA molecules containing a
24P4C12 polynudeotide.
The expression profile of 24P4C12 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 24P4C12 provides
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information useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The
invention provides methods and assays for determining 24P4C12 status and
diagnosing cancers that express 24P4C12, such as
cancers of the tissues listed in Table I. For example, because 24P4C12 mRNA is
so highly expressed in prostate and other
cancers relative to normal prostate tissue, assays that evaluate the levels of
24P4C12 mRNA transcripts or proteins in a biological
sample can be used to diagnose a disease associated with 24P4C12
dysregulation, and can provide prognostic information useful
in defining appropriate therapeutic options.
The expression status of 24P4C12 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 24P4C12 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 24P4C12 in a biological sample can be
examined by a number of well-known
procedures in the art. For example, the status of 24P4C12 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
24P4C12 expressing cells (e.g. those that express
24P4C12 mRNAs or proteins). This examination can provide evidence of
dysregulated cellular growth, for example, when
24P4C12-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 24P4C12 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 at at, Prostate 42(4): 315-317 (2000);Su etal., 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 24P4C12 gene
products by determining the status of
24P4C12 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 24P4C12 gene
products in a corresponding normal sample. The presence of aberrant 24P4C12
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 24P4C12 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
24P4C12 mRNA can, for example, be
evaluated in tissues including but not limited to those listed in Table I. The
presence of significant 24P4C12 expression in
any of these tissues is useful to indicate the emergence, presence and/or
severity of a cancer, since the corresponding
normal tissues do not express 24P4C12 mRNA or express it at lower levels.
In a related embodiment, 24P4C12 status is determined at the protein level
rather than at the nucleic acid level. For
example, such a method comprises determining the level of 24P4C12 protein
expressed by cells in a test tissue sample and
comparing the level so determined to the level of 24P4C12 expressed in a
corresponding normal sample. In one embodiment,
the presence of 24P4C12 protein is evaluated, for example, using
immunohistochemical methods. 24P4C12 antibodies or
binding partners capable of detecting 24P4C12 protein expression are used in a
variety of assay formats well known in the art for
this purpose.
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In a further embodiment, one can evaluate the status of 24P4C12 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 etal., 1999, J.
Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of
24P4C12 may be indicative of the presence or
promotion of a tumor. Such assays therefore have diagnostic and predictive
value where a mutation in 24P4C12 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
24P4C12 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 24P4C12 gene in a
biological sample. Aberrant
demethylation and/or hypermethylation of CpG islands in gene 5' regulatory
regions frequently occurs in immortalized and
transformed mils, 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 at, 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
etal., Cancer Epidemiol. Biomarkers Prey., 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 etal., 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 deave
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
24P4C12. 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
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 24P4C12 expression. The
presence of RT-PCR amplifiable 24P4C12 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
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at, 1997, Urol. Res. 25:373-384; Ghossein etal., 1995, J. Clin. Oncol. 13:1195-
2000; Heston etal., 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 24P4C12 mRNA or 24P4C12 protein in a
tissue sample, its presence indicating susceptibility to cancer, wherein the
degree of 24P4C12 mRNA expression correlates to the
degree of susceptibility. In a specific embodiment the presence of 24P4C12 in
prostate or other tissue is examined, with the
presence of 24P4C12 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 24P4C12 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 24P4C12 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 24P4C12 mRNA or
24P4C12 protein expressed by tumor cells,
comparing the level so determined to the level of 24P4C12 mRNA or 24P4C12
protein expressed in a corresponding normal
tissue taken from the same individual or a normal tissue reference sample,
wherein the degree of 24P4C12 mRNA or 24P4C12
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 24P4C12 is expressed in the tumor cells,
with higher expression levels indicating more aggressive tumors. Another
embodiment is the evaluation of the integrity of
24P4C12 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 overtime. In one embodiment, methods for observing the progression
of a malignancy in an individual over time
comprise determining the level of 24P4C12 mRNA or 24P4C12 protein expressed by
cells in a sample of the tumor, comparing
the level so determined to the level of 24P4C12 mRNA or 24P4C12 protein
expressed in an equivalent tissue sample taken from
the same individual at a different time, wherein the degree of 24P4C12 mRNA or
24P4C12 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 24P4C12 expression in the tumor cells over time,
where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity 24P4C12
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 24P4C12 gene and 24P4C12 gene products (or
perturbations in 24P4C12 gene and 24P4C12 gene
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 etal., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum.
Pathol. 26(2):223-9; Thorson etal., 1998, Mod.
Pathol. 11(6):543-51; Baisden etal., 1999, Am. J. Surg. Pathol. 23(8):918-24).
Methods for observing a coincidence between
the expression of 24P4C12 gene and 24P4C12 gene products (or perturbations in
24P4C12 gene and 24P4C12 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.
=
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In one embodiment, methods for observing a coincidence between the expression
of 24P4C12 gene and 24P4C12
gene products (or perturbations in 24P4C12 gene and 24P4C12 gene products) and
another factor associated with malignancy
entails detecting the overexpression of 24P4C12 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 24P4C12 mRNA or protein and PSA
mRNA or protein overexpression (or PSCA or PSM expression). In a specific
embodiment, the expression of 24P4C12 and PSA
mRNA in prostate tissue is examined, where the coincidence of 24P4C12 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 24P4C12 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 24P4C12 mRNA indude in situ hybridization using labeled
24P4C12 riboprobes, Northern blot and related
techniques using 24P4C12 polynudeotide probes, RT-PCR analysis using primers
specific for 24P4C12, 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 24P4C12 mRNA expression.
Any number of primers capable of amplifying
24P4C12 can be used for this purpose, including but not limited to the various
primer sets specifically described herein. In a
specific embodiment, polydonal or monoclonal antibodies specifically reactive
with the wild-type 24P4C12 protein can be used in
an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules That Interact With 24P4C12
The 24P4C12 protein and nucleic acid sequences disclosed herein allow a
skilled artisan to identify proteins, small
molecules and other agents that interact with 24P4C12, as well as pathways
activated by 24P4C12 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, etal.,
Nature 402: 4 November 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that
interact with 24P4C12 protein sequences.
In such methods, peptides that bind to 24P4C12 are identified by screening
libraries that encode a random or controlled
collection of amino adds. Peptides encoded by the libraries are expressed as
fusion proteins of bacteriophage coat proteins,
the bacteriophage particles are then screened against the 24P4C12 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
libraries and screening methods that can be used to identify molecules that
interact with 24P4C12 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.
Alteinatively, cell lines that express 24P4C12 are used to identify protein-
protein interactions mediated by
24P4C12. Such interactions can be examined using immunoprecipitation
techniques (see, e.g., Hamilton B.J., etal.
Biochem. Biophys. Res. Commun. 1999, 261:646-51). 24P4C12 protein can be
innmunoprecipitated from 24P4C12-
expressing cell lines using anti-24P4C12 antibodies. Alternatively, antibodies
against His-tag can be used in a cell line
engineered to express fusions of 24P4C12 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.
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Small molecules and ligands that interact with 24P4C12 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 24P4C12'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 24P4C12-related ion channel, protein pump, or cell
communication functions are identified and
used to treat patients that have a cancer that expresses 24P4C12 (see, e.g.,
HiIle, B., Ionic Channels of Excitable
Membranes 2nd Ed., Sinauer Assoc., Sunderland, MA, 1992). Moreover, ligands
that regulate 24P4C12 function can be
identified based on their ability to bind 24P4C12 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 24P4C12 and a
DNA-binding protein are used to co-express a fusion protein of a hybrid
ligandismall 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 24P4C12.
An embodiment of this invention comprises a method of screening for a molecule
that interacts with a 24P4C12
amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of
contacting a population of molecules with a
= 24P4C12 amino acid sequence, allowing the population of molecules and the
24P4C12 amino acid sequence to interact
under conditions that facilitate an interaction, determining the presence of a
molecule that interacts with the 24P4C12 amino
acid sequence, and then separating molecules that do not interact with the
24P4C12 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 24P4C12 amino acid sequence. The identified molecule can be
used to modulate a function performed by
24P4C12. In a preferred embodiment, the 24P4C12 amino acid sequence is
contacted with a library of peptides.
X. Therapeutic Methods and Compositions
The identification of 24P4C12 as a protein that is normally expressed in a
restricted set of tissues, but which is also
expressed in prostate and other cancers, opens a number of therapeutic
approaches to the treatment of such cancers. As
contemplated herein, 24P4C12 functions as a transcription factor involved in
activating tumor-promoting genes or repressing
genes that block tumorigenesis.
Accordingly, therapeutic approaches that inhibit the activity of a 24P4C12
protein are useful for patients suffering
from a cancer that expresses 24P4C12. These therapeutic approaches generally
fall into two classes. One class comprises
various methods for inhibiting the binding or association of a 24P4C12 protein
with its binding partner or with other proteins.
Another class comprises a variety of methods for inhibiting the transcription
of a 24P4C12 gene or translation of 24P4C12
mRNA.
X.A3 Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 24P4C12-related protein or
24P4C12-related nucleic acid. In
view of the expression of 24P4C12, cancer vaccines prevent and/or treat
24P4C12-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 etal., 1995, int. J. Cancer 63:231-237; Fong et al., 1997,
J. Immunol. 159:3113-3117).
CA 02503346 2009-10-08
=
Such methods can be readily practiced by employing a 24P4C12-related protein,
or a 24P4C12-encoding nucleic
acid molecule and recombinant vectors capable of expressing and presenting the
24P4C12 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
eta!,, Ann Med 1999 Feb 31(1):66-78; Maruyama
at al., Cancer Immunol lmmunother 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
=
immunoreactive epitope (e.g. an epitope present in a 24P4C12 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). In a preferred method, a 24P4C12 immunogen contains a
biological motif, see e.g., Tables VIII-XXI
and XXII-XLIX, or a peptide of a size range from 24P4C12 indicated in Figure
5, Figure 6, Figure 7, Figure 8, and Figure 9.
The entire 24P4C12 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 al., J. Clin. Invest. 95:341,
1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide)
("PLG") microspheres (see, e.g., Eldridge, et al.,
Malec. Immunol 28:287-294, 1991: Alonso at a)., Vaccine 12:299-306, 1994;
Jones etal., Vaccine 13:675-681, 1995),
peptide compositions contained in immune stimulating complexes (ISCOMS) (see,
e.g., Takahashi etal., Nature 344:873-
875, 1990; Flu at al., Clin Exp Immune!. 113:235-243, 1996), 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. et al., In: Concepts in vaccine development, Kaufmann, S. H.
E., ed., p. 379, 1996; Chakrabarti, S. eta!,,
Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et
at, AIDS Bio/Technology 4:790, 1986; Top, F.
H. eta)., 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. etal., J. Immunol Methods. 192:25, 1996; Eldridge, J. H. eta).,
Sem. Hematot 30:16, 1993; Falo, L. D., Jr. et al.,
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. etal., Vaccine 11:293, 1993), liposomes (Reddy, R. etal., J.
Immunol. 148:1585, 1992; Rock, K. L., Immunol
Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. etal.,
Science 259:1745, 1993; Robinson, H. L.,
Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. etal.,
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 24P4C12-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 24P4C12 protein that bind
corresponding HLA alleles.
In a preferred embodiment, a 24P4C12 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 motifisupermotif (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 11
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binding groove is essentially open ended; therefore a peptide of about 9 or
more amino acids can be bound by an HLA Class
II 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 acid 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 II epitopes are often 9, 10, 11, 12, 13,
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
24P4C12 protein) so that an immune
response is generated. A typical embodiment consists of a method for
generating an immune response to 24P4C12 in a
host by contacting the host with a sufficient amount of at least one 24P4C12 B
cell or cytotoxic 1-cell epitope or analog
thereof; and at least one periodic interval thereafter re-contacting the host
with the 24P4C12 B cell or cytotoxic 1-cell epitope
or analog thereof. A specific embodiment consists of a method of generating an
immune response against a 24P4C12-
related protein or a man-made multiepitopic peptide comprising: administering
24P4C12 immunogen (e.g. a 24P4C12
protein or a peptide fragment thereof, a 24P4C12 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 PADRETh peptide (Epimmune
Inc., San Diego, CA; see, e.g., Alexander
et aL, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander etal., Immunity
1994 1(9): 751-761 and Alexander etal.,
Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating
an immune response in an individual
against a 24P4C12 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 24P4C12 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 24P4C12, 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 24P4C12.
Constructs comprising DNA encoding a 24P4C12-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 24P4C12 protein/immunogen. Alternatively, a vaccine
comprises a 24P4C12-related protein.
Expression of the 24P4C12-related protein immunogen results in the generation
of prophylactic or therapeutic humoral and
cellular immunity against cells that bear a 24P4C12 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).
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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, lenfivirus, and sindbis virus (see, e.g.,
Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer
lnst 87:982-990(1995)). Non-viral delivery systems
can also be employed by introducing naked DNA encoding a 24P4C12-related
protein into the patient (e.g., intramuscularly or
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
al., 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 24P4C12-related nucleic acid
molecule. In one embodiment, the full-
length human 24P4C12 cDNA is employed. In another embodiment, 24P4C12 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
24P4C12 antigen to a patient's immune system. Dendrific
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 etal., 1996, Prostate 28:65-
69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be
used to present 24P4C12 peptides to T cells in
the context of MHC class I or II molecules. In one embodiment, autologous
dendritic cells are pulsed with 24P4C12 peptides
capable of binding to MHC class I and/or class ll molecules. In another
embodiment, dendritic cells are pulsed with the
complete 24P4C12 protein. Yet another embodiment involves engineering the
overexpression of a 24P4C12 gene in
dendritic cells using various implementing vectors known in the art, such as
adenovirus (Arthur et al., 1997, Cancer Gene
Ther. 4:17-25), retrovirus (Henderson etal., 1996, Cancer Res. 56:3763-3770),
lentivirus, adeno-associated virus, DNA
transfection (Ribas etal., 1997, Cancer Res. 57:2865-2869), or tumor-derived
RNA transfection (Ashley etal., 1997, J. Exp.
Med. 186:1177-1182). Cells that express 24P4C12 can also be engineered to
express immune modulators, such as GM-
CSF, and used as immunizing agents.
X.B.) 24P4C12 as a Target for Antibody-based Therapy
24P4C12 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 24P4C12 is expressed by
cancer cells of various lineages relative to
corresponding normal cells, systemic administration of 24P4C12-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 24P4C12 are useful to treat
24P4C12-expressing cancers systemically, either as conjugates with a toxin or
therapeutic agent, or as naked antibodies
capable of inhibiting cell proliferation or function.
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24P4C12 antibodies can be introduced into a patient such that the antibody
binds to 24P4C12 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
24P4C12, inhibition of ligand binding or signal transduction pathways,
modulation of tumor cell differentiation, alteration of
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 24P4C12 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 etal. 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. 24P4C12),
the cytotoxic agent will exert its known biological
effect (i.e. cytotcodcity) 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-
24P4C12 antibody) that binds to a marker (e.g. 24P4C12) expressed, accessible
to binding or localized on the cell surfaces.
A typical embodiment is a method of delivering a cytotoxic andfor therapeutic
agent to a cell expressing 24P4C12,
comprising conjugating the cytotoxic agent to an antibody that
immunospecifically binds to a 24P4C12 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-24P4C12 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
al., 1998, Grit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki etal.,
1997, Blood 90:3179-3186, Tsunenari etal.,
1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res.
52:2771-2776), B-cell lymphoma (Funakoshi
etal., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia
(Zhong etal., 1996, Leuk. Res. 20:581-589),
colorectal cancer (Moun etal., 1994, Cancer Res. 54:6160-6166; Velders et aL,
1995, Cancer Res. 55:4398-4403), and
breast cancer (Shepard et at, 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, 1DEC
Pharmaceuticals Corp. or BexxarTm, Coulter Pharmaceuticals), while others
involve co-administration of antibodies and other
therapeutic agents, such as Herceptinim (trastuzumab) with paclitaxel
(Genentech, Inc.). The antibodies can be conjugated
to a therapeutic agent. To treat prostate cancer, for example, 24P4C12
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.,
Mylotarg TM, 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 24P4C12 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
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tolerate the toxicity of the chemotherapeutic agent very well. Fan et at.
(Cancer Res. 53:4637-4642, 1993), Prewett et al.
(International J. of Onco. 9:217-224, 1996), and Hancock et at. (Cancer Res.
51:4575-4580, 1991) describe the use of
various antibodies together with chemotherapeutic agents.
Although 24P4C12 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.
Cancer patients can be evaluated for the presence and level of 24P4C12
expression, preferably using
immunohistochemical assessments of tumor tissue, quantitative 24P4C12 imaging,
or other techniques that reliably indicate
the presence and degree of 24P4C12 expression. lmmunohistochemical 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-24P4C12 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-24P4C12 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-24P4C12 mAbs that
exert a direct biological effect on tumor growth
are useful to treat cancers that express 24P4C12. 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-24P4C12 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 24P4C12 antigen with high affinity but exhibit low
or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single
anti-24P4C12 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-
24P4C12 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-
24P4C12 mAbs are administered in their "naked" or unconjugated form, or can
have a therapeutic agent(s) conjugated to
them.
Anti-24P4C12 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-24P4C12 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,
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.3, .4, .5, .6, .7, .8, .9., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25
mg/kg 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-
24P4C12 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 24P4C12 expression in the patient, the extent of circulating shed
24P4C12 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 24P4C12 in a given
sample (e.g. the levels of circulating
24P4C12 antigen and/or 24P4012 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).
Ant-idiotypic anti-24P4C12 antibodies can also be used in anti-cancer therapy
as a vaccine for inducing an
immune response to cells expressing a 24P4C12-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-24P4C12 antibodies that mimic
an epitope on a 24P4C12-related protein (see, for example, Wagner et al.,
1997, Hybridoma 16: 33-40; Foon etal., 1995, J.
Olin. Invest. 96:334-342; Herlyn etal., 1996, Cancer Immunol. lmmunother.
43:65-76). Such an anti-idiotypic antibody can
be used in cancer vaccine strategies.
X.C.) 24P4C12 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) oligonudeotides
has been found to increase CTL
responses 10- to 100-fold. (see, e.g. Davila and Celis, J. lmmunol. 165:539-
547(2000))
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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
at least partially immune to later development of cells that express or
overexpress 24P4C12 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
nudeic 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 (TM). 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 al., Science 278:1447-1450). Epitopes from one TM 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 IC50 of 500 nM or less, often 200 nM or
less; and for Class II an IC50 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 ll 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,
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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
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
add 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.1. 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 of aL,
J. immunot 162:3915-3925, 1999; An,
L. and Whitton, J. L., J. Viral. 71:2292, 1997; Thomson, S. A. etal., .1
Immunot 157:822, 1996; Whitton, J. L. et aL, J. Vim!.
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 24P4C12, the PADRE universal helper T
cell epitope or multiple HTL epitopes from
24P4C12 (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
TMs.
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.
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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
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.
coil 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. coil 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., LelF), 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-p) may be
beneficial in certain diseases.
Therapeutic quantities of plasmid DNA can be produced for example, by
fermentation in E. coil, 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
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also be used in the formulation (see, e.g., as described by WO 93/24640;
Mannino & Gould-Fogerite, Biorechniques 6(7):
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 (PING)
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.
lmmunogenicity 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.
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In certain embodiments, the T helper peptide is one that is recognized by T
helper cells present in a majority of a
genetically diverse population. This can be accomplished by selecting peptides
that bind to many, most, or all of the HLA
class ll molecules. Examples of such amino acid bind many HLA Class II
molecules include sequences from antigens such
as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 29),
Plasmodium falciparum circumsporozoite (CS)
protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 30), and
Streptococcus 18kD protein at positions
116-131 (GAVDS1LGGVATYGAA; SEQ ID NO: 31). 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: AKXVAAWTLKAAA (SEQ ID NO: 32), where "X" is either
cyclohexylalanine, phenylalanine, or tyrosine, and a
is either o-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 o-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 e-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 e- 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, etal., 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.
XC.4. Vaccine Compositions Comprising DC Pulsed with CTL andlor 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 Progenipoietinn1 (Pharmacia-
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. In
this embodiment': a vaccine comprises peptide-pulsed DCs which present the
pulsed peptide epitopes complexed with HLA
molecules on their surfaces.
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The DC can be pulsed ex vivo with a cocktail of peptides, some of which
stimulate CTL responses to 24P4C12.
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 24P4C12.
X.D. Adoptive Immunotherapy
Antigenic 24P4C12-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 24P4C12. 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
24P4C12. 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 24P4C12-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 TM-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
24P4C12, a vaccine comprising 24P4C12-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 pg to about 50,000 ig of peptide pursuant to a boosting regimen over weeks
to months may be administered depending
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upon the patients 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
pg to about 50,000 pg 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 volumelquantity
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
pg, generally 100-5,000 pg, 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 nudeic acid (0.1 to 1000 pg)
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.
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For antibodies, a treatment generally involves repeated administration of the
anti-24P4C12 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 10-500mg 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- 24P4C12 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 24P4C12 expression in the
patient, the extent of circulating shed 24P4C12 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 - 1mg, 1mg
- 50mg, 50mg - 100mg, 100mg - 200mg, 200mg - 300mg, 400mg - 500mg, 500mg -
600mg, 600mg - 700mg, 700mg -
800mg, 800mg - 900mg, 900mg - 1g, or 1mg - 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 ring/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, 1 to 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 polynudeotide 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 thatc
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 cells1m2 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
therapeutic or immunogenic compositions. Thus, liposomes either filled or
decorated with a desired peptide of the invention
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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, etal., 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 24P4C12.
As disclosed herein, 24P4C12 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 24P4C12 in normal tissues, and
patient specimens").
24P4C12 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 etal., J. Urol. 163(2):
503-5120 (2000); Polascik etal., J. Urol, Aug; 162(2):293-306 (1999) and
Fortier etal., 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 etal., Int J Mol Med 1999 Jul 4(1):99-102 and Minimoto etal.,
Cancer Detect Prey 2000;24(1):1-12). Therefore,
this disclosure of 24P4C12 polynucleotides and polypeptides (as well as
24P4C12 polynucleotide probes and anti-24P4C12
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 24P4C12
polynudeotides, polypeptides, reactive T
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 polynudeotides are used as probes
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(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 at 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 24P4C12
polynucleotides described herein can be utilized in the same way to detect
24P4C12 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 etal., Urology 55(4):560-3
(2000)) or the metastasis of prostate cells (see,
e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 24P4C12
polypeptides described herein can be utilized to
generate antibodies for use in detecting 24P4C12 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 24P4C12 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 24P4C12-expressing cells
(lymph node) is found to contain 24P4C12-expressing cells such as the 24P4C12
expression seen in LAPC4 and LAPC9,
xenografts isolated from lymph node and bone metastasis, respectively, this
finding is indicative of metastasis.
Altematively 24P4C12 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
24P4C12 or express 24P4C12 at a different level are
found to express 24P4C12 or have an increased expression of 24P4C12 (see,
e.g., the 24P4C12 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 24P4C12) such as PSA, PSCA etc. (see, e.g.,
Alanen et aL, Pathol. Res. Pract. 192(3): 233-
237 (1996)).
Just as PSA polynucleotide fragments and polynucleotide variants are employed
by skilled artisans for use in
methods of monitoring PSA, 24P4C12 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 etal., 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 24P4C12 in normal
tissues, and patient specimens," where a 24P4C12 polynucleotide fragment is
used as a probe to show the expression of
24P4C12 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 etal. 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 24P4C12 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.
24P4C12 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
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such as fusion proteins being used by practitioners (see, e.g., Current
Protocols In Molecular Biology, Volume 2, Unit 16,
Frederick M. Ausubel etal. 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
24P4C12 biological motifs discussed herein or a motif-bearing subsequence
which is readily identified by one of skill in the
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
24P4C12 polypeptide shown in Figure 3).
As shown herein, the 24P4C12 polynucleotides and polypeptides (as well as the
24P4C12 polynucleotide probes
and anti-24P4C12 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
24P4C12 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 etal., Pathol. Res.
Pract. 192(3): 233-237 (1996)), and consequently,
materials such as 24P4C12 polynucleotides and polypeptides (as well as the
24P4C12 polynucleotide probes and anti-
24P4C12 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 24P4C12
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 24P4C12 gene maps (see the Example entitled
"Chromosomal Mapping of 24P4C12"
below). Moreover, in addition to their use in diagnostic assays, the 24P4C12-
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, 24P4C12-related proteins or polynucleotides of the invention can
be used to treat a pathologic
condition characterized by the over-expression of 24P4C12. 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 24P4C12 antigen. Antibodies or
other molecules that react with 24P4C12 can be used to modulate the function
of this molecule, and thereby provide a
therapeutic benefit
XII.) Inhibition of 24P4C12 Protein Function
The invention includes various methods and compositions for inhibiting the
binding of 24P4C12 to its binding
partner or its association with other protein(s) as well as methods for
inhibiting 24P4C12 function.
XIIA.) Inhibition of 24P4C12 With Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies
that specifically bind to 24P4C12 are
introduced into 24P4C12 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-
24P4C12 antibody is expressed intrac,ellularly, binds to 24P4C12 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
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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 at al., 1995, Proc. Natl. Acad.
Sci. USA 92: 3137-3141; Beerli etal., 1994, J.
Biol. Chem. 289: 23931-23936; Deshane at 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
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 24P4C12 in the nucleus,
thereby preventing its activity within
the nucleus. Nuclear targeting signals are engineered into such 24P4C12
intrabodies in order to achieve the desired
targeting. Such 24P4C12 intrabodies are designed to bind specifically to a
particular 24P4C12 domain. In another
embodiment, cytosolic intrabodies that specifically bind to a 24P4C12 protein
are used to prevent 24P4C12 from gaining
access to the nucleus, thereby preventing it from exerting any biological
activity within the nucleus (e.g., preventing 24P4C12
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.1 Inhibition of 24P4C12 with Recombinant Proteins
In another approach:recombinant molecules bind to 24P4C12 and thereby inhibit
24P4C12 function. For example,
these recombinant molecules prevent or inhibit 24P4C12 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 24P4C12 specific antibody
molecule. In a particular embodiment, the 24P4C12 binding domain of a 24P4C12
binding partner is engineered into a dimeric
fusion protein, whereby the fusion protein comprises two 24P4C12 ligand
binding domains linked to the Fc 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 CH1 domain. Such dimeric fusion proteins are administered in soluble form
to patients suffering from a cancer associated with
the expression of 24P4C12, whereby the dimeric fusion protein specifically
binds to 24P4C12 and blocks 24P4C12 interaction
with a binding partner. Such dimeric fusion proteins are further combined into
multimeric proteins using known antibody linking
technologies.
XII.C.) Inhibition of 24P4C12 Transcription or Translation
The present invention also comprises various methods and compositions for
inhibiting the transcription of the
24P4C12 gene. Similarly, the invention also provides methods and compositions
for inhibiting the translation of 24P4C12
mRNA into protein.
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In one approach, a method of inhibiting the transcription of the 24P4C12 gene
comprises contacting the 24P4C12
gene with a 24P4C12 antisense polynudeotide. In another approach, a method of
inhibiting 24P4C12 mRNA translation
comprises contacting a 24P4C12 mRNA with an antisense polynucleotide. In
another approach, a 24P4C12 specific
ribozyme is used to cleave a 24P4C12 message, thereby inhibiting translation.
Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the 24P4C12 gene,
such as 24P4C12 promoter and/or enhancer
elements. Similarly, proteins capable of inhibiting a 24P4C12 gene
transcription factor are used to inhibit 24P4C12 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 24P4C12 by interfering with
24P4C12 transcriptional activation are
also useful to treat cancers expressing 24P4C12. Similarly, factors that
interfere with 24P4C12 processing are useful to treat
cancers that express 24P4C12. 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
polynudeotide molecules to tumor cells
synthesizing 24P4C12 (i.e., antisense, ribozyme, polynudeotides encoding
intrabodies and other 24P4C12 inhibitory molecules).
A number of gene therapy approaches are known in the art. Recombinant vectors
encoding 24P4C12 antisense polynudeotides,
ribozymes, factors capable of interfering with 24P4C12 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 24P4C12 to a binding partner, etc.
In vivo, the effect of a 24P4C12 therapeutic composition can be evaluated in a
suitable animal model. For example,
xenogenic prostate cancer models can be used, wherein human prostate cancer
evlants or passaged xenograft tissues are
introduced into immune compromised animals, such as nude or SCID mice (Klein
etal., 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
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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
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 24P4C12
=
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 Bid Screen 7:69 (2002); Zlokamik, et al., Science
279:84-8 (1998); Heid, Genome Res 6:986-
94,1996).
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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
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.
=
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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,
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 nudeic acid
that encodes a cancer protein of the invention.
In another embodiment, a library of candidate agents is tested on a plurality
of cells.
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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
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
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foci. Alternatively, labeling index with (3H)-thymidine at saturation density
is used to measure density limitation of growth,
similarly an MIT 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.
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. Natl. 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, Angioge-nesis, 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, Sem Cancer
Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et
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
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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
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. Natl.
Cancer Inst. 52:921 (1974)), a SCID mouse, a
thymectomized 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).
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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
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 Teflon 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.
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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.
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 Bindine to 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.
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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.
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 Polynudeotides to Down-regylate 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
oligonudeotides 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
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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)).
Ribozvmes
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
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., Nucl. 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 at., 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 US97/01019. 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.
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Methods of Identifying Characterizino_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
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, Bestht, 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.) KitslArticles of Manufacture
For use in the diagnostic and therapeutic applications described herein, kits
are also 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. 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 Figure 2-related protein or a Figure
2 gene or message, 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 and/or a
container comprising a reporter-means, such as a
biotin-binding protein, such as avidin or streptavidin, bound to a reporter
molecule, such as an enzymatic, florescent, or
radioisotope label. 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 molecules that encodes such amino acid sequences.
The kit of the invention will typically comprise the container described above
and one or more other containers
comprising 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 the container to indicate that the composition is
used for a specific therapy or non-therapeutic
application, such as a 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
induded on an insert(s) or label(s) which is induded with
or on the kit
The terms 'kit" and "article of manufacture" can be used as synonyms.
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
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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 or plastic.
The container can hold amino acid sequence(s), small molecule(s), nucleic acid
sequence(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.
The container can alternatively hold a composition which 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 24P4C12 and modulating the function
of 24P4C12.
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 I. The article of manufacture can further comprise
a second container comprising a
pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringers
solution and/ordextrose 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 are intended to limit the scope of the invention.
Example 1: SSH-Generated Isolation of cDNA Fragment of the 24P4C12 Gene
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 the LAPC-9 AD prostate cancer xenograft. The
gene 24P4C12 was derived from an LAPC-9 AD minus benign prostatic hyperplasia
experiment.
The 24P4C12 SSH cDNA of 160 bp is listed in Figure 1. The full length 24P4C12
cDNAs and ORFs are described
in Figure 2 with the protein sequences listed in Figure 3.
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 Clontech, Palo Alto, CA.
RNA Isolation:
Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL)
using 10 ml/ g tissue 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 (0.D. 260/280 nm) and analyzed by gel
electrophoresis.
Oligonucleotides:
The following HPLC purified oligonucleotides were used.
DPNCDN (cDNA synthesis primer):
5'ITTTGATCAAGCTT303 (SEQ ID NO: 33)
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Adaptor 1:
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 34)
3'GGCCCGTCCTAG5 (SEQ ID NO: 35)
Adaptor 2:
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 36)
3'CGGCTCCTAG5' (SEQ ID NO: 37)
PCR primer 1:
5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO: 38)
Nested primer (NP)1:
5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 39)
Nested primer (NP)2:
5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 40)
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 prostate cancer and normal tissues.
The gene 24P4C12 sequence was derived from LAPC-4AD prostate cancer xenograft
minus begnin prostatic
hyperplasia cDNA subtraction. The SSH DNA sequence (Figure 1) was identified.
The cDNA derived from a pool of normal tissues and benign prostatic
hyperplasia was used as the source of the "driver"
cDNA, while the cDNA from LAPC-4AD xenograft was used as the source of the
"tester" cDNA. Double stranded cDNAs
corresponding to tester and driver cDNAs were synthesized from 2 jig of
poly(A) + RNA isolated from the relevant xenograft tissue,
as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng
of oligonucleotide DPNCDN as primer. First-
and second-strand synthesis were carried out as desaped in the Kit's user
manual protocol (CLONTECH Protocol No. P11117-1,
Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at
37 C. Digested cDNA was extracted with
phenol/chloroform (1:1) and ethanol precipitated.
= = Driver cDNA was generated by combining in a 1:1 ratio Dpn II
digested cDNA from the relevant tissue source (see
above) with a mix of digested cDNAs derived from the nine normal tissues:
stomach, skeletal muscle, lung, brain, liver, kidney,
pancreas, small intestine, and heart.
Tester cDNA was generated by diluting 1 I of Dpn II digested cDNA from the
relevant tissue source (see above) (400
ng) in 5 jil of water. The diluted cDNA (2 I, 160 ng) was then ligated to 2
I of Adaptor 1 and Adaptor 2 (10 M), in separate
ligation reactions, in a total volume of 10 I at 16 C overnight, using 400 u
of 14 DNA ligase (CLONTECH). Ligation was
terminated with 1 I of 0.2 M EDTA and heating at 72 C for 5 min.
The first hybridization was performed by adding 1.5 I (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 I,
the samples were overlaid with mineral oil, denatured
in an MJ Research thermal cycler at 98 C for 1.5 minutes, and then were
allowed to hybridize for 8 hrs at 68 C. The two
hybridizations were then mixed together with an additional 1 I of fresh
denatured driver cDNA and were allowed to hybridize
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overnight at 68 C. The second hybridization was then diluted in 200 pl of 20
mM Hepes, pH 8.3, 50 mM NaCI, 0.2 mM EDTA,
heated at 70 C for 7 min. and stored at -20 C.
PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from
SSH:
To amplify gene fragments resulting from SSH reactions, two PCR amplifications
were performed. In the primary PCR
reaction 1 I of the diluted final hybridization mix was added to 1 jAl of PCR
primer 1 (10 p.M), 0.5 pl dNTP mix (10 pM), 2.5 pi 10
x reaction buffer (CLONTECH) and 0.5111 50 x Advantage cDNA polymerase Mix
(CLONTECH) in a final volume of 25 I. PCR 1
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 pl 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 94 C 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. coil 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 ul of bacterial culture using the conditions of
PCR1 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 Gen Bank, dBest, and NCI-CGAP
databases.
RT-PCR Expression Analysis:
First strand cDNAs can be generated from 1 pug 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 I 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: 41) and 5'agccacacgcagctcattgtagaagg
3' (SEQ ID NO: 42) to amplify I3-actin. First
strand cDNA (5 pl) were amplified in a total volume of 50 pl containing 0.4 pM
primers, 0.2 M each dNTPs, 1XPCR buffer
(Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCI, pH8.3) and 1X Klentaq DNA
polymerase (Clontech). Five pi 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 b.p. 8-actin bands from
multiple tissues were compared by visual inspection.
.Dilution factors for the first strand cDNAs were calculated to result in
equal 8-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 24P4C12 gene, 5 pi 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 24P4C12 SSH sequence and are
listed below:
24P4C12.1
5'- AGATGAGGAGGAGGACAAAGGTG -3' (SEQ ID NO: 43)
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24P4C12.2
5'- ACTGCTGGGAGGAGTACCGAGTG -3' (SEQ ID NO: 44)
Example 2: Isolation of Full Length 24P4C12 Encoding cDNA
The 24P4C12 SSH cDNA sequence was derived from a substraction consisting of
LAPC-4AD xenograft minus benign
prostatic hyperplasia. The SSH cDNA sequence (Figure 1) was designated
24P4C12.
The isolated gene fragment of 160 bp encodes a putative open reading frame
(ORF) of 53 amino acids and exhibits
significant homology to an EST derived from a colon tumor library. Two larger
cDNA clones were obtained by gene trapper
experiments, GTE9 and GTF8, The ORF revealed a significant homology to the
mouse gene NG22 and the C.elegans gene
CEESB82F. NG22 was recently identified as one of many ORFs within a genomic
BAG clone that encompasses the MHC class III
in the mouse genome. Both NG22 and CEESB82F appear to be genes that contain 12
transmembrane domains. This suggests
that the gene encoding 24P4C12 contains 12 transmembrane domains and is the
human homologue of mouse NG22 and C.
elegans CEESB82F. Functional studies in Ce. elegans may reveal the biological
role of these homologs. If 24P4C12 is a cell
surface marker, then it may have an application as a potential imaging reagent
and/or therapeutic target in prostate cancer.
The 24P4C12 v.1 of 2587 bp codes for a protein of 710 amino acids (Figure 2
and Figure 3). Other variants of 24P4C12
were also identified and these are listed in Figures 2 and 3.24P4C12 v.1, v.3,
v.5 and v.6 proteins are 710 amino acids in length
and differ from each other by one amino acid as shown in Figure 11. 24P4C12
v.2 and v.4 code for the same protein as 24P4C12
v.1. 24P4C12 v.7, v.8 and v.9 are alternative splice variants and code for
proteins of 598, 722 and 712 amino acids in length,
respectively.
Example 3: Chromosomal Mapping of 24P4C12
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
CorteII Institute (Camden, New Jersey), and genomic viewers utilizing BLAST
homologies to sequenced and mapped genomic
clones (NCBI, Bethesda, Maryland). 24P4C12 maps to chromosome 6p21.3 using
24P4C12 sequence and the NCB! BLAST tool.
Example 4: Expression Analysis of 24P4C12
Expression analysis by RT-PCR demonstrated that 24P4C12 is strongly expressed
in prostate and ovary cancer patient
specimens (Figure 14). First strand cDNA was generated from vital pool 1
(kidney, liver and lung), vital pool 2 (colon, pancreas and
stomach), a pool of prostate cancer xenografts (LAPC-4AD, LAPC-4A1, LAPC-9AD
and LAPC-9A1), prostate cancer pool, bladder
cancer pool, kidney cancer pool, colon cancer pool, ovary cancer pool, breast
cancer pool, and cancer metastasis pool.
Normalization was performed by PCR using primers to actin. Semi-quantitative
PCR, using primers to 24P4C12, was performed at 26
and 30 cycles of amplification. Results show strong expression of 24P4C12 in
prostate cancer pool and ovary cancer pool.
Expression was also detected in prostate cancer xenografts, bladder cancer
pool, kidney cancer pool, colon cancer pool, breast
cancer pool, cancer metastasis pool, vital pool 1, and vital pool 2.
Extensive northern blot analysis of 24P4C12 in multiple human normal tissues
is shown in Figure 15. Two multiple tissue
northern blots (Clontech) both with 2 pg of mRNA/lane were probed with the
24P4C12 SSH sequence. Expression of 24P4C12 was
detected in prostate, kidney and colon. Lower expression is detected in
pancreas, lung and placenta amongst all 16 normal tissues
tested.
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Expression of 24P4C12 was tested in prostate cancer xenografts and cell lines.
RNA was extracted from a panel of cell
lines and prostate cancer xenografts (PrEC, LAPC-4AD, LAPC-4A1, LAPC-9A0, LAPC-
9AI, LNCaP, PC-3, DU145, TsuPr, and LAPC-
4CL). Northern blot with 10 pg of total RNA/lane was probed with 24P4C12 SSH
sequence. Size standards in kilobases (kb) are
indicated on the side. The 24P4C12 transcript was detected in LAPC-4AD, LAPC-
4AI, LAPC-9AD, LAPC-9AI, LNCaP, and LAPC-4
CL
Expression of 24P4C12 in patient cancer specimens and human normal tissues is
shown in Figure 16. RNA was extracted
from a pool of prostate cancer specimens, bladder cancer specimens, colon
cancer specimens, ovary cancer specimens, breast
cancer specimens and cancer metastasis specimens, as well as from normal
prostate (NP), normal bladder (NB), normal kidney
(NK), and normal colon (NC). Northern blot with 10 pg of total RNA/lane was
probed with 24P4C12 SSH sequence. Size standards
in kilobases (kb) are indicated on the side. Strong expression of 24P4C12
transcript was detected in the patient cancer pool
specimens, and in normal prostate but not in the other normal tissues tested.
Expression of 24P4C12 was also detected in individual prostate cancer patient
specimens (Figure 17). RNA was
extracted from normal prostate (N), prostate cancer patient tumors (T) and
their matched normal adjacent tissues (Nat).
Northern blots with 10 pg of total RNA were probed with the 24P4C12 SSH
fragment. Size standards in kilobases are on the
side. Results show expression of 24P4C12 in normal prostate and all prostate
patient tumors tested.
Expression of 24P4C12 in colon cancer patient specimens is shown in figure 18.
RNA was extracted from colon
cancer cell lines (CL: Cob 205, LoVo, and SK-00-), normal colon (N), colon
cancer patient tumors (T) and their matched
normal adjacent tissues (Nat). Northern blots with 10 pg of total RNA were
probed with the 24P4C12 SSH fragment. Size
standards in kilobases are on the side. Results show expression of 24P4C12 in
normal colon and all colon patient tumors
tested. Expression was detected in the cell lines Colo 205 and SK-CO-, but not
in LoVo.
Figure 20 displays expression results of 24P4C12 in lung cancer patient
specimens. Ma was extracted from lung
cancer cell lines (CL: CALU-1, A427, NCI-H82, NCI-H146), normal lung (N), lung
cancer patient tumors (T) and their
matched normal adjacent tissues (Nat). Northern blots with 10 pg of total RNA
were probed with the 24P4C12 SSH
fragment Size standards in kilobases are on the side. Results show expression
of 24P4C12 in lung patient tumors tested,
but not in normal lung. Expression was also detected in CALU-1, but not in the
other cell lines A427, NCI-H82, and NCI-
H146.
24P4C12 was assayed in a panel of human stomach and breast cancers (T) and
their respective matched normal
tissues (N) on RNA dot blots. 24P4C12 expression was seen in both stomach and
breast cancers. 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 24P4C12 may be
expressed in early stage tumors.
The level of expression of 24P4C12 was analyzed and quantitated in a panel of
patient cancer tissues. First strand
cDNA was prepared from a panel of ovary patient cancer specimens (A), uterus
patient cancer specimens (B), prostate
cancer specimens (C), bladder cancer patient specimens (D), lung cancer
patient specimens (E), pancreas cancer patient
specimens (F), colon cancer specimens (G), and kidney cancer specimens (H).
Normalization was performed by PCR using
primers to actin. Semi-quantitative PCR, using primers to 24P4C12, was
performed at 26 and 30 cycles of amplification.
Samples were run on an agarose gel, and PCR products were quantitated using
the Alphalmager software. Expression was
recorded as absent, low, medium or strong. Results show expression of 24P4C12
in the majority of patient cancer
specimens tested, 73.3% of ovary patient cancer specimens, 83.3% of uterus
patient cancer specimens, 95.0% of prostate
cancer specimens, 61.1% of bladder cancer patient specimens, 80.6% of lung
cancer patient specimens, 87.5% of pancreas
cancer patient specimens, 87.5% of colon cancer specimens, 68.4% of clear cell
renal carcinoma, 100% of papillary renal
cell carcinoma.
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The restricted expression of 24P4C12 in normal tissues and the expression
detected in prostate cancer, ovary cancer,
bladder cancer, colon cancer, lung cancer pancreas cancer, uterus cancer,
kidney cancer, stomach cancer and breast
cancer suggest that 24P4C12 is a potential therapeutic target and a diagnostic
marker for human cancers.
Example 5: Transcript Variants of 24P4C12
=
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 dusters which show direct or indirect
identity with each other. Second, ESTs 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
variant for that gene. Even when a variant is identified that is not a full-
length done, 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., etal., Identification
of human chromosome 22 transcribed
sequences with ORF expressed sequence tags, Proc. Nati Aced 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, SO., et al., Albumin banks peninsula: a new termination variant
characterized by electrospray mass spectrometry,
Biochem Biophys Ada. 1999 Aug 17;1433(1-2):321-6; Ferranti P, etal.,
Differential splicing of pre-messenger RNA produces
multiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997 Oct
1;249(1):1-7. For PCR-based Validation:
VVellmann S, at al., Specific reverse transcription-PCR quantification of
vascular endothelial growth factor (VEGF) splice
variants by LightCycler technology, Clin Chem. 2001 Apr;47(4):654-60; Jia,
H.P., et 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, K.E., et 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.
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Disclosed herein is that 24P4C12 has a particular expression profile related
to cancer. Alternative transcripts and splice
variants of 24P4C12 may also be involved in cancers in the same or different
tissues, thus serving as tumor-associated
markers/antigens.
The exon composition of the original transcript, designated as 24P4C12 v.1, is
shown in Table LI. Using the full-
length gene and EST sequences, three transcript variants were identified,
designated as 24P4C12 v.7, v.8 and v.9.
Compared with 24P4C12 v.1, transcript variant 24P4C12 v.7 has spliced out
exons 10 and 11 from variant 24P4C12 v.1, as
shown in Figure 12. Variant 24P4C12 v.8 inserted 36 bp in between 1931 and
1932 of variant 24P4C12 v.1 and variant
24P4C12 v.9 replaced with 36 bp the segment 1136-1163 of variant 24P4C12 v.1.
Theoretically, each different combination
of exons in spatial order, e.g. exons 2 and 3, is a potential splice variant.
Figure 12 shows the schematic alignment of exons
of the four transcript variants.
Tables LII through LXIII are set forth on a variant by variant basis. Tables
LII, LVI, and LX show nucleotide
sequences of the transcript variant. Tables LIII, DAL and LXI show the
alignment of the transcript variant with the nucleic
acid sequence of 24P4C12 v.1. Tables LIV, LVIII, and LXII lay out the amino
acid translation of the transcript variant for the
identified reading frame orientation. Tables LV, LIX, and LXIII display
alignments of the amino acid sequence encoded by
the splice variant with that of 24P4C12 v.1.
Example 6: Single Nucleotide Polymorphisms of 24P4C12
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: NT, 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),
often in the context of one gene or in the context of several tightly linked
genes. SNPs that occur on a cDNA are called
cSNPs. These cSNPs may change amino acids of the protein encoded by the gene
and thus change the functions of the
protein. Some SNPs cause inherited diseases; others contribute to quantitative
variations in phenotype and reactions to
environmental factors including diet and drugs among individuals. Therefore,
SNPs 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. VVindemuth, "The predictive
power of haplotypes in clinical response," Pharmacogenomics. 2000 feb; 1(1):15-
26).
SNPs 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,
SNPs are 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 SNPs 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). SNPs
can be verified and genotype or haplotype of an individual can be determined
by a variety of methods including direct
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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, five SNPs were identified in the original
transcript, 24P4C12 v.1, at positions 542 (G/A),
564 (G/A), 818 (C/T), 981(NG) and 1312 (NC). The transcripts or proteins with
alternative alleles were designated as
variants 24P4C12 v.2, v.3, v.4, v.5 and v.6, respectively. Figure 10 shows the
schematic alignment of the SNP variants.
Figure 11 shows the schematic alignment of protein variants, corresponding to
nucleotide variants. Nucleotide variants that
code for the same amino acid sequence as variant 1 are not shown in Figure 11.
These alleles of the SNPs, though shown
separately here, can occur in different combinations (haplotypes) and in any
one of the transcript variants (such as 24P4C12
v.7) that contains the sequence context of the SNPs.
Example 7: Production of Recombinant 24P4C12 in Prokaryotic Systems
To express recombinant 24P4C12 and 24P4C12 variants in prokaryotic cells, the
full or partial length 24P4C12
and 24P4C12 variant cDNA sequences are cloned into any one of a variety of
expression vectors known in the art. The full
length cDNA, 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 24P4C12, variants, or analogs thereof are used.
A. In vitro transcription and translation constructs:
pCRII: To generate 24P4C12 sense and anti-sense RNA probes for RNA in situ
investigations, pCRII constructs
(Invitrogen, Carlsbad CA) are generated encoding either all or fragments of
the 24P4C12 cDNA. The pCRII vector has Sp6
and T7 promoters flanking the insert to drive the transcription of 24P4C12 RNA
for use as probes in RNA in situ hybridization
experiments. These probes are used to analyze the cell and tissue expression
of 24P4C12 at the RNA level. Transcribed
24P4C12 RNA representing the cDNA amino acid coding region of the 24P4C12 gene
is used in in vitro translation systems
such as the TnTTm Coupled Reticulolysate System (Promega, Corp., Madison, WI)
to synthesize 24P4C12 protein.
B. Bacterial Constructs:
pGEX Constructs: To generate recombinant 24P4C12 proteins in bacteria that are
fused to the Glutathione S-
transferase (GST) protein, all or parts of the 24P4C12 cDNA or variants are
cloned into the GST- fusion vector of the pGEX
family (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow
controlled expression of recombinant
24P4C12 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 codons to the cloning primer at the 3' end,
e.g., of the open reading frame (ORE). 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 24P4C12-related protein. The ampicillin
resistance gene and pBR322 origin permits selection
and maintenance of the pGEX plasmids in E. coil.
pMAL Constructs: To generate, in bacteria, recombinant 24P4C12 proteins that
are fused to maltose-binding
protein (MBP), all or parts of the 24P4C12 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 24P4C12 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
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tag from 24P4C12. 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.
pET Constructs: To express 24P4C12 in bacterial cells, all or parts of the
24P4C12 cDNA protein coding
sequence are cloned into the pET family of vectors (Novagen, Madison, WI).
These vectors allow tightly controlled
expression of recombinant 24P4C12 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-Tagm" 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
24P4C12 protein are expressed as amino-terminal fusions to NusA.
C. Yeast Constructs:
pESC Constructs: To express 24P4C12 in the yeast species Saccharomyces
cerevisiae for generation of
recombinant protein and functional studies, all or parts of the 24P4C12 cDNA
protein coding sequence are cloned into the
pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3,
TRP1, LEU2, and URA3 (Stratagene, La
Jolla, CA). These vectors allow controlled expression from the same plasmid of
up to 2 different genes or doned sequences
containing either FlagTM or Myc epitope tags in the same yeast cell. This
system is useful to confirm protein-protein
interactions of 24P4C12. 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 24P4C12 in the yeast species Saccharomyces pombe,
all or parts of the 24P4C12
cDNA protein coding sequence are cloned into the pESP family of vectors. These
vectors allow controlled high level of
expression of a 24P4C12 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 FIagTM epitope tag
allows detection of the recombinant protein with anti-
FlagTM antibody.
Example 8: Production of Recombinant 24P4C12 in Higher Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant 24P4C12 in eukaryotic cells, the full or partial length
24P4C12 cDNA sequences can be
cloned into any one of a variety of expression vectors known in the art. One
or more of the following regions of 24P4C12 are
expressed in these constructs, amino acids 1 to 710, 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 24P4C12 v.1 through v.6;
amino acids 1 to 598, 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 24P4C12 v.7;
amino acids 1 to 722, 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 24P4C12 v.8, amino acids 1 to 712, 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 24P4C12
v.9, 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-24P4C12 polydonal
serum, described herein.
pcDNA3.11MycHis Constructs: To express 24P4C12 in mammalian cells, a 24P4C12
ORF, or portions thereof,
of 24P4C12 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
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resistance gene and ColE1 origin permits selection and maintenance of the
plasmid in E. coll. Figure 24 demonstrates
expression of 24P4C12 from the pcDNA3.1/MycHis construct in transiently
transfected 2931 cells.
pcDNA4IHisMax Constructs: To express 24P4C12 in mammalian cells, a 24P4C12
ORF, or portions thereof, of
24P4C12 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA).
Protein expression is driven from the
cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The
recombinant protein has Xpresem 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 ColE1
origin permits selection and maintenance of the plasmid in E. coll.
pcDNA3.1ICT-GFP-TOPO Construct: To express 24P4C12 in mammalian cells and to
allow detection of the
recombinant proteins using fluorescence, a 24P4C12 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
pcDNA3.1CT-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
Neomycin resistance gene allows for selection of mammalian cells that express
the protein and the ampicillin resistance
gene and ColE1 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 24P4C12 protein.
pTag5: A 24P4C12 ORF, or portions thereof, were cloned into pTag-5. This
vector is similar to pAPtag but
without the alkaline phosphatase fusion. This construct generates 24P4C12
protein with an amino-terminal IgGic signal
sequence and myc and 6X His epitope tags at the carboxyl-terminus that
facilitate detection and affinity purification. The
resulting recombinant 24P4C12 protein were optimized for secretion into the
media of transfected mammalian cells, and is
used as immunogen or ligand to identify proteins such as ligands or receptors
that interact with the 24P4C12 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. coll.
Figure 26 shows expression of 24P4C12 from two different pTag5 constructs.
PAPtag: A 24P4C12 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 24P4C12 protein while fusing the IgGic
signal sequence to the amino-terminus. Constructs are also generated in which
alkaline phosphatase with an amino-
terminal IgGic signal sequence is fused to the amino-terminus of a 24P4C12
protein. The resulting recombinant 24P4C12
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 24P4C12 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. coil.
PsecFc: A 24P4C12 ORF, or portions thereof, is also cloned into psecFc. The
psecFc vector was assembled by
cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into
pSecTag2 (lnvitrogen, California). This
construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 24P4C12
proteins, while fusing the IgGK signal
sequence to N-terminus. 24P4C12 fusions utilizing the murine IgG1 Fc region
are also used. The resulting recombinant
24P4C12 proteins are optimized for secretion into the media of transfected
mammalian cells, and can be used as
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immunogens or to identify proteins such as ligands or receptors that interact
with 24P4C12 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. coll.
pSRa Constructs: To generate mammalian cell lines that express 24P4C12
constitutively, 24P4C12 ORF, or
portions thereof, of 24P4C12 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,
24P4C12, 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 ColE1 origin permit selection and
maintenance of the plasmid in E. coll. The retroviral vectors can thereafter
be used for infection and generation of various
cell lines using, for example, PC3, NIH 313, TsuPr1, 293 or rat-1 cells.
Figure 23 shows RNA expression of 24P4C12 driven
from the 24P4C12.pSRa construct in stably transduced PC3, 3T3 and 300.19
cells. Figure 25 shows 24P4C12 protein
expression in P03 cells stably transduced with 24P4C12.pSRa construct.
Additional pSRa constructs are made that fuse an epitope tag such as the FLAGN
tag to the carboxyl-terminus of
24P4C12 sequences to allow detection using anti-Flag antibodies. For example,
the FLAGN sequence 5' gat tac aag gat
gac gac gat aag 3' (SEQ ID NO: 45) 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 24P4C12
proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated
delivery and expression of 24P4C12.
High virus titer leading to high level expression of 24P4C12 is achieved in
viral delivery systems such as adenoviral vectors
and herpes amplicon vectors. A 24P4C12 coding sequences 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, 24P4C12 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 P03, NIH 3T3, 293 or rat-1 cells.
Regulated Expression Systems: To control expression of 24P4C12 in mammalian
cells, coding sequences of
24P4C12, 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 (Sratagene). These systems
allow the study of the temporal and concentration dependent effects of
recombinant 24P4C12. These vectors are thereafter
used to control expression of 24P4012 in various cell lines such as PC3, NIH
313, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 24P4C12 proteins in a baculovirus expression system,
24P4C12 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-24P4C12 is co-transfected with helper plasmid pBac-N-
Blue (Invitrogen) into SF9 (Spodoptera
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 24P4C12 protein is then generated by infection of HighFive insect
cells (Invitrogen) with purified
baculovirus. Recombinant 24P4C12 protein can be detected using anti-24P4C12 or
anti-His-tag antibody. 24P4C12 protein
can be purified and used in various cell-based assays or as immunogen to
generate polyclonal and monoclonal antibodies
specific for 24P4C12.
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Example 9: Antieenicite Profiles and Secondary Structure
Figures 5-9 depict graphically five amino acid profiles of the 24P4C12 variant
1, assessment available by
accessing the ProtScale website on the ExPasy
Molecular biology server.
These profiles: Figure 5, Hydrophilicity, (Hopp T.P., Woods KR., 1981. Proc.
Natl. Acad. Sci. U.S.A. 78:3824-
3828); Figure 6, Hydropathicity, (Kyle J., Doolittle RE., 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 the
24P4C12 protein. Each of the above amino acid profiles of 24P4C12 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
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 24P4C12 protein and of the 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-24P4C12 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 24P4C12 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 profile 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 profile 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 profile 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 Figure 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 24P4C12 variant 1, namely the predicted presence
and location of alpha helices,
extended strands, and random coils, are predicted from the respective primary
amino acid sequences using the HNN -
Hierarchical Neural Network method
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accessed from the ExPasy molecular biology server. The analysis indicates
that 24P4C12
variant 1 is composed of 53.94% alpha helix, 9.44% extended strand, and 36.62%
random coil (Figure 13a).
Analysis for the potential presence of transmembrane domains in 24P4C12
variants were carried out using a variety of
transmembrane prediction algorithms accessed from the ExPasy molecular biology
server .
Shown graphically are the results of analysis of variant 1 depicting the
presence and location of 10 transmembrane domains
using the TMpred program (Figure 13b) and TMHMM program (Figure 13c). The
results of each program, namely the amino
acids encoding the transmembrane domains are summarized in Table L.
Example 10: Generation of 24P4C12 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 the
full length 24P4C12 protein, 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"). 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 amino acid profiles that
indicate such regions of 24P4C12 and variants).
For example, 24P4C12 recombinant bacterial fusion proteins or peptides
containing hydrophilic, flexible, beta-turn
regions of 24P4C12 variant proteins are used as antigens to generate
polyclonal antibodies in New Zealand White rabbits.
For example, such regions include, but are not limited to, amino acids 1-34,
amino acids 118-135, amino acids 194-224,
amino acids 280-290, and amino acids 690-710, of 24P4C12 variants 1. 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
one embodiment, a peptide encoding amino acids 1-14 of 24P4C12 variant 1 was
conjugated to KLH and used to immunize
a rabbit. This antiserum exhibited a high titer to the peptide (>10,000) and
recognized 24P4C12 in transfected 293T cells by
Western blot and flow cylometry (Figure 24) and in stable recombinant PC3
cells by Western blot and immunohistochemistry
(Figure 25). Alternatively the immunizing agent may include all or portions of
the 24P4C12 variant proteins, analogs or
fusion proteins thereof. For example, the 24P4C12 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 gtutathione-S-transferase
(GST) and HIS tagged fusion proteins. Such fusion proteins are purified from
induced bacteria using the appropriate affinity
matrix.
In one embodiment, a GST-fusion protein encoding amino acids 379-453,
encompassing the third predicted
extracellular loop of variant 1, is produced, purified, and used as immunogen.
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 24P4C12 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,
L.(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 Example
entitled 'Production of Recombinant 24P4C12 in Eukaryotic Systems"), and
retains post-translational modifications such as
glycosylations found in native protein. In two embodiments, the predicted 1st
and third extracellular loops of variant 1,
amino acids 59-227 and 379-453 respectively, were each cloned into the Tag5
mammalian secretion vector and expressed
in 293T cells (Figure 26). Each recombinant protein is then purified by metal
chelate chromatography from tissue culture
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supernatants and/or lysates of 2931 cells stably expressing the recombinant
vector. The purified Tag5 24P4C12 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 pig, typically 100-200 pg, 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 lig, typically 100-200 pg, 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 a KLH-
conjugated peptide encoding amino acids 1-14 of variant 1, the full-length
24P4C12 variant 1 cDNA is cloned into pCDNA
3.1 myc-his or retroviral expression vectors (lnvitrogen, see the Example
entitled "Production of Recombinant 24P4C12 in
Eukaryotic Systems"). After transfection of the constructs into 2931 cells or
transduction of PC3 with 24P4C12 retrovirus,
cell lysates are probed with the anti-24P4C12 serum and with anti-His antibody
(Santa Cruz Biotechnologies, Santa Cruz,
CA) to determine specific reactivity to denatured 24P4C12 protein using the
Western blot technique. As shown in Figures 24
and 25 the antiserum specifically recognizes 24P4C12 protein in 2931 and PC3
cells. In addition, the immune serum is
tested by fluorescence microscopy, flow cytometry, and immunohistochemistry
(Figure 25) and immunoprecipitation against
293T and other recombinant 24P4C12-expressing cells to determine specific
recognition of native protein. Western blot,
immunoprecipitation, fluorescent microscopy, immunohistochemistry and flow
cytometric techniques using cells that
endogenously express 24P4C12 are also carried out to test reactivity and
specificity.
Anti-serum from rabbits immunized with 24P4C12 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 GST-
24P4C12 fusion protein encoding amino acids 379-453 of variant 1 is first
purified by passage over a column of GST protein
covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum
is then affinity purified by passage over a
column composed of a MBP-fusion protein also encoding amino acids 379-453
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 24P4C12 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 24P4C12 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 24P4C12 variants, for example those that would disrupt the
interaction with ligands and substrates or disrupt
its biological activity. Immunogens for generation of such mAbs include those
designed to encode or contain the entire
24P4C12 protein variant sequence, regions of the 24P4C12 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"). lmmunogens 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
24P4C12 variant, such as 293T-24P4C12 variant 1 or 300.19-24P4C12 variant
1murine Pre-B cells, are used to immunize
mice.
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To generate mAbs to a 24P4C12 variant mice are first immunized
intraperitoneally (IP) with, typically, 10-50 pg of
protein immunogen or 107 24P4C12-expressing cells mixed in complete Freund's
adjuvant Mice are then subsequently
immunized IP every 2-4 weeks with, typically, 10-50 jig of protein immunogen
or 107 cells mixed in incomplete Freund's
adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In one
embodiment, mice were immunized as above
with 300.19-24P4C12 cells in complete and then incomplete Freund's adjuvant,
and subsequently sacrificed and the spleens
harvested and used for fusion and hybridoma generation. As is can be seen in
Figure 27, 2 hybridomas were generated
whose antibodies specifically recognize 24P4C12 protein expressed in 293T
cells by flow cytometry. 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 24P4C12 variant sequence is used to immunize mice
by direct injection of the plasmid DNA.
In one embodiment, a Tag5 mammalian secretion vector encoding amino acids 59-
227 of the variant 1 sequence (Figure 26)
was used to immunize mice. Subsequent booster immunizations are then carried
out with the purified protein. In another
example, the same amino acids are cloned into an Fc-fusion secretion vector in
which the 24P4C12 variant 1 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 as above and with cells expressing the
respective 24P4C12 variant.
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, immunohistochemistry,
and flow cytometric analyses, fusion and
hybridoma generation is then carried out with established procedures well
known in the art (see, e.g., Hallow and Lane,
1988).
In one embodiment for generating 24P4C12 variant 8 specific monoclonal
antibodies, a peptide encoding amino
acids 643-654 (RNPITPTGHVFQ) (SEQ ID NO: 46) of 24P4C12 variant 8 is
synthesized, coupled to KLH and used as
immunogen. Balb C mice are initially immunized intraperitoneally with 25 pg of
the KLH-24P4C12 variant 8 peptide mixed in
complete Freund's adjuvant. Mice are subsequently immunized every two weeks
with 25 tig of the antigen mixed in
incomplete Freund's adjuvant for a total of three immunizations. ELISA using
the free peptide determines the reactivity of
serum from immunized mice. Reactivity and specificity of serum to full length
24P4C12 variant 8 protein is monitored by
Western blotting, immunoprecipitation and flow cytometry using 293T cells
transfected with an expression vector encoding
the 24P4C12 variant 8 cDNA compared to cells transfected with the other
24P4C12 variants (see e.g., the Example entitled
Production of Recombinant 24P4C12 in Eukaryotic Systems"). Other recombinant
24P4C12 variant 8-expressing cells or
cells endogenously expressing 24P4C12 variant 8 are also used. Mice showing
the strongest specific reactivity to 24P4C12
variant 8 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 SP0/2 myeloma cells using standard
procedures (Harlow and Lane, 1988).
Supematants from HAT selected growth wells are screened by ELISA, Western
blot, immunoprecipitation, fluorescent
microscopy, and flow cytometry to identify 24P4C12 variant 8-specific antibody-
producing clones. A similar strategy is also
used to derive 24P4C12 variant 9-specific antibodies using a peptide
encompassing amino acids 379-388 (PLPTQPATLG)
=
(SEQ ID NO: 47).
The binding affinity of a 24P4C12 monoclonal antibody is determined using
standard technologies. Affinity
measurements quantify the strength of antibody to epitope binding and are used
to help define which 24P4C12 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
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monitor bimolecular interactions in real time. BlAcore analysis conveniently
generates association rate constants,
dissociation rate constants, equilibrium dissociation constants, and affinity
constants.
Example 12: HLA Class 1 and Class II Binding Assays
HLA class I and class ll binding assays using purified HLA molecules are
performed in accordance with disclosed
protocols (e.g., PCT publications WO 94120127 and WO 94/03205; Sidney et al.,
Current Protocols in Immunology 18.3.1
(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 125I-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 [labeINFILA] and ICso-4HLA1, the measured IC50
values are reasonable
approximations of the true Ko values. Peptide inhibitors are typically tested
at concentrations ranging from 120 Alml to 1.2
nglml, 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 IC50 of a positive control for
inhibition by the IC50 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 IC50 nM values by dividing the IC50 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 supermofif 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 24P4C12 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 II supermotifs or
motifs are performed as follows.
All translated 24P4C12 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:
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'AG"a1,xa2Jxa ............... x an;
where a; is a coefficient which represents the effect of the presence of a
given amino acid (j) 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 ji to
the free energy of binding of the peptide
irrespective of the sequence of the rest of the peptide.
The method of derivation of specific algorithm coefficients has been described
in Gulukota et al., J. Mot Biol.
267:1258-126, 1997; (see also Sidney etal., Human ImmunoL 45:79-93, 1996; and
Southwood etal., J. Immunot 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] is calculated relative to the remainder of the
group, and used as the estimate of Ji. 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 24P4C12 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 24P4C12 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 5_ 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 bearine epitopes
The 24P4C12 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 87-supertype allele (i.e., the prototype B7 supertype
allele). Peptides binding 6*0702 with ICso of 5_500
nM are identified using standard methods. These peptides are then tested for
binding to other common B7-supertype
molecules (e.g., B*3501, 8*5101, B*5301, and 8*5401). Peptides capable of
binding to three or more of the five B7-
supertype alleles tested are thereby identified.
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Selection of Al and A24 motif-bearing epitopes
To further increase population coverage, HLA-Al and -A24 epitopes can also be
incorporated into vaccine
compositions. An analysis of the 24P4C12 protein can also be performed to
identify HLA-Al- and A24-motif-containing
sequences.
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-
Iymphoblastoid 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%
(v/v) 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 pg/m1
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 RPM' to remove most of the non-
adherent and loosely adherent cells. Three ml of
complete medium containing 50 rigid of GM-CSF and 1,000 U/ml of IL-4 are then
added to each well. TNFa 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+ 1-cells (enough for a 48-well plate culture). Briefly, the PBMCs
are thawed in RPMI with 30pg/m1 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
100p1/m1 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/m1 of
peptide at a cell concentration of 1-2x106/m1 in the presence of 3pg/m1 82-
microglobulin for 4 hours at 20 C. The DC are
then irradiated (4,200 rads), washed 1 time with medium and counted again.
Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1x106
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 ng/ml and rhuman IL-2 is
added 48 hours later at 10
Restimulation of the induction cultures with pepfide-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
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with RPM! and DNAse. The cells are resuspended at 5x106 cells/ml 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 RPM' by
tapping the plate gently to remove the nonadherent cells and the adherent
cells pulsed with 10pg/m1 of peptide in the
presence of 3 pg/ml a2 microglobulin in 0.25mIRPM1/5%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 11.2 is added the next day and again 2-3 days later at 501U/m1 (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 61Cr 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 61Cr release.
Seven days after the second restimulation, cytotoxicity is determined in a
standard (5 hr) 61Cr release assay by
assaying individual wells at a single E:T. Peptide-pulsed targets are prepared
by incubating the cells with 10pg/m1 peptide
overnight at 37 C.
Adherent target cells are removed from culture flasks with trypsin-EDTA.
Target cells are labeled with 200pCi of
61Cr 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/m1 (an NK-sensitive
eryihroblastoma 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 p1 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 61Cr release sample)/(cpm of
the maximal 61Cr release sample-
cpm of the spontaneous Kr 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
lmmulon 2 plates are coated with mouse anti-human IFNy monoclonal antibody (4
1.19/m1 0.1M NaHCO3, pH8.2)
'overnight at 4 C. The plates are washed with Ca2+, Mg2.-free PBS/0.05 A)
Tween 20 and blocked with PBS/10% FCS for two
hours, after which the CTLs (100 l/well) and targets (100 p1/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 1x106 cells/ml. The plates are incubated for 48 hours at
37 C with 5% CO2.
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
p1 of biotinylated mouse anti-human IFN-
gamma monoclonal antibody (2 microgram/m1 in PBS/3%FCS/0.05 /0 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 microliterlwell 1M
H3PO4 and read at 0D450. A culture is considered positive if it measured at
least 50 pg of 1FN-gamma/well above
background and is twice the background level of expression.
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CTL Expansion.
Those cultures that demonstrate specific lytic activity against peptide-pulsed
targets andlor tumor targets are
expanded over a two week period with anti-CD3. Briefly, 5x104 CD8+ cells are
added to a 125 flask containing the following:
1x106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2x105
irradiated (8,000 rad) EBV- transformed cells per
ml, and 0KT3 (anti-CD3) at 3Ong 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/m1 and every three days
thereafter with fresh media at 501U/ml. The cells are
split if the cell concentration exceeds 1x106/m1 and the cultures are assayed
between days 13 and 15 at E:T ratios of 30, 10,
3 and 1:1 in the 5ICr release assay or at 1x1 06/m1 in the in situ 1FNy assay
using the same targets as before the expansion.
Cultures are expanded in the absence of anti-0O3+ 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
jig/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.
Immunopenicity of A2 supermotif-bearino 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.
lmmunogenicity can also be confirmed using PBMCs isolated from patients
bearing a tumor that expresses
24P4C12. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-
pulsed nnonocytes and assayed for the
ability to recognize peptide-pulsed target cells as well as transfected cells
endogenously expressing the antigen.
Evaluation of A*03/A11 immunooenicity
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 immunocienicity
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., FILA-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 by
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 FILA 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, I, V. or M
at position 2, and I or V at the C-terminus.
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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.
Alternatively, a peptide is confirmed as binding one or all supertype members
arid 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 IC50 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
etal., J. Immunol 157:2539, 1996; and Pogue et al.,
Proc. Natl. 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 1ILA-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 lc 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 B7-supertype alleles can be
= improved, where possible, to achieve increased cross-reactive binding or
greater binding affinity or binding half life. 87
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 et aL (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.
Analoqing 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 11$
= 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 1-ILA-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 24P4C12-
expressing tumors.
Other analoginq strategies
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Another form of peptide analoging, unrelated to anchor positions, involves the
substitution of a cysteine with a-
amino butyric acid. Due to its chemical nature, cysteine has the propensity to
form disulfide bridges and sufficiently alter the
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 etal., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John
Wiley St 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 24P4C12-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-bearinq epitopes.
To identify 24P4C12-derived, HLA class II HTL epitopes, a 24P4C12 antigen is
analyzed for the presence of
sequences bearing an HLA-DR-motif or supermotit 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 etal., J. Immunot
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 DR1, DR4w4, and
DR7, can efficiently select DR cross-reactive
peptides.
The 24P4C12-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 DR4w15, 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. 24P4C12-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 24P4C12 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 pilvi or better, i.e., less than
1 M. Peptides are found that meet this binding criterion and qualify as HLA
class ll high affinity binders.
DR3 binding epitopes identified in this manner are included in vaccine
compositions with DR supermotif-bearing
peptide epitopes.
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Similarly to the case of HLA dass I motif-bearing peptides, the class II motif-
bearing peptides are analoged to
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: Immunooenicitv_of 24P4C12-derived HTL epitopes
This example determines immunogenic DR supermotif- and DR3 motif-bearing
epitopes among those identified
using the methodology set forth herein.
lmmunogenicity 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 24P4C12-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=1-(SQRT(1-
af)) (see, e.g., Sidney etal., Human Immunot 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=1-(1-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., tota1=A+B*(1-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,
6*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.
lmmunogenicity studies in humans (e.g., Bertoni etal., J. Clin. Invest.
100:503, 1997; Doolan et at, Immunity 7:97,
1997; and Threlkeld etal., J. Immunot 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.
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With a sufficient number of epitopes (as disclosed herein and from the art),
an average population coverage is
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 24P4C12 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with
peptide epitope recognize
endogenously synthesized 24P4C12 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-A1 and A24)
are being developed. HLA-DR1 and FLA-
0R3 mouse models have also been developed, which may be used to evaluate HTL
epitopes.
Example 20: Activity Of CTL-HTL Coniugated Epitopes In Transgenic Mice
This example illustrates the induction of CTLs and HTLs in transgenic mice, by
use of a 24P4C12-derived CTL and
HTL peptide vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient
with a 24P4C12-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 CTUHTL 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)
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In vitto 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 mediumfT25 flask.
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 1 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., 5x106 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 24P4C12-
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 24P4C12
clearance. The number of epitopes used depends on observations of patients who
spontaneously clear 24P4C12. For
example, if it has been observed that patients who spontaneously clear 24P4C12-
expressing cells generate an immune
response to at least three (3) epitopes from 24P4C12 antigen, then at least
three epitopes should be included for HLA class
= I. A similar rationale is used to determine HLA class II epitopes.
Epitopes are often selected that have a binding affinity of an IC50 of 500 nM
or less for an HLA class I molecule, or
for class II, an IC50 of 1000 nM or less; or HLA Class I peptides with high
binding scores from the BIMAS web site, at URL
bimas.dcrt.nih.gov/.
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.
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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
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 24P4C12, 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
24P4C12.
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, -87 supermotif-bearing peptide epitopes and FILA-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 24P4C12, are selected such that multiple supermotifs/motifs
are represented to ensure broad population
coverage. Similarly, HLA class II epitopes are selected from 24P4C12 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 HA 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 1g-fight 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.
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Overlapping oligonucleotides that can, for example, average about 70
nucleotides in length with 15 nucleotide
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 1 min.
For example, a minigene is prepared as follows. For a first PCR reaction, 5
p.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 1 reactions containing Pfu polymerase buffer
(lx= 10 mM KCL, 10 mM (NH4)2SO4, 20
mM Iris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 Eig/mIBSA), 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 nudeic 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., Sips et al., J.
ImmunoL 156:683-692, 1996; Demotz etal., 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 etal., 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 of aL, Immunity 1:751-761, 1994.
For example, to confirm the capacity of a DNA minigene construct containing at
least one 1-ILA-A2 supermotif
peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for example, are
immunized intramuscularly with 100 pig 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 FILA-A2 supermotif peptide
epitopes as does the polyepitopic peptide vaccine. A similar analysis is also
performed using other HLA-A3 and FILA-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 1.1.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 etal. Immunity 1:751-761, 1994).
The results indicate the magnitude of the NIL
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 etal., Aids Res. and Human Retroviruses 14, Supplement 3:S299-
S309, 1998) or recombinant vaccinia, for
example, expressing a minigene or DNA encoding the complete protein of
interest (see, e.g., Hanke etal., Vaccine 16:439-
445, 1998; Sedegah et aL, Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke
and McMichael, Immunot Letters 66:177-
181, 1999; and Robinson etal., 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 jig
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 jig 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 1 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 24P4C12
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 24P4C12-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 IQ, generally 100-5,000
jig, 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 24P4C12-
associated disease.
Alternatively, a composition typically comprising transfecting agents is used
for the administration of a nucleic acid-
based vaccine in accordance with methodologies known in the art and disclosed
herein.
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Example 25: Polyepitopic Vaccine Compositions Derived from Native 24P4C12
Sequences
A native 24P4C12 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-met 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
24P4C12 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 24P4C12, 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 Antiqens
The 24P4C12 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 24P4C12 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide
that incorporates multiple epitopes from 24P4C12 as well as tumor-associated
antigens that are often expressed with a
target cancer associated with 24P4C12 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 24P4C12. Such an analysis can be performed in a manner
described by Ogg etal., Science
279:2103-2106, 1998. In this Example, peptides in accordance with the
invention are used as a reagent for diagnostic or
prognostic purposes, not as an immunogen.
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In this example highly sensitive human leukocyte antigen tetrameric complexes
("tetramers") are used for a cross-
sectional analysis of, for example, 24P4C12 HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at
different stages of disease or following immunization comprising a 24P4C12
peptide containing an A*0201 motif. Tetrameric
complexes are synthesized as described (Musey etal., N. EngL J. Med. 337:1267,
1997). Briefly, purified HLA heavy chain
(A*0201 in this example) and 132-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, p2-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 1 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 glof cold phosphate-buffered saline. Tr-color analysis
is performed with the tetramer-phycoerythrin,
along with anti-COB-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 24P4C12 epitope, and
thus the status of exposure to 24P4C12, 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 24P4C12-associated
disease or who have been vaccinated with a 24P4C12 vaccine.
For example, the class I restricted CTL response of persons who have been
vaccinated may be analyzed. The
vaccine may be any 24P4C12 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/m1), streptomycin (50
Ag/m1), and Hepes (10mM) containing 10%
heat-inactivated human AB serum (complete RPM') and plated using microculture
formats. A synthetic peptide comprising
an epitope of the invention is added at 10 jAg/mIto each well and HBV core 128-
140 epitope is added at 1 pg/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 til/well of complete RPMI. On days 3 and 10, 100 pl 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, et at., Nature Med.
2:1104,1108, 1996; Rehermann et aL, J. Clin. Invest. 97:1655-1665,1996; and
Rehermann etal. J. Clin. Invest. 98:1432-
1440, 1996).
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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, etal. J. Vim!. 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 pM, and labeled with 100 pCi of 51Cr (Amersham Corp.,
Arlington Heights, IL) for 1 hour after which they
are washed four times with HBSS.
Cytolyfic 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)/maximum 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 24P4C12 or a 24P4C12 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 cellslwell and are stimulated with 10
pg/m1 synthetic peptide of the invention, whole
24P4C12 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
10U/m1 IL-2. Two days later, 1 pCi 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 pg of peptide composition;
Group II: 3 subjects are injected with placebo and 6 subjects are injected
with 50 fig peptide composition;
Group III: 3 subjects are injected with placebo and 6 subjects are injected
with 500 pg 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
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.
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Example 30: Phase II Trials In Patients Expressing 24P4C12
Phase II trials are performed to study the effect of administering the CTL-HTL
peptide compositions to patients
having cancer that expresses 24P4C12. The main objectives of the trial are to
determine an effective dose and regimen for
inducing CTLs in cancer patients that express 24P4C12, 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
24P4C12.
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 24P4C12-
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 lmmunogenicity," 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 nudeic 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 p,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 24P4C12 is generated.
Example 32: Administration of Vaccine Compositions Using Dendritic Cells (DC)
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
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and HTL then destroy or facilitate destruction, respectively, of the target
cells that bear the 24P4C12 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 CTUHTL responses
Alternatively, ex vivo CTL or HTL responses to 24P4C12 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. 24P4C12. 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
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 24P4C12 to isolate peptides corresponding to 24P4C12
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.
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CA 02503346 2009-10-08
As appreciated by one in the art, one can perform a similar analysis on a cell
bearing more than one NIA 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: Complementary Polynucleotides
Sequences complementary to the 24P4C12-encoding sequences, or any parts
thereof, are used to detect,
decrease, or inhibit expression of naturally occurring 24P4C12. 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 24P4C12. 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 24P4C12-encoding transcript.
Example 35: Purification of Naturally-occurring or Recombinant 24P4C12 Using
24P4C12-Specific Antibodies
Naturally occurring or recombinant 24P4C12 is substantially purified by
immunoaffinity chromatography using
antibodies specific for 24P4C12. An immunoaffinity column is constructed by
covalently coupling anti-24P4C12 antibody to
an activated chromatographic resin, such as CNBr-activated SEPHAROSETM
(Amersham Pharmacia Biotech). After the
coupling, the resin is blocked and washed according to the manufacturer's
instructions.
Media containing 24P4C12 are passed over the immunoaffinity column, and the
column is washed under
conditions that allow the preferential absorbance of 24P4C12 (e.g., high ionic
strength buffers in the presence of detergent).
The column is eluted under conditions that disrupt antibody/24P4C12 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 24P4C12
24P4C12, or biologically active fragments thereof, are labeled with 12.1 1
Bolton-Hunter reagent. (See, e.g., Bolton
etal. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in
the wells of a multi-well plate are incubated
=
with the labeled 24P4C12, washed, and any wells with labeled 24P4C12 complex
are assayed. Data obtained using
different concentrations of 24P4C12 are used to calculate values for the
number, affinity, and association of 24P4C12 with
the candidate molecules.
Example 37: In Vivo Assay for 24P4C12 Tumor Growth Promotion
The effect of the 24P4C12 protein on tumor cell growth is evaluated in vivo by
evaluating tumor development and
growth of cells expressing or lacking 24P4C12. For example, SCID mice are
injected subcutaneously on each flank with 1 x
106 of either 3T3, prostate, colon, ovary, lung, or bladder cancer cell lines
(e.g. PC3, Caco, PA-1, CaLu or J82 cells)
containing tkNeo empty vector or 24P4C12. At least two strategies may be used:
(1) Constitutive 24P4C12 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 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, tetracycline, etc.,
provided such promoters are compatible with the host cell systems. Tumor
volume is then monitored by caliper
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CA 02503346 2010-10-27
measurement at the appearance of palpable tumors and followed over time to
determine if 24P4C12-expressing cells grow
at a faster rate and whether tumors produced by 24P4C12-expressing cells
demonstrate characteristics of altered
aggressiveness (e.g. enhanced metastasis, vascularization, reduced
responsiveness to chemotherapeutic drugs). As shown
in figure 31 and Figure 32, 24P4C12 has a profound effect on tumor growth in
SCID mice. The prostate cancer cells PC3
' and PC3-24P4C12 were injected subcutaneously in the right flank of SCID
mice. Tumor growth was evaluated by caliper
measurements. An increase in tumor growth was observed in PC3-24P4C12 tumors
within 47 days of injection (fig 31). In .
addition, subcutaneous injection of 313-24P4C12 induced tumor formation in
SCID mice (Figure 32). This finding is
-significant as control 3T3 cells fail to form tumors, indicating that 24P4C12
has several tumor enhancing capabilities,
including transformation, as well as tumor initiation and promotion.
=
Example 38: 24P4C12 Monoclonal Antibody-mediated inhibition of Prostate Tumors
In Vivo.
The significant expression of 24P4C12 in cancer tissues, together with its
restrictive expression in normal tissues
and cell surface localization, make 24P4C12 a good target for antibody
therapy. Similarly, 24P4C12 is a target for T cell-
based immunotherapy. Thus, the therapeutic efficacy of anti-24P4C12 mAbs in
human prostate cancer xenograft mouse
models is evaluated by using recombinant cell lines such as PC3-24P4C12, and
3T3-24P4C12 (see, e.g., Kaighn, ME., et
al., Invest Urol, 1979. 17(1): p. 16-23), as well as human prostate xenograft
models such as LAPC9 (Saffran at a Proc Nati
Aced Sci U S A. 2001,98:2658). Similarly, anti-24P4C12 mAbs are evaluated in
xenograft models of human bladder cancer
colon cancer, ovarian cancer or lung cancer using recombinant cell lines such
as J82-24P4C12, Caco-24P4C12, PA-
24P4C1 or CaLu-24P4C12, respectively.
Antibody efficacy on tumor growth.and metastasis formation is studied, e.g.,
in a mouse orthotopic bladder cancer
xenograft model, and a mouse prostate cancer xenograft model. The antibodies
can be unconjugated, as discussed in this
Example, or can be conjugated to a therapeutic modality, as appreciated in the
art. Anti-24P4C12 mAbs inhibit formation of
prostate and bladder xenografts. Anti-24P4C12 mAbs also retard the growth of
established orthotopic tumors and prolonged
survival of tumor-bearing mice. These results indicate the utility of anti-
24P4C12 mAbs in the treatment of local and
advanced stages of prostate, colon, ovarian, lung and bladder cancer.
Administration of the anti-24P4C12 mAbs led lo 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 24P4C12 as an attractive target for immunotherapy and
demonstrate the therapeutic potential of anti-24P4C12
mAbs for the treatment of local and metastatic cancer. This example
demonstrates that unconjugated 24P4C12 monoclonal
antibodies are'effective to inhibit the growth of human prostate, colon,
ovarian, lung and bladder cancer tumor xenografts
grown in SCID mice; accordingly a combination of such efficacious monoclonal
antibodies is also effective.
Tumor inhibition using multiple unconiugated 24P4C12 mAbs
Materials and Methods
=
24P4C12 Monoclonal Antibodies:
Monoclonal antibodies are raised against 24P4C12 as described in the Example
entitled "Generation of 24P4C12
Monoclonal Antibodies (mAbs)." The antibodies are characterized by EUSA,
Western blot, FACS, and immunoprecipitation
for their capacity to bind 24P4C12. Epitope mapping data for the anti-24P4C12
mAbs, as determined by ELISA and Western
analysis, recognize epitopes on the 24P4C12 protein. Immunohistochemical
analysis of prostate cancer tissues and cells
with these antibodies is performed.
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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 SCABER, J82, A498, 769P, Ca0v1 or PA1 tumor
xenografts.
Cell Lines
The prostate, colon, ovarian, lung and bladder cancer carcinoma cell linesõ
Caco, PA-1, CaLu or J82 cells as well
as the fibroblast line NIH 3T3 (American Type Culture Collection) are
maintained in media supplemented with L-glutamine
and 10% FBS.
PC3-24P4C12, Caco-24P4C12, PA-24P4012, CaLu-24P4C12 or J82-24P4C12 cells and
3T3-24P4C12 cell
populations are generated by retroviral gene transfer as described in Hubert,
R.S., et al., Proc Watt Acad Sci U S A, 1999.
96(25): 14523.
Xenooraft Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 1 x 10 6 cancer 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. Tumor
sizes are determined by 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.
Orthotopic injections are performed under anesthesia by using
ketamine/xylazine. For bladder orthotopic studies,
an incision is made through the abdomen to expose the bladder, and tumor cells
(5 x 105) mixed with Matrigel are injected
into the bladder wall in a 10-pt volume. To monitor tumor growth, mice are
palpated and blood is collected on a weekly basis
to measure BTA levels. For prostate orthopotic models, an incision is made
through the abdominal musdes to expose the
bladder and seminal vesicles, which then are delivered through the incision to
expose the dorsal prostate. Tumor cells e.g.
LAPC-9 cells (5 x 105) mixed with Matrigel are injected into the prostate in a
10-pt volume (Yoshida Yet al, Anticancer Res.
1998, 18:327; Ahn et al, Tumour Biol. 2001, 22:146). To monitor tumor growth,
blood is collected on a weekly basis
measuring PSA levels. Similar procedures are followed for lung and ovarian
xenograft models. The mice are segregated
into groups for the appropriate treatments, with anti-24P4C12 or control mAbs
being injected i.p.
Anti-24P4C12 mAbs Inhibit Growth of 24P4C12-Expressing Xenograft-Cancer Tumors
The effect of anti-24P4C12 mAbs on tumor formation is tested on the growth and
progression of bladder, and
prostate cancer xenografts using PC3-24P4C12, Caco-24P4C12, PA-24P4C12, CaLu-
24P4C12 or J82-24P4C12 orthotopic
models. As compared with the s.c. tumor model, the orthotopic model, which
requires injection of tumor cells directly in the
mouse prostate, colon, ovari, lung and bladder, 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., at at., 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 organs, and 2 days later,
the mice are segregated into two
groups and treated with either: a) 200-500pg, of anti-24P4C12 Ab, orb) PBS
three times per week for two to five weeks.
A major advantage of the orthotopic cancer models is the ability to study the
development of metastases.
Formation of metastasis in mice bearing established orthotopic tumors is
studies by 1HC analysis on lung sections using an
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antibody against a tumor-specific cell-surface protein such as anti-CK20 for
bladder cancer, anti-STEAP-1 for prostate
cancer models (Lin Set al, Cancer Detect Prey. 200125:202; Saffran, D., et
al., PNAS supra).
Mice bearing established orthotopic tumors are administered 1000pg injections
of either anti-24P4C12 mAb or
PBS over a 4-week period. Mice in both groups are allowed to establish a high
tumor burden, to ensure a high frequency of
metastasis formation in mouse lungs. Mice then are killed and their bladders,
livers, bone and lungs are analyzed for the
presence of tumor cells by IHC analysis.
These studies demonstrate a broad anti-tumor efficacy of anti-24P4C12
antibodies on initiation and progression of
prostate and kidney cancer in xenograft mouse models. Anti-24P4C12 antibodies
inhibit tumor formation of tumors as well
as retarding the growth of already established tumors and prolong the survival
of treated mice. Moreover, anti-24P4C12
mAbs demonstrate a dramatic inhibitory effect on the spread of local bladder
and prostate tumor to distal sites, even in the
presence of a large tumor burden. Thus, anti-24P4C12 mAbs are efficacious on
major clinically relevant end points (tumor
growth), prolongation of survival, and health.
Example 39: Therapeutic and Diagnostic use of Anti-24P4C12 Antibodies in
Humans.
Anti-24P4C12 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-24P4C12 mAb show strong extensive staining in carcinoma but
significantly lower or undetectable levels in normal
tissues. Detection of 24P4C12 in carcinoma and in metastatic disease
demonstrates the usefulness of the mAb as a
diagnostic and/or prognostic indicator. Anti-24P4C12 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-24P4C12 mAb specifically binds to
carcinoma cells. Thus, anti-24P4C12
antibodies are used in diagnostic whole body imaging applications, such as
radioimmunoscintigraphy and
radioimnnunotherapy, (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 24P4C12. Shedding or release
of an extracellular domain of 24P4C12 into
the extracellular milieu, such as that seen for alkaline phosphodiesterase B10
(Meerson, N. R, Hepatology 27:563-568
(1998)), allows diagnostic detection of 24P4C12 by anti-24P4C12 antibodies in
serum and/or urine samples from suspect
patients.
Anti-24P4C12 antibodies that specifically bind 24P4C12 are used in therapeutic
applications for the treatment of
cancers that express 24P4C12. Anti-24P4C12 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-24P4C12 antibodies are
tested for efficacy of tumor prevention and growth inhibition in the SCID
mouse cancer xenograft models, e.g., kidney cancer
models AGS-K3 and AGS-K6, (see, e.g., the Example entitled "24P4C12 Monoclonal
Antibody-mediated Inhibition of
Bladder and Lung Tumors In Vivo"). Either conjugated and unconjugated anti-
24P4C12 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-24P4C12 Antibodies In vivo
Antibodies are used in accordance with the present invention which recognize
an epitope on 24P4C12, and are
used in the treatment of certain tumors such as those listed in Table I. Based
upon a number of factors, including 24P4C12
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.
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I.) Adjunctive therapy: In adjunctive therapy, patients are treated with
anti-24P4C12 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-24P4C12 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-24P4C12
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 (predinical).
II.) Monotherapy: In connection with the use of the anti-24P4C12 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-24P4C12
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 24P4C12. In connection with the
use of the anti-24P4C12 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 (111In)-24P4C12 antibody is used as an imaging agent in a
Phase I human clinical trial in patients
having a carcinoma that expresses 24P4C12 (by analogy see, e.g., Divgi et at.
J. Natl. Cancer Ind 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-24P4C12
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-24P4C12
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-24P4C12 antibodies that are
fully human antibodies, as compared to
the chimeric antibody, have slower clearance; accordingly, dosing in patients
with such fully human anti-24P4C12 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.
Three distinct delivery approaches are useful for delivery of anti-24P4C12
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 (CDPI
Overview: The COP follows and develops treatments of anti-24P4C12 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-24P4C12 antibodies. As will
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be appreciated, one criteria that can be utilized in connection with
enrollment of patients is 24P4C12 expression levels in
their tumors as determined by biopsy. =
As with any protein or antibody infusion-based therapeutic, safety concerns
are related primarily to (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 antibody
therapeutic, or HAHA response); and, (iii) toxicity to
normal cells that express 24P4C12. Standard tests and follow-up are utilized
to monitor each of these safety concerns.
Anti-24P4C12 antibodies are found to be safe upon human administration.
Example 41: Human Clinical Trial Adjunctive Therapy with Human Anti-24P4C12
Antibody and Chemotherapeutic
Agent
A phase I human clinical trial is initiated to assess the safety of six
intravenous doses of a human anti-24P4C12
= 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-24P4C12 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 includee delivery of six single doses of an anti-
24P4C12 antibody with dosage of antibody
escalating from approximately about 25 mg/m 2to about 275 mg/m 2over 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 24P4C12. 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.
The anti-24P4C12 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-24P4C12 Antibody
Anti-24P4C12 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-24P4C12
antibodies.
Example 43: Human Clinical Trial: Diagnostic Imaging with Anti-24P4C12
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-24P4C12 antibodies as a
diagnostic imaging agent. The protocol is
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designed in a substantially similar manner to those described in the art, such
as in Divgi etal. 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: Homology Comparison of 24P4C12 to Known Sequences
The 24P4C12 protein of Figure 3 has 710 amino acids with calculated molecular
weight of 79.3 kDa, and pl of 8.9.
Several variants of 24P4C12 have been identified, including 4 SNPs (namely
v.1, v.3, v.5, v.6) and 3 splice variants (namely
v.7, v.8 and v.9) (figures 10 and 11). 24P4C12 variants v.3, v.5, and v.6
differ from 24P4C12 v.1 by 1 amino acid each, at aa
positions 187, 326 and 436, respectively. Variant v.7 carries a deletion of
111 aa long starting at aa 237, while variant v.8
and v.9 contain insertions at aa 642 and 378, respectively. The 24P4C12
protein exhibits homology to a previously cloned
human gene, namely NG22 also known as chorine transporter-like protein 4 (gi
14249468). It shows 99% identity and 99%
homology to the CTL4 protein over the length of that protein (Figure 4).
24P4C12 is a multi-transmembrane protein,
predicted to carry 10, 11 or 13 transmembrane domains. Bioinformatic analysis
indicates that the 24P4C12 protein localizes
to the plasma membrane with some endoplasmic reticulum localization (see Table
L). Recent evidence indicates that the
24P4C12 protein is a 10 transmembrane protein that localizes to the cell
surface (O'Regan Set al PNAS 2000, 97:1835).
Choline as an essential component of cell membranes that plays an important
role in cell integrity, growth and survival
of normal and tumor cells. Choline accumulates at increased concentration in
tumor cells relative to their normal
counterparts and as such constitutes a tool for the detection of cancer cells
by magnetic resonance imaging (Kurhanewicz J
et at, J Magn Reson Imaging. 2002.). In addition to its role in maintaining
membrane integrity, choline mediates signal
transduction event from the membrane to the nucleus (Spiegel S, Milsfien S. J
Membr Biol. 1995, 146:225). Choline
metabolites include sphingosylphosphorylcholine and lysophosphatidylcholine,
both of which activate G-protein coupled
receptors (Xu F et al Biochim Biophys Acta 2002, 1582:81). In addition,
choline results in the activation of kinase pathways
including Raf-1 (Lee M, Han SS, Cell Signal 2002, 14:373.). Choline also plays
a role in regulating DNA methylation and
. regulation of gene expression. For example, choline methabolites regulate
the expression of cytokines and chemokines
essential for tumor growth (Schwartz BM et al, Gynecol Oncol. 2001, 81:291;
Denda A et al, Carcinogenesis. 2002, 23:245).
Due to its effect on cell signaling and gene expression, choline controls cell
growth and survival (Holmes-McNary MQet al, J
= Biol Chem. 2001, 276:41197; Albright et al, FASEB 1996, 10:510). Choline
deficiency results in cell death, apoptosis and
transformation, while accumulation of choline is associated with tumor growth
(Zeisel Set al, Carcinogenesis 1997, 18:731).
Accordingly, when 24P4C12 functions as a regulator of tumor formation, cell
proliferation, invasion or cell signaling,
24P4C12 is used for therapeutic, diagnostic, prognostic and/or preventative
purposes.
Example 45: Identification and Confirmation of Potential Signal Transduction
Pathways
Many mammalian proteins have been reported to interact with signaling
molecules and to participate in regulating
signaling pathways. (J Neurochem. 2001; 76:217-223). In particular, choline
have been reported to activate MAK cascades
as well as G proteins, and been associated with the DAG and ceramide and
sphingophosphorylcholine signaling pathway
(Cummings et al, above). In addition, choline transmit its signals by
regulating choline-kinase and phospholipase activity,
'resulting in enhance tumorigenic effect (Ramirez et al, Oncogene. 2002,
21:4317; Lucas et al, Oncogene. 2001, 20:1110;
Chung T et al, Cell Signal. 2000, 12:279).
Using immunoprecipitation and Western blotting techniques, proteins are
identified that associate with 24P4C12
and mediate signaling events. Several pathways known to play a role in cancer
biology can be regulated by 24P4C12,
including phospholipid pathways such as P13 K, AKT, etc, adhesion and
migration pathways, including FAK, Rho, Rac-1, etc,
as well. as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth
Differ. 2000,11:279; J Biol Chem. 1999,
274:801; Oncogene. 2000, 19:3003; J. Cell Biol, 1997, 138:913). Using Western
blotting and other techniques, the ability of
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24P4C12 to regulate these pathways is confirmed. Cells expressing or lacking
24P4C12 are either left untreated or
stimulated with cytokines, androgen and anti-integrin antibodies. Cell lysates
are analyzed using anti-phospho-spedfic
antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect
phosphorylation and regulation of ERK, p38, AKT,
P13K, PLC and other signaling molecules.
To confirm that 24P4C12 directly or indirectly activates known signal
transduction pathways in cells, luciferase (luc)
based transcriptional reporter assays are carried out in cells expressing
individual genes. These transcriptional reporters
contain consensus-binding sites for known transcription factors that lie
downstream of well-characterized signal transduction
pathways. The reporters and examples of these associated transcription
factors, signal transduction pathways, and
activation stimuli are listed below.
1. NFkB-luc, NFkB/Rel; ik-kinaselSAPK; growthlapoptosis/stress
2. SRE-luc, SRFITCF/ELK1; MAPIUSAPK; growth/differentiation
3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress
4. ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
7. TCF-Iuc, TCF/Lef; 0-catenin, Adhesion/invasion
Gene-mediated effects can be assayed in cells showing mRNA expression.
Luciferase reporter plasmids can be
introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase
activity, an indicator of relative transcriptional
activity, is measured by incubation of cell extracts with luciferin substrate
and luminescence of the reaction is monitored in a
luminometer.
Signaling pathways activated by 24P4C12 are mapped and used for the
identification and validation of therapeutic
targets. When 24P4C12 is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and/or
therapeutic purposes.
Example 46: 24P4C12 Functions as a Choline transporter
Sequence and homology analysis of 24P4C12 indicate that 24P4C12 carries a
transport domain and that 24P4C12
functions as a choline transporter. In order to confirm that 24P4C12
transports choline, primary and tumor cells, includeing
prostate, colon, bladder and lung lines, are grown in the presence and absence
of 3H-choline. Radioactive choline uptake is
measured by counting incorporated counts per minutes (cpm). Parental 24P4C12
negative cells are compared to 24P4C12-
expressing cells using this and similar assays. Similarly, parental and
24P4C12-expressing cells can be compared for
choline content using NMR spectroscopy. These assay systems can be used to
identify small molecules and antibodies that
interfere with choline uptake and/or with the function of 24P4C12.
Thus, compounds and small molecules designed to inhibit 24P4C12 function and
downstream signaling events are
used for therapeutic diagnostic, prognostic and/or preventative purposes.
Example 47: Regulation of Transcription
The cell surface localization of 24P4C12 and its ability to regulate DNA
methylation indicate that it is effectively
used as a modulator of the transcriptional regulation of eukaryotic genes.
Regulation of gene expression is confirmed, e.g.,
by studying gene expression in cells expressing or lacking 24P4C12. For this
purpose, two types of experiments are
performed.
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In the first set of experiments, RNA from parental and 24P4C12-expressing
cells are extracted and hybridized to
commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J
Cancer. 2000. 83:246). Resting cells as well as
cells treated with FBS, pheromones, or growth factors are compared.
Differentially expressed genes are identified in
accordance with procedures known in the art. The differentially expressed
genes are then mapped to biological pathways
(Chen K et al. Thyroid. 2001. 11:41.).
In the second set of experiments, specific transcriptional pathway activation
is evaluated using commercially
available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-
luc, ELK1-luc, ARE-Iuc, p53-luc, and CRE-Iuc.
These transcriptional reporters contain consensus binding sites for known
transcription factors that lie downstream of well-
characterized signal transduction pathways, and represent a good tool to
ascertain pathway activation and screen for
positive and negative modulators of pathway activation.
Thus, 24P4C12 plays a role in gene regulation, and it is used as a target for
diagnostic, prognostic, preventative
and/or therapeutic purposes.
Example 48: Involvement in Tumor Progression
The 24P4C12 gene can contribute to the growth of cancer cells. The role of
24P4C12 in tumor growth is confirmed
in a variety of primary and transfected cell lines including prostate, and
bladder cell lines, as well as NIFI 313 cells
engineered to stably express 24P4C12. Parental cells lacking 24P4C12 and cells
expressing 24P4C12 are evaluated for cell
growth using a well-documented proliferation assay (Fraser SP, et at.,
Prostate 2000;44:61, Johnson DE, Ochieng J, Evans
SL. Anticancer Drugs. 1996, 7:288). Such a study was performed on prostate
cancer cells and the results are shown in
figure 28. The growth of parental PC3 and PC3-24P4C12 cells was compared in
low (0.1%) and 10% FBS. Expression of
24P4C12 imparted a growth advantage to PC3 cells grown in 10% FBS. Similarly,
expression of 24P4C12 in NIH-3T3 cells
enhances the proliferation of these cells relative to control 3T3-neo cells.
The effect of 24P4C12 can also be observed on
cell cycle progression. Control and 24P4C12-expressing cells are grown in low
serum overnight, and treated with 10% FBS
for 48 and 72 hrs. Cells are analyzed for BrdU and propidium iodide
incorporation by FACS analysis.
To confirm the role of 24P4C12 in the transformation process, its effect in
colony forming assays is investigated.
Parental NIH-3T3 cells lacking 24P4C12 are compared to N11-1-3T3 cells
expressing 24P4C12, using a soft agar assay under
stringent and more permissive conditions (Song Z. et al. Cancer Res.
2000;60:6730).
To confirm the role of 24P4C12 in invasion and metastasis of cancer cells, a
well-established assay is used. A
non-limiting example is the use of an assay which provides a basement membrane
or an analog thereof used to detect
whether cells are invasive (e.g., a Transwell Insert System assay (Becton
Dickinson) (Cancer Res. 1999; 59:6010)). Control
cells, including prostate, and bladder cell lines lacking 24P4C12 are compared
to cells expressing 24P4C12. Cells are
loaded with the fluorescent dye, calcein, and plated in the top well of a
support structure coated with a basement membrane
analog (e.g. the Transwell insert) and used in the assay. Invasion is
determined by fluorescence of cells in the lower
chamber relative to the fluorescence of the entire cell population.
24P4C12 can also play a role in cell cycle and apopto-sis. Parental cells and
cells expressing 24P4C12 are
compared for differences in cell cycle regulation using a well-established
BrdU assay (Abdel-Malek ZA. J Cell Physiol.
1988, 136:247). In short, cells are grown under both optimal (full serum) and
limiting (low serum) conditions are labeled with
BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed
for entry into the G1, S, and G2M phases of
the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated
in control parental cells and cells expressing
24P4C12, including normal and tumor prostate, colon and lung cells. Engineered
and parental cells are treated with various
chemotherapeutic agents, such as etoposide, flutamide, etc, and protein
synthesis inhibitors, such as cycloheximide. Cells
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are stained with annexin V-FITC and cell death is measured by FACS analysis.
The modulation of cell death by 24P4C12
can play a critical role in regulating tumor progression and tumor load.
When 24P4C12 plays a role in cell growth, transformation, invasion or
apoptosis, it is used as a target for
diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 49: Involvement in Angiogenesis
Angiogenesis or new capillary blood vessel formation is necessary for tumor
growth (Hanahan D, Folkman J. Cell.
1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effect of
phsophodieseterase inhibitors on
endothelial cells, 24P4C12 plays a role in angiogenesis (DeFouw L et al,
Microvasc Res 2001, 62:263). Several assays
have been developed to measure angiogenesis in vitro and in vivo, such as the
tissue culture assays endothelial cell tube
formation and endothelial cell proliferation. Using these assays as well as in
vitro neo-vascularization, the role of 24P4C12
in angiogenesis, enhancement or inhibition, is confirmed.
For example, endothelial cells engineered to express 24P4C12 are evaluated
ising tube formation and
=
proliferation assays. The effect of 24P4C12 is also confirmed in animal models
in vivo. For example, cells either expressing
or lacking 24P4C12 are implanted subcutaneously in immunocompromised mice.
Endothelial cell migration and
angiogenesis are evaluated 5-15 days later using immunohistochemistry
techniques. 24P4C12 affects angiogenesis and it is
used as a target for diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 50: Involvement in Adhesion
Cell adhesion plays a critical role in tissue colonization and metastasis. The
presence of leucine rich and cysteine
rich motifs in 24P4C12 is indicative of its role in cell adhesion. To confirm
that 24P4C12 plays a role in cell adhesion, control
cells lacking 24P4C12 are compared to cells expressing 24P4C12, using
techniques previously described (see, e.g., Haler et
al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst.
1998,90:118). Briefly, in one embodiment, cells
labeled with a fluorescent indicator, such as calcein, are incubated on tissue
culture wells coated with media alone or with
matrix proteins. Adherent cells are detected by fluorimetric analysis and
percent adhesion is calculated. This experimental
system can be used to identify proteins, antibodies and/or small molecules
that modulate cell adhesion to extracellular matrix
and cell-cell interaction. Since cell adhesion plays a critical role in tumor
growth, progression, and, colonization, the gene
involved in this process can serves as a diagnostic, preventative and
therapeutic modality.
Example 51: Detection of 24P4C12 protein in cancer patient specimens
To determine the expression of 24P4C12 protein, specimens were obtained from
various cancer patients and
stained using an affinity purified polyclonal rabbit antibody raised against
the peptide encoding amino acids 1-14 of 24P4C12
variant 1 and conjugated to KLH (See, Example 10: Generation of 24P4C12
Polyclonal Antibodies.) This antiserum
exhibited a high titer to the peptide (>10,000) and recognized 24P4C12 in
transfected 2931 cells by Western blot and flow
cytometry (Figure 24) and in stable recombinant PC3 cells by Western blot and
immunohistochemistry (Figure 25). Formalin
fixed, paraffin embedded tissues were cut into 4 micron sections and mounted
on glass slides. The sections were dewaxed,
rehydrated and treated with antigen retrieval solution (0.1M Iris, pH10) at
high temperature. Sections were then incubated in
polydonal rabbit anti-24P4C12 antibody for 3 hours. The slides were washed
three times in buffer and further incubated with
DAKO EnVision+Tm peroxidase-conjugated goat anti-rabbit immunoglobulin
secondary antibody (DAKO Corporation,
Carpenteria, CA) for 1 hour. The sections were then washed in buffer,
developed using the DAB kit (SIGMA Chemicals),
counterstained using hematoxylin, and analyzed by bright field microscopy. The
results showed expression of 24P4C12 in
cancer patients' tissue (Figures 29 and 30). Tissue from prostate cancer
patients showed expression of 24P4C12 in the
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tumor cells and in the prostate epithelium of tissue normal adjacent to tumor
(Figure 29). Generally, expression of 24P4C12
was high in all prostate tumors and was expressed mainly around the cell
membrane indicating that 24P4C12 is membrane
associated in prostate tissues. All of the prostate samples tested were
positive for 24P4C12. Other tumors that were positive
for 24P4C12 included colon adenocarcinoma, breast ductal carcinoma, pancreatic
adenocarcinoma, lung adenocarcinoma,
bladder transitional cell carcinoma and renal clear cell carcinoma (Figure
30). Normal tissues investigated for expression of
24P4C12 included heart, skeletal muscle, liver, brain, spinal cord, skin,
adrenal, lymph node, spleen, salivary gland, small
intestine and placenta. None demonstrated any expression of 24P4C12 by
immunohistochemistry. Normal adjacent to tumor
tissues were also studied to determine the presence of 24P4C12 protein by
immunohistochemistry. These included breast,
- lung, colon, ileum, bladder, kidney and pancreas. In some of the tissues
from these organs there was weak expression of
24P4C12. This expression may relate to the fact that the samples were not
truly normal and may indicate a precancerous
change. The ability to identify malignancy in tissue that has not undergone
obvious morphological changes is an important
diagnostic modality for cancerous and precancerous conditions.
These results indicate that 24P4C12 is a target for diagnostic, prophylactic,
prognostic and therapeutic applications in
cancer.
Throughout this application, varjous 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 24P4C12:
a. Malignant Tissues
Prostate
Bladder
Kidney
Lung
Colon
Ovary
Breast
Uterus
Stomach
TABLE II: Amino Acid Abbreviations
SINGLE LETTER THREE LETTER FULL NAME
Phe phenylalanine
Leu leucine
Ser serine
Tyr tyrosine
=
Cys cysteine
Trp tryptophan
Pro proline
His histidine
Gin glutamine
kg arginine
Ile isoleucine
Met methionine
Thr threonine
Asn asparagine
Lys lysine
V Val valine
A Ala alanine
Asp aspartic acid
Glu glutamic acid
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
protein&
ACDEFGHIKLMNPQRSTVWY.
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
5 -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 E
6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3G
8 -3 -1 -3 -2 1-2 0 0 -1 -2 -3 -2 2H
4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3-1 1
-2 -1 0-1 1 2 0 -1 -2 -3 -2 F
4 2 -3 -3 -2 -2 -2 -1 1-2 -1L
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 -3P
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 2W
7Y
=
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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 TIL VMS FWY
A2 LIVMATQ IVMA TL
A3 VSMATL/ RK
A24 YFWIVLMT FIYWLM
B7 P VILFMWYA
B27 RHK FYLWMIVA
B44 ED FVVYLIMVA
B58 ATS FVVYLIVMA
B62 QLIVMP FVVYMIVLA
MOTIFS
Al TSM
Al DEAS
A2.1 LMVOAT VLIMAT
A3 LMVISATFCGD KYRHFA
All VTMLISAGNCDF KRYH
A24 YFWM FLIW
A*3101 MVTALIS RK
A*3301 MVALFIST RK
A*6801 AVTMSLI RK
B*0702 P LMFWYAIV
B*3501 P LMFWVIVA
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, .1, 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 10 anchor 1 2 3 4 5 1 anchor 6 7 8
9
1DR4 preferred FMYLIVW M T I VSTCPAL1M MH
deleterious W R WDE
DR1 preferred MFL1VWY PAMQ VMATSPLIC M AVM
deleterious C CH FD CWD ODE D
DR7 preferred MFL/VWY M W A IVMSACTPL M IV
deleterious C 0 GRD N G
DR3 MOTIFS 1 anchor 1 2 3 10 anchor 4 5 10 anchor 6
Motif a preferred LIVMFY
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 10 Anchor 10 Anchor
TILVMS FWY
A2 1 Anchor 10 Anchor
LIVMATQ LIVMAT
A3 Preferred 10 Anchor YFW YEW YEW P 10 Anchor
VSMATLI (415) (3/5) (4/5) (4/5) RK
deleterious DE (3/5); DE
P (5/5) (4/5)
A24 10 Anchor 1 Anchor
YFW/VLMT FIYWLM
B7 Preferred FWY (5/5) 10 Anchor FWY FVVY 1 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) (4/5) (4/5)
G(4/5);
A(3/5);
QN(3/5)
B27 10 Anchor 1 Anchor
RHK FYLWMIVA
844 1 Anchor 1 Anchor
ED FVVYLIMVA
B58 10 Anchor 10 Anchor
ATS FWYLIVMA
B62 10 Anchor 10 Anchor
QLJVMP FlNYMIVLA
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 1 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
Al preferred YFW 1 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
1 Anchor
10- DEAS Y
mer
deleterious RHK RHKDEPYFW P G PRHK ON
A2.1 preferred YFW 1 Anchor YFW STC YFW A P 1
Anchor
9-mer LMIVOAT VLIMAT
deleterious DEP DERKH RKH DERKH
POSITION:1 2 3 4 5 6 7 8 9 C-
Terminus
A2.1 preferred AYFW 1 Anchor LVIM G G FYVVL 1
Anchor
10- LMIVQAT VIM VLIMAT
mer
deleterious DEP DE RKHA P RKH DERK RKH
H
A3 preferred RHK 1 Anchor YFW PRHKYF A YFW P 1 Anchor
LMVISATFCGD W KYRHFA
deleterious DEP DE
All preferred A 1 Anchor YFW YFW A YFW YFW P l'Anchor
VTLMISAGNCD KRYH
F
deleterious DEP A G
A24 preferred YFWRHK 1 Anchor SIC YFW YFW 1 Anchor
9-mer YFWM FLIW
deleterious DEG DE G QNP DERH G AQN
K
A24 Preferred 1 Anchor P YFWP P 1 Anchor
10- YFWM FLIW
mer '
Deleterious GDE ON RHK DE A QN DEA
A310 Preferred RHK 1 Anchor YEW P YFW YFW AP
l'Anchor
1 MVTALIS RK
Deleterious DEP DE ADE DE DE DE
A330 Preferred 1 Anchor YFW AYFW l*Anchor
1 MVALFIST RK
Deleterious GP DE
A680 Preferred YFWSTC 1 Anchor YFWLIV YFW P 1 Anchor
1 AVTMSLI M RK
deleterious GP DEG RHK .A
6070 Preferred RHKFVVY 1 Anchor RHK RHK RHK RHK . PA 1
Anchor
2 P LMFWYA/
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 1 Anchor DEA YFW P DEQN YFW 1 Anchor
9-met STM
deleterious DE RHKLIVMP A G A
Al preferred GRHK ASTCLIVM 1 Anchor GSTC ASTC LIVM DE
1 Anchor
9-mer DEAS
deleterious A RHKDEPYFW DE PQN RHK PG GP
B350 Preferred FWYLIVM 1 Anchor FWY FWY 1 Anchor
1 P LMFVVYIV
A
deleterious AGP G G
B51 Preferred LIVMFWY 1 Anchor FWY SIC FWY G FWY 1 Anchor
LIVFWYA
deleterious AGPDER DE G DEQN GDE
HKSTC
B530 preferred LIVMFWY ?Anchor FWY SIC FWY LIVMFW FWY 1 Anchor
1 p Y IMFVVYAL
V
deleterious AGPQN G RHKQN DE
B540 preferred FWY 1 Anchor FVVYLIVM LIVM ALIVM FVVYA ?Anchor
1 P P All VLMF
WY
deleterious GPQNDE GDESTC RHKDE DE QNDGE DE
130
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TABLE IV (F):
Summary of HLA-supepes
Overall phenotypic frequencies of HLA-supertypes in different ethnic
populations
Specificity Phenoty I ic frequency
SupertypePosition 2 C-TerminusCaucasian N.A.
BlacktapaneseChineseHispanicAverage
87 P AILMVFVVY43.2 55.1 -57.1
43.0 49.3 49.5
A3 AILMVST RK 37.5 42.1 45.8 -52.7 43.1
44.2
A2 AILMVT AILMVT 45.8 39.0 -
42.4 45.9 43.0 -42.2
A24 YF (WIVLMT)F1(YWLM) 23.9 38.9 58.6 40.1 38.3 -40.0
B44 E (D) FVVYLIMVA43.0 21.2 42.9 39.1 -39.0 37.0
Al TI (LVMS) 'FVVY 47.1 16.1 21.8 14.7 26.3 25.2
B27 RHK FYL (WM1) 28.4 26.1 13.3 13.9 35.3 23.4
862 _QL (IVMP) VVY (MIV) 12.6 4.8 36.5 25,4 11.1 18.1
B58 ATS FVVY (LIV) 10.0 25.1 1.6 9.0 5.9 -10.3
TABLE IV (G):
Calculated population coverage afforded by different HLA-supertype
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
A2, A3 and 137 99,5 98.1 100.0 99.5 99.4 99.3
A2, A3, 87, A24,134499.9 99.6 100.0 99.8 99.9 99.8
and Al
A2, A3, B7, A24,
B44, Al, B27,1362,
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.
Table V: Frequently Occurring Motifs
avrg. %
Name Description Potential Function
identity
Nudeic acid-binding protein functions as
transcription factor, nuclear location
zf-C2H2 34% Zinc finger, C2H2 type probable
Cytochrome b(N- membrane bound oxidase, generate
cytochrome_b_N 268% terminal)/b6/petB superoxide
domains are one hundred amino acids
long and include a conserved
Ig 10 lmmunoglobulin 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 repeatprotein interaction
may function in targeting signaling
PDZ a23% PDZ domain molecules to sub-membranous
sites
LRR 28% Leucine Rich Repeat short sequence motifs
involved in
protein-protein interactions
conserved catalytic core common to
both serinelthreonine and tyrosine
protein kinases containing an ATP
Pkinase 23% Protein kinase domain binding site and a
catalytic site
131
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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 49% _polymerasel
Cytoplasmic protein, associates integral
Ank 25% eknk repeat membrane proteins to the
cytoskeleton
NADH- membrane associated. Involved in
Ubiquinonelplastoquinone proton translocation across the
Oxidored_q1 32% (complex I), various chains membrane
calcium-binding domain, consists of al 2
residue loop flanked on both sides by a
Efhand 24% EF hand 12 residue alpha-helical domain
Retroviral aspartyl Aspartyl or acid proteases,
centered on
Rvp 79% protease a catalytic aspartyl 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 42% (20 copies) polypeptide 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 domainponds
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: Motifs and Post-translational Modifications of 24P4C12
N-glycosylation site
29 - 32 NRSC (SEQ ID NO: 48)
69 - 72 NSTG (SEQ ID NO: 49)
155 - 158 NMTV (SEQ ID NO: 50)
197 - 200 NDTT (SEQ ID NO: 51)
298- 301 NLSA (SEQ ID NO: 52)
393- 396 NISS (SEQ ID NO: 53)
405 - 408 NTSC (SEQ ID NO: 54)
416 - 419 NSSC (SEQ ID NO: 55)
678 - 681 NGSL (SEQ ID NO: 56)
Protein kinase C phosphorylation site
22 - 24 SfR
218 - 220 SyK
430 - 432 SsK
494 - 496 TIR
573 - 575 SaK
619 - 621 SgR
Casein kinase II phosphorylation site
31 - 34 SCTD (SEQ ID NO: 57)
102- 105 SVAE (SEQ ID NO: 58)
119- 122 SCPE (SEQ ID NO: 59)
135- 138 TVGE (SEQ ID NO: 60)
304 - 307 SVQE (SEQ ID NO: 61)
132
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Tyrosine kinase phosphorylation site
6. 13 RDEDDEAY (SEQ ID NO: 62)
N-myristoylation site
72 - 77 GAYCGM (SEQ ID NO: 63)
76 - 81 GMGENK (SEQ ID NO: 64)
151 - 156 GVPWNM (SEQ ID NO: 65)
207 -212 GLIDSL (SEQ ID NO: 66)
272 - 277 GIYYCW (SEQ ID NO: 67)
287 - 292 GASISQ (SEQ ID NO: 68)
349- 354 GQMMST (SEQ ID NO: 69)
449 - 454 GLFWTL (SEQ ID NO: 70)
467 - 472 GAFASF (SEQ ID NO: 71)
Amidation site
695- 698 IGKK (SEQ ID NO: 72)
Leucine zipper pattern
245 - 266 LFILLLRLVAGPLVLVLILGVL (SEQ ID NO: 73)
Cysteine-rich region
536- 547 CIMCCFKCCLWC (SEQ ID NO: 74)
Table VII:
Search Peptides
Variant 1, 9-mers, 10-mers, 15-mers (SEQ ID NO: 75)
MGGKQRDEDD EAYGKPVKYD PSFRGPIKNR SCTDVICCVL FLLFILGYIV VGIVAWLYGD
PRQVLYPRNS TGAYCGMGEN KDKPYLLYFN IFSCILSSNI ISVAENGLQC PTPQVCVSSC
PEDPWTVGKN EFSQTVGEVF YTKNRNFCLP GVPWNMTVIT SLQQELCPSF LLPSAPALGR
CFPWTNVTPP ALPGITNDTT IQQGISGLID SLNARDISVK IFEDFAQSWY WILVALGVAL
VLSLLFILLL RLVAGPLVLV LILGVLGVLA YGIYYCWEEY RVLRDKGASI SQLGFTTNLS
AYQSVQETWL AALIVLAVLE AILLLMLIFL RQRIRIAIAL LKEASKAVGQ MMSTMFYPLV
TFVLLLICIA YWAMTALYLA TSGQPQYVLW ASNISSPGCE KVPINTSCNP TAHLVNSSCP
GLMCVFQGYS SKGLIQRSVF NLQIYGVLCL FWTLNWVLAL GQCVLAGAFA SFYWAFHKPQ
DIPTFPLISA FIRTLRYHTG SLAFGALILT LVQIARVILE YIDHKLRGVQ NPVARCIMCC
FKCCLWCLEK FIKFLNRNAY IMIAIYGKNF CVSAKNAFML LMRNIVRVVV LDKVTDLLLF
FGKLLVVGGV GVLSFFFFSG RIPGLGKDFK SPHLNYYWLP IMTSILGAYV IASGFFSVFG
MCVDTLFLCF LEDLERNNGS LDRPYYMSKS LLKILGKKNE APPDNKKRKK
Variant 3:
9-mers
GRCFPWTNITPPALPGI (SEQ ID NO: 76)
10-mers
LGRCFPWTNITPPALPGIT (SEQ ID NO: 37)
15-mers
PSAPALGRCFPWINITPPALPGITNDTTI (SEQ ID NO: 78)
Variant 5:
9-mers
VLEAILLLVLIFLRQRI (SEQ ID NO: 79)
10-mers
AVLEAILLLVLIFLRQRIR (SEQ ID NO: 80)
15-mers
ALIVLAVLEAILLLVLIFLRQRIRIAIAL (SEQ ID NO: 81)
Variant 6:
9-mers
GYSSKGLIPRSVFNLQI (SEQ ID NO: 82)
10-mers
QGYSSKGLIPRSVFNLQIY (SEQ ID NO: 83)
15-mers
133
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LMCVFQGYSSKGLIPRSVFNLQIYGVLGL (SEQ ID NO: 84)
Variant 7
9-mers
SWYWILVAVGQMMSTM (SEQ ID NO: 85)
10-mers
QSWYWILVAVGQMMSTMF (SEQ ID NO: 86)
15-mers
FEDFAQSWYWILVAVGQMMSTMFYPLVT (SEQ ID NO: 87)
Variant 8
9-mers
NYYWLPIMRNPITPTGHVFQTSILGAYV (SEQ ID NO: 88)
10-mers
LNYYWLPIMRNPITPTGHVFQTSItGAYVI (SEQ ID NO: 89)
15-mers
FKSPHLNYYWLPIMRNPITPTGHVFQTSILGAYVIASGFF (SEQ ID NO: 90)
Variant 9
9-mers
YWAMTALYPLPTQPATLGYVLWASNI (SEQ ID NO: 91)
10-mers
AYWAMTALYPLPTQPATLGYVLWASNIS (SEQ ID NO: 92)
15-mers
LLICIAYWAMTALYPLPTQPATLGYVLWASNISSPGCE (SEQ ID NO: 93)
134
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Tables VIII- XXI: Table VIII-V1 -HLA-A1-9mers-
Table VIII-V1-HLA-A1-9mers- -
24P4C12 24P4C12
Table VIII-V1-HLA-A1-9mers- Each peptide is a portion of SEQ
Each peptide is a portion of SEQ
24P4C12 , ID NO: 3; each start position is ID NO: 3;
each start position is
Each peptide is a portion of SEQ specified, the length of
peptide is specified, the length of peptide is
ID NO. 3; each start position is 9 amino acids, and the end
9 amino acids, and the end
specified, the length of peptide is position for each peptide is the
position for each peptide is the
9 amino acids, and the end , start position plus eight. start position
plus eight.
position for each peptide is the Start Subsequence Score I Start
Subsequence i Score
start position plus eight.
- _______________________ 593 KVTDULFF 0.500 133 SQTVGEVFY 1
0.150
Start Subsequence Score
321 AILLLMLIF _ 0.500 1 613 1 LSFFFFSGR 1 0.150
58 I YGDPRQVLY 125.000
36 1 ICCVLFLLF_ 0.500 L 132 1 FSQTVGEVF 1 0.150
662 1 CVDTLFLCF 25.000
1 50 VVGIVAWLY 0.500 488 ISAFIRTLR 1 0.150
77 1 MGENKDKPY 11.250 -
1 186 NVTPPALIDG 0.500 163 QQELCPSFL , 0.135
594JVTDLLLFFG 6.250
1 609 GVGVLSFFF 0.500 199 1 TTIQQGISG 0.125
698 KNEAPPDNK 4.500
287 GASISQLGF 0.500 485 FPLISAFIR 1 0.125
318 VLEAILLLM 4.500
[187 1 VIPPALPGI 0.500 607 1 VGGVGVLSF 1 0.125 1
363 VLLLICIAY 2.500
f 668 1 LCFLEDLER 0.500 134 1 QTVGEVFYT 0.125
489 SAFIRTLRY 2.500
: _______________________ [ 323 LLLMLIFLR 0.500 575 KNAFMLLMR 0.125
267 GVLAYGIYY 2.500 1 272 GIYYCWEEY 0.500 266 11
LGVLAYGIY 0.125
689 KSLLKILGK 1.500 -
521 YIDHKLRGV _ 0.500 40 LFLLFILGY I 0.125
470 ASFYWAFHK 1.500
- 253 1 VAGPLVLVL 0.500 196
TNDTTIQQG 0125
222 FEDFAQSWY 1.250
1 398 õI GCEKVPINT 1 0.450 610 VGVLSFFFF 0.125
32 1 CTDVICCVL 1.250
1 560 I YIMIAIYGK 1 0.400 360 VTFVLLLIC 0.125
QRDEDDEAY 1.250
I 338 1 IALLKEASK [ 0.400 156 1 MTVITSLQQ 0.125 _
121 PEDPVVTVGK 1.000
1 135 1 TVGEVFYTK 1 0.400 677 q NNGSLDRPY 11 0.125
379 - LATSGQPQY 1.000
349 , GQMMSTMFY 0.375 498 1 HTGSLAFGA 1 0.125
700 EAPPDNKKR 1.000 -
[ 118 SSCPEDPVVT 0.300 172 1 LPSAPALGR 0.125
558 _ NAYIMIAIY 1.000
I 305 1 VQETWLAAL 1 0.270 195 11 ITNDTTIQQ 11 0.1251
542 - KCCLWCLEK 1.000
1 629 FKSPHLNYY I 0.250 452 1 WTLNWVLAL 1 0.125 1
7 DEDDEAYGK 1.000 ' t
1 214 ARDISVKIF 0.250 353 1 STMFYPLVT 1 0.125
11 1 EAYGKPVKY 1.000 i
1 702 PPDNKKRKK 0.250 443 QIYGVLGLF 1 0.100 -
670 FLEDLERNN 0.900
1 641 IMTSILGAY 0.250 543 CCLWCLEKF 1 0.100
276 CWEEYRVLR 0.900
1 678 1 NGSLDRPYY 0.250 207 GLIDSLNAR 0.100
518 ' ILEYIDHKL 0.900 _
1 513 QIARVILEY 0.250 407 SCNPTAHLV 1 0.100
417 SSCPGLMCV 0.750
483 PTFPLISAF 0.250 . 180 RCFPWTNVT 1 0.100
437 RSVFNLQIY 0.750
120 CPEDPVVTVG 0.225 354 1 TMFYPLVIT 1 0.100
80 NKDKPYLLY 0.625 .
- 129 KNEFSQTVG 0.225 1
263 LGVLGVLAY 0.625
- 136 VGEVFYTKN 0.225 Table VIII-V3-HLA-A1-9mers-
546 WCLEKFIKF .1 0.500 24P4C12
170 FLLPSAPAL 0.200
243 SLLFILLLR 1 0.500
= 1 147 FCLPGVPWN 0.200 Each peptide is a portion of
SEQ '
238 VALVLSLLF 0 500 ID NO: 7; each start
position is
' NISSPGCEK 0.200
579 1 MLLMRNIVR 0 ' 500 specified, the length of
peptide is
I - I 464 VLAGAFASF 0.200 9 amino acids,
and the end
1
1 465 LAGAFASFY 0.500
1 517 VILEYIDHK 0.200 position for each peptide is the
I 421 GLMCVFQGY 1 0.500 start position plus eight.
424 CVFQGYSSK 0.200 -..-
508 ILTLVQIAR 1 0.500 IISSPGCEKV 0.150 Start I
Subsequence Score
394
, ,
135
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Table VIII-V3-HLA-A1-9mers- 6 GLIPRSVFN 0.200
Table VIIIN8-HLA-A1-9mers-
24P4C12 2 YSSKGLIPR . 0.075 24P4C12 .
Each peptide is a portion of SEQ 5 KGLIPRSVF 0.025 = Each
peptide is a portion of SEQ
ID NO: 7; each start position is I LIPRSVFNL 0.005 ID NO: 17;
each start position is
7
specified, the length of peptide is specified, the length of
peptide is
9 amino acids, and the end 3 I SSKGLIPRS 0.003 9 amino
acids, and the end
position for each peptide is the 4 I SKGLIPRSV 0.001
position for each peptide is the
start position plus eight. 9 I PRSVFNLQI 0.000 start
position plus eight.
Start Subsequence I Score 8 IPRSVFNLQ 0.000
Start I Subsequence Score
9 ITPPALPGI 0.500 1 I GYSSKGLIP 0.000
18 VFQTSILGA 0.003 _
8 I NITPPALPG I 0.500 10 I
NPITPTGHV 0.003
I 2 RCFPVVTNIT I 0.100 Table VIII-V7-HLA-A1-9mers-
15 1 TGHVFQTSI I 0.003
I 6 WTNITPPAL I 0.050 24P4C12 9 I
RNPITPTGH I 0.003
7 TNITPPALP 0.001 Each peptide is a
portion of SEQ 14 PTGHVFQTS 0.003
I 1 J GRCFPVVTNI I 0.001 1 ID NO: 15; each start
position is 7 IMRNPITPT 0.001
I
specified, the length of peptide is 3 CFPWTNITP I-air I 3
YWLPIMRNP 0.001
9 amino acids, and the end
I 5I PVVTNITPPA I 0.000 position for each peptide is
the 16 GHVFQTSIL I 0.001
1 4 FPWTNITPP I 0.000 start
position plus eight. I 2 I YYWLPIMRN 10.000
Start I Subsequence Score 6 PIMRNPITP 0.000
7 VAVGQMMST 0.050
Table VIII-V5-HLA-A1-9mers- 6 LVAVGQMMS 0.050 Table
VIII-V9-HLA-A1-9mers-
24P4C12 24P4C12
8 AVGQMMSTM 0.010
Each peptide is a portion of SEQ
5 ILVAVGQMM 0.010 Each peptide is a portion of SEQ
ID NO: 3; each start position is ID NO:
19; each start position is
specified, the length of peptide is I 4 WILVAVGQM 0.010
specified, the length of peptide is
9 amino acids, and the end I 3 YWILVAVGQ I 0.001 9 amino
acids, and the end
position for each peptide is the I 1 I SWYWILVAV 0.001
position for each peptide is the
start position plus eight.
= I 2 WYWILVAVG 0.000 start
position plus eight
I Start Subsequence I Score Start I Subsequence Score
1 I VLEAILLLV I 4.500 11
PTQPATLGY 6.250
Table VI1148-HLA-A1-9mers-
6 LLLVLIFLR 0.500 24P4C12 _____________ 4 I
MTALYPLPT I 0.125
4 AILLLVLIF 0.500 Each
peptide is a portion of SEQ 15 I ATLGYVLWA I 0.125
I 8 I LVLIFLRQR 0.100 ID NO:
17; each start position is 8 I YPLPTQPAT I 0.050
7 LLVLIFLRQ 0.050 specified, the length
of peptide is
I TALYPLPTQ I 0.020 1
I
5 ILLLVLIFL 0.050
9 amino acids, and the end
position for each peptide is the 2 WAMTALYPL 0.020
3 EAILLLVLI I 0.020 start position plus
eight 16 TLGYVLWAS 0.010
9 VLIFLRQRI 0,010 [Start Subsequence I
Score 6 ALYPLPTQP 0.010
2 LEAILLLVL 0.003 I 11 I PITPTGHVF I
0.100 13 QPATLGYVL 0.005
1 19 FQTSILGAY I 0.075 17 LGYVLWASN 0.005
Table VIII-V6-HLA-A1-9mers- I 20 QTSILGAYV 0.050 10
LPTQPATLG 0.003
24P4C12
17 HVFQTSILG 0.050 9 PLPTQPATL 0.002
Each peptide is a portion of SEQ
I 12 ITPTGHVFQ 0.050 14 PATLGYVLW 0.002
ID NO: 13; each start position is
specified, the length of peptide is I 1
NYYWLPIMR I 0.025 12 I TQPATLGYV 0.002
9 amino acids, and the end I 13 I TPTGHVFQT I 0.013 3
AMTALYPLP 0.001
position for each peptide is the I 8 MRNPITPTG I 0.010 18
I GYVLWASNI 0.001
start position plus eight
4 WLPIMRNPI 0.010 7 LYPLPTQPA 0.001
IStart I Subsequence I Score
5 LPIMRNPIT 0.005 1 I YWAMTALYP 0.000
136
=
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Table IX-V1-HLA-A1-10mers- Table IX-V1-HLA-A1-10mers-
24P4C12 24P4C12
Table IX-V1-HLA-A1-10mers- Each peptide is a portion of
SEQ Each peptide is a portion of SEQ
24P4C12 ID NO: 3; each start position is
ID NO: 3; each start position is
Each peptide is a portion of SEQ specified,
the length of peptide is specified, the length of peptide is
ID NO: 3; each start position is 10 amino acids, and the end 10 amino
acids, and the end
specified, the length of peptide is position for each peptide is the
position for each peptide is the
amino acids, and the end start position plus nine, start position plus
nine.
position for each peptide is the Start Subsequence Score = I
Start (Subsequence Score
start position plus nine.
609 GVGVLSFFFF 0.500 1 186 -1
NVTPPALPGI 1 0.200
[Start Subsequence I Score
353 1 STMFYPLVTF i 0.500 618 1 FSGRIPGLGK (0.150
594 VTDLLLFFGK 1125.000
464 1 VLAGAFASFY 0.500 1 173 1PSAPALGRCF_ 0.150
32 CTDVICCVLF [25.000
322 ILLLMLIFLR 0.500 118
1SSCPEDPVVTV 0.150
120 CPEDPVVTVGK 1_ 9.000
1 35 1 VICCVLFLLF 0.500 1 125
1VVTVGKNEFSQ1 0.125
518 ILEYIDHKLR 1 9.000
I 606 VVGGVGVLSF 0.500 676 1RNNGSLDRPY 0.125
680 SLDRPYYMSK 1 5.000
1 521 1 YIDHKLRGVQ 0.500 608 GGVGVLSFFF 0.125
698 KNEAPPDNKK 1 4.500
662 1 CVDTLFLCFL 0.500 286 KGASISQLGF I 0.125
318 VLEAILLLML 1 4.500
661 1 MCVDTLFLCF 0.500 80 I NKDKPYLLYF 0.125
488 ISAFIRTLRY 3.750
1 265 1 VLGVLAYGIY 0.500 1 360 VTFVLLLICI 1 0.125
39 VLFLLFILGY 2.500
49 IVVGIVAWLY 0.500 1 196
ThIDTTIQQGI 0.125
262 ILGVLGVLAY 1 2.500
667 1 FLCFLEDLER 0.500 1 198 1 DTTIQQGISG 0.125 ;
362 1 FVLLLICIAY 2.500
407 SCNPTAHLVN 0.500 1 293 1
LGFTTNLSAY 0.125
136 VGEVFYTKNR 2.250
165 1 ELCPSFLLPS 1 0.500 1 271 1 YGIYYCWEEY 0.125
221 IFEDFAQSVVY 2.250
77 1MGENKDKPYL 0.450 382 SGQPQYVLWA 0.125
700 EAPPDNKKRK 2.000
547 CLEKFIKFLN 0.450 467 GAFASFYWAF 0.100
9 DDEAYGKPVK 1 1.800
1 337 I AIALLKEASK 0.400 1 487 1 LISAFIRTLR 0.100
6 RDEDDEAYGK 1.800 I
j 512 VQIARVILEY 0.375 1 650 1
VIASGFFSVF 0.100 ,
417 SSCPGLMCVF_I 1.500
1 689 KSLLKILGKK 0.300 64 1VLYPRNSTGA 0.100
132 FSQTVGEVFY 1.500
305 1 VQETWLAALI 1 0.270 1 347 1AVGQMMSTMF 0.100
134 QTVGEVFYTK 1.000
18 1 KYDPSFRGPI 0.250 272 GIYYCWEEYR 1 0.100
1 469 FASFYWAFHK [ 1.000
76 GMGENKDKPY 0.250 1 333 RIRIAIALLK "I 0.100
369 IAYWAMTALY 1 1.000
I 557 RNAYIMIAIY 1 0.250 1 612
VLSFFFFSGR 1 0.100
378 YLATSGQPQY 1 1.000
1 590 1 VLDKVTDLLL 0.250 (147 FCLPGVPWNM 0.100 '
670 FLEDLERNNG 1 0.900
1 677 NNGSLDRPYY 0.250 1 216 - DISVKIFEDF 0.100
[-TO- VAENGLQCPT 0.900
578 1 FMLLMRNIVR 0.250 , 53 _
IVAWLYGDPR 0.100
277 WEEYRVLRDK 0.900
187 1 VTPPALPG1T 0.250 1 326 MLIFLRQRIR 0.100
242 LSLLFILLLR 0.750
1 463 1 CVLAGAFASF 0.200 , 1 544
CLWCLEKF1K 0.100
163 QQELCPSFLL 0.675
1 516 1 RVILEYINK 0.200
58 YGDPRQVLYP 1 0.625
74 YCGMGENKDK 0.200 =
1 266 LGVLAYGIYY (,0.625
72 GAYCGMGENK 0.200 Table IX-V3-
1-1LA-A1-10mers.
348 VGQMMSTMFY1 0.625 I 24P4C12 423 MCVFQGYSSK 0.200
171 LLPSAPALGR 1 0.500 Each
peptide is a portion of SEQ
621 RIPGLGKDFK 0.200
1 507 LILTLVQIAR 0.500 ID NO: 7;
each start position is
170 FLLPSAPALG 0.200 specified,
the length of peptide is
1 237 GVALVLSLLF 1 0.500
211 SLNARDISVK 0.200 10 amino acids,
and the end
1 320 EAILLLMLIF [ 0.500
161 1 SLQQELCPSF 0.200 position
for each peptide is the
1 208 L LIDSLNARD1 1 0.500 start position plus nine.
1 253 1 VAGPLVLVLI 0.200
137
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1 Start 1 Subsequence Score 1 4 I SSKGLIPRSV ,1-070-01- 2
NYYWLPIMRN 0.003
I 10 1 ITPPALPGIT " 0= .250 1 r 9 IPRSVFNLQII 0.001 16
TGHVFQTSIL 10.003
1 9 1 NITPPALPGI ' 0= .200 1 1 1 QGYSSKGLIP ' 0.001 17 1
GHVFQTSILG 11Ø003'
1 3 1 RCFPVVTNITP 0.050 I 8 1 LIPRSVFNLQ 1 0.001 I 6 f
LPIMRNPITP 1_0.001
1 8 1 TNITPPALPG 0.013 , 8 [
IMRNPITPTG [0.001
t 7 1 VVTNITPPALP 0.005 Table IX-V7-HLA-A1-10mers- 7 1
PIMRNPITPT 10.000
1 5 1 FPWTNITPPA 0.001 24P4C123
LYWLPIMRNP 115571
1 2 1GRCFPWTNIT I 0.001 Each peptide is a portion of SEQ
-
t 1 1 LGRCFPWTNI 0.000 ID NO: 15; each start position is
Table IX-V9-HLA-A1-10mers-
specified, the length of peptide is
6 1 PWTNITPPAL 0.000 10 amino acids, and the
end 24P4C12
, 4 [CFPWTNITPP 0.000 position for each peptide is the
Each peptide is a portion of SEQ
start position plus nine. ID NO:
19; each start position is
Table IX-V5-HLA-A1-10mers- Start Subsequence [Score
specified, the length of peptide is
amino acids, and the end
24P4C12 9 AVGQMMSTMF 0.100 position for each
peptide is the
Each peptide is a portion of SEQ 6 ILVAVGQMMS 0.050 start position
plus nine.
ID NO: 11; each start position is 1 7 L= VAVGQMMST [0.050
Start,( Subsequence (Score
specified, the length of peptide is 8 VAVGQMMSTM '10.010 1
ii LPTQPATLGY 10.625
10 amino acids, and the end
position for each peptide is the 1 5 - W= ILVAVGQMM [0.010
I 7 I ALYPLPTQPA Morff
start position plus nine. 1 QSWYWILVAV 0.0031 I 9 f
YPLPTQPATL 10.050
Start Subsequence f Score 2 SWYWILVAVG 10.001 5
MTALYPLPTQ 10.050
2 1 VLEAILLLVL 4:500 1 4 YWILVAVGQM
_10.001 1 12 ' PTQPATLGYV 10.025
6 1 ILLLVLIFLR 0.500 1 3 WYWILVAVGQ_ 0.000 1 4
AMTALYPLPT 10"."8-27"
_
4 EAILLLVLIF 1 0.500 16 ATLGYVLWAS
0.025
8 1 LLVLIFLRQR a_ 0.100, Table IX-V8-HLA-A1-10mers- 17
TLGYVLWASN 0.020
10 VLIFLRQRIR L0.100 24P4C12
15 1_, PATLGYVLWA 10.005'
7 LLLVLIFLRQ 0.050 Each peptide is a portion of SEQ 1
14 QPATLGYVLW 10.005
ID NO: 17; each start position is
1 1 AVLEAILLLV 0.050 13 1 TQPATLGYVL 10.003_1
specified, the length of peptide is
5 - AILLLVLIFL 0.050 10 amino acids, and
the end 18 LGYVLWASNI 0.003
9 1 LVLIFLRQRI 0.010 position for each peptide is
the 1 3 WAMTALYPLP 10.002
3 1 LEAILLLVLI 1 0.001 start position plus nine.
1 2 YWAMTALYPL 10.001
-
Table IX-V6-HLA-A1-10mers- I 1 I LNYYWLPIMR 0.1251 1 6 TALYPLPTQP
10.001,
24P4C12 1 13 ITPTGHVFQT 0.125 8 LYPLPTQPAT
10.001_
Each peptide is a portion of SEQ 1 21 QTSILGAYVI , 0.0501 1 19
GYVLWASNIS 1 0.001
ID NO: 13; each start position is 1_ 18 1 HVFQTSILGA 0.0501 1 1
AYWAMTALYP 110.0001
10 amino acids, and the end
start position plus nine. 12 1 PITPTGHVFQ 0.020 Table X-
V1-HLA-A0201-9mers-
1 7 GLIPRSVFNL 1 0.500 4 _ I YWLPIMRNPI 0.005 Each
peptide is a portion of SEQ
1 2 1 GYSSKGLIPR 1 0.025 9 1 MRNPITPTGH 0.005 ID NO: 3;
each start position is
specified, the length of peptide is
I 6 KGLIPRSVFN 0.005 20 1 FQTSILGAYV 0.003 9 amino
adds, and the end
1 5 SKGLIPRSVF 1 0.005 1 [ 15 1
PIGHVFQTSI 0.003 . position for each peptide is the
1 3 ' I YSSKGLIPRS 0.003 14 TPTGHVFQTS 0.003 start position plus
eight. t
1 10 1 PRSVFNLQIY 1 0.003 10 RNPITPTGHV 0.003 I Start
1 Subsequence (Score
138
CA 02503346 2005-04-21
WO 2004/050828 PCT/US2002/038264
Table X-V1-HLA-A0201-9mers- Table X-V1-HLA-A0201-9mers-
Table X-V1-HLA-A0201-9mers-
24P4C12 24P4C12 24P4C12
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ 1 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
9 amino acids, and the end 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 eight
I Start Subsequence Score Start I Subsequence (Score
Start I Subsequence (Score
3255. 1 1 37 638 I WLPIMTSIL 140.28
449 GLFWTLNWV
381 103.5 4019
- 456 WVLALGQCV 49 IVVGIVAWL
1699. 80 7
322 ILLLMLIFL
774 99.66 37.82
537 IMCCFKCCL 38 CVLFLLFIL
1006. 7 7
i 580 LLMRNIVRV
209 98.55 37.26
446 GVLGLFWTL 148 CLPGVPWNM
510.6 4 0
597 LLLFFGKLL =
04 88.04 36.31
257 LVLVLILGV 232 ILVALGVAL
476.2 3 6
544 CLWCLEKFI
57 85 30.45
660 GMCVDTLFL 1134'6 291 SQLGFTTNL
437.4 3
598 LLFFGKLLV
82
686 YMSKSLLKI 179'71
85 YLLYFNIFS
363.5 8 26.50 '
8
170 FLLPSAPAL
88 77.87 23.99
177 ALGRCFPVVT 506 ALILTLVQI
360.5 3 5
86 LLYFNIFSC
26 69.55 23.79
211 SLNARDISV 252 LVAGPLVLV
2 5
578 FMLLMRNIV 35299'5
107 GLQCPTPQV 169'55 233 LVALGVALV 23'79
309.0 2 5
244 LLFILLLRL
50 69.00 18.50
241 VLSLLFILL 525 KLRGVQNPV
292.0 1 1
41 FLLFILGYI
08 66.61 18.38
434 LIQRSVFNL 339 ALLKEASKA
271.9 3 2
95 ILSSNIISV
48 66.61 17.73
35 VICCVLFLL 265 VLGVLAYGI
271.9 3 6
260 VLILGVLGV
48 65.72 17.73
547 CLEKFIKFL 326 MLIFLRQRI
204.7 1 6
56 WLYGDPRQV
61 65.21 16.96
317 AVLEAILLL 340 LLKEASKAV
179.3 9 7
42 LLFILGYIV - .
68
240 LVLSLLFIL 164.630 445 YGVLGLFWT 16.41
179.1 8
650 VIASGFFSV 1-
61 54.79 14.89
=
302 YQSVQETWL 315 VLAVLEAIL
177.4 8 0
564 AIYGKNFCV
97 52.56 1 14.89
= , 309 WLAALIVLA
457 VLALGQCVL
131.9 1 0
239 ALVLSLLFI -
75 49.83 13.97
351 MMSTMFYPL 509 LTLVQIARV
.118.2 ,
4 5
604 LLVVGGVGV
38 46.45 13.961
365 LLICIAYWA 119 SCPEDPWTV
110.8 1 1
589 VVLDKVTOL
72 40.79 13.061
1 268 VLAYGIYYC 1106.8
45 ILGYIVVGI 2 366 LICIAYWAM
4
139
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Table X-V1-HLA-A0201-9mers- Table X-V1-HLA-A0201-9mers-
Table X45-HLA-A0201-9mers-
24P4C12 _ 24P4C12 24P4C12
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: 11; 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 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 eight.
Start 1 Subsequence 11-;;;; Start 1 Subsequence Score Start
1 Subsequence (Score
226 AQSVVYWILV 11098 1 536 1 CIMCCFKCC 4,802 1 4 1
AILLLVLIF 10.0361
u 1 246 1 FILLLRLVA 4.767 1 3 ,1
EAILLLVLI 10.0251
452 WILNWVLAL 111'61 1 357 YPLVTFVLL 4.510 1 8 1
LVLIFLRQR 0.014
426 1 FQGYSSKGL 19.963
Table X-V3-HLA-A0201-9mers- Table X-V6-HLA-A0201-9mers-
1 554 FLNRNAYIM 19.370 24P4C12 24P4C12
642 1 MTSILGAYV 9.032 Each peptide is a
portion of SEQ Each peptide is a portion of SEQ
164 1 QELCPSFLL 8.914 ID NO: 7; each
start position is ID NO: 13; each start position is
693 KILGKKNEA 8.846 specified, the
length of peptide is specified, the length of peptide is
251 RLVAGPLVL 18.759
9 amino acids, and the end 9 amino
acids, and the end
1
position for each peptide is the position for each peptide is
the
1 501 1 SLAFGALIL 18.759 start position
plus eight. start position plus eight.
1 487 1 LISAFIRTL 18.729 Start Subsequence lic-We'
Start Subsequence Score
1 442 LQIYGVLGL 8.469 6 1 VVTNITPPAL 1.365
'66.61
7 LIPRSVFNL
262 1 ILGVLGVLA 18.446 9 1 ITPPALPGI 0.567 3
1 521 1 YIDHKLRGV 18.094 2 1 RCFPVVTNIT 0.074 6 GLIPRSVFN
10.410
373 1 AMTALYLAT 18.073 8 NITPPALPG 0.010 1 4 SKGL1PRSV
0.019
242 1 LSLLF1LLL 7.666 4 FPWTNITPP 0.009 1 5
KGLIPRSVF 0.003
1 134 1 QTVGEVFYT 7.594 1 1 GRCFPVVTNI 10.002 1 2 YSSKGLIPR 0.001
1 191 1 ALPGITNDT 7.452 7 1 TNITPPALP 0.000 9 PRSVFNLQI
10.000
1 590 VLDKVTDLL 17.118 1 5 1 PWTNITPPA 10.0001 1 3
SSKGLIPRS 10.000
362 FVLLLICIA 6.977 3 1 CFPWTNITP 10.000 8 IPRSVFNLQ
[0.000
1 200 TIQQGISGL 6.756 1 1 GYSSKGLIP ,
0.000,
83 KPYLLYFNI 6.636 Table X-V5-HLA-A0201-9mers-
314 IVLAVLEAI 6.471 24P4C12
Table X-V7-HLA-A0201-9mers-
383 1 GQPQYVLWA 16.372 Each peptide is a portion of SEQ 24P4C12
1 225 FAQSWYWIL 6.295 ID NO: 11; each start position is
Each peptide is a portion of SEQ
specified, the length of peptide is ID NO: 15; each start position is
289 1 SISQLGFTT 5.943 9 amino acids,
and the end specified, the length of peptide is
364 LLLICIAYW 5.929 position for
each peptide is the 9 amino adds, and the end
596 1 DLLLFFGKL 15.5641 start position plus eight.
position for each peptide is the
1 611 1 GVLSFFFFS 15.557 Start Subsequence Score,
start position plus eight.
1 282 VLRDKGASI 5.526 ILLLVLIFL 1699. Start
Subsequence Score
5
1 154 WNMTVITSL [5.459 774 1 5 1LVAVGQMM
8.446
1 380 1 ATSGQPQYV 5.313 9 VLIFLRQRI 17.73 [ 4 WILVAVGQM
3.476
1
612 VLSFFFFSG 5.305 6 8 AVGQMMSTM
11.000
i 1 1 1 10.40511 1 oo 1 IISVAENGL 14.993 VLEAILLLV 7
VAVGQMMST ro
t
(158 V1TSLQQEL
6 1 LLLVLIFLR 1.251 1 1 SWYWILVAV 10.071
1 4.993
1
504 1 FGALILTLV 14.804 2 1 LEAILLLVL 0.666 6 LVAVGQMMS
10.011
7 LLVLIFLRQ 0.048
140
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PCT/US2002/038264
Table X-V7-HLA-A0201-9mers- Each peptide is a portion of
SEQ Table XI-V1-HLA-A0201-10mers-
Each peptide is a portion of SEQ specified, the length of
peptide is Each peptide is a portion of SEQ
ID NO: 15; each start position is 9 amino acids, and the end ID NO: 3;
each start position is
specified, the length of peptide is position for each peptide is
the specified, the length of peptide is
9 amino acids, and the end start position plus eight. 10 amino acids,
and the end
start position plus eight. 11.61 ' start position plus nine.
(Start Subsequence IScore 2 WAMTALYPL 5 Start
r Subsequence Score
..
2 WYWILVAVG [0.000 11.59 L 48
12 TQPATLGYV
3 1 YWILVAVGQ 10.000 7 485.3
1
41 FLLFILGYIV
15 ATLGYVLINA 13.23048
Table X-V8-HLA-A0201-9mers- I 16 TLGYVL WAS 1.778-6 641
IMTSILGAYV 469.6
24P4C12 1 8 YPLPTQPAT 0.828 69
Each peptide is a portion of SEQ 9 , PLPTQPATL 110.470 546
WCLEKFIKFL 467.7
ID NO: 17; each start position is 1 4 MTALYPLPT 10.176 ., 71
-
specified, the length of peptide is 437.4
13 QPATLGYVL ro:76-7 597 LLLFFGKLLV
9 amino acids, and the end 82
position for each peptide is the 1 6
ALYPLPTQP 1.031-6-1 412.5
start position plus eight. 1 3 L AMTALYPLP 10.016 598 LLFFGKLLW 46
Start Subsequence Score I 17 LGYVLWASN 10.004
665 TLFLCFLEDL 338.5-
47.99 5 TALYPLPTQ [0.0021 00
4 WLPIMRNPI 1 1 18 GYVLWASN I
F.F11 317.4
- 241 VLSLLFILLL
1 20 QTSILGAYV 5.313 7 LYPLPTQPA
10.001 03
1 7 IMRNPITPT 11.599 10 LPTQPATLG 10.001 649
YVIASGFFSV 308.5
1 13 TPTGHVFQT 0.649 1 1 YWAMTALYP
1_0.000 01
1 15 1 TGHVFOTSI 10.259 1 14 PATLGYVLW 10.000 433 GLIQRSVFNL 284.9
74
NPITPTGHV 0.059 11 I PTQPATLGY 10.000 271.9
1 5 1 LPIMRNPIT [0.034 508 ILTLVQIARV
48
1 18 I VFQTSILGA [0.013 Table XI-V1-HLA-A0201-10mers-
271.9
48
42 LLFILGYIVV
2 YYWLPIMRN [0.001 257.3
10 amino acids, and the end 339 ALLKEASKAV
9 I RNPITPTGH [0.000 start position plus nine. 449
GLFVVTLNWVL 243.051
11 PITPTGHVF [0.000 TMFYPLVTFV 2351. 244 LLFILLLRLV
354 42
14 PTGHVFQTS 16E7 109
- 181.7
1 8 MRNPITPTG [0.000 85 YLLYFNIFSC 1127. 243 SLLFILLLRL
94 '
969
1 3 YVVLPIMRNP [0.000' 1 11006. 364 LLLICIAYWA 171.8 1
NYYVVLPIMR 1_0.000 579 MLLMRNIVRV 68
209 . _
170.9
900.6 48 YIVVGIVAWL
Table X-V9-1-1LA-A0201-9mers- 603 KLLVVGGVGV
98 23 =
24P4C12159.9
735.8 251 RLVAGPLVLV
309 WLAALIVLAV 70
137.4
351 1 MMSTMFYPLV 1486.7 321 AILLLMLIFL
82
141
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WO 2004/050828 PCT/US2002/038264
Table XI-V1-HLA-A0201-10mers- Table XI-V1-HLA-A0201-10mers-
Table XI-V1-HLA-A0201-10mars-
24P4C12 24P4C12 24P4C12
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
10 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.
[ Start Subsequence IScore, Start Subsequence Score I Start I
Subsequence Score
-
128.9 9 478 KPQDIPTFPL 111.60
56 WLYGDPRQVL
26
315 VLAVLEAILLJ36' 31
238 VALVLSLLFI 11%2
116,8 6 9
239 ALVLSLLFIL
40 12
448 LGLFWTLNWV 36. 312
ALIVLAVLEA 11.42
108.4 6 6
350 QMMSTMFYPL
62 35.94 11.42
662 CVDTLFLCFL 459 ALGQCVLAGA
8 1 6
86 LLYFNIFSCI 107. ,
33 02
64 VLYPRNSTGA 27. 571
CVSAKNAFML 10.84
6 1
365 LLICIAYWAM 95.01
3
589 VVLDKVTDLL 23.62 563 IAIYGKNFCV 19.525
88.04 0 445 YGVLGLFWIL 9.141
259 LVLILGVLGV -
3 22.52
596 DLLLFFGKLL 379
LATSGQPQYV 9.032
7
162 LQQELCPSFL 83.03
327 1 LIFLRQRIRI 19.023
0 22.33
240 LVLSLLFILL 249
LLRLVAGPLV 8.986
- 82.50 9
580 LLMRNIVRVV 539
CCFKCCLWCL 18.900
9 20.74
357 YPLVTFVLLL
94 CILSSNIISV 81.38 4 513 QIARVILEYI 8.892
5510 I TLVQIARVIL 18.759
- 267 GVLAYGIYYC 156.--31
6 457 VLALGQCVLA 8.446
517 VILEYIDHKL 75/5
1
304 SVQETWLAAL 17.62 95 I ILSSNIISVA 7.964
.98
, 7
554 FLNRNAYIMI 657 SVFGMCVDTL 7.794
71 .
6
17.46
248 LLLRLVAGPL 225
FAQSVVYWILV 17.554
92 8
686 YMSKSLLKIL 66. 588
VVVLDKVTDL 17.309
56'15 17.37
302 YQSVQETWLA 593 1
KVTDLLLFFG 16.865
44 LFILGYIVVGI ' 8
5 17.14 368 1 CIAYWAMTAL 6.756
501 SLAFGALILT
55.43 0 562 MIAIYGKNFC 167371
133 SQTVGEVFYT 5 15.16 363 VLLLICIAYW 5.929
317 AVLEAILLLM
438 SVFNLQIYGV 51.79 ' I36 ICCVLFLLFI 5.565
0
590 VLDKVTDLLL 14.52 318 VLEAILLLML 15.346
49.99 6
231 WILVALGVAL 292 1
QLGFTTNLSA 4.968
3 14.49
45 ILGYIVVGIV 314
IVLAVLEAIL 4.821
49.13
235 ALGVALVLSL = 5 1 393
NISSPGCEKV Pal
4 13.05
659 FGMCVDTLFL 506
ALILTLVQIA 4.685
49.13 4
441 NLOIYGVLGL
4 13.04 260 1 VLILGVLGVL 14.452
456 VVVLALGQCVL
47.86 4 604 1 LLWGGVGVL 4.452
660 GMCVDTLFLC
4
148 CLPGVPWNMT 12.66 261 1 LILGVLGVLA [4.297
,-,
8 502 1 LAFGALILTL ' 4.292
325 LMLIFLRQRI 47'39
4 108 LQCPTPQVCV 11.98 147 1 FCLPGVPWNM F47714n)
I 536 1 CIMCCFKCCL 141.29 8 '
. ________________________________________ .
142
CA 02503346 2005-04-21
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PCT/US2002/038264
Table XI-V3-HLA-A0201-10mers- Each peptide is a portion of
SEQ 178.4
20 FQTSILGAYV
24P4C12 ID NO: 13; each start position is
11
Each peptide is a portion of SEQ specified, the length of
peptide is 14.05
ID NO: 7; each start position is 10 amino acids, and the end 5
WLPIMRNPIT 4
specified, the length of peptide is position for each peptide is the
13 ITPTGHVFQT 12.347
amino acids, and the end start position plus nine. _
position for each peptide is the Start ,1 Subsequence 'Score( 7
PIMRNPITPT 10192_
I
start position plus nine. 284.9 18
HVFQTSILGA 10.126
7
r Start Subsequence Score GLIPRSVFNL74 1 21 QTSILGAYVI 10.059
1 9 1 NITPPALPGI 3.299 [ 6 r KGLIPRSVFN 0.035 10
RNPITPTGHV 0.059,,
5 1 FPVVTNITPPA 11.238 9 IPRSVFNLQI 0.033 1 16 TGHVFQTSIL 10.057
1 1 1 LGRCFPVVTNI 0.015 [ 8 LIPRSVFNLQ 10.007 4 YWLPIMRNPI
0.025]
10 ITPPALPGIT 0.009 3 J YSSKGLIPRS 10.005 15
PTGHVFQTSI 0.012
7 1 VVTNITPPALP 0.001 4 SSKGLIPRSV 0.003 1 8 IMRNPITPTG 0.007
8 1 TNITPPALPG 0.000 [ 1 QGYSSKGLIP 10.000 1 14 TPTGHVFQTS 0.001
1 2 GRCFPVVTNIT 10.000 5 SKGLIPRSVF110.000 1 1
LNYYWLPIMR 0.001
3 RCFPVVTNITP 10.000 2 GYSSKGLIPR 0.000 1 12
PITPTGHVFQ 0.000
1 6 PVVTNITPPAL 10.000 10 PRSVFNLQIY 10.000 1 11
NPITPTGHVF 0.000
1 4 CFPVVTNITPP ' 0.000 6 LPIMRNPITP
0.000
,.
Table XI-V7-HLA-A0201-10mers- 2 NYYWLPIMRN 0.000
Table XI-V5-HLA-A0201-10mers- 24P4C12 17 1 GHVFOTSILG
0.0001
24P4C12 Each peptide is a portion of SEQ
3 1 YYWLPIMRNP 0.000
Each peptide is a portion of SEQ ID NO: 15; each start position
is 19 1 VFQTSILGAY 0.000
ID NO: 11; each start position is specified, the length of peptide is
9 MRNPITPTGH 0.000
specified, the length of peptide is 10 amino acids, and the end
10 amino acids, and the end position for each peptide is the
position for each peptide is the start position plus nine.
Table XI-V9-HLA-A0201-1 Omers-
start position plus nine. 1 Start 1 Subsequence {Score 24P4C12
Start Subsequence (Score WILVAVGQMM 11.62 Each peptide is a portion of
SEQ
. 5
212.3 6 ID NO: 19; each start position
is .
1 AVLEAILLLV
40 1 1 QSWYWILVAV 8.667
specified, the length of peptide is
1
10 amino acids, and the end
137.4 1 7 LVAVGQMMST 2.550
5 AILLLVLIFL position for
each peptide is the
82 1 8 1 VAVGQMMSTM 10.270 start position plus nine.
9 1 LVLIFLRQRI _ 5.742 1 6 ILVAVGQMMS
10.127 Start Subsequence (Score
2 VLEAILLLVL 2.1921 1 9 1 AVGQMMSTMF 10.007 15.89
7 ALYPLPTQPA
6 ILLLVLIFLR 1.251 4 YWILVAVGQM 10.001 a
3 1 LEAILLLVLI*1117.91 f 3 WYWILVAVGQ 10.000 1 4 1
AMTALYPLPT 15.382
1 7 1 LLLVLIFLRQ 1 0.178 2 1 SWYWILVAVG 0.000 9
YPLPTQPATL 12.373
= 8 LLVLIFLRQR 0.044 1 13 I
TQPATLGYVL 0.8881
10 VLIFLRQRIR 0.002 Table XI-V8-HLA-A0201-10mers-
1 18 LGYVLWASNI 157
1 4 1 EAILLLVLIF 110.0001 24P4C12 17 1
TLGYVLWASN 0.1271
Each peptide is a portion of SEQ 16 1 ATLGYVLWAS 0.066
Table XI-V6-HLA-A0201-10mers- ID NO: 17; each start
position is 12 1 PTQPATLGYV 0.035
24P4C12 specified, the length of peptide
is
2 1 YWAMTALYPL 0.031
' 10 amino acids, and the end
position for each peptide is the 15 I PATLGYVLWA 0.019
start position plus nine. 1 3 '
WAMTALYPLP 0.005
1 Start Subsequence (Score 1 8 1
LYPLPTQPAT 0.002
143
. .
CA 02503346 2005-04-21
WO 2004/050828
PCT/US2002/038264
1 6 TALYPLPTQP 10.001 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
1 Start r Subsequence (Score Start Subsequence Score
Table XII-V1-HLA.A3-9mers- 272 -1 GIYYCWEEY 6.000 1
501 SLAFGALIL 1.200
Each peptide is a portion of SEQ 470 1_ ASFYWAFHK 14.500 1 349
GQMMSTMFY 1.080
ID NO: 3; each start position is 449 I GLFWTLNWV 4.500 1
443 QIYGVLGLF 1.012
specified, the length of peptide is
9 amino acids, and the end
position for each peptide is the 446 1 GVLGLFWTL 3.645 590
VLDKVTDLL 0.900
start position plus eight. 660 GMCVDTLFL 3.600 326
MLIFLRQRI 0.900
Start 1 Subsequence (Score 633 1 HLNYYWLPI 3.600 1 268 VLAYGIYYC
0.900
..
81.00 542 KCCLWCLEK 13.600 1 107 GLQCPTPQV 0.900
421 GLMCVFQGY
0 241 VLSLLFILL 13.600 I 613
LSFFFFSGR 0.900
40.50
135 TVGEVFYTK 42 LLFILGYIV 3.000 1 318
VLEAILLLM . 0.900
o
- 393 NISSPGCEK 3.000 232
ILVALGVAL 0.900
27.00
207 GLIDSLNAR 1 325 LMLIFLRQR 2.700 r 518
ILEYIDHKL 0.900
0
27.00 ILGYIVVGI 2.700 1 452 VVTLNWVLAL 0.810
,
323 LLLMLIFLR
0 1 322 1 ILLLMLIFL 2.700 596 DLLLFFGKL
0.729
,
,
27.00 1 239 ALVLSLLFI 2.700 645 ILGAYVIAS 0.720
243 SLLFILLLR o 641 1 IMTSILGAY
12.700 1 258 VLVLILGVL 0.608
22.50 598 LLFFGKLLV 2.000 49 IVVGIVAWL 0.608
354 TMFYPLVTF o 260 1 VLILGVLGV 1.800 1
41 , FLLFILGYI 0.608
20.25
690 SLLKILGKK 265 1 VLGVLAYGI 1.800 1 54
VAWLYGDPR 10.600
0
513 1 QIARVILEY 11.800 1 665 TLFLCFLED 0.600
20.25
517 VILEYIDHK 609 1 GVGVLSFFF 1.800 1 95
ILSSNIISV - 0.600
0
537
18.00 I IMCCFKCCL 1.800 457 VLALGQCVL 10.600
363 VLLLICIAY
0 1 50 1 WGIVAWLY 17871 282
1 VLRDKGASI 1-0-B-61
18.00 1 686 I YMSKSLLKI 11,8001 1 554 FLNRNAYIM 10.6E1
585 IVRVWLDK o 1 251 I
RLVAGPLVL 1.800 I 39 I VLFLLFILG 0.600
13.50 593 1 KVTDLLLFF 11.800, 315 1 VLAVLEAIL 10.600
560 YIMIAIYGK o 1 358 PLVTFVLLL
1.620 638 1 WLPIMTSIL 10.600
12.00
508 ILTLVQIAR 544 CLWCLEKFI 11.500 1
434 LIQRSVFNL 10.540
o
= 12.00
579 MLLMRNIVR
0 i. 525 KLRGVQNPV 17.-3761 611
GVLSFFFFS row'
10.80 170 FLLPSAPAL 11.350 647 1 GAYVIASGF 0.450
267 GVLAYGIYY 0 I 547 CLEKFIKFL 11.350
1 580 LLMRNIVRV 1-675-51
10.00 I 597 LLLFFGKLL 1.350 364 1 LLLICIAYW
10.4501
424 CVFQGYSSK o 365 LLICIAYWA , 1.350 564
1 AIYGKNFCV 10.450-
1 244 LLFILLLRL 9.000 506 ALILTLVQI
1.350 237 I GVALVLSLL 10.4051
1 464 1 VLAGAFASF 9.000 14.8 CLPGVPWNM 1.350 38 1
CVLFLLFIL 10.405
144
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Table XII-V1-HLA-A3-9mers- Table XII-V5-HLA-A3-9mers- Table XII-V7-HLA-
A3-9mers-
24P4C12 24P4C12 24P4C12
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ I Start] Subsequence Score
ID NO: 3; each start position is ID NO: 11; each start
position is 1 8 AVGQMMSTM 0.030
specified, the length of peptide is specified, the length of peptide is
1 4 9.027
9 amino acids, and the end 9 amino acids, and the end WILVAVGQM
position for each peptide is the position for each peptide is
the 1 6 LVAVGQMMS 0.008
start position_plus eight. , start
position plus eight. 1 7 ' VAVGQMMST 0.007
Start 1 Subsequence (Score I Start I Subsequence Score, 1 SVVYWILVAV
0.002
204 GISGLIDSL 0.405 1 0 r 2
, VVYWILVAVG 0.000
35 VICCVLFIL 0.405 1 5 1 ILLLVLIFL 4.050 3
YWILVAVGQ 0.000
317_( AVLEAILLL 10.405 1 4 1 AILLLVLIF 11.800
240 LVLSLLF IL 10.405 1 9 VLIFLRQRI , 0.900 Table
XIIA/13-HLA-A3-9mers-
668 LCFLEDLER 0.400 1 1 VLEAILLLV 0.900 24P4C12
388 VLWASNISS 10.400 7 1 LLVLIFLRQ 10.270 Each
peptide i a portion of SEQ
489 SAFIRTLRY 10.400 8 LVLIFLRQR 0.270 ID
NO: 17; each start position is
specified, the length of peptide is
1 211 SLNARDISV , 0.400 1 2 1 LEAILLLVL 10.005 9
amino acids, and the end
85 YLLYFNIFS 10.360 3 1 EAILLLVLI 10.004
position for each peptide is the
start position plus eight. ,
Table XII-V3-HLA-A3-9mers- Table X1146-HLA-A3-9mers- Start I
Subsequence Score
24P4C12 24P4C12 4
WLPIMRNPI _ 0.600
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ f 7 1 IMRNPITPT 0.225
ID NO: 7; each start position is ID NO: 13; each start
position is 19 FQTSILGAY _ 10.081
specified, the length of peptide is specified, the length of peptide is
, 1I
NYYWLPIMR 10.040
9 amino adds, and the end 9 amino acids, and the end
position for each peptide is the position
for each peptide is the 11 I PITPTGHVF 10.030
start position plus eight. start position ilus eight. I 17
HVFQTSILG 10.020
,
Start Subsequence Score Start Subsequence Score 13 1 TPTGHVFQT 0.013
9 ITPPALPGI 0.068. . 1 7 LIPRSVFNL 0.540 20 1
QTSILGAYV 0.010
6 VVTNITPPAL 0.030 6 I GLIPRSVFN 10.135 16
GHVFQTSIL 0.003
2 1 RCFPWTNIT 0.022 2 I YSSKGLIPR 10.060 15 TGHVFQTSI 0.002
8 NITPPALPG 0.009 1 5 f KGLIPRSVF 10.013 5
LPIMRNPIT 0.002
1 1 GRCFPVVTNI [OAR 1 8 f IPRSVFNLQ 10.001 10
NPITPTGHV 10.001
1 4 1 FPWTNITPP 10.002 1 3 SSKGLIPRS 0.000 12 ITPTGHVFQ 10.001
7 TNITPPALP 0.000 9 PRSVFNLQI 10.000 14
PTGHVFQTS 0.001
3 CFPWTNITP 0.000 1 GYSSKGLIP
10.000 ' = 18 VFQTSILGA 0.001
1 5 1 PVVTNITPPA 0.000 1 4 SKGLIPRSV 10.000 6 PIMRNPITP 10.001
2 YYWLPIMRN 0.000
Table XII-V5-HLA-A3-9mers- Table X11-1/7-HLA-A3-9mers- r 9
RNPITPTGH 0.000,
24P4C12 24P4C12 8 MRNPITPTG 0.000 *
Each peptide is a portion of SEQ Start Subsequence Score 1
3 YWLPIMRNP 0.000
ID NO: 11; each start position is Each peptide is a portion of SEQ
specified, the length of peptide is ID NO: 15; each start position is
9 amino acids, and the end specified, the length of peptide is = Table
XII-V9-HLA-A3-9mers-
position for each peptide is the _______________________ 9 amino acids, and
the end 24P4C12
start position plus eight position for each peptide is the
I Start Subsequence ,ITCO-R start position plus eight.
6 1 LLLVLIFLR 127.00 1 5 1 ILVAVGQMM 11-67450
145
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Each peptide is a portion of SEQ Table XlII-V1-HLA-A3-10mers-
Table XIII-V1-HLA-A3-10mers-
ID NO: 19; each.start position is 24P4C12 24P4C12
specified, the length of peptide is' Each peptide is a portion of SEQ Each
peptide is a portion of SEQ
9 amino acids, and the end ID NO: 3; each start position is ID
NO: 3; each start position is
position for each peptide is the specified, the length of peptide is
specified, the length of peptide is
start position plus eight. 10 amino acids, and the end
10 amino acids, and the end
Start I Subsequence [Score position for each peptide is
the position for each peptide is the
15 1 ATLGYVLWA 10.405 start position plus nine, start position plus
nine.
16 I TLGYVLWAS 10.270
Start Subse. uence 'Score I Start Subsequence Score
6 J ALYPLPTQP [0.150 1 0 1 354 TMFYPLVTFV = 3.000
11 PTQPATLGY riTgl) 322 ILLLMLIFLR 27.00
1 72 1 GAYCGMGENK 13.000
9 PLPTQPATL 0.060 0 1 324 I LLMLIFLRQR
[2.700
-
27.00
2 WAMTALYPL 0.041 584 NIVRVVVLDK 1
660 GMCVDTLFLC 2.700
. 0
4 MTALYPLPT 10.030 24.30 467
1 GAFASFYVVAF 1 2.700
3 AMTALYPLP 0.020 433 GLIQRSVFNL
0 243 1 SLLFILLLRL 12.700
13 QPATLG-YVL 10.018 24.00 83
r KPYLLYFNIF 2.700
18 GYVLWASNI 10.008
262 ILGVLGVLAY0 42 1 LLFILGYIVV 2.000-
12 TQPATLGYV 10.003
272 GIYYCWEEYR 18.00 1 518 1 ILEYIDHKLR 12.000
8 YPLPTQPAT 11-67657 0 1 161
SLQQELCPSF 12.0001
5I TALYPLPfb 0.001 464 VLAGAFASFY 18.00 1
337 1 AIALLKEASK 2.000
7
LYPLPTQPA Fig
0 1 362 1 FVLLLICIAY 1.800 ,
13.50
LPTQPATLG 0.000 665 TLFLCFLEDL 1 650
1_VIASGFFSVF 1.800
0
14 .11 PATLGYVLW _10.000 '13.50 t. 606 1 VVGGVGVLSF 1.800
17 LGYVLWASN 0.000 516 RVILEYIDHK
0 1 507 1 LILTLVQIAR 11.800
1 YWANTrALYP 10.000 - -
13.50 1 329 1 FLRQRIRIAI 11.800
86 LLYFNIFSCI 0 1 318
VLEAILLLML 11.800
_
Table XIII-V1-HLA-A3-10mers- 171 LLPSAPALGR 12.00 [ 624
GLGKDFKSPH 11.800
24P4C12 0 1 309
WLAALIVLAV 1.800
Each peptide is a portion of SEQ 578 FMLLMRNIVR 12.00 1
312 I ALIVLAVLEA 11.800
ID NO: 3; each start position is 0 L
469 1 FASFYWAFHK 1.800
specified, the length of peptide is 1 76 GMGENKDKPY 1_9.000
[ 64 1 VLYPRNSTGA 1.500
10 amino acids, and the end 1 594 VTDLLLFFGK 9.000
position for each peptide is the [ 364 ILLICIAYWA 1.350
350 QMMSTMFYPL 8.100
start position plus nine.
657 1 SVFGMCVDTL 1.350
I Start I Subsequence !Score!. 1 667
FLCFLEDLER 8.000 85 , YLLYFNIFSC :1.350
544 CLWCLEKFIK 300.0 1
56 WLYGDPRQVL 6.750 1 220 [ KIFEDFAQSW 1.350
00 333 RIRIAIALLK 6.000 264 1 GVLGVLAYGI
1.215
180.0 1 609 GVGVLSFFFF 5.400
39 VLFLLRLGY
315 f VLAVLEAILL 1.200
00 1 241 VLSLLFILLL 5.400 .-
- 237 L GVALVLSLLF 1.200
1
120.0 561 IMIAIYGKNF 4.500
680 SLDRPYYMSK 554 FLNRNAYIMI 1.200
00
- - 1 239 ALVLSLLFIL
4.050 590 _. VLDKVTDLLL 1.200
612 VLSFFFFSGR 36.00 1 49 IVVGIVAWLY 4.050
0 1
265 VLGVLAYGIY Fig]
1 378 YLATSGQPQY 4.000 1
35 [ VICCVLFLLF 11.200
1 134 QTVGEVFYTK 3 37
5 I 441 NLQIYGVLGL 3.600 1
53 IVAWLYGDPR 11.200
30.00 235 1 ALGVALVLSL 3.600 447 VLGLFWILNW 11.2001
211 SLNARDISVK 0 598 1 LLFFGKLLVV 3.000 1
268 VLAYGIYYCW 10.900
1 449 ___________________ I GLFWTLNWVL 127.00 621 RIPGLGKDFK 3.000
, 413 HLVNSSCPGL 0.900
(
146
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WO 2004/050828 PCT/US2002/038264
Table XIII-V141LA-A3-10mers- Table X111-V3-HLA-A3-10mers-
Each peptide is a portion of SEQ
24P4C12 24P4C12 ID NO: 13;
each start position is
Each peptide is a portion of SEQ 1 Start 1 Subsequence [Score
specified, the length of peptide is
ID NO: 3; each start position is Each peptide is a portion of SEQ 10
amino acids, and the end
specified, the length of peptide is ID NO: 7; each start position is
position for each peptide is the
amino acids, and the end specified, the length of peptide is
start position plus nine.
position for each peptide is the 10 amino
acids, and the end Start 1 Subsequence Score
start position plus nine, position for each peptide is the
36.45
7 GLIPRSVFNL
Start Subsequence [Score start
position plus nine. 0
275 1 YCWEEYRVLR _10.900 1 9 1 NITPPALPGI 10.135 [ 2 1
GYSSKGLIPR 1 0.036
232 1 ILVALGVALV 10.900 1 5 1 FPVVTNITPPA 10.015 1 9 1
IPRSVFNLQI 10.036
325 1 LMLIFLRQRI 0.900 1 3 RCFPWTNITP 10.003 1 8 õ1
LIPRSVFNLQ 10.009
463 _ CVLAGAFASF 10.900 10 1 ITPPALPGIT 10.002 1 5 1
SKGLIPRSVF 10.003
525 _1 KLRGVQNPVA 10.900, 7 1 WTNITPPALP 10,002 1 10
i PRSVFNLQIY 10.001
506 1 ALILTLVQ1A " 0.900 1 1 , LGRCFPVVTN1 F.7-1 L 3 1 YSSKGLIPRS
0.000"
603 1 KLLVVGGVGV 10.900 I 2 GRCFPWTNIT 10.001 1
4 II SSKGLIPRSV 0.000
633 1 HLNYYWLPIV_I j1,010,0 I 8 1 TNITPPALPG 10.000 1 1 I
QGYSSKGLIP 1 0.000
510 TLVOIARVIL 10.900 1 6 1 PWTNITPPAL 10.000 1 6 1
KGL1PRSVFN 0.000
365 1 LLICIAYWAM 0.900, 1 4 1 CFPWTNITPP 10.000
1 41 FLLFILGYIV 0.900
Table X11147-HLA-A3-10mers-
1 512 1 VQIARVILEY 10.810 ' Table XIII-V5-HLA-A3-10mers- 24P4C12
1 604 1 LLVVGGVGVL 10.810 24P4C12 1 Start 1
Subsequence (Score
251 I RLVAGPLVLV 10.675, Each peptide is a portion of SEQ Each peptide
is a portion of SEQ
260 11 VLILGVLGVL 10.608
ID NO: 11; each start position is ID NO: 15; each start position is
1 specified, the length of peptide is
specified, the length of peptide is
1 44 1 FILGYIVVGI 10.608 10 amino acids, and the end 10 amino acids,
and the end
1 107 GLQCPTPQVC 10.600 position for each peptide is the
position for each peptide is the
1 327 LIFLRQRIRI 10.600 start position plus nine,
start position plus nine.
326 1 MLIFLRQRIR 10.600 [Start _ Subsequence (Score 9 1 AVGQMMSTMF
10.200
1 597 1 LLLFFGKLLV 0.600 6 ILLLVLIFLR 27.00 6
ILVAVGQMMS '10.120
1 5 1 487 1 LISAFIRTLR 10.600 - o
WILVAVGQMM 0.045
1
120 CPEDPWTVGK 10.600 8 LLVL1FLRQR 12.700 7 LVAVGQMMST 0.030
1 351 MMSTMFYPLV 10.6001
i 1 2 VLEAILLLVL [1.8001[1.80011 1
QSWY1NILVAV 0.011
1 k
I
240 LVLSLLFILL 0.540 10 1 VLIFLRQRIR F.T) 8 1
VAVGQMMSTM 0.007
5
I 252 LVAGPLVLVL 0.540 I AILLLVLIFL (17.4531
1 2 1 SINYWILVAVG 0.000
7
1 360 1 VTFVLLLICI 10.450' LLLVLIFLRQ 110.2701 1 4 YWILVAVGQM 0.000
1 1
I 363 VLLLICIAYW 0.450 AVLEA1LLLV 10.203 1 3
INYWILVAVGQ 10.0001
1 9 1
I 579 MLLMRNIVRV 10.45011 LVLIFLRQRI 10.0901
95 ILSSNIISVA 10.4501
i 1 4 EAILLLVLIF 0M TableTable X111-V8-HLA-A3-
10mers-
3 1 LEAILLLVLI 10.003 24P4C12
Each peptide is a portion of SEQ
Table XIII-V3-HLA-A3-10mers-
24P4C12 Table XIII-V6-HLA-A3-10mers- ID
NO: 17; each start position is
24P4C12 specified,
the length of peptide is
'Start I Subsequence [Score - 10 amino acids, and the end
position for each peptide is the
start position plus nine.
1 Start 1 Subsequence [Score
1 18 1 HVFQTSILGA 10.300
147
=
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Table XIII-V8-HLA-A3-10mers- Table XIII-V9-HLA-A3-10mers-
Table XIV-V1-HLA-A1101-9mers-
24P4C12 24P4C12 24P4C12
Each peptide is a portion of SEQ Each peptide is a portion of SEQ [
Start Subsequence Score
ID NO: 17; each start position is ID NO: 19; each start position is 1
689 KSLLKILGK 0.180 _
specified, the length of peptide is specified, the length of peptide is
516 RVILEYIDH 0.180
amino acids, and the end 10 amino acids, and the end ,
position for each peptide is the position for each peptide is the I 609
GVGVLSFFF 0.180
start position plus nine, start position plus nine. 1_ 485
FPLISAFIR 0.180
Start Subsequence Score I Start Subsequence Score I 446 GVLGLFWIL
0.180
5 WLPIMRNPIT 0,100 15 PATLGYVLWA 0.004 [ 267
GVLAYGIYY 0.180
1 21 QTSILGAYVI 10.0901 1 10 PLPTQPATLG 10.003 273
IYYCWEEYR 10.160
1 1 1 LNYYWLPIMR 10.080 1 2 YWAMTALYPL 10.003 I 508 ILTLVQIAR
10.160
1 13 ITPTGHVFOT 10,045, 1 5 MTALYPLPTQ 0.002 668
LCFLEDLER 0.160
11 NPITPTGHVF 1-6701 14 QPATLGYVLW 10.002 698
KNEAPPDNK 0.120
1 8 IMRNPITPTG 10.030 12 PTQPATLGYV 10.001 1
470 ASFYWAFHK Fill
1 15 PIGHVFQTSI 10.009 1 6 TALYPLPTQP 0.000 593
KVTDLLLFF 0.120
FQTSILGAYV 10.006 1 3 WAMTALYPLP 0.000 701
APPDNKKRK _0.100
7 PIMRNPITPT [0.003 I 1 AYWAMTALYP 0.000 595
TDLLLFFGK 0.090
14 TPTGHVFQTS [0.003 1 19 1 GYVLWASNIS 0.000 1 38
CVLFLLFIL 10.090
19 VFQTSILGAY 10.003 1 8
LYPLPTQPAT 0.000 L/!4 311 LVLSLLFIL 10.090
I 4 YWLPIMRNPI 10.001 54 VAWLYGDPR
[0.080 '
6 LPIMRNPITP 10.001 172
LPSAPALGR [0.080
16 TGHVFQTSIL 0.001 Table XIV-V1-HLA-A1101-9mers- I
349 GQMMSTMFY 0.072
24P4C12
2 NYYWLPIMRN 0.000 1 334 IRIAIALLK
10.060
1 Start ___________________________________ 11 Subsequence 'Score
9 MRNPITPTGH 0.000 545 LWCLEKFIK 10.060
Each peptide is a portion of SEQ
12 PITPTGHVFQ 0.000 [ 567 GKNFCVSAK
10.060
ID NO: 3; each start position is
17 I GHVFQTSILG 0.000 specified, the length of peptide is
1 317 AVLEAILLL [0.060'
10 RNPITPTGHV 0.000 9 amino acids, and the end
1 699 NEAPPDNKK [0.060
3I YYWLPIMRNP 10.000 position for each peptide is the1_ 151
GVPWNMTVI 0.060
start-position plus nine.
237 GVALVLSLL 10.060
000
Table XIII-V9-HLA-A3-10mers- 135 1 TVGEVFYTK 4. 257 LVLVLILGV
10.060
24P4C12 585 1 IVRVVVLDK 14.000
I 20 DPSFRGPIK 10.060
000GYSSK 4.
Each peptide is a portion of SEQ 424 CVFQ 1 575 KNAFMLLMR
10.048
ID NO: 19; each start position is 560 YIMIAIYGK 11.600
1 212 LNARDISVK 0.040
specified, the length of peptide is 685 1 YYMSKSLLK 1.600
10 amino acids, and the end 359 LVTFVLLLI
0.040
542 KCCLWCLEK 1.200
position for each peptide is the 16 PVKYDPSFR 0.040
start position plus nine. 690 1 SLLKILGKK 10.600
304 f SVQETWLAA 0.040
I Start I Subsequence Score 517 1 VILEYIDHK [0.600
[ 619 f SGRIPGLGK 0.040
7 1 ALYPLPTOPA 1778-61 73 1 AYCGMGENK 0.400
50 1 VVGIVAWLY 10.040
4 AMTALYPLPT PRI 393 1 NISSPGCEK 0.400
__________________________________________ - 681
LDRPYYMSK 0.040
11 LPTQPATLGY 10.080 1 207 I GLIDSLNAR 0.360
662 CVDTLFLCF 0.040
13 TQPATLGYVL 0.054 323 LLLMLIFLR 10.360
1 7 DEDDEAYGK 0.0361
16 1 ATLGYVLWAS 10.030 338 I IALLKEASK 0.300
[ 83 I KPYLLYFNI 0.036
17 1 TLGYVLWASN 10.020 I 579 I MLLMRNIVR 110.240
1 47 GYIVVGIVA 0.0361
9 1 YPLPTQPATL 15707-3- 243 1 SLLFILLLRH 0.240
1 251 RLVAGPLVL 0.036
IPGLGKDFK 10.200
18 1 LGYVLWASNI 10.009 -I 622 I 1 383 1
GQPQYVLWA 0.036
= 148
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Table XIV-V1-HLA-A1101-9mers- Table XIV-V6-HLA-A1101-9mers-
24P4C12 Table XIV-V3-HLA-A1101-9mers-
24P4C12
Start Subsequence Score 24P4C12 I Start I
Subsequence IScore
49 IVVGIVAWL 10.030 (Start Subsequence Score 7 1
LIPRSVFNL 0.012
314 IVLAVLEAI 10.030 Each peptide is a portion of SEQ 2
[ YSSKGLIPR 0.008
456 WVLALGQCV 10.030
ID NO: 7; each start position is
[ 1
specified, the length of peptide is 1 1 GYSSKGLIP 0.002
1 589 VVLDKVTDL 10.030 9 amino
acids, and the end 6 1 GLIPRSVFN 10.002
452 WTLNWVLAL 0.030 position for each peptide is the L
5 i KGLIPRSVF 10.001
141 YTKNRNFCL 10.030 start position plus eight. I
1 8
IPRSVFNLQ 0.000
1 498 HTGSLAFGA 10.030 9 ITPPALPGI
0.010 1 9 1 PRSVFNLQI FET
1 605 LVVGGVGVL 10.030 6 WTNITPPAL ITLIT 1 4
SKGLIPRSV 0.000
1 362 FVLLLICIA 0.030 1 2 RCFPVVTNIT
0.001 1 3 SSKGLIPRS 10.000
1 611 GVLSFFFFS 0.027 1 8 1 NITPPALPG 0.001
137 GEVFYTKNR 0.027 1 GRCFPVVTNI
0.001 Table XIV-V7-HLA-A1101-9mers-
564 AIYGKNFCV 0.024 1 4 1 FPVVTNITPP 10.000 24P4C12
272_ GIYYCWEEY 0.024 3 CFPWTNITP 10.000
Each peptide is a portion of SEQ
60 DPRQVLYPR 0.024 7 l' TNITPPALP 10.000 ID NO: 15;
each start position is
421 GLMCVFQGY 0.024 5 1 PVVTNITPPA FT specified,
the length of peptide is
9 amino adds, and the end
467 1 GAFASFYWA 0.024 position for each peptide is the
449 GLFVVTLNWV 0.024 Table XIV-V5-HLA-A1101-9mers-
start position plus eight.
24P4C12
660 , GMCVDTLFL 0.024 Start Subsequence Score
Each peptide is a portion of SEQ 8 I AVGQMMSTM 0.020
496 RYHTGSLAF 0.024 ID NO: 11; each start
position is
511 LVQIARVIL 0.020 specified, the length of peptide is
5 ILVAVGQMM 0.006
218 SVKIFEDFA 0.020 9 amino acids, and the end 1
4 WILVAVGQM 0.006
233 , LVALGVALV 0.0201 position for each
peptide is the 1 6 1 LVAVGQMMS 10.004
start position plus eight. , 1 2
1' WYWILVAVG 10.001
22 SFRGPIKNR 0.020
Start Subsequence Score I 7 1 VAVGQMMST 0.001
, 75 CGMGENKDK 0.020
1 6 LLLVLIFLR 0.360 1 1 SWYWILVAV 0.000
414 LVNSSCPGL 0.020
1 8 LVLIFLRQR 0.060 3 YWILVAVGQ 10.000
252 LVAGPLVLV 0.020
- - 4
571 CVSAKNAFM 0.020 1 AILLLVLIF 0.0125 ILLLVLIFL 0.012
347 1 AVGQMMSTM 0.020 Table XIV-V8-HLA-A1101-9mers-
534 ARCIMCCFK Ip.620
I 1 VLEAILLLV 10.008 24P4C12
g
9 VLIFLRQRI 10.006 Each peptide is a portion of SEQ
527 RGVQNPVAR 0.018
34 1 DVICCVLFL 0.018
2 LEAILLLVL 10.001 specified, the length of peptide is
3 1
I 461 GQCVLAGAF 0.018 EAILLLVLI 10.0011 position for
each peptide is the
4 KQRDEDDEA 0.018
start position plus eight.
1
Table X1V-V6-HLA-A1101-9mers- Start Subsequence (Score
I 331 1 RQRIRIAIA 0.018 24P4C12 1 I_
NYYWLPIMR 0.320
442 LQIYGVLGL 0,018 Each peptide is a portion of
SEQ 1 17 HVFQTSILG 0.008
1 255 GPLVLVLIL 0.018 ID NO: 13; each start
position is 1 19 FQTSILGAY 1-0..001
1 598 LLFFGKLLV 0.016 specified, the length of
peptide is
42 LLFILGYIV 0.016 9 amino
acids, and the end 1 18 1 VFQTSILGA 10.004
position for each peptide is the 1 4 WLPIMRNPI 10.004
244 LLFILLLRL 0.016 start
position plus eight. 1 10 1. NPITPTGHV 10.0031
1 327 LIFLRQRIR 0.016
149
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Table XIVA/8-HLA-A1101-9mers- Table XIV-V9-HLA-A1101-9mers- Table XV-V1-
A1101-10mers-
24P4C12 24P4C12 24P4C12
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ Each peptide is a portion of SEQ
ID NO: 17; each start position is ID NO: 19; 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
9 amino acids, and the end 9 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 eight. start position plus eight. start position
plus nine.
Start Subsequence Score 1 Start - Subsequence Score 1 Start Subsequence
Score,
2 1 YYWLPIMRN 0.002 1 14 PATLGYVLW 0.000 684
., PYYMSKSLLK 0.160
9 1 RNPITPTGH 10.001 1 17 LGYVLWASN 10.000 667 1 FLCFLEDLER 0.160
12 1 ITPTGHVFQ 0.001 1 1 YWAMTALYP 0.000 171 LLPSAPALGR 0.160.
16 1 GHVFQTSIL 0.001 6 RDEDDEAYGK 0.120
13 1 TPTGHVFQT 0.001 Table XV-V1-A1101-10mers-
698 1 KNEAPPDNKK 0.120
11 PITPTGHVF 0.000 24P4C12_
484 TFPLISAFIR 0.120
-
, 7 1 IMRNPITPT 0.000 Each peptide is a portion of
SEQ 237 1 GVALVLSLLF 0.120
,
ID NO: 3; each start position is _____________________________________ ,
LPIMRNPIT 0.000 1 74 YCGMGENKDK 0.100
specified, the length of peptide is
TGHVFQTSI 0.000 10 amino acids, and the end 1 689
KSLLKILGKK 0.090
6 PIMRNPITP 0.000 position for each peptide is the 649 1
YVIASGFFSV 0.090
14 PTGHVFQTS 0.000start position plus nine. 281
RVLRDKGASI 0.090
, .
8 MRNPITPTG 0.000 I Start Subsequence Score
z, 612 VLSFFFFSGR 0.080
3 YWLPIMRNP 0.000 516 I RVILEYIDHK 9.000 487
LISAFIRTLR 0.080-
1 594 - VTDLLLFFGK 3.000 275 YCWEEYRVLR 0.080'
Table XIV-V9-HLA-A1101-9mers- 1 134 1 QTVGEVFYTK 3.000 438 1 SVFNLQIYGV
0.080
24P4C12 ' 1 333 RIRIAIALLK 2.400
1 697 KKNEAPPDNK 0.060
Each peptide is a portion of SEQ 544 CLWCLEKFIK 2.400 1
392 1 SNISSPGCEK 0.060
ID NO: 19; each start position is 621 RIPGLGKDFK 1.200 571
CVSAKNAFML 0.060
.
specified, the length of peptide is 559 AYIMIAIYGK 1.200 1 259
LVLILGVLGV 0.060
9 amino acids, and the end
position for each peptide is the 1 72 GAYCGMGENK 1.200 49
1 IVVGIVAWLY 0.060_
start position plus eight 1 584 NIVRVVVLDK 1.200 240
LVLSLLFILL 10.060_
Start 1 Subsequence 'Score 680 SLDRPYYMSK 0.800 317
AVLEAILLLM 0.060.
_
15 1 ATLGYVLWA 10.030 469 FASFYWAFHK 0.600 362 FVLLLICIAY 10.060
18 GYVLWASNI 0.018 272 GIYYCWEEYR 10.480 433
GLIQRSVFNL 0.054
2 1 WAMTALYPL 0.008 1 428 GYSSKGLIQR , 0.480
449 1 GLFWTLNVVVL 0.048
12 TQPATLGYV 10.006 337
AIALLKEASK 0.400 1 493 1 RTLRYHTGSL 0.045
7 1 LYPLPTQPA 0.004 53 IVAWLYGDPR 10.400 518 1 ILEYIDHKLR 0.040
13 - QPATLGYVL 0.004 211 SLNARDISVK 10.400 252 1 LVAGPLVLVL 0.040
4 MTALYPLPT 0.002 322 ILLLMLIFLR 0.360
618 1 FSGRIPGLGK 0.040
. .
11 PTQPATLGY 0.002 423 MCVFQGYSSK 0.300 688
SKSLLKILGK 0.040
6 1 ALYPLPTQP 10.001 507 LILTLVQIAR 0.240 606 1 VVGGVGVLSF
0.040
16 TLGYVLWAS 0.001 578 FMLLMRNIVR 0.240 541
1 FKCCLWCLEK 0.040
3 AMTALYPLP 0.000 120 CPEDPWTVGK 0.200 657 1
SVFGMCVDTL 0.040
9 1 PLPTQPATL 10.000 533 1 VARCIMCCFK 0.200 1 360 1 VTFVLLLICI
0.040
8 1 YPLPTQPAT 10.000 15 1 KPVKYDPSFR 0.180 233 LVALGVALVL 0.040
5 TALYPLPTQ 10.000 264 1 GVLGVLAYGI 15.7173.61
331 1 RQRIRIAIAL 0.036
10 LPTQPATLG 0.000 609 1 GVGVLSFFFF 0.180 589
1 VVLDKVTDLL 10:01
150
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Table XV-V1-A1101-10mers- Table V-V1-A1101-10mers- Table XV-V5-HLA-
A1101-
24P4C12 24P4C12 10mers-24P4C12
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: 11; each start position is
specified, the length of peptide is specified, the length of
peptide is specified, the length of peptide is
10 amino acids, and the end 10 amino acids, and the end 10 amino adds,
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.
I Start - Subsequence "(Score I Start I Subsequence fi707;
I Start I Subsequence Score
157 TVITSLQQEL 10.030 267 GV1AYGIYYC 1-6.7-81 1
7 1 LLLVLIFLRQ 10.001
463 CVLAGAFASF 10.0301 3 r LEAILLLVLI
10.001
588 VVVLDKVTDL 10.030 Table XV-V3-HLA-A1101-
I 700 EAPPDNKKRK 0.030 lOmers-24P4C12 Table XV-V6-
HLA-A1101-
.
I 314 IVLAVLEAIL 10.0301 Each peptide is a portion of SEQ
10mers-24P4C12
ID NO: 7; each start position is
456 VWLALGQCVL 10.0301 Each peptide
is a portion of SEQ
specified, the length of peptide is ID NO: 13; each start position is
257 LVLVLILGVL 0.030 10 amino
acids, and the end specified, the length of peptide is
34 DVICCVLFLL 10.027 position for each peptide is
the 10 amino acids, and the end
611 GVLSFFFFSG , 0.027 start position plus nine,
position for each peptide is the
59 - GDPRQVLYPR 0.024 [ Start I Subsequence (Score start position plus
nine.
, ..
220 - KIFEDFAQSW 10.024 1 9 NITPPALPGI 10.004 I Start
Subsequence (Score
654 GFFSVFGMCV 10.024 1 5 FPVVTNITPPA 10.004 2
GYSSKGLIPR 10.480 =
_
548 - LEKFIKFLNR 10.024 1 3 I RCFPVVTNITP [0.002 7 GLIPRSVFNL 10.054
_
.:
467 GAFASFYWAF 10.024 1 7 I VVTNITPPALP 11757- 9
IPRSVFNLQI 1 0.004
674 - LERNNGSLDR 10.024 1 10 I ITPPALPGIT [0.001 8 LIPRSVFNLQ
10.000
1 347 AVGQMMSTMF 10.020 4 CFPWTNITPP 0.000 5 SKGLIPRSVF 10.000
1 566 YGKNFCVSAK 110.020 1 1 I LGRCFPWTNI 0.000 6 ,
KGLIPRSVFN 10.0001
I 353 _ STMFYPLVTF 0.020 1 8 I TNITPPALPG 0.000 1 1 QGYSSKGLIP 0.000
1 585 _ IVRVVVLDKV Pal 1 2 1 GRCFPWTNIT 10.000 1 10 PRSVFNLQIY 0.000
701 APPDNKKRKK 15:-0761 1 6 I PWTNITPPAL 1r07"0-815" 1
4 , SSKGLIPRSV ,10.000,
1 304 SVQETWLAAL 10.020 1 3 YSSKGLIPRS
10.000
380 ATSGQPQYVL 10.020 Table XV-V5-HLA-A1101-
I
,
662 0.020 , l0mers-24P4C12 Table ki-V7-HLA-A1101-
1 CVDTLFLCFL 1 I
414 LVNSSCPGLM 10.020
Each peptide is a portion of SEQ l0mers-24P4C12 ID NO: 11; each start
position is Each peptide is a portion of SEQ
19 YDPSFRGPIK IMFli specified, the length of peptide is
ID NO: 15; each start position is
(116 CVSSCPEDPW 11-072-6-1 10 amino
acids, and the end specified, the length of peptide is
1 186 NVIPPALPGI 10.0201 position for each peptide is
the 10 amino acids, and the end
start position plus nine.
1 642 MTSILGAYVI 1110.018 0.020 position for
each peptide is the
512
Start Subsequence (Score start
position plus nine.
1 1 VQIARVILEY
6 ILLLVLIFLR 10.360 IStart
Subsequence (Score
478 KPQDIPTFPL 10.018
I 47 GYIVVGIVAW 10.018
1 AVLEAILLLV 1676-01 9
AVGQMMSTMF 0.020
'
9 1 LVLIFLRQRI 10.030 5 WILVAVGQMM 0.006 461
GQCVLAGAFA 10.018
239 ALVLSLLF IL 10.018 r 8 LLVLIFLRQR [0.012 7
LVAVGQMMST 0.004
4 KQRDEDDEAY 10.018 10 VLIFLRQRIR 10.012 8
VAVGQMMSTM 10,003
603 KLLWGGVGV '0.018( AILLLVLIFL [0.012 6 ILVAVGQMMS
10.0011
1
1
1 553 KFLNRNAYIM 16.761 2 VLEAILLLVL 10,008 3
WYWILVAVGQ 0.001_
i
I 4
(163 1 QQELCPSFLL 11(7
EAILLLVLIF 1-61-671 1 QSVVYWILVAV 10.000
, -01-61
151
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-
Table XV-V7-HLA-A1101- Each peptide is a portion of SEQ
Table XVI-V1-HLA-A24-9mers-
10mers-24P4C12 ID NO: 19; each start position is
24P4C12
Each peptide is a portion of SEQ specified, the length of
peptide is Each peptide is a portion of SEQ
ID NO: 15; each start position is 10 amino acids, and the end
ID NO: 3; each start position is
specified, the length of peptide is position for each peptide is
the spedfied, the length of peptide is
amino acids, and the end start position plus nine. 9 amino acids, and
the end
position for each peptide is the Start I Subsequence Score
position for each peptide is the
startposition plus nine. 13 If TQPATLGYVL 10.012 start position plus
eight
I Start I Subsequence IScore 7 1 ALYPLPTQPA 10.008 Start Subsequence
'Score
4 1 YWILVAVGQM 10.000 11 LPTQPATLGY 10.00430.00
88 YFNIFSCIL
2 1 SVVYWILVAVG 0.000 9 1 YPLPTQPATL 0.003 0
___________________________________________________ p ________
. 30.00
16 1 ATLGYVLWAS 10.003 666 LFLCFLEDL 0
Table XV-V8-HLA-A1101- 14 I QPATLGYVLW 10.002 .
10mers-24P4C12 450
LFVVTLNVVVL 24.00
19 GYVLWASNIS
10.002 0
Each peptide is a portion of SEQ 1 I AYWAMTALYP 0.002 24.00
ID NO: 17; each start position is 503 AFGALILTL
specified, the length of peptide is 12 1 PTQPATLGYV 0.001, 0
10 amino acids, and the end 5 1 MTALYPLPTQ 10.001 21.60
84 PYLLYFNIF
position for each peptide is the 4 1 AMTALYPLPT 0.001 0
start position plus nine. 20. 0
8 LYPLPTQPAT 0.000 540 CFKCCLWCL
I Start Subsequence Score 0
18 1 LGYVLWASNI 10.000
1 18 HVFQTSILGA 0.080 20.00
15 I PATLGYVLWA 0.000 684 PYYMSKSLL
1 LNYYWLPIMR 0.032 0
17 1 TLGYVLWASN 0.000 _____ 20.00
21 QTSILGAYVI 0.020 617 FFSGRIPGL
3 WAMTALYPLP 0.000 0
FQTSILGAYV 0.006
1 2 1 YWAMTALYPL 0.000 20.00
11 NPITPTGHVF 10.003 658 VFGMCVDTL
6 TALYPLPTQP 0.000 ___ 0
13 ITPTGHVFQT 10.003
k ' 1 10 1 PLPTQPATLG 0.000 15.00
19 VFQTSILGAY 10.002 553 KFLNRNAYI
_________________________________________________________________ 0
2 NYYWLPIMRN 10.002 ' 12.00
Table XVI-V1-HLA-A24-9mers- 251 RLVAGPLVL
10 RNPITPTGHV
1070-61-1 24P4C12 0
15 PIGHVFQTSI 10.001 Each peptide is a portion of SEQ -
583 RN1VRVVVL 12.00
6 LPIMRNPITP 0.001 ID NO: 3; each
start position is 0
8 IMRNPITPTG 10.000 specified, the length of peptide is
484 TFPLISAFI 10.50
5 WLPIMRNPIT 10.000 9 amino acids, and the end _
0
position for each peptide is the 10.50
4 YWLPIMRNPI 10.000 start position plus eight. 47
GYIVVGIVA 0
14 TPTGHVFQTS 10.000 420.0- ______________ 301 0
356 FYPLVTFVL - __
16 TGHVFQTSIL 0.000 00 10.00
- 468 AFASFYVVAF
17 GHVFQTSILG 10.000 288.0 0
57 LYGDPRQVL
7 PIMRNPITPT 0.000 00
139 VFYTKNRNF 10.00
3 YYWLPIMRNP FORT 496 RYHTGSLAF 200.0 _______ - 0
00 518 ILEYIDHKL 9.240
12 PITPTGHVFQ 10.000' -
150.0 361 1 TFVLLLICI 9.000
648 AYVIASGFF
00 1 577 I AFMLLMRNI 19.000
Table XV-V9-HLA-A1101- 84.00
10mers-24P4C12 87 LYFNIFSCI 0 446 1 GVLGLFVVTL 18.640
75.00 258 I VLVLILGVL 18.400
386 QYVLWASNI 49 1
- 0 IVVGIVAWL 18.400
152
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Table XVIV1-HLA-A24-9mers- Table XVIA/1-HLA-A24-9mers-
Each peptide is a portion of SEQ
24P4C12 24P4C12 ID NO: 7; each start position is
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ specified, the length of peptide is
ID NO: 3; each start position is ID NO: 3; each start position
is 9 amino acids, and the end
specified, the length of peptide is specified, the length of peptide is
position for each peptide is the
9 amino acids, and the end 9 amino acids, and the end start position
plus eight.
position for each peptide is the position for each peptide is
the Start Subsequence (Score
start position plus eight. start position plus eight. 6
VVTNITPPAL FOF
Start Subsequence Score I Start Subsequence Score 9 1 ITPPALPGI 11.800,
154 WNMTVITSL 8.400 1 452 VVTLNVVVLAL
16.000 2 1 RCFPVVTNIT 10.288,
311 AALIVLAVL 18.400- 1 242 1 LSLLFILLL 16.000 1 1
GRCFPVVTNI 19.100
1 261 LILGVLGVL 18.400 605 1
LVVGGVGVL 16.000 [ 3 1 CFPVVTNITP 10.075
1 440 FNLQIYGVL 18,400 638 1 WLPIMTSIL 6.000 7 1
TNITPPALP 10.015
234 VALGVALVL 8.400 511 , LVQIARVIL 6.000 5 1
P1NTNITPPA 0.014
1 683 RPYYMSKSL 18.000 163 QQELCPSFL 6.000 8
NITPPALPG 10.012
I 33 1 RIRIAIALL 18.000 291 SQLGFTTNL 6.000 4
FPVVTNITPP 10.010
596 1 DLLLFFGKL 17.920 434 LIQRSVFNL 16.000
1 65 _1 LYPRNSTGA 17.500 432 1 KGLIQRSVF 16.000
Table VIA/5-HLA-A24-9mers-
328 1 IFLRORIRI 17.500 1 225 FAQSWYWIL 16.000
24P4C12
I 317 1 AVLEAILLL ,17.200 322 ILLLMLIFL 6.000
Each peptide is a portion of SEQ
1 255 1 GPLVLVLIL 17.200 593 KVTDLLLFF 5.760
ID NO: 11; each start position is
1 38 CVLFLLFIL 7.200 241 VLSLLFILL
5.760 specified, the length of peptide is9 amino acids, and the end
240 , LVLSLLFIL 7.200 253 1
VAGPLVLVL 15.760 position for each peptide is the
(232 1 ILVALGVAL 7.200 1 237 1 GVALVLSLL 5.600 start
position plus eight.
589 1 VVLDKVTDL 7.200 1 228 1 SVVYWILVAL 5.600 [ Start I Subsequence
(Score
170 FLLPSAPAL 7.200 249 I LLRLVAGPL 5.600 1 5 I
ILLLVLIFL 18.400
357 1 YPLVTFVLL 7.200 35 VICCVLFLL rgobl 1 4 1 AILLLVLIF
13.600
236 LGVALVLSL 7.200 32 CTDVICCVL 5.600 MI VLIFLRQRI 12.160
621 1 RIPGLGKDF 7.2001 1 590 1 VLDKVTDLL
15.600 1 3 1 EAILLLVLI 11.800
158 1 VITSLQQEL 6.336 217 1 ISVKIFEDF 15.040 2
LEAILLLVL 0.480
305 VQETWLAAL 6.000 224 - DFAQSWYWI 15.000 1 1
VLEA1LLLV 0.210
15 KPVKYDPSF 6.000 614 1 SFFFFSGRI 5.000 7
LLVLIFLRQ 10.025
547 CLEKFIKFL 6.000 274 YYCWEEYRV 5.000 6 LLLVLIFLR 0.018
597 LLLFFGKLL 6.000 636 1
YYWLPIMTS 5.000 8 1 LVLIFLRQR 1-0771
565 IYGKNFCVS 6.000 370 1 AYWAMTALY 5.000 __ _
34 DVICCVLFL 6.000 573 1
SAKNAFMLL 14.800 Table XV1-V6=HLA-A24-9mers-
308 TWLAALIVL 6.000 (351 1 MMSTMFYPL 4.800 24P4C12
184 WINVIPPAL 6.000 1 315 1 VLAVLEAIL 4.800 Each
peptide is a portion of SEQ
ID NO: 13; each start position is
316 LAVLEAILL 6.000 100 IISVAENGL
4.800 specified, the length of peptide is
200 TIQQGISGL 16.000 204 1 GISGLIDSL 14.800 9 amino
acids, and the end
635 NYYWLPIMT 6.000 687 MSKSLLKIL
4.800 position for each peptide is the
140 FYTKNRNFC 16.000 1 244 LLFILLLRL 4.800
start position plus eight.
673 DLERNNGSL 16.000' 499 TGSLAFGAL
14.800 I Start , Subsequence [Score
442 LQIYGVLGL 6.000 1 5 KGLIPRSVF
16.0001
1 414 LVNSSCPGL 6.000 Table XV1-1/3-1-ILA-A24-9mers-
7 LIPRSVFNL 16.000
I 444 IYGVLGLFW 16.000 24P4C12 I 1 GYSSKGLIP
FRT.
153
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,
Table XVI-V6-HLA-A24-9mers- Table XVI-V8-HLA-A24-9mers-
Table XVI-V9-HLA-A24-9mers-
- 24P4C12 24P4C12 24P4C12
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ Each peptide is a portion of SEQ
ID NO: 13; each start position is ID NO: 17; each start position
is ID NO: 19; 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 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
p_lus eight.
I Start I Subsequence !Score] Start I Subsequence Score I
Start Subsequence Score
I 6 1 GLIPRSVFN 1 0.180 1 1 NYYWLPIMli 0.600 5
TALYPLPTQ 10.015
1 3 SSKGLIPRS 0.120 11 -1 PITPTGHVF 0.240 1 6 1
ALYPLPTQP 10.014
1 8 1 IPRSVFNLQ 0.020 10 1 NPITPTGHV 0.150 1 3 1 AMTALYPLP 10.012
1 4 SKGLIPRSV 0.014 5 1 LPIMRNPIT 0.150 1 10 1
LPTQPATLG 0.010
2 YSSKGLIPR 0.010 19 FQTSILGAY
0.140 14 L PATLGYVLW 10.010
9 PRSVFNLQI 10.010 20 QTSILGAYV
0.1201 1 1 1 YVVAMTALYP 0.010 =
13 - TPTGHVFQT 0.100
Table XVI-V7-HLA-A24-9mers- 7 1 IMRNPITPT 0.100
Table Xiill-V1-HLA-A24-10mers-
24P4C12 9 1 RNPITPTGH 0.030 24P4C12
_
Each peptide is a portion of SEQ 1 3 I YWLPIMRNP__I0.025
Each peptide is a portion of SEQ
ID NO: 15; each start position is 14 PTGHVFQTS L0.020 ID
NO: 3; each start position is
specified, the length of peptide is specified, the length of
peptide is
9 amino acids, and the end 12 1 ITPTGHVFQ 10.015 10 amino
acids, and the end
position for each peptide is the 17 1 HVFQTSILG 10.0101
position for each peptide is the
start position plus eight. 8 MRNPITPTG 0.003 start position
plus nine.
. 1 Start Subsequence Score 6 PIMRNPITP 0.002 I Start I
Subsequence Score
5 ILVAVGQMM 1.260 360.0
356 FYPLVTFVLL
4 WILVAVGQM 0.750 ' Table XVI-V9-HLA-A24.9mers-
00
2 INYWILVAVG _ 0.600 24P4C12
301 AYQSVQETWL 300.0
1 8 AVGQMMSTM 0.500 Each peptide is a portion of SEQ
- 00
200.0
7 VAVGQMMST 0.150 ID NO: 19; each start position is
87 LYFNIFSCIL
00
1 1 SVVYWILVAV 10.140 specified, the length of peptide
is
P
9 amino acids, and the end 200.0
140 FYTKNRNFCL
1 6 LVAVGQMMS 10.100 position for each peptide is the
00
I 3 1 YWILVAVGQ 0.021 start position plus eight.
-
200.0
274 YYCWEEYRVL
I Start I Subsequence Score, 00
Table XVI-V8-HLA-A24-9mers- 75.00 200.0
18 GYVLWASNI 370 AYWAMTALYL
24P4C12 0 ___________ 00
_
Each peptide is a portion of SEQ 7 LYPLPTQPA 9.000_ 18
IMPSFRGPI 120.0
ID NO: 17; each start position is 2 WAMTALYPL 16.000 .
00
specified, the length of peptide is82.50 =
1 13 QPATLGYVL 14.800- 685 YYMSKSLLKI
9 amino acids, and the end 0
position for each peptide is the I 9 PLPTQPATL 0.600 -
70.00
start position plus eight. 8 YPLPTQPAT 10.180- 636
YYWLPIMTSI 0
Start Subsequence 'Score I 15 ' ATLGYVLWA 10.150 ' 42.00
1 2 YYWLPIMRN 5.000 1 12 TQPATLGYV 0.150 439
VFNLQIYGVL 0
1 4 1 WLPIMRNPI 11,800 16 TLGYVLWAS 10.140
133.6
355 MFYPLVTFVL
1 15 -1 TGHVFQTSI 11,000 1 17 LGYVLWASN 10.120 0
1 18 VFQTSILGA 10.750 1 4 MTALYPLPT 0.100 169
SFLLPSAPAL 30.00
1 16 GHVFQTSIL 10.600- 1 11 1 PTQPATLGY 0.018 0
1 425 VFQGYSSKGL 130.00
154
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6 4
Table XVII-V1-HLA-A24-10mers- Table XVII-V1-HLA-A24-10mers-
Table XVII-V1-HLA-A24-10mers-
24P4C12 24P4C12 24P4C12
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.
Start I Subsequence Score Start -I Subsequence [Score Start Subsequence
Score
I 0 65 " LYPRNSTGAY 7.500 500 5
GSLAFGALIL 16.000
20.00 553 I KFLNRNAYIM 7.500 1 83 f KPYLLYFNIF [5.760
616 FFFSGRIPGL o 254 AGPLVLVLIL 17.200 310
LAALIVLAVL [5.600
20.00
224 DFAQSWYWIL 304 SVQETWLAAL 7.200 233 1
LVALGVALVL [5.600
o
- 231 WILVALGVAL 17.200 227 QSWYWILVAL [5.600
14.40
478 KPQDIPTFPL 637 YWLP1MTSIL 7.200 661 1
MCVDTLFLCF 5.184
0
162 LQQELCPSFL 7.200 565 IYGKNFCVSA [5.000
14,00
131 EFSQTVGEVF o 239 ALVLSLLFIL 7.200 1 279
EYRVLRDKGA 15.000
14.00 318 VLEAILLLML 17.200 ' [ 635 NYYWLPIMTS 15.000
658 VFGMCVDTLF o 314 IVLAVLEAIL 17.200 273
IYYCWEEYRV 15.000
12.00 37 CCVLFLLFIL 7.200 1 444 IYGVLGLFWT 5.000
569 NFCVSAKNAF o 546 5 WCLEKFIKFL 7.200 686 I
YMSKSLLKIL 14.800
12.00
630 KSPHLNYYWL 350 QMMSTMFYPL 7.200 56 I
WLYGDPRQVL 117871
0
99 NIISVAENGL 7.200 235 ALGVALVLSL 14.800
12.00
493 RTLRYHTGSL 0 1 203 , QGISGLIDSL 17.200 1
252 5 LVAGPLVLVL 14.800
'
111'2 1 243 SLLFILLLRL 7.200 1 449
GLFWTLNVVVL 4.800
331 RQRIRIAIAL
0 229 WYWILVALGV 7.000 1 502
LAFGALILTL 4.800
11.08 1 31 SCTDVICCVL 6.720 1 625 LGKDFKSPHL 14.800
517 VILEYIDHKL
8 441 NLQIYGVLGL 6.000 1 498
HTGSLAFGAL 4.800
10.50 1 357 YPLVTFVLLL 6.000 572 VSAKNAFMLL 14.800
40 LFLLFILGYI o I 604 LLVVGGVGVL 6.000 542
KCCLWCLEKF [4.400
10.08
589 VVLDKVTDLL I 510 1 TLVQIARVIL 6.000 442 I
LQIYGVLGLF 4.200
0
1 596 DLLLFFGKLL 6.000 368 I CIAYWAMTAL RET
1767 1 TVITSLQQEL [9.504
536 -1 CIMCCFKCCL 6.000 1 241 VLSLLFILLL I4.671
I 520 EYIDHKLRGV 9.000
588 VVVLDKVTDL 6.000
386 QYVLWASNIS 9.000
433 GLIQRSVFNL 6.000 Table XVII-V3-HLA-A24-10mers-
1 445 YGVLGLFWTL 8.640
659 FGMCVDTLFL 6.000 24P4C12
1 240 I LVLSLLFILL 18.640
456 1 WVLALGQCVL 6.000 Each peptide is a portion of SEQ
248 LLLRLVAGPL 11.47 [ , 413 HLVNSSCPGL 6.000
ID NO: 7; each start position is
1
257 LVLVLILGVL 1-87001
specified, the length of peptide is
48 YIVVGIVAWL 18.400
290 1 ISQLGFTTNL 6.000 10 amino acids, and the end
260 VLILGVLGVL 18.400
321 1 AILLLMLIFL rEgol position for each peptide is the
1
236 LGVALVLSLL 18.400 1 316 1 LAVLEAILLL FYI
start position plus nine.
1 57
DVICCVLFLL 177a151 1 LYGDPRQVLY 16.000 Start I
Subsequence 'Score
I 34 1 ,
1 91 IFSCILSSNI [6.000 9 1 NITPPALPGI 11.200
1 683 RPYYMSKSLL 8.000
77 MGENKDKPYL 16.000 I 1 LGRCFPWTNI 11.000
648 AYVIASGFFS 7.500
1 47 GYIVVGIVAW 17.500 1 163 QQELCPSFLL 1701-1 6
PWTN1TPPAL 10.400
199 TTIQQGISGL FR 10 1 ITPPALPGIT 10.216
155
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Table XV1143-HLA-A24-10mers= Table XVII-V6-HLA-A24-10mers-
' 4 I. YWLPIMRNPI 2.160
24P4C12 24P4C12 - 1 19 VFQTSILGAY 1.050
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ 21 , QTSILGAYVI 1.000
ID NO: 7; each start position is ID NO: 13; each start position is
1 3 YYWLPIMRNP 10.700
specified, the length of peptide is specified, the length of peptide is
amino acids, and the end 10 amino acids, and the end
10 RNPITPTGHV 10.300
position for each peptide is the position for each peptide
is the 1 14 TPTGHVFQTS 10.202
start position plus nine, start position plus nine. 5
WLPIMRNPIT 10.150
-
I Start Subsequence rgoTe- Start Subsequence 'Score
13 1 ITPTGHVFQT 0.150
5 FPVVTNITPPA 0.140 . 6 1 KGLIPRSVFN 0.300 20
FQTSILGAYV 10.120
1 4 1 CFPWTNITPP 10.075 5 SKGLIPRSVF 0.200 18 HVFQTSILGA 10.100
I. 3 1 RCFPVVTNITP 0.024 1_ 4 SSKGLIPRSV 0.140 15 PIGHVFQTSI 0.100
8 TNITPPALPG 0.015 [ 3 YSSKGLIPRS 0.120 6 __
LPIMRNPITP 10.015
1 7 WTNITPPALP 0.015 8 LIPRSVFNLQ 0.030 7
PIMRNPITPT 0.015
2 GRCFPVVTNIT Fii" [ 1 QGYSSKGLIP 0.010 8
IMRNPITPTG 0.014
1 10 PRSVFNLQIY 0.001, 1
LNYYWLP1MR 10.012
Table XVII-V5-HLA-A24-10mers- 1 9 MRNPITPTGH 10.002
24P4C12 Table XVII-W-HLA-A24-10mers- 17 GHVFQTSILG 10.002
Each peptide is a portion of SEQ 24P4C12
12 PITPTGHVFQ 10.001
ID NO: 11; each start position is Each peptide is a portion of SEQ
specified, the length of peptide is ID NO: 15; each start position is
10 amino acids, and the end
specified, the length of peptide is Table XVII-V9-HLA-A24-10mers-
position for each peptide is the 10 amino acids, and
the end 24P4C12
start position plus nine, position for each peptide is the Each
peptide is a portion of SEQ
I Start I Subsequence "Score start position plus nine.
ID NO: 19; each start position is
5 AILLLVLIFL 18.400 Start
Subsequence fgccTe specified, the length of peptide is
10 amino acids, and the end
2 1 VLEAILLLVL 17.200 1 9 AVGQMMSTMF 2.000
position for each peptide is the
4 1 EAILLLVLIF 3.600 1. 5 WILVAVGQMM ri:27/1
start position plus nine.
I 9 I LVLIFLRQRI 2.160 1 4 YWILVAVGQM 0.750
Start Subsequence Score
I 1 I AVLEAILLLV 0.252 1, 8 VAVGQMMSTM FREI 19 GYVLWASNIS 9.000
1 3 I LEAILLLVLI 0.120 1_. 3 1
VVYWILVAVGQ 0.700 8 1 LYPLPTQPAT 17.500
1 7 1 LLLVLIFLRQ 0.025- 1 6 ILVAVGQMMS 10.150 13 TQPATLGYVL 17.200
1 6 ILLLVLIFLR 10.018 1 1 1 QSWYWILVAV 0.140 9
YPLPTQPATL 7.200
10 VLIFLRQR1R 10.015 7 1 LVAVGQMMST 171601 2
YWAMTALYPL 14.000
8 LLVLIFLRQR 170715 1 2 SVVYWILVAVG [NT 1 18
LGYVLWASNI 1.000
I 1 AYWAMTALYP 0.500
Table )0/11-V6-HLA-A24-10mers- Table XVII-V8-HLA-A24-10mers-
16 1 ATLGYVLWAS 10.210
24P4C12 24P4C12
1 ALYPLPTQPA 0.144
Each peptide is a portion of SEQ Each peptide is a portion of SEQ
1 17 TLGYVLWASN 0.120
ID NO: 13; each start position is ID NO: 17; each start position is
specified, the length of peptide is specified, the length of
peptide is 4 AMTALYPLPT 0.100
10 amino acids, and the end 10 amino acids, and the
end 14 QPATLGYVLW 0.100
position for each peptide is the position for each peptide
is the 11 LPTQPATLGY 0.100
start position plus nine. startposition plus nine.
1 12 PTQPATLGYV 0.018
I Start I Subsequence FO-r-e- [Start I Subsequence Score 1 -
1 6 TALYPLPTQP 0.018
I 7 1 GLIPRSVFNL 17.200- 1 2 NYYWLPIMRN 5.000
1 3I WAMTALYPLP 0.018
1 9 IPRSVFNLQI 1.000 1 16 1 TGHVFQTSIL 4.000
1 15 PATLGYVLWA 0.010
2 1 GYSSKGLIPR 10.500! 1 11 NPITPTGHVF isET
5I MTALYPLPTQ 0.010
156
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Table XVII-1/9-HIA-A24-10mers- Table XVIII-V1-HLA-B7-9mers-
Table XVIII-V1-HLA-B7-9mers-
24P4C12 24P4C12 24P4C12
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ 1 Each peptide is a portion of SEQ
ID NO: 19; 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
10 amino acids, and the end 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 nine, start position plus eight. start position plus
eight.
I Start I Subsequence (Score [ Start I Subsequence (Score
Start Subsequence Score
I 10 PLPTQPATLG 10.002 20.00 241 VLSLLFILL
14.000
240 LVLSLLFIL o 1 236 LGVALVLSL
14.000
Table XVIII-V1-1-11A-137-9mers- 605 LVVGGVGVL 20.00 1 440 ,
FNLQIYGVL 4,000
24P4C12 0
[184 WTNVTPPAL 4.000
Each peptide is a portion of SEQ 34 DVICCVLFL 20.00
597 LLLFFGKLL 4.000
ID NO: 3; each start position is 0
583 RNIVRVVVL 14.000
specified, the length of peptide is 20.00
589 VVLDKVTDL
9 amino acids, and the end 0 275 YCWEEYRVL 4.000
position for each peptide is the 170 FLLPSAPAL
4.000
start position plus eight 347 AVGQMMSTM 15.00 0 596
DLLLFFGKL 4.000
Start Subsequence Score 12.00 282 VLRDKGASI 14.000
573 SAKNAFMLL
80.00 0
255 GPLVLVLIL 158 1
VITSLQQEL 4.000
0 00
253 VAGPLVLVL 12. 537
1MCCFKCCL 4.000 .
00 0
631 SPHLNYYWL 80. 660
GMCVDTLFL 14.000
0 12.00
369 IAYWAMTAL 457 1
VLALGQCVL 14.000
80.00 o
357 YPLVTFVLL 499 o 12.00 TGSLAFGAL
14.000
225 FAQSVVYWIL
80.00 , o 66 YPRNSTGAY 14.000
683 RPYYMSKSL 0 12.00 141 I
YTKNRNFCL 14.000
213 NARDISVKI
60.00 o 1 555 LNRNAYIMI 14.000
317 AVLEAILLL o 12.00 426
FQGYSSKGL 4.000
514 IARVILEYI
I 249 LLRLVAGPL 40.00 o 244 LLFILLLRL
4.000
o 12.00
154 WNMTVITSL 242 1
LSLLFILLL 14.000
00 0
494 TLRYHTGSL 40. 12.00 487 1
LISAFIRTL 4,000
0
316 LAVLEAILL . 79
ENKDKPYLL 14,000
40.00 o
333 RIRIAIALL 0 12.00 351
MMSTMFYPL 4,000
234 VALGVALVL
36,00 0 442 LQIYGVLGL 4,000
311 AALIVLAVL 0 396 SPGCEKVPI 8.000 1 200
TIQQGISGL 14.000
0 83 I KPYLLYFNI 8.000 434 LIQRSVFNL 4.000
511 LVQIARVIL 13"
0
406 TSCNPTAHL 6.0001 1 501 SLAFGALIL
14.000
414 LVNSSCPGL 20.00 381 1 TSGQPQYVL 6.000 322
ILLLMLIFL , 4.000
0
. , 571 1 CVSAKNAFM , 5.000 251
RLVAGPLVL 14:665-1
20.00
38 CVLFLLFIL 0 261 1 LILGVLGVL 4.000 204
GISGLIDSL 14.000
315 1 VLAVLEAIL 4.000 572 VSAKNAFML
14.000
49 IVVGIVAWL -2(10m
291 1 SQLGFTTNL 4.0001 1 687 MSKSLLKIL
14.000
20.00 638 WLPIMTSIL 4.000 100 IISVAENGL 14,000
446 GVLGLFVVTL 0 258 VLVLILGVL 4.000 232
ILVALGVAL 4.000
237 GVALVLSLL 2000. 452 1 VVTLNWVLAL 4.000 302
YQSVQETWL 4.000
0
- _____________________ 1 28 11 KNRSCTDVI 4.000: 1 35 VICCVLFLL
4.000
-
157
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__________________ '
Table XVIII-V1-HLA-B7-9mers- Table XVIII-V3-HLA-B7-9mers-
Table XVIII-V6-HLA-B7-9mers-
24P4C12 24P4C12 24P4C12
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: 7; each start position
is ID NO: 13; each start position is
specified, the length of peptide is specified, the length of
peptide is specified, the length of peptide is
position for each peptide is the position for each peptide is
the position for each peptide is the
25 GPIKNRSCT 13.000 1 8 NITPPALPG 0.015 1 9
PRSVFNLQI 10.004
482 IPTFPLISA 13.000 3 ' CFPWTNITP 0.001 1
GYSSKGLIP 10.001 ,
344 1 ASKAVGQMM 13.000 1 5 1 PWTNITPPA 0.001
343 1 EASKAVGQM 13.000 Table XVIII-V7-HLA-B7-9mers-
149 i_. LPGVPWNMT 13.000 Table XVIII-V5-HLA-B7-9mer;1 24P4C12
581 LMRNIVRVV 1.-2761 24P4C12
Each peptide is a portion of SEQ
152 VPWNMTVIT 2.000 Each peptide is
a portion of SEQ ID NO: 15; each start position is
531 1 NPVARCIMC 2.000
1 9 amino acids, and the end
specified, the length of peptide is
188 TPPALPGIT 12.000 9 amino acids,
and the end position for each peptide is the
112 TPQVCVSSC 2.000 position for each peptide is
the start position plus eight.
60 DPRQVLYPR 12.000 start position
plus eight. 1 Start I Subsequence 1Score
525 I KLRGVQNPV , 2.000 I Start Subsequence Score
8 AVGQMMSTM 15.00
314 IVLAVLEAI 2.000 5 ILLLVLIFL 14.000. 0
167 CPSFLLPSA 2.000 3 1 EAILLLVLI 11.200 1 5 1 ILVAVGQMM
11.0001
151 1 GVPWNMTVI 12.000 9 1 VLIFLRQRI 10.600 4 WILVAVGQM 1.000
192 LPGITNDTT 12.000 1 2 1 LEAILLLVL 10.400 1 7 1
VAVGQMMST 0.300
I 359 LVTFVLLLI 12.000 4 1 AILLLVLIF 10.060 6
LVAVGQMMS 0.100
I 252 LVAGPLVLV 1.500 1 1 _1 VLEAILLLV 0.060 1
SVVYWILVAV 0.020
491 FIRTLRYHT 11.500 8 LVLIFLRQR 10.050 3 YWILVAVGQ 10.001
1 530 QNPVARCIM 1.500 1 7 1 LLVLIFLRQ 10.010 2
VVYWILVAVG ,10.001
I 305 VQETWLAAL 1.200 Table XVIII-V8-
HLA-B7-9mers-
24P4C12
Table XVIII-V6-HLA-B7-9mers-
24P4C12 Each
peptide is a portion of SEQ
Table XVIII-V3-HLA-B7-9mers- ID NO: 17; each start position
is
24P4C12 Each peptide is a portion of SEQ specified, the length of
peptide is
ID NO: 13; each start position is
Each peptide is a portion of SEQ 9 amino acids, and the end
specified, the length of peptide is
ID NO: 7; each start position is position for each peptide is
the
9 amino acids, and the end
9 amino acids, and the end Start I Subsequence (Score
start position plus eight.
position for each peptide is the
Start Subsequence Score 7 LIPRSVFNL 14.000 I 5 1
LPIMRNPIT 12.000
6 VVTNITPPAL 4.000 , I 8 IPRSVFNLQ 12.000 I 13
TPTGHVFQT 12.000
9 ITPPALPGI 0.400 5 1 KGLIPRSVF 10.045 I 7
IMRNPITPT 11.500
4 FPWTNITPP 0.200 1 6 GLIPRSVFN ,10.0201 1 4
WLPIMRNPI 10.600
1 2 1 RCFPWTNIT 0.100 4 SKGLIPRSV 0.020 I 15
ITGHVFQTSI 0.400
1 I GRCFPWTNI [0.7661 I 3 1 SSKGLIPRS 10.0201 1 1E-11 GHVFQTSIL 13.1761
I 7 1 TNITPPALP 0.015 121 YSSKGLIPR 10.0101 1-
120 1 QTSILGAYV Wall
158
,
CA 02503346 2005-04-21
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17 HVFQTSILG 0.050 Each peptide is a portion of SEQ I
Table XIX-V1-HLA-B7-10mers-
19 FQTSILGAY 0.020 ID NO: 3; each start position is
24P4C12
specified, the length of peptide is
18 VFQTSILGA 0.010 Each peptide is a portion of SEQ
10 amino acids, and the end ID NO: 3; each start position is
12 ITPTGHVFQ 10.010 position for each peptide is the
specified, the length of peptide is
9 RNPITPTGH 0,010 start position_plus nine. 10 amino acids,
and the end
6 PIMRNPITP 10.003 Start I Subsequence IScore position
for each peptide is the
2 YYWLPIMRN 10.003478 KPQDIPTFPL 1 start position
plus nine.
120.0
11 PITPTGHVF 10.002 00 Start Subsequence 'Score
14 PTGHVFQTS 0.002 683 RPYYMSKSLL 8U 1 0
o 12.00
3 YVVLPIMRNP 10.001 1 80.00 239 ALVLSLLFIL
0 8 MRNPITPTG 10.001 357 YPLVTFVLLL
-
_________________________________________ o 12.00
1 1 NYYWLPIMR 10.001. 659
FGMCVDTLFL
00 0
331 RQRIRIAIAL ro.
12.00
Table XVIII-V9-HLA-B7=9mers- 20.00 254 AGPLVLVLIL
0
24P4C12 571 CVSAKNAFML o 12.00
' Each peptide is a portion of SEQ 257 LVLVLILGVL 20.00 350
QMMSTMFYPL 0
ID NO: 19; each start position is o 12.00
specified, the length of peptide is 20.00 235 ALGVALVLSL
0
9 amino acids, and the end 456 WVLALGQCVL o 12.00
position for each peptide is the 536 CIMCCFKCCL
20.00 0
start position plus eight. 588 VVVLDKVTDL
_________________________________________ 0 12.00
I Start I Subsequence Score 316 LAVLEAILLL
20.00 o o 12.00
80.00 157 TVITSLQQEL ..
13 QPATLGYVL
0 310 LAALIVLAVL
20.00 0
36.00 240 LVLSLLFILL
2 WAMTALYPL o 10.00
o 585 IVRVVVLDKV
20.00 0
1 8 1 YPLPTQPAT 12.000 66 YPRNSTGAYC
_________________________________________ 0 1 56
WLYGDPRQVL 9.000
1 9 PLPTQPATL 10.400 20.00 192 LPGITNDTTI 18.000
1 10 1 LPTQPATLG 10.3001 314 __________ IVLAVLEAIL o
510 I TLVQIARVIL 16.000
1 15 ATLGYVLVVA 0.300 1
589 WLDKVTDLL 20.00 662 1 CVDTLFLCFL
6.000
1 12 TQPATLGYV 10.20010
20.00 329 1 FLRQRIRIAI 16.000
1 4 MTALYPLPT 10.100 252 LVAGPLVLVL o 405
NTSCNPTAHL 6.000
1 5 TALYPLPTQ 0.045
20.00 1 414 LVNSSCPGLM 15.000
1 18 GYVLWASNI 0.040 657 SVFGMCVDTL 0 413 I HLVNSSCPGL
14.000
3 1 AMTALYPLP 10.030 ,.
20.00 203 QGISGLIDSL 14.000
6 1 ALYPLPTQP 0.030 ___ 304 LSVQETVVLAAL 0 368 CIAYWAMTAL 4.000
1 17 1 LGYVLWASN 0.020 20.00 686 YMSKSLLKIL 14.000
1 16 1 TLGYVLWAS 0.020 34 DVICCVLFLL o
I 99 I NIISVAENGL 14710-
1 7 1 LYPLPTQPA 0.015 00.
233 LVALGVALVL 665 TLFLCFLEDL 4.000
1 14 1 PATLGYVLW 0.006 o
_________________________ - __________
18.00 290 I ISQLGFTTNL 4.000
1 11 1 PTQPATLGY 10.002 380 ATSGQPQYVL o 441I NLQIYGVLGL 14.0001
1 1 YWAMTALYP 0.001
15.00 630 KSPHLNYYVVL r4.00-01
317 AVLEAILLLM o 315 VLAVLEAILL 4.000
Table XIX-V1-HLA-B7-10mers-
12.00 236 LGVALVLSLL 4.000
24P4C12 321 AILLLMLIFL __ o 596 DLLLFFGKLL 14.0001
502 LAFGALILTL 112,00 60
DPRQVLYPRN 14.0001
159
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Table XIX-V1-HLA-B7-10mers- Table XIX-V1-HLA-137-10mers-
- Each peptide is a portion of SEQ
24P4C12 24P4C12 ID NO: 11; each start
position is
_
Each peptide is a portion of SEQ Each peptide is a portion of
SEQ specified, the length of peptide is
ID NO: 3; each start position is ID NO: 3; each start position
is 10 amino acids, and the end
specified, the length of peptide is specified, the length of peptide is
position for each peptide is the
amino acids, and the end 10 amino acids, and the end start position plus
nine.
position for each peptide is the position for each peptide is
the 1 Start Subsequence 1Score
start position plus nine, start position plus nine. '12.00
5 1 Start Subsequence Score AILLLVLIFL.
1 Start Subsequence Score 0
1 243 SLLFILLLRL 14.000 1 143 1 KNRNFCLPGV 12.000 9 ,
LVLIFLRQRI 3.000
1 37 CCVLFLLFIL 14.000 639 LPIMTSILGA 2.000 1 1
AVLEAILLLV FYI
I 449 GLFVVTLNWVL 4.000 1 249 1 LLRLVAGPLV _ 2.000 1 2 VLEAILLLVL
rifoir
1 162 UMELCPSFL 4.000 172 LPSAPALGRC 12.000 1 4
EAILLLVLIF 0.060
625 LGKDFKSPHL 4.000 485 FPLISAFIRT 2.000 3 1 LEAILLLVLI 0.040
1 227 QSVVYWILVAL 4.000 264 GVLGVLAYGI 2.000 1 7
LLLVLIFLRQ [0.010
1 498 HTGSLAFGAL 4.000 531 , NPVARCIMCC
12.000 6 ILLLVLIFLR 10.0101
1 48 YIVVGIVAWL 14.000 163 QQELCPSFLL 11.800 10
VLIFLRQRIR 10.010
1 604 LLVVGGVGVL 14.0001 529 VQNPVARCIM 11.500 8
LLVLIFLRQR 0.010
149 LPGVPWNMTV 14.000 576 1 NAFMLLMRNI 11.200
1 260 VLILGVLGVL 14.000 370 1
AYWAMTALYL _11.200 Table XIX-V6-HLA-B7-10mers-
1 493 RTLRYHTGSL 4.000 318 1 VLEAILLLML
11.200 24P4C12
248 LLLRLVAGPL
14.000 Each peptide is a portion of SEQ
ID NO: 13; each start position is
1 231 , WILVALGVAL 4.000 Table XIX-V3-HLA-B7-10mers- specified, the
length of peptide is
500 , GSLAFGALIL 14.000 24P4C12 10 amino acids,
and the end
1 546 WCLEKFIKFL 14.000 Each peptide
is a portion of SEQ position for each peptide is the
Subsequence Score
ID NO: 7; each start position is start position plus nine.
241 VLSLLFILLL 14.000 specified, the length of peptide is
Start
539 CCFKCCLWCL 121171 10 amino acids, and the end
1
1 445 YGVLGLFVVTL Rai position for each
peptide is the 9 IPRSVFNLQI
80.00
307 ETWLAALIVL 4.000 start position
plus nine. o
7
1 435 IQRSVFNLQI 4.000 1 Start 1
Subsequence Score GLIPRSVFNL 14.000
572 VSAKNAFMLL 4.000 1 LGRCFPWTNI 6.000 4
SSKGLIPRSV 10.200
1 433 GLIQRSVFNL 4.000 1 5 1
FPVVTNITPPA 2.000 6 1 KGLIPRSVFN 10.020
1 517 VILEYIDHKL 4.000 9 1 NITPPALPGI 0.400 I
3 YSSKGLIPRS 10.020
199 TTIQQGISGL RET 1 10 1
iTPPALPGIT 0.100 8 1 LIPRSVFNLQ 0.010
31 SCTDVICCVL 4.000 1 6 1 PWTNITPPAL 0.040 1
QGYSSKGLIP 10.010
178 LGRCFPVVTNV 13.000: I 8 1
TNITPPALPG 0.015 I 5 1 SKGLIPRSVF 10.0051
343 EASKAVGQMM 3.000 7 1 WTNITPPALP 0.015 2
GYSSKGLIPR 0.001
346 I<AVGQMMSTM 3.000 1 3 1 ,RCFPVVTNITP 0.010 10 1
PRSVFNLQIY 0.000
581 LMRNIVRVVV 3.000 1 2 LGRCFPWTNIT 0.010
573 SAKNAFMLLM 3.o00 1 4 1
CFPVVTNITPP 0.001 Table XIX-V7-HLA-B7-10mers-
24P4C12
652 ASGFFSVFGM
3.000 Each peptide is a portion of SEQ
402 VPINTSCNPT 2.000 Table XIX-V5-
HLA-B7-10mers- ID NO: 15; each start position is
24P4C12
182 FPWTNVTPPA
2.000 specified, the length of peptide is
528 GVQNPVARCI
2.0001 10 amino acids, and the end
position for each peptide is the
281 RVLRDKGASI FOR" start
position plus nine.
186 NVTPPALPGI 2.000
160
CA 02503346 2005-04-21
WO 2004/050828 PCT/US2002/038264
Start 1 Subsequence "Score Each peptide is a portion of
SEQ Table XX-V1 =HLA-B35-9meres-
8 VAVGQMMSTM 13.000 ID NO: 19; each start position is
24P4C12
. specified, the length of peptide is
1 WILVAVGQMM 1.000 Each peptide is a portion of
SEQ
amino acids, and the end
1 7 1 LVAVGQMMST 0.500 position for each peptide is the
ID NO: 3; each start position is
9 1 AVGQMMSTMF 0.300 start position plus nine,
specified, the length of peptide is9 amino acids, and the end
1 1 I QSWYWILVAV 0.200 1 Start
Subsequence Score position for each peptide is the
..
1 4 1 YWILVAVGQM 0.10080.00 start
position plus eight.
YPLPTQPATL
9 t -
I 6 ILVAVGQMMS 0.020 0 . Start
Subsequence 'Score
1 2 1 SWYWILVAVG 0.001 1 13 TQPATLGYVL 4.000 I 0
1 3 1 VVYWILVAVGQ 0.001 7 ALYPLPTQPA , 0.450,
357 YPLVTFVLL 20.00
11 LPTQPATLGY , 0.400 0
Table XIX-V8-HLA-B7-10mers- 14 QPATLGYVLW 0.400 255
GPLVLVLIL 20.00
24P4C12 2 YWAMTALYPL 0.400 0
Each peptide is a portion of SEQ 1 18 . LGYVLWASNI 10.400
631 SPHLNYYWL 20.00o
ID NO: 17; each start position is 1 4 AMTALYPLPT _10.300
specified, the length of peptide is
10 amino acids, and the end 3 WAMTALYPLP 10.090 83
KPYLLYFNI 16.00
0
position for each peptide is the 16 ATLGYVLWAS 10.060
687 MSKSLLKIL 15.00
start position plus nine, 6 TALYPLPTQP 10.030 0
I Start , Subsequence , Score 15 PATLGYVLWA 10.030 396
SPGCEKVPI 12.00
16 TGHVFQTSIL 4.000 1 12 1 PTQPATLGYV 10.020 o
18 HVFQTSILGA 10.500 I 17 1 TLGYVLWASN 0.020 69
NSTGAYCGM 10.00
11 NPITPTGHVF 10.400 5 MTALYPLPTQ
10.015 0
573
21 1 QTSILGAYVI 10.400 8 LYPLPTQPAT 0.010 SAKNAFMLL
19.000
533
14 I TPTGHVFOTS 10.400 1 AYWAMTALYP 0.003 VARCIMCCF
9.000
10 1 RNPITPTGHV 10.300 19 1 GYVLWASNIS 0.002 213 NARDISVKI 7.200
20 1 FQTSILGAYV 10.200 1 10 1 PLPTQPATLG 10.002 465 LAGAFASFY 16.000
1 6 LPIMRNPITP 0.200 11 EAYGKPVKY
6.000
13 ITPTGHVFQT 0.100 Table XX-V1-HLA-B35-9meres-
333 RIRIAIALL 6.000
8 1 IMRNPITPTG 10.100 24P4C12 343 EASKAVGQM
6.000
5 1 WLPIMRNPIT 10.100 Each peptide is a portion of SEQ
489 SAFIRTLRY 6.000
ID NO: 3; each start position is 79 ENKDKPYLL 6.000
4 YWLPIMRNPI [0.0601 specified, the length of peptide is
379 LATSGOPOY 6.000
7 1 PIMRNPITPT 10.045 9 amino acids, and the end 558
NAYIMIAIY 6.000
PIGHVFQTSI 0.040 position for each peptide is the
1 LNYYWLPIMR 0.010 start position plus eight. 630
KSPHLNYYW 5.000
2 NYYWLPIMRN 0.003 1 Start 1 Subsequence 11Score 381
TSGQPQYVL 5.000
19 VFQTSILGAY 0.002 66 YPRNSTGAY 120.0 217 ISVKIFEDF 5.000
17 GHVFQTSILG 10.001 00 132 FSQTVGEVF
5.000
3 YYWLPIMRNP 10.001 683 RPYYMSKSL 40.00 242 I
LSLLFILLL 5.0000 406 TSCNPTAHL 5.000
12 1 PITPTGHVFQ 10.001 40.00 572 VSAKNAFML 5.000
_________________________________________ 0
9 MRNPITPTGH 1-0-.07 15 KPVKYDPSF
__________________________ - _____________________________________ 316
LAVLEAILL 4.500
344 ASKAVGQMM 13 600
593 KVTDLLLFF 4.000
Table XIX-V9-HLA-B7-10mers-
24P4C12 20.00 514 1 IARVILEYI 3.600
437 RSVFNLQIY ____________________________ o 287
GASISOLGF 3.000
_ __________________________
1 679 I GSLDRPYYM +0.00 238 I VALVLSLLF 3.000
161
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Table XX-V1-HLA-B35-9meres- Table XX-V1-HLA-B35-9meres-
Table XX-V3-HLA-B35-9mers-
24P4C12 24P4C12 , 24P4C12
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: 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 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
startsosition plus eight. start position plus eight. start position plus
eight.
1 Start I Subsequence Score Start Subsequence 1Score
Start Subsequence FOR
1 311 AALIV1AVL 3.000 133 SQTVGEVFY 12.000 9
ITPPALPGI FRU
1 275 YCWEEYRVL 13.000 1 251 1 RLVAGPLVL 2.000 4
FPWTNITPP 10.200
253 VAGPLVLVL 13.000 , L 409 NPTAHLVNS 2.000 2 1
RCFPVVTNIT 10.200
1 651 1 IASGFFSVF 13.000, 347 AVGQMMSTM 2.000 1 1
GRCFPVVTNI 0.040
1 647 GAYVIASGF 13.000 634 1 LNYYWLPIM 12.000 7 1
TNITPPALP 0.010
1 225 1 FAQSVVYWIL 3.000 621 RIPGLGKDF 2.000 1 8 1
NITPPALPG 0.010
1 174 SAPALGRCF 13.000 1 643 TSILGAYVI
2.000 3 1 CFPVVTNITP 10.001
234 VALGVALVL 13.000 482 1 IPTFPLISA 2.000 1
5 1 PWTNITPPA 10.001
369 IAYWAMTAL 3.000 1 110 CPTPQVCVS 12.000
r 141 YTKNRNFCL 13.000 1 641 IMTSILGAY
2.000 Table XX-V5-HLA-B35-9mers-
494 , TLRYHTGSL 13.000 1 677 NNGSLDRPY 2.000 24P4C12
678 I NGSLDRPYY 3.000 1 421 GLMCVFQGY 2.000 Each
peptide is a portion of SEQ
249 LLRLVAGPL 13.000 162 LQQELCPSF
2.000 ID NO; 11; each start position is
specified, the length of peptide is
117 J VSSCPEDPW [2.500 149 LPGVPWNMT 2.000 9 amino acids, and
the end
282 1 VLRDKGASI 12.400 1 263 LGVLGVLAY 2.000 position
for each peptide is the
28 KNRSCTDVI 12.400 1 167 1 CPSFLLPSA 2.000 start
position plus eight.
317 AVLEAILLL 12.000 1 148 CLPGVPWNM
2.000 Start Subsequence Score
266 I LGVLAYGIY 2.000 571 CVSAKNAFM 2.000 3
EAILLLVLI 11.200
363 I VLLLICIAY 2.000 112 TPQVCVSSC 2.000 5
ILLLVLIFL 11.000
267 I GVLAYGIYY 2.000 1 384 QPQYVLWAS ' 2.000 4 1
AILLLVLIF 11.000
25 I GPIKNRSCT 2.000 500 1 GSLAFGALI '2.000 9 1 VLIFLRQRI 10.400
415 VNSSCPGLM 2.000 349 1 GQMMSTMFY 2.000 2
LEAILLLVL 0.100
50 VVGIVAWLY 2.000 653 1 SGFFSVFGM 2.000 1
VLEAILLLV 10.060
589 VVLDKVTDL 2.000 4 KQRDEDDEA _ 1.800 1 6
LLLVLIFLR 10.010
272 GIYYCWEEY 2.000 660 GMCVDTLFL 11.500 1 7 1 LLVLIFLRQ 10.010
188 TPPALPGIT 2.000 1 30 RSCTDVICC 11.500 1 8
LVLIFLRQR 0.010 .
432 KGLIQRSVF 2.000 ' 1 430 SSKGLIQRS 1.500
152 VPWNMTVIT 2.000 Table XX-
V6-HLA-B35-9mers-
- 24P4C12
192 I LPGITNDTT 2.000 Table XX-V3-HLA-B35-9mers-
531 NPVARCIMC 2.000 24P4C12
Each peptide is a portion of SEQ
ID NO: 13; each start position is
583 I RNIVRVVVL 2.000 Each peptide is a portion of SEQ
specified, the length of peptide is
ID NO: 7; each start position is
366 LICIAYWAM 2.000 9 amino acids,vand the end
specified, the length of peptide is
546 WCLEKFIKF 2.000 9 amino acids, and the end position
for each peptide is the
start position plus eight.
554 FLNRNAYIM 2.000 position for each peptide is the
513 I QIARVILEY 2.000 start position plus eight Start
Subsequence FOR
1 92 I FSCILSSNI 2.000 1 Start 1 Subsequence Score 5 1
KGLIPRSVF 12.0001
1 530 I QNPVARCIM 2.000 6 1 VVTNITPPAL 1.000 3 1 SSKGLIPRS IT.-
57101
162
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1 7 1 LIPRSVFNL 1.000_ Table XX-V8-HLA-B35-
9mers- ,
Table XXI-V1-HLA-B35-10mers-
1 8 IPRSVFNLQ 10.600 24P4C12
24P4C12 4.
6 1 GLIPRSVFN 0.100 Each
peptide is a portion of SEQ Each peptide is a portion of SEQ
2 YSSKGLIPR 10.050 ID NO: 17; each start position
is ID NO: 3; each start position is
specified, the length of peptide is specified, the length of peptide is
4 SKGLIPRSV _10.020 9 amino acids, and the
end 10 amino acids, and the end
9 PRSVFNLQI 0.004 position
for each peptide is the position for each peptide is the
1 GYSSKGLIP 0.001 start
position plus eight. start position plus nine. ,
Start Subsequence [Score 1 Start 1 Subsequence 1Scorel
Table XX-V7-HLA-B35-9mers- 18 VFQTSILGA 10.010
478 KPCIDIPTFPL 80.00
24P4C12 1 14 PTGHVFQTS 10410 _ 0
Each peptide is a portion of SEQ 1 17 HVFQTSILG [0.010 83
KPYLLYFNIF 40.00
ID NO: 15; each start position is 1 12 ITPTGHVFQ 0.010 -.
, 0
specified, the length of peptide is 40.00
6 1 PIMRNPITP [0.001 683 RPYYMSKSLL
9 amino acids, and the end o
position for each peptide is the 1 3 ,1 YWLPIMRNP 0.001 .
- -
36.00
start position plus eight. 1 8 1 MRNPITPTG. 0.001 4
KQRDEDDEAY o
=
. 1 Start 1 Subsequence (Score 1 1 1
NYYWLPIMR 0.001 20.00
123 DPWTVGKNEF
1 8 1 AVGQMMSTM 2.000 0
1 5 ILVAVGQMM 2.000 Table XX-V9-HLA-B35-9mers-
482 IPTFPLISAF 20.00
4 WILVAVGQM 2.000 24P4C12 1 0
1 7 VAVGQMMST 10.300 Each peptide is a portion of SEQ
I 357 YPLVTFVLLL 20.00
ID NO: 19; each start position is 0
1 6 LVAVGQMMS 0.100 specified, the length of peptide
is 18.00
1 SWYWILVAV 10.020 9 amino acids, and the
end 213 NARDISVKIF o
1 3 ,1 YWILVAVGQ 0.001
position for each peptide is the 18.00
2 VVYWILVAVG 10.001 start position plus
eight. 573 SAKNAFMLLM 0
Start Subsequence Score 12.00
Table XX-V8-11LA-B35-9mers- 13 QPATLGYVL 20.00 346
KAVGQMMSTM 0
24P4C12 0 12.00
Each peptide is a portion of SEQ 2 WAMTALYPL 3.000 79
ENKDKPYLLY
0
ID NO: 17; each start position is 8 YPLPTQPAT [2.000 -
10.00
652 ASGFFSVFGM
specified, the length of peptide is 11 1 PTQPATLGY 10.200 o
9 amino acids, and the end
12 TQPATLGYV 0.200 10.00
position for each peptide is the 488 ISAFIRTLRY
start position plus eight. 10 LPTQPATLG 0.200
0
1 Start 1 Subsequence Score 14 PATLGYVLW 0.150 132
FSQTVGEVFY 10.000
1 10 NPITPTGHV 14.000 15 1 ATLGYVLWA ,10.100
10.00
13 TPTGHVFQT 12.000 4 1 MTALYPLPT 0.100 175
APALGRCFPW o
1 19 , FQTSILGAY 12.000 16 1 TLGYVLWAS 0.100 10.00
1 5 1 LPIMRNPIT 2.000 17 1 LGYVLWASN 10.100 630 KSPHLNYYWL
. o
1 15 TGHVFQTSI 0.400 9 1
PLPTQPATL 0,100 192 1 LPGITNDTTI 18.000
1 4 WLPIMRNPI FR- 18 1 GYVLWASNI 110.040 I 551
1 FIKFLNRNAY 16.000
7 IMRNPITPT 0.300 I5 1
TALYPLPTQ 0.030 I 625 1 LGKDFKSPHL 16.000
20 I QTSILGAYV 0.200 7 LYPLPTQPA 10.0101 I 331
RQRIRIAIAL 16.000
11 PITPTGHVF 0.100_ 3
AMTALYPLP 10.0101 11171I EASKAVGQMM 6.000
1 16 1 GHVFQTSIL 10.100 6 1 ALYPLPTQP 10.0101 I 60 1 DPRQVLYPRN 16.000
9 RNPITPTGH 1-071571 I 1 Y1NAMTALYP 10.0011 I
66 YPRNSTGAYC [8.000
2 YYWLPIMRN (67671 369 _
IAYWAMTALY 6.000
163
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Table )00-V1-HLA-B35-10mers- Table XXI-V1-HLA-B35-10mers-
Table XXI-V1-HLA-B35-10mers-
24P4C12 24P4C12 24P4C12
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.
Start Subsequence Score Start I Subsequence Score I Start
I Subsequence Score
572 VSAKNAFMLL 5.000 396 [ SPGCEKVPIN 2.000 576 1
NAFMLLMRNI 1.200
227 QSVVYWILVAL 15.000 1 266 LGVLAYGIYY
12.000 1 435 1 IQRSVFNLQI 11.200
500 1 GSLAFGALIL 5.000 402 VPINTSCNPT [2.000
417 SSCPGLMCVF 5.000 378 1
YLATSGQPQY [2.07 Table XXI-V3-HLA-B35-10mers-
290 ISQLGFTTNL 5.000 365 I LLICIAYWAM 2.000 24P4C12
76 GMGENKDKPY 4.000 293
LGFTTNLSAY 2.000 Each peptide is a portion of SEQ
68 1 RNSTGAYCGM 4.000 1 262 1 ILGVLGVLAY 2.000 ID
NO: 7; each start position is
specified, the length of peptide is
317 AVLEAILLLM 4.000 1 286 1 KGASISQLGF 2.000 10 amino
acids, and the end
1 557 RNAYIMIAIY 4.000 I 529 VQNPVARCIM 2.000
position for each peptide is the
149 I LPGVPWNMTV 4.000 1 678 1 NGSLDRPYYM 2.000 start position plus
nine.
1 676 RNNGSLDRPY 4.000 49 1 IVVGIVAWLY 2.000 Start I
Subsequence Score
310 LAALIVLAVL 1515151 147 1
FCLPGVPWNM 1-2761 5 I FPWTNITPPA 2.000
316 LAVLEAILLL 1571 265 VLGVLAYGIY
2.000 1 1 1 LGRCFPVVTNI 1.200
1 320 EAILLLMLIF 13:01 1 304 1 SVQETWLAAL 2.000 9
NITPPALPGI 0.400
[ 467 GAFASFYWAF 13.000 1 464 1 VLAGAFASFY 2.000 I 10 1
ITPPALPGIT 0.100
395 SSPGCEKVPI 13.000 1 20 1 DPSFRGPIKN 2.000 3
RCFPVVTNITP 0.020
1 647 GAYVIASGFF 13.000 1 661 MCVDTLFLCF
Z000 8 , TNITPPALPG 0.010
677 NNGSLDRPYY 13.000 92 FSCILSSNII 2.000 1 6
PVVTNITPPAL 0.010
502 LAFGALILTL 3.000 512 1 VQIARVILEY 2.000 I 7 1
VVTNITPPALP 0.010 -
1 430 SSKGLIQRSV 3.000 1 182 I FPWTNVTPPA 2.000 2 1
GRCFPVVTNIT 0.010
381 TSGQPQYVLW 2.500 639 1
LPIMTSILGA 2.0001 4 1 CFP1NTNITPP 0.001
I 362 FVLLLICIAY 2.000 570 I FCVSAKNAFM 2.000
1 39 VLFLLFILGY 2.000 1 493 1 RTLRYHTGSL 2.000
Table XXI-V5-HLA-B35-10mers-
I 188 TPPALPGITN 2.000 633 HLNYYWLPIM 2.000
24P4C12
152 VPWNMTVITS 2.000 531 NPVARCIMCC
2.000 Each peptide is a portion of SEQ
r 348 VGQMMSTMFY Z000 1 622 IPGLGKDFKS 2.000 ID NO:
11; each start position is
31 SCTDVICCVL 2.000 1 485 1 FPLISAFIRT 2.000
specified, the length of peptide is
384 QPQYVLWASN 2.000 1 542 1 KCCLWCLEKF 2.000 10 amino
acids, and the end
1 409 NPTAHLVNSS 2.000 589 1 VVLDKVTDLL 2.000 position
for each peptide is the
start position plus nine.
1 613 LSFFFFSGRI 2.000 517 VILEYIDHKL
2.000 Start I Subsequence Score
17271 KIFEDFAQSW 2.000 I 414 LVNSSCPGLM 2.000 4 EAILLLVLIF
3.000
1 110 CPTPQVCVSS 2.000 1 344 ASKAVGQMMS 1.500 5
AILLLVLIFL 1.000
1 546 WCLEKFIKFL 2.000 465 1 LAGAFASFYW 1.500 1 9 I
LVLIFLRQRI 10,400,
1 271 YGIYYCWEEY 2.000 300 SAYQSVQETW 11.500, 1 1 1
AVLEAILLLV 10.400'
1 30 RSCTDVICCV 2.0001 659 1 FGMCVDTLFL 1.5001 i
1 2
VLEAILLLVL 0.300
[ 172 LPSAPALGRC FOR- 315 VLAVLEAILL 1.500 )
3 1
[ 162 LIQQELCPSFL 17651 118 1 SSCPEDPVVTV 1.500 LEAILLLVLI 10.040
164
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Table XXI-V5-HLA-B35-10mers- Table XXI-V7-HLA-835-10mers-
Table XXI-V9-HLA-B35-10mers-
.
24P4C12 24P4C12 24P4C12
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: 15; each start
position is ID NO: 19; each start position is
specified, the length of peptide is specified, the length of
peptide is specified, the length of peptide is
10 amino adds, 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.
Start I Subsequence Score Start Subsequence Score Start Subsequence
'Score
6 1 ILLLVLIFLR 10.010 4 1 YWILVAVGQM firiOT 2 YWAMTALYPL
1.000
10 I VLIFLRQRIR 0.010 6 1 ILVAVGQMMS 0.100 13 1 TQPATLGYVL 1.000
1 7 I LLLVLIFLRQ 10.010 7 LVAVGQMMST 0.100 9 YPLPTQPATL 11.000
1 8 LLVLIFLRQR 0.010 2 S1NYWILVAVG 0.001 18
LGYVLWASNI 11.000
3 WYVVILVAVGQ 0.001 14
1 QPATLGYVLW 0.500
Table XXI-V6-HLA-B35-10mers- 11 I LPTQPATLGY 10.200
24P4C12 Table XXI-V8-HLA-1335-
10mers- 16 1 ATLGYVLWAS 10.150
Each peptide is a portion of SEQ 24P4C12 1 19 GYVLWASN IS 10.100
ID NO: 13; each start position is Each peptide is a portion of
SEQ 1 4 1 AMTALYPLPT 10.100
specified, the length of peptide is ID NO: 17; each start
position is 8 1 LYPLPTQPAT 10.100
10 amino adds, and the end specified, the length of peptide is
position for each peptide is the 10 amino acids, and the end 17
I TLGYVLWASN 0.100
start position plus nine, position for each peptide is the 7
ALYPLPTQPA 0.100
I Start I Subsequence 'Score start position plus nine. 3 WAMTALYPLP
0.050
24.00 Start Subsequence Score =
9 IPRSVFNLQI 12 PTQPATLGYV
10.020 .
0 20.00 1
11 NPITPTGHVF 6
1 TALYPLPTQP 10.010
4 SSKGLIPRSV 13.000 0
1 MTALYPLPTQ 0.010
' 1 7 GLIPRSVFNL 1.000 14 TPTGHVFQTS 2.000
PATLGYVLWA 0.010
1 3 YSSKGLIPRS 0.500 16 TGHVFQTSIL 1.000
1 AYWAMTALYP 0.010
6 KGLIPRSVFN 0.200 21 QTSILGAYVI 0.400
1 10 PLPTQPATLG 0.005
5 1 SKGLIPRSVF 0.100 1 10 RNPITPTGHV 0.400 .
10 PRSVFNLQIY 0.020 1 6 LPIMRNPITP 0.200
8 LIPRSVFNLQ 167-6- 20 FQTSILGAYV 0.200 .
1 1 QGYSSKGLIP 10.010 19 , VFOTSILGAY 0.200
1 2 GYSSKGLIPR 0.001 13 ITPTGHVFQT 0.100
18 HVFQTSILGA 0.100
Table XXI-V7-HLA-B35-10mers- 5 WLPIMRNPIT , 0.100
24P4C12 15 PTGHVFQTSI 0.040
Each peptide is a portion of SEQ 4 YWLPIMRNPI 0.040
ID NO: 15; each start position is
8 IMRNPITPTG 0.030 =
specified, the length of peptide is
10 amino acids, and the end 2 NYYWLPIMRN 0.010
position for each peptide is the 7 PIMRNPITPT 0.010
start position plus nine. 1 LNYYWLPIMR 0.010
I Start Subsequence 'Score 17 GHVFQTSILG 0.001
1 8 1 VAVGQMMSTM 16.000 3 YYWLPIMRNP 10.001
1 5 WILVAVGQMM 12.000 12 I PITPTGHVFQ [W11
I 9 AVGQMMSTMF 1.000 I 9 I MRNPITPTGH 0.001 =
I 1 QS1NYWILVAV 1.000
165
. .
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Tables XXII-XLIX:
TableXXII-V1-HLA-A1-9mers-
24P4C12
Each peptide is a portion of
SEQ ID NO: 3; each start
position is specified, the length
of peptide is 9 amino acids,
and the end position for each
peptide is the start position
plus eight.
Pos 123456789 score
80 NKDKPYLLY 34
58 YGDPRQVLY 33
222 FEDFAQSWY 26
QRDEDDEAY 25
77 MGENKDKPY 25
263 LGVLGVLAY 24
489 SAFIRTLRY 23
513 Q1ARVILEY 23
628 DFKSPHLNY 22
40 LFLLF I LGY 21
267 GVLAYGJyY 21
363 VLLLICIAY 21
421 GLMCVFQGY 21
50 VVGIVAWLY 20
318 VLEAILLLM 20
629 FKSPHLNYY 20
133 SQTVGEVFY 19
437 RSVFNLQIY 19
662 CVDTLFLCF 19
11 EAYGKPVKY 18
370 AYWAMTALY 18
18 KYDPSFRGP 17
32 CTDVICCVL 17
66 YPRNSTGAY 17
277 WEEYRVLRD 17
379 LATSGQPQY 17
594 VIDLUFFG 17
165 ELCPSFLLP 16
353 STMFYPLVT 16
398 GCEKVPINT 16
552 IKFLNRNAY 16
590 VLDKVTDLL 16
678 NGSLDRPYY 16
TableXXII-V3-HLA-A1-9mers-
24P4C12
Each peptide is a portion of
SEO ID NO: 7; each start
posifion is specified, the length
of peptide is 9 amino acids, and
the end position for each
peptide is the start position plus
eight.
Pos 123456789 score
165
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TableXXII-V3-HLA-A1-9mers-
24P4C12
Each peptide is a portion of
SEQ ID NO: 7; each start
position is specified, the length
of peptide is 9 amino acids, and
the end position for each
peptide is the start position plus
eight.
Pos 123456789 score
8 NITPPALPG 11
9 ITPPALPGI 10
6 VVTNITPPAL 6
3 CFPWTNITP 5
6 LVAVGQMMS 3 TableXXIII-
V1-HLA-A0201-
TableXXII-V5-HLA-A1-9mers- 1 SVVYWILVAV 2 9mers-24P4C12
24P4C12 2 VVYWILVAVG 2 Each peptide is a
portion of
Each peptide is a portion of SEQ ID NO:
3; each start
position is specified, the length 24P4C12 of peptide is 9 amino acids,
and
of peptide is 9 amino acids, and Each peptide is a portion of SEQ the
end position for each
peptide is the start position plus specified, the length of peptide is
eight.
eight. 9 amino acids, and the end Pos 123456789 score
Pos 123456789 score position for each peptide is the
260 VLILGVLGV 31
1 VLEAILLLV 20 start position plus
eight. 244 LLFILLLRL 29
7 LLVLIFLRQ 10 SCOr 580 LLMRN1VRV 29
Pos 123456789
95 ILSSNIISV 28
TableXXII-V6-HLA-A1-9mers- 19 FQTSILGAY 16 204
GISGLIDSL 28
24P4C12 14 PTGHVFQTS 11 261 LILGVLGVL 28
Each peptide is a portion of SEQ 12 ITPTGHVFQ 8 322
ILLLMLIFL 28
ID NO: 13; each start position is 18 VFQTSILGA 7 506
AUL1LVQ1 28
specified, the length of peptide is 20 QTSILGAYV 7 170
FLLPSAPAL 27
9 amino acids, and the end 252
LVAGPLVLV 27
position for each peptide is the TableXXII-V9-HLA-A1-9mers-
449 GLFVVTLNVVV 27
start position plus eight. 24P4C12 487 LISAFIRTL 27
Pos 123456789 score Each
peptide is a portion of 604 LLVVGGVGV 27
2 YSSKGLIPR 12 SEQ ID NO: 19; each
start 45 ILGYIVVGI 26
GYSSKGL1P 7 position is specified, the length
232 ILVALGVAL 26
3 SSKGLIPRS 7 of peptide
is 9 amino acids, and 233 LVALGVALV 26
8 IPRSVFNLQ 7 the end position for
each 315 VLAVLEAIL 26
9 PRSVFNLQI 7 peptide is the start
position plus 501 SLAFGALIL 26
6 GLIPRSVFN 5 eight. 521
YIDHKLRGV 26
Pos 123456789 score 42 LLFILGYIV 25
TableXXII-V7-HLA-A1-9mers- 11 PTQPATLGY 31 107
GLQCPTPQV 25
24P4C12 15 ATLGYVLWA 16 200 TIQQGtSGL 25
Each peptide is a portion of 211 SLNARDISV
25
SEQ ID NO: 15; each start TableXXIII-V1-HLA-A0201- 239 ALVLSLLFI
25
position is specified, the length 9mers-24P4C12 257 LVLVLILGV 25
of peptide is 9 amino acids, and Each peptide is a portion of 258
VLVLILGVL 25
the end position for each SEQ ID NO: 3; each start 282 VLRDKGASI
25
peptide is the start position plus position is specified, the length 317
AVLEAILLL 25
eight. of peptide is 9 amino acids, and 457
vLALGgcvL 25
Pos 123456789 score the end position for
each 598 LLFFGKLLV 25
ILVAVGQMM 5 peptide is the start position plus
650 VIASGFFSV 25
3 YWILVAVGQ 4 eight 686
YMSKSLLKI 25
7 VAVGQMMST 4 Pos 123456789 score 41 FLLFILGYI
24
167
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TableXXIII-V1-HLA-A0201- TableXXIII-V1-HLA-A0201- TableXXIII-V1-HLA-
A0201-
9mers-24P4C12 9mers-24P4C12 9mers-24P4C12
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
peptide is the start position plus peptide is the start position
plus peptide is the start position plus
eight. eight eight
Pos 123456789 score Pos 123456789 score Pos 123456789 score
49 IVVGIVAWL 24 34 DVICCVLFL 20 514 IARVILEYI 18
310 LAALIVLAV 24 38 CVLFLLFIL 20 517 VILEYINK 18
311 AALIVLAVL 24 44 FILGYIVVG 20 583 RNIVRVVVL 18
333 RIRIAIALL 24 207 GLIDSLNAR 20 602 GKLLVVGGV 18
434 LIQRSVFNL 24 228 SWYWILVAL 20 645 ILGAYVIAS 18
509 LTLVQIARV 24 234 VALGVALVL 20 46 LGYIVVGIV 17
525 KLRGVQNPV 24 236 LGVALVLSL 20 128 GKNEFSQTV 17
564 AIYGKNFCV 24 242 LSLLFILLL 20 154 WNMTVITSL 17
581 LMRNIVRVV 24 319 LEAILLLML 20 177 ALGRCFPWT 17
596 DLLLFFGKL 24 326 MLIFLRQRI 20 184 WINVIPPAL 17
605 LVVGGVGVL 24 339 ALLKEASKA 20 213 NARDISVKI 17
35 VICCVLFLL 23 364 LLLICIAYW 20 246 FILLLRLVA 17
56 WLYGDPRQV 23 417 SSCPGLMCV 20 289 SISQLGFTT 17
240 LVLSLLFIL 23 503 AFGALILTL 20 300 SAYQSVQET 17
251 RLVAGPLVL 23 633 HLNYYWLPI 20 305 VQETWLAAL 17
253 VAGPLVLVL 23 644 SILGAYVIA 20 312 ALIVLAVLE 17
309 WLAALIVLA 23 673 DLERNNGSL 20 325 LMLIFLRQR 17
340 LLKEASKAV 23 690 SLLKILGKK 20 335 RIAIALLKE 17
358 PLVTFVLLL 23 48 YIVVGIVAW 19 354 TMFYPLVTF 17
494 TLRYHTGSL 23 245 LFILLLRLV 19 359 LVTFVLLLI 17
518 ILEYIDHKL 23 255 GPLVLVLIL 19 453 TLNVVVLALG 17
547 CLEKFIKFL 23 262 ILGVLGVLA 19 456 VVVLALGQCV 17
589 VVLDKVTDL 23 268 VLAYGIYYC 19 502 LAFGALILT 17
590 VLDKVTDLL 23 291 SQLGFTTNL 19 504 FGALILTLV 17
597 LLLFFGKLL 23 318 VLEAILLLM 19 513 QIARVILEY 17
100 IISVAENGL 22 323 LLLMLIFLR 19 554 FLNRNAYIM 17
241 VLSLLFILL 22 329 FLRQRIRIA 19 560 YIMIAIYGK 17
248 LLLRLVAGP 22 351 MMSTMFYPL 19 586 VRVVVLDKV 17
249 LLRLVAGPL 22 365 LLICIAYWA 19 642 MTSILGAYV 17
265 VLGVLAYGI 22 414 LVNSSCPGL 19 658 VFGMCVDTL 17
446 GVLGLFWTL 22 464 VLAGAFASF 19 31 SCTDVICCV 16
452 VVTLNWVLAL 22 544 CLWCLEKFI 19 43 LFILGYIVV 16
578 FMLLMRNIV 22 617 FFSGRIPGL 19 64 VLYPRNSTG 16
638 WLPIMTSIL 22 666 LFLCFLEDL 19 90 NIFSCILSS 16
660 GMCVDTLFL 22 86 LLYFNIFSC 18 119 SCPEDPWTV 16
158 VITSLQQEL 21 231 WILVALGVA 18 144 NRNFCLPGV 16
187 VIPPALPGI 21 235 ALGVALVLS 18 148 CLPGVPWNM 16
191 ALPGITNDT 21 243 SLLFILLLR 18 161 SLQQELCPS 16
237 GVALVLSLL 21 336 IAIALLKEA 18 230 YWILVALGV 16
247 ILLLRLVAG 21 355 MFYPLVTFV 18 254 AGPLVLVLI 16
313 LIVLAVLEA 21 369 IAYWAMTAL 18 308 TWLAALIVL 16
314 IVLAVLEAI 21 380 ATSGQPQYV 18 316 LAVLEAILL 16
442 LQIYGVLGL 21 394 ISSPGCEKV 18 320 EAILLLMLI 16
507 LILTLVQIA 21 439 VFNLQIYGV 18 357 YPLVTFVLL 16
537 IMCCFKCCL 21 459 ALGQCVLAG 18 362 FVLLLICIA 16
599 LFFGKLLVV 21 510 TLVQIARVI 18 373 AMTALYLAT 16
693 KILGKKNEA 21 511 LVQIARVIL 18 376 ALYLATSGQ 16
168
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TableXXIII-V1-EILA-A0201- TableXX1114/5-FILA-A0201-
Each peptide is a portion of
9mers-24P4C12 9mers-.24P4C12 SEQ ID NO: 17; each start
Each peptide is a portion of Each peptide is a portion of position is
specified, the length
SEQ ID NO: 3; each start SEQ ID NO: 11; each start of
peptide is 9 amino acids, and
position is specified, the length position is specified, the length the
end position for each
of peptide is 9 amino acids, and of peptide is 9 amino acids, and
peptide is the start position plus
the end position for each the end position for each eight.
peptide is the start position plus peptide is the start position plus
Pos 123456789 score
eight. eight. 4 WLPIMRNPI 19
Pos 123456789 score Pos 123456789 score 7 IMRNPITPT 19
407 SCN PTAHLV 16 1 VLEAILLLV 25 20
QTSILGAYV 17
458 LALGQCVLA 16 9 VLIFLRQRI 21 10 NPITPTGHV 15
637 YWLP I MTS I 16 2 LEAILLLVL 20 16
GHVFQTSIL 12
640 PIMTSILGA 16 6 LLLVLIFLR 19 15 TGHVFgTSI 11
52 GIVAWLYGD 15 3 EAILLLVLI 18 18 VFQTS1LGA 11
141 YTKNRNFCL 15 4 AILLLVLIF 18 12 ITPTGHVFQ 10
225 FAQSWYWIL 15 7 LLVLIFLRQ 13 5 LPIMRNPIT 9
250 LRLVAGPLV 15 8 LVLIFLRQR 13 13 TPTGHVFQT 9
264 GVLGVLAYG 15
275 YCWEEYRVL 15 TableXXIII-V9-HLA-A0201-
366 LICIAYWAM 15 TableXXIII-V6-HLA-A0201- 9mers-24P4C12
368 CIAYWAMTA 15 9mers-
24P4C12 Each peptide is a portion of
371 YWAMTALYL 15 Each peptide is a portion of SEQ
ID NO: 19; each start
374 MTALYLATS 15 SEQ ID NO: 13; each start
position is specified, the length
406 TSCNPTAHL 15 position is specified, the length
of peptide is 9 amino acids,
433 GLIQRSVFN 15 of peptide
is 9 amino acids, and and the end position for each
443 QIYGVLGLF 15 the end position for each
peptide is the start position plus
491 F I RTLRYHT 15 peptide is the start
position plus eight.
573 SAKNAFMLL 15 eight. Pos 123456789 score
657 SVFGMCVDT 15 Pos 123456789 score 9 PLPTQPATL 21
663 VDTLFLCFL 15 2 YSSKGLIPR 12 2 WAMTALYPL 20
1 GYSSKGLIP 7 15 ATLGYVLWA 20
TableXXIII-V3-HLA-A0201- 3 SSKGLIPRS 7 6 ALYPLPTQP 16
9mers-24P4C12 8 IPRSVFNLQ 7 12 TQPATLGYV 14
Each peptide is a portion of SEQ 9 PRSVFNLQI 7 13
QPATLGYVL 14
ID NO: 7; each start position is 6 GLIPRSVFN 5 16
TLGYVLWAS 14
specified, the length of peptide is 5 TALYPLPTQ
13
9 amino acids, and the end TableXXIII-V7-HLA-A0201- 4 MTALYPLPT 12
position for each peptide is the 9mers-24P4C12 8 YPLPTgPAT 12
start position plus eight. Each peptide is a portion
of SEQ 3 AMTALYPLP 11
Pos 123456789 score ID NO: 15; each start position is
9 ITPPALPGI 22 specified, the length of
peptide
6 WTNITPPAL 17 is 9 amino
acids, and the end TableXXIV-V1-HLA-A0203-
8 N ITPPALPG 11 position for each
peptide is the 9mers-24P4C12
2 RCFPVVTN IT 10 start position plus eight
Pos 1234567890 score
Pos 123456789 score NoResultsFound.
TableXXIII-V5-HLA-A0201- 1 SVVYWILVAV 20
9mers-24P4C12 4 WILVAVGQM 18 TableXXIV-V3-HLA-A0203-
Each peptide is a portion of 5 ILVAVGQMM 16 9mers-24P4C12
SEQ ID NO: 11; each start 7 VAVGQ_MMST 13 Pos 1234567890 score
position is specified, the length 8 AVGQMMSTM 12 NoResultsFound.
of peptide is 9 amino acids, and 6 LVAVGgMMS 10
the end position for each TableXXIV-
V5-HLA-A0203-
peptide is the start position plus TableXXIII-V8-HLA-A0201- 9mers-
24P4C12
eight. 9mers-24P4C12 Pos 1234567890 score
Pos 123456789 score NoResultsFound.
ILLLVLIFL 28
169
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TableXXIV-V6-HLA-A0203- TableXXV-V1-HLA-A3-9mers- TableXXV-V1-HLA-A3-
9mers-
9mers-24P4C12 24P 24P
Pos 1234567890 score Each peptide is a portion
of Each peptide is a portion of
NoResultsFound. SEQ ID NO: 3; each start SEQ ID NO: 3; each
start
TableXXIV-V7-HLA-A0203- of peptide is 9 amino acids, and of peptide is 9
amino acids, and
Pos 1234567890 score peptide is the start position plus
peptide is the start position plus
NoResultsFound. eight. eight.
Pos 123456789 spore Pos 123456789 score
TableXXIV-V8-HLA-A0203- 393 NISSPGCEK 21 457 VLALGQCVL 18
9mers-24P4C12 517 VILEYIDHK 21 564 AIYGKNFCV 18
Pos 1234567890 score 593 KVTDLLLFF 21 587 RWVLDKVT 18
NoResultsFound. 619 SGRIPGLGK 21 649 YVIASGFFS 18
621 RIPGLGKDF 21 10 DEAYGKPVK 17
TableXXIV-V9-HLA-A0203- 44 FILGYIVVG 20 63 QVLYPRNST 17
9mers-24P4C12 56 WLYGDPRQV 20 121 PEDP1NTVGK 17
Pos 1234567890 score 243 SLLFILLLR 20 177 ALGRCFPWT 17
NoResultsFound. 259 LVLILGVLG 20 211 SLNARDISV 17
TableXXV-V1-HLA-A3-9mers- 363 VLLLICIAY 20 235 ALGVALVLS 17
Each peptide is a portion of 501 SLAFGALIL .. 20 .. ' .. 252
.. LVAGPLVLV 17
SEQ ID NO: 3; each start 606 VVGGVGVLS 20 309 WLAALIVLA 17
position is specified, the length 689 KSLLKILGK .. 20 .. 335 ..
RIAIALLKE .. 17
of peptide is 9 amino acids, and 16 PVKYDPSFR 19 365
LLICIAYWA 17
the end position for each 170 FLLPSAPAL 19 368
CIAYWAMTA 17
peptide is the start position plus 186 NVTPPALPG 19 401
KVPINTSCN 17
eight - 207 GLIDSLNAR 19 421
GLMCVEgGY 17
Pos 123456789 score 246 FILLLRLVA 19 456 WVLALggCV 17
585 IVRVWLDK 29 249 LLRLVAGPL 19 459 ALGQCVLAG 17
424 CVFQGYSSK 27 260 VLILGVLGV 19 510 TLVQIARVI 17
'
64 VLYPRNSTG 26 262 ILGVLGVLA 19 542 KCCLWCLEK 17
135 'TVGEVFYTK 26 298 NLSAYVQ 19 562 MIAIYGKNF 17
251 RLVAGPLVL 26 317 AVLEAILLL 19 580 LLMRNIVRV 17
506 ALILTLVQI 24 333 RIRIAIALL 19 583 RNIVRVVVL 17
513 QIARVILEY 24 433 GLIQRSVFN 19 644
SILGAYVIA 17 '
603 KLLVVGGVG 24 508 ILTLVQIAR 19 657 SVFGMCVDT 17
690 SLLKILGKK 24 525 KLRGVQNPV 19 662 CVDTLFLCF 17
267 GVLAYGIYY 23 560 YIMIAIYGK 19 26 PIKNRSCTD 16
282 VLRDKGASI 23 588 VVVLDKVTD 19 34 DVICCVLFL 16
312 ALIVLAVLE 23 604 LLVVGGVGV 19 45 ILGYIVVGI 16
334 IRIAIALLK 23 605 LWGGVGVL - 19 86
LLYFNIFSC 16
102 SVAENGLQC 22 681 LDRPYYMSK 19 157 TVITSLQQE 16
232 ILVALGVAL 22 11 EAYGKPVKY 18 165 ELCPSFLLP 16
247 ILLLRLVAG 22 49 IWGIVAWL 18 237 GVALVLSLL 16
443 QIYGVLGLF 22 73 AYCGMGENK 18 258 VLVLILGVL 16
464 VLAGAFASF 22 220 KIFEDFAQS 18 289 SISQLGFTT 16
516 RVILEYIDH 22 248 LLLRLVAGP 18 304 SVQETWLAA 16
579 MLLMRNIVR 22 261 LILGVLGVL 18 323 LLLMLIFLR 16
50 WGIVAWLY 21 264 GVLGVLAYG 18 364 LLLICIAYW 16
212 LNARDISVK 21 272 GIYYCWEEY 18 470 ASFYWAFHK 16
281 RVLRDKGAS 21 278 EEYRVLRDK 18 494 TLRYHTGSL 16
321 AILLLMLIF 21 314 IVLAVLEAI 18 511 iNgIARviL 16
338 IALLKEASK 21 432 KGLIQRSVF 18 554 FLNRNAYIM 16
339 ALLKEASM 21 441 NLglYGVLG 18 571 CVSAKNAFM 16
376 ALYLATSGQ 21 446 GVLGLF1NTL 18 584 NIVRVVVLD 16
170
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TableXXV-V1-HLA-A3-9mers- TableXXV-V1-HLA-A3-9mers- Each peptide is a
portion of SEQ
24P 2415 ID NO: 13;
each start position is
Each peptide is a portion of Each
peptide is a portion of specified, the length of peptide is
SEQ ID NO: 3; each start SEQ ID NO: 3; each start 9 amino acids, and the
end
position is specified, the length position is specified, the
length position for each peptide is the
of peptide is 9 amino acids, and of peptide is 9 amino acids, and start
position plus eight.
the end position for each the end position for each Pos
123456789 score
peptide is the start position plus peptide is the start position plus
6 GLIPRSVFN 22
eight. eight. 5 KGLIPRSVF 18
Pos 123456789 score Pos 123456789 score 7 LIPRSVFNL 11
673 DLERNNGSL 16 527 RGVQNPVAR 14
693 KILGKKNEA 16 528 GygNPVARC 14 TableXXV-
V7-HLA-A3-9mers-
698 KNEAPPDNK 16 534 ARCIMCCFK 14 24P4C12
20 DPSFRGPIK 15 558 NAYIMIAIY 14 Each peptide is a
portion-of
48 YIVVGIVAW 15 567 GKNFCVSAK 14 SEQ ID
NO: 15; each start
58 YGDPRQYLY 15 596 DLLLFFGKL
14 position is specified, the length
99 NIISVAENG 15 609 GVGVLSFFF
14 of peptide is 9 amino acids, and
151 GVPWNMTVI 15 638 WLPIMTSIL 14 the end
position for each
191 ALPGITNDT 15 647 GAY VIASGF
14 peptide is the start position plus
231 WILVALGVA 15 665 TLFLCFLED 14 eight.
234 VALGVALVL 15 685 YYMSKSLLK 14 Pos
123456789 score
257 LVLVLILGV 15 694 ILGKKNEAP 14 8 AVGQMMSTM 20
318 VLEAILLLM 15 699 NEAPPDNKK 14 5 ILVAVGQMM 19
322 ILLLMLIFL 15 701 APPDNKKRK 14 6 LVAVG2L1MS 15
327 LIFLROIR 15 4 WILVAVGQM 14
329 FLRQRIRIA 15 TableXXV-V3-HLA-A3-9mers- 3 YWILVAVGQ 12
532 PVARCIMCC 15 24P4C12 1 SlNYWILVAV 10
589 VVLDKVIDL 15 Each peptide is a portion of
597 LLLFFGKLL 15 SEQ ID NO: 7; each start TableXXV-
V8-HLA-A3-9mers-
598 LLFFGKLLV 15 position is
specified, the length 24P4C12
622 IPGLGKDFK 15 of peptide
is 9 amino acids, and Each peptide is a portion of SEQ
645 ILGAYVIAS 15 the end
position for each ID NO: 17; each start position is
651 IASGFFSVF 15 peptide is
the start position plus specified, the length of peptide is
680 SLDRPYYMS 15 eight. 9 amino acids, and the end
691 LLKILGKKN 15 Pos 123456789 score
position for each peptide is the
7 DEDDEAYGK 14 8 NITPPALPG 17 start position
plus eight.
42 LLFILGYIV 14 Pos 123456789 score
53 IVAWLYGDP 14 TableXXV-V5-HLA-A3-9mers- 11 PITPTGHVF 22
81 KDKPYLLYF 14 24P4C12 6 PIMRNPITP 16
95 ILSSNIISV 14 Each peptide is a portion
of SEQ 4 WLPIMRNPI 12
148 CLPGVPWNM 14 ID NO: 11; each start position is
9 RNPITPTGH 11
171 LLPSAPALG 14 specified, the length of
peptide is 1 NYYWLPIMR 10
244 LLFILLLRL 14 9 amino acids, and the end
17 HVFQTSILG 10
311 AALIVLAVL 14 position for each peptide is the
315 VLAVLEAIL 14 start position plus eight.
324 LLMLIFLRQ 14 Pos 123456789 score
TableXXV-V9-HLA-A3-9mers-
326 MLIFLMRI 14 4 AILLLVLIF 21 24P4C12
337 AIALLKEAS 14 8 LVLIFLRQR 20 Each
peptide is a portion of SEQ
359 LVTFVLLLI 14 5 ILLLVLIFL 16 ID
NO: 19; each start position is
370 AYWAMTALY 14 6 LLLVLIFLR 16
specified, the length of peptide is
378 YLATSaQPQ 14 1 VLEAILLLV 15 9 amino acids,
and the end
388 VLWASNISS 14 7 LLVLIFLRQ 14
position for each peptide is the
. 453 TLNVVVLALG 14 9 VLIFLFIQRI 14 start position
plus eight.
465 LAGAFASFY 14 Pos 123456789 score
487 LISAFIRTL 14 TableXXV-V6-HLA-A3-9mers- 6 ALYPLPTQP 25
496 RYHTGSLAF 14 24P4C12 9 PLPTQPATL 18
523 DHKLRGVQN 14 11 PTQPATLGY 12
171
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16 TLGYVLWAS 12 TableXXVI-V1-HLA-A26-
Pos 123456789 score
9mers-24P4C12 7 LIPRSVFNL 16
Table)0(VI-V1-HLA-A26- Each peptide is a portion of 5 KGLIPRSVF
9
9mers-24P4C12 SEQ ID NO: 3; each start
Each peptide is a portion of position is specified, the
length TableXXVI-V7-HLA-A26-9mers-
SEQ ID NO: 3; each start of peptide is 9 amino acids, and 24P4C12
position is specified, the length the end position for each
Each peptide is a portion of SEQ ID
of peptide is 9 amino acids, and peptide is the start position plus NO:
15; each start position is
the end position for each eight
specified, the length of peptide is 9
peptide is the start position plus Pos 123456789 score
amino adds, and the end position
eight. 184 WTNVTPPAL 17 for each
peptide is the start position
Pos 123456789 score 216 DISVKIFED 17
plus eight.
34 DVICCVLFL 35 261 LILGVLGVL 17 Pos
123456789 score
49 IVVGIVAWL 28 358 PLVTFVLLL 17 8
AVGQMMSTM 12
483 PTFPLISAF 28 438 SVFNLQIYG 17 6
LVAVGQMMS 11 .
605 LVVGGVGVL 27 442 LQIYGVLGL 17 4
WILVAVGQM 10
593 KVTDLLLFF 26 443 QIYGVLGLF 17 1
SVVYWILVAV 8
317 AVLEAILLL 25 487 LISAFIRTL 17 5
ILVAVGQMM 6
592 DKVTDLLLF 25 608 GGVGVLSFF 17 2
VVYWILVAVG 5
138 EVFYTKNRN 24 664 DTLFLCFLE 17 7
VAVGQMMST 5
240 LVLSLLFIL 24
589 VVLDKVTDL 24 TableXXVI-V3-HLA-A26-9mers- TableXXVI-V8-HLA-A26-
9mers-
38 CVLFLLFIL 23 24P4C12 24P4C12
237 GVALVLSLL 23 Each
peptide is a portion of SEQ Each peptide is a portion of SEQ ID
11 EAYGKPVKY 22 ID NO: 7; each start position is
NO: 17; each start position is
267 GVLAYGIYY 22 specified,
the length of peptide is 9 specified, the length of peptide is 9
285 DKGASISQL 22 amino acids,
and the end position amino acids, and the end position
452 VVTLNWVLAL 22 for each
peptide is the start for each peptide is the start position
50 VVGIVAWLY 20 position plus eight.
plus eight.
79 ENKDKPYLL 20 Pos 123456789 score Pos 123456789 score
157 TVITSLQQE 20 6 1NTNITPPAL 17 19 FQTSILGAY 20
263 LGVLGVLAY 20 9 ITPPALPGI 13 11 PITPTGHVF 15
446 GVLGLFWTL 20 17 HVFQTSILG 15
628 DFKSPHLNY 20 TableXXVI-V5-HLA-A26-9mers-
16 GHVFQTSIL 13
641 IMTSILGAY 20 24P4C12 20 QTSILGAYV 10
662 CVDTLFLCF 20 Each peptide is a portion
of SEQ 14 PTGHVFQTS 9
236 LGVALVLSL 19 ID NO: 11; each start position is
258 VLVLILGVL 19
specified, the length of peptide is TableX)(VI-V9-HLA-A26-9mers-
307 ETWLAALIV 19 9 amino acids, and the end
24P4C12
320 EAILLLMLI 19
position for each peptide is the Each peptide is a portion of SEQ
414 LVNSSCPGL 19 start position
plus eight. ID NO: 19; each start position is
437 RSVFNLQIY 19 P os 123456789
SCOr specified, the length of peptide is 9
513 QIARVILEY 19 e amino
acids, and the end position
609 GVGVLSFFF 19 3 EAILLLVLI 19 for each peptide is
the start
673 DLERNNGSL 19 4 AILLLVLIF 18 position plus eight
32 CTDVICCVL 18 8 LVLIFLRQR 15 Pos
123456789 score
198 DTTIQQGIS 18 2 LEAILLLVL 14 11
PTQPATLGY 20
200 TIQQGISGL 18 5 ILLLVLIFL 13 15
ATLGYVLWA 13
204 GISGLIDSL 18 2 WAMTALYPL 12
244 LLFILLLRL 18 Table)0011-V6-HLA-A26-9mers-
13 QPATLGYVL 10
294 GFTTNLSAY 18 24P4C12 4 MTALYPLPT 9
354 TMFYPLVTF 18 Each peptide is a portion of
SEQ ID 9 PLPTQPATL 9
360 . VTFVLLLIC 18 NO: 13; each start position is
400 EKVPINTSC 18 specified, the length of
peptide 1s9 TableXXVII-V1-HLA-B0702-
511 LVQIARVIL 18 amino acids, and the end
position for 9mers-24P4C12
596 DLLLFFGKL 18 each peptide is the start position
102 SVAENGLQC 17 plus eight.
172
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Each peptide is a portion of SEQ TableXXVII-
V1-HLA-B0702- TableXXVII-V1-HLA-B0702-
ID NO: 3; each start position is 9mers-24P4C12 9mers-
24P4C12
specified, the length of peptide is 9 Each peptide is a portion of
SEQ Each peptide is a portion of SEQ
amino acids, and the end position ID NO: 3;
each start position is ID NO: 3; each start position is
for each peptide is the start
specified, the length of peptide is 9 specified, the length of peptide is 9
position plus eight. amino acids, and the end position amino acids, and
the end position
Pos 123456789 score for each peptide is the start for each
peptide is the start
255 GPLVLVLIL 23 position plus eight. position plus eight.
357 YPLVTFVLL 23 Pos 123456789 score Pos 123456789
score
683 RPYYMSKSL 21 170 FLLPSAPAL 13 104 AENGLQCPT 11
149 LPGVPWNMT 20 182 FPI/VT-NV.1*PP 13 107 GLQCPTPQV 11
396 SPGCEKVPI 20 228 SWYWILVAL 13 109 QCPTPQVCV 11
482 IPTFPLISA 20 241 VLSLLFILL 13 112 TPQVCVSSC 11
631 SPHLNYYWL 20 249 LLRLVAGPL 13 123 DPVVTVGKNE 11
15 KPVKYDPSF 19 261 LILGVLGVL 13 163 QQELCPSFL 11
152 VPWNMTVIT 19 302 YQSVQETWL 13 169 SFLLPSAPA 11
167 CPSFLLPSA 19 319 LEAILLLML 13 177 ALGRCFPWT 11
25 GPIKNRSCT 18 358 PLVTFVLLL 13 191 ALPGITNDT 11
172 LPSAPALGR 18 369 IAYWAMTAL 13 237 GVALVLSLL 11
83 KPYLLYFNI 17 371 YVVAMTALYL 13 239 ALVLSLLFI 11
188 TPPALPGIT 17 409 NPTAHLVNS 13 258 VLVLILGVL 11
192 LPGITNDTT 17 442 LQIYGVLGL 13 262 ILGVLGVLA 11
57 LYGDPRQVL 16 446 GVLGLFWTL 13 275 YCWEEYRVL 11
232 ILVALGVAL 16 478 KPQDIPTFP 13 310 LAALIVLAV 11
253 VAGPLVLVL 16 487 L1SAFIRTL 13 332 QRIRIAIAL 11
479 PQDIPTFPL 16 494 TLRYHTGSL 13 343 EASKAVGQM 11
503 AFGALILTL 16 501 SLAFGALIL 13 354 TMFYPLVTF 11
49 IVVGIVAWL 15 511 LVQIARVIL 13 384 QPQYVLWAS 11
120 CPEDPWTVG 15 590 VLDKVTDLL 13 414 LVNSSCPGL 11
175 APALGRCFP 15 622 IPGLGKDFK 13 426 FQGYSSKGL 11
189 PPALPGITN 15 651 IASGFFSVF 13 434 LIQRSVFNL 11
234 VALGVALVL 15 32 CTDVICCVL 12 440 FNLQIYGVL 11
251 RLVAGPLVL 15 78 GENKDKPYL 12 450 LFWTLNVVVL 11
381 TSGQPQYVL 15 154 WNMTVITSL 12 464 VLAGAFASF 11
406 TSCNPTAHL 15 184 WTNVTPPAL 12 518 ILEYIDHKL 11
583 RNIVRVVVL 15 242 LSLLFILLL 12 531 NPVARCIMC 11
617 FFSGRIPGL 15 244 LLFILLLRL 12 537 IMCCFKCCL 11
20 DPSFRGPIK 14 285 DKGAS1SQL 12 571 CVSAKNAFM 11
34 DVICCVLFL 14 305 VQETWLAAL 12 573 SAKNAFMLL 11
66 YPRNSTGAY 14 308 TWLAALIVL 12 574 AKNAFMLLM 11
204 GISGLIDSL 14 315 VLAVLEAIL 12 596 DLLLFFGKL 11
236 LGVALVLSL 14 322 ILLLMLIFL 12 597 LLLFFGKLL 11
252 LVAGPLVLV 14 356 FYPLVTFVL 12 599 LFFGKLLVV 11
291 SQLGFTTNL 14 373 AMTALYLAT 12 638 WLPIMTSIL 11
311 AALIVLAVL 14 380 ATSGQPQYV 12 663 VDTLFLCFL 11
317 AVLEAILLL 14 457 VLALGQCVL 12 686 YMSKSLLKI 11
333 RIRIAIALL 14 525 KLRGVQNPV 12 702 PPDNKKRKK 11
351 MMSTMFYPL 14 547 CLEKFIKFL 12
419 CPGLMCVFQ 14 572 VSAKNAFML 12 TableXXVII-
V3-HLA-B0702-
452 VVTLNWVLAL 14 589 VVLDKVTDL 12 9mers-24P4C12
499 TGSLAFGAL 14 591 . LDKVTDLLL 12
Each peptide is a portion of SEQ ID
605 LWGGVGVL 14 626 GKDFKSPHL 12 NO: 7; each
start position is
660 GMCVDTLFL 14 658 VFGMCVDTL 12 specified, the length of
peptide is 9
60 DPRQVLYPR 13 701 APPDNKKRK 12 amino acids, and the end
position
100 IISVAENGL 13 28 KNRSCTDVI 11 for each
peptide is the start position
110 CPTPQVCVS 13 45 ILGYIVVGI 11 = plus eight.
164 QELCPSFLL 13 79 ENKDKPYLL 11 Pos 123456789 score
173
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TableXXVII-V3-HLA-B0702- e
TableXXVIII-V1-HLA-B08-9mers
9mers-24P4C12 1 SWYVVILVAV 9 Each peptide is a
portion of SEQ
Each peptide is a portion of SEQ ID 5 ILVAVGQMM
9 ID NO: 3; each start position is
NO: 7; each start position is 8 AVGQMMSTM 9
specified, the length of peptide is 9
specified, the length of peptide is 9 7 VAVGQMMST
8 amino acids, and the end position
amino acids, and the end position 4 WILVAVGQM I for each
peptide is the start
for each peptide is the start position position plus eight.
plus eight. TableXXVII-V8-HLA-B0702-9mers-
Pos 123456789 score
Pos 123456789 score 24P4C12 589
WLDKVTDL 22
4 FPWTNITPP 12 Each peptide is a portion of SEQ
ID 333 RIRIAIALL 21
6 WTNITPPAL 12 NO: 17; each start position is 583 RNIVRVVVL
21
1 GRCFPVVTNI 10 specified, the length of peptide
is 9 591 LDKVTDLLL 21
2 RCFPWTNIT 9 amino acids, and the end position
626 GKDFKSPHL 21
PWTNITPPA 9 for each peptide is the start position
687 MSKSLLKIL 21
9 ITPPALPGI 9 plus eight. 340 LLKEASKAV 20
8 NITPPALPG 7 Pos
123456789 score 474 WAFHKPQDI 20
19 FQTSILGAY 20 523
DHKLRGVQN 20
TableXXVII-V5-HLA-B0702- 11 PITPTGHVF 15 540
CFKCCLWCL 20
9mers-24P4C12 17 HVFQTSILG 15 617
FFSGRIPGL 20
Each peptide is a portion of SEQ ID 16 GHVFQTSIL 13 2
GGKQRDEDD 19
NO: 11; each start position is 20 QTSILGAYV 10 232
ILVALGVAL 19
specified, the length of peptide is 9 14 PTGHVFQTS 9 255
GPLVLVLIL 19
amino acids, and the end position, 631 SPHLNYYWL 19
for each peptide is the start position 694 ILGKKNEAP 19
plus eight. TableXX'VII-V9-HLA-B0702-9mers-
139 VFYTKNRNF 18
Pos 123456789 score 24P4C12 170
FLLPSAPAL 18
2 LEAILLLVL 14 Each peptide is a portion of SEQ
ID 241 VLSLLFILL 18
5 ILLLVLIFL 12 NO: 19; each start position is 247 ILLLRLVAG
18
4 AILLLVLIF 11 specified, the length of peptide
is 9 258 VLVLILGVL 18
1 VLEAILLLV 9 amino acids, and the end position
315 VLAVLEAIL 18
3 EAILLLVLI 9 for each peptide is the start
position 322 ILLLMLIFL 18
9 VLIFLRQRI 7 plus eight. 357 YPLVTFVLL 18
Pos 123456789 score 457 VLALGQCVL 18
TableXXVII-V6-HLA-B0702- 13 QPATLGYVL 23 501 SLAFGALIL
18
9mers-24P4C12 8 YPLPTQPAT 19 514 IARVILEYI 18
Each peptide is a portion of SEQ ID 10 LPTQPATLG 14 518
ILEYIDHKL 18
NO: 13; each start position is 15 ATLGYVLWA 13 546
WCLEKFIKF 18
specified, the length of peptide is 9 2 WAMTALYPL 12 547
CLEKFIKFL 18
amino acids, and the end position 7 LYPLPTQPA 11 683
RPYYMSKSL 18
for each peptide is the start position 9 PLPTQPATL 11 11
EAYGKPVKY 17
plus eight 213 NARDISVKI 17
Pos 123456789 score TableXXVIII-V1-HLA-B08-9mers 216
DISVKIFED 17
8 IPRSVFNLQ 14 Each peptide is a portion of SEQ
358 PLVTFVLLL 17
5 KGLIPRSVF 12 ID NO: 3; each start position is 533
VARCIMCCF 17
7 LIPRSVFNL 11 specified, the length of peptide
is 9 590 VLDKVTDLL 17
9 PRSVFNLQI 10 amino adds, and the end position
596 DLLLFFGKL 17
4 SKGLIPRSV 7 for each peptide is the start 597 LLLFFGKLL
17
position plus eight 673 DLERNNGSL 17
TableXXVII-V7-HLA-B0702- Pos 123456789 score 691
LLKILGKKN 17
9mers-24P4C12 79 ENKDKPYLL 32 45
ILGYIVVGI 16
Each peptide is a portion of SEQ ID 141 YTKNRNFCL 29 64
VLYPRNSTG 16
NO: 15; each start position is 282 VLRDKGASI 29 81
KDKPYLLYF 16
specified, the length of peptide is 9 573 SAKNAFMLL 26 100
IISVAENGL 16
amino acids, and the end position 249 LLRLVAGPL 23 158
VITSLQQEL 16
for each peptide is the start position 494 TLRYHTGSL 23 204
GISGLIDSL 16
plus eight. 26 PIKNRSCTD 22 211 SLNARDISV 16
Pos 123456789 scor 329 FLRQRIRIA 22 244
LLFILLLRL 16
174
,
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TableXXVIII-V1-HLA-B08-9mers TableXXVIII-V5-1308-9mers-
TableXXVIII-V8-HLA-B08-9mers-
Each peptide is a portion of SEQ 24P4C12 24P4C12
ID NO: 3; each start position is Each peptide is a portion
of SEQ ID Each peptide is a portion of SEQ
specified, the length of peptide is 9 NO: 11; each start position is ID
NO: 17; each start position is
= amino acids, and the end position
specified, the length of peptide is 9 specified, the length of peptide is 9
for each peptide is the start amino acids, and the end
position amino acids, and the end position
= position plus eight.
for each peptide is the start position for each peptide is the start
Pos 123456789 score plus eight. position plus
eight
251 RLVAGPLVL 16 Pos 123456789 score Pos 123456789 score
253 VAGPLVLVL 16 2 LEAILLLVL 10 15 TGHVFQTSI 7
338 IALLKEASK 16 6 LLLVLIFLR 8
369 IAYWAMTAL 16 TableXXVIII-V9-HLA-
1308-
433 GLIQRSVFN 16 TableXXVIII-V6-HLA-B08-
9mers- 9mers-24P4C12
551 FIKFLNRNA 16 24P4C12
Each peptide is a portion of SEQ
638 WLPIMTSIL 16 Each peptide is a portion
of SEQ ID NO: 19; each start position is
702 PPDNKKRKK 16 ID NO: 13; each start
position is specified, the length of peptide is
35 VICCVLFLL 15 specified, the length of
peptide is 9 9 amino acids, and the end
200 TIQQGISGL 15 amino acids, and the end
position position for each peptide is the
225 FAQSVVYWIL 15 for each peptide is the start start
position plus eight.
234 VALGVALVL 15 position plus eight Pos 123456789
score
316 LAVLEAILL 15 Pos 123456789 score 9
PLPTQPATL 16
331 RQRIRIAIA 15 6 GLIPRSVFN 16 13 QPATLGYVL 16
396 SPGCEKVPI 15 7 LIPRSVFNL 15 2 WAMTALYPL 14
434 LIQRSVFNL 15 3 SSKGLIPRS 13 16 TLGYVLWAS 8
487 LISAFIRTL 15 8 IPRSVFNLQ 13 18 GYVLWASNI 8
553 KFLNRNAYI 15 1 GYSSKGLIP 11 8
YPLPTQPAT 7
564 AIYGKNFCV 15 9 PRSVFNLQI 8
579 MLLMRNIVR 15
TableXXIX-V1-HLA-B1510-9mers-
693 KILGKKNEA 15 TableXXV11147-HLA-B08-
9mers- 24P4C12
24P4C12
Each peptide is a portion of SEQ ID
TableXXVIII-V3-HLA-B08-9mers- Each peptide is a portion of SEQ NO: 3;
each start position is
24P4C12 ID NO: 15; each start
position is specified, the length of peptide is 9
Each peptide is a portion of SEQ specified, the length of
peptide is 9 amino acids, and the end position
ID NO: 7; each start position is amino acids, and the end
position for each peptide is the start position
specified, the length of peptide is 9 for each peptide is the start plus
eight.
amino acids, and the end position position plus eight. Pos
123456789 score
for each peptide is the start Pos 123456789 score 275
YCWEEYRVL 16
position plus eight 5 ILVAVGQMM 7 583 RNIVRVVVL
16
Pos 123456789 score 4 WILVAVGQM 6 57 LYGDPRQVL 15
6 WTNITPPAL 11 7 VAVGQMMST 5 232 ILVALGVAL 15
4 FPWTNITPP 8 1 SWYWILVAV 4 253 VAGPLVLVL 15
1 GRCFPWTNI 7 381 TSGQPQYVL 15
9 ITPPALPGI 7 TableXXVIII-V8-HLA-B08-
9mers- 487 LISAFIRTL 15
24P4C12 605 LVVGGVGVL 15
TableXXVIII-V5-608-9mers- Each peptide is a portion of SEQ 49
IVVGIVAWL 14
24P4C12 ID NO: 17; each start position is 78
GENKDKPYL 14
Each peptide is a portion of SEQ ID specified, the length of
peptide is 9 100 IISVAENGL 14
NO: 11; each start position is amino acids, and the end position 170
FLLPSAPAL 14
specified, the length of peptide is 9 for each peptide is
the start 184 WTNVTPPAL 14
amino acids, and the end position position plus eight 200 TIQQGISGL
14
for each peptide is the start position Pos 123456789 score
204 GISGLIDSL 14
plus eight. 5 LPIMRNPIT 15 251 RLVAGPLVL
14
Pos 123456789 score 4 WLPIMRNPI 12 357
YPLV'TFVLL 14
5 ILLLVLIFL 18 16 GHVFQTSIL 11 369 IAYWAMTAL 14
3 EAILLLVLI 14 11 PITPTGHVF 10 457 VLALGQCVL
14
9 VLIFLRQRI 13 7 IMRNPITPT 8 617 FFSGRIPGL
14
4 AILLLVLIF 12 13 TPTGHVFQT 7 32
CTDVICCVL 13
175
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TableXXIX-V1-HLA-B1510-9mers- TableXXIX-V1-HLA-B1510-9mers-
TableXXIX-V1-HLA-B1510-9mers-
24P4C12 24P4C12 24P4C12
Each peptide is a portion of SEQ ID Each peptide is a portion of
SEQ ID Each peptide is a portion of SEQ ID
NO: 3; each start position is NO: 3; each start position
is NO: 3; each start position is
specified, the length of peptide is 9 specified, the length of
peptide is 9 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 position for each peptide is the start
position for each peptide is the start position
plus eight plus eight. plus eight
Pos 123456789 score Pos 123456789 score Pos 123456789 score
79 ENKDKPYLL 13 236 LGVALVLSL 11 679 GSLDRPYYM 9
228 SWYVVILVAL 13 241 VLSLLF I LL 11 15
KPVKYDPSF 8
234 VALGVALVL 13 242 LSLLF I LLL 11 81
KDKPYLLYF 8
255 GPLVLVLIL 13 285 DKGASI SQL 11 132
FSQTVGEVF 8
261 LILGVLGVL 13 291 SQLGFTTNL 11 139
VFYTKNRNF 8
302 YQSVQETWL 13 319 LEAILLLML 11 148 CLPGVPWNM 8
308 TWLAALIVL 13 332 QRIRIAIAL 11 162
LQQELCPSF 8
440 FNLQIYGVL 13 333 RI RIAIALL 11 174
SAPALGRCF 8
446 GVLGLFWTL 13 351 MMSTMFYPL 11 287 GASISQLGF 8
499 TGSLAFGAL 13 354 TMFYPLVTF 11 415 VNSSCPGLM 8
511 LVQIARVIL 13 358 PLVTFVLLL 11 464
VLAGAFASF 8
518 ILEYIDHKL 13 414 LVNSSCPGL 11 468
AFASFYWAF 8
537 IMCCFKCCL 13 434 LIQRSVFNL 11 496 RYHTGSLAF 8
547 CLEKFIKFL 13 479 PQDIPTFPL 11 530
QNPVARCIM 8
572 VSAKNAFML 13 494 TLRYHTGSL 11 570 FCVSAKNAF 8
163 QQELCPSFL 12 590 VLDKVTDLL 11 608 GGVGVLSFF 8
237 GVALVLSLL 12 591 LDKVTDLLL 11 609
GVGVLSFFF 8
244 LLF I LLLRL 12 631 SPHLNYYWL 11 647
GAYVIASGF 8
258 VLVLILGVL 12 684 PYYMSKSLL 11 48 YIVVGIVAW 7
305 VQETWLAAL 12 35 VICCVLFLL 10 69 NSTGAYCGM 7
311 AALIVLAVL 12 38 CVLFLLFIL 10 214
ARDISVKIF 7
315 VLAVLEAIL 12 124 PWTVGKNEF 10 238 VALVLSLLF 7
317 AVLEAILLL 12 225 FAQSWYWIL 10 318 VLEAILLLM 7
322 ILLLMLIFL 12 240 LVLSLLFIL 10 321
AILLLMLIF 7
356 FYPLVTFVL 12 249 LLRLVAGPL 10 366
LICIAYWAM 7
371 YVVAMTALYL 12 316 LAVLEAILL 10 443 QIYGVLGLF 7
406 TSCN PTAH L 12 343 EASKAVGQM 10 533 VARC I
MCCF 7
412 AHLVNSSCP 12 418 SCPGLMCVF 10 546 WCLEKFIKF 7
442 LQIYGVLGL 12 426 FQGYSSKGL 10 554 FLNRNAYIM 7
450 LFVVTLNVVVL 12 477 HKPQDIPTF 10 562 MIAIYGKNF 7
452 VVTLNWVLAL 12 483 PTFPLISAF 10 571 CVSAKNAFM 7
476 FH KPQDI PT 12 540 CFKCCLWCL 10 574
AKNAFMLLM 7
497 YHTGSLAFG 12 573 SAKNAFMLL 10 593 KVTDLLLFF 7
501 SLAFGALIL 12 596 DLLLFFGKL 10 621
RIPGLGKDF 7
503 AFGALILTL 12 597 LLLFFGKLL 10 634
LNYYWLPIM 7
523 DHKLRGVQN 12 632 PHLNYYWLP 10 653 SGFFSVFGM 7
589 VVLDKVTDL 12 638 WLPIMTS IL 10
626 GKDFKSPHL 12 663 VDTLFLCFL 10 Table)0(1X-
V3-HLA-B1510-9mers-
651 IASGFFSVF 12 666 LFLCFLEDL 10 24P4C12
658 VFGMCVDTL 12 683 RPYYMSKSL 10 Each
peptide is a portion of SEQ ID
660 GMCVDTLFL 12 687 MSKSLLKIL 10 . NO: 7; each start
position is
673 DLERNNGSL 12 33 TDVICCVLF 9
specified, the length of peptide is 9
34 DVICCVLFL 11 36 ICCVLFLLF 9 amino
acids, and the end position
88 YFNIFSCIL 11 217 ISVKIFEDF 9 for each
peptide is the start position
141 YTKNRNFCL 11 347 AVGQMMSTM 9 plus eight.
154 WNMTVITSL 11 432 KGLIQRSVF 9 Pos 123456789 score
158 VITSLQQEL 11 461 GQCVLAGAF 9 6 WIN
ITPPAL 13
164 QELCPSFLL 11 607 VGGVGVLSF 9
176
=
=
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TableXXIX-V5-81510-9mers-
TableXXIX-V9-B1510-9mers-24P4C12 ' TableXXX-V1-HLA-B2705-9mers-
24P4C12 Each peptide is a portion of SEQ ID NO: * 24P4C12
Each peptide is a portion of SEQ 19; each start position is
specified, the Each peptide is a portion of SEQ ID
ID NO: 11; each start position is length of peptide is 9 amino
acids, and NO: 3; each start position is
specified, the length of peptide is 9 the end position for each
peptide is the specified, the length of peptide is 9
amino acids, and the end position start
position plus eight. amino acids, and the end position
for each peptide is the start Pos 123456789 score
for each peptide is the start position
position plus eight. 13 QPATLGYVL 13 plus eight.
Pos 123456789 score 9 PLPTQPATL 12 Pos 123456789
score
2 LEAILLLVL 13 2 WAMTALYPL 10 237 GVALVLSLL 17
ILLLVLIFL 12 242 LSLLFILLL 17
TableXXX-V1-HLA-B2705-9mers- 261 LILGVLGVL 17
TableXXIX-V6-61510-9mers- 24P4C12 287 GASISQLGF 17
24P4C12 Each peptide is a portion of SEQ ID 311
AALIVLAVL 17
Each peptide is a portion of SEQ ID NO: 3; each start position is
338 IALLKEASK 17
NO: 13; each start position is specified, the length of
peptide is 9 354 TMFYPLVTF 17
specified, the length of peptide is 9 amino acids, and the end
position 381 TSGQPQYVL 17
/ amino acids, and the end position for for each peptide is the
start position 429 YSSKGLIQR 17
each peptide is the start position plus plus eight. 477
HKPQDIPTF 17
eight. Pos 123456789 score 503
AFGALILTL 17
Pos 123456789 score 334 IRIAIALLK 26 516
RVILEYIDH 17
7 LIPRSVFNL 11 332 QRIRIAIAL 25 546 WCLEKFIKF 17
5 KGLIPRSVF 10 675 ERNNGSLDR 24 549 EKFIKFLNR 17
3 SSKGLIPRS 5 214 ARDISVKIF 23 605 LVVGGVGVL 17
6 GLIPRSVFN 5 534 ARCIMCCFK 21 621 RIPGLGKDF 17
620 GRIPGLGKD 21 11 EAYGKPVKY 16
TableXXIX-V7-61510-9mers- 5 QRDEDDEAY 20 23 FRGPIKNRS 16
24P4C12 204 GISGLIDSL 20 137 GEVFYTKNR 16
Each peptide is a portion of SEQ ID 446 GVLGLFWTL 20 139
VFYTKNRNF 16
NO: 15; each start position is specified, 689 KSLLKILGK 20 170
FLLPSAPAL 16
the length of peptide is 9 amino acids, 251 RLVAGPLVL 19 283
LRDKGASIS 16
and the end position for each peptide 424 CVFQGYSSK 19 285
DKGASISQL 16
is the start position plus eight. 436 QRSVFNLQI 19 321
AILLLMLIF 16
Pos 123456789 score 483 PTFPLISAF 19 322
ILLLMLIFL 16
8 AVGQMMSTM 9 583 RNIVRVVVL 19 323 LLLMLIFLR 16
4 WILVAVGQM 8 608 GGVGVLSFF 19 327 LIFLRQRIR 16
5 ILVAVGQMM 8 15 KPVKYDPSF 18 432 KGLIQRSVF 16
1 SWYWILVAV 3 22 SFRGPIKNR 18 440 FNLQIYGVL 16
2 VVYWILVAVG 3 179 GRCFPVVTNV 18 442 LQIYGVLGL 16
3 YWILVAVGQ 3 200 TIQQGISGL 18 443 QIYGVLGLF 16
6 LVAVGQMMS 3 207 GLIDSLNAR 18 457 VLALGQCVL 16
234 VALGVALVL 18 508 ILTLVQIAR 16
TableXXIX-V8-81510-9mers- 244 LLFILLLRL 18 517
VILEYIDHK 16
24P4C12 255 GPLVLVLIL 18 589 WLDKVTDL 16
Each peptide is a portion of SEQ ID 291 SQLGFTTNL 18 617
FFSGRIPGL 16
NO: 17; each start position is 317 AVLEAILLL 18 626
GKDFKSPHL 16
specified, the length of peptide 1s9 330 LRORIRIAI 18 699
NEAPPDNKK 16
amino acids, and the end position 333. RIRIAIALL 18 10
DEAYGKPVK 15
for each peptide is the start position 496 RYHTGSLAF 18 40
LFLLFILGY 15
plus eight. 527 RGVQNPVAR 18 60 DPRQVLYPR 15
Pos 123456789 score 647 GAYVIASGF 18 73 AYCGMGENK 15
16 GHVFQTSIL 21 668 LCFLEDLER 18 81
KDKPYLLY.F 15
11 PITPTGHVF 10 683 RPYYMSKSL 18 124 PWTVGKNEF 15
13 QPATLGYVL 13 690 SLLKILGKK 18 212 LNARDISVK 15
9 PLPTQPATL 12 49 IWGIVAWL 17 217 ISVKIFEDF 15
2 WAMTALYPL 10 78 GENKDKPYL 17 228 SVVYWILVAL 15
154 WNMTVITSL 17 236 LGVALVLSL 15
177
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TableXXX-V1-HLA-B2705-9mers- TableXXX-V1-HLA-B2705-9mers-
TableXXX-V1-HLA-B2705-9mers-
24P4C12 24P4C12 24P4C12
Each peptide is a portion of SEQ ID Each peptide is a portion of
SEQ ID Each peptide is a portion of SEQ ID
NO: 3; each start position is NO: 3; each start position is NO:
3; each start position is
specified, the length of peptide is 9 specified, the length of
peptide is 9 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 position for each peptide is the start
position for each peptide is the start position
plus eight. plus eight plus eight.
Pos 123456789 score Pos 123456789 score Pos 123456789
score
238 VALVLSLLF 15 464 VLAGAFASF 14 582 MRNIVRVVV 13
243 SLLFILLLR 15 485 FPLISAFIR 14 590 VLDKVTDLL 13
253 VAGPLVLVL 15 . 487 LISAFIRTL 14 592 DKVTDLLLF 13
258 VLVLILGVL 15 488 ISAFIRTLR 14 610 VGVLSFFFF 13
308 TWLAALIVL 15 489 SAFIRTLRY 14 637 YWLPIMTSI 13
316 LAVLEAILL 15 501 SLAFGALIL 14 648 AYVIASGFF 13
369 IAYWAMTAL 15 513 . QIARVILEY 14 653 SGFFSVFGM 13
461 GQCVLAGAF 15 515 ARVILEYID 14 666 LFLCFLEDL 13
470 ASFYWAFHK 15 552 IKFLNRNAY 14 681 LDRPYYMSK 13
518 ILEYIDHKL 15 556 NRNAYIMIA 14 682 DRPYYMSKS 13
542 KCCLWCLEK 15 558 NAYIMIAIY 14 685 YYMSKSLLK 13
543 CCLWCLEKF 15 560 YIMIAIYGK 14 686 YMSKSLLKI 13
547 CLEKFIKFL 15 575 KNAFMLLMR 14 29 NRSCTDVIC 12
567 GKNFCVSAK 15 585. IVRVVVLDK 14 32 CTDVICCVL 12
579 MLLMRNIVR 15 595 TDLLLFFGK 14 33 TDVICCVLF 12
586 VRVVVLDKV 15 613 LSFFFFSGR 14 35 VICCVLFLL 12
593 KVTDLLLFF 15 643 TSILGAYVI 14 57 LYGDPRQVL 12
596 DLLLFFGKL 15 659 FGMCVDTLF 14 58 YGDPRQVLY 12
607 VGGVGVLSF 15 660 GMCVDTLFL 14 79 ENKDKPYLL 12
609 GVGVLSFFF 15 679 GSLDRPYYM 14 80 NKDKPYLLY 12
622 IPGLGKDFK 15 700 EAPPDNKKR 14 93 SCILSSNII 12
651 IASGFFSVF 15 701 APPDNKKRK 14 100 IISVAENGL 12
684 PYYMSKSLL 15 702 PPDNKKRKK 14 121 PEOPWTVGK 12
698 KNEAPPDNK 15 7 DEDDEAYGK 13 132 FSQTVGEVF 12
34 DVICCVLFL 14 36 ICCVLFLLF 13 144 NRNFCLPGV 12
38 CVLFLLF IL 14 172 LPSAPALGR 13 151 GVPWNMTVI 12
61 PRQVLYPRN 14 241 VLSLLFILL 13 163 QQELCPSFL 12
75 CGMGENKDK 14 249 LLRLVAGPL 13 190 PALPGITND 12
83 KPYLLYFNI 14 250 LRLVAGPLV 13 193 PGITNDTTI 12
84 PYLLYFNIF 14 273 IYYCWEEYR 13 239 ALVLSLLFI 12
135 TVGEVFYTK 14 275 YCWEEYRVL 13 276 CWEEYRVLR 12
148 CLPGVPWNM 14 280 YRVLRDKGA 13 302 YQSVQETWL 12
158 VITSLQQEL 14 294 GFTTNLSAY 13 305 VQETWLAAL 12
162 LQQELCPSF 14 319 LEAILLLML 13 315 VLAVLEAIL 12
164 QELCPSFLL 14 347 AVGQMMSTM 13 320 EAILLLMLI 12
232 ILVALGVAL 14 348 VGQMMSTMF 13 328 IFLRQRIRI 12
240 LVLSLLF IL 14 349 GQMMSTMFY 13 343 EASKAVGQM 12
263 LGVLGVLAY 14 356 FYPLVTFVL 13 371 YWAMTALYL 12
267 GVLAYGIYY 14 357 YPLVTFVLL 13 386 QYVLWASNI 12
272 GIYYCWEEY 14 358 PLVTFVLLL 13 393 NISSPGCEK 12
278 EEYRVLRDK 14 363 VLLLICIAY 13 406 TSCNPTAHL 12
325 LMLIFLRQR 14 492 IRTLRYHTG 13 414 LVNSSCPGL 12
379 LATSGQPQY 14 495 LRYHTGSLA 13 421 GLMCVFQGY 12
418 SCPGLMCVF 14 506 ALILTLVQI 13 426 FQGYSSKGL
12
434 LIQRSVFNL 14 526 LRGVQNPVA 13 468 AFASFYWAF
12
437 RSVFNLQIY 14 545 LWCLEKFIK 13 490 AFIRTLRYH
12
450 LFWTLNVVVL 14 570 FCVSAKNAF 13 500 GSLAFGALI
12
452 VVTLNWVLAL 14 572 VSAKNAFML 13 510 TLVQIARVI
12
178
=
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TableXXX-V1-HLA-B2705-9mers- TableXXX-V6-HLA-B2705-9mers-
Each peptide is a portion of SEQ
24P4C12 24P4C12 ID NO: 19; each start
position is
Each peptide is a portion of SEQ ID Each peptide is a portion of SEQ
specified, the length of peptide is 9
NO: 3; each start position is ID NO: 13; each start position is
. amino acids, and the end position
specified, the length of peptide is 9 specified, the length of peptide
is 9 for each peptide is the start
amino acids, and the end position amino acids, and the end position
position plus eight.
for each peptide is the start position for each peptide is the
start Pos 123456789 score
plus eight. position plus eight 18 GYVLWASNI 15
Pos 123456789 score Pos 123456789 score 13
QPATLGYVL 13
519 LEYIDHKLR 12 9 PRSVFNLQI 19 2
WAMTALYPL 12
537 IMCCFKCCL 12 5 KGLIPRSVF 17 9
PLPTQPATL 12
540 CFKCCLWCL 12 2 YSSKGLIPR 16 11
PTQPATLGY 10
553 KFLNRNAYI 12 7 LIPRSVFNL 14 6 ALYPLPTQP
8
557 RNAYIMIAI 12 3 SSKGLIPRS 9 15
ATLGYVLWA 7
562 MIAIYGKNF 12
591 LDKVTDLLL 12 TableXXX-V7-
HLA-B2705- Tablek0a-V1-HLA-B2709-
597 LLLFFGKLL 12 9mers-24P4C12 9merse-24P4C12
614 SFFFFSGRI 12 Each
peptide is a portion of SEQ Each peptide is a portion of SEQ
619 SGRIPGLGK 12 ID NO: 15;
each start position is ID NO: 3; each start position is
628 DFKSPHLNY 12 specified,
the length of peptide is specified, the length of peptide is
631 SPHLNYYWL 12 9 amino
acids, and the end 9 amino acids, and the end
634 LNYYWLPIM 12 position
for each peptide is the position for each peptide is the
658 VFGMCVDTL 12 start position plus eight.
start position plus eight.
662 CVDTLFLCF 12 Pos 123456789 score Pos
123456789 score
663 VDTLFLCFL 12 8 AVGQMMSTM 13 332
QRIRIAIAL 23
673 DLERNNGSL 12 4 WILVAVGQM 12 179
GRCFPINTNV 22
687 MSKSLLKIL 12 5 ILVAVGQMM 11 250
LRLVAGPLV 21
3 YWILVAVGQ 6 214
ARDISVKIF 20
TableXXX-V3-HLA-B2705-9mers- 436
QRSVFNLQI 20
24P4C12 TableXXX-V8-HLA-B2705-9mers- 144 NRNFCLPGV
19
Each peptide is a portion of SEQ ID 24P4C12 330
LRQRIRIAI 19
NO: 7; each start position is Each peptide is a portion
of SEQ ID = 582 MRNIVRVVV 19
specified, the length of peptide is 9 NO: 17; each start
position is 586 VRVVVLDKV 19
amino acids, and the end position for specified, the length of peptide
is 9 255 GPLVLVLIL 17
each peptide is the start position plus amino acids, and the end position
583 RNIVRVVVL 17
eight. for each peptide is the start position 251
RLVAGPLVL 16
Pos 123456789 score plus eight. 683 RPYYMSKSL 16
1 GRCFPVVTNI 24 Pos 123456789 score 78
GENKDKPYL 15
6 WTNITPPAL 11 16 GHVFQTSIL 15 170
FLLPSAPAL 15
1 NYYWLPIMR 14 334 IRIAIALLK 15
TableXXX-V5-HLA-B2705-9mers- 8 MRNPITPTG
14 446 GVLGLFVVTL 15
24P4C12 9 RNPITPTGH 14 620 GRIPGLGKD 15
Each peptide is a portion of SEQ ID 11 PITPTGHVF 12 647
GAYVIASGF 15
NO: 11; each start position is 15 TGFIVFOTSI 11 660
GMCVDTLFL 15
specified, the length of peptide is 9 19 FQTSILGAY 10 49
IVVGIVAWL 14
amino acids, and the end position 2 YYWLPIMRN 8 228
SWYWILVAL 14
for each peptide is the start position 4 WLPIMRNPI 7 234
VALGVALVL 14
plus eight. 7 IMRNPITPT 7 244 LLFILLLRL 14
Pos 123456789 score 17 HVFQTSILG 7 317
AVLEAILLL 14
4 AILLLVLIF 17 333 RIRIAIALL
14
ILLLVLIFL 17 " 452
INTLNVVVLAL 14
6 LLLVLIFLR 16 TableXXX-V9-HLA-B2705-9mers-
602 GKLLVVGGV 14
2 LEAILLLVL 14 24P4C12 626
GKDFKSPHL 14
8 LVLIFLRQR 14 679
GSLDRPYYM 14
3 EAILLLVLI 12 23
FRGPIKNRS 13
9 VLIFLRQRI 11 34 DVICCVLFL
13
83 KPYLLYFNI 13
179
-
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TableXXXI-V1-HLA-B2709- TableXXXI-V1-HLA-B2709- TableXXXI-V1-HLA-B2709-
9merse-24P4C12 9 merse-24P4C12 9merse-24P4C12
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
9 amino acids, and the end 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 eight
Pos 123456789 score Pos 123456789 score Pos
123456789 score
107 GLQCPTPQV 13 518 ILEYIDHKL 12 509 LTLVQIARV 11
204 GISGLIDSL 13 553 KFLNRNAYI 12 510 TLVQIARVI 11
232 ILVALGVAL 13 593 KVTDLLLFF 12 511 LVQIARVIL 11
236 LGVALVLSL 13 596 DLLLFFGKL 12 526 LRGVQNPVA 11
237 GVALVLSLL 13 597 LLLFFGKLL 12 534 ARCIMCCFK 11
240 LVLSLLFIL 13 605 LWGGVGVL 12 537 IMCCFKCCL 11
242 LSLLFILLL 13 608 GGVGVLSFF 12 564 AIYGKNFCV 11
253 VAGPLVLVL 13 621 RIPGLGKDF 12 572 VSAKNAFML 11
291 SQLGFTTNL 13 637 YWLPIMTSI 12 591 LDKVTDLLL 11
311 AALIVLAVL 13 666 LFLCFLEDL 12 592 DKVTDLLLF 11
322 ILLLMLIFL 13 684 PYYMSKSLL 12 598 LLFFGKLLV 11
357 YPLVTFVLL 13 5 QRDEDDEAY 11 599 LFFGKLLW 11
358 PLVTFVLLL 13 28 KNRSCTDVI 11 609 GVGVLSFFF 11
369 IAYWAMTAL 13 29 NRSCTDVIC 11 614 SFFFFSGRI 11
440 FNLQIYGVL 13 32 CTDVICCVL 11 617 FFSGRIPGL 11
442 LQIYGVLGL 13 41 FLLFILGYI 11 631 SPHLNYYWL 11
449 GLFWTLNWV 13 42 LLFILGYIV 11 634 LNYYWLPIM 11
496 RYHTGSLAF 13 46 LGYIVVGIV 11 643 TSILGAYVI 11
500 GSLAFGALI 13 67 PRNSTGAYC 11 653 SGFFSVFGM 11
515 ARVILEYID 13 79 ENKDKPYLL 11 658 VFGMCVDTL 11
557 RNAYIMIAI 13 87 LYFNIFSCI 11 663 VDTLFLCFL 11
589 WLDKVTDL 13 100 IISVAENGL 11 675 ERNNGSLDR 11
15 KPVKYDPSF 12 128 GKNEFSQTV 11 687 MSKSLLKIL 11
38 CVLFLLFIL 12 139 VFYTKNRNF 11
. 45 ILGYIVVGI 12 151 GVPWNMTVI 11
TableXXXI-V3-HLA-B2709-
56 WLYGDPRQV 12 184 VVINVIPPAL 11 9mers-24P4C12
61 PRQVLYPRN 12 217 ISVKIFEDF 11
Each peptide is a portion of SEQ
81 KDKPYLLYF 12 225 FAQSWYWIL 11
ID NO: 7; each start position is
158 VITSLQQEL 12 230 YWILVALGV 11
specified, the length of peptide is
164 QELCPSFLL 12 238 VALVLSLLF 11
9 amino acids, and the end
258 VLVLILGVL 12 239 ALVLSLLFI 11
position for each peptide is the
261 LILGVLGVL 12 249 LLRLVAGPL 11 start
position plus eight
287 GASISQLGF 12 257 LVLVLILGV 11 Pos 123456789 score
308 TWLAALIVL 12 260 VLILGVLGV 11 1 GRCFPWTNI 22
316 ¨ LAVLEAILL 12 280 YRVLRDKGA 11 6 VVTNITPPAL
11
321 AILLLMLIF 12 283 LRDKGASIS 11 9 ITPPALPGI 11
328 IFLRQRIRI 12 285 DKGASISQL 11
355 MFYPLVTFV 12 297 TNLSAYQSV 11 TableXXXI-V5-62709-9mers-
371 YWAMTALYL 12 310 LAALIVLAV 11 24P4C12
414 LVNSSCPGL 12 314 IVLAVLEAI 11
Each peptide is a portion of
432 KGLIQRSVF 12 319 LEAILLLML 11
SEQ ID NO: 11; each start
434 LIQRSVFNL ' 12 351 MMSTMFYPL 11
position is specified, the length
461 GQCVLAGAF 12 354 TMFYPLVTF 11
of peptide is 9 amino adds,
492 IRTLRYHTG 12 381 TSGQPQYVL 11
and the end position for each
495 LRYHTGSLA 12 386 QYVLWASNI 11
peptide is the start position plus
501 SLAFGALIL 12 427 QGYSSKGLI 11 eight
= 503 AFGALILTL 12 480
QDIPTFPLI 11 Pos 123456789 score
506 ALILTLVQI 12 483 PTFPLISAF 11 4
AILLLVLIF 13
, 180
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ILLLVLIFL 13 Each peptide is a portion of TableXXXII-V1-HLA-B4402-
2 LEAILLLVL 11 SEQ ID NO: 19; each start 9mers-24P4C12
1 VLEAILLLV 10 position is specified, the Each
peptide is a portion of SEQ
3 EAILLLVLI 10 length of peptide is 9 amino ID NO: 3; each start
position is
9 VLIFLRQRI 10 acids, and the end position for specified,
the length of peptide is
each peptide is the start 9 amino acids, and the end
=
TableXXXI-V6-HLA-B2709- position plus eight position for each peptide
is the
9mers-24P4C12 Pos 123456789 score start position plus eight
Each peptide is a portion of SEQ 18 GYVLWASNI 14 Pos 123456789
score
ID NO: 13; each start position is 2 WAMTALYPL 11 629 FKSPHLNYY 16
specified, the length of peptide is 13 QPATLGYVL 11 699 NEAPPDNKK
16
9 amino acids, and the end 9 PLPTQPATL 10 34 DVICCVLFL
15
position for each peptide is the 12 TQPATLGYV 8 79 ENKDKPYLL 15
start position plus eight. 130 NEFSQTVGE 15
Pos 123456789 score TableXXXII-V1-HLA-B4402- 154 WNMTVITSL 15
9 PRSVFNLQI 20 9mers-24P4C12 204 GISGLIDSL 15
5 KGLIPRSVF 12 Each peptide is a portion of SEQ
234 VALGVALVL 15
7 LIPRSVFNL 12 ID NO: 3; each start position is 241
VLSLLFILL 15
4 SKGLIPRSV 9 specified, the length of peptide is
263 LGVLGVLAY 15
9 amino acids, and the end 278 EEYRVLRDK 15
TableXXXI-V7-HLA-B2709- position for each peptide is the 294
GFTTNLSAY 15
9mers-24P4C12 start position plus eight. 354
TMFYPLVTF 15
Each peptide is a portion of SEQ Pos 123456789 score 370
AYWAMTALY 15
ID NO: 15; each start position is 164 QELCPSFLL 22 399
CEKVPINTS 15
specified, the length of peptide is 319 LEAILLLML 22 442
LQIYGVLGL 15
9 amino acids, and the end 222 FEDFAQSWY 21 468
AFASFYWAF 15
position for each peptide is the 78 GENKDKPYL 20 477
HKPQDIPTF 15
start position plus eight. 306 QETWLAALI 20 499
TGSLAFGAL 15
Pos 123456789 score 483 PTFPLISAF 20 513 QIARVILEY 15
1 SVVYWILVAV 12 317 AVLEAILLL 19 547 CLEKFIKFL 15
4 WILVAVGQM 12 332 QRIRIAIAL 19 66 YPRNSTGAY 14
5 ILVAVGQMM 10 503 AFGALILTL 18 80 NKDKPYLLY 14
8 AVGQMMSTM 9 506 ALILTLVQI 18 84 PYLLYFNIF 14
552 IKFLNRNAY 18 93 SCILSSNII 14
TableXXXI-V8-HLA-B2709= 58 YGDPRQVLY 17 104 AENGLQCPT 14
9 mers-24P4C 12 170 FLLPSAPAL 17 193 PGITNDTTI 14
Each peptide is a portion of 214 ARDISVKIF 17 223
EDFAQSVVYW 14
SEQ ID NO: 17; each start 242 LSLLFILLL 17 239
ALVLSLLFI 14
position is specified, the length 583 RNIVRVVVL 17 244
LLFILLLRL 14
of peptide is 9 amino acids, 11 EAYGKPVKY 16 258
VLVLILGVL 14
and the end position for each 40 LFLLFILGY 16 261 LILGVLGVL
14
peptide is the Start position 48 YIVVGIVAW 16 285
DKGASISQL 14
plus eight. 81 KDKPYLLYF 16 291 SQLGFTTNL 14
Pos 123456789 score 121 PEDPWTVGK 16 301 AYQSVQETW 14
16 GHVFQTSIL 14 228 SVVYWILVAL 16 = 305 VQETWLAAL 14
8 MRNPITPTG 13 253 VAGPLVLVL 16 308 TWLAALIVL 14
11 PITPTGHVF 10 254 AGPLVLVLI 16 316 LAVLEAILL 14
NPITPTGHV 9 311 AALIVLAVL 16 322 ILLLMLIFL 14
4 WLPIMRNPI 8 320 EAILLLMLI 16 330 LRQRIRIAI 14
TGHVFQTSI 8 321 AILLLMLIF 16 333 RIRIAIALL 14
QTSILGAYV 8 363 VLLLICIAY 16 356 FYPLVTFVL 14
382 SGQPQYVLW 16 357 YPLVTFVLL 14
TableXXXI-V9-HLA-B2709- 452 WTLNWVLAL 16 358 PLVTFVLLL 14
9mers-24P4C12 480 QDIPTFPLI 16 364 LLLICIAYW 14
=
487 LISAFIRTL 16 418 SCPGLMCVF 14
489 SAFIRTLRY 16 432 KGLIQRSVF 14
617 FFSGRIPGL 16 446 GVLGLFINTL 14
181
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TableXXXII-V1-HLA-B4402- TableXXXII-V1-HLA-B4402- TableXXXII-V1-HLA-
B4402-
9mers-24P4C12 9mers-24P4C12 9mers-24P4C12
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
9 amino acids, and the end 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 eight
Pos 123456789 score Pos 123456789 score Pos 123456789 score
496 RYHTGSLAF 14 519 LEYIDHKLR 13 548 LEKFIKFLN 12
546 WCLEKFIKF 14 529 VQNPVARCI 13 553 KFLNRNAYI 12
558 NAYIMIAIY 14 543 CCLWCLEKF 13 557 RNAYIMIAI 12
573 SAKNAFMLL 14 570 FCVSAKNAF 13 562 MIAIYGKNF 12
577 AFMLLMRNI 14 589 VVLDKVTDL 13 572 VSAKNAFML 12
592 DKVTDLLLF 14 590 VLDKVTDLL 13 591 LDKVTDLLL 12
593 KVTDLLLFF 14 605 LVVGGVGVL 13 607 VGGVGVLSF 12
596 DLLLFFGKL 14 631 SPHLNYYWL 13 608 GGVGVLSFF 12
597 LLLFFGKLL 14 637 YWLPIMTSI 13 610 VGVLSFFFF 12
621 RIPGLGKDF 14 648 AYVIASGFF 13 630 KSPHLNYYW 12
641 IMTSILGAY 14 674 LERNNGSLD 13 638 WLPIMTSIL 12
643 TSILGAYVI 14 ' 687 MSKSLLKIL 13 647 GAYVIASGF 12
651 IASGFFSVF 14 33 TDVICCVLF 12 658 VFGMCVDTL 12
662 CVDTLFLCF 14 35 VICCVLFLL 12 659 FGMCVDTLF 12
671 LEDLERNNG 14 38 CVLFLLFIL 12 660 GMCVDTLFL 12
678 NGSLDRPYY 14 50 VVGIVAWLY 12 663 VDTLFLCFL 12
QRDEDDEAY 13 100 IISVAENGL 12 666 LFLCFLEDL 12
7 DEDDEAYGK 13 132 FSQTVGEVF 12 673 DLERNNGSL 12
32 CTDVICCVL 13 133 SQTVGEVFY 12 677 NNGSLDRPY 12
36 ICCVLFLLF 13 139 VFYTKNRNF 12 683 RPYYMSKSL 12
49 IVVGIVAWL 13 141 YTKNRNFCL 12 686 YMSKSLLKI 12
57 LYGDPRQVL 13 163 QQELCPSFL 12 10 DEAYGKPVK 11
77 MGENKDKPY 13 217 ISVKIFEDF 12 15 KPVKYDPSF 11
87 LYFNIFSCI 13 221 IFEDFAQSW 12 28 KNRSCTDVI 11
137 GEVFYTKNR 13 236 LGVALVLSL 12 37 CCVLFLLFI 11
146 NFCLPGVPW 13 240 LVLSLLFIL 12 41 FLLFILGYI 11
174 SAPALGRCF 13 249 LLRLVAGPL 12 45 ILGYIVVGI 11
176 PALGRCFPW 13 267 GVLAYGIYY 12 117 VSSCPEDPW 11
184 WINVIPPAL 13 269 LAYGIYYCW 12 124 PWTVGKNEF 11
187 VIPPALPGI 13 275 YCWEEYRVL 12 151 GVPWNMTVI 11
200 TIQQGISGL 13 287 GASISQLGF 12 197 NDTTIQQGI 11
209 IDSLNARDI 13 314 IVLAVLEAI 12 201 IQQGISGLI
11
' 213 NARDISVKI 13 326 MLIFLRQRI 12 266 LGVLAYGIY 11
232 ILVALGVAL 13 328 IFLRQRIRI 12 302 YQSVQETWL 11
237 GVALVLSLL 13 349 GQMMSTMFY 12 359 LVTFVLLLI 11
238 VALVLSLLF 13 369 IAYWAMTAL 12 361 TFVLLLICI 11
251 RLVAGPLVL 13 371 YWAMTALYL 12 379 LATSGQPQY 11
255 GPLVLVLIL 13 406 TSCNPTAHL 12 381 TSGQPQYVL 11
277 WEEYRVLRD 13 421 GLMCVFQGY 12 436 QRSVFNLQI 11
342 KEASKAVGQ 13 426 FQGYSSKGL 12 444 IYGVLGLFW 11
351 MMSTMFYPL 13 434 LIQRSVFNL 12 465 LAGAFASFY 11
440 FNLQIYGVL 13 437 RSVFNLQIY 12 474 WAFHKPQDI 11
443 QIYGVLGLF 13 450 LFVVTLNWVL 12 484 TFPLISAFI 11
' 448 LGLFWTLNW 13 457 VLALGQCVL 12 494
TLRYHTGSL 11
461 GQCVLAGAF 13 464 VLAGAFASF 12 533 VARCIMCCF 11
466 AGAFASFYW 13 479 PQDIPTFPL 12 538 MCCFKCCLW 11
501 SLAFGALIL 13 510 TLVQIARVI 12 540 CFKCCLWCL 11
518 ILEVIDHKL 13 511 LVQIARVIL 12 614 SFFFFSGRI 11
=
182
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TableXXXII-V1-HLA-B4402- 2 LEAILLLVL 23
Each peptide is a portion of SEQ ID
9mers-24P4C12 3 EAILLLVLI 17 NO: 19; each start position
is
Each peptide is a portion of SEQ 4 AILLLVLIF 17
specified, the length of peptide is 9
ID NO: 3; each start position is 5 ILLLVLIFL 14
amino acids, and the end position for
specified, the length of peptide is 9 VLIFLRQRI 12 each
peptide is the start position plus
9 amino acids, and the end eight.
position for each peptide is the TableMII-V6-HLA-B4402- Pos
123456789 score
start position plus eight. 9mers-24P4C12 11 PTQPATLGY 15
Pos 123456789 score Each peptide is a portion of SEQ 9
PLPTQPATL 14
626 GKDFKSPHL 11 ID NO: 13; each start position is 2
WAMTALYPL 13
628 DFKSPHLNY 11 specified, the length of peptide is 14
PATLGYVLW 13
684 PYYMSKSLL 11 9 amino acids, and the end 13 QPATLGYVL
12
19 YDPSFRGPI 10 position for each peptide is the 18
GYVLWASNI 10
83 KPYLLYFNI 10 start position plus eight. 6 ALYPLPTQP
8
88 YFNIFSCIL 10 Pos 123456789 score 15 ATLGYVLWA 7
158 VITSLQQEL 10 5 KGLIPRSVF 14
162 LQQELCPSF 10 7 LIPRSVFNL 13 TableXXXIII-V1-HLA-B5101-
225 FAQSWYWIL 10 9 PRSVFNLQI 11 9mers-24P4C12
272 GIYYCWEEY 10 6 GLIPRSVFN 8 . Each
peptide is a portion of SEQ
315 VLAVLEAIL 10 ID NO: 3; each start
position is
348 VGQMMSTMF 10 TableX)OCII-V7-HLA-B4402-
specified, the length of peptide is 9
386 QYVLWASNI 10 9mers-24P4C12 amino
acids, and the end position
396 SPGCEKVPI 10 Each peptide is a portion of SEQ for each peptide
is the start
414 LVNSSCPGL 10 ID NO: 15; each start position is position plus
eight
500 GSLAFGALI 10 specified, the length of peptide is Pos
123456789 score
514 IARVILEYI 10 9 amino acids, and the end 234 VALGVALVL
27
537 IMCCFKCCL 10 position for each peptide is the 213
NARDISVKI 25
544 CLWCLEKFI 10 start position plus eight. 46 LGYIVVGIV
24
555 LNRNAYIMI 10 Pos 123456789 score 83 KPYLLYFNI 24
609 GVGVLSFFF 10 1 SVVYWILVAV 6 311 AALIVLAVL 24
3 YWILVAVGQ 6 253 VAGPLVLVL 23
TableXXXII-V3-HLA-B4402- 8 AVGQMMSTM 4 310 LAALIVLAV 23
9mers-24P4C12 4 WILVAVGQM 3 357 YPLVTFVLL 23
Each peptide is a portion of 2 VVYWILVAVG 2 369 IAYWAMTAL
23
SEQ ID NO: 7; each start 474 WAFHKPQDI 23
position is specified, the length TableXXXII-V8-HLA-B4402-
514 IARVILEYI 23
of peptide is 9 amino acids, and 9mers-24P4C12 683 RPYYMSKSL 22
the end position for each Each peptide is a portion of SEQ 254
AGPLVLVLI 21
peptide is the start position plus ID NO: 17; each start
position is 255 GPLVLVLIL 21
eight. specified, the length of peptide is 9 320 EAILLLMLI 21
Pos 123456789 score amino acids, and the end position 396
SPGCEKVPI 21
6 VVTNITPPAL 13 for each peptide is the start 427
QGYSSKGLI 21
9 ITPPALPGI 13 position plus eight. 11 EAYGKPVKY
20
1 GRCFPWTNI 8 Pos 123456789 score 193
PGITNDTTI 20
2 RCFPWTNIT 7 11 PITPTGHVF 15 316 LAVLEAILL 20
7 TNITPPALP 6 19 FQTSILGAY 14 123 DPVVTVGKNE 19
8 NITPPALPG 6 4 WLPIMRNPI 11 236 LGVALVLSL 18
16 GHVFQTSIL 11 314 IVLAVLEAI 18
TableXXXII-V5-HLA-B4402- 15 TGHVFQTSI 8 599 LFFGKLLVV 18
9mers-24P4C12 686 YMSKSLLKI 18
Each peptide is a portion of SEQ TableXXXII-V9-HLA-B4402-9mers- 60
DPRQVLYPR 17
ID NO: 11; each start position is 24P4C12 150 PGVPWNMTV
17
specified, the length of peptide is 225 FAQSWYWIL 17
9 amino acids, and the end 261 LILGVLGVL 17
position for each peptide is the 269 LAYGIYYCW 17
start position plus eight. 300 SAYQSVQET 17
Pos 123456789 score 504 FGALILTLV 17
183
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TableXXXIII-V1-HLA-B5101- TableXXXIII-V1-HLA-B5101- Pos 123456789
score
9mers-24P4C12 9mers-24P4C12 4 FPWTNITPP 15
558 NAYIMIAIY 17 499 TGSLAFGAL 14 9 ITPPALPGI 14
573 SAKNAFMLL 17 509 LTLVQIARV 14 1 GRCFPVVTNI 11
651 IASGFFSVF 17 576 NAFMLLMRN 14 6 VVTNITPPAL 8
182 FPWINVIPP 16 586 VRVVVLDKV 14
192 LPGITNOTT 16 589 WLDKVTDL 14 TableXXXIII-V5-HLA-85101-9mers-
328 IFLRQRIRI 16 602 GKLLWGGV 14 24P4C12
355 MFYPLVTFV 16 605 LVVGGVGVL 14 Each
peptide is a portion of SEQ ID
359 LVTFVLLLI 16 639 LPIMTSILG 14 NO: 11; each start
position is
458 LALGQCVLA 16 701 APPDNKKRK 14
specified, the length of peptide is 9
502 LAFGALILT 16 702 PPDNKKRKK 14 amino acids, and the end
position for
505 GALILTLVQ 16 19 YDPSFRGPI 13 each peptide is the start
position plus
510 TLVQIARVI 16 28 KNRSCTDVI 13 eight.
581 LMRNIVRVV 16 34 DVICCVLFL 13 Pos 123456789 score
631 SPHLNYYWL 16 54 VAWLYGDPR 13 3 EAILLLVLI 22
9 DDEAYGKPV 15 66 YPRNSTGAY 13 5 ILLLVLIFL 14
45 ILGYIVVGI 15 112 TPQVCVSSC 13 2 LEAILLLVL 13
56 WLYGDPRQV 15 149 LPGVPWNMT 13 1 VLEAILLLV 12
110 CPTPQVCVS 15 174 SAPALGRCF 13 9 VLIFLRQRI 12
120 CPEDPWTVG 15 176 PALGRCFPW 13
151 GVPWNMTVI 15 187 VIPPALPGI 13 TableW111-V6-HLA-B5101-
172 LPSAPALGR 15 189 PPALPGITN 13 9mers-24P4C12
224 DFAQSINYWI 15 201 IQQGISGLI 13
Each peptide is a portion of SEQ
275 YCWEEYRVL 15 239 ALVLSLLFI 13 ID NO: 13; each start
position is
308 TWLAALIVL 15 252 LVAGPLVLV 13
specified, the length of peptide is 9
336 IAIALLKEA 15 282 VLRDKGASI 13 amino
acids, and the end position
338 IALLKEASK 15 285 DKGASISQL 13 for each peptide is the
start
375 TALYLATSG 15 293 LGFTTNLSA 13 position plus eight.
485 FPLISAFIR 15 322 ILLLMLIFL 13 Pos 123456789 score
529 VQNPVARCI 15 330 LRQRIRIAI 13 8 IPRSVFNLQ 16
564 AIYGKNFCV 15 340 LLKEASKAV 13 7 LIPRSVFNL 12
582 MRNIVRVVV 15 343 EASKAVGQM 13 9 PRSVFNLQI 12
596 DLLLFFGKL 15 356 FYPLVTFVL 13 5 KGLIPRSVF 11
637 YWLPIMTSI 15 361 TFVLLLICI 13 4 SKGLIPRSV 10
643 TSILGAYVI 15 384 QPQYVLWAS 13
647 GAYVIASGF 15 478 KPQDIPTFP 13 TableXXXIII-VT-HLA-B5101-
700 EAPPDNKKR 15 487 LISAFIRTL 13 9mers-24P4C12
20 DPSFRGPIK 14 489 SAFIRTLRY 13 Each
peptide is a portion of SEQ
41 FLLFILGYI 14 500 GSLAFGALI 13 ID NO: 15; each start
position is .
43 LFILGYIW 14 506 ALILTLVQI 13
specified, the length of peptide is 9
72 GAYCGMGEN 14 521 YIDHKLRGV 13 amino
acids, and the end position
87 LYFNIFSCI 14 531 NPVARCIMC 13 for each peptide is the
start
119 SCPEDPWTV 14 553 KFLNRNAYI 13 position plus eight.
152 VPWNMTVIT 14 555 LNRNAYIMI 13 Pos
123456789 score
188 TPPALPGIT 14 563 IAIYGKNFC 13 1 SWYWILVAV 14
190 PALPGITND 14 578 FMLLMRNIV 13 7 VAVGQMMST 12
209 IDSLNARDI 14 580 LLMRNIVRV 13 2 WYWILVAVG 6
230 YWILVALGV 14 3 YWILVAVGQ 6
238 VALVLSLLF 14 TableXXXIII-V3-HLA-B5101-
257 LVLVLILGV 14 9mers-24P4C12 TableXXXIII-V8-HLA-B5101-
9rners-
409 NPTAHLVNS 14 Each peptide is a portion of SEQ
24P4C12 =
411 TAHLVNSSC 14 ID NO: 7; each start position is
450 LFWTLNVVVL 14 specified, the length of peptide is
9
465 LAGAFASFY 14 amino acids, and the end position
467 GAFASFYWA 14 for each peptide is the start
482 IPTFPLISA 14 position plus eight.
184
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PCT/US2002/038264
Each peptide is a portion of SEQ ID TableXXXIV41-HLA-A1-10mers-
TableXXXIV-V3-HLA-A1-10mers-
NO: 17; each start position is 24P4C12 24P4C12
specified, the length of peptide is 9 Each peptide is a portion of
SEQ Each peptide is a portion of SEQ ID
amino acids, and the end position for ID NO: 3; each start position is =
NO: 7; each start position is
- each peptide is the start position plus
specified, the length of peptide is specified, the length of peptide is
eight. 10 amino acids, and the end 10 amino
acids, and the end
Pos 123456789 score
position for each peptide is the position for each peptide is the start
NPITPTGHV 21 start position plus nine, position
plus nine.
. 15 TGHVFQTSI 18 Pos 1234567890 score
Pos 1234567890 score
13 TPTGHVFQT 14 49 IVVGIVAWLY 18 10 ITPPALPGIT 10
4 WLPIMRNPI 13 378 YLATSGQPQY 18 3
RCFPVVTNITP 9
5 LPIMRNPIT 13 420 PGLMCVFQGY 18 7
VVTNITPPALP 8
464 VLAGAFASFY 18 8 TNITPPALPG 6
TableXXXIII-V9-HLA-135101- 10 DEAYGKPVKY 17 9
NITPPALPGI 4
9mers-24P4C12 57 LYGDPRQVLY 17
Each peptide is a portion of SEQ 121 PEDPVVTVGKN 17 TableXXXIV-
V5-HLA-A1-
ID NO: 19; each start position is 265 VLGVLAYGIY 17 10mers-
24P4C12
specified, the length of peptide is 9 271 YGIYYCWEEY 17 Each
peptide is a portion of
amino acids, and the end position 276 CWEEYRVLRD 17 SEQ ID NO: 11; each
start
for each peptide is the start 369 IAYWAMTALY 17 position is
specified, the length
position plus eight. 551 F1KFLNRNAY 17 of peptide is 10 amino
acids,
Pos 123456789 score 80 NKDKPYLLYF 16 and the end
position for each
13 QPATLGYVL 20 348 VGQMMSTMFY 16
peptide is the start position plus
2 WAMTALYPL 18 676 RNNGSLDRPY 16 nine.
5 TALYPLPTQ 16 677 NNGSLDRPYY 16 Pos
1234567890 score
8 YPLPTQPAT 15 4 KQRDEDDEAY 15 2
VLEAILLLVL 19
10 LPTQPATLG 14 18 KYDPSFRGPI 15 7
LLLVLIFLRQ 10
12 TQPATLGYV 13 65 LYPRNSTGAY 15 1
AVLEAILLLV 9
17 LGYVLWASN 12 76 GMGENKDKPY 15
9 PLPTQPATL 11 214 ARDISVKIFE 15
TablOOOCIV-V6-HLA-A1-10mers-
14 PATLGYVLW 11 293 LGFTTNLSAY 15 24P4C12
18 GYVLWASNI 11 436 QRSVFNLQIY 15 Each
peptide is a portion of SEQ
479 PQDIPTFPLI 15 ID NO: 13; each start position is
TableXXXIV-V1-HLA-A1-10mers- 557 RNAYIMIAIY 15 specified, the
length of peptide is
24P4C12 628 DFKSPHLNYY 15 10 amino
acids, and the end
Each peptide is a portion of SEQ 640 PIMTSIL¨GAY 15
position for each peptide is the
ID NO: 3; each start position is 664 DTLFLCFLED 15 start
position plus nine.
specified, the length of peptide is 283 LRDKGASISQ 14 Pos
1234567890 score
10 amino acids, and the end 521 YIDHKLRGVQ 14 10
PRSVFNLQIY 15
position for each peptide is the 673 DLERNNGSLD 14 1
QGYSSKGLIP 7
start position plus nine. 141 YTKNRNFCLP 13 4
SSKGLIPRSV 7
Pos 1234567890 score 305 VQETWLAALI 13 9
IPRSVFNLQI 7
221 IFEDFAQSWY 25 382 SGQPQYVLWA 13
488 ISAFIRTLRY 25 407 SCNPTAHLVN 13
TableXXXIV-W-HLA-A1-10mers-
39 VLFLLF1LGY 23 518 ILEYIDHKLR 13 24P4C12
58 YGDPRQVLYP 23 547 CLEKFIKFLN 13 Each
peptide is a portion of SEC),
79 ENKDKPYLLY 23 670 FLEDLERNNG 13 ID
NO: 15; each start position is
262 ILGVLGVLAY 23 680 SLDRPYYMSK 13
specified, the length of peptide is
512 VQIARV1LEY 22 7 DEDDEAYGKP 12 10
amino acids, and the end
627 KDFKSPHLNY 21 35 VICCVLFLLF 12
position for each peptide is the
132 FSQTVGEVFY 20 159 ITSLQQELCP 12 start
position plus nine.
266 LGVLAYGIYY 20 163 QQELCPSFLL 12
Pos 1234567890 score
362 FVLLLICIAY 20 242 LSLLFILLLR 12 1
Q5VVYWILVAV 4
590 VLDKVTDLLL 20 618 FSGRIPGLGK 12 2
SVVYWILVAVG 4
594 VTDLLLFFGK 20 626 GKDFKSPHLN 12 4
YWILVAVGQM 3
318 VLEAILLLML 19 698 KNEAPPDNKK 12 5
WILVAVGQMM 2
32 CTDVICCVLF 18 6 ILVAVGQMMS 2
185
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8 VAVGQMMSTM 2 Tab leX)0(V-V1-H LA-A0201- TableXXXV-V1-HLA-
A0201-
9 AVGQMMSTMF 2 l0mers-24P4C12 10mers-24P4C12
Each peptide is a portion of SEQ Each peptide is a portion of SEQ
TableXXXIV-V8-HLA-A1- ID NO: 3; each start position is ID NO: 3;
each start position is
lOmers-24P4C12 , specified, the length of
peptide is specified, the length of peptide is
Each peptide is a portion of SEQ 10 amino acids, and the end 10 amino
acids, and the end
ID NO: 17; each start position is position for each peptide is the
position for each peptide is the
specified, the length of peptide is start position plus nine, start
position plus nine.
10 amino acids, and the end Pos 1234567890 score Pos
1234567890 score
Pos 1234567890 score 517 VILEYIDHKL 26 238 VALVLSLLFI 20
19 VFQTSILGAY 16 603 KLLVVGGVGV 26 261 LILGVLGVLA 20
4 YWLPIMRNPI 7 604 LLVVGGVGVL 26 314 IVLAVLEAIL 20
13 ITPTGHVFQT 7 45 ILGYIVVGIV 25 325 LMLIFLRQRI 20
21 QTSILGAYVI 7 252 LVAGPLVLVL 25 329 FLRQRIRIAI 20
304 SVQETWLAAL 25 350 QMMSTMFYPL 20
TableXXXIV-V9-HLA-A1-10mers- 312 ALIVLAVLEA 25 358 PLVTFVLLLI 20
24P4C12 318 VLEAILLLML 25 368 CIAYWAMTAL 20
Each peptide is a portion of SEQ ID 486 PLISAFIRTL 25 . 393
NISSPGCEKV 20
nine. 310 LAALIVLAVL 24 34 DVICCVLFLL 19
Pos 1234567890 score 339 ALLKEASKAV 24 64 VLYPRNSTGA 19
11 LPTQPATLGY 21 597 LLLFFGKLLV 24 85 YLLYFNIFSC 19
12 PTQPATLGYV 10 41 FLLFILGYIV 23 186 NVIPPALPGI 19
42 LLFILGYIVV 23 233 LVALGVALVL 19
' 56 WLYGDPRQVL 23 264 GVLGVLAYGI 19
TableXXXV-V1-HLA-A0201- 231 WILVALGVAL 23 317 AVLEAILLLM 19
10mers-24P4C12 249 LLRLVAGPLV 23 327 LIFLRQRIRI 19
Each peptide is a portion of SEQ 256 PLVLVLILGV 23 335
RIAIALLKEA 19
, ID NO: 3; each start position is 313
LIVLAVLEAI 23 351 MMSTMFYPLV 19
specified, the length of peptide is 315 VLAVLEAILL 23 357
YPLVTFVLLL 19
10 amino acids, and the end 438 SVFNLglycv 23 363 VLLLICIAYW
19
Pos 1234567890 score 99 NIISVAENGL 22 380 ATSGQPQYVL 19
235 ALGVALVLSL 29 257 LVLVL1LGVL 22 457 VLALGQCVLA 19
44 FILGYIVVGI 28 354 TMFYPLVTFV 22 536 CIMCCFKCCL 19
232 ILVALGVALV 28 413 HLVNSSCPGL 22 588 VVVLDKVTDL 19
243 SLLFILLLRL 28 449 GLF1NTLNVVVL 22 633 HLNYYWLPIM 19
309 WLAALIVLAV 28 506 ALILTLVQIA 22 644 SILGAYVIAS 19
579 MLLMRNIVRV 28 510 TLVQIARVIL 22 39 VLFLLFILGY 18
244 LLFILLLRLV 27 513 QIARVILEYI 22 157 TVITSLQQEL 18
260 VLILGVLGVL 27 581 LMRNIVRVVV 22 203 QGISGLIDSL 18
433 GLIQRSVFNL 27 585 IVRVVVLDKV 22 208 LIDSLNARDI 18
508 ILTLVQIARV 27 590 VLDKVTDLLL 22 240 LVLSLLFILL 18
580 LLMRNIVRVV 27 199 TTIQQgSGL 21 246 FILLLRLVAG 18
598 LLFFGKLLW 27 247 ILLLRLVAGP 21 262 ILGVLGVLAY 18
48 YIVVGIVAVVL 26 253 VAGPLVLVLI 21 281 RVLRDKGASI 18
94 CILSSNIISV 26 316 LAVLEAILLL 21 322 ILLLMLIFLR 18
239 ALVLSLLFIL 26 501 SLAFGALILT 21 332 CRIRIAIALL 18
241 VLSLLFILLL 26 505 GALILTLVQI 21 360 VTFVLLLICI 18
251 RLVAGPLVLV 26 641 IMTSILGAYV 21 .
388 VLWASNISSP 18
321 AILLLMLIFL 26 86 LLYFNIFSCI 20 448 LGLFWTLNVVV 18
186
=
. .
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PCT/US2002/038264
TableXXXV-V1-HLA-A0201- TableXXXV-V1-HLA-A0201- Pos
1234567890 score
lOmers-24P4C12 lOmers-24P4C12 9 N ITPPALPG I 23
Each peptide is a portion of SEQ Each peptide is a portion of SEQ
10 ITPPALPG IT 12
specified, the length of peptide is specified, the length of peptide is
TableXXXV-V5-HLA-A0201-
amino acids, and the end 10 amino acids, and the end
lOmers-24P4C12
position for each peptide is the position
for each peptide is the Each peptide is a portion of SEQ
start position plus nine, start position plus nine. ID NO: 11;
each start position is
Pos 1234567890 score Pos 1234567890 score
specified, the length of peptide is
493 RTLRYHTGSL 18 323 LLLMLIFLRQ 15 10
amino acids, and the end
525 KLRGVQNPVA 18 340 LLKEASKAVG 15
position for each peptide is the
589 VVLDKVTDLL 18 378 YLATSGQPQY 15 start
position plus nine.
616 FFFSGRIPGL 18 379 LATSGQPQYV 15 Pos
1234567890 score
662 CVDTLFLCFL 18 430 SSKGLIQRSV 15 5
AILLLVLIFL 26
685 YYMSKSLLKI 18 464 VLAGAFASFY 15 1
AVLEAILLLV 25
130 NEFSQTVGEV 17 498 HTGSLAFGAL 15 2
VLEAILLLVL 25
143 KNRNFCLPGV 17 520 EYIDHKLRGV 15 3
LEAILLLVLI 18
148 CLPGVPWNMT 17 539 CCFKCCLWCL 15 6
ILLLVLIFLR 18
170 FLLPSAPALG 17 601 FGKLLWGGV 15 8
LLVLIFLRQR 16
211 SLNARDISVK 17 690 SLLKILGKKN 15 9
LVLIFLRQRI 16
227 QSVVYWILVAL 17 26 PI KN RSCTDV 14 7
LLLVLIFLRQ 15
254 AGPLVLVLIL 17 30 RSCTDVICCV 14 10
VLIFLRQRIR 12
296 TTNLSAYQSV 17 37 CCVLFLLFIL 14
324 LLMLIFLRQR 17 102 SVAENGLQCP 14 Table)000.1-
V6-HLA-A0201-
373 AMTALYLATS 17 149 LPGVPWNMTV 14 lOmers-24P4C12
481 DIPTFPLISA 17 153 PWNMTVITSL 14 Each
peptide is a portion of SEQ
546 WCLEKFIKFL 17 162 LQQELCPSFL 14 ID
NO: 13; each start position is
563 IAIYGKNFCV 17 165 ELCPSFLLPS 14
specified, the length of peptide is
582 MRNIVRVVVL 17 171 LLPSAPALGR 14 10
amino acids, and the end
40 LF LLF I LGYI 16 177 ALGRCFPWTN 14
position for each peptide is the
108 LQCPTPQVCV 16 220 KIFEDFAQSW 14 start
position plus nine.
118 SSCPEDPVVTV 16 273 IYYCWEEYRV 14 Pos
1234567890 score
169 SFLLPSAPAL 16 338 IALLKEASKA 14 7 GLIPRSVFNL 29
200 TIQQGISGLI 16 353 STMFYPLVTF 14 4 SSKGLIPRSV 15
207 GLIDSLNARD 16 370 AYWAMTALYL 14
212 LNARDISVKI 16 395 SSPGCEKVPI 14 TableXXXV-
V7-HLA-A0201-
236 LGVALVLSLL 16 416 NSSCPGLMCV 14 lOmers-24P4C12
292 QLGFTTNLSA 16 445 YGVLGLFINTL 14 Each
peptide is a portion of SEQ
307 ETWLAALIVL 16 483 PTFPLISAFI 14 ID
NO: 15; each start position is
319 LEAILLLMLI 16 500 GSLAFGALIL 14
specified, the length of peptide is
.337 AIALLKEASK 16 571 CVSAKNAFML 14 10
amino acids, and the end
366 LI C IAYWAMT 16 577 AFMLLMRNIV 14
position for each peptide is the
405 NTSCNPTAHL 16 595 TDLLLFFGKL 14 start position
plus nine.
451 FWTLNVVVLAL 16 606 VVGGVGVLSF 14 Pos
1234567890 score
456 WVLALGQCVL 16 639 LPIMTSILGA 14 1
QSWYWILVAV 4
458 LALGQCVLAG 16 680 SLDRPYYMSK 14 2
SVVYWILVAVG 4
503 AFGAULTLV 16 693 KILGKKNEAP 14 4
YW1LVAVGQM 3
509 LTLVQIARVI 16 694 ILGKKNEAPP 14 5
W1LVAVGQMM 2
637 YWLP I MTSI L 16 6 ILVAVGQMMS
2
33 TDVICCVLFL 15 TableXXXV-V3-HLA-A0201- 8
VAVGQMMSTM 2
36 I CCVLFLLF I 15 lOmers-24P4C12 9
AVGQMMSTMF 2
90 NIFSCILSSN 15 Each peptide is a portion of SEQ
161 SLQQELCPSF 15 ID NO: 7;
each start position is TableXXXV-V8-HLA-A0201-
225 FAQSVVYWILV 15 specified, the length of
peptide is lOmers-24P4C12
234 VALGVALVLS 15 10 amino acids, and the end
250 LRLVAGPLVL 15 position for each peptide is the
284 RDKGASISQL 15 start position plus nine.
187
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WO 2004/050828 PCT/US2002/038264
Each peptide is a portion of SEQ TableXXXVI-V1-HLA-A0203- TableXXXVI-V1-
HLA-A0203-
ID NO: 17; each start position is 10mers-24P4C12 1Omers-24P4C12
specified, the length of peptide Each peptide is a portion of SEQ ID
Each peptide is a portion of SEQ ID
is 10 amino acids, and the end NO: 3; each start position is
NO: 3; each start position is
position for each peptide is the specified, the length of peptide is 10
specified, the length of peptide is 10
start position plus nine, amino acids, and the end
position amino acids, and the end position
Pos 1234567890 score for each peptide is the
start position for each peptide is the start position
4 YWLPIMRNPI 15 plus nine, plus nine.
5 WLPIMRNPIT 15 Pos 1234567890 score Pos 1234567890 score
18 HVFQTSILGA 15 46 LGYIVVGIVA 10 218 SVKIFEDFAQ 9
7 PIMRNPITPT 14 64 VLYPRNSTGA 10 227 QSVVYWILVAL 9
13 ITPTGHVFQT 14 95 ILSSNIISVA 10 231 WILVALGVAL 9
8 IMRNPITPTG 13 166 LCPSFLLPSA 10 246 FILLLRLVAG 9
21 QTSILGAYVI 13 182 FPWTNVTPPA 10 262 ILGVLGVLAY 9
20 FQTSILGAYV 12 205 ISGLIDSLNA 10 280 YRVLRDKGAS 9
15 PTGHVFQTSI 11 217 ISVKIFEDFA 10 293 LGFTTNLSAY 9
10 RNPITPTGHV 10 226 AQSVVYWILVA 10 309 WLAALIVLAV 9
16 TGHVFQTSIL 10 230 YWILVALGVA 10 313 LIVLAVLEAI 9
12 PITPTGHVFQ 8 245 - LFILLLRLVA 10 329
FLRQRIRIAI 9
261 LILGVLGVLA 10 331
RgRIRIAIAL 9
TableXXXV-V9-HLA-A0201- 279 EYRVLRDKGA 10 336 IAIALLKEAS 9
10mers-24P4C12 292 QLGFTTNLSA 10 339 ALLKEASKAV 9
Each peptide is a portion of SEQ 302 YgSVQETWLA 10 362
FVLLLICIAY 9
ID NO: 19; each start position is 308 TWLAALIVLA 10 365
LLICIAYWAM 9
specified, the length of peptide is 312 ALIVLAVLEA 10 368
CIAYWAMTAL 9
10 amino acids, and the end 328 IFLRGIRIRIA 10 372 WAMTALYLAT
9
position for each peptide is the 335 RIAIALLKEA 10 383
GQPQYVLWAS 9
start position plus nine. 338 IALLKEASM 10 404
INTSCNPTAH 9
Pos 1234567890 score 361 TFVLLLICIA 10 451 FINTLNWVLAL 9
9 YPLPT2PATL 20 364 LLLICIAYWA 10 458 LALGQCVLAG 9
2 YWAMTALYPL 19 367 ICIAYWAMTA 10 460 LGQCVLAGAF 9
7 ALYPLPTQPA 19 371 YWAMTALYLA 10 462 QCVLAGAFAS 9
12 PTQPATLGYV 17 382 SGQPQYVLWA 10 467 GAFASFYWAF 9
16 ATLGYVLWAS 15 403 PINTSCNPTA 10 482 IPTFPLISAF 9
4 AMTALYPLPT 14 450 LFVVTLNWVLA 10 495 LRYHTGSLAF 9
5 MTALYPLPTQ 13 457 VIALGQCVLA 10 498 HTGSLAFGAL 9
17 TLGYVLWASN 13 466 AGAFASFYWA 10 507 LILTLVglAR 9
13 TQPATLGYVL 11 481 DIPTFPLISA 10 526 LRGVQNPVAR 9
18 LGYVLWASNI 11 494 TLRYHTGSLA 10 551 FIKFLNRNAY 9
15 PATLGYVLWA 9 497 YHTGSLAFGA 10 556 NRNAYIMIAI 9
506 ALILTLVQIA 10 566 YGKNFCVSAK 9
,
TableXXXVI-V1-HLA-A0203- 525 KLRGVQNPVA 10 569 NFCVSAKNAF 9
10mers-24P4C12 550 KFIKFLNRNA 10 640 PIMTSILGAY 9
Each peptide is a portion of SEQ ID 555 LNRNAYIMIA 10 644
SILGAYVIAS 9
NO: 3; each start position is 565 IYGKNFCVSA 10 693 KILGKKNEAP
9
specified, the length of peptide is 10 568 KNFCVSAKNA 10
amino acids, and the end position 639 LPIMTSILGA 10
for each peptide is the start position 643 TSILGAYVIA 10
TableXXXVI-V3-HLA-A0203-10mers-
plus nine. 692 LKILGKKNEA 10 24P4C12
Pos 1234567890 score 4 KgRDEDDEAY 9 Each peptide is a
portion of SEQ ID
303 QSVQETWLAA 19 47 GYIVVGIVAW 9 NO: 7; each
start position is specified,
168 PSFLLPSAPA 18 65 LYPRNSTGAY 9 the length
of peptide is 10 amino
330 LRQRIRIAIA 18 96 LSSNIISVAE 9 acids, and the
end position for each
459 ALGQCVLAGA 18 167 CPSFLLPSAP 9 peptide is
the start position plus nine.
461 GgCVLAGAFA 18 169 SFLLPSAPAL 9 Pos 1234567890 score
304 SVQETWLAAL 17 183 PWTNVTPPAL 9 5 FPWTNITPPA 10
3 GKQRDEDDEA 10 206 SGLIDSLNAR 9 6 PVVTNITPPAL 9
188
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TableXXXVI-V3-HLA-A0203-10mers- 16 ATLGYVLWAS 9 .
TableXXXVII-V1 -HLA-A3-10mers-
24P4C12 ' 9 YPLPTQPATL 8 24P4C12
Each peptide is a portion of SEQ ID 17 TLGYVLWASN 8
Each peptide is a portion of SEQ ID
NO: 7; each start position is specified, NO: 3; each start position is
specified,
the length of peptide is 10 amino TableXXXVII-V1-HLA-A3-10mers-
the length of peptide is 10 amino
acids, and the end position for each 24P4C12
acids, and the end position for each
peptide is the start position plus nine. Each peptide is a portion of
SEQ ID peptide is the start position plus nine.
acids, and the end position for each 347 AVGQMMSTMF 19
TableXXXVI-V5-HLA-A0203- peptide is the start position plus nine. 494
TLRYHTGSLA 19
lOmers-24P4C12 Pos
1234567890 score 605 LVVGGVGVLS 19
Pos 1234567890 score 333 RIRIAIALLK 32 618
FSGRIPGLGK 19
TableXXXVI-V7-HLA-A0203-10mers- 49 IVVGIVAWLY 23 258
VLVLILGVLG 18
24P4C12 463
CVLAGAFASF 23 324 LLMLIFLRQR 18
NO: 15; each start position is specified, 262 ILGVLGVLAY 22 532
PVARCIMCCF 18
the length of peptide is 10 amino acids, 376 ALYLATSGQP 22 72
GAYCGMGENK 17
and the end position for each peptide is 443 QIYGVLGLFW 22 86
LLYFNIFSCI 17
Pos 1234567890 score 587 RVVVLDKVTD 22 207
GL1DSLNARD 17
1 QSVVYWILVAV 9 603 KLLVVGGVGV 22 220 KIFEDFAQSW 17
2 SVVYWILVAVG 8 56 WLYGDPRQVL 21 232 ILVALGVALV 17
63 QVLYPRNSTG 21 249 LLRLVAGPLV 17
TableXXXVI-V8-HLA-A0203-10mers- 177 ALGRCFPVVTN 21 257 LVLVLILGVL 17
specified, the length of peptide is 10 53 IVAWLYGDPR 20 309
WLAALIVLAV 17
amino acids, and the end position for 171 LLPSAPALGR 20 326
MLIFLEQRIR 17
each peptide is the start position plus 251 RLVAGPLVLV 20 364
LLLICIAYWA 17
Pos 1234567890 score 282 VLRDKGASIS 20 392
SN1SSPGCEK 17
18 HVFQTSILGA 10 362 FVLLLICIAY 20 486
PLISAFIRTL 17
19 VFQTSILGAY 9 ' 378 YLATSPQY 20 506
ALILTLVQIA 17
20 FgrsILGAYV 8 544 CLWCLEKFIK 20 551
FIKFLNRNAY 17
TableXXXVI-V9-HLA-A0203-10mers- 95 ILSSNIISVA 19 598
LLFFGKLLVV 17
Each peptide is a portion of SEQ ID 191 ALPGITNDTT 19 624
GLGKDFKSPH 17
specified, the length of peptide is 10 248 LLLRLVAGPL 19 657
SVFGMCVDTL 17
amino acids, and the end position for 260 VLILGVLGVL 19 667
FLCFLEDLER 17
each peptide is the start position plus 261 LILGVLGVLA 19 684
PYYMSKSLLK 17
nine. 298
NLSAYOVQE 19 689 KSLLKILGKK 17
Pos 1234567890 score 312 ALIVLAVLEA 19 9 DDEAYGKPVK 16
7 ALYPLPTQPA 10 314 IVLAVLEAIL 19 44
FILGYIVVGI 16
15 PATLGYVLWA 10 317 AVLEAILLLM 19 126 TVGKNEFSQT 16
8 LYPLPTQPAT 9 322 ILLLMLIFLR 19 165
ELCPSFLLPS 16
189
=
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TablekOWII-V1-HLA-A3-10mers- Each peptide is a portion of SEQ
ID Pos 1234567890 score
24P4C12 NO: 7; each start position is 9
AVGQMMSTMF 19
Each peptide is a portion of SEQ ID specified, the length of peptide
is 10 6 I LVAVQQMM S 16
NO: 3; each start position is specified, amino acids, and the end position
for 5 WILVAVGQMM 14
the length of peptide is 10 amino each peptide is the start
position plus 7 LVAVGQLAM ST 14
adds, and the end position for each nine. 2
SVVYWILVAVG 12
peptide is the start position plus nine. Pos 1234567890 score 8
VAVGQMMSTM 9
Pos 1234567890 score 3 RCFPWTNITP 11
243 SLLF I LLLRL 16 9 N ITPPALPGI 11
TableXXXVII-V8-HLA-A3-10mers-
246 F I LLLRLVAG 16 8 TNITPPALPG 9 24P4C12
259 LVLILGVLGV 16 10 ITPPALPGIT 7 Each
peptide is a portion of SEQ ID
272 GIYYCWEEYR 16 7 VVIN ITPPALP 5 NO: 17; each start
position is
304 SVQETWLAAL 16
specified, the length of peptide is 10
318 VLEAILLLML 16 amino
acids, and the end position for
339 ALLKEASKAV 16 TableX=11145-HLA-A3-10mers-
each peptide is the start position plus
363 VLLLICIAYW 16 24P4C12 nine.
453 TLNVVVLALGQ 16 Each peptide is a portion of SEQ
ID Pos 1234567890 score
457 VLALGQQVLA 16 NO: 11; each start position is
specified, 12 PITPTGHVFQ 15
459 ALGQCVLAGA 16 the length of peptide is 10
amino acids, 11 NPITPTGHVF 14
487 LISAFIRTLR 16 and the end position for each
peptide is 18 HVFQTSILGA 13
508 ILTLVQ!ARV 16 the start position plus nine. 7
PIMRNPITPT 12
518 ILEYIDHKLR 16 Pos 1234567890 score 5
WLPIMRNPIT 11
559 AYIMIAIYGK 16 1 AVLEAILLLV 19 1 LNYYWLPIMR
10
566 YGKNFCVSAK 16 2 VLEAILLLVL 19 8
IMRNPITPTG 10
571 CVSAKNAFML 16 6 ILLLVLIFLR 19 21 QTSILGAYVI
10
579 M LLM RN IVRV 16 8 LLVLIFLRQR 18 9
MRNPITPTGH 9
596 DLLLFFGKLL 16 10 VLIFLBQRIR 17 6
LP1MRNPITP 8
640 P I MTSI LGAY 16 7 LLLVLIFLRQ 15 19
VFQTSILGAY 8
690 SLLKILGKKN 16 5 AILLLVLIFL 14
693 KILGKKN EAP 16 9 LVLIFLRQRI 14
TableXXXVII-V9-HLA-A3-10mers-
35 VI CCVLFLLF 15 4 EAILLLVLIF 11 24P4C12
41 FLLFILGYIV 15 Each
peptide is a portion of SEQ ID
42 LLF I LGYIVV 15 TableXXXVII-V6-HLA-A3-10mers-
NO: 19; each start position is
107 GLQCPTPQVC 15 24P4C12
specified, the length of peptide is 10
120 CPEDPWTVGK 15 Each peptide is a portion of SEQ
ID amino acids, and the end position for
180 RCFPWTNVTP 15 NO: 13; each start position is each
peptide is the start position plus
323 LLLMLIFLRQ 15 specified, the length of peptide
is 10 nine.
329 FLRQRIRIAI 15 amino acids, and the end
position for Pos 1234567890 score
367 ICIAYWAMTA 15 each peptide is the start
position plus 7 ALYPLPTQPA 20
369 IAYWAMTALY 15 nine. 17 TLGYVLWASN
15
423 MCVFQGYSSK 15 Pos 1234567890 score 10 PLPTQPATLG 14
446 GVLGLFVVTLN 15 7 GLIPRSVFNL 16 9 YPLPTQEATL
13
491 FIRTLRYHTG 15 5 SKGLIPRSVF 14 1 AYWAMTALYP 11
507 LILTLVQIAR 15 1 QGYSSKGLIP 12 18 LGYVLWASN I
10
510 TLVQIARVIL 15 8 LIPRSVFNLQ 11 4 AMTALYPLPT
9
585 IVRVVVLDKV 15 9 IPRSVFN LQI 11 11 LPTQPATLGY
9
597 LLLFFGKLLV 15 6 KGLIPRSVFN 10 13 TQPATLGYVL
9
604 LLWGGVGVL 15 4 SSKGLIPRSV 7
688 SKSLLKILGK 15
TableMON111-V1-HLA-A26-10mers-
694 ILGKKNEAPP 15 TableXXXVII-V7-HLA-A3-10mers-
24P4C12
697 KKNEAPPDNK 15 24P4C12 Each
peptide is a portion of SEQ ID
698 KNEAPPDNKK 15 Each peptide is a portion of SEQ
ID NO: 3; each start position is
NO: 15; each start position is
specified, the length of peptide is 10
Table)0(XVII-V3-HLA-A3-10mers- specified, the length of peptide
is 10 amino acids, and the end position for
24P4C12 amino acids, and the end
position for each peptide is the start position
each peptide is the start position plus plus nine.
nine. Pos
1234567890 score
190
CA 02503346 2005-04-21
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TableXXXVIII-V1-HLA-A26-10mers- TableXXXVIII-V1-HLA-A26-
10mers- 10 ITPPALPGIT 10
24P4C12 24P4C12 7
VVTNITPPALP 8
Each peptide is a portion of SEQ ID Each peptide is a portion
of SEQ ID 3 RCFPWTNITP 7
NO: 3; each start position is NO: 3; each start position is 8
TNITPPALPG 6
specified, the length of peptide is 10 specified, the length of
peptide is 10 4 CFPVYTNITPP 4
amino acids, and the end position for amino acids, and the end
position for
each peptide is the start position each peptide is the
start position TableXXXVIII-V5-HLA-A26-
plus nine, plus nine. 10mers-24P4C12
Pos 1234567890 score Pos 1234567890 score Each
peptide is a portion of
34 DVICCVLFLL 34 532 PVARCIMCCF 18 SEQ ID NO: 11;
each start
138 EVFYTKNRNF 32 549 EKFIKFLNRN 18
position is specified, the length
307 ETWLAALIVL 31 609 GVGVLSFFFF 18 of peptide is 10
amino acids,
657 SVFGMCVDTL 28 99 NIISVAENGL 17 and the end
position for each
199 TTIQQGISGL 26 102 SVAENGLQCP 17
peptide is the start position plus
304 SVQETWLAAL 26 156 MTVITSLQQE 17 nine.
588 WVLDKVTDL 26 236 LGVALVLSLL 17 Pos 1234567890
score
592 DKVTDLLLFF 25 260 VL1LGVLGVL 17 4 EAILLLVLIF 27
49 IVVGIVAVVLY 24 316 LAVLEAILLL 17 1 AVLEAILLLV 17
606 VVGGVGVLSF 24 317 AVLEAILLLM 17 5 AILLLVLIFL 17
157 TVITSLQQEL 23 321 AILLLMLIFL 17 2 VLEAILLLVL 13
252 LVAGPLVLVL 23 360 VTFVLLLICI 17
257 LVLVLILGVL 23 442 LQIYGVLGLF 17 TableXXXVIII-V6-
HLA-A26-
320 EAILLLMLIF 23 596 DLLLFFGKLL 17 10mers-24P4C12
628 DFKSPHLNYY 23 604 LLVVGGVGVL 17 Each peptide is
a portion of
79 ENKDKPYLLY 22 616 FFFSGRIPGL 17 SEQ ID NO: 13;
each start
353 STMFYPLVTF 22 664 DTLFLCFLED 17
position is specified, the length
362 FVLLLICIAY 22 665 TLFLCFLEDL 17 of peptide is 10
amino acids,
662 CVDTLFLCFL 22 682 DRPYYMSKSL 17 and the end
position for each
672 EDLERNNGSL 22 32 CTDVICCVLF 16
peptide is the start position plus
48 YIVVGIVAWL 20 37 CCVLFLLFIL 16 nine.
198 DTTIQQGISG 20 123 DPVVTVGKNEF 16 Pos 1234567890
score
216 DISVKIFEDF 20 165 ELCPSFLLPS 16 7 GL1PRSVFNL 17
240 LVLSLLFILL 20 186 NVIPPALPGI 16 10 PRSVFNLQIY 14
293 LGFTTNLSAY 20 224 DFAQSVVYWIL 16 5 SKGLIPRSVF 10
640 PIMTSILGAY 20 239 ALVLSLLFIL 16
DEAYGKPVKY 19 262 ILGVLGVLAY 16 TableXXXVIII-V7-HLA-
A26-
39 VLFLLFILGY 19 266 LGVLAYGIYY 16 10mers-24P4C12
131 EFSQTVGEVF 19 332 QRIRIAIALL 16 Each peptide is
a portion of SEQ
233 LVALGVALVL 19 359 LVTFVLLLIC 16 ID
NO: 15; each start position is
=
237 GVALVLSLLF 19 380 ATSGQPQYVL 16 specified, the
length of peptide is
347 AVGQMMSTMF 19 400 EKVPINTSCN 16 10 amino acids,
and the end
=
438 SVFNLQIYGV 19 405 NTSCNPTAHL 16 .
position for each peptide is the
463 CVLAGAFASF 19 424 CVFQGYSSKG 16 start position
plus nine.
498 HTGSLAFGAL 19 433 GL1QRSVFNL 16 Pos
1234567890 score
512 VQIARVILEY 19 539 CCFKCCLWCL 16 9 AVGQMMSTMF 19
520 EYIDHKLRGV 19 593 KVTDLLLFFG 16 7 LVAVGQMMST 11
571 CVSAKNAFML 19 4 YWILVAVGQM 10
589 WLDKVTDLL 19 TableXXXVIII-V3-HLA-A26-10mers-
33 TDVICCVLFL 18 24P4C12 TableXXXVIII-V8-HLA-
A26-
203 QGISGLIDSL 18 Each peptide is a portion of SEQ ID NO:
l0mers-24P4C12
314 IVLAVLEAIL 18 7; each start position is
specified, the Each peptide is a portion of SEQ ID
456 WVLALGQCVL 18 length of peptide is 10
amino acids, and NO: 17; each start position is
481 DIPTFPL1SA 18 ? the end position for each
peptide is the specified, the length of peptide is 10
486 PLISAFIRTL 18 start position plus nine, amino acids,
and the end position
493 RTLRYHTGSL 18 Pos 1234567890 score for each
peptide is the start position
502 LAFGALILTL 18 6 PWTNITPPAL 10 plus
nine.
516 RVILEY1DHK 18 9 NITPPALPGI = 10 Pos
1234567890 score
191
=
CA 02503346 2005-04-21
WO 2004/050828 PCT/US2002/038264
18 HVFQTSILGA 19 TableXXXIX-V1-11LA-B0702- TableXXXIX-V1-HLA-B0702-
19 VFQTSILGAY 16 lOmers-24P4C12 1Omers-24P4C12
11 NPITPTGHVF 13 Each peptide is a portion of SEQ Each peptide is
a portion of SEQ
13 ITPTGHVFQT 13 ID NO: 3; each start position is ID NO: 3; each
start position is
16 TGHVFQTSIL 10 specified, the length of peptide is specified,
the length of peptide is
15 FIGHVFQTS1 9 10 amino acids, and the end 10 amino acids, and
the end
position for each peptide is the position for each peptide
is the
TableXXXVIII-V9-HLA-A26-10mers- start position plus nine, start position
plus nine.
24P4C12 Pos 1234567890 score Pos 1234567890 score
Each peptide is a portion of SEQ ID 331 RQRIRIAIAL 14 316
LAVLEAILLL 12
NO: 19; each start position is 405 NTSCNPTAHL 14 409
NPTAHLVNSS 12
specified, the length of peptide is 10 451 FVVTLNWVLAL 14 419
CPGLMCVFQG 12
amino acids, and the end position for 502 LAFGALILTL 14 425
VFQGYSSKGL 12
each peptide is the start position plus 582 MRNIVRVVVL 14 456
VVVLALGQCVL 12
nine. 590 VLDKVTDLLL 14 493 RTLRYHTGSL 12
Pos 1234567890 score 15 KPVKYDPSFR 13 581 LMRNIVRVVV 12
12 PTQPATLGYV 14 60 DPRQVLYPRN 13 588 WVLDKVTDL 12
5 MTALYPLPTQ 13 66 YPRNSTGAYC 13 604 LLWGGVGVL 12
16 ATLGYVLWAS 13 110 CPTPQVCVSS 13 606 VVGGVGVLSF 12
2 YWAMTALYPL 12 120 CPEDPVVTVGK 13 622 IPGLGKDFKS 12
11 LPTQPATLGY 12 167 CPSFLLPSAP 13 637 YVVLPIMTSIL 12
9 YPLPTQPATL 10 172 LPSAPALGRC 13 662 CVDTLFLCFL 12
13 TQPATLGYVL 10 226 AQSVVYWILVA 13 701 APPDNKKRKK 12
15 PATLGYVLWA 6 227 QSWYWILVAL 13 18 IMPSFRGPI 11
231 WILVALGVAL 13 25 GP IKN RSCTD 11
TableXXXIX-V1-HLA-B0702. 250 LRLVAGPLVL 13 31 SCTDVICCVL 11
l0mers-24P4C12 284 RDKGASISQL 13 44 FILGYIVVGI 11
Each peptide is a portion of SEQ 290 ISQLGFTTNL 13 77
MGENKDKPYL 11
ID NO: 3; each start position is 301 AYQSVQETWL 13 78 GENKDKPYLL
11
specified, the length of peptide is 310 LAALIVLAVL 13 140
FYTKNRNFCL 11
amino acids, and the end 314 IVLAVLEAIL 13 152
VPWNMTVITS 11
position for each peptide is the 318 VLEAILLLML 13 153
PWNMTVITSL 11
start position plus nine. 321 AILLLMLIFL 13 162
LQQELCPSFL 11
Pos 1234567890 score 350 QMMSTMFYPL 13 188 TPPALPGITN 11
357 YPLVTFVLLL 23 355 MFYPLVTFVL 13 224 DFAQSWYWIL 11
478 KPQDIPTFPL 23 356 FYPLVTFVLL 13 236 LGVALVLSLL 11
683 RPYYMSKSLL 21 368 CIAYWAMTAL 13 240 LVLSLLFILL 11
182 FPWTNVTPPA 19 396 SPGCEKVPIN 13 248 LLLRLVAGPL 11
83 KPYLLYFNIF 18 441 NLQIYGVLGL 13 257 LVLVLILGVL 11
192 LPGITNDTTI 18 498 HTGSLAFGAL 13 260 VLILGVLGVL 11
482 IPTFPLISAF 18 500 GSLAFGALIL 13 274 YYCWEEYRVL 11
639 LPIMTSILGA 18 510 TLVQIARVIL 13 312 ALIVLAVLEA 11
¨
149 LPGVPWNMTV 17 525 KLRGVQNPVA 13 315 VLAVLEAILL 11
252 LVAGPLVLVL 17 571 CVSAKNAFML 13 332 QRIRIAIALL 11
380 ATSGQPQYVL 17 572 VSAKNAFMLL 13 384 QPQYVLWASN 11
402 VPINTSCNPT 17 657 SVFGMCVDTL 13 395 SSPGCEKVPI 11
485 FPLISAFIRT 17 686 YMSKSLLKIL 13 413 HLVNSSCPGL 11
123 DPVVTVGKNEF 16 20 DPSFRGPIKN 12 433 GLIQRSVFNL 11
235 ALGVALVLSL 16 48 YIVVGIVAVVL 12 = 435 IQRSVFNLQI 11
254 AGPLVLVLIL 15 169 SFLLPSAPAL 12 439 VFNLQIYGVL 11
370 AYWAMTALYL 15 183 PVVTNVTPPAL 12 445 YGVLGLFVVTL 11
659 FGMCVDTLFL 15 189 PPALPGITND 12 449 GLFWILNINVL 11
33 TDVICCVLFL 14 239 ALVLSLLFIL 12 503 AFGALILTLV 11
56 WLYGDPRQVL 14 243 SLLFILLLRL 12 531 NPVARCIMCC 11
175 APALGRCFPW 14 304 SVQETWLAAL 12 536 CIMCCFKCCL 11
233 LVALGVALVL 14 307 ETWLAALIVL 12 539 CCFKCCLWCL 11
241 VLSLLFILLL 14 309 WLAALIVLAV 12 546 WCLEKFIKFL 11
192
=
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WO 2004/050828 PCT/US2002/038264
TableXXXIX-V1-HLA-B0702- 24P4C12
10mers-24P4C12 Pos 1234567890 score
Each peptide is a portion of SEQ TableXXXIX-V7-HLA-B0702-
NoResultsFound.
ID NO: 3; each start position is l0mers-24P4C12
specified, the length of peptide is Each peptide is a portion of SEQ ID
TableXL-V3-HLA-1308-10mers-
amino acids, and the end NO: 15; each start
position is 24P4C12
position for each peptide is the specified, the length of
peptide is 10 Pos 1234567890 score
start position plus nine, amino acids, and the end
position for NoResultsFound.
Pos 1234567890 score each peptide is the start position plus
565 IYGKNFCVSA 11 nine. TableXL-V5-HLA-1308-10mers-
589 VVLDKVTDLL 11 Pos 1234567890 score 24P4C12
595 TDLLLFFGKL 11 9 AVGQMMSTMF 10 Pos 1234567890 score
616 FFFSGRIPGL 11 1 QSVVYWILVAV 9 NoResultsFound.
625 LGKDFKSPHL 11 8 VAVGQMMSTM 8
630 KSPHLNYYWL 11 4 YWILVAVGQM 7 TableXL-V6-HLA-B08-10mers-
672 EDLERNNGSL 11 7 LVAVGQMMST 7 24P4C12
5 VVILVAVGQMM 6 Pos 1234567890 score
TableXXXIX-V3-HLA-B0702- NoResultsFound.
10mers-24P4C12 TableX,XXIX-V8-HLA-B0702-
Each peptide is a portion of SEQ ID l0mers-24P4C12 TableXL-V7-
HLA-1308-10mers-
NO: 7; each start position is Each peptide is a portion of SEQ
24P4C12
specified, the length of peptide is 10 ID NO: 17; each start
position is Pos 1234567890 score
amino acids, and the end position specified, the length of peptide is
NoResultsFound.
for each peptide is the start position 10 amino acids, and the end
plus nine, position for each peptide is the TableXL-V13-HLA-
B08-10mers-
Pos 1234567890 score start position
plus nine. 24P4C12
5 FPWTNITPPA 19 Pos 1234567890 score Pos 1234567890 score
6 PWTNITPPAL 12 11 NPITPTGHVF 17 NoResultsFound.
1 LGRCFPWTNI 9 14 TPTGHVFQTS 13
16 TGHVFQTSIL 11 TableXL-V9-HLA-B08-
TableXXXIX-V5-HLA-B0702- 6 LPIMRNPITP 10 10mers-24P4C12
=
l0mers-24P4C12 4 YWLPIMRNPI 9 Pas 1234567890 score
Each peptide is a portion of SEQ 7 PIMRNPITPT 9 NoResultsFound.
ID NO: 11; each start position is 21 QTSILGAYVI 9
specified, the length of peptide is 10 RNPITPTGHV 8 TableXLI-V1-HLA-
B1510-10mers-
10 amino acids, and the end 13 ITPTGHVFQT 8 24P4C12
position for each peptide is the 15 PTGHVFQTSI 8 Pos
1234567890 score
start position plus nine. 18 HVFQTSILGA 8 NoResultsFound.
Pos 1234567890 score
2 VLEAILLLVL 14 TableXXXIX-V9-HLA-B0702- TableXLI-V3-HLA-B1510-
10mers-
5 AILLLVLIFL 13 10mers-24P4C12 24P4C12
1 AVLEAILLLV 10 Each peptide is a portion of SEQ Pos
1234567890 score
4 EAILLLVLIF 10 ID NO: 19; each start position is NoResultsFound.
3 LEAILLLVLI 9 specified, the length of peptide is
9 LVLIFLRQRI 7 10 amino acids, and the end
TableXLI-V5-HLA-B1510-1 Omers-
position for each peptide is the 24P4C12
TableXXXIX-V6-HLA-B0702- start position plus nine. Pos 1234567890
score
10mers-24P4C12 Pos 1234567890 score NoResultsFound.
Each peptide is a portion of SEQ ID 9 YPLPTQPATL 22
NO: 13; each start position is 11 LPTQPATLGY 13 TableXLI-V6-HLA-
B1510-
specified, the length of peptide is 10 14 QPATLGYVLW 13
lOmers-24P4C12
amino acids, and the end position 2 YWAMTALYPL 12 Pos
1234567890 score
for each peptide is the start position 4 AMTALYPLPT 12
NoResultsFound.
plus nine. 13 TQPATLGYVL 12
Pos 1234567890 score 7 ALYPLPTQPA 11 TableXLIN7-HLA-B1510-10mers-
9 IPRSVFNLQI 21 24P4C12
7 GLIPRSVFNL 12 Tabl eXL-V1-HLA-B08-10mers-
193
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WO 2004/050828 PCT/US2002/038264
Pos 1234567890 score TableXLIII-V3-HLA-B2709-10mers-
TableXLIV41-HLA-B4402-
NoResultsFound. 24P4C12 10mers-
24P4C12
Pos 1234567890 score Each
peptide is a portion of SEQ IC)
TableXLI-V8-HLA-B1510-10mers- NoResultsFound. NO: 3; each start
position is
24P4C12
specified, the length of peptide is 10
Pos 1234567890 score TableXLIII-V5-1ILA-B2709-
10mers- amino acids, and the end position
NoResultsFound. 24P4C12 for
each peptide is the start position
Pos 1234567890 score plus nine.
TableXLI-V9-HLA-B1510-10mers- NoResultsFound. Pos 1234567890 score
24P4C12 199 TTIQQGISGL 16
Pos 1234567890 score TableXLIII-V6-HLA-82709-10mers-
203 QGISGLIDSL 16
NoResultsFound. 24P4C12 260 VLILGVLGVL
16
Pos 1234567890 score 293 LGFTTNLSAY 16
NoResultsFound. 307 ETWLAALIVL 16
TableXLII-V1-HLA-B2705-10mers- 316 LAVLEAILLL
16
24P4C12 TableXLIII-W-HLA-B2709-10mers-24P4C12 380 ATSGQPQYVL
16
Pos 1234567890 score Pos 1234567890 score 546 WCLEKFIKFL 16
NoResultsFound. NoResultsFound. 657 SVFGMCVDTL 16
34 DVICCVLFLL
15
TableXLII-V3-HLA-B2705-10mers-24P4C12 TableXLIII-V8-HLA-B2709-10mers- 65
LYPRNSTGAY 15
Pos 1234567890 score 24P4C12 79 ENKDKPYLLY 15
NoResultsFound. Pos 1234567890 score 99 NIISVAENGL
15
NoResultsFound. 104 AENGLQCPTP 15
TableXLII-V5-HLA-B2705-10mers- 138 EVFYTKNRNF 15
24P4C12 TableXLIII-V9-HLA-B2709-10mers-
213 NARDISVKIF 15
Pos 1234567890 score 24P4C12 235 ALGVALVLSL 15
NoResultsFound. Pos 1234567890 score 239
ALVLSLLFIL 15
NoResultsFound. 278 EEYRVLRDKG 15
TableXLII-V6-HLA-B2705-10mers- 284 RDKGASISQL 15
24P4C12 TableXLIV-V1-HLA-B4402- 353 STMFYPLVTF 15
Pos 1234567890 score 10mers-24P4C12 355 MFYPLVTFVL 15
NoResultsFound. Each peptide is a portion of
SEQ ID 356 FYPLVTFVLL 15
NO: 3; each start position is 362 FVLLLICIAY
15
TableXLII-W-HLA-B2705-10mers- specified, the length of
peptide is 10 363 VLLLICIAYW 15
24P4C12 amino acids, and the end
position 370 AYWAMTALYL 15
Pos 1234567890 score for each peptide is the start
position 417 SSCPGLMCVF 15
NoResultsFound. plus nine. 442 LQIYGVLGLF
15
Pos 1234567890 score 451 FINTLNVVVLAL 15
TableXLII-V8-HLA-B2705-10mers- 10 DEAYGKPVKY 23 482
IPTFPLISAF 15
24P4C12 78 GENKDKPYLL 22 561
IMIAIYGKNF 15
Pos 1234567890 score 222 FEDFAQS1NYW 21 596
DLLLFFGKLL 15
NoResultsFound. 319 LEAILLLMLI 20 616
FFFSGRIPGL 15
47 GYIVVGIVAW 19 637 YWLPIMTSIL 15
TableXLII-V9-HLA-B2705-10mers- 332 QRIRIA1ALL 18 640
PIMTSILGAY 15
24P4C12 486 PLISAFIRTL 18 4
KQRDEDDEAY 14
Pos 1234567890 score 502 LAFGALILTL 18 18 KYDPSFRGPI 14
NoResultsFound. 620 GRIPGLGKDF 18 80
NKDKPYLLYF 14
39 VLFLLFILGY 17 83 KPYLLYFNIF
14
TableXL111V1-HLA-B270940mers- 241 VLSLLFILLL 17 130 NEFSQTVGEV 14
24P4C12 254 AGPLVLVLIL 17 131
EFSQTVGEVF 14
Pos 1234567890 score 320 EAILLLMLIF 17 157
TVITSLQQEL 14
NoResultsFound. 321 AILLLMLIFL 17 164
QELCPSFLLP 14
476 FHKPQDIPTF 17 173 PSAPALGRCF 14
TableXLIII-V3-HLA-B2709-10mers- 512 VQIARVILEY 17 175
APALGRCFPW 14
24P4C12 699 NEAPPDNKKR 17 183
PWTNVTPPAL 14
Pos 1234567890 score 121 PEDPWTVGKN 16 220 KIFEDFAQSW 14
169 SFLLPSAPAL 16 227 QSVVYWILVAL 14
194
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TableXLIV-V1-HLA-B4402- TableXLIV-V1-HLA-B4402= TableXLIV-V1-HLA-B4402-
10mers-24P4C12 10mers-24P4C12 1Omers-24P4C12
Each peptide is a portion of SEQ ID Each peptide is a portion of SEQ
ID Each peptide is a portion of SEQ ID
NO: 3; each start position is NO: 3; each start position
is NO: 3; each start position is
specified, the length of peptide is 10 specified, the length of peptide
is 10 specified, the length of peptide is 10
amino acids, and the end position amino acids, and the end position
amino acids, and the end position
for each peptide is the start position for each peptide is the start
position for each peptide is the start position
plus nine, plus nine, plus nine.
Pos 1234567890 score Pos 1234567890 score Pos
1234567890 score
231 WILVALGVAL 14 445 YGVLGLFVVTL 13 368 CIAYWAMTAL 12
233 LVALGVALVL 14 447 VLGLFWTLNW 13 - 369 IAYWAMTALY 12
240 LVLSLLFILL 14 449 GLFVVTLNWVL 13 378 YLATSGQPQY 12
243 SLLFILLLRL 14 460 LGQCVLAGAF 13 381 TSGQPQYVLW 12
250 LRLVAGPLVL 14 478 KPQDIPTFPL 13 395 SSPGCEKVPI 12
252 LVAGPLVLVL 14 483 PTFPLISAFI 13 420 PGLMCVFQGY 12
253 VAGPLVLVLI 14 493 RTLRYHTGSL 13 436 QRSVFNLQIY 12 .
262 ILGVLGVLAY 14 495 LRYHTGSLAF 13 439 VFNLQIYGVL 12
304 SVQETWLAAL 14 498 HTGSLAFGAL 13 443 QIYGVLGLFW 12
331 RQRIRIAIAL 14 500 GSLAFGALIL 13 456 VVVLALGQCVL 12
357 YPLVTFVLLL 14 517 VILEYIDHKL 13 463 CVLAGAFASF 12
431 SKGLIQRSVF 14 539 CCFKCCLWCL 13 464 VLAGAFASFY 12
433 GLIQRSVFNL 14 557 RNAYIMIAIY 13 488 ISAFIRTLRY 12
467 GAFASFYWAF 14 582 MRNIVRVVVL 13 505 GALILTLVQI 12
542 KCCLWCLEKF 14 590 VLDKVTDLLL 13 509 LTLVQIARVI 12
545 LWCLEKFIKF 14 591 LDKVTDLLLF 13 510 TLVQIARVIL 12
551 FIKFLNRNAY 14 592 DKVTDLLLFF 13 548 LEKFIKFLNR 12
569 NFCVSAKNAF 14 606 VVGGVGVLSF 13 556 NRNAYIMIAI 12
589 VVLDKVTDLL 14 659 FGMCVDTLFL 13 571 CVSAKNAFML 12
595 TDLLLFFGKL 14 661 MCVDTLFLCF 13 572 VSAKNAFMLL 12
627 KDFKSPHLNY 14 662 CVDTLFLCFL 13 576 NAFMLLMRNI 12
629 FKSPHLNYYW 14 671 LEDLERNNGS 13 588 VVVLDKVTDL 12
665 TLFLCFLEDL 14 672 EDLERNNGSL 13 604 LLVVGGVGVL 12
686 YMSKSLLKIL 14 682 DRPYYMSKSL 13 628 DFKSPHLNYY 12
7 DEDDEAYGKP 13 33 TDVICCVLFL 12 630 KSPHLNYYWL 12
31 SCTDVICCVL 13 37 CCVLFLLFIL 12 650 VIASGFFSVF 12
32 CTDVICCVLF 13 44 FILGYIVVGI 12 674 LERNNGSLDR 12
35 VICCVLFLLF 13 76 GMGENKDKPY 12 676 RNNGSLDRPY 12
49 IWGIVAWLY 13 123 DPWTVGKNEF 12 677 NNGSLDRPYY 12
56 WLYGDPRQVL 13 132 FSQTVGEVFY 12 685 YYMSKSLLKI 12
57 LYGDPRQVLY 13 150 PGVPWNMTVI 12 14 GKPVKYDPSF 11
87 LYFNIFSCIL 13 163 QQELCPSFLL 12 27 IKNRSCTDVI 11
145 RNFCLPGVPW 13 216 DISVKIFEDF 12 40 LFLLFILGYI 11
153 PWNMTVITSL 13 223 EDFAQSWYWI 12 48 YIWGIVAWL 11
186 NVIPPALPGI 13 236 LGVALVLSLL 12 77 MGENKDKPYL 11
237 GVALVLSLLF 13 266 LGVLAYGIYY 12 116 CVSSCPEDPW 11
248 LLLRLVAGPL 13 274 YYCWEEYRVL 12 137 GEVFYTKNRN 11
257 LVLVLILGVL 13 277 WEEYRVLRDK 12 161 SLQQELCPSF 11
271 YGIYYCWEEY 13 286 KGASISQLGF 12 162 LQQELCPSFL 11
301 AYQSVQETWL 13 290 ISQLGFTTNL 12 = 208 LIDSLNARDI 11
310 LAALIVLAVL 13 300 SAYQSVQETW 12 212 LNARDISVKI 11
315 VLAVLEAILL 13 306 QETWLAALIV 12 221 IFEDFAQS1NY 11
327 LIFLRQRIRI 13 313 LIVLAVLEAI 12 238 VALVLSLLFI
11
342 KEASKAVGQM 13 318 VLEAILLLML 12 264 GVLGVLAYGI 11
347 AVGQMMSTMF 13 329 FLRQRIRIAI 12 305 VQETWLAALI 11
405 NTSCNPTAHL 13 350 QMMSTMFYPL 12 314 IVLAVLEAIL 11
425 VFQGYSSKGL 13 358 PLVTFVLLLI 12 348 VGQMMSTMFY 11
441 NLQIYGVLGL 13 360 VTFVLLLICI 12 413 HLVNSSCPGL 11
195
DEMANDES OU BREVETS VOLUMINEUX
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COMPREND PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
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