Note: Descriptions are shown in the official language in which they were submitted.
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NOTE POUR LE TOME / VOLUME NOTE:
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
NUCLEIC ACID AND CORRESPONDING PROTEIN ENTITLED 213P1F11
USEFUL IN TREATMENT AND DETECTION OF CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
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 213P1F11,
expressed in certain cancers, and to diagnostic and therapeutic methods and
compositions useful in the
management of cancers that express 213P1F11.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of human death next to coronary disease.
Worldwide, millions of
people die from cancer every year. In the United States alone, as reported by
the American Cancer Society,
cancer causes the death of well over a half million people annually, with over
1.2 million new cases
diagnosed per year. While deaths from heart disease have been declining
significantly, those resulting from
cancer generally are on the rise. In the early part of the next century,
cancer is predicted to become the
leading cause of death.
Worldwide, several cancers stand out as the leading killers. In particular,
carcinomas of the lung,
prostate, breast, colon, pancreas, and ovary represent the primary causes of
cancer death. These and virtually
all other carcinomas share a common lethal feature. With very few exceptions,
metastatic disease from a
carcinoma is fatal. Moreover, even for those cancer patients who initially
survive their primary cancers,
common experience has shown that their lives are dramatically altered. Many
cancer patients experience
strong anxieties driven by the awareness of the potential for recurrence or
treatment failure. Many cancer
patients experience physical debilitations following treatment. Furthermore,
many cancer patients experience
a recurrence.
Worldwide, prostate cancer is the fourth most prevalent cancer in men. In
North America and
Northern Europe, it is by far the most common cancer in males and is the
second leading cause of cancer
death in men. In the United States alone, well over 30,000 men die annually of
this disease - second only to
lung cancer. Despite the magnitude of these figures, there is still no
effective treatment for metastatic prostate
cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy,
surgical castration and
chemotherapy continue to be the main treatment modalities. Unfortunately,
these treatments are ineffective
for many and are often associated with undesirable 'consequences.
On the diagnostic front, the lack of a prostate tumor marker that can
accurately detect early-stage,
localized tumors r_~mains a significant limitation in the diagnosis and
management of this disease. Although
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the serum prostate specific antigen (PSA) assay has been a very useful tool,
however its specificity and
general utility is widely regarded as lacking in several important respects.
Progress in identifying additional specific markers for prostate cancer has
been improved by the
generation of prostate cancer xenografts that can recapitulate different
stages of the disease in mice. The
LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts
that have survived passage in
severe combined immune deficient (SCID) mice and have exhibited the capacity
to mimic the transition from
androgen dependence to androgen independence (Klein et al., 1997, Nat. Med.
3:402). More recently
identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc.
Natl. Acad. Sci. USA 93: 7252),
prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996
Sep 2 (9): 1445-51), STEAP
(Hubert, et al., Proc Natl Acad Sci U S A. 1999 Dec 7; 96(25): 14523-8) and
prostate stem cell antigen
(PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
While previously identified markers such as PSA, PSM, PCTA and PSCA have
facilitated efforts to
diagnose and treat prostate cancer, there is need for the identification of
additional markers and therapeutic
targets for prostate and related cancers in order to further improve diagnosis
and therapy.
Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult
malignancies. Once
adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the
adult, the two principal malignant
renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of
the renal pelvis or ureter. The
incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases
in the United States, and more
than 11,600 patients died of this disease in 1998. Transitional cell carcinoma
is less frequent, with an
incidence of approximately 500 cases per year in the United States.
Surgery has been the primary therapy for renal cell adenocarcinoma for many
decades. Until
recently, metastatic disease has been refractory to any systemic therapy. With
recent developments in
systemic therapies, particularly immunotherapies, metastatic renal cell
carcinoma may be approached
aggressively in appropriate patients with a possibility of durable responses.
Nevertheless, there is a remaining
need for effective therapies for these patients.
Of all new cases of cancer in the United States, bladder cancer represents
approximately 5 percent in
men (fifth most common neoplasm) and 3 percent in women (eighth most common
neoplasm). The incidence
is increasing slowly, concurrent with an increasing older population. In 1998,
there was an estimated 54,500
cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence
in the United States is 32
per 100,000 for men and 8 per 100,000 in women. The historic male/female ratio
of 3:1 may be decreasing
related to smoking patterns in women. There were an estimated 11,000 deaths
from bladder cancer in 1998
(7,800 in men and 3,900 in women). Bladder cancer incidence and mortality
strongly increase with age and
will be an increasing problem as the population becomes more elderly.
Most bladder cancers recur in the bladder. Bladder cancer is managed with a
combination of
transurethral resection of the bladder (TUR) and intravesical chemotherapy or
immunotherapy. 'The
multifocal and recurrent nature of bladder cancer points out the limitations
of TUR. Most muscle-invasive
cancers are not cured by TUR alone. Radical cystectomy and urinary diversion
is the most effective means to
eliminate the cancer but carry an undeniable impact on urinary and sexual
function. There continues to be a
significant need for treatment modalities that are beneficial for bladder
cancer patients.
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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 iz~ women was 42.3 per
100,000.
Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,
accounting for 28% of all
cancer deaths. During 1992-1996, mortality from lung cancer declined
significantly among men (-1.7% per
year) while rates for women were still significantly increasing (0.9% per
year). Since 1987, more women
have died each year of lung cancer than breast cancer, which, for over 40
years, was the major cause of cancer
death in women. Decreasing lung cancer incidence and mortality rates most
likely resulted from decreased
smoking rates over the previous 30 years; however, decreasing smoking patterns
among women lag behind
those of men. Of concern, although the declines in adult tobacco use have
slowed, tobacco use in youth is
increasing again.
Treatment options for lung and bronchial cancer are determined by the type and
stage of the cancer
and include surgery, radiation therapy, and chemotherapy. For many localized
cancers, surgery is usually the
treatment of choice. Because the disease has usually spread by the time it is
discovered, radiation therapy and
chemotherapy are often needed in combination with surgery. Chemotherapy alone
or combined with
radiation is the treatment of choice for small cell lung cancer; on this
regimen, a large percentage of patients
experience remission, which in some cases is long lasting. There is however,
an ongoing need for effective
treatment and diagnostic approaches for lung and bronchial cancers.
An estimated 182,800 new invasive cases of breast cancer were expected to
occur among women in
the United States during 2000. Additionally, about 1,400 new cases ofbreast
cancer were expected to be
diagnosed in men in 2'000. 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.
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Taking into account the medical circumstances and the patient's preferences,
treatment of breast
cancer may involve lumpectomy (local removal of the tumor) and removal of the
lymph nodes under the arm;
mastectomy (surgical removal of the breast) and removal of the lymph nodes
under the arm; radiation
therapy; chemotherapy; or hormone therapy. Often, two or more methods are used
in combination.
Numerous studies have shown that, for early stage disease, long-term survival
rates after lumpectomy plus
radiotherapy are similar to survival rates after modified radical mastectomy.
Significant advances in
reconstruction techniques provide several options for breast reconstruction
after mastectomy. Recently, such
reconstruction has been done at the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of
surrounding normal
breast tissue may prevent the local recurrence of the DCIS. Radiation to the
breast and/or tamoxifen may
reduce the chance of DCIS occurring in the remaining breast tissue. This is
important because DCIS, if left
untreated, may develop into invasive breast cancer. Nevertheless, there are
serious side effects or sequelae to
these treatments. There is, therefore, a need for efficacious breast cancer
treatments.
There were an estimated 23,100 new cases of ovarian cancer in the United
States in 2000. It
accounts for 4% of all cancers among women and ranks second among gynecologic
cancers. During 1992-
1996, ovarian cancer incidence rates were significantly declining. Consequent
to ovarian Dancer, there were
an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any
other cancer of the female
reproductive system.
Surgery, radiation therapy, and chemotherapy are treatment options for ovarian
cancer. Surgery
usually includes the removal of one or both ovaries, the fallopian tubes
(salpingo-oophorectomy), and the
uterus (hysterectomy). In some very early tumors, only the involved ovary will
be removed, especially in
young women who wish to have children. In advanced disease, an attempt is made
to remove all intra-
abdominal disease to enhance the effect of chemotherapy. There continues to be
an important need for
effective treatment options for ovarian cancer.
There were an estimated 28,300 new cases of pancreatic cancer in the United
States in 2000. Over
the past 20 years, rates of pancreatic cancer have declined in men. Rates
among women have remained
approximately constant but may be beginning to decline. Pancreatic cancer
caused an estimated 28,200
deaths in 2000 in the United States. Over the past 20 years, there has been a
slight but significant decrease in
mortality rates among men (about -0.9% per year) ;r ~. ale 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.
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SUMMARY OF THE INVENTION
The present invention relates to a gene, designated 213P1F11, that has now
been found to be over-
expressed in the cancers) listed in Table I. Northern blot expression analysis
of 213P1F11 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 213P1F11 are provided. The tissue-
related profile of 213P1F11 in
normal adult tissues, combined with the over-expression observed in the
tissues listed in Table I, shows that
213P1F11 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
tissues) such as those listed in Table I.
The invention provides polynucleotides corresponding or complementary to all
or part of the
213P1F11 genes, mRNAs, and/or coding sequences, preferably in isolated form,
including polynucleotides
encoding 213P1F11-related proteins and fragments of4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35,
40, 45, 50, 55, 60, 65, 70, 80, 85,
90, 95, 100 or more than 100 contiguous amino acids of a 213P1F11-related
protein, as well as the
peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related
molecules, polynucleotides or
oligonucleotides complementary or having at least a 90% homology to the
213P1F11 genes or mRNA
sequences or parts thereof, and polynucleotides or oligonucleotides that
hybridize to the 213P1F11 genes,
mRNAs, or to 213P1F11-encoding polynucleotides. Also provided are means for
isolating cDNAs and the
genes encoding 213P1F11. Recombinant DNA molecules containing 213P1F11
polynucleotides, cells
transformed or transduced with such molecules, and host-vector systems for the
expression of 213P 1 F 11 gene
products are also provided. The invention further provides antibodies that
bind to 213P1F11 proteins and
polypeptide fragments thereof, including polyclonal and monoclonal antibodies,
marine 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 213P1F11
polynucleotides and proteins in various biological samples, as well as methods
for identifying cells that express
213P 1F11. A typical embodiment of this invention provides methods for
monitoring 213P1F11 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 213P1F11 such as cancers of tissues listed in
Table, I, including therapies aimed
at inhibiting the transcription, translation, processing or function of
213P1F11 as well as cancer vaccines. In
one aspect, the invention provides compositions, and methods comprising them,
for treating a cancer that
expresses 213P1F11 in a human subject wherein the composition comprises a
carrier suitable for human use
and a human unit dose of one or more than one agent that inhibits the
production or function of 213P1F11.
Preferably, the carrier is a uniquely human Garner. In another aspect of the
invention, the agent is a moiety
that is immunoreactive with 213P1F11 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
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thereof. The antibodies can be conjugated to a diagnostic or therapeutic
moiety. In another aspect, the agent
is a small molecule as defined herein.
In another aspect, the agent comprises one or more than one peptide which
comprises a cytotoxic T
lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to
elicit a CTL response to
213P1F11 and/or one or more than one peptide which comprises a helper T
lymphocyte (HTL) epitope which
binds an HLA class II molecule in a human to elicit an HTL response. The
peptides of the invention may be
on the same or on one or more separate polypeptide molecules. In a further
aspect of the invention, the agent
comprises one or more than one nucleic acid molecule that expresses one or
more than one of the CTL or
HTL response stimulating peptides as described above. In yet another aspect of
the invention, the one or
more than one nucleic acid molecule may express a moiety that is
immunologically reactive with 213P1F11
as described above. The one or more than one nucleic acid molecule may also
be, or encodes, a molecule that
inhibits production of 213P1F11. Non-limiting examples of such molecules
include, but are not limited to,
those complementary to a nucleotide sequence essential for production of
213P1F11 (e.g. antisense sequences
or molecules that form a triple helix with a nucleotide double helix essential
for 213P1F11 production) or a
ribozyme effective to lyse 213P1F11 mRNA.
BRIEF DESCRIPTION OF THE FIGURES
Figure i. The 213P1F11 SSH sequence of 166 nucleotides.
Figure 2. The cDNA and amino acid sequence of 213P1F11 variant 1 clone CASP14-
BrCi (also
called "213P1F11 v.l" or "213P1F11 variant 1" or "213P1F11") is shown in
Figure 2A. The start methionine
is underlined. The open reading frame extends from nucleic acid 404-1132
including the stop codon. The
cDNA and amino acid sequence of 213P1F11 variant 2 (also called "213P1F11
v.2") is shown in Figure 2B.
The codon for the start methionine is underlined. The open reading frame
extends from nucleic acid 409-
1096 including the stop codon. The cDNA and amino acid sequence of 213P1F11
variant 3 (also called
"213P 1F 11 v.3") is shown in Figure 2C. The codon for the start methionine is
underlined. The open reading
frame extends from nucleic acid 404-844 including the stop codon. The cDNA and
amino acid sequence of
213P1F11 variant 4 (also called "213P1F11 v.4") is shown in Figure 2D. The
codon for the start methionine
is underlined. The open reading frame extends from nucleic acid 1-966
including the stop codon. The cDNA
(SEQ ID. NO. and amino acid sequence of 213P1F11 variant S (also called
"213P1F11 v.5") is shown in
Figure 2E. The codon for the start methionine is underlined. The open reading
frame extends from nucleic
acid 404-1132 including the stop codon. The cDNA and amino acid sequence of
213P1F11 variant 6 (also
called "213P 1F11 variant v.6") is shown in Figure 2F. The codon for the start
methionine is underlined. The
open reading frame extends from nucleic acid 404-1132 including the stop
codon. The cDNA and amino
acid sequence of 213P1F11 variant 7 (also called "213P1F11 v.7") is shown in
Figure 2G. The codon for the
start methionine is underlined. The open reading frame extends from nucleic
acid 404-1132 including the
stop codon. The cDNA and amino acid sequence of 213P1F11 variant 8 (also
called "213P1F11 v.8") is
shown in Figure 2H. The codon for the start methionine is underlined. The open
reading frame extends from
nucleic acid 404-1132 including the stop codon. As used herein, a reference to
213P1F11 includes all
variants thereof, including those shown in Figure 10.
6
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Figure 3. Amino acid sequence of 213P1F11 v.l is shown in Figure 3A; it has
242 amino acids.
The amino acid sequence of 213P1F11 v.2 is shown in Figure 3B; it has 230
amino acids. The amino acid
sequence of 213P1F11 v.3 is shown in Figure 3C; it has 146 amino acids. The
amino acid sequence of
213P1F11 v.4 is shown in Figure 3D; it has 321 amino acids. The amino acid
sequence of 213P1F11 v.5 is
shown in Figure 3E; it has 242 amino acids. The amino acid sequence of
213P1F11 v.6 is shown in Figure
3F; it has 242 amino acids. As used herein, a reference to 213PIF11 includes
all variants thereof, including
those shown in Figure 11.
Figure 4. The nucleic acid sequence alignment of 213P1F11 v.l with human
Caspase-14 (gi
6912286) precursor mRNA is shown in Figure 4A. The amino acid sequence
alignment of 213P1F11 v.l
with human Caspase-14 (gi 6912286) mRNA is shown in Figure 4B. The amino acid
sequence alignment of
213P1F11 v.l with mouse Caspase-14 (gi 6753280) mRNA is shown in Figure 4C.
The amino acid sequence
alignment of 213P1F11 v.2 with human Caspase-14 (gi 6912286) mRNA is shown in
Figure 4D. The amino
acid sequence alignment of 213P1F11 v.3 with human Caspase-14 (gi 6912286)
mRNA is shown in
Figure 4E. The amino acid sequence alignment of 213P1F11 v.2 with mouse
caspase 14 (gi 6753280) mRNA
is shown in Figure 4F. The amino acid sequence alignment of 213P1F11 v.4 with
human Caspase-14 (gi
6912286) mRNA is shown in Figure 4G.
Figure 5. Hydrophilicity amino acid profile of A) 213P1F11 variant l, B)
213P1F11 variant 2,
C) 213P1F11 variant 3 and D) 213P1F11 variant 4, 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 at the World Wide Web
(.expasy.ch/cgi-bin/protscale.pl)
through the ExPasy molecular biology server.
Figure 6. Hydropathicity amino acid profile of A) 213P1F11 variant 1, B)
213P1F11 variant 2,
C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer
algorithm sequence analysis
using the method of Kyte and Doolittle (Kyle J., Doolittle R.F., 1982. J. Mol.
Biol. 157:105-132) accessed on
the ProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExFasy
molecular biology server.
Figure 7. Percent accessible residues amino acid profile ofA) 213P1F11 variant
1, B) 213P1F11
variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by
computer algorithm sequence
analysis using the method of Janin (Janin J., 1979 Nature 277:491-492)
accessed on the ProtScale website
located at the World Wide Web (.expasy.ch/cgi-bin/protscale.pl) through the
ExPasy molecular biology
server.
Figure 8. Average flexibility amino acid profile of A) 213P1F11 variant 1, B)
213P1F11 variant 2,
C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer
algorithm sequence analysis
using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy
P.K., 1988. Int. J. Pept.
Protein Res. 32:242-255) accessed on the ProtScale website located at the
World Wide Web (.expasy.ch/cgi-
bin/protscale.pl) through the ExPasy molecular biology server.
Figure 9. Beta-turn amino acid profile of A) 213P1F11 variant 1, B) 213P1F11
variant 2,
C) 213P1F11 variant 3 and D) 213P1F11 variant 4, 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 at the World Wide Web (.expasy.ch/cgi-
bin/protscale.pl) through the ExPasy
molecular biology server.
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Figure 10. Schematic display ofnucleotide variants of 213P1F11. Variants
213P1F11 v.2 and v.3
are splice variants. Variant 213P1F11 v.4 is an alternative transcript. Others
are Single Nucleotide
Polymorphism (also called "SNP") variants, which could also occur in any of
the transcript variants that
contains the base pairs. Numbers in "( )" underneath the box correspond to
those of 213P1F11 v.l. The
black boxes show the same sequence as 213P1F11 v.l. SNPs are indicated above
the box.
Figure 11. Schematic display of protein variants of 213P1F11. Nucleotide
variants 213P1F11 v.l
though v.6 in Figure 10 code for protein variants 213P1F11 v.l through
213P1F11 v.6, respectively. Variants
213P 1 F 11 v.7 through v.10 code the same protein as variant 213P 1 F 11 v. l
. Protein variants 213P 1 F 11 v.5
and v.6 are variants with single amino acid variations, which may exist in
transcript variants 213P1F11 v.2
through 4. 'The black boxes show the same sequence as 213PIF11 v.l. The
numbers in "()" underneath the
box correspond to those of 213P1F11 v.l. Single amino acid differences are
indicated above the box.
Figure 12. Secondary structure prediction for 213P1F11 variants 1 through 4.
The secondary
structures of 213P1F11 variant 1 (SEQ ID NO: 3) (A), variant 2 (SEQ ID NO: 5)
(B), variant 3 (SEQ ID
N0:7) (C), and variant 4 (SEQ ID NO: 9) (D) were predicted using the HNN -
Hierarchical Neural Network
method (Guermeur, 1997, http://pbil.ibcp.fr/cgi-bin/npsa automat.pl?page=npsa
nn.html), accessed from the
ExPasy molecular biology server located at the Wo~ld Wide Web
(.expasy.ch/tools/). 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 secondary structure is also
listed for each variant.
Figure 13. Exon compositions of transcript variants of 213P1F11. Variant
213P1F11 v.2 and v.3
are splice variants. Variant 213P1F11 v.4 is an alternative transcript.
Compared with 213P1F11 v.l,
213P1F11 v.2 has a longer (+74 by at 5' end) exon 6 and variant 213P1F11 v.3
has a longer (+68 by at 5'
end) exon 5. Variant 213P1F11 v.4 has three different exons. Relative
locations of exons from all variants
on the chromosome are shown at the bottom. Numbers in "( )" underneath the box
correspond to those of
213P1F11 v.l. Black boxes show the same sequence as 213P1F11 v.l. Intron
lengths are not proportional.
Figure 14. Expression of 213P1F11 by RT-PCR. First strand cDNA was prepared
from vital pool 1
(liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), LAPC
xenograft pool (LAPC-4AD,
LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, breast cancer pool, and
cancer metastasis pool.
Normalization was performed by PCR using primers to actin and GAPDH. Semi-
quantitative PCR, using
primers to 213P1F11, was performed at 26 and 30 cycles of amplification.
Results show strong expression of
213P 1F 11 in bladder cancer pool, breast cancer pool, xenograft pool, and
cancer metastasis pool.
Figure 15. Expression of 213P1F11 v.l compared to 213P1F11 v.2 in patient
cancer samples by
RT-PCR. To determine the relative expression of 213P1F11 v.l compared to
213P1F11 v.2 in human
cancers, primers were designed flanking the insertion in 213P1F11 v.2. Using
these primers, amplification of
213P1F11 v.l will generate a PCR fragment of 165 bp, whereas 213P1F11 v.2 will
generate a PCR fragment
of 249 by as depicted in the figure. First strand cDNA was prepared from vital
pool 1 (liver, lung and
kidney), bladder cancer pool, breast cancer pool, LAPC xenograft pool (LAPC-
4AD, LAPC-4AI, LAPC-9AD
and LAPC-9AI), and 213P1F11 v.l plasmid control. Normalization was performed
by PCR using primers to
actin and GAPDH. Semi-quantitative PCR, using primers depicted above, was
performed at 35 cycles of
amplification. Results show strong expression of 213P1F11 v.lin bladder cancer
pool, breast cancer pool,
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LAPC xenograft pool, and the plasmid positive control. A lower expression of
the 249 by 213P1F11 v.2
product was detected in breast cancer pool, LAPC xenograft pool, and to lower
extent in bladder cancer pool.
Figure 16. Expression of 213P1F11 in normal tissues. Three multiple tissue
northern blots (A and
B, Clontech; C, OriGene) with 2 ug of mRNA/lane were probed with the 213P1F11
SSH fragment. Size
standards in kilobases (kb) are indicated on the side. Results show strong
expression of 213P1F11 only in
skin tissue. A weak transcript is detected in normal thymus but not in the
other tissues tested.
Figure 17. Expression of 213P1F11 in bladder cancer patient tissues. RNA was
extracted from
normal bladder (N), bladder cancer cell lines (UM-UC-3 and SCaBER), bladder
cancer patient tumors (T) and
normal tissue adjacent to bladder cancer (NAT). Northern blots with 10 ug of
total RNA were probed with the
213P1F11 SSH fragment. Size standards in kilobases are indicated on the side.
Results show strong
expression of 213P1F11 in the bladder tumor tissues but not in normal bladder
nor in the bladder cancer cell
lines.
Figure 18. Expression of 213P1F11 in prostate cancer xenografts. RNA was
extracted from normal
prostate, LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI prostate cancer
xenografts. Northern blot with
ltg of total RNA/lane was probed with 213P1F11 SSH sequence. Size standards in
kilobases (kb) are
indicated on the side. The results show expression of 213P 1F 11 in the LAPC-
9AI xenograft, but not in the
other xenografts nor in normal prostate.
Figure 19. Expression of 213P1F11 in breast cancer patient tissues. RNA was
extracted from
normal breast (N), breast cancer cell lines (DU4475, MCF7 and CAMA-1), breast
cancer patient tumors (T)
and breast cancer metastasis to lymph node (Met). Northern blots with 10 ug of
total RNA were probed with
the 213P1F11 SSH fragment. Size standards in kilobases are indicated on the
side. Results show strong
expression of 213P1F11 in the breast tumor tissues as well as in the cancer
metastasis specimen. Weak
expression was also detected in the CAMA-1 cell line, but not in the other 2
breast cancer cell lines tested.
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
L) Definitions
IL) 213P1F11 Polynucleotides
ILA.) Uses of 213P1F11 Polynucleotides
ILA.1.) Monitoring of Genetic Abnormalities
ILA.2.) Antisense Embodiments
ILA.3.) Primers and Primer Pairs
ILA.4.) Isolation of 213P1F11-Encoding Nucleic Acid Molecules
ILA.S.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
IIL) 213P1F11-related Proteins
IILA.) Motif bearing Protein Embodiments
IILB.) Expression of 213P1F11-related Proteins
IILC.) Modifications of 213P1F11-related Proteins
IILD.) Uses of 213P1F11-related Proteins
IV.) 213P1F11 Antibodies
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V.) ~213P1F11 Cellular Immune Responses
VL) 213P1F11 Transgenic Animals
VIL) Methods for the Detection of 213P1F11
VIIL) Methods for Monitoring the Status of 213P1F11-related Genes and Their
Products
IX.) Identification of Molecules That Interact With 213P1Fi1
X.) Therapeutic Methods and Compositions
X.A.) Anti-Cancer Vaccines
X.B.) 213P1F11 as a Target for Antibody-Based Therapy
X.C.) 213P1F11 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 213P1F11.
XIL) Inhibition of 213P1F11 Protein Function '
XILA.) Inhibition of 213P1F11 With Intracellular Antibodies
XILB.) Inhibition of 213P1F11 with Recombinant Proteins
XILC.) Inhibition of 213P1Fi1 Transcription or Translation
XILD.) General Considerations for Therapeutic Strategies
XIIL) KITS
L) 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 eonstrued to
represent a substantial difference over what is generally understood in the
art. Many of the techniques and
procedures described or referenced herein are well understood and commonly
employed using conventional
methodology by those skilled in the art, such as, for example, the widely
utilized molecular cloning
methodologies described in Sambrook et al., Molecular Cloning: A Laboratory
Manual 2nd. edition ( 1989)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,
procedures involving the
use of commercially available kits and reagents are generally carned out in
accordance with manufacturer
defined protocols and/or parameters unless otherwise noted.
The terms "advanced prostate cancer", "locally advanced prostate cancer",
"advanced disease" and
"locally advanced disease" mean prostate cancers that have extended through
the prostate capsule, and are
meant to include stage C disease under the American Urological Association
(AUA) system, stage C1 - C2
disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease
under the TNM (tumor, node,
metastasis) system. In general, surgery is not recommended for patients with
locally advanced disease, and
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these patients have substantially less favorable outcomes compared to patients
having clinically localized
(organ-confined) prostate cancer. Locally advanced disease is clinically
identified by palpable evidence of
induration beyond the lateral border of the prostate, or asymmetry or
induration above the prostate base.
Locally advanced prostate cancer is presently diagnosed pathologically
following radical prostatectomy if the
tumor invades or penetrates the prostatic capsule, extends into the surgical
margin, or invades the seminal
vesicles.
"Altering the native glycosylation pattern" is intended for purposes herein to
mean deleting one or
more carbohydrate moieties found in native sequence 213P1F11 (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
213P1F11. 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 213P1F11-related protein). For
example an analog of a 213P1F11 protein
can be specifically bound by an antibody or T cell that specifically binds to
213P1F11.
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-213P1F11
antibodies comprise monoclonal and polyclonal antibodies as well as fragments
containing the antigen-binding
domain and/or one or more complementarity determining regions of these
antibodies.
An "antibody fragment" is defined as at least a portion of the variable region
of the immunoglobulin
molecule that binds to its target, i.e., the antigen-binding region. In one
embodiment it specifically covers
single anti-213P1F11 antibodies and clones thereof (including agonist,
antagonist and neutralizing antibodies) and
anti-213P1F11 antibody compositions withpolyepitopic 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
andlor optimization of GC content in addition to codon optimization are
referred to herein as an "expression
enhanced sequences."
The term "cytotoxic agent" refers to a substance that inhibits or prevents the
expression activity of
cells, function of cells and/or causes destruction of cells. The term is
intended to include radioactive isotopes
chemotherapeutic agents, and toxins such as small molecule toxins or
enzymatically active toxins of bacterial,
fungal, plant or animal origin, including fragments and/or variants thereof.
Examples of cytotoxic agents
include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin
A-chain, doxorubicin,
daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicine,
dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin
(PE) A, PE40, abrin, abrin
A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin,
phenomycin, enomycin, curicin,
crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid
and other chemotherapeutic agents,
as well as radioisotopes such as Atzl~, llsy Il2s, Y9o, Re~86, Re188, Sm~53,
Bizlz, Psz and radioactive isotopes of
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Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating
enzyme capable of converting
the pro-drug to its active form.
The term "homolog" refers to a molecule which exhibits homology to another
molecule, by for example,
having sequences of chemical residues that are the same or similar~at
corresponding positions.
"Human Leukocyte Antigen" or "HLA" is a human class I or class II Major
Histocompatibility
Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8T" 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% SDS1100 pg/ml ssDNA, in which temperatures for
hybridization are above
37 degrees C and temperatures for washing in O.1XSSC/0.1% SDS are above 55
degrees C.
The phrases "isolated" or "biologically pure" refer to material which is
substantially or essentially
free from components which normally accompany the material as it is found in
its native state. Thus, isolated
peptides in accordance with the invention preferably do not contain materials
normally associated with the
peptides in their in situ environment. For example, a polynucleotide is said
to be "isolated" when it is
substantially separated from contaminant polynucleotides that correspond or
are complementary to genes other
than the 213P1F11 genes or that encode polypeptides other than 213P1F11 gene
product or fragments thereof. A
skilled artisan can readily employ nucleic acid isolation procedures to obtain
an isolated 213P1F11
polynucleotide. A protein is said to be "isolated," for example, when
physical, mechanical or chemical methods
are employed to remove the 213P1F11 proteins from cellular constituents that
are normally associated with the
protein. A skilled artisan can readily employ standard purification methods to
obtain an isolated 213P 1F 11
protein. Alternatively, an isolated protein can be prepared by chemical means.
The term "mammal" refers to any organism classified as a mammal, including
mice, rats, rabbits, dogs,
cats, cows, horses and humans. In one embodiment of the invention, the mammal
is a mouse. In another
embodiment of the invention, the mammal is a human.
The terms "metastatic prostate cancer" and "metastatic disease" mean prostate
cancers that have
spread to regional lymph nodes or to distant sites, and are meant to include
stage D disease under the AUA
system and stage TxNxM+ under the TNM system. As is the case with locally
advanced prostate cancer,
surgery is generally not indicated for patients with metastatic disease, and
hormonal (androgen ablation)
therapy is a preferred treatment modality. Patients with metastatic prostate
cancer eventually develop an
androgen-refractory state within 12 to 18 months of treatment initiation.
Approximately half of these
androgen-refractory patients die within 6 months after developing that status.
The most common site for
prostate cancer metastasis is bone. Prostate cancer bone metastases are often
osteoblastic rather than
osteolytic (i.e., resulting in net bone formation). Bone metastases are found
most frequently in the spine,
followed by the femur, pelvis, rib cage, skull and humerus. Other common sites
for metastasis include lymph
nodes, lung, liver and brain. Metastatic prostate cancer is typically
diagnosed by open or laparoscopic pelvic
lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or
bone lesion biopsy.
The term "monoclonal antibody" refers to an antibody obtained from a
population of substantially
homogeneous antibodies, i.e., the antibodies comprising the population are
identical except for possible naturally
occurring mutations that are present in minor amounts.
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A "motif', as in biological motif of a 213P1F11-related protein, refers to any
pattern of amino acids
forming part of the primary sequence of a protein, that is associated with a
particular function (e.g. protein-
protein interaction, protein-DNA interaction, etc) or modification (e.g. that
is phosphorylated, glycosylated or
amidated), or localization (e.g. secretory sequence, nuclear localization
sequence, etc.) or a sequence that is
correlated with being immunogenic, either humorally or cellularly. A motif can
be either contiguous or
capable of being aligned to certain positions that are generally correlated
with a certain function or property.
In the context of HLA motifs, "motif' refers to the pattern of residues in a
peptide of defined length, usually a
peptide of from about 8 to about 13 amino acids for a class I HLA motif and
from about 6 to about 25 amino
acids for a class II HLA motif, which is recognized by a particular HLA
molecule. Peptide motifs for HLA
binding are typically different for each protein encoded by each human HLA
allele and differ in the pattern of
the primary and secondary anchor residues.
A "pharmaceutical excipient" comprises a material such as an adjuvant, a
Garner, pH-adjusting and
buffering agents, tonicity adjusting agents, wetting agents, preservative, and
the like.
"Pharmaceutically acceptable" refers to a non-toxic, inert, and/or composition
that is physiologically
compatible with humans or other mammals.
The term "polynucleotide" means a polymeric form of nucleotides of at least 10
bases or base pairs
in length, either ribonucleotides or deoxynucleotides or a modified form of
either type of nucleotide, and is
meant to include single and double stranded forms of DNA and/or RNA. In the
art, this term if often used
interchangeably with "oligonucleotide". A polynucleotide can comprise a
nucleotide sequence disclosed
herein wherein thymidine (T), as shown for example in Figure 2, can also be
uracil (IJ); 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 (II) instead of thymidine
(T).
The term "polypeptide" means a polymer of at least about 4, 5, 6, 7, or 8
amino acids. Throughout
the specification, standard three letter or single letter designations for
amino acids are used. In the art, this
term is often used interchangeably with "peptide" or "protein".
An HLA "primary anchor residue" is an amino acid at a specific position along
a peptide sequence
which is understood to provide a contact point between the immunogenic peptide
and the HLA molecule.
One to three, usually two, primary anchor residues within a peptide of defined
length generally defines a
"motif' for an immunogenic peptide. These residues are understood to fit in
close contact with peptide
binding groove of an HLA molecule, with their side chains buried in specific
pockets of the binding groove.
In one embodiment, for example, the primary anchor residues for an HLA class I
molecule are located at
position 2 (from the amino terminal position) and at the carboxyl terminal
position of a 8, 9, 10, 11, or 12
residue peptide epitope in accordance with the invention. In another
embodiment, for example, the primary
anchor residues of a peptide that will bind an HLA class II molecule are
spaced relative to each other, rather
than to .the termini of a peptide, where the peptide is generally of at least
9 amino acids in length. The
primary anchor positions for each motif and supermotif are set forth in Table
IV. For example, analog
peptides can be created by altering the presence or absence of particular
residues in the primary and/or
secondary anchor positions shown in Table IV. Such analogs are used to
modulate the binding affinity and/or
population coverage of a peptide comprising a particular HLA motif or
supermotif.
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A "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been
subjected to
molecular manipulation in vitro.
Non-limiting examples of small molecules include compounds that bind or
interact with 213P1F11,
ligands including hormones, neuropeptides, chemokines, odorants,
phospholipids, and functional equivalents
thereof that bind and preferably inhibit 213P1F11 protein function. Such non-
limiting small molecules
preferably have a molecular weight of less than about 10 kDa, more preferably
below about 9, about 8, about
7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules
physically associate with, or
bind, 213P1F11 protein; are not found in naturally occurring metabolic
pathways; and/or are more soluble in
aqueous than non-aqueous solutions
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art,
and generally is an empirical calculation dependent upon probe length, washing
temperature, and salt
concentration. In general, longer probes require higher temperatures for
proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on the ability
of denatured nucleic acid
sequences to reanneal when complementary strands are present in an environment
below.their melting
temperature. The higher the degree of desired homology between the probe and
hybridizable sequence, the
higher the relative temperature that can be used. As a result, it follows that
higher relative.temperatures
would tend to make the reaction conditions more stringent, while lower
temperatures less so. For additional
details and explanation of stringency of hybridization reactions, see Ausubel
et aZ., 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% Ficoll/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, sonicated salmon sperm DNA (50 ltg/ml), 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 overnight incubation at 37°C in a
solution comprising: 20% formamide, 5 x
SSC ( 150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),
5 x Denhardt's solution,
10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed
by washing the filters in
1 x SSC at about 37-50°C. The skilled artisan will recognize how to
adjust the temperature, ionic strength,
etc. as necessary to accommodate factors such as probe length and the like.
An HLA "supermotif ' is a peptide binding specificity shared by HLA molecules
encoded by two or
more HLA alleles.
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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-
242 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, 150, 155,
160, 165, 170, 175, 180, 185, 190,
195, 200, 225, or 242 or more peptides .of the invention. The peptides or
polypeptides can optionally be
modified, such as by lipidation, addition of targeting or other sequences. HLA
class I peptides of the
invention can be admixed with, or linked to, HLA class II peptides, to
facilitate activation of both cytotoxic T
lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-
pulsed antigen presenting
cells, e.g., dendritic cells.
The term "variant" refers to a molecule that exhibits a variation from a
described type or norm, such as a
protein that has one or more different amino acid residues in the
corresponding positions) of a specifically
described protein (e.g. the 213P 1F 11 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 "213P1F11-related proteins" of the invention include those specifically
identified herein, as well as
allelic variants, conservative substitution variants, analogs and homologs
that can be isolated/generated and
characterized without undue experimentation following the methods outlined
herein or readily available in the art.
Fusion proteins that combine parts of different 213P1F11 proteins or fragments
thereof, as well as fusion proteins
of a 213P1F11 protein and a heterologous polypeptide are also included. Such
213P1F11 proteins are collectively
referred to as the 213P1F11-related proteins, the proteins of the invention,
or 213P1F11. The term "213P1F11-
related protein" refers to a polypeptide fragment or a 213P 1F 11 protein
sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids;
or, at least 30, 35, 40, 45, 50, 55, 60,
65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180,
185, 190, 195, 200, 225, or 242 or more amino acids.
IL) 213P1F11 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or
complementary to all or part
of a 213P 1F11 gene, mRNA, and/or coding sequence, preferably in isolated
fom~, including polynucleotides
encoding a 213P1F11-related protein and fragments thereof, DNA, RNA, DNA/RNA
hybrid, and related
molecules, polynucleotides or oligonucleotides complementary to a 213P1F11
gene or mRNA sequence or a
part thereof, and polynucleotides or oligonucleotides that hybridize to a
213P1F11 gene, mRNA, or to a
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213P1F11 encoding polynucleotide (collectively, "213P1F11 polynucleotides").
In all instances when
referred to in this section, T can also be U in Figure 2.
Embodiments of a 213P1F11 polynucleotide include: a 213P1F11 polynucleotide
having the
sequence shown in Figure 2, the nucleotide sequence of 213P1F11 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 of213P1F11 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 2 , from nucleotide residue number 404 through nucleotide
residue number 1132,
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 404 through nucleotide
residue number 1096,
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 404 through nucleotide
residue number 844,
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 1 through nucleotide
residue number 966,
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 404 through nucleotide
residue number 1132,
including the stop codon, wherein T can also be U;
(VII) a polynucleotide comprising, consisting essentially of, or consisting of
the sequence as
shown in Figure 2F , from nucleotide residue number 404 through nucleotide
residue number 1132,
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 404 through nucleotide
residue number 1132,
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 404 through nucleotide
residue number 1132,
including the stop codon, wherein T can also be U;
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(X) -a polynucleotide that encodes a 213P1F11-related protein that is at least
90% homologous
to an entire amino acid sequence shown in Figure 2A-H ;
(XI) a polynucleotide that encodes a 213P1F11-related protein that is at least
90% identical to an
entire amino acid sequence shown in Figure 2A-H ;
(XII) a polynucleotide that encodes at least one peptide set forth in Tables V-
XIX;
(XIII) a polynucleotide that encodes a peptide region of at least 5 amino
acids of a peptide of
Figure 3A in any whole number increment up to 242 that includes an amino acid
position having a
value greater than 0.5 in the Hydrophilicity profile of Figure SA; or of
Figure 3B in any whole
number increment up to 230 that includes an amino acid position having a value
greater than 0.5 in
the Hydrophilicity profile of Figure SB; or of Figure 3C in any whole number
increment up to 146
that includes an amino acid position having a value greater than 0.5 in the
Hydrophilicity profile of
Figure SC; or of Figure 3D in any whole number increment up to 321 that
includes an amino acid
position having a value greater than 0.5 in the Hydrophilicity profile of
Figure SD;
(XN) a polynucleotide that encodes a peptide region of at least 5 amino acids
of a peptide of '
Figure 3A in any whole number increment up to 242 that includes an amino acid
position having a
~Vy"
value less than 0.5 in the Hydropathicity profile of Figure 6A; or of Figure
3B in any whole number
increment up to 230 that includes an amino acid position having a value less
than 0.5 in the
Hydropathicity profile of Figure 6B; or of Figure 3G in any whole number
increment up to 146 that
includes an amino acid position having a value less than 0.5 in the
Hydropathicity profile of Figure
6C; or of Figure 3D in any whole number increment up to 321 that includes an
amino acid position
having a value less than 0.5 in the Hydropathicity profile of Figure 6D;
(XV) a polynucleotide that encodes a peptide region of at least 5 amino acids
of a peptide of
Figure 3A in any whole number increment up to 242 that includes an amino acid
position having a
value greater than 0.5 in the Percent Accessible Residues profile of Figure
7A; or of Figure 3B in
any whole number increment up to 230 that includes an amino acid position
having a value greater
than 0.5 in the Percent Accessible Residues profile of Figure 7B; or of Figure
3C in any whole
number increment up to 146 that includes an amino acid position having a value
greater than 0.5 in
the Percent Accessible Residues profile of Figure 7C; or of Figure 3D in any
whole number
increment up to 321 that includes an amino acid position having a value
greater than 0.5 in the
Percent Accessible Residues profile of Figure 7D;
(XVI) a polynucleotide that encodes a peptide region of at least 5 amino acids
of a peptide of
Figure 3A in any whole number increment up to 242 that includes an amino acid
position having a
value greater than 0.5 in the Average Flexibility profile of Figure 8A; or of
Figure 3B in any whole
number increment up to 230 that includes an amino acid position having a value
greater than 0.5 in
the Average Flexibility profile of Figure 8B; or of Figure 3C in any whole
number increment up to
146 that includes an amino acid position having a value greater than 0.5 in
the Average Flexibility
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profile of FigureBC; or of Figure 3D in any whole number increment up to 321
that includes an
amino acid position having a value greater than 0.5 in the Average Flexibility
profile of Figure 8D;
(XVII) a polynucleotide that encodes a peptide region of at least 5 amino
acids of a peptide of
Figure 3A in any whole number increment up to 242 that includes an amino acid
position having a
value greater than 0.5 in the Beta-turn profile of Figure 9A; or of Figure 3B
in any whole number
increment up to 230 that includes an amino acid position having a value
greater than 0.5 in the Beta-
turn profile of Figure 9B; or of Figure 3C in any whole number increment up to
I46 that includes an
amino acid position having a value greater than 0.5 in the Beta-turn profile
of Figure9C; or of Figure
3D in any whole number increment up to 321 that includes an amino acid
position having a value
greater than 0.5 in the Beta-turn profile of Figure 9D;
(XVIII) a polynucleotide that is fully complementary to a polynucleotide of
any one of (I)-(XVII).
(XIX) a peptide that is encoded by any of (I)-(XVIII); and
(XXI) a polynucleotide of any of (I)-(XVIII) or peptide of (XIX) together with
a pharmaceutical
excipient and/or in a human unit dose form.
As used herein, a range is understood to specifically disclose all whole unit
positions thereof.
Typical embodiments of the invention disclosed herein include 213P1F11
polynucleotides that
encode specific portions of 213PIF11 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, 230, 235, 240, or 242 or more contiguous amino
acids of 213P1F11.
(b) 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, I40,
145, 150, 155, 160, 165, 170, 175,
180, 185, 190, 195, 200, 205, 210, 215, 220, 225, or 230 contiguous amino
acids of variant 2;
(c) 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, or 146 contiguous amino
acids of variant 3; or
(d) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2I, 22, 23,
24, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, I15, 120, 125, 130, 135, 140,
145, or 146 150, 155, 160, 165,
170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,
245, 250, 255, 260, 265, 270, 275,
280, 285, 290, 295, 300, 305, 310, 3I5, 320, or 321 contiguous amino acids of
variant 4.
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 213P1F11
protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino
acid 10 to about amino acid 20
of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides
encoding about amino acid 20 to
about amino acid 30 of the 213P1F11 protein shown in Figure 2 or Figure 3,
polynucleotides encoding about
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amino acid 30 to about amino acid 40 of the 213P1F11 protein shown in Figure 2
or Figure 3, polynucleotides
encoding about amino acid 40 to about amino acid 50 of the 213P1F11 protein
shown in Figure 2 or Figure 3,
polynucleotides encoding about amino acid 50 to about amino acid 60 of the
213P1F11 protein shown in
Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about
amino acid 70 of the 213P1F11
protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino
acid 70 to about amino acid 80
of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides
encoding about amino acid 80 to
about amino acid 90 of the 213P 1F11 protein shown in Figure 2 or Figure 3,
polynucleotides encoding about
amino acid 90 to about amino acid 100 of the 213P1F11 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 213P1F11
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 213P1F11 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 213P1F11 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 213P 1F11 sequence as shown in Figure 2.
Additional illustrative embodiments of the invention disclosed herein include
213P1F11
polynucleotide fragments encoding one or more of the biological motifs
contained within a 213P1F11 protein
"or variant" sequence, including one or more of the motif bearing subsequences
of a 213P1F11 protein "or
variant" set forth in Tables V-XIX.
Note that to determine the starting position of any peptide set forth in
Tables V-XIX (collectively
HLA Peptide Tables) respective to its parental protein, e.g., variant l,
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 XXIX. 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 XXIX. Accordingly if a Search Peptide begins at position "X", one
must add the value "X - 1" to
each position in Tables V-XIX 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 is
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 V-
XIX 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 V-XVIII and at
least once in table XIX, or an
oligonucleotide that encodes the HLA peptide.
Another embodiment of the invention is antibody epitopes which comprise a
peptide regions, or an
oligonucleotide encoding the peptide region, that has one two, three, four, or
five of the following
characteristics:
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i) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole number
increment up to the full length of that protein in Figure 3, that includes an
amino acid position having a value
equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to
1.0, in the Hydrophilicity profile of
Figure 5;
ii) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole number
increment up to the full length of that protein in Figure 3, that includes an
amino acid position having a value
equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0,
in the Hydropathicity profile of
Figure 6;
iii) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole
number increment up to the full length of that protein in Figure 3, that
includes an amino acid position having
a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value
equal to 1.0, in the Percent Accessible
Residues profile of Figure 7;
iv) a peptide region of at least 5 amino acids of a particular peptide of
Figure 3, in any whole
number increment up to the full length of that protein in Figure 3, that
includes an amino acid position having
a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value
equal to 1.0, in the Average Flexibility
profile of Figure 8; or
v) a peptide region of at least 5 amino acids of a particular peptide of
Figure f, 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.
In another embodiment, typical polynucleotide fragments of the invention
encode one or more of the
regions of 213P1F11 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 213P1F11 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.
ILA.) Uses of 213P1F11 Polynucleotides
ILA.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different
specific uses. The
human 213P1F11 gene maps to the chromosomal location set forth in the Example
entitled "Chromosomal
Mapping of 213P1F11." For example, because the 213P1F11 gene maps to this
chromosome,
polynucleotides that encode different regions of the 213P 1F11 proteins are
used to characterize cytogenetic
abnormalities of this chromosomal locale, such as abnormalities that are
identified as being associated with
various cancers. In certain genes, a variety of chromosomal abnormalities
including rearrangements have
been identified as frequent cytogenetic abnormalities in a number of different
cancers (see e.g. Krajinovic et
al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-
3914 (1995) and Finger et al.,
P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific
regions of the 213P1F11
proteins provide new tools that can be used to delineate, with greater
precision than previously possible,
cytogenetic abnormalities in the chromosomal region that encodes 213P1F11 that
may contribute to the
malignant phenotype. In this context, these polynucleotides satisfy a need in
the art for expanding the
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sensitivity of chromosomal screening in order to identify more subtle and less
common chromosomal
abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057
(1994)).
Furthermore, as 213P1F11 was shown to be highly expressed in bladder and other
cancers,
213P1F11 polynucleotides are used in methods assessing the status of 213P1F11
gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode specific
regions of the 213P1F11 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 213P1F11
gene, such as regions containing one
or more motifs. Exemplary assays include both RT-PCR assays as well as single-
strand conformation
polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol.
26(8): 369-378 (1999), both of
which utilize polynucleotides encoding specific regions of a protein to
examine these regions within the
protein.
ILA.2.) Antisense Embodiments
Other specifically contemplated nucleic acid related embodiments of the
invention disclosed herein are
genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic
acid molecules based on an
alternative backbone, or including alternative bases, whether derived from
natural sources or synthesized, and
include molecules capable of inhibiting the RNA or protein expression of
213P1F11. 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 213P1F11
polynucleotides and polynucleotide sequences disclosed herein.
Antisense technology entails the administration of exogenous oligonucleotides
that bind to a target
polynucleotide located within the cells. The term "antisense" refers to the
fact that such oligonucleotides are
complementary to their intracellular targets, e.g., 213P1F11. See for example,
Jack Cohen,
Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press,
1989; and Synthesis 1:1-5
(1988). The 213P1F11 antisense oligonucleotides of the present invention
include derivatives such as S-
oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen,
supra), which exhibit enhanced
cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates)
are isoelectronic analogs of an
oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate
group is replaced by a sulfur
atom. The S-oligos of the present invention can be prepared by treatment of
the corresponding O-oligos with
3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent.
See, e.g., Iyer, R. P. et al., J. Org.
Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-
1254 (1990). Additional
213P1F11 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 213P1F11 antisense oligonucleotides of the present invention typically can
be RNA or DNA
that is complementary to and stably hybridizes with the first 100 S' codons or
last 100 3' codons of a
213P1F11 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 213P1F11 mRNA and not to mRNA
specifying other
regulatory subunits of protein kinase. In one embodiment, 213P1F11 antisense
oligonucleotides of the
present invention are 15 to 30-mer fragments of the antisense DNA molecule
that have a sequence that
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hybridizes to 213P1F11 mRNA. Optionally, 213P1F11 antisense oligonucleotide is
a 30-mer oligonucleotide
that is complementary to a region in the first 10 5' codons or last 10 3'
codons of 213P1F11. Alternatively,
the antisense molecules are modified to employ ribozymes in the inhibition of
213P 1 F 11 expression, see, e.g.,
L. A. Couture & D. T. Stinchcomb; Treads Genet 12: 510-S 15 ( 1996).
ILA.3.) Primers and Primer Pairs
Further specific embodiments of this nucleotides of the invention include
primers and primer pairs,
which allow the specific amplification of polynucleotides of the invention or
of any specific parts thereof, and
probes that selectively or specifically hybridize to nucleic acid molecules of
the invention or to any part
thereof. Probes can be labeled with a detectable marker, such as, for example,
a radioisotope, fluorescent
compound, bioluminescent compound, a chemiluminescent compound, metal chelator
or enzyme. Such
probes and primers are used to detect the presence of a 213P1F11
polynucleotide in a sample and as a means for
detecting a cell expressing a 213P1F11 protein.
Examples of such probes include polypeptides comprising all or part of the
human 213P1F11 cDNA
sequence shown in Figure 2. Examples of primer pairs capable of specifically
amplifying 213P1F11 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
213P1F11 mRNA.
The 213P1F11 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 213P1F11 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 213P1F11
polypeptides; as tools for
modulating or inhibiting the expression of the 213P1F11 genes) and/or
translation of the 213P1F11
transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to
identify and isolate a
213P1F11 or 213P1F11 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.
ILA.4.) Isolation of 213P1F11-Encoding Nucleic Acid Molecules
The 213P1F11 cDNA sequences described herein enable the isolation of other
polynucleotides encoding
213P1F11 gene product(s), as well as the isolation of polynucleotides encoding
213P1F11 gene product
homologs, alternatively spliced isoforms, allelic variants, and mutant forms
of a 213P1F11 gene product as well
as polynucleotides that encode analogs of 213P 1F 11-related proteins. Various
molecular cloning methods that
can be employed to isolate full length cDNAs encoding a 213P1F11 gene are well
known (see, for example,
Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold
Spring Harbor Press, New York,
1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Whey 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
213P1F11 gene cDNAs can be
identified by probing with a labeled 213P 1F 11 cDNA or a fragment thereof.
For example, in one embodiment, a
213P1F11 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 213P1F11 gene. A 213P1F11
gene itself can be isolated
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by screening genomic DNA libraries, bacterial artificial chromosome libraries
(BACs), yeast artificial
chromosome libraries (YACs), and the like, with 213P 1 F 11 DNA probes or
primers.
ILA.S.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
The invention also provides recombinant DNA or RNA molecules containing a 213P
1F11
polynucleotide, a fragment, analog or homologue thereof, including but not
limited to phages, plasmids,
phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors
well known in the art, and cells
transformed or transfected with such recombinant DNA or RNA molecules. Methods
for generating such
molecules are well known (see, for example, Sambrook et al., 1989, supra).
The invention further provides a host-vector system comprising a recombinant
DNA molecule
containing a 213P1F11 polynucleotide, fragment, analog or homologue thereof
within a suitable prokaryotic or
eukaryotic host cell. Examples of suitable eukaryotic host cells include a
yeast cell, a plant cell, or an animal
cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-
infectible cell such as an Sf9 or HighFive
cell). Examples of suitable mammalian cells include various prostate cancer
cell lines such as DU145 and
TsuPrl, other transfectable or transducible prostate cancer cell lines,
primary cells (PrEC), as well as a
number of mammalian cells routinely used for the expression of recombinant
proteins (e.g., COS, CHO, 293,
293T cells). More particularly, a polynucleotide comprising the coding
sequence of 213P1F11 or a fragment,
analog or homolog thereof can be used to generate 213P1F11 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 213P 1F 11
proteins or fragments
thereof are available, see for example, Sambrook et al., 1989, supra; Current
Protocols in Molecular Biology,
1995, supra). Preferred vectors for mammalian expression include but are not
limited to pcDNA 3.1 myc-His-
tag (Invitrogen) and the retroviral vector pSRatkneo (Muller et al., 1991, MCB
11:1785). Using these
expression vectors, 213P1F11 can be expressed in several prostate cancer and
non-prostate cell lines,
including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl. The host-vector
systems of the invention are
useful for the production of a 213P1F11 protein or fragment thereof. Such host-
vector systems can be
employed to study the functional properties of 213P1F11 and 213P1F11 mutations
or analogs.
Recombinant human 213P1F11 protein or an analog or homolog or fragment thereof
can be
produced by mammalian cells transfected with a construct encoding a 213P1F11-
related nucleotide. For
example, 293T cells can be transfected with an expression plasmid encoding
213P1F11 or fragment, analog
or homolog thereof, a 213P1F11-related protein is expressed in the 293T cells,
and the recombinant
213P 1F11 protein is isolated using standard purification methods (e.g.,
affinity purification using anti-
213P1F11 antibodies). In another embodiment, a 213P1F11 coding sequence is
subcloned into the retroviral
vector pSRaMSVtkneo and used to infect various mammalian cell lines, such as
NIH 3T3, TsuPrl, 293 and
rat-1 in order to establish 213P1F11 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 213P1F11
coding sequence can be used for the generation of a secreted form of
recombinant 213P1F11 protein.
As discussed herein, redundancy in the genetic code permits variation in
213P1F11 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
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sequences of the desired host) replaced with higher frequency codons. Codon
preferences for a specific
species are calculated, for example, by utilizing codon usage tables available
on the INTERNET such as at
URL located at the World Wide Web .dna.affrc.go.jp/~nakamura/codon.html.
Additional sequence modifications are known to enhance protein expression in a
cellular host.
These include elimination of sequences encoding spurious polyadenylation
signals, exon/intron splice site
signals, transposon-like repeats, and/or other such well-characterized
sequences that are deleterious to gene
expression. The GC content of the sequence is adjusted to levels average for a
given cellular host, as
calculated by reference to known genes expressed in the host cell. Where
possible, the sequence is modified
to avoid predicted hairpin secondary mRNA structures. Other useful
modifications include the addition of a
translational initiation consensus sequence at the start of the open reading
frame, as described in Kozak, tYlol.
Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general
rule that eukaryotic ribosomes
initiate translation exclusively at the 5' proximal AUG codon is abrogated
only under rare conditions (see,
e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148
(1987)).
IIL) 213P1F11-related Proteins
Another aspect of the present invention provides 213P1F11-related proteins.
Specific embodiments
of 213P1F11 proteins comprise a polypeptide having all or part of the amino
acid sequence of human
213P 1 F 11 as shown in Figure 2 or Figure 3. Alternatively, embodiments of
213P 1F 11 proteins comprise
variant, homolog or analog polypeptides that have alterations in the amino
acid sequence of 213P1F11 shown
in Figure 2 or Figure 3.
In general, naturally occurring allelic variants of human 213P 1F11 share a
high degree of structural
identity and homology (e.g., 90% or more homology). Typically, allelic
variants of a 213P1F11 protein contain
conservative amino acid substitutions within the 213P1F11 sequences described
herein or contain a substitution of
an amino acid from a corresponding position in a homologue of 213P1F11. One
class of 213P1F11 allelic
variants are proteins that share a high degree of homology with at least a
small region of a particular 213P1F11
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, ortliology
and paralogy can be important concepts describing the relationship of members
of a given protein family in one
organism to the members of the same family in other organisms.
Amino acid abbreviations are provided in Table II. Conservative amino acid
substitutions can
frequently be made in a protein without altering either'the conformation or
the function of the protein.
Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15 conservative substitutions.
Such changes include substituting any of isoleucine (I), valine (V), and
leucine (L) for any other of these
hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice
versa; glutamine (Q) for asparagine
(N) and vice versa; and serine (S) for threonine (T) and vice versa. Other
substitutions can also be considered
conservative, depending on the environment of the particular amino acid and
its role in the three-dimensional
structure of the protein. For example, glycine (G) and alanine (A) can
frequently be interchangeable, as can
alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic,
can frequently be interchanged
with leucine and isoleucine, and sometimes with valine. Lysine (K) and
arginine (R) are frequently
24
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interchangeable in locations in which the significant feature of the amino
acid residue is its charge and the
differing pIC's of these two amino acid residues are not significant. Still
other changes can be considered
"conservative" in particular environments (see, e.g. Table III herein; pages
13-15 "Biochemistry" 2"d ED.
Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89
10915-10919; Lei et al., J Biol
Chem 1995 May 19; 270(20):11882-6).
Embodiments of the invention disclosed herein include a wide variety of art-
accepted variants or
analogs of 213P1F11 proteins such as polypeptides having amino acid
insertions, deletions and substitutions.
213P1F11 variants can be made using methods known in the art such as site-
directed mutagenesis, alanine
scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl.
Acids Res., 13:4331 (1986);
Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells
et al., Gene, 34:315 (1985)),
restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London
SerA, 317:415 (1986)) or other
known techniques can be performed on the cloned DNA to produce the 213P1F11
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, 213P1F11 variants, analogs or homologs, have the
distinguishing attribute of
having at least one epitope that is "cross reactive" with a 213P1F11 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 213P1F11 variant also specifically binds to a 213P1F11 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 213P 1F11
protein. Those skilled in the art understand that antibodies that recognize
proteins bind to epitopes of varying
size, and a grouping of the order of about four or five amino acids,
contiguous or not, is regarded as a typical
number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol
2000 165(12): 6949-6955;
Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol
(1985) 135(4):2598-608.
Other classes of 213P1F11-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
213P1F11 protein variants or analogs comprise one or more of the 213P1F11
biological motifs described
herein or presently known in the art. Thus, encompassed by the present
invention are analogs of 213P1F11
fragments (nucleic or amino acid) that have altered functional (e.g.
immunogenic) properties relative to the
starting fragment. It is to be appreciated that motifs now or which become
part of the art are to be applied to
the nucleic or amino acid sequences of Figure 2 or Figure 3.
As discussed herein, embodiments of the claimed invention include polypeptides
containing less
than the full amino acid sequence of a 213P1F11 protein shown in Figure 2 or
Figure 3. For example,
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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 213P 1 F I 1 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 213P1F11 protein
shown in Figure 2 or Figure 3,
polypeptides consisting of about amino acid 10 to about amino acid 20 of a
213P1F11 protein shown in
Figure 2 or Figure 3, polypeptides consisting of about amino acid 20 to about
amino acid 30 of a 213P1F11
protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino
acid 30 to about amino acid 40
of a 213P1F11 protein shown in Figure 2 or Figure 3, polypeptides consisting
of about amino acid 40 to about
amino acid 50 of a 213PIF11 protein shown in Figure 2 or Figure 3,
polypeptides consisting of about amino
acid 50 to about amino acid 60 of a 213P1F11 protein shown in Figure 2 or
Figure 3, polypeptides consisting
of about amino acid 60 to about amino acid 70 of a 213P1F11 protein shown in
Figure 2 or Figure 3,
polypeptides consisting of about amino acid 70 to about amino acid 80 of a
213P1F11 protein shown in
Figure 2 or Figure 3, polypeptides consisting of about amino acid 80 to about
amino acid 90 of a 213P1F11
protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino
acid 90 to about amino acid
100 of a 213P1F11 protein shown in Figure 2 or Figure 3, etc. throughout the
entirety of a 213P1F11 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 213P1F11 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.
213P 1F11-related proteins are generated using standard peptide synthesis
technology or using chemical
cleavage methods well known in the art. Alternatively, recombinant methods can
be used to generate nucleic acid
molecules that encode a 213P1F11-related protein. In one embodiment, nucleic
acid molecules provide a means
to generate defined fragments of a 213P1F11 protein (or variants, homologs or
analogs thereof).
IILA.) Motif bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include
213P1F11
polypeptides comprising the amino acid residues of one or more of the
biological motifs contained within a
213P1F11 polypeptide sequence set forth in Figure 2 or Figure 3. Various
motifs are known in the art, and a
protein can be evaluated for the presence of such motifs by a number of
publicly available Internet sites (see,
e.g., URL addresses located at the World Wide Web: pfam.wustl.edu/;
http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.u-
tokyo.ac.jp/; .cbs.dtu.dk/;
.ebi.ac.uk/interpro/scan.html; .expasy.ch/tools/scnpsitl.html; EpimatrixTM and
EpimerTM, Brown University,
.brown.edu/Research/ TB-HIV Lab/epimatrix/epimatrix.html; and BIMAS,
bimas.dcrt.nih.gov/.).
Motif bearing subsequences of all 213P1F11 variant proteins are set forth and
identified in Tables V-
XIX.
Table X~ sets forth several frequently occurring motifs based on pfam searches
(see URL address
pfam.wustl.edu/). The columns of Table XX list (1) motif name abbreviation,
(2) percent identity found
amongst the different member of the motif family, (3) motif name or
description and (4) most common
function; location information is included if the motif is relevant for
location.
Polypeptides comprising one or more of the 213P1F11 motifs discussed above are
useful in
elucidating the specific characteristics of a malignant phenotype in view of
the observation that the 213P 1F 11
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motifs discussed above are associated with growth dysregulation and because
213P1F11 is overexpressed in
certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-
dependent protein kinase, and Protein
Kinase C, for example, are enzymes known to be associated with the development
of the malignant
phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 ( 1998); Gaiddon
et al., Endocrinology 136( 10):
4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996);
Peterziel et al., Oncogene
18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)).
Moreover, both glycosylation
and myristoylation are protein modifications also associated with cancer and
cancer progression (see e.g.
Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp.
Cell Res. 235(1): 145-154
(1997)). Amidation is another protein modification also associated with cancer
and cancer progression (see
e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).
In another embodiment, proteins of the invention comprise one or more of the
immunoreactive
epitopes identified in accordance with art-accepted methods, such as the
peptides set forth in Tables V-XIX.
CTL epitopes can be determined using specific algorithms to identify peptides
within a 213P1F11 protein that are
capable of optimally binding to specified HLA alleles (e.g., Table IV;
EpimatrixTM and EpimerT"', Brown
University, URL located at the World Wide Web .brown.edu/ResearchfTB-HIV
Lab/epimatrixlepimatrix.html;
and BIMAS, URL bimas.dcrt.nih.gov/.) 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 carned out without undue experimentation. In
addition, processes for identifying
peptides that are immunogenic epitopes, are well known in the art, and are
carried out without undue
experimentation either in vitro or in vivo.
Also known in the art are principles for creating analogs of such epitopes in
order to modulate
immunogenicity. For example, one begins with an epitope that bears a CTL or
HTL motif (see, e.g., the HLA
Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is
analoged by substituting out an
amino acid at one of the specified positions, and replacing it with another
amino acid specified for that
position. For example, one can substitute out a deleterious residue in favor
of any other residue, such as a
preferred residue as defined in Table IV; substitute a less-preferred residue
with a preferred residue as defined
in Table IV; or substitute an originally-occurring preferred residue with
another preferred residue as defined
in Table IV. Substitutions can occur at primary anchor positions or at other
positions in a peptide; see, e.g.,
Table IV.
A variety of references reflect the art regarding the identification and
generation of epitopes in a
protein of interest as well as analogs thereof. See, for example, WO 9733602
to Chesnut et al.; Sette,
Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001.166(2):
1389-1397; Sidney et al.,
Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-
258; Sidney et al., J.
Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt
et al., Science 255:1261-3
(1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J.
Immunol. 152:163-75 (1994)); Kast et
al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-
278; Alexander et al., J.
Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI:
95202582; O'Sullivan et
al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):
751-761 and Alexander et
al., Immunol. Res. 1998 18(2): 79-92.
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Related embodiments of the invention include polypeptides comprising
combinations of the different
motifs set forth in Table XXI, and/or, one or more of the predicted CTL
epitopes of Tables V-XIX, and/or,
one or more of the T cell binding motifs known in the art. Preferred
embodiments contain no insertions,
deletions or substitutions either within the motifs or the intervening
sequences of the polypeptides. In
addition, embodiments which include a number of either N-terminal and/or C-
terminal amino acid residues on
either side of these motifs may be desirable (to, for example, include a
greater portion of the polypeptide
architecture in which the motif is located). Typically the number of N-
terminal and/or C-terminal amino acid
residues on either side of a motif is between about 1 to about 100 amino acid
residues, preferably 5 to about
50 amino acid residues.
213P1F11-related proteins are embodied in many forms, preferably in isolated
form. A purified
213P1F11 protein molecule will be substantially free of other proteins or
molecules that impair the binding of
213P1F11 to antibody, T cell or other ligand. The nature and degree of
isolation and purification will depend on
the intended use. Embodiments of a 213P1F11-related proteins include purified
213P1F11-related proteins
and functional, soluble 213P1F11-related proteins. In one embodiment, a
functional, soluble 213P1F11
protein or fragment thereof retains the ability to be bound by antibody, T
cell or other ligand.
The invention also provides 213P1F11 prmteins comprising biologically active
fragments of a
213P1F11 amino acid sequence shown in Figure 2 or Figure 3. Such proteins
exhibit properties of the
starting 213P1F11 protein, such as the ability to elicit the generation of
antibodies that specifically bind an
epitope associated with the starting 213P1F11 protein; to be bound by such
antibodies; to elicit the activation
of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically
bind to the starting protein.
213P1F11-related polypeptides that contain particularly interesting structures
can be predicted and/or
identified using various analytical techniques well known in the art,
including, for example, the methods of Chou-
Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-
Wolf analysis, or on the basis
of immunogenicity. Fragments that contain such structures are particularly
useful in generating subunit-specific
anti-213P1F11 antibodies, or T cells or in identifying cellular factors that
bind to 213P1F11. 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. J. Pept. Protein Res. 32:242-255.
Beta-turn profiles can be
generated, and immunogenic peptide fragments identified, using the method of
Deleage, G., Roux B., 1987,
Protein Engineering 1:289-294.
CTL epitopes can be determined using specific algorithms to identify peptides
within a 213P1F11
protein that are capable of optimally binding to specified HLA alleles (e.g.,
by using the SYFPEITHI site at
World Wide Web URL syfpeithi.bmi-heidelberg.com/; the listings in Table IV(A)-
(E); EpimatrixT"" and
EpimerT"', Brown University, located at the World Wide Web
(.brown.edu/Research/'TB-
HIV Lab/epimatrix/epimatrix.html);'and BIIVIAS, URL bimas.dcrt.nih.gov~.
Illustrating this, peptide epitopes
from 213P1F11 that are presented-in the context of human MHC Class I
molecules, e.g., HLA-A1, A2, A3,
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A11, A24, B7 and B35 were predicted (Tables V-XIX). Specifically, the complete
amino acid sequence of
the 213P1F11 protein and relevant portions of other variants, i.e., for HLA
Class I predictions 9 flanking
redisues on either side of a point mutation, and for HLA Class II predictions
14 flanking residues on either
side of a point mutation, were entered into the HLA Peptide Motif Search
algorithm found in the
Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above;
in addition to the site
SYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.
The HLA peptide motif search algorithm was developed by Dr. Ken Parker based
on binding of
specific peptide sequences in the groove of HLA Class I molecules, in
particular HLA-A2 (see, e.g., Falk et
al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker
et al., J. Immunol. 149:3580-7
(1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows
location and ranking of 8-mer,
9-mer (also refered to as "nonamer"), and 10-mer (also refered to as
"decamer") peptides from a complete
protein sequence for predicted binding to HLA-A2 as well as numerous other HLA
Class I molecules. Many
HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class
I HLA-A2, the epitopes
preferably contain a leucine (L) or methionine (M) at position 2 and a valine
(V) or leucine (L) at the C-
terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected
results of 213P1F1 l predicted
binding peptides are shown in Tables V-XIX herein. In Tables V-XIX, the
selected candidates, 9-mers and
10-mers, and 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 HLA expression on
the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate
30:73-8 (1997) and Peshwa et al.,
Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be
evaluated in vitro by stimulation of
CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells
such as dendritic cells.
It is to be appreciated that every epitope predicted by the BIMAS site,
EpimerTM and EpimatrixT"'
sites, or specified by the HLA class I or class II motifs available in the art
or which become part of the art
such as set forth in Table IV (or determined using World Wide Web site URL
syfpeithi.bmi-heidelberg.com/,
or BIIVIAS, bimas.dcrt.nih.govn are to be "applied" tn a 213P1F11 protein in
accordance with the invention.
As used in this context "applied" means that a 213P1F11 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
213P1F11 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA
Class I motif, or a subsequence of
9 or more amino acid residues that bear an HLA Class II motif are within the
scope of the invention.
IILB.1 Expression of 213P1F11-related Proteins
In an embodiment described in the examples that follow, 213P 1F11 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 213P1F11 with a C-terminal 6XHis and MYC tag
(pcDNA3.1/mycHIS,
Invitrogen or Tags, GenHunter Corporation, Nashville TN). The Tags vector
provides an IgGK secretion
signal that can be used to facilitate the production of a secreted 213P1F11
protein in transfected cells. The
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secreted HIS-tagged 213P1F11 in the culture media can be purified, e.g., using
a nickel column using
standard techniques.
IILC.) Modifications of 213P1F11-related Proteins
Modifications of 213P1F11-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
213P 1F 11 polypeptide with an organic derivatizing agent that is capable of
reacting with selected side chains
or the N- or C- terminal residues of a 213P1F11 protein. Another type of
covalent modification of a
213P1F11 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 213P1F11 comprises linking a
213P1F11 polypeptide to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol (PEG),
polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S.
Patent Nos. 4,640,835; 4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The 213P1F11-related proteins of the present invention can also be modified to
form a chimeric
molecule comprising 213P1F11 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 213P
1F11 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 213P1F11. A chimeric molecule can comprise a fusion of
a 213P1F11-related
protein with a polyhistidine epitope tag, which provides an epitope to which
immobilized nickel can
selectively bind, with cytokines or with growth factors. The epitope tag is
generally placed at the amino- or
carboxyl- terminus of a213P1F11 protein. In an alternative embodiment, the
chimeric molecule can comprise
a fusion of a 213P1F11-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 213P1F11 polypeptide
in place of at least one
variable region within an Ig molecule. In a preferred embodiment, the
immunoglobulin fusion includes the
hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI
molecule. For the production of
immunoglobulin fusions see, e.g., U.S. Patent No. 5,428,130 issued June 27,
1995.
IILD.1 Uses of 213P1F11-related Proteins
The proteins of the invention have a number of different specific uses. As
213P1F11 is highly
expressed in prostate and other cancers, 213P1F11-related proteins are used in
methods that assess the status
of 213P1F11 gene products in normal versus cancerous tissues, thereby
elucidating the malignant phenotype.
Typically, polypeptides from specific regions of a 213P1F11 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
213P1F11-related proteins
comprising the amino acid residues of one or more of the biological motifs
contained within a 213P1F11
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, 213P1F11-
related proteins that contain the
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amino acid residues of one or more of the biological motifs in a 213P1F11
protein are used to screen for
factors that interact with that region of 213P1F11.
213P1F11 protein fragments/subsequences are particularly useful in generating
and characterizing
domain-specific antibodies (e.g., antibodies recognizing an extracellular or
intracellular epitope of a 213P1F1 I
protein), for identifying agents or cellular factors that bind to 213P1F11 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 213P1F11 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 213P1F11 gene product.
Antibodies raised against a
213P1F11 protein or fragment thereof are useful in diagnostic and prognostic
assays, and imaging
methodologies in the management of human cancers characterized by expression
of 213P1F11 protein, such
as those listed in Table I. Such antibodies can be expressed intracellularly
and used in methods of treating
patients with such cancers. 213P1F11-related nucleic acids or proteins are
also used in generating HTL or
CTL responses.
Various immunological assays useful for the detection of 213P1F11 proteins are
used, including but not
limited to various types of radioimmunoassays, enzyme-linked immunosorbent
assays (ELISA), enzyme-linked
immunofiuorescent assays (ELIFA), immunocytochemical methods, and the like.
Antibodies can be labeled and
used as immunological imaging reagents capable of detecting 213P1F11-
expressing cells (e.g., in
radioscintigraphic imaging methods). 213P1F11 proteins are also particularly
useful in generating cancer
vaccines, as fiu~ther described herein.
IV.) 213P1F11 Antibodies
Another aspect of the invention provides antibodies that bind to 213P1F11-
related proteins. Preferred
antibodies specifically bind to a 213P1F11-related protein and do not bind (or
bind weakly) to peptides or proteins
that are not 213P1F11-related proteins. For example, antibodies that bind
213P1F11 can bind 213P1F11-related
proteins such as the homologs or analogs thereof.
213P1F11 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 usefiil in the treatment,
diagnosis, and/or prognosis of other cancers, to the extent 213P1F11 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 213P1F11
is involved, such as advanced
or metastatic prostate cancers.
The invention also provides various immunological assays usefiil for the
detection and quantification of
213P1F11 and mutant 213P1F11-related proteins. Such assays can comprise one or
more 213P1F11 antibodies
capable of recognizing and binding a 213P1F11-related protein, as appropriate.
These assays are performed
within various immunological assay formats well known in the art, including
but not limited to various types of
radioimmunoassays, enzyme-linked immunosorbent~assays (ELISA), enzyme-linked
immunofluorescent assays
(ELIFA), and the like.
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Immunoiogical 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 213P1F11 are also provided by the invention, including but not
limited to radioscintigraphic imaging
methods using labeled 213P1F11 antibodies. Such assays are clinically useful
in the detection, monitoring, and
prognosis of 213P1F11 expressing cancers such as prostate cancer.
213P1F11 antibodies are also used in methods for purifying a 213P1F11-related
protein and for isolating
213P1F11 homologues and related molecules. For example, a method ofpurifying a
213P1F11-related protein
comprises incubating a 213P1F11 antibody, which has been coupled to a solid
matrix, with a lysate or other
solution containing a 213P1F11-related protein under conditions that permit
the 213P1F11 antibody to bind to the
213P1F11-related protein; washing the solid matrix to eliminate impurities;
and eluting the 213P1F11-related
protein from the coupled antibody. Other uses of 213P1F11 antibodies in
accordance with the invention
include generating anti-idiotypic antibodies that mimic a 213P1F11 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 213P 1F 11-
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
213P1F11 can also be used, such as a 213P1F11 GST-fusion protein. In a
particular embodiment, a GST fission
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
213P1F11-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
213P1F11-related protein or 213P1F11 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 213P1F11 protein as shown in Figure 2 or Figure 3
can be analyzed to
select specific regions of the 213P1F11 protein for generating antibodies. For
example, hydrophobicity and
hydrophilicity analyses of a 213P1F11 amino acid sequence are used to identify
hydrophilic regions in the
213P1F11 structure. Regions of a 213P1F11 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
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 213P 1 Fl 1 antibodies are further illustrated by way of the
examples provided herein. Methods
for preparing a protein or polypeptide for use as an immunogen are well known
in the art. Also well known in the
art are methods for preparing immunogenic conjugates of a protein with a
carrier, such as BSA, KLH or other
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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 213P1F11 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.
213P 1F11 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 213P1F11-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 213P1F11 protein can also be
produced in the context of chimeric or
complementarity determining region (CDR) grafted antibodies of multiple
species origin. Humanized or human
213P1F11 antibodies can also be produced, and are preferred for use in
therapeutic contexts. Methods for
humanizing marine and other non-human antibodies,~y substituting one or more
of the non-human antibody
CDRs for corresponding human antibody sequences, are well known (see for
example, Jones et al., 1986, Nature
321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al.,
1988, Science 239: 1534-1536).
See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et
al., 1993, J. Immunol. 151: 2296.
Methods for producing fully human monoclonal antibodies include phage display
and transgenic
methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-
539). Fully human 213P1F11
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 (I993); Burton and
Barbas, Hurnan Antibodies from combinatorial libraries. Id., pp 65-82). Fully
human 213P1F11 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
al., published December 3,
1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S.
patents 6,162,963 issued 19
December 2000; 6,150,584 issued 12 November 2000; and, 6,114598 issued 5
September 2000). This method
avoids the in vitro manipulation required with phage display technology and
efficiently produces high affinity
authentic human antibodies.
Reactivity of 213P1F11 antibodies with a 213P1F11-related protein can be
established by a number
of well known means, including Western blot, immunoprecipitation, ELISA, and
FAGS analyses using, as
appropriate, 213P1F11-related proteins, 213P1F11-expressing cells or extracts
thereof. A 213P1F11 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 213P1F11 epitopes are generated using methods
generally known in the art.
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Homodimeric antibodies can also be generated by cross-linking techniques known
in the art (e.g., Wolff et
al., Cancer Res. 53: 2560-2565).
V.) 213P1F11 Cellular Immune Responses
The mechanism by which T cells recognize antigens has been delineated.
Efficacious peptide
epitope vaccine compositions of the invention induce a therapeutic or
prophylactic immune responses in very
broad segments of the world-wide population. For an understanding of the value
and efficacy of
compositions of the invention that induce cellular immune responses, a brief
review of immunology-related
technology is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand
recognized by HLA-
restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et
al., Nature 317:359, 1985; Townsend,
A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.
Immunol. 11:403, 1993).
Through the study of single amino acid substituted antigen analogs and the
sequencing of endogenously
bound, naturally processed peptides, critical residues that correspond to
motifs required for specific binding to
HLA antigen molecules have been identified and are set forth in Table IV (see
also, e.g., Southwood, et al., J. '
Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995;
Rammensee et al.,
SYFPEITHI, access via World Wide Web at URL syfpeithi.bmi-heidelberg.com/;
Sette, A. and Sidney, J.
Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol.
6:13, 1994; Sette, A. and Grey,
H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr.
Biol. 6:52, 1994; Ruppert et al.,
Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney
et al., .J. Immunol. 157:3480-
3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and
Sidney, J. Immunogenetics 1999
Nov; SO(3-4):201-12, Review).
Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have
revealed pockets
within the peptide binding cleftJgroove of HLA molecules which accommodate, in
an allele-specific mode,
residues borne by peptide ligands; these residues in turn determine the HLA
binding capacity of the peptides
in which they are present. (See, e.g., Madden, D.R. Annu. Rev. Immunol.
13:587, 1995; Smith, et al.,
Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al.,
Structure 2:245, 1994; Jones, E.Y.
Curr. Opin. Immunol. 9:75, 1997; Brown, J. H, et al., Nature 364:33, 1993;
Guo, H. C. et al., Proc. Natl.
Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nc:.~ ~,re 360:364, 1992;
Silver, M. L. et al., Nature 360:367,
1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell
70:1035, 1992; Fremont, D. H. et al.,
Science 257:919, 1992; Saper, M. A. , Bjorkman, P. J. and Wiley, D. C., J Mol.
Biol. 219:277, 1991.)
Accordingly, the definition of class I and class II allele-specific HLA
binding motifs, or class I or
class II supermotifs allows identification of regions within a protein that
are correlated with binding to
particular HLA antigen(s).
Thus, by a process of HLA motif identification, candidates for epitope-based
vaccines have been
identified; such candidates can be further evaluated by HLA-peptide binding
assays to determine binding
affinity and/or the time period of association of the epitope and its
corresponding HLA molecule. Additional
confirmatory work can be performed to select, amongst these vaccine
candidates, epitopes with preferred
characteristics in terms of population coverage, and/or immunogenicity.
Various strategies can be utilized to evaluate cellular immunogenicity,
including:
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1) Evaluation of primary T cell cultures from normal individuals (see, e.g.,
Wentworth, P. A. et al.,
Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA
91:2105, 1994; Tsai, V. et al., J.
Immunol. 158:1796, 1997; Kawashima, I. et al., 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 51
Cr-release assay involving peptide
sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J.
Immunol. 26:97,
1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et
al., J. Immunol. 159:4753, 1997).
For example, in such methods peptides in incomplete Freund's adjuvant are
administered subcutaneously to
HLA transgenic mice. Several weeks following immunization, splenocytes are
removed and cultured in vitro
in the presence of test peptide for approximately one week. Peptide-specific T
cells are detected using, e.g., a
5lCr-release assay involving peptide sensitized target cells and target cells
expressing endogenously
generated antigen.
3) Demonstration of recall T cell responses from immune individuals who have
been either
effectively vaccinated and/or from chronically ill patients (see, e.g.,
Rehermann, B. et al., J. Exp. Med.
181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al.,
J. Clin. Invest. 100:503, 1997;
Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al.,
J. Virol. 71:6011, 1997).
Accordingly, recall responses are detected by culturing PBL from subjects that
have been exposed to the
antigen due to disease and thus have generated an immune response "naturally",
or from patients who were
vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-
2 weeks in the presence of test
peptide plus antigen presenting cells (APC) to allow activation of "memory" T
cells, as compared to "naive"
T cells. At the end of the culture period, T cell activity is detected using
assays including 5lCr release
involving peptide-sensitized targets, T cell proliferation, or lymphokine
release.
VLl 213P1F11 Trans~enic Animals
Nucleic acids that encode a 213P1F11-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
213P1F11 can be used to clone
genomic DNA that encodes 213P1F11. The cloned genomic sequences can then be
used to generate
transgenic animals containing cells that express DNA that encode 213P1F11.
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 213P1F11
transgene incorporation with
tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 213P1F11 can be
used to examine
the effect of increased expression of DNA that encodes 213P1F11. 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
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reduced incidence of a pathological condition, compared to untreated animals
that bear the transgene, would
indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of 213P1F11 can be used to construct a
213P1F11 "knock
out" animal that has a defective or altered gene encoding 213P1F11 as a result
of homologous recombination
between the endogenous gene encoding 213P1F11 and altered genomic DNA encoding
213P1F11 introduced
into an embryonic cell of the animal. For example, cDNA that encodes 213P1F11
can be used to clone
genomic DNA encoding 213P1F11 in accordance with established techniques. A
portion of the genomic
DNA encoding 213P1F11 can be deleted or replaced with another gene, such as a
gene encoding a selectable
marker that can be used to monitor integration. Typically, several kilobases
of unaltered flanking DNA (both
at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and
Capecchi, Cell, 51:503 (1987) for a
description of homologous recombination vectors). The vector is introduced
into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced DNA has
homologously recombined with the
endogenous DNA are selected (see, e.g., Li et al., Cell, 69:915 (1992)). The
selected cells are then injected
into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation
chimeras (see, e.g., Bradley, in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.
Robertson, ed. (IRL, Oxford,
1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable
pseudopregnant female foster
animal, and the embryo brought to term to create a "knock out" animal. Progeny
harboring the homologously
recombined DNA in their germ cells can be identified by standard techniques
and used to breed animals in
which all cells of the animal contain the homologously recombined DNA. Knock
out animals can be
characterized, for example, for their ability to defend against certain
pathological conditions or for their
development of pathological conditions due to absence of a 213P1F11
polypeptide.
VB.) Methods for the Detection of 213P1F11
Another aspect of the present invention relates to methods for detecting
213P1F11 polynucleotides and
213P1F11-related proteins, as well as methods for identifying a cell that
expresses 213P1F11. The expression
profile of 213P1F11 makes it a diagnostic marker for metastasized disease.
Accordingly, the status of
213P1F11 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 213P1F11 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 213P 1F
11 polynucleotides in a
biological sample, such as serum, bone, prostate, and other tissues, urine,
semen, cell preparations, and the like.
Detectable 213P1F,11 polynucleotides include, for example, a 213P1F11 gene or
fragment thereof, 213P1F11
mRNA, alternative splice variant 213P1F11 mRNAs, and recombinant DNA or RNA
molecules that contain a
213P1F11 polynucleotide. A number of methods for amplifying and/or detecting
the presence of 213P1F11
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 213P1F11 mRNA in a biological
sample comprises
producing cDNA from the sample by reverse transcription using at least one
primer; amplifying the cDNA so
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produced using a 213P1F11 polynucleotides as sense'and antisense primers to
amplify 213P1F11 cDNAs
therein; and detecting the presence of the amplified 213P1F11 cDNA.
Optionally, the sequence of the
amplified 213P1F11 cDNA can be determined.
In another embodiment, a method of detecting a 213P1F11 gene in a biological
sample comprises
first isolating genomic DNA from the sample; amplifying the isolated genomic
DNA using 213P1F11
polynucleotides as sense and antisense primers; and detecting the presence of
the amplified 213PiF11 gene.
Any number of appropriate sense and antisense probe combinations can be
designed from a 213P1F11
nucleotide sequence (see, e.g., Figure 2) and used for this purpose.
The invention also provides assays for detecting the presence of a 213P1F11
protein in a tissue or other
biological sample such as serum, semen, bone, prostate, urine, cell
preparations, and the like. Methods for
detecting a 213P 1F11-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 213P1F11-related protein
in a biological sample
comprises first contacting the sample with a 213P1F11 antibody, a 213P1F11-
reactive fragment thereof, or a
recombinant protein containing an antigen binding region of a 213P1F11
antibody; and then detecting the
binding of 213P1F11-related protein in the sample.
Methods for identifying a cell that expresses 213P1F11 are also within the
scope of the invention. In
one embodiment, an assay for identifying a cell that expresses a 213P1F11 gene
comprises detecting the presence
of 213P1F 11 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 213P1F11 riboprobes, Northern blot and related techniques) and various
nucleic acid amplification assays
(such as RT-PCR using complementary primers specific for 213P1F11, 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 213P1F11 gene comprises detecting the
presence of 213P1F11-related protein
in the cell or secreted by the cell. Various methods for the detection of
proteins are well known in the art and are
employed for the detection of 213P1F11-related proteins and cells that express
213P1F11-related proteins.
213P1F11 expression analysis is also useful as a tool for identifying and
evaluating agents that modulate
213P 1F11 gene expression. For example, 213P1F11 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 213P1F11 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 213P1F11
expression by RT-PCR, nucleic
acid hybridization or antibody binding.
VIIL) Methods for Monitorins the Status of 213P1F11-related Genes and Their
Products
Oncogenesis is known to be a multistep process where cellular growth becomes
progressively
dysregulated and cells progress from a normal physiological state to
precancerous and then cancerous states
(see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,
Cancer Surv. 23: 19-32 (1995)). In
this context, examining a biological sample for evidence of dysregulated cell
growth (such as aberrant
213P 1F 11 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
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worse. In such examinations, the status of 213P1F11 in a biological sample of
interest can be compared, for
example, to the status of 213P1F11 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 213P 1F 11
in the biological sample (as compared to the normal sample) provides evidence
of dysregulated cellular
growth. In addition to using a biological sample that is not affected by a
pathology as a normal sample, one
can also use a predetermined normative value such as a predetermined normal
level of mRNA expression
(see, e.g., Grever et al., J. Comp. Neurol. 1996 Dec 9; 376(2): 306-14 and
U.S. Patent No. 5,837,501) to
compare 213P1F11 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 213P1F11 expressing cells) as well as the level,
and biological activity of expressed
gene products (such as 213P1F11 mRNA, polynucleotides and polypeptides).
Typically, an alteration in the
status of 213P1F11 comprises a change in the location of 213P1F11 and/or
213P1F11 expressing cells and/or
an increase in 213P1F11 mRNA and/or protein expression.
213P1F11 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
213P1F11 gene and gene products are found, for example in Ausubel et al. eds.,
1995, Current Protocols In
Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15
(Immunoblotting) and 18 (PCR
Analysis). Thus, the status of 213P1F11 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 213P1F11 gene), Northern analysis and/or PCR analysis of
213P1F11 mRNA (to examine,
for example alterations in the polynucleotide sequences or expression levels
of 213P1F11 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 213P1F11 proteins
and/or associations of 213P1F11 proteins with polypeptide binding partners).
Detectable 213P1F11
polynucleotides include, for example, a 213P1F11 gene or fragment thereof,
213P1F11 mRNA, alternative splice
variants, 213P1F11 mRNAs, and recombinant DNA or RNA molecules containing a
213P1F11 polynucleotide.
The expression profile of 213PiF11 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 213P 1F i l provides information useful for predicting
susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides methods and
assays for detern~ining 213P1F11
status and diagnosing cancers that express 213P1F11, such as cancers of the
tissues listed in Table I. For
example, because 213P1F11 mRNA is so highly expressed in prostate and other
cancers relative to normal
prostate tissue, assays that evaluate the levels of 213P1F11 mRNA transcripts
or proteins in a biological sample
can be used to diagnose a disease associated with 213P1F11 dysregulation, and
can provide prognostic
information useful in defining appropriate therapeutic options.
The expression status of 213P1F11 provides information including the presence,
stage and location of
dysplastic, precancerous and cancerous cells, predicting susceptibility to
various stages of disease, and/or for
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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 213P1F11 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 213P1F11 in a biological sample can be
examined by a number of
well-known procedures in the art. For example, the status of 213P1F11 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
213P1F11 expressing cells (e.g. those that express 213P1F11 mIRNAs or
proteins). This examination can
provide evidence of dysregulated cellular growth, for example, when 213P1F11-
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 213P1F11 in a biological sample are often associated with
dysregulated cellular growth.
Specifically, one indicator of dysregulated cellular growth is the metastases
of cancer cells from an organ of
origin (such as the prostate) to a different area of the body (such as a lymph
node). In this context, evidence
of dysregulated cellular growth is important for example because occult lymph
node metastases can be
detected in a substantial proportion of patients with prostate cancer, and
such metastases are associated with
known predictors of disease progression (see, e.g., Murphy et al., Prostate
42(4): 315-317 (2000);Su et al.,
Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 Aug
154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring 213P1F11 gene
products by
determining the status of 213P1F11 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
r
status so determined to the status of 213P1F11 gene products in a
corresponding normal sample. The
presence of aberrant 213P1F11 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 213P1F11 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
213P1F11 mItNA can, for example, be evaluated in tissues including but not
limited to those listed in Table I.
The presence of significant 213P1F11 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 213P1F11 mRNA
or express it at lower levels.
In a related embodiment, 213P1F11 status is determined at the protein level
rather than at the nucleic
acid level. For example, such a method comprises determining the level of
213P1F11 protein expressed by cells
in a test tissue sample and comparing the level so determined to the level of
213P1F11 expressed in a
corresponding normal sample. In one embodiment, the presence of 213P1F11
protein is evaluated, for
example, using immunohistochemical methods. 213P1F11 antibodies or binding
parhiers capable of detecting
213P1F11 protein expression are used in a variety of assay formats well known
in the art for this purpose.
In a further embodiment, one can evaluate the status of 213P1F11 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
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the nucleotide and amino acid sequences are observed in a large number of
proteins associated with a growth
dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol.
26(8):369-378). For example, a
mutation in the sequence of 213P1F11 may be indicative of the presence or
promotion of a tumor. Such assays
therefore have diagnostic and predictive value where a mutation in 213P1F11
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 213P1F11 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 213P1F11 gene in a
biological sample.
Aberrant demethylation and/or hypermethylation of CpG islands in gene S'
regulatory regions frequently occurs
in immortalized and transformed cells, and can result in altered expression of
various genes. For example,
promoter hypermethylation of the pi-class glutathione S-transferase (a protein
expressed in normal prostate
but not expressed in >90% of prostate carcinomas) appears to permanently
silence transcription of this gene
and is the most. frequently detected genomic alteration in prostate carcinomas
(De Marzo et al., Am. J. Pathol.
155(6): 1985-1992 (1999)). In addition, this alteration is present in at least
70% of cases of high-grade
prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol.
Biomarkers Prev., 1998, 7:531-
536). In another example, expression of the LAGS-I tumor specific gene (which
is not expressed in normal
prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-
azacytidine in lymphoblastoid
cells, suggesting that tumoral expression is due to demethylation (Lethe et
al., Int. J. Cancer 76(6): 903-908
( 1998)). A variety of assays for examining methylation status of a gene are
well known in the art. For example,
one can utilize, in Southern hybridization approaches, methylation-sensitive
restriction enzymes that cannot
cleave sequences that contain methylated CpG sites to assess the methylation
status of CpG islands. In addition,
MSP (methylation specific PCR) can rapidly profile the methylation status of
all the CpG sites present in a CpG
island of a given gene. This procedure involves initial modification of DNA by
sodium bisulfite (which will
convert all unmethylated cytosines to uracil) followed by amplification using
primers specific for methylated
versus unmethylated DNA. Protocols involving methylation interference can also
be found for example in
Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al.
eds., 1995.
Gene amplification is an additional method for assessing the status of
213P1F11. 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 213PiF11 expression.
The presence of RT-PCR
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amplifiable 213P1F11 mRNA provides an indication of the presence of cancer. RT-
PCR assays are well known
in the art. RT-PCR detection assays for tumor cells in peripheral blood are
currently being evaluated for use in
the diagnosis and management of a number of human solid tumors. In the
prostate cancer field, these include RT-
PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al.,
1997, Urol. Res. 25:373-384;
Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995,
Clin. Chem. 41:1687-1688).
A further aspect of the invention is an assessment of the susceptibilitythat
an individual has for
developing cancer. In one embodiment, a method for predicting susceptibility
to cancer comprises detecting
213P1F11 mRNA or 213P1F11 protein in a tissue sample, its presence indicating
susceptibility to cancer, wherein
the degree of 213P1F11 mRNA expression correlates to the degree of
susceptibility. In a specific embodiment,
the presence of 213P1F11 in prostate or other tissue is examined, with the
presence of 213P1F11 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 213P1F11 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 213P1F11 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
213P1F11 mRNA or 213P1F11 protein
expressed by tumor cells, comparing the level so determined to the level of
213P1F11 mRNA or 213P1F11
protein expressed in a corresponding normal tissue taken from the same
individual or a normal tissue reference
sample, wherein the degree of 213P1F11 mRNA or 213P1F11 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 213P1F11 is expressed in the
tumor cells, with higher expression
levels indicating more aggressive tumors. Another embodiment is the evaluation
ofthe integrity of 213P1F11
nucleotide and amino acid sequences in a biological sample, in order to
identify perturbations in the structure of
these molecules such as insertions, deletions, substitutions and the like. The
presence of one or more
perturbations indicates more aggressive tumors.
Another embodiment of the invention is directed to methods for observing the
progression of a
malignancy in an individual over time. In one embodiment, methods for
observing the progression of a
malignancy in an individual over time comprise detemining the level of
213P1F11 mRNA or 213P1F11 protein
expressed by cells in a sample of the tumor, comparing the level so determined
to the level of 213P1F11 mRNA
or 213P1F11 protein expressed in an equivalent tissue sample taken from the
same individual at a different time,
wherein the degree of 213P1F11 mRNA or 213P1F11 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 213P1F11 expression in the tumor cells over time, where
increased expression over time indicates
a progression of the cancer. Also, one can evaluate the integrity 213P1F11
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
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for observing a coincidence between the expression of'213P1F11 gene and
213P1F11 gene products (or
perturbations in 213P1F11 gene and 213P1F11 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 et al.,
1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9;
Thorson et al., 1998, Mod.
Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-
24). Methods for observing a
coincidence between the expression of 213P1F11 gene and 213P1F11 gene products
(or perturbations in
213P1F11 gene and 213P1F11 gene products) and another factor that is
associated with malignancy are useful, for
example, because the presence of a set of specific factors that coincide with
disease provides information crucial
for diagnosing and prognosticating the status of a tissue sample.
In one embodiment, methods for observing a coincidence between the expression
of 213P1F11 gene and
213P1F11 gene products (or perturbations in 213P1F11 gene and 213P1F11 gene
products) and another factor
associated with malignancy entails detecting the overexpression of 213P1F11
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 213P 1F 11 mRNA or protein and PSA mRNA or
protein overexpression (or PSCA
or PSM expression). In a specific embodiment, the expression of 213P1F11 and
PSA mRNA in prostate tissue is
examined, where the coincidence of 213P1F11 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 213P1F11 mRNA or
protein are described
herein, and standard nucleic acid and protein detection and quantification
technologies are well lrnown in the art.
Standard methods for the detection and quantification of 213P1F11 mRNA include
in situ hybridization using
labeled 213P1F11 riboprobes, Northern blot and related techniques using
213P1F11 polynucleotide probes, RT-
PCR analysis using primers-specific for 213P1F11, 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 213P1F11 mRNA expression. Any number of primers
capable of amplifying
213P1F11 can be used for this purpose, including but not limited to the
various primer sets specifically described
herein. In a specific embodiment, polyclonal or monoclonal antibodies
specifically reactive with the wild-type
213P1F11 protein can be used in an ixnmunohistochemical assay of biopsied
tissue.
IX.) Identification of Molecules That Interact With 213P1F11
The 213P1F11 protein and nucleic acid sequences disclosed herein allow a
skilled artisan to identify
proteins, small molecules and other agents that interact with 213P1F11, as
well as pathways activated by
213P 1F 11 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.
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Algorithms are also available in the art for genome-based predictions of
protein function (see, e.g., Marcotte,
et al., Nature 402: 4 November 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that
interact with 213P1F11
protein sequences. In such methods, peptides that bind to 213P1F11 are
identified by screening libraries that
encode a random or controlled collection of amino acids. Peptides encoded by
the libraries are expressed as
fusion proteins of bacteriophage coat proteins, the bacteriophage particles
are then screened against the
213P 1F 11 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 213P1F11 protein sequences are disclosed for example in U.S. Patent Nos.
5,723,286 issued 3 March
1998 and 5,733,731 issued 31 March 1998.
Alternatively, cell lines that express 213P1F11 are used to identify protein-
protein interactions
mediated by 213P1F11. Such interactions can be examined using
immunoprecipitation techniques (see, e.g.,
Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51).
213P1F11 protein can be
immunoprecipitated from 213P1F11-expressing cell lines using anti-213P1F11
antibodies. Alternatively,
antibodies against His-tag can be used in a cell line engineered to express
fusions of 213P 1F11 and a His-tag
(vectors mentioned above). The immunoprecipitated complex can be examined for
protein association by
procedures such as Western blotting, 35S-methionine labeling of proteins,
protein microsequencing, silver
staining and two-dimensional gel electrophoresis.
Small molecules and ligands that interact with 213P1F11 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 213P1F11'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 213P1F11-related
ion channel, protein pump, or cell communication functions are identified and
used to treat patients that have
a cancer that expresses 213P1F11 (see, e.g., Hille, B., Ionic Channels of
Excitable Membranes 2~d Ed.,
Sinauer Assoc., Sunderland, MA, 1992). Moreover, ligands that regulate
213P1F11 function can be
identified based on their ability to bind 213P1F11 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 213P1F11 and a DNA-binding protein
are used to co-express a
fusion protein of a hybrid ligand/small molecule .and a cDNA library
transcriptional activator protein. The
cells further contain a reporter gene, the expression of which is conditioned
on the proximity of the first and
second fusion proteins to each other, an event that occurs only if the hybrid
ligand binds to target sites on both
hybrid proteins. Those cells that express the reporter gene are selected and
the unknown small molecule or.
the unknown ligand is identified. This method provides a means of identifying
modulators which activate or
inhibit 213P1F11.
An embodiment of this invention comprises a method of screening for a molecule
that interacts with
a 213P1F11 amino acid sequence shown in Figure 2 or Figure 3, comprising the
steps of contacting a
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population of molecules with a 213P1F11 amino acid sequence, allowing the
population of molecules and the
213P1F11 amino acid sequence to interact under conditions that facilitate an
interaction, determining the
presence of a molecule that interacts with the 213P1F11 amino acid sequence,
and then separating molecules
that do not interact with the 213P1F11 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 213P1F11 amino acid sequence. The identified molecule can be used to
modulate a function
performed by 213P1F11. In a preferred embodiment, the 213P1F11 amino acid
sequence is contacted with a
library of peptides.
X.l Therapeutic Methods and Compositions
The identification of 213P1F11 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, 213P1F11 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 213P1F11
protein are useful for
patients suffering from a cancer that expresses 213P1F11. These therapeutic
approaches generally fall into
two classes. One class comprises various methods for inhibiting the binding or
association of a 213P 1-F 11
protein with its binding partner or with other proteins. Another class
comprises a variety of methods for
inhibiting the transcription of a 213P1F11 gene or translation of 213P1F11
mRNA.
X.A.1 Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 213P1F11-related protein
or 213P1F11-related
nucleic acid. In view of the expression of 213P1F11, cancer vaccines prevent
and/or treat 213P1F11-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
et al., 1995, Int. J. Cancer
63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).
Such methods can be readily practiced by employing a 213P 1F11-related
protein, or a 213P 1F11-
encoding nucleic acid molecule and recombinant vectors capable of expressing
and presenting the 213P1F11
immunogen (which typically comprises a number of antibody or T cell epitopes).
Skilled artisans understand
that a wide variety of vaccine systems for delivery of immunoreactive epitopes
are known in the art (see, e.g.,
Heryln et al., Ann Med 1999 Feb 31(1):66-78; Maruyama et al., Cancer Immunol
Immunother 2000 Jun
49(3):123-32) Briefly, such methods of generating an~immune response (e.g.
humoral and/or cell-mediated)
in a mammal, comprise the steps of: exposing the mammal's immune system to an
immunoreactive epitope
(e.g. an epitope present in a 213P1F11 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 213P1F11
immunogen contains a biological
motif, see e.g., Tables V-XIX, or a peptide of a size range from 213P1F11
indicated in Figure 5, Figure 6,
Figure 7, Figure 8, and Figure 9.
The entire 213P1F11 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,
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A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated
in poly(DL-lactide-co-glycolide)
("PLG") microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294,
1991: Alonso et al., Vaccine
12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide
compositions contained in immune
stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-
875, 1990; Hu et al., Clin Exp
Imntunol. 113:235-243, 1998), multiple antigen peptide systems (MAPS) (see
e.g., Tam, J. P., Proc. Natl.
Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196:17-
32, 1996), peptides formulated
as multivalent peptides; peptides for use in ballistic delivery systems,
typically crystallized peptides, viral
delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development,
Kaufmann, S. H. E., ed., p. 379,
1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature
320:537, 1986; Kieny, M.-P. et al.,
AIDS BiolTechnology 4:790, 1986; Top, F. H. et al., J . Infect. Dis. 124:148,
1971; Chanda, P. K. et al.,
Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler,
N. et al., .I. Immunol. Methods.
192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 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. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., .l. Immunol.
148:1585, 1992; Rock, K. L., .
Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (LTlmer, J.
B. et al., Science 259:1745,
1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;
Shiver, J. W. et al., In:
Coneepts in vaccine development, Kaufinann, S. H. E., ed., p. 423, 1996;
Cease, K. B., and Berzofsky, J. A.,
Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. 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 213P1F11-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 213P1F11 protein
that bind corresponding HLA alleles (see e.g., Table IV; EpimerT"' and
EpimatrixTM, Brown University (located at
the World Wide Web .brown.edu/Research/'TB-HIV Lab/epimatrix/epimatri.e.html);
and, BIIvIAS, (URL '
bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.corr~. In a
preferred embodiment, a
213P 1F11 immunogen contains one or more amino acid sequences identified using
techniques well known in
the art, such as the sequences shown in Tables V-XIX, or a peptide of 8, 9, 10
or 11 amino acids specified by
an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV
(E)) and/or a peptide of at
least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g.,
Table IV (B) or Table IV (C)). As
is appreciated in the art, the HLA Class I binding groove is essentially
closed ended so that peptides of only a
particular size range can fit into the groove and be bound, generally HLA
Class I epitopes are 8, 9, 10, or 11
amino acids long. In contrast, the HLA Class II binding groove is essentially
open ended; therefore a peptide
of about 9 or more amino acids can be bound by an HLA Class 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
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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
213P1F11 protein) so that an immune response is generated. A typical
embodiment consists of a method for
generating an immune response to 213P1F11 in a host, by contacting the host
with a sufficient amount of at
least one 213P1F11 B cell or cytotoxic T-cell epitope or analog thereof; and
at least one periodic interval
thereafter re-contacting the host with the 213P1F11 B cell or cytotoxic T-cell
epitope or analog thereof. A
specific embodiment consists of a method of generating an immune response
against a 213P1F11-related
protein or a man-made multiepitopic peptide comprising: administering 213P1F11
immunogen (e.g. a
213P1F11 protein or a peptide fragment thereof, a 213P1F11 fusion protein or
analog etc.) in a vaccine
preparation to a human or another mammal. Typically, such vaccine preparations
further contain a suitable
adjuvant (see, e.g., U.S. Patent No. 6,146,635) or a universal helper epitope
such as a PADRETM peptide
(Epimmune Inc., San Diego, CA; see, e.g., Alexander et al., J. Immunol. 2000
164(3); 164(3): 1625-1633;
Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol.
Res. 1998 18(2): 79-92). An
alternative method comprises generating an immune response in an individual
against a 213P1F11
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 213P1F11 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 213P1F1 l, 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 proteins) 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 213P1F11. Constructs comprising DNA encoding a
213P1F11-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 213P1F11
protein/immunogen. Alternatively, a vaccine comprises a 213P1F11-related
protein. Expression of the
213P 1F 11-related protein immunogen results in the generation of prophylactic
or therapeutic humoral and
cellular immunity against cells that bear a 213P1F11 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 located at the World Wide Web .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;
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5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98Y04720. Examples of DNA-based
delivery technologies
include "naked DNA", facilitated (bupivicaine, polymers, peptide-mediated)
delivery, cationic lipid
complexes, and particle-mediated ("gene gun") or pressure-mediated delivery
(see, e.g., U.S. Patent No.
5,922,687).
For therapeutic or prophylactic immunization purposes, proteins of the
invention can be expressed
via viral or bacterial vectors. Various viral gene delivery systems that can
be used in the practice of the
invention include, but are not limited to, vaccinia, fowlpox, canarypox,
adenovirus, influenza, poliovirus, adeno-
associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996,
Curr. Opin. Immunol. 8:658-663; Tsang et
al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can
also be employed by introducing
naked DNA encoding a 213P 1F11-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 213P1F11-related nucleic
acid molecule. In one
embodiment, the full-length human 213P1F11 cDNA is employed. In another
embodiment, 213P1F11 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 213P1F11 antigen to a
patient's immune system. Dendritic cells express MHC class I and II molecules,
B7 co-stimulator, and IL-12, and
are thus highly specialized antigen presenting cells. In prostate cancer,
autologous dendritic cells pulsed with
peptides of the prostate-specific membrane antigen (PSMA) are being used in a
Phase I clinical trial to
stimulate prostate cancer patients' immune systems (Tjoa et al., 1996,
Prostate 28:65-69; Murphy et al., 1996,
Prostate 29:371-380). Thus, dendritic cells can be used to present 213P1F11
peptides to T cells in the context
of MHC class I or II molecules. In one embodiment, autologous dendritic cells
are pulsed with 213P 1F 11
peptides capable of binding to MHC class I and/or class II molecules. In
another embodiment, dendritic cells
are pulsed with the complete 213P1F11 protein. Yet another embodiment involves
engineering the
overexpression of a 213P1F11 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 et al., 1996,
Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA
transfection (Ribas et al., 1997, Cancer
Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J.
Exp. Med. 186:1177-1182).
Cells that express 213P1F11 can also be engineered to express immune
modulators, such as GM-CSF, and
used as immunizing agents.
X.B.) 213P1F11 as a Target for Antibody-based Therany
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213P1F11 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 213P1F11 is expressed
by cancer cells of various lineages relative to corresponding normal cells,
systemic administration of
213P1F11-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 213P1F11 are
useful to treat 213P1F11-
expressing cancers systemically, either as conjugates with a toxin or
therapeutic agent, or as naked antibodies
capable of inhibiting cell proliferation or function.
213P1F11 antibodies can be introduced into a patient such that the antibody
binds to 213P1F11 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 213P1F11, 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 213P1F11 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., Slevers et al. 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. 213P1F11), the cytotoxic agent will exert its known biological
effect (i.e. cytotoxicity) on those
cells.
A wide variety of compositions and methods for using antibody-cytotoxic agent
conjugates to kill
cells are known in the art. In the context of cancers, typical methods entail
administering to an animal having
a tumor a biologically effective amount of a conjugate comprising a selected
cytotoxic and/or therapeutic
agent linked to a targeting agent (e.g. an anti-213P1F11 antibody) that binds
to a marker (e.g. 213P1F11)
expressed, accessible to binding or localized on the cell surfaces. A typical
embodiment is a method of
delivering a cytotoxic and/or therapeutic agent to a cell expressing 213P1F11,
comprising conjugating the
cytotoxic agent to an antibody that immunospecifically binds to a 213P1F11
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-213P1F11 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, Crit. Rev. Immunol. 18:133-138),
multiple myeloma (Ozaki et al.,
1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric
cancer (Kasprzyk et al.,
1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.
Immunother. Emphasis
Tumor Immunol. 19:93-101), leukemia (thong et al., 1996, Leuk. Res. 20:581-
589), colorectal cancer (Moon
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et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.
55:4398-4403), and breast cancer
(Shepard et al., 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 Y9~ or
I~3~ to anti-CD20 antibodies (e.g.,
ZevalinTM, IDEC Pharmaceuticals Corp. or BexxarT"', Coulter Pharmaceuticals),
while others involve co-
administration of antibodies and other therapeutic agents, such as HerceptinrM
(trastuzumab) with paclitaxel
(Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To
neat prostate cancer, for
example, 213P1F11 antibodies can be administered in conjunction with
radiation, chemotherapy or hormone
ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin
(e.g., MylotargTM, Wyeth-
Ayerst, Madison, NJ, a recombinant humanized IgG4 kappa antibody conjugated to
antitumor antibiotic
calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug,
TAP, platform, ImmunoGen,
Cambridge, MA, also see e.g., US Patent 5,416,064).
Although 213P1F11 antibody therapy is useful for all stages of cancer,
antibody therapy can be
particularly appropriate in advanced or metastatic cancers. Treatment with the
antibody therapy of the
invention is indicated for patients who have received one or more rounds of
chemotherapy. Alternatively,
antibody therapy of the invention is combined with a chemotherapeutic or
radiation regimen for patients who
have not received chemotherapeutic treatment. Additionally, antibody therapy
can enable .the use of reduced
dosages of concomitant chemotherapy, particularly for patients who do not
tolerate the toxicity of the
chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993),
Prewett et al. (International
J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580,
1991) describe the use of
various antibodies together with chemotherapeutic agents.
Although 213P1F11 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 213P1F11
expression, preferably
using immunohistochemical assessments of tumor tissue, quantitative 213P1F11
imaging, or other techniques
that reliably indicate the presence and degree of 213P1F11 expression.
Immunohistochemical analysis of
tumor biopsies or surgical specimens is preferred for this purpose. Methods
for immunohistochemical
analysis of tumor tissues are well known in the art.
Anti-213P1F11 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-213P i F 11
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-213P1F11 mAbs that exert a direct biological effect on tumor
growth are useful to treat cancers
that express 213P1F11. 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
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apoptosis. The mechanisms) by which a particular anti-213P1F11 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 213P1F11 antigen
with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single
anti-213P1F11 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-
213P1F11 mAbs can be
administered concomitantly with other therapeutic modalities, including but
not limited to various
chemotherapeutic agents, androgen-Mockers, immune modulators (e.g., IL-2, GM-
CSF), surgery or radiation.
The anti-213P1F11 mAbs are administered in their "naked" or unconjugated form,
or can have a therapeutic
agents) conjugated to them.
Anti-213P1F11 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-213P1F11 antibody preparation, via an acceptable
route of administration such as
intravenous injection (IV), typically at a dose in the range of about 0.1, .2,
.3, .4, .5, .6, .7, .8, .9., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, or 25 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 mglkg patient body weight TV,
followed by weekly doses of about
2 mg/kg IV of the anti-213P1F11 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
213P1F11 expression in the patient, the extent of circulating shed 213P1F11
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 213P 1 F 11 in a
given sample (e.g. the levels
of circulating 213P1F11 antigen and/or 213P1F11 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
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therapy, and are useful to gauge therapeutic success in combination with the
evaluation of other parameters
(for example, urine cytology and/or ImmunoCyt levels in bladder cancer
therapy, or by analogy, serum PSA
levels in prostate cancer therapy).
Anti-idiotypic anti-213P1F11 antibodies can also be used in anti-cancer
therapy as a vaccine for
inducing an immune response to cells expressing a 213P1F11-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-213P1F11 antibodies that mimic an epitope on a 213P1F11-related
protein (see, for example,
Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest.
96:334-342; Herlyn et al., 1996,
Cancer Immunol. Irninunother. 43:65-76). Such an anti-idiotypic antibody can
be used in cancer vaccine
strategies.
X.C.) 213P1F11 as a TarEet for Cellular Immune Responses
Vaccines and methods of preparing vaccines that contain an immunogenically
effective amount of
one or more HLA-binding peptides as described herein are further embodiments
of the invention.
Furthermore, vaccines in accordance with the invention encompass compositions
of one or more of the
claimed peptides. A peptide can be present in a vaccine individually.
Alternatively, the peptide can exist as a
homopolymer comprising multiple copies of the same peptide, or as a
heteropolymer of various peptides.
Polymers have the advantage of increased immunological reaction and, where
different peptide epitopes are
used to make up the polymer, the additional ability to induce antibodies
and/or CTLs that react with different
antigenic determinants of the pathogenic organism or tumor-related peptide
targeted for an immune response.
The composition can be a naturally occurring region of an antigen or can be
prepared, e.g., recombinantly or
by chemical synthesis.
Carriers that can be used with vaccines of the invention are well known in the
art, and include, e.g.,
thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino
acids such as poly L-lysine,
poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like.
The vaccines can contain a
physiologically tolerable (i.e., acceptable) diluent such as water, or saline,
preferably phosphate buffered
saline. The vaccines also typically include an adjuvant. Adjuvants such as
incomplete Freund's adjuvant,
aluminum phosphate, aluminum hydroxide, or alum are examples of materials well
known in the art.
Additionally, as disclosed herein, CTL responses can be primed by conjugating
peptides of the invention to
lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl- serine (P3CSS).
Moreover, an adjuvant such as a
synthetic cytosine-phosphorothiolated-guanine-containing (CpG)
oligonucleotides has been found to increase
CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol.
165:539-547 (2000))
Upon immunization with a peptide composition in accordance with the invention,
via injection,
aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other
suitable routes, the immune system
of the host responds to the vaccine by producing large amounts of CTLs and/or
HTLs specific for the desired
antigen. Consequently, the host becomes at least partially immune to later
development of cells that express
or overexpress 213P1F11 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
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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 PADRET"'
(Epimmune, San Diego, CA) molecule (described e.g., in U.S. Patent Number
5,736,142).
A vaccine of the invention can also include antigen-presenting cells (APC),
such as dendritic cells
(DC), as a vehicle to present peptides of the invention. Vaccine compositions
can be created in vitro,
following dendritic cell mobilization and harvesting, whereby loading of
dendritic cells occurs in vitro. For
example, dendritic cells are transfected, e.g., with a minigene in accordance
with the invention, or are pulsed
with peptides. The dendritic cell can then be administered to a patient to
elicit immune responses in vivo.
Vaccine compositions, either DNA- or peptide-based, can also be administered
in vivo in combination with
dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
Preferably, the following principles are utilized when selecting an array of
epitopes for inclusion in a
polyepitopic composition for use in a vaccine, or for selecting discrete
epitopes to be included in a vaccine
and/or to be encoded by nucleic acids such as a minigene. It is preferred that
each of the following principles
be balanced in order to make the selection. The multiple epitopes to be
incorporated in a given vaccine
composition may be, but need not be, contiguous in sequence in the native
antigen from which the epitopes
are derived.
1.) Epitopes are selected which, upon administration, mimic immune responses
that have been
observed to be correlated with tumor clearance. For HLA Class I this includes
3-4 epitopes that come from at
least one tumor associated antigen (TAA). For HLA Class II a similar rationale
is employed; again 3-4
epitopes are selected from at least one TAA (see, e.g., Rosenberg et al.,
Science 278:1447-1450). Epitopes
from one TAA may be used in combination with epitopes from one or more
additional TAAs to produce a
vaccine that targets tumors with varying expression patterns of frequently-
expressed TAAs.
2.) Epitopes are selected that have the requisite binding affinity established
to be correlated
with immunogenicity: for HLA Class I an ICS° of 500 nM or less, often
200 nM or less; and for Class II an
ICso of 1000 nM or less.
3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-
specific motif bearing
peptides, are selected to give broad population coverage. For example, it is
preferable to have at least 80%
population coverage. A Monte Carlo analysis, a statistical evaluation known in
the art, can be employed to
assess the breadth, or redundancy of, population coverage.
4.) When selecting epitopes from cancer-related antigens it is often useful to
select analogs
because the patient may have developed tolerance to the native epitope.
5.) Of particular relevance are epitopes referred to as "nested epitopes."
Nested epitopes occur
where at least two epitopes overlap in a given peptide sequence. A nested
peptide sequence can comprise B
cell, HLA class I and/or HLA class II epitopes. When providing nested
epitopes, a general objective is to
provide the greatest number of epitopes per sequence. Thus, an aspect is to
avoid providing a peptide that is
any longer than the amino terminus of the amino terminal epitope and the
carboxyl terminus of the carboxyl
terminal epitope in the peptide. When providing a mufti-epitopic sequence,
such as a sequence comprising
nested epitopes, it is generally important to screen the sequence in order to
insure that it does not have
pathological or other deleterious biological properties.
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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 acid residues can,
for example, be introduced to
avoid functional 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 functional 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 mufti-epitope minigenes is described below and in, Ishioka et al.,
J. Immun~1. 162:3915-
3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A.
et al., J. Immunol. 157:822,
1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine
16:426, 1998. For example, a
mufti-epitope DNA plasmid encoding supermotif and/or motif bearing epitopes
derived 213P 1F 11, the
PADRE~ universal helper T cell epitope or multiple HTL epitopes from 213P1F11
(see e.g., Tables V-XIX),
and an endoplasmic reticulum-translocating signal sequence can be engineered.
.A vaccine may also comprise
epitopes that are derived from other TAAs.
The immunogenicity of a mufti-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
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include: HLA class I epitopes, HLA class II epitopes; antibody epitopes, a
ubiquitination signal sequence,
and/or an endoplasmic reticulum targeting signal. In addition, HLA
presentation of CTL and HTL epitopes
may be improved by including synthetic (e.g. poly-alanine) or naturally-
occurring flanking sequences
adjacent to the CTL or HTL epitopes; these larger peptides comprising the
epitope(s) are within the scope of
the invention.
The minigene sequence may be converted to DNA by assembling oligonucleotides
that encode the
plus and minus strands of the minigene. Overlapping oligonucleotides (30-100
bases long) may be
synthesized, phosphorylated, purified and annealed under appropriate
conditions using well known
techniques. The ends of the oligonucleotides can be joined, for example, using
T4 DNA ligase. This
synthetic minigene, encoding the epitope polypeptide, can then be cloned into
a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are
preferably included in the
vector to ensure expression in the target cells. Several vector elements are
desirable: a promoter with a down-
stream cloning site for minigene insertion; a polyadenylation signal for
efficient transcription termination; an
E. coli origin of replication; and an E. coli selectable marker (e.g.
ampicillin or kanamycin resistance).
Numerous promoters can be used for this purpose, e.g., the human
cytomegalovirus (hCMV) promoter. See,
e.g., U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter
sequences.
Additional vector modifications may be desired to optimize minigene expression
and
immunogenicity. In some cases, introns are required for efficient gene
expression, and one or more synthetic
or naturally-occurring introns could be incorporated into the transcribed
region of the minigene. The
inclusion of mRNA stabilization sequences and sequences for replication in
mammalian cells may also be
considered for increasing minigene expression.
Once an expression vector is selected, the minigene is cloned into the
polylinker region downstream
of the promoter. This plasmid is transformed into an appropriate E. coli
strain, and DNA is prepared using
standard techniques. The orientation and DNA sequence of the minigene, as well
as all other elements
included in the vector, are confirmed using restriction mapping and DNA
sequence analysis. Bacterial cells
harboring the correct plasmid can be stored as a master cell bank and a
working cell bank.
In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role
in the
immunogenicity of DNA vaccines. These sequences may be included in the vector,
outside the minigene
coding sequence, if desired to enhance immunogenicity.
In some embodiments, a bi-cistronic expression vector which allows production
of both the
minigene-encoded epitopes and a second protein (included to enhance or
decrease immunogenicity) can be
used. Examples of proteins or polypeptides that could beneficially enhance the
immune response if co-
expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing
molecules (e.g., LeIF),
costimulatory molecules, or for HTL responses, pan-DR binding proteins
(PADRET"", Epimmune, San Diego,
CA). Helper (HTL) epitopes can be joined to intracellular targeting signals
and expressed separately from
expressed CTL epitopes; this allows direction of the HTL epitopes to a cell
compartment different than that of
the CTL epitopes. If required, this could facilitate more efficient entry of
HTL epitopes into the HLA class II
pathway, thereby improving HTL induction. In contrast to HTL or CTL induction,
specifically decreasing the
immune response by co-expression of immunosuppressive molecules (e.g. TGF-(3)
may be beneficial in
certain diseases.
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Therapeutic quantities of plasmid DNA can be produced for example, by
fermentation in E, coli,
followed by purification. Aliquots from the working cell banle are used to
inoculate growth medium, and
grown to saturation in shaker flasks or a bioreactor according to well-known
techniques. Plasmid DNA can
be purified using standard bioseparation technologies such as solid phase
anion-exchange resins supplied by
QIAGEN, Inc. (Valencia, California). If required, supercoiled DNA can be
isolated from the open circular
and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of
formulations. The simplest of
these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline
(PBS). This approach, known as
"naked DNA," is currently being used for intramuscular (IM) administration in
clinical trials. To maximize
the immunotherapeutic effects of minigene DNA vaccines, an alternative method
for formulating purified
plasmid DNA may be desirable. A variety of methods have been described, and
new techniques may become
available. Cationic lipids, glycolipids, and fusogenic liposomes can also be
used in the formulation (see, e.g.,
as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682
(1988); U.S. Pat No.
5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'1 Acad. Sci. USA
84:7413 (1987). In addition,
peptides and 'compounds referred to collectively as protective, interactive,
non-condensing compounds
(PINC) could also be complexed to purified plasmicLDNA 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 (SICr) labeled and used as target
cells for epitope-specific CTL
lines; cytolysis, detected by SICr 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, S~Cr-labeled
target cells using standard techniques.
Lysis of target cells that were sensitized by HLA loaded with peptide
epitopes, corresponding to minigene-
encoded epitopes, demonstrates DNA vaccine function for in vivo induction of
CTLs. Immunogenicity of
HTL epitopes is confirmed in transgenic mice in an analogous manner.
Alternatively, the nucleic acids can be administered using ballistic delivery
as described, for
instance, in U.S. Patent No. 5,204,253. Using this technique, particles
comprised solely of DNA are
administered. In a further alternative embodiment, DNA can be adhered to
particles, such as gold particles.
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Minigenes can also be delivered using other bacterial or viral delivery
systems well known in the art,
e.g., an expression construct encoding epitopes of the invention can be
incorporated into a viral vector such as
vaccinia.
X.C.2. Combinations of CTL Peptides with Helper Peptides
Vaccine compositions comprising CTL peptides of the invention can be modified,
e.g., analoged, to
provide desired attributes, such as improved serum half life, broadened
population coverage or enhanced
immunogenicity.
For instance, the ability of a peptide to induce CTL activity can be enhanced
by linking the peptide
to a sequence which contains at least one epitope that is capable of inducing
a T helper cell response.
Although a CTL peptide can be directly linked to a T helper peptide, often CTL
epitope/HTL epitope
conjugates are linked by a spacer molecule. The spacer is typically comprised
of relatively small, neutral
molecules, such as amino acids or amino acid mimetics, which are substantially
uncharged under
physiological conditions. The spacers are typically selected from, e.g., Ala,
Gly, or other neutral spacers of
nonpolar amino acids or neutral polar amino acids. It will be understood that
the optionally present spacer
need not be comprised of the same residues and thus may be a hetero- or homo-
oligomer. When present, the
spacer will usually be at least one or two residues, more usually three to six
residues and sometimes 10 or
more residues. The CTL peptide epitope can be linked to the T helper peptide
epitope either directly or via a
spacer either at the amino or carboxy terminus of the CTL peptide. The amino
terminus of either the
immunogenic peptide or the T helper peptide may be acylated.
In certain embodiments, the T helper peptide is one that is recognized by T
helper cells present in a
majority of a genetically diverse population. This can be accomplished by
selecting peptides that bind to
many, most, or all of the HLA class II molecules. Examples of such amino acid
bind many HLA Class II
molecules include sequences from antigens such as tetanus toxoid at positions
830-843 (QYII~ANSKFIGITE;
SEQ ID NO: 38), Plasmodium falciparum circumsporozoite (CS) protein at
positions 378-398
(DIEKKLAI~MEKASSVFNWNS; SEQ ID NO: 39), and Streptococcus lBkD protein at
positions 116-131
(GAVDSILGGVATYGAA; SEQ )D NO: 40). 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.,
PADRET"", Epimmune, Inc., San Diego, CA) are designed to most preferably bind
most HLA-DR (human
HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having
the formula:
aKXVAAWTLKAAa (SEQ II? NO: 41), where "X" is either cyclohexylalanine,
phenylalanine, or tyrosine,
and a is either D=alanine or L-alanine, has been found to bind to most HLA-DR
alleles, and to stimulate the '
response of T helper lymphocytes from most individuals, regardless of their
HLA type. An alternative of a
pan-DR binding epitope comprises all "L" natural amino acids and can be
provided in the form of nucleic
acids that encode the epitope.
HTL peptide epitopes can also be modified to alter their biological
properties. For example, they
can be modified to include D-amino acids to increase their resistance to
proteases and thus extend their serum
half life, or they can be conjugated to other molecules such as lipids,
proteins, carbohydrates, and the like to
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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 s-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 ~- and a- amino groups of Lys, which is attached via
linkage, e.g., Ser-Ser, to the
amino terminus of the immunogenic peptide.
As another example of lipid priming of CTL responses, E. coli lipoproteins,
such as tripalmitoyl-S-
glycerylcysteinlyseryl- serine (P3CSS) can be used to prime virus specific CTL
when covalently attached to
an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989).
Peptides of the invention can be
coupled to P3CSS, for example, and the lipopeptide administered to an
individual to specifically prime an
immune response to the target antigen. Moreover, because the induction of
neutralizing antibodies can also
be primed with P3CSS-conjugated epitopes, two such compositions can be
combined to more effectively elicit
both humoral and cell-mediated responses.
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
An embodiment of a vaccine composition in accordance with the invention
comprises ex vivo
administration of a cocktail of epitope-bearing peptides to PBMC, or isolated
DC therefrom, from the
patient's blood. A pharmaceutical to facilitate harvesting of DC can be used,
such as ProgenipoietinT"'
(Pharmacia-Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with
peptides and prior to
reinfusion into patients, the DC are washed to remove unbound peptides. In
this embodiment, a vaccine
comprises peptide-pulsed DCs which present the pulsed peptide epitopes
complexed with HLA molecules on
their surfaces.
The DC can be pulsed ex vivo with a cocktail of peptides, some of which
stimulate CTL responses to
213P1F11. 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 213P1F11.
X.D. Adoptive Immunotherapy
Antigenic 213P1F11-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
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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 Pronhvlactic Purposes
Pharmaceutical and vaccine compositions of the invention are typically used to
treat and/or prevent a
cancer that expresses or overexpresses 213P1F11. 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 213P1F11. 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 213P1F11-
associated cancer. 'This is followed by boosting doses until at least symptoms
are substantially abated and for
a period thereafter. The embodiment of the vaccine composition (i. e.,
including, but not limited to
embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes,
or TAA-specific CTLs or
pulsed dendritic cells) delivered to the patient may vary according to the
stage of the disease or the patient's
health status. For example, in a patient with a tumor that expresses 213P1F11,
a vaccine comprising
213P1F11-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 effectively stimulate 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 ~.g and the higher value is about
10,000; 20,000; 30,000; or
50,000 ~tg. Dosage values for a human typically range from about 500 gg to
about 50,000 ~.g per 70 kilogram
patient. Boosting dosages of between about 1.0 ~g to about 50,000 pg of
peptide pursuant to a boosting
regimen over weeks to months may be administered depending upon the patient's
response and condition as
determined by measuring the specific activity of CTL and HTL obtained from the
patient's blood.
Administration should continue until at least clinical symptoms or laboratory
tests indicate that the neoplasia,
has been eliminated or reduced and for a period thereafter. The dosages,
routes of administration, and dose
schedules are adjusted in accordance with methodologies known in the art.
In certain embodiments, the peptides and compositions of the present invention
are employed in
serious disease states, that is, life-threatening or potentially life
threatening situations. In such cases, as a
result of the minimal amounts of extraneous substances and the
relative.nontoxic nature of the peptides in
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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 ~.g and the higher value is about
10,000; 20,000; 30,000; or 50,000 p,g.
Dosage values for a human typically range from about 500 ~,g to about 50,000
~,g per 70 kilogram patient.
This is followed by boosting dosages of between about 1.0 ~tg 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
Garner, preferably an aqueous
carrier.
A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8%
saline, 0.3% glycine,
hyaluronic acid and the like. These compositions may be sterilized by
conventional, well-known sterilization
techniques, or may be sterile filtered. The resulting aqueous solutions may be
packaged for use as is, or
lyophilized, the lyophilized preparation being combined with a sterile
solution prior to administration.
The compositions may contain pharmaceutically acceptable auxiliary substances
as required to ,
approximate physiological conditions, such as pH-adjusting and buffering
agents, tonicity adjusting agents,
wetting agents, preservatives, and the like, for example, sodium acetate,
sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine
oleate, etc.
The concentration of peptides of the invention in the pharmaceutical
formulations can vary widely,
i.e., from less than about 0.1%, usually at or at least about 2% to as much as
20% to 50% or more by weight,
and will be selected primarily by fluid volumes, viscosities, etc., in
accordance with the particular mode of
administration selected.
A human unit dose form of a composition is typically included in a
pharmaceutical composition that
comprises a human unit dose of an acceptable carrier, in one embodiment an
aqueous carrier, and is
administered in a volume/quantity that is known by those of skill in the art
to be used for administration of
such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences,
17'~ 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 ~tg, generally 100-5,000 ltg,
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 m) in the amounts of 0.5-S mg
at multiple sites. The nucleic
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 5-10' to 5x109 pfu.
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For antibodies, a treatment generally involves repeated administration of the
anti-213P 1F11 antibody
preparation, via an acceptable route of administration such as intravenous
injection (IV), typically at a dose in
the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the
range of 10-500 mg mAb per
week are effective and well tolerated. Moreover, an initial loading dose of
approximately 4 mg/kg patient
body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-
213P1F11 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
213P1F11 expression in the
patient, the extent of circulating shed 213P1F11 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, SOOg.g - lmg, lmg - SOmg, SOmg - lOQmg,
100mg - 200mg, 200mg -
300mg, 400mg - SOOmg, SOOmg - 600mg, 600mg - 700mg, 700mg - 800mg, 800mg -
900mg, 900mg - lg, or
lmg - 700mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body
weight, e.g., with follow on
weekly doses of 1-3 mg/kg; O.Smg, 1, 2, 3, 4, 5, 6, 7, 8, 9, lOmg/kg body
weight followed, e.g., in two, three
or four weeks by weekly doses; 0.5 - lOmg/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 m' 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 lilee. 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, S00 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral
routes of administration may
require higher doses of polynucleotide compared to more direct application to
the nucleotide to diseased
tissue, as do polynucleotides of increasing length.
In one embodiment, human unit dose forms of T-cells comprise a suitable dosage
range or effective
amount that provides any therapeutic effect. As appreciated by one of ordinary
skill in the art, a therapeutic
effect depends on a number of factors. Dosages are generally selected by the
physician or other health care
professional in accordance with a variety of parameters known in the art, such
as severity of symptoms,
history of the patient and the like. A dose may be about 104 cells to about
106 cells, about 106 cells to about
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108 cells, about 10$ to about 10~ ~ cells, or about 108 to about 5 x
10~° cells. A dose may also about 106
cells/mz to about 10~° cells/mz, or about 106 cells/m' to about 108
cells/m' .
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
can be directed to the site of lymphoid cells, where the liposomes then
deliver the peptide compositions.
Liposomes for use in accordance with the invention are formed from standard
vesicle-forming lipids, which
generally include neutral and negatively charged phospholipids and a sterol,
such as cholesterol. The
selection of lipids is generally guided by consideration of, e.g., liposome
size, acid lability and stability of the
liposomes in the blood stream. A variety of methods are available for
preparing liposomes, as described in,
e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Patent
Nos. 4,235,871, 4,501,728,
4,837,028, and 5,019,369.
For targeting cells of the immune system, a ligand to be incorporated into the
liposome can include,
e.g., antibodies or fragments thereof specific for cell surface determinants
of the desired immune system cells.
A liposome suspension containing a peptide may be administered intravenously,
locally, topically, etc. in a
dose which varies according to, inter alia, the manner of administration, the
peptide being delivered, and the
stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used which
include, for
example, pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talcum,
cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral
administration, a pharmaceutically
acceptable nontoxic composition is formed by incorporating any of the normally
employed excipients, such as
those carriers previously listed, and generally 10-95% of active ingredient,
that is, one or more peptides of the
invention, and more preferably at a concentration of 25%-75%.
For aerosol administration, immunogenic peptides are preferably supplied in
finely divided form
along with a surfactant and propellant. Typical percentages of peptides are
about 0.01 %-20% by weight,
preferably about 1%-10%. The surfactant must, of course, be nontoxic, and
preferably soluble in the
propellant. Representative of such agents are the esters or partial esters of
fatty acids containing from about 6
to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,
linoleic, linolenic, olesteric and oleic
acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed
esters, such as mixed or natural
glycerides may be employed. The surfactant may constitute about 0.1%-20% by
weight of the composition,
preferably about 0.25-5%. The balance of the composition is ordinarily
propellant. A carrier can also be
included, as desired, as with, e.g., lecithin for intranasal delivery.
XI 1 Diagnostic and Prognostic Embodiments of 213P1F11.
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As disclosed herein, 213P1F11 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
213P1F11 in Normal Tissues and Patient Specimens").
213P1F11 can be analogized to a prostate associated antigen PSA, the
archetypal marker that has
been used by medical practitioners for years to identify and monitor the
presence of prostate cancer (see, e.g.,
Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol.
Aug; 162(2):293-306 (1999) and
Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of
other diagnostic markers are also
used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et
al., Int J Mol Med 1999 Jul 4(1):99-
102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, this
disclosure of 213P1F11
polynucleotides and polypeptides (as well as 213P1F11 polynucleotide probes
and anti-213P1F11 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 213P1F11
polynucleotides,
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 polynucleotides are used as probes (for example in Northern
analysis, see, e.g., Sharief et al.,
Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR
analysis, see, e.g., Okegawa
et 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
213P1F11 polynucleotides
described herein can be utilized in the same way to detect 213P1F11
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 et al.,
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
213P1F11 polypeptides described herein can be utilized to generate antibodies
for use in detecting 213P1F11
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 213P1F11
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 213P1F11-expressing cells (lymph node)
is found to contain 213P1F11-
expressing cells such as the 213P1F11 expression seen in LAPC4 and LAPC9,
xenografts isolated from
lymph node and bone metastasis, respectively, this finding is indicative of
metastasis.
Alternatively 213P1F11 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 213P1F11 or express
213P1F11 at a different level are found to express 213P1F11 or have an
increased expression of 213P1F11
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(see, e.g., the 213P1F11 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
213P1F11) 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, 213P1F11 polynucleotide fragments and
polynucleotide variants are
used in an analogous manner. In particular, typical PSA polynucleotides used
in methods of monitoring PSA
are probes or primers which consist of fragments of the PSA cDNA sequence.
Illustrating this, primers used
to PCR amplify a PSA polynucleotide must include less than the whole PSA
sequence to function in the
polymerase chain reaction. In the context of such PCR reactions, skilled
artisans generally create a variety of
different polynucleotide fragments that can be used as primers in order to
amplify different portions of a
polynucleotide of interest or to optimize amplification reactions (see, e.g.,
Caetano-Anolles, G. Biotechniques
25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-
154 (1998)). An additional
illustration of the use of such fragments is provided in the Example entitled
"Expression Analysis of
213P1F11 in Normal Tissues and Patient Specimens," where a 213P1F11
polynucleotide fragment is used as
a probe to show the expression of 213P1F11 RNAs in cancer cells. In addition,
variant polynucleotide
sequences are typically used as primers and probes for the corresponding mRNAs
in PCR and Northern
analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 Nov-Dec 11(6):407-
13 and Current Protocols In
Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)).
Polynucleotide fragments
and variants are useful in this context where they are capable of binding to a
target polynucleotide sequence
(e.g., a 213P1F11 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. 213P1F11 polypeptide
fragments and polypeptide analogs or variants can also be used in an analogous
manner. This practice of
using polypeptide fragments or polypeptide variants to generate antibodies
(such as anti-PSA antibodies or T
cells) is typical in the art with a wide variety of systems such as fusion
proteins being used by practitioners
(see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16,
Frederick M. Ausubel et al. eds.,
1995). In this context, each epitope(s) functions to provide the architecture
with which an antibody or T cell
is reactive. Typically, skilled artisans create a variety of different
polypeptide fragments that can be used in
order to generate immune responses specific for different portions of a
polypeptide of interest (see, e.g., U.S.
Patent No. 5,840,501 and U.S. Patent No. 5,939,533). For example it may be
preferable to utilize a
polypeptide comprising one of the 213P1F11 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
213P1F11 polypeptide shown in Figure 3).
As shown herein, the 213P1F11 polynucleotides and polypeptides (as well as the
213P1F11
polynucleotide probes and anti-213P1F11 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 213P1F11 gene
products, in order to evaluate the
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presence or onset of a disease condition described herein, such as prostate
cancer, are used to identify patients
for preventive measures or further monitoring, as has been done so
successfully with PSA. Moreover, these
materials satisfy a need in the art for molecules having similar or
complementary characteristics to PSA in
situations where, for example, a definite diagnosis of metastasis of prostatic
origin cannot be made on the
basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.
192(3): 233-237 (1996)), and
consequently, materials such as 213P1F11 polynucleotides and polypeptides (as
well as the 213P1F11
polynucleotide probes and anti-213P1F11 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 213P1F11
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 213P1F11 gene maps (see
the Example entitled
"Chromosomal Mapping of 213P1F11" below). Moreover, in addition to their use
in diagnostic assays, the
213P1F11-related proteins and polynucleotides disclosed herein have other
utilities such as their use in the
forensic analysis of tissues of unlrnown origin (see, e.g., Takahama K
Forensic Sci Int 1996 Jun 28;80(1-2):
63-9).
Additionally, 213P1F11-related proteins or polynucleotides of the invention
can be used to treat a
pathologic condition characterized by the over-expression of 213P1F11. 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 213P1F11 antigen. Antibodies or other molecules that react with
213P1F11 can be used to
modulate the function of this molecule, and thereby provide a therapeutic
benefit.
XIL) Inhibition of 213P1F11 Protein Function
The invention includes various methods and compositions for inhibiting the
binding of 213P1F11 to
its binding partner or its association with other proteins) as well as methods
for inhibiting 213P1F11
function.
XILA.I Inhibition of 213P1F11 With Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies
that specifically bind to
213P1F11 are introduced into 213P1F11 expressing cells via gene transfer
technologies. Accordingly, the
encoded single chain anti-213P1F11 antibody is expressed intracellularly,
binds to 213P1F11 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
comparhnent within the cell, providing control over where the inhibitory
activity of the treatment is focused.
This technology has been successfully applied in the art (for review, see
Richardson and Marasco, 1995,
TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the
expression of otherwise abundant
cell surface receptors (see, e.g., lRichardson et al., 1995, Proc. Natl. Acad.
Sci. USA 92: 3137-3141; Beerli et
al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.
1: 332-337).
Single chain antibodies comprise the variable domains of,the heavy and light
chain joined by a
flexible linker 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
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chain antibodies in order to precisely target 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 213P1F11 in the nucleus,
thereby preventing its
activity within the nucleus. Nuclear targeting signals are engineered into
such 213P1F11 intrabodies in order
to achieve the desired targeting. Such 213P1F11 intrabodies are designed to
bind specifically to a particular
213P1F11 domain. In another embodiment, cytosolic intrabodies that
specifically bind to a 213P1F11 protein
are used to prevent 213P1F11 from gaining access to the nucleus, thereby
preventing it from exerting any
biological activity within the nucleus (e.g., preventing 213P1F11 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 promoterlenhancer can be utilized (See, for example, U.S. Patent No.
5,919,652 issued 6 July 1999).
X_II B ) Inhibition of 213P1F11 with Recombinant Proteins
In another approach, recombinant molecules bind to 213P1F11 and thereby
inhibit 213P1F11
function. For example, these recombinant molecules prevent or inhibit 213P1F11
from accessing/binding to
its binding partners) or associating with other protein(s). Such recombinant
molecules can, for example,
contain the reactive parts) of a 213P1F11 specific antibody molecule. In a
particular embodiment, the 213P 1F11
binding domain of a 213P1F11 binding partner is engineered into a dimeric
fusion protein, whereby the fusion
protein comprises two 213P 1 Fl l ligand binding domains linked to the Fc
portion of a human IgG, such as human
IgGl. Such IgG portion can contain, for example, the C~2 and CH3 domains and
the hinge region, but not the
CHl domain. Such dimeric fusion proteins are administered in soluble form to
patients suffering from a cancer
associated with the expression of 213P1F11, whereby the dimeric fusion protein
specifically binds to 213P1F11
and blocks 213P1F11 interaction with a binding partner. Such dimeric fusion
proteins are further combined into
multimeric proteins using known antibody linking technologies.
XII C.1 Inhibition of 213P1F11 Transcription or Translation
The present invention also comprises various methods and compositions for
inhibiting the
transcription of the 213P1F11 gene. Similarly, the invention also provides
methods and compositions for
inhibiting the translation of 213P1F11 mRNA into protein.
In one approach, a method of inhibiting the transcription of the 213P1F11 gene
comprises contacting
the 213P1F11 gene with a 213P1F11 antisense polynucleotide. In another
approach, a method of inhibiting
213P1F11 mRNA translation comprises contacting a 213P1F11 mRNA with an
antisense polynucleotide. In
another approach, a 213P1F11 specific ribozyme is used to cleave a 213P1F11
message, thereby inhibiting
translation. Such antisense and ribozyme based methods can also be directed to
the regulatory regions of the
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213P1F11 gene, such as 213P1F11 promoter and/or enhancer elements. Similarly,
proteins capable of
inhibiting a 213P1F11 gene transcription factor are used to inhibit 213P1F11
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 213P1F11 by interfering with
213P1F11 transcriptional
activation are also useful to treat cancers expressing 213P1F11. Similarly,
factors that interfere with
213P1F11 processing are useful to treat cancers that express 213P1F11. Cancer
treatment methods utilizing
such factors are also within the scope of the invention.
XILD.1 General Considerations for Therapeutic Strateeies
Gene transfer and gene therapy technologies can be used to deliver therapeutic
polynucleotide molecules
to tumor cells synthesizing 213P1F11 (i.e., antisense, ribozyme,
polynucleotides encoding intrabodies and other
213P 1F11 inhibitory molecules). A number of gene therapy approaches are known
in the art. Recombinant
vectors encoding 213P1F11 antisense polynucleotides, ribozymes, factors
capable of interfering with 213P1F11
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 ~f 213P 1F 11 to a binding partner, etc.
In vivo, the effect of a 213P1F11 therapeutic composition can be evaluated in
a suitable animal model.
For example, xenogenic prostate cancer models can be used, wherein human
prostate cancer explants or passaged
xenograft tissues are introduced into immune compromised animals, such as nude
or SC1D mice (Klein et al.,
1997, Nature Medicine 3: 402-408). For example, PCT Patent Application
WO98/16628 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 vivv 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
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include, but are not limited to, any of a number of standard pharmaceutical
carriers such as sterile phosphate
buffered saline solutions, bacteriostatic water, and the like (see, generally,
Remington's Pharmaceutical
Sciences 16'h Edition, A. O'sal., 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.
X1IL) Kits
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
containers) comprising one of the
separate elements to be used in the method. For example, the containers) can
comprise a probe that is or can
be detectably labeled. Such probe can be an antibody or polynucleotide
specific for a 213P1F11-related
protein or a 213P1F11 gene or message, respectively. Where the method utilizes
nucleic acid hybridization to
detect the target nucleic acid, the kit can also have containers containing
nucleotides) 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 sequence of 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, 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, and can also indicate directions for either in
vivo or in vitro use, such as those
described above. Directions and or other information can also be included on
an insert which is included with the
kit.
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.
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Example 1: SSH-Generated Isolation of a cDNA Fragment of the 213P1F11 Gene
To isolate genes that are over-expressed in bladder cancer, Suppression
Subtractive Hybridization
(SSH) procedure using cDNA derived from bladder cancer tissues was performed,
including invasive
transitional cell carcinoma. The 213P1F11 SSH cDNA sequence was derived from a
bladder cancer pool
minus cDNAs derived from 9 normal tissues. The 213P1F11 cDNA was identified as
highly expressed in the
bladder cancer tissue pool, with no expression detected in normal tissues.
The SSH DNA sequence of 166 by (Figure 1) did not show homology to any known
gene.
213P 1F1 lv.l of 3336 by was identified and the open reading frame cloned from
bladder cancer cDNA,
revealing an ORF of 242 amino acids (Figure 2 and Figure 3). Other variants of
213PiFl 1, were also
identified and these are listed in Figures 2 and 3. 213P1F11 v.l reveals 100%
identity to caspase-14
precursor apoptosis-related cysteine protease protein (Figure 4).
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 (O.D.
260/280 nm) and analyzed by
gel electrophoresis.
Oligonucleotides:
The following HPLC purified oligonucleotides were used.
DPNCDN fcDNA synthesis primed:
5'TTTTGATCAAGCTT3o3' (SEQ ID NO: 42)
Adaptor 1:
S'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 43)
3'GGCCCGTCCTAGS' (SEQ ID NO: 44)
Adaptor 2:
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 45)
3'CGGCTCCTAGS' (SEQ ID NO: 46)
PCR primer 1:
5'CTAATACGACTCACTATAGGGC3' ~ ~ (SEQ ID NO: 47)
Nested primer (NP) 1:
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5'TCGAGCGGCCGCCCGGGCAGGA3' ~(SEQ ID NO: 48)
Nested primer (NP~2:
5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 49)
Suppression Subtractive Hybridization:
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs
corresponding to genes
that may be differentially expressed in bladder cancer. The SSH reaction
utilized cDNA from bladder cancer
and normal tissues.
'The gene 213P1F11 sequence was derived from a bladder cancer pool minus
normal tissue cDNA
subtraction. The SSH DNA sequence (Figure 1) was identified.
The cDNA derived from of pool of normal tissues was used as the source of the
"driver" cDNA,
while the cDNA from a pool of bladder cancer tissues was used as the source of
the "tester" cDNA. Double
stranded cDNAs corresponding to tester and driver cDNAs were synthesized from
2 ~.g of poly(A)+ RNA
isolated from the relevant xenograft tissue, as described above, using
CLONTECH's PCR-Select cDNA
Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and
second-strand synthesis were
carried out as described in the Kit's user manual protocol (CLONTECH Protocol
No. PTi 117-1, 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 ~tl of Dpn II digested cDNA from the
relevant tissue
source (see above) (400 ng) in 5 ~,l of water. The diluted cDNA (2 pl, 160 ng)
was then ligated to 2 ~,1 of
Adaptor 1 and Adaptor 2 (10 pM), in separate ligation reactions, in a total
volume of 10 ~1 at 16°C overnight
using 400 a of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 ~tl of
0.2 M EDTA and
heating at 72°C for 5 min.
The first hybridization was performed by adding 1.5 ~tl (600 ng) of driver
cDNA to each of two
tubes containing 1.5 pl (20 ng) Adaptor 1- and Adaptor 2- ligated tester cDNA.
In a final volume of 4 ~.1, 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 pl of fresh denatured driver cDNA and were allowed to
hybridize overnight at 68°C. The
second hybridization was then diluted in 200 ~l of 20 mM Hepes, pH 8.3, 50 mM
NaCl, 0.2 mM EDTA,
heated at 70°C for 7 min. and stored at -20°C.
PCR Amplification Cloning and Sequenci~ 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 ~tl of the diluted final hybridization mix was
added to 1 ~tl of PCR primer 1 (10
~M), 0.5 E~l dNTP mix ( 10 ~M), 2.5 ~tl 10 x reaction buffer (CLONTECH) and
0.5 ~tl 50 x Advantage cDNA
polymerase Mix (CLONTECH) in a final volume of 25 pl. PCR 1 was conducted
using the following
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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 ~1 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 ~M)
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.l using the T/A vector cloning kit
(Invitrogen).
Transformed E. coli were subjected to blue/white and ampicillin selection.
White colonies were picked and
arrayed into 96 well plates and were grown in liquid culture overnight. To
identify inserts, PCR amplification
was performed on 1 ml of bacterial culture using the conditions of PCRl and
NP1 and NP2 as primers. PCR
products were analyzed using 2% agarose gel electrophoresis.
Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA
was prepared,
sequenced, and subjected to nucleic acid homology searches of the GenBank,
dBest, and NCI-CGAP
databases.
RT-PCR Expression Anal:
First strand cDNAs can be generated from 1 ~g of mRNA with oligo (dT)12-18
priming using the
Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was
used which included an
incubation for 50 min at 42°C with reverse transcriptase followed by
RNAse H treatment at 37°C for 20 min.
After completing the reaction, the volume can be increased to 200 ~1 with
water prior to normalization. First
strand cDNAs from 16 different normal human tissues can be obtained from
Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by
using the primers
5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 50) and 5'agccacacgcagctcattgtagaagg
3' (SEQ ID NO: 51) to
amplify ~3-actin. First strand cDNA (5 itl) were amplified in a total volume
of 50 ~1 containing 0.4 ~M
primers, 0.2 pM 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 ~1 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.
(3-actin bands from multiple tissues
were compared by visual inspection. Dilution factors for the first strand
cDNAs were calculated to result in
equal ~3-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 deterniinc expression levels of the 213P1F11 gene, 5 ~l 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 213P1F11 SSH sequence and are listed below:
Z13P1F11.1
5'- GGATACCAGGGAACGCTTGGAG - 3' (SEQ iD NO: 52)
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213P1F11.2
5'- TTTGACCTTTCCTGCTCAAGTAACC - 3' (SEQ ID NO: 53)
A typical RT-PCR expression analysis is shown in Figure 14. First strand cDNA
was prepared from
vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and
stomach), LAPC xenograft pool
(LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, breast
cancer pool, and cancer
metastasis pool. Normalization was performed by PCR using primers to actin and
GAPDH. Semi-
quantitative PCR, using primers to 213P1F11, was performed at 26 and 30 cycles
of amplification. Results
show. strong expression of 213P1F11 in bladder cancer pool, breast cancer
pool, xenograft pool, and cancer
metastasis pool, but not in the vital pools.
E_xamnle 2~ Full Length Cloning of 213P1F11
The 213P1F11 SSH cDNA sequence was derived from a bladder cancer pool minus
normal tissues
cDNA subtraction. The SSH cDNA sequence (Figure 1) was designated 213P1F11.
'The SSH DNA sequence of 166 by (Figure 1) did not show homology to any known
gene. The full-
length cDNA 213P1F11 was cloned from bladder cancer cDNA. Variants of 213P1F11
were identified and
these are listed in Figures 2 and 3. 213P1F11 v.l reveals 100°f°
identity to caspase-14 precursor apoptosis-
related cysteine protease protein (Figure 4).
Example 3~ Chromosomal Manpins of 213PiF11
Chromosomal localization can implicate genes in disease pathogenesis. Several
chromosome
mapping approaches are available including fluorescent in situ hybridization
(FISH), human/hamster
radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22;
Research Genetics, Huntsville Al),
human-rodent somatic cell hybrid panels such as is available from the Coriell
Institute (Camden, New Jersey),
and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic
clones (NCBI,
Bethesda, Maryland).
213P1F11 maps to chromosome 19p13.1 using 213P1F11 sequence and the NCBI BLAST
tool:
located at the World Wide Web
(.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).
Example 4~ Exuression Analysis of 213P1F11 in Normal Tissues and Patient
Specimens
Expression analysis by RT-PCR demonstrated that 213P1F11 is strongly expressed
in bladder cancer
patient specimens (Figure 14). First strand cDNA was prepared from vital pool
1 (liver, lung and kidney),
vital pool 2 (pancreas, colon and stomach), LAPC.xenograft pool (LAPC-4AD,
LAPC-4AI, LAPC-9AD and
LAPC-9AI), bladder cancer pool, breast cancer pool, and cancer metastasis
pool. Normalization was
performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR,
using primers to 213P1F11,
was performed at 26 and 30 cycles of amplification. Results show strong
expression of 213P1F11 in bladder
cancer pool, breast cancer pool, xenograft pool, and cancer metastasis pool,
but not in the vital pools.
To determine the relative expression of 213P1F11 v.l compared to 213P1F11 v.2
in human cancers,
primers were designed flanking the insertion in 213P1F11 v.2 (Figure 15).
Using these primers, amplification
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of 213PIF11 v.l will generate a PCR fragment of 165 bp, whereas 213P1F11 v.2
will generate a PCR
fragment of 249 by as depicted in Figure 15. The PCR product of 165bp will
also correspond to the variants
213P1F11 v.3, v.4, v.5, v.6 and v.7. First strand cDNA was prepared from vital
pool I (liver, lung and
kidney), bladder cancer pool, breast cancer pool, LAPC xenograft pool (LAPC-
4AD, LAPC-4AI, LAPC-9AD
and LAPC-9AI), and 213PIF11 v.l plasmid control. Normalization was performed
by PCR using primers to
actin and GAPDH. Semi-quantitative PCR, using primers depicted above, was
performed at 35 cycles of
amplification. Results show strong expression of 213P1F11 v.l in bladder
cancer pool, breast cancer pool,
LAPC xenograft pool, and the plasmid positive control. A lower expression of
the 249 by 213P1F11 v.2
product was detected in breast cancer pool, LAPC xenograft pool, and to lower
extent in bladder cancer pool.
Altogether these data show that expression of 213P1F1 I v.l is more abundant
than 213P1F11 v.2 in human
patient cancer samples.
Extensive northern blot analysis of 213PIF11 in multiple human normal tissues
is shown in Figure
16. Strong expression was only detected in skin tissue. A weak transcript is
detected in normal thymus but
not in the other tissues tested.
RNA was extracted from normal prostate, LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-
9AI
prostate cancer xenografts. Northern blot with 10 ~.g of total RNA/lane was
probed with 213P1F11 SSH
sequence (Figure 18). Results show expression of 213P1F11 in the LAPC-9AI
xenograft, but not in the other
xenografts nor in normal prostate.
Expression of 213P1F11 in patient bladder cancer specimens is shown in Figure
17. RNA was
extracted from normal bladder (N), bladder cancer cell lines (UM-UC-3 and
SCaBER), bladder cancer patient
tumors (T) and normal tissue adjacent to bladder cancer (NpT). Northern blots
with 10 ug of total RNA were
probed with the 213P1F11 SSH fragment. Size standards in kilobases are
indicated on the side. Results show
strong expression of 213P1F11 in the bladder tumor tissues but not in normal
bladder, nor in the bladder
cancer cell lines.
Figure 19 shows that 213P1F11 was expressed in breast cancer patient tissues.
RNA was extracted
from normal breast (N), breast cancer cell lines (DU4475, MCF7 and CAMA-1),
breast cancer patient tumors
(T) and breast cancer metastasis to lymph node (Met). Northern blots with 10
ug of total RNA were probed
with the 213P1F11 SSH fragment. Results show strong expression of 213PIF11 in
the breast tumor tissues as
well as in the cancer metastasis specimen. Weak expression was also detected
in the CAMA-1 cell line, but
not in the other 2 breast cancer cell lines tested.
The restricted expression of 213P1F11 in normal tissues and the expression
detected in bladder
cancer, breast cancer, prostate cancer xenograft, and cancer metastases
suggest that 213P1F11 is a potential
therapeutic target and a diagnostic marker for human cancers.
Example 5~ Transcript Variants of 213P1F11
Transcript variants are variants of matured mRNA from the same gene 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 mufti-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.
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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 may encode proteins
with different functions, and
may be expressed in the same tissue at the same time, or at different tissue,
or at different times, proteins
encoded by transcript variants can have similar or different cellular or
extracellular localizations, i.e., be
secreted.
Transcript variants are identified by a variety of art-accepted methods. For
example, alternative
transcripts and splice variants are identified in a full-length cloning
experiment, or by use of full-length
transcript and EST sequences. First, all human ESTs were grouped into clusters
which show direct or indirect
identity with each other. Second, 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 sequences)
or other full-length sequences. Each consensus sequence is a potential.splice
variant for that gene (see, e.g.,
located at the World Wide Web (.doubletwist.com/products/cl l
agentsOverview.jhtml). Even when a
variant is identified that is not a full-length clone, that portion of the
variant is very useful for antigen
generation and for further cloning of the full-length splice variant, using
techniques known in the art.
Moreover, computer programs are available in the art that identify transcript
variants based on
genomic sequences. Genomic-based transcript variant identification programs
include FgenesH (A. Salamov
and V. Solovyev, "Ab initio gene fording in Drosophila genomic DNA," Genome
Research. 2000
Apri1;10(4):516-22); Grail (http://compbio.oml.gov/Grail-bin/EmptyGrailForm)
and GenScan
(http://genes.mit.edu/GENSCAN.html). 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., et al., Identification of human chromosome 22 transcribed
sequences with ORF expressed
sequence tags, Proc. Natl Acad Sci U S A. 2000 Nov 7; 97(23):12690-3.
To further confirm the parameters of a transcript variant, a variety of
techniques are available in the
art, such as full-length cloning, proteomic validation, PCR-based validation,
and 5' RACE validation, etc.
(see e.g., Proteomic Validation: Brennan, S.O., et al., Albumin banks
peninsula: a new termination variant
characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999
Aug 17;1433(1-2):321-6;
Ferranti P, et al., Differential splicing of pre-messenger RNA produces
multiple forms of mature caprine
alpha(sl)-casein, Eur J Biochem. 1997 Oct 1;249(1):1-7. For PCR-based
Validation: Wellinann S, et al.,
Specific reverse transcription-PCR quantification of vascular endothelial
growth factor (VEGF) splice
variants by LightCycler technology, Clin Chem. 2001 Apr;47(4):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 marine
reduced folate carrier gene and
identification of variant splice forms, Biochem Biophys Acta. 1997 Aug 7;
1353(2): 191-8).
It is known in the art that genomic regions are modulated in cancers. When the
genomic region, to
which a gene maps, is modulated in a particular cancer, the alternative
transcripts or splice variants of the
gene are modulated as well. Disclosed herein is that 213P1F11 has a particular
expression profile related to
cancer. Alternative transcripts and splice variants of 213P1F11 may also be
involved in cancers in the same
or different tissues, thus serving as tumor-associated markers/antigens. .
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The exon composition of the original transcript, designated as 213P1F11 v.l,
is shown in Table
XXIIIA. Using the full-length gene and EST sequences, two splice variants were
identified, designated as
213P1F11 v.2 and 213P1F11 v.3. Compared with 213P1F11 v.l, splice variant
213P1F11 v.2 had a longer
exon 6 while 213P1F11 had a longer exon 5. Using the computer program GenScan,
one alternative
transcript was identified, designated as 213P 1 F 11 v.4. This alternative
transcript had three different leading
exons in place of the first two exons of 213P 1 F 11 v. l . The exon
composition of the alternative transcript
213P1F11 v.4 is shown in Table XXIIIB. Since 213P1F11 v.4 shares the same
exons 5 and 6 as 213P1F11
v.l, splice variants of this alternative transcript with a longer exon 5 or a
longer exon 6, or both, may exist in
human tissues. In fact, each different combination of exons in spatial order,
e.g., exons 1, 2, 3, 4 and 7, is a
potential splice variant. Figure 13 shows the schematic alignment of exons of
the two transcripts (in addition
to variants 2 and 3).
Tables XXIV through XXVII are set forth herein on a variant-by-variant basis.
Table XXIV shows
the nucleotide sequences of transcript variant 2 through variant 4. Table XXV
shows the alignment of
transcript variant 2 through variant 4, each with the nucleic acid sequence of
213P1F11 variant 1. Table
XXVI lays out amino acid translation of transcript variant 2 through variant 4
for the identified reading frame
orientation. Table XXVII displays alignments of the amino acid sequences
encoded by splice variant 2
through variant 4, each with that of 213P1F11 variant 1. Table XXVIII displays
clustal alignments of
213P 1F 11 protein variant 1 through variant 6.
Examule 6~ SinEle Nucleotide Polvmoruhisms of 213P1F11
Single Nucleotide Polymorphism (SNP) is a single base pair variation in
nucleotide sequences. At a
specific point of the genome, there are four possible nucleotide base pairs:
A/T, C/G, G/C and T/A. Genotype
refers to the base pair make-up of one or more spots in the genome of an
individual, while haplotype refers to
base pair make-up of more than one varied spots on the same DNA molecule
(chromosome in higher .
organism). 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 and some 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 disearses and discovery of
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 polymoiphisms in the isolation of common disease
genes," Pharmacogenomics.
2000 Feb; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, "The
predictive power of haplotypes in
clinical response," Pharmacogenomics. 2000 feb; 1(1):15-26).
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
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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
sequencing and high throughput
microarrays (P. Y. Kwok, "Methods for genotyping single nucleotide
polyrnorphisms," 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, six SNPs were identified in the original
transcript, 213P1F11
v.l, at positions 473 (T/C), 737 (C/A), 2027 (C/T), 2037 (T/C), 2268 (A/G) and
3196 (A/T). The transcripts
or proteins with alternative alleles were designated as variants 213P1F11 v.5,
v.6, v.7, v.8, v.9, and v.10.
Figure 10 shows the schematic alignment of the nucleotide variants. Figure 11
shows the schematic
aligmnent 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 that
contains the sequence context of the SNPs , e.g., 213P1F11 v.2, 213P1F11 v.3
or 213P1f11 v.4.
Examule 7~ Production of Recombinant 213P1F11 in Prokaryotic Systems
To express recombinant 213P1F11 and 213P1F11 variants in prokaryotic cells,
the full or partial
length 213P1F11 and 213P1F11 variant cDNA sequences are cloned into any one of
a variety of expression
vectors known in the art. One or more of the following regions of 213P 1F 11
or 213P 1 F 11 variants are
expressed in these constructs, amino acids 1 to 242 of 213P1F11 variant 1,
amino acids 1-230 of variant 2,
amino acids 1-146 of variant 3, amino acids 1-321 of variant 4; 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 213P1F11, variants, or
analogs thereof.
A. In vitro transcription and translation constructs:
gCRII: To generate 213P1F11 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 213P1F11
cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive
the transcription of
213P1F11 RNA for use as probes in RNA in situ hybridization experiments. These
probes are used to
analyze. the cell and tissue expression of 213PiF11 at the RNA level.
Transcribed 213P1F11 RNA
representing the cDNA amino acid coding region of the 213P1F11 gene is used in
in vitro translation systems
such as the TnTTM Coupled Reticulolysate Sytem (Promega, Corp., Madison, WI)
to synthesize 213P1F11
protein.
B. Bacterial Constructs:
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~GEX Constructs: To generate recombinant 213P1F11 proteins in bacteria that
are fused to the
Glutathione S-transferase (GST) protein, all or parts of the T- fusion vector
of the pGEX family (Amersham
Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled
expression of recombinant 213P1F11
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 (ORF). A proteolytic cleavage site, such
as the PreScissionTM recognition
site in pGEX-6P-l, may be employed such that it permits cleavage of the GST
tag from 213P1F11-related
protein. The ampicillin resistance gene and pBR322 origin permits selection
and maintenance of the pGEX
plasmids in E. coli.
pMAL Constructs: To generate, in bacteria, recombinant 213P1F11 proteins that
are fused to
maltose-binding protein (MBP), all or parts of the 213P1F11 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 213P 1F 11 protein
sequences with MBP fused at
the amino-terminus and a 6X His epitope tag at the carboxyl-terminus. The MBP
and 6X His tags permit
purification of the recombinant protein from induced bacteria with the
appropriate affinity matrix and allow
recognition of the fusion protein with anti-MBP and anti-His antibodies. The
6X His epitope tag is generated
by adding 6 histidine codons to the 3' cloning primer. A Factor Xa recognition
site permits cleavage of the
pMAL tag from 213P1F11. 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 213P1F11 in bacterial cells, all or parts of the
213P1F11 cDNA protein
coding sequence are cloned into the pET family of vectors (Novagen, Madison,
WI). These vectors allow
tightly controlled expression of recombinant 213P1F11 protein in bacteria with
and without fusion to proteins
that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags,
such as 6X His and S-Tag TM
that aid purification and detection of the recombinant protein. For example,
constructs are made utilizing
pET NusA fusion system 43.1 such that regions of the 213P1F11 protein are
expressed as amino-terminal
fusions to NusA.
C. Yeast Constructs:
pESC Constructs: To express 213P1F11 in the yeast species Saccharomyces
cerevdsiae for
generation of recombinant protein and functional studies, all or parts of the
213P1F11 cDNA protein coding
sequence are cloned into the pESC family of vectors each of which contain 1 of
4 selectable markers, HIS3,
TRP 1, LEU2, and URA3 (Stratagene, La Jolla, CA). These vectors allow
controlled expression from the
same plasmid of up to 2 different genes or cloned sequences containing either
FlagrM or Myc epitope tags in
the same yeast cell. This system is useful to confirm protein-protein
interactions of 213P1F11. 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 213P1F11 in the yeast species Saccharomyces pombe,
all or parts of
the 213P1F11 cDNA protein coding sequence are cloned into the pESP family of
vectors. These vectors
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allow controlled high level of expression of a 213P1F11 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 FlagT~'~
epitope tag allows detection of the recombinant protein with anti- FIagTM
antibody.
Example 8~ Production of Recombinant 213P1F11 in Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant 213P1F11 in eukaryotic cells, the full or partial
length 213P1F11 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 213P1F11 are expressed in these constructs, amino acids 1
to 242, 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
213P1F11, variants, or analogs thereof. In certain embodiments a region of a
specific variant of 213P1F11 is
expressed that encodes an amino acid at a specific position which differs from
the amino acid of any other
variant found at that position. In other embodiments, a region of a variant of
213P1F11 is expressed that lies
partly or entirely within a sequence that is unique to that variant.
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-213P1F11
polyclonal serum, described
herein.
pcDNA4/HisMax Constructs: To express 213P1F11 in mammalian cells, a 213P1F11
ORF, or
portions thereof, of 213P1F11 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 XpressTM and six histidine (6X His) epitopes fused
to the amino-terminus. The
pcDNA4/HisMax vector also contains the bovine growth hormone (BGH)
polyadenylation signal and
transcription termination sequence to enhance mRNA stability along with the
SV40 origin for episomal
replication and simple vector rescue in cell lines expressing the large T
antigen. The Zeocin resistance gene
allows for selection of mammalian cells expressing the protein and the
ampicillin resistance gene and ColEl
origin permits selection and maintenance of the plasmid in E. coli.
pcDNA3.1/MycHis Constructs: To express 213P1F11 in mammalian cells, a 213P1F11
O1RF', or
portions thereof, of 213P1F11 with a consensus Kozak translation initiation
site are 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 terniination sequence to enhance mRNA
stability, along with the
SV40 origin for episomal replication and simple vector rescue in cell lines
expressing the large T antigen.
The Neomycin resistance gene can be used, as it allows for selection of
mammalian cells expressing the
protein and the ampicillin resistance gene and ColEl origin permits selection
and maintenance of the plasmid
in E. coli.
pcDNA3 1/CT-GFP-TOPO Construct: To express 213P1F11 in mammalian cells and to
allow
detection of the recombinant proteins using fluorescence, a 213P1F11 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
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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 ColEl origin permits selection and maintenance
of the plasmid in E. coli.
Additional constructs with an amino-terminal GFP fusion are made in
pcDNA3.1/NT-GFP-TOPO spanning
the entire length of a 213P1F11 protein.
PAPtaQ: A 213P1F11 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
213P1F11 protein while fusing the IgGx signal sequence to the amino-terminus.
Constructs are also
generated in which alkaline phosphatase with an amino-terminal IgGK signal
sequence is fused to the amino-
terminus of a 213P1F11 protein. The resulting recombinant 213P1F11. 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 213P1F11 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. coli.
tp a~5: A 213P1F11 ORF, or portions thereof, is cloned into pTag-5. This
vector is similar to
pAPtag but without the alkaline phosphatase fusion. This construct generates
213P1F11 protein with an
amino-terminal IgGK signal sequence and myc and 6X His epitope tags at the
carboxyl-terminus that facilitate
detection and affinity purification. The resulting recombinant 213P1F11
protein is 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 213P1F11 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. coli.
PsecFc: A 213P1F11 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
(Invitrogen, California). This construct generates an IgGl Fc fusion at the
carboxyl-terminus of the
213P1F11 proteins, while fusing the IgGK signal sequence to N-terminus.
213P1F11 fusions utilizing the
marine IgGl Fc region are also used. The resulting recombinant 213P1F11
proteins are optimized for
secretion into the media of transfected mammalian cells, and can be used as
immunogens or to identify
proteins such as ligands or receptors that interact with 213P1F11 protein.
Protein expression is driven from
the CMV promotez. 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. coli.
pSRa Constructs: To generate mammalian cell lines that express 213P1F11
constitutively,
213P1F11 ORF, or portions thereof, of 213P1F11 are cloned into pSRa
constructs. Amphotropic and
ecotropic retroviruses are generated by transfection of pSRa constructs into
the 293T-10A1 packaging line or
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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, 213P1F11, into the host cell-lines. Protein expression is
driven from a long terminal repeat
(LTR). The Neomycin resistance gene present in the vector allows for selection
of mammalian cells that
express the protein, and the ampicillin resistance gene and ColEl origin
permit selection and maintenance of
the plasmid in E. coli. The retroviral vectors can thereafter be used for
infection and generation of various
cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells.
Additional pSRa constructs are made that fuse an epitope tag such as the
FLAGTM tag to the
carboxyl-terminus of 213P1F11 sequences to allow detection using anti-Flag
antibodies. For example, the
FLAGTM sequence 5' gat tac aag gat gac gac gat aag 3' (SEQ ID NO: 54) 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 213P1F11 proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated
delivery and expression
of 213P1F11. High virus titer leading to high level expression of 213P1F11 is
achieved in viral delivery
systems such as adenoviral vectors and herpes amplicon vectors. A 213P1F11
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, 213PiF11 coding sequences or fragments thereof are cloned into
the HSV-1 vector (Imgenex)
to generate herpes viral vectors. The viral vectors are thereafter used for
infection of various cell lines such
as PC3, NIH 3T3, 293 or rat-1 cells.
R~e,.ulated Expression Systems: To control expression of 213P1F11 in mammalian
cells, coding '
sequences of 213P1F11, 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 213P1F11. These vectors are thereafter used to control expression
of 213P1F11 in various cell
lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 213P1F11 proteins in a baculovirus expression system,
213P1F11 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-213P1F11 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 213P1F11 protein is then generated by infection of HighFive insect
cells (Invitrogen)
with purified baculovirus. Recombinant 213P1F11 protein can be detected using
anti-213P1F11 or anti-His-
tag antibody. 213P1F11 protein can be purified and used in various cell-based
assays or as immunogen to
generate polyclonal and monoclonal antibodies specific for 213P1F11.
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Example 9' Antigenicity Profiles and Secondary Structure
Figure 5A-D, Figure 6A-D, Figure 7A-D, Figure 8A-D, and Figure 9A-D depict
graphically five
amino acid profiles of the 213P1F11 variants 1 through 4 respectively, each
assessment available by
accessing the ProtScale website located at the World Wide Web (.expasy.ch/cgi-
bin/protscale.pl) on the
ExPasy molecular biology server.
These profiles: Figure 5, Hydrophilicity, (Hopp T.P., Woods K.R., 1981. Proc.
Natl. Acad. Sci.
U.S.A. 78:3824-3828); Figure 6, Hydropathicity, (Kyte J., Doolittle R.F.,
1982. J. Mol. Biol. 157:105-132);
Figure 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492);
Figure 8, Average Flexibility,
(Bhaskaran R., and Ponnuswamy P.K., 1988. Int. J. Pept. Protein Res. 32:242-
255); Figure 9, Beta-turn
(Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally
others available in the art, such
as on the ProtScale website, were used to identify antigenic regions of the
213P1F11 protein. Each of the
above amino acid profiles of 213P 1F11 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 213P1F11 protein and of the variant proteins
indicated, e.g., by the
profiles set forth in Figure 5A-D, Figure 6A-D, Figure 7A-D, Figure 8A-D,
and/or Figure 9A-D are used to
prepare immunogens, either peptides or nucleic acids that encode them, to
generate therapeutic and diagnostic
anti-213P1F11 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 213P1F11 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 up to the full length of the respective variant's
sequence that includes an amino
acid position having a value greater than 0.5 in the Hydrophilicity profiles
of Figure 5A-D; a peptide region
of at least 5 amino acids of Figures 2 and 3 in any whole number increment up
to the full length of the
respective variant's sequence that includes an amino acid position having a
value less than 0.5 in the
Hydropathicity profile of Figures 6 A-D; a peptide region of at least 5 amino
acids of Figures 2 and 3 in any
whole number increment up to the full length of the respective variant's
sequence that includes an amino acid
position having a value greater than 0.5 in the Percent Accessible Residues
profiles of Figure 7 A-D; a
peptide region of at least 5 amino acids of Figures 2 and 3 in any whole
number increment up to the full
length of the respective variant's sequence that includes an amino acid
position having a value greater than
0.5 in the Average Flexibility profiles on Figure 8 A-D; and, a peptide region
of at least 5 amino acids of
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Figures 2 and 3 in any whole number increment up to the full length of the
respective variant's sequence that
includes an amino acid position having a value greater than 0.5 in the Beta-
turn profile of Figures 9 A-D.
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 structures of 213P1F11 variants 1 through 4, namely the
predicted presence and
location of alpha helices, extended strands, and random coils, are predicted
from the primary amino acid
sequence using the HNN - Hierarchical Neural Network method (Guermeur, 1997,
hrip://pbil.ibcp.fr/cgi-
bin/npsa_automat.pl?page=npsa nn.html), accessed from the ExPasy molecular
biology server located at the
World Wide Web (.expasy.ch/toolsn. The analysis indicates that 213P1F11
variant 1 is composed 47.93%
alpha helix, 11.57% extended strand, and 40.50% random coil (Figure 12A),
variant 2 is composed of 38.70%
alpha helix, 9.57% extended strand, and 51.74% random coil (Figure 12B),
variant 3 is composed of 50.68%
alpha helix, 6.85% extended strand, and 42.47% random coil (Figure 12C), and
variant 4 is composed of
39.25% alpha helix, 12.15% extended strand, and 48.60% random coil (Figure
12D).
Analysis for the potential presence of transmembrane domains in 213P1F11
variant 1 was carried
out using a variety of transmembrane prediction algorithms accessed from the
ExPasy molecular biology
server located at the World Wide Web (.expasy.ch/tools/) (Table XXII). The
programs do not predict the
presence of transmembrane domains in any of the 213P 1F 11 variants,
suggesting that each is a soluble
protein.
Example 10' Generation of 213P1F11 Polvclonal 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 213P1F11 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 and Secondary
Structure"). Such regions would be predicted to be hydrophilic, flexible, in
beta-turn conformations, and be
exposed on the surface of the protein (see, e.g., Figure SA-D, Figure 6 A-D,
Figure 7 A-D, Figure 8 A-D, or
Figure 9 A-D for amino acid profiles that indicate such regions of 213P1F11
and variants).
For example, 213P1F11 recombinant bacterial fusion proteins or peptides
containing hydrophilic,
flexible, beta-turn regions of 213P1F11 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 1-17,
amino acids 25-80, amino acids 88-108, amino acids 131-147, and 207-242 of
213P1F11 variant 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), sernm albumin, bovine thyroglobulin, and soybean trypsin
inhibitor. In one
embodiment, a peptide encoding amino acids 1-17 of 213P1F11 variant 1 is
conjugated to KLH and used to
immunize the rabbit. Alternatively the immunizing agent may include all or
portions of the 213P1F11 variant
proteins, analogs or fusion proteins thereof. For example, the 213P1F11
variant 1 amino acid sequence can
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be fused using recombinant DNA techniques to any one of a variety of fusion
protein partners that are well
known in the art, such as glutathione-S-transferase (GST) and HIS tagged
fusion proteins. Such fusion
proteins are purified from induced bacteria using the appropriate affinity
matrix.
In one embodiment, a GST-fusion protein encoding amino acids 1-147,
encompassing several
predicted antigenic regions, is produced and 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
213P1F11 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
Tags and Fc-fusion vectors
(see the section entitled "Production of Recombinant 213P1F11 in Eukaryotic
Systems"), and retain post-
translational modifications such as glycosylations found in native protein. In
one embodiment, the full
length sequence of variant 1, amino acids 1-242, is cloned into the Tags
mammalian secretion vector. The
recombinant protein is purified by metal chelate chromatography from tissue
culture supernatants of 293T
cells stably expressing the recombinant vector. The purified Tags 213P1F11
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 fig, typically
100-200 ltg, 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 pg,
typically 100-200 ~tg, 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 1-17 of
variant 1, the full-length
213P1F11 variant 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector
(Invitrogen, see the Example
entitled "Production of Recombinant 213P 1F11 in Eukaryotic Systems"). After
transfection of the constructs
into 293T cells, cell lysates are probed with the anti-213P1F11 serum and with
anti-His antibody (Santa Cruz
Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured
213P1F11 protein using the
Western blot technique. The immune serum is then tested by the Western blot
technique against 293T-
213P1F11 cells. In addition, the immune serum is tested by fluorescence
microscopy, flow cytometry and
immunoprecipitation against 293T and other recombinant 213P1F11-expressing
cells to determine specific
recognition of native protein. Western blot, immunoprecipitation, fluorescent
microscopy, and flow
cytometric techniques using cells that endogenously express 213P1F11 are also
carried out to test reactivity
and specificity.
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Anti-serum from rabbits immunized with 213P1F1lvariant 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-213P1F11 fusion protein encoding
amino acids 1-147 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 1-147 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 213P1F11 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 213P1F11 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 213P1F11 variants, for example
those that would disrupt the
interaction with ligands and binding partners. Immunogens for generation of
such mAbs include those
designed to encode or contain the entire 213P1F11 protein variant sequence,
regions of the 213P1F11 protein
variants predicted to be antigenic from computer analysis of the amino acid
sequence (see, e.g., Figure SA-D,
Figure 6 A-D, Figure 7 A-D, Figure 8 A-D, or Figure 9 A-D, and the Example
entitled "Antigenicity Profiles
and Secondary Structure"). Immunogens include peptides, recombinant bacterial
proteins, and mammalian
expressed Tag 5 proteins and human and marine IgG FC fusion proteins. In
addition, cells engineered to
express high levels of a respective 213P1F11 variant, such as 293T-213P1F11
variant 1 or 300.19-213P1F11
variant lmurine Pre-B cells, are used to immunize mice.
To generate mAbs to a 213P1F11 variant, mice are first immunized
intraperitoneally (IP) with,
typically, 10-50 ~g ofprotein immunogen or 10' 213P1F11-expressing cells mixed
in complete Freund's
adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with,
typically, 10-50 ~tg of protein
immunogen or 10' cells mixed in incomplete Freund's adjuvant. Alternatively,.
MPL-TDM adjuvant is used
in immunizations. In addition to the above protein and cell-based immunization
strategies, a DNA-based
immunization protocol is employed in which a mammalian expression vector
encoding a 213P 1F11 variant
sequence is used to immunize mice by direct injection of the plasmid DNA. For
example, the full length
variant 1. sequence, encoding amino acids 1-242, is cloned into the Tags
mammalian secretion vector and the
recombinant vector is used as immunogen. In another example the same amino
acids are cloned into an Fc-
fusion secretion vector in which the 213P1F11 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 marine IgG Fc region.
This recombinant vector is then used as immunogen. The plasmid immunization
protocols are used in
combination with purified proteins expressed from the same vector and with
cells expressing the respective
213P 1 F 11 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, and flow cytometric
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analyses, fusion and hybridoma generation is then carried out with established
procedures well known in the
art (see, e.g., Harlow and Lane, 1988).
In one embodiment for generating 213P1F11 monoclonal antibodies, a Tags-
213P1F11 variant 1
antigen encoding amino acids 1-242, is expressed and purified from stably
transfected 293T cells. Balb C
mice are initially immunized intraperitoneally with 25 ltg of the Tags-
213P1F11 variant 1 protein mixed in
complete Freund's adjuvant. Mice are subsequently immunized every two weeks
with 25 ltg of the antigen
mixed in incomplete Freund's adjuvant for a total of three immunizations.
ELISA using the Tags antigen
determines the titer of sernm from immunized mice. Reactivity and specificity
of serum to full length
213P1F11 variant protein is monitored by Western blotting, immunoprecipitation
and flow cytometry using
293T cells transfected with an expression vector encoding the 213P1F11 variant
1 cDNA (see e.g., the
Example entitled "Production of Recombinant 213P1F11 in Eukaryotic Systems").
Other recombinant
213P1F1lvariant 1-expressing cells or cells endogenously expressing 213P1F11
variant 1 are also used.
Mice showing the strongest reactivity are rested and given a final injection
of Tags antigen in PBS and then
sacrificed four days later. The spleens of the sacrificed mice are harvested
and fused to SPO/2 myeloma cells
using standard procedures (Harlow and Lane, 1988). Supernatants from HAT
selected growth wells are
screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy,
and flow cytometry to
identify 213P1F11 specific antibody-producing clones.
Monoclonal antibodies are also derived that react only with specific 213P1F11
variants. To this end,
immunogens are designed to encode amino acid regions specific to the
respective variant. For example, a
Tags immunogen encoding amino acids 175-230 of variant 2 is produced,
purified, and used to immunize
mice to generate hybridomas. In another example, a KLH-coupled peptide
encoding amino acids 135-146 of
variant 3 is produced and used as immunogen. In another example amino acids 1-
86 of variant 4 is fused to
GST and used as immunogen. Monoclonal antibodies raised to these immunogens
are then screened for
reactivity to cells expressing the respective variants but not to other
213P1F11 variants. These strategies for°
raising 213P1F11 variant specific monoclonal antibodies are also applied to
polyclonal reagents described in
the Example entitled "Generation of 213P1F11 Polyclonal Antibodies."
The binding affinity of a 213P1F11 monoclonal antibody is determined using
standard technologies.
Affinity measurements quantify the strength of antibody to epitope binding and
are used to help define which
213P1F11 monoclonal antibodies preferred for diagnostic or therapeutic use, as
appreciated by one of skill in
the art. The BIAcore system (Uppsala, Sweden) is a preferred method for
determining binding affinity. The
BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt..
Quant. Elect. 23:1; Morton and
Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular
interactions in real time. BIAcore
analysis conveniently generates association rate constants, dissociation rate
constants, equilibrium
dissociation constants, and affinity constants.
Example 12: HLA Class I and Class II Bindine Assays
HLA class I and class II binding assays using purified HLA molecules are
performed in accordance
with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94!03205;
Sidney et al., Current
Protocols in Immunology 18.3.1 (1998); Sidney, et al., .I. Immunol. 154:247
(1995); Sette, et al., Mol.
Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are
incubated with various
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unlabeled peptide inhibitors and 1-10 nM ~zSI-radiolabeled probe peptides as
described. Following
incubation, MHC-peptide complexes are separated from free peptide by gel
filtration and the fraction of
peptide bound is determined. Typically, in preliminary experiments, each MHC
preparation is titered in the
presence of fixed amounts of radiolabeled peptides to determine the
concentration of HLA molecules
necessary to bind 10-20% of the total radioactivity. All subsequent inhibition
and direct binding assays are
performed using these HLA concentrations.
Since under these conditions [label]<[HLA] and ICSO>_[HLA], the measured ICso
values are
reasonable approximations of the true ICD values. Peptide inhibitors are
typically tested at concentrations
ranging from 120 ~g/ml to 1.2 ng/ml, and are tested in two to four completely
independent experiments. To
allow comparison of the data obtained in different experiments, a relative
binding figure is calculated for each
peptide by dividing the ICSO of a positive control for inhibition by the ICso
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
ICso nM values by dividing the ICso 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~ots of purified MHC.
Binding assays as outlined above may be used to analyze HLA supermotif and/or
HLA motif
bearing peptides.
Example 13~ Identification of HLA Sunermotif and Motif Bearing CTL Candidate
Euitopes
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 aleorithms for identification of su~ermotif and/or motif
bearing epitoyes
The searches performed to identify the motif bearing peptide sequences in the
Example entitled
"Antigenicity Profiles and Secondary Structure" and Tables V-XIX employ the
protein sequence data from
the gene product of 213P1F11 set forth in Figures 2 and 3.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or
motifs are performed
as follows. All translated 213P1F11 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 dG) of peptide-HLA molecule interactions can be
approximated as a linear polynomial
function of the type:
"~G~~=a~;xa2;xa3;......xa",
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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 j; 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.
Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-
93, 1996; and Southwood et
al., J. Imnaunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor
and non-anchor alike, the
geometric mean of the average relative binding (AItB) of all peptides carrying
j is calculated relative to the
remainder of the group, and used as the estimate of j;. For Class II peptides,
if multiple alignments are
possible, only the highest scoring alignment is utilized, following an
iterative procedure. To calculate an
algorithm score of a given peptide in a test set, the ARB values corresponding
to the sequence of the peptide
are multiplied. If this product exceeds a chosen threshold, the peptide is
predicted to bind. Appropriate
thresholds are chosen as a function of the degree of stringency of prediction
desired.
Selection of HLA-A2 supertype cross-reactive peptides
Protein sequences from 213P1F11 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 afFmity equal
to or less than 500 nM to three or more HLA-A2 supertype molecules.
Selection of HLA-A3 s ~ermotif bearing epitopes
The 213P1F11 protein sequences) scanned above is also examined for the
presence of peptides with
the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3
supermotif bearing
sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*
1101 molecules, the
molecules encoded by the two most prevalent A3-supertype alleles. The peptides
that bind at least one of the
two alleles with binding affinities of <_500 nM, often <_ 200 nM, are then
tested for binding cross-reactivity to
the other common A3-supertype alleles (e.g., A*3101,'A*3301, and A*6801) to
identify those that can bind at
least three of the five HLA-A3-supertype molecules tested.
Selection of HLA-B7 sugermotif bearin -~epitopes
The 213P1F11 proteins) scanned above is also analyzed for the presence of 8-,
9- 10-, or 11-mer
peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized
and tested for binding to
HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e.,
the prototype B7
supertype allele). Peptides binding B*0702 with ICso of 5500 nM are identified
using standard methods.
These peptides are then tested for binding to other common B7-supertype
molecules (e.g., B*3501, B*5101,
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B*5301, and B*5401). Peptides capable of binding to three or more of the five
B7-supertype alleles tested
are thereby identified.
Selection of A1 and A24 motif bearing epitopes
To further increase population coverage, HLA-A1 and -A24 epitopes can also be
incorporated into
vaccine compositions. An analysis of the 213P1F11 protein can also be
performed to identify HLA-A1- 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 Immunosenicity
Cross-reactive candidate CTL A2-supermotif bearing peptides that are
identified as described herein
are selected to confirm in vitro immunogenicity. Confirmation is performed
using the following
methodology:
Target Cell Lines for Cellular Screening:
The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the
HLA-A, -B, -C null
mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded
target to measure activity of
HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium
supplemented with antibiotics,
sodium pyruvate, nonessential amino acids and 10% (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.
Primar~CTL Induction Cultures:
Generation of Dendritic Cells (DC): PBMCs are thawed in 1ZPMI with 30 pg/ml
DNAse, washed
twice and resuspended in complete medium (IRPMI-1640 plus S% 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
shaleing the plates and aspirating the supernatants. The wells are washed a
total of three times with 3 ml
RPMI to remove most of the non-adherent and loosely adherent cells. Three ml
of complete medium
containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each
well. TNFoc is added to the
DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on
day 7.
Induction of CTL with DC and Peptide. CD8+ T-cells are isolated by positive
selection with Dynal
immunomagnetic beads (Dynabeads~ M-450) and the detacha-bead~ reagent.
Typically about 200-250x106
PBMC are processed to obtain 24x106 CD8+ T-cells (enough for a 48-well plate
culture). Briefly, the PBMCs
are thawed in RPMI with 30pg/ml DNAse, washed once with PBS containing 1%
human AB serum and
resuspended in PBS/1% AB serum at a concentration of 20x106cells/ml. The
magnetic beads are washed 3
times with PBS/AB serum, added to the cells (140p1 beads/20x106 cells) and
incubated for 1 hour at 4°C with
continuous mixing. The beads and cells are washed 4x with PBS/AB serum to
remove the nonadherent cells
and resuspended at 100x106 cells/ml (based on the original cell number) in
PBS/AB serum containing
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100~.1/ml detacha-bead~J 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 40~g/ml of peptide at a cell concentration of 1-
2x106/ml in the presence of
3~g/ml l3~- microglobulin for 4 hours at 20°C. The DC are then
irradiated (4,200 reds), washed 1 time with
medium and counted again.
Setting up indz~ction cultures: 0.25 ml cytokine-generated DC (at 1x105
cells/ml) are co-cultured
with 0.25m1 of CD8+ T-cells (at 2x106 cell/ml) in each well of a 48-well plate
in the presence of 10 ng/ml of
IL-7. Recombinant human IL-10 is added the next day at a final concentration
of 10 ng/ml and rhuman IL-2
is added 48 hours later at 10 IU/ml.
Restimulation of the induction cultures with peptide pulsed adherent cells:
Seven and fourteen days
after the primary induction, the cells are restimulated with peptide-pulsed
adherent cells. The PBMCs are
thawed and washed twice with RPMI and DNAse. The cells are resuspended at
5x106 cells/ml and irradiated
at 4200 reds. The PBMCs are plated at 2x106 in 0.5 ml complete medium per well
and incubated for 2 hours
at 37°C. The plates are washed twice with RPMI by tapping the plate
gently to remove the nonadherent cells
and the adherent cells pulsed with 10~g/ml of peptide in the presence of 3
pg/ml 13z microglobulin in 0.25m1
RPMI/5%AB per well for 2 hours at 37°C. Peptide solution from each well
is aspirated and the wells are
washed once with RPMI. Most of the media is aspirated from the induction
cultures (CD8+ cells) and
brought to 0.5 ml with fresh media. The cells are then transferred to the
wells containing the peptide-pulsed
adherent cells. Twenty four hours later recombinant human IL-10 is added at a
final concentration of 10
ng/ml and recombinant human IL2 is added the next day and again 2-3 days later
at 50IU/ml (Tsai et al.,
Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the
cultures are assayed for CTL
activity in a S~Cr release assay. In some experiments the cultures are assayed
for peptide-specific recognition
in the in situ IFN~y 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 l~tic activity by SICr release.
Seven days after the second restimulation, cytotoxicity is determined in a
standard (5 hr) SICr release
assay by assaying individual wells at a single E:T. Peptide-pulsed targets are
prepared by incubating the cells
with l Opg/ml peptide overnight at 37°C.
Adherent target cells are removed from culture flasks with trypsin-EDTA.
Target cells are labeled
with 200pCi of SICr sodium chromate (Dupont, Wilirvngton, DE) for 1 hour at
37°C. Labeled target cells are
resuspended at 106 per rnl and diluted 1:10 with K562 cells at a concentration
of 3.3x106/ml (an NK-sensitive
erythroblastoma cell line used to reduce non-specific lysis). Target cells
(100 ~.l) and effectors (100p1) are
plated in 96 well round-bottom plates and incubated for 5 hours at
37°C. At that time, 100 ~1 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 SICr release sample)/(cpm of
the maximal S~Cr
release sample- cpm of the spontaneous 5 ~Cr 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-
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background) is 10% or higher in the case of individual wells and is 1S% 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 Reco nib' tion
Immulon 2 plates are coated with mouse anti-human IFNy monoclonal antibody (4
~tg/ml O.1M
NaHC03, pH8.2) overnight at 4°C. The plates are washed with Ca2+, Mgz+-
free PBS/0.05% Tween 20 and
blocked with PBS/10% FCS for two hours, after which the CTLs (100 pl/well) and
targets (100 ~1/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 S% COZ.
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 pl of
biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in
PBSl3%FCS/O.OS%
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 S-1S minutes. The reaction is stopped with SO
microliter/well 1M H3P04 and
read at OD4S0. A culture is considered positive if it measured at least SO pg
of IFN-gamma/well above
background and is twice the background level of expression.
CTL Expansion.
Those cultures that demonstrate specific lytic activity against peptide-pulsed
targets and/or tumor
targets are expanded over a two week period with anti-CD3. Briefly, Sx104 CD8+
cells are added to a T2S
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 OKT3 (anti-CD3) at
30ng per ml in RPMI-1640 '
containing 10% (v/v) human AB serum, non-essential amino acids, sodium
pyruvate, 25~M
2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human
IL2 is added 24 hours
later at a final concentration of 200ILJ/ml and every three days thereafter
with fresh media at SOILT/ml. The
cells are split if the cell concentration exceeds 1x106/ml and the cultures
are assayed between days 13 and 1S
at E:T ratios of 30, 10, 3 and 1:1 in the S~Cr release assay or at 1x106/ml in
the in situ IFNy assay using the
same targets as before the expansion.
Cultures are expanded in the absence of anti-CD3+ as follows. Those. cultures
that demonstrate
specific lytic activity against peptide and endogenous targets are selected
and SxlO$ CD8+ cells are added to a
T2S flask containing the following: 1x106 autologous PBMC per ml which have
been peptide-pulsed with 10
pg/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, 2SmM
2-ME, L-glutamine and gentamicin.
ImmunoQenicity of A2 supermotif bearing peptides
A2-supermotif cross-reactive binding peptides are tested in the cellular assay
for the ability to induce
peptide-specific CTL in normal individuals. In this analysis, a peptide is
typically considered to be an epitope
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if it induces peptide-specific CTLs in at least individuals, and preferably,
also recognizes the endogenously
expressed peptide.
Immunogenicity can also be confirmed using PBMCs isolated from patients
bearing a tumor that
expresses 213P1F11. Briefly, PBMCs are isolated from patients, re-stimulated
with peptide-pulsed
monocytes and assayed for the ability to recognize peptide-pulsed target cells
as well as transfected cells
endogenously expressing the antigen.
Evaluation of A*03/Al l immunoeenicity
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 immuno~enicity
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, g~.g., HLA-A1, HLA-A24 etc. are
also confirmed using
similar methodology
Examule 15~ Implementation of the Extended Supermotif to Improve the Bindins
Capacity of
N_ ative Enitopes by Creatins Analogs
HLA motifs and supermotifs (comprising primary and/or secondary residues) are
useful in the
identification and preparation of highly cross-reactive native peptides, as
demonstrated herein. Moreover, the
definition of HLA motifs and supermotifs also allows one to engineer highly
cross-reactive epitopes by
identifying residues within a native peptide sequence which can be analoged to
confer upon the peptide
certain characteristics, e.g. greater cross-reactivity within the group of HLA
molecules that comprise a
supertype, and/or greater binding affinity for some or all of those HLA
molecules. Examples of analoging
peptides to exhibit modulated binding affinity are set forth in this example.
Analogin~ 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.
To analyze the cross-reactivity of the analog peptides, each engineered analog
is initially tested for
binding to the prototype A2 supertype allele A*Q201, 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 and then analoged to
modulate binding affinity to any one (or more) of the supertype members to add
population coverage.
The selection of analogs for immunogenicity in a cellular screening analysis
is typically further
restricted by the capacity of the parent wild type (WT) peptide to bind at
least weakly, i.e., bind at an ICSO of
SOOOnM 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
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have been shown.to have increased immunogenicity and cross-reactivity by T
cells specific for the parent
epitope (see, e.g., Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue 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.
Analogin~ of HLA-A3 and B7-supermotif bearing peutides
Analogs of HLA-A3 supermotif bearing epitopes are generated using strategies
similar to those
employed in analoging HLA-A2 supermotif bearing peptides. For example,
peptides binding to 3/5 of the
A3-supertype molecules are engineered at primary anchor residues to possess a
preferred residue (V, S, M, or
A) at position 2.
The analog peptides are then tested for the ability to bind A*03 and A*11
(prototype A3 supertype
alleles). Those peptides that demonstrate <_ 500 nM binding capacity are then
confirmed as having A3-
supertype cross-reactivity.
Similarly to the A2- and A3- motif bearing peptides, peptides binding 3 or
more B7-supertype alleles
can be improved, where possible, to achieve increased cross-reactive binding
or greater binding affinity or
binding half life. B7 supermotif bearing peptides are, for example, engineered
to possess a preferred residue
(V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by
Sidney 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.
Analo .ins at Secondar~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 1 is analyzed. The
peptide is then analoged to, for
example, substitute L for F at position 1. The analoged peptide is evaluated
for increased binding affinity,
binding half life and/or increased cross-reactivity. Such a procedure
identifies analoged peptides with
enhanced properties.
Engineered analogs with sufficiently improved binding capacity or cross-
reactivity can also be tested
for immunogenicity in HLA-B7-transgenic mice, following for example, IFA
immunization or lipopeptide
immunization. Analoged peptides are additionally tested for the ability to
stimulate a recall response using
PBMC from patients with 213P1F11-expressing tumors.
Other analoein~ 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 et
al., In: Persistent Viral Infections,
Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).
Thus, by the use of single amino acid substitutions, the binding properties
and/or cross-reactivity of
peptide ligands for HLA supertype molecules can be modulated.
Example 16' Identification and confirmation of 213P1F11-derived sectuences
with HLA-DR
binding motifs
Peptide epitopes bearing an HLA class II supermotif or motif are identified
and confnmed as
outlined below using methodology similar to that described for HLA Class I
peptides.
Selection of HLA-DR-supermotif bearing epitopes.
To identify 213P1F11-derived, HLA class II HTL epitopes, a 213P1F11 antigen is
analyzed for the
presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-
mer sequences are selected
comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and
C-terminal flanking regions
(15 amino acids total).
Protocols for predicting peptide binding to DR molecules have been developed
(Southwood et al., J.
Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR
molecules, allow the scoring,
and ranking, of 9-mer core regions. Each protocol not only scores peptide
sequences for the presence of DR-
supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer
core, but additionally evaluates
sequences for the presence of secondary anchors. Using allele-specific
selection tables (see, e.g., Southwood
et al., ibiei.), 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 DRl, DR4w4, and DR7, can efficiently select DR
cross-reactive peptides.
The 213P1F11-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'priinary
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, DRSwl l, 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. 213P1F11-
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
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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 213P1F11 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 lltM or better, i.e., less than 1 ~tM. Peptides are found
that meet this binding criterion and
qualify as HLA class II high affinity binders.
DR3 binding epitopes identified in this manner are included in vaccine
compositions with DR
supermotif bearing peptide epitopes.
Similarly to the case of HLA class I motif bearing peptides, the class 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~ Immuno~enicitv of 213P1F11-derived HTL enitopes
This example determines immunogenic DR supermotif and DR3 motif bearing
epitopes among
those identified using the methodology set forth herein.
Immunogenicity of HTL epitopes are confirmed in a manner analogous to the
determination of
immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL
responses and/or by using
appropriate transgenic mouse models. Immunogenicity is determined by screening
for: 1.) in vitro primary
induction using normal PBMC or 2.) recall responses from patients who have
213P1F11-expressing tumors.
Example 18~ Calculation of uhenotvnic frecruencies of HLA-sunertvpes in
various ethnic
backsrounds 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 gel-(SQRT(1-af)) (see, e.g., Sidney et al., Human
Immunol. 45:79-93, 1996). To
obtain overall phenotypic frequencies, cumulative gene frequencies are
calculated, and the cumulative antigen
frequencies derived by the use of the inverse formula [ail-(1-Cgfj2].
Where frequency data is not available at the level of DNA typing,
correspondence to the
serologically defined antigen frequencies is assumed. To obtain total
potential supertype population coverage
no linkage disequilibrium is assumed, and only alleles confirmed to belong to
each of the supertypes are
included (minimal estimates). Estimates of total potential coverage achieved
by inter-loci combinations are
made by adding to the A coverage the proportion of the non-A covered
population that could be expected to
be covered by the B alleles considered (e.g., total=A+B*(1-A)). Confirmed
members of the A3-like
supertype are A3, A11, 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,
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A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7,
B*3501-03, B51, B*5301,
B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-
06, B*4201, and
B*5602).
Population coverage achieved by combining the A2-, A3- and B7-supertypes is
approximately 86%
in five major ethnic groups. Coverage may be extended by including peptides
bearing the A1 and A24
motifs. On average, A1 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 A1 and A24 are combined with the coverage of
the A2-, A3- and B7-
supertype alleles is >95%. An analogous approach can be used to estimate
population coverage achieved
with combinations of class II motif bearing epitopes.
Immunogenicity studies in humans (e.g., Bertoni et al., J. Clip. Invest.
100:503, 1997; Doolan et al.,
Immunity 7:97, 1997; and Threlkeld et al., J. Immunol. 159:1648, 1997) have
shown that highly cross-reactive
binding peptides are almost always recognized as epitopes. The use of highly
cross-reactive binding peptides
is an important selection criterion in identifying candidate epitopes for
inclusion in a vaccine that is
immunogenic in a diverse population.
With a sufficient number of epitopes (as disclosed herein and from the art),
an average population
coverage is 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 Recoenition Of Endosenouslv 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 SICr labeled Jurkat-A2.1/Kb target cells in the absence or presence of
peptide, and also tested on SICr
labeled target cells bearing the endogenously synthesized antigen, i.e. cells
that are stably transfected with
213P1F11 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with
peptide epitope recognize
endogenously synthesized 213P1F11 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 A11,
which may also be used to
evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g.,
transgenic mice for HLA-A1
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and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been
developed, which
may be used to evaluate HTL epitopes.
Example 20~ Activity Of CTL-HTL Coniu~ated Epitones In Trans~enic Mice
This example illustrates the induction of CTLs and HTLs in transgenic mice, by
use of a 213P1F11-
derived CTL and HTL peptide vaccine compositions. The vaccine composition used
herein comprise
peptides to be administered to a patient with a 213P1F11-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. Irnnzunol. 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 subcut~neously (base of the tail)
with a 0.1 ml of peptide in
Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated
CTL/HTL conjugate, in
DMSO/saline, or if the peptide composition is a polypeptide, in PBS or
Incomplete Freund's Adjuvant.
Seven days after priming, splenocytes obtained from these animals are
restimulated with syngenic irradiated
LPS-activated lymphoblasts coated with peptide.
Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat
cells transfected with the
HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)
In vitro CTL activation: One week after priming, spleen cells (30x106
cells/flask) are co-cultured at
37°C with syngeneic, irradiated (3000 rads), peptide coated
lymphoblasts (10x106 cells/flask) in 10 ml of
culture medium/T25 flask. 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 SICr. 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 siCr-
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)l(maximum release -
spontaneous release). To facilitate comparison between separate CTL assays run
under the same conditions,
% S~Cr 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 SICr 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% SICr
release is obtained at the effector (E):
target (T) ratio of 50:1 (i.e., 5x105 effector cells for 10,000 targets) in
the absence of peptide and 5:1 (i.e.,
5x104 effector cells for 10,000 targets) in the presence of peptide, the
specific lytic units would be:
I(vso,ooo)-(1/soo,ooo)~ ~ 106 = is Lu.
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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 213P1F11-
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
213P1F11 clearance. The number of epitopes used depends on observations of
patients who spontaneously
clear 213P1F11. For example, if it has been observed that patients who
spontaneously clear 213P1F11-
expressing cells generate an immune response to at least three (3) from
213P1F11 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 ICso of 500 nM
or less for an HLA class
I molecule, or for class II, an ICso of 1000 nM or less; or HLA Class I
peptides with high binding scores from
the BIIvIAS 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 ~0% population
coverage. A Monte Carlo analysis, a statistical evaluation known in the art,
can be employed to assess
breadth, or redundancy, of population coverage. .
When creating polyepitopic compositions, or a minigene that encodes same, it
is typically desirable
to generate the smallest peptide possible that encompasses the epitopes of
interest. The principles employed
are similar, if not the same, as 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
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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
213P1F11, thus avoiding the need
to evaluate any functional 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 213P1F11.
Example 22~ Construction of "MiniEene" Multi-Enitope 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 mlnigene expression plasmid typically includes multiple CTL and HTL peptide
epitopes. In the
present example, HLA-A2, -A3, -B7 supermotif bearing peptide epitopes and HLA-
A1 and -A24 motif
bearing peptide epitopes are used in conjunction with DR supermotif bearing
epitopes and/or DR3 epitopes.
HLA class I supermotif or motif bearing peptide epitopes derived 213P1F11, are
selected such that multiple
supermotifs/motifs are represented to ensure broad population coverage.
Similarly, HLA class II epitopes are
selected from 213P1F11 to provide broad population coverage, i.e. both HLA DR-
1-4-7 supermotif bearing
epitopes and HLA DR-3 motif bearing epitopes are selected for inclusion in the
minigene construct: The
selected CTL and HTL epitopes are then incorporated into a minigene for
expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL
epitopes to the endoplasmic
reticulum. For example, the Ii protein may be fused to one or more HTL
epitopes as described in the art,
wherein the CLIP sequence of the Ii protein is removed and replaced with an
HLA class II epitope sequence
so that HLA class II epitope is directed to the endoplasmic reticulum, where
the epitope binds to an HLA
class II molecules.
This example illustrates the methods to be used for construction of a minigene-
bearing expression
plasmid. Other expression vectors that may be used for minigene compositions
are available and known to
those of skill in the art.
The minigene DNA plasmid of this example contains a consensus Kozak sequence
and a consensus
marine kappa Ig-light chain signal sequence followed by CTL and/or HTL
epitopes selected in accordance
with principles disclosed herein. The sequence encodes an open reading frame
fused to the Myc and His
antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
Overlapping oligonucleotides that can, for example, average about 70
nucleotides in length with 15
nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides
encode the selected peptide
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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, S+6, and 7+8 are combined in 100 ~tl
reactions containing Pfu
polymerase buffer (lx= 10 mM KCL, 10 mM (NH4)ZS04, 20 mM Tris-chloride, pH
8.75, 2 mM MgS04,
0.1% Triton X-100, 100 ~g/ml BSA), 0.25 mM each dNTP, and Z.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 5
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 DeEree to Which It Induces
Immunosenicitv.
The degree to which a plasmid construct, for example a plasmid constructed in
accordance with the
previous Example, is able to induce immunogenicity is confirmed in vitro by
determining epitope
presentation by APC following transduction or transfection of the APC with an
epitope-expressing nucleic
acid construct. Such a study determines "antigenicity" and allows the use of
human APC. The assay
determines the ability of the epitope to be presented by the APC in a context
that is recognized by a T cell by
quantifying the density of epitope-HLA class I complexes on the cell surface.
Quantitation can be performed
by directly measuring the amount of peptide eluted from the APC (see, e.g.,
Sijts et al., J. Immunol. 156:683-
692, 1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-
HLA class I complexes can
be estimated by measuring the amount of lysis or lymphokine release induced by
diseased or transfected
target cells, and then determining the concentration of peptide necessary to
obtain equivalent levels of lysis or
lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576,
1995).
Alternatively, immunogenicity is confirmed through in vivo injections into
mice and subsequent in
vitro assessment of CTL and HTL activity, which are analyzed using
cytotoxicity and proliferation assays,
respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761, 1994.
For example, to confirm the capacity of a DNA minigene construct containing at
least one HLA-A2
supermotif peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for
example, are immunized
intramuscularly with 100 pg 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 5'Cr release assay. The results indicate the magnitude
of the CTL response directed
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against the A2-restricted epitope, thus indicating the in vivo immunogenicity
of the minigene vaccine and
polyepitopic vaccine.
It is, therefore, found that the minigene elicits immune responses directed
toward the HLA-A2
supermotif peptide epitopes as does the polyepitopic peptide vaccine. A
similar analysis is also performed
using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction
by HLA-A3 and HLA-
B7 motif or supermotif epitopes, whereby it is also found that the minigene
elicits appropriate immune
responses directed toward the provided epitopes.
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 ~tg of
plasmid DNA. As a means of
comparing the level of HTLs induced by DNA immunization, a group of control
animals is also immunized
with an actual peptide composition emulsified in complete Freund's adjuvant.
CD4+ T cells, i.e. HTLs, are
purified from splenocytes of immunized animals and stimulated with each of the
respective compositions
(peptides encoded in the minigene). The HTL response is measured using a 3H-
thymidine incorporation
proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994).
The results indicate the
magnitude of the HTL response, thus demonstrating the in vivo immunogenicity
of the minigene.
DNA minigenes, constructed as described in the previous Example, can also be
confirmed as a
vaccine in combination with a boosting agent using a prime boost protocol. The
boosting agent can consist of
recombinant protein (e.g., Bamett et al., Aids Res. and Human Retroviruses 14,
Supplement 3:S299-5309,
1998) or recombinant vaccinia, for example, expressing a minigene or DNA
encoding the complete protein of
interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al.,
Proc. Natl. Acad. Sci USA
95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and
Robinson et al., Nature
Med. 5:526-34, 1999).
For example, the efficacy of the DNA minigene used in a prime boost protocol
is initially evaluated
in transgenic mice. .In this example, A2.1/Kb transgenic mice are immunized IM
with 100 pg 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 10' pfu/mouse of a
recombinant vaccinia virus expressing the same sequence encoded by the DNA
minigene. Control mice are
immunized with 100 pg 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 l 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."
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Example 24~ Peptide Compositions for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 213P1F11
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 213P1F11-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 ~tg, generally 100-5,000 p.g, for a 70 kg patient. The
initial administration of vaccine
is followed by booster dosages at 4 weeks followed by evaluation of the
magnitude of the immune response
in the patient, by techniques that determine the presence of epitope-specific
CTL populations in a PBMC
sample. Additional booster doses are administered as required. The composition
is found to be both safe and
efficacious as a prophylaxis against 213P1F11-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.
Example 25~ Polyepitopic Vaccine Compositions Derived from Native 213P1F11
Sectuences
A native 213P1F11 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
is selected; it 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
sifted relative to one another). For example, with overlapping epitopes, two 9-
mer epitopes and one 10-mer
epitope can be present in a 10 amino acid peptide. Such a vaccine composition
is administered for therapeutic
or prophylactic purposes.
The vaccine composition will include, for example, multiple CTL epitopes from
213P1F11 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
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unknown. Furthermore, this embodiment (excluding an analoged embodiment)
directs the immune response
to multiple peptide sequences that are actually present in native 213P1F11,
thus avoiding the need to evaluate
any functional 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~ Polvepitopic Vaccine Compositions From Multiple Antigens
The 213P1F11 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 213P1F11 and such other antigens. For
example, a vaccine composition
can be provided as a single polypeptide that incorporates multiple epitopes
from 213P1F11 as well as tumor-
associated antigens that are often expressed with a target cancer associated
with 213P1F11 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 uentides to evaluate an immune resuonse
Peptides of the invention may be used to analyze an immune response for the
presence of specific
antibodies, CTL or HTL directed to 213P1F11. Such an analysis can be performed
in a manner described by
Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in
accordance with the invention are
used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
In this example highly sensitive human leukocyte antigen tetrameric complexes
("tetramers") are
used for a cross-sectional analysis of, for example, 213P1F11 HLA-A*0201-
specific CTL frequencies from
HLA A*0201-positive individuals at different stages of disease or following
immunization comprising a
213P1F11 peptide containing an A*0201 motif. Tetrameric complexes are
synthesized as described (Musey
et al., N. Engl. J. V~led. 337:1267, 1997). Briefly, purified HLA heavy chain
(A*0201 in this example) and
(32-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, (32-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 3008
for 5 minutes and resuspended in 50 ~l of cold phosphate-buffered saline: Tri-
color analysis is performed
with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38.
The PBMCs are incubated
with tetramer and antibodies on ice for 30 to 60 min and then washed twice
before formaldehyde fixation.
Gates are applied to contain >99.98a/o 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
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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
213P1F11 epitope, and thus the status of exposure to 213P1F11, or exposure to
a vaccine that elicits a
protective or therapeutic response.
Example 28: Use of Peutide 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
213P1F11-associated disease or who have been vaccinated with a 213P1F11
vaccine.
For example, the class I restricted CTL response of persons who have been
vaccinated may be
analyzed. The vaccine may be any 213P1F11 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
(SOU/ml), streptomycin (50
pg/ml), and Hepes (1 OmM) containing 10% heat-inactivated human AB serum
(complete RPMI) and plated
using microculture formats. A synthetic peptide comprising an epitope of the
invention is added at 10 pg/ml
to each well and HBV core 128-140 epitope is added at 1 ~g/ml to each well as
a source of T cell help during
the first week of stimulation.
In the microculture format, 4 x 105 PBMC are stimulated with 'peptide in 8
replicate cultures in 96-
well round bottom plate in 100 ~tl/well of complete RPMI. On days 3 and 10,
100 p l 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 5 'Cr
release, based on comparison with
non-diseased control subjects as previously described (Rehermann, et al.,
Nature Med. 2:1104,1108, 1996;
Rehermann et al., J. Clip. Invest. 97:1655-1665, 1996; and Rehermann et al. J.
Clip. Invest. 98:1432-1440,
1996).
Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are
either purchased
from the American Society for Histocompatibility and Immunogenetics (ASHI,
Boston, MA) or established
from the pool of patients as described (Guilhot, et al. J. d~irol. 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 p,M, and labeled with 100
p.Ci of 5'Cr (Amersham Corp.;
Arlington Heights, IL) for 1 hour after which they are washed four times with
HBSS.
Cytolytic activity is determined in a standard 4-h, split well 5'Cr 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-
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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 213P1F11 or a 213P1F11 vaccine.
Similarly, Class II restricted HTL responses may also be analyzed. Purified
PBMC are cultured in a
96-well flat bottom plate at a density of 1.5x105 cells/well and are
stimulated with 10 ~tg/ml synthetic peptide
of the invention, whole 213P 1F 11 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 l0U/ml IL-2. Two days later, 1 ~tCi 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 earned 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 ofpeptide
composition;
Group II: 3 subjects are injected with placebo and 6 subjects are injected
with 50 pg peptide
composition;
Group III: 3 subjects are injected with placebo and 6 subjects are injected
with 500 p.g of peptide
composition.
After 4 weeks following the first injection, all subjects receive a booster
inoculation at the same
dosage.
The endpoints measured in this study relate to the safety and tolerability of
the peptide composition
as well as its immunogenicity. Cellular immune responses to the peptide
composition are an index of the
intrinsic activity of this the peptide composition, and can therefore be
viewed as a measure of biological
efficacy. The following summarize the clinical and laboratory data that relate
to safety and efficacy
endpoints.
Safety: The incidence of adverse events is monitored in the placebo and drug
treatment group and,
assessed in terms of degree and reversibility.
Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects
are bled before and
after injection. Peripheral blood mononuclear cells are isolated from fresh
heparinized blood by Ficoll-
Hypaque .density gradient 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 ExpressinE 213P1F11
Phase II trials are performed to study the effect of administering the CTL-HTL
peptide compositions
to patients having cancer that expresses 213P1F11. The main objectives of the
trial are to determine an
effective dose and regimen for inducing CTLs in cancer patients that express
213P1F11, 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 213P1F11.
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 213P1F11-associated disease.
Example 31 ~ Induction of CTL Responses Usine a Prime Boost Protocol
A prime boost protocol similar in its underlying principle to that used to
confirm the efficacy of a
DNA vaccine in transgenic mice, such as described above in the Example
entitled "The Plasmid Construct
and the Degree to Which It Induces Immunogenicity," can also be used for the
administration of the vaccine
to humans. Such a vaccine regimen can include an initial administration of,
fo>: example, naked DNA
followed by a boost using recombinant virus encoding the vaccine, or
recombinant protein/polypeptide or a
peptide mixture administered in an adjuvant.
For example, the initial immunization may be performed using an expression
vector, such as that
constructed in the Example entitled "Construction of "Minigene" Multi-Epitope
DNA Plasmids" in the form
of naked nucleic acid administered IM (or SC or lD) in the amounts of 0.5-5 mg
at multiple sites. The nucleic
acid (0.1 to 1000 fig) 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 5-10'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 213P 1 F 11 is generated.
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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 and HTL
then destroy or facilitate
destruction, respectively, of the target cells that bear the 213P1F11 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 ProgenipoietinT"'
(Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides,
and prior to reinfusion into
patients, the DC are washed to remove unbound peptides.
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 10' 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
ProgenipoietinT"' are injected into
patients without purification of the DC. The total number of PBMC that are
administered often ranges from
10$ to 101°. 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 ProgenipoietinT"' 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 10$ peptide-loaded
PBMC. The percent DC mobilized by an agent such as ProgenipoietinT"' is
typically estimated to be between
2-10%, but can vary as appreciated by one of skill in the art.
Ex vivo activation of CTL/HTL responses
Alternatively, ex vivo CTL or HTL responses to 213P1F11 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 Identifvin~ and Confirmins 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
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the antigen of interest, e.g. 213P1F11. 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 213P1F11 to isolate
peptides corresponding to~213P1F11
that have been presented on the cell surface. Peptides obtained from such an
analysis will bear motifs) that
correspond to binding to the single HLA allele that is expressed in the cell.
As appreciated by one in the art, one can perform a similar analysis on a cell
bearing more than one
HLA allele and subsequently determine peptides specific for each HLA allele
expressed. Moreover, one of
skill would also recognize that means other than transfection, such as loading
with a protein antigen, can be
used to provide a source of antigen to the cell. ,
Example 34' Comnlementarv Polynucleotides
Sequences complementary to the 213P1F11-encoding sequences, or any parts
thereof, are used to
detect, decrease, or inhibit expression of naturally occurring 213P1F11.
Although use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with smaller ar
with larger sequence fragments. Appropriate oligonucleotides are designed
using, e.g., OLIGO 4.06 software
(National Biosciences) and the coding sequence of 213P1F11. 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 213P1F11-encoding transcript.
Example 35 Purification of Naturally-occurring or Recombinant 213P1F11 Using
213P1F11-
Specific Antibodies
Naturally occurring or recombinant 213P1F11 is substantially purified by
immunoaffmity
chromatography using antibodies specific for 213P1F11. An immunoaffinity
column is constructed by
covalently coupling anti-213P1F11 antibody to an activated chromatographic
resin, such as CNBr-activated
SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is
blocked and washed
according to the manufacturer's instructions.
Media containing 213P1F11 are passed over the immunoaffmity column, and the
column is washed
under conditions that allow the preferential absorbance of 213P1F11 (e.g.,
high ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibody/213P1F11 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.
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Examule 36' Identification of Molecules Which Interact with 213P1F11
213P1F11, or biologically active fragments thereof, are labeled with 121 1
Bolton-Hunter reagent.
(See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules
previously arrayed in the wells of
a mufti-well plate are incubated with the labeled 213P1F11, washed, and any
wells with labeled 213P1F11
complex are assayed. Data obtained using different concentrations of 213P1F11
are used to calculate values
for the number, affinity, and association of 213P1F11 with the candidate
molecules.
Examule 37' In Vivo Assay for 213P1Fi1 Tumor Growth Promotion
The effect of the 213P1F11 protein on tumor cell growth is evaluated in vivo
by evaluating tumor
development and growth of cells expressing or lacking 213P1F11. For example,
SCID mice are injected
subcutaneously on each flank with 1 x 106 of either prostate, bladder or
breast cancer cell lines (such as PC3,
DU145, UM-UC3, J82, MCF7) or NIH-3T3 cells containing tkNeo empty vector or
213P1F11. At least two
strategies may be used: (1) Constitutive 213P1F11 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, tet, etc., provided such promoters are compatible
with the host cell systems.
Tumor volume is then monitored at the appearance of palpable tumors and
followed over time to determine if
213P1F11-expressing cells grow at a faster rate and whether tumors produced by
213P1F11-expressing cells
demonstrate characteristics of altered aggressiveness (e.g. enhanced
metastasis, vascularization, reduced
responsiveness to chemotherapeutic drugs).
Additionally, mice can be implanted with 1 x 105 of the same cells
ortliotopically to determine if
213P 1F11 has an effect on local growth in the prostate or on the ability of
the cells to metastasize, specifically
to lungs, lymph nodes, and bone marrow.
The assay is also useful to determine the 213P1F11 inhibitory effect of
candidate therapeutic
compositions, such as for example, 213P1F11 intrabodies, 213P1F11 antisense
molecules and ribozymes.
Example 38~ 213P1F11 Monoclonal Antibody-mediated Inhibition of Tumors In Vivo
The significant expression of 213P1F11 in cancer tissues, together with its
restricted expression in
normal tissues, makes 213P1F11 an excellent target for antibody therapy. In
cases where the monoclonal
antibody target is a cell surface protein, antibodies have been shown to be
efficacious at inhibiting tumor
growth (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 or the World Wide
Web at
.pnas.org/cgi/doi/10.1073/pnas.051624698). In cases where the target is not on
the cell surface, such as for
213P1F11 and including PSA and PAP in prostate cancer, antibodies have still
been shown to recognize and
inhibit growth of cells expressing those proteins (Saffran, D.C., et al.,
Cancer and Metastasis Reviews, 1999.
18: p. 437-449). As with any cellular protein with a restricted expression
profile, 213P1F11 is a target for T
cell-based immunotherapy.
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Accordingly, the therapeutic efficacy of anti-213P1F11 mAbs in human prostate,
bladder and breast
cancer mouse models is investigated using in 213P1F11-expressing prostate and
bladder cancer xenografts as
well as prostate, bladder and breast cancer cell lines, such as those
described in the Example entitled "In ~ivo
Assay for 213P1F11 Tumor Growth Promotion," that have been engineered to
express 213P1F11.
Antibody efficacy on tumor growth and metastasis formation is confirmed, e.g.,
in a mouse
orthotopic prostate or bladder cancer xenograft models, as well as SCID mice
injected with prostate, bladder
and breast cancer cell lines, such as those described in the Example entitled
"In Vivo Assay for 213P1F11
Tumor Growth Promotion," designed to express or lack 213P1F11. Therapeutic
efficacy of anti-213P1F11
mAbs in prostate cancer is also evaluated in human prostate xenograft mouse
models sucha s the LAPC-9
xenografts (Craft, N., et al.,. Cancer Res, 1999. 59( 19): p. 5030-6). The
antibodies can be unconjugated, as
discussed in this Example, or can be conjugated to a therapeutic modality, as
appreciated in the art. It is
confirmed that anti-213P1F11 mAbs inhibit formation of 213P1F11-expressing
tumors. Anti-213P1F11
mAbs inhibit formation of the androgen-independent LAPC-9-AI tumor xenografts,
as well as PC3-
213P1F11, MCF7-213P1F11 and UM-UC3-213P1F11 tumors. Anti-213P1F11 mAbs also
retard the growth
of established orthotopic tumors and prolong survival of tumor-bearing mice.
These results indicate the
utility of anti-213P1F11 mAbs in the treatment of local and advanced stages of
prostate, bladder or breast
cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or the World Wide
Web at '
.pnas.org/cgi/doill0.1073/pnas.051624698)
Administration of anti-213P1F11 mAbs retard established orthotopic tumor
growth and inhibit
metastasis to distant sites, resulting in a significant prolongation in the
survival of tumor-bearing mice. These
studies indicate that 213P1F11 is an attractive target for immunotherapy and
demonstrate the therapeutic
potential of anti-213P1F11 mAbs for the treatment of local and metastatic
bladder cancer.
This example demonstrates that unconjugated 213P1F11 monoclonal antibodies
effectively to inhibit
the growth of human prostate, bladder and breast tumors grown in SCID mice;
accordingly a combination of
such efficacious monoclonal antibodies is also effective.
Tumor inhibition using multiple unconjugated 213P1F11 mAbs
Materials and Methods
213P1F11 Monoclonal Antibodies:
Monoclonal antibodies are raised against 213P1F11 as described in the Example
entitled
"Generation of 213P1F11 Monoclonal Antibodies (mAbs)." The antibodies are
characterized by ELISA,
Western blot, FACS, and immunoprecipitation , in accordance with techniques
known in the art, for their
capacity to bind 213P1F11. Epitope mapping data for the anti-213P1F11 mAbs, as
determined by ELISA and
Western analysis, recognize epitopes on the 213P1F11 protein.
Immunohistochemical analysis ofbladder
cancer tissues and cells with these antibodies is performed.
The monoclonal antibodies are purified from ascites or hybridoma tissue
culture supernatants by
Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized,
and stored at -20°C. Protein
determinations are performed by a Bradford assay (Bio-Rad, Hercules, CA). A
therapeutic monoclonal
antibody or a cocktail comprising a mixture of individual monoclonal
antibodies is prepared and used for the
treatment of mice receiving subcutaneous or orthotopic injections of bladder
tumor xenografts.
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Cancer Xeno~raft and Cell Lines
The LAPC-9 xenograft, which expresses a wild-type androgen receptor and
produces prostate-
specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe
combined immunodeficient (SCID)
mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., supra).
Prostate, bladder or breast cancer cell
lines (such as PC3, DU145, UM-UC3, J82, MCF7) expressing 213P1F11 are
generated by retroviral gene
transfer as described in Hubert, R.S., et al., STEAP: a prostate-specific cell-
surface antigen highly expressed
in human prostate tumors. Proc Natl Acad Sci U S A, 1999. 96(25):14523-8. Anti-
213PIF11 staining is
detected by using an FITC-conjugated goat anti-mouse antibody (Southern
Biotechnology Associates)
followed by analysis on a Coulter Epics-XL f low cytometer.
In Vivo Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 1 x 10 6 213P1F1 I-
expressing 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. In preliminary
studies, no difference is found between mouse IgG or PBS on tumor growth.
Tumor sizes are determined by
vernier caliper measurements, and the tumor volume is calculated as length x
width x height. Mice with s.c.
tumors greater than 1.5 cm in diameter are sacrificed. Circulating levels of
anti-213P1F11 mAbs are
determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, TX). (See,
e.g., (Saffran, D., et al.,
PNAS 10:1073-1078)
Orthotopic injections are performed, for example, in two alternative
embodiments, under anesthesia
by, for example, use of ketamine/xylazine. In a first embodiment, an
intravesicular injection of bladder
cancer cells is administered directly through the urethra and into the bladder
(Peralta, E. A., et al., J. Urol.,
1999. 162:1806-181 I). In a second embodiment, an incision is made through the
abdominal wall, the bladder
is exposed, and bladder tumor tissue pieces (1-2 mm in size) derived from a
s.c. tumor are surgically glued
onto the exterior wall of the bladder, termed "onplantation" (Fu, X., et al.,
Int. J. Cancer, 1991. 49: 938-939;
Chang, S., et al., Anticancer Res., 1997. 17: p. 3239-3242). For prostate
orthotopic studies, an incision is
made through the abdominal muscles to expose the bladder and seminal vesicles,
which then are delivered
through the incision to the exposed the dorsal prostate. Antibodies can be
administered to groups of mice at
the time of tumor injection or onplantation, or after 1-2 weeks to allow tumor
establishment.
Anti-213PIF11 mAbs Inhibit Growth of 213PIF11-Expressing Cancer Tumors
In one embodiment, the effect of anti-213PIF11 mAbs on tumor formation is
tested by using the
prostate and bladder orthotopic models. As compared with the s.c. tumor model,
the orthotopic model, which
requires surgical attachment of tumor tissue directly on the prostate or
bladder, results in a local tumor
growth, development of metastasis in distal sites, and subsequent death (Fu,
X., et al., Int. J. Cancer, 1991.
49: p. 938-939; Chang, S., et al., Anticancer Res., 1997. 17: p. 3239-3242).
This feature make the orthotopic
model more representative of human disease progression and allows one to
follow the therapeutic effect of
mAbs, as well as other therapeutic modalities, on clinically relevant end
points.
Accordingly, 213P1F11-expressing tumor cells are implanted orthotopically, and
2 days later, the
mice are segregated into two groups and treated with either: a) 50-2000~.g,
usually 200-SOO~g, of anti-
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213P1F11 Ab, or b) PBS, three times per week for two to five weeks. Mice are
monitored weekly for
indications of tumor growth.
As noted, a major advantage of the orthotopic prostate and bladder cancer
models is the ability to
study the development of metastases. Formation of metastasis in mice bearing
established orthotopic tumors
is studied by histological analysis of tissue sections, including lung and
lymph nodes (Fu, X., et al., Int. J.
Cancer, 1991. 49:938-939; Chang, S., et al., Anticancer Res., 1997.
17:3239=3242). Additionally, IHC
analysis using anti-213PIF11 antibodies can be performed on the tissue
sections.
Mice bearing established orthotopic 213PIF11-expressing tumors are
administered 1000pg
injections of either anti-213P1F11 mAb or PBS over a 4-week period. Mice in
both groups are allowed to
establish a high tumor burden (1-2 weeks growth), to ensure a high frequency
of metastasis formation in
mouse lungs and lymph nodes. Mice are then sacrificed and their local bladder
tumor and lung and lymph
node tissue are analyzed for the presence of tumor cells by histology and IHC
analysis.
These studies demonstrate a broad anti-tumor efficacy of anti-213PIF11
antibodies on initiation and
progression of bladder cancer in mouse models. Anti-213P1F11 antibodies
inhibit tumor formation and
retard the growth of already established tumors and prolong the survival of
treated mice. Moreover, anti-
213P1F11 mAbs demonstrate a dramatic inhibitory effect on the spread of local
prostate, bladder and breast
tumors to distal sites, even in the presence of a large tumor burden. Thus,
anti-213P1F11 mAbs are
efficacious on major clinically relevant end points including lessened tumor
growth, lessened metastasis, and
prolongation of survival.
Example 39: Therapeutic and Diaenostic use of Anti-213P1Fi1 Antibodies in
Humans.
Anti-213P1F11 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-213P1F11 mAb show strong extensive
staining in carcinoma but
significantly lower or undetectable levels in normal tissues. Detection of
213P1F11 in carcinoma and in
metastatic disease demonstrates the usefulness of the mAb as a diagnostic
and/or prognostic indicator. Anti-
213PIF11 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-213P1F11 mAb specifically binds to
carcinoma cells. Thus,
anti-213P1F11 antibodies are used in diagnostic whole body imaging
applications, such as
radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et.
al. Anticancer Res
20(2A):925-948 (2000)) for the detection of localized and metastatic cancers
that exhibit expression of
213P1F11. Shedding or release of an extracellular domain of 213P1F11 into the
extracellular milieu, such as
that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology
27:563-568 (1998)), allows
diagnostic detection of 213P1F11 by anti-213P1F11 antibodies in serum and/or
urine samples from suspect
patients.
Anti-213P1F11 antibodies that specifically bind 213P1F11 are used in
therapeutic applications for
the treatment of cancers that express 213P1F11. Anti-213PIF11 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,
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unconjugated and conjugated anti-213P1F11 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 "213P1F11 Monoclonal Antibody-
mediated Inhibition of Bladder
and Lung Tumors In ~ivo "). Conjugated and unconjugated anti-213P1F11
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 Diaenosis of Human
Carcinomas
through use of Human Anti-213P1F11 Antibodies In vivo
Antibodies are used in accordance with the present invention which recognize
an epitope on
213P 1F11, and are used in the treatment of certain tumors such as those
listedin Table I. Based upon a
number of factors, including 213P1F11 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.
L) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-
213P1F11
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-
213P1F11 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-213P1F11 antibodies are utilized in several
adjunctive clinical trials in
combination with the chemotherapeutic or antineoplastic agents adriamycin
(advanced prostrate carcinoma),
cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer),
and doxorubicin (preclinical).
IL) Monotherapy: In connection with the use of the anti-213P1F11 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
(hsy Y9o) to anti-
213P1F11 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
213P1F11. In connection with the use of the anti-213P1F11 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 ('I1 In)-213P1F11
antibody is used as an imaging agent in a Phase I human clinical trial in
patients having a carcinoma that
expresses 213P1F11 (by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst.
83:97-104 (1991)). Patients are
followed with standard anterior and posterior gamma camera. The results
indicate that primary lesions and
metastatic lesions are identified
Dose and Route of Administration
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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-
213P1F11 antibodies can be
administered with doses in the range of 5 to 400 mg/m Z , with the lower doses
used, e.g., in connection with
safety studies. The affinity of anti-213P1F11 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-213P1F11 antibodies that are fully human antibodies, as compared to the
chimeric antibody, have slower
clearance; accordingly, dosing in patients with such fully human anti-213P
1F11 antibodies can be lower,
perhaps in the range of 50 to 300 mg/mz , and still remain efficacious. Dosing
in mg/mz , 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-213P1F11
antibodies.
Conventional intravenous delivery is one standard delivery technique for many
tumors. However, in
connection with tumors in the peritoneal cavity, such as tumors of the
ovaries, biliary duct, other ducts, and
the like, intraperitoneal administration may prove favorable for obtaining
high dose of antibody at the tumor
and to also minimize antibody clearance. In a similar manner, certain solid
tumors possess vasculature that is
appropriate for regional perfusion. Regional perfusion allows for a high dose
of antibody at the site of a
tumor and minimizes short term clearance of the antibody.
Clinical Development Plan (CDP)
Overview: The CDP follows and develops treatments of anti-213P1F11 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-213P1F11 antibodies. As will be appreciated, one
criteria that can be utilized in
connection with enrollment of patients is 213P1F11 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 213P1F11. Standard tests and
follow-up are utilized to monitor each of these safety concerns. Anti-213P1F11
antibodies are found to be
safe upon human administration.
Example 41: Human Clinical Trial Adiunctive Therany with Human Anti-213P1F11
Antibody
and Chemotherapeutic Asent
A phase I human clinical trial is initiated to assess the safety of six
intravenous doses of a human
anti-213P1F11 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 and-213P1F11 antibodies
when utilized as an adjunctive.
therapy to an antineoplastic or chemotherapeutic agent, such as cisplatin,
topotecan, doxorubicin, adriamycin,
taxol, or the like, is assessed. The trial design includes delivery of six
single doses of an anti-213P1F11
antibody with dosage of antibody escalating from approximately about 25 mg/m 2
to about 275 mg/m 2 over
the course of the treatment in accordance with the following schedule:
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Day 0 Day 7 Day 14 Day 21 Day 28 Day 35
mAb Dose 25 75 125 175 225 275
mg/m z mg/m Z mg/m Z mglm 2 mg/m Z 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 213P1F11.
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-213P1F11 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-213P1F11
Antibody
Anti-213P1F11 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-
213P1F11 antibodies.
Example 43: Human Clinical Trial: Diaenostic Ima~in~ with Anti-213P1F11
Antibod
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-213P
1F11 antibodies as a diagnostic
imaging agent. The protocol is designed in a substantially similar manner to
those described in the art, such
as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are
found to be both safe and
efficacious when used as a diagnostic modality.
Example 44: Homolo~y Comparison of 213P1F11 to Known Seauences
The 213P1F11 gene is homologous to a previously cloned gene, namely the human
caspase 14
precursor (gi 6912286) (Hu S et al, J. Biol. Chem. 1998, 273:29648), also
known as mini-ICE (MICE). The
213P1F11 gene resulted in several protein variants, which share several
characteristics (Table XXII),
including homology to ICE family of cysteine proteases. Several variants of
213P1F11, namely 213P1F11-
v.2, -v.3 and -v.4, are novel proteins that maintain some homology to the
published caspase 14 precursor (gi
6912286). For example, 213P1F11-v.2 shows 100% identity to the human caspase
14 precursor (gi
6912286) over the first 174 as of the protein (Figure 4D), while differing
from the published caspase 14
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precursor protein' by 56 amino acids at its C-terminus, thus resulting in 76%
overall identity to caspase 14
precursor. 213P1F11-v.2 also maintains homology to the mouse caspase 14, and
shows 83% homology and
72% identity to that protein (gi 6753280) (Figure 4F). The 213P 1F11-v.3
variant protein show 100% identity
to the human caspase 14 precursor (gi 6912286) over 134 amino acids, while
differing from that protein by 12
as at its C-terminus. Similarly, 213P1F11-v.4 shows 97% identity with the
human caspase 14 precursor over
235 amino acids, while differing from the human caspase 14 precursor (gi
6912286) by 86 as at its N-
terminus (Figure 4G). 213P1F11-v.l consists of 242 amino acids, with
calculated molecular weight of 28.0
kDa, and pI of 5.4. 213P1F11-v.l is an intercellular protein, located in the
cytosol with potential localization
to the nucleus (Table XXII). Similar localization patterns are observed for
213P1F11 protein variants 1, 3,
and 4 (Table XXII). Bioinformatic analysis indicates that 213P1F1-v.2 may also
localize to the mitochondria
(Table XXII).
Caspases are a family of cyteine proteases that function as effectors of
apoptosis or programmed cell
death (Salvesen GS, Dixit V, Cell. 1997, 91:443; Thomberry N, Lazebnik Y,
Science. 281: 1312). These
proteases cleave different cellular substrates in an aspartate-specific
manner. Cleavage may result in
activation or inactivation of the cleaved cellular proteins, but not in
protein degradation (Nunez et al,
Oncogene. 1998, 17:3237; Stennicke HR, Salvesen GS, Cell Death Differ 1999,
6:1054). Caspases
traditionally exist as precursor proteins also known as single polypeptide
zymogens consisting of a pro-
domain, and 2 catalytic subunits, p20 and p 10 and contain a conserved QACXG
active site (Stennicke HR,
Salvesen GS, Cell Death Differ 1999, 6:1054; Cohen M. Biochem J 1997, 326:1).
Similar to other members
of the caspase family, 213P1F11 contains two catalytic subunits, p20 and p10,
in addition to the conserved
penta-peptide active site. In 213P1F11 v.l, p20 spans as 16-139 and p10 spans
as 155-241, while the active
site is located at as 129. Similarly, 213P1F11 v.4 carnes both p20 and p10
subunits, while 213P1F11 v.2 and
v.3 contain the p20 subunit only, indicating that all 4 variants of 213P1F11
can function in a similar manner.
Caspases are activated by proteolytic cleavage of their internal aspartate by
an upstream enzyme, often
another caspase. However, unlike other caspases with short pro-domains,
caspase 14 is not reported to
associate with known caspases (Hu S et al, J. Biol. Chem. 1998, 273:29648).
Caspase 14 has been shown to
be processed by caspase 8 and caspase 10 as well as granzyme B, resulting in
two catalytic subunits, p20 and
p10 (Ahmad M et al, Cancer Res. 1998, 58:5201). These 2 cleavage products are
detected in human
epidermis and in vitro during keratinocyte differentiation (Eckhart L et al,
J. Invest. Drmatol. 2000,
115:1148). Overexpression of caspase 14 in breast carcinoma cells MCF7
resulted in the apoptosis of these
cells, suggesting that caspase 14 participates in the process of programmed
cell death (Hu S et al, J. Biol.
Chem. 1998, 273:29648).
Our findings that 213P1F11 is highly expressed in several cancers while
showing a restricted
expression pattern in normal tissues suggests that the 213P1F11 gene plays an
important role in various
cancers, including cancers of the prostate, bladder and breast. Based on its
similarity to caspase 14 213P1F11
has the ability to control tumor growth, apoptosis, survival, differentiation
and progression. Accordingly,
when 213P1F11 functions as a regulator of cell growth and apoptosis, or
expression, 213P1F11 is used for
therapeutic, diagnostic, prognostic or preventative purposes.
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Our findings that 213P1F11 is highly expressed in several cancers while
showing a restricted
expression pattern in normal tissues suggests that the 213P1F11 gene plays an
important role in various
cancers, including cancers of the prostate, bladder and breast. Based on its
similarity to caspase 14 213P1F11
has the ability to control tumor growth, apoptosis, survival, differentiation
and progression. Accordingly,
when 213P1F11 functions as a regulator of cell growth and apoptosis, or
expression, 213P1F11 is used for
therapeutic, diagnostic, prognostic or preventative purposes.
Example 45~ Identification and Confirmation of 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). Caspases
participate in signal
transduction processes by getting recruited to signaling complexes and
cleaving specific cellular substrates
including other caspases and structural proteins, ultimately resulting in
morphologic changes that represent
the hallmark of apoptosis (Cohen GM. Biochem J. 1997, 326:1). Recent studies
have demonstrated that
caspases also cleave signaling molecules, such as the guanine nucleotide
exchange factor TIAM1, leading to
the inactivation of TIAM1 and thereby the Rac cascade (Qi H et al, Cell Growth
Differ. 2001, 12:603). Using
immunoprecipitation and Western blotting techniques, proteins are identified
that associate with 213P1F11
and mediate signaling events. Several pathways known to play a role in cancer
biology can be regulated by
213P1F11, including phospholipid pathways such as PI3K, survival pathways such
as AKT, NFkB, 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.). Bioinformatic analysis revealed that
213P1F11 can become
phosphorylated by serine/threonine as well as tyrosine kinases. Thus, the
phosphorylation of 213P1F11 is
provided by the present invention to lead to activation of the above listed
pathways.
Using, e.g., Western blotting techniques the ability of 213P1F11 to regulate
these pathways is
confn-med. Cells expressing or lacking 213P1F11 are either left untreated or
stimulated with cytokines,
hormones and anti-integrin antibodies. Cell lysates are analyzed using anti-
phospho-specific antibodies (Cell
Signaling, Santa Cruz Biotechnology) in order to detect phosphorylation and
regulation of ERK, p38, AKT,
PI3K, PLC and other signaling molecules. When 213P 1F 11 plays a role in the
regulation of signaling
pathways, whether individually or communally, it is used as a target for
diagnostic, prognostic, preventative
and therapeutic purposes.
To confirm that 213P1F11 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.
NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress
SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation
AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress
ARE-luc, androgen receptor; steroids/MAPK; growthldifferentiation/apoptosis
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p53-luc, p53; SAPK; growth/differentiation/apoptosis
CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
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 213P1F11 are mapped and used for the
identification and validation
of therapeutic targets. When 213P1F11 is involved in cell signaling, it is
used as target for diagnostic,
prognostic, preventative and therapeutic purposes.
E_xamnle 46' Involvement in Tumor Prosression
Some apoptosis intermediates, such as DcRl, FLICE and TRAIL-R3, function as
cellular inhibitors
of apoptosis by acting as decoys and interfering with normal function of the
apoptotic machinery (Sheikh MS
et al, Oncogene. 1999, 18:4153; Ashkenazi A, Dixit VIVI. Curr Opin Cell Biol.
1999, 11:255). When
213P1F11 functions as a decoy, it can contribute to the growth of cancer
cells. The role of 213P1F11 in
tumor growth is confirmed in a variety of primary and transfected cell lines
including prostate, bladder and
breast cell lines as well as NIH 3T3 cells engineered to stably express
213P1F11. Parental cells lacking
213P1F11 and cells expressing 213P1F11 are evaluated for cell growth using a
well-documented proliferation
assay (Fraser SP, Grimes JA, Djamgoz MB. Prostate. 2000;44:61, Johnson DE,
Ochieng J, Evans SL.
Anticancer Drugs. 1996, 7:288).
To confirm the role of 213P1F11 in the transformation process, its effect in
colony forming assays is
investigated. Parental NIFI3T3 cells lacking 213P1F11 are compared to NHI-3T3
cells expressing 213P1F11,
using a soft agar assay under stringent and more permissive conditions (Song
Z. et al. Cancer Res.12000,
60:6730).
To confirm the role of 213P1F11 in invasion and metastasis of cancer cells, a
well-established assay
is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res.
1999, 59:6010). Control cells,
including prostate, colon, bladder and kidney cell lines lacking 213P1F11 are
compared to cells expressing
213P1F11. Cells are loaded with the fluorescent dye, calcein, and plated in
the top well of the Transwell
insert coated with a basement membrane analog. Invasion is determined by
fluorescence of cells in the lower
chamber relative to the fluorescence of the entire cell population. a
213P1F11 can also play a role in cell cycle and apoptosis. Parental cells and
cells expressing
213P1F11 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 213P1F11, including normal and
tumor bladder cells. Engineered and parental cells are treated with various
chemotherapeutic agents, such as
paclitaxel, gemcitabine, etc, and protein synthesis inhibitors, such as
cycloheximide. Cells are stained with
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annexin V-FITC and cell death is measured by FACS analysis. The modulation of
cell death by 213P1F11
can play a critical role in regulating tumor progression and tumor load.
When 213P1F11 plays a role in cell growth, transformation, invasion or
apoptosis, it is used as a
target for diagnostic, prognostic, preventative and therapeutic purposes.
Examule 47: Involvement in An~ioEenesis
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).
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
effect of 213P1F11 on angiogenesis is confirmed. For example, endothelial
cells engineered to express
213P1F11 are evaluated using tube formation and proliferation assays. The
effect of 213P1F11 is also
confirmed in animal models in vivo. For example, cells either expressing or
lacking 213P1F11 are implanted
subcutaneously in immunocompromised mice. Endothelial cell migration and
angiogenesis are evaluated 5-
15 days later using immunohistochemistry techniques. When 213P1F11 affects
angiogenesis, it is used as a
target for diagnostic, prognostic, preventative and therapeutic purposes
Example 48: Regulation of Transcription
The localization of 213P1F11 to the cytoplasm with potential nuclear
localization (Table XXII),
support the present invention use of 213P1F11 based on its role in the
transcriptional regulation of eukaryotic
genes. Regulation of gene expression is confirmed, e.g., by studying gene
expression in cells expressing or
lacking 213P1F11. For this purpose, two types of experiments are performed.
In the first set of experiments, RNA from parental and 213P1F11-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 or androgen 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, ELKl-luc,
ARE-luc, p53-luc, and CRE-luc. 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. '
When 213P1F11 plays a role in gene regulation, it is used as a target for
diagnostic, prognostic,
preventative and therapeutic purposes.
Examule 49~ Subcellular Localization of 213P1F11
The cellular location of 213P1F11 can be assessed using subcellular
fractionation techniques widely
used in cellular biology (Storrie B, et al. Methods Enzymol. 1990;182:203-25).
A variety of cell lines,
including prostate, bladder and breast cell lines as well as cell lines
engineered to express 213P1F11 are
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separated into nuclear, cytosolic and membrane fractions. Gene expression and
location in nuclei, heavy
membranes (lysosomes, peroxisomes, and mitochondria), light membranes (plasma
membrane and
endoplasmic reticulum), and soluble protein fractions are tested using Western
blotting techniques.
Alternatively, 293T cells can be transfected with an expression vector
encoding individual genes,
HIS-tagged (PCDNA 3.1 MYC/HIS, Invitrogen) and the subcellular localization of
these genes is determined
as described above. In short, the transfected cells can be harvested and
subjected to a differential subcellular
fractionation protocol (Pemberton, P.A. et al, 1997, J of Histochemistry and
Cytochemistry, 45:1697-1706.).
Location of the HIS-tagged genes is followed by Western blotting.
Using 213P1F11 antibodies, it is possible to demonstrate cellular localization
by
immunofluorescence and immunohistochemistry. For example, cells expressing or
lacking 213P1F11 are
adhered to a microscope slide and stained with anti-213P1F11 specific Ab.
Cells are incubated with an FITC-
coupled secondary anti-species Ab, and analyzed by fluorescent microscopy.
When 213P1F11 is localized to specific cell compartments, it is used as a
target for diagnostic,
preventative and therapeutic purposes.
Example 50: 213P1F11 Proteolytic Activity
The similarity of 213P1F11 to casapase cysteine proteases supports the use of
213P1F11 as a~
protease. Protease activity can be confirmed using on in vitro protease assay
coupled to detection of protein
fragments by western blotting (Hu S et al, above; Slee E et al, J Biol Chem.
2001, 276:7320). In one
embodiment, recombinant 213P1F11 protein is incubated with apoptotic
substrates, including other caspases
known to associate with caspase 14, namely caspase 2 and caspase 4, as well as
recombinant RIP and
poly(ADP-ribose) polymerase (i.e. PARP) (Slee E et al, J Biol Chem. 2001,
276:7320; Hayakawa et al,
Apoptosis. 2002, 7:107). Proteins are separated by SDS-Page and analyzed by
western blotting with substrate
specific antibodies. In another embodiment, 213P1F11 activity is compared in
control cells lacking
213P1F11 and cells expressing 213P1F11. Cell lysates from control and 213P1F11
expressing cells are
incubated in the presence of the recombinant substrates listed above. Whole
proteins are analyzed by western
blotting with antibodies directed to the apoptotic substrates.
When 213P1F11 functions as a protease, it is used as a target for diagnostic,
preventative and
therapeutic purposes
Throughout this application, various website data content, publications,
patent applications and
patents are referenced. (Websites are referenced by their Uniform Resource
Locator, or URL, addresses on
the World Wide Web.)
The present invention is not to be limited in scope by the embodiments
disclosed herein, which are
intended as single illustrations of individual aspects of the invention, and
any that are functionally equivalent
are within the scope of the invention. Various modifications to the models and
methods of the invention, in
addition to those described herein, will become apparent to those skilled in
the art from the foregoing
description and teachings, and are similarly intended to fall within the scope
of the invention. Such
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modifications or other embodiments can be practiced without departing from the
true scope and spirit of the
invention.
119
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TABLE I: Tissues that Express 213P1F11 When Malignant
- Bladder
- Prostate
- Breast
TABLE II: Amino Acid Abbreviations
SINGLE LETTER THREE LETTER FULL NAME
F Phe hen lalanine
L Leu leucine
S Ser serine
y T osine
C s c steine
W T to han
1
p Pro roline
H His histidine
Gln lutamine
Ar ar mine
I Ile isoleucine
M Met methionine
T Thr threonine
N Asn as ara ine
g L s 1 sine
V Val valine
A Ala alanine
As ~ as artic acid
E Glu lutamic acid
G Gl 1 cine
120
SUBSTITUTE SHEET (RULE 26)
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TABLE III: Amino Acid Substitution Matrix
Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix
(block substitution
matrix). The higher the value, the more likely a substitution is found in
related, natural proteins. (See URL at
the World Wide Web (.ikp.unibe.ch/manual/blosum62.htm1 )
A D E F G H I K L M N P Q R S T V W Y
C
4 -2 -1-2 0 -2 -1-1 -1-1 -2-1 -1-1 1 0 0 -3 -2
0 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 -20 -31 -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 -11 3
F
6 -2 -4-2 -4-3 0 -2 -2-2 0 -2 -3-2 -3
G
8 -3-1 -3-2 1 -2 0 0 -1-2 -3-2 2
H
4 -3 2 1 -3-3 -3-3 -2-1 3 -3 -1
I
5 -2-1 0 -1 1 2 0 -1 -2-3 -2
K
4 2 -3-3 -2-2 -2-1 1 -2 -1
L
5 -2-2 0 -1 -1-1 1 -1 -1
M
6 -2 0 0 1 0 -3-4 -2
N
7 -1-2 -1-1 -2-4 -3
P
5 1 0 -1 -2-2 -1
Q
5 -1-1 -3-3 -2
R
4 1 -2-3 -2
S
5 0 -2 -2
T
4 -3 -1
V
11 2
W
7
Y
121
SUBSTITUTE SHEET (RULE 26)
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TABLE IV
HLA Class I/II Motifs/Supermotifs
TABLE IV (A): HLA Class I Supermotifs/Motifs
SUPERMOTIFS POSITION P05ITION POSITION
2 (Primary Anchor)3 (Primary Anchor)C Terminus (Primary
Anchor)
A1 TIGVMS
A2 LIVMA T IVMATL
A3 V SMATLI RK
A24 YFWIVLMT FIYWLM
B7 p VILFMWYA
B27 RHK FYLWMIVA
B44 ED FWYLIMVA
B58 ATS FWYLIVMA
B62 QLIVMP FWYMIVLA
MOTIFS
A1 TSM
A1 DEAS Y
A2.1 LMV IAT ~ ' VLIMAT
A3 LMVISATFCGD KYRHFA
Al l VTMLISAGNCDF KRYII
A24 YFWM FLIW
A*3101 MVTALIS
A*3301 MVALFIST RK
A*6801 AVTMSLI RK
B*0702 P LMFWYAIV
B*3501 P LMFWYIVA
B51 P LIVFWYAM
B*5301 P IMFWYAL V
B*5401 P ATIVLMFWY
Bolded residues are preferred, italicized residues are less preferred: A
peptide is considered motif bearing if
it has primary anchors at each primary anchor position for a motif or
supermotif as specified in the above
table.
TABLE IV (B): HLA Class II Supermotif
1 6 9
W, F, Y, V, .I, L A, V, I, L, P, A, V, I, L, C, S,
C, S, T T, M, Y
122
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
~ >
C7
z
~ ~x ~A ~~
U N '"
o 'J 4 ~' o U
U
~ U d 'i'
,_... j
U
O' o
~ ~~ dc~ ~r~
z
A
M F'~ ~ M m
U .b
."
N
N
4H
cV '~"'U ~ c~
U
~
.
w ~'
0
~
~ ~
U '~ U ~ ~ b
w-.W ,'Y~~1 N ~ ' N
~
~ ~ a a ~
c ~ w r, ~,
U ' ~'
d '
o ~ ~ '~ '
o o
::
.
4~ 4w .~ ~ U
~ ~ W ~
'r ~ ~ ~ '.
~ ~ ~ G
Ci,C1 p.
,~ ~ ,~
a
b
H ~
~
~ A A ~ A A
~ ~~
~
123
SUBSTITUTE EET (RULE
SH 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
d
0 o o o o ~ o o o o
H ~ a > ~
rr' 4:i .~ U V U V
w ~ ~ ~ ~ w.
~ H
d ~ d
'~
d d d d~ ~a o ,~ ~ ~
a .
U .. ~ ~ . r, ' ~ ,~ ~ '-'
a w w w w
a
.--,
~'"' AMA i
i
wV
z~
,.
w~
~. .r
~.,
w~
A M
d.
3~,w_~
M
~ ~ .~t'.r
A
d'
,
o o o N o 0 0 0 0 o
~ ~, U ~ ~ U U U U a
U U U ~ ~ U
~ ~
d d~ d~ d~ d~" Q~ dw d d
~ d ~
w
0
E
y., ~ ~~ .~,-;~~
-, ~ i ~~d mM
Acs ~~A~c~~a
wa
U
x O
a~
o
.-,
::
A
O a, a, b
b
W
a
W ~~ N M N I~ N V~'v1
~ '
~ d a d d w ra c~ r~ c~
~
124
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
,.o
U '-'
".'
.s
~~
o, c ~~. w (.
~~ d ~ a
~
V -. , 7
w A C7 ~ ~ ~ a'
~
~
,,
3
w' ~ ~
~
~ ~ d
a a ~
U
a. d ~ ~ a C7
d
p ~ ~ w
c'7 ~ ~ ~.
a~
~r ..~..,~ ~ A d d
d A
.
~
w ~ ~ '
A W A
C.1 ~ ~ d
A ~ ~ . ,
~
o ~~" o ~a~, off'
w .~ w
q a ~ U
3 d~
H ~w
~a
'_" w~A ~ d
c7
U Z ~u ~ -d b ~o -o
O ~ ~ ~ a ~
a~ a~ a~ a~
o o o
~, ,o ,o
E~ ~ i ~ ~ ~ ~ i
~
w ~. s,.., w 4..~ 4..,
~ a~ ~ .~. ~ ..... .,..
: d a~ a~ o ~ a~
~, ~ ~ ~. o
a. ~ b o.
b ~. ~
: . . . ,
W
a i _ _ i r.
i i
.
O~ d C ~ ~ O N
O d
v -. ..., av
H
12s
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO PCT/US02/10220
03/085121
dw
~;~ >
o ~ 0 0 0 0
ov ~ ~ ~'' ~ ~ w ~
~d~ d~ o ~'"'~,, Ad o
0o a. a, c7 ~ ~ a d A
w w
3
d ~ ~ ~,
w
A
~n w d d
3
v c7 ~ ~ ~ ~ c7 a, ~, a,
a,
A~ ~ A
a
~ ~ w
cn ~~~ a~~ ~~~ ~~ a3 aE~., ads
a ~~ ° ~~-" ° >Q d~ ° >"
a
c
0
a,
w~ wd w~ w w . C7
o d A"" A A A ~ G.1
s ~ b ~ -d ~ b ~ b v b ~ b a 'd
V a~ o a~ o a~ o a~ , o a~ o a~ o a~ o
H
w .~.. 4, Y w .r 4.~ ~.. w ~.. 4., ~ W +r
O ~. ~ p. C. p, C. R. ~ P.
b b 'C b ~C 'C 'C
P~
:~:
W
w
W N ~ ~"~ ~_ N ~ N ~ O O
do d d d~ do ~ d
E~
126
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
H
U
b
0 0 "' o v o ~ o 'J o ~L
a.i ~~ ~ Qw dw~ d~ dw~
r.
a a a,....,d '3
0o a, d ~ A w ~ ~ ~3 A
w
3 ~ z ~. a w a~
xa
w ~ ~ d d
° . a
~o ~ ~ C7 C7 ~ A H
t7 3 A ~ .°
w w
H
wU
w ~ ~ w '-' c.~7 ~
b
'b .°a ~ ,~ :a x .~ .~
~w ~w ~a, aa. aw o
d j d d d d d
a ,°-.~ d .°-~ ° ~ °., °~ °~ o
U ~ A, > 3 W U ~ z
0
A,
w a d a d
z ~ ~ b ~ ~o ~ b ~ b
o ~ o ~ o ~ .o ~ ,o ~ .o ~ o
O p. .~ u~ .d p. .~ s~ b a, .d p. .d
W o 0 0 .--~ o 0
d ~ 0.Ml ~ ~ ~ ;v
H H
127
SUBSTITUTE SHEET (RULE 26)
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WO 03/085121 PCT/US02/10220
TableV v.l-A1-9mers:3P1F11 Table V v.l-A1-9mers:
21 213P1F11
Pos 123456789 Score Pos123456789 Score
96 KGEDGEMVK 22.500 Portion 97 GEDGEMVKL 0.050 Portion
of of
215 EAELVQEGK 18.000 N~ D 86 LMAHGREGF 0.050 NO D
171 TVEGYIAYR 18.000 each 142TVGGDETVM 0.050 each
start start
191 LVDVFTKRK 10.000 position 37 DALEHMFRQ 0.050 position
is is
170 STVEGYIAY 6.250 specified, $0 SCAFVVLMA 0.050 specified,
35 DLDALEHMF 5.000 the 119ALRAKPKW 0.050 the
length length
75 REDPVSCAF 2.500 of each 11 KYDMSGARL 0.050 of each
id tid
i i
9 9
53 KRDPTAEQF 2.500 pept 81 CAFWLMAH 0.050 pep
e e
s s
amino a~no
162 YTDALHVYS 2.500 acids, 108LFEALNNKN 0.045 acids,
the the
189 QTLVDVFTK 2.500 end 133RGEQRDPGE 0.045 end
228 NPEIQSTLR 2.250 Position 74 SREDPVSCA 0.045 position
for for
206 LTEVTRRMA 2.250 each 7 LEEEKYDMS 0.045 each
id
i
6 SLEEEKYDM 1.800 Peptide 28 AREGSEEDL 0.045 e
is s
the Pept
start the
start
145 GDEIVMVIK 1.800 position 169YSTVEGYIA 0.030 position
154 DSPQTIPTY 1.500 plus 117CQALRAKPK 0.030 plus
eight eight
31 GSEEDLDAL 1.350 79 VSCAFVVLM 0.030
99 DGEMVKLEN 1.125 14 MSGARLALI 0.030
104 KLENLFEAL 0.900 36 LDALEHMFR . 0.025
57 TAEQFQEEL 0.900 46 LRFESTMKR 0.025
202 ILELLTEVT 0.900 141ETVGGDEIV 0.025
38 ALEHMFRQL 0.900 212RMAEAELVQ 0.025
232 QSTLRKRLY 0.750 40 EHMFRQLRF 0.025
144 GGDEIVMVI 0.625 13 DMSGARLAL 0.025
167 HVYSTVEGY 0.500 ~ 153KDSPQTIPT 0.025
71 AIDSREDPV 0.500 161TYTDALHW 0.025
19 LALILCVTK 0.400 8 EEEKYDMSG 0.022
218 LVQEGKARK 0.400 45 QLRFESTMK 0.020
136 QRDPGETVG 0.250 121RAKPKWII 0.020
179 RHDQKGSCF 0.250 49 ESTMKRDPT 0.015
157 QTIPTYTDA 0.250 129IQACRGEQR 0.015
226 KTNPEIQST 0.250 184GSCFIQTLV 0.015
62 QEELEKFQQ 0.225 199KGHILELLT 0.013
175 YIAYRHDQK 0.200 233STLRKRLYL 0.013
125 KWIIQACR 0.200 30 EGSEEDLDA 0.013
107 NLFEALNNK 0.200 77 DPVSCAFW 0.013
213 MAEAELVQE 0.180 106ENLFEALNN 0.013
59 EQFQEELEK 0.150 ~ I60PTYTDALHV 0.013
219 VQEGKARKT 0.135 128IIQACRGEQ 0.010
61 FQEELEKFQ 0.135 20 ALILCVTKA 0.010
33 EEDLDALEH 0.125 ~ 158TIPTYTDAL 0.010
217 ELVQEGKAR 0.100 18 RLALILCVT 0.010
204 ELLTEVTRR 0.100 164DALHWSTV 0.010
187 FIQTLVDVF 0.100 111ALNNKNCQA O.O10
21 LILCVTKAR 0.100 193DVFTKRKGH O.O10
190 TLVDVFTKR 0.100 85 VLMAHGREG 0.010
83 FWLMAHGR 0.100 205LLTEVTRRM 0.010
230 EIQSTLRKR 0.100 203LELLTEVTR 0.010
64 ELEKFQQAI 0.090 147EIVMVIKDS 0.010
1 MSNPRSLEE 0.075 110EALNNKNCQ 0.010
115 KNCQALRAK 0.050 23 LCVTKAREG O.O10
128
SUBSTITUTE SHEET (RULE 26)
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Table V v.2-A1-9mers: 213P1F11 Table V v.2-A1-9mers: 213P1F11
Pos 123456789 Score Pos123456789 Score
33 DTSPTDMIR 12.500 Portion 27 PLWNSQDTS 0.000 Portion
of of
12 FQDPLYLPS 3.750 SEQ 41 RKAHALSRP 0.000 SEQ
ID 1D
TVEGPTPFQ 1.800 NO: 28 LWNSQDTSP 0.000 NO:
S; S;
h
34 TSPTDMIRK 1.500 start SS RRGKDISWN 0.000 each
eac start
position iti
is i
pos
9 PTPFQDPLY 0.250 specified, 11 PFQDPLYLP 0.000 on
s
specified
,
4 STVEGPTPF 0.250 the 49 PWWMCSRRG 0.000 the
length length
21 EAPPNPPLW 0.200 of of each
each
36 PTDMIRKAH 0.125 Peptide peptide
is is
9 9
46 LSRPWWMCS 0.075 a~no amino
acids id
the h
, ac
31 SQDTSPTDM 0.075 end e
s, t
end
8 GPTPFQDPL 0.025 position position
for for
17 YLPSEAPPN 0.020 each each
19 PSEAPPNPP 0.014 peptide peptide
is is
22 APPNPPLWN 0.013 the the
start start
i
i
TPFQDPLYL 0.013 t position
on lus
Pos ei
plus ht
eight
42 KAHALSRPW 0.010 p
g
SEAPPNPPL O.O10
44 HALSRPWWM 0.010
50 WWMCSRRGK O.O10
47 SRPWWMCSR 0.005
3 YSTVEGPTP 0.003
NSQDTSPTD 0.003
IRKAHALSR 0.003
24 PNPPLWNSQ 0.003
56 RGKDISWNF 0.003
23 PPNPPLWNS 0.003
14 DPLYLPSEA 0.003
35 SPTDMIRKA 0.003
6 VEGPTPFQD 0.003
48 RPWWMCSRR 0.003
29 WNSQDTSPT 0.003
37 TDMIRKAHA 0.001
52 MCSRRGKDI 0.001
ALSRPWWMC 0.001
16 LYLPSEAPP 0.001
1 HVYSTVEGP 0.001
43 AHALSRPWW 0.001
51 WMCSRRGKD 0.001
32 QDTSPTDMI 0.001
38 DMIRKAHAL 0.001
18 LPSEAPPNP 0.001
2 VYSTVEGPT 0.001
54 SRRGKDISW 0.000
25 NPPLWNSQD 0.000
26 PPLWNSQDT 0.000
7 EGPTPFQDP 0.000
39 MIRKAHALS 0.000
$3 CSRRGKDIS 0.000
15 PLYLPSEAP 0.000
13 QDPLYLPSE 0.000
129
SUBSTITUTE SHEET (RULE 26)
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Table V v.3-A1-9mers: 213P1F11
Pos 123456789 Score
9 ATLPSPFPY 62.500 Portion
of
11 LPSPFPYLS 0.050 SEQ
ID
7 RGATLPSPF 0.025
TLPSPFPYL 0.020 each
start
osition
is
1 YIIQACRGA 0.010 p
specified,
2 IIQACRGAT 0.010 the
length
12 PSPFPYLSL 0.008 of
each
5 ACRGATLPS 0.005 Peptide
is
9
3 IQACRGATL 0.003 amino
acids
the
8 GATLPSPFP 0.002 ,
end
4 QACRGATLP 0.000 position
for
6 CRGATLPSP 0.000 each
peptide
is
the
start
position
lus
ei
ht
130
SUBSTITUTE SHEET (RULE 26)
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TableV v.4-A1-9mers:3P1F11 TableV v.4-A1-9mers:3P1F11
21 21
Pos 123456789 Score Pos123456789 Score
85 ASEEEKYDM 2.700 Portion 76 ISFRNSETS 0.002 Portion
of of
25 NGECGQTFR 2.250 SEQ 74 LSTSFRNSE 0.002 SEQ
ID ID
35 KEEQGRAFR 0.900 No' 48 HQKLVNDPR 0.002 NO:
~' 9;
h
83 TSASEEEKY 0.750 eac 13 VQPEKRTGL 0.002 each
start start
position position
is is
9 KSLSVQPEK 0.600 specified, 28 CGQTFRLKE 0.001 specified,
82 ETSASEEEK 0.500 the 84 SASEEEKYD 0.001 the
length length
34 LKEEQGRAF 0.450 of 69 IVGRDLSIS 0.001 of each
each
71 GRDLSISFR 0.250 Peptide 20 GLRDENGEC 0.001 Peptide
is is
9 9
14 QPEKRTGLR 0.225 amino 33 RLKEEQGRA 0.001 amino
acids id
the th
, ac
27 ECGQTFRLK 0.200 end 40 RAFRGSSVH 0,001 s,
e
end
80 NSETSASEE 0.135 position 65 GVGDIVGRD 0.001 position
for for
58 TQEVFGGGV 0.135 each 32 FRLKEEQGR 0.001 each
4 CQEYDKSLS 0.135 peptide 59 QEVFGGGVG 0.001 Peptide
is is
the the
52 VNDPRETQE 0.125 start 61 VFGGGVGDI 0.001 start
i
i
pos position
66 VGDIVGRDL 0.125 t 78 FRNSETSAS 0.001 Plus
on ei
plus ht
eight
SLSVQPEKR 0.100 67 GDIVGRDLS 0.001 g
12 SVQPEKRTG 0.100 23 DENGECGQT 0.001
45 SSVHQKLVN 0.075 79 RNSETSASE 0.001
68 DIVGRDLSI 0.050 2 GKCQEYDKS 0.001
64 GGVGDIVGR 0.050 ~ 39 GRAFRGSSV 0.001
86 SEEEKYDMS 0.045 17 KRTGLRDEN 0.001
22 RDENGECGQ 0.045 38 QGRAFRGSS 0.000
57 ETQEVFGGG 0.025 37 EQGRAFRGS 0.000
24 ENGECGQTF 0.025 29 GQTFRLKEE 0.000
18 RTGLRDENG 0.025 41 AFRGSSVHQ 0.000
21 LRDENGECG 0.025 49 QKLVNDPRE 0.000
44 GSSVHQKLV 0.015 8 DKSLSVQPE 0.000
11 LSVQPEKRT 0.015 81 SETSASEEE 0.000
70 VGRDLSISF 0.013 31 TFRLKEEQG 0.000
63 GGGVGDIVG 0.013 77 SFRNSETSA 0.000
51 LVNDPRETQ 0.010 53 NDPRETQEV 0.000
3 KCQEYDKSL 0.010 19 TGLRDENGE 0.000
73 DLSISFRNS 0.010 7 YDKSLSVQP 0.000
1 MGKCQEYDK O.O10 ~ 47 VHQKLVNDP 0.000
50 KLVNDPRET 0.010 16 EKRTGLRDE 0.000
75 SISFRNSET 0.010 15 PEKRTGLRD
42 FRGSSVHQK 0.010
55 PRETQEVFG 0.009
54 DPRETQEVF 0.003
5 QEYDKSLSV 0.003
56 RETQEVFGG 0.003
72 RDLSISFRN 0.003
EYDKSLSVQ 0.003
62 FGGGVGDIV 0.003
30 QTFRLKEEQ 0.003
36 EEQGRAFRG 0.003
26 GECGQTFRL 0.003
43 RGSSVHQKL 0.003
46 svHQKLVND 0.002
60 EVFGGGVGD 0.002
131
SUBSTITUTE SHEET (RULE 26)
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Table V v.5-A1-9mers: 213P1F11
Pos 123456789 Score
4 LALILRVTK 0.400 Portion
of
1 GARLALILR 0.050 SEQ
ID
ALILRVTKA O.O10 NO:
11;
h
eac
6 LILRVTKAR 0.010 start
position
is
3 RLALILRVT 0.010 specified,
2 ARLALjILRV 0.003 the
length
9 RVTKAREGS 0.001 of each
8 LRVTKAREG 0.001 Peptide
is
9
7 ILRVTKARE 0 a~no
000
. acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
(Table V v.6-A1-9mers: 213P1F11 I
Pos 123456789 Score
1 KLENLFEAM 0.900 Portion
of
4 NLFEAMI~1I~TK0.200 SEQ
ID
5 LFEAMNNKN 0.045 NO:
13;
h
3 ENLFEAMNN 0.013 start
eac
position
is
7 EAMNNKNCQ 0.010 specified,
8 AMNNKNCQA 0.005 the
length
2 LENLFEAMN 0.001 of each
6 FEAMNNKNC 0.001 Peptide
is
9
9 MNNKNCQAL 0.000 a~no
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
132
SUBSTITUTE SHEET (RULE 26)
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TableVI v.l-A1-lOmers:213P1F11 Ta ble VI v.l-A1-lOmers: 213PiF11
Pos 1234567890 Score Pos1234567890 Score
35 DLDALEHMFR 25.000 Portion 118QALRAKPKW 0.050 Portion
of of
228 NPEIQSTLRK 22.500 SEQ g0 SCAFWLMAH 0.050 SEQ
ID ID
202 ILELLTEVTR 18.000 N0:3; 106ENLFEALNNK 0.050 I'10:3;
h
38 ALEHMFRQLR 9.000 start 95 LKGEDGEMVK 0.050 each
eac start
position osition
is is
144 GGDEIVMVIK 5.000 specified, 58 AEQFQEELEK 0.050 p
specified,
169 YSTVEGYIAY 3.750 the 45 QLRFESTMKR 0.050 the
length length
162 YTDALHWST 2.500 of 108LFEALNNKNC 0.045 of
each each
104 KLENLFEALN 1.800 Peptide 62 QEELEKFQQA 0.045 Peptide
is is
171 TVEGYIAYRH 1.800 10 5 RSLEEEKYDM 0.030 10
amino amino
acids id
the th
, ac
206 LTEVTRRMAE 1.125 end 49 ESTMKRDPTA 0.030 s,
e
end
87 MAHGREGFLK 1.000 position 179RHDQKGSCFI 0.025 position
for for
71 AIDSREDPVS 1.000 each 11 KYDMSGARLA 0.025 each
215 EAELVQEGKA 0.900 Peptide 39 LEHMFRQLRF 0.025 peptide
is is
the the
213 MAEAELVQEG 0.900 start 166LHWSTVEGY 0.025 start
iti i
i
6 SLEEEKYDMS 0.900 on 33 EEDLDALEHM 0.025 t
Pos on
plus Pos
nine plus
nine
61 FQEELEKFQQ 0.675 227TNPEIQSTLR 0.025
75 REDPVSCAFV 0.500 139PGETVGGDEI 0.022
136 QRDPGETVGG 0.500 145GDEIVMVIKD 0.022
153 KDSPQTIPTY 0.500 142TVGGDEIVMV 0.020
191 LVDVFTKRKG 0.500 165ALHWSTVEG 0.020
170 STVEGYIAYR 0.500 185SCFIQTLVDV 0.020
96 KGEDGEMVKL 0.450 81 CAFWLMAHG 0.020
74 SREDPVSCAF 0.450 187FIQTLVDVFT 0.020
18 RLALILCVTK 0.400 158TIPTYTDALH 0.020
217 ELVQEGKARK 0.400 64 ELEKFQQAID 0.018
31 GSEEDLDALE 0.270 59 EQFQEELEKF 0.015
189 QTLVDVFTKR 0.250 1 MSNPRSLEEE 0.015
226 KTNPEIQSTL 0.250 154DSPQTIPTYT 0.015
53 KRDPTAEQFQ 0.250 159IPTYTDALHV 0.013
157 QTIPTYTDAL 0.250 3 NPRSLEEEKY 0.013
133 RGEQRDPGET 0.225 143VGGDEIVMVI 0.013
7 LEEEKYDMSG 0.225 111ALNNKNCQAL O.O10
32 SEEDLDALEH 0.225 19 LALILCVTKA 0.010
99 DGEMVKLENL 0.225 70 QAIDSREDPV O.O10
116 NCQALRAKPK 0.200 167HWSTVEGYI 0.010
190 TLVDVFTKRK 0.200 125KWIIQACRG 0.010
188 IQTLVDVFTK 0.150 114NKNCQALRAK O.O10
219 VQEGKARKTN 0.135 22 ILCVTKAREG 0.010
141 ETVGGDEIVM 0.125 149VMVIKDSPQT 0.010
160 PTYTDALHW 0.125 204ELLTEVTRRM 0.010
152 IKDSPQTIPT 0.125 . 110EALNNKNCQA 0.010
20 ALILCVTKAR 0.100 13 DMSGARLALI O.O10
128 IIQACRGEQR 0.100 23 LCVTKAREGS O.O10
$5 VLMAHGREGF 0.100 ' 175YIAYRHDQKG 0.010 '
57 TAEQFQEELE 0.090 148IVMVIKDSPQ 0.010
232 QSTLRKRLYL 0.075 84 WLMAHGREG 0.010
14 MSGARLALIL 0.075 230EIQSTLRKRL 0.010
231 IQSTLRKRLY 0.075 214AEAELVQEGK 0.010
79 VSCAFWLMA 0.075 201HILELLTEVT O.O10
121 RAKPKWIIQ 0.050 193DVFTKRKGHI 0.010
133
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableVI v.2-A1-lOmers:213P1F11 Ta ble VI v.2-A1-lOmers:
213P1F11
Pos 1234567890 Score Pos1234567890 Score
34 DTSPTDMIRK 25.000 Portion 3 VYSTVEGPTP 0.000 Portion
of of
9 GPTPFQDPLY 2.500 SEQ 55 SRRGKDISWN 0.000 SEQ
ID ID
22 EAPPNPPLWN 0.500 NO: 1 LHVYSTVEGP 0.000 NO:
S; S;
h h
6 TVEGPTPFQD 0.450 start 51 WWMCSRRGKD 0.000 start
eac eac
position position
is is
20 PSEAPPNPPL 0.270 specified, 28 PLWNSQDTSP 0.000 specified,
37 PTDMIRKAHA 0.250 the 41 IRKAHALSRP 0.000 the
length length
47 LSRPWWMCSR 0.150 of of each
each
4 YSTVEGPTPF 0.150 Peptide peptide
is is
13 FQDPLYLPSE 0.150 10 10 amino
amino acids
acids the
the
32 SQDTSPTDMI 0.075 , ,
end end
STVEGPTPFQ 0.050 position position
for for
33 QDTSPTDMIR 0.025 each each
43 KAHALSRPWW 0.020 Peptide peptide
is is
31 NSQDTSPTDM 0.015 the the
start start
iti i
i
35 TSPTDMIRKA O.O15 on pos
Pos t
plus on
nine plus
nine
PTPFQDPLYL 0.013
45 uAT.SRPWWMCO.O10
21 SEAPPNPPLW 0.010
17 LYLPSEAPPN 0.010
2 HVYSTVEGPT 0.010
40 MIRKAHALSR 0.005
52 WMCSRRGKDI 0.005
46 ALSRPWWMCS 0.005
48 SRPWWMCSRR 0.005
8 EGPTPFQDPL 0.003
26 NPPLWNSQDT 0.003
24 PPNPPLWNSQ 0.003
36 SPTDMIRKAH 0.003
23 APPNPPLWNS 0.003
18 YLPSEAPPNP 0.002
39 DMIRKAHALS 0.001
53 MCSRRGKDIS 0.001
54 CSRRGKDISW 0.001
56 RRGKDISWNF 0.001
42 RKAHALSRPW 0.001
44 AHALSRPWWM 0.001
14 QDPLYLPSEA 0.001
11 TPFQDPLYLP 0.001
38 TDMIRKAHAL 0.001
29 LWNSQDTSPT 0.001
7 VEGPTPFQDP 0.001
30 WNSQDTSPTD 0.001
12 PFQDPLYLPS 0.000
27 PPLWNSQDTS 0.000
19 LPSEAPPNPP 0.000
DPLYLPSEAP 0.000
PNPPLWNSQD 0.000
49 RPWWMCSRRG 0.000
16 PLYLPSEAPP 0.000
50 PWWMCSRRGK 0.000
134
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table VI v.3-A1-lOmers: 213P1F11
Pos 1234567890 Score
9 GATLPSPFPY 2.500 Portion
of
ATLPSPFPYL 0.500 SEQ
ID
12 LPSPFPYLSL 0.125
h
11 TLPSPFPYLS 0.020 start
eac
position
is
3 IIQACRGATL 0.020 specified,
2 YIIQACRGAT 0.010 the
length
7 CRGATLPSPF 0.005 of
each
5 QACRGATLPS 0.005 peptide
is
1 VYI IQACRGA0.001 IO
amino
acids
the
$ RGATLPSPFP 0.001 ,
end
6 ACRGATLPSP 0.000 position
for
4 IQACRGATLP 0.000 each
peptide
is
the
start
position
lus
nine
135
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableVI v.4-A1-lOmers:13P1F11 Ta ble VI v.4-A1-lOmers: 213P1F11
2
Pos 1234567890 Score Pos 1234567890 Score
85 ASEEEKYDMS 1.350 Portion 29 GQTFRLKEEQ 0.002 Portion
of of
82 ETSASEEEKY 1.250 SEQ 41 AFRGSSVHQK 0.001 SEQ
ID ID
52 VNDPRETQEV 1,250 NO' 20 GLRDENGECG O.OO1 NO:
~' 9;
h
t
rt
25 NGECGQTFRL 1.125 each 73 DLSISFRNSE 0.001 eac
start s
osition a
is position
is
34 LKEEQGRAFR 0.900 p 51 LVNDPRETQE 0.001 specified,
specified,
4 CQEYDKSLSV 0.675 the 46 SVHQKLVNDP 0.001 the
length length
35 KEEQGRAFRG 0.225 of each 47 VHQKLVNDPR 0.001 of
each
86 SEEEKYDMSG 0.225 Peptide 31 TFRLICEEQGR0.001 Peptide
is is
i
9 KSLSVQPEKR 0.150 10 amino 53 NDPRETQEVF 0.001 no
id l0
th am
acids
the
80 NSETSASEEE 0.135 ac 49 QKLVNDPRET 0,001 ,
s, end'
e
end
58 TQEVFGGGVG 0.135 position 2 GKCQEYDKSL 0.001 position
for for
66 VGDIVGRDLS 0.125 each 56 RETQEVFGGG 0.001 each
71 GRDLSISFRN 0.125 Peptide 6 EYDKSLSVQP 0.001 Peptide
is is
the the
12 SVQPEKRTGL 0.100 start 54 DPRETQEVFG 0.001 start
iti
44 GSSVHQKLVN 0.075 Position 39 GRAFRGSSVH 0.001 on
lus Pos
nine plus
nine
63 GGGVGDIVGR 0.050 p 42 FRGSSVHQKL 0.001
69 IVGRDLSISF 0.050 61 VFGGGVGDIV 0:001
22 RDENGECGQT 0.045 5 QEYDKSLSVQ 0.001
57 ETQEVFGGGV 0.025 72 RDLSISFRNS 0.001
24 ENGECGQTFR 0.025 ~ 17 KRTGLRDENG 0.001
21 LRDENGECGQ 0.025 36 EEQGRAFRGS 0.001
55 PRETQEVFGG 0.023 79 RNSETSASEE 0.000
84 SASEEEKYDM 0.020 1 MGKCQEYDKS 0.000
8 DKSLSVQPEK 0.020 38 QGRAFRGSSV 0.000
11 LSVQPEKRTG 0.015 19 TGLRDENGEC 0.000
13 VQPEKRTGLR 0.015 64 GGVGDIVGRD 0.000
74 LSISFRNSET 0.015 28 CGQTFRLKEE 0.000
62 FGGGVGDIVG 0.013 37 EQGRAFRGSS 0.000
14 QPEKRTGLRD O.O11 78 FRNSETSASE 0.000
SLSVQPEKRT 0.010 59 QEVFGGGVGD 0.000 ,
60 EVFGGGVGDI 0.010 77 SFRNSETSAS 0.000
75 SISFRNSETS 0.010 16 EKRTGLRDEN 0.000
65 GVGDIVGRDL 0.010 32 FRLKEEQGRA 0.000
3 KCQEYDKSLS 0.010 ~ 48 HQKLVNDPRE 0.000
68 DIVGRDLSIS 0.010 7 YDKSLSVQPE 0.000
81 SETSASEEEK O.O10 15 PEKRTGLRDE 0.000
26 GECGQTFRLK O.O10
33 RLKEEQGRAF 0.010
50 KLVNDPRETQ O.O10
23 DENGECGQTF 0.005
27 ECGQTFRLKE 0.005
45 ssvHQKLVND 0.003
67 GDIVGRDLSI 0.003
70 VGRDLSISFR 0.003
30 QTFRLKEEQG 0.003
18 RTGLRDENGE 0.003
43 RGSSVHQKLV 0.003
40 RAFRGSSVHQ 0.002
83 TSASEEEKYD 0.002
76 ISFRNSETSA 0.002
136
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table
VI
v.S-A1-lOmers:
213P1F11
Pos 1234567890 Score
4 RLALILRVTK 0.400 Portion
of
6 ALILRVTKAR 0.100 SEQ
ID
1 SGARLALILR 0 NO:11;
013
. each
start
S LALILRVTKA 0.010 position
is
2 GARLALILRV O.OOS specified,
8 ILRVTKAREG 0.001 the
length
9 LRVTKAREGS 0.001 of
each
3 ARLALILRVT 0.001 Peptide
is
i
10
7 LILRVTKARE 0.000 no
am
the
acids
RVTKAREGSE 0.000 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
Table
VI
v.6-A1-lOmers:
213P1F11
Pos 1234567890 Score
2 KLENLFEAMN 1.800 Portion
of
4 ENLFEAMNNK O.OSO SEQ
ID
6 LFEAMNNKNC 045 N0:13;
0
. each
start
$ EAMNNKNCQA 0.010 position
is
S NLFEAMNNKN 0.010 specified,
10 MNNKNCQALR O.OOS the
length
9 AMNNKNCQAL O.OOS of
each
3 LENLFEAMNN 0.003 Peptide
is
i
10
7 FEAMNNKNCQ 0.001 am
no
the
acids
1 VKLENLFEAM 0.001 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
137
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
ers: 21 3P1F11 Tab le VII v.l-A2-9mers: 213P1F11
9
A2
l
I
Tablem
V -
v.
-
I
123456789 Score P os 123456789 Score
Pos f
rti
P
20 ALILCVTKA 426 Portion 135 EQRDPGETV 0.081 on
11 of o
o
. SEQ
ID
201 HILELLTEV 345 SEQ 2Q$ EVTRRMAEA 0.075 0
11 ID 3
. 3 :
;
N
143 VGGDEIVMV 278 ; g0 SCAFWLMA 0.075 each
7 N0: start
. each
start
44 RQLRFESTM 7.082 n is 160 PTYTDALHV 0.068 position
iti is
188 IQTLVDVFT 064 pos 169 YSTVEGYIA 0.061 specified,
7 o
ecified
. , h
sp l
18 RLALILCVT 027 the 15 SGARLALIL 0.057 engt
7 length the
. of
each
205 LLTEVTRRM 6.925 of 216 AELVQEGKA 0.046 eptide
233 STLRKRLYL 038 P each 86 LMAHGREGF 0.045 is
6 eptide P 9
is amino
9
. amino
111 ALNNKNCQA 4 , 31 GSEEDLDAL 0.041 the
968 acids
. the ,
acids
104 KLENLFEAL 455 , 76 EDPVSCAFV 0.040 end
4 d
. en
183 KGSCFIQTL 266 position~ 223 KARKTNPEI 0.039 position
4 for fog
. each
231 IQSTLRKRL 682 each 153 KDSPQTIPT 0.036
3
. peptide
is
118 QALRAKPKV 574 PeP~de 57 TAEQFQEEL 0.035 t
3 is rt
h
. t e s
th a
t t
103 VKLENLFEA 374 ar 19g RKGHILELL 0.033 Position
2 e s
. position
71 AIDSREDPV 874 h 78 pVSCAFWL 0.032 plus
1 eight
. plus
eig
t
100 GEMVKLENL 732 ' 22 ILCVTKARE 0.025
1
.
158 TIPTYTDAL 439 163 TDALHVYST 0.024
1
.
6 SLEEEKYDM 304 12 YDMSGARLA 0.023
1
.
13 DMSGARLAL 157 187 FIQTLVDVF 0.022
1
.
50 MVIKDSPQT 108 182 QKGSCFIQT 0.020
1
1 .
226 KTNPEIQST 833 195 FTKRKGHIL 0.020
0
.
227 TNPEIQSTL 572 134 GEQRDPGET 0.019
0
.
107 NLFEALNNK 520 149 VMVIKDSPQ 0.018
0
.
3$ ALEHMFRQL 520 212 RMAEAELVQ 0.018
0
.
79 VSCAFWLM 482 211 RRMAEAELV 0.018
0
.
184 GSCFIQTLV 454 219 VQEGKARKT 0.016
0
.
0 FEALNNKNC 444 125 KWIIQACR 0.015
0
1 .
9
64 DALHVYSTV 402 141 ETVGGDEIV 0.015
0
1 .
97 GEDGEMVKL 382 214 AEAELVQEG 0.014
0
. 4
$7 MAHGREGFL 361 120 LRAKPKWI 0.01
0
.
151 VIKDSPQTI 350 21 LILCVTKAR 0.013
0
.
$ LKGEDGEMV 330 16 GARLALILC 0.012
0
9 .
155 SPQTIPTYT 268 162 YTDALHWS 0.010
0
. 0
14 MSGARLALI 266 189 QTLVDVFTK 0.01
0
.
50 STMKRDPTA 255 61 FQEELEKFQ 0.010
0
. 10
112 LNNKNCQAL 237 30 EGSEEDLDA 0.0
0
. 010
63 EELEKFQQA 209 204 ELLTEVTRR 0.
0
. 09
64 ELEKFQQAI 190 ~ 105 LENLFEALN 0.0
0
.
142 TVGGDEIVM 178 123 KPKWIIQA 0.009
0
.
199 KGHILELLT 170 218 LVQEGKARK 0.009
0
.
202 ILELLTEVT 163 . 34 EDLDALEHM 0.009
0
.
190 TLVDVFTKR 116 81 CAFWLMAH 0.009
0
.
186 CFIQTLVDV 111 5 RSLEEEKYD 0.008
0
.
144 GGDEIVMVI 105 ' 68 FQQAIDSRE 0.007
0
. 7
94 FLKGEDGEM 104 114 NKNCQALRA 0.00
0
. 6
7 TIPTYTDA 104 170 STVEGYIAY 0.00
0
15 Q .
140 GETVGGDEI 099 165 ALHWSTVE 0.006
0
. 6
85 VLMAHGREG 094 83 FWLMAHGR 0.00
0
. 006
17 ARLALILCV 082 176 IAYRHDQKG 0.
0
. 05
77 DPVSCAFW 0.081 121 RAKPKWII 0.0
138
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
V 9mers: 21 3P1F11 Table VII v.2-A2-9mers: 213P1F11
A2
2
II
Table-
-
v.
123456789 Score P os 123456789 Score
Pos f
i
P
45 ALSRPWWMC 63.342 Portion 28 LWNSQDTSP 0.000 on
of o
ort
SEQ
ID
TPFQDPLYL 2.838 SEQ 11 PFQDPLYLP 0.000 S
ID
S ;
NO NO:
38 DMIRKAHAL 1.157 : 54 SRRGKDISW 0.000
;
SEAPPNPPL 0.415 each 49 pWWMCSRRG 0.000 position
start is
osition
is
44 HALSRPWWM 0 p 19 PSEAPPNPP 0.000 specified,
358 ecified
s
. , h
p
17 YLPSEAPPN 0.343 the 40 IRKAHALSR 0.000 the
length lengt
$ GPTPFQDPL 0.260 of of
29 WNSQDTSPT 224 P each p each
0 eptide eptide
is is
9 9
ino
. amino am
31 SQDTSPTDM 201 the
0 acids
. the ,
acids
52 MCSRRGKDI 116 , end
0 d
. en
35 SPTDMIRKA 0.061 position p osition
for foi
ach
37 TDMIRKAHA 026 each e
0
. peptide
is
12 FQDPLYLPS 021 peptide rt
0 is h
. t t
rt e
h sta
14 DPLYLPSEA 009 e s osition
0 a
t
. position p
32 QDTSpTDMI 007 i plus
0 h eight
. plus
e
t
g
27 PLWNSQDTS 007
0
.
51 WMCSRRGKD 006
0
.
4 STVEGPTPF 004
0
.
26 PPLWNSQDT 004
0
.
6 VEGPTPFQD 003
0
.
22 APPNPPLWN 003
0
.
39 MIRKAHALS 001
0
.
48 RPWWMCSRR 001
0
.
42 KAI3ALSRPW 001
0
.
1$ LPSEAPPNP 001
0
.
56 RGKDISWNF 001
0
.
15 PLYLPSEAP 001
0
.
5 TVEGPTPFQ 000
0
.
3 YSTVEGPTP 000
0
.
NSQDTSPTD 000
0
.
43 AHALSRPWW 000
0
.
2 VYSTVEGPT 000
0
.
34 TSPTDMIRK 000
0
.
23 PPNPPLWNS 000
0
.
1 HVYSTVEGP 000
0
.
55 RRGKDISWN 000
0
.
46 LSRPWWMCS 000
0
.
25 NPPLWNSQD 00
0
.
21 EAPPNPPLW 000
0
.
41 RKAHALSRP 000
0
.
13 QDPLYLPSE 000
0
.
9 pTPFQDPLY 000 .
0
.
7 EGPTPFQDP 000
0
.
16 LYLPSEAPP 000
0
.
36 PTDMIRKAH 000
0
.
53 CSRRGKDIS 000
0
.
50 WWMCSRRGK 000
0
.
33 DTSPTDMIR 000
0
.
47 SRPWWMCSR 000
0
.
24 PNPPLWNSQ 0.000
139
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table
VII
v.3-A2-9mers:
213P1F11
Pos 123456789 Score
TLPSPFPYL 223.237 Portion
of
3 IQACRGATL 3.682 SEQ
ID
1 YIIQACRGA 0.628 N0:7;
each
start
2 IIQACRGAT 0.226 position
is
9 ATLPSPFPY 0.022 specified,
12 PSPFPYLSL 0.005 the
length
11 LPSPFPYLS 0.003 of
each
8 GATLPSPFP 0.001 Peptide
is
9
ino
7 RGATLPSPF 0.000 am
the
acids
4 QACRGATLP 0.000 ,
end
$ ACRGATLPS 0.000 position
for
6 CRGATLPSP 0.000 each
peptide
is
the
start
position
lus
ei
ht
140
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableVII v.4-A2-9mers:13P1F11 Ta ble VII rs: 213P1F11
2 v.4-A2-9me
Pos 123456789 Score Pos 123456789 Score
QEYDKSLSV 17.743 Portion 63 GGGVGDIVG 0.000 Portion
of of
13 VQPEKRTGL 15.096 SEQ 45 SSVHQKLVN 0.000 SEQ
ID ID
50 KLVNDPRET 5.216 N0:9; 24 ENGECGQTF 0.000 N0:9;
1 h h
26 GECGQTFRL 2.409 start gl SETSASEEE 0.000 start
eac eac
position position
is is
3 KCQEYDKSL 2.001 specified, 57 ETQEVFGGG 0.000 specified,
75 SISFRNSET 1.025 the 59 QEVFGGGVG 0.000 the
length length
44 GSSVHQKLV 0.454 of 49 QKLVNDPRE 0.000 of
each each
62 FGGGVGDIV 0.420 peptide 2 GKCQEYDKS 0.000 Peptide
is is
9 9
58 TQEVFGGGV 0.223 amino 52 VNDPRETQE 0.000 amino
acids acids
the the
20 GLRDENGEC 0.201 , 67 GDIVGRDLS 0.000 ,
end end'
43 RGSSVHQKL 0.139 position 78 FRNSETSAS 0.000 position
for for
68 DIVGRDLSI 0.108 each 47 VHQKLVNDP 0.000 each
53 NDPRETQEV 0.097 Peptide 32 FRLKEEQGR 0.000 Peptide
is is
the the
33 RLKEEQGRA 0.093 start 25 NGECGQTFR 0.000 start
iti iti
on pos
11 LSVQPEKRT 0.083 pos 42 FRGSSVHQK 0.000 on
plus plus
eight eight
56 RETQEVFGG 0.019 38 QGRAFRGSS 0.000
66 VGDIVGRDL 0.019 17 KRTGLRDEN 0.000
69 IVGRDLSIS 0.010 21 LRDENGECG 0.000
39 GRAFRGSSV 0.010 71 GRDLSISFR 0.000
85 ASEEEKYDM 0.009 ~ 34 LKEEQGRAF 0.000
SLSVQPEKR 0.007 82 ETSASEEEK 0.000
84 SASEEEKYD 0.005 80 NSETSASEE 0.000
51 LVNDPRETQ 0.004 1 MGKCQEYDK 0.000
61 VFGGGVGDI 0.004 8 DKSLSVQPE 0.000
29 GQTFRLKEE 0.003 7 YDKSLSVQP 0.000
46 SVHQKLVND 0.003 27 ECGQTFRLK 0.000
72 RDLSISFRN 0.002 54 DPRETQEVF 0.000
73 DLSISFRNS 0.002 31 TFRLKEEQG 0.000
65 GVGDIVGRD 0.002 22 RDENGECGQ 0.000
40 RAFRGSSVH 0.002 48 HQKLVNDPR 0.000
76 ISFRNSETS 0.001 14 QPEKRTGLR 0.000
23 DENGECGQT 0.001 41 AFRGSSVHQ 0.000
12 SVQPEKRTG 0.001 15 PEKRTGLRD 0.000
9 KSLSVQPEK 0.001 . 55 PRETQEVFG 0.000
36 EEQGRAFRG 0.001 6 EYDKSLSVQ 0.000
74 LSISFRNSE 0.001 16 EKRTGLRDE 0.000
18 RTGLRDENG 0.001
4 CQEYDKSLS 0.000
79 RNSETSASE 0.000
30 QTFRLKEEQ 0.000
28 CGQTFRLKE 0.000 '
60 EVFGGGVGD 0.000
19 TGLRDENGE 0.000
35 KEEQGRAFR 0.000 '
86 SEEEKYDMS 0.000
77 SFRNSETSA 0.000
70 VGRDLSISF 0.000
83 TSASEEEKY 0.000
64 GGVGDIVGR 0.000
37 EQGRAFRGS 0.000
141
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table VII v.5-A2-9mers: 21'~PtFt t
Pos 123456789 Score
ALILRVTKA 11.426 Portion
of
3 RLALILRVT 1.405 SEQ
ID
2 ARLALILRV 0.082 NO:
11;
6 LILRVTKAR 0.013 each
start
i
pos
9 RVTKAREGS 0 tion
003 is
. specified
,
7 ILRVTKARE 0.002 the
length
4 LALILRVTK 0.001 of each
1 GARLALILR 0.000 Peptide
is
9
8 LRVTKAREG 0.000 amino
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
t
Table VII v.6-A2-9mers: 213PIF11
Pos 123456789 Score
8 AMhINKNCQA 3.588 Portion
of
1 KLENLFEAM 1.036 SEQ
ID
4 NLFEANlhTNK0.520 N0:13;
6 FEAMNNKNC 0.444 each
start
i
i
i
pos
9 MI~1DTKNCQAL0 t
237 on
s
. specified,
2 LENLFEANIhT0.009 the
length
3 ENLFEANINN 0.000 of each
7 EAMNNKNCQ 0.000 Peptide
is
9
5 LFEAMI~TNKN0.000 ammo
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
142
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableVIII v.l-A2-lOmers: 213P1F11 TableVIII v.l-A2-lOmers:
213P1F11
Pos 1234567890 Score Pos 1234567890 Score
187 FIQTLVDVFT 25.924 Portion 15 SGARLALILC
of 0 Portion
075 of
111 ALNNKNCQAL 21.362 SEQ 165 ALHVYSTVEG . SEQ
ID 0 ID
075
86 LMAHGREGFL 18.753 N0:3; 125 KWIIQACRG . N0:3;
0
073
142 TVGGDEIVMV 13.997 each 167 HWSTVEGYI . each
start 0 start
o 071
iti
i
p . position
149 VMVIKDSPQT 9.149 s 104 KLENLFEALN 0 is
on 063
s
specified
, . specified,
117 CQALRAKPKV 7.052 the 56 PTAEQFQEEL 0 the
length 050 len
th
205 LLTEVTRRMA 6.925 of 30 EGSEEDLDAL . g
each 0 of each
048
94 FLKGEDGEMV 5,487 peptide 175 YIAYRHDQKG . Peptide
is 0 is
047
119 ALRAKPKWI 4.361 10 41 HMFRQLRFES . 10 amino
amino 0
id 039
h
ac .
185 SCFIQTLVDV 3.864 s t 209 VTRRMAEAEL 0 the
e 038 ac~
end
75 REDPVSCAFV 2.975 position 188 IQTLVDVFTK . end
for 0 ositi
034 f
. p
70 QAIDSREDPV 1.941 each 193 DVFTKRKGHI 0 on
033 or
each
183 KGSCFIQTLV 1.589 peptide 10 EKYDMSGARL . peptide
is 0 is
029
150 MVIKDSPQTI 1.552 the 22 ILCVTKAREG . the
start 0 start
025
13 DMSGARLALI 1.300 Position 225 RKTNPEIQST . Position
lus 0
nine 024
p . plus
107 NLFEALNNKN 1.130 154 DSPQTIPTYT 0 nine
020
226 KTNPEIQSTL 1.038 110 EALNNKNCQA .
0
019
19 LALILCVTKA 0.998 76 EDPVSCAFW .
0
017
218 LVQEGKARKT 0.909 156 PQTIPTYTDA .
0
017
63 EELEKFQQAI 0.877 179 RHDQKGSCFI .
~ 0
016
159 IPTYTDALHV 0.772 122 AKPKWIIQA .
0
016
232 QSTLRKRLYL 0.767 20 ALILCVTKAR .
0.015
103 VKLENLFEAL 0.712 18 RLALILCVTK 0
015
134 GEQRDPGETV 0.663 222 GKARKTNPEI .
0
014
12 YDMSGARLAL 0.505 6 SLEEEKYDMS .
0
014
RSLEEEKYDM 0.492 21 LILCVTKARE .
0
013
143 VGGDEIVMVI 0.448 43 FRQLRFESTM .
0
012
162 YTDALHWST 0.438 62 QEELEKFQQA .
0
012
102 MVKLENLFEA 0.345 72 IDSREDPVSC .
0
012
48 FESTMKRDPT 0.327 170 STVEGYIAYR .
0
011
96 KGEDGEMVKL 0.295 61 FQEELEKFQQ .
0
011
204 ELLTEVTRRM 0.276 198 RKGHILELLT .
0
010
140 GETVGGDEIV 0.272 194 VFTKRKGHIL .
0
010
182 QKGSCFIQTL 0.259 158 TIPTYTDALH .
0
010
190 TLVDVFTKRK 0.232 123 KPKWIIQAC .
0.009
85 VLMAHGREGF 0.230 81 CAFVVLMAHG 0.009
207 TEVTRRMAEA 0.222 148 IVMVIKDSPQ 0
008
230 EIQSTLRKRL 0.220 84 VVLMAHGREG .
0.008
16 GARLALILCV 0.169 77 DPVSCAFWL 0
008
27 KAREGSEEDL 0.159 152 IKDSPQTIPT .
0
007
157 QTIPTYTDAL 0.145 176 IAYRHDQKGS .
0.006
163 TDALHWSTV 0.145 . 44 RQLRFESTMK 0.006
37 DALEHMFRQL 0.128 100 GEMVKLENLF 0.005
79 VSCAFWLMA 0.127 ~ 196 TKRKGHILEL 0.005
200 GHILELLTEV 0.111 101 EMVKLENLFE 0.004
201 HILELLTEVT 0.106 203 LELLTEVTRR 0.004
212 RMAEAELVQE 0.102 181 DQKGSCFIQT 0.004
14 MSGARLALIL 0.097 38 ALEHMFRQLR 0.004
29 REGSEEDLDA 0.097 17 ARLALILCVT 0.004
78 PVSCAFWLM 0.084 I 169 YSTVEGYIAY 0.003
~ I
143
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableVIII v.2-A2-lOmers: 213P1F11 TableVIII v.2-A2-lOmers:
213P1F11
Pos 1234567890 Score Pos 1234567890 Score
52 WMCSRRGKDI 34.660 Portion a4 PPNPPLWNSQ 0.000 Portion
of of
32 SQDTSPTDMI 0.207 SEQ 3 VYSTVEGPTP 0.000 SEQ
ID ID
44 AHALSRPWWM 0.142 NO: 25 PNPPLWNSQD 0.000 NO:
S, S;
31 NSQDTSPTDM 0.133 each 48 SRPWWMCSRR 0.000 each
start start
ositi
is
p position
46 ALSRPWWMCS 0.127 on 41 IRKAHALSRP 0.000 is
specified if
d
, spec
45 HALSRPWWMC 0.111 the 50 PWWMCSRRGK 0.000 ie
length ,
the
length
38 TDMIRKAHAL 0.110 of of each
each
18 YLPSEAPPNP 0.069 peptide peptide
is is
26 NPPLWNSQDT 0.049 10 10 amino
amino
id
th
ac acids
PTPFQDPLYL 0.036 s, the
e
end
end
43 KAHALSRPWW 0.020 position osition
for for
8 EGPTPFQDPL 0.019 each p
each
35 TSPTDMIRKA 0.015 peptide peptide
is is
2 HVYSTVEGPT 0.009 the the
start start
23 APPNPPLWNS 0.008 Position position
lus
nine
p plus
14 QDPLYLPSEA 0.007 nine
13 FQDPLYLPSE 0.006
5 STVEGPTPFQ 0.005
39 DMIRKAHALS 0.004
28 PLWNSQDTSP 0.003
4 YSTVEGPTPF 0.002
36 SPTDMIRKAH 0.002
29 LWNSQDTSPT 0.002
21 SEAPPNPPLW 0.001
16 PLYLPSEAPP 0.001
7 VEGPTPFQDP 0.001
11 TPFQDPLYLP 0.001
49 RPWWMCSRRG 0.001
19 LPSEAPPNPP 0.001
37 PTDMIRKAHA 0.001
9 GPTPFQDPLY 0.000
6 TVEGPTPFQD 0.000
30 WNSQDTSPTD 0.000
22 EAPPNPPLWN 0.000
40 MIRKAHALSR 0.000 .
PSEAPPNPPL 0.000 ,
53 MCSRRGKDIS 0.000
56 RRGKDISWNF 0.000
17 LYLPSEAPPN 0.000
54 CSRRGKDISW 0.000
34 DTSPTDMIRK 0.000
47 LSRPWWMCSR 0.000
42 RKAHAI,SRPW 0.000
1 LHVYSTVEGP 0.000
27 PPLWNSQDTS 0.000
15 DPLYLPSEAP 0.000
55 SRRGKDISWN 0.000
33 QDTSPTDMIR 0.000
12 PFQDPLYLPS 0.000
51 WWMCSRRGKD 0.000
144
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
ITable VIII v.3-A2-lOmers: 213P1F11 I
Pos 1234567890 Score
ATLPSPFPYL 11.472 Portion
of
3 IIQACRGATL 4.993 SEQ
ID
2 YIIQACRGAT 0.613 N0:7;
12 LPSPFPYLSL 0.356 each
start
osition
is
11 TLPSPFPYLS 0.2133 p
specified
,
9 GATLPSPFPY 0.006 the
length
4 IQACRGATLP 0.003 of each
5 QACRGATLPS 0.001 Peptide
is
8 RGATLPSPFP 0.001 10 amino
id
th
ac
1 VYIIQACRGA 0.000 s,
e
end
6 ACRGATLPSP 0.000 position
for
7 CRGATLPSPF 0.000 each
peptide
is
the
start
position
lus
nine
145
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TabIeVIII v.4-A2-lOmers: 213P1F11 TabIeVIII v.4-A2-lOmers: 213P1F11
Pos 1234567890 Score Pos1234567890 Score
SLSVQPEKRT 7.452 Portion 11 LSVQPEKRTG 0.000 Portion
of of
12 SVQPEKRTGL 1.869 SEQ 24 ENGECGQTFR 0.000 SEQ
ID ID
65 GVGDIVGRDL 1.533 N0:9; 66 VGDIVGRDLS 0.000 N0:9;
h
43 RGSSVHQKLV 0.454 start 72 RDLSISFRNS 0.000 each
eac start
position osition
is is
4 CQEYDKSLSV 0.451 specified, 81 SETSASEEEK 0.000 p
specified
,
52 VNDPRETQEV 0.309 the 26 GECGQTFRLK 0.000 the
length length
84 SASEEEKYDM 0.283 of 23 DENGECGQTF 0.000 of each
each
76 ISFRNSETSA 0.204 Peptide g5 ASEEEKYDMS 0.000 Peptide
is is
57 ETQEVFGGGV 0.147 IO 22 RDENGECGQT 0.000 10 amino
amino id
acids h
th
, ac
74 LSISFRNSET 0.083 e 54 DPRETQEVFG 0.000 s, t
end e
end
60 EVFGGGVGDI 0.076 position 34 LKEEQGRAFR 0.000 position
for for
25 NGECGQTFRL 0.052 each 36 EEQGRAFRGS 0.000 each
38 QGRAFRGSSV 0.035 Peptide g2 ETSASEEEKY 0.000 peptide
is is
the the
2 GKCQEYDKSL 0.030 start 64 GGVGDIVGRD 0.000 start
i
i
pos position
50 KLVNDPRETQ 0.026 t 27 ECGQTFRLKE 0.000 lus
on nine
plus
nine
61 VFGGGVGDIV 0.016 58 TQEVFGGGVG 0.000 p
19 TGLRDENGEC 0.016 71 GRDLSISFRN 0.000
67 GDIVGRDLSI 0.014 1 MGKCQEYDKS 0.000
42 FRGSSVHQKL 0.014 53 NDPRETQEVF 0.000
GLRDENGECG 0.011 ' 17 KRTGLRDENG 0.000
69 IVGRDLSISF 0.011 78 FRNSETSASE 0.000
51 LVNDPRETQE 0.009 47 VHQKLVNl7PR 0.000
49 QKLVNDPRET 0.008 7 YDKSLSVQPE 0.000
3 KCQEYDKSLS 0.007 14 QPEKRTGLRD 0.000
75 SISFRNSETS 0.005 21 LRDENGECGQ 0.000
73 DLSISFRNSE 0.004 39 GRAFRGSSVH 0.000
5 QEYDKSLSVQ 0.004 77 SFRNSETSAS 0.000
46 SVHQKLVNDP 0.003 80 NSETSASEEE 0.000
33 RLKEEQGRAF 0.002 41 AFRGSSVHQK 0.000
35 KEEQGRAFRG 0.002 48 HQKLVNDPRE 0.000
QTFRLKEEQG 0.002 8 DKSLSVQPEK 0.000
32 FRLKEEQGRA 0.002 31 TFRLKEEQGR 0.000
13 VQPEKRTGLR 0.001 16 EKRTGLRDEN 0.000
62 FGGGVGDIVG 0.001 . 55 PRETQEVFGG 0.000
RAFRGSSVHQ 0.001 15 PEKRTGLRDE 0.000
29 GQTFRLKEEQ 0.001 6 EYDKSLSVQP 0.000
68 DIVGRDLSIS 0.001
70 VGRDLSISFR 0.001
9 KSLSVQPEKR 0.001
83 TSASEEEKYD 0.001
79 RNSETSASEE 0.000 '
86 SEEEKYDMSG 0.000
56 RETQEVFGGG 0.000
59 QEVFGGGVGD 0.000 '
37 EQGRAFRGSS 0.000
SSVHQKLVND 0.000
28 CGQTFRLKEE 0.000
63 GGGVGDIVGR 0.000
18 RTGLRDENGE 0.000
44 GSSVHQKLVN 0.000
I
146
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table VIII v.5-A2-lOmers: 213P1F11 I
Pos 1234567890 Score
$ LALILRVTKA 0.998 Portion
of
2 GARLALILRV 0.169 SEQ
ID
6 ALILRVTKAR 0.015 N~~
11;
h
4 RLALILRVTK 0 start
015 eac
. osition
is
7 LILRVTKARE 0.013 p
specified,
8 ILRVTKAREG 0.002 the
length
3 ARLALILRVT 0.001 of
each
1 SGARLALILR 0.000 Peptide
is
RVTKAREGSE 0 10
000 amino
. acids
the
9 LRVTKAREGS 0.000 ,
end
position
for
each
.
peptide
is
the
start
position
lus
nine
Table VIII v.6-A2-lOmers: 213P1F11
Pos 1234567890 Score
9 AMNNKNCQAL 15.428 Portion
of
5 NLFEAMNNKN 1.130 SEQ
ID
1 VKLENLFEAM 0.166 N0:13;
h
2 KLENLFEAMN 0 start
063 eac
. position
is
8 EANIrINKNCQA0.019 specified,
3 LENLFEAMNN 0.002 the
length
7 FEANIrINKNCQ0.001 of
each
6 LFEAMNNKNC 0.000 Peptide
is
10 NITINKNCQALR0.000 10
ammo
acid
th
s
4 ENLFEAMNNK 0.000 e
end
position
for
each
peptide
is
the
start
position
lus
nine
147
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableIX v.l-A3-9mers:13P1F11 Ta ble IX v.l-A3-9mers: 213P1F11
2
Pos 123456789 Score Pos 123456789 Score
107 NLFEALNNK 225.000 Portion 205 LLTEVTRRM 0.045 Portion
of of
190 TLVDVFTKR 27.000 SEQ gl CAFWLMAH 0.045 SEQ
ID ID
45 QLRFESTMK 20.000 N0:3; 228 NPEIQSTLR 0.040 N0:3;
h h
rt
189 QTLVDVFTK 13.500 start 39 LEHMFRQLR 0.036 sta
eac eac
position position
is is
125 KWIIQACR 9.000 specified, 226 KTNPEIQST 0.034 specified,
167 HVYSTVEGY 6.000 the 195 FTKRKGHIL 0.030 the
length length
204 ELLTEVTRR 5.400 of 149 VMVIKDSPQ 0.030 of
each each
104 KLENLFEAL 5.400 Peptide 44 RQLRFESTM 0.027 Peptide
is is
9 9
218 LVQEGKARK 3.000 amino 144 GGDEIVMVI 0.024 amino
acids acids
the the
191 LVDVFTKRK 3.000 , 157 QTIPTYTDA 0.022 ,
end end
171 TVEGYIAYR 2.700 position 31 GSEEDLDAL 0.020 position
for for
119 ALRAKPKW 2.000 each 71 AIDSREDPV 0.020 each
86 LMAHGREGF 2.000 peptide 51 TMKRDPTAE 0.020 Peptide
is is
the the
175 YIAYRHDQK 2.000 start 22 ILCVTKARE 0.020 start
i iti
i
59 EQFQEELEK 1.800 t 67 KFQQAIDSR 0.018 on
on Pos
Pos plus
plus eight
eight
6 SLEEEKYDM 0.900 115 KNCQALRAK 0.018
217 ELVQEGKAR 0.900 57 TAEQFQEEL 0.018
101 EMVKLENLF 0.900 203 LELLTEVTR 0.018
20 ALILCVTKA 0.900 229 PEIQSTLRK 0.018
170 STVEGYIAY 0.900 16 GARLALILC 0.018
83 FWLMAHGR 0.600 80 SCAFWLMA 0.018
35 DLDALEHMF 0.600 230 EIQSTLRKR 0.018
187 FIQTLVDVF 0.600 223 KARKTNPEI 0.018
64 ELEKFQQAI 0.540 78 PVSCAFWL O.O18
13 DMSGARLAL 0.540 193 DVFTKRKGH 0.015
21 LILCVTKAR 0.450 50 STMKRDPTA 0.015
19 LALILCVTK 0.300 150 MVIKDSPQT 0.015
117 CQALRAKPK 0.300 41 HMFRQLRFE 0.015
18 RLALILCVT 0.225 75 REDPVSCAF 0.013
111 ALNNKNCQA 0.200 97 GEDGEMVKL 0.012
3 NPRSLEEEK 0.200 100 GEMVKLENL 0.012
38 ALEHMFRQL 0.180 160 PTYTDALHV 0.010
15$ TIPTYTDAL 0.180 53 KRDPTAEQF 0.009
145 GDEIVMVIK 0.135 79 VSCAFVVLM 0.009
96 KGEDGEMVK 0.120 231 IQSTLRKRL 0.009
129 IQACRGEQR 0.120 208 EVTRRMAEA 0.009
202 ILELLTEVT 0.100 154 DSPQTIPTY 0.009
233 STLRKRLYL 0.090 ' 197 KRKGHILEL 0.008
94 FLKGEDGEM 0.090 183 KGSCFIQTL 0.008
234 TLRKRLYLQ 0.090 36 LDALEHMFR 0.008
215 EAELVQEGK 0.090 113 NNKNCQALR 0.008
.
121 RAKPKVYII 0.081 141 ETVGGDEIV 0.007
201 HILELLTEV 0.068 161 TYTDALHW 0.006
88 AHGREGFLK 0.060 ~ 140 GETVGGDEI 0.005
46 LRFESTMKR 0.060 60 QFQEELEKF 0.005
165 ALHWSTVE 0.060 188 IQTLVDVFT 0.005
151 VIKDSPQTI 0.060 184 GSCFIQTLV 0.005
212 RMAEAELVQ 0.060 148 IvMVIKDSP 0.005
142 TVGGDEIVM 0.060 14 MSGARLALI 0.005
123 KPKVYIIQA 0.054 102 MVKLENLFE 0.004
148
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableIX v.2-A3-9mers:3P1F11 Ta ble IX v.2-A3-9mers: 213P1F11
21
Pos 123456789 Score Pos 123456789 Score
45 ALSRPWWMC 0.900 Portion 7 EGPTPFQDP 0.000 Portion
of of
34 TSPTDMIRK 0.300 SEQ ~8 LWNSQDTSP 0.000 SEQ
ID ID
38 DMIRKAHAL 0.270 NO: lg PSEAPPNPP 0.000 NO:
S; S;
h
t
t
4 STVEGPTPF 0.225 each 11 PFQDPLYLP 0.000 eac
start s
osition ar
is position
is
48 RPWWMCSRR 0.200 p 24 PNPPLWNSQ 0.000 specified,
specified,
33 DTSPTDMIR 0.180 the 49 PWWMCSRRG 0.000 the
length length
8 GPTPFQDPL 0.081 of each of
each
TPFQDPLYL 0.060 Peptide peptide
is is
9 9
1 HVYSTVEGP 0.030 amino amino
the acids
id the
17 YLPSEAPPN 0.020 ac ,
s, end
end
27 PLWNSQDTS 0.020 position position
for for
9 PTPFQDPLY 0.020 each each
47 SRPWWMCSR 0.018 peptide peptide
is is
PLYLPSEAP 0.015 the the
start start
i iti
i
pos pos
56 RGKDISWNF 0.009 t on
on plus
lus eight
eight
44 HALSRPWWM 0.009 p
40 IRKAHALSR 0.008
51 WMCSRRGKD 0.006
31 SQDTSPTDM 0.006
5 TVEGPTPFQ 0.005
SEAPPNPPL 0.004
39 MIRKAHALS 0.004
12 FQDPLYLPS 0.004
50 WWMCSRRGK 0.003
52 MCSRRGKDI 0.003
46 LSRPWWMCS 0.002
32 QDTSPTDMI 0.001
14 DPLYLPSEA 0.001
21 EAPPNPPLW 0.001
36 PTDMIRKAH 0.001
22 APPNPPLWN 0.001
42 KAHALSRPW 0.001
NPPLWNSQD 0.001
54 SRRGKDISW 0.001
23 PPNPPLWNS 0.000
_
18 LPSEAPPNP 0.000 '
37 TDMIRKAHA 0.000
SPTDMIRKA 0.000
6 VEGPTPFQD 0.000
29 WNSQDTSPT 0.000
43 AHALSRPWW 0.000
53 CSRRGKDIS 0.000
26 PPLWNSQDT 0.000
3 YSTVEGPTP 0.000
30 NSQDTSPTD 0.000
13 QDPLYLPSE 0.000
16 LYLPSEAPP 0.000
2 VYSTVEGPT 0.000
55 RRGKDISWN 0.000
41 RKAHALSRP 0.000
149
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableIX v.3-A3-9mers:13P1F11
2
Pos 123456789 Score
TLPSPFPYL 2.700 Portion
of
9 ATLPSPFPY 1.350 SEQ
ID
3 IQACRGATL 0.018 N0:7;
h
t
t
11 LPSPFPYLS 0.005 eac
s
ar
position
is
1 YIIQACRGA 0.003 specified,
2 I IQACRGAT 0.003 the
length
7 RGATLPSPF 0.002 of each
5 ACRGATLPS 0.001 Peptide
is
9
i
12 PSPFPYLSL 0:001 no
am
the
acids
8 GATLPSPFP 0.001 ,
end
4 QACRGATLP 0.000 position
for
6 CRGATLPSP 0.000 each
peptide
is
the
start
position
lus
ei
ht
150
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table4-A3-9mers:3P1F11 Table IX v.4-A3-9mers: 213P1F11
I 21
X v
Pos . Score p os 123456789 Score
123456789
SLSVQPEKR 4.000 Portion 34 LKEEQGRAF 0.000 Portion
of of
9 KSLSVQPEK 0.675 SEQ 45 SSVHQKLVN 0.000 SEQ
ID ID
82 ETSASEEEK 0.300 NO' S3 NDPRETQEV 0.000 NO:
~' 9;
each
start
48 HQKLVNDPR 0.180 each 77 SFRNSETSA 0.000 osition
start is
GLRDENGEC 0.180 ppecifieds 67 GDIVGRDLS 0.000 specified
33 RLKEEQGRA 0.090 the 86 SEEEKYDMS 0.000 the
length length
68 DIVGRDLSI 081 of 84 SASEEEKYD 0.000 of each
0 each
42 FRGSSVHQK . Peptide 72 RDLSISFRN 0.000 Peptide
0.060 is is
9 9
i
1 MGKCQEYDK 0.060 amino 2 GKCQEYDKS 0.000 no
am
acids
the
50 KLVNDPRET 0.045 acids, 63 GGGVGDIVG 0.000 ,
the end'
end
64 GGVGDIVGR 0.041 position 28 CGQTFRLKE 0.000 position
for for
3 KCQEYDKSL 0.041 each 37 EQGRAFRGS 0.000 each
35 KEEQGRAFR 0.036 peptide g0 NSETSASEE 0.000 Peptide
is is
13 VQPEKRTGL 0.027 the 66 VGDIVGRDL 0.000 the
start start
osition
26 GECGQTFRL 0.024 [ 17 KRTGLRDEN 0.000 plus
~ eight
ht
i
83 TSASEEEKY 0.020 p 36 EEQGRAFRG 0.000
us
e
g
71 GRDLSISFR O.O1$ 52 VNDPRETQE 0.000 '
27 ECGQTFRLK 0.018 79 RNSETSASE 0.000
14 QPEKRTGLR 0.012 47 VHQKLVNDP 0.000
40 RAFRGSSVH O.O10 ~ 81 SETSASEEE 0.000
75 SISFRNSET 0.010 23 DENGECGQT 0.000
54 DPRETQEVF 0.009 78 FRNSETSAS 0.000
65 GVGDIVGRD 0.008 38 QGRAFRGSS 0.000
32 FRLKEEQGR 0.006 21 LRDENGECG 0.000
69 TVGRDLSIS 0.006 19 TGLRDENGE 0.000
S QEYDKSLSV 0.006 49 QKLVNDPRE 0.000
58 TQEVFGGGV O.OOS 41 AFRGSSVHQ 0.000
QTFRLKEEQ 0.005 59 QEVFGGGVG 0.000
85 ASEEEKYDM 0.005 31 TFRLKEEQG 0.000
60 EVFGGGVGD 0.005 7 YDKSLSVQP 0.000 .
70 VGRDLSISF 0.004 22 RDENGECGQ 0.000
25 NGECGQTFR 0.004 8 DKSLSVQPE 0.000
73 DLSISFRNS 0.004 15 PEKRTGLRD 0.000
51 LVNDPRETQ 0.003 . 6 EYDKSLSVQ 0.000
46 SVHQKLVND 0.003 S5 PRETQEVFG 0.000
24 ENGECGQTF 0.002 16 EKRTGLRDE 0.000
44 GSSVHQKLV 0.002
29 GQTFRLKEE 0.001
4 CQEYDKSLS 0.001
18 RTGLRDENG 0.001
76 ISFRNSETS 0.001
43 RGSSVHQKL 0.001
61 VFGGGVGDI 0.001
$7 ETQEVFGGG 0.001
39 GRAFRGSSV 0.001
11 LSVQPEKRT 0.001
56 RETQEVFGG 0.001
62 FGGGVGDIV 0.000
74 LSISFRNSE 0.000 ,
12 SVQPEKRTG 0.000
151
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table IX v.5-A3-9mers: 213P1F11 I
Pos 123456789 Score
ALILRVTKA 0.900 Portion
of
6 LILRVTKAR 0.450 SEQ
ID
1 GARLALILR 0.360 NO:
II;
h
4 LALILRVTK 0.300 start
eac
position
is
3 RLALILRVT 0.022 specified,
7 ILRVTKARE 0.020 the
length
9 RVTKAREGS 0.004 of
each
2 ARLALILRV 0.001 Peptide
is
9
8 LRVTKAREG 0.000 amino
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
Table IX v.6-A3-9mers: 213P1F11
Pos 123456789 Score
4 NLFEAMNNK 225.000 Portion
of
1 KLENLFEAM 1.800 SEQ
ID
8 AMNNKNCQA 0.200 N0:13;
rt
h
9 MNNKNCQAL 0.001 eac
sta
position
is
6 FEAMNNKNC 0.000 specified,
2 LENLFEAMN 0.000 the
length
7 EAMNNKNCQ 0.000 of
each
3 ENLFEAMNN 0.000 Peptide
is
9
5 LFEANINNKN 0 arruno
000
. acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
152
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Tablev.l-A3-lOmers TableX v.l-A3-
X l
Omers
Pos 1234567890 Score P os 1234567890 Score
190 TLVDVFTKRK 45.000 Portion 51 TMKRDPTAEQ 0.030 Portion
of of
18 RLALILCVTK 20.000 SEQ 209 VTRRMAEAEL 0.030 SEQ
ID ID
38 ALEHMFRQLR 12.000 N0:3; 193 DVFTKRKGHI 0.027 N0:3;
each
staff
217 ELVQEGKARK 9.000 each 153 KDSPQTIPTY 0.027 osition
start is
45 QLRFESTMKR 8.000 ppecifieds 106 ENLFEALNNK 0.027 specified,
188 IQTLVDVFTK 5.400 the 27 KAREGSEEDL 0.027 the
length length
20 ALILCVTKAR 4.500 of 100 GEMVKLENLF 0.027 of
each each
202 ILELLTEVTR 4.000 Peptide 201 HILELLTEVT 0.022 Peptide
is is
10
i
$5 VLMAHGREGF 3.000 10 158 TIPTYTDALH 0.020 no
amino am
the
acids
35 DLDALEHMFR 2.400 acids, 165 ALHWSTVEG 0.020 ,
the end
end
170 STVEGYIAYR 2.025 position 166 LHWSTVEGY 0.018 position
for for
189 QTLVDVFTKR 1.350 each 16 GARLALILCV 0.018 each
87 MAHGREGFLK 0.900 Peptide 7$ PVSCAFVVLM 0.018 Peptide
is is
44 RQLRFESTMK 0.900 the 101 EMVKLENLFE O.O18 the
start start
osition
119 ALRAKPKWI 0.900 Position 187 FIQTLVDVFT 0.015 P
i lus
l nine
p
41 HMFRQLRFES 0.600 ne 1$5 SCFIQTLVDV 0.015 .
us
n
P
111 ALNNKNCQAL 0.600 123 KPKWIIQAC 0.013
13 DMSGARLALI 0.405 141 ETVGGDEIVM 0.013
128 IIQACRGEQR 0.400 204 ELLTEVTRRM 0.013
.
228 NPEIQSTLRK 0.400 56 PTAEQFQEEL 0.013
94 FLKGEDGEMV 0.300 231 IQSTLRKRLY 0.012
144 GGDEIVMVIK 0.203 227 TNPEIQSTLR 0.012
157 QTIPTYTDAL 0.203 39 LEHMFRQLRF 0.012
226 KTNPEIQSTL 0.203 186 CFIQTLVDVF 0.009
86 LMAHGREGFL 0.180 79 VSCAFWLMA 0.009
104 KLENLFEALN 0.180 66 EKFQQAIDSR 0.009
107 NLFEALNNKN 0.150 216 AELVQEGKAR 0.009
160 PTYTDALHW 0.150 19 LALILCVTKA 0.009
149 VMVIKDSPQT 0.150 80 SCAFVVLMAH 0.009
59 EQFQEELEKF 0.135 230 EIQSTLRKRL 0.009
214 AEAELVQEGK 0.135 77 DPVSCAFWL 0.008
167 HVYSTVEGYI 0.135 181 DQKGSCFIQT 0.008
171 TVEGYIAYRH 0.135 112 LNNKNCQALR 0.008
58 AEQFQEELEK 0.120 233 STLRKRLYLQ 0.007
116 NCQALRAKPK 0.100 5 RSLEEEKYDM 0.007
174 GYIAYRHDQK 0.090 197 KRKGHILELL 0.006
150 MVIKDSPQT2 0.090 - 82 AFWLMAHGR 0.006
102 MVKLENLFEA 0.090 14 MSGARLALIL 0.006
95 LKGEDGEMVK 0.060 232 QSTLRKRLYL 0.006
6 SLEEEKYDMS 0.060 117 CQALRAKPKV 0.006
203 LELLTEVTRR 0.054 64 ELEKFQQAID 0.006
162 YTDALHWST 0.045 143 VGGDEIVMVI 0.005
142 TVGGDEIVMV 0.045 ~ 120 LRAKPKVYII 0.005
212 RMAEAELVQE 0.045 103 VKLENLFEAL 0.004
169 YSTVEGYIAY 0.040 159 IPTYTDALHV 0.004
2 SNPRSLEEEK 0.040 71 AIDSREDPVS 0.004
3 NPRSLEEEKY 040 63 EELEKFQQAI 0.004
0
118 QALRAKPKW . 74 SREDPVSCAF 0.003
0.030
125 KWIIQACRG 030 114 NKNCQALRAK 0.003
0
205 LLTEVTRRMA . 148 IVMVIKDSPQ 0.003
0.030
153
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table2-A3-lOmers:3P1F11 Tab le X v.2-A3-lOmers: 213P1F11
X 21
v
Pos . Score P os 1234567890 Score
1234567890
34 DTSPTDMIRK 1.350 Portion 41 IRKAHALSRP 0.000 Portion
of of
4Q MIRKAHALSR 0.800 SEQ 3 VYSTVEGPTP 0.000 SEQ
ID ID
52 WMCSRRGKDI 0.300 NO: 42 RKAHALSRPW 0.000 NO:
S; S;
each
start
46 ALSRPWWMCS 0.240 each 25 PNPPLWNSQD 0.000 osition
start is
9 GPTPFQDPLY 0.180 ppecifieds 51 WWMCSRRGKD 0.000 specified,
47 LSRPWWMCSR 0.135 the 12 PFQDPLYLPS 0.000 the
length length
32 SQDTSPTDMI 0.027 of of
each each
2 HVYSTVEGPT 0.022 Peptide peptide
is is
i
10
18 YLPSEAPPNP 0.020 10 am
amino no
the
acids
39 DMIRKAEiALS 0.018 acids, ,
the end
end
45 HALSRPWWMC 0.013 position position
for fo
16 PLYLPSEAPP 0.010 each each
4 YSTVEGPTPF 0.010 peptide peptide
is is
28 PLWNSQDTSP 0.010 the the
start start
osition
56 RRGKDISWNF 0.009 position p
i plus
l nine
6 TVEGPTPFQD 0.009 p
ne
us
n
33 QDTSPTDMIR 0.008
,
11 TPFQDPLYLP 0.007
PTPFQDPLYL 0.006
43 KAxALSRPww 0.006
13 FQDPLYLPSE 0.004
48 SRPWWMCSRR 0.004
5 STVEGPTPFQ 0.003
23 APPNPPLWNS 0.003
54 CSRRGKDISW 0.002
36 SPTDMIRKAH 0.002
50 PWWMCSRRGK 0.001
31 NSQDTSPTDM 0.001
37 PTDMIRKAHA 0.001
26 NPPLWNSQDT 0.001
3$ TDMIRKAHAL 0.001
21 SEAPPNPPLW 0.001
44 AHALSRPWWM 0.001
$ EGPTPFQDPL 0.001
PSEAPPNPPL 0.000 '
19 LPSEAPPNPP 0.000
7 VEGPTPFQDP 0.00
53 MCSRRGKDIS 0.000
22 EAPPNPPLWN 0.000
14 QDPLYLPSEA 0.000
35 TSPTDMIRKA 0.000
15 DPLYLPSEAP 0.000
29 LWNSQDTSPT 000
0
49 RPWWMCSRRG .
0.000
1 LHVYSTVEGP 000
0
27 PPLWNSQDTS .
0.000
55 SRRGKDISWN 0.000
17 LYLPSEAPPN 0.000
WNSQDTSPTD 0.000
24 PPNPPLWNSQ 0.000
154
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table 13P1F11
X
v.3-A3-lOmers:
2
Pos 1234567890 Score
11 TLPSPFPYLS 0.360 Portion
of
9 GATLPSPFPY 0.360 SEQ
ID
ATLPSPFPYL 0.304 N0:7;
each
start
3 IIQACRGATL 0.060 position
is
12 LPSPFPYLSL 0.027 specified,
2 YIIQACRGAT 0.005 the
length
7 CRGATLPSPF 0.002 of
each
5 QACRGATLPS 0.001 Peptide
is
i
10
4 IQACRGATLP 0.001 am
no
the
acids
6 ACRGATLPSP 0.000 ,
end
$ RGATLPSPFP 0.000 position
for
1 VYIIQACRGA 0.000 each
peptide
is
the
start
position
lus
nine
155
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table4-A3-lOmers:3P1F11 Tablev.4-A3-lOmers:3P1F11
X 21 X 21
v
Pos . Score P os 1234567890 Score
1234567890
69 IVGRDLSISF 0.400 Portion 14 QPEKRTGLRD 0.000 Portion
of of
33 RLKEEQGRAF 0.300 SEQ 27 ECGQTFRLKE 0.000 SEQ
ID ID
50 KLVNDPRETQ 0.135 NO' 37 EQGRAFRGSS 0.000 NO:
~' 9;
each
start
60 EVFGGGVGDI 0.121 each g5 ASEEEKYDMS 0.000 osition
start is
41 AFRGSSVHQK 0.090 p 71 GRDLSISFRN 0.000 specified,
ecifieds
12 SVQPEKRTGL 0.090 p 45 SSVHQKLVND 0.000 the
the length
length
9 KSLSVQPEKR 0.090 of 38 QGRAFRGSSV 0.000 of each
each
26 GECGQTFRLK 0.081 Peptide 64 GGVGDIVGRD 0.000 Peptide
is is
i
IO
SLSVQPEKRT 0.075 IO Sg TQEVFGGGVG 0.000 no
amino am
the
acids
GLRDENGEC 0.060 acids,G 80 NSETSASEEE 0.000 ,
the end'
end
81 SETSASEEEK 0.060 position 43 RGSSVHQKLV 0.000 position
for for
$2 ETSASEEEKY 0.060 each 17 KRTGLRDENG 0.000 each
13 VQPEKRTGLR 0.054 Peptide 32 FRLKEEQGRA 0.000 Peptide
is is
65 GVGDIVGRDL 0.027 the lg TGLRDENGEC 0.000 the
start start
63 GGGVGDIVGR 0.018 Position 54 DPRETQEVFG 0.000 justm'ne
i P
l
73 DLSISFRNSE 0.018 ne Sg QEVFGGGVGD 0.000
us
n
P
4 CQEYDKSLSV 0.012 56 RETQEVFGGG 0.000
84 SASEEEKYDM 0.009 79 RNSETSASEE 0.000
$ DKSLSVQPEK 0.009 62 FGGGVGDIVG 0.000
70 VGRDLSISFR 0.006 7 YDKSLSVQPE 0.000
47 VHQKLVNDPR 0.006 83 TSASEEEKYD 0.000
34 LKEEQGRAFR 0.006 77 SFRNSETSAS 0.000
46 SVHQKLVNDP 0.006 66 VGDIVGRDLS 0.000
67 GDIVGRDLSI 0.005 1 MGKCQEYDKS 0.000
76 ISFRNSETSA 0.005 21 LRDENGECGQ 0.000
QTFRLKEEQG 0.005 22 RDENGECGQT 0.000
57 ETQEVFGGGV 0.004 78 FRNSETSASE 0.000
68 DIVGRDLSIS 0.004 55 PRETQEVFGG 0.000
75 SISFRNSETS 0.004 72 RDLSISFRNS 0.000
31 TFRLKEEQGR 0.004 36 EEQGRAFRGS 0.000 .
24 ENGECGQTFR 0.004 28 CGQTFRLKEE 0.000
23 DENGECGQTF 0.003 11 LSVQPEKRTG 0.000
2 GKCQEYDKSL 0.003 49 QKLVNDPRET 0.000
51 LVNDPRETQE 0.002 . 6 EYDKSLSVQP 0.000
53 . NDPRETQEVF0.002 16 EKRTGLRDEN 0.000
25 NGECGQTFRL O.O02 15 PEKRTGLRDE 0.000
29 GQTFRLKEEQ 0.002
3 KCQEYDKSLS 0.002
RAFRGSSVHQ 0.001
18 RTGLRDENGE 0.001
42 FRGSSVHQKL 0.001
74 LSISFRNSET 0.001
44 GSSVHQKLVN 0.001
39 GRAFRGSSVH 0.001
48 HQKLVNDPRE 0.001
$2 VNDPRETQEV 001
0
35 KEEQGRAFRG .
001
0
86 SEEEKYDMSG .
001
0
61 VFGGGVGDIV .
0.000
5 QEYDKSLSVQ 0.000
156
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table 13P1F11
Xv.S-A3-lOmers:
2
Pos 1234567890 Score
RLALILRVTK 20.000 Portion
of
6 ALILRVTKAR 4.500 SEQ
ID
2 GARLALILRV 018 NO:11;
0
. each
start
1 SGARLALILR 0.012 position
is
LALILRVTKA 0.009 specified,
7 LILRVTKARE 0.003 the
length
$ ILRVTKAREG 0.002 of
each
RVTKAREGSE 001 Peptide
0 is
. mino
10
9 LRVTKAREGS 0.000 a
acids
the
3 ARLALILRVT 0.000 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
Table 13P1F11
X
v.6-A3-lOmers:
2
Pos 1234567890 Score
9 AMNNKNCQAL 0.600 Portion
of
2 KLENLFEAMN 0.180 SEQ
ID
13
5 NLFEAMNNKN 150 ;
0 NO:
. each
start
4 ENLFEAMNNK 0.027 position
is
10 MNNKNCQALR O.Op8 specified,
1 VKLENLFEAM 0.001 the
length
$ EAMNNKNCQA 0.001 of
each
3 LENLFEANIrIN0 Peptide
000 is
. 10
amino
6 LFEAMNNKNC 0.000 the
acids
FEAMNNKNCQ 0.000 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
157
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
bl l-A11-9mers:13P1F11 Tab le XI v.l-Als: 213P1F11
X 2 l-9mer
T I
e v. Score P os 123456789 Score
a 123456789
P
os QTLVDVFTK 4.500 Portion 141 ETVGGDEIV 0.005 Portion
189 of of
125 KVYI IQACR 2.400 SEQ 94 FLKGEDGEM 0.004 SEQ
ID ID
3
218 LVQEGKARK 2.000 N0:3; g0 SCAFVVLMA 0.004 ;
N0:
each
start
191 LVDVFTKRK 1.000 each 111 ALNNKNCQA 0.004 osition
start is
107 NLFEALNNK 0.800 s 102 MVKLENLFE 0.004 ppecified,
ecifieds
59 EQFQEELEK 0.720 p 86 LMAHGREGF 0.004 the
the length
length
HGR 600 of 148 IVMVIKDSP 0.004 of
0 each each
83 FVVLMA . Peptide 160 PTYTDALHV 0.004 Peptide
171 TVEGYIAYR 400 is is
0 9 9
ino
45 QLRFESTMK . a~no 187 FIQTLVDVF 0.004 am
0.400 the
acids
the ,
acids
175 YIAYRHDQK 0.400 , 151 VIKDSPQTI 0.004 end
d
en
19 LALILCVTK 300 position 161 TYTDALHVY 0.004 position
0 for for
. h
117 CQALRAKPK 300 each 71 AIDSREDPV 0.004 eac
0
. Peptide
is
3 NPRSLEEEK 200 Peptide 168 VYSTVEGYI 0.004 h
0 is
96 KGEDGEMVK . the 158 TIPTYTDAL 0.004 e start
120 start t
0 osition
. position p
129 IQACRGEQR 120 h 100 GEMVKLENL 0.004 Plus
0 i eight
190 TLVDVFTKR . t 226 KTNPEIQST 0.003
120 plus
0 e
g
.
67 KFQQAIDSR 120 150 MVIKDSPQT 0.003 .
0
.
$$ AHGREGFLK 060 118 QALRAKPKV 0.003
0
145 GDEIVMVIK . 186 CFIQTLVDV 0.003
060
0
.
215 EAELVQEGK 060 84 VVLMAHGRE 0.003
0
. .
21 LILCVTKAR 0.060 231 IQSTLRKRL 0.003
142 TVGGDEIVM 0.040 77 DPVSCAFVV 0.003
228 NPEIQSTLR 0.040 212 RMAEAELVQ 0.002
167 HVYSTVEGY 0.040 13 DMSGARLAL 0.002
204 ELLTEVTRR 036 10 EKYDMSGAR 0.002
0
.
233 STLRKRLYL 0.030 230 EIQSTLRKR 0.002
170 STVEGYIAY 0.030 57 TAEQFQEEL 0.002
44 RQLRFESTM 0.027 159 IPTYTDALH 0.002
50 STMKRDPTA 020 60 QFQEELEKF 0.002
0
217 ELVQEGKAR . 119 ALRAKPKVY 0.002
018
0
.
229 PEIQSTLRK 018 87 MAHGREGFL 0.002
0
.
203 LELLTEVTR 018 194 VFTKRKGHI 0.002
0
.
46 LRFESTMICR 016 78 PVSCAFVVL 0.002
0
.
157 QTIPTYTDA 015 24 CVTKAREGS 0.002
0
.
115 KNCQALRAK 012 174 GYIAYRHDQ 0.002
0
.
121 RAKPKVYII 012 135 EQRDPGETV 0.002
0
.
39 LEHMFRQLR 012 75 REDPVSCAF 0.002
0
.
104 KLENLFEAL 012 101 EMVKLENLF 0.002
0
.
123 KPKVYIIQA 012 140 GETVGGDEI 0.002
0
.
11 KYDMSGARL 012 97 GEDGEMVKL 0.002
0
.
195 FTKRKGHIL O10 16 GARLALILC 0.001
O
.
36 LDALEHMFR 008 211 RRMAEAELV 0.001
0
.
81 CAFVVLMAH 008 197 KRKGHILEL 0.001
0
. '
113 NNKNCQALR 008 ~ 144 GGDEIVMVI 0.001
0
.
6 SLEEEKYDM 008 18 RLALILCVT 0.001
0
.
201 HILELLTEV 006 64 ELEKFQQAI 0.001
0
.
193 DVFTKRKGH 006 172 VEGYIAYRH 0.001
0
.
20 ALILCVTKA 006 35 DLDALEHMF 0.001
0
.
223 KARKTNPEI 006 162 YTDALHVYS 0.001
0
.
208 EVTRRMAEA 0.006 206 LTEVTRRMA 0.001
158
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableI v.2-A11-9mers TableI v.2-Al1-
X X
9mers
~Pos123456789 Score P os 123456789 Score
4g RPWWMCSRR 0.240 Portion 3 YSTVEGPTP 0.000 Portion
of of
33 DTSPTDMIR 0.120 SEQ 2$ LWNSQDTSP 0.000 SEQ
ID ID
34 TSPTDMIRK 0.040 N0:5; 7 EGPTPFQDP 0.000 N0:5;
each
start
4 STVEGPTPF 0.015 each 24 pNPPLWNSQ 0.000 osition
start is
40 IRKAHALSR 0.008 specifieds 19 PSEAPPNPP 0.000 specified,
'
TPFQDPLYL 0.008 the 49 PWWMCSRRG 0.000 the
length length
DTSPTDM 0 of of
S 006 each each
31 Q . Peptide peptide
44 HALSRPWWM 0.006 is is
9 9
i
$ GPTPFQDPL 0.006 a~no am
no
the
acids
50 WWMCSRRGK 0.004 acids, ,
the end
end
1 HVYSTVEGP 0.004 position position
for for
47 SRPWWMCSR 0.004 each each
5 TVEGPTPFQ 0.002 peptide peptide
is is
3$ DMIRKAHAL 0.002 the the
start start
osition
56 RGKDISWNF 0.001 Position p
i plus
ht eight
l
12 FQDPLYLPS 0.001 g
us
e
p
5~ MCSRRGKDI 0.001
.
9 PTPFQDPLY 0.001
14 DPLYLPSEA 0.001
45 ALSRPWWMC 0.001
16 LYLPSEAPP 0.001
42 KAHALSRPW 0.001
21 EAPPNPPLW 0.001
SEAPPNPPL 0.001
36 PTDMIRKAH 0.001
2 VYSTVEGPT 0.000
22 APPNPPLWN 0.000
17 YLPSEAPPN 0.000
37 TDMIRKAHA 0.000
39 MIRKAHALS 0.000
51 WMCSRRGKD 0.000
54 SRRGKDISW 0.000
NPPLWNSQD 0.000
32 QDTSPTDMI 0.000
18 LPSEAPPNP 0.000
SPTDMIRKA 0.000
43 AHALSRPWW 0.000
6 VEGPTPFQD 0.000
15 PLYLPSEAP 0.000
27 PLWNSQDTS 0.000
41 RKAHALSRP 0.000
55 RRGKDISWN 000
0
29 WNSQDTSPT .
0.000
11 PFQDPLYLP 0.000
46 LSRPWWMCS 0.000
23 PPNPPLWNS 0.000
26 PPLWNSQDT 0.000
53 CSRRGKDIS 0
000
30 ~NSQDTSPTD .
000
0
13 QDPLYLPSE .
0.000
159
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table
XI
v.3-Al
l-9mers
Pos 123456789 Score
9 ATLPSPFPY 0.045 Portion
of
3 IQACRGATL 0.006 SEQ
ID
TLPSPFPYL 0.004 N0:7;
each
start
$ GATLPSPFP 0.001 position
is
1 YIIQACRGA 0.001 specified,
7 RGATLPSPF 0.001 the
length
11 LPSPFPYLS 0.000 of
each
ACRGATLPS 0.000 Peptide
is
9
o
i
2 IIQACRGAT 0.000 am
n
the
acids
4 QACRGATLP 0.000 ,
end
12 PSPFPYLSL 0.000 position
for
6 CRGATLPSP 0.000 each
peptide
is
the
start
position
lus
ei
ht
160
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
X 4-A11-9mers:13P1F11 TableI v.4-Al 13P1F11
bl 2 X l-9mers:
T I v 2
e . Score p os 123456789 Score
a 123456789
P
os ETSASEEEK 0.300 Portion 79 RNSETSASE 0.000 Portion
g2 of of
4$ HQKLVNDPR 0.120 SEQ 63 GGGVGDIVG 0.000 SEQ
ID ID
9 KSLSVQPEK 0.090 N~' 6 EYDKSLSVQ 0.000 NO:
~' 9;
each
start
SLSVQPEKR 0.080 each 24 ENGECGQTF 0.000 position
start is
osition
is
1 MGKCQEYDK 0.040 p 84 SASEEEKYD 0.000 specified,
ecified
s
14 QPEKRTGLR 0.040 , 67 GDIVGRDLS 0.000 the
p length
the
length
AFR 036 of 59 QEVFGGGVG 0.000 of each
0 each
35 KEEQGR . Peptide 17 KRTGLRDEN 0.000 Peptide
42 FRGSSVHQK 0.020 is is
9 9
i
o
64 GGVGDIVGR 0.018 amino 45 SSVHQKLVN 0.000 n
am
the
acids
71 GRDLSISFR 0.012 acids, 22 RDENGECGQ 0,.000 ,
the end
end
33 RLKEEQGRA 0.012 position 2 GKCQEYDKS 0.000 position
for for
40 RAFRGSSVH 0.012 each 86 SEEEKYDMS 0.000 each
32 FRLKEEQGR 0.006 Peptide 81 SETSASEEE 0.000 peptide
is is
h
65 GVGDIVGRD 0.006 the 36 EEQGRAFRG 0.000 e start
start t
osition
27 ECGQTFRLK 0.006 position 52 VNDPRETQE 0.000 p
l p]us
i eight
h
58 TQEVFGGGV 0.006 p 28 CGQTFRLKE 0.000
us
e
g
t
13 VQPEKRTGL 0.006 76 ISFRNSETS 0.000
26 GECGQTFRL 0.005 74 LSISFRNSE 0.000
25 NGECGQTFR 0.004 49 QKLVNDPRE 0.000
68 DIVGRDLSI 0.004 19 TGLRDENGE 0.000
3 KCQEYDKSL 003 47 VHQKLVNDP 0.000
0
18 RTGLRDENG . 66 VGDIVGRDL 0.000
0.003
5 QEYDKSLSV 0.002 38 QGRAFRGSS 0.000
77 SFRNSETSA 0.002 34 LKEEQGRAF 0.000
46 SVHQKLVND 0.002 80 NSETSASEE 0.000
51 LVNDPRETQ 0.002 7 YDKSLSVQP 0.000
30 QTFRLKEEQ 002 78 FRNSETSAS 0.000
0
69 IVGRDLSIS . 21 LRDENGECG 0.000
0.002
61 VFGGGVGDI 002 37 EQGRAFRGS 0.000
0
GLRDENGEC . 23 DENGECGQT 0.000 ,
0.001
60 EVFGGGVGD 0.001 11 LSVQPEKRT 0.000
29 GQTFRLKEE 001 15 PEKRTGLRD 0.000
0
43 RGSSVHQKL . 73 DLSISFRNS 0.000
0.001
4 CQEYDKSLS 0.001 . 8 DKSLSVQPE 0.000
39 GRAFRGSSV 0.001 55 PRETQEVFG 0.000
54 DPRETQEVF 001 16 EKRTGLRDE 0.000
0
56 RETQEVFGG .
0.001
75 SISFRNSET 0.000
85 ASEEEKYDM 000
0
70 VGRDLSISF .
000
0
44 GSSVHQKLV .
000
0
57 ETQEVFGGG .
000
0
72 RDLSISFRN .
000
0
41 AFRGSSVHQ .
000
0
53 NDPRETQEV .
000
0
31 TFRLKEEQG .
0
000
83 TSASEEEKY .
000
0
.
62 FGGGVGDIV 000
0
.
12 SVQPEKRTG 000
0
.
50 KLVNDPRET 0.000
161
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table
XI
v.5-A11-9mers:
213P1F11
Pos 123456789 Score
4 LALILRVTK 0.300 Portion
of
1 GARLALILR 0.240 SEQ
ID
6 LILRVTKAR 060 NO:11;
0
. each
start
9 RVTKAREGS 0.006 position
is
ALILRVTKA 0.006 specified,
2 ARLALILRV 0.001 the
length
7 ILRVTKARE 0.000 of
each
3 RLALILRVT 0.000 Peptide
is
9
8 LRVTKAREG 0.000 anuno
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
Table
XI
v.6-Al
l-9mers:
213P1F11
Pos 123456789 Score
4 NLFEAMNNK 0.800 Portion
of
1 KLENLFEAM 0.012 SEQ
ID
$ AMNNKNCQA 004 N0:13;
0
. each
start
9 MNNKNCQAL 0.000 position
is
5 LFEAMNNKN 0.000 specified,
7 EAMNNKNCQ 0.000 the
length
2 LENLFEAMN 0.000 of
each
3 ENLFEAMNN 0.000 Peptide
is
9
6 FEAMNNKNC 0.000 anuno
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
162
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXII v.l-A11-lOmers:213P1F11 TableCII v.l-A11-lOmers:213P1F11
3
Pos 1234567890 Score Pos 1234567890 Score
44 RQLRFESTMK 2.700 Portion 158 TIPTYTDALH 0.004Portion
of of
SEQ . SEQ ID
LD l NO:
NO: 3; each
3; start
each position
start is
' position specified,
is the
specified, ength
the of each
length peptide
of is 10
each amino
peptide acids,
is the end
10 position
amino for
acids, each
the peptide
end is the
position start
for position
each plus
peptide nine
is
the
start
.
174 GYIAYRHDQK 1.800 position 119 ALRAKPKVYI 0.004
18$ IQTLVDVFTK 1.800 plus 159 IPTYTDALHV 0.004.
nine
18 RLALILCVTK 1.200 148 IVMVIKDSPQ 0.004
87 MAHGREGFLK 0.600 59 EQFQEELEKF 0.004
228 NPEIQSTLRK 0.400 9 EEKYDMSGAR 0.004
190 TLVDVFTKRK 0.300 29 REGSEEDLDA 0.004
~
189 QTLVDVFTKR 0.300 100 GEMVKLENLF 0.004
170 STVEGYIAYR 0.300 83 FWLMAHGRE 0.003
217 ELVQEGKARK 0.180 233 STLRKRLYLQ 0.003
45 QLRFESTMKR 0.160 19 LALILCVTKA 0.003
5$ AEQFQEELEK 0.120 186 CFIQTLVDVF 0.003
116 NCQALRAKPK 0.100 70 QAIDSREDPV 0.003
3$ ALEHMFRQLR 0.080 41 HMFRQLRFES 0.002
202 ILELLTEVTR 0.080 66 EKFQQAIDSR 0.002
128 IIQACRGEQR 0.0$0 114 NKNCQALRAK 0.002
144 GGDEIVMVIK 0.060 50 STMKRDPTAE 0.002
102 MVKLENLFEA 0.060 3 NPRSLEEEKY 0.002
214 AEAELVQEGK 0.060 162 YTDALHVYST 0.002
82 AFVVLMAHGR 0.060 206 LTEVTRRMAE 0.002
20 ALILCVTKAR 0.060 195 FTKRKGHILE 0.002
35 DLDALEHMFR 0.048 194 VFTKRKGHIL 0.002
171 TVEGYIAYRH 0.040 24 CVTKAREGSE 0.002
2 SNPRSLEEEK 0.040 78 PVSCAFVVLM 0.002
95 LKGEDGEMVK 0.040 . 160 PTYTDALHW 0.002
167 HVYSTVEGYI 0.040 5 RSLEEEKYDM 0.002
150 MVIKDSPQTI 0.030 61 FQEELEKFQQ 0.002
226 KTNPEIQSTL 0.030 75 REDPVSCAFV 0.002
142 TVGGDEIVMV 0.020 ' 140 GETVGGDEIV 0.002
203 LELLTEVTRR 0.018 134 GEQRDPGETV 0.002
106 ENLFEALNNK 0.018 II8 QALRAKPKVY 0.002
157 QTIPTYTDAL O.O15 . 212 RMAEAELVQE 0.001
193 DVFTKRKGHI 0.012 ll KYDMSGARLA 0.001
16 GARLALILCV 0.012 12I RAKPKVYIIQ 0.001
125 KWIIQACRG 0.012 104 KLENLFEALN 0.001
.
209 VTRRMAEAEL 0.010 39 LEHMFRQLRF 0.001
93 GFLKGEDGEM 0.009 13 DMSGARLALI 0.001
216 AELVQEGKAR 0.009 32 SEEDLDALEH 0.001
163
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
141ETVGGDEIVM 0.009 56 PTAEQFQEEL 0.001
168VYSTVEGYIA 0.008 25 VTKAREGSEE 0.001
112LNNKNCQALR 0.008 200 GHILELLTEV 0.001
85 VLMAHGREGF 0.008 207 TEVTRRMAEA 0.001
227TNPEIQSTLR 0.008 77 DPVSCAFVVL 0.001
27 KAREGSEEDL 0.006 110 EALNNKNCQA 0.001
117CQALRAKPKV 0.006 12 YDMSGARLAL 0.001
111ALNNKNCQAL 0.004 107 NLFEALNNKN 0.001
80 SCAFVVLMAH 0.004 113 NNKNCQALRA 0.001
185SCFIQTLVDV 0.004 123 KPKVYIIQAC 0.001
94 FLKGEDGEMV 0.004 179 RHDQKGSCFI 0.001
86 LMAHGREGFL 0.004 I 129 IQACRGEQRD 0.001
I
164
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XII v.2-A11-lOmers: 213P1F11 Table XII v.2-Al l-lOmers: 213P1F11
Pos 1234567890 Score Pos1234567890 Score
34 DTSPTDMIRK 0.600 Portion 24 PPNPPLWNSQ 0.000 Portion
of of
40 MIRKAIiALSR0.160 SEQ 29 LWNSQDTSPT 0.000 SEQ
ID ID
33 QDTSPTDMIR 0.008 NO: 55 SRRGKDISWN 0.000 NO:
S; S;
43 KAHALSRPWW 0.006 each 35 TSPTDMIRKA 0.000 each
start start
osition osition
is is
9 GPTPFQDPLY 0.006 p 20 PSEAPPNPPL 0.000 p
specified, specified
,
6 TVEGPTPFQD 0.006 the 25 PNPPLWNSQD 0.000 the
length length
32 SQDTSPTDMI 0.006 of of each
each
48 SRPWWMCSRR 0.004 Peptide peptide
is is
47 LSRPWWMCSR 0.004 10 10 amino
amino id
acids h
the
, ac
2 HVYSTVEGPT 0.004 end s t
e
end
PTPFQDPLYL 0.002 position position
for for
52 WMCSRRGKDI 0.002 each each
5 STVEGPTPFQ 0.002 Peptide peptide
is is
56 RRGKDISWNF 0.001 the the
start start
i
i
37 PTDMIRKAHA 0.001 t position
on lus
Pos nine
plus
nine
36 SPTDMIRKAH 0.001 p
46 ALSRPWWMCS 0.001
11 TPFQDPLYLP 0.001
13 FQDPLYLPSE 0.001
45 HALSRPWWMC 0.001
17 LYLPSEAPPN 0.001
21 SEAPPNPPLW 0.001
50 PWWMCSRRGK 0.000
23 APPNPPLWNS 0.000
54 CSRRGKDISW 0.000
38 TDMIRKAHAL 0.000
18 YLPSEAPPNP 0.000
3 VYSTVEGPTP 0.000
44 AHALSRPWWM 0.000
4 YSTVEGPTPF 0.000
26 NPPLWNSQDT 0.000
14 QDPLYLPSEA 0.000
31 NSQDTSPTDM 0.000
19 LPSEAPPNPP 0.000
53 MCSRRGKDIS 0.000
39 DMIRKAHALS 0.000
49 RPWWMCSRRG 0.000
22 EAPPNPPLWN 0.000
DPLYLPSEAP 0.000
16 PLYLPSEAPP 0.000
28 PLWNSQDTSP 0.000
42 RKAHALSRPW 0.000
8 EGPTPFQDPL 0.000
7 VEGPTPFQDP 0.000
12 PFQDPLYLPS 0.000
30 WNSQDTS,PTD0.000
51 WWMCSRRGKD 0.000
1 LHVYSTVEGP 0.000
27 PPLWNSQDTS 0.000
41 IRKAHALSRP 0.000
165
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XII v.3-A11-lOmers: 213P1F11
Pos 1234567890 Score
9 GATLPSPFPY 0.018 Portion
of
ATLPSPFPYL 0.015 SEQ
ID
12 LPSPFPYLSL 0.004 N0:7;
3 IIQACRGATL 0.004 each
start
osition
is
11 TLPSPFPYLS 0.001 p
specified
,
1 VYIIQACRGA 0.001 the
length
4 IQACRGATLP 0.001 of each
2 YIIQACRGAT 0.001 Peptide
is
5 QACRGATLPS 0.000 10 amino
id
th
ac
6 ACRGATLPSP 0.000 s,
e
end
7 CRGATLPSPF 0.000 position
for
8 RGATLPSPFP 0.000 each
peptide
is
the
start
position
lus
nine
166
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXII v.4-A11-lOmers: 213P1F11 TableXII v.4-A11-lOmers:
213P1F11
Pos 1234567890 Score Pos1234567890 Score
41 AFRGSSVHQK 0.200 Portion 56 RETQEVFGGG 0.000 Portion
of of
13 VQPEKRTGLR 0.120 SEQ 6g DIVGRDLSIS 0.000 SEQ
ID ID
81 SETSASEEEK 0.060 NO' 23 DENGECGQTF 0.000 NO:
~' 9;
h
t
69 IVGRDLSTSF 0.040 each 37 EQGRAFRGSS 0.000 star
start eac
osition position
is is
31 TFRLKEEQGR ~ 0.040 p 79 RNSETSASEE 0.000 specified,
specified,
12 SVQPEKRTGL 0.020 the 44 GSSVHQKLVN 0.000 the
length length
9 KSLSVQPEKR 0.018 of 6 EYDKSLSVQP 0.000 of each
each
26 GECGQTFRLK 0.018 Peptide 73 DLSISFRNSE 0.000 Peptide
is is
63 GGGVGDIVGR 0.012 ]0 5 QEYDKSLSVQ 0.000 10 amino
amino acids
id the
th
60 EVFGGGVGDI 0.012 ac 86 SEEEKYDMSG 0.000 end
s,
e
end
4 CQEYDKSLSV 0.012 position 27 ECGQTFRLKE 0.000 position
for for
65 GVGDIVGRDL 0.006 each 64 GGVGDIVGRD 0.000 each
8 DKSLSVQPEK 0.006 Peptide 59 QEVFGGGVGD 0.000 Peptide
is is
the the
84 SASEEEKYDM 0.004 start 17 KRTGLRDENG 0.000 start
i iti
i
pos on
47 VHQKLVNDPR 0.004 t 22 RDENGECGQT 0.000 pos
on Plus
plus nine
nine
34 LKEEQGRAFR 0.004 54 DPRETQEVFG 0.000
70 VGRDLSISFR 0.004 62 FGGGVGDIVG 0.000
82 ETSASEEEKY 0.003 45 SSVHQKLVND 0.000
57 ETQEVFGGGV 0.003 19 TGLRDENGEC 0.000
18 RTGLRDENGE 0.003 ~ 74 LSISFRNSET 0.000
24 ENGECGQTFR 0.002 80 NSETSASEEE
30 QTFRLKEEQG 0.002 1 MGKCQEYDKS 0.000
46 SVHQKLVNDP 0.002 21 LRDENGECGQ 0.000
51 LVNDPRETQE 0.002 85 ASEEEKYDMS 0.000
61 VFGGGVGDIV 0.002 66 VGDIVGRDLS 0.000
67 GDIVGRDLSI 0.002 7 YDKSLSVQPE 0.000
50 KLVNDPRETQ 0.002 78 FRNSETSASE 0.000
29 GQTFRLKEEQ 0.002 83 TSASEEEKYD 0.000
20 GLRDENGECG 0.001 28 CGQTFRLKEE 0.000
40 RAFRGSSVHQ 0.001 72 RDLSISFRNS 0.000
33 RLICEEQGRAF0.001 55 PRETQEVFGG 0.000
39 GRAFRGSSVH 0.001 16 EKRTGLRDEN 0.000
3 KCQEYDKSLS 0.001 11 LSVQPEKRTG 0.000
25 NGECGQTFRL 0.001 49 QKLVNDPRET 0.000
58 TQEVFGGGVG 0.001 36 EEQGRAFRGS 0.000
48 HQKLVNDPRE 0.001 15 PEKRTGLRDE 0.000
35 KEEQGRAFRG 0.001
76 ISFRNSETSA 0.000
52 VNDPRETQEV 0.000
75 SISFRNSETS 0.000
14 QPEKRTGLRD 0.000
32 FRLKEEQGRA 0.000
43 RGSSVHQKLV 0.000
2 GKCQEYDKSL 0.000
77 SFRNSETSAS 0.000
~ SLSVQPEKRT0.000
42 FRGSSVHQKL 0.000
53 NDPRETQEVF 0.000
38 QGRAFRGSSV 0.000
71 GRDLSISFRN 0.000
167
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table
XII
v.5-A11-lOmers:
213P1F11
Pos 1234567890 Score
4 RLALILRVTK 1.200 Portion
of
6 ALILRVTKAR 0.060 SEQ
ID
2 GARLALILRV 012 ND~
0 11;
. each
start
1 SGARLALILR 0.008 position
is
RVTKAREGSE 0.006 specified,
5 LALILRVTKA 0.003 the
length
7 LILRVTKARE 0.001 of
each
8 ILRVTKAREG 0.000 Peptide
is
10
i
9 LRVTKAREGS 0 no
000 am
. the
acids
3 ARLALILRVT 0.000 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
Table
XII
v.6-Al
l-lOmers:
213P1F11
Pos 1234567890 Score
4 ENLFEAMNNK 0.018 Portion
of
10 NR9NKNCQALR 0.008 SEQ
ID
9 AMNNKNCQAL 004 1'10:13;
0
. each
start
2 KLENLFEAMN 0.001 position
is
$ EANIrINKNCQA0.001 specified,
5 NLFEAMNNKN 0.001 the
length
1 VKLENLFEAM 0.000 of
each
3 LENLFEAMNN 0.000 Peptide
is
i
0
6 LFEAMNNKNC 0.000 no
1
am
the
acids
7 FEAMNNKNCQ 0.000 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
168
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXIII v.l-A24-9mers: 213P1F11 TableXIII v.l-A24-9mers;
213P1F11
Pos 123456789 Score Pos 123456789 Score
11 KYDMSGARL 400.000Portion 226 KTNPEIQST 0.432 Portion
of of
168 WSTVEGYI 70.000 SEQ 17g RHDQKGSCF 0.400 SEQ
ID ID
60 QFQEELEKF 19.800 N0:3; 40 EHMFRQLRF 0.300 NO:3;
104 KLENLFEAL 17.280 each 18 RLALILCVT 0.280 each
start start
osition
is
p position
227 TNPEIQSTL 10.080 specified, 199 KGHILELLT 0.240 is
s
ecified
p
183 KGSCFIQTL 9.600 the 201 HILELLTEV 0.238 ,
length the
length
112 LNNKNCQAL 7.200 of 99 DGEMVKLEN 0.231 of each
each
31 GSEEDLDAL 7.200 Peptide 155 SPQTIPTYT 0.210 peptide
is is
9 9
38 ALEHMFRQL 7.200 amino 147 EIVMVIKDS 0.210 amino
id
th
ac acids,
57 TAEQFQEEL 6.600 s, 164 DALHVYSTV 0.210 the
e d
end
233 STLRKRLYL 6.000 position 123 KPKWIIQA 0.200 en
for position
for
158 TIPTYTDAL 6.000 each 157 QTIPTYTDA 0.180 each
161 TYTDALHW 6.000 Peptide 202 ILELLTEVT 0.180 Peptide
is is
the the
177 AYRHDQKGS 5.000 start 170 STVEGYIAY 0.180 start
i
os p
194 VFTKRKGHI 5.000 tion 20 ALILCVTKA 0.165
lus
ei
ht
p Plus
15 SGARLALIL 4.800 g 219 VQEGKARKT 0.165 eight
231 IQSTLRKRL 4.800 118 QALRAKPKV 0.165
101 EMVKLENLF 4.320 , 50 STMKRDPTA 0.150
87 MAHGREGFL 4.000 150 MVIKDSPQT 0.150
195 FTKRKGHIL 4.000 47 RFESTMKRD 0.150
13 DMSGARLAL 4.000 154 DSPQTIPTY 0.150
187 FIQTLVDVF 3.600 ~ 206 LTEVTRRMA 0.150
35 DLDALEHMF 2.400 106 ENLFEALNN 0.150
121 RAKPKWII 2.400 111 ALNNKNCQA 0.150
223 KARKTNPEI 2.200 67 KFQQAIDSR 0.150
86 LMAHGREGF 2.000 141 ETVGGDEIV 0.150
64 ELEKFQQAI 1.800 77 DPVSCAFW 0.150
144 GGDEIVMVI 1.680 180 HDQKGSCFI 0.150
44 RQLRFESTM 1.500 80 SCAFVVLMA 0.140
151 VIKDSPQTI 1.440 188 IQTLVDVFT 0.140
108 LFEALNNKN 1.188 184 GSCFIQTLV 0.140
198 RKGHILELL 1.120 30 EGSEEDLDA 0.120
14 MSGARLALI 1.000 143 VGGDEIVMV 0.120
6 SLEEEKYDM 0.900 162 YTDALHWS 0.120
197 KRKGHILEL 0.880 73 DSREDPVSC 0.120
205 LLTEVTRRM 0.840 135 EQRDPGETV 0.120'
126 WIIQACRG 0.750 208 EVTRRMAEA 0.110
174 GYIAYRHDQ 0.750 140 GETVGGDEI 0.110
186 CFIQTLVDV 0.750 232 QSTLRKRLY 0.100
75 REDPVSCAF 0.672 16 GARLALILC 0.100
42 MFRQLRFES 0.660 119 ALRAKPKW 0.100
100 GEMVKLENL 0.600 167 HWSTVEGY 0.100
28 AREGSEEDL 0.600 24 CVTKAREGS 0.100
94 FLKGEDGEM 0.550 49 ESTMKRDPT 0.100
79 VSCAFWLM 0.500 71 AIDSREDPV 0.100
142 TVGGDEIVM 0.500 . 120 LRAKPKWI 0.100
78 PVSCAFVVL 0.480 169 YSTVEGYIA 0.100
53 KRDPTAEQF 0.480 34 EDLDALEHM 0.090
97 GEDGEMVKL 0.440 82 AFVVLMAHG 0.090
210 TRRMAEAEL 0.440 93 GFLKGEDGE 0.075
I
169
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXIII v.2-A24-9mers:213P1F11 TableXIII v.2-A24-9mers:213P1F11
Pos 123456789 Score Pos 123456789 Score
2 VYSTVEGPT 7.000 Portion 13 QDPLYLPSE 0.002 Portion
of of
56 RGKDISWNF 6.720 SEQ 36 PTDMIRKAH 0.001 SEQ
ID ID
38 DMIRKAHAL 6.000 NO: 6 VEGPTPFQD 0.001 N0:5;
S; h
t
rt
$ GPTPFQDPL 4.800 each 40 IRKAHALSR 0.001 s
start a
osition eac
is position
is
TPFQDPLYL 4.000 p 49 PWWMCSRRG 0.001 specified,
specified,
4 STVEGPTPF 3.600 the 15 PLYLPSEAP 0.001 the
length length
52 MCSRRGKDI 1.000 of each of
each
16 LYLPSEAPP 0.900 Peptide , peptide
is is
9 9
44 HALSRPWWM 0.750 amino amino
id acids
th the
31 SQDTSPTDM 0.500 e ,
ac end
s,
end
SEAPPNPPL 0.480 position position
for for
42 KAHALSRPW 0.240 each each
14 DPLYLPSEA 0.198 Peptide peptide
is is
21 EAPPNPPLW 0.180 the the
start start
iti
22 APPNPPLWN 0.150 Position pos
lus on
ei plus
ht eight
17 YLPSEAPPN 0.150 g
p
12 FQDPLYLPS 0.144
35 SPTDMIRKA 0.132
46 LSRPWWMCS 0.120
45 ALSRPWWMC 0.100
29 WNSQDTSPT 0.100
39 MIRKAHALS 0.100
32 QDTSPTDMI 0.100
53 CSRRGKDIS 0.100
7 EGPTPFQDP 0.022
48 RPWWMCSRR 0.020
55 RRGKDISWN 0.020
26 PPLWNSQDT 0.018
23 PPNPPLWNS 0.018
NSQDTSPTD 0.018
9 PTPFQDPLY 0.015
50 WWMCSRRGK 0.015
25 NPPLWNSQD 0.015
34 TSPTDMIRK 0.015
28 LWNSQDTSP 0.015
37 TDMIRKAHA 0.015
5 TVEGPTPFQ 0.015
18 LPSEAPPNP 0.012
33 DTSPTDMIR 0.012
51 WMCSRRGKD 0.011
54 ' SRRGKDISW0.010
43 AHALSRPWW 0.010
27 PLWNSQDTS O.O10
3 YSTVEGPTP 0.010
1 HVYSTVEGP 0.010
11 PFQDPLYLP 0.009
24 PNPPLWNSQ 0.003
41 RKAHALSRP 0.002
19 PSEAPPNPP 0.002
47 SRPWWMCSR 0.002
170
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table XIII v.3-A24-9mers: 213P1F11 I
Pos 123456789 Score
TLPSPFPYL 7.200 Portion
of
7 RGATLPSPF 4.800 SEQ
ID
3 IQACRGATL 4.000 N0:7;
h
12 PSPFPYLSL 0.600 start
eac
position
is
9 ATLPSPFPY 0.180 specified,
2 IIQACRGAT 0.150 the
length
1 YIIQACRGA 0.150 of
each
11 LPSPFPYLS 0.120 Peptide
is
9
5 ACRGATLPS 0.100 amino
acids
the
4 QACRGATLP 0.010 ,
end
8 GATLPSPFP 0.010 position
for
6 CRGATLPSP 0.001 each
peptide
is
the
start
position
lus
ei
ht
171
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXIII v.4-A24-9mers: 213P1F11 TableXIII v.4-A24-9mers:
213P1F11
Pos 123456789 Score Pos123456789 Score
43 RGSSVHQKL 14.784 Portion 64 GGVGDIVGR 0.015 Portion
of of
3 KCQEYDKSL 14.400 SEQ 67 GDIVGRDLS 0.015 SEQ
ID ID
13 VQPEKRTGL 7.200 NO' 7$ FRNSETSAS O.O15 NO:
9' 9;
h t
h
rt
66 VGDIVGRDL 5.600 start 14 QPEKRTGLR 0.015 eac
eac a
osition s
is position
is
61 VFGGGVGDI 5.000 p 48 HQKLVNDPR 0.014 specified,
specified,
24 ENGECGQTF 2.880 the 30 QTFRLKEEQ 0.013 the
length length
70 VGRDLSISF 2.880 of 5 QEYDKSLSV 0.012 of each
each
54 DPRETQEVF 2.400 Peptide 52 VNDPRETQE 0.012 Peptide
is is
9 9
68 DIVGRDLSI 1.500 amino 27 ECGQTFRLK 0.012 a~no
id acids
the the
85 ASEEEKYDM 0.900 ac 84 SASEEEKYD 0.012 end
s,
end
77 SFRNSETSA 0.500 position 2 GKCQEYDKS 0.011 position
for for
6 EYDKSLSVQ 0.500 each 10 SLSVQPEKR 0.011 each
26 GECGQTFRL 0.400 Peptide 29 GQTFRLKEE 0.011 peptide
is is
the the
34 LKEEQGRAF 0.360 start 82 ETSASEEEK 0.011 start
i iti
i
50 KLVNDPRET 0.330 t 39 GRAFRGSSV 0.010 on
on Pos
Pos plus
plus eight
eight
33 RLKEEQGRA 0.240 46 SVHQKLVND 0.010
4 CQEYDKSLS 0.150 60 EVFGGGVGD O.O10
11 LSVQPEKRT 0.150 63 GGGVGDIVG 0.010
58 TQEVFGGGV 0.150 1 MGKCQEYDK 0.010
45 SSVHQKLVN 0.150 ' 35 KEEQGRAFR 0.003
62 FGGGVGDIV 0.140 22 RDENGECGQ 0.003
20 GLRDENGEC 0.132 47 VHQKLVNDP 0.002
37 EQGRAFRGS 0.120 56 RETQEVFGG 0.002
73 DLSISFRNS 0.120 32 FRLKEEQGR 0.002
83 TSASEEEKY 0.110 59 QEVFGGGVG 0.002
75 SISFRNSET 0.110 36 EEQGRAFRG 0.002
44 GssvHQKLV 0.100 49 QxLVNDPRE 0.002
38 QGRAFRGSS 0.100 8 DKSLSVQPE 0.001
69 IVGRDLSIS 0.100 7 YDKSLSVQP 0.001
76 ISFRNSETS 0.100 21 LRDENGECG 0.001
41 AFRGSSVHQ 0.050 81 ~ SETSASEEE 0.001
31 TFRLKEEQG 0.050 42 FRGSSVHQK 0.001
9 KSLSVQPEK 0.046 16 EKRTGLRDE 0.001
72 RDLSISFRN 0.042 71 GRDLSISFR 0.001
57 ETQEVFGGG 0.030 55 PRETQEVFG 0.000
17 KRTGLRDEN 0.026 15 PEKRTGLRD 0.000
79 RNSETSASE 0.024
40 RAFRGSSVH 0.020
18 RTGLRDENG 0.020
53 NDPRETQEV 0.020
86 SEEEKYDMS 0.018
12 SVQPEKRTG 0.018
74 LSISFRNSE 0.018 .
51 LVNDPRETQ 0.018
19 TGLRDENGE 0.018
65 ~ GVGDIVGRD0.017
28 CGQTFRLKE 0.017
80 NSETSASEE 0.017
23 DENGECGQT 0.015
25 NGECGQTFR 0.015
172
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
I Table XIII v.5-A24-9mers: 213P 1 F 11 I
Pos 123456789 Score
3 RLALILRVT 0.280 Portion
of
9 RVTKAREGS 0.200 SEQ
ID
ALILRVTKA 0.165 NO:11;
t
h
6 LILRVTKAR 0.021 star
eac
position
is
2 ARLALILRV 0.018 specified,
4 LALILRVTK 0.018 the
length
1 GARLALILR 0.010 of
each
7 ILRVTKARE 0.010 Peptide
is
9
8 LRVTKAREG 0.002 amino
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
Table
XIII
v.6-A24-9mers:
213P1F11
Pos 123456789 Score
9 MNNKNCQAL 7.200 Portion
of
1 KLENLFEAM 2.160 SEQ
ID
5 LFEAMNNKN 0.990 N0:13;
h
t
t
3 ENLFEAMNN 0.150 s
ar
eac
position
is
$ AMNNKNCQA 0.150 specified,
7 EAMNNKNCQ 0.018 the
length
2 LENLFEAMN 0.015 of
each
4 NLFEAMNNK 0.014 Peptide
is
9
6 FEAMNNKNC 0.010 amino
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
173
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXIV v.i-A24-lOmers: 213P1F11 TableXIV v.1-A24-lOmers:
213P1F11
Pos 1234567890 Score Pos 1234567890 Score
226 KTNPEIQSTL 20.160 Portion 52 MKRDPTAEQF 0.240 Portion
of of
194 VFTKRKGHIL 20.000 SEQ 178 YRHDQKGSCF 0.240 SEQ
ID ID
186 CFIQTLVDVF 18.000 N0:3; 63 EELEKFQQAI 0.216 N0:3;
96 KGEDGEMVKL 15.840 each 201 HILELLTEVT 0.216 each
start start
osition
is
p position
11 KYDMSGARLA 10.000 specified 67 KFQQAIDSRE 0.210 is
ecified
s
, p
27 KAREGSEEDL 9.600 the 187 FIQTLVDVFT 0.210 ,
length the
length
37 DALEHMFRQL 8.640 of 154 DSPQTIPTYT 0.210 of each
each
161 TYTDALHVYS 7.200 Peptide 179 RHDQKGSCFr 0.200 Peptide
is is
111 ALNNKNCQAL 7.200 10 39 LEHMFRQLRF 0.200 10 amino
amino
a
id
th
c acids,
230 EIQSTLRKRL 7.200 s, 218 LVQEGKARKT 0.198 the
e end
end
77 DPVSCAFVVL 7.200 position 107 NLFEALNNKN 0.190 position
for for
157 QTIPTYTDAL 7.200 each 6 SLEEEKYDMS 0.180 each
99 DGEMVKLENL 6.000 Peptide 7Q QAIDSREDPV 0.180 Peptide
is is
the the
177 AYRHDQKGSC 5.000 start 139 PGETVGGDEI 0.165 start
i
i
168 VYSTVEGYIA 5.000 t 19 LALILCVTKA 0.165 Position
on
Pos i
lus l
nine
14 MSGARLALIL 4.800 P 215 EAELVQEGKA 0.165 us n
ne
p
30 EGSEEDLDAL 4.800 47 RFESTMKRDP 0.150
209 VTRRMAEAEL 4.400 , 23 LCVTKAREGS 0.150
93 GFLKGEDGEM 4.125 118 QALRAKPKVY 0.150
232 QSTLRKRLYL 4.000 149 VMVIKDSPQT 0.150
86 LMAHGREGFL 4.000 110 EALNNKNCQA 0.150
85 VLMAHGREGF 3.000 219 VQEGKARKTN 0.150
59 EQFQEELEKF 2.200 79 VSCAFVVLMA 0.140
150 MVIKDSPQTI 1.800 41 HMFRQLRFES 0.132
RSLEEEKYDM 1.800 73 DSREDPVSCA 0.120
143 VGGDEIVMVI 1.680 181 DQKGSCFIQT 0.120
167 HVYSTVEGYI 1.400 205 LLTEVTRRMA 0.120
197 KRKGHILELL 1.120 16 GARLALILCV 0.120
204 ELLTEVTRRM 1.050 102 MVKLENLFEA 0.110
103 VKLENLFEAL 1.037 3 NPRSLEEEKY 0.110
119 ALRAKPKVYI 1.000 222 GKARKTNPEI 0.110
193 DVFTKRKGHI 1.000 117 CQALRAKPKV 0.110
13 DMSGARLALI 1.000 60 QFQEELEKFQ 0.108
141 ETVGGDEIVM 0.750 169 YSTVEGYIAY 0.100
108 LFEALNNKNC 0.750 176 IAYRHDQKGS 0.100
126 VYIIQACRGE 0.750 185 SCFIQTLVDV 0.100
174 GYIAYRHDQK 0.750 120 LRAKPKVYII 0.100
12 YDMSGARLAL 0.600 231 IQSTLRKRLY 0.100
42 MFRQLRFEST 0.600 113 NNKNCQALRA 0.100
56 PTAEQFQEEL 0.528 142 TVGGDEIVMV 0.100
74 SREDPVSCAF 0.504 94 FLKGEDGEMV 0.100
EKYDMSGARL 0.480 49 ESTMKRDPTA 0.100
182 QKGSCFIQTL 0.480 15 SGARLALILC 0.100
196 TKRKGHILEL 0.440 162 YTDALHVYST 0.100
100 GEMVKLENLF 0.432 71 AIDSREDPVS 0.100
34 EDLDALEHMF 0.432 . 159 IPTYTDALHV 0.100
123 KPKVYIIQAC 0.336 82 AFVVLMAHGR 0.07
5
133 RGEQRDPGET 0.330 43 FRQLRFESTM _
_
0.075
1_04KLENLFEALN 0.300 78 PVSCAFVVLM 0.050
~83 KGSCFIQTLV 0.280 33 EEDLDALEHM 0.050
174
SUBSTITUTE SHEET (RULE 26)
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Table XIV v.2-A24-lOmers: 213P1F11 Table XIV v.2-A24-lOmers: 213P1F11
Pos 1234567890 Score Pos1234567890 Score
17 LYLPSEAPPN 9.000 Portion 1 LHVYSTVEGP 0.002 Portion
of of
8 EGPTPFQDPL 6.000 SEQ 50 PWWMCSRRGK 0.001 SEQ
ID ID
4 YSTVEGPTPF 2.000 ~ 28 PLWNSQDTSP 0.001 D
a ~ aach
32 SQDTSPTDMI 1.000 s art 33 QDTSPTDMIR 0.001 st rt
position iti
is i
pos
52 WMCSRRGKDI 1.000 specified, 41 IRKAHALSRP 0.001 on
s
specified
,
31 NSQDTSPTDM 0.900 the 16 PLYLPSEAPP 0.001 the
length length
20 PSEAPPNPPL 0.600 of _ of each
each
PTPFQDPLYL 0.600 Peptide peptide
is is
38 TDMIRKAHAL 0.600 10 10 amino
amino id
acids
th
, ac
56 RRGKDISWNF 0.560 e s the
end end
3 VYSTVEGPTP 0.500 position position
for for
43 KAHALSRPWW 0.200 each each
26 NPPLWNSQDT 0.180 peptide peptide
is is
22 EAPPNPPLWN 0.180 the the
start start
i
i
pos position
35 TSPTDMIRKA 0.165 t lus
on nine
plus
nine
29 LWNSQDTSPT 0.150 p
23 APPNPPLWNS 0.150
39 DMIRKAHALS 0.150
45 HAT.SRPWWMC0.150
2 HVYSTVEGPT 0.140
9 GPTPFQDPLY 0.120
12 PFQDPLYLPS 0.108
46 ALSRPWWMCS 0.100
53 MCSRRGKDIS 0.100
54 CSRRGKDISW 0.100
44 AHALSRPWWM 0.050
42 RKAHALSRPW 0.024
49 RPWWMCSRRG 0.020
14 QDPLYLPSEA 0.020
5 STVEGPTPFQ 0.018
36 SPTDMIRKAH 0.017
51 WWMCSRRGKD 0.017
18 YLPSEAPPNP 0.015
6 TVEGPTPFQD 0.015
27 PPLWNSQDTS 0.015
DPLYLPSEAP 0.015
19 LPSEAPPNPP 0.014
13 FQDPLYLPSE 0.012
21 SEAPPNPPLW 0.012
34 DTSPTDMIRK 0.012
47 LSRPWWMCSR 0.012
55 SRRGKDISWN 0.010
37 PTDMIRKAHA 0.010
11 TPFQDPLYLP 0.010
30 WNSQDTSPTD 0.010
40 MIRKAHALSR 0.010
24 PPNPPLWNSQ 0.003
PNPPLWNSQD 0.002
7 VEGPTPFQDP 0.002
48 SRPWWMCSRR 0.002
175
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XIV v.3-A24-lOmers: 213PiF11
Pos 1234567890 Score
ATLPSPFPYL 8.640 Portion
of
1 VYIIQACRGA 7.500 SEQ
ID
3 IIQACRGATL 6.000 N0:7;
h
12 LPSPFPYLSL 4.800 start
eac
position
is
7 CRGATLPSPF 0.240 specified,
11 TLPSPFPYLS 0.150 the
length
2 YI IQACRGAT0.150 of
each
5 QACRGATLPS 0.100 Peptide
is
9 GATLPSPFPY 0.100 10
amino
acids
the
8 RGATLPSPFP 0.020 ,
end
4 IQACRGATLP 0.010 position
for
6 ACRGATLPSP 0.010 each
peptide
is
the
start
position
lus
nine
176
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXIV v.4-A24-lOmers: 213P1F11 TableXIV v.4-A24-lOmers:
213P1F11
Pos 1234567890 Score Pos 1234567890 Score
12 SVQPEKRTGL 7,200 Portion 11 LSVQPEKRTG 0.015 Portion
of of
65 GVGDIVGRDL 6.720 SEQ 14 QPEKRTGLRD 0.015 SEQ
ID ID
25 NGECGQTFRL 6.000 NO' 32 FRLKEEQGRA 0.015 NO:
g' 9;
t
h
33 RLKEEQGRAF 4.800 each 45 SSVHQKLVND 0.015 star
start eac
osition position
is is
69 IVGRDLSISF 2,400 p 46 SVHQKLVNDP 0.014 specified,
specified,
60 EVFGGGVGDI 1.000 the 71 GRDLSISFRN 0.014 the
length Iength
42 FRGSSVHQKL 0.739 of each 16 EKRTGLRDEN 0.013 of
each
61 VFGGGVGDIV 0.700 Peptide 29 GQTFRLKEEQ 0.013 Peptide
is is
84 SASEEEKYDM 0.600 ]0 amino 70 VGRDLSISFR 0.012 IO
id amino
the acids
the
6 EYDKSLSVQP 0.600 ac 73 DLSISFRNSE 0.012 ,
s end
end
77 SFRNSETSAS 0.500 position 20 GLRDENGECG 0.012 position
for for
2 GKCQEYDKSL 0.400 each 54 DPRETQEVFG 0.012 each
23 DENGECGQTF 0.360 Peptide 24 ENGECGQTFR 0.012 Peptide
is is
the the
3 KCQEYDKSLS 0.360 start 27 ECGQTFRLKE 0.011 start
i iti
i
Pos pos
53 NDPRETQEVF 0.300 t 30 QTFRLKEEQG 0.010 on
on Plus
lus nine
nine
43 RGSSVHQKLV 0.200 P 83 TSASEEEKYD 0.010
85 ASEEEKYDMS 0.180 63 GGGVGDIVGR O.O10
57 ETQEVFGGGV 0.180 48 HQKLVNDPRE 0.010
74 LSISFRNSET 0.165 62 FGGGVGDIVG 0.010
19 TGLRDENGEC 0.165 56 RETQEVFGGG 0.003_
52 VNDPRETQEV 0.158 35 KEEQGRAFRG 0.003
68 DIVGRDLSIS 0.150 47 VHQKLVNDPR 0.002
67 GDIVGRDLSI 0.150 17 KRTGLRDENG 0.002
4 CQEYDKSLSV 0.150 34 LKEEQGRAFR 0.002
82 ETSASEEEKY 0.110 86 SEEEKYDMSG 0.002
1 MGKCQEYDKS 0.110 8 DKSLSVQPEK 0.002
38 QGRAFRGSSV 0.100 59 QEVFGGGVGD 0.002
44 GSSVHQKLVN 0.100 78 FRNSETSASE 0.002
37 EQGRAFRGSS 0.100 26 GECGQTFRLK 0.001
76 ISFRNSETSA 0.100 7 YDKSLSVQPE 0.001
SLSVQPEKRT 0.100 21 LRDENGECGQ 0.001
66 VGDIVGRDLS 0.100 5 QEYDKSLSVQ 0.001
75 SISFRNSETS 0.100 81 SETSASEEEK 0.001
31 TFRLKEEQGR 0.060 39 GRAFRGSSVH 0.001
41 AFRGSSVHQK 0.050 55 PRETQEVFGG 0.000
~
72 RDLSISFRNS 0.036 15 PEKRTGLRDE 0.000
9 KSLSVQPEKR 0.033
22 RDENGECGQT 0.030
50 KLVNDPRETQ 0.030
79 RNSETSASEE 0.026
18 RTGLRDENGE 0.024
64 GGVGDIVGRD 0.021
40 RAFRGSSVHQ 0.020
36 EEQGRAFRGS 0.018
13 VQPEKRTGLR 0.018
51 ~ LVNDPRETQE0.018
28 CGQTFRLKEE 0.017
80 NSETSASEEE 0.017
49 QKLVNDPRET 0.017
58 TQEVFGGGVG 0.015 ~ ~
177
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table XIV v.5-A24-lOmers: 213P1F11 I
Pos 1234567890 Score
LALILRVTKA 0.165 Portion
of
2 GARLALILRV 0.120 SEQ
ID
4 RLALILRVTK 0 NO:
024 I1;
. h
t
rt
3 ARLALILRVT 0.021 eac
s
a
position
is
6 ALILRVTKAR 0.021 specified,
RVTKAREGSE 0.020 the
length
7 LILRVTKARE 0.015 of each
9 LRVTKAREGS 0.015 Peptide
is
8 ILRVTKAREG 0 10 amino
011
. the
acids
1 SGARLALILR 0.010 ,
end
position
for
each
peptide
is
the
start
position
plus
nine
Table XIV v.6-A24-lOmers: 213P1F11
Pos 1234567890 Score
9 AMNNKNCQAL 7.200 Portion
of
6 LFEAMNNKNC 0.750 SEQ
ID
2 KLENLFEAMN 0.300 N0:13;
t
h
t
5 NLFEANINNKN0.158 ar
s
eac
position
is
8 EANIhINKNCQA0.150 specified,
1 VKLENLFEAM 0.130 the
length
4 ENLFEAMNNK 0.018 of each
3 LENLFEAMNN 0.015 Peptide
is
10 MNNKNCQALR 0 10 amino
015
. the
acids
7 FEAMNNKNCQ 0.001 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
178
SUBSTITUTE SHEET (RULE 26)
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WO 03/085121 PCT/US02/10220
TableXV v.l-B7-9mers:13P1F11 TableXV v.l-B7-9mers:13P1F11
2 2
Pos 123456789 Score Po's123456789 Score
$7 MAHGREGFL 12.000 Portion 143 VGGDEIVMV 0.200 Portion
of of
223 KARKTNPEI 12.000 SEQ 201 HILELLTEV 0.200 SEQ
ID ID
13 DMSGARLAL 6.000 N0:3; 138 DPGETVGGD 0.200 N0:3;
h
t
rt
233 STLRKRLYL 6.000 each 141 ETVGGDEIV 0.200 eac
start s
osition a
is position
is
231 IQSTLRKRL 6.000 p 71 AIDSREDPV 0.180 specified,
specified,
142 TVGGDEIVM 5.000 the 24 CVTKAREGS O.1S0 the
length length
112 LNNKNCQAL 4.000 of 148 IVMVIKDSP O.1S0 of
each each
227 TNPEIQSTL 4.000 Peptide 4g ESTMKRDPT O.1S0 Peptide
is is
9 9
210 TRRMAEAEL 4.000 id 97 GEDGEMVKL 0.120 acidsnthe
nth
183 KGSCFIQTL 4.000 ac 144 GGDEIVMVI 0.120 end
s
e
end
77 DPVSCAFW 4.000 position 11 KYDMSGARL 0.120 position
for for
1S8 TIPTYTDAL 4.000 each 64 ELEKFQQAI 0.120 each
1S SGARLALIL 4.000 Peptide 199 KGHILELLT 0.100 peptide
is is
the the
19S FTKRKGHIL 4.000 start 80 SCAFWLMA 0.100 start
iti
position pos
S7 TAEQFQEEL 3.600 lus 34 EDLDALEHM 0.100 on
ei plus
ht eight
38 ALEHMFRQL 3.600 p 89 HGREGFLKG 0.100
g
16 GARLALILC 3.000 234 TLRKRLYLQ 0.100
13S EQRDPGETV 3.000 , 4S QLRFESTMK 0.100
3 NPRSLEEEK 2.000 188 IQTLVDVFT 0.100
1SS SPQTIPTYT 2.000 167 HVYSTVEGY 0.100
78 PVSCAFWL 2.000 18 RLALILCVT 0.100
123 KPKVYIIQA 2.000 226 KTNPEIQST 0.100
31 GSEEDLDAL 1.200 209 VTRRMAEAE 0.100
121 RAKPKWII 1.200 1S7 QTIPTYTDA 0.100
100 GEMVKLENL 1.200 169 YSTVEGYIA 0.100
~
104 KLENLFEAL 1.200 30 EGSEEDLDA 0.100
94 FLKGEDGEM 1.000 193 DVFTKRKGH 0.075
73 DSREDPVSC 1.000 177 AYRHDQKGS 0.060
79 VSCAFWLM 1.000 120 LRAKPKVYI 0.060
44 RQLRFESTM 1.000 211 RRMAEAELV 0.060
20S LLTEVTRRM 1.000 17 ARLALILCV 0.060
118 QALRAKPKV 0.600 228 NPEIQSTLR 0.060
164 DALHWSTV 0.600 218 LVQEGKARK O.OSO
119 ALRAKPKVY 0.600 12S KVYIIQACR O.OSO
208 EVTRRMAEA O.SOO 102 MVKLENLFE O.OSO
1S0 MVIKDSPQT O.S00 84 WLMAHGRE O.OSO
197 KRKGHILEL 0.400 83 FVVLMAHGR O.OSO
198 RKGHILELL 0.400 70 QAIDSREDP 0.045
14 MSGARLALI 0.400 206 LTEVTRRMA 0.045
1S1 VIKDSPQTI 0.400 140 GETVGGDEI 0.040
2$ AREGSEEDL 0.360 168 WSTVEGYI 0.040
131 ACRGEQRDP 0.300 194 VFTKRKGHI 0.040
27 KAREGSEED 0.300 180 HDQKGSCFI 0.040
111 ALNNKNCQA 0.300 86 LMAHGREGF 0.030
20 ALILCVTKA 0.300 19 LALILCVTK 0.030
6 SLEEEKYDM 0.300 , 37 DALEHMFRQ 0.030
SO STMKRDPTA 0.300 12 YDMSGARLA 0.030
SS DPTAEQFQE 0.200 16S ALHVYSTVE 0.030
184 GSCFIQTLV 0.200 8S VLMAHGREG 0.030
iS9 IPTYTDALH 0.200 216 AELVQEGKA 0.030
179
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXV v.2-B7-9mers TableXV v.2-B7-
9mers
Pos 123456789 Score Pos 123456789 Score
8 GPTPFQDPL 80.000 Portion 16 LYLPSEAPP 0.001 Portion
of of
TPFQDPLYL 80.000 SEQ 2g LWNSQDTSP 0.001 SEQ
ID ID
38 DMIRKAHAL 4.000 NO: 19 PSEAPPNPP 0.000 NO:
S; S;
h
t
t
44 HALSRPWWM 3.000 each 36 PTDMIRKAH 0.000 s
start ar
osition eac
is position
is
14 DPLYLPSEA 2.000 p 11 PFQDPLYLP 0.000 specified,
specified,
35 SPTDMIRKA 2.000 the 49 PWWMCSRRG 0.000 the
length length
22 APPNPPLWN 1.800 of each of
each
SEAPPNPPL 0.600 Peptide peptide
is is
9 9
45 ALSRPWWMC 0.450 amino amino
id acids
th the
52 MCSRRGKDI 0.400 ac ,
s, end
e
end
31 SQDTSPTDM 0.300 position position
for for
48 RPWWMCSRR 0.200 each each
46 LSRPWWMCS 0.200 Peptide peptide
is is
53 CSRRGKDIS 0.200 the the
start start
iti
39 MIRKAHALS 0.200 Position pos
lus on
eight plus
eight
NPPLWNSQD ' 0.200p
'
26 PPLWNSQDT 0.200
18 LPSEAPPNP 0.200
29 WNSQDTSPT 0.100
42 KAHALSRPW 0.060
32 QDTSPTDMI 0.060
23 PPNPPLWNS 0.060
21 EAPPNPPLW 0.060
1 HVYSTVEGP 0.050
37 TDMIRKAHA 0.030
5 TVEGPTPFQ 0.023
54 SRRGKDISW 0.020
56 RGKDISWNF 0.020
4 STVEGPTPF 0.020
17 YLPSEAPPN 0.020
7 EGPTPFQDP O.O15
51 WMCSRRGKD 0.015
33 DTSPTDMIR 0.010
2 VYSTVEGPT O.O10
34 TSPTDMIRIC 0.010
NSQDTSPTD O.O10
3 YSTVEGPTP 0.010
43 AHALSRPWW 0.009
12 FQDPLYLPS 0.006
50 WWMCSRRGK 0.005
9 PTPFQDPLY 0.002
55 RRGKDISWN 0.002
27 PLWNSQDTS 0.002
15 PLYLPSEAP 0.002
41 RKAZiALSRP 0.001
6 VEGPTPFQD 0.001
13 QDPLYLPSE 0.001
IRKAHALSR 0.001
47 SRPWWMCSR 0.001
24 PNPPLWNSQ 0.001
180
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XV v.3-B7-9mers: 213P1F11
Pos 123456789 Score
TLPSPFPYL 6.000 Portion
of
3 IQACRGATL 4.000 SEQ
ID
5 ACRGATLPS 0.600 N0:7;
each
12 PSPFPYLSL 0.600 start
position
is
11 LPSPFPYLS 0.400 specified,
2 IIQACRGAT 0.150 the
length
1 YI IQACRGA 0.100 of each
9 ATLPSPFPY 0.060 Peptide
is
9
8 GATLPSPFP 0.045 amino
acids
the
4 QACRGATLP 0.030 ,
end
7 RGATLPSPF 0.020 position
for
6 CRGATLPSP 0.001 each
peptide
is
the
start
position
lus
ei
ht
181
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXV v.4-B7-9mers:213P1F11 TableXV v.4-B7-9mers:213P1F11
Pos 123456789 Score Pos 123456789 Score
13 VQPEKRTGL 6.000 Portion 64 GGVGDIVGR. 0.010 Portion
of of
3 KCQEYDKSL 4.000 SEQ 1$ RTGLRDENG 0.010 SEQ
ID ID
54 DPRETQEVF 4.000 No' 16 EKRTGLRDE 0.010
~'
43 RGSSVHQKL 4.000 each ' 48 HQKLVNDPR 0.010 each
start start
osition
is
p
66 VGDIVGRDL 1.200 p 29 ' 0.010 osition
specified GQTFRLKEE is
s
ecified
, p
20 GLRDENGEC 1.000 the 79 RNSETSASE 0.010 ,
length the
length
85 ASEEEKYDM 0.900 of 19 TGLRDENGE 0.010 of each
each
26 GECGQTFRL 0.400 Peptide 31 TFRLKEEQG 0.010 Peptide
is is
9 9
68 DIVGRDLSI 0.400 amino 4 CQEYDKSLS 0.006 amino
id
th
ac acids
38 QGRAFRGSS 0.300 s 67 GDIVGRDLS 0.003 the
e d
end
62 FGGGVGDIV 0.200 position 17 KRTGLRDEN 0.003 en
for position
for
70 VGRDLSISF 0.200 each 52 VNDPRETQE 0.003 each
44 GSSVHQKLV 0.200 peptide 25 NGECGQTFR 0.003 Peptide
is is
the the
11 LSVQPEKRT 0.150 start 80 NSETSASEE 0.003 start
51 LVNDPRETQ 0.113 p ositi~n 2 GKCQEYDKS 0.002 p
lus l
ei
ht
77 SFRNSETSA 0.100 72 RDLSISFRN 0.002 p g
us
ei ht
69 IVGRDLSIS 0.100 78 FRNSETSAS 0.002
75 SISFRNSET 0.100 59 QEVFGGGVG 0.001
33 RLKEEQGRA 0.100 47 VHQKLVNDP 0.001
50 KLVNDPRET 0.100 ' 42 FRGSSVHQK 0.001
14 QPEKRTGLR 0.060 81 SETSASEEE 0.001
58 TQEVFGGGV 0.060 49 QKLVNDPRE 0.001
60 EVFGGGVGD 0.050 36 EEQGRAFRG 0.001
12 SVQPEKRTG O.O50 8 DKSLSVQPE 0.001
46 SVHQKLVND 0.050 32 FRLKEEQGR 0.001
65 GVGDIVGRD 0.050 7 YDKSL$VQP 0.001
61 VFGGGVGDI 0.040 56 RETQEVFGG 0.001
84 SASEEEKYD 0.030 34 LKEEQGRAF 0.001
41 AFRGSSVHQ 0.030 86 SEEEKYDMS 0.001
40 RAFRGSSVH 0.030 35 KEEQGRAFR 0.000
83 TSASEEEKY 0.020 6 EYDKSLSVQ 0.000
37 EQGRAFRGS 0.020 21 LRDENGECG 0.000
73 DLSISFRNS 0.020 71 GRDLSISFR 0.000
24 ENGECGQTF 0.020 22 RDENGECGQ 0.000
39 GRAFRGSSV 0.020 15 PEKRTGLRD 0.000
53 NDPRETQEV 0.020 55 PRETQEVFG 0.000
QEYDKSLSV 0.020
45 SsvHQKLVN 0.020
76 ISFRNSETS 0.020
74 LSISFRNSE 0.015
28 CGQTFRLKE 0.015
27 ECGQTFRLK 0.010
9 KSLSVQPEK O.O10
23 DENGECGQT O.O10
30 QTFRLKEEQ 0.010
l0 ~ SLSVQPEKRO.O10
1 MGKCQEYDK O.O10
63 GGGVGDIVG 0.010
82 ETSASEEEK 0.010
7 ETQEVFGGG 0.010
~
182
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table XV v.5-B7-9mers: 213P1F11 I
Pos123456789 Score
1 GARLALILR 0.300 Portion
of
ALILRVTKA 0.300 SEQ
ID
9 RVTKAREGS 0.150 NO:11;
7 ILRVTKARE 0 each
100 start
. osition
is
3 RLALILRVT 0.100 p
specified
,
2 ARLALILRV 0.060 the
length
4 LALILRVTK 0.045 of each
6 LILRVTKAR 0.010 Peptide
is
9
8 LRVTKAREG 0 amino
001
. acids,
the
end
position
for
each
peptide
is
the
start
position
I lus
ei
ht
Table XV v.6-B7-9mers: 213PiF11
Pos123456789 Score
9 MNNKNCQAL 4.000 Portion
of
8 AMNNKNCQA 0.300 SEQ
ID
1 KLENLFEAM 0.300 N0:13;
7 EAMNNKNCQ 0 each
090 start
. osition
is
3 ENLFEAMNN 0.020 p
specified,
6 FEAMNNKNC 0.010 the
length
4 NLFEANIhTNK 0.010 of each
2 LENLFEAMN 0.002 Peptide
is
9
5 LFEAMNNKN 0.001 a~no
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
183
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVI v.l-B7-lOmers: 213P1F11
TableXVI v.l-B7-lOmers: 213P1F11
Pos 1234567890 Score Pos 1234567890 Score
27 KAREGSEEDL 120.000 Portion 138 DPGETVGGDE 0.200 Portion
of of
77 DPVSCAFWL 80.000 SEQ SS DPTAEQFQEE 0.200 SEQ
ID ID
209 VTRRMAEAEL 40.000 N0:3; 210 TRRMAEAELV 0.200 N0:3;
119 ALRAKPKVYI 18.000 each 117 CQALRAKPKV 0.200 each
start start
osition
is
p position
111 ALNNKNCQAL 12.000 specified 205 LLTEVTRRMA 0.150 is
s
ecified
, p
37 DALEHMFRQL 12.000 the 148 IVMVIKDSPQ 0.150 ,
length the
length
16 GARLALILCV 6.000 of 135 EQRDPGETVG 0.100 of each
each
232 QSTLRKRLYL 6.000 peptide 42 MFRQLRFEST 0.100 Peptide
is is
230 EIQSTLRKRL 6.000 10 7g VSCAFWLMA 0.100 10 amino
amino
id
h
ac acids,
157 QTIPTYTDAL 4.000 s t 89 HGREGFLKGE 0.100 the
e d
end
3 NPRSLEEEKY 4.000 position 113 NNKNCQALRA 0.100 en
for position
for
226 KTNPEIQSTL 4.000 each 149 VMVIKDSPQT 0.100 each
196 TKRKGHILEL 4.000 peptide 181 DQKGSCFIQT 0.100 Peptide
is is
the the
159 IPTYTDALHV 4.000 start 93 GFLKGEDGEM 0.100 start
30 EGSEEDLDAL 4.000 Position 43 FRQLRFESTM 0.100 Position
lus
nine
p
14 MSGARLALIL 4.000 p 15 SGARLALILC 0.100 lus
nine
86 LMAHGREGFL 4.000 45 QLRFESTMKR 0.100
193 DVFTKRKGHI 2.000 187 FIQTLVDVFT 0.100
167 HWSTVEGYI 2.000 201 HILELLTEVT 0.100
150 MVIKDSPQTI 2.000 154 DSPQTIPTYT 0.100
123 KPKWIIQAC 2.000 49 ESTMKRDPTA 0.100
12 YDMSGARLAL 1.800 85 VLMAHGREGF 0.090
73 DSREDPVSCA 1.500 215 EAELVQEGKA 0.090
96 KGEDGEMVKL 1.200 228 NPEIQSTLRK 0.060
99 DGEMVKLENL 1.200 176 IAYRHDQKGS 0.060
RSLEEEKYDM 1.000 118 QALRAKPKVY 0.060
204 ELLTEVTRRM 1.000 84 VVLMAHGREG 0.050
142 TVGGDEIVMV 1.000 125 KWIIQACRG 0.050
141 ETVGGDEIVM 1.000 208 EVTRRMAEAE 0.050
70 QAIDSREDPV 0.600 24 CVTKAREGSE 0.050
78 PVSCAFWLM 0.500 83 FWLMAHGRE O.O50
102 MVKLENLFEA 0.500 63 EELEKFQQAI 0.040
218 LVQEGKARKT 0.500 120 LRAKPKWII 0.040
131 ACRGEQRDPG 0.450 222 GKARKTNPEI 0.040
EKYDMSGARL 0.400 130 QACRGEQRDP 0.030
13 DMSGARLALI 0.400 134 GEQRDPGETV 0.030
194 VFTKRKGHIL 0.400 23 LCVTKAREGS 0.030
103 VKLENLFEAL 0.400 165 ALHWSTVEG 0.030
182 QKGSCFIQTL 0.400 17 ARLALILCVT 0.030
143 VGGDEIVMVI 0.400 20 ALILCVTKAR 0.030
S6 PTAEQFQEEL 0.400 122 AKPKWIIQA 0.030
197 KRKGHILELL 0.400 81 CAFWLMAHG 0.030
223 KARKTNPEIQ 0.300 50 STMKRDPTAE 0.030
19 LALILCVTKA 0.300 164 DALHWSTVE 0.030
1SS SPQTIPTYTD 0.300 41 HMFRQLRFES 0.030
177 AYRHDQKGSC 0.300 87 MAHGREGFLK 0.030
110 EALNNKNCQA 0.300 121 RAKPKWIIQ 0.030
183 KGSCFIQTLV 0.200 33 EEDLDALEHM 0.030
94 FLKGEDGEMV 0.200 162 YTDALHWST 0.030
185 SCFIQTLVDV 0.200 133 RGEQRDPGET 0.030
184
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVI- v.2-B7-lOmers: 213P1
F11 TableXVI- v.2-B7-lOmers: 213P1F11
Pos 1234567890 Score Pos 1234567890 Score
8 EGPTPFQDPL 4.000 Portion 3 VYSTVEGPTP 0.001 Portion
of of
26 NPPLWNSQDT 2.000 SEQ 16 PLYLPSEAPP 0.001 SEQ
ID ID
23 APPNPPLWNS 1.800 NO: 28 pLWrTSQDTSP0.001 NO:
S; S;
38 TDMIRKAHAL 1.200 each 1 LHVYSTVEGP 0.001 each
start start
osition
is
p position
31 NSQDTSPTDM 1.000 specified 12 PFQDPLYLPS 0.000 is
if
d
, spec
2 HVYSTVEGPT 0.500 the 50 PWWMCSRRGK 0.000 ie
length ,
the
length
45 HALSRPWWMC 0.450 of of each
each
9 GPTPFQDPLY 0.400 Peptide peptide
is is
52 WMCSRRGKDI 0.400 IO 10 amino
amino
id
th
ac acids,
PTPFQDPLYL 0.400 s, the
e d
end
36 SPTDMIRKAH 0.300 position en
for position
for
DPLYLPSEAP 0.300 each each
11 TPFQDPLYLP 0.300 Peptide peptide
is is
44 AHALSRPWWM 0.300 the the
start start
19 LPSEAPPNPP 0.300 Position position
lus
nine
p plus
49 RPWWMCSRRG 0.200 nine
54 CSRRGKDISW 0.200
32 SQDTSPTDMI 0.180
PSEAPPNPPL 0.180
35 TSPTDMIRKA 0.100
40 MIRKAHALSR 0.100
47 LSRPWWMCSR 0.100
43 KAHALSRPWW 0.090
22 EAPPNPPLWN 0.090
46 ALSRPWWMCS 0.060
27 PPLWNSQDTS 0.040
4 YSTVEGPTPF 0.020
55 SRRGKDISWN 0.020
39 DMIRKAHALS 0.020
24 PPNPPLWNSQ 0.020
53 MCSRRGKDIS 0.020
6 TVEGPTPFQD O.O15
5 STVEGPTPFQ O.O15
WNSQDTSPTD O.O10
29 LWNSQDTSPT 0.010
18 YLPSEAPPNP O.O10
34 DTSPTDMIRK O.O10
14 QDPLYLPSEA O.O10
51 WWMCSRRGKD 0.005
13 FQDPLYLPSE 0.003
37 PTDMIRKAHA 0.003
56 RRGKDISWNF ~ 0.002
42 RKAHALSRPW 0.002
21 SEAPPNPPLW 0.002
17 LYLPSEAPPN 0.002
7 VEGPTPFQDP 0.002
48 SRPWWMCSRR 0.001
25 PNPPLWNSQD 0.001
33 QDTSPTDMIR 0.001
41 IRKAHALSRP 0.001
185
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table XVI v.3-B7-lOmers: 213P1F11 I
Pos1234567890 Score
12 LPSPFPYLSL 120.000Portion
of
ATLPSPFPYL 18.000 SEQ
ID
3 IIQACRGATL 4.000 N0:7;
6 ACRGATLPSP 0.300 each
start
osition
is
2 YIIQACRGAT 0.150 p
specified
,
9 GATLPSPFPY 0.060 the
length
5 QACRGATLPS 0.060 of each
11 TLPSPFPYLS 0.020 Peptide
is
8 RGATLPSPFP 0.015 10 amino
id
h
ac
4 IQACRGATLP 0.010 s, t
e
end
1 VYIIQACRGA 0.010 position
for
7 CRGATLPSPF 0.002 each
peptide
is
the
start
position
lus
nine
186
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVI v.4-B7-lOmers: 213P1F11
TableXVI v.4-B7-lOmers: 213P1F11
Pos 1234567890 Score Pos 1234567890 Score
12 SVQPEKRTGL 30.000 Portion 64 GGVGDIVGRD 0.010 Portion
of of
65 GVGDIVGRDL 20.000 SEQ 29 GQTFRLKEEQ 0.010 SEQ
ID ID
84 SASEEEKYDM 3,000 NO' 48 HQKLVNDPRE 0.010 NO:
~' 9;
60 EVFGGGVGDI 2.000 each 45 SSVHQKLVND 0.010 each
start start
osition
is
p position
54 DPRETQEVFG 2.000 specified 32 FRLKEEQGRA 0.010 is
s
ecified
, p
38 QGRAFRGSSV 2.000 the 63 GGGVGDIVGR 0,010 ,
length the
length
25 NGECGQTFRL 1.200 of . 18 RTGLRDENGE 0.010 of each
each
2 GKCQEYDKSL 0.400 Peptide 79 RNSETSASEE 0.010 Peptide
is is
42 FRGSSVHQKL 0.400 10 49 Q~,~pRET 0.010 10 amino
amino
id
th
ac acids,
43 RGSSVHQKLV 0.200 s 66 VGDIVGRDLS 0.009 the
e d
end
57 ETQEVFGGGV 0.200 position 80 NSETSASEEE 0.003 en
for position
for
SLSVQPEKRT 0.150 each 58 TQEVFGGGVG 0.003 each
74 LSISFRNSET 0.100 Peptide 22 RDENGECGQT 0.003 peptide
is is
the the
69 IVGRDLSISF 0.100 start 23 DENGECGQTF 0.002 start
i
i
76 ISFRNSETSA 0.100 os 53 NDPRETQEVF 0.002 Psition
t l
on
P
lus
nine
p
70 VGRDLSISFR 0.100 P 72 RDLSISFRNS 0.002 usnine
19 TGLRDENGEC 0.100 36 EEQGRAFRGS 0.002
GLRDENGECG 0.100 81 SETSASEEEK 0.001
14 QPEKRTGLRD 0.060 39 GRAFRGSSVH 0.001
52 VNDPRETQEV 0.060 7 YDKSLSVQPE 0.001
4 CQEYDKSLSV 0.060 26 GECGQTFRLK 0.001
46 SVHQKLVNDP 0.050 5 QEYDKSLSVQ 0.001
51 LVNDPRETQE 0.050 47 VHQKLVNDPR 0.001
67 GDIVGRDLSI 0.040 $ DKSLSVQPEK 0.001
41 AFRGSSVHQK 0.030 56 RETQEVFGGG 0.001
37 EQGRAFRGSS 0.030 7$ FRNSETSASE 0.001
40 RAFRGSSVHQ 0.030 17 KRTGLRDENG 0.001
16 EKRTGLRDEN 0.030 59 QEVFGGGVGD 0.001
50 KLVNDPRETQ 0.023 71 GRDLSISFRN 0.00
1
33 RLKEEQGRAF 0.020 34 LKEEQGRAFR _
0.000
77 SFRNSETSAS 0.020 21 LRDENGECGQ 0.000
44 GSSVHQKLVN 0.020 35 KEEQGRAFRG
75 SISFRNSETS 0.020 6 EYDKSLSVQP 0.000
68 DIVGRDLSIS 0.020 86 SEEEKYDMSG 0.000
3 KCQEYDKSLS 0.020 15 PEKRTGLRDE 0.000
82 ETSASEEEKY 0.020 $5 PRETQEVFGG 0.000
1 MGKCQEYDKS 0.020
61 VFGGGVGDIV 0.020
85 ASEEEKYDMS 0.018
27 ECGQTFRLKE 0.015
73 DLSISFRNSE O.O15
28 CGQTFRLKEE O.O10
24 ENGECGQTFR 0.010 .
9 KSLSVQPEKR 0.010
QTFRLKEEQG 0.010
31 ~ TFRLKEEQGR0.010
13 VQPEKRTGLR 0.010
83 TSASEEEKYD 0.010
11 LSVQPEKRTG 0.010
62 FGGGVGDIVG 0.010
187
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table XVI v.5-B7-lOmers: 213P1F11 I
Pos 1234567890 Score
2 GARLALILRV 6.000 Portion
of
LALILRVTKA 0.300 SEQ
ID
$ ILRVTKAREG 0.100 NO:
11;
t
t
h
RVTKAREGSE 0.050 s
ar
eac
position
is
6 ALILRVTKAR 0.030 specified,
3 ARLALILRVT 0.030 the
length
4 RLALILRVTK 0.015 of
each
7 LILRVTKARE 0.010 Peptide
is
i SGARLALILR 010 10
0 amino
. acids
the
9 LRVTKAREGS 0.003 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
Table XVI v.6-B7-lOmers: 213P1F11
Pos 1234567890 Score
9 AMNNKNCQAL 12.000 Portion
of
$ EAMNNKNCQA 0.900 SEQ
ID
1 VKLENLFEAM 0.100 N0:13;
t
t
h
5 NLFEAMNNKN 0.020 ar
s
eac
position
is
4 ENLFEANIrINK0.010 specified,
10 MNNKNCQALR 0.010 the
length
2 KLENLFEAMN 0.006 of
each
6 LFEAMNNKNC 0.003 Peptide
is
3 LENLFEAMNN 002 10
0 amino
. the
acids
7 FEAMNNKNCQ 0.001 ,
end
position
for
each
peptide
is
the
start
position
lus
nine
188
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXVII v.l-B3$-9mers: 213P1F11 TableXVII v.l-B35-9mers:
213P1F11
Pos 123456789 Score Pos123456789 Score
123 KPKVYIIQA 12.000 Portion 60 QFQEELEKF 0.300 Portion
of of
232 QSTLRKRLY 10.000 SEQ 35 DLDALEHMF 0.300 SEQ
ID ID
79 VSCAFVVLM 10.000 N0:3; 55 DPTAEQFQE 0.300 N0:3;
h
t
rt
154 DSPQTIPTY 10.000 each 210TRRMAEAEL 0.300 eac
start s
osition a
is position
is
94 FLKGEDGEM 9.000 p 5 RSLEEEKYD 0.300 specified,
specified,
121 RAKPKVYII 7.200 the 226KTNPEIQST 0.300 the
length length
223 KARKTNPEI 7.200 ofeach 3g ALEHMFRQL 0.300 of each
119 ALRAKPKVY 6.000 Peptide 30 EGSEEDLDA 0.300 Peptide
is is
9 9
73 DSREDPVSC 4.500 id 144GGDEIVMVI 0.240 ac dsnthe
h
31 GSEEDLDAL 4.500 ac 141ETVGGDEIV 0.200 end
s t
e
end
205 LLTEVTRRM 4.000 position 199KGHILELLT 0.200 position
for for
77 DPVSCAFVV 4.000 each 198RKGHILELL 0.200 each
170 STVEGYIAY 4.000 Peptide 1g RLALILCVT 0.200 Peptide
is is
the the
44 RQLRFESTM 4.000 start 159IPTYTDALH 0.200 start
iti
195 FTKRKGHIL 3.000 Position 150MVIKDSPQT 0.150 on
lus , Pos
eight plus
eight
142 TVGGDEIVM 3.000 p 106ENLFEALNN 0.150
87 MAHGREGFL 3.000 ~64ELEKFQQAI 0.120
151 VIKDSPQTI 2.400 . 24 CVTKAREGS 0.100
227 TNPEIQSTL 2.000 40 EHMFRQLRF 0.100
183 KGSCFIQTL 2.000 111ALNNKNCQA 0.100
167 HVYSTVEGY 2.000 188IQTLVDVFT 0.100
14 MSGARLALI 2.000 147EIVMVIKDS 0.100
155 SPQTIPTYT 2.000 78 PVSCAFVVL 0.100
6 SLEEEKYDM 1.800 208EVTRRMAEA 0.100
135 EQRDPGETV 1.200 100GEMVKLENL 0.100
231 IQSTLRKRL 1.000 20 ALILCVTKA 0.100
233 STLRKRLYL 1.000 157QTIPTYTDA 0.100
101 EMVKLENLF 1.000 80 SCAFVVLMA 0.100
158 TIPTYTDAL 1.000 50 STMKRDPTA 0.100
112 LNNKNCQAL 1.000 89 HGREGFLKG 0.060
187 FIQTLVDVF 1.000 228NPEIQSTLR 0.060
15 SGARLALIL 1.000 70 QAIDSREDP 0.060
184 GSCFIQTLV 1.000 95 LKGEDGEMV 0.060
86 LMAHGREGF 1.000 211RRMAEAELV 0.060
13 DMSGARLAL 1.000 37 DALEHMFRQ 0.060
16 GARLALILC 0.900 11 KYDMSGARL 0.060
57 TAEQFQEEL 0.900 71 AIDSREDPV 0.060
169 YSTVEGYIA 0.750 53 KRDPTAEQF 0.060
118 QALRAKPKV 0.600 179RHDQKGSCF 0.060
3 NPRSLEEEK 0.600 7S REDPVSCAF 0.060
104 KLENLFEAL 0.600 1 MSNPRSLEE 0.050
197 KRKGHILEL 0.600 9 EEKYDMSGA 0.045
164 DALHVYSTV 0.600 177AYRHDQKGS 0.045
143 VGGDEIVMV 0.600 51 TMKRDPTAE 0.045
49 ESTMKRDPT 0.500 45 QLRFESTMK 0.045
161 TYTDALHVY 0.400 . 102MVKLENLFE 0.045
138 DPGETVGGD 0.400 131ACRGEQRDP 0.045
34 EDLDALEHM 0.400 97 GEDGEMVKL 0.045
201 HILELLTEV 0.400 180HDQKGSCFI 0.040
27 KAREGSEED 0.360 168VYSTVEGYI 0.040
189
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVII v.2-B35-9mers: 213P1F11 Table XVII v.2-B35-9mers: 213P1F11
Pos 123456789 Score Pos123456789 Score
TPFQDPLYL 30.000 Portion 6 VEGPTPFQD 0.001 Portion
of of
8 GPTPFQDPL 20.000 SEQ 15 PLYLPSEAP 0.001 SEQ
ID ID
56 RGKDISWNF 12.000 NO: 2g LWNSQDTSP 0.001 NO:
S; S;
44 HALSRPWWM 6.000 each 36 PTDMIRKAH 0.000 each
start start
osition o
is iti
i
p p
35 SPTDMIRKA 4.000 specified, 11 PFQDPLYLP 0.000 s
on
s
specified
,
42 KAHALSRPW 3.000 the 49 PWWMCSRRG 0.000 the
length length
4 STVEGPTPF 2.000 of of each
each
22 APPNPPLWN 2.000 Peptide peptide
is is
9 9
14 DPLYLPSEA 2.000 amino amino
acids id
the h
, ac
46 LSRPWWMCS 1.500 end s, t
e
end
53 CSRRGKDIS 1.500 position position
for for
21 EAPPNPPLW 1.500 each each
38 DMIRKAHAL 1.000 peptide peptide
is is
31 SQDTSPTDM 0.600 the the
start start
i
i
48 RPWWMCSRR 0.400 t position
on lus
Pos ei
plus ht
eight
18 LPSEAPPNP 0.400 p
g
52 MCSRRGKDI ' 0.400
39 MIRKAHALS 0.300
23 PPNPPLWNS 0.200
9 PTPFQDPLY 0.200
26 PPLWNSQDT 0.200
25 NPPLWNSQD 0.200
54 SRRGKDISW 0.150
17 YLPSEAPPN 0.150
29 WNSQDTSPT 0.150
45 ALSRPWWMC 0.100
SEAPPNPPL 0.100
NSQDTSPTD 0.100
34 TSPTDMIRK 0.075
3 YSTVEGPTP 0.075
43 AHALSRPWW 0.050
32 QDTSPTDMI 0.040
55 RRGKDISWN 0.030
12 FQDPLYLPS 0.030
33 DTSPTDMIR 0.010
27 PLWNSQDTS O.O10
37 TDMIRKAHA O.O10
2 VYSTVEGPT O.O10
51 WMCSRRGKD O.O10
1 HVYSTVEGP 0.010
7 EGPTPFQDP 0.010
IRKAHALSR 0.003
5 TVEGPTPFQ 0.003
41 RFCAHALSRP 0.002
19 PSEAPPNPP 0.002
SO WWMCSRRGK 0.001
24 PNPPLWNSQ 0.001
13 QDPLYLPSE 0.001
47 SRPWWMCSR 0.001
16 LYLPSEAPP 0.001
1
190
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table XVII v.3-B35-9mers I
Pos 123456789 Score
11 LPSPFPYLS 2.000 Portion
of
9 ATLPSPFPY 2.000 SEQ
ID
7 RGATLPSPF 2.000 N0:7;
TLPSPFPYL 1.000 each
start
osition
is
3 IQACRGATL 1.000 p
specified,
12 PSPFPYLSL 0.500 the
length
5 ACRGATLPS 0.300 of
each
1 YIIQACRGA 0.100 Peptide
is
9
2 IIQACRGAT 0.100 amino
id
th
ac
4 QACRGATLP 0.030 s,
e
end
8 GATLPSPFP 0.030 position
for
6 CRGATLPSP 0.001 each
peptide
is
the
start
position
lus
ei
ht
191
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TableXVII v.4-B35-9mers TableXVII v.4-B35-9mers
Pos 123456789 Score Pos 123456789 Score
54 DPRETQEVF 120.000Portion 64 GGVGDIVGR 0.015 Portion
of of
83 TSASEEEKY 15.000 SEQ 78 FRNSETSAS 0.015 SEQ
ID ID
85 ASEEEKYDM 9.000 NO' 2 GKCQEYDKS 0.015 NO:
9' 9;
h
t
t
70 VGRDLSISF 6.000 each 46 SVHQKLVND 0.010 ar
start eac
osition s
is position
is
3 KCQEYDKSL 4.000 p 28 CGQTFRLKE 0.010 specified,
specified,
13 VQPEKRTGL 2.000 the 29 GQTFRLKEE 0.010 the
length length
43 RGSSVHQKL 2.000 of each 30 QTFRLKEEQ 0.010 of
each
24 ENGECGQTF 2.000 Peptide 60 EVFGGGVGD 0.010 Peptide
is is
9 9
33 RLKEEQGRA 1.800 a~no g2 ETSASEEEK 0.010 amino
id acids
th the
44 GSSVHQKLV 1.000 ac 10 SLSVQPEKR 0.010 ,
s, end
e
end
20 GLRDENGEC 0.900 position 27 ECGQTFRLK 0.010 position
for for
76 ISFRNSETS 0.500 each 63 GGGVGDIVG 0.010 each
45 SSVHQKLVN 0.500 Peptide 67 GDIVGRDLS 0.010 peptide
is is
the the
11 LSVQPEKRT 0.500 start 86 SEEEKYDMS 0.006 start
iti
68 DIVGRDLSI 0.400 Position 16 EKRTGLRDE 0.003 on
lus Pos
ei plus
ht eight
38 QGRAFRGSS 0.300 p 7 YDKSLSVQP 0.003
g
66 VGDIVGRDL 0.300 56 RETQEVFGG 0.003
50 KLVNDPRET 0.300 41 AFRGSSVHQ 0.003
62 FGGGVGDIV 0.200 0 31 TFRLKEEQG 0.003
69 IVGRDLSIS 0.150 52 VNDPRETQE 0.003
75 SISFRNSET 0.100 25 NGECGQTFR 0.003
37 EQGRAFRGS 0.100 32 FRLKEEQGR 0.002
73 DLSISFRNS 0.100 36 EEQGRAFRG 0.001
9 KSLSVQPEK 0.100 81 SETSASEEE 0.001
26 GECGQTFRL 0.100 8 DKSLSVQPE 0.001
84 SASEEEKYD 0.090 49 QKLVNDPRE 0.001
40 RAFRGSSVH 0.060 47 VHQKLVNDP 0.001
14 QPEKRTGLR 0.060 42 FRGSSVHQK 0.001
34 LKEEQGRAF 0.060 59 QEVFGGGVG 0.001
58 TQEVFGGGV 0.060 22 RDENGECGQ 0.001
74 LSISFRNSE 0.050 21 LRDENGECG 0.001
4 CQEYDKSLS 0.045 35 KEEQGRAFR 0.001
79 RNSETSASE 0.040 15 PEKRTGLRD 0.000
61 VFGGGVGDI 0.040 71 GRDLSISFR 0.000
QEYDKSLSV 0.040 6 EYDKSLSVQ 0.000
48 FIQKLVNDPR 0.030 55 PRETQEVFG 0.000
77 SFRNSETSA 0.030
53 NDPRETQEV 0.030
1 MGKCQEYDK 0.030
18 RTGLRDENG 0.020
51 LVNDPRETQ 0.020
39 GRAFRGSSV .020
0
72 RDLSISFRN _
0.020
57 ETQEVFGGG 0.020
17 KRTGLRDEN 0.020
65 ~ GVGDIVGRD0.020
23 DENGECGQT 0.015
19 TGLRDENGE 0.015
12 SVQPEKRTG 0.015
80 NSETSASEE 0.015
192
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVII v.5-B35-9mers: 213P1F11
Pos 123456789 Score
9 RVTKAREGS 0.200 Portion
of
3 RLALILRVT 0.200 SEQ
ID
ALILRVTKA 0.100 NO:11;
1 GARLALILR 0 each
090 start
. osition
is
7 ILRVTKARE 0.030 p
specified,
4 LALILRVTK 0.030 the
. length
2 ARLALILRV 0.020 of
each
6 LILRVTKAR 0.010 Peptide
is
9
8 LRVTKAREG 0.001 amino
acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
(Table XVII v.6-B35-9mers: 213P1F11 I
Pos 123456789 Score
1 KLENLFEAM 1.200 Portion
of
9 MNNKNCQAL 1.000 SECT
ID
3 ENLFEAMNN 0.150 N0:13;
h
8 ANIr7NKNCQA0 start
100 eac
. position
is
7 EAMNNKNCQ 0.030 specified,
4 NLFEAMNNK 0.020 the
length
6 FEAMNNKNC 0.010 of
each
2 LENLFEAMN 0.010 Peptide
is
9
5 LFEAMNNKN 0 a~no
003
. acids,
the
end
position
for
each
peptide
is
the
start
position
lus
ei
ht
193
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVIII v.l-B35-lOmers: 213P1F11
TableXVIII v.l-B3$-lOmers: 1
213P1F1
Pos 1234567890 Score Pos1234567890 Score
3 NPRSLEEEKY 180.000 Portion 181DQKGSCFIQT 0.300 Portion
of of
RSLEEEKYDM 60.000 SEQ 93 GFLKGEDGEM 0.300 SEQ
ID ID
27 KAREGSEEDL 36.000 NO:3; 160PTYTDALHW 0.200 NO.3,
77 DPVSCAFWL 20.000 each 55 DPTAEQFQEE 0.200 each
start start
osition
is
p position
123 KPKVYIIQAC 12.000 specified 10 EKYDMSGARL 0.200 is
s
ecified
, p
169 YSTVEGYIAY 10.000 the 34 EDLDALEHMF 0.200 ,
length the
length
37 DALEHMFRQL 6.000 of 117CQALRATCPKV 0.200 of each
each
118 QALRAKPKVY 6.000 Peptide 155SPQTIPTYTD 0.200 Peptide
is is
159 IPTYTDALHV 6.000 10 $6 PTAEQFQEEL 0.200 10 amino
amino
id
th
ac acids,
232 QSTLRKRLYL 5.000 s, 205LLTEVTRRMA 0.200 the
e d
end
14 MSGARLALIL 5.000 position 78 PVSCAFVVLM 0.200 en
for position
for
30 EGSEEDLDAL 3.000 each 218LVQEGKARKT 0.200 each
209 VTRRMAEAEL 3.000 Peptide 201HILELLTEVT 0.200 Peptide
is is
the the
141 ETVGGDEIVM 3.000 start 107NLFEALNNKN 0.200 start
73 DSREDPVSCA 3.000 osition 185SCFIQTLVDV 0.200 Position
P
lus
nine
p
204 ELLTEVTRRM 2.000 p 166LHVYSTVEGY 0.200 lus
nine
231 IQSTLRKRLY 2.000 103VKLENLFEAL 0.200
226 KTNPEIQSTL 2.000 178YRHDQKGSCF 0.200
96 KGEDGEMVKL 1.800 43 FRQLRFESTM 0.200
16 GARLALILCV 1.800 121RAKPKWIIQ 0.180
59 EQFQEELEKF 1.500 223KARKTNPEIQ 0.180
119 ALRAKPKWI 1.200 149VMVIKDSPQT 0.150
70 QAIDSREDPV 1.200 182QKGSCFIQTL 0.100
1S7 QTIPTYTDAL 1.000 187FIQTLVDVFT 0.100
85 VLMAHGREGF 1.000 186CFIQTLVDVF 0.100
86 LMAHGREGFL 1.000 12 YDMSGARLAL 0.100
111 ALNNKNCQAL 1.000 194VFTKRKGHIL 0.100
230 EIQSTLRKRL 1.000 39 LEHMFRQLRF 0.100
94 FLKGEDGEMV 0.900 100GEMVKLENLF 0.100
143 VGGDEIVMVI 0.800 41 HMFRQLRFES 0.100
197 KRKGHILELL 0.600 15 SGARLALILC 0.100
52 MKRDPTAEQF 0.600 23 LCVTKAREGS 0.100
154 DSPQTIPTYT 0.500 210TRRMAEAELV 0.090
79 VSCAFWLMA 0.500 215EAELVQEGKA 0.090
49 ESTMKRDPTA 0.500 133RGEQRDPGET 0.090
176 IAYRHDQKGS 0.450 63 EELEKFQQAI 0.080
193 DVFTKRKGHI 0.400 228NPEIQSTLRK 0.060
167 HWSTVEGYI 0.400 212RMAEAELVQE 0.060
183 KGSCFIQTLV 0.400 89 HGREGFLKGE 0.060
13 DMSGARLALI 0.400 135EQRDPGETVG 0.060
153 KDSPQTIPTY 0.400 1S1VIKDSPQTIP 0.060
138 DPGETVGGDE 0.400 33 EEDLDALEHM 0.060
150 MVIKDSPQTI 0.400 74 SREDPVSCAF 0.060
142 WGGDEIVMV 0.300 104KLENLFEALN 0.060
110 EALNNKNCQA 0.300 6 SLEEEKYDMS 0.060
196 TKRKGHILEL 0.300 1 MSNPRSLEEE O.O50
113 NNKNCQALRA 0.300 1 GSCFIQTLVD 0.050
84
19 LALILCVTKA 0.300 _ MAHGREGFLK 0.045
87
102 MVKLENLFEA 0.300 2$ VTKAREGSEE 0.045
DGEMVKLENL 0.300 I 130QACRGEQRDP 0.045
~ ~
194
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVIII v.2-B35-lOmers: 213P1F11 Table XVIII v.2-B35-lOmers: 213P1F11
Pos 1234567890 Score Pos 1234567890 Score
9 GPTPFQDPLY 40.000 Portion 51 WWMCSRRGKD 0.001 Portion
of of
31 NSQDTSPTDM 20.000 SEQ 33 QDTSPTDMIR 0.001 SEQ
ID ID
54 CSRRGKDISW 7.500 N0:5; 2g PLWNSQDTSP 0.001 N0:5;
4 YSTVEGPTPF 5.000 each 16 PLYLPSEAPP 0.001 each
start start
osition i
is i
i
p pos
43 KAHALSRPWW 3.000 specified, 1 LI~VYSTVEGP0.001 t
on
s
specified
,
23 APPNPPLWNS 2.000 the 50 PWWMCSRRGK 0.000 the
length length
26 NPPLWNSQDT 2.000 of of each
each
8 EGPTPFQDPL 1.000 Peptide peptide
is is
35 TSPTDMIRKA 0.500 10 10 amino
amino
id
th
ac acids,
19 LPSEAPPNPP 0.400 s, the
e end
end
52 WMCSRRGKDI 0.400 position position
for for
36 SPTDMIRKAH 0.400 each each
49 RPWWMCSRRG 0.400 Peptide peptide
is is
22 EAPPNPPLWN 0.300 the the
start start
i
i
45 HALSRPWWMC 0.300 t position
on l
Pos i
plus
nine
p
15 DPLYLPSEAP 0.200 us n
ne
56 RRGKDISWNF 0.200
11 TPFQDPLYLP 0.200
27 PPLWNSQDTS 0.200
44 AHALSRPWWM 0.200
PTPFQDPLYL 0.150
PSEAPPNPPL 0.150
47 LSRPWWMCSR 0.150
32 SQDTSPTDMI 0.120
38 TDMIRKAHAL 0.100
2 HVYSTVEGPT 0.100
46 ALSRPWWMCS 0.100
53 MCSRRGKDIS 0.100
39 DMIRKAHALS 0.100
42 RKAHALSRPW 0.100
21 SEAPPNPPLW 0.050
55 SRRGKDISWN 0.045
40 MIRKAHALSR 0.030
5 STVEGPTPFQ 0.020
24 PPNPPLWNSQ 0.020
17 LYLPSEAPPN 0.015
34 DTSPTDMIRK 0.015
29 LWNSQDTSPT 0.015
WNSQDTSPTD O.O10
18 YLPSEAPPNP 0.010
14 QDPLYLPSEA 0.010
41 IRKAHAI,SRP0.003
13 FQDPLYLPSE 0.003
TVEGPTPFQD 0.003
37 PTDMIRKAHA 0.003
12 PFQDPLYLPS 0.002
3 VYSTVEGPTP 0.002
25 PNPPLWNSQD 0.001
7 VEGPTPFQDP 0.001
~8 SRPWWMCSRR 0.001
1
195
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
(Table XVIII v.3-B35-lOmers: 213P1F11 I
Pos1234567890 Score
12 LPSPFPYLSL 20.000 Portion
of
9 GATLPSPFPY 6.000 SEQ
ID
ATLPSPFPYL 1.000 N0:7;
3 IIQACRGATL 1.000 each
start
osition
is
5 QACRGATLPS 0.300 p
specified,
7 CRGATLPSPF 0.100 the
length
11 TLPSPFPYLS 0.100 ofeach
2 YIIQACRGAT 0.100 Peptide
is
6 ACRGATLPSP 0.030 10 amino
id
th
ac
8 RGATLPSPFP 0.020 s,
e
end
4 IQACRGATLP 0.010 position
for
1 VYIIQACRGA 0.010 each
peptide
is
the
start
position
lus
nine
196
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVIII v.4-B35-lOmers: 213P1F11
TableXVIII v.4-B35-lOmers: 1
213P1F1
Pos 1234567890 Score Pos 1234567890 Score
84 SASEEEKYDM 18.000 Portion $0 NSETSASEEE 0.015 Portion
of of
33 RLKEEQGRAF 12.000 SEQ 49 QKLVNDPRET 0.015 SEQ
ID ID
82 ETSASEEEKY 3.000 NO' 32 FRLKEEQGRA 0.015 NO:
9' 9;
65 GVGDIVGRDL 2.000 each each
start 63 GGGVGDIVGR 0.015 start
osition
is
p position
54 DPRETQEVFG 1.200 specified 64 GGVGDIVGRD 0.010 is
s
if
d
, pec
12 SVQPEKRTGL 1.000 the 30 QTFRLKEEQG 0.010 ie
length ,
the
length
69 IVGRDLSISF 1.000 of 73 DLSISFRNSE 0.010 of each
each
38 QGRAFRGSSV 0.600 Peptide 2g CGQTFRLKEE 0.010 Peptide
is is
3 KCQEYDKSLS 0.600 10 29 GQTFRLKEEQ 0.010 10 amino
amino
id
h
ac
74 LSISFRNSET 0.500 s t 46 SVHQKLVNDP 0.010 acie
e dthe
end
76 ISFRNSETSA 0.500 position 62 FGGGVGDIVG 0.010 n
for position
for
44 GSSVHQKLVN 0.500 each 36 EEQGRAFRGS 0.010 each
1 MGKCQEYDKS 0.450 Peptide 27 ECGQTFRLKE 0.010 Peptide
is is
the the
57 ETQEVFGGGV 0.400 start 22 RDENGECGQT 0.009 start
43 RGSSVHQKLV 0.400 Position 31 TFRLKEEQGR 0.005 Position
lus
nine
p
60 EVFGGGVGDI 0.400 P 7 YDKSLSVQPE 0.003 lus
nine
85 ASEEEKYDMS 0.300 41 ~ AFRGSSVHQK0.003
25 NGECGQTFRL 0.300 58 TQEVFGGGVG 0.003
19 TGLRDENGEC 0.150 71 GRDLSISFRN 0.003
68 DIVGRDLSIS 0.150 ~ 56 RETQEVFGGG 0.002
2 GKCQEYDKSL 0.100 17 KRTGLRDENG 0.002
37 EQGRAFRGSS 0.100 5 QEYDKSLSVQ 0.002
75 SISFRNSETS 0.100 47 VHQKLVNDPR 0.001
SLSVQPEKRT 0.100 8 DKSLSVQPEK 0.001
53 NDPRETQEVF 0.100 26 GECGQTFRLK 0.001
9 KSLSVQPEKR 0.100 81 SETSASEEEK 0.001
42 FRGSSVHQKL 0.100 78 FRNSETSASE 0.001
23 DENGECGQTF 0.100 39 GRAFRGSSVH 0.001
52 VNDPRETQEV 0.090 59 QEVFGGGVGD 0.001
83 TSASEEEKYD 0.075 21 LRDENGECGQ 0.001
11 LSVQPEKRTG 0.075 34 LKEEQGRAFR 0.001
GLRDENGECG 0.060 86 SEEEKYDMSG 0.001
40 RAFRGSSVHQ 0.060 35 KEEQGRAFRG 0.001
14 QPEKRTGLRD 0.060 15 PEKRTGLRDE 0.000
70 VGRDLSISFR 0.060 6 EYDKSLSVQP 0.000
4 CQEYDKSLSV 0.060 55 PRETQEVFGG 0.000
45 SSVHQKLVND 0.050
77 SFRNSETSAS 0.045
9 RNSETSASEE 0.040
67 GDIVGRDLSI 0.040
18 RTGLRDENGE 0.030
6 EKRTGLRDEN 0.030
48 HQKLVNDPRE 0.030
66 VGDIVGRDLS 0.030
24 ENGECGQTFR 0.020
61 ~ VFGGGVGDIV0.020
72 RDLSISFRNS 0.020
S LVNDPRETQE 0.020
1
50 KLVNDPRETQ 0.020
13 VQPEKRTGLR 0.020
J
197
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
Table XVIII v.5-B35-lOmers: 213PIF11
Pos 1234567890 Score
2 GARLALILRV 1.800 Portion
of
LALILRVTKA 0.300 SEQ
ID
8 ILRVTKAREG 0.030 NO:11;
4 RLALILRVTK 0 each
020 start
. osition
is
RVTKAREGSE 0.020 p
specified
,
6 ALILRVTKAR 0.010 the
length
7 LILRVTKARE 0.010 of each
3 ARLALILRVT 0.010 peptide
is
9 LRVTKAREGS 0.010 10 amino
id
h
ac
s, t
e
1 SGARLALILR 0.010 end
position
for
each
peptide
is
the
start
position
plus
nine
Table XVIII-v.6-B35-lOmers: 213P1F11
Pos 1234567890 Score
9 AMNNKNCQAL 1.000 Portion
of
1 VKLENLFEAM 0.400 SEQ
ID
8 EAMNNKNCQA 0.300 NO:13;
5 NLFEAMNNKN 0 each
200 start
. osition
is
2 KLENLFEAMN 0.060 p
specified
,
3 LENLFEAMNN 0.015 the
length
4 ENLFEAMNNK 0.010 of each
10 MNNKNCQALR 0.010 Peptide
is
6 LFEAMNNKNC 0.003 10 amino
id
h
ac
s, t
e
7 FEAMNNKNCQ 0.001 end
position
for
each
peptide
is
the
start
position
lus
nine
198
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
TABLE 13P1F11 v. 1: HLA-A*0201amers
XIXA: non
MHC
Class
I
Analysis
of
213P Pos1 2 3 4 5 6 7 score
1 8 9
F
11
(9-mers).
Listed
are
scores
which
correlate 22 I L C V T _K A 14
with R E
the
ligation
strength
to
a
defined
HLA
type 50 S T M K R D P 14
for T A
a
sequence
of
amino
acids.
The
algorithms
"
used g5 V L M A H G R 14
are E G
based
on
the
book
MHC
Ligands
and
"
Peptide 95 L K G E D G E 14
Motifs M V
by
H.G.Rammensee,
J.Bachmann
and 112L N N K N C Q 14
S.Stevanovic. A L
The
probability
of
being
processed 187F I Q T L V D 14
and V F
presented
is
given
in
order
to
predict
T-cell 198R R G H I L E 14
epitopes. L L
210T R R M A E A 14
T E L
bl
XIX
a 227T N P E I Q S 14
e T L
A,
part
1:
MHC
Class
I
nonamer
l
i
f2
ana 11 K Y D M S G A 1
s R L
s
o
13P1F11
v.l
as
1-242
.
3
21
*
3P1F11 19 L A L I L C V 13
v. T K
1:
HLA-A
0201
nonamers
Pos 1 2 3 4 5 6 7 score 2g A R E G S E E 13
8 9 D L
201 H I L E L L T 29 Portion 41 H M F R Q 13
E V of L R F E
SE _
ID
20 A L I L C V T 24 Q 78 P V S C A F V 13
K A V L
each
NO:
3
104 K L E N L F E 22 ; 127Y I I Q A C R 13
A L G E
start
158 T I P T Y T D 22 160P T Y T D A L 13
A L H V
position
is
13 D M S G A R L 21 s 163T D A L H V Y 13
A L ecified S T
p
,
38 A L E H M F R 21 the 165A L H V Y S T 13
Q L length V E
17 A R L A L I L 20 of 190T L V D V F T 13
C V each K R
18 R L A L I L C 20 peptide 204E L L T E 13
V T is V T R R
9
_
71 A I D S R _E D 20 amino 212R M A E A E L 13
P V V Q
acids
the
233 S T L R K R L 2p , 214A E A E L V Q 13
Y L E G
end -
position
118 Q A L R A K P 1 79 V S C A F V V 12
K V g L M
for
each
121 R A K P K V Y 19 tid 80 S C A F V V L 12
I I i M A
pep
e
s
151 V I K D S P Q 19 the 119A L R A K P K 12
T I start V Y
197 K R K G H I L 19 position 12gI I Q A C R G 12
E L E Q
205 L L T E V T R 19 plus 140G E T V G G D 12
R M eight E I
6 S L E E E K Y 18 157Q T I P T Y T 12
D M D A
94 F L K G E D G 18 171T V E G Y I A 12
E M Y R
107 N L F E A L N 18 189Q T L V D V F 12
N R T R
111 A L N N K N C 18 218L V Q E G 12
Q A K A R R
_
143 V G G D E I V 18 231I Q S T L R K 12
M V R L
183 K G S C F I Q 18 27 K A R E G S E 11
T L E D
186 C F I Q T L V 18 35 D L D A L E H 11
D V M F
202 I L E L L _T E 18 74 S R E D P V 11
V T S C A
_
226 K T N P E I Q 18 135E Q R D P G E 11
S T T V
31 G S E E D L D 17 150M V I K D S P 11
A L Q T
97 G E D G E M V 17 170S T V E G Y I 11
K L A Y
,
100 G E M V K L E 17 175Y I A Y R H D 11
N L Q IC
164 D A L H V Y S 17 211R R M A E A E 11
T V L V
15 S G A R L A L 16 230E I Q S T 11
I L L R K R
_
57 T A E Q F _Q E 16 $1 C A F V V L M 10
E L A H
64 E L E K F Q Q 16 142T V G G D E I 10
A I V M
87 M A H G R E G 16 149V M V I K D S 10
F L P Q
195 F T K R K G H 16 167H V Y S T V E 10
I L G Y
223 K A R K T N P 16 180H D Q K G S C 10
E I F I
86 L M A H G R E 15 184G S C F I Q T 10
G F L V
103 V IC L E N L F 15 200G H I L E L L 10
E A T E
120 L R A K P K V 15 216A E L V Q E G 10
Y I K A
141 E T V G G D E 15 12 Y D M S G A R 9
I V L A
144 G G D E I V M 15 16 G A R L A L I 9
V I L C
234 T L R K R _L Y 15 44 R Q L R F E S 9
L Q T M
14 M S G A R L A 14 45 Q L R F E S T 9
L I M K
21 L I L C V T K 14 46 L R F E S T M 9
A R K R
199
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
213P1F11 I: namers 13P1F11 namers
v. HLA-A*0201 v.
no 1:
HLA-A*0201
no
Pos 1 23 4 7 9 score Pos1 3 4 56 7 89 score
5 8 2
6
76 E DP V A V 9 23 L V T KA R EG $
S F C
_C
77 D PV F V 9 32 S E D LD A E 5
S V E L
C
A
123 K PK I A 9 73 D R E D_ V SC 5
V Q S P
Y
I
148 I VM V P 9 82 A V LM G $
I F V A
K H
D
S
153 K DS P I T 9 108L E A LN N KN 5
Q P F
T
162 Y TD A V S 9 116N Q R A K $
L Y C A.L P
H
168 V S T G I 9 161T T D AL H V 5
Y V Y Y Y
E
176 I AY R Q 9 174G I A YR H DQ
H K Y
D G
206 L TE V R 9 178Y H D QK SC $
T M R G
R A
213 M AE A V E 9 193D F T KR K GH $
E Q V
_L
219 V QE GKA_RKT 9 7 LE E E K_ MS 4
YD
2 S NP R E E $ 24 C T K _ E GS 4
S E V A
L R
34 E DL D E $ 26 T R EG S EE 4
A H R
_L M A
51 T MK R T $ 30 E S E ED L DA 4
D A G
P E
52 M KR D A Q $ 36 L A EH M FR 4
P E D L
T
60 Q FQ E E F $ . 69 Q I DS R ED 4
E K Q
L A
125 K VY I A R $ 102M ~KL EN L FE 4
I C V
Q
147 E IV K S $ . 110E L N K CQ 4
M D A N N
V
I
191 L V V K K $ 129I C RG E QR 4
D F R Q
T A
194 V FT K G I $ 131A R G EQ R DP 4
R H C
K
203 L EL L V R $ 136Q P GE T VG 4
T T R
_E D
208 E VT R A A $ 182Q G S CF I QT 4
R E K
M
37 D AL E F Q 7 3 N R S LE E ER 3
H R P
_M
43 F RQ LR_F ES T- 7 5 RS L E E_ KY D 3
E
56 P TA E Q E 7 39 L H M F_ Q LR 3
Q E E R
F
67 K FQ Q D R 7 42 M R Q LR F ES 3
A S F
I
70 Q AI D E P 7 53 K D P TA QF 3
S D R E
R
83 F V L R 7 72 I S R ED P VS 3
V M D
A
H
G
84 V L M G E 7 ~ 7$ R D P VS C F 3
V A R E A
H
89 H GR E L 7 96 K E D GE M V 3
G K G R
_F G
90 G RE G K E 7 126V I I Q C RG 3
F G Y A
L
101 E M K N F 7 152I S P_ T IP 3
V L L It Q
E D
134 G EQ R G T 7 61 F E E LE K FQ 2
D E Q
P
137 R DP G G 7 _
E 65 L K F QQ A ID 2
T E
V
G
146 D EI V I D 7 $$ A R EG F LIC2
M K H
V G
166 L HV G 7 91 R G F L_ ED 2
Y E K
S G
T
V
E
188 I QT L V T 7 9$ E G E MV K LE 2
V F D
D
199 K G I L T 7 109F A N K NC 2
H L L 8 L N
E
217 E I~V Q K R 7 122A P K VY I IQ 2
E A K
G
222 G E 7 132C G E QR D PG 2
IC R
A
R
K
T
N
P
1 M SN P L E 6 145G E I V IR 2
R E D M
S V
25 V TK A G E 6 ~ 156P T I PT Y T 2
R S Q D
_E
63 E EL E Q A 6 159I T Y TD A H 2
K Q P L
F
93 G FL K D E 6 177A H DQ K GS 2
G G Y
_E R
105 L EN L A' N 6 192V V F TK R K 2
F L D G
E
114 N AN C A 6 196T R K GH I LE 2
Q IC
A
L
R
115 K C Q R K 6 229P I Q ST L R 2
N A A E R
L
130 Q AC R Q D 6 48 F S T MK DP 1
G R 8 R
E
138 D PG E~T D 6 49 E T M K D PT 1
V S R
G
G
154 D SP Q P Y 6 58 A Q F QE E LE 1
T T E
I
155 S PQ T T T 6 59 E F Q EE L ER 1
I Y Q
P
169 Y ST Y A 6 6$ F Q ID S RE 1
V I Q A
E
_G
185 S CF I L 6 . 99 D E M _ EN 1
Q V G V K
T D L
209 V TR R E E 6 113N K CQ A LR 1
M A N N
A
200
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 13P1F11
v. v.l:
I: HLA-A1
HLA-A*0201 nonamers
nonamers
Pos1 2 3 45 6 78 score Pos1 2 45 6 78 score
9 3 9
117C Q A LR A KP 1 139P G TV G GD 13
K E E
124P K V YI I QA 1 28 A R GS E _ED 12
C E L
133R G E QR D PG 1 35 D L AL E HM 12
E D F
172V E G YI A R 1 80 S C FV LM 12
Y H A V A
179R H D QK G SC 1 144G G EI V MV 12
F D I
207T E V TR R MA 1 145G D IV M I 12
E E V K
215E A E LV Q EG 1 160P T TD A H 12
K Y L V
220Q E G KR K 1 171T V GY I A_Y 12
A T E R
N
224A R K N _ EI 1 202I L LL T EV 12
T P Q E T
4 P R S LE _ EK -1 215E A LV Q EG 12
_E Y E K
$ E E E K D MS -1 7 L _E EK Y M 11
Y G E D S
62 Q E E LE K Q -1 8 E E KY D MS 11
F Q E G
92 E G F L_ ED -1 11 K MS G 11
K G Y A
G D R
L
106E N L FE _ LN -1 56 P T EQ F QE 11
A N A E
228N P E IQ S TL -1 61 F Q EL E KF 11
R E Q
232Q S T LR K L -1 64 E L K Q QA 11
R Y E F I
40 E H M FR Q LR -2 71 A I SR E DP 11
F D V
47 R F E ST M K -2 90 G R GF L KG 11
R E E
D
55 D P T A Q FQ -2 152I K SP Q TI 11
E E D P
66 E K F QQ A ID -2 I79R H QK SC 11
S D G F
173E G Y IA Y RH -2 185S _C IQ T LV 11
D F D
139P G E TV G GD -4 47 R F ST M K 10
E E R
D
221E G K K N -4 57 T QF Q EE 10
A T P A L
R E
62 Q _E LE K _FQ 10
E Q
13P1F11 nonamers 133R G QR D PG 10
v.l: E E
HLA-A1
Pos1 2 3 45 6 78 score 153K PQ T IP 10
9 D T
S
17 S T V EG Y IA 29 Portion 157Q T PT Y TD 10
Y of I A
232Q S T LR K L 21 SEQ 191L VF T KR 10
R Y ID V K
D
154D S P QT I PT 19 ND: 213M A L VQ 10
Y 3; A E E
each E
99 D G E M K LE 1 start 226K PE I QS 10
V N g _T T
N
119A L R AK P _V 18 Position 141E T G D EI 9
KY is V V
if G
d
162Y _ D A H V 18 ie 13 D M GA R LA 8
T L Y , S L
S spec
th
the
len
206L T E VT R _M 18 g 15 S G L A I $
RA of A L L
each R
4 P _ S LE E _K 17 peptide 50 S T KR D PT 8
R EY is M A
9
136Q R PG E TV 17 amino 79 V S AF V 8
D G C V
L
M
33 E _ D LD A E 16 acids, 114N K CQ A LR 8
E L H the N A
75 _ E D PV S CA 16 end 190T L DV F TK 8
R F position V R
161T Y T DA L H 16 for 195F T K I 8
V each K G L
Y R H
167H Y ST EG 16 Peptide 196T K KG H IL 8
V V Y is R E
t
th
t
38 A L E HM F RQ 15 ar 199K G IL E LL 8
L e s H T
position
233S T L RK LY I5 plus 17 A I LC 7
R L eight _R V
L
A
L
1 M S N PR S LE 14 25 V T AR E GS 7
E K 8
31 G S E ED L DA 14 102M _V LE N LF 7
L K E
32 S _ E DL D AL 14 122A K KV Y II 7
E E P Q
53 K R D PT A EQ 14 146D E VM V IK 7.
F I D
74 S R E DP V SC 14 169Y _S E G YI 7
A T A
V
104K _ E NL F EA 14 182Q K SC F IQ 7
L L G T
219V Q E GK K 14 189Q T VD V FT 7
A T L R
R
228N P E IQ S TL 14 197K G I LE 7
R R H L
K
6 S L E EE K YD 13 209V R A 7
M T M E
R A
E
$9 H/ G R EG LK 13 212R M EA E LV 7
F G A Q
96 K E DG E MV 13 12 Y D SG L 6
G K M A A
_R
97 G E D GE M VK 13 _ 14 M S L AL 6
L G I
A
R
108L F E AL N NK 13 16 G LA IL 6
N A L C
R
201
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.l: HLA-A1 nonamers ~213P1F11 v.l: HLA-A1 nonamers
Pos 1 23 4 56 7 8 score Pos1 2 56 7 89 score
9 3
4
30 E GS E ED L D 6 109F _E N K C 2
A A N N
L
40 E H F RQ L R 6 113N CQ A_LR 2
M F N
R
N
59 E QF Q EE L E 6 118Q A AK P K 2
K L V
R
66 E K Q QA I D 6 127Y I AC R GE 2
F S I
Q
106 E NL F EA N 6 130Q GE Q ~RD 2
L N A
C
R
142 T VG G DE I V 6 131A C EQ R DP 2
M R
G
184 G SC F IQ T L 6 135E Q PG E TV 2
V R
D
200 G HI L EL _LT 6 ~ 143V G EI V MV 2
E G
D
229 P EI Q ST _LR 6 147E I VI _K S 2
R V D
M
20 A _LI L CV _TK 5 155S P IP _TYT 2
A Q
T
49 E ST M K P 5 159I _P TD A LH 2
R T T
D_ Y
58 A _EQ F QE E LE 5 164DALH VY _STV 2
121 R AR P KV _YI 5 174G R H DQ 2
I Y
I
A
Y
123 K _PK V YI I Q 5 175Y I R D QR 2
A A H
Y
216 A EL V QE G K 5 176I A HD Q K 2
A Y G
R
225 R K N PE I Q 5 180H D GS C FI 2
T S Q
K
R SL E EE K Y 4 188I Q VD V FT 2
D T
L
43 F RQ L RF E S 4 198R K IL E LL 2
T G
H
73 D _SR E DP V S 4 204E L EV RR 2
C L _T
T
7$ P VS C AF _V 4 211R R EA LV 2
V M E
L A
88 A H_G R EG _FL 4 214A E LV Q EG
R A
E
165 A _LHVY S TVS 4 217E _LVQ EG _KA R 2
178 Y _RH QK S 4 218L V_ GK R 2
D G C Q A_
E R
39 L EH FR _QL 3 230E I TL _RK 2
M R Q R
S
63 E EL E KF Q Q 3 22 I L TK A R8 1
A C
V
85 V L G R E 3 24 C V R E GS 1
M G T
A K
H A
86 L MA H GR E G 3 26 T K EG S EE 1
F A
R
94 F LR G ED G E 3 42 M F LR F ES 1
M R
Q
98 E DG E MV K 3 48 F E MK DP 1
L S R
E T
111 A LN K _CQ 3 $2 M _K PT A EQ 1
N N A R
D
116 N _CQ A LR A K 3 68 F Q ID _SRE 1
P Q
A
126 V _YI I QA _CR 3 70 Q SR _EDP 1
G A_
I
D
132 C RG E QR D P 3 72 I D ED P VS 1
G S
R
140 G _ETVG G D EI 3 77 D _PVS CA _FVV 1
168 V YS T V G Y 3 82 A F LM A G 1
E I V H
V
187 F IQ T LV D V 3 83 F V MA GR 1
F V H
L
192 V DV F TK R K 3 84 V V H G RE 1
G L
M
A
205 L LT E VT R 3 87 M RE G FL 1
R A
M H
G
234 T LR K RL Y 3 92 E G KG E DG 1
L F
Q L
2 S NP R SL E E 2 103V K NL F EA 1
8 L
E
18 R L L IL C V 2 107N AL N_NK 1
A T L
F
E
21 L IL C VT _KA 2 120L _R PK V I 1
R A Y
K
27 K E GS E E 2 12$I _I CR G EQ 1
A D Q
R A
29 R _EG S EE D L 2 129I Q RG E QR 1
D A
C
41 H _MF R QL R F 2 137R ET V_GG'1
E D
P
G
45 Q LR F ES T M 2 ' 138D P TV _GGD 1
R G
E
46 L RF E S'T M K 2 149V M KD S PQ 1
R V
I
60 Q FQ E EL E K 2 151V I SP Q TI 1
F R
D
65 ~L ER F QQ I 2 156P Q PT Y TD 1
A D T
I
81 C AF V M A 2 158T I YT D A 1
V H P L
L T
93 G FL K E D G 2 163T D HV Y ST 1
G E A
L
95 L K E DG E M 2 172V _E IA Y RH 1
G V G
Y
101 E M_V K LE N_L 2 177A DQ K S 1
F Y G
R
H
105 L EN FE A L 2 181D Q SC F IQ 1
L N R
G
202
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
3P1F11 v.l: HLA-A1 nonamers ~213P1F11 v.l: HLA-A26 nonamers
Pos 1 23 4 56 7 8 score Pos1 23 4 7 89 score
9 5
6
193 D VF T K K G 1 100G EM V E NL 14
R H K
L
194 V FT K K G H 1 127Y II Q R GE 14
R I A
C
207 T EV T RR M A 1 - 151V IK Q TI 14
E D
S
P
220 Q EG K AR K T 1 179R HD Q S CF 14
N K
G
223 K R K N P E 1 186C FI Q V DV 14
A T I T
L
224 A RK T NP E I 1 189Q TL V F TK 14
Q D
V
231 I QS T LR K R 1 190T LV T K 14
1; D R
V
F
218L VQ E A RK 14
G
K
13P 1F11 rs 9 E EK S G 13
v.l: Y A
HLA-A26 D
noname M
Pos 1 23 4 56 7 8 score 18 R LA C VT 13
9 L
I
L
35 D LD A LE H M 27 Portion 37 D A E F RQ 13
F of L H
M
170 S TV E GY I A 27 SEQ 79 V SC A V LM 13
Y ID F
V
167 H VY 5 TV 26 NO: g2 A FV HG 13
E 3; V
G each L
Y M
A
187 F IQ T LV D V 25 start 138D PG E G GD 13
F i T
i V
60 Q FQ E EL E K 23 t 146D EI V I K 13
F on is M D
pos V
s
ecified
p
78 P VS C AF V V 23 , 162Y TD A V YS 13
L the L
length H
154 D SP Q TI P T 23 of each 183K S C Q TL 13
Y G F
I
104 K LE N LF E A 22 peptide 234T LR K Y LQ 13
L is R
9 L
208 E VT R RM 22 ammo 11 K M A RL 12
A Y S
E D G
A
230 E IQ S TL R K 22 acids, 24 C V K E GS 12
R the T A
R
end
38 A LE H F R Q 21 osition 25 V TK G SE 12
M L p A
R
E
34 E DL D AL E H 20 for 47 R FE S K RD 12
M each T
tid M
i
56 P TA E QF Q E 20 e 50 S TM K P T 12
E s R A
pep D
the
start
94 F LK ED G E 20 position 67 K FQ Q D SR 12
G M A
I
142 T VG G DE I V 20 plus 71 A ID S D PV 12
M eight R
E
147 E IV M VI K D 20 86 L MA E GF 12
S H
G
R
158 T IP T YT D A 20 98 E DG E K LE 12
L M
V
193 D VF T KR K G 20 175Y IA D QK 12
H Y
R
H
195 F TK R KG H I 20 191L VD V K RK 12
L F
T
119 A LR KP K V 19 198R K E LL 12
A Y G
H
I
L
204 E LL T EV 19 227T NP E S TL 12
T I
R Q
R
205 L LT E VT R R 19 4 P RS L E KY 11
M E
E
233 S TL R K L Y 19 21 L IL C K 11
R L V A
T R
6 S LE E EK Y D 18 28 A RE G E DL 11
M S
E
101 E MV K E N L 18 83 F V GR 11
L F V
L
M
A
H
141 E TV G GD E I 18 84 V VL M G RE 11
V A
H
157 Q TI P TY T D 18 102M VK L FE 11
A L
E
N
201 H IL E LL T E 18 112L NN K Q AL 11
V N
C
226 K N P EI Q S 18 125K I CR 11
T T V I
Y Q
A
13 D MS G AR L A 17 128I IQ A G EQ 11
L C
R
40 E HM F RQ L R 17 148I V SP 11
F M
V
I
K
D
64 E LE K FQ Q A 17 160P TY T L HV 11
I D
A
97 G ED G EM V K 17 164D H S TV 11
L A V
L Y
107 N LF E AL N N 17 173E GY I R HD 11
K A
Y
171 T VE G YI A Y 17 206L TE V R M 11
R T A
R
197 K K HI L E 16 209V TR R E A 11
R G L M E
A
217 E LV Q EG K A 16 15'S G L IL 10 '
R A
R
L
A
20 A LI L CV T K 15 22 I LC V RE 10
A T
K
A
31 G SE E DL D A 15 57 T Q E EL 10
L A F
E Q
63 E EL E KF Q Q 15 59 E QF Q L EK 10
A E
E
150 M VI K S P Q 15 66 E KF Q I DS 10
D T Q
A
161 T YT D AL H V 15 73 D SR E V SC 10
Y D
P
53 K D P T 14 87 M G G FL 10
R A A R
E H E
Q
F
75 R ED P VS C A 14 111A LN N C QA 10
F K
N
203
f~'"'r'
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.l: HLA-A26
nonam ers 13P1F11 rs
v.l:
HLA-A26
noname
Pos 1 23 4 56 7 8 9 score Pos1 23 4 56 7 89 score
181 D QK G SC F I Q 10 145G DE I V V IK S
M
210 T RR M AE A L 10 229P EI Q ST L RK 5
E
214 A EA E LV Q E G 10 16 G AR L AL I LC 4
231 I QS T LR K L 10 70 Q AI D SR E DP 4
R
232 Q ST L RK L Y 10 126V YI I QA C RG 4
R
8 E EE K YD M S G 9 219V QE G K K 4
A T
R
42 M FR Q LR F E S 9 _ M SN P RS L EE 3
1
44 R QL R FE S T M 9 3 N PR S LE E EK 3
45 Q LR F ES T M K 9 26 T K R EG S EE 3
A
76 E DP V SC A F V 9 27 K R E GS E ED 3
A
8S V LM A HG R E G 9 32 S EE D LD A LE 3
92 E GF L KG E D G 9 S2 M K D PT A EQ 3
R
202 I LE L LT E V 9 61 F QE E LE K FQ 3
T
7 L EE E K D M S 8 6S L EK F QQ A ID 3
Y
E KY D MS G A 8 9S L K E DG E MV 3
R G
30 E GS E ED L D A 8 116N CQ R A KP 3
A
L
33 E ED L DA L E H 8 120L R PK YI 3
A V
K
81 C AF V L M A $ 132C RG E QR D PG 3
V H
93 G FL K E D G E 8 133R GE Q RD P GE 3
G
108 L FE A LN N K 8 . 136Q RD P GE T VG 3
N
121 R K P K Y I I 8 1S3K DS P QT I PT 3
A V
144 G GD E IV V I 8 166L HV ST V E. 3
M Y G
16S A LH S T V E 8 178Y RH D QK G SC 3
V
Y
194 V FT K RK H I $ 18SS CF I QT L VD 3
G
221 E GK RK T N P 8 212R MA A L VQ 3
A E E
2 S NP R SL E E E 7 223K A K N P EI 3
R T
41 H F R QL R F E 7 S R SL E EE K 2
M Y
D
46 L RF E ST M K 7 19 L AL I LC V TK 2
R
49 E ST M K D P T 7 23 L CV T KA EG 2
R R
SS D PT EQ F Q E 7 36 L DA L EH M FR 2
A
77 D PV S CA F V 7 S1 T MK DP T A 2
V R E
89 H GR E GF L K G 7 S4 R DP T E Q FQ 2
A
90 G RE G FL K E 7 68 F QQ ID S RE 2
G A
99 D GE M VK E N 7 69 Q QA I DS R ED 2
L
103 V L E NL F E A 7 91 R EG F LK G ED 2
K
106 E NL F EA N 7 96 K E D GE M VK 2
L N G
13S E QR D PG E T V 7 109F EA N K NC 2
L N
143 V GG D EI V M V 7 113N K N CQ LR 2
N A
21S E A L VQ E G K 7 114N KN C QA R 2
E L A
74 S RE D PV S C A 6 118Q A R K P KV 2
L A
80 S C F V M 6 129I QA RG E QR 2
A V A C
L
110 E AL N K C Q 6 130Q C R GE Q R 2
N N A D
11S K C Q AL R 6 131A CR G EQ R DP 2
N A
K
122 A KP K VY I I Q 6 149V V I K S PQ 2
M D
123 K PK YI I Q A 6 1S2I K S PQ T IP 2
V D
124 P K Y II Q C 6 174G YI A YR H DQ 2
V A
163 T DA L HV S T 6 176I A R HD Q K 2
Y Y G
182 Q KG S CF I Q T 6 207T EV T RR M AE 2
200 G HI L EL L T E 6 216A EL V E G KA 2
Q
213 M E A EL V Q E 6 222G K KT N PE 2
A A
R
14 M SG A RL A I S 12 Y DM S G LA 1
L A
R
17 A L A I L C V 5 29 R EG S EE D LD 1
R L
43 F RQ L RF E S T S 39 L EH FR Q LR 1
M
137 R DP G ET V G G 5 58 A Q F QE E LE 1
E
204
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 13P1F11 rs
v.l: v.l:
HLA-A26 HLA-A3
nonamers noname
Pos 1 3 6 7 9 score Pos1 23 4 5 67 8 score
2 4 8 9
5
62 Q E K F Q 1 150M VI K SP Q 16
E L Q D T
E
72 I 5 D P S 1 187F IQ T L V V 16
D R V D F
E
105 L N E A N 1 193D VF T K RK G 16
E L L H
F
117 C A A K K 1 229P EI Q S TL R 16
Q L P K
R
134 G Q P G T 1 22 I LC V T KA R 15
E R E E
D
140 G T G D I 1 $5 V LM A GR E 15
E V E H G
G '
159 I T D A H 1 102M VK L E NL F 15
P Y L E
T
172 V G A H 1 104K L_EN L FE A 15
E Y Y L
I R
177 A R Q K S 1 129I QA_C R G_EQ 15
Y H G R
D
180 H Q S C I 1 151V IK D S P_QT 15
D K F I
G
188 I T D V T 1 179R HD Q K GS C 15
Q L F F
V
192 V V K R 1 203L E_LL T E_VT 15
D F K R
T G
196 T H I E 1 234T LR K R L_YL 15
K L Q
R
K
G
199 K H E L T 1 94 F LK E DG E 14
G I L G M
L
203 L L E V R 1 115K C Q A LR A 14
E L T N R
T
220 Q G R K N 1 128I IQ C RG E 14
E K T A Q
A
224 A K P E Q 1 148I V. V I KD S 14
R T I M P
N
225 R T E I S 1 3 N PR S L EE E 13
K N Q R
P
228 N E S T R 1 59 E QF Q E EL E 13
P I L R
Q
71 A ID S R E_ P 13
D V
13P1F11 nonamers 75 R ED P V SC A 13
v.l: F
HLA-A3
Pos 1 3 6 7 9 score 84 V L M _ _ R 13
2 4 8 V A G E
5 H
119 A R P K 30 Portion 212R A _ V 13
L A V of M E L Q
K Y A
E
45 Q R_ _S_T R 27 SEQ 6 S LE E E KY D 12
L F M ID M
E
218 L Q K A R 26 NO: 136Q RD P G ET V 12
V E R 3; G
G each
125 K Y Q R 25 stmt 201H IL E L LT E 12
V I A osition V
I C is
175 Y A H D R 24 p 205L LT E V TR R 12
I Y Q specified M
R
,
19 L L C V K 21 the 215E AE L V QE G 12
A I T length R
L
96 K E E M R 21 of 23_0E IQ S T LR K 12
G D V each R
G
107 N _F L N 21 peptide 10 E RY D M _SG A 11
L E N is R
A R 9
18 R A _LC T 2p amino 15 S G L A I 11
L L V A_ _L L
I R
167 H _Y V _E Y 20 acids, 24 C V_TK A _RE G 11
V S G the S
T
191 L _D T K R 20 end 33 E ED L D A E 11
V V R position _L H
F for
ea
h
88 A G G F R 19 c 64 E L_EK F QQ A 11
H R L peptide I
E is
171 T _E I A R 19 73 D SR E D P_VS 11
V G Y the C
Y start
20 A I V T 18 position 127Y II Q A CR G 11
L L K E
C A
78 P S F V L 18 plus 135E QR D P GE T 11
V C V eight V
A
83 F A R 18 161T YT H 11
V H D V
V G A'L Y
L
M
111 A N C A 18 170S TV E G YI A 11
L N Q Y
K
N
165 A H S T E 18 200G HI L E LL T 11
L V V E
Y
189 Q L V F R 18 232Q ST L R KR L 11
T V T Y
D
208 E _T M A 18 . 233S TL R K L Y 11
V R A R L
R E
217 E V G K 18 17 A RL A L IL C 10
L Q A V
E R
35 D D E H F 17 26 T RA E G_SE 10
L A M R E
L
117 C A A__K' R 17 137R D_PG E TV_G 10
Q L P G
R
190 T V F T R 17 154D SP Q T I_PT 10
L D K Y
V
202 I E T E 17 160P TY T D AL H 10
L L V V
L T
204 E L V T R 17 164D AL H YS T 10
L T R V V
E
21 L L K 16 209V TR R M A 10
I C. A A E
V R E
T
38 A E F R L 16 11 K YD M S GA R 9
L H Q L
M
44 R L E S 16 27 K AR E G SE E 9
Q R T D
F M
53 K D A E F 16 40 E HM F R QL R 9
R P Q F
T
142 T _G E I 16 ~ 67 K FQ Q A ID S
V G V R
D M
145 G E M V R 16 72 I DS R E DP V 9
D I I S
V
205
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.l: HLA-A3 nonamers ' 13P1F11 v.l: HLA-A3 nonamers
Pos 1 23 4 56 7 8 score Pos 1 23 4 5 89 score
9 6 7
89 H GR E GF L K 9 144 G GD E I VI 6
G V_
M
113 N N_KN C_QA L 9 153 K DS P Q PT 6
R T I
121 R AK P KV Y I 9 162 Y TD A L YS 6
I H V
147 E IV M VI K D 9 174 G YI A Y DQ 6
S R H
157 Q TI P TY T D 9 177 A R H D GS 6
A Y Q K
158 T IP T YT D A 9 210 T RR M A EL 6
L E A
159 I PT Y TD A L 9 216 A EL V Q KA 6
H E G
176 I A_YR HD Q K 9 219 V Q_EG K K 6
G A R T
185 S C_FI QT L V 9 221 E GK A R NP 6
D _K
_T
197 K RK G HI L E 9 223 K AR K T EI 6
L N P
211 R RM EA__E 9 228 N P_EI Q LR 6
A L S T
V
213 M A_EA _LV_ 9 12 Y DM S G LA $'
E Q A_
E R_
1 M SN P RS L E $ 28 A RE G S DL 5
E E E
25 V TK E G S $ 32 S EE D L LE 5
A E D A
R
86 L MA H GR E G $ 39 L EH M F LR 5
F R Q
106 E NL F EA L N $ 43 F RQ L R ST 5
N F E
131 A CR G EQ R D $ 55 D PT A E QE 5
P Q F
188 I QT L V V F $ 68 F QQ RE 5
D T A
I
D
S
195 F T_K K_G_H $ 74 S RE D P CA 5
R I V_
L _S
225 R R_TN P_EI Q $ $7 M AH G R FL 5
S E G
226 K N P EI _Q $ 91 R E_GF L ED 5
T S _K
T _G
227 T N_PE I_Q_S $ 92 E GF L K DG 5
T _G
L _E
4 P R_SL E_E_E 7 95 L ICG E D M 5
K G E V
Y
R S_LE EE _K 7 120 L RA YI 5
Y K
D P
K
V
13 D MS G AR L A 7 134 G EQ R D ET 5
L P G
14 M SG A RL A L 7 139 P GE T V DE 5
I G G
36 L DA L EH M F 7 140 G ET V G EI 5
R G D
50 S TM K D P T 7 149 V I K PQ 5
R A M D S
V
51 T K DP T A 7 156 P QT I P TD 5
M R E T Y
52 M RR D P_TA_ 7 173 E GY I A HD
E Y R
Q
60 Q FQ E EL E K 7 182 Q R_GS C QT 5
F _F
_I
63 E EL E K_F_Q 7 196 T ICR K G LS 5
Q H I
A
70 Q A_ID SR E D 7 207 T EV_T R AE $
P _R
_M
77 D P_VS CA_F V 7 214 A EA_ EG 5
V E
L
V
Q
80 S CA F V _L 7 222 G K PE 5
V_ M A_
A R
K
_T
N
81 C AF V VL M A 7 224 A K IQ 5
H R T
N
P
E
118 Q AL R AK P K 7 ~ 2 S NP R S EE 4
V L E
123 K K YI I Q 7 9 E EK G 4
P V A Y A
D
M
S
126 V YI I QA C R 7 16 G AR L A LC 4
G L I
130 Q AC R GE Q R 7 23 L CV T K EG 4
D A R
172 V EG Y IA__Y 7 29 R E_GS E LD 4
R _E
H D_
183 K S C FI Q T 7 30 E G_SE E DA 4
G L D L
186 C F_IQ TL V D 7 42 M FR Q L ES 4
V R F
199 K I L_E_L 7 47 R FE S T 4
G L M K
_H T R D
220 Q EG K A _K 7 54 R P T A FQ 4
_R T D _E
N _Q
46 L RF E ST _M 6 65 L EK F Q TD 4
K Q A_
R
62 Q EE L EK F Q 6 69 Q Q I D ED 4
Q A S R
79 V SC FV V L 6 93 G FL K G GE 4
A M E D
$2 A FV LM A H 6 99 D GE M V EN 4
V G K L
97 G ED G EM V K 6 101 E K L LF 4
L M E N
V
114 N RN C QA L R 6 105 L EN L F LN 4
A E A
116 N C_QA LR A K 6 122 A ICP K V IQ 4
P Y _I
133 R GE Q R_DP G 6 141 E TV G G IV 4
E _D
_E
143 V GG D EI V M 6 163 T DA ST 4
V L
H
V
Y
206
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
l3PiF11 v.l: HLA-A3 nonamers 13P1F11 v.l: HLA-B*0702 nonamers
Pos 1 3 4 56 7 89 score Pos1 3 4 5 78 score
2 2 6 9
166 L Y ST V EG 4 13 D S G LA 18 Portion
H M A L of
V_ R
178 Y H D Q_K_GSC 4 77 D V S C FV 17 SEQ
R P A V ID
198 R G H IL E LL 4 123K K Y IQ 17 NO:
K P V I A 3;
each
7 L E E KY D MS 3 155S Q T I TY 17 start
E P P T
8 E E K D M SG 3 7$ P S C A V 16 Position
E Y V F V is
L s
ecified
p
34 E L D AL E H 3 97 G D G E VK 15 ,
D M E M L the
length
37 D L E HM F RQ 3 197K K H LE 15 of each
A R G I L
41 H F R QL _RFE 3 11 K D M S AR 14 peptide
M Y G L is
9
$$ A _QF Q_E_ELE 3 28 A E G S ED 14 amino
E R E L
66 E _FQ QA _IDS 3 231I S T L KR 14 acids,
K Q R L the
76 E _PV SC A FV 3 15 S A R L I 13 end
D G A L position
L
103 V L E N_LF EA 3 38 A E H M RQ 13 for
K L F L each
tid
i
110 E L N K N_CQ 3 87 M G R GF 13 e
A N A E L s
H pep
the
start
146 D I V V I K 3 104K E N L EA 13 position
E M D L F L
152 I D S PQ T IP 3 120L A K P Y 13 plus
R R K I eight
V
155 S Q T IP T YT 3 135E R D P ET 13
P Q G V
169 Y T V G Y IA 3 183K S C F QT 13
S E G I L
181 D K G SC F IQ 3 210T R M AE 13
Q R A L
E
194 V T K RK I 3 233S L R K LY 13
F G T R L
H
231 I S T L_RK L 3 3 N R S L EE 12
Q R P E R
31 G _EE DL _DAL 2 112L K QA 12
S N N L
N C
49 E T M K D PT 2 138D G E T GG 12
S _R P V D
56 P A E Q_F_QEE 2 153K S P Q IP 12
T D T T
90 G E G F_LK E ~ 158T P T Y DA 12
R G I T L
108 L E A LN KN 2 159I T Y T AL 12
F N P D H
109 F NN K C 2 198R G H I EL 12
E N R L L
A
L
132 C G E QR D PG 2 17 A L A L LC 11
R R I V
138 D G E TV G GD ~ 30 E S E E LD 11
P G D A
168 V S T VE G YI 2 31 G E E D DA 11
Y S L L
180 H Q K S _CFI 2 55 D T FQ 11
D G P A E
E
Q
206 L _EV _RR MA 2 100G M V K EN 11
T T E L L
61 F E E L_EK Q 1 195F K K HI 11
Q F T R G L
98 E G E MV _KLE 1 223K R K T PE 11
D A N I
100 G M V K_LE NL 1 228N E I Q TL 11
E P S R
112 L K C Q 1 20 A I L C TK 10
N N A L V A
N L
124 P V Y II Q AC 1 40 E M F R LR 10
It H Q F
18 G C F IQ T LV 1 57 T E Q F EE 10
S A Q L
71 A D S R DP 10
I E V
74 S E D P SC 10
R V A
75 R D P V CA 10
E S F
79 V C A F VL 10
S V M
80 S F V LM 10
C V A
A
142T G G D IV 10
V E M
188I T L V VF 10
Q D T
227T P E I ST 10
N Q L
' 14 M G A R AL 9
S L I
18 R A L I CV 9
L L T
49 E T M K DP 9
S R T
50 S M K PT 9
T R A
D
53 K D P T Q 9
R A F
E
64 E E K F QA 9
L Q I
76 E P V S AF 9
D C V
121R K P K YI 9
A V I
141E V G G EI 9
T D V
207
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.l: HLA-B*0702 nonamer
s 13P 1F11 amers
v.l:
HLA-B*0702
non
Pos 1 23 4 5 7 score Pos 1 2 5 6 7 89 score
6 8 3 4
9
143 V GG D E V 9 33 E E D A L EH 4
I M D L
V
144 G GD E I 9 73 D S D P V SC 4
V R E
M
V
I
179 R HD Q K S 9 $$ A H E G F LR 4
G C G R
F
199 K GH I L L 9 136 Q R G E T VG 4
E L D P
T
202 I LE L L E 9 156 P Q P T Y TD 4
T V T I
T
211 R RM A E E 9 162 Y T H V YS 4
A L D A
V L
35 D LD A L H $ 165 A L Y S T VE 4
E M H V
F
63 E EL E K Q $ 177 A Y D Q K GS 4
F Q R H
A
86 L M G E $ 185 S C Q T L V 4
A R G F I D
H F
94 F LK E G $ 214 A E L Q EG 4
G D E A E V
M
95 L RG E D E $ 225 R R P E I QS 4
G M T N
V
101 E MV K N $ 1 M S R S L EE 3
L L N P
E F
111 A LN N K C $ 27 K A G S E ED 3
N Q R E
A
114 N RN C Q $ 42 M F L R F ES 3
A R Q
L
R
A
150 M I K P $ 45 Q L E S T MR 3
V D Q R F
S T
157 Q TI P T T $ 59 E Q E E L ER 3
Y D F Q
A
160 P TY T D $ $2 A F M A G 3
A V V H
L L
H
V
163 T DA L H $ $5 V L H G R EG 3
V M A
Y
S
T
168 V S T G $ 98 E D M V LE 3
Y V Y G E K
E I
180 H DQ K G C $ 102 M V E N L FE 3
S F K L
I
182 Q RG S C I $ 106 E N E A N 3
F Q L F L N
T
186 C FI Q T V $ 116 N C R KP 3
L D Q A A
V L
187 F IQ T L D $ 122 A R Y I IQ 3
V V P K
F V
208 E VT R R $ 128 I I C R G EQ 3
M Q A
A
E
A
216 A EL V G $ 129 I Q R G E QR 3
Q K A C
E A
219 V QE G K R $ 132 C R Q R D PG 3
A K G E
T
226 K TN P E Q $ 166 L H S T V EG 3
I S V Y
T
9 E EK S 7 171 T V Y I A YR 3
Y G E G
D A
M
12 Y DM S G 7 196 T R H I LE 3
A R K
R G
L
A
34 E DL D A E 7 204 E L E V T RR 3
L H L T
M
43 F RQ L R E 7 209 V T M AE 3
F S R R A
T E
44 R QL R F S 7 213 M A L V QS 3
E T E A
M E
52 M RR D P A 7 217 E L E G K AR 3
T E V Q
Q
118 Q R P 7 220 Q E A R K T 3
A A K G K N
L K V
140 G ET V G D 7 221 E G K NP 3
G E K A T
I R
169 Y ST V E Y 7 222 G R K T N PE 3
G I A R
A
184 G SC F I T 7 229 P E S T L RR 3
Q L I Q
V
194 V FT K G 7 234 T L L Y LQ 3
R H R K
K I R
201 H IL E L T 7 4 P R E E E K 2
L E S L Y
V
205 L LT E V R 7 $ E E D M SG 2
T R E K
M Y
206 L TE V T R 7 10 E R M S G A 2
R M Y D R
A
6 S LE E E 6 16 G A I LC 2
K R L
Y A L
D
M
60 Q FQ E E E 6 19 L A L C V TR 2
L K L I
F
103 V L E N F 6 ~21 L I V T K AR 2
R L E L C
A
119 A LR K K 6 22 I L T K E 2
A P V C V A
Y R
131 A CR G E R 6 24 C V E GS 2
Q D T K
P A R
134 G EQ R D G 6 26 T R E G S EE 2
P E ~ A R
T
137 R DP G E V 6 29 R E E E D LD 2
T G G S
G
151 V IK S Q 6 36 L D E H M FR 2
D P T A L
I
152 I RD S P T 6 48 F E M K DP 2
Q I S T R
P
164 D AL H V S 6 51 T M D P T AE 2
Y T K R
V
72 I DS R E P 5 54 R D Q FQ 2
D V P T
S A E
89 H GR E G L 5 56 P T Q F Q EE 2
F K A E
G
212 R M A L 5 58 A E Q E E LE 2
A E V Q F
E Q
208
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 13P1F11
v.l: v.l:
HLA-B*0702 HLA-B*0702
nonamers nonamers
Pos 1 2 34 5 67 8 9 score Pos ~.2 34 67 8 score
. 5 9
90 G R EG F LK G E 2 193 D V FT RK G 1
K H
91 R E GF L K E D 2 215 E A EL QE G 1
G V R
92 E G FL K GE D G 2 218 L V E K 1
Q G A
R
R
96 K G ED G EM V 2 230 E I QS LR K i
R T R
99 D G EM V KL E N 2
115 K CQ A LR A 2 13P1F11 :
N R v.l HLA-B*08
nonamers
117 C Q AL R K P R 2 Pos 1 2 34 67 8 score
A 5 9
125 K YI I Q C R 2 121 R P VY I 31 Portion
V A A R I of
K
133 R G EQ R DP G E 2 195 F T RR GH I 31 SEQ
R L ID
145 G D EI V M I R 2 119 A RA PK V 23 NO:
V L R Y 3;
each
148 I V MV I K S P 2 $7 M A HG EG F 22 start
D R L
. position
is
'
174 G Y IA RH D Q 2 100 G E LE N 22 specified,
Y M L
V
R
181 D Q K S CF I Q 2 197 K G IL E 22 the
G R H L length
R
190 T L VD V FT K R 2 233 S T LR RL Y 22 of each
R L
191 L V DV F TK R 2 151 V I R PQ T 21 peptide
R D I is
S 9
200 G H IL E LL T E 2 221 E G RA KT N 20 amino
R P
203 L E LL T EV T R 2 234 T L RK LY L 20 acids,
R Q the
207 T E VT R RM A E 2 25 V T RA EG S 19 end
R E position
for
each
224 A R KT N PE I Q 2 94 F L RG DG E 1 peptide
E M g is
R S LE E EK D 1 ~ 123 K P RV II Q 18 the
Y Y A start
25 V K EG S E 1 223 K RK NP E 18 position
T A A T I
R
32 S E ED L DA L E 1 104 K L EN FE A' 17 plus
L L eight
39 L E HM F RQ L R 1 111 A L N C Q 17
N A
R
N
41 H M FR Q LR F 8 1 210 T R RM EA E 17
A L
46 L R FE S TM K R 1 38 A EH FR Q 16
L M L
47 R F ES T MK D 1 40 E H F QL R 16
R M R F
61 F Q EE L EK F Q 1 179 R H DQ GS C 16
R F
65 L E KF Q Q I D 1 57 T Q QE E 15
A A F L
8
66 E R FQ Q AI D S 1 64 E L FK QQ A 15
F I
67 K F QQ ID S R 1 158 T I PT TD 15
A Y A
L
6$ F Q QA I DS R E 1 194 V F TK K 15
R G
H
I
69 Q Q AI D SR E D 1 14 M S G L 14
A A
R L
I
70 Q A ID S RE D P 1 31 G S EE LD A 14
D L
$1 C A FV LM A H 1 63 E E LE FQ Q 14
V R A
93 G F LK ED G 8 1 3 N P RS EE E 13
G L R
105 L 8 NL F EA L N 1 9 E E RY MS G 13
D A
108 L F EA N K 1 27 K E SE E 13
L N N A G D
R
109 F E AL N NK N C 1 92 E G FL GE D 13 ,
R G
110 E A L K C Q 1 175 Y I A HD Q 13
N N Y R
N R
113 N K C Q R 1 7 L E 8E Y M 12
N N A R D S
L
124 P R V I IQ A C 1 15 S G AR I 12
Y L L
A
L
139 P G ET G D E 1 16 G L I L 12
V G A A C
R L
146 D E IV I K 1 35 D L DA EH M 12
M D L F
V
147 E I VM IK S 1 45 Q L RF ST M 12
V D $ R
149 V M VI K DS P Q 1 49 E S TM R P 12
R D T
154 D S PQ T IP T Y 1 71 A I DS ED P 12
R V
161 T Y TD A H 1 97 G E DG MV K 12
L V E L
Y
167 H S T VE G Y 1 102 M L NL F 12
V V E E
Y R
170 S T VE G YI A 1 187 F I QT D V 12
Y L F
V
172 V GY I AY R H 1 227 T N PE QS T 12
E I L
173 E G YI A YR H D 1 231 I Q ST RK R 12
L L
175 Y I A R HD Q R 1 13 D M SG L 11
Y A A
R L
176 I A YR H DQ K 1 23 L C VT AR E 11
G R G
189 Q T LV VF T R 1 51 T M RR PT 11
D D A
E
209
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 ers 13P1F11 ers
v.l: v.l:
HLA-B*08 HLA-B*08
nonam nonam
Pos1 2 5 6 78 9 score Pos1 2 3 4 5 78 score
3 6 9
4
7$ P V A F VV L 11 215E A E L V EG 7
S Q FC
C
112L N C QA L 11 21 L I L C V KA 6
N T R
K
N
149V M K D SP Q 11 37 D A L E H FR 6
V M Q
I
183K G F I QT L 11 53 K R D P T EQ 6
S A F
C
193D V K R KG H 11 55 D P T A E FQ 6
F Q E
T
208E V R M AE A 11 77 D P V S C FV 6
T ' A V
R
219V Q K A RK 11 81 C A F V M 6
E T V A
G L H
222G K K T NP E 11 86 L M G EG 6
A A R F
R H
6 S L E K YD M 10 98 E D G E M K 6
8 V L
E E
11 K S G AR L 10 110E A L N N C 6
Y K Q
D N
M
28 A S E ED L 10 128I I Q A C GE 6
R R Q
E
G
43 F R R F ES T 10 131A C R G E RD 6
Q Q P
L
50 S T R D PT A 10 190T L V V TK 6
M D F R
K
65 L E Q Q AI D 10 196T K R K IL 6
K G E
F H
113N N C Q AL R 10 228N P E I Q TL 6
R S R
N
117C Q R A KP IL10 230E I Q S T RK 6
A L R
L
144G G I V V I 10 19 L A.L I L V 5
D M C T
E R
181D Q S C FI Q 10 170S T V E G IA 5
R . Y Y
G
198R K I L EL L 10 176I A H QK 5
G Y D G
H R
217E L E G K 10 213M E VQ 5
V A A L E
Q R E
A
224A R N P EI Q 10 30 E G S E E LD 4
It D A
T
1 M S R S LE E 9 33 E E D L D LE 4
N A H
P
73 D S D P VS C 9 61 F Q E E L KF 4
R E Q
E
89 H G G F LK G 9 70 Q I D S ED 4
R A R P
E
129I Q R G EQ R 9 103V K E N FE 4
A L L A
C
138D P T V GG D 9 109F E A N K 4
G L N N
E C
140G E G G DE I 9 118Q A L R A PK 4 I
T K V
V
204E L E V TR R 9 127Y I I Q A RG 4
L C E
T
207T E R R MA E 9 ' 130Q A C R G QR 4
V E D
T
232Q S R K RL Y 9 164D A L H ST 4
T V V
L Y
20 A C V TK $ 214A E A E L QE 4
L A V G
I
L
22 I L T K AR E $ 2 S N P R S EE 3
C L E
V
60 Q F E L EK F $ S R S L E E K 3
Q E Y
E D
$S V G RE G $ 10 E K M G 3
L Y S A
M D R
A
H
101E M E NL F 8 56 P T A E Q QE 3
V F E
K
L
107N L A L N $ 59 E Q F Q E LE 3
F N E IC
E R
133R G R D PG E $ 74 S R E D P SC 3
E V A
Q
13SE Q P G ET V $ 76 E D P V S AF 3
R ~C V
D
147E I I K S $ 79 V S C A F VL 3
V D V M
M
V
155S P I P TY T $ 80 S C A F V LM 3
Q V A
T
1S9I P T D AL H $ 124P K I Q 3
T V I A
Y Y C
201H I L L TE V $ . 143V G G D E VM 3
L I V
E
202I L L T EV $ 166L H S V 3
E T V T E
L Y G
205L L V T RR 8 188I Q T L V VF 3
T M D T
E
209V T M A E 8 $ E E E K MS 2
R A Y G
R E~ D
18 R L I L CV 7 32 S E E D L A 2
A T D L
L E
42 M F L R FE S 7 34 E D L D A EH 2
R L M
Q
52 M K P T AE Q 7 46 L R F E S MK 2
R T R
D
7S R E V S CA F 7 66 E K F Q Q ID 2
D A S
P.
120L R K Y I 7 $3 F V G 2
A V V R
K L
P M
A
H
16SA S TV E 7 90 G R E G F K 2
L L G
H E
V
Y
168V V E GY I 7 9$ L K G E D EM 2
Y G V
S
T
177A D Q KG S 7 . 106E N L F E N 2
Y A N
R L
H
180H D S CF I 7 132C R G E Q DP 2
Q R G
K
G
210
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
l3PiF11 v.l: HLA-B*08 nonamers I ~ 1213P1F11 v.l: HLA-B*1510 nonamers
Pos 1 23 4 56 7 89 score Pos1 2 3 56 7 89 score
4
134 G EQ R DP G ET 2 4 E H M RQ L RF 19 Portion
F of
141 E TV G GD E IV 2 179R H D KG S CF 17 SEQ
Q ID
145 G DE I V V IK 2 231I Q S LR K RL 16 NO:
M T 3;
each
146 D EI V V I KD 2 78 P V S AF V VL 15 start
M C
148 I VM V IK D SP 2 97 G E D EM V K 1 Position
G L S is
s
ecified
p
163 T DA L HV Y ST 2 13 D M S AR L A 14 ,
G L the
length
167 H Y S TV E GY 2 31 G S E DL D AL 14 of each
V E
172 V EG Y IA RH 2 S7 T A E FQ E EL 14 peptide
Y Q is
9
173 E GY I AY R HD 2 38 A L E MF R QL 13 amino
H
185 S CF I QT L 2 112L N N C Q AL 13 acids,
V K N the
D
189 Q TL V DV F TR 2 166L H V ST V EG 13 end
Y position
191 L VD V FT K R 2 183K G S FI Q TL 13 for
R C each
tid
i
200 G HI L EL L TE 2 197K R K HI L EL 13 e
G s
Pep
the
start
203 L EL L TE V TR 2 227T N P IQ S TL 13 position
E
212 R MA E AE L VQ 2 11 K Y D SG A RL 12 plus
M eight
218 L VQ E GK R 2 28 A R E SE E DL 12
A G
R
4 P RS L EE E K 1 100G E M KL E NL 12
Y V
17 A RL A LI L CV 1 104K L E LF E AL 12
N
26 T K EG S EE 1 158T I P YT D AL 12
A T
R
36 L DA L EH M FR 1 200G H I EL L TE 12
L
41 H MF R QL R FE 1 210T R R A EL 12
M A
E
47 R FE S TM K D 1 15 5 G A LA IL 11
R R L
48 F ES T P 1 87 M A H RE G FL 11
M G
K
R
D
62 Q EE L EK F QQ 1 205L L T VT R R 11
E M
67 K FQ Q AI D SR 1 233S T L K L YL 11
R R
68 F QQ ID S RE 1 $$ A H G EG F LR 10
A R
69 Q QA I DS R ED 1 142T V G DE I VM 10
G
72 I DS R ED P VS 1 195F T K K IL 10
R G
H
82 A FV LM G 1 198R R G IL E LL 10
V A H
H
$4 V L M A G RE 1 6 S L E EK Y M 9
V H E D
91 R EG F LK G ED 1 75 R E D VS C AF 9
P
93 G FL K E D GE 1 79 V S C FV L 9
G A V M
99 D GE M EN 1 86 L M A GR E GF 9
V H
K
L
105 L EN L FE N 1 187F I Q LV VF 9
A T D
L
108 L FE A LN K 1 34 E D L AL E HM $
N N D
115 K C Q AL R 1 53 K R D T QF $
N A . P A
R E
126 V YI I QA C RG 1 94 F L K ED G EM $
G
137 R P G ET V G 1 101E M V LE N LF $
D G K
152 I KD S PQ T IP 1 44 R Q L FE S T 7
R M
154 D SP Q TI P T 1 60 Q F Q EL E KF 7
Y E
162 Y TD A LH V YS 1 22 I L C TK E 6
V A
R
169 Y ST V G Y IA 1 35 D L D E H MF 6
E A L
171 T E G YI A 1 72 I D S ED P VS 6
V Y R
R
174 G YI A H DQ 1 120L R A PK YI 6
Y K V
R
184 G SC F IQ T LV 1 96 K G E GE M VR S
D
186 C FI Q TL V V 1 136Q R D GE T VG 5
D P
192 V V F TK K 1 144G G D IV I 5
D R G E M
V
206 L TE V TR R 1 145G D E VM IR $
M I V
A
216 A L V QE G K 1 202I L E LT E V 5
E A L T
220 Q EG K K N 1 206L T E TR R MA 5
A T V
R
226 K N P EI Q ST 1 218L V Q GK RR 5
T E A
12 Y D M G LA 4
S A
R
37 D A L H F RQ 4
E M
47 R F E TM K RD 4
S
211
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P 1F11 mers 13P 1F11 mers
v.l: v.l:
HLA-B*1510 HLA-B*1510~
nona nona
Pos 1 23 4 56 7 89 score Pos 1 23 4 5 7 8 score
6 9
48 F ES T MK R DP 4 162 Y TD A L V Y 3
H S
64 E LE K FQ Q AI 4 163 T DA L H Y S 3
V T
69 Q QA I DS R ED 4 165 A LH V Y T V 3
S E
73 D SR E DP V SC 4 170 S TV E G I A 3
Y Y
74 S RE D PV S CA 4 173 E GY I A R H 3
Y D
115 K C Q AL R A 4 178 Y RH D Q S 3
N R K C
G
127 Y II Q AC R GE 4 186 C FI Q T V 3
L D
V
128 I IQ CR G EQ 4 189 Q TL V D F T 3
A V K
135 E QR D PG E TV 4 191 L VD V F K R 3
T IC
140 G ET V GG D EI 4 192 V DV F T R K 3
K G
143 V GG D EI V MV 4 196 T RR K G I L 3
H E
148 I VM V IK SP 4 203 L EL L T V T 3
D E R
154 D SP Q TI P TY 4 207 T EV T R M A 3
R 8
161 T YT D A H V 4 208 E V R R A E 3
L Y T M A
171 T V G YI A R 4 213 M A A V 3
E Y E E Q
L E
188 I QT L VD V FT 4 217 E LV Q E K 3
G A
R
204 E LL T EV T RR 4 220 Q EG K A K T 3
R N
212 R A E A L VQ 4 223 K R K P E 3
M E A T I
N
214 A EA E LV Q EG 4 226 K N P E Q S 3
T I T
219 V QE G KA R KT 4 229 P EI Q S L R 3
T IC
1 M SN P RS L EE 3 230 E IQ 5 T R K 3
L R
$ E EE K D M SG 3 232 Q ST L R L 3
Y K Y
R
E KY D MS G A 3 7 L EE E K D M 2
R Y S
18 R LA L IL C VT 3 9 E EK Y D S G 2
M A
19 L AL I LC V TIt 3 24 C VT K E G 2
A S
R
23 L CV T KA R EG 3 32 S EE D L A L 2
D E
26 T RA R EG S EE 3 33 E ED L D E 2
A H
L
27 K A E GS E ED 3 41 H F R Q R F 2
R M L E
30 E GS E ED L DA 3 49 E ST M K P 2
R T
D
42 M FR Q LR F ES 3 61 F QE E L K F 2
E Q
50 S TM K P TA 3 62 Q EE L E F Q 2
R K Q
D
51 T K DP T E 3 63 E EL E K Q Q 2
M R A F A
52 M KR D PT A EQ 3 66 E RF Q Q I D 2
A S
56 P T E QF Q EE 3 67 K FQ Q D S 2
A A R
I
59 E QF Q EE L ER 3 68 F QQ A I S R 2
D E
85 V LM G R EG 3 70 Q AI D S E D 2
A R P
H
90 G RE G FL K E 3 76 E DP V S F 2
G C V
A
93 G FL K GE D GE 3 77 D PV S C F V 2
A V
98 E DG E MV K E 3 80 S C F V M 2
L A V A
L
99 D GE M K L EN 3 81 C AF V M 2
V V A
L H
103 V KL E NL F EA 3 82 A FV V L A H 2 '
M G
110 E A N K CQ 3 84 V L M G R 2
L N N V A E
H
119 A R KP K 3 89 H GR E G L K 2
L A V F G
Y
121 R A P K Y II 3 91 R EG F L E 2
K V K D
G
129 I QA C RG E QR 3 109 F E N K 2
A N N
L C
130 Q AC R GE Q RD 3 ~ 114 N K C Q R 2
N A A
L
131 A CR G EQ R P 3 118 Q AL R A P K 2
D K V
133 R GE Q RD P GE 3 123 K K V Y I Q 2
P I A
134 'G EQ R DP G ET 3 124 P KV Y I Q 2 '
I A
C
137 R DP G ET V GG 3 126 V YI I Q C R 2
A G
141 E TV G G E IV 3 132 C RG E Q D P 2
D R G
151 V IK SP Q TI 3 138 D PG E T G G 2
D V D
152 I ItD S PQ T IP 3 146 D EI V M I K 2
V D
153 K DS P QT I PT 3 147 E IV K D 2
M S
V
I
212
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 mers 13P1F11 mers
v.l: v.l:
HLA-B*1510 HLA-B*2705
nona nona
Pos1 2 3 4 78 score Pos1 3 45 6 78 9 score
5 9 2
6
150M V I K PQ 2 197K K GH I LE L 29 Portion
D T R of
S
156P Q T I YT 2 46 L F ES T MK R 28 5EQ
P D R ID
T
157Q T I P TD 2 53 K D PT A EQ F 25 NO:
T A R 3;
Y each
159I P T Y AL 2 28 A E GS E ED L 24 start
T H R iti
D is
169Y S T V YI 2 4 P S LE E EK 22 on
E A R Y Pos
G specified
,
172V E G Y R 2 210T R M E AE L 22 the
I H R A length
A
Y
174G Y I A HD 2 120L A KP K Y I 21 of
Y Q R V each
R
175Y I A DQ 2 97 G D GE M VK L 19 peptide
Y K E is
R 9
H
176I A Y R QK 2 17 A L I LC V 1$ amino
H G R A
D L
180H D Q K CF 2 59 E F QE E LE K 1 acids,
G I Q g the
S
185S C F I LV 2 67 K Q QA I DS R 1 end
Q D F g position
T h
f
190T L V TK 2 107N F EA NN K 18 or
D R L L eac
V tide
F is
e
194V F T K H 2 125K Y II Q AC R 18 P
R I V p
K the
G start
201H I L E TE 2 179R D QK SC F 18 position
L V H G
L
215E A E L EG 2 204E L TE V TR R 18 plus
V K L eight
Q
221E G K N 2 229P I QS T LR K 18
A P E
R
K
T
222G K R P 2 75 R D PV S CA F 17
A K E ~ E
T
N
224A K EI 2 100G M VK L EN L 17
R T Q E
N
P
234T L R K YL 2 218L Q EG K K 17
R Q V A
L R
2 S N P R EE 1 11 K D MS G AR L 16
S E Y
L
3 N P R S EE 1 44 R L RF E ST 16
L K Q M
E
4 P R S L EK 1 90 G E GF L K E 16 .
E Y R G
E
R S L E KY 1 96 K E DG E MV 16
E D G K
E
16 G A L IL 1 136Q D PG E TV G 16
R A C R
L
17 A L LC 1 171T E GY I A R 16
R A V V Y
L
I
20 A L I L TK 1 183K S CF I QT L 16
C A G
V
21 L I L C K 1 190T V V F TK 16
V A L D R
T R
36 L D A MF 1 198R G HI L EL L 16
L R K
E
H
39 L E H QL 1 211R M L V 16
M R R A
F E
R A
E
46 L R F E MK 1 227T P EI Q ST L 16
S R N
T
92 E G F L ED 1 19 L L IL C VT K 15
K G A
G
95 L K G E EM 1 31 G E ED L DA 15
D V S L
G
106E N F LN 1 40 E M FR Q LR F 15
L E N H
A
107N L F E NN 1 57 T QF Q EE L 15
A K A
L E
108L F E K 1 60 Q Q EE L EK F 15
A N F
L
N
N
116N C Q AK 1 101E KL E NL F 15
A P M
L V
R
117C Q L KP 1 115K C QA L RA K 15
A R K N
A
122A P K II 1 145G E IV VI K 15
K V Q D M
Y
139P G E T GD 1 154D P QT I PT Y 15
V E S
G
155S P Q T TY 1 203L L LT E VT R 15
I T E
P
164D A L H ST 1 45 Q R FE S TM K 14
V V L
Y
167H V S EG 1 81 C F V L MA H 14
Y T Y A V
V
168V Y S T GY 1 121R K PK I I 14
V I A V
E Y
181D Q K G FI 1 ~ 144G D EI V MV I 14
S Q G
C
184G S C F TL 1 189Q L V V FT K 14
I V T D
Q
193D V F T KG 1 215E E LV Q EG R 14
K H A
R
209V T R EA 1 217E V QE G K 14
R E L A
M R
A
216A E L V GK 1 231I S TL R K L 14 .
Q A Q R
E
225R K T N IQ 1 233S L RK LY L 14
P S T R
E
228N P E I TL 1 10 E Y DM S GA 13
Q R K R
S
13 D S G LA 13
M A L
R
15 S A RL A I L 13
G L
21 L L CV T K R 13
I A
36 L A LE H MF R 13
D
213
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 mers 13P1F11 mers
v.l: v.l:
HLA-B*2705 HLA-B*2705
nona nona
Pos1 2 3 4 56 7 score Pos1 3 4 78 9 score
8 2 5 6
9
74 S R E D PV S 13 151V K D QT I 8
C I S P
A
83 F V L MA 13 184G C F TL V $
V H S I Q
G
R
94 F L K G ED G 13 201H L E TE V $
E I L L
M
104K E N LF E 13 226K N P QS T $
L A T E I
L
113N N K CQ A 13 27 K R E EE D 7
N L A G S
R
170S T V E GY I 13 66 E ' F ID S 7
A K Q Q
Y A
172V E G Y IA 13 91 R G F GE D 7
Y E L K
R
H
187F I Q T LV D 13 106E L F N 7
V N E A N
F L
223K A R K TN P 13 123K K V IQ A 7
E P Y I
I
228N P E I QS T 13 150M I K PQ T 7
L V D S
R
230E I Q S TL R 13 168V S T GY I 7
K Y V E
R
3 N P R S LE E 12 212R LV Q 7
E M
R A
E
A
E
6 S L E E EK Y 12 225R T N IQ S 7
D R P E
M
33 E E D L DA 12 16 G R L IL C 6
L A A L
E
H
38 A L E H MF R 12 47 R E S KR D 6
Q F T M
L
43 F R Q L RF E 12 89 H R E LK 6
S G G F G
T
7$ P S C AF V 12 118Q L R PK 6
V V A A K V
L
86 L M H GR E 12 131A R G RD P 6
A G C E Q
F
$7 M G RE G 12 141E V G EI V 6
A F T G D
H L
$$ A G R EG F 12 ~ 146D I V IK D 6
H L E M V
R
112L N K NC Q 12 152I D S TI P 6
N A R P Q
L
117C Q A RA K 12 176I QK 6
L P A G
K Y
R
H
D
129I Q A C RG E 12 186C I Q VD V 6
Q F T L
R
140G E T V GG D 12 219V E G K 6
E Q K A T
I R
142T V G G DE I 12 8 E E K MS G 5
V E Y D
M
178Y R H D QK 12 22 I C V AR E 5
G L T K
S
C
191L V D V FT K 12 29 R G S DL D 5
R E E E
it
193D V F T KR K 12 37 D FR Q 5
G A
H L
E
H
M
205L L T E VT R 12 50 S M K PT A 5
R T R D
M
34 E D L D A E 11 63 E L E QQ A 5
L H E K F
M
35 D L D A E H 11 68 F Q A SR E 5
L M Q I D
F
119A R A KP K 11 72 I S R PV S 5
L V D E D
Y
132C R G E QR D 11 92 E F L ED G 5
P G K G
G
159I P T Y TD A 11 103V L E FE A 5
L R N L
H
167H S TV E 11 108L E A K 5
V G F L N N
Y Y N
175Y I A Y RH D 11 122A P K II Q 5
Q R V Y
R
180H D Q K GS C 11 124P V Y QA C 5
F R I I
I
195F T K K H 11 126V I I CR G 5 ,
R G I Y Q A
L
224A R K T NP E 11 143V G D VM V 5
I G E I
Q
39 L E H M FR Q 10 147E V M K S 5
L I V I D
R
64 E L E K FQ Q 10 157Q I P TD A 5
A T T Y
I
79 V S C A FV 10 164D L H ST 5
V A V Y V
L
M
158T I P T YT D 10 165A H V TV E 5
A L Y S
L
161T Y T D A H 10 174G I A D Q 5
L V Y Y R
Y H
200G H I L EL L 10 185S F I LV D 5
T C Q T
E
232Q S T L RK R 10 18$I T L VF T 5
L Q V D
Y
20 A L I L CV T 9 196T R K IL E 5
K R G H .
A
93 G F L K GE D 9 214A A E QE G 5
G E L V
E
194V F T K K 9 216A L V GK 5
R G E Q E A'~
H
I
R S L E EE K 8 221E K A N P 5
Y G R K
D T
14 M S G RL A 8 222G P E 5
A L R
I A
R
K
T
N
1$ R L A IL C $ 1 M N P LE E 4
L V S R S
T
133R G E Q RD P $ 23 L V T E G 4
G C K A
E R
137" D P G ET V $ 30 E S E LD A 4
R G G E D
G
214
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
3P1F11 v.l: HLA-B*2705 nonamers
13P1F11
v.l:
HLA-B*2705
nonamers
Pos 1 3 4 5 67 8 9 score pos1 23 4 67 8 score
2 5 9
41 H F R Q LR F E 4 138D PG E VG G 2
M T D
42 M R Q L RF E S 4 162Y TD A HV Y 2
F L S
54 R P T A EQ F Q 4 169Y ST V GY I 2
D E A
55 D T A E QF Q E 4 173E GY I YR H 2
P A D
62 Q E L E ICF Q Q 4 182Q KG S FI Q 2
E C T
82 A V V L MA H G 4 207T EV T. RM 2
F R A
E
84 V L M A HG R E 4 208E VT R MA E 2
V R A
99 D E M V K E N 4 9 E EK MS G 1
G L Y A
D
102 M K L E NL F E 4 25 V TK EG S 1
V A E
R
109 F A L N K C 4 48 F ES T K D 1
E N N M R P
110 E L N K C Q 4 49 E ST M D P 1
A N N K T
R
111 A N K C Q A 4 58 A EQ F EE L 1
L N N Q E
114 N N C Q AL R 4 71 A ID S ED P 1
K A R V
116 N Q A L RA K P 4 85 V LM GR E 1
C A G
H
127 Y I Q CR G E 4 95 L KG E GE M 1
I A D V
130 Q C R G EQ R D 4 139P GE T GG D 1
A V E
134 G Q R D PG E T 4 209V . R E A 1
E T R M E
A
148 I V I K S P 4
V D ~
M
149 V I K S P Q 4 l3PiF11
M D v.l:
V HLA-B*2709
nonamers
153 K S P Q TI P T 4 Pos1 23 4 67 8 score
D 5 9
160 P Y T D A H V 4 53 K P A Q 23 Portion
T L R T E F of
D
163 T A L H VY S T 4 197K RK G IL E 23 SEQ
D H L ID
166 L V Y S TV E G 4 211R RM A L 23 NO:
H A E V 3;
E each
192 V V F T K K 4 17 A RL IL C 22 start
D R G A V
L
199 K H I L EL L T 4 28 A E G EE D 21 Position
G R S L is
202 I E L L TE V 4 specified
L T 210T RR M EA 20 h
A E
L
t
213 M E A E LV Q E 4 120L RA K K Y 19 e length
A P V I of
each
234 T R K LY L Q 4 121R K P Y I 15 peptide
L R A K I is
V 9
2 S P R S LE E E 3 ~ 198R K LE L 15 amino
N G L
H
I
12 Y M S G L A 3 31 G SE E LD A 14 acids,
D A D L the
R
26 T A R E GS E E 3 44 R QL R ES T 14 end
K F M position
32 S E D L DA L E 3 97 G ED G MV K 14 for
E E L each
52 M R D P TA E Q 3 peptide
K 100G EM E N 14 is
V L
K
L
the
65 L K F Q QA I D 3 11 K M G 13 start
E Y S A ositio
D R
L
p
70 Q I D S RE D P 3 75 R ED P SC A 13 n
A V F plus
eight
73 D R E D PV S C 3 90 G RE G LK G 13
S F E
77 D V S C AF V V 3 144G G E V V 13
P D I M I
80 S A V M 3 160P TY T H 13
C F V A D V
L 'A
L
155 S Q T I PT Y T 3 233S TL R RL Y 13
P K L
156 P T I P TY T D 3 15 S G R A I 12
Q A L L L
177 A R D QK G S 3 38 A LE H FR Q 12
Y H M L
181 D K S CF I Q 3 . 46 L RF E TM K 12
Q G S R
220 Q G K A RK T N 3 104K LE N FE A 12
8 L L
7 L E E K YD M S 2 140G ET V GD E 12
8 G I
24 C T K RE' S 2 179R HD Q S C 12
V A G K F
G
51 T K D PT E 2 183K GS C IQ T 12
M R A F L
56 P A Q FQ E E 2 184G SC F QT L 12
T E I V
61 F E E L EK F Q 2 23 I QS T RK 12
Q 1 L R
L
69 Q A I. SR E D 2 _ D MS G L A 11
Q D 13 A L
R
76 E P V S CA F V 2 74 S RE D VS C 11
D P A
98 E G E M VK L E 2 77 D pV S F V 11
D C V
A
105 L N L F EA L N 2 78 P VS C FV 11
E A V
L
128 I Q A C RG E Q 2 . 118Q AL R KP K 11
I A V
135 E R D P GE T V 2 136Q RD P ET V 11
Q G G
215
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.l: HLA-B*2709 nonamers
13P1F11 amers
v.l:
HLA-B*2709
non
Pos 1 2 3 6 89 score Pos1 3 4 5 7 8 score
4 7 2 6 9
5
223 K N EI 11 145G E I V V I 4
A P D M K
R
K
T
224 A R K P IQ 11 150M I K P Q 4
T E V D T
N S
227 T N P Q TL 11 167H Y S T E G 4
E S V V Y
I
4 P R S E KY 10 172V G Y I Y R 4
L E E A H
E
34 E D L L HM 10 174G I A Y H D 4
D E Y R Q
A
40 E H M Q RF 1O 189Q L V D F T 4
F L T V K
R
43 F R Q F ST 10 204E L T E T R 4
L E L V R
R
57 T Q EL 10 16 G R L A I L 3
A E A L C
E
Q
F
71 A I D E PV 10 19 L L I L V T 3
S D A C K
R
79 V S C V LM 10 20 A I L C T K 3
A V L V A
F
87 M A H E FL 10 33 E D L D L E 3
G G E A H
R
112 L N N C AL 10 37 D L E H F R 3
K Q A M Q
N
132 C R G R PG 10 59 E F Q E L E 3
E D Q E K
Q
158 T I P T A 10 63 E L E K Q Q 3
T D L E F A
Y
164 D A L TV 10 66 E F Q Q I D 3
H K A S
V
Y
S
178 Y R H K SC 10 84 V M A G R 3
D G V H E
Q L
186 C F I L V 10 107N F E A N N 3
Q V L L K
T D
195 F T K G IL 10 114N C Q L R 3
R H K A A
K N
201 H I L L EV 10 123K K Y I Q 3
E T P V I A
L
205 L L T RM 10 126V I I Q C R 3
E Y A G
V
T
R
6 S L E K DM 9 153K S P Q I P 3
E Y D T T
E
95 L K G G MV 9 176I Y R H Q K 3
E E A D G
D
101 E M V E LF 9 185S F I Q L V 3
K N C T D
L
141 E T V IV 9 188I T L V V F 3
G Q D T
G
D
E
142 T V G E VM 9 199K H I L L L 3
G I G E T
D
143 V G G I V 9 203L L L T V T 3
D V E E R
E M
187 F I Q V VF 9 216A L V Q G K 3
T D E E A
L
194 V F T K HI 9 222G R K P 3
K G K T E
R A N
14 M S G L LI 8 226K N P E Q S 3
A A T I T
R
35 D L D E MF 8 229P I Q S L R 3
A H E T K
L
60 Q F Q L KF 8 1 M N P R L E 2
E E S S E
E
64 E L E Q AI 8 10 E D M G A 2
K Q K S R
F Y
76 E D P C FV 8 12 Y M S G L 2
V A D A A
S R
86 L M A R GF 8 21 L L C V K 2
H E I T A
G R
94 F L K D EM $ 22 I C V T R 2
G G L K E
E A
135 E Q R G TV 8 23 L V T K R E 2
D E C A G
P
151 V I K P TI 8 _ 27 K E G E E 2
D Q A S D
S R
168 V Y S YI 8 41 H F R Q R F 2
T M L E
V
E
G
180 H D Q S FI 8 55 D T E F Q 2
K C P A Q E
G
R S L E YD 6 67 K Q Q A D S 2
E K F I R
E
47 R F E D 6 68 F Q I S R 2
S Q A D E
T
M
K
R
225 R K T E QS 6 72 I S R E P V 2
N I D D S
P
18 R L A L VT 5 73 D R E D V S 2
L C S P C
I
29 R E G E LD 5 80 S A F V L M 2
S D C V A
E
93 G F L E GE 5 81 C F V M 2
K D A V A
G L H
106 E N L A NN $ 82 A V L 2
F L F V M
E A
H
G
125 K Q CR 5 83 F L M G 2
V A V A R
Y V H
I
I
133 R G E GE 5 92 E F L K E D 2
Q G G G
R
D
P
137 R D P T GG 5 96 K E D G M V 2
G V G E K
E
200 G H I L TE 5 103V E N F E 2
L L K L A
E L
212 R M A VQ $ 110E L N C 2
E A N Q
A K
E N
L
54 R D P FQ 4 111A N K C Q 2
T L N N A
A
E
Q
91 R E G K ED 4 115K C Q R 2
F G N A A
L L K
134 G E Q P ET 4 124P I Q A 2
R G K I C
D V
Y
216
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 mers 13P1F11
v.l: v.l:
HLA-B*2709 HLA-B*2709
nona nonamers
Pos1 2 3 45 6 7 8 score Pos1 2 3 4 56 8 score
9 7 9
129I Q A CR G E Q 2 207T E V T RR A 1
R M E
146D E I VM V I K 2 208E V T R RM E 1
D A A
148I V M VI K D S 2 209V T R R MA A 1
P E E
152I K D SP Q T I 2 217E L V Q EG A 1
P K R
156P Q T IP T Y T 2 218L V Q E GK R 1
D A K
157Q T I PT Y T D 2 21 V Q E G K K 1
A A T
R
159I P T YT D A L 2 221E G K A K N 1
H R T P
163T D A LH V Y S 2 23 E I Q S TL K 1
T R R
166L H V YS T V E 2 232Q S T L RK L 1
G R Y
169Y S T VE G Y I 2 23 T L R K RL L 1
A Y Q
173E G Y IA R H 2
Y D
177A R HD Q K G 2 13P1Fi1v.l:
Y S HLA-B*4402
nonamers
182Q K G SC F I Q 2 Pos1 2 3 4 56 8 score
T 7 9
193D V F TK K 2 75 R E D P VS A 26 Portion
R G C F of
H
213M A E AE L V Q 2 97 G E D G EM K 24 SEQ
E V L ID
214A E A EL V Q E 2 100G E M K N 22 NO:
G V L L 3;
E each
3 N P R SL E E EK 1 140G E T V GGD EI lg start
9 E E K M S G 1 53 K D P T Q 17 Position
Y A R A F is
D E ecified
s
p
24 C V T K R E G 1 119A L R KP 17 ,
A S A K the
V length
Y
30 E G S EE D L D 1 13 D S G R A 16 of
A M A L L each
49 E S T MK R D P 1 33 E E D L DA E 16 peptide
T L H is
9
50 S T M K D P T 1 38 A E H F Q 16 amino
R A L M R L
51 T M K D P T A 1 146D E I V V K 16 acids,
R E M I D the
58 A E Q FQ E E L 1 183K G S C FI T 16 nd
E Q L position
62 Q E E LE K F Q 1 197K R K G HI E 16 for
Q L L each
tid
i
69 Q Q A ID S R E 1 40 E H M F RQ R 15 e
D L F s
pep
the
start
70 Q A I DS R E D 1 63 E E L E KF Q 15 position
P Q A
88 A G RE G F L 1 104K L E N LF A 15 plus
H K E L eight
89 H G R EG F L K 1 154D S P Q TI T 15
G P Y
98 E D G EM K 1 158T I P T YT A 15
V L D L
E
99 D G E MV K L E 1 207T E V T RR A 15
N M E
102M V K LE N L F 1 216A E L V E K 15
E Q G A
109F E A LN N K N 1 229P E I Q ST R 15
C L K
117C Q A R K P 1 231I Q S T LR 15
L A K K
R
L
119A L R A P K V 1 233S T L R K Y 15
K Y R L
L
122A K P KV Y I I 1 15 S G A R LA I 14
Q L L
127Y I I Q R G 1 28 A E G SE D 14
A E R E L
C
128I I Q AC R G E 1 58 A E Q F QE L 14
Q E E
130Q A C RG E Q R 1 78 P V S C AF 14
D V
V
L
131A C R GE Q R 1 101E M K E L 14
D V L N F
P
138D P G ET G G 1 109F E A N N 14
V D L N C
K
147E I V I K 1 161T Y T D AL V 14
M D H Y
V S
149V V IK S P 1 170S T V E GY A 14
M D Q I Y
154D S P QT I P T 1 203L E L L TE T 14
Y V R
155S P Q TI P T Y 1 214A E A LV E 14
T E Q G
161T Y T DA H 1 220Q E G K T 14
L V A N
Y R
K
162Y T D AL H 1 232Q S T L RK L 14
V R Y
Y
S
165A L H V S T V 1 4 P R S L EE K 13
Y E E Y
170'S T V EG Y I A 1 31 G S E E DL A 13
Y D L
175Y I A YR H D Q 1 32 S E E D LD 13
K A
L
E
190T L V DV F T K 1 48 F E S T MK D 13
R R P
191L V D VF T K R 1 112L N K C A 13
K N N Q L
192V V FT K R K 1 187F I Q T LV V 13
D G D F
~02I L E LL T E V ~ ~ 198R R G H IL L 13
I T 1 E L
217
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 mers 13P1F11 mers
v.l: v.l:
HLA-B*4402 HLA-B*4402
nona nona
Pos1 3 4 56 7 89 score Pos1 2 3 45 6 7 8 score
2 9
227T P E IQ S TL 13 70 Q A I DS R E D 5
N P
$ E E K D M SG 12 71 A I D SR E D P 5
E Y V
9 E K DM S GA 12 81 C A F V L M A 5
E Y V H
11 K D M SG RL 12 88 A H G RE G F L 5
Y A K
35 D D A LE H MF 12 92 E G F LK G E D 5
L G
39 L H M FR Q LR 12 106E N L FE A L N 5
E N
60 Q Q E EL E KF 12 107N L F EA L N N 5
F K
62 Q E L EK F QQ 12 111A N NK C Q 5
E L N A
64 E E K FQ Q AI 12 115K C QA L R A 5
L N It
$7 M H G RE G FL 12 117C Q A LR A K P 5
A R
1OSL N L FE A LN 12 122A P K Y I I 5
E IC V Q
121R K P K II 12 131A C R GE Q R D $
A V P
Y
134G Q R DP G ET 12 136Q R D PG E T V 5
E G
144G D E IV I 12 143V G G DE I V M 5
G M V
V
172V G Y IA Y RH 12 174G Y I AY R H D 5
E Q
195F K R K H IL 12 177A HD Q K G 5 I
T G Y S
R
14 M G L A LI 11 186C F I QT L V D 5 I
S A V
R
29 R G S EE D LD 11 2O4E L L TE V T R 5
E R
86 L H GR E GF 11 230E I Q ST L R K 5
M R
A
151V K D SP Q TI 11 2 S N P RS L E E 4
I E
167H Y S TV E GY 11 12 Y D M SG L 4
V A A
R
179R D Q K S CF 11 16 G A LA I L 4 ,
H G R L C
7 L E E K MS 10 18 R L A LI L C V 4
E Y T
D
57 T E Q FQ E EL 10 30 E G S EE D L D 4
A A
65 L K F QQ A ID 10 46 L R F ES T M K 4
E R
91 R G F LK G ED 10 50 S T M KR D P T 4
E A
168V S T VE G YI 10 51 T RD P T A 4
Y M E
K
210T R M AE A EL 10 67 K F Q Q I D S 4
R A R
20 A I L CV KA 9 90 G R E GF L K G 4
L T E
120L A K PK V YI 9 126V Y I IQ A C R 4
R G
194V T K RK G HI 9 127Y I I Q C R G 4
F A E
223K K N P EI 9 135E Q R P G E T 4
A T D V
R
17 A L A I L C $ 137R D P GE T V G 4
R L V G
180H Q K S C FI 8 150M I K S P Q 4
D G V D T
147E V I K DS 7 155S P Q TI P T Y 4
I M T
V
153K S P QT I PT 7 165A H VY S T V 4
D L E
2O0G I L EL L TE 7 171T V E GY I A Y 4
H R
226K N P EI Q ST 7 191L V VF T K R 4
T D K
21 L L C V K 6 208E V T RR M A E 4
I T A A
R
66 EK F Q Q,A I DS 6 209VT R RMA E AE 4
98 E G E M K E 6 219V Q E GK K 4
D V L A T
R
110E L N NICN CQ 6 5 R S L EE E K Y 3
A D
123K K YI I QA 6 19 L A IL C V T 3 .
P V L R
124P Y II Q C 6 ~ 23 L C TK R E 3
K A V A G
V
157Q I P TY T DA 6 43 F R Q LR F E S 3
T T
185S F I QT L VD 6 44 R Q L RF E S T 3
C M
193D F T K K GH 6 49 E S T MK R D P 3
V R ~ T
217E V Q EG K 6 72 I D S RE D P V 3
L A S
R
224A K T P E IQ 6 74 S R E DP V S C 3
R N A
1 M N P RS L EE 5 76 E D P VS C A F 3
S V
E Y D MS G AR 5 79 V S C A'FV L 3
R V M
34 E L D A E H 5 80 S C FV L M 3
D L M A V A
41 H F R QL R FE 5 82 A F V L M A 3
M V H
G
59 E F Q EE L ER 5 83 F V LM A H G 3
Q V R
218
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P 1F11 amers 213P1F11 amers
v.l: v.l:
HLA-B*4402 HLA-B*4402
non non
Pos 1 23 4 7 8 score Pos1 3 4 6 7 89 score
5 6 9 2 5
89 H GR E L K 3 25 V K E G SE 1
G F G T A
R
96 K GE D M V 3 26 T A R G S EE 1
G E R R E
103 V RL E F E 3 27 K R E S E ED 1
N L A A G
113 N NK N A L 3 36 L A L H M FR 1
C Q R D E
114 N KN C L R 3 37 D E M F RQ 1
Q A A A H
L
116 N CQ A A K 3 42 M R Q R F ES 1
L R P F L
118 Q AL R P K 3 55 D T A Q F QE 1
A K V P E
141 E TV G E I 3 56 P A E F Q EE 1
G D V T Q
142 T V I V 3 68 F Q A D S RS 1
G M Q I
G
D
E
160 P TY T L H 3 69 Q A I S R ED 1
D A V Q D
173 E GY I R H 3 84 V M H G RE 1
A Y D V A
L
182 Q RG S I Q 3 93 G L K E D GE 1
C F T F G
189 Q TL V F T 3 94 F K D G EM 1
D V R L G
E
190 T LV D T K 3 99 D E M K L EN 1
V F R G V
199 K G L L 3 128I Q R G EQ 1
H T I A
I C
L
E
202 I LE L E V 3 130Q C R E Q RD 1
L T T A G
211 R RM A E L 3 132C G E R D PG 1
E A V R Q
213 M AE A V Q 3 13 R E Q D P G8 1
E L E 3 G R
215 E AE L E G 3 _ D G E V G GD 1
V Q R 138P T
221 E GK A T N 3 ~ 13 P E T G DE 1
R K P G V
G
222 G RA R N P 3 145G E I V IR 1
K T E D V
M
225 R KT N I Q 3 152I S Q T IP 1
P E S K P
D
234 T LR K Y L 3 15 I T Y D A H 1
R L Q P T L
47 R FE S K R 2 17$Y H K SC 1
T M D R D G
Q
52 M KR D A E 2 181D K C F IQ 1
P T Q Q G
S
54 R DP T Q F 2
A E Q
61 F QE E K F 2 13P1F11
L E Q v.i:
HLA-B*5101
nonamers
73 D SR E V S 2 Pos1 3 4 6 7 89 score
D P C 2 5
77 D PV S F V 2 16 D L H S T 28 Portion
C A V A V V of
Y
85 V LM A R E 2 77 D V S A F V 26 SEQ
H G G P C V ID
102 M K L L F 2 121R K P Y II 23 NO:
V E N E A K 3;
V each
108 L FE A N K 2 144G D E V VI 23 start
L N N G I M
125 K Y I A C 2 223K R K P EI 23 Position
V I Q R A T is
N
129 I QA C E Q 2 specified,
R G R 118Q L R K P K 22 h
A A V l
t
148 I V S 2 37 D L E M F RQ 19 e
M P A H ength
V of
I each
K
D
156 P QT I Y T 2 138D G E G G 19 peptide
P T D P T D is
V 9
162 Y TD A Y 2 $7 M H G E G FL 1$ amino
L H S A R
V
163 T DA L Y S 2 143V G D I V V 18 acids,
H V T G E M the
164 D A S T 2 176I Y R D Q K 1 end
L V A H G g position
H
V
Y
166 L HV Y V E 2 19 L L I C V K 17 for
S T G A L T each
175 Y IA Y D Q 2 57 T E Q Q E EL 17 peptide
R H K A F is
176 I A Q K 2 the
Y G 151V K P Q TI 16 start
R I D osition
H S
D
184 G SC F T L 2 160P Y T A L H 16 p
I Q V T D V plus
eight
188 I QT L V F 2 55 D T A Q F QE 15
V D T P E
192 V DV F K 2 183K S C I Q TL 15
T K G G F
R
196 T KR K I L 2 15 S L IL 14
G H E G
A
R
L
A
201 H IL E T E 2 81 C F V L M H 14
L L V A V A
205 L LT E R R 2 120L A K K V YI 14
V T M R P
206 L TE V R M 2 168V S T G YI 14
T R A Y V
E
212 R L V 2 201H L E L T EV 14
M Q I L
A
E
A
E
228 N PE I T L 2 213M A L V QE 14
Q S R A E
E
3 N PR S E E 1 14 M G L A LI 13
L E R S A
R
6 S LE E D 1 17 A L A I L C 13
E K M R L V
Y
24 C VT K E G 1 99 D E M K L EN 13
A R S G V
219
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 mers 13P1F11 mers
v.l: v.l:
HLA-B*5101 HLA-B*5101
nona nona
Pos1 2 3 45 6 78 score Pos1 2 3 4 5 6 7 score
9 8 9
110E A L N K C 13 104K L E N L F E 7
N N Q A L
123K K VY I IQ 13 108L F E A L N N 7
P A K N
155S P Q TI P TY 13 119A R A K P K 7
T L V Y
159I P T YT D AL 13 167H V Y S T V E 7
H G Y
194V F T K K H 13 187F I Q T L V D 7
R G I V F
13 D M S GA R LA 12 198R K G H I L E 7
L L L
.
16 G A R LA IL 12 199K H L E L L 7
L C G T
I
27 K EG S EE 12 204E L L T E V T 7
A D R R
R
7Q Q A I DS R ED 12 210T R R M A E A 7
P E L
$9 H G R EG F LK 12 219V Q E G K A R 7
G K T
95 L K G ED G EM 12 10 E K Y D M S G 6
V A R
173E G Y IA Y RH 12 23 L C V T K A R 6
D E G
215E A E LV Q EG 12 63 E E L E K F Q 6
K Q A
227T N P EI Q ST 12 98 E D G E M V K 6
L L E
228N P E IQ S TL 12 103V K L E N L F 6
R E A
3 N P R SL E EE 11 107N L F E A L N 6
K N K
64 E L E KF Q QA 11 133R G.E Q R D P 6
I G E
7$ P V S CA F VV 11 142T V G G D E I 6
L V M
130Q C RG E QR 11 . 161T Y T D A L H 6
A D V Y
135E Q R DP G ET 11 181D Q K 6
V G
S
C
F
I
Q
180H D Q K S CF 11 189Q T L V D V F 6
G I T K
186C F I QT L V 11 212R E A E L 6
D M V Q
V A
231I Q S TL R K 11 5 R S L E E E K 5
R Y D
L
76 E D P VS C AF 10 7 L E E E K Y D 5
V M S
96 K G E DG E MV 10 20 A I L C V T 5
K L K A
97 G E D GE M VK 10 34 E D L D A L E 5
L H M
140G E T VG G DE 10 35 D L D A L E H 5
I M F
146D E I V V IK 10 60 Q F Q E E L E 5
M D K F
158T I P TY T DA 10 ~ 72 I D S R E D P 5
L V S
211R R M E A EL 10 79 V S C 5
A V A
F
V
V
L
M
233S T L RK R LY 10 80 S C A F V V L 5
L M A
30 E G S EE D LD 9 122A K P K V Y I 5
A I Q
31 G S E ED L DA 9 165A L H 5
L V
Y
S
T
V
E
3$ A E H F RQ 9 185S C F I Q T L 5
L M L V D
112L N KN C QA 9 188I Q T L V D V 5
N L F T
139P G E T G GD 9 192V V F T K R 5
V E D K G
141E T V GG D EI 9 202I L E L L T E 5
V V T
154D S P QT I PT 9 218L V Q E G~K A 5
Y R K
184G S C FI Q TL 9 230E I Q S T L R 5
V K R
197K K I LE 9 12 Y D M S G A R 4
R G L L A
H
11 K D MS G AR 8 18 R L A L I L C 4
Y L V T
28 A E GS E ED 8 22 I L C V T K A 4
R L R E
46 L R F ES T MK 8 44 R Q L R F E S 4
R T M
71 A I D SR E DP 8 48 F E S T M K R 4
V D P
73 D S R ED P VS 8 61 F Q E E L E K 4
C F Q
92 E G F LK ED 8 84 V V L M A H G 4
G G R E
100G E M K EN 8 106E N F E A L 4
V L L L N N
190T L V V F TK 8 114N K C Q A L 4
D R N R A
193D V F T. R KG 8 116N C Q A L R A 4
K H K P
195F T K K G HI 8 125K V I I Q A 4
R L . C R
Y
203L E L LT E VT 8 136Q R D P G E T 4
R V G
205L L T EV T RR 8 147E I V 4
M M
V
I
K
D
S
221E G K AR K N $ ~ 162Y T D A L H V 4
T P Y S
21 L I L CV T KA 7 166L H 4
R V
Y
S
T
V
E
G
220
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 mers' 13P1F11
v.l: v.l:
HLA-B*5101 HLA-B*5101
nona nonamers
Pos1 3 45 6 78 9 score Pos1 2 3 45 6 78 score
2 9
171T E GY I A R 4 101E M KL E NL 2
V Y V F
191L VF T K K 4 105L E N LF E A 2
V R L
D N
200G I LE L LT E 4 111A L N NK CQ 2
H N A
206L E VT R RM A 4 115K N C QA L RA 2
T R
214A A EL V QE G 4 117C Q A R A KP 2
E L K
216A L VQ E GK 4 129I Q A CR G EQ 2
E A R
220Q G K KT N 4 131A C R GE Q RD 2
E A P
R
2 S P RS L EE E 3 132C R G EQ R DP 2
N G
41 H F RQ L RF E 3 148I V M VI K S 2
M D P
47 R E ST M K D 3 149V M IK SP 2
F R V D Q
50 S M K D PT A 3 150M V I KD 5 PQ 2
T R T
51 T K D P TA 3 156P Q T IP T YT 2
M R E D
56 P A EQ F QE E 3 157Q T I PT Y TD 2
T A
66 E F QQ A ID S 3 169Y S T E G YI 2
R V A
68 F Q AI D SR E 3 174G Y I AY R HD 2
Q Q
74 S E DP V SC A 3 175Y I A YR H DQ 2
R R
83 F LM G R 3 182Q ItG SC F IQ 2
V A T
V H
85 V M A G RE G 3 207T E V TR R MA 2
L H E
90 G E GF L KG E 3 222G R A RK NP 2
R T E
93 G L KG E DG E 3 224A R K N P EI 2
F T Q
102M K LE N LF E 3 24 C V T KA R EG 1
V S
109F A LN K C 3 26 T K,A E G SE 1
E N N R E
126V I IQ CR G 3 32 S E E DL D AL 1
Y A E
127Y I QA RG E 3 42 M F R QL R FE 1
I C S
128I Q AC R GE Q 3 45 Q L R FE S TM 1
I It
137R P GE T VG G 3 49 E S T MK DP 1
D R T
145G E IV I R 3 67 K F Q Q I DS 1
D M A R
V
152I D SP Q TI P 3 69 Q Q A ID S RE 1
K D
163T A H V S T 3 82 A F V VL M 1
D L Y A
H
G
170S V EG Y IA 3 113N K C Q AL 1
T Y N N R
172V G YI A R 3 124P R YI I QA 1
E Y H V C
178Y H DQ K GS C 3 134G E Q R P GE 1
R D T
196T R K IL E 3 153K S PQ T IP 1
FC G D T
H
209V R RM A EA E 3 177 <
T
229P I QS T LR 3
E R
234T R KR L YL Q 3 TABLE s
L XIXA, I
part nonamer
2:
MHC
Clas
1 M N PR S LE E 2 ~ anal 213P1F11 (aa
S sis v.2 1-230
of
4 P S LE E EK Y 2 13P1F11
R v.2:
HLA-A*0201
nonamers
6 S E EE K 2 Pos1 2 3 45 6 78 score
L Y 9
D
M
$ E E K MS G 2 38 D M I RK 22 Portion
E Y A_ of
D H
A
L
25 V K E GS E 2 20 S E A P N PP 1$ SEQ
T A P L ID
R
33 E D LD A E H 2 8 G P T PF Q DP. 13 NO:
E L L 5;
each
start
36 L A E H MF R 2 17 Y L P SE A P 13 position
D L P N is
39 L H MF R QL R 2 45 A L S RP W WM 13 specified,
E C
40 E M FR Q LR F 2 10 T P F QD P LY 12 the
H L length
43 F Q LR F ES T 2 35 S P T DM I RK 12 of
R A each
52 M R DP T AE Q 2 1 H V Y ST V G 10 peptide
K E P is
9
53 K D PT Q F 2 4 S T V G P TP 10 amino
R A E F
E
54 R P T QF Q 2 15 P L Y LP S EA 10 acids,
D A P the
E sition
d
59 E F QE E LE R 2 27 P L W NS _QDT 10 po
Q S en
for
each
65 L K FQ Q AI D 2 32 Q D T SP T DM 10 peptide
E I is
75 R D PV S CA F 2 39 M I R KA L 10 the
E H S start
A
86 L A G EG F 2 44 H A L SR P WW 10 position
M H R M
94 F K GE D GE M 2 51 W M C SR R GK 10 plus
L D eight
221
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
213P1F11 mers 13P1F11
v.2: v.2:
HLA-A*0201 HLA-A1
nona nonamers
Pos1 2 34 67 8 9 score pos1 2 34 5 67 8 score
5 9
$ T EG T_P F Q 9 9 P T PF Q DP L 25 Portion
V P Y of
13 Q D PL LP S E 8 12 F Q DP L YL P 21 SEQ
Y S ID
16 L Y LP EA P P 8 36 P T DM I RK 17 NO:
S A 5;
H each
14 D P LY PS E A 7 19 P S EA P PN P 15 start
L P iti
n is
37 T D MI K 7 5 T V G P TP F 12 o
R A E Q Pos
H specified
A
52 M C SR GK I 7 31 S Q DT S PT D 12 the
R D M length
54 S R RG I S W 7 4 S T VE G PT P 10 of
K F each
D
29 W N SQ _TS P T 6 33 D T SP T DM I 10 peptide
D R is
9
31 S Q DT PT D 6 20 S E AP P N_PP $ amino
S M L
41 R K S R P 6 22 A _PPN P P_LW g acids,
A N the
H
A
_L
42 K A HA 5R P W 6 34 T _SPT D MI R g end
L R position
h
f
2 V Y ST _EG P T 5 46 L S RP W WM_C 8 or
V S eac
tide
is
e
12 F Q DP Y_L P S 5 3 Y _STV E GP T 7 P
L P p
the
start
18 L P SE PP N P 5 54 S R RG K I S 7 position
A D W
23 P P NP LW N S 5 10 T P FQ D PL Y 6 plus
P L eight
28 L W NS DT S P 5 21 E A P N PP L 6
Q P W
33 D T SP M I R 5 40 I R L S 6
T R R
D A
H
A
43 A L RP W W 5 53 C S RR G KD I 6
H S S
A
3 Y S TV _GP T P 4 6 V GP T PF Q 5
E E D
11 P F QD LY L P 4 23 P _PNP P LW N 5
P S
21 E A PP PP L W 4 7 E _GPT P FQ D 4
N P
22 A PN PL W 4 $ G P TP F QD_P 4
P P N L
30 N S QD _SP T D 4 16 L Y LP S EA_P 4
T P
40 I R KA L S R 4 24 P N PP L WN S 4
H Q
A_
46 L S RP WM C S 4 30 N S QD T SP T 4
W D
47 S R PW C S R 4 35 S P TD M IR K 4
W A
M
55 R R GK IS W 4 45 A SR P WW M 4
D N L C
6 V E GP PF Q D 3 2 V ST G P 3
T Y V T
E
24 P N PP W S Q 3 11 P F QD P LY L 3
L N P
26 P P LW _SQ D T 3 17 Y _LPS E AP P 3
N N
50 W MC RR G IC2 51 W CS R RG K 3
W S M_ D
53 C S R _K I S 2 15 P L YL P S_EA 2
R D P
G
9 P T PF D_P L Y 1 28 L _WNS Q DT S 2
Q P
36 P T DM RK H 1 32 Q D TS P T_DM 2
I A I
48 R P WW _CS R R 1 39 M _IRK A HA L 2
M S
7 E G PT FQ D P -2 43 A L S RP W 2
P H W
A
19 P S EA PN P P -2 47 S R PW W MC S 2
P R
49 P W WM SR R G -2 1 H YS T VE G 1
C V P
13 Q PL Y LP S 1
D E
25 N P PL W NS Q 1
D
27 P L W S QD T i
N S
50 W _W C S RR G 1
M R
$2 M C SR R GK 1
D
I
222
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 rs 13P1F11
v.2: v.2:
HLA-A26 HLA-A3
noname nonamers
Pos 1 23 4 56 7 89 score Pos1 3 4 56 7 8 score
2 9
9 P TP F QD P LY 23 Portion 4 I K A HA__LS 17 Portion
of R R of
4 S TV E GP T PF 22 SEQ 45 A S R PW W M 17 SEQ
ID L C ID
33 D TS P TD M IR 19 NO: 15 P Y L PS E A 16 NO:
5; L P 5;
each each
38 D MI R K L 18 start 27 p W N SQ D T 15 start
A i L S iti
H i i
A i
on pos
1 H VY S TV E GP 16 pos 1 H Y S TV E G 14 on
t V P s
s specified
ecified
s
p ,
T VE G PT P FQ 13 the 39 M R K A 14 the
length I A L length
H S
56 R GK IS W F 13 of each 17 Y P S EA P P 13 of each
D N L N
7 E GP T PF Q DP 12 peptide 5 T _EG P_T_PF 12 peptide
is V Q is
9 9,
20 S EA P PN P PL 12 amino 3g D I R K H_ 11 amino
M A_ A
L
36 P TD M IR K 12 acids, 4g R W W MC S R 11 acids,
A the P R the
H
end - end
39 M IR K A S 12 position 50 W C S_RR G 11 position
H W R
A _M f
L h
T PF Q DP L YL 11 for 4 S V E G_P_TP 10 or eac
each T F tide
i is
tid e
11 P FQ D PL Y LP 11 e 34 T P T D_MI R 10 p
s S R p
pep the
the start
start
17 Y LP S EA PN 11 position 56 R K IS W N 10 position
P G D F
8 G PT P FQ D PL 10 plus 22 A P N PP L W 9 plus
eight P N eight
21 E AP P NP P LW 10 54 S R G K I S 9
R D W
31 S QD T SP T DM 10 16 L L P SE A P 8
Y P
14 D PL Y LP S EA 9 33 D S P TD M I 8
T R
P LY L PS E AP 9 41 R A H A_LS R 8
R P
27 P LW SQ D TS 9 52 M S R RG K 8
N C D
I
44 H AL S RP W WM 9 53 C R R GK I 8
S D S
45 A LS R PW MC 9 55 R G K DI _SW 8 .
W R N
12 F QD P LY L PS 8 3 Y T E_GP T 7
S V P
13 Q DP L YL P SE - 6 V _GP TP F Q 7
7 8 D
23 P PN P PL W S 7 25 N P L WN S Q 7
N P D
41 R KA L S RP 7 43 A SR P W 7
H H W
A A
L
34 T SP T DM I RK 6 46 L R P WW M C 7
S S
35 S PT D MI R K 6 47 S P W WM C S 7
A R R
47 S RP W M C SR 6 9 P P F QD P L 6
W T Y
55 R RG K I S WN 6 12 F D P LY _LP 6
D Q S
24 P NP P LW N SQ 5 14 D L Y L_P_SE 6
P A
16 L YL P SE A PP 4 36 P D M IR K A 6
T H
18 L PS E AP P NP 4 37 T M I RK 6
D A
H
A
54 S RR G KD I SW 4 44 H S RP W W 6
A M
L
6 V EG P TP F QD 3 13 Q P L Y_L_PS 5
D 8
N PP L WN S QD 3 20 S P P P P 5
S N L
A
28 L WN S QD T SP 3 24 P P P LW N S 5
N Q
43 A L SR P WW 3 30 N Q D TS P T 5
H S D
A
46 L SR P WW CS 3 42 K S R P 5
M A W
H
A
L
29 W S Q DT S PT 2 10 T F Q DP L Y 4
N P L
N SQ D TS P TD 2 21 E _PP NP _PL 4
A W
32 Q DT S PT MI 2 51 W _CS RR _GK 4
D M D
37 T DM I RK A 2 8 G _TP F_QD P 3
H P L
A
I RK L SR 2 19 P E A PP N P 3
A S P
H
A
48 R PW W C S RR 2 23 P N P PL W N 3
M P S
W WM C SR R GK 2 28 L N S Q T S 3
W D P
51 W MC S RR G K 2 29~W S Q DT S P 3
D N T
3 Y ST V G P TP 1 31 . D T SP T D 3
E S M
Q
22 A PP N PP L WN 1 32 Q T S PT D M 3
D I
26 P PL W S Q DT 1 2 V S T V G P 2
N Y E T
42 K A S R PW 1 18 L S E 'AP P N 2
H P P
A
L
49 P WW M CS R G 1 26 P L W NS _QD 2
R P T
35 S T D MI _RK 2
P A
11 P Q D PL Y L 1
F P
223
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11
v.2:
HLA-B*08
nonamers
13P1F11 : 2 mers pos1 2 5 6 78 9 score
v.2 HLA-B*070 nona 3
4
Pos1 3 45 6 7 8 score 38 D M K A A L 21 Portion
2 9 2 H of
R
$ G T PF Q D P 22 Portion 54 S R R D IS W 18 SEQ
P L of R ID
G
T F QD P L Y 22 SEQ g G P F Q DP L 17 NO:
P L ID T S;
P each
S A PP N P P 17 NO: 10 T P D P LY L 16 start
E L 5; F i
each Q iti
22 A P NP P L W 17 start 56 R G I S WN F 16 on
P N i K s
i D pos
i specified
pos ,
14 D L YL P S E 16 t 52 M C R G K I 15 the
P A on S D length
s R
s
ecified
26 P L WN S Q D 16 p 20 S E P N PP L 13 of
P T , A each
the P
length
35 S T DM I R K 16 of 44 H A R P WW 13 peptide
P A each' L M is
S 9
23 P N PP L W N 14 peptide 40 I R H A LS R 11 amino
P S is IC
9 A
18 L S EA P P N 13 amino 4 S T G P TP F 10 acids,
P P V the
E
3$ D I RK 11 acids, 39 M I S 10 end
M A the R position
H K h
A A f
L H
A
L
48 R W WM C S R 11 end 35 S P M I RK A 9 or
P R position T eac
D tide
is
e
N P LW N S Q 10 for 14 D P L P SE A $ P
P D each L p
tide Y the
is start
e
29 W S QD T S P 10 p 15 P L P S EA P 8 position
N T p Y
the L
start
32 Q T SP T D M 10 position 18 L P A P P P 8 plus
D I S N eight
E
2 V S TV E G P 9 plus 25 N P W N SQ D $
Y T eight P
L
52 M S RR G K 9 37 T D R K A $
C D M A
I I H
4 S V EG P T P 8 51 W M R R GK $ i
T F C D
S
5 T E GP T P F $ 17 Y L E A PP N 6
V Q P
S
31 S D TS P T D $ 21 E A N ~PPL 6
Q M P W
P
37 T M IR K $ 22 A P P P LW ,6
D A P N
H N
A
45 A S RP W W M 7 23 P P P L WN S 6
L C N
P
56 R K I S W N 7 26 P P S QD T 6
G D F L
W
N
44 H L SR P W W 6 27 P L S Q DT S 6
A M W
N
7 E P TP F Q D 5 32 Q D P T DM I 6
G P T
S
12 F D PL Y L P 5 45 A P W WM C 6
Q S L
S
R
19 P E AP P P 5 46 L S W W MC S 6
S N P R
P
43 A A LS R P W 5 48 R P M C SR R 6
H W W
W
15 P Y LP S E A 4 53 C S G K I S 6
L P R D
R
39 M R KA H A 4 42 K S RP W 4
I L A
S H
A
L
40 I K AH S 4 1 H V T V EG P 2
R A R Y
L S
54 S R GK I S 4 7 E G P F Q P 2
R D W P D
T
11 P Q DP L Y L 3 31 S Q S P TD M 2
F P D
T
16 L L PS E A P 3 36 P T I R K 2
Y P D A
M H
21 E P PN P P L 3 47 S R M CS R 2
A W P
W
W
33 D S PT D M I 3 2 V GP T 1
T R Y
S
T
V
E
36 P D MI R K 3 3 Y S G PT P 1
T A T
H V
E
42 K H S R P 3 6 V E T P FQ D 1
A A W G
L P
46 L R PW W M C 3 12 F Q L Y LP S 1
S S D
P
55 R G K I S W 3 13 Q D Y L PS E 1
R D N P
L
3 Y T VE G P T 2 43 A H S R PW W 1
S P A
L
6 V G PT P F Q 2 55 R R D I SW N 1
E D G
K
17 Y P SE A P P 2
L N
41 R A L S R 2
R H P
A
50 W M CS R R G 2
W R
53 C R RG K I 2 '
S D S
1 H Y ST G 1
V V P
E
9 P P FQ D P L 1
T Y
13 ~Q P LY L P S 1
D E
24 P P PL W S 1
N N Q
28 L SQ D T S 1
W P
N
N Q DT S P T 1
S D
~1 W C SR R G K 1
M D
224
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.2: HLA-B*1510 nonamers
Pos 1 23 4 56 7 9 score 13P1F1I amers
8 v.2:
HLA-B*2705
non
2 S EA P PN P L 15 Portion Pos1 23 4 56 7 89 score
P of
$ G PT P FQ D L 13 SEQ 4 I RK A L SR 24 Portion
P ID A of
H
T PF Q DP L L 13 NO: 47 S RP W WM C SR 21 SEQ
Y 5; ID
each
43 A HA L SR P W 13 start 55 R RG K I S WN 20 NO:
W i D 5;
i each
pos start
38 D MI R KA 11 t 56 R GK IS W NF 19
H on is D
A s
L ecified
p position
4 5 TV E GP T F 9 , 48 R PW W MC S RR 17 is
P the if
length d
31 S QD T SP T 7 of each 4 S TV GP T PF 16 s ec
D E ie
M p
the
length
44 H AL S RP W 7 peptide 8 G PT P FQ D PL 16 of each
W is
M 9
56 R GK IS W F 7 amino 10 T PF Q DP L YL 16 peptide
D N is
9
21 E AP P NP P W 6 acids, 54 S RR G KD I SW 16 amino
L the
end
$ T VE G PT P Q 5 osition 20 S EA P PN P PL 14 acids
F p the
3 Y ST V EG P P 4 for 38 D I R K H A 14 end
T each M A L position
a tide
is
18 L PS E AP P P 4 phe 33 D TS P T M IR 13 t d
N start D li
p p
34 T SP T DM I IC4 position 34 T SP T DM I RR 13 e
R s
the
start
35 S PT D MI R A 4 plus 31 S QD T SP T DM 11 position
K eight
49 P wW M CS R G 4 44 H AL S RP W WM 11 plus
R eight
6 V EG P TP F D 3 50 W M C SR R GIt11
Q W
19 P SE A PP N P 3 9 P TP F QD P LY 10
P
23 P PN P PL W S 3 36 P TD M IR K H 9
N A
30 N SQ D TS P D 3 41 R It H L S RP $
T A A
33 D TS P TD M R 3 32 Q DT S PT D MI 7
I
41 R RA L S P 3 23 P PN P PL W NS 6
H R
A
50 W W C SR R R 3 52 M CS R RG K I 6
M G D
7 E GP T PF Q P 2 14 D PL Y LP S EA 5
D
11 P FQ D PL Y P 2 15 P LY L PS E A 5
L P
12 F QD P LY L S 2 16 L YL P SE A PP 5
P
14 D PL Y LP S A 2 35 S PT MI R K 5
E D A
P LY L PS E 2 5 T VE G PT P FQ 4
A
P
16 L YL P SE A P 2 13 Q DP L YL P SE 4
P
17 Y LP S EA P N 2 18 L PS E AP P NP 4
P
22 A PP N PP L 2 24 P NP P LW N SQ 4
W
N
24 P NP P LW N Q 2 25 N PP L W S QD 4
S N
29 W NS Q DT S T 2 27 P LW N SQ D TS 4
P
36 P TD M IR K H 2 28 L S QD T SP 4
A W
N
37 T DM I RK 2 42 K H A S R PW 4
A A L
H
A
40 I RK R 2 43 A H L SR P WW 4
A A
H
A
L
S
42 K AH A LS R W 2 1 H S T E GP 3
P V V
Y
45 A LS R PW W C 2 3 Y ST EG P TP 3
M V
46 L SR P WW M S 2 11 P FQ D PL Y LP 3
C
48 R PW C S R 2 12 F QD P LY L PS 3
W R
M
53 C SR R GK S 2 17 Y LP S EA P PN 3
D
I
55 R RG K I S 2 22 A P N PP L WN 3
D W P
N
1 H VY S TV E P 1 26 P PL W NS Q DT 3
G
2 V S T VE G T 1 29 W S Q DT S PT 3
Y P N
9 P TP F QD P Y 1 30 N SQ D TS P TD 3
L
13 Q DP L YL P E 1 39 M IR K AH A LS 3
S
26 P PL W NS Q T 1 . 45 A S R PW W MC 3
D L
27 P LW N SQ D S 1 49 P W CS R RG 3
T W
M
32 Q DT S PT D I 1 53 C SR GK IS 3
M R D
39 M IR K S 1 6 V G P TP F QD 2
A E
H
A
L
47 S RP W WM C R 1 19 P SE A~PP N PP 2
S
51 W MC S R G 1 21 E AP P NP P LW 2
R K
D
52 M CS R G K I 1 37 T DM I RK A 2
R D A
H
54 S RR G K I W 1 I 46 L SR P WW M CS 2
I D S
225
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.2: HLA-B*2705 nonamers
Pos 1 2 34 5 67 8 9 score 13P1F11
v.2:
HLA-B*4402
nonamers
51 W M CS R RG K D 2 Pos 1 2 3 45 6 8 score
7 9
2 V Y ST V EG P T 1 2 S E A PP N P 24 Portion
P L of
7 E G PT P FQ D P 1 21 E A P PN P L 19 SEQ
P W ID
3$ D' I RK lg NO:
M A S;
H each
A
L
l3Pi_ 6 V G PT P Q 15 start
2: E F D
HLA-B*2709
nonamers
F11
v
. ps'tion
is
Pos 1 2 34 5 67 8 9 score 43 A LS R W 15 specified,
t H P W
A
$ G P TP F QD P L 15 Portion 10 T P F QD P Y 14 the
of L L length
55 R R GK D IS W N 15 SEQ $ G P T PF Q P 13 of each
ID D L
T P FQ D PL Y L 14 NO: 52 M C S RR G D 13 peptide
5; K I is
each 9
56 R G KD I 5W N F 14 std 54 S R R GK D S 13 amino
I W
position 4 S T V EG P P 12 acids,
is T F the
40 I R K AL S R 13 specified, end
A position
H
S E AP P NP P L 12 the 9 P T P FQ D L 12 f
length P Y h
38 D M IR K L 12 of 42 K H A S P 12 or eac
A each A L R W peptide
H is
A
44 H A LS R PW W M 11 peptide 56 R G K DI S 12 the
is W start
9 N
F
32 Q D TS P TD M I 10 amino 32 Q D T SP T M 9 position
D I
47 S R PW W MC S R 10 acids, 22 A P P NP P W 7 plus
the L N eight
nd
54 S R RG K I 5 W 10 osition 35 S P T DM I K 7
D P R A
4 S T VE G PT P F g for 36 P T D MI R 7
each K
A
H
Peptide
is
31 S Q DT S PT D M $ 7 E G P TP F D 5
the Q P
start
52 M C SR R GK D I 8 position 12 F Q D PL Y P 5
L S
41 R K AH A LS R P 5 plus 24 P N P PL W S 5
eight N Q
48 R P WW M CS R R 5 45 A L S RP W M 5
W C
1 H V YS T VE G P 4 16 L Y L PS E P 4
A P
16 L Y LP S EA P P 3 23 P P N PP L N 4
W S
17 ~YL PS E AP P N 3 25 N P P LW N Q 4
S D
3 Y S TV E GP T P 2 31 S Q D TS P D 4
T M
6 V E GP T PF Q D 2 33 D T S PT D I 4
M R
12 F Q DP L YL P S 2 11 P F Q DP L L 3
Y P
14 D P LY L PS E A 2 13 Q D P LY L S 3
P E
15 P L YL P SE A P 2 15 P L Y LP S A 3
E P
18 L P SE A PP N P 2 30 N S Q DT S T 3
P D
21 E A PP N PP L W 2 , 34 T S P TD M R 3 ,
I IC
22 A P PN P PL W N 2 44 H L SR P W 3
A W M
23 P P NP P LW N S 2 46 L S R PW W C 3
M S '
26 P P LW N SQ D T 2 50 W W M CS R G 3
R IC
34 T S PT MI R K 2 51 W C SR R K 3
D M G D
42 K A L SR P W 2 55 R R G KD I W 3
A S N
H
43 A S RP W W 2 2 V Y S T P 2
H V T
A E
L G
5 T V G P TP F Q 1 5 T V E GP T F 2
E P Q
9 P T PF Q DP L Y 1 14 D P L YL P E 2
S A
11 P F QD P LY L P 1 17 Y L P SE A P 2
P N
13 Q D PL Y LP S E 1 18 L P S EA P N 2
P P
24 P N PP L WN S Q 1 19 P S E AP P P 2
N P
N P PL W S Q D 1 26 P P L W S 2
N N Q
D
T
2$ L W S Q DT S P 1 27 P L W S Q T 2
N N D S
29 W N SQ D TS P T 1 29 W S QD T P 2
N S T
N S Q T SP T D 1 37 T D M IR K 2
D A
H
A
33 D T SP T DM I R 1 40 I R K S 2
A R
H
A
L
S P T M IR K A 1 47 S R P WW M S 2
D C R
36 P T DM I RK H 1 1 H V ST G 1
A Y V P
E
A L SR P WW M C 1 3 Y S T VE G T 1
P P
46 L S RP W WM C S 1 39 M I R KA H 1
A
L
S
49 P W W C SR R G 1 41 R R A HA R 1
M L P
S
51 W M CS R RG K D 1 49 P W W MC S R 1
R G
226
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.2: HLA-B*4402 nonamers
TABLE
XIXA,
part
3:
MHC
Class
I
nonamer
Pos1 2 3 45 78 9 score anal 1F11 (aa
6 sis v.3 1-146
of213P
53 C S R RG I S 1 13P1Fi1 v.3: A*0201
K HLA- nonamers
D
Pos 1 2 45 6 78 9 score
3
13P1F11 v.2: mers 1 T L 5P F PY L 22 Portion
HLA-B*5101 P of
nona
Pos1 2 3 45 78 9 score 3 I Q CR G AT L l SEQ
6 A g ID
1 T P F QD LY L 20 Portion1 Y T QA C RG A 17 NO:
P of I 7;
each
14 D P L YL SE A 17 SEQ 2 I I AC R GA 15 start
P ID Q T
8 G P T PF DP L 16 NO: g A T PS P FP Y 10 position
Q 5; L is
each
35 S P T DM RK A 16 start 12 P S FP Y LS L 10 specified,
I P
the
Position len
is h
h
18 L P S EA PN P 15 specified,5 A C GA LP S 7 of
P R T ea
22 A P P NP LW N 13 the 6 C R T _LPS P 7 peptide
P length G is
A 9
25 N P P LW SQ D 13 of each4 Q A RG A_TL P 6 amino
N C
44 H A L SR WW 13 peptide8 G A LP S PF P 6 acids,
P M is T the
9
48 R P W W SR R 13 amino 11 L P PF P YL S 6 end
M S position
C
21 E A P PN PL W 12 acids, 7 R G TL P SP F 3 for
P the A . each
nd position - peptide
26 P P L WN QD T 12 is
S
for the
each start
38 D I RK A L 12 osition
M A s l
H
42 K H AL RP W 12 phe ~ us
A S art ei
ht
52 M C 5 RR K I 12 position
G D
23 P P N PP WN S 11 plus 13P1F11 nonamers
L eight v.3:
HLA-A1
32 Q D T SP M Z 10 Pos 1 2 45 6 78 9 score
T 3
D
7 E G P TP QD P 9 A _T PS P FP Y 26 Portion
F L of
1 H V Y ST EG P 7 _ SEQ
V ID
20 S E A PP PP L 7 NO:
N 7;
each
56 R G K DI WN F 7 start
S
16 L Y L PS AP P 5 position
E is
specified
30 N S Q DT PT D 5 the
5 length
33 D T S PT MI R 5 of
D each
34 T S P TD IR K 5 a tide
M is
9
40 I R K AH LS R 5 1 P S FP Y LS L 11 amino
A P
2 V y S TV GP T 4 5 A C GA T LP S 8 acids,
E R the
3 Y S T VE PT P 4 11 L P PF P YL S 6 end
G S
4 S T V EG TP F 4 1 T L SP F PY L 5 position
P P
15 P L Y LP EA P 4 Q RG A TL P 4 for
S A each
C
17 Y L P SE PP N 4 1 Y I QA C RG A 2 peptide
A I is
27 P L W NS DT S 4 2 I I AC R G 1 the
Q Q A start
T
31 S Q D TS TD M 4 3 I _Q CR G AT L 1 position
P A
6 V E G PT FQ D 3 8 G LP S _F P I plus
P A P eight
T
13 Q D P LY PS E 3
L
19 P S E AP P P 3
P
N
28 L W SQ TS P 3
N D
4 P W W MC RR G 3
S
S R R GK IS W 3
D
5 T G P FQ</
V T
E P
t
227
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
213P1F11 13P1 F11
v.3: v.3:
HLA-A26 HLA-t3~U70Z
nonamers nonamers
Pos 1 2 3 4 7 score Pos 1 2 3 4 5 6 7 score
6 8 8 9
9
A T L P F 23 Portion 10 T L P S P F P 15 Portion
S P P of Y L of
Y
T L P S P 23 SEQ I2 p S P F P Y L 15 SEQ
P F Y ID S L ID
L
Y I I Q R 14 NO: 3 I Q A C R G A 14 NO:
A C G 7; T L 7;
A each each
1 start
12 P S P F L 14 start 11 L p S P F P Y 13
P Y S L S
L
L P S 13 Position 2 I I Q A C R G 10 position
P is A T is
F
7 R G A T s
ecified
2 I I Q A G 11 the 5 A C R G A T L 10 the
C R A length P S length
T
3 I Q A C A 10 of 7 R G A T L P S 9 of
R G T each P F each
L
6 C R G A P 6 peptide 1 Y I I Q A C R 6 peptide
T L S is G A is
P 9 9
5 A C R G L 2 amino $ G A T L P S P 5 amino
A T P F P
S
acids, g A T L P S P F 4 acids,
the P Y the
end 6 C R G A T L P 3 end
position S P position
h 4 Q A C R G A T 1 p
L p p
each
peph'de a
s tide
is
the the
start start
position position
plus lus
eight ei
ht
11 L P S P Y 2
F P L
S
13P1 Fii
v.3:
HLA-B*08
nonamers
Pos 1 2 3 4 5 6 7 score
8 9
3 I Q A C R G A 19 Portion
T L of
10 T L P S P F P 16 SEQ
Y L ID
12 P S P F P Y L 10 NO:
. S L 7;
each
2 I I Q A C R G 6 start
A T
$ G A T L P 1 position
P S F A T L P S 6 is
P
5 A C R G specified,
7 R G A T L P S 6 the
P F length
13P 1F11 nonamers T L P S P F P 6 h
v3:
HLA-A3
g G A of
eac
Pos 1 2 3 4 7 score 11 L P S P F P Y 6 peptide
5 6 8 L S is
9 9
2 I I Q A G 15 Portion 1 y I I Q A C R 4 a~no
C R A of G A
T
3 I Q A C A 15 SEQ G A T L P 4 acids,
R G T ID the
L .
9 A T L P F 15 NO: 4 Q A C R nd
S P P 7; position
Y each
7 R G A T S 13 for
L P P each
F
1 y I I Q R 11 position peptide
A C G is is
A
specified, the
. start
4 Q A C R T 11 the
G A L length
P
5 A C R G L 11 of position,
A T P each
S
lus
ei
ht
10 T. L P S P 10 peptide
P F Y is
L 9
amino
12 P S P F L 5 the 13P 1F11
P Y S acids v.3:
L I3LA-B*1510
nonamers
6 C R G A P 4 ,
T L S
- P
-
- end Pos 1 2 3 4 5 6 7 score
positio 8 9
for 3 I Q A C R G A 14 Portion
each T L of
peptide 10 T L P S P F P 13 SEQ
is Y L ID
the 12 p S p F P Y L 11 NO:
start S L 7;
each
11 L P S P Y 4 position start
F P L
S
G A T L P 1 lus position
P S F ei is
P ht
specified,
the
length
of
each
peptide
is
9
amino
acids,
R G A T L P S 8 the
P F end
1 Y I I Q A C R 4 position
G A
I I Q A C R G 4 for
A T each
11 L P S P F P Y 4 peptide
L S is
g G A T L P S P 3 the
F P start
A T L P S P F 3 position
P Y
Q A C R G A T 1 plus
L P eight
5 A C R G A T L 1
P S
228
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 13P1F 11
v.3: v.3:
HLA-B*1510 HLA-B*4402
nonamers nonamers
Pos1 2 3 45 6 78 9 score Pos 1 2 3 67 8 9 score
4
5
C R G AT L PS P 1 9 A T L PF P Y 18 Portion
P of
S
3 I Q A G T L 12 SEQ
C A ID
R
13P1F11 7 R G A PS P F 12 NO:
v.3: T 7;
HLA-B*2705 L each
nonamers
Pos1 2 3 45 6 78 9 score 10 T L P FP Y L 12 start
S
P
Position
is
7 R G A TL P SP F 16 Portion 12 P S P YL S L 12 specified,
of F
P
3 I Q A CR G AT L 13 SE 5 A C R TL P S 6 the
ID G length
Q A
6 C R G AT L PS P 13 NO: 11 L P S PY L S 6 of
7; P each
each F
9 A T L PS P FP Y 13 start 1 y I I CR G A ~ peptide
Q 4 is
A 9
12 P S P FP Y LS L 13 position 2 I I Q RG A T 3 amino
is A
C
specified, acids,
P F PY L 12 4 Q A C A L P 3 the
R T
G
T L P S the end
length position
$ G A T LP S PF P 7 of 6 C R G LP S P 1 for
each A each
T
4 Q A C RG A TL P 5 peptide 8 G A T SP F P 1 peptide
is , L is
9 P
1 Y I I QA C RG A 4 ammo the
start
5 A C R GA T LP S 4 acids, position
the
11 L P S PF P YL S 2 end lus
position ei
ht
for
each
2 I I Q AC R GA T 1 peptide
is
13P1F11 :
the v.3 HLA-B*5101
start nonamers
position Pos 1 2 3 67 8 9 score
4
5
lus 11 L P -S PY L S 13 Portion
ei P of
ht F
4 Q C T L P 12 SEQ
A R ID
G
A
13P1F11 v.3: A-B*2709 3 I Q G T L 10 N~~
HL nonamers A A 7;
C each
R
Pos1 2 3 45 6 78 9 score 8 G SP F P 10 start
A Position
T is
L
P
7 R G A TL P SP F 12 Portion 10 T L P FP Y L 9 specified,
of S
P
10 T L P SP F PY L 12 SE 7 R G PS P F 8 the
ID A length
Q T
L
3 I Q A CR G AT L 11 NO: 12 p S p YL S L 8 of
7; F each
each P
6 C R G AT L PS P 11 start 9 A T L PF P Y 4 peptide
P is
S 9
12 P S P FP Y LS L 11 position 1 y I I CR G A 3 amino
is Q
A
specified, acids,
the
$ G A T LP S PF P 5 the end
length position
9 A T L PS P FP Y 4 of for
each each
5 A R G T LP S 2 peptide peptide
C A is is
9
11 L P S PF P YL S 2 amino the
start
1 Y I I QA C RG 1 acids, position
A the
end lus
position ei
ht
for
each
peptide Tp~LE
is XIXA,
part
4:
MHC
Class
I
nonamer
the
start gal 213P1F11 as )
sis v. 1-321
of
2 I I Q AC R GA T 1 position *
Q C RG A L P 1 Ius 13P 1F11 v.4: 0201
A T ei HLA-A nonamers
ht
Pos 1 2 3 67 8 9 score
4
5
6$ D I V DL S I 20 Portion
G of
R
50 K L V PR E T 17 SEQ
N ID
D
61 V F G G D I 17 NO:
G 9;
G each
V
Q 8 Y SL S V 16 start
D '
K ~
',
position
is
3 K C Q DK S L 15 specified,
E '
Y
13 V P T G L 15 the
Q E length
K
_R
26 G 8 C _TF R L'15 of
G each
Q
33 R L K QG R 15 peptide
E A is
E 9
75 S I S S E T 15 amino
F
R
N
10 S L S PE K 14 acids,
V R the
Q
20 G L R NG E C 14 end
D position
E
for
each
39 G R GS S V 14 peptide
A is
F
R
53 N D P TQ E V 14 the
R start
E
46 S V _LV D 13 position
H N
Q
K
62 F G G _GD I V 13 plus
G eight
V
43 R G S Q K 12
S L
v
H
229
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
213P1F11 v.4: HLA-A*0201mers 13P1F11 v.4: HLA-A~UZUl
nona nonamers
s 1 2 3 4 5 6 7 core Pos1 2 3 4 5 6 7 score
P 8 9 s 8 9
o L V N D P R E 11 6 E Y D K S L S 1
51 T Q V Q
5$ T Q E V F G G 11 14 Q P E K R _T G 1
G V L R
66 V G D I V G R 11 24 E N G E C G Q 1
D L T F
12 S V Q P E K R 10 25 N G E C G Q T 1
T G F R
44 G S S V H Q K 10 31 T F R L K E E 1
L V Q G
64 G G V G D I V 10 54 D P R E T Q E 1
G R V F
65 G V G D I V G 10 5 Q E V F G G G 1
R D V G
69 I V G R D _L S 10 8 N S E T S A_ S 1
I S E E
60 E V F G G _G V 9 3 E E Q G R A_ F -1
G D R G
73 D L S I S F R 9 48 H Q K L V N D -1
N S P R
30 Q T F R L K E 8 4 C Q E Y D K S -2
E Q L S
74 L S I S F R N 8 37 E Q G R A _F R -2
S E G S
77 S F R N S _E T 8 55 P R E T Q E V -2
S A F G
85 A S E E E K Y 8 15 P E K R T G L -3
D M R D
40 R A F R G S S 7 2 E C G Q T F R -3
V H L K
47 V H Q K L V N 7
D P
84 S A S E E E K 7 13P1F11 v.4: HLA-A1
Y D nonamers
9 K S L S V Q P 6 Pos1 2 3 4 5 6 7 score
E K 8 9
11 E K R T 6 83 T _S A S E E E 21 Portion
L S V Q P K Y of
23 _ 6 45 S S V H Q K _L 15 SEQ
D E N G E _C G V N ID
Q T NO:
9;
each
41 A F R G S S V 6 52 V N_ D P R E T 15 t
H Q Q E t
42 F R G S S V H 6 85 A _S E E E K Y 15 ar
Q K D M s
pesition
is
79 S A S E 6 66 V G D I V G R 14 specified,
R N S E T D L
17 _ 5 80 N S E T S A _S 14 the
K R T G L R D E E length
E N
67 G D I V G R D 5 34 L K E E Q G R 13 of
L S A F each
70 V G R D L S I 5 35 K E E Q G R A 13 peptide
S F F R is
9
71 G R D L S I S 5 86 S E E E K Y D 13 amino
F R M S
76 I S F R N S E 5 4 C Q E Y D K S 12 acids,
T S L S the
end
positior
78 F R N S E T S 5 14 Q P E K R T G 12 for
A S L R each
7 Y D K 5 L S V 4 22 R D E N G E _C 12 peptide
Q P G Q is
1 R T G L R D E 4 25 N G E C G Q T 12 the
g N G F R start
19 T G L R D E 'N 4 55 P R E T Q E V_ 12 position
G E F G
21 ; L R D E N G 4 6 E Y_ D K S L S 11 plus
E C G V Q eight
2g ~,C G Q T F R 4 21 L R D E N G _E 11
L K E C G
29 I G Q T F R L 4 57 E T Q E V F G 1.1
K E E G G
32 F R L K E E Q 4 58 T Q E V F G G 11
G R G V
34 L K E E Q G R 4 71 G R D L S I S 10
A F F R
35 K E E Q G R A 4 28 C G Q T F R L 9
F R K E
56 R E T Q E V F 4 10 S L S V Q P E 7
G G K,R
57 E T Q E V F G 4 15 P E K R T G L 7
G G R D
g3 T S A S E E E 4 63 G _G G V G D I 7
K Y V G
g6 S 8 E E K Y D 4 68 D I V G R D L 7
M S S I
2 G K C Q E Y D 3 5 Q E Y D K S _L 6
K S S V
g Q P E 3 9 K S L S V Q _P 6
D K S L S V E K
16 _ 3 12 S V Q P E K R_ 6
L R D E T G
E K R T G
3g _ 3 18 R T G L R D E 6
Q G R A F R G N G
S S
45 S S V H Q K L 3 30 Q T F R L K E 6
V N E Q
49 Q K L V N D P 3 44 G S S V H Q K 6
R E L V
52 V N D P R E T 3 82 E T S A S E E 6
Q E E R
63 G G G V G D I 3 11 L S V Q P E K 5
V G R T
$1 S E T S A S E 3 27 E C G Q 'T F R 5
E E L K
g2 E T S A S E E 3 61 V _F G G G V G 5
E K D I
22 R D E N G E C 2 62 F G G G V G D_ 5
G Q I V
72 R D L S I S F 2 67 G D I V G R D 5
R N L S
230
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.4: HLA-A1 nonamers I 1213P1F11 v.4: HLA-A26 nonamers
Pos1 2 34 5 67 8 9 score Pos1 2 34 5 67 8 9 score
70 V G RD L SI S F 5 5 E T QE V FG G G 23 Portion
of
74 L S IS F RN_S E 5 60 E V FG G GV G D 22 SEQ
ID
43 R G SS V Q K L 4 73 D L SI S FR N S 20 NO:
H 9;
each
59 Q E VF G GG V G 4 24 E N GE C GQ T F 1g start
~
60 E V FG G GV D 4 68 D I V R DL S I 19 Position
G G is
s
ecified
p
73 D L SI S FR N S 4 46 S V HQ K LV 18 ,
N the
D length
76 I S FR N SE T S 4 54 D P RE T QE V F 18 of
each
26 G E CG Q TF R L 3 82 E T SA S EE E K 18 peptide
is
9
37 E _QGR A FR G S 3 65 G V GD I VG R D 16 amino
40 R A_FR G SS V 3 69 I V GR D LS I S 15 acids,
H the
48 H Q RL V P R 3 6 E Y DK S LS V Q 14 end
N position
D_
75 S _ISF R N_SE T 3 30 Q T FR L K E Q 14 for
E each
e
tid
i
p
1 M G RC Q EY D R 2 34 L K EE Q GR A F 14 p
e
s
the
start
2 G K CQ E YD K S 2 13 V Q PE K T G L 13 position
R
7 Y D RS L SV Q P 2 70 V G RD L SI S F 13 plus
eight
13 V Q PE K RT G L 2 83 T S AS E EE K 13
Y
16 E K RT G LR D E 2 $ D K SL S VQ P E 12
17 K R TG L RD E N 2 12 S V QP E KR T G 12
20 G L RD E N_GE C 2 26 G E CG Q TF R L 12
41 A _FRG S S_VH Q 2 ~ 33 R L KE E QG R A 12
42 F _RGS S V Q R 2 61 V F GG G G D I 12
H V
46 S V Q K LV_ D 2 75 S I SF R NS E T 12
H N
47 V QK L V_ND P 2 3 K C QE Y DK S L 11
_H
51 L _VND P RE T Q 2 18 R T GL R DE N G 11
77 S F RN S ET S A 2 27 E C GQ T FR L K 11
81 S E TS A SE E E 2 37 E Q GR A FR G S 11
84 S A SE E EK D 2 41 A F RG S SV Q 11
Y H
3 K C QE Y DK S L 1 43 R G SS V Q K L 11
H
$ D K SL S VQ P E 1 51 L V P RE T Q 11
N
D
23 D _ENG E CG Q T 1 10 S L SV PE K 10
Q R
24 E N GE C G_QT F 1 16 E K RT G LR D E 10
32 F _RLK E EQ G R 1 20 G L RD E NG E C 10
33 R _LRE E Q_GR 1 85 A S EE E KY D M 10
A
36 E E QG R _FR G 1 23 D E N E CG Q T
A G
38 Q _GRA F R_GS S 1 36 E E QG R F R G 9
A
39 G R AF R GS S V 1 50 K L V D PR E T 9
N
50 K V PR E T 1 66 V G DI V R L 9
L N G D
D
$4 D P RE T QE V F 1 31 T F RL K EE Q G 8
56 R E TQ E VF G G 1 64 G G D IV R 8
V G
G
64 G G VG D IV G R 1 77 S F RN S ET S A 8
65 G V GD I V_GR D 1 86 S E EE K D M S 8
Y
78 F R NS E TS A S 1 42 F R GS S VH Q K 7
71 G R DL S IS F R 7
2 G K CQ E YD K S 6
47 V H QK L V P 6
N
D
56 R E TQ E VF G G 6
7 Y D KS L SV Q P 5
29 G Q TF R LK E E 5
17 K TG L RD E N 4
R
52 V DP R ET Q E 4
N
74 L S IS F RN S E 4
79 R N SE T SA S E 4
5 Q E YD K SL S V 3
9 K S LS V QP E K 3
231
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.4: HLA-A26 nonamers
13P1F11 s
v.4:
HLA-A3
nonamer
Pos 1 23 4 6 7 8 9 score Pos1 23 4 5 67 8 9 score
5
21 L RD E G E C G 3 38 Q GR F RG S S 11
N A
32 F RL K E Q G R 3 39 G R F R GS S V 11
E A_
39 G RA F G S S V 3 73 D LS I S FR N S 11
R
40 R F R S S V H 3 1S P EK T GL R D 10
A G R
S3 N P R T Q E V 3 27 E CG Q T FR L R 10
D E
78 F RN S T S A S 3 64 G GV G.D IV G R 10
E
8 N SE T A S E E 3- 70 V GR D L SI S F 10
S
1 M GK C E Y D K 2 79 R NS E T _SA S E 10
Q
11 L SV Q E K T 2 14 Q PE K TG L R 9
P R R
1S P EK R G L R D 2 34 L RE E Q GR A F 9
T
3S K E Q R A F R 2 S2 V P R _ET Q E 9
E G N
D
4 Q KL V P R E 2 76 I SF R N S_ET S 9
N
D
SS P RE T E V F G 2 7 Y D_KS L S_VQ P $
Q
62 F GG G D I V 2 31 T FR L K EE Q G $
V
G
67 G I V R D L S 2 4S S SV H Q KL V $
D G N
72 R L S S F R 2 71 G RD L S IS F R $
D I N
7 I SF R S E T S 2 77 S F~R S ET S A $
N N
81 S ET S S E.E E 2 6 E Y K S LS V 7
A D Q
8 S AS E E K Y D 2 18 R TG L R D_EN G 7
E
C QE Y K S L S 1 22 R D_EN G EC G Q 7
D
1 Q PE K T G L R 1 2S N GE C G _Q_TF R 7
R
1 T GL R E N G E 1 36 E EQ G R _FR G 7
D A
22 R DE N E C G Q 1 48 H Q_K V _DP R 7
G L N
28 C GQ T R L K E 1 83 T SA S E _EE K 7
F Y
38 Q GR R G S S 1 3 K CQ E Y K S L 6
A D
F
4S S SV K L V N 1 17 K T G L R E N 6
H R D
Q
48 H QK L N D P R 1 23 D EN G E CG Q T 6
V
S8 T QE V G G G V 1 32 F RL K E EQ G R 6
F
62 F GG G D I V 6
V
G
13P1F11 63 G GG G I V
v.4: V D_ G
HLA-A3
nonamers
Pos 1 23 4 6 7 8 9 score 67 G DI V R_DL S 6
5 G
6 E V_FG G V G D 21 Portion 72 R DL S I S_FR 6
G of N
40 R F R S S V H 20 SEQ 74 L SI S F _RN S E 6
A G ID
12 S VQ P K R T G 18 NO: g0 N SE T S A_SE S 6
E 9;
each
69 I VG R _LS I S 18 std 16 E RR T G LR D E S
D iti
i
33 R LK E Q G R 17 on 19 T GL R EN G E S
E A s D
pos
specified
,
68 D IV G' L S I 17 the 30 Q TF R L KE E Q S
R length
D
9 K SL S Q P E R 16 of 43 R GS S V' Q K S
V each H L
S LS V P E K R 16 peptide 61 V FG G G V D I S
Q is G
9
46 S V Q V D 16 amino 66 V I V R D L 5
H K N G G
L D
S L N_D R E T Q 16 acids, 13 V QP E K _TG L 4
1 V P the _R
end
G LR D N G E C 1S osition 21 L R_DE N GE C G 4
E p
SO K L_V P _RE T 1$ for 28 C G_QT F RL K 4
N each E
D tide
is
e
7S S I_SF N_S E T 14 p 49 Q R_LV P R S 4
R p N
the D
start
S Q E_YD S L S V 13 position SS P RE T Q EV F G 4
K
3S K _EQ R A F R 13 plus S6 R ET Q E VF G G 4
E G eight
42 F RG S V H Q R 13 S8 T QE V F GG G V 4
S
24 E N E G Q T F 12 78 F R S E TS A S 4
G C N
41 A FR G S V H Q 12 8S A SE E E KY D 4
S M
S4 D PR E Q E V F 12 86 S EE E K YD M S 4
T
S9 Q EV F G G V G 12 4 C QE Y KS L S 3
G D
6S G _GD V G R D 12 37 E QG R FR G S 3
V I . A
82 E T_SA E E E R 12 S3 N P R E TQ E V 3
S D
1 M GK C E Y D R 11 S7 E TQ E V FG G G 3
Q
232
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 13P1F11
v.4: v.4:
HLA-A3 HLA-B*0702
nonamers nonamers
Pos 1 23 5 7 ~ Pos 1 2 3 45 6 78 9 - I
4 6 8 score ~
9
score
26 G EC Q F 2 7 Y D K SL S VQ p 2
G T R
L
29 G QT R K 2 9 K S L SV Q PE R 2 j
F L E
B
84 S AS E K 2 10 S L S VQ P EK 2
E E Y R
D
$ D RS S Q I 18 R ' G LR D EN G 2
L V P T
E
81 S ET A E 1 20 G L R DE N GE C 2
S S E
E
21 L R D EN G EC G 2 I
13P1F11 amers 25 N G E CG Q TF R 2
v.4:
HLA-B*0702
non
Pos 1 23 5 7 score 31 T F R LK E EQ G 2
4 6 8
9
D PR T E 19 Portion 40 R A F RG S SV 2
E Q V of H
F
26 G EC Q F 13 SEQ 42 F R G SS V HQ R 2
G T R ID
L
43 R GS V Q 13 NO: 46 S V QK L VN 2
S H K 9; H D
L each
13 V QP K T 12 start 47 V Q K V P 2
E R G H L N
L D
14 Q PE G position
K L 12 is 56 R E T QE V FG G 2
R R
T
specified,
66 V D V R 12 the 57 E T Q EV F GG G 2
G I G D length
L
3 K CQ Y K 11 of 59 Q E V FG G GV G 2
E D S each
L
61 V FG G G 10 peptide 63 G G G D IV 2
G V D is V G
I 9 G
62 F GG V D 10 amino 65 G V G DI V GR 2
G G I D
V
68 D IV R L 10 acids, 69 I V RD L SI S 2
G D S the G
I
nd
11 L SV P K 9 osition 71 G R D LS I SF R 2
Q E R P
T
24 E N C Q 9 for 7$ F R SE T SA S 2
G G T each N
E F
Peptide
is
77 S FR S T the 84 S A S EE E K D 2
N E S start Y
A
5 Q 8Y K L 8 position 12 S V Q PE K T G 1
D S S R
V
23 D EN E G $ plus 22 R D E NG E CG Q I
G C Q eight
T
34 L RE Q R $ 32 F R L K E QG R 1
E G A E
F
39 G R R S $ 48 H Q K LV N DP R 1
A G S
F V
41 A FR S V $ 49 Q R L V D PR E 1
G S H N
Q
44 G SS K $ 67 G D I V R DL S 1
V L G
H V
Q
70 V GR L I $ 72 R D L SI S FR' 1
D S S N
F
75 S IS R S $ 74 L S I SF R S E 1
F N E N
T
85 A SE E $ 76 I S F RN S ET S 1
E K
Y
D
M
33 R LK E G 7 ~ T S A SE E EK ~
E Q R $3 Y 1
A ~
50 K V D R 7
L N P E
T
53 N P Q 7 13P1F11
D R E v.4:
E v HLA-B*08
T nonamers
58 T QE F G 7 Pos 1 2 3 45 6 78 9 score
V G G
V
60 E VF G V 6 13 V Q P ER R TG L 23 Portion
G G G of
D
6 E Y S 4 54 D P R ET Q EV F 20 SEQ
D V ID
K Q
S
L
$ D RS S 4 ~ 33 R L R E8 Q GR 1$ NO:
L V A 9;
Q each
P
E
17 K RTG LR DEN 4 68 D I VG R D LS I lg start
35 K EE G 4 31 T F R LR E EQ G 17 Position
Q R is
A
F
R
38 Q G 4 specified
R 75 S I S FR SE T 16
A N
F
R
G
S
S
the
length
51 L V P E 4 20 G L R DE N GE C 15 of
N R T each
D Q
64 G G V 4 14 Q P E K T GL R 14 peptide
V G R is
G R 9
D
I
79 R S T A 4 86 S E E ER Y DM S 14 amino
N E S S
S
82 E TS S E 4 3 K C Q EY KS L 13 acids,
A E E D the
R
P EK T L 3 46 S V QR L V D 13 end
R G R H N position
D
16 E R G R 3 48 H Q R LV DP R 13 for
R L D N each
T E
27 E CG T R 3 70 V R L S IS F 13 peptide
Q F L G D is
R
the
start
28 C GQ F L 3 7 Y D R SL S VQ P 11 position
T R K
E
36 E EQ R 3 26 G E C GQ T FR L 11 plus
G A eight
F
R
G
37 E QG R 3 29 G Q T FR L KE E 11
R G
A S
F
45 S SV Q L 3 66 V D IV RD L 11
H K V G G
N
52 V D R T 3 _ 1 M G R CQ E Y R 10
N P E Q D
E
55 P RE Q V 3 5 Q E Y DR S LS V 10
T E F
G
73 D LS S R 3 15 P E R RT G LR D 10
I F N
S
233
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 HLA-B*OS ~ 13P1F11
v.4: nonamers_ v.4:
HLA-B*08
nonamers
Pos 1 3 4 67 8 score Pos1 3 4 5 6 score
2 5 9 2 7 8 9
24 E G E GQ T 10 $0 N E T S A 1
N C F S S E E
36 E Q G F R 10
E R G
A
43 R S S Q K 10 13P1F11 HLA-B*1510
.G V L v.4: nonamers
H
S S V PE K 9 Pos1~ 3 4 5 6 scoreI
L Q R 2 7 8 9
18 R G L DE N 9 2 G C G Q T 14 Portion
T R G E F R L of
'
34 L E E GR A 9 13 V P E K R 12 SEQ
K Q F Q T G L ID
39 G A F GS S 9 47 V Q K L V 12 NO:
R R V H N D P 9;
each
52 V D P ET Q 9 66 V D I V G 12 start
N R E G R D L
$4 S S E EK Y~D 9 3 p Q E Y D 11 sition
A E K K S L is
C
specified,
16 E R T LR D $ 34 L E E Q G i the
K G E A R A F l length
61 V G G VG D $ 43 R S S V H 11 of
F G I G Q K L each
77 S R N ET S $ 24 E G E C G 9 peptide
F S A N Q T F is
9
41 A R G SV H 7 54 D R E T Q 9 amino
F S Q P E V F
50 K V N PR E 7 85 A E E E K g acids,
L D T S Y D M the
3$ Q R A RG S 6 70 V R D L S 7 nd
G F S G I S F position
for
each
73 D S I FR N 6 12 S Q P E K 6 peptide
L S S V R T G is
40 R F R SS V 4 49 Q L V N D 5 the
A G H It P R E start
57 E Q E FG G 4 60 E F G G G 5 position
T V G V V G D
44 G S V QK L 3 63 G 5 plus
S H V G eight
G
V
G
D
I
V
G
60 E F G GV G 3 64 G V G D I 5
V G D G V G R
64 G V G IV G 3 - 65 G G D I V 5
G D R V G R D
71 G D L IS F 3 6 E D K S L 4
R S R Y S V Q
$1 S T S SE E 3 11 L V Q P E 4
E A E S K R T
$2 E' S A EE E 3 16 E 4
T S K A
R
T
G
L
R
D
8
6 ~ D K LS V 2 27 E G Q T F 4
E S Q C R __L R
Y
$ D S L VQ P 2 35 K E Q G R 4
K S. E E A F R
12 S Q P KR T 2 36 E .Q G R A 4
V F G E F R G
21 Ir D E E C 2 $0 K V N D P 4
R N G L R E T
G
27 E G Q.T FR L 2 51 L ,
C IC V 4
N
D
P
R
E
T
Q
37 E G R FR G 2 67 G I V G,R 4
Q A S D D L S
42 F G S VH Q 2 , 76 I F R N S 4
R -S R S E T S
45 S V H KL V 2 7 Y K S L S 3
S Q N D V Q P
47 V Q K V 2 10 S S V Q P 3
H L N L E K R
D
P
63 G G V DI V 2 17 K T G L R 3
G G G R D E N
65 G G D V 2 20 G R D E N 3
V I G L G E C
R
D
67 G I V RD L 2 33 R K E E Q 3
D G S L G R A
7$ F N S TS A 2 37 E G R A F 3
R S S Q R G S
79 R S E SA S 2 40 R F R G S 3
N T S A S V H
_ $5 A 8 E KY D 2 41 A R G S S 3
S E M F V H Q
2 G C Q'S Y 1 44 G S V H Q 3
K D S K L V
K
S
4 C 8 Y KS L 1 45 S V H Q K 3
Q D S S L V N
9 K L S P E 1 46 S 3
S V R V
Q H
Q
K
L
V
N
D
11 L V Q EK R 1 55 P E T Q E 3
S P T R V F G
17 K T G RD E 1 58 T E V F G 3
R L N ~ Q G G V
30 Q F R ~KE E 1 59 Q V F G G 3
T L Q E G V G
3 F L K EQ G 1 61 V G G G V 3
R E R F G D I
4 Q L V P R 1 73 D S I S F 3
K N E L R N S
D
53 N P R TQ E 1 82 E S A S E 3
D 8 V T E E It
55 ' E T EV F 1 2 G C Q E Y 2
P Q G Ft D K S
R
5 R T Q VF G 1 4 C E Y D K 2
E E G Q S L S
5$ T E V GG G 1 8 D S L S V 2
Q F V ~ R Q P E
62 F G G GD I 1 9 K L S V Q 2
G V V S P E K
6 I G R LS I 1 14 Q E K R T 2
V D S P G L R
7 I F R SE T 1 15 P K R T G 2
S N S E L R D
234
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 13P1FI1 : mers
v.4: v.4 HLA-B*2705nona
HLA-B*1510
nonamers
Pos 1 23 4 56 7 89 store Pos 1 23 4 56 7 8 score
9
21 L RD E NG E CG 2 14 Q PE K T G L 12
R R
22 R DE N GE C GQ 2 24 E N E CG Q T 12
G F
2j N GE C GQ T FR 2 27 E CG Q TF R L 12
' R
29 G QT F RL K EE 2 68 D IV D L S 12
G I
R
30 Q TF R LK EQ 2 78 F RN S ET S A 12
E S
39 G RA F RG S SV 2 82 E TS A SE E E 12 '
R
j2 V P RE T QE 2 . 1 M GK C QE Y D 11
N R
D
J6 R ET Q EV F GG 2 21 L RD E NG E C 11
G
j7 E TQ E VF G GG 2 34 L RE E QG R A 11
F
69 I VG R DL S IS 2 55 P RE T QE V F 11
G
71 G RD L SI S FR 2 61 V FG G GV G D 11
I
72 R L S IS F R 2 66 V G I VG R D 11
D N D L
7j S IS F R S ET 2 83 T SA S EE E K 11
N Y
79 R NS E TS A E 2 33 R LK E EQ G R 10
S A
80 N SE T SA S EE 2 72 R DL S IS F R 10
N
E3 T SA S EE E K 2 2 G RC Q EY D K 8
Y S
84 S AS E EE K 2 18 R TG L RD E N 8
Y G
D
86 S EE E K MS 2 56 R ET Q EV F G $
Y G
D
j Q EY D KS L SV 1 60 E VF G GG V G 8
D
19 T GL R E N GE 1 63 G GG V D I V $
D G G
23 D EN EC G QT 1 79 R S E TS A S $
G N E
2$ C GQ T FR L K 1 46 S V Q KL V 7
E H N
D
31 T FR L K E QG 1 65 G D IV G R 7
E V D
G
32 F RL K EE Q GR 1 67 G I V R D L 7
D G S
3$ Q GR A FR G SS 1 5 Q EY D KS L S 6
V
42 F RG S SV QR 1 6 E YD K SL S V 6
H Q
53 N DP R ET Q E 1 11 L SV Q PE K 6
V R
T
62 F GG G G IV 1 20 G LR EN G E 6
V D D C
6$ D IV G R L SI 1 22 R DE N GE C G 6
D Q
74 L SI S FR N SE 1 30 Q TF R LK E E 6
Q
7$ F R S ET S AS 1 41 A FR G SS V 6
N H
Q
81 S- ET S AS E EE 1 49 Q RL V D P R 6
N E
76 I SF R NS E T 6
S
13P 1F11 29 G QT F R'L K 5
v.4: E
HLA-B*2705 E
nonamers
Pos 1 23 4 56 7 89 score 36 E EQ G R F R S
A G
71 G RD L SI S FR 29 Portion 44 G SS V Q K L 5
of H V
32 F RL K E Q GR 25 SEQ 47 V Q K V 5
E ID H L N
D
P
42 F RG S SV Q 23 NO: 7 Y DK S LS V Q 4
H R 9; P
each
40 R F R S S V 20 std 12 S VQ P EK T 4
A G H R G
64 G GV DI V R 20 position 23 D EN EC G Q 4
G G is ' G T
d
9 K SL S V P ER 19 , 45 S SV QK V 4
Q specifie H L N
the
len
th
26 G EC G QT F RL 17 g 50 K LV N P R E 4
of D T
each
35 K E Q GR FR 17 peptide 52 V P RE T Q 4
E A is N E
9 D
43 R GS S V Q K 17 amino 59 Q EV F GG G V 4
H L G
17 K T G LR D EN 16 acids, 74 L SI S FR N S 4
R the E
25 N GE C GQ T FR T6 end 77 S FR SE T S 4
positio N A
3 K CQ E YD K SL 15 for 8p N SE T SA S E 4
each E
39 G F R S V 15 peptide 8 D RS L SV Q P 3
R G S is E
A rt
h
13 V QP E K T GL 14 t 15 P EK TG L R 3
R e sta R D
osition
54 D PR E TQ E VF 14 p 1 E RR T GL R 3
. D
plus E
eight
70 V R D LS I SF 14 1 T GL R E N 3
G ~D G
E
4$ H QK D PR 13 2 C GQ T FR L K 3
L E
V
N
85 A E E EK 13 3 1 FR L K E Q 3
S Y T E G
D
M
S LS V QP E K 12 6 2 G G VG D I 3
R F G V
235
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 v.4: 13P1F11
HLA-B*2705 v.4:
nonamers HLA-B*2709
nonamers
Pos 1 2 3 56 7 8 9 score pos 1 23 4 56 7 9 score
4 8
6 I V G DL S I S 3 7 Y DK S LS V P 3
R Q
73 D L S SF R N S 3 19 T GL R DE N E 3
I G
7S S I S RN S E T 3 20 G LR D EN G C 3
F E
C Q E DK S L S 2 60 E~ VF G GG V D 3
Y G
51 L V N PR E T Q 2 63 G GG V GD I G 3
D V
S3 N D P ET Q E V 2 76 I SF R NS E S 3
R T
S7 E T Q VF G G G 2 11 L SV Q P.E K T 2
E R
81 S E T AS E E E 2 1S P EK R TG L D 2
S R
$ S A S EE K Y D 2 30 Q TF R LK E Q 2
E E
$ S E E K D M S 2 35 K E Q GR R 2
E Y ~ E A
F
3$ Q G R R G S S 1 36 E EQ G R F G 2
A F A R
41 A FR G SS V Q 2
H
13P 1F11 4S S SV QK 2
v.4: H L
HLA-B*2709 V
nonamers N
Pos 1 2 3 56 7 8 9 score 46 S VH Q K V D 2
4 L N
3 G R A RG S S V 21 Portion S2 V N P RE T E 2
F of D Q
43 R G S VH Q K L 15 SEQ 69 I VG R DL S S 2
S ID I
26 G E C QT F R L 14 ND~ 74 L SI S FR N E 2
G 9; S
each
17 KRTG LR D E N 13 start gl S ET S AS EE E 2
osition
is
42 F R G SV H Q K 13 P 1 M GK C QE Y K 1
S specified D
,
71 G R D SI S F R 13 the 4 C QE Y DK S S 1
L length L
3 K C Q YD K S L 12 of 8 D KS L SV Q E 1
E each P
32 F R L E Q G R 12 peptide 12 S VQ P EK R G 1
K E is T
9
S Q E Y KS L S V 11 amino 23 D EN G EC G T 1
D Q
44 G S S HQ K L V 11 acids, 2g C GQ T FR L E 1
V the K
SS P R E QE V F G 11 nd 31 T FR L K E G 1
T positio E Q
h
f
78 F R N ET S A S 11 or 47 V Q K LV P 1
S eac H N
eptide D
is
13 V Q P K T G L 10 p S9 Q EV F GG G G 1
E R the V
start
21 L R D NG E C G 10 position 73 D LS I SF R S 1
E N
66 V G D VG R L 10 plus 80 N SE T SA S E 1
I D eight E
68 - D I RD L S I 10 83 T SA S EE E Y 1
V G K
8S A S E EK D M 10
E Y
61 V F G GV D I 9 13P 1F11
G G v.4:
HI,A-B*4402
nonamers
72 R D L IS F R N 9 Pos 1 23 4 56 7 9 score
S 8
24 E N G CG Q T F 8 2 G EC G QT F L 22 Portion
E R of
34 L K E QG R F 8 36 E EQ G R F G 1S SEQ
E A A R ID
h
9
NO
S3 N D P ET Q E V 8 3 K Q E YD K L 13 ; eac
R C S :
start
54 D P R TQ E V F 8 24 E N E CG Q F 13 Position
E G T is
S$ T Q E FG G G V 8 _ 34 L KE E QG R F 13 specified
V A
62 F G G I V 8 . 5 Q EY D KS L V 12 the
G V S length
G D
70 V G R LS I S F 8 13 V QP E K T L 12 of
D R G each
9 K S L VQ P E K 6 1S P EK TG L D 12 peptide
S R R is
9
40 R A F GS S V 6 23 D E G EC G T 12 amino
R H N Q h
id
S6 R E T EV F G G 6 3S K EE Q GR R 12 e
Q A s,
F t
ac
osition
nd
64 G G V I V G R 6 43 R GS S V Q 12 p
G D H K for
L each
6S G V G IV R D 6 66 V GD I VG R L 12 peptide
D G D is
18 R T G R E N G 5 70 V GR D LS I F 12 the
L D S start
33 R L K EQ G R A 5 54 D PR E TQ E F 11 position
E V
22 R D E E C G Q 4 56 R ET Q EV F G 11 plus
N G ~ G eight
29 G Q T RL K E 4 ' Q EV F GG G 11
F E S9 V
G
49 Q K L ND P R E 4 68 D IV RD L I 11
V G S
SO K L V P R E T 4 81 S ET S AS E E 11
N D E
67 G D I R L S 4 83 T SA S EE E 11
V G D K
Y
79 R N S TS A S E 4 86 S EE E K D S 11
E Y M
2 G K C EY D K S 3 61 V FG G GV I 10
Q G
D
236
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
l3PiF11 v.4: HLA-B*4402mers 13P14: HLA-B*~4U~'nonamers
nona F11 v.
_ -
Pos 1 2 3 4 5 6 7 score Pos1 2 3 4 5 6 7 score
8 9 8 9
12 S V Q P E K R 6 22 R D E N G E C 1~
T G G Q
52 V N D P R E T 6 25 N G E C G Q T 1
Q E F R
60 E V F G G G V 6 32 F R L K E E Q 1
G D G R
6 E Y D K S L S 5 38 Q G R A F R G 1
V Q S S
16 E R R T G L R 5 3 G R A F R G S 1
D E S V
27 E C G Q T F . 5 48 H Q K L V N D 1
R L R P R
37 E Q G R A F R 5 6 F G G G V G D 1
G S I V
41 A F R G S S V 5 7 R N S E T S A 1
H Q S E
64 G G V G D I V 5
G R
67 G D I V G R D 5 13P1F11 v.4: HLA-B*5101
L S ' nonamers
71 G R D L S I S 5 Pos2 2 3 4 5 6 7 score
F R 8 9
74 _ 5 5 D P R E T Q E 20 Portion
L S I S F R N V F of
S E
76 I S F R N S E 5 62 F G G G V G D 18 SEQ
T S I V ID
_ 4 43 R G S S V H Q 16 NO:
S L S V Q P E K L 9;
K R each
28 C G Q T F R L 4 66 V G D I V G R 16 start
K S D L ition
is
o
29 G Q T F R L K 4 68 D I V G R D L 16 s
E E S I p
specified
,
40 R A F R G S S 4 5 Q E~ Y D K S L 15 the
V H S V length
44 G S S V H Q K, 4 40 R A F R G S S 14 of
L V V H each
50 K L V N D P R 4 61 V F G G G V G 14 peptide
E T D I is
9
53 N D P R E T Q 4 84 S A S E E E K 13 amino
E V Y D
84 S A S E E E K 4 13 V Q P E K R T 12 acids,
Y D G L the
i
i
d
$ D R S L S V Q 3 14 Q P E K R T G 11 t
P E L R or
Pos
n
h
f
14 Q P E K R T G 3 3 K C Q E Y D K 10 or
L R S L eac
peptide
is
17 K R T G L R D 3 19 T G L R D E N 10 the
E N G 8 start
19 T G L R D E N 3 28 C G Q T F R L 10 position
G E K E
30 Q T F R L K E 3 53 N D P R E T Q 10 plus
E Q E V eight
42 F R G S S V H 3 63 G G G V G D I 10
Q K V G
45 S S V H Q K L 3 44 G S S V H Q K 9
V N L V
46 S V H Q K L V 3 ~ 58 T Q E V F G G 9
N D G V
51 L V N D P R E 3 64 G G V G D I V 9
T Q G R
57 E T Q E V F G 3 , 70 V G R D L S I 9
G G S F
65 G V G D I V G 3 25 N G E C G Q T $
R D F R
73 D L S I S F R 3 26 G E C G Q T F 8
N S R L
75 S I S F R N S 3 39 G R A F R G S $
E T S V
7$ F R N S E T S 3 1 M G K C Q E Y 7
A S D R
80 N S E T S A S 3 $ D R S L S V Q 7
E E P E
$2 E T S A S E E 3 38 Q G R A F R G 7
E R S S
85 A S E E E K Y 3 73 D L S I S~ F R 7
D M N S
7 Y D K S L S V 2 6 E Y D K S L S 6
Q P V Q
9 K S L S V Q. P 2 23 D E N G E C G 5
E R Q T
11 L S V Q P E K 2 34 L I~ E E Q G R 5
R T A F
1g R T G L R D E 2 ~ 47 V H Q K L V N 5
N G D P
21 L R D E N G E 2 51 L V N D P R E 5
C G T Q
31 T F R L K E E 2 57 E T Q E V F G 5
Q G G G
4 V H Q K L V N 2 76 I S F R N S E 5
D P T S
4 Q It L V N D P 2 9 K S L S V Q P 4
R E E R
55 P R E T Q E V 2 11 L S V Q P E K 4
F G R T
63 G G G V G D I 2 21 L R D E N G E 4
V G C G
6 I V G R D L S 2 24 E N G E C G Q 4
I S T F
72 R D L S I S F 2 32 F R L K E E Q 4
R N G R
7 S F R N S E T 2 41 A F R G S S V 4
S A H Q
2 G FC C Q E Y D 1 42 F R G S S V H 4
K S . Q IC
C Q E Y D K S 1 45 S S V H Q K L 4
L S V N
2 G L R D E N G 1 65 G V G D I V G 4
E C R D
237
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 ~ 13P1F11
v.4: v.4:
HLA-B*5101 RT1.AI
nonamers nonamers
Pos1 2 3 45 6 78 9 score Posl 2 3 6 8 score
4 7 9
5
80 N S E TS A SE E 4 7 I S F S T 18 Portion
R E ~S of
N
7 Y D K SL 5 VQ P 3 83 T S A E K 1$ SEQ
S E Y ID
E
S L S VQ P EK 3 60 E V F G G 17 NO:
R G V D 9;
G each
12 S V Q PE K RT G 3 85 A S E K D 17 start
E Y M iti
E i
P E K T G LR D 3 40 R A F S V 16 on
R R S H s
G ps
specified
16 E K TG L RD E 3 9 K S L Q E 14 the
R S P K length
V
27 E C G QT F RL K 3 30 Q T F K E 13 of
R E Q each
L
33 R L K E Q GR A 3 24 E N G G T 12 peptide
E E Q F is
C 9
36 E E Q GR A FR G 3 54 D _PR Q V 12 amino
E E F
T
46 S V H QK VN D 3 12 S V Q K T 11 acids,
L P R G the
E
4$ H Q K LV DP R 3 34 L _KE G A 11 end
N E R F position
Q h
f
49 Q K L V PR E 3 5 Q E Y S S 10 or
N D L V eac
D K tide
is
e
59 Q E V FG G GV G 3 70 V _GR S S 10 p
D I F p
L the
start
60 E V F GG G VG D 3 51 L V N R T 9 position
D E Q
P
69 I V RD L SI S 3 84 S A S E Y 9 plus
G E K D eight
E
72 R D L SI S FR 3 3 K C Q S $
N E L
Y
D
K
74 L S I SF R NS E 3 13 V Q P G $
E L
K
R
T
79 R S ET S AS E 3 32 F R L E G $
N K Q R
E
83 T S A SE E EK 3 45 S _SV K V $
Y H L N
Q
86 5 E E EK DM S 3 46 S V L $
Y H V
Q N
K D
2 G K C QE Y DK S 2 74 L S I R S $
S N E
F
G L R DE N GE C 2 11 L _SV E 7
Q K
P R
T
29 G Q T FR L KE E 2 19 T _GL E G 7
R N E
D
31 T F R LK E EQ G 2 26 G E C T R 7
G F L
Q
37 E Q G RA F RG S 2 66 V G D G D 7
I R L
V
50 K L V P RE T 2 69 I V G L I 7
N R S S
D D
52 V N PR E TQ E 2 80 N S E A E 7
D T S E
S
55 P R E TQ E VF G 2 43 R G S K 6
S L
V
H
Q
56 R E T QE V FG G 2 44 G S S Q 6
V K
H L
V
71 G R LS I SF R 2 49 Q _K D R 6
D L P E
V
N
77 S F R NS E TS A 2 65 G V_ V R 6
G G D
D
I
82 E T S AS E EE K 2 72 R D L S R 6
S F N
I
85 A S E EE K D M 2 17 K T R E 5
Y _R G D N
L
17 K T GL R DE N 1 2 G K C Y K 3
R Q D S
E
Q T F RL K E Q 1 35 K E R F 3
E _E Q A R
G
75 S I S FR N SE T 1 50 K P E 3
L R T
V
N
D
7$ F R SE T SA S 1 ~ 81 S E T S E 3
N S E E
A
86 S E S M 3
E S
K
Y
D
4 C Q E K L 2
Y S S
D
10 S L S P K 2
V E R
Q
1$ R _TG D N 2
L E G
R
20 G L R N E 2
D G C
E
21 L _RDEN GE CG 2
23 D E N C Q 2
G G T
E
28 C _GQ R K 2
T L E
F
31 T _FRLK EE QG 2
33 R L R Q R 2
E G A
E
38 Q G R R S 2
A G S
F
41 A F R S 2
G V
S H
Q
47 V H Q V D 2
K N P
L
48 H Q K P 2
L R
V
N
D
53 N P T E 2
D R Q V
E
56 R E T V G 2
Q F G
E
61 V F G V D 2
G G I
G
238
SUBSTITUTE SHEET (RULE 26)
CA 02480811 2004-09-29
WO 03/085121 PCT/US02/10220
13P1F11 13P1F11
v.4: v.5:
RT1.A1 HLA-A26
nonamers nonamers
Pos 1 2 4 6 7 score Pos 1 3 5 6 7 score
3 5 8 9 2 4 8 9
62 F G G G D 2 5 A I R V T 15 Portion
G V I V L L K A of
64 V G I V 2 3 R A I L R 13 SEQ
D G R L L V T ID
G G
67 _ V R D 2 9 R T 12 NO:
G D G L S V K 11;
I A
R
E
G
S
75 S I F N S 2 6 L L V T K 11 each
S R E T I R A R start
position
is
82 E T A E E 2 7 I R T K A 10 specified,
S S E K L V R E
1 M G C E Y 1 2 A L L I L 6 the
K Q D K R A R V length
6 E Y K L S 1 1 G R A L I 4 of
D S V Q A L L R each
8 D L V Q 1 4 L L L R V 2 peptide
K S S P E A I T K is
9
14 _ K T G 1 8 L V K A R 2 amino
Q P R L R R T E G
E
16 E K T L R acids,
R G D E the
end
position
for
each
peptide
is
TABLE : MHC the
XIXA, Class start
part I nonamer
analysis position
of
213P1F11
v.5
(aa
1-242
13P1F11 lus
v.5: ei
HLA-A*0201 ht
nonamers
Pos 1 2 4 6 7 score'
3 5 8 9
5 A L L V T 24 Portion l3P iF11 :
I R K A of v.5 HLA-A3
nonamers
2 A R A I L 20 SEQ pos 1 3 5 6 7 score
L L R V ID 2 4 8 9
3 R L L R 20 NO: L 'L L R V 22 Portion
A V T 11; A I T R of
L
I
6 L I R _ 14 each 3 R A I ' L 20 SEQ
L V T K start L L R V T ID
A R
position NO:
7 I L V K A 14 is 5 A I R V T 20 11
R T R E L L K A
s ecified
a
4 L A I R V 13 the 6 L L V T K 19 os
L L ' T length I R A R tion
K is
p
1 G A L 9 of 7 I R T K A 18 specified,
R A each L V R E
L
I
L
R
$ L R T 5 peptide 9 R T 15 the
V K is V K length
A 9 A
R R
E E
G G
S
9 R V K R E 4 ammo 2 A L L I L 10 of
T A G S R A R V each
_ acids, 1 G R I L R 8 peptide
the A L A L is
9
end 8 L _ _ 4 amino
position R V K A R
T E G
for - - - acids,
each the
peptide end
is positio
the for
start each
position peptide
is
lus the
ei start
ht
position
13P1F11 lus
v.5: ei
HLA-A1 ht
nonamers
Pos 1 2 4 6 7 score
3 5 8 9
2 A A L R 7 Portion 13P 1F11 amers
R L L v of v.5:
I HLA-B*0702
non
_ _ SEQ pos 1 3 5 6 7 score
ID 2 4 8 9
NO: 2 A L 11 Portion
11; R A of
L
I
L
R
V
each SEQ
start ID
position NO:
is 11
specified, each
start
the position
length is
of
each specified,
a tide the
is length
9
1 G A L L I 6 amino of
R A L R each
5 A L L V T 5 acids, a tide
I R IC the is
A
3 R L L L R 2 end 5 A I R V T 10 9 amino
A I V T L L K A
L I R T K 2 position 3 R A I L R 9 acids,
L V A R fo L L V T the
4 L A I R V 1 reach I R T K A 4 end
L L T K L V R E
7 I _L K A_ 1 peptide L L R V 3 position
R R E is A T R
V L
T I
V T K 1 the R T A R E 3 for
R A start V K G S each
R
_E
G
S
_ position 1 G A L I 2 peptide
A L R is
R
L
lus L L V T K 2 the
ei I R A R start
ht
position
lus
ei
ht
239
SUBSTITUTE SHEET (RULE 26)
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 239
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
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VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 239
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