Note: Descriptions are shown in the official language in which they were submitted.
CA 02742088 2011-06-01
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THAN ONE VOLUME.
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NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02742088 2011-06-01
78895-24D
ANTIBODIES THAT BIND PSCA PROTEINS FOR DIAGNOSIS OF CANCER
This is a divisional application of Canadian Patent Application Serial
No. 2,567,449 filed on May 17, 2005.
It is to be understood that the expression "the present invention" or the
like used in this specification encompasses not only the subject-matter of
this
divisional application but that of the parent also.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is related to pending United States patent
application number 10/857,484, filed 28-May-2004, which claims priority to
United
States Provisional patent application number 60/475,064, filed 30-May-2003.
This
applications is related to United States Provisional Patent Application
No.: 60/616,381, filed 05-October-2004; United States Provisional Patent
Application
No.: 60/617,881, filed 12-October-2004; and United States Provisional Patent
Application No.: 60/621,310, filed 21-October-2004; United States Provisional
Patent
Application No.: 60/633,077, filed 02-December-2004; and United States
Provisional
Patent Application No.: Not Yet Assigned, filed 14-April-2005. This
application is
related to PCT Patent Application No.: PCT/US2004/017231, filed 28-May-2004.
The
contents of each application listed in this paragraph are fully incorporated
by
reference herein.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The invention described herein relates to antibodies, as well as
binding
fragments thereof and molecules engineered therefrom, that bind proteins,
termed
PSCA. The invention further relates to diagnostic, prognostic, prophylactic
and
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therapeutic methods and compositions useful in the treatment of cancers that
express PSCA.
BACKGROUND OF THE INVENTION
[0004 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
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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.
[00051 Worldwide, several cancers stand out as the leading killers. In
particular, carcinomas
of the lung, prostate, breast, colon, pancreas, ovary, and bladder 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.
[00061 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.
[00071 On the diagnostic front, the lack of a prostate tumor marker that can
accurately detect
early-stage, localized tumors remains a significant limitation in the
diagnosis and management
of this disease. Although the serum prostate specific antigen (PSA) assay has
been a very useful
tool, however its specificity and general utility is widely regarded as
lacking in several important
respects.
[00081 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
(SC1D) 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),
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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).
[0009] 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.
[00101 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.
[0011] 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.
[0012] Of all new cases of cancer in the United States, bladder cancer
represents
approximately 5 percent in men (fifth most common neoplasm) and 3 percent in
women (eighth
most common neoplasm). The incidence is increasing slowly, concurrent with an
increasing
older population. In 1998, there was an estimated 54,500 cases, including
39,500 in men and
15,000 in women. The age-adjusted incidence in the United States is 32 per
100,000 for men
and eight per 100,000 in women. The historic male/female ratio of 3:1 may be
decreasing
related to smoking patterns in women. There were an estimated 11,000 deaths
from bladder
cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and
mortality
strongly increase with age and will be an increasing problem as the population
becomes more
elderly.
[0013] 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
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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.
[0014] 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.
[0015] 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.
[0016] There were an estimated 164,100 new cases of lung and bronchial cancer
in 2000,
accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung
and bronchial
cancer is declining significantly in men, from a high of 86.5 per 100,000 in
1984 to 70.0 in
1996. In the 1990s, the rate of increase among women began to slow. In 1996,
the incidence
rate in women was 42.3 per 100,000.
[0017] 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.
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[0018] 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.
[0019] An estimated 182,800 new invasive cases of breast cancer were expected
to occur
among women in the United States during 2000. Additionally, about 1,400 new
cases of breast
cancer were expected to be diagnosed in men in 2000. After increasing about 4%
per year in the
1980s, breast cancer incidence rates in women have leveled off in the 1990s to
about 110.6 cases
per 100,000.
[0020] 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.
[0021] 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.
[0022] 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.
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Nevertheless, there are serious side effects or sequelae to these treatments.
There is, therefore, a
need for efficacious breast cancer treatments.
[00231 There were an estimated 23,100 new cases of ovarian cancer in the
United States in
2000. It accounts for 4% of all cancers among women and ranks second among
gynecologic
cancers. During 1992-1996, ovarian cancer incidence rates were significantly
declining.
Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000.
Ovarian cancer
causes more deaths than any other cancer of the female reproductive system.
[00241 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.
[0025] There were an estimated 28,300 new cases of pancreatic cancer in the
United States
in 2000. Over the past 20 years, rates of pancreatic cancer have declined in
men. Rates among
women have remained approximately constant but may be beginning to decline.
Pancreatic
cancer caused an estimated 28,200 deaths in 2000 in the United States. Over
the past 20 years,
there has been a slight but significant decrease in mortality rates among men
(about -0.9% per
year) while rates have increased slightly among women.
[00261 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 cancers. These include the use of
antibodies, vaccines,
and small molecules as treatment modalities. Additionally, there is also a
need to use these
modilities as research tools to diagnose, detect, monitor, and further the
state of the art in all
areas of cancer treatment and studies.
[0027] The therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler and
C. Milstein,
Nature 256:495-497 (1975)) is being realized. Monoclonal antibodies have now
been approved
as therapies in transplantation, cancer, infectious disease, cardiovascular
disease and
inflammation. Different isotypes have different effector functions. Such
differences in function
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are reflected in distinct 3-dimensional structures for the various
immunoglobulin isotypes (P.M.
= Alzari et al., Annual Rev. Immunol., 6:555-580 (1988)).
[0028) Because mice are convenient for immunization and recognize most human
antigens
as foreign, mAbs against human targets with therapeutic potential have
typically been of murine
origin. However, murine mAbs have inherent disadvantages as human
therapeutics. They
require more frequent dosing as mAbs have a shorter circulating half-life in
humans than human
antibodies. More critically, the repeated administration of murine antibodies
to the human
immune system causes the human immune system to respond by recognizing the
mouse protein
as a foreign and generating a human anti-mouse antibody (HAMA) response. Such
a HAMA
response may result in allergic reaction and the rapid clearing of the murine
antibody from the
system thereby rendering the treatment by murine antibody useless. To avoid
such affects,
attempts to create human immune systems within mice have been attempted.
100291 Initial attempts hoped to create transgenic mice capable of responding
to antigens
with antibodies having human sequences (See Bruggemann et al., Proc. Nat'l.
Acad. Sci. USA
86:6709-6713 (1989)), but were limited by the amount of DNA that could be
stably maintained
by available cloning vehicles. The use of yeast artificial chromosome (YAC)
cloning vectors
led the way to introducing large germline fragments of human Ig locus into
transgenic
mammals. Essentially a majority of the human V, D, and J region genes arranged
with the same
spacing found in the human genome and the human constant regions were
introduced into mice
using YACs. One such transgenic mouse strain is known as XenoMouse(r) mice and
is
commercially available from Abgenix, Inc. (Fremont CA).
SUMMARY OF THE INVENTION
[0030] The invention provides antibodies as well as binding fragments thereof
and
molecules engineered therefrom, that bind to PSCA proteins and polypeptide
fragments of
PSCA proteins. The invention comprises polyclonal and monoclonal antibodies,
murine and
other mammalian antibodies, chimeric antibodies, humanized and fully human
antibodies, and
antibodies labeled with a detectable marker or therapeutic agent. In certain
embodiments, there
is a proviso that the entire nucleic acid sequence of Figure 3 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 3 is encoded and/or the entire amino acid sequence of
Figure 2 is prepared,
either of which are in respective human unit dose forms.
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100311 The invention further provides methods for detecting the presence and
status of
PSCA polynucleotides and proteins in various biological samples, as well as
methods for
identifying cells that express PSCA. An embodiment of this invention provides
methods for
monitoring PSCA gene products in a tissue or hematology sample having or
suspected of having
some form of growth dysregulation such as cancer.
[00321 The invention further provides various immunogenic or therapeutic
compositions and
strategies for treating cancers that express PSCA such as cancers of tissues
listed in Table I,
including therapies aimed at inhibiting the transcription, translation,
processing or function of
PSCA as well as cancer vaccines. In one aspect, the invention provides
compositions, and
methods comprising them, for treating a cancer that expresses PSCA 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 PSCA.
Preferably, the carrier is a
uniquely human carrier. In another aspect of the invention, the agent is a
moiety that is
immunoreactive with PSCA protein. Non-limiting examples of such moieties
include, but are
not limited to, antibodies (such as single chain, monoclonal, polyclonal,
humanized, chimeric, or
human antibodies), functional equivalents thereof (whether naturally occurring
or synthetic), and,
combinations thereof. The antibodies can be conjugated to a diagnostic or
therapeutic moiety.
In another aspect, the agent is a small molecule as defined herein.
BRIEF DESCRIPTION OF THE FIGURES
[00331 Figure 1. The cDNA and amino acid sequence of PSCA (also called "PSCA
v.1" or
"PSCA variant 1") is shown in Figure IA. The start methionine is underlined.
The open
reading frame extends from nucleic acid 18-389 including the stop codon.
[0034] The cDNA and amino acid sequence of PSCA variant 2 (also called "PSCA
v.2") is
shown in Figure 1 B. The codon for the start methionine is underlined. The
open reading frame
extends from nucleic acid 56-427 including the stop codon.
[00351 The cDNA and amino acid sequence of PSCA variant 3 (also called "PSCA
v.3") is
shown in Figure I C. The codon for the start methionine is underlined. The
open reading frame
extends from nucleic acid 423-707 including the stop codon.
[0036] The cDNA and amino acid sequence of PSCA variant 4 (also called "PSCA
v.4") is
shown in Figure I D. The codon for the start methionine is underlined. The
open reading frame
extends from nucleic acid 424-993 including the stop codon.
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[0037] The cDNA and amino acid sequence of PSCA variant 5 (also called "PSCA
v.5") is
shown in Figure IE. The codon for the start methionine is underlined. The open
reading frame
extends from nucleic acid 910-1479 including the stop codon.
[00381 The cDNA and amino acid sequence of PSCA variant 6 (also called "PSCA
v.6") is
shown in Figure IF. The codon for the start methionine is- underlined. The
open reading frame
extends from nucleic acid 83-427 including the stop codon.
[0039] Figure 1 G. SNP variants of PSCA v.2, PSCA v.7 through v.18. The PSCA
v.7
through v.18 proteins have 123 amino acids. Variants PSCA v.7 through v.18 are
variants with
single nucleotide difference from PSCA v.2, and code for the same protein as
v.2. Though these
SNP variants are shown separately, they can also occur in any combinations and
in any of the
transcript variants listed above in Figures IA through IF.
[0040] Figure 1H. SNP variants of PSCA v.4, PSCA v.19 through v.30. The PSCA
v.19
through v.30 proteins have 189 amino acids. Variants PSCA v.19 through v.30
are variants with
single nucleotide difference from PSCA v.4. PSCA v.9, v.10, v.11, v.24 and
v.25 proteins differ
from PSCA v.1 by one amino acid. PSCA v.23, v.28, v.29 and v.30 code for the
same protein as
v.4. Though these SNP variants are shown separately, they can also occur in
any combinations
and in any of the transcript variants v.3 and v.4.
[0041] Figure 11, Expression of PSCA variants. (1 I(a)) Primers were designed
to
differentiate between the variants PSCA v.1/v.2/v.4, PSCA v.3 and PSCA v.5.
Primers A and B,
indicated by small arrows above exons in the figure result in a PCR product of
425 bp for PSCA
v.1/v.2/v4, a PCR product of 300 bp for PSCA v3 and a PCR product of 910 bp
for PSCA v.5.
(11(b)) First strand cDNA was prepared from normal bladder, brain, heart,
kidney, liver, lung,
prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, pools of
prostate cancer,
bladder cancer, kidney cancer, colon cancer, lung cancer, ovary cancer, breast
cancer, cancer
metastasis, and pancreas cancer. Normalization was performed by PCR using
primers to actin.
Semi-quantitative PCR, using the variant specific primers was performed at 30
cycles of
amplification. Results show expression of PSCA v.5 mainly in breast cancer,
cancer metastasis,
and pancreas cancer, and at lower level in colon cancer and lung cancer. PSCA
v.1/v.2/v.4 PCR
product was detected in prostate cancer, bladder cancer, kidney cancer, colon
cancer, lung
cancer, ovary cancer, breast cancer, cancer metastasis, and pancreas cancer.
Amongst normal
tissues, PSCA v.1/v.2/v.4 PCR product was detected only in prostate, stomach
and at lower level
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in kidney and lung, whereas PSCA v. 5 was not detected in any normal tissue.
PSCA v.3 PCR
detected product was not detected in any of the samples tested.
10042] Figure I J. Expression of PSCA v.4 and PSCA v.5. IJ(a) Primers were
designed to
differentiate between PSCA v.4 and PSCA v.5 as indicated by the arrows labeled
B and C in the
figure. Primers specific for PSCA v.4 lead to a PCR product of 460 bp, whereas
primers
specific for PSCA v.5 leads to a PCR product of 945 bp in size. 1J(b) First
strand cDNA was
prepared from normal bladder, brain, heart, kidney, liver, lung, prostate,
spleen, skel. muscle,
testis, pancreas, colon, stomach, pools of prostate cancer, bladder cancer,
and multi-xenograft
pool (prostate cancer, kidney cancer and bladder cancer xenografts).
Normalization was
performed by PCR using primers to actin. Semi-quantitative PCR, using the
variant specific
primers was performed at 30 cycles of amplification. Results show expression
of PSCA v.4 in
prostate cancer, bladder cancer, and multi-xenograft pool, normal kidney and
prostate. PSCA v.5
was detected only in normal prostate and bladder cancer.
100431 Figure 2. Amino Acid Sequences of PSCA antibodies. Figure 2A. The amino
acid
sequence of Hal-4.117 VH. Underlined is the heavy chain constant region.
Figure 2B. The
amino acid sequence of Ha1-4.117 VL. Underlined is the light chain constant
region. Figure
2C. The amino acid sequence of Hal-4.120 VH. Figure 2D. The amino acid
sequence of Hal-
4.120 VL. Underlined is the light chain constant region. Figure 2E. The amino
acid sequence of
Hal-5.99 VH. Underlined is the heavy chain constant region. Figure 2F. The
amino acid
sequence of Hal-5.99 VL. Underlined is the light chain constant region. Figure
2G. The amino
acid sequence of Hal -4.121 VH. Underlined is the heavy chain constant region.
Figure 2H.
The amino acid sequence of Hal-4.121 VL c.5. Underlined is the light chain
constant region.
Figure 21. The amino acid sequence of Hal -4.121 VL c.26. Underlined is the
light chain
constant region. Figure 2J. The amino acid sequence of Hal-1.16 VH. Underlined
is the heavy
chain constant region. Figure 2K. The amino acid sequence of Hal-1.16 VL.
Underlined is the
light chain constant region. Figure 2L. The amino acid sequence of Hal-4.5 VH.
Underlined is
the heavy chain constant region. Figure 2M. The amino acid sequence of Hal -
4.5 VL.
Underlined is the light chain constant region. Figure 2N. The amino acid
sequence of Hal-4.40
VH. Underlined is the heavy chain constant region. Figure 2O. The amino acid
sequence of
Hal-4.40 VL. Underlined is the light chain constant region. Figure 2P. The
amino acid
sequence of Hal-4.37 VH. Underlined is the heavy chain constant region. Figure
2Q. The
amino acid sequence of Hal-4.37 VL. Underlined is the light chain constant
region. Figure 2R.
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The amino acid sequence of Hal-1.43 VH. Underlined is the heavy chain constant
region.
Figure 2S. The amino acid sequence of Hal-1.43 VL. Underlined is the light
chain constant
region. Figure 2T. The amino acid sequence of Hal-1.152 VH. Underlined is the
heavy chain
constant region. Figure 2U. The amino acid sequence of Hal-1.152 VL.
Underlined is the light
chain constant region.
(0044] Figure 3. Nucleotide and Amino Acid sequences of PSCA antibodies.
Figure 3A.
The cDNA and amino acid sequence of Hal -4.117 VH. Underlined is the heavy
chain constant
region. Figure 3B. The cDNA and amino acid sequence of Hal-4.117 VL.
Underlined is the
light chain constant region. Figure 3C. The cDNA and amino acid sequence of
Hal-4.120 VH.
Underlined is the heavy chain constant region. Figure 3D. The cDNA and amino
acid sequence
of Hal-4.120 VL. Underlined is the light chain constant region. Figure 3E. The
eDNA and
amino acid sequence of Hal-5.99 VH. Underlined is the heavy chain constant
region. Figure
3F. The cDNA and amino acid sequence of Hal-5.99 VL. Underlined is the light
chain
constant region. Figure 3G. The cDNA and amino acid sequence of Ha1-4.121 VH.
Underlined
is the heavy chain constant region. Figure 3H. The cDNA and amino acid
sequence of Hal-
4.121 VL c.5. Underlined is the light chain constant region. Figure 31. The
cDNA and amino
acid sequence of Hal -4.121 VL c.26. Underlined is the light chain constant
region. Figure 3J.
The eDNA and amino acid sequence of Hal-1.16 VH. Underlined is the heavy chain
constant
region. Figure 3K. The cDNA and amino acid sequence of Hal-1.16 VL. Underlined
is the
light chain constant region. Figure 3L. The cDNA and amino acid sequence of
Hal-4.5 VH.
Underlined is the heavy chain constant region. Figure 3M. The cDNA and amino
acid sequence
of Hal -4.5 VL. Underlined is the light chain constant region. Figure 3N. The
cDNA and amino
acid sequence of Hal-4.40 VH. Underlined is the heavy chain constant region.
Figure 30. The
cDNA and amino acid sequence of Hal-4.40 VL. Underlined is the light chain
constant region.
Figure 3P. The cDNA and amino acid sequence of Hal-4.37 VH. Underlined is the
heavy chain
constant region. Figure 3Q. The cDNA and amino acid sequence of Hal-4.37 VL.
Underlined
is the light chain constant region. Figure 3R. The eDNA and amino acid
sequence of Hal-1.43
VH. Underlined is the heavy chain constant region. Figure 3S. The eDNA and
amino acid
sequence of Hal-1.43 VL. Underlined is the light chain constant region. Figure
3T. The eDNA
and amino acid sequence of Hal -1.152 VH. Underlined is the heavy chain
constant region.
Figure 3U. The eDNA and amino acid sequence of Hal-1.152 VL. Underlined is the
light chain
constant region.
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[00451 Figure 4. Alignment of PSCA antibodies to germline V-D-J Sequences.
Figure 4A.
Alignment of Hal-4.117 VH (SEQ ID NO: 13) to human VH4-31. Figure 4B.
Alignment of
Hal-4.1 17 VL (SEQ ID NO: 14) to humanL19 Figure 4C. Alignment of Hal-4.120 VH
(SEQ
ID NO: 15) to human VH4-31 Figure 4D. Alignment of Hal-4.120 VL (SEQ ID NO:
16) to
human 02 Figure 4E. Alignment of Hal-5.99 VH (SEQ ID NO: 17) to human VH4-34
Figure
4F. Alignment of Hal-5.99 VL (SEQ ID NO: 18) to humanA27. Figure 4G. Alignment
of
Hal-4.121 VH (SEQ ID NO: 19) to human VH4-34. Figure 4H. Alignment of Hal-
4.121 c.5
VL (SEQ ID NO: 20) to human 08. Figure 41. Alignment of Hal-4.121 c.26 VL (SEQ
ID
NO: 21) to humanA3. Figure 4J. Alignment of Hal-1.16 VH (SEQ ID NO: 22) to
human
VH6-1. Figure 4K. Alignment of Hal-1.16 VL (SEQ ID NO: 23) to human B3. Figure
4L.
Alignment of Hal-4.37 VH (SEQ ID NO: 28) to human VH4-31. Figure 4M. Alignment
of
Hal-4.37 VL (SEQ ID NO: 29) to human 02.
[00461 Figure 5. Expression of PSCA protein in recombinant murine, rat and
human cell
lines. The indicated murine, rat, and human cell lines were infected with
retroviruses carrying
the human PSCA cDNA and a neomycin resistance gene or control virus with only
the
neomycin resistance gene. Stable recombinant cell lines were selected in the
presence of G418.
PSCA expression was determined by FACS staining with the 1 G8 anti-PSCA MAb (5
ug/ml).
Shown is the FACS profile of each cell line demonstrating a fluorescent shift
only in the PSCA
infected line indicative of cell surface PSCA expression. These lines are
useful in MAb
development as immunogens, MAb screening reagents, and in functional assays.
[0047] Figure 6. Purification of PSCA protein from E.Coli. E. coli. Strain
BL21 pLysS was
transformed with pET-21b vector encoding amino acids 21-94 of the PSCA cDNA.
PSCA
protein was expressed by induction of log phase cultures with IPTG and
purified by affinity
chromatography from either the soluble or insoluble fractions of the lysed
bacteria. Shown are
SDS-PAGE Coomasie blue stained gels of the eluted fractions. This protein is
useful as a MAb
and pAb immunogen and as an antibody screening reagent.
100481 Figure 7. Purification of recombinant glycoslated PSCA protein
expressed from
293T cells. 293Tcells were transfected with the psecTag2 vector carrying a
PSCA cDNA
encoding amino acids 28-100. A stable recombinant PSCA-secreting cell line was
created by
drug selection with hygromycin B. PSCA protein present in conditioned culture
medium was
purified by affinity chromatography using the I G8 MAb. Shown is a Coomasie
blue stained
SDS-PAGE gel of the low pH eluted fractions. The broad molecular weight smear
of the protein
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demonstrates glycosylation of recombinant PSCA protein as is seen in
endogenously expressed
PSCA.
[0049] Figure 8. Purification GST-PSCA protein from E.Coli. E. coli strain
BL21 DE3 was
transformed with pGEX-2T encoding amino acids 18-98 of PSCA fused to
glutathione-S-
transferase (GST). GST-PSCA protein was induced with isopropyl-beta-D-
thiogalactopyranoside (IPTG) from log phase cultures and purified from lysed
bacteria by
affinity chromatography with glutathione agarose matrix. Shown is an SDS-PAGE
Coomasie
blue stained gel of the glutathione eluted fractions containing GST-PSCA.
Indicated are the
intact GST-PSCA fusion protein and a minor degradation product containing GST.
This protein
is useful as a MAb and pAb immunogen and as an Ab screening reagent.
[0050] Figure 9. Screening for human PSCA antibodies by FACS. Antibody
concentration
from supernatants was determined by ELISA. 50u1/well of (neat) was added to 96-
well FACS
plates and serially diluted. PSCA-expressing cells were added (endogenous or
recombinant,
50,000 cells/well) and the mixture incubated at 4 C for two hours. Following
incubation, the
cells were washed with FACS Buffer and further incubated with 100ul of
detection antibody
(anti-hIgG-PE) for 45 minutes at 4 C. At the end of incubation, the cells were
washed with
FACS Buffer, fixed with formaldehyde and analyzed using FACScan. Data were
analyzed
using CellQuest Pro software. Solid histograms represent data from negative
control cells and
open histograms indicate data from PSCA-positive cells.
[0051] Figure 10. Relative affinity ranking of PSCA MAbs by FACS. 21 serial
1:2
dilutions of each PSCA antibody was incubated with SW780 cells (50,000 cells
per well) over
night at 4 C ( final MAb concentrations ranged from 40nM to 0.038pM). At the
end of the
incubation, cells were washed and incubated with anti-hIgG-PE detection
antibody. After
washing the unbound secondary antibodies, the cells were analyzed by FACS and
the mean
fluorescence intensity of each point obtained using CellQuest Pro software.
Affinity was
calculated withGraphpad Prism software using a Sigmoidal Dose-Response
(variable slope)
equation. A representative FACS analysis depicting the binding titration for
PSCA MAb 4.121
is presented in the figure.
[0052] Figure 11. Expression of murine and cynomolgus monkey PSCA in 293T
cells and
recognition by anti-human PSCA MAbs. 293T cells were transiently transfected
with
pCDNA3.1 vector carrying the murine PSCA cDNA, the simian PSCA cDNA, or with
empty
vector (neo). Two days following transfection, cells were harvested and
stained with either
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human anti-PSCA MAb Hal-4.117 or murine MAb 1G8 (5 ug/ml). Shown is the FACS
profile
demonstrating that MAb Hal -4.117 raised to the human PSCA protein binds to
both murine and
simian PSCA protein expressed in 293T cells. The murine 1G8 MAb binds to
simian PSCA but
not to murine PSCA. Such results demonstrate the ability to select MAbs that
crossreact with
antigen from other species. Crossreacting MAbs will be useful for studying
expression and
toxicity in those species.
10053] Figure 12. Internalization of PSCA following incubation with Mab 4.121.
PSCA
MAb 4.121 was incubated with PC3-PSCA cells at 4 C for 90 min to allow binding
of the
antibodies to the cell surface. The cells were then split into two aliquots
and incubated at either
37 C (to allow antibody internalization) or t 4 C (control no
internalization). Following
incuabation at 37 C or 4 C , the remaining PSCA MAb 4.121 bound to the cell
surface was
removed with an acid wash. Subsequent permeablization and incubation with a
secondary
detection antibody allowed detection of internalized PSCA MAb 4.121. Cells
were analyzed
using FACS or observed under the fluorescence microscope. Approximately 30% of
PSCA Mab
4.121 was internalized after incubation at 37 C for two hours.
100541 Figure 13. Antibodies to PSCA mediate saporin dependent killing in PSCA
expressing cells. B300.19 cells (750 cells/well) were seeded into a 96 well
plate on day 1. The
following day an equal volume of medium containing a 2X concentration of the
indicated
primary antibody together with a 2 fold excess of anti-human (Hum-Zap) or anti-
goat (Goat-
Zap) polyclonal antibody conjugated with saporin toxin (Advanced Targeting
Systems, San
Diego, CA) was added to each well. The cells were allowed to incubate for 5
days at
37 degrees C. At the end of the incubation period, MTS (Promega) was added to
each well and
incubation continued for an additional 4 hours. The OD at 450 nM was
determined. The results
in Figure 13(A) show that PSCA antibodies HA1-4.121 and HAI-4.117 mediated
saporin
dependent cytotoxicity in B300.19-PSCA cells while a control, nonspecific IgG1
antibody, had
no effect. The results in Figure 13(B) show that the addition of a secondary
saporin conjugated
antibody that did not recognize human Fc, failed to mediate cytotoxicity.
10055] Figure 14. Complement mediated cytotoxicity of PSCA Mabs. PSCA
antibodies (0-
50 g/ml) were diluted with RHB buffer (RPMI 1640, Gibco Life Technologies, 20
mM
HEPES). B300.19-PSCA expressing cells were washed in RHB buffer and
resuspended at a
density of 106 cells/ml. In a typical assay, 50 l of PSCA antibody, 50 pl of
diluted rabbit
complement serum (Cedarlane, Ontario, Can), and 50 l of a cell suspension
were added
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together into a flat-bottom tissue culture 96-well plate. The mixture was
incubated for 2 hr. at
37 C in a 5% C02 incubator to facilitate complement-mediated cell lysis. 50 gl
of Alamar Blue
(Biosource Intl. Camarillo, CA) was added to each well and incubation
continued for an
additional 4-5 hr at 37 C. The fluorescence in each well was read using a 96-
well fluorometer
with excitation at 530 nm and emission at 590 nm. The results show that PSCA
antibodies
having an IgG i (HA I-4.121) or an IgG2 isotype (HA I-5.99.1) but not an IgG4
isotype (HA 1-
6.46) were able to mediate complement dependent lysis of target cells.
[0056] Figure 15. Generation of F(ab')2 fragments of MAb Hal -4.121 by pepsin
digestion.
20 mgs of MAb Hal -4.121 in 20 mM sodium acetate buffer pH 4.5 was incubated
with and
without immobilized pepsin (Pierce. Rockford IL) for the indicated times.
Intact MAb and
digested Fc fragments were removed by protein A chromatography. Shown is a SDS-
PAGE
Coomasie stained gel of intact undigested unreduced MAb, unreduced aliquots of
digested
material taken at the indicated times, and a reduced sample of the final
digested F(ab')2 product.
[0057] Figure 16. Binding of recombinant anti-PSCA human mAb to PSCA by flow
cytometry. (16A) 293T cells were transfected with expression constructs
encoding the heavy
and light chains of anti-PSCA human mAb. Supernatant was collected after 48
hours and
assayed for binding to PSCA. (16B) Anti-PSCA human mAb was purified from
hybridoma
supernatant and used for PSCA binding assays. PSCA binding was tested as
follows. PC3
parental or PC3-PSCA cells were incubated with the anti-PSCA human mAbs
described above
for 30 minutes on ice. The cells were washed and incubated with PE-conjugated
anti-human Ig
for 30 minutes on ice. The cells were washed and then assayed by flow
cytometry.
[0058] Figure 17. Detection of PSCA protein by immunohistochemistry.
Expression of
PSCA protein in tumor specimens from cancer patients was detected using the
antibody HA 1-
4.117. Formalin fixed, paraffin embedded tissues were cut into 4 micron
sections and mounted
on glass slides. The sections were dewaxed, rehydrated and treated with
antigen retrieval
solution (Antigen Retrieval Citra Solution; BioGenex, 4600 Norris Canyon Road,
San Ramon,
CA, 94583) at high temperature. Sections were then incubated in fluorescein
conjugated human
monoclonal anti-PSCA antibody, Hal -4.117, for 16 hours at 4 C. The slides
were washed three
times in buffer and further incubated with Rabbit anti-Fluorescein for 1 hour
and, after washing
in buffer, immersed in DAKO EnVision+TM peroxidase-conjugated goat anti-rabbit
immunoglobulin secondary antibody (DAKO Corporation, Carpenteria, CA) for 30
minutes.
The sections were then washed in buffer, developed using the DAB kit (SIGMA
Chemicals),
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counterstained using hematoxylin, and analyzed by bright field microscopy. The
results show
expression of PSCA in the tumor cells of prostate adenocarcinoma (Panel A,
Panel B), bladder
transitional carcinoma (Panel C) and pancreatic ductal adenocarcinoma (Panel
D). These results
indicate that PSCA is expressed in human cancers and that antibodies directed
to this antigen are
useful as diagnostic reagents.
[00591 Figure 18. PSCA MAb Hal-4.120 Inhibits the Growth of Subcutaneous
Prostate
Cancer Xenografts. LAPC-9AI tumor cells (2.0 x 106 cells) were injected
subcutaneously into
male SCID mice. The mice were randomized into groups (n=10 in each group) and
treatment
initiated intraperitoneally (i.p.) on Day 0 with HA1-4.120 or isotype MAb
control as indicated.
Animals were treated twice weekly for a total of 7 doses until study day 28.
Tumor growth was
monitored using caliper measurements every 3 to 4 days as indicated. The
results show that
Human anti-PSCA monoclonal antibody Hal -4.120 significantly inhibited the
growth of human
prostate cancer xenografts implanted subcutaneously in SCID mice (p< 0.05).
[00601 Figure 19. PSCA MAb Hal -5.99 Inhibits the Growth of Established
Prostate Cancer
Xenografts in SCID mice. LAPC-9AI tumor cells (2.0 x 106 cells) were injected
subcutaneously
into male SCID mice. When tumor volume reached 50 mm3, the mice were
randomized into
groups (n=10 in each group) and treatment initiated intraperitoneally (i.p.)
with HA1-5.99.1 or
isotype MAb control as indicated. Animals were treated twice weekly for a
total of 5 doses until
study day 14. Tumor growth was monitored using caliper measurements every 3 to
4 days as
indicated. The results show that Fully human anti-PSCA monoclonal antibody Hal-
5.99
significantly inhibited the growth of established androgen-independent human
prostate cancer
xenografts implanted subcutaneously in SCID mice (p<0.05).
[00611 Figure 20. PSCA Mab HAI-4.121 Inhibits the Growth of Established
Androgen-
dependent Human Prostate Cancer Xenografts. LAPC-9AD tumor cells (2.5 x 106
cells) were
injected subcutaneously into male SCID mice. When the tumor volume reached 40
mm3, the
mice were randomized into groups (n=10 in each group) and treatment was
initiated
intraperitoneally (i.p.) with increasing concentrations of HA1-4.121 or
isotype MAb control as
indicated. Animals were treated twice weekly for a total of 7 doses until
study day 21. Tumor
growth was monitored using caliper measurements every 3 to 4 days as
indicated. The results
from this study demonstrated that HAI-4.121 inhibited the growth of
established subcutaneous
human androgen-dependent prostate xenografts in SCID mice. The results were
statistically
significant for the 300 ug dose group on days 14, 17 and 21 (p< 0.05, Kruskal-
Wallis test, two-
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sided with a=0.05) and for the 700 ug dose group on days 10, 14, 17 and 21 (p<
0.05, Kruskal-
Wallis test, two-sided with a=0.05).
[0062] Figure 21. Patient-derived, androgen-dependent, LAPC-9AD tumor cells
(2.0 x 106
cells) were injected into the dorsal lobes of the prostates of male SCID mice.
The tumors were
allowed to grow for approximately 10 days at which time the mice were
randomized into groups.
Treatment with human 500 ug of HA1-4.117, HA1-4.121 or Isotype control MAb was
initiated
days after tumor implantation. Antibodies were delivered intraperitoneally two
times a week
for a total of 7 doses. Four days after the last dose, animals were sacrificed
and primary tumors
excised and weighed. The results show that Human anti-PSCA monoclonal
antibodies Hal -
4.121 (p< 0.01) and Hal-4.117 (p< 0.05) significantly inhibited the growth of
LAPC-9AD
prostate cancer xenografts orthotopically implanted in SCID mice.
[0063] Figure 22. PSCA MAb HA1-4.121 Prolongs the Survival of SCID Mice with
Established Orthotopic Human Androgen-Dependent Prostate Tumors. Patient-
derived,
androgen-dependent, LAPC-9AD tumor cells (2.0 x 106 cells) were injected into
the dorsal
lobes of the prostates of male SCID mice. The tumors were allowed to grow for
approximately
9 days at which time the mice were randomized into groups. The animals
randomized into the
survival groups include 11 mice in the isotype MAb control and 12 mice in the
HA1-4.121
treated group. Animals were treated i.p. with 1000 ug Hal-4.121 or 1000 ug of
isotype MAb
control twice weekly for a total of 9 doses. The results demonstrated that HA
1-4.121
significantly (log-rank test: p<0.01) prolonged the survival of SCID mice with
human androgen-
dependent prostate tumors. Two mice in the HA1-4.121 treated group remained
free of palpable
tumors 110 days after the last treatment.
[0064] Figure 23. Enhanced Inhibition of Prostate Tumor Growth with HAI-4.21
and
Taxotere Combination Therapy. LAPC-9AI tumor cells (2 x 106 cells per animal)
were injected
subcutaneously into male SCID mice. When the tumor volume reached 65 mm3,
animals were
randomized and assigned to four different groups (n=l 0 in each group) as
indicated. Beginning
on day 0, Hal-4.121 or isotype MAb control were administered i.p. two times a
week at a dose
of 500 ug for a total of 6 doses. The last dose was given on day 17. Taxotere
was given
intravenously at a dose of 5 mg/kg on days 0, 3 and 7. Tumor growth was
monitored every 3-4
days using caliper measurements. The results from this study demonstrate that
HA1-4.121 as a
single agent inhibited the growth of androgen-independent prostate xenografls
in SCID mice by
45% when compared to control antibody treatment alone on day 28 (ANOVA/Tukey
test:
.17
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p<0.05). Administration of the isotype MAb control plus taxotere inhibited
tumor growth by
28% when compared to control antibody treatment alone, which was not
statistically significant.
Administration of HA1-4.121 in combination with Taxotere, enhanced the effect
and resulted in
a 69% inhibition of tumor growth when compared to control antibody alone
(ANOVA/Tukey
test: p<0.01). A statistically significant difference was also demonstrated
when the HAI-4.121
plus Taxotere combination group was compared to either the HA1-4.121 or
isotype MAb control
plus Taxotere groups (ANOVA/Tukey test: p<0.05).
[0065] Figure 24. Human PSCA MAbs Inhibit the Growth of Pancreatic Cancer
Xenografts
in SCID mice/ Human HPAC. Pancreatic cancer cells (2 x 106/ mouse) were
injected
subcutaneously into immunodeficient ICR SCID mice (Taconic Farm, Germantown,
NY). The
mice were randomized into groups (n= 10 animals/group) and treatment with the
indicated
humanPSCA monoclonal antibody initiated the same day. Antibodies (500
mg/mouse) were
delivered intraperitoneally two times a week for a total of 8 doses. The
results demonstrated that
human anti-PSCA monoclonal antibodies Hal-4.121, Hal-4.117 and Hal-1.16
significantly
inhibited the growth of human pancreatic cancer xenografts subcutaneously
implanted in SCID
mice. Statistical analyses were performed using a t test (two-sided, a=0.05).
[0066] Figure 25. PSCA MAb HA 1-4.121 Inhibits the Growth of Pancreatic Tumors
Implanted Orthotopically in SCID Mice. HPAC cells (3.0 x 106 cells) were
implanted
orthotopically into the pancreata of SCID mice. Mice were randomly assigned to
three groups
(n=9 in each group) as indicated. Treatment with HAI-4.121 (250 ug or 1000 ug)
or isotype
MAb control (1000 ug) was initiated on the day of implantation. Antibodies
were administered
i.p. twice weekly for a total of 10 doses. Thirteen days after the last dose,
animals were
sacrificed and primary tumors excised and weighed. The results from this study
demonstrated
that HAI-4.121 significantly inhibited the orthotopic growth of human
pancreatic cancer
xenografts in SCID mice at both dose levels examined. Treatment with 250 ug
and 1000 ug
AGS-PSCA inhibited tumor growth by 66% and 70%, respectively (Kruskal-
Wallis/Tukey test:
p<0.01 and p<0.01, respectively).
[0067] Figure 26. PSCA MAb HA I-4.121 inhibits matastases. At autopsy, visible
metastases to lymph nodes and distant organs were observed in the control
antibody treated
group. No visible metastases were observed in both HAI-4.121 treated groups.
Lymph nodes,
lungs and livers were removed from all animals and examined histologically for
the presence of
metastatic tumor. Sections from the lungs and lymph nodes removed from each
animal were
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stained for human cytokeratin and the number of metastases determined
microscopically. The
results from the histologic analysis demonstrated a significant reduction in
lymph node (LN)
metastases in animal treated with HA1-4.121 (p= 0.0152 as detected by Fishers
exact test). The
incidence of metastasis and invasion was also decreased significantly in
animals treated with
both concentrations of HA1-4.121 (p= 0.0152 as detected by Fishers exact
test). The number of
lung metastases decreased significantly in mice treated with the 1.0 mg dose
of HAl-4.121 only
(p= 0.0498 as detected by Fishers exact test).
[00681 Figure 27. Human PSCA MAbs Inhibit the Growth of SW780 Bladder Tumors
in
SCID Mice. Human SW780 bladder cancer cells (2 x 106/ mouse) were injected
subcutaneously
into immunodeficient ICR SCID mice (Taconic Farm, Germantown, NY). The mice
were
randomized into groups (n= 10 animals/group) and treatment with the indicated
human PSCA
MAb initiated the same day. Antibodies (250 mg/mouse) were delivered
intraperitoneally two
times a week for a total of 7 doses. The results demonstrated that HA1-4.117
(p= 0.014), HAl-
4.37 (p= 0.0056), HA I -1.78 (p= 0.001), Hal-5.99 (p=0.0002) and HA I -4.5 (p=
0.0008)
significanctly inhibited the growth of SW780 bladder tumors implanted
subcutaneously in SCID
mice. Statistical analyses were performed using a t test (two-sided, a=0.05).
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) PSCA Polynucleotides
II.A.) Uses of PSCA Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
II.A.2.) Antisense Embodiments
II.A.3.) Primers and Primer Pairs
II.A.4.) Isolation of PSCA-Encoding Nucleic Acid Molecules
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
III.) PSCA-related Proteins
III.A.) Motif-bearing Protein Embodiments
III.B.) Expression of PSCA-related Proteins
III.C.) Modifications of PSCA-related Proteins
11I.D.) Uses of PSCA-related Proteins
IV.) PSCA Antibodies
V.) PSCA Cellular Immune Responses
VI.) PSCA Transgenic Animals
VII.) Methods for the Detection of PSCA
VIII.) Methods for Monitoring the Status of PSCA-related Genes and Their
Products
IX.) Identification of Molecules That Interact With PSCA
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X.) Therapeutic Methods and Compositions
X.A.) Anti-Cancer Vaccines
X.B.) PSCA as a Target for Antibody-Based Therapy
X.C.) PSCA as a Target for Cellular Immune Responses
X.C. 1. Minigene Vaccines
X.C.2. Combinations of CTL Peptides with Helper Peptides
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL
Peptides
X.D.) Adoptive Immunotherapy
X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes
XI.) Diagnostic and Prognostic Embodiments of PSCA.
XII.) Inhibition of PSCA Protein Function
XII.A.) Inhibition of PSCA With Intracellular Antibodies
XII.B.) Inhibition of PSCA with Recombinant Proteins
XII.C.) Inhibition of PSCA Transcription or Translation
XII.D.) General Considerations for Therapeutic Strategies
XIII.) Identification, Characterization and Use of Modulators of PSCA
XIV.) RNAi and Therapeutic use of small interfering RNA (siRNAs)
XV.) KITS/Articles of Manufacture
I.) Definitions:
[00691 Unless otherwise defined, all terms of art, notations and other
scientific terms or
terminology used herein are intended to have the meanings commonly understood
by those of
skill in the art to which this invention pertains. In some cases, terms with
commonly understood
meanings are defined herein for clarity and/or for ready reference, and the
inclusion of such
definitions herein should not necessarily be construed to represent a
substantial difference over
what is generally understood in the art. Many of the techniques and procedures
described or
referenced herein are well understood and commonly employed using conventional
methodology by those skilled in the art, such as, for example, the widely
utilized molecular
cloning methodologies described in Sambrook et al., Molecular Cloning: A
Laboratory Manual
2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. As
appropriate, procedures involving the use of commercially available kits and
reagents are
generally carried out in accordance with manufacturer defined protocols and/or
parameters
unless otherwise noted.
[00701 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
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Urological Association (AUA) system, stage Cl - C2 disease under the Whitmore-
Jewett
system, and stage T3 - T4 and N+ disease under the TNM (tumor, node,
metastasis) system. In
general, surgery is not recommended for patients with locally advanced
disease, and these
patients have substantially less favorable outcomes compared to patients
having clinically
localized (organ-confined) prostate cancer. Locally advanced disease is
clinically identified by
palpable evidence of induration beyond the lateral border of the prostate, or
asymmetry or
induration above the prostate base. Locally advanced prostate cancer is
presently diagnosed
pathologically following radical prostatectomy if the tumor invades or
penetrates the prostatic
capsule, extends into the surgical margin, or invades the seminal vesicles.
[0071] "Altering the native glycosylation pattern" is intended for purposes
herein to mean
deleting one or more carbohydrate moieties found in native sequence PSCA
(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
PSCA. 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.
[0072] The term "analog" refers to a molecule which is structurally similar or
shares similar
or corresponding attributes with another molecule (e.g. a PSCA-related
protein). For example,
an analog of a PSCA protein can be specifically bound by an antibody or T cell
that specifically
binds to PSCA.
[0073] The term "antibody" is used in the broadest sense unless clearly
indicated otherwise.
Therefore, an "antibody" can be naturally occurring or man-made such as
monoclonal antibodies
produced by conventional hybridoma technology. Anti-PSCA 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. As used
herein, the term
"antibody" refers to any form of antibody or fragment thereof that
specifically binds PSCA
and/or exhibits the desired biological activity and specifically covers
monoclonal antibodies
(including full length monoclonal antibodies), polyclonal antibodies,
multispecific antibodies
(e.g., bispecific antibodies), and antibody fragments so long as they
specifically bind PSCA
and/or exhibit the desired biological activity. Any specific antibody can be
used in the methods
and compositions provided herein. Thus, in one embodiment the term "antibody"
encompasses
a molecule comprising at least one variable region from a light chain
immunoglobulin molecule
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WO 2005/118864 PCT/US2005/017412
and at least one variable region from a heavy chain molecule that in
combination form a specific
binding site for the target antigen. In one embodiment, the antibody is an IgG
antibody. For
example, the antibody is a IgG1, IgG2, IgG3, or IgG4 antibody. The antibodies
useful in the
present methods and compositions can be generated in cell culture, in phage,
or in various
animals, including but not limited to cows, rabbits, goats, mice, rats,
hamsters, guinea pigs,
sheep, dogs, cats, monkeys, chimpanzees, apes. Therefore, in one embodiment,
an antibody of
the present invention is a mammalian antibody. Phage techniques can be used to
isolate an
initial antibody or to generate variants with altered specificity or avidity
characteristics. Such
techniques are routine and well known in the art. In one embodiment, the
antibody is produced
by recombinant means known in the art. For example, a recombinant antibody can
be produced
by transfecting a host cell with a vector comprising a DNA sequence encoding
the antibody.
One or more vectors can be used to transfect the DNA sequence expressing at
least one VL and
one VH region in the host cell. Exemplary descriptions of recombinant means of
antibody
generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL
TECHNIQUES (Wiley, 1997); Shephard, et al., MONOCLONAL ANTIBODIES (Oxford
University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND
PRACTICE (Academic Press, 1993); CURRENT PROTOCOLS IN IMMUNOLOGY (John
Wiley & Sons, most recent edition). An antibody of the present invention can
be modified by
recombinant means to increase greater efficacy of the antibody in mediating
the desired
function. Thus, it is within the scope of the invention that antibodies can be
modified by
substitutions using recombinant means. Typically, the substitutions will be
conservative
substitutions. For example, at least one amino acid in the constant region of
the antibody can be
replaced with a different residue. See, e.g., U.S. Patent No. 5,624,821, U.S.
Patent No.
6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30:
105-08 (1993).
The modification in amino acids includes deletions, additions, substitutions
of amino acids. In
some cases, such changes are made to reduce undesired activities, e.g.,
complement-dependent
cytotoxicity. Frequently, the antibodies are labeled by joining, either
covalently or non-
covalently, a substance which provides for a detectable signal. A wide variety
of labels and
conjugation techniques are known and are reported extensively in both the
scientific and patent
literature. These antibodies can be screened for binding to normal or
defective PSCA. See e.g.,
ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996).
Suitable antibodies with the desired biologic activities can be identified the
following in vitro
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assays including but not limited to: proliferation, migration, adhesion, soft
agar growth,
angiogenesis, cell-cell communication, apoptosis, transport, signal
transduction, and the
following in vivo assays such as the inhibition of tumor growth. The
antibodies provided herein
can also be useful in diagnostic applications. As capture or non-neutralizing
antibodies, they can
be screened for the ability to bind to the specific antigen without inhibiting
the receptor-binding
or biological activity of the antigen. As neutralizing antibodies, the
antibodies can be useful in
competitive binding assays. They can also be used to quantify the PSCA or its
receptor.
100741 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-PSCA antibodies and clones
thereof (including
agonist, antagonist and neutralizing antibodies) and anti-PSCA antibody
compositions with
polyepitopic specificity. The antibody of the present methods and compositions
can be
monoclonal or polyclonal. An antibody can be in the form of an antigen binding
antibody
fragment including a Fab fragment, F(ab')2 fragment, a single chain variable
region, and the like.
Fragments of intact molecules can be generated using methods well known in the
art and include
enzymatic digestion and recombinant means.
[00751 As used herein, any form of the "antigen" can be used to generate an
antibody that is
specific for PSCA. Thus, the eliciting antigen may be a single epitope,
multiple epitopes, or the
entire protein alone or in combination with one or more immunogenicity
enhancing agents
known in the art. The eliciting antigen may be an isolated full-length
protein, a cell surface
protein (e.g., immunizing with cells transfected with at least a portion of
the antigen), or a
soluble protein (e.g., immunizing with only the extracellular domain portion
of the protein). The
antigen may be produced in a genetically modified cell. The DNA encoding the
antigen may
genomic or non-genomic (e.g., eDNA) and encodes at least a portion of the
extracellular
domain. As used herein, the term "portion" refers to the minimal number of
amino acids or
nucleic acids, as appropriate, to constitute an immunogenic epitope of the
antigen of interest.
Any genetic vectors suitable for transformation of the cells of interest may
be employed,
including but not limited to adenoviral vectors, plasmids, and non-viral
vectors, such as cationic
lipids. In one embodiment, the antibody of the methods and compositions herein
specifically
bind at least a portion of the extracellular domain of the PSCA of interest.
[00761 The antibodies or antigen binding fragments thereof provided herein may
be
conjugated to a "bioactive agent." As used herein, the term "bioactive agent"
refers to any
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synthetic or naturally occurring compound that binds the antigen and/or
enhances or mediates a
desired biological effect to enhance cell-killing toxins.
[00771 In one embodiment, the binding fragments useful in the present
invention are
biologically active fragments. As used herein, the term "biologically active"
refers to an
antibody or antibody fragment that is capable of binding the desired the
antigenic epitope and
directly or indirectly exerting a biologic effect. Direct effects include, but
are not limited to the
modulation, stimulation, and/ or inhibition of a growth signal, the
modulation, stimulation, and/
or inhibition of an anti-apoptotic signal, the modulation, stimulation, and/
or inhibition of an
apoptotic or necrotic signal, modulation, stimulation, and/ or inhibition the
ADCC cascade, and
modulation, stimulation, and/ or inhibition the CDC cascade.
[0078] "Bispecific" antibodies are also useful in the present methods and
compositions. As
used herein, the term "bispecific antibody" refers to an antibody, typically a
monoclonal
antibody, having binding specificities for at least two different antigenic
epitopes. In one
embodiment, the epitopes are from the same antigen. In another embodiment, the
epitopes are
from two different antigens. Methods for making bispecific antibodies are
known in the art. For
example, bispecific antibodies can be produced recombinantly using the co-
expression of two
immunoglobulin heavy chain/light chain pairs. See, e.g., Milstein et al.,
Nature 305:537-39
(1983). Alternatively, bispecific antibodies can be prepared using chemical
linkage. See, e.g.,
Brennan, et al., Science 229:81 (1985). Bispecific antibodies include
bispecific antibody
fragments. See, e.g., Hollinger, et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-
48 (1993), Gruber,
et al., J. Immunol. 152:5368 (1994).
[00791 The monoclonal antibodies herein specifically include "chimeric"
antibodies in
which a portion of the heavy and/or light chain is identical with or
homologous to corresponding
sequences in antibodies derived from a particular species or belonging to a
particular antibody
class or subclass, while the remainder of the chain(s) is identical with or
homologous to
corresponding sequences in antibodies derived from another species or
belonging to another
antibody class or subclass, as well as fragments of such antibodies, so long
as they specifically
bind the target antigen and/or exhibit the desired biological activity (U.S.
Pat. No. 4,816,567;
and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).
100801 The term "Chemotherapeutic Agent" refers to all chemical compounds that
are
effective in inhibiting tumor growth. Non-limiting examples of
chemotherapeutic agents include
alkylating agents; for example, nitrogen mustards, ethyleneimine compounds and
alkyl
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sulphonates; antimetabolites; for example, folic acid, purine or pyrimidine
antagonists; mitotic
inhibitors; for example, vinca alkaloids and derivatives of podophyllotoxin,
cytotoxic
antibiotics, compounds that damage or interfere with DNA expression, and
growth factor
receptor antagonists. In addition, chemotherapeutic agents include cytotoxic
agents (as defined
herein), antibodies, biological molecules and small molecules.
[00811 The term "codon optimized sequences" refers to nucleotide sequences
that have been
optimized for a particular host species by replacing any codons having a usage
frequency of less
than about 20%. Nucleotide sequences that have been optimized for expression
in a given host
species by elimination of spurious polyadenylation sequences, elimination of
exon/intron
splicing signals, elimination of transposon-like repeats and/or optimization
of GC content in
addition to codon optimization are referred to herein as an "expression
enhanced sequences."
[00821 A "combinatorial library" is a collection of diverse chemical compounds
generated
by either chemical synthesis or biological synthesis by combining a number of
chemical
"building blocks" such as reagents. For example, a linear combinatorial
chemical library, such
as a polypeptide (e.g., mutein) library, is formed by combining a set of
chemical building blocks
called amino acids in every possible way for a given compound length (i.e.,
the number of
amino acids in a polypeptide compound). Numerous chemical compounds are
synthesized
through such combinatorial mixing of chemical building blocks (Gallop et al.,
J. Med. Chem.
37(9): 1233-1251 (1994)).
[00831 Preparation and screening of combinatorial libraries is well known to
those of skill in
the art. Such combinatorial chemical libraries include, but are not limited
to, peptide libraries
(see, e.g., U.S. Patent No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493
(1991), Houghton
et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735),
encoded
peptides (PCT Publication WO 93/20242), random bio- oligomers (PCT Publication
WO
92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as
hydantoins,
benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA
90:6909-6913 (1993)),
vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568
(1992)), nonpeptidal
peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer.
Chem. Soc.
114:9217-9218 (1992)), analogous organic syntheses of small compound libraries
(Chen et al.,
J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbarnates (Cho, et al., Science
261:1303 (1993)),
and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)).
See, generally,
Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see,
e.g., Stratagene, Corp.),
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peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody
libraries (see, e.g.,
Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and
PCT/US96/10287),
carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996),
and U.S. Patent
No. 5,593,853), and small organic molecule libraries (see, e.g.,
benzodiazepines, Baum, C&EN,
Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588;
thiazolidinones and
metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos.
5,525,735 and
5,519,134; morpholino compounds, U.S. Patent No. 5,506, 337; benzodiazepines,
U.S. Patent
No. 5,288,514; and the like).
[0084] Devices for the preparation of combinatorial libraries are commercially
available
(see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville KY; Symphony,
Rainin,
Woburn, MA; 433A, Applied Biosystems, Foster City, CA; 9050, Plus, Millipore,
Bedford,
NIA). A number of well-known robotic systems have also been developed for
solution phase
chemistries. These systems include automated workstations such as the
automated synthesis
apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and
many robotic
systems utilizing robotic arms (Zymate H, Zymark Corporation, Hopkinton,
Mass.; Orca,
Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic
operations performed by
a chemist. Any of the above devices are suitable for use with the present
invention. The nature
and implementation of modifications to these devices (if any) so that they can
operate as
discussed herein will be apparent to persons skilled in the relevant art. In
addition, numerous
combinatorial libraries are themselves commercially available (see, e.g.,
ComGenex, Princeton,
NJ; Asinex, Moscow, RU; Tripos, Inc., St. Louis, MO; ChemStar, Ltd, Moscow,
RU; 3D
Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; etc.).
[0085] As used herein, the term "conservative substitution" refers to
substitutions of amino
acids are known to those of skill in this art and may be made generally
without altering the
biological activity of the resulting molecule. Those of skill in this art
recognize that, in general,
single amino acid substitutions in non-essential regions of a polypeptide do
not substantially
alter biological activity (see, e.g., Watson, et al., MOLECULAR BIOLOGY OF THE
GENE,
The Benjamin/Cummings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary
substitutions
are preferably made in accordance with those set forth in Table(s) III(a-b).
For example, 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
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substitutions can also be considered conservative, depending on the
environment of the
particular amino acid and its role in the three-dimensional structure of the
protein. For example,
glycine (G) and alanine (A) can frequently be interchangeable, as can alanine
(A) and valine
(V). Methionine (M), which is relatively hydrophobic, can frequently be
interchanged with
leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R)
are frequently
interchangeable in locations in which the significant feature of the amino
acid residue is its
charge and the differing pK's of these two amino acid residues are not
significant. Still other
changes can be considered "conservative" in particular environments (see, e.g.
Table 111(a)
herein; pages 13-15 "Biochemistry" 2nd ED. Lubert Stryer ed (Stanford
University); Henikoff
et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;
270(20):11882-
6). Other substitutions are also permissible and may be determined empirically
or in accord with
known conservative substitutions.
[0086] The term "cytotoxic agent" refers to a substance that inhibits or
prevents the
expression activity of cells, function of cells and/or causes destruction of
cells. The term is
intended to include radioactive isotopes, chemotherapeutic agents, and toxins
such as small
molecule toxins or enzymatically active toxins of bacterial, fungal, plant or
animal origin,
including fragments and/or variants thereof. Examples of cytotoxic agents
include, but are not
limited to auristatins, auristatin e, auromycins, maytansinoids, yttrium,
bismuth, ricin, ricin A-
chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin,
taxol, cispiatin,
cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,
vinblastine,
colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin,
Pseudomonas exotoxin
(PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin,
mitogellin,
retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,
Sapaonaria officinalis
inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as
radioisotopes such as
At211' II31' 1125' Y90' Re'xb, Re'88, Sm153'BI 212 or 213' P32 and radioactive
isotopes of Lu including
Lu177. Antibodies may also be conjugated to an anti-cancer pro-drug activating
enzyme capable
of converting the pro-drug to its active form.
100871 As used herein, the term "diabodies" refers to small antibody fragments
with two
antigen-binding sites, which fragments comprise a heavy chain variable domain
(VH) connected
to a light chain variable domain (Vi,) in the same polypeptide chain (VH-VL).
By using a linker
that is too short to allow pairing between the two domains on the same chain,
the domains are
forced to pair with the complementary domains of another chain and create two
antigen-binding
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sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161;
and Hollinger
et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).
[0088] The "gene product" is used herein to indicate a peptide/protein or
mRNA. For
example, a "gene product of the invention" is sometimes referred to herein as
a "cancer amino
acid sequence", "cancer protein", "protein of a cancer listed in Table I", a
"cancer mRNA",
"mRNA of a cancer listed in Table I", etc. In one embodiment, the cancer
protein is encoded by
a nucleic acid of Figure 1. The cancer protein can be a fragment, or
alternatively, be the full-
length protein encoded by nucleic acids of Figure 1. In one embodiment, a
cancer amino acid
sequence is used to determine sequence identity or similarity. In another
embodiment, the
sequences are naturally occurring allelic variants of a protein encoded by a
nucleic acid of
Figure 1. In another embodiment, the sequences are sequence variants as
further described
herein.
[0089] "Heteroconjugate" antibodies are useful in the present methods and
compositions.
As used herein, the term "heteroconjugate antibody" refers to two covalently
joined antibodies.
Such antibodies can be prepared using known methods in synthetic protein
chemistry, including
using crosslinking agents. See, e.g., U.S. Patent No. 4,676,980.
[0090] "High throughput screening" assays for the presence, absence,
quantification, or
other properties of particular nucleic acids or protein products are well
known to those of skill in
the art. Similarly, binding assays and reporter gene assays are similarly well
known. Thus, e.g.,
U.S. Patent No. 5,559,410 discloses high throughput screening methods for
proteins; U.S. Patent
No. 5,585,639 discloses high throughput screening methods for nucleic acid
binding (i.e., in
arrays); while U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high
throughput methods of
screening for ligand/antibody binding.
[0091] In addition, high throughput screening systems are commercially
available (see, e.g.,
Amersham Biosciences, Piscataway, NJ; Zymark Corp., Hopkinton, MA; Air
Technical
Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision
Systems, Inc.,
Natick, MA; etc.). These systems typically automate entire procedures,
including all sample and
reagent pipetting, liquid dispensing, timed incubations, and final readings of
the microplate in
detector(s) appropriate for the assay. These configurable systems provide high
throughput and
rapid start up as well as a high degree of flexibility and customization. The
manufacturers of
such systems provide detailed protocols for various high throughput systems.
Thus, e.g.,
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Zymark Corp. provides technical bulletins describing screening systems for
detecting the
modulation of gene transcription, ligand binding, and the like.
[0092] 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.
[0093] In one embodiment, the antibody provided herein is a "human antibody."
As used
herein, the term "human antibody" refers to an antibody in which essentially
the entire
sequences of the light chain and heavy chain sequences, including the
complementary
determining regions (CDRs), are from human genes. In one embodiment, human
monoclonal
antibodies are prepared by the trioma technique, the human B-cell technique
(see, e.g., Kozbor,
et al., Immunol. Today 4: 72 (1983) , EBV transformation technique (see, e.g.,
Cole et al.
MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985)), or using phage
display (see, e.g., Marks et al., J. Mol. Biol. 222:581 (1991)). In a specific
embodiment, the
human antibody is generated in a transgenic mouse. Techniques for making such
partially to
fully human antibodies are known in the art and any such techniques can be
used. According to
one particularly preferred embodiment, fully human antibody sequences are made
in a
transgenic mouse engineered to express human heavy and light chain antibody
genes. An
exemplary description of preparing transgenic mice that produce human
antibodies found in
Application No. WO 02/43478 and United States Patent 6,657,103 (Abgenix) and
its progeny.
B cells from transgenic mice that produce the desired antibody can then be
fused to make
hybridoma cell lines for continuous production of the antibody. See, e.g.,
U.S. Patent Nos.
5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Jakobovits,
Adv. Drug Del.
Rev. 31:33-42 (1998); Green, et al., J. Exp. Med. 188:483-95 (1998).
[0094] "Human Leukocyte Antigen" or "HLA" is a human class I or class II Major
Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,
IMMUNOLOGY, 8TH ED.,
Lange Publishing, Los Altos, CA (1994).
[0095] As used herein, the term "humanized antibody" refers to forms of
antibodies that
contain sequences from non-human (e.g., murine) antibodies as well as human
antibodies. Such
antibodies are chimeric antibodies which contain minimal sequence derived from
non-human
immunoglobulin. In general, the humanized antibody will comprise substantially
all of at least
one, and typically two, variable domains, in which all or substantially all of
the hypervariable
loops correspond to those of a non-human immunoglobulin and all or
substantially all of the FR
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regions are those of a human immunoglobulin sequence. The humanized antibody
optionally
also will comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of
a human immunoglobulin. See e.g., Cabilly U.S. Patent No. 4,816,567; Queen et
al. (1989)
Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODY ENGINEERING: A
PRACTICAL APPROACH (Oxford University Press 1996).
[00961 The terms "hybridize", "hybridizing", "hybridizes" and the like, used
in the
context of polynucleotides, are meant to refer to conventional hybridization
conditions,
preferably such as hybridization in 50% formamide/6XSSC/0.1 % SDS/100 gg/ml
ssDNA, in
which temperatures for hybridization are above 37 degrees C and temperatures
for washing in
0.1XSSC/0.1% SDS are above 55 degrees C.
[00971 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
PSCA genes or
that encode polypeptides other than PSCA gene product or fragments thereof. A
skilled artisan
can readily employ nucleic acid isolation procedures to obtain an isolated
PSCA polynucleotide.
A protein is said to be "isolated," for example, when physical, mechanical or
chemical methods
are employed to remove the PSCA proteins from cellular constituents that are
normally
associated with the protein. A skilled artisan can readily employ standard
purification methods
to obtain an isolated PSCA protein. Alternatively, an isolated protein can be
prepared by
chemical means.
[00981 Suitable "labels" include radionuclides, enzymes, substrates,
cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic particles, and the
like. Patents
teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752;
3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. In addition, the antibodies
provided herein can
be useful as the antigen-binding component of fluorobodies. See e.g., Zeytun
et al., Nat.
Biotechnol. 21:1473-79 (2003).
[00991 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.
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[01001 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.
[01011 The term "modulator" or "test compound" or "drug candidate" or
grammatical
equivalents as used herein describe any molecule, e.g., protein, oligopeptide,
small organic
molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity
to directly or
indirectly alter the cancer phenotype or the expression of a cancer sequence,
e.g., a nucleic acid
or protein sequences, or effects of cancer sequences (e.g., signaling, gene
expression, protein
interaction, etc.) In one aspect, a modulator will neutralize the effect of a
cancer protein of the
invention. By "neutralize" is meant that an activity of a protein is inhibited
or blocked, along
with the consequent effect on the cell. In another aspect, a modulator will
neutralize the effect
of a gene, and its corresponding protein, of the invention by normalizing
levels of said protein.
In preferred embodiments, modulators alter expression profiles, or expression
profile nucleic
acids or proteins provided herein, or downstream effector pathways. In one
embodiment, the
modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint.
In another
embodiment, a modulator induced a cancer phenotype. Generally, a plurality of
assay mixtures
is run in parallel with different agent concentrations to obtain a
differential response to the
various concentrations. Typically, one of these concentrations serves as a
negative control, i.e.,
at zero concentration or below the level of detection.
101021 Modulators, drug candidates or test compounds encompass numerous
chemical
classes, though typically they are organic molecules, preferably small organic
compounds
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having a molecular weight of more than 100 and less than about 2,500 Daltons.
Preferred small
molecules are less than 2000, or less than 1500 or less than 1000 or less than
500 D. Candidate
agents comprise functional groups necessary for structural interaction with
proteins, particularly
hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl
or carboxyl
group, preferably at least two of the functional chemical groups. The
candidate agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures
substituted with one or more of the above functional groups. Modulators also
comprise
biomolecules such as peptides, saccharides, fatty acids, steroids, purines,
pyrimidines,
derivatives, structural analogs or combinations thereof. Particularly
preferred are peptides. One
class of modulators are peptides, for example of from about five to about 35
amino acids, with
from about five to about 20 amino acids being preferred, and from about 7 to
about 15 being
particularly preferred. Preferably, the cancer modulatory protein is soluble,
includes a non-
transmembrane region, and/or, has an N-terminal Cys to aid in solubility. In
one embodiment,
the C-terminus of the fragment is kept as a free acid and the N-terminus is a
free amine to aid in
coupling, i.e., to cysteine. In one embodiment, a cancer protein of the
invention is conjugated to
an immunogenic agent as discussed herein. In one embodiment, the cancer
protein is conjugated
to BSA. The peptides of the invention, e.g., of preferred lengths, can be
linked to each other or
to other amino acids to create a longer peptide/protein. The modulatory
peptides can be digests
of naturally occurring proteins as is outlined above, random peptides, or
"biased" random
peptides. In a preferred embodiment, peptide/protein-based modulators are
antibodies, and
fragments thereof, as defined herein.
[01031 Modulators of cancer can also be nucleic acids. Nucleic acid modulating
agents can
be naturally occurring nucleic acids, random nucleic acids, or "biased" random
nucleic acids.
For example, digests of prokaryotic or eukaryotic genomes can be used in an
approach
analogous to that outlined above for proteins.
[0104] The term "monoclonal antibody", as used herein, refers to an antibody
obtained from
a population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising
the population are identical except for possible naturally occurring mutations
that may be
present in minor amounts. Monoclonal antibodies are highly specific, being
directed against a
single antigenic epitope. In contrast, conventional (polyclonal) antibody
preparations typically
include a multitude of antibodies directed against (or specific for) different
epitopes. In one
embodiment, the polyclonal antibody contains a plurality of monoclonal
antibodies with
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different epitope specificities, affinities, or avidities within a single
antigen that contains
multiple antigenic epitopes. The modifier "monoclonal" indicates the character
of the antibody
as being obtained from a substantially homogeneous population of antibodies,
and is not to be
construed as requiring production of the antibody by any particular method.
For example, the
monoclonal antibodies to be used in accordance with the present invention may
be made by the
hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or
maybe made by
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies"
may also be isolated from phage antibody libraries using the techniques
described in Clackson
et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-
597 (1991), for
example. These monoclonal antibodies will usually bind with at least a Kd of
about 1 M, more
usually at least about 300 nM, typically at least about 30 nM, preferably at
least about 10 nM,
more preferably at least about 3 nM or better, usually determined by ELISA.
101051 A "motif', as in biological motif of a PSCA-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. Frequently occurring motifs are set forth in Table
V.
101061 A "pharmaceutical excipient" comprises a material such as an adjuvant,
a carrier, pH-
adjusting and buffering agents, tonicity adjusting agents, wetting agents,
preservative, and the
like.
[01071 "Pharmaceutically acceptable" refers to a non-toxic, inert, and/or
composition that is
physiologically compatible with humans or other mammals.
101081 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
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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 1, can also be uracil (U); this definition
pertains to the differences
between the chemical structures of DNA and RNA, in particular the observation
that one of the
four major bases in RNA is uracil (U) instead of thymidine (T).
[0109] 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".
[0110] An HLA "primary anchor residue" is an amino acid at a specific position
along a
peptide sequence which is understood to provide a contact point between the
immunogenic
peptide and the HLA molecule. One to three, usually two, primary anchor
residues within a
peptide of defined length generally defines a "motif' for an immunogenic
peptide. These
residues are understood to fit in close contact with peptide binding groove of
an HLA molecule,
with their side chains buried in specific pockets of the binding groove. In
one embodiment, for
example, the primary anchor residues for an HLA class I molecule are located
at position 2
(from the amino terminal position) and at the carboxyl terminal position of a
8, 9, 10, 11, or 12
residue peptide epitope in accordance with the invention. Alternatively, in
another embodiment,
the primary anchor residues of a peptide binds an HLA class II molecule are
spaced relative to
each other, rather than to the termini of a peptide, where the peptide is
generally of at least 9
amino acids in length. The primary anchor positions for each motif and
supermotif are set forth
in Table IV(a). 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.
(0111] "Radioisotopes" include, but are not limited to the following (non-
limiting exemplary
uses are also set forth in Table IV(I)).
(0112] By "randomized" or grammatical equivalents as herein applied to nucleic
acids and
proteins is meant that each nucleic acid and peptide consists of essentially
random nucleotides
and amino acids, respectively. These random peptides (or nucleic acids,
discussed herein) can
incorporate any nucleotide or amino acid at any position. The synthetic
process can be designed
to generate randomized proteins or nucleic acids, to allow the formation of
all or most of the
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possible combinations over the length of the sequence, thus forming a library
of randomized
candidate bioactive proteinaceous agents.
[01131 In one embodiment, a library is "fully randomized," with no sequence
preferences or
constants at any position. In another embodiment, the library is a "biased
random" library. That
is, some positions within the sequence either are held constant, or are
selected from a limited
number of possibilities. For example, the nucleotides or amino acid residues
are randomized
within a defined class, e.g., of hydrophobic amino acids, hydrophilic
residues, sterically biased
(either small or large) residues, towards the creation of nucleic acid binding
domains, the
creation of cysteines, for cross-linking, prolines for SH-3 domains, serines,
threonines, tyrosines
or histidines for phosphorylation sites, etc., or to purines, etc.
[0114] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has
been
subjected to molecular manipulation in vitro.
[0115] As used herein, the term "single-chain Fv" or "scFv" or "single chain"
antibody
refers to antibody fragments comprising the VH and VL domains of antibody,
wherein these
domains are present in a single polypeptide chain. Generally, the Fv
polypeptide further
comprises a polypeptide linker between the VH and VL domains which enables the
sFv to form
the desired structure for antigen binding. For a review of sFv, see Pluckthun,
THE
PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds.
Springer-Verlag, New York, pp. 269-315 (1994).
[0116] Non-limiting examples of "small molecules" include compounds that bind
or interact
with PSCA, ligands including hormones, neuropeptides, chemokines, odorants,
phospholipids,
and functional equivalents thereof that bind and preferably inhibit PSCA
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, PSCA protein;
are not found
in naturally occurring metabolic pathways; and/or are more soluble in aqueous
than non-aqueous
solutions.
[0117] As used herein, the term "specific" refers to the selective binding of
the antibody
to the target antigen epitope. Antibodies can be tested for specificity of
binding by comparing
binding to appropriate antigen to binding to irrelevant antigen or antigen
mixture under a given
set of conditions. If the antibody binds to the appropriate antigen at least
2, 5, 7, and preferably
times more than to irrelevant antigen or antigen mixture then it is considered
to be specific.
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In one embodiment, a specific antibody is one that only binds the PSCA
antigen, but does not
bind to the irrelevent antigen. In another embodiment, a specific antibody is
one that binds
human PSCA antigen but does not bind a non-human PSCA antigen with 70%, 75%,
80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid
homology with
the PSCA antigen. In another embodiment, a specific antibody is one that binds
human PSCA
antigen and binds murine PSCA antigen, but with a higher degree of binding the
human antigen.
In another embodiment, a specific antibody is one that binds human PSCA
antigen and binds
primate PSCA antigen, but with a higher degree of binding the human antigen.
In another
embodiment, the specific antibody binds to human PSCA antigen and any non-
human PSCA
antigen, but with a higher degree of binding the human antigen or any
combination thereof.
[01181 "Stringency" of hybridization reactions is readily determinable by one
of ordinary
skill in the art, and generally is an empirical calculation dependent upon
probe length, washing
temperature, and salt concentration. In general, longer probes require higher
temperatures for
proper annealing, while shorter probes need lower temperatures. Hybridization
generally
depends on the ability of denatured nucleic acid sequences to reanneal when
complementary
strands are present in an environment below their melting temperature. The
higher the degree of
desired homology between the probe and hybridizable sequence, the higher the
relative
temperature that can be used. As a result, it follows that higher relative
temperatures would tend
to make the reaction conditions more stringent, while lower temperatures less
so. For additional
details and explanation of stringency of hybridization reactions, see Ausubel
et al., Current
Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
[01191 "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.I %
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
NaCl, 0.075 M
sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate,
5 x Denhardt's
solution, sonicated salmon sperm DNA (50 pg/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.
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"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 65 C in a solution comprising:
I% bovine serum
albumin, 0.5M sodium phosphate pH7.5, 1.25mM EDTA, and 7% SDS 5 x SSC (150 mM
NaCl,
15 mM trisodium citrate), followed by washing the filters in 2 x SSC/1% SDS at
50 C and 0.2 X
SSC/0.1% SDS at 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.
[01201 An HLA "supermotif' is a peptide binding specificity shared by HLA
molecules
encoded by two or more HLA alleles. Overall phenotypic frequencies of HLA-
supertypes in
different ethnic populations are set forth in Table IV (f). The non-limiting
constituents of
various supertypes are as follows:
A2: A*0201, A*0202, A*0203, A*0204, A* 0205, A*0206, A*6802, A*6901, A*0207
A3: A3, All, A31, A*3301, A*6801, A*0301, A*1101, A*3101
B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*6701,
B*7801, B*0702, 13*5101, B*5602
B44: B*3701, B*4402, B*4403, B*60 (13*4001), B61 (B*4006)
A]: A*0102, A*2604, A*3601, A*4301, A*8001
A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003
B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901, B*3902,
B*3903-04, B*4801-02, B*7301, 13*2701-08
B58: B*1516, B*1517, B*5701, B*5702, B58
B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)
Calculated population coverage afforded by different HLA-supertype
combinations are set forth
in Table IV(g).
[01211 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; as is readily appreciated in the art, full eradication of disease is
a preferred out albeit
not a requirement for a treatment act.
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[0122] 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.
[0123] As used herein, an HLA or cellular immune response "vaccine" is a
composition that
contains or encodes one or more peptides of the invention. There are numerous
embodiments of
such vaccines, such as a cocktail of one or more individual peptides; one or
more peptides of the
invention comprised by a polyepitopic peptide; or nucleic acids that encode
such individual
peptides or polypeptides, e.g., a minigene that encodes a polyepitopic
peptide. The "one or more
peptides" can include any whole unit integer from 1-150 or more, e.g., at
least 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of
the invention. The
peptides or polypeptides can optionally be modified, such as by lipidation,
addition of targeting
or other sequences. HLA class I peptides of the invention can be admixed with,
or linked to,
HLA class II peptides, to facilitate activation of both cytotoxic T
lymphocytes and helper T
lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting
cells, e.g.,
dendritic cells.
[0124] The term "variant" refers to a molecule that exhibits a variation from
a described type
or norm, such as a protein that has one or more different amino acid residues
in the
corresponding position(s) of a specifically described protein (e.g. the PSCA
protein shown in
Figure 1. An analog is an example of a variant protein. Splice isoforms and
single nucleotides
polymorphisms (SNPs) are further examples of variants.
[0125] The "PSCA-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 PSCA proteins or fragments thereof, as well as fusion proteins of a
PSCA protein and a
heterologous polypeptide are also included. Such PSCA proteins are
collectively referred to as
the PSCA-related proteins, the proteins of the invention, or PSCA. The term
"PSCA-related
protein" refers to a polypeptide fragment or a PSCA 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,
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35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125,
130, 135, 140, 145,
150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300,
325, 330, 335, 339 or
more amino acids.
II.) PSCA Polynucleotides
[01261 One aspect of the invention provides polynucleotides corresponding or
complementary to all or part of a PSCA gene, mRNA, and/or coding sequence,
preferably in
isolated form, including polynucleotides encoding a PSCCA-related protein and
fragments
thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or
oligonucleotides complementary to a PSCA gene or mRNA sequence or a part
thereof, and
polynucleotides or oligonucleotides that hybridize to a PSCA gene, mRNA, or to
a PSCA
encoding polynucleotide (collectively, "PSCA polynucleotides"). In all
instances when referred
to in this section, T can also be U in Figure 1.
[01271 Embodiments of a PSCA polynucleotide include: a PSCA polynucleotide
having the
sequence shown in Figure 1, the nucleotide sequence of PSCA as shown in Figure
1 wherein T
is U; at least 10 contiguous nucleotides of a polynucleotide having the
sequence as shown in
Figure l ; or, at least 10 contiguous nucleotides of a polynucleotide having
the sequence as
shown in Figure 1 where T is U.
[01281 Polynucleotides encoding relatively long portions of a PSCA 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
PSCA protein "or
variant" shown in Figure 1 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 PSCA
sequence as
shown in Figure 1.
II.A.) Uses of PSCA Polynucleotides
11.A.1. Monitoring of Genetic Abnormalities
101291 The polynucleotides of the preceding paragraphs have a number of
different specific
uses. The human PSCA gene maps to the chromosomal location set forth in the
Example
entitled "Chromosomal Mapping of PSCA." For example, because the PSCA gene
maps to this
chromosome, polynucleotides that encode different regions of the PSCA 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
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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 PSCA
proteins provide new tools that can be used to delineate, with greater
precision than previously
possible, cytogenetic abnormalities in the chromosomal region that encodes
PSCA that may
contribute to the malignant phenotype. In this context, these polynucleotides
satisfy a need in
the art for expanding the sensitivity of chromosomal screening in order to
identify more subtle
and less common chromosomal abnormalities (see e.g. Evans et al., Am. J.
Obstet. Gynecol
171(4): 1055-1057 (1994)).
[0130] Furthermore, as PSCA was shown to be highly expressed in prostate and
other
cancers, PSCA polynucleotides are used in methods assessing the status of PSCA
gene products
in normal versus cancerous tissues. Typically, polynucleotides that encode
specific regions of
the PSCA 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
PSCA gene, such as regions containing one or more motifs. Exemplary assays
include both RT-
PCR assays as well as single-strand conformation polymorphism (SSCP) analysis
(see, e.g.,
Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize
polynucleotides
encoding specific regions of a protein to examine these regions within the
protein.
II.A.2. Antisense Embodiments
[0131] 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 PSCA. 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
PSCA polynucleotides and polynucleotide sequences disclosed herein.
[0132] 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., PSCA. See for
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example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene
Expression, CRC
Press, 1989; and Synthesis 1:1-5 (1988). The PSCA 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-l,l-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 PSCA 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).
[01331 The PSCA antisense oligonucleotides of the present invention typically
can be RNA
or DNA that is complementary to and stably hybridizes with the first 100 5'
codons or last 100
3' codons of a PSCA 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
PSCA mRNA and not to mRNA specifying other regulatory subunits of protein
kinase. In one
embodiment, PSCA antisense oligonucleotides of the present invention are 15 to
30-mer
fragments of the antisense DNA molecule that have a sequence that hybridizes
to PSCA mRNA.
Optionally, PSCA 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 PSCA.
Alternatively, the antisense
molecules are modified to employ ribozymes in the inhibition of PSCA
expression, see, e.g., L.
A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
II.A.3. Primers and Primer Pairs
[01341 Further specific embodiments of these nucleotides of the invention
include primers
and primer pairs, which allow the specific amplification of polynucleotides of
the invention or of
any specific parts thereof, and probes that selectively or specifically
hybridize to nucleic acid
molecules of the invention or to any part thereof. Probes can be labeled with
a detectable
marker, such as, for example, a radioisotope, fluorescent compound,
bioluminescent compound,
a chemiluminescent compound, metal chelator or enzyme. Such probes and primers
are used to
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detect the presence of a PSCA polynucleotide in a sample and as a means for
detecting a cell
expressing a PSCA protein.
[0135] Examples of such probes include polypeptides comprising all or part of
the human
PSCA cDNA sequence shown in Figure 1. Examples of primer pairs capable of
specifically
amplifying PSCA 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 PSCA
mRNA.
[0136] The PSCA 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 PSCA 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 PSCA polypeptides; as tools for modulating or inhibiting the
expression of the
PSCA gene(s) and/or translation of the PSCA transcript(s); and as therapeutic
agents.
[01371 The present invention includes the use of any probe as described herein
to identify
and isolate a PSCA or PSCA related nucleic acid sequence from a naturally
occurring source,
such as humans or other mammals, as well as the isolated nucleic acid sequence
per se, which
would comprise all or most of the sequences found in the probe used.
II.A.4. Isolation of PSCA-Encoding Nucleic Acid Molecules
[0138] The PSCA cDNA sequences described herein enable the isolation of other
polynucleotides encoding PSCA gene product(s), as well as the isolation of
polynucleotides
encoding PSCA gene product homologs, alternatively spliced isoforms, allelic
variants, and
mutant forms of a PSCA gene product as well as polynucleotides that encode
analogs of PSCA-
related proteins. Various molecular cloning methods that can be employed to
isolate full length
cDNAs encoding a PSCA gene are well known (see, for example, Sambrook, J. et
al., Molecular
Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York,
1989;
Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons,
1995). For
example, lambda phage cloning methodologies can be conveniently employed,
using
commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene).
Phage clones
containing PSCA gene cDNAs can be identified by probing with a labeled PSCA
cDNA or a
fragment thereof. For example, in one embodiment, a PSCA cDNA (e.g., Figure 1)
or a portion
thereof can be synthesized and used as a probe to retrieve overlapping and
full-length cDNAs
corresponding to a PSCA gene. A PSCA gene itself can be isolated by screening
genomic DNA
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libraries, bacterial artificial chromosome libraries (BACs), yeast artificial
chromosome libraries
(YACs), and the like, with PSCA DNA probes or primers.
II.A.5. Recombinant Nucleic Acid Molecules and Host-Vector Systems
[01391 The invention also provides recombinant DNA or RNA molecules containing
a
PSCA 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).
[01401 The invention further provides a host-vector system comprising a
recombinant DNA
molecule containing a PSCA 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 DU 145 and TsuPrl,
other transfeetable 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
PSCA or a
fragment, analog or homolog thereof can be used to generate PSCA proteins or
fragments
thereof using any number of host-vector systems routinely used and widely
known in the art.
[0141] A wide range of host-vector systems suitable for the expression of PSCA
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
pSROtkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors,
PSCA can be
expressed in several prostate cancer and non-prostate cell lines, including
for example 293,
293T, rat-l, NIH 3T3 and TsuPrl. The host-vector systems of the invention are
useful for the
production of a PSCA protein or fragment thereof. Such host-vector systems can
be employed
to study the functional properties of PSCA and PSCA mutations or analogs.
[01421 Recombinant human PSCA protein or an analog or homolog or fragment
thereof can
be produced by mammalian cells transfected with a construct encoding a PSCA-
related
nucleotide. For example, 293T cells can be transfected with an expression
plasmid encoding
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PSCA or fragment, analog or homolog thereof, a PSCA-related protein is
expressed in the 293T
cells, and the recombinant PSCA protein is isolated using standard
purification methods (e.g.,
affinity purification using anti-PSCA antibodies). In another embodiment, a
PSCA 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 PSCA
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
PSCA coding
sequence can be used for the generation of a secreted form of recombinant PSCA
protein.
[0143] As discussed herein, redundancy in the genetic code permits variation
in PSCA gene
sequences. In particular, it is known in the art that specific host species
often have specific
codon preferences, and thus one can adapt the disclosed sequence as preferred
for a desired host.
For example, preferred analog codon sequences typically have rare codons
(i.e., codons having a
usage frequency of less than about 20% in known sequences of the desired host)
replaced with
higher frequency codons. Codon preferences for a specific species are
calculated, for example,
by utilizing codon usage tables available on the INTERNET such as at URL
dna.affrc.gojp/-nakamura/codon.html.
[0144] Additional sequence modifications are known to enhance protein
expression in a
cellular host. These include elimination of sequences encoding spurious
polyadenylation
signals, exon/intron splice site signals, transposon-like repeats, and/or
other such well-
characterized sequences that are deleterious to gene expression. The GC
content of the sequence
is adjusted to levels average for a given cellular host, as calculated by
reference to known genes
expressed in the host cell. Where possible, the sequence is modified to avoid
predicted hairpin
secondary mRNA structures. Other useful modifications include the addition of
a translational
initiation consensus sequence at the start of the open reading frame, as
described in Kozak, Mol.
Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general
rule that eukaryotic
ribosomes initiate translation exclusively at the 5' proximal AUG codon is
abrogated only under
rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR
15(20):
8125-8148 (1987)).
III.) PSCA-related Proteins
[0145] Another aspect of the present invention provides PSCA-related proteins.
Specific
embodiments of PSCA proteins comprise a polypeptide having all or part of the
amino acid
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sequence of human PSCA as shown in Figure 1, preferably Figure IA.
Alternatively,
embodiments of PSCA proteins comprise variant, homolog or analog polypeptides
that have
alterations in the amino acid sequence of PSCA shown in Figure 1.
[0146] Embodiments of a PSCA polypeptide include: a PSCA polypeptide having a
sequence shown in Figure 1, a peptide sequence of a PSCA as shown in Figure 1
wherein T is
U; at least 10 contiguous nucleotides of a polypeptide having the sequence as
shown in Figure 1;
or, at least 10 contiguous peptides of a polypeptide having the sequence as
shown in Figure 1
where T is U.
[0147] 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.
[0148] Embodiments of the invention disclosed herein include a wide variety of
art-accepted
variants or analogs of PSCA proteins such as polypeptides having amino acid
insertions,
deletions and substitutions. PSCA 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 PSCA variant DNA.
[0149] 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.
[0150] As defined herein, PSCA variants, analogs or homologs, have the
distinguishing
attribute of having at least one epitope that is "cross reactive" with a PSCA
protein having an
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amino acid sequence of Figure 1. As used in this sentence, "cross reactive"
means that an
antibody or T cell that specifically binds to a PSCA variant also specifically
binds to a PSCA
protein having an amino acid sequence set forth in Figure 1. A polypeptide
ceases to be a
variant of a protein shown in Figure 1, when it no longer contains any epitope
capable of being
recognized by an antibody or T cell that specifically binds to the starting
PSCA 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.
[01511 Other classes of PSCA-related protein variants share 70%, 75%, 80%, 85%
or 90%
or more similarity with an amino acid sequence of Figure 1, or a fragment
thereof. Another
specific class of PSCA protein variants or analogs comprises one or more of
the PSCA
biological motifs described herein or presently known in the art. Thus,
encompassed by the
present invention are analogs of PSCA 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 1.
[01521 As discussed herein, embodiments of the claimed invention include
polypeptides
containing less than the full amino acid sequence of a PSCA protein shown in
Figure 1. For
example, representative embodiments of the invention comprise
peptides/proteins having any 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a PSCA
protein shown in
Figure 1.
[01531 PSCA-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 PSCA-related
protein. In one
embodiment, nucleic acid molecules provide a means to generate defined
fragments of a PSCA
protein (or variants, homologs or analogs thereof).
III.A.) Motif-bearing Protein Embodiments
[0154] Additional illustrative embodiments of the invention disclosed herein
include PSCA
polypeptides comprising the amino acid residues of one or more of the
biological motifs
contained within a PSCA polypeptide sequence set forth in Figure 1. Various
motifs are known
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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: pfam.wustl.edu/;
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/scnpsit1.htmI; EpimatrixTM and
EpimerTM, Brown
University, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,
bimas.dert.nih.gov/.).
[01551 Motif bearing subsequences of all PSCA variant proteins are set forth
and identified
in Tables V-XVIII and XXII-LI.
[01561 Table IV(h) sets forth several frequently occurring motifs based on
pfam searches
(see URL address pfam.wustl.eduJ). The columns of Table IV(h) 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.
[01571 Polypeptides comprising one or more of the PSCA motifs discussed above
are useful
in elucidating the specific characteristics of a malignant phenotype in view
of the observation
that the PSCA motifs discussed above are associated with growth dysregulation
and because
PSCA 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)).
[01581 In another embodiment, proteins of the invention comprise one or more
of the
immunoreactive epitopes identified in accordance with art-accepted methods,
such as the
peptides set forth in Tables V-XVIII and XXII-LI. CTL epitopes can be
determined using
specific algorithms to identify peptides within a PSCA protein that are
capable of optimally
binding to specified HLA alleles (e.g., Table IV; EpimatrixTM and EpimerTM,
Brown University,
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=
URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.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 carried out without undue experimentation. In
addition, processes for
identifying peptides that are immunogenic epitopes, are well known in the art,
and are carried
out without undue experimentation either in vitro or in vivo.
[01591 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 11 motifs/supermotifs of Table
IV). The
epitope is analoged by substituting out an amino acid at one of the specified
positions, and
replacing it with another amino acid specified for that position. For example,
on the basis of
residues defined in Table IV, one can substitute out a deleterious residue in
favor of any other
residue, such as a preferred residue; substitute a less-preferred residue with
a preferred residue;
or substitute an originally-occurring preferred residue with another preferred
residue.
Substitutions can occur at primary anchor positions or at other positions in a
peptide; see, e.g.,
Table IV.
101601 A variety of references reflect the art regarding the identification
and generation of
epitopes in a protein of interest as well as analogs thereof. See, for
example, WO 97/33602 to
Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J.
Immunol. 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.
[01611 Related embodiments of the invention include polypeptides comprising
combinations
of the different motifs set forth in Table(s) IV(a), IV(b), IV(c), IV(d), and
IV(h), and/or, one or
more of the predicted CTL epitopes of Tables V-XVIII and XXII-LI, and/or, one
or more of the
predicted HTL epitopes of Tables XLVIII-LI, and/or, one or more of the T cell
binding motifs
known in the art. Preferred embodiments contain no insertions, deletions or
substitutions either
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within the motifs or within the intervening sequences of the polypeptides. In
addition,
embodiments which include a number of either N-terminal and/or C-terminal
amino acid
residues on either side of these motifs may be desirable (to, for example,
include a greater
portion of the polypeptide architecture in which the motif is located).
Typically, the number of
N-terminal and/or C-terminal amino acid residues on either side of a motif is
between about I to
about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
[01621 PSCA-related proteins are embodied in many forms, preferably in
isolated form. A
purified PSCA protein molecule will be substantially free of other proteins or
molecules that
impair the binding of PSCA to antibody, T cell or other ligand. The nature and
degree of
isolation and purification will depend on the intended use. Embodiments of a
PSCA-related
proteins include purified PSCA-related proteins and functional, soluble PSCA-
related proteins.
In one embodiment, a functional, soluble PSCA protein or fragment thereof
retains the ability to
be bound by antibody, T cell or other ligand.
[0163] The invention also provides PSCA proteins comprising biologically
active fragments
of a PSCA amino acid sequence shown in Figure 1. Such proteins exhibit
properties of the
starting PSCA protein, such as the ability to elicit the generation of
antibodies that specifically
bind an epitope associated with the starting PSCA 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.
[0164] PSCA-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-Fasmnan, Gamier-Robson, Kyte-Doolittle,
Eisenberg,
Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity.
Fragments that
contain such structures are particularly useful in generating subunit-specific
anti-PSCA
antibodies or T cells or in identifying cellular factors that bind to PSCA.
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.,
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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.
[0165] CTL epitopes can be determined using specific algorithms to identify
peptides within
a PSCA 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); EpimatrixTM and EpimerTM, Brown University, URL
(brown.edu/Research/TB-
HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/).
Illustrating this,
peptide epitopes from PSCA that are presented in the context of human MHC
Class I molecules,
e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables V-
XVIII, XXII-
LI). Specifically, the complete amino acid sequence of the PSCA protein and
relevant portions
of other variants, i.e., for HLA Class I predictions 9 flanking residues on
either side of a point
mutation or exon juction, and for HLA Class II predictions 14 flanking
residues on either side of
a point mutation or exon junction corresponding to that variant, were entered
into the HLA
Peptide Motif Search algorithm found in the Bioinformatics and Molecular
Analysis Section
(BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL
syfpeithi.bmi-
heidelberg.com/.
[0166] The HLA peptide motif search algorithm was developed by Dr. Ken Parker
based on
binding of specific peptide sequences in the groove of HLA Class I molecules,
in particular
HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science
255:1261-3
(1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J.
Immunol. 152:163-75
(1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and I 0-
mer peptides from
a complete protein sequence for predicted binding to HLA-A2 as well as
numerous other HLA
Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or I 1-
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 PSCA predicted binding peptides are
shown in Tables
V-XVIII and XXII-LI herein. In Tables V-XVIII and XXII-XLVIII, selected
candidates, 9-mers
and I0-mers, for each family member are shown along with their location, the
amino acid
sequence of each specific peptide, and an estimated binding score. In Tables
XLVIII-LI,
selected candidates, 15-mers, for each family member are shown along with
their location, the
amino acid sequence of each specific peptide, and an estimated binding score.
The binding
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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.
[01671 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.
[0168] It is to be appreciated that every epitope predicted by the BIMAS site,
EpimerTM and
EpimatrixTM sites, or specified by the HLA class I or class II motifs
available in the art or which
become part of the art such as set forth in Table IV (or determined using
World Wide Web site
URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be
"applied" to a
PSCA protein in accordance with the invention. As used in this context
"applied" means that a
PSCA 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 PSCA
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.
III.B.) Expression of PSCA-related Proteins
101691 In an embodiment described in the examples that follow, PSCA 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 PSCA with a C-terminal
6XHis and
MYC tag (pcDNA3.1/mycHlS, Invitrogen or Tag5, GenHunter Corporation, Nashville
TN).
The Tag5 vector provides an IgGK secretion signal that can be used to
facilitate the production
of a secreted PSCA protein in transfected cells. The secreted HIS-tagged PSCA
in the culture
media can be purified, e.g., using a nickel column using standard techniques.
III.C.) Modifications of PSCA-related Proteins
101701 Modifications of PSCA-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 PSCA polypeptide with an organic derivatizing agent
that is capable of
reacting with selected side chains or the N- or C- terminal residues of a PSCA
protein. Another
type of covalent modification of a PSCA polypeptide included within the scope
of this invention
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comprises altering the native glycosylation pattern of a protein of the
invention. Another type of
covalent modification of PSCA comprises linking a PSCA 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.
[01711 The PSCA-related proteins of the present invention can also be modified
to form a
chimeric molecule comprising PSCA 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 PSCA 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 1. Such a chimeric molecule can comprise multiples of the same
subsequence of
PSCA. A chimeric molecule can comprise a fusion of a PSCA-related protein with
a
polyhistidine epitope tag, which provides an epitope to which immobilized
nickel can selectively
bind, with cytokines or with growth factors. The epitope tag is generally
placed at the amino- or
carboxyl- terminus of a PSCA protein. In an alternative embodiment, the
chimeric molecule can
comprise a fusion of a PSCA-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 Fe region of an IgG molecule.
The Ig fusions
preferably include the substitution of a soluble (transmembrane domain deleted
or inactivated)
form of a PSCA polypeptide in place of at least one variable region within an
Ig molecule. In a
preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and
CH3, or the
hinge, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of
immunoglobulin
fusions see, e.g., U.S. Patent No. 5,428,130 issued June 27, 1995.
III.D.) Uses of PSCA-related Proteins
[01721 The proteins of the invention have a number of different specific uses.
As PSCA is
highly expressed in prostate and other cancers, PSCA-related proteins are used
in methods that
assess the status of PSCA gene products in normal versus cancerous tissues,
thereby elucidating
the malignant phenotype. Typically, polypeptides from specific regions of a
PSCA 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
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antibodies or T cells targeting PSCA-related proteins comprising the amino
acid residues of one
or more of the biological motifs contained within a PSCA 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, PSCA-related proteins that
contain the amino
acid residues of one or more of the biological motifs in a PSCA protein are
used to screen for
factors that interact with that region of PSCA.
[0173] PSCA protein fragments/subsequences are particularly useful in
generating and
characterizing domain-specific antibodies (e.g., antibodies recognizing an
extracellular or
intracellular epitope of a PSCA protein), for identifying agents or cellular
factors that bind to
PSCA 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.
[0174] Proteins encoded by the PSCA 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
PSCA gene product.
Antibodies raised against a PSCA protein or fragment thereof are useful in
diagnostic and
prognostic assays, and imaging methodologies in the management of human
cancers
characterized by expression of PSCA protein, such as those listed in Table I.
Such antibodies
can be expressed intracellularly and used in methods of treating patients with
such cancers.
PSCA-related nucleic acids or proteins are also used in generating HTL or CTL
responses.
[01751 Various immunological assays useful for the detection of PSCA proteins
are used,
including but not limited to various types of radioimmunoassays, enzyme-linked
immunosorbent
assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA),
immunocytochemical
methods, and the like. Antibodies can be labeled and used as immunological
imaging reagents
capable of detecting PSCA-expressing cells (e.g., in radioscintigraphic
imaging methods).
PSCA proteins are also particularly useful in generating cancer vaccines, as
further described
herein.
IV.) PSCA Antibodies
[01761 Another aspect of the invention provides antibodies that bind to PSCA-
related
proteins. Preferred antibodies specifically bind to a PSCA-related protein and
do not bind (or
bind weakly) to peptides or proteins that are not PSCA-related proteins under
physiological
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conditions. In this context, examples of physiological conditions include: 1)
phosphate buffered
saline; 2) Tris-buffered saline containing 25mM Tris and 150 mM NaCl; or
normal saline (0.9%
NaCl); 4) animal serum such as human serum; or, 5) a combination of any of 1)
through 4);
these reactions preferably taking place at pH 7.5, alternatively in a range of
pH 7.0 to 8.0, or
alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place
at a temperature
between 4 C to 37 C. For example, antibodies that bind PSCA can bind PSCA-
related proteins
such as the homologs or analogs thereof.
[0177] PSCA antibodies of the invention are particularly useful in cancer
(see, e.g., Table I)
diagnostic and prognostic assays, and imaging methodologies. Similarly, such
antibodies are
useful in the treatment, diagnosis, and/or prognosis of prostate and other
cancers, to the extent
PSCA 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 PSCA is involved, such as advanced or metastatic
prostate cancers or
other advanced or metastatic cacners.
[0178] The invention also provides various immunological assays useful for the
detection
and quantification of PSCA and mutant PSCA-related proteins. Such assays can
comprise one
or more PSCA antibodies capable of recognizing and binding a PSCA-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.
[0179] Immunological non-antibody assays of the invention also comprise T cell
immunogenicity assays (inhibitory or stimulatory) as well as major
histocompatibility complex
(MHC) binding assays.
[0180] In addition, immunological imaging methods capable of detecting
prostate cancer
and other cancers expressing PSCA are also provided by the invention,
including but not limited
to radioscintigraphic imaging methods using labeled PSCA antibodies. Such
assays are
clinically useful in the detection, monitoring, and prognosis of PSCA
expressing cancers such as
prostate cancer.
[0181] PSCA antibodies are also used in methods for purifying a PSCA-related
protein and
for isolating PSCA homologues and related molecules. For example, a method of
purifying a
PSCA-related protein comprises incubating a PSCA antibody, which has been
coupled to a solid
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matrix, with a lysate or other solution containing a PSCA-related protein
under conditions that
permit the PSCA antibody to bind to the PSCA-related protein; washing the
solid matrix to
eliminate impurities; and eluting the PSCA-related protein from the coupled
antibody. Other
uses of PSCA antibodies in accordance with the invention include generating
anti-idiotypic
antibodies that mimic a PSCA protein.
[0182] 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 PSCA-
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 PSCA can also
be used, such
as a PSCA GST-fusion protein. In a particular embodiment, a GST fusion protein
comprising
all or most of the amino acid sequence of Figure 1 is produced, then used as
an immunogen to
generate appropriate antibodies. In another embodiment, a PSCA-related protein
is synthesized
and used as an immunogen.
[0183] In addition, naked DNA immunization techniques known in the art are
used (with or
without purified PSCA-related protein or PSCA expressing cells) to generate an
immune
response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann.
Rev. Immunol.
15: 617-648).
[0184] The amino acid sequence of a PSCA protein as shown in Figure 1 can be
analyzed to
select specific regions of the PSCA protein for generating antibodies. For
example,
hydrophobicity and hydrophilicity analyses of a PSCA amino acid sequence are
used to identify
hydrophilic regions in the PSCA structure. Regions of a PSCA 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
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methods is within the scope of the present invention. Preferred methods for
the generation of
PSCA antibodies are further illustrated by way of the examples provided
herein. Methods for
preparing a protein or polypeptide for use as an immunogen are well known in
the art. Also well
known in the art are methods for preparing immunogenic conjugates of a protein
with a carrier,
such as BSA, KLH or other carrier protein. In some circumstances, direct
conjugation using, for
example, carbodiimide reagents are used;.in other instances linking reagents
such as those
supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration
of a PSCA
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.
[0185] PSCA 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 PSCA-
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.
[0186] The antibodies or fragments of the invention can also be produced, by
recombinant
means. Regions that bind specifically to the desired regions of a PSCA protein
can also be
produced in the context of chimeric or complementarity-determining region
(CDR) grafted
antibodies of multiple species origin. Humanized or human PSCA antibodies can
also be
produced, and are preferred for use in therapeutic contexts. Methods for
humanizing murine and
other non-human antibodies, by substituting one or more of the non-human
antibody CDRs for
corresponding human antibody sequences, are well known (see for example, Jones
et al., 1986,
Nature 321: 522-525; Riechmann et 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.
[0187) 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 PSCA 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
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libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and
Therapeutic
Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993);
Burton and
Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully
human PSCA
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.
[01881 Reactivity of PSCA antibodies with a PSCA-related protein can be
established by a
number of well known means, including Western blot, immunoprecipitation,
ELISA, and FACS
analyses using, as appropriate, PSCA-related proteins, PSCA-expressing cells
or extracts
thereof. A PSCA 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 PSCA epitopes are generated using methods generally known in the art.
Homodimeric
antibodies can also be generated by cross-linking techniques known in the art
(e.g., Wolff et al.,
Cancer Res. 53: 2560-2565).
[01891 In one embodiment, the invention provides for monoclonal antibodies
identified as
Hal-1.16, Hal-5.99, Hal-4.117, Hal-4.20, Hal-4.121, Hal-4.37 were sent (via
Federal
Express) to the American Type Culture Collection (ATCC), P.O. Box 1549,
Manassas, VA
20108 on 04-May-2005 and assigned Accession numbers PTA-6698 and PTA-6703 and
PTA-
6699 and PTA-6700 and PTA-6701 and PTA-6702 respectively.
V.) PSCA Cellular Immune Responses
[01901 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.
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[0191] 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
(134.2.96.221/scripts.hlaserver.dll/home.htm); 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; 50(3-4):201-12, Review).
[0192] Furthermore, x-ray crystallographic analyses of HLA-peptide complexes
have
revealed pockets within the peptide binding cleft/groove of HLA molecules
which
accommodate, in an allele-specific mode, residues borne by peptide ligands;
these residues in
turn determine the HLA binding capacity of the peptides in which they are
present. (See, e.g.,
Madden, D.R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,
1996;
Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994;
Jones, E.Y. Curr.
Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H.
C. et al., Proc.
Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992;
Silver, M. L.
et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992;
Madden et al., Cell
70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A. ,
Bjorkman, P. J. and
Wiley, D. C., J. Mol. Biol. 219:277, 1991.)
[0193] Accordingly, the definition of class 1 and class II allele-specific HLA
binding motifs,
or class I or class 11 supermotifs allows identification of regions within a
protein that are
correlated with binding to particular HLA antigen(s).
101941 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
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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.
10195] Various strategies can be utilized to evaluate cellular immunogenicity,
including:
1) Evaluation of primary T cell cultures from normal individuals (see, e.g.,
Wentworth, P. A. et al., Mol. 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 51Cr-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
51 Cr-release assay
involving peptide sensitized target cells and target cells expressing
endogenously generated
antigen.
3) Demonstration of recall T cell responses from immune individuals who have
been either effectively vaccinated and/or from chronically ill patients (see,
e.g., Rehermann, B.
et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97,
1997; 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 51 Cr release involving peptide-sensitized targets, T cell
proliferation, or lymphokine
release.
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VI.) PSCA Transgenic Animals
[01961 Nucleic acids that encode a PSCA-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, eDNA
encoding PSCA can be used to clone genomic DNA that.encodes PSCA. The cloned
genomic
sequences can then be used to generate transgenic animals containing cells
that express DNA
that encode PSCA. 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 PSCA transgene incorporation with
tissue-specific
enhancers.
[01971 Transgenic animals that include a copy of a transgene encoding PSCA can
be used to
examine the effect of increased expression of DNA that encodes PSCA. Such
animals can be
used as tester animals for reagents thought to confer protection from, for
example, pathological
conditions associated with its overexpression. In accordance with this aspect
of the invention,
an animal is treated with a reagent and a reduced incidence of a pathological
condition,
compared to untreated animals that bear the transgene, would indicate a
potential therapeutic
intervention for the pathological condition.
101981 Alternatively, non-human homologues of PSCA can be used to construct a
PSCA
"knock out" animal that has a defective or altered gene encoding PSCA as a
result of
homologous recombination between the endogenous gene encoding PSCA and altered
genomic
DNA encoding PSCA introduced into an embryonic cell of the animal. For
example, eDNA that
encodes PSCA can be used to clone genomic DNA encoding PSCA in accordance with
established techniques. A portion of the genomic DNA encoding PSCA 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
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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 PSCA polypeptide.
VII.) Methods for the Detection of PSCA
101991 Another aspect of the present invention relates to methods for
detecting PSCA
polynucleotides and PSCA-related proteins, as well as methods for identifying
a cell that
expresses PSCA. The expression profile of PSCA makes it a diagnostic marker
for metastasized
disease. Accordingly, the status of PSCA 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 PSCA
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.
[0200] More particularly, the invention provides assays for the detection of
PSCA
polynucleotides in a biological sample, such as serum, bone, prostate, and
other tissues, urine,
semen, cell preparations, and the like. Detectable PSCA polynucleotides
include, for example, a
PSCA gene or fragment thereof, PSCA mRNA, alternative splice variant PSCA
mRNAs, and
recombinant DNA or RNA molecules that contain a PSCA polynucleotide. A number
of
methods for amplifying and/or detecting the presence of PSCA polynucleotides
are well known
in the art and can be employed in the practice of this aspect of the
invention.
[0201] In one embodiment, a method for detecting a PSCA mRNA in a biological
sample
comprises producing eDNA from the sample by reverse transcription using at
least one primer;
amplifying the eDNA so produced using a PSCA polynucleotides as sense and
antisense primers
to amplify PSCA cDNAs therein; and detecting the presence of the amplified
PSCA eDNA.
Optionally, the sequence of the amplified PSCA eDNA can be determined.
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[02021 In another embodiment, a method of detecting a PSCA gene in a
biological sample
comprises first isolating genomic DNA from the sample; amplifying the isolated
genomic DNA
using PSCA polynucleotides as sense and antisense primers; and detecting the
presence of the
amplified PSCA gene. Any number of appropriate sense and antisense probe
combinations can
be designed from a PSCA nucleotide sequence (see, e.g., Figure 1) and used for
this purpose.
[02031 The invention also provides assays for detecting the presence of a PSCA
protein in a
tissue or other biological sample such as serum, semen, bone, prostate, urine,
cell preparations,
and the like. Methods for detecting a PSCA-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 PSCA-related protein in a biological sample comprises first contacting the
sample with a
PSCA antibody, a PSCA-reactive fragment thereof, or a recombinant protein
containing an
antigen-binding region of a PSCA antibody; and then detecting the binding of
PSCA-related
protein in the sample.
[02041 Methods for identifying a cell that expresses PSCA are also within the
scope of the
invention. In one embodiment, an assay for identifying a cell that expresses a
PSCA gene
comprises detecting the presence of PSCA 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 PSCA
riboprobes,
Northern blot and related techniques) and various nucleic acid amplification
assays (such as RT-
PCR using complementary primers specific for PSCA, 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 PSCA gene comprises detecting
the presence of
PSCA-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 PSCA-
related proteins
and cells that express PSCA-related proteins.
[0205) PSCA expression analysis is also useful as a tool for identifying and
evaluating
agents that modulate PSCA gene expression. For example, PSCA 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 PSCA expression
or over-expression
in cancer cells is of therapeutic value. For example, such an agent can be
identified by using a
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screen that quantifies PSCA expression by RT-PCR, nucleic acid hybridization
or antibody
binding.
VIII.) Methods for Monitoring the Status of PSCA-related Genes and Their
Products
[0206] 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 PSCA expression in
cancers) allows for
early detection of such aberrant physiology, before a pathologic state such as
cancer has
progressed to a stage that therapeutic options are more limited and or the
prognosis is worse. In
such examinations, the status of PSCA in a biological sample of interest can
be compared, for
example, to the status of PSCA 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 PSCA 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
PSCA status in
a sample.
[0207] 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
PSCA expressing cells) as well as the level, and biological activity of
expressed gene products
(such as PSCA mRNA, polynucleotides and polypeptides). Typically, an
alteration in the status
of PSCA comprises a change in the location of PSCA and/or PSCA expressing
cells and/or an
increase in PSCA mRNA and/or protein expression.
[0208] PSCA 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 PSCA gene and gene
products are
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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 PSCA 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 PSCA gene), Northern analysis and/or PCR analysis of PSCA
mRNA (to
examine, for example alterations in the polynucleotide sequences or expression
levels of PSCA
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 PSCA proteins and/or associations of PSCA
proteins with
polypeptide binding partners). Detectable PSCA polynucleotides include, for
example, a PSCA
gene or fragment thereof, PSCA mRNA, alternative splice variants, PSCA mRNAs,
and
recombinant DNA or RNA molecules containing a PSCA polynucleotide.
[0209] The expression profile of PSCA 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 PSCA provides information
useful for predicting
susceptibility to particular disease stages, progression, and/or tumor
aggressiveness. The
invention provides methods and assays for determining PSCA status and
diagnosing cancers that
express PSCA, such as cancers of the tissues listed in Table I. For example,
because PSCA
mRNA is so highly expressed in prostate and other cancers relative to normal
prostate tissue,
assays that evaluate the levels of PSCA mRNA transcripts or proteins in a
biological sample can
be used to diagnose a disease associated with PSCA dysregulation, and can
provide prognostic
information useful in defining appropriate therapeutic options.
[0210] The expression status of PSCA provides information including the
presence, stage
and location of dysplastic, precancerous and cancerous cells, predicting
susceptibility to various
stages of disease, and/or for gauging tumor aggressiveness. Moreover, the
expression profile
makes it useful as an imaging reagent for metastasized disease. Consequently,
an aspect of the
invention is directed to the various molecular prognostic and diagnostic
methods for examining
the status of PSCA 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.
[0211] As described above, the status of PSCA in a biological sample can be
examined by a
number of well-known procedures in the art. For example, the status of PSCA in
a biological
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sample taken from a specific location in the body can be examined by
evaluating the sample for
the presence or absence of PSCA expressing cells (e.g. those that express PSCA
mRNAs or
proteins). This examination can provide evidence of dysregulated cellular
growth, for example,
when PSCA-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
PSCA 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).
[0212] In one aspect, the invention provides methods for monitoring PSCA gene
products by
determining the status of PSCA gene products expressed by cells from an
individual suspected
of having a disease associated with dysregulated cell growth (such as
hyperplasia or cancer) and
then comparing the status so determined to the status of PSCA gene products in
a corresponding
normal sample. The presence of aberrant PSCA 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.
[0213] In another aspect, the invention provides assays useful in determining
the presence of
cancer in an individual, comprising detecting a significant increase in PSCA
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 PSCA mRNA can, for example, be
evaluated in tissues
including but not limited to those listed in Table I. The presence of
significant PSCA 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 PSCA mRNA or express it
at lower
levels.
[0214] In a related embodiment, PSCA status is determined at the protein level
rather than at
the nucleic acid level. For example, such a method comprises determining the
level of PSCA
protein expressed by cells in a test tissue sample and comparing the level so
determined to the
level of PSCA expressed in a corresponding normal sample. In one embodiment,
the presence
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of PSCA protein is evaluated, for example, using immunohistochemical methods.
PSCA
antibodies or binding partners capable of detecting PSCA protein expression
are used in a
variety of assay formats well known in the art for this purpose.
102151 In a further embodiment, one can evaluate the status of PSCA nucleotide
and amino
acid sequences in a biological sample in order to identify. perturbations in
the structure of these
molecules. These perturbations can include insertions, deletions,
substitutions and the like.
Such evaluations are useful because perturbations in the nucleotide and amino
acid sequences
are observed in a large number of proteins associated with a growth
dysregulated phenotype
(see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For
example, a mutation in the
sequence of PSCA may be indicative of the presence or promotion of a tumor.
Such assays
therefore have diagnostic and predictive value where a mutation in PSCA
indicates a potential
loss of function or increase in tumor growth.
[02161 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 PSCA 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).
102171 Additionally, one can examine the methylation status of a PSCA gene in
a biological
sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene
5' regulatory
regions frequently occurs in immortalized and transformed cells, and can
result in altered
expression of various genes. For example, promoter hypermethylation of the pi-
class
glutathione S-transferase (a protein expressed in normal prostate but not
expressed in >90% of
prostate carcinomas) appears to permanently silence transcription of this gene
and is the most
frequently detected genomic alteration in prostate carcinomas (De Marzo et
al., Am. J. Pathol.
155(6): 1985-1992 (1999)). In addition, this alteration is present in at least
70% of cases of
high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer
Epidemiol.
Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-
I tumor
specific gene (which is not expressed in normal prostate but is expressed in
25-50% of prostate
cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting
that tumoral
expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-
908 (1998)). A
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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.
[0218] Gene amplification is an additional method for assessing the status of
PSCA. 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.
[0219] 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 PSCA
expression. The presence of RT-PCR amplifiable PSCA 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).
[0220] A further aspect of the invention is an assessment of the
susceptibility that an
individual has for developing cancer. In one embodiment, a method for
predicting susceptibility
to cancer comprises detecting PSCA mRNA or PSCA protein in a tissue sample,
its presence
indicating susceptibility to cancer, wherein the degree of PSCA mRNA
expression correlates to
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the degree of susceptibility. In a specific embodiment, the presence of PSCA
in prostate or other
tissue is examined, with the presence of PSCA 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 PSCA 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 PSCA
gene products in
the sample is an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0221] 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
PSCA mRNA or PSCA protein expressed by tumor cells, comparing the level so
determined to
the level of PSCA mRNA or PSCA protein expressed in a corresponding normal
tissue taken
from the same individual or a normal tissue reference sample, wherein the
degree of PSCA
mRNA or PSCA 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 PSCA is expressed in the tumor cells, with
higher expression
levels indicating more aggressive tumors. Another embodiment is the evaluation
of the integrity
of PSCA 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.
[0222] Another embodiment of the invention is directed to methods for
observing the
progression of a malignancy in an individual over time. In one embodiment,
methods for
observing the progression of a malignancy in an individual over time comprise
determining the
level of PSCA mRNA or PSCA protein expressed by cells in a sample of the
tumor, comparing
the level so determined to the level of PSCA mRNA or PSCA protein expressed in
an equivalent
tissue sample taken from the same individual at a different time, wherein the
degree of PSCA
mRNA or PSCA 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 PSCA expression in the tumor cells over time, where increased
expression over
time indicates a progression of the cancer. Also, one can evaluate the
integrity PSCA 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.
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[02231 The above diagnostic approaches can be combined with any one of a wide
variety of
prognostic and diagnostic protocols known in the art. For example, another
embodiment of the
invention is directed to methods for observing a coincidence between the
expression of PSCA
gene and PSCA gene products (or perturbations in PSCA gene and PSCA 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 PSCA gene
and PSCA
gene products (or perturbations in PSCA gene and PSCA 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.
[02241 In one embodiment, methods for observing a coincidence between the
expression of
PSCA gene and PSCA gene products (or perturbations in PSCA gene and PSCA gene
products)
and another factor associated with malignancy entails detecting the
overexpression of PSCA
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 PSCA
mRNA or
protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In
a specific
embodiment, the expression of PSCA and PSA mRNA in prostate tissue is
examined, where the
coincidence of PSCA 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.
[02251 Methods for detecting and quantifying the expression of PSCA mRNA or
protein are
described herein, and standard nucleic acid and protein detection and
quantification technologies
are well known in the art. Standard methods for the detection and
quantification of PSCA
mRNA include in situ hybridization using labeled PSCA riboprobes, Northern
blot and related
techniques using PSCA polynucleotide probes, RT-PCR analysis using primers
specific for
PSCA, 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 PSCA mRNA expression. Any number of primers capable of
amplifying
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PSCA 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 PSCA protein can be used in an
immunohistochemical
assay of biopsied tissue.
IX.) Identification of Molecules That Interact With PSCA
[02261 The PSCA protein and nucleic acid sequences disclosed herein allow a
skilled artisan
to identify proteins, small molecules and other agents that interact with
PSCA, as well as
pathways activated by PSCA via any one of a variety of art accepted protocols.
For example,
one can utilize one of the so-called interaction trap systems (also referred
to as the "two-hybrid
assay"). In such systems, molecules interact and reconstitute a transcription
factor which directs
expression of a reporter gene, whereupon the expression of the reporter gene
is assayed. Other
systems identify protein-protein interactions in vivo through reconstitution
of a eukaryotic
transcriptional activator, see, e.g., U.S. Patent Nos. 5,955,280 issued 21
September 1999,
5,925,523 issued 20 July 1999, 5,846,722 issued 8 December 1998 and 6,004,746
issued 21
December 1999. Algorithms are also available in the art for genome-based
predictions of
protein function (see, e.g., Marcotte, et al., Nature 402: 4 November 1999, 83-
86).
[02271 Alternatively one can screen peptide libraries to identify molecules
that interact with
PSCA protein sequences. In such methods, peptides that bind to PSCA 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 PSCA protein(s).
102281 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 PSCA 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.
102291 Alternatively, cell lines that express PSCA are used to identify
protein-protein
interactions mediated by PSCA. Such interactions can be examined using
immunoprecipitation
techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51).
PSCA protein can be immunoprecipitated from PSCA-expressing cell lines using
anti-PSCA
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antibodies. Alternatively, antibodies against His-tag can be used in a cell
line engineered to
express fusions of PSCA 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.
[02301 Small molecules and ligands that interact with PSCA 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
PSCA'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 PSCA-related ion channel, protein
pump, or cell
communication functions are identified and used to treat patients that have a
cancer that
expresses PSCA (see, e.g., Hille, B., Ionic Channels of Excitable Membranes
2nd Ed., Sinauer
Assoc., Sunderland, MA, 1992). Moreover, ligands that regulate PSCA function
can be
identified based on their ability to bind PSCA 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 PSCA
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 PSCA.
[02311 An embodiment of this invention comprises a method of screening for a
molecule
that interacts with a PSCA amino acid sequence shown in Figure 1, comprising
the steps of
contacting a population of molecules with a PSCA amino acid sequence, allowing
the population
of molecules and the PSCA amino acid sequence to interact under conditions
that facilitate an
interaction, determining the presence of a molecule that interacts with the
PSCA amino acid
sequence, and then separating molecules that do not interact with the PSCA
amino acid
sequence from molecules that do. In a specific embodiment, the method further
comprises
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purifying, characterizing and identifying a molecule that interacts with the
PSCA amino acid
sequence. The identified molecule can be used to modulate a function performed
by PSCA. In
a preferred embodiment, the PSCA amino acid sequence is contacted with a
library of peptides.
X.) Therapeutic Methods and Compositions
[0232] The identification of PSCA as a protein that is normally expressed in a
restricted set
of tissues, but which is also expressed in cancers such as those listed in
Table I, opens a number
of therapeutic approaches to the treatment of such cancers.
[0233] Of note, targeted antitumor therapies have been useful even when the
targeted protein
is expressed on normal tissues, even vital normal organ tissues. A vital organ
is one that is
necessary to sustain life, such as the heart or colon. A non-vital organ is
one that can be
removed whereupon the individual is still able to survive. Examples of non-
vital organs are
ovary, breast, and prostate.
[0234] For example, Herceptin is an FDA approved pharmaceutical that consists
of an
antibody which is immunoreactive with the protein variously known as HER2,
HER2/neu, and
erb-b-2. It is marketed by Genentech and has been a commercially successful
antitumor agent.
Herceptin sales reached almost $400 million in 2002. Herceptin is a
treatment for HER2
positive metastatic breast cancer. However, the expression of HER2 is not
limited to such
tumors. The same protein is expressed in a number of normal tissues. In
particular, it is known
that HER2/neu is present in normal kidney and heart, thus these tissues are
present in all human
recipients of Herceptin. The presence of HER2/neu in normal kidney is also
confirmed by Latif,
Z., et al., B.J.U. International (2002) 89:5-9. As shown in this article
(which evaluated whether
renal cell carcinoma should be a preferred indication for anti-HER2 antibodies
such as
Herceptin) both protein and mRNA are produced in benign renal tissues.
Notably, HER2/neu
protein was strongly overexpressed in benign renal tissue.
[0235] Despite the fact that HER2/neu is expressed in such vital tissues as
heart and kidney,
Herceptin is a very useful, FDA approved, and commercially successful drug.
The effect of
Herceptin on cardiac tissue, i.e., "cardiotoxicity," has merely been a side
effect to treatment.
When patients were treated with Herceptin alone, significant cardiotoxicity
occurred in a very
low percentage of patients. To minimize cariotoxicity there is a more
stringent entry
requirement for the treatment with HER2/neu. Factors such as predisposition to
heart condition
are evaluated before treatment can occur.
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[02361 Of particular note, although kidney tissue is indicated to exhibit
normal expression,
possibly even higher expression than cardiac tissue, kidney has no appreciable
Herceptin side
effect whatsoever. Moreover, of the diverse array of normal tissues in which
HER2 is
expressed, there is very little occurrence of any side effect. Only cardiac
tissue has manifested
any appreciable side effect at all. A tissue such as kidney,. where HER2/neu
expression is
especially notable, has not been the basis for any side effect.
102371 Furthermore, favorable therapeutic effects have been found for
antitumor therapies
that target epidermal growth factor receptor (EGFR); Erbitux (ImClone). EGFR
is also
expressed in numerous normal tissues. There have been very limited side
effects in normal
tissues following use of anti-EGFR therapeutics. A general side effect that
occurs with the
EGFR treatment is a severe skin rash observed in 100% of the patients
undergoing treatment.
[02381 Thus, expression of a target protein in normal tissue, even vital
normal tissue, does
not defeat the utility of a targeting agent for the protein as a therapeutic
for certain tumors in
which the protein is also overexpressed. For example, expression in vital
organs is not in and of
itself detrimental. In addition, organs regarded as dispensible, such as the
prostate and ovary,
can be removed without affecting mortality. Finally, some vital organs are not
affected by
normal organ expression because of an immunoprivilege. Immunoprivileged organs
are organs
that are protected from blood by a blood-organ barrier and thus are not
accessible to
immunotherapy. Examples of immunoprivileged organs are the brain and testis.
[0239[ Accordingly, therapeutic approaches that inhibit the activity of a PSCA
protein are
useful for patients suffering from a cancer that expresses PSCA. These
therapeutic approaches
generally fall into three classes. The first class modulates PSCA function as
it relates to tumor
cell growth leading to inhibition or retardation of tumor cell growth or
inducing its killing. The
second class comprises various methods for inhibiting the binding or
association of a PSCA
protein with its binding partner or with other proteins. The third class
comprises a variety of
methods for inhibiting the transcription of a PSCA gene or translation of PSCA
mRNA.
X.A.) Anti-Cancer Vaccines
102401 The invention provides cancer vaccines comprising a PSCA-related
protein or PSCA-
related nucleic acid. In view of the expression of PSCA, cancer vaccines
prevent and/or treat
PSCA-expressing cancers with minimal or no effects on non-target tissues. The
use of a tumor
antigen in a vaccine that generates cell-mediated humoral immune responses as
anti-cancer
therapy is well known in the art and has been employed in prostate cancer
using human PSMA
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and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong
et al., 1997,
J. Immunol. 159:3113-3117).
[0241] Such methods can be readily practiced by employing a PSCA-related
protein, or a
PSCA-encoding nucleic acid molecule and recombinant vectors capable of
expressing and
presenting the PSCA immunogen (which typically comprises a number of T-cell
epitopes or
antibody). 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. cell-mediated and/or
humoral) in a
mammal, comprise the steps of: exposing the mammal's immune system to an
immunoreactive
epitope (e.g. an epitope present in a PSCA protein shown in Figure 1 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).
[0242] The entire PSCA protein, immunogenic regions or epitopes thereof can be
combined
and delivered by various means. Such vaccine compositions can include, for
example,
lipopeptides (e.g., Vitiello, A. 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 Immunol.
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
Bio/Technology 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., J.
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 at., Vaccine 11:293, 1993),
liposomes
(Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today
17:131, 1996), or,
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naked or particle absorbed eDNA (Ulmer, 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: Concepts in
vaccine development, Kauftnann, 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.
[02431 In patients with PSCA-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:
[02441 CTL epitopes can be determined using specific algorithms to identify
peptides within
PSCA protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM
and
EpimatrixTM, Brown University (URL brown.edu/Research/TB-
HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/;
SYFPEITHI at
URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a PSCA
immunogen contains
one or more amino acid sequences identified using techniques well known in the
art, such as the
sequences shown in Tables V-XVIII and XXII-LI 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 1 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 attached to the amino and/or
carboxyl termini of a
motif-bearing sequence. HLA Class 11 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.
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[02451 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 PSCA protein) so that an immune
response is
generated. A typical embodiment consists of a method for: generating an immune
response to
PSCA in a host, by contacting the host with a sufficient amount of at least
one PSCA B cell or
cytotoxic T-cell epitope or analog thereof, and at least one periodic interval
thereafter re-
contacting the host with the PSCA B cell or cytotoxic T-cell epitope or analog
thereof. A
specific embodiment consists of a method of generating an immune response
against a PSCA-
related protein or a man-made multiepitopic peptide comprising: administering
PSCA
immunogen (e.g. a PSCA protein or a peptide fragment thereof, a PSCA 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 PSCA
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 PSCA 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 PSCA, in order to
generate a response to
the target antigen.
Nucleic Acid Vaccines:
[02461 Vaccine compositions of the invention include nucleic acid-mediated
modalities.
DNA or RNA that encode protein(s) of the invention can be administered to a
patient. Genetic
immunization methods can be employed to generate prophylactic or therapeutic
humoral and
cellular immune responses directed against cancer cells expressing PSCA.
Constructs
comprising DNA encoding a PSCA-related protein/immunogen and appropriate
regulatory
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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 PSCA
protein/immunogen.
Alternatively, a vaccine comprises a PSCA-related protein. Expression of the
PSCA-related
protein immunogen results in the generation of prophylactic or therapeutic
humoral and cellular
immunity against cells that bear a PSCA protein. Various.prophylactic and
therapeutic genetic
immunization techniques known in the art can be used (for review, see
information and
references published at Internet address genweb.com). Nucleic acid-based
delivery is described,
for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Patent
Nos. 5,580,859;
5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples
of DNA-
based delivery technologies include "naked DNA", facilitated (bupivicaine,
polymers, peptide-
mediated) delivery, cationic lipid complexes, and particle-mediated ("gene
gun") or pressure-
mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
[02471 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 eta!. J. Natl. Cancer
Inst. 87:982-990
(1995)). Non-viral delivery systems can also be employed by introducing naked
DNA encoding
a PSCA-related protein into the patient (e.g., intramuscularly or
intradermally) to induce an anti-
tumor response.
[02481 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 a!., 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.
102491 Thus, gene delivery systems are used to deliver a PSCA-related nucleic
acid
molecule. In one embodiment, the full-length human PSCA cDNA is employed. In
another
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embodiment, PSCA nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL)
and/or antibody epitopes are employed.
Ex Vivo Vaccines
[0250) 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 PSCA 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 PSCA peptides to T
cells in the
context of MHC class I or II molecules. In one embodiment, autologous
dendritic cells are
pulsed with PSCA peptides capable of binding to MHC class I and/or class II
molecules. In
another embodiment, dendritic cells are pulsed with the complete PSCA protein.
Yet another
embodiment involves engineering the overexpression of a PSCA 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 PSCA can also be engineered to express immune modulators, such as GM-
CSF, and
used as immunizing agents.
X.B.) PSCA as a Target for Antibody-based Therapy
[0251] PSCA 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 PSCA is expressed by cancer cells of various lineages relative to
corresponding normal
cells, systemic administration of PSCA-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 PSCA are useful to treat PSCA-expressing cancers
systemically, either
as conjugates with a toxin or therapeutic agent, or as naked antibodies
capable of inhibiting cell
proliferation or function.
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(0252] PSCA antibodies can be introduced into a patient such that the antibody
binds to
PSCA 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 PSCA, inhibition of ligand binding or signal transduction
pathways, modulation of
tumor cell differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
Examples include Rituxan for Non-Hodgkins Lymphoma, Herceptin for metastatic
breast
cancer, and Erbitux for colorectal cancer.
[0253] 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 PSCA
sequence shown in
Figure 1. 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. PSCA), the
cytotoxic agent will
exert its known biological effect (i.e. cytotoxicity) on those cells.
[0254] 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-
PSCA antibody) that binds to a marker (e.g. PSCA) 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 PSCA, comprising conjugating the
cytotoxic agent to an
antibody that immunospecifically binds to a PSCA 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.
[0255] Cancer immunotherapy using anti-PSCA 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,
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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 (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun
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 Y91 or I131 to
anti-CD20 antibodies (e.g., ZevalinTM, IDEC Pharmaceuticals Corp. or BexxarTM,
Coulter
Pharmaceuticals) respectively, while others involve co-administration of
antibodies and other
therapeutic agents, such as HerceptinTM (trastuzuMAb) with paclitaxel
(Genentech, Inc.). The
antibodies can be conjugated to a therapeutic agent. To treat prostate cancer,
for example,
PSCA 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) or
Auristatin E (Nat Biotechnol. 2003 Jul; 21(7):778-84. (Seattle Genetics)).
[02561 Although PSCA 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.
[02571 Although PSCA 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
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chemotherapy, particularly for patients who do not tolerate the toxicity of
the chemotherapeutic
agent very well.
[02581 Cancer patients can be evaluated for the presence and level of PSCA
expression,
preferably using immunohistochemical assessments of tumor tissue, quantitative
PSCA imaging,
or other techniques that reliably indicate the presence and:degree of PSCA
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.
102591 Anti-PSCA 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-PSCA 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 Fe portion of the immunoglobulin molecule for
interaction with effector
cell Fc receptor sites on complement proteins. In addition, anti-PSCA MAbs
that exert a direct
biological effect on tumor growth are useful to treat cancers that express
PSCA. Mechanisms by
which directly cytotoxic MAbs act include: inhibition of cell growth,
modulation of cellular
differentiation, modulation of tumor angiogenesis factor profiles, and the
induction of apoptosis.
The mechanism(s) by which a particular anti-PSCA 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.
[02601 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 PSCA antigen
with high
affinity but exhibit low or no antigenicity in the patient.
[02611 Therapeutic methods of the invention contemplate the administration of
single anti-
PSCA 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
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effects. In addition, anti-PSCA MAbs can be administered concomitantly with
other therapeutic
modalities, including but not limited to various chemotherapeutic agents,
androgen-blockers,
immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-PSCA
MAbs are
administered in their "naked" or unconjugated form, or can have a therapeutic
agent(s)
conjugated to them.
[02621 Anti-PSCA 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-PSCA 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.
[02631 Based on clinical experience with the HerceptinTM MAb in the treatment
of
metastatic breast cancer, an initial loading dose of approximately 4 mg/kg
patient body weight
IV, followed by weekly doses of about 2 mg/kg IV of the anti-PSCA 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
PSCA expression in the patient, the extent of circulating shed PSCA 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.
[02641 Optionally, patients should be evaluated for the levels of PSCA in a
given sample
(e.g. the levels of circulating PSCA antigen and/or PSCA expressing cells) in
order to assist in
the determination of the most effective dosing regimen, etc. Such evaluations
are also used for
monitoring purposes throughout therapy, and are useful to gauge therapeutic
success in
combination with the evaluation of other parameters (for example, urine
cytology and/or
ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in
prostate cancer
therapy).
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[0265] Anti-idiotypic anti-PSCA antibodies can also be used in anti-cancer
therapy as a
vaccine for inducing an immune response to cells expressing a PSCA-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-PSCA antibodies that
mimic an epitope on
a PSCA-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. Immunother.
43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine
strategies.
[0266] An object of the present invention is to provide PSCA antibodies, which
inhibit or
retard the growth of tumor cells expressing PSCA. A further object of this
invention is to
provide methods to inhibit angiogenesis and other biological functions and
thereby reduce tumor
growth in mammals, preferably humans, using such PSCA antibodies, and in
particular using
such PSCA antibodies combined with radiation and chemotherapy or both.
[0267] In one embodiment, there is synergy when tumors, including human
tumors, are
treated with PSCA antibodies in conjunction with chemotherapeutic agents or
radiation or
combinations thereof. In other words, the inhibition of tumor growth by a PSCA
antibody is
enhanced more than expected when combined with chemotherapeutic agents or
radiation or
combinations thereof. Synergy may be shown, for example, by greater inhibition
of tumor
growth with combined treatment than would be expected from a treatment of only
PSCA
antibodies or the additive effect of treatment with a PSCA antibody and a
chemotherapeutic
agent or radiation. Preferably, synergy is demonstrated by remission of the
cancer where
remission is not expected from treatment either from a naked PSCA antibody or
with treatment
using an additive combination of a PSCA antibody and a chemotherapeutic agent
or radiation.
[0268] The method for inhibiting growth of tumor cells using a PSCA antibody
and a
combination of chemotherapy or radiation or both comprises administering the
PSCA antibody
before, during, or after commencing chemotherapy or radiation therapy, as well
as any
combination thereof (i.e. before and during, before and after, during and
after, or before, during,
and after commencing the chemotherapy and/or radiation therapy). For example,
the PSCA
antibody is typically administered between I and 60 days, preferably between 3
and 40 days,
more preferably between 5 and 12 days before commencing radiation therapy
and/or
chemotherapy. However, depending on the treatment protocol and the specific
patient needs, the
method is performed in a manner that will provide the most efficacious
treatment and ultimately
prolong the life of the patient.
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[02691 The administration of chemotherapeutic agents can be accomplished in a
variety of
ways including systemically by the parenteral and enteral routes. In one
embodiment, the PSCA
antibody and the chemotherapeutic agent are administered as separate
molecules. In another
embodiment, the PSCA antibody is attached, for example, by conjugation, to a
chemotherapeutic
agent. (See the Example entitled "Human Clinical Trials for the Treatment and
Diagnosis of
Human Carcinomas through use of Human Anti-PSCA Antibodies in vivo") and (See
section
entitled "PSCA as a Target for Antibody-based Therapy"). Particular examples
of
chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC),
dactinomycin,
mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine
(BCNU),
lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine,
mitomycin,
cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine,
bleomycin,
paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan,
carboplatin,
cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide,
interferon alpha,
leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane,
pegaspargase,
pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide,
testolactone,
thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, taxol and
combinations thereof.
[02701 The source of radiation, used in combination with a PSCA antibody, can
be either
external or internal to the patient being treated. When the source is external
to the patient, the
therapy is known as external beam radiation therapy (EBRT). When the source of
radiation is
internal to the patient, the treatment is called brachytherapy (BT).
[02711 The radiation is administered in accordance with well known standard
techniques
using standard equipment manufactured for this purpose, such as AECL Theratron
and Varian
Clinac. The dose of radiation depends on numerous factors as is well known in
the art. Such
factors include the organ being treated, the healthy organs in the path of the
radiation that might
inadvertently be adversely affected, the tolerance of the patient for
radiation therapy, and the
area of the body in need of treatment. The dose will typically be between 1
and 100 Gy, and
more particularly between 2 and 80 Gy. Some doses that have been reported
include 35 Gy to
the spinal cord, 15 Gy to the kidneys, 20 Gy to the liver, and 65-80 Gy to the
prostate. It should
be emphasized, however, that the invention is not limited to any particular
dose. The dose will
be determined by the treating physician in accordance with the particular
factors in a given
situation, including the factors mentioned above.
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[02721 The distance between the source of the external radiation and the point
of entry into
the patient may be any distance that represents an acceptable balance between
killing target cells
and minimizing side effects. Typically, the source of the external radiation
is between 70 and
100 cm from the point of entry into the patient.
[02731 Brachytherapy is generally carried out by placing the source of
radiation in the
patient. Typically, the source of radiation is placed approximately 0-3 cm
from the tissue being
treated. Known techniques include interstitial, intercavitary, and surface
brachytherapy. The
radioactive seeds can be implanted permanently or temporarily. Some typical
radioactive atoms
that have been used in permanent implants include iodine-125 and radon. Some
typical
radioactive atoms that have been used in temporary implants include radium,
cesium-137, and
iridium- 192. Some additional radioactive atoms that have been used in
brachytherapy include
americium-241 and gold-198.The dose of radiation for brachytherapy can be the
same as that
mentioned above for external beam radiation therapy. In addition to the
factors mentioned above
for determining the dose of external beam radiation therapy, the nature of the
radioactive atom
used is also taken into account in determining the dose of brachytherapy.
X.C.) PSCA as a Target for Cellular Immune Responses
[02741 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.
[02751 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
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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)).
[0276] 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
PSCA antigen, or
derives at least some therapeutic benefit when the antigen was tumor-
associated.
[0277] In some embodiments, it may be desirable to combine the class I peptide
components
with components that induce or facilitate neutralizing antibody and or helper
T cell responses
directed to the target antigen. A preferred embodiment of such a composition
comprises class I
and class II epitopes in accordance with the invention. An alternative
embodiment of such a
composition comprises a class I and/or class II epitope in accordance with the
invention, along
with a cross reactive HTL epitope such as PADRETM (Epimmune, San Diego, CA)
molecule
(described e.g., in U.S. Patent Number 5,736,142).
[0278] 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.
[0279] 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
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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 IC50 of 500 nM or less,
often 200 nM or
less; and for Class II an IC50 of 1000 nM or less.
3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-
specific
motif-bearing peptides, are selected to give broad population coverage. For
example, it is
preferable to have at least 80% population coverage. A Monte Carlo analysis, a
statistical
evaluation known in the art, can be employed to assess the breadth, or
redundancy of, population
coverage.
4.) When selecting epitopes from cancer-related antigens it is often useful to
select
analogs because the patient may have developed tolerance to the native
epitope.
5.) Of particular relevance are epitopes referred to as "nested epitopes."
Nested
epitopes occur where at least two epitopes overlap in a given peptide
sequence. A nested
peptide sequence can comprise B cell, HLA class I and/or HLA class 11
epitopes. When
providing nested epitopes, a general objective is to provide the greatest
number of epitopes per
sequence. Thus, an aspect is to avoid providing a peptide that is any longer
than the amino
terminus of the amino terminal epitope and the carboxyl terminus of the
carboxyl terminal
epitope in the peptide. When providing a multi-epitopic sequence, such as a
sequence
comprising nested epitopes, it is generally important to screen the sequence
in order to insure
that it does not have pathological or other deleterious biological properties.
6.) If a polyepitopic protein is created, or when creating a minigene, an
objective is
to generate the smallest 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
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against the need to integrate any spacer sequences between epitopes in the
polyepitopic protein.
Spacer amino acid residues can, for example, be introduced to avoid junctional
epitopes (an
epitope recognized by the immune system, not present in the target antigen,
and only created by
the man-made juxtaposition of epitopes), or to facilitate cleavage between
epitopes and thereby
enhance epitope presentation. Junctional epitopes are generally to be avoided
because the
recipient may generate an immune response to that non-native epitope. Of
particular concern is
a junctional epitope that is a "dominant epitope." A dominant epitope may lead
to such a
zealous response that immune responses to other epitopes are diminished or
suppressed.
7.) Where the sequences of multiple variants of the same target protein are
present,
potential peptide epitopes can also be selected on the basis of their
conservancy. For example, a
criterion for conservancy may define that the entire sequence of an HLA class
I binding peptide
or the entire 9-mer core of a class II binding peptide be conserved in a
designated percentage of
the sequences evaluated for a specific protein antigen.
X.C.1. Minigene Vaccines
[02801 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.
[02811 The use of multi-epitope minigenes is described below and in, Ishioka
et al., J.
Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292,
1997; Thomson, S.
A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et at., J. Virol. 67:348,
1993; Hanke, R.
et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid
encoding supermotif-
and/or motif-bearing epitopes derived PSCA, the PADRETM universal helper T
cell epitope or
multiple HTL epitopes from PSCA (see e.g., Tables V-XVIII and XXII to LI), and
an
endoplasmic reticulum-translocating signal sequence can be engineered. A
vaccine may also
comprise epitopes that are derived from other TAAs.
[02821 The immunogenicity of a multi-epitopic minigene can be confirmed in
transgenic
mice to evaluate the magnitude of CTL induction responses against the epitopes
tested. Further,
the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the
in vitro
responses of specific CTL lines against target cells transfected with the DNA
plasmid. Thus,
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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.
[0283] For example, to create a DNA sequence encoding the selected epitopes
(minigene)
for expression in human cells, the amino acid sequences of the epitopes may be
reverse
translated. A human codon usage table can be used to guide the codon choice
for each amino
acid. These epitope-encoding DNA sequences may be directly adjoined, so that
when translated,
a continuous polypeptide sequence is created. To optimize expression and/or
immunogenicity,
additional elements can be incorporated into the minigene design. Examples of
amino acid
sequences that can be reverse translated and included in the minigene sequence
include: HLA
class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination
signal sequence,
and/or an endoplasmic reticulum targeting signal. In addition, HLA
presentation of CTL and
HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or
naturally-occurring
flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides
comprising the
epitope(s) are within the scope of the invention.
[0284] 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.
[0285] 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.
[0286] 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.
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[0287] 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.
[0288] 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.
[0289] In some embodiments, a bi-cistronic expression vector which allows
production of
both the minigene-encoded epitopes and a second protein (included to enhance
or decrease
immunogenicity) can be used. Examples of proteins or polypeptides that could
beneficially
enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-
12, GM-CSF),
cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL
responses, pan-
DR binding proteins (PADRETM, Epimmune, San Diego, CA). Helper (HTL) epitopes
can be
joined to intracellular targeting signals and expressed separately from
expressed CTL epitopes;
this allows direction of the HTL epitopes to a cell compartment different than
that of the CTL
epitopes. If required, this could facilitate more efficient entry of HTL
epitopes into the HLA
class II pathway, thereby improving HTL induction. In contrast'to HTL or CTL
induction,
specifically decreasing the immune response by co-expression of
immunosuppressive molecules
(e.g. TGF-(3) may be beneficial in certain diseases.
[0290] Therapeutic quantities of plasmid DNA can be produced for example, by
fermentation in E. cols, followed by purification. Aliquots from the working
cell bank are used
to inoculate growth medium, and grown to saturation in shaker flasks or a
bioreactor according
to well-known techniques. Plasmid DNA can be purified using standard
bioseparation
technologies such as solid phase anion-exchange resins supplied by QIAGEN,
Inc. (Valencia,
California). If required, supercoiled DNA can be isolated from the open
circular and linear
forms using gel electrophoresis or other methods.
[0291] 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
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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 Feigner, et al., Proc. Nat'l Acad. Sci.
USA 84:7413
(1987). In addition, peptides and compounds referred to collectively as
protective, interactive,
non-condensing compounds (PINC) could also be complexed to purified plasmid
DNA to
influence variables such as stability, intramuscular dispersion, or
trafficking to specific organs or
cell types.
[02921 Target cell sensitization can be used as a functional assay for
expression and HLA
class I presentation of minigene-encoded CTL epitopes. For example, the
plasmid DNA is
introduced into a mammalian cell line that is suitable as a target for
standard CTL chromium
release assays. The transfection method used will be dependent on the final
formulation.
Electroporation can be used for "naked" DNA, whereas cationic lipids allow
direct in vitro
transfection. A plasmid expressing green fluorescent protein (GFP) can be co-
transfected to
allow enrichment of transfected cells using fluorescence activated cell
sorting (FACS). These
cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-
specific CTL lines;
cytolysis, detected by 51Cr release, indicates both production of, and HLA
presentation of,
minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in
an
analogous manner using assays to assess HTL activity.
[02931 In vivo immunogenicity is a second approach for functional testing of
minigene DNA
formulations. Transgenic mice expressing appropriate human HLA proteins are
immunized with
the DNA product. The dose and route of administration are formulation
dependent (e.g., IM for
DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days
after
immunization, splenocytes are harvested and restimulated for one week in the
presence of
peptides encoding each epitope being tested. Thereafter, for CTL effector
cells, assays are
conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using
standard techniques.
Lysis of target cells that were sensitized by HLA loaded with peptide
epitopes, corresponding to
minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo
induction of CTLs.
Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous
manner.
[02941 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
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solely of DNA are administered. In a further alternative embodiment, DNA can
be adhered to
particles, such as gold particles.
[0295] 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
[0296] 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.
[0297] 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.
[0298] HTL peptide epitopes can also be modified to alter their biological
properties. For
example, they can be modified to include D-amino acids to increase their
resistance to proteases
and thus extend their serum half life, or they can be conjugated to other
molecules such as lipids,
proteins, carbohydrates, and the like to increase their biological activity.
For example, a T
helper peptide can be conjugated to one or more palmitic acid chains at either
the amino or
carboxyl termini.
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
[0299) 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
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lymphocytes. Lipids have been identified as agents capable of priming CTL in
vivo. For
example, palmitic acid residues can be attached to the E-and a- amino groups
of a lysine residue
and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-,
Ser, Ser-Ser, or the
like, to an immunogenic peptide. The lipidated peptide can then be
administered either directly
in a micelle or particle, incorporated into a liposome, or emulsified in an
adjuvant, e.g.,
incomplete Freund's adjuvant. In a preferred embodiment, a particularly
effective immunogenic
composition comprises palmitic acid attached to E-and a- amino groups of Lys,
which is
attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic
peptide.
[03001 As another example of lipid priming of CTL responses, E. coli
lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl- serine (P3CSS) can be used to prime
virus specific CTL
when covalently attached to an appropriate peptide (see, e.g., Deres, et al.,
Nature 342:561,
1989). Peptides of the invention can be coupled to P3CSS, for example, and the
lipopeptide
administered to an individual to prime specifically an immune response to the
target antigen.
Moreover, because the induction of neutralizing antibodies can also be primed
with P3CSS-
conjugated epitopes, two such compositions can be combined to more effectively
elicit both
humoral and cell-mediated responses.
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL
Peptides
[0301] An embodiment of a vaccine composition in accordance with the invention
comprises ex vivo administration of a cocktail of epitope-bearing peptides to
PBMC, or isolated
DC therefrom, from the patient's blood. A pharmaceutical to facilitate
harvesting of DC can be
used, such as ProgenipoietinTM (Pharmacia-Monsanto, St. Louis, MO) or GM-
CSF/IL-4. After
pulsing the DC with peptides and prior to reinfusion into patients, the DC are
washed to remove
unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs
which present
the pulsed peptide epitopes complexed with HLA molecules on their surfaces.
[0302] The DC can be pulsed ex vivo with a cocktail of peptides, some of which
stimulate
CTL responses to PSCA. 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 PSCA.
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X.D.) Adoptive Immunotherapy
[03031 Antigenic PSCA-related peptides are used to elicit a CTL and/or HTL
response ex
vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in
patients that do not
respond to other conventional forms of therapy, or will not respond to a
therapeutic vaccine
peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL
responses to a
particular antigen are induced by incubating in tissue culture the patient's,
or genetically
compatible, CTL or HTL precursor cells together with a source of antigen-
presenting cells
(APC), such as dendritic cells, and the appropriate immunogenic peptide. After
an appropriate
incubation time (typically about 7-28 days), in which the precursor cells are
activated and
expanded into effector cells, the cells are infused back into the patient,
where they will destroy
(CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a
tumor cell). Transfected
dendritic cells may also be used as antigen presenting cells.
X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes
[03041 Pharmaceutical and vaccine compositions of the invention are typically
used to treat
and/or prevent a cancer that expresses or overexpresses PSCA. 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.
[03051 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 PSCA. 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.
103061 For therapeutic use, administration should generally begin at the first
diagnosis of
PSCA-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
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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 PSCA, a vaccine comprising
PSCA-specific
CTL may be more efficacious in killing tumor cells in patient with advanced
disease than
alternative embodiments.
[03071 It is generally important to provide an amount of the peptide epitope
delivered by a
mode of administration sufficient to stimulate effectively a cytotoxic T cell
response;
compositions which stimulate helper T cell responses can also be given in
accordance with this
embodiment of the invention.
[03081 The dosage for an initial therapeutic immunization generally occurs in
a unit dosage
range where the lower value is about 1, 5, 50, 500, or 1,000 pg and the higher
value is about
10,000; 20,000; 30,000; or 50,000 jig. Dosage values for a human typically
range from about
500 jig to about 50,000 pg per 70 kilogram patient. Boosting dosages of
between about 1.0 pg
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.
103091 In certain embodiments, the peptides and compositions of the present
invention are
employed in serious disease states, that is, life-threatening or potentially
life threatening
situations. In such cases, as a result of the minimal amounts of extraneous
substances and the
relative nontoxic nature of the peptides in preferred compositions of the
invention, it is possible
and may be felt desirable by the treating physician to administer substantial
excesses of these
peptide compositions relative to these stated dosage amounts.
[03101 The vaccine compositions of the invention can also be used purely as
prophylactic
agents. Generally the dosage for an initial prophylactic immunization
generally occurs in a unit
dosage range where the lower value is about 1, 5, 50, 500, or 1000 pg and the
higher value is
about 10,000; 20,000; 30,000; or 50,000 pg. Dosage values for a human
typically range from
about 500 pg to about 50,000 pg per 70 kilogram patient. This is followed by
boosting dosages
of between about 1.0 pg to about 50,000 pg of peptide administered at defined
intervals from
about four weeks to six months after the initial administration of vaccine.
The immunogenicity
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of the vaccine can be assessed by measuring the specific activity of CTL and
HTL obtained from
a sample of the patient's blood.
[0311] The pharmaceutical compositions for therapeutic treatment are intended
for
parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or
topical ointment)
administration. Preferably, the pharmaceutical compositions are administered
parentally, e.g.,
intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the
invention provides
compositions for parenteral administration which comprise a solution of the
immunogenic
peptides dissolved or suspended in an acceptable carrier, preferably an
aqueous carrier.
[0312] 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.
[0313] 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.
[0314] 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.
[0315] A human unit dose form of a composition is typically included in a
pharmaceutical
composition that comprises a human unit dose of an acceptable carrier, in one
embodiment an
aqueous carrier, and is administered in a volume/quantity that is known by
those of skill in the
art to be used for administration of such compositions to humans (see, e.g.,
Remington's
Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing
Co., Easton,
Pennsylvania, 1985). For example a peptide dose for initial immunization can
be from about 1
to about 50,000 g, generally 100-5,000 g, for a 70 kg patient. For example,
for nucleic acids
an initial immunization may be performed using an expression vector in the
form of naked
nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at
multiple sites. The
nucleic acid (0.1 to 1000 g) can also be administered using a gene gun.
Following an
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incubation period of 3-4 weeks, a booster dose is then administered. The
booster can be
recombinant fowlpox virus administered at a dose of 5-107 to 5x109 pfu.
[0316] For antibodies, a treatment generally involves repeated administration
of the anti-
PSCA 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- PSCA 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 iminunogenicity of a substance, the degree of
PSCA expression in
the patient, the extent of circulating shed PSCA 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,
500gg - Img, 1mg
- 50mg, 50mg - I00mg, 100mg - 200mg, 200mg - 300mg, 400mg - 500mg, 500mg -
600mg,
600mg - 700mg, 700mg - 800mg, 800mg - 900mg, 900mg - 1 g, or 1 mg - 700mg. In
certain
embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with
follow on weekly
doses of 1-3 mg/kg; 0.5mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/kg body weight
followed, e.g., in two,
three or four weeks by weekly doses; 0.5 - 10mg/kg body weight, e.g., followed
in two, three or
four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400mg m2 of
body area weekly;
1-600mg m2 of body area weekly; 225-400mg m2 of body area weekly; these does
can be
followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.
[0317] In one embodiment, human unit dose forms of polynucleotides comprise a
suitable
dosage range or effective amount that provides any therapeutic effect. As
appreciated by one of
ordinary skill in the art a therapeutic effect depends on a number of factors,
including the
sequence of the polynucleotide, molecular weight of the polynucleotide and
route of
administration. Dosages are generally selected by the physician or other
health care professional
in accordance with a variety of parameters known in the art, such as severity
of symptoms,
history of the patient and the like. Generally, for a polynucleotide of about
20 bases, a dosage
range may be selected from, for example, an independently selected lower limit
such as about
0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,
400 or 500 mg/kg up to
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an independently selected upper limit, greater than the lower limit, of about
60, 80, 100, 200,
300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000
or 10,000
mg/kg. For example, a dose may be about any of the following: 0.1 to 100
mg/kg, 0.1 to 50
mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, Ito 500 mg/kg, 100 to 400 mg/kg, 200
to 300 mg/kg,
I to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to
1000 mg/kg,
500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of
administration may
require higher doses of polynucleotide compared to more direct application to
the nucleotide to
diseased tissue, as do polynucleotides of increasing length.
[0318] In one embodiment, human unit dose forms of T-cells comprise a suitable
dosage
range or effective amount that provides any therapeutic effect. As appreciated
by one of
ordinary skill in the art, a therapeutic effect depends on a number of
factors. Dosages are
generally selected by the physician or other health care professional in
accordance with a variety
of parameters known in the art, such as severity of symptoms, history of the
patient and the like.
A dose may be about 104 cells to about 106 cells, about 106 cells to about 108
cells, about 108 to
about 1011 cells, or about 108 to about 5 x 1010 cells. A dose may also about
106 cells/m2 to
about 1010 cells/m2, or about 106 cells/m2 to about 108 cells/m2.
[0319] 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,
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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.
[0320] 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.
[0321] 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%.
[0322] For aerosol administration, immunogenic peptides are preferably
supplied in finely
divided form along with a surfactant and propellant. Typical percentages of
peptides are about
0.01 %-20% by weight, preferably about 1 %-10%. The surfactant must, of
course, be nontoxic,
and preferably soluble in the propellant. Representative of such agents are
the esters or partial
esters of fatty acids containing from about 6 to 22 carbon atoms, such as
caproic, octanoic,
lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with
an aliphatic polyhydric
alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural
glycerides may be
employed. The surfactant may constitute about 0.1 %-20% by weight of the
composition,
preferably about 0.25-5%. The balance of the composition is ordinarily
propellant. A carrier
can also be included, as desired, as with, e.g., lecithin for intranasal
delivery.
XI.) Diagnostic and Prognostic Embodiments of PSCA.
[0323] As disclosed herein, PSCA 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
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s
described for example in the Example entitled "Expression analysis of PSCA in
normal tissues,
and patient specimens").
[0324] PSCA 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 PSCA
polynucleotides and
polypeptides (as well as PSCA polynucleotide probes and anti-PSCA 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.
[0325] Typical embodiments of diagnostic methods which utilize the PSCA
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 PSCA polynucleotides
described herein
can be utilized in the same way to detect PSCA 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 PSCA polypeptides described herein can
be utilized to
generate antibodies for use in detecting PSCA overexpression or the metastasis
of prostate cells
and cells of other cancers expressing this gene.
[0326] 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
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PSCA 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
PSCA-expressing
cells (lymph node) is found to contain PSCA-expressing cells such as the PSCA
expression seen
in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis,
respectively,
this finding is indicative of metastasis.
[03271 Alternatively PSCA 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
PSCA or express PSCA at a different level are found to express PSCA or have an
increased
expression of PSCA (see, e.g., the PSCA 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 PSCA) such as
PSA, PSCA etc.
(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).
[03281 The use of immunohistochemistry to identify the presence of a PSCA
polypeptide
within a tissue section can indicate an altered state of certain cells within
that tissue. It is well
understood in the art that the ability of an antibody to localize to a
polypeptide that is expressed
in cancer cells is a way of diagnosing presence of disease, disease stage,
progression and/or
tumor aggressiveness. Such an antibody can also detect an altered distribution
of the
polypeptide within the cancer cells, as compared to corresponding non-
malignant tissue.
[03291 The PSCA polypeptide and immunogenic compositions are also useful in
view of the
phenomena of altered subcellular protein localization in disease states.
Alteration of cells from
normal to diseased state causes changes in cellular morphology and is often
associated with
changes in subcellular protein localization/distribution. For example, cell
membrane proteins
that are expressed in a polarized manner in normal cells can be altered in
disease, resulting in
distribution of the protein in a non-polar manner over the whole cell surface.
[03301 The phenomenon of altered subcellular protein localization in a disease
state has been
demonstrated with MUCI and Her2 protein expression by use of
immunohistochemical means.
Normal epithelial cells have a typical apical distribution of MUC1, in
addition to some
supranuclear localization of the glycoprotein, whereas malignant lesions often
demonstrate an
apolar staining pattern (Diaz et al, The Breast Journal, 7; 40-45 (2001);
Zhang et al, Clinical
Cancer Research, 4; 2669-2676 (1998): Cao, et al, The Journal of
Histochemistry and
Cytochemistry, 45: 1547-1557 (1997)). In addition, normal breast epithelium is
either negative
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for Her2 protein or exhibits only a basolateral distribution whereas malignant
cells can express
the protein over the whole cell surface (De Potter, et al, International
Journal of Cancer, 44; 969-
974 (1989): McCormick, et al, 117; 935-943 (2002)). Alternatively,
distribution of the protein
may be altered from a surface only localization to include diffuse cytoplasmic
expression in the
diseased state. Such an example can be seen with MUC I (Diaz, et al, The
Breast Journal, 7: 40-
45 (2001)).
[0331] Alteration in the localization/distribution of a protein in the cell,
as detected by
immunohistochemical methods, can also provide valuable information concerning
the
favorability of certain treatment modalities. This last point is illustrated
by a situation where a
protein may be intracellular in normal tissue, but cell surface in malignant
cells; the cell surface
location makes the cells favorably amenable to antibody-based diagnostic and
treatment
regimens. When such an alteration of protein localization occurs for PSCA, the
PSCA protein
and immune responses related thereto are very useful. Accordingly, the ability
to determine
whether alteration of subcellular protein localization occurred for 24P4C12
make the PSCA
protein and immune responses related thereto very useful. Use of the PSCA
compositions
allows those skilled in the art to make important diagnostic and therapeutic
decisions.
[03321 Immunohistochemical reagents specific to PSCA are also useful to detect
metastases
of tumors expressing PSCA when the polypeptide appears in tissues where PSCA
is not
normally produced.
[03331 Thus, PSCA polypeptides and antibodies resulting from immune responses
thereto
are useful in a variety of important contexts such as diagnostic, prognostic,
preventative and/or
therapeutic purposes known to those skilled in the art.
[0334] Just as PSA polynucleotide fragments and polynucleotide variants are
employed by
skilled artisans for use in methods of monitoring PSA, PSCA 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.
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Biol. 98:121-154 (1998)). An additional illustration of the use of such
fragments is provided in
the Example entitled "Expression analysis of PSCA in normal tissues, and
patient specimens,"
where a PSCA polynucleotide fragment is used as a probe to show the expression
of PSCA
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 PSCA polynucleotide shown in Figure 1 or
variant thereof)
under conditions of high stringency.
103351 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. PSCA 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
PSCA 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 PSCA
polypeptide shown in
Figure 1).
103361 As shown herein, the PSCA polynucleotides and polypeptides (as well as
the PSCA
polynucleotide probes and anti-PSCA 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 PSCA gene
products, in order
to evaluate the presence or onset of a disease condition described herein,
such as prostate cancer,
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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 PSCA polynucleotides and polypeptides (as well as the PSCA
polynucleotide probes and
anti-PSCA antibodies used to identify the presence of these molecules) need to
be employed to
confirm a metastases of prostatic origin.
[0337] Finally, in addition to their use in diagnostic assays, the PSCA
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
PSCA gene maps (see the Example entitled "Chromosomal Mapping of PSCA" below).
Moreover, in addition to their use in diagnostic assays, the PSCA-related
proteins and
polynucleotides disclosed herein have other utilities such as their use in the
forensic analysis of
tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun
28;80(1-2): 63-9).
[0338] Additionally, PSCA-related proteins or polynucleotides of the invention
can be used
to treat a pathologic condition characterized by the over-expression of PSCA.
For example, the
amino acid or nucleic acid sequence of Figure 1, or fragments of either, can
be used to generate
an immune response to a PSCA antigen. Antibodies or other molecules that react
with PSCA
can be used to modulate the function of this molecule, and thereby provide a
therapeutic benefit.
XII.) Inhibition of PSCA Protein Function
103391 The invention includes various methods and compositions for inhibiting
the binding
of PSCA to its binding partner or its association with other protein(s) as
well as methods for
inhibiting PSCA function.
XII.A.)Inhibition of PSCA With Intracellular Antibodies
[0340] In one approach, a recombinant vector that encodes single chain
antibodies that
specifically bind to PSCA are introduced into PSCA expressing cells via gene
transfer
technologies. Accordingly, the encoded single chain anti-PSCA antibody is
expressed
intracellularly, binds to PSCA protein, and thereby inhibits its function.
Methods for
engineering such intracellular single chain antibodies are well known. Such
intracellular
antibodies, also known as "intrabodies", are specifically targeted to a
particular compartment
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e
within the cell, providing control over where the inhibitory activity of the
treatment is focused.
This technology has been successfully applied in the art (for review, see
Richardson and
Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually
eliminate the
expression of otherwise abundant cell surface receptors (see, e.g., Richardson
et al., 1995, Proc.
Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289:
23931-23936;
Deshane et al., 1994, Gene Ther. 1: 332-337).
103411 Single chain antibodies comprise the variable domains of the heavy and
light chain
joined by a flexible linker polypeptide, and are expressed as a single
polypeptide. Optionally,
single chain antibodies are expressed as a single chain variable region
fragment joined to the
light chain constant region. Well-known intracellular trafficking signals are
engineered into
recombinant polynucleotide vectors encoding such single chain antibodies in
order to target
precisely the intrabody to the desired intracellular compartment. For example,
intrabodies
targeted to the endoplasmic reticulum (ER) are engineered to incorporate a
leader peptide and,
optionally, a C-terminal ER retention signal, such as the KDEL amino acid
motif. Intrabodies
intended to exert activity in the nucleus are engineered to include a nuclear
localization signal.
Lipid moieties are joined to intrabodies in order to tether the intrabody to
the cytosolic side of
the plasma membrane. Intrabodies can also be targeted to exert function in the
cytosol. For
example, cytosolic intrabodies are used to sequester factors within the
cytosol, thereby
preventing them from being transported to their natural cellular destination.
[03421 In one embodiment, intrabodies are used to capture PSCA in the nucleus,
thereby
preventing its activity within the nucleus. Nuclear targeting signals are
engineered into such
PSCA intrabodies in order to achieve the desired targeting. Such PSCA
intrabodies are designed
to bind specifically to a particular PSCA domain. In another embodiment,
cytosolic intrabodies
that specifically bind to a PSCA protein are used to prevent PSCA from gaining
access to the
nucleus, thereby preventing it from exerting any biological activity within
the nucleus (e.g.,
preventing PSCA from forming transcription complexes with other factors).
[03431 In order to specifically direct the expression of such intrabodies to
particular cells,
the transcription of the intrabody is placed under the regulatory control of
an appropriate tumor-
specific promoter and/or enhancer. In order to target intrabody expression
specifically to
prostate, for example, the PSA promoter and/or promoter/enhancer can be
utilized (See, for
example, U.S. Patent No. 5,919,652 issued 6 July 1999).
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XII.B.) Inhibition of PSCA with Recombinant Proteins
[03441 In another approach, recombinant molecules bind to PSCA and thereby
inhibit PSCA
function. For example, these recombinant molecules prevent or inhibit PSCA
from
accessing/binding to its binding partner(s) or associating with other
protein(s). Such
recombinant molecules can, for example, contain the reactive part(s) of a PSCA
specific
antibody molecule. In a particular embodiment, the PSCA binding domain of a
PSCA binding
partner is engineered into a dimeric fusion protein, whereby the fusion
protein comprises two
PSCA ligand binding domains linked to the Fc portion of a human IgG, such as
human IgG1.
Such IgG portion can contain, for example, the CH2 and CH3 domains and the
hinge region, but
not the CHI domain. Such dimeric fusion proteins are administered in soluble
form to patients
suffering from a cancer associated with the expression of PSCA, whereby the
dimeric fusion
protein specifically binds to PSCA and blocks PSCA interaction with a binding
partner. Such
dimeric fusion proteins are further combined into multimeric proteins using
known antibody
linking technologies.
XII.C.) Inhibition of PSCA Transcription or Translation
10345] The present invention also comprises various methods and compositions
for
inhibiting the transcription of the PSCA gene. Similarly, the invention also
provides methods
and compositions for inhibiting the translation of PSCA mRNA into protein.
[0346] In one approach, a method of inhibiting the transcription of the PSCA
gene
comprises contacting the PSCA gene with a PSCA antisense polynucleotide. In
another
approach, a method of inhibiting PSCA mRNA translation comprises contacting a
PSCA mRNA
with an antisense polynucleotide. In another approach, a PSCA specific
ribozyme is used to
cleave a PSCA message, thereby inhibiting translation. Such antisense and
ribozyme based
methods can also be directed to the regulatory regions of the PSCA gene, such
as PSCA
promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a
PSCA gene
transcription factor are used to inhibit PSCA 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.
103471 Other factors that inhibit the transcription of PSCA by interfering
with PSCA
transcriptional activation are also useful to treat cancers expressing PSCA.
Similarly, factors
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that interfere with PSCA processing are useful to treat cancers that express
PSCA. Cancer
treatment methods utilizing such factors are also within the scope of the
invention.
XII.D.)General Considerations for Therapeutic Strategies
103481 Gene transfer and gene therapy technologies can be used to deliver
therapeutic
polynucleotide molecules to tumor cells synthesizing PSCA (i.e., antisense,
ribozyme,
polynucleotides encoding intrabodies and other PSCA inhibitory molecules). A
number of gene
therapy approaches are known in the art. Recombinant vectors encoding PSCA
antisense
polynucleotides, ribozymes, factors capable of interfering with PSCA
transcription, and so forth,
can be delivered to target tumor cells using such gene therapy approaches.
[03491 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.
[03501 The anti-tumor activity of a particular composition (e.g., antisense,
ribozyme,
intrabody), or a combination of such compositions, can be evaluated using
various in vitro and
in vivo assay systems. In vitro assays that evaluate therapeutic activity
include cell growth
assays, soft agar assays and other assays indicative of tumor promoting
activity, binding assays
capable of determining the extent to which a therapeutic composition will
inhibit the binding of
PSCA to a binding partner, etc.
[03511 In vivo, the effect of a PSCA 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 xenografl tissues are introduced into
immune compromised
animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-
408). For
example, PCT Patent Application W098/16628 and U.S. Patent 6,107,540 describe
various
xenograft models of human prostate cancer capable of recapitulating the
development of primary
tumors, micrometastasis, and the formation of osteoblastic metastases
characteristic of late stage
disease. Efficacy can be predicted using assays that measure inhibition of
tumor formation,
tumor regression or metastasis, and the like.
[03521 In vivo assays that evaluate the promotion of apoptosis are useful in
evaluating
therapeutic compositions. In one embodiment, xenografts from tumor bearing
mice treated with
the therapeutic composition can be examined for the presence of apoptotic foci
and compared to
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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.
[0353] The therapeutic compositions used in the practice of the foregoing
methods can be
formulated into pharmaceutical compositions comprising a carrier suitable for
the desired
delivery method. Suitable carriers include any material that when combined
with the therapeutic
composition retains the anti-tumor function of the therapeutic composition and
is generally non-
reactive with the patient's immune system. Examples include, but are not
limited to, any of a
number of standard pharmaceutical carriers such as sterile phosphate buffered
saline solutions,
bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical
Sciences 16th
Edition, A. Osal., Ed., 1980).
[0354] 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
polyvinyl chloride 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 bacteriostatie water (containing for example, benzyl
alcohol
preservative) or in sterile water prior to injection.
[0355] Dosages and administration protocols for the treatment of cancers using
the
foregoing methods will vary with the method and the target cancer, and will
generally depend on
a number of other factors appreciated in the art.
XIII.) Identification, Characterization and Use of Modulators of PSCA
Methods to Identify and Use Modulators
[03561 In one embodiment, screening is performed to identify modulators that
induce or
suppress a particular expression profile, suppress or induce specific
pathways, preferably
generating the associated phenotype thereby. In another embodiment, having
identified
differentially expressed genes important in a particular state; screens are
performed to identify
modulators that alter expression of individual genes, either increase or
decrease. In another
embodiment, screening is performed to identify modulators that alter a
biological function of the
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expression product of a differentially expressed gene. Again, having
identified the importance
of a gene in a particular state, screens are performed to identify agents that
bind and/or modulate
the biological activity of the gene product.
[0357] In addition, screens are done for genes that are induced in response to
a candidate
agent. After identifying a modulator (one that suppresses a cancer expression
pattern leading to
a normal expression pattern, or a modulator of a cancer gene that leads to
expression of the gene
as in normal tissue) a screen is performed to identify genes that are
specifically modulated in
response to the agent. Comparing expression profiles between normal tissue and
agent-treated
cancer tissue reveals genes that are not expressed in normal tissue or cancer
tissue, but are
expressed in agent treated tissue, and vice versa. These agent-specific
sequences are identified
and used by methods described herein for cancer genes or proteins. In
particular these
sequences and the proteins they encode are used in marking or identifying
agent-treated cells. In
addition, antibodies are raised against the agent-induced proteins and used to
target novel
therapeutics to the treated cancer tissue sample.
Modulator-related Identification and Screening Assays:
Gene Expression-related Assays
[0358] Proteins, nucleic acids, and antibodies of the invention are used in
screening assays.
The cancer-associated proteins, antibodies, nucleic acids, modified proteins
and cells containing
these sequences are used in screening assays, such as evaluating the effect of
drug candidates on
a "gene expression profile," expression profile of polypeptides or alteration
of biological
function. In one embodiment, the expression profiles are used, preferably in
conjunction with
high throughput screening techniques to allow monitoring for expression
profile genes after
treatment with a candidate agent (e.g., Davis, GF, et al, J Biol Screen 7:69
(2002); Zlokarnik,
et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94,1996).
[0359] The cancer proteins, antibodies, nucleic acids, modified proteins and
cells containing
the native or modified cancer proteins or genes are used in screening assays.
That is, the present
invention comprises methods for screening for compositions which modulate the
cancer
phenotype or a physiological function of a cancer protein of the invention.
This is done on a
gene itself or by evaluating the effect of drug candidates on a "gene
expression profile" or
biological function. In one embodiment, expression profiles are used,
preferably in conjunction
with high throughput screening techniques to allow monitoring after treatment
with a candidate
agent, see Zlokamik, supra.
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[03601 A variety of assays are executed directed to the genes and proteins of
the invention.
Assays are run on an individual nucleic acid or protein level. That is, having
identified a
particular gene as up regulated in cancer, test compounds are screened for the
ability to modulate
gene expression or for binding to the cancer protein of the invention.
"Modulation" in this
context includes an increase or a decrease in gene expression. The preferred
amount of
modulation will depend on the original change of the gene expression in normal
versus tissue
undergoing cancer, with changes of at least 10%, preferably 50%, more
preferably 100-300%,
and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-
fold increase in
cancer tissue compared to normal tissue, a decrease of about four-fold is
often desired; similarly,
a 10-fold decrease in cancer tissue compared to normal tissue a target value
of a 10-fold increase
in expression by the test compound is often desired. Modulators that
exacerbate the type of gene
expression seen in cancer are also useful, e.g., as an upregulated target in
further analyses.
[03611 The amount of gene expression is monitored using nucleic acid probes
and the
quantification of gene expression levels, or, alternatively, a gene product
itself is monitored, e.g.,
through the use of antibodies to the cancer protein and standard immunoassays.
Proteomics and
separation techniques also allow for quantification of expression.
Expression Monitoring to Identify Compounds that Modify Gene Expression
[03621 In one embodiment, gene expression monitoring, i.e., an expression
profile, is
monitored simultaneously for a number of entities. Such profiles will
typically involve one or
more of the genes of Figure 1. In this embodiment, e.g., cancer nucleic acid
probes are attached
to biochips to detect and quantify cancer sequences in a particular cell.
Alternatively, PCR can
be used. Thus, a series, e.g., wells of a microtiter plate, can be used with
dispensed primers in
desired wells. A PCR reaction can then be performed and analyzed for each
well.
[03631 Expression monitoring is performed to identify compounds that modify
the
expression of one or more cancer-associated sequences, e.g., a polynucleotide
sequence set out
in Figure 1. Generally, a test modulator is added to the cells prior to
analysis. Moreover,
screens are also provided to identify agents that modulate cancer, modulate
cancer proteins of
the invention, bind to a cancer protein of the invention, or interfere with
the binding of a cancer
protein of the invention and an antibody or other binding partner.
[03641 In one embodiment, high throughput screening methods involve providing
a library
containing a large number of potential therapeutic compounds (candidate
compounds). Such
"combinatorial chemical libraries" are then screened in one or more assays to
identify those
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library members (particular chemical species or subclasses) that display a
desired characteristic
activity. The compounds thus identified can serve as conventional "lead
compounds," as
compounds for screening, or as therapeutics.
[0365] In certain embodiments, combinatorial libraries of potential modulators
are screened
for an ability to bind to a cancer polypeptide or to modulate activity.
Conventionally, new
chemical entities with useful properties are generated by identifying a
chemical compound
(called a "lead compound") with some desirable property or activity, e.g.,
inhibiting activity,
creating variants of the lead compound, and evaluating the property and
activity of those variant
compounds. Often, high throughput screening (HTS) methods are employed for
such an
analysis.
[0366] As noted above, gene expression monitoring is conveniently used to test
candidate
modulators (e.g., protein, nucleic acid or small molecule). After the
candidate agent has been
added and the cells allowed to incubate for a period, the sample containing a
target sequence to
be analyzed is, e.g., added to a biochip.
[0367] If required, the target sequence is prepared using known techniques.
For example, a
sample is treated to lyse the cells, using known lysis buffers,
electroporation, etc., with
purification and/or amplification such as PCR performed as appropriate. For
example, an in
vitro transcription with labels covalently attached to the nucleotides is
performed. Generally,
the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
[0368] The target sequence can be labeled with, e.g., a fluorescent, a
chemiluminescent, a
chemical, or a radioactive signal, to provide a means of detecting the target
sequence's specific
binding to a probe. The label also can be an enzyme, such as alkaline
phosphatase or
horseradish peroxidase, which when provided with an appropriate substrate
produces a product
that is detected. Alternatively, the label is a labeled compound or small
molecule, such as an
enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme.
The label also can be
a moiety or compound, such as, an epitope tag or biotin which specifically
binds to streptavidin.
For the example of biotin, the streptavidin is labeled as described above,
thereby, providing a
detectable signal for the bound target sequence. Unbound labeled streptavidin
is typically
removed prior to analysis.
[0369] As will be appreciated by those in the art, these assays can be direct
hybridization
assays or can comprise "sandwich assays", which include the use of multiple
probes, as is
generally outlined in U.S. Patent Nos. 5, 681,702; 5,597,909; 5,545,730;
5,594,117; 5,591,584;
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5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118;
5,359,100; 5,124,
246; and 5,681,697. In this embodiment, in general, the target nucleic acid is
prepared as
outlined above, and then added to the biochip comprising a plurality of
nucleic acid probes,
under conditions that allow the formation of a hybridization complex.
[0370] A variety of hybridization conditions are used in the present
invention, including
high, moderate and low stringency conditions as outlined above. The assays are
generally run
under stringency conditions which allow formation of the label probe
hybridization complex
only in the presence of target. Stringency can be controlled by altering a
step parameter that is a
thermodynamic variable, including, but not limited to, temperature, formamide
concentration,
salt concentration, chaotropic salt concentration pH, organic solvent
concentration, etc. These
parameters may also be used to control non-specific binding, as is generally
outlined in U.S.
Patent No. 5,681,697. Thus, it can be desirable to perform certain steps at
higher stringency
conditions to reduce non-specific binding.
[0371] The reactions outlined herein can be accomplished in a variety of ways.
Components
of the reaction can be added simultaneously, or sequentially, in different
orders, with preferred
embodiments outlined below. In addition, the reaction may include a variety of
other reagents.
These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc.
which can be used to
facilitate optimal hybridization and detection, and/or reduce nonspecific or
background
interactions. Reagents that otherwise improve the efficiency of the assay,
such as protease
inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used
as appropriate,
depending on the sample preparation methods and purity of the target. The
assay data are
analyzed to determine the expression levels of individual genes, and changes
in expression
levels as between states, forming a gene expression profile.
Biological Activity-related Assays
[03721 The invention provides methods identify or screen for a compound that
modulates the
activity of a cancer-related gene or protein of the invention. The methods
comprise adding a test
compound, as defined above, to a cell comprising a cancer protein of the
invention. The cells
contain a recombinant nucleic acid that encodes a cancer protein of the
invention. In another
embodiment, a library of candidate agents is tested on a plurality of cells.
103731 In one aspect, the assays are evaluated in the presence or absence or
previous or
subsequent exposure of physiological signals, e.g. hormones, antibodies,
peptides, antigens,
cytokines, growth factors, action potentials, pharmacological agents including
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chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell
contacts). In another
example, the determinations are made at different stages of the cell cycle
process. In this way,
compounds that modulate genes or proteins of the invention are identified.
Compounds with
pharmacological activity are able to enhance or interfere with the activity of
the cancer protein
of the invention. Once identified, similar structures are evaluated to
identify critical structural
features of the compound.
[03741 In one embodiment, a method of modulating ( e.g., inhibiting) cancer
cell division is
provided; the method comprises administration of a cancer modulator. In
another embodiment,
a method of modulating ( e.g., inhibiting) cancer is provided; the method
comprises
administration of a cancer modulator. In a further embodiment, methods of
treating cells or
individuals with cancer are provided; the method comprises administration of a
cancer
modulator.
[03751 In one embodiment, a method for modulating the status of a cell that
expresses a gene
of the invention is provided. As used herein status comprises such art-
accepted parameters such
as growth, proliferation, survival, function, apoptosis, senescence, location,
enzymatic activity,
signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is
an antibody as
discussed above. In another embodiment, the cancer inhibitor is an antisense
molecule. A
variety of cell growth, proliferation, and metastasis assays are known to
those of skill in the art,
as described herein.
High Throughput Screening to Identify Modulators
[03761 The assays to identify suitable modulators are amenable to high
throughput
screening. Preferred assays thus detect enhancement or inhibition of cancer
gene transcription,
inhibition or enhancement of polypeptide expression, and inhibition or
enhancement of
polypeptide activity.
[03771 In one embodiment, modulators evaluated in high throughput screening
methods are
proteins, often naturally occurring proteins or fragments of naturally
occurring proteins. Thus,
e.g., cellular extracts containing proteins, or random or directed digests of
proteinaceous cellular
extracts, are used. In this way, libraries of proteins are made for screening
in the methods of the
invention. Particularly preferred in this embodiment are libraries of
bacterial, fungal, viral, and
mammalian proteins, with the latter being preferred, and human proteins being
especially
preferred. Particularly useful test compound will be directed to the class of
proteins to which the
target belongs, e.g., substrates for enzymes, or ligands and receptors.
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Use of Soft Agar Growth and Colony Formation to Identify and Characterize
Modulators
[0378] Normal cells require a solid substrate to attach and grow. When cells
are
transformed, they lose this phenotype and grow detached from the substrate.
For example,
transformed cells can grow in stirred suspension culture or suspended in semi-
solid media, such
as semi-solid or soft agar. The transformed cells, when transfected with tumor
suppressor genes,
can regenerate normal phenotype and once again require a solid substrate to
attach to and grow.
Soft agar growth or colony formation in assays are used to identify modulators
of cancer
sequences, which when expressed in host cells, inhibit abnormal cellular
proliferation and
transformation. A modulator reduces or eliminates the host cells' ability to
grow suspended in
solid or semisolid media, such as agar.
[0379] Techniques for soft agar growth or colony formation in suspension
assays are
described in Freshney, Culture of Animal Cells a Manual of Basic Technique
(3rd ed., 1994).
See also, the methods section of Garkavtsev et at. (1996), supra.
Evaluation of Contact Inhibition and Growth Density Limitation to Identify and
Characterize Modulators
[0380] Normal cells typically grow in a flat and organized pattern in cell
culture until they
touch other cells. When the cells touch one another, they are contact
inhibited and stop growing.
Transformed cells, however, are not contact inhibited and continue to grow to
high densities in
disorganized foci. Thus, transformed cells grow to a higher saturation density
than
corresponding normal cells. This is detected morphologically by the formation
of a disoriented
monolayer of cells or cells in foci. Alternatively, labeling index with (3H)-
thymidine at
saturation density is used to measure density limitation of growth, similarly
an MTT or Alamar
blue assay will reveal proliferation capacity of cells and the ability of
modulators to affect same.
See Freshney (1994), supra. Transformed cells, when transfected with tumor
suppressor genes,
can regenerate a normal phenotype and become contact inhibited and would grow
to a lower
density.
[0381] In this assay, labeling index with 3H)-thymidine at saturation density
is a preferred
method of measuring density limitation of growth. Transformed host cells are
transfected with a
cancer-associated sequence and are grown for 24 hours at saturation density in
non-limiting
medium conditions. The percentage of cells labeling with (3H)-thymidine is
determined by
incorporated cpm.
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103821 Contact independent growth is used to identify modulators of cancer
sequences,
which had led to abnormal cellular proliferation and transformation. A
modulator reduces or
eliminates contact independent growth, and returns the cells to a normal
phenotype.
Evaluation of Growth Factor or Serum Dependence to Identify and Characterize
Modulators
[0383] Transformed cells have lower serum dependence than their normal
counterparts (see,
e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp.
Med 131:836-879
(1970)); Freshney, supra. This is in part due to release of various growth
factors by the
transformed cells. The degree of growth factor or serum dependence of
transformed host cells
can be compared with that of control. For example, growth factor or serum
dependence of a cell
is monitored in methods to identify and characterize compounds that modulate
cancer-associated
sequences of the invention.
Use of Tumor-specific Marker Levels to Identify and Characterize Modulators
[0384] Tumor cells release an increased amount of certain factors (hereinafter
"tumor
specific markers") than their normal counterparts. For example, plasminogen
activator (PA) is
released from human glioma at a higher level than from normal brain cells
(see, e.g., Gullino,
Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor
Growth, in
Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly,
Tumor
Angiogenesis Factor (TAF) is released at a higher level in tumor cells than
their normal
counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem. Cancer Biol.
(1992)), while
bFGF is released from endothelial tumors (Ensoli, B et al.).
[0385] Various techniques which measure the release of these factors are
described in
Freshney (1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4295-
4305 (1974);
Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J.
Cancer 42:305 312
(1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential
Interference with Tumor
Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985);
Freshney,
Anticancer Res. 5:111-130 (1985). For example, tumor specific marker levels
are monitored in
methods to identify and characterize compounds that modulate cancer-associated
sequences of
the invention.
Invasiveness into Matrigel to Identify and Characterize Modulators
[0386] The degree of invasiveness into Matrigel or an extracellular matrix
constituent can be
used as an assay to identify and characterize compounds that modulate cancer
associated
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sequences. Tumor cells exhibit a positive correlation between malignancy and
invasiveness of
cells into Matrigel or some other extracellular matrix constituent. In this
assay, tumorigenic
cells are typically used as host cells. Expression of a tumor suppressor gene
in these host cells
would decrease invasiveness of the host cells. Techniques described in Cancer
Res. 1999;
59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion
of host cells is
measured by using filters coated with Matrigel or some other extracellular
matrix constituent.
Penetration into the gel, or through to the distal side of the filter, is
rated as invasiveness, and
rated histologically by number of cells and distance moved, or by prelabeling
the cells with 1251
and counting the radioactivity on the distal side of the filter or bottom of
the dish. See, e.g.,
Freshney (1984), supra.
Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
[0387] Effects of cancer-associated sequences on cell growth are tested in
transgenic or
immune-suppressed organisms. Transgenic organisms are prepared in a variety of
art-accepted
ways. For example, knock-out transgenic organisms, e.g., mammals such as mice,
are made, in
which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-
out transgenic
mice are made by insertion of a marker gene or other heterologous gene into
the endogenous
cancer gene site in the mouse genome via homologous recombination. Such mice
can also be
made by substituting the endogenous cancer gene with a mutated version of the
cancer gene, or
by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.
[0388] To prepare transgenic chimeric animals, e.g., mice, a DNA construct is
introduced
into the nuclei of embryonic stem cells. Cells containing the newly engineered
genetic lesion
are injected into a host mouse embryo, which is re-implanted into a recipient
female. Some of
these embryos develop into chimeric mice that possess germ cells some of which
are derived
from the mutant cell line. Therefore, by breeding the chimeric mice it is
possible to obtain a
new line of mice containing the introduced genetic lesion (see, e.g., Capecchi
et al., Science
244:1288 (1989)). Chimeric mice can be derived according to US Patent
6,365,797, issued 2
April 2002; US Patent 6,107,540 issued 22 August 2000; Hogan et al.,
Manipulating the Mouse
Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and
Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press,
Washington, D.C.,
(1987).
[0389] Alternatively, various immune-suppressed or immune-deficient host
animals can be
used. For example, a genetically athymic "nude" mouse (see, e.g., Giovanella
et al., J. Natl.
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Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectornized mouse, or an
irradiated mouse
(see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et at., Br. J.
Cancer 41:52 (1980))
can be used as a host. Transplantable tumor cells (typically about 106 cells)
injected into
isogenic hosts produce invasive tumors in a high proportion of cases, while
normal cells of
similar origin will not. In hosts which developed invasive tumors, cells
expressing cancer-
associated sequences are injected subcutaneously or orthotopically. Mice are
then separated into
groups, including control groups and treated experimental groups) e.g. treated
with a modulator).
After a suitable length of time, preferably 4-8 weeks, tumor growth is
measured (e.g., by volume
or by its two largest dimensions, or weight) and compared to the control.
Tumors that have
statistically significant reduction (using, e.g., Student's T test) are said
to have inhibited growth.
In Vitro Assays to Identify and Characterize Modulators
[03901 Assays to identify compounds with modulating activity can be performed
in vitro.
For example, a cancer polypeptide is first contacted with a potential
modulator and incubated for
a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the
cancer
polypeptide levels are determined in vitro by measuring the level of protein
or mRNA. The
level of protein is measured using immunoassays such as Western blotting,
ELISA and the like
with an antibody that selectively binds to the cancer polypeptide or a
fragment thereof. For
measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization
assays, e. g.,
Northern hybridization, RNAse protection, dot blotting, are preferred. The
level of protein or
mRNA is detected using directly or indirectly labeled detection agents, e.g.,
fluorescently or
radioactively labeled nucleic acids, radioactively or enzymatically labeled
antibodies, and the
like, as described herein.
[03911 Alternatively, a reporter gene system can be devised using a cancer
protein promoter
operably linked to a reporter gene such as luciferase, green fluorescent
protein, CAT, or P-gal.
The reporter construct is typically transfected into a cell. After treatment
with a potential
modulator, the amount of reporter gene transcription, translation, or activity
is measured
according to standard techniques known to those of skill in the art (Davis GF,
supra; Gonzalez,
J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).
[03921 As outlined above, in vitro screens are done on individual genes and
gene products.
That is, having identified a particular differentially expressed gene as
important in a particular
state, screening of modulators of the expression of the gene or the gene
product itself is
performed.
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[0393] In one embodiment, screening for modulators of expression of specific
gene(s) is
performed. Typically, the expression of only one or a few genes is evaluated.
In another
embodiment, screens are designed to first find compounds that bind to
differentially expressed
proteins. These compounds are then evaluated for the ability to modulate
differentially
expressed activity. Moreover, once initial candidate compounds are identified,
variants can be
further screened to better evaluate structure activity relationships.
Binding Assays to Identify and Characterize Modulators
[0394] In binding assays in accordance with the invention, a purified or
isolated gene
product of the invention is generally used. For example, antibodies are
generated to a protein of
the invention, and immunoassays are run to determine the amount and/or
location of protein.
Alternatively, cells comprising the cancer proteins are used in the assays.
[0395] Thus, the methods comprise combining a cancer protein of the invention
and a
candidate compound such as a ligand, and determining the binding of the
compound to the
cancer protein of the invention. Preferred embodiments utilize the human
cancer protein; animal
models of human disease of can also be developed and used. Also, other
analogous mammalian
proteins also can be used as appreciated by those of skill in the art.
Moreover, in some
embodiments variant or derivative cancer proteins are used.
[0396] Generally, the cancer protein of the invention, or the ligand, is non-
diffusibly bound
to an insoluble support. The support can, e.g., be one having isolated sample
receiving areas (a
microtiter plate, an array, etc.). The insoluble supports can be made of any
composition to
which the compositions can be bound, is readily separated from soluble
material, and is
otherwise compatible with the overall method of screening. The surface of such
supports can be
solid or porous and of any convenient shape.
[0397] Examples of suitable insoluble supports include microtiter plates,
arrays, membranes
and beads. These are typically made of glass, plastic (e.g., polystyrene),
polysaccharide, nylon,
nitrocellulose, or TeflonTM, etc. Microtiter plates and arrays are especially
convenient because a
large number of assays can be carried out simultaneously, using small amounts
of reagents and
samples. The particular manner of binding of the composition to the support is
not crucial so
long as it is compatible with the reagents and overall methods of the
invention, maintains the
activity of the composition and is nondiffusable. Preferred methods of binding
include the use
of antibodies which do not sterically block either the ligand binding site or
activation sequence
when attaching the protein to the support, direct binding to "sticky" or ionic
supports, chemical
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crosslinking, the synthesis of the protein or agent on the surface, etc.
Following binding of the
protein or ligand/binding agent to the support, excess unbound material is
removed by washing.
The sample receiving areas may then be blocked through incubation with bovine
serum albumin
(BSA), casein or other innocuous protein or other moiety.
[0398) Once a cancer protein of the invention is bound to the support, and a
test compound
is added to the assay. Alternatively, the candidate binding agent is bound to
the support and the
cancer protein of the invention is then added. Binding agents include specific
antibodies, non-
natural binding agents identified in screens of chemical libraries, peptide
analogs, etc.
[0399] Of particular interest are assays to identify agents that have a low
toxicity for human
cells. A wide variety of assays can be used for this purpose, including
proliferation assays,
cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic
mobility shift
assays, immunoassays for protein binding, functional assays (phosphorylation
assays, etc.) and
the like.
[0400] A determination of binding of the test compound (ligand, binding agent,
modulator,
etc.) to a cancer protein of the invention can be done in a number of ways.
The test compound
can be labeled, and binding determined directly, e.g., by attaching all or a
portion of the cancer
protein of the invention to a solid support, adding a labeled candidate
compound (e.g., a
fluorescent label), washing off excess reagent, and determining whether the
label is present on
the solid support. Various blocking and washing steps can be utilized as
appropriate.
[04011 In certain embodiments, only one of the components is labeled, e.g., a
protein of the
invention or ligands labeled. Alternatively, more than one component is
labeled with different
labels, e.g., I125, for the proteins and a fluorophor for the compound.
Proximity reagents, e.g.,
quenching or energy transfer reagents are also useful.
Competitive Binding to Identify and Characterize Modulators
[0402] In one embodiment, the binding of the "test compound" is determined by
competitive
binding assay with a "competitor." The competitor is a binding moiety that
binds to the target
molecule (e.g., a cancer protein of the invention). Competitors include
compounds such as
antibodies, peptides, binding partners, ligands, etc. Under certain
circumstances, the
competitive binding between the test compound and the competitor displaces the
test compound.
In one embodiment, the test compound is labeled. Either the test compound, the
competitor, or
both, is added to the protein for a time sufficient to allow binding.
Incubations are performed at
a temperature that facilitates optimal activity, typically between four and 40
C. Incubation
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periods are typically optimized, e.g., to facilitate rapid high throughput
screening; typically
between zero and one hour will be sufficient. Excess reagent is generally
removed or washed
away. The second component is then added, and the presence or absence of the
labeled
component is followed, to indicate binding.
[0403] In one embodiment, the competitor is added first, followed by the test
compound.
Displacement of the competitor is an indication that the test compound is
binding to the cancer
protein and thus is capable of binding to, and potentially modulating, the
activity of the cancer
protein. In this embodiment, either component can be labeled. Thus, e.g., if
the competitor is
labeled, the presence of label in the post-test compound wash solution
indicates displacement by
the test compound. Alternatively, if the test compound is labeled, the
presence of the label on
the support indicates displacement.
[0404] In an alternative embodiment, the test compound is added first, with
incubation and
washing, followed by the competitor. The absence of binding by the competitor
indicates that
the test compound binds to the cancer protein with higher affinity than the
competitor. Thus, if
the test compound is labeled, the presence of the label on the support,
coupled with a lack of
competitor binding, indicates that the test compound binds to and thus
potentially modulates the
cancer protein of the invention.
[0405] Accordingly, the competitive binding methods comprise differential
screening to
identity agents that are capable of modulating the activity of the cancer
proteins of the invention.
In this embodiment, the methods comprise combining a cancer protein and a
competitor in a first
sample. A second sample comprises a test compound, the cancer protein, and a
competitor. The
binding of the competitor is determined for both samples, and a change, or
difference in binding
between the two samples indicates the presence of an agent capable of binding
to the cancer
protein and potentially modulating its activity. That is, if the binding of
the competitor is
different in the second sample relative to the first sample, the agent is
capable of binding to the
cancer protein.
[0406] Alternatively, differential screening is used to identify drug
candidates that bind to
the native cancer protein, but cannot bind to modified cancer proteins. For
example the structure
of the cancer protein is modeled and used in rational drug design to
synthesize agents that
interact with that site, agents which generally do not bind to site-modified
proteins. Moreover,
such drug candidates that affect the activity of a native cancer protein are
also identified by
screening drugs for the ability to either enhance or reduce the activity of
such proteins.
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[04071 Positive controls and negative controls can be used in the assays.
Preferably control
and test samples are performed in at least triplicate to obtain statistically
significant results.
Incubation of all samples occurs for a time sufficient to allow for the
binding of the agent to the
protein. Following incubation, samples are washed free of non-specifically
bound material and
the amount of bound, generally labeled agent determined. For example, where a
radiolabel is
employed, the samples can be counted in a scintillation counter to determine
the amount of
bound compound.
[04081 A variety of other reagents can be included in the screening assays.
These include
reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which
are used to facilitate
optimal protein-protein binding and/or reduce non-specific or background
interactions. Also
reagents that otherwise improve the efficiency of the assay, such as protease
inhibitors, nuclease
inhibitors, anti-microbial agents, etc., can be used. The mixture of
components is added in an
order that provides for the requisite binding.
Use of Polynucleotides to Down-regulate or Inhibit a Protein of the Invention.
[04091 Polynucleotide modulators of cancer can be introduced into a cell
containing the
target nucleotide sequence by formation of a conjugate with a ligand-binding
molecule, as
described in WO 91/04753. Suitable ligand-binding molecules include, but are
not limited to,
cell surface receptors, growth factors, other cytokines, or other ligands that
bind to cell surface
receptors. Preferably, conjugation of the ligand binding molecule does not
substantially.
interfere with the ability of the ligand binding molecule to bind to its
corresponding molecule or
receptor, or block entry of the sense or antisense oligonucleotide or its
conjugated version into
the cell. Alternatively, a polynucleotide modulator of cancer can be
introduced into a cell
containing the target nucleic acid sequence, e.g., by formation of a
polynucleotide-lipid
complex, as described in WO 90/10448. It is understood that the use of
antisense molecules or
knock out and knock in models may also be used in screening assays as
discussed above, in
addition to methods of treatment.
Inhibitory and Antisense Nucleotides
104101 In certain embodiments, the activity of a cancer-associated protein is
down-regulated,
or entirely inhibited, by the use of antisense polynucleotide or inhibitory
small nuclear RNA
(snRNA), i.e., a nucleic acid complementary to, and which can preferably
hybridize specifically
to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the
invention, mRNA, or a
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subsequence thereof. Binding of the antisense polynucleotide to the mRNA
reduces the
translation and/or stability of the mRNA.
[0411] In the context of this invention, antisense polynucleotides can
comprise naturally
occurring nucleotides, or synthetic species formed from naturally occurring
subunits or their
close homologs. Antisense polynucleotides may also have altered sugar moieties
or inter-sugar
linkages. Exemplary among these are the phosphorothioate and other sulfur
containing species
which are known for use in the art. Analogs are comprised by this invention so
long as they
function effectively to hybridize with nucleotides of the invention. See,
e.g., Isis
Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.
[0412] Such antisense polynucleotides can readily be synthesized using
recombinant means,
or can be synthesized in vitro. Equipment for such synthesis is sold by
several vendors,
including Applied Biosystems. The preparation of other oligonucleotides such
as
phosphorothioates and alkylated derivatives is also well known to those of
skill in the art.
[0413] Antisense molecules as used herein include antisense or sense
oligonucleotides.
Sense oligonucleotides can, e.g., be employed to block transcription by
binding to the anti-sense
strand. The antisense and sense oligonucleotide comprise a single stranded
nucleic acid
sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA
(antisense)
sequences for cancer molecules. Antisense or sense oligonucleotides, according
to the present
invention, comprise a fragment generally at least about 12 nucleotides,
preferably from about 12
to 30 nucleotides. The ability to derive an antisense or a sense
oligonucleotide, based upon a
eDNA sequence encoding a given protein is described in, e.g., Stein &Cohen
(Cancer Res.
48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).
Ribozymes
[0414] In addition to antisense polynucleotides, ribozymes can be used to
target and inhibit
transcription of cancer-associated nucleotide sequences. A ribozyine is an RNA
molecule that
catalytically cleaves other RNA molecules. Different kinds of ribozymes have
been described,
including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P,
and axhead
ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317
(1994) for a general
review of the properties of different ribozymes).
[0415] The general features of hairpin ribozymes are described, e.g., in
Hampel et al., Nucl.
Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S.
Patent No.
5,254,678. Methods of preparing are well known to those of skill in the art
(see, e.g., WO
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94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993);
Yamada et al.,
Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci. USA
92:699- 703
(1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et
al., Virology
205: 121-126 (1994)).
Use of Modulators in Phenotypic Screening
[04161 In one embodiment, a test compound is administered to a population of
cancer cells,
which have an associated cancer expression profile. By "administration" or
"contacting" herein
is meant that the modulator is added to the cells in such a manner as to allow
the modulator to
act upon the cell, whether by uptake and intracellular action, or by action at
the cell surface. In
some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a
peptide) is put into a
viral construct such as an adenoviral or retroviral construct, and added to
the cell, such that
expression of the peptide agent is accomplished, e.g., PCT US97/01019.
Regulatable gene
therapy systems can also be used. Once the modulator has been administered to
the cells, the
cells are washed if desired and are allowed to incubate under preferably
physiological conditions
for some period. The cells are then harvested and a new gene expression
profile is generated.
Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce
or suppress, the cancer
phenotype. A change in at least one gene, preferably many, of the expression
profile indicates
that the agent has an effect on cancer activity. Similarly, altering a
biological function or a
signaling pathway is indicative of modulator activity. By defining such a
signature for the
cancer phenotype, screens for new drugs that alter the phenotype are devised.
With this
approach, the drug target need not be known and need not be represented in the
original
gene/protein expression screening platform, nor does the level of transcript
for the target protein
need to change. The modulator inhibiting function will serve as a surrogate
marker
[0417] As outlined above, screens are done to assess genes or gene products.
That is, having
identified a particular differentially expressed gene as important in a
particular state, screening
of modulators of either the expression of the gene or the gene product itself
is performed.
Use of Modulators to Affect Peptides of the Invention
[0418] Measurements of cancer polypeptide activity, or of the cancer phenotype
are
performed using a variety of assays. For example, the effects of modulators
upon the function of
a cancer polypeptide(s) are measured by examining parameters described above.
A
physiological change that affects activity is used to assess the influence of
a test compound on
the polypeptides of this invention. When the functional outcomes are
determined using intact
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cells or animals, a variety of effects can be assesses such as, in the case of
a cancer associated
with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone
release,
transcriptional changes to both known and uncharacterized genetic markers
(e.g., by Northern
blots), changes in cell metabolism such as cell growth or pH changes, and
changes in
intracellular second messengers such as cGNIP.
Methods of Identifying Characterizing Cancer-associated Sequences
104191 Expression of various gene sequences is correlated with cancer.
Accordingly,
disorders based on mutant or variant cancer genes are determined. In one
embodiment, the
invention provides methods for identifying cells containing variant cancer
genes, e.g.,
determining the presence of, all or part, the sequence of at least one
endogenous cancer gene in a
cell. This is accomplished using any number of sequencing techniques. The
invention
comprises methods of identifying the cancer genotype of an individual, e.g.,
determining all or
part of the sequence of at least one gene of the invention in the individual.
This is generally
done in at least one tissue of the individual, e.g., a tissue set forth in
Table 1, and may include the
evaluation of a number of tissues or different samples of the same tissue. The
method may
include comparing the sequence of the sequenced gene to a known cancer gene,
i.e., a wild-type
gene to determine the presence of family members, homologies, mutations or
variants. The
sequence of all or part of the gene can then be compared to the sequence of a
known cancer gene
to determine if any differences exist. This is done using any number of known
homology
programs, such as BLAST, Bestfit, etc. The presence of a difference in the
sequence between
the cancer gene of the patient and the known cancer gene correlates with a
disease state or a
propensity for a disease state, as outlined herein.
104201 In a preferred embodiment, the cancer genes are used as probes to
determine the
number of copies of the cancer gene in the genome. The cancer genes are used
as probes to
determine the chromosomal localization of the cancer genes. Information such
as chromosomal
localization finds use in providing a diagnosis or prognosis in particular
when chromosomal
abnormalities such as translocations, and the like are identified in the
cancer gene locus.
XIV.) RNAi and Therapeutic Use of Small Interfering RNA (siRNAs)
[04211 The present invention is also directed towards siRNA oligonucleotides,
particularly
double stranded RNAs encompassing at least a fragment of the PSCA coding
region or 5" UTR
regions, or complement, or any antisense oligonucleotide specific to the PSCA
sequence. In one
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embodiment such oligonucleotides are used to elucidate a function of PSCA, or
are used to
screen for or evaluate modulators of PSCA function or expression. In another
embodiment,
gene expression of PSCA is reduced by using siRNA transfection and results in
significantly
diminished proliferative capacity of transformed cancer cells that
endogenously express the
antigen; cells treated with specific PSCA siRNAs show reduced survival as
measured, e.g., by a
metabolic readout of cell viability, correlating to the reduced proliferative
capacity. Thus, PSCA
siRNA compositions comprise siRNA (double stranded RNA) that correspond to the
nucleic
acid ORF sequence of the PSCA protein or subsequences thereof; these
subsequences are
generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29,
30,31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides in length and
contain
sequences that are complementary and non-complementary to at least a portion
of the mRNA
coding sequence In a preferred embodiment, the subsequences are 19-25
nucleotides in length,
most preferably 21-23 nucleotides in length.
[0422] RNA interference is a novel approach to silencing genes in vitro and in
vivo, thus
small double stranded RNAs (siRNAs) are valuable therapeutic agents. The power
of siRNAs to
silence specific gene activities has now been brought to animal models of
disease and is used in
humans as well. For example, hydrodynamic infusion of a solution of siRNA into
a mouse with
a siRNA against a particular target has been proven to be therapeutically
effective.
[0423] The pioneering work by Song et al. indicates that one type of entirely
natural nucleic
acid, small interfering RNAs (siRNAs), served as therapeutic agents even
without further
chemical modification (Song, E., et al. "RNA interference targeting Fas
protects mice from
fulminant hepatitis" Nat. Med. 9(3): 347-51(2003)). This work provided the
first in vivo
evidence that infusion of siRNAs into an animal could alleviate disease. In
that case, the authors
gave mice injections of siRNA designed to silence the FAS protein (a cell
death receptor that
when over-activated during inflammatory response induces hepatocytes and other
cells to die).
The next day, the animals were given an antibody specific to Fas. Control mice
died of acute
liver failure within a few days, while over 80% of the siRNA-treated mice
remained free from
serious disease and survived. About 80% to 90% of their liver cells
incorporated the naked
siRNA oligonucleotides. Furthermore, the RNA molecules functioned for 10 days
before losing
effect after 3 weeks.
[0424] For use in human therapy, siRNA is delivered by efficient systems that
induce long-
lasting RNAi activity. A major caveat for clinical use is delivering siRNAs to
the appropriate
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cells. Hepatocytes seem to be particularly receptive to exogenous RNA. Today,
targets located
in the liver are attractive because liver is an organ that can be readily
targeted by nucleic acid
molecules and viral vectors. However, other tissue and organs targets are
preferred as well.
[0425] Formulations of siRNAs with compounds that promote transit across cell
membranes
are used to improve administration of siRNAs in therapy. Chemically modified
synthetic
siRNA, that are resistant to nucleases and have serum stability have
concomitant enhanced
duration of RNAi effects, are an additional embodiment.
[0426] Thus, siRNA technology is a therapeutic for human malignancy by
delivery of
siRNA molecules directed to PSCA to individuals with the cancers, such as
those listed in Table
1. Such administration of siRNAs leads to reduced growth of cancer cells
expressing PSCA,
and provides an anti-tumor therapy, lessening the morbidity and/or mortality
associated with
malignancy.
[0427] The effectiveness of this modality of gene product knockdown is
significant when
measured in vitro or in vivo. Effectiveness in vitro is readily demonstrable
through application
of siRNAs to cells in culture (as described above) or to aliquots of cancer
patient biopsies when
in vitro methods are used to detect the reduced expression of PSCA protein.
XV.) Kits/Articles of Manufacture
[0428] For use in the laboratory, prognostic, prophylactic, diagnostic and
therapeutic
applications described herein, kits are within the scope of the invention.
Such kits can comprise
a carrier, package, or container that is compartmentalized to receive one or
more containers such
as vials, tubes, and the like, each of the container(s) comprising one of the
separate elements to
be used in the method, along with a label or insert comprising instructions
for use, such as a use
described herein. For example, the container(s) can comprise a probe that is
or can be detectably
labeled. Such probe can be an antibody or polynucleotide specific for a
protein or a gene or
message of the invention, respectively. Where the method utilizes nucleic acid
hybridization to
detect the target nucleic acid, the kit can also have containers containing
nucleotide(s) for
amplification of the target nucleic acid sequence. Kits can comprise a
container comprising a
reporter, such as a biotin-binding protein, such as avidin or streptavidin,
bound to a reporter
molecule, such as an enzymatic, fluorescent, or radioisotope label; such a
reporter can be used
with, e.g., a nucleic acid or antibody. The kit can include all or part of the
amino acid sequences
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in Figure 1, Figure 2, or Figure 3 or analogs thereof, or a nucleic acid
molecule that encodes
such amino acid sequences.
[04291 The kit of the invention will typically comprise the container
described above and
one or more other containers associated therewith that comprise materials
desirable from a
commercial and user standpoint, including buffers, diluents, filters, needles,
syringes; carrier,
package, container, vial and/or tube labels listing contents and/or
instructions for use, and
package inserts with instructions for use.
10430] A label can be present on or with the container to indicate that the
composition is
used for a specific therapy or non-therapeutic application, such as a
prognostic, prophylactic,
diagnostic or laboratory application, and can also indicate directions for
either in vivo or in vitro
use, such as those described herein. Directions and or other information can
also be included on
an insert(s) or label(s) which is included with or on the kit. The label can
be on or associated
with the container. A label a can be on a container when letters, numbers or
other characters
forming the label are molded or etched into the container itself; a label can
be associated with a
container when it is present within a receptacle or carrier that also holds
the container, e.g., as a
package insert. The label can indicate that the composition is used for
diagnosing, treating,
prophylaxing or prognosing a condition, such as a neoplasia of a tissue set
forth in Table I.
[04311 The terms "kit" and "article of manufacture" can be used as synonyms.
[04321 In another embodiment of the invention, an article(s) of manufacture
containing
compositions, such as amino acid sequence(s), small molecule(s), nucleic acid
sequence(s),
and/or antibody(s), e.g., materials useful for the diagnosis, prognosis,
prophylaxis and/or
treatment of neoplasias of tissues such as those set forth in Table I is
provided. The article of
manufacture typically comprises at least one container and at least one label.
Suitable containers
include, for example, bottles, vials, syringes, and test tubes. The containers
can be formed from
a variety of materials such as glass, metal or plastic. The container can hold
amino acid
sequence(s), small molecule(s), nucleic acid sequence(s), cell population(s)
and/or antibody(s).
In one embodiment, the container holds a polynucleotide for use in examining
the mRNA
expression profile of a cell, together with reagents used for this purpose. In
another embodiment
a container comprises an antibody, binding fragment thereof or specific
binding protein for use
in evaluating protein expression of PSCA in cells and tissues, or for relevant
laboratory,
prognostic, diagnostic, prophylactic and therapeutic purposes; indications
and/or directions for
such uses can be included on or with such container, as can reagents and other
compositions or
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tools used for these purposes. In another embodiment, a container comprises
materials for
eliciting a cellular or humoral immune response, together with associated
indications and/or
directions. In another embodiment, a container comprises materials for
adoptive
immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL),
together with
associated indications and/or directions; reagents and other compositions or
tools used for such
purpose can also be included.
[0433] The container can alternatively hold a composition that is effective
for treating,
diagnosis, prognosing or prophylaxing a condition and can have a sterile
access port (for
example the container can be an intravenous solution bag or a vial having a
stopper pierceable
by a hypodermic injection needle). The active agents in the composition can be
an antibody
capable of specifically binding PSCA and modulating the function of PSCA.
[0434] The article of manufacture can further comprise a second container
comprising a
pharmaceutical] y-acceptable buffer, such as phosphate-buffered saline,
Ringer's solution and/or
dextrose solution. It can further include other materials desirable from a
commercial and user
standpoint, including other buffers, diluents, filters, stirrers, needles,
syringes, and/or package
inserts with indications and/or instructions for use.
EXAMPLES:
[0435] Various aspects of the invention are further described and illustrated
by way of the
several examples that follow, none of which is intended to limit the scope of
the invention.
Example 1
Expression Analysis of PSCA Variants in Normal Tissues and Patient Specimens
[0436] Previously, PSCA, herein referred to as PSCA v.1, was identified as an
antigen
expressed in prostate cancer. Its expression was detected in greater than 80%
of primary
prostate cancers and in the majority of prostate metastasis. It has also been
shown to be
expressed in bladder cancer, ovary cancer, and pancreatic cancer; these
cancers are listed in
Table I. By immunohistochemical analysis, PSCA has been shown to be
overexpressed on the
cell surface of most urothelial transitional carcinoma, and in 60% of primary
pancreatic
adenocarcinomas. The PSCA expression data has been reported in patent
publications
(PCT/US98/04664, PCT/US/28883, PCT/US00/19967) and in peer-reviewed articles
(Saffran
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et al., Proc Nat] Acad Sci U S A. 2001 Feb 27; 98(5): 2658-2663; Amara et al.,
Cancer Res.
2001 Jun 15; 61(12): 4660-65; Reiter et al., Proc Nat! Acad Sci USA. 1998 Feb
17; 95(4): 1735-
40; Argani et al., Cancer Res. 2001 Jun 1; 61(11): 4320-24).
[0437] Specific expression of different PSCA variants is studied in normal and
cancer
patient specimens. Primers were designed to differentiate between PSCA
v.1/v.2/v.4, PSCA v.3
and PSCA v.5. PSCA v.1/v.2/v.4 lead to a PCR product of 425 bp, PSCA v.3 leads
to a PCR
product of 300 bp, whereas PSCA v.5 leads to a PCR product of 910 bp in size
(Figure I I(a).
[04381 First strand cDNA was prepared from normal bladder, brain, heart,
kidney, liver,
lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach,
pools of prostate cancer,
bladder cancer, kidney cancer, colon cancer, lung cancer, ovary cancer, breast
cancer, cancer
metastasis, and pancreas cancer (Figure 11(b). Normalization was performed by
PCR using
primers to actin. Semi-quantitative PCR, using the variant specific primers
was performed at 30
cycles of amplification.
104391 Results show expression of PSCA v.5 mainly in breast cancer, cancer
metastasis, and
pancreas cancer, and at lower level in colon cancer and lung cancer. PSCA
v.l/v.2/v.4 PCR
product was detected in prostate cancer, bladder cancer, kidney cancer, colon
cancer, lung
cancer, ovary cancer, breast cancer, cancer metastasis, and pancreas cancer.
Amongst normal
tissues, PSCA v.l/v.2/v.4 PCR product was detected only in prostate, stomach
and at lower level
in kidney and lung, whereas PSCA v. 5 was not detected in any normal tissue.
PSCA v.3 PCR
detected product was not detected in any of the samples tested.
[0440] Primers were designed to differentiate between PSCA v.4 and PSCA v.5
(Figure
IJ(a). PSCA v.4 lead to a PCR product of 460 bp, whereas PSCA v.5 lead to a
PCR product of
945 bp in size.
[04411 First strand cDNA was prepared from normal bladder, brain, heart,
kidney, liver,
lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach,
pools of prostate cancer,
bladder cancer, and multi-xenograft pool (prostate cancer, kidney cancer and
bladder cancer
xenografts) (Figure IJ(b). Normalization was performed by PCR using primers to
actin. Semi-
quantitative PCR, using the variant specific primers was performed at 30
cycles of amplification.
[04421 Results show expression of PSCA v.4 in prostate cancer, bladder cancer,
and multi-
xenograft pool, normal kidney and prostate. PSCA v.5 was detected only in
normal prostate and
bladder cancer.
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[0443] The restricted expression of PSCA variants in normal tissues and the
expression
detected in cancer patient specimens indicate that PSCA variants are
therapeutic, prognostic,
laboratory, prophylactic, and diagnostic targets for human cancers.
Example 2
Splice Variants of PSCA
[0444] As used herein, the term variant includes transcript variants and
single nucleotide
polymorphisms (SNPs). Transcript variants are variants of mature mRNA from the
same gene
which arise by alternative transcription or alternative splicing. Alternative
transcripts are
transcripts from the same gene but start transcription at different points.
Splice variants are
mRNA variants spliced differently from the same transcript. In eukaryotes,
when a multi-exon
gene is transcribed from genomic DNA, the initial RNA is spliced to produce
functional mRNA,
which has only exons and is used for translation into an amino acid sequence.
Accordingly, a
given gene can have zero to many alternative transcripts and each transcript
can have zero to
many splice variants. Each transcript variant has a unique exon makeup, and
can have different
coding and/or non-coding (5' or 3' end) portions, from the original
transcript. Transcript
variants can code for the same, similar or different proteins, such proteins
having the same or a
similar function or a different function. The variant proteins can be
expressed in the same tissue
at the same time, in a different tissue at the same time, or in the same
tissue at different times, or
in a different tissue at a different time. Proteins encoded by a transcript
variant can have similar
or different subcellular or extracellular localizations (e.g., secreted versus
intracellular).
[0445] Transcript variants are identified by a variety of art-accepted
methods. For example,
alternative transcripts and splice variants are identified by full-length
cloning, 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 sequence(s) or other full-length sequences. Each
consensus
sequence is a potential splice variant for that gene. Several confirmation
modalities are known
in the art, such as identification of the variant by Northern analysis, full
length cloning or by use
of probe libraries, etc.. Even when a variant is identified that is not yet a
full-length clone, that
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portion of the variant is very useful as a research tool, e.g., for antigen
generation or for further
cloning of the full-length splice variant, using techniques known in the art.
[04461 Moreover, computer programs are available in the art that identify
transcript variants
based on genomic sequences. Genomic-based transcript variant identification
programs include
FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in Drosophila
genomic DNA,"
Genome Research. 2000 April; 10(4):516-22); Grail (URL compbio.ornl.gov/Grail-
bin/EmptyGrailForm) and GenScan (URL 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.
[04471 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: Wellmann S, et al., Specific
reverse
transcription-PCR quantification of vascular endothelial growth factor (VEGF)
splice variants
by LightCycler technology, Clin Chem. 2001 Apr;47(4):654-60; Jia, H.P., et
al., Discovery of
new human beta-defensins using a genomics-based approach, Gene. 2001 Jan 24;
263(1-2):211-
8. For PCR-based and 5' RACE Validation: Brigle, K.E., et al., Organization of
the murine
reduced folate carrier gene and identification of variant splice forms,
Biochem Biophys Acta.
1997 Aug 7; 1353(2): 191-8).
104481 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 PSCA
has a particular expression profile related to cancer (see, e.g., Table I).
Alternative transcripts
and splice variants of PSCA are also involved in cancers, for example in one
or more of these
tissues and in certain additional tissues as well. The variants thus serve as
tumor-associated
markers/antigens.
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(0449] Using the full-length PSCA gene together with EST sequences, four
additional
transcript variants were identified, designated as PSCA v.2, v.3, v.4, and
v.5. The boundaries of
exons in the original transcript, PSCA v.1 were shown in Table VI. The
sequences for PSCA
and the PSCA variants are set forth in Figure 1.
Example 3
Single Nucleotide Polymorphisms of PSCA
[0450] A Single Nucleotide Polymorphism (SNP) is a single base pair variation
in a
nucleotide sequence at a specific location. At any given point of the genome,
there are four
possible nucleotide base pairs: A/T, C/G, G/C, and T/A. As used herein, an
allele is one of a
series of alternative forms of a given gene, differing in DNA sequence, and
affecting a product
(RNA and/or protein).
10451] A SNP that occurs on a cDNA is called a cSNP. This cSNP may change
amino acids
of the protein encoded by the gene and thus change the function of the
protein. Some SNPs
cause inherited diseases; others contribute to quantitative variations in
phenotype and reactions
to environmental factors including diet and drugs among individuals.
Therefore, the existence
of a SNP and/or combinations of alleles (called haplotypes) have many useful
applications, such
as diagnosis of inherited diseases, determination of drug reactions and
dosage, identification of
genes responsible for diseases, and analysis of the genetic relationship
between individuals (P.
Nowotny, J. M. Kwon and A. M. Goate, " SNP analysis to dissect human traits,"
Curr. Opin.
Neurobiol. 2001 Oct; 11(5):637-641; M. Pirmohamed and B. K. Park, "Genetic
susceptibility to
adverse drug reactions," Trends Pharmacol. Sci. 2001 Jun; 22(6):298-305; J. H.
Riley, C. J.
Allan, E. Lai and A. Roses, "The use of single nucleotide polymorphisms in the
isolation of
common disease genes," Pharmacogenomics. 2000 Feb; 1(l):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).
[0452] SNPs are identified by a variety of art-accepted methods (P. Bean, "The
promising
voyage of SNP target discovery," Am. Clin. Lab. 2001 Oct-Nov; 20(9):18-20; K.
M. Weiss, "In
search of human variation," Genome Res. 1998 Jul; 8(7):691-697; M. M. She,
"Enabling large-
scale pharmacogenetic studies by high-throughput mutation detection and
genotyping
technologies," Clin. Chem. 2001 Feb; 47(2):164-172). For example, SNPs are
identified by
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sequencing DNA fragments that show polymorphism by gel-based methods such as
restriction
fragment length polymorphism (RFLP) and denaturing gradient gel
electrophoresis (DGGE).
They are also 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 also
discovers 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 the genotype or haplotype of an individual can be
determined by a variety of
methods including direct sequencing and high throughput microarrays (P. Y.
Kwok, "Methods
for genotyping single nucleotide polymorphisms," Annu. Rev. Genomics Hum.
Genet. 2001;
2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B.
Hines and A.
Duesterhoeft, "High-throughput SNP genotyping with the Masscode system," Mol.
Diagn. 2000
Dec; 5(4):329-340).
[0453] Using the methods described above, thirteen SNP were identified in the
transcript for
PSCA v.2. Variant 2 was used, rather than for example variant 1, as it had
fewer ambiguous
bases than variant 1. Accordingly, SNPs were identified in PSCA v.2, at
positions 57 (t/c), 367
(c/t), 424 (a/c), 495 (c/g), 499 (c/t), 563 (c/t), 567 (g/a), 627 (g/a), 634
(t/g), 835 (g/a), 847 (g/a),
878 (g/a), and 978 (c/g). The transcripts or proteins with alternative alleles
were designated as
variant PSCA v.6 through v.18, as shown in Figure 1B and Figure 1G.
[0454] The nucleotide change in v.6 changed the start codon of v.1 and thus,
the translation
would not start until the next ATG (AUG in mRNA), resulting in a protein 9 AA
shorter than
v.1 protein. The nucleotide changes for v.7 and v.8 were silent at the protein
level.
[0455] Twelve of these 13 SNPs were also present in variant 4. The 12 SNP
variants
relative to PSCA v. 4 are designated PSCA v. 19 through v.30. Variants 19
through 27 encode
alternative amino acids as shown in Figure 1H.
Example 4
Production of Recombinant PSCA in Prokaryotic Systems
[04561 To express recombinant PSCA and PSCA variants in prokaryotic cells, the
full or
partial length PSCA and PSCA 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
PSCA variants are
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expressed: the full length sequence presented in Figure 1, 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 PSCA,
variants, or analogs thereof.
A. In vitro transcription and translation constructs:
[0457] pCRII: To generate PSCA 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 PSCA cDNA. The pCRII vector has Sp6 and T7 promoters flanking
the insert
to drive the transcription of PSCA RNA for use as probes in RNA in situ
hybridization
experiments. These probes are used to analyze the cell and tissue expression
of PSCA at the
RNA level. Transcribed PSCA RNA representing the eDNA amino acid coding region
of the
PSCA gene is used in in vitro translation systems such as the TnTTM Coupled
Reticulolysate
System (Promega, Corp., Madison, WI) to synthesize PSCA protein.
B. Bacterial Constructs:
[0458] pGEX Constructs: To generate recombinant PSCA proteins in bacteria that
are fused
to the Glutathione S-transferase (GST) protein, all or parts of the PSCA cDNA
protein coding
sequence are cloned into the pGEX family of GST-fusion vectors (Amersham
Pharmacia
Biotech, Piscataway, NJ). These constructs allow controlled expression of
recombinant PSCA
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-1, maybe
employed such that it permits cleavage of the GST tag from PSCA-related
protein. The
ampicillin resistance gene and pBR322 origin permits selection and maintenance
of the pGEX
plasmids in E. coli.
[0459] pMAL Constructs: To generate, in bacteria, recombinant PSCA proteins
that are
fused to maltose-binding protein (MBP), all or parts of the PSCA 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 PSCA 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
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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 PSCA. 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.
[0460] pET Constructs: To express PSCA in bacterial cells, all or parts of the
PSCA cDNA
protein coding sequence are cloned into the pET family of vectors (Novagen,
Madison, WI).
These vectors allow tightly controlled expression of recombinant PSCA 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 PSCA protein are expressed as amino-terminal fusions to NusA.
C. Yeast Constructs:
[0461] pESC Constructs: To express PSCA in the yeast species Saccharomyces
cerevisiae
for generation of recombinant protein and functional studies, all or parts of
the PSCA cDNA
protein coding sequence are cloned into the pESC family of vectors each of
which contain I of 4
selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, CA).
These vectors
allow controlled expression from the same plasmid of up to 2 different genes
or cloned
sequences containing either F1agTM or Myc epitope tags in the same yeast cell.
This system is
useful to confirm protein-protein interactions of PSCA. In addition,
expression in yeast yields
similar post-translational modifications, such as glycosylations and
phosphorylations, that are
found when expressed in eukaryotic cells.
[0462] pESP Constructs: To express PSCA in the yeast species Saccharomyces
pombe, all
or parts of the PSCA cDNA protein coding sequence are cloned into the pESP
family of vectors.
These vectors allow controlled high level of expression of a PSCA protein
sequence that is fused
at either the amino terminus or at the carboxyl terminus to GST which aids
purification of the
recombinant protein. A FIagTM epitope tag allows detection of the recombinant
protein with
anti- FIagTM antibody.
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Example 5
Production of Recombinant PSCA in Higher Eukaryotic Systems
A. Mammalian Constructs:
[04631 To express recombinant PSCA in eukaryotic cells, the full or partial
length PSCA
cDNA sequences, or variants thereof, can be cloned into any one of a variety
of expression
vectors known in the art. One or more of the following regions of PSCA are
expressed in these
constructs, amino acids I to 123, 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 PSCA v.1,
PSCA variants, or
analogs thereof.
[04641 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-
PSCA polyclonal
serum, described herein.
[04651 pcDNA4/HisMax Constructs: To express PSCA in mammalian cells, a PSCA
ORF,
or portions thereof, of PSCA are cloned into pcDNA4/HisMax Version A
(Invitrogen, Carlsbad,
CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and
the SP 16
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.
[04661 pcDNA3.1/MycHis Constructs: To express PSCA in mammalian cells, a PSCA
ORF, or portions thereof, of PSCA with a consensus Kozak translation
initiation site was cloned
into peDNA3.I/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression
is driven
from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc
epitope
and 6X His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector
also
contains the bovine growth hormone (BGH) polyadenylation signal and
transcription
termination sequence to enhance mRNA stability, along with the SV40 origin for
episomal
replication and simple vector rescue in cell lines expressing the large T
antigen. The Neomycin
resistance gene can be used, as it allows for selection of mammalian cells
expressing the protein
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and the ampicillin resistance gene and CoIEI origin permits selection and
maintenance of the
plasmid in E. coli.
[04671 pcDNA3.1/CT-GFP-TOPO Construct: To express PSCA in mammalian cells and
to
allow detection of the recombinant proteins using fluorescence, a PSCA ORF, or
portions
thereof, with a consensus Kozak translation initiation site are cloned into
pcDNA3.1/CT-GFP-
TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus
(CMV)
promoter. The recombinant proteins have the Green Fluorescent Protein (GFP)
fused to the
carboxyl-terminus facilitating non-invasive, in vivo detection and cell
biology studies. The
pcDNA3.1 CT-GFP-TOPO vector also contains the bovine growth hormone (BGH)
polyadenylation signal and transcription termination sequence to enhance mRNA
stability along
with the SV40 origin for episomal replication and simple vector rescue in cell
lines expressing
the large T antigen. The Neomycin resistance gene allows for selection of
mammalian cells that
express the protein, and the ampicillin resistance gene and CoIEl 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 PSCA protein.
104681 PAPtag: A PSCA 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 PSCA 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 PSCA protein. The resulting
recombinant PSCA
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 PSCA
proteins. Protein
expression is driven from the CMV promoter and the recombinant proteins also
contain myc and
6X His epitopes fused at the carboxyl-terminus that facilitates detection and
purification. The
Zeocin resistance gene present in the vector allows for selection of mammalian
cells expressing
the recombinant protein and the ampicillin resistance gene permits selection
of the plasmid in
E. coll.
[0469] tp ag5: A PSCA ORF, or portions thereof, was cloned into pTag-5. This
vector is
similar to pAPtag but without the alkaline phosphatase fusion. This construct
generates PSCA
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
PSCA protein is optimized for secretion into the media of transfected
mammalian cells, and is
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used as immunogen or ligand to identify proteins such as ligands or receptors
that interact with
the PSCA proteins. Protein expression is driven from the CMV promoter. The
Zeocin
resistance gene present in the vector allows for selection of mammalian cells
expressing the
protein, and the ampicillin resistance gene permits selection of the plasmid
in E. coll.
104701 PsecFc: A PSCA ORF, or portions thereof, was- cloned into psecFc. The
psecFc
vector was assembled by cloning the human immunoglobulin GI (IgG) Fe (hinge,
CH2, CH3
regions) into pSecTag2 (Invitrogen, California). This construct generates an
IgGl Fc fusion at
the carboxyl-terminus of the PSCA proteins, while fusing the IgGK signal
sequence to N-
terminus. PSCA fusions utilizing the murine IgGI Fc region are also used. The
resulting
recombinant PSCA 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 PSCA protein. Protein expression is driven from
the CMV promoter.
The hygromycin resistance gene present in the vector allows for selection of
mammalian cells
that express the recombinant protein, and the ampicillin resistance gene
permits selection of the
plasmid in E. coll.
[04711 Figure 8 shows expression and purification of PSCA.psecFc protein from
293T cells.
[04721 pSRa Constructs: To generate mammalian cell lines that express PSCA
constitutively, PSCA ORF, or portions thereof, of PSCA were cloned into pSRa
constructs.
Amphotropic and ecotropic retroviruses were generated by transfection of pSRa
constructs into
the 293T-I OA1 packaging line or co-transfection of pSRa and a helper plasmid
(containing
deleted packaging sequences) into the 293 cells, respectively. The retrovirus
is used to infect a
variety of mammalian cell lines, resulting in the integration of the cloned
gene, PSCA, 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. coll. 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.
[04731 Figure 6 shows expression of PSCA in recombinant murine, rat and human
cell lines
using the PSCA.pSRa construct. The indicated murine, rat, and human cell lines
were infected
with retrovirus carrying the human PSCA cDNA and a neomycin resistance gene or
with only
the neomycin resistance gene. Stable recombinant cell lines were created by
G41 8 drug
selection. PSCA expression was determined by FACS staining with the I G8 anti-
PSCA MAb
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(5 ug/ml). Shown is the FACS profile of each cell line showing a fluorescent
shift only in the
PSCA infected line indicating cell surface PSCA expression. These lines are
useful in MAb
development as immunogens, MAb screening reagents, and in functional assays.
[04741 Additional pSRa constructs are made that fuse an epitope tag such as
the
FLAGTM tag to the carboxyl-terminus of PSCA 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:76) 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 PSCA proteins.
[0475] Additional Viral Vectors: Additional constructs are made for viral-
mediated delivery
and expression of PSCA. High virus titer leading to high level expression of
PSCA is achieved
in viral delivery systems such as adenoviral vectors and herpes amplicon
vectors. A PSCA
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,
PSCA 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.
[0476] Regulated Expression Systems: To control expression of PSCA in
mammalian cells,
coding sequences of PSCA, 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 PSCA. These vectors are
thereafter used to
control expression of PSCA in various cell lines such as PC3, NIH 3T3, 293 or
rat-1 cells.
B. Baculovirus Expression Systems
[0477] To generate recombinant PSCA proteins in a baculovirus expression
system, PSCA
ORF, or portions thereof, are cloned into the baculovirus transfer vector
pB]ueBac 4.5
(Invitrogen), which provides a His-tag at the N-terminus. Specifically,
pBlueBac-PSCA 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.
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[0478] Recombinant PSCA protein is then generated by infection of HighFive
insect cells
(Invitrogen) with purified baculovirus. Recombinant PSCA protein can be
detected using anti-
PSCA or anti-His-tag antibody. PSCA protein can be purified and used in
various cell-based
assays or as immunogen to generate polyclonal and monoclonal antibodies
specific for PSCA.
C. Expression Vectors for PSCA Orthologs
[0479] Mouse and monkey orthologs of PSCA were 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. These vectors allow expression of PSCA orthologs to
assay cross-
reactivity of monoclonal anti-human PSCA antibodies.
[0480] Mouse and monkey orthologs of PSCA were also cloned into pSRa
constructs. The
pSRa constructs allow for the generation of mammalian cell lines that express
PSCA orthologs
constitutively. 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.
These vectors allow expression of PSCA orthologs to assay cross-reactivity of
monoclonal anti-
human PSCA antibodies and to study functional activity of PSCA orthologs.
Amphotropic and
ecotropic retroviruses were generated by transfection of pSRa constructs into
the 293T-10A1
packaging line or co-transfection of pSRa and a helper plasmid (containing
deleted packaging
sequences) into the 293 cells, respectively. The retrovirus is used to infect
a variety of
mammalian cell lines, resulting in the integration of the cloned gene, PSCA
ortholog, into the
host cell-lines.
[0481] Figure 7 shows expression of mouse and simian PSCA.pcDNA3.1/MycHis
following
transfection into 293T cells. 293T cells were transfected with either mouse
PSCA.pcDNA3.1/MycHis or simian PSCA.pcDNA3.1/MycHis or pcDNA3.1/MycHis vector
control. Forty hours later, cells were collected and analyzed by flow
cytometry using anti-PSCA
monoclonal antibodies.
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Example 6
Anti eg nicity Profiles and Secondary Structure
[04821 Amino acid profiles of PSCA variants 1, 3, and 4, were found accessing
the
ProtScale website on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl)
on the ExPasy
molecular biology server.
[04831 These profiles: Hydrophilicity, (Hopp T.P., Woods K.R., 1981. Proc.
Natl. Acad. Sci.
U.S.A. 78:3824-3828); Hydropathicity, (Kyte J., Doolittle R.F., 1982. J. Mol.
Biol. 157:105-
132); Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492);
Average Flexibility,
(Bhaskaran R., and Ponnuswamy P.K., 1988. Int. J. Pept. Protein Res. 32:242-
255); Beta-turn
(Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally
others available in
the art, such as on the ProtScale website, were used to identify antigenic
regions of each of the
PSCA variant proteins. Each of the above amino acid profiles of PSCA variants
were generated
using the following ProtScale parameters for analysis: 1) A window size of 9;
2) 100% weight
of the window edges compared to the window center; and, 3) amino acid profile
values
normalized to lie between 0 and 1.
[0484 Hydrophilicity, Hydropathicity, and Percentage Accessible Residues
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.
104851 Average Flexibility and Beta-turn 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.
104861 Antigenic sequences of the PSCA variant proteins indicated, e.g., by
the profiles
described above are used to prepare immunogens, either peptides or nucleic
acids that encode
them, to generate therapeutic and diagnostic anti-PSCA 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
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the PSCA protein variants listed in Figure 1 of which the amino acid profiles
can be inferred
because the variant contains sequence that is the same as a variant depicted.
In particular,
peptide immunogens of the invention can comprise, a peptide region of at least
5 amino acids of
Figure 1 in any whole number increment that includes an amino acid position
having a value
greater than 0.5 in the Hydrophilicity profile; a peptide region of at least 5
amino acids of Figure
1 in any whole number increment that includes an amino acid position having a
value less than
0.5 in the Hydropathicity profile; a peptide region of at least 5 amino acids
of Figure 1 in any
whole number increment that includes an amino acid position having a value
greater than 0.5 in
the Percent Accessible Residues profiles; a peptide region of at least 5 amino
acids of Figures 1
in any whole number increment that includes an amino acid position having a
value greater than
0.5 in the Average Flexibility profiles; and, a peptide region of at least 5
amino acids of Figure 1
in any whole number increment that includes an amino acid position having a
value greater than
0.5 in the Beta-turn profile. Peptide immunogens of the invention can also
comprise nucleic
acids that encode any of the forgoing.
[0487] 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.
[0488] The secondary structure of PSCA protein variants 1, 3, 4, and 6, namely
the predicted
presence and location of alpha helices, extended strands, and random coils, is
predicted from the
primary amino acid sequence using the HNN - Hierarchical Neural Network method
(NPS@:
Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150
Combet C.,
Blanchet C., Geourjon C. and Deleage G., http://pbil.ibcp.fr/cgi-
bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular
biology server
located on the World Wide Web at (.expasy.ch/tools/). The analysis indicates
that PSCA variant
1 is composed of 30.89% alpha helix, 21.95% extended strand, and 47.15% random
coil. PSCA
protein variant 3 is composed of 14.89% alpha helix, 8.51 % extended strand,
and 76.60%
random coil. PSCA protein variant 4 is composed of 9.52% alpha helix, 8.99%
extended strand,
and 81.48% random coil. PSCA protein variant 6 is composed of 24.56% alpha
helix, 21.93%
extended strand, and 53.51% random coil.
[04891 Analysis for the potential presence of transmembrane domains in the
PSCA variant
proteins was carried out using a variety of transmembrane prediction
algorithms accessed from
the ExPasy molecular biology server located on the World Wide Web at
(.expasy.ch/tools/).
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Example 7
Generation of PSCA Polyclonal Antibodies
[0490] Polyclonal antibodies can be raised in a mammal, for example, by one or
more
injections of an immunizing agent and, if desired, an adjuvant. Typically, the
immunizing agent
and/or adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal
injections. In addition to immunizing with a full length PSCA protein variant,
computer
algorithms are employed in design of immunogens that, based on amino acid
sequence analysis
contain characteristics of being antigenic and available for recognition by
the immune system of
the immunized host (see the Example entitled "Antigenicity Profiles and
Secondary Structure").
Such regions would be predicted to be hydrophilic, flexible, in beta-turn
conformations, and be
exposed on the surface of the protein.
[0491] For example, recombinant bacterial fusion proteins or peptides
containing
hydrophilic, flexible, beta-turn regions of PSCA protein variants are used as
antigens to generate
polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as
described in
the Example entitled "Generation of PSCA Monoclonal Antibodies (MAbs)". For
example, in
PSCA variant 1, such regions include, but are not limited to, amino acids 28-
56 and amino acids
66-94. For variant 3, such regions include, but are not limited to, amino
acids 7-39 and amino
acids 70-94. For variant 4 such regions include, but are not limited to, amino
acids 6-18, amino
acids 27-39, amino acids 103-133, and 177-189. For variant 6, such regions
include, but are not
limited to, amino acids 19-35 and amino acids 57-85. It is useful to conjugate
the immunizing
agent to a protein known to be immunogenic in the mammal being immunized.
Examples of
such immunogenic proteins include, but are not limited to, keyhole limpet
hemocyanin (KLH),
serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one
embodiment, a
peptide encoding amino acids 103-133 of PSCA variant 4 is conjugated to KLH
and used to
immunize a rabbit. Alternatively the immunizing agent may include all or
portions of the PSCA
variant proteins, analogs or fusion proteins thereof. For example, the PSCA
variants amino acid
sequences can be fused using recombinant DNA techniques to any one of a
variety of fusion
protein partners that are well known in the art, such as glutathione-S-
transferase (GST) and HIS
tagged fusion proteins. In one embodiment, the PSCA variant I sequence, amino
acids 18-98
was fused to GST using recombinant techniques in the pGEX expression vector,
expressed,
purified and used to immunize both rabbits and mice to generate polyclonal and
monoclonal
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antibodies respectively. Such fusion proteins are purified from induced
bacteria using the
appropriate affinity matrix.
104921 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 PSCA 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).
[04931 In addition to bacterial derived fusion proteins, mammalian expressed
protein
antigens are also used. These antigens are expressed from mammalian expression
vectors such
as the Tag5 and Fe-fusion vectors (see the section entitled "Production of
Recombinant PSCA in
Eukaryotic Systems"), and retain post-translational modifications such as
glycosylations found
in native protein. In one embodiment, the eDNA of PSCA variant 1, minus the N-
terminal
leader peptide and C-terminal GPI anchor was cloned into the Tag5 mammalian
secretion
vector, and expressed in 293T cells. The recombinant protein was purified by
metal chelate
chromatography from tissue culture supernatants of 293T cells stably
expressing the
recombinant vector. The purified Tag5 PSCA protein was then used as immunogen.
104941 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).
104951 In a typical protocol, rabbits are initially immunized subcutaneously
with up to
200 g, typically 100-200 g, of fusion protein or peptide conjugated to KLH
mixed in
complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously
every two weeks
with up to 200 g, typically 100-200 g, of the immunogen in incomplete
Freund's adjuvant
(IFA). Test bleeds are taken approximately 7-10 days following each
immunization and used to
monitor the titer of the antiserum by ELISA.
[04961 To test reactivity and specificity of immune serum, such as rabbit
serum derived from
immunization with a GST-fusion of PSCA variant 3 or 4 protein, the respective
full-length
PSCA variant eDNA is cloned into pCDNA 3.1 myc-his expression vector
(Invitrogen, see the
Example entitled "Production of Recombinant PSCA in Eukaryotic Systems").
After
transfection of the constructs into 293T cells, cell lysates are probed with
the anti-variant serum
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and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to
determine specific
reactivity to denatured variant protein using the Western blot technique. In
addition, the
immune serum is tested by fluorescence microscopy, flow cytometry and
immunoprecipitation
against 293T and other recombinant PSCA variant-expressing cells to determine
specific
recognition of native protein. Western blot, immunoprecipitation, fluorescent
microscopy, and
flow cytometric techniques using cells that endogenously express PSCA are also
carried out to
test reactivity and specificity.
[04971 Anti-serum from rabbits immunized with PSCA variant fusion proteins,
such as GST
and MBP fusion proteins, are purified by depletion of antibodies reactive to
the fusion partner
sequence by passage over an affinity column containing the fusion partner
either alone or in the
context of an irrelevant fusion protein. For example, antiserum derived from a
GST-PSCA
variant I fusion protein 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-PSCA fusion protein covalently coupled
to Affigel
matrix. The serum is then further purified by protein G affinity
chromatography to isolate the
IgG fraction. Sera from other His-tagged antigens and peptide immunized
rabbits as well as
fusion partner depleted sera are affinity purified by passage over a column
matrix composed of
the original protein immunogen or free peptide.
Example 8
Generation of PSCA Monoclonal Antibodies (MAbs)
[0498] In one embodiment, therapeutic Monoclonal Antibodies ("MAbs") to PSCA
and
PSCA variants comprise those that react with epitopes specific for each
protein or specific to
sequences in common between the variants that would bind, internalize, disrupt
or modulate the
biological function of PSCA or PSCA 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 extracellular domain or the
entire PSCA protein
sequence, regions predicted to contain functional motifs, and regions of the
PSCA protein
variants predicted to be antigenic from computer analysis of the amino acid
sequence.
Immunogens include peptides, recombinant bacterial proteins such as GST-PSCA
fusion
proteins (Figure 8) and His tagged PSCA pET vector protein (Figure 6) and
mammalian
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expressed purified His tagged proteins (Figure 7) and human and murine IgG FC
fusion
proteins. In addition, Cells engineered through retroviral transduction to
express high levels of
PSCA variant 1, such as RATI-PSCA. 293T-PSCA, 3T3-PSCA or 300.19-PSCA are used
to
immunize mice (Figure 5).
[04991 To generate Monoclonal Antibodies to PSCA, mice were first immunized in
the foot
pad (FP) with, typically, 5-50 pg of protein immunogen or between 106 and 107
PSCA-
expressing cells mixed in a suitable adjuvant. Examples of suitable adjuvants
for FP
immunizations are TiterMax (Sigma) for the initial FP injection followed by
alum gel with
Immuneasy (Qiagen). Following the initial injection mice were subsequently
immunized twice
a week until the time they are sacrificed and B cells obtained from the lymph
node used for
fusion.
[05001 During the immunization protocol, test bleeds were taken to monitor the
titer and
specificity of the immune response. In most cases, once appropriate reactivity
and specificity
was obtained as determined by ELISA, Western blotting, immunoprecipitation,
fluorescence
microscopy or flow cytometric analyses, fusion and hybridoma generation was
then carried out
using electrocell fusion (BTX, ECM2000).
105011 In one embodiment, the invention provides for monoclonal antibodies
designated,
Hal-1.16, Hal-5.99, Hal -4.117, Hal-4.120, Hal-4.121, and Hal-4.37. The
antibodies were
identified and are shown to react and bind with cell surface or immobilized
PSCA.
105021 MAbs to PSCA were generated using XenoMouse technology wherein the
murine
heavy and kappa light chain loci have been inactivated and a majority of the
human heavy and
kappa light chain immunoglobulin loci have been inserted: Hal-1.16 was
generated after
immunizing human gamma 1 producing Xenomice (13 times with PSCA-GST); Hal-5.99
was
generated after immunizing human gamma 2 producing Xenomice 6 times with Ratl-
PSCA
cells followed by two injections with PSCA-tag5; Hal-4.117, HAI-4.37, Hal-
4.120, and Hal-
4.121 were generated after immunizing human gamma I producing Xenomice) 6
times with
Rat] -PSCA cells followed by 4 injections with PSCA-tag5. The anti-PSCA MAbs,
Hal-1.16,
Hal-5.99, Hal-4.117, Hal-4.120, and Hal-4.121 bind endogenous cell surface
PSCA expressed
in prostate cancer xenograft cells.
[05031 The antibodies designated Hal-5.99, Hal-4.117, Hal-4.120, Hal-4.37, Hal-
1.16 and
Hal-4.121 were sent (via Federal Express) to the American Type Culture
Collection (ATCC),
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P.O. Box 1549, Manassas, VA 20108 on 04-May-2004 and assigned Accession
numbers PTA-
6703 and PTA-6699 and PTA-6700 and PTA-6702 and PTA-6698 and PTA-6701
respectively.
[0504] DNA coding sequences for anti PSCA MAbs Hal-1.16, Hal-5.99, Hal-4.117,
Hal-
4.120, Hal-4.121, and Hal-4.37, were determined after isolating mRNA from the
respective
hybridoma cells with Trizol reagent (Life Technologies, Gibco BRL). Total RNA
was purified
and quantified. First strand cDNAs was generated from total RNA with oligo
(dT)12 18
priming using the Gibco BRL Superscript Preamplification system. First strand
cDNA was
amplified using human immunoglobulin variable heavy chain primers, and human
immunoglobulin variable light chain primers. PCR products were cloned into the
pCRScript
vector (Stratagene, La Jolla). Several clones were sequenced and the variable
heavy and light
chain regions determined. The nucleic acid and amino acid sequences of the
variable heavy and
light chain regions are listed in Figure 2 and Figure 3. Alignment of PSCA
antibodies to
germline V-D-J Sequences is shown in Figure 4A - Figure 4M.
Example 9
Screening and Identification of PSCA Antibodies
[0505] Antibodies generated using the procedures set forth in the example
entitled
"Generation of PSCA Monoclonal Antibodies (MAbs)" were screened and identified
using a
combination of assays including ELISA, FACS, epitope grouping, and affinity
for PSCA
expressed on the cell surface.
A. PSCA human MAb screening by FACS.
[0506] Primary hybridoma screening for MAbs toPSCA was performed by FACS. The
protocol was as follows: 50ul/well of hybridoma supernatant (neat) or purified
antibodies (in
serial dilutions) were added to 96-well FACS plates and mixed with PSCA-
expressing cells
(endogenous or recombinant, 50,000 cells/well). The mixture was incubated at 4
C for two
hours. At the end of incubation, the cells were washed with FACS Buffer and
incubated with
100ul of detection antibody (anti-hIgG-PE) for 45 minutes at 4 C. At the end
of incubation, the
cells were washed with FACS Buffer, fixed with Formaldehyde and analyzed using
FACScan.
Data were analyzed using CellQuest Pro software. Filled histograms indicate
data from negative
control cells, and open histograms represent data from PSCA-positive cells
(Figure 9).
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[0507] Positive hybridomas identified from primary screens were transferred to
24-well
plates and supernatants collected for confirmatory screens. Confirmatory
screens included
FACs analysis on B300.19-PSCA/300.I9-neo, Ratl-PSCA/Ratl-neo, PC3-PSCA/neo,
SW780
(bladder cancer cell line), LAPC9AI (prostate cancer cell line), HPAC
(pancreatic cancer cell
line), and ELISA assay using Tag5-PSCA, GST-PSCA, GST-PSCA N-term, Med. C-
Term, and
pET-PSCA.
B. PSCA human MAb Relative Affinity Analysis
[0508] The hybridoma supernatants were tested to determine their relative
binding affinity to
cell surface PSCA. Hybridoma supernatants were serially diluted in FACS Buffer
(FB), from
g/ml to sub-ng/ml; and evaluated in a FACS binding assay using LAPC9AI cells.
High affinity
antibodies gave high MFI values. MFI values of each point were obtained using
CellQuest Pro
software and used for affinity calculation using Graphpad Prism software
(Table VII and Table
VIII): Sigmoidal Dose-Response (variable slope) equation. The results of the
relative affinity
analysis is set forth in Figure 10.
C. Epitope Grouping
[0509] PSCA antibodies were grouped according to epitope by evaluating their
binding
pattern on LAPC9AI cells. In brief, a small amount of each of the antibodies
was biotinylated;
then each of the biotinylated antibodies were incubated with LAPC9AI in the
presence of excess
(100 x) amount of non-biotinylated antibodies at 4 C for 1 hour during the
incubation.
Generally, an excess amount of antibodies will compete with biotinylated
antibodies if they bind
to the same epitope. At the end of incubation, cells were washed and incubated
with
Streptavidin-PE for 45 min at 4 C. After washing off the unbound streptavidin-
PE, the cells
were analyzed using FACS. MFI determinations were used for data analysis
(Table VII). As
shown in Table XI, cells highlighted in yellow indicate self-competition (100%
competition), the
MFI in these cells are background control for each biotinylated antibody.
Cells with no color
indicate that the two antibodies compete each other (low MFI), high MFI
(highlighted in blue)
indicate that the two antibodies bind to two distinct epitopes. The antibodies
that have same
binding pattern bind to same epitope among the antibodies. There are 6 epitope
groups within
the antibodies tested. Table XI shows that PSCA 4.121 binds to its unique
epitope.
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Example 10
Characterization and Expression of PSCA Antibodies
A. Cross Reactivity with Monkey PSCA and Mouse PSCA
[0510] MAbs were screened and characterized for their ability to react with
PSCA from
mouse and simian origin. This property is useful to understand the
consequences of MAb
engagement of PSCA on cells and tissues when using mouse and simian animal
models. The
Cynomolgus monkey and mouse PSCA genes were cloned, expressed in a retrovirus
and
transiently infected into 293-T cells. were incubated with the respective
antibodies using the following protocol. Test antibodies were incubated with
293-T cells
expressing either Cynomolgus monkey or murine PSCA or 293T cells expressing
the-neo gene
gene only as a negative control. Specific recognition was determined using
anti-hIgG-PE
secondary detection antibody. A representative histogram depicting specied
cross-reactivity is
presented in Figure 11. The summary presented in Table X shows that all but
one of the anti-
human PSCA antibodies cross-react with monkey PSCA and only one anti-human
PSCA
antibody cross-reacts with mouse PSCA.
B. Affinity Determination by FAGS
[0511] A panel of seven (7) anti-human PSCA antibodies was tested for their
binding
affinity to PSCA on SW780 cells, a human bladder cancer cell line expressing
high level of
PSCA. 23 serial 1:2 dilutions of purified antibodies were incubated with SW780
cells (50,000
cells per well) over night at 4 C with final concentration of 167nM to O.OlpM.
At the end of the
incubation, cells were washed and incubated with anti-hIgG-PE detection
antibody for 45 min at
4 C. After washing the unbound detection antibodies, the cells were analyzed
by FACS; MFI
values of each point were obtained using CellQuest Pro software and were used
for affinity
calculation using Graphpad Prism software: Sigmoidal Dose-Response (variable
slope) equation
(Table VII and Table VIII). Affinity values of the seven (7) antibodies are
set forth in Table IX.
Example 11
PSCA Antibody Internalization
[0512] Internalization of Hal -4.121 was studied using PC3-PSCA cells. In
brief, Hal -4.121
was incubated with cells at 4 C for 90 min to allow binding of the antibodies
to the cell surface.
The cells were then split into two groups and incubation continued at either
37 C to allow
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I
antibody internalization or at 4 C as controls (no internalization). An acid-
wash after 37 C/4 C
incubation was employed to remove PSCA 4.121 bound on cell surface. Subsequent
permeablization allowed detection of antibodies bound to internalized PSCA.
After incubation
with secondary detection antibodies, cells were analyzed using FACS or
observed under
fluorescence microscope. Approximately 30% of Hal -4.121 internalized after
incubation at
37 C for two hours (Figure 12 ).
Example 12
Antibody mediated secondary killing
[0513] Antibodies to PSCA mediate saporin dependent killing in PSCA expressing
cells.
B300.19 -PSCA expressing cells (750 cells/well) were seeded into a 96 well
plate on day 1.
The following day an equal volume of medium containing 2X concentration of the
indicated
primary antibody together with a 2 fold excess of anti-human (Hum-Zap) or anti-
goat (Goat-
Zap) polyclonal antibody conjugated with saporin toxin (Advanced Targeting
Systems, San
Diego, CA) was added to each well. The cells were allowed to incubate for 5
days at
37 degrees C. At the end of the incubation period, MTS (Promega) was added to
each well and
incubation continued for an additional 4 hours. The OD at 450 nM was
determined. The results
in Figrue 13(A) show that PSCA antibodies HA1-4.121 and HA1-4.117 mediated
saporin
dependent cytotoxicity in B300.19-PSCA cells while a control, nonspecific
human IgGl,
antibody had no effect. The results in Figure 13(B) show that addition of a
secondary saporin
conjugated antibody that did not recognize human Fc, failed to mediate
cytotoxicity (Figure
13(A) and Figure 13(B)). These results indicate that drugs or cytotoxic
proteins can selectively
be delivered to PSCA expressing cells using an appropriate anti-PSCA MAb.
Example 13
Antibody Immune Mediated Cytotoxicity
[0514] PSCA antibodies were evaluated to determine their ability to mediate
immune
dependent cytotoxicity. PSCA antibodies (0-50 gg/ml) were diluted with RHB
buffer (RPMI
1640, Gibco Life Technologies, 20 mM HEPES). B300.19-PSCA expressing cells
were washed
in RHB buffer and resuspended at a density of 106 cells/mi. In a typical
assay, 50 l of PSCA
antibody, 50 tl of diluted rabbit complement serum (Cedarlane, Ontario, Can),
and 50 l of a
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cell suspension were added together into a flat-bottom tissue culture 96-well
plate. The mixture
was incubated for 2 hr. at 37 C in a 5% C02 incubator to facilitate complement-
mediated cell
lysis. Then, 50 l of Alamar Blue (Biosource Intl. Camarillo, CA) was added to
each well and
incubation continued for an additional 4-5 hr at 37 C. The fluorescence in
each well was read
using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm.
The results
show that PSCA antibodies having an Igl (HA1-4.121) or an IgG2 isotype (HA1-
5.99.1) but not
an IgG4 isotype (HA1-6.46) were able to mediate complement dependent lysis
(Figure 14).
105151 ADCC (Antibody-Dependent Cellular Cytotoxicity) is an immune mediate
lytic
attack on cells bound with an antibody targeted to a specific cell surface
antigen. In this case it
is PSCA. Immune cells recognize the Fc portion of the antibody through binding
to Fey
receptors on the surface of leukocyte, monocyte and NK cells triggering a
lytic attack that results
in cell death. The ability of PSCA antibodies to mediate this reaction can be
evaluated by
labeling tumor cells in vitro with 51chromium, europium or a fluorescent
molecule and
incubating them in the presence of human PSCA MAbs together with peripheral
blood
mononuclear cells. Specific lysis of the tumor cells can be determined by
measuring the per
cent lysis of the targeted tumor cells. Common endpoints that are determined
include release of
radioactivity, europium or fluorescent dye from the dead cells using an
appropriate detection
method. Alternatively, the release of an intracellular enzyme such as lactate
dehydrogenase
(LDH) can be measured.
Example 14
Generation of F(Ab')2 Fragments
[05161 Generation of F(Ab')2 fragments of MAbs is useful to study the effects
of MAb
molecules that retain their bivalent anitgen binding site but lack the immune
effector Fc domain
in in vitro and in vivo therapeutic models. 20 mgs of MAb Hal-4.121 in 20 mM
sodium acetate
buffer pH 4.5 was incubated with and without immobilized pepsin (Pierce.
Rockford IL) for the
indicated times. Intact MAb and digested Fe fragments were removed by protein
A
chromatography. Shown in Figure 15 is a SDS-PAGE Coomasie stained gel of
intact undigested
unreduced MAb, unreduced aliquots of digested material taken at the indicated
times, and a
reduced sample of the final digested F(ab')2 product. This reagent can be used
to treat animals
bearing PSCA expressing tumors. The anti-tumor activity observed with this
antibody fragment
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can distinguish intrinsic biologic activity from activity mediated by immune
dependent
mechanisms.
Example 15
Expression of Human Antibodies using Recombinant DNA Methods
[0517] To express anti-PSCA MAbs recombinantly in transfected cells, anti-PSCA
variable
heavy and light chain sequences were cloned upstream of the human heavy chain
IgGl and light
chain Igx constant regions respectively. The complete anti-PSCA human heavy
chain and light
chain cassettes were cloned downstream of the CMV promoter/enhancer in a
cloning vector. A
polyadenylation site was included downstream of the MAb coding sequence. The
recombinant
anti-PSCA MAb expressing constructs were transfected into 293T, Cos and CHO
cells. The
HA1-4.121 antibody secreted from recombinant 293-T cells was evaluated for
binding to cell
surfacef PSCA in Figure Pia-3A and compared with the same antibody produced
from the
original hybridoma (Figure 16).
Example 16
HLA Class I and Class II Binding Assays
[0518] 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.,
J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)).
Briefly, purified
MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide
inhibitors and 1-10
nM 1251-radiolabeled probe peptides as described. Following incubation, MHC-
peptide
complexes are separated from free peptide by gel filtration and the fraction
of peptide bound is
determined. Typically, in preliminary experiments, each MHC preparation is
titered in the
presence of fixed amounts of radiolabeled peptides to determine the
concentration of HLA
molecules necessary to bind 10-20% of the total radioactivity. All subsequent
inhibition and
direct binding assays are performed using these HLA concentrations.
[0519] Since under these conditions [label]<[HLA] and IC50 [HLA], the measured
IC50
values are reasonable approximations of the true KD 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
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A completely independent experiments. To allow comparison of the data obtained
in different
experiments, a relative binding figure is calculated for each peptide by
dividing the IC50 of a
positive control for inhibition by the IC50 for each tested peptide (typically
unlabeled versions of
the radiolabeled probe peptide). For database purposes, and inter-experiment
comparisons,
relative binding values are compiled. These values can subsequently be
converted back into
IC50 nM values by dividing the IC50 nM of the positive controls for inhibition
by the relative
binding of the peptide of interest. This method of data compilation is
accurate and consistent for
comparing peptides that have been tested on different days, or with different
lots of purified
MHC.
[0520] Binding assays as outlined above may be used to analyze HLA supermotif
and/or
HLA motif-bearing peptides (see Table IV).
Example 17
Construction of "Minigene" Multi-Epitope DNA Plasmids
[0521] 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.
[0522] A minigene expression plasmid typically includes multiple CTL and HTL
peptide
epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide
epitopes and
HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR
supermotif-
bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing
peptide
epitopes derived PSCA, are selected such that multiple supermotifs/motifs are
represented to
ensure broad population coverage. Similarly, HLA class 11 epitopes are
selected from PSCA 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.
[0523] 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
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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 molecule.
[0524] 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.
[0525] The minigene DNA plasmid of this example contains a consensus Kozak
sequence
and a consensus murine kappa Ig-light chain signal sequence followed by CTL
and/or HTL
epitopes selected in accordance with principles disclosed herein. The sequence
encodes an open
reading frame fused to the Myc and His antibody epitope tag coded for by the
pcDNA 3.1 Myc-
His vector.
[0526] Overlapping oligonucleotides that can, for example, average about 70
nucleotides in
length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The
oligonucleotides
encode the selected peptide epitopes as well as appropriate linker
nucleotides, Kozak sequence,
and signal sequence. The final multiepitope minigene is assembled by extending
the
overlapping oligonucleotides in three sets of reactions using PCR. A
Perkin/Elmer 9600 PCR
machine is used and a total of 30 cycles are performed using the following
conditions: 95 C for
15 sec, annealing temperature (5 below the lowest calculated Tm of each
primer pair) for 30
see, and 72 C for 1 min.
[0527] For example, a minigene is prepared as follows. For a first PCR
reaction, 5 g of
each of two oligonucleotides are annealed and extended: In an example using
eight
oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6,
and 7+8 are
combined in 100 l reactions containing Pfu polymerise buffer (lx= 10 mM KCL,
10 mM
(NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1 % Triton X-100, 100
g/ml
BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerise. 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.
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Example 18
The Plasmid Construct and the Degree to Which It Induces Immuno enicity.
105281 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).
[0529] 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.
[05301 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.]/Kb
transgenic mice,
for example, are immunized intramuscularly with 100 tg of naked eDNA. As a
means of
comparing the level of CTLs induced by eDNA 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.
[05311 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 51 Cr release assay.
The results indicate
the magnitude of the CTL response directed against the A2-restricted epitope,
thus indicating the
in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.
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[0532] 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.
[0533] To confirm the capacity of a class II epitope-encoding minigene to
induce HTLs in
vivo, DR transgenic mice, or for those epitopes that cross react with the
appropriate mouse MHC
molecule, I-Ab-restricted mice, for example, are immunized intramuscularly
with 100 g of
plasmid DNA. As a means of comparing the level of HTLs induced by DNA
immunization, a
group of control animals is also immunized with an actual peptide composition
emulsified in
complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from
splenocytes of
immunized animals and stimulated with each of the respective compositions
(peptides encoded
in the minigene). The HTL response is measured using a 3H-thymidine
incorporation
proliferation assay, (see, e.g., Alexander et at. Immunity 1:751-761, 1994).
The results indicate
the magnitude of the HTL response, thus demonstrating the in vivo
immunogenicity of the
minigene.
[0534] DNA minigenes, constructed as described in the previous Example, can
also be
confirmed as a vaccine in combination with a boosting agent using a prime
boost protocol. The
boosting agent can consist of recombinant protein (e.g., Barnett et al., Aids
Res. and Human
Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for
example,
expressing a minigene or DNA encoding the complete protein of interest (see,
e.g., Hanke et al.,
Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-
53, 1998; Hanke
and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature
Med. 5:526-
34, 1999).
[0535] For example, the efficacy of the DNA minigene used in a prime boost
protocol is
initially evaluated in transgenic mice. In this example, A2.1/Kb transgenic
mice are immunized
IM with 100 g of a DNA minigene encoding the immunogenic peptides including
at least one
HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-
9 weeks), the
mice are boosted IP with 10' pfu/mouse of a recombinant vaccinia virus
expressing the same
sequence encoded by the DNA minigene. Control mice are immunized with 100 gg
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
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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.
[0536] It is found that the minigene utilized in a prime-boost protocol
elicits greater immune
responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an
analysis can
also be performed using HLA-Al I or HLA-B7 transgenic mouse models to assess
CTL
induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime
boost
protocols in humans is described below in the Example entitled "Induction of
CTL Responses
Using a Prime Boost Protocol."
Example 19
Polyepitopic Vaccine Compositions from Multiple Antigens
[0537] The PSCA 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 PSCA and such
other antigens.
For example, a vaccine composition can be provided as a single polypeptide
that incorporates
multiple epitopes from PSCA as well as tumor-associated antigens that are
often expressed with
a target cancer associated with PSCA 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 20
Use of peptides to evaluate an immune response
[0538] Peptides of the invention may be used to analyze an immune response for
the
presence of specific antibodies, CTL or HTL directed to PSCA. 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.
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[05391 In this example highly sensitive human leukocyte antigen tetrameric
complexes
("tetramers") are used for a cross-sectional analysis of, for example, PSCA
HLA-A*0201-
specific CTL frequencies from HLA A*0201 -positive individuals at different
stages of disease
or following immunization comprising a PSCA peptide containing an A*0201
motif.
Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J.
Med. 337:1267,
1997). Briefly, purified HLA heavy chain (A*0201 in this example) and (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 I
mg/ml. The resulting
product is referred to as tetramer-phycoerythrin.
[05401 For the analysis of patient blood samples, approximately one million
PBMCs are
centrifuged at 300g for 5 minutes and resuspended in 50 l of cold phosphate-
buffered saline.
Tri-color analysis is performed with the tetramer-phycoerythrin, along with
anti -CD 8-Tn'color,
and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for
30 to 60 min
and then washed twice before formaldehyde fixation. Gates are applied to
contain >99.98% of
control samples. Controls for the tetramers include both A*0201-negative
individuals and
A*0201-positive non-diseased donors. The percentage of cells stained with the
tetramer is then
determined by flow cytometry. The results indicate the number of cells in the
PBMC sample
that contain epitope-restricted CTLs, thereby readily indicating the extent of
immune response to
the PSCA epitope, and thus the status of exposure to PSCA, or exposure to a
vaccine that elicits
a protective or therapeutic response.
Example 21
Induction of Immune Responses Using a Prime Boost Protocol
[05411 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
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for the administration of the vaccine to humans. Such a vaccine regimen can
include an initial
administration of, for example, naked DNA followed by a boost using
recombinant virus
encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture
administered in
an adjuvant.
[0542] For example, the initial immunization may be performed using an
expression vector,
such as that constructed in the Example entitled "Construction of "Minigene"
Multi-Epitope
DNA Plasmids" in the form of naked nucleic acid administered IM (or SC or ID)
in the amounts
of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 g) can also be
administered using a
gene gun. Following an incubation period of 3-4 weeks, a booster dose is then
administered.
The booster can be recombinant fowlpox virus administered at a dose of 5-107
to 5x109 pfu. An
alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-
associated
virus, can also be used for the booster, or the polyepitopic protein or a
mixture of the peptides
can be administered. For evaluation of vaccine efficacy, patient blood samples
are obtained
before immunization as well as at intervals following administration of the
initial vaccine and
booster doses of the vaccine. Peripheral blood mononuclear cells are isolated
from fresh
heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted
in freezing
media and stored frozen. Samples are assayed for CTL and HTL activity.
[0543] Analysis of the results indicates that a magnitude of response
sufficient to achieve a
therapeutic or protective immunity against PSCA is generated.
Example 22
Complementary Polynucleotides
[0544] Sequences complementary to the PSCA-encoding sequences (Figure 1 or
Figure 3),
or any parts thereof, are used to detect, decrease, or inhibit expression of
naturally occurring
PSCA. Although use of oligonucleotides comprising from about 15 to 30 base
pairs is
described, essentially the same procedure is used with smaller or with larger
sequence
fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06
software
(National Biosciences) and the coding sequence of PSCA. 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 PSCA-encoding
transcript.
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Example 23
Purification of Naturally-occurring or Recombinant PSCA Using PSCA-Specific
Antibodies
[0545] Naturally occurring or recombinant PSCA is substantially purified by
immunoaffinity chromatography using antibodies specific for PSCA. An
immunoaffinity
column is constructed by covalently coupling anti-PSCA 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.
[0546] Media containing PSCA are passed over the immunoaffinity column, and
the column
is washed under conditions that allow the preferential absorbance of PSCA
(e.g., high ionic
strength buffers in the presence of detergent). The column is eluted under
conditions that disrupt
antibody/PSCA binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope,
such as urea or thiocyanate ion), and GCR.P is collected.
Example 24
Identification of Molecules Which Interact with PSCA
[0547] PSCA, or biologically active fragments thereof, are labeled with 121 1
Bolton-Hunter
reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate
molecules previously
arrayed in the wells of a multi-well plate are incubated with the labeled
PSCA, washed, and any
wells with labeled PSCA complex are assayed. Data obtained using different
concentrations of
PSCA are used to calculate values for the number, affinity, and association of
PSCA with the
candidate molecules.
Example 25
In Vivo Assay for PSCA Tumor Growth Promotion
[0548] The effect of the PSCA protein on tumor cell growth is evaluated in
vivo by
evaluating tumor development and growth of cells expressing or lacking PSCA.
For example,
SCID mice are injected subcutaneously on each flank with 1 x 106 of either
3T3, or prostate
cancer cell lines (e.g. PC3 cells) containing tkNeo empty vector or PSCA. At
least two
strategies may be used: (1) Constitutive PSCA expression under regulation of a
promoter such
as a constitutive promoter obtained from the genomes of viruses such as
polyoma virus, fowlpox
virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2),
bovine
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papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-
B virus and
Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the
actin promoter
or an immunoglobulin promoter, provided such promoters are compatible with the
host cell
systems, and (2) Regulated expression under control of an inducible vector
system, such as
ecdysone, tetracycline, etc., provided such promoters are compatible with the
host cell systems.
Tumor volume is then monitored by caliper measurement at the appearance of
palpable tumors
and followed over time to determine if PSCA-expressing cells grow at a faster
rate and whether
tumors produced by PSCA-expressing cells demonstrate characteristics of
altered aggressiveness
(e.g. enhanced metastasis, vascularization, reduced responsiveness to
chemotherapeutic drugs).
[05491 Additionally, mice can be implanted with I x 105 of the same cells
orthotopically to
determine if PSCA has an effect on local growth in the prostate, and whether
PSCA affects the
ability of the cells to metastasize, specifically to lymph nodes, and bone
(Miki T et al, Oncol
Res. 2001;12:209; Fu X et al, Int J Cancer. 1991, 49:938). The effect of PSCA
on bone tumor
formation and growth may be assessed by injecting prostate tumor cells
intratibially.
[05501 The assay is also useful to determine the PSCA inhibitory effect of
candidate
therapeutic compositions, such as for example, PSCA intrabodies, PSCA
antisense molecules
and ribozymes.
Example 26
PSCA Monoclonal Antibody-mediated Inhibition of Tumors In Vivo
[05511 The significant expression of PSCA on the cell surface of tumor
tissues, together
with its restrictive expression in normal tissues makes PSCA a good target for
antibody therapy.
Similarly, PSCA is a target for T cell-based immunotherapy. Thus, the
therapeutic efficacy of
anti-PSCA MAbs in human prostate cancer xenograft mouse models and human
pancreatic
cancer xenografl mouse models is evaluated by using recombinant cell lines
such as PC3-PSCA,
and 3T3-PSCA (see, e.g., Kaighn, M.E., et al., Invest Urol, 1979. 17(1): 16-
23), as well as
human prostate xenograft models such as LAPC 9AD (Saffran et al PNAS 1999,
10:1073-1078).
[05521 Antibody efficacy on tumor growth and metastasis formation is studied,
e.g., in a
mouse orthotopic prostate or pancreatic cancer xenograft models. The
antibodies can be
unconjugated, as discussed in this Example, or can be conjugated to a
therapeutic modality, as
appreciated in the art. Anti-PSCA MAbs inhibit formation of both pancreatic
and prostate
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xenografts. Anti-PSCA MAbs also retard the growth of established orthotopic
tumors and
prolonged survival of tumor-bearing mice. These results indicate the utility
of anti-PSCA MAbs
in the treatment of local and advanced stages prostate cancer, pancreatic
cancer and those
cancers set forth in Table I. (See, e.g., Saffran, D., et al., PNAS 10:1073-
1078 or world wide
web URL pnas.org/cgi/doi/ 10.1073/pnas.051624698).
[05531 Administration of the anti-PSCA MAbs led to retardation of established
orthotopic
tumor growth and inhibition of metastasis to distant sites, resulting in a
significant prolongation
in the survival of tumor-bearing mice. These studies indicate that PSCA is an
attractive target
for immunotherapy and demonstrate the therapeutic potential of anti-PSCA MAbs
for the
treatment of local and metastatic prostate and pancreatic cancer. This example
demonstrates that
unconjugated PSCA monoclonal antibodies are effective to inhibit the growth of
human prostate
tumor xenografts grown in SCID mice; accordingly a combination of such
efficacious
monoclonal antibodies is also effective.
Tumor inhibition using multiple PSCA MAbs
Materials and Methods
PSCA Monoclonal Antibodies:
[05541 Monoclonal antibodies were raised against PSCA as described in the
Example
entitled "Generation of PSCA Monoclonal Antibodies (MAbs)." The antibodies are
characterized by ELISA, Western blot, FACS, and immunoprecipitation for their
capacity to
bind PSCA. Epitope mapping data for the anti-PSCA MAbs, as determined by ELISA
and
Western analysis, recognize epitopes on the PSCA protein. Immunohistochemical
analysis of
prostate cancer tissues and cells with these antibodies is performed.
[05551 The monoclonal antibodies are purified from ascites or hybridoma tissue
culture
supernatants by Protein-G or Protein-A 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 LAPC9 AD and HPAC tumor xenografts.
Cell Lines and Xeno rg afts
[05561 The prostate cancer cell lines, PC3 and LNCaP cell line as well as the
fibroblast line
NIH 3T3 (American Type Culture Collection) are maintained in RPMI and DMEM
respectively,
supplemented with L-glutamine and 10% FBS.
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105571 PC3-PSCAand 3T3-PSCA cell populations are generated by retroviral gene
transfer
as described in Hubert, R.S., et al., Proc Natl Acad Sci U S A, 1999. 96(25):
14523.
(0558] 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., Nat Med.
1999, 5:280). Single-cell suspensions of LAPC-9 tumor cells are prepared as
described in Craft,
et al.
Xenograft Mouse Models.
(0559] Subcutaneous (s.c.) tumors are generated by injection of 1 x 106 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.e. 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 caliper measurements, and the
tumor volume is
calculated as length x width x height. Mice with Subcutaneous tumors greater
than 1.5 cm in
diameter are sacrificed.
(0560] Orthotopic injections are performed under anesthesia by using
ketamine/xylazine.
For prostate orthotopic studies, an incision is made through the abdomen to
expose the prostate
and LAPC or PC3 tumor cells (2 x 106) mixed with Matrigel are injected into
the prostate
capsule in a 10-pl volume. To monitor tumor growth, mice are palpated and
blood is collected
on a weekly basis to measure PSA levels. The mice are segregated into groups
for the
appropriate treatments, with anti-PSCA or control MAbs being injected i.p.
Anti-PSCA MAbs Inhibit Growth of PSCA-Expressing Xenograft-Cancer Tumors
(0561] The effect of anti-PSCA MAbs on tumor formation is tested by using HPAC
and
LAPC9 orthotopic models. As compared with the s.c. tumor model, the orthotopic
model,
which requires injection of tumor cells directly in the mouse pancreas or
prostate, respectively,
results in a local tumor growth, development of metastasis in distal sites,
deterioration of mouse
health, and subsequent death (Saffran, D., et al., PNAS supra). These features
make the
orthotopic model more representative of human disease progression and allowed
us to follow the
therapeutic effect of MAbs on clinically relevant end points.
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[0562] Accordingly, tumor cells are injected into the mouse prostate, and 2
days later, the
mice are segregated into two groups and treated with either: a) 250-1000 g, of
anti-PSCA Ab,
or b) control antibody three times per week for two to five weeks.
[0563] A major advantage of the orthotopic cancer models is the ability to
study the
development of metastases. Formation of metastasis in mice bearing established
orthotopic
tumors is studies by IHC analysis on lung sections using an antibody against a
tumor-specific
cell-surface protein such as anti-CK20 for prostate cancer (Lin et al., Cancer
Detect Prev. (2001)
25:202).
[0564] Another advantage of xenograft cancer models is the ability to study
neovascularization and angiogenesis. Tumor growth is partly dependent on new
blood vessel
development. Although the capillary system and developing blood network is of
host origin, the
initiation and architecture of the neovasculature is regulated by the
xenograft tumor (Davidoff
et al., Clin Cancer Res. (2001) 7:2870; Solesvik et al., Eur J Cancer Clin
Oncol. (1984)
20:1295). The effect of antibody and small molecule on neovascularization is
studied in
accordance with procedures known in the art, such as by IHC analysis of tumor
tissues and their
surrounding microenvironment.
[0565) Mice bearing established orthotopic tumors are administered injections
of either anti-
PSCA MAb or Control antibody over a 4-week period. Mice in both groups are
allowed to
establish a high tumor burden, to ensure a high frequency of metastasis
formation in mouse
lungs. Mice then are killed and their bladders, livers, bone and lungs are
analyzed for the
presence of tumor cells by IHC analysis. These studies demonstrate a broad
anti-tumor efficacy
of anti-PSCA antibodies on initiation and progression of prostate cancer in
xenograft mouse
models. Anti-PSCA antibodies inhibit tumor formation of tumors as well as
retarding the
growth of already established tumors and prolong the survival of treated mice.
Moreover, anti-
PSCA MAbs demonstrate a dramatic inhibitory effect on the spread of local
prostate tumor to
distal sites, even in the presence of a large tumor burden. Thus, anti-PSCA
MAbs are
efficacious on major clinically relevant end points (tumor growth),
prolongation of survival, and
health.
Effect of PSCA MAbs on the Growth of Human Prostate Cancer in mice
[0566] Using the above methodology, LAPC-9A1 tumor cells (2.0 x 106 cells)
were injected
subcutaneously into male SCID mice. The mice were randomized into groups (n=10
in each
group) and treatment initiated intraperitoneally (i.p.) on Day 0 with HA1-
4.120 or isotype MAb
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control as indicated. Animals were treated twice weekly for a total of 7 doses
until study day 28.
Tumor growth was monitored using caliper measurements every 3 to 4 days as
indicated. The
results show that Human anti-PSCA monoclonal antibody Hal-4.120 significantly
inhibited the
growth of human prostate cancer xenografts implanted subcutaneously in SCID
mice (p< 0.05)
(Figure 18).
[05671 In another experiment, LAPC-9AI tumor cells (2.0 x 106 cells) were
injected
subcutaneously into male SCID mice. When tumor volume reached 50 mm3, the mice
were
randomized into groups (n=10 in each group) and treatment initiated
intraperitoneally (i.p.) with
HA1-5.99.1 or isotype MAb control as indicated. Animals were treated twice
weekly for a total
of 5 doses until study day 14. Tumor growth was monitored using caliper
measurements every 3
to 4 days as indicated. The results show that Fully human anti-PSCA monoclonal
antibody Hal -
5.99 significantly inhibited the growth of established androgen-independent
human prostate
cancer xenografts implanted subcutaneously in SCID mice (p<0.05). (Figure 19).
[05681 In another experiment, LAPC-9AD tumor cells (2.5 x 106 cells) were
injected
subcutaneously into male SCID mice. When the tumor volume reached 40 mm3, the
mice were
randomized into groups (n=10 in each group) and treatment was initiated
intraperitoneally (i.p.)
with increasing concentrations of HA1-4.121 or isotype MAb control as
indicated. Animals
were treated twice weekly for a total of 7 doses until study day 21. Tumor
growth was
monitored using caliper measurements every 3 to 4 days as indicated. The
results from this
study demonstrated that HA1-4.121 inhibited the growth of established
subcutaneous human
androgen-dependent prostate xenografts in SCID mice. The results were
statistically significant
for the 300 ug dose group on days 14, 17 and 21 (p< 0.05, Kruskal-Wallis test,
two-sided with
a=0.05) and for the 700 ug dose group on days 10, 14, 17 and 21 (p< 0.05,
Kruskal-Wallis test,
two-sided with (x=0.05) (Figure 20).
[05691 In another experiment, patient-derived, androgen-dependent, LAPC-9AD
tumor cells
(2.0 x 106 cells) were injected into the dorsal lobes of the prostates of male
SCID mice. The
tumors were allowed to grow for approximately 10 days at which time the mice
were
randomized into groups. Treatment with human 500 mg of HA1-4.117, HA1-4.121 or
Isotype
control MAb was initiated 10 days after tumor implantation. Antibodies were
delivered
intraperitoneally two times a week for a total of 7 doses. Four days after the
last dose, animals
were sacrificed and primary tumors excised and weighed. The results show that
Human anti-
PSCA monoclonal antibodies Hal -4.121 (p< 0.01) and Hal -4.117 (p< 0.05)
significantly
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inhibited the growth of LAPC-9AD prostate cancer xenografts orthotopically
implanted in SCID
mice (Figure 21).
[05701 In another experiment, Patient-derived, androgen-dependent, LAPC-9AD
tumor cells
(2.0 x 106 cells) were injected into the dorsal lobes of the prostates of male
SCID mice. The
tumors were allowed to grow for approximately 9 days at which time the mice
were randomized
into groups. The animals randomized into the survival groups include 11 mice
in the isotype
MAb control and 12 mice in the HA1-4.121 treated group. Animals were treated
i.p. with 1000
ug Hal-4.121 or 1000 ug of isotype MAb control twice weekly for a total of 9
doses. The
results demonstrated that HAI-4.121 significantly (log-rank test: p<0.01)
prolonged the survival
of SCID mice with human androgen-dependent prostate tumors. Two mice in the
HA1-4.121
treated group remained free of palpable tumors on day 110, the last day of the
experiment
(Figure 22).
Effect of PSCA MAbs in combination with Taxotere in mice
105711 In another experiment, LAPC-9AI tumor cells (2 x 106 cells per animal)
were
injected subcutaneously into male SCID mice. When the tumor volume reached 65
mm3,
animals were randomized and assigned to four different groups (n=10 in each
group) as
indicated. Beginning on day 0, Hal -4.121 or isotype MAb control were
administered i.p. two
times a week at a dose of 500 ug for a total of 6 doses. The last dose was
given on day 17.
Taxotere was given intravenously at a dose of 5 mg/kg on days 0, 3 and 7.
Tumor growth was
monitored every 3-4 days using caliper measurements. The results from this
study demonstrate
that HA 1-4.121 as a single agent inhibited the growth of androgen-independent
prostate
xenografts in SCID mice by 45% when compared to control antibody treatment
alone on day 28
(ANOVA/Tukey test: p<0.05)_ Administration of the isotype MAb control plus
taxotere
inhibited tumor growth by 28% when compared to control antibody treatment
alone, which was
not statistically significant. Administration of HA1-4.121 in combination with
Taxotere,
enhanced the effect and resulted in a 69% inhibition of tumor growth when
compared to control
antibody alone (ANOVA/Tukey test: p<0.01). A statistically significant
difference was also
demonstrated when the HA 1-4.121 plus Taxotere combination group was compared
to either the
HAl-4.121 or isotype MAb control plus Taxotere groups (ANOVA/Tukey test:
p<0.05) (Figure
23).
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Effect of PSCA MAbs on the Growth of Human Pancreatic Cancer in mice
[0572] In another experiment, Human HPAC pancreatic cancer cells (2 x 106/
mouse) were
injected subcutaneously into immunodeficient ICR SCID mice (Taconic Farm,
Germantown,
NY). The mice were randomized into groups (n= 10 animals/group) and treatment
with the
indicated humanPSCA monoclonal antibody initiated the same day. Antibodies
(500
mg/mouse) were delivered intraperitoneally two times a week for a total of 8
doses. The results
demonstrated that human anti-PSCA monoclonal antibodies Hal-4.121, Hal-4.117
and Hal-
1.16 significantly inhibited the growth of human pancreatic cancer xenografts
subcutaneously
implanted in SCID mice. Statistical analyses were performed using a t test
(two-sided, a=0.05)
(Figure 24).
[0573] In another experiment, HPAC cells (3.0 x 106 cells) were implanted
orthotopically
into the pancreata of SCID mice. Mice were randomly assigned to three groups
(n=9 in each
group) as indicated. Treatment with HAI-4.121 (250 ug or 1000 ug) or isotype
MAb control
(1000 ug) was initiated on the day of implantation. Antibodies were
administered i.p. twice
weekly for a total of 10 doses. Thirteen days after the last dose, animals
were sacrificed and
primary tumors excised and weighed. The results from this study demonstrated
that HAI-4.121
significantly inhibited the orthotopic growth of human pancreatic cancer
xenografts in SCID
mice at both dose levels examined. Treatment with 250 ug and 1000 ug AGS-PSCA
inhibited
tumor growth by 66% and 70%, respectively (Kruskal-Wallis/Tukey test: p<0.01
and p<0.01,
respectively) (Figure 25).
[0574] At autopsy, visible metastases to lymph nodes and distant organs were
observed in
the control antibody treated group. No visible metastases were observed in
both HA1-4.121
treated groups. Lymph nodes, lungs and livers were removed from all animals
and examined
histologically for the presence of metastatic tumor. Sections from the lungs
and lymph nodes
removed from each animal were stained for human cytokeratin and the number of
metastases
determined microscopically. The results from the histologic analysis
demonstrated a significant
reduction in lymph node (LN) metastases in animal treated with HA1-4.121 (p=
0.0152 as
detected by Fishers exact test). The incidence of metastasis and invasion was
also decreased
significantly in animals treated with both concentrations of HA 1-4.121 (p=
0.0152 as detected
by Fishers exact test). The number of lung metastases decreased significantly
in mice treated
with the 1.0 mg dose of HA1-4.121 only (p= 0.0498 as detected by Fishers exact
test)
(Figure 26).
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Effect of PSCA MAbs on the Growth of Human Bladder Cancer in mice
[0575] In another experiment, Human SW780 bladder cancer cells (2 x 106/
mouse) were
injected subcutaneously into immunodeficient ICR SCID mice (Taconic Farm,
Germantown,
NY). The mice were randomized into groups (n= 10 animals/group) and treatment
with the
indicated human PSCA MAb initiated the same day. Antibodies (250 mg/mouse)
were delivered
intraperitoneally two times a week for a total of 7 doses. The results
demonstrated that HA 1-
4.117 (p= 0.014), HA1-4.37 (p= 0.0056), HAI-1.78 (p= 0.001), Hal-5.99
(p=0.0002) and HAl-
4.5 (p= 0.0008) significanctly inhibited the growth of SW780 bladder tumors
implanted
subcutaneously in SCID mice. Statistical analyses were performed using a.t
test (two-sided,
a=0.05) (Figure 27).
[0576] The results of these experiments show that PSCA MAbs can be used for
therapeutic
and diagnostic purposes to treat and manage cancers set forth in Table I.
Example 27
Therapeutic and Diagnostic use of Anti-PSCA Antibodies in Humans.
[0577] Anti-PSCA 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-
PSCA MAb
show strong extensive staining in carcinoma but significantly lower or
undetectable levels in
normal tissues. Detection of PSCA in carcinoma and in metastatic disease
demonstrates the
usefulness of the MAb as a diagnostic and/or prognostic indicator. Anti-PSCA
antibodies are
therefore used in diagnostic applications such as immunohistochemistry of
kidney biopsy
specimens to detect cancer from suspect patients.
[0578] As determined by flow cytometry, anti-PSCA MAb specifically binds to
carcinoma
cells. Thus, anti-PSCA 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 PSCA. Shedding or release of an extracellular
domain of PSCA into
the extracellular milieu, such as that seen for alkaline phosphodiesterase B
10 (Meerson, N. R.,
Hepatology 27:563-568 (1998)), allows diagnostic detection of PSCA by anti-
PSCA antibodies
in serum and/or urine samples from suspect patients.
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105791 Anti-PSCA antibodies that specifically bind PSCA are used in
therapeutic
applications for the treatment of cancers that express PSCA. Anti-PSCA
antibodies are used as
an unconjugated modality and as conjugated form in which the antibodies are
attached to one of
various therapeutic or imaging modalities well known in the art, such as a
prodrugs, enzymes or
radioisotopes. In preclinical studies, unconjugated and conjugated anti-PSCA
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 "PSCA Monoclonal Antibody-mediated Inhibition of Tumors In Vivo").
Either
conjugated and unconjugated anti-PSCA 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 28
Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas
through use of
Human Anti-PSCA Antibodies In vivo
[05801 Antibodies are used in accordance with the present invention which
recognize an
epitope on PSCA, and are used in the treatment of certain tumors such as those
listed in Table I.
Based upon a number of factors, including PSCA 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.
[05811 I.) Adjunctive therapy: In adjunctive therapy, patients are treated
with anti-
PSCA 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-PSCA 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-PSCA 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).
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[0582] II.) Monotherapy: In connection with the use of the anti-PSCA
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.
[05831 III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or
yttrium (I131,
Y90) to anti-PSCA 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 PSCA. In connection with the use of the
anti-PSCA
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 ("'In)-PSCA antibody is
used as an
imaging agent in a Phase I human clinical trial in patients having a carcinoma
that expresses
PSCA (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
[0584] 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-
PSCA antibodies can be administered with doses in the range of 5 to 400 mg/m2,
with the lower
doses used, e.g., in connection with safety studies. The affinity of anti-PSCA
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-PSCA antibodies that
are fully human
antibodies, as compared to the chimeric antibody, have slower clearance;
accordingly, dosing in
patients with such fully human anti-PSCA antibodies can be lower, perhaps in
the range of 50 to
300 mg/m2, and still remain efficacious. Dosing in mg/m2, as opposed to the
conventional
measurement of dose in mg/kg, is a measurement based on surface area and is a
convenient
dosing measurement that is designed to include patients of all sizes from
infants to adults.
[05851 Three distinct delivery approaches are useful for delivery of anti-PSCA
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
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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)
[0586] Overview: The CDP follows and develops treatments of anti-PSCA
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-PSCA
antibodies. As will be
appreciated, one criteria that can be utilized in connection with enrollment
of patients is PSCA
expression levels in their tumors as determined by biopsy.
[05871 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 PSCA. Standard tests and follow-up are utilized to
monitor each of
these safety concerns. Anti-PSCA antibodies are found to be safe upon human
administration.
Example 29
Human Clinical Trial: Monotherapy with Human Anti-PSCA Antibody
[0588] Anti-PSCA 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-PSCA antibodies.
Example 30
Human Clinical Trial: Diagnostic Imaging with Anti-PSCA Antibody
[0589] 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-PSCA
antibodies as a diagnostic imaging agent. The protocol is designed in a
substantially similar
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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 31
Human Clinical Trial Adjunctive Therapy with Human Anti-PSCA Antibody and
Chemotherapeutic, radiation, and/or hormone ablation therapy
[0590] A phase I human clinical trial is initiated to assess the safety of six
intravenous doses
of a human anti-PSCA antibody in connection with the treatment of a solid
tumor, e.g., a cancer
of a tissue listed in Table I. In the study, the safety of single doses of
anti-PSCA antibodies
when utilized as an adjunctive therapy to an antineoplastic or
chemotherapeutic or hormone
ablation agent as defined herein, such as, without limitation: cisplatin,
topotecan, doxorubicin,
adriarnycin, taxol, Lupron, Zoladex, Eulexin, Casodex, Anandron or the like,
is assessed. The
trial design includes delivery of approximately six single doses of an anti-
PSCA antibody with
dosage of antibody escalating from approximately about 25 mg/m2 to about 275
mg/m2 over the
course of the treatment in accordance with the following or similar schedule:
Day 0 Day 7 Day 14 Day 21 Day 28 Day 35
MAb Dose 25 mg/m2 75 mg/m2 125 mg/m2 175 mg/m2 225 mg/m2 275mg/m2
Chemotherapy + + + + + +
(standard dose)
[0591] 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 PSCA. 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.
[0592] The anti-PSCA antibodies are demonstrated to be safe and efficacious.
Phase II trials
confirm the efficacy and refine optimum dosing.
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Example 32
RNA interference (RNAi)
[0593] RNA interference (RNAi) technology is implemented to a variety of cell
assays
relevant to oncology. RNAi is a post-transcriptional gene silencing mechanism
activated by
double-stranded RNA (dsRNA). RNAi induces specific mRNA degradation leading to
changes
in protein expression and subsequently in gene function. In mammalian cells,
these dsRNAs
called short interfering RNA (siRNA) have the correct composition to activate
the RNAi
pathway targeting for degradation, specifically some mRNAs. See, Elbashir
S.M., et al.,
Duplexes of 21 -nucleotide RNAs Mediate RNA interference in Cultured Mammalian
Cells,
Nature 411(6836):494-8 (2001). Thus, RNAi technology is used successfully in
mammalian
cells to silence targeted genes.
[0594] Loss of cell proliferation control is a hallmark of cancerous cells;
thus, assessing the
role of PSCA in cell survival/proliferation assays is relevant. Accordingly,
RNAi was used to
investigate the function of the PSCA antigen. To generate siRNA for PSCA,
algorithms were
used that predict oligonucleotides that exhibit the critical molecular
parameters (G:C content,
melting temperature, etc.) and have the ability to significantly reduce the
expression levels of the
PSCA protein when introduced into cells. In accordance with this Example, PSCA
siRNA
compositions are used that comprise siRNA (double stranded, short interfering
RNA) that
correspond to the nucleic acid ORF sequence of the PSCA protein or
subsequences thereof.
Thus, siRNA subsequences are used in this manner are generally 5, 6, 7, 8, 9,
10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34,
35 or more than 35
contiguous RNA nucleotides in length. These siRNA sequences are complementary
and non-
complementary to at least a portion of the mRNA coding sequence. In a
preferred embodiment,
the subsequences are 19-25 nucleotides in length, most preferably 21-23
nucleotides in length.
In preferred embodiments, these siRNA achieve knockdown of PSCA antigen in
cells
expressing the protein and have functional effects as described below.
[0595] The selected siRNA (PSCA.b oligo) was tested in numerous cell lines in
the
survival/proliferation MTS assay (measures cellular metabolic activity).
Tetrazolium-based
colorimetric assays (i.e., MTS) detect viable cells exclusively, since living
cells are
metabolically active and therefore can reduce tetrazolium salts to colored
formazan compounds;
dead cells, however do not. Moreover, this PSCA.b oligo achieved knockdown of
PSCA
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antigen in cells expressing the protein and had functional effects as
described below using the
following protocols.
[0596] Mammalian siRNA transfections: The day before siRNA transfection, the
different
cell lines were plated in media (RPMI 1640 with 10% FBS w/o antibiotics) at
2x103 cells/well in
80 1 (96 well plate format) for the survival/MTS assay. In parallel with the
PSCA specific
siRNA oligo, the following sequences were included in every experiment as
controls: a) Mock
transfected cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and
annealing buffer (no
siRNA); b) Luciferase-4 specific siRNA (targeted sequence: 5'-
AAGGGACGAAGACGAACACUUCTT-3') (SEQ ID NO:77); and c) Eg5 specific siRNA
(targeted sequence: 5'-AACTGAAGACCTGAAGACAATAA-3') (SEQ ID NO:78). SiRNAs
were used at l OnM and I pg/ml Lipofectamine 2000 final concentration.
[0597] The procedure was as follows: The siRNAs were first diluted in OPTIMEM
(serum-
free transfection media, Invitrogen) at O.luM pM (10-fold concentrated) and
incubated 5-10 min
RT. Lipofectamine 2000 was diluted at 10 g/ml (10-fold concentrated) for the
total number
transfections and incubated 5-10 minutes at room temperature (RT). Appropriate
amounts of
diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 with diluted 10-
fold
concentrated siRNA and incubated at RT for 20-30" (5-fold concentrated
transfection solution).
20 1s of the 5-fold concentrated transfection solutions were added to the
respective samples and
incubated at 37 C for 96 hours before analysis.
[0598] MTS assay: The MTS assay is a colorimetric method for determining the
number of
viable cells in proliferation, cytotoxicity or chemosensitivity assays based
on a tetrazolium
compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-
tetrazolium, inner salt; MTS(b)] and an electron coupling reagent (phenazine
ethosulfate; PES).
Assays were performed by adding a small amount of the Solution Reagent
directly to culture
wells, incubating for 1-4 hours and then recording absorbance at 490nm with a
96-well plate
reader. The quantity of colored formazan product as measured by the amount of
490nm
absorbance is directly proportional to the mitochondrial activity and/or the
number of living
cells in culture.
[0599] In order to address the function of PSCA in cells, PSCA is silenced by
transfecting
the endogenously expressing PSCA cell lines.
[0600] Another embodiment of the invention is a method to analyze PSCA related
cell
proliferation is the measurement of DNA synthesis as a marker for
proliferation. Labeled DNA
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precursors (i.e. 3H-Thymidine) are used and their incorporation to DNA is
quantified.
Incorporation of the labeled precursor into DNA is directly proportional to
the amount of cell
division occurring in the culture. Another method used to measure cell
proliferation is
performing clonogenic assays. In these assays, a defined number of cells are
plated onto the
appropriate matrix and the number of colonies formed after a period of growth
following siRNA
treatment is counted.
[0601] In PSCA cancer target validation, complementing the cell
survival/proliferation
analysis with apoptosis and cell cycle profiling studies are considered. The
biochemical
hallmark of the apoptotic process is genomic DNA fragmentation, an
irreversible event that
commits the cell to die. A method to observe fragmented DNA in cells is the
immunological
detection of histone-complexed DNA fragments by an immunoassay (i.e. cell
death detection
ELISA) which measures the enrichment of histone-complexed DNA fragments (mono-
and
oligo-nucleosomes) in the cytoplasm of apoptotic cells. This assay does not
require pre-labeling
of the cells and can detect DNA degradation in cells that do not proliferate
in vitro (i.e. freshly
isolated tumor cells).
[0602] The most important effector molecules for triggering apoptotic cell
death are
caspases. Caspases are proteases that when activated cleave numerous
substrates at the carboxy-
terminal site of an aspartate residue mediating very early stages of apoptosis
upon activation.
All caspases are synthesized as pro-enzymes and activation involves cleavage
at aspartate
residues. In particular, caspase 3 seems to play a central role in the
initiation of cellular events
of apoptosis. Assays for determination of caspase 3 activation detect early
events of apoptosis.
Following RNAi treatments, Western blot detection of active caspase 3 presence
or proteolytic
cleavage of products (i.e. PARP) found in apoptotic cells further support an
active induction of
apoptosis. Because the cellular mechanisms that result in apoptosis are
complex, each has its
advantages and limitations. Consideration of other criteria/endpoints such as
cellular
morphology, chromatin condensation, membrane blebbing, apoptotic bodies help
to further
support cell death as apoptotic. Since not all the gene targets that regulate
cell growth are anti-
apoptotic, the DNA content of permeabilized cells is measured to obtain the
profile of DNA
content or cell cycle profile. Nuclei of apoptotic cells contain less DNA due
to the leaking out
to the cytoplasm (sub-GI population). In addition, the use of DNA stains
(i.e., propidium
iodide) also differentiate between the different phases of the cell cycle in
the cell population due
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to the presence of different quantities of DNA in G0/G 1, S and G2/M. In these
studies the
subpopulations can be quantified.
[0603] For the PSCA gene, RNAi studies facilitate the understanding of the
contribution of
the gene product in cancer pathways. Such active RNAi molecules have use in
identifying
assays to screen for MAbs that are active anti-tumor therapeutics. Further,
siRNA are
administered as therapeutics to cancer patients for reducing the malignant
growth of several
cancer types, including those listed in Table 1. When PSCA plays a role in
cell survival, cell
proliferation, tumorigenesis, or apoptosis, it is used as a target for
diagnostic, prognostic,
preventative and/or therapeutic purposes.
Example 33
Detection of PSCA protein in cancer patient specimens by IHC
[0604] Expression of PSCA protein in tumor specimens from cancer patients was
detected
using the antibody HA 1-4.117. Formalin fixed, paraffin embedded tissues were
cut into 4
micron sections and mounted on glass slides. The sections were dewaxed,
rehydrated and
treated with antigen retrieval solution (Antigen Retrieval Citra Solution;
BioGenex, 4600 Norris
Canyon Road, San Ramon, CA, 94583) at high temperature. Sections were then
incubated in
fluorescein conjugated human monoclonal anti-PSCA antibody, Hal -4.117, for 16
hours at 4 C.
The slides were washed three times in buffer and further incubated with Rabbit
anti-Fluorescein
for 1 hour and, after washing in buffer, immersed in DAKO EnVision+TM
peroxidase-conjugated
goat anti-rabbit immunoglobulin secondary antibody (DAKO Corporation,
Carpenteria, CA) for
30 minutes. The sections were then washed in buffer, developed using the DAB
kit (SIGMA
Chemicals), counterstained using hematoxylin, and analyzed by bright field
microscopy. The
results show expression of PSCA in the tumor cells of prostate adenocarcinoma
(A, B), bladder
transitional carcinoma (C) and pancreatic ductal adenocarcinoma (D). These
results indicate
that PSCA is expressed in human cancers and that antibodies directed to this
antigen are useful
as diagnostic reagents (Figure 17).
[0605] These results indicate that PSCA is a target for diagnostic, prognostic
and therapeutic
applications in cancer.
10606] Throughout this application, various website data content,
publications, patent
applications and patents are referenced. (Websites are referenced by their
Uniform Resource
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Locator, or URL, addresses on the World Wide Web.) The disclosures of each of
these
references are hereby incorporated by reference herein in their entireties.
[06071 The present invention is not to be limited in scope by the embodiments
disclosed
herein, which are intended as single illustrations of individual aspects of
the invention, and any
that are functionally equivalent are within the scope of the invention.
Various modifications to
the models and methods of the invention, in addition to those described
herein, will become
apparent to those skilled in the art from the foregoing description and
teachings, and are
similarly intended to fall within the scope of the invention. Such
modifications or other
embodiments can be practiced without departing from the true scope and spirit
of the invention.
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Tables
Table I: Tissues that express PSCA when malignant.
Prostate
Pancreas
Bladder
Kidney
Colon
Lung
Ovary
Breast
TABLE II: Amino Acid Abbreviations
SINGLE LETTER THREE LETTER FULL NAME
F Phe phenylalanine
L Leu leucine
S Ser serine
Y Tyr tyrosine
C Cys cysteine
W Tr t to han
P Pro praline
H His histidine
Q Gin glutamine
R Arg arginine
I Ile isoleucine
M Met methionine
T Thr threonine
N Asn as ara gine
K Lys lysine
V Val valine
A Ala alanine
D Asp aspartic acid
E Glu lutamic acid
G Gly glycine
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TABLE III: Amino Acid Substitution Matrix
Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix
(block substitution matrix). The higher the
value, the more likely a substitution is found in related, natural proteins.
(See world wide web URL
ikp.unibe.ch/manuaVblosum62.html )
A C D E F G H I K L M N P Q R S T V W Y.
4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A
9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C
6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D
-3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2 E
6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3 F
6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G
8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2 H
4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 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
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TABLE IV:
HLA Class I/Il Motifs/Supermotifs
TABLE IV (A): HLA Class I Supermotifs/Motifs
SUPERMOTIF POSITION POSITION POSITION
2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary
Anchor)
Al TILVMS FWY
A2 LIVMATQ IVMATL
A3 VSMATLI RK
A24 YFWIVLMT FI YWLM
B7 P VILFMWYA
B27 RHK FYLWMIVA
B44 ED FWYLIMVA
B58 ATS FWYLIVMA
B62 QLIVMP FWYMIVLA
MOTIFS
Al TSM Y
Al DEAS Y
A2.1 LMVQIAT VLIMAT
A3 LMVISATFCGD KYRHFA
All VTMLISAGNCDF KRYH
A24 YFWM FLIW
A*3101 MVTALIS RK
A*3301 MVALFIST RK
A*6801 AVTMSLI RK
B*0702 P LMFWYAIV
B*3501 P LMFWYAVA
B51 P LIVF WYAM
B*5301 P IMFWYALV
B*5401 P ATIVLMFWY
Bolded residues are preferred, italicized residues are less preferred: A
peptide is considered motif-bearing if it has primary
anchors at each primary anchor position for a motif or supermotif as specified
in the above table.
TABLE IV (B): HLA Class II Supermotif
1 6 9
W, F, Y, V, .I,L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y
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TABLE IV (C): HLA Class II Motifs
MOTIFS 1 anchor 1 2 3 4 5 1 anchor 6 7 8 9
DR4 preferred FMYLIVW M T I VSTCPALIM MH MH
deleterious w R WDE
DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM
deleterious C CH FD CWD GDE D
DR7 preferred MFLIVWY M W A IVMSACTPL M IV
deleterious C G GRD N G
DR3 MOTIFS 1 anchor 1 2 3 1 anchor 4 5 1 anchor 6
Motif a preferred LIVMFY D
Motif b preferred LIVMFAY DNQEST KRH
DR Supermotif MFLIVWY VMSTACPLI
Italicized residues indicate less preferred or "tolerated" residues
TABLE IV (D): HLA Class I Supermotifs
POSITION: 1 2 3 4 5 6 7 8 C-terminus
SUPER-
MOTIFS Al 1 Anchor 1 0 Anchor
TILVMS FWY
A2 1 0 Anchor 1 Anchor
LIVMATQ LIVMAT
A3 Preferred 1 Anchor YFW YFW YFW P 1 Anchor
VSMATLI (4/5) (3/5) (4/5) (4/5) RK
deleterious DE (3/5); DE
P (5/5) (4/5)
A24 1 0 Anchor 1 Anchor
YFWIVLMT FIY WLM
B7 Preferred FWY (5/5) 10 Anchor FWY FWY 1 Anchor
LIVM (3/5) P (4/5) (3/5) VILFMWYA
deleterious DE (3/5); DE G ON DE
P(5/5); (3/5) (4/5) (4/5) (4/5)
G(4/5);
A(3/5);
QN(3/5)
B27 10 Anchor 1 Anchor
RHK FYLWMIVA
B44 1 Anchor 1 0 Anchor
ED FWYLIMVA
B58 1 0 Anchor 10 Anchor
ATS FWYLIVMA
B62 1 0 Anchor 1 Anchor
QLIVMP FWYMIVLA
Italicized residues indicate less preferred or "tolerated" residues
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TABLE IV (E): HLA Class I Motifs
POSITION 1 2 3 4 5 6 7 8 9 C-
terminus
or
C-terminus
Al preferred GFYW 1 Anchor DEA YFW P DEAN YFW l Anchor
9-mer STM y
deleterious DE RHKLIVMP A G A
Al preferred GRHK ASTCLIVM l Anchor GSTC ASTC LIVM DE 1 Anchor
9-mer DEAS Y
deleterious A RHKDEPYFW DE PQN RHK PG GP
Al preferred YFW 1 Anchor DEAQN A YFWQN PASTC GDE P l Anchor
10- STM Y
mer
deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A
Al preferred YFW STCLIVM l Anchor A YFW PG G YFW 1 Anchor
10- DEAS Y
mer
deleterious RHK RHKDEPYFW P G PRHK ON
A2.1 preferred YFW 1 Anchor YFW STC YFW A P 1 Anchor
9-mer LMIVQAT VLIMAT
deleterious DEP DERKH RKH DERKH
A2.1 preferred AYFW 1 Anchor LVIM G G FYWL 1 Anchor
10- LMIVQAT VIM VLIMAT
mer
deleterious DEP DE RKHA P RKH DERK RKH
H
A3 preferred RHK l Anchor YFW PRHKYF A YFW P 1 Anchor
LMVISATFCGD W KYRHFA
deleterious DEP DE
All preferred A 1 Anchor YFW YFW A YFW YFW P 1 Anchor
VTLMISAGNCD KRYH
F
deleterious DEP A G
A24 preferred YFWRHK 1 Anchor STC YFW YFW 1 Anchor
9-mer YFWM FLIW
deleterious DEG DE G QNP DERH G AQN
K
A24 Preferred 1 Anchor P YFWP P 1 Anchor
10- YFWM FLIW
mer
Deleterious GDE ON RHK DE A ON DEA
A310l Preferred RHK 1 Anchor YFW P YFW YFW AP 1 Anchor
MVTALIS RK
DeleteriousDEP DE ADE DE DE DE
A3301 Preferred 1 Anchor YFW AYFW 1 Anchor
MVALFIST RK
DeleteriousGP DE
A680lPreferred YFWSTC 1 Anchor YFWLIV YFW P 1 Anchor
AVTMSLI M RK
deleterious GP DEG RHK A
B0702Preferred RHKFWY l Anchor RHK RHK RHK RHK PA 1 Anchor
P LMFWYAI
V
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POSITION 1 2 3 4 5 6 7 8 9 C-
terminus
or
C-terminus
deleterious DEQNP DEP DE DE GDE QN DE
83501 Preferred FWYLIVM 1 Anchor FWY FWY 1 *Anchor
P LMFWYIV
A
deleteriousAGP G G
B51 Preferred LIVMFWY 1 Anchor FWY STC FWY G FWY 1 Anchor
P LIVFWYA
M
deleterious AGPDER DE G DEAN GDE
HKSTC
B5301preferred LIVMFWY 1 Anchor FWY STC FWY LIVMFW FWY 1 Anchor
P Y IMFWYAL
V
deleterious AGPQN G RHKQN DE
B5401preferred FWY 1 Anchor FWYLIVM LIVM ALIVM FWYA 1 Anchor
P P ATIVLMF
WY
deleteriousGPQNDE GDESTC RHKDE DE QNDGE DE
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TABLE IV (F):
Summary of HLA-supertypes
Overall phenotypic frequencies of HLA-supertypes in different ethnic
populations
Specificity Phenotypic frequency
Supertype Position 2 C-Terminus Caucasian N.A. Black Japanese Chinese Hispanic
Average
B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5
A3 AILMVST RK 37.5 42.1 45.8 52.7 43.1 44.2
A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2
A24 YF (WIVLMT) FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0
B44 E (D) FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0
Al TI (LVMS) FWY 47.1 16.1 21.8 14.7 26.3 25.2
B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4
B62 CL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1
B58 ATS FWY (LIV) 10.0 25.1 1.6 9.0 5.9 10.3
TABLE IV (G):
Calculated population coverage afforded by different HLA-supertype
combinations
HLA-supertypes Phenotypic frequency
Caucasian N.A Blacks Japanese Chinese Hispanic Average
83.0 86.1 87.5 88.4 86.3 86.2
A2, A3 and B7 99.5 98.1 100.0 99.5 99.4 99.3
A2, A3, B7, A24, 99.9 99.6 100.0 99.8 99.9 99.8
B44 and Al
A2, A3, B7, A24,
844, Al, B27,
B62, and B 58
Motifs indicate the residues defining supertype specificites. The motifs
incorporate residues determined on the basis of
published data to be recognized by multiple alleles within the supertype.
Residues within brackets are additional
residues also predicted to be tolerated by multiple alleles within the
supertype.
Table V: Frequently Occurring Motifs
Name vrg' % escription Potential Function
identity
Nucleic acid-binding protein functions as
transcription factor, nuclear location
f-C2H2 14% inc finger, C2H2 type Drobable
Cytochrome b(N- nembrane bound oxidase, generate
cytochrome-b-N 8% erminal /b6/ etB 3u peroxide
omains are one hundred amino acids
long and include a conserved
19% ImmunO lobulin domain intradomain disulfide bond.
andem repeats of about 40 residues,
each containing a Trp-Asp motif.
Function in signal transduction and
040 18% D domain, G-beta repeat rotein interaction
nay function in targeting signaling
DZ 3% PDZ domain nolecules to sub-membranous sites
LRR 8% Leucine Rich Repeat hort sequence motifs involved in
rotein-protein interactions
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onserved catalytic core common to
th serine/threonine and tyrosine
rotein kinases containing an ATP
Pkinase 3% Protein kinase domain indin site and a catalytic site
leckstrin homology involved in
ntracellular signaling or as constituents
PH 16% PH domain f the c oskeleton
10-40 amino-acid long found in the
xtracellular domain of membrane-
EGF 34% EGF-like domain and proteins or in secreted proteins
everse transcriptase
RNA-dependent DNA
Rvt 9% of merase)
oplasmic protein, associates integral
k 5% k repeat membrane proteins to the cytoskeleton
ADH- embrane associated. Involved in
biquinonefplastoquinone roton translocation across the
xidored_ 1 2% complex 1), various chains nembrane
alcium-binding domain, consists of a12
esidue loop flanked on both sides by a
Efhand 4% EF hand 12 residue alpha-helical domain
Retroviral aspartyl spartyl or acid proteases, centered on
Rvp 9% Protease catalytic as a l residue
xtracellular structural proteins involved
in formation of connective tissue. The
Collagen triple helix repeat sequence consists of the G-X-Y and the
ollagen 2% (20 copies) I pe tide chains forms a triple helix.
Located in the extracellular ligand-
inding region of receptors and is about
00 amino acid residues long with two
airs of cysteines involved in disulfide
Fn3 0% Fibronectin type III domain bonds
even hydrophobic transmembrane
regions, with the N-terminus located
transmembrane receptor 3xtracellularly while the C-terminus is
tm_1 19% (rhodopsin family) oplasmic. Signal through G proteins
Table VI: Exon boundriaries of transcript PSCA v.1
Exon Number Start End Length
1 10 69 60
2 70 177 108
3 178 985 808
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Table VII: MFI values of each data points used for affinity calculation.
MFI Values
PDP3 PDP3
nM PSCA PSCA T=8 +4' T=8 +4
40 869.3 777.06 795.24 661.66
20 875.19 835.94 816.34 824.07
856.28 847.83 777.85 842.72
5 866.94 817.45 758.15 818.83
2.5 835.47 769.79 742.45 783.5
1.25 813.12 782.84 806.2 792.44
0.625 766.52 689.3 683.64 666.28
0.3125 588.3 541.25 549.03 499.22
0.15625 389.95 354.24 366.82 355.83
0.07813 234.85 230.37 225.82 211.77
0.03906 138.05 132.14 134.25 134.07
0.01953 84.35 78.49 77.62 80.14
0.00977 48.13 48.38 50.93 46.87
0.00488 33.49 30.58 30.71 30.04
0.00244 21.77 20.33 18.14 20.44
0.00122 14.45 13.59 13.82 13.11
0.00061 11.07 10.14 9.52 10.35
0.00031 8.61 8.3 8.57 9.09
0.00015 7.45 7.17 7.28 7.85
0.00008 6.91 6.73 7.32 7.86
0.00004 6.94 6.41 6.81 6.51
Table VIII: Affinity calculated using Graphpad Prism software: Sigmoidal Dose-
Response (variable slope) equation.
Kd Values
PDP3 PDP3
PSCA PSCA T=8 +4 T=8 +4
Equation 1
Best-fit values
BMAX 896.6 843.9 816.4 820.3
KD 0.1784 0.1849 0.1678 0.1839
Std. Error
BMAX 10.83 10.92 12.63 20.12
KD 0.01152 0.01275 0.01397 0.02405
95% Confidence Intervals
873.9 to 821.0 to 790.0 to 778.2 to
BMAX 919.3 866.7 842.9 862.4
0.1386 0.1336
0.1543 to 0.1583 to to to
KD 0.2025 0.2116 0.1971 0.2343
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Table IX: FACS based affinity on fully human PSCA MAbs
FACS-Based Affinity
Sample ID Kd (nM)
Hal -4.37 0.23
Hal-4.121 0.26
Hal -5.99.1 . 0.28
Hal-4.117 0.32
Hal-4.120 0.41
Hal-4.5 1.00
Hal-1.16.1 6.91
Table X: Antibodies that cross-react with Monkey PSCA and/or Mouse PSCA.
Hybridoma ID Cross-react with Monkey- Cross-react with Mouse-
PSCA PSCA
H1-1.10 -
Hat-1.16 + -
Hal-1.78 + -
Hal -1.41 +
Hal-4.5 + -
Hal-4.37 + -
Hat-4.117 + +
Hai -4.120 + -
Ha1-4.121 + -
Hal -5.99 +
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