Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS OF USE OF TARGETING PEPTIDES FOR
DIAGNOSIS AND THERAPY OF HUMAN CANCER
BACKGROUND OF THE INVENTION
This application is a continuation in part of PCT patent application entitled,
"Compositions and Methods of Use of Targeting Peptides Against Placenta and
Adipose Tissues," by Pasqualini, Arap and Kolonin, filed August 30, 2002,
which was a
continuation in part of PCT/LTSO1/27692, filed on September 7, 2001. This
application
is also a continuation in part of PCT/USO1/28044, filed on September 7, 2001
and of
PCT/LTS01/27702, filed on September 7, 2001. The entire texts of all the above-
cited
applications are incorporated herein by reference. This invention was made
with U.S.
government support under grants grants CA90270, ' 1R1CA90810-01 and
1R01CA82976-01 from the National Institutes of Health. The U.S. government has
certain rights in this invention.
Field of the Invention
The present invention concerns the fields of cancer diagnostics and targeted
delivery of therapeutic agents to cancer cells. More specifically, the present
invention
relates to compositions and methods for identification and use of peptides
that
selectively target cancer cell receptors, such as the IL-11 receptor and/or
the GRP78
receptor. In particular embodiments, the targeted receptors are preferentially
expressed
in prostate cancer, especially in metastatic prostate cancer. In certain
embodiments, the
invention concerns compositions and methods of use of novel phage-based gene
delivery vectors.
Description of Related Art
Therapeutic treatment of many disease states is limited by the systemic
toxicity
of the therapeutic agents used. . Cancer therapeutic agents in particular
exhibit a very
low therapeutic index, with rapidly growing normal tissues such as skin and
bone
marrow affected at concentrations of agent that are not much higher than the
concentrations used to kill tumor cells. Treatment of cancer and other organ,
tissue or
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cell type confined . disease states would be greatly facilitated by the
development of
compositions and methods for targeted delivery to a desired organ, tissue or
cell type of
a therapeutic agent.
Recently, an ire vivo selection system was developed using phage display
libraries to identify targeting peptides for various organs, tissues or cell
types in a
mouse model system. Phage display libraries expressing transgenic peptides on
the
surface of bacteriophage were initially developed to map epitope binding sites
of
immunoglobulins (Smith and Scott, 1986, 1993). Such libraries can be generated
by
inserting random oligonucleotides into cDNAs encoding a phage surface protein,
generating collections of phage particles displaying unique peptides in as
many as 109
permutations. (Pasqualini and Ruoslahti, 1996, Arap et al, 1998a; Arap et al
1998b).
Intravenous administration of phage display libraries to mice was followed by
the recovery of phage from individual organs (Pasqualini and Ruoslahti, 1996).
Phage
were recovered that were capable of selective homing to the vascular beds of
different
mouse organs, tissues or cell types, based on the specific targeting peptide
sequences
expressed on the outer surface of the phage (Pasqualini and Ruoslahti, 1996).
A variety
of organ and tumor-homing peptides have been identified by this method
(Rajotte et al.,
1998, 1999; Koivunen et al., 1999a; Burg et al., 1999; Pasqualini, 1999). Each
of those
targeting peptides bound to different receptors that were selectively
expressed on the
vasculature of the mouse target tissue (Pasqualini, 1999; Pasqualini et al.,
2000;
Folkman, 1995; Folkman 1997). Tumor-homing peptides bound to receptors that
were
upregulated in the tumor angiogenic vasculature of mice (Brooks et al., 1994b;
Pasqualini et al., 2000). In addition to identifying individual targeting
peptides
selective for an organ, tissue or cell type (Pasqualini and Ruoslahti, 1996;
Arap et al,
1998a; I~oivunen et al., 1999b), this system has been used to identify
endothelial cell
surface markers that are expressed in mice in vivo (Rajotte and Ruoslahti,
1999).
This relative success notwithstanding, cell surface selection of phage
libraries
has been plagued by technical difficulties. A high number of non-binder and
non-
specific binder clones are recovered using previous ire vivo methods,
particularly with
components of the reticuloendothelial system such as spleen and liver. Removal
of this
background phage binding by repeated washes is both labor-intensive and
inefficient.
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Cells and potential ligands are frequently lost during the many washing steps
required.
Methods that have been successful with animal model systems are unsatisfactory
for
identifying human targeting peptides, which may differ from those obtained in
mouse
or other animal model systems.
Attachment of therapeutic agents to targeting peptides has resulted in the
selective delivery of the agent to a desired organ, tissue or cell type in the
mouse model
system. Targeted delivery of chemotherapeutic agents and proapoptotic peptides
to
receptors located in tumor angiogenic vasculature resulted in a marked
increase in
therapeutic efficacy and a decrease in systemic toxicity in tumor-bearing
mouse models
(Arap et al., 1998a, 1998b; Ellerby et al., 1999). However, the targeted
delivery of anti-
cancer agents in humans has not yet been demonstrated. The targeted receptors
reported in previous studies may be present in angiogenic normal tissues as
well as in
tumor tissues and may or may not be of use in distinguishing between normal
tissues,
non-metastatic cancers and metastatic cancer. A need exists for tumor
targeting
peptides that are selective against human cancers, as well as for targeting
peptides that
can distinguish between metastatic and non-metastatic human cancers.
Attempts have been made to target delivery of gene therapy vectors to specific
organs, tissues or cell types in vivo. Directing such vectors to the site of
interest would
enhance therapeutic effects and diminish adverse systemic immunologic
responses.
Adenovirus type 5 (Ad5)-based vectors have been commonly used for gene
transfer
studies (Weitzman et al., 1997; Zhang, 1999). The attachment of Ad5 to the
target cell
is mediated by the capsid's fiber knob region, which interacts with cell
surface
receptors, including the coxsackie adenovirus receptor (CAR) and possibly with
MHC
class I (Bergelson et al., 1997; Hong et al., 1997). Upon systemic
administration ih
vivo, binding of virus to CAR can result in unintended enrichment of vectors
in non-
targeted but CAR-expressing tissues. Conversely, target cells that express
little or no
CAR are inefficiently transduced. A need exists to develop novel gene therapy
vectors
to allow more selective delivery of gene therapy agents.
A need also exists to identify receptor-ligand pairs in organs, tissues or
cell
types. Previous attempts to identify targeted receptors and ligands binding to
receptors
have largely targeted a single ligand at a time for investigation.
Identification of
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previously unknown receptors and previously uncharacterized ligands has been a
very
slow and laborious process. Such novel receptors and ligands may provide the
basis for
new therapies for a variety of disease states, such as cancer and/or
metastatic prostate
cancer.
SUMMARY OF THE INVENTION
The present invention solves a long-standing need in the art by providing
compositions and methods of preparation and use of targeting peptides that are
selective
and/or specific for human cancer tissues, such as metastatic prostate cancer.
In some
embodiments, the invention concerns particular targeting peptides selective or
specific
for prostate cancer, including but not limited to SEQ m NO:S-35, SEQ m N0:37,
SEQ
m N0:39-67 and SEQ m NO:83-129. Other embodiments concern such targeting
peptides attached to therapeutic agents. In other embodiments, cancer
targeting
peptides may be used to selectively or specifically deliver therapeutic agents
to target
tissues, such as prostate cancer and/or metastatic prostate cancer. In certain
embodiments, the subject methods concern the preparation and identification of
targeting peptides selective or specific for a given target cell, tissue or
organ, such as
prostate cancer.
One embodiment of the invention concerns isolated peptides of 100 amino acids
or less in size, comprising at least 3 contiguous amino acids of a targeting
peptide
sequence, selected from any of SEQ m N0:5-35, SEQ m N0:37, SEQ m N0:39-67
and SEQ m N0:83-129. In a preferred embodiment, the isolated peptide is 50
amino
acids or less, more preferably 30 amino acids or less, more preferably 20
amino acids or
less, more preferably 10 amino acids or less, or even more preferably 5 amino
acids or
less in size. In other preferred embodiments, the isolated peptide may
comprise at least
4, 5, 6, 7, 8 or 9 contiguous amino acids of a targeting peptide sequence,
selected from
any of SEQ ID N0:5-35, SEQ m N0:37, SEQ m N0:39-67 and SEQ m N0:83-129.
In certain embodiments, the isolated peptide may be attached to a molecule. In
preferred embodiments, the attachment is a covalent attachment. In various
embodiments, the molecule is a drug, a chemotherapeutic agent, a radioisotope,
a pro-
apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth
factor, a
cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab
fragment of an
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antibody, a survival factor, an anti-apoptotic factor, a hormone antagonist,
an imaging
agent, a nucleic acid or an antigen. Those molecules are representative only
and
virtually any molecule may be attached to a targeting peptide and/or
administered to a
subject within the scope of the invention. In preferred embodiments, the pro-
aptoptosis
agent is gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK)2 (SEQ m
NO:1), (KLAKKLA)Z (SEQ m N0:2), (KAAI~KAA)Z (SEQ JD N0:3) or
(KLGKKLG)3 (SEQ >D N0:4). In other preferred embodiments, the anti-angiogenic
agent is angiostatin5, pigment epithelium-derived factor, angiotensin, laminin
peptides,
fibronectin peptides, plasminogen activator inhibitors, tissue
metalloproteinase
inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro-13,
thrombospondin,
2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101,
Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-
alpha,
herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide,
pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, docetaxel,
polyamines, a
proteasome inhibitor, a kinase inhibitor, a signaling inhibitor (SU5416,
SU666~, Sugen,
South San Francisco, CA), accutin, cidofovir, vincristine, bleomycin, AGM-
1470,
platelet factor 4 or minocycline. In further preferred embodiments, the
cytokine is
interleukin 1 (IL-1), lL.-2, IL-5, IL-10, IL-11, IL-12, IL-1~, interferon-'y
(IF-~y), IF-a, IF-
13, tumor necrosis factor-a (TNF-a), or GM-CSF (granulocyte macrophage colony
stimulating factor). Such examples are representative only and are not
intended to
exclude other pro-apoptosis agents, anti-angiogenic agents or cytokines known
in the
art.
In various embodiments, targeting peptides attached to one or more therapeutic
agents may be administered to a subject, such as a human subject. Such
administration
may be of use for the treatment of various disease states, such as prostate
cancer. In
certain embodiments, cancer-targeting peptides attached to a cytocidal, pro-
apoptotic,
anti-angiogenic or other therapeutic agent may be of use in methods to treat
human
cancer.
In other embodiments of the invention, the isolated peptide may be attached to
a
macromolecular complex. In preferred embodiments, the macromolecular complex
is a
virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic
bead, a
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yeast cell, a mammalian cell, a cell or a microdevice. These are
representative
examples only and macromolecular complexes within the scope of the present
invention may include virtually any complex that may be attached to a
targeting peptide
and administered to a subject. In other preferred embodiments, the isolated
peptide
may be attached to a eukaryotic expression vector, more preferably a gene
therapy
vector.
Various embodiments concern novel targeted gene therapy vectors, comprising
targeting peptides expressed on the surface of a gene therapy vector. In
particular
embodiments, the targeted gene therapy vector is a chimeric phage-based vector
containing elements from adeno-associated virus (AAV), the modified vector
being
referred to as an adeno-associated phage (AAP) vector.
In another embodiment, the targeting peptides may be attached to a solid
support, preferably magnetic beads, Sepharose beads, agarose beads, a
nitrocellulose
membrane, a nylon membrane, a column chromatography matrix, a high performance
liquid chromatography (HPLC) matrix or a fast performance liquid
chromatography
(FPLC) matrix. Such immobilized peptides may be used, for example, for
affinity
purification of various components, such as receptor proteins or circulating
antibodies
that bind to the peptides.
Additional embodiments of the present invention concern fusion proteins
comprising at least 3 contiguous amino acids of a sequence selected from any
of SEQ
m NO:S-35, SEQ m N0:37, SEQ m N0:39-67 and SEQ m N0:83-129. In some
embodiments, larger contiguous sequences, up to a full-length sequence
selected from
any of SEQ m N0:5-35, SEQ m N0:37, SEQ m N0:39-67 and SEQ m N0:83-129
may be used.
Certain other embodiments concern compositions comprising the claimed
isolated peptides or fusion proteins in a pharmaceutically acceptable carrier.
Further
embodiments concern kits comprising the claimed isolated peptides or fusion
proteins
in one or more containers.
Other embodiments concern methods of targeted delivery comprising selecting a
targeting peptide for a desired organ, tissue or cell type, such as prostate
cancer,
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attaching said targeting peptide to a molecule, macromolecular complex or gene
therapy
vector, and providing said peptide attached to said molecule, complex or
vector to a
subject. Preferably, the targeting peptide is selected to include at least 3
contiguous
amino acids from any of selected from any of SEQ m N0:5-35, SEQ m N0:37 and
SEQ m N0:83-129. In other preferred embodiments, the molecule attached to the
targeting peptide is a chemotherapeutic agent, an antigen or an imaging agent.
In
various embodiments, methods of targeted delivery may utilize antibodies
against
particular peptide sequences, such as SEQ m N0:39-67. Such antibodies may be
attached to a molecule, macromolecular complex or gene therapy vector and
administered to a subject. The skilled artisan will realize that the targeting
moiety is
not limited to antibodies, but may comprise any molecule or complex that binds
to a
receptor located in a target tissue, including but not limited to antibodies,
genetically
engineered antibodies, antibody fragments, single-chain antibodies, humanized
antibodies, chimeric antibodies, binding proteins and native ligands or
homologs
thereof. In preferred embodiments of the invention, the targeted receptor is
GRP78 or
IL-llRa.
In certain embodiments, the cancer targeting peptides and/or antibodies
disclosed herein may be of use for the detection, diagnosis and/or prognosis
of human
cancer, such as prostate cancer. In preferred embodiments, the cancer
targeting
peptides may be used to differentially diagnose metastatic and non-metastatic
prostate
cancer.
Other embodiments of the present invention concern isolated nucleic acids of
300 nucleotides or less in size, encoding a targeting peptide. In preferred
embodiments,
the isolated nucleic acid is 250, 225, 200, 175, 150, 125, 100, 75, 50, 40,
30, 20 or even
nucleotides or less in size. In other preferred embodiments, the isolated
nucleic acid
is incorporated into a eukaryotic or a prokaryotic expression vector. In even
more
preferred embodiments, the vector is a plasmid, a cosmid, a yeast artificial
chromosome
(YAC), a bacterial artificial chromosome (BAC), a virus or a bacteriophage. In
other
preferred embodiments, the isolated nucleic acid is operatively linked to a
leader
sequence that localizes the expressed peptide to the extracellular surface of
a host cell.
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Additional embodiments of the present invention concern methods of treating a
disease state, such as cancer, comprising selecting a targeting peptide and/or
antibody
against a selected peptide that targets cells associated with the disease
state, attaching
one or more molecules effective to treat the disease state to the peptide, and
administering the peptide to a subject with the disease state. Preferably, the
peptide
includes at least three contiguous amino acids selected from any of selected
from any of
SEQ ID N0:5-35, SEQ ID N0:37, SEQ ID N0:39-67 and SEQ m N0:83-129.
In certain embodiments, the methods concern Biopanning and Rapid Analysis of
Selective Interactive Ligands (BRASIL), a novel method for phage display that
results
in decreased background of non-specific phage binding, while retaining
selective
binding of phage to cell receptors. In preferred embodiments, targeting
peptides are
identified by exposing a subject to a phage display library, collecting
samples of one or
more organs, tissues or cell types, separating the samples into isolated cells
or small
clumps of cells suspended in an aqueous phase, layering the aqueous phase over
an
organic phase, centrifuging the two phases so that the cells are pelleted at
the bottom of
a centrifuge tube and collecting phage from the pellet. In an even more
preferred
embodiment, the organic phase is dibutylphtalate.
In other embodiments, phage that bind to a target organ, tissue or cell type,
for
example to prostate cancer, may be pre-screened or post-screened against a
subject
lacking that organ, tissue or cell type, such as a female subject. Phage that
bind to a
control subject are removed from the library prior to screening in subjects
possessing
the organ, tissue or cell type.
In preferred embodiments, targeting phage may be recovered from specific cell
types or sub-types present in an organ or tissue after selection of the cell
type by PALM
(Positioning and Ablation with Laser Microbeams). PALM allows specific cell
types to
be selected from, for example, a thin section of an organ or tissue. Phage may
be
recovered from the selected sample.
In another embodiment, a phage display library displaying the antigen binding
portions of antibodies from a subject is prepared, the library is screened
against one or
more antigens and phage that bind to the antibodies are collected. In more
preferred
embodiments, the antigen is a targeting peptide.
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In certain embodiments, the methods and compositions may be used. to identify
one or more receptors for a targeting peptide. In alternative embodiments, the
compositions and methods may be used to identify naturally occurring ligands
for
known or newly identified receptors. In preferred embodiments, the receptor
may be
selectively or specifically expressed in prostate cancer. In some embodiments,
expression of the receptor may be up regulated in prostate cancer compared to
normal
prostate, and/or in metastatic compared to non-metastatic prostate cancer.
Methods of
diagnosis and/or prognosis of cancer, such as prostate cancer, may comprise
detection
and/or quantification of such disease-state selective or specific receptors in
tissue
samples. In some embodiments, detection and/or quantification may take place
in situ
within an intact subject, for example by attaching an imaging agent to an
antibody or
equivalent molecule that binds to the receptor.
In some embodiments, the methods may comprise contacting a targeting peptide
to an organ, tissue or cell containing a receptor of interest, allowing the
peptide to bind
to the receptor, and identifying the receptor by its binding to the peptide.
In preferred
embodiments, the targeting peptide contains at least three contiguous amino
acids
selected from any of selected from any of SEQ m NO:S-35, SEQ m N0:37 and SEQ
m NO:83-129. In other preferred embodiments, the targeting peptide may
comprise a
portion of an antibody against the receptor. In more preferred embodiments,
the
antibody or antibody portion may bind to SEQ m N0:39-67.
In alternative embodiments, the targeting peptide may contain a random amino
acid sequence. The skilled artisan will realize that the contacting step can
utilize intact
organs, tissues or cells, or may alternatively utilize homogenates or
detergent extracts of
the organs, tissues or cells. In certain embodiments, the cells to be
contacted may be
genetically engineered to express a suspected receptor for the targeting
peptide. In a
preferred embodiment, the targeting peptide is modified with a reactive moiety
that
allows its covalent attachment to the receptor. In a more preferred
embodiment, the
reactive moiety is a photoreactive group that becomes covalently attached to
the
receptor when activated by light. In another preferred embodiment, the peptide
is
attached to a solid support and the receptor is purified by affinity
chromatography. In
other preferred embodiments, the solid support comprises magnetic beads,
Sepharose
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beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column
chromatography matrix, a high performance liquid chromatography (HPLC) matrix
or a
fast performance liquid chromatography (FPLC) matrix.
In certain embodiments, the targeting peptide may inhibit the activity of a
receptor upon binding to the receptor. The skilled artisan will realize that
receptor
activity can be assayed by a variety of methods known in the art, including
but not
limited to catalytic activity and binding activity. In other embodiments,
binding of a
targeting peptide to a receptor may inhibit a transport activity of the
receptor.
In alternative embodiments, one or more ligands for a receptor of interest may
be identified by the disclosed methods and compositions. One or more targeting
peptides that mimic part or all of a naturally occurring ligand may be
identified by
phage display and biopanning ih vivo or in vitro. A naturally occurring ligand
may be
identified by homology with a single targeting peptide that binds to the
receptor, or a
consensus motif of sequences that bind to the receptor. In other alternative
embodiments, an antibody may be prepared against one or more targeting
peptides that
bind to a receptor of interest. Such antibodies may be used for identification
or
immunoaffinity purification of the native ligand.
In certain embodiments, the targeting peptides of the present invention are of
use for the selective delivery of therapeutic agents, including but not
limited to gene
therapy vectors and fusion proteins, to specific organs, tissues or cell
types. The skilled
artisan will realize that the scope of the claimed methods of use include any
disease
state that can be treated by targeted delivery of a therapeutic agent to a
desired organ,
tissue or cell type. Although such disease states include those where the
diseased cells
are confined to a specific organ, tissue or cell type, other disease states
may be treated
by an organ, tissue or cell type-targeting approach. In particular
embodiments, the
organ, tissue or cell type may comprise prostate cancer.
Certain embodiments concern methods of obtaining antibodies against an
antigen. In preferred embodiments, the antigen comprises one or more targeting
peptides. The targeting peptides may be prepared and immobilized on a solid
support,
serum-containing antibodies is added and antibodies that bind to the targeting
peptides
may be collected.
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BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to further demonstrate certain aspects of the present invention. The invention
may be
better understood by reference to one or more of these drawings in combination
with
the detailed description of specific embodiments presented herein.
FIG. 1. IHC (irnmunohistochemistry) localization of IL-llRa in benign
prostate glands. (A) Normal glands from the peripheral zone showed predominant
nuclear staining of the basal and luminal cell layers (x 200). (B) A similar
pattern of
staining was observed in normal glands from the central zone (x 200).
FIG. Z. IHC staining of IL-llRa expression in primary androgen dependent
prostate cancer of low, intermediate, and high Gleason scores (FIG. 2A-C,
respectively). (A) Gleason score 6 prostate adenocarcinoma showed homogeneous
2+
staining (x 200). (B) Prostate carcinoma (arrowheads) showing 1+ and 2+
heterogeneous staining. Note negative staining in the luminal cells of the
contiguous
benign glands (black arrows) (x 100). (C) Strong 3+ positive staining in high-
grade
prostatic adenocarcinoma (x 100). (D) Negative control including benign glands
from
the peripheral zone and a few neoplastic acini (x 100).
FIG. 3. Immunodetection of IL-llRa in advanced, androgen independent,
prostate cancer. (A) Homogeneous 3+ expression of IL-llRa in prostate cancer
metastatic to the bone (x 100). (B) A higher power view of a bone metastasis
showing
2+ and 3+ expression of the receptor in the tumor cells (x 200). (C) Positive
staining in
the small vessels around the tumor nodules in the bone matrix (x 200). (D)
CD31
staining of the previous area confirming endothelial cell reactivity (x 200)
(E) High-
grade, androgen-independent primary tumor showing strong (3+) and homogeneous
expression of IL-llRa (x 100). (F) Negative control from the same area as (B)
(x 100).
FIG. 4. Selection of peptide library on immunoglobulins from the serum of
metastatic prostate cancer patients. Each successive round of panning
demonstrates an
increase in selectivity as measured by the increase in total number of
transducing units
for cancer patients relative to the serum of control volunteers. Three
metastatic
androgen-independents (patients A, B, and D) serum samples and one metastatic
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androgen-dependent (patient C) serum sample were examined. Standard error of
the
mean (S.E.M.) from triplicate plating is shown.
FIG. 5. Selection of peptide library on immunoglobulins from the serum of
metastatic prostate cancer patients. A series of 100-fold dilutions (1:100-
1:1200) was
performed for each patient's serum to test specific binding of cancer
antibodies to
immobilized GST-fusion proteins by ELISA.
FIG. 6. Reactivity between the serum from prostate cancer patients or control
men and the selected peptide is stage-specific. Serum samples derived from a
large
panel of prostate cancer patients (n=108) were divided into four groups: organ-
confined
(n=17), locally advanced (n=31), and metastatic androgen-dependent (n=31), and
metastatic androgen-independent (n=29). Serum samples derived from 71 age-
matched
blood-donor men served as a negative control group. Serial dilutions were
performed
for each serum to determine optimal reactivity by ELISA.
FIG. 6A. Distribution of reactivity is shown as the ratio of GST-peptide to
GST
alone for the four prostate cancer groups and control. Positive reactivity was
defined by
a ratio of GST-peptide to GST alone equal to or greater than 2.
FIG. 6B. Distribution of reactivity is shown as a percentage of positive
reactivity for each group. GST, glutathione S-transferase; A.D., androgen-
dependent;
A.L, androgen-independent.
FIG. 6C. Correlation between overall survival and serological reactivity
against
the CNVSDKSC (SEQ ID NO:39) peptide . The same prostate cancer patient
population was used to generate the Kaplan-Meier survival curves shown. Log-
Rank
tests were used to detect significant differences in survival time between
patients
positively reacting versus non-reacting to the peptide. A significant
correlation was
observed between .poor survival outcome and positive serum reactivity against
the
peptide CNVSDKSC (SEQ ID N0:39).
FIG. 7. Tmmunohistological analysis of tumors from a prostate cancer patient.
T_mmunostairiing of sections from prostate cancer metastatic to the bone
marrow of the
patient whose screening yielded CNVSDKSC (SEQ ID N0:39) and of normal prostate
are shown. (A) Strong staining was observed on metastatic tumor with the
autologous
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immunopurified IgGs. (B) Strong staining was also observed with a rabbit
polyclonal
antibody raised against the synthetic form of the CNVSDKSC (SEQ DJ N0:39)
soluble
peptide. (C) No staining was observed with the rabbit pre-immune serum. (D) No
staining was observed with secondary antibody alone. (E) A recombinant
CNVSDKSC
(SEQ ID N0:39) fusion protein inhibited staining under the same conditions
used in
FIG. 7B. (F) Weak staining was observed in normal prostate with the same
rabbit
polyclonal antibody used in FIG. 7B. Scale bar is 50 ,um.
FIG. 8. Cross-inhibition of patient serum antibodies by (A) GRP78 or (B)
GST-CNVSDKSC (SEQ ID N0:39). Recombinant GRP78 or GST-CNVSDKSC
(SEQ ID N0:39) were coated on microtiter well plates and various
concentrations of
patient serum, anti-GRP78 antibody and anti-CNVSDKSC (SEQ ID N0:39) antibodies
were added and analyzed by ELISA. Pre-incubation of the patient serum
antibodies
with GRP78 or GST-CNVSDKSC (SEQ ID N0:39) inhibited the reaction. The data
shows means ~ SD of triplicate wells.
FIG. 9. Reactivity against GRP78 is a serum marker of prostate cancer. (A)
Microtiter wells were coated with recombinant GRP78 and triplicates of serum
samples
were added at a 1:50 dilution. Serum samples from the same prostate cancer
population
presented in FIG. 6 were examined. For this assay, male (n=155) and female
(n=48)
donors served as negative controls. Positive reactivity by ELISA was defined
as an
Absorbance equal to or greater than 0.95 as determined by a statistical method
"CART". To test whether reactivity against GRP78 was restricted to prostate
cancer,
three additional non-prostate cancer tumor types are shown as controls:
metastatic non-
small cell (N.S.C.) lung cancer (n=31), metastatic breast cancer (n=32) and
advanced
ovarian cancer (32). Percentages of positive reactivity are shown. (B) CART
test for
comparative survival of GRP78 reactive (lower line) versus non-reactive (upper
line)
individuals with prostate cancer.
FIG. 10. Expression pattern of GRP78 by immunohistochemistry.
Immunostaining of normal prostate tissue and bone metastasis by anti-GRP78
antibody
and anti-CNVSDKSC (SEQ ID NO:39) antibodies are shown. (A) Strong staining was
observed in bone metastasis. (B) Weak staining was observed in the normal
prostate.
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(C) Recombinant GRP7~ can inhibit staining. (D) Recombinant GST-CNVSDKSC
(SEQ ID NQ:39) also inhibits staining. The magnification=100x.
FIG. 11. Scheme of the construction of phage with a targeting domain and a
mammalian reporter gene cassette. Replicative forms of the phage-derived RGD-
4C
and the fd-tet derived fMCS 1 DNA were digested with Sac II and Bam HI.
Ligation of
the fMCS 1 fragment with the RGD-4C plasmid fragment resulted in a chimeric
RGD-
4C-fMCS 1 phage vector with a multicloning site containing a Pst I site. The
Ps~t I-
digested (3-gal gene cassette was cloned into the Pst I site of the chimeric
vector RGD-
4C-fMCSl. The mammalian transgene cassette contains a CMV promoter, a [3-
galactosidase ((3-gal) gene, and an SV40 polyadenylation signal (SV40 polyA).
The
other targeted and control phage vectors presented in this study were
constructed by the
same general strategy.
FIG. 12. Transgene expression in mammalian cells after transfection of single-
stranded phage DNA into 293 cells. (3-gal expression was analyzed by an X-gal
staining after 24 hours. (A) Positive control plasmid pCMV[3-gal. (B) Negative
control
plasmid without the reporter gene cassette. (C) Single-stranded DNA extracted
from
phage with a forward orientation of the transgene cassette. (D) Single-
stranded DNA
extracted from phage with a reverse orientation of the transgene cassette.
FIG.13. Transduction of tumor cells by targeted phage is specific. Tumor cells
were incubated with targeted phage. (3-gal expression was evaluated after 72
hours. An
anti (3-gal antibody (Sigma) was used for the staining. (A, B) KS 1767 cells
with
HWGF-(3-gal phage, (C, D) MDA-MB-435 cells with RGD-4C-(3-gal phage, (E, F)
control insertless phage (fd-tet-(3-gal). The left side (A, C, E) shows only
Texas Red-
positive ((3-gal infected) cells. The right side (B, D, E) shows the total
number of cells
in identical fields. Magnification: x200.
FIG. 14. Quantitative analysis of cell transduction by targeted and control
phage. Phage were incubated with tumor cell lines as described in the legend
to FTG.
13. (A) An anti-(3-gal antibody was used for staining. Gene expression was
detected by
immunofluorescence and results are expressed in % of [3-gal positive cells. In
each
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case, standard error of the mean (SEM) was calculated after counting 10 fields
under
the microscope in three independent experiments. (B) Inhibition of HWGF-(3-gal
phage
transduction by the synthetic CTTHWGFTLC (SEQ ID N0:69) peptide. (C)
Inhibition
of RGD-4C-~3-gal phage transduction by the synthetic RGD-4C peptide. Unrelated
control peptides did not inhibit transduction of the tumor cells by the
targeted phage;
non-specific transduction levels were determined by using control insertless
phage.
Shown are mean ~ SEM obtained from duplicate wells.
FIG. 15. Specific transduction irz vivo by tumor-targeting phage.
Imrnunohistochemical analysis of (3-gal expression after systemic
administration of
targeted or control phage into tumor-bearing mice was performed. RGD-4C-(3-gal
(A,
D, and G), HWGF-(3-gal (B, E, and H), or control phage (C, F, and I) were
injected
intravenously into mice bearing KS 1767-derived Kaposi's sarcoma xenografts.
At
seven days post-administration, tumors and control organs were removed, fixed
in 4%
paraformaldehyde, embedded in paraffin, and sectioned. An antibody anti-(3-gal
(Sigma) was used for staining. Liver (D, E, and F) and brain (G, H, and I) are
shown
as control organs. Magnification: x400. Arrows point to anti-(3-gal
immunoreactivity.
FIG. 16. Tumor-selective targeting by RGD-4C (3-Gal phage, compared to
control insertless phage. The ability of different tissues to be infected by
the tumor
targeting versus control phage was examined for tumor, kidney, lung, brain,
liver and
spleen tissues. Although a comparatively high level of RGD-4C phage were
localized
to kidney, the difference between tumor-targeting and control phage
distribution was
not significant. Only tumor tissue showed a significant enhancement of phage
localization for the RGD-4C phage compared to control phage.
FIG. 17. Specific transduction in vivo by lung-targeting phage. Lung (targeted
organ) and liver (control organ) were evaluated for ~i-gal expression after
systemic
administration of GFE-phage or control phage into C57B1/6 immunocompetent
mice.
At 14 days post-administration lungs and livers were removed and processed as
described in the text. [3-gal enzymatic activity in the tissue cell lysates
was measured by
chemiluminescence. Shown are mean ~ SEM (n=5 mice per group).
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FIG. 18. Enhancement of transduction by genotoxic agents or genetic trans-
complementation. Semi-confluent MDA-MB-435 cells were infected with 105 TU of
phage per cell for four hours. Next, the cells were incubated for 36 hours
followed by
addition of genotoxic drugs (topotecan, 10 ,uM; cisplatin, 10 p,M) or
application of
physical agents such as ultraviolet radiation (UV; 15 J/m2). A phage mixture
of RGD-
4C-(3ga1 forward and reverse clones (molar ratio = 1; termed For/Rev) at the
same
number of phage TU of RGD-4C-(3gal phage was also tested. At 72 hours post-
infection, the cells were analyzed for expression of a reporter transgene.
Shown are
mean ~ SEM (n = 3) normalized green fluorescent protein (GFP) expression
relative to
controls.
FIG. 19. AAP vectors markedly improve gene transduction stability. Vectors
were constructed by cloning a full-length 2.8 kb fragment of pAAV-eGFP (Green
Fluorescent Protein, Stratagene) from inverted terminal repeat (TTR) to ITR
into the
blunted PstI site of the construct presented in FIG. 11. An engineered
chimeric vector
composed of an RGD-4C targeted phage and AAV genetic cis-elements was
incubated
with cells and analyzed for GFP gene expression 72 hours after infection as
indicated.
Either synthetic RGD-4C peptide or control unrelated peptide (CKDRFERC, SEQ ID
N0:41) was pre-incubated with cells to confirm specificity of targeted gene
transduction.
FIG. 20. GRP expression in cells infected with an AAV-GFP vector, in the
presence or absence of RGD-4C peptide or control peptide. For GFP detection,
cells in
each experiment were analyzed by fluorescence activated cell sorting (FAGS)
and
photographed under a fluorescence microscope.
FIG. 21. Time course of gene transduction. Cells were plated at 3 x 105
cells/well, infected with 105 TU of phage per cell for 4 hours, and sorted
based on GFP
expression by FACS at seven days post-infection. GFP-positive cells were
plated and
GFP expression was monitored. Robust GFP expression is shown at days 0, 15,
30, and
45.
FIG. 22. AAP vectors promote AAV integration. Viral rescue experiments.
GFP-expressing cells were detected after 48 hours. AAV particles can be
detected after
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adenoviral rescue in AAP-transduced 293 cells but not in control uninfected
293 cells
incubated with culture medium.
FIG. 23. Validation of adipose homing peptides. Phage bearing targeting
peptides were injected into obese mice and their recovery from adipose tissue
was
compared to control fd-tet phage without targeting sequences.
FIG. 24. In vivo fat homing of the CKGGRAKDC (SEQ m N0:81) motif in
genetically obese mice. (A) and (B) Anti-phage immunohistochemistry in
paraffin
sections of subcutaneous white fat from leptin-deficient mice intravenously
injected 6
hr prior to tissue processing. (C) and (D) Peptide-FTTC immunofluorescence in
paraffin sections of subcutaneous white fat from leptin-deficient mice
intravenously
injected 6 hr prior to tissue processing. Mice were injected with (A)
CKGGRAKDC
(SEQ ID NO:81) phage, (B) control insertless phage, (C) CKGGRAKDC (SEQ ID
N0:81) linked to FITC peptide, or (D) control CARAC (SEQ ID NO:71) linked to
FTTC peptide. Homing of the CKGGRAKDC (SEQ ID N0:81) peptide to fat blood
vessels (arrows) and its uptake by fat endothelium are indicated. Bar: 10 p,m.
FIG. 25. Ir2 vivo fat homing of the CKGGRAKDC (SEQ m N0:81) motif in
wild-type mice. (a), (C) and (E) Peptide-FITC immunofluorescence or (B), (D)
and (F)
lectin-rhodamine immunofluorescence in blood vessels of (A), (B), (E) and (F)
subcutaneous white fat or (C) and (D) pancreas controls detected in paraffin-
sectioned
tissues from c57b1/6 mice intravenously co-injected 5 min prior to tissue
processing.
Mice were injected with (A), (B), (C) and (D) CKGGRAKDC (SEQ m N0:81) linked
to FITC peptide and lectin-rhodamine; or (E) and (F) control CARAC (SEQ ID
N0:71)
linked to FITC peptide and lectin-rhodamine. (B), (D) and (F) Arrows show
endothelium marked with lectin. (A) Arrows show homing of the CKGGRAKDC
(SEQ ID N0:81) peptide to fat endothelium. Bar: 10 ~,m.
FIG. 26. Treatment of mouse obesity with fat vasculature-targeted apoptosis.
Three cohorts (n=3) of (A) high-fat cafeteria diet-fed obese c57b1/6 mice; or
(B) regular
diet-fed old (~lyear) c57b1/6 mice were each subcutaneously injected daily
with
equimolar amounts of the indicated peptides. Mouse body mass measurement was
taken on days when injections were performed (injections were skipped on days
for
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which body mass measurement is not shown). Error bars are SEM for the
measurements in three mice.
FIG. 27. Fat resorption induced by fat vasculature-targeted apoptosis. (A)
Representative high-fat cafeteria diet-fed obese c57b1/6 mice; (B) and (C)
representative regular diet-fed old (~lyear) c57b1/6 mice; or (D) epididymal
fat from
representative regular diet-fed old c57b1/6 mice from the experiment described
in FIG.
10. Whole mice (A), subcutaneous fat (B), peritoneal fat (C) and total
epididymal fat
(D) from the corresponding indicated treatments were photographed 1 week (A)
or 3
weeks (B), (C) and (D) after the beginning of subcutaneous injections. The
injected
peptides were CKGGRAKDC (SEQ ID N0:81) linked to (KLAKLAK)2 (SEQ ID
NO:1) (left column), CARAC (SEQ ID N0:71) linked to (KLAKLAK)2 (SEQ >D
N0:1) (middle column), and CKGGRAKDC (SEQ m N0:81) co-administered with
(KLAKLAK)2 (SEQ ID N0:1) (right column).
FIG. 28. Destruction of fat blood vessels as a result of targeted apoptosis.
(A)
Tunnel immunohistochemistry, (B) secondary antibody only negative tunnel
staining
control and (C) and (D) hematoxylin/eosin staining of white fat of mice. (A),
(B) and
(C) Mice were treated with CKGGRAKDC (SEQ ID N0:81) linked to (KLAKLAK)2
(SEQ ID NO:1). (D) Mice were treated with CARAC (SEQ ID N0:71) linked to
(KLAKLAK)Z (SEQ ID NO:1). Apoptosis (arrows, (A)) and necrosis/lymphocyte
infiltration (arrows, (C)) in response to CKGGRAKDC (SEQ m N0:81) linked to
(KLAKLAK)2 (SEQ ID NO:1) treatment are indicated. Bar: 10 p,m.
FIG. 29. Expression of prohibitin in human tissues. Prohibitin expression was
determined by immunohistochemistry of fixed human paraffin-embedded thin
tissue
sections with rabbit polyclonal antibodies against prohibitin. Arrows indicate
prohibitin staining in: (A) normal human white fat tissue; (B) normal human
breast
tissue; (C) a low grade human lipoma; (D) a high grade human lipoma; (E) a
myxoid
liposarcoma; and (F) a dedifferentiated liposarcoma.
FIG. 30. Expression of prohibitin in human tissues. Prohibitin expression was
determined by immunohistochemistry of fixed human paraffin-embedded thin
tissue
sections with rabbit polyclonal antibodies against prohibitin. Arrows indicate
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prohibitin staining in normal human tissues of: (A) white fat; (B) skin; (C)
prostate;
(E) bone; and (F) muscle; and (F) skeletal muscle. A fat staining control is
shown in
(D).
FIG. 31. Model of prohibitin function in fat vasculature.
FIG. 32. AAP construction. The AAP vector was constructed as disclosed in
Example 6.
FIG. 33. Distribution of IL-llRa expression in primary androgen-dependent
prostate carcinoma by immunohistochemical score, according to Gleason grade
and
pathological stage.
FIG. 34. Screening procedure for biopanning against ovarian cancer ascites.
FIG. 35. Specificity of phage binding to ovarian cancer IgG vs. BSA or control
IgGs.
FIG. 36. Validation of ovarian cancer targeting by competition for binding to
IgGs isolated from ovarian cancer ascites to immobilized GST fusion peptides
versus
the corresponding synthetic peptide (CVPELGHEC, SEQ ID N0:132).
FIG. 37. Reactivity between GST- CVPELGHEC (SEQ ID N0:132) fusion
peptide and ascites from patients with different stages of ovarian cancer
versus non-
ovarian cancer or non-malignant conditions. Positive reactivity is indicated
as the ratio
between binding to GST-fusion peptide compared to GST alone.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
As used herein in the specification, "a" or "an" may mean one or more. As used
herein in the claim(s), in conjunction with the word "comprising," the words
"a" or
"an" may mean one or more than one. As used herein "another" may mean at least
a
second or more of an item.
A "targeting peptide" is a peptide comprising a contiguous sequence of amino
acids, which is characterized by selective localization to an organ, tissue or
cell type.
Selective localization may be determined, for example, by methods disclosed
below,
wherein the putative targeting peptide sequence is incorporated into a protein
that is
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displayed on the outer surface of a phage. Administration to a subject of a
library of
such phage that have been genetically engineered to express a multitude of
such
targeting peptides of different amino acid sequence is followed by collection
of one or
more organs, tissues or cell types from the subject and identification of
phage found in
that organ, tissue or cell type. A phage expressing a targeting peptide
sequence is
considered to be selectively localized to a tissue or organ if it exhibits
greater binding in
that tissue or organ compared to a control tissue or organ. Preferably,
selective
localization of a targeting peptide should result in a two-fold or higher
enrichment of
the phage in the target organ, tissue or cell type, compared to a control
organ, tissue or
cell type. Selective localization resulting in at least a three-fold, four-
fold, five-fold,
six-fold, seven-fold, eight-fold, nine-fold, ten-fold or higher enrichment in
the target
organ compared to a control organ, tissue or cell type is more preferred.
Alternatively,
a phage expressing . a targeting peptide sequence that exhibits selective
localization
preferably shows an increased enrichment in the target organ compared to a
control
organ when phage recovered from the target organ are reinjected into a second
host for
another round of screening. Further enrichment may be exhibited following a
third
round of screening. Another alternative means to determine selective
localization is
that phage expressing the putative target peptide preferably exhibit a two-
fold, more
preferably a three-fold or higher enrichment in the target organ compared to
control
phage that express a non-specific peptide or that have not been genetically
engineered
to express any putative target peptides. Another means to determine selective
localization is that localization to the target organ of phage expressing the
target peptide
is at least partially blocked by the co-administration of a synthetic peptide
containing
the target peptide sequence. "Targeting peptide" and "homing peptide" are used
synonymously herein.
A "phage display library" means a collection of phage that have been
genetically
engineered to express a set of putative targeting peptides on their outer
surface. In
preferred embodiments, DNA sequences encoding the putative targeting peptides
are
inserted in frame into a gene encoding a phage capsule protein. In other
preferred
embodiments, the putative targeting peptide sequences are in part random
mixtures of
all twenty amino acids and in part non-random. In certain preferred
embodiments the
CA 02496938 2005-02-23
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putative targeting peptides of the phage display library exhibit one or more
cysteine
residues at fixed locations within the targeting peptide sequence. Cysteines
may be
used, for example, to create a cyclic peptide.
A "macromolecular complex" refers to a collection of molecules that may be
random, ordered or partially ordered in their arrangement. The term
encompasses
biological organisms such as bacteriophage, viruses, bacteria, unicellular
pathogenic
organisms, multicellular pathogenic organisms and prokaryotic or eukaryotic
cells. The
term also encompasses non-living assemblages of molecules, such as liposomes,
microcapsules, microparticles, magnetic beads and microdevices. The only
requirement
is that the complex contains more than one molecule. The molecules may be
identical,
or may differ from each other.
A "receptor" for a targeting peptide includes but is not limited to any
molecule
or macromolecular complex that binds to a targeting peptide. Non-limiting
examples of
receptors include peptides, proteins, glycoproteins, lipoproteins, epitopes,
lipids,
carbohydrates, multi-molecular structures, a specific conformation of one or
more
molecules and a morphoanatomic entity. In preferred embodiments, a "receptor"
is a
naturally occurring molecule or complex of molecules that is present on the
lumenal
surface of cells forming blood vessels within a target organ, tissue or cell
type.
A "subject" refers generally to a mammal. In certain preferred embodiments,
the subject is a mouse or rabbit. In even more preferred embodiments, the
subject is a
human.
Prostate Cancer Detection and Diagnosis
A particular problem in cancer detection and diagnosis occurs with prostate
cancer. Carcinoma of the prostate (PCA) is the most frequently diagnosed
cancer
among men in the United States. Although relatively few prostate tumors
progress to
clinical significance during the lifetime of the patient, those which are
progressive in
nature are likely to have metastasized by the time of detection. Survival
rates for
individuals with metastatic prostate cancer are quite low. Between these
extremes are
patients with prostate tumors that will metastasize but have not yet done so,
for whom
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surgical prostate removal is curative. Determination of which group a patient
falls
within is critical in determining optimal treatment and patient survival.
Serum prostate specific antigen (PSA) is widely used as a biomarker to detect
and monitor therapeutic response in prostate cancer patients (Badalament et
al., 1996;
ODowd et al., 1997). Although PSA has been widely used since 1988 as a
clinical
marker of prostate cancer (Partin and Oesterling, 1994), screening programs
utilizing
PSA alone or in combination with digital rectal examination (DRE) have not
been
successful in improving the survival rate for men with prostate cancer (Partin
and
Oesterling, 1994). PSA is produced by normal and benign as well as malignant
prostatic tissue, resulting in a high false-positive rate for prostate cancer
detection
(Partin and Oesterling, 1994). While an effective indicator of prostate cancer
when
serum levels are relatively high, PSA serum levels are more ambiguous
indicators of
prostate cancer when only modestly elevated. The specificity of the PSA assay
for
prostate cancer detection at low serum PSA levels remains a problem.
Other markers that have been used for prostate cancer detection include
prostatic acid phosphatase (PAP) (Brawn et al., 1996), prostate secreted
protein (PSP)
(Huang et al., 1993), prostate specific membrane antigen (PSMA) (Murphy et
al.,
1995), human kallekrein 2 (HK2) (Piironen et al., 1996), prostate specific
transglutaminase (pTGase) and interleukin 8 (IL-8) (Veltri et al., 1999). None
of these
has yet been demonstrated to provide a more sensitive and discriminating test
for
prostate cancer than PSA.
In addition to these protein markers for prostate cancer, genetic changes
reported to be associated with prostate cancer, include allelic loss (Bova, et
al., 1993);
DNA hypermethylation (Isaacs et al., 1994); point mutations or deletions of
the
retinoblastoma (Rb), p53 and KAll genes (Isaacs et al., 1991); aneuploidy and
aneusomy of chromosomes detected by fluorescence ifz situ hybridization (FISH)
(Macoska et al., 1994) and differential expression of HER2/rzeu oncogene
receptor (An
et al., 1998). None of these has been reported to exhibit sufficient
sensitivity and
specificity to be useful as general screening tools for asymptomatic prostate
cancer.
In current clinical practice, the serum PSA assay and digital rectal exam
(DRE)
is used to indicate which patients should have a prostate biopsy (Orozco et
al., 1998).
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Histological examination of the biopsied tissue is used to make the diagnosis
of prostate
cancer. It is estimated that over a half million prostate biopsies are
performed annually
in the United States (Orozco et al., 1998). A need exists for a serological
test that is
sensitive enough to detect small and early stage prostate tumors, that also
has sufficient
specificity to exclude a greater portion of patients with noncancerous
conditions such as
BPH.
There remain deficiencies in the prior art with respect to the identification
of
markers linked with the progression of prostate cancer and the development of
diagnostic methods to monitor disease progression. The identification of
novel,
prostate selective or specific markers that are differentially expressed in
metastatic
and/or non-metastatic prostate cancer, compared to non-malignant prostate
tissue,
would represent a major, unexpected advance for the diagnosis, prognosis and
treatment
of prostate cancer. As discussed below, one approach to identifying novel
prostate
cancer markers involves the phage dislay technique. The skilled artisan will
realize that
although various embodiments of the invention are discussed in terms of
prostate
cancer, the disclosed methods and/or compositions may be of use to identify
markers
(targeting peptides) for other types of cancer within the scope of the
invention.
Phage Display
Recently, an iu vivo selection system was developed using phage display
libraries to identify organ, tissue or cell type-targeting peptides in a mouse
model
system. Phage display libraries expressing transgenic peptides on the surface
of
bacteriophage were initially developed to map epitope binding sites of
immunoglobulins (Smith, GP and Scott, JK, 1985. Science, 228:1315-1317, Smith,
GP
and Scott, JK, 1993. Meth. Ehzymol. 21:228-257). Such libraries can be
generated by
inserting random oligonucleotides into cDNAs encoding a phage surface protein,
generating collections of phage particles displaying unique peptides in as
many as 109
permutations. (Pasqualini, R. and Ruoslahti, E. 1996, Nature, 380: 364-366;
Arap et al,
1998a; Arap et al., 1998b, Curr. Opin. Oncol. 10:560-565).
Intravenous administration of phage display libraries to mice was followed by
the recovery of phage from individual organs (Pasqualini and Ruoslahti, 1996).
Phage
were recovered that were capable of selective homing to the vascular beds of
different
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mouse organs, tissues or cell types, based on the specific targeting peptide
sequences
expressed on the outer surface of the phage (Pasqualini and Ruoslahti, 1996).
A variety
of organ and tumor-homing peptides have been identified by this method
(Rajotte et al.,
1998, J. Clin. Invest. 102:430-437; Rajotte et al, 1999, T. Biol. Che»i.
274:11593-
11598; Koivunen et al., 1999x, Nature Biotechfiol. 17: 768-774; Burg M, et
al., 1999a,
Cancer Res. 58:2869-2874; Pasqualini" 1999, Quart. J. Nucl. Med. 43:159-162).
Each
of those targeting peptides bound to different receptors that were selectively
expressed
on the vasculature of the mouse target tissue (Pasqualini, 1999; Pasqualini et
al., 2000;
Folkman J. Nature Biotechnol. 15:510, 1997; Folkman J. Nature Med 1:27-31,
1995).
In addition to identifying individual targeting peptides selective for an
organ, tissue or
cell type (Pasqualini and Ruoslahti, 1996; Arap et al, 1998x; Koivunen et al.,
Methods
Mol. Biol. 129: 3-17, 1999b), this system has been used to identify
endothelial cell
surface markers that are expressed in mice in vivo (Rajotte and Ruoslahti,
1999).
Attachment of therapeutic agents to targeting peptides resulted in the
selective
delivery of the agent to a desired organ, tissue or cell type in the mouse
model system.
Targeted delivery of chemotherapeutic agents and proapoptotic peptides to
receptors
located in tumor angiogenic vasculature resulted in an increase in therapeutic
efficacy
and a decrease in systemic toxicity in tumor bearing mouse models (Arap et
al., 1998x,
1998b; Ellerby et al., Nature Med 9:1032-1038, 1999).
The methods described herein for identification of targeting peptides involve
the
if2 vivo administration of phage display libraries. Various methods of phage
display and
methods for producing diverse populations of peptides are well known in the
art. For
example, U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods
for
preparing a phage library. The phage display technique involves genetically
manipulating bacteriophage so that small peptides can be expressed on their
surface
(Smith and Scott, 1985, 1993). The past decade has seen considerable progress
in the
construction of phage-displayed peptide libraries and in the development of
screening
methods in which the libraries are used to isolate peptide ligands. For
example, the use
of peptide libraries has made it possible to characterize interacting sites
and receptor-
ligand binding motifs within many proteins, such as antibodies involved in
inflammatory reactions or integrins that mediate cellular adherence. This
method has
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also been used to identify novel peptide ligands that serve as leads to the
development
of peptidomimetic drugs or imaging agents (Arap et al., 1998a). In addition to
peptides, larger protein domains such as single-chain antibodies can also be
displayed
on the surface of phage particles (Arap et al., 1998a).
Targeting peptides selective for a given organ, tissue or cell type can be
isolated
by "biopanning" (Pasqualini and Ruoslahti, 1996; Pasqualini, 1999). In brief,
a library
of phage containing putative targeting peptides is administered to an animal
or human
and samples of organs, tissues or cell types containing phage are collected.
In preferred
embodiments utilizing filamentous phage, the phage may be propagated in vitro
between rounds of biopanning in pilus-positive bacteria. The bacteria are not
lysed by
the phage but rather secrete multiple copies of phage that display a
particular insert.
Phage that bind to a target molecule can be eluted from the target organ,
tissue or cell
type and then amplified by growing them in host bacteria. If desired, the
amplified
phage can be administered to a host and samples of organs, tissues or cell
types again
collected. Multiple rounds of biopanning can be performed until a population
of
selective binders is obtained. The amino acid sequence of the peptides is
determined by
sequencing the DNA corresponding to the targeting peptide insert in the phage
genome.
The identified targeting peptide can then be produced as a synthetic peptide
by standard
protein chemistry techniques (Arap et al., 1998a, Smith and Scott, 1985). This
approach allows circulating targeting peptides to be detected in an unbiased
functional
assay, without any preconceived notions about the nature of their target. Once
a
candidate target is identified as the receptor of a targeting peptide, it can
be isolated,
purified and cloned by using standard biochemical methods (Pasqualini, 1999;
Rajotte
and Ruoslahti, 1999).
In certain embodiments, a subtraction protocol may be used with biopanning to
further reduce background phage binding. The purpose of subtraction is to
remove
phage from the library that bind to cells other than the cell of interest, or
that bind to
inactivated cells. In alternative embodiments, the phage library may be
prescreened
against a subject who does not possess the targeted cell, tissue or organ. For
example,
prostate and/or prostate cancer binding peptides may be identified after
prescreening a
library against female subjects. After subtraction, the library may be
screened against
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the cell, tissue or organ of interest. In another alternative embodiment, an
unstimulated,
quiescent cell type, tissue or organ may be screened against the library and
binding
phage removed. The cell line, tissue or organ is then activated, for example
by
administration of a hormone, growth factor, cytokine or chemokine and the
activated
cell type, tissue or organ screened against the subtracted phage library.
Other methods
of subtraction protocols are known and may be used in the practice of the
present
invention, for example as disclosed in U.S Patent Nos. 5,840,841, 5,705,610,
5,670,312
and 5,492,807.
Choice of phage display system.
Previous in vivo selection studies performed in mice preferentially employed
libraries of random peptides expressed as fusion proteins with the gene III
capsule
protein in the fUSE5 vector (Pasqualini and Ruoslahti, 1996). The number and
diversity of individual clones present in a given library is a significant
factor for the
success of in vivo selection. It is preferred to use primary libraries, which
are less likely
to have an over-representation of defective phage clones (Koivunen et al.,
1999b). The
preparation of a library should be optimized to between 10$-109 transducing
units
(T.U.)/ml. In certain embodiments, a bulk amplification strategy is applied
between
each round of selection.
Phage libraries displaying linear, cyclic, or double cyclic peptides may be
used
within the scope of the present invention. However, phage libraries displaying
3 to 10
random residues in a cyclic insert (CX3_loC) are preferred, since single
cyclic peptides
tend to have a higher affinity for the target organ than linear peptides.
Libraries
displaying double-cyclic peptides (such as CX3C X3CX3C; Rojotte et al., 1998)
have
been successfully used. However, the production of the cognate synthetic
peptides,
although possible, can be complex due to the multiple conformers with
different
disulfide bridge arrangements.
Identification of homing peptides ahd receptors by ire vivo plzage display ifz
mice.
Irz vivo selection of peptides from phage-display peptide libraries
administered
to mice has been used to identify targeting peptides selective for normal
mouse brain,
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kidney, lung, skin, pancreas, retina, intestine, uterus, prostate, and adrenal
gland
(Pasqualini and Ruoslahti, 1996; Pasqualini, 1999; Rajotte et al., 1998).
These results
show that the vascular endothelium of normal organs is sufficiently
heterogeneous to
allow differential targeting with peptide probes (Pasqualini and Ruoslahti,
1996;
Rajotte et al., 1998). A panel of peptide motifs that target the blood vessels
of tumor
xenografts in nude mice has been assembled (Arap et al., 1998a; reviewed in
Pasqualini, 1999). These motifs include the sequences RGD-4C, NGR, and GSL.
The
RGD-4C peptide has previously been identified as selectively binding av
integrins and
has been reported to home to the vasculature of tumor xenografts in nude mice
(Arap et
al., 1998a, 1998b; Pasqualini et al., Nature Biotechnol 15: 542-546, 1997).
The receptors for the tumor homing RGD4C targeting peptide has been
identified as av integrins (Pasqualini et al., 1997). The av integrins play an
important
role in angiogenesis. The av(33 and av~i5 integrins are absent or expressed at
low
levels in normal endothelial cells and are induced in angiogenic vasculature
of tumors
(Brooks PC, Clark RA, Cheresh DA. Science, 264: 569-571, 1994a; Hammes HP,
Brownlee M, Jonczyk A, Sutter A, and Preissner I~T. Nature Meel. 2: 529-533,
1996.).
Aminopeptidase N/CD 13 has recently been identified as an angiogenic receptor
for the
NGR motif (Burg, M.A., et al. Cancer Res. 59, 2869-2874, 1999.).
Aminopeptidase
N/CD13 is strongly expressed not only in the angiogenic blood vessels of
prostate
cancer in TRAMP mice but also in the normal epithelial prostate tissue.
Tumor-homing phage co-localize with their receptors in the angiogenic
vasculature of tumors but not in non-angiogenic blood vessels in normal
tissues (Arap
et al., 1998b). Immunohistochemical evidence shows that vascular targeting
phage
bind to human tumor blood vessels in tissue sections (Pasqualini et al., 2000)
but not to
normal blood vessels. A negative control phage with no insert (fd phage) did
not bind
to normal or tumor tissue sections. The expression of the angiogenic receptors
was
evaluated in cell lines, in non-proliferating blood vessels and in activated
blood vessels
of tumors and other angiogenic tissues such as corpus luteum. Flow cytometry
and
immunohistochemistry showed that these receptors are expressed in a number of
tumor
cells and in activated HUVECs (data not shown). The angiogenic receptors were
not
detected in the vasculature of normal organs of mouse or human tissues.
27
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The distribution of these receptors was analyzed by immunohistochemistry in
tumor cells, tumor vasculature, and normal vasculature. Alpha v integrins, CD
13,
aminopeptidase A, NG2, and MMP-2/MZVB'-9 - the known receptors in tumor blood
vessels - are specifically expressed in angiogenic endothelial cells and
pericytes of both
human and murine origin. Angiogenic neovasculature expresses markers that are
either
expressed at very low levels or not at all in non-proliferating endothelial
cells (not
shown).
The markers of angiogenic endothelium include receptors for vascular growth
factors, such as specific subtypes of VEGF and basic FGF receptors, and av
integrins,
among many others (Mustonen T and Alitalo K. J. Cell Biol. 129:895-898,
1995.).
Thus far, identification and isolation of novel molecules characteristic of
angiogenic
wasculature has been slow, mainly because endothelial cells undergo dramatic
phenotypic changes when grown in culture (Watson et al., Science, 268:447-448,
1995).
Many of these tumor vascular markers are proteases and some of the markers
also serve as viral receptors. Alpha v integrins are receptors for
adenoviruses
(Wickham et al., Cahcer Immuf2ol. Immunother. 45:149-151, 1997c) and CD13 is a
receptor for coronaviruses (Look et al. N. J. Cli~c. Invest. 83:1299-1307,
1989.).
MMP-2 and MMP-9 are receptors for echoviruses (Koivunen et al., 1999a).
Aminopeptidase A also appears to be a viral receptor. Bacteriophage may use
the same
cellular receptors as eukaryotic viruses. These findings suggest that
receptors isolated
by ifa vivo phage display will have cell internalization capability, a key
feature for
utilizing the identified peptide motifs as targeted gene therapy carriers.
Targeted delivery
Peptides that home to tumor vasculature have been coupled to cytotoxic drugs
or proapoptotic peptides to yield compounds that were more effective and less
toxic
than the parental compounds in experimental models of mice bearing tumor
xenografts
(Arap et al., 1998a; Ellerby et al, 1999). The insertion of the RGD-4C peptide
into a
surface protein of an adenovirus has produced an adenoviral vector that may be
of use
for tumor targeted gene therapy (Arap et al., 1998b). A need exists for
improved gene
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therapy vectors capable of targeted delivery in human subjects, particularly
for
improved vectors that exhibit prolonged expression of therapeutic genes in the
transfected cells.
BRASIL
In preferred embodiments, separation of phage bound to the cells of a target
organ, tissue or cell type from unbound phage is achieved using the BRASIL
technique
(PCT Patent Application PCT/LTS01/2~124 entitled, "Biopanning and Rapid
Analysis
of Selective Interactive Ligands (BRASIL)" by Arap et al., filed September 7,
2001,
incorporated herein by reference in its entirety). In BRAS1L (Biopanning and
Rapid
Analysis of Soluble Interactive Ligands), an organ, tissue or cell type is
gently separated
into cells or small clumps of cells that are suspended in an aqueous phase.
The aqueous
phase is layered over an organic phase of appropriate density and centrifuged.
Cells
attached to bound phage are pelleted at the bottom of the centrifuge tube,
while
unbound phage remain in the aqueous phase. This allows a more efficient
separation of
bound from unbound phage, while maintaining the binding interaction between
phage
and cell. BRASIL may be performed in an in vivo protocol, in which organs,
tissues or
cell types are exposed to a phage display library by intravenous
administration, or by an
ex vivo protocol, where the cells are exposed to the phage library in the
aqueous phase
before centrifugation. A non-limiting exemplary application of the BRASIL
technique
is disclosed in the Examples below.
Preparation of large scale primary libraries
In certain embodiments, primary phage libraries are amplified before injection
into a human subject. A phage library is prepared by ligating targeting
peptide-
encoding sequences into a phage vector, such as fUSES. The vector is
transformed into
pilus negative host E. coli such as strain MC1061. The bacteria are grown
overnight
and then aliquots are frozen to provide stock for library production. Use of
pilus
negative bacteria avoids the bias in libraries that arises from differential
infection of
pilus positive bacteria by different targeting peptide sequences.
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To freeze, bacteria are pelleted from two thirds of a primary library culture
(5
liters) at 4000 x g for 10 min. Bacteria are resuspended and washed twice with
500 ml
of 10% glycerol in water, then frozen in an ethanol/dry ice bath and stored at
-80°C.
For amplification, 1.5 ml of frozen. bacteria are inoculated into 5 liters of
LB
medium with 20 p,g/ml tetracycline and grown overnight. Thirty minutes after
inoculation, a serial dilution is plated on LB/tet plates to verify the
viability of the
culture. If the number of viable bacteria is less than 5-10 times the number
of
individual clones in the library (1-2 x 10$) the culture is discarded.
After growing the bacterial culture overnight, phage are precipitated. About
1/4
to 1/3 of the bacterial culture is kept growing overnight in 5 liters of fresh
medium and
the cycle is repeated up to 5 times. Phage are pooled from all cycles and used
for
injection into human subjects.
Human Subjects
The methods used for phage display biopanning in the mouse model system
require substantial improvements for use with humans. Techniques for
biopanning in
human subjects are disclosed in PCT Patent Application PCT/USO1/28044, filed
September 7, 2001, the entire text of which is incorporated herein by
reference. In
general, humans suitable for use with phage display are either brain dead or
terminal
wean patients. The amount of phage library (preferably primary library)
required for
administration must be significantly increased, preferably to 1014 TU or
higher,
preferably administered intravenously in approximately 200 ml of Ringer
lactate
solution over about a 10 minute period.
The amount of phage required for use in humans has required substantial
improvement of the mouse protocol, increasing the amount of phage available
for
injection by five orders of magnitude. To produce such large phage libraries,
the
transformed bacterial pellets recovered from up to 500 to 1000 transformations
are
amplified up to 10 times in the bacterial host, recovering the phage from each
round of
amplification and adding LB Tet medium to the bacterial pellet for collection
of
additional phage. The phage inserts remain stable under these conditions and
phage
may be pooled to form the large phage display library required for humans.
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Samples of various organs and tissues are collected starting approximately 15
minutes after injection of the phage library. Samples are processed as
described below
and phage collected from each organ, tissue or cell type of interest for DNA
sequencing
to determine the amino acid sequences of targeting peptides.
With humans, the opportunities for enrichment by multiple rounds of
biopanning are severely restricted, compared to the mouse model system. A
substantial
improvement in the biopanning technique involves polyorgan targeting.
Polyorgah targeting
In the standard protocol for phage display biopanning, phage from a single
organ are collected, amplified and injected into a new host, where tissue from
the same
organ is collected for phage rescue and a new round of biopanning. This
protocol is
feasible in animal subjects. However, the limited availability and expense of
processing samples from humans requires an improvement in the protocol.
It is possible to pool phage collected from multiple organs after a first
round of
biopanning and inject the pooled sample into a new subject, where each of the
multiple
organs may be collected again for phage rescue. The polyorgan targeting
protocol may
be repeated for as many rounds of biopanning as desired. In this manner, it is
possible
to significantly reduce the number of subjects required for isolation of
targeting
peptides for multiple organs, while still achieving substantial enrichment of
the organ-
homing phage.
In preferred embodiments, phage are recovered from human organs, tissues or
cell types after injection of a phage display library into a human subject. In
certain
embodiments, phage may be recovered by exposing a sample of the organ, tissue
or cell
type to a pilus positive bacterium, such as E. coli K91. In alternative
embodiments,
phage may be recovered by amplifying the phage inserts, ligating the inserts
to phage
DNA and producing new phage from the ligated DNA.
Proteins and Peptides
In certain embodiments, the present invention concerns novel compositions
comprising at least one protein or peptide. As used herein, a protein or
peptide
generally refers, but is not limited to, a protein of greater than about 200
amino acids up
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WO 2004/020999 PCT/US2002/034987
to a full length sequence translated from a gene; a polypeptide of about 100
to 200
amino acids; and/or a peptide of from about 3 to about 100 amino acids. For
convenience, the terms "protein," "polypeptide" and "peptide are used
interchangeably
herein.
In certain embodiments the size of at least one protein or peptide may
comprise,
but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about 130,
about 140,
about 150, about 160, about 170, about 180, about 190, about 200, about 210,
about
220, about 230, about 240, about 250, about 275, about 300, about 325, about
350,
about 375, about 400, about 425, about 450, about 475, about 500, about 525,
about
550, about 575, about 600, about 625, about 650, about 675, about 700, about
725,
about 750, about 775, about 800, about 825, about 850, about 875, about 900,
about
925, about 950, about 975, about 1000, about 1100, about 1200, about 1300,
about
1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater
amino
acid residues.
As used herein, an "amino acid residue" refers to any naturally occurring
amino
acid, any amino acid derivative or any amino acid mimic known in the art. In
certain
embodiments, the residues of the protein or peptide are sequential, without
any non-
amino acid interrupting the sequence of amino acid residues. In other
embodiments, the
sequence may comprise one or more non-amino acid moieties. In particular
embodiments, the sequence of residues of the protein or peptide may be
interrupted by
one or more non-amino acid moieties.
Accordingly, the term "protein or peptide" encompasses amino acid sequences
comprising at least one of the 20 common amino acids found in naturally
occurring
proteins, or at least one modified or unusual amino acid, including but not
limited to
those shown on Table 1 below.
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TABLE
1
Modified
and
Unusual
Amino
Acids
Amino Acid Abbr. Amino Acid
bbr.
2-Aminoadipic acid EtAsn N-Ethylasparagine
ad
3- Aminoadipic acid Hyl Hydroxylysine
aad
[3-alanine, (3-Amino-propionicAHyI allo-Hydroxylysine
ala acid
2-Aminobutyric acid 3Hyp 3-Hydroxyproline
bu
4- Aminobutyric acid, 4Hyp 4-Hydroxyproline
Abu piperidinic acid
6-Aminocaproic acid Ide Isodesmosine
cp
2-Aminoheptanoic acid AIIe allo-Isoleucine
he
2-Aminoisobutyric acid MeGly N-Methylglycine,
ib sarcosine
3-Aminoisobutyric acid Melle N-Methylisoleucine
aib
2-Aminopimelic acid MeLys 6-N-Methyllysine
pm
2,4-l~iaminobutyric acid MeVal N-Methylvaline
bu
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TABLE
1
Modified
and
Unusual
Amino
Acids
Amino Acid Abbr. Amino Acid
bbr.
Desmosine Nva Norvaline
es
2,2'-Diaminopimelic acid Nle Norleucine
pm
2,3-Diaminopropionic acid Orn Ornithine
pr
N-Ethylglycine
tGly
Proteins or peptides may be made by any technique known to those of skill in
the art, including the expression of proteins, polypeptides or peptides
through standard
molecular biological techniques, the isolation of proteins or peptides from
natural
sources, or the chemical synthesis of proteins or peptides. The nucleotide and
protein,
polypeptide and peptide sequences corresponding to various genes have been
previously disclosed, and may be found at computerized databases known to
those of
ordinary skill in the art. One such database is the National Center for
Biotechnology
Information's Genbank and GenPept databases (http:llwww.ncbi.nlm.nih.~. The
coding regions for known genes may be amplified and/or expressed using the
techniques disclosed herein or as would be know to those of ordinary skill in
the art.
Alternatively, various commercial preparations of proteins, polypeptides and
peptides
are known to those of skill in the art.
Peptide mimetics
Another embodiment for the preparation of polypeptides according to the
invention is the use of peptide mimetics. Mimetics are peptide-containing
molecules
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that mimic elements of protein secondary structure. See, for example, Johnson
et al.,
"Peptide Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al.,
Eds., Chapman and Hall, New York (1993), incorporated herein by reference. The
underlying rationale behind the use of peptide mimetics is that the peptide
backbone of
proteins exists chiefly to orient amino acid side chains in such a way as to
facilitate
molecular interactions, such as those of antibody and antigen. A peptide
mimetic is
expected to permit molecular interactions similar to the natural molecule.
These
principles may be used to engineer second generation molecules having many of
the
natural properties of the targeting peptides disclosed herein, but with
altered and even
improved characteristics.
Fusion proteins
Other embodiments of the present invention concern fusion proteins. These
molecules generally have all or a substantial portion of a targeting peptide,
linked at the
N- or C-terminus, to all or a portion of a second polypeptide or protein. For
example,
fusions may employ leader sequences from other species to permit the
recombinant
expression of a protein in a heterologous host. Another useful fusion includes
the
addition of an irnmunologically active domain, such as an antibody epitope, to
facilitate
purification of the fusion protein. Inclusion of a cleavage site at or near
the fusion
junction will facilitate removal of the extraneous polypeptide after
purification. Other
useful fusions include linking of functional domains, such as active sites
from enzymes,
glycosylation domains, cellular targeting signals or transmembrane regions. In
preferred embodiments, the fusion proteins of the instant invention comprise a
targeting
peptide linked to a therapeutic protein or peptide. Examples of proteins or
peptides that
may be incorporated into a fusion protein include cytostatic proteins,
cytocidal proteins,
pro-apoptosis agents, anti-angiogenic agents, hormones, cytokines, growth
factors,
peptide drugs, antibodies, Fab fragments antibodies, antigens, receptor
proteins,
enzymes, lectins, MHC proteins, cell adhesion proteins and binding proteins.
These
examples are not meant to be limiting and it is contemplated that within the
scope of
the present invention virtually and protein or peptide could be incorporated
into a
fusion protein comprising a targeting peptide. Methods of generating fusion
proteins
are well known to those of skill in the art. Such proteins can be produced,
for example,
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
by chemical attachment using bifunctional cross-linking reagents, by de novo
synthesis
of the complete fusion protein, or by attachment of a DNA sequence encoding
the
targeting peptide to a DNA sequence encoding the second peptide or protein,
followed
by expression of the intact fusion protein.
Protein purifccation
In certain embodiments a protein or peptide may be isolated or purified.
Protein
purification techniques are well known to those of skill in the art. These
techniques
involve, at one level, the homogenization and crude fractionation of the
cells, tissue or
organ to polypeptide and non-polypeptide fractions. The protein or polypeptide
of
interest may be further purified using chromatographic and electrophoretic
techniques
to achieve partial or complete purification (or purification to homogeneity).
Analytical
methods particularly suited to the preparation of a pure peptide are ion-
exchange
chromatography, gel exclusion chromatography, polyacrylamide gel
electrophoresis,
affinity chromatography, immunoaffinity chromatography, reverse phase
chromatography and isoelectric focusing. An example of receptor protein
purification
by affinity chromatography is disclosed in U.S. Patent No. 5,206,347, the
entire text of
which is incorporated herein by reference. A particularly efficient method of
purifying
peptides is fast performance liquid chromatography (FPLC) or even high
performance
liquid chromatography (HPLC).
A purified protein or peptide is intended to refer to a composition,
isolatable
from other components, wherein the protein or peptide is purified to any
degree relative
to its naturally-obtainable state. An isolated or purified protein or peptide,
therefore,
also refers to a protein or peptide free from the environment in which it may
naturally
occur. Generally, "purified" will refer to a protein or peptide composition
that has been
subjected to fractionation to remove various other components, and which
composition
substantially retains its expressed biological activity. Where the term
"substantially
purified" is used, this designation will refer to a composition in which the
protein or
peptide forms the major component of the composition, such as constituting
about 50%,
about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins
in
the composition.
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Various methods for quantifying the degree of purification of the protein or
peptide are known to those of skill in the art in light of the present
disclosure. These
include, for example, determining the specific activity of an active fraction,
or assessing
the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred
method for assessing the purity of a fraction is to calculate the specific
activity of the
fraction, to compare it to the specific activity of the initial extract, and
to thus calculate
the degree of purity therein, assessed by a "-fold purification number." The
actual units
used to represent the amount of activity will, of course, be dependent upon
the
particular assay technique chosen to follow the purification, and whether or
not the
expressed protein or peptide exhibits a detectable activity.
Various techniques suitable for use in protein purification are well known to
those of skill in the art. These include, for example, precipitation with
ammonium
sulphate, PEG, antibodies and the like, or by heat denaturation, followed by:
centrifugation; chromatography steps such as ion exchange, gel filtration,
reverse phase,
hydroxylapatite and affinity chromatography; isoelectric focusing; gel
electrophoresis;
and combinations of these and other techniques. As is generally known in the
art, it is
believed that the order of conducting the various purification steps may be
changed, or
that certain steps may be omitted, and still result in a suitable method for
the
preparation of a substantially purified protein or peptide.
There is no general requirement that the protein or peptide always be provided
in their most purified state. Indeed, it is contemplated that less
substantially purified
products will have utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or by utilizing
different
forms of the same general purification scheme. For example, it is appreciated
that a
cation-exchange column chromatography performed utilizing an HPLC apparatus
will
generally result in a greater "-fold" purification than the same technique
utilizing a low
pressure chromatography system. Methods exhibiting a lower degree of relative
purification may have advantages in total recovery of protein product, or in
maintaining
the activity of an expressed protein.
Affinity chromatography is a chromatographic procedure that relies on the
specific affinity between a substance to be isolated and a molecule to which
it can
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specifically bind. This is a receptor-ligand type of interaction. The column
material is
synthesized by covalently coupling one of the binding partners to an insoluble
matrix.
The column material is then able to specifically adsorb the substance from the
solution.
Elution occurs by changing the conditions to those in which binding will not
occur
(e.g., altered pH, ionic strength, temperature, etc.). The matrix should be a
substance
that itself does not adsorb molecules to any significant extent and that has a
broad range
of chemical, physical and thermal stability. The ligand should be coupled in
such a way
as to not affect its binding properties. The ligand should also provide
relatively tight
binding. And it should be possible to elute the substance without destroying
the sample
or the ligand.
Synthetic Peptides
Because of their relatively small size, the targeting peptides of the
invention can
be synthesized in solution or on a solid support in accordance with
conventional
techniques. Various automatic synthesizers are commercially available and can
be used
in accordance with known protocols. See, for example, Stewart and Young, Solid
Phase Peptide Synthesis, 2d ed. Pierce Chemical Co., 1984; Tam et al., J. Am.
Chem.
Soc., 105:6442, 1983; Merrifield, Scief2ce, 232: 341-347, 1986; and Barany and
Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New
York, pp.
1-284, 1979, each incorporated herein by reference. Short peptide sequences,
usually
from about 6 up to about 35 to 50 amino acids, can be readily synthesized by
such
methods. Alternatively, recombinant DNA technology may be employed wherein a
nucleotide sequence which encodes a peptide of the invention is inserted into
an
expression vector, transformed or transfected into an appropriate host cell,
and
cultivated under conditions suitable for expression.
Antibodies
In certain embodiments, it may be desirable to make antibodies against the
identified targeting peptides or their receptors. The appropriate targeting
peptide or
receptor, or portions thereof, may be coupled, bonded, bound, conjugated, or
chemically-linked to one or more agents via linkers, polylinkers, or
derivatized amino
acids. This may be performed such that a bispecific or multivalent composition
or
vaccine is produced. It is further envisioned that the methods used in the
preparation of
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WO 2004/020999 PCT/US2002/034987
these compositions are familiar to those of skill in the art and should be
suitable for
administration to humans, i.e., pharmaceutically acceptable. Preferred agents
are the
carriers are keyhole limpet hemocyanin (I~LLH) or bovine serum albumin (BSA).
The term "antibody" is used to refer to any antibody-like molecule that has an
antigen binding region, and includes antibody fragments such as Fab', Fab,
F(ab')~,
single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
Techniques
for preparing and using various antibody-based constructs and fragments are
well
known in the art. Means for preparing and characterizing antibodies are also
well
known in the art (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,
Cold
Spring Harbor Laboratory, 1988; incorporated herein by reference).
In various embodiments of the invention, circulating antibodies from one or
more individuals with a disease state may be obtained and screened against
phage
display libraries. Targeting peptides that bind to the circulating antibodies
may act as
mimeotopes of a native antigen, such as a receptor protein located on an
endothelial cell
surface of a target tissue. For example, circulating antibodies in an
individual with
prostate cancer may bind to antigens specifically or selectively localized in
prostate
tumors. As discussed in more detail below, targeting peptides against such
antibodies
may be identified by phage display. Such targeting peptides may be used to
identify the
native antigen recognized by the antibodies, for example by using known
techniques
such as immunoaffinity purification, Western blotting, electrophoresis
followed by
band excision and protein/peptide sequencing and/or computerized homology
searches.
The skilled artisan will realize that antibodies against disease specific or
selective
antigens may be of use for various applications, such as detection, diagnosis
and/or
prognosis of a disease state, imaging of diseased tissues and/or targeted
delivery of
therapeutic agents.
lynagihg agents and radioisotopes
In certain embodiments, the claimed peptides or proteins of the present
invention may be attached to imaging agents of use for imaging and diagnosis
of
various diseased organs, tissues or cell types. For example, a prostate cancer
selective
targeting peptide may be attached to an imaging agent, provided to a subject
and the
precise boundaries of the cancer tissue may be determined by standard imaging
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techniques, such as CT scanning, MRI, PET scanning, etc. Alternatively, the
presence
or absence and location in the body of metastatic prostate cancer may be
determined by
imaging using one or more targeting peptides that are selective for metastatic
prostate
cancer. Targeting peptides that bind to normal as well as cancerous prostate
tissues
may still be of use, as such peptides would not be expected to be selectively
localized
anywhere besides the prostate in disease-free individuals. Naturally, the
distribution of
a prostate or prostate cancer selective targeting peptide may be compared to
the
distribution of one or more non-selective peptides to provide even greater
discrimination for detection and/or localization of diseased tissues.
Many appropriate imaging agents are known in the art, as are methods for their
attachment to proteins or peptides (see, e.g., U.S. patents 5,021,236 and
4,472,509, both
incorporated herein by reference). Certain attachment methods involve the use
of a
metal chelate complex employing, for example, an organic chelating agent such
a
DTPA attached to the protein or peptide (U.S. Patent 4,472,509). Proteins or
peptides
also may be reacted with an enzyme in the presence of a coupling agent such as
glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared
in the
presence of these coupling agents or by reaction with an isothiocyanate.
Non-limiting examples of paramagnetic ions of potential use as imaging agents
include chromium (III), manganese (II), iron (111), iron (II), cobalt (II),
nickel (II),
copper (II), neodymium (Ill), samarium (I~, ytterbium (III), gadolinium (III),
vanadium
(In, terbium (III), dysprosium (III), holmium (111) and erbium (11T), with
gadolinium
being particularly preferred. Ions useful in other contexts, such as X-ray
imaging,
include but are not limited to lanthanum (111), gold (111), lead (II), and
especially
bismuth (111).
Radioisotopes of potential use as imaging or therapeutic agents include
astatine2y l4carbon, Slchromium, 36chlorine, S~cobalt, SBCObalt, copper6~,
iszEu,
gallium6~, 3hydrogen, iodine123, iodinelas, iodineisy indiumll, s9iron,
3aphosphorus,
rheniumls6, rheniuml88, ~$selenium, 35sulphur, technicium99m and
yttrium9°. izsl is often
being preferred for use in certain embodiments, and technicium9~m and indiums
are
also often preferred due to their low energy and suitability for long range
detection.
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Radioactively labeled proteins or peptides of the present invention may be
produced according to well-known methods in the art. For instance, they can be
iodinated by contact with sodium or potassium iodide and a chemical oxidizing
agent
such as sodium hypochlorite, or an enzymatic oxidizing agent, such as
lactoperoxidase.
Proteins or peptides according to the invention may be labeled with technetium-
99m by
ligand exchange process, for example, by reducing pertechnate with stannous
solution,
ch~lating the reduced technetium onto a Sephadex column and applying the
peptide to
this column or by direct labeling techniques, e.g., by incubating pertechnate,
a reducing
agent such as SNCl2, a buffer solution such as sodium-potassium phthalate
solution,
and the peptide. Intermediary functional groups that are often used to bind
radioisotopes that exist as metallic ions to peptides are
diethylenetriaminepenta-acetic
acid (DTPA) and ethylene diaminetetra-acetic acid (EDTA). Also contemplated
for use
are fluorescent labels, including rhodamine, fluorescein isothiocyanate and
renographin.
In certain embodiments, the claimed proteins or peptides may be linked to a
secondary binding ligand or to an enzyme (an enzyme tag) that will generate a
colored
product upon contact with a chromogenic substrate. Examples of suitable
enzymes
include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and
glucose
oxidase. Preferred secondary binding ligands are biotin and avidin or
streptavidin
compounds. The use of such labels is well known to those of skill in the art
in light and
is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350;
3,996,345;
4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
IrYOSS-~112kG'YS
The targeting peptides, ligands, receptor proteins and other molecules of
interest
may be attached to surfaces or to therapeutic agents and other molecules using
a variety
of known cross-linking agents. Methods for covalent or non-covalen attachment
of
proteins or peptides are well known in the art. Such methods may include, but
are not
limited to, use of chemical cross-linkers, photoactivated cross-linkers and/or
bifunctional cross-linking reagents. Exemplary methods for cross-linking
molecules
are disclosed in U.S. Patent 5,603,872 and U.S. Patent 5,401,511, incorporated
herein
by reference. Non-limiting examples of cross-linking reagents of potential use
include
glutaraldehyde, bifunctional oxirane, ethylene glycol diglycidyl ether,
carbodiimides
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such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide or
dicyclohexylcarbodiimide,
bisimidates, dinitrobenzene, N-hydroxysuccinimide ester of suberic acid,
disuccinimidyl tartarate, dimethyl-3,3'-dithio-bispropionimidate,
azidoglyoxal, N-
succinimidyl-3-(2-pyridyldithio)propionate and 4-(bromoadminoethyl)-2-
nitrophenylazide.
Homobifunctional reagents that carry two identical functional groups are
highly
efficient in inducing cross-linking. Heterobifunctional reagents contain two
different
functional groups. By taking advantage of the differential reactivities of the
two
different functional groups, cross-linking can be controlled both selectively
and
sequentially. The bifunctional cross-linking reagents can be divided according
to the
specificity of their functional groups, e.g., amino, sulfhydryl, guanidino,
indole,
carboxyl specific groups. Of these, reagents directed to free amino groups
have become
especially popular because of their commercial availability, ease of synthesis
and the
mild reaction conditions under which they can be applied.
In certain embodiments, it may be appropriate to link one or more targeting
peptides to a liposome or other membrane-bounded particle. For example,
targeting
peptides cross-linked to liposomes, microspheres or other such devices may be
used to
deliver larger volumes of a therapeutic agent to a target organ, tissue or
cell type.
Various ligands can be covalently bound to liposomal surfaces through the
cross-
linking of amine residues. Liposomes containing phosphatidylethanolamine (PE)
may
be prepared by established procedures. The inclusion of PE provides an active
functional amine residue on the liposomal surface.
In another non-limiting example, heterobifunctional cross-linking reagents and
methods of use axe disclosed in U.S. Patent 5,889,155, incorporated herein by
reference. The cross-linking reagents combine a nucleophilic hydrazide residue
with an
electrophilic maleimide residue, allowing coupling in one example, of
aldehydes to
free thiols. The cross-linking reagent can be modified to cross-link various
functional
groups.
Other techniques of general use for proteins or peptides that are known in the
art
have not been specifically disclosed herein, but may be used in the practice
of the
claimed subject matter.
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Nucleic Acids
In certain embodiments, nucleic acids may encode a targeting peptide, a
receptor
protein, a fusion protein or other protein or peptide. The nucleic acid may be
derived
from genomic DNA, complementary DNA (cDNA) or synthetic DNA. Where
incorporation into an expression vector is desired, the nucleic acid may also
comprise a
natural intron or an intron derived from another gene. Such engineered
molecules are
sometime referred to as "mini-genes." In various embodiments of the invention,
targeting peptides may be incorporated into gene therapy vectors via nucleic
acids.
A "nucleic acid" as used herein includes single-stranded and double-stranded
molecules, as well as DNA, RNA, chemically modified nucleic acids and nucleic
acid
analogs. It is contemplated that a nucleic acid within the scope of the
present invention
may be of almost any size, determined in part by the length of the encoded
protein or
peptide.
It is contemplated that targeting peptides, fusion proteins and receptors may
be
encoded by any nucleic acid sequence that encodes the appropriate amino acid
sequence. The design and production of nucleic acids encoding a desired amino
acid
sequence is well known to those of skill in the art, using standardized codon
tables (see
Table 2 below). In preferred embodiments, the codons selected for encoding
each
amino acid may be modified to optimize expression of the nucleic acid in the
host cell
of interest. Codon preferences for various species of host cell are well known
in the art.
TABLE 2
Amino Acid Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C ~ UGC UGU
Aspartic acid Asp D ~ GAC GAU
Glutamic acid Glu E I GAA GAG
Phenylalanine Phe F ~ UUC UUU
Glycine Gly G I GGA GGC GGG GGU
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Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gln Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
In addition to nucleic acids encoding the desired peptide or protein, the
present
invention encompasses complementary nucleic acids that hybridize under high
stringency conditions with such coding nucleic acid sequences. High stringency
conditions for nucleic acid hybridization are well known in the art. For
example,
conditions may comprise low salt and/or high temperature conditions, such as
provided
by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to
about 70°C. It is
understood that the temperature and ionic strength of a desired stringency are
determined in part by the length of the particular nucleic acid(s), the length
and
nucleotide content of the target sequence(s), the charge composition of the
nucleic
acid(s), and to the presence or concentration of formamide,
tetramethylammonium
chloride or other solvents) in a hybridization mixture.
Nucleic acids for use in the disclosed methods and compositions may be
produced by any method known in the art, such as chemical synthesis (e.g.
Applied
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Biosystems Model 3900, Foster City, CA), purchase from commercial sources
(e.g.
Midland Certified Reagents, Midland, TX) and/or standard gene cloning methods.
A
number of nucleic acid vectors, such as expression vectors and/or gene therapy
vectors,
may be commercially obtained (e.g., American Type Culture Collection,
Rockville,
MD; Promega Corp., Madison, WI; Stratagene, La Jolla, CA).
Vectors for Cloning, Gene Transfer and Expression
In certain embodiments expression vectors are employed to express the
targeting
peptide or fusion protein, which can then be purified and used. In other
embodiments,
the expression vectors are used in gene therapy. Expression requires that
appropriate
signals be provided in the vectors, and which include various regulatory
elements, such
as enhancers/promoters from both viral and mammalian sources that drive
expression
of the genes of interest in host cells. Elements designed to optimize
messenger RNA
stability and translatability in host cells also are known.
Regulatory Elements
The terms "expression construct" or "expression vector" are meant to include
any type of genetic construct containing a nucleic acid coding for a gene
product in
which part or all of the nucleic acid coding sequence is capable of being
transcribed. In
preferred embodiments, the nucleic acid encoding a gene product is under
transcriptional control of a promoter. A "promoter" refers to a DNA sequence
recognized by the synthetic machinery of the cell, or introduced synthetic
machinery,
required to initiate the specific transcription of a gene. The phrase "under
transcriptional control" means that the promoter is in the correct location
and
orientation in relation to the nucleic acid to control RNA polymerise
initiation and
expression of the gene.
The particular promoter employed to control the expression of a nucleic acid
sequence of interest is not believed to be important, so long as it is capable
of directing
the expression of the nucleic acid in the targeted cell. Thus, where a human
cell is
targeted, it is preferable to position the nucleic acid coding region adjacent
and under
the control of a promoter that transcriptionally active in human cells.
Generally
speaking, such a promoter might include either a human or viral promoter.
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In various embodiments, the human cytomegalovirus (CMV) immediate early
gene promoter, the SV40 early promoter, the Rouse sarcoma virus long terminal
repeat,
rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase promoter
can be
used to obtain high-level expression of the coding sequence of interest. The
use of
other viral or mammalian cellular or bacterial phage promoters that are known
in the art
to achieve expression of a coding sequence of interest is contemplated as
well, provided
that the levels of expression are sufficient for a given purpose.
Where a cDNA insert is employed, one will typically include a polyadenylation
signal to effect proper polyadenylation of the gene transcript. The nature of
the
polyadenylation signal is not believed to be crucial to the successful
practice of the
invention, and any such sequence may be employed, such as human growth hormone
and SV40 polyadenylation signals. Also contemplated as an element of the
expression
construct is a terminator. These elements can serve to enhance message levels
and to
minimize read through from the construct into other sequences.
Selectable Markers
In certain embodiments of the invention, the cells containing nucleic acid
constructs of the present invention may be identified ifa vitro or in vivo by
including a
marker in the expression construct. Such markers would confer an identifiable
change
to the cell permitting easy identification of cells containing the expression
construct.
Usually the inclusion of a drug selection marker aids in cloning and in the
selection of
transformants. For example, genes that confer resistance to neomycin,
puromycin,
hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers.
Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or
chloramphenicol acetyltransferase (CAT) may be employed. Immunologic markers
also can be employed. The selectable marker employed is not believed to be
important,
so long as it is capable of being expressed simultaneously with the nucleic
acid
encoding a gene product. Further examples of selectable markers are well known
to
one of skill in the art.
Delivery of Expressio~e Vectors
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There are a number of ways in which expression vectors may introduced into
cells. In certain embodiments of the invention, the expression construct
comprises a
virus or engineered construct derived from a viral genome. The ability of
certain
viruses to enter cells via receptor-mediated endocytosis, to integrate into
host cell
genome, and express viral genes stably and efficiently have made them
attractive
candidates for the transfer of foreign genes into mammalian cells (Ridgeway,
1988;
Nicolas and Rubinstein, Ih: Vectors: A survey of molecular cloning vectors and
their
uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988.;
Baichwal and Sugden, Baichwal, In: Gene Transfer, Kucherlapati R, ed., New
York,
Plenum Press, pp. 117-148, 1986. 1986; Temin, In: Gene Transfer, Kucherlapati,
R. ed.,
New York, Plenum Press, pp. 149-188, 1986). Preferred gene therapy vectors are
generally viral vectors.
In using viral delivery systems, one will desire to purify the virion
sufficiently to
render it essentially free of undesirable contaminants, such as defective
interfering viral
particles or endotoxins and other pyrogens such that it will not cause any
untoward
reactions in the cell, animal or individual receiving the vector construct. A
preferred
means of purifying the vector involves the use of buoyant density gradients,
such as
cesium chloride gradient centrifugation.
DNA viruses used as gene vectors include the papovaviruses (e.g., simian virus
40, bovine papilloma virus, and polyoma) (Ridgeway, pp 467-492, 1988; Baichwal
and
Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
An exemplary method for in vivo delivery involves the use of an adenovirus
expression vector. Although adenovirus vectors have a low capacity for
integration into
genomic DNA, this feature is counterbalanced by the high efficiency of gene
transfer
afforded by these vectors. "Adenovirus expression vector" is meant to include,
but is
not limited to, constructs containing adenovirus sequences sufficient to (a)
support
packaging of the construct and (b) to express an antisense or a sense
polynucleotide that
has been cloned therein.
Generation and propagation of adenovirus vectors that are replication
deficient
depend on a helper cell line, such as the 293 cell line, which was transformed
from
human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses
E1
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proteins (Graham et al., J. Geu. Virol., 36:59-72, 1977.). Since the E3 region
is
dispensable from the adenovirus genome (Jones and Shenk, Cell, 13:181-188,
1978),
adenovirus vectors, with the help of 293 cells, carry foreign DNA in either
the E1, the
E3, or both regions (Graham and Prevec, Irz: Methods ifZ Molecular Biology:
Gene
Trafzsfer and Expression Protocol, E.J. Murray, ed., Humana Press, Clifton,
NJ,
7:109-128, 1991.).
Helper cell lines may be derived from human cells such as human embryonic
kidney cells, muscle cells, hematopoietic cells or other human embryonic
mesenchymal
or epithelial cells. Alternatively, the helper cells may be derived from the
cells of other
mammalian species that are permissive for human adenovirus. Such cells
include, e.g.,
Vero cells or other monkey embryonic mesenchymal or epithelial cells. Racher
et al.,
(Biotechnol. Tech. 9:169-174, 1995) disclosed methods for culturing 293 cells
and
propagating adenovirus.
Adenovirus vectors have been used in eukaryotic gene expression (Levrero et
al., GefZe, 101:195-202, 1991; Gomez-Foix et al., J. Biol. Chem., 267:25129-
25134,1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and
Prevec, 1991). Animal studies have suggested that recombinant adenovirus could
be
used for gene therapy (Stratford-Perricaudet and Perricaudet, Ih: Human Gene
Transfer,
O. Cohen-Haguenauer et al, eds. John Libbey Eurotext, France, pp. 51-61, 1991;
Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256, 1990; Rich et al.,
Hum. Gene.
Ther. 4:461-476, 1993). Studies in administering recombinant adenovirus to
different
tissues include trachea instillation (Rosenfeld et al., Science, 252: 431-434,
1991;
Rosenfeld et al., Cell, 68: 143-155, 1992), muscle injection (Ragot et al.,
Nature,
361:647-650, 1993), peripheral intravenous injections (Herz and Gerard, Proc.
Natl.
Acad. Sci. USA, 90:2812-2816, 1993) and stereotactic innoculation into the
brain (Le
Gal La Salle et al., Science, 259:988-990,1993).
In preferred embodiments, gene therapy vectors are based upon adeno-
associated virus (AAV), discussed in more detail in the Examples below.
Other gene transfer vectors may be constructed from retroviruses. (Coffin, In:
Virology, Fields et al., eds., Raven Press, New York, pp. 1437-1500, 1990.)
The
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retroviral genome contains three genes, gag, pol, and erav. that code for
capsid proteins,
polymerase enzyme, and envelope components, respectively. A sequence found
upstream from the gag gene contains a signal for packaging of the genome into
virions.
Two long terminal repeat (LTR) sequences are present at the 5 ~ and 3 0 ends
of the
viral genome. These contain strong promoter and enhancer sequences, and also
are
required for integration in the host cell genome (Coffin, 1990).
In order to construct a retroviral vector, a nucleic acid encoding protein of
interest is inserted into the viral genome in the place of certain viral
sequences to
produce a virus that is replication-defective. In order to produce virions, a
packaging
cell line containing the gag, pol, and ehv genes, but without the LTR and
packaging
components, is constructed (Mann et al., Cell, 33:153-159, 1983). When a
recombinant
plasmid containing a cDNA, together with the retroviral LTR and packaging
sequences
is introduced into this cell line (by calcium phosphate precipitation for
example), the
packaging sequence allows the RNA transcript of the recombinant plasmid to be
packaged into viral particles, which are then secreted into the culture media
(Nicolas
and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing
the
recombinant retroviruses is then collected, optionally concentrated, and used
for gene
transfer. Retroviral vectors are capable of infecting a broad variety of cell
types.
However, integration and stable expression require the division of host cells
(Paskind et
al., Virology, 67:242-248, 1975).
Other viral vectors may be employed as expression constructs. Vectors derived
from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden,
1986;
Coupar et al., Gene 68:1-10, 1988), adeno-associated virus (AAV) (Ridgeway,
1988;
Baichwal and Sugden, 1986; Hermonat and Muzycska, Proc. Natl. Acad. Sci. USA,
81:
6466-6470, 1984), and herpes viruses may be employed. They offer several
attractive
features for various mammalian cells (Friedmann, Science, 244:1275-1281, 1989;
Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et
al., J.
Virol., 64:642-650,1990).
Several non-viral methods for the transfer of expression constructs into
cultured
mammalian cells also are contemplated by the present invention. These include
calcium phosphate precipitation (Graham and van der Eb, Virology, 52:456-467,
1973.;
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Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987.; Rippe et al., Mol. Cell
Biol.
10: 689-695, 1990; DEAF dextran (Gopal, et al. Mol. Cell. Biol., 5:1188-
1190,1985),
electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et
al., Proc.
Natl. Acad. Sci. USA, 81: 7161-7165, 1984), direct microinjection, DNA-loaded
liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment
using high velocity microprojectiles, and receptor-mediated transfection (Wu
and Wu,
J. Biol. Chem. 262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892,
1988).
Some of these techniques may be successfully adapted for in vivo or ex vivo
use.
In a further embodiment of the invention, the expression construct may be
entrapped in a liposome. Liposome-mediated nucleic acid delivery and
expression of
foreign DNA in vitro has been very successful. Wong et al., (Gene, 10:87-94,
1980)
demonstrated the feasibility of liposome-mediated delivery and expression of
foreign
DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al.,
(Methods
Enzymol., 149:157-176, 1987.) accomplished successful liposome-mediated gene
transfer in rats after intravenous injection.
Pharmaceutical compositions
Where clinical applications are contemplated, it may be necessary to prepare
pharmaceutical compositions - expression vectors, virus stocks, proteins,
antibodies and
drugs - in a form appropriate for the intended application. Generally, this
will entail
preparing compositions that are essentially free of impurities that could be
harmful to
humans or animals.
One generally will desire to employ appropriate salts and buffers to render
delivery vectors stable and allow for uptake by target cells. Aqueous
compositions of
the present invention may comprise an effective amount of a protein, peptide,
fusion
protein, recombinant phage and/or expression vector, dissolved or dispersed in
a
pharmaceutically acceptable carrier or aqueous medium. Such compositions also
are
referred to as inocula. The phrase "pharmaceutically or pharmacologically
acceptable"
refers to molecular entities and compositions that do not produce adverse,
allergic, or
other untoward reactions when administered to an animal or a human. As used
herein,
"pharmaceutically acceptable carrier" includes any and all solvents,
dispersion media,
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coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents
and the like. The use of such media and agents for pharmaceutically active
substances
is well known in the art. Except insofar as any conventional media or agent is
incompatible with the proteins or peptides of the present invention, its use
in
therapeutic compositions is contemplated. Supplementary active ingredients
also can
be incorporated into the compositions.
The active compositions of the present invention may include classic
pharmaceutical preparations. Administration of these compositions according to
the
present invention are via any common route so long as the target tissue is
available via
that route. This includes oral, nasal, buccal, rectal, vaginal or topical.
Alternatively,
administration may be by orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal, intraarterial or intravenous injection. Such compositions
normally
would be administered as pharmaceutically acceptable compositions, described
supra.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of
sterile injectable solutions or dispersions. In all cases the form must be
sterile and must
be fluid to the extent that easy syringability exists. It must be stable under
the
conditions of manufacture and storage and must be preserved against the
contaminating
action of microorganisms, such as bacteria and fungi. The carrier can be a
solvent or
dispersion medium containing, for example, water, ethanol, polyol (for
example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the like),
suitable
mixtures thereof, and vegetable oils. The proper fluidity can be maintained,
for
example, by the use of a coating, such as lecithin, by the maintenance of the
required
particle size in the case of dispersion and by the use of surfactants. The
prevention of
the action of microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. In many cases, it is preferable to include isotonic
agents, for
example, sugars or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of agents
delaying
absorption, for example, aluminum monostearate and gelatin.
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Sterile injectable solutions are prepared by incorporating the active
compounds
in the required amount in the appropriate solvent with various other
ingredients
enumerated above, as required, followed by filtered sterilization. Generally,
dispersions
are prepared by incorporating the various sterilized active ingredients into a
sterile
vehicle which contains the basic dispersion medium and the required other
ingredients
from those enumerated above. In the case of sterile powders for the
preparation of
sterile injectable solutions, the preferred methods of preparation are vacuum-
drying and
freeze-drying techniques which yield a powder of the active ingredient plus
any
additional desired ingredient from a previously sterile-filtered solution
thereof.
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Therapeutic agents
In certain embodiments, therapeutic agents may be attached to a targeting
peptide
or fusion protein for selective delivery to, for example, non-metastatic
and/or metastatic
prostate cancer. Agents or factors suitable for use may include any chemical
compound
that induces apoptosis, cell death, cell stasis and/or anti-angiogenesis or
otherwise affects
the survival and/or growth rate of a cancer cell.
Regulators of Programmed Cell Death
Apoptosis, or programmed cell death, is an essential process for normal
embryonic development, maintaining homeostasis in adult tissues, and
suppressing
carcinogenesis (Kerr et al., 1972). The Bcl-2 family of proteins and ICE-like
proteases
have been demonstrated to be important regulators and effectors of apoptosis
in other
systems. The Bcl-2 protein, discovered in association with follicular
lymphoma, plays
a prominent role in controlling apoptosis and enhancing cell survival in
response to
diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985;
Tsujimoto et
al., 1985). The evolutionarily conserved Bcl-2 protein now is recognized to be
a
member of a family of related proteins, which can be categorized as death
agonists or
death antagonists.
Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell
death
triggered by a variety of stimuli. Also, it now is apparent that there is a
family of Bcl-2
cell death regulatory proteins that share in common structural and sequence
homologies. These different family members have been shown to either possess
similar
functions to Bcl-2 (e.g., Bcl~,, Bclw, Bcls, Mcl-1, A1, Bfl-1) or counteract
Bcl-2
function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad,
Harakiri).
Non-limiting examples of pro-apoptosis agents contemplated within the scope
of the present invention include gramicidin, magainin, mellitin, defensin,
cecropin,
(KLAKLAK)a (SEQ ID N0:1), (KLAKKLA)2 (SEQ ll~ N0:2), (KAAKKAA)2 (SEQ
ID N0:3) or (KLGKKLG)3 (SEQ ll~ N0:4).
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Angiogenic inhibitors
In certain embodiments the present invention may concern administration of
targeting peptides attached to anti-angiogenic agents, such as angiotensin,
laminin
peptides, fibronectin peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4,
IP-10, Gro-13,
thrombospondin, 2-methoxyoestradiol, proliferin-related protein,
carboxiamidotriazole,
CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),
interferon-
alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide,
thalidomide,
pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin,
angiostatin,
cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.
Proliferation of tumors cells relies heavily on extensive tumor
vascularization,
which accompanies cancer progression. Thus, inhibition of new blood vessel
formation
with anti-angiogenic agents and targeted destruction of existing blood vessels
have been
introduced as an effective and relatively non-toxic approach to tumor
treatment. (Arap
et al., Science 279:377-380, 1998a; Arap et al., Curr. Opin. Oncol. 10:560-
565, 1998b;
Ellerby et al., Nature Med. 5:1032-1038, 1999). A variety of anti-angiogenic
agents
and/or blood vessel inhibitors are known. (E.g., Folkman, In: Cancer:
Principles and
Practice, eds. DeVita et al., pp. 3075-3085, Lippincott-Raven, New York, 1997;
Eliceiri and Cheresh, Curr. Opin. Cell. Biol. 13, 563-568, 2001).
Cytotoxic Agents
A wide variety of anticancer agents are well known in the art and any such
agent
may be coupled to a cancer targeting peptide for use within the scope of the
present
invention. Exemplary cancer chemotherapeutic (cytotoxic) agents of potential
use
include, but are not limited to, 5-fluorouracil, bleomycin, busulfan,
camptothecin,
carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin,
daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16),
farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide,
mechlorethamine,
melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine,
raloxifene,
tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum,
vinblastine
and methotrexate, vincristine, or any analog or derivative variant of the
foregoing.
Most chemotherapeutic agents fall into the categories of alkylating agents,
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antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic
inhibitors, and
nitrosoureas, hormone agents, miscellaneous agents, and any analog or
derivative
variant thereof.
Chemotherapeutic agents and methods of administration, dosages, etc. are well
known to those of skill in the art (see for example, the "Physicians Desk
Reference",
Goodman & Gilman's "The Pharmacological Basis of Therapeutics" and "Remington:
The Science and Practice of Pharmacy," 20th edition, Gennaro, Lippincott,
2000, each
incorporated herein by reference in relevant parts), and may be combined with
the
invention in light of the disclosures herein. Some variation in dosage will
necessarily
occur depending on the condition of the subject being treated. The person
responsible
for administration will, in any event, determine the appropriate dose for the
individual
subject. Of course, all of these dosages and agents described herein are
exemplary
rather than limiting, and other doses or agents may be used by a skilled
artisan for a
specific patient or application. Any dosage in-between these points, or range
derivable
therein is also expected to be of use in the invention.
Alkylatifzg agents
Alkylating agents are drugs that directly interact with genomic DNA to prevent
cells from proliferating. This category of chemotherapeutic drugs represents
agents that
affect all phases of the cell cycle, that is, they are not phase-specific. An
alkylating
agent, may include, but is not limited to, nitrogen mustard, ethylenimene,
methylmelamine, alkyl sulfonate, nitrosourea or triazines. They include but
are not
limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan),
dacarbazine,
ifosfamide, mechlorethamine (mustargen), and melphalan.
Ahtimetabolites
Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents,
they specifically influence the cell cycle during S phase. Antimetabolites can
be
differentiated into various categories, such as folic acid analogs, pyrimidine
analogs and
purine analogs and related inhibitory compounds. Antimetabolites include but
are not
limited to, 5-fluorouracil (5-FLT, cytarabine (Ara-C), fludarabine,
gemcitabine, and
methotrexate.
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Natural Products
Natural products generally refer to compounds originally isolated from a
natural
source, and identified as having a pharmacological activity. Such compounds,
analogs
and derivatives thereof may be, isolated from a natural source, chemically
synthesized
or recombinantly produced by any technique known to those of skill in the art.
Natural
products include such categories as mitotic inhibitors, antitumor antibiotics,
enzymes
and biological response modifiers.
Mitotic inhibitors include plant alkaloids and other natural agents that can
inhibit either protein synthesis required for cell division or mitosis. They
operate
during a specific phase during the cell cycle. Mitotic inhibitors include, for
example,
docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine,
vincristine, and
vinorelbine.
Taxoids are a class of related compounds isolated from the bark of the ash
tree,
Taxus brevifolia. Taxoids include but are not limited to compounds such as
docetaxel
and paclitaxel. Paclitaxel binds to tubulin (at a site distinct from that used
by the vinca
alkaloids) and promotes the assembly of microtubules.
Antibiotics
Certain antibiotics have both antimicrobial and cytotoxic activity. These
drugs
also interfere with DNA by chemically inhibiting enzymes and mitosis or
altering
cellular membranes. These agents are not phase specific so they work in all
phases of
the cell cycle. Examples of cytotoxic antibiotics include, but are not limited
to,
bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin); plicamycin
(mithramycin) and idarubicin.
Miscellaneous Agents
Miscellaneous cytotoxic agents that do not fall into the previous categories
include, but are not limited to, platinum coordination complexes,
anthracenediones,
substituted ureas, methyl hydrazine derivatives, amsacrine, L-asparaginase,
and
tretinoin. Platinum coordination complexes include such compounds as
carboplatin
and cisplatin (cis-DDP). An exemplary anthracenedione is mitoxantrone. An
exemplary substituted urea is hydroxyurea. An exemplary methyl hydrazine
derivative
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is procarbazine (N-methylhydrazine, MII~. These examples are not limiting and
it is
contemplated that any known cytotoxic, cytostatic or cytocidal agent may be
attached to
targeting peptides and administered to a targeted organ, tissue or cell type
within the
scope of the invention.
Cytokihes ahd chemokihes
In certain embodiments, it may be desirable to couple specific bioactive
agents
to one or more targeting peptides for targeted delivery to an organ, tissue or
cell type.
Such agents include, but are not limited to, cytokines and/or chemokines.
The term "cytokine" is a generic term for proteins released by one cell
population that act on another cell as intercellular mediators. Examples of
cytokines are
lymphokines, monokines, growth factors and traditional polypeptide hormones.
Included among the cytokines are growth hormones such as human growth hormone,
N-
methionyl human growth hormone, and bovine growth hormone; parathyroid
hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones
such as
follicle stimulating hormone (FSIT), thyroid stimulating hormone (TSIT), and
luteinizing hormone (LIB; hepatic growth factor; prostaglandin, fibroblast
growth
factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-
alpha. and -beta;
mullerian-inhibiting substance; mouse gonadotropin-associated peptide;
inhibin;
activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO);
nerve
growth factors such as NGF-.beta.; platelet-growth factor; transforming growth
factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth factor-I and -
II;
erythropoietin (EPO); osteoinductive factors; interferons such as interferon-
a, -.(3, and -
y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-
macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such
as
IL-1, IL-l.alpha., 1L-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-
11, IL-12; lL-
13, IL-14, IL-15, IL-16, IL-17, IIrl8, LIF, G-CSF, GM-CSF, M- CSF, EPO, kit-
ligand
or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and
LT. As
used herein, the term cytokine includes proteins from natural sources or from
recombinant cell culture and biologically active equivalents of the native
sequence
cytokines.
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Chemokines generally act as chemoattractants to recruit immune effector cells
to the site of chemokine expression. It may be advantageous to express a
particular
chemokine gene in combination with, for example, a cytokine gene, to enhance
the
recruitment of other immune system components to the site of treatment.
Chemokines
include, but are not limited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-
10.
The skilled artisan will recognize that certain cytokines are also known to
have
chemoattractant effects and could also be classified under the term
chemokines.
Dosages
The skilled artisan is directed to "Remington: The Science and Practice of
Pharmacy," 20th edition, Gennaro, Lippincott (2000). Some variation in dosage
will
necessarily occur depending on the condition of the subject being treated. The
person
responsible for administration will, in any event, determine the appropriate
dose for the
individual subject. Moreover, for human administration, preparations should
meet
sterility, pyrogenicity, and general safety and purity standards as required
by the FDA
Office of Biologics standards.
EXAMPLES
The following examples are included to demonstrate preferred embodiments of
the invention. It should be appreciated by those of skill in the art that the
techniques
disclosed in the examples which follow represent techniques discovered by the
inventors to function well in the practice of the invention, and thus can be
considered to
constitute preferred modes for its practice. However, those of skill in the
art should, in
light of the present disclosure, appreciate that many changes can be made in
the specific
embodiments which are disclosed and still obtain a like or similar result
without
departing from the spirit and scope of the invention.
Example 1. Biopanning with Phage Display Libraries Using Human
Patients
Certain of the methods and compositions of the present invention concern
identification of targeting peptides for human organs, tissues or cell types
by ih vivo
biopanning. Generally, protocols used in animal subjects, such as mice, are
not suited
for humans. Further, ethical considerations play a large role in human
protocols. The
5~
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following novel methods are preferred for use with humans, although the
skilled artisan
will realize that variations on the methods and compositions disclosed herein
may be
used within the scope of the present invention.
Human p~epaYatioh
Patients were selected for the protocol according to inclusion and exclusion
criteria. Inclusion criteria include: (1) patient legally declared brain dead
or terminal
wean patient; (2) approval of attending and/or treating physicians; and (3)
approved
written informed consent form signed by the patient's legally responsible
family
member. Exclusion criteria were: (1) the absence of a responsible family
member; (2)
HIV positive patient; (3) patient with active tuberculosis infection; (4)
acute or chronic
hepatitis B or C infections; or (5) patient was a potential organ transplant
donor. In
preferred embodiments, the patient was not on antibiotics for at least the
previous 6 hrs,
preferably the last 24 hrs, in order to avoid detrimental effects on the
bacterial hosts
used to propagate the phage used for the peptide display library.
After informed consent and before the patient was prepared for the procedure,
relatives of the patient were asked to leave the room the patient was in. The
patient had
a well running IV line (preferably central) with nothing but saline running
through the
channel of application of the phage library. Personnel required for the
procedure were
notified (i.e., intervention radiologist, internist, surgeon, nurse, possibly
neurologist or
neuroradiologist). Materials needed for biopsies were collected: bone marrow
aspiration needle, lumbar puncture kit, skin biopsy kit, materials for taking
biopsies of
any organ, tissue or cell type used for targeted peptide identification, such
as liver, fat
and tumor, materials for transabdominal prostate biopsy, 50 ml syringe with 40
ml
saline for blood sample, 10 ml tube containing heparin and 10 ml serum
collection tube
to draw blood sample for lab tests. Before phage library injection, blood
samples were
drawn for routine screening of liver function, bicarbonate, electrolytes and
blood count,
unless test results from the day of the injection were available.
In the laboratory, 120 large dishes with LB-tet/kan agar as well as 200
regular
LB tetlkan plates (100 mm) were prepared (tetracycline concentration = 40
~.g/ml,
kanamycin concentration = 50 ~,g/ml). E. coli K91 kan were grown in 10
independent
tubes, each containing 10 ml TB medium plus supplements. Growth of bacteria
was
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started approx. 15-60 min prior to beginning the biopsies. About 1014 TU of
the
(preferably primary) phage library were diluted in 200 ml ringer lactate at
room
temperature and aspirated under clean but not necessarily sterile conditions
into four 50
ml syringes. LB-tet/kan dishes or plates were warmed in a 37°C
incubator. One liter of
LB medium containing 0.2 ,ug/ml tet and 100 ,ug/ml kan was warmed in the
waterbath
at 37°C. One liter LB medium containing 40 ~,g/ml tet and 100 ,ug/ml
kan was warmed
to 37°C and 8 more liters were prepared at room temperature. Thirty
glass grinders A
and B size as well as suitable glass tubes were autoclaved. Three 50 ml Falcon
tubes
were prepared for each of the organs for which biopsies were to be taken.
Tubes were
filled with 10 ml DMEM-PI - DMEM containing PMSF (1 mM), aprotinin (20,ug/ml)
and leupeptin (l,ug/ml) - and put on ice approximately 15 minutes before
tissue
collection. For each of the 4 teams taking over in the lab after the tissue
samples were
collected, one autoclaved set of surgicals (i.e., at least one forceps and one
pair of
scissors and a scalpel) were prepared in order to trim, divide or mince organ
samples.
Phage library injection
All drugs running through the intended port of application of the phage
library
were discontinued during library injection. If possible without compromising
the
patient's hemodynamic stability, all IV drugs running through different ports
were
discontinued during library injection as well. A running saline infusion
ensured that the
IV line for the library injection was open and was left running during the
injection.
The 200 ml library solution was manually injected over a period of 10 minutes
while monitoring and protocoling the patient's vital functions such as
breathing (if not
mechanically ventilated), heart rate and blood pressure. The injection was
stopped any
time the running saline infusion stopped dripping, indicating obstruction of
the line.
Fifteen minutes after beginning the injection, tissue sample collection
(biopsies) was
initiated. Biopsy sites included bone marrow aspirate, liver, prostate, skin,
skeletal
muscle, tumor (if applicable), adipose tissue, blood (as positive control),
blood (for
red/white blood cells) and cerebral-spinal fluid (CSF).
The samples were taken under very clean if not sterile conditions to reduce
contamination with bacteria. To the extent possible, the different samples
were taken
simultaneously. For small samples, triplicate biopsies were preferred. The
time elapsed
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between beginning of injection and the collection of a particular tissue
sample was
recorded. Tissue samples were placed in the prepared 50 ml tubes containing
DMEM-
PI and stored on ice. For bone marrow, a regular diagnostic sample (undiluted
into a
syringe with heparin) was taken in addition to the samples diluted in 40 ml
saline to
confirm aspiration of bone marrow as opposed to blood. If needed, all IV
drugs,
including antibiotics, were continued after removal of tissue samples.
All organ samples that were not taken in triplicate were divided under clean
conditions to obtain three different pieces of tissue. The three samples of
each organ
were handled as follows. One piece was stored at -80°C as a backup. One
piece was
forwarded to the histology/pathology department to cut cryosections (or to
make smears
for bone marrow) and perform HE staining (Pappenheim staining for bone marrow)
as
well' as phage staining to confirm that the samples contained the organ of
interest. In
some cases the histology sample was divided in two - one for regular HE
staining and
one for LCM (laser capture microscopy) or LPC (laser pressure catapulting).
The last
of the three original pieces was processed for bacterial infection to recover
phage.
After freezing of backup tissue and saving material for pathology, samples for
phage rescue were weighed. Samples were kept on ice at all times. Sample was
transferred to 1ml DMEM-PI in a glass tube and homogenized with a grinder.
Some
organs such as bone marrow, blood, or CSF do not require homogenization,
whereas
other organs like muscle need to be minced before they can be efficiently
homogenized.
Lysis of erythrocytes for blood samples was preferred. Homogenized samples
were
transferred to autoclaved 2 ml Eppendorf tubes.
Tissue samples were washed 3 times with ice cold DMEM-PI containing
1°l0
BSA by mixing the tissue with DMEM-PI and vortexing for 30 seconds. After
centrifugation at 4,000 rpm for 3 min, supernatant was carefully discarded,
leaving the
tissue pellet undisturbed. A small amount of medium was left on the surface of
the
pellet. Samples were vortexed again for 30 seconds before adding more medium
to
facilitate resuspension of the tissue. After adding 1.5 ml of DMEM-PI plus BSA
the
samples were centrifuged again. When processing multiple samples, the tissues
were
kept on ice at all times.
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After 3 washes, the pellet was briefly vortexed and the dissolved pellet was
warmed briefly to 37°C before adding bacteria. The washed tissue
samples were
incubated with 1.5 ml of competent K91-kan bacteria (OD6oo 0.2 in 1:10 dil.)
for one
hour at room temperature, then transferred to Falcon tubes containing 10 ml of
LB
medium with 0.2 ~,glml tetracycline. After 20 min at RT, multiple aliquots
were plated
on LB tetlkan plates or dishes containing 40 ~.glml of tetracycline and 100
~,g/ml
kanamycin. The following quantities (per organ sample) were plated: 2 dishes
with 3
ml; 2 dishes with 1 ml; 3 dishes with 300 ~,1; 3 dishes with 100 ,ul; 3 dishes
with 30 ~,1.
The beads that were used for plating were passed on to two subsequent 10 cm
LB tet/kan plates to recover every potentially phage infected bacterial clone
that might
be trapped on the 'bead surface. Dishes were incubated overnight at
37°C.
The remaining 2-3 ml of infected culture (including the homogenized tissue)
was transferred to 10 ml of LB medium containing 40~,g/ml tetracycline and
100~,g/ml
kanamycin (LB tet/kan) and shaken at 37°C for 2 hr. This approximately
12 ml culture
was transferred to 1 liter LB tet/kan and grown overnight in a 37°C
shaker.
The next day, phage were rescued from the bulk amplified bacterial culture
according to standard protocols and saved for a potential second round of in
vivo
selection. From the plates/dishes in the incubator, 1500 well separated
colonies were
picked for each organ plated and transfered to 96 well plates containing 20
~,l PBS/well
for sequencing. This assumed a readout of about 2 out of 3 picked colonies to
obtain
1000 sequences.
After picking 1500 colonies, the remainder of colonies on the dishes/plates
were
grown in 1000 ml LB tet/kan overnight in the 37°C shaker. Then phage
were harvested
as before for a second round of selection. Alternatively, the plates were
stored in the
refrigerator and 1000-2000 individual colonies grown at a time. Alternatively,
the
remainder of colonies were transferred to PBS and stored frozen to infect and
amplify
as needed.
Numerous non-limiting examples of human organ, tissue or cell type selective
targeting peptides have been identified by in vivo biopanning using the
present
methods, as disclosed below.
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Example 2. Mapping the Human Vasculature by Ih vivo Phage Display
The ih vivo selection method discussed above was used to screen a phage
library
in a human subject. A pattern recognition analysis program was used to survey
47,160
tripeptide motifs within peptides that localized to the human bone marrow,
fat, skeletal
muscle, prostate, or skin. The results of this large-scale screening indicated
that the
distribution of circulating peptide motifs to different organs is non-random.
High-
throughput analysis of peptide motifs enriched in individual tissues revealed
similarities
to sequences present in candidate ligands for differentially expressed
vascular receptors.
These data represent a major step towards the construction of a ligand-
receptor
map of human vasculature and may have broad implications for the development
of
targeted therapies. Many therapeutic targets may be expressed in very
restricted--but
highly specific and accessible--locations in the vascular endothelium.
Potential targets
for intervention may be overlooked in high-throughput DNA sequencing or in
gene
arrays because these approaches do not usually take into account cellular
location and
anatomical, and functional context. The human ih vivo phage display screening
methods disclosed herein are uniquely suited to identification of naturally
occurring
ligand-receptor pairs that may provide the basis for highly selective
therapies against
various disease states.
Materials aged Methods
A 4~ year-old male Caucasian patient who had been diagnosed with
Waldenstrom macroglobulinemia (a B cell malignancy) was previously treated by
splenectomy, systemic chemotherapy (fludarabine, mitoxantrone, and
dexamethasone),
and immunotherapy (anti-CD20 monoclonal antibody). In the few months prior to
his
admission, the disease became refractory to treatment and clinical progression
occurred
with retroperitoneal lymphadenopathy, pancytopenia, and marked bone marrow
infiltration by tumor cells. The patient was admitted with massive
intracranial bleeding
secondary to thrombocytopenia. Despite prompt craniotomy and surgical
evacuation of
a cerebral hematoma, the patient remained comatose with progressive and
irreversible
loss of brainstem function until the patient met the formal criteria for brain-
based
determination of death, as evaluated by an independent clinical neurologist.
Because of
his advanced cancer, the patient was rejected as transplant organ donor. After
surrogate
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written informed consent was obtained from the legal next of kin, in vivo
phage display
was performed.
lyz Vivo Phage Display
A large-scale preparation of a CX~C (C, cysteine; X, any amino acid residue)
phage display random peptide library was optimized to create the highest
possible insert
diversity (Pasqualini et al., 2000). The diversity of the library was about 2
x 10$ and its
final titer was about 1012 transducing units (TU)/ml. For biopanning with
human
subjects, use of a large-scale phage display library (diversity about 2 x 108)
is
advantageous compared to the smaller scale libraries used in mouse studies.
Short-term
intravenous infusion of the phage library (a total dose of 1014 phage TU
suspended in
100 ml of saline) into the patient was followed by multiple representative
tissue
biopsies. Prostate and liver samples were obtained by needle biopsy under
ultrasonographic guidance. Skin, fat tissue, and skeletal muscle samples were
obtained
by surgical excision. Bone marrow needle aspirates and core biopsies were also
obtained. Histopathological diagnosis was determined by examination of frozen
sections processed from tissues obtained at the bedside.
Triplicate samples were processed for host bacterial infection, phage
recovery,
and histopathological analysis. In brief, tissues were weighed, ground with a
glass
Dounce homogenizes, suspended in 1 ml of Dulbecco Modified Eagle's medium
(DMEM) containing proteinase inhibitors (DMEM-prin; 1 mM PMSF, 20 ~.g/ml
aprotinin, and 1 ,ug/ml leupeptin), vortexed, and washed three times with DMEM-
prin.
The human tissue homogenates were incubated with 1 ml of host bacteria (log
phase E.
coli K9lkan; OD6oo ~ 2). Aliquots of the bacterial culture were plated onto
Luria-
Bertani agar plates containing 40 ~,glml tetracycline and 100 ~,g/ml of
kanamycin.
Plates were incubated overnight at 37°C. Bacterial colonies were
processed for
sequencing of phage inserts recovered from each tissue and from unselected
phage
library. Human samples were handled with universal blood and body fluid
precautions.
Statistical Analysis
A high-throughput character pattern recognition program (M.D. Anderson
Cancer Center, Biostatistics, Houston, TX) was developed to automate the
analysis of
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the peptide motifs derived from phage screenings. By using SAS (version 8, SAS
Institute) and Perl (version 5.0), the program conducts an exhaustive amino
acid residue
sequence count and tracks the relative frequencies of N distinct tripeptide
motifs
representing all possible n3 overlapping tripeptide motifs in both directions
(N« n3).
This was applied for phage recovered from each target tissue and for the
unselected
CX~C random phage display peptide library.
With "p" defined as the probability of observing a particular tripeptide motif
under total randomness, and q=1-p, the probability of observing K sequences
characterized as a particular tripeptide motif out of n3 total tripeptide
motif sequences is
binomial (n3, p). That probability may be approximated by the formula: px _
~[(k+1)/sqrt(n3 pq)] - ~[k/sqrt(n3 pq)], where ~ is the cumulative Gaussian
probability. The value pK may be treated as a P-value in testing for total
randomness of
observing exactly K sequences of a particular tripeptide motif. However, this
test
requires exact knowledge of the true value of p, which it is difficult to
obtain in
practice.
In order to identify the motifs that were enriched in the screening, the count
for
each tripeptide motif within each tissue was compared with the count for that
tripeptide
motif within the unselected library. Starting from a CX~C peptide insert,
counts were
recorded for all overlapping interior tripeptide motifs, subject only to
reflection and
single-voting restrictions. No peptide was allowed to contribute more than
once for a
single tripeptide motif (or a reflected tripeptide motifj. Each peptide
contributed five
tripeptide motifs. Tripeptide motif counts were conditioned on the total
number for all
motifs being held fixed within a tissue. The significance of association of a
given
allocation of counts was assessed by Fisher's exact test (one-tailed). Results
were
considered statistically significant at P < 0.05. In summary, to test for
randomness of
distribution, the relative frequencies of a particular tripeptide motif from
each target
was compared to the frequencies of the motifs from the unselected library.
This
approach is a large-scale contingency table association test.
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Results
Phage localizing to human prostate tissue exhibited targeting peptide
sequences
as disclosed in Table 3, minus the terminal cysteine residues on each end of
the
peptides.
Table 3. Human Prostate Tar~etin~ Peutides
GRRAGGS (SEQ ID N0:5) LLAGGVL (SEQ ID N0:18)
TRRAGGG (SEQ >D NO:6) LWSAGG (SEQ m N0:19)
SRAGGLG (SEQ ID NO:7) RTQAGGV (SEQ m N0:20)
SYAGGLG (SEQ ID NO:B) AGGFGEQ (SEQ ID N0:21)
DVAGGLG (SEQ ID N0:9) AGGLIDV (SEQ ID NO:22)
GAGGLGA (SEQ >D N0:10) AGGSTWT (SEQ ~ NO:23)
GAGGWGV (SEQ ID NO:11) AGGDWWW (SEQ ID N0:24)
AGGTFI~I' (SEQ ID N0:12) AGGGLLM (SEQ m N0:25)
LGEVAGG (SEQ m NO:13) VAAGGGL (SEQ m N0:26)
GSNDAGG (SEQ m N0:14) LYGAGGS (SEQ ID NO:27)
YRGIAGG (SEQ m N0:15) CALAGGC (SEQ ID N0:28)
AGGVAGG (SEQ m N0:16) IGAGGVH (SEQ ID N0:29)
GGLAGGF (SEQ m NO:17)
To determine the distribution of the peptide inserts homing to specific sites
after
intravenous administration, the relative frequencies of every tripeptide motif
from
prostate tissue were compared to the frequencies from the unselected library.
The 1,018
phage inserts recovered from representative samples of prostate and from the
unselected
library were analyzed. Tripeptide motifs were chosen for the phage insert
analysis
because three amino acid residues appear to provide the minimal framework for
structural formation and protein-protein interaction (Vendruscolo et al.,
2001).
Examples of biochemical recognition units and binding of tripeptide ligand
motifs to
receptors include RGD (Ruoslahti, 1996), LDV (Ruoslahti, 1996), and LLG
(Koivunen
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et al., 2001) to integrins, NGR (Pasqualini et al., 2000) to aminopeptidase
N/CD13, and
GFE (Rajotte and Ruoslahti, 1999) to membrane dipeptidase.
Each phage insert analyzed contained seven amino acid residues and contributed
five potential tripeptide motifs. Comparisons of the motif frequencies in
prostate tissue
are shown in Table 4. The AGG (SEQ ID N0:30) motif was found only in prostate
homing phage, while the other tripeptide motifs were all found in at least one
other
tissue. Table 4 lists motifs occurring in peptides isolated from prostate but
not from the
unselected phage library (Fisher's exact test, one-tailed; P < 0.05).
Table 4. Peptide Motifs Isolated from Prostate by Ih Vivo Phage Display in
Humans
Motif Motif Freguency P-value
AGG (SEQ ID N0:30) 2.5 0.0340
EGR (SEQ ID N0:31) 1.0 0.0185
GER (SEQ ID N0:32) 0.9 0.0382
GVL (SEQ ID NO:33) 2.3 0.0079
The ClustalW program (European Molecular Biology Laboratory; EMBL) was
used to analyze the original cyclic phage peptide inserts of seven amino acid
residues
containing the tripeptide motifs. The analysis revealed five to six residue
motifs that
were shared among multiple peptides isolated from prostate (Table 5),
including
RRAGGS (SEQ ll~ N0:34) and RRAGG (SEQ ID NO:35). On-line databases were
searched for each of the motifs (including BLAST, SWISSPROT, PROSITE,
PRODOM, and BLOCKS) through the NCBI website
(http:/lwww.ncbi.nlm.nih.~ov/blast/html/blastcgihelp). These motifs are likely
to
represent sequences present in circulating ligands (either secreted proteins
or surface
receptors in circulating cells) that home to vascular receptors in prostate.
Candidate
human proteins potentially mimicked by the selected peptide motifs are
presented in
Table 5.
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Table 5: Examples of human proteins potentially mimicked by peptide motifs
Extended motif Human protein Protein description Accession number
Prostate ~ NP_000632
RRAGGS Interleukin 11 cytokine
(SEQ m N0:34)
RRAGG Smad6 Smad family member AAB94137
(SEO m N0.35)
Table 5 shows sequences corresponding to regions of 100% sequence identity
between the peptide selected and the candidate protein. The identified
homologous
proteins may represent natural ligands for the human receptors that bound
targeting
phage. For example, interleukin 11 has been reported to interact with
receptors within
endothelium and prostate epithelium (Mahboubi et al., 2000). IL-11 may be
mimicked
by a targeting peptide recovered from the prostate (Table 5). These results
were
confirmed by ire situ staining, using polyclonal antibodies against 1L-11
receptor alpha.
IL-11 is a cytokine that is apparently mimicked by the peptide motif RRAGGS
(SEQ m
N0:34), a human prostate targeting peptide. This suggests that the IL-11
receptor alpha
(IL-llRa) should be overexpressed in prostate blood vessels. Studies with
cultured
cells have indicated that 1L-11 interacts with receptors in endothelium and
prostate
epithelium (Mahboubi et al., 2000; Campbell et al., 2001). However, expression
of IL-
llRa in prostate blood vessels has not previously been examined.
Immunostaining of prostate thin sections with antibodies against IL-llRa
showed that IL-llRa is present in the luminal prostate epithelium and in
prostate blood
vessels (not shown). This result validates the human biopanning results and
shows that
the presence of cell surface receptors identified by targeting peptide binding
can be
confirmed by antibodies against the receptor protein.
A considerable advantage of the present method is that the selected targeting
peptides bind to native receptors, as they are expressed if2 vivo. Even if a
ligand-
receptor interaction is mediated through a conformational (rather than a
linear) epitope,
it is still possible to select binders in the screening. As it is difficult to
ensure that
transmembrane proteins expressed by recombinant systems (such as in protein
arrays)
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maintain the correct structure and folding after purification ih vitro,
peptides selected ih
vivo are likely to be more suitable to clinical applications, such as
identification of
novel inhibitors or activators of native receptor proteins.
The skilled artisan will realize that the prostate-targeting peptide sequences
identified in the present Example will be of use for numerous applications
within the
scope of the present invention, including but not limited to targeted delivery
of
therapeutic agents or gene therapy, irz vivo imaging of normal or diseased
organs,
tissues or cell types, identification of receptors and receptor ligands in
organs, tissues or
cell types, and therapeutic treatment of human diseases, such as benign
prostatic
hyperplasia (BPH) and/or prostate cancer. -
Example 3. The IL-11 Receptor as a Therapeutic and Diagnostic Target in
Cancer
The preceding Example identified prostate-targeting motifs (RRAGGS, SEQ m
N0:34 and RRAGG, SEQ ID N0:35) in normal human prostate tissue. The homology
of the RRAGGS (SEQ m N0:34) motif with human IL-11 suggests that the native
prostate receptor for binding of RRAGGS (SEQ ~ N0:34) may be the IL-11
receptor.
The present Example determined whether the IL-11 receptor could be targeted in
prostate cancer, including metastatic prostate cancer.
To test the tissue specificity of the 1L-11 peptide mimic, a phage overlay
assay
was developed to evaluate receptor-ligand interactions in tissue sections,
using the
motif RRAGGS (SEQ m N0:34) (Arap et al., Nature Med. 8:121-127, 2002). Phage
overlay on human tissue sections showed that the prostate-homing phage
displaying an
1L-11 peptide mimic specifically bound to the endothelium and epithelium of
normal
prostate, but not to control organs, such as skin (data not shown). In
contrast, a control
phage that localized to skin tissue, displaying the motif HGGVG (SEQ m NO:36),
did
not bind to prostate tissue (not shown). However, the control phage
specifically
recognized blood vessels in the skin (not shown).
The immunostaining pattern obtained with an antibody against human IL-llRa
(IL-11 receptor alpha) on normal prostate tissue was indistinguishable from
that of a
CGRRAGGSC (SEQ ID N0:37)-displaying phage overlay (not shown). In contrast, a
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control antibody showed no staining in prostate tissue (not shown). These
findings
were recapitulated in multiple tissue sections obtained from several different
patients
(Arap et al., 2002).
Using a ligand-receptor binding assay in vitro, the interaction of the
CGRRAGGSC (SEQ 1D N0:37)-displaying phage with immobilized IL-llRa was
demonstrated at the protein-protein level (not shown). Recombinant IL-llRa,
vascular
endothelial growth factor receptor-1 (VEGFR1) or leptin receptor (OB-R) were
incubated with phage displaying the CGRRAGGSC (SEQ m N0:37) peptide.
VEGFR1 was used as a representative vascular receptor, while OB-R was used
because
it shares a co-receptor with IL-llRa. An unrelated phage clone displaying the
peptide
CRVDFSKGC (SEQ ID N0:38) and insertless fd-tet phage were used as controls.
Only the IL,-llRa receptor protein exhibited a significant amount of binding
to
CGRRAGGSC (SEQ m N0:37)-phage (not shown). Neither OB-R nor VEGFR1
showed binding to CGRRAGGSC (SEQ iD N0:37)-phage above control levels (not
shown). Neither of the control phage exhibited selective binding to IL-llRa
(not
shown). Binding of CGRRAGGSC (SEQ ID N0:37)-phage to IL-llRa was specific,
since it was inhibited by the native IL-11 ligand in a concentration-dependent
manner
(not shown). Close to 100% inhibition of CGRRAGGSC (SEQ m N0:37)-phage
binding was observed at a peptide concentration of about 0.1 nM (not shown).
These
observations with normal prostate tissues were followed by an examination of
the
expression of IL-llRa in tumors, as discussed in the present Example. IL-11R
expression was found to be upregulated in human prostate cancer (see below).
Characteristics of IL-IIReceptor
IL-llRa is a member of the gp130-dependent family of proteins, along with
receptors for IL6, oncostatin M, leukemia inhibitory factor, and cilliary
neurotrophic
factor (Du and Williams, Blood 89:3897-3908, 1997). 1L-11 initiates signaling
via
binding to the unique 1L-llRa chain, The complex of IL-11 and IL-llRa then
binds to
and induces clustering of gp130, leading to the activation of associated Janus
kinases
(JAKs) and translocation to the nucleus of the signal transducers and
activators of
transcription (STAT) proteins 3 and 1 (Lutticken et al., Science 263:89, 1994;
CA 02496938 2005-02-23
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Campbell et al., Am. J. Pathol. 158:25-32, 2001). STAT3 has been reported to
be
constitutively activated in prostate cancer (Ni et al., J. Urol. 167:1859-62,
2002). IL,-
llRa expression was reported to be increased in primary prostatic carcinoma
compared
to non-malignant prostate tissue (Campbell et al., 2001). No previous reports
have
characterized IL-llRa expression in metastatic cancer.
Other signaling systems that may be activated by IL-llRa include MAP kinase,
and the ribosomal S6 protein kinase pp90rsk, src-family tyrosine kinases
including
p60src and p62yes, and phosphatidylinositol-3 kinase. 1L-llRa has been
characterized
on human solid tumors such as breast, colon, ovary, and melanoma (Douglas et
al.,
Oncogene 14:661-69, 1997; Gupta et al., Proc. Am.Assn. Cancer Res. 38:554,
1997;
Paglia et al., J. Interf. Cytokine Res., 15:455-460, 1995; Campbell et al,
Gynecol.
Oncol. 80:121-27, 2001), although its functional role and prognostic
significance were
unknown.
Distributioyt of IL-IIRc~In Primary and Metastatic PYOState Cancer
Imrnunohistochemical (IHC) analysis was performed to examine the distribution
of IL-llRoc in primary prostate cancer and metastatic prostate cancer. The
present
Example represents the first report of IL-llRoc distribution in metastatic
cancer of any
kind. Normal tissues from different areas in the prostate were also examined.
Tissues
from 99 archival formalin-fixed paraffin-embedded human primary and metastatic
prostate cancers and the corresponding adjacent non-neoplastic tissues were
obtained
from 90 patients and evaluated. Samples consisted of 81 primary
adenocarcinomas (71
androgen-dependent [AD] obtained from radical prostatectomy without prior
treatment,
and 10 androgen-independent [AI] obtained either from radical prostatectomy,
cystoprostatectomy, or pelvic exenteration) and 18 lymph node and bone
metastases
and were selected to reflect: 1) stages in prostate cancer progression; 2)
different
Gleason scores; 3) hormonal dependence: AD and AI tumours; and 4) tonal
origin:
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peripheral zone and transition zone. Additional blocks from the same
specimens,
including benign prostatic tissue from peripheral zone, transition zone, and
central
zone, and the seminal vesicle/ejaculatory duct, were included when available.
The samples were stained within two weeks of cutting to minimize loss of
immunoreactivity. Four-~,m sections were conventionally deparaffinized and
rehydrated, blocked for endogenous peroxidases, antigen-retrieved in a
microwave oven
by treatment with EDTA solution (pH 8.0; Zymed, San Francisco, CA), and biotin
and
protein blocked (both from DAKO Corp., Carpinteria, CA). Incubation with anti-
human
IL-llRa K-20 (1:15 for 45 minutes at room temperature; Santa Cruz
Biotechnology,
Santa Cruz, CA) followed. The LSAB+ kit (DAKO) was used for immunostaining and
development. All sections from each specimen were from the same staining run
to
avoid interassay variability.
Competition experiments with the antigenic peptide (5:1 w/w absorption; Santa
Cruz) were performed to confirm specificity. Paraffin sections of the HeLa
cell line
were used as immunopositive controls. Negative controls included omission of
the
primary antibody, and substitution of primary antibody with non-immune goat
serum at
equivalent immunoglobulin concentration. Endothelial cells were immunostained
by
JC/70A (anti-CD31, DAKO) monoclonal antibody. IL-llRa staining was evaluated
both in tumour and non-tumour tissues, including pathologic conditions as
benign
prostatic hyperplasia (BPH) and transitional metaplasia, and high-grade
prostatic
intraepithelial neoplasia (PIN).
Positive cases were defined by the presence of cytoplasmic staining, as seen
in
the positive controls. Intensity in benign and malignant tissues was scored as
0
(negative), 1+ (weak), 2+ (moderate), or 3+ (strong). IL-llRcc expression in
benign
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glands was generally observed in the basal cell compartment with/without
staining of
the luminal cells. We evaluated benign glands from 1+ (no/weak luminal cell
staining)
to 3+, taking into account then the highest intensity of staining in the
luminal
compartment, and compared this score with the most prevalent one observed in
the
cancerous tissue from the corresponding area. Due to heterogeneity in
intensity among
and within tumour samples, a total immunostaining score was calculated as the
sum of
the products of percentage of cells (in 10% units) per intensity level (up to
a maximum
score of 300) to evaluate differences in expression among cancerous specimens.
All
analyses were done with S-PLUS 2000 (Math Soft, Inc.).
Table 6. Clinical and histopathological characteristics and ILllRa expression
Specimen Number Median score (range)*
p
of cases
Normal prostate
Peripheral zone 62 1+ (1-2) NS
Transition zone 51 1+ (1-2)
Central zone 40 1+ (1-2)
Seminal vesicle / Ejaculatory43 / 3 2+ (2-3) / 2+ (2) ..
Duct
Benign pathologic conditions
Benign prostatic hyperplasia15 1+ (1-2) ..
Stromal nodule 2 1+ (1-2) ..
Atrophy 10 2+ (1-2) ..
Transitional metaplasia 18 2+ (1-2) ..
Prostatic intraepithelial neoplasia (PIN) 23 2+ (1-3) ..
Primary prostate cancer
Androgen-dependent 71 2+ (1-3) / 180 (50-290)
Zonal origin
Peripheral zone 55 190 (50-290) 0.0003~~
Transition zone 16 135 (50-250)
Gleason score
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<_ 7 (3+4) 26 150 (50-260) 0.0049[
>_ 7 (4+3) 38 200 (100-290)
Pathological stage fi
pT2_pT3a 42 175 (50-290) 0.0469[
pT3b-pT~ypNi
22 210 (100-280)
PSA (ng/mL) fi
< 10 48 180 (50-280) NS9[
>_ 10 14 200 (100-290)
Androgen-independent 10 250 (80-300) ..
Metastatic prostate cancer
Lymph nodes
Androgen-dependent 4 235 (200-290) NS~
Androgen-independent 8 235 (190-300)
Bone 6 270 (140-290) ..
NS= non-significant. * Categories 1+-3+ were used for evaluation of benign
prostatic
tissues and comparison to prostatic intraepithelial neoplasia and primary
prostate
cancer. A combined intensity per percentage of immunostained tumour cells
scoring
system was used to evaluate differences in expression among cancerous
specimens (see
text). ~ Only the predominant tumour focus in each case was considered (64/71
cases).
~ Wilcoxon signed rank test. ~~ Mann-Whitney rank sum test. 9[ Spearman
correlation
test.
No differences were observed in IL,-llRoc expression between normal glands in
the different prostatic areas (Table 6). Some background, distinct to a
frequent stromal
staining, was observed in the epithelium of seminal vesicles and ejaculatory
ducts.
Expression in PIN and AD samples examined was significantly higher than in
their
benign counterparts from the same areas (p<0.0001 in both cases, Wilcoxon
signed
rank test), but no differences were observed between P1N and AD (p=0.5, signed
rank
test). Among primary AD specimens, IIrllRa immunoreactivity was increased in
cancers from the peripheral vs. transition zone (p=0.0003), in Gleason >7
(4+3) vs.
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Gleason <_7 (3+4) (p= 0.004), and, more marginally, in pT3b-pTanypNi tumours
vs. pT2-
pT3a (p=0.046) (Table 6).
Primary AI specimens showed a more homogeneous pattern of staining, with
more than 80% cells displaying moderate/strong intensity in 80% of the
samples.
However, no significant increase in expression was observed in AI vs. AD cases
matched by Gleason score (p=0.15, rank-sum test), likely because of the small
number
of samples. Expression in 6 regional (4 AD and 2 AI) and 6 distant lymph node
metastases (6 AI) was also intense in a high percentage of tumour cells.
Cancer cells
displayed a homogeneous moderate to strong intensity of staining in 5 out of 6
specimens from bone metastases (all An. Both osteoblasts and osteoclasts
stained
moderately, and were used as internal positive controls. Interestingly, blood
vessels in
bone and lymph node metastases and in primary cases with previous treatment,
showed
an occasionally striking ILllRa immunoreactivity that was confirmed by CD31
staining on consecutive slides, as opposed to a more random pattern in the
other benign
and malignant tissues analysed.
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The results show that IL-llRa expression correlates with tumor progression
(FTG. 1 and FIG. 2). FIG. 1 shows localization of 1L-llRa in benign prostate
glands.
Normal prostate glands in the peripheral (FIG. 1A) or central (FIG. 1B) zones
showed
predominantly nuclear staining of the basal and luminal cell layers.
FIG. 2 illustrates IHC staining for lL-llRa in primary androgen dependent
prostate cancer of low (FIG. 2A), intermediate (FIG. 2B) and high (FIG. 2C)
Gleason
grade prostate tumors. FIG. 2A shows IL-llRa distribution in a Gleason score 6
prostate adenocarcinoma (homogeneous 2+ staining). FIG. 2B shows 1L-llRa
distribution in prostate carcinoma (arrowheads) (1+ and 2+ staining). The
prostate
carcinoma exhibited elevated staining for IL-llRa compared to adjacent luminal
cells
of benign prostate (arrows). Strong (3+) staining for IL-llRa was observed in
high-
grade prostate adenocarcinoma (FTG. ZC). FIG. 2D shows that benign prostate
glands
from the peripheral zone, containing a few neoplastic acini, exhibited little
or no
staining for 1L-llRa compared to prostate cancer.
IL-llRa expression was strongly up-regulated in metastatic prostate cancer
(FIG. 3). FIG. 3A shows strong homogenous (3+) staining in prostate cancer
that had
metastasized to bone. A higher power magnification of the same sample shows 2+
and
3+ staining in tumor cells (FIG. 3B). FIG. 3C shows that small blood vessels
around
tumor nodules in the bone matrix also exhibited strong staining for IL-llRa.
CD31
staining of the same sample (FIG. 3D) confirmed the endothelial cell
reactivity of the
1L-llRa IHC staining. A high-grade, androgen-independent primary prostate
tumor
also exhibited strong (3+) staining for IL-llRa (FIG. 3E). A negative control
of benign
prostate tissue from the same area as FIG. 3B exhibited little or no staining
for 1L-llRa
(FIG. 3F). FIG. 33 shows the distribution of 1L-llRa expression in primary
androgen-
dependent prostate carcinoma by immunohistochemical score, according to
Gleason
grade and pathological stage.
1L-llRa expression was examined in blood vessels of prostate tissue samples.
Although staining was observed in some prostate blood vessels, it was not
observed in
others. A sub-group of cases displayed a stronger and more consistent staining
in blood
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vessels. The majority of such cases were androgen-independent, including both
primary and metastatic androgen-independent tumors (17 of 24 AI cases). Blood
vessel
staining in AI tumors could result from either a shift in hormonal dependence
or
exposure to previous treatment. No common modality of treatment was observed
in
such cases, with the exception of hormone ablation. A few cases had been
treated with
radiotherapy and the rest with different type of chemotherapy. In some cases
the
systemic treatments had been administered a long time before sample analysis
for IL-
llRa expression. It is concluded that androgen independence is correlated with
high
levels of IL-llRa expression in blood vessels. The skilled artisan will
realize that IL-
llRa staining may be of use to distinguish androgen-dependent from androgen-
independent cases and therefore to assist in tailoring therapeutic treatment
to the status
of the tumor as androgen-dependent or androgen-independent.
It is concluded that expression of IL-llRa is of use as a specific marker for
metastatic prostate cancer in bone tissue. The skilled artisan will realize
that IL-llRa
staining may be used for detection, diagnosis and/or imaging of metastatic
prostate
cancer in bone and/or other tissues, such as lymph nodes.
Clinical Sig~if-'ccahce
Approximately half of presently hospitalized cancer patients will die of their
disease despite optimal management. Given such a high failure rate, estimates
of
potentially curative treatment based on the risk of recurrence remain
difficult to
extrapolate for an individual cancer patient. There is a clear need for
improved
biomarkers of cellular growth potential and targets in cancer. Based on the
present
results, expression of IL-llRa in prostate cancer appears to be one such
biomarker.
The molecular observations reported herein may be confirmed in a clinical
context by following patient outcome in prostatic cancers with varying levels
of lL-
llRa expression, using known methods. For example, probabilities of survival
for
each group of patients may be analyzed by the Kaplan-Meier method. Log-Rank
test
may be used to determine statistical differences between groups. A Cox
proportional
hazards model may be used to analyze the effect of single and multiple risk
factors in
association with survival. Martingale residual plots may be used to assess the
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proportional hazard assumption. Results may be considered statistically
significant at P
< 0.05.
Such observations may be validated in archived pathological material. Group
stratification of cancer patients based on a panel of one or more such markers
would
allow less aggressive tumors to be effectively eradicated, while patients with
more
aggressive tumors could be offered experimental therapies earlier in their
clinical
progression. Without reliable ways of predicting which tumors will progress,
many
cases are treated aggressively on the chance of cure, but often at the price
of potentially
devastating treatment-associated side effects. There is a clear need for
markers of
cellular growth potential, such as 1L-llRa, as diagnostic and therapeutic
targets in
cancer patients. The skilled artisan will realize that expression of IL-llRa
may be
useful in other types of tumors besides prostate cancer, so long as 1L-llRa is
correlated
with tumor growth and/or metastatic potential. Exemplary tumors in which IL-
llRa
may be of use for detection, diagnosis and/or prognosis of cancer include
prostate,
breast, colon, ovary and melanoma.
Example 4. Biopanning Circulating Immunoglobulins In Human Prostate
Cancer Patients
A phage display library was screened against a pool of circulating antibodies
obtained from a human prostate cancer patient. The biopanning procedure
resulted in
the identification of a novel marker for prostate cancer that is diagnostic
for disease
progression in metastatic prostate cancer. In this embodiment, the antibody
pool
provides a structural sampling of ligands targeted to naturally occuring
receptors, some
of which may constitute novel disease markers. Biopanning against an antibody
pool
may be used to identify disease markers and to further characterize the
molecular events
underlying the disease state.
The present Example shows the feasibility of this approach by identifying a
novel marker for prostate cancer. The results further show that this marker
has
prognostic value for predicting which individuals with prostate cancer are
likely to have
an unfavorable clinical outcome, resulting in death of the patient. As
discussed above,
there is a great need in the field of prostate cancer for a reliable method to
separate
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those individuals whose prostate cancer will prove lethal (and therefore are
candidates
for more agressive therapeutic intervention) from individuals who will not die
from
prostate cancer. The present Example represents a significant advance in
prostate
cancer prognosis and illustrates the utility of the claimed methods and
compositions.
The skilled artisan will realize that although the present Example deals with
prostate cancer, the methods and compositions disclosed are suitable for use
with any
disease state or condition in which the host immune system is likely to
produce
antibodies against a molecular marker associated with the disease or
condition.
The repertoire of circulating antibodies from the serum of prostate cancer
patients with advanced disease was used to screen a phage display library.
Certain
peptides binding to those antibodies correspond to tumor antigens expressed in
bone
metastasis of prostate cancer. A panel of prostate cancer serum samples from
patients
with recorded clinical outcome was screened by an ELISA assay against those
peptides.
The results show that reactivity against one particular peptide ("peptide C")
can be used
to identify patients with metastatic androgen-independent prostate cancer.
Moreover,
patients with detectable levels of circulating antibodies against peptide C
exhibited
decreased survival compared to individuals without such antibodies.
Methods
Sera was selected from patients diagnosed with androgen-dependent and
androgen-independent prostate cancer. A CX6C peptide library was screened
against
this pool of IgGs in a two-step procedure. First, the peptide library was pre-
cleared
against a pool of purified IgGs from normal serum samples using Protein G
affinity
chromatography. This step removed peptides from the phage display library that
bound
to immunoglobulins from patients without prostate cancer. Next, the pre-
cleared
peptide library was screened against the pool of purified IgGs from the serum
of
prostate cancer patients. This step selected peptides binding specifically to
IgGs elicited
against prostate cancer.
Human seta ahd tissue samples
Human plasma samples were prospectively collected from 91 patients with
locally advanced, metastatic androgen-dependent and metastatic androgen-
independent
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adenocarcinoma of the prostate. In each case, the patient was evaluated in
reference to
tumor staging (locally advanced or metastatic disease) and hormone
responsiveness of
the disease (androgen-dependent or androgen-independent). Criteria for
enrollment
consisted of a combination of the TNM classification and histological grading.
Patients
diagnosed with adenocarcinoma of the prostate with stage Tlc or T2 with
Gleason
score less than or equal to 7 and serum PSA < 10 ng/ml were considered to have
clinically organ-confined prostate cancer. Study entry in the locally advanced
group
required appropriate primary tumor staging (stage Tl~ or T2 with Gleason score
greater
than 7; or clinical stage T2b-2c with Gleason score equal to or greater than 7
and serum
PSA > 10 ng/ml; or clinical stage T3) and no regional (No) or distant (Mo)
metastases.
Study entry in the metastatic group required evidence of regional (Nl) and/or
distant
(Ml) metastases in radionuclide bone scan, chest radiography, or computed
tomography
of the abdomen and pelvis. Androgen-independence was defined as serum
testosterone
lower than 50 ng/dl and serially rising serum PSA; index patients 1, 2, and 4
were
androgen-independent, while index patient 3 was androgen-dependent at the time
their
serum samples were obtained.
For biopanning, sera was examined from three metastatic androgen-independent
and one metastatic androgen-dependent prostate cancer patients. Plasma from 34
healthy individual donors (eleven males) was obtained from the Blood Bank at
the
University of Texas M. D. Anderson Cancer Center (UTMDACC). Archived tissue
paraffin blocks were obtained from the Department of Pathology at UTMDACC. The
blood samples were initially allowed to clot at room temperature and then
centrifuged
to separate the cellular component from the supernatant. Aliquots of
supernatant were
promptly frozen and stored at -~0°C until assayed.
Biopanhihg.
A 6-mer cyclic peptide (CX(C) phage display library was used for the
biopanning. To select peptides specific to the serum antibodies of cancer
patients, a
pre-clearing stage was employed to remove non-specific peptides by pre-
absorbing the
peptide library onto purified IgGs from pooled normal serum (five healthy male
individuals). The pre-cleared peptide library was screened onto the purified
IgGs from
CA 02496938 2005-02-23
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the serum of prostate cancer patients. In brief, 109 transducing units (T.U.)
of a CX(C
cyclic peptide phage library were incubated with IgG antibodies from 50 ~,l of
normal
serum immobilized on 50 ~.l of protein G (Gibco BRL) for 1 hour at 4°C.
This was
followed by affinity selection on the immobilized IgG antibodies from prostate
cancer
patient serum for 2 hours at 4°C. Phage peptides specifically bound to
IgGs elicited
against prostate cancer were eluted with 100 ~1 of 0.1 M glycine buffer, pH
2.2,
neutralized by the addition of 10 ~,1 1M Tris pH 9.0, and used to infect E.
coLi strain
K91. Ten-fold serial dilutions of the infected solution were spread onto agar
plates
containing 40 ~g/ml of tetracycline and grown overnight. Two hundred colonies
were
picked, amplified, and precipitated for a subsequent round of panning. A total
of three
rounds were performed. Individual phage clones were picked for PCR and the
insert
DNA was sequenced.
Enzyme-linked inzmunosorbent assay and peptide inhibition study.
A 20 [ug/ml solution of purified GST or GST-fusion proteins in 0.1M NaHC03
was used to coat maxisorp mufti-well plates (Nalge Nunc International
Corporation)
and incubated overnight at 4°C. The plates were blocked in a blocking
buffer (4%
milk, 2% casein, and 0.05% Tween-20) for 3-4 hours. A series of 100-fold
dilutions
(1:100-1:1200) of sera from prostate cancer patients or healthy individuals
was added
and incubated for 1.5 hours and then washed five times with washing buffer (1%
milk,
0.5% casein, and 0.025% Tween-20), followed by incubation at 4°C with
anti-human
alkaline phosphatase-conjugated antibodies (Gibco). The plates were then
washed six
times in washing buffer and developed using p-NPP (Sigma) as a substrate. An
automatic ELISA plate reader (BIO-TEK instrument) recorded the results at
OD405
nm.
Antibody biotihylation.
GST-fusion proteins containing the peptide sequence from patient C were
coated on mufti-well plates. After incubating the plates with the patient's
serum, the
plates were washed. The bound IgG antibodies were eluted with 50 ~.l of 0.1 M
glycine
buffer, pH 2.2, neutralized by addition of 10 p,l 1 M Tris pH 9.0, and
dialyzed in PBS
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overnight followed by concentration of the antibody using Centricon-30
(Millipore)
filters. The purified antibody (500 p.g) was coupled to biotin according to
the
manufacturer's instructions (Vector). The biotinylated antibody was analyzed
by SDS-
gel electrophoresis.
Immufzohistological staining.
Paraffin sections (4 Vim) were stained with purified biotinylated antibodies
and
peptide antibodies by immunoperoxidase detection using the Dako antigen
retrieval kit
and DAB (diaminobenzidine) as a substrate. All of the sections were counter-
stained
with hematoxylin. Purified IgGs were coupled to biotin and resolved by SDS-
PAGE.
The biotinylated immunopurified antibodies were used at a dilution of 1:60.
Peptide C
antibodies and purified pre-immune antibodies were used at 0.01 p,g/~1. For
the
inhibition staining, peptide C antibodies were pre-incubated for 30 minutes at
room
temperature with the corresponding GST-peptide C (500 fig) prior to staining.
For the
GRP78 immunostaining, anti-GRP78 antibody (C-20) was used at 1:350 (Santa Cruz
Biotechnology, Santa Cruz, CA). Peptide antibodies were generated in rabbits
and
purified using a T-gel immunoglobulin purification kit and protein G column
(Pierce).
Protein purification, mass spectrometry, ayzd immuhoprecipitatioh
DU-145 prostate cancer cells (American Type Culture Collection), which
express the native antigen (data not shown), were used for protein
purification. Cells
were grown to 70% confluence, harvested in PBS, and treated with TM buffer
(100 mM
Tris-Cl, 2 mM MgCl2, 1% Triton-X100). Cells were sheared to separate nuclei
from
cytoplasm and other organelles. The cytosolic/membrane fraction was
centrifuged.
The supernatant was collected, resolved on 4.-20% gradient SDS-PAGE, probed by
rabbit anti-peptide antibodies on Western blots and detected by enhanced
chemiluminescence (ECL; Pharmacia). The band containing the protein recognized
by
the anti-serum was excised and used for protein sequencing. Mass spectrometry
analysis was compared to databases containing known protein sequences by BLAST
homology search.
For immunoprecipitation, 200 ,ul of protein G agarose beads (Pierce) were
coupled to anti-GRP78 or rabbit anti-peptide antibodies, and the recombinant
GRP78
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(Stressgen) was added at 150 ~Cg, and incubated for 4 hours. As a negative
control,
protein G agarose beads alone were used. The immunoprecipitates were recovered
by
centrifugation, rinsed with wash buffer (0.05% Tween-20 in PBS), and resolved
by
SDS-PAGE.
A Western blot was probed with either anti-CNVSDKSC (SEQ ID N0:39) or
anti-GRP78 antibodies (each at 1:200 dilution) and detected by ECL. For
detection of
GRP78 in the normal prostate and bone metastasis, whole lysates from frozen
tissue
samples were prepared by grinding the tissue in a dounce homogenizer in a 2m1
of
Tissue Protein Extraction Reagent (Pierce) per sample with protease inhibitors
(10
,ug/ml of leupeptin and aprotonin). The homogenate was incubated on ice for 10
minutes prior to repeated grinding. The homogenate was spun at 610g for 5
minutes and
the supernatant was removed and protein concentration was measured using the
Protein
DC Assay (BIO-RAD). 20 ,ug of protein from the normal prostate and bone
metastasis
lysates were resolved on a 4-20% SDS-PAGE, probed by anti-GRP78 antibody on
Western blots and detected by ECL.
Cross-itzhibitioh assays
Microtiter 96-well plates were coated with 10 ~,g/ml recombinant GRP78
(Stressgen) or GST attached to CNVSDKSC (SEQ ID N0:39) in 100 mM NaHC03
overnight at 4°C, washed and then blocked with blocking buffer (2%
milk, 1 % casein,
0.05% Tween-20 in PBS) for 2 hours at 37°C. To determine the inhibitory
activities of
GRP78 or GST-CNVSDKSC (SEQ ID N0:39), patient serum (1:50), anti-GRP78
(1:1000), and anti-GST-CNVSDKSC (1:20) were incubated with GRP78 (50-100 ~,g)
or GST-CNVSDKSC (SEQ ID N0:39) (100-300 ~.g). The mixtures were incubated for
1 hour at 37°C prior to adding to the coated wells. After 1 hour of
incubation at room
temperature the wells were washed several times with PBST buffer (0.05% Tween-
20
in PBS). Secondary antibodies conjugated to horseradish peroxidase were added
at
1:5000 dilution, incubated for 30 minutes at room temperature and washed five
times
with PBST buffer. Finally, the substrate 3,3',5,5'-Tetramethylbenzidine (TMB;
Calbiochem) was added and incubated for 15 minutes at room temperature before
stopping the reaction by addition of 0.5M H2S04. Absorbance at 450 nm was
determined in an automated ELISA reader (Bio-Tek).
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Statistical analysis.
Probabilities of survival for each group were estimated using the Kaplan-Meier
method. A log-rank test was implemented in order to detect significant
differences
between the groups. Reactivity was considered to be detected if the ratio
between GST-
peptide and GST alone was greater or equal to two by the ELISA data. A Log-
Rank test
was used to determine statistical differences between groups. A statistical
CART
analysis (Classification And Regression Tree) was used to identify the best
cut-off point
for determining reactivity to GRP78. In this method, the censored survival
data were
transformed into a single uncensored data value (the so-called "null
martingale
residual"), which was used as input into a standard regression tree algorithm.
A cut-off
point of 0.95 was determined by this program
Results
After three rounds of selection, a striking enrichment (log scale) was
observed
in three out of the four serum samples examined (FIG. 4). In the fourth
patient sample,
no enrichment was observed and this patient was not studied further.
Individual phage
clones from the second and third rounds of selection from serum samples A, B,
and C
were sequenced. The peptide motifs CHQKPWEC (SEQ ID N0:40) from patient A and
CKDRFERC (SEQ ID N0:41) from patient B represented 100% of the clones analyzed
from those patients. In patient C, the peptide CNVSDKSC (SEQ ID N0:39)
appeared
in 55% of the clones analyzed. The remaining clones identified in patient C
were
CNWTDKTC (SEQ ID N0:43), representing 33.3% of the clones in round II and 7%
of
the clones in round III, CNITQKSC (SEQ ID N0:44), representing 33.3% of the
clones
in round II and 0% in round III, and CNKTDKGC (SEQ ID N0:45), representing
16.7% of the clones in round II and 0% in round III.
ELISA was performed to assess if the peptides could be specifically recognized
by the antibodies present in the serum of the patients selected for the
screenings.
Peptides A (CHQKPWEC, SEQ ID NO:40), B (CKDRFERC, SEQ ID N0:41), and C
(CNVSDKSC, SEQ ID N0:39) were produced as GST-fusion proteins and
immobilized onto microtiter wells, along with GST alone as a negative control.
For
each sample tested, a series of 100-fold dilutions was performed. Little
reactivity
occurred with the GST control, whereas strong reactivity occurred with the GST-
fusion
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peptides (FIG. 5). The reactivity of each serum against peptides A, B and C
was
inhibited by the corresponding synthetic peptides (data not shown).
Characterization of the Peptide CNYSDKSC (SEQ ID N0:39) and Cli~eical
Correlatio~s
Having shown selective binding of GST-CNVSDKSC (SEQ ll7 N0:39) fusion
peptides to prostate cancer patient serum, the reactivity profile against the
peptide
CNVSDKSC (SEQ ID NO:39) was assessed in a population of 108 sera obtained from
clinically annotated prostate cancer patients and 71 age-matched healthy men
(negative
control). Among the control serum samples tested, a small percentage of
positive
reactivity (7%) was detected with the selected peptide (FIG. 6A and 6B). In
contrast,
positive serum reactivity from the 108 sera samples from prostate cancer
patients
correlated positively with the natural progression of the disease (Fig. 6A and
6B).
Thus, positive serum reactivity against CNVSDKSC (SEQ ID N0:39) correlated
with
late-stage prostate cancer and androgen-independence. For example, only 6% of
the
organ-confined patients' sera reacted against the peptide CNVSDKSC (SEQ ID
N0:39), whereas patients with androgen-dependent tumors reacted against the
peptide
in 29% of the samples (FIG. 6B). Most notably, 76% of the samples obtained
from
patients with metastatic androgen-independent prostate cancer reacted to the
sequence
CNVSDKSC (SEQ ID N0:39) (FIG. 6B).
Kaplan-Meier curve estimates (Kaplan and Meier, J. Am. Statist. Assoc. 53:457-
481, 1958) were applied to compare survival between the positive reactive and
negative
reactive groups (FIG. 6C). Reactivity against the peptide CNVSDKSC (SEQ ID
N0:39) (n=42) was associated with a significantly shorter patient survival
(FIG. 6C,
Log-Rank test, P=0.02). The median survival in the positive reactivity group
was
reached after 32.7 months while the median survival in the non-reactivity
group was not
reached (FIG. 6C). The data show a strong correlation between positive
reactivity
against the peptide CNVSDKSC (SEQ ID N0:39), development of metastatic
androgen-independent prostate cancer (the most advanced stage of the disease),
and
decreased survival.
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Identification of the Corresponding Native Turraor-Associated Antigen.
Antibodies against the peptide sequence CNVSDKSC (SEQ ID N0:39) were
used to determine whether they could specifically recognize tumor-associated
targets in
tissue sections by immunohistochemistry. Normal prostate tissue and metastatic
prostate cancer from bone marrow in samples obtained from patient C were
subject to
IHC staining using autologous immunopurified IgGs or a rabbit polyclonal
antibody
against CNVSDKSC (SEQ m NO:39). Strong staining was observed using immuno-
purified antibodies from the autologous patient serum (FIG. 7A). Specific
immunostaining was also observed using a rabbit polyclonal antibody raised
against the
synthetic peptide CNVSDKSC (SEQ ID NO:39) (FTG. 7B). No immunostaining was
observed with the pre-immune antibodies (FIG. 7C) or a secondary antibody
alone
(FIG. 7D). The immunohistochemical signal observed in FIG. 7B was mostly
inhibited
by a fusion protein containing the sequence CNVSDKSC (SEQ ID NO:39)
demonstrating the specificity of the staining protocol (FIG. 7E). Normal
prostate from
the same individual only exhibited weak staining using the antibody against
CNVSDKSC (SEQ ID N0:39) (FIG. 7F).
The target antigen mimicked by the peptide sequence CNVSDKSC (SEQ ID
N0:39) was identified by standard biochemical techniques. An extract of the
DU145
prostate cell line, containing cytosolic and cell membrane fractions, was
prepared as
disclosed above and reacted with anti-CNVSDKSC (SEQ ll~ N0:39) polyclonal
antibody, prepared by injecting rabbits with CNVSDKSC (SEQ ID N0:39)
conjugated
to KLH. A single 80 KDa protein was identified by Western blotting (not
shown).
The 80 kDa band was excised for protein sequencing. Five peptide sequences
were obtained from the protein excised from SDS gels. All five peptides
matched
portions of the 78 kDa glucose regulated protein (Table 6, GRP78, SEQ ID
NO:42,
GenBank Accession Numbers CAB71335 and XM 044202). The locations of the five
sequenced peptides within GRP78 are indicated in Table 7 in bold font. A
commercial
antibody against GRP78 (Santa Cruz Biotechnology, Santa Cruz, CA) reacted on
Western blotting with the purified 80 kDa peptide C antigen from DU145 cells
(not
shown). The original peptide C sequence (SEQ ID NO:39) is not found within the
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GRP78 sequence (SEQ ID N0:42), indicating that the epitope recognized i~z vivo
by
anti-peptide C antibodies is formed from discontiguous regions of the GRP78
protein.
Table 7. Sequence of Human GRP78 (SEQ ID N0:42)
MKT=SLVAAMLLLLSAARAEEEDKKEDVGTVVGIDLGTTYSCVG
VFKNGRVEIIANDQGNRITPSYVAFTPEGERLIGDAAKNQLTSNPENTV
FDAKRLIGRTWNDPS VQQDIKFLPFKV VEKKTKPYIQVDIGGGQTKTFA
PEEISAMVLTKMKETAEAYLGKKVTHAVVTVPAYFNDAQRQATKDAG
TIAGLNVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTID
NGVFEVVATNGDTHLGGEDFDQRVMEHFIKLYKKKTGKDVRKDNRAV
QKLRREVEKAKRALSSQHQAR1EIESFYEGEDFSETLTRAKFEELNMDLF
RSTMKPVQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKEPS
RGINPDEAVAYGAAVQAGVLSGDQDTGDLVLLDVCPLTLGIETVGGVM
TKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDL
TGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNK1TITNDQNRLTP
EElERMVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGG
KLSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISKL
YGSAGPPPTGEEDTAEKDEL
The molecular mimicry between the selected peptide and GRP78 was shown by
reciprocal co-immunoprecipitation with either anti-GRP78 antibody or anti-
peptide
CNVSDKSC (SEQ ID N0:39) antibody (not shown). Whole lysates were made from
frozen tissue samples of normal prostate and bone metastasis. Equivalent
amounts of
protein (20 ug) were resolved on 4-20% SDS-PAGE and probed with an anti-GRP78
antibody on Western blots. GRP78 was weakly expressed in normal prostate
tissue,
whereas it was highly expressed in the bone metastasis from a patient with
prostate
cancer (not shown). Recombinant GRP78 or the GST-CNVSDKSC (SEQ ID N0:39)
fusion protein were capable of blocking binding to the 80 kDa protein of the
patient's
serum antibodies, the anti-GRP78 antibody, and polyclonal antibodies raised
against the
peptide CNVSDKSC (SEQ ID NO:39) (FIG. 8). Collectively, these data demonstrate
that GRP78 is the endogenous antigen against which circulating antibodies are
present
in a high percentage of metastatic prostate cancer patients.
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Prognosis and Predictive Value of Serum Reactivity to GRP78.
GRP78 functions in antigen presentation (Melnick & Argon, Immuhol. Today
16:243-50 1995). Its stress-responsive promoter is strongly induced in
response to
glucose deprivation, acidosis, and chronic hypoxia Lee, Trends Biochem. Sci.
26:504-
510, 2001). Since such conditions are generally present in poorly vascularized
solid
tumors, it was determined whether GRP78 is a general biomarker of the tumor
microenvironment or whether its expression is specific to prostate cancer.
The reactivity of serum samples obtained from prostate cancer patients and
controls was evaluated against GRP78. Using a cut-off point of 0.95 absorbance
as
determined by the "CART" (Classification And Regression Tree) statistical
method, a
26-52% positive reactivity was observed in a population of patients with
advanced
prostate cancer in contrast to only 6% in age-matched control men and 0% in
the organ-
confined group (FIG. 9A). GRP78 reactivity was also examined in the serum of
three
groups of non-prostate cancer patients (FIG. 9A). Significantly less
reactivity against
GRP78 was observed in serum from patients with metastatic non-small cell lung
cancer
(P < 0.001), metastatic breast cancer (P < 0.001) and advanced ovarian cancer
(P <
0.001) (FIG. 9A).
A survival curve was applied to compare the overall survival between the
positive reactivity and non-reactivity groups (n=108) for GRP78 (FIG. 9B).
Positive
reactivity to GRP78 was associated with a shorter survival outcome (Log-Rank
test,
P=0.07) (FIG. 9B, lower line). Taken together, these data strongly suggest
that
reactivity against GRP78 is a preferential serological marker of prostate
cancer relative
to other malignant tumors.
Expression of GRP78 irz bone metastasis and normal prostate tissue.
The presence of circulating antibodies against GRP78 was associated with the
most aggressive stage of prostate cancer (metastatic androgen-independent
disease).
The expression of GRP78 was examined by immunohistochemical analysis in normal
prostate tissue and bone marrow metastasis from a prostate cancer. The GRP78
antigen
was highly expressed in bone marrow metastasis as shown by strong
immunostaining
(FIG. l0A), whereas weak staining was observed in normal prostate tissue (FIG.
10B).
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These results confirm the Western analysis using the same tissue samples noted
above
(FIG. 7F). To show specificity, staining was inhibited using recombinant GRP78
(FIG.
lOC) or the peptide fusion protein GST-CNVSDKSC (SEQ ID N0:39) (FIG. 10D).
These data demonstrate that GRP78 is highly expressed in prostate cancer
metastases to
bone marrow and weakly expressed in normal prostate tissue.
Discussion
The present Example shows that it is possible to identify molecular markers of
disease progression and survival without prior knowledge of the antigens
related to the
disease. In cases where the tumor antigen is unknown, disease-specific
antigens
identified by this approach could be employed to define common or unique
features in
the immune response of individuals to the same disease, i.e., to fingerprint
the immune
response against a given antigen. The approach presented here is based on
selection of
immunoglobulin-binding peptides that mimic tumor-related antigens from phage
libraries. Serum samples from human prostate cancer were screened and an
antibody-
binding peptide ligand was validated by using a large panel of patient serum
samples.
The corresponding tumor antigen eliciting the immune response was identified
as
GRP78, a molecular marker of use for detection, diagnosis and/or prognosis of
metastatic prostate cancer. The GRP78 protein is highly expressed in bone
marrow
metastasis and the high prevalence of circulating antibodies against GRP78 is
associated with metastatic androgen-independent disease and poor prognosis.
GRP78 (also known as Hsp70 protein 5) expression is induced by cellular
stress and hypoxia, conditions associated with prostate cancer. Recently, this
protein
has been shown to be abundant in malignant prostate tumor by two-dimensional
electrophoresis and mass spectrometry (Alaiya et al., Cell Mol. Life Sci.
58:307-11,
2001). In addition to GRP78, other heat shock proteins, such as 90, 72, and
27, are
highly expressed in malignant prostate tissue (Thomas et al., Br. J. Urol.
77:367-72,
1996). GRP78 associates with the major histocompatibility complex (MHC) class
I on
the cell surface and its presence on the cell surface is not dependent on MHC
class I
expression (Triantafilou et al., Hum. Immunol. 62:764-70, 2001). Cancer-
derived
HSP-peptide complexes are being used as HSP vaccine in human cancer (Tamura et
al.,
Science 278:117-120, 1997). A recent study showed that the expression of heat
shock
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proteins could independently determine the clinical outcome of individual
prostate
cancers (Tamura et al., 1997).
Although phage peptide libraries have been used to identify various
pathological
and disease-related agents in patients including Lyme disease, hepatitis, HIV-
1, and
autoimmune diseases, this is the first report in which sera from prostate
cancer patients
have been used to identify new markers for this cancer.
Example 5. Biopanning Circulating Antibodies in Prostate Cancer:
Antibody Progression Corresponds to Disease Progression
The present Example illustrates a further embodiment of the invention, using
phage display library screening to examine the progression in circulating
antibodies
accompanying disease progression in prostate cancer.
The methods used were similar to those described in Example 4. A subtraction
protocol was used, in which IgG from a normal individual was coupled to
protein G
chromatography beads. A cyclic CX6C phage display library, prepared as
described
above, was exposed to the normal IgG's. Phage that did not bind to the normal
IgG
pool were collected and used for the next step. Antibodies from patient M
(prostate
cancer patient) were attached to fresh protein G chromatograpy beads. The
phage
display library that had been pre-exposed to normal IgG's was exposed to the
IgG pool
from patient M. After thorough washing of the column, the phage that bound to
the
prostate cancer IgG (but did not bind to normal IgG) was eluted and amplified.
This
procedure was followed for three rounds of screening and targeting peptides
against
patient M's antibodies were obtained.
Serum samples from the same patient were obtained from archival specimens
and used to obtain targeting peptides. Patient M's serum from 1994 (early
stage
cancer), 1998 (intermediate stage) and 2000 (late stage) were used to obtain
antibody
targeting peptides as described above. These peptides were shown in Table 8.
The
numbers in parentheses indicate the number of phage exhibiting the sequence
out of the
total number of clones obtained.
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Table 8. Peptides identified after three rounds of panning on purified
immunoglobulins from the serum of prostate cancer patient M.
1994 Serum 1998 Serum 2000 Serum
CTFAGSSC (6122) CTFAGSSC (12/20) CTFAGSSC (26/29)
(SEQ ID N0:46) (SEQ ID N0:46) (SEQ TD N0:46)
CNSAFAGC (1/22) CSKKFVTC (3/20) CNSAFAGC (1/29)
(SEQ ID N0:47) (SEQ ID N0:62) (SEQ ID N0:47)
CSYTFAGC (1/22) CNSAFAGC (1/20) CFPKRVTC (1/29)
(SEQ ID N0:48) (SEQ ID N0:47) (SEQ ID N0:66)
CSTFAGSC (1/22) CKNKHTTC (1/20) CPRSAKNC (1/29)
(SEQ ID N0:49) (SEQ ID N0:63) (SEQ ID N0:67)
CRDGYHHC (1/22) CFETFAGC (1/20)
(SEQ ll~ N0:50) (SEQ ID NO:64)
CSASDLSC (2/22) CNNMYAGC (1/20)
(SEQ ID N0:51) (SEQ ID N0:65)
CQNQYPEC (1/22) CQNQYPEC (1/20)
(SEQ ID N0:52) (SEQ ID NO:52)
CRASAMVC (1/22)
(SEQ ID N0:53)
CIDMTHQC (1/22)
(SEQ ID N0:54)
CISSPSNC (1/22)
(SEQ ID NO:55)
CNQSMWSC (1/22)
(SEQ ID N0:56)
CQFENGTC (1/22)
(SEQ ID NO:57)
CAVKSVTC (1/22)
(SEQ ID NO:58)
CNGFMGYC (1/22)
(SEQ ID N0:59)
CLTSENAC (1/22)
(SEQ ID N0:60)
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CRASAMVC (1/22)
(SEQ ID N0:61)
It is apparent that one sequence, CTFAGSSC (SEQ ID N0:46) was the
predominant antibody-binding peptide in all three samples. Further, the
frequency of
this targeting peptide as a fraction of the total pool of targeting peptides
increased with
time, suggesting that the antibody that bound this peptide also became more
prevalent
with tumor progression. It is also apparent that the diversity of targeting
peptides
binding to circulating antibodies decreased with disease progression,
indicating that
there was a corresponding decrease in antibody diversity.
It is not unusual for tumor cells to shed antigens into the circulation.
Leukocytes may also be exposed to tumor antigens ifz situ. It is therefore
expected that
cancer patients in general will exhibit cirfculating antibodies against tumor
antigens.
Phage display libraries may be screened against cancer patient samples to
identify
targeting peptides that bind to antibodies against tumor specific or tumor
associated
antigens. The identified targeting peptides may be used, for example, to
purify anti-
tumor antibodies using affinity chromatograpy or other well-known techniques.
The
purified anti-tumor antibodies can be used in diagnostic kits to identify
individuals with
cancer. Alternatively, they could be attached to various therapeutic moieties,
such as
chemotherapeutic agents, radioisotopes, anti-angiogenic agents or pro-
apoptosis agents
and used for cancer therapy. The targeting peptides against anti-tumor
antibodies may
also be used to identify novel tumor specific or tumor-associated antigens, of
diagnostic
or therapeutic use. Phage display antibody libraries may also be constructed
and
screened against tumor targeting peptides. By this method, it is possible to
isolate and
purify large quantities of antibodies specific for tumor antigens.
The skilled artisan will realize that the CTFAGSSC (SEQ ID N0:46) peptide
could be used for ELISA or other immunoassays to screen the blood of
individuals at
risk for prostate cancer. The presence of an antibody that bound to SEQ ID
N0:46 in
the serum of a patient would be indicative of prostate cancer. The peptide may
also be
used to prepare monoclonal or polyclonal antibodies that are of use for tumor
diagnosis,
imaging or therapy.
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Example 6. Targeted Phage-Based Vectors for Systemic Gene Delivery
Certain embodiments of the present invention concern gene therapy vectors for
treatment of various cell, tissue or organ-localized disease states, such as
prostate
cancer. Targeting peptides may be incorporated into or attached to therapeutic
vectors
and administered to patients with the disease, decreasing the systemic
toxicity of the
therapeutic agent and increasing its targeting to the diseased tissue, thereby
increasing
efficacy. In particular embodiments, the gene therapy vectors of use include,
but are
not limited to, modified adeno-associated virus (AAV) vectors, referred to
herein as
adeno-associated phage (AAP) vectors. The AAP vector enables systemic and
local
gene delivery and robust long-term transgene expression. The vector
specifically
homes to receptors that have been well characterized for selective expression
on the
vascular endothelium. The AAP vector can deliver genes to angiogenic or tissue-
specific receptors. It results in markedly increased transduction stability
and duration of
gene expression
The development of vectors for systemic targeted delivery is required for
successful gene therapy. Commonly used approaches rely on ablating the native
tropism of viral vectors and/or retargeting them to alternative receptors.
Thus far, a
major drawback of these approaches has been that the expression of the
receptors is not
restricted to the target tissues.
Many malignant, cardiovascular, and inflammatory diseases have a marked
angiogenic component. In cancer, tumor vasculature is a suitable target for
intervention
because the vascular endothelium is composed of non-malignant cells that are
genetically stable but epigenetically diverse (St. Croix, B. et al., Science
289:1197-
1202, 2000; Kolonin et al., Curr. Opin. Chem. Biol. 5:308-313, 2001). In vivo
phage
display has been used to isolate probes that home selectively to different
vascular beds
and target receptors expressed only on certain blood vessels. Both tissue-
specific and
angiogenesis-related vascular ligand-receptor pairs have been identified with
this
technology. Targeted delivery of cytotoxic drugs (Arap et al., Science 279:377-
380,
1998a), proapoptotic peptides (Ellerby et al. Nat. Med. 5:1032-1038, 1999),
fluorophores (Hong & Clayman, Cancer Res. 60:6551-6556, 2000) or cytokines
(Curnis
et al., Nat. Biotechnol. 18:1185-1190, 2000) to the vasculature generally
improved
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selectivity and/or therapeutic windows in animal models. Vascular receptors
are
attractive targets for systemic delivery of gene therapy. Such receptors are
readily
accessible through the circulation and often can mediate internalization of
ligands by
cells (Kolonin et al., 2001).
While incorporation of vascular homing peptides derived from ifZ vivo phage
display screenings into viral vectors has been attempted, this strategy has
proven quite
challenging because the structure of the capsid and the targeting properties
of the
peptides can be adversely affected (Wickham, Gene Tlaer. 7:110-114, 2000).
However,
gene expression in mammalian cells is possible if phage vectors are processed
in the
correct trafficking pathway (Foul & Marks, J. Mol. Biol. 288:203-211, 1999).
In theory, phage vectors have several advantages over mammalian viruses
conventionally used for gene therapy. Receptors for prokaryotic viruses such
as
untargeted (wild-type) phage are not expressed on mammalian cells. Receptor-
mediated internalization by mammalian cells does occur if re-targeted phage
vectors
display certain peptide ligands (Larocca et al., Faseb T. 13:727-734, 1999).
There is
substantial evidence suggesting that phage can be safely administered to
patients, as
bacteriophage were given to humans during the pre-antibiotic era with no
adverse
effects (Barrow & Soothill, Trends Microbiol. 5:268-271, 1997). Because homing
phage have been pre-selected to home to vascular receptors in an ih vivo
screening,
there is no need for further targeting modifications. The localization of gene
expression
if2 vivo recapitulates previous observations using immunohistochemistry for
phage
localization (Rajotte et al., 1998; Rajotte & Ruoslahti, 1999; Pasqualini et
al., 1997).
The parental tumor-homing phage used in the present Example are known to
target
receptors expressed in the activated blood vessels of multiple types of human
and
murine tumors, including carcinomas, melanomas, and sarcomas in mouse models
(Pasqualini et al., 1997; Arap et al., 1998; Koivunen et al., 1999a). The lung-
homing
phage and its corresponding receptor expressed in the lung vasculature have
also been
well characterized in mice (Rajotte et al., 1998; Rajotte & Ruoslahti, 1999).
Based on the rationale outlined above, targeted systemic gene delivery to the
vascular endothelium may be accomplished with phage particles homing to cell
surface
receptors on blood vessels while meeting receptor requirements for selective
tissue
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expression and vector accessibility. The results presented herein demonstrate
the
feasibility of this approach.
A new generation of targeted phage-based vectors is provided that enables
systemic gene delivery and robust long-term transgene expression. A novel
chimeric
phage-based vector containing the inverted terminal repeat (ITR) sequences
from
adeno-associated virus (AAV) has been designed, constructed, and evaluated. As
demonstrated below, these vectors (i) specifically home to receptors that have
been well
characterized for selective expression on the vascular endothelium, (ii) can
deliver
genes to angiogenic or tissue-specific blood vessels, and (iii) markedly
increase
transductiun stability and duration of gene expression. These data indicate
that targeted
phage-based vectors and their derivatives are of use for clinical
applications, such as
targeted delivery to prostate cancer.
Materials and Methods
Reagents, cells, ahd tissue culture
All of the restriction enzymes (New England Biolabs, Beverly, MA), T4 DNA
ligase (Ruche, Indianapolis, IN), topotecan (Sigma Chemical Company, St.
Louis, MO),
and cisplatin (Sigma) were obtained commercially. The fMCS 1 plasmid was
obtained
from Dr. George P. Smith (University of Missouri, MO). DNA sequence analysis
was
performed with the Big Dye" terminator sequence kit (Perkin ElmerlABI Systems,
Norwalk, CT). All peptides used in this Example were synthesized at greater
than 95%
purity, cyclized, and analyzed by HPLC and mass spectrometry (AnaSpec, San
Jose,
CA). Targeting peptides used in this Example included the GFE (CGFECVRQCPERC,
SEQ ID N0:68); HWGF (CTTHWGFTLC, SEQ ID N0:69) and RGD-4C
(CDCRGDCFC, SEQ ID N0:70) peptides.
The human cell lines used were Kaposi's sarcoma (KS1767), 293 embryonic
kidney (ATCC; Manassas, VA), and MDA-MB-435 breast carcinoma. Cell lines were
maintained in minimal essential medium (MEM; Irvine Scientific, Santa Ana, CA)
supplemented with 10% fetal calf serum (FCS; Gibco-BRL, Rockville, MD) plus
sodium pyruvate, L-glutamine, and penicillin/streptomycin (Gibco-BRL).
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Construction of phage-based targeted expressiofa vectors
The FUSES-based filamentous phage display vector was modified by inserting
into an intergenic region of the phage genome a (3-galactosidase ([3-gal)
coding
sequence under the control of a CMV promoter, Targeted RGD4C-(3-gal phage
vector
was engineered in a two-step process that included the generation of an
intermediate
construct (termed RGD-4C-fMCS1) and subsequent production of RGD-4C-[3-gal.
The
overall construction scheme is illustrated in FIG. 11. RGD-4C-fMCS 1 contained
the
oligonucleotide insert encoding the RGD-4C targeting peptide, inserted into
the S, fi I
site of the gene III minor coat protein of the FUSES phage, and a fragment of
the
fMCS 1 plasmid that had a multicloning site (MCS) for insertion of transgenes.
RGD-
4C phage-derived fUSES DNA and fd-tet phage-derived fMCS 1 DNA were purified
from lysates of host bacteria (E. coli MC1061). The intermediate RGD-4C-fMCSl
vector was constructed by ligating a 5.4-kb BamHIlSaclI fragment of the RGD-4C
plasmid to the 4.1 kb BamHIlSacII fragment of the fMCS 1 plasmid. Next, a 14
kb
RGD-4C-(3-gal phage plasmid was obtained by insertion of a 4.5 kb PstI CMV-~3-
gal
fragment derived from pCMV(3 (Clontech, Palo Alto, CA) into the PstI site of
RGD-
4C-fMCS 1. This strategy allowed cloning of the CMV-(3-gal cassette in either
forward
or reverse orientation.
Orientations of resulting vectors were differentiated by EcoRV restriction
analysis and by DNA sequencing. Targeted phage vectors were designated fRGD4C-
(3-
gal (forward) and rRGD4C-(3-gal (reverse). Other targeting (HWGF-(3-gal, GFE-
~i-gal)
phage and insertless control (fd-(3-gal) phage were constructed through the
same
strategy. The targeting phage were designed to target integrins (RGD-4C) and
the
MMP-2 and MMP-9 matrix metalloproteinases (HWGF), expressed in angiogenic
vasculature. The GFE phage were designed to target membrane dipeptidase (MDP)
expressed in lung vasculature.
A targeted phage/AAV chimeric vector was created by cloning a 2.8 kb
fragment of pAAV-eGFP (enhanced GFP; Stratagene) from TTR (inverted terminal
repeat) to ITR into the PstI site of RGD-fMSC. Briefly, pAAV was digested with
PacI
to release a 2.8 kb fragment, which was blunted with DNA polymerise and cloned
into
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the blunted PstI site of RGD-fMSC (thus destroying the PstI restriction site).
The final
AAP vector construct is illustrated in FIG. 32. The 12.3 kb DNA contains a
targeting
motif inserted into gene III, a gene of interest (e.g., (3-gal) inserted
between the AAV
ITR elements under control of a CMV promotor and with a poly A terminator. The
locations of the deleted Pst I sites are also shown (crossed out). In each of
the
constructs, correct orientation of insert was verified by restriction
analysis. Single
clones in each orientation were sequenced. . Unless otherwise stated, the
forward
vectors were used.
Phage DNA trahsfectiou into mammalia~z cells
The double-stranded DNAs of the replicative forms of targeted (RGD4C-[3-gal,
HWGF-[3-gal, GFE-(3-gal) and insertless control (fd-(3-gal) constructs were
prepared by
using the Plasmid Maxi kit (Qiagen). The single-stranded DNAs of the infective
forms
of the phage vectors were extracted from the phage capsid proteins by using
the
Strataclean resin (Stratagene), followed by two ethanol precipitations. DNA
was
quantified by spectrophotometry with 1.0 A26o equal to 40 ~ug/ml for single-
stranded
DNA or 50 ~,g/ml for double-stranded DNA. The 293 recipient cells were
transfected
with 5 p,g of either double-stranded or single-stranded phage DNA into 5 x105
cells by
using the SuperFect" reagent (Qiagen) according to the manufacture's protocol.
Both
the gene expression and enzyme activity of (3-gal were evaluated at least 48
hours post-
transfection. Cells were incubated with the X-gal substrate for 3 hours at
37°C and
enzyme activity was visualized by using an ifz situ (3-galactosidase staining
kit
(Stratagene) according to the manufacturer's instructions.
Vector production, purification, and titratiofz.
Phage vectors were isolated and purified from the culture supernatant as
disclosed (Pasqualini et al., in Phage Display: A Laboratory Mauual (Barbas et
al.,
eds.), chap. 22, pp. 1-24, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
NY, 2000). Phage were re-suspended in Tris-buffered saline (pH 7.4) and re-
centrifuged to remove residual bacteria and debris. The resulting supernatant
containing the phage in suspension was filtered through a 0.45 p,m filter and
titered
according to standard protocols (Pasqualini et al., 2000).
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Targeted phage vector transduction and specific inhibition by using synthetic
peptides. MDA-MB-435 breast cancer and KS1767 Karposi's sarcoma cells were
cultured on 8-well chamber glass slides. The culture media was replaced by 200
,ul of
MEM with 2% FCS and 5 X101° TU of RGD-4C-(3-gal, HWGF-~3-gal, or fd-
(3-gal
phage vectors (at 105 transducing units/cell in each case). Both cell lines
express high
levels of the integrin and MMP receptors for those targeting peptides. Phage
were
incubated with cells for 3 hr at 37°C, followed by a medium change to
MEM plus 10%
FCS. The cells were incubated for 72 hr at 37°C to allow for (3-gal
gene expression.
In the peptide inhibition studies, MDA-MB-435 cells were cultured on 12-well
plates and then incubated with 10 ~,g of RGD-4C peptide or control peptides
(CARAC,
SEQ ID N0:71 or CKDRFERC, SEQ ID N0:41) in normal growth media for 30
minutes. KS 1767 cells were grown on 12-well plates and then incubated with 40
~,g
CTTHWGFTLC (SEQ ID N0:69) or control peptides in normal growth media for 30
minutes. The growth media were replaced by 500 ~,1 of MEM containing 2% FCS
and
x 101° transducing units (TU) of either RGD-4C-(3-gal, HWGF-(3-gal, or
control fd-(3-
gal phage. Phage vectors were incubated on peptide-treated cells (three hours
at 37°C,
5% C02) followed by a media change to MEM plus 10% FCS. Transduced cells were
maintained in a cell incubator for 72 hours (37°C, 5% COZ).
In the cell culture transduction assay, (3-gal expression was analyzed by
immunofluorescence. For quantification of expression in cell culture, the
transduced
cells were washed with PBS and permeabilized with 0.2% Triton X-100 for five
minutes on ice, followed by blocking with 1% BSA in PBS. An anti-~i-gal
antibody
(Sigma) diluted to 1:2,000 in blocking solution was then incubated with the
cells
overnight. Next, a Texas Red-conjugated secondary antibody (Caltag,
Burlingame,
CA) diluted to 1:600 in PBS was incubated with the cells for 1 hour. The
degree of (3-
gal gene expression was determined by counting fluorescent cells in at least
ten fields
under an inverted microscope (Nikon, Japan). Quantification of the [3-gal
activity in
cell culture was measured as relative light units (RLU) in a luminometer and
then
normalized to the amount of protein in micrograms, as determined by the Lowry
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method in a protein assay kit (Bio-Rad Protein Assay ~; Hercules, CA).
Subsequently,
blue cells were counted under an inverted microscope (Nikon).
In the peptide inhibition assays, (3-gal activity in cell lysates was detected
by the
Galacto-Star ~ chemiluminescent reporter gene system (Tropix, Bedford, MA)
according to the manufacturer's protocol. In other peptide inhibition assays,
293 cells
were plated at 3 x 105 cells/well and incubated with either 1 mg/ml of RGD-4C
peptide
or unrelated control peptides (CARAC, SEQ >D N0:71 or CKDRFERC, SEQ >D
N0:41). After 30 minutes, cells were washed and 105 TU of phage per cell were
added
for 4 hours in serum free media. After the 4 hours, 10% FCS supplemented
medium
was added. Cells were analyzed for GFP gene expression at 72 hours post
infection.
For GFP detection, cells were analyzed by fluorescence activated cell sorting
(FACS) in
a FACScan (Becton-Dickinson, San Jose, CA) or counted and photographed under a
fluorescence microscope (Nikon).
For time course of gene expression assays, cells were plated at 3 x 105
cells/well
and infected with 105 TU of phage per cell for 4 hours in serum-free media.
After 4
hours, 10% FCS supplemented medium was added. Cells were visualized 72 hours
post-infection and sorted by FAGS for GFP expression 7 days after infection.
GFP-
positive cells were plated in T75 tissue culture flasks and serial assays of
GFP
expression as described above were made weekly for the next 60 days.
Gefzotoxic agents.
Semi-confluent MDA-MB-435 cells were infected with 105 TU of phage per
cell for 4 hours in serum free media, after which fresh medium supplemented
with FCS
was added (no phage were washed out or removed). In some experiments, a phage
admixture of forward and reverse clones at 101° TU (forward/reverse
molar ratio = 1)
was tested. Next, cells were incubated for 36 hours followed by the addition
of
genotoxic drugs (topotecan, 10 ~,M; cisplatin, 10 ~.M) or administration of UV
radiation (15 J/m2) with a cross-linker apparatus (UV Stratalinker Model 2400;
Stratagene). At 72 hours post-infection, the cells were analyzed for
transduction of a
reporter gene ((3-gal or GFP), and gene expression was normalized per cell
number
relative to controls.
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In vivo transduction of tumor xenografts and no~nal lung in mouse models.
Female 4-month old nude mice and female 4-month old immunocompetent
C57B1/6 mice (Harlan Sprague Dawley, San Diego, CA) were used in this study.
Avertin (0.015 ml/g) was used as an anesthetic. Tumor xenografts derived from
human
Kaposi's sarcoma KS1767 cells were established by injecting tumor cells (106
cells per
mouse in 200 ~l of serum-free MEM) into the mammary fat pad of nude mice.
Tumor-
bearing mice with matched tumor sizes were used for systemic gene transfer
experiments 20 to 40 days afterwards when tumors reached 0.5 to 1.5 cm in
diameter.
In tumor transduction experiments, RGD-4C-[3-gal, HWGF-[3-gal, and fd-(3-gal
phage (109 TU/mouse) were injected intravenously (tail vein) into female nude
mice
carrying subcutaneous tumor xenografts. One week after vector administration
of the
targeted or control phage, tumors and control organs (liver, brain) were
surgically
harvested under deep anesthesia and the mice euthanized. (3-gal expression in
the
tumor and control tissues was detected by an anti-(3-gal antibody by using a
peroxidase-
based immunodetection kit (Vector Labs, Burlingame, CA).
In lung transduction studies, GFE-(3-gal phage and fd-~3-gal control phage
(109
TU/mouse) were injected intravenously into female C57B1/6 mice. Lungs and
livers
were harvested two weeks after vector administration. For in vivo experiments
involving tissue extracts, (3-gal activity in the lung and control tissues
were detected by
a chemiluminescent assay system (Tropix). Several assays for ~3-galactosidase
were
used in different studies to ensure that the results were not assay-dependent
and were
reproduced with distinct methods.
AAP VeCtoY fOY DeliveYy of Therapeutic Genes to Tumors
The efficacy of the AAP vector to deliver therapeutic genes to Karposi's
sarcoma tumors in nude mice was evaluated. The most frequently used system of
gene
delivery consists of transferring the Herpes simplex virus type 1 (HSV-1)
thymidine
kinase (TK) gene into tumor cells, followed by treatment with Ganciclovir
(GCV).
This guanosine analogue is specifically monophosphorilated by the viral kinase
and
then converted by cellular enzymes into the triphosphate derivative, which,
upon
incorporation into elongating DNA, induce cell death, by premature chain
termination.
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To determine whether AAP vector could be used for systemic gene therapy
delivery, the
(3-gal cassette was replaced with a "suicide" gene (thymidine kinase - TK).
The
resulting RGD-4C-AAP-TK vector was injected intravenously in nude mice bearing
human KS 1767 Kaposi's sarcoma xenografts. Targeting, internalization and
transduction of the therapeutic AAP vector into the tumor cells, followed by
treatment
with GCV should result in cell death.
Molecular characterization of AAP vectors
Viral rescue experiments were performed in AAP-transduced 293 cells by
infecting them with Ad5 (MOI of 10 particles/cell). After 48 hours the cells
were
processed to obtain a crude viral lysate, then heat inactivated to remove
contaminating
adenovirus. The resulting material was next used to infect 293 cells and 8431
cells.
GFP-expressing cells were detected after 48 hours. PCR analysis was performed
by
analysis of genomic DNA extracted from AAP-transduced 293 cells and from
control
cells. Genomic DNA (200 ~,g/reaction) was reacted with GFP specific primers
(GFP-
N, by 143-164; GFP-C, by 654-676). After 30 PCR cycles, the presence of a
diagnostic
490 by band is evaluated. To ensure specific amplification, pCMV-GFP DNA was
used as a positive control and pCMV DNA was used as a negative control.
Southern
Blot analysis was performed with Eco RI-digested genomic DNA extracted from
AAP-
transduced 293 cells and controls. The digests were electrophoresed and
hybridized
with a 32P-labeled cDNA fragment containing an AAV-specific probe. The
presence of
a diagnostic 2.3 kb band was evaluated.
Results
Targeted phage vectors designed to drive gene expression iu eukaryotic cells.
The fUSES-based filamentous phage display vector (Smith & Scott, 1993) was
modified by inserting the (3-galactosidase ((3-gal)-encoding gene under the
control of a
CMV promoter into an intergenomic region of the phage genome to construct a
fCTSES-
(3-gal backbone vector. Next, DNA olignonucleotide sequences encoding the
targeting
peptides CDCRGDCFC (SEQ ll~ N0:70, "RGD-4C"), CTTHWGFTLC (SEQ ID
NO:69, "HWGF") or CGFECVRQCPERC (SEQ ID N0:68, "GFE") were inserted into
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the Sfz I site of the gene III minor coat protein (p111J of the phage. Phage
produced in
this manner display 3-5 copies of the targeting peptides per viral particle.
The resulting viral constructs (RGD-4C-(3-gal, HWGF-(3-gal, and GFE-(3-gal)
were used for production of targeted phage particles that display each of the
targeting
peptides and carry a CMV-[3-gal transgene (Fig. 11). RGD-4C-(3-gal and HWGF-(3-
gal
were designed to target av integrins and matrix metalloproteinases (M1VVIP-2
and 1~-
9), respectively, expressed in angiogenic vasculature. GFE-(3-gal was designed
to target
membrane dipeptidase (IVVIDP) expressed in lung vasculature. The strategy
depicted in
Figure 11 was used to construct the other targeting and control vectors.
Phage DNA cof2text permits transgene expression ih mammaliaya cells
To determine whether the inserted (3-gal cassette was functional, embryonic
human kidney cells were transfected with the infective forms of the phage DNA,
constructed to contain the reporter transgene in either forward or reverse
orientation. A
CMV-driven mammalian expression vector was used as a positive control (FIG.
12A)
and an empty vector as a negative control (FIG. 12B) for (3-gal expression.
Transfer of
the modified single-stranded DNA of the phage infective form promoted
transgene
expression in mammalian cells. Furthermore, the orientation of the transgene
cassette
did not significantly influence the level of gene expression (FIG. 12C vs.
FIG. 12D).
All subsequent experiments used the vector with the [3-gal expression cassette
in the
forward orientation. Given that single-stranded DNA does not support gene
expression
in mammalian cells and that the infective forms of the phage genome are single-
stranded, these results strongly suggest that the single-stranded phage genome
must be
first converted to double-stranded DNA in recipient cells before allowing gene
expression.
Consistent with this hypothesis, DNA from replicative forms of the phage,
which are double-stranded, expressed the (3-gal transgene several fold more
efficiently
at levels comparable to the mammalian expression vector used as the positive
control
(data not shown).
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Receptor-mediated inter~zalizatiofz afad specific tra~zsduction of recipient
cells by
targeted phage vectors ifz vitro.
Having shown that the transgene constructs were functional, transduction of
human cell lines expressing the receptors targeted by RGD-4C-(3-gal and HWGF-
(3-gal
phage vectors was examined. The untargeted fUSES-derived control phage vector
(fd-
(3-gal) was used as a negative control. RGD-4C-(3-gal phage (FIG. 13C-D) and
HWGF-(3-gal phage (FIG. 13A-B) were incubated with breast cancer (FIG. 13C-D)
and
Kaposi's sarcoma (FIG. 13A-B) cells (MDA-MB-435 and KS1767 lines),
respectively.
Both cell lines express high levels of the RGD-4C-receptors av(33 and av~i5
integrins
and of the HWGF receptors MMP-2 and MMP-9.
(3-gal transduction was observed of 14 ~ 2 % (mean ~ standard error of the
mean; SEM) of MDA-MB-435 cells incubated with RGD-4C-[3-gal phage and 12 ~ 2%
(mean ~ SEM) of the KS 1767 cells incubated with HGWF-(3-gal (FIG. 14A).
Comparable transduction results were also obtained by incubating HWGF-~i-gal
on
MDA-MB-435 cells and RGD-4C-[3-gal on KS 1767 cells (data not shown). Control
phage (fd-[3-gal) were not internalized when incubated with either cell line
and only
minimal (3-gal transduction (~0.1 % of the tumor cells) could be detected
(FIG. 14A).
To demonstrate specificity, transduction with RGD-4C-(3-gal and HWGF-(3-gal
phage was blocked by pre-incubating the target cells with the corresponding
synthetic
peptides (FIG. 14B-C). Tn each case, almost complete inhibition of
transduction was
observed, of greater than 99% with RGD-4C peptide (FIG. 14C) and greater than
90%
with CTTHWGFTLC (SEQ ID N0:69) peptide (FIG. 14B) in a dose-dependent
manner. Pre-incubation with nonspecific negative control peptides had no
significant
effects on transduction of the recipient cells (FIG. 14 B-C). These data show
that
transduction of mammalian cells by internalized phage vectors i~z vitro is
substantial,
specific, and mediated by ligand-receptor mechanisms.
Targeted transductiofZ of tissue-specific and tumor vasculature upon systemic
adfnihistration iyz vivo
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To determine whether the targeted RGD-4C(3-gal and HWGF-(3-gal phage
vectors could selectively transduce tumors upon systemic administration, each
vector
was administered intravenously into nude mice bearing human KS 1767 Kaposi's
sarcoma xenografts. KS1767 cells are suitable because they form well-
vascularized
tumors and the receptor expression profiles in tumor cells and tumor-
associated blood
vessels has been characterized (Pasqualini et al., 1997; Arap et al., 1998a,
1998b;
Koivunen et al., 1999a). . The av integrins and gelatinases (MMP-2 and -9)
receptors
for the targeting peptides are highly expressed on the KS 1767-derived tumor
xenografts
and their angiogenic vasculature. Phage displaying RGD-4C and HWGF peptides
target KS 1767 tumors efficiently and specifically in vivo. Tumor targeted
phage were
not detected in control tissues studied, including brain, kidney, pancreas,
adrenal, skin,
muscle, intestine, lymph nodes, uterus, prostate, and fat (Pasqualini et al.,
1997; Arap et
al., 1998a, 1998b; Koivunen et al., 1999a).
Tumors and control organs were surgically harvested one week after
administration of the vectors. Tumor and control organs (liver and brain) were
imrnunostained with an anti-(3-gal antibody. The RGD-4C-(3-gal (FIG. 15A, D
and G),
HWGF-(3-gal (FIG. 15B, E, and IT), and control fd-(3-gal (FIG. 15C, F, and I)
vectors
were analyzed.
Strong [3-gal immunostaining was observed in tumor tissues (FTG. 15A and B),
with negligible immunostaining observed in control liver and brain organs
(FIG. 15D,
E, G and IT). In contrast, tissues recovered from mice that received
untargeted negative
control fd-(3-gal phage vector did not show detectable [3-gal expression in
either the
tumor (FIG. 15C) or the control organs (FIG. 15F and I). In each case, (3-gal
reactivity
matched the corresponding immunostaining pattern of phage targeting to the
vascular
endothelium of blood vessels in tumors (Pasqualini et al., 1997; Arap et al.,
1998;
Koivunen et al., 1999). A non-(3-gal-containing phage produced no staining in
the liver
(data not shown). Measuring (3-gal activity produced results consistent with
the
immunohistochemistry data used for detection of targeted gene transduction
(FIG. 16).
Targeted gene delivery was also evaluated in vivo by using GFE-[3-gal, a phage
vector targeted to MDP in the vascular endothelium of lung blood vessels
(Rajotte &
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Ruoslahti, 1998). The lung-homing GFE-[3-gal vector was injected intravenously
into
immunocompetent C57B1/6 mice. Substantial (3-gal activity was seen in the
lungs of
mice injected with GFE-[3-gal phage but not in the lungs of mice injected with
fd-(3-gal
control (FIG. 17). In contrast, the (3-gal activity in the liver of mice
injected with the
GFE-(3-gal phage was similar to that of background (3-gal activity from mice
injected
with control phage (FIG. 17). Taken together, these results show ih vivo
systemic gene
delivery and transduction targeted to and mediated by vascular receptors
selectively
expressed in tumors and in normal organs.
Ifzcrease ih trahsduction by genetic traf2s-complementation.
Because the genome from the infective form of M13-derived phage is single-
stranded, conversion to double-stranded DNA is required to allow gene
expression. It
was hypothesized that genotoxic agents that promote DNA repair would enhance
the
transduction of genes carried by single-stranded phage vectors. To test this
hypothesis,
cells infected by targeted phage vectors were challenged with genotoxic agents
such as
ultraviolet (UV) radiation and cancer chemotherapy drugs (topotecan and
cisplatin).
This approach consistently resulted in gene transduction several fold higher
than
various controls (FIG. 18). Interestingly, an equal mixture of forward and
reverse
single-stranded phage clones showed a two-fold increase in gene expression
relative to
the same molar concentrations of either forward or reverse phage (FIG. 18). It
is
postulated that the presence of both sense and anti-sense of the reporter gene
allowed
hybridization of the strands to occur. Such facilitation in gene expression is
consistent
with the requirement for double-stranded DNA. The enhancement of gene
expression
by DNA lesions or genetic trans-complementation indicates that conversion to
double-
stranded DNA is a rate-limiting step in developing of effective phage vectors.
These
data also suggest the possibility of synergism if cytotoxic agents commonly
utilized in
clinical applications are used in combination with phage-derived vectors.
PhagelAAV cl2imeric vectors markedly improve gene trahsductiorz stability.
To solve the DNA conversion problem described above, it was determined
whether the incorporation of genetic cis-elements derived from AAV (a single-
stranded
mammalian virus) into targeted phage-based constructs would affect gene
transduction.
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First, chimeric vectors composed of a targeted phage and an AAV genome from
inverted terminal repeat (ITR) to ITR was designed and engineered. Vectors
were
constructed by cloning a full-length 2.8 kb fragment of pAAV-eGFP (Green
Fluorescent Protein, Stratagene) from inverted terminal repeat (ITR) to ITR
into the
blunted PstI site of the construct presented in FIG. 11. The targeting
properties of the
resulting chimeric vectors were not altered by insertion of AAV genetic
elements.
Specific inhibition by the corresponding synthetic peptide was again observed
(FIG. 20)
indicating that the phage targeting features were intact.
Having established that the tropism of the targeted vector was preserved, the
effects of AAV genome insertion on transgene expression was assessed. While
the
levels of gene expression remained unchanged (data not shown), the duration of
gene
transduction was markedly prolonged relative to the parental targeted phage.
Robust
long-term expression of the reporter gene was observed beyond eight weeks
(FIG. 21).
This finding is in clear contrast to the one-week transgene expression usually
observed
with the parental targeted phage vector ih vitro (Table 9). Table 9 represents
relative
reporter gene expression in 293 cells transfected with targeted phage versus
AAP, using
triplicate wells for each time point. Results represent averaged independent
determinations by two different investigators. Expression in the standard
targeted
phage vector disappeared after 10 days, while some expression was observed in
AAP
vectors for at least 60 days.
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Tumor targeted transgene expression was also observed in vivo after systemic
AAP administration in mice bearing MDA-MB-435 xenografts. Such transduction
was
specific because it was blocked by co-administration of cognate--but not
unrelated
control--synthetic peptides (not shown).
The combination of genotoxic agents plus insertion of AAV cis-elements
appears to be at least additive if not synergistic (data not shown). Gene
expression in
cells transduced with targeted phage/AAV chimeric vectors has been
systematically
followed for up to 60 days (Table 8). Expression of GFP has been detected for
as long
as 90 days (not shown). To rule out the possibility of genetic complementation
by
trans-acting factors (for example, FA. or f6) in the permissive 293 cell line,
the
transduction of HepG2 (liver carcinoma-derived) and MDA-MB-435 cells was
examined. Similar levels and duration of gene expression were observed (data
not
shown).
To characterize the AAP chimeric vectors, studies were performed to detect AAV
elements in cells transduced with AAP vectors and to demonstrate excision,
amplification, and integration (FIG. 22). Adenoviral rescue (FIG. 22), PCR
(not
shown) and Southern Blot analysis (not shown) demonstrate that (i) AAV
particles can
be generated using the supernatant from cells infected with AAP; and (ii) AAV
elements integrate within the genome in cells transduced with targeted AAP,
but not
control phage vectors (targeted or untargeted).
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These data indicate that phage/AAV chimeric (AAP) vectors may be readily
constructed and used with no apparent losses in their targeted acquired
tropism and
with substantial enhancement in the long-term stability of the genes
transduced.
Delivery of Therapeutic Genes Ifzto Turraors
An AAP vector designed to contain a "suicide" TK gene was constructed as
described above and injected into nude mice containing Karposi's sarcoma human
tumors. Seven (7) days after vector injection, mice received daily
intraperitoneal
injections of GCV (5 mg/Kg/day) for 7 days. Tumors in animals injected with
RGD-
4C-TK-AAP vector, followed by GCV treatment, showed significant growth
reduction,
comparing to tumors in the control animals which were injected with insertless
fd-
AAP-TK vector prior to GCV treatment. These results demonstrate the
feasibility of
using AAP vectors for targeted delivery of therapeutic genes to tumors and
other tissues
for which selective and/or specific targeting peptide sequences have been
identified.
The skilled artisan will realize that the AAP vector described herein is not
limited to
targeted delivery to tumor tissues, but may be used for targeted gene therapy
of a wide
variety of organs, tissues or cell types.
Discussion
The present Example shows for the first time that systemic gene delivery can
be
achieved by genetically adapting targeted phage clones selected from
screenings of
phage display random peptide libraries. The characteristics of an efficient
phage-based
gene therapy vector include: [1] selectivity towards target tissues; [2]
receptor-mediated
cell internalization; and [3] long-term duration of gene transduction upon
delivery.
Each of these characteristics was exhibited by the AAP vectors disclosed
herein.
Targeting peptides can be integrated into conventional gene therapy vectors or
even used as bi-functional molecular adaptors (Larocca et al., 1999; Wickham,
2000;
Grifman et al., Mol. Ther. 6:964-975, 2001; Trepel et al., Hum. Gene Ther.
11:1971-
81, 2000). These strategies have proven to be technically challenging and not
necessarily efficient. Issues of specificity and efficiency have been
addressed by taking
advantage of peptide ligands selected from phage libraries i~z vitro and i~c
vivo. The
targeting phage obtained in screenings performed ih vivo are often selected
using a 3-
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minute circulation timeframe. Thus, it is unlikely that the phage exits the
circulation.
The selection strategy is designed to favor vascular targeting and the
isolation of phage
that target markers that are accessible to circulating ligands (i.e.,
expressed in cells
forming vascular endothelium).
Given that selection has already occurred for those particular clones, it is
likely
that such phage would meet criteria for tissue targeting. Here, it was
demonstrated that
the null-tropism of wild-type phage towards mammalian cells can be modified to
target
and deliver genes to receptors expressed on the vascular endothelium of normal
organs
(such as the lung) and tumors. Thus, the phage vectors introduced by this
study have a
number of potential advantages. Their targeting to selective vascular beds is
based on
receptor expression patterns that are known and characterized. The receptors
are
accessible to circulating probes. These ligand-receptor pairs provide
internalization of
the vector into targeted cells.
While it has been shown that phage can promote gene expression ih vitro, gene
transduction i~ vivo after systemic administration of a targeted phage vector
has not as
yet been reported. A major limitation in the practical use of phage vectors
has been
poor levels of transduction achieved in vivo. A possible cause of this is the
low
efficiency of conversion from single-stranded to double-stranded DNA occurring
in
mammalian cells. To solve this problem two independent strategies were
applied: (i)
enhancement of gene transduction by genotoxic agents (cytotoxic drugs and W
radiation) which cause strand breaks and promote DNA repair; and (ii) genetic
incorporation of AAV cis-elements into targeted phage vectors. The strategies
are not
mutually exclusive and may be used together to further improve the efficiency
of gene
therapy ih vivo.
The term adeno-associated phage (AAP) is used for the new class of vectors for
gene delivery described here. The biological features of AAP are distinct from
either
targeted phage or AAV. While the enhanced duration of gene transduction by AAP
is
similar to the long-term expression patterns associated with AAV transduction,
the
receptor-mediated targeting is characteristic of phage clones selected in
screenings.
Thus, AAP are endowed with several advantages as a gene therapy vector. AAP
are
easy to produce in high titers in host bacteria. No helper viruses or trans-
acting factors
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are needed. The native tropism of AAV for human cells is eliminated because
there is
no AAV capsid formation. The AAP vectors are presumed targeted because they
incorporate peptides that have been isolated in vivo and are defined by their
ability to
home to selective vascular beds. Gene transduction stability was compared
between a
targeted phage and AAP vectors. Targeted gene delivery specific to the ligand-
receptor
pair to which the phage is directed is possible, and gene expression is
maintained for
over two months (possibly because of DNA integration).
The results reported herein demonstrate that the targeting properties are
preserved in the hybrid AAP (a feature conferred by the phage) and that gene
expression elicited by such vectors is robust (a feature conferred by the AAV
elements).
Data with the AAP in vivo appear to confirm these contentions (not shown).
In summary, genetically modified phage have potential to be adapted as
targeted
gene delivery vectors to mammalian cells after systemic administration. Based
on the
favorable targeting properties and long-term duration of gene transduction of
AAP,
these vectors are of use as superior gene delivery tools.
The skilled artisan will realize that the AAP vectors are not limited to the
targeting peptides used in the present Example, but rather may take advantage
of any of
the targeting peptides known in the art or disclosed herein, such as the
prostate cancer
targeting peptides described above. Such AAP gene therapy vectors, designed to
contained cytostatic, cytotoxic, pro-apoptotic, anti-angiogenic or other
therapeutic
genes may be selectively and/or specifically targeted to tissues, such as
cancer tissues,
prostate cancer tissues, and/or metastatic prostate cancer tissues to provide
a high
efficacy of tumor treatment, while exhibiting little or no systemic toxicity.
Example 7. Identification of mouse adipose targeting peptides
The present Example concerns compositions and uses of novel adipose targeting
peptides and receptors. In certain embodiments, the peptides and receptor
targets may
be of use for targeted delivery of therapeutic agents to tumors and/or normal
adipose
tissues.
Adipose targeting peptides
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A substractive phage display protocol (see Example 8 below) was used to
isolate fat targeting peptides from a genetically obese mouse (Zhang et al.,
Nature,
372:425-432, 1994; Pelleymounter et al., Science 269:540-543, 1994). Phage
that had
been subjected to biopanning in obese mice were post-cleared in a normal
mouse. The
fat-targeting peptides isolated included TRNTGNI (SEQ ID N0:72), FDGQDRS (SEQ
ID N0:73); WGPKRL (SEQ ID NO:74); WGESRL (SEQ ID N0:75); VMGSVTG
(SEQ ID N0:76), KGGRAKD (SEQ ID N0:77), RGEVLWS (SEQ ID N0:78),
TREVHRS (SEQ ID N0:79) and HGQGVRP (SEQ ID N0:80).
Homology searches identified several candidate proteins as the endogenous
analogs of the fat targeting peptides, including stem cell growth factor
(SCGF)
(KGGRAKD, SEQ ID N0:77), attractin (mahogany) (RGEVLWS, SEQ ID N0:78),
angiopoietin-related adipose factor (FIAF) (TREVHRS, SEQ ID N0:79),
adipophilin
(ADRP) (VMGSVTG, SEQ ID N0:76), Flt-1 or procollagen type XVII (TRNTGNI,
SEQ ID N0:72) and fibrillin 2 or transferrin-like protein p97 (HGQGVRP, SEQ ID
NO:80)
Validati~s2 of aelipose targeting peptides
The fat homing peptides were validated by in vivo homing, as shown in FIG. 23.
The fat homing clones selected were: FA - KGGRAKD (SEQ ID N0:77), FC -
RGEVLWS (SEQ ID N0:78), FE - TREVHRS (SEQ ID NO:79) and FX -
VMGSVTG (SEQ ID N0:76). As seen in FIG. 23, all of these clones exhibited some
elevation of homing to adipose tissue, with clone FX showing several orders of
magnitude higher adipose localization than control fd-tet phage. Clone FX also
exhibited substantially higher localization than the other selected fat homing
clones.
However, by analogy with the placental homing peptides disclosed above, the
skilled
artisan will realize that fat homing clones exhibiting lower levels of adipose
tissue
localization may still be of use for targeted delivery of therapeutic agents.
The skilled artisan will realize that targeting peptides selective for
angiogenic
vasculature in adipose tissue could be of use for weight reduction or for
preventing
weight gain. By attaching anti-angiogenic or toxic moieties to an adipose
targeting
peptide, the blood vessels supplying new fat tissue could be selectively
inhibited,
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preventing the growth of new deposits of fat and potentially killing existing
fat
deposits.
Example 8: CKGGRAKDC (SEQ ID N0:81) homes to white fat in ob/ob
mice
Materials and Methods
Experimental animals
C57BL/6 mice were purchased from Harlan Teklad. Leptin-deficient (ob/ob)
(stock 000632) and leptin receptor-deficient (stock 000642) mice were
purchased from
Jackson Laboratories (Bar Harbor, ME). Anesthesia was performed with Avertin
(0.015 ml/g) administered intraperitoneally (Arap, et al., 1998; Pasqualini &
Rouslahti,
1996).
Irc vivo phage library screening
Ih vivo phage-display screening of the CX~C library (C, cysteine; X, any amino
acid) (Pasqualini et al., 2000; Arap et al., Nature Meel. 8:121-127, 2002) for
fat-homing
peptides was performed (Pasqualini & Rouslahti 1996, Pasqualini et al., 2000).
In each
biopanning round, an adult ob/ob mouse was injected intravenously (tail vein)
with lOlo
transducing units (TU) of the library. Phage 0300 TU/g in round 1 increased to
104
TU/g in round 3) were recovered after 5 min of circulation by grinding
subcutaneous
white fat with a glass Dounce homogenizer, suspending the homogenate in
4°C
Dulbecco's Modified Eagle's medium (DMEM) containing proteinase inhibitors
- (DMEM-prim 1 mM PMSF, 20 ,ug/ml aprotinin, and 1 ~,g/ml leupeptin) and
washing
with DMEM-prin. The lipid phase was discarded during the washes and only the
solid-
phase cellular material was used. Washed homogenates were incubated with host
bacteria (log phase E. coli K9lkan; OD6oo ~ 2). Bacterial cultures were plated
onto
Luria-Bertani agar plates containing 40 ~.g/ml tetracycline and 100 ~.g/ml
kanamycin,
incubated overnight at 37°C and selected clones were bulk-amplified and
used to
precipitate phage for a subsequent round of biopanning. The sub-library
amplified after
the third round of panning was enriched for fat-specific binders using a
subtraction step.
A lean C57BL/6 female was injected (tail vein) with 10~ TU of phage selected
in round
3. After 5 min of circulation, the unbound phage were recovered from plasma
and
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amplified for the fourth and final round of biopanning. In this protocol,
phage that
bound to tissues other than adipose were removed from the sub-library,
increasing the
selectivity of the recovered phage for binding to adipose tissue.
Peptide locali,zatiou ih tissues
Staining of formalin-fixed, paraffin-embedded mouse tissue sections was
performed (Pasqualini & Rouslahti, 1996; Pasqualini et al., 2000). For phage-
peptide
immunolocalization, 101° TU of CKGGRAKDC (SEQ ID N0:81)-phage or a
control
insertless phage was injected intravenously. Phage immunohistochemistry was
performed using a rabbit anti-fd phage antibody (Sigma Chemicals, St. Louis,
MO)
used at 1:1,000 dilution and a secondary horseradish peroxidase (HRP)-
conjugated
antibody. Apoptosis was detected using standard TUNEL immunohistochemistry and
an HRP-conjugated antibody. For ifz viv~ peptide homing validation, stocks of
5-
carboxyfluorescein (FITC)-conjugated CKGGRAKDC (SEQ ID N0:81) or CARAC
(SEQ ID N0:71) were chemically synthesized, cyclized using the terminal
cysteines
and HPLC-purified to > 90% purity by Anaspec (San Jose, CA). Lyophilized
peptides
were dissolved in DMSO to a concentration of 20 mM. Ten ,ul of 1 mM peptide-
FITC
solution in PBS was injected 5 min prior to tissue extraction. For blood
vessel
localization, 10 ,ul of 2 mg/ml of rhodamine-conjugated lectin-I (RL-1102,
Vector
Laboratories, Burlingame, CA) was co-injected. All immunohistochemistry and
FITC
immunofluorescence images were captured using an Olympus IX70 microscope and
digital camera setup (Melville, NY).
A~zti-obesity therapy
Stocks of CKGGRAKDC (SEQ ll~ N0:81) fused to (KLAKLAK)~ (SEQ ID
NO:1); (KLAKLAK)2 (SEQ ID NO:1) alone; CARAC (SEQ ID N0:71) fused to
(KLAKLAK)Z (SEQ ID N0:1); and CKGGRAKDC (SEQ ID NO:81) peptide were
chemically synthesized, cyclized using the terminal cysteines and HPLC-
purified to >
90% (Anaspec). Lyophilized peptides were dissolved in DMSO to a concentration
of
65 mM to make stock solutions. A total of 150 ~l of 0.65 mM peptide solution
in PBS
was subcutaneously injected daily in the back of C57BL16 males, after body
mass was
measured each day. High-fat cafeteria diet for obesity induction (TD97366:
25.4% fat,
21.79% protein, 38.41% carbohydrate) was purchased from Harlan Teklad. Mice
were
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pre-fed with TD97366 prior to the initiation of treatment with adipose
targeting
peptides to induce diet-related obesity. The high-fat diet resulted in an
average weight
of 50 g before treatment.
Results
Ih vivo phage display (Pasqualini and Ruoslahti,. Nature 380:364-366, 1996;
Kolonin et al., Curr. Opin. Chem. Biol. 5:308-313, 2001; Pasqualini et al., Iu
Vivo
Phage Display, In Phage Display: A Laboratory Mafzual, eds. Barbas et al., pp.
1-24.
Cold Spring Harbor Laboratory Press, New York, 2000) was used as described
above to
obtain a peptide targeting the fat vasculature. A phage-display library was
screened for
peptide motifs that home to the vasculature of subcutaneous white fat in
morbidly obese
leptin-deficient (ob/ob) mice (Zhang et al.. Nature 372:425-432, 1994). This
model
provides a convenient source of adipose tissue. Four rounds of panning were
followed
by a fat-specific ifz vivo subtraction to restrict ligands to those binding to
adipose-
specific endothelial receptors. The DNA encoding the corresponding phage-
displayed
peptides was then sequenced to obtain the targeting peptide amino acid
sequences.
Statistical analysis of selected motifs using SAS software (version 8, SAS
Institute)
revealed that the motif CKGGRAKDC (SEQ ID N0:81) constituted 4.5% of all
clones
identified in the screen. Intravenous administeration of this clone into oblob
mice
showed that CKGGRAKDC (SEQ ID N0:81)-phage accumulated in subcutaneous fat
to a higher level than a control insertless phage (data not shown).
The tropism of CKGGRAKDC (SEQ ID N0:81)-phage for adipose tissue was
confirmed by immunohistochemistry: CKGGRAKDC (SEQ ID N0:81)-phage showed
marked localization to the vasculature of subcutaneous and peritoneal white
fat (FIG.
24a, arrows), whereas the control phage was undetectable in fat blood vessels
(FIG.
24b). To test whether targeting of the CKGGRAKDC (SEQ ID N0:81) motif to the
fat
vasculature would also occur when the peptide is outside of the context of the
phage,
the ih vivo distribution of intravenously injected CKGGRAKDC (SEQ ID NO:81)
peptide fused to fluorescent (FITC) was determined. Immunofluorescence in
subcutaneous and peritoneal fat from peptide-injected ob/ob mice showed that
CKGGRAKDC (SEQ ID N0:81)-FITC localized to and was internalized by cells of
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white adipose vasculature (FTG. 24c, arrows), whereas a control CARAC (SEQ ll~
N0:71)-FITC conjugate was undetectable in adipose tissue (FIG. 24d).
CKGGRAKDC (SEQ ID NO: SI ) homes to white fat iyZ wild-type mice
The mutation in leptin that leads to the extreme proliferation of white
adipose
tissue in mice (Zhang et al., 1994) is not frequently encountered in humans
(Ozata et
al., J. Clih. Endocrinol. Metab. 84:3686-3695. 1999). Thus, this animal model
may not
be representative of the typical pattern of obesity in humans. To exclude the
possibility
that CKGGRAKDC (SEQ ID N0:81) homing to fat is limited to oblob mice and to
demonstrate the general applicability of adipose-targeting peptides for
naturally-
occurring obesity, the CKGGRAKDC (SEQ ID N0:81) peptide was tested in wild-
type
mice.
FIG. 25 shows that the CKGGRAKDC (SEQ ID NO:81)-FITC fusion peptide
intravenously injected into C57BL/6 (leptin +/+) mice specifically localized
to blood
vessels of subcutaneous and peritoneal white fat (FIG. 25A, FIG. 25B). A
lectin-
rhodamine peptide was used to visualize blood vessel endothelium (arrows, FIG.
25B,
FIG. 25D, FIG. 25F). The CKGGRAKDC (SEQ ID NO:81)-FITC fusion peptide co-
localized with lectin-rhodamine in adipose tissue (arrows, FIG. 25A and FIG.
25B). No
such co-localization was observed in control pancreatic tissue (FIG. 25C and
FIG. 25D)
or other control organs (data not shown). The control CARAC (SEQ ID N0:71)-
FITC
peptide was not detectable in white fat vasculature (FIG. 25E and FIG. 25F).
These in
vivo localization data show that the adipose-targeting CKGGRAKDC (SEQ ID
N0:81)
peptide targets the white adipose vasculature in genetically normal obese mice
as well
as in leptin deficient mice, demonstrating the general applicability of
adipose targeting
using such peptides.. The uptake of CKGGRAKDC (SEQ ID N0:81)-FITC by the
endothelium of fat tissue suggests that the motif targets a receptor
selectively expressed
in the adipose vasculature that could provide a mechanism for directed
delivery of
therapeutic compounds to fat.
Design and use of fat-targeted pYO-apoptotic peptide
It was next determined whether proliferation of adipose tissue could be
controlled via targeted destruction of the fat vasculature. The pro-apoptotic
peptide
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KLAKLAKKLAKLAK (SEQ m NO:1) (Ellerby et al., Nature Med. 5:1032-38, 1999),
designated (KLAKLAK)2 (SEQ ID NO:1), which disrupts mitochondrial membranes to
induce apoptosis, has been targeted to receptors in tumor vasculature via a
conjugated
homing peptide (Ellerby et al 1999, Arap, et al., Proc. Natl. Acad. Sci. U. S.
A.
99:1527-1531, 2002). The (KLAKLAK)2 (SEQ ID N0:1) peptide was conjugated to
the fat targeting CKGGRAKDC (SEQ m N0:81) peptide for targeted delivery to fat
vasculature in adipose tissue. The D enantiomer of (KLAKLAK)2 (SEQ ID NO:1),
which is resistant to proteolysis but still exhibits pro-apoptotic activity,
was conjugated
to the CKGGRAKDC (SEQ ID N0:81) peptide via a glycinylglycine bridge. The
conjugated fat-targeting, pro-apoptotic peptide was administered to mice and
the effect
on adipose tissue was monitored.
A non-genetic mouse obesity model was initially used. A cohort of C57BL/6
(wild-type) mice, in which obesity had been induced by a high-fat cafeteria
diet, were
subcutaneously injected with CKGGRAKDC (SEQ ID N0:81)-(KLAKLAK)2 (SEQ ID
N0:1) peptide and weighed daily over a period of two weeks. Cafeteria dieting
continued throughout the experiment. As shown in FIG. 26A, injections of
CKGGRAKDC (SEQ ID N0:81) conjugated to (KLAKLAK)2 (SEQ ID NO:1)
prevented obesity development and surprisingly caused a rapid decrease in body
mass
of up to 20%. In contrast, obese mice injected with two negative controls (an
equimolar
amount of either unconjugated CKGGRAKDC (SEQ ll~ N0:81) and (KLAKLAK)2
(SEQ ID N0:1) or a control CARAC (SEQ ID N0:71)-(KLAKLAK)2 (SEQ ID N0:1)
conjugate) did not show a significant body mass decrease and continued to
increase in
weight (FIG. 26A).
The effectiveness of the CKGGRAKDC (SEQ ID N0:81)-(KLAKLAK)~ (SEQ
ID N0:1) conjugate was also examined in wild-type mice fed on a regular diet
(FIG.
26B). C57BL/6 mice that had developed a considerable amount of subcutaneous
and
peritoneal fat due to old age were subcutaneously injected with the CKGGRAKDC
(SEQ ll~ N0:81)-(KLAKLAK)2 (SEQ ID N0:1) conjugate or control peptides over a
period of one month. As in the diet-induced obesity model, targeting of
(KLAKLAK)2
(SEQ ID NO:1) to fat by conjugation with CKGGRAKDC (SEQ ID N0:81) resulted in
greater than 35% reduction in body mass at a rate of 10% per week (FIG. lOB).
No
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toxicity of the conjugated peptide was detected under these conditions (data
not shown).
In fact, the CKGGRAKDC (SEQ ID N0:81)-(KLAKLAK)2 (SEQ ID NO:1) treated
mice became more active and agile following body mass reduction and appeared
healthier than prior to treatment (data not shown). The control untargeted
(KLAKLAK)2 (SEQ m NO:1) treatments resulted in only a slight body mass
reduction
(FIG. 26B), possibly due to low levels of nonspecific toxicity. The control
mice did not
exhibit the increased activity and/or agility seen in treated mice (data not
shown).
Fat resoYptiorc with CKGGRAKDC (SEQ ID N0:81)-(KLAKLAK)a ~(SEQ ID
NO:1 ) is mediated by apoptosis
In both diet-induced and age-related obesity, the effect of CKGGRAKDC (SEQ
m N0:81)-(KLAKLAK)~ (SEQ ID N0:1) treatment on body mass was due to fat
resorption, which was visually apparent by the end of treatment (FIG. 27).
Wild-type
mice were fed on a high fat cafeteria diet (FIG. 27A). Alternatively, wild-
type fed on a
regular diet became obese as a consequence of old age (FIG. 27B, FIG. 27C,
FIG. 27D).
Mice were treated with CKGGRAKDC (SEQ ID N0:81) conjugated to (KLAKLAK)2
(SEQ ll~ NO:1) (left side of FIG. 27), with CARAC (SEQ ID N0:71) conjugated to
(KLAKLAK)a (SEQ ID NO:1) (middle of figure), or with unconjugated
CKGGRAKDC (SEQ ID N0:81) and (KLAKLAK)~ (right side of FIG. 27).
Gross inspection of mouse organs revealed that both subcutaneous (FIG. 27B)
and visceral (FIG. 27C) fat exhibited marked resorption upon treatment with
CKGGRAKDC (SEQ m N0:81) conjugated to (KLAKLAK)~ (SEQ m NO:1) (right
side of FIG. 27). Quantification of fat resorption after three weeks of
treatment by
weighing a specific fat depot (epididymal fat, FIG. 27D) showed a greater than
3-fold
reduction in fat mass compared with controls (FTG. 27D, left side of figure
compared to
middle and right side).
Histopathological analysis of tissues from mice treated with CKGGRAKDC
(SEQ m N0:81) conjugated to (KLAKLAK)2 (SEQ ID NO:1) showed vascular
apoptosis (FIG. 28A, arrows) and resulting fat necrosis with lymphocyte
infiltration
(FTG. 28C, arrows) in adipose tissue. following treatment. In contrast, mice
treated
with a control fusion peptide comprising CARAC (SEQ m N0:71) conjugated to
(KLAKLAK)2 (SEQ ll~ NO:1) showed no vascular apoptosis or fat necrosis (FIG.
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28D). No abnormalities in other organs treated with CKGGRAKDC (SEQ ID N0:81)
conjugated to (KLAKLAK)2 (SEQ ID N0:1) (data not shown).
Injection of CKGGRAKDC (SEQ m N0:81) conjugated to (KLAKLAK)2
(SEQ ID N0:1) into genetically obese mice, but not into normal obese mice, was
occasionally observed to result in mortality within a few days of injection.
It is not
clear what the mechanism might be for inducing death in genetically obese
mice,
although development of pulmonary or cardiac fat embolism or rapid drop of
serum
calcium due to saponification by released lipids are possibilities. However,
these
results suggest that treatment of grossly obese subjects might result in
sufficient adipose
cell death and necrosis to adversely affect the health of the subject,
indicating that lower
dosages andlor use of a time release formulation of the adipose targeting
conjugate may
be preferred in cases of excessive obesity.
Aelip~se receptor protei~z for CKGGRAKDC (SEQ ID 1V0: 81
A band of approximately 35,000 Daltons (35 kDa) was isolated from mouse
adipose tissue extract that bound to CKGGRAKCDC (SEQ ID N0:81) conjugated to
(KLAKLAK)2 (SEQ ID N0:1) (not shown). There was much less binding of the 35
kDa fraction to the control peptide CARAC (SEQ ID N0:71) conjugated to
(KLAKAK)a (SEQ ID N0:1) (data not shown). The 35 kDa band was analyzed by mass
spectrometry, which identified three proteins present in the sample.
The three proteins included predominately a B cell receptor associated protein
(prohibitin), apolipoprotein E, and the voltage dependent anion channel
(VDAC).
Further studies were performed by immunoprecipitation, using either CKGGRAKDC
(SEQ ID NO:81) or CARAC (SEQ ID N0:71) conjugated to (KLAKAK)2 (SEQ ID
NO:1) and precipitating with commercially available antibodies.
SDS-polyacrylamide gel electrophoresis of the immunoprecipitated protein
showed that only the prohibitin receptor protein complex was substantially
enriched by
binding to CKGGRAKDC (SEQ ID NO:81) (data not shown), with over a ten-fold
enrichment in the CKGGRAKDC (SEQ ID N0:81) precipitated fraction compared to
the CARAC (SEQ ID N0:71) precipitated fraction (data not shown). The CARAC
(SEQ ID N0:71)-(KLAKAK)2 (SEQ ID NO:1) fusion peptide exhibited low levels of
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non-specific binding to all three proteins (VDAC, prohibitin and
apolipoprotein E). It
is unknown whether those proteins bound to the CARAC (SEQ ID N0:71) moiety or
to
(KLAKAK)2 (SEQ ID NO:1).
It is concluded that the adipose tissue endothelial receptor for CKGGRAKDC
(SEQ ID N0:81) is prohibitin (Genbank Accession No. NM 008831). Probitin is
expressed in mitochrondria of various cell types and in the cell membrane of B
lymphocytes, where it is associated with the IgM receptor (McClung et al.,
Exp.
Gerontol. 30:99-124, 1995). Based on these results, it is concluded that pro-
apoptosis
agents conjugated to targeting peptides that bind to a prohibitin receptor
protein
complex may be effective to induce adipose cell death and weight loss in obese
subjects. The skilled artisan will realize that other prohibitin-binding
targeting
peptides, antibodies, etc. may be used within the scope of the claimed methods
and
compositions to control weight and/or to induce weight loss. Further, other
known
cytocidal, cytotoxic and/or cytostatic agents may be used in place of
(KLAKAK)2 (SEQ
ID N0:1) to control weight or induce weight loss within the scope of the
claimed
subject matter.
The results obtained in a mouse model system were confirmed in human tissue
sections. Rabbit polyclonal antibodies against prohibitin were commercially
purchased
(RDI-PROHIBTT, Research Diagnostics, Inc., Flanders, NJ). Immunohistochemistry
on
sections of fixed human paraffin-embedded tissues was performed using the
LSAB+
peroxidase kit from Dako (Carpinteria, CA). Comparison of prohibitin
expression in
mouse versus human white fat tissue showed that prohibitin is highly expressed
in
blood vessels of both mouse and human white fat tissues (not shown).
Prohibitin is expressed in the vascular endothelium of a number of human
organs (FIG. 30, arrows), including white fat tissue (FIG. 30A), skin (FIG.
30B),
prostate (FIG. 30C) and bone (FIG. 30E). However, the level of prohibitin
expression
in white fat blood vessels is much higher than in other types of human tissues
(FIG. 30).
Prohibitin expression appears to be inversely correlated with the degree of
malignancy in human adipose tissues (FIG. 29). The arrows indicate prohibitin
staining
in normal human white fat tissue (FIG. 29A), normal human breast tissue (FIG.
29B), a
low grade human lipoma (FIG. 29C), a high grade human lipoma (FIG. 29D), a
myxoid
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liposarcoma (FIG. 29E) and a dedifferentiated liposarcoma (FIG. 29F). For each
tumor
sample, prohibitin expression was also evaluated in a control organ from the
same
patient (data not shown) to verify that prohibitin was specifically
downregulated in the
vasculature of the tumor. FIG. 29 shows that prohibitin expression is
progressively lost
in the blood vessels of fat tissue, parallel to fat transformation into
malignant
liposarcoma tissues. Thus, prohibitin is a negative indicator of malignancy in
adipose
tissues, as prohibitin expression is inversely correlated with the degree of
malignancy of
the tissue.
A model for prohibitin function in fat vasculature is presented in FIG. 31.
The
KARGG (SEQ ID N0:82) motif, found in reverse orientation in the prohibitin
binding
peptide CKGGRAKDC (SEQ ID N0:81), shows homology with the human stem cell
growth factor (SCGF) protein (FIG. 31), a member of the C-type lectin
superfamily.
SCGF in combination with VEGF has been reported to cause differentiation of
CD34(+) progenitor cells into endothelial cells, with characteristics of
vascular
endothelium (Gehling et al., Blood, 95:3106-12, 2000). SCGF expression also
appears
to be associated with B lymphopoiesis (Witte et al., Eur. J. Immunol. 32:1809-
17,
1993). It is proposed that binding of SCGF protein to a complex of prohibitin
and the
IgM receptor protein in B lymphocytes may mediate B cell differentiation (FIG.
31). It
is further suggested that binding of CKGGRAKDC (SEQ ID N0:81) to prohibitin in
adipose blood vessel cells may potentially mimic an endogenous SCGF dependent
signaling pathway, perhaps related to endothelial cell differentiation (FIG.
31).
Example 9: Novel Prostate Tumor Targeting Peptides
DU145 prostate tumor cells were injected subcutaneously into the right fat pad
of nude mice. A large phage library (X2CX14CX~) was prepared as discussed
above and
109 phage were injected into male tumor-bearing nude mice. After 24 hr
circulation,
tumors were removed and phage recovered from the tumors using the bulk method
disclosed above. The recovered phage were amplified, titered and reinjected
into a new
set of tumor bearing nude mice. The biopanning protocol was repeated for a
total of
three rounds. Ninety-six phage clones recovered from the third round of
biopanning
were selected for sequencing. Translated sequences were obtained for 76 of the
96
clones.
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Targeting peptides recovered from DU145 xenograftic tumors are listed in
Table 10. The primary prostate tumor targeting peptides recovered were
YRCTLNSPFFWEDMTHECHA (SEQ ID N0:83) and
LGCMASMLREFEGATHACTQ (SEQ ID N0:84). The numbers in parentheses
indicate the number of times the same targeting peptide sequence was obtained.
As
indicated in Table 9, the YRCTLNSPFFWEDMTHECHA (SEQ ll~ N0:83) targeting
sequence was recovered in 11 out of 76 colonies, while the
LGCMASMLREFEGATHACTQ (SEQ ID NO:84) peptide was recovered in 8 out of
76 colonies. No obvious homologies were observed between the prostate tumor
targeting peptides listed in Table 10 and any known protein sequence.
Table 10. Prostate Tumor-Targeting
Peptides Recovered From DU145
Xenografts
YRCTLNSPFFWEDMTHECHA (11) SEQ 1D N0:83
LGCMASMLREFEGATHACTQ (8) SEQ 1D N0:84
RGCTEAAGLVIGITTHQCGN (3) SEQ ID NO:85
IGCNHPSPLGSTVVPTYCFK (3) SEQ ID N0:86
GTCPRQFFHMQEFWPSDCSR (3) SEQ 1D N0:87
DRCVLVRPEFGRGDARLCHS (2) SEQ 1D N0:88
EGCSDI1VINTAAERVTGDCSY (2) SEQ ll~ N0:89
VFCCGSYCGGVEMLASRCGH (2) SEQ ID N0:90
RECGRTVHRYPWGSPESCER (2) SEQ ID NO:91
DACSRFLGERVDATAAGCSR (2) SEQ ID N0:92
GNCMGLQVSELFMGPYKCRQ (2) SEQ ID NO:93
SRCHALRSQSVSTSAGACIS (1) SEQ ID NO:94
YSCTRLNGTGLQNPPSACDR (1) SEQ ID N0:95
WVCTSASQDTRLKEPGMCIA (1) SEQ ID N0:96
MHCTSQTLRGTPSLAPKCSD (1) SEQ ID N0:97
QHCVKGQFPFRESVTITCNS (1) SEQ 117 N0:98
HTCWGARDVAQPSGTVRCLK (1) SEQ ID N0:99
ARCREDTGFMGLGSANICTD (1) SEQ ll~ NO:100
RTCEEVRNRALEELTNFCPY (1) SEQ ID N0:101
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RTCQVRSNNISPRMALACVT (1) SEQ ID NO:102
RSCVNSDTGVLQRGAPSCLF (1) SEQ ID N0:103
RGCWRDSTAWHVSYPVECLA (1) SEQ ID N0:104
NRCMPGFLDDADSAASPCGS (1) SEQ ID N0:105
NQCSSLLTYQGWKRTKDCIP (1) SEQ ID N0:106
NDCSAHAQPGWDEVPPMCNQ (1) SEQ ID N0:107
NNCPVEGSQQNYSGATWCRA (1) SEQ ID N0:108
TTCNKSMSSQPMRDSRECHR (1) SEQ ID NO:109
TSCVRTGHDENLLKAAYCSS (1) SEQ ~ NO:110
TECRGASSGSVSGAATDCRD (1) SEQ ID NO:111
TLCPPASMGLGREKPRLCSV (1) SEQ ID N0:112
TLCRSLEHEVGLFKPRECPF (1) SEQ ID N0:113
LRCPLEVDRPNRDPAFLCSQ (1) SEQ ID N0:114
LGCNKGRYWLSTRLSVSCAL (1) SEQ ID N0:115
VACDISAVERLPASARSCKT (1) SEQ ID N0:116
VVCFMERQMGTDVVSPMCVN (1) SEQ ID N0:117
VECVMASASTDGTAAHPCKP (1) SEQ ll~ N0:118
VRCNEAQLQDSGTVPHPCLR (1) SEQ ID N0:119
PNCDLDDIVLNPYTAGPCGT (1) SEQ ID N0:120
PNCYSGDGEISSHIPVQCLM (1) SEQ ID N0:121
PGCVVSPFALSAQGTSVCTI (1) SEQ ID N0:122
GDCETNNVTKVGGITRNCVG (1) SEQ ~ N0:123
GYCLTVVGGAVLTIALLCVT (1) SEQ ID N0:124
GPCAATGVNPGDHGAAVCDQ (1) SEQ ID N0:125
GDCETNNVTKVGGITRNCVG (1) SEQ ID N0:126
KSCGKYGLIVGQPFAEHCPP (1) SEQ ID N0:127
KLCYRSSAGSELRPPEKCAY (1) SEQ ID NO:128
KICPVTNMWTTPSWAHKCGM (1) SEQ ll~ N0:129
To determine the specificity of
the prostate tumor targeting
peptides, 109 phage
carrying the targeting peptide
sequences YRCTLNSPFFWEDMTHECHA
(SEQ ID
N0:83) and LGCMASMLREFEGATHACTQ
(SEQ ID N0:84) were injected
into nude
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mice bearing DU145 xenografts. After 24 hour circulation, tissue samples were
obtained from tumor and control organs (kidney, brain, lung and spleen).
Tissue
samples were washed immediately in DMAM and fixed in 10% formalin for 48 hours
at room temperature. Thin sections were stained for phage using anti-phage
antibody
(1:500 dilution) and detected using the DAKO LSAB+ system. The DU145 tumor
showed very heavy staining with anti-phage antibodies (data not shown). No
staining
was observed for control kidney, brain or lung tissues (not shown). A low
level of anti-
phage staining was observed in normal spleen tissue (not shown). This may be
due to
the tendency of spleen tissue to trap phage and other foreign particles in
general as part
of the reticuloendothelial system. In a separate study, samples of the MDA-MB-
435
breast carcinoma showed no apparent localization of phage bearing the
YRCTLNSPFFWEDMTHECHA (SEQ m N0:83) sequence.
Competition studies with the YRCTLNSPFFWEDMTHECHA (SEQ m
NO:83) peptide were performed to determine whether it could inhibit
localization of
phage bearing the same targeting sequence to DU145 tumors. Nude mice bearing
the
prostate tumor xenograft were simultaneously injected with 300 ~g of synthetic
YRCTLNSPFFWEDMTHECHA (SEQ m N0:83) peptide and 10~ phage bearing the
same targeting peptide sequence. A control tumor-bearing mouse was co-injected
with
fd insertless phage plus synthetic peptide. After 24 hours of circulation,
tumor tissue
samples were removed, washed, fixed, sectioned and stained as disclosed above.
Co-
administration of synthetic peptide with the same targeting sequence inhibited
the
ability of YRCTLNSPFFWEDMTHECHA (SEQ m N0:83)-phage to localize to
prostate carcinoma tissue (not shown). No staining of prostate carcinoma with
control
fd-tet phage was observed (not shown)
Receptor Purification
The prostate homing receptor for the B2 clone
(YRCTLNSPFFWEDMTHECHA SEQ m N0:83) was identified. Nude mice bearing
DU145 xenografts were prepared and tissue samples from tumor, kidney and liver
were
removed. The tissue samples were immediately washed with PBS and three parts
of
homogenization buffer (PBS, 250 mM sucrose, 1 mM EDTA, protease inhibitors)
was
added to one part of tissue sample (about 4 ml). The tissue with homogenized
with an
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electric grinder, then further homogenized with a dounce homogenizer. After
sonication for 1 min on ice, the homogenate was centrifuged at 8000 x g for 5
min. The
supernatant was removed and the pellet was analyzed for receptor content.
The YRCTLNSPFFWEDMTHECHA (SEQ ll~ NO:83) peptide was
biotinylated and coupled to NeutrAvidin beads (Molecular Probes, Eugene, OR)
using
standard methods. About 500 ~,g of biotinylated peptide was incubated with 1
ml of
NeutrAvidin beads in binding buffer (0.5 M NaCI in PBS) overnight at
4°C in a 2 ml
column. The column was agitated using a rotator. Uncoupled peptides were
removed
and the beads washed three times with binding buffer and protease inhibitors.
Approximately 1 mg of tissue extract was added to the biotinylated peptide
conjugated
beads. The material was resuspended in 2 ml of binding buffer and incubated
overnight
at 4°C on a rotator. The material was centrifuged and supernatant was
removed.
The beads were washed four times with wash buffer (0.1% Triton X-100 in PBS
with protease inhibitors) and the bound material eluted with 8 M guanidine
HCI.
Eluted proteins were analyzed on a 4-20% SDS-PAGE denaturing gel. Protein (40
fig)
from the tumor and kidney were run as controls. Bands that showed apparent
enrichment for binding to the YRCTLNSPFFWEDMTHECHA (SEQ ll~ N0:83)
peptide were cut out for protein sequencing.
MALDI analysis of the excised bands identified HSP90 and an unidentified
protein. HSP90 is known to be overexpressed in prostate cancer and to be
associated
with MHC-I on the cell surface. It is concluded that the endogenous receptor
for the
YRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) peptide is HSP90oc (GenBank
Accession No. NM005348).
Example 10: Novel Ovarian Cancer Targeting Peptides
Background
Carcinomas that arise from the ovarian surface epithelium represent a great
challenge in gynecologic oncology (Rosenthal & Jacobs, Semin Oncol, 1998.
25:315-
25). Ovarian cancer is the sixth most common cancer in women and the deadliest
of all
gynecologic malignancies, resulting in about 14,000 deaths annually in the
United
States. Although the prognosis of ovarian cancer is influenced by many factors
capable
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of predicting clinical outcome, including tumor stage, pathological grade,
patient
performance status and amount of residual disease following primary debulking
surgery, the biological aspects of ovarian cancer are not completely
understood,
implying that there may be other predictive indicators that could be used.
Tumor
markers have the potential to contribute to cancer screening, diagnosis,
monitoring, and
prognosis as well as provide targets for anti-tumor therapy. The most
extensively
researched tumor marker in ovarian carcinoma is CA125. CA125 levels have been
used
as indicators of treatment response or progression. In monitoring response to
therapy,
CA125 is able to reflect progression or regression in over 90% of patients who
had
elevated preoperative levels. Still, in respect to persistent disease, CA125
only has an
accuracy of 60-80% and normal values often do not exclude active disease.
Thus, the
identification of additional markers with biological relevance would be
desirable.
Neoplasms of the ovary represent a diverse group. They can be divided into
four major histological classes based on their origin: coelomic epithelial,
germ cell,
specialized gonadal-stromal, and non-specific mesenchymal. The neoplasms
derived
from coelomic epithelium are the most common, comprising over 80% of all
ovarian
tumors. In becoming neoplastic, the coelomic epithelium exhibits a variety of
Mullerian type differentiation, such as serous, mutinous, endometroid, and
clear cell,
which comprise the different histological subtypes.
The molecular and cellular events leading to the development of ovarian cancer
are not Completely understood and it is unclear whether ovarian cancer follows
a
stepwise pattern of progression, as no pre-malignant lesion has yet been
identified. One
proposed theory is that in early stages the cancer is confined to small
epithelial
inclusion cysts in the ovary. With time, the tumor penetrates through the
surface
capsule and malignant cells enter the peritoneal Cavity. Here, exfoliation and
implantation are the primary modes of spread of ovarian cancer. Within the
peritoneal
cavity, the Cells follow the natural pattern of peritoneal fluid circulation,
leaving all
peritoneal surfaces at risk for tumor cell implantation. Likewise, ovarian
cancer may
spread by lymphatic dissemination and less commonly by hematogenous route to
areas
such as the liver and lungs.
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The standard staging system for ovarian cancer is based on surgical
exploration
and clinical examination. Stage I is confined to the ovaries; stage II is
confined to the
pelvis; stage III has spread throughout the peritoneal cavity; and stage IV is
occult
distant metastasis, including parenchymal liver and lung metastasis.
Currently, the
most powerful determinant of prognosis in ovarian cancer is the extent to
which the
tumor has disseminated from the primary site at the time of diagnosis. If
diagnosed and
treated while the cancer has not spread outside the ovary, the five-year
survival rate is
95%. However, only 25%~ of all ovarian cancers are found at this early stage
due to
vague symptomatology and lack of effective screening strategies. Moreover,
older
women with ovarian cancer tend to have a poorer prognosis than younger ones.
The
overall primary treatment response rate is 80-90%, however, the clinical
complete
response rate is only 40-50% and the pathological response rate is even lower,
about
20-40%. Thus, even with optimal cytoreduction and chemotherapy, many patients
remain at risk for the development of recurrent disease.
Since ovarian malignancy may result in the accumulation of ascites in the
peritoneal cavity that contains tumor cells as well as tumor-associated
immunoglobulins, probing the antibody repertoire in the ascites of ovarian
cancer
patients may result in the identification of peptide epitopes resembling tumor
antigens.
The identified peptide epitopes would correspond to primary sequences found in
tumor
antigens or mimetopes of such antigens and could potentially serve as markers
for the
ovarian cancer. Such markers may be of use for the detection, diagnosis and/or
prognosis of ovarian and/or other cancers of the female reproductive tract.
The phage display methods disclosed above were used to identify novel tumor
markers for ovarian cancer. Random peptide phage library were screened against
IgGs
isolated from the ascites of ovarian cancer patients to enrich for phage that
bind to
ovarian cancer patient IgGs and identify ovarian cancer peptide epitopes.
Biochemical
methods are employed to identify the antigen eliciting the antibody response.
The
identified peptide epitopes and corresponding antigens are tested to determine
whether
they are linked to disease progression and survival. To assess the value of
each motif
and corresponding antigen, banked ascites and serum from ovarian cancer
patients are
screened by an enzyme linked immunosorbent assay (ELISA) protocol.
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Materials and Methods
Experimental samples from patients with ovarian cancer were obtained from the
M.D. Anderson Cancer Center Specialized Program of Research Excellence (SPORE)
Ovarian Tumor Bank. Control serum samples were obtained from healthy blood-
donor
age-matched women. Ascites samples were collected into sterile containers and
subjected to centrifugation to separate the cell free fraction from the
cellular fraction.
The fluid was stored in aliquots at -20 °C and the remaining cellular
fraction processed
to purify the cells involved in the immune response as well as ovarian cancer
tumor
cells. All blood samples were allowed to clot at room temperature and then
centrifuged. They were promptly aliquoted and frozen at -20 °C. Protein
G beads
(Pierce) were used for immunoaffinity purification of IgG from serum and
ascites
samples. Archived paraffin-embedded tissue blocks and slides (malignant and
non-
malignant) collected from the Department of Pathology at the M.D. Anderson
Cancer
Center were also utilized.
To identify peptide epitopes specific for the anti-tumor immune response in
ovarian cancer, a two-step screening procedure was followed (FIG. 34). The
peptide
library was initially pre-cleared on IVIg (intravenous imrnunoglobulins) to
remove non-
specific peptides. The pre-cleared peptide phage library was then incubated
with IgGs
isolated from the ascites of ovarian cancer patients. Phage bound to the
ovarian cancer
IgGs were recovered, amplified, and precipitated for subsequent rounds of
biopanning.
Following enrichment of a phage population that bound to ovarian cancer
patient IgGs,
individual phage clones were picked for sequence analysis to evaluate
enrichment of the
most consistently binding peptide sequences. Phage display biopanning was
performed
as described above.
Once the selection rounds were completed as determined by enrichment of
phage capable of binding cancer patient IgGs over control at least 3 fold,
sequencing
and evaluation of the DNA phage insert were undertaken. To sequence the FUSES
ssDNA directly, phage ssDNA was prepared using the StrataCleanTM resin
(Stratagene).
StrataClean bead slurry was placed in a microcentrifuge tube containing phage.
The
mixture was vortexed strongly for 30 seconds and then incubated at room
temperature
for one minute. The tubes were centrifuged at 2,000 x g for 1 minute. The
supernatant
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containing the ssDNA was placed into a fresh microcentrifuge tube. A total of
3
extractions were performed. The DNA was then precipated with ethanol and used
as a
template for the sequencing reaction utilizing the primer. Sequencing was done
by the
chain termination method on an ABI Prism~ 3700 (Applied Biosystems/Hitachi.
The
commercially available computer program, DNA Strider, was used in the analysis
of the
sequences.
Analysis of the distribution of inserts from the random peptide library used a
program based on SAS (version 8; SAS Institute) and Perl (version 5.0). The
program
is a high-throughput pattern recognition software used to analyze short amino-
acid
residue sequences. The program conducts an exhaustive amino-acid residue
sequence
count and keeps track of the relative frequencies of n distinct tripeptide
motifs
representing all possible n3 overlapping tripeptide motifs in both directions
(n«n3).
Counts were recorded for all interior tripeptide motifs, subject only to
reflection and
single-voting restrictions. No peptide, in the program, is allowed to
contribute more
than once for a single tripeptide motif (or a reversed tripeptide motif).
Tripeptide
motifs were chosen for the phage insert analysis because three amino-acid
residues
seem to provide the minimal framework for structural formation and protein-
protein
interaction. Each phage insert analyzed contained seven amino-acid residues
and
contributed to ten potential tripeptide motifs.
The Clustal W software from the European Molecular Biology Laboratory was
adopted to analyze the cyclic phage peptides. Clustal W is a general purpose
multiple
sequence alignment program for DNA or proteins and produces biologically
meaningful
multiple sequence alignments of divergent sequences. It calculates the best
match for
the selected sequences, and lines them up so that the identities, similarities
and
differences can be seen.
Construction and ,-purification of GST-fusion ~e tt~ ides. Peptide coding
sequences were amplified using colony PCR with the following forward
(5'AGGCTCGAGGATCCTCGGCCGACGGGGCT-3', SEQ ID N0:130) and reverse
(5'-AGGTCTAGAATTCGCCCCAGCGGCCCC-3', SEQ ID N0:131) primers that
contain BamHI and EcoRI sites (shown in bold), respectively. The amplified
sequence,
containing the peptide coding sequence, was cloned into the BamHI-EcoRI sites
of the
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GST vector, pGEX-2TK (Amersham/Pharmacia), and automated sequencing used for
verification of positive clones. Positive clones were transformed into the
bacterial
expression host strain, BL21 (DE3) pLys (Stratagene), by electroporation.
Expression
of the GST-fusion proteins was induced with 200 ~,M isopropylthiogalactoside
(IPTG).
Expression of the GST-fusion constructs was compared to uninserted pGEX-2TK
vector to select for positive clones that produced the greatest amount of
fusion proteins.
GST-fusion proteins were expressed from selected clones and affinity purified
from
bacterial lysates by affinity chromatography to immobilized glutathione using
glutathione Sepharose 4B resin (Amersham/Pharmacia).
Testing of individual phase clones (binding assa~g-patient derived or
control I_~ Binding of individual phage clones to cancer patient IgGs was
studied
by a microtiter assay. Antibodies from the ascites or donor were purified by
standard
techniques. The antibodies were used to coat MaxiSorp 96-well plates (Nalge
Nunc
International Corporation) at a concentration of 10-100 ~,g/ml. Coating of
plates was
carried out at 4 °C overnight. The plates were blocked with 3% Bovine
serum
albumin/phosphate buffered saline (BSA/PBS). For the binding reaction, 109 TU
of
phage was added to the coated and blocked plates. The binding was performed at
room
temperature for 2 hours. After the binding reaction, the wells were washed
four times
with 3% BSA/PBS. Addition of K9lKan bacterial culture and incubation at room
temperature for 30 min were used to rescue bound phage. The bacteria were
diluted in
ml of LB culture media supplemented with 0.2 ~,g/ml tetracycline and incubated
for
another 30 min at room temperature. Serial dilutions of this bacterial culture
were
plated on LB plates containing 40 ,ug/ml tetracycline. Plates were incubated
at 37 °C
overnight before counting colonies. Binding of control (insertless) phage was
also
assessed.
Inhibition of binding assays. Both GST fusion proteins and synthetic peptides
corresponding the sequence displayed were used for inhibitory studies.
Inhibition
studies were performed in a similar manner as the binding assays described
above with
the exception that either GST fusion protein or synthetic peptide
corresponding to the
phage clone were added to the experiment. Where the peptide is mediating the
interaction with the immunoglobulins then an inhibition of phage binding
should be
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observed in a dose dependent manner versus the synthetic peptide. GST alone
and a
control peptide containing unrelated amino acids were tested at identical
concentrations.
Purification of peptide specific antibodies and immunohistochemistry. To test
if
the antigen is indeed specifically expressed in ovarian cancer and tumor-
associated, the
specific immunoglobulins capable of reacting with the identified peptide
epitopes were
purified and immunohistochemical staining performed on tissue sections from
the
patient in whom the initial screening was performed. GST fusion proteins made
from
inserting recombinant peptide sequences of interest in an expression vector
were coated
on MaxiSorp multi-well plates (Nalge Nunc International Corporation). The
plates
were incubated with the ascites fluid from which the peptide was originally
isolated.
Following a washing procedure to remove unbound IgGs, bound IgGs were eluted
with
0.1 M glycine buffer, pH2.2, neutralized with 1 M Tris-Cl, pH9.0, and dialyzed
in PBS
overnight. To concentrate the IgG, centricon-30 columns (Millipore) were used.
The
purified antibody was coupled to biotin according to the manufacturer's
instructions
(Vector). The biotinylated antibody was analyzed by SDS-gel electrophoresis.
Tumor
paraffin sections were deparaffinized in xylene, rehydrated in ethanol, and
treated with
an antigen retrieval reagent (DAKO) in 10 mM sodium citrate, pH 7.5 in a steam
bath.
Non-specific sites on the tissue were blocked by incubating the deparaffinized
slide in a
casein blocking buffer. Affinity purified biotinylated ovarian cancer ascites
fluid IgGs
was applied to the sections. A rinsing step and the addition of strepavidin
conjugated to
horseradish peroxidase followed. Positive staining cells were visualized by
the addition
of diamino benzidine and sections with phase contrast microscopy with an
Olympus
IX70 Inverted microscope. All sections were additionally counterstained with
hematoxylin.
Protein homology searches. Database searches may be helpful in the
identification of the antigen for a given peptide sequence. Validated peptide
epitope(s)
were searched in online databases (through the National Center for
Biotechnology
Information (NCBI; http://www.ncbi.nlm.nih.govBLAST/) and candidate tumor
antigens were identified by homology with known human proteins.
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Analysis of patients eliciting an immune response against the identified
a tide s .
An ELISA protocol was used to examine the presence of antibodies for the
selected markers in a panel of ovarian cancer patients. Normal serum and non-
malignant ascites were also tested to help show whether or not the immune
response to
the marker was associated with evaluated patient characteristics.
ELISA. Peptide sequences of interest were expressed as GST fusion proteins
(described above) and used to screen banked ascites and serum to determine the
role of
the humoral response against these markers. The purified GST-fusion proteins
were
used to coat a 96-well plate at 100 ng/well at room temperature (RT) or at 4
°C
overnight. Following coating, the wells were emptied, rinsed, and non-specific
sites
were blocked with 200 p,l 3% BSA/PBS at RT for 1-2 hours. Cancer patient
ascites
and/or sera were applied to each coated and blocked well at 1:100 dilution and
then
incubated at RT for 1 hour. The wells were rinsed 3x with 3% BSA/PBS
containing
0.01 % Tween 20, and then incubated for 1 hour with 50 ~,1 each of anti-human
alkaline
phosphatase at 1:2000 dilution. Signals were detected in the presence of p-
nitrophenyl
phosphate by measuring OD4os at specific intervals to follow the course of
color
development. A positive control was the cancer ascites the peptide was
identified from,
and a negative control was donor sera and/or BSA.
Results
FIG. 35 shows the results of biopanning a CX~C phage display library against
ascites taken from an ovarian cancer patient after 2 and 3 rounds of
biopanning. As can
be seen, after two rounds of biopanning the targeting phage specificity was
fairly low,
exhibiting higher levels of binding to the BSA and control immunoglobulins.
However, after a third round of biopanning the phage exhibited a very high
degree of
selectivity for binding to the ovarian cancer patient's immunoglobulins,
compared to
control IgGs or BSA.
The primary peptide sequence recovered against ovarian cancer patient ascites
exhibited the targeting sequence CVPELGHEC (SEQ ll~ N0:132). This peptide
represented ~6% (73 of ~5) of the phage clones that were sequenced. Additional
studies were carried out to validate the ovarian ascites targeting specificity
of this
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peptide sequence. FIG. 36 shows that antibodies isolated from the ascites of
an ovarian
cancer patient bound specifically to the targeting peptide sequence CVPELGHEC
(SEQ
ID N0:132). Purified ascites immunoglobulins were exposed to microtiter plates
containing immobilized GST-CVPELGHEC (SEQ ID N0:132) fusion proteins.
Antibody binding to the immobilized fusion protein was competitively inhibited
in a
dose-dependent fashion by the synthetic CVPELGHEC (SEQ ID N0:132) peptide, but
was unaffected by a control peptide (FIG. 36).
Ascites from patients with different stages of ovarian cancer, non-ovarian
cancer
or non-malignant conditions was screened against GST-CVPELGHEC (SEQ ID
N0:132) fusion proteins using serial dilutions to determine the optimal
reactivity of
immunoglobulins present in each sample. The results, presented in FIG. 37,
show that
peptide binding to immunoglobulins is stage dependent, with ascites from Stage
IV
ovarian cancers showing a higher reactivity than ascites from Stage III
ovarian cancer.
Some reactivity was also observed with ascites from non-ovarian cancer, but
not with
ascites from patients with non-malignant conditions (FIG. 37).
These results demonstrate the utility of the CVPELGHEC (SEQ ID N0:132)
peptide for the detection, diagnosis, staging and/or prognosis of ovarian
cancer. The
present of antibodies reactive with the CVPELGHEC (SEQ ID N0:132) peptide in
ascites from suspected ovarian cancer patients is indicative of the presence
of a high
stage ovarian cancer. The skilled artisan will realize that the presence of
anti-
CVPELGHEC (SEQ ID N0:132) antibodies in patient ascites may also be indicative
of
the presence of non-ovarian cancers. The artisan will further realize that the
CVPELGHEC (SEQ ID N0:132) peptide may be of use as a mimeotope of an ovarian
cancer selective endogenous protein. As discussed above, the endogenous
mimeotope
of the CVPELGHEC (SEQ ID N0:132) peptide may be identified by protein homology
searches of the CVPELGHEC (SEQ ID N0:132) peptide against standard databases.
Alternatively, as disclosed above, antibodies binding to the CVPELGHEC (SEQ ID
NO:132) peptide may be purified by immunoaffinity chromatography and used to
identify the endogenous mimeotope. Also alternatively, monoclonal antibodies
reactive
with the CVPELGHEC (SEQ ID N0:132) peptide may be prepared by standard
methods and used to identify the endogenous mimeotope. A preliminary BLAST
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search against the NCBI database did not reveal any obvious homologies with
known
protein sequences, indicating that the ovarian cancer targeting peptide may
mimic an
epitope comprised of two or more portions of the primary sequence of the
endogenous
mimeotope.
Immunohistochemical analysis against ovarian cancer thin sections from the
same patient whose ascites was screened for reactive antibodies demonstrated
that an
endogenous mimeotope was in fact present in the ovarian tumor (not shown).
Autologous immunopurified immunoglobulins used for IHC versus a primary
ovarian
lesion as well as a metastatic peritoneal nodule showed the presence of strong
immunoreactive staining (not shown). Negative controls ~ using secondary
antibody
alone, or in combination with immunoglobulins obtained from a pool of non-
cancer
patients showed no lHC staining under identical conditions (not shown). A
recombinant GST-CVPELGHEC (SEQ ID NO:132) fusion protein inhibited staining
with autologous immunoglobulins. These results demonstrate that IgGs from the
ascites of ovarian cancer patients are reactive against an endogenous ovarian
cancer
antigen that is of use for ovarian cancer detection, diagnosis and/or staging.
* *
All of the COMPOSITIONS, METHODS and APPARATUS disclosed and
claimed herein can be made and executed without undue experimentation in light
of the
present disclosure. While the compositions and methods of this invention have
been
described in terms of preferred embodiments, it are apparent to those of skill
in the art
that variations maybe applied to the COMPOSITIONS, METHODS and APPARATUS
and in the steps or in the sequence of steps of the methods described herein
without
departing from the concept, spirit and scope of the invention. More
specifically, it are
apparent that certain agents that are both chemically and physiologically
related may be
substituted for the agents described herein while the same or similar results
would be
achieved. All such similar substitutes and modifications apparent to those
skilled in the
art are deemed to be within the spirit, scope and concept of the invention as
defined by
the appended claims.
133
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Irmnunol. Immunother. 45:149-151, 1997c.
Wickham, T.J. Targeting adenovirus. Gene Ther 7, 110-114, 2000.
WICKHAM, T.J., CARRION, M.E., and KOVESDI, I. (1995). Targeting of
adenovirus penton base to new receptors through replacement of its RGD motif
with
other receptor-specific peptide motifs. Gene Ther. 2; 750-756.
WICKHAM, T.J., LEE, G., TITUS, J., SCONOCCHIA, G., BAKACS, T.,
KOVESDI, L, and SEGAL, D. (1997x). Targeted adenovirus-mediated gene delivery
to
T-cells via CD3. J. Virol. 71; 7663-7669.
WICKHAM, T.J., MATHIAS, P., CHERESH, D.A., and NEMEROW, G.R.
(1993). Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus
internalization
but not attachment. Cell 73; 309-319.
WICKHAM, T.J., ROELVINK, P.W., BROUGH, D.E., and KOVESDI, I.
(1996b). Adenovirus targeted to heparan-containing receptors increases its
gene
delivery efficiency to multiple cell types. Nature Biotechnol. 14; 1570-1573.
WICKIiAM, T.J., SEGAL, D.M., ROELVINK, P.W., CARRION, M.E.,
LIZONOVA, A., LEE, G.M., and KOVESDI, I. (1996x). Targeted adenovirus gene
transfer to endothelial and smooth muscle cells by using bispecific
antibodies. J. Virol.
70; 6831-6838.
WICKHAM, T.J., TZENG, E., SHEARS II, L.L., ROELVINK, P.E., LI, Y.,
LEE, G.M., BROUGH, D.E., LIZONOVA, A., and KOVESDI, I. (1997b). Increased in
vitro and in vivo gene transfer by adenovirus vectors containing chimeric
fiber proteins.
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Wu and Wu, BioehemistYy, 27: 887-892, 1988.
Wu and Wu, J. Biol. Chem., 262: 4429-4432, 1987.
Zetter BR. Angiogenesis and tumor metastasis. Ann Rev Med 49:407-424,
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Zhang et al., Nature 372: 425-432, 1994.
Zhang J and Russell S. Vectors for cancer gene therapy.Cancer Met. Rev. 3:385-
401, 1996.
ZHANG, W. (1999). Development and application of adenoviral vectors for
gene therapy of cancer. Cancer Gene Ther. 6; 113-138.
Zini, S., Fournie-Zaluski, M.C., Chauvel, E., Rogues, B., Corvol, P. and
Cortes-
Llorens, C. (1996) Identification of metabolic pathways of brain angiotensin
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using specific aminopeptidase inhibitors: predominant role of angiotensin III
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157
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SEQUENCE LISTING
<110> Board of Regents, The University of Texas System (applicant for the
purposes
of all designated states except US)
Arap, Wadih (applicant for the purpose of the United States of America only)
Kolonin, Mikhail G.(applicant for the purpose of the United States of America
only)
Mintz, Paul J.(applicant for the purpose of the United States of America only)
Pasqualini, Renata (applicant for the purpose of the United States of America
only)
Zurita, Amado J.(applicant for the purpose of the United States of America
only)
<120> Compositions and Methods of Use of Targeting Peptides for Diagnosis and
Therapy of Human Cancer
<130> 005774.POlOPCT
<140> Unknown
<141> 2002-10-30
<150> PCT/US02/27836
<151> 2001-08-30
<160> 132
<170> PatentIn version 3.1
<210> 1
<211> 14
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 1
Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys
1 5 10
<210> 2
<211> 14
<212> PRT
<213> Artificial
<220>
1
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<223> Synthetic Peptide
<400> 2
Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala
1 5 10
<210> 3
<211> 14
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 3
Lys Ala Ala Lys Lys Ala Ala Lys Ala Ala Lys Lys Ala Ala
1 5 10
<210> 4
<211> 21
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 4
Lys Leu Gly Lys Lys Leu Gly Lys Leu Gly Lys Lys Leu Gly Lys Leu
1 5 10 15
Gly Lys Lys Leu Gly
<210> 5
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 5
2
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Gly Arg Arg Ala Gly Gly Ser
1 5
<210> 6
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 6
Thr Arg Arg Ala Gly Gly Gly
1 5
<210> 7
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 7
Ser Arg Ala Gly Gly Leu Gly
1 5
<210> 8
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 8
Ser Tyr Ala Gly Gly Leu Gly
1 5
<210> 9
<211> 7
3
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 9
Asp Val Ala Gly Gly Leu Gly
1 5
<210> 10
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 10
Gly Ala Gly Gly Leu Gly Ala
1 5
<210> 11
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 11
Gly Ala Gly Gly Trp Gly Val
1 5
<210> 12
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 12
4
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Ala Gly Gly Thr Phe Lys Pro
1 5
<210> 13
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 13
Leu Gly Glu Val Ala Gly Gly
1 5
<210> 14
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 14
Gly Ser Asn Asp Ala Gly Gly
1 5
<210> 15
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 15
Tyr Arg Gly Ile Ala Gly Gly
1 5
<210> 16
<211> 7
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 16
Ala Gly Gly Val Ala Gly Gly
1 5
<210> 17
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 17
Gly Gly Leu Ala Gly Gly Phe
1 5
<210> 18
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 18
Leu Leu Ala Gly Gly Val Leu
1 5
<210> 19
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 19
6
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Leu Val Val Ser Ala Gly Gly
1 5
<210> 20
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 20
Arg Thr Gln Ala Gly Gly Val
1 5
<210> 21
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 21
Ala Gly Gly Phe Gly Glu Gln
1 5
<210> 22
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 22
Ala Gly Gly Leu lle Asp Val
1 5
<210> 23
<211> 7
7
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 23
Ala Gly Gly Ser Thr Trp Thr
1 5
<210> 24
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 24
Ala Gly Gly Asp Trp Trp Trp
1 5
<210> 25
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 25
Ala Gly Gly Gly Leu Leu Met
1 5
<210> 26
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 26
8
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Val Ala Ala Gly Gly Gly Leu
1 5
<210> 27
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 27
Leu Tyr Gly Ala Gly Gly Ser
1 5
<210> 28
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 28
Cys Ala Leu Ala Gly Gly Cys
1 5
<210> 29
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 29
lle Gly Ala Gly Gly Val His
1 5
<210> 30
<211> 3
9
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 30
Ala Gly Gly
1
<210> 31
<211> 3
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 31
Glu Gly Arg
1
<210> 32
<211> 3
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 32
Gly Glu Arg
1
<210> 33
<211> 3
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 33
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Gly Val Leu
1
<210> 34
<211> 6
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 34
Arg Arg Ala Gly Gly Ser
1 5
<210> 35
<211> 5
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 35
Arg Arg Ala Gly Gly
1 5
<210> 36
<211> 5
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 36
His Gly Gly Val Gly
1 5
<210> 37
<211> 9
11
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 37
Cys Gly Arg Arg Ala Gly Gly Ser Cys
1 5
<210> 38
<211> 9
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 38
Cys Arg Val Asp Phe Ser Lys Gly Cys
1 5
<210> 39
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 39
Cys Asn Val Ser Asp Lys Ser Cys
1 5
<210> 40
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 40
12
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Cys His Gln Lys Pro Trp Glu Cys
1 5
<210> 41
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 41
Cys Lys Asp Arg Phe Glu Arg Cys
1 5
<210> 42
<211> 654
<212> PRT
<213> Homo Sapiens
<400> 42
Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Ala Ala
1 5 10 15
Arg Ala Glu Glu Glu Asp Lys Lys Glu Asp Val Gly Thr Val Val Gly
20 25 30
Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Phe Lys Asn Gly
35 40 45
Arg Val Glu lle lle Ala Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser
50 55 60
Tyr Val Ala Phe Thr Pro Glu Gly Glu Arg Leu Ile Gly Asp Ala Ala
65 70 75 80
Lys Asn Gln Leu Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala Lys
85 90 95
13
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Arg Leu Ile Gly Arg Thr Trp Asn Asp Pro Ser Val Gln Gln Asp lle
100 105 110
Lys Phe Leu Pro Phe Lys Val Val Glu Lys Lys Thr Lys Pro Tyr Ile
115 120 125
Gln Val Asp Ile Gly Gly Gly Gln Thr Lys Thr Phe Ala Pro Glu Glu
130 135 140
Ile Ser Ala Met Val Leu Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr
145 150 155 160
Leu Gly Lys Lys Val Thr His Ala Val Val Thr Val Pro Ala Tyr Phe
165 170 175
Asn Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Thr Ile Ala Gly
180 185 190
Leu Asn Val Met Arg lle lle Asn Glu Pro Thr Ala Ala Ala Ile Ala
195 200 205
Tyr Gly Leu Asp Lys Arg Glu Gly Glu Lys Asn Ile Leu Val Phe Asp
210 215 220
Leu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Thr Ile Asp Asn Gly
225 230 235 240
Val Phe Glu Val Val Ala Thr Asn Gly Asp Thr His Leu Gly Gly Glu
245 250 255
Asp Phe Asp Gln Arg Val Met Glu His Phe Ile Lys Leu Tyr Lys Lys
260 265 270
Lys Thr Gly Lys Asp Val Arg Lys Asp Asn Arg Ala Val Gln Lys Leu
275 280 285
14
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Arg Arg Glu Val Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln His Gln
290 295 300
Ala Arg Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu Asp Phe Ser Glu
305 310 315 320
Thr Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Met Asp Leu Phe Arg
325 330 335
Ser Thr Met Lys Pro Val Gln Lys Val Leu Glu Asp Ser Asp Leu Lys
340 345 350
Lys Ser Asp Ile Asp Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile
355 360 365
Pro Lys lle Gln Gln Leu Val Lys Glu Phe Phe Asn Gly Lys Glu Pro
370 375 380
Ser Arg Gly Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val
385 390 395 400
Gln Ala Gly Val Leu Ser Gly Asp Gln Asp Thr Gly Asp Leu Val Leu
405 410 415
Leu Asp Val Cys Pro Leu Thr Leu Gly Ile Glu Thr Val Gly Gly Val
420 425 430
Met Thr Lys Leu Ile Pro Arg Asn Thr Val Val Pro Thr Lys Lys Ser
435 440 445
Gln lle Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr Val Thr Ile Lys
450 455 460
Val Tyr Glu Gly Glu Arg Pro Leu Thr Lys Asp Asn His Leu Leu Gly
465 470 475 480
Thr Phe Asp Leu Thr Gly lle Pro Pro Ala Pro Arg Gly Val Pro Gln
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
485 490 495
Ile Glu Val Thr Phe Glu Ile Asp Val Asn Gly Ile Leu Arg Val Thr
500 505 510
Ala Glu Asp Lys Gly Thr Gly Asn Lys Asn Lys Ile Thr Ile Thr Asn
515 520 525
Asp Gln Asn Arg Leu Thr Pro Glu Glu Ile Glu Arg Met Val Asn Asp
530 535 540
Ala Glu Lys Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu Arg Ile Asp
545 550 555 560
Thr Arg Asn Glu Leu Glu Ser Tyr Ala Tyr Ser Leu Lys Asn Gln Ile
565 570 575
Gly Asp Lys Glu Lys Leu Gly Gly Lys Leu Ser Ser Glu Asp Lys Glu
580 585 590
Thr Met Glu Lys Ala Val Glu Glu Lys Ile Glu Trp Leu Glu Ser His
595 600 605
Gln Asp Ala Asp lle Glu Asp Phe Lys Ala Lys Lys Lys Glu Leu Glu
610 615 620
Glu Ile Val Gln Pro Ile Ile Ser Lys Leu Tyr Gly Ser Ala Gly Pro
625 630 635 640
Pro Pro Thr Gly Glu Glu Asp Thr Ala Glu Lys Asp Glu Leu
645 650
<210> 43
<211> 8
<212> PRT
<213> Artificial
<220>
16
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<223> Synthetic Peptide
<400> 43
Cys Asn Trp Thr Asp Lys Thr Cys
1 5
<210> 44
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 44
Cys Asn Ile Thr Gln Lys Ser Cys
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 45
Cys Asn Lys Thr Asp Lys Gly Cys
1 5
<210> 46
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 46
Cys Thr Phe Ala Gly Ser Ser Cys
1 5
17
<210> 43
<211> 8
<212> PRT
<213> A
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 47
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 47
Cys Asn Ser Ala Phe Ala Gly Cys
1 5
<210> 48
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 48
Cys Ser Tyr Thr Phe Ala Gly Cys
1 5
<210> 49
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 49
Cys Ser Thr Phe Ala Gly Ser Cys
1 5
<210> 50
<211> 8
<212> PRT
<213> Artificial
<220>
18
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<223> Synthetic Peptide
<400> 50
Cys Arg Asp Gly Tyr His His Cys
1 5
<210> 51
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 51
Cys Ser Ala Ser Asp Leu Ser Cys
1 5
<210> 52
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 52
Cys Gln Asn Gln Tyr Pro Glu Cys
1 5
<210> 53
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 53
Cys Arg Ala Ser Ala Met Val Cys
1 5
19
<210> 43
<211> 8
<212> PRT
<213> A
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 54
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 54
Cys Ile Asp Met Thr His Gln Cys
1 5
<210> 55
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 55
Cys Ile Ser Ser Pro Ser Asn Cys
1 5
<210> 56
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 56
Cys Asn Gln Ser Met Trp Ser Cys
1 5
<210> 57
<211> 8
<212> PRT
<213> Artificial
<220>
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<223> Synthetic Peptide
<400> 57
Cys Gln Phe Glu Asn Gly Thr Cys
1 5
<210> 58
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 58
Cys Ala Val Lys Ser Val Thr Cys
1 5
<210> 59
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 59
Cys Asn Gly Phe Met Gly Tyr Cys
1 5
<210> 60
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 60
Cys Leu Thr Ser Glu Asn Ala Cys
1 5
21
<210> 43
<211> 8
<212> PRT
<213> A
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 61
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 61
Cys Arg Ala Ser Ala Met Val Cys
1 5
<210> 62
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 62
Cys Ser Lys Lys Phe Val Thr Cys
1 5
<210> 63
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 63
Cys Lys Asn Lys His Thr Thr Cys
1 5
<210> 64
<211> 8
<212> PRT
<213> Artificial
<220>
22
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<223> Synthetic Peptide
<400> 64
Cys Phe Glu Thr Phe Ala Gly Cys
1 5
<210> 65
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 65
Cys Asn Asn Met Tyr Ala Gly Cys
1 5
<210> 66
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 66
Cys Phe Pro Lys Arg Val Thr Cys
1 5
<210> 67
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 67
Cys Pro Arg Ser Ala Lys Asn Cys
1 5
23
<210> 43
<211> 8
<212> PRT
<213> A
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 68
<211> 13
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 68
Cys Gly Phe Glu Cys Val Arg Gln Cys Pro Glu Arg Cys
1 5 10
<210> 69
<211> 10
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 69
Cys Thr Thr His Trp Gly Phe Thr Leu Cys
1 5 10
<210> 70
<211> 9
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 70
Cys Asp Cys Arg Gly Asp Cys Phe Cys
1 5
<210> 71
<211> 5
<212> PRT
<213> Artificial
<220>
24
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<223> Synthetic Peptide
<400> 71
Cys Ala Arg Ala Cys
1 5
<210> 72
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 72
Thr Arg Asn Thr Gly Asn Ile
1 5
<210> 73
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 73
Phe Asp Gly Gln Asp Arg Ser
1 5
<210> 74
<211> 6
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 74
Trp Gly Pro Lys Arg Leu
1 5
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 75
<211> 6
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 75
Trp Gly Glu Ser Arg Leu
1 5
<210> 76
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 76
Val Met Gly Ser Val Thr Gly
1 5
<210> 77
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 77
Lys Gly Gly Arg Ala Lys Asp
1 5
<210> 78
<211> 7
<212> PRT
<213> Artificial
<220>
26
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<223> Synthetic Peptide
<400> 78
Arg Gly Glu Val Leu Trp Ser
1 5
<210> 79
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 79
Thr Arg Glu Val His Arg Ser
1 5
<210> 80
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 80
His Gly Gln Gly Val Arg Pro
1 5
<210> 81
<211> 9
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 81
Cys Lys Gly Gly Arg Ala Lys Asp Cys
1 5
27
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 82
<211> 5
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 82
Lys Ala Arg Gly Gly
1 5
<210> 83
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 83
Tyr Arg Cys Thr Leu Asn Ser Pro Phe Phe Trp Glu Asp Met Thr His
1 5 10 15
Glu Cys His Ala
<210>84
<211>20
<212>PRT
<213>Artificial
<220>
<223> Synthetic Peptide
<400> 84
Leu Gly Cys Met Ala Ser Met Leu Arg Glu Phe Glu Gly Ala Thr His
1 5 10 15
Ala Cys Thr Gln
28
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 85
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 85
Arg Gly Cys Thr Glu Ala Ala Gly Leu Val Ile Gly Ile Thr Thr His
1 5 10 15
Gln Cys Gly Asn
<210> 86
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 86
Ile Gly Cys Asn His Pro Ser Pro Leu Gly Ser Thr Val Val Pro Thr
1 5 10 15
Tyr Cys Phe Lys
<210> 87
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 87
Gly Thr Cys Pro Arg Gln Phe Phe His Met Gln Glu Phe Trp Pro Ser
29
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
1 5 10 15
Asp Cys Ser Arg
<210> 88
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 88
Asp Arg Cys Val Leu Val Arg Pro Glu Phe Gly Arg Gly Asp Ala Arg
1 5 10 15
Leu Cys His Ser
<210> 89
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 89
Glu Gly Cys Ser Asp Ile Met Asn Thr Ala Ala Glu Arg Val Thr Gly
1 5 10 15
Asp Cys Ser Tyr
<210> 90
<211> 20
<212> PRT
<213> Artificial
<220>
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<223> Synthetic Peptide
<400> 90
Val Phe Cys Cys Gly Ser Tyr Cys Gly Gly Val Glu Met Leu Ala Ser
1 5 10 15
Arg Cys Gly His
<210> 91
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 91
Arg Glu Cys Gly Arg Thr Val His Arg Tyr Pro Trp Gly Ser Pro Glu
1 5 10 15
Ser Cys Glu Arg
<210> 92
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 92
Asp Ala Cys Ser Arg Phe Leu Gly Glu Arg Val Asp Ala Thr Ala Ala
1 5 10 15
Gly Cys Ser Arg
<210> 93
31
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 93
Gly Asn Cys Met Gly Leu Gln Val Ser Glu Leu Phe Met Gly Pro Tyr
1 5 10 15
Lys Cys Arg Gln
<210> 94
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 94
Ser Arg Cys His Ala Leu Arg Ser Gln Ser Val Ser Thr Ser Ala Gly
1 5 10 15
Ala Cys Ile Ser
<210> 95
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 95
Tyr Ser Cys Thr Arg Leu Asn Gly Thr Gly Leu Gln Asn Pro Pro Ser
1 5 10 15
32
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Ala Cys Asp Arg
<210> 96
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 96
Trp Val Cys Thr Ser Ala Ser Gln Asp Thr Arg Leu Lys Glu Pro Gly
1 5 10 15
Met Cys lle Ala
<210> 97
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 97
Met His Cys Thr Ser Gln Thr Leu Arg Gly Thr Pro Ser Leu Ala Pro
1 5 10 15
Lys Cys Ser Asp
<210> 98
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 98
33
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Gln His Cys Val Lys Gly Gln Phe Pro Phe Arg Glu Ser Val Thr Ile
1 5 10 15
Thr Cys Asn Ser
<210> 99
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 99
His Thr Cys Trp Gly Ala Arg Asp Val Ala Gln Pro Ser Gly Thr Val
1 5 10 15
Arg Cys Leu Lys
<210> 100
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 100
Ala Arg Cys Arg Glu Asp Thr Gly Phe Met Gly Leu Gly Ser Ala Asn
1 5 10 15
Ile Cys Thr Asp
<210> 101
<211> 20
<212> PRT
<213> Artificial
34
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<220>
<223> Synthetic Peptide
<400> 101
Arg Thr Cys Glu Glu Val Arg Asn Arg Ala Leu Glu Glu Leu Thr Asn
1 5 10 15
Phe Cys Pro Tyr
<210> 102
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 102
Arg Thr Cys Gln Val Arg Ser Asn Asn lle Ser Pro Arg Met Ala Leu
1 5 10 15
Ala Cys Val Thr
<210> 103
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 103
Arg Ser Cys Val Asn Ser Asp Thr Gly Val Leu Gln Arg Gly Ala Pro
1 5 10 15
Ser Cys Leu Phe
35
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 104
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 104
Arg Gly Cys Trp Arg Asp Ser Thr Ala Trp His Val Ser Tyr Pro Val
1 5 10 15
Glu Cys Leu Ala
<210> 105
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 105
Asn Arg Cys Met Pro Gly Phe Leu Asp Asp Ala Asp Ser Ala Ala Ser
1 5 10 15
Pro Cys Gly Ser
<210> 106
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 106
Asn Gln Cys Ser Ser Leu Leu Thr Tyr Gln Gly Trp Lys Arg Thr Lys
1 5 10 15
36
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Asp Cys Ile Pro
<210> 107
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 107
Asn Asp Cys Ser Ala His Ala Gln Pro Gly Trp Asp Glu Val Pro Pro
1 5 10 15
Met Cys Asn Gln
<210> 108
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 108
Asn Asn Cys Pro Val Glu Gly Ser Gln Gln Asn Tyr Ser Gly Ala Thr
1 5 10 15
Trp Cys Arg Ala
<210> 109
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
37
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<400> 109
Thr Thr Cys Asn Lys Ser Met Ser Ser Gln Pro Met Arg Asp Ser Arg
1 5 10 15
Glu Cys His Arg
<210> 110
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 110
Thr Ser Cys Val Arg Thr Gly His Asp Glu Asn Leu Leu Lys Ala Ala
1 5 10 15
Tyr Cys Ser Ser
<210> 111
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 111
Thr Glu Cys Arg Gly Ala Ser Ser Gly Ser Val Ser Gly Ala Ala Thr
1 5 10 15
Asp Cys Arg Asp
<210> 112
<211> 20
38
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 112
Thr Leu Cys Pro Pro Ala Ser Met Gly Leu Gly Arg Glu Lys Pro Arg
1 5 10 15
Leu Cys Ser Val
<210> 113
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 113
Thr Leu Cys Arg Ser Leu Glu His Glu Val Gly Leu Phe Lys Pro Arg
1 5 10 15
Glu Cys Pro Phe
<210> 114
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 114
Leu Arg Cys Pro Leu Glu Val Asp Arg Pro Asn Arg Asp Pro Ala Phe
1 5 10 15
Leu Cys Ser Gln
39
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 115
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 115
Leu Gly Cys Asn Lys Gly Arg Tyr Trp Leu Ser Thr Arg Leu Ser Val
1 5 10 15
Ser Cys Ala Leu
<210> 116
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 116
Val Ala Cys Asp Ile Ser Ala Val Glu Arg Leu Pro Ala Ser Ala Arg
1 5 10 15
Ser Cys Lys Thr
<210> 117
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 117
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Val Val Cys Phe Met Glu Arg Gln Met Gly Thr Asp Val Val Ser Pro
1 5 10 15
Met Cys Val Asn
<210> 118
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 118
Val Glu Cys Val Met Ala Ser Ala Ser Thr Asp Gly Thr Ala Ala His
1 5 10 15
Pro Cys Lys Pro
<210> 119
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 119
Val Arg Cys Asn Glu Ala Gln Leu Gln Asp Ser Gly Thr Val Pro His
1 5 10 15
Pro Cys Leu Arg
<210> 120
<211> 20
<212> PRT
<213> Artificial
41
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<220>
<223> Synthetic Peptide
<400> 120
Pro Asn Cys Asp Leu Asp Asp Ile Val Leu Asn Pro Tyr Thr Ala Gly
1 5 10 15
Pro Cys Gly Thr
<210> 121
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 121
Pro Asn Cys Tyr Ser Gly Asp Gly Glu Ile Ser Ser His Ile Pro Val
1 5 10 15
Gln Cys Leu Met
<210> 122
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 122
Pro Gly Cys Val Val Ser Pro Phe Ala Leu Ser Ala Gln Gly Thr Ser
1 5 10 15
Val Cys Thr lle
42
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<210> 123
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 123
Gly Asp Cys Glu Thr Asn Asn Val Thr Lys Val Gly Gly Ile Thr Arg
1 5 10 15
Asn Cys Val Gly
<210> 124
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 124
Gly Tyr Cys Leu Thr Val Val Gly Gly Ala Val Leu Thr Ile Ala Leu
1 5 10 15
Leu Cys Val Thr
<210> 125
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 125
Gly Pro Cys Ala Ala Thr Gly Val Asn Pro Gly Asp His Gly Ala Ala
1 5 10 15
43
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
Val Cys Asp Gln
<210> 126
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 126
Gly Asp Cys Glu Thr Asn Asn Val Thr Lys Val Gly Gly Ile Thr Arg
1 5 10 15
Asn Cys Val Gly
<210> 127
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 127
Lys Ser Cys Gly Lys Tyr Gly Leu lle Val Gly Gln Pro Phe Ala Glu
1 5 10 15
His Cys Pro Pro
<210> 12~
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
44
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
<400> 12S
Lys Leu Cys Tyr Arg Ser Ser Ala Gly Ser Glu Leu Arg Pro Pro Glu
1 5 10 15
Lys Cys Ala Tyr
<210> 129
<211> 20
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 129
Lys lle Cys Pro Val Thr Asn Met Trp Thr Thr Pro Ser Trp Ala His
1 5 10 15
Lys Cys Gly Met
<210> 130
<211> 29
<212> DNA
<213> Artificial
<220>
<223> Synthetic Oligonucleotide
<400> 130
aggctcgagg atcctcggcc gacggggct 29
<210> 131
<211> 27
<212> DNA
<213> Artificial
<220>
<223> Synthetic Oligonucleotide
<400> 131
CA 02496938 2005-02-23
WO 2004/020999 PCT/US2002/034987
aggtctagaa ttcgccccag cggcccc 2~
<210> 132
<211> 9
<212> PRT
<213> Artificial
<220>
<223> Synthetic Peptide
<400> 132
Cys Val Pro Glu Leu Gly His Glu Cys
1 5
46
