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
AGAINST PLACENTA AND ADIPOSE TISSUES
BACKGROUND OF THE INVENTION
[0001] This application is a continuation in part of PCT application
PCT/US01/27692, filed on September 7, 2001, the entire text of which is
incorporated
herein by reference. This application claims priority from U.S. Provisional
Patent
Application No. 60/231,266 filed September 8, 2000, and U.S. Patent
Application No.
09/765,101, filed January 17, 2001. This invention was made with U.S.
government
support under grants grants CA90270, 1R1CA90810-01 and 1RO1CA82976-01 from the
National Institutes of Health. The U.S. government has certain rights in this
invention.
Field of the Invention
[0002] The present invention concerns the fields of molecular medicine and
targeted
delivery of therapeutic agents. More specifically, the present invention
relates to
compositions and methods for identification and use of peptides that
selectively target
white adipose tissue and placenta in vivo or i.~a vitro. In other embodiments,
the invention
concerns compositions and methods for screening potential teratogenic agents.
Description of Related Art
[0003] Phage display is a technique in which a phage library expresses, for
example,
a set of random peptide sequences of defined length, incorporated into a phage
coat
protein (e.g., Smith and Scott, Science 228:1315-17, 1985; Smith and Scott,
Meth.
Enzymol. 21:228-57, 1993). Peptide sequences that bind to a target molecule,
cell, tissue
or organ may be identified by incubating a phage display library with the
target and
selecting for bound peptides (biopanning) (e.g., Pasqualini and Ruoslahti,
Nature
380:364-66, 1996; Arap et al., SciefZCe 279:377-80, 1998a). Unbound phage may
be
washed away and bound phage eluted and collected. The collected phage may be
amplified and taken through further binding/amplification cycles to enrich the
pool of
peptides for those that selectively and/or specifically bind to the target.
With each cycle,
the proportion of phage in the pool that contain targeting peptides for the
target of
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interest is enriched. After several cycles, individual phage clones may be
characterized
by DNA sequencing to identify the targeting peptide sequences.
[0004] Targeting peptides that exhibit selective and/or specific binding for
placenta
or adipose tissues have not been previously reported in the literature.
Targeting peptides
against placenta or adipose tissues would have a variety of potential uses.
Targeting
peptides against adipose tissue could be used to control obesity and related
conditions.
Adipose-targeting peptides would also be of potential use to treat HIV related
adipose
malformations such as lipodystrophia and/or hyperlipidemia (see, e.g., Zhang
et al., J.
Clin. Endocrin. Metab. 84:4274-77, 1999; Jain et al., Antiviral Res. 51:151-
177, 2001;
Raolin et al., Prog. Lipid Res. 41:27-65, 2002). Targeting peptides against
placental
tissue could be used to reduce harmful effects by tetragenic agents, to
deliver therapeutic
agents to the placenta and/or the fetus and to induce labor or spontaneous
abortion.
Placental receptors identified through the use of placental targeting peptides
could be
used to screen for potential teratogens.
[0005] Presently available methods for control of weight include dieting and
surgical
procedures. These often exhibit adverse effects and may not result in long-
term weight
loss. Dieting includes both popular (fad) diets and the use of weight loss and
appetite
supplements. Fad diets are only good for short-term weight loss and do not
achieve long-
term weight control. They are often unhealthy, since many important nutrients
are
missing from the diet. In addition, rapid weight loss can result in
dehydration. After
losing weight, the dieters typically return to their original eating habits.
This often
results in weight gain that can exceed the subject's weight before dieting (yo
yo effect).
[0006] Appetite suppressants such as Phentermen HCI, Meridia, Xernical, Adipex-
P,
Bontril and Ionomin may have adverse effects, such as addiction, dry mouth,
nausea,
irritability, and constipation. These supplements can also lead to more
serious problems
like eating disorders. Weight control through use of such supplements is
ineffective, with
only limited weight loss achieved. Effective drugs for controlling weight,
such as
fenfluramine, were withdrawn from the market due to cardiotoxicity.
Surgical methods for weight reduction, such as liposuction and gastric bypass
surgery, have many risks. Liposuction removes subcutaneous fat through a
suction tube
inserted into a small incision in the skin. Risks and complications may
include scarring,
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bleeding, infection, change . in skin sensation, pulmonary complications, skin
loss,
chronic pain, etc. In gastric bypass surgery, the patient has to go through
the rest of his
or her life with a drastically altered stomach that can hold just two or three
ounces of
food. Side effects may include nausea, diarrhea, bleeding, infection, bowel
blockage
caused by scar tissue, hernia and adverse reactions to general anesthesia. The
most
serious potential risk is leakage of fluid from the stomach or intestines,
which may result
in abdominal infection and the need for a second surgery. None of the
presently
available methods for weight control is satisfactory and a need exists for
improved
methods of weight loss and control.
Another adipose related disease state is lipodystrophy syndromes) related to
HIV
infection (e.g., Jain et al., Antiviral Res. 51:151-177, 2001). Mortality
rates from HIV
infection have decreased substantially following use of highly active
antiretroviral
therapy (HAART) (Id.) However, treatment with protease inhibitors as part of
the
HAART protocol appears to result in a number of lipid-related symptoms, such
as
hyperlipidemia, fat redistribution with accumulation of abdominal and cervical
fat,
diabetes mellitus and insulin resistance (Jain et al., 2001; Yanovski et al.,
J. Clin.
Endocrin. Metab. 84:1925-1931; Raulin et al., Prog. Lipid Res. 41:27-65,
2002).
Although of minor significance compared to the underlying HIV infection and
possible
development of AIDS related complex (ARC) and/or AIDS, lipodystrophy syndrome
adversely affects quality of life and may be associated with increased risk of
coronary
artery disease, heart attack, stroke and other adverse side affects of
increased blood
lipids. While treatment with metformin, an insulin-sensitizing aget, has been
reported to
provide some alleviation of symptoms (Hadigan et al., J. Amer. Med. Assn.
284:472-
477, 2000), a need exists for more effective methods of treating HIV related
lypodystrophy.
[0007] Teratogens fall into two classes. The first class includes compounds
that are
actively or passively transferred through the materno-fetal barrier. Those
target fetal
development by altering cell-signaling pathways that control essential
processes in the
developing embryo, such as angiogenesis (D'Amato, R.J., et al 1994. Proc.
Natl. Acad.
Sci. USA 91: 4082-4085; Finnell, R.H.1999. J. Allergy Clirz. Inamufzol.
103:337-342).
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[0010] Teratogens of the second class interfere with fetal development by
affecting
the delivery of nutrients to the embryo through the placenta (Maranghi, F., et
al. 1998.
Adv. Exp. Med. Biol. 444: 129-136; Rugh, R. 1990. The Mouse: Its Reproduction
and
Development, Oxford Science Publications, Oxford). Materno-fetal molecule
exchange
occurs by filtration of blood from the maternal to the fetal side of the
placenta through
several distinct cell layers. Teratogens that target the placenta are thought
to function by
blocking receptors required for transport of nutrients to the fetus (Beckman,
D.A., et al.
1990. Teratology 41: 395-404). Present methods of treatment primarily involve
avoiding
exposure of the pregnant woman to teratogens. Such methods are ineffective
where the
mother is unaware of her pregnancy, or for novel teratogens whose effect on
fetal
development have not yet been characterized. Because teratogens are identified
by in
vivo animal testing, differences in placental receptors between humans and
test animals,
such as mice, may result in the failure to identify teratogenic effects until
multiple birth
defects are reported, such as in the thalidomide tragedy. A need exists for
methods of
identifying the placental receptors for teratogens, in order to allow more
accurate
teratogen screening procedures.
SUMMARY OF THE INVENTION
[0011] 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 white adipose tissue or placenta. In some embodiments, the
invention
concerns particular targeting peptides selective or specific for adipose or
placental tissue,
including but not limited to SEQ >D N0:5-11, SEQ ID N0:13-22 and SEQ ID
N0:144.
Other embodiments concern such targeting peptides attached to therapeutic
agents. In
other embodiments, placental, adipose or other targeting peptides may be used
to
selectively or specifically deliver therapeutic agents to target tissues, such
as white
adipose tissue, placenta or fetal tissue. 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 adipose or placenta.
[0012] 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 ID NO:S-11, SEQ )D N0:13-22 and SEQ ID
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N0:144. 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 m
N0:5-11, SEQ m N0:13-22 and SEQ m N0:144.
[0013] 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
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 ll~ NO:1),
(KLAKKLA)2 (SEQ ll~ NO:2), (KAAKKAA)2 (SEQ m NO:3) or (KLGKKLG)3 (SEQ
m 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, SU6668, 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), IL-2,
IL-5, IL-10,
IL.-11, lL-12, IL-18, 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.
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[0014] In various embodiments, targeting peptides attached to one or more
therapeutic agents may be administered to a subject, such as an animal,
mammal, cat,
dog, cow, pig, horse, sheep or human subject. Such administration may be of
use for the
treatment of various disease states. In certain embodiments, adipose-targeting
peptides
attached to a cytocidal, pro-apoptotic, anti-angiogenic or other therapeutic
agent may be
of use in methods to treat obesity, induce weight loss and/or to treat highly
active
antiretroviral therapy (HAART) associated lipodystrophy syndrome. In other
embodiments, placenta-targeting peptides attached to such agents may be used,
for
example, to induce labor or to terminate pregnancy.
[0015] 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
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.
[0016] In another embodiment, the isolated peptide 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.
[0017] 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
N0:5-11, SEQ m N0:13-22 and SEQ m NO:144. In some embodiments, larger
contiguous sequences, up to a full-length sequence selected from any of SEQ m
N0:5-
11, SEQ m N0:13-22 and SEQ m N0:144 may be used.
[0018] 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.
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[0019] Other embodiments concern methods of targeted delivery comprising
selecting a targeting peptide for a desired organ, tissue or cell type,
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 ID N0:5-11, SEQ ID N0:13-22 and SEQ ID
N0:144. In certain preferred embodiments, the organ, tissue or cell type is
white adipose
or placenta. In other preferred embodiments, the molecule attached to the
targeting
peptide is a chemotherapeutic agent, an antigen or an imaging agent.
[0020] 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 baeteriophage. 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.
[0021] Additional embodiments of the present invention concern methods of
treating
a disease state comprising selecting a targeting 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 targeting peptide includes at least three contiguous amino acids selected
from any of
selected from any of SEQ ID N0:5-11, SEQ ID N0:13-22 and SEQ ID N0:144. In
preferred embodiments the disease state is obesity, lipodystrophy or a related
condition.
[0022] 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
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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.
[0023] In other embodiments, phage that bind to a target organ, tissue or cell
type,
for example to adipose tissue or placenta, may be pre-screened or post-
screened against a
subject lacking that organ, tissue or cell type. Phage that bind to the
subject lacking the
target organ, tissue or cell type are removed from the library prior to
screening in
subjects possessing the organ, tissue or cell type.
[0024] 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.
[0025] 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 antigens are collected. In
more preferred
embodiments, the antigen is a targeting peptide.
[0026] 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 a
placental
receptor for teratogens. In some embodiments, the placental teratogen
receptors)
identified may be used for screening of potential teratogens for receptor
binding.
[0027] 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 N0:5-11, SEQ n7 N0:13-22
and SEQ m N0:144. In other preferred embodiments, the targeting peptide may
comprise a portion of an antibody against the receptor.
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[0028] 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 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.
[0029] 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.
[0030] 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 in vivo or in vitro. A naturally occun~ing 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.
[0031] 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
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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 white adipose tissue or placenta.
[0032] Certain embodiments concern methods of obtaining antibodies against an
antigen. In preferred embodiments, the antigen comprises one or more targeting
peptides. The targeting peptides are prepared and immobilized on a solid
support,
serum-containing antibodies is added and antibodies that bind to the targeting
peptides
are collected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] 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.
[0034] FIG. 1. Validation of placenta homing phage. Phage bearing targeting
peptides were injected into pregnant mice and their recovery from placenta was
compared to control fd-tet phage without targeting sequences. The placenta
homing
phage clones were: PA - TPKTSVT (SEQ m N0:5), PC - RAPGGVR (SEQ m N0:7),
PE - LGLRSVG (SEQ m N0:10), PF - YIRPFTL (SEQ m NO:9).
[0035] FIG. 2. 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. 3. In viva homing of the TPKTSVT (SEQ m N0:5) motif to the mouse
vinous yolk sac (vys). (A) and (B) anti-phage immunohistochemistry; (C) and
(D) anti-
GST immunohistochemistry; (E) and (F) FITC immunofluorescence in paraffin
sections
of placentas from 18 dpc (days post-conception) pregnant mice injected
intravenously 6
h prior to tissue processing with: (A) control insertless phage, (B) TPKTSVT
(SEQ m
N0:5) phage, (C) control GST peptide, (D) TPKTSVT (SEQ m N0:5) linked to GST
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peptide, (E) control FITC-peptide, or (F) TPKTSVT (SEQ ID N0:5) linked to FITC
peptide. Homing of the TPKTSVT (SEQ ID N0:5) peptide to the vys (dark arrows,
D)
and translocation to the embryonic capillaries (light arrow, D) are indicated.
Only the
vys is shown in (C -F). Bar:100 ~,m (A -B); 20 ~.m (C -F).
FIG. 4. The TPKTSVT (SEQ ID N0:5) peptide specifically binds a placental
transporter. (A) Recovery of indicated phage from embryos carried by 18 dpc
pregnant
mice intravenously injected (tail vein) with 101° TU of the indicated
phage 6 h prior to
phage recovery or immunohistochemistry. Control phage (light cross-hatch)
showed no
selective targeting of placenta. TPKTSVT (SEQ ID N0:5) phage were administered
alone (dark cross-hatched) or co-administered with control-GST (black) or
TPKTSVT
(SEQ ID NO:S) linked to GST (white bar). Shown are mean +/- SEM (standard
error)
from different embryos. (B -D) Anti-phage HRP immunohistochemistry
(arrowheads) in
paraffin sections of the vys from the corresponding mice, as indicated.
Asterisks mark
embryonic capillaries. Bar:20 ~,m.
FIG. 5. The TPKTSVT (SEQ ~ NO:S) peptide blocks placental IgG
transcytosis. A 18 dpc pregnant mouse was intravenously injected with: (A) 100
mg of
control GST fusion or (B) TPKTSVT (SEQ ID N0:5) linked to GST. The IgG
distribution in the placenta after 6 h of peptide circulation was detected by
anti-mouse
IgG immunohistochemistry in paraffin sections. Strong immunostaining is noted
in the
vys of mice injected with a control GST fusion (arrowheads) and in the
labyrinth of wild-
type mice injected with either TPKTSVT (SEQ ID N0:5) linked to GST or control
GST
fusion (asterisks), but not in the vys of TPKTSVT (SEQ ID N0:5) linked to GST
fusion.
Bar: 100 ~,m. (C) and (D) A hypothetical model for the TPKTSVT (SEQ ID N0:5)
peptide function. (C) Normally, the FcRn/(32m complex transports IgG from
maternal
circulation through the labyrinth layer and then the yolk sac placenta (vys)
and into the
embryo. (D) Targeting of the FcRn/(32m with TPKTSVT (SEQ ID N0:5) peptide can
block the receptor complex and the transport of IgG or phage into the embryo.
FIG 6. Placental targeting by the TPKTSVT (SEQ ID NO:S) peptide requires a
functional FcRn/(3~m receptor complex. (A) Relative phage recovery (placenta
TU/ml to
blood TU/ml ratio) from placentas derived from 18 dpc pregnant wild-type or
(3~m-
deficient mice injected with 101° TU of control insertless phage
(black) or TPKTSVT
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(SEQ ID N0:5) phage (white) 6 h prior to phage recovery or
immunohistochemistry.
Shown are mean +/- SEM from individual placentas. (B) and (C) Anti-phage
immunohistochemistry in paraffin sections of the placenta from 18 dpc pregnant
(32m-
null mice intravenously injected with (B) TPKTSVT (SEQ ID N0:5) phage or (C)
control placenta-homing phage displaying the YIRPFTL (SEQ m NO:9) peptide 6 h
prior to tissue processing. Staining of a placenta-homing phage displaying the
unrelated
peptide YIRPFTL (SEQ ID N0:9) is detected in the labyrinth blood vessels
(arrowheads). Bar:100 ~,m.
FIG. 7. TPKTSVT (SEQ ID N0:5) peptide inhibits mouse pregnancy and is
teratogenic. (A) Pregnancy courses (representative from 5 independent
experiments) in
mice subcutaneously injected with the indicated phage or peptides were
monitored by
daily weighing of the mice upon each injection (arrows). (B) Appearance of a
normally
developed 20 dpc embryo (left) for control GST fusion treatment compared to a
representative 20 dpc embryo (right) resulting from TPKTSVT (SEQ ID NO:S)
linked to
GST fusion treatment. A severely teratogenic phenotype is observed with the
placental
targeting fusion peptide. (C) and (D) Hematoxylin/eosin staining of 20 dpc
paraffin-
embedded placentas derived from mice injected for 7 days with (C) control GST
fusion
or (D) the TPKTSVT (SEQ ID N0:5) linked to GST fusion peptide. Note hemorrhage
(black asterisks), necrosis (white asterisks), and fibrosis (arrowheads).
Bar:500 ~,m (50
p,m for insets).
FIG. 8. Irz vivo fat homing of the CKGGRAKDC (SEQ ID N0:22) 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-FITC 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 m
N0:22)
phage, (B) control insertless phage, (C) CKGGRAKDC (SEQ m N0:22) linked to
FITC
peptide, or (D) control CARAC (SEQ m N0:12) linked to FITC peptide. Homing of
the
CKGGRAKDC (SEQ m N0:22) peptide to fat blood vessels (arrows) and its uptake
by
fat endothelium are indicated. Bar: 10 ~.m.
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[0036] FIG. 9. In vivo fat homing of the CKGGRAKDC (SEQ m N0:22) 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 ID N0:22) linked
to FITC peptide and lectin-rhodamine; or (E) and (F) control CARAC (SEQ ID
N0:12)
linked to FTTC peptide and lectin-rhodamine. (B), (D) and (F) Arrows show
endothelium marked with lectin. (A) Arrows show homing of the CKGGRAKDC (SEQ
m NO:22) peptide to fat endothelium. Bar: 10 ~,m.
FIG. 10. 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
which
body mass measurement is not shown). Error bars are SEM for the measurements
in
three mice.
[0037] FIG. 11. 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 c57b116 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 ~ N0:22) linked to (KLAKLAK)2 (SEQ ID NO:1) (left column),
CARAC (SEQ ID N0:12) linked to (KLAKLAK)2 (SEQ ID NO:1) (middle column), and
CKGGRAKDC (SEQ ID NO:22) co-administered with (KLAKLAK)Z (SEQ ID N0:1)
(right column).
[0038] FIG. 12. 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)
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Mice were treated with CKGGRAKDC (SEQ ID N0:22) linked to (KLAKLAK)2 (SEQ m
N0:1). (D) Mice were treated with CARAC (SEQ m N0:12) linked to (KLAKLAK)Z
(SEQ m NO:1). Apoptosis (arrows, (A)) and necrosis/lymphocyte infiltration
(arrows,
(C)) in response to CKGGRAKDC (SEQ m NO:22) linked to (KLAKLAK)2 (SEQ >D
NO:1) treatment are indicated. Bar: 10 Vim.
[0039] FIG. 13. Spleen targeting an vitro using BRASIL. Binding of Fab clones
#2,
#6, #10, #12 and control Fab clone NPC-3TT were directly compared to each
other.
[0040] FIG. 14. Spleen targeting in vivo using BRASIL. Binding of Fab clones
#2,
#6, #10 and #12 to spleen tissue was compared to binding of Fab control clone
NPC-
3TT.
[0041] FIG. 15. Spleen targeting in vivo using BRASIL. Binding of Fab clones
#2,
#6, #10, #12 was compared to binding of Fd-tet phage.
[0042] FIG. 16. Spleen targeting in vivo using BRASIL. Binding of Fab clone
#10
to spleen tissue was compared to binding of Fab control clone NPC-3TT and Fd-
tet
phage.
[0043] FIG. 17. Binding of Fab clone #10 to spleen versus bone marrow in
comparison to Fd-tet phage.
FIG. 18. Binding of 133 cytoplasmic domain-selected phage to immobilized
proteins. GST fusion proteins or GST alone were coated on microtiter wells at
10 ~,g/ml
and used to bind phage-expressing endostatin targeting peptides. Each phage is
identified
by the peptide sequence it displayed: GLDTYRGSP (SEQ ID N0:30); YDWWYPWSW
(SEQ >D N0:29); CLRQSYSYNC (SEQ m N0:38); SDNRYIGSW (SEQ )D NO:31);
CEQRQTQEGC (SEQ ID N0:27); CFQNRC (SEQ m N0:36). The data represent the
mean colony counts from triplicate wells, with standard error of less than 10%
of the
mean.
[0044] FIG. 19. Binding of 135 cytoplasmic domain-selected phage to
immobilized
proteins. GST fusion proteins or GST alone were coated on microtiter wells at
10 ,ug/ml
and used to bind phage-expressing endostatin binding peptides. Each phage is
identified
by the peptide sequence it displayed: (A) DEEGYYNEVIR (SEQ m N0:44); (B)
KQFSYRYLL (SEQ ID N0:45); (C) CEPYWDGWFC (SEQ ID N0:40); (D)
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VVISYSMPD (SEQ ID N0:46); and (E) CYIWPDSGLC (SEQ ID N0:39). The data
represent the mean colony counts from triplicate wells, with standard error
less than 10%
of the mean.
[0045] FIG. 20. Binding of the cytoplasmic-domain binding phage to 133
immobilized protein and inhibition with the synthetic peptide. Phage were
incubated on
wells coated with GST-133cyto in the presence of increasing concentrations of
the
corresponding synthetic peptide or a control peptide. The data represent the
mean colony
counts from triplicate wells, with standard error less than 10% of the mean.
[0046] FIG. 21. Binding of the cytoplasmic-domain binding phage to 135
immobilized protein and inhibition with the synthetic peptide. Phage were
incubated on
wells coated with GST-135cyto in the presence of increasing concentrations of
the
corresponding synthetic peptide or a control peptide. The data represent the
mean colony
counts from triplicate wells, with standard error less than 10% of the mean.
[0047] FIG. 22. Binding of phage to immobilized l33-GST and (35-GST after
phosphorylation. Phage were phosphorylated with Fyn kinase. Insertless phage
were
used as a control. Phage were incubated on wells coated with GST-133cyto or
GST-
133cyto. The data represent the mean colony counts from triplicate wells, with
standard
error less than 10% of the mean.
[0048] FIG. 23. Binding of phage to immobilized GST fusion proteins after
phosphorylation. Phages were phosphorylated with Fyn kinase. Insertless phage
was
used as a control. Phage were incubated on wells coated with GST-cytoplasmic
domains. The data represent the mean of colony counts from triplicate wells,
with
standard error less than 10% of the mean.
[0049] FIG. 24. Effect of integrin cytoplasmic domain binding peptides on cell
proliferation. Serum-deprived cells were cultured for 24 h. and the
proliferation was
determined by [3H] thymidine (l,uCi/ml) uptake measurements. In a positive
control,
VEGF was added back to serum-starved cells. Each experiment was performed
three
times with triplicates, and the results were expressed as the mean +/- SD.
[0050] FIG. 25. Effect of penetratin peptide chimeras on endothelial cell
migration.
Cell migration assay were performed in a 48-well microchemotaxis chamber. Five
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random high-power fields (magnitude 40~e) were counted in each well. The
results show
that both (33-integrin cytoplasmic domain-binding peptides (Y-18 and TYR-11)
increase
cell migration while penetratin does not affect the cells.
[0051] FIG. 26. Penetratin peptide chimera binding to the 135 cytoplasmic
domain
induces programmed cell death. 106 IiiJVEC cells were harvested in complete
media and
15 ,uM penetratin peptide chimeras were added to the cells. After four, eight
and twelve
hours the cells were stained with Propidium Iodide (PI) and induction of
apoptosis was
analyzed by cytometric analysis. (a) Profile obtained with starved cells after
24 h. (b)
Confluent cells in complete media. (c) 15 ~,M of penetratin after four hours.
(d) 15~,M of
VISY-penetratin chimera after four hours. Cells analyzed after eight and
twelve hours
showed similar profiles for the percentage of G~/Gl.
[0052] FIG. 27. Specificity of the antibodies raised against 133- or 135-
selected phage
(ELISA). Increasing dilutions of sera obtained after three immunizations with
GLDTYRGSP (SEQ ID N0:30) or SDNRYIGSW (SEQ m NO:31) conjugated to KLH
were incubated on microtiter wells coated with 10 ~,g of SDNRYIGSW (SEQ m
N0:31,
Y-18), GLDTYRGSP (SEQ ID N0:30, TYR-11) or control peptides. Preimmune sera
were used as controls. After incubation with HRP-goat anti-rabbit, OD was
measured at
405 nm. The data represent the means from triplicate wells, with standard
error less than
10%.
[0053] FIG. 28. Specificity of the antibodies raised against 133- or 135-
selected phage
(ELISA). Sera obtained after three immunizations with SDNRYIGSW (SEQ >D N0:31,
Y-18) or GLDTYRGSP (SEQ ID NO:30, TYR-11) conjugated to I~LH were incubated
in microtiter wells coated with 10 ~,g of TYR-11 or Y-18. GLDTYRGSP (SEQ m
N0:30) or SDNRYIGSW (SEQ ID N0:31) and control peptides were added in
solution.
After incubation with HRP goat anti-rabbit, OD was measured at 405 nm. The
data
represent the means from triplicate wells, with standard error less than 10%.
Peptides
added in solution specifically block the reactivity with the immobilized
peptides.
[0054] FIG. 29A. Competitive binding of Annexin V to (35 integrin with VISY
peptide. Binding assays were performed by ELISA.
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[0055] FIG. 29B. Relative levels of binding of anti-Annexin V antibody to
purified
Annexin V protein and VISY peptide.
[0056] FIG. 30. Chimeric peptide containing VISY peptide linked to penetratin
(antennapedia) induces apoptosis. VISY induced apoptosis was inhibited by
addition of
a caspase inhibitor (zVAD).
[0057] FIG. 31. APA-binding phage specifically bind tumors. Equal amounts of
phage were injected into the tail veins of mice bearing MDA-MB-435-derived
tumors
and phage were recovered after perfusion. Mean values for phage recovered from
the
tumor or control tissue (brain) and,the standard error from triplicate
platings are shown.
[0058] FIG. 32. CPRECESIC (SEQ ID N0:56) is a specific inhibitor of APA
activity. APA enzyme activity was assayed in the presence of increasing
concentrations
of either GACVRLSACGA (SEQ ID N0:57) (control) or CPRECESIC (SEQ ID N0:56)
peptide. The ICSO for APA inhibition by CPRECESIC (SEQ ID N0:56) was estimated
at
800 ~,M. Error bars are the standard error of the means of triplicate wells.
The
experiment was repeated three times with similar results.
[0059] FIG. 33. CPRECESIC (SEQ ID NO:56) inhibits HCTVEC migration.
HUVECs were stimulated with VEGF-A (10 ng/ml). The assay was performed in a
Boyden microchemotaxis chamber, and cells were allowed to migrate through an 8-
~,m
pore filter for 5 h at 37°C. GACVRLSACGA (SEQ ID N0:57) (control) and
CPRECESIC (SEQ ID N0:56) peptides were tested at 1 mM concentration. Migrated
cells were stained and five high-power fields (magnitude 100x) for each
microwell were
counted. Error bars are the standard error of the means of triplicate
microwells.
[0060] FIG. 34. CPRECESIC (SEQ ID NO:56) inhibits IiLJVEC proliferation.
Cells were stimulated with VEGF-A (10 ng/ml), and growth was evaluated at the
indicated times by a colorimetric assay based on crystal violet staining.
Error bars are the
standard error of the means of triplicate wells. Each experiment was repeated
at least
twice with similar results.
[0061] FIG. 35. Protocol for ira vivo biopanning for phage targeted in mouse
pancreas, kidneys, liver, lungs and adrenal gland.
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[0062] FIG. 36. Protocol for recovery of phage by infection of E. coli or
recovery
of phage DNA by amplification and subcloning.
[0063] FIG. 37. Pancreatic islet targeting peptides and homologous proteins.
Candidate endogenous proteins mimicked by the pancreatic islet targeting
peptides
CVSNPRWKC (SEQ ID N0:131), CVPRRWDVC (SEQ ID N0:128), CQHTSGRGC
(SEQ ID N0:129) and CRARGWLLC (SEQ ID N0:130), identified by standard
homology searches.
[0064] FIG. 38. Pancreatic islet targeting peptides and homologous proteins.
Candidate endogenous proteins mimicked by the pancreatic islet targeting
peptides
CGGVHALRC (SEQ ID N0:98), CFNRTWIGC (SEQ ID N0:132) and CWSRQGGC
(SEQ ID N0:134, identified by standard homology searches.
[0065] FIG. 39. Pancreatic islet targeting peptides and homologous proteins.
Candidate endogenous proteins mimicked by the pancreatic islet targeting
peptides
CLASGMDAC (SEQ ID N0:138), CHDERTGRC (SEQ m N0:139), CAHHALMEC
(SEQ ID NO:140) and CMQGARTSC (SEQ ID N0:142), identified by standard
homology searches.
[0066] FIG. 40. Pancreatic islet targeting peptides and homologous proteins.
Candidate endogenous proteins mimicked by the pancreatic islet targeting
peptides
CHVLWSTRC (SEQ ID NO:135), CMSSPGVAC (SEQ ID N0:137) and
CLGLLMAGC (SEQ ID N0:136), identified by standard homology searches.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0067] 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.
[0068] 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
displayed on the outer surface of a phage. Administration to a subject of a
library of
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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.
[0069] 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 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.
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[0070] 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.
[0071] 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
lurnenal
surface of cells forming blood vessels within a target organ, tissue or cell
type.
[0072] 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.
Phage Display
[0073] Recently, an in 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, JIB, 1985. Scie~zce, 228:1315-1317, Smith, GP and Scott, JIB, 1993.
Meth.
Erzzyjj2ol. 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 10~ 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).
[0074] Intravenous administration of phage display libraries to mice was
followed by
the recovery of phage from individual organs (Pasqualini and Ruoslahti, 1996).
Phage
CA 02458047 2004-02-19
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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, J. Clin. Ifzvest. 102:430-437; Rajotte et al, 1999, J. Biol. Chefn.
274:11593-11598;
I~oivunen et al., 1999a, Nature Biotechnol. 17: 768-774; Burg M, et al., 1999,
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 Biotechf2ol. 15:510, 1997; Folkman J. Nature Med 1:27-31, 1995).
Tumor-
homing peptides bound to receptors that were upregulated in the tumor
angiogenic
vasculature of mice (Brooks, P.C., et al. Cell 79:1157-1164, 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;
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).
[0075] 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 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., Nature Med 9:1032-1038, 1999).
[0076] The methods described herein for identification of targeting peptides
involve
the in 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 potential range of applications for this
technique is
quite broad, and the past decade has seen considerable progress in the
construction of
phage-displayed peptide libraries and in the development of screening methods
in which
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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 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).
[0077] 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).
[0078] In certain embodiments, a subtraction protocol is used with 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
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alternative embodiments, the phage library may be prescreened against a
subject who
does not possess the targeted cell, tissue or organ. For example, placenta-
binding
peptides may be identified after prescreening a library against a male or non-
pregnant
female subject After subtraction the library may be screened against 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.
[0079] 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.
[0080] Previous ifs vivo selection studies performed in mice preferentially
employed
libraries of random peptides expressed as fusion proteins with the gene ffI
capsule
protein in the fIJSE5 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 ifa
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$-10~ transducing units
(T.U.)/ml. In
certain embodiments, a bulk amplification strategy is applied between each
round of
selection.
[0081] 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
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 .
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Identi, fication of homing peptides and receptors by i~ vivo phage display is2
mice.
[0082] In vivo selection of peptides from phage-display peptide libraries
administered to mice has been used to identify targeting peptides selective
for normal
mouse brain, 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 means of identifying peptides that
home to the
angiogenic vasculature of tumors has been devised, as described below. 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 shown to home to
the
vasculature of tumor xenografts in nude mice (Arap et al., 1998a, 1998b;
Pasqualini et
al., Nature Bioteclznol 15: 542-546, 1997).
[0083] The receptors for the tumor homing RGD4C targeting peptide has been
identified as ocv integrins (Pasqualini et al., 1997). The av integrins play
an important
role in angiogenesis. The ocv(33 and ocv~35 integrins are absent or expressed
at low levels
in normal endothelial cells but are induced in angiogenic vasculature of
tumors (Brooks
PC, Clark RA, Cheresh DA. Science, 264: 569-571, 1994, 1994; Hammes HP,
Brownlee
M, Jonczyk A, Sutter A, and Preissner KT. Nature Med. 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. Cafacer 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.
[0084] 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
24
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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 HIJVECs (data not shown). The angiogenic receptors were
not
detected in the vasculature of normal organs of mouse or human tissues.
[0085] 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/MMP-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).
[0086] The markers of angiogenic endothelium include receptors for vascular
growth
factors, such as specific subtypes of VEGF and basic FGF receptors, and ocv
integrins,
among many others (Mustonen T and Alitalo K. J. Cell Bi.ol. 129:895-898,
1995.). Thus
far, identification and isolation of novel molecules characteristic of
angiogenic
vasculature has been slow, mainly because endothelial cells undergo dramatic
phenotypic changes when grown in culture (Watson et al., Sciefzce, 268:447-
448, 1995).
[0087] 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., Caf2cer Immuhol. Immunot7ier. 45:149-151, 1997c) and CD13 is a
receptor for
coronaviruses (Look et al. N. J. Claf2. hZVest. 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 ih vivo phage
display will have
cell internalization capability, a key feature for utilizing the identified
peptide motifs as
targeted gene therapy carriers.
Targeted delivery
[0088] 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
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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 used
for tumor
targeted gene therapy (Arap et al., 1998b).
BRASIL
[0089] 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/US01/28124 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 BRASIL (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.
Preparation of large scale primary libraries
[0090] In certain embodiments, primacy 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 fCTSES. The vector is
transformed into
pilus negative host E. c~li. 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.
[0091] 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.
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[0100] For amplification, 1.5 ml of frozen bacteria are inoculated into 5
liters of LB
medium with 20 ~.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.
[0101] 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
[0102] 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/LJSOl/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.
[0103] 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.
[0104] 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.
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[0105] 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.
Polyorgan targeting
[0106] 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.
[0107] 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.
[0108] 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
[0109] 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 to a full
length sequence translated from a gene; a polypeptide of greater than about
100 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.
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[0110] 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.
[0111] 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 rnay 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.
[0112] 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
Abbr.Amino Acid Abbr. Amino Acid
Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine
Baad 3- Aminoadi is acid Hyl Hydroxylysine
Bala (3-alanine, (3-Amino-propionicAHyI allo-Hydroxylysine
acid
Abu 2-Aminobutyric acid 3Hy 3-Hydroxyproline
4Abu 4- Aminobutyric acid, piperidinic4Hyp 4-Hydroxyproline
acid
Acp 6-Aminocaproic acid Ide Isodesmosine
Ahe 2-Aminoheptanoic acid AIIe alto-Isoleucine
Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,
sarcosine
Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine
Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine
Dbu 2,4-Diaminobutyric acid MeVal N-Methylvaline
Des Desmosine Nva Norvaline
Dpm 2,2'-Diamino imelic acid Nle Norleucine
Dpr 2,3-Diaminopropionic acid Orn Ornithine
EtGlyN-Ethyl lycine
[0113] 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:l/www.ncbi.nlm.nih.~ov/). 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.
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Peptide niimetics
[0114] Another embodiment for the preparation of polypeptides according to the
invention is the use of peptide mimetics. Mimetics are peptide-containing
molecules 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 mimeties 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
[0115] 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 immunologically 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
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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, by
chemical
attachment using bifunctional cross-linking reagents, by de ~zovo 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 purij~catio~a
[0116] 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 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).
[0117] 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°Io,
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about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins
in the
composition.
[0118] 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.
[0119] 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.
[0120] 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
canon-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.
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[0121] 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
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
[0122] 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, Science, 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
[0123] 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
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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
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 (KLH) or bovine serum albumin (BSA).
[0124] 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')Z,
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 LaboratorX Manual, Cold Spring
Harbor
Laboratory, 1988; incorporated herein by reference).
Cytokines and claemokines
[0125] 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, chemokines, pro-
apoptosis
factors and anti-angiogenic factors. The term "cytokine" is a generic term for
proteins
released by one cell population that act on another cell as intercellular
mediators.
[0126] Examples of such 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 (FSH),
thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); 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-cc, -.(3, and -'y; colony stimulating factors (CSFs) such as
macrophage-CSF (M-
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CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, 1L-l.alpha., IL-2, IL-3, 1L,-4, IL-5, IL-6,
IL-7, IL-8, IL-9,
IL,-10, IL-11, IL-12; IL-13, IL,-14, IL-15, IL.-16, IL,-17, IL.-18, 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.
[0127] 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, M1P1-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.
Imaging agents ahd radioisotopes
[0128] 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. 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.
[0129] Non-limiting examples of paramagnetic ions of potential use as imaging
agents include chromium (III), manganese (I>), iron (III), iron (II), cobalt
(II), nickel (II),
copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium
(III, vanadium
(II), terbium (III), dysprosium (la), holmium (III) and erbium (III), with
gadolinium
being particularly preferred. Ions useful in other contexts, such as X-ray
imaging,
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include but are not limited to lanthanum (III), gold (~, lead (II), and
especially bismuth
(
[0130] Radioisotopes of potential use as imaging or therapeutic agents include
astatine2y l4carbon, Slchromium, 36chlorine, 57cobalt, SBCObalt, copper67,
isaEu,
gallium~7, 3hydrogen, iodine123, iodinel2s, iodinel3y indiums, S~iron,
32phosphorus,
rheniumls~, rheniumlg8, 7sselenium, 35sulphur, technicium~~m and
yttrium°. lass is often
being preferred for use in certain embodiments, and technicium~~m and indiums
are also
often preferred due to their low energy and suitability for long range
detection.
[0131] 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-
9~m by
ligand exchange process, for example, by reducing pertechnate with stannous
solution,
chelating 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 SNC12, 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.
[0132] 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.
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Cross-linkers
[0133] Bifunctional cross-linking reagents have been extensively used for a
variety
of purposes including preparation of affinity matrices, modification and
stabilization of
diverse structures, identification of ligand and receptor binding sites, and
structural
studies. Homobifunctional reagents that carry two identical functional groups
proved to
be highly efficient in inducing cross-linking between identical and different
macromolecules or subunits of a macromolecule, and linking of polypeptide
ligands to
their specific binding sites. 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. A majority of heterobifunctional cross-linking
reagents
contains a primary amine-reactive group and a thiol-reactive group.
[0134] Exemplary methods for cross-linking ligands to liposomes are described
in
U.S. Patent 5,603,872 and U.S. Patent 5,401,511, each specifically
incorporated herein
by reference in its entirety). Various ligands can be covalently bound to
liposomal
surfaces through the cross-linking of amine residues. Liposomes, in
particular,
multilamellar vesicles (MLV) or unilamellar vesicles such as microemulsified
liposomes
(MEL) and large unilamellar liposomes (LUVET), each containing
phosphatidylethanolamine (PE), have been prepared by established procedures.
The
inclusion of PE in the liposome provides an active functional residue, a
primary amine,
on the liposomal surface for cross-linking purposes. Ligands such as epidermal
growth
factor (EGF) have been successfully linked with PE-liposomes. Ligands are
bound
covalently to discrete sites on the liposome surfaces. The number and surface
density of
these sites are dictated by the liposome formulation and the liposome type.
The
liposomal surfaces may also have sites for non-covalent association. To form
covalent
conjugates of ligands and liposomes, cross-linking reagents have been studied
for
effectiveness and biocompatibility. Cross-linking reagents include
glutaraldehyde
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(GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE),
and a
water soluble carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide
(EDC). Through the complex chemistry of cross-linking, linkage of the amine
residues
of the recognizing substance and liposomes is established.
[0135] In another example, heterobifunctional cross-linking reagents and
methods of
using the cross-linking reagents are described (U.S. Patent 5,889,155,
specifically
incorporated herein by reference in its entirety). 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.
Nucleic Acids
[0136] Nucleic acids according to the present invention 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."
[0137] 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.
[0138] 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.
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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 GAA GAG
Phenylalanine Phe F UUC UUU
Glycine Gly G GGA GGC GGG GGU
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
[0139] In addition
to nucleic acids
encoding the desired
peptide or protein,
the
present invention
encompasses complementary
nucleic acids that
hybridize under
high
stringency conditionssuch coding
with nucleic
acid sequences.
High stringency
conditions for nucleichybridization
acid are well
known
in the
art. For
example,
conditions may comprisesalt and/or
low high temperature
conditions,
such as
provided
by
about 0.02 M to
about 0.15 M NaCI
at temperatures
of about 50C to
about 70C. 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 de, tetramethylammonium
of formami chloride
or other
solvents)
in a
hybridization mixture.
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Vectors for Cloning, Gene Transfer and Expression
[0140] 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 enhancerslpromoters 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
[0141] 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 polymerase initiation and expression of the gene.
[0142] 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.
[0143] 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 well-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.
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[0144] 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
[0145] In certain embodiments of the invention, the cells containing nucleic
acid
constructs of the present invention may be identified in 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~a Vectors
[0146] 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 clofZing vectors and their-
uses, Rodriguez
and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988.; Baichwal and
Sugden,
Baichwal, Irz: Gef2e Transfer, I~ucherlapati R, ed., New York, Plenum Press,
pp. 117-148,
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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.
[0147] 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.
[0148] 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).
[0149] One of the preferred methods for ifz vivo delivery involves the use of
an
adenovirus expression vector. Although adenovirus vectors are known to 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.
[0150] Generation and propagation of adenovirus vectors that are replication
deficient depend on a unique helper cell line, designated 293, which is
transformed from
human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses
E1
proteins (Graham et al., J. Gef2. Virol., 36:59-72, 1977.). Since the E3
region is
dispensable from the adenovirus genome (Jones and Shenk, Cell, 13:181-188,
1978), the
current adenovirus vectors, with the help of 293 cells, carry foreign DNA in
either the
E1, the E3, or both regions (Graham and Prevec, ha: Methods in Molecular
Biology:
Gefte Transfer afzd Expressiof2 Protocol, E.J. Murray, ed., Humana Press,
Clifton, NJ,
7:109-128, 1991.).
[0151] 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.,
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CA 02458047 2004-02-19
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Vero cells or other monkey embryonic mesenchymal or epithelial cells. As
discussed,
the preferred helper cell line is 293. Racher et al., (Biotechnol. Tech. 9:169-
174, 1995)
disclosed improved methods for culturing 293 cells and propagating adenovirus.
[0152] Adenovirus vectors have been used in eukaryotic gene expression
(Levrero et
al., Gene, 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, In: 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.
Aead. Sci. ZISA, 90:2812-2816, 1993) and stereotactic innoculation into the
brain (Le Gal
La Salle et al., Science, 259:988-990,1993).
[0153] Other gene transfer vectors may be constructed from retroviruses.
(Coffin, I32:
Virology, Fields et al., eds., Raven Press, New York, pp. 1437-1500, 1990.)
The retroviral
genome contains three genes, gag, pol, and env. 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' 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).
[0154] 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 env 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
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CA 02458047 2004-02-19
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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., Viz°ology, 67:242-248, 1975).
[0155] 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).
[0156] 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.;
Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987.; Rippe et al., Mol. Cell
Biol. 10:
689-695, 1990; DEAE 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,
T. Biol.
Chem. 262:4429-4432, 1987; Wu and Wu, Biochezyzistry, 27:887-892, 1988). Some
of
these techniques may be successfully adapted for izz vivo or ex vivo use.
[0157] 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., (Gezze, 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
CA 02458047 2004-02-19
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Euzymol., 149:157-176, 1987.) accomplished successful liposome-mediated gene
transfer in rats after intravenous injection.
Pharmaceutical compositions
[0158] Where clinical applications are contemplated, it may be necessary to
prepare
pharmaceutical compositions - expression vectors, virus stocks, proteins,
antibodies and
chugs - 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.
[0159] One generally will desire to employ appropriate salts and buffers to
render
delivery vectors stable and allow for uptake by target cells. Buffers also are
employed
when recombinant cells are introduced into a patient. 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 innocula. 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,
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.
[0160] 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.
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[0161] 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.
[0162] 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.
Therapeutic agents
[0163] In certain embodiments, therapeutic agents may be attached to a
targeting
peptide or fusion protein for selective delivery to, for example, white
adipose tissue.
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Agents or factors suitable for use may include any chemical compound that
induces
apoptosis, cell death, cell stasis andlor anti-angiogenesis.
Regulators of Prograrmned Cell Deatla
[0164] 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; Cleary et
al., 1986;
Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). 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.
[0165] 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).
[0166] Non-limiting examples of pro-apoptosis agents contemplated within the
scope
of the present invention include gramicidin, magainin, mellitin, defensin,
cecropin,
(I~LAKLAK)~ (SEQ ID NO:1), (KLAKKLA)2 (SEQ ID N0:2), (I~AAI~KAA)Z (SEQ ID
N0:3) or (KLGI~KLG)3 (SEQ >D NO:4).
Ar2giogerzic ifzhibitors
[0167] 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,
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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., Scief2ce 279:377-380, 1998; Arap et al., Curr. Opin. Oncol. 10:560-565,
1998;
Ellerby et al., Nature Meel. 5:1032-1038, 1999). A variety of anti-angiogenic
agents
and/or blood vessel inhibitors are known. (E.g., Folkman, In: Cancer:
Prineiples 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).
White fat represents a unique tissue that, like tumors, can quickly
proliferate and
expand (Wasserman, In: Handbook of Physiology, eds. Renold and Cahill, pp. 87-
100,
American Physiological Society, Washington, D.C., 1965; Cinti, Eat. Weight.
Disord.
5:132-142, 2000). Studies of adipose tissue reveal that it is highly
vascularized.
Multiple capillaries make contacts with every adipocyte, suggesting the
importance of
the vasculature for maintenance of the fat mass (Crandall et al.,
Microcirculation 4:211-
232, 1997). A hypothesis underlying the present invention is that adipose
tissue
proliferation might rely on angiogenesis similarly to tumors. If so,
destruction of fat
neovasculature could prevent the development of obesity, whereas targeting
existing
adipose blood vessels could potentially result in fat regression. Methods of
use of
adipose targeting peptides may include induction of weight loss, treatment of
obesity
and/or treatment of HIV related lipodystrophy.
Cytotoxic Agents
[0168] 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
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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, antimetabolites, antitumor antibiotics,
corticosteroid
hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous
agents,
and any analog or derivative variant thereof.
[0169] 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
in
"Remington's Pharmaceutical Sciences" 15~' ed., pp 1035-1038 and 1570-1580,
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. Examples of specific chemotherapeutic agents and dose regimes are
also
described herein. 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.
Alkylating agents
[0170] 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, a nitrogen mustard, an
ethylenimene,
a methylmelamine, an alkyl sulfonate, a nitrosourea or a triazines. They
include but are
not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan),
dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.
Antinaetabolites
[0171] 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
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limited to, 5-fluorouracil (5-FLJ), cytarabine (Ara-C), fludarabine,
gemcitabine, and
methotrexate.
Natural Products
[0172] 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.
[0173] 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.
[0174] 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.
[0175] Vinca alkaloids are a type of plant alkaloid identified to have
pharmaceutical
activity. They include such compounds as vinblastine (VLB) and vincristine.
Antibiotics
[0176] 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.
Miscellatzeous Agents
[0177] Miscellaneous cytotoxic agents that do not fall into the previous
categories
include, but are not limited to, platinum coordination complexes,
anthracenediones,
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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 is procarbazine
(N-
methylhydrazine, MIIT). 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.
Dosages
[0178] The skilled artisan is directed to "Remington's Pharmaceutical
Sciences" 15th
Edition, chapter 33, and in particular to pages 624-652. 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
[0179] 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. Identification of mouse placenta targeting peptides
Identificatio~e of placenta horft.zf2g peptides
[0180] Peptides homing to the mouse placenta were identified by a post-
clearing
protocol using a phage display library. A first round of biopanning was
performed on
pregnant mice. Samples of placenta were removed and phage rescued according to
protocols described below, with one modification. In the typical biopanning
protocol,
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thousands of phage may be recovered from a single organ, tissue or cell type.
Typically,
between 200 and 300 individual colonies are selected from plated phage and
these are
amplified and pooled to form the phage display library for the second or third
rounds of
biopanning. In the present Example, all phage rescued from the first round of
biopanning were amplified in bulk on solid medium and then pooled to form the
phage
display library for the second round of biopanning. That is, there was no
restriction of
the rescued phage from the first round of,biopanning. This ifz vivo biopanning
without
restriction was performed for three rounds (rounds I-III), then a post-
clearing procedure
was used. .
[0181] In a post-clearing protocol (round IV), phage were administered to a
non-
pregnant mouse. Phage that bound to tissues other than placenta were absorbed
from the
circulation. Remaining phage were recovered from the plasma of the non-
pregnant
mouse. This protocol was designed to isolate phage that bound to placenta but
not to
other mouse organs, tissues or cell types. The following placenta-targeting
peptides
were identified, along with their frequencies. A search of the GenBank
database
disclosed that none of the sequences listed below was 100% homologous with any
known peptide sequence.
TPKTSVT (SEQ ID N0:5) 7.4% in round III, 8.5% in round IV
RMDGPVR (SEQ ID N0:6) 3.1% in round III, 8.5% in round IV
RAPGGVR (SEQ ID N0:7) <1% in round III, 8.5% in round IV
VGLHARA (SEQ ID N0:8) 4.2% in round III, 7.4% in round IV
YIRPFTL (SEQ ID N0:9) 2.1% in round III, 5.3% in round IV
LGLRSVG (SEQ ID NO:10) <1% in round III, 5.3% in round IV
PSERSPS (SEQ ID NO:11) (data not available)
[0182] As can be seen, the use of a post-clearing procedure resulted in a
substantial
enrichment of phage bearing placenta targeting peptides. Although this
procedure was
used for placenta, the skilled artisan will realize that post-clearance can be
performed for
any organ, tissue or cell type where a phage library can be administered to a
subject
lacking that organ, tissue or cell type. For example, a post-clearing
procedure for
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prostate or testicle targeting peptides could be performed in a female
subject, and for
ovary, vagina or uterus in a male subject.
[0183] A homology search identified several candidate proteins as endogenous
analogs of the placental targeting peptides, including TCR gamma-1 (TPI~TSVT,
SEQ
ID N0:5), tenascin (RMDGPVR, SEQ ID N0:6 and RAPGGVR, SEQ ID N0:7),
angiotensin I (YIRPFTL, SEQ ID N0:9) and MHC H2-D-q alpha chain (VGLHARA,
SEQ ID N0:8).
Validatiozz of placenta horzzihg peptides azzd inhibition of pregzza>zcy
[0184] The placenta homing peptides were validated iz2 vivo by injection into
pregnant mice and recovery from the placenta. FIG. 1 shows the results of the
validation studies for selected placenta homing phage. The phage clones are
identified
as: PA - TPKTSVT (SEQ ID N0:5), PC - RAPGGVR (SEQ ID N0:7), PE - LGLRSVG
(SEQ ID NO:10), PF - YIRPFTL (SEQ ID N0:9). It can be seen that the PA clone
exhibited placental homing more than an order of magnitude greater than
observed with
control fd-tet phage. The PC clone also showed substantially higher placental
localization, while the PE and PF clones were not substantially enriched in
placenta
compared to control phage.
[0185] Despite the absence of apparent enrichment of the PF clone in placental
tissue, both the PA and PF peptides showed anti-placental activity. Table 3
shows the
effects of the PA and PF placental targeting peptides injected into pregnant
mice,
attached to FTTC (fluorescein isothiocyanate), GST (glutathion S-transferase)
or to
phage. At lower dosages (450 ~g total), FITC conjugated PA and PF showed a
slight
effect on pregnancy (Table 3). At higher dosages (800 to 1000 ~,g protein or
4.5 x 1010
phage), both protein and phage conjugated PA and PF peptides substantially
interfered
with fetal development (Table 3), apparently resulting in death of the fetuses
in most
cases. The CARAC peptide (SEQ ID N0:12), an adipose targeting peptide (FE,
TREVHRS, SEQ ID N0:13) or fd-tet phage were used as non-placental targeting
controls.
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Table 3. Effect of Placental Tar~etin~ Peptides on Fetal Development
Inhibition with FITC conjugates -I
1 mouse injected iv (predominantly) or ip every other day, day 1-day 18,
9 times, Total 450 mM (~450~.g)
Peptide Injected Pregnancy Outcome Peptide Effect on
Embryo
CARAC-FITC (- control) Delivery: 18d, 5 normal pups No effect
PA-FTTC (placenta homer) Delivery: 19d, 8 normal pups No effect
PF-FTTC (placenta homer) Delivery: 21d, 1 dead pup Development delay,
toxicity
Inhibition with FITC conjugates -II
1 mouse injected sc (predominantly) or iv every other day, day 4-day 17,
times, Total 1 M (~lmg)
Peptide Injected Pregnancy Outcome Peptide Effect on
Embryo
CARAC-FITC (- control) Delivery: 20d, 5 pups, 1-dead Slight toxicity?
PA-FITC (placenta homer) No fetuses inside after 21 d Pregnancy
termination
PF-FTTC (placenta homer) No fetuses inside after 21 d Pregnancy
termination
Inhibition with phage conjugates -I
1 mouse injected iv (predominantly) or ip every other day, day 1-day 18,
9 times, Total 4.5x101° TU
Peptide Injected Pregnancy Outcome Peptide Effect on
Embryo
Fd-Tet (- control) Avertin OD=>death. fetuses-OK
PA-phage (placenta homer) Delivery: 24d, 4 pups, 1-dead Development delay, t
PF-phage (placenta homer) Delivery: 25d, 8 pups, all dead Development
delay, toxicity
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Inhibition with GST conjugates -I
1 mouse injected sc (predominantly) or iv every other day, day 4-day 17,
times, Total 800 ~g
Peptide Injected Pregnancy Outcome Peptide
Effect on
Embryo
GST-FE (- control) Delivery: 20d, 2 pups,OK No effect
GST-PA (placenta homer) No delivery or fetuses after 21 d Pregnancy
termination
GST-PF (placenta homer) Day 15: no fetuses inside, uterus necrotic Pregnancy
termination
[0186] These results validate the placental targeting peptide sequences
identified
above. They further demonstrate that even in the absence of substantial
enrichment of
phage bearing the targeting sequence to the target organ (e.g. peptide PF,
FIG. 1), the
targeting peptide may nevertheless provide for targeted delivery of
therapeutic agents to
the target organ. In this study, it appeared that at lower dosages the PF
peptide was more
effective than the PA peptide at interfering with pregnancy, despite the
observation that
the PA peptide produced a many-fold higher level of phage localization to
placenta.
[0187] The skilled artisan will realize that the disclosed methods and
peptides may
be of use for targeted delivery of therapeutic agents to the fetus through the
placenta, as
well as for novel approaches to terminating pregnancy and/or inducing labor.
Example 2. Localization of the TPKTSVT (SEQ ID NO:S) Peptide in Mouse
Placenta
Material arad Methods
Animals Staged pregnant 18 days postconception (dpc) C57BL/6 female mice
were purchased from Harlan Teklad (Indianapolis, IN). Congenic pregnant (32m-
null
females (stock 002087) mice were purchased from The Jackson Laboratories (Bar
Harbor, ME). Anesthesia was performed with Avertin (0.015 ml/g) administered
intraperitoneally (Pasqualini and Ruoslahti, 1996, Nature 380:364-366; Rajotte
et al.,
1998, J. Clin. Ifivest. 102: 430-437).
Phase Library Screening In vivo screening of an M13 page-display CX7C library
(Pasqualini et al., 2000, in Phage Display: A Laboratory Manual, eds. Barbas
et al.,
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Cold Spring Harbor Laboratory Press, New York, NY, pp. 22.1-24; Arap et al.,
2002,
Nature Med. 8:121-127), for placenta-homing peptides was performed as
described
(Pasqualini et al., 2000; Pasqualini and Ruoslahti, 1996) with novel
modifications
described below. In each biopanning round, an 18 dpc C57BL/6 female was
injected
intravenously (tail vein) with 101° transducing units (TU) of the
library. Increasing
amounts of phage (from 103 TU in round 1 to 104 TU in round 4) were recovered
from
the placentas after 5 min of circulation. Recovered phage were bulk-amplified
for
subsequent rounds of screening. In a procedure introduced here for the first
time, the
sub-library that was amplified after the third round of panning was cleared of
nonspecific
binders in a subtraction step. A virgin C57BL/6 female was infused through the
tail vein
with 10~ TU of phage selected in round 3. After 5 min, the unbound circulating
phage
were recovered from plasma. The plasma contained approximately 107 TU of pre-
cleared phage. The precleared phage population, representing less than 1% of
the
injected pool, was recovered and amplified for the final round of biopanning.
Phage Recovery Mouse placentas and embryonic livers were individually
weighed, ground with a glass Dounce homogenizer and suspended in 1 ml of
Dulbecco's
Modified Eagle 's Medium (DMEM) containing proteinase inhibitors (DMEM-prin -
1
mM PMSF, 20 ~,g/ml aprotinin, and 1 ~,g/ml leupeptin). The suspension was
vortexed
and washed three times with DMEM-prin. Tissue homogenates (or 10 ml of blood
for
normalization of phage titer in placenta against circulating phage titer) were
incubated
with 1 ml of host bacteria (log phase E. coli K9lkan; OD600 ~2). Aliquots of
the
bacterial culture were plated onto Luria-Bertani agar plates containing 40
~,g/ml
tetracycline and 100 ~,g/ml kanamycin. Plates were incubated overnight at
37°C.
Triplicate samples were processed for host bacterial infection, phage
recovery, and
histological analysis.
Fusion and Recombinant Peptides Carboxyfluorescein (FTTC)-conjugated
CTPKTSVTC (SEQ ID N0:144) or control peptide CARAC (SEQ ID N0:12), formed
into cyclic peptides using the flanking cysteines, were chemically synthesized
and
HPLC-purified to >90% purity by Anaspec (San Jose, CA). The FTTC-peptide
stocks
were made by dissolving lyophilized peptides in DMSO to a concentration of 20
mM,
after which the peptides were diluted to 1 mM with PBS (phosphate buffered
saline) and
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aliquots were frozen until use. The CTPKTSVTC (SEQ ID N0:144) peptide and an
unrelated control peptide, CTREVHRSC (SEQ ID N0:87), fused in-frame with GST
at
the amino-terminus were purified to approximately 90°7o purity using
the BugBuster GST
Bind Kit (Novagen, Madison, WI). Purified peptides were buffer-exchanged into
PBS
using Centricon PL-10 columns (Millipore, Bedford, MA) and the aliquots were
frozen
until use. For in vivo peptide homing validation, 10 ~,1 of FITC-peptide
stocks diluted
20-fold with PBS or 250 ,u1 of 5 mglml GST-peptides were injected. For phage
homing
competition and IgG transcytosis blocking experiments, 500 ~,l of 5 mg/ml GST-
peptides
were administered intravenously.
Peptide Localization in Tissues Immunohistochemistry on sections of formalin-
fixed, paraffin-embedded mouse tissue was performed as described (Pasqualini
et al.,
2000; Pasqualini and Ruoslahti, 1996). For phage-peptide immunolocalization, a
rabbit
anti-fd phage antibody (Sigma Chemicals, St. Louis, MO) was used at 1:1,000
dilution
and detected with a secondary horseradish peroxidase (HRP)-conjugated
antibody. For
GST-peptide immunolocalization, a goat anti-GST antibody (Amersham,
Piscataway,
NJ) was used at 1:1,000 dilution and detected with a secondary alkaline
phosphatase
(AP)-conjugated antibody. For mouse IgG immunolocalization, the ARK Peroxidase
Kit
(DAKO, Carpinteria, CA) was used. All immunohistochemistry and FITC
immunofluorescence images were captured using an Olympus IX70 microscope and
digital camera setup.
Peptide Embryotoxicity For peptide embryotoxicity studies, agents were
injected
at the following daily doses: GST-peptides 0.1 mg (~3 nMoles), FITC-peptides
50 ~,g
(~30 nMoles),and phage-peptides 1011 TU. All agents were dissolved in PBS.
Mice
were injected subcutaneously in the back (5-10 injections per course).
Results
Localization of the TPKTSVT (SEQ ID N0:5) Peptide in Mouse Placenta The
tissue distribution of recombinant phage injected into pregnant mice was
examined by
immunohistochemistry (FIG. 3). While a control phage barely localized to
placental
tissues (FIG. 3A), the TPKTSVT (SEQ ID N0:5) phage homed to the placenta and
showed marked localization to the villi of the visceral yolk sac (vys)
endoderm (FIG. 3B,
arrows). The vys is a layer of epithelial cells which surrounds the embryonic
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microvasculature and functions as the final barrier during transport of
molecules, such as
immunoglobulin G (IgG), from the labyrinth layer of the placenta into the
fetus (Rugh,
1990; Beckman et al., 1990; Lyden et al., 2001, J. Immunol. 166:3882-3889;
Jollie,
1990, Teratology 41:361-381).
To verify that targeting of the TPKTSVT (SEQ ID NO:S) motif to the vys
endoderm also occurs if the peptide is outside of the context of the phage,
the homing of
TPKTSVT (SEQ ID N0:5) fused with glutathione S-transferase (GST) protein to
placenta was also tested. GST fused to either TPKTSVT (SEQ ID N0:5) or a
control
peptide TREVHRS (SEQ ID N0:13) with a similar overall charge were injected
into
pregnant mice and the tissue distribution of each peptide was examined. No
localization
of the control GST fusion peptide to the placenta was observed (FIG. 3C). In
contrast,
accumulation of the TPKTSVT (SEQ D7 N0:5) GST fusion peptide was readily
detectable in the apical cytoplasm of the vys (FIG. 3D, arrows) and matched
that
observed for TPKTSVT (SEQ ID NO:S)-phage (FIG. 3B, arrows).
Similarly, fluorescein (FTTC)-conjugated TPKTSVT (SEQ ID N0:5) injected
intravenously into pregnant mice also specifically localized to the apical vys
cytoplasm
(FIG. 3F). A control peptide FTTC conjugate was not detectable in the placenta
(FIG.
3E). Localization of TPKTSVT (SEQ ID N0:5)-phage, TPKTSVT (SEQ ~ N0:5)-
GST, or TPKTSVT (SEQ ID NO:S)-FTTC to control organs, such as brain and
pancreas,
was not detected (data not shown). Together, these data show that the TPKTSVT
(SEQ
ID N0:5) peptide targets the placenta, with the strongest homing noticed in
the vys (FIG.
3B, FIG. 3D and FIG. 3F ).
The TPKTSVT (SEQ ID N0~5) Peptide Binds to a Placental Transporter The
TPKTSVT (SEQ ID N0:5) peptide localizes to the vys (FIG. 3), which is the
tissue
primarily responsible for materno-fetal transport in mice. This motif was
tested to see if
it would promote phage transport into the embryo. Either TPKTSVT (SEQ ID N0:5)-
phage or control phage were injected into pregnant mice and the recovery of
phage from
embryos was determined. Specific accumulation of TPKTSVT (SEQ ID N0:5)-phage
in
embryos was observed to be up to 1,000-fold greater than that of control phage
(FIG.
4A). The TPKTSVT (SEQ ID NO:S) peptide was observed to apparently bind to a
specific placental transporter. The materno-fetal transfer of TPKTSVT (SEQ ID
NO:S)-
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phage was blocked by an excess of co-injected TPKTSVT (SEQ ID N0:5) peptide,
but
not the control GST fusion peptide (FIG. 4A), suggesting transport by a
specific receptor
protein as opposed to non-specific uptake.
Phage immunocytochemistry confirmed that the uptake of TPKTSVT (SEQ ID
N0:5)-phage by the vys cells was not affected by the control GST fusion
peptide (FIG.
4A, FIG. 4B and FIG. 4C). FIG. 4B shows that TPKTSVT (SEQ ID N0:5)-phage
administered alone are localized to the vys (arrows). FIG. 4C shows that in
the presence
of control-GST fusion peptides, the TPKTSVT (SEQ ID N0:5)-phage still localize
to the
vys (arrows). In contrast, the addition of TPKTSVT (SEQ ID N0:5)-GST fusion
peptide
prevented internalization of TPKTSVT (SEQ ID N0:5)-phage into the vys
epithelium
(FIG. 4D). Together, these results show that the TPKTSVT (SEQ ID NO:S) peptide
is
actively transported through the placenta into the embryo by binding to a
receptor
located on endothelial cells in the vys.
The TPKTSVT (SEQ ID N0:5) Peptide Blocks Placental I~G Transport The
pattern of the TPKTSVT (SEQ ID N0:5) localization to the placenta (FIG. 3) is
reminiscent of that observed for IgG in that tissue (Parr and Parr, 1985, J.
Reprod.
Izzzzrzuuol. 8: 153-171). Moreover, both IgG and TPKTSVT (SEQ ID N0:5) appear
to
undergo a receptor-mediated transport into the embryo during pregnancy. It was
hypothesized that IgG and TPKTSVT (SEQ ID N0:5) bind to a common receptor in
the
placenta. The results presented herein show that the TPKTSVT (SEQ ID NO:S)
peptide
competes for the placental transport of IgG.
Intravenous administration of a control GST-fusion peptide did not affect the
placental transfer of IgG to the vys (FIG. 5A, arrowheads). In contrast, co-
administration of an equimolar dose of TPKTSVT (SEQ ID N0:5)-GST blocked
translocation of IgG through the placenta (FIG. 5B). Although IgG localization
to the
labyrinthine blood vessels in the embryo-distal placental compartments was
still
detectable (FIG. 5B, asterisks), IgG staining in the vys epithelium was
markedly
decreased (FIG. 5B). The vys levels of IgG in the presence of TPKTSVT (SEQ ID
N0:5)-GST were similar to those observed in (32m-null mice (not shown), which
are
genetically deficient in IgG transcytosis (Israel et al., 1995, J. Iznmuzzol.
154:6246-6251;
Zijlstra, et al., 1990, Nature 344:742-746). Based on these data, it is
proposed that the
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TPKTSVT (SEQ ID N0:5) peptide selectively blocks transport through an
immunoglobulin Fc receptor that mediates the uptake of IgG by the yolk sac
(FIG. 5C
and FIG. 5D).
The TPKTSVT (SEQ ID N0:5) Receptor is Associated with the (32m Protein
FcRn is a (32m-associated Class I major histocompatibility complex (MHC-I)
homologue.
FcRN appears to regulate placental IgG transport (Ghetie and Ward, 2000, Ann.
Rev.
Inamunol. 18:739-766; Simister and Story, 1997, J. Reprod. Inununol. 37:1-23).
This is
supported by the observation that IgG species which are incapable of binding
to FcRn
are not transported across the human placenta in an ex vivo model (Firan et
al., 2001, Int.
Immunol. 13:993-1002). In addition, FcRn expression patterns in the placenta
resemble
the pattern of placental IgG localization (Saji et al., 1999. Rev. Reprod.
4:81-89). A
search of the mouse protein database using BLAST software (NCBI;
http://www.ncbi.nlm.nih.gov/BLAST/) revealed the similarity of the TPKTSVT
(SEQ
ID NO: 5) placental targeting peptide to the sequence PPKTTVT (amino acids 192-
198
of the mouse MHC-I; Genbank accession AAD43175). Moreover, the corresponding
conserved human MHC-I sequence, PPKTHVT, is exposed on the surface of the MHC-
I
oc3 chain immediately adjacent to H-192 residue, a known (32m contact site in
the a3
domain of MHC-I homologues (Tysoe-Calnon et al., 1991, Bioclzem. J. 277:359-
369).
This makes the TPKTSVT (SEQ ID N0:5) motif an apparent mimeotope of FcRn.
Homing of the TPKTSVT (SEQ ID N0:5) peptide to the placenta, despite
expression of
FcRn in other tissues, may be due to either differential association of
additional receptor
subunits, or by altered accessibility of the receptor to the circulating
ligand in the
placenta. This is consistent with previous reports of FcRn interacting with
IgG in the
placenta through a mechanism different from that in other tissues (Ghetie and
Ward,
Ann. Rev. Immunol. 18:739-766, 2000; Simister and Story, J. Reprod. Immunol.
37:1-23,
1997).
The TPKTSVT (SEQ ID NO: 5) motif was tested to determine if it targets the
FcRn/[32m receptor complex in the placenta. The (32m-deficient mouse strain,
in which
FcRn is not functional (Isreal et al., 1995, J. Imnzunol. 154:6246-51;
Zijlstra et al., 1990,
Nature 344:742-746), was used as a model system to test whether the TPKTSVT
(SEQ
ll~ N0:5) peptide is a ligand for the FcRn/(32m receptor complex. Phage
displaying the
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TPKTSVT (SEQ ID N0:5) peptide were intravenously injected into pregnant (32m-
null
mice and the recovery of phage from the placenta was assayed (FIG. 6). While
the
TPKTSVT (SEQ ID N0:5)-phage (FIG. 6A, white bars) homed to the wild-type (+/+)
placenta relative to control phage (FIG. 6A, black bars), no such homing was
detectable
in (32m-deficient mice (FIG. 6A). Immunohistochemical analysis of phage
accumulation
in [32m wild-type (FIG. 3B) and (32m-null (FIG. 6B) placentas confirmed that
TPKTSVT
(SEQ ID N0:5)-phage were not taken up by the vys epithelium in the (32m-
deficient
mice. In contrast, in vivo localization of a control phage displaying a
different placenta
homing peptide, YIRPFTL (SEQ ID N0:9), which, unlike TPKTSVT (SEQ B~ N0:5)
peptide, homes to the vasculature of the labyrinthine placenta, rather than to
the vys, was
not affected in (32m-null mice (FIG. 6C, arrowheads). Together, these
observations
strongly suggest that the TPKTSVT (SEQ ID N0:5) motif targets the placenta by
binding to FcRn/(32m.
The TPKTSVT (SEQ B~ NO: 5) Peptide Interferes with Mouse Pre~nancy
Because FcRn/(32m regulates materno-fetal exchange, the above observations
suggested
that TPKTSVT (SEQ ID NO:S) might interfere with placental transport and
embryonic
development. TPKTSVT (SEQ ID N0:5) peptide was administered to pregnant mice
in
three different forms - displayed on the phage capsid, fused to GST or fused
to FITC.
The phage or fusion peptides were subcutaneously injected and the progression
of
pregnancy was compared with mice injected with control phage, control
peptides, or
saline. Multiple injections of the TPKTSVT (SEQ ID N0:5) peptide were
administered,
starting at mid-pregnancy (~ 12 days postconception, dpc) in doses non-toxic
to the
mother.
The effect of TPKTSVT (SEQ ID N0:5) peptide on fetal development is
illustrated in FIG. 7A and FIG. 7B. TPKTSVT (SEQ ID N0:5) peptide inhibited
the
progression of pregnancy, as evidenced by diminished weight gain in mice
injected with
the TPKTSVT (SEQ ID N0:5) phage or GST fusion peptide compared to control
phage
or GST-peptide (FIG.7A). The course of pregnancy courses in mice injected with
control peptides were undistinguishable from those in saline-injected mice
(data not
shown).
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Examination of embryos on the 20th day of pregnancy (the delivery day for the
control) revealed that the TPKTSVT (SEQ ID N0:5) peptide has a severe effect
on
embryonic development. Growth-retarded, dead, or partially resorbed embryos
were
observed in mice injected with either TPKTSVT (SEQ ID N0:5)-phage, TPKTSVT
(SEQ ID N0:5)-GST, or TPKTSVT (SEQ ID N0:5)-FTTC (FIG.7B, embryo to right of
figure). The normal development of a mouse embryo injected with control
peptide is
shown on the left side of FIG. 7B. Administration of TPKTSVT (SEQ ID NO:S)
fusion
peptides to pregnant mice resulted in 43% complete embryo resorption and 21%
dead or
malformed conceptuses (FIG. 7B). The extent of embryo resorption and frequency
of
complete pregnancy abortion increased with prolonged TPKTSVT (SEQ m N0:5)
treatment (data not shown), suggesting that the peptide effect is dose-
dependent. Embryo
death or morbidity were not observed in any of the control groups.
Morphologic inspection of tissues from TPKTSVT (SEQ ID N0:5) peptide-
injected mice revealed that, in contrast to controls, placentas were edematous
and grossly
deformed. Histopathological examination of the placentas showed that the
TPKTSVT
(SEQ ID NO:S) treatment induced massive dilation of blood vessels and
intraplacental
bleeding, as well as widespread hemorrhagic necrosis (FIG. 7D). Hematoxylin
staining
of the placental epithelium after seven days of peptide administration showed
that most
nuclei in the yolk sac and many in the labyrinthine compartment were degraded
(FIG.
7D). Also, massive fibrosis was evident in the yolk sac cavity and in the
labyrinthine
portion of the placenta (FIG. 7D). In contrast, placentas from mice injected
with control
peptides (FIG. 7C) were indistinguishable from untreated placentas at
corresponding
stages of pregnancy. The effect of TPKTSVT (SEQ ID N0:5) peptide on the
reproductive system was strikingly specific, as histological examination of
control
organs revealed no pathological changes or necrosis, and no signs of peptide
toxicity to
the mother were observed (data not shown).
The teratogenicity observed with TPKTSVT (SEQ ID N0:5) is probably not
caused by the disruption of the FcRn/~i2M receptor function, as the fertility
of mice is not
significantly affected by (32M deficiency (Zijlstra et al., Nature 344:742-46,
. 1990).
Rather, embryotoxicity is likely secondary to placental thrombosis and
ischemia. An
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alternative mechanism could be activation of complement and an immune response
against the targeting peptide itself.
These results have important implications for the development of pregnancy-
safe
therapeutics, as it appears that a substance can cause embryotoxicity by
merely homing
to a placental cell surface marker without inactivating the receptor function.
This creates
the basis for a high throughput identification system based on placental
receptors prone
to teratogen binding. Systematic screening of potential teratogens for binding
to such
receptors could be used to identify teratogenic activity and decrease the risk
of teratogen
induced birth defects.
Example 3. Identification of mouse adipose targeting peptides
Adipose targeting peptides
[0188] A similar protocol to that disclosed in Example 1 was used to isolate
fat
targeting peptides from a genetically obese mouse (Zhang et al., Nature,
372:425-432,
1994; Pelleymounter et al., Seience 269:540-543, 1995). 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 N0:14), FDGQDRS (SEQ ID N0:15);
WGPKRT. (SEQ ID N0:16); WGESRL (SEQ ID N0:17); VMGSVTG (SEQ ID
NO:18), KGGRAI~1D (SEQ m NO:19), RGEVLWS (SEQ m N0:20), TREVHRS (SEQ
m N0:13) and HGQGVRP (SEQ ll~ N0:21).
[0189] Homology searches identified several candidate proteins as the
endogenous
analogs of the fat targeting peptides, including stem cell growth factor
(SCGF)
(I~GGRAKD, SEQ m N0:19), attractin (mahogany) (RGEVLWS, SEQ ~ N0:20),
angiopoitin-related adipose factor (FIAF) (TREVHRS, SEQ m N0:13), adipophilin
(ADRP) (VMGSVTG, SEQ ID N0:18), Flt-1 or procollagen type XVII (TRNTGNI,
SEQ ID N0:14) and fibrillin 2 or transferrin-like protein p97 (HGQGVRP, SEQ ID
NO:21)
Validation of adipose targeting peptades
[0190] The fat homing peptides were validated by ifz vivo homing, as shown in
FIG.
2. The fat homing clones selected were: FA - KGGRAKD (SEQ m NO:19), FC -
RGEVLWS (SEQ ID N0:20), FE - TREVHRS (SEQ m N0:13) and FX - VMGSVTG
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(SEQ ID N0:18). As seen in FIG. 2, 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.
[0191] 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,
preventing the growth of new deposits of fat and potentially killing existing
fat deposits.
Example 4: CI~GGRAKDC (SEQ ID NO: 22) homes to white fat in ob/ob mice
Materials and Methods
Experimental afziynals
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).
In vivo p7zage library screenit2g
In vivo phage-display screening of the CX7C library (C, cysteine; X, any amino
acid) (Pasqualini et al., 2000; Arap et al., Nature Med. 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 1010
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-grin: 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
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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 109 TU of phage selected
in round
3. After 5 min of circulation, the unbound phage were recovered from plasma
and
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 localization in 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 N0:22)-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 in vivo peptide homing validation, stocks of 5-
carboxyfluorescein (FTTC)-conjugated CKGGRAKDC (SEQ ID NO:22) or CARAC
(SEQ ID N0:12) were chemically synthesized, cyclized using the terminal
cysteines and
HPLC-purified to > 90°7o purity by Anaspec (San Jose, CA). Lyophilized
peptides were
dissolved in DMSO to a concentration of 20 mM. Ten ~1 of 1 mM peptide-FTTC
solution in PBS was injected 5 min prior to tissue extraction. For blood
vessel
localization, 10 ,u1 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).
Afati-obesity therapy
Stocks of CKGGRAKDC (SEQ ID N0:22) fused to (KLAKLAK)2 (SEQ ID
NO:l); (KLAKLAK)Z (SEQ DJ N0:1) alone; CARAC (SEQ D7 NO:12) fused to
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(KLAKLAK)2 (SEQ ID N0:1); and CKGGRAKDC (SEQ ll~ N0:22) 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 ~,1 of 0.65 mM peptide solution in
PBS was
subcutaneously injected daily in the back of C57BL/6 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
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
Ifi vivo phage display (Pasqualini and Ruoslahti,. Nature 380:364-366, 1996;
Kolonin et. al., Curr. Opih. Clzem. Biol. 5:308-313, 2001; Pasqualini et al.,
In Vivo Phage
Display, In Plzage Display: A Laboratory Mafaual, 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 in 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 N0:22) constituted 4.5% of all clones identified
in
the screen. Intravenous administeration of this clone into ob/ob mice showed
that
CKGGRAKDC (SEQ ID N0:22)-phage accumulated in subcutaneous fat to a higher
level than a control insertless phage (data not shown).
The tropism of CKGGRAKDC (SEQ ID N0:22)-phage for adipose tissue was
confirmed by immunohistochemistry: CKGGRAKDC (SEQ m N0:22)-phage showed
marked localization to the vasculature of subcutaneous and peritoneal white
fat (FIG. 8a,
arrows), whereas the control phage was undetectable in fat blood vessels (FIG.
8b). To
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test whether targeting of the CKGGRAKDC (SEQ ID N0:22) motif to the fat
vasculature would also occur when the peptide is outside of the context of the
phage, the
ifZ vivo distribution of intravenously injected CKGGRAKDC (SEQ ID N0:22)
peptide
fused to fluorescent (FTTC) was determined. Irnmunofluorescence in
subcutaneous and
peritoneal fat from peptide-injected ob/ob mice showed that CKGGRAKDC (SEQ ID
N0:22)-FITC localized to and was internalized by cells of white adipose
vasculature
(FIG. 8c, arrows), whereas a control CARAC (SEQ ID N0:12)-FTTC conjugate was
undetectable in adipose tissue (FIG. 8d).
CKGGRAKDC (SEQ ID N0:22) homes to wl2ite fat in 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. Clip. Endocri~2ol. 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 N0:22) homing to fat is limited to ob/ob mice and to
demonstrate the general applicability of adipose-targeting peptides for
naturally-
occurring obesity, the CKGGRAKDC (SEQ ID NO:22) peptide was tested in wild-
type
mice.
FIG. 9 shows that the CKGGRAKDC (SEQ ID N0:22)-FITC fusion peptide
intravenously injected into C57BL/6 (leptin +/+) mice specifically localized
to blood
vessels of subcutaneous and peritoneal white fat (FIG. 9A, FIG. 9B). A lectin-
rhodamine peptide was used to visualize blood vessel endothelium (arrows, FIG.
9B,
FIG. 9D, FIG. 9F). The CKGGRAKDC (SEQ ID N0:22)-FITC fusion peptide co-
localized with lectin-rhodamine in adipose tissue (arrows, FIG. 9A and FIG.
9B). No
such co-localization was observed in control pancreatic tissue (FIG. 9C and
FIG. 9D) or
other control organs (data not shown). The control CARAC (SEQ ID N0:12)-FITC
peptide was not detectable in white fat vasculature (FIG. 9E and FIG. 9F).
These ih vivo
localization data show that the adipose-targeting CKGGRAKDC (SEQ ID N0:22)
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 N0:22)-FTTC by the
endothelium of fat tissue suggests that the motif targets a receptor
selectively expressed
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in the adipose vasculature that could provide a mechanism for directed
delivery of
therapeutic compounds to fat.
Design and use of fat-targeted pro-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
KLAKLAKKLAKLAK (SEQ ID N0:1) (Ellerby et al., Nature Med. 5:1032-38, 1999)
(designated KLAKLAK)2), which disrupts mitochondria) 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. Nat). 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 ID NO:22) peptide for targeted delivery to fat vasculature in
adipose tissue. The D enantiomer of (KLAKLAK)Z (SEQ ll~ NO:1), which is
resistant
to proteolysis but still exhibits pro-apoptotic activity, was conjugated to
the
CKGGRAKDC (SEQ ID N0:22) 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 N0:22)-(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. 10A, injections of
CKGGRAKDC (SEQ ID NO:22) conjugated to (KLAKLAK)2 (SEQ ~ N0:1)
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 ID N0:22) and (KLAKLAK)2
(SEQ ID N0:1) or a control CARAC (SEQ ID N0:12)-(KLAKLAK)2 (SEQ ID NO:1)
conjugate) did not show a significant body mass decrease and continued to
increase in
weight (FIG. 10A).
The effectiveness of the CKGGRAKDC (SEQ ID N0:22)-(KLAKLAK)2 (SEQ
ID N0:1) conjugate was also examined in wild-type mice fed on a regular diet
(FIG.
10B). C57BL/6 mice that had developed a considerable amount of subcutaneous
and
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peritoneal fat due to old age were subcutaneously injected with the CKGGRAKDC
(SEQ
ID N0:22)-(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 NO:22) resulted in greater
than 35% reduction in body mass at a rate of 10% per week (FIG. 10B). No
toxicity of
the conjugated peptide was detected under these conditions (data not shown).
In fact, the
CKGGRAKDC (SEQ ID N0:22)-(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)Z (SEQ ID N0:1)
treatments resulted in only a slight body mass reduction (FIG. 10B), 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 resorptioyi witl2 CKGGRAKDC (SEQ ID N0:22)-(I~LAI~LAK)Z (SEQ ID
NO:1 ) is mediated by apoptosis
In both diet-induced and age-related obesity, the effect of CKGGRAKDC (SEQ
ID N0:22)-(KLAKLAK)2 (SEQ ID NO:1) treatment on body mass was due to fat
resoiption, which was visually apparent by the end of treatment (FIG. 11).
Wild-type
mice were fed on a high fat cafeteria diet (FIG. 11A). Alternatively, wild-
type fed on a
regular diet became obese as a consequence of old age (FIG. 11B, FIG. 11C,
FIG. 11D).
Mice were treated with CKGGRAKDC (SEQ ID NO:22) conjugated to (KLAKLAK)2
(SEQ ID NO:1) (left side of FIG. 11), with CARAC (SEQ ID N0:12) conjugated to
(KLAKLAK)2 (SEQ ID NO:1) (middle of figure), or with unconjugated CKGGRAKDC
(SEQ ID NO:22) and (KLAKLAK)2 (right side of FIG. 11).
Gross inspection of mouse organs revealed that both subcutaneous (FIG. 11B)
and visceral (FIG. 11C) fat exhibited marked resorption upon treatment with
CKGGRAKDC (SEQ ID N0:22) conjugated to (KLAKLAK)2 (SEQ ID N0:1) (right
side of FIG. 11). Quantification of fat resorption after three weeks of
treatment by
weighing a specific fat depot (epididymal fat, FIG. 11D) showed a greater than
3-fold
reduction in fat mass compared with controls (FIG. 11D, left side of figure
compared to
middle and right side).
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Histopathological analysis of tissues from mice treated with CKGGRAKDC
(SEQ ID N0:22) conjugated to (KLAKLAK)Z (SEQ ID N0:1) showed vascular
apoptosis (FIG. 12A, arrows) and resulting fat necrosis with lymphocyte
infiltration
(FIG. 12C, arrows) in adipose tissue. following treatment. In contrast, mice
treated with
a control fusion peptide comprising CARAC (SEQ ID N0:12) conjugated to
(KLAKLAK)2 (SEQ ID NO:1) showed no vascular apoptosis or fat necrosis (FIG.
12D).
No abnormalities in other organs treated with CKGGRAKDC (SEQ ID N0:22)
conjugated to (KLAKLAK)2 (SEQ ID NO:1) (data not shown).
Injection of CKGGRAKDC (SEQ 117 N0:22) conjugated to (KLAKLAK)2 (SEQ
ID NO: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 and/or
use of a time release formulation of the adipose targeting conjugate may be
preferred in
cases of excessive obesity.
Adipose receptor protein for CKGGRAKDC (SEQ ID N0:22)
A band of approximately 35,000 Daltons (35 kDa) was isolated from mouse
adipose tissue extract that bound to CKGGRAKCDC (SEQ ID N0:22) conjugated to
(KLAKLAK)2 (SEQ ID NO:1). There was much less binding of the 35 kDa fraction
to
the control peptide CARAC (SEQ ID NO:12) conjugated to (KLAKAK)2 (SEQ m
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
N0:22) or CARAC (SEQ ID N0:12) conjugated to (KLAKAK)2 (SEQ ID NO:1) and
precipitating with commercially available antibodies.
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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 N0:22) (data not shown), with over a ten-fold
enrichment in the CKGGRAKDC (SEQ ID N0:22) precipitated fraction compared to
the
CARAC (SEQ ID N0:12) precipitated fraction (data not shown). The CARAC (SEQ ID
NO:12)-(KLAKAK)2 (SEQ ID NO:1) fusion peptide exhibited low levels of non-
specific
binding to all three proteins (VDAC, prohibitin and apolipoprotein E). It is
unknown
whether those proteins bound to the CARAC (SEQ ID N0:12) moiety or to
(KLAKAK)2
(SEQ ID N0:1).
It is concluded that the adipose tissue endothelial receptor for CKGGRAKDC
(SEQ ID N0:22) is prohibitin (Genbank Accession No. NM_008831). Probitin is
expressed in mitochrondria of various cell types and in the cell membrane of B
lymphocytes. Immunohistochemical analysis shows that prohibitin is expressed
in blood
vessels of adipose tissues but not of other organs (data not shown). Based on
these
results, it is concluded that pro-apoptosis agents conjugated to targeting
peptides that
bind to a prohibitin receptor protein complex are 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.
Example 5: Screening an alpha-spleen antibody library ih vivo by BRASIL
[0192] The following Examples are illustrative of general techniques that may
be of
use in various embodiments of the claimed invention. As part of the reticulo-
endothelial
system, biopanning against spleen tissue is complicated by the high background
of non-
specific phage localization to spleen. The decreased background observed in
biopanning
with the BRASIL method is advantageous for identifying targeting peptides
against
tissues such as spleen.
[0193] This example demonstrates an illustrative embodiment of the BRASIL
method. A phage library based on imrnunoglobulins derived against the target
organ
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(mouse spleen) was developed and then subjected to in vivo biopanning. To
construct
the immunoglobulin library, mouse spleen was injected into a chicken. After
boosting,
the chicken spleen was collected and immunoglobulin variable domain sequences
were
obtained by PCRTM amplification of chicken spleen mRNA. The amplified
immunoglobulin variable sequences were inserted into a phage display library
(oc-library)
that was then used for ifz vivo biopanning against mouse spleen. Thus, the
spleen
targeting peptide sequences obtained from phage localized to mouse spleen ifz
vivo were
derived from antibody fragments produced in the chicken in response to mouse
spleen
antigens. The success of this example further shows the broad utility of the
BRASIL
method. The skilled artisan will realize that the present invention is not
limited to the
embodiments disclosed herein and that many further developments of the BRASIL
methodology are included in the scope of the present invention.
Materials and Methods
Library constructiof2
[0194] A white leghorn chicken was immunized with spleen homogenate (about 150
mg per injection) from a perfused (10 ml MEM) Balb/c mouse. The chicken
received
spleen homogenate boosters at 4 weeks and 8 weeks after the initial
immunization.
Immune response to mouse spleen by FACS analysis showed that the chicken
immune
serum contained antibodies against a mouse cell-line (TRAMP-C1). The chicken
was
sacrificed and its spleen was removed to TRI Reagent (Molecular Research
Center, Inc.,
Cincinnati, OH) 12 weeks after the first immunization.
[0195] Total RNA was prepared from the chicken spleen using the manufacturer's
protocol for the TRI reagent. cDNA was prepared from the total RNA using oligo
(dT)-
primers and Superscript enzyme (Life Technologies, Gaithersburg, MD). cDNAs
encoding chicken spleen immunoglobulin variable regions were amplified by
CHybVH
and ChybIgB (V heavy) or by CSCVI~ and CHHybL-B (V kappa) primers according to
standard techniques. Light chain variable regions and constant regions were
PCRTM
amplified together using CSC-F and lead-B primers and Vkappa and C kappa
templates.
Heavy chain variable regions and constant regions were PCRTM amplified
together using
dp-seq and lead-F primers and Vhea~y and C heavy templates. Heavy- and light -
chain
fragments were PCRTM amplified together with CSC-F and dp-Ex primers. PCR
primers
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were purchased from Genosys (The Woodlands, TX) or GenBase (St. Lucia,
Queensland, Australia), using primer sequences listed in the Cold Spring
Harbor
laboratory course manual, "Phage Display of Combinatorial Antibody Libraries"
(Barbas
et al., 2000), the relevant text of which is incorporated herein by reference.
[0196] After digestion with Sfi I, the amplification products were ligated to
SfiI -
digested pComb3x for insertion into the phage library. Ligated pComb3-123
plasmid
was electroporated into ER2537 -E.coli and phage production was started with
subsequent VCM13 (helper phage) infection. The resulting library size was
about 5 x 10~
cfu.
1>z vivo screeniyzg of a spleen library using BRASIL
[0197] Four rounds of irz vivo screening in mice were performed using the
chicken oc-
spleen library. About 0.8 to 2.0 x 101° TU were injected into a Balblc
mouse. The library
was allowed to circulate for 5 minutes. After sacrifice, the mouse spleen was
recovered
and a single cell suspension was prepared by pressing the spleen through a 70
~m cell
strainer nylon mesh. The single cell suspension was centrifuged over oil (9:1
dibutyl
phtalate: cyclohexane) using the BRASIL technique and 200 ~.l of log phase
ER2537 E.
coli were infected with the pellet. Amplified phage recovered from the mouse
spleen
was used for the subsequent round of screening. No obvious enrichment in the
screening
rounds was seen in the number of phage homing to spleen and brain compared
with the
conventional biopanning method, using a piece of spleen obtained prior to
BRASIL.
[0198] Phage localized in mouse spleen from the fourth round of screening of
the
chicken Fab inserts were PCRTM amplified and the PCR product was digested with
fist I.
Half of the clones out of 90 analyzed produced a similar restriction pattern.
Of those, 20
clones were sequenced from which only two had an identical restriction
pattern. Four of
the antibody based phage clones (numbers 2, 6, 10 and 12) were subjected to
further
analysis using binding and localization assays.
Testing the clones izz vitro using BRASIL
[0199] A singe cell suspension was prepared from two mouse spleens. The
suspension was divided into five tubes and incubated on ice with 3x10 TU of
Fab clones
#2, #6, #10, #12 and 2x10 TU tet-phage. Phage bound to mouse spleen cells were
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recovered by BRASIL,. 200 p,1 of log phase ER2537 E.coli was infected with the
pellet
and serial dilutions were plated on LB/carbenicillin and LB/tetracycline
plates for
assessment of phage binding. Fd-tet was used as an internal control to
normalize all the
phage homing experiments.
Testitzg clones izz vivo with BRASIL
Phage (3x100 of Fab clones #2, #6, #10, #12 and 2x10 TU tet-phage were
injected into the tail veins of Balb/c mice and allowed to circulate for 5
minutes. The
spleens were recovered and single cell suspensions were prepared on ice from
whole
spleens. Cell bound phage were recovered by BRASIL. 200 ~.1 of log phase
ER2537
E.coli was infected with the pellet and serial dilutions were plated on
LB/carbenicillin
and LB/tetracycline plates for assessment of the phage recovery.
Testing clozze #10 versus, control phage NPC-3TT in vivo with BRASIL
Phage (3x10 TU) of Fab clone #10 and NPC-3TT (control Fab phage) and 1x10
TU of control Fd-tet -phage were injected to mice (2 mice for NPC-3TT, 2 mice
for
clone #10) and allowed to circulate for 5 minutes. Spleens were recovered and
single
cell suspensions were prepared on ice. Cell-bound phage were recovered by
BRAS1L.
200 ~,l of log phase ER2537 E.coli was infected with the pellet and serial
dilutions were
plated on LB/carbenicillin and LB/tetracycline plates. The NPC-3TT phage is a
human
anti-tetanus toxin Fab fragment displaying phage.
Hoznirzg of Fab clone #10 to spleezz versus bone marrow
Phage (3x10 TU) of Fab clone #10 and NPC-3tt control and 1x10 TU of Fd-tet
control phage were injected into mice (2 mice for NPC-3TT, 2 mice for clone
#10) and
allowed to circulate for 5 minutes. The spleens were recovered and single cell
suspensions were prepared. Bone marrow was recovered from the same mice (both
femurs) as a control for organ specific homing. Cell-bound phage were
recovered by
BRASTL.
Fab -fragmeht productiofz
[0200] The plasmid pComb3 containing the chicken Fab inserts was
electroporated
into ER2537 bacteria. Serial dilutions were plated onto LB/carbenicillin
plates and
incubated overnight at 37°C. Fab production culture (in super broth
with 100 ~,g/ml
CA 02458047 2004-02-19
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carbenicillin) was started from a single plated colony. Fab production was
induced with
1 mM IPTG for 7 hours at 30°C. The Fab fragment was purified from the
periplasmic
fraction SN2 by affinity purification after determination of the Fab
concentration in
bacteria supernatant, periplasmic fractions SNl and SN2 and in the bacteria
lysate by
ELISA. An a-Fab-Protein G -column was coupled (2 mg/ml) with
dimethylpimelimidate (DMP) using standard protocols (Harlow and Lane,
Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, NY, 1988).
[0201] For purifying Fab fragments the following method was used. The SN2
fraction was loaded into a 1 ml HiTrap-protein G-oc-Fab-column (Amersham
Pharmacia
Biotech, Piscataway, NJ) either over 2 hours (if using lower than 50 ml volume
with
superloop) or overnight (with more than 50 ml volume using a peristaltic
pump). The
column was washed with 10-20 ml of PBS (phosphate buffered saline). The Fab
fragments were eluted with 10 ml of 20 mM glycine buffer, pH 2.2, 150 mM NaCl
and 1
ml fractions were collected. Fractions are neutralized with 1 M Tris
immediately after
elution. Protein concentrations were quantified by A2so.
Intravascular staizzirzg
[0202] To determine iz~ vivo distribution of the recovered Fab fragments, 50
to 60 ~,g
of Fab fragment (Fab#10, NPC3-tt or R#16) was injected into the tail vein of a
Balb/c
mouse and allowed to circulate for 8 minutes. 50 ~,g of L. esculezztuzn lectin-
FTTC was
injected into the mouse and the mouse tissues were fixed by perfusion with 25
to 30 m1
of 4% paraformaldehyde/PBS after 2 minutes of lectin circulation. Tissues were
removed and post-fixed in 4% paraformaldehyde for 1 hour. Fixed tissues were
incubated in 30% sucrose/PBS overnight at 4°C, changing the solution at
least twice.
The tissues were embedded in the freezing media and frozen on dry ice.
[0203] Fixed tissue sections were stained for Fab as follows. Frozen tissue
sections
(55 ~,m) were cut on a microtome and washed 3x with PBS. The thin sections
were
blocked with PBSl0.3%TritonX-100/5% goat serum for 1 hr at room temperature.
Sections were incubated overnight at room temperature with 1:400 Cy3
conjugated cc-
human anti-Fab antibody. The conjugated sections were washed 6x with PBS/0.3%
Triton X-100, 3x with PBS, and fixed with 4% paraformaldehyde for 15 minutes.
After
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fixation the sections were washed again 2x with PBS and 2x with distilled
water, then
mounted on slides using VectorShield.
Results
[0204] The if2 vitro localization to mouse spleen cells of phage clones
expressing
chicken Fab fragments was examined by BRASIL. As shown in FIG. 13, the Fab
phage
clones isolated by BRASIL showed differential binding to mouse spleen cells
compared
to Fd-tet insertless control phage. Clone #6 showed the lowest degree of
binding, similar
to the control phage NPC-3TT, which contained a Fab fragment but was not
isolated
from mouse spleen. Clones #2, #10 and #12 all showed selective binding to
mouse
spleen cells compared to the Fd-tet control, with at least a two-fold
increased binding
observed for clones #2 and #10 (FIG. 13). The amino acid sequences determined
for the
clone inserts were:
Clone #2:
CQPAMAAVTLDES GGGLQTPGGALSLV CKAS GFTFNS YPMGW VRQAPG
KGLEWVAVISSSGTTWYAPAVKGRATISRDNGQSTVRLQLSNLRAED
(SEQ ID NO:23)
Clone #6:
CQPAMAAVTLDES GGGLQTPGGTLSLV CKAS GISIGYGMNW VRQAPGK
GLEYVASISGDGNFAHYGAPVKGRATISRDDGQNTVTLQLNNLR (SEQ
ID NO:24)
Clone #10:
CQPAMAAVTLDES GGGLQTPGGTLSLV CKGS GFIFSRYDMAW VRQAPG
KGLEWVAGIDDGGGYTTLYAPAVKGRATITSRDNGQSTVRLQLNNLR
(SEQ ID NO:25)
Clone #12:
ANQPWPPLTLDESGGGLQTPGGALSLVCKASGFTMSSYDMFWVRQAPG
KGLEFVAGIS S S GS STEYGAAVKGRATISRDNGQSTVRLQLNNLRAED
(SEQ ID N0:26)
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[0205] A direct comparison was made of in vitro phage binding for the Fab
clones
compared to NPC-3TT. As shown in FIG. 14, clones #2 and #10 exhibited the
highest
levels of binding to mouse spleen cells ifa vitro. Clones #6 and #12 showed
levels of
binding to mouse spleen that were only slightly higher than the binding of
phage NPC-
3TT.
[0206] The preferential binding of the chicken Fab phage clones was confirmed
by ire
vivo studies using BRASIL. As shown in FIG. 15, selective localization to
mouse spleen
was even more dramatic ire vivo, with Fab clones #2, #6 and #10 showing many-
fold
increased binding to spleen compared to Fd-tet phage. In contrast, Fab clone
#12 did not
exhibit significantly elevated binding to mouse spleen compared to Fd-tet
phage. These
results show that in vitro results obtained with spleen targeting phage are
confirmed ifa
vivo.
[0207] Fab clone #10 was selected for additional characterization by in vivo
localization to mouse spleen. The results, shown in FIG. 16, confirm that Fab
clone #10
exhibited 3 to 10 fold enrichment in spleen compared to Fd-tet. This effect
was not due
to general Fab binding, since the Fab control phage NPC-3TT did not exhibit
selective
localization in spleen compared to Fd-tet insertless phage.
[0208] Binding of Fab clone #10 was organ specific, as demonstrated in FIG.
17.
Phage from Fab clone #10 and NPC-3TT control were recovered from spleen and
bone
marrow tissue from the same injected mice. It can be seen in FIG. 17 that Fab
clone #10
exhibited selective localization to spleen but not to bone marrow tissue. The
control
phage did not exhibit selective localization to bone marrow (FIG. 17) or
spleen (not
shown).
[0209] These results show that Fab phage clone #10 selectively targets mouse
spleen
tissue for binding both in vitro and in vivo. These results were further
validated by
vascular staining for in vivo phage distribution. Control phage used for this
study were
clones NPC-3TT (Fab fragment) and clone R#16 (isolated from angiogenic retina
screening).
[0210] Fab clone #10 was observed to bind to mouse spleen tissue in vivo by
fluorescent staining (not shown). The control phage NPC-3TT and R#16 did not
stain
spleen tissue under identical conditions (not shown). The clone #10 and NPC-
3TT
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phage were observed to intensively stain kidneys of injected animals, perhaps
due to
glomerular filtration (not shown). Other control organs (lung, brain, liver,
heart and
skeletal muscle) did not show staining with clone #10 (not shown).
[0211] These results demonstrate that spleen targeting phage peptides can be
identified by the BRASIL method. They further show the feasibility of the
phage display
technique using antibody fragments against a target organ, tissue or cell type
to obtain a
starting phage library. The ability to obtain targeting peptides against
spleen, a tissue
that has proven refractory to biopanning using standard phage display
protocols because
of the high non-specific background, shows the advantages of the BRASIL
method.
Example 6. Identification of Receptor/Ligand Pairs: Targeting Peptides against
Integrin Receptors
[0212] Certain embodiments of the present invention concern the identification
of
receptor/ligand pairs for various applications. Targeting peptides selective
for organs,
tissues or cell types bind to receptors (as defined above), normally located
on the
lumenal surface of blood vessels within the target. In certain embodiments,
targeting
peptides may be used to identify or characterize such receptors, either
directly or
indirectly. In addition to their use as targets for delivery of gene therapy
vectors, other
therapeutic agents or imaging agents for ifz vivo imaging, such naturally
occurring
receptors are of use as potential targets for development of new therapeutic
agents
directed against the receptor itself, for development of vaccines directed
against the
receptor, and for understanding the molecular mechanisms underlying various
disease
states. Naturally, the targeting peptides themselves may serve as the basis
for new
therapeutic agents directed against the receptors.
[0213] Targeting peptides may frequently act as mimeotopes of endogenous
ligands
that bind to the targeted receptor. In other embodiments, the endogenous
ligands may be
identified and characterized using the disclosed methods. Such ligands are
also of
potential use as targets for development of new therapeutic agents, etc.
[0214] The present example illustrates one embodiment related to
identification of
receptor/ligand pairs, in this case, integrin receptors. Non-limiting examples
of
applications of targeting peptides directed against integrins include
regulation of cell
proliferation and chemotaxis, pro-apoptosis and anti-angiogenesis. In this
embodiment,
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purified integrins attached to a solid substrate were used to screen phage
display libraries
to identify targeting peptides directed against integrins.
Background
[0215] Integrin function is regulated by cytokines and other soluble factors
in a
variety of biological systems. Most commonly, exposure to such factors leads
to
conformational alterations that result in changes in the activation state of
the receptors
(i.e., increased or decreased affinity for a given ligand and/or receptor
clustering in the
plasma membrane). Changes in integrin-dependent adhesion ultimately activate
various
complex signal transduction pathways. At the molecular level, the induced co-
localization of cytoskeleton proteins with integrin cytoplasmic domains
controls signal
transduction.
[0216] Cytoplasmic domains are key regulators of integrin function (reviewed
in
Hynes, Cell 69:11-25, 1992; Ruoslahti, Ann. Rev. Cell Dev. Biol. 12:697-715,
1996).
Individual a and 13 subunit cytoplasmic domains are highly conserved among
different
species (Hemler et al., In: Integrins: Tl2e Biological PYOblems, ed. Takada,
CRC Press,
Inc. Boca Raton, FL, pp. 1-35, 1994). Although the cytoplasmic domains of
various 13
subunits share similar primary structures, they differ in certain functional
characteristics.
Experiments with chimeric integrins have shown that the cytoplasmic domains of
13
chains are responsible for regulating receptor distribution and recruitment to
focal
adhesion sites (Pasqualini and Hemler, J. Cell. Biol. 125:447-460, 1994).
Thus, certain
cytoplasmic domains are critical for integrin-mediated signaling into the cell
(outside-in
signaling) and activation of integrin-ligand binding activity (inside-out
signaling)
(Hemler et al., 1994).
[0217] The integrins av133 and av135 are selectively expressed in angiogenic
vasculature but not in normal vasculature (Brooks et al., 1994a, 1994b;
Pasqualini et al.,
1997; Arap et al., 1998). Moreover, ccv integrin antagonists have been shown
to block
the growth of neovessels (Brooks et al., 1994a, 1994b, 1995; Hammes et al.,
1996). In
these experiments, endothelial cell apoptosis was identified as the mechanism
for the
inhibition of angiogenesis (Brooks et al., 1994a, 1994b, 1995). Angiogenesis
initiated
by bFGF can be inhibited by an anti-ocv133 blocking antibody, whereas VEGF-
mediated
angiogenesis can be prevented by a blocking antibody against av135. The
integrins ocv133
CA 02458047 2004-02-19
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and av135 have been reported to be preferentially displayed in different types
of ocular
neovascular disease (Friedlander et al., 1995, 1996). Thus, distinct cytokine-
induced
pathways that lead to angiogenesis seem to depend on specific av integrins.
[0218] The search for ocv integrin-associated molecules has been hampered by
technical difficulties. First, the physical associations involved are likely
to rely on an
assembly of multimeric ligands that no longer occurs when cells are not
intact. Second,
their association to integrins is usually of low affinity. Finally, changes in
the
conformation and phosphorylation states of the associating proteins may add a
further
level of complexity in these transiently modulated interactions. Because of
these
problems, only a limited number of proteins that bind to integrin cytoplasmic
domains
have been identified. These proteins, such as paxillin and ICAP-1, mainly
associate with
the 131 chain (Shattil and Ginsberg, 1997). Cytohesin-1 and filamin associate
with the
cytoplasmic domain of 132.
[0219] The disclosed methods have several advantages over previous approaches:
(i)
the ability to characterize the intracellular molecules that directly or
indirectly interact
with integrin cytoplasmic domains; (ii) the development of antibodies against
molecules
that bind to integrin cytoplasmic domains in very low amounts; and (iii) the
phage
display library screenings will lead to the identification of peptides that
mimic
cytoplasmic-domain binding proteins.
Methods
Two di~t,ensiofaal cell culture
[0220] Three human endothelial cell lines that express 133 and 135 integrins
were
used: KS1767 cells (Herndier et al., 1996), IiLJVECs (ATCC), and BCE cells
(Solowska
et al., 1991). Sterile glass coverslips covered with different proteins (i.e.
vitronectin,
fibronectin, collagen, or laminin) were used as substrates. After cells
attached and
spread, the monolayers were rendered quiescent by a 12-hour incubation in
medium
containing 0.05% fetal calf serum. Peptides were introduced into the cells
using the
penetratin membrane-permeable tag (see below). The cells were plated onto ECM
proteins for adhesion and spreading. The monolayer was stimulated for 6 hours
with
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each of the growth factors involved in av-mediated angiogenesis, including
bFGF,
TNFa, VEGF, and TGF(3. Untreated cells were the negative controls.
Three-Dimensiofial Cell Culture:
[0221] 150 ~ul of Matrigel were added per well of 24-well tissue culture
plates and
allowed to gel at 37°C for 10 min. HCTVECs starved for 24 h in M199
medium
supplemented with 2% FCS before being trypsinized were used. 104 cells were
gently
added to each of the triplicate wells and allowed to adhere to the gel coating
for 30 min
at 37°C. Then, medium was replaced with peptides in complete medium.
The plates
were monitored and photographed after 24 h with an inverted microscope
(Canon).
Cheuaotaxis Assay:
[0222] Cell migration assays were performed as follows: 48-well
microchemotaxis
chambers were used. Polyvinylpyrrolidone-free polycarbonate filters
(Nucleopore,
Cambridge, MA) with 8-,um pores were coated with 1 % gelatin for 10 min at
room
temperature and equilibrated in M199 medium supplemented with 2% FCS. Peptides
were placed in the lower compartment of a Boyden chamber in M199 supplemented
with
2% FCS, 20 ng/ml VEGF-A (R&D System), and 1 U/ml heparin. Overnight-starved
subconfluent cultures were quickly trypsinized, and resuspended in M199
containing 2%
FCS at a final concentration of 2106 cellslml. After the filter was placed
between lower
and upper chambers, 50 ~l of the cell suspension was seeded in the upper
compartment.
Cells were allowed to migrate for 5 h at 37°C in a humidified
atmosphere with 5% C02.
The filter was then removed, and cells on the upper side were scraped with a
rubber
policeman. Migrated cells were fixed in methanol and stained with Giemsa
solution
(Diff-Quick, Baxter Diagnostics, Rome, Italy). Five random high-power fields
(magnitude 40x) were counted in each well.
Proliferation Assay:
[0223] Cell proliferation was measured as described (Pasqualini and Hemler,
1994).
Briefly, 4x104 HUVECs were incubated in 24-wells plates. The cells were
starved for 24
h, and then the medium was removed and replaced in the presence of VEGF and 15
,uM
of each peptide and incubated for 18 h. Then, 50 ~,1 of media containing
[3H]thymidine
(1 p,Ci/ml) was added to the wells, and after 6 additional hours of incubation
at 37°C, the
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medium was removed and the cells were fixed in 10% TCA for 30 min at
4°C, washed
with ethanol, and solubilized in 0.5 N NaOH. Radioactivity was counted by
liquid
scintillation with an LS 6000SC Beckman scintillation counter. Each experiment
was
performed three times with triplicates, and the results are expressed as the
mean ~ SD.
Apoptosis Assay (propidiufra iodide staif2i~2g subdiploid populatioiZ)
[0224] Approximately 1 x 106 cells were harvested in complete media and 15 ,uM
of
peptide added for 4, 8, or 12 h. The cells were then washed in PBS and
resuspended in
0.5 ml propidium iodide solution (50~.g/ml PI, 0.1 % Triton X-100, 0.1 %
sodium citrate).
After a 24-h incubation at 4°C, cells were counted with a XL Coulter
(Coulter
Corporation) with a 488-nm laser; 12,000 cells were counted for each
histogram, and cell
cycle distributions were analyzed with Multicycle program.
[0225] After microinjection or penetratin-mediated internalization of the
peptides
and appropriate controls, cell apoptosis was monitored using the ApopTag kit.
Experiments were performed in the presence of caspase inhibitors and
antibodies against
specific caspases.
Cytokine- and Tuynor-Iyaduced Angiogehesis Assays
[0226] Angiogenic factors and tumor cells implanted into CAM stimulate growth
of
new capillaries. Angiogenesis was induced in CAMs from 10-day chicken embryos
by
VEGF or bFGF filters implanted in regions that were previously avascular.
Different
treatments (penetratin peptides and controls) were applied topically, and
after 3 days, the
filters and surrounding CAMS were resected and fixed in formalin. The number
of blood
vessels entering the disk was quantified within the focal plane of the CAM
with a
stereomicroscope. The mean number of vessels and standard errors from 8 CAMs
in
each group were compared.
Phosplzorylatiofz afzd pararaifag of phosphorylated phage libraries
[0227] Phosphorylation of peptide libraries with src family protein kinases
(Fyn, c-
Src, Lyn, and Syc) and serine/threonine kinases such as a MAP kinase were
performed
as described previously (Schmitz et al., 1996; Dente et al., 1997; Gram et
al., 1997).
Briefly, phage particles were collected from culture supernatants by double
precipitation
with 20% polyethylene glycol 8000 in 2.5 M NaCI. Particles were dissolved at
lOlz
83
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particles/ml. Purified phage (10 ~ul) were incubated for 3 hours at room
temperature with
different concentrations (35 to 3,500 units) of protein kinases in a reaction
buffer volume
of 50 ~,1. The reaction mixtures were transferred to tubes containing 10 ~,g
of agarose-
conjugated anti-P-Tyr, anti-P-Ser, or anti-P-Thr monoclonal antibodies to
select phage
displaying phosphorylated peptides. Bound phage were eluted by washing the
column
with 0.3 ml of elution buffer (0.1 M NaCllglycine/1 mg/ml BSA, pH 2.35). The
eluates
were neutralized with 2 M Tris-base and incubated with 2 ml of a mid-log
bacteria
culture. Aliquots of 20 ~,1 were removed for plating, and phage were harvested
as
described. The phosphorylation-selection step was repeated. Phosphorylated
peptides
binding to 133 and 135 cytoplasmic domains were analyzed as described in the
previous
section.
[0228] Matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass
spectrometry was used to map in vitro phosphorylation sites on the (33 and (35
cytoplasmic domains and cytoplasmic domain-binding peptides. The fusion
proteins or
peptides were phosphorylated i~2 vitro as described and purified by RP-HPLC or
RP
microtip columns. Phosphorylated peptides were identified by three methods:
(1) 80-Da
mass shifts after kinase reactions; (2) loss of 80 Da after phosphatase
treatment; or (3)
loss of 80 Da or 98 Da in reflector vs. linear mode for tyrosine
phosphorylated or serine,
threonine phosphorylated peptides, respectively. Where needed, peptides were
purified
by RP=HPLC and subjected to carboxypeptidase and aminopeptidase digestions to
produce sequence ladders. This was particularly useful where one peptide may
harbor
two or more phosphorylation sites.
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Pafanifzg on phosphorylated GST- fusion proteins.
[0229] GST fusion proteins were phosphorylated iu vitro as described (Schmitz
et
al., 1996; Dente et al., 1997; Gram et al., 1997). Briefly, 10 lCg/ml was
incubated for 3 h
at room temperature with 5.5 units of Fyn protein kinase in reaction buffer
(50 mM Tris,
5mM MgCl2, 500 ,uM Na3V04, 500 p,M ATP in a total volume of 50 ~,1). The
reaction
was stopped by adding 40% of TCA. After the kinase substrate protein was
precipitated,
it was resuspended in PBS and coated on microtiter wells at 10 ~.glwell. An
aliquot of
CX7C library (2.5x1011 transducing units) was incubated on the GST fusion
proteins.
Phage were sequenced from randomly selected clones.
Mass Spectrometry Studies
[0230] Mass spectrometric peptide mass mapping was used to identify novel
ligands
for f33 and/or 135 cytoplasmic domains. Polyclonal and monoclonal antibodies
raised
against the cytoplasmic domain-binding peptides were used to purify target
proteins
(cytoskeletal or signaling molecules). These proteins were resolved by SDS-
PAGE, cut
out from the SDS gels, and digested in-gel with trypsin. After extraction of
the peptides,
MALDI-TOF mass spectrometry analysis was performed to produce a list of
peptide
masses. This list of peptide masses, in combination with protease specificity,
produces a
relatively specific "signature" that can be used to search sequence databases.
If the
protein sequence is present in a database, the protein can be identified with
high
confidence by this method. The lower detection limit for this approach is
currently 1
pmol, at least 10-20- fold better than N-terminal Edman sequencing methods.
Results
Pafzaif2g of phage peptide libraries on J33 or f35 cytoplasmic domaifis.
[0231] 133 and 135 cytoplasmic domain-binding peptides were isolated by
screening
multiple phage libraries with recombinant GST fusion proteins that contained
either
GST-(33cyto or GST-135cyto coated onto microtiter wells. Immobilized GST was
used as
a negative control for enrichment during the panning of each cytoplasmic
domain.
Phage were sequenced from randomly selected clones after three rounds of
panning as
disclosed elsewhere (I~oivunen et al., Biotechnology 13:265-270, 1995;
Pasqualini et al.,
1995). Distinct sequences were isolated that interacted specifically with the
133 or with
CA 02458047 2004-02-19
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the 135 cytoplasmic domains (Table 4). Randomly selected clones from panning
rounds
II and III were sequenced. Amino acid sequences of the phagemid encoded
peptides were
deduced from nucleotide sequences. The most frequent motifs found after
panning with
the indicated libraries are shown in Table 4. The ratios were calculated by
dividing the
number of colonies recovered from (33-GST-coated wells and those recovered
from GST
or BSA.
Table 4. Sequences displayed by phage binding to 133 or X35 integrin
cytoplasmic
domain
Peptide motif SEQ ID NO [33/GST [33BSA
Ratio Ratio
CX~ Libr
CEQRQTQEGC SEQ ID NO:27 4.3 14
CARLEVLLPC SEQ ID NO:28 2.8 18.7
X4YX4 I-ibr
YDWWYPWSW SEQ ID N0:29 5.6 163
GLDTYRGSP SEQ ID N0:30 4.1 48
SDNRYIGSW SEQ ID N0:31 3.3 32
YEWWYWSWA SEQ ID N0:32 2.2 28.1
KVSWYLDNG SEQ ID N0:33 2.1 20
SDWYYPWSW SEQ ID N0:34 2.1 157
AGWLYMSWI~ SEQ ID N0:35 1.8 2.4
Pool Cyclic Libraries
CFQNRC SEQ ID NO:36 3.1 16
CNLSSEQC SEQ ID NO:37 2.7 62
CLRQSYSYNC SEQ ID N0:38 2.4 3.2
Peptide motif SEQ ID NO [35/GST (35BSA
Ratio Ratio
Pool Cyclic Libraries
CYIWPDSGLC SEQ ID N0:39 5.2 193
CEPYWDGWFC SEQ ID N0:40 3.1 400
CKEDGWLMTC SEQ ID NO:41 2.3 836
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CKLWQEDGY SEQ ID N0:42 1.8 665
CWDQNYLDDC SEQ ID NO:43 1.5 100
XYX4 Libr
DEEGYYM1VIR SEQ ID NO:44 11.5 29
KQFSYRYLL SEQ ID NO:45 4.5 8
VVISYSMPD SEQ ID N0:46 3.8 28
SDWYYPWSW SEQ ID N0:34 2.4 304
[0232] The specificity of the interaction with 133 or 135 cytoplasmic domains
was
determined by calculating the ratios of phage bound to the cytoplasmic domain
containing-fusion proteins (133 or 135) versus GST alone (negative control).
FIG. 18
shows the results from binding assays performed with the GST-133cyto binding
phage.
Six phage were tested that displayed the motifs most frequently found during
the second
and third rounds of panning. Each panel shows the results from binding assays
for the
phage displaying different peptides that bind to the 133 cytoplasmic domain,
as indicated.
Insertless phage or unselected libraries were used as negative controls and
did not show
binding above background. Two plating dilutions were shown for each assay.
[0233] A similar strategy was used to determine the specificity of the phage
isolated
in the screenings involving the 135 cytoplasmic domain fusion protein. The
binding
assays were performed with individually amplified phage, shown in FIG. 19.
Five phage
were tested that displayed the motifs found most frequently during the second
and third
rounds of panning. Each panel shows the binding assays for the phage
displaying
peptides that bind to the 135 cytoplasmic domain. Insertless phage or
unselected libraries
were used as negative controls and did not show binding above background in
these
assays.
[0234] To determine whether the binding of the selected motifs was specific
for each
cytoplasmic domain, binding assays were performed comparing the interaction of
individual phage motifs with 131, 133, or 135 cytoplasmic domain fusion
proteins. ELISA
with anti-GST antibodies showed that the three proteins can be coated onto
plastic at
equivalent efficiency, and thus the differences in binding do not reflect
differences in
coating concentrations (not shown). Both the 133- and 135-selected phage
selectively
87
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WO 03/022991 PCT/US02/27836
interacted with the proteins on which they were originally selected, with
average binding
selectivities observed of (33/(31 = 3.9, [33/(35 = 3.7, (35/(31 = 4.8, and
(35/(33 = 6.9 (not
shown). The average selectivity for integrin cytoplasmic domains versus BSA
was about
one to two orders of magnitude (not shown). None of the phage tested seemed to
bind
strongly to the 131 cytoplasmic domain (not shown).
Characterization of syyathetic peptides correspofZding to the sequences
displayed
by the integrin-cytoplasmi,c douzaifi-bif2difag pl2age.
[0235] Specific phage were selected for further studies on the basis of their
binding
properties. Synthetic peptides corresponding to the sequence displayed by each
phage
were used to perform binding inhibition studies. This assay determined whether
phage
binding was entirely mediated by the targeting peptide displayed by the phage
or whether
it also included a non-specific component. As expected, the synthetic peptides
inhibited
the binding of the corresponding phage in a dose-dependent manner (FIG. 20 and
FIG.
21). A control peptide containing unrelated amino acids had no effect on phage
binding
when tested at identical concentrations.
Phosphorylatioya evef~ts f-nodulate the irtteraetion of the selected peptides
with
cytoplasrraic dot~zains
[0236] Events involving phosphorylation are important in regulating signal
transduction. The phage display system was used to evaluate the effect of
tyrosine
phosphorylation at twa levels. First, recombinant fusion proteins containing
133 or 135
cytoplasmic domains were used for panning of phage libraries displaying
tyrosine-
containing peptides. Second, the cytoplasmic domains themselves were
phosphorylated
before phage selection was performed. Experiments were performed to
investigate the
capacity of specific tyrosine kinases to modulate the interaction of the
selected peptides
with the cytoplasmic domains. The results obtained in the panning of phage
libraries
displaying tyrosine-containing peptides axe shown in Table 5.
[0237] Randomly selected clones from rounds ITI and IV were sequenced from a
X4YX4 phosphorylated library with Fyn. Amino acid sequences of the phagemid
encoded peptides were deduced from nucleotide sequences. Table 5 shows the
motifs
found most frequently after the indicated libraries were panned with (33 or
[35. The ratio
88
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of binding to (33 or (35 was calculated by dividing the number of (33 or (35
colonies by
GST or BSA colonies found after panning. The ratio of binding to 133 or 135
with
phosphorylated phage by Fyn versus unphosphorylated phage was calculated by
dividing
the number of colonies found after the panning.
Table 5. Sequences displayed by phosphorylated phage binding to integrin
cytoplasmic domains.
Peptide Motif Phos/Unphos (33 or [35/GST(33 or [35BSA
j33 c.~toRlasmic
GGGSYRHVE SEQ ID N0:4913.2 1.5 5.3
RAILYRLAN SEQ ID N0:502.8 1.3 20
MLLGYRFEK SEQ ID NO:512.5 3.5 2.7
X35 cytoplasmic
TMLRYTVRL SEQ ID NO:5214.3 3.4 2.2
TMLRYFMF'P SEQ ID NO:534.2 2.3 3.8
TLRKYFHSS SEQ ID N0:543.8 3.8 15.2
[0238] The effect of phosphorylation on the affinity and specificity of the
cytoplasmic domain-binding was examined. Phage displaying the 133 and 135
cytoplasmic
domain-binding peptides were phosphorylated in vitro as previously described
(Schmitz
et al., 1996; Dente et al., 1997; Gram et al., 1997), using Fyn kinase.
Specific
phosphorylation of the tyrosine-containing peptide on the surface of the phage
was
confirmed by using 32P-gamma dATP in the kinase reaction and by separating the
phage
pllI protein by SDS-PAGE.
[0239] Phage phosphorylated i~z vitro showed increased binding affinity and
specificity to the l33 integrin cytoplasmic domain (FIG. 22). The TLRKYFHSS
(SEQ ID
N0:54) phage was also tested in assays that included other GST-cytoplasmic
domain
fusion proteins to determine specificity (FIG. 23).
Sequence similarity of ihtegrifz bizzdirag peptides with k~zown cytoskeletal
a~zd
signalifzg proteins.
[0240] The peptides displayed by integrin cytoplasmic domain-binding phage
were
similar to certain regions found within cytoskeletal proteins and proteins
involved in
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signal transduction (Table 6). The similarity of some of the isolated peptides
to a region
of mitogen-activated protein kinase 5 (MAPKS, amino acids 227-234) was
particularly
interesting. A connection involving the MAPK cascade, cell adhesion, migration
and
proliferation has been proposed (Lin et al., 1997)
Table 6. Sequence similarity of integrin binding peptides with known
cytoskeletal
and signaling proteins.
Isolated MotifCandidate Proteins .Region Homology
(AA #) Io
J33 c~plasmic
GLDTYRGSP Ras-related protein 124-133 75
(SEQ ID N0:30)Ser/Thr kinase (K-11) 18-25 75
SDNRYIGSW PDGF receptor 985-992 85
(SEQ ID N0:31)Phosphatidylinositol 4 phosphatase233-241 85
5
Receptor protein kinase 185-191 85
Protein kinase clk2 71-79 63
CEQRQTQEGC MAPK5 227-234 75
(SEQ ID N0:27)Phosphatidylinositol 3-kinase494-503 78
CLRQSYSYNC Cyclin-dependent kinase 5 230-239 75
(cdk5)
(SEQ ID N0:38)
X35 c~toplasmic
VVISYSMPD Ser/Thr kinase 479-485 83
(SEQ ID N0:46)IEN ((3 chain) 27-35 70
Actin 240-248 67
DEEGYYMMR Focal adhesion kinase 43-51 75
(SEQ ID N0:44)Tubulin 60-66 100
Putative Ser/Thr kinase 292-299 86
Me~zbrane permeable peptides
[0241] Penetratin is a peptide that can translocate hydrophilic compounds
across the
plasma membrane. Fusion to the penetrating moiety allows oligopeptides to be
targeted
directly to the cytoplasm, nucleus, or both without apparent degradation
(Derossi et al.,
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
1994). This membrane-permeable peptide consists of 16 residues
(RQIK1WFQNRRMI~WKK, SEQ ID N0:55) corresponding to amino acids 43-58 of the
homeodomain of Antennapedia, a Drosophila transcription factor (Juliet et al.,
1991a,
1991b; Le Roux et al., 1993). Internalization mediated by penetratin occurs at
both 37°C
and 4°C, and the internalized peptide can be retrieved intact from
cells.
[0242] Peptides were designed containing penetratin sequences fused to the
sequences of motifs found to bind 133 or 135 cytoplasmic domains. The peptides
were
synthesized on a 431 Applied Biosystems peptide synthesizer using p-
hydroxymethylphenoxy methyl polystyrene (HMP) resin and standard Fmoc
chemistry.
Peptide internalization and visualization was performed as described (Derossi
et al.,
1994; Hall et al., 1996; Theodore et al., 1995).
[0243] Briefly, 10-50 ~,g/ml of the biotinylated peptide was added to cells in
culture.
Peptides were incubated with plated cells. After 2-4 hours, the cultures were
washed
three times with tissue culture media, fixed and permeabilized using
ethanol:acetic acid
(9:1) for 5 min at -20°C. Nonspecific protein binding sites were
blocked by incubating
the cultures for 30 min with Tris-buffered saline (TBS) containing 10% fetal
calf serum
(FCS) and 0.02% Tween. The cultures were incubated in the same buffer
containing
FTTC-conjugated Streptavidin (1:200 dilution) and washed with TBS before being
mounted for viewing by confocal microscopy. The penetratin-linked peptides
were
internalized quite efficiently (data not shown).
[0244] Functional data showed that the cytoplasmic domain-binding peptides
selected on (33 or (35 can interfere with integrin-mediated signaling and
subsequent
cellular responses (i.e., endothelial cell adhesion, spreading, proliferation,
migration). A
commercial panel of "internalizable" versions of the synthetic motifs found by
phage
screenings (SDNRYIGSW (SEQ ID N0:31); CEQRQTQEGC (SEQ ID N0:27); 133
binding peptides and VVISYSMPD (SEQ ID NO:46); a 135-binding peptide) were
obtained. These complex chimeric peptides consist of the most selective of the
(33 or (35-
cytoplasmic domain-binding peptides coupled to penetratin, plus a biotin
moiety to allow
the peptides to be tracked once they were internalized into intact cells.
These membrane-
permeable forms of the peptides are internalized, may affect (33 and [35 post-
ligand
binding cellular events and can induce massive apoptosis (data not shown).
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Endothelial cell proliferation, chemotaxis and apoptosis
[0245] The effect of 133 and 135 integrin cytoplasmic domain-binding motifs on
endothelial cell proliferation was evaluated after stimulation with factors
that activate
endothelial cells (FIG. 24). Cell proliferation was measured according to
Pasqualini and
Hemler (1994). Briefly, 4x104 HLTVECs were incubated in 24-well plates and
starved for
24 h, after which the medium was removed and replaced in the presence of VEGF
and 15
~,M of each peptide. After another 18 h of incubation, 50 ~,1 of medium
containing
[3H]thymidine (l,uCi/rnl) was added to the wells. After 6 additional hours of
incubation
at 37°C, the medium was removed and the cells were fixed in 10% TCA for
30 min at
4°C, washed with ethanol and solubilized in 0.5 N NaOH. Radioactivity
was counted by
liquid scintillation by using a LS 6000SC Beckman scintillation counter. Each
experiment was performed three times with triplicates, and the results were
expressed as
the mean ~ SD.
[0246] The effect of 133 and 135 integrin cytoplasmic domain-binding motifs in
endothelial cell migration was evaluated after stimulation with factors that
activate
endothelial cells. The peptides tested affected cell function in a dose-
dependent and
specific way. Their properties seem to be intrinsic to the 133 or to the 135
cytoplasmic
domain (FIG: 25).
Claemotaxis Assay.
[0247] Cell migration was assayed in a 48-well microchemotaxis chamber.
Polyvinylpyrrolidone-free polycarbonate filters with 8-~,m pores were coated
with 1%
gelatin for 10 min at room temperature and equilibrated in M199 medium
supplemented
with 2% FCS. Peptides were placed in the lower compartment of a Boyden chamber
in
M199 supplemented with 2% FCS, 20 ng/ml VEGF-A (R&D System), and 1 U/ml
heparin. Overnight-starved subconfluent cultures were quickly trypsinized, and
resuspended in M199 containing 2% FCS at a final concentration of 2x106
cells/ml.
After the filter was placed between lower and upper chambers, 50 ~,1 of the
cell
suspension was seeded in the upper compartment. Cells were allowed to migrate
for 5 h
at 37°C in a humidified atmosphere with 5% C02. The filter was then
removed, and
cells on the upper side were scraped with a rubber policeman. Migrated cells
were fixed
in methanol and stained with Giemsa solution. Five random high-power fields
92
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
(magnitude 40~e) were counted in each well. The results showed that both [33-
integrin
cytoplasmic domain binding peptides increased cell migration but penetratin
did not
affect the cells (data not shown).
Apoptosis assay (Propidium Iodide (PI) staining subdiploid population).
[0248] Approximately 1x10 cells were harvested in complete medium, and 15 ~,M
of peptide was added for 4, 8, or 12 hours. The cells were then washed in PBS
and
resuspended in 0.5 ml propidium iodide solution (50 ,ug/ml PI, 0.1% Triton X-
100, 0.1%
sodium citrate). After a 24-h incubation at 4°C, the cells were counted
with an XL
Coulter (Coulter Corporation) with a 488 nm laser; 12,000 cells were counted
for each
histogram, and cell cycle distributions were analyzed with the Multicycle
program.
[0249] Treatment of cells with VISY-penetratin chimera resulted in induction
of
apoptosis (FIG. 26, panel d). Pro-apoptotic effects were not observed when the
cells
were exposed to other growth factors (not shown). Penetratin alone and the
other
penetratin chimeras also could not induce similar effects (FIG. 26, panel c).
This finding
shows that novel approaches for inhibiting angiogenesis can be developed based
on the
use of integrin targeting peptides.
Immunization with cytoplasmic domain binding peptides and characterization of
the resulting antibodies
[0250] Polyclonal antibodies that recognize av133 and ccvl35-binding peptides
were
generated using KLH conjugates made with the synthetic peptides, according to
standard
techniques. Antibodies against two different synthetic peptides have been
produced
(FIG. 27). The sera not only recognize the immobilized peptides, but also
recognize
specific proteins in total cell extracts, as shown by western blot analysis
(FIG. 28).
[0251] Rabbits were immunized with SDNRYIGSW (SEQ ID N0:31) or
GLDTYRGSP (SEQ ID N0:30) -I~LH conjugates. Each rabbit was injected with 200
p,g
of peptide conjugated with KLH in Complete Freund's Adjuvant. Between 20 and
60
days later, the rabbits were injected with 100 ,ug Incomplete Freund's
Adjuvant. After
the third immunization, sera was collected. Pre-immune serum obtained before
the first
immunization was used as an additional control in the experiments.
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CA 02458047 2004-02-19
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[0252] The polyclonal antibodies were tested by ELISA, Western blot and
immunoprecipitation. In the ELISA assays, microtiter well plates were coated
with 10
,ug/ml of peptides. The plates were dried at 37°C, blocked with
PBS+3°Io BSA, and
incubated with different serum dilutions in PBS+1% BSA. After washing and
incubation with the secondary antibody, an alkaline phosphate substrate was
added and
antibody binding detected colorimetrically at 405 nm. The reactivity observed
both in
the mouse and rabbit polyclonal sera was highly specific. In all cases,
antibody binding
could be abrogated by preincubation with the corresponding peptide that was
used for
immunization, but not by a control peptide (FIG. 27 and FIG. 28). Antibodies
raised
against two of the (33 cytoplasmic domain binding peptides recognize specific
bands on
total cell extracts and in immunoprecipitation experiments using 35S-labeled
extracts.
Similar results were obtained with polyclonal sera and purified IgGs (not
shown).
[0253] The present example shows that targeting peptides against specific
domains
of cell receptors can be identified by phage display. Such peptides may be
used to
identify the endogenous ligands for cell receptors, such as endostatin. In
addition, the
peptides themselves may have therapeutic effects, or may serve as the basis
for
identification of more effective therapeutic agents. The endostatin targeting
peptides
identified herein, when introduced into cells, showed effects on cell
proliferation,
chemotaxis and apoptosis. The skilled artisan will realize that the present
invention is
not limited to the disclosed peptides or therapeutic effects. Other cell
receptors and
ligands, as well as inhibitors or activators thereof, may be identified by the
disclosed
methods.
Example 7. Induction of Apoptosis with Integrin Binding Peptides (Endothanos)
[0254] Example 6 showed that the VISY peptide (VVISYSMPD, SEQ ID NQ:46),
imported into cells by attachment to penetratin, could induce apoptosis in
HZJVEC cells.
Antibodies raised against the VISY peptide were used to identify the
endogenous cell
analog of the peptide, identified herein as Annexin V. The results indicate
that Annexin
V is an endogenous ligand for the integrins that is involved in a novel
pathway for
apoptosis.
Methods
Protein purifzcatiof2
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[0255] Polyclonal antibodies against the VISY peptide (VVISYSMPD, SEQ ID
N0:46) were prepared using the methods described in Example 6. MDA-MB-435
breast
carcinoma cells were used for purification of the endogenous VISY peptide
analog.
Cells were washed three times with ice cold PBS and lysed with chilled water
for 20
min. Cell extracts were centrifuged for 30 min at 100,000 x g to separate the
cytoplasmic fraction from the membrane fraction. The cytoplasmic fraction was
subjected to column chromatography on a gel filtration column (10-50kDa) and
an anion
exchange column (mono Q). The anion exchange column was eluted with a salt
gradient
from 50 mM to 1 M NaCI. One ml fractions were collected, run on SDS-PAGE and
tested by Western blotting for the presence of endogenous proteins reactive
with the anti-
VISY antibody. The fraction of interest, containing a 36 kDa antibody reactive
band,
eluted at about 300 mM NaCl.
[0256] The 36 kDa always appeared in fractions that showed positive reactivity
with
the anti-VISY antibody. The fractions were analyzed by SDS-PAGE and 2-D gel
electrophoresis, followed by Western blotting. A substantial enrichment of the
36 kDa
protein was seen after column chromatography (not shown). The 36 kDa peptide
was cut
from the SDS-PAGE gel and analyzed by mass spectroscopy to obtain its
sequence. All
five peptide sequences that were obtained by mass spectroscopy showed 100%
homology to the reported sequence of Annexin V (GenBank Accession No. GI
468888).
In addition to its presence in 435 cells, the 36 kDa band was also seen in
I~aposi
sarcoma, SKOV and HUVEC cells (not shown).
[0257] Commercial antibodies against Annexin V were obtained (Santa Cruz
Biologics, Santa Cruz, CA). Comparative Western blots were performed using the
anti-
VISY antibody and the anti-Annexin V antibody. Both antibodies showed
reactivity
with the 36 kDa protein (not shown). These results indicate that the
endogenous protein
analog of the VISY peptide is Annexin V.
Proteifa proteifz interaction with Annexin V and,/35 cytoplasmic domain.
[0258] Competitive binding assays were performed to examine the binding of
Annexin V to (35 integrin and the effect of the VISY peptide. Plates were
coated with
GST fusion proteins of the cytoplasmic domains of various integrins and
Annexin V was
added to the plates. Binding of Annexin V was determined using anti-Annexin V
CA 02458047 2004-02-19
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antibodies. As shown in FIG. 29A, Annexin V did not bind to either the GST-(31
or
GST-(33 integrins. Annexin V bound strongly to the GST-(35 integrin, but
binding was
dependent on the buffer used (FIG. 29A). Low binding was observed in Tris-
buffered
saline (TBS), while high binding was observed in "cytoplasmic buffer" (100 mM
KCI, 3
mM NaCI, 3.5 mM MgCl2, 10 mM PIPES, 3 mM DTT) with or without added calcium
(2 mM) (FIG. 29A). Calcium was used because Annexin V activity has been
reported to
be modulated by calcium. Binding of Annexin V to GST-(35 was blocked by
addition of
the VISY peptide (FIG. 29A). FIG. 29B shows the relative levels of binding of
anti-
Annexin V antibody to purified Annexin V and to VISY peptide.
[0259] A reciprocal study was performed, using Annexin V to coat plates and
adding
GST fusion proteins of integrin cytoplasmic domains. Binding was assessed
using anti-
GST fusion protein antibodies. As expected, only GST-(35 showed substantial
binding to
Annexin V, while GST-(31 and GST-(33 showed low levels of Annexin V binding
(not
shown). In some studies, calcium ion appeared to interfere with the binding
interaction
between GST-(35 and Annexin V, with decreased binding observed in the presence
of
calcium (not shown). A greater degree of inhibition of Annexin V binding to
GST-(35 by
the VISY peptide was observed in the presence of calcium (67°7o
inhibition) than in the
absence of calcium (45%) (FIG. 29A).
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Penetratin peptide chimera binding to the f35 cytoplasmic domain induces
programmed cell death.
[0260] The induction of apoptosis by VISY peptide was shown in Example 6 was
confirmed. 10~ HCTVEC were treated with 15 ~,M of VISY antennapedia
(penetratin)
chimera or 15 ~,M of antennapedia peptide (pentratin) alone for 2-4 hours and
chromatin
fragmentation was analyzed by electrophoresis in an agarose gel. FIG. 30 shows
the
induction of apoptosis by VISY-Ant (penetratin), as indicated by chromatin
fragmentation. Neither VISY nor penetratin alone induced apoptosis. Induction
of
apoptosis was inhibited up to 70% when a caspase inhibitor (zVAD, caspase
inhibitor I,
Calbiochem #627610, San Diego, CA) was added to the media at the same time as
the
VISY chimeric peptide.
[0261] A distinction between the mechanism of cell death induced by VISY
peptide
and other pro-apoptosis agents is that other apoptotic mechanisms evaluated in
cell
culture typically involve detachment of the cells from the substrate, followed
by cell
death. In contrast, in VISY induced cell death, the cells do not detach from
the substrate
before dying. Thus, endothanos (death from inside) appears to differ from
anoikis
(homelessness).
[0262] The present results show that VISY peptides activate an integrin
dependent
apoptosis pathway. The present example shows that the endogenous analog for
VISY
peptide is Annexin V. These results demonstrate the existence of a novel
apoptotic
pathway, mediated through an interaction between Annexin V and (35 integrin
and
dependent on caspase activity. This novel apoptotic mechanism is termed
endothanos.
The skilled artisan will realize that the existence of a novel mechanism for
inducing or
inhibiting apoptosis is of use for a variety of applications, such as cancer
therapy.
Example 8. Identification of Receptor/Ligand Pairs: Aminopeptidase A regulates
endothelial cell function and angiogenesis
[0263] Endothelial cells in tumor vessels express specific angiogenic markers.
Aminopeptidase A (APA, EC 3.4.11.7) is upregulated in microvessels undergoing
angiogenesis. APA is a homodimeric, membrane-bound zinc metallopeptidase that
hydrolyzes N-terminal glutamyl or aspartyl residues from oligopeptides (Nanus
et. al.,
1993). In vivo, APA converts angiotensin II to angiotensin III. The renin-
angiotensin
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system plays an important role in regulating several endocrine,
cardiovascular, and
behavioral functions (Ardaillou, 1997; Stroth and Unger, 1999). Recent studies
also
suggest a role for angiotensins in angiogenesis (Andrade et al., 1996), but
the function of
APA in the angiogenic process has not been investigated so far.
[0264] In the present example, targeting peptides capable of binding APA were
identified by screening phage libraries on APA-expressing cells. APA-binding
peptides
containing the motif CPRECESIC (SEQ ID N0:56) specifically inhibited APA
enzyme
activity. Soluble CPRECESIC (SEQ ID N0:56) peptide inhibited migration,
proliferation, and morphogenesis of endothelial cells in vitro and interfered
with iT2 vivo
angiogenesis in a chick embryo chorioallantoic membrane (CAM) assay.
Furthermore,
APA null mice had a decreased amount of retinal neovascularization compared to
wild
type (wt) mice in hypoxia-induced retinopathy in premature mice. These results
may
lead to a better understanding of the role of APA in angiogenesis and to
development of
new anti-tumor therapeutic strategies.
Materials and Methods
Cell cultures
[0265] The renal carcinoma cell line SIB-RC-49 was transfected with an
expression
vector encoding full-length APA cDNA (Geng et al., 1998). Cells were
maintained in
MEM (Irvine Scientific, Santa Ana, CA), supplemented with 2 mM glutamine, 1%
nonessential amino acids, 1% vitamins (Gibco BRL), 100 U/ml streptomycin, 100
U/ml
penicillin (Irvine Scientific), 10 mM sodium pyruvate (Sigma-Aldrich), and 10%
fetal
calf serum (FCS) (Tissue Culture Biological, Tulare, CA). Stably transfected
cells were
maintained in 6418-containing medium. HWECs were isolated by collagenase
treatment and used between passages 1 to 4. Cells were grown on gelatin-coated
plastic
in M199 medium (Sigma) supplemented with 20% FCS, penicillin (100 U/ml),
streptomycin (50 ~.g/ml), heparin (50 ~,g/ml), and bovine brain extract (100
~,g/ml). All
media supplements were obtained commercially (Life Technologies, Inc., Milan,
Italy).
Antibodies aT2d peptides
[0266] The anti-APA mAb RC38 (Schlingemann et. al., 1996) was used to
immunocapture APA from transfected cell lysates. CPRECESIC (SEQ ID N0:56) and
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GACVRLSACGA (SEQ ID N0:57) cyclic peptides were chemically synthesized,
spontaneously cyclized in non-reducing conditions, and purified by mass
spectrometry
(AnaSpec San Jose, CA). The mass spectrometer analysis of the CPRECESIC (SEQ
ID
N0:56) peptide revealed six different peaks, possibly reflecting different
positions of
disulfide bounds and the formation of dimers. Due to the similar biochemical
behavior of
the different fractions on APA enzyme activity, a mix of the six peaks was
used in all
procedures described below.
APA inanzuhocapture
[0267] Cells were scraped from semi-confluent plates in cold PBS containing
100
mM N-octyl-(3-glucopyranoside (Calbiochem), lysed on ice for 2 h, and
centrifuged at
13,000 x g for 15 min. Microtiter round-bottom wells (Falcon) were coated with
2 ~,g of
RC38 for 4 h at room temperature and blocked with PBS/3% BSA (InterGen,
Purchase,
NY) for 1 h at room temperature, after which 150 ~1 of cell lysate (1 mg/ml)
was
incubated on the mAb-coated wells overnight at 4°C, washed five times
with PBS/0.1%
Tween-20 (Sigma), and washed twice with PBS.
APA er-azyjne assay
[0268] Cells and immunocaptured proteins were tested for specific enzyme
activity
according to Liln et al. (1998). Briefly, adherent cells or RC38-
irnmunocaptured cell
extracts were incubated for 2 h at 37°C with PBS containing 3 mM a-L-
glutamyl-p-
nitroanilide (Fluka) and 1 mM CaCl2. Enzyme activity was determined by reading
the
optical absorbance (0.D.) at 405 nm in a microplate reader (Molecular Devices,
Sunnyvale, CA).
Cell panhirzg
[0269] A CX3CX3CX3C (C, cysteine; X, any amino acid) library was prepared
(Rajotte et al., 1998). Amplification and purification of phage particles and
DNA
sequencing of phage-displayed inserts were performed as described. Cells were
detached
by incubation with 2.5 mM EDTA in PBS, washed once in binding medium (DMEM
high glucose supplemented with 20 mM HEPES and 2% FCS), and resuspended in the
same medium at a concentration of 2x106 cells/ml. 101° TU of phage were
added to 500
~.l of the cell suspension, and the mixture was incubated overnight (first
round) or for 2 h
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(successive rounds) at 4°C with gentle rotation. Cells were washed five
times in binding
medium at room temperature and resuspended in 100 ~.l of the same medium.
Phage
were rescued by adding 1 ml of exponentially growing K9lKan Escher ichia coli
bacteria
and incubating the mixture for 1 h at room temperature. Bacteria were diluted
in 10 ml of
LB medium supplemented with 0.2 ~g/ml tetracycline and incubated for another
20 min
at room temperature. Serial dilutions were plated on LB plates containing 40
~,g/ml
tetracycline, and plates were incubated at 37°C overnight before
colonies were counted.
Phage bifzdirzg specificity assay
[0270] The cell binding assay was performed with an input of 109 TU as
described
for the cell panning. The specificity was confirmed by adding CPRECESIC (SEQ
ID
N0:56) peptide to the binding medium in increasing concentrations. For phage
binding
on immunocaptured APA, wells were blocked for 1 h at room temperature with
PBS/3%
BSA and incubated with 109 TU for 1 h at room temperature in 50 ~,1 PBS/3%
BSA.
After eight washes in PBS/1% BSA/0.01% Tween-20 and two washes in PBS, phage
were rescued by adding 200 ~,1 of exponentially growing K9lKan E. coli. Each
experiment was repeated at least three times.
Izz vivo tu~rzor hozniz2g of APA-binding phage
[0271] MDA-MB-435-derived tumor xenografts were established in female nude
mice 2 months old (Jackson Labs, Bar Harbor, Maine). Mice were anesthetized
with
Avertin and injected intravenously through the tail vein with 109 TU of the
phage in a
200 ~1 volume of DMEM. The phage were allowed to circulate for 5 min, and the
animals were perfused through the heart with 5 ml of DMEM. The tumor and brain
were
dissected from each mouse, weighed, and equal amounts of tissue were
homogenized.
The tissue homogenates were washed three times with ice-cold DMEM containing a
proteinase inhibitor cocktail and 0.1% BSA. Bound phage were rescued and
counted as
described for cell panning. Fd-tet phage was injected at the same input as a
control. The
experiment was repeated twice. In parallel, part of the same tissue samples
were fixed in
Bouin solution, and imbedded in paraffin for preparation of tissue sections.
An antibody
to M-13 phage (Amersham-Pharmacia) was used for the staining.
Cell growth assay
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[0272] HUVECs were seeded in 48-well plates (104 cells/well) and allowed to
attach
for 24 h in complete M199 medium. The cells were then starved in M199 medium
containing 2% FCS for 24 h. CPRECESIC (SEQ ID N0:56) or control
GACVRLSACGA (SEQ ID N0:57) peptide (1 mM) was added to the wells in medium
containing 2% FCS and 10 ng/ml VEGF-A (R&D System, Abingdom, LTK). After
incubation for the indicated times, cells were fixed in 2.5% glutaraldehyde,
stained with
0.1% crystal violet in 20% methanol, and solubilized in 10% acetic acid. All
treatments
were done in triplicate. Cell growth was evaluated by measuring the O.D. at
590 nm in a
microplate reader (Bio-Rad Laboratories, Hercules, CA). A calibration curve
was
established and a linear correlation between O.D. and cell counts was observed
between
103 and 105 cells.
Chernotaxis assay
[0273] A cell migration assay was performed in a 48-well microchemotaxis
chamber
(NeuroProbe, Gaithersburg, MD) according to Bussolini et al. (1995).
Polyvinylpyrrolidone-free polycarbonate filters (Nucleopore, Cambridge, MA)
with 8-
~m pores were coated with 1 % gelatin for 10 min at room temperature and
equilibrated
in M199 medium supplemented with 2% FCS. CPRECESIC (SEQ ID N0:56) or control
GACVRLSACGA (SEQ ID N0:57) peptide (1 mM) was placed in the lower
compartment of a Boyden chamber in M199 medium supplemented with 2% FCS and 10
ng/ml VEGF-A (R&D System). Subconfluent cultures that had been starved
overnight
were harvested in PBS containing 2.5 mM EDTA, washed once in PBS, and
resuspended
in M199 medium containing 2% FCS at a final concentration of 2x10 cells/ml.
After
the filter was placed between the lower and upper chambers, 50 ~1 of the cell
suspension
was seeded in the upper compartment, and cells were allowed to migrate for 5 h
at 37°C
in a humidified atmosphere with 5% C02. The filter was then removed, and cells
on the
upper side were scraped with a rubber policeman. Migrated cells were fixed in
methanol
and stained with Giemsa solution (Diff-Quick, Baxter Diagnostics, Rome,
Italy). Five
random high-power fields (magnitude 100x) were counted in each well. Each
assay was
run in triplicate.
Three-dinZensional cell cultuYe
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[0274] Matrigel (Collaborative Research, Bedford, MA) was added at 100 ~.l per
well to 48-well tissue culture plates and allowed to solidify for 10 min at
37°C.
HLIVECs were starved for 24 h in M199 medium supplemented with 2% FCS before
being harvested in PBS containing 2.5 mM EDTA. 104 cells were gently added to
each
of the triplicate wells and allowed to adhere to the gel coating for 30 min at
37°C. Then,
medium was replaced with indicated concentrations of CPRECESIC (SEQ ID N0:56)
or
GACVRLSACGA (SEQ ID N0:57) peptides in complete medium. The plates were
photographed after 24 h with an inverted microscope (Canon). The assay was
repeated
three times.
CAM assay
[0275] In vivo angiogenesis was evaluated by a CAM assay (Ribatti et al.,
1994).
Fertilized eggs from White Leghorn chickens were maintained in constant
humidity at
37°C. On the third day of incubation, a square window was opened in the
eggshell and
2-3 ml of albumen was removed to detach the developing CAM from the shell. The
window was sealed with a glass plate of the same size and the eggs were
returned to the
incubator. At day 8, 1 mm3 sterilized gelatin sponges (Gelfoam, Upjohn Co,
Kalamazoo,
Milan) were adsorbed with VEGF-A (20 ng, R&D System) and either CPRECESIC
(SEQ ID NO:56) or control GACVRLSACGA (SEQ m N0:57) peptide (1 mM) in 3 ~,1
PBS and implanted on the top of the growing CAMS under sterile conditions.
CAMS
were examined daily until day 12 and photographed ifa ovo with a Leica
stereomicroscope. Capillaries emerging from the sponge were counted. The assay
was
repeated twice.
Inductiofz of retifzal heovascularizatio~z
[0276] APA null mice have been described (Lin et al., 1998). Mice pups on P7
(7tn
day postpartum) with their nursing mothers were exposed to 75% oxygen for 5
days.
Mice were brought back to normal oxygen (room air) on P12. For histological
analysis
mice were killed between P17 and P21 and eyes were enucleated and fixed in 4%
paraformaldehyde in PBS overnight at 4°C. Fixed eyes were imbedded in
paraffin and 5
~.m serial sections were cut. Sections were stained with hematoxylin/eosin
solution.
Neovascular nuclei on the vitreous side of the internal limiting membrane were
counted
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from 20 h/e-stained sections per each eye. The average number of neovascular
nuclei
per section was calculated and compared between animal groups using Student's
t-test.
Results
Cell panfzing with plaage display select an APA-bindifig motif
[0277] To identify a peptide capable of binding to APA, cells were screened
with a
random peptide phage library. First, SK-RC-49 renal carcinoma cells, which do
not
express APA, were transfected with full-length APA cDNA to obtain a model of
APA
expression in the native conformation. APA expressed as a result of
transfection was
functionally active, as evidenced by an APA enzyme assay (not shown), but
parental SK-
RC-49 cells showed neither APA expression nor activity (not shown).
[0278] The CX3CX3CX3C phage library (101° transducing units [TLJ]) was
preadsorbed on parental SK-RC-49 cells to decrease nonspecific binding.
Resuspended
SK-RC-49/APA cells were screened with phage that did not bind to the parent
cells. SK-
RC-49/APA-bound phage were amplified and used for two consecutive rounds of
selection. An increase in phage binding to SK-RC-49/APA cells relative to
phage
binding to SK-RC-49 parental cells was observed in the second and third rounds
(not
shown).
[0279] Subsequent sequencing of the phage revealed a specific enrichment of a
peptide insert, CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID N0:58),
with a tandem repetition of the general library sequence CX3CX3CX3C. This
sequence
represented 50°7o of 18 randomly selected phage inserts from round 2
and 100% of phage
inserts from round 3. Four peptide inserts derived from round 2 shared
sequence
similarity with the tandem phage (Table 7, in bold font). Several other
apparently
conserved motifs were observed among round 2 peptides (Table 7, underlined or
italicized). One of these overlapped in part with the tandem repeated
sequence. A
search for sequence homology of the selected peptides against human databases
did not
yield a significant match.
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Table 7. APA-binding peptide sequences.
Peptide sequences Round 2 (%)l 3 (°7o)
CYNLCIRECESICGADGACWTWCADGCSRSC 50/100
(SEQ ID N0:58)
CLGQCASICVNDC (SEQ ID N0:59) 5/-
CPKVCPRECESNC (SEQ ID N0:60) 5/-
CGTGCAVECEVVC (SEQ ID N0:61) 5/-
CAVACWADCQLGC (SEQ ID N0:62) 5/-
CSGLCTVQCLEGC (SEQ ID N0:63) 5/-
CSMMCLEGCDDWC (SEQ ID NO:64) 5/-
OTHER 20/-
Selected phage inserts are specific APA ligan.ds.
[0280] Phage displaying the peptide inserts
CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID NO:58),
CPKVCPRECESNC (SEQ ID N0:60) or CLGQCASICVNDC (SEQ ID N0:59) were
individually tested for APA binding. All three phage specifically bound to the
surface of
SK-RC-49/APA cells (not shown), with a similar pattern of 6-fold enrichment
relative to
SK-RC-49 parental cells. Control, insertless phage showed no binding
preference (not
shown). CGTGCAVECEVVC (SEQ ID N0:61) and the other phage selected in round 2
showed no selective binding to SK-RC-49/APA cells (data not shown). A soluble
peptide, CPRECESIC (SEQ 117 NO:56) containing a consensus sequence reproducing
the
APA-binding phage inserts was synthesized.
[0281] Binding assays were performed with CPKVCPRECESNC (SEQ DJ N0:60)
phage in the presence of the CPRECESIC (SEQ ID N0:56) peptide. Soluble
CPRECESIC (SEQ ID N0:56) peptide competed with CPKVCPRECESNC (SEQ ID
N0:60) phage for binding to SK-RC-49/APA cells, but had no effect on
nonspecific
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binding to SK-RC-49 parental cells (not shown). The unrelated cyclic peptide
GACVRLSACGA (SEQ ID N0:57) had no competitive activity (not shown). Binding
of CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID N0:58) phage was also
displaced by CPRECESIC (SEQ ID NO:56) peptide, but the binding of
CLGQCASICVNDC (SEQ ID N0:59) phage was not affected (data not shown).
[0282] To further confirm the substrate specificity of the selected peptide
inserts,
APA was partially purified from APA-transfected cell extracts by immunocapture
with
mAb RC38. The APA protein immobilized on RC38-coated microwells was
functional,
as confirmed by enzyme assay (not shown). The
CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID N0:58),
CPKVCPRECESNC (SEQ ID N0:60), and CLGQCASICVNDC (SEQ ID N0:59)
phage selectively bound immunocaptured APA, with a 10- to 12-fold enrichment
compared to phage binding to RC38-immunocaptured cell lysates from SK-RC-49
parental cells (not shown).
APA-bifadihg phage target tumors in vivo.
[0283] The ability of the identified peptide to home to tumors was evaluated,
using
nude mice implanted with human breast tumor xenografts as a model system.
Phage
were injected into the tail vein of tumor-bearing mice, and targeting was
evaluated by
phage recovery from tissue homogenates. CPKVCPRECESNC (SEQ ID N0:60) phage
was enriched 4-fold in tumor xenografts compared to brain tissue, which was
used as a
control (FIG. 31). Insertless phage did not target the tumors (FIG. 31).
Neither
CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID N0:58) nor
CLGQCASICVNDC (SEQ ID N0:59) phage showed any tumor-homing preference
(data not shown).
[0284] The homing of CPKVCPRECESNC (SEQ ID N0:60) was confirmed by anti-
M13 immunostaining on tissue sections (not shown). Strong phage staining was
apparent in tumor vasculature but not in normal vasculature (not shown).
Insertless
phage did not bind to tumor vessels.
CPRECESIC (SEQ ID NO: 56) is a specific ifzhibitor of APA activity.
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[0285] To investigate the effect of CPRECESIC (SEQ ID NO:56) on APA enzyme
activity, SK-RC-49/APA cells were incubated with the APA specific substrate a-
glutamyl-p-nitroanilide in the presence of increasing concentrations of either
CPRECESIC (SEQ ID N0:56) or control GACVRLSACGA (SEQ ID N0:57) peptides.
Enzyme activity was evaluated by a colorimetric assay after 2 h incubation at
37°C.
CPRECESIC (SEQ ID N0:56) inhibited APA enzyme activity, reducing the activity
by
60% at the highest concentration tested (FIG. 32). The ICSO of CPRECESIC (SEQ
m
N0:56) for enzyme inhibition was calculated to be 800 ~M. CPRECESIC (SEQ ID
N0:56) did not affect the activity of a closely related protease,
aminopeptidase N (data
not shown).
CPRECESIC (SEQ ID N0:56) izzlzibits migration a>zd proliferatiofz of
endothelial
cells.
[0286] The potential use of CPRECESIC (SEQ ID N0:56) peptide as an anti-
angiogenic drug was determined. First, the effect of APA inhibition by
CPRECESIC
(SEQ ID N0:56) peptide in vitro on the migration and proliferation of human
umbilical
vein endothelial cells (HUVECs) stimulated with VEGF-A (10 ng/ml) was
examined.
The presence of functional APA on HUVECs was evaluated by enzyme assay (not
shown). At the highest concentration tested (1 mM), CPRECESIC (SEQ ID N0:56)
peptide inhibited chemotaxis of HUVECs by 70% in a Boyden chamber assay (FIG.
33).
At the same peptide concentration, cell proliferation was inhibited by 50%
(FIG. 34).
Lower concentrations of CPRECESIC (SEQ ID NO:56) peptide or the
GACVRLSACGA (SEQ ID N0:57) control peptide had no significant effect on cell
migration or proliferation (not shown).
~'PRECESIC (SEQ ID NO: 56) inhibits angiogefzesis in vitro arid in vivo
[0287] The inhibitory effect of CPRECESIC (SEQ ID N0:56) peptide in different
i~2
vitro and irz vivo models of angiogenesis was examined. HLJVECs plated on a
three
dimensional matrix gel differentiate into a capillary-like structure,
providing an ifz vitro
model for angiogenesis. Increasing concentrations of CPRECESIC (SEQ ID N0:56)
peptide resulted in a progressive impairment of the formation of this network
(not
shown). At a peptide concentration of 1 mM, vessel-like branching structures
were
significantly fewer and shorter, and as a result, the cells could not form a
complete
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network organization (not shown). The control peptide GACVRLSACGA (SEQ ID
NO:57) did not affect HLTVEC morphogenesis (not shown).
[0288] A commonly used model of simplified in vivo angiogenesis is the chicken
chorioallantoic membrane (CAM), in which neovascularization can be stimulated
during
embryonic development. An appropriate stimulus, adsorbed on a gelatin sponge,
induces
microvessel recruitment to the sponge itself, accompanied by remodeling and
ramification of the new capillaries. Eight-day-old chicken egg CAMS were
stimulated
with VEGF-A alone (20 ng) or with VEGF-A plus CPRECESIC (SEQ ID N0:56) or
GACVRLSACGA (SEQ ID N0:57) (1 mM) peptides. The CAMS were photographed at
day 12. Neovascularization induced by VEGF-A was inhibited by CPRECESIC (SEQ
ID N0:56) by 40% based on the number of capillaries emerging from the sponge
(Table
8). The neovessels did not show the highly branching capillary structures
typically seen
after VEGF-A stimulation (not shown). Treatment with control peptide
GACVRLSACGA (SEQ ID NO:57) or with lower peptide concentrations of
CPRECESIC (SEQ ID NO:56) had no effect on the number of growing vessels (not
shown).
Table 8. CAM assay for angiogenesis
TREATMENT BLOOD VESSEL NUMBERS
No VEGF-A 12.0 ~ 2.82
VEGF-A . 57.0 ~ 1.41 ~
VEGF-A + control 56.5 ~ 2.12
VEGF-A + CPRECESIC (SEQ ID N0:56) 5.5 ~ 1.41'
*p< 0.01 with the Student-Newman-Keuls test. The results are expressed as the
mean
and standard error from two independent experiments.
APA-deficient mice show irepaired neovasczzlarizatiozz
[0289] The ability of APA+~- and APA-~- null mice to undergo
neovascularization was
examined in a model of hypoxic retinopathy in premature mice. Induction of
retinal
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neovascularization by relative hypoxia was already present in APA+~- mice
compared to
wild type mice (not shown). Neovascularization was almost undetectable in APA
null
mice (not shown). Neovascularization was quantified by counting vitreous
protruding
neovascular nuclei from 20 sections of hypoxic eyes. Significant induction of
retinal
neovascularization (16.17 ~ 1.19 neovascular nuclei/eye section) was seen in
the wild
type mice on postnatal day 17 (P17) after 75% oxygen treatment from P7 to P12.
Decreased amounts of neovascular nuclei were seen in the retinas of APA+~-
(10.76 ~
1.03 neovascular nuclei/eye section) and APA null (4.25 ~ 0.45 neovascular
nuclei/eye
section) mice on P17 after exposure to 75% oxygen from P7 to P12.
Discussion
[0290] In vivo, APA is overexpressed by activated microvessels, including
those in
tumors, but it is barely detectable in quiescent vasculature, making it a
suitable target for
vessel-directed tumor therapy. The present example identified a novel
targeting peptide
ligand for APA, CPRECESIC (SEQ 1D N0:56). Soluble CPRECESIC (SEQ 1D N0:56)
peptide inhibited APA enzyme activity with an ICSO of 800 ~.M.
[0291] Using cultured HCTVECs as an in vitro model of angiogenesis, soluble
CP1RECESIC (SEQ ID N0:56) peptide inhibited VEGF-A-induced migration and
proliferation of HU-VECs. These data are consistent with a requirement for
migration
and proliferation of endothelial cells during angiogenesis. CPRECESIC (SEQ 1D
N0:56) also blocked the formation of capillary-like structures in a Matrigel
model and
inhibited angiogenesis in VEGF-A-stimulated CAMs.
[0292] APA was shown to be an important player in neovascularization induced
by
relative hypoxia, since APA null mice had significantly less retinal
neovascularization
compared to wt mice. These results strengthen the potential of using APA as a
specific
target for the inhibition of tumor angiogenesis.
[0293] In summary, the soluble peptide CPRECESIC (SEQ ID N0:56) is a selective
APA ligand and inhibitor. The inhibition of APA by CPRECESIC (SEQ ID N0:56)
led
to the inhibition of angiogenesis in different ifz vitro and irv vivo assays,
demonstrating
for the first time a prominent role for APA in the angiogenic process.
Furthermore,
APA-binding phage can home to tumor blood vessels, suggesting possible
therapeutic
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uses of CPRECESIC (SEQ ID N0:56) as an inhibitor of tumor neovascularization.
The
endogenous analog of CPRECESIC (SEQ ID N0:56) may be identified by antibody
based purification or identification methods, similar to those disclosed.
Example 9. Screening Phage Libraries by PALM
[0294] In certain embodiments, it is desirable to be able to select specific
cell types
from a heterogeneous sample of an organ or tissue. One method to accomplish
such
selective sampling is by PALM (Positioning and Ablation with Laser
Microbeams).
[0295] The PALM Robot-Microbeam uses a precise, computer-guided laser for
microablation. A pulsed ultra-violet (W) laser is interlaced into a microscope
and
focused through an objective to a beam spot size of less than 1 micrometer in
diameter.
The principle of laser cutting is a locally restricted ablative
photodecomposition process
without heating (Hendrix, 1999). The effective laser energy is concentrated on
the
minute focal spot only and most biological objects are transparent for the
applied laser
wavelength. This system appears to be the tool of choice for recovery of
homogeneous
cell populations or even single cells or subcellular structures for subsequent
phage
recovery. Tissue samples may be retrieved by circumcising a selected zone or a
single
cell after phage administration to the subject. A clear-cut gap between
selected and non-
selected area is typically obtained. The isolated tissue specimen can be
ejected from the
object plane and catapulted directly into the cap of a common micro centrifuge
tube in an
entirely non-contact manner. The basics of this so called Laser Pressure
Catapulting
(LPC) method is believed to be the laser pressure force that develops under
the
specimen, caused by the extremely high photon density of the precisely focused
laser
microbeam. This tissue harvesting technique allows the phage to survive the
microdissection procedure and be rescued.
(0296] PALM was used in the present example to select targeting phage for
mouse
pancreatic tissue, as described below.
Materials and Methods
Ira vivo and in situ Panning
[0297] A CX7C peptide phage library (10~ TLT) was injected into the tail vein
of a
C57BL/6 male mouse, and the pancreas was harvested to recover the phage by
bacterial
infection. Phage from 246 colonies were grown separately in 5 ml LB/kanamycin
(100
109
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
~,g/ml)/tetracycline (40 ~,g/ml) at 37°C in the dark with agitation.
Overnight cultures
were pooled and the phage purified by NaCl/PEG precipitation for another round
of in
vivo bio-panning. Three hundred colonies were picked from the second round of
panning, and the phage were recovered by precipitation. Phage from the second
bio-
panning round was then used for another round of in vivo panning and also was
incubated with thawed frozen murine pancreatic sections for one in situ
panning round.
[0298] For the third if2 vivo panning round, 109 TU phage from the second
round
were injected into a third mouse and allowed to circulate for six minutes,
followed by an
intravenous injection of 50 ~l of FITC-lectin (Vector Laboratories, Inc.).
After a two-
minute circulation, the mouse was perfused through the left ventricle with 3
ml MEM
Earle salts. The pancreas was harvested, frozen at -80 °C in Tissue Tek
(Sakura), and
sectioned onto prepared slides.
[0299] For the third in situ round, purified phage, isolated from the second
round,
were incubated with 4-14 ~,m thawed murine pancreatic sections on ice for 30
minutes.
Sections were rinsed with 100 ~,1 ice-cold PBS 8x at room temperature (RT).
Bound
phage were recovered from each section by adding 100 ~,1 K91 KanR (OD~oo =
2.03) to
infect at RT for 30-60 minutes. Infected K91 KanR were withdrawn from each
section
and allowed to recover in 10 ml LB/Kan/Tet (0.2 ~,g/ml) for 20 minutes in the
dark.
Aliquots from the each culture were plated out onto LB/Kan/Tet (40 ~,g/ml)
plates and
incubated overnight in the dark at 37 °C. The tetracycline
concentration of the remainder
of each culture was increased to 40 ~,g/ml and the cultures were incubated
overnight at
37 °C in the dark with agitation for phage amplification and
purification.
DNA Afnplificatiofa
[0300] Phage were recovered from cryo-preserved FTTC-lectin stained mouse
pancreatic islets and surrounding acinar cells that were microdissected from
14 ~,m
sections using the PALM (Positioning and Ablation with Laser Microbeams) cold
laser
pressure catapulting system. Pancreatic islet and control sections were
catapulted into 1
mM EDTA, pH 8, and frozen at -20 °C until enough material was collected
for PCR
amplification. Phage DNA was amplified with fUSES primers: forward primer 5'
TAA
TAC GAC TCA CTA TAG GGC AAG CTG ATA AAC CGA TAC AATT 3' (SEQ ID
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N0:65), reverse primer 5' CCC TCA TAG TTA GCG TAA CGA TCT 3' (SEQ ID
N0:66). The PCR products were subjected to another round of PCR using a nested
set
of primers. The 3' end of the second primer set was tailed with the M13
reverse primer
for sequencing purposes. The nested primer set used was: forward nested primer
5'
CCTTTCTATTCTCACTCGGCCG 3' (SEQ ID N0:67), reverse nested primer 5'
CAGGAAACAGCTATGACCGCTAAACAACTTTCAACAGTTTCGGC 3' (SEQ ID
N0:68). ~To generate peptide insert sequence containing flanking SfiI
restriction sites,
two more primers were used: forward library primer 5' CACTCGGCCGACGGGGC 3'
(SEQ ID NO:69), reverse primer 5' CAGTTTCGGCCCCAGCGGCCC 3' (SEQ ID
N0:70). PCR products generated from the nested primers were gel purified
(Qiagen),
and confirmed for the presence of a CX7C peptide insert sequence using the M13
reverse
primer by automated sequencing. PCR products generated from the library
primers were
gel purified (Qiagen), ligated into CsCl2 purified fUSES/SfiI, electroporated
into
electrocompetent MC1061 cells, and plated onto LB/streptomycin (100
~.g/ml)/tetracycline (40 ~,g/ml) agar plates. Single colonies were subjected
to colony
PCR using the fUSES primers to verify the presence of a CX7C insert sequence
by gel
electrophoresis. Positive clones were sequenced using BigDye terminators
(Perkin
Elmer)
Plaage Infection
[0301] Pancreatic islet and control sections were catapulted into 1 mM AEBSF,
20
~,glml aprotinin, 10 ~,g/ml leupeptin, 1 mM elastase inhibitor I, 0.1 mM TPCK,
1 nM
pepstatin A in PBS, pH 7.4, and frozen for 48 hours or less until enough
material was
collected. The sections were thawed on ice and the volume adjusted to 200 ~,1
with PBS,
pH 7.4. Samples were incubated with 1 ml K91 KanR (OD = 0.22) for two hours at
RT
on a nutator. Each culture was transferred to 1.2 ml LB/Kan/Tet (0.2 ~,g/ml)
and
incubated in the dark at RT for 40 minutes. The tetracycline concentration was
increased
to 40 ~,g/ml for each culture, and the cultures were incubated overnight at 37
°C with
agitation. Each culture was plated out the following day onto LB/Kan/Tet agar
plates
and incubated for 14 hours at 37 °C in the dark. Positive clones were
picked for colony
PCR and automated sequencing.
Results
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[0302] The general scheme for if2 vivo panning using PALM is illustrated in
FTG. 35.
After an initial round of ifz vivo selection, phage were either bulk amplified
or else single
colonies of phage from pancreas, kidney, lung and adrenal glands were
amplified and
subjected to additional rounds of ih vivo screening. Both bulk amplified and
colony
amplified phage from mouse pancreas showed successive enrichment with
increasing
rounds of selection (not shown). After three rounds of selection, the colony
amplified
phage showed almost an order of magnitude higher enrichment than bulk
amplified
phage (not shown).
[0303] Table 9 lists selected targeting sequences and consensus motifs
identified by
pancreatic screening.
Table 9. Pancreatic targeting peptides and motifs
Motif Peptide Sequence
GGL CVPGLGGLC (SEQ m N0:71)
(SEQ m N0:111) CGGLDVRMC (SEQ m N0:72)
CDGGLDWVC (SEQ ~ N0:73)
LGG CVPGLGGLC (SEQ m N0:71)
(SEQ m NO:112) CTWLGGREC (SEQ ~ N0:74)
CSRWGLGGC (SEQ m N0:75)
CPPLGGSRC (SEQ )D N0:48)
VRG CVGGVRGGC (SEQ m N0:76)
(SEQ m NO:113) CVGNDVRGC (SEQ ~ N0:77)
CESRLVRGC (SEQ m N0:78)
CGGRPVRGC (SEQ m N0:79)
AGG CTPFIAGGC (SEQ 1D NO:80)
(SEQ m NO:114) CREWMAGGC (SEQ m N0:81)
CAGGSLRVC (SEQ m NO:82)
VVG CEGVVGIVC (SEQ m N0:83)
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(SEQ ID N0:115) CDSVVGAWC (SEQ m N0:84)
CRTAVVGSC (SEQ ID NO:85)
VGG CVGGARALC (SEQ ID NO:86)
(SEQ ID N0:116) CVGGVRGGC (SEQ ID N0:76)
CLAHRVGGC (SEQ ID N0:88)
GGL CWALSGGLC (SEQ ~ N0:89)
(SEQ ID N0:117) CGGLVAYGC (SEQ ID NO:90)
CGGLATTTC (SEQ ID NO:91)
GRV CGRVNSVAC (SEQ ID N0:92)
(SEQ ID N0:118) CAGRVALRC (SEQ ID NO:93)
GGA CWNGGARAC (SEQ ID N0:94)
(SEQ ID N0:119) CLDRGGAHC (SEQ ID N0:95)
GVV CELRGVVVC (SEQ ID N0:96)
(SEQ ID N0:120)
GGV CIGGVHYAC (SEQ ID N0:97)
(SEQ ID N0:121) CGGVHALRC (SEQ ID N0:98)
GMWG CIREGMWGC (SEQ ID N0:99)
(SEQ ID N0:122) CIRKGMWGC (SEQ ID NO:100)
ALR CGGVHALRC (SEQ ID N0:98)
(SEQ ID NO:123) CAGRVALRC (SEQ ID NO:93)
CEALRLRAC (SEQ ID N0:101)
ALV CALVNVHLC (SEQ ID N0:102)
(SEQ ID N0:124) CALVMVGAC (SEQ ID N0:103)
GGVH CGGVHALRC (SEQ m N0:98)
(SEQ ID N0:125) CIGGVHYAC (SEQ ID N0:97)
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VSG CMVSGVLLC (SEQ ID N0:104)
(SEQ ID N0:126) CGLVSGPWC (SEQ ID N0:105)
CLYDVSGGC (SEQ ~ N0:106)
GPW CSKVGPWWC (SEQ ID N0:107)
(SEQ ID N0:127) CGLVSGPWC (SEQ ID N0:,108)
none CAHHALMEC (SEQ ID N0:109)
CERPPFLDC (SEQ ID N0:110)
[0304] FIG. 36 shows a general protocol for recovery of phage insert sequences
from
PALM selected thin section materials. As indicated, phage may be recovered by
direct
infection of E. coli host bacteria, after protease digestion of the thin
section sample.
Alternatively, phage inserts may be recovered by PCR amplification and cloned
into new
vector DNA, then electroporated or otherwise transformed into host bacteria
for cloning.
[0305] Both methods of PALM recovery of phage were successful in retrieving
pancreatic targeting sequences. Pancreatic sequences recovered by direct
bacterial
infection included CVPRRWDVC (SEQ I~ N0:128), CQHTSGRGC (SEQ ID N0:129),
CRARGWLLC (SEQ ID N0:130), CVSNPRWKC (SEQ m NO:131), CGGVHALRC
(SEQ m NO:98), CFNRTWIGC (SEQ ID NO:132) and CSRGPAWGC (SEQ ID
N0:133). Pancreatic targeting sequences recovered by amplification of phage
inserts
and cloning into phage include CWSRGQGGC (SEQ ID N0:134), CHVLWSTRC (SEQ
ID NO:135), CLGLLMAGC (SEQ ID N0:136), CMSSPGVAC (SEQ ID N0:137),
CLASGMDAC (SEQ ID N0:138), CHDERTGRC (SEQ ID N0:139), CAHHALMEC
(SEQ ID N0:140), CMQGAATSC (SEQ ID N0:141), CMQGARTSC (SEQ ID
N0:142) and CVRDLLTGC (SEQ ID N0:143).
[0306] FIG. 37 through FIG. 40 show sequence homologies identified for
selected
pancreatic targeting sequences. Several proteins known to be present in
pancreatic
tissues are identified. The results of this example show that the PALM method
may be
used for selecting cell types from tissue thin sections and recovering
targeting phage
sequences. The skilled artisan will realize that this method could be used
with virtually
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any tissue to obtain targeting sequences directed to specific types of cells
in heterologous
organs or tissues.
All of the COMPOSTTIONS, 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
0
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.
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U.S. Patent No. 5,401,511
U.S. Patent
U.S. Patent No. 5,622,699
U.S. Patent No. 5,889,155
U.S. Patent No. 6,068,829
Varmus et al., Cell, 25:23-36, 1981.
Veikkola, T. and Alitalo, K. (1999) VEGFs, receptors and angiogenesis. Seminar
Cancer Bio.l, 9, 211-220.
VIGNE, E., MAHFOUZ, L, DEDIEU, J.F., BRIE, A., PERRICAUDET, M., and
YEH, P. (1999). RGD inclusion in the hexon monomer provides adenovirus type 5-
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Vu, T.H. et al. MMP-9/gelatinase B is a key regulator of growth plate
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Vuori K. Ruoslahti E. Association of insulin receptor substrate-1 with
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WATKINS, S.J., MESYANZHINOV, V.V., KUROCHKINA, L.P., and
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WICKHAM, T.J., MATHIAS, P., CHERESH, D.A., and NEMEROW, G.R.
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Llorens, C. (1996) Identification of metabolic pathways of brain angiotensin
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control of vasopressin release. Proc Natl Acael Sci USA, 93, 11968-11973.
136
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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)
Pasqualini, Renata (applicant for the purpose of the United States of America
only)
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)
<120> Compositions and Methods of Use of Targeting Peptides Against Placenta
and Adipose Tissues
<130> 5774.P009PCT
<140> Unknown
<141> 2002-08-30
<150> PCT/LTS01/27692
<151> 2001-09-07
<160> 144
<170> Patentln version 3.1
<210> 1
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
1
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<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 Sequence
<220>
<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 Sequence
<220>
<223> Synthetic Peptide
<400> 3
Lys Ala Ala Lys Lys Ala Ala Lys Ala Ala Lys Lys Ala Ala
1 5 10
2
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<210> 4
<211> 21
<212> PRT
<213> Artificial Sequence
<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 Sequence
<220>
<223> Synthetic Peptide
<400> 5
Thr Pro Lys Thr Ser Val Thr
1 5
<210> 6
3
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 6
Arg Met Asp Gly Pro Val Arg
1 5
<210> 7
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 7
Arg Ala Pro Gly Gly Val Arg
1 5
<210> 8
<211> 7
<212> PRT
<213> Artificial Sequence
4
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 8
Val Gly Leu His Ala Arg Ala
1 5
<210> 9
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 9
Tyr Ile Arg Pro Phe Thr Leu
1 5
<210> 10
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 10
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Leu Gly Leu Arg Ser Val Gly
1 5
<210> 11
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 11
Pro Ser Glu Arg Ser Pro Ser
1 5
<210> 12
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 12
Cys Ala Arg Ala Cys
1 5
<210> 13
6
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 13
Thr Arg Glu Val His Arg Ser
1 5
<210> 14
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 14
Thr Arg Asn Thr Gly Asn Ile
1 5
<210> 15
<211> 7
<212> PRT
<213> Artificial Sequence
7
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 15
Phe Asp Gly Gln Asp Arg Ser
1 5
<210> 16
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 16
Trp Gly Pro Lys Arg Leu
1 5
<210> 17
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 17
8
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Trp Gly Glu Ser Arg Leu
1 5
<210> 18
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 18
V al Met Gly S er V al Thr Gly
1 5
<210> 19
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 19
Lys Gly Gly Arg Ala Lys Asp
1 5
<210> 20
<211> 7
9
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 20
Arg Gly Glu Val Leu Trp Ser
1 5
<210> 21
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 21
His Gly Gln Gly Val Arg Pro
1 5
<210> 22
<211> 9
<212> PRT
<213> Artificial Sequence
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 22
Cys Lys Gly Gly Arg Ala Lys Asp Cys
1 5
<210> 23
<211> 95
<212> PRT
<213> Mus musculus
<400> 23
Cys Gln Pro Ala Met Ala Ala Val Thr Leu Asp Glu Ser Gly Gly Gly
1 5 10 15
Leu Gln Thr Pro Gly Gly Ala Leu Ser Leu Val Cys Lys Ala Ser Gly
20 25 30
Phe Thr Phe Asn Ser Tyr Pro Met Gly Trp Val Arg Gln Ala Pro Gly
35 40 45
Lys Gly Leu Glu Trp Val Ala Val Ile Ser Ser Ser Gly Thr Thr Trp
50 55 60
Tyr Ala Pro Ala Val Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly
65 70 75 80
Gln Ser Thr Val Arg Leu Gln Leu Ser Asn Leu Arg Ala Glu Asp
85 90 95
<210> 24
11
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 92
<212> PRT
<213> Mus musculus
<400> 24
Cys Gln Pro Ala Met Ala Ala Val Thr Leu Asp Glu Ser Gly Gly Gly
1 5 10 15
Leu Gln Thr Pro Gly Gly Thr Leu Ser Leu Val Cys Lys Ala Ser Gly
20 25 30
Ile Ser Ile Gly Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys
35 40 45
Gly Leu Glu Tyr Val Ala Ser Ile Ser Gly Asp Gly Asn Phe Ala His
50 55 60
Tyr Gly Ala Pro Val Lys Gly Arg Ala Thr lle Ser Arg Asp Asp Gly
65 70 75 80
Gln Asn Thr Val Thr Leu Gln Leu Asn Asn Leu Arg
85 90
<210> 25
<211> 95
<212> PRT
<213> Mus musculus
<400> 25
12
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Cys Gln Pro Ala Met Ala Ala Val Thr Leu Asp Glu Ser Gly Gly Gly
1 5 10 15
Leu Gln Thr Pro Gly Gly Thr Leu Ser Leu Val Cys Lys Gly Ser Gly
20 25 30
Phe Ile Phe Ser Arg Tyr Asp Met Ala Trp Val Arg Gln Ala Pro Gly
35 40 45
Lys Gly Leu Glu Trp Val Ala Gly Ile Asp Asp Gly Gly Gly Tyr Thr
50 55 60
Thr Leu Tyr Ala Pro Ala Val Lys Gly Arg Ala Thr Ile Thr Ser Arg
65 70 75 80
Asp Asn Gly Gln Ser Thr Val Arg Leu Gln Leu Asn Asn Leu Arg
85 90 95
<210> 26
<211> 96
<212> PRT
<213> Mus musculus
<400> 26
Ala Asn Gln Pro Trp Pro Pro Leu Thr Leu Asp Glu Ser Gly Gly Gly
1 5 10 15
Leu Gln Thr Pro Gly Gly Ala Leu Ser Leu Val Cys Lys Ala Ser Gly
20 25 30
Phe Thr Met Ser Ser Tyr Asp Met Phe Trp Val Arg Gln Ala Pro Gly
35 40 45
13
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Lys Gly Leu Glu Phe Val Ala Gly Ile Ser Ser Ser Gly Ser Ser Thr
50 55 60
Glu Tyr Gly Ala Ala Val Lys Gly Arg Ala Thr lle Ser Arg Asp Asn
65 70 75 80
Gly Gln Ser Thr Val Arg Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp
85 90 95
<210> 27
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 27
Cys Glu Gln Arg Gln Thr Gln Glu Gly Cys
1 5 10
<210> 28
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
14
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<400> 28
Cys Ala Arg Leu Glu Val Leu Leu Pro Cys
1 5 10
<210> 29
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 29
Tyr Asp Trp Trp Tyr Pro Trp Ser Trp
1 5
<210> 30
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 30
Gly Leu Asp Thr Tyr Arg Gly Ser Pro
1 5
<210> 31
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 31
Ser Asp Asn Arg Tyr Ile Gly Ser Trp
1 5
<210> 32
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 32
Tyr Glu Trp Trp Tyr Trp Ser Trp Ala
1 5
<210> 33
<211> 9
<212> PRT
<213> Artificial Sequence
16~
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 33
Lys Val Ser Trp Tyr Leu Asp Asn Gly
1 5
<210> 34
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 34
Ser Asp Trp Tyr Tyr Pro Trp Ser Trp
1 5
<210> 35
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 35
17
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Ala Gly Trp Leu Tyr Met Ser Trp Lys
1 5
<210> 36
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 36
Cys Phe Gln Asn Arg Cys
1 5
<210> 37
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 37
Cys Asn Leu Ser Ser Glu Gln Cys
1 5
<210> 38
18
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 3~
Cys Leu Arg Gln Ser Tyr Ser Tyr Asn Cys
1 5 10
<210> 39
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 39
Cys Tyr Ile Trp Pro Asp Ser Gly Leu Cys
1 5 10
<210> 40
<211> 10
<212> PRT
<213> Artificial Sequence
19
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 40
Cys Glu Pro Tyr Trp Asp Gly Trp Phe Cys
1 5 10
<210> 41
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 41
Cys Lys Glu Asp Gly Trp Leu Met Thr Cys
1 5 10
<210> 42
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 42
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Cys Lys Leu Trp Gln Glu Asp Gly Tyr
1 5
<210> 43
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 43
Cys Trp Asp Gln Asn Tyr Leu Asp Asp Cys
1 5 10
<210> 44
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 44
Asp Glu Glu Gly Tyr Tyr Met Met Arg
1 5
<210> 45
<211> 9
21
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 45
Lys Gln Phe Ser Tyr Arg Tyr Leu Leu
1 5
<210> 46
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 46
Val Val Ile Ser Tyr Ser Met Pro Asp
1 5
<210> 47
<211> 9
<212> PRT
<213> Artificial Sequence
22
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 47
Ser Asp Trp Tyr Tyr Pro Trp Ser Trp
1 5
<210> 48
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 48
Cys Pro Pro Leu Gly Gly Ser Arg Cys
1 5
<210> 49
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 49
Gly Gly Gly Ser Tyr Arg His Val Glu
23
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
1 5
<210> 50
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 50
Arg Ala Ile Leu Tyr Arg Leu Ala Asn
1 5
<210> 51
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 51
Met Leu Leu Gly Tyr Arg Phe Glu Lys
1 5
<210> 52
<211> 9
24
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 52
Thr Met Leu Arg Tyr Thr Val Arg Leu
1 5
<210> 53
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 53
Thr Met Leu Arg Tyr Phe Met Phe Pro
1 5
<210> 54
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<223> Synthetic Peptide
<400> 54
Thr Leu Arg Lys Tyr Phe His Ser Ser
1 5
<210> 55
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 55
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys
1 5 10 15
<210> 56
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 56
Cys Pro Arg Glu Cys Glu Ser Ile Cys
1 5
26
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<210> 57
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 57
Gly Ala Cys Val Arg Leu Ser Ala Cys Gly Ala
1 5 10
<210> 58
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 58
Cys Tyr Asn Leu Cys Ile Arg Glu Cys Glu Ser Ile Cys Gly Ala Asp
1 5 ~ 10 15
Gly Ala Cys Trp Thr Trp Cys Ala Asp Gly Cys Ser Arg Ser Cys
20 25 30
<210> 59
27
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 59
Cys Leu Gly Gln Cys Ala Ser Ile Cys Val Asn Asp Cys
1 5 10
<210> 60
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 60
Cys Pro Lys Val Cys Pro Arg Glu Cys Glu Ser Asn Cys
1 5 10
<210> 61
<211> 12
<212> PRT
<213> Artificial Sequence
2~
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 61
Cys Gly Thr Gly Cys Ala Val Glu Cys Val Val Cys
1 5 10
<210> 62
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 62
Cys Ala Val Ala Cys Trp Ala Asp Cys Gln Leu Gly Cys
1 5 10
<210> 63
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 63
29
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Cys Ser Gly Leu Cys Thr Val Gln Cys Leu Glu Gly Cys
1 5 10
<210> 64
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 64
Cys Ser Met Met Cys Leu Glu Gly Cys Asp Asp Trp Cys
1 5 10
<210> 65
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 65
taatacgact cactataggg caagctgata aaccgataca att 43
<210> 66
<211> 24
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 66
ccctcatagt tagcgtaacg atct 24
<210> 67
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 67
cctttctatt ctcactcggc cg 22
<210> 68
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 68
31
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
caggaaacag ctatgaccgc taaacaactt tcaacagttt cggc 44
<210> 69
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 69
cactcggccg acggggc 17
<210> 70
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 70
cagtttcggc cccagcggcc c 21
<210> 71
<211> 9
<212> PRT
<213> Artificial Sequence
32
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 71
Cys Val Pro Gly Leu Gly Gly Leu Cys
1 5
<210> 72
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 72
Cys Gly Gly Leu Asp Val Arg Met Cys
1 5
<210> 73
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 73
33
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Cys Asp Gly Gly Leu Asp Trp Val Cys
1 5
<210> 74
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 74
Cys Thr Trp Leu Gly Gly Arg Glu Cys
1 5
<210> 75
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 75
Cys Ser Arg Trp Gly Leu Gly Gly Cys
1 5
<210> 76
34
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 76
Cys Val Gly Gly Val Arg Gly Gly Cys
1 5
<210> 77
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 77
Cys Val Gly Asn Asp Val Arg Gly Cys
1 5
<210> 78
<211> 9
<212> PRT
<213> Artificial Sequence
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 78
Cys Glu Ser Arg Leu Val Arg Gly Cys
1 5
<210> 79
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 79
Cys Gly Gly Arg Pro Val Arg Gly Cys
1 5
<210> 80
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 80
36
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Cys Thr Pro Phe Ile Ala Gly Gly Cys
1 5
<210> 81
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 81
Cys Arg Glu Trp Met Ala Gly Gly Cys
1 5
<210> 82
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 82
Cys Ala Gly Gly Ser Leu Arg Val Cys
1 5
<210> 83
<211> 9
37
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 83
Cys Glu Gly Val Val Gly Ile Val Cys
1 5
<210> 84
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 84
Cys Asp Ser Val Val Gly Ala Trp Cys
1 5
<210> 85
<211> 9
<212> PRT
<213> Artificial Sequence
38
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 85
Cys Arg Thr Ala Val Val Gly Ser Cys
1 5
<210> 86
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 86
Cys Val Gly Gly Ala Arg Ala Leu Cys
1 5
<210> 87
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 87
Cys Thr Arg Glu Val His Arg Ser Cys
39
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
1 5
<210> 88
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 88
Cys Leu Ala His Arg Val Gly Gly Cys
1 5
<210> 89
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 89
Cys Trp Ala Leu Ser Gly Gly Leu Cys
1 5
<210> 90
<211> 9
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 90
Cys Gly Gly Leu Val Ala Tyr Gly Cys
1 5
<210> 91
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 91
Cys Gly Gly Leu Ala Thr Thr Thr Cys
1 5
<210> 92
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
41
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<223> Synthetic Peptide
<400> 92
Cys Gly Arg Val Asn Ser Val Ala Cys
1 5
<210> 93
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 93
Cys Ala Gly Arg Val Ala Leu Arg Cys
1 5
<210> 94
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 94
Cys Trp Asn Gly Gly Ala Arg Ala Cys
1 5
42
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<210> 95
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 95
Cys Leu Asp Arg Gly Gly Ala His Cys
1 5
<210> 96
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 96
Cys Glu Leu Arg Gly Val Val Val Cys
1 5
<210> 97
<211> 9
<212> PRT
43
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 97
Cys lle Gly Gly Val His Tyr Ala Cys
1 5
<210> 98
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 98
Cys Gly Gly Val His Ala Leu Arg Cys
1 5
<210> 99
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
44
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<223> Synthetic Peptide
<400> 99
Cys Ile Arg Glu Gly Met Trp Gly Cys
1 5
<210> 100
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 100
Cys Ile Arg Lys Gly Met Trp Gly Cys
1 5
<210> 101
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 101
Cys Glu Ala Leu Arg Leu Arg Ala Cys
1 5
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<210> 102
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 102
Cys Ala Leu Val Asn Val His Leu Cys
1 5
<210> 103
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 103
Cys Ala Leu Val Met Val Gly Ala Cys
1 5
<210> 104
<211> 9
<212> PRT
46
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 104
Cys Met Val Ser Gly Val Leu Leu Cys
1 5
<210> 105
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 105
Cys Gly Leu Val Ser Gly Pro Trp Cys
1 5
<210> 106
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
47
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<400> 106
Cys Leu Tyr Asp Val Ser Gly Gly Cys
1 5
<210> 107
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 107
Cys Ser Lys Val Gly Pro Trp Trp Cys
1 5
<210> 108
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 108
Cys Gly Leu Val Ser Gly Pro Trp Cys
1 5
48
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<210> 109
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 109
Cys Ala His His Ala Leu Met Glu Cys
1 5
<210> 110
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 110
Cys Glu Arg Pro Pro Phe Leu Asp Cys
1 5
<210> 111
<211> 3
<212> PRT
<213> Artificial Sequence
49
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223>Synthetic
Peptide
<400>111
Gly
Gly
Leu
1
<210>112
<211>3
<212>PRT
<213>Artificial
Sequence
<220>
<223>Synthetic
Peptide
<400>112
Leu y Gly
Gl
1
<210>113
<211>3
<212>PRT
<213>Artificial
Sequence
<220>
<223>Synthetic
Peptide
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<400> 113
Val Arg Gly
1
<210> 114
<211> 3
<212> PRT
<213> Artificial Sequence
<220>.
<223> Synthetic Peptide
<400> 114
Ala Gly Gly
1
<210> 115
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 115
Val Val Gly
1
<210> 116
51
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 116
V al Gly Gly
1
<210> 117
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 117
Gly Gly Leu
1
<210> 118
<211> 3
<212> PRT
<213> Artificial Sequence
52
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223>Synthetic Peptide
<400>118
Gly
Arg
Val
1
<210>119
<211>3
<212>PRT
<213>Artificial
Sequence
<220>
<223>Synthetic Peptide
<400>119
Gly
Gly
Ala
1
<210>120
<211>3
<212>PRT
<213>Artificial
Sequence
<220>
<223>Synthetic Peptide
<400>120
53
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Gly Val Val
1
<210> 121
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 121
Gly Gly V al
1
<210> 122
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 122
Gly Met Trp Gly
1
<210> 123
54
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 123
Ala Leu Arg
1
<210> 124
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 124
Ala Leu Val
1
<210> 125
<211> 4
<212> PRT
<213> Artificial Sequence
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 125
Gly Gly Val His
1
<210> 126
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 126
Val Ser Gly
1
<210> 127
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 127
56
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
Gly Pro Trp
1
<210> 128
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 128
Cys Val Pro Arg Arg Trp Asp Val Cys
1 5
<210> 129
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 129
Cys Gln His Thr Ser Gly Arg Gly Cys
1 5
<210> 130
<211> 9
57
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 130
Cys Arg Ala Arg Gly Trp Leu Leu Cys
1 5
<210> 131
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 131
Cys Val Ser Asn Pro Arg Trp Lys Cys
1 5
<210> 132
<211> 9
<212> PRT
<213> Artificial Sequence
58
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<220>
<223> Synthetic Peptide
<400> 132
Cys Phe Asn Arg Thr Trp Ile Gly Cys
1 5
<210> 133
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 133
Cys Ser Arg Gly Pro Ala Trp Gly Cys
1 5
<210> 134
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 134
Cys Trp Ser Arg Gly Gln Gly Gly Cys
59
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
1 5
<210> 135
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 135
Cys His Val Leu Trp Ser Thr Arg Cys
1 5
<210> 136
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 136
Cys Leu Gly Leu Leu Met Ala Gly Cys
1 5
<210> 137
<211> 9
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 137
Cys Met Ser Ser Pro Gly Val Ala Cys
1 5
<210> 138
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 138
Cys Leu Ala Ser Gly Met Asp Ala Cys
1 5
<210> 139
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
61
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<223> Synthetic Peptide
<400> 139
Cys His Asp Glu Arg Thr Gly Arg Cys
1 5
<210> 140
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 140
Cys Ala His His Ala Leu Met Glu Cys
1 5
<210> 141
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 141
Cys Met Gln Gly Ala Ala Thr Ser Cys
1 5
62
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<210> 142
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 142
Cys Met Gln Gly Ala Arg Thr Ser Cys
1 5
<210> 143
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 143
Cys Val Arg Asp Leu Leu Thr Gly Cys
1 5
<210> 144
<211> 9
<212> PRT
63
CA 02458047 2004-02-19
WO 03/022991 PCT/US02/27836
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 144
Cys Thr Pro Lys Thr Ser Val Thr Cys
1 5
64
