U.S. patent application number 10/956748 was filed with the patent office on 2005-07-28 for compounds and methods for modulating adhesion molecule function.
This patent application is currently assigned to Adherex Technologies, Inc.. Invention is credited to Blaschuk, Orest W., Doherty, Patrick, Gour, Barbara J..
Application Number | 20050163786 10/956748 |
Document ID | / |
Family ID | 22352647 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050163786 |
Kind Code |
A1 |
Doherty, Patrick ; et
al. |
July 28, 2005 |
Compounds and methods for modulating adhesion molecule function
Abstract
Modulating agents and methods for enhancing or inhibiting
cadherin-mediated functions are provided. The modulating agents
comprise at least an HAV binding motif, an analogue or
peptidomimetic thereof, or an antibody or fragment thereof that
specifically binds to such a motif. Modulating agents may
additionally comprise one or more cell adhesion recognition
sequences recognized by cadherins and/or other adhesion molecules.
Such modulating agents may, but need not, be linked to a targeting
agent, drug and/or support material.
Inventors: |
Doherty, Patrick;
(Twickenham, GB) ; Blaschuk, Orest W.; (Westmount,
CA) ; Gour, Barbara J.; (Kemptville, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Adherex Technologies, Inc.
Ottawa
CA
|
Family ID: |
22352647 |
Appl. No.: |
10/956748 |
Filed: |
October 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10956748 |
Oct 1, 2004 |
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10193653 |
Jul 10, 2002 |
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6806255 |
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10193653 |
Jul 10, 2002 |
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09351048 |
Jul 9, 1999 |
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6472368 |
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09351048 |
Jul 9, 1999 |
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09113977 |
Jul 10, 1998 |
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6277824 |
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Current U.S.
Class: |
424/178.1 ;
514/19.1; 514/44R; 514/8.3 |
Current CPC
Class: |
C07K 14/705 20130101;
A61P 43/00 20180101; A61K 38/12 20130101; A61P 17/02 20180101; A61P
25/00 20180101; C07K 7/06 20130101; A61P 35/00 20180101; C07K 7/64
20130101 |
Class at
Publication: |
424/178.1 ;
514/044; 514/012 |
International
Class: |
A61K 048/00; A61K
039/395; A61K 038/16 |
Claims
1. (canceled)
2. A cell adhesion modulating agent, comprising: (a) an HAV-BM
sequence or peptidomimetic thereof; (b) a polynucleotide encoding
an HAV-BM sequence; or (c) an antibody or antigen-binding fragment
thereof that specifically binds to an HAV-BM sequence; wherein the
agent modulates a cadherin-mediated process.
3. A modulating agent according to claim 2, wherein the HAV-BM
sequence is: (a)
Ile/Val-Phe-Aaa-Ile-Baa-Caa-Daa-Ser/Thr-Gly-Eaa-Leu/Met (SEQ ID
NO:3), wherein Aaa, Baa, Caa, Daa and Eaa are independently
selected from the group consisting of amino acid residues; (b)
Trp-Leu-Aaa-Ile-Asp/Asn-- Baa-Caa-Daa-Gly-Gln-Ile (SEQ ID NO:4),
wherein Aaa, Baa, Caa and Daa are independently selected from the
group consisting of amino acid residues; or (c) an analogue of any
of the foregoing sequences that retains at least seven consecutive
amino acid residues, wherein the ability of the analogue to
modulate a cadherin-mediated process is not diminished.
4. A modulating agent according to claim 3, wherein the HAV-BM
sequence comprises INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23),
IDPVSGQ (SEQ ID NO:24) or IDPVNGQ (SEQ ID NO:94).
5. A cell adhesion modulating agent according to claim 3, wherein
the HAV-BM sequence is selected from the group consisting of:
IFIINPISGQL (SEQ ID NO:5), IFILNPISGQL (SEQ ID NO:6), VFAVEKETGWL
(SEQ ID NO:7), VFSINSMSGRM (SEQ ID NO:8), VFIIERETGWL (SEQ ID
NO:9), VFTIEKESGWL (SEQ ID NO:10), VFNIDSMSGRM (SEQ ID NO:11),
WLKIDSVNGQI (SEQ ID NO:12), WLKIDPVNGQI (SEQ ID NO:13), WLAMDPDSGQV
(SEQ ID NO:14), WLHINATNGQI (SEQ ID NO:15), WLEINPDTGAI (SEQ ID
NO:16), WLAVDPDSGQI (SEQ ID NO:17), WLEINPETGAI (SEQ ID NO:18),
WLHINTSNGQI (SEQ ID NO:19), NLKIDPVNGQI (SEQ ID NO:20), LKIDPVNGQI
(SEQ ID NO:21) and analogues of the foregoing sequences that retain
at least seven consecutive residues, wherein the ability of the
analogue to modulate a cadherin-mediated process is not
diminished.
6. A modulating agent according to claim 2, wherein the HAV-BM
sequence comprises at least five consecutive residues of a peptide
selected from the group consisting of INPISGQ (SEQ ID NO:22),
LNPISGQ (SEQ ID NO:23), NLKIDPVNGQI (SEQ ID NO:20) and WLKIDPVNGQI
(SEQ ID NO:13).
7. A modulating agent according to claim 6, wherein the agent
comprises a sequence selected from the group consisting of PISGQ
(SEQ ID NO:26), PVNGQ (SEQ ID NO:27), PVSGR (SEQ ID NO:28), IDPVN
(SEQ ID NO:29), INPIS (SEQ ID NO:30) and KIDPV (SEQ ID NO:31).
8. A modulating agent according to claim 2, wherein the agent
comprises an HAV-BM sequence or an analogue thereof present within
a peptide ranging in size from 6 to 16 amino acid residues.
9. A modulating agent according to claim 2, wherein the agent
comprises an HAV-BM sequence that is present within a linear
peptide.
10. A modulating agent according to claim 2, wherein the agent
comprises an HAV-BM sequence that is present within a cyclic
peptide.
11.-23. (canceled)
24. A modulating agent according to claim 2, wherein the HAV-BM
sequence is present within a peptide comprising an N-terminal or
C-terminal modification.
25. A modulating agent according to claim 2, wherein the agent
comprises an antibody or antigen binding fragment thereof that
binds to an HAV-BM sequence.
26.-38. (canceled)
39. A method for modulating a cadherin-mediated function,
comprising contacting a cadherin-expressing cell with a cell
adhesion modulating agent according to claim 2.
40.-62. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/193,653 filed Jul. 10, 2002, now allowed; which is a
continuation of U.S. Ser. No. 09/351,048 filed Jul. 9, 1999, now
U.S. Pat. No. 6,472,368; which is a continuation-in-part of U.S.
Ser. No. 09/113,977, filed Jul. 10, 1998, now U.S. Pat. No.
6,277,824.
TECHNICAL FELD
[0002] The present invention relates generally to methods for
modulating cadherin-mediated processes, and more particularly to
the use of modulating agents comprising a cadherin cell adhesion
recognition sequence, or an antibody that specifically recognizes
such a sequence, for inhibiting or enhancing functions such as cell
adhesion.
BACKGROUND OF THE INVENTION
[0003] Cell adhesion is a complex process that is important for
maintaining tissue integrity and generating physical and
permeability barriers within the body. All tissues are divided into
discrete compartments, each of which is composed of a specific cell
type that adheres to similar cell types. Such adhesion triggers the
formation of intercellular junctions (i.e., readily definable
contact sites on the surfaces of adjacent cells that are adhering
to one another), also known as tight junctions, gap junctions and
belt desmosomes. The formation of such junctions gives rise to
physical and permeability barriers that restrict the free passage
of cells and other biological substances from one tissue
compartment to another. For example, the blood vessels of all
tissues are composed of endothelial cells. In order for components
in the blood to enter a given tissue compartment, they must first
pass from the lumen of a blood vessel through the barrier formed by
the endothelial cells of that vessel. Similarly, in order for
substances to enter the body via the gut, the substances must first
pass through a barrier formed by the epithelial cells of that
tissue. To enter the blood via the skin, both epithelial and
endothelial cell layers must be crossed.
[0004] Cell adhesion is mediated by specific cell surface adhesion
molecules (CAMs). There are many different families of CAMs,
including the immunoglobulin, integrin, selectin and cadherin
superfamilies, and each cell type expresses a unique combination of
these molecules. Cadherins are a rapidly expanding family of
calcium-dependent CAMs (Munro et al., In: Cell Adhesion and
Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RG Landes
Co. (Austin Tex., 1996). The classical cadherins (abbreviated CADs)
are integral membrane glycoproteins that generally promote cell
adhesion through homophilic interactions (a CAD on the surface of
one cell binds to an identical CAD on the surface of another cell),
although CADs also appear to be capable of forming heterotypic
complexes with one another under certain circumstances and with
lower affinity. Cadherins have been shown to regulate epithelial,
endothelial, neural and cancer cell adhesion, with different CADs
expressed on different cell types. N (neural)--cadherin is
predominantly expressed by neural cells, endothelial cells and a
variety of cancer cell types. E (epithelial)--cadherin is
predominantly expressed by epithelial cells. Other CADs are P
(placental)--cadherin, which is found in human skin and R
(retinal)--cadherin. A detailed discussion of the classical
cadherins is provided in Munro S B et al., 1996, In: Cell Adhesion
and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34 (RG
Landes Company, Austin Tex.).
[0005] The structures of the CADs are generally similar. As
illustrated in FIG. 1, CADs are composed of five extracellular
domains (EC1-EC5), a single hydrophobic domain (TM) that
transverses the plasma membrane (PM), and two cytoplasmic domains
(CP1 and CP2). The calcium binding motifs DXNDN (SEQ ID NO:1), DXD
and LDRE (SEQ ID NO:2) are interspersed throughout the
extracellular domains. The first extracellular domain (EC1)
contains the classical cadherin cell adhesion recognition (CAR)
sequence, HAV (His-Ala-Val), along with flanking sequences on
either side of the CAR sequence that may play a role in conferring
specificity. Synthetic peptides containing the CAR sequence and
antibodies directed against the CAR sequence have been shown to
inhibit CAD-dependent processes (Munro et al., supra; Blaschuk et
al., J. Mol. Biol. 211:679-82, 1990; Blaschuk et al., Develop.
Biol. 139:227-29, 1990; Alexander et al., J. Cell. Physiol.
156:610-18, 1993). However, the determination of the
three-dimensional solution and crystal structures of the EC1 domain
of classical cadherins (Overduin et al., Science 267:386-389, 1995;
Shapiro et al., Nature 374:327-337, 1995) suggest that amino acid
residues other than HAV may be directly involved in mediating the
interactions between cadherins.
[0006] Although cell adhesion is required for certain normal
physiological functions, there are situations in which the level of
cell adhesion is undesirable. For example, many pathologies (such
as autoimmune diseases, cancer and inflammatory diseases) involve
abnormal cellular adhesion. Cell adhesion may also play a role in
graft rejection. In such circumstances, modulation of cell adhesion
may be desirable.
[0007] In addition, permeability barriers arising from cell
adhesion create difficulties for the delivery of drugs to specific
tissues and tumors within the body. For example, skin patches are a
convenient tool for administering drugs through the skin. However,
the use of skin patches has been limited to small, hydrophobic
molecules because of the epithelial and endothelial cell barriers.
Similarly, endothelial cells render the blood capillaries largely
impermeable to drugs, and the blood/brain barrier has hampered the
targeting of drugs to the central nervous system. In addition, many
solid tumors develop internal barriers that limit the delivery of
anti-tumor drugs and antibodies to inner cells.
[0008] Attempts to facilitate the passage of drugs across such
barriers generally rely on specific receptors or carrier proteins
that transport molecules across barriers in vivo. However, such
methods are often inefficient, due to low endogenous transport
rates or to the poor functioning of a carrier protein with drugs.
While improved efficiency has been achieved using a variety of
chemical agents that disrupt cell adhesion, such agents are
typically associated with undesirable side-effects, may require
invasive procedures for administration and may result in
irreversible effects.
[0009] Accordingly, there is a need in the art for compounds that
modulate cell adhesion and improve drug delivery across
permeability barriers without such disadvantages. The present
invention fulfills this need and further provides other related
advantages.
SUMMARY OF THE INVENTION
[0010] The present invention provides compositions and methods for
modulating cadherin-mediated functions. Within certain aspects, the
present invention provides cell adhesion modulating agents capable
of binding to the cadherin CAR sequence HAV, wherein the agent does
not comprise an antibody or antigen-binding fragment thereof.
[0011] Within related aspects, the present invention provides cell
adhesion modulating agents, comprising: (a) an HAV-BM sequence or
peptidomimetic thereof; (b) a polynucleotide encoding an HAV-BM
sequence; or (c) an antibody or antigen-binding fragment thereof
that specifically binds to an HAV-BM sequence; wherein the agent
modulates a cadherin-mediated process. Within certain specific
embodiments, the HAV-BM sequence is: (a)
Ile/Val-Phe-Aaa-Ile-Baa-Caa-Daa-Ser/Thr-Gly-Eaa-L- eu/Met (SEQ ID
NO:3), wherein Aaa, Baa, Caa, Daa and Eaa are independently
selected from the group consisting of amino acid residues; (b)
Trp-Leu-Aaa-Ile-Asp/Asn-Baa-Caa-Daa-Gly-Gln-Ile (SEQ ID NO:4),
wherein Aaa, Baa, Caa and Daa are independently selected from the
group consisting of amino acid residues; or (c) an analogue of any
of the foregoing sequences that retains at least seven consecutive
amino acid residues, wherein the ability of the analogue to
modulate a cadherin-mediated process is not diminished. For
example, a cell adhesion modulating agent may comprise an HAV-BM
sequence is selected from the group consisting of: IFIINPISGQL (SEQ
ID NO:5), IFILNPISGQL (SEQ ID NO:6), VFAVEKETGWL (SEQ ID NO:7),
VFSINSMSGRM (SEQ ID NO:8), VFIIERETGWL (SEQ ID NO:9), VFTIEKESGWL
(SEQ ID NO:10), VFNIDSMSGRM (SEQ ID NO:11), WLKIDSVNGQI (SEQ ID
NO:12), WLKIDPVNGQI (SEQ ID NO:13), WLAMDPDSGQV (SEQ ID NO:14),
WLHINATNGQI (SEQ ID NO:15), WLEINPDTGAI (SEQ ID NO:16), WLAVDPDSGQI
(SEQ ID NO:17), WLEINPETGAI (SEQ ID NO:18), WLHINTSNGQI (SEQ ID
NO:19), NLKIDPVNGQI (SEQ ID NO:20), LKIDPVNGQI (SEQ ID NO:21) and
analogues of the foregoing sequences that retain at least seven
consecutive residues (e.g., INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID
NO:23), IDPVSGQ (SEQ ID NO:24) or KIDPVNGQ (SEQ ID NO:25)), wherein
the ability of the analogue to modulate a cadherin-mediated process
is not diminished. Alternatively, a modulating agent may comprise
an HAV-BM sequence that comprises at least five consecutive
residues of a peptide selected from the group consisting of INPISGQ
(SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), NLKIDPVNGQI (SEQ ID NO:20)
and WLKIDPVNGQI (SEQ ID NO:13). For example, the agent may comprise
a sequence selected from the group consisting of PISGQ (SEQ ID
NO:26), PVNGQ (SEQ ID NO:27), PVSGR (SEQ ID NO:28), IDPVN (SEQ ID
NO:29), INPIS (SEQ ID NO:30) and KIDPV (SEQ ID NO:31). Within such
modulating agents, an HAV-BM sequence may be present within a
linear peptide or a cyclic peptide. Certain modulating agents
comprise a cyclic peptide having one of the formulas: 1
[0012] wherein X.sub.1, and X.sub.2 are optional, and if present,
are independently selected from the group consisting of amino acid
residues and combinations thereof in which the residues are linked
by peptide bonds, and wherein X.sub.1 and X.sub.2 independently
range in size from 0 to 10 residues, such that the sum of residues
contained within X.sub.1 and X.sub.2 ranges from 1 to 12; wherein
Y.sub.1 and Y.sub.2 are independently selected from the group
consisting of amino acid residues, and wherein a covalent bond is
formed between residues Y.sub.1 and Y.sub.2; and wherein Z.sub.1
and Z.sub.2 are optional, and if present, are independently
selected from the group consisting of amino acid residues and
combinations thereof in which the residues are linked by peptide
bonds. Such cyclic peptides may contain modifications. For example,
Y.sub.1 may comprise an N-acetyl group and/or Y.sub.2 may comprise
a C-terminal amide group. Cyclization may be achieved in any of a
variety of ways, such as covalent linkage of Y.sub.1 and Y.sub.2
via a disulfide, amide or thioether bond.
[0013] Within certain embodiments, modulating agents as described
above may be linked to one or more of a drug, a solid support, a
detectable marker or a targeting agent.
[0014] Within other embodiments, a modulating agents as described
above may further comprise one or more of: (a) a cell adhesion
recognition sequence other than an HAV-BM sequence, wherein the
cell adhesion recognition sequence is separated from any HAV-BM
sequence(s) by a linker; and/or (b) an antibody or antigen-binding
fragment thereof that specifically binds to a cell adhesion
recognition sequence other than an HAV-BM sequence. For example,
the adhesion molecule may be selected from the group consisting of
cadherins, integrins, occludin, N--CAM, desmogleins, desmocollins,
fibronectin, laminin and other extracellular matrix proteins.
[0015] Within further aspects, the present invention provides
pharmaceutical compositions comprising a cell adhesion modulating
agent as described above, in combination with a pharmaceutically
acceptable carrier. Such compositions may further comprise one or
more of a drug and/or a modulator of cell adhesion, wherein the
modulator comprises one or more of: (a) a peptide comprising a cell
adhesion recognition sequence other than an HAV-BM sequence; and/or
(b) an antibody or antigen-binding fragment thereof that
specifically binds to a cell adhesion recognition sequence other
than an HAV-BM sequence. For example, the adhesion molecule may be
selected from the group consisting of cadherins, integrins,
occludin, N-CAM, desmogleins, desmocollins, fibronectin, laminin
and other extracellular matrix proteins.
[0016] The present invention further provides, within other
aspects, methods for modulating a cadherin-mediated function,
comprising contacting a cadherin-expressing cell with a cell
adhesion modulating agent as described above. Cadherin-mediated
functions include cell adhesion, neurite outgrowth, Schwann cell
migration and synaptic stability. Cadherin-expressing cells include
epithelial cells, endothelial cells, neural cells, tumor cells and
lymphocytes. Within such aspects, the cell adhesion modulating
agent may inhibit or enhance a cadherin-mediated function.
[0017] Within other aspects, the present invention provides methods
for reducing unwanted cellular adhesion in a mammal, comprising
administering to a mammal a modulating agent as described above,
wherein the modulating agent inhibits cadherin-mediated cell
adhesion. The cell may be selected from the group consisting of
epithelial cells, endothelial cells, neural cells, tumor cells and
lymphocytes.
[0018] The present invention further provides, within other
aspects, methods for enhancing the delivery of a drug through the
skin of a mammal, comprising contacting epithelial cells of a
mammal with a drug and a modulating agent as described above,
wherein the step of contacting is performed under conditions and
for a time sufficient to allow passage of the drug across the
epithelial cells, and wherein the modulating agent inhibits
cadherin-mediated cell adhesion. The modulating agent may, but need
not, be linked to the drug and may, within certain embodiments,
pass into the blood stream of the mammal. The step of contacting
may be performed via a skin patch comprising the modulating agent
and the drug.
[0019] Within further aspects, methods are provided for enhancing
the delivery of a drug to a tumor in a mammal, comprising
administering to a mammal a modulating agent as described above,
wherein the modulating agent inhibits cadherin-mediated cell
adhesion. Suitable tumors include, but are not limited to, bladder
tumors, ovarian tumors and melanomas, and the modulating agent may
be administered to the tumor or systemically.
[0020] Within other aspects, the present invention provides methods
for treating cancer and/or inhibiting metastasis in a mammal,
comprising administering to a mammal a modulating agent as
described above, wherein the modulating agent inhibits
cadherin-mediated cell adhesion. The mammal may be afflicted with a
cancer such as a carcinoma, leukemia or melanoma, and the
modulating agent may be administered to the tumor or
systemically.
[0021] The present invention further provides, within other
aspects, methods for inducing apoptosis in a cadherin-expressing
cell, comprising contacting a cadherin-expressing cell with a
modulating agent as described above, wherein the modulating agent
inhibits cadherin-mediated cell adhesion.
[0022] Within other aspects, methods are provided for inhibiting
angiogenesis in a mammal, comprising administering to a mammal a
modulating agent as described above, wherein the modulating agent
inhibits cadherin-mediated cell adhesion.
[0023] The present invention further provides, within other
aspects, methods for enhancing drug delivery to the central nervous
system of a mammal, comprising administering to a mammal a
modulating agent as described above, wherein the modulating agent
inhibits cadherin-mediated cell adhesion.
[0024] Within further aspects, the present invention provides
methods for facilitating wound healing in a mammal, comprising
contacting a wound in a mammal with a modulating agent as described
above, wherein the modulating agent enhances cadherin-mediated cell
adhesion.
[0025] Methods are also provided, within other aspects, for
enhancing adhesion of foreign tissue implanted within a mammal,
comprising contacting a site of implantation of foreign tissue in a
mammal with a modulating agent as described above, wherein the
modulating agent enhances cadherin-mediated cell adhesion. Such
foreign tissue may be a skin graft or organ implant. Within certain
embodiments, the modulating agent is linked to a support
material.
[0026] The present invention further provides, in other aspects,
methods for enhancing and/or directing neurite outgrowth,
comprising contacting a neuron with a modulating agent as described
above, wherein the modulating agent enhances cadherin-mediated cell
adhesion.
[0027] Within other aspects, the present invention provides methods
for treating spinal cord injuries in a mammal, comprising
administering to a mammal a modulating agent as described above,
wherein the modulating agent enhances cadherin-mediated cell
adhesion.
[0028] Methods are also provided, within further aspects, for
treating a demyelinating neurological disease such as multiple
sclerosis in a mammal, comprising administering to a mammal a
modulating agent as described above. Within certain embodiments,
the modulating agent is administered by implantation with Schwann
cells, oligodendrocyte progenitor cells and/or
oligodendrocytes.
[0029] Within further aspects, methods are provided for modulating
the immune system of a mammal, comprising administering to a mammal
a modulating agent as described above, wherein the modulating agent
inhibits cadherin-mediated cell adhesion.
[0030] Within other aspects, the present invention provides methods
for preventing pregnancy in a mammal, comprising administering to a
mammal a modulating agent as described above, wherein the
modulating agent inhibits cadherin-mediated cell adhesion.
[0031] Methods are further provided for increasing vasopermeability
in a mammal, comprising administering to a mammal a modulating
agent as described above, wherein the modulating agent inhibits
cadherin-mediated cell adhesion.
[0032] Within further aspects, the present invention provides
methods for inhibiting synaptic stability in a mammal, comprising
administering to a mammal a modulating agent as described above,
wherein the modulating agent inhibits cadherin-mediated cell
adhesion.
[0033] The present invention further provides methods for detecting
the presence of cadherin-expressing cells in a sample, comprising:
(a) contacting a sample with an antibody or antigen-binding
fragment thereof that binds to an HAV-BM sequence under conditions
and for a time sufficient to allow formation of an
antibody-cadherin complex; and (b) detecting the level of
antibody-cadherin complex, and therefrom detecting the presence of
cadherin expressing cells in a sample. The antibody may be linked
to a support material or a detectable marker such as a fluorescent
marker. In certain embodiments, the step of detecting is performed
using fluorescence activated cell sorting.
[0034] The present invention also provides, within further aspects,
kits for enhancing transdermal drug delivery, comprising: (a) a
skin patch; and (b) a modulating agent as described above. The skin
patch may be impregnated with the modulating agent, and the kit may
further comprise a drug.
[0035] Kits for detecting the presence of cadherin-expressing cells
in a sample are also provided. Such kits may comprise: (a) an
antibody or antigen-binding fragment thereof that specifically
binds to an HAV-BM sequence; and (b) a detection reagent.
[0036] Within other aspects, the present invention provides methods
for identifying a compound capable of modulating cadherin-mediated
cell adhesion, comprising: (a) contacting an antibody or
antigen-binding fragment thereof that specifically binds to an
HAV-BM sequence with a test compound; and (b) detecting the level
of antibody or fragment that binds to the test compound, and
therefrom identifying a compound capable of modulating
cadherin-mediated cell adhesion.
[0037] Methods are also provided, within other aspects, for
facilitating blood sampling in a mammal, comprising contacting
epithelial cells of a mammal with a cell adhesion modulating agent
as described above, wherein the modulating agent inhibits
cadherin-mediated cell adhesion, and wherein the step of contacting
is performed under conditions and for a time sufficient to allow
passage of one or more blood components across the epithelial
cells. The step of contacting may be performed via a skin patch
comprising the modulating agent, and the skin patch may further
comprise a reagent for detecting a blood component of interest.
Within certain embodiments, the epithelial cells are skin cells or
gum cells.
[0038] Within related aspects, the present invention provides kits
for sampling blood via the skin or gum of a mammal, comprising: (a)
a skin patch; (b) a cell adhesion modulating agent comprising a
cyclic peptide that comprises a cadherin CAR sequence; and (c) a
reagent for detecting a blood component of interest. The skin patch
may be impregnated with the cell adhesion modulating agent.
[0039] Within other aspects, the present invention provides methods
for screening for a compound that interacts with an HAV-BM
sequence, comprising the steps of: (a) contacting a candidate
compound with an HAV-BM sequence; and (b) evaluating the ability of
the candidate compound to bind to the HAV-BM sequence, and
therefrom determining whether the candidate compound interacts with
an HAV-BM sequence. Within certain embodiments, the ability of the
candidate compound to bind to the HAV-BM sequence is evaluated
using an affinity column or a Western blot analysis. Within certain
embodiments, the candidate compound is encoded by a polynucleotide
in an expression library, or the candidate compound is a cellular
protein, and step (b) is performed using whole cells.
[0040] These and other aspects of the invention will become evident
upon reference to the following detailed description and attached
drawings. All references disclosed herein are hereby incorporated
by reference in their entirety as if each were individually noted
for incorporation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a diagram depicting the structure of classical
CADs. The five extracellular domains are designated EC1-EC5, the
hydrophobic domain that transverses the plasma membrane (PM) is
represented by TM, and the two cytoplasmic domains are represented
by CP1 and CP2. The calcium binding motifs are shown by DXNDN (SEQ
ID NO:1), DXD and LDRE (SEQ ID NO:2). The CAR sequence, HAV, is
shown within EC1. The sequences, INPISGQ (SEQ ID NO:22)and
LKIDPVNGQI (SEQ ID NO:21) are shown within EC1 and EC4,
respectively. Cytoplasmic proteins .beta.-catenin (.beta.),
.alpha.-catenin (.alpha.) and .alpha.-actinin (ACT), which mediate
the interaction between CADs and microfilaments (MF) are also
shown.
[0042] FIG. 2 provides the amino acid sequences of mammalian
classical cadherin EC1 domains: human N-cadherin (SEQ ID NO:32),
mouse N-cadherin (SEQ ID NO:33), cow N-cadherin (SEQ ID NO:34),
human E-cadherin (SEQ ID NO:35), mouse E-cadherin (SEQ ID NO:36),
human P-cadherin (SEQ ID NO:37), mouse P-cadherin (SEQ ID NO:38),
human R-cadherin (SEQ ID NO:39) and mouse R-cadherin (SEQ ID
NO:40).
[0043] FIG. 3 provides the amino acid sequences of mammalian
classical cadherin EC4 domains: human N-cadherin (SEQ ID NO:41),
mouse N-cadherin (SEQ ID NO:42), cow N-cadherin (SEQ ID NO:43),
human E-cadherin (SEQ ID NO:44), mouse E-cadherin (SEQ ID NO:45),
human P-cadherin (SEQ ID NO:46), mouse P-cadherin (SEQ ID NO:47),
human R-cadherin (SEQ ID NO:48) and mouse R-cadherin (SEQ ID
NO:49).
[0044] FIGS. 4A-4E provide structures of representative cyclic
peptide modulating agents (SEQ ID NOs: 51-63 and 85).
[0045] FIG. 5 is a graph showing the mean neurite length measured
for neurons cultured on monolayers of 3T3 cells or 3T3 cells
expressing N-cadherin in media containing varying concentrations of
the linear peptide H-WLKIDPVNGQI-OH (SEQ ID NO:13; designated
N-CAD-CHD2).
[0046] FIG. 6 is a graph showing the mean neurite length measured
for neurons cultured on monolayers of either 3T3 cells, 3T3 cells
expressing N-cadherin, 3T3 cells expressing NCAM, or 3T3 cells
expressing L1 in media containing the linear peptide
H-WLKIDPVNGQI-OH (SEQ ID NO:13; designated N-CAD-CHD2) at a
concentration of 250 .mu.g/ml.
[0047] FIG. 7 is a graph illustrating the binding of the peptide H-
WLKIDPVNGQI-OH (SEQ ID NO:13) at various concentrations to a flow
cell coated with an N-cadherin-Fc chimera or human IgG1. The
peptide H-WLKIDPVNGQI-OH (SEQ ID NO:13) was passed over both flow
cells at a concentration of either 250, 500 or 1000 .mu.g/ml. The
results show the association of the peptide to the flow cell coated
with the N-cadherin Fc chimera, with the binding to the control
flow cell (coated with human IgG1) automatically subtracted.
[0048] FIGS. 8A and 8B are photographs showing monolayer cultures
of human ovarian cancer cells (SKOV3) in the presence (FIG. 8B) and
absence (FIG. 8A) of the peptide N-Ac-INPISGQ-NH.sub.2 (SEQ ID
NO:22). FIG. 8B shows the cells 24 hours after being cultured in
the presence of 1 mg/mL of N-Ac-INPISGQ (SEQ ID NO:22).
DETAILED DESCRIPTION OF THE INVENTION
[0049] As noted above, the present invention provides methods for
modulating cadherin-mediated processes, such as cell adhesion. The
present invention is based upon the identification of a previously
unknown cell adhesion recognition (CAR) sequence in classical
cadherins. This CAR sequence is referred to herein as the "HAV
binding motif" (or "HAV-BM"). The HAV-BM appears to interact
directly with the HAV CAR sequence and/or flanking regions in
homophilic and heterophilic interactions.
[0050] In general, to modulate cadherin-mediated cell adhesion, a
cell that expresses a classical cadherin is contacted with a cell
adhesion modulating agent (also referred to herein as a "modulating
agent") either in vivo or in vitro. A modulating agent may comprise
one or more HAV-BM sequences (which may be native sequences or
analogues thereof) or a peptidomimetic of such a sequence, with or
without one or more additional CAR sequences (which may be derived
from classical cadherins or from other adhesion molecules), as
described below. HAV-BM sequences may be present within a linear or
cyclic peptide. Alternatively, or in addition, a modulating agent
may comprise a polynucleotide encoding a peptide comprising one or
more HAV-BM sequences (such that the encoded peptide is produced in
vivo) and/or a substance (such as an antibody or antigen-binding
fragment thereof) that specifically binds an HAV-BM sequence.
[0051] Certain methods provided herein employ cell adhesion
modulating agents for inhibiting or enhancing cadherin-mediated
cell adhesion. Inhibition of cell adhesion may generally be used,
for example, to treat diseases or other conditions characterized by
undesirable cell adhesion or to facilitate drug delivery to a
specific tissue or tumor. Within other aspects, the methods
provided herein may be used to enhance cell adhesion (e.g., to
supplement or replace stitches or to facilitate wound healing).
Within still further aspects, methods are provided for enhancing
and/or directing neurite outgrowth.
[0052] Cell Adhesion Modulating Agents
[0053] As noted above, the term "cell adhesion modulating agent,"
as used herein, generally refers to a compound that is capable of
binding to a classical cadherin (i.e., the compound interacts
detectably with one or more amino acid residues within a classical
cadherin such that a cadherin-mediated process is modulated, as
described herein). Preferably, a modulating agent binds in or near
a classical cadherin CAR sequence HAV (i.e., the agent interacts
detectably with one or more amino acid residues present within the
HAV sequence and/or one or more amino acid residues present within
ten amino acid residues, and more preferably within five amino acid
residues, of the HAV sequence in a native cadherin). Within
specific embodiments, a modulating agent comprises at least one of
the following:
[0054] (a) an HAV-BM sequence (i.e., a native HAV-BM sequence or an
analogue thereof), or a peptidomimetic thereof;
[0055] (b) a polynucleotide encoding an HAV-BM sequence; or
[0056] (c) an antibody or antigen-binding fragment thereof that
specifically binds to an HAV-BM sequence.
[0057] A modulating agent may consist entirely of an HAV-BM
sequence (within a linear or cyclic peptide), peptidomimetic,
polynucleotide or antibody, or may additionally comprise further
peptide and/or non-peptide regions.
[0058] An "HAV-BM sequence" is an HAV-binding sequence that exists
in a naturally occurring cadherin, or an analogue of such a
sequence in which the ability to modulate a cadherin-mediated
process is not diminished. Such sequences generally comprise at
least five amino acid residues, preferably 6-16 amino acid
residues, and may be identified based on sequence homology to known
HAV-BM sequences, which are provided herein, and based on the
ability of a peptide comprising such a sequence to bind to an HAV
sequence and modulate a cadherin-mediated function, within a
representative assay as described herein. Within certain
embodiments, the HAV-BM sequence is:
[0059] (a) Ile/Val-Phe-Aaa-Ile-Baa-Caa-Daa-Ser/Thr-Gly-Eaa-Leu/Met
(SEQ ID NO:3), wherein Aaa, Baa, Caa, Daa and Eaa are independently
selected from the group consisting of amino acid residues;
[0060] (b) Trp-Leu-Aaa-Ile-Asp/Asn-Baa-Caa-Daa-Gly-Gln-Ile (SEQ ID
NO:4), wherein Aaa, Baa, Caa and Daa are independently selected
from the group consisting of amino acid residues; or
[0061] (c) an analogue of any of the foregoing sequences that
retains at least seven consecutive amino acid residues.
[0062] Representative known HAV-BM sequences are provided in Table
I. These sequences are not intended to limit the scope of HAV-BM
sequences encompassed by the present invention. In particular, a
modulating agent may comprise a portion or other analogue of such
sequences, provided that the ability of the analogue to modulate a
cadherin-mediated function is not substantially diminished.
1TABLE I Representative HAV-BM Sequences Cadherin HAV-BM EC1
Domains BTCADHN IFIINPISGQL (SEQ ID NO:5) HSNCADHER IFILNPISGQL
(SEQ ID NO:6) HSPCAD VFAVEKETGWL (SEQ ID NO:7) HUMCA4A VFSINSMSGRM
(SEQ ID NO:8) HUMUVOECAD VFIIERETGWL (SEQ ID NO:9) MMCADHP
VFTIEKESGWL (SEQ ID NO:10) MMECADH VFIIERETGWL (SEQ ID NO:9)
MMRCADA VFNIDSMSGRM (SEQ ID NO:11) MUSCADNA IFIINPISGQL (SEQ ID
NO:5) CONSENSUS IFXIXXXSGXL (SEQ ID NO:3) V T M EC4 Domains BTCADHN
WLKIDSVNGQI (SEQ ID NO:12) HSNCADHER WLKIDPVNGQI (SEQ ID NO:13)
HSPCAD WLAMDPDSGQV (SEQ ID NO:14) HUMCA4A WLHINATNGQI (SEQ ID
NO:15) HUMUVOECAD WLEINPDTGAI (SEQ ID NO:16) MMCADHP WLAVDPDSGQI
(SEQ ID NO:17) MMECADH WLEINPETGAI (SEQ ID NO:18) MMRCADA
WLHINTSNGQI (SEQ ID NO:19) MUSCADNA WLKIDPVNGQI (SEQ ID NO:13)
CONSENSUS WLXIDXXXGQI (SEQ ID NO:4) N
[0063] Within certain specific embodiments, the HAV-BM sequence
comprises INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ
(SEQ ID NO:24) or KIDPVNGQ (SEQ ID NO:25). For example, HAV-BM
sequences include, but are not limited to N-Ac-NLKIDPVNGQI-NH.sub.2
(SEQ ID NO:20) and H-LKIDPVNGQI-OH (SEQ ID NO:21).
[0064] Within other embodiments, an HAV-BM sequence may comprise at
least five consecutive residues of one of the following peptides:
INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), NLKIDPVNGQI (SEQ ID
NO:20) and WLKIDPVNGQI (SEQ ID NO:13). For example, a modulating
agent may comprise the sequence PISGQ (SEQ ID NO:26), PVNGQ (SEQ ID
NO:27), PVSGR (SEQ ID NO:28), KIDPV (SEQ ID NO:31), IDPVN (SEQ ID
NO:29), INPIS (SEQ ID NO:30) or KIDPVN (SEQ ID NO:50). As noted
above, within any of the above embodiments, an HAV-BM sequence may
be present within a cyclic peptide, such as PVNGQ (SEQ ID NO:51),
PISGQ (SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54),
KIDPVN (SEQ ID NO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57),
CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID
NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ ID NO:62), CINPISC
(SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ ID NO:86) or
CINPIC (SEQ ID NO:87), in which cyclization is indicated by the
underline.
[0065] Other HAV-BM sequences include sequences in which a native
sequence is modified. For example, the peptides H-LKIDPANGQI-OH
(SEQ ID NO:64) and H-LKIDAVNGQI- OH (SEQ ID NO:65) comprise HAV-BM
sequences.
[0066] As noted above, the present invention further contemplates
native HAV-BM sequences from other cadherins not specifically
recited herein. Additional native HAV-BM sequences may be
identified based upon sequence similarity to one or more of the
native HAV-BMs provided herein. In general, a native HAV-BM
sequence should retain at least three amino acid residues of a
native HAV-BM provided herein, and a total of at least seven amino
acid residues should be identical or contain conservative
substitutions. A "conservative substitution" is one in which an
amino acid is substituted for another amino acid that has similar
properties, such that one skilled in the art of peptide chemistry
would expect the secondary structure and hydropathic nature of the
polypeptide to be substantially unchanged. Amino acid substitutions
may generally be made on the basis of similarity on polarity,
charge, solubility, hydrophobicity, hydrophilicity and/or the
amphipathic nature of the residues. For example, negatively charged
amino acids include aspartic acid and glutamic acid; positively
charged amino acids include lysine and arginine; and amino acids
with uncharged polar head groups having similar hydrophilicity
values include leucine, isoleucine and valine; glycine and alanine;
asparagine and glutamine; and serine, threonine, phenylalanine and
tyrosine. Other groups of amino acids that may represent
conservative changes include: (1) ala, pro, gly, glu, asp, gln,
asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,
phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. The critical
determining features of a native HAV-BM are the ability to bind to
an HAV sequence and the ability to modulate a cadherin-mediated
function. Such abilities may be evaluated using the representative
assays provided herein.
[0067] As noted above, modulating agents as described herein may
comprise a native HAV-BM sequence, or an analogue or peptidomimetic
thereof. An analogue generally retains at least three amino acid
residues of a native HAV-BM, and binds to an HAV sequence and
modulates a cadherin-mediated function as described below. In
particular, an analogue should bind to a classical cadherin and
modulate a cadherin-mediated function at least as well as a native
HAV-BM sequence within at least one of the assays provided herein.
A peptidomimetic is a non-peptide compound that is structurally
similar to an HAV-BM sequence, such that it binds to HAV sequences
and modulates a cadherin-mediated function as described below. Such
peptidomimetics may be designed based on techniques that evaluate
the three dimensional structure of a peptide. For example, nuclear
magnetic resonance (NMR) and computational techniques may be used
to determine the conformation of an HAV-BM sequence. NMR is widely
used for structural analyses of both peptidyl and non-peptidyl
compounds. Nuclear Overhauser Enhancements (NOE's), coupling
constants and chemical shifts depend on the conformation of a
compound. NOE data provides the interproton distance between
protons through space and can be used to calculate of the lowest
energy conformation for the HAV-BM sequence. This information can
then be used to design peptidomimetics of the preferred
conformation. Linear peptides in solution exist in many
conformations. By using conformational restriction techniques it is
possible to fix the peptide in the active conformation.
Conformational restriction can be achieved by i) introduction of an
alkyl group such as a methyl which sterically restricts free bond
rotation; ii) introduction of unsaturation which fixes the relative
positions of the terminal and geminal substituents; and/or iii)
cyclization, which fixes the relative positions of the sidechains.
Peptidomimetics of an HAV-BM sequence may be synthesized where one
or more of the amide linkages has been replaced by isosteres,
substituents or groups which have the same size or volume such as,
but not limited to, --CH.sub.2NH--, --CSNH--, --CH.sub.2S--,
--CH.dbd.CH--, --CH.sub.2CH.sub.2--, --CONMe-- and others. These
backbone amide linkages can be also be part of a ring structure
(i.e., lactam). Peptidomimetics of an HAV-BM sequence may be
designed where one or more of the side chain functionalities of the
HAV-BM sequence can be replaced by groups that do not necessarily
have the same size or volume, but have similar chemical and/or
physical properties which produce similar biological responses. It
should be understood that, within embodiments described below, an
analogue or peptidomimetic may be substituted for an HAV-BM
sequence.
[0068] Modulating agents, or peptide portions thereof, may
generally comprise from 5 to about 1000 amino acid residues,
preferably from 6 to 50 residues. When non-peptide linkers are
employed, each CAR sequence of the modulating agent is present
within a peptide that generally ranges in size from 5 to 50
residues in length, preferably from 5 to 25 residues, more
preferably from 5 to 16 residues and still more preferably from 5
or 6 to 10 residues.
[0069] Modulating agents, or peptide portions thereof, may be
linear or cyclic peptides. The term "cyclic peptide," as used
herein, refers to a peptide or salt thereof that comprises (1) an
intramolecular covalent bond between two non-adjacent residues and
(2) at least one HAV-BM sequence or an analogue thereof present
within the peptide ring. The intramolecular bond may be a backbone
to backbone, side-chain to backbone or side-chain to side-chain
bond (i.e., terminal functional groups of a linear peptide and/or
side chain functional groups of a terminal or interior residue may
be linked to achieve cyclization). Preferred intramolecular bonds
include, but are not limited to, disulfide, amide and thioether
bonds. As noted above, in addition to one or more HAV-BM sequence
or analogue thereof, a modulating agent may comprise additional CAR
sequences, which may or may not be cadherin CAR sequences, and/or
antibodies or fragments thereof that specifically recognize a CAR
sequence. Antibodies and antigen-binding fragments thereof are
typically present in a non-cyclic portion of a modulating
agent.
[0070] The size of a cyclic peptide ring generally ranges from 4 to
about 15 residues, preferably from 5 to 10 residues. Additional
residue(s) may be present on the N-terminal and/or C-terminal side
of an HAV-BM sequence, and may be derived from sequences that flank
a native HAV-BM sequence, with or without amino acid substitutions
and/or other modifications. Additional residue(s) that may be
present on the N-terminal and/or C-terminal side of an HAV-BM
sequence may be derived from sequences that flank the HAV-BM
sequence within one or more naturally occurring cadherins, with or
without amino acid substitutions and/or other modifications.
Flanking sequences for endogenous N-, E-, P- and R-cadherin HAV-BMs
are shown in FIGS. 2 and 3, and SEQ ID NOs: 32 to 49.
Alternatively, additional residues present on one or both sides of
the CAR sequence(s) may be unrelated to an endogenous sequence
(e.g., residues that facilitate cyclization, purification or other
manipulation and/or residues having a targeting or other
function).
[0071] In certain preferred embodiments, a modulating agent
comprises a cyclic peptide having one of the following structures:
2
[0072] In these structures, X.sub.1, and X.sub.2 are optional, and
if present, are independently selected amino acid residues and
combinations thereof in which the residues are linked by peptide
bonds. In general, X.sub.1 and X.sub.2 independently range in size
from 0 to 10 residues, such that the sum of residues contained
within X.sub.1 and X.sub.2 ranges from 1 to 12. Y.sub.1 and Y.sub.2
are independently selected amino acid residues, and a covalent bond
is formed between residues Y.sub.1 and Y.sub.2. Z.sub.1 and Z.sub.2
are optional, and if present, are independently selected amino acid
residues and combinations thereof in which the residues are linked
by peptide bonds. Representative examples of such structures are
provided in FIGS. 4A-4D.
[0073] A modulating agent that contains sequences that flank the
HAV-BM sequence on one or both sides may be specific for cell
adhesion mediated by one or more specific cadherins, resulting in
tissue and/or cell-type specificity. Suitable flanking sequences
for conferring specificity include, but are not limited to,
endogenous sequences present in one or more naturally occurring
cadherins. Modulating agents having a desired specificity may be
identified using the representative screens provided herein.
[0074] As noted above, multiple CAR sequences may be present within
a modulating agent. The total number of CAR sequences present
within a modulating agent may range from 1 to a large number, such
as 100, preferably from 1 to 10, and more preferably from 1 to 5.
CAR sequences that may be included within a modulating agent are
any sequences specifically bound by an adhesion molecule (i.e., a
molecule that mediates cell adhesion via a receptor on the cell's
surface). Adhesion molecules include members of the cadherin gene
superfamily that are not classical cadherins (e.g., proteins that
do not contain an HAV sequence and/or one or more of the other
characteristics recited above for classical cadherins), such as
desmogleins (Dsg) and desmocollins (Dsc); integrins; members of the
immunoglobulin supergene family, such as N-CAM; and other
uncategorized transmembrane proteins, such as occludin, as well as
extracellular matrix proteins such as laminin, fibronectin,
collagens, vitronectin, entactin and tenascin. Within certain
embodiments, preferred CAR sequences for inclusion within a
modulating agent include Arg-Gly-Asp (RGD), which is bound by
integrins (see Cardarelli et al., J. Biol. Chem. 267:23159-64,
1992); Tyr-Ile-Gly-Ser-Arg (YIGSR; SEQ ID NO:66), which is bound by
.alpha.6.beta.1 integrin; KYSFNYDGSE (SEQ ID NO:67), which is bound
by N-CAM; the N-CAM heparin sulfate-binding site IWKHKGRDVILKKDVRF
(SEQ ID NO:68), the occludin CAR sequence LYHY (SEQ ID NO:70); a
junctional adhesion molecule CAR sequence DPK and/or one or more
nonclassical cadherin CAR sequences, such as the VE-cadherin CAR
sequence DAE, the Dsc CAR sequences IEK, VER and IER, the Dsg CAR
sequences INQ, INR and LNK; and the claudin CAR sequence IYSY (SEQ
ID NO:95).
[0075] Within certain embodiments, another preferred CAR sequence
is the OB-cadherin CAR sequence DDK. A variety of peptides
comprising this sequence may be included, such as IDDK (SEQ ID
NO:71), DDKS (SEQ ID NO:72), VIDDK (SEQ ID NO:73), IDDKS (SEQ ID
NO:74), VIDDKS (SEQ ID NO:75), DDKSG (SEQ ID NO:76), IDDKSG (SEQ ID
NO:77), VIDDKSG (SEQ ID NO:78), FVIDDK (SEQ ID NO:79), FVIDDKS (SEQ
ID NO:80), FVIDDKSG (SEQ ID NO:81), IFVIDDK (SEQ ID NO:82),
IFVIDDKS (SEQ ID NO:83), or IFVIDDKSG (SEQ ID NO:84). In certain
preferred embodiments, at least one terminal amino acid residue of
such a peptide is modified (e.g., the N-terminal amino group is
modified by, for example, acetylation or alkoxybenzylation and/or
an amide or ester is formed at the C-terminus). Certain preferred
modulating agents contain modifications at the N- and C-terminal
residues, such as N-Ac-IFVIDDKSG-NH.sub.2 (SEQ ID NO:84). Analogues
of any of the foregoing sequences may also be used. An analogue
generally retains at least 50% of a native OB-cadherin CAR
sequence, and modulates OB-cadherin-mediated cell adhesion.
[0076] Linkers may, but need not, be used to separate CAR sequences
and/or antibody sequences within a modulating agent. Linkers may
also, or alternatively, be used to attach one or more modulating
agents to a support molecule or material, as described below. A
linker may be any molecule (including peptide and/or non-peptide
sequences as well as single amino acids or other molecules), that
does not contain a CAR sequence and that can be covalently linked
to at least two peptide sequences. Using a linker,
HAV-BM-containing peptides and other peptide or protein sequences
may be joined head-to-tail (i.e., the linker may be covalently
attached to the carboxyl or amino group of each peptide sequence),
head-to-side chain and/or tail-to-side chain. Modulating agents
comprising one or more linkers may form linear or branched
structures. Within one embodiment, modulating agents having a
branched structure comprise three different CAR sequences, such as
RGD, YIGSR (SEQ ID NO:66) and an HAV-BM sequence. Within another
embodiment, modulating agents having a branched structure may
comprise RGD, YIGSR (SEQ ID NO:66), an HAV-BM sequence and
KYSFNYDGSE (SEQ ID NO:67). In a third embodiment, modulating agents
having a branched structure comprise an HAV-BM sequence, one or
more Dsc CAR sequences, one or more Dsg CAR sequence and LYHY (SEQ
ID NO:70).
[0077] Linkers preferably produce a distance between CAR sequences
between 0.1 to 10,000 mn, more preferably about 0.1-400 nm. A
separation distance between recognition sites may generally be
determined according to the desired function of the modulating
agent. For inhibitors of cell adhesion, the linker distance between
HAV-BM sequences should be small (0.1-400 nm). For enhancers of
cell adhesion, the linker distance between HAV-BM sequences should
be 400-10,000 nm. One linker that can be used for such purposes is
(H.sub.2N(CH.sub.2).sub.nCO.sub.2H).sub.m, or derivatives thereof,
where n ranges from 1 to 10 and m ranges from 1 to 4000. For
example, if glycine (H.sub.2NCH.sub.2CO.sub.2H) or a multimer
thereof is used as a linker, each glycine unit corresponds to a
linking distance of 2.45 angstroms, or 0.245 nm, as determined by
calculation of its lowest energy conformation when linked to other
amino acids using molecular modeling techniques. Similarly,
aminopropanoic acid corresponds to a linking distance of 3.73
angstroms, aminobutanoic acid to 4.96 angstroms, aminopentanoic
acid to 6.30 angstroms and amino hexanoic acid to 6.12 angstroms.
Other linkers that may be used will be apparent to those of
ordinary skill in the art and include, for example, linkers based
on repeat units of 2,3-diaminopropanoic acid, lysine and/or
ornithine. 2,3-Diaminopropanoic acid can provide a linking distance
of either 2.51 or 3.11 angstroms depending on whether the
side-chain amino or terminal amino is used in the linkage.
Similarly, lysine can provide linking distances of either 2.44 or
6.95 angstroms and ornithine 2.44 or 5.61 angstroms. Peptide and
non-peptide linkers may generally be incorporated into a modulating
agent using any appropriate method known in the art.
[0078] Modulating agents that inhibit cell adhesion typically
contain one HAV-BM sequence or multiple HAV-BM sequences, which may
be adjacent to one another (i.e., without intervening sequences) or
in close proximity (i.e., separated by peptide and/or non-peptide
linkers to give a distance between the CAR sequences that ranges
from about 0.1 to 400 nm). Within one such embodiment, a modulating
agent contains two HAV-BM sequences. Such a modulating agent may
additionally comprise a CAR sequence for one or more different
adhesion molecules (including, but not limited to, other CAMs)
and/or one or more antibodies or fragments thereof that bind to
such sequences. Linkers may, but need not, be used to separate such
CAR sequence(s) and/or antibody sequence(s) from the HAV-BM
sequence(s) and/or each other. Such modulating agents may generally
be used within methods in which it is desirable to simultaneously
disrupt cell adhesion mediated by multiple adhesion molecules.
Within certain preferred embodiments, the second CAR sequence is
derived from fibronectin and is recognized by an integrin (i.e.,
RGD; see Cardarelli et al., J. Biol. Chem. 267:23159-23164, 1992),
or is an occludin CAR sequence (e.g., LYHY; SEQ ID NO:70). One or
more antibodies, or fragments thereof, may similarly be used within
such embodiments.
[0079] Modulating agents that enhance cell adhesion may contain
multiple HAV-BM sequences, and/or antibodies that specifically bind
to such sequences, joined by linkers as described above.
Enhancement of cell adhesion may also be achieved by attachment of
multiple modulating agents to a support molecule or material, as
discussed further below. Such modulating agents may additionally
comprise one or more CAR sequence for one or more different
adhesion molecules (including, but not limited to, other CAMs)
and/or one or more antibodies or fragments thereof that bind to
such sequences, to enhance cell adhesion mediated by multiple
adhesion molecules.
[0080] As noted above, modulating agents may be polypeptides or
salts thereof, containing only amino acid residues linked by
peptide bonds, or may contain non-peptide regions, such as linkers.
Peptide regions of a modulating agent may comprise residues of
L-amino acids, D-amino acids, or any combination thereof. Amino
acids may be from natural or non-natural sources, provided that at
least one amino group and at least one carboxyl group are present
in the molecule; .alpha.- and .beta.-amino acids are generally
preferred. The 20 L-amino acids commonly found in proteins are
identified herein by the conventional three-letter or one-letter
abbreviations.
[0081] A modulating agent may also contain rare amino acids (such
as 4-hydroxyproline or hydroxylysine), organic acids or amides
and/or derivatives of common amino acids, such as amino acids
having the C-terminal carboxylate esterified (e.g., benzyl, methyl
or ethyl ester) or amidated and/or having modifications of the
N-terminal amino group (e.g., acetylation or alkoxycarbonylation),
with or without any of a wide variety of side-chain modifications
and/or substitutions (e.g., methylation, benzylation, t-butylation,
tosylation, alkoxycarbonylation, and the like). Preferred
derivatives include amino acids having a C-terminal amide group.
Residues other than common amino acids that may be present with a
modulating agent include, but are not limited to,
2-mercaptoaniline, 2-mercaptoproline, omithine, diaminobutyric
acid, .alpha.-aminoadipic acid, m-aminomethylbenzoic acid and
.alpha.,.beta.-diaminopropionic acid.
[0082] Peptide modulating agents (and peptide portions of
modulating agents) as described herein may be synthesized by
methods well known in the art, including chemical synthesis and
recombinant DNA methods. For modulating agents up to about 50
residues in length, chemical synthesis may be performed using solid
phase peptide synthesis techniques, in which a peptide linkage
occurs through the direct condensation of the .alpha.-amino group
of one amino acid with the .alpha.-carboxy group of the other amino
acid with the elimination of a water molecule. Peptide bond
synthesis by direct condensation, as formulated above, requires
suppression of the reactive character of the amino group of the
first and of the carboxyl group of the second amino acid. The
masking substituents must permit their ready removal, without
inducing breakdown of the labile peptide molecule.
[0083] Solid phase peptide synthesis uses an insoluble polymer for
support during organic synthesis. The polymer-supported peptide
chain permits the use of simple washing and filtration steps
instead of laborious purifications at intermediate steps.
Solid-phase peptide synthesis may generally be performed according
to the method of Merrifield et al., J. Am. Chem. Soc. 85:2149,
1963, which involves assembling a linear peptide chain on a resin
support using protected amino acids. Solid phase peptide synthesis
typically utilizes either the Boc or Fmoc strategy. The Boc
strategy uses a 1% cross-linked polystyrene resin. The standard
protecting group for .alpha.-amino functions is the
tert-butyloxycarbonyl (Boc) group. This group can be removed with
dilute solutions of strong acids such as 25% trifluoroacetic acid
(TFA). The next Boc-amino acid is typically coupled to the amino
acyl resin using dicyclohexylcarbodiimide (DCC). Following
completion of the assembly, the peptide-resin is treated with
anhydrous HF to cleave the benzyl ester link and liberate the free
peptide. Side-chain functional groups are usually blocked during
synthesis by benzyl-derived blocking groups, which are also cleaved
by HF. The free peptide is then extracted from the resin with a
suitable solvent, purified and characterized. Newly synthesized
peptides can be purified, for example, by gel filtration, HPLC,
partition chromatography and/or ion-exchange chromatography, and
may be characterized by, for example, mass spectrometry or amino
acid sequence analysis. In the Boc strategy, C-terminal amidated
peptides can be obtained using benzhydrylamine or
methylbenzhydrylamine resins, which yield peptide amides directly
upon cleavage with HF.
[0084] In the procedures discussed above, the selectivity of the
side-chain blocking groups and of the peptide-resin link depends
upon the differences in the rate of acidolytic cleavage. Orthoganol
systems have been introduced in which the side-chain blocking
groups and the peptide-resin link are completely stable to the
reagent used to remove the .alpha.-protecting group at each step of
the synthesis. The most common of these methods involves the
9-fluorenylmethyloxycarbonyl (Fmoc) approach. Within this method,
the side-chain protecting groups and the peptide-resin link are
completely stable to the secondary amines used for cleaving the
N-.alpha.-Fmoc group. The side-chain protection and the
peptide-resin link are cleaved by mild acidolysis. The repeated
contact with base makes the Merrifield resin unsuitable for Fmoc
chemistry, and p-alkoxybenzyl esters linked to the resin are
generally used. Deprotection and cleavage are generally
accomplished using TFA.
[0085] Those of ordinary skill in the art will recognize that, in
solid phase synthesis, deprotection and coupling reactions must go
to completion and the side-chain blocking groups must be stable
throughout the entire synthesis. In addition, solid phase synthesis
is generally most suitable when peptides are to be made on a small
scale.
[0086] Acetylation of the N-terminus can be accomplished by
reacting the final peptide with acetic anhydride before cleavage
from the resin. C-amidation is accomplished using an appropriate
resin such as methylbenzhydrylamine resin using the Boc
technology.
[0087] Following synthesis of a linear peptide, with or without
N-acetylation and/or C-amidation, cyclization may be achieved if
desired by any of a variety of techniques well known in the art.
Within one embodiment, a bond may be generated between reactive
amino acid side chains. For example, a disulfide bridge may be
formed from a linear peptide comprising two thiol-containing
residues by oxidizing the peptide using any of a variety of
methods. Within one such method, air oxidation of thiols can
generate disulfide linkages over a period of several days using
either basic or neutral aqueous media. The peptide is used in high
dilution to minimize aggregation and intermolecular side reactions.
This method suffers from the disadvantage of being slow but has the
advantage of only producing H.sub.2O as a side product.
Alternatively, strong oxidizing agents such as I.sub.2 and
K.sub.3Fe(CN).sub.6 can be used to form disulfide linkages. Those
of ordinary skill in the art will recognize that care must be taken
not to oxidize the sensitive side chains of Met, Tyr, Trp or His.
Cyclic peptides produced by this method require purification using
standard techniques, but this oxidation is applicable at acid pHs.
Oxidizing agents also allow concurrent deprotection/oxidation of
suitable S-protected linear precursors to avoid premature,
nonspecific oxidation of free cysteine.
[0088] DMSO, unlike I.sub.2 and K.sub.3Fe(CN).sub.6, is a mild
oxidizing agent which does not cause oxidative side reactions of
the nucleophilic amino acids mentioned above. DMSO is miscible with
H.sub.2O at all concentrations, and oxidations can be performed at
acidic to neutral pHs with harmless byproducts.
Methyltrichlorosilane-diphenylsulfoxide may alternatively be used
as an oxidizing agent, for concurrent deprotection/oxidation of
S-Acm, S-Tacm or S-t-Bu of cysteine without affecting other
nucleophilic amino acids. There are no polymeric products resulting
from intermolecular disulfide bond formation. Suitable
thiol-containing residues for use in such oxidation methods
include, but are not limited to, cysteine, .beta.,.beta.-dimethyl
cysteine (penicillamine or Pen), .beta.,.beta.-tetramethylene
cysteine (Tmc), .beta.,.beta.-pentamethylene cysteine (Pmc),
.beta.-mercaptopropionic acid (Mpr),
.beta.,.beta.-pentamethylene-.beta.-mercaptopropionic acid (Pmp),
2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline.
[0089] Within another embodiment, cyclization may be achieved by
amide bond formation. For example, a peptide bond may be formed
between terminal functional groups (i.e., the amino and carboxy
termini of a linear peptide prior to cyclization). Within another
such embodiment, the linear peptide comprises a D-amino acid.
Alternatively, cyclization may be accomplished by linking one
terminus and a residue side chain or using two side chains, with or
without an N-terminal acetyl group and/or a C-terminal amide.
Residues capable of forming a lactam bond include lysine, ornithine
(Orn), .alpha.-amino adipic acid, m-aminomethylbenzoic acid,
.alpha.,.beta.-diaminopropionic acid, glutamate or aspartate.
[0090] Methods for forming amide bonds are well known in the art
and are based on well established principles of chemical
reactivity. Within one such method, carbodiimide-mediated lactam
formation can be accomplished by reaction of the carboxylic acid
with DCC, DIC, EDAC or DCCI, resulting in the formation of an
O-acylurea that can be reacted immediately with the free amino
group to complete the cyclization. The formation of the inactive
N-acylurea, resulting from ON migration, can be circumvented by
converting the O-acylurea to an active ester by reaction with an
N-hydroxy compound such as 1-hydroxybenzotriazole,
1-hydroxysuccinimide, 1-hydroxynorbornene carboxamide or ethyl
2-hydroximino-2-cyanoacetate. In addition to minimizing O.fwdarw.N
migration, these additives also serve as catalysts during
cyclization and assist in lowering racemization. Alternatively,
cyclization can be performed using the azide method, in which a
reactive azide intermediate is generated from an alkyl ester via a
hydrazide. Hydrazinolysis of the terminal ester necessitates the
use of a t-butyl group for the protection of side chain carboxyl
functions in the acylating component. This limitation can be
overcome by using diphenylphosphoryl acid (DPPA), which furnishes
an azide directly upon reaction with a carboxyl group. The slow
reactivity of azides and the formation of isocyanates by their
disproportionation restrict the usefulness of this method. The
mixed anhydride method of lactam formation is widely used because
of the facile removal of reaction by-products. The anhydride is
formed upon reaction of the carboxylate anion with an alkyl
chloroformate or pivaloyl chloride. The attack of the amino
component is then guided to the carbonyl carbon of the acylating
component by the electron donating effect of the alkoxy group or by
the steric bulk of the pivaloyl chloride t-butyl group, which
obstructs attack on the wrong carbonyl group. Mixed anhydrides with
phosphoric acid derivatives have also been successfully used.
Alternatively, cyclization can be accomplished using activated
esters. The presence of electron withdrawing substituents on the
alkoxy carbon of esters increases their susceptibility to
aminolysis. The high reactivity of esters of p-nitrophenol,
N-hydroxy compounds and polyhalogenated phenols has made these
"active esters" useful in the synthesis of amide bonds. The last
few years have witnessed the development of
benzotriazolyloxytris-(dimeth- ylamino)phosphonium
hexafluorophosphonate (BOP) and its congeners as advantageous
coupling reagents. Their performance is generally superior to that
of the well established carbodiimide amide bond formation
reactions.
[0091] Within a further embodiment, a thioether linkage may be
formed between the side chain of a thiol-containing residue and an
appropriately derivatized .alpha.-amino acid. By way of example, a
lysine side chain can be coupled to bromoacetic acid through the
carbodiimide coupling method (DCC, EDAC) and then reacted with the
side chain of any of the thiol containing residues mentioned above
to form a thioether linkage. In order to form dithioethers, any two
thiol containing side-chains can be reacted with dibromoethane and
diisopropylamine in DMF.
[0092] For longer modulating agents, recombinant methods are
preferred for synthesis. Within such methods, all or part of a
modulating agent can be synthesized in living cells, using any of a
variety of expression vectors known to those of ordinary skill in
the art to be appropriate for the particular host cell. Suitable
host cells may include bacteria, yeast cells, mammalian cells,
insect cells, plant cells, algae and other animal cells (e.g.,
hybridoma, CHO, myeloma). The DNA sequences expressed in this
manner may encode portions of an endogenous cadherin or other
adhesion molecule. Such sequences may be prepared based on known
cDNA or genomic sequences (see Blaschuk et al., J. Mol. Biol.
211:679-682, 1990), or from sequences isolated by screening an
appropriate library with probes designed based on the sequences of
known cadherins. Such screens may generally be performed as
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,
1989 (and references cited therein). Polymerase chain reaction
(PCR) may also be employed, using oligonucleotide primers in
methods well known in the art, to isolate nucleic acid molecules
encoding all or a portion of an endogenous adhesion molecule. To
generate a nucleic acid molecule encoding a desired modulating
agent, an endogenous cadherin sequence may be modified using well
known techniques. For example, portions encoding one or more CAR
sequences may be joined, with or without separation by nucleic acid
regions encoding linkers, as discussed above. Alternatively,
portions of the desired nucleic acid sequences may be synthesized
using well known techniques, and then ligated together to form a
sequence encoding the modulating agent.
[0093] As noted above, instead of (or in addition to) an HAV-BM
sequence, a modulating agent may comprise an antibody, or
antigen-binding fragment thereof, that specifically binds to an
HAV-BM sequence. As used herein, an antibody, or antigen-binding
fragment thereof, is said to "specifically bind" to an HAV-BM
sequence (with or without flanking amino acids) if it reacts at a
detectable level with a peptide containing that sequence, and does
not react detectably with peptides containing a different CAR
sequence or a sequence in which the order of amino acid residues in
the cadherin CAR sequence and/or flanking sequence is altered. Such
antibody binding properties may be assessed using an ELISA, as
described by Newton et al., Develop. Dynamics 197:1-13, 1993.
[0094] Polyclonal and monoclonal antibodies may be raised against
an HAV-BM sequence using conventional techniques. See, e.g., Harlow
and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988. In one such technique, an immunogen comprising
the HAV-BM sequence is initially injected into any of a wide
variety of mammals (e.g., mice, rats, rabbits, sheep or goats). The
smaller immunogens (i.e., less than about 20 amino acids) should be
joined to a carrier protein, such as bovine serum albumin or
keyhole limpet hemocyanin. Following one or more injections, the
animals are bled periodically. Polyclonal antibodies specific for
the CAR sequence may then be purified from such antisera by, for
example, affinity chromatography using the modulating agent or
antigenic portion thereof coupled to a suitable solid support.
[0095] Monoclonal antibodies specific for the HAV-BM sequence may
be prepared, for example, using the technique of Kohler and
Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements
thereto. Briefly, these methods involve the preparation of immortal
cell lines capable of producing antibodies having the desired
specificity from spleen cells obtained from an animal immunized as
described above. The spleen cells are immortalized by, for example,
fusion with a myeloma cell fusion partner, preferably one that is
syngeneic with the immunized animal. Single colonies are selected
and their culture supernatants tested for binding activity against
the modulating agent or antigenic portion thereof. Hybridomas
having high reactivity and specificity are preferred.
[0096] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies, with or without the use of various
techniques known in the art to enhance the yield. Contaminants may
be removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation and extraction.
Antibodies having the desired activity may generally be identified
using immunofluorescence analyses of tissue sections, cell or other
samples where the target cadherin is localized.
[0097] Within preferred embodiments, such monoclonal antibodies are
specific for particular cadherins (e.g., the antibodies bind to
E-cadherin, but do not bind significantly to N-cadherin, or vise
versa). Such antibodies may be prepared as described above, using
an immunogen that comprises (in addition to a minimal HAV-BM
sequence) sufficient flanking sequence to generate the desired
specificity. To evaluate the specificity of a particular antibody,
representative assays as described herein and/or conventional
antigen-binding assays may be employed. Such antibodies may
generally be used for therapeutic, diagnostic and assay purposes,
as described herein. For example, such antibodies may be linked to
a drug and administered to a mammal to target the drug to a
particular cadherin-expressing cell.
[0098] Within certain embodiments, the use of antigen-binding
fragments of antibodies may be preferred. Such fragments include
Fab fragments, which may be prepared using standard techniques.
Briefly, immunoglobulins may be purified from rabbit serum by
affinity chromatography on Protein A bead columns (Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988; see especially page 309) and digested by papain to yield Fab
and Fc fragments. The Fab and Fc fragments may be separated by
affinity chromatography on protein A bead columns (Harlow and Lane,
1988, pages 628-29).
[0099] Within certain embodiments, antibodies may be used within
methods in which enhanced cell adhesion is desired, as described
above. For example, antibodies may be used within the above methods
for enhancing and/or directing neurite outgrowth in vitro or in
vivo. Antibodies may be used within the lumen of a tubular nerve
guide or may be attached to a fiber nerve guide, suture or other
solid support and used as described above for peptide modulating
agents. Antibody dosages are sufficient to enhance or direct
neurite outgrowth, and will vary with the method of administration
and the condition to be treated.
[0100] Antibodies may also be used as a "biological glue," as
described above to bind multiple cadherin-expressing cells within a
variety of contexts, such as to enhance wound healing and/or reduce
scar tissue, and/or to facilitate cell adhesion in skin grafting or
prosthetic implants. In general, the amount of matrix-linked
antibody administered to a wound, graft or implant site varies with
the severity of the wound and/or the nature of the wound, graft, or
implant, but may vary as discussed above. Antibodies may also be
linked to any of a variety of support materials, as described
above, for use in tissue culture or bioreactors.
[0101] Antibodies (or, preferably, antigen-binding fragments
thereof) may also be used in situations where inhibition of cell
adhesion is desired. Such antibodies or fragments may be used, for
example, for treatment of demyelinating diseases, such as MS, or to
inhibit interactions between tumor cells, as described above. The
use of Fab fragments is generally preferred.
[0102] Evaluation of Modulating Agent Activity
[0103] As noted above, native HAV-BM sequences, as well as
analogues and mimetics thereof, bind to a classical cadherin,
preferably within or near an HAV sequence, and modulate a
cadherin-mediated response. The ability to bind to a cadherin
sequence may generally be evaluated using any binding assay known
to those of ordinary skill in the art. For example, a Pharmacia
Biosensor machine may be used, as discussed in Jonsson et al.,
Biotechniques 11:520-27, 1991. A specific example of the technology
that measures the interaction of peptides with molecules can be
found in Williams et al., J. Biol. Chem. 272:8539-8545, 1997.
Real-time BIA (Biomolecular Interaction Analysis) uses the optical
phenomenon surface plasmon resonance to monitor biomolecular
interactions. The detection depends upon changes in the mass
concentration of macromolecules at the biospecific interface, which
in turn depends upon the immobilization of test molecule or peptide
(referred to as the ligand) to the surface of a Biosensor chip,
followed by binding of the interacting molecule (referred to as the
analyte) to the ligand. Binding to the chip is measured in
real-time in arbitrary units of resonance (RU).
[0104] For example, surface plasmon resonance experiments may be
carried out using a BIAcore X.TM. Biosensor (Pharmacia Ltd.,
BIAcore, Uppsala, Sweden). Parallel flow cells of CM 5 sensor chips
may be derivatized, using the amine coupling method, with
streptavidin (200 .mu.g/ml) in 10 mM Sodium Acetate, pH 4.0,
according to the manufacturer's protocol. Approximately 2100-2600
resonance units (RU) of ligand may be immobilized, corresponding to
a concentration of about 2.1-2.6 ng/mm.sup.2. The chips may then
coated be with a peptide comprising a known or putative HAV-BM, or
analogue or mimetic thereof. Any non-specifically bound peptide is
removed.
[0105] To determine binding, test analytes (e.g., cadherin
peptides, such as HAV-containing peptides) may be placed in running
buffer and passed simultaneously over test and control flow cells.
After a period of free buffer flow, any analyte remaining bound to
the surface may be removed with, for example, a pulse of 0.1% SDS
bringing the signal back to baseline. Specific binding to the
derivatized sensor chips may be determined automatically by the
system by subtraction of test from control flow cell responses. In
general, an HAV-BM, or a mimetic or analogue thereof, binds an
HAV-containing peptide at a detectable level within such as assay.
Preferably, the level of binding is at least that observed for a
native HAV-BM as provided herein under similar conditions.
[0106] The ability to modulate a cadherin-mediated function may be
evaluated using any of a variety of in vitro assays designed to
measure the effect of the peptide on a typical cadherin response.
As noted above, modulating agents may be capable of enhancing or
inhibiting a cadherin-mediated function. The ability of an agent to
modulate cell adhesion may generally be evaluated in vitro by
assaying the effect on one or more of the following: (1) neurite
outgrowth, (2) Schwann cell-astrocyte adhesion, (3) Schwann cell
migration on astrocyte monolayers, (4) adhesion between endothelial
cells, (5) adhesion between epithelial cells (e.g., normal rat
kidney cells and/or human skin) and/or (6) adhesion between cancer
cells. In general, a modulating agent is an inhibitor of cell
adhesion if, within one or more of these representative assays,
contact of the test cells with the modulating agent results in a
discernible disruption of cell adhesion. Modulating agents that
enhance cell adhesion (e.g., agents comprising multiple HAV-BM
sequences and/or linked to a support material) are considered to be
modulators of cell adhesion if they are capable of enhancing
neurite outgrowth as described below or are capable of promoting
cell adhesion, as judged by plating assays to assess epithelial
cell adhesion to a modulating agent attached to a support material,
such as tissue culture plastic.
[0107] Within a representative neurite outgrowth assay, neurons may
be cultured on a monolayer of cells (e.g., 3T3 fibroblasts) that
express N-cadherin. Neurons grown on such cells (under suitable
conditions and for a sufficient period of time) extend neurites
that are typically, on average, twice as long as neurites extended
from neurons cultured on 3T3 cells that do not express N-cadherin.
For example, neurons may be cultured on monolayers of 3T3 cells
transfected with cDNA encoding N-cadherin essentially as described
by Doherty and Walsh, Curr. Op. Neurobiol. 4:49-55, 1994; Williams
et al., Neuron 13:583-594, 1994; Hall et al., Cell Adhesion and
Commun. 3:441-450, 1996; Doherty and Walsh, Mol. Cell. Neurosci.
8:99-111, 1994; and Safell et al., Neuron 18:231-242, 1997.
Briefly, monolayers of control 3T3 fibroblasts and 3T3 fibroblasts
that express N-cadherin may be established by overnight culture of
80,000 cells in individual wells of an 8-chamber well tissue
culture slide. 3000 cerebellar neurons isolated from post-natal day
3 mouse brains may be cultured for 18 hours on the various
monolayers in control media (SATO/2% FCS), or media supplemented
with various concentrations of the modulating agent or control
peptide. The cultures may then be fixed and stained for GAP43 which
specifically binds to the neurons and their neurites. The length of
the longest neurite on each GAP43 positive neuron may be measured
by computer assisted morphometry.
[0108] A modulating agent that modulates N-cadherin-mediated cell
adhesion may inhibit or enhance such neurite outgrowth. Under the
conditions described above, the presence of 500 .mu.g/mL of a
modulating agent that disrupts neural cell adhesion should result
in a decrease in the mean neurite length by at least 50%, relative
to the length in the absence of modulating agent or in the presence
of a negative control peptide. Alternatively, the presence of 500
.mu.g/mL of a modulating agent that enhances neural cell adhesion
should result in an increase in the mean neurite length by at least
50%.
[0109] The effect of a modulating agent on Schwann cell adhesion to
astrocytes may generally be evaluated using a cell adhesion assay.
Briefly, Schwann cells fluorescently labeled with Di-I may be
plated onto an astrocytic surface (e.g., a glass coverslip coated
with a monolayer of astrocytes) and incubated on a shaking platform
(e.g., 25 rpm for 30 minutes) in the presence and absence of
modulating agent at a concentration of approximately 1 mg/mL. Cells
may then be washed (e.g., in Hanks medium) to remove non-attached
cells. The attached cells may then be fixed and counted (e.g.,
using a fluorescent microscope). In general, 1 mg/mL of a
modulating agent results in an increase or decrease in cell
adhesion of at least 50%. This assay evaluates the effect of a
modulating agent on N-cadherin mediated cell adhesion.
[0110] Schwann cell migration may generally be evaluated using a
micro-inverted-coverslip assay. In this assay, a dense Schwann cell
culture is established on coverslip fragments and Schwann cell
migration away from the fragment edge is measured. Briefly, Schwann
cells fluorescently labeled with Di-I may be plated on polylysine-
and laminin-coated fragments of a glass coverslip and allowed to
bind to the surface for 16-18 hours. Cells may then be washed
(e.g., in Hanks medium) to remove non-attached cells, and then
inverted, with cells facing downward onto an astrocyte-coated
surface. Cultures are then incubated further for 2 days in the
presence or absence of modulating agent at a concentration of
approximately 1 mg/mL and fixed. The maximum migration distance
from the edge of the coverslip fragment may then be measured. At a
level of 1 mg/mL, a modulating agent results in an increase or
decrease in the maximum migration distance of at least 50%. This
assay evaluates the effect of a modulating agent on N-cadherin
mediated cell adhesion.
[0111] Within certain cell adhesion assays, the addition of a
modulating agent to cells that express a cadherin results in
disruption of cell adhesion. A "cadherin-expressing cell," as used
herein, may be any type of cell that expresses at least one
cadherin on the cell surface at a detectable level, using standard
techniques such as immunocytochemical protocols (e.g., Blaschuk and
Farookhi, Dev. Biol. 136:564-567, 1989). Cadherin-expressing cells
include endothelial, epithelial and/or cancer cells. For example,
such cells may be plated under standard conditions that, in the
absence of modulating agent, permit cell adhesion. In the presence
of modulating agent (e.g., 500 .mu.g/mL), disruption of cell
adhesion may be determined visually within 24 hours, by observing
retraction of the cells from one another.
[0112] For use within one such assay, bovine pulmonary artery
endothelial cells may be harvested by sterile ablation and
digestion in 0.1% collagenase (type II; Worthington Enzymes,
Freehold, N.J.). Cells may be maintained in Dulbecco's minimum
essential medium supplemented with 10% fetal calf serum and 1%
antibiotic-antimycotic at 37.degree. C. in 7% CO.sub.2 in air.
Cultures may be passaged weekly in trypsin-EDTA and seeded onto
tissue culture plastic at 20,000 cells/cm.sup.2. Endothelial
cultures may be used at 1 week in culture, which is approximately.
3 days after culture confluency is established. The cells may be
seeded onto coverslips and treated (e.g., for 30 minutes) with
modulating agent or a control compound at, for example, 500
.mu.g/ml and then fixed with 1% paraformaldehyde. As noted above,
disruption of cell adhesion may be determined visually within 24
hours, by observing retraction of the cells from one another. This
assay evaluates the effect of a modulating agent on N-cadherin
mediated cell adhesion.
[0113] Within another such assay, the effect of a modulating agent
on normal rat kidney (NRK) cells may be evaluated. According to a
representative procedure, NRK cells (ATCC #1571-CRL) may be plated
at 10-20,000 cells per 35 mm tissue culture flasks containing DMEM
with 10% FCS and sub-cultured periodically (Laird et al., J. Cell
Biol. 131:1193-1203, 1995). Cells may be harvested and replated in
35 mm tissue culture flasks containing 1 mm coverslips and
incubated until 50-65% confluent (24-36 hours). At this time,
coverslips may be transferred to a 24-well plate, washed once with
fresh DMEM and exposed to modulating agent at a concentration of,
for example, 1 mg/mL for 24 hours. Fresh modulating agent may then
be added, and the cells left for an additional 24 hours. Cells may
be fixed with 100% methanol for 10 minutes and then washed three
times with PBS. Coverslips may be blocked for 1 hour in 2% BSA/PBS
and incubated for a further 1 hour in the presence of mouse
anti-E-cadherin antibody (Transduction Labs, 1:250 dilution).
Primary and secondary antibodies may be diluted in 2% BSA/PBS.
Following incubation in the primary antibody, coverslips may be
washed three times for 5 minutes each in PBS and incubated for 1
hour with donkey anti-mouse antibody conjugated to fluorescein
(diluted 1:200). Following further washes in PBS (3.times.5 min)
coverslips can be mounted and viewed by confocal microscopy.
[0114] In the absence of modulating agent, NRK cells form
characteristic tightly adherent monolayers with a cobblestone
morphology in which cells display a polygonal shape. NRK cells that
are treated with a modulating agent that disrupts E-cadherin
mediated cell adhesion may assume a non-polygonal and elongated
morphology (i.e., a fibroblast-like shape) within 48 hours of
treatment with 1 mg/mL of modulating agent. Gaps appear in
confluent cultures of such cells. In addition, 1 mg/mL of such a
modulating agent reproducibly induces a readily apparent reduction
in cell surface staining of E-cadherin, as judged by
immunofluorescence microscopy (Laird et al., J. Cell Biol.
131:1193-1203, 1995), of at least 75% within 48 hours.
[0115] A third cell adhesion assay involves evaluating the effect
of a modulating agent on permeability of adherent epithelial and/or
endothelial cell layers. For example, the effect of permeability on
human skin may be evaluated. Such skin may be derived from a
natural source or may be synthetic. Human abdominal skin for use in
such assays may generally be obtained from humans at autopsy within
24 hours of death. Briefly, a modulating agent (e.g., 500 .mu.g/ml)
and a test marker (e.g., the fluorescent markers Oregon Green.TM.
and Rhodamine Green.TM. Dextran) may be dissolved in a sterile
buffer (e.g., phosphate buffer, pH 7.2), and the ability of the
marker to penetrate through the skin and into a receptor fluid
(e.g., phosphate buffer) may be measured using a Franz Cell
apparatus (Franz, Curr. Prob. Dermatol. 7:58-68, 1978; Franz, J.
Invest. Dermatol. 64:190-195, 1975). The penetration of the markers
through the skin may be assessed at, for example, 6, 12, 24, 36,
and 48 hours after the start of the experiment. In general, a
modulating agent that enhances the permeability of human skin
results in a statistically significant increase in the amount of
marker in the receptor compartment after 6-48 hours in the presence
of 500 .mu.g/mL modulating agent. This assay evaluates the effect
of a modulating agent on E-cadherin mediated cell adhesion.
[0116] Modulating Agent Modification and Formulations
[0117] A modulating agent as described herein may, but need not, be
linked to one or more additional molecules. In particular, as
discussed below, it may be beneficial for certain applications to
link multiple modulating agents (which may, but need not, be
identical) to a support material, such as a support molecule (e.g.,
keyhole limpet hemocyanin) or a solid support, such as a polymeric
matrix (which may be formulated as a membrane or microstructure,
such as an ultra thin film), a container surface (e.g., the surface
of a tissue culture plate or the interior surface of a bioreactor),
or a bead or other particle, which may be prepared from a variety
of materials including glass, plastic or ceramics. For certain
applications, biodegradable support materials are preferred, such
as cellulose and derivatives thereof, collagen, spider silk or any
of a variety of polyesters (e.g., those derived from hydroxy acids
and/or lactones) or sutures (see U.S. Pat. No. 5,245,012). Within
certain embodiments, modulating agents and molecules comprising
other CAR sequence(s) (e.g., HAV, RGD or LYHY (SEQ ID NO:70)) may
be attached to a support such as a polymeric matrix, preferably in
an alternating pattern.
[0118] Suitable methods for linking a modulating agent to a support
material will depend upon the composition of the support and the
intended use, and will be readily apparent to those of ordinary
skill in the art. Attachment may generally be achieved through
noncovalent association, such as adsorption or affinity or,
preferably, via covalent attachment (which may be a direct linkage
between a modulating agent and functional groups on the support, or
may be a linkage by way of a cross-linking agent). Attachment of a
modulating agent by adsorption may be achieved by contact, in a
suitable buffer, with a solid support for a suitable amount of
time. The contact time varies with temperature, but is generally
between about 5 seconds and 1 day, and typically between about 10
seconds and 1 hour.
[0119] Covalent attachment of a modulating agent to a molecule or
solid support may generally be achieved by first reacting the
support material with a bifunctional reagent that will also react
with a functional group, such as a hydroxyl or amino group, on the
modulating agent. For example, a modulating agent may be bound to
an appropriate polymeric support or coating using benzoquinone, by
condensation of an aldehyde group on the support with an amine and
an active hydrogen on the modulating agent or by condensation of an
amino group on the support with a carboxylic acid on the modulating
agent. A preferred method of generating a linkage is via amino
groups using glutaraldehyde. A modulating agent may be linked to
cellulose via ester linkages. Similarly, amide linkages may be
suitable for linkage to other molecules such as keyhole limpet
hemocyanin or other support materials. Multiple modulating agents
and/or molecules comprising other CAR sequences may be attached,
for example, by random coupling, in which equimolar amounts of such
molecules are mixed with a matrix support and allowed to couple at
random.
[0120] Although modulating agents as described herein may
preferentially bind to specific tissues or cells, and thus may be
sufficient to target a desired site in vivo, it may be beneficial
for certain applications to include an additional targeting agent.
Accordingly, a targeting agent may also, or alternatively, be
linked to a modulating agent to facilitate targeting to one or more
specific tissues. As used herein, a "targeting agent," may be any
substance (such as a compound or cell) that, when linked to a
modulating agent enhances the transport of the modulating agent to
a target tissue, thereby increasing the local concentration of the
modulating agent. Targeting agents include antibodies or fragments
thereof, receptors, ligands and other molecules that bind to cells
of, or in the vicinity of, the target tissue. Known targeting
agents include serum hormones, antibodies against cell surface
antigens, lectins, adhesion molecules, tumor cell surface binding
ligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes
and those drugs and proteins that bind to a desired target site.
Among the many monoclonal antibodies that may serve as targeting
agents are anti-TAC, or other interleukin-2 receptor antibodies;
9.2.27 and NR-ML-05, reactive with the 250 kilodalton human
melanoma-associated proteoglycan; and NR-LU-10, reactive with a
pancarcinoma glycoprotein. An antibody targeting agent may be an
intact (whole) molecule, a fragment thereof, or a functional
equivalent thereof. Examples of antibody fragments are F(ab')2,
-Fab', Fab and F[v] fragments, which may be produced by
conventional methods or by genetic or protein engineering. Linkage
is generally covalent and may be achieved by, for example, direct
condensation or other reactions, or by way of bi- or
multi-functional linkers. Within other embodiments, it may also be
possible to target a polynucleotide encoding a modulating agent to
a target tissue, thereby increasing the local concentration of
modulating agent. Such targeting may be achieved using well known
techniques, including retroviral and adenoviral infection.
[0121] For certain embodiments, it may be beneficial to also, or
alternatively, link a drug to a modulating agent. As used herein,
the term "drug" refers to any bioactive agent intended for
administration to a mammal to prevent or treat a disease or other
undesirable condition. Drugs include hormones, growth factors,
proteins, peptides and other compounds. The use of certain specific
drugs within the context of the present invention is discussed
below.
[0122] Within certain aspects of the present invention, one or more
modulating agents as described herein may be present within a
pharmaceutical composition. A pharmaceutical composition comprises
one or more modulating agents in combination with one or more
pharmaceutically or physiologically acceptable carriers, diluents
or excipients. Such compositions may comprise buffers (e.g.,
neutral buffered saline or phosphate buffered saline),
carbohydrates (e.g., glucose, mannose, sucrose or dextrans),
mannitol, proteins, polypeptides or amino acids such as glycine,
antioxidants, chelating agents such as EDTA or glutathione,
adjuvants (e.g., aluminum hydroxide) and/or preservatives. Within
yet other embodiments, compositions of the present invention may be
formulated as a lyophilizate. One or more modulating agents (alone
or in combination with a targeting agent and/or drug) may, but need
not, be encapsulated within liposomes using well known technology.
Compositions of the present invention may be formulated for any
appropriate manner of administration, including for example,
topical, oral, nasal, intravenous, intracranial, intraperitoneal,
subcutaneous, or intramuscular administration.
[0123] For certain embodiments, as discussed below, a
pharmaceutical composition may further comprise a modulator of cell
adhesion that is mediated by one or more molecules other than
cadherins. Such modulators may generally be prepared as described
above, incorporating one or more non-cadherin CAR sequences and/or
antibodies thereto in place of the HAV-BM sequences and antibodies.
Such compositions are particularly useful for situations in which
it is desirable to inhibit cell adhesion mediated by multiple
cell-adhesion molecules, such as other members of the cadherin gene
superfamily that are not classical cadherins (e.g., Dsg and Dsc);
integrins; members of the immunoglobulin supergene family, such as
N--CAM; and other uncategorized transmembrane proteins, such as
occludin, as well as extracellular matrix proteins such as laminin,
fibronectin, collagens, vitronectin, entactin and tenascin.
Preferred CAR sequences for use within such a modulator include
HAV, RGD, YIGSR (SEQ ID NO:66), KYSFNYDGSE (SEQ ID NO:67), a Dsc or
Dsg CAR sequence, a claudin CAR sequence, a JAM CAR sequence and/or
LYHY (SEQ ID NO:70).
[0124] A pharmaceutical composition may also, or alternatively,
contain one or more drugs, which may be linked to a modulating
agent or may be free within the composition. Virtually any drug may
be administered in combination with a modulating agent as described
herein, for a variety of purposes as described below. Examples of
types of drugs that may be administered with a modulating agent
include analgesics, anesthetics, antianginals, antifungals,
antibiotics, anticancer drugs (e.g., taxol or mitomycin C),
antiinflammatories (e.g., ibuprofen and indomethacin),
anthelmintics, antidepressants, antidotes, antiemetics,
antihistamines, antihypertensives, antimalarials, antimicrotubule
agents (e.g., colchicine or vinca alkaloids), antimigraine agents,
antimicrobials, antiphsychotics, antipyretics, antiseptics,
anti-signaling agents (e.g., protein kinase C inhibitors or
inhibitors of intracellular calcium mobilization), antiarthritics,
antithrombin agents, antituberculotics, antitussives, antivirals,
appetite suppressants, cardioactive drugs, chemical dependency
drugs, cathartics, chemotherapeutic agents, coronary, cerebral or
peripheral vasodilators, contraceptive agents, depressants,
diuretics, expectorants, growth factors, hormonal agents,
hypnotics, immunosuppression agents, narcotic antagonists,
parasympathomimetics, sedatives, stimulants, sympathomimetics,
toxins (e.g., cholera toxin), tranquilizers and urinary
antiinfectives.
[0125] For imaging purposes, any of a variety of diagnostic agents
may be incorporated into a pharmaceutical composition, either
linked to a modulating agent or free within the composition.
Diagnostic agents include any substance administered to illuminate
a physiological function within a patient, while leaving other
physiological functions generally unaffected. Diagnostic agents
include metals, radioactive isotopes and radioopaque agents (e.g.,
gallium, technetium, indium, strontium, iodine, barium, bromine and
phosphorus-containing compounds), radiolucent agents, contrast
agents, dyes (e.g., fluorescent dyes and chromophores) and enzymes
that catalyze a colorimetric or fluorometric reaction. In general,
such agents may be attached using a variety of techniques as
described above, and may be present in any orientation.
[0126] The compositions described herein may be administered as
part of a sustained release formulation (i.e., a formulation such
as a capsule or sponge that effects a slow release of modulating
agent following administration). Such formulations may generally be
prepared using well known technology and administered by, for
example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release
formulations may contain a modulating agent dispersed in a carrier
matrix and/or contained within a reservoir surrounded by a rate
controlling membrane (see, e.g., European Patent Application
710,491 A). Carriers for use within such formulations are
biocompatible, and may also be biodegradable; preferably the
formulation provides a relatively constant level of modulating
agent release. The amount of modulating agent contained within a
sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated or prevented.
[0127] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). Appropriate dosages and a suitable duration and
frequency of administration will be determined by such factors as
the condition of the patient, the type and severity of the
patient's disease and the method of administration. In general, an
appropriate dosage and treatment regimen provides the modulating
agent(s) in an amount sufficient to provide therapeutic and/or
prophylactic benefit. Within particularly preferred embodiments of
the invention, a modulating agent or pharmaceutical composition as
described herein may be administered at a dosage ranging from 0.001
to 50 mg/kg body weight, preferably from 0.1 to 20 mg/kg, on a
regimen of single or multiple daily doses. For topical
administration, a cream typically comprises an amount of modulating
agent ranging from 0.00001% to 1%, preferably 0.0001% to 0.002%.
Fluid compositions typically contain about 10 ng/ml to 5 mg/ml,
preferably from about 10 .mu.g to 2 mg/mL modulating agent.
Appropriate dosages may generally be determined using experimental
models and/or clinical trials. In general, the use of the minimum
dosage that is sufficient to provide effective therapy is
preferred. Patients may generally be monitored for therapeutic
effectiveness using assays suitable for the condition being treated
or prevented, which will be familiar to those of ordinary skill in
the art.
[0128] Therapeutic Methods Employing Modulating Agents
[0129] In general, the modulating agents and compositions described
herein may be used for modulating the adhesion of
cadherin-expressing cells (i.e., cells that express one or more of
E-cadherin, N-cadherin, P-cadherin, R-cadherin and/or other
cadherin(s) containing the HAV-BM sequence, including as yet
undiscovered cadherins). Such modulation may be performed in vitro
and/or in vivo, preferably in a mammal such as a human. As noted
above, modulating agents for purposes that involve the disruption
of cadherin-mediated cell adhesion may comprise an HAV-BM sequence,
multiple HAV-BM sequences in close proximity and/or an antibody (or
an antigen-binding fragment thereof) that recognizes an HAV-BM
sequence. When it is desirable to also disrupt cell adhesion
mediated by other adhesion molecules, a modulating agent may
additionally comprise one or more CAR sequences bound by such
adhesion molecules (and/or antibodies or fragments thereof that
bind such sequences), preferably separated from each other and from
the HAV-BM sequence by linkers. As noted above, such linkers may or
may not comprise one or more amino acids. For enhancing cell
adhesion, a modulating agent may contain multiple HAV-BM sequences
or antibodies (or fragments), preferably separated by linkers,
and/or may be linked to a single molecule or to a support material
as described above.
[0130] Certain methods involving the disruption of cell adhesion as
described herein have an advantage over prior techniques in that
they permit the passage of molecules that are large and/or charged
across barriers of cadherin-expressing cells. As described in
greater detail below, modulating agents as described herein may
also be used to disrupt or enhance cell adhesion in a variety of
other contexts. Within each of the methods described herein, one or
more modulating agents may generally be administered alone, or
within a pharmaceutical composition. In each specific method
described herein, as noted above, a targeting agent may be employed
to increase the local concentration of modulating agent at the
target site.
[0131] In general, within methods for modulating cell adhesion, a
cadherin-expressing cell is contacted with a modulating agent under
conditions and for a time sufficient to permit inhibition or
enhancement of a cadherin-mediated function. Cadherin-expressing
cells include, but are not limited to, epithelial cells,
endothelial cells, neural cells, tumor cells and lymphocytes. Such
contact may be achieved in vitro, or in vivo by administration of a
pharmaceutical composition as provided herein.
[0132] Within certain aspects, methods are provided in which cell
adhesion is diminished. In one such aspect, the present invention
provides methods for reducing unwanted cellular adhesion by
administering a modulating agent as described herein. Unwanted
cellular adhesion can occur between tumor cells, between tumor
cells and normal cells or between normal cells as a result of
surgery, injury, chemotherapy, disease, inflammation or other
condition jeopardizing cell viability or function. Preferred
modulating agents for use within such methods include those
comprising one or more of the sequences INPISGQ (SEQ ID NO:22),
LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID
NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID
NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID
NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID
NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ ID NO:59), CPVSGRC
(SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ ID NO:62),
CINPISC (SEQ ID NO:63) or CKIDPVC (SEQ ID NO:85), CINPC (SEQ ID
NO:86) or CINPIC (SEQ ID NO:87) in which cyclization is indicated
by an underline. Modulating agents may alternatively, or in
addition, comprise a derivative of one of the foregoing sequences.
In addition, a modulating agent may comprise the sequence RGD,
which is bound by integrins, the sequence LYHY (SEQ ID NO:70),
which is bound by occludin, a JAM CAR sequence, a claudin CAR
sequence and/or one or more of HAV and/or a non-classical cadherin
CAR sequence. Preferably, such sequences are separated from the
HAV-BM sequence via a linker. Alternatively, a separate modulator
of cell adhesion (e.g., integrin- and/or occludin-mediated) may be
administered in conjunction with the modulating agent(s), either
within the same pharmaceutical composition or separately. Topical
administration of the modulating agent(s) is generally preferred,
but other means may also be employed. Preferably, a fluid
composition for topical administration (comprising, for example,
physiological saline) comprises an amount of modulating agent as
described above, and more preferably from 10 g/mL to 1 mg/mL.
Creams may generally be formulated as described above. Topical
administration in the surgical field may be given once at the end
of surgery by irrigation of the wound or as an intermittent or
continuous irrigation with the use of surgical drains in the
post-operative period or by the use of drains specifically inserted
in an area of inflammation, injury or disease in cases where
surgery does not need to be performed. Alternatively, parenteral or
transcutaneous administration may be used to achieve similar
results.
[0133] Within another such aspect, methods are provided for
enhancing the delivery of a drug through the skin of a mammal.
Transderrnal delivery of drugs is a convenient and non-invasive
method that can be used to maintain relatively constant blood
levels of a drug. In general, to facilitate drug delivery via the
skin, it is necessary to perturb adhesion between the epithelial
cells (keratinocytes) and the endothelial cells of the
microvasculature. Using currently available techniques, only small,
uncharged molecules may be delivered across skin in vivo. The
methods described herein are not subject to the same degree of
limitation. Accordingly, a wide variety of drugs may be transported
across the epithelial and endothelial cell layers of skin, for
systemic or topical administration. Such drugs may be delivered to
melanomas or may enter the blood stream of the mammal for delivery
to other sites within the body.
[0134] To enhance the delivery of a drug through the skin, a
modulating agent as described herein and a drug are contacted with
the skin surface. Preferred modulating agents for use within such
methods include those comprising one or more of the sequences
INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID
NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ
ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ
ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ
ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC
(SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61),
CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID
NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87) in which
cyclization is indicated by an underline. Modulating agents may
alternatively, or in addition, comprise a derivative of one of the
foregoing sequences. Multifunctional modulating agents comprising
the cadherin CAR sequence HAV-BM linked to one or more of the Dsc
or Dsg CAR sequences may also be used to disrupt epithelial cell
adhesion. Such modulating agents may also, or alternatively,
comprise the fibronectin CAR sequence RGD, which is recognized by
integrins, a JAM CAR sequence, a claudin CAR sequence and/or the
occludin CAR sequence LYHY (SEQ ID NO:70). Alternatively, a
separate modulator of cell adhesion may be administered in
conjunction with the modulating agent(s), either within the same
pharmaceutical composition or separately.
[0135] Contact may be achieved by direct application of the
modulating agent, generally within a composition formulated as a
cream or gel, or using any of a variety of skin contact devices for
transdermal application (such as those described in European Patent
Application No. 566,816 A; U.S. Pat. No. 5,613,958; U.S. Pat. No.
5,505,956). A skin patch provides a convenient method of
administration (particularly for slow-release formulations). Such
patches may contain a reservoir of modulating agent and drug
separated from the skin by a membrane through which the drug
diffuses. Within other patch designs, the modulating agent and drug
may be dissolved or suspended in a polymer or adhesive matrix that
is then placed in direct contact with the patient's skin. The
modulating agent and drug may then diffuse from the matrix into the
skin. Modulating agent(s) and drug(s) may be contained within the
same composition or skin patch, or may be separately administered,
although administration at the same time and site is preferred. In
general, the amount of modulating agent administered via the skin
varies with the nature of the condition to be treated or prevented,
but may vary as described above. Such levels may be achieved by
appropriate adjustments to the device used, or by applying a cream
formulated as described above. Transfer of the drug across the skin
and to the target tissue may be predicted based on in vitro studies
using, for example, a Franz cell apparatus, and evaluated in vivo
by appropriate means that will be apparent to those of ordinary
skill in the art. As an example, monitoring of the serum level of
the administered drug over time provides an easy measure of the
drug transfer across the skin.
[0136] Transdermal drug delivery as described herein is
particularly useful in situations in which a constant rate of drug
delivery is desired, to avoid fluctuating blood levels of a drug.
For example, morphine is an analgesic commonly used immediately
following surgery. When given intermittently in a parenteral form
(intramuscular, intravenous), the patient usually feels sleepy
during the first hour, is well during the next 2 hours and is in
pain during the last hour because the blood level goes up quickly
after the injection and goes down below the desirable level before
the 4 hour interval prescribed for re-injection is reached.
Transdermal administration as described herein permits the
maintenance of constant levels for long periods of time (e.g.,
days), which allows adequate pain control and mental alertness at
the same time. Insulin provides another such example. Many diabetic
patients need to maintain a constant baseline level of insulin
which is different from their needs at the time of meals. The
baseline level may be maintained using transdermal administration
of insulin, as described herein. Antibiotics may also be
administered at a constant rate, maintaining adequate bactericidal
blood levels, while avoiding the high levels that are often
responsible for the toxicity (e.g., levels of gentamycin that are
too high typically result in renal toxicity).
[0137] Drug delivery by the methods of the present invention also
provide a more convenient method of drug administration. For
example, it is often particularly difficult to administer
parenteral drugs to newborns and infants because of the difficulty
associated with finding veins of acceptable caliber to catheterize.
However, newborns and infants often have a relatively large skin
surface as compared to adults. Transdermal drug delivery permits
easier management of such patients and allows certain types of care
that can presently be given only in hospitals to be given at home.
Other patients who typically have similar difficulties with venous
catheterization are patients undergoing chemotherapy or patients on
dialysis. In addition, for patients undergoing prolonged therapy,
transdermal administration as described herein is more convenient
than parenteral administration.
[0138] Transdermal administration as described herein also allows
the gastrointestinal tract to be bypassed in situations where
parenteral uses would not be practical. For example, there is a
growing need for methods suitable for administration of therapeutic
small peptides and proteins, which are typically digested within
the gastrointestinal tract. The methods described herein permit
administration of such compounds and allow easy administration over
long periods of time. Patients who have problems with absorption
through their gastrointestinal tract because of prolonged ileus or
specific gastrointestinal diseases limiting drug absorption may
also benefit from drugs formulated for transdermal application as
described herein.
[0139] Further, there are many clinical situations where it is
difficult to maintain compliance. For example, patients with mental
problems (e.g., patients with Alzheimer's disease or psychosis) are
easier to manage if a constant delivery rate of drug is provided
without having to rely on their ability to take their medication at
specific times of the day. Also patients who simply forget to take
their drugs as prescribed are less likely to do so if they merely
have to put on a skin patch periodically (e.g., every 3 days).
Patients with diseases that are without symptoms, like patients
with hypertension, are especially at risk of forgetting to take
their medication as prescribed.
[0140] For patients taking multiple drugs, devices for transdermal
application such as skin patches may be formulated with
combinations of drugs that are frequently used together. For
example, many heart failure patients are given digoxin in
combination with furosemide. The combination of both drugs into a
single skin patch facilitates administration, reduces the risk of
errors (taking the correct pills at the appropriate time is often
confusing to older people), reduces the psychological strain of
taking "so many pills," reduces skipped dosage because of irregular
activities and improves compliance.
[0141] The methods described herein are particularly applicable to
humans, but also have a variety of veterinary uses, such as the
administration of growth factors or hormones (e.g., for fertility
control) to an animal.
[0142] As noted above, a wide variety of drugs may be administered
according to the methods provided herein. Some examples of drug
categories that may be administered transdermally include
anti-inflammatory drugs (e.g., in arthritis and in other condition)
such as all NSAID, indomethacin, prednisone, etc.; analgesics
(especially when oral absorption is not possible, such as after
surgery, and when parenteral administration is not convenient or
desirable), including morphine, codeine, Demerol, acetaminophen and
combinations of these (e.g., codeine plus acetaminophen);
antibiotics such as Vancomycin (which is not absorbed by the GI
tract and is frequently given intravenously) or a combination of
INH and Rifampicin (e.g., for tuberculosis); anticoagulants such as
heparin (which is not well absorbed by the GI tract and is
generally given parenterally, resulting in fluctuation in the blood
levels with an increased risk of bleeding at high levels and risks
of inefficacy at lower levels) and Warfarin (which is absorbed by
the GI tract but cannot be administered immediately after abdominal
surgery because of the normal ileus following the procedure);
antidepressants (e.g., in situations where compliance is an issue
as in Alzheimer's disease or when maintaining stable blood levels
results in a significant reduction of anti-cholinergic side effects
and better tolerance by patients), such as amitriptylin, imipramin,
prozac, etc.; antihypertensive drugs (e.g., to improve compliance
and reduce side effects associated with fluctuating blood levels),
such as diuretics and beta-blockers (which can be administered by
the same patch; e.g., furosemide and propanolol); antipsychotics
(e.g., to facilitate compliance and make it easier for care giver
and family members to make sure that the drug is received), such as
haloperidol and chlorpromazine; and anxiolytics or sedatives (e.g.,
to avoid the reduction of alertness related to high blood levels
after oral administration and allow a continual benefit throughout
the day by maintaining therapeutic levels constant).
[0143] Numerous other drugs may be administered as described
herein, including naturally occurring and synthetic hormones,
growth factors, proteins and peptides. For example, insulin and
human growth hormone, growth factors like erythropoietin,
interleukins and inteferons may be delivered via the skin.
[0144] Kits for administering a drug via the skin of a mammal are
also provided within the present invention. Such kits generally
comprise a device for transdermal application (e.g., a skin patch)
in combination with, or impregnated with, one or more modulating
agents. A drug may additionally be included within such kits.
[0145] Within a related aspect, the use of modulating agents as
described herein to increase the permeability of endothelial and
epithelial cell layers, thereby facilitating sampling of the blood
compartment by passive diffusion. Such methods permit the detection
and/or measurement of the levels of specific molecules circulating
in the blood. In general, to sample the blood compartment, it is
necessary to perturb adhesion between the epithelial cells
(keratinocytes) and the endothelial cells of the microvasculature.
Using currently available techniques, only small, uncharged
molecules may be detected across skin in vivo. The methods
described herein are not subject to the same degree of limitation.
Accordingly, a wide variety of blood components may be sampled
across epithelial and endothelial cell layers. Such sampling may be
achieved across any such cell layers, including skin and gums.
[0146] For example, application of one or more modulating agents to
the skin, via a skin patch as described herein, permits the patch
to function like a sponge to accumulate a small quantity of fluid
containing a representative sample of the serum. The patch is then
removed after a specified amount of time and analyzed by suitable
techniques for the compound of interest (e.g., a medication,
hormone, growth factor, metabolite or marker). Alternatively, a
patch may be impregnated with reagents to permit a color change if
a specific substance (e.g., an enzyme) is detected. Substances that
can be detected in this manner include, but are not limited to,
illegal drugs such as cocaine, HIV enzymes, glucose and PSA. This
technology is of particular benefit for home testing kits.
[0147] To facilitate sampling of blood in a patient, a modulating
agent as described herein is contacted with the skin surface.
Multifunctional modulating agents comprising an HAV-BM sequence
linked to one or more of the OB-cadherin CAR sequence DDK, a
claudin CAR sequence, the Dsc and/or Dsg CAR sequences may also be
used to disrupt epithelial cell adhesion. Such modulating agents
may also, or alternatively, comprise the fibronectin CAR sequence
RGD, which is recognized by integrins, a claudin CAR sequence, a
JAM CAR sequence and/or the occludin CAR sequence LYHY (SEQ ID
NO:70). Alternatively, a separate modulator of non-classical
cadherin-mediated cell adhesion may be administered in conjunction
with the modulating agent(s), either within the same pharmaceutical
composition or separately.
[0148] Contact may be achieved as described herein for transdermal
drug delivery. Modulating agent(s) and reagents for assaying blood
components may, but need not, be contained within the same
composition or skin patch. In general, the amount of modulating
agent administered via the skin may vary as described above. Such
levels may be achieved by appropriate adjustments to the device
used, or by applying a cream formulated as described above.
Transfer of the blood component across the skin may be predicted
based on in vitro studies using, for example, a Franz cell
apparatus, and evaluated in vivo by appropriate means that will be
apparent to those of ordinary skill in the art.
[0149] Kits for sampling blood component via, for example, the skin
or gums of a mammal, are also provided within the present
invention. Such kits generally comprise a device for transdermal
application (i.e., skin patch) in combination with, or impregnated
with, one or more modulating agents. A reagent for detection of a
blood component may additionally be included within such kits.
[0150] Within a further aspect, methods are provided for enhancing
delivery of a drug to a tumor in a mammal, comprising administering
a modulating agent in combination with a drug to a tumor-bearing
mammal. Modulating agents for use within such methods include those
designed to disrupt E-cadherin and/or N-cadherin mediated cell
adhesion, such as those comprising one or more of the sequences
INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID
NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ
ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ
ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ
ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC
(SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61),
CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID
NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87) in which
cyclization is indicated by an underline. Modulating agents may
alternatively, or in addition, comprise a derivative of one of the
foregoing sequences. Bi-functional modulating agents that comprise
an HAV-BM sequence with flanking E-cadherin-specific sequences
joined via a linker to an HAV-BM sequence with flanking
N-cadherin-specific sequences are also preferred. Preferably, the
peptide portion(s) of a modulating agent comprises 6-16 amino
acids, since longer peptides are difficult to dissolve in aqueous
solution and are more likely to be degraded by peptidases.
[0151] In one particularly preferred embodiment, a modulating agent
is capable of disrupting cell adhesion mediated by multiple
adhesion molecules. For example, a single branched modulating agent
(or multiple agents linked to a single molecule or support
material) may disrupt E-cadherin, N-cadherin, occludin, Dsc and Dsg
mediated cell adhesion, thereby disrupting adherens junctions,
tight junctions and desmosomes. Such an agent may comprise one or
more of the HAV-BM sequence, as well as a Dsg or Dsc CAR sequence;
a JAM CAR sequence; a claudin CAR sequence; the OB-cadherin CAR
sequence DDK; and the occludin CAR sequence LYHY (SEQ ID NO:70).
Such agents serve as multifunctional disrupters of cell adhesion.
Alternatively, a separate modulator of non-classical
cadherin-mediated cell adhesion may be administered in conjunction
with the modulating agent(s), either within the same pharmaceutical
composition or separately. Preferred antibody modulating agents
include Fab fragments directed against either the N-cadherin HAV-BM
sequence or E-cadherin HAV-BM sequence. Fab fragments directed
against the occludin CAR sequence
GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO:69) may
also be employed, either incorporated into a modulating agent or
within a separate modulator that is administered concurrently.
[0152] Preferably, the modulating agent and the drug are formulated
within the same composition or drug delivery device prior to
administration. In general, a modulating agent may enhance drug
delivery to any tumor, and the method of administration may be
chosen based on the type of target tumor. For example, injection or
topical administration as described above may be preferred for
melanomas and other accessible tumors (e.g., metastases from
primary ovarian tumors may be treated by flushing the peritoneal
cavity with the composition). Other tumors (e.g., bladder tumors)
may be treated by injection of the modulating agent and the drug
(such as mitomycin C) into the site of the tumor. In other
instances, the composition may be administered systemically, and
targeted to the tumor using any of a variety of specific targeting
agents. Suitable drugs may be identified by those of ordinary skill
in the art based upon the type of cancer to be treated (e.g.,
mitomycin C for bladder cancer). In general, the amount of
modulating agent administered varies with the method of
administration and the nature of the tumor, within the typical
ranges provided above, preferably ranging from about 1 .mu.g/mL to
about 2 mg/mL, and more preferably from about 10 .mu.g/mL to 1
mg/mL. Transfer of the drug to the target tumor may be evaluated by
appropriate means that will be apparent to those of ordinary skill
in the art. Drugs may also be labeled (e.g., using radionuclides)
to permit direct observation of transfer to the target tumor using
standard imaging techniques.
[0153] Within a related aspect, the present invention provides
methods for treating cancer and/or inhibiting metastasis in a
mammal. Cancer tumors are solid masses of cells, growing out of
control, which require nourishment via blood vessels. The formation
of new capillaries is a prerequisite for tumor growth and the
emergence of metastases. Administration of modulating agents as
described herein may disrupt the growth of such blood vessels,
thereby providing effective therapy for the cancer and/or
inhibiting metastasis. Modulating agents may also be used to treat
leukemias. Preferred modulating agents for use within such methods
include those that disrupt N-cadherin and/or E-cadherin mediated
cell adhesion, such as those comprising one or more of the
sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ
(SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26),
KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52),
PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55),
IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58),
CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID
NO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC
(SEQ ID NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in
which cyclization is indicated by an underline. Modulating agents
may alternatively, or in addition, comprise a derivative of one of
the foregoing sequences. Preferably, the peptide portion(s) of such
modulating agents comprise 6-16 amino acids. In addition, a
modulating agent may comprise the sequence RGD, which is recognized
by integrins, a JAM CAR sequence, a claudin CAR sequence, the
occludin CAR sequence LYHY (SEQ ID NO:70), the OB-cadherin CAR
sequence DDK, Dsc or Dsg CAR sequences, and/or the occludin CAR
sequence LYHY (SEQ ID NO:70). Preferably such sequences are
separated from the HAV-BM sequence via a linker. Alternatively, a
separate modulator of integrin- and/or occludin-mediated cell
adhesion may be administered in conjunction with the modulating
agents(s), either within the same pharmaceutical composition or
separately.
[0154] A modulating agent may be administered alone (e.g., via the
skin) or within a pharmaceutical composition. For melanomas and
certain other accessible tumors, injection or topical
administration as described above may be preferred. For ovarian
cancers, flushing the peritoneal cavity with a composition
comprising one or more modulating agents may prevent metastasis of
ovarian tumor cells. Other tumors (e.g., bladder tumors, bronchial
tumors or tracheal tumors) may be treated by injection of the
modulating agent into the cavity. In other instances, the
composition may be administered systemically, and targeted to the
tumor using any of a variety of specific targeting agents, as
described above. In general, the amount of modulating agent
administered varies depending upon the method of administration and
the nature of the cancer, but may vary within the ranges identified
above. The effectiveness of the cancer treatment or inhibition of
metastasis may be evaluated using well known clinical observations,
such as the level of serum tumor markers (e.g., CEA or PSA).
[0155] In yet another related aspect, the present invention
provides methods for inducing apoptosis in a cadherin-expressing
cell. In general, patients afflicted with cancer may benefit from
such treatment. Certain preferred modulating agents for use within
such methods comprise one or more of the sequences INPISGQ (SEQ ID
NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ
(SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ
(SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV
(SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ ID NO:56), INPIS
(SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ ID NO:59),
CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ ID
NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ
ID NO:86) or CINPIC (SEQ ID NO:87), in which cyclization is
indicated by an underline. Modulating agents may alternatively, or
in addition, comprise a derivative of one of the foregoing
sequences. Modulating agents comprising an additional CAR sequence
(e.g., HAV, RGD, a Dsc CAR sequence, a Dsg CAR sequence, a JAM CAR
sequence, a claudin CAR sequence and/or LYHY (SEQ ID NO:70)) are
also preferred. As noted above, such additional sequences may be
separated from the HAV-BM sequence via a linker. Alternatively, a
separate modulator of integrin-mediated cell adhesion may be
administered in conjunction with the modulating agent(s), either
within the same pharmaceutical composition or separately.
Administration may be topical, via injection or by other means, and
the addition of a targeting agent may be beneficial, particularly
when the administration is systemic. Suitable modes of
administration and dosages depend upon the location and nature of
the cells for which induction of apoptosis is desired but, in
general, dosages may vary as described above. A biopsy may be
performed to evaluate the level of induction of apoptosis.
[0156] Within a further related aspect, a modulating agent may be
used to inhibit angiogenesis (i.e., the growth of blood vessels
from pre-existing blood vessels) in a mammal. Inhibition of
angiogenesis may be beneficial, for example, in patients afflicted
with diseases such as cancer or arthritis. Preferred modulating
agents for inhibition of angiogenesis include those comprising one
or more of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID
NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ
ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ
ID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ
ID NO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ
ID NO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC
(SEQ ID NO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63),
CKIDPVC (SEQ ID NO:85), CINPC (SEQ ID NO:86) or CIPIC (SEQ ID
NO:87), in which cyclization is indicated by an underline.
Modulating agents may alternatively, or in addition, comprise a
derivative of one of the foregoing sequences. In addition, a
modulating agent for use in inhibiting angiogenesis may comprise
the sequence RGD, which is recognized by integrins, the OB-cadherin
CAR sequence DDK, a JAM CAR sequence, a claudin CAR sequence and/or
the occludin CAR sequence LYHY (SEQ ID NO:70), separated from the
HAV-BM sequence via a linker. Alternatively, a separate modulator
of integrin- or occludin-mediated cell adhesion may be administered
in conjunction with the modulating agent(s), either within the same
pharmaceutical composition or separately.
[0157] The effect of a particular modulating agent on angiogenesis
may generally be determined by evaluating the effect of the agent
on blood vessel formation. Such a determination may generally be
performed, for example, using a chick chorioallantoic membrane
assay (Iruela-Arispe et al., Molecular Biology of the Cell
6:327-343, 1995). Briefly, a modulating agent may be embedded in a
mesh composed of vitrogen at one or more concentrations (e.g.,
ranging from about 1 to 100 .mu.g/mesh). The mesh(es) may then be
applied to chick chorioallantoic membranes. After 24 hours, the
effect of the modulating agent may be determined using computer
assisted morphometric analysis. A modulating agent should inhibit
angiogenesis by at least 25% at a concentration of 33
.mu.g/mesh.
[0158] The addition of a targeting agent as described above may be
beneficial, particularly when the administration is systemic.
Suitable modes of administration and dosages depend upon the
condition to be prevented or treated but, in general,
administration by injection is appropriate. Dosages may vary as
described above. The effectiveness of the inhibition may be
evaluated grossly by assessing the inability of the tumors to
maintain their growth and microscopically by observing an absence
of nerves at the periphery of the tumor.
[0159] The present invention also provides methods for enhancing
drug delivery to the central nervous system of a mammal. The
blood/brain barrier is largely impermeable to most neuroactive
agents, and delivery of drugs to the brain of a mammal often
requires invasive procedures. Using a modulating agent as described
herein, however, delivery may be by, for example, systemic
administration of a modulating agent-drug-targeting agent
combination, injection of a modulating agent (alone or in
combination with a drug and/or targeting agent) into the carotid
artery or application of a skin patch comprising a modulating agent
to the head of the patient. Certain preferred modulating agents for
use within such methods comprise one or more of the sequences
INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID
NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ
ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ
ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ
ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC
(SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61),
CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID
NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in which
cyclization is indicated by an underline. Modulating agents may
alternatively, or in addition, comprise a derivative of one of the
foregoing sequences. Also preferred are multi-functional modulating
agents comprising an HAV-BM sequence and the occludin CAR sequence
LYHY (SEQ ID NO:70), a JAM CAR sequence and/or a claudin CAR
sequence, preferably joined by a linker. Alternatively, a separate
modulator of occludin-mediated cell adhesion may be administered in
conjunction with the modulating agent(s), either within the same
pharmaceutical composition or separately. Preferably, the peptide
portion(s) of such modulating agents comprise 6-16 amino acids.
Modulating agents may further comprise antibodies or Fab fragments
directed against an N-cadherin HAV-BM sequence. Fab fragments
directed against the occludin CAR sequence
GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO:69) may
also be employed, either incorporated into the modulating agent or
administered concurrently as a separate modulator.
[0160] In general, the amount of modulating agent administered
varies with the method of administration and the nature of the
condition to be treated or prevented, but typically varies as
described above. Transfer of the drug to the central nervous system
may be evaluated by appropriate means that will be apparent to
those of ordinary skill in the art, such as magnetic resonance
imaging (MRI) or PET scan (positron emitted tomography).
[0161] In certain other aspects, the present invention provides
methods for enhancing adhesion of cadherin-expressing cells. Within
certain embodiments, a modulating agent may be linked to a solid
support, resulting in a matrix that comprises multiple modulating
agents. Within one such embodiment, the support is a polymeric
matrix to which modulating agents and molecules comprising other
CAR sequence(s) are attached (e.g., modulating agents and molecules
comprising an RGD sequence may be attached to the same matrix,
preferably in an alternating pattern). Such matrices may be used in
contexts in which it is desirable to enhance adhesion mediated by
multiple cell adhesion molecules. Alternatively, the modulating
agent itself may comprise multiple HAV-BM sequences or antibodies
(or fragments thereof), separated by linkers as described above.
Either way, the modulating agent(s) function as a "biological glue"
to bind multiple cadherin-expressing cells within a variety of
contexts.
[0162] Within one aspect, such modulating agents may be used to
enhance wound healing and/or reduce scar tissue in a mammal.
Peptides that may be linked to a support, and/or to one another via
a linker, to generate a suitable modulating agent include, but are
not limited to, those comprising one or more of the sequences
INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID
NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ
ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO.52), PVSGR (SEQ
ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ
ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC
(SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61),
CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID
NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in which
cyclization is indicated by an underline. Modulating agents may
alternatively, or in addition, comprise a derivative of one of the
foregoing sequences. Modulating agents that are linked to a
biocompatible and biodegradable matrix such as cellulose or
collagen are particularly preferred. For use within such methods, a
modulating agent should have a free amino or hydroxyl group.
Multi-functional modulating agents comprising the HAV-BM sequence,
the fibronectin CAR sequence RGD, which is recognized by integrins,
the OB-cadherin CAR sequence DDK, a JAM CAR sequence and/or a Dsc
or Dsg CAR sequence may also be used as potent stimulators of wound
healing and/or to reduce scar tissue. Such agents may also, or
alternatively, comprise the occludin CAR sequence LYHY (SEQ ID
NO:70) and/or a claudin CAR sequence. Alternatively, one or more
separate modulator of integrin-, Dsc-, Dsg- and/or
occludin-mediated cell adhesion may be administered in conjunction
with the modulating agent(s), either within the same pharmaceutical
composition or separately.
[0163] The modulating agents are generally administered topically
to the wound, where they may facilitate closure of the wound and
may augment, or even replace, stitches. Similarly, administration
of matrix-linked modulating agents may facilitate cell adhesion in
foreign tissue implants (e.g., skin grafting and prosthetic
implants) and may prolong the duration and usefulness of collagen
injection. In general, the amount of matrix-linked modulating agent
administered to a wound, graft or implant site varies with the
severity of the wound and/or the nature of the wound, graft, or
implant, but may vary as discussed above.
[0164] Within another aspect, one or more modulating agents may be
linked to the interior surface of a tissue culture plate or other
cell culture support, such as for use in a bioreactor. Such linkage
may be performed by any suitable technique, as described above.
Modulating agents linked in this fashion may generally be used to
immobilize cadherin-expressing cells. For example, dishes or plates
coated with one or more modulating agents may be used to immobilize
cadherin-expressing cells within a variety of assays and screens.
Within bioreactors (i.e., systems for large scale production of
cells or organoids), modulating agents may generally be used to
improve cell attachment and stabilize cell growth. Modulating
agents may also be used within bioreactors to support the formation
and function of highly differentiated organoids derived, for
example, from dispersed populations of fetal mammalian cells.
Bioreactors containing biomatrices of modulating agent(s) may also
be used to facilitate the production of specific proteins.
[0165] Modulating agents as described herein may be used within a
variety of bioreactor configurations. In general, a bioreactor is
designed with an interior surface area sufficient to support large
numbers of adherent cells. This surface area can be provided using
membranes, tubes, microtiter wells, columns, hollow fibers, roller
bottles, plates, dishes, beads or a combination thereof. A
bioreactor may be compartmentalized. The support material within a
bioreactor may be any suitable material known in the art;
preferably, the support material does not dissolve or swell in
water. Preferred support materials include, but are not limited to,
synthetic polymers such as acrylics, vinyls, polyethylene,
polypropylene, polytetrafluoroethylene, nylons, polyurethanes,
polyamides, polysulfones and poly(ethylene terephthalate);
ceramics; glass and silica.
[0166] The present invention also provides, within further aspects,
methods for enhancing and/or directing neurological growth. In one
such aspect, neurite outgrowth may be enhanced and/or directed by
contacting a neuron with one or more modulating agents. Preferred
modulating agents for use within such methods are linked to a
polymeric matrix or other support and/or contain multiple HAV-BM
sequences separated by one or more linkers. Peptides that may be
linked to a support material (and/or to one another via a linker to
generate a suitable modulating agent) include, but are not limited
to, those comprising one or more of the sequences INPISGQ (SEQ ID
NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ
(SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ
(SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV
(SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ ID NO:56), INPIS
(SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ ID NO:59),
CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ ID
NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ
ID NO:86) or CINPIC (SEQ ID NO:87), in which cyclization is
indicated by an underline. Certain preferred modulating agents
comprise one or more of N-Ac-INPISGQ-NH.sub.2 (SEQ ID NO:22),
H-INPISGQ-OH (SEQ ID NO:22), N-Ac-NLKIDPVNGQI-NH.sub.2 (SEQ ID
NO:20), H- WLKIDPVNGQI-OH (SEQ ID NO:13), H-LKIDPVNGQI-OH (SEQ ID
NO:21), H-LKIDPANGQI- OH (SEQ ID NO:64) or H-LKIDAVNGQI-OH (SEQ ID
NO:65).
[0167] Modulating agents may alternatively, or in addition,
comprise a derivative of one of the foregoing sequences. In
addition, a modulating agent comprising HAV, RGD and/or YIGSR (SEQ
ID NO:66), which are bound by integrins, and/or the N-CAM CAR
sequence KYSFNYDGSE (SEQ ID NO:67) may further facilitate neurite
outgrowth. Modulating agents comprising antibodies, or fragments
thereof, may be used within this aspect of the present invention
without the use of linkers or support materials. Preferred antibody
modulating agents include Fab fragments directed against an
N-cadherin HAV-BM sequence. Fab fragments directed against the
N-CAM CAR sequence KYSFNYDGSE (SEQ ID NO:67) may also be employed,
either incorporated into the modulating agent or administered
concurrently as a separate modulator.
[0168] The method of achieving contact and the amount of modulating
agent used will depend upon the location of the neuron and the
extent and nature of the outgrowth desired. For example, a neuron
may be contacted (e.g., via implantation) with modulating agent(s)
linked to a support material such as a suture, fiber nerve guide or
other prosthetic device such that the neurite outgrowth is directed
along the support material. Alternatively, a tubular nerve guide
may be employed, in which the lumen of the nerve guide contains a
composition comprising the modulating agent(s). In vivo, such nerve
guides or other supported modulating agents may be implanted using
well known techniques to, for example, facilitate the growth of
severed neuronal connections and/or to treat spinal cord injuries.
It will be apparent to those of ordinary skill in the art that the
structure and composition of the support should be appropriate for
the particular injury being treated. In vitro, a polymeric matrix
may similarly be used to direct the growth of neurons onto
patterned surfaces as described, for example, in U.S. Pat. No.
5,510,628.
[0169] Within another aspect, one or more modulating agents may be
used for therapy of a demyelinating neurological disease in a
mammal. There are a number of demyelinating diseases, such as
multiple sclerosis, characterized by oligodendrocyte death. Since
Schwann cell migration on astrocytes is inhibited by N-cadherin,
modulating agents that disrupt N-cadherin mediated cell adhesion as
described herein, when implanted with Schwann cells into the
central nervous system, may facilitate Schwann cell migration and
permit the practice of Schwann cell replacement therapy.
[0170] Multiple sclerosis patients suitable for treatment may be
identified by criteria that establish a diagnosis of clinically
definite or clinically probable MS (see Poser et al., Ann. Neurol.
13:227, 1983). Candidate patients for preventive therapy may be
identified by the presence of genetic factors, such as HLA-type
DR2a and DR2b, or by the presence of early disease of the relapsing
remitting type.
[0171] Schwann cell grafts may be implanted directly into the brain
along with the modulating agent(s) using standard techniques.
Preferred peptide modulating agents for use within such methods
include those comprising one or more of the sequences INPISGQ (SEQ
ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ
(SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ
(SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV
(SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ ID NO:56), INPIS
(SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ ID NO:59),
CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ ID
NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ
ID NO:86) or CINPIC (SEQ ID NO:87), in which cyclization is
indicated by an underline. Modulating agents may alternatively, or
in addition, comprise a derivative of one of the foregoing
sequences. Modulating agents may further comprise HAV, RGD and/or
YIGSR (SEQ ID NO:66), which are bound by integrins, and/or the
N-CAM CAR sequence KYSFNYDGSE (SEQ ID NO:67). Preferred antibody
modulating agents include Fab fragments directed against the
N-cadherin CAR sequence KYSFNYDGSE (SEQ ID NO:67). Such antibodies
and fragments can be prepared using standard techniques, as
discussed above. Suitable amounts of modulating agent generally
range as described above, preferably from about 10 .mu.g/mL to
about 1 mg/mL.
[0172] Alternatively, a modulating agent may be implanted with
oligodendrocyte progenitor cells (OPs) derived from donors not
afflicted with the demyelinating disease. The myelinating cell of
the CNS is the oligodendrocyte. Although mature oligodendrocytes
and immature cells of the oligodendrocyte lineage, such as the
oligodendrocyte type 2 astrocyte progenitor, have been used for
transplantation, OPs are more widely used. OPs are highly motile
and are able to migrate from transplant sites to lesioned areas
where they differentiate into mature myelin-forming
oligodendrocytes and contribute to repair of demyelinated axons
(see e.g., Groves et al., Nature 362:453-55, 1993; Baron-Van
Evercooren et al., Glia 16:147-64, 1996). OPs can be isolated using
routine techniques known in the art (see e.g., Milner and
French-Constant, Development 120:3497-3506, 1994), from many
regions of the CNS including brain, cerebellum, spinal cord, optic
nerve and olfactory bulb. Substantially greater yields of OP's are
obtained from embryonic or neonatal rather than adult tissue. OPs
may be isolated from human embryonic spinal cord and cultures of
neurospheres established. Human fetal tissue is a potential
valuable and renewable source of donor OP's for future, long range
transplantation therapies of demyelinating diseases such as MS.
[0173] OPs can be expanded in vitro if cultured as "homotypic
aggregates" or "spheres" (Avellana-Adalid et al, J. Neurosci. Res.
45:558-70, 1996). Spheres (sometimes called "oligospheres" or
"neurospheres") are formed when OPs are grown in suspension in the
presence of growth factors such as PDGF and FGF. OPs can be
harvested from spheres by mechanical dissociation and used for
subsequent transplantation or establishment of new spheres in
culture. Alternatively, the spheres themselves may be transplanted,
providing a "focal reservoir" of OPs (Avellana-Adalid et al, J.
Neurosci. Res. 45:558-70, 1996).
[0174] An alternative source of OP may be spheres derived from CNS
stem cells. Recently, Reynolds and Weiss, Dev. Biol. 165:1-13, 1996
have described spheres formed from EGF-responsive cells derived
from embryonic neuroepithelium, which appear to retain the
pluripotentiality exhibited by neuroepithelium in vivo. Cells
dissociated from these spheres are able to differentiate into
neurons, oligodendrocytes and astrocytes when plated on adhesive
substrates in the absence of EGF, suggesting that EGF-responsive
cells derived from undifferentiated embryonic neuroepithelium may
represent CNS stem cells (Reynolds and Weiss, Dev. Biol. 165:1-13,
1996). Spheres derived from CNS stem cells provide an alternative
source of OP which may be manipulated in vitro for transplantation
in vivo. Spheres composed of CNS stem cells may further provide a
microenvironment conducive to increased survival, migration, and
differentiation of the OPs in vivo.
[0175] The use of neurospheres for the treatment of MS may be
facilitated by modulating agents that enhance cell migration from
the spheres. In the absence of modulating agent, the cells within
the spheres adhere tightly to one another and migration out of the
spheres is hindered. Modulating agents that disrupt N-cadherin
mediated cell adhesion as described herein, when injected with
neurospheres into the central nervous system, may improve cell
migration and increase the efficacy of OP replacement therapy.
[0176] Neurosphere grafts may be implanted directly into the
central nervous system along with the modulating agent(s) using
standard techniques. Preferred peptide modulating agents for use
within such methods include those comprising one or more of the
sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ
(SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26),
KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52),
PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55),
IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58),
CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID
NO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC
(SEQ ID NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in
which cyclization is indicated by an underline. Modulating agents
may alternatively, or in addition, comprise a derivative of one of
the foregoing sequences. Modulating agents comprising one or more
of these sequences or derivatives thereof are also preferred.
Preferred antibody modulating agents include Fab fragments directed
against an N-cadherin HAV-BM sequence (e.g., INPISGQ (SEQ ID
NO:22)). Such antibodies and fragments can be prepared using
standard techniques, as discussed above. Suitable amounts of
modulating agent generally range as described above, preferably
from about 10 .mu.g/mL to about 1 mg/mL.
[0177] Alternatively, a modulating agent may be administered alone
or within a pharmaceutical composition. The duration and frequency
of administration will be determined by such factors as the
condition of the patient, and the type and severity of the
patient's disease. Within particularly preferred embodiments of the
invention, the modulating agent or pharmaceutical composition may
be administered at a dosage ranging from 0.1 mg/kg to 20 mg/kg
although appropriate dosages may be determined by clinical trials.
Methods of administration include injection, intravenous or
intrathecal (i.e., directly in cerebrospinal fluid). A modulating
agent or pharmaceutical composition may further comprise a drug
(e.g., an immunomodulatory drug).
[0178] Effective treatment of multiple sclerosis may be evidenced
by any of the following criteria: EDSS (extended disability status
scale), appearance of exacerbations or MRI (magnetic resonance
imaging). The EDSS is a means to grade clinical impairment due to
MS (Kurtzke, Neurology 33:1444, 1983), and a decrease of one full
step defines an effective treatment in the context of the present
invention (Kurtzke, Ann. Neurol. 36:573-79, 1994). Exacerbations
are defined as the appearance of a new symptom that is attributable
to MS and accompanied by an appropriate new neurologic abnormality
(Sipe et al., Neurology 34:1368, 1984). Therapy is deemed to be
effective if there is a statistically significant difference in the
rate or proportion of exacerbation-free patients between the
treated group and the placebo group or a statistically significant
difference in the time to first exacerbation or duration and
severity in the treated group compared to control group. MRI can be
used to measure active lesions using gadolinium-DTPA-enhanced
imaging (McDonald et al. Ann. Neurol. 36:14, 1994) or the location
and extent of lesions using T.sub.2-weighted techniques. The
presence, location and extent of MS lesions may be determined by
radiologists using standard techniques. Improvement due to therapy
is established when there is a statistically significant
improvement in an individual patient compared to baseline or in a
treated group versus a placebo group.
[0179] Efficacy of the modulating agent in the context of
prevention may be judged based on clinical measurements such as the
relapse rate and EDSS. Other criteria include a change in area and
volume of T2 images on MRI, and the number and volume of lesions
determined by gadolinium enhanced images.
[0180] Within further aspects, modulating agents as described
herein may be used for modulating the immune system of a mammal in
any of several ways. Cadherins are expressed on immature B and T
cells (thymocytes and bone marrow pre-B cells), as well as on
specific subsets of activated B and T lymphocytes and some
hematological malignancies (see Lee et al., J. Immunol.
152:5653-5659, 1994; Munro et al., Cellular Immunol. 169:309-312,
1996; Tsutsui et al., J. Biochem. 120:1034-1039, 1996; Cepek et
al., Proc. Natl. Acad. Sci. USA 93:6567-6571, 1996). Modulating
agents may generally be used to modulate specific steps within
cellular interactions during an immune response or during the
dissemination of malignant lymphocytes.
[0181] For example, a modulating agent as described herein may be
used to treat diseases associated with excessive generation of
otherwise normal T cells. Without wishing to be bound by any
particular theory, it is believed that the interaction of cadherins
on maturing T cells and B cell subsets contributes to protection of
these cells from programmed cell death. A modulating agent may
decrease such interactions, leading to the induction of programmed
cell death. Accordingly, modulating agents may be used to treat
certain types of diabetes and rheumatoid arthritis, particularly in
young children where the cadherin expression on thymic pre-T cells
is greatest.
[0182] Modulating agents may also be administered to patients
afflicted with certain skin disorders (such as cutaneous
lymphomas), acute B cell leukemia and excessive immune reactions
involving the humoral immune system and generation of
immunoglobulins, such as allergic responses and antibody-mediated
graft rejection. In addition, patients with circulating
cadherin-positive malignant cells (e.g., during regimes where
chemotherapy or radiation therapy is eliminating a major portion of
the malignant cells in bone marrow and other lymphoid tissue) may
benefit from treatment with a modulating agent. Such treatment may
also benefit patients undergoing transplantation with peripheral
blood stem cells.
[0183] Preferred modulating agents for use within such methods
include those that disrupt E-cadherin and/or N-cadherin mediated
cell adhesion, such as those comprising one or more of the
sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ
(SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26),
KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52),
PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55),
IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58),
CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID
NO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC
(SEQ ID NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in
which cyclization is indicated by an underline. Modulating agents
may alternatively, or in addition, comprise a derivative of one of
the foregoing sequences. In addition, a preferred modulating agent
may comprise one or more additional CAR sequences, such as HAV, RGD
and/or KYSFNYDGSE (SEQ ID NO:67). As noted above, such additional
sequence(s) may be separated from the HAV-BM sequence via a linker.
Alternatively, a separate modulator of integrin-mediated cell
adhesion may be administered in conjunction with the modulating
agent(s), either within the same pharmaceutical composition or
separately.
[0184] Within the above methods, the modulating agent(s) are
preferably administered systemically (usually by injection) or
topically. A modulating agent may be linked to a targeting agent.
For example, targeting to the bone marrow may be beneficial. A
suitable dosage is sufficient to effect a statistically significant
reduction in the population of B and/or T cells that express
cadherin and/or an improvement in the clinical manifestation of the
disease being treated. Typical dosages generally range as described
above.
[0185] Within further aspects, the present invention provides
methods and kits for preventing pregnancy in a mammal. In general,
disruption of E-cadherin function prevents the adhesion of
trophoblasts and their subsequent fusion to form
syncitiotrophoblasts. In one embodiment, one or more modulating
agents as described herein may be incorporated into any of a
variety of well known contraceptive devices, such as sponges
suitable for intravaginal insertion (see, e.g., U.S. Pat. No.
5,417,224) or capsules for subdermal implantation. Other modes of
administration are possible, however, including transdermal
administration, for modulating agents linked to an appropriate
targeting agent. Preferred modulating agents for use within such
methods include those comprising one or more of the sequences
INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID
NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ
ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ
ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ
ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC
(SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61),
CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID
NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in which
cyclization is indicated by an underline. Modulating agents may
alternatively, or in addition, comprise a derivative of one of the
foregoing sequences. In addition, a preferred modulating agent may
comprise additional CAR sequences, such as HAV, DDK and/or RGD. As
noted above, such additional sequences may be separated from the
HAV-BM sequence via a linker. Alternatively, a separate modulator
of integrin-mediated cell adhesion may be administered in
conjunction with the modulating agent(s), either within the same
pharmaceutical composition or separately.
[0186] Suitable methods for incorporation into a contraceptive
device depend upon the type of device and are well known in the
art. Such devices facilitate administration of the modulating
agent(s) to the uterine region and may provide a sustained release
of the modulating agent(s). In general, modulating agent(s) may be
administered via such a contraceptive device at a dosage ranging
from 0.1 to 50 mg/kg, although appropriate dosages may be
determined by monitoring hCG levels in the urine. hCG is produced
by the placenta, and levels of this hormone rise in the urine of
pregnant women. The urine hCG levels can be assessed by
radio-immunoassay using well known techniques. Kits for preventing
pregnancy generally comprise a contraceptive device impregnated
with one or more modulating agents.
[0187] Alternatively, a sustained release formulation of one or
more modulating agents may be implanted, typically subdermally, in
a mammal for the prevention of pregnancy. Such implantation may be
performed using well known techniques. Preferably, the implanted
formulation provides a dosage as described above, although the
minimum effective dosage may be determined by those of ordinary
skill in the art using, for example, an evaluation of hCG levels in
the urine of women.
[0188] The present invention also provides methods for increasing
vasopermeability in a mammal by administering one or more
modulating agents or pharmaceutical compositions. Within blood
vessels, endothelial cell adhesion (mediated by N-cadherin) results
in decreased vascular permeability. Accordingly, modulating agents
as described herein that decrease N-cadherin mediated adhesion may
be used to increase vascular permeability. Particularly preferred
modulating agents include those comprising one or more of the
sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ
(SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26),
KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52),
PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55),
IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58),
CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID
NO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC
(SEQ ID NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in
which cyclization is indicated by an underline. Modulating agents
may alternatively, or in addition, comprise a derivative of one of
the foregoing sequences. In addition, a preferred modulating agent
may comprise an occludin CAR sequence LYHY (SEQ ID NO:70), a JAM
CAR sequence, a claudin CAR sequence and/or an OB-cadherin CAR
sequence DDK. As noted above, such an additional sequence may be
separated from the HAV sequence via a linker. Alternatively, a
separate modulator of occludin mediated cell adhesion may be
administered in conjunction with one or modulating agents, either
within the same pharmaceutical composition or separately.
[0189] Within certain embodiments, preferred modulating agents for
use within such methods include peptides capable of decreasing both
endothelial and tumor cell adhesion. Such modulating agents may be
used to facilitate the penetration of anti-tumor therapeutic or
diagnostic agents (e.g., monoclonal antibodies) through endothelial
cell permeability barriers and tumor barriers. For example, a
modulating agent may further comprise an E-cadherin HAV or HAV-BM
sequence. Alternatively, separate modulating agents capable of
disrupting N- and E-cadherin mediated adhesion may be administered
concurrently.
[0190] In one particularly preferred embodiment, a modulating agent
is further capable of disrupting cell adhesion mediated by multiple
adhesion molecules. Such an agent may comprise an HAV-BM sequence,
as well as an RGD sequence, a Dsc CAR sequence, a Dsg CAR sequence
and/or the occludin CAR sequence LYHY (SEQ ID NO:70).
Alternatively, a separate modulator cell adhesion that comprises a
CAR sequence other than an HAV-BM sequence may be administered in
conjunction with the modulating agent(s), either within the same
pharmaceutical composition or separately.
[0191] Treatment with a modulating agent may be appropriate, for
example, prior to administration of an anti-tumor therapeutic or
diagnostic agent (e.g., a monoclonal antibody or other
macromolecule), an antimicrobial agent or an anti-inflammatory
agent, in order to increase the concentration of such agents in the
vicinity of the target tumor, organism or inflammation without
increasing the overall dose to the patient. Modulating agents for
use within such methods may be linked to a targeting agent to
further increase the local concentration of modulating agent,
although systemic administration of a vasoactive agent even in the
absence of a targeting agent increases the perfusion of certain
tumors relative to other tissues. Suitable targeting agents include
antibodies and other molecules that specifically bind to tumor
cells or to components of structurally abnormal blood vessels. For
example, a targeting agent may be an antibody that binds to a
fibrin degradation product or a cell enzyme such as a peroxidase
that is released by granulocytes or other cells in necrotic or
inflamed tissues.
[0192] Administration via intravenous injection or transdermal
administration is generally preferred. Effective dosages are
generally sufficient to increase localization of a subsequently
administered diagnostic or therapeutic agent to an extent that
improves the clinical efficacy of therapy of accuracy of diagnosis
to a statistically significant degree. Comparison may be made
between treated and untreated tumor host animals to whom equivalent
doses of the diagnostic or therapeutic agent are administered. In
general, dosages range as described above.
[0193] Within a further aspect, modulating agents as described
herein may be used for controlled inhibition of synaptic stability,
resulting in increased synaptic plasticity. Within this aspect,
administration of one or more modulating agents may be advantageous
for repair processes within the brain, as well as learning and
memory, in which neural plasticity is a key early event in the
remodeling of synapses. Cell adhesion molecules, particularly
N-cadherin and E-cadherin, can function to stabilize synapses, and
loss of this function is thought to be the initial step in the
remodeling of the synapse that is associated with learning and
memory (Doherty et al., J. Neurobiology, 26:437-446, 1995; Martin
and Kandel, Neuron, 17:567-570, 1996; Fannon and Colman, Neuron,
17:423-434, 1996). Inhibition of cadherin function by
administration of one or more modulating agents that inhibit
cadherin function may stimulate learning and memory. Preferred
modulating agents for use within such methods include those that
disrupt E-cadherin and/or N-cadherin mediated cell adhesion, such
as those comprising one or more of the sequences INPISGQ (SEQ ID
NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ
(SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ
(SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV
(SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ ID NO:56), INPIS
(SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ ID NO:59),
CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ ID
NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ
ID NO:86) or CINPIC (SEQ ID NO:87), in which cyclization is
indicated by an underline. Modulating agents may alternatively, or
in addition, comprise a derivative of one of the foregoing
sequences. In addition, a preferred modulating agent may comprise
one or more additional CAR sequences, such as the sequence RGD,
which is bound by integrins and/or the N-CAM CAR sequence
KYSFNYDGSE (SEQ ID NO:67). As noted above, such additional
sequence(s) may be separated from the HAV-BM sequence via a linker.
Alternatively, a separate modulator of integrin and/or N-CAM
mediated cell adhesion may be administered in conjunction with the
modulating agent(s), either within the same pharmaceutical
composition or separately. For such aspects, administration may be
via encapsulation into a delivery vehicle such as a liposome, using
standard techniques, and injection into, for example, the carotid
artery. Alternatively, a modulating agent may be linked to a
disrupter of the blood-brain barrier. In general dosages range as
described above.
[0194] Assay Employing Anti-HAV-BM Antibodies
[0195] Other aspects of the present invention provide methods that
employ antibodies raised against an HAV-BM sequence for diagnostic
and assay purposes. Such polyclonal and monoclonal antibodies may
be raised against a peptide using conventional techniques and as
described above. Assays employing antibodies typically involve
using an antibody to detect the presence or absence of a cadherin
(free or on the surface of a cell), or proteolytic fragment
containing the EC1 or EC4 domain in a suitable biological sample,
such as tumor or normal tissue biopsies, blood, lymph node, serum
or urine samples, or other tissue, homogenate, or extract thereof
obtained from a patient.
[0196] There are a variety of assay formats known to those of
ordinary skill in the art for using an antibody to detect a target
molecule in a sample. See, e.g., Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For
example, the assay may be performed in a Western blot format,
wherein a protein preparation from the biological sample is
submitted to gel electrophoresis, transferred to a suitable
membrane and allowed to react with the antibody. The presence of
the antibody on the membrane may then be detected using a suitable
detection reagent, as described below.
[0197] In another embodiment, the assay involves the use of
antibody immobilized on a solid support to bind to the target
cadherin, or a proteolytic fragment containing the EC1 or EC4
domain and encompassing the CAR sequence, and remove it from the
remainder of the sample. The bound cadherin may then be detected
using a second antibody or reagent that contains a reporter group.
Alternatively, a competitive assay may be utilized, in which a
cadherin is labeled with a reporter group and allowed to bind to
the immobilized antibody after incubation of the antibody with the
sample. The extent to which components of the sample inhibit the
binding of the labeled cadherin to the antibody is indicative of
the reactivity of the sample with the immobilized antibody, and as
a result, indicative of the level of the cadherin in the
sample.
[0198] The solid support may be any material known to those of
ordinary skill in the art to which the antibody may be attached,
such as a test well in a microtiter plate, a nitrocellulose filter
or another suitable membrane. Alternatively, the support may be a
bead or disc, such as glass, fiberglass, latex or a plastic such as
polystyrene or polyvinylchloride. The antibody may be immobilized
on the solid support using a variety of techniques known to those
in the art, which are amply described in the patent and scientific
literature.
[0199] In certain embodiments, the assay for detection of a
cadherin in a sample is a two-antibody sandwich assay. This assay
may be performed by first contacting an antibody that has been
immobilized on a solid support, commonly the well of a microtiter
plate, with the biological sample, such that the cadherin within
the sample is allowed to bind to the immobilized antibody (a 30
minute incubation time at room temperature is generally
sufficient). Unbound sample is then removed from the immobilized
cadherin-antibody complexes and a second antibody (containing a
reporter group such as an enzyme, dye, radionuclide, luminescent
group, fluorescent group or biotin) capable of binding to a
different site on the cadherin is added. The amount of second
antibody that remains bound to the solid support is then determined
using a method appropriate for the specific reporter group. The
method employed for detecting the reporter group depends upon the
nature of the reporter group. For radioactive groups, scintillation
counting or autoradiographic methods are generally appropriate.
Spectroscopic methods may be used to detect dyes, luminescent
groups and fluorescent groups. Biotin may be detected using avidin,
coupled to a different reporter group (commonly a radioactive or
fluorescent group or an enzyme). Enzyme reporter groups may
generally be detected by the addition of substrate (generally for a
specific period of time), followed by spectroscopic or other
analysis of the reaction products. Standards and standard additions
may be used to determine the level of cadherin in a sample, using
well known techniques.
[0200] The present invention also provides kits for use in such
immunoassays. Such kits generally comprise one or more antibodies,
as described above. In addition, one or more additional
compartments or containers of a kit generally enclose elements,
such as reagents, buffers and/or wash solutions, to be used in the
immunoassay.
[0201] Within further aspects, modulating agents or antibodies (or
fragments thereof) may be used to facilitate cell identification
and sorting in vitro or imaging in vivo, permitting the selection
of cells expressing different cadherins (or different cadherin
levels). Preferably, the modulating agent(s) or antibodies for use
in such methods are linked to a detectable marker. Suitable markers
are well known in the art and include radionuclides, luminescent
groups, fluorescent groups, enzymes, dyes, constant immunoglobulin
domains and biotin. Within one preferred embodiment, a modulating
agent linked to a fluorescent marker, such as fluorescein, is
contacted with the cells, which are then analyzed by fluorescence
activated cell sorting (FACS).
[0202] Antibodies or fragments thereof may also be used within
screens of combinatorial or other nonpeptide-based libraries to
identify other compounds capable of modulating cadherin-mediated
cell adhesion. Such screens may generally be performed using an
ELISA or other method well known to those of ordinary skill in the
art that detect compounds with a shape and structure similar to
that of the modulating agent. In general, such screens may involve
contacting an expression library producing test compounds with an
antibody, and detecting the level of antibody bound to the
candidate compounds. Compounds for which the antibody has a higher
affinity may be further characterized as described herein, to
evaluate the ability to modulate cadherin-mediated cell
adhesion.
[0203] Identification of HAV-BM Binding Compounds
[0204] The present invention further provides methods for
identifying compounds that bind to an HAV-BM sequence. Such agents
may generally be identified by contacting a polypeptide as provided
herein with a candidate compound or agent under conditions and for
a time sufficient to allow interaction with a polypeptide
comprising an HAV-BM sequence. Any of a variety of well known
binding assays may then be performed to assess the ability of the
candidate compound to bind to the polypeptide. In general, a
candidate compound that binds to the polypeptide at a significantly
greater level than a similar polypeptide that does not contain an
HAV-BM sequence, is considered a compound that binds to an HAV-BM
sequence. Preferably, the candidate compound generates a signal
within a binding assay that is at least three standard deviations
above the level of signal detected for a polypeptide that does not
contain an HAV-BM sequence. Depending on the design of the assay, a
polypeptide comprising an HAV-BM sequence may be free in solution,
affixed to a solid support, present on a cell surface or located
within the cell. Large scale screens may be performed using
automation.
[0205] Within certain embodiments, the polypeptide may be
immobilized onto a solid support material, and used to affinity
purify binding compounds from, for example, cell or tissue
extracts. The solid support material may be any material known to
those of ordinary skill in the art to which the polypeptide may be
attached. For example, the solid support may be a test well in a
microtiter plate or a nitrocellulose filter or other suitable
membrane. Alternatively, the support may be a bead or disc, such as
glass, fiberglass, latex or a plastic material such as polystyrene
or polyvinylchloride. The polypeptide may be immobilized on the
solid support using a variety of techniques known to those in the
art, which are amply described in the patent and scientific
literature. In the context of the present invention, the term
"immobilization" refers to both noncovalent association, such as
adsorption, and covalent attachment (which may be a direct linkage
between the polypeptide and functional groups on the support or may
be a linkage by way of a cross-linking agent). Adsorption may be
achieved by contacting the polypeptide, in a suitable buffer, with
the solid support for a suitable amount of time. The contact time
varies with temperature, but is typically between about 1 hour and
1 day. Covalent attachment of polypeptide to a solid support may
also generally be achieved by first reacting the support with a
bifunctional reagent that will react with both the support and a
functional group, such as a hydroxyl or amino group, on the
polypeptide using well known techniques.
[0206] Alternatively, a polypeptide may be incubated with whole
cells, and interacting proteins may then be cross-linked to the
polypeptide using standard techniques. Such polypeptides may be
labeled with a detectable marker (e.g., a radionuclide) or may be
subsequently detected using a detection reagent (e.g., an antibody)
that is linked to such a marker. Within other assays, cDNA
expression libraries may be screened with a labeled polypeptide to
identify polynucleotides encoding proteins that interact with the
labeled polypeptide. Similarly, a yeast two-hybrid system may be
employed to identify interacting proteins. Other assays may be
performed in a Western blot format, wherein a protein preparation
from a biological sample such as a cell or tissue extract is
submitted to gel electrophoresis, transferred to a suitable
membrane and allowed to react with the polypeptide. The presence of
the polypeptide on the membrane may then be detected using a label
linked to the polypeptide or to a suitable detection reagent, such
as an antibody. All of the above assays are well known to those of
ordinary skill in the art, and may be performed according to
standard protocols. These assays are representative only, and it
will be apparent that other assays designed to evaluate binding may
also be employed.
[0207] Following identification of a compound that binds to an
HAV-BM sequence (or a polynucleotide encoding such a compound),
standard structural analyses may be performed. In general, a
polynucleotide may be sequenced using well known techniques
employing such enzymes as Klenow fragment of DNA polymerase I,
Sequenase.RTM. (US Biochemical Corp., Cleveland Ohio) Taq
polymerase (Perkin Elmer, Foster City Calif.) or thermostable T7
polymerase (Amersham, Chicago, Ill.). An automated sequencing
system may be used, using instruments available from commercial
suppliers such as Perkin Elmer and Pharmacia. Proteins may be
partially sequenced using standard techniques, and the sequence
information used to retrieve a cDNA molecule encoding the protein
(e.g., using PCR or hybridization screens employing degenerate
oligonucleotides).
[0208] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Preparation of Representative Modulating Agents
[0209] This Example illustrates the solid phase synthesis of
representative peptide modulating agents.
[0210] The peptides were synthesized on a 431A Applied Biosystems
peptide synthesizer using p-Hydroxymethylphenoxymethyl polystyrene
(HMP) resin and standard Fmoc chemistry. After synthesis and
deprotection, the peptides were de-salted on a Sephadex G-10 column
and lyophilized. The peptides were analyzed for purity by
analytical HPLC, and in each case a single peak was observed.
Peptides were made as stock solutions at 10 to 25 mg/mL in
dimethylsulfoxide (DMSO) or water and stored at -20.degree. C.
before use.
Example 2
Disruption of the Ability of Mouse Cerebellar Neurons to Extend
Neurites
[0211] N-cadherin and N-CAM are established as CAMs that can
regulate neurite outgrowth (Doherty and Walsh, Curr. Op. Neurobiol.
4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994; Hall et
al., Cell Adhesion and Commun. 3:441-450, 1996; Doherty and Walsh,
Mol. Cell. Neurosci. 8:99-111, 1996; Saffell et al., Neuron
18:231-242, 1997). Neurons cultured on monolayers of 3T3 cells that
have been transfected with cDNAs encoding N-cadherin or N-CAM
extend longer neurites than neurons cultured on the untransfected
parental 3T3 cells (commonly referred to as the control 3T3 cells).
It has been determined that the neurite response stimulated by
transfected N-CAM and N-cadherin initially depends upon a trans
homophilic binding interaction between the transfected CAM in the
3T3 cell and the corresponding CAM in the neuron. This Example
illustrates the use of representative modulating agents to disrupt
neurite outgrowth stimulated by N-cadherin.
[0212] Neurons were cultured on monolayers of 3T3 cells transfected
with cDNA encoding N-cadherin essentially as described by Doherty
and Walsh, Curr. Op. Neurobiol. 4:49-55, 1994; Williams et al.,
Neuron 13:583-594, 1994; Hall et al., Cell Adhesion and Commun.
3:441-450, 1996; Doherty and Walsh, Mol. Cell. Neurosci. 8:99-111,
1994; Safell et al., Neuron 18:231-242, 1997. Briefly, monolayers
of control 3T3 fibroblasts and 3T3 fibroblasts that express
N-cadherin were established by overnight culture of 80,000 cells in
individual wells of an 8-chamber well tissue culture slide. 3000
cerebellar neurons isolated from post-natal day 3 mouse brains were
cultured for 18 hours on the various monolayers in control media
(SATO/2% FCS), or media supplemented with various concentrations of
the test peptide to be evaluated. The cultures were then fixed and
stained for GAP43 which specifically binds to the neurons and their
neurites. The length of the longest neurite on each GAP43 positive
neuron was then measured by computer assisted morphometry. For each
data point, measurements were made from 100-160 neurons, and the
given values show the mean +/- the standard error of the mean.
[0213] One modulating agent was H-WLKIDPVNGQI-OH (SEQ ID NO:13), an
11 amino acid peptide containing the ECD4 HAV-BM from human
N-cadherin plus some flanking sequence. This peptide is designated
N-CAD-CHD2. FIG. 5 shows the neurite outgrowth response for neurons
cultured on monolayers of 3T3 cells or 3T3 cells expressing
N-cadherin in media containing varying concentrations of the
modulating agent. In the absence of the peptide, neurites were
considerably longer on the N-cadherin monolayers. The peptide fully
inhibited the N-cadherin response at a concentration of 125 and 250
.mu.g/ml.
[0214] The mean neurite length was further measured for neurons
cultured on monolayers of 3T3 cells, 3T3 cells expressing
N-cadherin, 3T3 cells expressing NCAM, and 3T3 cells expressing L1
in media containing the linear peptide H-WLKIDPVNGQI- OH (SEQ ID
NO:13; designated N-CAD-CHD2) at a concentration of 250 .mu.g/ml.
The graph shown in FIG. 6 summarizes the results. Neurite outgrowth
was approximately twice as long on 3T3 cells expressing either of
the cell adhesion molecules N-cadherin, N-CAM, or L1, as compared
to outgrowth on 3T3 cells in the absence of the peptide. Only
neurite outgrowth on 3T3 cells expressing N-cadherin was inhibited
by the peptide H-WLKIDPVNGQI-OH (SEQ ID NO:13). This peptide is
therefore a specific inhibitor of N-cadherin function.
[0215] The ability of different modulating agents to inhibit
neurite outgrowth was also evaluated. Table II shows the effects of
N-Ac-INPISGQ-NH.sub.2 (SEQ ID NO:22; derived from EC1 of human
N-cadherin), and certain control peptides with changes in specific
amino acids (N-Ac-INPASGQ-NH.sub.2 (SEQ ID NO:88), N-Ac-
INAISGQ-NH.sub.2 (SEQ ID NO:89) and N-Ac-LNPISGQ-NH.sub.2 (SEQ ID
NO:90)) on neurite outgrowth. The peptides were tested at different
concentrations. Rat neurons were grown for 20 hours on monolayers
of either 3T3 cells, or 3T3 cells expressing N-cadherin. The
cultures were then fixed, and the mean neurite length was
determined by making measurements on at least 150 neurons for each
treatment. The results are presented as the percentage inhibition
of neurite outgrowth over 3T3 cells expressing N-cadherin. The
peptides did not inhibit neurite outgrowth over 3T3 cells not
expressing N-cadherin.
2TABLE II Effect (Percent Inhibition) of Representative Modulating
Agents on Neurite Outgrowth on 3T3 Cells Expressing N-cadherin
Sequence 100 .mu.g/ml 33 .mu.g/ml 10 .mu.g/ml 3 .mu.g/ml
N--Ac-INPISGQ-NH.sub.2 94.5 .+-. 4.8 73.0 .+-. 4.6 47.8 .+-. 4.1
19.5 .+-. 4.3 (SEQ ID NO: 22) N--Ac-INPASGQ-NH.sub.2 7.2 .+-. 3.2
(SEQ ID NO: 88) N--Ac-INAISGQ-NH.sub.2 37.2 .+-. 2.3 12.3 .+-. 6.2
(SEQ ID NO: 89) N--Ac-LNPISGQ-NH.sub.2 19.2 .+-. 7.7 (SEQ ID NO:
90)
[0216] Similar experiments, illustrated in Table III, were
performed to evaluate the effect of various cyclic peptide
modulating agents derived from EC1 of human N-cadherin on neurite
outgrowth.
Table III
Effect of Cyclic Peptide Modulating Agents on Neurite Outgrowth
[0217]
3 on 3T3 Cells Expressing N-cadherin Percent Inhibition of Neurite
Outgrowth Sequence EC.sub.50 (mM) 100 .mu.g/ml 33 .mu.g/ml 10
.mu.g/ml 3 .mu.g/ml N--Ac-CINPC-NH.sub.2 0.0143 98.1 .+-. 8.2 71.0
.+-. 6.5 51.9 .+-. 2.5 17.4 .+-. 5.7 (SEQ ID NO: 86)
N--Ac-CINPIC-NH.sub.2 0.0382 94.5 .+-. 10.5 56.2 .+-. 6.9 31.5 .+-.
7.5 7.5 .+-. 3.2 (SEQ ID NO: 87) N--Ac-CINPISC-NH.sub.2 0.0381 73.4
.+-. 2.2 39.5 .+-. 4.3 21.8 .+-. 1.2 (SEQ ID NO: 63)
[0218] To further evaluate the specificity of these peptide
modulating agents, inhibition of neurite outgrowth was assessed, at
100 .mu.g/ml, on monolayers of 3T3 cells transfected with various
molecules responsible for neurite outgrowth. The results, shown in
Table IV, illustrate a high level of specificity for N-cadherin
mediated neurite outgrowth.
4TABLE IV Effect (Percent Inhibition) of Representative Peptide
Modulating Agents on Neurite Outgrowth over 3T3 Cells Expressing
Various Protein Involved in Neurite Outgrowth Transfected
N--Ac-INPISGQ-NH.sub.2 N--Ac-CINPIC-NH.sub.2 Protein (SEQ ID NO:
22) (SEQ ID NO: 86) N-cadherin 94.5 .+-. 4.8 94.5 .+-. 10.5 NCAM
6.2 .+-. 3.0 13.7 .+-. 7.1 FGFR 1.9 .+-. 5.1 L1 0.3 .+-. 1.8 7.8
.+-. 3.8
[0219] Further experiments were performed as described above to
assess the activity of peptide modulating agents derived from EC4
of human N-cadherin. Table V shows the effect on neurite outgrowth
of agents comprising the sequence IDPVN (SEQ ID NO:29), as well as
agents containing substitutions within this sequence. The EC.sub.50
(mM) for N-Ac-WLKIDPVNGQI-NH.sub.2 (SEQ ID NO:13) was 0.046.
5TABLE V Effect of Peptide Modulating Agents on Neurite Outgrowth
Percent Inhibition of Neurite Outgrowth Sequence 250 .mu.g/ml 100
.mu.g/ml 33 .mu.g/ml .mu.g/ml N-Ac-WLKIDPVNGQI-NH.sub.2 83.5 .+-.
6.6 66.9 .+-. 3.7 19.8 .+-. 5.0 (SEQ ID NO:13)
N-Ac-WLKADPVNGQI-NH.sub.2 54.7 .+-. 1.2 22.2 .+-. 11.0 (SEQ ID
NO:91) N-Ac-WLKIDAVNGQI-NH.sub.2 52.3 .+-. 10.3 12.4 .+-. 7.6 (SEQ
ID NO:92) N-Ac-WLKADAVNGQI-NH.sub.2 19.7 .+-. 5.2 10.5 .+-. 4.1
(SEQ ID NO:93) N-Ac-IDPVNGQ-NH.sub.2 85.6 .+-. 7.3 56.4 .+-. 7.7
34.3 .+-. 2.4 0.6 .+-. 10.0 (SEQ ID NO:94) H-WLKIDPVNGQI-OH 76.1
.+-. 7.3 (SEQ ID NO:13) N-Ac-NLKIDPVNGQI-NH.sub.2 86.8 .+-. 8.2
67.8 .+-. 7.7 25.6 .+-. 6.7 H-LKIDPVNGQI-OH 46.0 .+-. 10.0 (SEQ ID
NO:21) H-LKIDPANGQI-OH 56.8 .+-. 1.2 (SEQ ID NO:64) H-LKIDAVNGQI-OH
103.8 .+-. 8.8 (SEQ ID NO:65) N-Ac-CIDPVNC-NH.sub.2 96.4 .+-. 7.3
75.8 .+-. 2.0 40.6 .+-. 6.3 15.4 .+-. 8.9 (SEQ ID NO:62)
[0220] These results demonstrate that modulating agents comprising
an HAV-BM sequence are effective and specific inhibitors of
N-cadherin function.
Example 3
Modulating Agent Binding to N-Cadherin
[0221] This Example illustrates the ability of a representative
modulating agent to bind to N-cadherin.
[0222] The peptide H-WLKIDPVNGQI-OH (SEQ ID NO:13) was passed over
flow cells coated with an N-cadherin-Fc chimera or human IgG1 at a
concentration of either 250, 500 or 1000 .mu.g/ml. FIG. 7 is a
graph illustrating the association of the peptide to the flow cell
coated with the N-cadherin Fc chimera, with the binding to the
control flow cell (coated with human IgG1) automatically
subtracted.
Example 4
Effect of a Representative Modulating Agent on Tumor Cell
Adhesion
[0223] This Example illustrates the ability of a modulating agent
to disrupt tumor cell adhesion.
[0224] Monolayer cultures of human ovarian cancer cells (SKOV3)
were grown in the presence and absence of the peptide
N-Ac-INPISGQ-NH.sub.2 (SEQ ID NO:22). FIG. 8A shows the cells grown
in the absence of peptide. FIG. 8B shows the cells 24 hours after
being cultured in the presence of 1 mg/mL of N-Ac-INPISGQ (SEQ ID
NO:22). The SKOV3 cells retract from one another and round-up when
cultured in the presence of the peptide.
[0225] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0226] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
95 1 5 PRT Artificial Sequence Description of Artificial Sequence
Solid Phase Synthesis 1 Asp Xaa Asn Asp Asn 1 2 4 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis 2
Leu Asp Arg Glu 1 3 11 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 3 Xaa Phe Xaa Ile Xaa Xaa
Xaa Xaa Gly Xaa Xaa 1 5 10 4 11 PRT Artificial Sequence Description
of Artificial Sequence Solid Phase Synthesis 4 Trp Leu Xaa Ile Xaa
Xaa Xaa Xaa Gly Gln Ile 1 5 10 5 11 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 5 Ile Phe
Ile Ile Asn Pro Ile Ser Gly Gln Leu 1 5 10 6 11 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis 6
Ile Phe Ile Leu Asn Pro Ile Ser Gly Gln Leu 1 5 10 7 11 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 7 Val Phe Ala Val Glu Lys Glu Thr Gly Trp Leu 1 5 10 8 11
PRT Artificial Sequence Description of Artificial Sequence Solid
Phase Synthesis 8 Val Phe Ser Ile Asn Ser Met Ser Gly Arg Met 1 5
10 9 11 PRT Artificial Sequence Description of Artificial Sequence
Solid Phase Synthesis 9 Val Phe Ile Ile Glu Arg Glu Thr Gly Trp Leu
1 5 10 10 11 PRT Artificial Sequence Description of Artificial
Sequence Solid Phase Synthesis 10 Val Phe Thr Ile Glu Lys Glu Ser
Gly Trp Leu 1 5 10 11 11 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 11 Val Phe Asn Ile Asp
Ser Met Ser Gly Arg Met 1 5 10 12 11 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 12 Trp Leu
Lys Ile Asp Ser Val Asn Gly Gln Ile 1 5 10 13 11 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis
13 Trp Leu Lys Ile Asp Pro Val Asn Gly Gln Ile 1 5 10 14 11 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 14 Trp Leu Ala Met Asp Pro Asp Ser Gly Gln Val 1 5 10 15
11 PRT Artificial Sequence Description of Artificial Sequence Solid
Phase Synthesis 15 Trp Leu His Ile Asn Ala Thr Asn Gly Gln Ile 1 5
10 16 11 PRT Artificial Sequence Description of Artificial Sequence
Solid Phase Synthesis 16 Trp Leu Glu Ile Asn Pro Asp Thr Gly Ala
Ile 1 5 10 17 11 PRT Artificial Sequence Description of Artificial
Sequence Solid Phase Synthesis 17 Trp Leu Ala Val Asp Pro Asp Ser
Gly Gln Ile 1 5 10 18 11 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 18 Trp Leu Glu Ile Asn
Pro Glu Thr Gly Ala Ile 1 5 10 19 11 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 19 Trp Leu
His Ile Asn Thr Ser Asn Gly Gln Ile 1 5 10 20 11 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis
20 Asn Leu Lys Ile Asp Pro Val Asn Gly Gln Ile 1 5 10 21 10 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 21 Leu Lys Ile Asp Pro Val Asn Gly Gln Ile 1 5 10 22 7
PRT Artificial Sequence Description of Artificial Sequence Solid
Phase Synthesis 22 Ile Asn Pro Ile Ser Gly Gln 1 5 23 7 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 23 Leu Asn Pro Ile Ser Gly Gln 1 5 24 7 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis
24 Ile Asp Pro Val Ser Gly Gln 1 5 25 8 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 25 Lys Ile
Asp Pro Val Asn Gly Gln 1 5 26 5 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 26 Pro Ile
Ser Gly Gln 1 5 27 5 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 27 Pro Val Asn Gly Gln 1
5 28 5 PRT Artificial Sequence Description of Artificial Sequence
Solid Phase Synthesis 28 Pro Val Ser Gly Arg 1 5 29 5 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 29 Ile Asp Pro Val Asn 1 5 30 5 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 30 Ile Asn
Pro Ile Ser 1 5 31 5 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 31 Lys Ile Asp Pro Val 1
5 32 108 PRT Homo sapiens Description of Artificial Sequence Solid
Phase Synthesis 32 Asp Trp Val Ile Pro Pro Ile Asn Leu Pro Glu Asn
Ser Arg Gly Pro 1 5 10 15 Phe Pro Gln Glu Leu Val Arg Ile Arg Ser
Asp Arg Asp Lys Asn Leu 20 25 30 Ser Leu Arg Ile Arg Val Thr Gly
Pro Gly Ala Asp Gln Pro Pro Thr 35 40 45 Gly Ile Phe Ile Leu Asn
Pro Ile Ser Gly Gln Leu Ser Val Thr Lys 50 55 60 Pro Leu Asp Arg
Gln Gln Asn Ala Arg Phe His Leu Gly Ala His Ala 65 70 75 80 Val Asp
Ile Asn Gly Asn Gln Val Glu Thr Pro Ile Asp Ile Val Ile 85 90 95
Asn Val Ile Asp Met Asn Asp Asn Arg Pro Glu Phe 100 105 33 108 PRT
Mus musculus Description of Artificial Sequence Solid Phase
Synthesis 33 Asp Trp Val Ile Pro Pro Ile Asn Leu Pro Glu Asn Ser
Arg Gly Pro 1 5 10 15 Phe Pro Gln Glu Leu Val Arg Ile Arg Ser Asp
Arg Asp Lys Asn Leu 20 25 30 Ser Leu Arg Tyr Ser Val Thr Gly Pro
Gly Ala Asp Gln Pro Pro Thr 35 40 45 Gly Ile Phe Ile Ile Asn Pro
Ile Ser Gly Gln Leu Ser Val Thr Lys 50 55 60 Pro Leu Asp Arg Glu
Leu Ile Ala Arg Phe His Leu Arg Ala His Ala 65 70 75 80 Val Asp Ile
Asn Gly Asn Gln Val Glu Asn Pro Ile Asp Ile Val Ile 85 90 95 Asn
Val Ile Asp Met Asn Asp Asn Arg Pro Glu Phe 100 105 34 108 PRT Bos
taurus Description of Artificial Sequence Solid Phase Synthesis 34
Asp Trp Val Ile Pro Pro Ile Asn Leu Pro Glu Asn Ser Arg Gly Pro 1 5
10 15 Phe Pro Gln Glu Leu Val Arg Ile Arg Ser Asp Arg Asp Lys Asn
Leu 20 25 30 Ser Leu Arg Tyr Ser Val Thr Gly Pro Gly Ala Asp Gln
Pro Pro Thr 35 40 45 Gly Ile Phe Ile Ile Asn Pro Ile Ser Gly Gln
Leu Ser Val Thr Lys 50 55 60 Pro Leu Asp Arg Glu Leu Ile Ala Arg
Phe His Leu Arg Ala His Ala 65 70 75 80 Val Asp Ile Asn Gly Asn Gln
Val Glu Asn Pro Ile Asp Ile Val Ile 85 90 95 Asn Val Ile Asp Met
Asn Asp Asn Arg Pro Glu Phe 100 105 35 108 PRT Homo sapiens
Description of Artificial Sequence Solid Phase Synthesis 35 Asp Trp
Val Ile Pro Pro Ile Ser Cys Pro Glu Asn Glu Lys Gly Pro 1 5 10 15
Phe Pro Lys Asn Leu Val Gln Ile Lys Ser Asn Lys Asp Lys Glu Gly 20
25 30 Lys Val Phe Tyr Ser Ile Thr Gly Gln Gly Ala Asp Thr Pro Pro
Val 35 40 45 Gly Val Phe Ile Ile Glu Arg Glu Thr Gly Trp Leu Lys
Val Thr Glu 50 55 60 Pro Leu Asp Arg Glu Arg Ile Ala Thr Tyr Thr
Leu Phe Ser His Ala 65 70 75 80 Val Ser Ser Asn Gly Asn Ala Val Glu
Asp Pro Met Glu Ile Leu Ile 85 90 95 Thr Val Thr Asp Gln Asn Asp
Asn Lys Pro Glu Phe 100 105 36 108 PRT Mus musculus Description of
Artificial Sequence Solid Phase Synthesis 36 Asp Trp Val Ile Pro
Pro Ile Ser Cys Pro Glu Asn Glu Lys Gly Glu 1 5 10 15 Phe Pro Lys
Asn Leu Val Gln Ile Lys Ser Asn Arg Asp Lys Glu Thr 20 25 30 Lys
Val Phe Tyr Ser Ile Thr Gly Gln Gly Ala Asp Lys Pro Pro Val 35 40
45 Gly Val Phe Ile Ile Glu Arg Glu Thr Gly Trp Leu Lys Val Thr Gln
50 55 60 Pro Leu Asp Arg Glu Ala Ile Ala Lys Tyr Ile Leu Tyr Ser
His Ala 65 70 75 80 Val Ser Ser Asn Gly Glu Ala Val Glu Asp Pro Met
Glu Ile Val Ile 85 90 95 Thr Val Thr Asp Gln Asn Asp Asn Arg Pro
Glu Phe 100 105 37 108 PRT Homo sapiens Description of Artificial
Sequence Solid Phase Synthesis 37 Asp Trp Val Val Ala Pro Ile Ser
Val Pro Glu Asn Gly Lys Gly Pro 1 5 10 15 Phe Pro Gln Arg Leu Asn
Gln Leu Lys Ser Asn Lys Asp Arg Asp Thr 20 25 30 Lys Ile Phe Tyr
Ser Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu 35 40 45 Gly Val
Phe Ala Val Glu Lys Glu Thr Gly Trp Leu Leu Leu Asn Lys 50 55 60
Pro Leu Asp Arg Glu Glu Ile Ala Lys Tyr Glu Leu Phe Gly His Ala 65
70 75 80 Val Ser Glu Asn Gly Ala Ser Val Glu Asp Pro Met Asn Ile
Ser Ile 85 90 95 Ile Val Thr Asp Gln Asn Asp His Lys Pro Lys Phe
100 105 38 108 PRT Mus musculus Description of Artificial Sequence
Solid Phase Synthesis 38 Glu Trp Val Met Pro Pro Ile Phe Val Pro
Glu Asn Gly Lys Gly Pro 1 5 10 15 Phe Pro Gln Arg Leu Asn Gln Leu
Lys Ser Asn Lys Asp Arg Gly Thr 20 25 30 Lys Ile Phe Tyr Ser Ile
Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu 35 40 45 Gly Val Phe Thr
Ile Glu Lys Glu Ser Gly Trp Leu Leu Leu His Met 50 55 60 Pro Leu
Asp Arg Glu Lys Ile Val Lys Tyr Glu Leu Tyr Gly His Ala 65 70 75 80
Val Ser Glu Asn Gly Ala Ser Val Glu Glu Pro Met Asn Ile Ser Ile 85
90 95 Ile Val Thr Asp Gln Asn Asp Asn Lys Pro Lys Phe 100 105 39
108 PRT Homo sapiens Description of Artificial Sequence Solid Phase
Synthesis 39 Asp Trp Val Ile Pro Pro Ile Asn Val Pro Glu Asn Ser
Arg Gly Pro 1 5 10 15 Phe Pro Gln Gln Leu Val Arg Ile Arg Ser Asp
Lys Asp Asn Asp Ile 20 25 30 Pro Ile Arg Tyr Ser Ile Thr Gly Val
Gly Ala Asp Gln Pro Pro Met 35 40 45 Glu Val Phe Ser Ile Asn Ser
Met Ser Gly Arg Met Tyr Val Thr Arg 50 55 60 Pro Met Asp Arg Glu
Glu His Ala Ser Tyr His Leu Arg Ala His Ala 65 70 75 80 Val Asp Met
Asn Gly Asn Lys Val Glu Asn Pro Ile Asp Leu Tyr Ile 85 90 95 Tyr
Val Ile Asp Met Asn Asp Asn His Pro Glu Phe 100 105 40 108 PRT Mus
musculus Description of Artificial Sequence Solid Phase Synthesis
40 Asp Trp Val Ile Pro Pro Ile Asn Val Pro Glu Asn Ser Arg Gly Pro
1 5 10 15 Phe Pro Gln Gln Leu Val Arg Ile Arg Ser Asp Lys Asp Asn
Asp Ile 20 25 30 Pro Ile Arg Tyr Ser Ile Thr Gly Val Gly Ala Asp
Gln Pro Pro Met 35 40 45 Glu Val Phe Asn Ile Asp Ser Met Ser Gly
Arg Met Tyr Val Thr Arg 50 55 60 Pro Met Asp Arg Glu Glu Arg Ala
Ser Tyr His Leu Arg Ala His Ala 65 70 75 80 Val Asp Met Asn Gly Asn
Lys Val Glu Asn Pro Ile Asp Leu Tyr Ile 85 90 95 Tyr Val Ile Asp
Met Asn Asp Asn Arg Pro Glu Phe 100 105 41 107 PRT Homo sapiens
Description of Artificial Sequence Solid Phase Synthesis 41 Ala Pro
Asn Pro Lys Ile Ile Arg Gln Glu Glu Gly Leu His Ala Gly 1 5 10 15
Thr Met Leu Thr Thr Phe Thr Ala Gln Asp Pro Asp Arg Tyr Met Gln 20
25 30 Gln Lys Tyr Leu Arg Tyr Thr Lys Leu Ser Asp Pro Ala Asn Trp
Leu 35 40 45 Lys Ile Asp Pro Val Asn Gly Gln Ile Thr Thr Ile Ala
Val Leu Asp 50 55 60 Arg Glu Ser Pro Asn Val Lys Asn Asn Ile Tyr
Asn Ala Thr Phe Leu 65 70 75 80 Ala Ser Asp Asn Gly Ile Pro Pro Met
Ser Gly Thr Gly Thr Leu Gln 85 90 95 Ile Tyr Leu Leu Asp Ile Asn
Asp Asn Ala Pro 100 105 42 106 PRT Mus musculus Description of
Artificial Sequence Solid Phase Synthesis 42 Ala Pro Asn Pro Lys
Ile Ile Arg Gln Glu Glu Gly Leu His Ala Gly 1 5 10 15 Thr Met Leu
Thr Thr Leu Thr Ala Gln Asp Pro Asp Arg Tyr Met Gln 20 25 30 Gln
Asn Ile Arg Tyr Thr Lys Leu Ser Asp Pro Ala Asn Trp Leu Lys 35 40
45 Ile Asp Pro Val Asn Gly Gln Ile Thr Thr Ile Ala Val Leu Asp Arg
50 55 60 Glu Ser Pro Tyr Val Gln Asn Asn Ile Tyr Asn Ala Thr Phe
Leu Ala 65 70 75 80 Ser Asp Asn Gly Ile Pro Pro Met Ser Gly Thr Gly
Thr Leu Gln Ile 85 90 95 Tyr Leu Leu Asp Ile Asn Asp Asn Ala Pro
100 105 43 106 PRT Bos taurus Description of Artificial Sequence
Solid Phase Synthesis 43 Ala Pro Asn Pro Lys Ile Ile Arg Gln Glu
Glu Gly Leu His Ala Gly 1 5 10 15 Thr Val Leu Thr Thr Phe Thr Ala
Gln Asp Pro Asp Arg Tyr Met Gln 20 25 30 Gln Asn Ile Arg Tyr Thr
Lys Leu Ser Asp Pro Ala Asn Trp Leu Lys 35 40 45 Ile Asp Ser Val
Asn Gly Gln Ile Thr Thr Ile Ala Val Leu Asp Arg 50 55 60 Glu Ser
Pro Asn Val Lys Ala Asn Ile Tyr Asn Ala Thr Phe Leu Ala 65 70 75 80
Ser Asp Asn Gly Ile Pro Pro Met Ser Gly Thr Gly Thr Leu Gln Ile 85
90 95 Tyr Leu Leu Asp Ile Asn Asp Asn Ala Pro 100 105 44 107 PRT
Homo sapiens Description of Artificial Sequence Solid Phase
Synthesis 44 Val Pro Pro Glu Lys Arg Val Glu Val Ser Glu Asp Phe
Gly Val Gly 1 5 10 15 Gln Glu Ile Thr Ser Tyr Thr Ala Gln Glu Pro
Asp Thr Phe Met Glu 20 25 30 Gln Lys Ile Thr Tyr Arg Ile Trp Arg
Asp Thr Arg Asn Trp Leu Glu 35 40 45 Ile Asn Pro Asp Thr Gly Ala
Ile Ser Thr Arg Ala Glu Leu Asp Arg 50 55 60 Glu Asp Phe Glu His
Val Lys Asn Ser Thr Tyr Thr Ala Leu Ile Ile 65 70 75 80 Ala Thr Asp
Asn Gly Ser Pro Val Ala Thr Gly Thr Gly Thr Leu Leu 85 90 95 Leu
Ile Leu Ser Asp Val Asn Asp Asn Ala Pro 100 105 45 107 PRT Mus
musculus Description of Artificial Sequence Solid Phase Synthesis
45 Met Pro Ala Glu Arg Arg Val Glu Val Pro Glu Asp Phe Gly Val Gly
1 5 10 15 Gln Glu Ile Thr Ser Tyr Thr Ala Arg Glu Pro Asp Thr Phe
Met Asp 20 25 30 Gln Lys Ile Thr Tyr Arg Ile Trp Arg Asp Thr Ala
Asn Trp Leu Glu 35 40 45 Ile Asn Pro Glu Thr Gly Ala Ile Phe Thr
Arg Ala Glu Met Asp Arg 50 55 60 Glu Asp Ala Glu His Val Lys Asn
Ser Thr Tyr Val Ala Leu Ile Ile 65 70 75 80 Ala Thr Asp Asp Gly Ser
Pro Ile Ala Thr Gly Thr Gly Thr Leu Leu 85 90 95 Leu Val Leu Leu
Asp Val Asn Asp Asn Ala Pro 100 105 46 106 PRT Homo sapiens
Description of Artificial Sequence Solid Phase Synthesis 46 Val Pro
Pro Ser Lys Val Val Glu Val Gln Glu Gly Ile Pro Thr Gly 1 5 10 15
Glu Pro Val Cys Val Tyr Thr Ala Glu Asp Pro Asp Lys Glu Asn Gln 20
25 30 Lys Ile Ser Tyr Arg Ile Leu Arg Asp Pro Ala Gly Trp Leu Ala
Met 35 40 45 Asp Pro Asp Ser Gly Gln Val Thr Ala Val Gly Thr Leu
Asp Arg Glu 50 55 60 Asp Glu Gln Phe Val Arg Asn Asn Ile Tyr Glu
Val Met Val Leu Ala
65 70 75 80 Met Asp Asn Gly Ser Pro Pro Thr Thr Gly Thr Gly Thr Leu
Leu Leu 85 90 95 Thr Leu Ile Asp Val Asn Asp His Gly Pro 100 105 47
106 PRT Mus musculus Description of Artificial Sequence Solid Phase
Synthesis 47 Val Pro Pro Ser Lys Val Ile Glu Ala Gln Glu Gly Ile
Ser Ile Gly 1 5 10 15 Glu Leu Val Cys Ile Tyr Thr Ala Gln Asp Pro
Asp Lys Glu Asp Gln 20 25 30 Lys Ile Ser Tyr Thr Ile Ser Arg Asp
Pro Ala Asn Trp Leu Ala Val 35 40 45 Asp Pro Asp Ser Gly Gln Ile
Thr Ala Ala Gly Ile Leu Asp Arg Glu 50 55 60 Asp Glu Gln Phe Val
Lys Asn Asn Val Tyr Glu Val Met Val Leu Ala 65 70 75 80 Thr Asp Ser
Gly Asn Pro Pro Thr Thr Gly Thr Gly Thr Leu Leu Leu 85 90 95 Thr
Leu Thr Asp Ile Asn Asp His Gly Pro 100 105 48 106 PRT Homo sapiens
Description of Artificial Sequence Solid Phase Synthesis 48 Pro Ser
Asn His Lys Leu Ile Arg Leu Glu Glu Gly Val Pro Pro Gly 1 5 10 15
Thr Val Leu Thr Thr Phe Ser Ala Val Asp Pro Asp Arg Phe Met Gln 20
25 30 Gln Ala Val Arg Tyr Ser Lys Leu Ser Asp Pro Ala Ser Trp Leu
His 35 40 45 Ile Asn Ala Thr Asn Gly Gln Ile Thr Thr Val Ala Val
Leu Asp Arg 50 55 60 Glu Ser Leu Tyr Thr Lys Asn Asn Val Tyr Glu
Ala Thr Phe Leu Ala 65 70 75 80 Ala Asp Asn Gly Ile Pro Pro Ala Ser
Gly Thr Gly Thr Leu Gln Ile 85 90 95 Tyr Leu Ile Asp Ile Asn Asp
Asn Ala Pro 100 105 49 106 PRT Mus musculus Description of
Artificial Sequence Solid Phase Synthesis 49 Pro Ser Asn His Lys
Leu Ile Arg Leu Glu Glu Gly Val Pro Ala Gly 1 5 10 15 Thr Ala Leu
Thr Thr Phe Ser Ala Val Asp Pro Asp Arg Pro Met Gln 20 25 30 Gln
Ala Val Arg Tyr Ser Lys Leu Ser Asp Pro Ala Asn Trp Leu His 35 40
45 Ile Asn Thr Ser Asn Gly Gln Ile Thr Thr Ala Ala Ile Leu Asp Arg
50 55 60 Glu Ser Leu Tyr Thr Lys Asn Asn Val Tyr Glu Ala Thr Phe
Leu Ala 65 70 75 80 Ala Asp Asn Gly Ile Pro Pro Ala Ser Gly Thr Gly
Thr Leu Gln Ile 85 90 95 Tyr Leu Ile Asp Ile Asn Asp Asn Ala Pro
100 105 50 6 PRT Artificial Sequence Description of Artificial
Sequence Solid Phase Synthesis 50 Lys Ile Asp Pro Val Asn 1 5 51 5
PRT Artificial Sequence Description of Artificial Sequence Solid
Phase Synthesis 51 Pro Val Asn Gly Gln 1 5 52 5 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis
52 Pro Ile Ser Gly Gln 1 5 53 5 PRT Artificial Sequence Description
of Artificial Sequence Solid Phase Synthesis 53 Pro Val Ser Gly Arg
1 5 54 5 PRT Artificial Sequence Description of Artificial Sequence
Solid Phase Synthesis 54 Lys Ile Asp Pro Val 1 5 55 6 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 55 Lys Ile Asp Pro Val Asn 1 5 56 5 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis
56 Ile Asp Pro Val Asn 1 5 57 5 PRT Artificial Sequence Description
of Artificial Sequence Solid Phase Synthesis 57 Ile Asn Pro Ile Ser
1 5 58 7 PRT Artificial Sequence Description of Artificial Sequence
Solid Phase Synthesis 58 Cys Pro Val Asn Gly Gln Cys 1 5 59 7 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 59 Cys Pro Ile Ser Gly Gln Cys 1 5 60 7 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis
60 Cys Pro Val Ser Gly Arg Cys 1 5 61 8 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 61 Cys Lys
Ile Asp Pro Val Asn Cys 1 5 62 7 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 62 Cys Ile
Asp Pro Val Asn Cys 1 5 63 7 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 63 Cys Ile Asn Pro Ile
Ser Cys 1 5 64 10 PRT Artificial Sequence Description of Artificial
Sequence Solid Phase Synthesis 64 Leu Lys Ile Asp Pro Ala Asn Gly
Gln Ile 1 5 10 65 10 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 65 Leu Lys Ile Asp Ala
Val Asn Gly Gln Ile 1 5 10 66 5 PRT Artificial Sequence Description
of Artificial Sequence Solid Phase Synthesis 66 Tyr Ile Gly Ser Arg
1 5 67 10 PRT Artificial Sequence Description of Artificial
Sequence Solid Phase Synthesis 67 Lys Tyr Ser Phe Asn Tyr Asp Gly
Ser Glu 1 5 10 68 17 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 68 Ile Trp Lys His Lys
Gly Arg Asp Val Ile Leu Lys Lys Asp Val Arg 1 5 10 15 Phe 69 48 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 69 Gly Val Asn Pro Thr Ala Gln Ser Ser Gly Ser Leu Tyr
Gly Ser Gln 1 5 10 15 Ile Tyr Ala Leu Cys Asn Gln Phe Tyr Thr Pro
Ala Ala Thr Gly Leu 20 25 30 Tyr Val Asp Gln Tyr Leu Tyr His Tyr
Cys Val Val Asp Pro Gln Glu 35 40 45 70 4 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 70 Leu Tyr
His Tyr 1 71 4 PRT Artificial Sequence Description of Artificial
Sequence Solid Phase Synthesis 71 Ile Asp Asp Lys 1 72 4 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 72 Asp Asp Lys Ser 1 73 5 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 73 Val Ile
Asp Asp Lys 1 5 74 5 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 74 Ile Asp Asp Lys Ser 1
5 75 6 PRT Artificial Sequence Description of Artificial Sequence
Solid Phase Synthesis 75 Val Ile Asp Asp Lys Ser 1 5 76 5 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 76 Asp Asp Lys Ser Gly 1 5 77 6 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 77 Ile Asp
Asp Lys Ser Gly 1 5 78 7 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 78 Val Ile Asp Asp Lys
Ser Gly 1 5 79 6 PRT Artificial Sequence Description of Artificial
Sequence Solid Phase Synthesis 79 Phe Val Ile Asp Asp Lys 1 5 80 7
PRT Artificial Sequence Description of Artificial Sequence Solid
Phase Synthesis 80 Phe Val Ile Asp Asp Lys Ser 1 5 81 8 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 81 Phe Val Ile Asp Asp Lys Ser Gly 1 5 82 7 PRT
Artificial Sequence Description of Artificial Sequence Solid Phase
Synthesis 82 Ile Phe Val Ile Asp Asp Lys 1 5 83 8 PRT Artificial
Sequence Description of Artificial Sequence Solid Phase Synthesis
83 Ile Phe Val Ile Asp Asp Lys Ser 1 5 84 9 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 84 Ile Phe
Val Ile Asp Asp Lys Ser Gly 1 5 85 7 PRT Artificial Sequence
Description of Artificial Sequence Solid Phase Synthesis 85 Cys Lys
Ile Asp Pro Val Cys 1 5 86 5 PRT Artificial Sequence Description of
Artificial Sequence Solid Phase Synthesis 86 Cys Ile Asn Pro Cys 1
5 87 6 PRT Artificial Sequence Description of Artificial Sequence
Solid Phase Synthesis 87 Cys Ile Asn Pro Ile Cys 1 5 88 7 PRT
Artificial Sequence Description of Artificial Sequence Control
Peptide derived for EC1 of human N-cadherin with single amino acid
change from SEQ ID NO. 22 88 Ile Asn Pro Ala Ser Gly Gln 1 5 89 7
PRT Artificial Sequence Description of Artificial Sequence Control
Peptide derived for EC1 of human N-cadherin with single amino acid
change from SEQ ID NO. 22 89 Ile Asn Ala Ile Ser Gly Gln 1 5 90 7
PRT Artificial Sequence Description of Artificial Sequence Control
Peptide derived for EC1 of human N-cadherin with single amino acid
change from SEQ ID NO. 22 90 Leu Asn Pro Ile Ser Gly Gln 1 5 91 11
PRT Artificial Sequence Description of Artificial Sequence Peptide
modulating agent derived from EC4 of human N-cadherin comprising
the sequence IDPVN containing an amino acid substitution 91 Trp Leu
Lys Ala Asp Pro Val Asn Gly Gln Ile 1 5 10 92 11 PRT Artificial
Sequence Description of Artificial Sequence Peptide modulating
agent derived from EC4 of human N-cadherin comprising the sequence
IDPVN containing an amino acid substitution 92 Trp Leu Lys Ile Asp
Ala Val Asn Gly Gln Ile 1 5 10 93 11 PRT Artificial Sequence
Description of Artificial Sequence Peptide modulating agent derived
from EC4 of human N-cadherin comprising the sequence IDPVN
containing amino acid substitutions 93 Trp Leu Lys Ala Asp Ala Val
Asn Gly Gln Ile 1 5 10 94 7 PRT Artificial Sequence Description of
Artificial Sequence Peptide modulating agent derived from EC4 of
human N-cadherin comprising the sequence IDPVN 94 Ile Asp Pro Val
Asn Gly Gln 1 5 95 4 PRT Artificial Sequence Claudin cell adhesion
recognition sequence 95 Ile Tyr Ser Tyr 1
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