U.S. patent application number 10/885482 was filed with the patent office on 2005-02-17 for compounds and methods for cancer therapy.
This patent application is currently assigned to Adherex Technologies, Inc.. Invention is credited to Alexander, J. Steven, Blaschuk, Orest W., Gour, Barbara J., Symonds, James Matthew.
Application Number | 20050037973 10/885482 |
Document ID | / |
Family ID | 32993244 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050037973 |
Kind Code |
A1 |
Blaschuk, Orest W. ; et
al. |
February 17, 2005 |
Compounds and methods for cancer therapy
Abstract
Methods for using modulating agents to enhance or inhibit
occludin-mediated cell adhesion in a variety of in vivo and in
vitro contexts are provided. Within certain embodiments, the
modulating agents may be used for cancer therapy or to increase
immune cell infiltration into tumors. The modulating agents
comprise at least one occludin cell adhesion recognition sequence
or an antibody or fragment thereof that specifically binds the
occludin cell adhesion recognition sequence. Modulating agents may
additionally comprise one or more cell adhesion recognition
sequences recognized by other adhesion molecules. Such modulating
agents may, but need not, be linked to a targeting agent, drug
and/or support material.
Inventors: |
Blaschuk, Orest W.;
(Westmount, CA) ; Symonds, James Matthew; (Ottawa,
CA) ; Gour, Barbara J.; (Kemptville, CA) ;
Alexander, J. Steven; (Shreveport, LA) |
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: |
32993244 |
Appl. No.: |
10/885482 |
Filed: |
July 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10885482 |
Jul 6, 2004 |
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09450073 |
Nov 29, 1999 |
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6797807 |
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09450073 |
Nov 29, 1999 |
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09222373 |
Dec 29, 1998 |
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6110747 |
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09222373 |
Dec 29, 1998 |
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09001511 |
Dec 31, 1997 |
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6248864 |
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Current U.S.
Class: |
424/130.1 ;
514/19.1; 514/19.3 |
Current CPC
Class: |
C07K 14/705 20130101;
A61P 35/00 20180101; C07K 14/47 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/017 ;
514/018; 514/016 |
International
Class: |
A61K 038/06; A61K
038/05; A61K 038/08 |
Claims
What is claimed is:
1. A method for enhancing the delivery of a drug to a tumor in a
mammal, comprising administering to a mammal a cell adhesion
modulating agent and a drug, wherein said modulating agent
comprises the sequence LYHY (SEQ ID NO:1), and wherein said
modulating agent inhibits occludin-mediated cell adhesion.
2. A method for enhancing the delivery of a drug to a tumor in a
mammal, comprising administering to a mammal a cell adhesion
modulating agent and a drug, wherein said modulating agent
comprises an antibody or fragment thereof that specifically binds
to an occludin cell adhesion recognition sequence, and wherein said
modulating agent inhibits occludin-mediated cell adhesion.
3. A method according to claim 1 or claim 2, wherein the tumor is
selected from the group consisting of bladder tumors, ovarian
tumors and melanomas.
4. A method according to claim 1 or claim 2, wherein said
composition is administered to said tumor.
5. A method according to claim 1 or claim 2, wherein said
composition is administered systemically.
6. A method according to claim 1, wherein said modulating agent
comprises a sequence selected from the group consisting of
QYLYHYCVVD (SEQ ID NO:2), YLYHYCVVD (SEQ ID NO:12), LYHYCVVD (SEQ
ID NO:13), QYLYHYC (SEQ ID NO:14), YLYHYC (SEQ ID NO:15), LYHYC
(SEQ ID NO:16), QYLYHY (SEQ ID NO:17), YLYHY (SEQ ID NO:18), CLYHYC
(SEQ ID NO:3), CYLYHYC (SEQ ID NO:40), CQYLYHYC (SEQ ID NO:41),
KQYLYHYD (SEQ ID NO:42), YLYHY (SEQ ID NO:43), QYLYHY (SEQ ID
NO:44), KLYHYD (SEQ ID NO:45) and derivatives of the foregoing
sequences having one or more C-terminal, N-terminal and/or side
chain modifications.
7. A method according to claim 1 or claim 2, wherein said
modulating agent is linked to a targeting agent.
8. A method according to claim 1 or claim 2, wherein said
modulating agent is linked to said drug.
9. A method according to claim 1 or claim 2, wherein said
modulating agent further comprises one or more of: (a) a cell
adhesion recognition sequence bound by an adhesion molecule other
than an occludin, wherein said cell adhesion recognition sequence
is separated from any LYHY (SEQ ID NO:1) sequence(s) by a linker;
and/or (b) an antibody or antigen-binding fragment thereof that
binds to a cell adhesion recognition sequence bound by an adhesion
molecule other than an occludin.
10. A method according to claim 9, wherein said cell adhesion
recognition sequence comprises one or more sequences selected from
the group consisting of HAV, NQK, NRN, NKD, EKD, ERD, RGD, DDK,
EEY, EAQ, IYSY (SEQ ID NO:49), TSSY (SEQ ID NO:50), VTAF (SEQ ID
NO:51) and VSAF (SEQ ID NO:52).
11. A method according to claim 9, wherein said antibody or
antigen-binding fragment thereof binds to a cell adhesion
recognition sequence comprising a sequence selected from the group
consisting of HAV, NQK, NRN, NKD, EKD, ERD, RGD, DDK, EEY, EAQ,
IYSY (SEQ ID NO:49), TSSY (SEQ ID NO:50), VTAF (SEQ ID NO:51) and
VSAF (SEQ ID NO:52).
12. A method according to claim 1 or claim 2, wherein said
modulating agent and said drug are present within a pharmaceutical
composition comprising a pharmaceutically acceptable carrier.
13. A method according to claim 12, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion
comprising one or more of: (a) a cell adhesion recognition sequence
bound by an adhesion molecule other than an occludin; and/or (b) an
antibody or antigen-binding fragment thereof that binds to a cell
adhesion recognition sequence bound by an adhesion molecule other
than an occludin.
14. A method according to claim 13, wherein said cell adhesion
recognition sequence comprises one or more sequences selected from
the group consisting of HAV, NQK, NRN, NKD, EKD, ERD, RGD, DDK,
EEY, EAQ, IYSY (SEQ ID NO:49), TSSY (SEQ ID NO:50), VTAF (SEQ ID
NO:51) and VSAF (SEQ ID NO:52).
15. A method according to claim 13, wherein said antibody or
antigen-binding fragment thereof binds to a cell adhesion
recognition sequence comprising a sequence selected from the group
consisting of HAV, NQK, NRN, NKD, EKD, ERD, RGD, DDK, EEY, EAQ,
IYSY (SEQ ID NO:49), TSSY (SEQ ID NO:50), VTAF (SEQ ID NO:51) and
VSAF (SEQ ID NO:52).
16. A method for treating cancer in a mammal, comprising
administering to a mammal a cell adhesion modulating agent, wherein
said modulating agent comprises the sequence LYHY (SEQ ID NO:1),
and wherein said modulating agent inhibits occludin-mediated cell
adhesion.
17. A method for treating cancer in a mammal, comprising
administering to a mammal a cell adhesion modulating agent, wherein
said modulating agent comprises an antibody or fragment thereof
that specifically binds to an occludin cell adhesion recognition
sequence, and wherein said modulating agent inhibits
occludin-mediated cell adhesion.
18. A method according to claim 16 or claim 17, wherein said cancer
is selected from the group consisting of carcinomas, leukemia and
melanomas.
19. A method according to claim 16, wherein said modulating agent
comprises a sequence selected from the group consisting of
QYLYHYCVVD (SEQ ID NO:2), YLYHYCVVD (SEQ ID NO:12), LYHYCVVD (SEQ
ID NO:13), QYLYHYC (SEQ ID NO:14), YLYHYC (SEQ ID NO:15), LYHYC
(SEQ ID NO:16), QYLYHY (SEQ ID NO:17), YLYHY (SEQ ID NO:18), CLYHYC
(SEQ ID NO:3), CYLYHYC (SEQ ID NO:40), CQYLYHYC (SEQ ID NO:41),
KQYLYHYD (SEQ ID NO:42), YLYHY (SEQ ID NO:43), QYLYHY (SEQ ID
NO:44), KLYHYD (SEQ ID NO:45) and derivatives of the foregoing
sequences having one or more C-terminal, N-terminal and/or side
chain modifications.
20. A method according to claim 16 or claim 17, wherein said
modulating agent is linked to a targeting agent.
21. A method according to claim 16 or claim 17, wherein said
modulating agent further comprises one or more of: (a) a cell
adhesion recognition sequence bound by an adhesion molecule other
than an occludin, wherein said cell adhesion recognition sequence
is separated from any LYHY (SEQ ID NO:1) sequence(s) by a linker;
and/or (b) an antibody or antigen-binding fragment thereof that
binds to a cell adhesion recognition sequence bound by an adhesion
molecule other than an occludin.
22. A method according to claim 21, wherein said cell adhesion
recognition sequence comprises a sequence selected from the group
consisting of HAV, NQK, NRN, NKD, EKD, ERD, RGD, DDK, EEY, EAQ,
IYSY (SEQ ID NO:49), TSSY (SEQ ID NO:50), VTAF (SEQ ID NO:51) and
VSAF (SEQ ID NO:52).
23. A method according to claim 16 or claim 17, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
24. A method according to claim 16 or claim 17, wherein said
pharmaceutical composition further comprises a modulator of cell
adhesion comprising one or more of: (a) a cell adhesion recognition
sequence bound by an adhesion molecule other than an occludin;
and/or (b) an antibody or antigen-binding fragment thereof that
binds to a cell adhesion recognition sequence bound by an adhesion
molecule other than an occludin.
25. A method according to claim 24, wherein said cell adhesion
recognition sequence comprises a sequence selected from the group
consisting of HAV, NQK, NRN, NKD, EKD, ERD, RGD, DDK, EEY, EAQ,
IYSY (SEQ ID NO:49), TSSY (SEQ ID NO:50), VTAF (SEQ ID NO:51) and
VSAF (SEQ ID NO:52).
26. A method for enhancing immune cell infiltration into a tumor in
a mammal, comprising administering to a mammal a cell adhesion
modulating agent and a drug, wherein said modulating agent
comprises the sequence LYHY (SEQ ID NO:1), and wherein said
modulating agent inhibits occludin-mediated cell adhesion.
27. A method for enhancing immune cell infiltration into a tumor in
a mammal, comprising administering to a mammal a cell adhesion
modulating agent and a drug, wherein said modulating agent
comprises an antibody or fragment thereof that specifically binds
to an occludin cell adhesion recognition sequence, and wherein said
modulating agent inhibits occludin-mediated cell adhesion.
28. A method according to claim 26 or claim 27, wherein the tumor
is selected from the group consisting of bladder tumors, ovarian
tumors and melanomas.
29. A method according to claim 26 or claim 27, wherein said
composition is administered to said tumor.
30. A method according to claim 26 or claim 27, wherein said
composition is administered systemically.
31. A method according to claim 26, wherein said modulating agent
comprises a sequence selected from the group consisting of
QYLYHYCVVD (SEQ ID NO:2), YLYHYCVVD (SEQ ID NO:12), LYHYCVVD (SEQ
ID NO:13), QYLYHYC (SEQ ID NO:14), YLYHYC (SEQ ID NO:15), LYHYC
(SEQ ID NO:16), QYLYHY (SEQ ID NO:17), YLYHY (SEQ ID NO:18), CLYHYC
(SEQ ID NO:3), CYLYHYC (SEQ ID NO:40), CQYLYHYC (SEQ ID NO:41),
KQYLYHYD (SEQ ID NO:42), YLYHY (SEQ ID NO:43), QYLYHY (SEQ ID
NO:44), KLYHYD (SEQ ID NO:45) and derivatives of the foregoing
sequences having one or more C-terminal, N-terminal and/or side
chain modifications.
32. A method according to claim 26 or claim 27, wherein said
modulating agent is linked to a targeting agent.
33. A method according to claim 26 or claim 27, wherein said
modulating agent and said drug are present within a pharmaceutical
composition comprising a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 09/450,073 filed Nov. 29, 1999, now allowed, which is a
continuation-in-part of U.S. application Ser. No. 09/222,373, filed
Dec. 29, 1998, now U.S. Pat. No. 6,110,747, which is a
continuation-in-part of U.S. application Ser. No. 09/001,511, filed
Dec. 31, 1997, now U.S. Pat. No. 6,248,864.
TECHNICAL FIELD
[0002] The present invention relates generally to methods for
regulating occludin-mediated processes, and more particularly to
the use of modulating agents comprising an occludin cell adhesion
recognition sequence and/or an antibody that specifically
recognizes such a sequence for inhibiting functions such as cell
adhesion and the formation of tissue permeability barriers.
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, spot
desmosomes 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, R G Landes
Co. (Austin Tex., 1996). The cadherins (abbreviated CADs) are
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). Cadherins have
been shown to regulate epithelial, endothelial, neural and cancer
cell adhesion, with different CADs expressed on different cell
types. For example, 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. VE (vascular endothelial)--cadherin is
predominantly expressed by endothelial cells. Other CADs are P
(placental)--cadherin, which is found in human skin, and R
(retinal)--cadherin. A detailed discussion of the 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.) and Lampugnani and Dejana, Curr. Opin. Cell Biol.
9:674-682, 1997.
[0005] CAD-mediated cell adhesion triggers a cascade of events that
lead to the formation of intercellular junctions, and ultimately to
the establishment of permeability barriers between tissue
compartments. The intercellular junction that is directly
responsible for the creation of permeability barriers that prevent
the diffusion of solutes through paracellular spaces is known as
the tight junction, or zonula occludens (Anderson and van Itallie,
Am. J. Physiol. 269:G467-G475, 1995; Lampugnani and Dejaila, Curr.
Opin. Cell Biol. 9:674-682, 1997).
[0006] Occludin is a transmembrane component of tight junctions
(Furuse et al., J. Cell Biol. 123:1777-1788, 1993; Furuse et al.,
J. Cell Sci. 109:429-435, 1996). This protein appears to be
expressed by all endothelial cell types, as well as by most
epithelial cell types. Occludin is an integral membrane protein
(FIG. 1) that is composed of two extracellular domains (EC1 and
EC2), four hydrophobic domains (TM1-TM4) that transverse the plasma
membrane, and three cytoplasmic domains (CP1-CP3). The structures
of all known mammalian occludins are similar (FIG. 2; Ando-Akatsuka
et al., J. Biol. Chem. 133:43-47, 1996). Occludin is believed to be
directly involved in cell adhesion and the formation of tight
junctions (Furuse et al., J. Cell Sci. 109:429-435, 1996; Chen et
al., J. Cell Biol. 138:891-899, 1997). It has been proposed that
occludin promotes cell adhesion through homophilic interactions (an
occludin on the surface of one cell binds to an identical occludin
on the surface of another cell). A detailed discussion of occludin
structure and function is provided by Lampugnani and Dejana, Curr.
Opin. Cell Biol. 9:674-682, 1997.
[0007] 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 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.
[0008] 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.
[0009] 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.
[0010] Further, internal barriers developed by tumors inhibit the
immune system's ability to attack tumor cells. Immune cells such as
leukocytes often cannot infiltrate a tumor, and thus tumor cells
are protected from the body's natural defenses. There are presently
no available methods for enhancing immune cell infiltration of
solid tumors.
[0011] Accordingly, there is a need in the art for compounds that
modulate cell adhesion, improve drug delivery across permeability
barriers and permit immune cell infiltration of solid tumors. The
present invention fulfills this need and further provides other
related advantages.
SUMMARY OF THE INVENTION
[0012] The present invention provides compounds and methods for
modulating occludin-mediated cell adhesion and the formation of
permeability barriers. Within certain aspects, compounds provided
herein comprise an occludin CAR sequence, or variant thereof that
retains the ability to modulate occludin-mediated cell adhesion.
Certain compounds are cyclic peptides that comprise the sequence
LYHY (SEQ ID NO:1). Within certain embodiments, such cyclic
peptides have the formula: 1
[0013] 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 comprise modifications such as an
N-acetyl or N-alkoxybenzyl group and/or a C-terminal amide or ester
group. Cyclic peptides may be cyclized via, for example, a
disulfide bond; an amide bond between terminal functional groups,
between residue side-chains or between one terminal functional
group and one residue side chain; a thioether bond or
.delta..sub.1.delta..sub.1-ditryptophan, or a derivative
thereof.
[0014] Within other embodiments, such compounds may be linear
peptides comprising the sequence LYHY (SEQ ID NO:1) or a variant
thereof. Such peptides are preferably 4-30 amino acid residues in
length, preferably 5-16 amino acid residues, and more preferably
6-9 amino acid residues.
[0015] Within further aspects, the present invention provides cell
adhesion modulating agents that comprise a cyclic or linear peptide
as described above. Within specific embodiments, such modulating
agents may be linked to one or more of a targeting agent, a drug, a
solid support or support molecule, or a detectable marker. Within
further specific embodiments, cell adhesion modulating agents are
provided that comprise a sequence selected from the group
consisting of QYLYHYCVVD (SEQ ID NO:2), YLYHYCVVD (SEQ ID NO:12),
LYHYCVVD (SEQ ID NO:13), QYLYHYC (SEQ ID NO:14), YLYHYC (SEQ ID
NO:15), LYHYC (SEQ ID NO:16), QYLYHY (SEQ ID NO:17), YLYHY (SEQ ID
NO:18) and derivatives of the foregoing sequences having one or
more C-terminal, N-terminal and/or side chain modifications.
[0016] Within further related aspects, cell adhesion modulating
agents are provided which comprise an antibody or antigen-binding
fragment thereof that specifically binds to a cell adhesion
recognition sequence bound by an occludin.
[0017] In addition, any of the above cell adhesion modulating
agents may further comprise one or more of: (a) a cell adhesion
recognition sequence that is bound by an adhesion molecule other
than an occludin, wherein said cell adhesion recognition sequence
is separated from any LYHY (SEQ ID NO:1) sequence(s) by a linker;
and/or (b) an antibody or antigen-binding fragment thereof that
specifically binds to a cell adhesion recognition sequence bound by
an adhesion molecule other than an occludin.
[0018] The present invention further provides pharmaceutical
compositions comprising a cell adhesion modulating agent as
described above, in combination with a pharmaceutically acceptable
carrier. Such compositions may further comprise a drug. In
addition, or alternatively, such compositions may further comprise
one or more of: (a) a peptide comprising a cell adhesion
recognition sequence that is bound by an adhesion molecule other
than an occludin; and/or (b) an antibody or antigen-binding
fragment thereof that specifically binds to a cell adhesion
recognition sequence bound by an adhesion molecule other than an
occludin.
[0019] Within further aspects, methods are provided for modulating
cell adhesion, comprising contacting a cadherin-expressing cell
with a cell adhesion modulating agent as described above.
[0020] The present invention further provides methods for enhancing
the delivery of a drug to a tumor in a mammal, comprising
administering to a mammal a cell adhesion modulating agent as
provided above and a drug, wherein the modulating agent inhibits
occludin-mediated cell adhesion.
[0021] Within further aspects, the present invention provides
methods for treating cancer in a mammal, comprising administering
to a mammal a cell adhesion modulating agent as provided above,
wherein the modulating agent inhibits occludin-mediated cell
adhesion.
[0022] Within further aspects, the present invention provides
methods for enhancing immune cell infiltration of a tumor in a
mammal, comprising administering to a mammal a cell adhesion
modulating agent as provided above, wherein the modulating agent
inhibits occludin-mediated cell adhesion.
[0023] 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
[0024] FIG. 1 is a diagram depicting the structure of a human
occludin. The two extracellular domains are designated EC1 and EC2,
the four hydrophobic domains that transverse the plasma membrane
are represented by TM1-TM4, and the three cytoplasmic domains are
denoted CP1-CP3. The occludin cell adhesion recognition sequence,
LYHY (Leu-Tyr-His-Tyr; SEQ ID NO:1), along with flanking amino acid
residues is shown within EC2 and is indicated by .cndot..
[0025] FIG. 2 provides the amino acid sequences of representative
mammalian occludin EC2 domains: human (SEQ ID NO:5), mouse (SEQ ID
NO:6), dog (SEQ ID NO:7), and rat-kangaroo (SEQ ID NO:8), as
indicated, along with the consensus sequence obtained using a
Clustal W protein sequence alignment. The occludin cell adhesion
recognition sequence, LYHY (Leu-Tyr-His-Tyr; SEQ ID NO:1), along
with flanking amino acid residues is shown in bold.
[0026] FIGS. 3A-3E provide the structures of representative cyclic
peptide modulating agents.
[0027] FIGS. 4A and 4B are immunofluorescence photographs of
monolayer cultures of human aortic endothelial cells immunolabeled
for occludin (red color) and VE-cadherin (green color).
Colocalization of occludin and VE-cadherin is indicated by the
yellow color. Arrows indicate gaps between the cells. The cells
were either not treated (FIG. 4A), or exposed for 1 hour to 100
.mu.g/ml H-QYLYHYCVVD-OH (Peptide 3; SEQ ID NO:2; FIG. 4B).
[0028] FIG. 5 is a photograph of the shaved back of a rat that
received duplicate subdermal injections of either phosphate
buffered saline, phosphate buffered saline containing
acetyl-QYLYHYCVVD-NH.sub.2 (SEQ ID NO:2; Peptide 1)
H-QYLYHYCVVD-NH.sub.2 (SEQ ID NO:2; Peptide 2), or H-QYLYHYCVVD-OH
(SEQ ID NO:2; Peptide 3) at a concentration of 100 .mu.g/ml,
followed 15 minutes later by a single injection of Evans blue into
the tail vein. The photograph was taken 15 minutes after injection
of the dye.
[0029] FIG. 6 is a histogram depicting the optical densities of
dimethylformamide extracts prepared from the excised injection
sites shown in FIG. 5, and showing that more dye was extracted from
the sites injected with H-QYLYHYCVVD-OH (SEQ ID NO:2; Peptide 3),
than from sites injected with either phosphate buffered saline,
acetyl-QYLYHYCVVD-NH.sub.- 2 (SEQ ID NO:2; Peptide 1) or
H-QYLYHYCVVD-NH.sub.2 (SEQ ID NO:2; Peptide 2).
[0030] FIG. 7 is a series of photographs of the shaved back of a
rat that received duplicate subdermal injections of either
phosphate buffered saline, phosphate buffered saline containing
acetyl-CLYHYC-NH.sub.2 (SEQ ID NO:3; Peptide 4), or H-CLYHYC-OH
(SEQ ID NO:3; Peptide 5) at a concentration of 100 .mu.g/ml,
followed 15 minutes later by a single injection of Evans blue into
the tail vein. The photographs were taken 15 minutes after
injection of the dye.
[0031] FIG. 8 is a histogram depicting the optical densities of
dimethylformamide extracts prepared from the excised sites of the
shaved back of a rat that received duplicate subdermal injections
of either phosphate buffered saline, phosphate buffered saline
containing acetyl-CLYHYC-NH.sub.2 (SEQ ID NO:3; Peptide 4), or
H-CLYHYC-OH (SEQ ID NO:3; Peptide 5) at a concentration of 100
.mu.g/ml, followed 15 minutes later by a single injection of Evans
blue into the tail vein.
[0032] FIG. 9 is a histogram depicting the mean electrical
resistance across MDCK cell monolayers cultured for 24 hours in
medium alone (Control), or medium containing H-QYLYHYCVVD-NH.sub.2
(Peptide 2), H-QYLYHYCVVD-COOH (Peptide 3) or N-Ac-CLYHYC-NH.sub.2
(Peptide 4) at a concentration of 0.5 mg/ml. Duplicate measurements
were taken, and error bars represent the standard deviation.
[0033] FIG. 10 is a graph illustrating the percent fMLP-stimulated
migration for neutrophils in the presence of different levels of
peptide 76 (H-CLYHYC-OH; SEQ ID NO:3), as indicated. Values
presented are means.+-.SE. *p<0.01 vs. control with fMLP.
[0034] FIG. 11 is a graph showing the effect of peptide 76
(H-CLYHYC-OH; SEQ ID NO:3) on neutrophil migration over time, as
indicated. Values presented are means.+-.SE. *p<0.01 vs. control
with fMLP. Data were analyzed using one-way ANOVA with Bonferroni's
correction for multiple comparisons. Significance was accepted at
p<0.05.
[0035] FIGS. 12A and 12B are photographs illustrating the results
of immunofluorescence analysis of occludin in HUVEC grown on glass
coverslips and treated with vehicle (FIG. 12A) or 200 .mu.g/mL
peptide 76 (FIG. 12B). Cells were fixed with methanol and acetone
and stained for occludin. Immunofluorescent staining was performed
with anti-occludin polyclonal antibody and Cy3-conjugated goat
anti-rabbit secondary antibody, and analyzed by fluorescence
microscope. FIG. 12B shows the gaps between adjacent endothelial
cells and the lack of junctional proteins at these gaps (arrows).
Bar=25 .mu.m.
[0036] FIG. 13 is a histogram illustrating the effect of peptide 76
(H-CLYHYC-OH; SEQ ID NO:3) on transendothelial permeability. Values
presented are means.+-.SE.
DETAILED DESCRIPTION OF THE INVENTION
[0037] As noted above, the present invention provides cell adhesion
modulating agents comprising peptides that are capable of
modulating occludin-mediated processes, such as cell adhesion. In
general, to modulate occludin-mediated cell adhesion, an
occludin-expressing cell is contacted with a cell adhesion
modulating agent (also referred to herein as a "modulating agent")
either in vivo or in vitro. It has been found, within the context
of the present invention, that the second extracellular domain
(EC2) of occludin contains a CAR sequence that promotes the
formation of permeability barriers. Accordingly, a modulating agent
may comprise at least one peptide (which may, but need not, be
cyclic) that contains an occludin cell adhesion recognition (CAR)
sequence and/or an antibody or fragment thereof that specifically
binds to an occludin CAR sequence. In humans and certain other
mammals, the CAR sequence is LYHY (Leu-Tyr-His-Tyr; SEQ ID NO:1;
see FIG. 2 and SEQ ID NOs:5-8). However, the present invention
further contemplates occludin CAR sequences from other organisms.
Such CAR sequences may be identified based upon sequence similarity
to the sequences provided herein, and the ability to modulate an
occludin-mediated function may be confirmed as described herein. A
modulating agent may further comprise one or more additional CAR
sequences and/or antibodies (or antigen-binding fragments thereof)
that specifically bind to an occludin CAR sequence. Alternatively,
or in addition, a modulating agent may further comprise one or more
CAR sequences for a CAM other than an occludin and/or an antibody
or antigen-binding fragment thereof that specifically binds to such
a CAM.
[0038] Certain modulating agents described herein inhibit cell
adhesion. Such modulating agents 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 of the present
invention, certain modulating agents may be used to enhance cell
adhesion (e.g., to supplement or replace stitches or to facilitate
wound healing). Certain modulating agents provided herein have the
ability to stimulate the formation of tight junctions in epithelial
cells, but not in endothelial cells. Such agents may be used, for
example, for treating diarrhea.
[0039] Cell Adhesion Modulating Agents
[0040] The term "cell adhesion modulating agent," as used herein,
refers to a molecule comprising at least one of the following
components:
[0041] (a) a linear or cyclic peptide sequence that is at least 50%
identical to an occludin CAR sequence (i.e., an occludin CAR
sequence or an analogue thereof that retains at least 50% sequence
identity);
[0042] (b) a mimetic (e.g., peptidomimetic or small molecule mimic)
of an occludin CAR sequence;
[0043] (c) a substance, such as an antibody or antigen-binding
fragment thereof, that specifically binds an occludin CAR sequence;
and/or
[0044] (d) a polynucleotide encoding a polypeptide that comprises
an occludin CAR sequence or analogue thereof A modulating agent may
consist entirely of one or more of the above elements, or may
additionally comprise further peptide and/or non-peptide regions.
Additional peptide or polynucleotide regions may be derived from
occludin (preferably an extracellular domain that comprises a CAR
sequence) and/or may be heterologous. Certain modulating agents
comprise the occludin CAR sequence LYHY (SEQ ID NO:1) or an
analogue thereof. Within certain preferred embodiments, such a
modulating agent contains 4-30 consecutive amino acid residues,
preferably 5-16 consecutive amino acid residues and more preferably
6-9 consecutive amino acid residues, present within an
occludin.
[0045] An "occludin CAR sequence," as used herein, refers to an
amino acid sequence that is present within in a naturally occurring
occludin and that is capable of detectably modulating an
occludin-mediated function, such as cell adhesion, as described
herein. In other words, contacting an occludin-expressing cell with
a peptide comprising a CAR sequence results in a detectable change
in an occludin-mediated function using at least one of the
representative assays provided herein. CAR sequences may be of any
length, but generally comprise 4-16 amino acid residues, and
preferably 5-8 amino acid residues. As noted above, the four amino
acid sequence LYHY (SEQ ID NO:1) is an occludin CAR sequence.
[0046] As an alternative to comprising a native occludin CAR
sequence, modulating agents as described herein may comprise an
analogue or mimetic of an occludin CAR sequence. Within the
specific embodiments described herein, it should be understood that
an analogue or mimetic may be substituted for a native CAR sequence
within any modulating agent. An analogue generally retains at least
50% identity to a native occludin CAR sequence, and modulates an
occludin-mediated function as described herein. Such analogues
preferably contain at least three residues of, and more preferably
at least five residues of, an occludin CAR sequence. An analogue
may contain any of a variety of amino acid substitutions,
additions, deletions and/or modifications (e.g., side chain
modifications). Preferred amino acid substitutions are
conservative. 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 in 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 feature of an occludin CAR sequence analogue is the
ability to modulate an occludin-mediated function, which may be
evaluated using the representative assays provided herein.
[0047] Alternatively, a modulating agent may comprise one or more
bioisoteres. "Bioisoteres" are substituents or groups that have
chemical or physical similarities and which produce broadly similar
biological properties. For example, preferred substitutions for the
imidazole ring of histidine in the aforementioned modulators are
triazole, pyrazole, thiatriazole, triazolone, benzoxadiazole,
pyrazine, pyrimidine, oxadiazole, tetraazole, aminopyridine,
triazine, benzodioxole, benzodiazole or benzoxadiazole.
[0048] A mimetic is a non-peptidyl compound that is
conformationally similar to an occludin CAR sequence, such that it
modulates an occludin-mediated function as described below. Such
mimetics may be designed based on techniques that evaluate the
three dimensional structure of the peptide. For example, Nuclear
Magnetic Resonance spectroscopy (NMR) and computational techniques
may be used to determine the conformation of an occludin CAR
sequence within a cyclic peptide. 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 the lowest energy conformation for the occludin
CAR sequence. This information can then be used to design mimetics
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.
Mimetics 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 --CH.sub.2NH--, --CSNH--,
--CH.sub.2S--, --CH.dbd.CH--, --CH.sub.2CH.sub.2--, --CONMe- and
others. These backbone amide linkages can also be part of a ring
structure (e.g., lactam). Mimetics may be designed where one or
more of the side chain functionalities of the occludin CAR sequence
are 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. Other mimetics may be
small molecule mimics, which may be readily identified from small
molecule libraries, based on the three-dimensional structure of the
CAR sequence. It should be understood that, within embodiments
described below, an analogue or mimetic may be substituted for an
occludin CAR sequence.
[0049] A portion of a modulating agent that comprises an occludin
CAR sequence, or analogue or mimetic thereof, may be a linear or
cyclic peptide. 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 occludin CAR sequence. 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.
[0050] In addition to one or more of the above components, a
modulating agent may comprise one or more additional CAR sequences,
which may or may not be occludin CAR sequences, and/or one or more
antibodies or fragments thereof that specifically recognize a CAR
sequence. Additional CAR sequences may be present within a cyclic
peptide containing an occludin CAR sequence, within a separate
cyclic peptide component of the modulating agent and/or in a
non-cyclic portion of the modulating agent. Antibodies and
antigen-binding fragments thereof are typically present in a
non-cyclic portion of the modulating agent.
[0051] Within certain embodiments in which inhibition of cell
adhesion is desired, a modulating agent may contain one occludin
CAR sequence or analogue thereof. Alternatively, such an agent may
comprise multiple occludin CAR 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). For example, a modulating agent with adjacent LYHY
sequences may comprise the peptide LYHYLYHY (SEQ ID NO:9). A
representative modulating agent with LYHY sequences in close
proximity may comprise the sequence QLYHYQLYHYQLYHY (SEQ ID NO:10).
One or more antibodies, or fragments thereof, may similarly be used
within such embodiments, either alone or in combination with one or
more CAR sequences.
[0052] In certain embodiments, a modulating agent as described
above may enhance cell adhesion among epithelial cells, but not
among endothelial cells. It has been found, within the context of
the present invention, that certain modulating agents comprising an
LYHY sequence affect endothelial and epithelial cells differently,
stimulating the formation of tight junctions in epithelial cells.
Such agents include H-QYLYHYCVVD-COOH (SEQ ID NO:2) and
N-Ac-CLYHYC-NH.sub.2 (SEQ ID NO:3). Terminal functional groups may
influence the activity of peptide modulating agents in epithelial
and endothelial cells.
[0053] Within other embodiments in which enhancement of cell
adhesion is desired, a modulating agent may generally contain
multiple occludin CAR 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.
[0054] A modulating agent as described herein 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 LYHY 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. As used herein, an
"adhesion molecule" is any molecule that mediates cell adhesion via
a receptor on the cell's surface. Adhesion molecules include
classical cadherins; a typical cadherins such as cadherin-11 (OB
cadherin), cadherin-5 (VE-cadherin), cadherin-6 (K-cadherin),
cadherin-7, cadherin-8, cadherin-12 (Br-cadherin, cadherin-14,
cadherin-15, and PB-cadherin; other nonclassical cadherins such as
desmocollins (dsc) and desmogleins (dsg); claudin; integrins; and
members of the immunoglobulin supergene family, such as N-CAM and
PECAM). Preferred CAR sequences for inclusion within a modulating
agent include: (a) His-Ala-Val (HAV), which is bound by classical
cadherins; (b) Arg-Gly-Asp (RGD), which is bound by integrins (see
Cardarelli et al., J. Biol. Chem. 267:23159-23164, 1992); (c)
KYSFNYDGSE (SEQ ID NO:11), which is bound by N-CAM; (d) claudin CAR
sequences comprising at least four consecutive amino acids present
within a claudin region that has the formula:
Trp-Lys/Arg-Aaa-Baa-Ser/Ala-Tyr/Ph- e-Caa-Gly (SEQ ID NO:47),
wherein Aaa, Baa and Caa indicate independently selected amino acid
residues; Lys/Arg is an amino acid that is lysine or arginine;
Ser/Ala is an amino acid that is serine or alanine; and Tyr/Phe is
an amino acid that is tyrosine or phenylalanine; and (e)
nonclassical cadherin CAR sequences comprising at least three
consecutive amino acids present within a nonclassical cadherin
region that has the formula:
Aaa-Phe-Baa-Ile/Leu/Val-Asp/Asn/Glu-Caa-Daa-Ser/Thr/Asn-Gly (SEQ ID
NO:48), wherein Aaa, Baa, Caa and Daa are independently selected
amino acid residues; Ile/Leu/Val is an amino acid that is selected
from the group consisting of isoleucine, leucine and valine,
Asp/Asn/Glu is an amino acid that is selected from the group
consisting of aspartate, asparagine and glutamate; and Ser/Thr/Asn
is an amino acid that is selected from the group consisting of
serine, threonine or asparagine. Representative claudin CAR
sequences include IYSY (SEQ ID NO:49), TSSY (SEQ ID NO:50), VTAF
(SEQ ID NO:51) and VSAF (SEQ ID NO:52). Representative nonclassical
cadherin CAR sequences include the VE-cadherin CAR sequence DAE;
the OB-cadherin CAR sequences DDK, EEY and EAQ; the dsg CAR
sequences NQK, NRN and NKD and the dsc CAR sequences EKD and
ERD.
[0055] 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, LYHY (SEQ ID NO:1)-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, LYHY (SEQ ID NO:1) and HAV. Within another embodiment,
modulating agents having a branched structure may comprise, for
example, LYHY (SEQ ID NO:1), along with one or more of a claudin
CAR sequence; a VE-cadherin CAR sequence; a dsg CAR sequence and/or
a dsc CAR sequence. Linkers preferably produce a distance between
CAR sequences between 0.1 to 10,000 nm, 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 should be small (0.1-400 nm). For enhancers of cell
adhesion, the linker distance 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 run, 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
omithine. 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.
[0056] The total number of CAR sequences (including occludin CAR
sequence(s), with or without other CAR sequences derived from one
or more adhesion molecules) 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. Peptide modulating agents
comprising multiple CAR sequences typically contain from 4 to about
1000 amino acid residues, preferably from 4 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 4 to 50 residues in length, preferably from 4 to 25
residues, more preferably from 4 to 16 residues and still more
preferably from 4 to 15 residues. Additional residue(s) that may be
present on the N-terminal and/or C-terminal side of a CAR sequence
may be derived from sequences that flank the LYHY sequence within
naturally occurring occludins with or without amino acid
substitutions and/or other modifications. Flanking sequences for
mammalian occludins are shown in FIG. 2, and in SEQ ID NOs:5-8.
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 purification or other manipulation
and/or residues having a targeting or other function).
[0057] A modulating agent may contain sequences that flank the
occludin CAR sequence on one or both sides, to enhance potency or
specificity. A suitable flanking sequence for enhancing potency
includes, but is not limited to, an endogenous sequence present in
an occludin (shown in, for example, FIG. 2).
[0058] To facilitate the preparation of modulating agents having a
desired potency, nuclear magnetic resonance (NMR) and computational
techniques may be used to determine the conformation of a peptide
that confers a known potency. NMR is widely used for structural
analysis of molecules. Cross-peak intensities in nuclear Overhauser
enhancement (NOE) spectra, coupling constants and chemical shifts
depend on the conformation of a compound. NOE data provide the
interproton distance between protons through space. This
information may be used to facilitate calculation of the lowest
energy conformation for the LYHY (SEQ ID NO:1) sequence.
Conformation may then be correlated with tissue specificity to
permit the identification of peptides that are similarly tissue
specific or have enhanced tissue specificity.
[0059] 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 indicated in
Table 1, and the corresponding D-amino acids are designated by a
lower case one letter symbol.
1TABLE 1 Amino acid one-letter and three-letter abbreviations A Ala
Alanine R Arg Arginine D Asp Aspartic acid N Asn Asparagine C Cys
Cysteine Q Gln Glutamine E Glu Glutamic acid G Gly Glycine H His
Histidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met
Methionine F Phe Phenylalanine P Pro Proline S Ser Serine T Thr
Threonine W Trp Tryptophan Y Tyr Tyrosine V Val Valine
[0060] 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, ornithine, diaminobutyric
acid, .alpha.-aminoadipic acid, m-aminomethylbenzoic acid and
.alpha.,.beta.-diaminopropionic acid.
[0061] Certain preferred modulating agents for use within the
present invention comprise at least one of the following sequences:
QYLYHYCVVD (SEQ ID NO:2), YLYHYCVVD (SEQ ID NO:12), LYHYCVVD (SEQ
ID NO:13), QYLYHYC (SEQ ID NO:14), YLYHYC (SEQ ID NO:15), LYHYC
(SEQ ID NO:16), QYLYHY (SEQ ID NO:17), YLYHY (SEQ ID NO:18), and/or
LYHY (SEQ ID NO:1), wherein each amino acid residue may, but need
not, be modified as described above. Within other embodiments, a
modulating agent may comprise a cyclic peptide of one of the
following sequences: CLYHYC (SEQ ID NO:3), CYLYHYC (SEQ ID NO:40),
CQYLYHYC (SEQ ID NO:41), KQYLYHYD (SEQ ID NO:42), YLYHY (SEQ ID
NO:43), QYLYHY (SEQ ID NO:44) or KLYHYD (SEQ ID NO:45). Modulating
agents comprising derivatives of any of the sequences recited
herein (i.e., sequences having one or more C-terminal. N-terminal
and/or side chain modifications) are also encompassed by the
present invention.
[0062] 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
standard solution or 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.
[0063] In solution phase synthesis, a wide variety of coupling
methods and protecting groups may be used (see Gross and
Meienhofer, eds., "The Peptides: Analysis, Synthesis, Biology,"
Vol. 1-4 (Academic Press, 1979); Bodansky and Bodansky, "The
Practice of Peptide Synthesis," 2d ed. (Springer Verlag, 1994)). In
addition, intermediate purification and linear scale up are
possible. Those of ordinary skill in the art will appreciate that
solution synthesis requires consideration of main chain and side
chain protecting groups and activation method. In addition, careful
segment selection is necessary to minimize racemization during
segment condensation. Solubility considerations are also a
factor.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] N-acetylation of the N-terminal residue can be accomplished
by reacting the final peptide with acetic anhydride before cleavage
from the resin. C-amidation may be accomplished using an
appropriate resin such as methylbenzhydrylamine resin using the Boc
technology.
[0068] 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 occludin 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 known occludin
sequences. 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
occludin 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.
[0069] As noted above, a modulating agent may comprise one or more
cyclic peptides. Such cyclic peptides may contain only one CAR
sequence, or may additionally contain one or more other adhesion
molecule binding sites, which may or may not be CARs. Such
additional sequences may be separated by a linker (i.e., one or
more peptides not derived from a CAR sequence or other adhesion
molecule binding site, as described previously). Within one such
embodiment, a modulating agent comprises a cyclic peptide
containing two LYHY (SEQ ID NO:1) sequences. Within another
embodiment, a cyclic peptide contains one LYHY (SEQ ID NO:1) and
one CAR sequence recognized by a different CAM. In certain
preferred embodiments, the second CAR sequence is derived from
fibronectin (i.e., RGD); a classical cadherin (i.e., HAV); a
claudin or a nonclassical cadherin as described above.
[0070] Cyclic peptides containing at least one occludin CAR
sequence may be covalently linked to either cyclic or linear
peptides containing at least one CAR sequence recognized by a
different CAM, as described previously. Using a linker, cyclic
LYHY-containing peptides and other cyclic or linear 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 multiple different CAR
sequences, such as various combinations of LYHY (SEQ ID NO:1), RGD,
HAV, claudin CAR sequence(s) and/or nonclassical cadherin CAR
sequence(s).
[0071] In addition to the CAR sequence(s), cyclic peptides
generally comprise at least one additional residue, such that the
size of the cyclic peptide ring ranges from 5 to about 15 residues,
preferably from 5 to 10 residues. Such additional residue(s) may be
present on the N-terminal and/or C-terminal side of a CAR sequence,
and may be derived from sequences that flank the endogenous
occludin CAR sequence with or without amino acid substitutions
and/or other modifications. 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).
[0072] Within certain preferred embodiments, as discussed below,
relatively small cyclic peptides that do not contain significant
sequences flanking the LYHY sequence are preferred for modulating
occludin mediated cell adhesion. Such peptides may contain an
N-acetyl group and a C-amide group (e.g., the 6-residue ring
N-Ac-CLYHYC-NH.sub.2 (SEQ ID NO:3). Within the context of the
present invention, underlined peptide sequences indicate cyclic
peptides, wherein the cyclization is performed by any suitable
method as provided herein.
[0073] Within other preferred embodiments, a cyclic peptide may
contain sequences that flank the LYHY (SEQ ID NO:1) sequence in a
native occludin molecule on one or both sides. Such sequences may
result in increased potency. Suitable flanking sequences include,
but are not limited to, the endogenous sequence present in
naturally occurring occludin. To facilitate the preparation of
cyclic peptides having increased potency, nuclear magnetic
resonance (NMR) and computational techniques may be used to
determine the conformation of a peptide that confers increased
potency, as described above.
[0074] Cyclic peptides as described herein may comprise residues of
L-amino acids, D-amino acids, or any combination thereof. A cyclic
peptide may also contain one or more 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 an N-acetyl group (such that
the amino group that represents the N-terminus of the linear
peptide prior to cyclization is acetylated) and/or a C-terminal
amide group (i.e., the carboxy terminus of the linear peptide prior
to cyclization is amidated). Residues other than common amino acids
that may be present with a cyclic peptide include, but are not
limited to, penicillamine, .beta.,.beta.-tetramethylene cysteine,
.beta.,.beta.-pentamethylene cysteine, .beta.-mercaptopropionic
acid, .beta.,.beta.-pentamethylene-.beta.-mercaptopropionic acid,
2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, omithine,
diaminobutyric acid, .alpha.-aminoadipic acid, m-aminomethylbenzoic
acid and .alpha.,.beta.-diaminopropionic acid.
[0075] Cyclic peptides as described herein may be synthesized by
methods well known in the art, including recombinant DNA methods
and chemical synthesis. Following synthesis of a linear peptide
(utilizing methods described herein), with or without N-acetylation
and/or C-amidation, cyclization may be achieved 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. By way of example, strong
oxidizing agents can be used to perform the cyclization shown below
(SEQ ID NOs:19 and 20), in which the underlined portion is
cyclized:
2
FmocCysAsp(t-Bu)GlyTyr(t-Bu)ProLys(Boc)Asp(t-Bu)CysLys(t-Bu)Gly-O-
Me .fwdarw. FmocCysAsp(t-Bu)GlyTyr(t-Bu)ProLys(Boc)Asp(t--
Bu)CysLys(t-Bu)Gly-OMe
[0076] Oxidizing agents also allow concurrent
deprotection/oxidation of suitable S-protected linear precursors to
avoid premature, nonspecific oxidation of free cysteine, as shown
below (SEQ ID Nos:21 and 22), where X and Y=S-Trt or S-Acm:
3 BocCys(X)GlyAsnLeuSer(t-Bu)Thr(t-Bu)Cys(Y)MetLeuGlyOH .fwdarw.
BocCysGlyAsnLeuSer(t-Bu)Thr(t-Bu)CysMetLeuGlyOH
[0077] 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. In the example below
(SEQ ID NOs:23 and 24), X is Acm, Tacm or t-Bu:
4 H-Cys(X)TyrIleGlnAsnCys(X)ProLeuGly-NH.sub.2 .fwdarw.
H-CysTyrIleGlnAsnCysProLeuGly-NH.sub.2
[0078] 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.
Peptides containing such residues are illustrated by the following
representative formulas, in which the underlined portion is
cyclized, N-acetyl groups are indicated by N-Ac and C-terminal
amide groups are represented by --NH.sub.2:
5 i) N-Ac-Cys-Leu-Tyr-His-Tyr-Cys-NH.sub.2 (SEQ ID NO: 25) ii)
H-Cys-Leu-Tyr-His-Tyr-Cys-OH (SEQ ID NO: 26) iii)
N-Ac-Cys-Gln-Tyr-Leu-Tyr-His-Tyr-Cys-NH.sub.2 (SEQ ID NO: 27) iv)
H-Cys-Gln-Tyr-Leu-Tyr-His-Tyr-Cys-OH (SEQ ID NO: 28) v)
N-Ac-Cys-Tyr-Leu-Tyr-His-Tyr-Cys-NH.sub.2 (SEQ ID NO: 29) vi)
H-Cys-Tyr-Leu-Tyr-His-Tyr-Cys-OH (SEQ ID NO: 30) vii)
N-Ac-Cys-Leu-Tyr-His-Tyr-Pen-NH.sub.2 (SEQ ID NO: 31) viii)
N-Ac-Tmc-Leu-Tyr-His-Tyr-Cys-NH.sub.2 (SEQ ID NO: 32) ix)
N-Ac-Pmc-Leu-Tyr-His-Tyr-Cys-NH.sub.2 (SEQ ID NO: 33) x)
N-Ac-Mpr-Leu-Tyr-His-Tyr-Cys-NH.sub.2 (SEQ ID NO: 34) xi)
N-Ac-Pmp-Leu-Tyr-His-Tyr-Cys-NH.sub.2 (SEQ ID NO: 35) xii) 2 xiii)
3
[0079] It will be readily apparent to those of ordinary skill in
the art that, within each of these representative formulas, any of
the above thiol-containing residues may be employed in place of one
or both of the thiol-containing residues recited.
[0080] 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). Two such cyclic
peptides are YLYHY (SEQ ID NO:18) and QYLYHY (SEQ ID NO:17). Within
another such embodiment, the cyclic peptide comprises a D-amino
acid (e.g., yLYHY; SEQ ID NO:18). Alternatively, cyclization may be
accomplished by linking one terminus and a residue side chain or
using two side chains, as in KLYHYD (SEQ ID NO:36) or KQYLYHYD (SEQ
ID NO:37), 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.
[0081] 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 O.fwdarw.N 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-(dimethylamino)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.
[0082] 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. Examples of thiol-containing linkages are
shown below: 4
[0083] Cyclization may also be achieved using
.delta..sub.1,.delta..sub.1'- -Ditryptophan (i.e.,
Ac-Trp-Gly-Gly-Trp-OMe) (SEQ ID NO:38), as shown below: 5
[0084] Representative structures of cyclic peptides are provided in
FIG. 3. Within FIG. 3, certain cyclic peptides having the ability
to modulate cell adhesion (shown on the left) are paired with
similar inactive structures (on the right). The structures and
formulas recited herein are provided solely for the purpose of
illustration, and are not intended to limit the scope of the cyclic
peptides described herein.
[0085] As noted above, instead of (or in addition to) an occludin
CAR sequence, a modulating agent may comprise an antibody, or
antigen-binding fragment thereof, that specifically binds to a
occludin CAR sequence. As used herein, an antibody, or
antigen-binding fragment thereof, is said to "specifically bind" to
a occludin CAR 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 occludin 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.
[0086] Polyclonal and monoclonal antibodies may be raised against
an occludin CAR 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 occludin CAR 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.
[0087] Monoclonal antibodies specific for the occludin CAR 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.
[0088] 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 occludin is localized.
[0089] 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).
[0090] Evaluation of Modulating Agent Activity
[0091] As noted above, modulating agents as described herein are
capable of modulating occludin-mediated cell adhesion. The ability
of an agent to modulate cell adhesion may generally be evaluated in
vitro by assaying the effect on endothelial and/or epithelial cell
adhesion using, for example, any of a variety of immunostaining
protocols and/or plating assays. In general, a modulating agent is
an inhibitor of cell adhesion if contact of the test cells with the
modulating agent results in a discernible disruption of cell
adhesion using one or more representative assays provided herein.
Modulating agents that enhance cell adhesion (e.g., agents
comprising multiple LYHY (SEQ ID NO:1) sequences and/or linked to a
support molecule or material) are considered to be modulators of
cell adhesion if they are capable of promoting cell adhesion, as
judged by plating assays to assess either endothelial or epithelial
cell adhesion to a modulating agent attached to a support material,
such as tissue culture plastic.
[0092] The ability of an agent to modulate cell adhesion may
generally be evaluated in vivo by assessing the effect on vascular
permeability utilizing the Miles assay (McClure et al., J.
Pharmacological & Toxicological Methods 32:49-52, 1994).
Briefly, a candidate modulating agent may be dissolved in phosphate
buffered saline (PBS) at a concentration of 100 .mu.g/ml. Adult
rats may be given 100 .mu.l subdermal injections of each peptide
solution into their shaved backs, followed 15 minutes later by a
single 250 .mu.l injection of 1% Evans blue dissolved in PBS into
their tail veins. The subdermal injection sites may be visually
monitored for the appearance of blue dye. Once the dye appears
(about 15 minutes after injection), each subdermal injection site
may be excised, weighed, and placed in 1 ml dimethylformamide for
24 hours to extract the dye. The optical density of the dye
extracts may then be determined at 620 nm. In general, the
injection of 0.1 ml of modulating agent (at a concentration of 0.1
mg/ml) into the backs of rats causes an increase of dye
accumulation at the injection sites of at least 50%, as compared to
dye accumulation at sites into which PBS has been injected.
[0093] The effect of a modulating agent on endothelial cell
adhesion may generally be evaluated using immunolocalization
techniques. Human aortic endothelial cells (HAEC) may be cultured
on fibronectin-coated coverslips (fibronectin may be obtained from
Sigma, St. Louis, Mo.) according to the procedures of Jaffe et al.,
J. Clin. Invest. 52:2745-2756, 1973. Briefly, human endothelial
cells may be maintained in EGM (endothelial cell growth medium;
Clonetics, San Diego, Calif.) and used for experiments at passage
4. Confluent cultures of HAEC may be exposed to either a candidate
modulating agent (final concentration 100 .mu.g/ml EGM), or EGM
alone for 1 hour. The cells are then be fixed for 30 minutes at
4.degree. C. in 95% ethanol, followed by fixation in acetone for 1
minute at 4.degree. C. (Furuse et al., J. Cell Biol. 123:1777-1788,
1993). After fixation, the cells may be probed with either mouse
anti-VE-cadherin antibodies (Hemeris, Sassenage, France; diluted
1:250 in 0.1% dried skim milk powder dissolved in PBS), or rabbit
anti-occludin antibodies (Zymed, South San Francisco, Calif.;
diluted 1:300 in 0.1% dried skim milk powder dissolved in PBS) for
1 hour at 37.degree. C. The cells may then be washed with 0.1%
dried skim milk powder dissolved in PBS (three washes, 5
minutes/wash), and probed with secondary antibodies (donkey
anti-mouse Cy3, or donkey anti-rabbit Cy5 diluted 1:250 in 0.1%
dried skim milk powder dissolved in PBS; Jackson Immunoresearch
Laboratories Inc., Westgrove, Pa.) for 1 hour at 37.degree. C. The
cells may then be washed again with in 0.1% dried skim milk powder
dissolved in PBS and mounted in a solution composed of 50% glycerol
and 50% PBS to which phenylenediamine (Sigma, St. Louis, Mo.) has
been added to a final concentration of 1 mg/ml. The sample may then
be analyzed using a Bio-Rad MRC 1000 confocal microscope with Laser
Sharp software version 2.1T (Bio-Rad, Hercules, Calif.). In
general, 0.1 mg/ml of modulating agent results in the appearance of
intercellular gaps within the monolayer cultures and a decrease of
at least 50% in the surface expression of occludin and VE-cadherin,
as compared to monolayer cultures that were not exposed to the
modulating agent.
[0094] Within certain cell adhesion assays, the addition of a
modulating agent to cells that express occludin results in
disruption of cell adhesion. An "occludin-expressing cell," as used
herein, may be any type of cell that expresses occludin 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). Occludin-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., 100 .mu.g/mL), disruption of cell adhesion
may be determined visually within 24 hours, by observing retraction
of the cells from one another.
[0095] 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, 0.1 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
rabbit anti-occludin antibody ((Zymed, South San Francisco, Calif.)
and 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 Cy3 and donkey anti-rabbit Cy5
(Jackson Immunoresearch Laboratories Inc., Westgrove, Pa.) for 1
hour at 37.degree. C. Following further washes in PBS (3.times.5
min) coverslips can be mounted and viewed by confocal
microscopy.
[0096] 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 occludin-mediated
cell adhesion may assume a non-polygonal and elongated morphology
(i.e., a fibroblast-like shape) within 48 hours of treatment with
0.1 mg/mL of modulating agent. Gaps appear in confluent cultures of
such cells. In addition, 0.1 mg/mL of such a modulating agent
reproducibly induces a readily apparent reduction in cell surface
staining of occludin and E-cadherin, as judged by
immunofluorescence microscopy (Laird et al., J. Cell Biol.
131:1193-1203, 1995), of at least 75% within 48 hours.
[0097] A third cell adhesion assay involves evaluating the effect
of a modulating agent on permeability of adherent endothelial cell
monolayers. The effects of a modulating agent on the permeability
of endothelial cell monolayers may be assessed utilizing the
protocols of Ehringer et al., J. Cell. Physiol. 167:562-569, 1996.
HAEC can be seeded onto inserts in 24-well plates
(Becton-Dickenson, Franklin Lake, N.J.) and cultured in EGM.
Confluent cell monolayers may be exposed to either modulating agent
(final concentration 100 .mu.g/ml EGM), or EGM alone for 1 hour.
The inserts may then be transferred to 24-chamber plates
(Becton-Dickenson) for permeability assays. Perfusate (0.5% bovine
serum albumin, fraction V (Sigma) dissolved in 15 mM HEPES, pH 7.4)
and FITC-Dextran (50 .mu.g/ml HEPES buffer; MW 12 kDa; Sigma) may
be added to each well (1 ml/well and 50 .mu.l/well, respectively),
and the cells incubated at 37.degree. C. for 30 min. Aliquots of
100 .mu.l may then be removed from the lower chamber and the
optical density of the solution determined at a wavelength of 450
nm. In general, the presence of 100 .mu.g/mL modulating agent that
enhances the permeability of endothelial cell monolayers results in
a statistically significant increase in the amount of marker in the
receptor compartment after 1 hour.
[0098] Yet another assay evaluates the effect of an occludin
modulating agent on the electrical resistance across a monolayer of
cells. For example, Madin Darby canine kidney (MDCK) cells can be
exposed to the modulating agent dissolved in medium (e.g., at a
final concentration of 0.5 mg/ml for a period of 24 hours). The
effect on electrical resistance can be measured using standard
techniques. This assay evaluates the effect of a modulating agent
on tight junction formation in epithelial cells. In general, the
presence of 500 .mu.g/mL modulating agent should result in a
statistically significant increase or decrease in electrical
resistance after 24 hours.
[0099] A further assay for evaluating modulating agent activity
involves determining an effect of a candidate agent on neutrophil
migration on endothelial cells. Within such assays, endothelial
cells (e.g., HUVEC) may be harvested and cultured using standard
techniques. Cells are grown to confluence on 8.0 .mu.m pore cell
culture inserts (Falcon, Franklin Lake, N.J.) essentially as
described by Ohno et al., Inflammation 21(3):313-24, 1997.
Radiolabeled human neutrophils (polymorphonuclear leukocytes, PMN)
are added to the apical surface of HUVEC monolayers and allowed to
migrate. Migration assays are generally performed in the presence
of 10.sup.-7 M N-formyl-methionyl-leucyl-phenylalanine (fMLP), a
chemoattractant that causes the leukocytes to undergo diapedesis.
PMN migration may be quantitated as follows: 1 % Migration = Lower
chamber ( cpm ) [ Upper chamber ( cpm ) + lower chamber ( cpm )
]
[0100] In general, the presence of 500 .mu.g/mL modulating agent
should result in a statistically significant increase or decrease
in percent migration after 1 hour.
[0101] Within another assay, a candidate modulating agent may be
evaluated for the ability to increase transendothelial
permeability. Endothelial barrier function may be evaluated by
measuring the transendothelial flux of FITC dextran using
monolayers of human umbilical vein endothelial cells. To perform
solute flux (permeability) measurements, monolayers may be
incubated in the presence and absence of candidate modulating agent
in peptide culture dishes, such that the monolayer divides the
medium into two compartments. FITC-dextran (10 kD) may be added to
one compartment, and incubated, after which the contents of the
other compartment may be assayed spectrophotometrically. In
general, the presence of 500 .mu.g/mL modulating agent should
result in a statistically significant increase in permeability
after 1 hour.
[0102] Modulating Agent Modification and Formulations
[0103] 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 single 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., an HAV sequence) may be attached to a
support such as a polymeric matrix, preferably in an alternating
pattern.
[0104] 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.
[0105] 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, thiol, carboxyl,
ketone 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.
[0106] 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').sub.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.
[0107] 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.
[0108] 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.
[0109] 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
occludin. Such modulators may generally be prepared as described
above, incorporating one or more non-occludin CAR sequences and/or
antibodies thereto in place of the occludin CAR sequence 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 cadherins
(e.g., classical cadherins, E-cadherin, Dsg and Dsc); integrins;
members of the immunoglobulin supergene family (such as N-CAM and
PECAM); and claudins. Preferred CAR sequences for use within such a
modulator include HAV, RGD, and CAR sequences of claudins,
VE-cadherin, dsg and dsc.
[0110] 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.
[0111] 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 calorimetric 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.
[0112] 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.
[0113] 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 from 0.0001% to 0.2%
and more preferably from 0.01% to 0.1%. Fluid compositions
typically contain an amount of modulating agent ranging from 10
ng/ml to 5 mg/ml, preferably from 10 .mu.g to 2 mg/mL. 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.
[0114] Modulating Agent Methods of Use
[0115] In general, the modulating agents and compositions described
herein may be used for modulating the adhesion of
occludin-expressing cells in vitro and/or in vivo. As noted above,
modulating agents for purposes that involve the disruption of
occludin-mediated cell adhesion may comprise an occludin CAR
sequence, multiple occludin CAR sequences in close proximity and/or
an antibody (or an antigen-binding fragment thereof) that
recognizes the occludin CAR 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 occludin CAR 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 occludin CAR 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.
[0116] 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 occludin-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.
[0117] 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 a 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.
[0118] 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.
[0119] Within certain aspects, the present invention provides
methods in which cell adhesion is diminished. In one such aspect,
methods for reducing unwanted cellular adhesion by administering a
modulating agent are provided. 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 H-QYLYHYCVVD-OH (SEQ ID NO:2) and H-CLYHYC-OH
(SEQ ID NO:3) and modulating agents comprising such sequences or
derivatives thereof. Preferred antibody modulating agents include
Fab fragments directed against either H-QYLYHYCVVD-OH (SEQ ID NO:2)
or H-CLYHYC-OH (SEQ ID NO:3). In addition, a modulating agent may
comprise one or more of a claudin CAR sequence, a CAR sequence for
a nonclassical cadherin (such as VE-cadherin, OB-cadherin, dsc or
dsg), RGD sequence, and/or HAV sequence separated from an occludin
CAR sequence via a linker. Alternatively, separate modulators of
cadherin- and integrin-mediated cell adhesion 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 .mu.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.
[0120] 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. Preferred modulating agents for use within such methods
include H-QYLYHYCVVD-OH (SEQ ID NO:2) and H-CLYHYC-OH (SEQ ID NO:3)
and modulating agents comprising such sequences or derivatives
thereof. Preferred antibody modulating agents include Fab fragments
directed against either H-QYLYHYCVVD-OH (SEQ ID NO:2) or
H-CLYHYC-OH (SEQ ID NO:3). 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 occludin,
classical cadherin, integrin, and nonclassical cadherin (e.g., Dsc
and Dsg) mediated cell adhesion, thereby disrupting tight
junctions, adherens junctions, and desmosomes. Multifunctional
modulating agents comprising an occludin CAR sequence linked to an
RGD sequence and/or CAR sequence(s) for one or more of a classical
cadherin, claudin or nonclassical cadherin (e.g., OB-cadherin,
VE-cadherin, dsc or dsg) may be used to disrupt cell adhesion.
Alternatively, a separate modulator of non-occludin-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 that may be used
in conjunction with the occludin modulating agents include Fab
fragments directed against either an N-cadherin CAR sequence (such
as FHLRAHAVDINGNQV-NH.sub.2; SEQ ID NO:4) or an E-cadherin CAR
sequence LFSHAVSSNG-NH.sub.2 (SEQ ID NO:39).
[0121] 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.
[0122] 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.
[0123] Preferred modulating agents for use within such methods
include H-QYLYHYCVVD-OH (SEQ ID NO:2) and H-CLYHYC-OH (SEQ ID NO:3)
and modulating agents comprising such sequences or derivatives
thereof. Preferred antibody modulating agents include Fab fragments
directed against either H-QYLYHYCVVD-OH (SEQ ID NO:2) or
H-CLYHYC-OH (SEQ ID NO:3). 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 occludin,
classical cadherin, integrin, Dsc and Dsg mediated cell adhesion,
thereby disrupting tight junctions, adherens junctions, focal
contacts and desmosomes. Multifunctional modulating agents
comprising the occludin CAR sequence linked to one or more of a
claudin CAR sequence, VE-cadherin CAR sequence, dsc CAR sequence,
dsg CAR sequence, RGD sequence, OB-cadherin CAR sequence and/or HAV
sequence may be used to disrupt cell adhesion. Alternatively, a
separate modulator of non-occludin-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 that may be used in conjunction with the
occludin modulating agents include Fab fragments directed against
either an N-cadherin CAR sequence, such as FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO:4), or an E-cadherin CAR sequence, such as
LFSHAVSSNG-NH.sub.2 (SEQ ID NO:39). 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 monitoring the level of serum
tumor markers (e.g., CEA or PSA).
[0124] It has further been found, within the context of the present
invention, that modulating agents provided herein may be used to
increase immune cell (e.g., leukocyte) infiltration into tumors,
resulting in cancer immunotherapy. Modulating agents may also be
used to treat leukemias. Preferred modulating agents for use within
such methods include H-QYLYHYCVVD-OH (SEQ ID NO:2) and H-CLYHYC-OH
(SEQ ID NO:3) and modulating agents comprising such sequences or
derivatives thereof. Preferred antibody modulating agents include
Fab fragments directed against either H-QYLYHYCVVD-OH (SEQ ID NO:2)
or H-CLYHYC-OH (SEQ ID NO:3). 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 occludin,
classical cadherin, integrin, Dsc and Dsg mediated cell adhesion,
thereby disrupting tight junctions, adherens junctions, focal
contacts and desmosomes. Multifunctional modulating agents
comprising the occludin CAR sequence linked to one or more of a
claudin CAR sequence, VE-cadherin CAR sequence, dsc CAR sequence,
dsg CAR sequence, RGD sequence, OB-cadherin CAR sequence and/or HAV
sequence may be used to disrupt cell adhesion. Alternatively, a
separate modulator of non-occludin-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 that may be used in conjunction with the
occludin modulating agents include Fab fragments directed against
either an N-cadherin CAR sequence, such as FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO:4), or an E-cadherin CAR sequence, such as
LFSHAVSSNG-NH.sub.2 (SEQ ID NO:39). 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. 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.
[0125] 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 use within such methods include H-QYLYHYCVVD-OH (SEQ ID
NO:2) and H-CLYHYC-OH (SEQ ID NO:3) and modulating agents
comprising such sequences or derivatives thereof. Preferred
antibody modulating agents include Fab fragments directed against
either H-QYLYHYCVVD-OH (SEQ ID NO:2) or H-CLYHYC-OH (SEQ ID NO:3).
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 occludin, classical cadherin, and integrin mediated
cell adhesion, thereby disrupting tight junctions, adherens
junctions, and focal contacts. Multifunctional modulating agents
comprising the occludin CAR sequence linked to one or more of a
claudin CAR sequence, VE-cadherin CAR sequence, dsc CAR sequence,
dsg CAR sequence, RGD sequence, and/or HAV sequence may be used to
disrupt cell adhesion. Alternatively, a separate modulator of
non-occludin-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 that may be used in conjunction with the occludin
modulating agents include Fab fragments directed against an
N-cadherin CAR sequence, such as FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID
NO:4).
[0126] 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 5 to 50 .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 50
.mu.g/mesh.
[0127] 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.
[0128] In yet another related aspect, the present invention
provides methods for inducing apoptosis in an occludin-expressing
cell. In general, patients afflicted with cancer may benefit from
such treatment. Preferred modulating agents for use within such
methods include H-QYLYHYCVVD-OH (SEQ ID NO:2) and H-CLYHYC-OH (SEQ
ID NO:3) and modulating agents comprising such sequences or
derivatives thereof. Preferred antibody modulating agents include
Fab fragments directed against either H-QYLYHYCVVD-OH (SEQ ID NO:2)
or H-CLYHYC-OH (SEQ ID NO:3). 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 occludin,
classical cadherin, and integrin mediated cell adhesion, thereby
disrupting tight junctions, adherens junctions, and focal contacts.
Multifunctional modulating agents comprising the occludin CAR
sequence linked to one or more of a claudin CAR sequence,
nonclassical cadherin CAR sequence (e.g., VE-cadherin, OB-cadherin,
dsc or dsg), RGD sequence, and/or HAV sequence may be used to
disrupt cell adhesion. Alternatively, a separate modulator of
non-occludin-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 that may be used in conjunction with the occludin
modulating agents include Fab fragments directed against either an
N-cadherin CAR sequence, such as FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID
NO:4), or an E-cadherin CAR sequence, such as LFSHAVSSNG-NH.sub.2
(SEQ ID NO:39).
[0129] Administration of modulating agents to induce apoptosis 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.
[0130] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLE 1
Preparation of Representative Cyclic Peptides
[0131] This Example illustrates the solid phase synthesis of
representative linear and cyclic peptides as modulating agents.
[0132] The peptides were assembled on methylbenzhydrylamine resin
(MBHA resin) for the C-terminal amide peptides. The traditional
Merrifield resins were used for any C-terminal acid peptides. Bags
of a polypropylene mesh material were filled with the resin and
soaked in dichloromethane. The resin packets were washed three
times with 5% diisopropylethylamine in dichloromethane and then
washed with dichloromethane. The packets are then sorted and placed
into a Nalgene bottle containing a solution of the amino acid of
interest in dichloromethane. An equal amount of
diisopropylcarbodiimide (DIC) in dichloromethane was added to
activate the coupling reaction. The bottle was shaken for one hour
to ensure completion of the reaction. The reaction mixture was
discarded and the packets washed with DMF. The N-.alpha.-Boc was
removed by acidolysis using a 55% TFA in dichloromethane for 30
minutes leaving the TFA salt of the .alpha.-amino group. The bags
were washed and the synthesis completed by repeating the same
procedure while substituting for the corresponding amino acid at
the coupling step. Acetylation of the N-terminal was performed by
reacting the peptide resins with a solution of acetic anhydride in
dichloromethane in the presence of diisopropylethylamine. The
peptide was then side-chain deprotected and cleaved from the resin
at 0.degree. C. with liquid HF in the presence of anisole as a
carbocation scavenger.
[0133] The crude peptides were purified by reversed-phase
high-performance liquid chromatography. Purified linear precursors
of the cyclic peptides were solubilized in 75% acetic acid at a
concentration of 2-10 mg/mL. A 10% solution of iodine in methanol
was added dropwise until a persistent coloration was obtained. A 5%
ascorbic acid solution in water was then added to the mixture until
discoloration. The disulfide bridge containing compounds were then
purified by HPLC and characterized by analytical HPLC and by mass
spectral analysis.
EXAMPLE 2
Establishment of a Model System for Assessing Endothelial Cell
Adhesion
[0134] This Example illustrates an endothelial cell adhesion assay
for evaluating the effects of occludin-modulating agents on
endothelial cell adhesion.
[0135] A. Cell Culture
[0136] Human aortic endothelial cells (HAEC) were cultured on
fibronectin (Sigma, St. Louis, Mo.) according to the procedures of
Jaffe et al., J. Clin. Invest. 52:2745-2756, 1973. Cells were
maintained in EGM (endothelial cell growth medium; Clonetics, San
Diego, Calif.) and used for experiments at passage 4.
[0137] B. Occludin and VE-Cadherin Immunolocalization Methods
[0138] HAEC were cultured on fibronectin-coated coverslips.
Confluent cultures of HAEC were exposed to linear peptides (final
concentration 100 .mu.g/ml EGM), or EGM alone for 1 hour. The cells
were then fixed for 30 minutes at 4.degree. C. in 95% ethanol,
followed by fixation in acetone for 1 minute at 4.degree. C.
(Furuse et al., J. Cell Biol. 123.1777-1788, 1993). After fixation,
the cells were allowed to air dry at room temperature. The cells
were probed with either mouse anti-VE-cadherin antibodies (Hemeris,
Sassenage, France; diluted 1:250 in 0.1% dried skim milk powder
dissolved in PBS), or rabbit anti-occludin antibodies (Zymed, South
San Francisco, Calif.; diluted 1:300 in 0.1% dried skim milk powder
dissolved in PBS) for 1 hour at 37.degree. C. The cells were then
washed with 0.1% dried skim milk powder dissolved in PBS (three
washes, 5 minutes/wash), and probed with secondary antibodies
(donkey anti-mouse Cy3, or donkey anti-rabbit Cy5 diluted 1:250 in
0.1% dried skim milk powder dissolved in PBS; Jackson
Immunoresearch Laboratories Inc., Westgrove, Pa.) for 1 hour at
37.degree. C. The cells were washed again with in 0.1% dried skim
milk powder dissolved in PBS and mounted in a solution composed of
50% glycerol and 50% PBS to which phenylenediamine (Sigma, St.
Louis, Mo.) had been added to a final concentration of 1 mg/ml. The
samples were analyzed using a Bio-Rad MRC 1000 confocal microscope
with Laser Sharp software version 2.1T (Bio-Rad, Hercules, Calif.).
Staining for occludin was assigned the pseudo-color red, whereas
VE-cadherin staining was assigned pseudo-color green using Confocal
Assistant 4.02 software. Immunofluorescence photographs of
monolayer cultures of human aortic endothelial cells immunolabeled
for occludin (red color) and VE-cadherin (green color) are shown in
FIGS. 4A and 4B. Colocalization of occludin and VE-cadherin is
indicated by the yellow color. Arrows indicate gaps between the
cells. Note that the endothelial cells retract from one another
when cultured in the presence of H-QYLYHYCVVD-OH (SEQ ID NO:2; FIG.
4B), indicating that adhesion is decreased between the cells.
Furthermore, the cells do not form cobblestone-like monolayers when
exposed to this peptide. Also note that surface expression of both
VE-cadherin and occludin is greatly reduced in the cells treated
with H-QYLYHYCVVD-OH (SEQ ID NO:2), as compared to the VE-cadherin
and occludin levels expressed by untreated cells.
EXAMPLE 3
Effect of Representative Modulating Agents on Vasopermeability
[0139] This Example illustrates a vasopermeability assay for
evaluating the effects of occludin-modulating agents on endothelial
cell permeability in vivo.
[0140] A. Miles Assay for Vascular Permeability
[0141] The ability of cyclic and linear peptides to increase
vascular permeability was assessed utilizing the Miles assay
(McClure et al., J. Pharmacological & Toxicological Meth.
32:49-521994). The peptides were dissolved in phosphate buffered
saline (PBS) at a concentration of 100 .mu.g/ml. Adult rats were
given 100 .mu.l subdermal injections of each peptide solution into
their shaved backs, followed 15 minutes later by a single 250 .mu.l
injection of 1% Evans blue dissolved in PBS into their tail veins.
The subdermal injection sites were visually monitored for the
appearance of blue dye. Once the dye appeared (15 minutes after
injection), each subdermal injection site was excised, weighed, and
placed in 1 ml dimethylformamide for 24 hours to extract the dye.
The optical density of the dye extracts was determined at 620 nm.
The effects of injecting either phosphate buffered saline,
phosphate buffered saline containing acetyl-QYLYHYCVVD-NH.sub.2
(SEQ ID NO:2) H-QYLYHYCVVD-NH.sub.2 (SEQ ID NO:2), or
H-QYLYHYCVVD-OH (SEQ ID NO:2) into sites along the shaved back of a
rat on the accumulation of Evans blue at the injection sites is
shown in FIG. 5. Note that more blue dye has accumulated at the
sites where the peptide H-QYLYHYCVVD-OH (SEQ ID NO:2) was injected,
as opposed to the sites where either phosphate buffered saline,
phosphate buffered saline containing acetyl-QYLYHYCVVD-NH.sub.2
(SEQ ID NO:2), or H-QYLYHYCVVD-NH.sub.2 (SEQ ID NO:2) were
injected.
[0142] FIG. 6 shows a histogram depicting the optical densities of
dimethylformamide extracts prepared from the excised injection
sites shown in FIG. 5. Note that more dye was extracted from the
sites injected with H-QYLYHYCVVD-OH (SEQ ID NO:2), than from sites
injected with either phosphate buffered saline,
acetyl-QYLYHYCVVD-NH.sub.2 (SEQ ID NO:2), or H-QYLYHYCVVD-NH.sub.2
(SEQ ID NO:2).
[0143] The effects of injecting either phosphate buffered saline,
phosphate buffered saline containing acetyl-CLYHYC-NH.sub.2 (SEQ ID
NO:3) or H-CLYHYC-OH (SEQ ID NO:3) into sites along the shaved back
of a rat on the accumulation of Evans blue at the injection sites
is shown in FIG. 7. FIG. 8 shows a histogram depicting the optical
densities of dimethylformamide extracts prepared from the excised
sites of the shaved back of a rat that received injections of
either phosphate buffered saline, phosphate buffered saline
containing acetyl-CLYHYC-NH.sub.2 (SEQ ID NO:3), or H-CLYHYC-OH
(SEQ ID NO:3) at a concentration of 100 .mu.g/ml, followed 15
minutes later by a single injection of Evans blue into the tail
vein. Note that more dye was extracted from the sites injected with
H-CLYHYC-OH (SEQ ID NO:3), than from sites injected with either
phosphate buffered saline, or acetyl-CLYHYC-NH.sub.2 (SEQ ID
NO:3).
EXAMPLE 4
Effect of Representative Modulating Agents on Electrical Resistance
Across Cell Monolayer
[0144] This Example illustrates an electrical resistance assay for
evaluating the effects of occludin-modulating agents on epithelial
cell adhesion.
[0145] Madin Darby canine kidney (MDCK) cells were plated in
Millicells (Millipore, Bedford, Mass.), at a density of 300,000
cells per Millicell, and cultured in Dulbecco's Modified Eagle
Medium (DMEM; Sigma, St. Louis, Mo.) containing 5% fetal calf serum
(Sigma, St. Louis, Mo.). Monolayers were exposed to the modulating
agent dissolved in medium at a final concentration of 0.5 mg/ml for
a period of 24 hours. The electrical resistance was measured using
the EVOM device (World Precision Instruments, Sarasota, Fla.). At
the time of measurement, fresh medium, with or without the
modulating agent, may be added to the Millicells.
[0146] FIG. 9 is a histogram depicting the mean electrical
resistance across MDCK cell monolayers cultured for 24 hours in
medium alone (Control), or medium containing H-QYLYHYCVVD-NH.sub.2
(SEQ ID NO:2; Peptide 2), H-QYLYHYCVVD-COOH (SEQ ID NO:2; Peptide
3) or N-Ac-CLYHYC-NH.sub.2 (SEQ ID NO:3; Peptide 4) at a
concentration of 0.5 mg/ml. Duplicate measurements were taken, and
error bars represent the standard deviation. Peptide 2 was found to
reduce the electrical resistance, while peptides 3 and 4 increased
the electrical resistance across the monolayer, relative to the
control. These results demonstrate the ability of occludin
modulating agents to modulate the formation of tight junctions in
epithelial cells. In particular, certain agents (such as peptides 3
and 4, above) stimulate the formation of tight junctions in
epithelial cells.
EXAMPLE 5
Effect of Representative Modulating Agents on Neutrophil Migration
on Endothelial Cells
[0147] This Example illustrates the use of peptide modulating
agents as provided herein to increase leukocyte infiltration into
tumors.
[0148] Human umbilical vein endothelial cells (HUVEC) were
harvested from umbilical cords by 0.2% collagenase treatment
essentially as described by Yoshida et al., Am. J. Physiol.
262:H1891-1898, 1992. The cells were cultured in Endothelial Cell
Growth Medium (EGM; Clonetics, San Diego, Calif.) supplemented with
10% heat-inactivated fetal calf serum (FCS; Hyclone, Logan, Md.),
heparin sodium (10 IU/ml; Sigma, St. Louis, Mo.), 1 IU/ml
penicillin, 1 mg/ml streptomycin, and 12.5 .mu.g amphotericin B,
and endothelial cell growth factor (80 ng/ml; Biomedical
Technologies, Stoughton, Mass.). The cell cultures were incubated
at 37.degree. C. in a 100% humidified atmosphere with 5% CO.sub.2
and expanded by brief trypsinization (0.25% trypsin in
phosphate-buffer saline containing 0.02% EDTA. Primary passage
HUVEC were seeded into fibronectin coated (5 .mu.g/ml) tissue
culture plates and used when confluent. Culture medium was replaced
every second day. Only first-passage cultures were used for the
studies. Cells were identified as endothelial cells by their
cobblestone appearance at confluence, and by positive labeling with
(I) acetylated low density lipoprotein labeled with Dil-Ac-LDL
(Biomedical Technologies), and (2) mouse antihuman factor VIII
(Calbiochem, San Diego, Calif.).
[0149] Human neutrophils (polymorphonuclear leukocytes, PMN) were
isolated from venous blood of healthy adults using standard dextran
sedimentation and gradient separation on Histoparque-1077 (Sigma)
essentially as described by Harlan et al., Lab. Invest.
52(2):141-50, 1985. This procedure yielded a PMN population that
was 95-98% viable (by trypan blue exclusion) and 98% pure (by
acetic acid--crystal violet staining).
[0150] For neutrophil diapedesis experiments, HUVEC were grown to
confluence on 8.0 .mu.m pore cell culture inserts (Falcon, Franklin
Lake, N.J.) essentially as described by Ohno et al., Inflammation
21(3):313-24, 1997. Isolated neutrophils were suspended in PBS and
radiolabeled by incubating PMN at 2.times.10.sup.7 cells/ml with 30
.mu.Ci Na.sup.51CrO.sub.4/ml (New England Nuclear, Natick, Mass.)
at 37.degree. C. for 60 minutes. The cells were then washed twice
with cold phosphate-buffered saline (PBS), spun at 250 g for 8
minutes to remove unincorporated radioactivity and resuspended in
PBS. The labeled neutrophils were added to the apical surface of
HUVEC monolayers and allowed to migrate into the lower chamber for
one hour. Migration assays were performed in the presence of
10.sup.-7 M N-formyl-methionyl-leucyl-p- henylalanine (fMLP), a
chemoattractant that causes the leukocytes to undergo
diapedesis.
[0151] PMN migration was quantitated as follows: 2 % Migration =
Lower chamber ( cpm ) [ Upper chamber ( cpm ) + lower chamber ( cpm
) ]
[0152] FIGS. 10-11 present the results of these experiments to
evaluate the effect of modulating agents and control peptides on
neutrophil transendothelial migration. In FIG. 10, the percent
fMLP-stimulated migration is shown for neutrophils in the presence
of different levels of peptide 76 (H-CLYHYC-OH; SEQ ID NO:3), as
indicated. Values presented are means.+-.SE. *p<0.01 vs. control
with fMLP. FIG. 11 shows the effect of peptide 76 (H-CLYHYC-OH; SEQ
ID NO:3) on neutrophil migration over time, as indicated. Values
presented are means.+-.SE. *p<0.01 vs. control with fMLP. Data
were analyzed using one-way ANOVA with Bonferroni's correction for
multiple comparisons. Significance was accepted at p<0.05. These
results indicate that the representative modulating agent
H-CLYHYC-OH (SEQ ID NO:3) can increase fMLP-stimulated
transendothelial migration.
[0153] For immunofluorescence analysis of occludin, HUVEC grown on
glass coverslips were treated with vehicle (FIG. 12A) or 200
.mu.g/mL peptide 76 (FIG. 12B). Cells were fixed with methanol and
acetone and stained for occludin (Kevil et al., Microcirculation
5:197-210, 1998). Immunofluorescent staining was performed with
anti-occludin polyclonal antibody (Zymed, San Francisco, Calif.,
used at a concentration of 4 .mu.g/ml) and Cy3-conjugated goat
anti-rabbit secondary Ab (Jackson Labs, Westgrove, Pa., used at a
1:250 dilution), and analyzed by fluorescence microscope. FIG. 12B
shows the gaps between adjacent endothelial cells and the lack of
junctional proteins at these gaps (arrows). The photographs shown
are representative of at least 20 different fields observed in each
experiment and of three independent experiments. Bar=25 .mu.m.
EXAMPLE 6
Effect of Representative Modulating Agents on Transendothelial
Permeability
[0154] This Example illustrates the use of peptide modulating
agents as provided herein to increase transendothelial
permeability.
[0155] Endothelial barrier function was evaluated by measuring the
transendothelial flux of FITC dextran using monolayers of human
umbilical vein endothelial cells cultured to confluency on 8 .mu.m
pore transwell inserts (Falcon). The surface area of these inserts
was 0.32 cm.sup.2. The total volume for the medium inside of this
insert was 0.5 ml. These inserts with cells were cultured inside of
a 24 well tissue culture plate adapted for transwell inserts
(Falcon) and the total volume in the `outer` compartment was 1 ml.
Cells were cultured to 100% confluency under these conditions in
medium consisting of endothelial growth medium-2 (EGM-2,
Biowhittaker) for 72 hours. Cells were used for solute flux
protocols at this point.
[0156] To perform solute flux (permeability) measurements,
monolayers were washed with Hank's balanced salts solution (HBSS)
and incubated in either HBSS alone (control) or HBSS+peptide 76
(H-CLYHYC-OH; SEQ ID NO:3) at a concentration of 200 .mu.g/ml for
60 minutes in peptide culture dishes. These chamber inserts were
transferred to in the `outer` compartment. FITC-dextran (10 kD) was
added to the upper compartment to a final concentration of 0.45 mM
(0.45%) and incubated at 37.degree. C. for an additional 60
minutes. At the end of this incubation period, the insert was
removed and 200 .mu.l of the lower chamber contents aspirated and
the absorbance measured spectrophotometrically. These experiments
were performed such that n=4. Statistical significance was
determined using one-way analysis of variance (ANOVA) with
Bonferroni post testing to test for significance between groups.
The results are presented in FIG. 13, which shows the ability of
peptide 76 (H-CLYHYC-OH; SEQ ID NO:3) to increase transendothelial
permeability.
[0157] 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.
[0158] 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
52 1 4 PRT Artificial Sequence Description of Artificial Sequence
Occludin cell adhesion recognition sequence 1 Leu Tyr His Tyr 1 2
10 PRT Artificial Sequence Description of Artificial Sequence Cell
adhesion modulating agent 2 Gln Tyr Leu Tyr His Tyr Cys Val Val Asp
1 5 10 3 6 PRT Artificial Sequence Description of Artificial
Sequence Cell adhesion modulating agent 3 Cys Leu Tyr His Tyr Cys 1
5 4 15 PRT Artificial Sequence Description of Artificial Sequence
N-cadherin cell adhesion recognition sequence 4 Phe His Leu Arg Ala
His Ala Val Asp Ile Asn Gly Asn Gln Val 1 5 10 15 5 48 PRT Homo
sapiens 5 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 6 48 PRT Mus musculus 6 Gly Val
Asn Pro Thr Ala Gln Ala Ser Gly Ser Met Tyr Gly Ser Gln 1 5 10 15
Ile Tyr Met Ile Cys Asn Gln Phe Tyr Thr Pro Gly Gly 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 7 48 PRT Canis sp. 7 Gly Val Asn Pro Thr Ala Gln Ala
Ser Gly Ser Leu Tyr Ser Ser Gln 1 5 10 15 Ile Tyr Ala Met Cys Asn
Gln Phe Tyr Ala Ser Thr Ala Thr Gly Leu 20 25 30 Tyr Met Asp Gln
Tyr Leu Tyr His Tyr Cys Val Val Asp Pro Gln Glu 35 40 45 8 50 PRT
dipodomys sp. 8 Gly Val Asn Pro Arg Ala Gly Leu Gly Ala Ser Ser Gly
Ser Leu Tyr 1 5 10 15 Tyr Asn Gln Met Leu Met Leu Cys Asn Gln Met
Met Ser Pro Val Ala 20 25 30 Gly Gly Ile Met Asn Gln Tyr Leu Tyr
His Tyr Cys Met Val Asp Pro 35 40 45 Gln Glu 50 9 8 PRT Artificial
Sequence Description of Artificial Sequence Representative occludin
cell adhesion recognition sequence 9 Leu Tyr His Tyr Leu Tyr His
Tyr 1 5 10 15 PRT Artificial Sequence Description of Artificial
Sequence Representative occludin cell adhesion recognition sequence
10 Gln Leu Tyr His Tyr Gln Leu Tyr His Tyr Gln Leu Tyr His Tyr 1 5
10 15 11 10 PRT Artificial Sequence Description of Artificial
Sequence Cell adhesion recognition sequence bound by N-cell
adhesion molecules 11 Lys Tyr Ser Phe Asn Tyr Asp Gly Ser Glu 1 5
10 12 9 PRT Artificial Sequence Description of Artificial Sequence
Cell adhesion modulation agent 12 Tyr Leu Tyr His Tyr Cys Val Val
Asp 1 5 13 8 PRT Artificial Sequence Description of Artificial
Sequence Cell adhesion modulation agent 13 Leu Tyr His Tyr Cys Val
Val Asp 1 5 14 7 PRT Artificial Sequence Description of Artificial
Sequence Cell adhesion modulation agent 14 Gln Tyr Leu Tyr His Tyr
Cys 1 5 15 6 PRT Artificial Sequence Description of Artificial
Sequence Cell adhesion modulation agent 15 Tyr Leu Tyr His Tyr Cys
1 5 16 5 PRT Artificial Sequence Description of Artificial Sequence
Cell adhesion modulation agent 16 Leu Tyr His Tyr Cys 1 5 17 6 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 17 Gln Tyr Leu Tyr His Tyr 1 5 18 5 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 18 Tyr Leu Tyr His Tyr 1 5 19 10 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 19 Cys Asp Gly Tyr Pro Lys Asp Cys Lys
Gly 1 5 10 20 10 PRT Artificial Sequence Description of Artificial
Sequence Cell adhesion modulation agent 20 Cys Asp Gly Tyr Pro Lys
Asp Cys Lys Gly 1 5 10 21 10 PRT Artificial Sequence Description of
Artificial Sequence Cell adhesion modulation agent 21 Cys Gly Asn
Leu Ser Thr Cys Met Leu Gly 1 5 10 22 10 PRT Artificial Sequence
Description of Artificial Sequence Cell adhesion modulation agent
22 Cys Gly Asn Leu Ser Thr Cys Met Leu Gly 1 5 10 23 9 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 23 Cys Tyr Ile Gln Asn Cys Pro Leu Gly 1
5 24 9 PRT Artificial Sequence Description of Artificial Sequence
Cell adhesion modulation agent 24 Cys Tyr Ile Gln Asn Cys Pro Leu
Gly 1 5 25 6 PRT Artificial Sequence Description of Artificial
Sequence Cell adhesion modulation agent 25 Cys Leu Tyr His Tyr Cys
1 5 26 6 PRT Artificial Sequence Description of Artificial Sequence
Cell adhesion modulation agent 26 Cys Leu Tyr His Tyr Cys 1 5 27 8
PRT Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 27 Cys Gln Tyr Leu Tyr His Tyr Cys 1 5 28
8 PRT Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 28 Cys Gln Tyr Leu Tyr His Tyr Cys 1 5 29
7 PRT Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 29 Cys Tyr Leu Tyr His Tyr Cys 1 5 30 7
PRT Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 30 Cys Tyr Leu Tyr His Tyr Cys 1 5 31 6
PRT Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 31 Cys Leu Tyr His Tyr Xaa 1 5 32 6 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 32 Xaa Leu Tyr His Tyr Cys 1 5 33 6 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 33 Xaa Leu Tyr His Tyr Cys 1 5 34 6 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 34 Xaa Leu Tyr His Tyr Cys 1 5 35 6 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 35 Xaa Leu Tyr His Tyr Cys 1 5 36 6 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 36 Lys Leu Tyr His Tyr Asp 1 5 37 8 PRT
Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 37 Lys Gln Tyr Leu Tyr His Tyr Asp 1 5 38
4 PRT Artificial Sequence Description of Artificial Sequence Cell
adhesion modulation agent 38 Trp Gly Gly Trp 1 39 10 PRT Artificial
Sequence Description of Artificial Sequence E-cadherin cell
adhesion recognition sequence 39 Leu Phe Ser His Ala Val Ser Ser
Asn Gly 1 5 10 40 7 PRT Artificial Sequence Description of
Artificial Sequence Cell adhesion modulating agent 40 Cys Tyr Leu
Tyr His Tyr Cys 1 5 41 8 PRT Artificial Sequence Description of
Artificial Sequence Cell adhesion modulating agent 41 Cys Gln Tyr
Leu Tyr His Tyr Cys 1 5 42 8 PRT Artificial Sequence Description of
Artificial Sequence Cell adhesion modulating agent 42 Lys Gln Tyr
Leu Tyr His Tyr Asp 1 5 43 5 PRT Artificial Sequence Description of
Artificial Sequence Cell adhesion modulating agent 43 Tyr Leu Tyr
His Tyr 1 5 44 6 PRT Artificial Sequence Description of Artificial
Sequence Cell adhesion modulating agent 44 Gln Tyr Leu Tyr His Tyr
1 5 45 6 PRT Artificial Sequence Description of Artificial Sequence
Cell adhesion modulating agent 45 Lys Leu Tyr His Tyr Asp 1 5 46 51
PRT Homo sapiens MOD_RES (8) Where Xaa is any amino acid residue 46
Gly Val Asn Pro Thr Ala Gln Xaa Gly Ala Ser Ser Gly Ser Leu Tyr 1 5
10 15 Xaa Ser Gln Ile Tyr Xaa Xaa Cys Asn Gln Phe Tyr Xaa Pro Xaa
Ala 20 25 30 Thr Gly Leu Tyr Xaa Asp Gln Tyr Leu Tyr His Tyr Cys
Val Val Asp 35 40 45 Pro Gln Glu 50 47 8 PRT Artificial Sequence
Description of Artificial Sequence Claudin cell adhesion
recognition sequence 47 Trp Xaa Xaa Xaa Xaa Xaa Xaa Gly 1 5 48 9
PRT Artificial Sequence Description of Artificial Sequence
Non-classical cell adhesion recognition sequence 48 Xaa Phe Xaa Xaa
Xaa Xaa Xaa Xaa Gly 1 5 49 4 PRT Artificial Sequence Description of
Artificial Sequence Representative claudin cell adhesion
recognition sequence 49 Ile Tyr Ser Tyr 1 50 4 PRT Artificial
Sequence Description of Artificial Sequence Representative claudin
cell adhesion recognition sequence 50 Thr Ser Ser Tyr 1 51 4 PRT
Artificial Sequence Description of Artificial Sequence
Representative claudin cell adhesion recognition sequence 51 Val
Thr Ala Phe 1 52 4 PRT Artificial Sequence Description of
Artificial Sequence Representative claudin cell adhesion
recognition sequence 52 Val Ser Ala Phe 1
* * * * *