U.S. patent application number 10/119537 was filed with the patent office on 2003-02-06 for compounds and methods for modulating junctional adhesion molecule-mediated functions.
This patent application is currently assigned to Adherex Technologies, Inc.. Invention is credited to Blaschuk, Orest W., Gour, Barbara J., Symonds, James Matthew.
Application Number | 20030027761 10/119537 |
Document ID | / |
Family ID | 23264040 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027761 |
Kind Code |
A1 |
Blaschuk, Orest W. ; et
al. |
February 6, 2003 |
Compounds and methods for modulating junctional adhesion
molecule-mediated functions
Abstract
Methods for using modulating agents to enhance or inhibit
junctional adhesion molecule (JAM)-mediated cell adhesion in a
variety of in vivo and in vitro contexts are provided. The
modulating agents comprise at least one JAM cell adhesion
recognition sequence or an antibody or fragment thereof that
specifically binds thee JAM 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) |
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: |
23264040 |
Appl. No.: |
10/119537 |
Filed: |
April 8, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10119537 |
Apr 8, 2002 |
|
|
|
09324541 |
Jun 2, 1999 |
|
|
|
6391855 |
|
|
|
|
Current U.S.
Class: |
435/6.14 ;
514/19.1; 514/9.3; 514/9.4; 530/324 |
Current CPC
Class: |
C07K 5/1024 20130101;
C07K 14/705 20130101; C07K 5/101 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/12 ;
530/324 |
International
Class: |
A61K 038/17; C07K
014/435 |
Claims
What is claimed is:
1. A cell adhesion modulating agent that: (a) comprises a JAM CAR
sequence; and (b) contains 3-16 amino acid residues linked by
peptide bonds.
2. A modulating agent that: (a) comprises at least five consecutive
amino acid residues of a JAM CAR sequence having the formula:
Ser--Phe--Thr--Ile--Asp--Pro--Lys--Ser--Gly (SEQ ID NO:1); and (b)
contains no more than 50 consecutive amino acid residues present
within the JAM.
3. A modulating agent that: (a) comprises at least seven
consecutive amino acid residues of a JAM CAR sequence having the
formula: Ser--Phe--Thr--Ile--Asp--Pro--Lys--Ser--Gly (SEQ ID NO:1);
and (b) contains no more than 50 consecutive amino acid residues
present within the JAM.
4. A modulating agent that: (a) comprises at least eight
consecutive amino acid residues of a JAM CAR sequence having the
formula; Ser--Phe--Thr--Ile--Asp--Pro--Lys--Ser--Gly (SEQ ID NO:1);
and (b) contains no more than 50 consecutive amino acid residues
present within the JAM.
5. A modulating agent according to claim 2, wherein the agent is a
peptide ranging in-size from 3 to 50 amino acid residues.
6. A modulating agent according-to claim 1 or claim 2, wherein the
agent is a peptide ranging in size from 4 to 16 amino acid
residues.
7. A modulating agent according to claim 1 or claim 2, wherein the
agent comprises an N-terminal acetyl group.
8. A modulating agent according to claim 1 or claim 2, wherein the
CAR sequence is present within a cyclic peptide.
9. A modulating agent according to claim 8, wherein the cyclic
peptide has the formula: 5wherein 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.
10. A modulating agent according to claim 9, wherein Y.sub.1 and
Y.sub.2 are covalently linked via a disulfide bond.
11. A modulating agent according to claim 10, wherein Y.sub.1 and
Y.sub.2 are each independently selected from the group consisting
of penicillamine, .beta.,.beta.-tetramethylene cysteine,
.beta.,.beta.-pentamethylene cysteine, .beta.-mercaptopropionic
acid, .beta.,.beta.-pentamethylene-.beta.-mercaptopropionic acid,
2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline.
12. A modulating agent according to claim 10, wherein Y.sub.1 and
Y.sub.2 are cysteine residues.
13. A modulating agent according to claim 9, wherein Y.sub.1 and
Y.sub.2 are covalently linked via an amide bond.
14. A modulating agent according to claim 13, wherein the amide
bond is formed between terminal functional groups.
15. A modulating agent according to claim 13, wherein the amide
bond is formed between amino acid residue side-chains.
16. A modulating agent according to claim 13, wherein the amide
bond is formed between one terminal functional group and one amino
acid residue side chain.
17. A modulating agent according to claim 13, wherein: (a) Y.sub.1
is lysine or ofnithine and Y.sub.2 is aspartate or glutamate; or
(b) Y.sub.2 is lysine or ornithine and Y.sub.1 is aspartate or
glutamate.
18. A modulating agent according to claim 9, wherein Y.sub.1 and
Y.sub.2 are covalently linked via a thioether bond.
19. A modulating agent according to claim 9, wherein Y.sub.1 and
Y.sub.2 are each tryptophan or a derivative thereof, such that the
covalent bond generates a .delta..sub.1.delta..sub.1-ditryptophan,
or a derivative thereof.
20. A polynucleotide encoding a modulating agent according to claim
1 or claim 2.
21. An expression vector comprising a polynucleotide according to
claim 20.
22. A host cell transformed or transfected with an expression
vector according to claim 21.
23. A modulating agent comprising an antibody or antigen-binding
fragment thereof that specifically binds to a JAM CAR sequence and
modulates a JAM-mediated function, wherein the JAM CAR sequence
comprises the sequence: Ser--Phe--Thr--Ile--Asp--Pro--Lys--Ser--Gly
(SEQ ID NO:1).
24. A modulating agent comprising a mimetic of a JAM CAR sequence
that comprises at least three consecutive amino acid residues of a
JAM CAR sequence having the formula:
Ser--Phe--Thr--Ile--Asp--Pro--Lys--Ser--Gly (SEQ ID NO:1); wherein
the mimetic is capable of modulating a JAM-mediated function.
25. A modulating agent comprising a mimetic of a JAM CAR sequence
that comprises at least five consecutive amino acid residues of a
JAM CAR sequence having the formula:
Ser--Phe--Thr--Ile--Asp--Pro--Lys--Ser--Gly (SEQ ID NO:1); wherein
the mimetic is capable of modulating a JAM-mediated function.
26. A modulating agent according to any one of claims 1, 2 or 23
linked to a drug.
27. A modulating agent according to any one of claims 1, 2 or 23
linked to a detectable marker.
28. A modulating agent according to any one of claims 1, 2 or 23
linked to a targeting agent.
29. A modulating agent according to any one of claims 1, 2 or 23
linked to a support material.
30. A modulating agent according to claim 29, wherein the support
material is a polymeric matrix.
31. A modulating agent according to claim 29, wherein the support
material is selected from the group consisting of plastic dishes,
plastic tubes, sutures, membranes, ultra thin films, bioreactors
and microparticles.
32. A cell adhesion modulating agent according to any one of claims
1, 2 or 23, further comprising one or more of: (a) a cell adhesion
recognition sequence that is bound by an adhesion molecule other
than a JAM, wherein the cell adhesion recognition sequence is
separated from any JAM CAR 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 a JAM.
33. A cell adhesion modulating agent according to claim 32, wherein
the adhesion molecule is selected from the group consisting of
integrins, cadherins, occludin, A claudins; members of the
immunoglobulin family such as N-CAM and PECAM; fibronectin, laminin
and other extracellular matrix proteins.
34. A pharmaceutical composition comprising a adhesion modulating
agent according to any one of claims 1, 2 or 23, in combination
with a pharmaceutically acceptable carrier.
35. A composition according to claim 34, further comprising a
drug.
36. A composition, according to claim 34, wherein the cell adhesion
modulating agent is present within a sustained-release
formulation.
37. A composition according to claim 34, further comprising one or
more of: (a) a peptide comprising a cell adhesion recognition
sequence that is bound by an adhesion molecule other than a JAM;
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 a JAM.
38. A composition according to claim 37, wherein the adhesion
molecule is selected from the group consisting of integrins,
cadherins, occludin, claudins, members of the immunoglobulin family
such as N-CAM, PECAM, fibronectin, laminin and other extracellular
matrix proteins.
39. A modulating agent according to claim 1 or claim 2, wherein the
agent comprises a linear peptide having the sequence SFTIDPKSG (SEQ
ID NO:1).
40. A method for modulating cell adhesion, comprising contacting a
JAM-expressing cell with a cell adhesion modulating agent according
to claim 1 or claim 2.
41. A method for increasing vasopermeability in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent according to claim 1 or claim 2, wherein the modulating agent
inhibits JAM-mediated cell adhesion.
42. A method for reducing unwanted cellular adhesion in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent according to claim 1 or claim 2, wherein the modulating agent
inhibits JAM-mediated cell adhesion.
43. A method for enhancing the delivery of a drug through the skin
of a mammal, comprising contacting epithelial cells of a mammal
with a cell adhesion modulating agent according to claim 1 or claim
2 and a drug, wherein the modulating agent inhibits JAM-mediated
cell adhesion, and wherein the step of contacting is performed
under conditions and for a time sufficient to allow passage of the
drug across the epithelial cells.
44. 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 according to claim 1 or claim 2 and a drug,
wherein the modulating agent inhibits JAM-mediated cell
adhesion.
45. A method for treating cancer in a mammal, comprising
administering to a mammal a cell adhesion modulating agent
according to claim 1 or claim 2, wherein the modulating agent
inhibits JAM-mediated cell adhesion.
46. A method for inhibiting angiogenesis in a mammal, comprising
administering to a mammal a cell adhesion modulating agent
according to claim 1 or claim 2, wherein the modulating agent
inhibits JAM-mediated cell adhesion.
47. A method for enhancing drug delivery to the central nervous
system of a mammal, comprising administering to a mammal a cell
adhesion modulating agent according to claim 1 or claim 2, wherein
the modulating agent inhibits JAM-mediated cell adhesion.
48. A method for enhancing wound healing in a mammal, comprising
contacting a wound in a mammal with a cell adhesion modulating
agent according to claim 1 or claim 2, wherein the modulating
agent, enhances JAM-mediated cell adhesion.
49. A method for enhancing adhesion of foreign tissue implanted
within a mammal, comprising contacting a site of implantation of
foreign tissue in a mammal with a cell adhesion modulating agent
according to claim 1 or claim 2, wherein the modulating agent
enhances JAM-mediated cell adhesion.
50. A method for inducing apoptosis in a JAM-expressing cell,
comprising contacting a JAM-expressing cell with a cell adhesion
modulating agent according to claim 1 or claim 2, wherein the
modulating agent inhibits JAM mediated cell adhesion.
51. A method for modulating monocyte traffic in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent according to claim 1 or claim 2, and thereby modulating
monocyte traffic in the mammal.
52. A method for detecting the presence of JAM-expressing cells in
a sample, comprising: (a) contacting a sample with a modulating
agent according to claim 23 under conditions and for a time
sufficient to allow formation of a modulating agent-JAM complex;
and (b) detecting the level of modulating agent-JAM complex, and
therefrom detecting the presence of JAM-expressing cells in the
sample.
53. A kit for detecting the presence of JAM-expressing cells in a
sample, comprising: (a) a modulating agent according to claim 23;
and (b) a detection reagent.
54. A kit for enhancing transdermal drug delivery, comprising: (a)
a skin patch; and (b) a cell adhesion modulating agent-according to
claim 1 or claim 2, wherein the modulating agent inhibits
JAM-mediated cell adhesion.
Description
TECHNICAL FIELD
[0001] The present invention relates generally to methods for
regulating processes mediated by junctional adhesion molecule
(JAM), and more particularly to the use of modulating agents
comprising a JAM 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
[0002] 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.
[0003] 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 (R G Landes Company,
Austin Tex.) and Lampugnani and Dejana, Curr. Opin. Cell Biol.
9:671-682, 1997.
[0004] 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 Dejana, Curr.
Opin. Cell Biol. 9:674-682, 1997).
[0005] The transmembrane component of tight junctions that has been
the most studied is occludin (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
believed to be directly involved in cell adhesion and the formation
of tight junctions (Furuse et al., J. Cell Sci. 109:429435, 1996;
Chen et al., J. Cell Biol. 138:891-899, 1997). A detailed
discussion of occludin structure and function is provided by
Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682, 1997.
[0006] More recently, junctional adhesion molecule (JAM) has been
identified as an immunoglobulin gene superfamily member that is a
component of tight junctions (Martin-Padura et al., J. Cell. Biol.
142:117-127, 1998). This protein is selectively concentrated at
intercellular junctions of endothelial and epithelial cells of
different origins, and has been shown to play a role in regulating
monocyte transmigration.
[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] Accordingly, there is a need in the art for compounds that
modulate cell adhesion and improve drug delivery across
permeability barriers without such disadvantages. The present
invention fulfills this need and further provides other related
advantages.
SUMMARY OF THE INVENTION
[0011] The present invention provides compounds and methods for
modulating JAM-mediated cell adhesion and the formation of
permeability barriers. Within certain aspects, the present
invention provides cell adhesion modulating agents that inhibit or
enhance JAM-mediated cell adhesion. Certain modulating agents are
4-16 amino acid peptides (which may be linear or cyclic) that
comprise the sequence Asp--Pro--Lys (DPK). Within other
embodiments, a modulating agent may (a) comprise at least five or
seven consecutive amino acid residues of a JAM CAR sequence having
the formula:
Ser--Phe--Thr--Ile--Asp--Pro--Lys--Ser--Gly (SEQ ID NO:1)
[0012] and (b) contain no more than 50 consecutive amino acid
residues present within a JAM.
[0013] Within certain embodiments, a modulating agent as described
above comprises a JAM CAR sequence that is present within a cyclic
peptide. The cyclic peptide may have the formula: 1
[0014] 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. In certain embodiments, Y. comprises an N-acetyl group
and/or Y.sub.2 comprises a C-terminal amide group. Y.sub.1 and
Y.sub.2 may be covalently linked via any suitable bond, including a
disulfide bond, an amide bond or a thioether bond.
[0015] The present invention further provides, within other
aspects, polynucleotides encoding a modulating agent as provided
above, expression vectors comprising such a polynucleotide, and
host cells transformed or transfected with such an expression
vector. Within further aspects, the present invention provides
modulating agents that comprise an antibody or antigen-binding
fragment thereof that specifically binds to a JAM CAR sequence as
described above and modulates a JAM-mediated function.
[0016] The present invention further provides modulating agents
comprising a mimetic of a JAM CAR sequence provided above, wherein
the mimetic is capable of modulating a JAM-mediated function.
[0017] Within other aspects, modulating agents as described above
may be linked to one or more of a drug, a detectable marker, a
targeting agent and/or a support material. Alternatively, or in
addition, modulating agents as described above may further comprise
one or more of: (a) a cell adhesion recognition sequence that is
bound by an adhesion molecule other than a JAM; 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 a JAM. Such adhesion molecules may be selected
from the group consisting of integrins, cadherins, occludin, N-CAM,
claudins, PE-CAM, desmogleins, desmocollins, fibronectin, laminin
and other extracellular matrix proteins.
[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 a JAM; 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 a JAM.
[0019] Within further aspects, methods are provided for modulating
cell adhesion, comprising contacting a JAM-expressing cell with a
cell adhesion modulating agent as described above.
[0020] Within one such aspect, the present invention provides
methods for increasing vasopermeability in a mammal, comprising
administering to a mammal a cell adhesion modulating agent as
provided above, wherein the modulating agent inhibits JAM-mediated
cell adhesion.
[0021] Within another aspect, methods are provided for reducing
unwanted cellular adhesion in a mammal, comprising administering to
a mammal a cell adhesion modulating agent as provided above,
wherein the modulating agent inhibits JAM-mediated cell
adhesion.
[0022] In yet another aspect, the present invention provides
methods for enhancing the delivery of a drug through the skin of a
mammal, comprising contacting epithelial cells of a mammal with a
cell adhesion modulating agent as provided above and a drug,
wherein the modulating agent inhibits JAM-mediated cell adhesion,
and wherein the step of contacting is performed under conditions
and for a time sufficient to allow passage of the drug across the
epithelial cells.
[0023] 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
JAM-mediated cell adhesion.
[0024] 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 JAM-mediated cell
adhesion.
[0025] The present invention further provides methods for
inhibiting angiogenesis in a mammal, comprising administering to a
mammal a cell adhesion modulating agent as provided above, wherein
the modulating agent inhibits JAM-mediated cell adhesion.
[0026] Within further aspects, the present invention provides
methods for enhancing drug delivery to the central nervous system
of a mammal, comprising administering to a mammal a cell adhesion
modulating agent as provided above, wherein the modulating agent
inhibits JAM-mediated cell adhesion.
[0027] The present invention further provides methods for enhancing
wound healing in a mammal, comprising contacting a wound in a
mammal with a cell adhesion modulating agent as provided above,
wherein the modulating agent enhances JAM-mediated cell
adhesion.
[0028] Within a related aspect, the present invention provides
methods for enhancing adhesion of foreign tissue implanted within a
mammal, comprising contacting a site of implantation of foreign
tissue in a mammal with a cell adhesion modulating agent as
provided above, wherein the modulating agent enhances JAM-mediated
cell adhesion.
[0029] The present invention further provides methods for inducing
apoptosis in a JAM-expressing cell, comprising contacting a
JAM-expressing cell with a cell adhesion modulating agent as
provided above, wherein the modulating agent inhibits JAM-mediated
cell adhesion.
[0030] The present invention further provides methods for
modulating monocyte traffic in a mammal, comprising administering
to a mammal a cell adhesion modulating agent as provided above.
[0031] The present invention further provides methods for
identifying an agent capable of modulating JAM-mediated cell
adhesion. One such method comprises the steps of (a) culturing
cells that express a JAM in the presence and absence of a candidate
agent, under conditions and for a time sufficient to allow cell
adhesion; and (b) visually evaluating the extent of cell adhesion
among the cells.
[0032] Within another embodiment, such methods may comprise the
steps of: (a) culturing normal rat kidney cells in the presence and
absence of a candidate agent, under conditions and for a time
sufficient to allow cell adhesion; and (b) comparing the level of
cell surface JAM and E-cadherin for cells cultured in the presence
of candidate agent to the level for cells cultured in the absence
of candidate agent.
[0033] Within a further embodiment, such methods may comprise the
steps of: (a) culturing human aortic endothelial cells in the
presence and absence of a candidate agent, under conditions and for
a time sufficient to allow cell adhesion; and (b) comparing the
level of cell surface JAM and N-cadherin for cells cultured in the
presence of candidate agent to the level for cells cultured in the
absence of candidate agent.
[0034] Within yet another embodiment, such methods comprise the
steps of: (a) contacting an antibody that binds to a modulating
agent comprising a JAM CAR sequence with a test compound; and (b)
detecting the level of antibody that binds to the test
compound.
[0035] The present invention further provides methods for detecting
the presence of JAM-expressing cells in a sample, comprising: (a)
contacting a sample with an antibody that binds to a JAM CAR
sequence under conditions and for a time sufficient to allow
formation of an antibody-JAM complex; and (b) detecting the level
of antibody-JAM complex, and therefrom detecting the presence of
JAM-expressing cells in the sample.
[0036] Within further aspects, the present invention provides kits
for detecting the presence of JAM-expressing cells in a sample,
comprising: (a) an antibody that binds to a modulating agent
comprising a JAM CAR sequence; and (b) a detection reagent.
[0037] The present invention further provides, within other
aspects, kits for enhancing transdermal drug delivery, comprising:
(a) a skin patch; and (b) a cell adhesion modulating agent, wherein
the modulating agent comprises a JAM CAR sequence, and wherein the
modulating agent inhibits JAM-mediated cell adhesion.
[0038] 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
[0039] FIG. 1 provides the amino acid sequence of the second
extracellular immunoglobulin-like domain of a representative mouse
JAM (SEQ ID NO:2). The JAM cell adhesion recognition region is
shown in bold.
[0040] FIG. 2 is a schematic diagram illustrating the structure of
JAM, a membrane glycoprotein, passing through the plasma membrane
(PM). It possesses two extracellular immunoglobulin-like repeats
(EC1, EC2), a transmembrane domain and a cytoplasmic region. The
JAM CAR sequence SFTIDPKSG (SEQ ID NO:1) is present in EC2, where
letters correspond to IUPAC amino acid symbols. `N` represents the
amino-terminus and `C` represents the carboxy-terminus.
DETAILED DESCRIPTION OF THE INVENTION
[0041] As noted above, the present invention provides cell adhesion
modulating agents comprising peptides that are capable of
modulating JAM-mediated processes, such as cell adhesion. The
present invention is based on the identification of a previously
unknown cell adhesion recognition (CAR) sequence present in JAM. A
modulating agent may generally comprise one or more JAM CAR
sequences (or analogues or mimetics thereof), with or without one
or more additional CAR sequences, as described below. Peptide CAR
sequences may be present within a linear or cyclic peptide.
Alternatively, or in addition, a modulating agent may comprise a
polynucleotide encoding a peptide comprising one or more JAM CAR
sequences and/or a modulating agent may comprise a substance (such
as an antibody or antigen-binding fragment thereof) that
specifically binds to a JAM CAR sequence.
[0042] In general, to modulate JAM-mediated cell adhesion, a
JAM-expressing cell is contacted with a cell adhesion modulating
agent (also referred to herein as a "modulating agent") either in
vivo or in vitro. JAM-expressing cells may be readily identified
using any of a variety of techniques well known in the art (such
as, for example, hybridization, PCR or immunohistochemical
techniques), and include endothelial and epithelial cells, as well
as cancer cells, such as carcinoma cells. Within certain aspects,
the methods provided herein inhibit a JAM-mediated function. Such
methods include, for example, methods for treating diseases or
other conditions characterized by undesirable cell adhesion or for
facilitating drug delivery to a specific tissue or tumor. Certain
methods may inhibit cell adhesion (e.g., cancer cell adhesion), as
well as cancer invasion and metastasis. Alternatively, a modulating
agent may, such as when linked to a matrix or to another modulating
agent via a linker, be used to enhance a JAM-mediated function,
such as cell adhesion. Such conjugates may be used, for example, to
facilitate wound healing or the adhesion of implants.
[0043] Cell Adhesion Modulating Agents
[0044] The term "cell adhesion modulating agent," as used herein,
refers to a molecule comprising at least one of the following
components:
[0045] (a) a linear or cyclic peptide sequence that is at least 50%
identical to a JAM CAR sequence (i.e., a JAM CAR sequence or an
analogue thereof that retains at least 50% identity);
[0046] (b) a mimetic (e.g., peptidomimetic or small molecule mimic)
of a JAM CAR sequence;
[0047] (c) a substance such as an antibody or antigen-binding
fragment thereof that specifically binds a JAM CAR sequence;
and/or
[0048] (d) a polynucleotide encoding a polypeptide that comprises a
JAM CAR sequence or analogue thereof
[0049] 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 regions may be
derived from a JAM (preferably an extracellular domain that
comprises a CAR sequence) and/or may be heterologous. Within
certain preferred embodiments, a modulating agent contains no more
than 85 consecutive amino acid residues, and preferably no more
than 50 consecutive amino acid residues, present within a JAM.
[0050] A modulating agent is further capable of modulating a
function mediated by a JAM. Such activity may generally be assessed
using, for example, representative assays provided herein. Certain
modulating agents inhibit an interaction between JAM molecules
and/or between a JAM and a different adhesion molecule. For
functions (e.g., cell adhesion) that are inhibited by a full
length, soluble JAM, such a modulating agent may inhibit the
function with an activity that is not substantially diminished
relative to the full length JAM (i.e., the modulating agent
inhibits the function at least as well as soluble JAM, when
contacted with cells that express the JAM). For example, a
modulating agent may be as effective as soluble JAM in preventing
and/or disrupting adhesion of JAM-expressing cells. Alternatively,
to enhance adhesion of JAM-expressing cells, a modulating agent may
comprise an antibody or antigen-binding fragment thereof and/or
multiple peptides or mimetics linked to a support material. Such
modulating agents may function as a biological glue to bind
JAM-expressing cells, and should result in a detectable enhancement
of cell adhesion (preferably an enhancement that is at least as
great as that observed for immobilized JAM or antibody directed
against the JAM).
[0051] The term "JAM," as used herein, refers to an integral
membrane protein that is selectively concentrated at intercellular
junctions of endothelial and epithelial cells. JAM appears to have
an extracellular domain containing two immunoglobulin-like loops, a
transmembrane domain and a cytoplasmic domain, and has a molecular
weight of approximately 36-43 kD. Mouse JAM has been reported by
Martin-Padura et al., J. Cell Biol. 142:117-127, 1998. JAMs from
other species may be identified based on similarity in sequence to
mouse JAM, and should generally display at least 50% sequence
identity to mouse JAM. Such JAMs may further display structural
similarity, such as similar numbers of immunoglobulin repeats,
based on the generic JAM structure provided in FIG. 2, and may also
display similar localization.
[0052] A JAM CAR sequence, as used herein, is an amino acid
sequence that is present in a naturally occurring JAM and that is
capable of detectably modulating a JAM-mediated function, such as
cell adhesion, as described herein. In other words, contacting a
JAM-expressing cell with a peptide comprising a CAR sequence
results in a detectable change in a JAM-mediated function using at
least one of the representative assays provided herein. CAR
sequences may be of any length, but generally comprise at least
three amino acid residues, preferably 4-16 amino acid residues, and
more preferably 5-8 amino acid residues. A peptide modulating agent
may comprise any number of amino acid residues, but preferred
agents comprise 3-50 residues, preferably 4-16 residues. Within
certain embodiments, a peptide modulating agent preferably
comprises an N-acetyl group (i.e., the amino group present on the
amino terminal residue of the peptide is acetylated). It has been
found, within the context of the present invention, that the
presence of such an acetyl group may enhance peptide modulating
activity for certain applications.
[0053] JAM CAR sequences are generally physically located within
the JAM molecule in or near the binding site of an adhesion
molecule (i.e., within 10 amino acids, and preferably within 5
amino acids). The location of a binding site may generally be
determined using well known techniques, such as evaluating the
ability of a portion of the JAM to bind to the same JAM or to
another molecule. Any standard binding assay may be employed for
such an evaluation. Recognition of a CAR sequence by the JAM or
other molecule results in a measurable effect on a JAM function,
such as cell adhesion. Peptides comprising a CAR sequence generally
inhibit such a function unless linked, as described herein, to form
an enhancer of adhesion molecule function. It has been found,
within the context of the present invention, that certain JAM CAR
sequences comprise the sequence:
Ser--Phe--Thr--Ile--Asp--Pro--Lys--Ser--Gly (SEQ ID NO:1).
[0054] Modulating agents provided herein may include at least a
portion of such a CAR sequence. Additional JAM CAR sequences may be
identified based on sequence homology to the JAM CAR sequence
provided herein, and based on the ability of a peptide comprising
such a sequence to modulate a JAM-mediated function within a
representative assay described herein. Within certain embodiments,
a modulating agent comprises at least three consecutive residues,
preferably at least five consecutive residues and more preferably
at least seven consecutive residues, of a JAM CAR sequence that
satisfies the above consensus sequence. Preferred modulating agents
comprise the sequence DPK.
[0055] Peptides comprising a greater number of consecutive residues
derived from a JAM CAR sequence may have a greater specificity for
that JAM. In addition, further flanking sequences may be included
to enhance specificity. Such flanking sequences may be identified
based on the sequences provided in FIG. 1, or based on published
sequences.
[0056] As noted above, certain preferred modulating agents comprise
a peptide (containing a JAM, CAR sequence or an analogue thereof)
in which at least one terminal amino acid residue is modified
(e.g., the N-terminal amino group is modified by, for example,
acetylation or alkoxybenzylation and/or an amide or ester is formed
at the C-terminus). It has been found, within the context of the
present invention, that the addition of at least one such group to
a linear or cyclic peptide modulating agent may improve the ability
of the agent to modulate a JAM-mediated function. Certain preferred
modulating agents contain modifications at the N- and C-terminal
residues, such as N--Ac--SFTIDPKSG--NH.sub.2 (SEQ ID NO:1)
[0057] As noted above, the present invention further contemplates
JAM 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 a JAM-mediated function may be
confirmed as described herein.
[0058] As noted above, modulating agents as described herein may
comprise an analogue or mimetic of a JAM CAR sequence. An analogue
generally retains at least 50% identity to a native JAM CAR
sequence, and modulates a JAM-mediated function as described
herein. Such analogues preferably contain at least three residues
of, and more preferably at least five residues of, a JAM 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 a JAM CAR sequence analogue is the ability
to modulate a JAM-mediated function, which may be evaluated using
the representative assays provided herein.
[0059] A mimetic is a non-peptidyl compound that is
conformationally similar to a JAM CAR sequence, such that it
modulates a JAM-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 a JAM CAR sequence. NMR is
widely used for structural analyses of both peptidyl and
non-peptidyl compounds. Nuclear Overhauser Enhancements (NOE's),
coupling constants and chemical shifts depend on the conformation
of a compound. NOE data provides the interproton distance between
protons through space and can be used to calculate the lowest
energy conformation for the JAM 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 JAM 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 a
JAM CAR sequence.
[0060] Modulating agents, or peptide portions thereof, may be
linear or cyclic peptides. The term "cyclic peptide," as used
herein, refers to a peptide or salt thereof that comprises (1) an
intramolecular covalent bond between two non-adjacent residues,
forming a peptide ring and (2) at least one JAM CAR sequence or an
analogue thereof present within the peptide ring. The
intramolecular bond may be a backbone to backbone, side-chain to
backbone or side-chain to side-chain bond (Le., 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. Any of the above JAM CAR
sequences, or an analogue or mimetic thereof, may be incorporated
into a cyclic peptide, with or without one or more other adhesion
molecule CAR sequences. Additional adhesion molecule CAR sequences
are described in greater detail below.
[0061] The size of a cyclic peptide ring generally ranges from 5 to
about 15 residues, preferably from 5 to 10 residues. Additional
residue(s) may be present on the N-terminal and/or C-terminal side
of a JAM CAR sequence, and may be derived from sequences that flank
a 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,
purification or other manipulation and/or residues having a
targeting or other function).
[0062] Within certain embodiments, a modulating agent may comprise
a cyclic peptide that contains a JAM CAR sequence as provided
herein (or a portion of such a CAR sequence). Certain cyclic
peptides have the formula: 2
[0063] Within this formula, 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; 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 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.
[0064] Cyclic peptides may comprise any of the above CAR
sequence(s). Such cyclic peptides may be used as modulating agents
without modification, or may be incorporated into a modulating
agent. Within the context of the present invention, underlined
sequences are cyclized using any suitable method, as described
herein.
[0065] Within certain embodiments, as discussed below, cyclic
peptides that contain small CAR sequences (e.g., five residues,
such as CDPKC (SEQ ID NO:3), without significant flanking
sequences) are preferred for modulating JAM-mediated functions.
Such peptides may contain an N-acetyl group and a C-amide group.
Small cyclic peptides may generally be used to specifically
modulate adhesion of cancer and/or other cell types by topical
administration or by systemic administration, with or without
linking a targeting agent to the peptide, as discussed below.
Within other preferred embodiments, a cyclic peptide may contain
sequences that flank the JAM CAR sequence on one or both sides,
which may result in increased potency and/or specificity. Suitable
flanking sequences include, but are not limited to, an endogenous
sequence present in a naturally occurring JAM. 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.
[0066] Within embodiments in which inhibition of a JAM interaction
is desired, a modulating agent may contain one JAM CAR sequence, or
multiple CAR sequences that are 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 JAM CAR sequences that ranges from about 0.1 to 400
nm). For example, a modulating agent with adjacent DPK sequences
may comprise the peptide DPKDPK (SEQ ID NO:4). A linker may be any
molecule (including peptide and/or non-peptide sequences) that does
not contain a CAR sequence and that can be covalently linked to at
least two peptide sequences. Using a linker, CAR
sequence-containing peptides and other peptide or protein sequences
may be joined end-to-end (i.e., the linker may be covalently
attached to the carboxyl or amino group of each peptide sequence),
and/or via side chains. One linker that can be used for such
purposes is (H.sub.2N(CH.sub.2).sub.nCO.sub.2H), or derivatives
thereof, where n ranges from 1 to 4. Other linkers that may be used
will be apparent to those of ordinary skill in the art. Peptide and
non-peptide linkers may generally be incorporated into a modulating
agent using any appropriate method known in the art.
[0067] Within embodiments in which enhancement of cell adhesion
mediated by a JAM is desired, a modulating agent may contain
multiple JAM CAR sequences, or antibodies that specifically bind to
such sequences, joined by linkers as described above. For enhancers
of JAM function, the linker distance should generally 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.2-NCH.sub.2CO.sub.2H) or a multimer
thereof is used as a linker, each glycine unit corresponds to a
linking distance of 2.45 angstroms, or 0.245 nm, as determined by
calculation of its lowest energy conformation when linked to other
amino acids using molecular modeling techniques. Similarly,
aminopropanoic acid corresponds to a linking distance of 3.73
angstroms, aminobutanoic acid to 4.96 angstroms, aminopentanoic
acid to 6.30 angstroms and amino hexanoic acid to 6.12 angstroms.
Enhancement of cell adhesion may also be achieved by attachment of
multiple modulating agents to a support material, as discussed
further below.
[0068] 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 JAM CAR 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. For cyclic
peptides, additional CAR sequences may be present within the cyclic
peptide ring, 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.
[0069] As used herein, an "adhesion molecule" is any molecule that
mediates cell adhesion via a receptor on the cell's surface.
Adhesion molecules include cell adhesion proteins (e.g., classical
cadherins, other members of the cadherin gene superfamily that are
not classical cadherins (such as a typical cadherins (e.g.,
VE-cadherin and PB-cadherin), desmogleins (Dsg) and desmocollins
(Dsc)); integrins; occludin; claudins; and members of the
immunoglobulin supergene family, such as N-CAM and PECAM).
Preferred CAR sequences for inclusion within a modulating agent
include His--Ala--Val (HAV), which is bound by classical cadherins
(Munro S B et al., 1996, In: Cell Adhesion and Invasion in Cancer
Metastasis, P. Brodt, ed., pp.17-34 (R G Landes Company, Austin
Tex.); Arg--Gly--Asp (RGD), which is bound by integrins (see
Cardarelli et al., J. Biol. Chem. 267:23159-64, 1992); KYSFNYDGSE
(SEQ ID NO:5), which is bound by N-CAM; the occludin CAR sequence
LYHY (SEQ ID NO:6); and/or one or more nonclassical cadherin CAR
sequences, such as the VE-cadherin CAR sequence DAE, the Dsc CAR
sequences IEK, VER and IER, the Dsg CAR sequences INQ, INR and LNK;
and the claudin CAR sequence IYSY (SEQ ID NO:7).
[0070] Using linkers, JAM CAR sequence-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 may
comprise a JAM CAR sequence and an occludin CAR sequence.
Alternatively, a branched structure may comprise three different
CAR sequences, such as RGD, a JAM CAR sequence and HAV. Within
another embodiment, modulating agents having a branched structure
comprise a JAM CAR sequence, along with one or more of HAV, RGD,
LYHY (SEQ ID NO:6), DAE, IEK, VER, IER, INQ, INR, IYSY (SEQ ID
NO:7) and/or LNK.
[0071] Other combinations of CAR sequences are also possible.
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, as
discussed above.
[0072] The total number of CAR sequences (including JAM 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 6 to about
1000 amino acid residues, preferably from 6 to 50 residues. When
non-peptide linkers are employed, each CAR sequence of the
modulating agent is present within a peptide that generally ranges
in size from 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.
[0073] As noted above, modulating agents may be polypeptides or
salts thereof, containing only amino acid residues linked by
peptide bonds, or may contain non-peptide regions, such as linkers.
Peptide regions of a modulating agent may comprise residues of
L-amino acids, D-amino acids, or any combination thereof. Amino
acids may be from natural or non-natural sources, provided that at
least one amino group and at least one carboxyl group are present
in the molecule; .alpha.- and .beta.-amino acids are generally
preferred. The 20 L-amino acids commonly found in proteins are
identified herein by the conventional three-letter or one-letter
abbreviations, and the corresponding D-amino acids are designated
by a lower case one letter symbol.
[0074] 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.
[0075] 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
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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] Acetylation of the N-terminus can be accomplished by
reacting the final peptide with acetic anhydride before cleavage
from the resin. C-amidation is accomplished using an appropriate
resin such as methylbenzhydrylamine resin using the Boc
technology.
[0081] Following synthesis of a linear peptide, with or without
N-acetylation and/or C-amidation, cyclization may be achieved if
desired by any of a variety of techniques well known in the art.
Within one embodiment, a bond may be generated between reactive
amino acid side chains. For example, a disulfide bridge may be
formed from a linear peptide comprising two thiol-containing
residues by oxidizing the peptide using any of a variety of
methods. Within one such method, air oxidation of thiols can
generate disulfide linkages over a period of several days using
either basic or neutral aqueous media. The peptide is used in high
dilution to minimize aggregation and intermolecular side reactions.
This method suffers from the disadvantage of being slow but has the
advantage of only producing H.sub.2O as a side product.
Alternatively, strong oxidizing agents such as I.sub.2 and
K.sub.3Fe(CN).sub.6 can be used to form disulfide linkages. Those
of ordinary skill in the art will recognize that care must be taken
not to oxidize the sensitive side chains of Met, Tyr, Trp or His.
Cyclic peptides produced by this method require purification using
standard techniques, but this oxidation is applicable at acid pHs.
Oxidizing agents also allow concurrent deprotection/oxidation of
suitable S-protected linear precursors to avoid premature,
nonspecific oxidation of free cysteine.
[0082] DMSO, unlike I.sub.2 and K.sub.3Fe(CN).sub.6, is a mild
oxidizing agent which does not cause oxidative side reactions of
the nucleophilic amino acids mentioned above. DMSO is miscible with
H.sub.2O at all concentrations, and oxidations can be performed at
acidic to neutral pHs with harmless byproducts.
Methyltrichlorosilane-diphenylsulfoxide may alternatively be used
as an oxidizing agent, for concurrent deprotection/oxidation of
S--Acm, S--Tacm or S--t--Bu of cysteine without affecting other
nucleophilic amino acids. There are no polymeric products resulting
from intermolecular disulfide bond formation. Suitable
thiol-containing residues for use in such oxidation methods
include, but are not limited to, cysteine, .beta.,.beta.-dimethyl
cysteine (penicillamine or Pen), .beta.,.beta.-tetramethylene
cysteine (Tmc), .beta.,.beta.-pentamethylene cysteine (Pmc),
.beta.-mercaptopropionic acid (Mpr),
.beta.,.beta.-pentamethylene-.beta.-mercaptopropionic acid (Pmp),
2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline.
[0083] Within another embodiment, cyclization may be achieved by
amide bond formation. For example, a peptide bond may be formed
between terminal functional groups (i.e., the amino and carboxy
termini of a linear peptide prior to cyclization). Within another
such embodiment, the cyclic peptide comprises a D-amino acid.
Alternatively, cyclization may be accomplished by linking one
terminus and a residue side chain or using two side chains, with or
without an N-terminal acetyl group and/or a C-terminal amide.
Residues capable of forming a lactam bond include lysine, ornithine
(Orn), .alpha.-amino adipic acid, m-aminomethylbenzoic acid,
.alpha.,.beta.-diaminopropionic acid, glutamate or aspartate.
[0084] 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.
[0085] 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: 3
[0086] Cyclization may also be achieved using
.delta..sub.1,.delta..sub.1-- Ditryptophan (i.e.,
Ac--TEp--Gly--Gly--Trp--OMe) (SEQ ID NO:8), as shown below: 4
[0087] 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 JAM or other adhesion
molecule. Such sequences may be prepared based on known cDNA or
genomic sequences, or from sequences isolated by screening an
appropriate library with probes designed based on known JAM
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
JAM 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.
[0088] As noted above, polynucleotides may also function as
modulating agents. In general, such polynucleotides should be
formulated to permit expression of a polypeptide modulating agent
following administration to a mammal. Such formulations are
particularly useful for therapeutic purposes, as described below.
Those of ordinary skill in the art will appreciate that there are
many ways to achieve expression of a polynucleotide within a
mammal, and any suitable method may be employed. For example, a
polynucleotide may be incorporated into a viral vector such as, but
not limited to, adenovirus, adeno-associated virus, retrovirus, or
vaccinia or other pox virus (e.g., avian pox virus). Techniques for
incorporating DNA into such vectors are well known to those of
ordinary skill in the art. A retroviral vector may additionally
transfer or incorporate a gene for a selectable marker (to aid in
the identification or selection of transfected cells) and/or a
targeting moiety, such as a gene that encodes a ligand for a
receptor on a specific target cell, to render the vector target
specific. Targeting may also be accomplished using an antibody, by
methods known to those of ordinary skill in the art. Other
formulations for polynucleotides for therapeutic purposes include
colloidal dispersion systems, such as macromolecule complexes,
nanocapsules; microspheres, beads, and lipid-based systems
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes. A preferred colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (i.e., an artificial
membrane vesicle). The preparation and use of such systems is well
known in the art.
[0089] As noted above, instead of (or in addition to) a JAM CAR
sequence, a modulating agent may comprise an antibody, or
antigen-binding fragment thereof, that specifically binds to a JAM
CAR sequence. As used herein, an antibody, or antigen-binding
fragment thereof, is said to "specifically bind" to a JAM 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 JAM 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.
[0090] Polyclonal and monoclonal antibodies may be raised against a
JAM 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 JAM 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.
[0091] Monoclonal antibodies specific for the JAM 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.
[0092] 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 JAM is localized.
[0093] 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).
[0094] Evaluation of Modulating Agent Activity
[0095] As noted above, modulating agents as described herein are
capable of modulating JAM-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 or cells transfected with JAM cDNA, such as L cells
(Furuse et al. J Cell Biol. 143:391-401, 1998), 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 JAM CAR
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.
[0096] An initial screen for the ability to modulate one or more
JAM-mediated functions may be performed by evaluating the ability
of a modulating agent to bind to a JAM using any binding assay
known to those of ordinary skill in the art. For example, a
Pharmacia Biosensor machine may be used, as discussed in Jonsson et
al., Biotechniques 11:520-27, 1991. For example, a modulating agent
may comprise a CAR sequence that binds to a JAM. A specific example
of a technology that measures the interaction of peptides with
molecules can be found in Williams et al., J. Biol. Chem. 272,
22349-22354, 1997. Alternatively, real-time BIA (Biomolecular
Interaction Analysis) uses the optical phenomenon surface plasmon
resonance to monitor biomolecular interactions. The detection
depends upon changes in the mass concentration of macromolecules at
the biospecific interface, which in turn depends upon the
immobilization of test molecule or peptide (referred to as the
ligand) to the surface of a Biosensor chip, followed by binding of
the interacting molecule (referred to as the analyte) to the
ligand. Binding to the chip is measured in real-time in arbitrary
units of resonance (RU).
[0097] By way of example, surface plasmon resonance experiments may
be carried out using a BIAcore X.TM. Biosensor (Pharmacia Ltd.,
BIAcore, Uppsala, Sweden). Parallel flow cells of CM 5 sensor chips
may be derivatized, using the amine coupling method, with
streptavidin (200 .mu.g/ml) in 10 mM Sodium Acetate, pH 4.0,
according to the manufacturer's protocol. Approximately 2100-2600
resonance units (RU) of ligand may be immobilized, corresponding to
a concentration of about 2.1-2.6 ng/mm.sup.2. The chips may then
coated be with JAM derivatized to biotin. Any non-specifically
bound protein is removed.
[0098] To determine binding, test analytes (e.g., peptides
containing the JAM CAR sequence) may be placed in running buffer
and passed simultaneously over test and control flow cells. After a
period of free buffer flow, any analyte remaining bound to the
surface may be removed with, for example, a pulse of 0.1% SDS
bringing the signal back to baseline. Specific binding to the
derivatized sensor chips may be determined automatically by the
system by subtraction of test from control flow cell responses. In
general, a modulating agent binds to a JAM at a detectable level
within such as assay. The level of binding is preferably at least
that observed for the full length JAM under similar conditions.
[0099] 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.
[0100] 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 anti-JAM
antibodies (prepared as described by Martin-Padura et al., J. Cell.
Biol. 142:117-127, 1998) diluted 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-rat 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 JAM and VE-cadherin, as compared to monolayer
cultures that were not exposed to the modulating agent.
[0101] Another such assay evaluates leukocyte (e.g., monocyte)
transmigration through an endothelial cell layer (see Martin-Padura
et al., J. Cell. Biol. 142:117-127, 1998). Human umbilical vein
endothelial cells for use in such assays may be cultured using
standard techniques, such as in 24 well format 8 mm inserts
(Falcon). A fresh 24 well plate may be filled with 1 mL HBSS (Hanks
balanced salts solution) in each of the lower wells. In all wells,
a chemoattractant is then added (e.g., IL-8 added to a final
concentration of 1 .mu.M). Medium is removed from the upper well
and HBSS plus modulating agent are added to the upper wells of the
insert plates (e.g., at 200 .mu.g/ml) and pre-incubated (e.g., for
1 hour). Leukocytes may be labeled by, for example, incubation with
30.mu.l of .sup.51Cr (5 mCi/5 ml, NEN, Natick, Mass.) for 1 hour at
37.degree. C., with 3 washing steps with HBSS. The concentration of
the leukocyte suspension may be adjusted to about 2.times.10.sup.7
cells/mL before starting the assay. This leukocyte suspension
(e.g., 50 mL) is added to the upper well and the inserts incubated
in the new chemoattractant supplemented tray for 60 minutes. The
inserts may then be removed and discarded. The contents from each
well are aspirated and placed in a separate gamma counter tube.
Wells may be washed twice with 1N NaOH, and the wash added to the
same tube. Samples may be read by gamma counting for .sup.51Cr and
may be expressed as a percentage of the total leukocytes added to
each upper well chamber. In general, 200 .mu.g/ml modulating agent
should result in an increase in transmigration by at least 50%.
[0102] Within certain cell adhesion assays, the addition of a
modulating agent to cells that express JAM results in disruption of
cell adhesion. A "JAM-expressing cell," as used herein, may be any
type of cell that expresses JAM 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). JAM-expressing cells include endothelial and epithelial
cells, as well as megakaryocytes and mesothelial 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.
[0103] 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
anti-JAM antibody 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 1hour at 37.degree. C. Following further washes
in PBS (3.times.5 min) coverslips can be mounted and viewed by
confocal microscopy.
[0104] 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 JAM-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 JAM and E-cadherin, as judged by immunofluorescence
microscopy (Laird al., J. Cell Biol. 131:1193-1203, 1995),
preferably at least 75% within 48 hours.
[0105] 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 24chamber 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.
[0106] Alternatively, cells that do not naturally express a JAM may
be used within such assays. Such cells may be stably transfected
with a polynucleotide (e.g., a cDNA) encoding a JAM of interest,
such that the JAM is expressed on the surface of the cell.
Transfection of cells for use in cell adhesion assays may be
performed using standard techniques and published JAM sequences.
Expression of the JAM may be confirmed by assessing adhesion of the
transfected cells, in conjunction with immunocytochemical
techniques using antibodies directed against the JAM of interest,
The stably transfected cells that aggregate, as judged by light
microscopy, following transfection express sufficient levels of the
JAM. Preferred cells for use in such assays include L cells, which
do not detectably adhere in the absence of transfection (Nagafuchi
et al., Nature 329:341-343, 1987). Following transfection of L
cells with a cDNA encoding a JAM, cell adhesion may be observed
(Furuse et al J Cell Biol. 143:391-401, 1998). Modulating agents
that detectably inhibit such aggregation may be used to modulate
functions mediated by the JAM. Such assays have been used for
numerous nonclassical cadherins including OB-cadherin (Okazaki et
al., J. Biol. Chem. 269:12092-98, 1994), cadherin-5 (Breier et al.,
Blood 87:630-641, 1996), cadherin-6 (Mbalaviele et al., J. Cell.
Biol. 141:1467-1476, 1998), cadherin-8 (Kido et al., Genomics
48:186-194, 1998), cadherin-15 (Shimoyama et al., J. Biol. Chem.
273:10011-10018, 1998), PB-cadherin (Sugimoto et al., J. Biol.
Chem. 271:11548-11556, 1996), LI-cadherin (Kreft et al., J. Cell.
Biol. 136:1109-1121, 1997), protocadherin 42 and 43 (Sano et al.,
EMBO J. 12:2249-2256, 1993) and desmosomal cadherins (Marcozzi et
al., J. Cell. Sci. 111:495-509, 1998). It will be apparent to those
of ordinary skill in the art that assays may be performed in a
similar manner for JAMs. In general, a modulating agent that is
derived from a particular JAM CAR sequence (i.e., comprises such a
CAR sequence, or an analog or mimetic thereof, or an antibody that
specifically recognizes such a CAR sequence) and that modulates
adhesion of a cell that expresses the same JAM is considered to
modulate a function mediated by the JAM.
[0107] Yet another assay evaluates the effect of a 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 decrease in electrical resistance after 24 hours.
[0108] Modulating Agent Modification and Formulations
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
For example, in receptor-mediated delivery, a modulating agent may
be linked to a ligand that recognizes a specific receptor on the
surface of a target cell. In certain instances, modulating agent is
released within the cell following cleavage with intracellular
enzymes. Among the many monoclonal antibodies that may serve as
targeting agents are anti-TAC, or other interleukin-2 receptor
antibodies; 9.2.27 and NR-ML-05, reactive with the 250 kilodalton
human melanoma-associated proteoglycan; and NR-LU-10, reactive with
a pancarcinoma glycoprotein. An antibody targeting agent may be an
intact (whole) molecule, a fragment thereof, or a functional
equivalent thereof Examples of antibody fragments are F(ab')2,
-Fab', Fab and F[v] fragments, which may be produced by
conventional methods or by genetic or protein engineering. Linkage
is generally covalent and may be achieved by, for example, direct
condensation or other reactions, or by way of bi- or
multi-functional linkers.
[0113] 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.
[0114] 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.
[0115] 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 JAM.
Such modulators may generally be prepared as described above,
except that one or more non-JAM CAR sequences and/or antibodies
thereto are substituted for the JAM CAR sequence. Such compositions
are particularly useful for situations in which it is desirable to
inhibit cell adhesion mediated by multiple cell adhesion molecules,
such as other members of the cadherin gene superfamily (e.g.,
classical cadherins such as E-cadherin and/or nonclassical
cadherins such as VE-cadherin, Dsg and Dsc); integrins; claudins;
and members of the immunoglobulin supergene family, such as N-CAM
and PECAM. Preferred CAR sequences for use within such a modulator
include HAV, RGD, DDK, EEY, EAQ (OB-cads), DAE (VE-cad), IEK, VER,
IER, INQ, INR and/or LNK. Also preferred is the occludin CAR
sequence LYHY (SEQ ID NO:6) and the claudin CAR sequence IYSY (SEQ
ID NO:7).
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] Modulating Agent Methods of Use
[0121] In general, the modulating agents and compositions described
herein may be used for modulating the adhesion of JAM-expressing
cells in vitro and/or in vivo, preferably in a mammal such as a
human, by contacting the JAM-expressing cell with the modulating
agent. As noted above, modulating agents for purposes that involve
the disruption of JAM-mediated cell adhesion may comprise a JAM CAR
sequence, multiple JAM CAR sequences in close proximity and/or an
antibody (or an antigen-binding fragment thereof) that recognizes
the JAM 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 JAM 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 JAM 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. When it is desirable to also enhance 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 JAM CAR sequence by linker.
[0122] 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 JAM-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.
[0123] Within certain aspects, the present invention provides
methods for modulating monocyte traffic through endothelial cells.
Such cells may, but need not, be within a mammal. In general, a
modulating agent that disrupts JAM-mediated cell adhesion increases
monocyte traffic. In one particularly preferred embodiment, a
modulating agent for use within such methods is capable of
disrupting cell adhesion mediated by multiple adhesion molecules.
Alternatively, a separate modulator of non-JAM-mediated cell
adhesion may be administered in conjunction with the modulating
agent(s), either within the same pharmaceutical composition or
separately.
[0124] The present invention provides, within certain aspects,
methods for increasing vasopermeability in a mammal by
administering one or more modulating agents or pharmaceutical
compositions. Endothelial cell adhesion may be disrupted by linear
and cyclic peptides containing a JAM CAR sequence. Within blood
vessels, endothelial cell adhesion results in decreased vascular
permeability. Accordingly, modulating agents that disrupt
JAM-mediated cell adhesion as described herein, can increase
vascular permeability and thus may facilitate drug delivery to
previously inaccessible tissues, such as the brain. In one
particularly preferred embodiment, a modulating agent for use
within such methods 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 JAM, occludin, claudin and cadherin
mediated cell adhesion, thereby disrupting tight junctions and
adherens junctions. Multi-functional modulating agents comprising a
JAM CAR sequence joined to the cadherin CAR sequence HAV, the
VE-cadherin CAR sequence DAE, the claudin CAR sequence IYSY (SEQ ID
NO:7) and the occludin CAR sequence LYHY (SEQ ID NO:6), preferably
by a linker, are also preferred. Alternatively, a separate
modulator of non-JAM-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 JAM
modulating agents include Fab fragments directed against an
N-cadherin CAR sequence such as FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID
NO:9), a VE-cadherin CAR sequence such as VFRVDAETG-NH.sub.2 (SEQ
ID NO:10), a claudin CAR sequence such as WKIYSYAG-NH.sub.2 (SEQ ID
NO:11), an occludin CAR sequence such as QYLYHYCVVD-NH.sub.2 (SEQ
ID NO:12) or an OB-cadherin CAR sequence such as IFVIDDKSG-NH.sub.2
(SEQ ID NO:13).
[0125] Within certain embodiments, preferred modulating agents for
use within such methods include peptides capable of decreasing both
endothelial and tumor cell adhesion. Such modulating agents may be
used to facilitate the penetration of anti-tumor therapeutic or
diagnostic agents (e.g., monoclonal antibodies) through endothelial
cell permeability barriers and tumor barriers. 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 JAM, occludin,
claudin, classical cadherin, integrin, and nonclassical cadherin
(e.g., Dsc and/or Dsg) mediated cell adhesion, thereby disrupting
tight junctions, adherens junctions, focal contacts and desmosomes
Multifunctional modulating agents comprising a JAM CAR sequence
linked to one or more of the classical cadherin CAR sequence HAV;
the sequence RGD, which is bound by integrins; the sequence LYHY
(SEQ ID NO:6), which is bound by occludin, the sequence IYSY (SEQ
ID NO:7) which is bound by claudins and/or a nonclassical cadherin
CAR sequence, such as a VE-cadherin CAR sequence DAE, a Dsc CAR
sequence (IEK, VER or IER) and/or a Dsg CAR sequence (INQ, INR or
LNK), may be used to disrupt cell adhesion. Alternatively, a
separate modulator of non-JAM-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
JAM modulating agents include Fab fragments directed against either
an N-cadherin CAR sequence such as FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID
NO:9), an E-cadherin CAR sequence such as LFSHAVSSNG-NH.sub.2 (SEQ
ID NO:14). an occludin CAR sequence such as QYLYHYCVVD-NH.sub.2
(SEQ ID NO:12), a VE-cadherin CAR sequence such as
VFRVDAETG-NH.sub.2 (SEQ ID NO:10), a claudin CAR sequence such as
WKIYSYAG-NH.sub.2 (SEQ ID NO:11) or an OB-cadherin CAR sequence,
such as IFVIDDKSG-NH.sub.2 (SEQ ID NO:13).
[0126] Treatment with a modulating agent may be appropriate, for
example, prior to administration of an anti-tumor therapeutic or
diagnostic agent (e.g., a monoclonal antibody or other
macromolecule), an antimicrobial agent or an anti-inflammatory
agent, in order to increase the concentration of such agents in the
vicinity of the target tumor, organism or inflammation without
increasing the overall dose to the patient. Modulating agents for
use within such methods may be linked to a targeting agent to
further increase the local concentration of modulating agent,
although systemic administration of a vasoactive agent even in the
absence of a targeting agent increases the perfusion of certain
tumors relative to other tissues. Suitable targeting agents include
antibodies and other molecules that specifically bind to tumor
cells or to components of structurally abnormal blood vessels. For
example, a targeting agent may be an antibody that binds to a
fibrin degradation product or a cell enzyme such as a peroxidase
that is released by granulocytes or other cells in necrotic or
inflamed tissues.
[0127] 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.
[0128] Within certain aspects, methods are provided in which cell
adhesion is diminished. In one such aspect, the present invention
provides methods for reducing unwanted cellular adhesion in a
mammal by administering a modulating agent as described herein.
Unwanted cellular adhesion can occur, for example, 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. Certain
preferred modulating agents for use within such methods comprise
one or more of the JAM CAR sequences provided herein. In one
particularly preferred embodiment, a modulating agent is further
capable of disrupting cell adhesion mediated by multiple adhesion
molecules. Such an agent may comprise, in addition to one or more
JAM CAR sequences, CAR sequences such as the classical cadherin CAR
sequence HAV sequence, an RGD sequence, which is bound by
integrins; an IYSY (SEQ ID NO:7) sequence which is bound by
claudins and/or the occludin CAR sequence LYHY (SEQ ID NO:6),
preferably separated from the JAM CAR sequence via a linker.
Alternatively, separate modulators of cell adhesion mediated by
other adhesion molecules may be administered in conjunction with
the modulating agent(s), either within the same pharmaceutical
composition or separately.
[0129] 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.
[0130] Within another such aspect, methods are provided for
enhancing the delivery of a drug through the skin of a mammal.
Transdermal delivery of drugs is a convenient and non-invasive
method that can be used to maintain relatively constant blood
levels of a drug. In general, to facilitate drug delivery via the
skin, it is necessary to perturb adhesion between the epithelial
cells (keratinocytes) and the endothelial cells of the
microvasculature. Using currently available techniques, only small,
uncharged molecules may be delivered across skin in vivo. The
methods described herein are not subject to the same degree of
limitation. Accordingly, a wide variety of drugs may be transported
across the epithelial and endothelial cell layers of skin, for
systemic or topical administration. Such drugs may be delivered to
melanomas or may enter the blood stream of the mammal for delivery
to other sites within the body.
[0131] To enhance the delivery of a drug through the skin, a
modulating agent as described herein and a drug are contacted with
the skin surface. Within certain embodiments, multifunctional
modulating agents comprising a JAM CAR sequence linked to one or
more of the classical cadherin CAR sequence HAV; the sequence RGD
which is bound by integrins; the sequence IYSY (SEQ ID NO:7) which
is bound by claudins; the sequence LYHY (SEQ ID NO:6) which is
bound by occludin and/or a nonclassical cadherin CAR sequence, such
as a VE-cadherin CAR sequence DAE, a Dsc CAR sequence (EEK, VER or
IER) and/or a Dsg CAR sequence (INQ, INR or LNK), may also be used
to disrupt cell adhesion. Alternatively, a separate modulator of
non-JAM-mediated cell adhesion may be administered in conjunction
with the modulating agent(s), either within the same pharmaceutical
composition or separately. Contact may be achieved by direct
application of the modulating agent, generally within a composition
formulated as a cream or gel, or using any of a variety of skin
contact devices for transdermal application (such as those
described in European Patent Application No. 566,816 A; U.S. Pat.
Nos. 5,613,958; 5,505,956). A skin patch provides a convenient
method of administration (particularly for slow-release
formulations). Such patches may contain a reservoir of modulating
agent and drug separated from the skin by a membrane through which
the drug diffuses. Within other patch designs, the modulating agent
and drug may be dissolved or suspended in a polymer or adhesive
matrix that is then placed in direct contact with the patient's
skin. The modulating agent and drug may then diffuse from the
matrix into the skin. Modulating agent(s) and drug(s) may be
contained within the same composition or skin patch, or may be
separately administered, although administration at the same time
and site is preferred. In general, the amount of modulating agent
administered via the skin varies with the nature of the condition
to be treated or prevented, but may vary as described above. Such
levels may be achieved by appropriate adjustments to the device
used, or by applying a cream formulated as described above.
Transfer of the drug across the skin and to the target tissue may
be predicted based on in vitro studies using, for example, a Franz
cell apparatus, and evaluated in vivo by appropriate means that
will be apparent to those of ordinary skill in the art. As an
example, monitoring of the serum level of the administered drug
over time provides an easy measure of the drug transfer across the
skin.
[0132] Transdermal drug delivery as described herein is
particularly useful in situations in which a constant rate of drug
delivery is desired, to avoid fluctuating blood levels of a drug.
For example, morphine is an analgesic commonly used immediately
following surgery. When given intermittently in a parenteral form
(intramuscular, intravenous), the patient usually feels sleepy
during the first hour, is well during the next 2 hours and is in
pain during the last hour because the blood level goes up quickly
after the injection and goes down below the desirable level before
the 4 hour interval prescribed for re-injection is reached.
Transdermal administration as described herein permits the
maintenance of constant levels for long periods of time (e.g.,
days), which allows adequate pain control and mental alertness at
the same time. Insulin provides another such example. Many diabetic
patients need to maintain a constant baseline level of insulin
which is different from their needs at the time of meals. The
baseline level may be maintained using transdermal administration
of insulin, as described herein. Antibiotics may also be
administered at a constant rate, maintaining adequate bactericidal
blood levels, while avoiding the high levels that are often
responsible for the toxicity (e.g., levels of gentamycin that are
too high typically result in renal toxicity).
[0133] Drug delivery by the methods of the present invention also
provide a more convenient method of drug administration. For
example, it is often particularly difficult to administer
parenteral drugs to newborns and infants because of the difficulty
associated with finding veins of acceptable caliber to catheterize.
However, newborns and infants often have a relatively large skin
surface as compared to adults. Transdermal drug delivery permits
easier management of such patients and allows certain types of care
that can presently be given only in hospitals to be given at home.
Other patients who typically have similar difficulties with venous
catheterization are patients undergoing chemotherapy or patients on
dialysis. In addition, for patients undergoing prolonged therapy,
transdermal administration as described herein is more convenient
than parenteral administration.
[0134] Transdermal administration as described herein also allows
the gastrointestinal tract to be bypassed in situations where
parenteral uses would not be practical. For example, there is a
growing need for methods suitable for administration of therapeutic
small peptides and proteins, which are typically digested within
the gastrointestinal tract. The methods described herein permit
administration of such compounds and allow easy administration a
over long periods of time. Patients who have problems with
absorption through their gastrointestinal tract because of
prolonged ileus or specific gastrointestinal diseases limiting drug
absorption may also benefit from drugs formulated for transdermal
application as described herein.
[0135] Further, there are many clinical situations where it is
difficult to maintain compliance. For example, patients with mental
problems (e.g., patients with Alzheimer's disease or psychosis) are
easier to manage if a constant delivery rate of drug is provided
without having to rely on their ability to take their medication at
specific times of the day. Also patients who simply forget to take
their drugs as prescribed are less likely to do so if they merely
have to put on a skin patch periodically (e.g., every 3 days).
Patients with diseases that are without symptoms, like patients
with hypertension, are especially at risk of forgetting to take
their medication as prescribed.
[0136] For patients taking multiple drugs, devices for transdermal
application such as skin patches may be formulated with
combinations of drugs that are frequently used together. For
example, many heart failure patients are given digoxin in
combination with furosemide. The combination of both drugs into a
single skin patch facilitates administration, reduces the risk of
errors (taking the correct pills at the appropriate time is often
confusing to older people), reduces the psychological strain of
taking "so many pills," reduces skipped dosage because of irregular
activities and improves compliance.
[0137] The methods described herein are particularly applicable to
humans, but also have a variety of veterinary uses, such as the
administration of growth factors or hormones (e.g., for fertility
control) to an animal.
[0138] As noted above, a wide variety of drugs may be administered
according to the methods provided herein. Some examples of drug
categories that may be administered transdermally include
anti-inflammatory drugs (e.g., in arthritis and in other condition)
such as all NSAID, indomethacin, prednisone, etc.; analgesics
(especially when oral absorption is not possible, such as after
surgery, and when parenteral administration is not convenient or
desirable), including morphine, codeine, Demerol, acetaminophen and
combinations of these (e.g., codeine plus acetaminophen);
antibiotics such as Vancomycin (which is not absorbed by the GI
tract and is frequently given intravenously) or a combination of
INH and Rifampicin (e.g., for tuberculosis); anticoagulants such as
heparin (which is not well absorbed by the GI tract and is
generally given parenterally, resulting in fluctuation in the blood
levels with an increased risk of bleeding at high levels and risks
of inefficacy at lower levels) and Warfarin (which is absorbed by
the GI tract but cannot be administered immediately after abdominal
surgery because of the normal ileus following the procedure);
antidepressants (e.g., in situations where compliance is an issue
as in Alzheimer's disease or when maintaining stable blood levels
results in a significant reduction of anti-cholinergic side effects
and better tolerance by patients), such as amitriptylin, imipramin,
prozac, etc.; antihypertensive drugs (e.g., to improve compliance
and reduce side effects associated with fluctuating blood levels),
such as diuretics and beta-blockers (which can be administered by
the same patch; e.g., furosemide and propanolol); antipsychotics
(e.g., to facilitate compliance and make it easier for care giver
and family members to make sure that the drug is received), such as
haloperidol and chlorpromazine, and anxiolytics or sedatives (e.g.,
to avoid the reduction of alertness related to high blood levels
after oral administration and allow a continual benefit throughout
the day by maintaining therapeutic levels constant).
[0139] Numerous other drugs may be administered as described
herein, including naturally occurring and synthetic hormones,
growth factors, proteins and peptides. For example, insulin and
human growth hormone, growth factors like erythropoietin,
interleukins and inteferons may be delivered via the skin.
[0140] Kits for administering a drug via the skin of a mammal are
also provided within the present invention. Such kits generally
comprise a device for transdermal application (e.g., a skin patch)
in combination with, or impregnated with, one or more modulating
agents. A drug may additionally be included within such kits.
[0141] Within a related aspect, the use of modulating agents as
described herein to increase skin permeability may also facilitate
sampling of the blood compartment by passive diffusion, permitting
detection and/or measurement of the levels of specific molecules
circulating in the blood. For example, application of one or more
modulating agents to the skin, via a skin patch as described
herein, permits the patch to function like a sponge to accumulate a
small quantity of fluid containing a representative sample of the
serum. The patch is then removed after a specified amount of time
and analyzed by suitable techniques for the compound of interest
(e.g., a medication, hormone, growth factor, metabolite or marker).
Alternatively, a patch may be impregnated with reagents to permit a
color change if a specific substance (e.g., an enzyme) is detected.
Substances that can be detected in this manner include, but are not
limited to, illegal drugs such as cocaine, HIV enzymes, glucose and
PSA. This technology is of particular benefit for home testing
kits.
[0142] 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. 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 JAM, occludin, claudin, cadherin (classical
and/or nonclassical, such as Dsc, Dsg, OB-cadherin and/or
VE-cadherin) and integrin mediated cell adhesion, thereby
disrupting tight junctions, adherens junctions, and desmosomes.
Multifunctional modulating agents comprising the JAM CAR sequence
DPK linked to one or more of the classical cadherin CAR sequence
HAV; the sequence RGD, which is bound by integrins; and/or a
nonclassical cadherin CAR sequence, such as a Dsc CAR sequence
(IEK, VER or ER), a Dsg CAR sequence (INQ, INR or LNK), an occludin
CAR sequence (LYHY; SEQ ID NO:6), a claudin CAR sequence (IYSI (SEQ
ID NO:7), an OB-cadherin CAR sequence (PDK, EEY or EAQ) and/or the
VE-cadherin CAR sequence DAE, may be used to disrupt cell adhesion.
Alternatively, a separate modulator of non-JAM-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 JAM modulating agents include Fab fragments
directed against either an N-cadherin CAR sequence (such as
FHLRAHAVDINGNQV-NH.sub.2; SEQ ID NO:9) or an E-cadherin CAR
sequence LFSHAVSSNG-NH.sub.2 (SEQ ID NO:14), an occludin CAR
sequence, such as QYLYHYCVVD-NH.sub.2 (SEQ ID NO:12), an
OB-cadherin CAR sequence, such as IFVIDDKSG-NH.sub.2 (SEQ ID
NO:13), a VE-cadherin CAR sequence such as VFRVDAETG-NH.sub.2 (SEQ
ID NO:10) or a claudin CAR sequence such as WKIYSYAG-NH.sub.2 (SEQ
ID NO:11).
[0143] 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.
[0144] Within a related aspect, the present invention provides
methods for treating cancer and/or inhibiting metastasis in a
mammal. Cancer tumors are solid masses of cells, growing out of
control, which require nourishment via blood vessels. The formation
of new capillaries is a prerequisite for tumor growth and the
emergence of metastases. Administration of modulating agents as
described herein may disrupt the growth of such blood vessels,
thereby providing effective therapy for the cancer and/or
inhibiting metastasis. Modulating agents may also be used to treat
leukemias. 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 JAM, cadherin and integrin mediated cell
adhesion, thereby disrupting tight junctions, adherens junctions,
focal contacts and desmosomes. Multifunctional modulating agents
comprising a JAM CAR sequence linked to one or more of the
classical cadherin CAR sequence HAV; the sequence RGD, which is
bound by integrins; and/or a nonclassical cadherin CAR sequence,
such as a Dsc CAR sequence (IEK, VER and ER), a Dsg CAR sequence
(INQ, INR and/or LNK), an OB-cadherin CAR sequence (DDK, EEY or
EAQ), an occludin CAR sequence (LYHY; SEQ ID NO:6), and/or the
VE-cadherin CAR sequence DAE, may be used to disrupt cell adhesion.
Alternatively, a separate modulator of non-JAM-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 JAM modulating agents include Fab fragments
directed against either an N-cadherin CAR sequence, such as
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO:9), an E-cadherin CAR sequence,
such as LFSHAVSSNG-NH.sub.2 (SEQ ID NO:14), an occludin CAR
sequence, such as QYLYHYCVWD-NH.sub.2 (SEQ ID NO:12), an
OB-cadherin CAR sequence, such as IFVIDDKSG-NH.sub.2 (SEQ ID NO:13)
or a claudin CAR sequence such as WKIYSYAG-NH.sub.2 (SEQ ID
NO:11).
[0145] 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).
[0146] 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 Of arthritis. 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 JAM, occludin,
classical cadherin, and integrin mediated cell adhesion, thereby
disrupting tight junctions adherens junctions, and focal contacts.
Multifunctional modulating agents comprising a JAM CAR sequence
linked to one or more of the classical cadherin CAR sequence HAV,
the sequence RGD, which is bound by integrins, the OB-cadherin CAR
sequence (DDK, EEY or EAQ), the occludin CAR sequence (LYHY; SEQ ID
NO:6), a claudin CAR sequence IYSY (SEQ ID NO:7) and/or the
VE-cadherin CAR sequence DAE may be used to disrupt cell adhesion.
Alternatively, a separate modulator of non-JAM-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 JAM modulating agents include Fab fragments
directed against an N-cadherin CAR sequence, such as
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO:9). an occludin CAR sequence,
such as QYLYHYCVVD-NH.sub.2 (SEQ ID NO:12), an OB-cadherin CAR
sequence, such as IFVIDDKSG-NH.sub.2 (SEQ ID NO:13), a VE-cadherin
CAR sequence such as VFRVDAETG-NH.sub.2 (SEQ ID NO:10) or a claudin
CAR sequence such as WKIYSYAG-NH.sub.2 (SEQ ID NO:11).
[0147] 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.
[0148] 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.
[0149] In yet another related aspect, the present invention
provides methods for inducing apoptosis in a JAM-expressing cell.
In general, patients afflicted with cancer may benefit from such
treatment. 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 JAM, occludin, claudin, classical cadherin,
and integrin mediated cell adhesion, thereby disrupting tight
junctions, adherens junctions, and focal contacts. Multifunctional
modulating agents comprising a JAM CAR sequence linked to one or
more of the cadherin CAR sequence HAV and/or the sequence RGD,
which is bound by integrins, a claudin CAR sequence IYSY (SEQ ID
NO:7), an OB-cadherin CAR sequence DDK and/or an occludin CAR
sequence LYHY (SEQ ID NO:6) may be used to disrupt cell adhesion.
Alternatively, a separate modulator of non-JAM-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 JAM modulating agents include Fab fragments
directed against either an N-cadherin CAR sequence, such as
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO:9), or an E-cadherin CAR
sequence, such as LFSHAVSSNG-NH.sub.2 (SEQ ID NO:14), an occludin
CAR sequence, such as QYLYHYCVVD-NH.sub.2 (SEQ ID NO:12), an
OB-cadherin CAR sequence, such as IFVIDDKSG-NH.sub.2 (SEQ ID NO:13)
or a claudin CAR sequence such as WKIYSYAG-NH.sub.2 (SEQ ID
NO:11).
[0150] 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.
[0151] The present invention also plies methods for enhancing drug
delivery to the central nervous system of a mammal. The blood/brain
barrier is largely impermeable to most neuroactive agents, and
delivery of drugs to the brain of a mammal often requires invasive
procedures. Using a modulating agent as described herein, however,
delivery may be by, for example, systemic administration of a
modulating agent-drug-targeting agent combination, injection of a
modulating agent (alone or in combination with a drug and/or
targeting agent) into the carotid artery or application of a skin
patch comprising a modulating agent to the head of the patient. 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 JAM, occludin and cadherin mediated cell adhesion,
thereby disrupting tight junctions and adherens junctions.
Multi-functional modulating agents comprising a JAM CAR sequence
linked to one or more of the classical cadherin CAR sequence HAVE
the occludin CAR sequence LYHY (SEQ ID NO:6), a claudin CAR
sequence IYSY (SEQ ID NO:6), the OB-cadherin CAR sequence (DDK, EEY
or EAQ) and/or the VE-cadherin CAR sequence DAE, preferably by way
of a linker, are also preferred. Alternatively, a separate
modulator of non-JAM-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 JAM
modulating agents include Fab fragments directed against the
N-cadherin CAR sequence FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO:9), an
occludin CAR sequence, such as QYLYHYCVVD-NH.sub.2 (SEQ ID NO:12),
an OB-cadherin CAR sequence, such as IFVIDDKSG-NH.sub.2 (SEQ ID
NO:13), a VE-cadherin CAR sequence such as VFRVDAETG-NH.sub.2 (SEQ
ID NO:10) or a claudin CAR sequence such as WKIYSYAG-NH.sub.2(SEQ
ID NO:11).
[0152] In general, the amount of modulating agent administered
varies with the method of administration and the nature of the
condition to be treated or prevented, but typically varies as
described above. Transfer of the drug to the central nervous system
may be evaluated by appropriate means that will be apparent to
those of ordinary skill in the art, such as magnetic resonance
imaging (MRI) or PET scan positron emitted tomography).
[0153] In certain other aspects, the present invention provides
methods for enhancing adhesion of JAM-expressing cells. Within
certain embodiments, a modulating agent may be linked to a solid
support, resulting in a matrix that comprises multiple modulating
agents. Within one such embodiment, the support is a polymeric
matrix to which modulating agents and molecules comprising other
CAR sequence(s) are attached (e.g., modulating agents and molecules
comprising HAV and RGD sequences may be attached to the same
matrix, preferably in an alternating pattern). Such matrices may be
used in contexts in which it is if desirable to enhance adhesion
mediated by multiple cell adhesion molecules. Alternatively, the
modulating agent itself may comprise multiple JAM CAR sequences or
antibodies (or fragments thereof), separated by linkers as
described above. Either way, the modulating agent(s) function as a
"biological glue" to bind multiple JAM-expressing cells within a
variety of contexts.
[0154] Within one such aspect, modulating agents comprising
multiple JAM CAR sequences and/or multiple modulating agents linked
to a single molecule or support material may be used to enhance
wound healing and/or reduce scar tissue in a mammal. Modulating
agents that are linked to a biocompatible and biodegradable matrix
such as cellulose or collagen are particularly preferred. For use
within such methods, a modulating agent should have a free amino or
hydroxyl group. The modulating agents are generally administered
topically to the wound, where they may facilitate closure of the
wound and may augment, or even replace, stitches. Similarly,
administration of matrix-linked modulating agents may facilitate
cell adhesion in skin grafting and prosthetic implants, and may
prolong the duration and usefulness of collagen injection. In
general, the amount of matrix-linked modulating agent administered
to a wound, graft, or implant site varies with the severity of the
wound and/or the nature of the wound, graft, or implant, but may
vary as discussed above. Multi-functional modulating agents
comprising the JAM CAR sequence DPK, the claudin CAR sequence IYSY
(SEQ ID NO:7), the classical cadherin CAR sequence (HAV), the
integrin CAR sequence (RGD), the occludin CAR sequence LYHY (SEQ ID
NO:6), as well as a nonclassical cadherin CAR sequence, such as the
OB-cadherin CAR sequence (DDK, EEY or EAQ), the VE-cadherin CAR
sequence DAE and/or one or more of the Dsc and Dsg CAR sequences
IEK, VER, IER, INQ, INR and/or LNK, may also be used as potent
stimulators of wound healing and/or to reduce scar tissue.
Alternatively, one or more separate modulator of cadherin-,
integrin-, and/or nonclassical cadherin-mediated cell adhesion may
be administered in conjunction with the modulating agent(s), either
within the same pharmaceutical composition or separately.
[0155] Within another aspect, one or more modulating agents may be
linked to the interior surface of a tissue culture plate or other
cell culture support, such as for use in a bioreactor. Such linkage
may be performed by any suitable-technique, as described above.
Modulating agents linked in this fashion may generally be used to
immobilize JAM-expressing cells. For example, dishes or plates
coated with one or more modulating agents may be used to immobilize
JAM-expressing cells within a variety of assays and screens. Within
bioreactors (i.e., systems for large scale production of cells or
organoids), modulating agents may generally be used to improve cell
attachment and stabilize cell growth.
[0156] Modulating agents may also be used within bioreactors to
support the information and function of highly differentiated
organoids derived,-for example, from dispersed populations of fetal
mammalian cells. Bioreactors containing biomatrices of modulating
agent(s) may also be a used to facilitate the production of
specific proteins.
[0157] Modulating agents as described herein may be used within a
variety of bioreactor configurations. In general, a bioreactor is
designed with an interior surface area sufficient to support large
numbers of adherent cells. This surface area can be provided using
membranes, tubes, microtiter wells, columns, hollow fibers, roller
bottles, plates, dishes, beads or a combination thereof. A
bioreactor may be compartmentalized. The support material within a
bioreactor may be any suitable material known in the art;
preferably, the support material does not dissolve or swell in
water. Preferred support materials include, but are not limited to,
synthetic polymers such as acrylics, vinyls, polyethylene,
polypropylene, polytetrafluoroethylene, nylons, polyurethanes,
polyamides, polysulfones and poly(ethylene terephthalate);
ceramics; glass and silica.
[0158] Within further aspects, the present invention provides
methods for reducing backleak in kidney transplant patients. This
is a problem encountered by kidney transplant patients who are
experiencing renal failure, in which the filtrate in the tubules of
the kidney leaks back into the kidney. Backleak appears to be the
result of impaired integrity of tight junctions (see Kwon et al.,
J. Clin Invest. 101:2054-2064, 1998). Modulating agents that
enhance JAM-mediated cell adhesion may be administered to the
patient to alleviate backleak. The modulating agents may be
administered topically at the time of transplant, or systemically
(e.g., intravenously). Multi-functional modulating agents
comprising the JAM CAR sequence DPK in combination with CAR
sequence(s) of adhesion molecules that are involved in tight
junction formation may also be used. Such CAR sequences include the
claudin CAR sequence IYSY (SEQ ID-NO:7), the classical cadherin CAR
sequence (HAV) and the occludin CAR sequence LYHY (SEQ ID NO:6).
Alternatively, one or more separate modulator of cadherin-,
occludin-, and/or claudin-mediated cell adhesion may be
administered in conjunction with the modulating agent(s), either
within the same pharmaceutical composition or separately.
[0159] Other aspects of the present invention provide methods that
employ antibodies raised against the modulating agents for
diagnostic and assay purposes. Assays typically involve using an
antibody to detect the presence or absence of JAM (free or on the
surface of a cell), or proteolytic fragment containing the EC1
domain in a suitable biological sample, such as tumor or normal
tissue biopsies, blood, lymph node, serum or urine samples, or
other tissue, homogenate, or extract thereof obtained from a
patient.
[0160] There are a variety of assay formats known to those of
ordinary skill in the art for using an antibody to detect a target
molecule in a sample. See, e.g., Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For
example, the assay may be performed in a Western blot format,
wherein a protein preparation from the biological sample is
submitted to gel electrophoresis, transferred to a suitable
membrane and allowed to react with the antibody. The presence of
the antibody on the membrane may then be detected using a suitable
detection reagent, as described below.
[0161] In another embodiment, the assay involves the use of
antibody immobilized on a solid support to bind to the target JAM,
or a proteolytic fragment containing the EC1 domain and
encompassing the CAR sequence, and remove it from the remainder of
the sample. The bound JAM may then be detected using a second
antibody or reagent that contains a reporter group. Alternatively,
a competitive assay may be utilized, in which the JAM is labeled
with a reporter group and allowed to bind to the immobilized
antibody after incubation of the antibody with the sample. The
extent to which components of the sample inhibit the binding of the
labeled JAM to the antibody is indicative of the reactivity of the
sample with the immobilized antibody, and as a result, indicative
of the level of the JAM in the sample.
[0162] The solid support may be any material known to those of
ordinary skill in the art to which the antibody may be attached,
such as a test well in a microtiter plate, a nitrocellulose filter
or another suitable membrane. Alternatively, the support may be a
bead or disc, such as glass, fiberglass, latex or a plastic such as
polystyrene or polyvinylchloride. The antibody may be immobilized
on the solid support using a variety of techniques known to those
in the art, which are amply described in the patent and scientific
literature.
[0163] In certain embodiments, the assay for detection of JAM in a
sample is a two-antibody sandwich assay. This assay may be
performed by first contacting an antibody that has been immobilized
on a solid support, commonly the well of a microtiter plate, with
the biological sample, such that the JAM within the sample is
allowed to bind to the immobilized antibody (a 30 minute incubation
time at room temperature is generally sufficient). Unbound sample
is then removed from the immobilized JAM-antibody complexes and a
second antibody (containing a reporter group such as an enzyme,
dye, radionuclide, luminescent group, fluorescent group or biotin)
capable of binding to a different site on the JAM is added. The
amount of second antibody that remains bound to the solid support
is then determined using a method appropriate for the specific
reporter group. The method employed for detecting the reporter
group depends upon the nature of the reporter group. For
radioactive groups, scintillation counting or autoradiographic
methods are generally appropriate. Spectroscopic methods may be
used to detect dyes, luminescent groups and fluorescent groups.
Biotin may be detected using avidin, coupled to a different
reporter group (commonly a radioactive or fluorescent group or an
enzyme). Enzyme reporter groups may generally be detected by the
addition of substrate (generally for a specific period of time),
followed by spectroscopic or other analysis of the reaction
products. Standards and standard additions may be used to determine
the level of JAM in a sample, using well known techniques.
[0164] The present invention also provides kits for use in such
immunoassays. Such kits generally comprise one ogre antibodies, as
described above. In addition, one or more additional compartments
or containers of a kit generally enclose elements, such as
reagents, buffers and/or wash solutions, to be used in the
immunoassay.
[0165] Within further aspects, modulating agents or antibodies (or
fragments thereof) may be used to facilitate cell identification
and sorting in vitro or imaging in vivo, permitting the-selection
of cells expressing JAM (or different JAM levels). Preferably, the
modulating agent(s) or antibodies for use in such methods are
linked to a detectable marker. Suitable markers are well known in
the art and include radionuclides, luminescent groups, fluorescent
groups, enzymes, dyes, constant immunoglobulin domains and biotin.
Within one preferred embodiment, a modulating agent linked to a
fluorescent marker, such as fluorescein, is contacted with the
cells, which are then analyzed by fluorescence activated cell
sorting (FACS).
[0166] Antibodies or fragments thereof may also be used within
screens of combinatorial or other nonpeptide-based libraries to
identify other compounds capable of modulating JAM-mediated cell
adhesion. Such screens may generally be performed using an ELISA or
other method well known to those of ordinary skill in the art that
detect compounds with a shape and structure similar to that of the
modulating agent. In general, such screens may involve contacting
an expression library producing test compounds with an antibody,
and detecting the level of antibody bound to the candidate
compounds. Compounds for which the antibody has a higher affinity
may be further characterized as described herein, to evaluate the
ability to modulate JAM-mediated cell adhesion.
[0167] The following Example is offered by way of illustration and
not by way of limitation.
EXAMPLE 1
Preparation of Representative Cyclic Peptides
[0168] This Example illustrates the solid phase synthesis of
representative linear and cyclic peptides as modulating agents.
[0169] The peptides are assembled on, methylbenzhydrylamine resin
(MBHA resin) for the C-terminal amide peptides. The traditional
Merrifield resins are used for any C-terminal acid peptides. Bags
of a polypropylene mesh material are filled with the resin and
soaked in dichloromethane. The resin packets are 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 is added to activate the coupling
reaction. The bottle is shaken for one hour to ensure completion of
the reaction. The reaction mixture is discarded and the packets
washed with DMF. The N-.alpha.-Boc is removed by acidolysis using a
55% TFA in dichloromethane for 30 minutes leaving the TFA salt of
the ic-amino group. The bags are 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 is performed by reacting the peptide resins with a
solution of acetic anhydride in dichloromethane in the presence of
diisopropylethylamine. The peptide is 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.
[0170] The crude peptides are purified by reversed-phase
high-performance liquid chromatography. Purified linear precursors
of the cyclic peptides are solubilized in 75% acetic acid at a
concentration of 2-10 mg/mL. A 10% solution of iodine in methanol
is added dropwise until a persistent coloration was obtained. A 5%
ascorbic acid solution in water is then added to the mixture until
discoloration. The disulfide bridge containing compounds are then
purified by HPLC and characterized by analytical HPLC and by mass
spectral analysis.
[0171] From the foregoing, it will be evident that although
specific embodiments of the invention have been described herein
for the purpose of illustrating the invention, 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
14 1 9 PRT Artificial Sequence Juntion adhesion molecule cell
adhesion recognition sequence 1 Ser Phe Thr Ile Asp Pro Lys Ser Gly
1 5 2 59 PRT Mus musculus 2 Ser Glu His Asp Gly Ser Pro Pro Ser Glu
Tyr Ser Trp Phe Lys Asp 1 5 10 15 Gly Ile Ser Met Leu Thr Ala Asp
Ala Lys Lys Thr Arg Ala Phe Met 20 25 30 Asn Ser Ser Phe Thr Ile
Asp Pro Lys Ser Gly Asp Leu Ile Phe Asp 35 40 45 Pro Val Thr Ala
Phe Asp Ser Gly Glu Tyr Tyr 50 55 3 5 PRT Artificial Sequence
Cyclic CAR sequence for modulating JAM-mediated funtions 3 Cys Asp
Pro Lys Cys 1 5 4 6 PRT Artificial Sequence Modulating agent with
JAM cell adhesion recognition sequences 4 Asp Pro Lys Asp Pro Lys 1
5 5 10 PRT Artificial Sequence Adhesion Molecule which is bound by
N-CAM 5 Lys Tyr Ser Phe Asn Tyr Asp Gly Ser Glu 1 5 10 6 4 PRT
Artificial Sequence Occludin cell adhesion recognition sequence 6
Leu Tyr His Tyr 1 7 4 PRT Artificial Sequence Claudin cell adhesion
recognition sequence 7 Ile Tyr Ser Tyr 1 8 4 PRT Artificial
Sequence Synthetic peptide - Example of cyclization process 8 Trp
Gly Gly Trp 1 9 15 PRT Artificial Sequence Fab fragment directed
against N-cadherin cell adhesion recognition sequence 9 Phe His Leu
Arg Ala His Ala Val Asp Ile Asn Gly Asn Gln Val 1 5 10 15 10 9 PRT
Artificial Sequence Fab fragment directed against VE-cadherin cell
adhesion recognition sequence 10 Val Phe Arg Val Asp Ala Glu Thr
Gly 1 5 11 8 PRT Artificial Sequence Fab fragment directed against
claudin cell adhesion recognition sequence 11 Trp Lys Ile Tyr Ser
Tyr Ala Gly 1 5 12 10 PRT Artificial Sequence Fab fragment directed
against occludin cell adhesion recognition sequence 12 Gln Tyr Leu
Tyr His Tyr Cys Val Val Asp 1 5 10 13 9 PRT Artificial Sequence Fab
fragment directed against OB-cadherin cell adhesion recognition
sequence 13 Ile Phe Val Ile Asp Asp Lys Ser Gly 1 5 14 10 PRT
Artificial Sequence Fab fragment directed against E-cadherin cell
adhesion recognition sequence 14 Leu Phe Ser His Ala Val Ser Ser
Asn Gly 1 5 10
* * * * *