U.S. patent application number 09/778026 was filed with the patent office on 2003-01-16 for compounds and methods for regulating cell adhesion.
Invention is credited to Blaschuk, Orest W., Gour, Barbara J..
Application Number | 20030013655 09/778026 |
Document ID | / |
Family ID | 25473850 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030013655 |
Kind Code |
A1 |
Blaschuk, Orest W. ; et
al. |
January 16, 2003 |
Compounds and methods for regulating cell adhesion
Abstract
Methods for using modulating agents to enhance or inhibit
cadherin-mediated cell adhesion in a variety of in vivo and in
vitro contexts are provided. In particular, the modulating agents
may be used in the therapy of multiple sclerosis and other
demyelinating diseases. The modulating agents comprise at least one
cadherin cell adhesion recognition sequence (HAV) or an antibody or
fragment thereof that specifically binds to a cadherin 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) ; Gour, Barbara J.; (Beaconsfield,
CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Family ID: |
25473850 |
Appl. No.: |
09/778026 |
Filed: |
February 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09778026 |
Feb 5, 2001 |
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08939853 |
Sep 29, 1997 |
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6203788 |
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Current U.S.
Class: |
424/184.1 ;
514/17.9; 514/19.1; 514/19.6 |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 7/06 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/14 ; 514/15;
514/16; 514/17; 514/18 |
International
Class: |
A61K 038/08; A61K
038/10; A61K 038/06 |
Claims
What is claimed is:
1. A method for treating a demyelinating neurological disease in a
mammal, comprising administering to a mammal a cell adhesion
modulating agent, wherein said modulating agent comprises the
sequence His-Ala-Val, and wherein said modulating agent inhibits
cadherin-mediated cell adhesion.
2. A method according to claim 1, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), AHAVSE (SEQ ID NO: 27), AHAVDI (SEQ ID NO: 28) and
derivatives of the foregoing sequences having one or more
C-terminal, N-terminal and/or side chain modifications.
3. A method for treating a demyelinating neurological disease in a
mammal, comprising administering to a mammal a cell adhesion
modulating agent, wherein said modulating agent comprises an
antibody or fragment thereof that specifically binds to a cadherin
cell adhesion recognition sequence, and wherein said modulating
agent inhibits cadherin-mediated cell adhesion.
4. A method according to claim 1 or claim 3, wherein said
modulating agent is linked to a targeting agent.
5. A method according to claim 1 or claim 3, wherein said
modulating agent is linked to a drug.
6. A method according to claim 1 or claim 3, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin.
7. A method according to claim 1 or claim 3, wherein said
modulating agent is administered by implantation with Schwann
cells.
8. A method according to claim 1 or claim 3, wherein said
modulating agent is administered by implantation with
oligodendrocyte progenitor cells and/or oligodendrocytes.
9. A method according to claim 1 or claim 3, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
10. A method according to claim 1 or claim 3, wherein said disease
is multiple sclerosis.
11. A method for reducing unwanted cellular adhesion in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises the sequence
His-Ala-Val, and wherein said modulating agent inhibits unwanted
cadherin-mediated cell adhesion resulting from surgery, injury,
disease or inflammation.
12. A method according to claim 11, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO:
27), AHAVDI (SEQ ID NO: 28), SHAVSS (SEQ ID NO: 29), LFSHAVSSNG
(SEQ ID NO: 19) and derivatives of the foregoing sequences having
one or more C-terminal, N-terminal and/or side chain
modifications.
13. A method for reducing unwanted cellular adhesion in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence, and wherein said modulating agent
inhibits cadherin-mediated cell adhesion.
14. A method according to claim 11 or claim 13, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin, and wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val sequence(s) by a
linker.
15. A method according to claim 14, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
16. A method according to claim 11 or claim 13, wherein said
modulating agent is linked to a targeting agent.
17. A method according to claim 11 or claim 13, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
18. A method according to claim 17, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion
comprising at least one cell adhesion recognition sequence bound by
an adhesion molecule other than a classical cadherin.
19. A method according to claim 18, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
20. 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 and a drug, wherein said
modulating agent comprises the sequence His-Ala-Val, wherein said
modulating agent inhibits cadherin-mediated cell adhesion, and
wherein the step of contacting is performed under conditions and
for a time sufficient to allow passage of said drug across said
epithelial cells.
21. 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 and a drug, wherein said
modulating agent comprises an antibody or fragment thereof that
specifically binds to a cadherin cell adhesion recognition
sequence, wherein said modulating agent inhibits cadherin-mediated
cell adhesion and wherein the step of contacting is performed under
conditions and for a time sufficient to allow passage of said drug
across said epithelial cells.
22. A method according to claim 20 or claim 21, wherein said
modulating agent passes into the blood stream of said mammal.
23. A method according to claim 20, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO:
27), AHAVDI (SEQ ID NO: 28), SHAVSS (SEQ ID NO: 29), LFGHAVSENG
(SEQ ID NO: 20), LFSHAVSSNG (SEQ ID NO: 19), GHAVSE (SEQ ID NO: 26)
and derivatives of the foregoing sequences having one or more
C-terminal, N-terminal and/or side chain modifications.
24. A method according to claim 23, wherein said modulating agent
is AHAVSE-NH.sub.2 (SEQ ID NO: 27).
25. A method according to claim 20 or claim 21, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin, and wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val sequence(s) by a
linker.
26. A method according to claim 25, wherein said cell adhesion
recognition sequence comprises a sequence selected from the group
consisting of YAT, FAT, YAS, RAL, QSSGSLYGSQ (SEQ ID NO: 16) and
QYLYHYCVVD (SEQ ID NO: 17).
27. A method according to claim 20 or claim 21, wherein said
modulating agent is linked to a targeting agent.
28. A method according to claim 20 or claim 21, wherein said
modulating agent is linked to said drug.
29. A method according to claim 20 or claim 21, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
30. A method according to claim 29, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion
comprising one or more of: (a) a cell adhesion recognition sequence
bound by an adhesion molecule other than a classical cadherin;
and/or (b) an antibody or antigen-bin ding fragment thereof that
specifically binds to a cell adhesion recognition sequence bound by
an adhesion molecule other than a classical cadherin.
31. A method according to claim 30, wherein said cell adhesion
recognition sequence comprises a sequence selected from the group
consisting of YAT, FAT, YAS, RAL, QSSGSLYGSQ (SEQ ID NO: 16) and
QYLYHYCVVD (SEQ ID NO: 17).
32. A method according to claim 30, wherein said antibody or
fragment thereof specifically binds to the sequence
GVNPTAQSSGSLYGSQI YALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO:
15).
33. A method according to claim 20 or claim 21, wherein the step of
contacting is performed via a skin patch comprising said modulating
agent and said drug.
34. A method for enhancing the delivery of a drug to a tumor in a
mammal, comprising administering to a mammal a cell adhesion
modulating agent and a drug, wherein said modulating agent
comprises 3-16 amino acid residues, including the sequence
His-Ala-Val, and wherein said modulating agent inhibits
cadherin-mediated cell adhesion.
35. A method for enhancing the delivery of a drug to a tumor in a
mammal, comprising administering to a mammal a cell adhesion
modulating agent and a drug, wherein said modulating agent
comprises an antibody or fragment thereof that specifically binds
to a cadherin cell adhesion recognition sequence, and wherein said
modulating agent inhibits cadherin-mediated cell adhesion.
36. A method according to claim 34 or claim 35, wherein the tumor
is selected from the group consisting of bladder tumors, ovarian
tumors and melanomas.
37. A method according to claim 34 or claim 35, wherein said
composition is administered to said tumor.
38. A method according to claim 34 or claim 35, wherein said
composition is administered systemically.
39. A method according to claim 34, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO:
27), AHAVDI (SEQ ID NO: 28), SHAVSS (SEQ ID NO: 29), LYSHAVSSNG
(SEQ ID NO: 18) and derivatives of the foregoing sequences having
one or more C-terminal, N-terminal and/or side chain
modifications.
40. A method according to claim 39, wherein said modulating agent
is AHAVSE-NH.sub.2 (SEQ ID NO: 27).
41. A method according to claim 34 or claim 35, wherein said
modulating agent is linked to a targeting agent.
42. A method according to claim 34 or claim 35, wherein said
modulating agent linked to said drug.
43. A method according to claim 34 or claim 35, wherein said
modulating agent further comprises one or more of: (a) a cell
adhesion recognition sequence bound by an adhesion molecule other
than a classical cadherin, wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val sequence(s) by a linker;
and/or (b) an antibody or antigen-binding fragment thereof that
binds to a cell adhesion recognition sequence bound by an adhesion
molecule other than a classical cadherin.
44. A method according to claim 43, wherein said cell adhesion
recognition sequence comprises a sequence selected from the group
consisting of YAT, FAT, YAS, RAL, QSSGSLYGSQ (SEQ ID NO: 16) and
QYLYHYCVVD (SEQ ID NO: 17).
45. A method according to claim 44, wherein said antibody or
antigen-binding fragment thereof binds to the sequence
GVNPTAQSSGSLYGSQI YALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO:
15).
46. A method according to claim 33 or claim 34, wherein said
modulating agent and said drug are present within a pharmaceutical
composition comprising a pharmaceutically acceptable carrier.
47. A method according to claim 46, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion
comprising one or more of: (a) a cell adhesion recognition sequence
bound by an adhesion molecule other than a classical cadherin;
and/or (b) an antibody or antigen-binding fragment thereof that
binds to a cell adhesion recognition sequence bound by an adhesion
molecule other than a classical cadherin.
48. A method according to claim 47, wherein said cell adhesion
recognition sequence comprises a sequence selected from the group
consisting of YAT, FAT, YAS, RAL, QSSGSLYGSQ (SEQ ID NO: 16) and
QYLYHYCVVD (SEQ ID NO: 17).
49. A method according to claim 47, wherein said antibody or
antigen-binding fragment thereof binds to the sequence
GVNPTAQSSGSLYGSQI YALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO:
15).
50. A method for treating cancer in a mammal, comprising
administering to a mammal a cell adhesion modulating agent, wherein
said modulating agent comprises 3-16 amino acid residues, including
the sequence His-Ala-Val, and wherein said modulating agent
inhibits cadherin-mediated cell adhesion.
51. A method for treating cancer in a mammal, comprising
administering to a mammal a cell adhesion modulating agent, wherein
said modulating agent comprises an antibody or fragment thereof
that specifically binds to a cadherin cell adhesion recognition
sequence, and wherein said modulating agent inhibits
cadherin-mediated cell adhesion.
52. A method according to claim 50 or claim 51, wherein said cancer
is selected from the group consisting of carcinomas, leukemia and
melanomas.
53. A method according to claim 50, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), AHAVSE (SEQ ID NO: 27), AHAVDI (SEQ ID NO: 28),
SHAVSS (SEQ ID NO: 29), LYSHAVSSNG (SEQ ID NO: 18), LFSHAVSSNG (SEQ
ID NO: 19) and derivatives of the foregoing sequences having one or
more C-terminal, N-terminal and/or side chain modifications.
54. A method according to claim 50 or claim 51, wherein said
modulating agent is linked to a targeting agent.
55. A method according to claim 50 or claim 51, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin, and wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val sequence(s) by a
linker.
56. A method according to claim 55, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
57. A method according to claim 50 or claim 51, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
58. A method according to claim 51, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion, wherein
said modulator comprises a cell adhesion recognition sequence bound
by an adhesion molecule other than a classical cadherin.
59. A method according to claim 58, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
60. A method for inhibiting angiogenesis in a mammal, comprising
administering to a mammal a cell adhesion modulating agent, wherein
said modulating agent comprises the sequence His-Ala-Val, and
wherein said modulating agent inhibits cadherin-mediated cell
adhesion.
61. A method according to claim 60, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), AHAVSE (SEQ ID NO: 27), AHAVDI (SEQ ID NO: 28) and
derivatives of the foregoing sequences having one or more
C-terminal, N-terminal and/or side chain modifications.
62. A method for inhibiting angiogenesis in a mammal, comprising
administering to a mammal a cell adhesion modulating agent, wherein
said modulating agent comprises an antibody or fragment thereof
that specifically binds to a cadherin cell adhesion recognition
sequence, and wherein said modulating agent inhibits
cadherin-mediated cell adhesion.
63. A method according to claim 60 or claim 62, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin, and wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val sequence(s) by a
linker.
64. A method according to claim 63, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
65. A method according to claim 60 or claim 62, wherein said
modulating agent is linked to a target agent.
66. A method according to claim 60 or claim 62, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
67. A method according to claim 66, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion, wherein
said modulator comprises a cell adhesion recognition sequence bound
by an adhesion molecule other than a classical cadherin.
68. A method according to claim 67, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
69. A method for enhancing drug delivery to the central nervous
system of a mammal, comprising administering to a mammal a cell
adhesion modulating agent, wherein said modulating agent comprises
3-16 amino acid residues, including the sequence His-Ala-Val, and
wherein said modulating agent inhibits cadherin-mediated cell
adhesion.
70. A method according to claim 69, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), AHAVSE (SEQ ID NO: 27), AHAVDI (SEQ ID NO: 28) and
derivatives of the foregoing sequences having one or more
C-terminal, N-terminal and/or side chain modifications.
71. A method for enhancing drug delivery to the central nervous
system of a mammal, comprising administering to a mammal a cell
adhesion modulating agent, wherein said modulating agent comprises
an antibody or fragment thereof that specifically binds to a
cadherin cell adhesion recognition sequence, and wherein said
modulating agent inhibits cadherin-mediated cell adhesion.
72. A method according to claim 69 or claim 71 wherein said
modulating agent farther comprises one or more of: (a) a cell
adhesion recognition sequence bound by an adhesion molecule other
than a classical cadherin, wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val 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 classical cadherin.
73. A method according to claim 72, wherein said cell adhesion
recognition sequence is QSSGSLYGSQ (SEQ ID NO: 16) or QYLYHYCVVD
(SEQ ID NO: 17).
74. A method according to claim 72, wherein said antibody or
fragment thereof specifically binds to the sequence
GVNPTAQSSGSLYGSQI YALCNQFYT?AATGLYVDQYLYHYCVVDPQE (SEQ ID NO:
15).
75. A method according to claim 69 or claim 71, wherein said
modulating agent is linked to a targeting agent.
76. A method according to claim 69 or claim 71, wherein said
modulating agent linked to a drug.
77. A method according to claim 69 or claim 71, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
78. A method according to claim 77, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion,
comprising one or more of: (a) a cell adhesion recognition sequence
bound by an adhesion molecule other than a classical cadherin;
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 classical cadherin.
79. A method according to claim 78, wherein said cell adhesion
recognition sequence is QSSGSLYGSQ (SEQ ID NO: 16) or QYLYHYCVVD
(SEQ ID NO: 17).
80. A method according to claim 78, wherein said antibody or
fragment thereof specifically binds to the sequence
GVNPTAQSSGSLYGSQI YALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO:
15).
81. A method for enhancing wound healing in a mammal, comprising
contacting a wound in a mammal with a cell adhesion modulating
agent, wherein said modulating agent comprises the sequence
His-Ala-Val, and wherein said modulating agent enhances
cadherin-mediated cell adhesion.
82. A method according to claim 81, wherein said modulating agent
comprises at least two His-Ala-Val sequences separated by a
linker.
83. A method according to claim 81, wherein said modulating agent
comprises a sequence selected from the group consisting of
LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO: 27), SHAVSS (SEQ ID
NO: 29), LFSHAVSSNG (SEQ ID NO: 19), LFGHAVSENG (SEQ ID NO: 20),
GHAVSE (SEQ ID NO: 26) and derivatives of the foregoing sequences
having one or more C-terminal, N-terminal and/or side chain
modifications.
84. A method according to claim 83, wherein said modulating agent
is LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19), AHAVSE-NH.sub.2 (SEQ ID NO:
27), LFGHAVSENG-NH.sub.2 (SEQ ID NO: 20) or GHAVSE-NH.sub.2 (SEQ ID
NO: 26).
85. A method for enhancing wound healing in a mammal, comprising
contacting a wound in a mammal with a cell adhesion modulating
agent, wherein said modulating agent comprises an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence, and wherein said modulating agent
enhances cadherin-mediated cell adhesion.
86. A method according to claim 81 or 85, wherein said modulating
agent further comprises at least one cell adhesion recognition
sequence bound by an adhesion molecule other than a classical
cadherin, and wherein said cell adhesion recognition sequence is
separated from any His-Ala-Val sequence(s) by a linker.
87. A method according to claim 86, wherein said cell adhesion
recognition sequence comprises one or more of the sequences
Arg-Gly-Asp, Tyr-Ala-Thr, Phe-Ala-Thr, Tyr-Ala-Ser or
Arg-Ala-Leu.
88. A method according to claim 81 or claim 85, wherein said
modulating agent is linked to a targeting agent.
89. A method according to claim 81 or claim 85, wherein said
modulating agent is linked to a support material.
90. A method according to claim 81 or claim 85, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
91. A method according to claim 90, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion,
comprising one or more of: (a) a cell adhesion recognition sequence
bound by an adhesion molecule other than a classical cadherin;
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 classical cadherin.
92. A method according to claim 91, wherein said cell adhesion
recognition sequence is selected from the group consisting of
Arg-Gly-Asp, Tyr-Ala-Thr, Phe-Ala-Thr, Tyr-Ala-Ser or
Arg-Ala-Leu.
93. 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,
wherein said modulating agent comprises the sequence His-Ala-Val,
and wherein said modulating agent enhances cadherin-mediated cell
adhesion.
94. A method according to claim 93, wherein said modulating agent
comprises at least two His-Ala-Val sequences separated by a
linker.
95. A method according to claim 93, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO:
27), AHAVDI (SEQ ID NO: 28), SHAVSS (SEQ ID NO: 29), LFSHAVSSNG
(SEQ ID NO: 19) and derivatives of the foregoing sequences having
one or more C-terminal, N-terminal and/or side chain
modifications.
96. 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,
wherein said modulating agent comprises an antibody or fragment
thereof that specifically binds to a cadherin cell adhesion
recognition sequence, and wherein said modulating agent enhances
cadherin-mediated cell adhesion.
97. A method according to claim 93 or claim 96, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin, and wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val sequence(s) by a
linker.
98. A method according to claim 97, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
99. A method according to claim 93 or claim 96 wherein said
modulating agent is linked to a targeting agent.
100. A method according to claim 93 or claim 96, wherein said
modulating agent is linked to a support material.
101. A method according to claim 93 or claim 96 wherein said
foreign tissue is a skin graft or organ implant.
102. A method according to claim 93 or claim 96, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
103. A method according to claim 102, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion a
modulator of cell adhesion, wherein said modulator comprises a cell
adhesion recognition sequence bound by an adhesion molecule other
than a classical cadherin.
104. A method according to claim 103, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
105. A method for inducing apoptosis in a cadherin-expressing cell,
comprising contacting a cadherin-expressing cell with a cell
adhesion modulating agent, wherein said modulating agent comprises
the sequence His-Ala-Val, and wherein said modulating agent
inhibits cadherin-mediated cell adhesion.
106. A method according to claim 105, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO:
27), AHAVDI (SEQ ID NO: 28), SHAVSS (SEQ ID NO: 28), LFSHAVSSNG
(SEQ ID NO: 19) and derivatives of the foregoing sequences having
one or more C-terminal, N-terminal and/or side chain
modifications.
107. A method for inducing apoptosis in a cadherin-expressing cell,
comprising contacting a cadherin-expressing cell with a cell
adhesion modulating agent, wherein said modulating agent comprises
an antibody or fragment thereof that specifically binds to a
cadherin cell adhesion recognition sequence, and wherein said
modulating agent inhibits cadherin-mediated cell adhesion.
108. A method according to claim 105 or claim 107, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin.
109. A method according to claim 108, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
110. A method according to claim 105 or claim 107, wherein said
modulating agent is linked to a targeting agent.
111. A method according to claim 105 or claim 107, wherein said
modulating agent is linked to a drug.
112. A method according to claim 105 or claim 107, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
113. A method according to claim 112, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion, wherein
said modulator comprises a cell adhesion recognition sequence bound
by an adhesion molecule other than a classical cadherin.
114. A method according to claim 113, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
115. A method for modulating the immune system of a mammal,
comprising administering to a mammal a pharmaceutical composition
comprising a cell adhesion modulating agent, wherein said
modulating agent comprises the sequence His-Ala-Val, and wherein
said modulating agent inhibits cadherin-mediated cell adhesion.
116. A method according to claim 115, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO:
27), AHAVDI (SEQ ID NO: 28), SHAVSS (SEQ ID NO: 29), LFSHAVSSNG
(SEQ ID NO: 19) and derivatives of the foregoing sequences having
one or more C-terminal, N-terminal and/or side chain
modifications.
117. A method for modulating the immune system of a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence, and wherein said modulating agent
inhibits cadherin-mediated cell adhesion.
118. A method according to claim 115 or claim 117, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin.
119. A method according to claim 118, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
120. A method according to claim 115 or claim 117, wherein said
modulating agent is linked to a targeting agent.
121. A method according to claim 115 or claim 117, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
122. A method according to claim 121, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion, wherein
said modulator comprises a cell adhesion recognition sequence bound
by an adhesion molecule other than a classical cadherin.
123. A method according to claim 122, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
124. A method for preventing pregnancy in a mammal, comprising
administering to a mammal a cell adhesion modulating agent, wherein
said modulating agent comprises the sequence His-Ala-Val, and
wherein said modulating agent inhibits cadherin-mediated cell
adhesion.
125. A method according to claim 124, wherein said modulating agent
comprises a sequence selected from the group consisting of
LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO: 27), SHAVSS (SEQ ID
NO: 29), LFSHAVSSNG (SEQ ID NO: 19) and derivatives of the
foregoing sequences having one or more C-terminal, N-terminal
and/or side chain modifications.
126. A method for preventing pregnancy in a mammal, comprising
administering to a mammal a cell adhesion modulating agent, wherein
said modulating agent comprises an antibody or fragment thereof
that specifically binds to a cadherin cell adhesion recognition
sequence, and wherein said modulating agent inhibits
cadherin-mediated cell adhesion.
127. A method according to claim 124 or claim 126, wherein said
modulating agent further comprises at least one cell adhesion
recognition sequence bound by an adhesion molecule other than a
classical cadherin, and wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val sequence(s) by a
linker.
128. A method according to claim 127, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
129. A method according to claim 124 or claim 126, wherein said
modulating agent is linked to a targeting agent.
130. A method according to claim 124 claim 126, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
131. A method according to claim 130, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion, wherein
said modulator comprises a cell adhesion recognition sequence bound
by an adhesion molecule other than a classical cadherin.
132. A method according to claim 131, wherein said cell adhesion
recognition sequence comprises the sequence Arg-Gly-Asp.
133. A method according to claim 124 or 126, wherein the step of
administration is performed by intravaginal insertion of a device
containing said modulating agent.
134. A method for increasing vasopermeability in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises the sequence
His-Ala-Val, and wherein said modulating agent inhibits
cadherin-mediated cell adhesion.
135. A method according to claim 134, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), AHAVSE (SEQ ID NO: 27), AHAVDI (SEQ ID NO: 28) and
derivatives of the foregoing sequences having C-terminal,
N-terminal and/or side chain modifications.
136. A method for increasing vasopermeability in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence, and wherein said modulating agent
inhibits cadherin-mediated cell adhesion.
137. A method according to claim 134 or claim 136, wherein said
modulating agent is linked to a targeting agent.
138. A method according to claim 134 or claim 136 wherein said
modulating agent further comprises one or more of: (a) a cell
adhesion recognition sequence bound by an adhesion molecule other
than a classical cadherin, wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val 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 classical cadherin.
139. A method according to claim 138, wherein said cell adhesion
recognition sequence is QSSGSLYGSQ (SEQ ID NO: 16) or QYLYHYCVVD
(SEQ ID NO: 17).
140. A method according to claim 139, wherein said antibody or
fragment thereof specifically binds to the sequence
GVNPTAQSSGSLYGSQI YALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO:
15).
141. A method according to claim 134 or claim 136, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
142. A method according to claim 141, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion,
comprising one or more of: (a) a cell adhesion recognition sequence
bound by an adhesion molecule other than a classical cadherin;
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 classical cadherin.
143. A method according to claim 142, wherein said cell adhesion
recognition sequence is an occludin cell adhesion recognition
sequence.
144. A method according to claim 142, wherein said antibody or
fragment thereof specifically binds to an occludin cell adhesion
recognition sequence.
145. A method for enhancing and/or directing neurite outgrowth,
comprising contacting a neuron with a cell adhesion modulating
agent, wherein said modulating agent comprises the sequence
His-Ala-Val, and wherein said modulating agent enhances
cadherin-mediated cell adhesion.
146. A method according to claim 145, wherein said modulating agent
comprises at least two His-Ala-Val sequences separated by a
linker.
147. A method according to claim 145, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), AHAVSE (SEQ ID NO: 27), AHAVDI (SEQ ID NO: 28) and
derivatives of the foregoing sequences having one or more
C-terminal, N-terminal and/or side chain modifications.
148. A method for enhancing and/or directing neurite outgrowth,
comprising contacting a neuron with a cell adhesion modulating
agent, wherein said modulating agent comprises an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence, and wherein said modulating agent
enhances cadherin-mediated cell adhesion.
149. A method according to claim 145 or claim 148 wherein said
modulating agent is linked to a targeting agent.
150. A method according to claim 145 or claim 148 wherein said
modulating agent further comprises one or more of: (a) a cell
adhesion recognition sequence bound by an adhesion molecule other
than a classical cadherin, wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val 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 classical cadherin.
151. A method according to claim 150, wherein said cell adhesion
recognition sequence is selected from the group consisting of
Arg-Gly-Asp, Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 12) and
Lys-Tyr-Ser-Phe-Asn-Tyr-Asp-Gly-Ser-Glu (SEQ ID NO: 13).
152. A method according to claim 150, wherein said antibody or
fragment thereof specifically binds to an N-CAM cell adhesion
recognition sequence.
153. A method according to claim 145 or claim 148, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
154. A method according to claim 153, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion,
comprising one or more of: (a) a cell adhesion recognition sequence
bound by an adhesion molecule other than a classical cadherin;
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 classical cadherin.
155. A method according to claim 154, wherein said cell adhesion
recognition sequence is selected from the group consisting of
Arg-Gly-Asp, Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 12) and
Lys-Tyr-Ser-Phe-Asn-Tyr-Asp-Gly-Ser-Glu (SEQ ID NO: 13).
156. A method according to claim 154, wherein said antibody or
fragment thereof specifically binds to an N-CAM cell adhesion
recognition sequence.
157. A method according to claim 145 or claim 148, wherein said
modulating agent is linked to a solid support.
158. A method for treating spinal cord injuries in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises the sequence
His-Ala-Val, and wherein said modulating agent enhances
cadherin-mediated cell adhesion.
159. A method according to claim 158, wherein said modulating agent
comprises at least two His-Ala-Val sequences separated by a
linker.
160. A method for treating spinal cord injuries in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence, and wherein said modulating agent
enhances cadherin-mediated cell adhesion.
161. A method according to claim 158 or claim 160, wherein said
modulating agent further comprises one or more of: (a) a cell
adhesion recognition sequence bound by an adhesion molecule other
than a classical cadherin; 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 classical cadherin.
162. A method according to claim 161, wherein said cell adhesion
recognition sequence comprises a sequence selected from the group
consisting of Arg-Gly-Asp, Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 12) and
Lys-Tyr-Ser-Phe-Asn-Tyr-Asp-Gly-Ser-Glu (SEQ ID NO: 13).
163. A method according to claim 161, wherein said antibody or
fragment thereof specifically binds to an N-CAM cell adhesion
recognition sequence.
164. A method according to claim 158, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), AHAVSE (SEQ ID NO: 27), AHAVDI (SEQ ID NO: 28) and
derivatives of the foregoing sequences having one or more
C-terminal, N-terminal and/or side chain modifications.
165. A method according to claim 158 or claim 160, wherein said
modulating agent is linked to a targeting agent.
166. A method according to claim 158 or claim 160, wherein said
modulating agent is present within a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
167. A method according to claim 166, wherein said pharmaceutical
composition further comprises a modulator of cell adhesion,
comprising one or more of: (a) a cell adhesion recognition sequence
bound by an adhesion molecule other than a classical cadherin;
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 classical cadherin.
168. A method according to claim 167, wherein said cell adhesion
recognition sequence is selected from the group consisting of
Arg-Gly-Asp, Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 12) and
Lys-Tyr-Ser-Phe-Asn-Tyr-Asp-Gly-Ser-Glu (SEQ ID NO: 13).
169. A method according to claim 167, wherein said antibody or
fragment thereof specifically binds to an N-CAM cell adhesion
recognition sequence.
170. A method according to claim 158 or claim 160, wherein said
modulating agent is linked to a solid support.
171. A method for inhibiting synaptic stability in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises the sequence
His-Ala-Val, and wherein said modulating agent inhibits
cadherin-mediated cell adhesion.
172. A method according to claim 171, wherein said modulating agent
comprises a sequence selected from the group consisting of
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), LYSHAVSSNG (SEQ ID NO: 18), AHAVSE (SEQ ID NO:
27), AHAVDI (SEQ ID NO: 28), SHAVSS (SEQ ID NO: 29), LFSHAVSSNG
(SEQ ID NO: 19) and derivatives of the foregoing sequences having
C-terminal, N-terminal and/or side chain modifications.
173. A method for inhibiting synaptic stability in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent, wherein said modulating agent comprises an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence, and wherein said modulating agent
enhances cadherin-mediated cell adhesion.
174. A method according to claim 171 or claim 173, wherein said
modulating agent further comprises one or more of: (a) a cell
adhesion recognition sequence bound by an adhesion molecule other
than a classical cadherin, wherein said cell adhesion recognition
sequence is separated from any His-Ala-Val 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 classical cadherin.
175. A method according to claim 174, wherein said cell adhesion
recognition sequence is Lys-Tyr-Ser-Phe-Asn-Tyr-Asp-Gly-Ser-Glu
(SEQ ID NO: 12).
176. A method according to claim 174, wherein said antibody or
fragment thereof specifically binds to an N-CAM cell adhesion
recognition sequence.
177. A method for identifying an agent capable of modulating
cadherin-mediated cell adhesion, comprising: (a) contacting Schwann
cells with an astrocytic surface in the presence of candidate
modulating agent; (b) washing said astrocytic surface to remove
non-attached cells; and (c) comparing the number of Schwann cells
attached to said astrocytic surface with the number of Schwann
cells attached to an astrocytic surface in the absence of candidate
modulating agent, and therefrom identifying an agent capable of
modulating cadherin-mediated cell adhesion.
178. A method for identifying an agent capable of modulating
cadherin-mediated cell adhesion, comprising: (a) contacting Schwann
cells with polylysine- and/or laminin-coated surface in the
presence of candidate modulating agent; (b) washing said surface to
remove non-attached cells; (c) contacting attached Schwann cells
with an astrocyte-coated surface; and (d) comparing the migration
of said attached Schwann cells with the migration in the absence of
candidate modulating agent, and therefrom identifying an agent
capable of modulating cadherin-mediated cell adhesion.
179. A method for identifying an agent capable of modulating
cadherin-mediated cell adhesion, comprising: (a) culturing neurons
on a monolayer of cells that express N-cadherin in the presence and
absence of a candidate agent, under conditions and for a time
sufficient to allow neurite outgrowth, wherein said cells are
transfected with a polynucleotide encoding N-cadherin and wherein
said cells do not express a detectable level of N-cadherin in the
absence of transfection with such a polynucleotide; (b) determining
a mean neurite length for said neurons; and (c) comparing the mean
neurite length for neurons cultured in the presence of candidate
agent to the neurite length for neurons cultured in the absence of
candidate agent, and therefrom identifying a modulating agent
capable of modulating cell adhesion.
180. A method for identifying an agent capable of modulating
cadherin-mediated cell adhesion, comprising: (a) culturing cells
that express a cadherin 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 said cells, and therefrom identifying an agent capable of
modulating cell adhesion.
181. A method according to claim 180, wherein said cells are
selected from the group consisting of endothelial, epithelial and
cancer cells.
182. A method for identifying an agent capable of modulating
cadherin-mediated cell adhesion, comprising: (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 E-cadherin for cells
cultured in the presence of candidate agent to the level for cells
cultured in the absence of candidate agent, and therefrom
identifying an agent capable of modulating cell adhesion.
183. A method for identifying an agent capable of modulating
cadherin-mediated cell adhesion, comprising: (a) contacting an
epithelial surface of skin with a test marker in the presence and
absence of candidate agent; and (b) comparing the amount of test
marker that passes through said skin in the presence of candidate
agent to the amount that passes through skin in the absence of
candidate agent, and therefrom identifying an agent capable of
modulating cell adhesion.
184. A method for detecting the presence of cadherin-expressing
cells in a sample, comprising: (a) contacting a sample with an
antibody that binds to a modulating agent comprising the sequence
His-Ala-Val under conditions and for a time sufficient to allow
formation of an antibody-cadherin complex; and (b) detecting the
level of antibody-cadherin complex, and therefrom detecting the
presence of cadherin expressing cells in a sample.
185. A method according to claim 184, wherein said antibody is
linked to a support material.
186. A method according to claim 184, wherein said antibody is
linked to a detectable marker.
187. A method according to claim 186, wherein said detectable
marker is a fluorescent marker, and wherein the step of detecting
is performed using fluorescence activated cell sorting.
188. A kit for detecting the presence of cadherin-expressing cells
in a sample, comprising: (a) an antibody that binds to a modulating
agent comprising the sequence His-Ala-Val; and (b) a detection
reagent.
189. A kit for enhancing transdermal drug delivery, comprising: (a)
a skin patch; and (b) a cell adhesion modulating agent, wherein
said modulating agent comprises the sequence His-Ala-Val, and
wherein said modulating agent inhibits cadherin-mediated cell
adhesion.
190. A kit according to claim 189, further comprising a drug.
191. A method for identifying a compound capable of modulating
cadherin-mediated cell adhesion, comprising: (a) contacting an
antibody that binds to a modulating agent comprising the sequence
His-Ala-Val with a test compound; and (b) detecting the level of
antibody that binds to the test compound, and therefrom identifying
a compound capable of modulating cadherin-mediated cell adhesion.
Description
TECHNICAL FIELD
[0001] The present invention relates generally to methods for
modulating cadherin-mediated processes, and more particularly to
the use of modulating agents comprising a cadherin cell adhesion
recognition sequence, or an antibody that specifically recognizes
such a sequence, for inhibiting or enhancing functions such as cell
adhesion.
BACKGROUND OF THE INVENTION
[0002] Multiple sclerosis (MS) is a chronic neurological disease
that affects approximately 250,000 individuals in the United
States. In a patient afflicted with MS, axons become demyelinated
and oligodendrocytes die. Although the clinical course can vary,
the most common form is manifested by relapsing neurological
deficits, including paralysis, sensory deficits, and visual
problems.
[0003] In MS and other demyelinating diseases, Schwann cells are
generally excluded from areas of demyelination and, following axon
damage, regeneration generally fails at Schwann cell-astrocyte
boundaries (Carlstedt et al., Brain Res. Bulletin 22:93-102, 1989).
Inhibition of Schwann cell migration and boundary formation by
astrocytes appears to play a significant part in limiting
spontaneous repair processes in the damaged central nervous system
(CNS).
[0004] In theory, Schwann cells from the peripheral nervous system
could be used to replace damaged oligodendrocytes in the CNS.
However, the efficacy of such treatment has been limited by poor
Schwann cell migration and by boundary formation. When Schwann
cells are grafted into the adult CNS, they can migrate along blood
vessels and meningeal surfaces, but form boundaries where they meet
astrocytes. These boundaries can present an obstacle for
regenerating axons. Thus, recruitment of regenerating axons into
Schwann cell grafts is frequently poor, and axons remaining in the
grafts fail to grow back into CNS tissue unless their target
neurons are immediately adjacent (Brecknell et al., Neurosci.
74:775-784, 1996; Liuzzi and Lasak, Science 237:642-645, 1987).
Transplanted Schwann cells have been found to be capable of
remyelinating central axons of normal (Blakemore, Nature 266:68-69,
1977) or myelin deficient rats (Duncan et al., J. Neurocytol.
17:351-360, 1988), but in both of these cases the area of
remyelination is limited to the region close to the transplantation
site.
[0005] Other approaches to developing a definitive treatment for MS
have also been largely unsuccessful. Corticosteroids and ACTH may
hasten recovery from acute exacerbations, but they do not prevent
future attacks, the development of additional disabilities or
chronic progression of MS. In addition, the substantial side
effects of steroid treatments make these drugs undesirable for
long-term use. Other toxic compounds, such as azathioprine, a
purine antagonist, cyclophosphamide and cyclosporine have also been
used to treat symptoms of MS. Like corticosteroids, however, these
drugs are beneficial at most for a short term and are highly toxic.
More recently, cytokines such as IFN-.gamma. and IFN-.beta. have
been administered in attempts to alleviate the symptoms of MS, but
such treatment has led to a clinical exacerbation for some
patients. Betaseron has also been employed, but with no effect on
the rate of clinical deterioration, and side effects were commonly
observed.
[0006] Accordingly, there is a need in the art for methods for
treating MS that are effective and are not associated with the
disadvantages of the present treatments. The present invention
fulfills this need and further provides other related
advantages.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods for modulating
cadherin-mediated cell adhesion. Within one aspect, methods are
provided for treating a demyelinating neurological disease, such as
multiple sclerosis, in a mammal, comprising administering to a
mammal a cell adhesion modulating agent that inhibits
cadherin-mediated cell adhesion. The modulating agent may comprise
the sequence His-Ala-Val or may comprise an antibody or fragment
thereof that specifically binds to a cadherin cell adhesion
recognition sequence. A modulating agent may be administered by
implantation with Schwann cells or oligodendrocyte progenitor cells
and/or may be administered within a pharmaceutical composition.
[0008] Within further aspects, the present invention provides
methods for reducing unwanted cellular adhesion in a mammal,
comprising administering to a mammal a cell adhesion modulating
agent that inhibits unwanted cadherin-mediated cell adhesion
resulting from surgery, injury, disease or inflammation. The
modulating agent may comprise the sequence His-Ala-Val or may
comprise an antibody or fragment thereof that specifically binds to
a cadherin cell adhesion recognition sequence.
[0009] The present invention further 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 that inhibits cadherin-mediated cell adhesion and
a drug, wherein the step of contacting is performed under
conditions and for a time sufficient to allow passage of said drug
across said epithelial cells. The modulating agent may comprise the
sequence His-Ala-Val or may comprise an antibody or fragment
thereof that specifically binds to a cadherin cell adhesion
recognition sequence.
[0010] Within further aspects, methods are provided for enhancing
the delivery of a drug to a tumor in a mammal, comprising
administering to a mammal a cell adhesion modulating agent that
inhibits cadherin-mediated cell adhesion and a drug. The modulating
agent may comprise 3-16 amino acid residues including the sequence
His-Ala-Val or may comprise an antibody or fragment thereof that
specifically binds to a cadherin cell adhesion recognition
sequence.
[0011] In a related aspect, the present invention provides methods
for treating cancer in a mammal, comprising administering to a
mammal a cell adhesion modulating agent that inhibits
cadherin-mediated cell adhesion and a drug. The modulating agent
may comprise 3-16 amino acid residues including the sequence
His-Ala-Val or may comprise an antibody or fragment thereof that
specifically binds to a cadherin cell adhesion recognition
sequence.
[0012] Within further aspects, methods are provided for inhibiting
angiogenesis in a mammal, comprising administering to a mammal a
cell adhesion modulating agent that inhibits cadherin-mediated cell
adhesion. The modulating agent may comprise the sequence
His-Ala-Val or may comprise an antibody or fragment thereof that
specifically binds to a cadherin cell adhesion recognition
sequence.
[0013] The present invention further provides methods for enhancing
drug delivery to the CNS of a mammal, comprising administering to a
mammal a cell adhesion modulating agent that inhibits
cadherin-mediated cell adhesion. The modulating agent may comprise
3-16 amino acid residues including the sequence His-Ala-Val or may
comprise an antibody or fragment thereof that specifically binds to
a cadherin cell adhesion recognition sequence.
[0014] Within further aspects, the present invention provides
methods for enhancing wound healing in a mammal, comprising
contacting a wound in a mammal with a cell adhesion modulating
agent that enhances cadherin-mediated cell adhesion. The modulating
agent may comprise the sequence His-Ala-Val or may comprise an
antibody or fragment thereof that specifically binds to a cadherin
cell adhesion recognition sequence.
[0015] In a related aspect, methods are provided 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 that enhances
cadherin-mediated cell adhesion. The modulating agent may comprise
the sequence His-Ala-Val or may comprise an antibody or fragment
thereof that specifically binds to a cadherin cell adhesion
recognition sequence.
[0016] Within further aspects, the present invention provides
methods for inducing apoptosis in a cadherin-expressing cell,
comprising contacting a cadherin-expressing cell with a cell
adhesion modulating agent that inhibits cadherin-mediated cell
adhesion. The modulating agent may comprise the sequence
His-Ala-Val or may comprise an antibody or fragment thereof that
specifically binds to a cadherin cell adhesion recognition
sequence.
[0017] The present invention further provides methods for
modulating the immune system of a mammal, comprising administering
to a mammal a pharmaceutical composition comprising a cell adhesion
modulating agent that inhibits cadherin-mediated cell adhesion. The
modulating agent may comprise the sequence His-Ala-Val or may
comprise an antibody or fragment thereof that specifically binds to
a cadherin cell adhesion recognition sequence.
[0018] Within another aspect, the present invention provides
methods for preventing pregnancy in a mammal, comprising
administering to a mammal a cell adhesion modulating agent that
inhibits cadherin-mediated cell adhesion. The modulating agent may
comprise the sequence His-Ala-Val or may comprise an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence.
[0019] In still further aspects, the present invention provides
methods for increasing vasopermeability in a mammal, comprising
administering to a mammal a cell adhesion modulating agent that
inhibits cadherin-mediated cell adhesion. The modulating agent may
comprise the sequence His-Ala-Val or may comprise an antibody or
fragment thereof that specifically binds to a cadherin cell
adhesion recognition sequence.
[0020] The present invention also provides, within further aspects,
methods for enhancing and/or directing neurite outgrowth,
comprising contacting a neuron with a cell adhesion modulating
agent that enhances cadherin-mediated cell adhesion. The modulating
agent may comprise the sequence His-Ala-Val or may comprise an
antibody or fragment thereof that specifically binds to a cadherin
cell adhesion recognition sequence.
[0021] Within related aspects, methods are provided for treating
spinal cord injuries in a mammal, comprising administering to a
mammal a cell adhesion modulating agent that enhances
cadherin-mediated cell adhesion. The modulating agent may comprise
the sequence His-Ala-Val or may comprise an antibody or fragment
thereof that specifically binds to a cadherin cell adhesion
recognition sequence.
[0022] The present invention also provides methods for inhibiting
synaptic stability in a mammal, comprising administering to a
mammal a cell adhesion modulating agent that inhibits
cadherin-mediated cell adhesion. The modulating agent may comprise
the sequence His-Ala-Val or may comprise an antibody or fragment
thereof that specifically binds to a cadherin cell adhesion
recognition sequence.
[0023] In still further aspects, methods are provided for
identifying an agent capable of modulating cadherin-mediated cell
adhesion. One such method comprises the steps of: (a) contacting
Schwann cells with an astrocytic surface in the presence of
candidate modulating agent; (b) washing the astrocytic surface to
remove non-attached cells; and (c) comparing the number of Schwann
cells attached to the astrocytic surface with the number of Schwann
cells attached to an astrocytic surface in the absence of candidate
modulating agent. Another method for identifying an agent capable
of modulating cadherin-mediated cell adhesion comprises the steps
of: (a) contacting Schwann cells with polylysine- and/or
laminin-coated surface in the presence of candidate modulating
agent; (b) washing the surface to remove non-attached cells; (c)
contacting attached Schwann cells with an astrocyte-coated surface;
and (d) comparing the migration of the attached Schwann cells with
the migration in the absence of candidate modulating agent.
[0024] A further method for identifying an agent capable of
modulating cadherin-mediated cell adhesion comprises the steps of:
(a) culturing neurons on a monolayer of cells that express
N-cadherin in the presence and absence of a candidate agent, under
conditions and for a time sufficient to allow neurite outgrowth,
wherein the cells are transfected with a polynucleotide encoding
N-cadherin and wherein the cells do not express a detectable level
of N-cadherin in the absence of transfection with such a
polynucleotide; (b) determining a mean neurite length for the
neurons; and (c) comparing the mean neurite length for neurons
cultured in the presence of candidate agent to the neurite length
for neurons cultured in the absence of candidate agent.
[0025] Yet another method for identifying an agent capable of
modulating cadherin-mediated cell adhesion comprises the steps of:
(a) culturing cells that express a cadherin 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.
[0026] A further method for identifying an agent capable of
modulating cadherin-mediated cell adhesion comprises the steps of:
(a) culturing normal rat kidney (NRK) 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 E-cadherin for cells cultured in the presence of
candidate agent to the level for cells cultured in the absence of
candidate agent.
[0027] Another method for identifying an agent capable of
modulating cadherin surface mediated cell adhesion comprises the
steps of: (a) contacting an epithelial of skin with a test marker
in the presence and absence of candidate agent; and (b) comparing
the amount of test marker that passes through the skin in the
presence of candidate agent to the amount that passes through skin
in the absence of candidate agent.
[0028] Within further aspects, the present invention provides
methods for detecting the presence of cadherin-expressing cells in
a sample, comprising: (a) contacting a sample with an antibody that
binds to a modulating agent comprising the sequence His-Ala-Val
under conditions and for a time sufficient to allow formation of an
antibody-cadherin complex; and (b) detecting the level of
antibody-cadherin complex.
[0029] Within a related aspect, the present invention provides kits
for detecting the presence of cadherin-expressing cells in a
sample, comprising: (a) an antibody that binds to a modulating
agent comprising the sequence His-Ala-Val; and (b) a detection
reagent.
[0030] The present invention also provides, within a further
aspect, kits for enhancing transdermal drug delivery, comprising:
(a) a skin patch; and (b) a cell adhesion modulating agent, wherein
the modulating agent comprises the sequence His-Ala-Val, and
wherein the modulating agent inhibits cadherin-mediated cell
adhesion.
[0031] Within another aspect, the present invention provides
methods for identifying a compound capable of modulating
cadherin-mediated cell adhesion, comprising: (a) contacting an
antibody that binds to a modulating agent comprising the sequence
His-Ala-Val with a test compound; and (b) detecting the level of
antibody that binds to the test compound, and therefrom identifying
a compound capable of modulating cadherin-mediated cell
adhesion.
[0032] 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
[0033] FIG. 1 is a diagram depicting the structure of classical
cadherins. The five extracellular domains are designated EC1-EC5,
the hydrophobic domain that transverses the plasma membrane (PM) is
represented by TM, and the two cytoplasmic domains are represented
by CP1 and CP2. The calcium binding motifs are shown by DXNDN (SEQ
ID NO: 1), DXD and LDRE (SEQ ID NO. 2). The cell adhesion
recognition (CAR) sequence, HAV, is shown within EC1. Cytoplasmic
proteins .beta.-catenin (.beta.), .alpha.-catenin (.alpha.) and
.alpha.-actinin (ACT), which mediate the interaction between
cadherins and microfilaments (MF) are also shown.
[0034] FIG. 2 provides the amino acid sequences of mammalian
classical cadherin EC1 domains: human N-cadherin (SEQ ID NO: 3),
mouse N-cadherin (SEQ ID NO: 4), cow N-cadherin (SEQ ID NO: 5),
human P-cadherin (SEQ ID NO: 6), mouse P-cadherin (SEQ ID NO: 7),
human E-cadherin (SEQ ID NO: 8) and mouse E-cadherin (SEQ ID NO:
9).
[0035] FIGS. 3A-3D are photographs showing cellular boundaries
generated in culture. FIGS. 3A and 3C represent Schwann
cell-astrocyte boundaries. FIG. 3D is the fluorescent counterpart
of 3C. Astrocytes (green) are stained with anti-GFAP and Schwann
cells (orange) are stained with anti GAP-43. Notice the Schwann
cells surrounding one lone astrocyte and only occasional
"finger-like" processes of Schwann cells overlapping the
astrocytes. FIG. 3B depicts a typical Schwann cell-skin fibroblast
"boundary"; note the lack of a defined territorial margin with
clear cellular overlap. Scale bar 50 .mu.m.
[0036] FIGS. 4A and 4B are graphs depicting Schwann cell migration
upon a variety of cellular substrates as determined by the inverted
coverslip migration assay. 1 mm.times.2 mm glass fragments laden
with fluorescently labeled Schwann cells were inverted onto various
substrates and left for 2 days. The number of migrated Schwann
cells per unit distance was recorded and is shown as FIG. 4A; the
furthest distance of migration by the Schwann cells from the edge
of the fragment is shown as FIG. 4B. Schwann cells migrated poorly
upon primary astroglial cultures and upon the astrocytic cell line
A7 compared to fibroblastic or meningeal cell substrates. The most
migration was found upon the non-cellular substrate, laminin. All
data are expressed as the mean.+-.S.E.C. of at least three separate
determinations; each determination consisting of at least 8-12
fragments per group. One way analysis of variance performed upon
the maximum migratory distances revealed a statistical
difference<0.001 between at least one of the groups. A post hoc
multiple comparisons test (Tukey test) revealed significant
differences (p<0.01) between the groups marked with (*) and the
control astrocytes.
[0037] FIGS. 5A and 5B are graphs depicting the migration of
Schwann cells on astrocyte matrix as determined by the
inverted-fragmented-coverslip migration assay. 1 mm.times.2 mm
glass fragments laden with fluorescently labeled Schwann cells were
inverted onto whole astrocyte surfaces, astrocyte matrix or laminin
substrates and left for 2 days. The number of Schwann cells per
unit distance is given as FIG. 5A, with the maximum migration
values as FIG. 5B. Schwann cells migrated poorly upon astrocytes
but considerably better upon astrocyte matrix or laminin. All data
are expressed as the mean.+-.S.E.M. of at least three separate
determinations, each determination consisting of at least 8-12
fragments per group. One way analysis of variance (ANOVA) performed
upon the maximum migratory distances revealed a statistical
difference<0.001 between at least one of the groups. A post hoc
multiple comparisons test (Tukey test) revealed significant
differences (p<0.01) between the groups marked with (*) and the
control astrocytes.
[0038] FIGS. 6A and 6B are graphs depicting the migration of
Schwann cells on laminin, and the effects of astroglial soluble
factors as determined by the inverted-fragmented-coverslip
migration assay. 1 mm.times.2 mm glass fragments laden with
fluorescently labeled Schwann cells were inverted onto laminin, and
left for 2 days in either serum-free (SF) medium or astrocyte
conditioned medium (ACM). The number of Schwann cells per unit
distance is given as FIG. 6A, with the maximum migration values as
FIG. 6B. Schwann cells were found to migrate less well upon laminin
in the absence of serum (c.f. FIG. 1) and considerably better in
ACM. All data are expressed as the mean.+-.S.E.M. of at least three
separate determinations, each determination consisting of at least
8-12 fragments per group. A student's t-test was performed giving
p<0.001 (indicated by *).
[0039] FIGS. 7A and 7B are graphs, and FIG. 7C is a diagram,
depicting the results of timelapse analysis. FIG. 7A shows the mean
migration rates of single Schwann cells plated onto astrocytic,
fibroblastic or laminin substrates and filmed for a period of 6
hours. The position of the cell body was recorded (represented
diagramatically as FIG. 7C) every 30 minutes and the displacement
calculated. The number of cells recorded was 30 upon astrocytes, 25
upon fibroblasts and 30 upon laminin. Schwann cells were found to
migrate fastest on laminin, slowest on astrocytes and slightly
faster on fibroblasts. One way ANOVA performed on the data revealed
a statistical difference (p<0.001) between at least one of the
groups. A post hoc multiple comparisons test (Tukey test) revealed
significant differences (p<0.01) between the groups marked with
(*) and the control astrocytes. FIG. 7B shows the duration of
interaction between Schwann cells and astrocytes and Schwann cells
and fibroblasts. The durations of 50 Schwann cell-astrocyte
interactions and 45 Schwann cell-fibroblast interactions were
recorded. Interactions between Schwann cells and astrocytes were
found to be almost 5 times longer than those with fibroblasts. A
student's t-test was performed and p<0.001 (*). All data are
expressed as the mean.+-.S.E.M. Scale bar in 5C is 50 .mu.m.
[0040] FIGS. 8A-E are photographs illustrating Schwann cells
colliding with sparsely plated astroglia. This series of
photographs was obtained from the timelapse videomicroscopy
apparatus, each picture preceding the next by a period of 2 hours.
In FIG. 8A, a group of two Schwann cells encounters astroglia
(labeled *). The growth cone of one Schwann cell contacts an
astrocyte (arrow indicates first contact). In FIG. 8B, the first
Schwann cell process continues to explore the astrocyte surface
whilst the perikarya of the second Schwann cell contacts another
astrocyte (arrow). The first contact persists beyond the 8 hours of
recording. The second contact is more short-lived, although
astrocyte and Schwann cell remain in close approximation. Scale bar
20 .mu.m.
[0041] FIG. 9 is a histogram depicting the adhesion of Schwann
cells to various substrates. 20,000 DiI-labelled Schwann cells were
plated onto a 13 mm glass coverslip coated with laminin, a complete
monolayer of astrocytes, fibroblasts or Schwann cells and then
placed onto a shaking (25 rpm) platform for 30 minutes. After
washing, the number of cells found still to be attached were
counted. More cells were found to have stuck on the astrocytic
surfaces than on fibroblasts or laminin whereas even more had stuck
to Schwann cells. Note that the speed of migration as determined by
the inverted-fragmented-coverslip migration assay is inversely
proportional to the adhesivity of the substrate. All data are
normalized to control and expressed as the mean.+-.S.E.M. of at
least three separate determinations. One way analysis of variance
(ANOVA) was performed and revealed a statistical
difference<0.001 between at least one of the groups. A post hoc
multiple comparisons test (Tukey test) revealed significant
differences (p<0.01) between the groups marked with (*) and the
control astrocytes.
[0042] FIGS. 10A-C are graphs depicting the effect of lowering
extracellular calcium in reducing Schwann cell-astrocyte adhesion
and improving Schwann cell migration on astrocytes. FIG. 10A shows
Schwann cell-astrocyte adhesion in reduced calcium solutions. The
adhesion assay was performed on astrocytes in normal calcium
solution (control), in 0.2 mM calcium (low calcium) solution and in
the presence of 1.6 mM EGTA. Adhesion was greatly reduced in both
the latter cases. All data are normalized to control and expressed
as the mean.+-.S.E.M. of at least three separate determinations.
One way analysis of variance (ANOVA) was performed and revealed a
statistical difference<0.001 between at least one of the groups.
A post hoc multiple comparisons test (Tukey test) revealed
significant differences (p<0.01) between the groups marked with
(*) and the control astrocytes. FIGS. 10B and C: Schwann cell
migration on astrocytes with reduced extracellular calcium as
determined by the inverted coverslip migration assay. The number of
cells migrated per unit distance is shown in FIG. 10B with maximum
distances represented in FIG. 10C. Schwann cells were found to have
migrated further on astrocytes in the presence of reduced calcium
than on astrocytes in the presence of normal calcium levels. A
student's t-test revealed significant difference between the
maximum distances (*p<0.001).
[0043] FIGS. 11A-D are graphs depicting the effect of cadherin
disruption by representative modulating agents in reducing
intercellular adhesion and promoting Schwann cell migration on
astrocytes. FIG. 11A shows Schwann cell-astrocyte adhesion in the
presence of representative modulating agents. The adhesion assay
was performed in the presence of modulating agent or a similar
(control) peptide without the HAV sequence. The modulating agents
were found to significantly reduce Schwann cell-astrocyte adhesion
compared to either the non-HAV peptide or control. FIG. 11B shows
the ability of N-cadherin blocking antibodies (rabbit
anti-N-cadherin CAR sequence antibodies; designated as L7) to
reduce Schwann cell adhesion to astrocytes and Schwann cells.
Neither a rabbit polyclonal antibody directed against N-CAM nor the
rabbit serum were found to alter intercellular adhesion. All data
in FIGS. 11A and B are normalized with respect to control and
expressed as the mean.+-.S.E.M. of at least three separate
determinations. One way analysis of variance (ANOVA) was performed
and revealed a statistical difference<0.001 between at least one
of the groups. A post hoc multiple comparisons test (Tukey test)
revealed significant differences (p<0.0l) between the groups
marked with (*) and the control astrocytes. FIGS. 11C and 11D:
Increased Schwann cell migration on astrocytes in the presence of
cadherin-function blocking antibodies (L7). FIG. 11C represents
number of cells migrated per unit distance, with FIG. 11D
representing maximum migration distance. Only the anti-cadherin CAR
sequence antibody L7 caused a significant difference as determined
by post hoc analysis (Tukey test) following one way ANOVA
(*p<0.001). The L7 antibody increased migration on astroctyes up
to three fold compared to control.
[0044] FIGS. 12A-D are photographs showing Schwann cell migration
as visualized by the inverted coverslip migration assay. 1
mm.times.2 mm glass fragments laden with fluorescently labeled
Schwann cells were inverted onto various substrates and left for
2-3 days. FIG. 12A shows a fluorescent photograph of Schwann cell
migration normally observed on control astroctyes, with little
spread from the edge of the inverted fragment. FIG. 12B shows a
fluorescent photograph of Schwann cell migration on skin
fibroblasts. Note the considerable number of cells leaving the edge
of the inverted fragment. FIGS. 12C and 12D show a phase and
fluorescent photograph, respectively, of Schwann cell migration on
astroctyes in the presence of the anti-cadherin CAR sequence
antiserum (L7). Notice the astrocyte monolayer in FIG. 12C is
intact. Scale bar for FIGS. 12A and B are 40 .mu.m; for FIGS. 12C
and 12D the scale bar is 100 .mu.m.
DETAILED DESCRIPTION OF THE INVENTION
[0045] As noted above, the present invention provides methods for
modulating cadherin-mediated processes, such as cell adhesion. In
general, to modulate cadherin-mediated cell adhesion, a
cadherin-expressing cell is contacted with a cell adhesion
modulating agent (also referred to herein as a "modulating agent")
either in vivo or in vitro. A modulating agent may comprise the
classical cadherin cell adhesion recognition (CAR) sequence HAV
(i.e., His-Ala-Val), with or without one or more additional CAR
sequences, as described below. Alternatively, or in addition, a
modulating agent may comprise an antibody, or antigen-binding
fragment thereof, that specifically binds to a cadherin CAR
sequence. Within certain aspects, the methods provided herein
inhibit cell adhesion. Such methods may generally be used, for
example, to treat diseases or other conditions characterized by
undesirable cell adhesion or to facilitate drug delivery to a
specific tissue or tumor. Within other aspects, the methods
provided herein may be used to enhance cell adhesion (e.g., to
supplement or replace stitches or to facilitate wound healing).
Within still further aspects, methods are provided for enhancing
and/or directing neurite outgrowth. Within one such aspect, the
present invention provides methods for treating a demyelinating
disorder, such as multiple sclerosis.
[0046] Certain aspects of the present invention are based on the
discovery that cadherin-mediated cell adhesion is involved in
regulating Schwann cell adhesion to astrocytes and in limiting
Schwann cell migration. Cadherins are a rapidly expanding family of
cell adhesion molecules (CAMs). The classical cadherins are
integral membrane glycoproteins that generally promote cell
adhesion through homophilic interactions (a cadherin on the surface
of one cell binds to an identical cadherin on the surface of
another cell), although cadherins also appear to be capable of
forming heterotypic complexes with one another under certain
circumstances and with lower affinity. Cadherins have been shown to
regulate epithelial, endothelial, neural and cancer cell adhesion,
with different cadherins expressed on different cell types. N
(neural)-cadherin is predominantly expressed by neural cells,
endothelial cells and a variety of cancer cell types. E
(epithelial)-cadherin is predominantly expressed by epithelial
cells. Other cadherins are P (placental)-cadherin, which is found
in human skin and R (retinal)-cadherin. A detailed discussion of
the classical cadherins is provided in Munro et al., 1996, In: Cell
Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp.
17-34 (R G Landes Company, Austin Tex.).
[0047] The structures of the classical cadherins are generally
similar. As illustrated in FIG. 1, cadherins are composed of five
extracellular domains (EC1-EC5), a single hydrophobic domain (TM)
that transverses the plasma membrane (PM), and two cytoplasmic
domains (CP1 and CP2). The calcium binding motifs DXNDN (SEQ ID NO:
1), DXD and LDRE (SEQ ID NO: 2) are interspersed throughout the
extracellular domains. The first extracellular domain (EC1)
contains the classical cadherin CAR sequence, HAV (His-Ala-Val),
along with flanking sequences on either side of the CAR sequence
that appear to play a role in conferring specificity. The
three-dimensional solution and crystal structures of the EC1 domain
have been determined (Overduin et al., Science 267:386-389, 1995;
Shapiro et al., Nature 374:327-337, 1995). Sequences of the EC1
domain of some naturally occurring cadherins are shown in FIG. 2
and SEQ ID NOs: 3 to 9.
[0048] Cell Adhesion Modulating Agents
[0049] The term "cell adhesion modulating agent," as used herein,
refers to a molecule comprising at least one cadherin CAR sequence,
generally HAV (His-Ala-Val), and/or an antibody (or antigen-binding
fragment thereof) that specifically binds a cadherin CAR sequence.
Within embodiments in which inhibition of cell adhesion is desired,
a modulating agent may contain one HAV sequence or multiple HAV
sequences, which may be adjacent to one another (i.e., without
intervening sequences) or in close proximity (i.e., separated by
peptide and/or non-peptide linkers to give a distance between the
CAR sequences that ranges from about 0.1 to 400 nm). For example, a
modulating agent with adjacent HAV sequences may comprise the
peptide HAVHAV (SEQ ID NO: 10). A representative modulating agent
with HAV sequences in close proximity may comprise the sequence
SHAVSHAVSHAVS (SEQ ID NO: 11). One or more antibodies, or fragments
thereof, may similarly be used within such embodiments, either
alone or in combination with one or more CAR sequences.
[0050] 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 HAV sequence(s) and/or each
other. Such modulating agents may generally be used within methods
in which it is desirable to simultaneously disrupt cell adhesion
mediated by multiple adhesion molecules. As used herein, an
"adhesion molecule" is any molecule that mediates cell adhesion via
a receptor on the cell's surface. Adhesion molecules include
members of the cadherin gene superfamily that are not classical
cadherins (e.g., proteins that do not contain an HAV sequence
and/or one or more of the other characteristics recited above for
classical cadherins), such as desmogleins (Dsg) and desmocollins
(Dsc); integrins; members of the immunoglobulin supergene family,
such as N-CAM; and other uncategorized transmembrane proteins, such
as occludin) as well as extracellular matrix proteins such as
laminin, fibronectin, collagens, vitronectin, entactin and
tenascin. Preferred CAR sequences for inclusion within a modulating
agent include Arg-Gly-Asp (RGD), which is bound by integrins (see
Cardarelli et al., J. Biol. Chem. 267:23159-64, 1992);
Tyr-Ile-Gly-Ser-Arg (YIGSR; SEQ ID NO: 12), which is bound by
.alpha.6.beta.1 integrin; KYSFNYDGSE (SEQ ID NO: 13), which is
bound by N-CAM; the N-CAM heparan sulfate-binding site
IWKHKGRDVILKKDVRF (SEQ ID NO: 14), the putative Dsc CAR sequences
YAT, FAT and YAS; the putative Dsg CAR sequence RAL; and/or the
putative occludin CAR sequence
GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQ E (SEQ ID NO: 15),
or derivatives thereof such as QSSGSLYGSQ (SEQ ID NO: 16) and
QYLYHYCVVD (SEQ ID NO: 17).
[0051] A linker may be any molecule (including peptide and/or
non-peptide sequences as well as single amino acids or other
molecules), that does not contain a CAR sequence and that can be
covalently linked to at least two peptide sequences. Using a
linker, HAV-containing peptides and other peptide or protein
sequences may be joined head-to-tail (i.e., the linker may be
covalently attached to the carboxyl or amino group of each peptide
sequence), head-to-side chain and/or tail-to-side chain. Modulating
agents comprising one or more linkers may form linear or branched
structures. Within one embodiment, modulating agents having a
branched structure comprise three different CAR sequences, such as
RGD, YIGSR and HAV. Within another embodiment, modulating agents
having a branched structure comprise RGD, YIGSR (SEQ ID NO: 12),
HAV and KYSFNYDGSE (SEQ ID NO: 13). In a third embodiment,
modulating agents having a branched structure comprise HAV, YAT,
FAT, YAS and RAL. Bi-functional modulating agents that comprise an
HAV sequence with flanking E-cadherin-specific sequences joined via
a linker to an HAV sequence with flanking N-cadherin-specific
sequences are also preferred for certain embodiments. Linkers
preferably produce a distance between CAR sequences between 0.1 to
10,000 nm, more preferably about 0.1-400 nm. A separation distance
between recognition sites may generally be determined according to
the desired function of the modulating agent. For inhibitors of
cell adhesion, the linker distance should be small (0.1-400 nm).
For enhancers of cell adhesion, the linker distance should be
400-10,000 nm. One linker that can be used for such purposes is
(H.sub.2N(CH.sub.2).sub.nCO.sub.2H)- .sub.m, or derivatives
thereof, where n ranges from 1 to 10 and m ranges from 1 to 4000.
For example, if glycine (H.sub.2NCH.sub.2CO.sub.2H) or a multimer
thereof is used as a linker, each glycine unit corresponds to a
linking distance of 2.45 angstroms, or 0.245 nm, as determined by
calculation of its lowest energy conformation when linked to other
amino acids using molecular modeling techniques. Similarly,
aminopropanoic acid corresponds to a linking distance of 3.73
angstroms, aminobutanoic acid to 4.96 angstroms, aminopentanoic
acid to 6.30 angstroms and amino hexanoic acid to 6.12 angstroms.
Other linkers that may be used will be apparent to those of
ordinary skill in the art and include, for example, linkers based
on repeat units of 2,3-diaminopropanoic acid, lysine and/or
omithine. 2,3-Diaminopropanoic acid can provide a linking distance
of either 2.51 or 3.11 angstroms depending on whether the
side-chain amino or terminal amino is used in the linkage.
Similarly, lysine can provide linking distances of either 2.44 or
6.95 angstroms and ornithine 2.44 or 5.61 angstroms. Peptide and
non-peptide linkers may generally be incorporated into a modulating
agent using any appropriate method known in the art.
[0052] Within embodiments in which enhancement of cell adhesion is
desired, a modulating agent may contain multiple HAV sequences, or
antibodies that specifically bind to such sequences, joined by
linkers as described above. Enhancement of cell adhesion may also
be achieved by attachment of multiple modulating agents to a
support material, as discussed further below.
[0053] The total number of CAR sequences (including HAV, 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 3 to about 1000 amino
acid residues, preferably from 4 to 50 residues. When non-peptide
linkers are employed, each CAR sequence of the modulating agent is
present within a peptide that generally ranges in size from 3 to 50
residues in length, preferably from 3 to 25 residues, more
preferably from 3 to 16 residues and still more preferably from 4
to 16 residues. Additional residue(s) that may be present on the
N-terminal and/or C-terminal side of a CAR sequence may be derived
from sequences that flank the HAV sequence within one or more
naturally occurring cadherins (e.g., N-cadherin, E-cadherin,
P-cadherin, R-cadherin or other cadherins containing the HAV
sequence) with or without amino acid substitutions and/or other
modifications. Flanking sequences for endogenous N-, E-, P- and
R-cadherin are shown in FIG. 2, and in SEQ ID NOs: 3 to 9.
Alternatively, additional residues present on one or both sides of
the CAR sequence(s) may be unrelated to an endogenous sequence
(e.g., residues that facilitate purification or other manipulation
and/or residues having a targeting or other function).
[0054] A modulating agent may contain sequences that flank the HAV
sequence on one or both sides that are designed to confer
specificity for cell adhesion mediated by one or more specific
cadherins, resulting in tissue and/or cell-type specificity.
Suitable flanking sequences for conferring specificity include, but
are not limited to, endogenous sequences present in one or more
naturally occurring cadherins. Modulating agents having a desired
specificity may be identified using the representative screens
provided herein. Within preferred embodiments, the addition of
appropriate endogenous sequences may result in modulating agents
that specifically disrupt N-cadherin, P-cadherin or E-cadherin
mediated cell adhesion. For example, the peptide modulating agent
LYSHAVSSNG (SEQ ID NO: 18) or LFSHAVSSNG (SEQ ID NO: 19) may be
used to disrupt E-cadherin mediated function, the peptide
modulating agent LFGHAVSENG (SEQ ID NO: 20) may be used to disrupt
P-cadherin mediated function, and the peptide LRAHAVDING (SEQ ID
NO: 21) may be used to disrupt N-cadherin mediated function.
[0055] To facilitate the preparation of modulating agents having a
desired specificity, nuclear magnetic resonance (NMR) and
computational techniques may be used to determine the conformation
of a peptide that confers a known specificity. NMR is widely used
for structural analysis of molecules. Cross-peak intensities in
nuclear Overhauser enhancement (NOE) spectra, coupling constants
and chemical shifts depend on the conformation of a compound. NOE
data provide the interproton distance between protons through
space. This information may be used to facilitate calculation of
the lowest energy conformation for the HAV sequence. Conformation
may then be correlated with tissue specificity to permit the
identification of peptides that are similarly tissue specific or
have enhanced tissue specificity.
[0056] Modulating agents may be polypeptides or salts thereof,
containing only amino acid residues linked by peptide bonds, or may
contain non-peptide regions, such as linkers. Peptide regions of a
modulating agent may comprise residues of L-amino acids, D-amino
acids, or any combination thereof. Amino acids may be from natural
or non-natural sources, provided that at least one amino group and
at least one carboxyl group are present in the molecule; .alpha.-
and .beta.-amino acids are generally preferred. The 20 L-amino
acids commonly found in proteins are identified herein by the
conventional three-letter or one-letter abbreviations indicated in
Table 1, and the corresponding D-amino acids are designated by a
lower case one letter symbol.
1TABLE 1 Amino acid one-letter and three-letter abbreviations A Ala
Alanine R Arg Arginine D Asp Aspartic acid N Asn Asparagine C Cys
Cysteine Q Gln Glutamine E Glu Glutamic acid G Gly Glycine H His
Histidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met
Methionine F Phe Phenylalanine P Pro Proline S Ser Serine T Thr
Threonine W Trp Tryptophan Y Tyr Tyrosine V Val Valine
[0057] 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.
[0058] Certain preferred modulating agents for use within the
present invention comprise at least one of the following sequences:
LRAHAVDING (SEQ ID NO: 21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING
(SEQ ID NO: 23), HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV
(SEQ ID NO: 25), LYSHAVSSNG (SEQ ID NO: 18), LFSHAVSSNG (SEQ ID
NC): 19), LFGHAVSENG (SEQ ID NO: 20), GHAVSE (SEQ ID NO: 26),
AHAVSE (SEQ ID NO: 27), AHAVDI (SEQ ID NO: 28) and/or SHAVSS (SEQ
ID NO: 29), wherein each amino acid residue may, but need not, be
modified as described above. Within one particularly preferred
group, modulating agents comprise an N-terminal acetyl group and/or
a C-terminal amide group. Representative modulating agents
comprising a C-terminal amide group include: LRAHAVDING-NH.sub.2
(SEQ ID NO: 21), LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22),
MRAHAVDING-NH.sub.2 (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ
ID NO: 24), FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25),
LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), LFSHAVSSNG-NH.sub.2 (SEQ ID
NO: 19), LFGHAVSENG-NH.sub.2 (SEQ ID NO: 20), GHAVSE-NH.sub.2 (SEQ
ID NO: 26), AHAVSE-NH.sub.2 (SEQ ID NO: 27), AHAVDI-NH.sub.2 (SEQ
ID NO: 28), SHAVSS-NH.sub.2 (SEQ ID NO: 29) and compounds
comprising such sequences or derivatives thereof. Representative
modulating agents comprising a N-terminal acetyl group and a
C-terminal amide group include: N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID
NO: 21), N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22),
N-Ac-MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25),
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-LFSHAVSSNG-NH.sub.2
(SEQ ID NO: 19), N-Ac-LFGHAVSENG-NH.sub.2 (SEQ ID NO: 20),
N-Ac-GHAVSE-NH.sub.2 (SEQ ID NO: 26), N-Ac-AHAVSE-NH.sub.2 (SEQ ID
NO: 27), N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28), N-Ac-SHAVSS-NH.sub.2
(SEQ ID NO: 29) and compounds comprising such sequences or
derivatives thereof.
[0059] Within certain other preferred embodiments, as discussed
below, relatively small modulating agents that do not contain
significant sequences flanking the HAV sequence (e.g.,
AHAVSE-NH.sub.2; SEQ ID NO: 27) are preferred for modulating
N-cadherin and E-cadherin mediated cell adhesion. Such modulating
agents can be thought of as "master keys" that fit into peptide
binding sites of each of the different classical cadherins, and are
capable of disrupting cell adhesion of neural cells, endothelial
cells, epithelial cells and/or certain cancer cells. Such
modulating agents may generally by used to specifically modulate
cell adhesion of neural or other cell types by topical
administration or by linking a suitable targeting agent to the
peptide, as discussed below. Such peptides may, but need not,
contain an N-acetyl group and/or a C-amide group (e.g.,
N-Ac-AHAVSE-NH.sub.2; SEQ ID NO: 27).
[0060] Peptide modulating agents (and peptide portions of
modulating agents) as described herein may be synthesized by
methods well known in the art, including chemical synthesis and
recombinant DNA methods. For modulating agents up to about 50
residues in length, chemical synthesis may be performed using solid
phase peptide synthesis techniques, in which a peptide linkage
occurs through the direct condensation of the .alpha.-amino group
of one amino acid with the .alpha.-carboxy group of the other amino
acid with the elimination of a water molecule. Peptide bond
synthesis by direct condensation, as formulated above, requires
suppression of the reactive character of the amino group of the
first and of the carboxyl group of the second amino acid. The
masking substituents must permit their ready removal, without
inducing breakdown of the labile peptide molecule.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] N-acetylation of the N-terminal residue can be accomplished
by reacting the final peptide with acetic anhydride before cleavage
from the resin. C-amidation may be accomplished using an
appropriate resin such as methylbenzhydrylamine resin using the Boc
technology.
[0065] For longer modulating agents, recombinant methods are
preferred for synthesis. Within such methods, all or part of a
modulating agent can be synthesized in living cells, using any of a
variety of expression vectors known to those of ordinary skill in
the art to be appropriate for the particular host cell. Suitable
host cells may include bacteria, yeast cells, mammalian cells,
insect cells, plant cells, algae and other animal cells (e.g.,
hybridoma, CHO, myeloma). The DNA sequences expressed in this
manner may encode portions of an endogenous cadherin or other
adhesion molecule. Such sequences may be prepared based on known
cDNA or genomic sequences (see Blaschuk et al., J. Mol. Biol.
211:679-682, 1990), or from sequences isolated by screening an
appropriate library with probes designed based on the sequences of
known cadherins. Such screens may generally be performed as
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,
1989 (and references cited therein). Polymerase chain reaction
(PCR) may also be employed, using oligonucleotide primers in
methods well known in the art, to isolate nucleic acid molecules
encoding all or a portion of an endogenous adhesion molecule. To
generate a nucleic acid molecule encoding a desired modulating
agent, an endogenous cadherin sequence may be modified using well
known techniques. For example, portions encoding one or more CAR
sequences may be joined, with or without separation by nucleic acid
regions encoding linkers, as discussed above. Alternatively,
portions of the desired nucleic acid sequences may be synthesized
using well known techniques, and then ligated together to form a
sequence encoding the modulating agent.
[0066] As noted above, instead of (or in addition to) an HAV
sequence, a modulating agent may comprise an antibody, or
antigen-binding fragment thereof, that specifically binds to a
cadherin CAR sequence. As used herein, an antibody, or
antigen-binding fragment thereof, is said to "specifically bind" to
a cadherin CAR sequence (with or without flanking amino acids) if
it reacts at a detectable level (within, for example, an ELISA, as
described by Newton et al., Develop. Dynamics 197:1-13, 1993) with
a peptide containing that sequence, and does not react detectably
with peptides containing a different CAR sequence or a sequence in
which the order of amino acid residues in the cadherin CAR sequence
and/or flanking sequence is altered.
[0067] Within certain aspects of the present invention, modulating
agents comprising polyclonal or monoclonal antibodies may be used
to enhance and/or direct neurite outgrowth. Modulating agents
comprising antibodies or antigen-binding fragments thereof (e.g.,
Fab fragments) may also be used, within other aspects, to inhibit
cell adhesion in a variety of contexts. For example, such
modulating agents may be used for treatment of demyelinating
diseases, such as MS, or to inhibit interactions between tumor
cells. Within further aspects, modulating agents comprising
antibodies or antigen-binding fragments thereof that are linked to
one or more linkers, or to a single molecule or support material
may be used to enhance cell adhesion.
[0068] Polyclonal and monoclonal antibodies may be raised against a
cadherin 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 cadherin 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.
[0069] Monoclonal antibodies specific for the cadherin 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.
[0070] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies, with or without the use of various
techniques known in the art to enhance the yield. Contaminants may
be removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction.
Antibodies having the desired activity may generally be identified
using immunofluorescence analyses of tissue sections, cell or other
samples where the target cadherin is localized.
[0071] Within preferred embodiments, such monoclonal antibodies are
specific for particular cadherins (e.g., the antibodies bind to
E-cadherin, but do not bind significantly to N-cadherin, or vise
versa). Such antibodies may be prepared as described above, using
an immunogen that comprises (in addition to the HAV sequence)
sufficient flanking sequence to generate the desired specificity
(e.g., 6 amino acids on each side is generally sufficient). One
representative immunogen is the 15-mer FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO: 25), linked to KLH (see Newton et al., Dev. Dynamics
197:1-13, 1993). To evaluate the specificity of a particular
antibody, representative assays as described herein and/or
conventional antigen-binding assays may be employed. Such
antibodies may generally be used for therapeutic, diagnostic and
assay purposes, as described herein. For example, such antibodies
may be linked to a drug and administered to a mammal to target the
drug to a particular cadherin-expressing cell, such as a leukemic
cell in the blood.
[0072] 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).
[0073] Evaluation of Modulating Agent Activity
[0074] As noted above, modulating agents as described herein are
capable of enhancing or inhibiting cadherin-mediated cell adhesion.
The ability of an agent to modulate cell adhesion may generally be
evaluated in vitro by assaying the effect on one or more of the
following: (1) Schwann cell-astrocyte adhesion, (2) Schwann cell
migration on astrocyte monolayers, (3) neurite outgrowth, (4)
adhesion between endothelial cells, (5) adhesion between epithelial
cells (e.g., normal rat kidney cells and/or human skin) and/or (6)
adhesion between cancer cells. In general, a modulating agent is an
inhibitor of cell adhesion if, within one or more of these
representative assays, contact of the test cells with the
modulating agent results in a discernible disruption of cell
adhesion. Modulating agents that enhance cell adhesion (e.g.,
agents comprising multiple HAV sequences and/or linked to a support
material) are considered be modulators of cell adhesion if they are
capable of enhancing neurite outgrowth as described below or are
capable of promoting cell adhesion, as judged by plating assays to
assess epithelial cell adhesion to a modulating agent attached to a
support material, such as tissue culture plastic.
[0075] The effect of a modulating agent on Schwann cell adhesion to
astrocytes may generally be evaluated using a cell adhesion assay.
Briefly, Schwann cells fluorescently labeled with Di-I may be
plated onto an astrocytic surface (e.g., a glass coverslip coated
with a monolayer of astrocytes) and incubated on a shaking platform
(e.g., 25 rpm for 30 minutes) in the presence and absence of
modulating agent (e.g., LRAHAVDING (SEQ ID NO: 21) at a
concentration of 1 mg/mL). Cells may then be washed (e.g., in Hanks
medium) to remove non-attached cells. The attached cells may then
be fixed and counted (e.g., using a fluorescent microscope). In
general, 1 mg/mL of a modulating agent results in an increase or
decrease in cell adhesion of at least 50%. This assay evaluates the
effect of a modulating agent on N-cadherin mediated cell
adhesion.
[0076] Schwann cell migration may generally be evaluated using a
micro-inverted-coverslip assay. In this assay, a dense Schwann cell
culture is established on coverslip fragments and Schwann cell
migration away from the fragment edge is measured. Briefly, Schwann
cells fluorescently labeled with Di-I may be plated on polylysine-
and laminin-coated fragments of a glass coverslip and allowed to
bind to the surface for 16-18 hours. Cells may then be washed
(e.g., in Hanks medium) to remove non-attached cells, and then
inverted, with cells facing downward onto an astrocyte-coated
surface. Cultures are then incubated further for 2 days in the
presence or absence of modulating agent (e.g., LRAHAVDING (SEQ ID
NO: 21) at a concentration of 1 mg/mL) and fixed. The maximum
migration distance from the edge of the coverslip fragment may then
be measured. At a level of 1 mg/mL, modulating agent results in an
increase or decrease in the maximum migration distance of at least
50%. This assay evaluates the effect of a modulating agent on
N-cadherin mediated cell adhesion.
[0077] Within a representative neurite outgrowth assay, neurons may
be cultured on a monolayer of cells (e.g., 3T3) that express
N-cadherin. Neurons grown on such cells (under suitable conditions
and for a sufficient period of time) extend longer neurites than
neurons cultured on cells that do not express N-cadherin. For
example, neurons may be cultured on monolayers of 3T3 cells
transfected with cDNA encoding N-cadherin essentially as described
by Doherty and Walsh, Curr. Op. Neurobiol. 4:49-55, 1994; Williams
et al., Neuron 13:583-594, 1994; Hall et al., Cell Adhesions and
Commun. 3:441-450, 1996; Doherty and Walsh, Mol. Cell. Neurosci.
8:99-111, 1994; and Safell et al., Neuron 18:231-242, 1997.
Briefly, monolayers of control 3T3 fibroblasts and 3T3 fibroblasts
that express N-cadherin may be established by overnight culture of
80,000 cells in individual wells of an 8-chamber well tissue
culture slide. 3000 cerebellar neurons isolated from post-natal day
3 mouse brains may be cultured for 18 hours on the various
monolayers in control media (SATO/2% FCS), or media supplemented
with various concentrations of the modulating agent or control
peptide. The cultures may then be fixed and stained for GAP43 which
specifically binds to the neurons and their neurites. The length of
the longest neurite on each GAP43 positive neuron may be measured
by computer assisted morphometry.
[0078] A modulating agent that modulates N-cadherin-mediated cell
adhesion may inhibit or enhance such neurite outgrowth. Under the
conditions described above, the presence of 500 .mu.g/mL of a
modulating agent that disrupts neural cell adhesion should result
in a decrease in the mean neurite length by at least 50%, relative
to the length in the absence of modulating agent or in the presence
of a negative control peptide. Alternatively, the presence of 500
.mu.g/mL of a modulating agent that enhances neural cell adhesion
should result in an increase in the mean neurite length by at least
50%.
[0079] Within certain cell adhesion assays, the addition of a
modulating agent to cells that express a cadherin results in
disruption of cell adhesion. A "cadherin-expressing cell," as used
herein, may be any type of cell that expresses at least one
cadherin on the cell surface at a detectable level, using standard
techniques such as immunocytochemical protocols (e.g., Blaschuk and
Farookhi, Dev. Biol. 136:564-567, 1989). Cadherin-expressing cells
include endothelial, epithelial and/or cancer cells. For example,
such cells may be plated under standard conditions that, in the
absence of modulating agent, permit cell adhesion. In the presence
of modulating agent (e.g., 500 .mu.g/mL), disruption of cell
adhesion may be determined visually within 24 hours, by observing
retraction of the cells from one another.
[0080] For use within one such assay, bovine pulmonary artery
endothelial cells may be harvested by sterile ablation and
digestion in 0.1% collagenase (type II; Worthington Enzymes,
Freehold, N.J.). Cells may be maintained in Dulbecco's minimum
essential medium supplemented with 10% fetal calf serum and 1%
antibiotic-antimycotic at 37.degree. C. in 7% CO.sub.2 in air.
Cultures may be passaged weekly in trypsin-EDTA and seeded onto
tissue culture plastic at 20,000 cells/cm.sup.2. Endothelial
cultures may be used at 1 week in culture, which is approximately 3
days after culture confluency is established. The cells may be
seeded onto coverslips and treated (e.g., for 30 minutes) with
modulating agent or a control compound at, for example, 500
.mu.g/ml and then fixed with 1% paraformaldehyde. As noted above,
disruption of cell adhesion may be determined visually within 24
hours, by observing retraction of the cells from one another. This
assay evaluates the effect of a modulating agent on N-cadherin
mediated cell adhesion.
[0081] Within another such assay, the effect of a modulating agent
on normal rat kidney (NRK) cells may be evaluated. According to a
representative procedure, NRK cells (ATCC #1571-CRL) may be plated
at 10-20,000 cells per 35 mm tissue culture flasks containing DMEM
with 10% FCS and sub-cultured periodically (Laird et al., J. Cell
Biol. 131:1193-1203, 1995). Cells may be harvested and replated in
35 mm tissue culture flasks containing 1 mm coverslips and
incubated until 50-65% confluent (24-36 hours). At this time,
coverslips may be transferred to a 24-well plate, washed once with
fresh DMEM and exposed to modulating agent at a concentration of,
for example, 1 mg/mL for 24 hours. Fresh modulating agent may then
be added, and the cells left for an additional 24 hours. Cells may
be fixed with 100% methanol for 10 minutes and then washed three
times with PBS. Coverslips may be blocked for 1 hour in 2% BSA/PBS
and incubated for a further 1 hour in the presence of mouse
anti-E-cadherin antibody (Transduction Labs, 1:250 dilution).
Primary and secondary antibodies may be diluted in 2% BSA/PBS.
Following incubation in the primary antibody, coverslips may be
washed three times for 5 minutes each in PBS and incubated for 1
hour with donkey anti-mouse antibody conjugated to fluorescein
(diluted 1:200). Following further washes in PBS (3.times.5 min)
coverslips can be mounted and viewed by confocal microscopy.
[0082] In the absence of modulating agent, NRK cells form
characteristic tightly adherent monolayers with a cobblestone
morphology in which cells display a polygonal shape. NRK cells that
are treated with a modulating agent that disrupts E-cadherin
mediated cell adhesion may assume a non-polygonal and elongated
morphology (i.e., a fibroblast-like shape) within 48 hours of
treatment with 1 mg/mL of modulating agent. Gaps appear in
confluent cultures of such cells. In addition, 1 mg/mL of such a
modulating agent reproducibly induces a readily apparent reduction
in cell surface staining of E-cadherin, as judged by
immunofluorescence microscopy (Laird et al., J. Cell Biol.
131:1193-1203, 1995), of at least 75% within 48 hours.
[0083] A third cell adhesion assay involves evaluating the effect
of a modulating agent on permeability of adherent epithelial and/or
endothelial cell layers. For example, the effect of permeability on
human skin may be evaluated. Such skin may be derived from a
natural source or may be synthetic. Human abdominal skin for use in
such assays may generally be obtained from humans at autopsy within
24 hours of death. Briefly, a modulating agent (e.g., 500 .mu.g/ml)
and a test marker (e.g., the fluorescent markers Oregon Green.TM.
and Rhodamine Green.TM. Dextran) may be dissolved in a sterile
buffer (e.g., phosphate buffer, pH 7.2), and the ability of the
marker to penetrate through the skin and into a receptor fluid
(e.g., phosphate buffer) may be measured using a Franz Cell
apparatus (Franz, Curr. Prob. Dermatol. 7:58-68, 1978; Franz, J.
Invest. Dermatol. 64:190-195, 1975). The penetration of the markers
through the skin may be assessed at, for example, 6, 12, 24, 36,
and 48 hours after the start of the experiment. In general, a
modulating agent that enhances the permeability of human skin
results in a statistically significant increase in the amount of
marker in the receptor compartment after 6-48 hours in the presence
of 500 .mu.g/mL modulating agent. This assay evaluates the effect
of a modulating agent on E-cadherin mediated cell adhesion.
[0084] Modulating Agent Modification and Formulations
[0085] 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 RGD sequence) may be attached to a
support such as a polymeric matrix, preferably in an alternating
pattern.
[0086] 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.
[0087] 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.
[0088] Although modulating agents as described herein may
preferentially bind to specific tissues or cells, and thus may be
sufficient to target a desired site in vivo, it may be beneficial
for certain applications to include an additional targeting agent.
Accordingly, a targeting agent may also, or alternatively, be
linked to a modulating agent to facilitate targeting to one or more
specific tissues. As used herein, a "targeting agent," may be any
substance (such as a compound or cell) that, when linked to a
modulating agent enhances the transport of the modulating agent to
a target tissue, thereby increasing the local concentration of the
modulating agent. Targeting agents include antibodies or fragments
thereof, receptors, ligands and other molecules that bind to cells
of, or in the vicinity of, the target tissue. Known targeting
agents include serum hormones, antibodies against cell surface
antigens, lectins, adhesion molecules, tumor cell surface binding
ligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes
and those drugs and proteins that bind to a desired target site.
Among the many monoclonal antibodies that may serve as targeting
agents are anti-TAC, or other interleukin-2 receptor antibodies;
9.2.27 and NR-ML-05, reactive with the 250 kilodalton human
melanoma-associated proteoglycan; and NR-LU-10, reactive with a
pancarcinoma glycoprotein. An antibody targeting agent may be an
intact (whole) molecule, a fragment thereof, or a functional
equivalent thereof. Examples of antibody fragments are F(ab')2,
-Fab', Fab and F[v] fragments, which may be produced by
conventional methods or by genetic or protein engineering. Linkage
is generally covalent and may be achieved by, for example, direct
condensation or other reactions, or by way of bi- or
multi-functional linkers. Within other embodiments, it may also be
possible to target a polynucleotide encoding a modulating agent to
a target tissue, thereby increasing the local concentration of
modulating agent. Such targeting may be achieved using well known
techniques, including retroviral and adenoviral infection.
[0089] 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.
[0090] 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.
[0091] For certain embodiments, as discussed below, a
pharmaceutical composition may further comprise a modulator of cell
adhesion that is mediated by one or more molecules other than
cadherins. Such modulators may generally be prepared as described
above, incorporating one or more non-cadherin CAR sequences and/or
antibodies thereto in place of the cadherin CAR sequences and
antibodies. Such compositions are particularly useful for
situations in which it is desirable to inhibit cell adhesion
mediated by multiple cell-adhesion molecules, such as other members
of the cadherin gene superfamily that are not classical cadherins
(e.g., Dsg and Dsc); integrins; members of the immunoglobulin
supergene family, such as N-CAM; and other uncategorized
transmembrane proteins, such as occludin, as well as extracellular
matrix proteins such as laminin, fibronectin, collagens,
vitronectin, entactin and tenascin. Preferred CAR sequences for use
within such a modulator include RGD, YIGSR (SEQ ID NO: 12),
KYSFNYDGSE (SEQ ID NO: 13), IWKHKGRDVILKKDVRF (SEQ ID NO: 14), YAT,
FAT, YAS, RAL and/or GVNPTAQSSGSLYGSQIYALCNQFYTP
AATGLYVDQYLYHYCVVDPQE (SEQ ID NO: 15), or derivatives thereof such
as QSSGSLYGSQ (SEQ ID NO: 16) and QYLYHYCVVD (SEQ ID NO: 17).
[0092] 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.
[0093] For imaging purposes, any of a variety of diagnostic agents
may be incorporated into a pharmaceutical composition, either
linked to a modulating agent or free within the composition.
Diagnostic agents include any substance administered to illuminate
a physiological function within a patient, while leaving other
physiological functions generally unaffected. Diagnostic agents
include metals, radioactive isotopes and radioopaque agents (e.g.,
gallium, technetium, indium, strontium, iodine, barium, bromine and
phosphorus-containing compounds), radiolucent agents, contrast
agents, dyes (e.g., fluorescent dyes and chromophores) and enzymes
that catalyze a colorimetric or fluorometric reaction. In general,
such agents may be attached using a variety of techniques as
described above, and may be present in any orientation.
[0094] 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.
[0095] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). Appropriate dosages and a suitable duration and
frequency of administration will be determined by such factors as
the condition of the patient, the type and severity of the
patient's disease and the method of administration. In general, an
appropriate dosage and treatment regimen provides the modulating
agent(s) in an amount sufficient to provide therapeutic and/or
prophylactic benefit. Within particularly preferred embodiments of
the invention, a modulating agent or pharmaceutical composition as
described herein may be administered at a dosage ranging from 0.001
to 50 mg/kg body weight, preferably from 0.1 to 20 mg/kg, on a
regimen of single or multiple daily doses. For topical
administration, a cream typically comprises an amount of modulating
agent ranging from 0.00001% to 1%, preferably 0.0001% to 0.002%.
Fluid compositions typically contain 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.
[0096] Modulating Agent Methods of Use
[0097] In general, the modulating agents and compositions described
herein may be used for modulating the adhesion of
cadherin-expressing cells (i.e., cells that express one or more of
E-cadherin, N-cadherin, P-cadherin, R-cadherin and/or other
cadherin(s) containing the HAV sequence, including as yet
undiscovered cadherins) in vitro and/or in vivo. As noted above,
modulating agents for purposes that involve the disruption of
cadherin-mediated cell adhesion may comprise an HAV sequence,
multiple HAV sequences in close proximity and/or an antibody (or an
antigen-binding fragment thereof) that recognizes a cadherin 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 HAV sequence by linkers. As noted above, such linkers may or
may not comprise one or more amino acids. For enhancing cell
adhesion, a modulating agent may contain multiple HAV 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.
[0098] Certain methods involving the disruption of cell adhesion a,
described herein have an advantage over prior techniques in that
they permit the passage of molecules that are large and/or charged
across barriers of cadherin-expressing cells. As described in
greater detail below, modulating agents as described herein may
also be used to disrupt or enhance cell adhesion in a variety of
other contexts. Within each of the methods described herein, one or
more modulating agents may generally be administered alone, or
within a pharmaceutical composition. In each specific method
described herein, as noted above, a targeting agent may be employed
to increase the local concentration of modulating agent at the
target site.
[0099] Within one aspect, one or more modulating agents may be used
for therapy of a demyelinating neurological disease in a mammal.
There are a number of demyelinating diseases, such as multiple
sclerosis, characterized by oligodendrocyte death. It has been
found, within the context of the present invention, that Schwann
cell migration on astrocytes is inhibited by N-cadherin. Modulating
agents that disrupt N-cadherin mediated cell adhesion as described
herein, when implanted with Schwann cells into the central nervous
system, may facilitate Schwann cell migration and permit the
practice of Schwann cell replacement therapy.
[0100] Multiple sclerosis patients suitable for treatment may be
identified by criteria that establish a diagnosis of clinically
definite or clinically probable MS (see Poser et al., Ann. Neurol.
13:227, 1983). Candidate patients for preventive therapy may be
identified by the presence of genetic factors, such as HLA-type
DR2a and DR2b, or by the presence of early disease of the relapsing
remitting type.
[0101] Schwann cell grafts may be implanted directly into the brain
along with the modulating agent(s) using standard techniques.
Preferred peptide modulating agents for use within such methods
include LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), LRAHAVDVNG-NH.sub.2
(SEQ ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO: 25), AHAVSE-NH.sub.2 (SEQ ID NO: 27), AHAVDI-NH.sub.2
(SEQ ID NO: 28), N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2
(SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), N-Ac-AHAVSE-NH.sub.2
(SEQ ID NO: 27), N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28), derivatives
of such sequences and modulating agents comprising any one of these
sequences or derivatives thereof. Preferred antibody modulating
agents include Fab fragments directed against the N-cadherin CAR
sequence FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25). Such antibodies
and fragments can be prepared using standard techniques, as
discussed above. Suitable amounts of modulating agent generally
range as described above, preferably from about 10 .mu.g/mL to
about 1 mg/mL.
[0102] Alternatively, a modulating agent may be implanted with
oligodendrocyte progenitor cells (OPs) derived from donors not
afflicted with the demyelinating disease. The myelinating cell of
the CNS is the oligodendrocyte. Although mature oligodendrocytes
and immature cells of the oligodendrocyte lineage, such as the
oligodendrocyte type 2 astrocyte progenitor, have been used for
transplantation, OPs are more widely used. OPs are highly motile
and are able to migrate from transplant sites to lesioned areas
where they differentiate into mature myelin-forming
oligodendrocytes and contribute to repair of demyelinated axons
(see e.g., Groves et al., Nature 362:453-55, 1993; Baron-Van
Evercooren et al., Glia 16:147-64, 1996). OPs can be isolated using
routine techniques known in the art (see e.g., Milner and
French-Constant, Development 120:3497-3506, 1994), from many
regions of the CNS including brain, cerebellum, spinal cord, optic
nerve and olfactory bulb. Substantially greater yields of OP's are
obtained from embryonic or neonatal rather than adult tissue. OPs
may be isolated from human embryonic spinal cord and cultures of
neurospheres established. Human fetal tissue is a potential
valuable and renewable source of donor OP's for future, long range
transplantation therapies of demyelinating diseases such as MS.
[0103] OPs can be expanded in vitro if cultured as "homotypic
aggregates" or "spheres" (Avellana-Adalid et al, J. Neurosci. Res.
45:558-70, 1996). Spheres (sometimes called "oligospheres" or
"neurospheres") are formed when OPs are grown in suspension in the
presence of growth factors such as PDGF and FGF. OPs can be
harvested from spheres by mechanical dissociation and used for
subsequent transplantation or establishment of new spheres in
culture. Alternatively, the spheres themselves may be transplanted,
providing a "focal reservoir" of OPs (Avellana-Adalid et al, J.
Neurosci. Res. 45:558-70, 1996).
[0104] An alternative source of OP may be spheres derived from CNS
stem cells. Recently, Reynolds and Weiss, Dev. Biol. 165:1-13, 1996
have described spheres formed from EGF-responsive cells derived
from embryonic neuroepithelium, which appear to retain the
pluripotentiality exhibited by neuroepithelium in vivo. Cells
dissociated from these spheres are able to differentiate into
neurons, oligodendrocytes and astrocytes when plated on adhesive
substrates in the absence of EGF, suggesting that EGF-responsive
cells derived from undifferentiated embryonic neuroepithelium may
represent CNS stem cells (Reynolds and Weiss, Dev. Biol. 165:1-13,
1996). Spheres derived from CNS stem cells provide an alternative
source of OP which may be manipulated in vitro for transplantation
in vivo. Spheres composed of CNS stem cells may further provide a
microenvironment conducive to increased survival, migration, and
differentiation of the OPs in vivo.
[0105] The use of neurospheres for the treatment of MS may be
facilitated by modulating agents that enhance cell migration from
the spheres. In the absence of modulating agent, the cells within
the spheres adhere tightly to one another and migration out of the
spheres is hindered. Modulating agents that disrupt N-cadherin
mediated cell adhesion as described herein, when injected with
neurospheres into the central nervous system, may improve cell
migration and increase the efficacy of OP replacement therapy.
[0106] Neurosphere grafts may be implanted directly into the
central nervous system along with the modulating agent(s) using
standard techniques. Preferred peptide modulating agents for use
within such methods include LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID
NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), AHAVSE-NH.sub.2 (SEQ ID
NO: 27), AHAVDI-NH.sub.2 (SEQ ID NO: 28), N-Ac-LRAHAVDING-NH.sub.2
(SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22),
N-Ac-MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.s- ub.2 (SEQ ID NO: 25),
N-Ac-AHAVSE-NH.sub.2 (SEQ ID NO: 27), N-Ac-AHAVDI-NH.sub.2 (SEQ ID
NO: 28) and derivatives of such sequences. Modulating agents
comprising one or more of these sequences or derivatives thereof
are also preferred. Preferred antibody modulating agents include
Fab fragments directed against the N-cadherin CAR sequence
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25). Such antibodies and
fragments can be prepared using standard techniques, as discussed
above. Suitable amounts of modulating agent generally range as
described above, preferably from about 10 .mu.g/mL to about 1
mg/mL.
[0107] Alternatively, a modulating agent may be administered alone
or within a pharmaceutical composition. The duration and frequency
of administration will be determined by such factors as the
condition of the patient, and the type and severity of the
patient's disease. Within particularly preferred embodiments of the
invention, the modulating agent or pharmaceutical composition may
be administered at a dosage ranging from 0.1 mg/kg to 20 mg/kg
although appropriate dosages may be determined by clinical trials.
Methods of administration include injection, intravenous or
intrathecal (i.e., directly in cerebrospinal fluid). A modulating
agent or pharmaceutical composition may further comprise a drug
(e.g., an immunomodulatory drug).
[0108] Effective treatment of multiple sclerosis may be evidenced
by any of the following criteria: EDSS (extended disability status
scale), appearance of exacerbations or MRI (magnetic resonance
imaging). The EDSS is a means to grade clinical impairment due to
MS (Kurtzke, Neurology 33:1444, 1983), and a decrease of one fill
step defines an effective treatment in the context of the present
invention (Kurtzke, Ann. Neurol. 36:573-79, 1994). Exacerbations
are defined as the appearance of a new symptom that is attributable
to MS and accompanied by an appropriate new neurologic abnormality
(Sipe et al., Neurology 34:1368, 1984). Therapy is deemed to be
effective if there is a statistically significant difference in the
rate or proportion of exacerbation-free patients between the
treated group and the placebo group or a statistically significant
difference in the time to first exacerbation or duration and
severity in the treated group compared to control group. MRI can be
used to measure active lesions using gadolinium-DTPA-enhanced
imaging (McDonald et al. Ann. Neurol. 36:14, 1994) or the location
and extent of lesions using T.sub.2-weighted techniques. The
presence, location and extent of MS lesions may be determined by
radiologists using standard techniques. Improvement due to therapy
is established when there is a statistically significant
improvement in an individual patient compared to baseline or in a
treated group versus a placebo group.
[0109] Efficacy of the modulating agent in the context of
prevention may be judged based on clinical measurements such as the
relapse rate and EDSS. Other criteria include a change in area and
volume of T2 images on MRI, and the number and volume of lesions
determined by gadolinium enhanced images.
[0110] Within other aspects, methods are provided in which cell
adhesion is diminished. In one such aspect, the present invention
provides methods for reducing unwanted cellular adhesion by
administering a modulating agent as described herein. Unwanted
cellular adhesion can occur between tumor cells, between tumor
cells and normal cells or between normal cells as a result of
surgery, injury, chemotherapy, disease, inflammation or other
condition jeopardizing cell viability or function. Preferred
modulating agents for use within such methods include
LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), LRAHAVDVNG-NH.sub.2 (SEQ ID
NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO: 25), LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18),
AHAVSE-NH.sub.2 (SEQ ID NO: 27), AHAVDI-NH.sub.2 (SEQ ID NO: 28),
SHAVSS-NH.sub.2 (SEQ ID NO: 29), LFSHAVSSNG-NH.sub.2 (SEQ ID NO:
19), derivatives of such sequences (e.g., N-Ac-LRAHAVDING-NH.sub.2
(SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22),
N-Ac-MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25),
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-LFSHAVSSNG-NH.sub.2
(SEQ ID NO: 19), N-Ac-AHAVSE-NH.sub.2 (SEQ ID NO: 27),
N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28), N-Ac-SHAVSS-NH.sub.2 (SEQ ID
NO: 29)) and modulating agents comprising such sequences or
derivatives thereof. Preferred antibody modulating agents include
Fab fragments directed against either the N-cadherin CAR sequence
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25) or E-cadherin CAR sequence
LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19). In addition, a modulating
agent may comprise the sequence RGD, which is bound by integrins,
separated from the HAV sequence via a linker. Alternatively, a
separate modulator of integrin-mediated cell adhesion may be
administered in conjunction with the modulating agent(s), either
within the same pharmaceutical composition or separately. 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.
[0111] 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.
[0112] To enhance the delivery of a drug through the skin, a
modulating agent as described herein and a drug are contacted with
the skin surface. Preferred modulating agents for use within such
methods include LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID
NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), LYSHAVSSNG-NH.sub.2 (SEQ
ID NO: 18), AHAVSE-NH.sub.2 (SEQ ID NO: 27), AHAVDI-NH.sub.2 (SEQ
ID NO: 28), SHAVSS-NH.sub.2 (SEQ ID NO: 29), LFGHAVSENG-NH.sub.2
(SEQ ID NO: 20), LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19),
GHAVSE-NH.sub.2 (SEQ ID NO: 26), derivatives of such sequences
(e.g., N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2
(SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25),
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-LFSHAVSSNG-NH.sub.2
(SEQ ID NO: 19), N-Ac-AHAVSE-NH.sub.2 (SEQ ID NO: 27),
N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28), N-Ac-SHAVSS-NH.sub.2 (SEQ ID
NO: 29)) and modulating agents comprising such sequences or
derivatives thereof. Preferred antibody modulating agents include
Fab fragments directed against either the N-cadherin CAR sequence
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), P-cadherin CAR sequence
LFGHAVSENG-NH.sub.2 (SEQ ID NO: 20) or E-cadherin CAR sequence
LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19). Multifunctional modulating
agents comprising the cadherin CAR sequence HAV linked to one or
more of the Dsc CAR sequences YAT, FAT and YAS and/or the Dsg CAR
sequence RAL may also be used to disrupt epithelial cell adhesion.
Alternatively, a separate modulator of non-classical
cadherin-mediated cell adhesion may be administered in conjunction
with the modulating agent(s), either within the same pharmaceutical
composition or separately. Contact may be achieved by direct
application of the modulating agent, generally within a composition
formulated as a cream or gel, or using any of a variety of skin
contact devices for transdermal application (such as those
described in European Patent Application No. 566,816 A; U.S. Pat.
No. 5,613,958; U.S. Pat. No. 5,505,956). A skin patch provides a
convenient method of administration (particularly for slow-release
formulations). Such patches may contain a reservoir of modulating
agent and drug separated from the skin by a membrane through which
the drug diffuses. Within other patch designs, the modulating agent
and drug may be dissolved or suspended in a polymer or adhesive
matrix that is then placed in direct contact with the patient's
skin. The modulating agent and drug may then diffuse from the
matrix into the skin. Modulating agent(s) and drug(s) may be
contained within the same composition or skin patch, or may be
separately administered, although administration at the same time
and site is preferred. In general, the amount of modulating agent
administered via the skin varies with the nature of the condition
to be treated or prevented, but may vary as described above. Such
levels may be achieved by appropriate adjustments to the device
used, or by applying a cream formulated as described above.
Transfer of the drug across the skin and to the target tissue may
be predicted based on in vitro studies using, for example, a Franz
cell apparatus, and evaluated in vivo by appropriate means that
will be apparent to those of ordinary skill in the art. As an
example, monitoring of the serum level of the administered drug
over time provides an easy measure of the drug transfer across the
skin.
[0113] 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).
[0114] 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.
[0115] Transdermal administration as described herein also allows
the gastrointestinal tract to be bypassed in situations where
parenteral uses would not be practical. For example, there is a
growing need for methods suitable for administration of therapeutic
small peptides and proteins, which are typically digested within
the gastrointestinal tract. The methods described herein permit
administration of such compounds and allow easy administration over
long periods of time. Patients who have problems with absorption
through their gastrointestinal tract because of prolonged ileus or
specific gastrointestinal diseases limiting drug absorption may
also benefit from drugs formulated for transdermal application as
described herein.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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 propranolol); 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).
[0120] 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.
[0121] 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.
[0122] 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.
[0123] Within a further aspect, methods are provided for enhancing
delivery of a drug to a tumor in a mammal, comprising administering
a modulating agent in combination with a drug to a tumor-bearing
mammal. Modulating agents for use within such methods include those
designed to disrupt E-cadherin and/or N-cadherin mediated cell
adhesion, such as AHAVDI-NH.sub.2 (SEQ ID NO: 28), which is
specific for N-cadherin, SHAVSS-NH.sub.2 (SEQ ID NO: 29) and
LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), which are specific for
E-cadherin, AHAVSE-NH.sub.2 (SEQ ID NO: 27) and derivatives
thereof. Other preferred modulating agents include
LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), LRAHAVDVNG-NH.sub.2 (SEQ ID
NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO: 25), LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), derivatives
of such sequences (e.g., N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2
(SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.s- ub.2 (SEQ ID NO: 25),
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-LFSHAVSSNG-NH.sub.2
(SEQ ID NO: 19), N-Ac-AHAVSE-NH.sub.2 (SEQ ID NO: 27),
N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28), N-Ac-SHAVSS-NH.sub.2 (SEQ ID
NO: 29)) and modulating agents comprising such sequences or
derivatives thereof. Bi-functional modulating agents that comprise
an HAV sequence with flanking E-cadherin-specific sequences joined
via a linker to an HAV sequence with flanking N-cadherin-specific
sequences are also preferred. Preferably, the peptide portion(s) of
a modulating agent comprises 3-16 amino acids, since longer
peptides are difficult to dissolve in aqueous solution and are more
likely to be degraded by peptidases. To achieve specificity for N-
or E-cadherin mediated cell adhesion, the peptide portion(s)
preferably comprise 4-16 amino acids, and more preferably 6-16
amino acids.
[0124] In one particularly preferred embodiment, a modulating agent
is capable of disrupting cell adhesion mediated by multiple
adhesion molecules. For example, a single branched modulating agent
(or multiple agents linked to a single molecule or support
material) may disrupt E-cadherin, N-cadherin, occludin, Dsc and Dsg
mediated cell adhesion, thereby disrupting adherens junctions,
tight junctions and desmosomes. Such an agent may comprise the
cadherin CAR sequence, HAV, as well as the putative Dsc CAR
sequences YAT, FAT, and YAS; the putative Dsg CAR sequence RAL; and
the putative occludin CAR sequence GVNPTAQSSGSLYGSQIYALCNQFYTP
AATGLYVDQYLYHYCVVDPQE (SEQ ID NO: 15) or a derivative thereof such
as QSSGSLYGSQ (SEQ ID NO: 16) or QYLYHYCVVD (SEQ ID NO: 17). Such
agents serve as multifunctional disrupters of cell adhesion.
Alternatively, a separate modulator of non-classical
cadherin-mediated cell adhesion may be administered in conjunction
with the modulating agent(s), either within the same pharmaceutical
composition or separately. Preferred antibody modulating agents
include Fab fragments directed against either the N-cadherin CAR
sequence FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25) or E-cadherin CAR
sequence LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 18). Fab fragments
directed against the occludin CAR sequence
GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO: 15)
may also be employed, either incorporated into a modulating agent
or within a separate modulator that is administered
concurrently.
[0125] 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.
[0126] Within a related aspect, the present invention provides
methods for treating cancer and/or inhibiting metastasis in a
mammal. Cancer tumors are solid masses of cells, growing out of
control, which require nourishment via blood vessels. The formation
of new capillaries is a prerequisite for tumor growth and the
emergence of metastases. Administration of modulating agents as
described herein may disrupt the growth of such blood vessels,
thereby providing effective therapy for the cancer and/or
inhibiting metastasis. Modulating agents may also be used to treat
leukemias. Preferred modulating agents for use within such methods
include those that disrupt N-cadherin and/or E-cadherin mediated
cell adhesion, such as LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID
NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), AHAVSE-NH.sub.2 (SEQ ID
NO: 27), AHAVDI-NH.sub.2 (SEQ ID NO: 28), LFSHAVSSNG-NH.sub.2 (SEQ
ID NO: 19), SHAVSS-NH.sub.2 (SEQ ID NO: 29), LYSHAVSSNG-NH.sub.2
(SEQ ID NO: 18), derivatives of such sequences (e.g.,
N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH.sub.2
(SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.s- ub.2 (SEQ ID NO: 25),
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-LFSHAVSSNG-NH.sub.2
(SEQ ID NO: 19), N-Ac-AHAVSE-NH.sub.2 (SEQ ID NO: 27),
N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28), N-Ac-SHAVSS-NH.sub.2 (SEQ ID
NO: 29)) and modulating agents comprising such sequences or
derivatives thereof. Preferably, the peptide portion(s) of such
modulating agents comprise 3-16 amino acids, more preferably 4-16
amino acids, since longer peptides are difficult to dissolve in
aqueous solution and are more likely to be degraded by peptidases.
Preferred antibody modulating agents include Fab fragments directed
against either the N-cadherin CAR sequence FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO: 25) or E-cadherin CAR sequence LFSHAVSSNG-NH.sub.2 (SEQ
ID NO: 19). In addition, a modulating agent may comprise the
sequence RGD, which is recognized by integrins, separated from the
HAV sequence via a linker. 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).
[0127] Within a further related aspect, a modulating agent may be
used to inhibit angiogenesis (i.e., the growth of blood vessels
from pre-existing blood vessels) in a mammal. Inhibition of
angiogenesis may be beneficial, for example, in patients afflicted
with diseases such as cancer or arthritis. Preferred modulating
agents for inhibition of angiogenesis include LRAHAVDING-NH.sub.2
(SEQ ID NO: 21), LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22),
MRAHAVDING-NH.sub.2 (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ
ID NO: 24), FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25),
AHAVDI-NH.sub.2 (SEQ ID NO: 28), derivatives of such sequences
(e.g., N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2
(SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.s- ub.2 (SEQ ID NO: 25),
N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28)) and modulating agents
comprising such sequences or derivatives thereof. Preferred
antibody modulating agents include Fab fragments directed against
the N-cadherin CAR sequence FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO:
25). In addition, a modulating agent for use in inhibiting
angiogenesis may comprise the sequence RGD, which is recognized by
integrins, separated from the HAV sequence via a linker.
Alternatively, a separate modulator of integrin-mediated cell
adhesion may be administered in conjunction with the modulating
agent(s), either within the same pharmaceutical composition or
separately. 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.
[0128] 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.
[0129] In yet another related aspect, the present invention
provides methods for inducing apoptosis in a cadherin-expressing
cell. In general, patients afflicted with cancer may benefit from
such treatment. Certain preferred modulating agents for use within
such methods comprise the sequence LRAHAVDING-NH.sub.2 (SEQ ID NO:
21), LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ
ID NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 25),
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), LYSHAVSSNG-NH.sub.2 (SEQ
ID NO: 18), AHAVSE-NH.sub.2 (SEQ ID NO: 27), AHAVDI-NH.sub.2 (SEQ
ID NO: 28), SHAVSS-NH.sub.2 (SEQ ID NO: 29), LFSHAVSSNG-NH.sub.2
(SEQ ID NO: 19), derivatives of such sequences (e.g.,
N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH.sub.2
(SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.s- ub.2 (SEQ ID NO: 25),
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-AHAVSE-NH.sub.2 (SEQ
ID NC): 27), N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28),
N-Ac-SHAVSS-NH.sub.2 (SEQ ID NO: 29), N-Ac-LFSHAVSSNG-NH.sub.2 (SEQ
ID NO: 19)) and modulating agents comprising such sequences or
derivatives thereof. In addition, a preferred modulating agent may
comprise the an additional CAR sequences, such as the sequence RGD,
which is recognized by integrins. As noted above, such additional
sequences may be separated from the HAV sequence via a linker.
Alternatively, a separate modulator of integrin-mediated cell
adhesion may be administered in conjunction with the modulating
agent(s), either within the same pharmaceutical composition or
separately. Preferred antibody modulating agents include Fab
fragments directed against either the N-cadherin CAR sequence
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25) or E-cadherin CAR sequence
LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18). Administration may be topical,
via injection or by other means, and the addition of a targeting
agent may be beneficial, particularly when the administration is
systemic. Suitable modes of administration and dosages depend upon
the location and nature of the cells for which induction of
apoptosis is desired but, in general, dosages may vary as described
above. A biopsy may be performed to evaluate the level of induction
of apoptosis.
[0130] The present invention also provides methods for enhancing
drug delivery to the central nervous system of a mammal. The
blood/brain barrier is largely impermeable to most neuroactive
agents, and delivery of drugs to the brain of a mammal often
requires invasive procedures. Using a modulating agent as described
herein, however, delivery may be by, for example, systemic
administration of a modulating agent-drug-targeting agent
combination, injection of a modulating agent (alone or in
combination with a drug and/or targeting agent) into the carotid
artery or application of a skin patch comprising a modulating agent
to the head of the patient. Certain preferred modulating agents for
use within such methods are LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID
NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), AHAVSE-NH.sub.2 (SEQ ID
NO: 27), AHAVDI-NH.sub.2 (SEQ ID NO: 28), derivatives of such
sequences (e.g., N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2
(SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), N-Ac-AHAVSE-NH.sub.2
(SEQ ID NO: 27), N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28)) and
modulating agents comprising such sequences or derivatives thereof.
Also preferred are bi-functional modulating agents comprising a
cadherin CAR sequence and the putative occludin CAR sequence
GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYV DQYLYHYCVVDPQE (SEQ ID NO: 15),
or derivatives or portions thereof such as QSSGSLYGSQ (SEQ ID NO:
16) and QYLYHYCVVD (SEQ ID NO: 17), preferably joined by a linker.
Alternatively, a separate modulator of occludin-mediated cell
adhesion may be administered in conjunction with the modulating
agent(s), either within the same pharmaceutical composition or
separately. Preferably, the peptide portion(s) of such modulating
agents comprise 3-16 amino acids, more preferably 4-16 amino acids.
Preferred antibody modulating agents include Fab fragments directed
against the N-cadherin CAR sequence FHLRAHAVDINGNQV-NH.sub.2 (SEQ
ID NO: 25). Fab fragments directed against the occludin CAR
sequence GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLY VDQYLYHYCVVDPQE (SEQ ID
NO: 15) may also be employed, either incorporated into the
modulating agent or administered concurrently as a separate
modulator. 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).
[0131] The present invention also provides, within further aspects,
methods for enhancing and/or directing neurological growth. In one
aspect, neurite outgrowth may be enhanced and/or directed by
contacting a neuron with one or more modulating agents. Preferred
modulating agents for use within such methods are linked to a
polymeric matrix or other support and/or contain multiple HAV
sequences separated by one or more linkers. Peptides that may be
linked to a support material (and/or to one another via a linker to
generate a suitable modulating agent) include, but are not limited
to, LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), LRAHAVDVNG-NH.sub.2 (SEQ
ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO: 25), AHAVDI-NH.sub.2 (SEQ ID NO: 28), derivatives of
such sequences (e.g., N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2
(SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), N-Ac-AHAVDI-NH.sub.2
(SEQ ID NO: 28)) and modulating agents comprising such sequences or
derivatives thereof. In addition, a modulating agent comprising RGD
and/or YIGSR (SEQ ID NO: 12), which are bound by integrins, the
cadherin CAR sequence HAV, and/or the N-CAM CAR sequence KYSFNYDGSE
(SEQ ID NO: 13) may further facilitate neurite outgrowth.
Modulating agents comprising antibodies, or fragments thereof, may
be used within this aspect of the present invention without the use
of linkers or support materials. Preferred antibody modulating
agents include Fab fragments directed against the N-cadherin CAR
sequence FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25). Fab fragments
directed against the N-CAM CAR sequence KYSFNYDGSE (SEQ ID NO: 13)
may also be employed, either incorporated into the modulating agent
or administered concurrently as a separate modulator.
[0132] The method of achieving contact and the amount of modulating
agent used will depend upon the location of the neuron and the
extent and nature of the outgrowth desired. For example, a neuron
may be contacted (e.g., via implantation) with modulating agent(s)
linked to a support material such as a suture, fiber nerve guide or
other prosthetic device such that the neurite outgrowth is directed
along the support material. Alternatively, a tubular nerve guide
may be employed, in which the lumen of the nerve guide contains a
composition comprising the modulating agent(s). In vivo, such nerve
guides or other supported modulating agents may be implanted using
well known techniques to, for example, facilitate the growth of
severed neuronal connections and/or to treat spinal cord injuries.
It will be apparent to those of ordinary skill in the art that the
structure and composition of the support should be appropriate for
the particular injury being treated. In vitro, a polymeric matrix
may similarly be used to direct the growth of neurons onto
patterned surfaces as described, for example, in U.S. Pat. No.
5,510,628.
[0133] In certain other aspects, the present invention provides
methods for enhancing adhesion of cadherin-expressing cells. Within
certain embodiments, a modulating agent may be linked to a solid
support, resulting in a matrix that comprises multiple modulating
agents. Within one such embodiment, the support is a polymeric
matrix to which modulating agents and molecules comprising other
CAR sequence(s) are attached (e g., modulating agents and molecules
comprising an RGD sequence may be attached to the same matrix,
preferably in an alternating pattern). Such matrices may be used in
contexts in which it is desirable to enhance adhesion mediated by
multiple cell adhesion molecules. Alternatively, the modulating
agent itself may comprise multiple HAV sequences or antibodies (or
fragments thereof), separated by linkers as described above. Either
way, the modulating agent(s) function as a "biological glue" to
bind multiple cadherin-expressing cells within a variety of
contexts.
[0134] Within one such aspect, modulating agents comprising
multiple HAV 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. Peptides that may be
linked to a support, and/or to one another via a linker, to
generate a suitable modulating agent include, but are not limited
to, LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), AHAVSE-NH.sub.2 (SEQ ID
NO: 27), SHAVSS-NH.sub.2 (SEQ ID NO: 29), LFSHAVSSNG-NH.sub.2 (SEQ
ID NO: 19), derivatives of such sequences (e.g,
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-AHAVSE-NH.sub.2 (SEQ
ID NO: 27), N-Ac-SHAVSS-NH.sub.2 (SEQ ID NO: 29),
N-Ac-LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19)) and modulating agents
comprising such sequences or derivatives thereof. Preferred
antibody modulating agents include Fab fragments directed against
the E-cadherin CAR sequence LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19).
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 cadherin CAR sequence, HAV, the
integrin CAR sequence, RGD, as well as the putative Dsc and Dsg CAR
sequences YAT, FAT, YAS and RAL may also be used as potent
stimulators of wound healing and/or to reduce scar tissue.
Alternatively, one or more separate modulator of integrin-, Dsc-
and/or Dsg-mediated cell adhesion may be administered in
conjunction with the modulating agent(s), either within the same
pharmaceutical composition or separately.
[0135] Within another aspect, one or more modulating agents may be
linked to the interior surface of a tissue culture plate or other
cell culture support, such as for use in a bioreactor. Such linkage
may be performed by any suitable technique, as described above.
Modulating agents linked in this fashion may generally be used to
immobilize cadherin-expressing cells. For example, dishes or plates
coated with one or more modulating agents may be used to immobilize
cadherin-expressing cells within a variety of assays and screens.
Within bioreactors (i.e., systems for large scale production of
cells or organoids), modulating agents may generally be used to
improve cell attachment and stabilize cell growth. Modulating
agents may also be used within bioreactors to support the formation
and function of highly differentiated organoids derived, for
example, from dispersed populations of fetal mammalian cells.
Bioreactors containing biomatrices of modulating agent(s) may also
be used to facilitate the production of specific proteins.
[0136] 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.
[0137] Within further aspects, modulating agents as described
herein may be used for modulating the immune system of a mammal in
any of several ways. Cadherins are expressed on immature B and T
cells (thymocytes and bone marrow pre-B cells), as well as on
specific subsets of activated B and T lymphocytes and some
hematological malignancies (see Lee et al., J. Immunol.
152:5653-5659, 1994; Munro et al., Cellular Immunol. 169:309-312,
1996; Tsutsui et al., J. Biochem. 120:1034-1039, 1996; Cepek et
al., Proc. Natl. Acad. Sci. USA 93:6567-6571, 1996). Modulating
agents may generally be used to modulate specific steps within
cellular interactions during an immune response or during the
dissemination of malignant lymphocytes.
[0138] For example, a modulating agent as described herein may be
used to treat diseases associated with excessive generation of
otherwise normal T cells. Without wishing to be bound by any
particular theory, it is believed that the interaction of cadherins
on maturing T cells and B cell subsets contributes to protection of
these cells from programmed cell death. A modulating agent may
decrease such interactions, leading to the induction of programmed
cell death. Accordingly, modulating agents may be used to treat
certain types of diabetes and rheumatoid arthritis, particularly in
young children where the cadherin expression on thymic pre-Tcells
is greatest.
[0139] Modulating agents may also be administered to patients
afflicted with certain skin disorders (such as cutaneous
lymphomas), acute B cell leukemia and excessive immune reactions
involving the humoral immune system and generation of
immunoglobulins, such as allergic responses and antibody-mediated
graft rejection. In addition, patients with circulating
cadherin-positive malignant cells (e.g., during regimes where
chemotherapy or radiation therapy is eliminating a major portion of
the malignant cells in bone marrow and other lymphoid tissue) may
benefit from treatment with a modulating agent. Such treatment may
also benefit patients undergoing transplantation with peripheral
blood stem cells.
[0140] Preferred modulating agents for use within such methods
include those that disrupt E-cadherin and/or N-cadherin mediated
cell adhesion, such as LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID
NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), LYSHAVSSNG-NH.sub.2 (SEQ
ID NO: 18), AHAVSE-NH.sub.2 (SEQ ID NO: 27), AHAVDI-NH.sub.2 (SEQ
ID NO: 28), SHAVSS-NH.sub.2 (SEQ ID NO: 29), LFSHAVSSNG-NH.sub.2
(SEQ ID NO: 19), derivatives of such sequences (e.g.,
N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH.sub.2
(SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.s- ub.2 (SEQ ID NO: 25),
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-AHAVSE-NH.sub.2 (SEQ
ID NO: 27), N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28),
N-Ac-SHAVSS-NH.sub.2 (SEQ ID NO: 29), N-Ac-LFSHAVSSNG-NH.sub.2 (SEQ
ID NO: 19)) and modulating agents comprising such sequences or
derivatives thereof. In addition, a preferred modulating agent may
comprise one or more additional CAR sequences, such as the sequence
RGD, which is bound by integrins. As noted above, such additional
sequence(s) may be separated from the HAV sequence via a linker.
Alternatively, a separate modulator of integrin-mediated cell
adhesion may be administered in conjunction with the modulating
agent(s), either within the same pharmaceutical composition or
separately. Preferred antibody modulating agents include Fab
fragments directed against either the N-cadherin CAR sequence
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25) or E-cadherin CAR sequence
LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18). Within the above methods, the
modulating agent(s) are preferably administered systemically
(usually by injection) or topically. A modulating agent may be
linked to a targeting agent. For example, targeting to the bone
marrow may be beneficial. A suitable dosage is sufficient to effect
a statistically significant reduction in the population of B and/or
T cells that express cadherin and/or an improvement in the clinical
manifestation of the disease being treated. Typical dosages
generally range as described above.
[0141] Within further aspects, the present invention provides
methods and kits for preventing pregnancy in a mammal. In general,
disruption of E-cadherin function prevents the adhesion of
trophoblasts and their subsequent fusion to form
syncitiotrophoblasts. In one embodiment, one or more modulating
agents as described herein may be incorporated into any of a
variety of well known contraceptive devices, such as sponges
suitable for intravaginal insertion (see, e.g., U.S. Pat. No.
5,417,224) or capsules for subdermal implantation. Other modes of
administration are possible, however, including transdermal
administration, for modulating agents linked to an appropriate
targeting agent. Preferred modulating agents for use within such
methods include LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18),
AHAVSE-NH.sub.2 (SEQ ID NO: 27), SHAVSS-NH.sub.2 (SEQ ID NO: 29),
LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19), derivatives of such sequences
(e.g, N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18),
N-Ac-AHAVSE-NH.sub.2 (SEQ ID NO: 27), N-Ac-SHAVSS-NH.sub.2 (SEQ ID
NO: 29) and N-Ac-LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19)) and
modulating agents comprising such sequences or derivatives thereof.
In addition, a preferred modulating agent may comprise additional
CAR sequences, such as the sequence RGD, which is bound by
integrins. As noted above, such additional sequences may be
separated from the HAV sequence via a linker. Alternatively, a
separate modulator of integrin-mediated cell adhesion may be
administered in conjunction with the modulating agent(s), either
within the same pharmaceutical composition or separately. Preferred
antibody modulating agents include Fab fragments directed against
the E-cadherin CAR sequence LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19).
Suitable methods for incorporation into such a device depend upon
the type of device and are well known in the art. Such devices
facilitate administration of the modulating agent(s) to the uterine
region and may provide a sustained release of the modulating
agent(s). In general, modulating agent(s) may be administered via
such a contraceptive device at a dosage ranging from 0.1 to 50
mg/kg, although appropriate dosages may be determined by monitoring
hCG levels in the urine. hCG is produced by the placenta, and
levels of this hormone rise in the urine of pregnant women. The
urine hCG levels can be assessed by radio-immunoassay using well
known techniques. Kits for preventing pregnancy generally comprise
a contraceptive device impregnated with one or more modulating
agents.
[0142] Alternatively, a sustained release formulation of one or
more modulating agents may be implanted, typically subdermally, in
a mammal for the prevention of pregnancy. Such implantation may be
performed using well known techniques. Preferably, the implanted
formulation provides a dosage as described above, although the
minimum effective dosage may be determined by those of ordinary
skill in the art using, for example, an evaluation of hCG levels in
the urine of women.
[0143] The present invention also provides methods for increasing
vasopermeability in a mammal by administering one or more
modulating agents or pharmaceutical compositions. Within blood
vessels, endothelial cell adhesion (mediated by N-cadherin) results
in decreased vascular permeability. Accordingly, modulating agents
as described herein that decrease N-cadherin mediated adhesion may
be used to increase vascular permeability. Particularly preferred
modulating agents include LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID
NO: 23), HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), AHAVDI-NH.sub.2 (SEQ ID
NO: 28), derivatives of such sequences (e.g.,
N-Ac-LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH.sub.2
(SEQ ID NO: 22), N-Ac-MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25), AHAVDI-NH.sub.2 (SEQ
ID NO: 28)) and modulating agents comprising such sequences or
derivatives thereof. Modulating agents comprising antibodies, or
fragments thereof, may also be used within this aspect of the
present invention. Preferred antibody modulating agents include Fab
fragments directed against the N-cadherin CAR sequence
FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25).
[0144] Within certain embodiments, preferred modulating agents for
use within such methods include peptides capable of decreasing both
endothelial and tumor cell adhesion. Such modulating agents may be
used to facilitate the penetration of anti-tumor therapeutic or
diagnostic agents (e.g., monoclonal antibodies) through endothelial
cell permeability barriers and tumor barriers. For example, a
modulating agent may further comprise a sequence such as
SHAVSS-NH.sub.2 (SEQ ID NO: 29), LFSHAVSSNG-NH.sub.2 (SEQ ID NO:
19), AHAVSE-NH.sub.2 (SEQ ID NO: 27), LYSHAVSSNG-NH.sub.2 (SEQ ID
NO: 18), and/or one or more derivatives of such sequences (e.g.,
N-Ac-SHAVSS-NH.sub.2 (SEQ ID NO: 29), N-Ac-LFSHAVSSNG-NH.sub.2 (SEQ
ID NO: 19), N-Ac-AHAVSE-NH.sub.2 (SEQ ID NO: 27) or
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18)). Bi-functional modulating
agents that comprise an HAV sequence with flanking
E-cadherin-specific sequences joined via a linker to an HAV
sequence with flanking N-cadherin-specific sequences are also
preferred. Alternatively, separate modulating agents capable of
disrupting N- and E-cadherin mediated adhesion may be administered
concurrently. Preferably, the peptide portion(s) of a modulating
agent comprises 3-16 amino acids, since longer peptides are
difficult to dissolve in aqueous solution and are more likely to be
degraded by peptidases.
[0145] 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 the cadherin CAR
sequence, HAV, as well as and RGD sequence and/or the putative
occludin CAR sequence GVNPTAQSSGSLYGSQIYALCNQFYTP
AATGLYVDQYLYHYCVVDPQE (SEQ ID NO: 15) or a derivative thereof such
as QSSGSLYGSQ (SEQ ID NO: 16) or QYLYHYCVVD (SEQ ID NO: 17).
Alternatively, a separate modulator of non-classical
cadherin-mediated cell adhesion may be administered in conjunction
with the modulating agent(s), either within the same pharmaceutical
composition or separately. Preferred antibody modulating agents
include Fab fragments directed against either the N-cadherin CAR
sequence FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 25) or E-cadherin CAR
sequence LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 18). Fab fragments
directed against the occludin CAR sequence
GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVV DPQE (SEQ ID NO: 15)
may also be employed, either incorporated into a modulating agent
or within a separate modulator that is administered
concurrently.
[0146] 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.
[0147] 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.
[0148] Within a further aspect, modulating agents as described
herein may be used for controlled inhibition of synaptic stability,
resulting in increased synaptic plasticity. Within this aspect,
administration of one or more modulating agents may be advantageous
for repair processes within the brain, as well as learning and
memory, in which neural plasticity is a key early event in the
remodeling of synapses. Cell adhesion molecules, particularly
N-cadherin and E-cadherin, can function to stabilize synapses, and
loss of this function is thought to be the initial step in the
remodeling of the synapse that is associated with learning and
memory (Doherty et al., J. Neurobiology, 26:437-446, 1995; Martin
and Kandel, Neuron, 17:567-570, 1996; Fannon and Colman, Neuron,
17:423-434, 1996). Inhibition of cadherin function by
administration of one or more modulating agents that inhibit
cadherin function may stimulate learning and memory. Preferred
modulating agents for use within such methods include those that
disrupt E-cadherin and/or N-cadherin mediated cell adhesion, such
as LRAHAVDING-NH.sub.2 (SEQ ID NO: 21), LRAHAVDVNG-NH.sub.2 (SEQ ID
NO: 22), MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH.sub.2
(SEQ ID NO: 25), LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18),
AHAVSE-NH.sub.2 (SEQ ID NO: 27), AHAVDI-NH.sub.2 (SEQ ID NO: 28),
SHAVSS-NH.sub.2 (SEQ ID NO: 29), LFSHAVSSNG-NH.sub.2 (SEQ ID NO:
19), derivatives of such sequences (e.g., N-Ac-LRAHAVDING-NH.sub.2
(SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH.sub.2 (SEQ ID NO: 22),
N-Ac-MRAHAVDING-NH.sub.2 (SEQ ID NO: 23),
N-Ac-HLGAHAVDINGNQVET-NH.sub.2 (SEQ ID NO: 24),
N-Ac-FHLRAHAVDINGNQV-NH.s- ub.2 (SEQ ID NO: 25),
N-Ac-LYSHAVSSNG-NH.sub.2 (SEQ ID NO: 18), N-Ac-AHAVSE-NH.sub.2 (SEQ
ID NO: 27), N-Ac-AHAVDI-NH.sub.2 (SEQ ID NO: 28),
N-Ac-SHAVSS-NH.sub.2 (SEQ ID NO: 29), N-Ac-LFSHAVSSNG-NH.sub.2 (SEQ
ID NO: 19)) and modulating agents comprising such sequences or
derivatives thereof. In addition, a preferred modulating agent may
comprise one or more additional CAR sequences, such as the sequence
RGD, which is bound by integrins and/or the N-CAM CAR sequence
KYSFNYDGSE (SEQ ID NO: 12). As noted above, such additional
sequence(s) may be separated from the HAV sequence via a linker.
Alternatively, a separate modulator of integrin and/or N-CAM
mediated cell adhesion may be administered in conjunction with the
modulating agent(s), either within the same pharmaceutical
composition or separately. Preferred antibody modulating agents
include Fab fragments directed against either the N-cadherin CAR
sequence FHLRAHAVDINGNQV-NH.sub.2 (SEQ ID NO: 15) or E-cadherin CAR
sequence LFSHAVSSNG-NH.sub.2 (SEQ ID NO: 19). For such aspects,
administration may be via encapsulation into a delivery vehicle
such as a liposome, using standard techniques, and injection into,
for example, the carotid artery. Alternatively, a modulating agent
may be linked to a disrupter of the blood-brain barrier. In general
dosages range as described above.
[0149] 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 a cadherin (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.
[0150] 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.
[0151] In another embodiment, the assay involves the use of
antibody immobilized on a solid support to bind to the target
cadherin, or a proteolytic fragment containing the EC1 domain and
encompassing the CAR sequence, and remove it from the remainder of
the sample. The bound cadherin may then be detected using a second
antibody or reagent that contains a reporter group. Alternatively,
a competitive assay may be utilized, in which a cadherin is labeled
with a reporter group and allowed to bind to the immobilized
antibody after incubation of the antibody with the sample. The
extent to which components of the sample inhibit the binding of the
labeled cadherin to the antibody is indicative of the reactivity of
the sample with the immobilized antibody, and as a result,
indicative of the level of the cadherin in the sample.
[0152] 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.
[0153] In certain embodiments, the assay for detection of a
cadherin in a sample is a two-antibody sandwich assay. This assay
may be performed by first contacting an antibody that has been
immobilized on a solid support, commonly the well of a microtiter
plate, with the biological sample, such that the cadherin within
the sample is allowed to bind to the immobilized antibody (a 30
minute incubation time at room temperature is generally
sufficient). Unbound sample is then removed from the immobilized
cadherin-antibody complexes and a second antibody (containing a
reporter group such as an enzyme, dye, radionuclide, luminescent
group, fluorescent group or biotin) capable of binding to a
different site on the cadherin is added. The amount of second
antibody that remains bound to the solid support is then determined
using a method appropriate for the specific reporter group. The
method employed for detecting the reporter group depends upon the
nature of the reporter group. For radioactive groups, scintillation
counting or autoradiographic methods are generally appropriate.
Spectroscopic methods may be used to detect dyes, luminescent
groups and fluorescent groups. Biotin may be detected using avidin,
coupled to a different reporter group (commonly a radioactive or
fluorescent group or an enzyme). Enzyme reporter groups may
generally be detected by the addition of substrate (generally for a
specific period of time), followed by spectroscopic or other
analysis of the reaction products. Standards and standard additions
may be used to determine the level of cadherin in a sample, using
well known techniques.
[0154] The present invention also provides kits for use in such
immunoassays. Such kits generally comprise one or more antibodies,
as described above. In addition, one or more additional
compartments or containers of a kit generally enclose elements,
such as reagents, buffers and/or wash solutions, to be used in the
immunoassay.
[0155] Within further aspects, modulating agents or antibodies (or
fragments thereof) may be used to facilitate cell identification
and sorting in vitro or imaging in vivo, permitting the selection
of cells expressing different cadherins (or different cadherin
levels). Preferably, the modulating agent(s) or antibodies for use
in such methods are linked to a detectable marker. Suitable markers
are well known in the art and include radionuclides, luminescent
groups, fluorescent groups, enzymes, dyes, constant immunoglobulin
domains and biotin. Within one preferred embodiment, a modulating
agent linked to a fluorescent marker, such as fluorescein, is
contacted with the cells, which are then analyzed by fluorescence
activated cell sorting (FACS).
[0156] Antibodies or fragments thereof may also be used within
screens of combinatorial or other nonpeptide-based libraries to
identify other compounds capable of modulating cadherin-mediated
cell adhesion. Such screens may generally be performed using an
ELISA or other method well known to those of ordinary skill in the
art that detect compounds with a shape and structure similar to
that of the modulating agent. In general, such screens may involve
contacting an expression library producing test compounds with an
antibody, and detecting the level of antibody bound to the
candidate compounds. Compounds for which the antibody has a higher
affinity may be further characterized as described herein, to
evaluate the ability to modulate cadherin-mediated cell
adhesion.
[0157] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLE 1
Preparation of Representative Modulating Agents
[0158] This Example illustrates the solid phase synthesis of
representative peptide modulating agents.
[0159] The peptides were assembled on either methylbenzhydrylamine
(MBHA) resin (for the C-terminal amide peptides) or the traditional
Merrifield resins (for any C-terminal acid peptides). Bags of a
polypropylene mesh material were filled with the resin and soaked
in dichloromethane. The resin packets were washed three times with
5% diisopropylethylarnine in dichloromethane and then washed with
dichloromethane. The packets are then sorted and placed into a
Nalgene bottle containing a solution of the amino acid of interest
in dichloromethane. An equal amount of diisopropylcarbodiimide
(DIC) in dichloromethane was added to activate the coupling
reaction. The bottle was shaken for one hour to ensure completion
of the reaction. The reaction mixture was discarded and the packets
washed with DMF. The N-.alpha.-Boc was removed by acidolysis using
a 55% TFA in dichloromethane for 30 minutes leaving the TFA salt of
the .alpha.-amino group. The bags were washed and the synthesis
completed by repeating the same procedure while substituting for
the corresponding amino acid at the coupling step. Acetylation of
the N-terminal., where desired, was performed by reacting the
peptide resins with a solution of acetic anhydride in
dichloromethane in the presence of diisopropylethylamine. The
peptide was then side-chain deprotected and cleaved from the resin
at 0.degree. C. with liquid HF in the presence of anisole as a
carbocation scavenger.
[0160] The crude peptides were purified by reversed-phase
high-performance liquid chromatography and characterized by
analytical HPLC and by mass spectral analysis.
EXAMPLE 2
Establishment of a Model System for Schwann Cell-Astrocyte
Interactions
[0161] This Example illustrates a cell boundary assay for use in
evaluating interactions between Schwann cells and astrocytes.
[0162] A. Cell Culture
[0163] All cells were cultured in Dulbecco's modified Eagle's
medium (DMEM; Gibco, Grand Island, N.Y.) supplemented with
penicillin/streptomycin (100 U/ml; Gibco) and either 10% fetal calf
serum (FCS) or serum-free (SF) medium; a modification of
Bottenstein's and Sato's (Proc. Natl. Acad. Sci USA 76:514-517,
1979) defined medium with supplements of insulin (5 .mu.g/ml;
Sigma, St. Louis, Mo.), transferrin (100 .mu.g/ml; Sigma),
glutamine (1 mM; ICN/Flow), progesterone (60 ng/ml; Sigma),
putrescine (16 .mu.g/ml; Sigma), selenium (160 ng/ml; Sigma), T4
(500 ng/ml; Sigma), T3 (10 ng/ml; Sigma), BSA (0.035%; Sigma) and
dexamethasone (38 rig/ml; Sigma).
[0164] Schwann cells were cultured from neonatal day 2 (P2) sciatic
nerve, a variation of the procedure described by Brockes et al.,
Brain Res. 165:105-118, 1979. Nerves were removed and placed in
L-15 medium, cleaned of any blood vessels, musculature and their
epineurial sheaths and placed into a 34 mm diameter plastic dish
containing Trypsin (0.1%; Sigma) and collagenase (0.03%; Sigma).
The nerves were cut very finely using dissection scissors and
placed in an incubator at 37.degree. C. and 10% CO.sub.2 for 30
minutes. Following this incubation an equal volume of triturating
solution (300 mg BSA; Sigma, 1 mg DNAse; Sigma, 50 mg Trypsin
inhibitor; Sigma per 100 ml HBSS) was added and the whole mixture
gently triturated using a flamed glass pasteur. Having spun down
the cells into a pellet by centrifugation at 1000 rpm for 3-5
minutes, the cells were then resuspended in DMEM with 10% FCS and
plated on poly-lysine (0.01% Sigma) at a density of 5000
cells/mm.sup.2. On the following day cells were treated with
Cytosine arabinoside (Ara-C 1.times.10.sup.-5M; Sigma) for three
days. Following a period of two days in normal untreated FCS the
ARA-C was again applied for a further three days. The few remaining
fibroblast contaminants were then killed via complement mediated
lysis using rabbit serum (a gift from R. Oldroyd) and the IgM class
anti-Thyl.l (1:1000 Serotec, Kidlington, Oxford, UK). Subsequently,
the Schwann cells (>98% pure) were maintained in FCS
supplemented with bovine pituitary extract (BPE; 10 .mu.g/ml;
Sigma) and forskolin (2 .mu.M, Sigma). These cells were maintained
for experiments until two weeks after the treatment with
complement.
[0165] Primary astrocyte cultures were obtained from neonatal rats
(P2) as described by McCarthy and de Vellis, J. Cell Biol.
85:890-902, 1980. The brains were removed, de-membraned, chopped
and then incubated with 0.1% trypsin for 30 minutes. The mixture
was then triturated in triturating solution and the cells were
centrifuged down into a pellet. Having resuspended the cells in FCS
they were plated onto poly-lysine coated plastic at a density of
two brains per 75 cc Falcon flask. After 6-10 days, the majority of
cells of the oligodendrocyte lineage were removed by shaking the
culture overnight. Skin fibroblasts were obtained from a flap of
skin removed from P2 rat neonates. The tissue was chopped using a
sterile blade and then enzymatically dissociated with trypsin and
collagenase for 45 minutes. After trituration, the cells were
resuspended in DMEM containing 10% FCS.
[0166] Meningeal cell cultures were obtained from the meningeal
cell layer which was dissected from P2 brains, then treated as
described for the astrocytic cultures.
[0167] A7 cells, an astrocyte cell line derived from postnatal
brain and shown to support axon growth more readily than primary
astrocytes (Fok-Seang et al., Brain Res. 698:207-223, 1995), were
grown in DMEM containing 10% FCS.
[0168] B. Immunofluorescent Staining
[0169] Schwann cells were identified by indirect immunofluorescent
labeling using polyclonal anti-growth associated protein 43
(GAP-43; a generous gift from G. Wilkie) and astrocytes were
identified by the mouse monoclonal anti-glial fibrillary acidic
protein (GFAP; Boehringer, Laval, Quebec). The tissue was fixed in
4% paraformaldehyde for 30 minutes, blocked with PBS-Triton X-100
(0.2%) and 5% goat serum and then given one hour of incubation with
the primary antibody. Rhodamine-conjugated anti-rabbit antibodies
(Jackson Immunoresearch Labs, Inc. Westgrove, Pa.; 1:200) and
fluorescein conjugated anti-mouse (1:200) allowed visualization.
Fibroblasts were identified with the mouse monoclonal anti-Thyl.l
(Serotec, Kidlington, Oxford, UK; 1:1000) using the same staining
technique.
[0170] C. The Generation of Schwann Cell and Astrocyte Cellular
Boundaries
[0171] A cell boundary assay was used to study the behavior of two
cell populations which have the ability to divide and migrate
freely, meeting head on as continuous cellular frontiers. Schwann
cells were prepared as a dense cell suspension consisting of
2.times.10.sup.6 cells per ml of solution. 70 .mu.l of this
suspension was placed as a drop at one end of a 2 mm polylysine
coated coverslip. A glass 10 mm.times.5 mm fragment was taken with
a pair of forceps and the drop was smeared towards the center of
the coverslip so as to generate a straight edge to the drop. An
equal number of a different type of cells suspended in an equal
volume as the first drop was then placed at the opposite end of the
coverslip. Using a different glass fragment (of similar dimensions)
this second drop was again smeared towards the center of the
coverslip so that the straight edged boundary of this new drop was
as close as possible and parallel to the edge of the first drop
without the two drops mixing. The cells were allowed to attach for
2-3 hours before washing three times in Hanks to remove any
non-attached cells. These cultures were then grown for three days
in medium supplemented with serum, BPE and forskolin to provide a
maximal mitotic stimulus to the Schwann cells. The cultures were
then fixed in 4% paraformaldehyde for 20 minutes prior to
immunohistochemistry. In this way, interactions between populations
of Schwann cells and astroglia, and between populations of Schwann
cells and fibroblasts were studied with respect to the morphology
of their cellular territories.
[0172] Once confluent cultures of two cell types were established
(approximately 200 .mu.m away from one another), the cultures
expanded and interacted with one another along a straight front.
The interactions between the two opposing cell types were then
analyzed over the course of several days. The establishment of
territories between Schwann cells and astrocytic cells, and between
Schwann cells and fibroblasts was studied. In each case, the two
populations of cells generally came into contact after two days.
Cultures consisting of Schwann cells and astrocytes were taken for
immunohistochemical analysis (n=18). In all cultures, it was
evident that Schwann cell and astroglial territories remained
largely exclusive. The Schwann cells at the boundary were seen in
two orientations. In some areas the long axes of the Schwann cells
were parallel to the astrocytic boundary. Here the territories
occupied by the two cell types were completely exclusive. In other
areas the boundary was more complex. Groups of Schwann cells had
their long axes at right angles to the cell interface, making
finger-like projections, and there was often a slight degree of
overlap between the two territories (FIG. 1D). Time lapse
observations indicated that the astrocytes were constantly
advancing, sending processes under the Schwann cells, which would
then retreat as a group (data not shown). Apart from the
distinctive territorial arrangements, it was observed that
astrocytes in contact with Schwann cells displayed a more intense
staining with GFAP and showed hypertrophy of the perikarya, as
reported by previous authors both in vivo and in vitro (Brook et
al., Glia 9:292-304, 1993; Ghimikar and Eng, Glia 11:367-377,
1994). Schwann cells and astrocytes cultured by this method were
therefore able to establish a structure similar to the peripheral
nerve entry zones seen in vivo.
[0173] To determine whether the development of these distinctive
patterns were a common feature of the manipulations peculiar to
this technique, or unique for the cell types, the assay was
repeated using Schwann cells and fibroblasts, cells normally
associated with Schwann cell migration in damaged peripheral nerve.
None of the Schwann cell-fibroblast cultures (n=15) displayed the
clear territorial exclusion seen in Schwann cell-astrocyte
cultures. Similarly, none of the cultures displayed the parallel
Schwann cell alignment at the boundary or the finger-like
projections. Indeed, Schwann cells were seen to cluster together
rather irregularly and to overlie the fibroblasts. Phase contrast
photographs showing the parallel alignment commonly seen in the
Schwann cell-astrocyte co-cultures and the irregular clustering of
the Schwann cells upon the fibroblasts are presented in FIGS. 3A
and 3B.
[0174] D. Migration of Schwann Cells on Laminin and Monolayers
[0175] In order to assess the rates of Schwann cell migration on
different surfaces, the micro-inverted-coverslip migration assay
was employed. This is a variation of the technique first described
by Fok-Seang et al., Dev. Biol. 171:1-15, 1995. Schwann cells
fluorescently labeled with Di-I (25 .mu.g/ml) were evenly plated
onto polylysine and laminin coated fragments of glass coverslip
(1.times.2 mm). After 16-18 hours, the pieces of glass coated with
Di-I labeled Schwann cells were dipped into Hanks three times to
remove any loosely attached cells and then inverted with cells
facing downwards onto laminin-coated tissue culture surfaces or
onto cell monolayers. These cultures were then incubated for a
further two days and fixed for 20 minutes with 4% paraformaldehyde.
The maximum migration distance was measured, and the number of
cells in bands of 0.1 mm progressing outwards from the edge of the
coverslip were counted.
[0176] In this assay, a dense Schwann cell culture is established
on coverslip fragments and their migration away from its edge
measured. The assay therefore measures the ability of Schwann cells
to migrate on a surface, and their ability to migrate away from a
confluent Schwann cell monolayer. The migration front of the
foremost cells was measured, and the number of cells against
distance of migration plotted. Schwann cell-laden fragments were
placed on laminin to give a baseline migration rate on a favorable
defined surface, and laminin controls were done for comparison on
each assay. The average distance of migration on laminin was 1.02
mm.+-.0.06 (mean.+-.S.E.M.) over three days. Migration assays were
done on four different cell monolayers: (1) astrocytes cultured
from postnatal brain, (2) A7 astrocyte cell line, (3) fibroblasts,
and (4) meningeal cells. These results are presented in FIG.
4A.
[0177] The mean maximum distance covered by the Schwann cells on an
astroglial monolayer over 3 days was found to be 0.33 mm.+-.0.02.
Migration on fibroblasts was 0.99 mm.+-.0.04 (FIG. 4B). Schwann
cells can therefore migrate on fibroblasts almost as rapidly as on
laminin, while migration on astrocytes is much more limited. FIGS.
12A and 12B compare the migration of Schwann cells upon astrocyte
surfaces to that upon fibroblast surfaces, showing the migration of
fluorescently labeled cells from the edge of the fragment.
[0178] Primary cortical astrocyte cultures purified in the manner
described have been shown to yield type-I astrocyte purities
greater than 95%. Contaminating cell types may include microglial
cells, meningeal cells or cells of the oligodendrocyte-lineage. In
order to be certain that the restricted migration of Schwann cells
on astrocyte cultures was not due to the presence of small numbers
of meningeal cells, which inhibit oligo-precursor migration
(Fok-Seang et al., Dev. Biol. 171:1-15, 1995), purified meningeal
cell cultures extracted from neonatal brain were used as a
migratory substrate. The average distance of migration by Schwann
cells on a meningeal cell monolayer was found to be 0.90
mm.+-.0.04. This is a degree of Schwann cell migration similar to
that achieved on fibroblasts and laminin, and much greater than on
astrocytes.
[0179] In order to determine whether non-astrocytic contaminants
were responsible for the non-permissive behavior, the astrocyte
cell line A7 was used as a migratory substrate. A homogenous
astroglial population permitted only 0.40 mm.+-.0.03 of Schwann
cell migration over the two day period, very similar to that seen
on primary astrocyte cultures.
[0180] Thus, when confluent cultures of Schwann cells and
astrocytes were placed so as to confront one another a clear
division of territory resulted, comparable to the peripheral nerve
entry zones. Several mechanisms could be responsible for the
segregation of two different cell types and their failure to
migrate over or through one another. The simplest is an inhibitory
interaction, as is seen when axon growth cones meet
oligodendrocytes, when axons from CNS and PNS meet, or when
oligodendrocyte precursors meet meningeal cells. However, in such
instances, the exploratory cell process undergoes a sudden and
catastrophic collapse within a few minutes of cell contact, leading
to withdrawal of the migrating cell. This "growth cone collapse"
did not occur when Schwann cells met either astrocytes or
fibroblasts. A second reason for failure of cells to mix could be a
lack of complementary adhesion molecules; however Schwann cells
adhere more strongly to astrocytes than to fibroblasts or laminin,
both of which support migration.
[0181] The data presented herein demonstrate that Schwann cells
form prolonged and firm contacts with astrocytes. Schwann cells are
unable to move until these contacts are broken. This behavior is
very similar to that seen when oligodendrocyte precursors encounter
astrocytes or when a Schwann cell process encounters an axonal
growth cone in the presence of external calcium. Meetings, with
fibroblasts result in much shorter lived contacts. The results
suggest that a secreted or cell-associated factor may be involved
in this interaction.
EXAMPLE 3
Identification of Cell Membrane Associated Molecules as Factors
Inhibiting Schwann Cell Migration
[0182] This Example illustrates the identification of molecules
responsible for differential rates of Schwann cell migration.
[0183] A. Effects of Cell Matrix and Diffusible Factors on Schwann
Cell Migration
[0184] We examined whether the differential rates of Schwann cell
migration on different cell types could be due either to secreted
molecules, to the different properties of the extracellular
matrices, or to cell membrane associated molecules (i.e.,
cadherin). In order to determine whether matrix or secreted
molecules were responsible, we assayed Schwann cell migration on
extracellular matrix and in the presence of conditioned medium. The
micro inverted coverslip migration assay was employed. Surfaces
laden with astroglial matrix were produced by lysing astrocytes
grown on coverslips with PBS and Triton X-100 (0.1%). Schwann
cell-covered fragments were inverted onto the matrix preparations
and the maximum migratory distances of the cells were assessed.
Control experiments were performed utilizing laminin as substrate.
Schwann cells were found to migrate distances of 0.79 mm.+-.0.02 on
astroglial matrix, slightly less than that seen upon laminin (1.09
mm.+-.0.05), but further than on whole cells (0.24 mm.+-.0.03; FIG.
5).
[0185] In order to assess the contribution of diffusible factors,
astrocyte-conditioned serum free medium (ACM) was used to conduct
Schwann cell migration assays from laminin. Schwann cells were
found to have migrated distances up to 10.04 mm.+-.0.02 upon
laminin in the presence of ACM whereas migration upon laminin in
serum free (SF) medium alone was 0.59 mm.+-.0.04 (FIG. 6). It
appears that pro-migratory factors exist in serum and paradoxically
ACM.
[0186] These experiments suggest that neither astrocyte matrix nor
secreted molecules are inhibitory to Schwann cell migration. The
inhibition must therefore be cell surface mediated.
[0187] B Movement of Single Schwann Cells on Laminin and
Monolayers
[0188] The inverted coverslip migration assay described in the
previous section involves a number of different cell interactions,
namely Schwann cell-Schwann cell interactions and the adhesion
between the Schwann cells and the overlying glass fragment. In
order to analyze a simpler situation, time lapse videomicroscopy
was used to determine the migration of single Schwann cells on
differing cellular and proteinaceous substrates.
[0189] Cells were plated onto a 35 mm tissue culture dish and were
filmed on a Nikon Diaphot inverted microscope mounted in a chamber
maintained at 37.degree. C. and at a humidified atmosphere of 10%
CO.sub.2 in air. The events were recorded on a Panasonic 8051 video
recorder at 8 frames every 30 seconds for a period of 14-25 hours.
Three types of culture were established:
[0190] 1. Astrocyte, fibroblast or meningeal monolayers were grown
to confluence within 35 mm tissue culture dishes and filled with 2
ml of DMEM supplemented with 10% FCS. A 50 .mu.l Schwann cell
suspension containing approximately 1000 cells was then added to
the dish which was then transferred to the timelapse chamber.
Filming was initiated immediately and Schwann cells were clearly
identifiable landing and attaching to the underlying monolayer.
Movement of the Schwann cell body was recorded every 30 minutes for
6 hour periods by marking the position of its nucleus onto an
acetate sheet covering the monitor. Pathways of migration were
therefore constructed. Distance moved by the cell body every half
hour was measured and used to generate an average speed of
migration for the cells.
[0191] 2. Cultures from which interactions between single cells
colliding as they moved on a laminin surface could be filmed were
generated as follows: 1 ml of solution containing 1000 Schwann
cells was placed into a 35 mm culture dish followed by a further 1
ml of an equal number of either astroglia or fibroblasts. The dish
was transferred to a timelapse chamber and a field of view in which
cells of each type were close but not yet touching was selected.
The nature and duration of interactions between the different cell
types were recorded.
[0192] 3. Confrontation assays, in which an expanding monolayer of
astrocytes or fibroblasts would come into contact with an expanding
monolayer of Schwann cells, were established using the cell
boundaries generated as above.
[0193] Within one such study, single Schwann cells were plated onto
astrocytic, fibroblastic or laminin substrates and their cell body
movement was observed over a time period of 6 hours. The position
of the cell body after 30 minute intervals was noted onto an
acetate sheet covering the monitor and displacement diagrams were
obtained for twenty cells upon each substrate. From these diagrams,
the distance moved every 30 minutes was obtained and used to
generate the average migratory rates of the single cells for each
of the conditions. A selection of displacement diagrams are
presented as FIG. 5C. It was found that Schwann cells migrate the
slowest on astrocytes with an average speed of 16.2
.mu.m/hr.+-.1.12. They move faster on fibroblasts (31.8
.mu.m/hr.+-.1.39) and attain their fastest speed on laminin (64.8
.mu.m/hr.+-.2.88). Therefore, the same trend as seen with the
population migration experiment is seen with single Schwann cells.
This data is presented graphically as FIG. 7A.
[0194] C Interactions Between Single Schwann Cells, Astrocytes and
Fibroblasts
[0195] Sparse mixed cultures of Schwann cells and either astrocytes
or fibroblasts were established. Regions where single Schwann cells
were in contact with isolated astrocytes or fibroblasts were
filmed. Astrocyte-Schwann cell (n=50) and fibroblast-Schwann cell
(n=40) interactions were observed. When a Schwann cell process
encountered an astrocyte, the exploratory growth cone first
appeared to attach firmly to the astrocytic surface and then expand
in area, with active lamellipodia exploring the perikarya. The
growth cone could be seen to become anchored to the astrocyte
whilst the cell body would move away, resulting in a very long
process connecting the two cells. The average process length was
found to be 33.0 .mu.m.+-.3.0 (mean.+-.S.E.M.). In contrast Schwann
cells encountering fibroblasts did so via an exploratory growth
cone which did not expand on contact. Furthermore, the average
process length between Schwann cells and fibroblasts was found to
be 11.8 .mu.m.+-.1.85. The longer processes developing between
Schwann cells and astrocytes implies greater tension between the
cells.
[0196] Contacts between Schwann cells and astrocytes were of much
longer duration than those between Schwann cells and fibroblasts.
Most (80%) of the Schwann cell-astrocyte interactions were longer
than 90 minutes. In comparison, only 5% of the Schwann
cell-fibroblast interactions were as long as this. The average
length of interaction between Schwann cells and astrocytes was
found to be 257 min.+-.41 minutes whereas the average Schwann
cell-fibroblast-interaction was found to be 48 min.+-.5 (FIG. 7B).
A sequence of encounters between a Schwann cell and an astrocyte
captured from a time lapse recording is presented in FIG. 6. Each
consecutive frame represents a time interval of 2 hours. Thus,
Schwann cells appear to interact with astrocytes and fibroblasts
differently at the single cell level. Schwann cells display an
exploratory behavior as well as a static form of interaction with
astrocytes. Contact with fibroblasts seems only to involve simple
exploration with little interruption of Schwann cell migratory
movement.
[0197] D. Adhesion of Schwann Cells to Laminin and Monolayers
[0198] In order to test whether the migratory behavior of Schwann
cells on different cell types was a function of adhesivity to the
substratum, DiI-labeled Schwann cells were plated onto either
astrocytic, fibroblasts, Schwann cell or laminin surfaces. 20,000
DiI-labeled Schwann cells were placed in a 15 mm diameter well in
0.5 ml of medium over a 13 mm glass coverslip coated with laminin,
or a complete monolayer of astrocytes, fibroblasts or Schwann cells
and then placed onto a shaking platform (25 rpm) for 30 minutes in
an incubator. The coverslips were washed three times in Hanks after
30 minutes to remove any non-attached cells and the remainder were
fixed for 20 minutes in 4% paraformaldehyde. The number of
DiI-labeled Schwann cells that were attached to the coverslip were
counted using a Leitz Diaplan fluorescent microscope under
rhodamine optics.
[0199] The data was normalized by setting Schwann cell adhesion to
astrocytes at the arbitrary value of 1.+-.0.03 (mean.+-.S.E.M.).
More than twice the number of cells adhered to the astrocytic
surfaces as compared to either the fibroblastic (0.49.+-.0.02) or
laminin surfaces (0.35.+-.0.02). The most adhesive substrate was
found to be Schwann cell monolayers, with adhesion values of
1.58.+-.0.1, compared to astrocytes (FIG. 7). The anti-migratory
astrocyte surface is therefore more adhesive to Schwann cells than
are fibroblastic surfaces with in turn are slightly more adhesive
than laminin-coated surfaces. There is therefore an inverse
correlation between rate of Schwann cell migration and
adhesion.
[0200] The results presented herein show that ACM promoted the
migration of Schwann cells in the absence of serum, and astrocyte
matrix is only a little less good than laminin as a migratory
surface. This suggests that the majority of the anti-migratory
activity displayed by astrocytes is due to interactions with cell
surface associated molecules.
EXAMPLE 4
Effect of Representative Modulation Agents on Schwann Cell Adhesion
and Migration
[0201] The cadherins are known to mediate calcium-dependent cell
adhesion (Redies and Takeichi, Dev. Biol. 180:413-423, 1996; Munro
and Blaschuk, "The Structure, Function and Regulation of
Cadherins," in Cell Adhesion and Cancer Metastasis (P. Brodt ed.)
pp. 17-34 (R. G. Landes Co., 1996). Lowering the external calcium
to 0.2 mM has been shown to disrupt cadherin-mediated interactions
between Schwann cells and other cell types (Letourneau et al.,
Neurobiol. 22:707-720, 1991).
[0202] This example illustrates the use of calcium or two
representative modulating agents to disrupt cadherin function and
increase Schwann cell migration.
[0203] A. The Effect of Lowering External Calcium Concentrations on
Schwann Cell Adhesion and Migration
[0204] In order to reduce extracellular calcium, DMEM was replaced
by S-MEM (Joklik's modification; Gibco) with 0.2 mM calcium
chloride added or the calcium buffer EGTA (Sigma) was employed.
Adhesion assays in the absence of external calcium were performed
using Schwann cells and astrocytes. Either a low calcium solution
(S-MEM in place of DMEM with 0.2 mM calcium chloride) or a calcium
buffer (EGTA in a normal DMEM medium) was used in these assays.
Various concentrations of EGTA were tested; the optimal
concentration for Schwann cell migration was found to be 1.6 mM.
EGTA concentrations less than 1.3 mM had little effect upon Schwann
cell migration whereas those above 1.8 mM caused disruption of the
astrocytic monolayer (data not shown). Adhesion of Schwann cells to
astrocytes in the presence of the standard DMEM based medium was
taken as the control and assigned the normalized value of
1.0.+-.0.03. Low calcium solutions reduced intercellular adhesion
to 0.47.+-.0.09 and the addition of 1.6 mM EGTA to DMEM reduced
adhesion to 0.39.+-.0.05 (FIG. 10A). EGTA, being the more effective
adhesion inhibitor, was incorporated into the migration assay and
found to increase the extent of Schwann cell migration upon
astrocyte monolayers to 0.86 mm.+-.0.06 compared to control
migration (0.25.+-.0.03; FIGS. 10B and 10C).
[0205] B. The Effect of Representative Modulating Agents on
Adhesion and Migration
[0206] The following modulating agents were employed at
concentrations of 1 mg/ml, LRAHAVDING-NH.sub.2 (SEQ ID NO: 21),
MRAHAVDING-NH.sub.2 (SEQ ID NO: 23), and the control peptide
LRAHGVDING-NH.sub.2 (SEQ ID NO: 21). The former two peptide
modulating agents harbor the cadherin CAR sequence, HAV. Cadherin
function was also blocked utilizing the rabbit anti-cadherin CAR
sequence antiserum designated as L7 (1:20). Normal rabbit serum
(NRS; Sigma, St. Louis, Mo.) and the goat anti-neural cell adhesion
molecule (NCAM) antiserum (Santa Cruz Biotechnology Inc., Santa
Cruz, Calif.) were also used at a dilution of 1:20 as controls.
[0207] The modulating agents LRAHAVDING-NH.sub.2 (SEQ ID NO: 21)
and MRAHAVDING-NH.sub.2 (SEQ ID NO: 23) were found to reduce
Schwann cell-astrocyte adhesion (0.38.+-.0.07 and 0.39.+-.0.04,
respective) as compared to the normalized adhesion in the absence
of peptide (1.0.+-.0.05). The control peptide LRAHGVDING-NH.sub.2
(SEQ ID NO: 21) did not significantly alter Schwann cell-astrocyte
adhesion (0.78.+-.0.10; p>0.05; FIG. 11A). Furthermore, the
rabbit antiserum L7, shown to be specific for the cadherin CAR
sequence (Alexander et al., J. Cell Physiol. 156:610-618, 1993) and
reported to block N-cadherin mediated adhesion (Newton et al., Dev.
Dynamics 197:1-13, 1993) reduced Schwann cell-astrocyte adhesion to
0.39.+-.0.06 as compared to the normalized control adhesion
1.0.+-.0.09 in the absence of antibody. This effect was not due to
non-specific factors within the antibody sera as rabbit serum had
little effect upon intercellular adhesion (0.96.+-.0.05). The
control and NCAM antibody also did not affect intracellular
adhesion (0.99.+-.0.08).
[0208] As a further control, the entire adhesion experiment was
repeated using Schwann cell monolayers as the adhesive substrate,
thereby assaying Schwann cell-Schwann cell adhesion. The antiserum
L7 was found to disrupt Schwann cell-Schwann cell adhesion to a
value of 0.5.+-.0.05 compared to the normalized control adhesion
1.0.+-.0.16. The addition of NRS and the polyclonal NCAM antibody
yielded adhesion values of 0.96.+-.0.06 and 0.99.+-.0.08,
respectively (FIG. 11B). Having shown the ability of L7 to disrupt
both Schwann cell-astrocyte and Schwann cell-Schwann cell adhesion,
the antibody was employed within the migration assay. Schwann cells
were found to migrate poorly on astrocytes in the presence of
control medium (0.16 mm.+-.0.03), NRS (0.12 mm.+-.0.02) or
polyclonal anti-NCAM (0.15 mm.+-.0.02). In comparison treatment of
the cultures with the L7 antiserum more than tripled the maximum
migration distance of Schwann cells on astrocytes (0.51 mm.+-.0.04;
FIGS. 9C, 9D, 10C, and 10D). This effect was not due to disruption
of the astroglial monolayer which remained intact (FIG. 10C).
[0209] Thus, disrupting cadherin function alters Schwann cell
adhesion and migration. Schwann cells adhere to astrocytes more
strongly than to fibroblasts and laminin, and nearly as strongly as
to other Schwann cells. In the above experiments, the number of
cells adhering to astrocytes was halved by subjecting cultures to
calcium withdrawal or by treating the cells either with modulating
agents containing the cadherin CAR sequence or with the L7
antiserum which is directed against the CAR sequence. Schwann
cell-Schwann cell adhesion was also reduced by L7 antiserum. Both
calcium withdrawal and the presence of L7 antiserum increased the
rate of Schwann cell migration on astrocytes approximately
three-fold.
[0210] These results demonstrate that modulating agents containing
the cadherin CAR sequence and antibodies directed against that
sequence are capable of disrupting cadherin function. These results
also indicate that the main family of CAMs involved in Schwann cell
adhesion and migration are the cadherins, and that blocking
cadherin mediated adhesive interactions provides a viable approach
for enhancing Schwann cell migration within the CNS. Modulating
agents capable of interfering with cadherin function may be used to
facilitate the grafting of Schwann cells into the CNS to promote
remyelination of axon regeneration, and for other purposes where a
modulation of cell adhesion is desired.
[0211] 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.
Sequence CWU 1
1
* * * * *