U.S. patent application number 10/420029 was filed with the patent office on 2003-08-21 for methods for determining cell responses through ephb receptors.
Invention is credited to Daniel, Thomas O., Stein, Elke.
Application Number | 20030157712 10/420029 |
Document ID | / |
Family ID | 22002596 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157712 |
Kind Code |
A1 |
Daniel, Thomas O. ; et
al. |
August 21, 2003 |
Methods for determining cell responses through EphB receptors
Abstract
The present invention provides a method for initiating,
promoting and/or directing cell attachment to a matrix or to
another cell, comprising contacting an EphB receptor-expressing
cell with a tetrameric EphB receptor-binding ligand, whereby
binding of the tetrameric ligand promotes multimerization of the
EphB receptor, thereby initiating, promoting and directing cell
attachment to a matrix or to another cell. Also provided is a
method for promoting angiogenesis, comprising contacting EphB
receptor-expressing cells which are associated with angiogenesis
with a multimeric EphB receptor-binding ligand, whereby binding of
the tetrameric ligand promotes multimerization of the EphB
receptor, thereby promoting angiogenesis.
Inventors: |
Daniel, Thomas O.;
(Nashville, TN) ; Stein, Elke; (San Francisco,
CA) |
Correspondence
Address: |
NEEDLE & ROSENBERG P C
127 PEACHTREE STREET N E
ATLANTA
GA
30303-1811
US
|
Family ID: |
22002596 |
Appl. No.: |
10/420029 |
Filed: |
April 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10420029 |
Apr 17, 2003 |
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09485653 |
Feb 14, 2000 |
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6555321 |
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09485653 |
Feb 14, 2000 |
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PCT/US98/17157 |
Aug 19, 1998 |
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60056164 |
Aug 19, 1997 |
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Current U.S.
Class: |
435/366 ;
435/368 |
Current CPC
Class: |
C07K 14/705 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/366 ;
435/368 |
International
Class: |
C12N 005/08 |
Goverment Interests
[0002] The present invention was funded in part by Public Health
Service awards DK8517 and DK47-48 and National Cancer Institute
award CA 68485. The government has certain rights in this
invention.
Claims
What is claimed is:
1. A method for initiating, promoting and/or directing cell
attachment to a matrix or to another cell, comprising contacting an
EphB receptor-expressing cell with a tetrameric EphB
receptor-binding ligand, whereby binding of the tetrameric ligand
promotes multimerization of the EphB receptor, thereby initiating,
promoting and/or directing cell attachment to a matrix or to
another cell.
2. The method of claim 1, wherein the receptor is EphB1 and the
ligand is ephrin-B1.
3. The method of claim 1, wherein the cell is selected from the
group consisting of endothelial cells, neuron progenitor cells,
fibroblasts, embryonic kidney cells and teratocarcinoma cells.
4. A method for initiating and/or promoting cell migration,
comprising contacting an EphB receptor-expressing cell with a
tetrameric EphB receptor-binding ligand, whereby binding of the
tetrameric ligand promotes multimerization of the EphB receptor,
thereby initiating and/or promoting cell migration.
5. The method of claim 4, wherein the receptor is EphB1 and the
ligand is ephrin-B1.
6. The method of claim 4, wherein the cell is selected from the
group consisting of endothelial cells, neuron progenitor cells,
fibroblasts, embryonic kidney cells and teratocarcinoma cells.
7. A method for promoting endothelialization of a prosthesis,
comprising contacting the prosthesis with EphB receptor-expressing
endothelial cells with a tetrameric EphB receptor-binding ligand
whereby binding of the tetrameric ligand promotes multimerization
of the EphB receptor, thereby promoting endothelialization of the
prosthesis.
8. The method of claim 7, wherein the receptor is EphB1 and the
ligand is ephrin-B1.
9. A method for promoting expression of a molecule on the surface
of an ephB receptor-expressing cell, wherein the expressed molecule
affects leukocyte or platelet attachment and migration, comprising
contacting the cell with a tetrameric Eph receptor-binding ligand,
whereby binding of the tetrameric ligand promotes multimerization
of the EphB receptor, thereby promoting expression of a molecule on
the surface of the cell which affects leukocyte or platelet
attachment and migration.
10. The method of claim 9, wherein the receptor is EphB1 and the
ligand is ephrin-B1.
11. The method of claim 9, wherein the molecule is selected from
the group consisting of .alpha..sub.v.beta..sub.3,
.alpha..sub.5.beta..sub.1, ICAM-1 and E-selectin.
12. A method for promoting function of a molecule on the surface of
an ephB receptor-expressing cell, wherein the expressed molecule
affects leukocyte or platelet attachment and migration, comprising
contacting the cell with a tetrameric EphB receptor-binding ligand
whereby binding of the tetrameric ligand promotes multimerization
of the EphB receptor, thereby promoting function of a molecule on
the surface of the cell which affects leukocyte or platelet
attachment and migration.
13. The method of claim 12, wherein the receptor is EphB1 and the
ligand is ephrin-B1.
14. The method of claim 12, wherein the molecule is selected from
the group consisting of .alpha..sub.v.beta..sub.3,
.alpha..sub.5.beta..sub.1, ICAM-1 and E-selectin.
15. A method for promoting migration, survival and/or targeting of
an EphB receptor-expressing neural cell, comprising contacting the
cell with a tetrameric EphB receptor-binding ligand, whereby
binding of the tetrameric ligand promotes multimerization of the
EphB receptor, thereby promoting migration, survival and/or
targeting of the neural cell.
16. The method of claim 15, wherein the receptor is EphB1 and the
ligand is ephrin-B1.
17. A method for targeting and/or promoting endothelial cell
incorporation at a site of endothelial cell injury or new blood
vessel formation in a subject, comprising contacting an EphB
receptor-expressing endothelial cell at the site of endothelial
cell injury or new blood vessel formation in the subject with a
tetrameric EphB receptor-binding ligand, whereby binding of the
tetrameric ligand promotes multimerization of the receptor, thereby
targeting and/or promoting endothelial cell incorporation at the
site of endothelial cell injury or new blood vessel formation in
the subject.
18. The method of claim 17, wherein the receptor is EphB1 and the
ligand is ephrin-B1.
19. A method for inhibiting cell attachment to a matrix or to
another cell, comprising contacting an EphB receptor-expressing
cell, which is stimulated to attach to a matrix or to another cell
upon binding a tetrameric Eph receptor-binding ligand, with a
substance which prevents binding of the tetrameric ligand to the
EphB receptor, thereby inhibiting cell attachment to a matrix or to
another cell.
20. A method for inhibiting cell migration, comprising contacting
an EphB receptor-expressing cell, which is stimulated to migrate
upon binding a tetrameric EphB receptor-binding ligand, with a
substance which prevents binding of the tetrameric ligand to the
EphB receptor, thereby inhibiting cell migration.
21. A method for inhibiting expression of a molecule on the surface
of an EphB receptor-expressing cell, wherein the expressed molecule
affects leukocyte or platelet attachment and migration, comprising
contacting an EphB receptor-expressing cell, which is stimulated to
express a surface molecule which affects leukocyte or platelet
attachment and migration upon binding a tetrameric EphB
receptor-binding ligand, with a substance which prevents binding of
the tetrameric ligand, thereby inhibiting expression of a molecule
on the surface of the cell which affects leukocyte or platelet
attachment and migration.
22. A method for inhibiting function of a molecule on the surface
of an EphB receptor-expressing cell, wherein the molecule affects
leukocyte or platelet attachment and migration, comprising
contacting an EphB receptor-expressing cell, which is stimulated to
promote function of a surface molecule which affects leukocyte or
platelet attachment and migration upon binding a tetrameric EphB
receptor-binding ligand, with a substance which prevents binding of
the tetrameric ligand, thereby inhibiting function of a molecule on
the surface of the cell which affects leukocyte or platelet
attachment and migration.
23. A method for inhibiting migration, survival and/or targeting of
a neural cell which expresses an EphB receptor, comprising
contacting an EphB receptor-expressing neural cell, which is
stimulated to promote migration, survival or targeting of the
neural cell upon binding a tetrameric EphB receptor-binding ligand,
with a substance which prevents binding of the tetrameric ligand,
thereby inhibiting migration, survival and/or targeting of the
neural cell.
24. A composition comprising an isolated tetrameric EphB receptor
ligand.
25. A composition comprising an isolated tetrameric EphB
receptor.
26. A composition comprising an isolated tetrameric EphB receptor
ligand/tetrameric EphB receptor complex.
27. A method for screening an EphB receptor-binding ligand for the
ability to initiate, promote and/or direct cell attachment to a
matrix or to another cell when in multimeric form, comprising: a)
contacting a multimeric EphB receptor-binding ligand with an EphB
receptor-expressing cell under conditions whereby the ligand can
bind the receptor; and b) detecting attachment of cells which have
bound multimeric ligand as compared to attachment of cells which
have not bound multimeric ligand, whereby attachment of cells which
have bound multimeric ligand and no attachment of cells which have
not bound multimeric ligand identifies an EphB receptor-binding
ligand with the ability to initiate, promote and/or direct cell
attachment to a matrix or to another cell when in multimeric
form.
28. A method for screening an EphB receptor-binding ligand for the
ability to promote cell migration when in multimeric form,
comprising: a) contacting a multimeric EphB receptor-binding ligand
with an EphB receptor-expressing cell under conditions whereby the
ligand can bind the receptor; and b) detecting migration of cells
which have bound multimeric ligand as compared to migration of
cells which have not bound multimeric ligand, whereby migration of
cells which have bound multimeric ligand and no migration of cells
which have not bound multimeric ligand identifies an EphB
receptor-binding ligand with the ability to promote cell migration
when in multimeric form.
29. A method for screening an EphB receptor-binding ligand for the
ability to promote expression of a molecule on the surface of an
EphB receptor-expressing cell, wherein the expressed molecule
affects leukocyte or platelet attachment and migration, when in
multimeric form, comprising: a) contacting a multimeric EphB
receptor-binding ligand with a cell which expresses an EphB
receptor under conditions whereby the ligand can bind the receptor;
and b) detecting expression of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells which
have bound multimeric ligand as compared to expression of a
molecule which affects leukocyte or platelet attachment and
migration on the surface of cells which have not bound multimeric
ligand, whereby expression of a molecule which affects leukocyte or
platelet attachment and migration on the surface of cells which
have bound multimeric ligand and no expression of a molecule which
affects leukocyte or platelet attachment and migration on the
surface of cells which have not bound multimeric ligand identifies
a ligand with the ability to promote the expression of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells, when the ligand is in multimeric form.
30. A method for screening an EphB receptor-binding ligand for the
ability to promote function of a molecule on the surface of an EphB
receptor-expressing cell, wherein the function of the molecule
affects leukocyte or platelet attachment and migration, when in
multimeric form, comprising: a) contacting a multimeric EphB
receptor-binding ligand with an EphB receptor-expressing cell under
conditions whereby the ligand can bind the receptor; and b)
detecting function of a molecule which affects leukocyte or
platelet attachment and migration on the surface of cells which
have bound multimeric ligand as compared to function of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells which have not bound multimeric ligand, whereby
function of a molecule which affects leukocyte or platelet
attachment and migration on the surface of cells which have bound
multimeric ligand and no function of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells which have not bound multimeric ligand identifies a ligand
with the ability to promote function of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells, when the ligand is in multimeric form.
31. A method for screening an EphB receptor-binding ligand for the
ability to promote migration, survival and/or targeting of a neural
cell when in multimeric form, comprising: a) contacting a
multimeric EphB receptor-binding ligand with an EphB
receptor-expressing neural cell under conditions whereby the ligand
can bind the receptor; and b) determining migration, survival
and/or targeting of a neural cell which has bound multimeric ligand
as compared to migration, survival or targeting of a neural cell
which has not bound multimeric ligand, whereby migration, survival
or targeting of a neural cell which has bound multimeric ligand and
no migration, survival, or targeting of a neural cell which has not
bound multimeric ligand identifies a ligand with the ability to
promote migration, survival or targeting of a neural cell when in
multimeric form.
32. A method for screening an EphB receptor for the ability to
initiate, promote and/or direct cell attachment to a matrix or to
another cell when the receptor is in multimeric form, comprising:
a) producing a multimeric EphB receptor on the surface of a cell
which expresses an EphB receptor; and b) detecting attachment of
cells with a multimeric EphB receptor as compared to attachment of
cells without a multimeric EphB receptor, whereby attachment of
cells with a multimeric EphB receptor and no attachment of cells
without a multimeric EphB receptor identifies an EphB receptor with
the ability to initiate, promote and/or direct cell attachment to a
matrix or to another cell when in multimeric form.
33. A method for screening an EphB receptor for the ability to
promote cell migration when the receptor is in multimeric form,
comprising: a) producing a multimeric EphB receptor on the surface
of a cell which expresses an EphB receptor; and b) detecting
migration of cells with a multimeric EphB receptor as compared to
migration of cells without a multimeric EphB receptor, whereby
migration of cells with a multimeric EphB receptor and no migration
of cells without a multimeric EphB receptor identifies an EphB
receptor with the ability to promote cell migration when in
multimeric form.
34. A method for screening an EphB receptor for the ability to
promote expression of a molecule on the surface of an EphB
receptor-expressing, wherein the expressed molecule affects
leukocyte or platelet attachment and migration, when the receptor
is in multimeric form, comprising: a) producing a multimeric EphB
receptor on the surface of a cell which expresses an EphB receptor;
and b) detecting expression of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells with a
multimeric EphB receptor as compared to expression of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells without a multimeric EphB receptor, whereby
expression of a molecule which affects leukocyte or platelet
attachment and migration on the surface of cells with a multimeric
EphB receptor and no expression of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells without a multimeric EphB receptor identifies an EphB
receptor with the ability to promote expression of a molecule on
the surface of a cell which expresses an EphB receptor, wherein the
expressed molecule affects leukocyte or platelet attachment and
migration, when the EphB receptor is in multimeric form.
35. A method for screening an Eph receptor for the ability to
promote function of a molecule on the surface of an EphB
receptor-expressing cell, wherein the function of the molecule
affects leukocyte or platelet attachment and migration, when the
receptor is in multimeric form, comprising: a) producing a
multimeric EphB receptor on the surface of a cell which expresses
an EphB receptor; and b) detecting function of a molecule which
affects leukocyte or platelet attachment and migration on the
surface of cells with a multimeric EphB receptor as compared to
function of a molecule which affects leukocyte or platelet
attachment and migration on the surface of cells without a
multimeric EphB receptor, whereby function of a molecule which
affects leukocyte or platelet attachment and migration on the
surface of cells with a multimeric EphB receptor and no function of
a molecule which affects leukocyte or platelet attachment and
migration on the surface of cells without a multimeric EphB
receptor identifies an EphB receptor with the ability to promote
function of a molecule on the surface of a cell which expresses an
EphB receptor, wherein the expressed molecule affects leukocyte or
platelet attachment and migration, when the EphB receptor is in
multimeric form.
36. A method for screening an EphB receptor for the ability to
promote migration, survival and/or targeting of a neural cell when
the receptor is in multimeric form, comprising: a) producing a
multimeric EphB receptor on the surface of a neural cell which
expresses an EphB receptor; and b) detecting migration, survival or
targeting of neural cells with a multimeric EphB receptor as
compared to migration, survival or targeting of neural cells
without a multimeric EphB receptor, whereby migration, survival
and/or targeting of neural cells with a multimeric EphB receptor
and no migration, survival or targeting of neural cells without a
multimeric EphB receptor identifies an EphB receptor with the
ability to promote migration, survival and/or targeting of neural
cells when in multimeric form.
37. A method for screening a substance for the ability to inhibit
the binding of a multimeric EphB receptor-binding ligand to an EphB
receptor comprising: a) contacting the substance with a cell
expressing an EphB receptor; b) contacting the cell of step (a)
with a multimeric EphB receptor-binding ligand under conditions
whereby the multimeric ligand can bind the receptor; and c)
detecting the binding of the multimeric ligand to the receptor,
whereby no binding of the multimeric ligand to the receptor
identifies a substance with the ability to inhibit the binding of a
multimeric EphB receptor-binding ligand to an EphB receptor.
38. The method of claim 38, wherein the detection of binding of the
multimeric EphB receptor-binding ligand to the EphB receptor is by
detecting an activity selected from the group consisting of cell
attachment to a matrix or to another cell, cell migration,
expression of a surface molecule affecting leukocyte or platelet
attachment and migration, promotion of function of a surface
molecule affecting leukocyte or platelet attachment and migration
and promotion of migration, survival or targeting of a neural
cell.
39. A method for promoting angiogenesis, comprising contacting EphB
receptor-expressing cells which are associated with angiogenesis
with a multimeric EphB receptor-binding ligand, whereby binding of
the tetrameric ligand promotes multimerization of the EphB
receptor, thereby promoting angiogenesis.
40. A method for disrupting angiogenesis comprising contacting an
EphB receptor-expressing cell which promotes angiogenesis upon
binding a multimeric EphB receptor-binding ligand with a substance
which prevents formation of a multimeric EphB receptor-binding
ligand or inhibits binding of the multimeric EphB receptor-binding
ligand to the receptor, thereby disrupting angiogenesis.
41. A method for treating a disease associated with pathological
angiogenesis in a subject, comprising contacting an EphB
receptor-expressing cell of the subject which promotes angiogenesis
upon binding a tetrameric EphB receptor-binding ligand with a
substance which prevents binding of the tetrameric ligand, thereby
disrupting angiogenesis and treating a disease associated with
pathological angiogenesis.
42. A method for treating a condition associated with interruption
of angiogenic processes in a subject, comprising contacting an EphB
receptor-expressing cell of the subject with a tetrameric EphB
receptor-binding ligand, whereby binding of the tetrameric ligand
promotes multimerization of the EphB receptor, thereby promoting
angiogenesis and treating a condition associated with interruption
of angiogenic processes.
43. A method for screening a substance for the ability to inhibit
angiogenesis, comprising: a) contacting the substance with a cell
expressing an EphB receptor; b) contacting the cell of step (a)
with a multimeric EphB receptor-binding ligand, which promotes
angiogenesis, under conditions whereby the multimeric ligand can
bind the receptor; and c) detecting angiogenesis in cells contacted
with the substance, as compared to angiogenesis in cells not
contacted with the substance, whereby no angiogenesis in cells
contacted with the substance and angiogenesis in cells not
contacted with the substance identifies a substance having the
ability to inhibit angiogenesis.
44. A method for screening an EphB-receptor binding ligand for the
ability to promote angiogenesis when in multimeric form,
comprising: a) contacting a multimeric EphB receptor-binding ligand
with a cell which expresses an EphB receptor under conditions
whereby the ligand can bind the receptor; and b) detecting
angiogenesis of cells which have bound multimeric ligand as
compared to angiogenesis of cells which have not bound multimeric
ligand, whereby angiogenesis of cells which have bound multimeric
ligand and no angiogenesis of cells which have not bound multimeric
ligand identifies an EphB receptor-binding ligand with the ability
to promote angiogenesis when in multimeric form.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of and claims
benefit of priority from U.S. Ser. No. 09/485,653, filed Feb. 14,
2000 (now allowed), which is a 35 U.S.C. .sctn. 371 national phase
application filed from and claiming benefit of priority from
international application PCT/US98/17157, filed Aug. 19, 1998,
which claims benefit of priority from U.S. provisional application
60/056,164, filed Aug. 19, 1997, all of which applications are
herein incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods and compositions
for initiating, promoting and directing cell attachment, migration
and cell to cell assembly in response to multimerization of ligands
and/or receptors. In particular, the present invention provides
multimeric ligands which bind receptors of the Eph receptor
tyrosine kinase family to selectively modulate specific cell
activities such as cell attachment and cell to cell assembly by
promoting the formation of multimeric receptors of specific
composition.
[0005] 2. Background Art
[0006] Developmental organization and subsequent remodeling of
vasculature obligates vascular endothelial cells and their
progenitors to use cell-cell recognition and targeting machinery,
both in the initial stages of vasculogenesis and in the angiogenic
remodeling required for organogenesis, wound healing and tumor
growth. Similar to the developing neural system, vascular cells
must migrate, discriminate and assemble with appropriate partner
cells to establish and remodel highly integrated and interconnected
cellular networks. Early developmental "vasculogenic" assembly of
blood vessels requires that endothelial progenitor cells migrate in
response to yet unidentified cues, then discriminate among cells
they contact to assemble vascular structures with similarly
programmed endothelial progenitors (32). During the vascular
remodeling occurring during organogenesis, wound healing and tumor
growth, endothelial cells in existing vessels receive angiogenic
cues to invade their basement membrane and sprout long migrating
cellular processes that resemble axons (33,34). Cultured
microvascular endothelial cells extend similar processes that track
along fibrillar strands of connective tissue matrix to contact and
engage appropriate partner cells to form cord and tube structures
during in vitro capillary-like morphogenesis. In vivo (and in
vitro), these processes encounter and discriminate among cells
until appropriate partners for assembly are identified. On
engagement of processes extended from existing, efferent limb
vessels, specialized interendothelial cellular connections are
established to ultimately interconnect lumen (35). This entire
scenario is quite similar to the axonal extension, migration,
discrimination and targeting processes that direct correct assembly
of neural networks.
[0007] An enlarging body of evidence has assigned members of the
Eph family of receptor tyrosine kinases crucial roles in neural
targeting, and early data also support their function in the
assembly of vascular structures (1, 2, 3, 7, 25). As a class, the
Eph family of receptors and their ligands are tissue restricted in
their distribution and are highly diverse, with over 13 distinct
receptors interacting with distinct ligands. During development,
specific Eph family receptors are expressed in distinct tissue
sites that are bounded by adjacent tissues expressing their
membrane-bound ligands in a reciprocal compartmentation pattern
(26). A topographically defined gradient of one ligand, ephrin-A2
(ELF-1), on membranes in the tectum directs the correct targeting
of retinal axons that express differential levels of its receptor,
EphA3 (Mek 4) (36). Interestingly, regional overexpression of
ephrin-A2 (ELF-1) misdirects these projections (37). These and
other experiments emphasize the important role these Eph family
receptors and ligands play in directing neuronal cell-cell
interactions in developing nervous tissue.
[0008] Eph family receptors are subdivided into two functional
classes by their affinities for membrane-bound ligands of two
different structural types. Receptors of the EphA subfamily,
including EphA3 (Mek 4), EphA5 (Ehk-1) and others, bind ligands
that are membrane-associated through glycerophosphatidylinositol
(GPI) linkages, and may be released by phospholipases C and D (41).
The GPI-linked ligands characterized to date are ephrin-A1,
ephrin-A3, ephrin-A4, ephrin-A2 and ephrin-A5 (formerly called
LERKs 1, 3, 4, 6, & 7) (2, 7, 36, 38). The EphB receptor
subfamily members show overlapping high affinities for ligands that
are transmembrane proteins, including ephrin-B1, ephrin-B2 and
ephrin-B3 (formerly called LERKs 2, 5 & 8) (9, 10, 30, 39). The
transmembrane spanning ligands show remarkable amino acid
conservation on the carboxy terminus, implying conservation of
structure important in their function, and clouding the distinction
between receptors and ligands.
[0009] Recent evidence suggests the capacity of these "ligands" to
signal through engagement of "receptor" extracellular domains.
Engagement of ephrin-B1("ligand") by recombinant EphB2 (Nuk)
("receptor") ectodomain initiates tyrosine phosphorylation of
ephrin-B1 (28). Moreover, guidance of hippocampal neurons appears
directed by their expression of "ligands" for EphB2 (Nuk), a
"receptor" ectodomain they engage during the course of decussation
through the anterior commissure during development (23). Thus,
bidirectional signaling may be initiated by juxtacrine
ligand-receptor engagement on cell-cell contact. Such bidirectional
signaling is attractive as an intermediate step in cell-cell
recognition and commitment to assemble multicellular
structures.
[0010] Both EphA2 (Eck) and EphB1 (ELK) have been implicated as
important intermediaries during angiogenesis. The primary ligand
for EphA2 (Eck) was first cloned as a TNF.alpha.-induced product of
cultured human umbilical vein endothelial cells (ephrin-A1,
previously called B-61 or LERK-1) (39, 40). Ephrin-A1 is a
GPI-linked membrane protein that is also released into a soluble
fraction where it may promote migration of bovine endothelial cells
through its interaction with EphA2 (Eck) (7). Antibodies against
ephrin-A1 interrupt TNF.alpha.-induced angiogenic responses in the
rat cornea assay, consistent with their interruption of ephrin-A1
to promote angiogenesis through EphA2 (Eck) (7).
[0011] EphB1 (ELK) and ephrin-B1 (LERK-2) are both expressed on
mesenchymal progenitors of vascular cells, on glomerular capillary
endothelial cells in mature kidney and on human umbilical vein
endothelial cells (8). The early expression of ephrin-B1 and EphB1
in renal glomerular microvascular progenitor cells has suggested
their participation in targeting and capillary assembly in this
specialized microcirculation (8). Ephrin-B1 promotes assembly of
human renal microvascular endothelial cells (HRMEC) into
capillary-like structures (8), yet human umbilical vein endothelial
cells (HUVEC) are not responsive, despite their expression of
EphB1, and its tyrosine phosphorylation in response to ephrin-B1.
In contrast, ephrin-A1 (LERK-1) has no capillary-assembly activity
for HRMEC, yet promotes capillary-like assembly of HUVEC (8). Thus,
the downstream signaling responses in vascular endothelial cells
from different microcirculations distinguish among Eph receptor
ligands to determine different cellular responses.
[0012] The present invention provides methods for selectively
modulating cell attachment, cell migration, cell to cell assembly
and other activities regulated by Eph receptor activation through
the promotion or inhibition of multimerization of the receptors
which regulate these functions by signal transduction.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method for initiating,
promoting and/or directing cell attachment to a matrix or to
another cell, comprising contacting an EphB receptor-expressing
cell with a tetrameric EphB receptor-binding ligand, whereby
binding of the tetrameric ligand promotes multimerization of the
EphB receptor, thereby initiating, promoting and/or directing cell
attachment to a matrix or to another cell.
[0014] Also provided is a method for initiating and/or promoting
cell migration, comprising contacting an EphB receptor-expressing
cell with a tetrameric EphB receptor-binding ligand, whereby
binding of the tetrameric ligand promotes multimerization of the
EphB receptor, thereby initiating and/or promoting cell
migration.
[0015] A method for promoting endothelialization of a prosthesis is
also provided, comprising contacting the prosthesis with EphB
receptor-expressing endothelial cells with a tetrameric EphB
receptor-binding ligand whereby binding of the tetrameric ligand
promotes multimerization of the EphB receptor, thereby promoting
endothelialization of the prosthesis.
[0016] In addition, the present invention provides a method for
promoting expression of a molecule on the surface of an ephB
receptor-expressing cell, wherein the expressed molecule affects
leukocyte or platelet attachment and migration, comprising
contacting the cell with a tetrameric Eph receptor-binding ligand,
whereby binding of the tetrameric ligand promotes multimerization
of the EphB receptor, thereby promoting expression of a molecule on
the surface of the cell which affects leukocyte or platelet
attachment and migration.
[0017] Further provided is a method for promoting function of a
molecule on the surface of an ephB receptor-expressing cell,
wherein the expressed molecule affects leukocyte or platelet
attachment and migration, comprising contacting the cell with a
tetrameric EphB receptor-binding ligand whereby binding of the
tetrameric ligand promotes multimerization of the EphB receptor,
thereby promoting function of a molecule on the surface of the cell
which affects leukocyte or platelet attachment and migration.
[0018] The present invention also provides a method for promoting
migration, survival and/or targeting of an EphB receptor-expressing
neural cell, comprising contacting the cell with a tetrameric EphB
receptor-binding ligand, whereby binding of the tetrameric ligand
promotes multimerization of the EphB receptor, thereby promoting
migration, survival and/or targeting of the neural cell.
[0019] The present invention additionally provides a method for
targeting and/or promoting endothelial cell incorporation at a site
of endothelial cell injury or new blood vessel formation in a
subject, comprising contacting an EphB receptor-expressing
endothelial cell at the site of endothelial cell injury or new
blood vessel formation in the subject with a tetrameric EphB
receptor-binding ligand, whereby binding of the tetrameric ligand
promotes multimerization of the receptor, thereby targeting and/or
promoting endothelial cell incorporation at the site of endothelial
cell injury or new blood vessel formation in the subject.
[0020] Furthermore, the present invention provides a method for
inhibiting cell attachment to a matrix or to another cell,
comprising contacting an EphB receptor-expressing cell, which is
stimulated to attach to a matrix or to another cell upon binding a
tetrameric Eph receptor-binding ligand, with a substance which
prevents binding of the tetrameric ligand to the EphB receptor,
thereby inhibiting cell attachment to a matrix or to another
cell.
[0021] A method for inhibiting cell migration is additionally
provided, comprising contacting an EphB receptor-expressing cell,
which is stimulated to migrate upon binding a tetrameric EphB
receptor-binding ligand, with a substance which prevents binding of
the tetrameric ligand to the EphB receptor, thereby inhibiting cell
migration.
[0022] Additionally provided is a method for inhibiting expression
of a molecule on the surface of an EphB receptor-expressing cell,
wherein the expressed molecule affects leukocyte or platelet
attachment and migration, comprising contacting an EphB
receptor-expressing cell, which is stimulated to express a surface
molecule which affects leukocyte or platelet attachment and
migration upon binding a tetrameric EPhB receptor-binding ligand,
with a substance which prevents binding of the tetrameric ligand,
thereby inhibiting expression of a molecule on the surface of the
cell which affects leukocyte or platelet attachment and
migration.
[0023] The present invention also provides a method for inhibiting
function of a molecule on the surface of an EphB
receptor-expressing cell, wherein the molecule affects leukocyte or
platelet attachment and migration, comprising contacting an EphB
receptor-expressing cell, which is stimulated to promote function
of a surface molecule which affects leukocyte or platelet
attachment and migration upon binding a tetrameric EphB
receptor-binding ligand, with a substance which prevents binding of
the tetrameric ligand, thereby inhibiting function of a molecule on
the surface of the cell which affects leukocyte or platelet
attachment and migration.
[0024] The present invention additionally provides a method for
inhibiting migration, survival and/or targeting of a neural cell
which expresses an EphB receptor, comprising contacting an EphB
receptor-expressing neural cell, which is stimulated to promote
migration, survival or targeting of the neural cell upon binding a
tetrameric EphB receptor-binding ligand, with a substance which
prevents binding of the tetrameric ligand, thereby inhibiting
migration, survival and/or targeting of the neural cell.
[0025] The present invention also provides a composition comprising
an isolated tetrameric EphB receptor ligand, a composition
comprising an isolated tetrameric EphB receptor and a composition
comprising an isolated tetrameric EphB receptor ligand/tetrameric
EphB receptor complex.
[0026] Furthermore, the present invention provides a method for
screening an EphB receptor-binding ligand for the ability to
initiate, promote and/or direct cell attachment to a matrix or to
another cell when in multimeric form, comprising:
[0027] a) contacting a multimeric EphB receptor-binding ligand with
an EphB receptor-expressing cell under conditions whereby the
ligand can bind the receptor; and
[0028] b) detecting attachment of cells which have bound multimeric
ligand as compared to attachment of cells which have not bound
multimeric ligand, whereby attachment of cells which have bound
multimeric ligand and no attachment of cells which have not bound
multimeric ligand identifies an EphB receptor-binding ligand with
the ability to initiate, promote and/or direct cell attachment to a
matrix or to another cell when in multimeric form.
[0029] A method for screening an EphB receptor-binding ligand for
the ability to promote cell migration when in multimeric form is
additionally provided, comprising:
[0030] a) contacting a multimeric EphB receptor-binding ligand with
an EphB receptor-expressing cell under conditions whereby the
ligand can bind the receptor; and
[0031] b) detecting migration of cells which have bound multimeric
ligand as compared to migration of cells which have not bound
multimeric ligand, whereby migration of cells which have bound
multimeric ligand and no migration of cells which have not bound
multimeric ligand identifies an EphB receptor-binding ligand with
the ability to promote cell migration when in multimeric form.
[0032] Also provided is a method for screening an EphB
receptor-binding ligand for the ability to promote expression of a
molecule on the surface of an EphB receptor-expressing cell,
wherein the expressed molecule affects leukocyte or platelet
attachment and migration, when in multimeric form, comprising:
[0033] a) contacting a multimeric EphB receptor-binding ligand with
a cell which expresses an EphB receptor under conditions whereby
the ligand can bind the receptor; and
[0034] b) detecting expression of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells which have bound multimeric ligand as compared to expression
of a molecule which affects leukocyte or platelet attachment and
migration on the surface of cells which have not bound multimeric
ligand, whereby expression of a molecule which affects leukocyte or
platelet attachment and migration on the surface of cells which
have bound multimeric ligand and no expression of a molecule which
affects leukocyte or platelet attachment and migration on the
surface of cells which have not bound multimeric ligand identifies
a ligand with the ability to promote the expression of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells, when the ligand is in multimeric form.
[0035] The present invention further provides a method for
screening an EphB receptor-binding ligand for the ability to
promote function of a molecule on the surface of an EphB
receptor-expressing cell, wherein the function of the molecule
affects leukocyte or platelet attachment and migration, when in
multimeric form, comprising:
[0036] a) contacting a multimeric EphB receptor-binding ligand with
an EphB receptor-expressing cell under conditions whereby the
ligand can bind the receptor; and
[0037] b) detecting function of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells which
have bound multimeric ligand as compared to function of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells which have not bound multimeric ligand, whereby
function of a molecule which affects leukocyte or platelet
attachment and migration on the surface of cells which have bound
multimeric ligand and no function of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells which have not bound multimeric ligand identifies a ligand
with the ability to promote function of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells, when the ligand is in multimeric form.
[0038] The present invention also provides a method for screening
an EphB receptor-binding ligand for the ability to promote
migration, survival and/or targeting of a neural cell when in
multimeric form, comprising:
[0039] a) contacting a multimeric EphB receptor-binding ligand with
an EphB receptor-expressing neural cell under conditions whereby
the ligand can bind the receptor; and
[0040] b) determining migration, survival and/or targeting of a
neural cell which has bound multimeric ligand as compared to
migration, survival or targeting of a neural cell which has not
bound multimeric ligand, whereby migration, survival or targeting
of a neural cell which has bound multimeric ligand and no
migration, survival, or targeting of a neural cell which has not
bound multimeric ligand identifies a ligand with the ability to
promote migration, survival or targeting of a neural cell when in
multimeric form.
[0041] Additionally provided is a method for screening an EphB
receptor for the ability to initiate, promote and/or direct cell
attachment to a matrix or to another cell when the receptor is in
multimeric form, comprising:
[0042] a) producing a multimeric EphB receptor on the surface of a
cell which expresses an EphB receptor; and
[0043] b) detecting attachment of cells with a multimeric EphB
receptor as compared to attachment of cells without a multimeric
EphB receptor, whereby attachment of cells with a multimeric EphB
receptor and no attachment of cells without a multimeric EphB
receptor identifies an EphB receptor with the ability to initiate,
promote and/or direct cell attachment to a matrix or to another
cell when in multimeric form.
[0044] Further provided is a method for screening an EphB receptor
for the ability to promote cell migration when the receptor is in
multimeric form, comprising:
[0045] a) producing a multimeric EphB receptor on the surface of a
cell which expresses an EphB receptor; and
[0046] b) detecting migration of cells with a multimeric EphB
receptor as compared to migration of cells without a multimeric
EphB receptor, whereby migration of cells with a multimeric EphB
receptor and no migration of cells without a multimeric EphB
receptor identifies an EphB receptor with the ability to promote
cell migration when in multimeric form.
[0047] The present invention also provides a method for screening
an EphB receptor for the ability to promote expression of a
molecule on the surface of an EphB receptor-expressing, wherein the
expressed molecule affects leukocyte or platelet attachment and
migration, when the receptor is in multimeric form, comprising:
[0048] a) producing a multimeric EphB receptor on the surface of a
cell which expresses an EphB receptor; and
[0049] b) detecting expression of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells with a multimeric EphB receptor as compared to expression of
a molecule which affects leukocyte or platelet attachment and
migration on the surface of cells without a multimeric EphB
receptor, whereby expression of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells with a
multimeric EphB receptor and no expression of a molecule which
affects leukocyte or platelet attachment and migration on the
surface of cells without a multimeric EphB receptor identifies an
EphB receptor with the ability to promote expression of a molecule
on the surface of a cell which expresses an EphB receptor, wherein
the expressed molecule affects leukocyte or platelet attachment and
migration, when the EphB receptor is in multimeric form.
[0050] The present invention additionally provides a method for
screening an Eph receptor for the ability to promote function of a
molecule on the surface of an EphB receptor-expressing cell,
wherein the function of the molecule affects leukocyte or platelet
attachment and migration, when the receptor is in multimeric form,
comprising:
[0051] a) producing a multimeric EphB receptor on the surface of a
cell which expresses an EphB receptor; and
[0052] b) detecting function of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells with a
multimeric EphB receptor as compared to function of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells without a multimeric EphB receptor, whereby
function of a molecule which affects leukocyte or platelet
attachment and migration on the surface of cells with a multimeric
EphB receptor and no function of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells
without a multimeric EphB receptor identifies an EphB receptor with
the ability to promote function of a molecule on the surface of a
cell which expresses an EphB receptor, wherein the expressed
molecule affects leukocyte or platelet attachment and migration,
when the EphB receptor is in multimeric form.
[0053] A method for screening an EphB receptor for the ability to
promote migration, survival and/or targeting of a neural cell when
the receptor is in multimeric form is also provided,
comprising:
[0054] a) producing a multimeric EphB receptor on the surface of a
neural cell which expresses an EphB receptor; and
[0055] b) detecting migration, survival or targeting of neural
cells with a multimeric EphB receptor as compared to migration,
survival or targeting of neural cells without a multimeric EphB
receptor, whereby migration, survival and/or targeting of neural
cells with a multimeric EphB receptor and no migration, survival or
targeting of neural cells without a multimeric EphB receptor
identifies an EphB receptor with the ability to promote migration,
survival and/or targeting of neural cells when in multimeric
form.
[0056] In addition, the present invention provides a method for
screening a substance for the ability to inhibit the binding of a
multimeric EphB receptor-binding ligand to an EphB receptor
comprising:
[0057] a) contacting the substance with a cell expressing an EphB
receptor;
[0058] b) contacting the cell of step (a) with a multimeric EphB
receptor-binding ligand under conditions whereby the multimeric
ligand can bind the receptor; and
[0059] c) detecting the binding of the multimeric ligand to the
receptor, whereby no binding of the multimeric ligand to the
receptor identifies a substance with the ability to inhibit the
binding of a multimeric EphB receptor-binding ligand to an EphB
receptor.
[0060] Also provided herein is a method for promoting angiogenesis,
comprising contacting EphB receptor-expressing cells which are
associated with angiogenesis with a multimeric EphB
receptor-binding ligand, whereby binding of the tetrameric ligand
promotes multimerization of the EphB receptor, thereby promoting
angiogenesis.
[0061] A method for disrupting angiogenesis is additionally
provided, comprising contacting an EphB receptor-expressing cell
which promotes angiogenesis upon binding a multimeric EphB
receptor-binding ligand with a substance which prevents formation
of a multimeric EphB receptor-binding ligand or inhibits binding of
the multimeric EphB receptor-binding ligand to the receptor,
thereby disrupting angiogenesis.
[0062] Further provided is a method for treating a disease
associated with pathological angiogenesis in a subject, comprising
contacting an EphB receptor-expressing cell of the subject which
promotes angiogenesis upon binding a tetrameric EphB
receptor-binding ligand with a substance which prevents binding of
the tetrameric ligand, thereby disrupting angiogenesis and treating
a disease associated with pathological angiogenesis.
[0063] The present invention also provides a method for treating a
condition associated with interruption of angiogenic processes in a
subject, comprising contacting an EphB receptor-expressing cell of
the subject with a tetrameric EphB receptor-binding ligand, whereby
binding of the tetrameric ligand promotes multimerization of the
EphB receptor, thereby promoting angiogenesis and treating a
condition associated with interruption of angiogenic processes.
[0064] The present invention additionally provides a method for
screening a substance for the ability to inhibit angiogenesis,
comprising:
[0065] a) contacting the substance with a cell expressing an EphB
receptor;
[0066] b) contacting the cell of step (a) with a multimeric EphB
receptor-binding ligand, which promotes angiogenesis, under
conditions whereby the multimeric ligand can bind the receptor;
and
[0067] c) detecting angiogenesis in cells contacted with the
substance, as compared to angiogenesis in cells not contacted with
the substance, whereby no angiogenesis in cells contacted with the
substance and angiogenesis in cells not contacted with the
substance identifies a substance having the ability to inhibit
angiogenesis.
[0068] Finally, the present invention provides a method for
screening an EphB-receptor binding ligand for the ability to
promote angiogenesis when in multimeric form, comprising:
[0069] a) contacting a multimeric EphB receptor-binding ligand with
a cell which expresses an EphB receptor under conditions whereby
the ligand can bind the receptor; and
[0070] b) detecting angiogenesis of cells which have bound
multimeric ligand as compared to angiogenesis of cells which have
not bound multimeric ligand, whereby angiogenesis of cells which
have bound multimeric ligand and no angiogenesis of cells which
have not bound multimeric ligand identifies an EphB
receptor-binding ligand with the ability to promote angiogenesis
when in multimeric form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] FIGS. 1A, 1B and 1C. Ligand multimers elicit different
responses. FIG. 1A: In an in vitro angiogenesis assay, HRMEC (12)
were plated on Matrigel-coated dishes in defined medium in the
absence (NA), or presence of indicated agonists, including phorbol
myristate acetate (PMA, 20 ng ml.sup.-1). Ephrin-B1/Fc (at
indicated concentrations) or a control Fc fusion protein, ORF/Fc
(9) (500 ng ml.sup.-1), were presented as either dimers, or
preclustered multimers (+anti-Fc). Cells were photographed 8 h
after plating. Insert: Phosphotyrosine immunoblots of EphB1
immunoprecipitates from HRMEC plated on Matrigel-coated dishes.
FIG. 1B: Attachment of HRMEC to either Matrigel- or
fibronectin-coated dishes 90 min after plating in medium
supplemented with dimeric or multimeric (+anti-Fc) Fc fusion
proteins. (Mean+/-SEM of three independent determinations are
displayed (*p<0.05, **p<0.001, ***p<0.0001)). FIG. 1C:
Attachment of P19 cells to fibronectin coated dishes and
phosphotyrosine immunoblots of EphB1 (insert) after plating in
medium supplemented with dimeric, or multimeric (+anti-Fc), ORF/Fc
or ephrin-B1/Fc.
[0072] FIGS. 2A, 2B and 2C. Ephrin-B1/Fc tetramers recruit LMW-PTP
to EphB1 and promote attachment. FIG. 2A: Ephrin-B1/Fc dimers
(ephrin-B1/Fc, 50 .mu.g) or preclustered multimers (ephrin-B1/Fc,
50 .mu.g+anti-Fc, 5 .mu.g) were separated by exclusion
chromatography in PBS on a Sepharose 6 column (Pharmacia)
calibrated with standards of 445 kDa (ferritin), 272 kDa (urease)
and 150 kDa (IgG). FIGS. 2B and 2C: Fractions containing 500 ng
ml.sup.-1 protein from the indicated A.sub.278 peaks (Fxn "A," Fxn
"B," Fxn "C") were analyzed for activity to promote tyrosine
phosphorylation of EphB1 and recruitment of LMW-PTP to EphB1
complexes (FIG. 2B) and to promote attachment of HRMEC and P19
cells (FIG. 2C). Complex stoichiometry was confirmed by comparison
of each fraction to mixed standards of Ephrin-B1/Fc and anti-Fc
separated by SDS-PAGE, transferred to Immobilon-P and stained with
amido black. Tetramers (one anti-Fc molecule complexing two
ephrin-B1/Fc dimers) showed activity in both assays.
[0073] FIGS. 3A, 3B and 3C. Ephrin-B1/Fc adsorbed to
nitrocellulose-coated surfaces stimulates .alpha..sub.v.beta..sub.3
integrin-mediated endothelial attachment at defined densities. FIG.
3A: Microvascular endothelial attachment to fibrinogen is
stimulated by precoating of surfaces with nitrocellulose-bound
ephrin-B1/Fc. EphrinB1/Fc (300 ng/cm.sup.2 surface area) was
adsorbed overnight at 4.degree. C. to 48-well plates precoated with
either nitrocellulose, fibrinogen, or both, in combination with
ephrin-B1/Fc (300 ng/cm.sup.2) or an Fc control, human IgG.sub.1
(300 ng/cm.sup.2), as indicated. Wells were washed, blocked with
solution containing 1% bovine albumin, plated with 7.times.10.sup.5
endothelial cells for 1 h, then assayed for cell attachment, as
described herein. FIG. 3B: Effect of adsorbed fibrinogen density on
endothelial attachment. Indicated amounts of fibrinogen were
adsorbed to surfaces coated with no additive (Fbg alone) or
ephrinB1/Fc (300 ng/cm.sup.2), and endothelial attachment was
assayed, as described herein. FIG. 3C: Adsorbed ephrin-B1/Fc
density effect on endothelial attachment. Indicated amounts of
ephrin-B1/Fc were adsorbed to surfaces coated with fibrinogen (2
mg/cm.sup.2), and endothelial attachment was assayed, as described
herein. EphB1 receptor activation was determined by phosphotyrosine
immunoblot of immunoprecipitated EphB1 receptors.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present invention provides the surprising discovery that
distinctions among ligand multimers are critical determinants of
biological response such as cell attachment, cell migration and
cell to cell assembly. Thus, the methods of this invention provide
for the formation of ligand multimers which promote the formation
of receptor multimers on the surface of cells for inducing specific
biological responses. It is understood that in these methods,
multimeric receptors can be produced by binding multimeric ligands
as well as by any other mechanism or with any other substance which
can produce multimeric receptors.
[0075] Thus, the present invention provides a method for
initiating, promoting and/or directing cell attachment, comprising
contacting cells with a substance which promotes multimerization of
receptors associated with initiating, promoting and/or directing
cell attachment. Thus, it is understood that the method of this
invention provides for one or more of the processes of initiation,
promotion and direction as these processes pertain to cell
attachment. The substance can be a multimeric ligand and in a
preferred embodiment can be a tetrameric ligand and the
multimerization of the receptor can result in the formation of
tetrameric receptors. The multimeric ligand can also be a multimer
of ligands greater than four which promotes multimerization of
receptors.
[0076] Further provided is a method for initiating and promoting
cell migration comprising contacting cells with a substance which
promotes multimerization of receptors associated with initiating
and promoting cell migration. The substance can be a multimeric
ligand such as a tetrameric ligand which promotes the formation of
tetrameric receptors. The cells can be contacted with the substance
in vivo or ex vivo as described above.
[0077] In addition, the present invention provides a method for
promoting cell to cell assembly, comprising contacting cells with a
substance which promotes multimerization of receptors associated
with cell to cell assembly. The substance can be a multimeric
ligand such as a tetrameric ligand which promotes the formation of
tetrameric receptors. The cells can be contacted with the substance
in vivo or ex vivo as described above.
[0078] Further provided is a method for producing multimeric
receptors comprising contacting receptors with a substance which
promotes multimerization. The receptors can be isolated according
to methods standard in the art or the receptors can be on the
surface of cells. The substance which promotes multimerization of
receptors can be a multimeric ligand, such as a tetrameric ligand
which produces tetrameric receptors. In addition, the multimeric
receptors can be produced in vivo, ex vivo or in vitro.
[0079] The receptor associated with cell migration, cell attachment
and cell to cell assembly can be a member of the ELK receptor
family, which can be, but is not limited to member of the Eph
receptor subclass, such as EphA1, EphA2, EphA3, EphA4, EphA5,
EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4 and EphB5.
[0080] The ligand which can be used to promote multimerization of
receptors can be, but is not limited to, ephrin-A1, ephrin-A2,
ephrin-A3, ephrin-A4, ephrin-A5, ephrin-B1, ephrin-B2 and
ephrin-B3.
[0081] Thus, the present invention also provides a method for
initiating, promoting and/or directing the attachment and
differentiation of cells, comprising contacting cells with a ligand
which initiates, promotes and/or directs the attachment and
differentiation of cells when in multimeric form. The ligand
multimers can initiate, promote and/or direct these activities by
producing multimeric receptors which are activated when in
multimeric form to trigger these activities in the cell. The ligand
multimers of the present invention are preferably tetramers, but
can be multimers of greater numbers, such as octamers, etc., which
initiate, promote and/or direct the attachment and differentiation
of cells. Alternatively, the ligand multimers of this invention can
block the initiation, promotion and/or direction of cell attachment
and differentiation of cells.
[0082] Furthermore, the present invention provides a method for
initiating and/or promoting cell migration, comprising contacting
cells with a ligand which initiates and/or promotes cell migration
when in multimeric form. The ligand multimers can initiate and/or
promote cell migration by producing multimeric receptors which are
activated when in multimeric form to trigger cell migration. The
ligand multimers of the present invention are preferably tetramers,
but can be multimers of greater numbers which initiate and/or
promote cell migration. Alternatively, the ligand multimers of this
invention can block the initiation and/or promotion of cell
migration.
[0083] Also provided is a method for promoting cell to cell
assembly, comprising contacting cells with a ligand which promotes
cell to cell assembly when in multimeric form. The multimeric
ligand can promote cell to cell assembly by promoting the formation
of multimeric receptors which activate cell to cell assembly upon
formation into multimeric receptors. As stated above, the ligand
multimers can be tetramers or larger multimers which promote
cell-cell assembly. Alternatively, the ligand multimers of this
invention can block the promotion of cell to cell assembly.
[0084] In particular, the present invention provides a method for
initiating, promoting and/or directing cell attachment to a matrix
or to another cell, comprising contacting an EphB
receptor-expressing cell with a tetrameric EphB receptor-binding
ligand which promotes multimerization of the receptor, whereby
binding of the tetrameric ligand promotes multimerization of the
EphB receptor, thereby initiating, promoting and/or directing cell
attachment to a matrix or to another cell.
[0085] As used herein, "matrix" can mean any solid or semi-solid
surface or substrate, which can be either biological or
non-biological, to which a cell can attach, as would be well known
in the art. Examples of a matrix of this invention can include, but
is not limited to, peptides containing an RGD sequence],
vitronectin, fibrinogen, fibronectin and like compounds, as would
be known to one of skill in the art.
[0086] The present invention also provides a method for initiating
and promoting cell migration, comprising contacting an EphB
receptor-expressing cell with a tetrameric EphB receptor-binding
which promotes multimerization of the receptor, whereby binding of
the tetrameric ligand promotes multimerization of the EphB
receptor, thereby initiating and promoting cell migration.
[0087] The EphB receptor of these methods can be EphB1 or EphB2 and
the ligand of this method can be ephrinB1 or ephrinB2. In addition,
the cells of these methods can be, but are not limited to,
epithelial cells, endothelial cells, neuron progenitor cells,
fibroblasts, embryonic kidney cells and teratocarcinoma cells.
[0088] For the purposes of the present invention, cell to cell
assembly, cell attachment and cell migration are distinct from cell
proliferation and tyrosine phosphorylation. Also, as used herein,
the term "binding" describes the interaction between a receptor and
a ligand specific for that receptor; "signaling" describes cellular
events or processes which are mediated by binding of ligand to
receptor; and "attachment" describes the interaction between a cell
and a matrix or between a cell and another cell.
[0089] The cells of the present invention can include, but are not
limited to, mature and progenitor forms of cells with
differentiated characteristics of neurons of all types,
endothelial, pericyte, smooth muscle and vascular cells,
osteoclasts and osteoblasts, epithelial cell types including
keratinocytes, intestinal and other alimentary epithelial cells and
corneal epithelial cells.
[0090] Furthermore, the cells can be contacted with the substance
which promotes these activities by producing multimeric ligands and
receptors either in vivo or ex vivo. For in vivo administration,
the substance can be administered to any subject having cells which
express the receptors of this invention. The subject can be an
animal and is preferably a human. The substance can be administered
to the subject in a pharmaceutically acceptable carrier and can be
orally or parenterally administered to the subject. By
"pharmaceutically acceptable" is meant a material that is not
biologically or otherwise undesirable, i.e., the material may be
administered to a subject along with the selected substance without
causing any undesirable biological effects or interacting in a
deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained.
[0091] Suitable carriers for parenteral administration of the
substance in a sterile solution or suspension can include sterile
saline that may contain additives, such as ethyl oleate or
isopropyl myristate, and can be injected, for example,
intravenously, as well as into subcutaneous or intramuscular
tissues.
[0092] Suitable carriers for oral administration include one or
more substances which may also act as flavoring agents, lubricants,
suspending agents, or as protectants. Suitable solid carriers
include calcium phosphate, calcium carbonate, magnesium stearate,
sugars, starch, gelatin, cellulose, carboxypolymethylene, or
cyclodextrans. Suitable liquid carriers may be water, pyrogen free
saline, pharmaceutically accepted oils, or a mixture of any of
these. The liquid can also contain other suitable pharmaceutical
additions such as buffers, preservatives, flavoring agents,
viscosity or osmo-regulators, stabilizers or suspending agents.
Examples of suitable liquid carriers include water with or without
various additives, including carboxypolymethylene as a pH-regulated
gel.
[0093] The substance which promotes these activities by producing
multimeric receptors and ligands can be administered to the subject
in amounts sufficient to modulate the activity of the receptor in
the subject as desired. Optimal dosages used will vary according to
the individual, on the basis of age, size, weight, condition, etc,
as well as the particular modulating effect being induced. One
skilled in the art will realize that dosages are best optimized by
the practicing physician and methods for determining dosage are
described, for example, in Remington's Pharmaceutical Sciences
(31). Treatment can be continued for an indefinite period of time,
as indicated by monitoring of the signs, symptoms and clinical
parameters associated with a particular activity of the
receptor.
[0094] The ligands of this invention which initiate, promote and/or
direct cell attachment and/or initiate and promote cell migration,
cell differentiation and/or cell-cell assembly can be, but are not
limited to, ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5,
ephrin-B1, ephrin-B2 and ephrin-B3, as well as any other ligand now
known or identified in the future to initiate, promote and direct
the attachment, migration and differentiation of cells and/or
promote cell-cell assembly, particularly by promoting
multimerization of the receptors which trigger these
activities.
[0095] It is also understood that the receptors and ligands of the
present invention can be adfixed to solid surfaces to carry out the
methods of this invention. For example, the receptors and ligands
can be engineered with adapters which allow them to be adfixed to a
solid surface as multimers, such as a prosthetic vascular graft and
endothelial cells can be added to the graft, allowing for the
biological processes of cell attachment, cell migration and cell to
cell assembly to proceed on the solid surface as facilitated by the
presence of the multimeric ligands and receptors. The differential
activity of tetrameric and dimeric ligands on a solid surface is
described in the Examples section provided herein.
[0096] Thus, the present invention also provides a method for
promoting endothelialization of a prosthesis, comprising contacting
the prosthesis with endothelial cells and a ligand which promotes
endothelialization of cells when in multimeric form under
conditions whereby binding of the ligand to the cells can occur and
endothelialization is promoted. Specifically, the present invention
provides a method for promoting endothelialization of a prosthesis,
comprising contacting the prosthesis with EphB receptor-expressing
endothelial cells with a tetrameric EphB receptor-binding ligand
which promotes multimerization of the EphB receptor, whereby
binding of the tetrameric ligand promotes multimerization of the
EphB receptor, thereby promoting endothelialization of the
prosthesis.
[0097] As used herein, "endothelialization" means the process by
which a confluent and continuous endothelial monolayer assembles,
either de novo or in repair of inflicted damage. Endothelialization
also means the spreading and attachment of endothelial cells to a
prosthetic surface to promote spreading and covering the surface
with an endothelial monolayer. The multimeric ligands can promote
endothelialization of a prosthesis by promoting multimerization of
receptors which promote endothelialization when in multimeric
receptor form. Also as used herein, "prosthesis" means any
biologically acceptable material which can be used to replace,
restore or facilitate a biological function. For example, the
prosthesis of this invention can be a GORTEX endovascular graft, or
other similar materials as would be well known in the art.
[0098] Furthermore, the present invention provides methods for
promoting migration and attachment of cells, as well as methods for
promoting expression of surface molecules affecting leukocyte or
platelet attachment and migration. All of these methods comprise
contacting cells of the subject with a ligand which promotes
migration and attachment of cells and/or promotes expression of
surface molecules affecting leukocyte or platelet attachment and
migration when in multimeric (e.g., tetrameric) form. The
multimeric ligands can promote these activities in cells by
promoting multimerization of receptors which promote these
activities when in multimeric receptor form.
[0099] Specifically, provided herein is a method for promoting
expression of a molecule on the surface of an ephB
receptor-expressing cell, wherein the expressed molecule affects
leukocyte or platelet attachment and migration, comprising
contacting the cell with a tetrameric EphB receptor-binding ligand
which promotes multimerization of the EphB receptor, whereby
binding of the tetrameric ligand promotes multimerization of the
EphB receptor, thereby promoting expression of a molecule on the
surface of the cell which affects leukocyte or platelet attachment
and migration. The receptor of this method can be EphB1 and the
ligand of this method can be ephrin-B1.
[0100] The present invention also provides a method for promoting
function of a molecule on the surface of an ephB
receptor-expressing cell, wherein the expressed molecule affects
leukocyte or platelet attachment and migration, comprising
contacting the cell with a tetrameric ligand which binds an EphB
receptor and promotes multimerization of the EphB receptor, whereby
multimerization of the EphB receptor promotes function of a
molecule on the surface of the cell which affects leukocyte or
platelet attachment and migration, under conditions whereby the
tetrameric ligand can bind the EphB receptor and promote
multimerization of the EphB receptor, thereby promoting function of
a molecule on the surface of the cell which affects leukocyte or
platelet attachment and migration. The receptor of this method can
be EphB1 and the ligand of this method can be ephrin-B1.
[0101] As used herein, a molecule which affects leukocyte or
platelet attachment or migration is a molecule which can initiate,
promote and/or direct leukocyte or platelet attachment or
migration, as well as a molecule which can inhibit and/or disrupt
leukocyte or platelet attachment. Such molecules are well known in
the art. For example, the molecule on the surface of a cell which
affects leukocyte or platelet attachment and migration as set forth
in this invention can be, but is not limited to,
.alpha..sub.v.beta..sub.3, .alpha..sub.5.beta..sub.1, ICAM-1 and
E-selectin.
[0102] The methods described above for promoting these various
activities can be directed to specific locations in a subject. For
example, the above-listed activities can be promoted according to
the methods of the present invention in any tissues which include
vascular and neural cells. In particular, the specific location
could be tumor beds, renal glomeruli, central nervous system
capillaries, or any location at which the promotion of these
activities would be desirable.
[0103] A method is also provided herein for stimulating endothelial
cell attachment, migration and/or cell to cell assembly, comprising
contacting endothelial cells with a ligand which stimulates
endothelial cell attachment and migration when in multimeric (e.g.,
tetrameric) form. The ligand can be any of the ligands of the
present invention which have been shown to stimulate endothelial
cell attachment and migration when in multimeric form. The
endothelial cells can be any of the endothelial cells of the
present invention which can be stimulated to attach and migrate
upon contact with the multimeric ligands of this invention. The
mechanism by which the multimeric ligands stimulate endothelial
cell attachment and migration can be by promoting multimerization
of receptors which promote endothelial cell attachment and
migration when in multimeric receptor form.
[0104] The present invention further provides a method for
initiating, promoting and directing the attachment, migration,
differentiation and repair of neural cells, comprising contacting
neural cells with a ligand with initiates, promotes and directs the
attachment, migration, differentiation and repair of neural cells
when in multimeric form. As used herein, "repair of neural cells"
means integration to the extent that normal function of a damaged
neural cell is recovered. The multimeric ligands can initiate,
promote and direct these activities in cells by promoting
multimerization of receptors associated with these activities and
which are activated to promote these activities in cells when in
multimeric receptor form.
[0105] Thus, the present invention further provides a method for
promoting migration, survival and/or targeting of an EphB
receptor-expressing neural cell, comprising contacting the cell
with a tetrameric EphB receptor-binding ligand which promotes
multimerization of the EphB receptor, whereby binding of the
tetrameric ligand promotes multimerization of the EphB receptor,
thereby promoting migration, survival or targeting of the neural
cell. The receptor of any of these methods can be EphB1 and the
ligand of this method can be ephrin-B1.
[0106] Also provided in the present invention is a method for
treating endothelial cell injury in a subject, comprising
contacting a site of endothelial cell injury in the subject with a
ligand which stimulates endothelial cell attachment and migration
when present in multimeric form. As used herein, "endothelial cell
injury" is damage or injury to an endothelial cell which causes
disruption of normal function and viability.
[0107] The endothelial cell injury can be in any of the endothelial
cells of the present invention and can be the result of a variety
of conditions including, but not limited to, hypoxic insult,
thermal insult, immunological insult and toxic insult. Such injury
can also be the result of an interruption of angiogenic processes
due to a variety of conditions, such as, for example, cancer,
inflammatory arthritis, thrombotic microangiopathies, diabetes,
hypertension, viral and rickettsial diseases, peripheral vascular
disease, atherosclerotic vascular disease and the like. The
treatment of endothelial cell injury by multimeric ligand contact
at the site of injury can be via a mechanism of promoting
multimerization of receptors associated with repair of endothelial
cell injury which are activated to initiate the repair response
when in multimeric receptor form.
[0108] Thus, the present invention further provides a method for
targeting and promoting endothelial cell incorporation at a site of
endothelial cell injury or new blood vessel formation in a subject,
comprising contacting an EphB receptor-expressing endothelial cell
at the site of endothelial cell injury or new blood vessel
formation in the subject with a tetrameric EphB receptor-binding
ligand which promotes multimerization of the EphB receptor, whereby
binding of the tetrameric ligand promotes multimerization of the
receptor, thereby targeting and promoting endothelial cell
incorporation at the site of endothelial cell injury or new blood
vessel formation in the subject. The receptor of this method can be
EphB1 and the ligand of this method can be ephrin-B1.
[0109] In a further embodiment, the present invention also provides
methods for inhibiting the above-listed activities promoted by the
multimeric ligands of this invention, comprising contacting
multimeric ligands with a substance which disrupts the ligand
(e.g., antibodies, modified Eph receptor ectodomains or other
substances which can disrupt the multimer or inhibit multimer
formation), thereby inhibiting the formation of multimeric
receptors and thus, the multimeric receptor-promoted activity. The
mechanism by which these activities can be inhibited can be by
disrupting multimeric receptors or preventing the formation of
multimeric receptors, thereby inhibiting multimeric
receptor-promoted activity. It is also contemplated that the
multimeric ligands and receptors themselves may inhibit these
activities.
[0110] The present invention also provides a method for inhibiting
cell attachment, comprising contacting cells which are stimulated
to attach upon binding a ligand which promotes cell attachment when
in multimeric form, with a substance which prevents binding of the
ligand. Such a substance can be an antibody which binds the
receptor or which binds the ligand, having the net effect of
preventing binding of the ligand and receptor. Other substances can
include proteins, peptides, or other molecules which interfere with
the binding of ligand and receptor, either directly or indirectly.
For example, the substance can be a dimeric ligand which has
antagonistic activity, as well as other small molecules which
interrupt the receptor interfaces that engage in multimer binding
to activate Eph receptor in oligomeric forms that do not permit the
signals evoked by ligand multimers to be generated (i.e., a
pseudo-agonist effect). Additionally, the substance can be a ligand
or receptor ectodomain which binds its respective target and
occupies the necessary binding site, thereby inhibiting the binding
of ligand and receptor.
[0111] In addition, the inhibition of cell attachment can be by
disrupting multimeric receptors which promote or maintain cell
attachment, as well as by inhibiting the formation of multimeric
receptors associated with cell attachment. Such inhibition can be
induced by contacting the receptors with a substance that disrupts
or inhibits formation of multimeric receptors. The substance can
be, for example, a substance, as described above, which disrupts or
inhibits the formation of multimeric ligands or which disrupts or
inhibits receptor/ligand binding.
[0112] Further provided is a method for inhibiting cell-cell
assembly, comprising contacting cells which are stimulated to
assemble upon binding a ligand which promotes cell-cell assembly
when in multimeric form, with a substance which prevents binding of
the ligand. The inhibition of cell to cell assembly may be by
inhibiting the formation of multimeric receptors which promote cell
to cell assembly when in multimeric receptor form.
[0113] The present invention also provides a method for inhibiting
cell migration, comprising contacting cells which are stimulated to
migrate upon binding a ligand which promotes cell migration when in
multimeric form, with a substance which prevents binding of the
ligand. The inhibition of cell migration may be by inhibiting the
formation of multimeric receptors which promote cell migration when
in multimeric receptor form.
[0114] In the same context, the present invention also contemplates
methods for inhibiting migration of cells and/or expression of
surface molecules affecting leukocyte attachment and migration in a
subject comprising contacting cells of the subject with a substance
which prevents binding of a ligand which promotes migration and/or
expression of surface molecules affecting leukocyte attachment and
migration in a subject when in multimeric form.
[0115] Also provided is a method for inhibiting endothelial cell
attachment, migration and/or cell to cell assembly, comprising
contacting endothelial cells with a substance which prevents
binding of a ligand which promotes endothelial cell attachment,
migration and/or cell to cell assembly when in multimeric form.
[0116] In addition, a method is provided for inhibiting or
interrupting neural cell migration and repair, comprising
contacting neural cells with a substance which prevents binding of
a ligand which promotes neural cell migration and repair when in
multimeric form.
[0117] In a particular embodiment, the present invention provides a
method for inhibiting cell attachment to a matrix or to another
cell, comprising contacting an EphB receptor-expressing cell, which
is stimulated to attach to a matrix or to another cell upon binding
a tetrameric Eph receptor-binding ligand, with a substance which
prevents formation of a tetrameric ligand or prevents formation of
a tetrameric receptor or prevents binding of a tetrameric ligand to
the EphB receptor, thereby inhibiting cell attachment to a matrix
or to another cell.
[0118] Also provided is a method for inhibiting cell migration,
comprising contacting an EphB receptor-expressing cell, which is
stimulated to migrate upon binding a tetrameric EphB
receptor-binding ligand, with a substance which prevents formation
of a tetrameric ligand, formation of a tetrameric receptor or
binding of a tetrameric ligand to the EphB receptor, thereby
inhibiting cell migration.
[0119] Furthermore, the present invention provides a method for
inhibiting expression and/or function of a molecule on the surface
of an EphB receptor-expressing cell, wherein the expression and/or
promotion of function of the molecule affects leukocyte or platelet
attachment and migration, comprising contacting an EphB
receptor-expressing cell, which is stimulated to express or promote
function a surface molecule which affects leukocyte or platelet
attachment and migration upon binding a tetrameric EphB
receptor-binding ligand, with a substance which prevents formation
of a tetrameric ligand or prevents formation of a tetrameric
receptor or prevents binding of a tetrameric ligand to the EphB
receptor, thereby inhibiting expression and/or function of a
molecule on the surface of the cell which affects leukocyte or
platelet attachment and migration.
[0120] A method for inhibiting migration, survival or targeting of
a neural cell which expresses an EphB receptor is also provided
herein, comprising contacting an EphB receptor-expressing neural
cell, which is stimulated to promote migration, survival or
targeting of the neural cell upon binding a tetrameric EphB
receptor-binding ligand, with a substance which prevents formation
of a tetrameric ligand or prevents formation of a tetrameric
receptor or prevents binding of a tetrameric ligand to the EphB
receptor, thereby inhibiting migration, survival or targeting of
the neural cell.
[0121] The above described methods for inhibiting various cell
activities can be by disrupting or inhibiting the formation of
multimeric receptors which promote or maintain these activities
when present in the cells in multimeric receptor form. The
inhibition can be caused by a substance which can be an antibody
which binds the receptor or which binds the ligand, having the net
effect of preventing binding of the ligand and receptor. Other
substances can include proteins, peptides, or other molecules which
interfere with the binding of ligand and receptor, either directly
or indirectly. For example, the substance can be a dimeric ligand
which has antagonistic activity, as well as other small molecules
which interrupt the receptor interfaces that engage in multimer
binding to activate Eph receptor in oligomeric forms that do not
permit the signals evoked by ligand multimers to be generated
(i.e., a pseudo-agonist effect). Additionally, the substance can be
a ligand or receptor ectodomain which binds its respective target
and occupies the necessary binding site, thereby inhibiting the
binding of ligand and receptor.
[0122] In addition, the present invention provides an isolated
multimeric Eph receptor binding ligand having more than two ligand
subunits. For example, the multimeric Eph receptor ligand can have
four ligand subunits, six ligand subunits, eight ligand subunits,
etc. It is further contemplated that the multimeric Eph receptor
ligand can be bound to a receptor protein, thus providing a
multimeric Eph receptor ligand/receptor complex, which can be an
isolated complex. Thus, the present invention specifically provides
a composition comprising an isolated tetrameric EphB receptor
ligand, a composition comprising an isolated tetrameric EphB
receptor and a composition comprising an isolated tetrameric EphB
receptor ligand/tetrameric EphB receptor complex. The composition
of this invention can be a tetrameric EphB1 receptor or a
tetrameric EphB2 receptor or a tetrameric EphB3 receptor. The
composition of this invention can also be a tetrameric ephrin-B1
ligand, a tetrameric ephrin-B2 ligand or a tetrameric ephrin-B3
ligand. Furthermore, the composition of this invention can be a
tetrameric EphB1 receptor/tetrameric ephrin-B1 ligand complex, a
tetrameric EphB2 receptor/ephrin-B2 ligand complex, a tetrameric
EphB3 receptor/ephrin-B3 ligand complex, or a tetrameric EphB
receptor/ephrin-B ligand complex having any combination of EphB
receptor and ephrin-B ligand.
[0123] The production of such multimeric Eph receptor ligands or
multimeric Eph receptors can be carried out by methods known in the
art and as provided herein for producing a multimeric Eph receptor
binding ligand having more than two ligand subunits or a multimeric
Eph receptor having more than two receptor subunits. For example,
for the production of multimeric Eph receptor ligands, proteins are
engineered in a two step approach to permit defined
oligomerization. A number of natural examples exist for interaction
"cassettes" to be incorporate in tandem arrangements in multidomain
proteins. For the most part, these serve as adapter molecules that
serve as scaffolds to associate different proteins to accomplish
complex sequential reactions. This tandem adapter function is
exploited to generate a series of adapter proteins that serve as
binding scaffolds. Protein ligands are expressed as recombinant
proteins containing the ectodomain linked to a high affinity
epitope recognition binding site for the adapter domain to create a
fusion protein ligand monomer. Adapter proteins are engineered to
contain, in tandem, multiple defined copies (1, 2, 4, 6) of a
selected interaction motif (e.g., SH2, SH3, PDB, PDZ, etc.).
Adapter proteins and epitope tagged ligand recombinant proteins are
expressed independently, in bacterial expression systems (adapter)
or eukaryotic cell (CHO or sf9 insect cells) expression systems
(ephrin ectodomain-tag). The epitope tagged ephrin ectodomains are
expressed as secreted fusion proteins containing the recognition
motif for the adapter cassette on the C-terminus, analogous to the
position of the current ectodomain fusions. In cases where SH2 or
PDB domains have been used, the recombinant ligand-epitope fusion
will require tyrosine phosphorylation of the epitope to create the
high affinity binding site.
[0124] These recombinant proteins are mixed in defined ratios and
subsequently characterized as described in the Examples provided
herein to determine the oligomeric state of the respective ligands.
This approach permits strict definition of the ligand oligomers and
provides a method for rigorously evaluating biological functions,
as agonists and antagonists for specific receptor and their
associated activities. Additional motifs may be engineered on
either N or C termini to facilitate attachment of these adapter
proteins to solid phase surfaces, for both purification and for
solid phase attachment (e.g., for endothelialization of a
prosthesis).
[0125] The multimeric ligands of this invention can be multimers of
ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5, ephrin-B1,
ephrin-B2 and ephrin-B3, as well as any other ligand now known or
identified in the future to promote the activities of this
invention when in multimeric (e.g., tetrameric) form. Thus, the
present invention provides tetrameric ephrin-A1, tetrameric
ephrin-A2, tetrameric ephrin-A3, tetrameric ephrin-A4, tetrameric
ephrin-A5, tetrameric ephrin-B1, tetrameric ephrin-B2 and
tetrameric ephrin-B3.
[0126] Further provided in the present invention is an isolated
multimeric Eph receptor. The receptor can be isolated and purified,
if desired, according to standard protein isolation and/or
purification protocols well known in the art. The multimeric
receptor of this invention can be, but is not limited to multimers
of EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1,
EphB2, EphB3, EphB4 and EphB5, as well as any other receptor now
known or identified in the future to promote the activities of this
invention when in multimeric (e.g. tetrameric) form. Thus, the
present invention provides tetrameric EphA1, tetrameric EphA2,
tetrameric EphA3, tetrameric EphA4, tetrameric EphA5, tetrameric
EphA6, tetrameric EphA7, tetrameric EphA8, tetrameric EphB1,
tetrameric EphB2, tetrameric EphB3, tetrameric EphB4 and tetrameric
EphB5.
[0127] The present invention also provides a method for screening
Eph receptor binding ligands for the ability to promote cell
attachment when in multimeric form, comprising: producing a
multimeric Eph receptor binding ligand; contacting the multimeric
ligand with cells which express Eph receptors; and determining an
amount of cell attachment and migration in the presence of the
multimeric ligand as compared to the amount of cell attachment and
migration without the multimeric ligand, whereby an increase in
cell attachment and migration in cells contacted with the
multimeric ligand as compared with the amount of cell attachment
and migration in cells not contacted with the multimeric ligand
identifies an Eph receptor binding ligand having the ability to
promote cell attachment and migration when in multimeric form.
[0128] Additionally provided is a method for screening Eph receptor
binding ligands for the ability to promote cell-cell assembly when
in multimeric form, comprising: producing a multimeric Eph receptor
binding ligand; contacting the multimeric ligand with cells which
express Eph receptors; and determining an amount of cell-cell
assembly in the presence of the multimeric ligand as compared to
the amount of cell-cell assembly without the multimeric ligand,
whereby an increase in cell to cell assembly of cells contacted
with the multimeric ligand as compared with the amount of cell to
cell assembly in cells not contacted with the multimeric ligand
identifies a ligand having the ability to promote cell to cell
assembly when in multimeric form.
[0129] A method is also provided herein for screening Eph receptor
binding ligands for the ability to promote cell migration and/or
expression of surface molecules affecting leukocyte attachment and
migration when in multimeric form, comprising: producing a
multimeric Eph receptor binding ligand; contacting the multimeric
ligand with cells which express Eph receptors; and determining an
amount of migration and/or expression of surface molecules
affecting leukocyte attachment and migration in the presence of the
multimeric ligand as compared to the amount of migration and/or
expression of surface molecules affecting leukocyte attachment and
migration without the multimeric ligand, whereby an increase in the
amount of migration and/or expression of surface molecules
affecting leukocyte attachment and migration in the presence of the
multimeric ligand as compared to the amount in the absence of the
multimeric ligand indicates an Eph receptor binding ligand having
the ability to promote migration and/or expression of surface
molecules affecting leukocyte attachment and migration when in
multimeric form.
[0130] Further provided is a method for screening Eph receptor
binding ligands for the ability to initiate, promote and direct
neural cell migration and repair when in multimeric form,
comprising: producing a multimeric Eph receptor binding ligand;
contacting the multimeric ligand with neural cells which express
Eph receptors; and determining an amount of neural cell migration
and repair in the presence of the multimeric ligand as compared to
the amount of neural cell migration and repair without the
multimeric ligand, whereby an increase in the amount of neural cell
migration and repair in the presence of the multimeric ligand as
compared to the amount in the absence of the multimeric ligand
indicates an Eph receptor binding ligand which can initiate,
promote and direct neural cell migration and repair.
[0131] The present invention also provides a method for screening
Eph receptors for the ability to promote cell attachment and/or
when in multimeric form, comprising: producing a multimeric Eph
receptor on the surface of cells which express Eph receptors; and
determining an amount of cell attachment and/or migration in the
presence of the multimeric receptor as compared to the amount of
cell attachment and/or migration in cells without the multimeric
receptor, whereby an increase in cell attachment and/or migration
in cells with the multimeric receptor as compared with the amount
of cell attachment and/or migration in cells not contacted with the
multimeric receptor identifies an Eph receptor having the ability
to promote cell attachment and/or migration when in multimeric
form.
[0132] Additionally provided is a method for screening Eph
receptors for the ability to promote cell-cell assembly when in
multimeric form, comprising: producing a multimeric Eph receptor of
the surface of cells which express Eph receptors; and determining
an amount of cell-cell assembly in the presence of the multimeric
receptor as compared to the amount of cell-cell assembly in cells
without the multimeric receptor, whereby an increase in cell to
cell assembly of cells with the multimeric receptor as compared
with the amount of cell to cell assembly in cells without the
multimeric receptor identifies a receptor having the ability to
promote cell to cell assembly when in multimeric form.
[0133] A method is also provided herein for screening Eph receptors
for the ability to promote migration and/or expression of surface
molecules affecting leukocyte attachment and migration when in
multimeric form, comprising: producing a multimeric Eph receptor on
the surface of cells which express Eph receptors; and determining
an amount of migration and/or expression of surface molecules
affecting leukocyte attachment and migration in the presence of the
multimeric receptor as compared to the amount of migration and/or
expression of surface molecules affecting leukocyte attachment and
migration without the multimeric receptor, whereby an increase in
the amount of migration and/or expression of surface molecules
affecting leukocyte attachment and migration in the presence of the
multimeric receptor as compared to the amount in the absence of the
multimeric receptor identifies an Eph receptor having the ability
to promote migration and/or expression of surface molecules
affecting leukocyte attachment and migration when in multimeric
form.
[0134] Further provided is a method for screening Eph receptors for
the ability to initiate, promote and direct neural cell migration
and repair when in multimeric form, comprising: producing a
multimeric Eph receptor on neural cells which express Eph
receptors; and determining an amount of neural cell migration and
repair in the presence of the multimeric receptor as compared to
the amount of neural cell migration and repair without the
multimeric receptor, whereby an increase in the amount of neural
cell migration and repair in the presence of the multimeric
receptor as compared to the amount in the absence of the multimeric
receptor indicating an Eph receptor which can initiate, promote and
direct neural cell migration and repair. The multimeric Eph
receptor can be produced on the neural cells by inducing the
formation of defined multimeric receptors according to the methods
provided herein.
[0135] In another embodiment, the present invention provides a
method for screening substances for the ability to inhibit the
binding of a multimeric Eph receptor binding ligand to an Eph
receptor comprising: contacting the substance with cells expressing
an Eph receptor; contacting the cells with a multimeric Eph
receptor binding ligand under conditions whereby the multimeric
ligand can bind the receptor; and determining the activity induced
by the cells contacted with the substance as compared to the
activity of cells not contacted with the substance, whereby a
reduction in activity of cells contacted with the substance as
compared to the amount of activity of cells not contacted with the
substance indicating a substance having the ability to inhibit the
binding of a multimeric Eph receptor binding ligand to an Eph
receptor.
[0136] In a specific embodiment, the present invention provides the
following screening methods:
[0137] A. A method for screening an EphB receptor-binding ligand
for the ability to initiate, promote and direct cell attachment to
a matrix or to another cell when in multimeric form,
comprising:
[0138] a) contacting a multimeric EphB receptor-binding ligand with
an EphB receptor-expressing cell under conditions whereby the
ligand can bind the receptor; and
[0139] b) detecting attachment of cells which have bound multimeric
ligand as, compared to attachment of cells which have not bound
multimeric ligand, whereby attachment of cells which have bound
multimeric ligand and no attachment of cells which have not bound
multimeric ligand identifies an EphB receptor-binding ligand with
the ability to initiate, promote and direct cell attachment to a
matrix or to another cell when in multimeric form. Detection of
cell attachment can be according to standard protocols known in the
art and as set forth in the Examples provided herein. As used
herein, no cell attachment means the absence of cell attachment or
a lesser degree of cell attachment in a control sample, relative to
the detection of attachment in the experimental sample.
[0140] B. A method for screening an EphB receptor-binding ligand
for the ability to promote cell migration when in multimeric form,
comprising:
[0141] a) contacting a multimeric EphB receptor-binding ligand with
an EphB receptor-expressing cell under conditions whereby the
ligand can bind the receptor; and
[0142] b) detecting migration of cells which have bound multimeric
ligand as compared to migration of cells which have not bound
multimeric ligand, whereby migration of cells which have bound
multimeric ligand and no migration of cells which have not bound
multimeric ligand identifies an EphB receptor-binding ligand with
the ability to promote cell migration when in multimeric form.
Detection of cell migration can be according to methods well known
in the art and as set forth in the Examples provided herein. As
used herein, no migration means the absence of migration or a
lesser amount of migration in a control sample, relative to the
detection of cell migration in an experimental sample.
[0143] C. A method for screening an EphB receptor-binding ligand
for the ability to promote expression of a molecule on the surface
of an EphB receptor-expressing cell, wherein the expressed molecule
affects leukocyte or platelet attachment and migration, when in
multimeric form, comprising:
[0144] a) contacting a multimeric EphB receptor-binding ligand with
a cell which expresses an EphB receptor under conditions whereby
the ligand can bind the receptor; and
[0145] b) detecting expression of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells which have bound multimeric ligand as compared to expression
of a molecule which affects leukocyte or platelet attachment and
migration on the surface of cells which have not bound multimeric
ligand, whereby expression of a molecule which affects leukocyte or
platelet attachment and migration on the surface of cells which
have bound multimeric ligand and no expression of a molecule which
affects leukocyte or platelet attachment and migration on the
surface of cells which have not bound multimeric ligand identifies
a ligand with the ability to promote the expression of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells, when the ligand is in multimeric form. The
expression of a molecule of this invention can be detected
according to methods well known in the art and as set forth in the
Examples provided herein. As used herein, no expression means the
absence of expression or a lesser amount of expression detected in
a control sample, relative to the detection of expression in an
experimental sample.
[0146] D. A method for screening an EphB receptor-binding ligand
for the ability to promote function of a molecule on the surface of
an EphB receptor-expressing cell, wherein the function of the
molecule affects leukocyte or platelet attachment and migration,
when in multimeric form, comprising:
[0147] a) contacting a multimeric EphB receptor-binding ligand with
a cell which expresses an EphB receptor under conditions whereby
the ligand can bind the receptor; and
[0148] b) detecting function of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells which
have bound multimeric ligand as compared to function of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells which have not bound multimeric ligand, whereby
function of a molecule which affects leukocyte or platelet
attachment and migration on the surface of cells which have bound
multimeric ligand and no function of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells which have not bound multimeric ligand identifies a ligand
with the ability to promote function of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells, when the ligand is in multimeric form. The detection of
function of a molecule of this invention can be carried out by
methods standard in the art and as set forth in the Examples
provided herein. As used herein, no function means the absence of
function or a lesser amount of function detected in a control
sample, relative to detection of function of a molecule in an
experimental sample.
[0149] E. A method for screening an EphB receptor-binding ligand
for the ability to promote migration, survival or targeting of a
neural cell when in multimeric form, comprising:
[0150] a) contacting a multimeric EphB receptor-binding ligand with
a neural cell which expresses an EphB receptor under conditions
whereby the ligand can bind the receptor; and
[0151] b) determining migration, survival or targeting of a neural
cell which has bound multimeric ligand as compared to migration,
survival or targeting of a neural cell which has not bound
multimeric ligand, whereby migration, survival or targeting of a
neural cell which has bound multimeric ligand and no migration,
survival, or targeting of a neural cell which has not bound
multimeric ligand identifies a ligand with the ability to promote
migration, survival or targeting of a neural cell when in
multimeric form. Detection of migration, survival or targeting of a
neural cell can be carried out according to methods standard in the
art and as set forth in the Examples provided herein. As used
herein, no migration, survival or targeting of a neural cell means
absence of migration, survival or targeting or detection of a
lesser amount of inigration, survival, or targeting of a neural
cell in a control sample, relative to detection of migration,
survival or targeting of a neural cell in an experimental
sample.
[0152] F. A method for screening an EphB receptor for the ability
to initiate, promote and direct cell attachment to a matrix or to
another cell when the receptor is in multimeric form,
comprising:
[0153] a) producing a multimeric EphB receptor on the surface of a
cell which expresses an EphB receptor; and
[0154] b) detecting attachment of cells with a multimeric EphB
receptor as compared to attachment of cells without a multimeric
EphB receptor, whereby attachment of cells with a multimeric EphB
receptor and no attachment of cells without a multimeric EphB
receptor identifies an EphB receptor with the ability to initiate,
promote and direct cell attachment to a matrix or to another cell
when in multimeric form. Detection of cell attachment can be
according to standard protocols known in the art and as set forth
in the Examples provided herein. As used herein, no cell attachment
means the absence of cell attachment or a lesser degree of cell
attachment in a control sample, relative to the detection of
attachment in the experimental sample.
[0155] G. A method for screening an EphB receptor for the ability
to promote cell migration when the receptor is in multimeric form,
comprising:
[0156] a) producing a multimeric EphB receptor on the surface of a
cell which expresses an EphB receptor; and
[0157] b) detecting migration of cells with a multimeric EphB
receptor as compared to migration of cells without a multimeric
EphB receptor, whereby migration of cells with a multimeric EphB
receptor and no migration of cells without a multimeric EphB
receptor identifies an EphB receptor with the ability to promote
cell migration when in multimeric form. Detection of cell migration
can be according to methods well known in the art and as set forth
in the Examples provided herein. As used herein, no migration means
the absence of migration or a lesser amount of migration in a
control sample, relative to the detection of cell migration in an
experimental sample.
[0158] H. A method for screening an EphB receptor for the ability
to promote expression of a molecule on the surface of an EphB
receptor-expressing, wherein the expressed molecule affects
leukocyte or platelet attachment and migration, when the receptor
is in multimeric form, comprising:
[0159] a) producing a multimeric EphB receptor on the surface of a
cell which expresses an EphB receptor; and
[0160] b) detecting expression of a molecule which affects
leukocyte or platelet attachment and migration on the surface of
cells with a multimeric EphB receptor as compared to expression of
a molecule which affects leukocyte or platelet attachment and
migration on the surface of cells without a multimeric EphB
receptor, whereby expression of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells with a
multimeric EphB receptor and no expression of a molecule which
affects leukocyte or platelet attachment and migration on the
surface of cells without a multimeric EphB receptor identifies an
EphB receptor with the ability to promote expression of a molecule
on the surface of a cell which expresses an EphB receptor, wherein
the expressed molecule affects leukocyte or platelet attachment and
migration, when the EphB receptor is in multimeric form. The
expression of a molecule of this invention can be detected
according to methods well known in the art and as set forth in the
Examples provided herein. As used herein, no expression means the
absence of expression or a lesser amount of expression detected in
a control sample, relative to the detection of expression in an
experimental sample.
[0161] I. A method for screening an EphB receptor for the ability
to promote function of a molecule on the surface of an EphB
receptor-expressing, wherein the function of the molecule affects
leukocyte or platelet attachment and migration, when the receptor
is in multimeric form, comprising:
[0162] a) producing a multimeric EphB receptor on the surface of a
cell which expresses an EphB receptor; and
[0163] b) detecting function of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells with a
multimeric EphB receptor as compared to function of a molecule
which affects leukocyte or platelet attachment and migration on the
surface of cells without a multimeric EphB receptor, whereby
function of a molecule which affects leukocyte or platelet
attachment and migration on the surface of cells with a multimeric
EphB receptor and no function of a molecule which affects leukocyte
or platelet attachment and migration on the surface of cells
without a multimeric EphB receptor identifies an EphB receptor with
the ability to promote function of a molecule on the surface of a
cell which expresses an EphB receptor, wherein the expressed
molecule affects leukocyte or platelet attachment and migration,
when the EphB receptor is in multimeric form. The detection of
function of a molecule of this invention can be carried out by
methods standard in the art and as set forth in the Examples
provided herein. As used herein, no function means the absence of
function or a lesser amount of function detected in a control
sample, relative to detection of function of a molecule in an
experimental sample.
[0164] J. A method for screening an EphB receptor for the ability
to promote migration, survival or targeting of a neural cell when
the receptor is in multimeric form, comprising:
[0165] a) producing a multimeric EphB receptor on the surface of a
neural cell which expresses an EphB receptor; and
[0166] b) detecting migration, survival or targeting of neural
cells with a multimeric EphB receptor as compared to migration,
survival or targeting of neural cells without a multimeric EphB
receptor, whereby migration survival or targeting of neural cells
with a multimeric EphB receptor and no migration, survival or
targeting of neural cells without a multimeric EphB receptor
identifies an EphB receptor with the ability to promote migration,
survival or targeting of neural cells when in multimeric form.
Detection of migration, survival or targeting of a neural cell can
be carried out according to methods standard in the art and as set
forth in the Examples provided herein. As used herein, no
migration, survival or targeting of a neural cell means absence of
migration, survival or targeting or detection of a lesser amount of
migration, survival, or targeting of a neural cell in a control
sample, relative to detection of migration, survival or targeting
of a neural cell in an experimental sample.
[0167] The activity induced by the binding of multimeric ligand to
receptor on the cell surface can be tyrosine phosphorylation, cell
proliferation, cell attachment, cell to cell assembly or any other
activity as described in the examples provided herein. The
conditions whereby the multimeric ligand can bind the receptor can
be the conditions as set forth in the examples described herein, as
well as any other conditions as would be able to be readily
determined by an artisan, under which a receptor and ligand can
bind one another and form a complex.
[0168] The present invention further provides a method for
screening a substance for the ability to inhibit the binding of a
multimeric EphB receptor-binding ligand to an EphB receptor
comprising:
[0169] a) contacting the substance with a cell expressing an EphB
receptor;
[0170] b) contacting the cell of step (a) with a multimeric EphB
receptor-binding ligand under conditions whereby the multimeric
ligand can bind the receptor; and
[0171] c) detecting the binding of the multimeric ligand to the
receptor, whereby no binding of the multimeric ligand to the
receptor identifies a substance with the ability to inhibit the
binding of a multimeric EphB receptor-binding ligand to an EphB
receptor.
[0172] The detection of binding of the multimeric EphB
receptor-binding ligand to the EphB receptor can be by detecting an
activity such as cell attachment to a matrix or to another cell,
cell migration, expression of a surface molecule affecting
leukocyte or platelet attachment and migration, promotion of
function of a surface molecule affecting leukocyte or platelet
attachment and migration and promotion of migration, survival or
targeting of a neural cell.
[0173] The inhibitory substance of this invention can be an
antibody or other molecule or compound which binds the receptor or
which binds the ligand, having the net effect of preventing
formation of a multimeric ligand, preventing formation of a
multimeric receptor or preventing binding of the ligand and
receptor. Other substances can include proteins, peptides, or other
molecules or compounds which interfere with the binding of ligand
and receptor, either directly or indirectly. For example, the
substance can be a monomeric or dimeric ligand or higher order
multimeric ligand which has antagonistic activity, as well as other
small molecules which interrupt the receptor interfaces that engage
in multimer binding to activate Eph receptor in oligomeric forms
that do not permit the signals evoked by ligand multimers to be
generated (i.e., a pseudo-agonist effect). Additionally, the
substance can be a ligand or receptor ectodomain which binds its
respective target and occupies the necessary binding site, thereby
inhibiting the binding of ligand and receptor.
[0174] The activity induced by the cells expressing an Eph
receptor, upon contact with a multimeric Eph binding ligand can be,
but is not limited to, cell attachment, cell-cell assembly,
migration, expression of surface molecules affecting leukocyte
attachment and migration, neural cell migration and repair and
endothelial cell proliferation.
[0175] The present invention also provides a method for promoting
angiogenesis, comprising contacting cells which are associated with
angiogenesis with a substance which promotes multimerization of
receptors associated with promoting angiogenesis. The substance can
be a multimeric ligand, for example, a tetrameric ligand, which
induces the formation of tetrameric receptors. The cells can be
contacted with the substance either in vivo or ex vivo as described
herein. Angiogenesis can also be promoted upon solid surfaces, as
also described herein. The promotion of angiogenesis upon formation
of multimeric receptors can be determined according to protocols
well known in the art. An example of the promotion of angiogenesis
in vivo with a multimeric receptor is described in the Examples
herein.
[0176] In addition, the present invention provides a method for
promoting angiogenesis comprising contacting cells having an Eph
receptor with an Eph receptor binding ligand which promotes
angiogenesis upon binding the Eph receptor of the cells. The
promotion of angiogenesis can be at a vascular bed-specific site at
which the promotion of angiogenic processes is desired because, for
example, these processes have been interrupted due to a variety of
conditions such as cancer, inflammatory arthritis and injury to
endothelium due to conditions such as hypoxic injury, thermal
injury, immunologic injury and toxic injury.
[0177] To target the promotion of angiogenesis to a vascular
bed-specific site, cells can be engineered in vitro to express an
Eph receptor which promotes angiogenesis upon binding an Eph
receptor binding ligand, delivered to the site where promotion of
angiogenesis is desired and contacted with the Eph receptor binding
ligand to promote angiogenesis at the specific site. Cells could be
delivered to the specific sites of interest by intravenous
injection, local tissue infiltration or via a slow release
implantable delivery system.
[0178] In addition, the present invention provides a method for
disrupting and interrupting angiogenesis (i.e., promoting vascular
regression) comprising contacting cells expressing an Eph receptor
with a substance which inhibits binding of an Eph receptor binding
ligand which promotes angiogenesis when bound to the Eph receptor
expressed by the cells.
[0179] The angiogenesis to be targeted for disruption and
interruption can be caused by such conditions as cancer, arthritis,
proliferative retinopathy, ischemic tissue injury, reproductive
activity (e.g., ovulation and menstruation) and any other condition
associated with angiogenesis. Furthermore, the angiogenesis can be
targeted for disruption and interruption at a specific site in a
subject, such as, for example, a tumor, the retina, an inflammatory
condition and any site of vascular malformation.
[0180] The substance which disrupts and/or interrupts angiogenesis
by inhibiting binding of an Eph receptor binding ligand which
promotes angiogenesis when bound to Eph receptor can be antibodies
which bind the ligand, thereby inhibiting binding of the ligand to
the receptor, or antibodies which bind the receptor, thereby
inhibiting binding of the ligand to the receptor. Additional
substances applicable for this method of inhibiting angiogenesis
can include proteins, peptides, or other molecules which interfere
with the binding of ligand and receptor, either directly or
indirectly. For example, the substance can be a dimeric ligand
which has antagonistic activity, as well as other small molecules
which interrupt the receptor interfaces that engage in multimer
binding to activate Eph receptor in oligomeric forms that do not
permit the signals evoked by ligand multimers to be generated
(i.e., a pseudo-agonist effect). Additionally, the substance can be
a ligand or receptor ectodomain which binds its respective target
and occupies the necessary binding site, thereby inhibiting the
binding of ligand and receptor.
[0181] Also contemplated in the present invention is a method for
treating a disease associated with pathological angiogenesis in a
subject comprising contacting cells of the subject, which are
undergoing pathological angiogenesis and which have an Eph
receptor, with a substance which inhibits binding of an Eph
receptor binding ligand which promotes angiogenesis upon binding
the Eph receptor. The substance can be an antibody which binds the
Eph receptor and inhibits ligand binding to the receptor or an
antibody which binds the Eph receptor binding ligand and inhibits
ligand binding. Additional substances applicable for this method of
inhibiting angiogenesis can include proteins, peptides, or other
molecules which interfere with the binding of ligand and receptor,
either directly or indirectly. For example, the substance can be a
dimeric ligand which has antagonistic activity, as well as other
small molecules which interrupt the receptor interfaces that engage
in multimer binding to activate Eph receptor in oligomeric forms
that do not permit the signals evoked by ligand multimers to be
generated (i.e., a pseudo-agonist effect). Additionally, the
substance can be a ligand or receptor ectodomain which binds its
respective target and occupies the necessary binding site, thereby
inhibiting the binding of ligand and receptor.
[0182] The disease associated with pathological angiogenesis can be
a variety of conditions, such as cancer, destructive arthritis and
proliferative retinopathy.
[0183] A method is also provided for treating a condition
associated with interruption of angiogenic processes in a subject,
such as cancer, inflammatory arthritis and the like, comprising
contacting cells of the subject, which are involved with the
interruption of angiogenic processes and which have an Eph
receptor, with an Eph receptor binding ligand which promotes
angiogenesis upon binding the Eph receptor of the cells.
[0184] In particular, the present invention provides the following
methods:
[0185] K. A method for promoting angiogenesis, comprising
contacting EphB receptor-expressing cells which are associated with
angiogenesis with a multimeric EphB receptor-binding ligand,
whereby binding of the tetrameric ligand promotes multimerization
of the EphB receptor, thereby promoting angiogenesis.
[0186] L. A method for disrupting angiogenesis comprising
contacting an EphB receptor-expressing cell which promotes
angiogenesis upon binding a multimeric EphB receptor-binding ligand
with a substance which prevents formation of a multimeric EphB
receptor-binding ligand or inhibits binding of the multimeric EphB
receptor-binding ligand to the receptor, thereby disrupting
angiogenesis.
[0187] M. A method for treating a disease associated with
pathological angiogenesis in a subject, comprising contacting an
EphB receptor-expressing cell of the subject which promotes
angiogenesis upon binding a tetrameric EphB receptor-binding ligand
with a substance which prevents binding of the tetrameric ligand,
thereby disrupting angiogenesis and treating a disease associated
with pathological angiogenesis.
[0188] N. A method for treating a condition associated with
interruption of angiogenic processes in a subject, comprising
contacting an EphB receptor-expressing cell of the subject with a
tetrameric EphB receptor-binding ligand, whereby binding of the
tetrameric ligand promotes multimerization of the EphB receptor,
thereby promoting angiogenesis and treating a condition associated
with interruption of angiogenic processes.
[0189] Furthermore, the present invention provides a method for
screening a substance for the ability to inhibit angiogenesis
comprising: contacting the substance with cells having an Eph
receptor; contacting the cells with an Eph receptor binding ligand
which promotes angiogenesis upon binding the Eph receptor of the
cells, under conditions whereby the ligand can bind the cell
receptor; and detecting an amount of angiogenesis in cells
contacted with the substance and the ligand as compared to the
amount of angiogenesis in cells not contacted with the substance
and contacted with the ligand, whereby a decrease in the amount of
angiogenesis in cells contacted with the substance as compared to
the amount of angiogenesis in cells not contacted with the
substance identifying a substance having the ability to inhibit
angiogenesis.
[0190] Specifically provided is a method for screening a substance
for the ability to inhibit angiogenesis, comprising:
[0191] a) contacting the substance with a cell expressing an EphB
receptor;
[0192] b) contacting the cell of step (a) with a multimeric EphB
receptor-binding ligand, which promotes angiogenesis, under
conditions whereby the multimeric ligand can bind the receptor;
and
[0193] c) detecting angiogenesis in cells contacted with the
substance, as compared to angiogenesis in cells not contacted with
the substance, whereby no angiogenesis in cells contacted with the
substance and angiogenesis in cells not contacted with the
substance identifies a substance having the ability to inhibit
angiogenesis.
[0194] Further provided is a method for screening Eph receptor
binding ligands for the ability to promote angiogenesis,
comprising: contacting an Eph receptor binding ligand with cells
having an Eph receptor; and detecting angiogenesis in the cells
contacted with the ligand as compared to angiogenesis in cells not
contacted with the ligand.
[0195] Also specifically provided is a method for screening an
EphB-receptor binding ligand for the ability to promote
angiogenesis when in multimeric form, comprising:
[0196] a) contacting a multimeric EphB receptor-binding ligand with
a cell which expresses an EphB receptor under conditions whereby
the ligand can bind the receptor; and
[0197] b) detecting angiogenesis of cells which have bound
multimeric ligand as compared to angiogenesis of cells which have
not bound multimeric ligand, whereby angiogenesis of cells which
have bound multimeric ligand and no angiogenesis of cells which
have not bound multimeric ligand identifies an EphB
receptor-binding ligand with the ability to promote angiogenesis
when in multimeric form.
[0198] The cells which can be used in the screening methods of this
invention can include endothelial cells and derived cell lines with
origin in any human or animal vascular bed. For example, the cells
can express the ephrin A1 receptor and be contacted with the Eph-A2
ligand.
[0199] The angiogenesis of the screening methods of this invention
can be detected with a variety of experimental systems, including
but not limited to, corneal pocket assay (44), chick
chorioallantoic membrane assay (43), hamster skin flap windows, in
vitro endothelial assembly (e.g. by detection of migration, cell
attachment, cell to cell assembly, etc.) and Matrigel injection
into animal tissues.
[0200] The following examples are intended to illustrate, but not
limit, the invention. While the protocols described are typical of
those that might be used, other procedures known to those skilled
in the art may be alternatively employed.
EXAMPLES
Example 1
[0201] Eph Receptors Discriminate Specific Ligand Oligomers to
Determine Alternative Signaling Complexes, Attachment and Assembly
Responses.
[0202] Reagents. Anti-EphB1 (15) and anti-EphB2 (28) sera were
described previously. Monoclonal phosphotyrosine antibody (4G10)
was from Upstate Biotechnology Inc., Lake Placid, N.Y. Monoclonal
anti-human IgG1 (anti-Fc) antibody was from The Binding Site,
Birmingham, United Kingdom. Polyclonal rabbit antiserum to LMW-PTP
was generated against the entire coding region (11) and affinity
purified by adsorption and elution, using 2M glycine pH2.5, from
recombinant LMW-PTP protein immobilized on Immobilin P
membranes.
[0203] Ligand stimulation and immunoprecipitation of endogenous
EphB receptors. Primary HRMEC were used at passages 3-6, as
described (12). P19 cells were cultured as recommended (ATCC,
Rockville, Md.). Ninety min prior to ligand stimulation, cells
(2.times.10.sup.5cells/60 mm dish) were plated on
Matrigel-(Collaborative Biomedical Products, Becton Dickinson,
Bedford, Mass., as recommended) or fibronectin-coated (29) dishes.
Ligand (ephrin-B1/Fc) or control (ORF/Fc) (30) multimers were
generated by preincubation with anti-Fc at a fixed ratio of
0.1.times. the indicated concentrations of Fc fusion proteins [50
ng ml.sup.-1 anti-Fc for 500 ng ml.sup.-1 Fc fusion protein]. If
not otherwise indicated, Fc fusion proteins (ephrinB1/Fc or ORF/Fc)
were used at 500 ng ml.sup.-1 in the absence or presence of
preclustering anti-Fc at 50 ng ml.sup.-1. Cells were incubated with
agonists at 37.degree. C. for 10 min, or as described in the figure
legends. Immunoprecipitations were conducted on either cell lysates
(P19) or on Triticum vulgaris lectin affinity purified fractions of
HRMEC (15) (needed to reduce the background from Matrigel).
Precipitated proteins were analyzed by immunoblot (15).
[0204] Endothelial assembly into capillary-like structures. Twelve
well plates (Falcon) were coated with thin layers of Matrigel.
HRMEC were plated at a density of 4.times.10.sup.4 cells/well in
DME containing 1% fetal bovine serum. Indicated agonists, control
peptides, alone, or in combination with the EphB1/Fc antagonist
(1000 ng ml.sup.-1) (FIG. 5) were added at the time of plating.
Cells were incubated at 37.degree. C. for 8 h and photographed
using phase microscopy (magnification 100.times.) (Diaphot-TMD,
Nikon).
[0205] Cell adhesion assay. Six well plates (Falcon) were coated
with thin layers of Matrigel or with 0.5 .mu.g cm.sup.2 fibronectin
(29). Growth medium was replaced 48 h before harvest with binding
medium, either DMEM (HRMEC) or .varies.-MEM (P19) containing 1%
bovine albumin. Cells were recovered by trypsinization (HRMEC) or
vigorous tituration (P19) and washed three times with binding
medium and plated at 1.times.10.sup.5 cells per well. Ligands were
added coincident with plating at concentrations indicated above.
After 90 min, unattached cells were dislodged by applying 4 brisk
slaps of the plate on a horizontal surface, and the attached cell
layer was carefully washed once with PBS containing calcium and
magnesium to collect the remaining unattached cells. Attached cells
were collected after incubation in Dispase (Collaborative
Biomedical Products). Separate fractions were recovered by
centrifugation, washed once and trypan blue excluding cells counted
visually using a hemocytomer. The ratio of attached to total number
of cells recovered were calculated for each of three wells. Data
are expressed as mean+/-SEM and are representative of three
independent experiments.
[0206] Transfections. P19 cells were transfected with a total of 10
.mu.g plasmid DNA per P100 plate using the Lipofectamine method
(Gibco/BRL). As indicated, pSR.varies.-EphB1/HA or
pSR.varies.-EphB1-Y929F/HA were mixed prior to transfection at the
ratio indicated in FIG. 4. Forty h after transfection, cells from
the same transfections were used in attachment assays and in
immunoprecipitation experiments. Displayed data represent those
from 3 independent experiments.
[0207] GST-EphB1 affinity binding assays. Recombinant GST-EphB1cy
and GST-EphB1-Y929F/HA proteins were expressed in Sf9 cells as
described (15), premixed in defined ratios where indicated, bound
to glutathione-sepharose, kinased and used as an affinity matrix to
evaluate LMW-PTP binding, as described in the figure legend.
[0208] Eph family receptor tyrosine kinases (including EphA3,
EphB4) direct pathfinding of neurons within migratory fields of
cells expressing gradients of their membrane-bound ligands, ephrins
(ephrin-A2,ephrin-B2) (1-6). EphB1 and EphA2 direct vascular
network assembly, affecting endothelial migration, capillary
morphogenesis and angiogenesis (7,8). To explore how ephrins could
provide positional labels for cell targeting, EphB1 (ELK) and EphB2
(Nuk) receptors were tested to determine whether they discriminate
between variably oligomerized forms of an ephrin-B1/Fc fusion
ligand (9,10) presented to P19 and endothelial cells.
[0209] To test if variably oligomerized forms of ephrin-B1 evoke
alternative signals through EphB receptors, a disulfide-linked
immunoglobulin Fc fusion form of ephrin-B1 (ephrin-B1/Fc) (9) was
used. Ephrin-B1/Fc dimers, or anti-Fc clustered multimers were
presented to each of two cell types that express endogenous EphB1
receptors, human renal microvascular endothelial cells (HRMEC) (12)
and teratocarcinoma-derived, pluripotent murine cells, P19 (13)
(ATCC accession number CRL 1825). As shown in FIG. 1A, both dimeric
and multimeric forms of ephrin-B1/Fc promoted endothelial EphB1
activation and tyrosine phosphorylation at concentrations from
15-1000 ng ml.sup.-1.
[0210] However, in sharp contrast to this observation, marked
differences in cellular behavior were observed (FIG. 1A).
Preclustered, multimeric ephrin-B1/Fc promoted endothelial
capillary-like assembly in a two dimensional in vitro assay (12),
while dimeric ephrin-B1/Fc did not, at concentrations that
stimulated receptor tyrosine phosphorylation. Dimeric and
preclustered multimeric forms of an irrelevant open reading frame
Fc fusion protein (ORF/Fc) were inactive at these concentrations
(FIG. 1A). In a second assay, ephrin-B1/Fc multimers promoted HRMEC
attachment to Matrigel- and fibronectin-coated surfaces, while
dimers did not (FIG. 1B).
[0211] Differential responses were also noted in P19 cells (FIG.
1C). Preclustered ephrin-B1/Fc multimers promoted P19 attachment to
fibronectin, while ephrin-B1/Fc dimers had a modest effect to
decrease fibronectin attachment of a small subpopulation of cells,
notably at concentrations where multimers increased attachment.
Thus, two different cell types show specific attachment and
assembly responses that depend upon the oligomeric form of
ephrin-B1/Fc presented.
[0212] Given that other EphB subclass receptors share a similar
affinity for ephrin-B1 (14), ephrin-B1 dimers and multimers were
evaluated to determine whether they could variably activate or
recruit other EphB subclass receptors (14). HRMEC were screened for
EphB2, EphB3 and EphB4 mRNA using specific primers and RT-PCR.
Among these, only EphB2 was expressed at detectable levels.
[0213] EphB1 and EphB2 immunoprecipitates from HRMEC stimulated
with dimeric or multimeric (+anti-Fc) ephrin-B1/Fc were analyzed
for receptor activation (anti-PY), and coprecipitation of
heteromeric receptors (anti-EphB1, or anti-EphB2). Coprecipitating
proteins were identified by immunoblots, using anti-pTyr,
anti-EphB2 and anti-EphB1 antibodies, of EphB1 receptor
immunoprecipitates from HRMEC or P19 cells and EphB2 receptor
immunoprecipitates (from HRMEC) from cells exposed to no addition,
or Fc fusion proteins (500 ng ml.sup.-1), ORF/Fc or EphB1/Fc, in
either dimeric (-anti-Fc) or multimeric (+anti-Fc) forms.
[0214] These studies demonstrated that ephrin-B1/Fc dimers or
preclustered multimers (+anti-Fc) stimulated tyrosine
phosphorylation of EphB2, similar to that seen with EphB1.
Immunoprecipitation of either EphB1 or EphB2 failed to recover the
other receptor, or the activating ephrin-B1/Fc, in a stable complex
following activation. Although more than one EphB subclass receptor
was activated, each receptor (EphB1 and EphB2) was similarly
activated by ephrin-B1 dimers and multimers.
[0215] It was reasoned that alternative signaling responses
mediated through EphB receptors might reflect differential
association of additional receptor signaling complex components.
Recruitment of SH2-containing adapter proteins, Grb2 and Grb10 (15)
and Nck to EphB1 complexes did not differ between dimeric and
multimeric ephrin-B1/Fc ligands. However, analysis of low molecular
weight species recovered in EphB1 and EphB2 immunoprecipitates
revealed an 18 kDa tyrosine phosphoprotein recruited by multimeric,
but not by dimeric, ephrin-B1/Fc. A recent report demonstrated that
the low molecular weight, human cytoplasmic phosphotyrosine
phosphatase (LMW-PTP) is itself a substrate for v-src tyrosine
phosphorylation (16). Accordingly, EphB1 immunoprecipitates were
analyzed for LMW-PTP immunoreactivity (11,17). These studies
demonstrated that affinity purified LMW-PTP antibodies recognized
the 18 kDa tyrosine phosphoprotein in both EphB1 and EphB2
multimer-activated signaling complexes.
[0216] LMW-PTP is structurally distinct from the better studied
tyrosine phosphatases (18), is expressed in a wide range of cell
types, and structural homologues are expressed in yeast (19). A
catalytically inactive LMW-PTP functions as a dominant negative
protein that binds and precipitates tyrosine phosphorylated PDGF
receptors and to promote cell proliferation (20). At least two
alternatively spliced LMW-PTP isoforms exist. The A isoform (18) is
the dominant species and it precipitates with EphB1 in response to
multimeric, but not dimeric, ephrin-B1. EphB1 receptor tyrosine
kinase phosphorylates LMW-PTP in vitro, and time courses have shown
that LMW-PTP immunoreactivity is tyrosine phosphorylated as soon as
it is detected in EphB1 complexes.
[0217] Whether LMW-PTP interacts directly with EphB1 was tested
using yeast two hybrid and in vitro coprecipitation assays of
recombinant proteins. Recombinant GST fusions containing EphB1
cytoplasmic domain (GST-EphB1cy), or a kinase-inactive mutant
(GST-EphB1cyK652R), were immobilized on glutathione-sepharose,
incubated in kinase buffer in the presence (+) or absence (-) of
ATP (15), incubated with HRMEC extracts or recombinant LMW-PTP
protein, washed extensively, then analyzed by immunoblot, using
anti-GST and anti-LMW-PTP antibodies. Recombinant GST-EphB1cy and
HA-epitope tagged GST-EphB1cy-Y929F were mixed in (%) ratios of
100:0, 75:25, 50:50, 25:75 and 0:100, bound to
glutathione-sepharose and assayed as described herein. P19 cells
were transfected with pSR.varies. expression constructs (27)
encoding wild-type EphB1 (hEphB1/HA) or mutant (hEphB1-Y929F/HA) at
(%) ratios of 100:0, 80:20, 60:40, 20:80 and 0:100, stimulated with
precomplexed ephrin-B1/Fc, and assayed for either attachment, or
LMW-PTP recruitment to anti-HA immunoprecipitated EphB1.
[0218] These experiments demonstrated that a recombinant GST-EphB1
cytoplasmic domain fusion protein (GST-EphB1.sub.cy) binds purified
recombinant LMW-PTP (A isoform) and endogenous LMW-PTP from crude
endothelial cell lysates following self-phosphorylation in vitro.
Kinase inactive (GST-EphB1.sub.cyK652R) and unphosphorylated EphB1
cytoplasmic domains (GST-EphB1.sub.cy, (-ATP) did not bind LMW-PTP.
Yeast two hybrid interaction between LMW-PTP and EphB1.sub.cy
required EphB1 residues 883-984, the carboxyterminal conserved
sterile alpha motif shared by Eph family receptors (21). This
domain includes a single tyrosine residue required for Grb10
binding (15). A site directed point mutation of this tyrosine
residue (Y929F) disrupted LMW-PTP/EphB1.sub.cy interaction in two
hybrid assays and in the recombinant protein interaction assay.
Stoichiometric amounts of GST-EphB1.sub.cy (Y929F) blocked the
capacity of GST-EphB1 to acquire LMW-PTP binding activity upon
self-phosphorylation, providing strong evidence that Y929 is
involved in the interaction, and suggesting that cooperative
interaction between multiple EphB1.sub.cy subunits is required.
[0219] These findings led to an evaluation of the functional
importance of LMW-PTP recruitment for the EphB1 mediated increases
in P19 adhesion seen with multimeric ephrinB1/Fc. P19 cells were
cotransfected with varying ratios of expression plasmids driving
high level expression of epitope-tagged versions of EphB1, either
wild type or (Y929F) mutant. Expression of increasing ratios of
mutant (Y929F) receptor blocked both LMW-PTP recruitment to
immunoprecipitated EphB1 complexes and the subsequent increases in
attachment attending stimulation with multimeric ephrin-B1/Fc. This
finding suggests that LMW-PTP may play a role in coupling receptor
activation with attachment responses.
[0220] Because the ratio of preclustering monoclonal anti-Fc
antibody to ephrin-B1/Fc was a critical determinant of cellular
responses, studies were conducted to examine which ephrin-B1/Fc
multimeric species determines recruitment of LMW-PTP to EphB1 and
EphB2 complexes to promote cellular adhesion. Gel filtration
chromatography separated 2 distinct peaks of size and composition
consistent with ephrin-B1 tetramers and hexamers (Peaks "B" &
"C"), as well as a broad peak containing higher order multimers
(Peak "A") (FIG. 2A). Exposure of HRMEC and P19 cells to different
dilutions of these fractions showed each was active to promote
EphB1 receptor tyrosine phosphorylation, yet ephrin-B1/Fc multimers
from fractions of peak "C" (complexes composed of two ephrin-B1/Fc
dimers and one anti-Fc monoclonal antibody) uniquely promoted
LMW-PTP recruitment (FIG. 2B) and increased adhesion (FIG. 2C).
[0221] In light of evidence that EphB3 is activated upon contact
with ephrin-B1 expressing cells (22), and that HRMEC express
ephrin-B1 and ephrin-B2 at high constitutive levels (8), the
following were addressed: 1) whether EphB receptors are activated
during endothelial capillary-like assembly, 2) whether this
assembly is sensitive to inhibition of EphB1 engagement, and 3)
whether the EphB1 complexes that form during juxtacrine activation
include LMW-PTP.
[0222] HRMEC were plated in media supplemented with no addition
(NA), PMA (20 ng ml.sup.-1) or ephrinB1/Fc multimers (500 ng
ml.sup.-1+anti-Fc), in the absence or presence of the ectodomain
competitor, EphB1/Fc (1000 ng ml.sup.-1). Cells were harvested at
15 min (NA, ephrin-B1/Fc) or 100 min (PMA), and EphB1 or EphB2
complexes were analyzed by immunoblot, using anti-EphB2, anti-pTyr
and anti-LMW-PTP antibodies. EphB1/Fc interrupts the EphB1
activation and LMW-PTP recruitment.
[0223] In a parallel experiment, HMREC were treated as above in the
absence or presence of EphB1/Fc (1000 ng ml.sup.-1), then
photographed after 8 h. ELK/Fc interrupts the capillary-like
assembly.
[0224] EphB1 and EphB2 are tyrosine phosphorylated and LMW-PTP is
recruited during capillary-like endothelial assembly stimulated by
the angiogenic agent, phorbol myristate acetate. EphB1 and EphB2
activation occurs through juxtacrine engagement, requiring that
cells are plated at sufficient cell density to promote juxtacrine
contact. Addition of exogenous receptor ectodomain, EphB1/Fc, at
concentrations of 2-8 nM inhibited not only EphB1 and EphB2
tyrosine phosphorylation and LMW-PTP recruitment, but also
capillary-like assembly. These findings argue strongly that native
endothelial EphB1 and EphB2 ligands are presented to receptors
through juxtacrine engagement to assemble receptor signaling
complexes similar to those invoked by exogenous tetrameric
ephrin-B1/Fc.
[0225] The specific response of EphB1 and EphB2 receptors to
multimeric ephrinB1, to mediate assembly of alternative complexes
and to promote cell attachment and cell-cell assembly, suggests
that ligand oligomerization may be a control point involved in
juxtacrine receptor responses required for cell targeting. Recent
data demonstrated tyrosine phosphorylation of the ephrin-B1
cytoplasmic domain in response to engagement by NUK receptor
ectodomain fusions (23,24), and ephrin-B1 has been implicated in
developmental targeting of hippocampal neurons (25). These results
suggested "outside-in" signaling through ephrin-B proteins.
Alternatively, these data suggest that conserved cytoplasmic domain
sequences among ephrin-B proteins (26) may participate in
oligomerization of these "ligands" to provide positional labels and
direct targeting of migrating vascular and neural cells as they
assemble integrated networks. The surprising capacity of
"activated" EphB receptors to direct alternative signaling events
through discrete pathways uncovers an additional level of
discrimination that is relevant for receptor tyrosine kinases that
interact with soluble and membrane bound forms of ligand.
[0226] In summary, the studies described herein demonstrate that
both dimeric and clustered tetrameric forms of ephrin-B1/Fc
stimulated EphB1 & EphB2 activation (tyrosine phosphorylation),
yet only ephrin-B1/Fc tetramers promoted cell attachment,
capillary-like endothelial assembly and receptor recruitment of the
low molecular weight phosphotyrosine phosphatase, LMW-PTP (11). The
EphB1 binding site for LMW-PTP is required for downstream cell
attachment responses. During capillary-like assembly, endogenous
endothelial ephrins engaged EphB1 to recruit LMW-PTP, a process
inhibited by an EphB1 ectodomain competitor (EphB1/Fc). EphB
receptors signal different attachment and cell-cell assembly
responses based upon their engagement of specific ephrin oligomers
that may serve as positional labels to determine targeting.
[0227] A differential signaling effect of multimeric as compared
with dimeric Ephrin-B1/Fc with regard to attachment responses to
fibronectin-coated surfaces has also been demonstrated.
Specifically, nitrocellulose coated surfaces were preadsorbed with
500 ng/well of non-denatured ephrin-B1/Fc, either as dimer
(LERK-2/Fc) or preclustered multimer (LERK2/FC+anti-Fc) or control
protein (anti-Fc, BSA), with fibronectin (500 ng/well). P19 cells
were plated and the % attached cells were assayed after a 90 min
incubation at 37.degree. C.
[0228] These data showed approximately 40% attachment of cells in
wells preadsorbed with ephrin-B1/Fc dimers as compared with
approximately 60% attachment of cells in wells preadsorbed with
ephrin-B1/Fc tetramers. These data demonstrate that Eph receptors
are competent to engage ephrin-B1 displayed as an attached protein,
as on a membrane. Furthermore, Eph discriminates the oligomeric
state on a solid surface, similar to the manner in which it may be
presented on cell surfaces through juxtacrine contact.
[0229] Vascular bed-specific promotion of angiogenesis with Eph
ligands. On the basis of differential tissue distributions of Eph
receptors and ligands, ephrin ligands from different classes
(GPI-linked vs. transmembrane proteins) were examined for the
ability to evoke different responses in endothelial cells derived
from different vascular beds.
[0230] For these studies, HRMEC or HUVEC were plated on
Matrigel-coated surfaces in defined medium in a two dimensional
capillary-like assembly assay. Ephrin-B1 promoted capillary-like
assembly of HRMEC, but not HUVEC, while ephrin-A1 promoted assembly
of HUVEC but not HRMEC, in identical media at concentrations
appropriate for their respective affinities for EphB1 and EphA2. In
each cell type, ephrin-B1 and ephrin-A1 promoted tyrosine
phosphorylation of 130 kDa EphB1 and EphA2, both of which are
expressed in each system. These findings show that endothelial
cells cultured from different vascular beds are variably responsive
to different ligands, despite similar capacity to activate
receptors of both EphB and EphA subclasses.
[0231] The biological activity of multimeric ephrin-B1 has also
been confirmed in an in vivo system. Specifically, tetramers of
ephrin-B1 have been shown to promote angiogenesis in a chick
chorioallantoic membrane (CAM) assay system as described by Folkman
et al. (43) and in a mouse corneal model as described by Kenyon et
al. (44) and the content of both of these references is
incorporated in the entirety by reference herein.
Example 2
[0232] EphB Receptors Signal Activation of Cell-Matrix Attachment
through .alpha..sub.v.beta..sub.3 and .alpha..sub.5.beta..sub.1
integrins.
[0233] Matrix proteins, peptides and antibodies. EphrinB1/Fc was
provided by Immunex (Seattle, Wash.), monoclonal anti-human IgG1
(anti-Fc) was from the Binding Site (Birmingham, UK) and monoclonal
anti-HA (12CA5) from Boehringer Mannheim (Indianapolis, Ind.).
Human IgG1 and plasma fibrinogen were from Sigma (St Louis, Mo.).
Human plasma fibronectin and bovine serum albumin (BSA) were from
Life Technologies (Gaithersburg, Md.). The GRGDTP (SEQ ID NO:1) and
GRGESP (SEQ ID NO:2) peptides were from Calbiochem (La Jolla,
Calif.), the GRGDSPK (SEQ ID NO:3) peptide from American Peptide
Company (Sunnyvale, Calif.). The following anti-integrin blocking
mAbs from Chemicon (Temecula, Calif.) were used: LM609
(.alpha..sub.v.beta..sub.3), P1F6 (.alpha..sub.5.beta..sub.1), JBS5
(.alpha..sub.v.beta..sub.5).
[0234] Cell culture and transfection. Human embryonic kidney cells,
HEK293, were passaged in minimum essential medium (Life
Technologies) supplemented with 10% fetal bovine serum (Hyclone
Laboratories, Logan, Utah). HRMEC were cultured as described (12).
HEK293 cells were transfected with Lipofectamine Plus (Life
Technologies) as described by the manufacturer. The expression
constructs pSR.alpha.-hEphB1-HA, pSR.alpha.-hEphB1-HA-K652R,
pSR.alpha.-hEphB1-HA-Y596F and pSR.alpha.-hEphB1-HA-Y929F have been
described previously (45,46). Cell attachment assays were performed
48 hours after transfection.
[0235] Solid phase cell attachment assays. Forty-eight-well plates
(Falcon) were coated with a layer of nitrocellulose (Schleicher
& Schuell) and allowed to air dry as described (47). Coated
wells were incubated overnight at 4.degree. C. with PBS containing
matrix proteins at the indicated concentrations alone (no addition,
NA) or in combination with dimeric EphrinB1/Fc. Two hours prior to
assay, wells were washed twice then blocked at 37.degree. C. with
1% BSA. Serum starved cells were harvested in 2 mM EDTA in PBS
(HEK293) or recovered by trypsinization (HRMEC), washed twice in
serum free medium containing 1% BSA, then plated at a density of
0.7-1.times.10.sup.5 cells per well. After incubation at 37.degree.
C. for 1 hour (or as specified), unattached cells were dislodged by
5 brisk slaps of the plate on a horizontal surface. Wells were
gently washed with PBS until cells were not adherent to
albumin-coated wells. Adherent cells were fixed with 2%
glutaraldehyde, stained with 0.5% crystal violet (in EtOH), and
quantified by OD reading at 570 nm. In experiments using
competitive peptides or blocking antibodies, cells were
preincubated with the indicated peptides (100 .mu.M) or antibodies
(10 .mu.g/ml) for 15 min at RT before plating. Results
representative of three independent experiments are given as OD
values and represent the mean of duplicate or triplicate
wells.+-.SEM.
[0236] In experiments to quantitate the mass of EphrinB1/Fc
adsorbed, 12-well plates were coated with nitrocellulose,
fibrinogen and increasing amounts of EphrinB1/Fc (supplemented with
biotin-conjugated EphrinB1/Fc at a molar ratio of 1:10) as
described. After an overnight incubation at 4.degree. C.,
supernatants were recovered, and wells were washed twice with PBS,
prior to extraction of adsorbed proteins in boiling
SDS-electrophoresis sample buffer. Biotinylated proteins in
supernatant and adsorbed fractions were quantitated by comparison
to standards of biotinylated ephrin-B1/Fc following electrophoresis
and blotting using Streptavidin-HRP (Jackson, Westgrove, Pa.).
[0237] Surface biotinylation and integrin immunoprecipitation. To
determine if surface integrin expression was altered by
transfection and/or exposure to ephrinB1/Fc, cells were
biotinylated for 30 min at 4.degree. C. with 0.5 mg/ml
Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) before plating onto 60
mm dishes coated with nitrocellulose, fibrinogen and EphrinB1/Fc.
After a 60 min incubation at 37.degree. C., cells were harvested in
WG buffer (15) and immunoprecipitation performed with 2 ug of the
integrin antibodies used in the competition experiments. After
SDS-PAGE (non-reducing conditions) and Western blotting,
biotinylated proteins were detected by enhanced chemiluminescence
using ECL Western Blotting Detection (Amersham).
[0238] To reconstitute the surface display of ephrin-B1 in a native
form similar to that encountered on membrane surfaces contacted by
migratory cells and cell processes, the capacity of nitrocellulose
to bind a recombinant fusion protein ligand, ephrin-B1/Fc, was
exploited. As shown in FIGS. 3A-3C, primary human renal
microvascular endothelial cells that express endogenous EphB1 and
EphB2 receptors (45) adhere poorly to plastic or
nitrocellulose-coated plastic in serum-free medium, but attach
effectively to fibrinogen-coated surfaces. Pre-coating of plates
with ephrinB1/Fc, alone or in the presence of fibrinogen, using
standard procedures, did not promote cell attachment. However, when
adsorbed to nitrocellulose-coated surfaces under non-denaturing
conditions, ephrin-B1/Fc was presented in a form that promoted
endothelial attachment (FIG. 3A), compared with an Fc control,
human IgG.sub.1. This effect required that extracellular matrix
components, either fibrinogen (FIG. 3B) or vitronectin (not shown),
be adsorbed to the nitrocellulose surface, suggesting roles for
integrins in the attachment. Although the surface density of
adsorbed fibrinogen required to promote attachment showed a
threshold at approximately 1 ug/cm.sup.2, increasing fibrinogen
surface density above that level showed modest impact on cell
attachment in the absence of ephrin-B1.
[0239] In sharp contrast, a biphasic response to the adsorbed
surface density of displayed ephrin-B1/Fc was apparent (FIG. 3B).
Maximal cell attachment was evoked by surfaces coated with 300
ng/cm.sup.2 ephrin-B1/Fc (FIG. 3C), and a sharp decline in
adsorption was evident at higher densities. This predicts a peak
effect at a maximal surface density of 5-18.times.10.sup.6
molecules/.mu..sup.2 if all adsorbed ligand is active. It is
noteworthy that EphB1 receptor activation, determined by
phosphotyrosine immunoblot of immunoprecipitated EphB1, was
stimulated at adsorption densities greater than 100 ng/cm.sup.2 and
did not show a decrement at densities greater than 300 ng/cm.sup.2.
(A minimum, calculated on the basis of labeled ephrin-B1/Fc
recovered from the adsorbed surface is 10 fold lower).
[0240] To determine if this ephrin-B1 surface density effect was
mediated through integrins, a defined RGD peptide, GRGDSPK (SEQ ID
NO:3), was applied to the surfaces in the absence and presence of
ephrin-B1/Fc, or IgG.sub.1. Attachment assays of serum-starved
microvascular endothelial cells to GRGDSPK (SEQ ID NO:3)-coated
48-well-plates were performed as described herein. Cells were
preincubated for 15 min at 22.degree. C. with blocking peptides
(100 uM) or anti-integrin antibodies (10 ug/ml) before plating.
Similar results were obtained using fibrinogen (1 ug/cm.sup.2).
HEK293 cells were transiently transfected with pSR.alpha.-EphB1 and
attachment assays were performed after 48 hours as described
herein.
[0241] These experiments demonstrated that the RGD peptide was
sufficient to support the ephrin-B1 induced increase in endothelial
attachment. Moreover, the increase in ephrinB1-induced attachment
was sensitive to antibodies that block RGD engagement by
.alpha..sub.v.beta..sub.3 integrin (LM609), but not
.alpha..sub.v.beta..sub.5 or .alpha..sub.5.beta..sub.1 integrins.
Alpha.sub.v.beta..sub.3 integrin is an important mediator of
endothelial survival, participates in crucial steps in tumor
angiogenesis, and has been implicated in angiogenic responses
stimulated by bFGF and other angiogenic agonists (49,50).
[0242] At the outset, it was not apparent whether ephrin-B1
functions as a mechanical tether in this integrin-mediated increase
in endothelial attachment, or as an inducer of signals that
activate integrins engagement of extracellular matrix determinants.
To address this issue further, a cell line, HEK293, was identified
that does not express endogenous EphB1 or EphB2 receptors and that
displayed no ephrin-B1 dependent change in attachment to fibrinogen
or RGD peptide under basal conditions. Transient expression of
EphB1 in HEK293 cells conferred ephrin-B1 dependent increases in
attachment to fibrinogen. Antibody competition experiments showed
that, in contrast to endothelial cells, ephrin-B1 dependent EphB1
activation in HEK293 cells increased cell attachment through
.alpha..sub.5.beta..sub.1 integrin, rather than
.alpha..sub.v.beta..sub.3, which is expressed at very low levels in
HEK293. Ephrin-B1 did not increase the surface expression of
endothelial .alpha..sub.v.beta..sub.3 integrin, or HEK293
.alpha..sub.5.beta..sub.1, as assayed by surface protein
biotinylation and immunoprecipitation experiments.
[0243] Recent reports demonstrated that activated EphB1 receptors
recruit Nck and LMW-PTPs through specific tyrosine residues (Y596
and Y929, respectively), and that integrity of receptor tyrosine
kinase activity and each of these residues is required for coupling
receptor activation to increases in fibronectin attachment. Point
mutant EphB1 receptors (Y596F) are defective in binding the SH2
domain of Nck, and in activating c-Jun kinase (51). Residue Y929 in
the EphB1 carboxyterminal sterile alpha motif is required for
LMW-PTP recruitment (45).
[0244] To show that EphB1 tyrosine kinase activity and specific
tyrosine residues are required to signal increased attachment
through .alpha..sub.5.beta..sub.1 integrin, the following
experiments were carried out. HEK293 cells were transfected with
wild type (wt), kinase inactive (K652R), Nck recruitment defective
(Y596F), or LMW-PTP recruitment defective (Y929F) EphB1 expression
plasmids. Forty-eight hours after transfection, solid phase
attachment assays were conducted on fibrinogen-coated 48-well
plates displaying ephrin-B1/Fc (300 ng/cm.sup.2), as described
herein. Quantitatively similar levels of EphB1 receptor expression
were confirmed by immunoblot using monoclonal anti-HA antibody.
[0245] These experiments demonstrated that transient HEK293
expression of kinase inactive (K652R), and point mutant EphB1
receptors (Y596F & Y929F), at similar levels, failed to alter
.alpha..sub.5.beta..sub.1 integrin-mediated attachment. Thus,
signaling competent receptors are required to mediate the ephrin-B1
attachment response, and the capacity for receptors to recruit both
Nck and LMW-PTP is necessary. Moreover, some change in integrin
function, other than surface expression, is required to mediate the
response. The ephrin-B1 induced increases in cell attachment
appears to reflect "inside-out" activation of
.alpha..sub.5.beta..sub.1 integrin in HEK293 cells and
.alpha..sub.v.beta..sub.3 integrin in endothelial cells.
[0246] Although the present process has been described with
reference to specific details of certain embodiments thereof, it is
not intended that such details should be regarded as limitations
upon the scope of the invention except as and to the extent that
they are included in the accompanying claims.
[0247] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference-into this
application in order to more fully describe the state of the art to
which this invention pertains.
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Sequence CWU 1
1
3 1 6 PRT Artificial Sequence Description of Artificial Sequence;
Note = synthetic construct 1 Gly Arg Gly Asp Thr Pro 1 5 2 6 PRT
Artificial Sequence Description of Artificial Sequence; Note =
synthetic construct 2 Gly Arg Gly Glu Ser Pro 1 5 3 7 PRT
Artificial Sequence Description of Artificial Sequence; Note =
synthetic construct 3 Gly Arg Gly Asp Ser Pro Lys 1 5
* * * * *