U.S. patent application number 11/712995 was filed with the patent office on 2007-09-20 for methods of modulation of the immune system.
This patent application is currently assigned to The Hospital For Sick Children. Invention is credited to Andrew Freywald, Thomas Grunberger, Eyal Grunebaum, Chaim M. Roifman, Nigel Sharfe.
Application Number | 20070218070 11/712995 |
Document ID | / |
Family ID | 22637191 |
Filed Date | 2007-09-20 |
United States Patent
Application |
20070218070 |
Kind Code |
A1 |
Roifman; Chaim M. ; et
al. |
September 20, 2007 |
Methods of modulation of the immune system
Abstract
Manipulation of the EphB6 receptor and its active Eph partners
allow for regulation of T cell responses, including TCR signalling,
T cell proliferation, and induction of T cell death. Methods of
modulating EphB6 are described as well as various therapeutic
applications.
Inventors: |
Roifman; Chaim M.; (North
York, CA) ; Freywald; Andrew; (Thornhill, CA)
; Sharfe; Nigel; (Toronto, CA) ; Grunberger;
Thomas; (Toronto, CA) ; Grunebaum; Eyal;
(Toronto, CA) |
Correspondence
Address: |
JAGTIANI + GUTTAG
10363-A DEMOCRACY LANE
FAIRFAX
VA
22030
US
|
Assignee: |
The Hospital For Sick
Children
Toronto
CA
|
Family ID: |
22637191 |
Appl. No.: |
11/712995 |
Filed: |
March 2, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10169520 |
Oct 26, 2002 |
7205113 |
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PCT/CA01/00004 |
Jan 5, 2001 |
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11712995 |
Mar 2, 2007 |
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60174710 |
Jan 6, 2000 |
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Current U.S.
Class: |
424/171.1 ;
424/173.1; 424/184.1; 435/375; 435/7.8 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 37/00 20180101; C07K 14/52 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/171.1 ;
424/173.1; 424/184.1; 435/375; 435/007.8 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; C12N 5/06 20060101
C12N005/06; C12N 5/08 20060101 C12N005/08; G01N 33/53 20060101
G01N033/53; G01N 33/564 20060101 G01N033/564 |
Claims
1-22. (canceled)
23. A method of modulating apoptosis, said method comprising
administering to the cell an effective amount of a substance that
modulates EphB6 function, thereby modulating apoptosis.
24. The method according to claim 23, wherein the substance that
modulates EphB6 function is ephrin-B1, ephrin-B2, an antibody or a
fragment thereof capable of binding EphB6, or an antisence molecule
to EphB6.
25. The method according to claim 24 wherein the substance that
modulates EphB6 function is Ephrin B1 or Ephrin B2.
26. The method according to claims 23 wherein a substance which
stimulates T-cell expression of EphB6 is co-administered.
27. The method according to claim 26, wherein the substance which
modulates T-cell expression of EphB6 is ephrin B1, ephrin B2, or a
combination thereof.
28. The method according to claim 27 wherein the catalytically
active member of the EphB subfamily is EphB1.
29. The method according to claim 23 wherein the substance that
modulates T-cell expression of EphB6 is ephrin-B1, an EphB6
receptor, ephrin- B2, an antibody or an antibody fragment capable
of binding EphB6, or an antisense molecule to EphB6.
30. The method according to claim 29, wherein the substance that
modulates T-cell expression of EphB6 is Ephrin-B1 or Eprin-B2.
31. The method according to claim 29, wherein the T-cell is a human
T-cell.
32. A method of modulating cell proliferation, comprising
administering to a population of cells comprising T-cells an
effective amount of a substance which modulates EphB6 function.
33. The method according to claim 32, wherein the substance that
modulates EphB6 function is ephrin-B1, an antibody or fragment
thereof capable of binding EphB6, or an antisense molecule to
EphB6.
34. The method according to claim 32, wherein the EphB6 function is
further defined as T-cell EphB6 function of a human T-cell.
35. The method according to claim 32, wherein the T-cell is a human
T-cell.
36. A method of modulating a T-cell response in an animal,
comprising administering to the animal an effective amount of a
substance that modulates T-cell expression of EphB6 such that the
T-cell response is modulated.
37. The method according to claim 36, wherein the substance that
modulates T-cell expression of EphB6 is ephrin-B1, ephrin -B2, an
antibody or antibody fragment capable of binding EphB6, or an
antisense molecule to EphB6.
38. The method according to claim 37, wherein the substance which
modulates T-cell expression of EphB6 is Ephrin-B1 or Ephrin B2.
39. The method according to claim 36, wherein the EphB6 function is
further defined as T-cell EphB6 function of a human T-cell.
40. A method of treating a disorder of T-cell proliferation, an
autoimmune disorder, a cell-associated autoimmune disorder, an
allergic disorder in an animal, or a host verses transplant
rejection, comprising modulating T-cell expression of EphB
receptor.
41. The method according to claim 40, wherein T-cell expression of
EphB receptor is modulated by ephrin-B1, ephrin -B2, an antibody or
antibody fragment capable of binding EphB6, or an antisense
molecule to EphB6.
42. The method according to claim 40, wherein the cell-associated
autoimmune disorder is multiple sclerosis, lupus, arthritis,
thyroiditis, diabetes, psoriasis, Crohn's disease or colitis.
43. The method according to claim 40, wherein the allergic disorder
is asthma, hyper-IgE syndrome, eosinophilic syndrome, or a T
cell-dependent graft-verses-host disease.
44. The method according to claim 40, wherein the EphB6 function is
further defined as EphB6 function of a human T-cell.
45. A method of promoting an anti-viral immune response in an
animal, comprising modulating T-cell expression of EphB receptor
thereby promoting the antiviral response in the animal.
46. The method according to claim 45, wherein modulating T-cell
expression of EphB receptor comprises administering ephrin-B1,
ephrin- B2, an antibody or antibody fragment capable of binding
EphB6, or an antisense molecule to EphB6.
47. The method according to claim 45 wherein the substance is
soluble stimulatory or inhibitory ephrin and/or a soluble EphB6
receptor.
48. A method for identifying a substance which is capable of
binding to a purified and isolated EphB6 protein, comprising
reacting the protein with at least one substance which potentially
can bind with the protein under conditions which permit the
formation of complexes between the substance and the protein, and
assaying for complexes, for free substance, for non-complexed
protein, or for activation of the protein.
49. A method for assaying a medium for the presence of an agonist
or antagonist of the interaction of a purified and isolated a EphB6
protein and a substance which binds to the protein which comprises
reacting the protein with a substance which is capable of binding
to the protein and a suspected agonist or antagonist substance
under conditions which permit the formation of complexes between
the substance and the protein, and assaying for complexes, for free
substance, for non-complexed protein, or for activation of the
protein.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the field of immunology and is c
with protein tyrosine kinases and, more particularly, to
Eph-related receptor tyrosine kinases, specifically the EphB6
receptor, and its active partners, and methods of its manipulation
for the modulation of cellular processes.
BACKGROUND OF THE INVENTION
[0002] The regulation of development and cell proliferation in
higher o involves signaling through receptor tyrosine kinases
(RTK). Ligand binding to the extracellular domain of RTKs induces
receptor dimerization or oligomerization and stimulates their
intrinsic tyrosine kinase activity (Honegger et al. (1990); Kashles
et al. (1991); Ueno et al. (1991); Yarden and Schlessinger (1987);
Yarden and Schlessinger (1987a)). As a consequence, RTKs undergo
autophosphorylation, causing further changes in receptor
configuration and providing specific docking sites for cytoplasmic
signaling proteins containing Src-homology 2 (SH2) or
phosphotyrosine binding (PTB) domains (Kavanaugh et al. (1995);
Koch et al. (1991); Songyang et al. (1993)).
[0003] RTKs are divided into families on the basis of their
structural organization (van der Geer et al. (1994)), Eph receptors
forming the largest known family, with at least 14 members
(Pasquale (1997); Zhou (1998); Ziach and Pasquale (1997)). Ephs
bind a group of ligands known as ephrins (Eh family receptor
interacting), eight of which are currently known, all membrane
anchored either by glycosylphosphatidylinositol (GPI)
(ephrinA1-A5), or a trans-membrane domain (ephrhiB1-B3) (Drescher
(1997); Pasquale (1997)). Eph receptors are divided into two groups
based upon their ligand binding characteristics, EphA or EphB,
according to the class of ephrin bound (Brambilla et al. (1995);
Ciossek and Ulrrich (1997); Gale et al. (1996); Koziosky et al.
(1995); Park and Sanchez (1997)); although receptor-ligand
specificity is degenerate within a group (Zhou (1998)). It is a
characteristic of the Eph receptor family that their ligands must
be membrane bound in order to be active (Davis et al. (1994);
Sakano et al. (1996); Winslow et al. (1995)). This absolute
requirement for membrane anchorage of the ligand makes the
formation of cell-cell contact an obligatory event in activation of
the Eph receptors. Consequently, activated receptors are
concentrated in areas of cell-cell contact.
[0004] The Eph receptors and their ligands are typically most
highly expressed in neural and endothelial cells (Zhou (1998)) and
most descriptions of their function corn the development of the
nervous system and angiogenesis (Drescher et al. (1995); Friedman
et al. (1996); Horoberger et al. (1999); Gao et al. (1999); Ciossek
et al. (1998); Daniel et al. (1996); O'Leary et al. (1999); Pandey
et al. (1995); Adams et al. (1999); Wang et al. (1998); Yue et al.
(1999)). Upon the formation of cell-cell contact, Eph receptor
signaling results in reorganization of the actin cytoskeleton and
integrin activation (Becker et al. (2000); Miao et al. (2000); Zou
et al. (1999); Holland et al. (1997); Huynh-Do et al. (1999)). As a
result, Eph receptors generate adhesive or repulsive signals and in
the neural system can guide the movement of axonal growth cones,
cell migration and synapse formation (Drescher et al. (1995);
Hornberger et al. (1999); Ciossek et al. (1998); Yue et al. (1999);
Bohme et al. (1996); Flanagan et al. (1998); Hsueh et al. (1998);
Krull et al. (1997); Monschau et al. (1997); Nakamoto et al.
(1996); Mellitzer et al. (1999); Smith et al. (1997); Xu et al.
(1999); Torres et al. (1998)).
[0005] The most recently identified member of the Eph family is the
orphan EphB6 receptor, with a structure typical of the EphB
subfamily (Gurniak et al. (1996); Matsuoka et al. (1997)). While
structural analysis of EphB6 reveals conservation of the major EphB
receptor autophosphorylation sites (Y638 and Y644), there are
several critical alterations in the tyrosine kinase domain. These
include substitution of a crucial lysine residue in the ATP binding
site, resulting in a receptor that does not demonstrate detectable
kinase activity (Gurniak et al. (1996); Matsuoka et al. (1997)).
This casts doubt upon the ability of EphB6 to undergo tyrosine
phosphorylation upon ligand stimulation and thus to initiate
signaling cascades in the cytoplasm However, analogy with EphB3, a
well-characterized catalytically inactive member of the EGF
receptor family, suggests that EphB6 may form hetero-oligiomers
with catalytically active family members. And similarly, as a
result of trans-phosphorylation by these active receptors, EphB6
may recruit cytoplasmic signal transducing molecules.
[0006] Unlike other receptor tyrosine kinases, EphB6 is
predominantly expressed in the thymus (Gurniak et al. (1996)),
suggesting that it may play an important role in T cell
differentiation. Current evidence suggests that Eph receptors may
directly interact with the TCR (T cell receptor) signaling pathway.
Eph receptors can regulate integrin activation and cytoskeletal
rearrangement (Becker et al. (2000); Miao et al. (2000); Zou et al.
(1999); Holland et al. (1997); Huynh-Do et al. (1999)), both
crucial events in TCR induced responses (Holsinger et al. (1998);
Abraham et al. (1999); Bleijs et al. (1999); (Ticchioni et al.
(1993); Valitutti et al. (1995); Wulfing et al. (1998); Wulfing et
al. (1998); Snapper et al. (1998); Viola et al. (1999);
Vivinus-Nebot et al. (1999)). Moreover, several Eph receptors also
bind the T cell kinase Fyn (Choi et al. (1999); Ellis et al.
(1996)). Indeed, high levels of EphB6 expression have been detected
in a population of human peripheral T lymphocytes, but not in B
cells (Shimoyama et al. (2000)). Despite its lack of kinase
activity, ephrin-B1-stimulated EphB6 undergoes typrosine
phosphorylation, which is provided by a catalytically active member
of the EphB subfamily. This initiates its downstream signaling. The
Jun N-terminal kinase (jNK) cascade (Becker et al. (2000)) is the
major pathway downstream of the Eph receptor family, and is one of
the key regulators of T cell apoptosis (Sabapathy et al. (1999);
Baker et al. (1998)). It is currently not clear whether the Eph
receptor family or any members, including the EphB6 receptor, have
a role in such apoptosis. Regulation of this aspect of the immune
system continues to be desirable.
SUMMARY OF THE INVENTION
[0007] The present inventors have demonstrated that manipulation of
the kinase-inactive EphB6 receptor and its active partners allows
for regulation of T cell responses preferably cell signalling and T
cell proliferation
[0008] Further, the present inventors have determined that despite
its lack of kinase activity, stimulated EphB6 undergoes tyrosine
phosphorylation, and that modulation of EphB6 provides a method for
modulating apoptosis, preferably for the induction of Activation
induced Cell Death (AICD).
[0009] Accordingly, in its broad aspect the present invention
provides a method of modulating the immune system of an animal
comprising administering to the animal an effective amount of a
substance that modulates the expression, or activity of EphB6, or
Its active partner thereby modulating the immune system.
[0010] In another aspect of the present invention there is provided
a method of modulating of a cell comprising administering to the
cell, an effective amount of a substance that modulates the
expression, or activity of EphB6, or its active partner thereby
modulating the apoptosis.
[0011] According to one embodiment of the methods of the invention
the substances which may be used to modulate are preferably
ephrin-B1, an oligomeric or monomeric soluble EphB6 receptor, a
soluble EphB6 ligand, ephrin-B2, an antibody capable of binding
EphB6, an antibody fragment which is agonistic or antagonistic to
EphB6, a physiological or synthetic EphB6 ligand, a soluble active
EphB6 partner, an antibody or fragments thereof to an EphB6 active
partner, an antisense molecule to EphB6 or its active partners, or
a physiological or synthetic ligand for an EphB6 active partner,
more preferably the substance is Ephrin -B1 or Ephlin B2.
[0012] According to another embodiment of the present invention
there is provided a method of modulating cell proliferation
comprising administering to the cell an effective amount of a
substance which modulates the expression or activity of an EphB6
receptor or its active partners. Preferably the substance is
ephrin-B1, an oligomeric or monomeric soluble EphB6 receptor, a
soluble EphB6 ligand, ephrin-B2, an antibody capable of binding
EphB6, an antibody fragment which is agonistic or antagonistic to
EphB, a physiological or synthetic EphB6 ligand, a soluble active
EphB6 partner, an antibody or fragments thereof to an EphB6 active
partner, an antisense molecule to EphB6 or its active partners, or
a physiological or synthetic ligand for an EphB6 active
partner.
[0013] According to yet another embodiment of the present invention
there is provided a method of modulating a T cell response in an
animal comprising administering to the animal an effective amount
of a substance that modulates EphB6 expression or activity or that
of its partner such that the T cell response is modulated.
Preferably the substance is ephrin-B1, an oligomeric or monomeric
soluble EphB6 receptor, a soluble EphB6 ligand, ephrin-B2, an
antibody capable of birding EphB6, an antibody fragment which is
agonistic or antagonistic to EphB6, a physiological or synthetic
EphB6 ligand, a soluble active EphB6 partner, an antibody or
fragments thereof to an EphB6 active partner, an antisense molecule
to EphB6 or its active partners, or a physiological or synthetic
ligand for an EphB6 active partner, more preferably the substance
is Ephrin-B1 or Ephrin B2.
[0014] According to other embodiments of the methods of the present
invention a substance which stimulates EphB6 is co-administered,
preferably the substance is ephrin B1, ephrin B2, or a
catalytically active member of the EphB subfamily, more preferably
the catalytically active member of the EphB subfamily is EphB1.
[0015] According to another embodiment of the present invention
there is provided a method of treating a disorder of T-cell
proliferation, an autoimmune disorder, a cell-associated autoimmune
disorder, an allergic disorder in an animal, or a host versus
transplant reaction comprising administering to the animal an
effective amount of a combination of inhibitory or stimulatory
soluble EphB6 ligand and/or soluble EphB6 receptor, or a ligand to
an EphB6 active partner or soluble partner, thereby treating the
disorder. Preferably the substance is ephrin-B1, an oligomeric or
monomeric soluble EphB6 receptor, a soluble EphB6 ligand,
ephrin-B2, an antibody capable of binding EphB6, an antibody
fragment which is agonistic or antagonistic to EphB6, a
physiological or synthetic EphB6 ligand, a soluble active EphB6
partner, an antibody or fragments thereof to an EphB6 active
partner, an antisense molecule to EphB6 or its active partners, or
a physiological or synthetic ligand for an EphB6 active partner.
According to a preferred embodiment the cell-associated
autoimmunity is multiple sclerosis, lupus, arthritis, thyroiditis,
diabetes, psoriasis, Crohn's disease or colitis. According to
another preferred embodiment of the method of the present
invention, the allergic disorder is asthma, hyper-IgE syndrome,
eosinophilic syndrome, or a T-cell dependent graft-verus-host
disease.
[0016] According to another embodiment of the present invention
there is provided a method of promoting an anti-viral immune
response in an animal comprising administering to the animal an
effective amount of a substance that modulates the expression or
activity of EphB6 or its active Eph partner thereby promoting the
antiviral response in the animal. Preferably the substance is
ephrin-B1, an oligomeric or monomeric soluble EphB6 receptor, a
soluble EphB6 ligand, ephrin-B2, an antibody capable of binding
EphB6, an antibody fragment which is agonistic or antagonistic to
EphB6, a physiological or synthetic EphB6 ligand, a soluble active
EphB6 partner, an antibody or fragments thereof to an EphB6 active
partner, an antisense molecule to EphB6 or its active partners, or
a physiological or synthetic ligand for an EphB6 active partner,
more preferably the substance is soluble stimulatory or inhibitory
ephrin and/or a soluble EphB6 receptor.
[0017] According to another aspect of the methods of the present
invention the animal which is subject of the methods is a mammal,
preferably human.
[0018] According to another aspect of the present invention there
is provided a method for identifying a substance which is capable
of binding to a purified and isolated EphB6 protein, comprising
reacting the protein with at least one substance which potentially
can bind with the protein under conditions which permit the
formation of complexes between the substance and the protein, and
assaying for complexes, for free substance, for complexed protein,
or for activation of the protein.
[0019] According to yet another aspect of the present invention
there is provided a method for assaying a medium for the presence
of an agonist or antagonist of the interaction of a purified and
isolated a EphB6 protein and a substance which binds to the protein
which comprises reacting the protein with a substance which is
capable of binding to the protein and a suspected agonist or
antagonist substance under conditions which permit the formation of
complexes between the substance and the protein, and assaying for
complexes, for free substance, for non-complexed protein, or for
activation of the protein.
[0020] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples while indicating preferred embodiments of the
invention are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The invention will now be described in relation to the
drawings in which:
[0022] FIG. 1A is a photograph of an immunoblot illustrating
EphB6-M phosphorylation in COS-7 cells upon Ephrin B1
stimulation.
[0023] FIG. 1B is a photograph of an immunoblot illustrating
EphB6-M phosphorylation in Hek-295 and NIH 3T3 cells upon Ephrin B1
stimulation.
[0024] FIG. 1C is an immunoblot illustrating time dependent
phosphorylation of EphB6.
[0025] FIG. 1D is an immunoblot illustrating the effects of varying
ligand concentration on phosphorylation of EphB6.
[0026] FIG. 1B is an immunoblot illustrating the effect of soluble
EphB6 receptor an ephrin-B1 induced EphB6 phosphorylation.
[0027] FIG. 2A is an immunoblot illustrating the phosphorylation of
EphB6-M upon co-expression of EphB1 in COS7 cells.
[0028] FIG. 2B is an immunoblot illustrating the phosphorylation of
transfected EphB6-M co-transfected with EphB1 or T-7 tagged kinase
inactive EphB1 (B1-KD).
[0029] FIG. 2C is an Immunoblot illustrating the ligand dependent
phosphorylation of EphB6-M.
[0030] FIG. 2D is an immunoblot illustrating the induction of EphB6
trans-phosphorylation by truncated EphB1.
[0031] FIG. 2E is another view of the immunoblot of FIG. 2D
illustrating the induction of phosphorylation of EphB6 by truncated
EphB1.
[0032] FIG. 3 is an agarose gel illustrating the expression of
EphA2, EphB1, EphB2 and EphB6 receptors in human thymocytes and T
cells.
[0033] FIG. 4A is an immunoblot illustrating pp115 co-precipitates
with EphB6 in human thymocytes.
[0034] FIG. 4B Is an immunoblot illustrating the time course of
pp115 association with EphB6.
[0035] FIG. 4C is an immunoblot illustrating pp115 as having the
same electrophoretic mobility as c-Cbl.
[0036] FIG. 4D is an immunoblot illustrating EphB6 association with
Cbl in samples immunoprecipitated from thymocyte lysates.
[0037] FIG. 4E is an immunoblot illustrating EphB6 association with
Cbl in transfected cells.
[0038] FIG. 4F is an immunoblot illustrating that the G306B loss
-of-function Cbl mutant does not bind EphB6.
[0039] FIG. 5A is an immunoblot illustrating EphB6 mediated
downregulation of Zap-70.
[0040] FIG. 5B is an immunoblot illustrating that phosphorylation
of Y493F Zap-70 is not altered by EphB6.
[0041] FIG. 5C is an immunoblot illustrating the stable expression
of EphB6-M in transfected Jurkat.
[0042] FIG. 5D is an immunoblot illustrating the phosphorylation of
EphB6-M in Jurkat with stimulation by ephrin-B1.
[0043] FIG. 5E is an immunoblot illustrating EphB6 downregulation
of the phosphorylation of Zap-70.
[0044] FIG. 5F is an immunoblot illustrating EphB6 downregulation
of the phosphorylation of Zap-70 associated CD3.zeta. in
Jurkat.
[0045] FIG. 6A is an immunoblot illustrating the effect of
ephrin-B1 on TCR induced activation of Lck.
[0046] FIG. 6B is an immunoblot illustrating the effect of EphB6 an
TCR induced activation of Lck.
[0047] FIG. 7A provides a series of graphs illustrating the effect
of ephrin-B1 on TCR mediated upregulation of CD25.
[0048] FIG. 7B provides a series of graphs illustrating the effect
of overexpression of EphB6 on TCR mediated upregulation of
CD25.
[0049] FIG. 8A is a histogram illustrating the enhancement of CD25
upregulation by dominant negative EphB6.
[0050] FIG. 8B is a histogram illustrating the enhancement of CD25
upregulation by dominant negative EphB6.
[0051] FIG. 8C is a further histogram illustrating the inhibition
of CD25 regulation.
[0052] FIG. 9A is an immunoblot illustrating stable expression of
EphB6 receptor in the mature T cell line Jurkat.
[0053] FIG. 9B is a series of figures illustrating the effect of
overexpression of EphB6 upon induction of apoptosis in T-cells.
[0054] FIG. 10A is a histogram illustrating the EphB6-dependent
increase in expression of TNF.alpha..
[0055] FIG. 11A is a histogram illustrating that the ephrin-B1
inhibits expression of TNFR II, but not TNFR I.
[0056] FIG. 11B is a histogram illustrating that the overexpressed
EphB6 receptor inhibits expression of TNFR II, but not TNFR I.
[0057] FIG. 12 are immunoblots illustrating that EphB6 receptor is
able to prevent activation of p54 JNK.
[0058] FIG. 13 provides an illustration of a model of EphB6
receptor interaction with the TCR signaling pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0059] As stated above, the present inventors have demonstrated
that modulation of the kinase-inactive EphB6 receptor allows for
modulation of the immune system.
[0060] In particular, the inventors have determined that despite
its lack of kinase activity, the EphB6 undergoes tyrosine
phosphorylation upon stimulation with a substance, preferably
membrane-bound or soluble oligomerized ephrin-B1. They have also
demonstrated that EphB6 can be trans-phosphorylated by
catalytically active members of the EphB subfamily, in particular,
by EphB1.
[0061] The present inventors also demonstrate that the EphB6
receptor associates with c-Cbl, a protein central to the regulation
of TCR signaling. Cbl binding to EphB6 is constitutive, but is lost
upon introduction of a Cbl G306E loss of function mutation. In
contrast, oncogenic 70-Z Cbl binds EphB6 essentially like wild type
Cbl.
[0062] The present inventors have also determined that,
overexpression of the EphB6 receptor in T cells resulted in
inhibition of anti-CD3 dependent phosphorylation of the
TCR-associated kinase Zap-70 and its associated CD3 chain. This
appeared to be mediated by a primary inhibition of the activity of
the src-like kinase Lck. Ultimately, this blockage in TCR signaling
results in a failure of T cell response, inhibiting upregulation of
CD25 expression.
[0063] Stable overexpression of the EphB6 receptor was also found
to significantly enhance TCR-mediated apoptosis in an
ephrin-B1-dependent manner, thus demonstrating that modulation of
EphB6 provides a method for regulating the induction of AICD.
[0064] As used herein "Behaviour of cells of the immune system"
means the sum of the ability of cells to respond to a given
stimulus and to interact with their environment, in particular, the
rate at which they undergo proliferation, differentiation and cell
death and develop immune responses.
[0065] As used herein "in conjunction with" or "co-adminstration"
means concurrently, before or following adminstration of a first
substance.
[0066] As used herein "animal" means any member of the animal
kingdom, including, preferably, humans.
[0067] As used herein, administration of an "effective amount" of a
substance or compound(s) of the present invention is defined as an
amount effective, at dosages and for periods of time necessary to
achieve the desired result. The effective amount of a compound of
the invention may vary according to factors such as the disease
state, age, sex, and weight of the animal. Dosage regima may be
adjusted to provide the optimum therapeutic response. For example,
several divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0068] The term "active partner" as used herein means any EphB6
interacting tyrosine kinase receptor, preferably a member of the
Eph family of tyrosine receptor linases. The EphB6 receptor
[0069] The standard features of the EphB6 receptor place it in the
EphB subfamily (Gumiak et al. (1996); Matsuoka et al. (1997)). EphB
receptors are stimulated by membrane bound ephrin-B ligands
demonstrating highly degenerate specificity, with ephrin-B1 and
ephrin-B2 activating most EphB receptors (Zhou et al. (1998)).
[0070] The murine EphB6 receptor was reported to be expressed
predominantly in thymocytes (Gurniak et al. (1996)), suggesting
that it may have an important role in T cell differentiation. By
RT-PCR, the present inventors detected EphB6 expression in both
human thymocytes and mature peripheral blood T cells, as well as in
the T cell line Jurkat (FIG. 3). Two catalytically active members
of the Eph family, EphB1 and EphB2, were also expressed throughout
the T cell lineage, while the EphA2 receptor could only be detected
in thymocytes. The persistent expression of EphB6 across the T cell
lineage suggested it might not only be important during
differentiation, but also in mature T cell function.
[0071] Due to the membrane bound nature of both the Eph receptor
and ephrin ligand, an important feature of receptor-ligand
interaction is the necessity for the formation of cell-cell
contact. As activation of the TCR complex occurs in an area of
T-cell contact with an antigen-presenting cell, activated TCR
complexes may potentially be brought into close proximity with EphB
receptors. TCR signaling responses are dependent upon
re-organization of the actin cytoskeleton and signals transmitted
via integrin receptors, both processes regulated by activated Eph
receptors in a variety of cells (Holsinger et al. (1998); Abraham
et al. (1999); Bleijs et al. (1999); Ticchioni et al. (1993);
Valitutti et al. (1995); Wulfing and Davies (1998); Wulfing et al.
(1998); Snapper et al. (1998); Viola et-al. (1999); Vivinus-Nebot
et al. (1999)). The potential for productive interaction between
these two receptor pathways therefore appeared high.
[0072] The inventors demonstrate that activation of the T cell
receptor results in association of phosphorylated c-Cbl protein
with the EphB6 receptor. The ability of Cbl to bind EphB6 suggests
that analogous to the EGF receptor, EphB6 expression may be
regulated by Cbl mediated modification. It is now clear that Cbl is
responsible for the physical downregulation of many receptors
through induction of receptor ubiquitination (Levkowitz et al.
(1998), Wang et al. (1999), Lee et al (1999), Miyake et al. (1999).
Addition of ubiquitin moieties to the lysine residues of a protein
targets it for degradation (Hershko et al. (1998), either in
cytoplasmic proteasomes or in lysosomes. Cbl binding induces
ubiquitination of the EGF, ErbB1, PDGF and CSF receptors, an
ability derived on its ring finger domain (mutated in 70-Z Cbl).
The ring-finger domain appears to be an E3 ubiquitin-ligase
(Joazeiro et al. (1999)), responsible for the transfer of ubiquitin
from a carrier protein (E2) to the target, thus controlling the
specificity of degradation. As all receptors known to bind Cbl
undergo ubiquitination, it is likely that EphB6 function will be
similarly regulated. This may provide a potential mechanism for
regulating the effective cell surface expression level of the EphB6
receptor.
[0073] The inventors also demonstrate that overexpression or
activation of the EphB6 receptor in T-cells can modulate signaling
through the T-cell antigen receptor. Overexpression of the EphB6
receptor results in an inhibition of anti-CD3 induced activation of
the Src-family kinase Ick and subsequently phosphorylation of
Zap-70 kinase and its associated CD3 chain. Stimulation of the TCR
leads in particular to induction of both IL-2 production and CD25
(IL-2R.alpha.) expression; thus potentially inducing expansion of
activated T-cell populations (Chambers et al. (1997)). TCR mediated
induction of CD25 requires activation of the TCR-associated
kinases. The investigators demonstrate that supression of the early
events of TCR signaling by EphB6 ultimately translates into an
inhibition of T-cell response, in particular, CD25 upregulation. In
agreement, Ephrin-B1 stimulation of thymocytes, which naturally
express high levels of EphB6, prevents TCR mediated upregulation of
IL-2 receptor expression. The inhibitory effect of the endogenous
EphB6 receptor upon the TCR complex was confirmed by the ability of
a dominant negative form of EphB6 to enhance the TCR-induced
upregulation of CD25 in T-cells. T lymphocyte homeostasis is
precisely regulated, with numerous TCR co-stimulatory events
required for finely tuned control of cell fate, these signals
regulating both proliferative and apoptic pathways (Chambers et al.
(1997), Janeway et al. (1994)). Clearly EphB6 acts as an effective
TCR co-receptor, influencing the response of cells to TCR
stimulation.
[0074] Expression of CD25 is central to the IL-2 driven clonal
expansion that occurs upon exposure of mature T-cells to antigen.
Failure to express the high affinity IL-2R complex composed of the
.alpha. (CD25), .beta. and .gamma. chains prevents the development
of the necessary IL-2 autocrine proliferative loop (Nelson and
Willerford, 1998). Thus, while not wishing to be bound by any one
theory, one of the biological functions of EphB6, in conjunction
with other EphB receptors, may be to control the clonal expansion
of antigen activated T-cells through suppression of antigen-induced
CD25 expression and associated events. Several alternative models
of EphB6 function also become apparent. Under physiological
conditions, stimulation of the EphB6 receptor may serve to maintain
activation of the TCR signaling pathway below a certain threshold,
preventing premature activation by inappropriate low affinity TCR
interactions. Or alternatively, the presence of varying ephrin-B
ligands may modify the ability of T-cells to respond to antigens
presented on different cell-surfaces. Failure to correctly regulate
TCR signaling may lead to uncontrolled activation or undesirable
activation upon very low affinity interaction with antigen. The
consequences of these events may be multiple but include autoimmune
reactions, as low affinity self-self interactions are not properly
regulated, or recognition by the T-cell of inappropriate target
cells due to the absence of appropriate targeting by Eph receptor
engagement, or inadvertent activation of bystander cells due to
cytokine overproduction by uncontrolled activated cells.
Modulation of EphB6
Antibodies
[0075] Antibodies represent a class of substances that may be used
advantageously to modulate the activity of the EphB6 receptor.
Antibodies may be used to either inhibit, or stimulate the EphB6
receptor. Antibodies can be prepared which bind a distinct epitope
in an unconserved region of the protein. An unconserved region of
the protein is one that does not have substantial sequence homology
to other proteins.
[0076] Conventional methods can be used to prepare the antibodies.
For example, by using a peptide of the EphB6 receptor, polyclonal
antisera or monoclonal antibodies can be made using standard
methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be
immunized with an immunogenic form of the peptide which elicits an
antibody response in the mammal. Techniques for conferring
immunmogenicity on a peptide include conjugation to carriers or
other techniques well known in the art. For example, the protein or
peptide can be administered in the presence of adjuvant. The
progress of immunization can be monitored by detection of antibody
titers in plasma or serum. Standard ELISA or other immunoassay
procedures can be used with the immunogen as antigen to assess the
levels of antibodies. Following immunization, antisera can be
obtained and, if desired, polyclonal antibodies isolated from the
sera.
[0077] To produce monoclonal antibodies, antibody producing cells
(lymphocytes) can be harvested from an immunized animal and fused
with myeloma cells by standard somatic cell fusion procedures thus
immortalizing these cells and yielding hybridoma cells. Such
techniques are well known in the art, (e.g., the hybridoma
technique originally developed by Kohler and Milstein (Nature 256,
495-497 (1975)) as well as other techniques such as the human
B-cell hybridoma technique (Kozbor et al., Immunol Today 4, 72
(1983)), the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy
(1985) Alen R Bliss, Inc., pages 77-96), and screening of
combinatorial antibody libraries (Huse et al., Science 246, 1275
(1989)). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with the peptide and
the monoclonal antibodies can be isolated. Therefore, the invention
also contemplates hybridoma cells secreting monoclonal antibodies
with specificity for the EphB6 receptor as described herein.
[0078] The term "antibody" as used herein is intended to include
fragments thereof which also specifically react with an EphB6
receptor, or peptide thereof, having the activity of the EphB6
receptor. Antibodies can be fragmented using conventional
techniques and the fragments screened for utility in the same
manner as described above. For example, F(ab')2 fragments can be
generated by treating antibody with pepsin. The resulting F(ab')2
fragment can be treated to reduce disulfide bridges to produce Fab'
fragments.
[0079] Chimeric antibody derivatives, i.e., antibody molecules that
combine a non-human animal variable region and a human constant
region are also contemplated within the scope of the invention.
Chimeric antibody molecules can include, for example, the antigen
binding domain from an antibody of a mouse, rat, or other species,
with human constant regions. Conventional methods may be used to
make chimeric antibodies containing the immunoglobulin variable
region which recognizes the gene product of EphB6 antigens of the
invention (See, for example, Morrison et al., Proc. Natl Acad. Sci
U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452 (1985),
Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No.
4,816,397; Tanaguchi et al., European Patent Publication EF171496;
European Patent Publication 0173494, United Kingdom patent GB
2177096B). It is expected that chimeric antibodies would be less
immunogenic in a human subject than the corresponding non-chimeric
antibody.
[0080] Monoclonal or chimeric antibodies specifically reactive with
a protein of the invention as described herein can be further
humanized by producing human constant region chimeras, in which
parts of the variable regions, particularly the conserved framework
regions of the antigen-binding domain, are of human origin and only
the hypervariable regions are of non-human origin. Such
immunoglobulin molecules may be made by techniques known in the
art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80,
7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983);
Olason et al., Meth. Enzymol., 92, 3-16 (1982)), and PCT
Publication WO92/06193 or EP 0239400). Humanized antibodies can
also be commercially produced (Scotgen Limited, 2 Holly Road,
Twickenham, Middlesex, Great Britain.)
[0081] Specific antibodies, or antibody fragments, reactive against
EphB6 receptor proteins may also be generated by screening
expression libraries encoding immunoglobulin genes, or portions
thereof, expressed in bacteria with peptides produced from the
nucleic acid molecules of the EphB6 receptor. For example, complete
Fab fragments, VH regions and FV regions can be expressed in
bacteria using phage expression libraries (See for example Ward et
al., Nature 341, 544-546: (1989); Huse et al., Science 246,
1275-1281 (1989); and McCafferty et al. Nature 348, 552-554
(1990)). Alternatively, a SCID-hu mouse, for example the model
developed by Genpharm, can be used to produce antibodies or
fragments thereof.
[0082] Antibodies specifically reactive with the EphB6 receptor, or
derivatives thereof, such as enzyme conjugates or labeled
derivatives, may be used to detect the EphB6 receptor in various
biological materials, for example they may be used in any known
immunoassays which rely on the binding interaction between an
antigenic determinat of the EphB6 receptor, and the antibodies.
Examples of such assays are radioimmunoassays, enzyme immunoassays
(e.g. ELISA), immunofluorescence, immunoprecipitation, latex
agglutination, hemagglutination and histocherrical tests. Thus, the
antibodies may be used to detect and quantify the EphB6 receptor in
a sample in order to determine its role in particular cellular
events or pathological states, and to diagnose and treat such
pathological states.
[0083] In particular, the antibodies of the invention may be used
in immuno-histochemical analyses, for example, at the cellular and
sub-subcellular level, to detect the EphB6 receptor, to localise it
to particular cells and tissues and to specific subcellular
locations, and to quantitate the level of expression.
[0084] Cytochemical techniques known in the art for localizing
antigens using light and electron microscopy may be used to detect
the EphB6 receptor. Generally, an antibody of the invention may be
labelled with a detectable substance and the EphB6 receptor may be
localised in tissue based upon the presence of the detectable
substance. Examples of detectable substances include various
enzymes, fluorescent materials, luminescent materials and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, biotin, alkaline phosphatase,
0-galactosidase, or acetylcholinesterase; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichaorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; and examples of suitable
radioactive material include radioactive iodine I-125, I-131 or
3-H. Antibodies may also be coupled to electron dense substances,
such as ferritin or colloidal gold, which are readily visualised by
electron microscopy.
[0085] Indirect methods may also be employed in which the primary
antigen-antibody reaction is amplified by the introduction of a
second antibody, having specificity for the antibody reactive
against the EphB6 receptor. By way of example, if the antibody
having specificity against the EphB6 receptor is a rabbit IgG
antibody, the second antibody may be goat anti-rabbit
gamma-globulin labelled with a detectable substance as described
herein.
[0086] Where a radioactive label is used as a detectable substance,
the EphB6 receptor may be localized by autoradiography. The results
of autoradiography may be quantitated by determining the density of
particles in the autoradiographs by various optical methods, or by
counting the grains.
[0087] Soluble proteins represent another class of substance that
may be used advantageously to modulate the activity of the EphB6
receptor. Soluble proteins can be prepared by a number of
conventional methodologies. GST fusion proteins of Eph receptor and
ephrin extracellular domains, or activated or inactive variants
thereof, can be created in the pGBX vector series (Pharmacia
Blotech, Uppsala). When the vectors containing the cDNAs are
transformed into bacteria by heat shock uptake, expression of the
GST fusion proteins can be induced with 1 mM IPTG. After growth
bacteria can be lysed by sonication and the addition of mild
detergents the resulting supernatant can be clarified by
centrifugation and the released GST-fusion proteins purified by
binding to glutathione-sepharose. After extensive washing these
complexes can be checked for purity and quantitated by reference to
standard proteins of similar molecular weight after staining with
coomassie blue. Alternatively fusions of the Eph or ephrin proteins
with MBP, His, thioHis, Fc, Myc tag, HA tag or other epitopes or
domains may be used to allow other purification procedures to be
utilized which may result in preferable activity of the purified
protein.
[0088] It would also be apparent to one skilled in the art that the
above described methods may be used to study the expression of the
EphB6 receptor and, accordingly, will provide further insight into
the role of the EphB6 receptor in cells.
Antisense Oligonucleotides
[0089] Antisense oligonucleotides that are complimentary to a
nucleic acid sequence from the EphB6 receptor can also be used in
the methods of the present invention to modulate the expression
and/or activity EphB6 receptors.
[0090] Accordingly, the present invention provides a method of
modulating the immune system by modulating the expression and/or
activity EphB6 receptors comprising administering an effective
amount of an antisense oligonucleotide that is complimentary to a
nucleic acid sequence from the EphB6 receptor to an animal in need
thereof.
[0091] The term "antisense oligonucleotide" as used herein means a
nucleotide sequence that is complimentary to its target.
[0092] The term "oligonudeotide" refers to an oligomer or polymer
of nucleotide or nucleoside monomers consisting of naturally
occurring bases, sugars, and intersugar (backbone) linkages. The
term also includes modified or substituted oligomers comprising
non-naturally occurring momars or portions thereof, which function
similarly. Such modified or substituted oligonucleotides may be
preferred over naturally occurring forms because of properties such
as enhanced cellular uptake, or increased stability in the presence
of nucleases. The term also includes chimeric oligonucleotides
which contain two or more chemically distinct regions. For example,
chimeric oligonucleotides may contain at least one region of
modified nucleotides that confer beneficial properties (e.g.
increased nuclease resistance, increased uptake into cells), or two
or more oligonucleotides of the invention may be joined to form a
chimeric oligonucleotide.
[0093] The antisense oligonucleotides of the present invention may
be ribonucleic or deoxyribonucleic acids and may contain naturally
occurring bases including adenine, guanine, cytosine, thymidine and
uracil. The oligonucleotides may also contain modified bases such
as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and
other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil,
6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil,
8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl
adenines, 8-hydroxyl adenine and other 8-substituted adenines,
8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl
guarines, 8-hydroxyl guanine and other 8-substituted guanines,
other aza and deaza uracils, thymidines, cytosines, adenines, or
guanines, 5trifluoromethyl uracil and 5-trifluoro cytosine.
[0094] Other antisense oligonucleotides of the invention may
contain modified phosphorous, oxygen heteroatoms in the phosphate
backbone, short chain alkyl or cycloalkyl intersugar linkages or
short chain heteroatomic or heterocyclic intersugar linkages. For
example, the antisense oligonucleotides may contain
phosphorothioates, phosphotriesters, methyl phosphonates, and
phosphorodithioates. In an embodiment of the invention there are
phosphorothioate bonds links between the four to six 3'-terminus
bases. In another embodiment phosphorothioate bonds link all the
nucleotides.
[0095] The antisense oligonucleotides of the invention may also
comprise nucleotide analogs that may be better suited as
therapeutic or experimental reagents. An example of an
oligonucleotide analogue is a peptide nucleic acid (PNA) wherein
the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA),
is replaced with a polyamide backbone which is similar to that
found in peptides (P. B. Nielsen, et al Science 1991, 254, 1497).
PNA analogues have been shown to be resistant to degradation by
enzymes and to have extended lives in vivo and in vitro. PNAs also
bind stronger to a complimentary DNA sequence due to the lack of
charge repulsion between the PNA strand and the DNA strand. Other
oligonucleotides may contain nucleotides containing polymer
backbones, cyclic backbones, or acyclic backbones. For example, the
nucleotides may have morpholino backbone structures (U.S. Pat. No.
5,034,506). Oligonucleotides may also contain groups such as
reporter groups, a group for improving the pharmacokinetic
properties of an oligonucleotide, or a group for improving the
pharmacodynamic properties of an antisense oligonucleotide.
Antisense oligonucleotides may also have sugar mimetics.
[0096] The antisense nucleic acid molecules may be constructed
using chemical synthesis and enzymatic ligation reactions using
procedures known in the art. The antisense nucleic acid molecules
of the invention or a fragment thereof, may be chemically
synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed with mRNA or the native gene e.g. phosphorothioate
derivatives and acridine substituted nucleotides. The antisense
sequences may be produced biologically using an expression vector
introduced into cells in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense sequences are
produced under the control of a high efficiency regulatory region,
the activity of which may be determined by the cell type into which
the vector is introduced.
[0097] The antisense oligonucleotides may be introduced into
tissues or cells using techniques in the art including vectors
(retroviral vectors, adenoviral vectors and DNA virus vectors) or
physical techniques such as microinjection. The antisense
oligonucleotides may be directly administered in vivo or may be
used to transfect cells in vitro which are then administered in
vivo. If one embodiment, the antisense oligonucleotide may be
delivered to macrophages and/or endothelial cells in a liposome
formulation.
Modulation of EphB6 Promoter
[0098] As would be readily apparent to those skilled in the art, it
is also possible to modulate EphB6 through manipulation of its
promoter. One or more alterations to a promoter sequence of the
EphB6 may increase or decrease promoter activity, or increase or
decrease the magnitude of the effect of a substance able to
modulate the promoter activity.
[0099] "Promoter activity" is used to refer to the ability to
initiate transcription. The level of promoter activity is
quantifiable for instance by assessment of the amount of mRNA
produced by transcription from the promoter or by assessment of the
amount of protein product produced by translation of mRNA produced
by transcription from the promoter. The amount of a specific mRNA
present in an expression system may be determined for example using
specific oligonucleotides which are able to hybridise with the mRNA
and which are labelled or may be used in a specific amplification
reaction such as the polymerase chain reaction.
[0100] Substances which affect the EphB6 promoter's activity may
also be identified using the methods of the invention by comparing
the pattern and level of expression of a reporter gene, in cells in
the presence, and in the absence of the substance. Accordingly a
method for assaying for the presence of an agonist or antagonist of
EphB6 promoter activity is provided comprising providing a cell
containing a reporter gene under the control of the promoter with a
substance which is a suspected agonist or antagonist under
conditions which permit interaction and assaying for the increase
or decrease of reporter gene product.
Apoptosis of Cells
[0101] Activation induced apoptosis (programmed cell death)
maintains homeostasis and immune tolerance by regulating the number
and type of antigen stimulated T-cells in circulation. Activation
induced cell death (ACID) can be provoked in antigen-stimulated
T-cells to eliminate potentially harmful cells and excessive
monotypes, thus preserving the functional balance of the immune
system, preventing autoimmune and lympho-proliferative disorders
(Park et al. (1997); Davis et al. (1994); Sakano et al.
(1996)).
[0102] The investigators demonstrate that stable overexpression of
the EphB6 receptor significantly enhances TCR-mediated apoptosis in
an ephrin-B1-dependent manner in the mature T-cell line Jurkat; a
commonly used model of pre-stimulated mature T cells in ACID
studies. Active T-cell apoptosis is driven by the antigen-induced
expression of the FASL and TNF death cytokines (Friedman et al.
(1996); Hombeeger et al. (1999); Gao et al. (1999); Clossek et al.
(1998)). The increased apoptosis observed in EphB6 overexpressing
cells appears to be due in part to increased TNF production.
Although TNF efficiently activates both the TNFR-I and TNFR-II
receptors, studies suggest that only TNFR-I is coupled to a caspase
cascade (Koziosky et al. (1995)) and thus may be the predominant
transmitter of the apoptic signal (Daniel et al. (1996); O'Leary et
al. (1999)). Expression of TNFR-II, but not TNFR-I, is suppressed
upon incubation of control and EphB6 overexpressing cells with
ephrin-B1. Although activation of the TCR overrides this effect in
control cells, overexpression of EphB6 maintains the
ephrin-B1-induced down regulation of TNFR-II. Anti-CD3 stimulation
of EphB6 overexpressing cells also reduces TNFR-II expression,
while it has no effect upon the receptor in control cells,
suggesting that the basal activity of overexpressed EphB6 receptor
is sufficient to make cells more sensitive to the induction of
apoptosis. The EphB6-induced imbalance in TNFR-I and TNFR-II
expression is interestingly similar to the situation observed in
the T-cells of aging humans, where TNFR-I is constitutively
expressed and TNFR-II is downregulated. These T-cells are
hypersensitive to TNF-induced apoptosis, which is probably
responsible for increasing T-cell deficiency in old-age (Pandey et
al. (1995)). Eph receptors could potentially be responsible for
this alteration in TNF receptor expression and modulation of their
activity could improve TNFR-II expression.
[0103] TNFR-I and TNFR-II employ only partially distinct signaling
pathways, both initiating the n-terminal JUN kinase cascade
(Kozlosky et al. (1995)). Activation of the JNK pathway is required
to protect cells from TNF-mediated apoptosis (Adams et al. (1999),
Wang et al. (1998)). Overexpression of EphB6 strongly inhibits
long-term anti-CD3 induced JNK stimulation. This effect is highly
specific, several other potentially anti-apoptic pathways,
including Akt activation and Bcl-2 expression, are not affected.
The elimination of JNK-JUN signaling reportedly enhances
TNF-induced apoptosis (Adams et al. (1999); Wang et al. (1998)),
suggesting that selective inhibition of the JUN kinase pathway
could further the promotion of AICD by EphB6.
[0104] Thus, and while not wishing to be bond by any particular
theory, the increase in inducible programmed cell death in EphB6
overexpressing cells can be attributed at least in part to
increased TNF production, complemented by an alteration in the
balance between TNFR-I and TNFR-II expression to favor the
pro-apoptic TNFR-I. Accordingly, the present invention provides a
method of regulating the immune system, preferably regulating
lymphocyte apoptosis, preferably AICD, by providing an effective
amount of a substance capable of modulation of EphB6 and its active
partners, thereby modulating the immune system.
[0105] The high level of EphB6 expression in thymocytes also
suggests that EphB6 may play an important role in vivo in the
negative and positive selection of thymocytes, regulating the
induction of the apoptic pathway in cells that fall to be
positively selected. Failure to properly regulate negative
selection can lead to the emergence of auto-reactive T-cells in the
periphery leading to the development of autoimmune diseases. In the
peripheral blood, failure to eliminate activated T-cells may result
in T-cell lymphoproliferative disorders or auto-immune disorders as
the result of an inability to eliminate self reactive T-cells.
Therapeutic Uses
[0106] As just discussed, the EphB6 receptor of the invention is
likely involved in the regulation of cell signalling pathways that
control cell death. Accordingly, the present invention provides a
method of modulating cell death or apoptosis comprising
administering to a cell or animal in need thereof, an effective
amount of a substance that modulates EphB6, in order to modulate
the cell death. Examples of substance which may be used to modulate
EphB6 Include antibodies, soluble EphB6, soluble ephrins, antisense
nucleic adds, organic substances that modulate the interaction of
EphB6 with ligands and active partners either alone or in
combination. The term "effective amount" as used herein means an
amount effective, at dosages and for periods of time necessary to
achieve the desired results.
[0107] In another aspect the present invention provides a method of
modulation of cell proliferation. In one embodiment, the invention
provides a method of inhibiting or reducing cell proliferation,
such as in neoplasia, by administering to a cell or animal an
effective amount of an agent that promotes the expression or the
biological activity of the EphB6 receptor or its active Eph
partners, such that there is an inhibition or reduction in cell
prolifereation.
[0108] In another embodiment, the present invention provides a
method of inducing cell proliferation by administering to a cell or
an animal an effective amount of a substance that inhibits the
expression or the biological activity of the EphB6 receptor, or
blocks the phosphorylation of the said receptor or its active Eph
partners, such that there is an induction of cell proliferation.
Substances that inhibit the activity of the EphB6 receptor include
antibodies to EphB6 receptor. Substances that inhibit the
expression of the EphB6 gene include antisense oligonucleotides to
an EphB6 receptor nucleic acid sequence.
[0109] In addition to antibodies and antisense oligonucleotides,
other substances that modulate EphB6 receptor expression or
activity may also be identified, as well as substances that block
the phosphorylation of EphB6. Substances that affect EphB6 receptor
activity can be identified based on their ability to bind to the
EphB6 receptor.
[0110] Substances which can bind with the EphB6 receptor of the
invention may be identified by reacting the EphB6 receptor with a
subsequence which potentially binds to the EphB6 receptor, and
assaying for complexes, for free substance, or for nonomplexed
EphB6 receptor, or for activation of the EphB6 receptor. In
particular, a yeast two hybrid assay system may be used to identify
proteins which interact with the EphB6 receptor (Fields, S. and
Song, O., 1989, Nature, 340:245-247) or a ligand binding or ligand
replacement assay system (Blechman, J. M. et al. (1993); Blechman,
J. M. et al. (1995); Lev et al. (1993)). Systems of analysis which
also may be used include ELISA, BIAcore(Bartley, T. D., et al.
(1994)).
[0111] A protein ligand for the Eph receptors can be isolated by
using the extraceulular domain of the receptor as an affinity
reagent. Concentrated cell culture supernatants can be screened for
receptor binding activity using immobilied receptor in a surface
plasmon resonance detection system (BIAcore). Supernatants from
selected cell lines can then be fractionated directly by receptor
affinity chromatography.
[0112] Conditions which permit the formation of substance and EphB6
receptor complexes maybe selected having regard to factors such as
the nature and amounts of the substance and the protein.
[0113] The substance-protein complex, free substance or
noncomplexed proteins may be isolated by conventional isolation
techniques, for example, salting out, chromatography,
electrophoresis, gel filtration, fractionation, absorption,
polyacrylamide gel electrophoresis, agglutination, or combinations
thereof. To facilitate the assay of the components, antibody
against the EphB6 receptor or the substance, or labelled EphB6
receptor, or a labelled substance may be utilized. The antibodies,
proteins, or substances may be labelled with a detectable substance
as described above.
[0114] Substances which bind to and activate the EphB6 receptor of
the invention may be identified by assaying for phosphorylation of
the tyrosine residues of the protein.
[0115] Substances which bind to and inactivate the EphB6 receptor
of the invention may be identified by assaying for reduction in
phosphorylation of the protein.
[0116] The EphB6 receptor, or the substance used in the method of
the invention may be insolubilized. For example, the EphB6 receptor
or substance may be bound to a suitable carrier. Examples of
suitable carriers are agarose, cellulose, dextran, Sephadex,
Sepharose, carboxymethyl cellulose polystyrene, filter paper,
ionexchange resin, plastic film, plastic tube, glass beads,
polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid
copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The
carrier may be in the shape of, for example, a tube, test plate,
beads, disc, sphere etc.
[0117] The insolubilized protein or substance may be prepared by
reacting the material with a suitable insoluble carrier using known
chemical or physical methods, for example, cyanogen broxnide
coupling.
[0118] The proteins or substance may also be expressed an the
surface of a cell using the methods described herein.
[0119] The invention also contemplates a method for assaying for an
agonist or antagonist of the EphB6 receptor. The agonist or
antagonist may be an endogenous physiological substance or it may
be a natural or synthetic substance. Substances that are capable of
binding the EphB6 receptor may be identified using the methods set
forth herein.
[0120] The invention also contemplates assaying for an antagonist
or agonist of the EphB receptor and its active partner or partners
preferably an Eph receptor.
[0121] It will be understood that the agonists and antagonists that
can be assayed using the methods of the invention may act on one or
more of the binding sites on the protein or substance including
agonist binding sites, competitive antagonist binding sites,
non-competitive antagonist binding sites or allosteric sites.
[0122] The invention also makes it possible to screen for
antagonists that inhibit the effects of an agonist of the EphB6
receptor or its active partners. Thus, the invention may be used to
assay for a substance that competes for the same binding site of
the EphB6 receptor or its active partners.
[0123] The methods described above may be used to identify a
substance which is capable of binding to an activated EphB6
receptor or its active partners, and to assay for an agonist or
antagonist of the binding of activated EphB6 receptor or its
partners, with a substance which is capable of binding with
activated EphB6 receptor or its partners. An activated, (i.e.
phosphorylated) the EphB6 receptor may be prepared using the
methods described (for example in Reedijk et al. The EMBO Journal
11(4):1365, 1992) for producing a tyrosine phosphorylated
protein.
[0124] It will also be appreciated that intracellular substances
which are capable of binding to EphB6 or its active partners may be
identified using the methods described herein.
[0125] The invention further provides a method for assaying for a
substance that affects an EphB6 receptor regulatory pathway
comprising administering to a human or animal or to a cell, or a
tissue of an animal, a substance suspected of affecting a EphB6
receptor regulatory pathway, and quantitating the EphB6 receptor or
nucleic acids encoding the EphB6 receptor, or examining the pattern
and/or level of expression of EphB6 receptor, in the human or
animal or tissue, or cell EphB6 receptor may be quantitated and its
expression may be examined using the methods described herein.
[0126] The substances identified by the methods described herein
may be used for modulating EphB6 receptor regulatory pathways and
accordingly may be used in the treatment of conditions involving
perturbation of EphB6 receptor signaling pathways. In particular,
the substances may be particularly useful in the treatment of
disorders of cell death.
[0127] As stated previously, EphB6 receptor may be involved in
modulating cell proliferation and stimulators and inhibitors of the
EphB6 receptor may be useful in modulating disorders involving cell
proliferation such as neoplasia and autoimmunity, such as for
example, substances that stimulate the EphB6 receptor (for example,
identified using the methods of the invention) may be used to
stimulate cell death or apoptosis, and inhibitors could be used
where an increase in T cell proliferation would be
advantageous.
Peptide Mimetics
[0128] The present invention also includes peptide minetics of the
EphB6 receptor of the invention. For example, a peptide derived
from a binding domain of an EphB6 protein will interact directly or
indirectly with an associated molecule in such a way as to mimic
the native binding domain. Such peptides may include competitive
inhibitors, enhancers, peptide mimetics, and the like. All of these
peptides as well as molecules substantially homologous,
complementary or otherwise functionally or structurally equivalent
to these peptides may be used for purposes of the present
invention.
[0129] "Peptide mimetics" are structures which serve as substitutes
for peptides in interactions between molecules (See Morgan et al
(1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide
mimetics include synthetic structures which may or may not contain
amino acids and/or peptide bonds but retain the structural and
functional features of a peptide, or enhancer or inhibitor of the
invention. Peptide mimetics also include peptoids, oligopeptolds
(Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide
libraries containing peptides of a designed length representing all
possible sequences of amino acids corresponding to a peptide of the
invention.
[0130] Peptide mimetics may be designed based on information
obtained by systematic replacement of L-amino acids by D-amino
acids, replacement of side chains with groups having different
electronic properties, and by systematic replacement of peptide
bonds with amide bond replacements. Local conformational
constraints can also be introduced to determine conformational
requirements for activity of a candidate peptide mimetic. The
mimetics may include isosteric amide bands, or Daminuo acids to
stabilize or promote reverse turn conformations and to help
stabilize the molecule. Cyclic amino acid analogues may be used to
constram amino acid residues to particular conformational states.
The mimetics can also include mimics of inhibitor peptide secondary
structures. These structures can model the 3-dimensional
orientation of amino acid residues into the known secondary
conformations of proteins. Peptoids may also be used which are
oligomers of N-substituted amino adds and can be used as motifs for
the generation of chemically diverse libraries of novel
molecules.
[0131] Peptides of the invention may also be used to identify lead
compounds for drug development. The structure of the peptides
described herein can be readily determined by a number of methods
such as NMR and X-ray crystallography. A comparison of the
structures of peptides similar in sequence, but differing in the
biological activities they elicit in target molecules can provide
information about the structure-activity relationship of the
target. Information obtained from the examination of
structure-activity relationships can be used to design either
modified peptides, or other small molecules or lead compounds which
can be tested for predicted properties as related to the target
molecule. The activity of the lead compounds can be evaluated using
assays similar to those described herein.
[0132] Information about structure-activity relationships may also
be obtained from co-crystallization studies. In these studies, a
peptide with a desired activity is crystallized in association with
a target molecule, and the X-ray structure of the complex is
determined. The structure can then be compared to the structure of
the target molecule in its native state, and information from such
a comparison maybe used to design compounds expected to
possess.
[0133] The invention also makes it possible to screen for
antagonists that inhibit the effects of an EphB6 receptor. Thus,
the invention may be used to assay for a substance that
anatagonizes or blocks the action of the receptor.
[0134] The invention further provides a method for assaying for a
substance that affects the EphB6 receptor, comprising administering
to a nonhuman animal or to a tissue of an animal, a substance
suspected of affecting the activity or action of the receptor and
quantitating the effect on CD25 expression in the human animal or
tissue. CD25 may be quantitated and its expression may be examined
using the methods described herein.
[0135] Substances identified by the methods described herein, may
be used for modulating EphB6 receptor activity or action and
accordingly may be used in the treatment of conditions involving
perturbation of the protein. In particular, the substances may be
particularly useful in the treatment of disorders of T-cell
proliferation. In addition, the application of a proper combination
of inhibitory or stimulatory soluble ligand or soluble receptors
should prevent T lymphocyte-target cell interaction and decrease
host reaction versus transplant, thus inhibiting transplant
rejection. As well by virtue of the methods and substances of the
present invention, the employment of inhibitory or stimulatory
soluble ligands and soluble receptors may be used for treatment or
slowing of autoimmune disorders. Such autoimmune disorders may
include cell-associated autoimmunities such as multiple sclerosis,
lupus, arthritis, thyroiditis, diabetes, psoriasis and Crohn's
disease and colitis. In addition, the methods and substances may be
used to treat allergic disorders such as asthma and hyper-IgE and
eosinophilic syndromes and T-cell dependent graft-verus-host
reactions. As well, by virtue of the substances and methods
described herein, soluble stimulatory or inhibitory ephrins and
soluble receptors could promote both T lymphocyte adhesion and T
cell response to infected cells, thus accelerating and increasing
anti-viral immune response.
[0136] It is also envisaged that the DNA sequences of the EphB6
receptor or its active partners might be determined in order to
assay for changes, preferably disease-causing mutations that may be
used as indicators of disease prognosis or as aids to inform
treatment of these diseases.
Pharmaceutical Compositions
[0137] The above described substances may be formulated into
pharmaceutical compositions for adminstration to subjects in a
biologically compatible form suitable for administration in vivo.
By "biologically compatible form suitable for administration in
vivo" is meant a form of the substance to be administered in which
any toxic effects are outweighed by the therapeutic effects. The
substances may be administered to living organisms including
humans, and animals.
[0138] Administration of a therapeutically active amount of
pharmaceutical compositions of the present invention is defined as
an amount effective, at dosages and for periods of time necessary
to achieve the desired result. For example, a therapeutically
active amount of a substance may vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of the substance to elicit a desired response in the
individual. Dosage regima may be adjusted to provide the optimum
therapeutic response. For example, several divided doses may be
administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation.
[0139] An active substance may be administered in a convenient
manner such as by injection (subcutaneous, intravenous, eta), oral
administration, inhalation, transdermal application or rectal
administration. Depending an the route of administration, the
active substance may be coated in a material to protect the
compound from the action of enzymes, acids and other natural
conditions which may inactivate the compound. If the active
substance is a nucleic acid encoding, for example, a modified EphB6
receptor it may be delivered using techniques known in the art.
[0140] The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an
effective quantity of the active substance is combined in a mixture
with a pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example, in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985) or Handbook of Pharmaceutical Additives
(compiled by Michael and Irene Ash, Gower Publishing Limited,
Aldershot, England (1995)). On this basis, the compositions
include, albeit not exclusively, solutions of the substances in
association with one or more pharmaceutically acceptable vehicles
or diluents, and may be contained in buffered solutions with a
suitable pH and/or be isoosmotic with physiological fluids. In this
regard, reference can be made to U.S. Pat. No. 5,843,456. As will
also be appreciated by those skilled, administration of substances
described herein may be by an inactive viral carrier.
Experimental Models
[0141] The invention also provides methods for studying the
function of EphB6 and its impact on cells of the immune system.
[0142] The following non-limiting examples are illustrative of the
present invention:
EXAMPLES
General Methods for Examples 1-5
Antibodies and Recombinant Proteins.
[0143] Monoclonal anti-phosphotyrosine was obtained from Upstate
Biotechnology, Inc. (Lake Placid, N.Y.). Antibodies to EphB6, MYC,
Zap-70 and LCK were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, Calif.). Soluble dimerized Ephrin-B1 and soluble EphB6
receptors were purchased from R&D Systems. Anti-human CD3 was
purchased from Serotec (UK) and anti-T7 from Novagen.
Immunoprecipitation and Western Blotting
[0144] Cells were quickly resuspended in ice cold lysis buffer
consisting of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1%
Triton X-100, 1 mM ethylene glycol-bis
(.beta.-aminoethylether)-N,N,N'-N'-tetraacetic acid (EGTA), 10
.mu.g/ml leupeptin, 10 .mu.g/ml aprotinin, 1 mm PMSF, 1 mM
Na-orthovanadate and 50 mM NaF. after solubilization on ice for 15
minutes, debris was removed by centrifugation at 12,000 g for 10
minutes at 4.degree. C. antibodies and 20 .mu.l of 50% protein G
sepharose were added to cleared lysates and incubated at 4.degree.
C. with constant shaking for 12-16 hours. Immunoprecipitates were
collected by a brief centrifugation and washed 3-4 times in lysis
buffer (without PMSF) before addition of SDS sample buffer. Samples
were separated on SDS-polyacrylamide gels and transferred to
nitrocellulose membranes (Amersham, Arlington Heights, Ill.).
Membranes were blocked overnight at 4.degree. C. with 7% blotting
grade non-fat milk (Biorad, Richmond, Calif.) in PBS.
Immunoblotting antibodies were added at optimal dilutions in PBS-T
(0.1% Tween-20) and incubated at 4.degree. C. overnight. After
extensive washing with PBS-T, bound antibodies were detected using
horseradish-peroxidase conjugated donkey anti-rabbit or sheep
anti-mouse antibodies (Amersham, Arlington Heights, Ill.) and
lumiglo chemiluminescent reagents (Kirkegaard and Perry,
Mass.).
Kinase Assays.
[0145] Kinase immunoprecipitates were prepared as above in 1%
Triton X-100 lysis buffer, given one wash in kinase buffer before
incubation in 50 .mu.l of kinase buffer (20 mM HEPES pH 7.6, 10 mM
MgCl.sub.2) in the presence of 4 .mu.g of the synthetic substrate
peptide raytide EL (Oncogene) and .gamma.[.sup.33p]-ATP for 15 min
at room temperature. The kinase buffer containing the labeled
peptide was collected and loaded onto phosphocellulose paper discs.
The paper was washed 3 times with 0.5% phosphoric acid to remove
unncorporated .sup.33P-ATF and one with acetone, dried and counted
in a .beta.-counter. Results are shown in arbitrary units and each
represents one of four independent experiments. The presence of Lck
was determined by immunoblotting lck immunoprecipitates run an
non-reducing SDS page with anti-Lck (not shown).
Subcloning and Mutation of ZAP-70, Cbl, EphB6 and EPHB1
[0146] cDNAs for Zap-70, Cbl EphB1, EphB6, ephrin-A1 and ephrin-B1
were cloned from normal human thymocyte RNA by RT-PCR into the
expression vector pcDNA3 (Invitrogen, Calif.) and sequenced.
Mutants of these molecules (Zap:Y493P, Cbl G306E, 70-Z) (EphB1:
truncation of 102 C-terminal amino acids, kinase-null K651Q)
(EphB6-DN: deletion of cytoplasmic tail) were created using the
overlapping PCR technique to introduce the required base changes,
using cloned cDNAs as the template. Kinase-null B1 was created by
mutating lysine 651 to glutamine (K651Q). The resulting cDNAs were
cloned and sequenced to confirm the mutations. Myc-tagged versions
of EphB6 and of the truncated EphB1 receptor were generated by
insertion of a Myc tag and construts verified by sequencing.
Expression of wild type proteins and mutants were examined by
transfection in COS-7 cells and western blotting with appropriate
antibodies. All mutations were expressed as well as respective wild
types. The truncated form of EphB1 was an active kinase, like the
wild type protein. Kinase-null EphB1 had no detectable kinase
activity.
Transfection of Cell Lines.
[0147] Adherent COS-7, HEK 293 and NIH 3T3 cells were routinely
transiently transfected using the lipid reagent lipofectamine (Life
Technologies, Grand Island, N.Y.). The DNA-lipid mixtures were
applied to the cells for 5 hours in the absence of serum before the
addition of complete medium. Cells were given 72 hours to express
the transfected proteins before harvest.
[0148] To raise stable EphB6 overexpressing cells, the mature human
T-cell line jurkat was transfected with empty pcDNA3, EphB6-M, or
DN-EphB6. The jurkat cells were electroporated in 400 .mu.l
complete RPMI medium with 30 .mu.g of DNA by pulsing once for 65
msec at 260V (BTK electro square porator, BTX, division of
Genetronics Inc, San Diego, Calif.). Cells were incubated at
37.degree. C. for 24 hours before addition of G418 to the medium.
After 30 days of selection the resulting oligoclonal cell
populations were screened by immunoprecipitation with anti-MYC and
western blotting with anti-myc or anti-EphB6 and the highest EphB6
expressing cell population (B6-J) selected.
Isolation of Truman Thymocymes.
[0149] Thymuses were obtained from children undergoing open heart
surgery. Mononuclear cells were isolated by Ficoll-hypaque gradient
centrifugation. Adherent cells were removed by incubation to
plastic dishes for 60 minutes at 37.degree. C. The resulting
thymocytes are typically >95% CD3.
Stimulation of EphB6 Receptor Transfected Cells with Membrane Bound
and soluble Ligand.
[0150] To assay for stimulation with membrane bound forms of the
ephrin ligands, receptor-expressing cells were resuspended using 25
mM EDTA and after washing, overlaid on a confluent monolayer of
control or ligand-expressing cells. After incubation at 3M for 1
hour, all the cells were solubilized in 1% Triton lysis buffer.
Soluble ephrin-B1-Fc fusion-protein dimer was purchased from
R&D Systems (Minneapolis, Minn.). The dimeric ephrin-B1 fusion
protein was pre-complexed with F(ab)'.sub.2goat anti-human Fc
(pierce) to form oligomers. F(ab)'.sub.2 goat anti-human Fc was
used as a control (no stimulation) where necessary. Although murine
ephrin-B1 was utilized, this effectively induced human EphB6
phosphorylation.
Analaysis of CD25 Expression by Flow Cytometry.
[0151] Cells were incubated in 0.5% seram for 24 hours with or
without 54 .mu.g/ml soluble oligonerized or immobilized ephrin-B1
and immobilized anti-CD3 antibody. Anti-human-CD19 antibody was
used as an irrelevant protein control for immobilized ephrin-B1
where necessary. The expression of CD25 was then analyzed by
staining with pre-labeled anti-CD25 and isotype control
(Immunotech).
Example 1
[0152] To determine if the catalytically inactive EphB6 receptor
could be tyrosine phosphorylated in response to ephrin-B ligand
stimulation, we transiently expressed human EphB6 in COS-7 cells
and exposed those cells to ephrin-B1. The EphB6 receptor was
expressed as a C-terminal myc-tagged protein (EphB6-M). To provide
cell surface expressed ligands, we transfected COS-7 cells with
pcDNA3 expression vector containing either ephrin-A1 or ephrin-B1
cDNA. Ligand expression was verified by immunoblotting (not shown).
EphB6 receptor expressing cells were overlaid on cells transfected
with ephrin-B1, ephrin-A1 or empty vector, and coincubated for an
hour at 37.degree. C. The EphB6 receptor was then precipitated with
anti-myc and immunoblotted with anti-phosphotyrosine antibody.
Stimulation of EphB6 with ephrin-B1-expressing cells resulted in a
major increase in EphB6 tyrosine phosphorylation, while
co-incubation with ephrin-A1-expressing or control cells had no
effect (FIG. 1A). The increase in EphB6 receptor tyrosine
phosphorylation caused by co-incubation with ephrin-B1-expressing
cells was also observed upon transfection of NIH 3T3 fibroblasts
and HRK 293 human embryonic kidney cells (FIG. 1B), indicating the
effect was not cell specific. Stimulation of EphB6 receptor
tyrosine phosphorylation was both time and ligand concentration
dependent (FIGS. 1C,D).
[0153] In contrast to soluble monomers of ephrin, which can inhibit
Eph receptor signaling, dimerized or oligomerezed forms can
stimulate receptor autophosphorylation and signaling (Davis et al.
(1994); Sakano et al. (1996)). A soluble dimerized form of the
ephrin-B1 ligand was also found to induce EphB6 phosphorylation.
Although recombinant murine ephrin-B1 was utilized, it induced
EphB6 phosphorylation as effectively as membrane expressed human
ephrin-B1. Moreover, this epluin-B1 induced phosphorylation could
be completely inhibited by the addition of soluble EphB6 receptor
to the medium (FIG. 1B), strongly suggesting the existence of a
direct interaction between ephrin-B1 and EphB6 receptor.
Example 2
[0154] To demonstrate that EphB6 is trans-phosphorylated upon
hetero-oligomerization with catalytically active members of the Eph
family, EphB6 receptor was coexpressed with human EphB1 receptor in
COS-7 cells. The EphB1 receptor was found to be constitutively
activated when overexpressed. EphB6 underwent significant tyrosine
phosphorylation upon coexpression with the EphB1 receptor,
trans-phosphorylated in a manner analogous to ErbB-3 (FIG. 2A). In
contrast, catalytically inactive EphB1 (K651Q, B1-KD) was unable
induce EphB6 phosphorylation (FIG. 2b). In NIH 3T3 fibroblasts,
where the basal activity of EphB1 was determined to be much lower
than in 293 or COS-7 cells, EphB6 trans-phosphorylation occurred in
a ligand dependent manner (FIG. 2C).
[0155] As EphB1 and EphB6 have essentially the same electrophoretic
mobility, the observed phosphorylation of EphB6 could, however, be
due to co-precipitating phosphorylated EphB1 in this
over-expressing system. Therefore, to unambiguously distinguish
between the two receptors, a myc-tagged truncated EphB1 receptor
lacking 102 C-terminal residues was constructed, but with its
kinase domain intact (B1-Tr). Like the wild type receptor,
truncated EphB1 was constitutively tyrosine phosphorylated, but now
clearly smaller than EphB6. Co-expression of truncated EphB1 also
resulted in dramatically increased EphB6 phosphorylation (FIG.
2D,E), demonstrating that EphB6 phosphorylation can be provided by
a catalytically active EphB receptor and suggesting that ephrin-B1
induced EphB6 phosphorylation may similarly result from
trans-phosphorylation.
Example 3
[0156] In RT-PCR analysis, we detected EphB6 expression in human
thymocytes as well as in mature peripheral blood T cells and in the
T cell line Jurkat (FIG. 3). Two catalytically active members of
the Eph family, EphB1 and EphB2, were also found to be expressed
throughout the T cell lineage, while EphA2 could only be detected
in thymocytes. The persistent expression of EphB6 across the T cell
lineage suggested it might be important both during differentiation
and in mature T cell function.
[0157] Single cell suspensions of human thymocytes were stimulated
with anti-CD3 for 10 minutes, the receptor immunoprecipitated with
anti-EphB6 antibodies and blotted with anti-phosphotyrosine.
Polyclonal antibodies to EphB6 were raised against a peptide from
the extreme C-terminal of EphB6, a unique sequence not present in
any other known Eph receptor (see Experimental Procedures). This
efficiently immunoprecipitated and Western blotted myc-tagged EphB6
(not shown). While tyrosine phosphorylation of the EphB6 receptor
itself was not detected in response to anti-CD3 stimulation, a
tyrosine phosphorylated protein of approximately 115 kDa (pp115)
was co-precipitated (FIG. 4A).
[0158] pp115 was the only highly tyrosine phosphorylated protein
consistently associated with EphB6 and remained for at least 20
minutes after anti-D3 stimulation (FIG. 4B). The electrophoretic
mobility of pp115 appeared similar to that of the c-Cbl
proto-oncogene, which is highly phosphorylated after TCR/CD3
stimulation (Tsygankov et al. (1996)) (FIG. 4C). Western blotting
with anti-EphB6 revealed the presence of a band of the expected
molecular weight in Cbl immunoprecipitates, but not in
immnunoprecipitates of FAK or Vav (FIG. 4D). Pre-immune serum
control blotting was also negative. This association was not
noticeably altered by addition of anti-CD3, indicating that TCR/CD3
stimulation primarily induced Cbl phosphorylation, rather than
increasing its recruitment to the EphB6 receptor. Cbl is central to
signaling pathways from many receptors, functioning as a regulator
of receptor tyrosine kinase activity, through initiation of
receptor ubiquitination, and inducibly binding a variety of signal
transducing molecules (Tsygankov et al. (1996); Pournel et al.
(1996); Lupher et al. (1996); Lupher et al. (1997); Lupher et al.
(1998); Ota et al. (1997); Thien et al. (1999); van Leeuwen (1999);
Lee et al. (1999); Levkowitz et al. (1998); Miyake et al.
(1998)).
[0159] This was confirmed by co-expressing human Cbl with either
the EphB6 or EphB1 receptor in COS-7 cells. Cbl appeared to
specifically co-precipitate with EphB6, as association with the
catalytically active receptor EphB1 could not be detected (FIG.
4E). Stimulation with ephrin-B1 expressing cells did not alter the
level of EphB6-Cbl association, nor Cbl tyrosine phosphorylation
(not shown). To further characterize this interaction, the binding
of two mutants of Cbl to EphB6 were examined. The first, G306E
(Cbl*) was initially identified as a mutation in the C.elegans Cbl
orthologue, sli-1 (ongeward et al. (1995)) and causes loss of Cbl
binding to the ErbB1 and PDGF receptors by disruption of the Cbl
phosphotyrosine binding domain (Bonita et al. (1997); Thien et al.
(1997)). The second, 70-Z Cbl (Cbl**), isolated as an oncogene from
a murine B cell line (Blake et al. (1991)), contains an internal 17
amino acid deletion in the Cbl RING finger domain. While the 70-Z
mutation only slightly decreased Cbl binding to the EphB6 receptor,
the G306H point mutation completely abolished assocation (FIG. 4P);
confirming the specificity of binding and drawing a parallel
between Cbl binding to the EGP and PDGF receptors and its
association with EphB6.
Example 4
[0160] Co-expression of Zap-70 with EphB6 or EphB1 receptors in
COS-7 cells revealed a selective downregulation of Zap-70
phosphorylation by EphB6 (FIG. 5A). The EphB6 receptor inhibited
Zap-70 tyrosine phosphorylation, while no significant change was
observed upon EphB1 co-expression. This effect was ligand
responsive, as a further decrease in Zap-70 phosphorylation occured
upon incubation of EphB6 co-transfected cells with ephrin-B1
expressing cells (FIG. 5A). The induction of EphB6 receptor
tyrosine phosphorylation by ephrin-B1 probably contributes to the
inhibition of Zap-70 through increased recruitment of effector
proteins to the receptor.
[0161] Stimulation of the TCR complex leads to Zap-70 linase
phosphorylation by the Fyn and Lck arc-like tyrosine kinases and
subsequent Zap-70 activation, primarily through phosphorylation of
tyrosine residue 493 in the Zap-70 catalytic domain (Wange et al.
(1995) Mege et al. (1996); Kong et al. (1996)). The removal of Y493
results in a level of Zap-70 phosphorylation essentially reflecting
its own basal kinase activity. A Y493F Zap-70 mutant (Zap*) was
constructed and while demonstrating significantly lower tyrosine
phosphorylation than wild type, Y493F Zap-70 was unaffected by
EphB6 co-expression (FIG. 5B). This suggested that EphB6 might
specifically affect phosphorylation of tyrosine residues
characteristic of activated Zap-70.
[0162] The ability of EphB6 to alter signaling in T cells was
demonstrated as follows. Stable overexpression of the myc-tagged
EphB6 receptor in the mature T cell line Jurkat (B6-J) (FIG. 5C)
was established. The transfected EphB6 receptor appeared to be
functional, undergoing tyrosine phosphorylation upon stimulation of
transfected T-cells with ephrin-B1 (FIG. 5D). TCR surface
expression on control and B6-J cells was found to be equivalent by
staining with anti CD3.epsilon.-FITC.
[0163] Zap-70 was immunoprecipitated from control and EphB6
transfected T cells and its phosphorylation status examined by
Western blotting. In agreement with our previous results,
expressing Y493F Zap-70 in 293 cells, the basal phosphorylation of
Zap-70 was not significantly affected by EphB6 overexpression.
However, the induction of Zap-70 phosphorylation in response to
TCR/CD3 stimulation was strongly inhibited (FIG. 5E). The amount of
phosphorylated CD3.zeta. chain associated with Zap-70 was also
decreased by EphB6 overexpression (FIG. 5F). The src-family kinase
Lck is primarily responsible for phosphorylation of the CD3 chain
upon TCR stimulation and subsequenttly regulates Zap-70 recruitment
to the CD3 receptor complex, in addition to its activation of
Zap-70 by direct phosphorylation Lck activation by the CD45
phosphatase is one of, if not the, earliest events following TCR
ligation and Lck kinase activity was significantly elevated after 5
minutes anti-CD3 stimulation of the control Jurkat cells. However,
Lck activation was constitutively inhibited in EphB6 overexpressing
B6-J cells (FIGS. 6A,B). Ephrin-B1 treatment of anti-CD3 stimulated
control cells also partially inhibited Lck activation, but had no
further effect upon Lck in EphB6 overexpressing cells, either
alone, or in then presence of anti-CD3. The absence of ligand
effect in B6-J cells suggests that the basal activity of
overexpressed EphB6 alone is sufficient to prevent Lck activation.
In sum, these results indicate that the EphB6 receptor modulates
TCR signaling, by regulating the tyrosine phosphorylation and
activity of TCR-associated kinases.
Example 5
[0164] The EphB6 receptor could downregulate both Lck and Zap-70
kinases, suggesting that EphB6 inhibition of TCR mediated CD25
upregulation was demonstrated as follows. pcDNA3 control and 5J
Jurkat cells were stimulated with anti-CD3 and the EphB6 ligand
ephrin-B1. The ligand had no effect upon resting control cells, and
demonstrated only a small and variable inhibition of TCR mediated
CD25 upregulation (see FIG. 7A). In contrast, overexpression of
EphB6, although variably affecting the basal level of CD25
expression, completely inhibited the ability of TCR stimulation to
induce CD25 upregulation (FIG. 7B). In parallel with the ability of
EphB6 to inhibit lck activation, no further inhibition of CD25
expression was observed upon addition of the ephrin-B1 ligand.
[0165] EphB6 is naturally highly expressed in thymocytes and when
induction of CD25 in response to TCR activation was examined in
these cells, ephrin-B1 co-stimulation caused a strong inhibition of
CD25 upregulation, while ephrin-B1 alone had little effect (see
FIG. 8C).
[0166] To confirm this role of endogenous EphB6, Jurkat cell lines
overexpressing a dominant-negative form of EphB6, namely
eliminating the cytoplasmic domain of the receptor (DN-J) were
created. In constrast to overpression of wild type EphB6, the
dominant-negative receptor did not prevent anti-CD3 mediated
induction of CD25 expression and a further enhancement of anti-CD3
induced upregulation of CD25 was observed upon ephrin-B1
co-stimulation; presumably due to the removal of inhibitory input
from the endogenous EphB6 receptor (see FIGS. 8A and 8B).
[0167] In summary, the results from Examples 1-5 provide support
for methods of modulating T cells by suppressing antigen induced
CD25 expression through manipulation of EphB6 receptors. The
following Examples 6-8 further support methods of modulating the
immune system through manipulation of EphB6 by demonstrating that
modulation of EphB6 provides a method to modulate antigen induced
cell death (AICD).
Discussion of Examples 1-5
[0168] Although the structure of EphB6 is typical of the EphB
receptors, its kinase domain contains numerous alterations to
critical catalytic residues and neither murine nor human EphB6
demonstrates kinase activity (Gurniak et al. (1996); Matsuoka et
al. (1997)). Despite these structural abnormalities the human EphB6
receptor responds to ephrin-B1 stimulation by undergoing tyrosine
phosphorylation. Further, the EphB6 tyrosine phosphorylation can be
provided by a catalytically active partner, in particular, by the
EphB1 receptor. While EphB1 receptor can trans-phosphorylate EphB6
upon co-transfection, in vivo EphB6 may potentially interact with
multiple members of the EphB subfamily. Lacking catalytic activity,
EphB6 is unlikely to operate as an independent receptor, but rather
as part of a hetero-oligomeric complex with the active EphB
receptors. Catalytically active EphB1 and EphB2 are both
co-expressed with EphB6 throughout the T cell lineage, raising the
possibility that EphB6 may interact with both receptors.
[0169] Until now, ErbB3 of the EGF receptor family (Pinkas et al.
(1996)) has been the only example of a trans-phosphorylated
kinase-inactive receptor. However, without wanting to be bound by
any particular theory, our findings suggest that this is a
universal mechanism for signaling through catalytically inactive
receptor tyrosine kinases. Two other kinase-inactive orphan
receptors, Klg and Vik, (Chou et al. (1991); Hovens et al. (1992);
Kelman et al. (1993); (Paul et al. (1992); Tamagnone et al. (1993);
Stacker et al. (1993)) may signal in a similar manner and have
catalytically active partners, as yet undescribed ErbB-3 acts to
modulate the intensity and duration of signaling by its active
partner (Pinkas et al. (1996); Levkowitz et al. (1998)) and
trans-phosphorylation results in recruitment of Shc and
phosphatidylinositol 3-kinase specifically to the ErB-3 receptor
chain (Kim et al. (1994); Waterman et al. (1999)). In similar
fashion, the catalytically inactive EphB6 receptor may recruit
specific cytoplasmic signaling molecules, as Cbl appears to
specifically bind EphB6 and not an active EphB1 partner (FIG.
4E).
[0170] Unusually for a receptor tyrosine kinase, and particularly
for an Eph receptor, EphB6 is most highly expressed in the thymus
(Gurriak et al. (1996)). Several lines of evidence suggested a
potential role for EphB6 in modulation of T-cell responses. First,
several Eph family members interact with the src-like kinase Fyn
(Choi et al. (1999); Ellis et al. (1996); Hock et al. (1998)), a
TCR-associated kinase critical for the development of T-cell
responses (Utting et al. (1998)). Secondly, Eph receptors can
regulate re-organization of the actin cytoskeleton (Meima et al.
(1997); Meima et al. (1997a)), an important event in TCR signaling;
as disruption of actin with cytochalasin D or Clostridium botulinum
toxin inhibits T lymphocyte responses to antigen (Valitutti et al.
(1995). And finally, the Eph receptors can modulate
integrin-mediated cell attachment (Becker et al. (2000); Huynh-Do
et al. (1999)), integrins functioning as TCR co-receptors to
modulate responses in both mature T cells and thymocytes (Abraham
et al. (1999); Bleijs et al. (1999); Ticchioni et al. (1993);
Wulfing and Davis (1998); Vivinus-Nebot et al. (1999.)).
[0171] In accordance with this hypothesis, it was shown that both
Zap-70 and Lck stimulation were decreased upon overexpression of
EphB6 in T cells (FIGS. 5 and 6). EphB6 did not affect a Zap-70
mutant lacking the activating tyrosine 493 residue when c-pressed
in COS-7 cells, appearing to prevent phosphorylation primarily of
residues phosphorylated in the activated state. Therefore,
decreased Zap-70 phosphorylation in COS-7 cells most likely
reflects inhibition of endogenous src-family kinases; while in
Jurkat, the primary inhibition of Lck activation by EphB6 is
probably responsible for the absence of Zap-70 stimulation. This
inhibition would reflect both a lack of phosphorylated CD3.zeta.
chain to recruit Zap-70 to the signaling complex, and a decrease in
the direct modification of Zap-70 by Lck. Without wishing to be
bound by any particular theory, the decrease in TCR stimulated Lck
kinase activity is in all probability the consequence of EphB6
induced re-arrangement of the cytoskeleton, sequestering lck away
from the TCR/CD3 receptor complex. In support of our hypothesis,
stimulation of .beta.1-integrins with either soluble ligand or
antibody has previously been shown to inhibit TCR mediated
activation of Lck and Zap-70 Nary et al. (1999)).
[0172] Inhibition of the early events of TCR signaling by
overexpression of EphB6 was found to ultimately translate into an
inhibition of T cell response, such as the induction of CD25
(IL-2R.alpha.) expression. Expression of CD25 is essential in the
IL-2 driven clonal expansion that occurs upon exposure to antigen.
Failure to express the high affinity IL-2R complex composed of the
.alpha., .beta. and .gamma. chains prevents the development of the
necessary IL-2 autocrine proliferative loop. Thus without wishing
to be bound by any one theory, one of the biological functions of
EphB6, in conjunction with other EphB receptors, may be to control
the clonal expansion of antigen activated T cells by suppressing
antigen induced CD25 expression and associated events. Several
alternative models of EphB6 function also become apparent. Under
more physiological conditions, ligation of the EphB6 receptor may
serve to maintain activation of the TCR signaling pathway below a
certain threshold, preventing premature activation by inappropriate
low affinity TCR interactions. Or, alternatively, the presence of
varying ephrin-B ligands may modify the ability of T-cells to
respond to antigens presented an different cell-surfaces.
[0173] EphB6, like the ErbB1 and PDGF receptors, was found to
physically associate with Cbl. The G315E mutation of the C.elegans
Cbl orthologue Sli-1 prevents interaction with the nematode ErbB
protein (let-23) (Jongeward et al. (1995)) and the analogous Cbl
mutation disrupts binding to PDGF and ErbB-1 receptors (Bonita et
al. (1997); Thien et al. (1997)). This mutation also abolished Cbl
association with EphB6 (FIG. 4F). The G306B mutation disrupts the
Cbl phosphotyrosine-binding domain, suggesting that phosphorylation
of EphB6 or an intermediate docking protein may be important for
Cbl binding to the receptor. Although EGF stimulation of the ErbB-1
receptor induces tyrosine phosphorylation of Cbl (Levkowitz et al.
(1996)), increased Cbl phosphorylation upon stimulation of the
EphB6 receptor with ephrin-B1 (not shown) was not detected. This
lack of Cbl phosphorylation probably reflects the absence of EphB6
catalytic activity, suggesting that EphB6 may simply recruit Cbl to
the cell membrane, rather than modifying its function by
phosphorylation. The failure to observe Cbl phosphorylation upon
ephrin-B1 stimulation also suggests that it is not a substrate of
the catalytically active EphB6 partner. TCR/CD3 stimulation of T
cells resulted in phosphorylation of EphB6-associated Cbl, although
EphB6 itself did not undergo detectable phosphorylation, suggesting
that Cbl phosphorylation is probably mediated by TCR/CD3 associated
cytoplasmic kinases.
[0174] The ability of Cbl to bind EphB6 raises the possibility that
EphB6 expression may be regulated by cbl mediated modification. It
is now clear that Cbl is responsible for the physical
downregulation of many receptors through induction of receptor
ubiquitination (Lee et al. (1999); Levkowitz et al. (1998); Miyake
et al. (1998)). The addition of multiple ubiquitin moieties to the
lysine residues of a protein targets it for degradation, either in
cytoplasmic proteasomes or the lysosomal compartment (Hershko et
al. (1998)). As all receptors binding to Cbl undergo
ubiquitination, it is likely that EphB6 will be similarly
regulated. This activity of Cbl is normally accompanied by its
phosphorylation, suggesting that anti-CD3 induced phosphorylation
of EphB6-associated Cbl may trigger EphB6 downregulation (FIG. 8).
While EphB6 may maintain activation of the TCR signaling pathway
below a certain threshold, preventing premature responses to
inappropriate stimulus, eliminations of the inhibitory EphB6-Cbl
complex from the plasma membrane may be an obligatory event for
maximal activation of the TCR signaling pathway.
[0175] To this point, Eph receptor function has been addressed
primarily in the development and function of the nervous system,
where they were shown to participate in targeting neurons and
growth cones, as well as in synapse formation (Zhou et al. (1998);
Flanagan et al. (1998)). This biological activity is essentially
due to the ability of Eph receptors to reorganize the actin
cytoskeleton and to control cell attachment by regulation of
integrin receptors (Becker et al. (2000); Holland et al. (1997);
Huynh-Do et al. (1999). Proper activation of T lymphocytes by
antigen-presenting cells requires stimulation not only of the TCR,
but the combined and coordinated engagement of its co-receptors.
Most TCR co-receptors bind cell-surface ligands and are
concentrated in areas of cell-cell contact, forming what has been
termed an immunological synapse (Grakoui et al (1999); Dustin et
al. (1999)). Assembly of these synapses and subsequent T cell
responses are strictly dependent upon cell attachment 101, actin
cytoskeleton re-organization (Holsinger et al. (1998); Valitutti et
al. (1995); Wulfing and Davis (1998)) and integrin receptor
signaling (Abraham et al. (1999); Bleijs et al. (1999); Ticchioni
et al. (1993); Wulfing et al (1998); Vivinus-Nebot et al. (1999)).
Without wishing to be bound by any particular theory, EphB
receptors and in particular EphB6, may be involved in coordination
of T cell attachment and formation of the immunological synapse and
thus may be important modulators of both thymocte selection and
T-cell responses.
Example 6
[0176] To explore the potential role of EphB6 in regulation of
AICD, we generated stable expression of myc-tagged human EphB6 in
the mature T cell line Jurkat (FIG. 9A); a commonly used model of
pre-stimulated mature T cells in AICD studies. AICD was induced in
EphB6 and control pcDNA transfected cells by overnight stimulation
with immobilized anti-CD3 antibody. To activate the EphB6 receptor,
cells were also treated with the EphB6 ligand, ephrin-B1. Stable
overexpression of the EphB6 receptor was found to significantly
enhance TCR-mediated apoptosis in a ephrin-B1-dependent manner
(FIG. 9B), thus confirming its potential to regulate the induction
of AICD.
Example 7
[0177] Active T cell apoptosis is driven by the antigen-induced
expression of the FASL and TNF death cytolines. In resting cells,
both FAS-L and TNF are weakly induced by TCR stimulation, but in
pre-activated cells these cytokines are highly expressed upon
stimulation. In vitro experiments suggest that CD4.sup.+ cells are
primarily eliminated by FAS-L, while AICD of CDS cells is
predominantly triggered by TNF. We therefore examined the
production of TNF by the control and EphB6 overexpressing CD4.sup.+
jurkat cells. Ephrin-B1 alone did not induce TNF production, nor
did it significantly alter the response to anti-CD3. Anti-CD3
stimulation induced significant TNF production in EphB6
overexpressing Jurkat but not in control cells (FIG. 10). Thus, the
increased apoptosis observed in EphB6 overexpressing cells may be
due in part to increased TNF production
Example 8
[0178] TNF efficiently activates both TNFR-I and TNFR-II. However,
previous studies have suggested that only TNFR-I is coupled to a
caspase cascade (Kozlosky et al. (1995)) and thus it may be the
predominant transmitter of the TNF apoptic signal (Daniel et al.
(1996); O'Leary and Wilkinson (1999)). We therefore examined the
expression of the two TNF receptors an EphB6 and control Jurkat
cells. Expression of TNFR-II, but not of INFR-I, was suppressed
upon incubation of both control and EphB6 overexpressing cells with
ephrin-B1 (FIG. 11). Activation of the TCR overrode this effect in
control cells, maintaining high INFR-II expression despite
ephrin-B1 stimulation. However, overexpression of EphB6 maintained
the ephrin-B1-induced down regulation of TNFR-II in the presence of
anti-CD3 stimulation. Interestingly, anti-CD3 stimulation of EphB6
overexpressing cells also reduced TINFR-II expression, while it had
no effect upon the receptor in control cells. This is probably
responsible for the greater degree of anti-CD3 induced apoptosis
observed in EphB6 overexpressing cells and suggests that the basal
activity of the EphB6 receptor is sufficient to make the cells more
sensitive to the induction of apoptosis. Activation of the EphB6
receptor by ephrin-B1 co-stimulation with anti-CD3 resulted in a
further decrease in TNFR-II expression, which is reflected in an
increase in induction of apoptosis.
[0179] TNFR-I and TNFR-II employ only partially distinct signaling
pathways, both initiating the n-terminal JUN kinase cascade
(Kozlosky et al. (1995)). Activation of the JNK pathway is required
to protect cells from TNF-mediated apoptosis (Adams et al. (1999);
Wang et al. (1998)). We examined the influence of EphB6 upon the
JNK cascade by following the threonine-tyrosine phosphorylation
(Thr183/Tyr185) of JNK upon anti-CD3 and ephrin-B1 stimulation.
Overexpression of EphB6 not only resulted in an alteration in the
balance of TNFR expression in favor of TNFR-I, but it also strongly
inhibited long-term antis induced JNK stimulation (FIG. 12). This
effect was highly specific, as none of the other potentially
anti-apoptic pathways examined, including AKT activation and Bcl-2
expression, was affected the suppression of JNK activation appeared
to be ligand-independent, suggesting that the basal activity of
overexpressed EphB6 was sufficient for JUN kinase inhibition the
elimination of JNK-JUN signaling was previously reported to enhance
TNF-induced apoptosis (Adams et al. (1999); Wang et al. (1998)),
suggesting that the selective inhibition of the JUN kinase pathway
observed here could further the promotion of AICD by EphB6.
[0180] Interestingly, while addressing the role of EphB6 in
apoptosis, we observed that overexpression of a dominant negative
form of EphB6 (cytoplasmic domain deleted) also increased the
induction of AICD. This is surprising in light of the ability of
wild type EphB6 to also promote AICD and probably reflects the
ability of the DN receptor to enhance TCR mediated responses, as
observed when examining CD25 expression. While not wishing to be
bound by any particular theory, this presumably occurs as the
result of removing TCR inhibitory input from the endogenous EphB6
receptor. However, this effectively overrides the actual inhibition
of EphB6-specific apoptic effects, such as TNF-R modulation and TNF
production, by the dominant negative receptor. While the apoptic
contribution of the endogendas EphB6 receptor is removed, apoptosis
still appears to increase due increased sensitivity to induction
through the TCR.
[0181] In sum, our findings conclusively demonstrate that the EphB6
receptor serves an important role as a TCR co-receptor in the
induction of AICD in mature activated T cells. Although driven by
different factors to AICD, and proceeding via a different
mechanism, the negative selection of thymocytes is also
predominantly, a TCR induced apoptic process. As the EphB6 receptor
is strongly expressed in thymocytes, it is therefore likely that it
also has an important role in regulating negative selection.
Materials and Methods for Examples 6-8
Western Blotting
[0182] Cells were quickly resuspended in ice cold lysis buffer
consisting of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1%
Triton X-100, 1 mM ethylene glycol-bis
(.beta.-aminoethylether)-N,N,N'-N'-tetraacetic acid (EGTA), 10
.mu.g/ml leupeptin, 10 .mu.g/ml aprotinin, 1 mM PMSP, 1 mM
Na-orthovanadate and 50 mM NaF. After solubilization on ice for 15
minutes, debris was removed by centrifugation at 12,000 g for 10
minutes at 4.degree. C. and SDS sample buffer added. Samples were
separated on SDS-polyacrylamide gels and transferred to
nitrocellulose membranes (Amersham, Arlington Heights, Ill.).
Membranes were blocked overnight at 4.degree. C. with 7% blotting
grade non-fat milk (Biorad, Richmond, Calif.) in PBS.
Immunoblotting antibodies were added at optimal dilutions in PBS-T
or TT (0.1% Tween-20) and incubated at 4.degree. C. overnight.
After extensive washing with PBS-T, bound antibodies were detected
using horseradish-peroxidase conjugated donkey anti-rabbit or sheep
anti-mouse antibodies (Amersham, Arlington Heights, Ill.) and
LumiGlo chemiluminescent reagents (Kirkegaard and Perry,
Mass.).
Subcloning of EphB6
[0183] CDNA for EphB6 was cloned from normal human thymocyte RNA by
RT-PCR into the expression vector pcDNA3 (Invitrogen, Calif.) and
sequenced. Myc-tagged version of EphB6 was generated by insertion
of an in frame Myc tag and construct verified by sequencing.
Expression of the tagged protein was examined by transfection in
COS-7 cells and Western blotting with appropriate antibodies.
EphB6 Stable Expression
[0184] To raise stable EphB6 expressing cells, the mature human
T-cell line Jurkat was transfected with empty pcDNA3 or EphB6-M.
The Jurkat cells were electroporated in 400 .mu.l complete RPMI
medium with 30 .mu.g of DNA by pulsing once for 65 sec at 260 V
(BTK electro square porator, BTX. Division of Genetronics Inc, San
Diego, Calif.). Cells were incubated at 37.degree. C. for 24 hours
before addition of G418 to the medium. After 30 days of selection
expression of the EphB6 receptor in resulting cell population was
confirmed by immunoprecipitation with anti-Myc and western blotting
with anti-Myc or anti-EphB6.
Stimulation of EphB6 Overexpressing and Control Cells
[0185] Soluble ephrin-B1-Fc fusion-protein dimers were purchased
from R&D Systems (Minneapolis, Minn.). The dimeric ephrin-B1
fusion protein was precomplexed with F(ab)'.sub.2 goat anti-human
Fc Pierce) to form oligomers. F(ab)'.sub.2 goat anti-human Fc was
used as a control (no stimulation) where necessary. Although murine
ephrin-B1 was used this effectively induced human EphB6
phosphorylation. Anti-CD3 (PharMingene, Canda) were immobilized on
24-well plates at 20 .mu.g/ml for 4 hours at room temperatue,
plates were rinsed once with PBS and cells stimulated for 24 hours,
37.degree. C.
Analysis of TNFR I and TNFR II Expression by Flow Cytometry
[0186] EphB6 and pcDNA3 Jurkat cells were incubated in 0.5% sennmm
for 24 hours with or without 5 .mu.g/ml soluble oligomerized
ephrin-B1 and immobilized anti-CD3 antibody. The expression of
TNF.alpha., TNFR I and TNFR II were then analyzed by staining with
corresponding PE-labeled antibody and isotype control. Anti-TNFR-I
and anti-TNFR II were from R&D Systems, Minn.
Analysis of Apoptosis
[0187] Cells were resuspended in RPM medium with 0.5% serum and
supplements as indicated. After 24 hours incubation the percentage
of apoptic cells was assessed by Annexin-V-FITC (Boehringer
Mannheim, Indianapolis, Ind.) binding and Propidium iodide (PI)
staining. Cells were analyzed on an Epics Elite V Flow Cytometer
(Coulter Electronics).
[0188] While the present invention has been described with
reference to what are presently considered to be the preferred
examples, it is to be understood that the invention is not limited
to the disclosed examples. To the contrary, the invention is
intended to cover various modifications and equivalent arrangements
included within the spirit and scope of the appended claims.
[0189] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
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DETAILED FIGURE LEGENDS
[0319] FIG. 1 Tyrosine Phosphorylation of the EphB6 Receptor is
Induced by Ephrin-B1 Ligand Stimulation.
[0320] (a) COS-7 cells transiently transfected with EphB6-M
encoding expression vector (pcDNA3) were stimulated by
co-incubation for 1 hour at 37oC with COS7 cells transfected with
empty vector (-), ephrin-A1 (A1), or ephrin-B1 (B1) cDNAs in
pcDNA3. Receptor phosphorylation was monitored by immunoblotting
anti-Myc immunoprecipitates with anti-phosphotyrosine (PY). EphB6-M
expression was determined by blotting with anti-Myc.
[0321] (b) HEK-293 and NIH 3T3 cells transiently expressing EphB6-M
were co-incubated for 1 hour with ligand expressing HEK-293 or NIH
3T3 cells respectively and receptor tyrosine phosphorylation and
expression levels determined as above.
[0322] (c) Time dependent phosphorylation of EphB6.
EphB6-M-expressing COS-7 cells were co-incubated with ephrin-B1
transfected COS-7 cells for the indicated time periods. EphB6-M
receptor phosphorylation and expression were determined as in
(a).
[0323] (d) Ligand concentration dependent EphB6 phosphorylation.
EphB6-M expressing COS-7 cells were co-incubated for 1 hour with
COS-7 cells transfected with 5 .mu.g of pcDNA3 (-), or varying
amounts of ephrin-B1-pcDNA3 (B1) as indicated.
[0324] (e) Soluble EphB6 receptor blocks ephrin-B1 induced EphB6
phosphorylation. Control (pcDNA3) or EphB6-M transfected cells were
stimulated with 1 .mu.g/ml soluble oligomerized ephrin-B1 (B1) in
the presence (+B6-R) or absence of Sig/ml soluble EphB6 receptor
for 30 minutes at 37oC. Cells were lysed by boiling in 1% SDS.
Phosphorylation of the membrane expressed myc-tagged EphB6 receptor
was examined by immunoprecipitation with anti-phosphotyrosine and
Western blotting with anti-Myc.
[0325] FIG. 2. EphB6 Receptor Phosphorylation is induced by
Co-expression of Catalytically Active EphB1 Receptor.
[0326] (a) COS-7 cells transiently transfected with either EphB1,
EphB6-M (B6-M), or both receptors were co-incubated for 1 hour at
37oC with control (-) or ephrin-B1 expressing (+) cells. Cells were
lysed and immunoprecipitation performed with anti-phosphotyrosine.
The presence of phosphorylated EphB6-M was detected by
immunoblotting with anti-Myc. EphB1 and EphB6-M expression levels
were quantitated by Western blotting with anti-EphB1 and anti-Myc,
respectively.
[0327] (b) COS-7 cells were transiently transfected with EphB6-M
(B6-M), EphB1, T-7 tagged kinase inactive EphB1 (B1-KD) or
co-transfected with EphB6-M and EphB1 or B1-KD. After 72 hours the
cells were lysed and immunoprecipitation performed with
anti-phosphotyrosine. The presence of EphB6-M in immunoprecipitates
was detected as in (a). Expression of the transfected proteins was
examined by western blotting.
[0328] (c) NIH 3T3 cells were transiently transfected with EphB6-M
alone, or in combination with EphB1. Cells were stimulated with 1
.mu.g/ml of soluble oligomerized ephrin-B1, lysed and EphB6
receptor precipitated with anti-Myc. Phosphorylation of EphB6-M was
monitored by immunoblotting with anti-phosphotyrosine.
[0329] (d) Truncated EphB1 receptor induces phosphorylation of
EphB6. COS-7 cells transiently transfected with EphB6-M, truncated
myc-tagged EphB1 (B1-Tr), or both receptors, were incubated for 1
hour with control (-) or ephrin-B1 ligand expressing (+) COS-7.
Cells were lysed and precipitation with anti-phosphotyrosine
performed. The presence of EphB6-M in immunoprecipitates and the
EphB6-M expression level were determined by anti-Myc Western blot,
as in (a).
[0330] (e) EphB6-M and B1-Tr were expressed in COS-7 cells as
indicated and analyzed as in (c).
[0331] FIG. 3. Eph Receptor Expression in Human Thymocytes and T
cells.
[0332] Expression of the EphA1, EphB1, EphB2 and EphB6 receptors
was examined by RT-PCR in human thymocytes, peripheral blood
T-lymphocytes and the mature T-cell line Jurkat Control
.beta.-actin primers were included in each reaction. The expected
product sizes are: .beta.-cactin-660 bp, EphA1-279 bp, EphB1-309
bp, EphB2-375 bp, EphB6-294 bp. The identity of the PCR products
was confirmed by sequencing. Water controls (no DNA) were all
negative (not shown). A 100 bp size ladder is shown on the right
(Gibco, BRL).
[0333] FIG. 4. EphB6 Associates with c-Cbl.
[0334] (a) pp115 co-precipitates with EphB6 in human thymocytes.
Thymocytes were stimulated with 1 .mu.g/ml anti-CD3 in the presence
of 5 .mu.g/ml crosslinking antibody for 10 minutes. Cells were then
lysed and precipitated with anti-EphB6 (358) or pre-immune serum
(PI). Phosphorylated proteins in these complexes were detected by
blotting with anti-phosphotyrosine. Preimmune (PI) antisera did not
precipitate phosphorylated pp115.
[0335] (b) Time course of pp115 association with EphB6. Thymocytes
were stimulated with anti-CD3 for the indicated time periods,
precipitated with anti-EphB6 (358) and blotted with
anti-phosphotyrosine.
[0336] (c) pp115 has the same electrophoretic mobility as c-Cbl.
Thymocytes were stimulated with 1 .mu.g/ml anti-CD3 in the presence
of 5 .mu.g/ml of crosslinking antibody for 10 min and Cbl and EphB6
immunoprecipitated. Immunocomplexes were resolved by SDS PACE,
transferred to the nitrocellulose and blotted with
anti-phosphotyrosine.
[0337] (d) Cbl, Vav, and PAK were immunoprecipitated from thymocyte
lysates after anti-CD3 stimulation and immunocomplexes Western
blotted with anti-EphB6 (358) or pre-immune serum as indicated.
[0338] (e) EphB6, but not EphB1, co-precipitates with Cbl. COS-7
cells were transiently transfected with Cbl and EphB6-M (B6-M), or
Cbl and EphB1 as indicated. After 72 hours, Cbl was precipitated
and association with EphB6-M and EphB1 examined by blotting with
anti-Myc and anti-EphB1respectively.
[0339] (f) The G306E loss-of-function Cbl mutant does not bind
EphB6. COS-7 cells were transiently transfected with wild type Cbl,
G306E Cbl(Cbl*), or the ancogenic 70-Z Cbl mutant (Cbl**), either
alone or in combination with EphB6-M as indicated. After 72 hours
EphB6-M association with Cbl was examined by immunoblotting Cbl
immunoprecipitates with anti-Myc. Expression of each form of Cbl
and EphB6 was confirmed by Western blotting of cell lysates.
[0340] FIG. 5. EphB6 Downregulates the Zap-70 Kinase.
[0341] (a) EphB6 downregulates Zap-70 tyrosine phosphorylation.
Zap-70 was transiently expressed in COS-7 cells, alone, or in
combination with EphB6-M (B-M) or EphB1 receptors. To activate
EphB6, cells were incubated for 1 hour with ephrin-B1 ligand (+)
expressing cells. Zap-70 phosphorylation was then analyzed by
immunoblotting Zap-70 immunoprecipitates with
anti-phosphotyrosine.
[0342] (b) Phosphorylation of Y493F Zap-70 is not altered by EphB6.
Zap-70 or Y493F Zap-70 (Zap*) were expressed in COS-7 cells, alone,
or with EphB6-M (B6-M. The phosphorylation status of Zap-70 and
Zap* were analyzed by anti-phosphotyrosine blotting and expression
by anti-Zap-70 blot. EphB6 expression was determined by Western
blot of lysates.
[0343] (c,d) Transfected EphB6-M is tyrosine phosphorylated in
Jurkat upon stimulation with ephrin-B1. The mature human T-cell
line Jurkat was transfected with empty pCDNA3 or EphB6-M. After 30
days of Geneticin selection the resulting oligodonal cell
populations were screened by immunoprecipitation with anti-Myc and
western blotting with anti-Myc or anti-EphB6 and the highest
expressing cell population (B6-D selected. B6-J and pcDNA3 Jurkat
cells were stimulated with 1 .mu.g/ml soluble ephrin-B1 for 15
minutes at 37oC, cells lysed, EphB6-M immunoprecipitated with
anti-Myc and its phosphorylation examined.
[0344] (e,f) Overexpression of EphB6 downregulates phosphorylation
of Zap-70 and Zap-70 associated CD3.zeta. in Jurkat. Transfected
Jurkat cells were stimulated 1 .mu.g/ml soluble dimerized ephrin-B1
for 15 minutes at 37.degree. C. and then costimulated for 7 minutes
with 4 .mu.g/ml anti-CD3. Zap-70 and CD3.zeta. tyrosine
phosphorylation was then examined by anti-phosphotyrosine Western
blotting of kinase immnunoprecipitates. Results shown represent
four independent experiments.
[0345] FIG. 6. The EphB6 Receptor Inhibits TCR Induced Activation
of Lck
[0346] (a,b) Lck immunoprecipitates were prepared from pcDNA3 and
B6-J Jurkat cells stimulated as in FIG. 5e. Immunocomplexes were
incubated in 50 .mu.l of kinase buffer in the presence of 4 .mu.g
of the synthetic substrate peptide Raytide EL and .gamma.[32P]-ATP
for 15 min at room temperature. The kinase buffer containing the
peptide was collected and loaded onto phosphocellulose paper. The
paper was washed 3 times with 0.5% phosphoric acid and once with
acetone, dried and counted in a .beta.-counter. Results are shown
in arbitrary units and represent one of four independent
experiments. The presence of Lck was determined by immunoblotting
of Lck immunoprecipitates run on non-reducing SDS PAGE with
anti-lck (not shown).
[0347] FIG. 7. EphB6 Overexpression Prevents TCR Mediated
Upregulation of CD25.
[0348] (a,b) B6-J and pcDNA3 Jurkat cells were incubated in 0.5%
serum for 24 hours with or without 5 .mu.g/ml soluble oligomerized
ephrin-B1 and immobilized anti-CD3 antibody as indicated. The
expression of CD25 was then analyzed by staining with PE-labeled
anti-CD25. The percentage of CD25 expressing cells is given in each
case after subtraction of the isotype control.
[0349] FIG. 8. Endogenous EpbB6 Downregulates CD25
Upregulation.
[0350] (A). Dominant negative (DN) EphB6 receptor expressing Jurkat
cells (DN-J) were generated as in FIG. 4A. Expression of the DN
receptor was assessed by Western Blot (see insert). DN-J cells were
stimulated as in FIG. 7 and CD25 expression analyzed by flow
cytometry. Results represent one of three independent
experiments.
[0351] (B) A further view of dominant negative (DN) EphB6 receptor
expressing Jurkat cells (DN-J) were generated as in FIG. 4A.
Expression of the DN receptor was assessed by Western Blot (see
insert). DN-J cells were stimulated as in FIG. 7 and CD25
expression analyzed by flow cytometry. Results represent one of
three independent experiments.
[0352] (C). Purified thymocytes were starved for 24 hours,
resuspended in 05% serum and stimulated with plate-immobilized
anti-CD3 and ephrin-B1 as indicated. Expression of CD25 was
analyzed by flow cytometry upon staining with PB-labeled anti CD25
antibody. The percentage of CD25 expressing cells is given after
subtraction of the isotype control. Results represent one of three
independent experiments.
[0353] FIG. 9. The EphB6 receptor enhances TCR mediated apoptosis.
a, Stable expression of EphB6 receptor. The mature T cell line
Jurkat was transfected with empty pcDNA3 expression vector or
myc-tagged EphB6. After 30 days of Geneticin selection EphB6
expression in the selected cells was confirmed by
immunoprecipitation with anti-myc and blotting with either anti-myc
or anti-EphB6. Equivalent expression of TCR/CD3 on EphB6 and
control cells was confirmed by flow cytometry (not shown). b, EphB6
overexpressing (B6-13) and control pcDNA3 transfected cells were
incubated in 0.5% serum for 24 hours with or without 5 .mu.g/ml
soluble oligomerized ephrin-B1 (B1) and immobilized anti-CD3
antibody as indicated. Induction of apoptosis was analyzed by
annexin-V binding. The percentage of apoptic cells is given in each
case. The results shown represent four independent experiments.
[0354] FIG. 1. The EphB6-dependent increase in activation induced
cell death is accompanied by increased TNF.alpha. production. EphB6
overexpressing and control Jurkat cells were stimulated for 24
hours as in FIG. 1. TNF.alpha. production was quantitated by
chemiluminescent immunoassay of the cell culture supernatant.
[0355] FIG. 11. The EphB6 receptor inhibits expression of TNFR II
but not TNFR I. Control (a) and EphB6 overexpressing (b) Jurkat T
cells were stimulated as in FIG. 1 and expression of TNFR I and
TNFR II determined by staining with PE-labeled anti-TNFR I or
anti-TNFR II antibodies accordingly. TNFR I and TNFR II expression
is given in arbitrary units (AU) after subtraction of the isotype
control. The results shown represent three independent
experiments.
[0356] FIG. 12. The EphB6 receptor prevents activation of p54 JNK.
EphB6 and pcDNA3 control cells were stimulated as in FIG. 1. Cells
were lysed, clarified by centrifugation and the lysates resolved by
SDS PAGE. Phosphorylation of Jun kinase a and Akt and expression of
Bcl-2, were analyzed by Western Blotting with the appropriate
antibody as indicated. The results shown represent three
independent experiments.
[0357] FIG. 13. Model of EphB6 Receptor Interaction with the TCR
Signaling Pathway.
[0358] Binding of the transmembrane ephrin-B family ligand induces
trans-phosphorylation of the catalytically inactive EphB6 receptor
by its active EphB partner and brings the Eph6-Cbl complex into the
proximity of the T cell receptor. The recruitment of EphB6 to the
immunological synapse downregulates activity of the TCR associated
kinases lck and Zap-70, possibly by affecting cytoskeleton and TCR
complex formation. This raises the threshold for T cell activation,
which may serve to prevent T cell activation by low-affinity
TCR-antigen interaction. However, strong and sustained TCR
stimulation causes phosphorylation of EphB6-associated Cbl
resulting in EphB6 ubqiutination (Ub) and consequent
downregulation. The removal of EphB6 from the membrane allows
complete activation o the TCR signaling pathway and subsequently,
of T cell responses.
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