Note: Descriptions are shown in the official language in which they were submitted.
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Description
MODULATION OF ENDOTHELIAL CELL SURFACE RECEPTOR
ACTIVITY IN THE REGULATION OF ANGIOGENESIS
Cross Reference to Related Applications
This application is a continuation-in-part of co-pending U.S. Patent
application serial number 09/152,160, filed September 10, 1998, herein
incorporated by reference in its entirety.
Grant Statement
This work was supported by NIH grants DK38517 and CA 68485. The
U.S. Government has certain rights in the invention.
Technical Field
The present invention relates generally to the modulation of the activity
of an endothelial cell surFace receptor in the regulation of endothelial cell
proliferation and migration and in the regulation of angiogenesis. More
particularly, the present invention relates to the modulation of ECRTP/DEP-1
activity in the regulation of endothelial cell proliferation and migration and
in the
regulation of angiogenesis.
Background of the Invention
As used herein, the term "angiogenesis" means the generation of new
blood vessels into a tissue or organ. Under normal physiological conditions,
humans or animals undergo angiogenesis only in very specific restricted
situations. For example, angiogenesis is normally observed in wound healing,
fetal and embryonal development and formation of the corpus luteum,
endometrium and placenta. The term "endothelium" means a thin layer of flat
epithelial cells that lines serous cavities, lymph vessels, and blood vessels.
The
term "endothelial modulating activity" means the capability of a molecule to
modulate angiogenesis in general and, for example, to stimulate or inhibit the
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growth of endothelial cells in culture. Both controlled and uncontrolled
angiogenesis are thought to proceed in a similar manner. Endothelial cells and
pericytes, surrounded by a basement membrane, form capillary blood vessels.
Angiogenesis begins with the erosion of the basement membrane by enzymes
released by endothelial cells and leukocytes. The endothelial cells, which
line
the lumen of blood vessels, then protrude through the basement membrane.
Angiogenic stimulants induce the endothelial cells to migrate through the
eroded basement membrane. The migrating cells form a "sprout" off the parent
blood vessel, where the endothelial cells undergo mitosis and proliferate. The
endothelial sprouts merge with each other to form capillary loops, creating
the
new blood vessel.
Persistent, unregulated angiogenesis occurs in a multiplicity of disease
states, and abnormal growth by endothelial cells and supports the pathological
damage seen in these conditions. The diverse pathological disease states in
which unregulated angiogenesis is present have been grouped together as
angiogenic dependent or angiogenic associated diseases.
It is also recognized that angiogenesis plays a major role in the
metastasis of a cancer. If this angiogenic activity could be repressed or
eliminated, then the tumor, although present, would not grow. In the disease
state, prevention of angiogenesis could avert the damage caused by the
invasion of the new microvascular system. Therapies directed at control of the
angiogenic processes could lead to the abrogation or mitigation of these
diseases.
The development of renal glomerular capillaries is anatomically
segregated and temporally staged in a multi-step process. The process
involves recruitment of endothelial progenitors from adjacent mesenchyme,
assembly of an arborized branching network, and maturation and specialization
of endothelial cells adjacent to mesangial and visceral epithelial cells.
Receptors for extracellular matrix components, cell surface molecules and
growth factors have been assigned roles to mediate steps in this assembly
process. See e.a., Wallner et al., Microsc Res Tech 39:261-284 (1997);
Takahashi et al., Kidney Int 53:826-835 (1998).
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Vascular endothelial growth factor (VEGF) is an important participant,
as it is induced in S stage developing glomerular epithelial cells, and
endothelial progenitors that are recruited to glomerular capillaries from the
adjacent metanephric mesenchyme express the VEGF receptor, flk-1. Robert
et al., Am J Physio1271:F744-F753 (1996).
Neutralizing VEGF antibodies interrupt postnatal murine glomerular
capillary development. Kitamoto et al., J Clin Invest 99:2351-2357 (1997).
Deletion of either PDGF~i receptor or PDGF~i genes in mice causes defective
recruitment of mesangial cell precursors with failure of
glomerulardevelopment.
Soriano, P., Genes Dev8:1888-1896 (1994); Leveen et al., Genes Dev8:1875-
1887 (1994). TGF~i1 expression and type II TGF~i receptors appear critical for
vascular development in the embryonic yolk sac (prior to renal development),
and type II receptors mediate in vitro capillary morphogenesis of endothelial
cells derived from bovine glomeruli. Choime et al., J Biol Chem 270:21144
21150 (1995).
Early evidence suggests that Eph family receptors and their ephrin
ligands participate in glomerular vascular development. EphB1 receptors are
expressed in isolated mesenchymal cells in a pattern similarto that of flk-1,
and
high level expression of ephrin-B1 is seen at the vascular cleft of developing
glomeruli, as well as in capillary endothelial cells of mature glomeruli.
Daniel
et al., Kidney Int 50:S-73-S-81 (1996). Oligomerized forms of ephrin-B1
stimulate in vitro assembly of human renal microvascular endothelial cells
(HRMEC) into capillary-like structures. Stein et al., Genes Dev 12:667-678
(1998).
A selected subclass of receptor tyrosine phosphatases, including
DPTP10D, serve important roles in directing axonal migration and neural
network assembly. Desai et al., Cell 84:599-609 (1996). Recent data has
identified mRNA expression of a related receptor phosphatase, ECRTP/DEP-1,
in arterial sites in mammalian kidney. Borges et al., Circulation Research
79:570-580 (1996). To date, however, there has been no evidence to implicate
receptor tyrosine phosphatases in microvascular or glomerular capillary
assembly or maturation.
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Vascular endothelial cells display a diverse range of vascular bed
specific properties (Gumkowski et al., Blood Vessels 24:11-13 (1987)), yet
the'
requirement to maintain a continuous, antithrombotic monolayer lining the
vascular space imposes rigorous requirements that their proliferation,
migration
and differentiation be regulated by interendothelial contacts. Specialized
intercellular contacts permit communication among interacting endothelial
cells
(Lampugnani et al., J Cell Biol 129:203-217 (1995)) yet the mechanisms
regulating arrest of proliferation and migration in response to
interendothelial
contact have not been elucidated. Tight regulatory control over proliferation
imposed by interendothelial cell contact is apparent in the low basal mitotic
index among endothelial cells in existing vessels. Engerman et al., Laboratory
Investigation 17:738-744 (1967). This is in contrast with the proliferative
endothelial responses that are evoked by mechanical disruption of large
vessels. More et al., J Patho 172:287-292 (1994). Similar proliferation and
migration responses are stimulated at the margin of a confluent endothelial
monolayer by "wounding", or physical removal cells from the packed
monolayer. Coomber, J CeI18iochem 52:289-296 (1993).
The molecular basis for effects of interendothelial contact on migratory
and proliferative responses is not defined, yet studies of cultured cells have
shown that endothelial, fibroblast, and epithelial cells grow to confluency at
a
predictable density, then arrest proliferation (density arrest). Augenlicht
and
Baserga, Exp Cell Res 89:255-262 (1974); Beekhuizen and van Furth, J
Vascular Res 31:230-239 (1994); Rijksen et al., J Cell Physiol 154:393-401
(1993). This phenomenon can be very relevant to the behavior of endothelial
cells in vascular sites in situ. Indeed, model culture systems of endothelial
"wounding" have shown that endothelial cells at the edge of an imposed
"wound" rapidly extend lamellae, spread, migrate and proliferate to replace
the
deficit created by mechanical disruption of the monolayer. Coomber, J Cell
Biochem 52:289-296 (1993).
Pallen and Tong observed that membrane-associated tyrosine
phosphatase activity recovered from cultured Swiss 3T3 cells increased eight
(8)-fold (expressed as activitylmg protein) as cells approached a density of 5
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x 104/cm2, while soluble fraction tyrosine phosphatase was unaffected by cell
density. Pallen and Tong, Proc Natl Acad Sci USA 88:6996-7000 (1991 ).
Ostman et al. determined that the abundance of a receptor tyrosine
phosphatase cloned from HeLa cells and named DEP-1, is increased as cells
approach high density. Ostman et al., Proc Natl Acad Sci USA 91:9680-9684
(1994). However, no links between molecules that evoke proliferation arresf
and receptor tyrosine phosphatases have been made.
To date, available information does not indicate what sort of receptor
ligand interaction might mediate a cell surface generated signal for density
or
contact arrest. The identification of such a receptor-ligand interaction is
therefore needed in that it will serve as a basis for intervention in a
disorder
wherein density or contact arrest, or the preclusion of density or contact
arrest,
has therapeutic value. Such disorders include disorders characterized by
undesired angiogenesis, such as angiogenesis associated with tumor growth.
Thus, what is also needed is a composition and method which can inhibit the
unwanted growth of blood vessels, especially into tumors. The composition and
method should attenuate the formation of the capillaries in the tumors thereby
inhibiting the growth of the tumors.
Summar~r of the Invention
In accordance with the present invention, a method of modulating
angiogenesis in a vertebrate subject is provided. The method comprises
administering to the vertebrate subject an ECRTP/DEP-1 activity modulating
amount of a composition, whereby an ECRTP/DEP-1 within the vertebrate
subject is contacted by the composition; and modulating angiogenesis through
the contacting of the ECRTP/DEP-1 with the composition.
In accordance with the present invention a method of modulating
endothelial cell migration and proliferation in a vertebrate subject is also
provided. The method comprises administering to the vertebrate subject an
ECRTP/DEP-1 activity-modulating amount of a composition, whereby an
ECRTP/DEP-1 within the vertebrate subject is contacted by the composition;
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and modulating endothelial cell migration and proliferation through the
contacting of the ECRTP/DEP-1 with the composition.
In accordance with the present invention there is also provided an
antibody which preferentially binds the ECRTPIDEP-1. Optionally, the antibody
comprises a monoclonal antibody or fragment or derivative thereof which
preferentially binds the ECRTP/DEP-1.
In accordance with the present invention, a method for isolating an
endogenous ligand for an ECRTP/DEP-1 is also provided. The method
comprises the steps of contacting cells or cell lysates having the ligand with
ECRTP/DEP-1; and isolating the ligand which binds with ECRTP/DEP-1.
A method of screening candidate substances for an ability to modulate
ECRTP/DEP-1 biological activity is also disclosed. The method comprises
establishing test samples comprising an ECRTP/DEP-1 polypeptide or
fragment thereof; administering a candidate substance to the test samples; and
measuring the interaction, effect, or combination thereof, of the candidate
substance on the test sample to thereby determine the ability of the candidate
substance to modulate ECRTP/DEP-1 biological activity.
In accordance with the present invention there are also provided
methods for performing a screening assay for identifying a compound that
modulates an activity of an ECRTP/DEP-1 in both a cell-based and a cell-free
assay. In a cell-based assay, the method comprises the steps of establishing
replicate test and control cultures of cells that express the ECRTP/DEP-1;
administering a candidate compound to the cells in the test culture but not
the
control culture; measuring ECRTP/DEP-1 activity in cells in the test and the
control cultures; and determining that the candidate compound modulates the
ECRTP/DEP-1 activity in a cell if the ECRTP/DEP-1 activity measured for the
test culture is greater or less than the ECRTP/DEP-1 activity measured for the
control culture.
In a cell-free system, the method comprises the steps of establishing a
control system comprising an ECRTP/DEP-1 and a ligand wherein the
ECRTP/DEP-1 is capable of binding to the ligand; establishing a test system
comprising the ECRTP/DEP-1, the ligand, and a candidate compound;
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measuring the binding affinity of the ECRTP/DEP-1 and the ligand in the
control and the test systems; and determining that the candidate compound
modulates ECRTP/DEP-1 activity in a cell-free system if the binding affinity
measured foi- the test system is less than or greater than the binding
affinity
-measured for the control system.
In another embodiment, the screening assay methods of the present
invention pertain to comparing the effect of a candidate compound to inhibit
growth of cells expressing exogenous ECRTP/DEP-1 compared with those not
expressing ECRTP/DEP-1 and determining that the effect of altering
ECRTP/DEP-1 activity is responsible by demonstrating the lack of activity of
the candidate compound on cells not expressing ECRTP/DEP-1. Thus, the
screening assays of the present invention show changes in growth that are
responsive to changes in ECRTP/DEP-1 activity.
In accordance with the present invention there is also provided a method
for delivering a therapeutic composition to a tissue in a patient, wherein the
tissue is characterized as having undesirable endothelial cell proliferation.
The
method comprises the steps of introducing into the patient a biologically
effective amount of an antibody operatively linked to a selected therapeutic
agent, the antibody preferentially binding to an ECRTP/DEP-1 on the surface
of the endothelial cells, whereby an ECRTP/DEP-1 within the vertebrate
subject is contacted by the antibody; and delivelring the therapeutic
composition
to the tissue through the contacting of the ECRTP/DEP-1 with the composition.
It is therefore an object of the present invention to localize and
characterize a receptor-ligand interaction which mediates a cell surface
generated signal for cell growth and survival.
It is another object of the present invention to provide forthe modulation
of a cell surface receptor activity in endothelial cells to mediate a cell
surface-
generated signal for cell growth and survival.
It is still another object of the present invention to provide for the
modulation of a cell surface receptor activity for use in the inhibition or
stimulation of angiogenesis.
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It is yet another object of the present invention to identify compounds
which modulate a receptor-ligand interaction which mediates a cell surface-
generated signal for density or contact arrest.
Some of the aspects and objects of the invention having been stated
hereinabove, other aspects and objects will become evident as the description
proceeds, when taken in connection with the accompanying Drawings and
Examples as best described hereinbelow.
Brief Description of the Drawings
Figure lAdepicts recognition by antibodies ECRTPAb-1 & ECRTPAb-2
of recombinant and over-expressed ECRTP/DEP-1 and is an autoradiograph
depicting recombinant proteins representing extracellular (Ec) or cytoplasmic
(Cy) domains of ECRTP/DEP-1 were expressed in bacteria and purified.
Proteins (100 ng) were separated on a 15% SDS-polyacrylamide gels,
transferred to PVDF membrane and probed with monoclonal antibodies
ECRTPAb-1 or ECRTPAb-2, as indicated.
Figure 1 B depicts recognition by antibodies ECRTPAb-1 & ECRTPAb-2
of recombinant and over-expressed ECRTP/DEP-1 and is an autoradiograph
depicting MDCK cells cultured in 100 mm dishes were transfected with 14 pg
of empty pSRa vector (SRa) or pSRa-ECRTP/DEP-1/HA (SRa-ECRTP/HA)
expression constructs and harvested at43 hours aftertransfection. Membrane
receptor proteins were recovered by WGA lectin-conjugated agarose from 150
pg of lysate protein. Lectin-adsorbed, eluted proteins were subjected to 7%
SDS-PAGE, transferred to a PVDF membrane and probed with ECRTPAb-1,
ECRTPAb-2, or anti-HA (HAAb) monoclonal antibodies, as indicated.
Figure 1 C is a series of photographs depicting MDCK cells stably
transfected with the pSRa-ECRTP/DEP-1/HA plasmid were fixed with cold
methanol and stained with ECRTPAb-2 (panels a, c & d) or a class matched
control antibody (panel b). ECRTPAb-2 labeled ~ lateral borders of cells in
contact. Preincubation of ECRTPAb-2 with 50 pg of recombinant immunogen
(Ec) blocked this staining (panel c), while an irrelevant recombinant protein
(Cy)
did not (panel d).
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Figures 2A-2E are a series of photographs depicting the abundance of
ECRTP/DEP-1 in endothelial cells of adult human kidney. Acetone fixed frozen
sections (5 pm thickness) of human kidney were incubated with ECRTPAb-1
(panels A-D) or a class matched control monoclonal antibody (panel E) and
bound antibody was detected by epifluorescence microscopy, as described in
Methods. ECRTPAb-1 prominently labeled glomerular, peritubular and arterial
endothelial cells. Magnifications were A) x 100; B) x 600; C) x 600; D x 400;
and E) x 100.
Figure 3 depicts confocal localization of ECRTPIDEP-1 and VE cadherin
in human kidney vasculature. Acetone fixed kidney sections were
simultaneously labeled with ECRTPAb-1 and a polyclonal goat antibody
against VE cadherin. Bound antibodies were detected using fluorescein
conjungated anti-mouse (panels A, B, E, F) or rhodamine-conjugated anti-goat
(panels C, D, E, F) Ig antibodies. ECRTPAb-1 (green) staining distributed over
the entire endothelial membrane in large artery and glomerular capillaries (A,
B) while VE cadherin labeling (red) is restricted to endothelial junctions (C,
D).
Overlapping confocal images demonstrated colocalization of ECRTP with VE
cadherin at inter-endothelial junctions. (magnification x 600).
Figure 4 is a series of photographs depicting ECRTP/DEP-1 expression
in developing murine glomeruli. Cryostat kidney sections of embryonic day 14
(A), day 16 (B), postnatal day 6 (C) and adult mice (D) were immunolabeled
with ECRTPAb-1 as described in the Methods of Example 1. In panels A & B;
ECRTPAb-1 binds to cells dispersed in the mesenchymal area (arrow), to
endothelial precursor cells (arrowhead) migrating to the vascular cleft of
comma-shaped glomeruli and to endothelium of capillary stage glomeruli (G).
In panels C & D, ECRTPAb-1 preferentially labels endothelial cells of the
glomerulus (G), artery (A) and peritubular capillaries (arrow) in mature
kidney.
(Original magnification; A) x 400; B) x 200; C) x 200; and D) x 350.
Figure 5A depicts distribution of ECRTP/DEP-1 of inter-endothelial
contacts in cultured human endothelial cells, but ECRTP/DEP-1 does not
dissociate from junctions with VE cadherin and is a series of photographs
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depicting Methanol fixed HRMEC cells were labeled with ECRTPAb-2 as
described in Methods of Example 1. ECRTP/DEP-1 is distributed between
points of inter-endothelial membrane contact and punctate regions of the
apical
membrane in serial confocal images.
Figure 5B depicts distribution of ECRTP/DEP-1 of inter-endothelial
contacts in cultured human endothelial cells, but ECRTP/DEP-1 does not
dissociate from junctions with VE cadherin and is a series of photographs
depicting HMEC-1 cells were grown to confluency, then incubated with media
containing 5mM EGTA for 0 min (panels a & c) or 20 min (panels b & d), prior
to fixation. The distribution of ECRTPAb-2 and VE cadherin labeling was
examined as described in Methods of Example 1 at each time. While the
distribution of ECRTP/DEP-1 immunoreactivity was not altered in the low Ca~+
medium, functional VE cadherin staining dissipated, consistentwith
dissociation
of VE cadherin junctions and redistribution across the cell membrane.
Figure 6A shows that endothelial cell density imposes growth arrest and
increases lectin recoverable tyrosine phosphatase activity and is a line graph
showing identical numbers of human renal microvascular endothelial cells
(HRMEC) were plated in growth medium on 100 (1 x), 60 (2.9x) or 35 (8.1 x) mm
diameter plastic dishes, effecting the indicated fold differences in cell
density
at the time of plating. Medium was replaced with growth medium at points
indicated by arrows. Cells were counted in a Coulter counter and means of
quadruplicate samples are displayed. Proliferation was arrested in cells at
8.1 x
density after a single cell doubling, and after approximately 3 doublings in
cells
plated at 2.9x density.
Figure 6B shows that endothelial cell density imposes growth arrest and
increases lectin recoverable tyrosine phosphatase activity and is a bar graph
showing cells plated for the indicated times at the indicated densities were
lysed, and receptor tyrosine phosphatase activity, including that attributable
to
ECRTP/DEP-1, was recovered by lectin affinity chromatography and assayed
as described in Methods of Example 2 in the absence or presence of the
tyrosine phosphatase inhibitor, sodium orthovanadate (V04, 100 pM).
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Figure 7 is an autoradiograph and a bar graph showing that increased
cell density. imposes increases in activity, but not amount, of
immunoprecipitated ECRTP/DEP-1. Identical numbers of HRMEC were plated
as in Figure 6 at the indicated cell densities. Monospecific affinity purified
rabbit polyclonal antibodies were used to immunoprecipitate ECRTP/DEP-1
from cells treated for 10 min immediately before harvest with 1 mM
peroxyvanadate (+V04) orvehicle (-V04) at 36 hours after plating, as described
in Methods of Example 2. Recovered ECRTP/DEP-1 antigen was quantitated
by immunoblot with the monospecific antibody and its endogenous
phosphotyrosine content assessed by phosphotyrosine immunoblot using the
4610 monoclonal antibody. Phosphatase activity in immunoprecipitated
samples was assayed using pNPP as substrate in the absence (-V04) or
presence (V04) of sodium orthovanadate, as described. Data are displayed as
optical density of the product in triplicate samples +/- SEM.
Figure 8A shows that ECRTP/DEP-1 overexpression, or bivalent
antibody against ECRTP/DEP-1, ECRTPAb-1, imposes proliferation arrest on
HRMEC and is a graph showing transient transfection of HMREC with
ECRTP/DEP-1 cDNA imposes a growth inhibition at low cell densities.
Approximately 3 x 105 HRMEC were cotransfected with 1.7~,g pSRa (vector
control) or HA epitope tagged (hemagglutinin) pSRa-ECRTP/DEP-1 (pSRa-
ECRTP), as indicated, and 0.4~,g pEGFP (Clontech) to permit scoring of BrdU
labeling of transfected cells, as described in Methods of Example 2. At 24
hours, transfected cells were replated on p35 dishes in the numbers indicated.
Thirty six hours later, S phase cells were labeled for 30 min with BrdU, as
described in Methods of Example 2, and +GFP positive cells were scored for
BrdU incorporation. Data represent means +/- SEM for quadruplicate
determinations.
Figure 8B shows that ECRTP/DEP-1 overexpression, or bivalent
antibody against ECRTP/DEP-1, ECRTPAb-1, imposes proliferation arrest on
HRMEC and is a line graph showing that ECRTPAb-1 inhibits endothelial
proliferation and migration. HRMEC (3 x 104) were plated in p35 dishes at time
0: Growth medium was replaced at 24h, cells were counted, and either IgG
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control (10p,g/ml) or ECRTPAb1 (10~g/ml) antibodies were added. Replicate
samples (5) of cells were counted on day 4, and are expressed as means +/-
SEM.
Figure 8C is a data point plot depicting that equal numbers of HRMEC
were plated at time 0, and antibodies or Fab fragments added at the
concentrations indicated. Replicate plates were harvested on day 1, to confirm
homogeneous plating efficiency in each condition, and on day 6 to assess cell
proliferation, respectively. Data points represent mean values of five
replicates
+ SEM.
Figure 9A depicts inhibition of endothelial migration by ECRTPAb-1 and
is a series of photographs depicting monolayers of HRMEC were transiently
transfected with plasmid pSRaECRTP/DEP-1/HA, or pSRaEphB1/HA, as
indicated. Forty eight hours later, "wounds" were created in the confluent
monolayers and permitted to close over the ensuing 30 h. Monolayers were
then stained with the monoclonal hemagglutinin antibody, 12CA5, to detect the
positions of cells transiently expressing high levels of ECRTP/DEP-1/HA or
EphB1/HA, respectively. Only rare ECRTP/DEP-1 overexpressing cells
migrated to close the "wound".
Figure 9B depicts inhibition of endothelial migration by ECRTPAb-1 and
is a line graph reflecting analysis of 300 to 420 ~.m diameter "wounds" which
were created in HRMEC confluent monolayers at time 0, as medium was
exchanged to serum-free medium supplemented by either no addition (NA), or
phorbol myristate acetate (20ng/ml) in the presence of the indicated
antibodies
orfragments, including a class matched IgG control (IgG,1 Op,g/ml), ECRTPAb1
(10~g/ml), or Fab fragments of ECRTPAb1 (3~,g/ml, molar equivalency).
Triplicate wounds were used to generate microscopic images at the indicated
times, and the residual "wound" area calculated and expressed as a fraction
of the original wound, by an automated capture sequence using Bioquant
Image Analysis Software. Each data point represents the mean ~ SEM of
three determinations.
Figure 9C depicts inhibition of endothelial migration by ECRTPAb-1 and
is a line graph analyzing data produced by the same assay procedure as
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Figure 9B. Using the same assay procedure, migration rates were calculated
by linear regression of mean values determined in cells exposed to IgG
control,
ECRTPAb1, or ECRTPAb1/Fab, using three independent time points. r2 values
> 0.90 for each data point plotted. The open square (~) indicates the
migration
rate for closure of unstimulated cells.
Figure 10 is line graph depicting that ECRPTAb1 Fab fragments
attenuate endothelial density mediated growth arrest. HMEC-1 cells of the
indicated numbers were plated in on coverslips in 12 well dishes at time 0 in
growth media supplemented by no addition (NA) or ECRTPAb1 (67nM).
Twenty four hours later BrdU staining was assayed as described in Methods
of Example 2 and the percentage of BrdU positive cells scored by counting of
five independent fields for each condition (greater than 400 cells/point).
Data
represent means ~ SEM.
Figure 11 is a series of photographs depicting that ECRTPAb1 inhibits
corneal pocket angiogenesis responses to bFGF. Hydron pellets were
impregnated with the angiogenesis stimulant, basic FGF (90ng), alone, or
supplemented with a class matched control monoclonal antibody (IgG, 200ng)
or ECRTPAb1 (200ng), and placed in a pocket created in the corneal
epithelium of anesthetized mice. Five days after implantation, angiogenic
responses were scored, and photographed. Representative examples show
inclusion of the ECRTPAb1 inhibits the zone of proliferation around the
implanted pellet.
Figure 12 is an autoradiograph showing that ECRTPAb-1 binds peptide
sequence QSRDTEVL of ECRTP/DEP-1 ectodomain. A "peptides on paper"
(Research Genetics Inc., Huntsville, Alabama) array was generated using the
351 amino acid sequence of ECRTP which ECRTPAb-1 binds. The array
comprises 96 peptides of 8 - 10 amino acids spanning the entire region in
overlapping sequences. A single peptide sequence (#41 ) in the series
represents amino acid residues in n-QSRDTEVL-c.
Figure 13A is a combination of autoradiographs and a graph depicting
that ECRTPAB-1 promotes dephosphorylation of ECRTP and arrests growth
of transfected CHO cells expressing wild type ECRTP, but not mutant ECRTP
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proteins, C/S (catalytically inactivated point mutant) or cy (a cytoplasmic
domain deletion). CHO cells were replated at 24 hours in the presence of
ECRTPAb-1, ECRTPAb-1-Fab, or control IgG1 and assayed for cell number
at 48 hours after transfection. In right panels, cells were transfected,
cultured
for 48 hours in serum-containing medium (5%), and exposed to 70nM of
ECRTPAb-1 or ECRTPAb-1-Fab for the times indicated. ECRTP was
immunoprecipitated and assayed by immunoblot for phosphotyrosine confient
(anti-PY) and antigen recovery (anti-HA), as indicated. ECRTPAb-1, but not
ECRTPAb-1-Fab, promoted acute dephosphorylation of wt but not catalytically
inactive ECRTP.
Figure 13B is a combination of autoradiographs and a graph
demonstrating that cotransfection of wild type ECRTP with either C/S or cy
forms abrogates the dephosphorylation of ECRTI? imposed by exposure of
cells to ECRTPAb-1. These mutant forms function as dominant negative
proteins to block the ECRTPAb-1-induced formation of catalytically active
ECRTP dimers that arrest cell growth and promote ECRTP dephosphorylation.
Detailed Description of the Invention
A mammalian transmembrane protein gene product called DEP-1 (for
density enhanced phosphatase), ECRTP, PTPRJ, HPTPr), CD148, BYP,
depending upon species and cDNA origin), was initially cloned from fibroblasts
and was subsequently shown to be expressed (hereinafter referred to as an
"ECRTPIDEP-1 ") on all hematopoietic lineages (de la Fuente-Garcia et al.,
Blood 91:2800-2809 (1998), including erythroid progenitor cells,
megakaryocytes and platelets, lymphocytes, polymorphononuclear leukocytes
and platelets, and very prominently in endothelial cells. Borges et al.,
Circulation Research 79:570-580 (1996), Schoecklmann et al., J Am Soc
Nephro15:730 (1994)(abstract). This gene product has been shown to promote
differentiation of erythroid progenitor cells (Kumet et al., J Biol Chem
271:30916-30921 (1996)), to modulate lymphocyte function when crosslinked
with other signaling proteins (de la Fuente-Garcia et al., Blood 91:2800-2809
(1998)); and to inhibit clonal expression of breast cancer cell lines
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overexpressing the protein (Keane et al., Cancer Research 56:4236-4243
(1996)).
I n accordance with the present invention, it has been demonstrated that
antibodies specific for ectodomain epitopes of the ECRTP/DEP-1 block
endothelial migration and proliferation in response to phorbol myristate
acetate
and fetal bovine serum respectively. It is recognized that the biological
activity
to inhibit endothelial proliferation and migration is a strong indicator of
angiogenesis inhibitory activity. Accordingly, the ECRTP/DEP-1 is also a
mediator of inhibitory signals that block angiogenesis.
In accordance with the present invention, then, antibodies that
aggregate the ECRTP/DEP-1, including monoclonal antibody ECRTPAb-1
described herein, inhibifiangiogenesis. Indeed, monoclonal antibodies against
the ectodomain of ECRTP/DEP-1 inhibit proliferation (as demonstrated by
BrdU uptake experiments) and migration of endothelial cells. Fab fragments
of the same monoclonal have no such activity. Accordingly, such monoclonal
ECRTP/DEP-1 antibodies described herein and derivatives thereof, have
biological activity as angiogenesis inhibitors.
An endogenous ligand for the receptor ectodomain signals endothelial
growth arrest. Therefore, in accordance with the present invention, a method
of screening for the endogenous ligand is provided. For example, the
endogenous ligand is isolated through the preparation of fusion proteins of
the
ECRTP/DEP-1 ectodomain as affinity reagents to identify, establish assays for,
and clone the putative natural ligand expressed on endothelial cells. The
purified and isolated endogenous ligand thus also has therapeutic application
as an angiogenesis inhibitor.
In accordance with the present invention, synthetic peptides and
peptidomimetics can also be used to contact the ECRTP/DEP-1 to activate
ECRTP/DEP-1 activity.
The ECRTP/DEP-1 is expressed on the luminal and interendothelial
membranes of endothelial cells in microvascular and large arterial vessels of
kidney and other organs, including but not limited to heart, spleen, muscle
and
skin. The ECRTP/DEP-1 localizes to interendothelial contacts in cultured
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endothelial cells, and in regions that overlap, but localization is not
limited to
the VE cadherin rich functional complexes. ECRTP/DEP-1 activity (tyrosine
phosphatase activity) increases approximately two times in confluent cells
anticipating density mediated growth arrest. Moreover, over-expression of
ECRTP/DEP-1 confers growth arrest on subconfluent endothelial cells. Thus,
in accordance with the present invention, a method of modulating
ECRTP/DEP-1 activity by contacting an ECRTP/DEP-1 with an ECRTP/DEP-1
modulating composition is contemplated. A method of screening for such a
composition is also contemplated. Finally, a method of targeting a therapeutic
composition to interendothelial contacts by preparing an antibody which
preferably binds the ECRTP/DEP-1 and which is bound to the therapeutic
composition in provided in accordance with the present invention.
A. General Considerations
The present invention relates generally to the discovery that
angiogenesis is modulated by the ECRTP/DEP=1 and that activation of
ECRTP/DEP-1 function inhibits angiogenesis. This discovery is important
because of the role that angiogenesis plays in a variety of disease processes.
By modulating angiogenesis, one can intervene in the disease, ameliorate the
symptoms, and in some cases cure the disease.
Where the growth of new blood vessels is the cause of, or contributes
to, the pathology associated with a disease, inhibition of angiogenesis will
reduce the deleterious effects of the disease. Examples include rheumatoid
arthritis, diabetic retinopathy, and the like. Where the growth of new blood
vessels is required to support growth of a deleterious tissue, inhibition of
angiogenesis will reduce the blood supply to the tissue and thereby contribute
to reduction in tissue mass based on blood supply requirements. Examples
include growth of tumors where neovascularization is a continual requirement
in order that the tumor grow beyond a few millimeters in thickness, and for
the
establishment of solid tumor metastases.
The methods of the present invention are effective in part because the
therapy is highly selective for angiogenesis and not other biological
processes.
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As shown in the Examples, the ECRTP/DEP-1 localizes to endothelial cells and
thus, primarily new vessel growth contains substantial ECRTP/DEP-1, and
therefore the therapeutic methods do not adversely effect mature vessels.
Furthermore, the ECRTP/DEP-1 is not widely distributed in normal tissues, but
rather is found selectively on the surface of endothelial cells, thereby
assuring
that the therapy can be selectively targeted.
The discovery that binding the ECRTPIDEP-1 will effectively inhibit
angiogenesis allows for the development of therapeutic compositions with
potentially high specificity, and therefore relatively low toxicity. Thus
although
the invention discloses the preferred use of an anti-ECRTP/DEP-1 monoclonal
antibody, one can design reagents which selectively bind ECRTP/DEP-1, and
therefore do not have the side effect of modulating other biological processes
other that those mediated by ECRTP/DEP-1.
As shown by the present teachings, it is possible to,prepare monoclonal
antibodies highly selective for immunoreaction with the ECRTP/DEP-1 that are
similarly selective for modulation of ECRTP/DEP-1 function. In addition,
peptides can be designed to be selective for binding to ECRTP/DEP-1, as
described further herein. Prior to the discoveries of the present invention,
it
was not known that angiogenesis could be modulated in vivo by the use of
reagents that modulate the biological function of ECRTP/DEP-1 or other
receptor tyrosine phosphatase.
Other related methods are described in U.S. Patent Nos. 5,753,230;
5,733,876; 5,762,918; 5,776,427; 5,766,591; and 5,660,827, the entire
contents of each of which are herein incorporated by reference.
Following long-standing patent law convention, the terms "a" and "an"
mean "one or more" when used in this application, including the claims.
B. Methods For Modulating of Angiogenesis
The invention provides for a method for the modulation of angiogenesis
in a tissue, and thereby modulating events in the tissue which depend upon
angiogenesis. Generally, the method comprises administering to the tissue a
composition comprising an angiogenesis-modulating amount of an
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ECRTPIDEP-1 modulator. As disclosed herein the term "modulate" is meant
to encompass both the inhibition or stimulation of angiogenesis. Thus, the
therapeutic methods of the present invention pertain to both the inhibition or
stimulation of angiogenesis, depending on the disorder to be treated.
Angiogenesis includes a variety of processes involving
neovascularization of a tissue including "sprouting", vasculogenesis, or
vessel
enlargement, al! of which angiogenesis processes are mediated by and
dependent upon the expression of ECRTP/DEP-1. With the exception of
traumatic wound healing, corpus luteum formation and embryogenesis, it is
believed that many angiogenesis processes are associated with disease
processes.
There are a variety of diseases in which angiogenesis is believed to be
important, referred to as angiogenic diseases, including but not limited to,
inflammatory disorders such as immune and non-immune inflammation,
chronic articular rheumatism and psoriasis, disorders associated with
inappropriate or inopportune invasion of vessels such as diabetic retinopathy,
neovascular glaucoma, capillary proliferation in atherosclerotic plaques and
osteoporosis, and cancer associated disorders, such as solid tumors, solid
tumor metastases, angiofibromas, retrolental fibroplasia, hemangiomas,
Karposi's sarcoma and the like cancers which require neovascularization to
support tumor growth.
Thus, methods which inhibit angiogenesis in a diseased tissue
ameliorates symptoms of the disease and, depending upon the disease, can
contribute to cure of the disease. In one embodiment, the invention pertains
to inhibition of angiogenesis, per se, in a tissue. The extent of angiogenesis
in
a tissue, and therefore the extent of inhibition achieved by the present
methods, can be evaluated by a variety of methods, such as are described in
the Examples for detecting an ECRTP/DEP-1-immunopositive immature and
nascent vessel structures by immunohistochemistry.
As described herein, any of a variety of tissues, or organs comprised of
organized tissues, can support angiogenesis in disease conditions including
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skin, muscle, gut, connective tissue, joints, bones and the like tissue in
which
blood vessels can invade upon angiogenic stimuli.
Thus, in one related embodiment, a tissue to be treated is an inflamed
tissue and the angiogenesis to be inhibited is inflamed tissue angiogenesis
where there is neovascularization of inflamed tissue. In this class the method
contemplates inhibition of angiogenesis in arthritic tissues, such as in a
patient
with chronic articular rheumatism, in immune or non-immune inflamed tissues,
in psoriatic tissue and the like.
The patienfi treated in the present invention in its many embodiments is
desirably a human patient, although it is to be understood that the principles
of
the invention indicate that the invention is effective with respect to all
vertebrate
species, including mammals, which are intended to be included in the term
"patient". In this context, a mammal is understood to include any mammalian
species in which treatment of diseases associated with angiogenesis is
desirable, particularly agricultural and domestic mammalian species.
The methods of the present invention are particularly useful in the
treatment of warm-blooded vertebrates. Therefore, the invention concerns
mammals and birds.
More particularly, contemplated is the treatment of mammals such as
humans, as well as those mammals of importance due to being endangered
(such as Siberian tigers), of economical importance (animals raised on farms
for consumption by humans) and/or social importance (animals kept as pets or
in zoos) to humans, for instance, carnivores other than humans (such as cats
and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle,
oxen,
sheep, giraffes, deer, goats, bison, and camels), and horses. Also
contemplated is the treatment of birds, including the treatment of those kinds
of birds that are endangered, kept in zoos, as well as fowl, and more
particularly domesticated fowl, i.e., poultry, such as turkeys, chickens,
ducks,
geese, guinea fowl, and the like, as they are also of economical importance to
humans. Thus, contemplated is the treatment of livestock, including, but not
limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry,
and
the like.
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In another related embodiment, a tissue to be treated is a retinal tissue
of a patient with diabetic retinopathy, macular degeneration or neovascular
glaucoma and the angiogenesis to be inhibited is retinal tissue angiogenesis
where there is neovascularization of retinal tissue.
!n an additional related embodiment, a tissue to be treated is a tumor
tissue of a patient with a solid tumor, a metastases, a skin cancer, a
hemangioma or angiofibroma and the like cancer, and the angiogenesis to be
inhibited is tumor tissue angiogenesis where there is neovascularization of a
tumor tissue.
Inhibition of tumor tissue angiogenesis is a particularly preferred
embodiment because of the important role neovascularization plays in tumor
growth. In the absence of neovascularization of tumor tissue, the tumor tissue
does not obtain the required nutrients, slows in growth, ceases additional
growth, regresses and ultimately becomes necrotic resulting in killing of the
tumor. Stated differently, the present invention provides for a method of
modulating tumor neovascularization by modulating tumor angiogenesis
according to the present methods. Similarly, the invention provides a method
of modulating tumor growth by practicing the angiogenesis-modulating
methods.
The methods are also particularly effective against the formation of
metastases because (1 ) their formation requires vascularization of a primary
tumor so that the metastatic cancer cells can exit the primary tumor and (2)
their establishment in a secondary site requires neovascularization to support
growth of the metastases.
In a related embodiment, the invention pertains to the practice of the
method in conjunction with othertherapies such as conventional chemotherapy
or surgery directed against solid tumors and for control of establishment of
metastases. The administration of angiogenesis inhibitor can be conducted
before, during or after chemotherapy or surgery. For example, the
angiogenesis inhibition methods of the present invention can be practiced for
chronic maintenance. As additional example, the angiogenesis inhibition
methods of the present invention can be practiced after a regimen of
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chemotherapy at times where the tumor tissue will be responding to the toxic
assault by inducing angiogenesis to recover by the provision of a blood supply
and nutrients to the tumor tissue. As a further example, the angiogenesis
inhibition methods of the present invention can be practiced after surgery
where solid tumors have been removed as a prophylaxis against metastases.
The present method for modulating angiogenesis in a tissue
contemplates contacting a tissue in which angiogenesis is occurring, or is at
risk for occurring, with a composition comprising a therapeutically effective
amount of an ECRTP/DEP-1 modulator capable of binding the ECRTP/DEP-1.
Thus, the method comprises administering to a patient a therapeutically
effective amount of a physiologically tolerable composition containing an
ECRTP/DEP-1 modulator of the invention.
The dosage ranges for the administration of the ECRTP/DEP-1
modulator depend upon the form of the modulator, and its potency, as
described further herein, and are amounts large enough to produce the desired
effect in which angiogenesis and the disease symptoms mediafied by
angiogenesis are ameliorated. The dosage should not be so large as to cause
adverse side effects, such as hyperviscosity syndromes, pulmonary edema,
congestive heart failure, and the like. Generally, the dosage will vary with
the
age, condition, sex and extent of the disease in the patient and can be
determined by one of skill in the art. The dosage can also be adjusted by the
individual physician in the event of any complication.
A therapeutically effective amount is an amount of an ECRTP/DEP-1
receptor modulator sufficient to produce a measurable inhibition of
angiogenesis in the tissue being treated, i.e., an angiogenesis-modulating
amount. Inhibition of angiogenesis can be measured in situ by
immunohistochemistry, as described herein, or by other methods known to one
skilled in the art.
Insofar as an ECRTP/DEP-1 modulator can take the form of an
ECRTPIDEP-1 ligand mimetic, and an anti-ECRTP/DEP-1 monoclonal
antibody, or fragment thereof, it is to be appreciated that the potency, and
therefore an expression of a "therapeutically effective" amount can vary.
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However, as shown by the present assay methods, one skilled in the art can
readily assess the potency of a candidate ECRTP/DEP-1 modulator of the
present invention.
ECRTP/DEP-1 modulator can be measured by a variety of means
including inhibition of angiogenesis in the mouse corneal assay for
angiogenesis described herein, binding of natural ligand' or monoclonal
antibody to an ECRTP/DEP-1 as described herein, and the like assays,
A preferred ECRTP/DEP-1 modulator has the ability to substantially bind
to an ECRTP/DEP-1 in solution at modulator concentrations of less than one
(1 ) micro molar (pM), preferably less than 0.1 pM, and more preferably less
than 0.01 pM. By "substantially" is meant that at least a 50 percent reduction
in endothelial cell proliferation and migration is observed by modulation in
the
presence of the an ECRTP/DEP-1 modulator, and at 50% reduction is referred
to herein as an IC50 value.
AtherapeuticallyeffectiveamountofanECRTP/DEP-1 modulatorofthe
present invention in the form of a monoclonal antibody, or fragment thereof,
is
typically an amount such that when administered in a physiologically tolerable
composition is sufficient to achieve a plasma concentration of from about 0.01
microgram (ug) per milliliter (mL) to about 100 ug/mL, preferably from about 1
ug/mL to about 5 ug/mL, and usually about 5 ug/mL. For example, for Mab
ECRTP/DEP-1 (MW = about 150kDa), 10 pg/mL ~ 67 X 10-9 M. Stated
differently, the dosage can vary from about 0.1 mg/kg to about 300 mg/kg,
preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from
about 0.5 mg/kg to about 20 mglkg, in one or more dose administrations daily,
for one or several days.
AtherapeuticallyeffectiveamountofanECRTP/DEP-1 modulatorofthe
present invention in the form of a polypeptide is typically an amount of
polypeptide such that when administered in a physiologically tolerable
composition is sufficient to achieve a plasma concentration of from about
0.001
microgram (pg) per milliliter (mL) to about 10 pg/mL, preferably from about
0.05
pg/mL to about 1.0 ug/mL. Based on a polypeptide having a mass of about
15,000 grams per mole (i.e. 15,000 Da), the preferred plasma concentration in
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molarity is from about 0.0001 micro molar (pM) to about 1 milli molar (mM).
Stated differently, the dosage per body weight can vary from about 0.01 mg/kg
to about 30 mg/kg, and preferably from about 0.05 mg/kg to about 20 mg/kg,
in one or more dose administrations daily, for one or several days.
The monoclonal antibodies or polypeptides of the invention can be
administered parenterally by injection or by gradual infusion over time.
Although the tissue to be treated can typically be accessed in the body by
systemic administration and therefore most often treated by intravenous
administration of therapeutic compositions, other tissues and delivery means
are contemplated where there is a likelihood that the tissue targeted contains
the target molecule. Thus, monoclonal antibodies or polypeptides of the
invention can be administered intravenously, intraperitoneally,
intramuscularly,
subcutaneously, intra-cavity, transdermally, and can be delivered by
peristaltic
means.
The therapeutic compositions containing a monoclonal antibody or a
polypeptide of the present invention . are conventionally administered
intravenously, as by injection of a unit dose, for example. The term "unit
dose"
when used in reference to a therapeutic composition of the present invention
refers to physically discrete units suitable as unitary dosage for the
subject,
each unit containing a predetermined quantity of active material calculated to
produce the desired therapeutic effect in association with the required
diluent;
i.e'., carrier or vehicle.
The compositions are administered in a manner compatible with the
dosage formulation, and in a therapeutically effective amount. The quantity to
be administered depends on the subject to be treated, capacity of the
subject's
system to utilize the active ingredient, and degree of therapeutic effect
desired.
Precise amounts of active ingredient required to be administered depend on
the judgement of the practitioner and are peculiar to each individual.
However,
suitable dosage ranges for systemic application are disclosed herein and
depend on the route of administration. Suitable regimes for administration are
also variable, but are typified by an initial administration followed by
repeated
doses at one or more hour intervals by a subsequent injection or other
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administration. Alternatively, continuous intravenous infusion sufficient to
maintain concentrations in the blood in the ranges specified for in vivo
therapies are contemplated.
C. Therapeutic Compositions
The present invention contemplates therapeutic compositions useful for
practicing the therapeutic methods described herein. Therapeutic
compositions of the present invention contain a physiologicallytolerable
carrier
together with an ECRTP/DEP-1 modulator as described herein, dissolved or
dispersed therein as an active ingredient. In a preferred embodiment, the
therapeutic ECRTP/DEP-1 modulator composition is not immunogenic when
administered to a mammal or human patient for therapeutic purposes.
As used herein, the terms "pharmaceutically acceptable",
"physiologically tolerable" and grammatical variations thereof, as they refer
to
compositions, carriers, diluents and reagents, are used interchangeably and
represent that the materials are capable of administration to or upon a mammal
without the production of undesirable physiological effects such as nausea,
dizziness, gastric upset and the like.
The preparation of a pharmacological composition that contains active
ingredients dissolved or dispersed therein is well understood in the art~and
need not be limited based on formulation. Typically such compositions are
prepared as injectables either as liquid solutions or suspensions; however,
solid forms suitable for solution, or suspensions, in liquid prior to use can
also
be prepared. The preparation can also be emulsified.
The active ingredient can be mixed with excipients which are
pharmaceutically acceptable and compatible with the active ingredient and in
amounts suitable for use in the therapeutic methods described herein. Suitable
excipients are, for example, wafier, saline, dextrose, glycerol, ethanol orthe
like
and combinations thereof. In addition, if desired, the composition can contain
minor amounts of auxiliary substances such as wetting or emulsifying agents,
pH buffering agents and the like which enhance the effectiveness of the active
ingredient.
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The therapeutic composition. of the present invention can include
pharmaceutically acceptable salts of the components therein.
Pharmaceutically acceptable salts include the acid addition salts (formed with
the free amino groups of the polypeptide) that are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such organic acids
as acetic, tartaric, mandelic and the like. Salts formed with the free
carboxyl
groups can also be derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium or ferric hydroxides, and such organic bases
as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine
and the like.
Physiologically tolerable carriers are well known in the art. Exemplary
of liquid carriers are sterile aqueous solutions that contain no materials in
addition to the active ingredients and water, or contain a buffer such as
sodium
phosphate at physiological pH value, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous carriers can contain more
than one buffer salt, as well as salts 'such as sodium and potassium
chlorides,
dextrose, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in addition to and to
the exclusion of v~iater. Exemplary of such additional liquid phases are
glycerin,
vegetable oils such as cottonseed oil, and water-oil emulsions.
A therapeutic composition contains an angiogenesis-modulating amount
of an ECRTP/DEP-1 modulator of the present invention, typically formulated
to contain an amount of at least 0.1 weight percent of modulator per weight of
total therapeutic composition. A weight percent is a ratio by weight of
modulator to total composition. Thus, for example, 0.1 weight percent is 0.1
grams of inhibitor per 100 grams of total composition.
D. Modulators of ECRTP/DEP-1
ECRTP/DEP-1 modulators are used in the present methods for
modulating ECRTP/DEP-1 activity in tissues, including modulating
angiogenesis in tissues. Thus, as used herein, the terms "modulate",
"modulating", and "modulator" are meant to be construed to encompass
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inhibiting, blocking, promoting, stimulating, agonising, antagonizing, or
otherwise affecting ECRTP/DEP-1 activity in tissues.
Such modulators can take a variety of forms that include compounds
which interact with the ECRTP/DEP-1 in a manner such that functional
interactions with natural ECRTP/DEP-1 ~ligands are mimicked, stimulated
and/or ~ inhibited, such as, for example, dimerization of ECRTP/DEP-1.
Exemplary modulators include analogs of an ECRTP/DEP-1 natural ligand
binding site on an ECRTP/DEP-1, mimetics of a natural ligand of an
ECRTP/DEP-1 that mimic the structural region involved in an ECRTP/DEP-1-
receptor ligand binding interactions, polypeptides having a sequence
corresponding to the domain of a natural ligand of an ECRTP/DEP-1, and
antibodies which immunoreact with either an ECRTP/DEP-1 or the natural
ligand, all of which exhibit modulator activity as defined herein.
1. Polypeptides
In one embodiment, the invention contemplates ECRTP/DEP-1
modulators in the form of polypeptides. A polypeptide (peptide) ECRTP/DEP-1
modulator interacts with the extracellular domain of ECRTP/DEP-1 and
promotes dimerization of ECRTP/DEP-1. A preferred ECRTP/DEP-1
modulator peptide corresponds in sequence to the natural ligand and promotes
or antagonizes dimerization of ECRTP/DEP-1 ECRTP/DEP-1.
In one embodiment, a polypeptide of the present invention comprises
no more than about 100 amino acid residues, preferably no more than about
60 residues, more preferably no more than about 30 residues. Peptides can be
linear or cyclic. Thus, it should be understood that a subject polypeptide
need
not be identical to the amino acid residue sequence of an ECRTP/DEP-1
natural ligand, so long as it includes required binding sequences and is able
to
function as an ECRTP/DEP-1 modulator in an assay such as is described
herein.
A subject polypeptide includes any analog, fragment or chemical
derivative of a polypeptide which is an ECRTP/DEP-1 modulator. Such a
polypeptide can be subject to various changes, substitutions, insertions, and
deletions where such changes provide for certain advantages in its use. I n
this
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regard, an ECRTP/DEP-1 modulator polypeptide of the present invention
corresponds to, rather than is identical to, the sequence of the natural
ligand
where one or more changes are made and it retains the ability to function as
an ECRTP/DEP-1 modulator in one or more of the assays as defined herein.
Thus, a polypeptide can be in any of a variety of forms of peptide
derivatives,
that include amides, conjugates with proteins, cyclized peptides, polymerized
peptides, analogs, fragments, chemically modified peptides, and the like
derivatives.
The term "analog" includes any polypeptide having an amino acid
residue sequence substantially identical to a sequence of the natural ligand
of
the ECRTP/DEP-1 in which one or more residues have been conservatively
substituted with a functionally similar residue and which displays the
ECRTP/DEP-1 modulator activity as described herein. Examples of
conservative substitutions include the substitution of one non-polar
(hydrophobic) residue such as isoleucine, valine, leucine or methionine for
another; the substitution of one polar (hydrophilic) residue for another such
as
between arginine and lysine, between glutamine and asparagine, between
glycine and serine; the substitution of one basic residue such as lysine,
arginine or histidine for another; orthe substitution of one acidic residue,
such
as aspartic acid or glutamic acid for another.
The phrase "conservative substitution" also includes the use of a
chemically derivatized residue in place of a non-derivatized residue provided
that such polypeptide displays~the requisite inhibition activity.
"Chemical derivative" refers to a subject polypeptide having one or more
residues chemically derivatized by reaction of a functional side group. Such
derivatized molecules include for example, those molecules in which free
amino groups have been derivatized to form amine hydrochlorides, p-toluene
sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl
groups or formyl groups. Free carboxyl groups can be derivatized to form
salts,
methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole
nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Also
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included as chemical derivatives are those peptides which contain one or more
naturally occurring amino acid derivatives of the twenty standard amino acids.
For examples: 4-hydroxyproline can be substituted for proline; 5-hydroxylysine
can be substituted for lysine; 3-methylhistidine can be substituted for
histidine;
homoserine can be substituted for serine; and ornithine can be substituted for
lysine. Polypeptides of the present invention also include any polypeptide
having one or more additions and/or deletions or residues relative to the
sequence of a polypeptide whose sequence is shown herein, so long as the
requisite activity is maintained. The term "fragment" refers to any subject
polypeptide having an amino acid residue sequence shorter than that of a
polypeptide disclosed herein.
When a polypeptide of the present invention has a sequence that is not
identical to the sequence of an EC~RTP/DEP-1 natural ligand, it is typically
because one or more conservative or non-conservative substitutions have been
made, usually no more than about 30 number percent, and preferably no more
than 10 number percent of the amino acid residues are substituted. Additional
residues can also be added at either terminus of a polypeptide for the purpose
of providing a "linker" by which the polypeptides of the present invention can
be conveniently affixed to a label or solid matrix, or carrier. Labels, solid
matrices and carriers that can be used with the polypeptides of the present
invention are described hereinbelow.
Amino acid residue linkers are usually at feast one residue and can be
40 or more residues, ,, more often 1 to 10 residues, but do not form
ECRTP/DEP-1 ligand epitopes. Typical amino acid residues used for linking
are tyrosine, cysteine, lysine, glutamic and aspartic acid, orthe like. In
addition,
a subject polypeptide can differ, unless otherwise specified, from the natural
sequence of an ECRTP/DEP-1 ligand by the sequence being modified by
terminal-NH2 acylation, e.g., acetylation, or thioglycolic acid amidation, by
terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like
terminal modifications. Terminal modifications are useful, as is well known,
to
reduce susceptibility by proteinase digestion, and therefore serve to prolong
half life of the polypeptides in solutions, particularly biological fluids
where
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proteases can be present. In this regard, polypeptide cyclization is also a
useful terminal modification, and is particularly preferred also because of
the
stable structures formed by cyclization and in view of the biological
activities
observed for such cyclic peptides as described herein.
Any peptide of the present invention can be used in the form of a
pharmaceutically acceptable salt. Suitable acids which are capable of the
peptides with the peptides of the present invention include inorganic acids
such
as trifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid,
perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric
acetic acid,
propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic
acid,
succinic acid, malefic acid, fumaric acid, anthranilic acid, cinnamic acid,
naphthalene sulfonic acid,' sulfanilic acid or the like. NCI and TFA salts are
particularly preferred.
Suitable bases capable of forming salts with the peptides of the present
invention include inorganic bases such as sodium hydroxide, ammonium
hydroxide, potassium hydroxide and the like; and organic bases such as mono-
di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropyl amine,
methyl
amine, dimethyl amine and the like), and optionally substituted ethanolamines
(e.g. ethanolamine, diethanolamine and the like).
A peptide of the present invention, also referred to herein as a subject
polypeptide, can be synthesized by any of the techniques that are known to
those skilled in the polypeptide art, including recombinant DNA techniques.
Synthetic chemistry techniques, such as a solid-phase Merrifield-type
synthesis, are preferred for reasons of purity, antigenic specificity, freedom
from undesired side products, ease of production and the like. An excellent
summary of the many techniques available can be found in Steward et al.,
"Solid Phase Peptide Synthesis", W. H. Freeman Co., San Francisco, 1969;
Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition,
1976; J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46,
Academic Press (NewYork),1983; Merrifield,AdvEnzymol, 32:221-96, 1969;
Fields et al., Int. J. Peptide Protein Res., 35:161-214, 1990; and U.S. Pat.
No.
4,244,946 for solid phase peptide synthesis, and Schroder et al., "The
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Peptides", Vol. 1, Academic Press (New York), 1965 for classical solution
synthesis, each of which is incorporated herein by reference. Appropriate
protective groups usable in such synthesis are described in the above texts
and
in J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press,
New York, 1973, which is incorporated herein by reference.
In general, the solid-phase synthesis methods contemplated comprise
the sequential addition of one or more amino acid residues or suitably
protected amino acid residues to a growing peptide chain. Normally, either the
amino or carboxyl group of the first amino acid residue is protected by a
suitable, selectively removable protecting group. a different, selectively
removable protecting group is utilized foramino acids containing a reactive
side
group such as lysine.
Using a solid phase synthesis as exemplary, the protected orderivatized
amino acid is attached to an inert solid support through its unprotected
carboxyl
or amino group. The protecting group of the amino or carboxyl group is then
selectively removed and the next amino acid in the sequence having the
complimentary (amino or carboxyl) group suitably protected is admixed and
reacted under conditions suitable forforming the amide linkage with the
residue
already attached to the solid support. The protecting group of the amino or
carboxyl group is then removed from this newly added amino acid residue, and
the next amino acid (suitably protected) is then added, and so forth. After
all
the desired amino acids have been linked in the proper sequence, any
remaining terminal and side group protecting groups (and solid support) are
removed sequentially or concurrently, to afford the final linear polypeptide.
The resultant linear polypeptides prepared for example as described
above can be reacted to form their corresponding cyclic peptides. An
exemplary method for cyclizing peptides is described by Zimmer et al.,
Peptides 1992, pp. 393-394, ESCOM Science Publishers, B. V., 1993.
Typically, tertbutoxycarbonyl protected peptide methyl ester is dissolved in
methanol and sodium hydroxide solution are added and the admixture is
reacted at 20°C to hydrolytically remove the methyl ester protecting
group.
After evaporating the solvent, the tertbutoxycarbonyl protected peptide is
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extracted with ethyl acetate from acidified aqueous solvent. The
tertbutoxycarbonyl protecting group is then removed under mildly acidic
conditions in dioxane cosolvent. The unprotected linear peptide with free
amino
and carboxytermini so obtained is converted to its corresponding cyclic
peptide
by reacting a dilute solution of the linear peptide, in a mixture of
dichloromethane and dimethylformamide, with dicyclohexylcarbodiimide in the
presence of 1-hydroxybenzotriazole and N-methylmorpholine. The resultant
cyclic peptide is then purified by chromatography.
2. Antibodies
The present invention describes, in one embodiment, ECRTP/DEP-1
modulators in the form of antibodies, including monoclonal antibodies, which
immunoreactwith an ECRTP/DEP-1 and bind the ECRTP/DEP-1 to modulate
receptor activity as described herein. The invention also describes cell lines
which produce the antibodies, methods for producing the cell lines, and
methods for producing the antibodies, including monoclonal antibodies.
An antibody of the present invention can comprise an antibody molecule
that 1 ) immunoreact with isolated ECRTP/DEP-1, and 2) bind to the
ECRTP/DEP-1 to modulate its biological function. Preferably, an antibody of
the present invention preferentially binds the ECRTP/DEP-1 ectodomain, which
comprises amino acids 1-351 of ECRTP/DEP-1. More preferably, an antibody
of the present invention preferentially binds an eight amino acid epitope
having
the sequence n-QSRDTEVL-c, or an eight amino acid epitope having an
analog sequence of the sequence n-QSRDTEVL-c, the term "analog" as
defined herein, of the ECRTP/DEP-1 ectodomain.
Preferred monoclonal antibodies which preferentially bind to
ECRTP/DEP-1 include a monoclonal antibody having the immunoreaction
characteristics of Mab ECRTPAb-1, having molecular weight of about 150 KDa
respectively and which binds to the ectodomain of the ECRTP/DEP-1, as is
described herein below. Mab ECRTPAb-1 is preferably secreted by hybridoma
cell line ATCC HB12570. The hybridoma cell line ATCC HB12570 was
deposited pursuant to Budapest Treaty requirements with the American Type
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Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia,
20110-2209, U.S.A., on September 18, 1998.
The term "antibody or antibody molecule" in the various gramrriatical
forms is used herein as a collective noun that refers to a population of
immunoglobulin molecules and/or immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antibody combining
site or paratope. An "antibody combining site" is that structural portion of
an
antibody molecule comprised of heavy and light chain variable and
hypervariable regions that specifically binds antigen.
Exemplary antibodies for use in the present invention are intact
immunoglobulin molecules, substantially intact immunoglobulin molecules,
single chain immunoglobulins or antibodies, those portions of an
immunoglobulin molecule that contain the paratope, including those portions
known in the art as Fab, Fab', F(ab')2 and F(v), and also referred to as
antibody fragments.
Indeed, as described in the Examples set forth below, an Fab fragment,
that is, a monovalent fragment, of the Mab ECRTPAb-1 releases density arrest.
Thus, it is contemplated to be within the scope of the present invention that
such a monovalent modulator is used to promote angiogenesis, or to promote
endothelial cell migration and proliferation, or to release inhibitory
influences
on endothelial cells to serve as an adjunctive to other angiogenic stimuli.
Thus,
the terms "modulate", "modulating", and "modulator" are meant to be construed
to encompass such promotion.
The phrase "monoclonal antibody" in its various grammatical forms
refers to a population of antibody molecules that contain only one species of
antibody combining site capable of immunoreacting with a particular epitope.
A monoclonal antibody thus typically displays a single binding affinity for
any
epitope with which it immunoreacts. A monoclonal antibody can therefore
contain an antibody molecule having a plurality of antibody combining sites,
each immunospecific for a different epitope, e.g., a bispecific monoclonal
antibody.
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A monoclonal antibody is typically composed of antibodies produced by
clones of a single cell called a hybridoma that secretes (produces) only one
kind of antibody molecule. The hybridoma cell is formed by fusing an antibody-
producing cell and a myeloma or other self-perpetuating cell line. The
preparation of such antibodies was first described by Kohler and Milstein,
Nature 256:495-497 (1975), which description is incorporated by reference.
Additional methods are described by Zola, MonoclonaiAntibodies: a Manual
of Techniques, CRC Press, Inc. (1987). The hybridoma supernates so
prepared can be screened for the presence of antibody molecules that
immunoreact with an ECRTP/DEP-1 and for inhibition of an ECRTP/DEP-1 to
activate its biological function.
Briefly, to form the hybridoma from which the monoclonal antibody
composition is produced, a myeloma or other self-perpetuating cell line is
fused
with lymphocytes obtained from the spleen of a mammal hyperimmunized with
a source of an ECRTP/DEP-1, as described by Cheresh et al., J. Biol Chem,
262:17703-17711 (1987).
It is preferred that the myeloma cell line used to prepare a hybridoma be
from the same species as the lymphocytes. Typically, a mouse of the strain
129 GIX+ is the preferred mammal, Suitable mouse myelomas for use in the
present invention include the hypoxanthine-aminopterin-thymidine-sensitive
(HAT) cell lines P3X63-Ag8.653, and Sp2/0-Ag14 that are available from the
ATCC, Manassas, Virginia, under the designations CRL 1580 and CRL 1581,
respectively.
Splenocytes are typically fused with myeloma cells using polyethylene
glycol (PEG) 1500. Fused hybrids are selected by their sensitivity to HAT.
Hybridomas producing a monoclonal antibody of the present invention are
identified using the enzyme linked immunosorbent assay (ELISA) described in
the Examples.
A monoclonal antibody of the present invention can also be produced
by initiating a monoclonal hybridoma culture comprising a nutrient medium
containing a hybridoma that secretes antibody molecules of the appropriate
specificity. The culture is maintained under conditions and for a time period
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sufficient for the hybridoma to secrete the antibody molecules into the
medium.
The antibody-containing medium is then collected. The antibody molecules can
then be further isolated by well known techniques. Media useful for the
preparation of these compositions are both well known in the art and
commercially available and include synthetic culture media, inbred mice and
the like. An exemplary synthetic medium is Dulbecco's minimal essential
medium (DMEM - Dulbecco et al., Virol 8:396 (1959)) supplemented with 4.5
gm/1 glucose, 20 mM glutamine, and 20% fetal calf serum. An exemplary
inbred mouse strain is the Balb/C.
Other methods of producing a monoclonal antibody, a hybridoma cell,
or a hybridoma cell culture are also well known. See, for example, the method
of isolating monoclonal antibodies from an immunological repertoire as
described by Sastry, et al., Proc Natl Acad Sci USA 86:5728-5732 (1989); and
Huse et al., Science 246:1275-1281 (1989).
Also contemplated by the present invention is the hybridoma cell, and
cultures containing a hybridoma cell that produce a monoclonal antibody of the
present invention. Particularly preferred is the hybridoma cell line that
secretes
monoclonal antibody Mab ECRTPAb-1 as described in the Examples
presented below and as designated ATCC HB12570. Mab ECRTPAb-1 was
prepared as described in the Examples. The invention thus contemplates, in
one embodiment, a monoclonal antibody that has the immunoreaction
characteristics of Mab ECRTPAb-1.
It is also possible to determine, without undue experimentation, if a
monoclonal antibody has the same (i.e., equivalent) specificity
(immunoreaction
characteristics) as a monoclonal antibody of the present invention by
ascertaining whether the former prevents the latter from binding to a
preselected target molecule. Ifthe monoclonal antibody being tested competes
with the monoclonal antibody of the invention, as shown by a decrease in
binding by the monoclonal antibody of the invention in standard competition
assays for binding to the target molecule when present in the solid phase,
then
it is likely that the two monoclonal antibodies bind to the same, or a closely
related, epitope. A preferred target molecule comprises a polypeptide fragment
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of the ECRTP/DEP-1 ectodomain includes an eight amino acid epitope having
the sequence n-QSRDTEVL-c, or an eight amino acid epitope having an
analog sequence of the sequence n-QSRDTEVL-c, the term "analog" as
defined herein.
Still another way to determine whether a monoclonal antibody has the
specificity of a monoclonal antibody of the invention is to pre-incubate the
monoclonal antibody of the invention with the target molecule with which it is
normally reactive, and then add the monoclonal antibody being tested to
determine if the monoclonal antibody being fiested is inhibited in its ability
to
bind the target molecule. If the monoclonal antibody being tested is inhibited
then, in all likelihood, it has the same, or functionally equivalent, epitopic
specificity as the monoclonal antibody of the invention. A preferred target
molecule comprises a polypeptide fragment of the ECRTP/DEP-1 ectodomain
includes an eight amino acid epitope having the sequence n-QSRDTEVL-c, or
an eight amino acid epitope having an analog sequence of the sequence n-
QSRDTEVL-c, the term "analog" as defined herein.
An additional way to determine whether a monoclonal antibody has the
specificity of a monoclonal antibody of the invention is to determine the
amino
acid residue sequence of the CDR regions of the antibodies in question.
Antibody molecules having identical, or functionally equivalent, amino acid
residue sequences in their CDR regions have the same binding specificity.
Methods for sequencing polypeptides are well known in the art.
The immunospecificity of an antibody, its target molecule binding
capacity, and the attendant affinity the antibody exhibits for the epitope,
are
defined by the epitope with which the antibody immunoreacts. The epitope
specificity is defined at least in part by the amino acid residue sequence of
the
variable region of the heavy chain of the immunoglobulin that comprises the
antibody, and in part by the light chain variable region amino acid residue
sequence. Use of the terms "having the binding specificity of or "having the
binding preference of indicates that equivalent monoclonal antibodies exhibit
the same or similar immunoreaction (binding) characteristics and compete for
binding to a preselected target molecule. Preferably, an antibody of the
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present invention preferentially binds an eight amino acid epitope having the
sequence n-QSRDTEVL-c, or an eight amino acid epitope having an analog
sequence of the sequence n-QSRDTEVL-c, the term "analog" as defined
herein, of the ECRTP/DEP-1 ectodomain.
Humanized monoclonal antibodies offer particular advantages over
murine monoclonal antibodies, particularly insofar as they can be used
therapeutically in humans. Specifically, human antibodies are not cleared from
the circulation as rapidly as "foreign" antigens, and do not activate the
immune
system in the same manner as foreign antigens and foreign antibodies.
Methods of preparing "humanized" antibodies are generally well known in the
art, and can readily be applied to the antibodies of the present invention.
Thus,
the invention contemplates, in one embodiment, a monoclonal antibody of the
present invention that is humanized by grafting to introduce components of the
human immune system without substantially interfering with the ability of the
antibody to bind antigen. Humanized antibodies can also be produced using
animals engineering to produce humanized antibodies, such as those available
from Medarex of Annandale, New Jersey (mice) and Abgenix, Inc., of Fremont,
California (mice).
The use of a molecular cloning approach to generate antibodies,
particularly monoclonal antibodies, and more particularly single chain
monoclonal antibodies, is also contemplated. The production of single chain
antibodies has been described in the art, see e.a., U.S. Patent No. 5,260,203,
the contents of which are herein incorporated by reference. For this,
combinatorial immunoglobulin phagemid or phage-displayed libraries are
prepared from RNA isolated from the spleen of the immunized animal, and
phagemids expressing appropriate antibodies are selected by panning on
endothelial tissue. This approach can also be used to prepared humanized
antibodies. The advantages of this approach over conventional hybridoma
techniques are that approximately 104 times as many antibodies can be
produced and screened in a single round, and that new specificities are
generated by H and L chain combination in a single chain, which further
increases the chance of finding appropriate antibodies. Thus, an antibody of
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the present invention, or a "derivative" of an antibody of the present
invention
pertains to a single polypeptide chain binding molecule which has binding
specificity and affinity substantially similar to the binding specificity and
affinity
of the light and heavy chain aggregate variable region of an antibody
described
herein, such as ECRTP/DEP-1. Preferably, an antibody of the present
invention preferentially binds an eight amino acid epitope having the sequence
n-QSRDTEVL-c, or an eight amino acid epitope having an analog sequence of
the sequence n-QSRDTEVL-c, the term "analog" as defined herein, of the
ECRTP/DEP-1 ectodomain.
"Fv" is the minimum antibody fragment which contains a complete
antigen-recognition and -binding site. In a two-chain Fv species, this region
consists of a dimer of one heavy- and one light-chain variable domain in
tight,
non-covalent association. In a single-chain Fv species (scFv), one heavy- and
one light-chain variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a "dimeric"
structure
analogous to that in a two-chain Fv species. It is in this configuration that
the
three CDRs of each variable domain interact to define an antigen-binding site
on the surface of the VH-VL dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single variable
domain (or half of an Fv comprising only three CDRs specific for an antigen)
has the ability to recognize and bind antigen, although at a lower affinity
than
the entire binding site. For a review of scFv see Pluckthun, in The
PharmacologyofMonoclonalAntibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
Using a phage-displayed approach forthe production of antibodies, scFv
antibody clones that bind to the ECRTP/DEP-1 ectodomain have been
identified, This was accomplished by competing off those phage displayed
antibodies using ECRTPAb-1 disclosed herein. Fv regions are sequenced an
bivalent functional reagents are designed and tested in a screening assay of
the present invention disclosed herein below. Thus, a preferred source for an
antibody, or derivative or fragment thereof, is a recombinant phage-displayed
antibody library. The recombinant phage can comprise antibody encoding
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nucleic acids isolated from any suitable vertebrate species, including
mammalian species such as mouse and rat; but preferably comprises antibody
encoding nucleic acids isolated from human. Such antibodies are thus already
"humanized".
3. Other Modulators
It is also envisioned that previously described and newly discovered
angiogenesis inhibiting or endothelial cell growth suppressing chemical
compounds are modulators of ECRTP/DEP-1 activity in tissues. Examples of
such compounds include, but are not limited to, angiostatin, endostatin and
thrombospondin. Accordingly, such compounds can be used in the modulation
of ECRTP/DEP-1 activity in tissues, according to the methods of the present
invention.
Given the disclosure of the ECRTP/DEP-1 activity in tissues herein, it
is also contemplated that as yet undefined chemical compounds can be used
to modulate ECRTP/DEP-1 activity in tissues in accordance with the methods
of the present invention. In one embodiment, a modulator of the present
invention interacts with an eight amino acid epitope having the sequence n-
QSRDTEVL-c of the ECRTP/DEP-1 ectodomain. In another embodiment, a
modulator of the present invention interacts with an intracellular catalytic
domain of ECRTP/DEP-1. The identification of such modulators is provided
through the description of screening assays directed to ECRTP/DEP-1 activity
in tissues presented herein.
D. Screening Assa~r
Skilled artisans will understand that the disclosure herein of the
localization and function of the ECRTP/DEP-1, and in vitro assays relating to
such localization and function, provides opportunities to screen for compounds
that modulate, whether partially or completely, the functional activity of the
ECRTP/DEP-1. In this context, "modulate" is intended to mean that the subject
compound increases or decreases one or more functional activities of the
ECRTP/DEP-1, such as but not limited to ECRTP/DEP-1 activity in cell growth,
cell survival and angiogenesis.
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Further, the screening assays illustrated in the Examples below include
biochemical assays (e.g., measuring effects of anti-ECRTP/DEP-1 monoclonal
antibodies on ECRTP/DEP-1 activity), and cellular in vitro assays (e.g.,
measuring the effects of ECRTP/DEP-1 over-expression on endothelial cell
proliferation and migration and/or evaluating ECRTP/DEP-1 phosphorylation).
The illustrative biochemical assays can be particularly useful in screening
for
compounds modulating an ECRTP/DEP-1 activity, while the cellular assays can
be particularly useful in screening for compounds completely altering an
ECRTP/DEP-1 activity. Thus, until the disclosure herein of the role of the
ECRTP/DEP-1 in regulating cell growth, cell survival, endothelial cell
proliferation and migration and in regulating angiogenesis, a motivation to
screen for compounds that modulate ECRTPIDEP-1 activity was lacking in the
prior art.
Those skilled in the art will understand that binding of a ligand at a
molecular binding site can be modulated in a direct matter (e.g., by blocking
the
site), as well as modulated in an indirect manner (e.g., by conformational
changes induced following binding of a second, i.e., different, ligand at a
distant
site). In this regard, it is likely that the binding site specificity of an
ECRTP/DEP-1 for its endogenous ligand can be completely modulated or
altered (i.e., to bind a different ligand) by agents that bind at distant
sites in the
ECRTP/DEP-1. Examples of compounds that can be screened in the latter
several assays include at least nucleic acids (e.g., DNA oligonucleotide
aptamers that bind proteins and alter their functions), proteins, antibodies
and
antibody fragments, carbohydrates, lectins, organic chemicals, and the like.
Such screening assays can be useful for identifying candidate therapeutic
agents that can provide drugs useful in animals and humans.
It is still further understood that due to the significance of the
ECRTP/DEP-1 in cell growth, cell survival, endothelial cell migration and
proliferation, in density induced growth arrest, and in modulation of
angiogenesis, innate regulatory mechanisms exist in cells for regulating their
activity by binding to an ECRTP/DEP-1, or to complexes containing an
ECRTP/DEP-1. Such regulatory factors can include, at least: (a) cofactors that
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bind to the complex and exert regulatory action by destabilizing or
stabilizing
the complex; (b) agents that modulate or alter the activity of the complex by
inducing conformational changes in the ECRTP/DEP-1 as they are bound in
a complex; (c) enzymes that inactivate one or both members of a complex; and
(d) cellular control factors (e.g., signal transduction second messengers,
transcription regulating factors, DNA replication factors and the like) that
bind
an ECRTP/DEP-1 or ECRTP/DEP-1 complexes and modulate or alter
functional activity. Those skilled in the art will recognize that the
functional
regions of an ECRTP/DEP-1 represent particularly attractive targets for three-
dimensional molecular modeling and for construction of mimetic compounds,
e.g., organic chemicals constructed to mimic the three-dimensional
interactions
between the ECRTP/DEP-1 and its endogenous binding partner, or other
binding partner.
Utilizing the methods and compositions of the present invention,
screening assays for the testing of candidate substances can be derived. A
candidate substance is a substance which potentially can modulate endothelial
cell growth, cell survival, cell migration and proliferation, density induced
growth
arrest and/or angiogenesis, and/or ECRTP/DEP-1 phosphorylation, by binding
or other intramolecular interaction, with an ECRTP/DEP-1 that modulates
endothelial cell growth, cell survival, cell migration and proliferation,
density
induced growth arrest and angiogenesis.
Thus, a method of screening candidate substances for an ability to
modulate ECRTP/DEP-1 biological activity is also disclosed. The method
comprises establishing a test sample comprising an ECRTP/DEP-1 polypeptide
or fragment thereof; administering a candidate substance to the test sample;
and measuring the interaction, effect, or combination thereof, of the
candidate
substance on the test sample to thereby determine the ability of the candidate
substance to modulate ECRTP/DEP-1 biological activity.
The present invention also provides a process of screening substances
for their ability to modulate or alter cell growth, cell survival, endothelial
cell
migration and proliferation, density induced growth arrest and/or angiogenesis
and/or ECRTP/DEP-1 phosphorylation comprising the steps of providing a cell
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that contains a functional ECRTP/DEP-1 and testing the ability of selected
subsfiances to modulate or alter cell growth, cell survival, migration or
proliferation of that cell, density induced growth arrest of the cell, or
initiation
of angiogenesis in the cell and/or evaluating ECRTP/DEP-1 phosphorylation
in the cell.
A screening assay of the present invention generally involves
determining the ability of a candidate substance to affect cell growth, cell
survival, endothelial cell migration and proliferation, density induced growth
arrest and/or angiogenesis, and/or ECRTP/DEP-1 phosphorylation in a target
cell, such as the screening of candidate substances to identify those that
modulate or alter cell growth, cell survival, endothelial cell migration and
proliferation, density induced growth arrest and/or angiogenesis, and/or
ECRTP/DEP-1 phosphorylation. Target cells can be either naturally occurring
cells known to contain an ECRTP/DEP-1 or transfected cell produced in
accordance with a process of transfection set forth herein and as are known in
the art.
Thus, in one embodiment a method of screening a candidate substance
for an ability to modulate a receptor tyrosine phosphatase in accordance with
the present invention comprises establishing test samples comprising a
receptor tyrosine phosphatase polypeptide; and administering a candidate
substance to the test samples; measuring the interaction, effect, or
combination thereof, of the candidate substance on the test sample to thereby
determine the ability of the candidate substance to modulate receptor tyrosine
phosphatase biological activity. A preferred receptor tyrosine phosphatase
comprises ECRTP/DEP-1.
In another embodiment, a method of screening a candidate substance
for an ability to modulate a receptor tyrosine phosphatase in accordance with
the present invention comprises establishing a test sample comprising a
receptortyrosine phosphatase; administering a candidate substance to the test
sample; and measuring a receptor tyrosine phosphatase biological activity in
the test sample; detecting phosphotyrosine residues on the receptor tyrosine
phosphatase; and determining that the candidate substance modulates the
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receptor tyrosine phosphatase if the receptor tyrosine phosphatase biological
activity measured for the test sample is greater or less than the receptor
tyrosine phosphatase biological activity measured for a control sample and if
the amount of phosphotyrosine residues on the receptor tyrosine phosphatase
is greater or less than an amount of phosphotyrosine residues on a receptor
tyrosine phosphate derived from a control sample. A preferred receptor
tyrosine phosphatase comprises ECRTP/DEP-1.
In yet another embodiment, a method of screening a candidate
substance for an ability to modulate a receptor tyrosine phosphatase in
accordance with the present invention comprises establishing replicate test
and
control cultures of cells that express the ECRTP/DEP-1; administering a
candidate compound to the cells in the test culture but not the control
culture;
measuring ECRTP/DEP-1 activity in cells in the test and the control cultures;
and determining that the candidate compound modulates the ECRTP/DEP-1
activity in a cell if the ECRTPIDEP-1 activity measured for the test culture
is
greater or less than the ECRTP/DEP-1 activity measured forthe control culture.
The screening assay methods of the present invention also pertain to
comparing the effect of a candidate compound to inhibit growth of cells
expressing exogenous ECRTPI,DEP-1 compared with those not expressing
ECRTP/DEP-1 and determining that the effect of altering ECRTP/DEP-1
activity is responsible by demonstrating the lack of activity of the candidate
compound on cells not expressing ECRTP/DEP-1. The screening assays of
the present invention show changes in growth that are responsive to changes
in ECRTP/DEP-1 activity. For example, the screening methods of the present
invention are used to screen for biologically active counter-receptors
(ligands)
by screening for growth inhibitory activity in biological fractions (plasma,
cell
lysates, cell membrane extracted proteins) by comparing growth inhibition on
CHO/ECRTP/DEP-1 to CHO parent cell lines.
In a cell-free system, a method of screening a candidate substance for
an ability to modulate a recepfior tyrosine phosphatase in accordance with the
present invention comprises establishing a control system comprising an
ECRTP/DEP-1, or fragment thereof and a ligand wherein the ECRTP/DEP-1
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is capable of binding to the ligand; establishing a test system comprising the
ECRTP/DEP-1, the ligand, and a candidate compound; measuring the binding
affinity of the ECRTP/DEP-1 and the ligand in the control and the test
systems;
and determining that the candidate compound modulates ECRTP/DEP-1
activity in a cell-free system if the binding affinity measured for the test
system
is less than or greater than the binding affinity measured forthe control
system.
Optionally, the iigand comprises an antibody which preferentially binds the
ECRTP/DEP-1. In this .case, it is preferred that the ligand comprise a
monoclonal antibody.
In accordance with the present invention, a method of affinity screening
of candidate modulator substances, including but not limited to antibodies, is
provided. The method comprises: (a) contacting a candidate modulator
substance with an ECRTP/DEP-1 polypeptide or fragment thereof under
conditions favorable to binding the candidate modulator substance with an
ECRTP/DEP-1 polypeptide or fragment thereof to form a complex
therebetween; and (b) detecting the complex.
The complex can be detected in any suitable manner. For example, the
complex can be defected via a label conjugated to the ECRTP/DEP-1
polypeptide or fragment thereof; via a labeled reagent that specifically binds
to the complex subsequent to its formation; or via a competition assay with a
substance . The ECRTP/DEP-1 polypeptide fragment can be a ECRTP/DEP-1
ectodomain fragment. Preferably, the ECRTP/DEP-1 ectodomain fragment
comprises an eight amino acid epitope having the sequence n-QSRDTEVL-c,
or an eight amino acid epitope having an analog sequence of the sequence n-
QSRDTEVL-c, the term "analog" as defined herein. Optionally, the
ECRTP/DEP-1 polypeptide orfragmentthereof is conjugated with a detectable
label. In this case, the detecting step comprises: (i) separating the complex
from unbound labeled binding substance; and (ii) detecting the detectable
label
which is present in the complex or which is unbound.
An antibody, or derivative or fragment thereof, can be screened as a
candidate modulator substance. As noted above, a preferred source for an
antibody, or derivative or fragment thereof, is a recombinant phage-displayed
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antibody library. The recombinant phage can comprise antibody encoding
nucleic acids isolated from any suitable vertebrate species, including
mammalian species such as mouse and rat; but preferably comprises antibody
encoding nucleic acids isolated from human. Such antibodies are thus already
"humanized".
In another aspect, the present invention pertains to a kit for use in the
aforementioned affinity screening method. The kit comprises a binding agent
comprising a polypeptide fragment of the ECRTP/DEP-1 ectodomain that
comprises an eight amino acid epitope having the sequence n-QSRDTEVL-c,
or an eight amino acid epitope having an analog sequence of the sequence n-
QSRDTEVL-c, the term "analog" as defined herein, contained in a first
container. Optionally, the binding agent can be immobilized to a solid phase
support, or the kit can also comprise a solid phase support contained in a
second container.
The kit can further comprise a reagent or indicator that comprises a
detectable label, the indicator containing in another container.
Alternatively,
the binding agent can comprise a detectable label or indicator. Preferably,
the
indicator is a radioactive label or an enzyme, or other suitable indicator.
Another technique for drug screening which can be used provides for
high throughput screening of compounds having suitable binding affinity to the
protein of interest as described in published PCT application WO 84/03564,
herein incorporated by reference. In this method, as applied to the
ECRTP/DEP-1 polypeptide, large numbers of different small test compounds
are synthesized on a solid substrate, such as plastic pins or some other
surface. The test compounds are reacted with the ECRTP/DEP-1 polypeptide,
or fragments thereof, and washed. Bound ECRTP/DEP-1 polypeptide is then
detected by methods well known in the art. Purified ECRTP/DEP-1 polypeptide
can also be coated directly onto plates for use in the aforementioned drug
screening techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
A screening assay of the present invention can also involve determining
the ability of a candidate substance to modulate, i.e. inhibit or promote
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ECRTP/DEP-1 biological activity and preferably, to thereby modulate the
ECRTP/DEP-1 biological activity in target cells. Target cells can be either
naturally occurring cells known to contain a polypeptide of the present
invention
or transfected cells produced in accordance with a process of transfection set
forth herein above. The test samples can further comprise a cell or cell line
that expresses the ECRTP/DEP-1; the present invention also contemplates a
recombinant cell line suitable for use in the exemplary method. Such cell
lines
can be mammalian, or human, or they can from another organism, including
but not limited to yeast. Exemplary assays including genetic screening assays
and molecular biology screens such as a yeast two-hybrid screen that will
effectively identify.ECRTP/DEP-1 -interacting genes important for endothelial
cell migration and proliferation, density induced growth arrest, angiogenesis
or
- other ECRTP/DEP-1-mediated cellular process. One version of the yeast two
hybrid system has been described (Chien et al., 1991, Proc. Natl. Acad. Sci.
USA, 88:9578-9582) and is commercially available from Clontech (Palo Alto,
California).
A method of identifying modulators of the ECRTP/DEP-1 by rational
drug design is also provided in accordance with the present invention. The
method comprises the steps of designing a potential modulator for the
ECRTP/DEP-1 that will form non-covalent bonds with amino acids in the
substrate binding site in the catalytic domain (an intracellular site) or with
the
ECRTP/DEP-1 ectodomain based upon the structure of the ECRTPIDEP-1;
synthesizing the modulator; and determining whether the potential modulator
modulates the activity of the ECRTP/DEP-1.
Thus, the present invention pertains to screening and rational drug
design methods for both ectodomain interactions, as well as catalytic
modulators. For example, catalytic antagonists that release cells from growth
arrest through modulation of the catalytic function independent of the
ectodomain can be screened and designed. Additionally, a modulator of the
present invention can be screened for interaction with an eight amino acid
epitope having the sequence n-QSRDTEVL-c, or an eight amino acid epitope
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having an analog sequence of the sequence n-QSRDTEVL-c, the term
"analog" as defined herein, of the ECRTP/DEP-1 ectodomain.
Modulators can be synthesized using techniques disclosed herein and
as are known in the art. The determination of whether the modulator
modulates the biological activity of the ECRTP/DEP-1 is made in accordance
with the screening methods disclosed herein, or by other screening methods
known in the art.
As is well known in the art, a screening assay provides a cell under
conditions suitable for testing modulation or alteration of cell growth,
cellendothelial cell migration and proliferation, density induced growth
arrest,
angiogenesis, and/or ECRTP/DEP-1 phosphorylation. These conditions
include but are not limited to pH, temperature, tonicity, the presence of
relevant
factors involved in the cell cycle (e.g., growth factors), and relevant
modifications to the polypeptide such as glycosylation or prenylation. It is
contemplated that an ECRTP/DEP-1 can be expressed and utilized in a
prokaryotic or eukaryotic cell. The host cell can also be fractionated into
sub-cellular fractions where the receptor can be found. For example, cells
expressing the polypeptide can be fractionated into the nuclei, the
endoplasmic
reticulum, vesicles, or the membrane surfaces of the cell.
pH is preferably from about a value of 6.0 to a value of about 8.0, more
preferably from about a value of about 6.8 to a value of about 7.8 and, most
preferably about 7.4. In a preferred embodiment, temperature is from about
20°C to about 50°C, more preferably from about 30°C to
about 40°C and,
even more preferably about 37°C. Osmolality is preferably from about 5
milliosmols per liter (mosm/L) to about 400 mosm/I and, more preferably from
about 200 milliosmols per liter to about 400 mosm/I and, even more preferably
from about 290 mosm/L to about 310 mosm/L. The presence of factors can be
required for the proper testing of endothelial cell migration and
proliferation,
density induced growth arrest and/or angiogenesis in specific cells. Such
factors include, for example, the presence and absence (withdrawal) of growth
factor, interleukins, or colony stimulating factors.
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E. Methods For Identifying Modulators of an ECRTP/DEP-1
The invention thus also pertains to assay methods for identifying
candidate ECRTP/DEP-1 modulators. In these assay methods candidate
molecules are evaluated for their potency in agonising an ECRTP/DEP-1
binding to natural ligands, and furthermore are evaluated for their potency in
modulating angiogenesis in a tissue.
An exemplary assay measures angiogenesis in the chick chorioallantoic
membrane (CAM) and is referred to as the CAM assay. The CAM assay has
be described in detail by others, and further has been used to measure both
angiogenesis and neovascularization of tumor tissues. See Ausprunk et al.,
Am J Pathol 79:597-618 (1975) and Ossonski et al., Cancer Res
40:2300-2309 (1980).
The CAM assay is a well recognized assay model for in vivo
angiogenesis because neovascularization of whole tissue is occurring, and
actual chick embryo blood vessels are growing into the CAM or into the tissue
grown on the CAM. The CAM assay illustrates inhibition of neovascularization
based on both the amount and extent of new vessel growth. Furthermore, it
is easy to monitor the growth of any tissue transplanted upon the CAM, such
as a tumor tissue. Finally, the assay is particularly useful because there is
an
internal control for toxicity in the assay system. The chick embryo is exposed
to any test reagent, and therefore the health of thre embryo is an indication
of
toxicity.
F. Preparation of Targeting Agent/toxin Compounds. Includin.a_
Immunotoxins
Methods for the production of the target agent/toxin agent compounds
of the invention are described herein. The targeting agents, such as
antibodies,
of the invention can be linked, or operatively attached, to the toxins of the
invention by eithercrosslinking orvia recombinant DNA techniques, to produce,
for example, targeted immunotoxins.
While the preparation of immunotoxins is, in general, well known in the
art (see e.g_, U.S. Pat. Nos. 4,340,535 and 5,776,427, and EP 44167, each of
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which incorporated herein by reference), certain advantages can be achieved
through the application of certain preferred technology, both in the
preparation
of the immunotoxins and in their purification for subsequent clinical
administration. For example, while numerous types of disulfide-bond
containing linkers are known which can successfully be employed to conjugate
the toxin moiety with the targeting agent, certain linkers will generally be
preferred over other linkers, based on differing pharmacologic characteristics
and capabilities. For example, linkers that contain a disulfide bond that is
sterically "hindered" are to be preferred, due to their greater stability in
vivo,
thus preventing release of the toxin moiety prior to binding at the site of
action.
A wide variety of cytotoxic agents are known that can be conjugated to
anti-endothelial cell antibodies. Examples include numerous useful plant-,
fungus- or even bacteria-derived toxins, which, by way of example, include
various A chain toxins, particularly ricin A chain, ribosome inactivating
proteins
such as saporin or gelonin, a -sarcin, aspergillin, restrictocin,
ribonucleases
such as placental ribonuclease, angiogenic, diphtheria toxin, and
pseudomonas exotoxin, to name just a few.
However, it can be desirable from a pharmacologic standpoint to employ
the smallest molecule possible that nevertheless provides an appropriate
biological response. One can thus desire to employ smaller-chain peptides
which will provide an adequate anti-cellular response.
Alternatively, one can find that the application of recombinant DNA
technology to the toxin moiety will provide additional significant benefits in
accordance the invention. For example, the cloning and expression of
biologically active toxin candidates has now been described through the
publications of others (O'Hare et al., FEBS Lett 210:731 (1987); Lamb et al.,
Eur Jrnl Biochem 148:265-270 (1985); Hailing et al., Nucl Acids Res
13:8019-8033 (1985)), it is now possible to identify and prepare smaller or
otherwise variant peptides which nevertheless exhibit an appropriate toxin
activity. Moreover, the use of cloned toxin candidates allows the application
of
site-directed mutagenesis, through which one can readily prepare and screen
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for mutated peptides and obtain additional useful moieties for use in
connection
with the present invention.
In cases where a releasable toxin is contemplated, one desires to have
a conjugate that will remain intact under conditions found everywhere in the
body except the intended site of action, at which point it is desirable that
the
conjugate have good "release" characteristics. Therefore, the particular cross-
linking scheme, including the particular cross-linking reagent used and the
structures that are cross-linked, will be of some significance.
Cross-linking reagents are used to form molecular bridges that tie
together functional groups of two different proteins (e.g., a toxin and a
binding
agent). To link two different proteins in a step-wise manner,
heterobifunctional
cross-linkers can be used which eliminate the unwanted homopolymer
formation. An exemplary heterobifunctional cross-linker contains two reactive
groups: one reacting with primary amine group (e.g., N-hydroxy succinimide)
and the other reacting with a thiol group (e.g., pyridyl disulfide,
maleimides,
halogens, etc.). Through the primary amine reactive group, the cross-linker
can react with the lysine residues) of one protein (e.g., the selected
antibody
or fragment) and through the thiol reactive group, the cross-linker, already
tied
up to the first protein, reacts with the cysteine residue (free sulfhydryl
group)
of the other protein.
The spacer arm between these two reactive groups of any cross-linkers
can have various length and chemical composition. A longer spacer arm
. allows a better flexibility of the conjugate components while some
particular
components in the bridge (e.g., benzene group) can lend extra stability to the
reactive group or an increased resistance of the chemical fink to the action
of
various aspects (e.g., disulfide bond resistant to reducing agents).
An exemplary cross-linking reagent is SMPT, which is a bifunctional
cross-linker containing a disulfide bond that is "sfierically hindered" by an
adjacent benzene ring and methyl groups. It is believed that stearic hindrance
of the disulfide bond serves a function of protecting the bond from attack by
thiolate anions such as glutathione which can be present in tissues and blood,
and thereby help in preventing decoupling of the conjugate prior to ifs
delivery
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to the site of action by the binding agent. The SMPT cross-linking reagent, as
with many other known cross-linking reagents, lends the ability to cross-link
functional groups such as the SH of cysteine or primary amines (e.g., the
epsilon amino group of lysine). Another possible type of cross-linker includes
the heterobifunctional photoreactive phenylazides containing a cleavable
disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3'-
dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino
groups and the phenylazide (upon photolysis) reacts non-selectively with any
amino acid residue.
Although the "hindered" cross-linkers will generally be preferred in the
practice of the invention, non-hindered linkers can be employed and
advantages in accordance herewith nevertheless realized. Other useful cross
linkers, not considered to contain or generate a protected disulfide, include
SATA, SPDP and 2-iminothiolane (Thorpe et al., Cancer Res 47:5924-5931
(1987)). The use of such cross-linkers is well understood in the art.
Once conjugated, it will be important to purify the conjugate so as to
remove contaminants such as unconjugated toxin or targeting agent. It is
important to remove unconjugated targeting agent to reduce undesired toxicity
and to avoid the possibility of competition for the antigen between conjugated
and unconjugated species. In general, the most preferred purification
technique will incorporate the use of Blue-Sepharose with a gel filtration or
gel
permeation step. Blue-Sepharose is a column matrix composed of Cibacron
Blue 3GA and agarose, which has been found to be useful in the purification
of immunoconjugates (Knowles & Thorpe, Anal. 8iochem 120:440-4.43
(1987)). The use of Blue-Sepharose combines the properties of ion exchange
with toxin binding to provide good separation of conjugated toxin from non-
conjugated toxin. The Blue-Sepharose column allows the elimination of the
free (non-conjugated) targeting agent (e.g., the antibody or fragment) from
the
conjugate preparation. To eliminate the free (non-conjugated) toxin a
molecular exclusion chromatography step is preferred using either conventional
gel filtration procedure or high performance liquid chromatography.
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Standard recombinant DNA techniques that are well known to those of
skill in the art can be utilized to express nucleic acids encoding the
targeting
agent/toxin compounds of the invention. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques and in vivo
recombinationigenetic recombination. DNA and RNA synthesis can,
additionally, be performed using an automated synthesizers (see, for example,
the techniques described in Sambrook et al., Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, New York (1989); and Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing Associates and
Wiley lnterscience, New York (1989).
When produced via recombinant DNA techniques such as those
described herein, the targeting agent/toxin compounds of the invention can be
referred to herein as "fusion proteins". It is to be understood that such
fusion
proteins contain at least a targeting agent and a toxic moiety operatively
attached, such that the fusion protein can be used in accordance with the
methods of the present invention. The fusion proteins can also include
additional peptide sequences, such as peptide spacers which operatively
attach the targeting agent and toxin compound, as long as such additional
sequences do not appreciably affect the targeting or toxin activities of the
fusion protein.
Depending on the specific toxin compound used as part of the fusion
protein, it can be necessary to provide a peptide spacer operatively attaching
the targeting agent and the toxin compound which is capable of folding into a
disulfide-bonded loop structure. Proteolytic cleavage within the loop would
then yield a heterodimeric polypeptide wherein the targeting agent and the
toxin compound are linked by only a single disulfide bond. See e. ., Lord et
al.,
In Genetically Engineered Toxins (Ed. A. Frank, M. Dekker Publ., p. 183)
(1992). An example of such a toxin is a Ricin A-chain toxin.
When certain other toxin compounds are utilized, a non-cleavable
peptide spacer can be provided to operatively attach the targeting agent and
the toxin compound of the fusion protein. Toxins which can be used in
conjunction with non-cleavable peptide spacers are those which can,
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themselves, be converted by proteolytic cleavage, into a cytotoxic disulfide-
bonded form (see e.ct., Ogata et al., J Biol Chem 256:20678-20685 (1990)).
An example of such a toxin compound is a Pseudomonas exotoxin compound.
Nucleic acids that can be utilized herein comprise nucleic acid
sequences that encode a targeting agent of interest and nucleic acid
sequences that encode a toxin agent of interest. Such target agent-encoding
and toxin agent-encoding nucleic acid sequences are attached in a manner
such that translation of the nucleic acid yields the targeting agent/toxin
compounds of the invention.
Standard techniques, such as those described above can be used to
construct expression vectors containing the above-described nucleic acids and
appropriate transcriptional/translational control sequences. A variety of host-
expression vector systems can be utilized. These include but are not limited
to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed
with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing targeting agent/toxin coding sequences; yeast (e.g.,
Saccharomyces, Pichia) transformed with recombinant yeast expression
vectors containing targeting agent/toxin coding sequences; insect cell systems
infected with recombinant virus expression vectors (e.g., baculovirus)
containing the targeting agent/toxin coding sequences; plant cell systems
infected with recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed viiith recombinant
plasmid expression vectors (e.g., Ti plasmid) containing the targeting
agent/toxin coding sequences coding sequence; or mammalian cell systems
(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs
containing promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late
promoter; the vaccinia virus 7.5K promoter; lentiviral vectors).
In bacterial systems a number of expression vectors can be
advantageously selected depending upon the use intended for the targeting
agent/toxin compound being expressed. For example, when large quantities
of targeting agenfi/toxin compound are to be produced for the generation of
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antibodies or to screen peptide libraries, vectors which direct the expression
of
high levels of fusion protein products that are readily purified can be
desirable.
Such vectors include but are not limited to the E. coli expression vector
pUR278 (Ruther et al., EM80 J 2:1791 (1983)), in which the targeting
agent/toxin coding sequence can be ligated individually into the vector in
frame
with the lac Z coding region so that a fusion protein additionally containing
a
portion of the lac Z product is provided; pIN vectors (Inouye et al., Nucleic
Acids Res 13:3101-3109 (1985); Van Heeke et al., J Biol Chem
264:5503-5509 (1989)); and the like. pGEX vectors can also be used to
express foreign polypeptides, such as the targeting agent/toxin compounds as
fusion proteins additionally containing glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be purified from
lysed
cells by adsorption to glutathione-agarose beads followed by elution in the
presence of free glutathione. The pGEX vectors are designed to include
thrombin or factor Xa protease cleavage sites so that the targeting
agent/toxin
protein of the fusion protein can be released from the GST moiety.
In an insect system, Autograph californica nuclear polyhidrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The targeting agent/toxin coding sequences can
be cloned into non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example the
polyhedrin promoter). Successful insertion of the targeting agent/toxin coding
sequences will result in inactivation of the polyhedrin gene and production of
non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat
coded for by the polyhedrin gene). These recombinant viruses are then used
to infect Spodoptera frugiperda cells in which the inserted gene is expressed
(see e. ., Smith et al., J Virol 46:584 (1983); U.S. Pat. No. 4,215,051 ).
In mammalian host cells, a number of viral based expression systems
can be utilized. In cases where an adenovirus is used as an expression vector,
the targeting agent/toxin coding sequences can be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter and
tripartite
leader sequence. This chimeric gene can then be inserted in the adenovirus
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genome by in vitro or in vivo recombination. Insertion in a non-essential
region
of the viral genome (e.g., region E1 or E3) will result in a recombinant virus
that
is viable and capable of expressing targeting agent/toxin proteins in infected
hosts (see e.a., Logan et al., Proc Natl Acad Sci USA 81:3655-3659 (1984)).
Specific initiation signals can also be required for efficient translation of
inserted targeting agent/toxin coding sequences. These signals include the
ATG initiation codon and adjacent sequences. Exogenous translational control
signals, including the ATG initiation codon, can additionally need to be
provided. One of ordinary skill in the art would readily be capable of
determining this and providing the necessary signals. Furthermore, the
initiation codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These exogenous
translational control signals and initiation codons can be of a variety of
origins,
both natural and synthetic. The efficiency of expression can be enhanced by
the inclusion of appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol 153:516-544 (1987)).
In addition, a host cell strain can be chosen which modulates the
expression of the inserted sequences, or modifies and processes the gene
product in the specific fashion desired. Such modifications (e.g.,
glycosylation)
and processing (e.g., cleavage) of protein products can be important for the
function of the protein. Different host cells have characteristic and specific
mechanisms forthe post-translational processing and modification of proteins.
Appropriate cells lines or host systems can be chosen to ensure the correct
modification and processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for proper
processing of the primary transcript, glycosylation, and phosphorylation of
the
gene product can be used. Such mammalian host cells include, but are not
limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, etc. For
long-term, high-yield production of recombinant proteins, stable expression is
preferred. For example, cell lines which stably express constructs encoding
the
targeting agent/toxin compounds can be engineered. Rather than using
expression vectors which contain viral origins of replication, host cells can
be
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transformed with targeting agent/toxin DNA controlled by appropriate
expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker.
Following the introduction of foreign DNA, engineered cells can be allowed to
grow for one or two (1-2) days in an enriched media, and then are switched to
a selective media. The selectable marker in the recombinant plasmid confers
resistance to the selection and allows cells to stably integrate the plasmid
into
their chromosomes and grow to form foci which in turn can be cloned and
expanded into cell fines.
A number of selection systems can be used, including, but not limited,
to the herpes simplex virus thymidine kinase (Wigler et al., Ce1111:223
(1977)),
hypoxanthine-guanine phosphoriboxyltransferase (Szybalska et al., Proc Natl
Acad Sci USA 48:2026 (1962)), and adenine phosphoribosyltransferase genes
(Lowy et al., Cell 22:817 (1980)) can be employed in tk-, hgprt- or aprt-
cells,
respectively. Also, antimetabolite resistance can be used as the basis of
selection for dhfr, which confers resistance to methotrexate (Wigler et al.,
Proc
Natl Acad Sci USA 77:3567 (1980); O'Hare et al., Proc Natl Acad Sci USA
78:1527 (1981 )); gpt, which confers resistance to mycophenolic acid (Mulligan
et al., Proc NaflAcad Sci USA 78:2072 (1981 )); neo, which confers resistance
to the aminoglycoside G-418 (Colberre-Garapin et al., J Mol Biol 750:7 (1981
));
and hygro, which confers resistance to hygromycin (Santerre et al., Gene
30:147 (1984)).
After a sufficiently purified compound has been prepared, one will desire
to prepare it into a pharmaceutical composition that can be administered
parenterally. This is done by using for the last purification step a medium
with
a suitable pharmaceutical composition.
Suitable pharmaceutical compositions in accordance with the invention
will generally comprise from about 10 to about 100 mg of the desired conjugate
admixed with an acceptable pharmaceutical diluent or excipient, such as a
sterile aqueous solution, to give a final concentration of about 0.25 to about
2.5
mg/mL with respect to the conjugate. Such formulations will typically include
buffers such as phosphate buffered saline (PBS), or additional additives such
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as pharmaceutical excipients, stabilizing agents such as BSA or HSA, or salts
such as sodium chloride. For parenteral administration it is generally
desirable
to further render such compositions pharmaceutically acceptable by insuring
their sterility, non-immunogenicity and non-pyrogenicity. Such techniques are
generally well known in the art as exemplified by Remington's Pharmaceutical
Sciences, 16th Ed. Mack Publishing Company (1980), incorporated herein by
reference. It should be appreciated that endotoxin contamination should be
kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
Moreover, for human administration, preparations should meet sterility,
pyrogenicity, general safety and purify standards as required by FDA Office of
Biological Standards.
A preferred parenteral formulation of the targeting agent/toxin
compounds, including immunotoxins, in accordance with the present invention
is 0.25 to 2.5 mg conjugate/mL in 0.15M NaCI aqueous solution at pH 7.5 to
9Ø The preparations can be stored frozen at -10° C to -70° C
for at least one
(1 ) year.
G. Attachment of Other Agents to Targeting Agents
It is contemplated that most therapeutic applications of the present
invention will involve the targeting of a toxin moiety to the endothelium,
particularly tumor endothelium. This is due to the much greater ability of
most
toxins to deliver a cell killing effect as compared to other potential agents.
However, there can be circumstances, such as when the target antigen does
not internalize by a route consistent with efficient intoxication by targeting
agent/toxin compounds, such as immunotoxins, where one will desire to target
chemotherapeutic agents such as antitumor drugs, other cytokines,
antimetabolites, alkylating agents, hormones, and the like. The advantages of
these agents over their non-targeting agent conjugated counterparts is the
added selectivity afforded by the targeting agent, such as an antibody.
Exemplary agents include, but are not limited to, such as steroids, cytosine
arabinoside, methotrexate, aminopterin, anthracyclines, mitomycin C, vinca
alkaloids, demecolcine, etoposide, mithramycin, and the like. This list is, of
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course, merely exemplary in that the technology for attaching pharmaceutical
agents to targeting agents, such as antibodies, for specific delivery to
tissues
is well established.
It is proposed that particular benefits can be achieved through the
application of the invention to tumor imaging. Imaging of the tumor
vasculature
is believed to provide a major advantage when compared to present imaging
techniques, in that the cells are readily accessible. Moreover, the technology
for attaching paramagnetic, radioactive and even fluorogenic ions to targeting
agents, such as antibodies, is well established. Many of these methods involve
the use of a metal chelate complex employing, for example, an organic
chelating agent such a DTPA attached to the antibody. See e. ., U.S. Pat. No.
4,472,509. In the context of the present invention the selected ion is thus
targeted to the tumor endothelium by the targeting agent, such as an antibody,
allowing imaging to proceed by means of the attached ion.
A variety of chemotherapeutic and other pharmacologic agents have
now been successfully conjugated to antibodies and shown to function
pharmacologically (see e.a., Vaickus et al., Cancer Invest 9:195-209 (1991 )).
Exemplary antineoplastic agents that have been investigated include
doxorubicin, daunomycin, methotrexate, vinblastine, and various others.
Dillman et al., Antibody Immunocon Radiopharm 1:65-77 (1988); Pietersz et
al., Antibody Immunoconj Radiopharm 1:79-103 (1988). Moreover, the
attachment of other agents such as neocarzinostatin (Kimura et al.,
Immunogenetics 11:373-381 (1980)), macromycin, trenimon (chose et al.,
Meth. Enzymology 93:280-333 (1983)) and a-amanitin has been described.
In addition to chemotherapeutic agents, the invention is contemplated
to be applicable to the specific delivery of a wide variety of other agents to
tumor vasculature. For example, under certain circumstances, one can desire
to deliver a coagulant such as Russell's Viper Venom, activated Factor IX,
activated Factor X or thrombin to the tumor vasculature. This will result in
coagulation of the tumor's blood supply. One can also envisage targeting a
cell
surface lytic agent such as phospholipase C, (Flickinger & Trost, Eu. J.
Cancer
12(2):159-60 (1976)) or cobra venom factor (CVF) (Vogel & Muller-Eberhard,
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Anal. Biochem 118(2):262-268 (1981 )) which should lyse the tumor endothelial
cells directly. The operative attachment of such structures to targeting
agents,
such as antibodies, can be readily accomplished, for example, by protein-
protein coupling agents such as SMPT. Moreover, one can desire to target
growth factors, other cytokines or even bacterial endotoxin or the lipid A
moiety
of bacterial endotoxin to a selected cell type, in order, e.g., to achieve
modulation of cytokine release. The attachment of such substances is again
well within the skill in the art as exemplified by Ghose et al., CRC Critical
Reviews in Therapeutic Drug Carrier Sysfiems 3:262-359 (1987).
Thus, it is generally believed to be possible to conjugate to antibodies
any pharmacologic agent that has a primary or secondary amine group,
hydrazide or hydrazine group, carboxyl alcohol, phosphate, or alkylating group
available for binding or cross-linking to the amino acids or carbohydrate
groups
of the antibody. In the case of protein structures, this is most readily
achieved
by means of a cross linking agent as described above. In the case of
doxorubicin and daunomycin, attachment can be achieved by means of an acid
labile acyl hydrazone or cis aconityl linkage between the drug and the
antibody.
Finally, in the case of methotrexate or aminopterin, attachment is achieved
through a peptide spacer such as L-Leu-L-Ala-L-Leu-L-Ala, between the y
carboxyl group of the drug and an amino acid of the antibody.
Alternatively, any such structures which are nucleic acid-encoded
structures can be operatively attached to the targeting agents of the
invention
by standard recombinant DNA techniques, such as, for example, those
discussed above.
EXAMPLES
The following Examples have been included to illustrate preferred
modes of the invention. Certain aspects of the following Examples are
described in terms of techniques and procedures found or contemplated by the
present inventors to work well in the practice of the invention. These
Examples
are exemplified through the use of standard laboratory practices of the
inventors. In light of the present disclosure and the general level of skill
in the
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art, those of skill will appreciate that the following Examples are intended
to be
exemplary only and that numerous changes, modifications and alterations can
be employed without departing from the spirit and scope of the invention.
EXAMPLE 1
Endothelial Localization of Receptor T~rrosine Phosphatase. ECRTP/DEP-1.
In Developing and Mature Renal Vasculature
Developmental assembly of the renal microvasculature is a precise
process requiring spatially and temporally coordinated migration, assembly,
differentiation and maturation of endothelial cells in the context of adjacent
epithelial and mesangial cells. Molecular determinants of assembly are largely
undefined, yet requirements for cell surface receptors to direct context
appropriate endothelial responses are anticipated. Endothelial expression and
distribution of the receptor tyrosine phosphatase, ECRTP/DEP-1, were
evaluated during developmental assembly of the renal microvasculature,
Monoclonal antibodies generated against ECRTP/DEP-1 ectodomain epitopes
localize its expression to membrane surfaces of endothelial cells in
glomerular,
peritubular capillary and arterial renal circulations of mature human and
murine
kidney. During kidney development, ECRTP/DEP-1 immunostaining is evident
on a subpopulation of metanephric mesenchymal cells and on putative
progenitors of glomerular capillary endothelial cells early in their
recruitment to
developing glomeruli. ECRTP/DEP-1 is prominently displayed on luminal
membrane surfaces with punctate accumulations at inter-endothelial contacts
that overlap, but do not co-localize with VE cadherin. I n vitro studies show
that
ECRTP/DEP-1 is recruited to positions of inter-endothelial contact in
confluent
cultured human renal and dermal microvascular endothelial cells, where its
distribution overlaps, but again does not coincide with VE cadherin.
Experimental dissociation of VE cadherin from endothelial functional complexes
does not redistribute ECRTP/DEP-1 away from inter-endothelial contacts.
These findings indicate that ECRTP/DEP-1 ectodomains interact with proteins
that are expressed on surfaces of endothelial cells and that are engaged by
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cell-cell contact, to convey signals for cell recognition, or arrest of
migration or
proliferation.
In orderto identify receptor tyrosine phosphatases expressed in human
renal microvascular endothelial cells (HRMEC), degenerate oligonucleotide
primers derived from conserved phosphatase domains were used to amplify
and sequence cDNAs representing expressed messages, according to
methods described in Schoecklmann et al., J Am Soc Nephrol 5:730
(1994)(abstract). Among putative receptor cDNAs identified was one that was
designated ECRTP (endothelial cell receptor tyrosine phosphatase), a product
virtually identical to the DEP-1 (for density enhanced phosphatase) cDNA
cloned by Ostman et al. from HeLa cells and regulated in abundance by cell
density in W I-38 cells. Ostman et al., Proc Natl Acad Sci USA 91:9680-9684
(1994). ECRTP/DEP-1 (also called byp-1, HPTPh, and CD148) expression has
been identified in neonatal smooth muscle cells, in breast and thryoid cancer
cell lines, and in all hematopoietic lineages. Keane et al., Cancer Research
56:4236-4243 (1996); de la Fuente-Garcia et al., Blood 91:2800-2809 (1998).
Although ECRTP/DEP-1 expression was identified in arterial endothelial cells
of the kidney, in sifiu hybridization experiments failed to detect glomerular
capillary localization of ECRTP/DEP-1 mRNA. Borges et al., Circulation
Research 79:570-580 (1996). The developmental timing and distribution of its
expression have not been previously reported.
Like other members of the Class III receptor tyrosine phosphatase
family, including GLEPP-1, SAP-1, and DPTP 10D, ECRTP/DEP-1 is a type I
membrane protein characterized by a large extracellular domain containing
eight or more fibronectin type III repeats and a single cytoplasmic domain
phosphatase catalytic domain. Ostman et al., Proc Natl Acad Sci USA
91:9680-9684 (1994). The GLEPP-1 receptor tyrosine phosphatase is
structurally similar to ECRTP/DEP-1, yet shows renal expression limited to
glomerular visceral epithelial cells, where it has been implicated in podocyte
integrity. Thomas et al., J Biol Chem 269:19953-19962 (1994). Unlike the
MAM domain containing receptors, PTP ~, ~c and ~,, available data do not
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support parfiicipation of class III receptors in homophilic binding, and
iigands
have not yet been identified.
Monoclonal antibodies were developed against ECRTP/DEP-1
ectodomain epitopes to characterize its distribution in the renal circulation
of
mature and developing kidney. ECRTP/DEP-1 is expressed at high levels in
glomerular, peritubular and renal arterial endothelia! cells and shows a
pattern
of distribution in vivo and in vitro that suggests it contributes to cell-cell
recognition required for capillary assembly and maintenance.
METHODS
Cell lines and cell culture - Primary human renal microvascular
endothelial cells (HRMEC) were isolated, cultured, and used at third or fourth
passage after thawing, as described. Martin et al., In Vitro Cell Dev 8io1
33:261-269 (1997). Human dermal microvascular endothelial cells (HMEC-1
cells, CDC) were grown in MCDB131 media (Sigma Chemical Co. of St. Louis,
Missouri) containing 15% fetal bovine serum (Hyclone Laboratories, Logan
Utah, USA), 10 ng/ml epidermal growth factor (Collaborative Biomedical
Products, Becton Dickinson, Bedford, Massachusetts), and 1 mg/ml
hydrocortisone (Sigma Chemical Co. of St. Louis, Missouri) Ades et al., J
Invest Dermatol. 99:683-690 (1992). Madin Darby Canine Kidney (MDCK) cells
(kindly provided by L. Limbird, Vanderbilt Pharmacology) were grown in
Dulbecco's minimal essential medium (DMEM, GIBCO BRL, Rockville,
Maryland) containing 4.5% D-glucose and supplemented with 10% fetal bovine
serum. All growth medium was supplemented with 1 mM L-glutamine (GIBCO
BRL, Rockville, Maryland), 100 unitslml penicillin and 100mg/ml streptomycin
(GIBCO BRL, Rockville, Maryland).
Generation of antibodies to recombinant ECRTP/DEP-1 proteins -
Ectodomain (amino acids 175-536) and catalytic domain (amino acids 1048-
1338) sequences of human, ECRTP/DEP-1. Ostman et al., Proc Natl Acad Sci
USA 91:9680-9684 (1994), were subcloned into the pRSET vector (Invitrogen,
Carlsbad, California). Recombinant fusion proteins were expressed in bacteria,
purified by a kit sold underthe registered trademark NI-AGAROSE AFFINITYT"'
by Invitrogen of Carlsbad, California, and characterized by SDS-PAGE as
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greater than 95% homogeneous proteins of 40 and 36 kDa, respectively.
Mouse hybridoma antibodies (ECRTP-Ab1, ECRTP-Ab2) were generated
against ECRTP/DEP-1 ectodomain (ECRTP/DEP-1e~) protein by intra-
peritoneal immunization, fusion with SP2-0 cells, ELISA screening, selection,
expansion and purification by affinity chromatography on PROTEIN A-
AGAROSE (Sigma Chemical Co. of St. Louis, Missouri).
Immunodetection of exo~eneously expressed ECRTP/DEP-1 - MDCK
cells grown in 100mm plastic dishes (sold under the registered trademark
FALCON~ by Becton, Dickinson and Company, Franklin Lakes, New Jersey)
were transfected with an expression plasmid pSRa DEP-1/3xHA that drives
high level expression of the human ECRTP/DEP-1 receptor modified by
addition of three repeats of a hemagglutinin peptide (HA) to the carboxy
terminus, using cationic lipid (LIPOFECTAMINET"", GIBCO BRL, Rockville,
Maryland) according to the manufacturer's protocol. Forty eight hours after
transfection, cells were placed on ice, washed twice with ice cold PBS(-) and
immediately lysed in 0.5m1 lysis buffer (50mM HEPES pH 7.5, 50mM NaCI,
5mM EDTA, 2 pg/mL aprotinin, 1 pg/mL leupeptin, 1 mM PMSF). Lysates were
clarified by centrifugation, and membrane receptors were recovered by batch
adsorption to WGA-Agarose (Sigma Chemical Co. of St. Louis, Missouri) for
4 hours at 4°C. The resultant precipitates were resolved by 7% SDS-PAGE
under reducing conditions, transferred to Immobilon-P transfer membranes
(Millipore Corporation, Bedford, Massachusetts), and blocked in 5% non -fat
dry milk in Tris-buffered saline (50mM Tris HCI pH 7.5, 137 mM NaCI)
containing 0.2% Tween 20 (TBST) overnight at 4°C. Blots were incubated
with
murine monoclonal ECRTPAbs 1 or 2 (10 pg/mL) or anti-HA (2.5 pg/mL)
antibodyfollowed by incubation with horseradish peroxidase-conjugated rabbit
anti-mouse IgG antibody (Boehringer Mannheim, Indianapolis, Indiana).
Membranes were washed with TBST, then developed using a
chemiluminescent substrate (ECL, Amersham, Buckinghamshire, England)
according to the manufacturer's instructions.
Generation of Stable Transfected MDCK Cells and Cell Stainina -
MDCK cells were transfected with an expression plasmid pCDNA3 DEP-
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1/3xHA (Invitrogen) using cationic lipids (LipofectamineT"', GIBCO BRL,
Rockville, Maryland) according to the manufacturer's protocol. Stable
transfectants were selected by addition of 6418 (GIBCO BRL, Rockville,
Maryland) to culture media at a final concentration of 800 pg/mL, and a single
colony was obtained by limited dilution cloning. The cells were grown on glass
coverslips (Fisher Scientific, Pittsburgh, Pennsylvania) and fixed with 100%
methanol for 10 min at -20'C. Coverslips were washed with phosphate
buffered saline, blocked with 5% goat serum for 30 min at room temperature,
incubated with ECRTPAb-2 (10 pg/mL) for 60 min, washed, then incubated
with FITC conjugated goat anti- mouse IgG (Jackson Immunoresearch
Laboratory, Westgrove, Pennsylvania) for 60 min. Coverslips were mounted
and analyzed by confocal microscopy (equipment sold under the registered
trademarkZEISS~ LSM410T"" byZeiss, Oberkochen, Germany). To preabsorb
the immunoreactivity of ECRTP/DEP-1-Ab, 50 pg of ECRTP/DEP-1 proteins
(Ec or Cy) were preincubated with ECRTPAb-2 for 4 hours at 4'C,
microcentrifuged at 15,000 rpm for 20 min and the resultant supernatant was
used to stain cells.
Tissue immunolocalization - Human kidney tissue was snap-frozen in
a dry ice-acetone bath. Cryostat sections (4mm) were fixed in acetone at
-20°C for 10 min, washed with phosphate buffered saline, and pre-
adsorbed
with avidin-biotin blocking reagents (Vector Laboratories, Inc. of Burlingame,
California) according to manufacturer's instructions. Sections were washed
with
phosphate buffered saline, blocked with 5% goat serum, incubated with
monoclonal ECRTP/DEP-1 antibody (ECRTP-Ab1,10 pg/mL,10 min), washed,
incubated with biotinylated goat anti-mouse IgG (Vector Laboratories, Inc. of
Burlingame, California, 7.5 pg/mL, 60 min), washed, incubated with fluorescein
isothiocyanate (FITC)-conjugated streptavidin (Pierce Chemical Company of
Rockford, Illinois, 4 pg/ml, 30 min) and finally washed with phosphate
buffered
saline. Coverslips were mounted (sold under the trademark VectashieldT"" by
Vector Laboratories, Inc. of Burlingame, California) and analyzed by confocal
microscopy (equipment sold underthe registered trademark ZEISS~ LSM41 OT"~
by Zeiss, Oberkochen, Germany). For colocalization experiments, acetone
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fixed frozen sections were blocked with 5% donkey serum, and incubated with
mixture of ECRTP/DEP-1 antibody (10 pg/mL) and goat VE cadherin antibody
(5 pg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, California) at room
temperature for 60 minutes. Specific antibodies were detected using a mixture
of FITC-conjugated donkey anti-mouse and rhodamine conjugated donkey anti-
goat antibodies (Jackson Immunoresearch Laboratory, Westgrove,
Pennsylvania) at room temperature for 60 minutes. Specific immunostaining
for each antigen was identified in overlapping images generated by analysis of
the same section at 488 nm and 568 nm wavelengths, respectively, on a
ZEISS~ LSM410TM confocal microscope.
Immunolabeled murine kidney sections showed high background and
required an alternative technique. The anti-ECRTP/DEP-1 mAb, ECRTP-Ab1,
was directly coupled to FITC. Briefly, ECRTP-Ab1 (0.55 mL of 0.94 mg IgG/mL
in 0.1 M sodium carbonate buffer, pH 9.0) was conjugated to 0.03 mL FITC
solution (Sigma Chemical Co._ of St. Louis, Missouri, 1.0 mg/ml in DMSO)
overnight at 4°C. The reaction was stopped by adding ammonium chloride
to
50 mM final concentration. Following incubation for 2 hours at 4°C, the
mixture
was dialyzed exhaustively against phosphate buffered saline to remove
unbound FITC. A mouse monoclonal IgG against rat glomerular basement
membrane coupled to FITC using the identical protocol was used as a control.
Hyink et al., Am J Physiol 270:F886-F899 (1996). Acetone fixed sections
were blocked with 0.5M ammonium chloride, incubated for 30 min with MoAb-
FITC conjugates, washed, and examined by epifluorescence microscopy. In
some additional control experiments, the anti-DEP-FITC conjugate was mixed
with a molar excess of the immunization peptide before incubation with the
sections.
Immunoblots and immunoc~~tochemistry of human endothelial cell lines
Human endothelial cells grown in 60 mm dishes were lysed at confluency in 0.5
mL of lysis buffer (20mM TrisCl pH7.5, 50mM NaCI, 1 mM EDTA, 0.5% Triton
X-100, 0.5% SDS, 0.5% deoxycholate, 2 pg/mL aprotinin, 1 ~rg/mL leupeptin,
1 mM phenylmethylsulfonylfluoride) on ice for 30 minutes. Cleared lysate
protein, 150 erg, was incubated with 10 pg/mL of affinity purified rabbit
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ECRTP/DEP-1 antibody or rabbit IgG (Sigma Chemical Co. of St. Louis,
Missouri) at 4'C for 4 hours, and immunoprecipitates were recovered using
Protein-a Sepharose (Sigma Chemical Co. of St. Louis, Missouri). SDS-PAGE,
and immunoblotting procedures were carried out as described above.
Endothelial cells were grown on uncoated glass coverslips (Fisher Scientific,
Pittsburgh, Pennsylvania), then fixed with 50% methanol for 10 min at 4'C.
Coverslips were washed with PBS, blocked with 5% goat serum for 30 min at
room temperature, incubated with ECRTPAb-2 monoclonal antibody (10 pg/ml)
or VE cadherin monoclonal antibody (2 pg/mL, BD Transduction Laboratory,
Lexington, Kentucky) for 60 min, washed, then incubated with biotinylated goat
anti-mouse IgG (Vector Laboratories of Burlingame, California) for 60 min,
washed, and finally incubated with fluorescein conjugated (FITC) streptavidin
(4 pg/ml, Pierce Chemical Company of Rockford, Illinois) for 30 min.
Coverslips were mounted and analyzed by confocal microscopy (equipment
sold under the registered trademark ZEISS~ LSM41 OTM by Zeiss, Oberkochen,
Germany).
Calcium chelation to disrupt inter-endothelial cadherin complexes -
Confluent HMEC-1 cells grown on glass coverslips in DMEM media
supplemented with 15% fetal bovine serum were exposed to addition of EGTA
(ethylene glycol-bis(b-aminoethylether)-N,N,N',N',-tetraacetic acid, Sigma
Chemical Co. of St. Louis, Missouri) to reach a final concentration of SmM.
Cells were incubated for an additional 20 min, then fixed with 50% methanol
at 4'C for 10 min, washed with phosphate buffered saline, and stained with
monoclonal ECRTP/DEP-1 antibody (10 pg/ml) or VE cadherin monoclonal
antibody (2 pg/mL, BD Transduction Laboratory, Lexington, Kentucky), as
described above.
RESULTS - Monoclonal antibodies recognize recombinant and
expressed ECRTP/DEP-1. Recombinant fusion proteins representing either
ectodomain (Ec) or cytoplasmic domain (Cy) ECRTP/DEP-1 sequences were
expressed in bacteria and used to immunize rabbits and/or mice. Shown in
Figure 1A, monoclonal antibodies, ECRTPAb-1 and ECRTPAb-2, specifically
identify the ectodomain but not the cytoplasmic domain recombinant proteins.
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To ascertain whether these antibodies recognize the full length protein
expressed in mammalian cells, MDCK cells were transiently transfected with
either an empty expression plasmid (SRa) or one driving expression of a full
length ECRTPIDEP-1 tagged on the carboxy terminus with a hemagglutinin
epitope (SRa DEP-1/HA). Cell lysates from transfected cells were
immunoprecipitated using the epitope-specific monoclonal anti-HA antibody,
then probed with the antibodies indicated, including ECRTPAb-1 and
ECRTPAb-2 (Figure 1 B). Both recognized the 220 kDa HA-tagged
ECRTP/DEP-1.
Finally, capacity of the,monoclonal antibodies to specifically recognize
the ECRTP/DEP-1 expressed in intact cells was assessed using MDCK cells
stably transfected with ECRTP/DEP-1. Indirect epifluorescence staining with
ECRTPAb-2 localized ECRTP/DEP-1 to lateral cell membranes (Figure 1 C,
Panel a), a finding confirmed in confocal Z plane sections of MDCK cells grown
to confluence on permeable membrane supports. Competition with the
immunizing peptide (Ec) blocked immunostaining (Figure 1 C, Panel c)whilethe
irrelevant cytoplasmic domain fusion peptide (Cy) did not (Figure 1 C, Panel
d).
ECRTP/DEP-1 immunoreactivity localizes to endothelial cells of
glomerular capillaries, peritubular capillaries and renal arteries. To
determine
the distribution of ECRTP/DEP-1 in mature mammalian kidney, indirect or
direct immunofluorescence staining experiments were conducted on frozen
sections from human and mouse sources. Shown in Figure 2, ECRTP-Ab2
immunolocalizes ECRTP/DEP-1 expression to arterial, glomerular and
peritubular capillaries, and in particular, to the endothelial cells in these
sites.
Higher magnification frames show predominant ECRTP/DEP-1 labeling along
the luminal membranes of endothelial cells, at least in the arterial sites
where
endothelial membrane definition is most reliable (Figure 3).
The punctate characteristic of the staining in the glomerular
microcirculation led to the evaluation of whether ECRTP/DEP-1 was engaged
in inter-endothelial functional complexes. In double labeling studies using
ECRTP-Ab1 and VE-cadherin antibodies, some overlap was evident (Figure
3). In addition to the luminal endothelial membrane staining, a regional
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accumulation of ECRTP/DEP-1 was evident at points of inter-endothelial
contact, overlapping, but not limited, to the endothelial functional complexes
that include VE cadherin. Lampugnani et al., J Cell Biol 129:203-217 (1995)
This pattern was evident in both arterial and peritubular capillaries. In
extra-
s renal sites, capillary and large vessel endothelial cells of brain, lung,
liver and
spleen was identified and endocardial staining were also apparent.
Based on the prominent ECRTP/DEP-1 expression in vascular
endothelium of mature kidney, temporal and spatial expression ofthis receptor
during renal vascular development in mouse embryos was evaluated. Shown
in Figure 4, ECRTPAb-1 binds as an antigen, its marine ECRTP/DEP-1, based
on its similar pattern of staining is mature marine and human kidneys, and
based on the effect of the recombinant human immunogen (Ec) to block
staining of the mouse tissue. In developing mouse kidneys at E14, E16, and
postnatal day 6, (Figure 4A-C) conjugates of ECRTP-Ab1-FITC displayed a
pattern of immunoreactivity that was strikingly similar to the pattern
observed
previously using antibodies against the VEGF receptor, flk-1, and the
EphB1/ephrin-B1 receptor-ligand. Daniel et al., Kidney Int 50:S-73-S-81
(1996). Notably, ECRTP-Ab1-FITC bound to endothelial cells of developing
glomeruli and microvessels in the fetal kidney cortex. Small but intense foci
of
bound antibodywere observed on isolated cortical mesenchymal cells believed
to be angioblasts (Figures 4A & 4B). Within vascular clefts of comma- and S-
shaped developing glomeruli, a subpopulation of cells consistent with
glomerular endothelial precursors were labeled (Figures 4A & 4B).
Immunolabeling for ECRTP/DEP-1 on sections of neonatal kidney
produced a distinct vascular labeling pattern (Figure 4C). Arteriolar,
glomerular, and peritubular capillary endothelia all labeled intensely (Figure
3C). Glomerular endothelial cells were also brightly labeled in adult mouse
kidney (Figure 4D), as they were in sections of human kidney. Other cells
within the immature and mature kidneys did not bind ECRTP-Ab1-FITC, and
sections labeled with control monoclonal IgG-FITC conjugates, or mixtures of
ECRTP-Ab1-FITC and the immunization peptide (Ec) showed no staining.
Independent immunoblot and immunofluorescence staining experiments
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using ECRTP-Ab1 showed high level expression in endothelial cells cultured
from a range of different vascular sites, including the HRMEC from which it
was
cloned, a dermal microvascular endothelial cell line, HMEG-1 (Ades et al., J
Invest Dermatol 99:683-690 (1992); human umbilical vein endothelial cells;
and a HUVEC derived cell line, Eahy926 (Bauer et al., J Cell Physiol 153:437-
449 (1992). Epitopes recognized by this antibody were not detected in non-
endothelial cell lines; including HEK293 cells, glomerular mesangial cells,
vascular smooth muscle cells, and P19 embryonic carcinoma cells.
Shown in Figure 5 are patterns of ECRTP/DEP-1 localization in human
renal microvascular endothelial cells, HRMEC (Panel A), and human dermal
microvascular endothelial cells, HMEC (Panel B). Confluent HRMEC cultures
displayed prominent staining with ECRTP-Ab2 at points of inter-endothelial
contact. In addition, there were punctate accumulations of apical membrane
staining in confocal planes capturing the apical surface (Panel A), but not on
the basal membrane surface. Endothelial cells plated at sufficiently low
density
to be isolated from contact with one another did not show-the prominent
pattern
of cell border staining seen in contacting cells. It should be noted that
ECRTP
Ab1 did not demonstrate the inter-endothelial localization seen with ECRPT
Ab2, but stained only the subpopulation of receptors evident on the apical
surface.
This apparent accumulation of ECRTP/DEP-1 at sites of endothelial cell-
cell contact is consistent with the punctate accumulations of staining seen in
intact mature vessels, and suggests that a subpopulation of receptors
distribute
to points of inter-endothelial contact. Thus, the distribution of ECRTP/DEP-1
was compared with that of VE cadherin. Confocal localization of ECRTP/DEP-1
and VE cadherin immunoreactivity in double labeling experiments of confluent
HMEC cultures again showed modest overlap of ECRTP/DEP-1 staining with
the VE-cadherin localized in inter-endothelial junctions. Similar patterns of
colocalization were seen in double labeled sections of human kidney tissue
(Figure 3). Finally, experiments were conducted to ascertain whether the
intercellular accumulation of ECRTP/DEP-1 immunoreactivity required the
integrity of VE-cadherin interactions. Shown in Figure 5B, EGTA treatment of
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the HMEC-1 cells dissociates VE cadherin from the inter-endothelial functional
complexes, but has no apparent effect on ECRTP/DEP-1 localization over the
20-30 minute time period of the experiment. This result suggests that any
inter-endothelial junctions that can retain ECRTP/DEP-1 do not require
cadherin integrity. Furthermore, these data are consistent with the
observations that ECRTP/DEP-1 and VE cadherin overlap, but do not co-
localize precisely in intact vessel endothelium (Figure 3).
DISCUSSION - Several of the observations presented here provide
new insights about the ECRTP/DEP-1 tyrosine phosphatase in vascular
development and in endothelial cell-cell interactions. The significance of the
initial identification of ECRTP/DEP-1 as a transcript expressed in cultured
human renal microvascular endothelial cells has been confirmed at several
levels. Schoecklmann et al., J Am Soc Nepf~rol 5:730 (1994)(abstract).
Cultured HRMEC's express the protein on cell membranes, just as glomerular
and peritubular capillaries do in intact kidney tissue. Indeed, capillary and
arterial endothelium appear to be the dominant cellular sources of
ECRTP/DEP-1 expression in mature human and mouse kidney. In contrast
with the previous in situ experiments in rat kidney kidneys, described in
Borges
et al., Circulation Research 79:570-580 (1996), high level expression were
found in glomeruli of both mouse and human.
Careful evaluation of the sites of membrane to which ECRTP/DEP-1
distributes has shown prominent apical membrane staining in arterial
endothelium in addition to the inter-endothelial membrane staining that
appears
responsible for the somewhat granular staining pattern in the glomerular
capillaries. The lateral cell membrane distribution of ECRTP/DEP-1 in the
artificial MDCK epithelial cell system and in contacting cultured HRMEC
(Figure
5), led to the formally evaluation of the relationship of lateral ECRTP/DEP-1
membrane accumulation with VE cadherin complex integrity. The in situ
overlap of ECRTPIDEP-1 and VE cadherin immunostaining is modest (Figure
3), and is restricted to very focal regions of inter-endothelial contact in
some,
but not all functional complexes. As ECRTP/DEP-1 lateral membrane
distribution is maintained in cultured endothelial cells in which VE cadherin
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complexes have been dissociated by calcium chelation, it is concluded that
there is neither anatomical co-localization nor functional correlation of
ECRTP/DEP-1 distribution with maintenance of inter-endothelial complexes.
These findings, however, cannot exclude the possibility that lateral
ECRTP/DEP-1 membrane distribution can function to establish conditions
permissive to assembly of inter-endothelial complexes containing VE cadherin.
Alternatively, the lateral membrane distribution can reflect interaction of
the ECRTP/DEP-1 extracellular domain with a putative ligand expressed on
contacting membranes that is capable of redistributing receptors or
stabilizing
them in ligand-receptor complexes created through juxtacrine engagement.
Certainly there is available evidence that membrane associated receptor
tyrosine phosphatase activity is increased in cultured cells, including
endothelial cells, that are in close contact. Pallen and Tong, Proc Natl Acad
Sci USA 88:6996-7000 (1991 ); Batt et al., J Biol Chem 273:3408-3414 (1998).
In the culture systems presented in this Example, an increase in ECRTP/DEP-1
activity that correlates with cell density and with cell-density mediated
growth
arrest has been demonstrated.
The apical membrane distribution of ECRTP/DEP-1 in arterial and
apparently in capillary endothelium is intriguing, particularly ih the context
of
data showing that platelets and all hematopoietic lineages express the
ECRTP/DEP-1. Palou et al., Immunol Lett 57:101-103 (1997). Homophilic
interactions between ECRTP/DEP-1 s of endothelial cells and circulating cells
that can encounter them on luminal membranes of intact vessels suggest that
it is likely that regulatory factors, or co-receptors on each of the engaging
cells
are important in modulating any downstream responses.
Finally, the data assessing the developmental pattern of ECRTP/DEP-1
expression on cells that contribute to assembly of the glomerular capillary
network offers insight about roles for this receptor in this coordinated
process.
Receptor tyrosine phosphatases of the ECRTP/DEP-1 subclass, including
DPTP10D, have been assigned important roles in the targeting of neurons to
correct destinations during development. Desai et al., Ce1184:599-609 (1996).
Previous reports have identified expression in hematopoietic progenitors,
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including erythroid, lymphoid and myeloid series lineages. Palou et al.,
Immunol Lett. 57:101-103 (1997). With the accumulating evidence that
hemangioblasts serve as common precursors of both hematopoietic and
vascular endothelial lineages, it now appears that ECRTP/DEP-1 expression
is initiated early in the ontogeny of these precursors. Furthermore, it
appears
that ECRTP/DEP-1 can function to promote differentiation of erythroid lineage
cells that express it. Kumet et al., J Biol Chem. 271:30916-30921 (1996).
EXAMPLE 2
ECRTP/DEP-1 Mediates S~nals for Endothelial Growth Arrest and
Migration Inhibition
Powerful endogenous inhibitors of angiogenesis, such as
thrombospondin, angiostatin and endostatin, inhibit the proliferation and
migration of cultured endothelial cells in vitro. Such angiogenesis inhibitory
controls appear to signal arrest of endothelial growth and migration by
engaging endothelial surface receptors. one of the most powerful growth
inhibitory signals for cultured endothelial cells is imposed by cell-cell
contact,
which is described in the art as "density mediated growth arrest" or "contact
mediated growth arrest". High level expression of the receptor tyrosine
phosphatase, ECRTP/DEP-1, at inter-endothelial contacts in microvascularand
large vessel endothelium of human kidney and other organs is described in
Example 1.
In this Example, the ECRTP/DEP-1 has been determined to mediate
endothelial growth and migration arrest signals. The ECRTP/DEP-1 is
catalytically activated in conjunction with cell-cell contact. Transient
overexpression of full length ECRTP/DEP-1 arrests endothelial growth and
migration. Bivalent forms of a monoclonal antibody, ECRTPAb-1, that binds
the ECRTP/DEP-1 ectodomain inhibits endothelial proliferation and migration,
while Fab fragments are inactive. This antibody imposes inhibition on corneal
angiogenic responses in a mouse system. These findings indicate that the
ECRTP/DEP-1 signals endothelial growth and migration arrest upon
engagement of its ligand on the surfaces of contacting endothelial cells, and
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fihat surrogate activators, or modulators, of endothelial growth arrest
signals are
viable candidates for angiogenesis inhibifiors.
METHODS
Cell Culture - Primary human renal microvascular endothelial cells,
HRMEC, were isolated, cultured, and used at third or fourth passage after
thawing, as described in Martin et al., In Vitro Cell Dev Bio133:261-269
(1997).
Human dermal microvascular endothelial cells (HMEC-1 cells, CDC) were
grown in MCDB131 media (Sigma Chemical Co. of St. Louis, Missouri)
containing 15% fetal bovine serum (Hyclone Laboratories, Logan Utah, USA),
10 ng/mL epidermal growth factor (Collaborative Biomedical Products; Becton
Dickinson, Bedford, Massachusetts), and 1 p,g/mL hydrocortisone (Sigma
Chemical Co. of St. Louis, Missouri). Ades et al., J Invest Dermatol 99:683-
690 (1992). All growth media were supplemented with 1 mM L-glutamine
(GIBCO BRL, Rockville, Maryland), 100 units/mL penicillin and 100~.g/mL
streptomycin (GIBCO BRL, Rockville, Maryland).
Antibodies - Ectodomain (ECRTP/DEP-1e~, amino acids 175-536) and
cafialyfiic domain (ECRTP/DEP-1 ~Y, amino acids 1048-1338) sequences of
human ECRTP/DEP-1 (Ostman efi al., Proc Natl Acad Sci USA 91:9680-9684
(1994)were subcloned into the pRSETvector(Invitrogen, Carlsbad, California).
Recombinant fusion proteins were expressed in bacteria, purified by Ni-
agarose affinity (Invitrogen, Carlsbad, California), and characterized by SDS-
PAGE as greater than 95% homogeneous proteins of 40 and 36 kDa,
respectively. Rabbit antiserum to the ECRTP/DEP-1 ~Y protein was generated
by repetitive immunization, and was affinity purified, as described in Koenig
et
al., J Clin Immunol13:204-211 (1993). Mouse hybridoma antibody ECRTPAb-
1 was obtained following immunization with ECRTP/DEP-1e~ protein by
intraperitoneal immunization, fusion with SP2-0 cells, ELISA screening,.
selection, expansion and purification by affinity chromatography on protein A-
agarose (Sigma Chemical Co. of St. Louis, Missouri).
Assays for ECRTP/DEP-1 Abundance and Tyrosine Phosphatase
Activity - Cells plated at the densities and harvested at the times indicated
in
the Figure Descriptions were washed repeafiedly with iced phosphate buffered
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saline before in situ addition of 2 ml of buffer containing 50mM Hepes (pH
7.5),
50mM NaCI, 5mM EDTA, 1 mM PMSF, 1 mM a-mercaptoethanol, 1 % Triton X-
100. Detergent solubiiized cells were incubated for 15 min at 4°C and
insoluble material was removed by repeated microcentrifugation (two times) at
13,000 x g, 10 min, 4°C. Proteins in solubilized fractions were
quantitated
using a modified BCA assay (Pierce Chemical Company of Rockford, Illinois).
In some experiments, batch adsorption and elution from triticum vulgaris
lectin
(WGA) conjugated to agarose (Sigma Chemical Co, of St. Louis, Missouri) was
conducted as described in Stein et al., J Biol Chem 271:23588-23593 (1996).
Final elution for fractions subjected to phosphatase assays was in buffer
containing 25 mM imidazole (pH 7.2), 0.1 mg/ml bovine albumin, 10mM
dithiothreitol (phosphatase assay buffer) , plus 3mM N,N',N"
triacetylchitotriose
(Sigma Chemical Co. of St. Louis, Missouri).
s2P_labeled, phosphorylated substrate (Raytide) was prepared by the
manufacturer's recommendations as described (Oncogene Sciences of
Uniondale, New York) to achieve specific activities of (dpm/fmol). Phosphatase
activity in lectin purified fractions was assayed in triplicate at 30°C
for times
indicated in the Figure Descriptions in 200,1 volumes of phosphatase buffer
using 300 ng/ml substrate in the presence or absence of Na3V04, as described.
Released phosphate was quantitated by scintillation counting and data are
expressed as mean cpm +/- SEM. Assays were linear over 1-10 min periods.
For determination of ECRTP/DEP-1 activity and abundance (Figure 8)
HRMEC cells were plated at cell densities indicated in the Figure
Descriptions.
At 36 hours after plating, a subset of cells, as indicated, was treated for 10
min
with pervanadate (1 mM Hz02 + 1 mM Na3V04), then cells were lysed in buffer
containing 50mM HEPES/pH7.5, 150mM NaCI, 5mM EDTA,1% Triton X-100,
0.1 mM, 5~g/ml aprotinin, 1 ~.g/ml leupeptin, 1 mM PMSF, clarified by
centrifugation and equivalent lysate proteins(150p.g)were immunoprecipitated
by incubation with affinity-purified monospecific ECRTP/DEP-1 rabbit antibody
(12.5 ~g/mL) overnight at 4°C, and collected on protein A-sepharose
(Sigma
Chemical Co. of St. Louis, Missouri).
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The washed immunocomplexes were assayed for PTP activity with p-
nitrophenylphosphate, pNPP (Sigma Chemical Co. of St. Louis, Missouri) as
previously described in Wang, Y. and Pallen, C.J., J Biol Chem 267:16696-
16702 (1992). Briefly, the immunocomplexes were incubated with reaction
mixture (50mM sodium acetate/pH5.5, 0.5mg/mL bovine albumin, 0.5mM DTT,
5mM pNPP) at 30°C for 30 min in the absence or presence of 1 mM Na3V04.
Reactions were stopped by addition of 2N NaOH, and the absorbance at
410nm was measured.
For quantification of ECRTP/DEP-1 abundance, immunoprecipitated
fractions were also resolved by 7% SDS-PAGE under reducing conditions,
transferred to PVDF membranes (sold under the trademark Immobilon-PT"" by
Millipore Corporation, Bedford, Massachusetts), and blocked in 5% non -fat dry
milk in Tris-buffered saline (50mM Tris/HCI pH7.5, 137 mM NaCI) containing
0.2% Tween 20 (TBST) overnight at 4°C. Blots were incubated with
ECRTPAb1 (10 ~,g/mL) or phosphotyrosine monoclonal antibody, 4610, (1.0
p.g/mL, Upstate Biotechnology) and bound antibodies detected with ~.
horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody
(Boehringer) and a chemiluminescent reagent (ECL; Amersham,
Buckinghamshire, England).
Proliferation Assays - In initial assays of HRMEC proliferation (Figure 6),
cells were plated at the indicated density, harvested at the indicated times
and
counted in quintriplicate. Data represent means ~ SEM. In other experiments
(Figures 8 and 9), HMEC-1 cells were grown on a 35mm diameter dish (sold
underthe registered trademark FALCON~ by Becton, Dickinson and Company,
Franklin Lakes, New Jersey) and cotransfected with ECRTP/DEP-1 expression
plasmids (either parent vector, pSRa, or pSRa-ECRTP/DEP-1/3xHA, driving
high level expression of a carboxyterminal hemagglutinin (HA) epitope tagged
human ECRTP/DEP-1, 1.8p,g) and a green fluorescence protein expression
plasmid (pEGFP, Clontech, 0.4~,g). An adenovirus-assisted lipofectamine
procedure that transfects 40-50% of HMEC-1 cells under these conditions was
used, as is also described in Example 1. Transfected cells were harvested 48h
after transfection and replated on glass coverslips in individual wells of a
12
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well plate at densities indicated in the Figure Descriptions (range 2-10 x
104),
to achieve attached cell confluencies of 20-90+%). Proliferating cells were
labeled by addition of 10p,M 5-Bromo-2'-deoxy-uridine (BrdU) to culture media
for 30 min at 70 hours after transfection. BrdU incorporation was
immunocytochemically detected using a monoclonal BrdU antibody and
rhodamine-conjugated anti-mouse IgG, according to manufacturer's protocol
(Boehringer Mannheim, Indianapolis, Indiana). The cells of at least five
independent fields were observed under epifluorescence microscopy (Nikon
ECLIPSE~ E600T"") and the frequency of BrdU labeling in GFP positive cells
was scored.
Planar Endothelial Migration Assay - A planar endothelial migration
assay was developed to assess the rate of endothelial closure of circular
"wounds" of 300-500p, diameter. A rotating silicon-flipped bit attached to a
drill
press was used to generate 3-5 "wounds" in confluent endothelial monolayers
within individual wells of multi-well plates. At the time of "wounding",
medium
in individual wells were supplemented with agents at concentrations indicated
in the Figure Descriptions. Residual areas of individual wounds in
photomicroscopic images captured at the indicated times (4 & 8 hours) were
quantitated using a Bioquant (Nashville, Tennessee) software package
calibrated to a Nikon DIAPHOT~ microscope. Expressed in this manner, the
rates of wound closure are remarkably linear, with linear regression r2 values
> 0.985. Each data point displayed here represent the mean ~ SEM of three
or more individual determinations from the same well. Each experiment
described is representative of findings from three or more independent
observations.
In situ Transfection Assay for Migration - Confluent HMEC-1 cells grown
on 6 well culture plate were transfected with 2.2 ~,g of expression plasmids,
pSRa ECRTP/DEP-1/3xHA, or pSRa-EphB1/3xHA (Stein et al., Genes Dev
12:667-678 (1998))and circular wounds were prepared at 48 hours after
transfection as described above. When the wounds were almost closed (12 h
after wounding), monolayers were fixed with 2% paraformaldehyde for 20 min,
washed with phosphate buffered saline, permeabilized with 0.02% saponin for
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60 min, blocked with 5% goat serum and incubated with 5 p,g/mL of monoclonal
HA antibody, 12CA5, (Berkeley Antibody Company (BAbCo), Richmond,
California) for 60 min. Coverslips were then washed with phosphate buffered
saline, incubated with biotinylated goat anti-mouse IgG (Vector Laboratories,
Inc. of Burlingame, California, 7.5p,g/mL) for 60 min, washed, incubated with
HRP conjugated avidin-biotin complexes (Vector Laboratories, Inc: of.
Burlingame, California) for 30 min and finally developed using 6 mg/mL of 3,
3'-diaminobenzidine (Sigma Chemical Co. of St. Louis, Missouri).
Cornea PocketAngiogenesisAssay-Agents to be tested forangiogenic
or anti-angiogenic activity were immobilized in a slow release form in an
inert
hydron pellet of approximately 0.2,1 volume, as described in Kenyon, Voest,
et al. (1996). That pellet is implanted into the corneal epithelium of an
anesthetized C57BL mice in a pocket created by micro-dissection. Over a 5
to 7 day period angiogenic factors stimulate the ingrowth of vessels from the
adjacent vascularized corneal limbus. A photographic record is generated
using slit lamp photography. The appearance, density and extent of these
vessels are evaluated and scored. In some cases the time course of the
progression is followed in anesthetized animals, priorto sacrifice. Vessels
are
evaluated for length, density and the radial surface of the limbus from which
they emanate (expressed as clock-face hours).
Results - Initial experiments were conducted to establish the cell density
(cell number/surface area) at which human renal microvascular endothelial
cells (HRMEC) display growth arrest in serum supplemented growth medium.
In situ experiments have shown high level expression of ECRTP/DEP-1 in
glomerular and extraglomerular microvascular endothelial cells of human
kidney, as well as in arteries and a wide range of other tissues. In Figure
6A,
identical numbers of HRMEC were plated on cell culture plates of 9.6, 28.3, or
78.5 cm2, representing 1, 2.9, or 8.1 fold the surface area of a 35mm diameter
dish, as indicated. Growth medium was replaced every 3 days. Depending
upon passage number, HRMEC reached growth arrest at a density of
approximately 1.3-6 x 104 cells/cm2, a response that supercedes responses to
maximal growth stimuli. Doubling time under density unrestricted conditions
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is approximately 44 hours. The established human dermal microvascular
endothelial cell line, HMEC-1, similarly displayed density-mediated growth
arrest properties.
Increasing fibroblast cell density was previously associated with
increases in tyrosine phosphatase activity recovered from membrane-
associated fractions. See e.a., Pallen, C.J. and Tong, P. H. Proc Natl Acad
Sci
USA 88:6996-7000 (1991 ). Among membrane-associated proteins, can
surface receptors, including ECRTP/DEP-1, are modified by N-linked
glycosylation of the ectodomain region and can be recovered using lectin
affinity chromatography. Honda et al., Blood 84:4186-4194 (1994). Shown in
Figure 6B, tyrosine phosphatase activity of the triticum vulgaris (wheat germ
agglutinin, WGA) lectin fraction recovered from identical numbers of HRMEC
plated was analyzed for the indicated times at densities determined by the
culture dish surface area. As early as 15 hours after plating, marked
differences in vanadate-sensitive tyrosine phosphatase activity were evident.
Lectin-recovered receptor-associated tyrosine phosphatase activity was 2 fold
higher in cells plated at a density sufficient to impose growth arrest (8.1x),
compared with those plated at lower density (1x). As cells plated at lower
densities (2.9 and 1 x) proliferated, increases in activity were seen,
eliminating
the marked difference. The increased lectin-recovered activity was evident at
times anticipating the imposition of proliferation arrest, suggesting that
either
the prevalence of specific tyrosine phosphatases was increasing, that the
activity of pre-existing phosphatases was increased, or that tyrosine
phosphatases were being recruited to associate with lectin recovered proteins.
The previous report that DEP-1 receptor prevalence increased with increasing
cell density lead us to evaluate the activity and distribution of DEP-1.
Ostman
et al., Proc Natl Acad Sci USA 91:9680-9684 (1994).
Shown in Figure 7, differences in the amount of immunoprecipitated
ECRTP/DEP-1 antigen could not be detected when cells plated for 33 hours
at different densities were compared. Additional experiments failed to show a
change in the ratio of Triton X-100 soluble to insoluble fractions at these
densities. However,1.8 fold increases in the vanadate-sensitive ECRTP/DEP-
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1 associated tyrosine phosphatase activity were recovered by
immunoprecipitation from cells plated at the highest (8.1x) compared with the
lowest (1x) cell density. Shown in the lower panel immunoblot (Figure 7),
immunoprecipitated ECRTP/DEP-1 is itself a tyrosine phosphoprotein in cells
pretreated with vanadate. Moreover, the level of intrinsic phosphotyrosine is
decreased in the immunoprecipitated ECRTP/DEP-1 recovered from cells
plated at high density, correlating with the increased tyrosine phosphatase
activity in that fraction. These findings indicate that the abundance of
ECRTP/DEP-1 does not change acutely in endothelial cells plated at high
density, but that the ECRTP/DEP-1-associated phosphatase activity does
increase. Efforts to demonstrate by in gel zymographic phosphatase assays
that the increased activity is intrinsic to ECRTP/DEP-1 have not been
successful.
To further pursue the possibility that ECRTP/DEP-1 mediates signals
capable of arresting endothelial proliferation and migration, HMEC-1 cells
were
cotransfected with an expression plasmid driving high level expression of an
epitope-tagged ECRTP/DEP-1, and with a piasmid driving expression of green
fluorescent protein to mark transfected cells. Using adenovirus-assisted
transfection method, transfection of 40-50% of HMEC-1 cells that display
survival, migration and proliferation properties similar to nontransfected
cells
was routinely accomplished. Shown in Figure 8A, high level expression of a
full
length ECRTP/DEP-1 imposes marked suppression of BrdU incorporation
across a range of plating densities of transfected cells when compared with
the
empty expression vector.
~ ECRTP/DEP-1 overexpression imposed similar effects upon endothelial
migration as those observed with proliferation. Shown in Figure 9A, HMEC-1
cells transfected with plasmids driving expression of hemagglutinin epitope
(HA) tagged versions of either ECRTP/DEP-1 /HA or a receptortyrosine kinase,
EphB1/HA, were plated at densities to permit them to rapidly attain a
confluent
monolayer. A circular "wound" of approximately 500~,m diameter was
generated, and migration of transfected and non-transfected cells to close the
"wound" was determined after 33 hours, by staining for the expressed protein
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HA epitope. Unlike cells transfected with the EphB1/HA control, ECRTP/DEP-
1 /HA expressing cells did not migrate to contribute to the wound closure.
While
forced overexpression of ECRTP/DEP-1 can be informative about the potential
for this receptor to affect proliferation or migration, this approach is much
less
discriminatory than use of high affinity reagents interacting with
endogenously
expressed ECRTP/DEP-1 s. To this end, a panel of monoclonal antibodies
generated against ECRTP/DEP-1 ectodomain sequences was screened for
activity.
Shown in Figure 8B, bivalent forms of the monoclonal, ECRTPAb1,
imposed a marked inhibitory effect on proliferation of HRMEC plated at low
density, in spite of repeated growth medium exchanges. Equivalent
concentrations of a class matched monoclonal control antibody were inactive.
Because oligomerization is a critical determinant of activation of many
receptor
tyrosine kinases and phosphatases (Weiss, A. and Schlessinger, J., Cell
94:277-280 (1998)), ECRTPAb1 Fab fragments were prepared to test whether
bivalency of the interacting monoclonal was required for activity. Also shown
in Figures 8B and 8C, equimolar concentrations of the ECRTPAb1 Fab
fragments were inactive as growth inhibitors in endothelial cells plated at
subconfluent densities in serum-containing growth medium.
Additional endothelial "wound" closure assays, similar in design to those
presented in Figure 9A, were conducted to evaluate effects of bivalent and
monovalent ECRTPAb1 on endothelial migration. Displayed in Figure 9B are
the residual fractions of original wound areas remaining at the times
indicated.
Phorbol myrisate acetate (PMA) markedly accelerated the rate of migration and
wound closure, compared with unstimulated cells in serum-free medium.
Bivalent ECRTPAb1 displayed marked activity to inhibit the PMA stimulated
migration, while equimolar concentrations of monovalent Fab fragments, and
a control monoclonal were inactive. The linear characteristics of time
dependent "wound" closure in this assay permitted us to determine relative
migration rates for the population, expressed in Figure 9C as fractional
closure/hr. Effective concentrations of bivalent ECRTPAb1 (67 and 200 nM)
were similar to those active as inhibitors of proliferation (Figure 8C).
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In aggregate, these findings suggested that engagement of endogenous
ECRTP/DEP-1 s by bivalent antibodies can function like a "surrogate ligand" to
emulate responses normally evoked by an endogenous membrane-associated
ligand upon cell-cell contact. Since the ECRTPAb1 Fab fragments were
inactive as "surrogate ligands" to inhibit migration and proliferation in
subconfluent cells, it was asked whether they can have activity as antagonists
of endogenous ligand engagement of ECRTP/DEP-1 in cells plated at high
density. It was reasoned that Fab fragments can interrupt endogenous ligand-
receptor engagement and subsequent growth arrest signals in~ cells plated at
high density. Shown in Figure 10, ECRTPAb-1 Fab had a marked effect to
release cells from the density-imposed inhibition of BrdU uptake that marks S
phase entry.
As a final test to determine whether ECRTPAb-1 functions to induce an
angiostatic signal, it was tested whether this antibody modified angiogenic
responses to basic FGF in the mouse corneal pocket assay. Shown in Figure
11, inclusion of ECRTPAb-1, but not a control IgG, in the implanted slow
release hydron pellet inhibited angiogenesis, scored by reducing the length of
capillary sprouts as they approached the source of the angiogenic stimulus.
This attenuation of capillary length, without effect on radial distribution of
new
vessels, suggests that pro-angiogenic basic FGF can diffuse more rapidly from
the slow release pellet than ECRTPAb-1, permitting brisk initiation of
angiogenesis with subsequent attenuation.
EXAMPLE 3
Method of Screening for Endogenous Li~gand of ECRTP/DEP-1
Labeled ECRTP/DEP-1 is used to perform binding studies to identify
cells with ECRTP/DEP-1 ligands using Scatchard analysis; and to perform
cross-linking studies which demonstrate the ECRTP/DEP-1 ligand(s) on
polyacrylamide gels. These initial characterization methods are used to
identify
cells with high and low numbers of ECRTP/DEP-1 ligand(s) for purification and
isolation studies. Once a cell line with high levels of ECRTP/DEP-1 ligand has
been identified, then the protein is purified by the following approaches:
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Approach A: Biochemical purification
A cell line which expresses high levels of ECRTP/DEP-1 ligand is lysed
and the protein from lysates or membrane preparations is purified by gel
filtration followed by purification of the ligand with a column containing the
ECRTP/DEP-1 bound to a solid phase such as sepharose. The purified ligand
protein can then be microsequenced and the gene cloned using degenerate
oligonucleotides derived form the protein sequence.
Approach B: cDNA librar~purification
The ECRTP/DEP-1 is radiolabeled with '251 and then used to screen cell
lines or tissues by Scratchard analysis for specific binding of ligand. Once
such ligand binding is identified, a cDNA library is constructed from that
tissue
or cell line and transfected into a cell line that does not exhibit specific
binding.
These transfected cells are then screened for newly acquired specific binding
which indicates they have been transfected with a construct containing the
gene for the ECRTP/DEP-1 ligand. Plasmid DNA from positive clones is then
isolated and sequenced for identification. A single construct is then
transfected
back into the null cells to verify that binding between ligand and receptor is
mediated by the transfected gene. Kluzen et al. Proc Natl Acad Sci USA
89:4618-4622 (1992).
Alternatively, chimeric ECRTP/DEP-1 and immunoglobulin Fc molecules
are constructed. LaRochelle et al., J Cell Biol 129:357-366 (1995). The
chimeric molecules can then be used to screen for binding to ECRTP/DEP-1
ligand on whole cells via flow cytometry. Alternatively, due to the presence
of
the immunoglobulin component of the molecule, cell lysates are screened by
immunoblotting or by immunoprecipitation of metabolically labeled cells. This
technique can identify ECRTP/DEP-1 binding proteins by a variety of different
methods. Peptide digests of the identified proteins are then generated so that
peptides can be sequenced and the whole molecule cloned by the
degenerative oligonucleotide approach.
Thus, this Example pertains to a method for isolating a ligand for an
ECRTP/DEP-1, and to a purified and isolated ligand for an ECRTP/DEP-1.
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The method comprises contacting cells or cell lysates having the ligand or
suspected of having the ligand with ECRTP/DEP-1; and isolating the ligand
which binds with ECRTP/DEP-1. The cells having the ligand are identified by
labeling the ECRTP/DEP-1; screening cell cultures with the labeled
ECRTP/DEP-1; and isolating cells that bind an elevated amount of the labeled
ECRTP/DEP-1.
The ligand is isolated by lysing the cells and passing the cell lysate over
a column containing the ECRTP/DEP-1 bound to a solid phase matrix within
the column. Alternatively, the ligand is isolated by constructing a cDNA
library
from the cells binding the ligand; transfecting the cDNA library into a cell
line
that does not exhibit binding of the ligand; screening the cell line for newly
acquired specific binding; isolating DNA form cells exhibiting specific
binding;
and sequencing the isolated DNA to determine the DNA sequence for the
ligand.
The ECRTP/DEP-1 is optionally labeled by binding the ECRTP/DEP-1
to an immunoglobulin. In this case, the ligand is isolated by
immunoprecipitation of the ECRTP/DEP-1-ligand-immunoglobulin complex.
Alternatively, the ligand of the ECRTP/DEP-1 is isolated using flow cytometry.
EXAMPLE 4
Identification of Binding Epitope for ECRTPAb-1
A series of 96 eight to nine amino acid peptides was generated. The
eight to nine amino acid peptides span in overlapping epitopes the 351 amino
acid sequence against which ECRTPAb-1 was derived was generated. These
peptides were generated and immobilized in a defined array on the surface of
a membrane that was probed with ~ECRTPAb-1: Binding of ECRTPAb-1 to
peptide #41 in the array was identified using a peroxidase-conjugated anti-
mouse IgG second antibody, by chemiluminescence autoradiography (Figure
12). The sequence of this peptide derived from the array is n-QSRNDEVL-c.
The 8-amino acid epitope represents the target sequence within the ECRTP
ectodomain against which functional ECRTP agonists and antagonists interact,
based on biological activities of ECRTPAb-1. Antibodies, including humanized
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antibodies, and other peptides with high affinity for this defined amino acid
sequence have biological activities comparable to those demonstrated herein
using ECRTPAb-1.
EXAMPLE 5
Assay for Scoring Biological Activity of Antibodies,
Proteins and Peptides that Bind ECRTP
In an effort to develop a simplified reconstitution assay system capable
of scoring biological activities mediated through ECRTP, Chinese Hamster
Ovary (CHO) cells that do not express endogenous ECRTP were transfected
with a plasmid construct driving high level expression of ECRTP (pSRa-
ECRTP/HA), or catalytically inactive mutated forms (pSRa-ECRTP/HA/C-S,
pSRa-ECRTPIHAocy), in these cells. Transiently transfected CHO cells were
then dispersed and plated on 24 well plates and exposed to either control IgG,
or ECRTPAb-1, or monovalent forms of ECRTP (ECRTPAb-1-Fab).
Proliferation of cells transfected with wild type ECRTP, but not with either
mutant form, was inhibited (approximately 40%) by incubation with ECRTPAb-
1, but not with either control IgG~ or ECRTPAb-1-Fab.
In parallel, ECRTP was immunoprecipitated from CHO cells transfected
with these plasmids and expressing equivalent amounts of ECRTP or mutant
forms (shown in the inserts above Figures 13A and 13B) following a time
course of exposure (0 - 30 min) to either ECRTPAb-1 or ECRTPAb-1-Fab.
Shown in the immunoblot panels on the right in Figures 13A and 13B, Ab1
evoked a rapid de-phosphorylation of tyrosine residues on the wild type (WT)
ECRTP, but had no effect on catalytically inactive C/S mutant forms. These
findings provide primary evidence that the catalytic phosphatase function is
critical to the action of ECRTPAb-1 to inhibit proliferation and to provoke de-
phosphorylation of ECRTP.
Finally, co-expression of either catalytically inactive C/S or Cy deleted
forms of ECRTP with the functional ECRTP form abrogates inhibitory effects
of the ECRTPAb-1 on CHO proliferation (Figure 13B). This proliferation assay
thus evaluates whether putative counter-receptors (ligands) are competent to
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function through ECRTP to inhibit cell growth. In each case, the monovalent
and bivalent forms of ECRTPAb-1 provide a reagent that can be used in the
assay to define functional counter-receptors.
EXAMPLE 6
Transgenic Animal Having Catalyticall I~ve ECRTP/DEP-1
A transgenic mouse expressing a catalytically inactive form of
ECRTP/DEP-1 was prepared. The mouse was prepared using a "knockout"
approach with respect to the ECRTP/DEP-1 gene. Embryonic death was
observed in homozygous "knockout" animals. Heterozygous animals displayed
significant vascularization malformations. Thus, the transgenic mouse further
established the role of ECRTPIDEP-1 in cell growth, cell survival and
angiogenesis.
REFERENCES
The references listed below as well as all references cited in the
specification are incorporated herein by reference to the extent that they
supplement, explain, provide a background for or teach methodology,
techniques and/or compositions employed herein.
Ades et al., J Invest Dermatol 99:683-690,(1992).
Augenlicht and Baserga, Exp Cell Res 89:255-262 (1974).
Ausprunk et al., Am J Pathol, 79:597-618 (1975).
Batt et al., J Biol Chem 273:3408-3414 (1998).
Bauer et al., J Cell Physiol 153:437-449 (1992).
Beekhuizen, H. and van Furth, R. J Vascular Res 31:230-239 (1994).
Billard, C., et al., Blood (2000 Feb 1 ) 95(3):965-72.
Bittner et al., Methods in Enzymol 153:516-544 (1987).
Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition,
(1976).
Borges efi al., Circulation Research 79:570-580 (1996).
Cheresh et al., J Biol Chem, 262:17703-17711 (1987).
Choime et al., J Biol Chem 270:21144-21150 (1995).
CA 02401621 2002-08-27
WO 01/64750 PCT/USO1/06178
-85-
Coomber, B.L. J Cell Biochem 52:289-296 (1993).
Daniel et al., Kidney Int 50:S-73-S-81 (1996).
de la Fuente-Garcia et al., Blood 91:2800-2809 (1998).
Desai et al., Cell 84:599-609 (1996).
Dillman et al., Antibody Immunocon Radiopharm 1:65-77 (1988).
Dulbecco et al., Virol 8:396 (1959).
Eijgenraam, F., Science 261:883-884 (1993).
Engerman et al., Laboratory Investigation 17:738-744 (1967).
EP 44167
Fields et al., lnt. J. Peptide Protein Res 35:161-214 (1990).
Flickinger & Trost, Eu, J, Cancer 12(2):159-60 (1976).
Ghose et al., CRC Critical Reviews in Therapeutic Drug Carrier
Systems 3:262-359 (1987).
Ghose et al., Meth. Enzymology 93:280-333 (1983).
Gumkowski et al., Blood Vessels 24:11-23 (1987).
Hailing et al., Nucl Acids Res 13:8019-8033 (1985).
Honda et al., Blood 84:4186-4194 (1994).
Huse et al., Science 246:1275-1281 (1989).
Hyink et al., Am J Physiol 270:F886-F899 (1996).
Inouye et al., Nucleic Acids Res 13:3101-3109 (1985).
Keane et al., Cancer Research 56:4236-4243 (1996).
Kimura et al., Immunogenetics 11:373-381 (1980).
Kitamoto et al., J Clin invest 99:2351-2357 (1997).
Kluzen et al. Proc Natl Acad Sci USA 89:4618-4622 (1992).
Knowles & Thorpe, Anal. Biochem 120:440-443 (1987).
Koenig et al., J Clin Immunol 13:204-211 (1993).
Kohler and Milstein, Nature 256:495-497 (1975).
Kumet et al., J Biol Chem 271:30916-30921 (1996).
Lamb et al., EurJrnlBiochem 1-48:265-270 (1985).
Lampugnani et al., J Cell Biol 129:203-217 (1995).
LaRochelle et al., J Cell Biol 129:357-366 (1995).
Leveen et al., Genes Dev 8:1875-1887 (1994).
CA 02401621 2002-08-27
WO 01/64750 PCT/USO1/06178
-86-
Logan et al., Proc Natl Acad Sci USA 81:3655-3659 (1984).
Lord et al., In Genetically Engineered Toxins (Ed. A. Frank, M. Dekker Publ.,
p. 183) (1992)
Lowy et al., Cell 22:817 (1980).
Martin et al., In Vitro Cell Dev Biol 33:261-269 (1997).
McOmie, J. F. W., "Protective Groups in Organic Chemistry", Plenum Press,
New York, (1973).
Meienhofer, J., "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic
Press (New York) (1983).
Merrifield, Adv Enzymol, 32:221-96 (1969).
More et al., J Patho 172:287-292 (1994).
Mulligan et al., Proc Natl Acad Sci USA 78:2072 (1981 ).
O'Hare et al., FEBS Lett 210:731 (1987).
Ogata et al., J Biol Chem 256:20678-20685 (1990).
Ossonski et al., Cancer Res 40:2300-2309 (1980)
Ostman et al., Proc Natl Acad Sci USA 91:9680-9684 (1994).
Pallen, C.J. and Tong, P.H. Proc Natl Acad Sci USA 88:6996-7000 (1991 ).
Palou et al., Immunol Lett 57:101-103 (1997).
Pietersz et al., Antibody, Immunoconj Radiopharm 1:79-103 (1988).
Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company,
(1980)
.Rijksen et al., J Cell Physiol 154:393-401 (1993).
Robert et al., Am J Physiol 271:F744-F753 (1996).
Robert et al., Am J Physiol 275:F164-F172 (1998).
Ruther et al., EMBO J 2:1791 (1983).
Sastry, et al., Proc Natl Acad Sci USA 86:5728-5732 (1989).
Schoecklmann et al., J Am Soc Nephrol 5:730 (1994) (abstract).
Scholz et al., Cell Tissue Res 290:623-631 (1997).
Schroder et al., "The Peptides", Vol. 1, Academic Press (New York) (1965).
Smith et al., J Virol46:584 (1983).
CA 02401621 2002-08-27
WO 01/64750 PCT/USO1/06178
-87-
Soriano, P., Genes Dev 8:1888-1896 (1994).
Stein et al., J Biol Chem 271:23588-23593 (1996)
Stein et al., Genes Dev 12:667-678 (1998).
Steward et al., "Solid Phase Peptide Synthesis", W. H. Freeman Co.,
San Francisco, (1969).
Szybalska et al., Proc Nafl Acad Sci USA 48:2026 (1962).
Takahashi et al., Kidney Int 53:826-835 (1998).
Thomas et al., J Biol Chem 269:19953-19962 (1994).
Thorpe et al., Cancer Res 47:5924-5931 (1987).
Tsiotra et al., J Biol Chem 271:29216-29222 (1996).
U.S. Pat. No. 4,244,946
U.S. Pat. No. 4,215,051
U.S. Pat. No. 4,340,535
U.S. Pat. No. 4,472,509
U.S. Pat. No. 5,660,827
U.S. Pat. No. 5,733,876
U.S. Pat. No. 5,753,230
U.S. Pat. No. 5,762,918
U.S. Pat. No. 5,766,591
U.S. Pat. No. 5,776,427
Vaickus et al., Cancer Invest 9:195-209 (1991 ).
Van Heeke et al., J Biol Chem 264:5503-5509 (1989).
Vogel & Muller-Eberhard, Anal. Biochem 118(2):262-268 (1981 ).
Wallner et al., Microsc Res Tech 39:261-284 (1997).
Wang, Y. and Pallen, C.J., J Biol Chem 267:16696-16702 (1992).
Weiss, A. and Schlessinger, J., Ce1194:277-280 (1998).
Wigler et al., Cell 11:223 (1977).
W O 84/03564
Zimmer et al., Peptides (1992) pp. 393-394, ESCOM Science Publishers,
B.V., (1993).
Zola, MonoclonalAntibodies: a Manual of Techniques, CRC Press, lnc. (1987).
CA 02401621 2002-08-27
WO 01/64750 PCT/USO1/06178
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It will be understood that various details of the invention can be changed
without departing from the scope of the invention. Furthermore, the foregoing
description is for the purpose of illustration only, and not for the purpose
of
limitation--the invention being defined by the claims.
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INDICATIONS RELATING TO A DEPOSITED lVItCROO~GA~
(PCT Rule l3bis)
A. The indications made below
relate to the microorganism referred
to in the description
on page 31 , line 30
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
American Type Culture Collection
(ATCC)
Address of depOSitary lnstitutiori
(including postal code and county)
10801 University Boulevard
Manassas, Virginia 20110-2209
UNITED STATES OF AMERICA
Date of deposit Accession Number
18 September 1998 (18.09.98) ATCC HB12570
C. ADDITIONAL INDICATIONS (leave
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additional sheet
Deposit is a mouse hybridoma cell
line. Applicant respectfully
requests that access to the deposited
matexial
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possible under applicable treaties,
laws and/or regulations of each
of the
Designated States of the present
International Application, iilcluding
but not limited to restricting
access only
to an expert designated by the
National Patent Office of a Designated
State. Applicant also requests
that to
the extent permissible under applicable
treaties, laws and/or regulations,
notification of requests for
the
deposited material be transmitted
to applicant's agent listed in
the present International Application
by the
National Patent Office of the
Desi ated States.
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if tlae
indications are not for all designated
States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if noz applicable)
The indications listed below will
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nature of the indicatiora, e.g.,
"Accession
Number of Deposit')
For receiving Office use only ~ For International Bureau use
This sheet was received with the international application ~ ~ ~ This sheet
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Form PCT/R01134 (duly 1992)
