Note: Descriptions are shown in the official language in which they were submitted.
' CA 02351585 2001-05-16
Influencing Angiogenesis Using CD66a
The invention relates to a pharmaceutical composition for
influencing angiogenesis. In one case, angiogenesis may be
improved by administration of CD66a or substances initiating
the formation of CD66a, while in the other case angiogenesis
may be inhibited by using substances preventing interaction
between CD66a and CD66a ligands.
The formation of blood vessels (angiogenesis) is in many
diseases an important step which may contribute to curing, on
the one hand, or shall desirably be prevented in other cases.
Improving angiogenesis is very desirable e.g. for
cardiovascular diseases to treat angina pectoris or heart
attacks or cerebral infarctions, for example. On the other
hand, the inhibition of the vascular supply of malignant
solid tumors in humans and animals is a promising approach in
tumor therapy. Angiogenesis inhibitors, such as endostatin,
directly attack normal and thus genetically stable
endothelial cells of the blood ve~:els supplying a humor,
cause them to die off and thus stop the supply of the tumor
cells with nutrient-containing blood (cf. Kerbel, R., Nature,
390, p. 335 et seq., 1997). This leads to a regression of
blood vessels and tumor mass. Since contrary to the tumor
cells the endothelial cells are genetically stable,
resistances do not form as is the case e.g. in a cytostatic
therapy aiming directly at the tumor cells. By inhibiting
angiogenesis the growth of human tumors could be blocked in
experimental models. Some angiogenesis inhibitors are
meanwhile tested clinically (cf.~Hanahan et al., Cell 86,
353-364, (1996)).
The supply of tissues with new vessP~.s is a complex process
in which a number of biomolecules are involved. Tumors
produce soluble mediators, for example, which initiate the
formation of new vessels. When angiogenesis proceeds,
adhesion molecules play a central part. They control the
communication of vessel cells with ~:,r.u~ another and with the
CA 02351585 2001-05-16
surrounding connective tissue. Finally, various proteinases
are also involved in the neovascularization.
It is the object of the present invention to provide a
possibility of improving or inhibiting angiogenesis as
desired. In case angiogenesis is inhibited, a form of cancer
therapy without the development of resistances .shall thus be
provided, i.e. in particular tumor-accompanying angiogenesis
shall be influenced within the meaning of a reduction of
inhibition.
According to the invention this is achieved by the subject
matters defined in the claims.
The subject matter of the present application is in
particular a pharmaceutical composition suitable to regulate
angiogenesis. Such a composition comprises:
(a) for positive regulation
one or more bodies of
. CD66a, CD66a -fragments or CD66a-derived glycostructures,-
or CD66a ligands, ligand fragments or structures derived
therefrom, as well as substances inducing the expression
of CD66a or CD66a ligand,
or
(b) for negative regulation
one or more bodies of
substances which inhibit the interaction between CD66a
and CD66a ligands or substances which inhibit the
expression of CD66a or CD66a ligand.
The protein CD66a which is also referred to as biliary
glycoprotein (BGP), transmembrane carbonembryonic antigen or
human C-CAM is a special adhesion molecule. The term CD66a is
used below. The gene coding for CD66a has already been cloned
(Hinoda et al., PNAS 85, 6959-6963, 1988). The applicants of
the present application described in 1991 already the only
CA 02351585 2001-05-16
CD66a-specific monoclonal antibody world-widd (Drzeniek et
al., Cancer Letters 56, 173-179 (1991); Stoffel et al., J.
Irnmunoi. 150, 4978-4984 (19y3)). This antibody is referred to
as 4D1/C2 and was deposited with DSMZ (Deutsche Sammlung von
Mikroorganismen and Zellkulturen [German-type Collection of
Microorganisms and Cell Cultures], Mascheroder Weg,
Braunschweig, under accession number DSM ACC2371 on October
22, 1998.
It has now been found surprisingly that the CD66a factor is
expressed in tumor capillaries whereas the blood vessels of
the corresponding normal tissues are negative.
In a human Leydig cell tumor, the individual stages of the
neovascularization could be traced accurately. In this
connection, it was found that certain stages of
neovascularization can be correlated with the occurrence of
the following factors:
1. proliferation of endothelial cells: VEGF (vascular
endothelial growth factor), VBGF receptors
2. formation of vascular lumens: CD66a
3. next differentiation step: endostatin
4. next differentiation step: angiostatin
In recent experiments conducted by the inventors using
chicken embryos it could be shown that CD66a is a potent
angiogenetic factor and improves the neovascularization of
normal and tumoral tissues.
It could also be shown that CD66a was blocked by an antibody
directed against CD66a and the formation of capillaries
necessary for tumor growth is inhibited. Tumor growth can no
longer take place.
CD66a can be detected in human tumors in newly formed blood
vessels (capillaries) in a defined differentiation window,
namely in the stage of lumen formation. In an in vitro
differentiation model a monoclonal CD66a antibody inhibits
CA 02351585 2001-05-16
the formation of vascular tubes (tube formation) by human
endothelial cells. These results prove that CD66a plays an
essential part in angiogenesis. It follows from the
expression of CD66a in tumoral vessels and the in vitro
inhibition of capillary structure formation by a monoclonal
CD66a antibody that tumor angiogenesis can be inhibited by
functionally blocking CD66a.
_ Experiments conducted with transfectomas have shown that
CD66a binds to itself (homeotypical binding) and to other
members of the CD66 family. The localization of CD66a in
newly formed endotheliums at the basal cell pole and the
inhibition of capillary formation indicate that CD66a
interacts with components of the extracellular matrix.
Antibodies, peptides, proteins or other -agents which bind
specifically to one or more functional domains of CD66a or
its ligands are particularly suitable for the CD66a
inhibition desired according to the invention in one respect.
Monoclonal antibodies which are directed against adhesive,
.- functior_ally significant domains of CD66a axe preferah7.y
used. Furthermore, CD66a has glycostructures which may have
an angiogenetic effect, e.g. LewisX and sialyl-LewisX groups.
The above-mentioned monoclonal CD66a antibody 4D1/C2 is
preferably used. This leads to an inhibition of tumor
angiogenesis via a functional inactivation of CD66a. CD66a is
in this connection functionally inactivated by inhibiting the
interaction between CD66a and possible ligands. Here,
structures which mediate interaction are blocked.
Furthermore, soluble ligands or soluble ligand domains may
also be used to block the interaction. The invention also
relates to the use of recombinant domains which correspond to
a CD66a fragment and to fragments of antibodies which react
substantially with the epitope of CD66a. As a result, the
signal chain starting from CD66a is blocked. The employed
compounds may also be modified suitably to bind e.g.
irreversibly to the receptor.
In particular CD66a as a whole molecule, CD66a domains as
CA 02351585 2001-05-16
well as specific glycostructures of CD66a ark suitable for
the angiogenesis improvement desired according to the
invention in one respect. In these cases, the soluble
molecule form is applied to the sites of the body where
angiogenesis shall be triggered (e. g. in the cardiac muscle).
A DNA may also be used which codes for CD66a or parts of the
CD66a protein. The DNA may also be integrated in vectors
which are common in gene therapy (e. g. adenoviruses).
Synthesis of the protein may also be achieved by
administration of simple plasmid DNA. The positive influence
~of angiogenesis is effected by improving interactions between
CD66a and CD96a ligands.
Methods of obtaining the above-mentioned antibodies which may
be used for inhibiting angiogenesis are known to a person
skilled in the art and comprise e.g. as to polyclonal
antibodies the use of CD66a or a fragment thereof as
immunogen for immunizing suitable animals and obtaining
serum. The person skilled in the art is also familiar with
methods of producing monoclonal antibodies. For this purpose,
e.g. cell hybrids a~-e produced from antibody-producing cells
and bone marrow tumor cells (myeloma cells) and cloned.
Thereafter, a clone is selected which produces an antibody
specific to CD66a. This antibody is then produced according
to standard methods. Examples of cells which ,,produce
antibodies are spleen cells, lymph node cells, B lymphocytes,
etc. Examples of animals which may be immunized for this
purpose are mice, rats, horses, goats and rabbits. The
myeloma cells may be obtained from mice, rats, humans or
other sources. The cell fusion may be carried out e.g. by the
generally known method of Kohler and Milstein. The hybridomas
obtained by cell fusion are screened using CD66a according to
the enzyme-antibody method or according to a similar method.
Clones are obtained e.g. with the boundary dilution method.
The resulting clones are implanted intraperitoneally into
BALB/c mice. Ascites is removed from the mouse after 10 to 14
days, and the monoclonal antibody is purified by known
methods (e.g. ammonium sulfate fractionation, PEG
fractionation, ion exchange chromatography, gel
CA 02351585 2001-05-16
chromatography or affinity chromatography). 'The collected
antibody may be used directly or a fragment thereof may be
employed. In this connection, the term "fragment" means all
parts of the antibody (e.g. Fab, Fv or single chain Fv
fragments) which have an epitope specificity the same as that
of the complete antibody.
In one embodiment, said monoclonal antibody is an antibody
originating from an animal (e. g. mouse), a humanized
antibody, a chimeric~antibody or a fragment thereof. Chimeric
~antibodies~which are similar to human antibodies or humanized
antibodies have a reduced potential antigenicity but their
affinity over the target is not lowered. The production of
chimeric and humanized antibodies or of antibodies similar to
human antibodies was discussed in detail (Noguchi, Nippon
Rinsho, 1997, 55(6) pp. 1543-1556; van Hogezand, Scand. J.
Gastroenterol. Suppl., 1997, 223, pp. 105-107). Humanized
immunoglobulins have variable framework regions which
originate substantially from a human immunoglobulin
(designated acceptor immunoglobulin) and the complementarity
of the determining regions which originate substantially fwo~n
non-human immunoglobulin (e. g. from mouse) (designated donor
immunoglobulin). The constant regions) originate(s), if
available, also substantially from a human immunoglobulin.
When administered to human patients, humanized (and human)
anti-CD66a antibodies according to the invention offer a
number of advantages over antibodies from mice or other
species: (a) the human immune system should not regard the
framework or the constant region of the humanized antibody as
foreign and therefore the antibody response to such an
injected antibody should be less than that to a fully foreign
mouse antibody or a partially foreign chimeric antibody; (b)
since the effector region of the humanized antibody is human
it might interact in a better way with other parts of the
human immune system, and (c) injected humanized antibodies
have a half life substantially equivalent to that of
naturally occurring human antibodies, which permits
administering smaller and less frequent doses as compared to
antibodies of other species.
CA 02351585 2001-05-16
v
The above described conventional technology may also be
supplemented or replaced using recombinant phage libraries
(Felici et al., Biotechnol. Rev. 1, pp. 149-183 (1995;
Hoogenboom et al., Immunotechnology 4, pp. 1-20 (1998)).
Recombinant phage libraries may have random peptide
structures in the antigen-binding regions of the phage-
presented antibody fragments. The advantage of this
technology is inter alia that in cloned phages the
information on the amino acid sequence of the antigen binding
~structures~is directly available.
The domains of CD66a or the CD66a ligands, whose blocking
effects a functional inactivation of CD66a, may be recombined
in any way and be used while being introduced into molecules
which are suitable for therapeutic purposes (e. g. to achieve
better immunological compatibility). The reactive domains may
also be expressed according to molecular-biological standard
methods, e.g. bacterially or in insect cells.
Interaction "between CD66a and potential ligands may
preferably be inhibited in the following ways (negative
regulation)
inhibition by antibodies and antibody fragments against
the functional domain of CD66a,
- inhibition by antibodies and antibody fragments against
the functional domains of the CD66a ligands,
- inhibition by the functional domain of CD66a,
- inhibition by the functional domain of the CD66a
ligands,
- inhibition of the endogenous formation of CD66a or CD66a
ligands using anti-sense oligonucleotides.
Interaction between CD66a and potential ligands may
preferably be improved in the following ways (positive
regulation):
- ap~;la~:ation of the native molecule purified by means of
CA 02351585 2001-05-16
biochemical methods, '
- application of recombinant CD66a fragments,
- application of angiogenetically active glycostructures
isolated from CD66a,
- application of glycostructures prepared in a fully
synthetic or partially synthetic way, whose structure
was derived from angiogenetically active glycostructures
of CD66a,
- application of a DNA, which codes for the complete CD66a
protein thereof, in the form of suitable vectors or
. plasmids,
- application of a DNA, which codes for isoforms or
fragments of CD66a, in the form of suitable vectors or
plasmids.
The pharmaceutical compositions according to the invention
may be administered in any way suitable to reach the desired
tissue. The administration is preferably carried out
parenterally, particularly orally, intravenously or
intratumorally. For the purpose of administration, the
substance is used in a fQrrn«lation suitable for the
respective kind of administration using corresponding common
pharmaceutical excipients. Orally applicable pharmacons are
developed in two ways. On the one hand, interaction between
ligand and receptor may be modelled e.g. by X-ray structural
analysis or NMR spectroscopy. On the other hand, chemical
combinatorial libraries (Myers, Current Opinion in
Biotechnology 8, pp. 701-717 (1997) may be used. Here, the
interaction of the ligand or receptor is examined with
initally largely randomly combined chemical compounds. If
binding was detected, the binding properties can be defined
in more detail by selecting similar compounds.
Dosage and posology of the administration of the compounds
according to the invention are determined by a physician on
the basis of the patient-specific parameters, such as age,
weight, sex, severity of the disease, etc.
According to the kind of administration, the medicament is
CA 02351585 2001-05-16
formulated suitably, e.g. in the form bf solutions,
suspensions, as a powder, tablet or capsule or injection
preparations which are produced according to common galenic
methods.
The infusion or injection solutions are preferably aqueous
solutions or suspensions, it being possible to produce them
prior to use, e.g. from lyophilized preparations which
contain the active substance as such or together with a
carrier, such as mannitol, lactose, glucose, albumin or the
-like. The ready-to-use solutions are sterilized and
optionally mixed with auxiliary agents, e.g. with
preservatives, stabilizers, emulsifiers, solubilizers,
buffers and/or salts for regulating the osmotic pressure. The
sterilization may be obtained by sterile filtration through
filters having a small pore size, whereupon the composition
may optionally be lyophilized. Antibiotics may also be added
to help maintaining sterility.
The pharmaceutical compositions contain a therapeutically
act.~:~re arnount of one or more of the above-mentioned active. .
substances together with common auxiliary agents and carrier
substances. They are preferably organic or inorganic liquid
pharmaceutically compatible carriers which are suitable for
the desired administration and which do not interact
negatively with the active components.
The pharmaceutical preparations according to the invention
are sold as unit dosage forms, e.g. as ampoules.
The invention also relates to -a method of producing a
pharmaceutical composition, which is characterized in that
the compound according to the invention is mixed with a
pharmaceutically compatible carrier.
"Substances inhibiting the expression of CD66a or CD66a
ligand" are administered preferably by means of gene therapy
introducing into tumor cells e.g. anti-sense oligonucleotides
ro CD66a and,!or CD66a ligand. These oligonucleotides are
CA 02351585 2001-05-16
derived from the known sequences for CD66a oY CD66a ligand
(Hinoda et al., Proc. Natl. Acad. Sci. U.S.A. 85, p. 6959
(1988)). The anti-sense oligonucleotides may also reach the
size of a DNA which is complementary to regions of the gene
mRNA and binds thereto. Then, a duplex molecule forms which
is taken away from the translation of the mRNA. Inhibition of
the gene expression can be achieved in this way. The term
"anti-sense oligonucleotide" comprises any DNA or RNA
_ molecule which is complementary to regions of the CD66a RNA
or CD66a ligand RNA,~in particular mRNA and most particularly
regulatory elements thereof, and effects inhibition of the
gene expression by binding to these regions. The anti-sense
oligonucleotides may be available as such or, if they are
relatively long, in the form of a vector or vector construct
coding for them, which is sometimes also referred to as
"minigene". Such a vector may be a common expression vector.
It may be favorable for the expression of the sequence coding
for them to be controlled by a constitutive or inducible
promoter, such as a tissue-specific or tumor-specific
promoter. The anti-sense molecules may be introduced by
common methods. If the anti-sense oligonucleotides are
available as such or in the form of a vector coding for them,
e.g. transfection techniques or packaging in liposomes is
suitable.
"Substances which induce the expression of CD66a or CD66a
ligand" are e.g. DNA molecules which code for CD66a or
angiogenetically active CD66a fragments or for CD66a ligands
or angiogenetically active ligand fragments. The expression
is controlled by suitable regulatory sequences. The DNA is
administered according to protocols known to a person skilled
in the field of gene therapy. Thus, e.g. packaging of the DNA
in viral particles (e. g. adenoviruses) or the administration
of naked plasmid DNA is an consideration.
According to the invention the growth of all solid tumors of
the body may be inhibited with the angiogenesis-inhibiting
pharmaceutical composition. Examples are epithelial tumors
(e. g. squamous epirheli~::a, columnar epithelium, glandular
CA 02351585 2001-05-16
epithelium, transitional epithelium), mesenchymal tumors
(e. g. fibers, muscles, cartilages, and bone tissues), mixed
tumors (mixed epithelial, mixed mesenchymal, epithelial-
mesenchymal), tumors of the hematopoietic and lymphatic
tissues (bone marrow, lymphatic. tissue), tumors of the serous
cavities (e. g. pulmonary pleura, heart sac, abdominal
membrane, synovial membrane), tumors of the nervous system
(e. g. ganglion cells, neuroepithelium, neroglia, meninges,
sympathicus, peripheral nerves), tumors of the gastro-
intestinal tract and~tumors of individual organs. The growth
.of tumors of the bronchi and the lungs, breast, liver, bile,
pancreas, kidneys and urinary tracts, stomach, large
intestine, straight intestine, prostate and uterus are
preferred according to the invention.
According to the invention the neovascularization may be
induced by the angiogenesis-improving pharmaceutical
composition in diseases in which the disease-dependent
occlusion of vessels results in a insufficient supply of the
tissue with oxygen and nutrients. Cardiac diseases or
insufficient blood supplies of the extremities in diabetics,
heavy smokers or patients suffering from hypertension are to
be mentioned as examples.
The invention is described in more detail by means of the
figures:
Figure 1 Localization of CD66a in the vessels of a human
Leydig cell tumor. The immunohistochemical staining
was carried out using the 4D1/C2 antibody.
Figure la: One of the stained tumor capillaries
is marked by an arrow (x350)
Figure lb: Enlargement of a region from figure
la. The arrow points to the staining
of an endothelial cell (x950).
Figure 2 Chemotactic effect of CD66a (= BGP) on HDMEC
CA 02351585 2001-05-16
Figure 3 Proliferation of HDMEC following stimulation using
CD66a (= BGP)
Figure 4 Effect of CD66a on the formation of capillary-like
vascular tubes in cell culture
The invention is explained in more detail by .means of the
following examples.-
Example 1
Localization of CD66a in tumor capillaries
Tumors were stained immunohistochemically using the
monoclonal anti-CD66a antibody 4D1/C2 and investigated by
means of an optical microscope. For this purpose, an
intensifying method using nickel and glucose oxidase was used
in addition to the previously employed immunohistochemical
methods (Prall et aI. (1996), J. Histochem. Cytochem. 44, 35-
41). Furthermore, electron-microscopic analyses were carried
out following immunohistochemical staining using the
monoclonal 4D1/C2 antibody (see figure 1).
Human testicular tumors, brain tumors as well as prostate,
bladder and kidney carcinomas were examined
immunohistochemically. CD66a was localized in endothelial
cells and in the basal membrane of the tumor capillaries.
Mature, non-proliferating resting vessels of the examined
organs were negative. In case the tumor is divided into
different zones in accordance with functional aspects, namely
tumor cells, tumor margin and tumor environment, the positive
immune response can be found in the newly formed tumor
capillaries on the tumor margin. This indicates a function of
CD66a in very early stages of neovascularization
(neoangiogenesis).
Example 2
CA 02351585 2001-05-16
v
Effect of CD66a on the proliferation and chemotaxis of
cultured endothelial cells
In order to test the effect of CD66a on the proliferation and
chemotaxis of cultured endothelial cells, the glycoprotein
was purified from membrane fractions of human:granulocytes.
The membrane fraction was isolated in accordance with
established methods (Drzeniek et aI. (1991), Cancer Letters
56, 173-179; Stoffel et aI. (1993), J. Immunol. 150, 4978-
4984). After extracting the membrane glycoproteins with a
non-ionic detergent, they were bound to an immobilized
monoclonal CD66 antibody and eluted using glycin-HC1 at pH
2.2. Following neutralization the eluate was further
separated by means of gel chromatography on Superdex 200
(Pharmacia). The CD66a-positive fractions were pooled.
Contaminations in the low-molecular region were separated by
means of ultrafiltration using a filter having an exclusion
of 100 kD. In combination with a Western blot it was shown by
means of SDS-PAGE in silver gel that the supernatant
exclusively contained CD66a. This traction was used for cea_1
culture experiments with endothelial cells.
The experiments were carried out with two different human
endothelial cell forms, namely with HUVEC (human umbelical
vein endothelial cells) and HDMEC (human dermal microvascular
endothelial cells).
The effect of CD66a on the proliferation was checked in a
monolayer culture. Endothelial cells were seeded in a defined
number on a microtitration plate. After 72 hours, the number
of endothelial cells in stimulated and non-stimulated
cultures was compared. It turned out that CD66a stimulated
the proliferation of both cell lines ~r: dose-dependent
manner.
The effect of CD66a on chemotaxis was investigated in a two-
chamber culture system (what is called a Boyden chamber). The
cells are cultured in the top chamber. The bottom chamber
13
CA 02351585 2001-05-16
contains chemotactic subtances. Both chambers~are separated
by a polycarbonate filter permitting passage of the cells.
After adding CD66a to the bottom chamber, a dose-dependent
chemotactic effect showed on both endothelial cell lines. The
effect of CD66a could be compared with the effect of VEGF. As
evident from figure 2, CD66a (= BGP) has a chemotactic effect
from a concentration of 100 ng/ml. With a concentration of
150 ng/ml the chemotactic effect is only slightly less than
that of VEGF (vascular endothelial growth factor).
The chemotactic effect of CD66a was also analyzed in
combination with VEGF and bFGF (basic fibroblast growth
factor). The chemotactic effect of VEGF or bFGF was increased
by CD66a by about 30 % each.
Cultured human microvascular dermatofibroblasts were
incubated with CD66a (= BGP) in concentrations of 50, 100,
200, 400 and 600 ng/ml. From a concentration of 200 ng/ml a
proliferation-increasing effect of CD66a could be detected.
This is shown in figure 3.
Due to the positive effect on proliferation and chemotaxis
CD66a fulfills the main criteria of angiogenesis factors.
Example 3
Effect of CD66a on the formation of capillary-like vascular
tubes in cell culture.
The test results described in Example 2 suggest that CD66a is
causally involved in the formation of new vessels
(neoangiogenesis). In order to check this hypothesis, animal
experiments would be most suitable. However, since CD66a is a
human glycoprotein, it has to be expected that due to the
differences in the species the effect in the experimental
animal shows no or only slight expression. The finding that
the monoclonal anti-CD66a 4D1/C2 antibody shows good reaction
in human tissues supports this assumption. The reaction is
_...,.... ,
CA 02351585 2001-05-16
weak in the corresponding tissues of rats and mice and can be
distinguished only with difficulty from a non-specific
background reaction. The 4D1/C2 antibody obviously binds to
an antigenic structure which does not occur in rodents in
this form.
In order to circumvent the problems caused by the differences
regarding the species, cell culture models are used in which
endothelial cells grow under certain conditions into vascular
tubes which correspond to newly formed capillaries (tube
formation). For this purpose, the cells are cultured in the
presence of specific growth factors such as VEGF (vascular
endothelial growth factor) or FGF-2 (fibroblast growth
factor) in a connective tissue matrix. This culture form
represents a good approach to in vivo conditions.
In order to investigate the significance of CD66a for the
formation of capillaries, HUVEC and HDMEC cells were cultured
in three-dimensional collagen I gels. In the presence of
growth factors such as VEGF and FGF-2, the endothelial cells
form tubular structures which c~rre~pond to newly formed
capillaries. In the presence of the monoclonal CD66a 4D1/C2
antibody, the formation of vascular tubes was inhibited. A
second monoclonal antibody which is directed against another
epitope on CD66a, had no effect on the formation of tubes.
These experiments prove a functional correlation between the
expression of CD66a and the neoformation of capillary-like
vascular tubes. The functional domain of CD66a is also
defined by means of the antibody.
The results of the above experiments are shown in figure 4:
In the presence of the angiogenesis factor VEGF (50 ng/ml),
capillary-like structures develop (see figure 4a). Capillary-
like structures manifest themselves by way of tubes in which
the longitudinal endothelial cells are arranged parallel.
These tubes can be compared to fish schools. In the middle of
figure 4a there is a region in which the endothelial cells
are rounded. They are no tubes.
rs
CA 02351585 2001-05-16
t
Figure 4b shows the result of an experiment in which the
capillary formation was investigated in the presence of VEGF
(50 ng/ml) and CD66a (150 ng/ml) . As compared to figure 4a,
almost all endothelial cells are involved in the formation of
tubes. Furthermore, a branching pattern can be seen which
supports the further differentiation of the angiogenesis
process. CD66a thus intensifies the angiogenetic effect of
VEGF.
In figure 4c, the endothelial cells were cultured in the
presence of CD66a (300 ng/ml) and in the absence of VEGF.
Capillary-like structures appear.
Figure 4d shows the result of an experiment in which the
endothelial cells were cultured in the presence of the
monoclonal 4D1/C2 antibody. The formation of capillaries is
fully inhibited. It follows from this experiment that the
4D1/C2 antibody binds to a domain of CD66a which is essential
for the formation of capillaries.
