Note: Descriptions are shown in the official language in which they were submitted.
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USE OF EMMPRIN ANTAGONISTS FOR THE TREATMENT OF
DISEASES ASSOCIATED WITH EXCESSIVE ANGIOGENESIS
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a method of using antagonists of EMMPRIN
(Extracellular Matrix Metalloproteinase Inducer) to treat pathological
processes associated with
proliferative diseases, such as cancer, by specifically preventing or
inhibiting the ability of proliferating
tissue to develop a blood supply. The invention more specifically relates to
methods of treating such
diseases by the use of EMMPRIN antagonists such as antibodies directed toward
EMMPRIN, including
specified portions or variants, specific for at least one protein or fragment
thereof, in an amount effective
to inhibit angiogenesis.
Background of the Invention
EMMPRIN
Angiogenesis is the process of new vessel formation. In adults, angiogenesis
occurs only
locally and transiently under physiological conditions such as wound healing,
menstruation and
pregnancy. In contrast, excessive angiogenesis occurs in more than 70 disease
conditions such as
cancer, atherosclerosis, diabetic blindness, age-related macular degeneration,
rheumatoid arthritis, and
psoriasis. On the other hand, insufficient angiogenesis underlies diseases
such as coronary artery
disease, stroke, and delayed wound healing.
Matrix metalloproteinases (MMPs), a family of more than twenty endopeptidases
that are
capable of cleaving all of the extracellular matrix components, play critical
roles in angiogenesis
[Klagsbrun and Moses 1999]. Angiogenesis initiates as the breakdown of blood
vessel basement
membrane by capillary endothelial cells activated by angiogenic stimulators
derived from tumors,
inflammation sites, or tissues undergo other pathological conditions. The
activated endothelial cells
express increased MMPs, which in turn, enable disseminated endothelial cells
to migrate away from their
parental vessels. Only after the cells escape, do they respond to various
growth factors to proliferate,
and eventually go through a complex differentiation process to form new
vessels. Depletion of MMPs,
such as MMP-2 or MMP-9, results in a significant inhibition of tumor
angiogenesis, supporting the critical
role of MMPs in this process.[Bergers et al. 2000; Fang et al. 2000].
Extracellular matrix metalloproteinase inducer (EMMPRIN) (also known as CD
147) is a
58 kDa glycoprotein, originally purified from the plasma membrane of cancer
cells and was designated
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tumor collagenase stimulating factor (TCSF) because of its ability to
stimulate collagenase-1 (MMP-1)
synthesis by tumor stromal fibroblast cells [Biswas et al. 1995; Ellis et al.
1989]. It was demonstrated to
be identical to the M6 antigen and human Basigen (Biswas et al, Cancer Res.55:
434, 1995).
Subsequent studies further demonstrated that EMMPRIN also induced fibroblast
synthesis of MMP-2,
MMP-3, as well as the membrane-type 1 MMP (MT1-MMP) and MT2-MMP that function
as endogenous
activator for MMP-2 [Guo et al. 1997; Kataoka et al. 1993; Sameshima et al.
2000b]. Several clinical
studies have demonstrated that the expression level of EMMPRIN in tumor
tissues is significantly higher
than that in peritumoral stromal tissues. These tumors include lung [Polette
et al. 1997], breast [Polette et
al. 1997], bladder [Javadpour and Guirguis 1992; Muraoka et al. 19931, and
glioma [Sameshima et al.
2000a]. Examination of EMMPRIN expression in these clinical samples by a
variety of means, including
Northern blot, in situ hybridization and immunostaining, revealed that EMMPRIN
is expressed by tumor
cells, but not by the neighboring stromal cells. On the other hand, MMPs are
expressed by peritumoral
stromal cells. The role of EMMPRIN in tumor growth and metastasis was directly
illustrated using
EMMPRIN-overexpressing human breast cancer cells. MDA MB 436 cells are
normally slow growing
cells when they are implanted into nude mice. However, when these cells were
transfected with
EMMPRIN, they adopted a more aggressive growth pattern, with both accelerated
growth rate and
metastatic phenotypes [Zucker et al. 2001].
Disorders associated with inappropriate angiogenesis
Angiogenesis is the process of generating new capillary blood vessels, and it
results from
activated proliferation of endothelial cells. Neovascularization is tightly
regulated, and occurs only during
embryonic development, tissue remodeling, wound healing and periodic cycle of
corpus luteum
development (Folkman and Cotran, Relation of vascular proliferation to tumor
growth, Int. Rev. Exp.
Pathol.'16, 207-248(1976)).
Endothelial cells normally proliferate much more slowly than other types of
cells in the
body. However, if the proliferation rate of these cells becomes unregulated,
pathological angiogenesis
can result. Pathological angiogenesis is involved in many diseases. For
example, cardiovascular
diseases such as angioma, angiofibroma, vascular deformity, atherosclerosis,
synechia and edemic
sclerosis; and opthalmological diseases such as neovascularization after
cornea implantation,
neovascular glaucoma, diabetic retinopathy, angiogenic corneal disease,
macular degeneration,
pterygium, retinal degeneration, retrolental fibroplasias, and granular
conjunctivitis are related to
angiogenesis. Chronic inflammatory diseases such as arthritis; dermatological
diseases such as
psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis, venous
ulcers, acne, rosacea (acne
rosacea or erythematosa), warts (verrucas), eczema, hemangiomas,
lymphangiogenesis are also
angiogenesis-dependent.
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Vision can be impaired or lost because of various ocular diseases in which the
vitreous
humor is infiltrated by capillary blood. Diabetic retinopathy can take one of
two forms, non-proliferative or
proliferative. Proliferative retinopathy is characterized by abnormal new
vessel formation
(neovascularization), which grows on the vitreous surface or extends into the
vitreous cavity. In advanced
disease, neovascular membranes can occur, resulting in a traction retinal
detachment. Vitreous
hemorrhages may result from neovascularization. Visual symptoms vary. A sudden
severe loss of vision
can occur when there is intravitreal hemorrhage. Visual prognosis with
proliferative retinopathy is more
guarded if associated with severe retinal ischemia, extensive
neovascularization, or extensive fibrous
tissue formation. Macular degeneration, likewise takes two forms, dry and wet.
In exudative macular
degeneration (wet form), which is much less common, there is formation of a
subretinal network of
choroidal neovascularization often associated with intraretinal hemorrhage,
subretinal fluid, pigment
epithelial detachment, and hyperpigmentation. Eventually, this complex
contracts and leaves a distinct
elevated scar at the posterior pole. Both forms of age-related macular
degeneration are often bilateral
and are preceded by drusen in the macular region. Another cause of loss of
vision related to angiogenic
etiologies are damage to the iris. The two most common situations that result
in the iris being pulled up
into the angle are contraction of a membrane such as in neovascular glaucoma
in patients with diabetes
or central retinal vein occlusion or inflammatory precipitates associated with
uveitis pulling the iris up into
the angle (Ch. 99. The Merck Manual 17'h Ed. 1999).
Rheumatoid arthritis, an inflammatory disease, also results in inappropriate
angiogenesis.
The growth of vascular endothelial cells in the synovial cavity is activated
by the inflammatory cytokines,
and results in cartilage destruction and replacement with pannus in the
articulation (Koch AK, Polverini PJ
and Leibovich SJ, Arth; 15 Rhenium, 29, 471-479(1986); Stupack DG, Storgard CM
and Cheresh DA,
Braz. J. Med. Biol. Res., 32, 578-581(1999); Koch AK, Arthritis Rheum, 41, 951
962(1998)).
Psoriasis is caused by uncontrolled proliferation of skin cells. Fast growing
cell requires
sufficient blood supply, and abnormal angiogenesis is induced in psoriasis
(Folkman J., J. Invest.
Derrnatol., 59, 40- 48(1972)).
There is now considerable evidence that tumor growth and cancer progression
requires
angiogenesis, the formation of new blood vessels in order to provide tumor
tissue with nutrients and
oxygen, to carry away waste products and to act as conduits for the metastasis
of tumor cells to distant
sites (Folkman, et al. N Engl J Med 285: 1181-1186, 1971 and Folkman, et al. N
Engl J Med 333: 1757-
1763, 1995).
A number of factors are involved in processes and events leading to
angiogenesis: cell
adhesion molecules, integrins, vascular endothelial growth factor (VEGF),
TNFalpha, bFGF, and
cytokines including IL-6 and IL-12. For example, the closely related but
distinct integrins aV03 and aV05
have been shown to mediate independent pathways in the angiogenic process. An
antibody generated
against aVG33 blocked basic fibroblast growth factor (bFGF) induced
angiogenesis, whereas an antibody
specific to aV(35 inhibited vascular endothelial growth factor (VEGF) induced
angiogenesis (Eliceiri, et al.,
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J. Clin. Invest.103: 1227-1230 (1999); Friedlander et al., Science 270: 1500-
1502 (1995)). IL-6 is
elevated in tissues undergoing angiogenesis and can induce VEGF in A431 cells,
a human epidermoid
carcinoma cell line (Cohen, et al. J. Biol. Chem. 271: 736-741, 1996).
Thus, angiogenesis is known to be a contributing factor in a number of
pathological
conditions including the ability of tumors to grow and metastasize, disorders
of the eye including
retinopathies, and disorders of the skin including Kaposi's Sarcoma. While
numerous factors have been
shown to be associated with these processes, it has not heretofore been
demonstrated that EMMPRIN
directly stimulates VEGF production, stimulates endothelial cells, in addition
to local fibroblast cells, to
express MMPs and therefore facilitate tumor angiogenesis, growth, invasion and
metastasis.
SUMMARY OF THE INVENTION
The present invention relates to a method of using antagonists of EMMPRIN,
including
antibodies directed toward EMMPRIN, and specified portions or variants thereof
specific for at least one
EMMPRIN protein or fragment thereof, to inhibit angiogenesis in disease
conditions associated with
abnormal angiogenesis. Such EMMPRIN antagonists such as antibodies can act
through their ability to
prevent the ability of EMMPRIN from stimulating MMP expression by
microvascular endothelial cells, the
cells involved in angiogenesis, in a dose-dependent fashion. Secondly, such
antagonists or antibodies
can act by limiting EMMPRIN induction of VEGF in the local environment thereby
reducing the
angiongenic potential of the tissue. By interfering with angiogenesis, such
antagonists can prevent
events associated with the initiation or progression of cancer tissue
including events involved with
angiogenesis and the metastatic spread of cancer. Based on the aforementioned
action of the
EMMPRIN antagonists of the invention, these antagonists can be best described
as anti-angiogenic
EMMPRIN antagonists.
Thus, in accordance with the invention, we have, for the first time,
demonstrated that
EMMPRIN can directly stimulate MMP-1 expression by microvascular endothelial
cells, the cells involved
in angiogenesis, in a dose-dependent fashion. This stimulation is specifically
inhibited by function-
blocking anti-EMMPRIN monoclonal antibodies. Since MMPs are essential for
angiogenesis, such
EMMPRIN antagonists can be useful as therapeutics for such diseases as cancer,
diabetic blindness,
age-related macular degeneration, rheumatoid arthritis, and psoriasis.
In one embodiment, the EMMPRIN antagonist is an capable of preventing the
production
of EMMPRIN by cells, such as an siRNA or a shRNA molecule.
In a particular embodiment, the EMMPRIN antagonist is an antibody that
specifically
binds EMMPRIN. A particular advantage of such antibodies is that they are
capable of binding EMMPRIN
in a manner that prevents its action systemically. The method of the present
invention thus employs
antibodies having the desirable neutralizing property which makes them ideally
suited for therapeutic and
preventative treatment of metastatic disease states associated with various
forms of cancer in human or
nonhuman patients. Accordingly, the present invention is directed to a method
of treating a disease or
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condition which is dependent on angiogenesis in a patient in need of such
treatment which comprises
administering to the patient an amount of a neutralizing EMMPRIN antibody to
inhibit angiogenesis.
In a particularly preferred embodiment of the EMMPRIN antagonist antibody of
the
invention, the antibody is known as CNT0146 and is a murine anti-human EMMPRIN
of the IgG1 k class
which has distinguished capability of inhibiting EMMPRIN-induced MMP
production, including inhibiting
MMP-1 production in fibroblast stimulated with recombinant EMMPRIN, as well as
inhibiting MMP
production in the co-culture of tumor cells and fibroblast cells.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. Schematic illustration of the central role of EMMPRIN in diseases
involving
abnormal angiogenesis.
Fig. 2. Recombinant EMMPRIN dose-dependently stimulated MMP-1 production by
HMVEC-L cells.
Fig. 3. Inhibition of EMMPRIN-induced MMP-1 production in HMVEC-L cells by a
neutralizing anti-EMMPRIN monoclonal antibody.
Fig. 4. is a set of bar graphs showing (A) the relative endothelial cell
migration induced by
Fig. 5 is a bar graph showing the average final tumor weights of tumors
produced by MDA MB231 human
breast tumor cells manipulated to express greater or lesser amounts of EMMPRIN
than normal (WT) and
(B) is a bar graph showing the relative reduction in the migration of
endothelial cells induced by WT cells
in the presence of increasing concentrations of anti-VEGF antibody.
Fig. 5A is a bar graph showing the average final tumor weights of tumors
produced by
MDA MB231 human breast tumor cells manipulated to express greater (S1-3) or
lesser (AS1-5) and
(AS2-5) amounts of EMMPRIN than normal (WT) or Vector control cells. 5B is a
micrograph showing the
difference in angiogenic structures between tumors produced by implantation of
mice with WT versus S1-
3 cells. 5C is a set of bar graphs shoinw the amount of human VEGF (left
panel) and mouse VEGF (right
panel) in tumors produced by MDA MB231 human breast tumor cell types.
Fig. 6A is a bar graph showing the amount of human EMMMPRIN in tissue extracts
from
xenograft tumors derived from WT, Vector control, S1-3, or AS EMMPRIN
engineered human tumor cells.
B. is a photo of a zymography gel showing MMP expression profile in tissue
extracts from the same
tumors containing where 10 g of total protein was added to each lane. C. is a
bar graph showin the
quantitative determination of human and mouse MMP-2 levels in xenograft
tumors. D. is a pair of bar
graphs showing quantitative determination of human (left panel) and mouse
(right panel) MMP-9 levels in
xenograft tumors.
Fig. 7. Photographs showing increased angiogenesis evidenced by numerous new
capillary blood vessels in tumors derived from sense cells expressing EMMPRIN,
but not in tumors
derived from WT OR AS cells.
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Fig. 8 shows photographs of tumors after immunohistochemical analysis of MMP,
VEGF,
EMMPRIN: A. H&E staining of MDA-MB-231 xenograft tumors; B. Mouse MMP-9
staining; C. Mouse
EMMPRIN staining; D. Blood vessel staining with anti-CD31 antibodies. Left
panels - Vector control
tumors; right panels - S1-3 tumors.
DETAILED DESCRIPTION OF THE INVENTION
EMMPRIN expressed by cells in diseased tissues directly stimulates neighboring
endothelial cells, which results in an increase in MMP expression, i.e., MMP-
1. (See FIG. 1) These
MMPs, in turn, mediate the breakdown of basement membrane of existing blood
vessels; promote
endothelial cells to migrate away from parental vessels; stimulate the
expression and release of
angiogenic growth factors; enable endothelial cells to respond to angiogenesis
stimulatory factors leading
to cell proliferation; and facilitate the remodeling of extracellular matrix
for endothelial cell differentiation
and assembly of new vessels. All these changes lead to an increase in
angiogenesis and further
contribute to the overall disease progression.
The anti-angiogenic EMMPRIN antagonists of the invention are useful in
inhibiting and
preventing angiogenesis in so far as they block the stimulatory effects of
EMMPRIN on endothelial cells,
reduce VEGF production by endothelial cell, reduce endothelial cell division,
decrease endothelial cell
migration, and impair the activity of the proteolytic enzymes secreted by the
endothelium. A number of
pathologies including various forms of solid primary tumors and the
metastases, lesions of the eye and
disorders of the skin are improved by treatment with EMMPRIN antagonists in
the method of the present
invention.
Cancer
Both benign and malignant tumors, including various cancers such as, cervical,
anal and
oral cancers, stomach, colon, bladder, rectal, liver, pancreatic, lung,
breast, cervix uteri, corpus uteri,
ovary, prostate, testis, renal, brain/cns (e.g., gliomas), head and neck, eye
or ocular, throat, skin
melanoma, acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's
Sarcoma, Kaposi's
Sarcoma, basal cell carinoma and squamous cell carcinoma, small cell lung
cancer, choriocarcinoma,
rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms Tumor,
neuroblastoma,
mouth/pharynx, esophageal, larynx, kidney and lymphoma, among others may be
treated using anti-
EMMPRIN antibodies of the present invention. In addition, conditions such as
neurofibromatosis,
tuberous sclerosis (each of which conditions produces benign tumors of the
skin), hemangiomas and
lymphangiogenesis, among others, may be treated effectively with EMMPRIN
antagonists according to
the present invention
A secondary tumor, a metastasis, is a tumor which originated in a primary site
elsewhere
in the body, but has now spread to a distant organ. The common routes for
metastasis are direct growth
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into adjacent structures, spread through the vascular or lymphatic systems,
and tracking along tissue
planes and body cavaties with, for example, peritoneal fluid or cerebrospinal
fluid. Secondary hepatic
tumors are one of the most common causes of death in cancer patients and are
by far and away the most
common form of liver tumor. Although virtually any malignancy can metastasize
to the liver, tumors which
are most likely to spread to the liver include: cancer of the stomach, colon,
and pancreas; melanoma;
tumors of the lung, oropharynx, and bladder; Hodgkin's and non- Hodgkin's
lymphoma; tumors of the
breast, ovary, and prostate. Secondary lung, brain, and bone tumors are common
to advanced stage
breast, prostate and lung cancers. Any cancer may metastasize to bone, but
metastases from
carcinomas are the most common, particularly those arising in the breast,
lung, prostate, kidney, and
thyroid. Carcinoma of the lung is very commonly accompanied by hematogenous
metastatic spread to
the liver, brain, adrenals, and bone and may occur early, resulting in
symptoms at those sites before
obvious pulmonary symptom. Metastases to the lungs are common from primary
cancers of the breast,
colon, prostate, kidney, thyroid, stomach, cervix, rectum, testis, and bone
and from melanoma. Each one
of the above-named secondary tumors may be treated by the antibodies of the
present invention.
In addition to tumors, numerous other non-tumorigenic angiogeneis-dependent
diseases
which are characterized by the abnormal growth of blood vessels may also be
treated with the anti-
angiogenic EMMPRIN antagonists of the present invention.
Representative examples of such non-tumorigenic angiogenesis-dependent
diseases
include corneal neovascularization, hypertrophic scars and keloids,
proliferative diabetic retinopathy,
rheumatoid arthritis, arteriovenous malformations (discussed above),
atherosclerotic plaques, delayed
wound healing, hemophilic joints, nonunion fractures, Osler-Weber syndrome,
psoriasis, pyogenic
granuloma, scieroderma, tracoma, menorrhagia (discussed above) and vascular
adhesions.
Angiogenic Conditions of the Eyes
The cornea is a tissue which normally lacks blood vessels. In certain
pathological
conditions, however, capillaries may enter the cornea from the pericorneal
vascular plexus of the limbus.
When the cornea becomes vascularized, it also becomes clouded, resulting in a
decline in the patient's
visual acuity. Visual loss may become complete if the cornea completely
opacitates.
Blood vessels can enter the cornea in a variety of patterns and depths,
depending upon
the process which incites the neovascularization. These patterns have been
traditionally defined by
ophthalmologists in the following types: pannus trachomatosus, pannus
leprosus, pannus phylctenulosus,
pannus degenerativus, and glucomatous pannus. The corneal stroma may also be
invaded by branches
of the anterior ciliary artery (called interstitial vascularization) which
causes several distinct clinical
lesions: terminal loops, a "brush-like" pattern, an umbel form, a lattice
form, interstitial arcades (from
episcleral vessels), and aberrant irregular vessels.
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Corneal neovascularization can result from corneal ulcers. A wide variety of
etiologies
may produce corneal ulcers including for example corneal infections (trachoma,
herpes simplex keratitis,
leishmaniasis and onchocerciasis), immunological processes (graft rejection
and Stevens-Johnson's
syndrome), alkali bums, trauma, inflammation (of any cause), toxic and Vitamin
A or protein deficiency
states, and as a complication of wearing contact lenses.
While the cause of corneal neovascularization may vary, the response of the
cornea to
the insult and the subsequent vascular ingrowth is similar regardless of the
cause. Several angiogenic
factors are likely involved in this process, many of which are products of the
inflammatory response.
Indeed neovascularization of the cornea appears to only occur in association
with an inflammatory cell
infiltrate, and the degree of angiogenesis is proportional to the extent of
the inflammatory reaction.
Corneal edema further facilitates blood vessel ingrowth by loosening the
corneal stromal framework
through which the capillaries grow.
Topical therapy with EMMPRIN antibodies may also be useful prophylactically in
corneal
lesions which are known to have a high probability of inducing an angiogenic
response (such as chemical
burns). In these instances the treatment, likely in combination with steroids,
may be instituted immediately
to help prevent subsequent complications.
Such methods may also be utilized in a similar fashion to prevent capillary
invasion of
transplanted corneas. Use in combination with a steroid is also contemplated.
Neovascular glaucoma is a pathological condition wherein new capillaries
develop in the
iris of the eye. The angiogenesis usually originates from vessels located at
the pupillary margin, and
progresses across the root of the iris and into the trabecular meshwork.
Fibroblasts and other connective
tissue elements associate with the capillary growth and a fibrovascular
membrane develops which
spreads across the anterior surface of the iris eventually forming a scar. The
scar formation prevents
adequate drainage of aqueous humor resulting in an increase in intraocular
pressure that may result in
blindness.
Neovascular glaucoma generally occurs as a complication of diseases in which
retinal
ischemia is predominant. In particular, about one third of the patients with
this disorder have diabetic
retinopathy. Other causes include chronic retinal detachment, end-stage
glaucoma, carotid artery
obstructive disease, retrolental fibroplasia, sickle-cell anemia, intraocular
tumors, and carotid cavernous
fistulas.
Angiogenic Conditions of the Skin
Within another aspect of the present invention, methods are provided for
treating
hypertrophic scars and keloids, comprising the step of administering one of
the above-described anti-
angiogenic compositions to a hypertrophic scar or keloid.
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Healing of wounds and scar formation occurs in three phases: inflammation,
proliferation,
and maturation. The first phase, inflammation, occurs in response to an injury
which is severe enough to
cause tissue damage and vascular leaking. During this phase, which lasts 3 to
4 days, blood and tissue
fluid form an adhesive coagulum and fibrinous network which serves to bind the
wound surfaces together.
This is then followed by a proliferative phase in which there is ingrowth of
capillaries and connective
tissue from the wound edges, and closure of the skin defect. Finally, once
capillary and fibroblastic
proliferation has ceased, the maturation process begins wherein the scar
contracts and becomes less
cellular, less vascular, and appears flat and white. This final phase may take
between 6 and 12 months.
Overproduction of connective tissue at the wound site causes a persistently
cellular and
possible red and raised scar to be formed. If the scar remains within the
boundaries of the original wound
it is referred to as a hypertrophic scar, but if it extends beyond the
original scar and into the surrounding
tissue, the lesion is referred to as a keloid. Hypertrophic scars and keloids
are produced during the
second and third phases of scar formation. Several wounds are particularly
prone to excessive
endothelial and fibroblastic proliferation, including burns, open wounds, and
infected wounds. With
hypertrophic scars, some degree of maturation occurs and gradual improvement
occurs. In the case of
keloids however, an actual tumor is produced which can become quite large.
Spontaneous improvement
in such cases rarely occurs. Administration of an anti-EMMPRIN antibody in the
method of the present
invention to inhibit angiogenesis in such conditions can thus inhibit the
formulation of such keloid scars.
Anti-angiogenic Combinations with EMMPRIN Antagonists
Angiogenesis is characterized by the invasion, migration and proliferation of
smooth
muscle and endothelial cells. The avP3 integrin (also known as the vitronectin
receptor) is known to play a
role in various conditions or disease states including tumor metastasis, solid
tumor growth (neoplasia),
osteoporosis, Paget's disease, humoral hypercalcemia of malignancy,
angiogenesis, including tumor
angiogenesis, retinopathy, including macular degeneration, arthritis,
including rheumatoid arthritis,
periodontal disease, psoriasis and smooth muscle cell migration (e.g.
restenosis).
The adhesion receptor integrin avP3 binds vitronectin, fibrinogen, von
Willebrand Factor,
laminin, thrombospondin, and other like ligands. It was identified as a marker
of angiogenic blood vessels
in chick and man and plays a critical role in angiogenesis or
neovascularization. Antagonists of avP3
inhibit this process by selectively promoting apoptosis of cells in
neovasculature. Therefore, av03
antagonists would be useful therapeutic targets for treating such conditions
associated with
neovascularization (Brooks et al., Science, Vol. 264, (1994), 569-571).
Additionally, tumor cell invasion
occurs by a three step process: 1) tumor cell attachment to extracellular
matrix; 2) proteolytic dissolution
of the matrix; and 3) movement of the cells through the dissolved barrier.
This process can occur
repeatedly and can result in metastases at sites distant from the original
tumor. The avP3 integrin has
been shown to play a role in tumor cell invasion as well as angiogenesis.
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As the antagonists of av03 and neutralizing anti-EMMPRIN antibodies both
target
neovasculature but act through different mechanisms, the combination of anti-
integrin antibodies with
anti-EMMPRIN antibodies should result in a particularly potent and effective
combination therapy with
little normal tissue toxicity. Thus, in one embodiment of the present
invention, there is provided a method
of treating a disease or condition associated with angiogenesis which
comprises administering a
combination of an integrin antagonist and an anti-EMMPRIN antibody to inhibit
angiogenesis in a patient
in need of such treatment. Other antibodies which selectively bind integrins
or integrin subunits,
especially those that bind the alphaV subunit, are disclosed in U.S. Patents
5,985,278 and 6,160,099.
Mabs that inhibit binding of alphaVbeta3 to its natural ligands containing the
tripeptide argininyl-glycyl-
aspartate (RGD) are disclosed in US 5,766,591 and W00078815.
A preferred combination of antibodies is the anti-alphaVbeta3 and anti-
alphaVbeta5 Mab
described in applicant's co-pending application U.S. serial no. 09/092,026 and
an anti-EMMPRIN
antibody, as disclosed herein. Both of the foregoing applications are
incorporated by reference into the
present application and form part of the disclosure hereof. In accordance with
the invention, other known
anti-angiogenesis agents such as thalidomide may also be employed in
combination with an anti-
EMMPRIN antibody.
Methods of Evaluating Anti-Angiogenic Activity
Widely accepted functional assays of angiogenesis and, hence, anti- angiogenic
agents
are the chick chorio-allantoic membrane assay (CAM) assay and the corneal
micropocket assay of
neovascularization.
For the CAM assay, fertilized chick embryos are removed from their shell on
day 3 (or 4)
and incubated in a Petri dish in high humidity and 5% C02. On day 6, a
methylcellulose disc (10 microL )
containing the test substance is implanted on the chorioallantoic membrane.
The embryos were
examined 48 hours later, and if a clear avascular zone appears around the
methylcellulose disc, the
diameter of that zone is measured. The larger the zone, the more effective the
antibody. India ink can be
injected into the heart of some embryos just before formalin fixation so that
vessels are visible near the
edge of the avascular zone in histological sections. Histologic cross-sections
of the chorioallantoic are
examined to determine whether the test substance prevents normal development
of the capillaries. This
method, described in U.S. Pat. No. 5,001,116 which is also specifically
incorporated herein by reference,
showed the test useful in the selection of anti-angiogenic compounds or
combinations of compounds.
The corneal micropocket assay of neovascularization may be practiced using rat
or rabbit
corneas. This in vivo model is widely accepted as being generally predictive
of clinical effect, as
described in many review articles and papers such as O'Reilly et. al. Cell 79:
315-328.
Briefly, a plug or pellet containing the recombinant bFGF (Takeda
Pharmaceuticals-
Japan) is implanted into corneal micropockets of each eye of an anesthetized
female New Zealand white
CA 02560903 2006-09-25
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rabbit, 2 mm from the limbus followed by topical application of erythromycin
ointment onto the surface of
the cornea. The animals are dosed with the test compounds and examined with a
slit lamp every other
day by a corneal specialist. Various mathematical models are utilized to
determine the amount of
vascularized cornea and this formula was found to provide the most accurate
approximation of the area of
the band of neovascularization that grows towards the pellet.
The method may also be practiced using rats.
In the present invention, the corneal micropocket assay may be used to
demonstrate the
anti-angiogenesis effect of anti-EMMPRIN antibodies. This is evidenced by a
significant reduction in
angiogenesis, as represented by a consistently observed and preferably marked
reduction in the number
of blood vessels within the cornea.
Endothelial and Non-Endothelial Cell Proliferation
It is important to establish which cell types are involved in the angiogenic
processes
specific for tumor vascularization. Tumor vessels are generally primitive,
that is, contain only endothelial
cells. Other cell types found in more mature vessels include: smooth muscle
cells, retinal pigment
epithelial cells, fibroblasts, and epithelial cells, as well as tumor cells
such as hemangioendothelioma
cells or carcinoma cells. One example of an angiogenesis inhibitor that
specifically inhibits endothelial
cell proliferation is ANGIOSTATINO protein. (O'Reilly et al., 1994 supra).
Various representative cell lines are available for testing. Bovine aortic
smooth muscle
(SMC), bovine retinal pigment epithelial (RPE), mink lung epithelial (MLE),
Lewis lung carcinoma (LLC),
and EOMA hemangioendothelioma cells and 3T3 fibroblasts. For the proliferation
assays, cells are
washed with PBS and dispersed in a 0.05% solution of trypsin. Optimal
conditions for the cell
proliferation assays are established for each different cell type. Generally,
cells are trypsinized and re-
seeded in growth medium in the presence and absence of EMMPRIN and anti-
EMMPRIN neutralizing
Mab. After approximately 72 hours, the change in cell number is assessed by
using a vital stain such as
a tetrazolium dye or by LDH release(Promega, Madison WI) or by individual cell
counting.
EMMPRIN Antagonists
As used herein, the term "EMMPRIN antagonists" refers to a substance which
inhibits or
neutralizes the angiogenic activity of EMMPRIN. Such antagonists accomplish
this effect in a variety of
ways. One class of EMMPRIN antagonists will bind to EMMPRIN protein with
sufficient affinity and
specificity to neutralize the angiogenic effect of EMMPRIN. Included in this
class of molecules are
antibodies and antibody fragments (such as for example, F(ab) or F(ab')2
molecules). Another class of
EMMPRIN antagonists are fragments of EMMPRIN protein, muteins or small organic
molecules i.e.
peptidomimetics, that will bind to EMMPRIN or EMMPRIN binding partners,
thereby inhibiting the
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angiogenic activity of EMMPRIN. The EMMPRIN antagonist may be of any of these
classes as long as it
is a substance that inhibits EMMPRIN angiogenic activity. EMMPRIN antagonists
include EMMPRIN
antibody, EMMPRIN receptor antibody, modified EMMPRIN, antisense EMMPRIN and
partial peptides of
EMMPRIN or EMMPRINR.
Anti-EMMPRIN Antibodies
Neutralizing antibodies to soluble factors that mediate inflammation and tumor
proliferation, such as TNFalpha, have proved to highly effective therapeutics.
REMICADE (infliximab)
sold by Centocor, Malvern, PA an anti-TNFalpha MAb is prescribed for RA and
Crohn's Disease and
RITUXAN (rituximab) an anti-CD20 Mab sold by Genentech, San Bruno, CA is used
to treat B-cell
lymphoma. "Neutralizing" Mabs not only bind their target but also inhibit its
biological activity, usually by
preventing its interaction with its cognate cell surface receptor. In certain
cases, the target protein will
comprise more than one active domain and exhibit multiple actions due to
binding to more than one
ligand or receptor. EMMPRIN is such a molecule and exhibits two immunoglobulin-
like domains in the
extracellular portion of the molecule, the Ig-like C2-type domain from aa 22-
103 of basigin isoform 2
(NCBI accession # NP_940991) domain and the Ig-like V-type domain at 105-199
of the same isoform
(Biswas, Zhang, DeCastro, Guo, Nakamura, Kataoka and Nabeshima, (1995), Cancer
Res 55: 434-9).
Monoclonal antibodies raised to EMMPRIN from cancer cells are capable of
inhibiting EMMPRIN-induced
MMP production in fibroblast cells, indicating neutralizing activity (Ellis,
Nabeshima and Biswas, (1989),
Cancer Res 49: 3385-91). These antibodies were subsequent shown to bind to
EMMPRIN in the region
34-99 which lies within the C2-type domain. In contrast, CBL1, a murine IgM,
anti-human lymphoblastoid
monoclonal antibody that was raised in Balb/c mice immunized with the T cell
acute lymphoblastic
leukemia cell line (T-ALL) CEM. The latter MAb has been tested clinically in
patients with graft versus
host disease (Hesiop, H. et al. (1995) Lancet 346: 805-806). W09945031 teaches
that antibodies with
activities similar to CBL1 share a consensus binding sequence located in a
region more C-terminal than
the V-type domain, that is RVSR (residues 201-204 of NP_940991) and of a panel
of MAbs made to the
extracellular domain of EMMPRIN only one, designated M-6/6, is capable of
inhibiting OKT3-induced T-
cell activation and binds to a region in the C2-type domain Koch, C. et al.
(1999) Internat. Immunol. 11:
777-786; Staffler, G. et al. (2003) J. Immunol. 171: 1707-1714). Therefore,
selection of a uniquely anti-
angiogenic anti-EMMPRIN Mab can be achieved by using a specific set of in
vitro assays as screening
tools.
Any of the anti-EMMPRIN antibodies known in the art which are anti-angiongenic
EMMPRIN antagonists may be employed in the method of the present invention.
Murine monocolonal
antibodies to EMMPRIN are known as in, for example, in Ellis et al, 1989 supra
and Koch, et al. 1999
Internat. Immunol. 11 (5): 777-786.
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Accordingly, as used herein, an "EMMPRIN antibody', "anti-EMMPRIN antibody,"
"anti-
EMMPRIN antibody portion," or "anti-EMMPRIN antibody fragment' and/or "anti-
EMMPRIN antibody
variant" and the like include any protein or polypeptide containing molecule
that comprises at least a
portion of an immunoglobulin molecule, such as but not limited to at least one
complementarity
determining region (CDR) of a heavy or light chain or a ligand binding portion
thereof, a heavy chain or
light chain variable region, a heavy chain or light chain constant region, a
framework region, or any
portion thereof, or at least one portion of an EMMPRIN binding protein derived
from a EMMPRIN protein
or peptide, which can be incorporated into an antibody for use in the present
invention. Such antibody
optionally further affects a specific ligand, such as but not limited to where
such antibody modulates,
decreases, increases, antagonizes, agonizes, mitigates, alleviates, blocks,
inhibits, abrogates and/or
interferes with EMMPRIN angiogenic activity, in vitro, in situ and/or in vivo.
As a non-limiting example, a
suitable anti-EMMPRIN antibody, specified portion or variant of the present
invention can bind at least
one EMMPRIN protein or peptide, or specified portions, variants or domains
thereof. A suitable anti-
EMMPRIN antibody, specified portion, or variant affects EMMPRIN angiogenic
function in a variety of
ways, such as but not limited to, RNA, DNA or protein synthesis, EMMPRIN
release, EMMPRIN receptor
signaling, EMMPRIN receptor binding, EMMPRIN production and/or synthesis. The
term "antibody "is
further intended to encompass antibodies, digestion fragments, specified
portions and variants thereof,
including antibody mimetics or comprising portions of antibodies that mimic
the structure and/or function
of an antitbody or specified fragment or portion thereof, including single
chain antibodies and fragments
thereof. Functional fragments include antigen-binding fragments that bind to a
mammalian EMMPRIN.
For example, antibody fragments capable of binding to EMMPRIN or portions
thereof, including, but not
limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion
and partial reduction) and F(ab')2
(e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by
pepsin or plasmin digestion),
Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or
scFv (e.g., by molecular biology
techniques) fragments, are encompassed by the invention (see, e.g., Colligan,
Immunology, supra).
Such fragments can be produced by enzymatic cleavage, synthetic or recombinant
techniques, as known in the art and/or as described herein. Antibodies can
also be produced in a variety
of truncated forms using antibody genes in which one or more stop codons have
been introduced
upstream of the natural stop site. For example, a combination gene encoding a
F(ab')2 heavy chain
portion can be designed to include DNA sequences encoding the CH1 domain
and/or hinge region of the
heavy chain. The various portions of antibodies can be joined together
chemically by conventional
techniques, or can be prepared as a contiguous protein using genetic
engineering techniques.
The anti-EMMPRIN antibody may be a primate, rodent, or human antibody or a
chimeric
or humanized antibody. As used herein, the term "human antibody" refers to an
antibody in which
substantially every part of the protein (e.g., CDR, framework, CL, CH domains
(e.g., CH1, CH2, CH3),
hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor
sequence changes or
variations. Similarly, antibodies designated primate (monkey, baboon,
chimpanzee, etc.), rodent (mouse,
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rat, rabbit, guinea pig, hamster, and the like) and other mammals designate
such species, sub-genus,
genus, sub-family, family specific antibodies. Further, chimeric antibodies of
the invention can include
any combination of the above. Such changes or variations optionally and
preferably retain or reduce the
immunogenicity in humans or other species relative to non-modified antibodies.
Thus, a human antibody
is distinct from a chimeric or humanized antibody. It is pointed out that a
human antibody can be
produced by a non-human animal or prokaryotic or eukaryotic cell that is
capable of expressing
functionally rearranged human immunoglobulin (e.g., heavy chain and/or light
chain) genes. Further,
when a human antibody is a single chain antibody, it can comprise a linker
peptide that is not found in
native human antibodies. For example, a Fv can comprise a linker peptide, such
as 2 to about 8 glycine
or other amino acid residues, which connects the variable region of the heavy
chain and the variable
region of the light chain. Such linker peptides are considered to be of human
origin.
Bispecific, heterospecific, heteroconjugate or similar antibodies can also be
used that
are monoclonal, preferably human or humanized, antibodies that have binding
specificities for at least two
different antigens. In the present case, one of the binding specificities is
for at least one EMMPRIN
protein, the other one is for any other antigen. Methods for making bispecific
antibodies are known in the
art. Traditionally, the recombinant production of bispecific antibodies is
based on the co-expression of two
immunoglobulin heavy chain-light chain pairs, where the two heavy chains have
different specificities
(Milstein and Cuello, Nature 305:537 (1983)). Because of the random assortment
of immunoglobulin
heavy and light chains, these hybridomas (quadromas) produce a potential
mixture of 10 different
antibody molecules, of which only one has the correct bispecific structure.
The purification of the correct
molecule, which is usually done by affinity chromatography steps, is rather
cumbersome, and the product
yields are low. Similar procedures are disclosed, e.g., in WO 93/08829, US
Patent Nos, 6210668,
6193967, 6132992, 6106833, 6060285, 6037453, 6010902, 5989530, 5959084,
5959083, 5932448,
5833985, 5821333, 5807706, 5643759, 5601819, 5582996, 5496549, 4676980, WO
91/00360, WO
92/00373, EP 03089, Traunecker et al., EMBO J. 10:3655 (1991), Suresh et al.,
Methods in Enzymology
121:210 (1986), each entirely incorporated herein by reference.
Anti-EMMPRIN antibodies useful in the methods and compositions of the present
invention can optionally be characterized by high affinity binding to EMMPRIN
and optionally and
preferably having low toxicity. In particular, an antibody, specified fragment
or variant of the invention,
where the individual components, such as the variable region, constant region
and framework,
individually and/or collectively, optionally and preferably possess low
immunogenicity, is useful in the
present invention. The antibodies that can be used in the invention are
optionally characterized by their
ability to treat patients for extended periods with measurable alleviation of
symptoms and low and/or
acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as
well as other suitable
properties, can contribute to the therapeutic results achieved. "Low
immunogenicity" is defined herein as
raising significant HAHA, HACA or HAMA responses in less than about 75%, or
preferably less than
about 50% of the patients treated and/or raising low titres in the patient
treated (less than about 300,
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preferably less than about 100 measured with a double antigen enzyme
immunoassay) (Elliott et al.,
Lancet 344:1125-1127 (1994), entirely incorporated herein by reference).
Suitable antibodies include those that compete for binding to human EMMPRIN
with the
commercially available monoclonal antibody CD147-RDI/clone UM-8D6 (Research
Diagnostics, Inc.,
Flanders, NJ).
Compositions and Their Uses
In accordance with the invention, the neutralizing anti-EMMPRIN monoclonal
antibodies
described herein can be used to inhibit angiogenesis and thus prevent or
impair tumor growth and
prevent or inhibit metastases. Additionally, such monoclonal antibodies can be
used to inhibit angiogenic
inflammatory diseases amenable to such treatment, which may include but are
not limited to rheumatoid
arthritis, diabetic retinopathy, psoriasis, and macular degeneration. The
individual to be treated may be
any mammal and is preferably a primate, a companion animal which is a mammal
and most preferably a
human patient. The amount of monoclonal antibody administered will vary
according to the purpose it is
being used for and the method of administration.
The anti-angiogenic anti-EMMPRIN antibodies may be administered by any number
of
methods that result in an effect in tissue in which angiogenesis is desired to
be prevented or halted.
Further, the anti-antiangiongenic anti-EMMPRIN antibodies need not be present
locally to impart an anti-
angiogenic effect, therefore, they may be administered wherever access to body
compartments or fluids
containing EMMPRIN is achieved. In the case of inflamed, malignant, or
otherwise compromised
tissues, these methods may include direct application of a formulation
containing the antibodies. Such
methods include intravenous administration of a liquid composition,
transdermal administration of a liquid
or solid formulation, oral, topical administration, or interstitial or inter-
operative administration.
Adminstration may be affect by the implantation of a device whose primary
function may not be as a drug
delivery vehicle as, for example, a vascular stent.
In particular, within one aspect of the present invention methods are provided
for treating
corneal neovascularization (including corneal graft neovascularization),
comprising the step of
administering a therapeutically effective amount of an anti-angiogenic EMMPRIN
antibody of the
invention directly to the cornea or systemically to the patient, such that the
formation of blood vessels is
inhibited.
Within another aspect of the present invention methods are provided for
treating
neovascular glaucoma, comprising the step of administering a therapeutically
effective amount of an anti-
angiogenic neutralizing anti-EMMPRIN antibodies directly to the eye or
systemically to the patient, such
that the formation of blood vessels is inhibited.
In another embodiment of the present invention either an anti-angiogenic
EMMPRIN
antibody of the invention alone, or in combination with another anti-
angiogenic agent are directly injected
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into a hypertrophic scar or keloid in order to prevent the progression of
these lesions. This therapy is of
particular value in the prophylactic treatment of conditions which are known
to result in the development
of hypertrophic scars and keloids such as burns. Therapy may be effective when
begun after the
proliferative phase has had time to progress (approximateiy 14 days after the
initial injury), but before
hypertrophic scar or keloid development.
Administration may also be oral or by local injection into a tumor or tissue
but generally,
the monoclonal antibody is administered intravenously. Generally, the dosage
range is from about 0.05
mg/kg to about 12.0 mg/kg. This may be as a bolus or as a slow or continuous
infusion which may be
controlled by a microprocessor controlled and programmable pump device.
Alternatively, DNA encoding preferably a fragment of said monoclonal antibody
may be
isolated from hybridoma cells and administered to a mammal. The DNA may be
administered in naked
form or inserted into a recombinant vector, e.g., vaccinia virus in a manner
which results in expression of
the DNA in the cells of the patient and delivery of the antibody.
The monoclonal antibody used in the method of the present invention may be
formulated
by any of the established methods of formulating pharmaceutical compositions,
e.g. as described in
Remington's Pharmaceutical Sciences, 1985. For ease of administration, the
monoclonal antibody will
typically be combined with a pharmaceutically acceptable carrier. Such
carriers include water,
physiological saline, or oils.
Formulations suitable for parenteral administration include aqueous and non-
aqueous
sterile injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes which render
the formulation isotonic with the blood of the intended recipient; and aqueous
and non-aqueous sterile
suspensions which may include suspending agents and thickening agents. Except
insofar as any
conventional medium is incompatible with the active ingredient and its
intended use, its use in any
compositions is contemplated.
The formulations may be presented in unit- dose or multi-dose containers, for
example,
sealed ampules and vials, and may be stored in a freeze-dried (lyophilized)
condition requiring only the
addition of the sterile liquid carrier, for example, water for injections,
immediately prior to use.
Abbreviations
Abs antibodies, polyclonal or monoclonal
aV integrin subunit alpha V
b3 integrin subunit beta 3
bFGF basic fibroblast growth factor
IFN interferon
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Ig immunoglobulin
IgG immunoglobulin G
IL interleukin
MMP-1
EMMPRIN extracellular matric metalloproteinase inducer
EMMPRINR receptor
sEMMPRINR soluble EMMPRIN receptor
Mab monoclonal antibody
VEGF vascular endothelial growth factor
MMP matrix metallopoteinase
While having described the invention in general terms, the embodiments of the
invention
will be further disclosed in the following examples.
EXAMPLE 1
Recombinant EMMPRIN stimulates MMP-1 production by human microvascular
endothelial cells
from the lung (HMVEC-L)
The effect of EMMPRIN on endothelial cells was investigated using
microvascular
endothelial cells, cells that are directly involved in angiogenesis process in
vivo.
HMVEC-L cells were obtained from Clonetics, Walkersville, Maryland (Cat# CC-
2527,
Lot# 8F1528). HMVEC-L cells were cultured under conditions recommended by the
supplier. Briefly, cells
were cultured in Endothelium Cell Growth Medium MV (EGM-2 MV, Clonetics,
Cat#CC-3202) containing
human epithelial growth factor (hEGF), hydrocortisone, human basic fibroblast
growth factor (hFGF-B),
vascular endothelial growth factor (VEGF), human insulin-like growth factor-1
(hIGF-1), ascorbic acid,
gentamicin, 5% FBS, at 37 C, 5% CO2.
Early passage cells (less than passage 3) were trypsinized and washed with
RPMI-1640
once. Cells were resuspended in Dilution Medium (DM - Fibroblast Basic Medium
+ 2% FBS) at a
concentration of 5X 100,000 cells/ml. 100 NI of the cell suspension containing
50,000 cells was added
into each well in a 96 well cell culture plate. These wells were preloaded
with soluble recombinant human
EMMPRIN with final concentrations of 20 Ng/ml, 6.67 Ng/mi, 2.22 Ng/mI, 0.74
ug/mI, 0.25 Ng/mi, 0.08
Ng/ml, and 0 Ng/mi. Cells were incubated at 37 C, 5% C02, in a humidified
incubator for 1 day and 3
days. Conditioned medium was collected from each well and subjected to MMP-1
activity assay.
Quantitative detection of MMP-1 activity in the conditioned medium was carried
out using
Human MMP-1 Activity Kit (R&D Systems, Minneapolis, Minnesota) (Cat#F1 M00).
Briefly, MMP-1 in 150
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NI of standard or sample was captured by anti-MMP-1 antibodies immobilized at
the bottom of each well.
Captured MMP-1 was subsequently activated by 4-aminophenylmercuric acetate
(APMA). MMP substrate
added into each well was cleaved by active MMP-1 and the resulting
fluorescence was determined using
SpectraFluor Plus Plate Reader (TECAN, Zurich, Switzerland) (Cat# F129005,
Ser# 94747) with the
following parameters: excitation wavelength at 320 nm and emission wavelength
at 405nm.
HMVEC-L cells were challenged with different concentrations of recombinant
EMMPRIN to
stimulate MMP-1 production. As shown in Figure 2, EMMPRIN dose-dependently
stimulated MMP-1
production in endothelial cells. HMVEC-L cells produced approximately 40 ng/ml
MMP-1 when treated
with 20 Ng/ml EMMPRIN. This response of HMVEC-L to EMMPRIN stimulation was
even stronger than
that by NHLF cells, which produced only half of that amount of MMP-1 in
response to the same treatment.
The stimulation of MMP-1 production was first observed after one-day challenge
and sustained for at
least three days.
Results shown in Fig. 2, for the first time, demonstrated that EMMPRIN can
directly stimulate
MMP-1 expression by microvascular endothelial cells, the cells directly
involved in angiogenesis, in a
dose-dependent fashion.
EXAMPLE 2
Inhibition of EMMPRIN-induced MMP-1 production by an anti-EMMPRIN mAb in HMVEC-
L cells
To further confirm the specificity of EMMPRIN-induced MMP-1 production,
monoclonal
antibodies against human EMMPRIN were included in the assay 15 minutes after
cells were stimulated
with EMMPRIN. At 10 pg/ml, the CD147-RDI/clone UM-8D6 (Research Diagnostics,
Inc., Flanders, NJ)
significantly inhibited MMP-1 production by fibroblast cells induced by
EMMPRIN (5 pg/mi) (Fig. 3).
However, the other anti-EMMPRIN mAb (mouse anti-human CD147/EMMPRIN, clone
HIM6, BD
Pharmingen, San Diego, CA) was not able to inhibit MMP-1 production induced by
EMMPRIN.
Our results demonstrated that the stimulation of MMP-1 production in HMVEC-L
cells by
EMMPRIN is specifically mediated by a unique epitope on EMMPRIN recognized by
UM-8D6 but not
HIM6.
EXAMPLE 3
Effects of EMMPRIN on Human Endothelial Cell Migration
The role of EMMPRIN in angiogenesis can also be directly investigated using in
vitro cell
migration and invasion assays. Human endothelial cells derived from primary
tissue (umbilical cord)
HUVEC cells were used in an in vitro system wherein endothelial cells are
seeded in the top wells of the
transwell system, in cell medium containing 1% FBS. In the bottom wells,
culturing medium with 10%
FBS will serve as a chemotactic source to induce cell migration or invasion.
The top and bottom wells are
separated by a membrane with pores of 8 pm in diameter. The membrane is either
uncoated or coated
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with various extracellular matrix proteins, i.e., collagen, fibronectin,
vitronectin, or Matrigel, for determining
cell migration or invasion, respectively.
Materials and Methods MDA-MB-231 human breast cancer cells were purchased from
ATCC (Manassas,
VA). Methods for transfection and establishment of MDA-MB-231 cells stably
expressing different levels
of EMMPRIN have been described previously (Tang, Y. et al. (2004) Mol. Cancer
Res. 2:73-80). The
cells were transfected with the cDNA corresponding to human EMMPRIN open
reading frame sense
(MDA MB231 S1-3) or an antisense strand of the same ORF (MDA MB231 AS1-5 and
MDA MB231 AS2-
5) in pcDNA3.1 TOPO vector (Invitrogen, Carlsbad, CA). Cells transfected wit
the empty vector were
used as a second control (Vector).
Endothelial cell (HUVEC) migration was evaluated using QCMT""-Collagen I
Quantitative
cell migration assay kit (Chemicon, Temecula, CA). HUVEC cells (100,000 in 100
l serum-free medium)
were added to the top compartment. Serum-free media conditioned by MDA MB231
cells: WT, Vector,
S1-3, AS1 -5, or AS2-5 was used as the chemoattactant source in the bottom
compartment of chamber. In
a second experiment, anti-VEGF mAb (R&D Systems, Minneapolis, MN) was added
into the bottom
compartment at various concentrations to neutralize VEGF biological activity.
Cell migration assays were
carried out at 37 C for 6 hours. Insert filters were fixed and cells remained
in the top compartment were
removed. Filters were stained with Gentsian violet and the number of migrated
cells determined using a
microscopic imaging system (Pro-Plus 3D Imaging System, ).
Fig. 4A shows the relative level of HUVEC cell migration induced by
conditioned medium
derived from the various MDA-MB-231 cell constructs. WT cell-induced migration
was assigned 100%.
Error bars represent standard deviations of triplicate data points.
Significant differences by Students T-
test (*) was at the p<0.01 value compared to endothelial cell migration
induced by WT cells. Fig. 4B
shows that a neutralizing antibodies to VEGF inhibited endothelial cell
migration stimulated by serum-free
medium conditioned by MDA-MB-231 EMMPRIN S1-3 tumor cells, assigned as 100%,
in a dose-
dependent manner. Error bars represent standard deviations of triplicate data
points; * p<0.01 compared
to endothelial cell migration in the absence of the anti-VEGF mAb.
These data demonstrate the involvement of VEGF in EMMPRIN-induced endothelial
cell
migration.
EXAMPLE 4
Effects of EMMPRIN on HMVEC-L cell tube formation
The role of EMMPRIN in angiogenesis can be shown using in vitro tube formation
assays. When seeded on Matrigel, HMVEC cells initiate a spontaneous
differentiation process to form
capillary-like tube structure. This in vitro differentiation mimics in vivo
angiogenesis process and is often
employed in angiogenesis studies.
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We predict that EMMPRIN will change the properties of endothelial cells by
stimulating
MMP expression, and therefore to stimulate cell migration and invasion. An
enhanced tube formation will
occur when these cells are stimulated with EMMPRIN.
The specificity of EMMPRIN in tube formation will be investigated using
monoclonal
antibodies against human EMMPRIN.
EXAMPLE 5
Effects of EMMPRIN on angiogenesis in vivo - Matrigel plug assay
The role of EMMPRIN in angiogenesis will be directly investigated in vivo
using Matrigel plug
assays. Matrigel is a solubilized basement membrane preparation extracted from
the Engel-Holm-Swarm
(EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. The major
component is laminin, but
Matrigel also contains trace amounts of fibroblast growth factor, TGF-beta,
tissue plasminogen activator,
and other growth factors that occur naturally in the EHS tumor. Matrigel is
the basis for several types of
tumor cell invasion assays and provides the necessary substrate for the study
of angiogenesis. Matrigel
forms a soft gel plug when injected subcutaneously into mice or rats and
supports an intense vascular
response when supplemented with angiogenic factors.
Matrigel plugs containing suboptimal doses of angiogenic growth factors, such
as basic
fibroblast growth factor (FGF), or vascular endothelial cell growth factor
(VEGF) can be implanted into
mice to induce angiogenesis in vivo. Some of these plugs are supplemented with
various doses of
recombinant EMMPRIN. Since EMMPRIN induces endothelial cell migration and MMP
production by
endothelial cells, we expect to observe an increase in angiogenesis due to
enhanced cell migration and
invasion through Matrigel.
These effects of EMMPRIN as tested in the Matrigel plug angiogenesis assay can
be used to
demonstrate the activity of EMMPRIN antagonists such as siRNA or anti-EMMPRIN
antibodies in
preventing angiongenesis.
EXAMPLE 6
Effects of EMMPRIN on angiogenesis in vivo - corneal pocket assay
Similarly, the role of EMMPRIN in angiogenesis will be directly investigated
in vivo using
corneal pocket assays.
Polymer discs containing angiogenic growth factors, such as basic fibroblast
growth
factor (FGF), or vascular endothelial cell growth factor (VEGF) will be
implanted into a corneal pocket in
order to evoke vascular outgrowth from the peripherally located limbal
vasculature. We will use a
combination of suboptimal doses of angiogenic growth factors supplemented with
various doses of
recombinant EMMPRIN. Since EMMPRIN will induce MMP production by endothelial
cells, we expect to
observe an increase in angiogenesis due to enhanced endothelial cell migration
and invasion.
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The specificity of EMMPRIN in corneal pocket angiogenesis assay will be
investigated
using EMMPRIN antagonists such as siRNA or anti-EMMPRIN antibodies.
EXAMPLE 7
Effects of EMMPRIN on angiogenesis -stimulation of VEGF production and release
mediated by
MMP
It has been reported that EMMPRIN also stimulates the expression of membrane-
type
matrix metalloproteinase 1 (MT1-MMP) [Sameshima et al. 2000b]. MT1-MMP in
turns stimulates
expression of VEGF, one of the most potent angiogenic growth factors,
resulting in enhanced
angiogenesis [Deryugina et al. 2002; Sounni et al. 2002]. However, the direct
link between EMMPRIN
and VEGF expression, and angiogenesis has yet to be established.
Using either recombinant EMMPRIN or tumor cells expressing altered levels of
EMMPRIN, the link between EMMPRIN and VEGF, in both in vitro and in vivo
settings can be
demonstrated. As an increase in the VEGF level promotes angiogenesis, in
addition to EMMPRIN-
induced endothelial cell migration and invasion, the resulting effect on tumor
invasiveness and growth
rate is made evident.
Materials and Methods MDA-MB-231 human breast cancer cells were purchased from
ATCC (Manassas,
VA). Methods for transfection and establishment of MDA-MB-231 cells stably
expressing different levels
of EMMPRIN have been described previously (Tang, Y. et al. (2004) Mol. Cancer
Res. 2:73-80). The
cells were transfected with the cDNA corresponding to human EMMPRIN open
reading frame sense
(MDA MB231 S1-3) or an antisense strand of the same ORF (MDA MB231 AS1-5 and
MDA MB231 AS2-
5) in pcDNA3.1 TOPO vector (Invitrogen, Carlsbad, CA).
Normal human lung or dermal fibroblast cells (NHLF or NHDF), and human
microvascular endothelial cells from the lung (HMVEC-L) or human umbilical
vein endothelial cells
(HUVEC) were obtained from (Clonetics, Walkersville, MD) and were cultured in
Fibroblast Growth
Medium or Endothelial Growth Medium-2 (EGM-2) respectively.
For co-culture studies of cancer and fibroblast cells, 100,000 cancer cells
(MDI MB231
WT, S1-3, AS1-5, or AS 2-5) were cultured together with 200,000 NHDF cells in
a six-well culture plate in
complete DMEM. After 24 h, the medium was replaced with serum-free DMEM and
the cultures
continued for 2 days. The medium was replaced with fresh serum free DMEM and
the cultures
maintained for an additional 3 days at which time the medium was collected and
analyzed. The cells
were lysed with Tris-buffered saline plus 1% NP40 to determine cell-associated
EMMPRIN.
The relative amount of EMMPRIN expressed in 10 ug of total cell protein was
determined
by Western blot analysis using scanning densitometry, by quantitative ELISA
using anti-EMMPRIN
antibody (RDI-147, Research diagnostics) as described (Tang et al. 2004) and
on the cell surface by
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fluorescence activated cell analysis (FAC analysis). The FAC analysis
confirmed that cell surface
EMMPRIN was absent on cells transfected with antisense constructs (data not
shown). The presence of
MMP-2 and MMP-9 in serum-free medium or tumor extracts was determined by
substrate SDS-PAGE
zymography using 10 ug of total protein. Proteolytic activities on the gel
were detected as clear bands on
a blue background of undigested and stained gelatin. ELISA measurements of
human or mouse MMP-2,
MMP-9 and VEGF concentrations were performed using Quantikine ELISA kits from
R&D Systems,
according to the manufacturer's instructions. Each sample was analyzed in
triplicates. Briefly, MMP-2,
MMP-9 or VEGF contained in 100 l of standard or samples (equivalent of 50 g
of total protein) were
captured by anti-MMP-2-, anti-MMP-9-, or anti-VEGF-antibodies immobilized on
the bottom of assay
wells. After washing, the MMP or VEGF specific antibody was used to quantitate
the amount present.
Results The transfected cells had altered levels of total EMMPRIN when grown
in cell
culture conditions (Table 1). S1-3 cells had approximately twice the level of
WT cells and 4-fold that of
the AS cells.
TABLE 1.
MDA MB231 cell EMMPRIN Expression MMP Detected VEGF (pg/ml)
(Rel. Amount)
WT 100% None 208.1
Vector ND 175.5
S1-3 190% MMP-2 (weak) 310.1
AS1-5 47% 64.6
AS2-5 62% 108.7
TABLE 2.
MDA MB231 cell MMP Detected VEGF (pg/mI)
WT MMP-2 306.3
MMP-9
WT+1,10 PA ND 240
WT + anti-CD147 ND 220
None (NHDF MMP-2 19.5
only)
S1-3 MMP-2 416.1
MMP-9
AS1-5 None 134.7
AS2-5 None 154.3
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These data show a relationship between tumor cell expression of EMMPRIN, MMP
expression, and VEGF levels in the engineered tumor cells alone. The co-
culture data (TABLE 2 )show a
supra-additive amount of VEGF is produced when NHDF are present with either WT
human breast tumor
cells or those overexpressing EMMPRIN (S1-3).
EXAMPLE 8
Stimulation of in vivo tumor angiogenesis by EMMPRIN
The stimulatory effects of tumor cell derived EMMPRIN on angiogenesis, the
formation of
new blood vessels, was directly assessed in vivo. Human breast cancer cells,
MDA MB 231, were
engineered to express different levels of EMMPRIN protein using recombinant
DNA technology. Sense
EMMPRIN cells were created that represent a cell population derived from a
single cell clone that was
stably transfected with a mammalian expression vector encoding the full-length
human EMMPRIN.
Antisense cells were generated by transfecting MDA MB 231 cells with a
mammalian expression vector
encoding the full-length human EMMPRIN in the antisense orientation. Sense
cells constitutively express
increased levels of EMMPRIN, and antisense cells express decreased levels of
EMMPRIN due to
inhibition of protein translation by the antisense RNA (See Example 7). These
cells, together with wild-
type cells, were implanted subcutaneously into nude mice. Tumor angiogenesis
were assessed in tumors
derived from these cells.
All procedures involving animals and their care were conducted in conformity
with the
company ICAUC guidelines that are in compliance with the NIH standard. Four-
week-old female CD1
Nu/Nu mice were obtained from Charles River Laboratories, and acclimated for
10-14 days prior to the
experiment.
Comparing with tumors derived from wild type or vector control tumor cells, a
5-fold
increase in final tumor weight was seen in S1-3 tumors produced by the EMMPRIN-
overexpressing cells
(Fig. 5A). AS1-5 and AS2-5 cells produced significantly smaller size (p=0.0242
and 0.0439, respectively)
compared to the unaltered control cells, WT, or those transfected with empty
vector, Vector, during the
same period of time (Fig. 5A).
As shown in Fig. 5B, increased angiogenesis was evidenced by numerous new
capillary
blood vessels in tumors derived from sense cells, but not in tumors derived
from wild-type and antisense
cells.
Human VEGF level was 2.6-fold higher in xenografts tissue produced by S1-3
cells
(235.3 pg/ug total protein) as compared to WT (92.4) and Vector control cells
(86.0 pg/ug total protein (p=
0.0043 sense vs wild type) (Figure 5C). Tumor tissue from AS cells, with
suppressed EMMPRIN levels
had 40.4% less human VEGF or 55 pg/mg total protein (p= 0.0177 compared WT).
More importantly, the impact of increased EMMPRIN level on tumor cell surface
to VEGF
expression in tumor tissues extended beyond tumor cells. Concomitant with
stimulation of tumor VEGF
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production, mouse stromal VEGF production also increased in mice with S1-3
tumors. Levels of host-
derived VEGF escalated 2.1-fold from 23 and 24 pg/mg total protein in wild
type and vector control
tumors to 48 pg/ug total protein in sense tumors (p=0.00009 sense vs WT)
(Figure 5C). Mouse tissue
VEGF from mice given AS cells had less VEGF by -56.6% or 10 pg/mg total
protein (p=0.00013
compared with WT).
Thus, both tumor and host cell-derived VEGF levels followed EMMPRIN-levels in
EMMPRIN-modified cell derived tumors and these trends. These observations
support a new paradigm
in which tumor EMMPRIN mediates an active interaction between tumor and
stromal compartments to
stimulate VEGF production and subsequently tumor angiogenesis and growth in
vivo.
EXAMPLE 9
Effect of tumor EMMPRIN on tumor tissue environment with respect to MMPs and
VEGF
Human breast tumor cells described in Example 3, were used to assess the
effect of
increased of decreased EMMPRIN on tumor tissue and tumor stroma (fibroblasts,
endothelial cells, and
other ancillary cells) in vivo.
On day 0, at approximately 6 weeks of age, mice were assigned to each of 5
groups
consisting of 8 mice per group. Animals were inoculated with 10' cells in 0.1
mL of cell suspension
subcutaneously in the right flank region. Tumor growth was monitored weekly by
caliper measurement
and tumor volume (mm) were calculated based on the formula [length x width x
width]/2. At termination
of the experiment, all animals were euthanized via CO2 asphyxiation. Primary
tumors were excised,
weighed, rinsed in ice-cold PBS and processed for histological/microscopic
examination. Tissue
specimens and sections were also snap-frozen in liquid nitrogen for protein
extraction and biochemical
analysis.
Human EMMPRIN levels were quantitatively assessed with ELISA analysis, which
demonstrated a considerable gain of EMMPRIN level in sense tumors (109.8 pg/ug
of total protein), and
conversely a greatly suppressed level in antisense tumors (26.0 pg/ug of total
protein), compared with
59.0 pg/ug of total protein in wild type tumors (p=0.000048 and 0.000077 for
sense and antisense tumors
vs wild type respectively) (Figure 6A). This stable effect of EMMPRIN
expression on transfected tumor
cells was subsequently translated into influences on MMP expression in vivo.
As expected, substrate
zymography analysis of tumor tissue extracts revealed increased levels of MMP-
2 and MMP-9 activities in
EMMPRIN sense tumors, and lowered levels when EMMPRN expression was suppressed
(Figure 6B).
MMP levels in both tumor and host compartments were then quantitatively
determined by biochemical
analysis. When EMMPRIN was over-expressed in tumor cells, both human MMP-2 and
human MMP-9
expression levels in the resulting xenograft tumors were elevated by
approximately 2.5-fold (p= 0.0068
and 0.0056 compared with wild type tumors respectively) (Figure 6C).
Conversely, a 2-fold decrease in
the expression of these two MMPs was observed when EMMPRIN was inhibited in
antisense tumors (p=
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0.0026 and 0.0035 compared with wild type tumors respectively) (Figure 6C).
The effect of tumor
EMMPRIN expression on host MMP-9 activity associated with stromal cells was
even greater than that on
tumor MMPs.
Thus, the change in tumor cell surface EMMPRIN were capable of inducing a 3.3-
fold
increase or a 59.3% decrease in mouse MMP-9 expression in sense or antisense
tumor nodules,
respectively (p=0.00013 and 0.0047 compared with wild type tumors) (Figure
6D).
Visualization of tumor EMMPRIN-MMP systems in vivo
Tumor
The difference in angiogenic activity is between tumors produced by the cells
overexpressing EMMPRIN and the WT or under expressing cells, AS, were clearly
visible (Fig. 7).
The effect of tumor EMMPRIN expression on host EMMPRIN-MMP system was further
studied in immunohistochemical analysis of the xenograft tumors. In tumors
produced by tumor cells that
over-express EMMPRIN (MDA MB231 S1-3), up-regulation of both mouse MMP-9 and
EMMPRIN was
detected in stromal cells. The expression of these two proteins was restricted
to mouse cells and was not
detected in xenograft human tumor cells (Fig.8). Interestingly, in addition to
the staining found in
capsules surrounding the tumor or in fibroblast cells in the stromal
compartments infiltrated into the tumor
tissues, both mouse EMMPRIN and MMP-9 were highly up-regulated around blood
vessel-like structures
(Fig. 8). Co-localization of MMP-9 and EMMPRIN around angiogenic blood vessels
was further
supported by overlapping distribution of mouse MMP-9, EMMPRIN and that of
CD31, a blood vessel
marker (Fig. 8). In contrast, there were only minimal levels of MMP-9 and
EMMPRIN expression in
tumors produced by Vector control tumor cells. In these tumors, MMP-9 was
mainly detected in
macrophage-like cells, and EMMPRIN was detected at very low levels in some
fibroblast cells (Fig. 8).
EXAMPLE 10
Production and Characterization of Anti-Angiogenic Anti-EMMPRIN Monoclonal
Antibody
Anti-angiogenic anti-EMMPRIN antibodies can be prepared using standard
procedures and
screened using the properties described herein for anti-angiogenic anti-
EMMPRIN antagonists.
Materials and Methods Three 12-14 week old Balb/c mice were obtained from
Charles River
Laboratories. Two mice each received combination intradermal and
intraperitoneal injections of 25 g
rHuEMMPRIN (R&D Systems) (12.5 g/site) in 75 L PBS emulsified in an equal
amount of Freund's
complete adjuvant on day 0, and 25 g rHuEMMPRIN in 75 L PBS emulsified in an
equal amount of
Freund's incomplete adjuvant on days 14, 28 and 51. The third mouse received
an initial injection of 25
g of rHuEMMPRIN + 0.33 x 105 U murine IFNa + 0.33 x 105 U murine IFN(3
(Biosource) in 100 i PBS
administered S.Q. at the base of the tail. On days 2 and 3, the mouse received
additional injections of
0.33 x 105 U IFNa + 0.33 x 105 U IFNR in 100 L PBS administered S.Q. at the
base of the tail. Several
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weeks later, the mouse was boosted with 25 g EMMPRIN + 100 g anti-murine
CD40 agonist Mab
(R&D Systems) administered S.Q. at the base of the tail.
The mice were bled at various time-points throughout the immunization
schedule. Blood
collections were performed by retro-orbital puncture and serum was collected
for titer determination by
solid phase EIA. Once titer plateau was obtained, the mice received their
final booster of 25 g of
EMMPRIN in PBS given intraveneously (IV). Three days later the mice were
euthanized by CO2
asphyxiation, and the spleens were aseptically removed and immersed in 10 mL
cold PBS containing 100
U/mL penicillin, 100 g/mL streptomycin, and 0.25 g/mL amphotericin B
(PBS/PSA). Lymphocytes were
harvested by sterilely passing cells though a wire mesh screen immersed in
cold PBS/PSA. The cells
were washed once in cold PSA/PBS, counted using Trypan blue dye exclusion and
resuspended in 10
mL PBS.
Characterization of Anti-Human EMMPRIN Antibodies
Enzyme immunoassays (EIAs} were used to test hybridoma cell supernatants for
the
presence of human anti-EMMPRIN Mabs. Briefly, plates (Nunc-Maxisorp) were
coated overnight with
human EMMPRIN at 1 g/mL in PBS. After washing in 0.15 M saline containing
0.02%(w/v) Tween 20,
the wells were blocked with 1%(w/v) bovine serum albumin (BSA) in PBS for 1 hr
at 37 C. Undiluted
hybridoma supernatants were incubated on coated plates for 1 hour at 37 C. The
plates were washed
and then incubated with HRP-labeled goat anti-murine IgG, Fc specific (Sigma)
diluted 1:10,000 in 1%
BSA/PBS for 30 minutes at 37 C. Plates were again washed then incubated for 15
minutes at RT with
100 Uwell of citrate-phosphate substrate solution (0.1 M citric acid and 0.2
M sodium phosphate, 0.01%
H202, and 1 mg/mL o-phenylenediamine dihydrochloride). Substrate development
was stopped by the
addition of 4N sulfuric acid at 25 Uwell and the absorbance was measured at
490nm via an automated
plate spectrophotometer. All reactive hybrid cell lines were subcloned twice
by limiting dilution at 1
cell/well in cloning plates. The homogeneous cell lines were cryopreserved in
freezing medium (90%
FBS, 10% DMSO) and stored in liquid nitrogen.
To identify the isotype of the murine anti-human EMPRIN antibodies, the
Monoclonal
Antibody Isotyping Kit-IsoStrip, Dipstick Format (Roche) was used as per the
manufacturer instructions.
Briefly, culture supernatant was diluted 1:10 in PBS and added to the
development tube. The dipstick
was added to the development tube and incubated at RT for approximately ten
minutes. Isotypes were
determined by visual assessment following incubation. A list of eighteen
different hybridoma clones that
secrete a murine IgG Mab that specifically binds to human EMMPRIN is shown in
Table 3.
The biologic activity of recombinant EMMPIRN used as antigen protein was
assayed by its ability
to stimulate production of MMP-1 from EMMPRIN stimulated in fibroblast cells
was performed as
described (Guo, Zucker, Gordon, Toole and Biswas, (1997), J Biol Chem 272: 24-
7)(24)), modified by
using highly homogenous primary human fibroblast cells of less than three
passages and modified
stimulation conditions. Only highly pure fibroblast cells that were confirmed
being negative for cytokeratin
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18, cytokeratin 19, factor VII-related antigen, and alpha actin were used in
the assay. The magnitude of
response to EMMPRIN stimulation was dependent on the passage of fibroblast
cells. Cells of earlier
passages responded more potently in producing increased amounts of MMP,
compared to cells that have
been cultured for more than three passages. In addition, a new cell challenge
method was used. Instead
of adding recombinant EMMPRIN to adherent cells, we preloaded recombinant
EMMPRIN in testing
wells. Suspended cells were then added into these wells and were directly
exposed to recombinant
EMMPRIN. This new challenge procedure ensures the maximal exposure of cell
surface receptors, which
are likely expressed on the basolateral surfaces and could be out of access in
adherent cells, to
EMMPRIN for optimal assay sensitivity.
Recombinant EMMPRIN corresponding to the extracellular domain of human EMMPRIN
protein was produced in NSO cells (R&D Systems, Minneapolis, MN). MMP-1
activity in serum-free
medium conditioned by fibroblast cells treated with different amounts of
recombinant EMMPRIN protein
was quantitatively determined using an MMP-1 Activity Assay Kit according to
product manual (R&D
Systems, Minneapolis, MN). Briefly, MMP-1 contained in 150 l of standards or
samples was captured by
anti-MMP-1 antibodies immobilized on the bottom of assay wells. Captured MMP-1
was subsequently
activated by 4-aminophenylmercuric acetate (APMA). MMP substrate added into
each well was cleaved
by activated MMP-1 and the resulting fluorescence was determined using
SpectraFluor Plus Plate
Reader (TECAN, Research Triangle Park, NC) with the following parameters:
excitation wavelength at
320 nm and emission wavelength at 405 nm. To determine the inhibitory activity
of anti-EMMPRIN
antibodies, antibodies were added into cell culture after cells were
stimulated with recombinant
EMMPRIN for 15 minutes.
The panel of monoclonal antibodies were all screened for these two activities
in addition to
Isotyping (TABLE 3).
TABLE 3.
CNTO# lsotype MMP-1 Co-culture
1111 IgG2bk N P
2169 IgG1 k N N
120 IgG1k N N
5125 IgG1 k N N
627 IgG1 k N/A N/A
828 IgG2bk N N
146 IgG1 k P P
314 IgG1 k N P
1310 IgG1k N N
1412 IgG2bk N N/A
1513 IgG1k N N
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1611 IgG1 k N* N
610 IgGlk N N/A
1134 IgGlk N N/A
4153 IgG1 k N N/A
3632 IgG1 k N N/A
1193 IgG1 k N N/A
4161 IgG1 k N N/A
N/A (Anti- IG1 k P P
CD147) g
The antibody designated CNTO 146 met the initial selection criteria for an
anti-
angiongenic anti-EMMPRIN Mab.
Inhibition of MMP-2 production in co-culture of tumor cells and fibroblast
cells
The co-culture assay was performed as previously described above using normal
human
dermal fibroblasts and human melanoma tumor cells (G361) were used and either
the commercial
antibody RDI CD147 or CNTO 146 were added to the cultures. Three days after
the last change of
serum free medium, the amount of MMP-2 was quantitated. The data showed that
CNTO 146 was
capable of inhibiting MMP-2 production in these co-cultures as did the
commercial antibody.
28
