U.S. patent application number 10/137351 was filed with the patent office on 2003-04-10 for genetically modified cells and methods for expressing recombinant heparanase and methods of purifying same.
Invention is credited to Ayal-Hershkovitz, Maty, Pecker, Iris, Yacoby-Zeevi, Oron.
Application Number | 20030068806 10/137351 |
Document ID | / |
Family ID | 26752433 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030068806 |
Kind Code |
A1 |
Ayal-Hershkovitz, Maty ; et
al. |
April 10, 2003 |
Genetically modified cells and methods for expressing recombinant
heparanase and methods of purifying same
Abstract
A method of purifying a recombinant heparanase from
overexpressing cells or growth medium which comprises adsorbing
said recombinant heparanase on an ion-exchange column under low
salt conditions, washing said column with low salt solution thereby
eluting other proteins, and eluting the recombinant heparanase from
said column by a salt gradient or higher salt concentration.
Inventors: |
Ayal-Hershkovitz, Maty;
(Herzlia, IL) ; Pecker, Iris; (Rishon LeZion,
IL) ; Yacoby-Zeevi, Oron; (Meitar, IL) |
Correspondence
Address: |
G.E. EHRLICH (1995) LTD.
c/o ANTHONY CASTORINA
SUITE 207
2001 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Family ID: |
26752433 |
Appl. No.: |
10/137351 |
Filed: |
May 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10137351 |
May 3, 2002 |
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09635923 |
Aug 10, 2000 |
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6426209 |
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09635923 |
Aug 10, 2000 |
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09487716 |
Jan 19, 2000 |
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09487716 |
Jan 19, 2000 |
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09260038 |
Mar 2, 1999 |
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6348344 |
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09260038 |
Mar 2, 1999 |
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09071618 |
May 1, 1998 |
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09071618 |
May 1, 1998 |
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09071739 |
May 1, 1998 |
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6177545 |
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09071739 |
May 1, 1998 |
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08922170 |
Sep 2, 1997 |
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5968822 |
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Current U.S.
Class: |
435/201 ;
435/69.1 |
Current CPC
Class: |
C12Y 302/01166 20130101;
G01N 2333/988 20130101; C12N 9/2402 20130101; A61K 38/00 20130101;
G01N 33/573 20130101; G01N 2400/40 20130101; C07K 16/40
20130101 |
Class at
Publication: |
435/201 ;
435/69.1 |
International
Class: |
C12N 009/26; C12P
021/02 |
Claims
What is claimed is:
1. A method of purifying a recombinant heparanase from
overexpressing cells or growth medium comprising adsorbing said
recombinant heparanase on an ion-exchange column under low salt
conditions, washing said column with low salt solution thereby
eluting other proteins, and eluting the recombinant heparanase from
said column by a salt gradient or higher salt concentration.
2. The method of claim 1, wherein said ion-exchange column is a
Source-S column.
3. The method of claim 1, wherein said recombinant heparanase
includes an amino acid sequence at least 70% homologous to SEQ ID
NO:2.
4. The method of claim 1, wherein said recombinant heparanase
includes an amino acid sequence as set forth in SEQ ID NO:2.
5. A method of affinity purifying heparanase comprising: (a)
contacting a heparanase preparation with an affinity substrate
including a solid matrix and an anti-heparanase antibody being
immobilized thereto, said anti-heparanase antibody being capable of
specifically binding at least one epitope of said heparanase; (b)
washing said affinity substrate; and (c) eluting heparanase
molecules being adsorbed on said affinity substrate via said
anti-heparanase antibody.
6. The method of claim 5, wherein said heparanase includes an amino
acid sequence at least 70% homologous to SEQ ID NO:2.
7. The method of claim 5, wherein said heparanase includes an amino
acid sequence as set forth in SEQ ID NO:2.
Description
[0001] This is a continuation of U.S. patent application Ser. No.
09/635,923, filed Aug. 10, 2000, which is a continuation of U.S.
patent application Ser. No. 09/487,716, filed Jan. 19, 2000, which
is a continuation of U.S. patent application Ser. No. 09/260,038,
filed Mar. 2, 1999, now U.S. Pat. No. 6,348,344, issued Feb. 19,
2002, which is a continuation-in-part of U.S. patent application
Ser. No. 09/071,618, filed May 1, 1998, now abandoned, which is a
continuation-in-part of U.S. patent application Ser. No.
09/071,739, filed May 1, 1998, now U.S. Pat. No. 6,177,545, issued
Jan. 23, 2002, which is a continuation-in-part of U.S. patent
application Ser. No. 08/922,170, filed Sep. 2, 1997, now U.S. Pat.
No. 5,968,822, issued Oct. 19, 1999.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to genetically modified cells
overexpressing recombinant heparanase, to methods of overexpressing
recombinant heparanase in cellular systems and to methods of
purifying recombinant heparanase. The invention further relates to
nucleic acid constructs for directing the expression of modified
heparanase species to which a protease recognition and cleavage
sequence has been introduced, to the modified heparanase species
expressed therefrom and to their proteolytic products. The
invention further relates to in vivo methods of inhibiting
heparanase activity.
[0003] The extracellular matrix (ECM) acts both as a structural
scaffold and as an informational medium. Its dynamic status is
determined by cells that secrete both its constituent molecules and
enzymes that catalyze the degradation of these molecules. A stasis
between ECM degrading enzymes and their inhibitors maintains the
integrity of the matrix. While controlled ECM remodeling is
fundamental to normal processes, uncontrolled disruption underlies
diverse pathological conditions.
[0004] Among the integral constituents of basement membrane and ECM
are cell adhesion molecules such as laminin and fibronectin,
structural components like collagen and ellastin, and proteoglycans
including sydecan, serglican, proteoglycan I and II versican
(1-2).
[0005] Brief Overview on Recombinant Gene Expression:
[0006] For biochemical characterization of a protein and
pharmaceutical applications, it is often necessary to overproduce
and purify large quantities of the protein. A major consideration
when setting up a production scheme for a recombinant protein is
whether the product should be expressed intracellularly or if a
secretion system can be used to direct the protein to the growth
medium. The inherent properties of the protein and the intended
applications dictate the expression system of choice. Another
consideration when attempting the production of recombinant
eukaryotic proteins are the folding and post translational
modification processes associated with their natural
expression.
[0007] Preferably, production is carried out in a cellular system
that supports appropriate transcription, translation, and
post-translation modification of the protein of interest. Thus,
cultured mammalian cells are widely used in applied biotechnology
as well as in different disciplines of basic sciences of cellular
and molecular biology for producing recombinant proteins of
mammalian origin.
[0008] One of the most widely used cells for recombinant protein
expression, particularly for biotechnological applications, is the
Chinese hamster ovary cell line (CHO). Alternatively, baby hamster
kidney cells (BHK21), Namalwa cells, Dauidi cells, Raji cells,
Human 293 cells, Hela cells, Ehrlich's ascites cells, Sk-Hepl
cells, MDCK.sub.1 cells, MDBK.sub.1 cells, Vero cells, Cos cells,
CV-1 cells, NIH3T3 cells, L929 cells and BLG cells (mouse melanoma)
have also been shown to consecutively express large quantities of
recombinant proteins.
[0009] These cells are easily transfected with foreign DNA, that
can integrate into the host genome to create stable cell lines,
with new acquired characteristics (i.e. expression of recombinant
proteins). These new cell lines originate from a single cell that
has undergone foreign DNA incorporation and are therefore referred
to as "cellular clones".
[0010] Since integration of foreign DNA in host cell genome is
relatively inefficient, the isolation of cellular clones requires a
selection system that discriminates between the stably transformed
and the primary cells.
[0011] Dihydrofolate reductase deficiency in CHO cells (CHO
dhfr-cell line) offers a particularly convenient selection system
for cellular clones. Transfection of the dhfr gene along with the
gene of interest, results in the survival of clones in a growth
medium containing methotrexate (MTX). The higher the number of
foreign dhfr gene copies in the cellular clone, the higher the MTX
concentration the cells can survive. It has been demonstrated that
integration events of foreign DNA into host cell genome often
maintain all the components of the transfected DNA. e.g., the
selection marker as well as the gene of interest (67).
[0012] In contrast to mammalian expression systems, that inherently
express limited quantities of recombinant proteins, other
expression systems, such as bacteria, yeast, and virus infected
insect cells are widely used.
[0013] Using such cellular gene expression systems, large amounts
of either active or non-active protein can be obtained and used for
biochemical analysis of protein properties, structure function
relationship, kinetic studies, identification of, screening for, or
production of specific inhibitors, production of poly- and
monoclonal antibodies recognizing the protein, pharmaceutical
applications and the like.
[0014] Bacteria are the most powerful tool for the production of
recombinant proteins. A recombinant protein that is overproduced in
a bacterial system might constitute up to 30% of the total protein
content of the cells. The recombinant protein accumulates in
inclusion bodies where it is relatively pure (comprises up to 50%
of the protein content of the bodies) and protected from protease
degradation.
[0015] Inclusion bodies enable the accumulation of up to 0.2 grams
of protein per liter fermentation culture.
[0016] Using specific expression vectors, bacteria can also be
directed to produce and secrete proteins into the periplasm and
therefrom into the growth medium. Although the reported production
quantities are not as high as in inclusion bodies, purification of
the expressed protein may be simpler (68).
[0017] These advantages and the relative simple growth conditions
required for bacteria to thrive, made bacteria a powerful and
widely used cellular expression system for the production of
recombinant proteins of interest (e.g., human o-interferon, human
.beta.-interferon, GM-CSF, G-CSF, human LNF-.gamma., IL-2, IL-3,
IL-6, TNF, human insulin, human growth hormone, etc.).
[0018] Furthermore, non-active bacterialy produced recombinant
proteins due to inappropriate folding and disulfide bonding may be
reduced and/or denatured and thereafter deoxidized and/or refolded
to acquire the catalytically active conformation.
[0019] However, when glycosylation of the protein is essential for
its activity or uses, eukaryotic expression systems are
required.
[0020] Yeasts are eukaryotic microorganisms which are widely used
for commercial production of recombinant proteins. Examples include
the production of insulin, human GM-CSF and hepatitis B antigens
(for vaccination) by the yeast Saccharomyces cerevisiae. The
relatively simple growth conditions and the fact that yeasts are
eukaryotes make the yeast gene expression system highly suitable
for the production of recombinant proteins, primarily those with
pharmaceutical relevance.
[0021] In recent years methylotrophic yeasts (e.g., Pichia
pastoris, Hansenula polymorpha) became widely used, thus replacing
in many cases the more traditionally used yeast Saccharomyces
cerevisiae.
[0022] Methylotrophic yeasts can grow to a high cellular density,
and express and if appropriately, secrete, high levels of
recombinant proteins. Quantities of the secreted, correctly-folded
recombinant protein can accumulate up to several grams per liter
culture. These advantages make Pichia pastoris suitable for an
efficient production of recombinant proteins (69).
[0023] One aspect of the present invention thus concerns the
expression of recombinant heparanase in cellular systems.
[0024] Heparan Sulfate Proteoglycans (HSPGs):
[0025] HSPGs are ubiquitous macromolecules associated with the cell
surface and extracellular matrix (ECM) of a wide range of cells of
vertebrate and invertebrate tissues (3-7). The basic HSPG structure
consists of a protein core to which several linear heparan sulfate
chains are covalently attached. The polysaccharide chains are
typically composed of repeating hexuronic and D-glucosamine
disaccharide units that are substituted to a varying extent with N-
and O-linked sulfate moieties and N-linked acetyl groups (3-7).
Studies on the involvement of ECM molecules in cell attachment,
growth and differentiation revealed a central role of HSPGs in
embryonic morphogenesis, angiogenesis, metastasis, neurite
outgrowth and tissue repair (3-7). The heparan sulfate (HS) chains,
which are unique in their ability to bind a multitude of proteins,
ensure that a wide variety of effector molecules cling to the cell
surface (6-8). HSPGs are also prominent components of blood vessels
(5). In large vessels they are concentrated mostly in the intima
and inner media, whereas in capillaries they are found mainly in
the subendothelial basement membrane where they support
proliferating and migrating endothelial cells and stabilize the
structure of the capillary wall. The ability of HSPGs to interact
with ECM macromolecules such as collagen, laminin and fibronectin,
and with different attachment sites on plasma membranes suggests a
key role for this proteoglycan in the self-assembly and
insolubility of ECM components, as well as in cell adhesion and
locomotion. Cleavage of HS may therefore result in disassembly of
the subendothelial ECM and hence may play a decisive role in
extravasation of normal and malignant blood-borne cells (9-11). HS
catabolism is observed in inflammation, wound repair, diabetes, and
cancer metastasis, suggesting that enzymes which degrade HS play
important roles in pathologic processes.
[0026] Heparanase:
[0027] Heparanase is a glycosylated enzyme that is involved in the
catabolism of certain glycosaminoglycans. It is an
endo-.beta.-glucuronidase that cleaves heparan sulfate at specific
intrachain sites (12-15). Interaction of T and B lymphocytes,
platelets, granulocytes, macrophages and mast cells with the
subendothelial extracellular matrix (ECM) is associated with
degradation of heparan sulfate by heparanase activity (16).
Connective tissue activating peptide III (CTAP), an
.beta.-chemokine, was found to have heparanase-like activity.
Placenta heparanase acts as an adhesion molecule or as a
degradative enzyme depending on the pH of the microenvironvent
(17).
[0028] Heparanase is released from intracellular compartments
(e.g., lysosomes, specific granules) in response to various
activation signals (e.g., thrombin, calcium ionophores, immune
complexes, antigens and mitogens), suggesting its regulated
involvement in inflammation and cellular immunity responses
(16).
[0029] It was also demonstrated that heparanase can be readily
released from human neutrophils by 60 minutes incubation at
4.degree. C. in the absence of added stimuli (18).
[0030] Gelatinase, another ECM degrading enzyme which is found in
tertiary granules of human neutrophils with heparanase, is secreted
from the neutrophils in response to phorbol 12-myristate 13-acetate
(PMA) treatment (19-20).
[0031] In contrast, various tumor cells appear to express and
secrete heparanase in a constitutive manner in correlation with
their metastatic potential (21).
[0032] Degradation of heparan sulfate by heparanase results in the
release of heparin-binding growth factors, enzymes and plasma
proteins that are sequestered by heparan sulfate in basement
membranes, extracellular matrices and cell surfaces (22-23).
[0033] Purification of Natural Heparanase:
[0034] Heparanase activity has been described in a number of cell
types including cultured skin fibroblasts, human neutrophils,
activated rat T-lymphocytes, normal and neoplastic murine
B-lymphocytes, human monocytes and human umbilical vein endothelial
cells, SK hepatoma cells, human placenta and human platelets.
[0035] A procedure for purification of natural heparanase was
reported for SK hepatoma cells and human placenta (U.S. Pat. No.
5,362,641) and for human platelets derived enzymes (62).
Purification was performed by a combination of ion exchange and
various affinity columns including Con-A Sepharose, Blue A-agarose,
Zn.sup.++-chelating agarose and Heparin-Sepharose. Evidently, the
amounts of active heparanase recovered by these methods is low.
[0036] Cloning and Expression of the Heparanase Gene:
[0037] A purified fraction of heparanase isolated from human
hepatoma cells was subjected to tryptic digestion. Peptides were
separated by high pressure liquid chromatography (HPLC) and micro
sequenced. The sequence of one of the peptides was used to screen
data bases for homology to the corresponding back translated DNA
sequence. This procedure led to the identification of a clone
containing an insert of 1020 base pairs (bp) which included an open
reading frame of 963 bp followed by 27 bp of 3' untranslated region
and a poly A tail. The new gene was designated hpa. Cloning of the
missing 5' end of hpa was performed by PCR amplification of DNA
from placenta cDNA composite. The joined hpa cDNA (also referred to
as phpa) fragment contained an open reading frame which encodes a
polypeptide of 543 amino acids with a calculated molecular weight
of 61,192 daltons. Cloning an extended 5' sequence was enabled from
the human SK-hepI cell line by PCR amplification using the Marathon
RACE system. The 5' extended sequence of the SK-hepI hpa cDNA was
assembled with the sequence of the hpa cDNA isolated from human
placenta. The assembled sequence contained an open reading frame
which encodes a polypeptide of 592 amino acids with a calculated
molecular weight of 66,407 daltons. The cloning procedures are
described in length in U.S. patent application Ser. Nos.
08/922,170, 09/109,386, and 09/071,618, entitled POLYNUCLEOTIDE
ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY AND EXPRESSION OF
SAME IN GENETICALLY MODIFIED CELLS, which is a continuation-in-part
of PCT/US98/17954, filed Aug. 31, 1998, all of which are
incorporated herein by reference.
[0038] The ability of the hpa gene product to catalyze degradation
of heparan sulfate (HS) in vitro was examined by expressing the
entire open reading frame of hpa in High five and Sf21 insect
cells, and the mammalian human 293 embryonic kidney cell line
expression systems. Extracts of infected cells were assayed for
heparanase catalytic activity. For this purpose, cell lysates were
incubated with sulfate labeled, ECM-derived HSPG (peak I), followed
by gel filtration analysis (Sepharose 6B) of the reaction mixture.
While the substrate alone consisted of high molecular weight
material, incubation of the HSPG substrate with lysates of cells
infected with hpa containing virus resulted in a complete
conversion of the high molecular weight substrate into low
molecular weight labeled heparan sulfate degradation fragments
(see, for example, U.S. patent application Ser. No. 09/071,618,
which is incorporated herein by reference.
[0039] In subsequent experiments, the labeled HSPG substrate was
incubated with the culture medium of infected High Five and Sf21
cells. Heparanase catalytic activity, reflected by the conversion
of the high molecular weight HSPG substrate into low molecular
weight HS degradation fragments, was found in the culture medium of
cells infected with the pFhpa virus, but not the control pF1
virus.
[0040] Altogether, these results indicate that the heparanase
enzyme is expressed in an active form by cells infected with
Baculovirus or mammalian expression vectors containing the newly
identified human hpa gene.
[0041] In other experiments, it was demonstrated that the
heparanase enzyme expressed by cells infected with the pFhpa virus
is capable of degrading HS complexed to other macromolecular
constituents (e.g., fibronectin, laminin, collagen) present in a
naturally produced intact ECM (see U.S. patent application Ser. No.
09/109,386, which is incorporated herein by reference), in a manner
similar to that reported for highly metastatic tumor cells or
activated cells of the immune system (7, 8)
[0042] Involvement of Heparanase in Tumor Cell Invasion and
Metastasis:
[0043] Circulating tumor cells arrested in the capillary beds often
attach at or near the intercellular junctions between adjacent
endothelial cells. Such attachment of the metastatic cells is
followed by rupture of the junctions, retraction of the endothelial
cell borders and migration through the breach in the endothelium
toward the exposed underlying base membrane (BM) (24). Once located
between endothelial cells and the BM, the invading cells must
degrade the subendothelial glycoproteins and proteoglycans of the
BM in order to migrate out of the vascular compartment. Several
cellular enzymes (e.g., collagenase IV, plasminogen activator,
cathepsin B, elastase, etc.) are thought to be involved in
degradation of BM (25). Among these enzymes is heparanase that
cleaves HS at specific intrachain sites (16,11). Expression of a HS
degrading heparanase was found to correlate with the metastatic
potential of mouse lymphoma (26), fibrosarcoma and melanoma (21)
cells. Moreover, elevated levels of heparanase were detected in
sera from metastatic tumor bearing animals and melanoma patients
(21) and in tumor biopsies of cancer patients (12).
[0044] The inhibitory effect of various non-anticoagulant species
of heparin on heparanase was examined in view of their potential
use in preventing extravasation of blood-borne cells. Treatment of
experimental animals with heparanase inhibitors markedly reduced
(>90%) the incidence of lung metastases induced by B16 melanoma,
Lewis lung carcinoma and mammary adenocarcinoma cells (12, 13, 28).
Heparin fractions with high and low affinity to anti-thrombin III
exhibited a comparable high anti-metastatic activity, indicating
that the heparanase inhibiting activity of heparin, rather than its
anticoagulant activity, plays a role in the anti-metastatic
properties of the polysaccharide (12).
[0045] Finally, heparanase externally adhered to B16-F1 melanoma
cells increased the level of lung metastases in C57BL mice as
compared to control mice (see U.S. patent application Ser. No.
09/260,037, entitled INTRODUCING A BIOLOGICAL MATERIAL INTO A
PATIENT, which is a continuation in part of U.S. patent application
Ser. No. 09/140,888, and is incorporated herein by reference.
[0046] Possible Involvement of Heparanase in Tumor
Angiogenesis:
[0047] Fibroblast growth factors are a family of structurally
related polypeptides characterized by high affinity to heparin
(29). They are highly mitogenic for vascular endothelial cells and
are among the most potent inducers of neovascularization (29-30).
Basic fibroblast growth factor (bFGF) has been extracted from a
subendothelial ECM produced in vitro (31) and from basement
membranes of the cornea (32), suggesting that ECM may serve as a
reservoir for bFGF. Immunohistochemical staining revealed the
localization of bFGF in basement membranes of diverse tissues and
blood vessels (23). Despite the ubiquitous presence of bFGF in
normal tissues, endothelial cell proliferation in these tissues is
usually very low, suggesting that bFGF is somehow sequestered from
its site of action. Studies on the interaction of bFGF with ECM
revealed that bFGF binds to HSPG in the ECM and can be released in
an active form by HS degrading enzymes (33, 32, 34). It was
demonstrated that heparanase activity expressed by platelets, mast
cells, neutrophils, and lymphoma cells is involved in release of
active bFGF from ECM and basement membranes (35), suggesting that
heparanase activity may not only function in cell migration and
invasion, but may also elicit an indirect neovascular response.
These results suggest that the ECM HSPG provides a natural storage
depot for bFGF and possibly other heparin-binding growth promoting
factors (36,37). Displacement of bFGF from its storage within
basement membranes and ECM may therefore provide a novel mechanism
for induction of neovascularization in normal and pathological
situations.
[0048] Recent studies indicate that heparin and HS are involved in
binding of bFGF to high affinity cell surface receptors and in bFGF
cell signaling (38, 39). Moreover, the size of HS required for
optimal effect was similar to that of HS fragments released by
heparanase (40). Similar results were obtained with vascular
endothelial cells growth factor (VEGF) (41), suggesting the
operation of a dual receptor mechanism involving HS in cell
interaction with heparin-binding growth factors. It is therefore
proposed that restriction of endothelial cell growth factors in ECM
prevents their systemic action on the vascular endothelium, thus
maintaining a very low rate of endothelial cells turnover and
vessel growth. On the other hand, release of bFGF from storage in
ECM as a complex with HS fragment, may elicit localized endothelial
cell proliferation and neovascularization in processes such as
wound healing, inflammation and tumor development (36,37).
[0049] Recombinant Heparanase for Screening Purposes:
[0050] Put together, the accumulated evidences indicate that a
reliable and high throughput (HTS) screening system for heparanase
inhibiting compounds may be applied to identify and develop
non-toxic drugs for the treatment of cancer and metastasis.
Research aimed at identifying and developing inhibitors of
heparanase catalytic activity has been handicapped by the lack of a
consistent and constant source of a purified and highly active
heparanase enzyme and of a reliable screening system. Such a HTS
system is described in U.S. patent application Ser. No. 09/113,168,
which is incorporated herein by reference. To this end, however,
methods are required for obtaining high quantities of highly pure
and active heparanase, so as to enable to study the kinetics of
heparanase per se and in the presence of potential inhibitors. The
recent cloning, expression and purification of the human
heparanase-encoding gene offer, for the first time, a most
appropriate and reliable source of active recombinant enzyme for
screening of anti-heparanase antibodies and compounds which may
inhibit the enzyme and hence be applied to identify and develop
drugs that may inhibit tumor metastasis, autoimmune
[0051] and inflammatory diseases.
[0052] Screening for Specific Inhibitors Using a Combinatorial
Library:
[0053] A new approach aimed at rational drug discovery was recently
developed for screening for specific biological activities.
According to the new approach, a large library of chemically
diverged molecules are screened for the desired biological
activity. The new approach has become an effective and hence
important tool for the discovery of new drugs. The new approach is
based on "combinatorial" synthesis of a diverse set of molecules in
which several components predicted to be associated with the
desired biological activity are systematically varied. The
advantage of a combinatorial library over the alternative use of
natural extracts for screening for desired biologically active
compounds is that all the components comprising the library are
known in advance (60).
[0054] In combinatorial screening, the number of hits discovered is
proportional to the number of molecules tested. This is true even
when knowledge concerning the target is unavailable. The large
number of compounds, which may reach thousands of compounds tested
per day, can only be screened, provided that a suitable assay
involving a high throughput screening technique, in which
laboratory automation and robotics may be applied, exists.
[0055] Expression of Heparanase by Cells of the Immune System:
[0056] Heparanase catalytic activity correlates with the ability of
activated cells of the immune system to leave the circulation and
elicit both inflammatory and autoimmune responses. Interaction of
platelets, granulocytes, T and B lymphocytes, macrophages and mast
cells with the subendothelial ECM is associated with degradation of
heparan sulfate (HS) by heparanase catalytic activity (10). The
enzyme is released from intracellular compartments (e.g.,
lysosomes, specific granules) in response to various activation
signals (e.g., thrombin, calcium ionophore, immune complexes,
antigens, mitogens), suggesting its regulated involvement and
presence in inflammatory sites and autoimmune lesions. Heparan
sulfate degrading enzymes released by platelets and macrophages are
likely to be present in atherosclerotic lesions (42).
[0057] Treatment of experimental animals with heparanase
alternative substrates (e.g., non-anticoagulant species of low
molecular weight heparin) markedly reduced the incidence of
experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis
and graft rejection (10, 43) in experimental animals, indicating
that heparanase inhibitors may be applied to inhibit autoimmune and
inflammatory diseases (10,43).
[0058] The involvement of Heparanase in Other Physiological
Processes and its Potential Therapeutic Applications:
[0059] Apart from its involvement in tumor cell metastasis,
inflammation and autoimmunity, mammalian heparanase may be applied
to modulate bioavailability of heparin-binding growth factors (45);
cellular responses to heparin-binding growth factors (e.g., bFGF,
VEGF) and cytokines (IL-8) (44, 41); cell interaction with plasma
lipoproteins (49); cellular susceptibility to certain viral and
some bacterial and protozoa infections (45-47); and disintegration
of amyloid plaques (48).
[0060] Viral Infection:
[0061] The presence of heparan sulfate on cell surfaces have been
shown to be the principal requirement for the binding of Herpes
Simplex (45) and Dengue (46) viruses to cells and for subsequent
infection of the cells. Removal of the cell surface heparan sulfate
by heparanase may therefore abolish virus infection. In fact,
treatment of cells with bacterial heparitinase (degrading heparan
sulfate) or heparinase (degrading heparan) reduced the binding of
two related animal herpes viruses to cells and rendered the cells
at least partially resistant to virus infection (45). There are
some indications that the cell surface heparan sulfate is also
involved in HIV infection (47).
[0062] Neurodegenerative Diseases:
[0063] Heparan sulfate proteoglycans were identified in the prion
protein amyloid plaques of Genstmann-Straussler Syndrome,
Creutzfeldt-Jakob disease and Scrape (48). Heparanase may
disintegrate these amyloid plaques which are also thought to play a
role in the pathogenesis of Alzheimer's disease.
[0064] Restenosis and Atherosclerosis:
[0065] Proliferation of arterial smooth muscle cells (SMCs) in
response to endothelial injury and accumulation of cholesterol rich
lipoproteins are basic events in the pathogenesis of
atherosclerosis and restenosis (50). Apart from its involvement in
SMC proliferation as a low affinity receptor for heparin-binding
growth factors, HS is also involved in lipoprotein binding,
retention and uptake (51). It was demonstrated that HSPG and
lipoprotein lipase participate in a novel catabolic pathway that
may allow substantial cellular and interstitial accumulation of
cholesterol rich lipoproteins (49). The latter pathway is expected
to be highly atherogenic by promoting accumulation of apoB and apoE
rich lipoproteins (e.g., LDL, VLDL, chylomicrons), independent of
feed back inhibition by the cellular cholesterol content. Removal
of SMC HS by heparanase is therefore expected to inhibit both SMC
proliferation and lipid accumulation and thus may halt the
progression of restenosis and atherosclerosis.
[0066] In summary, Heparanase may thus prove useful for conditions
such as wound healing, angiogenesis, restenosis, atherosclerosis,
inflammation, neurodegenerative diseases and viral infections.
Mammalian heparanase can be used to neutralize plasma heparin, as a
potential replacement of protamine. Anti-heparanase antibodies may
be applied for immunodetection and diagnosis of micrometastases,
autoimmune lesions and renal failure in biopsy specimens, plasma
samples, and body fluids. Common use in basic research is
expected.
[0067] ECM Proteases and Their Involvement in Tumor Progression and
Metastasis:
[0068] The cooperation with pericellular proteolysis cascades is
required for vascular remodeling during angiogenesis, inflammatory
processes, tumor s progression and metastasis. In particular, the
invasive processes that occur during tumor progression--local
invasion, intravasation, extravasation and metastasis
formation--involve extracellular matrix (ECM) degradation by
proteases.
[0069] Four classes of proteases, are known to correlate with
malignant phenotype: (i) cysteine proteases including cathepsin B
and L; (ii) aspartyl protease cathepsin D; (iii) serine proteases
including plasmin, tissue-type plasminogen activator (tPA) and
urokinase-type plasminogen activator (uPA), (iv) Matrix
metalloproteinases (MMPs) including collagenases, gelatinases A and
B (MMP2 and MMP9) and stromelysin (MMP3).
[0070] Cathepsins are a family of proteases that are found inside
cells in normal physiological conditions. Secretion of cathepsins
correlates with various pathological conditions, such as arthritis,
Alzheimer's disease and cancer progression (52).
[0071] The lysosomal cystein proteases cthepsin B and L have been
suggested to play a role in tumor cell invasion and spread, either
by directly cleaving extracellular matrix proteins or indirectly by
activating other proteases (53).
[0072] Cathepsin B was found to have elevated expression levels in
cancer cells. Furthermore, the intracellular distribution of the
protein differed between invasive and non-invasive cancer cells. In
invasive cells, cathepsin B was found in the plasma membrane,
whereas in non-invasive cells it was confined to the lysosomes
(56). In human tumor cells cathepsin B was secreted from the cells
(53) and was shown to degrade extracellular matrix components (54).
Cathepsin B and L have been shown to degrade type IV collagen,
laminin and fibronectin in vitro at both acid and neutral pH (54).
Both enzymes are able to activate the proenzyme form of the
urokinase-type plasminogen activator (pro-uPA), which is secreted
by tumor cells and can bind to receptors on the tumor cell surface
(55). In this cascade mechanism, the lysosomal cysteine proteases
may function as effective mediators of tumor associated
proteolysis.
[0073] MMPs are a family of zinc dependent endopeptidases. They are
secreted as inactive proenzymes and are activated by limited
proteolysis (57). During human pregnancy, cytotrophoblasts adopt
tumor-like properties: they attach the conceptus to the endometrium
by invading the uterus and they initiate blood flow to the placenta
by breaching maternal vessels. Matrix metalloproteinase MMP-9 (a
type IV collagenase/gelatinase) was shown to be upregulated during
cytotrophoblast differentiation along the invasive pathway.
Furthermore, it was shown that the activity of that protease
specified the ability of the cells to degrade ECM components in
vitro (58).
[0074] Large body of evidence suggests that the matrix
metalloproteinases MMP-2 and MMP-9 play an important role in tumor
invasion process (59, 58).
[0075] There is clearly a widely recognized need for, and it would
be highly advantageous to have, genetically modified cells
overexpressing recombinant heparanase or modified species thereof,
methods of overexpressing recombinant heparanase in cellular
systems and methods of purifying recombinant heparanase, so as to
enable, a search for heparanase inhibitors using a high throughput
assay and a combinatorial approach.
SUMMARY OF THE INVENTION
[0076] According to one aspect of the present invention there is
provided a recombinant cell comprising a polynucleotide sequence
encoding a polypeptide having heparanase catalytic activity, the
cell expressing recombinant heparanase.
[0077] According to a further aspect of the present invention,
there is provided a method of obtaining recombinant heparanase
comprising the steps of genetically modifying a cell with an
expression vector including a polynucleotide sequence encoding a
polypeptide having heparanase catalytic activity, the cell
expressing recombinant heparanase.
[0078] According to still further features in the described
preferred embodiments the polynucleotide sequence is as set forth
in SEQ ID NO:1 or a functional part thereof, the part encodes the
polypeptide having the heparanase catalytic activity.
[0079] According to still further features in the described
preferred embodiments the polypeptide includes an amino acid
sequence as set forth in SEQ ID NO:2 or a functional part thereof
having the heparanase catalytic activity. The functional part may
be the result of either genetic engineering natural processing by
the transduced cell.
[0080] According to still further features in the described
preferred embodiments the polynucleotide sequence is selected from
the group consisting of double stranded DNA, single stranded DNA
and RNA.
[0081] According to still further features in the described
preferred embodiments the cell is a bacterial cell.
[0082] According to still further features in the described
preferred embodiments the cell is E. coli.
[0083] According to still further features in the described
preferred embodiments the cell is an animal cell.
[0084] According to still further features in the described
preferred embodiments the animal cell is an insect cell.
[0085] According to still further features in the described
preferred embodiments the insect cell is selected from the group
consisting of High five and Sf21 cells.
[0086] According to still further features in the described
preferred embodiments the animal cell is a mammalian cell,
selected, for example, from the group consisting of a Chinese
hamster ovary cell line (CHO), baby hamster kidney cells (BHK21),
Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela
cells, Ehrlich's ascites cells, Sk-Hep1 cells, MDCK.sub.1 cells,
MDBK.sub.1 cells, Vero cells, Cos cells, CV-1 cells, NIH3T3 cells,
L929 cells and BLG cells (mouse melanoma).
[0087] According to still further features in the described
preferred embodiments the cell is a yeast cell.
[0088] According to still further features in the described
preferred embodiments the yeast cell is a methylotrophic yeast.
[0089] According to still further features in the described
preferred embodiments the yeast cell is selected from the group
consisting of Pichia pastoris, Hansenula polymorpha and
Saccharomyces cerevisiae.
[0090] According to still further features in the described
preferred embodiments the heparanase is human recombinant
heparanase.
[0091] According to still further features in the described
preferred embodiments the polynucleotide sequence is integrated in
the cell's genome rendering the cell a stably transduced.
[0092] According to still further features in the described
preferred embodiments the polynucleotide sequence is external to
the cell's genome, rendering the cell transiently transduced.
[0093] According to still further features in the described
preferred embodiments the polynucleotide sequence forms a part of a
viral genome infective to the cell, be it bacterial or animal
cell.
[0094] According to still further features in the described
preferred embodiments the polynucleotide sequence encodes, in
addition, a signal peptide for protein secretion.
[0095] According to still further features in the described
preferred embodiments the method further comprising the step of
subjecting the cell to a substance which induces secretion into the
growth medium of secretable proteins, thereby inducing secretion of
the recombinant heparanase into the growth medium.
[0096] According to still further features in the described
preferred embodiments the substance is selected from the group
consisting of thrombin, calcium ionophores, immune complexes,
antigens and mitogens.
[0097] According to still further features in the described
preferred embodiments the calcium ionophore is calcimycin (A23
187)
[0098] According to still further features in the described
preferred embodiments the substance is phorbol 12-myristate
13-acetate (PMA).
[0099] According to still further features in the described
preferred embodiments the method further comprising the step of
purifying the recombinant heparanase.
[0100] According to still further features in the described
preferred embodiments the purification is effected in part by an
ion exchange (e.g., Source-S) column.
[0101] According to still further features in the described
preferred embodiments the purification is from the cell.
[0102] According to still further features in the described
preferred embodiments the purification is from a growth medium in
which the cell is grown.
[0103] According to still further features in the described
preferred embodiments the cell is grown in a large biotechnological
scale of at least half a liter growth medium.
[0104] According to another aspect of the present invention
provided is a method of purifying a recombinant heparanase from
overexpressing cells or growth medium comprising the steps of
adsorbing the recombinant heparanase on an ion exchange (e.g.,
Source-S) column under low salt conditions, washing the column with
low salt solution thereby eluting other proteins, and eluting the
recombinant heparanase from the column by a salt gradient or a
higher salt concentration.
[0105] According to a further aspect of the present invention there
is provided a method of activating a heparanase enzyme comprising
the step of digesting the heparanase enzyme by a protease.
[0106] According to still further features in the described
preferred embodiments the protease is selected from the group
consisting of a cysteine protease, an aspartyl protease, a serine
protease and a meatlloproteinase.
[0107] According to still further features in the described
preferred embodiments the step of digesting the heparanase enzyme
by a protease is effected at a pH in which the protease is active,
preferably most active.
[0108] According to a further aspect of the present invention there
is provided a method of in vivo inhibition of proteolytic
processing of heparanase comprising the step of in vivo
administering a protease inhibitor.
[0109] According to still further features in the described
preferred embodiments the protease inhibitor is selected from the
group consisting of a cysteine protease inhibitor, an aspartyl
protease inhibitor, a serine protease inhibitor and a
meatlloproteinase inhibitor.
[0110] According to a further aspect of the present invention there
is provided a nucleic acid construct comprising a first nucleic
acid segment encoding for an upstream portion of heparanase, a
second, in frame, nucleic acid sequence encoding a recognition and
cleavage sequence of a protease and a third, in frame, nucleic acid
sequence encoding for a downstream portion of heparanase, wherein
the second nucleic acid sequence is in between the first nucleic
acid sequence and the third nucleic acid sequence.
[0111] According to still further features in the described
preferred embodiments the protease is selected having no
recognition and cleavage sequences in the upstream and the
downstream portions of heparanase.
[0112] According to still further features in the described
preferred embodiments the third nucleic acid sequence encodes for a
catalytically active heparanase when correctly folded.
[0113] According to a further aspect of the present invention there
is provided a precursor heparanase protein comprising an upstream
portion of heparanase, a mid portion of a recognition and cleavage
sequence of a protease and a downstream portion of heparanase,
wherein the protease is selected having no recognition and cleavage
sequences in the upstream and the downstream portions of
heparanase.
[0114] According to a further aspect of the present invention there
is provided a heparanase protein resulting by digesting the
precursor heparanase protein described herein.
[0115] According to a further aspect of the present invention there
is provided a method of obtaining a homogeneously processed, active
heparanase, the method comprising the steps of (a) expressing the
precursor heparanase protein in a cell which secretes the precursor
heparanase protein into the growth medium to obtain a conditioned
growth medium, the precursor heparanase protein including an
upstream portion of heparanase, a mid portion of a recognition and
cleavage sequence of a protease and a downstream portion of
heparanase, wherein the protease is selected having no recognition
and cleavage sequences in the upstream and the downstream portions
of heparanase; (b) treating the precursor heparanase protein with
the protease; and (c) purifying a proteolytic heparanase product
having heparanase catalytic activity.
[0116] According to a further aspect of the present invention there
is provided an antibody comprising an immunoglobulin elicited
against recombinant native heparanase.
[0117] According to a further aspect of the present invention there
is provided an affinity substrate comprising a solid matrix and an
immunoglobulin elicited against recombinant native heparanase being
immobilized thereto.
[0118] According to a further aspect of the present invention there
is provided a method of affinity purifying heparanase comprising
the steps of (a) loading a heparanase preparation on an affinity
substrate including a solid matrix and an immunoglobulin elicited
against recombinant native heparanase being immobilized thereto;
(b) washing the affinity substrate; and (c) eluting heparanase
molecules being adsorbed on the affinity substrate via the
immunoglobulin.
[0119] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
cells and methods for expressing recombinant heparanase, methods
for purifying the recombinant heparanase and modified heparanase
precursor species which can be processed to yield highly active
heparanase. Other features and advantages of the various
embodiments of the present invention are further addressed
hereinunder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0120] The invention herein described, by way of example only, with
reference to the accompanying drawings, wherein:
[0121] FIG. 1 demonstrates the expression of recombinant heparanase
in E. coli BL21(DE3)pLysS cells. Insoluble fractions of induced E.
coli cells containing expression constructs for heparanase were
analyzed on 10% SDS-PAGE. Following electrophoresis the gel was
stained with commassie blue. Lane 1-cells transformed with pRSET
(negative control), lanes 2 and 3-cells transformed with pRSEThpaSl
(two different colonies). Molecular size in kDa is shown to the
left (Prestained SDS-PAGE standards, Bio-Rad, CA).
[0122] FIG. 2 is a schematic presentation of the expression vector
pPIC3.5K-Sheparanase. Relative positions of some restriction
enzymes and genes are indicated. For the construction and utilities
of pPIC3.5K-Sheparanase, see Example 2 in the Examples section
below.
[0123] FIG. 3 is a schematic presentation of the expression vector
pPIC9K-PP2. Positions of some restriction enzymes and genes are
indicated. For the construction and utilities of
pPIC3.5K-Sheparanase, see Example 2 in the Examples section
below.
[0124] FIG. 4 demonstrates the secretion of human heparanase by
transformed Pichia pastoris yeast cells. Western blot analysis
using a rabbit anti-heparanase polyclonal antibody (disclosed in
U.S. patent application Ser. No. 09/071,618, which is incorporated
by reference as if fully set forth herein) was performed on culture
supernatants of different transformants (with and without selection
for G-418 resistance). Lane 1-pPIC3.5K-Sheparanase transformant,
lane 2-pPIC3.5K transformant (negative control), lanes 3-6,
transformants selected on 4 mg/ml of G-418. Molecular size is shown
on the right as was determined using prestained SDS-PAGE standards,
Bio-Rad, CA.
[0125] FIGS. 5a-e are schematic presentations of heparanase
expression vectors adapted to direct heparanase expression in
animal cells. hpa containing plasmids pShpa, pShpaCdhfr, pS1hpa,
pS2hpa and pChpa are of 5374 bp, 7090 bp, 6868 bp, 6892 bp and 6540
bp, respectively. SV40 prom--SV40 early promoter, CMV
prom--Citomegalovirus promoter, dhfr--mouse dihydrofolate reductase
gene, PPT--preprotrypsin signal peptide, hpa--heparanase cDNA
sequence, hpa' and hpa"--truncated hpa sequences.
[0126] FIGS. 6a-b show Western blot analysis of hpa transfected
cells. Cell extracts (40 .mu.g of CHO cells or 8 .mu.g of 293
cells) were separated on 4-20% gradient SDS-PAGE and transferred to
PVDF membranes. Detection of hpa gene products was performed with a
rabbit anti-heparanase polyclonal antibody (disclosed in U.S.
patent application Ser. No. 09/071,739) followed by ECL detection
(Amersham, UK). FIG. 6a--CHO stable cellular clones (lanes 1-3) and
transiently transfected 293 human cells (lane 4). FIG. 6b--Mock
transfected CHO cells (lane 3), CHO cells performing stable or
transient expression (lanes 1 and 2, respectively). Molecular size
in kDa is shown to the right, as was determined using prestained
SDS-PAGE standards, Bio-Rad, CA.
[0127] FIGS. 7a-b demonstrate recombinant heparanase secretion
induced by calcium ionophore and PMA. Cells of a stable CHO clone
(2TT1) were induced with either calcium ionophore (FIG. 7a) or PMA
(FIG. 7b).
[0128] Condition media were collected and 20 ml loaded on SDS
polyacrylamide gel followed by Western blot analysis with a rabbit
anti-heparanase polyclonal antibody (disclosed in U.S. patent
application Ser. No. 09/071,739) followed by ECL detection
(Amersham, UK). Molecular size in kDa is shown on the right, as was
determined using prestained SDS-PAGE standards, Bio-Rad, CA.
[0129] FIG. 7c demonstrates recombinant heparanase secretion by
human 293 cells. Conditioned media of human 293 cells transfected
with pS1hpa (lanes 3 and 4), pS2hpa (lanes 5 and 6) or control,
untransfected cells (lanes 1 and 2), were loaded on a denaturative
4-20% polyacrylamide gel (lanes 1, 3 and 5), or 5 fold concentrated
by 10 kDa ultrafiltration tube (Intersep U.K.) (lanes 4 and 6).
Heparanase was detected by Western blot analysis with a rabbit
anti-heparanase polyclonal antibody (disclosed in U.S. patent
application Ser. No. 09/071,618) followed by ECL detection
(Amersham, UK). Molecular size in kDa is shown on the left, as was
determined using prestained SDS-PAGE standards, Bio-Rad, CA.
[0130] FIG. 8a demonstrates heparanase activity as expressed by the
ability to degrade heparin. Following overnight incubation with 50
ml unconcentrated (lanes 3, 6), 20.times. concentrated (lanes 4 and
7) or 40.times. concentrated (lanes 5 and 8) conditioned media,
from untreated (lanes 3-5) versus treated (lanes 6-8, 2 hours of
incubation with 1 mg/ml calcium ionophore) stable clones, samples
were electrophoretically separated on 7.5% polyacrylamide gel.
Undegraded and degraded (by purified natural human heparanase)
controls are shown in lanes 1 and 2 respectively.
[0131] FIGS. 8b-c demonstrate recombinant heparanase activity
following secretion induced by calcium ionophore as determined by
the soluble .sup.35S-ECM degradation assay. 8b--the heparanase
activity in one ml untreated conditioned media (c60), compared to
one ml conditioned media treated with 100 ng/ml calcium ionophore
for 24 hours (p70) from stable CHO clones was determined by the
soluble .sup.35S-ECM degradation assay. 8c--the heparanase activity
in one ml untreated conditioned media (c45), compared to one ml
conditioned media treated with 1 mg/ml calcium s ionophore for two
hours (p52) from stable CHO clones was determined by the soluble
.sup.35S-ECM degradation assay. Degraded substrates shift to the
right.
[0132] FIGS. 8c-g show the relative heparanase activity of p70 and
p52 (see FIGS. 8b-c) by comparing the ability of diluted (.times.2,
.times.4 or .times.8) conditioned media to degrade.sup.35S-ECM.
[0133] FIG. 9 demonstrates glucose consumption record of heparanase
producing cells in a large scale, 0.5 liters, Spinner-Basket
bioreactor.
[0134] FIG. 10 demonstrates degradation of soluble sulfate labeled
HSPG substrate by lysates of High five cells infected with pFhpa2
virus. Lysates of High five cells that were infected with pFhpa2
virus (.circle-solid.) or control pF2 virus (.quadrature.) were
incubated (18 h, 37.degree. C.) with sulfate labeled ECM-derived
soluble HSPG (peak I). The incubation medium was then subjected to
gel filtration on Sepharose 6B. Low molecular weight HS degradation
fragments (peak II) were produced only during incubation with the
pFhpa2 infected cells, but there was no degradation of the HSPG
substrate (.diamond.) by lysates of pF2 infected cells.
[0135] FIGS. 11a-b demonstrate degradation of soluble sulfate
labeled HSPG substrate by the growth medium of pFhpa2 and pFhpa4
infected cells. Culture media of High five cells infected with
pFhpa2 (11a) or pFhpa4 (11b) viruses (.circle-solid.), or with
control viruses (.quadrature.) were incubated (18 h, 37.degree. C.)
with sulfate labeled ECM-derived soluble HSPG (peak I, .diamond.).
The incubation media were then subjected to gel filtration on
Sepharose 6B. Low molecular weight HS degradation fragments (peak
II) were produced only during incubation with the hpa gene
containing viruses. There was no degradation of the HSPG substrate
by the growth medium of cells infected o with control viruses.
[0136] FIG. 12 presents size fractionation of heparanase activity
expressed by pFhpa2 infected cells. Growth medium of pFhpa2
infected High five cells was applied onto a 50 kDa cut-off
membrane. Heparanase activity (conversion of the peak I substrate,
(.diamond.) into peak II HS degradation fragments) was found in the
high (>50 kDa) (.circle-solid.), but not low (<50 kDa)
(.smallcircle.) molecular weight compartment.
[0137] FIGS. 13a-b demonstrate the effect of heparin on heparanase
activity expressed by pFhpa2 and pFhpa4 infected High five cells.
Culture media of pFhpa2 (13a) and pFhpa4 (13b) infected High five
cells were incubated (18 h, 37.degree. C.) with sulfate labeled
ECM-derived soluble HSPG (peak I, .diamond.) in the absence
(.circle-solid.) or presence (.DELTA.) of 10 .mu.g/ml heparin.
Production of low molecular weight HS degradation fragments was
completely abolished in the presence of heparin, a potent
competitor for heparanase activity.
[0138] FIGS. 14a-b demonstrate degradation of sulfate labeled
intact ECM by virus infected High five and Sf2l cells. High five
(14a) and Sf21 (14b) cells were plated on sulfate labeled ECM and
infected (48 h, 28.degree. C.) with pFhpa4 (.circle-solid.) or
control pF1 (.quadrature.) viruses. Control non-infected Sf21 cells
(R) were plated on the labeled ECM as well. The pH of the cultured
medium was adjusted to 6.0-6.2 followed by 24 h incubation at
37.degree. C. Sulfate labeled material released into the incubation
medium was analyzed by gel filtration on Sepharose 6B. HS
degradation fragments were produced only by cells infected with the
hpa containing virus.
[0139] FIGS. 15a-b demonstrate degradation of sulfate labeled
intact ECM by virus infected cells. High five (15a) and Sf21 (15b)
cells were plated on sulfate labeled ECM and infected (48 h,
28.degree. C.) with pFhpa4 (.circle-solid.) or control pF1
(.quadrature.) viruses. Control non-infected Sf21 cells (R) were
plated on labeled ECM as well. The pH of the cultured medium was
adjusted to 6.0-6.2, followed by 48 h incubation at 28.degree. C.
Sulfate labeled degradation fragments released into the incubation
medium was analyzed by gel filtration on Sepharose 6B. HS
degradation fragments were produced only by cells infected with the
hpa containing virus.
[0140] FIGS. 16a-b demonstrate degradation of sulfate labeled
intact ECM by the growth medium of pFhpa4 infected cells. Culture
media of High five (16a) and Sf21 (16b) cells that were infected
with pFhpa4 (.circle-solid.) or control pF1 (.quadrature.) viruses
were incubated (48 h, 37.degree. C., pH 6.0) with intact sulfate
labeled ECM. The ECM was also incubated with the growth medium of
control non-infected Sf21 cells (R). Sulfate labeled material
released into the reaction mixture was subjected to gel filtration
analysis. Heparanase activity was detected only in the growth
medium of pFhpa4 infected cells.
[0141] FIGS. 17a-b demonstrate the effect of heparin on heparanase
activity in the growth medium of pFhpa4 infected cells. Sulfate
labeled ECM was incubated (24 h, 37.degree. C., pH 6.0) with growth
medium of pFhpa4 infected High five (17a) and Sf21 (17b) cells in
the absence (.circle-solid.) or presence (V) of 10 .mu.g/ml
heparin. Sulfate labeled material released into the incubation
medium was subjected to gel filtration on Sepharose 6B. Heparanase
activity (production of peak II HS degradation fragments) was
completely inhibited in the presence of heparin.
[0142] FIG. 18 demonstrate the purification of recombinant
heparanase by a Source-S column. Lanes 1-14, 40 ml of fractions
1-14 eluted from a Source-S column. Samples were analyzed on 8-16%
gradient SDS-PAGE. Gel was stained with commassie blue.
[0143] FIG. 19 demonstrate Western blot analysis of fractions 1-14
of FIG. 18. Fractions 1-14 eluted from a Source-S column were
analyzed following blotting onto nitrocellulose membrane with a
rabbit anti-heparanase polyclonal antibody (disclosed in U.S.
patent application Ser. No. 09/071,739) followed by ECL detection
(Amersham, UK).
[0144] FIG. 20 is a schematic presentation of plasmid pCdhffr that
contains the mouse dhfr gene under CMV promoter regulation. This
vector does not express heparanase and serves as negative
control.
[0145] FIG. 21a demonstrates the production of heparanase in pS1hpa
transfected BHK21 cells. Cell extracts (2.times.10.sup.5 BHK21
cells) were separated on 8-16% gradient SDS-PAGE and transferred to
PVDF membranes. Detection of hpa gene products was performed with a
mouse anti-heparanase monoclonal antibody No. HP-117 (disclosed in
U.S. patent application Ser. No. 09/071,739) followed by ECL
detection (Amersham, UK). Molecular size in kDa is shown to the
right, as was determined using prestained SDS-PAGE standards,
Bio-Rad, CA. Lane 1 pS1hpa transfected BHK21 cells. Lane 2 control,
pCdhfr transfected, BHK21 cells.
[0146] FIG. 21b demonstrates heparanase activity in human 293 cell
extract. Cells were collected and concentrated by centrifugation
(2750.times. g for 5 min). The pellets were passed through three
cycles of 5 minutes freezing in liquid nitrogen and thawing at
37.degree. C. Cell lysate was centrifuged for 15 minutes at
3000.times. g, and the supernatant was collected for analysis.
Increasing amounts of supernatant, between 0 and 5 .mu.g protein
per assay were assayed using the DMB activity assay described
herein (see also U.S. patent application Ser. No. 09/113,168).
[0147] FIG. 22a demonstrates recombinant heparanase constitutive
secretion by CHO cells transfected with pS1hpa (clone S1PPT-8).
Conditioned media (20 .mu.l) of untreated cells (lane 2), mock
treated cells (lane 3) and calcium ionophore treated cells (0.1
.mu.g/ml for 24 hours; lane 4) were electrophoresed next to a
cellular extract of 1.times.10.sup.5 cells from clone 2TT1 (CHO
cells transfected with pShpaCdhfr, lane 1). Samples were separated
on a 4-20% gradient SDS-PAGE, followed by Western blot analysis
with a rabbit anti-heparanase polyclonal antibody (disclosed in
U.S. patent application Ser. No. 09/071,739) and by ECL detection
(Amersham, UK). Molecular size in kDa is shown on the right, as was
determined using prestained SDS-PAGE standards, Bio-Rad, CA.
[0148] FIG. 22b demonstrates recombinant heparanase constitutive
secretion by CHO cells transfected with pShpaCdhfr (2TT1 clones).
Conditioned media (150 .mu.l, concentrated by 10 kDa
ultrafiltration tube) of 2TT1-2 clone (lane 2) and of clone 2TT1-8
(lane 3) were electrophoresed next to a cellular extract of
1.times.10.sup.5 cells from clone 2TT1 (lane 1). Samples were
separated on a 4-20% gradient SDS-PAGE, followed by Western blot
analysis with a rabbit anti-heparanase polyclonal antibody
(disclosed in U.S. patent application Ser. No. 09/071,739) and by
ECL detection (Amersham, UK). Molecular size in kDa is shown on the
right, as was determined using prestained SDS-PAGE standards,
Bio-Rad, CA.
[0149] FIG. 23a demonstrates purification of recombinant heparanase
from a mammalian cellular extract by ion exchange chromatography.
2TT1-8 CHO cells (1.times.10.sup.8) were extracted in 2.5 ml of 10
mM phosphate citrate buffer pH 5.4. The extract was centrifuged at
2750.times. g for 5 minutes and the supernatant was collected for
heparanase enzyme purification using a cation exchange
chromatography column. The chromatography column (mono-S HR 5/5,
Pharmacia Biotech) was equilibrated with 20 mM sodium phosphate
buffer, pH 6.8, and the mixture was loaded atop thereof. Proteins
were eluted from the column using a linear gradient of 0 to 1 M
sodium chloride in 20 mM sodium phosphate buffer, pH 6.8. The
gradient was carried out in 20 column volumes at a flow rate of one
ml per minute. The elution of proteins was monitored at 214 nm and
fractions of 1 ml each were collected, starting with the first
fraction (1) which was eluted after 13 minutes and which is
identified by the arrowhead mark.
[0150] FIG. 23b demonstrates the presence of immunologically active
recombinant heparanase in the mammalian cellular extract. An
aliquot from each fraction that was collected was analyzed for the
presence of the heparanase enzyme by Western blot analysis. 20
.mu.l from each fraction, numbered 1-26, were separated on a 4-20%
SDS-PAGE. The proteins were transferred from the gel to a PVDF
membrane and were detected with a monoclonal antibody No. HP-117
(disclosed in U.S. patent application Ser. No. 09/071,739) followed
by ECL detection (Amersham, UK). Molecular size in kDa is shown to
the right, as was determined using SDS-PAGE standards (M). St--a
purified recombinant heparanase enzyme from CHO cells.
[0151] FIG. 23c demonstrates the presence of catalytically active
recombinant heparanase in mammalian cellular extract fractions. An
aliquot (30 .mu.l) from each fraction that was collected was
analyzed for heparanase activity by the DMB assay. Load--extract
prior to purification. 5-7 and 16-26 correspond to fraction
Nos.
[0152] FIG. 23d demonstrates a heparanase dose response. Increasing
amounts from fraction No. 20, which exhibited the highest activity
using the DMB assay (FIG. 23c), were analyzed for heparanase
activity using the tetrazolium assay, as disclosed in U.S. patent
application Ser. No. 09/113,168.
[0153] FIG. 24a demonstrates the purification of heparanase from a
mammalian cellular extract by an affinity column. A cellular
extract from CHO 2TT1-8 cells was loaded on an affinity column
containing antibodies elicited against native (non-denatured)
recombinant heparanase. Western blot analysis of different
fractions (1-6) using a monoclonal antibody No. HP-117 (disclosed
in U.S. patent application Ser. No. 09/071,739) followed by ECL
detection (Amersham, UK) is shown. Molecular size in kDa is shown
to the left, as was determined using SDS-PAGE standards (M).
A--recombinant heparanase enzyme purified from CHO 2TT1-8 cells on
mono-S column; B--extract of 2TT1-8 cells; C--unbound, flow through
proteins; and D--wash fraction proteins.
[0154] FIG. 24b demonstrates the purification of heparanase from a
mammalian cellular extract by an affinity column. A cellular
extract from CHO 2TT1-8 cells was loaded on an affinity column
containing antibodies elicited against native (non-denatured)
recombinant heparanase. Heparanase activity in affinity column
fraction Nos. 1-9 was determined using the DMB assay. Load--extract
prior to purification; C--unbound, flow through proteins; and
D--wash fraction proteins.
[0155] FIGS. 25a-b demonstrates proteolytic processing of
heparanase from insect cells conditioned medium by protease
impurities. FIG. 25a shows a Western blot analysis of heparanase,
following processing of the enzyme expressed in insect cells.
Heparanase expressed in insect cells, partially purified on a
Source-S column, was incubated for one week at 4.degree. C. in
either, 20 mM phosphate citrate buffer pH 7, containing 5% PEG 300
(lane A ), 20 mM phosphate citrate buffer pH 4, containing 5% PEG
300 and 1.times. protease inhibitors cocktail (Boehringer Mannheim,
Cat. No.1836170, lane B), or 20 mM phosphate citrate buffer pH 4,
containing 5% PEG 300 (lane C). M--Molecular weight markers (NEB
Cat. No. 7708S). FIG. 25b shows the results of DMB heparanase
activity assays for the proteins.
[0156] FIGS. 25c-d demonstrate the effect of a panel of protease
inhibitors on proteolytic processing and activation of heparanase
expressed in insect cells. Heparanase expressed in insect cells,
partially purified on a Source-S column, was incubated for one week
at 4.degree. C. in 20 mM phosphate citrate buffer, pH 4, containing
5% PEG 300 and one of the different protease inhibitors:
A--antipain; B--bestatin; C--chymostatin; D--E-64; E--leupeptin;
F--pepstatin; G--phosphoramidon; H--EDTA; I--aprotinin. The treated
samples were either subjected to western blot analysis (FIG. 25c)
or to heparanase DMB activity assay (FIG. 25d). J--positive
control, incubated in the absence of a protease inhibitor at pH 4;
K--negative control, heparanase incubated with the same buffer at
pH 7. M--Molecular weight marker (NEB Cat. No. 7708S).
[0157] FIG. 26a demonstrates proteolytic processing of heparanase
secreted from insect cells by trypsin. 10 .mu.g of heparanase,
expressed in insect cells, and partially purified on a Source-S
column, was incubated with increasing concentrations of trypsin (0,
1.5, 5, 10, 15 units/test, Cat. No. T-8642, Sigma USA) for 10
minutes at 25.degree. C. Following incubation, reaction tubes were
placed on ice and 1.7 .mu.g/ml aprotinin (trypsin inhibitor) was
added. Activity was determined using the DMB assay.
[0158] FIG. 26b demonstrates a Western blot analysis of heparanase
following trypsin treatment. 10 .mu.g of heparanase, expressed in
insect cells, and partially purified on a Source-S column, was
incubated without (lane 1) or with 150 or 500 units of trypsin
(lanes 2 and 3, respectively). A processed heparanase sample
treated as described in FIG. 25a-b, lanes J (lane 4), and
heparanase from a CHO 2TT1 cell extract (lane 5) served as
controls.
[0159] FIG. 27 proteolytic processing of heparanase secreted from
CHO cells by trypsin. Conditioned medium of CHO cells transfected
with pS1hpa (clone S1PPT-8) that secrete heparanase in a
constitutive manner was subjected to proteolysis by trypsin.
Unpurified CHO conditioned medium containing heparanase (0.5 .mu.g
heparanase per reaction) in 20 mM phosphate buffer, pH 6.8, was
incubated with 0, 1.5, 15 or 150 units of trypsin for 10 minutes,
at 37.degree. C. Reactions were stopped by transferring the
reaction tubes into ice and adding 2 .mu.g/ml aprotinin. Tryptic
digest products were assayed for heparanase activity using the DMB
assay.
[0160] FIG. 28a-b demonstrates proteolytic processing of p70-bac
heparanase by cathepsin L. Partially purified heparanase from
insect cells (10 .mu.g) was subjected to proteolysis by 1.6 mU
cathepsin L (Cat. No. 219412, Calbiochem) for 3 hours, at
30.degree. C., in 20 mM citrate-phosphate buffer, pH 5.4.
Heparanase catalytic activity and immunoreactivity before (1) and
after (2) processing with cathepsin L as were determined using the
DMB heparanase activity assay and Western blot analysis with
monoclonal antibody No. HP-117 (disclosed in U.S. patent
application Ser. No. 09/071,739) followed by ECL detection
(Amersham, UK), FIGS. 28a-b, respectively.
[0161] FIG. 29a demonstrates a hydropathy plot of SEQ ID NO:2
predicted for heparanase as calculated by the Kyte-Doolittle method
for calculating hydrophilicity, using the Wisconsin University GCG
DNA analysis software. I and II point at peaks of most hydrophilic
regions of the enzyme.
[0162] FIG. 29b is a schematic depiction of modified heparanase
species (pre-p56' and pre-p52') that contain a unique protease
recognition and cleavage sequence of factor
Xa--Ile-Glu-Gly-Arg.dwnarw.--or of
enterokinase--Asp-Asp-Asp-Asp-Lys.dwnarw. (shaded regions, located
between amino acids 119 and 120 or 157 and 158 of the heparanase
enzyme depicted in SEQ ID NO:2, which acids are located within
peaks I and II, respectively, of FIG. 29a) which enable proteolytic
processing by the respective proteases to obtain homogeneously
processed and highly active heparanase species (p56' and p52',
respectively).
[0163] FIG. 29c is a schematic depiction of the steps in
constructing nucleic acid constructs harboring a unique protease
recognition and cleavage sequence of factor
Xa--Ile-Glu-Gly-Arg.dwnarw.--or of
enterokinase--Asp-Asp-Asp-Asp-Lys.dwnarw..
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0164] The present invention is of genetically modified cells
overexpressing recombinant heparanase and of methods for
overexpressing recombinant heparanase in cellular systems, which
can be used to obtain purified recombinant heparanase in large
quantities. Specifically, the present invention can be used to
provide a scheme for biotechnological large scale recombinant
heparanase production. The invention further relates to the
activation of heparanase precursors by proteolysis and further to
methods of in vivo inhibition of heparanase activity.
[0165] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0166] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description or illustrated in the drawings. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0167] In one aspect, the present invention provides a genetically
modified cell transduced with a polynucleotide sequence encoding a
polypeptide having heparanase catalytic activity, designed to
direct expression of recombinant heparanase by the cell.
[0168] In another aspect, the present invention provides a method
of obtaining recombinant heparanase by genetically modifying a cell
with an expression vector including a polynucleotide sequence
encoding a polypeptide having heparanase catalytic activity,
designed to direct expression of recombinant heparanase by the
cell.
[0169] As used herein in the specification and in the claims
section below, the phrase "genetically modified cell" refers to a
cell that includes a recombinant gene. As further detailed below
the cell may be a eukaryotic or prokaryotic cell.
[0170] As used herein in the specification and in the claims
section below, the term "transduced" refers to the result of a
process of inserting nucleic acids into cells. The insertion may,
for example, be effected by transformation, viral infection,
injection, transfection, gene bombardment, electroporation or any
other means effective in introducing nucleic acids io into cells.
Following transduction the nucleic acid is either integrated in all
or part, to the cell's genome (DNA), or remains external to the
cell's genome, thereby providing stably transduced or transiently
transduced cells.
[0171] As used herein in the specification and in the claims
section below, the phrase "polynucleotide sequence" also means a
nucleic acid sequence, typically a DNA sequence.
[0172] As used herein in the specification and in the claims
section below, the term "polypeptide" also means a protein.
[0173] As used herein in the specification and in the claims
section below, the phrase "heparanase catalytic activity" refers to
an animal endoglycosidase hydrolyzing activity which is specific
for heparin or heparan sulfate proteoglycan substrates, as opposed
to the activity of bacterial enzymes (heparinase I, II and III)
which degrade heparin or heparan sulfate by means of
.beta.-elimination.
[0174] As used herein in the specification and in the claims
section below, the term "expression" refers to the processes
executed by cells while producing and/or secreting proteins,
including where applicable, but not limited to, for example,
transcription, translation, folding and post translational
modification and processing.
[0175] As used herein in the specification and in the claims
section below, the terms "vector" and "construct" are
interchangeably used herein and refer to any vehicle suitable for
genetically modifying cells, including, but not limited to, viruses
(e.g., bacoluvirus), phages, plasmids, phagemids, bacmids, cosmids,
artificial chromosomes and the like.
[0176] As used herein in the specification and in the claims
section below, the phrase "a polynucleotide sequence encoding a
polypeptide having heparanase catalytic activity" refers to the
potential of the polypeptide to have heparanase catalytic activity
when correctly folded. Thus, this phrase refers to any
catalytically active or inactive conformant of a polypeptide which
may acquire at least one active conformation having heparanase
catalytic activity.
[0177] According to a preferred embodiment of the present
invention, the polynucleotide sequence is as set forth in SEQ ID
NO:1 or a functional part thereof. The functional part encodes a
polypeptide having heparanase catalytic activity. However, the
scope of the present invention is not limited to SEQ ID NO:1 or a
functional part thereof, as natural and man made innocuous
variations thereof (e.g., mutations, such as point mutations) may
also encode a protein having heparanase catalytic activity.
Furthermore, it is shown hereinunder that a 52 kDa (formerly
referred to as 45-50 kDa) protein, naturally processed from a 70
kDa (formerly referred to as 60 or 60-70 kDa) protein encoded by
SEQ ID NO:1, has heparanase catalytic activity. The polynucleotide
sequence may be a cDNA, a genomic DNA and a composite DNA
(including at least one intron derived from heparanase or any other
gene) as further detailed in U.S. patent application Ser. No.
09/071,618, entitled POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING
HEPARANASE ACTIVITY AND EXPRESSION OF SAME IN GENETICALLY MODIFIED
CELLS, which is incorporated herein by reference. Similarly it can
be derived from any animal including mammalians and avians because,
as shown in U.S. patent application Ser. No. 09/258,892, heparanase
sequences derived from species other than human beings are readily
hybridizeable with the human sequence, allowing for isolation of
such sequences by methods known in the art.
[0178] The functional part may be either man induced by genetic
engineering or post translation artificial processing (e.g., by a
protease) or naturally processed, depending on the cellular system
employed.
[0179] According to another preferred embodiment of the present
invention, the polypeptide includes an amino acid sequence as set
forth in SEQ ID NO:2 or a functional part thereof having heparanase
catalytic activity. However, the scope of the present invention is
not limited to SEQ ID NO:2 or a functional part thereof, as natural
and man made innocuous variations thereof (e.g., mutations, such
single amino acid substitution) may also have heparanase catalytic
activity. Polypeptides corresponding to species other than human
and having heparanase catalytic activity are also within the scope
of the present invention.
[0180] As used herein in the specification and in the claims
section below, the term "functional part thereof" refers to a part
of a nucleic acid sequence which encodes a polypeptide having
heparanase catalytic activity or a part of a polypeptide sequence
having heparanase catalytic activity.
[0181] In this context, it is important to remember that in many
cases truncated or naturally processed polypeptides exhibit a
catalytic activity similar to that of the natural polypeptide of
the preprocessed polypeptide, respectively. Apparently, in many
cases, not all of the amino acids of a protein are essential for
its catalytic function, some may be responsible for other features,
such as secretion, stability, interaction with other
macromolecules, etc., whereas other may be replaced without
affecting activity to a great extent. In many cases the processed
protein exerts higher catalytic activity as compared with its
unprocessed counterpart.
[0182] According to yet another preferred embodiment of the present
invention, the polynucleotide sequence is selected from the group
consisting of double stranded DNA, single stranded DNA and RNA.
[0183] According to still another preferred embodiment of the
present invention, the cell is a bacterial cell, preferably E.
coli.
[0184] According to a preferred embodiment of the present
invention, the cell is an animal cell.
[0185] The animal cell may be a mammalian cell, such as, but not
limited to, Chinese hamster ovary cell line (CHO), baby hamster
kidney cells (BHK21), Namalwa cells, Dauidi cells, Raji cells,
Human 293 cells, Hela cells, Ehrlich's ascites cells, Sk-Hep1
cells, MDCK.sub.1 cells, MDBK.sub.1 cells, Vero cells, Cos cells,
CV-1 cells, NIH3T3 cells, L929 cells or BLG cells (mouse
melanoma).
[0186] Alternatively, the animal cell may be a mammalian cell, such
as, but not limited to, High five or Sf21.
[0187] According to another preferred embodiment of the present
invention, the cell is a yeast cell, preferably a methylotrophic
yeast, such as, but not limited to, Pichia pastoris and Hansenula
polymorpha. Another preferred yeast is Saccharomyces
cerevisiae.
[0188] The specified bacterial, yeast and animal cells are of
specific advantage and interest since they are widely used in large
scale biotechnological production of proteins and therefore
knowledge has accumulated with respect to their large scale
propagation, maintenance and with respect to recombinant protein
purification therefrom.
[0189] According to another preferred embodiment of the present
invention, the recombinant heparanase is human recombinant
heparanase.
[0190] According to another preferred embodiment of the present
invention, the polynucleotide sequence encodes, in addition, a
signal peptide for protein secretion. The signal peptide may be the
natural signal peptide of heparanase or any other suitable signal
peptide, one non-limiting example is given under the Examples
section hereinunder. The signal peptide sequence is fused
downstream of and in frame with the heparanase sequence.
[0191] According to yet another preferred embodiment of the present
invention, the method is further effected by purifying the
recombinant heparanase. As further detailed hereinunder efficient
purification (e.g., 90 % purified) of recombinant heparanase may be
effected by a single step ion exchange (e.g., Source-S) column.
[0192] The purification may be from the cells themselves. To this
end the cells are collected, e.g., by centrifugation, homogenated
and the recombinant heparanase is purified from the homogenate. If
the recombinant heparanase is secreted by the cells to the growth
medium, then purification is preferably from the growth medium
itself.
[0193] According to yet another preferred embodiment of the present
invention, the method further includes a step of subjecting the
cell to a substance which induces secretion into the growth medium
of secretable proteins, thereby inducing secretion of the
recombinant heparanase into the growth medium. Preferably, the
substance is selected from the group consisting of thrombin,
calcium ionophores, immune complexes, antigens and mitogens, all
are known to induce secretion of native heparanase from expressing
cells. As shown in the Examples section below, the calcium
ionophore calcimycin (A23187) and phorbol 12-myristate 13-acetate,
are effective in inducing secretion of recombinant heparanase from
transduced cells into their media.
[0194] According to yet another preferred embodiment of the present
invention, the cell is grown to a large biotechnological scale of
at least half a liter, preferably at least 5, 7 or 35 liters of
growth medium, in a bioreactor, such as but not limited to,
Spinner-Basket bioreactor.
[0195] Further according to the present invention there is provided
a method of purifying a recombinant heparanase from overexpressing
cells or growth medium in which they grow by adsorbing the
recombinant heparanase on a Source-S column under low salt
conditions (e.g., about 50 mM NaCl), washing said column with low
salt solution thereby eluting other proteins, and eluting the
recombinant heparanase from the column by a salt gradient (e.g., 50
mM to 1 M NaCl) or a higher concentration of salt (e.g., about 0.4
M).
[0196] According to a further aspect of the present invention there
is provided an antibody comprising an immunoglobulin elicited
against recombinant native heparanase. The immunoglobulin therefore
recognizes and binds native (i.e., non denatured) natural or
recombinant heparanase.
[0197] As used herein in the specification and in the claims
section below, the term "antibody" include serum immunoglobulins,
polyclonal antibodies or fragments thereof or monoclonal antibodies
or fragments thereof. The antibodies are preferably elicited
against a surface determinant of the particulate. Monoclonal
antibodies or purified fragments of the monoclonal i o antibodies
having at least a portion of an antigen binding region, including
such as Fv, F(ab1)2, Fab fragments (63), single chain antibodies
(U.S. Pat. No. 4,946,778), chimeric or humanized antibodies (64-65)
and complementarily determining regions (CDR) may be prepared by
conventional procedure. Purification of the serum immunoglobulins
antibodies or fragments can be accomplished by a variety of methods
known to those of skill including, but not limited to,
precipitation by ammonium sulfate or sodium sulfate followed by
dialysis against saline, ion exchange chromatography, affinity or
immunoaffinity chromatography as well as gel filtration, zone
electrophoresis, etc. (see 66).
[0198] According to a further aspect of the present invention there
is provided an affinity substrate comprising a solid matrix and an
immunoglobulin elicited against recombinant native heparanase being
immobilized thereto. Methods of immobilizing immunoglobulins to
solid matrices, such as cellulose, polymeric beads including
magnetic beads, are well known in the art. One such method is
described in the Examples section that follows. The solid support
according to the present invention can be packed into an affinity
column.
[0199] According to a further aspect of the present invention there
is provided a method of affinity purifying heparanase. The method
is effected by (a) loading a heparanase preparation on an affinity
column including a solid matrix and an immunoglobulin elicited
against recombinant native heparanase being immobilized thereto;
(b) washing the affinity column, e.g., using low, say 0-500 mM,
salt solution; and (c) eluting heparanase molecules being adsorbed
on the affinity column via the immunoglobulin, e.g., using a high,
say 0.5-1.5 M, salt solution.
[0200] According to a further aspect of the present invention there
is provided a method of activating a heparanase enzyme comprising
the step of digesting the heparanase enzyme by a protease. The
heparanase enzyme according to this aspect of the present invention
can be natural or recombinant, purified, partially purified or
non-purified. The protease can be of any type, including, but not
limited to, a cysteine protease, an aspartyl protease, a serine
protease and a meatlloproteinase. Examples of specific proteases
associated with the above listed protease families are provided in
the Background section. The use of other proteases for which
heparanase includes a recognition and cleavage sequence is
envisaged. According to a preferred embodiment digesting the
heparanase enzyme by the protease is effected at a pH in which the
protease is active, preferably most active. It is known that some
proteases are most active in acidic pH values whereas other
proteases are most active in basic pH values. The pH value at which
a specific protease is most active can be readily determined by one
ordinarily skilled in the art.
[0201] According to a further aspect of the present invention there
is provided a method of in vivo inhibition of proteolytic
processing of heparanase. The method according to this aspect of
the present invention is effected by in vivo administering a
protease inhibitor. The protease inhibitor can be, for example, a
cysteine protease inhibitor, an aspartyl protease inhibitor, a
serine protease inhibitor or a meatlloproteinase inhibitor.
Examples of suitable inhibitors are provided in the Examples
section that follows. Some protease inhibitors are used
pharmaceutically for treatment of various conditions. In vivo
inhibition of proteolytic processing of heparanase by a protease
inhibitor can be used for treatment of cancer, metastatic cancers
in particular, in which heparanase activity is involved, because,
as further exemplified in the Examples section that follows, the
preheparanase (non-processed, p70 heparanase) is characterized by
lower activity as compared to its processed counterpart (p52
heparanase).
[0202] According to a further aspect of the present invention there
is provided a nucleic acid construct comprising a first nucleic
acid segment encoding for an upstream (N terminal) portion of
heparanase, a second, in frame, nucleic acid sequence encoding a
recognition and cleavage sequence of a protease and a third, in
frame, nucleic acid sequence encoding for a downstream portion (C
terminal) of heparanase, wherein the second nucleic acid sequence
is in between the first nucleic acid sequence and the third nucleic
acid sequence. Examples of such constructs are provided in the
Examples section that follows. Preferably, the protease is selected
having no recognition and cleavage sequences in the upstream and
the downstream portions of heparanase, such that when expressed the
modified heparanase is digested only at the introduced recognition
and cleavage sequence of the protease. Preferably, the third
nucleic acid sequence encodes for a catalytically active heparanase
when correctly folded. However, embodiments wherein the second
nucleic acid sequence is so positioned such that when expressed the
modified heparanase protein is digestible into portions lacking
catalytic activity are also envisaged. Such embodiments can be used
to provide a heparanase species having a shorter half life, in, for
example, physiological conditions, as compared with the
non-modified enzyme. One ordinarily skilled in the art would know
how to select locations for introduction of the recognition and
cleavage sequence such that the sequence will not hamper the
catalytic activity of the enzyme prior to cleavage thereof by the
protease.
[0203] The above construct, when introduced into a cell expression
system can be used to provide a precursor heparanase protein
comprising an upstream portion of heparanase, a mid portion of a
recognition and cleavage sequence of a protease and a downstream
portion of heparanase, wherein the protease is selected having no
recognition and cleavage sequences in the upstream and the
downstream portions of heparanase. The recognition and cleavage
sequence of the protease is composed either entirely from amino
acids which are not present in natural heparanase, or from amino
acids which are not present in natural heparanase in part, and
further from adjacent amino acids which are present in natural
heparanase. Further according to the present invention there is
provided a heparanase protein resulting by digesting the precursor
heparanase protein described herein.
[0204] According to a further aspect of the present invention there
is provided a method of obtaining a homogeneously processed, active
heparanase. The method according to this aspect of the present
invention is effected by (a) expressing the precursor heparanase
protein in a cell which secretes the precursor heparanase protein
into the growth medium to obtain a conditioned growth medium, the
precursor heparanase protein including an upstream portion of
heparanase, a mid portion of a recognition and cleavage sequence of
a protease and a downstream portion of heparanase, wherein the
protease is selected having no recognition and cleavage sequences
in the upstream and the downstream portions of heparanase; (b)
treating the precursor heparanase protein with the protease; and
(c) purifying a proteolytic heparanase product having heparanase
catalytic activity.
[0205] It will be appreciated that the various heparanase species
described herein, either activated and/or precursors can be used to
produce pharmaceutical compositions, including, in addition to
heparanase, a pharmaceutically acceptable carrier. Affinity
purified and protease treated, modified, recombinant heparanase is
of particular interest for pharmaceutical applications due to its
homogeneity and purity.
[0206] The present invention has advantages because it provides
means for expressing, purifying and activating recombinant/natural
heparanase. Such heparanase can be used in pharmaceutical
compositions (see, for example, U.S. patent application Ser. No.
09/046,465, in which heparanase is used in the treatment of CF), or
as a source of enzyme for high throughput heparanase activity
assay, which can be used for efficient screening of specific
heparanase inhibitors (see, for example, U.S. patent application
Ser. No. 09/113,168). By identifying the heparanase proteolytic
activation process, novel indirect methods of in vivo heparanase
inhibition by administration of protease inhibitors were conceived
and tested in vitro. By identifying the heparanase proteolytic
activation process, novel constructs encoding novel heparanase
species has been constructed and can be used to direct the
expression of a heparanase which is homogeneously processed and
activated or alternatively neutralized by a dedicated protease.
EXAMPLES
[0207] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non-limiting fashion.
[0208] Generally, the nomenclature used herein and the laboratory
procedures in recombinant DNA technology described below are those
well known and commonly employed in the art. Standard techniques
are used for cloning, DNA and RNA isolation, amplification and
purification. Generally enzymatic reactions involving DNA ligase,
DNA polymerase, restriction endonucleases and the like are
performed according to the manufacturers' specifications.
Similarly, standard techniques are used for the proteolysis of
heparanase by various proteases. These techniques and various other
techniques used while reducing the present invention to practice
are generally performed according to Sambrook et al., Molecular
Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1989), which is incorporated herein be
reference. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Expression of Recombinant Human Heparanase in Bacteria
Experimental Methods
[0209] Construction of Expression Vector:
[0210] A 1.6 kb fragment of hpa cDNA (SEQ ID NO:1) was amplified
from pfasthpa (hpa cDNA cloned in pFastBac, see U.S. patent
application Ser. No. 08/922,170) by PCR using specific sense
primer: Hpu-550Nde -5'-CGCATATGCAGGACGTCGTGG ACCTG-3' (SEQ ID NO:4)
and a vector specific antisense primer: 3'pFast
5'-TATGATCCTCTAGTACTTCTCGAC-3' (SEQ ID NO:5). PCR conditions were:
denaturation -94.degree. C., 40 seconds, first cycle 3 minutes;
annealing -58.degree. C., 60 seconds; and elongation -72.degree.
C., 2.5 minutes, total of 5 cycles, and then denaturation
-94.degree. C., 40 seconds; annealing -68.degree. C., 60 seconds;
and elongation -72.degree. C., 2.5 minutes, for additional 25
cycles.
[0211] The Hpu-550Nde primer introduced an NdeI site and an in
frame ATG codon preceding nucleotide 168 of hpa. The PCR product
was digested by NdeI and BamHI and its sequence was confirmed with
vector specific and gene specific primers, using an automated DNA
sequencer (Applied Biosystems, model 373A).
[0212] A 1.3 kb BamHI-KpnI fragment was cut out of pFasthpa. The
two fragments were ligated with the pRSET bacterial expression
vector (Invitrogen, CA.).
[0213] The resulting plasmid, designated pRSEThpaS1, encoded an
open reading frame of 508 amino acids (36-543, SEQ ID NO:2) of the
heparanase protein, lacking the N-terminal 35 amino acids which are
predicted to be a signal peptide.
[0214] Transformation:
[0215] Transformation of E. coli BL21(DE3)pLysS cells (Stratagene)
was performed following Stratagene's protocol. Briefly, using
.beta.-mercaptoethanol in the transformation buffer cells were
transformed by five seconds of heat shock at 42.degree. C.
[0216] Expression of Recombinant Heparanase:
[0217] E. coli BL21(DE3)pLysS cells transformed with the
recombinant plasmid were grown at 37.degree. C. overnight in Luria
broth (LB) medium containing 100 .mu.g/ml ampicillin and 34
.mu.g/ml chloramphenicol. Cells were diluted {fraction (1/10)} in
the same medium, and the cultures were grown to an OD600 of
approximately 0.5. Isopropyl-thiogalactoside (IPTG) (Promega) was
added to a final concentration of 1 mM and the culture was
incubated at 37.degree. C. for 3 hours. Cells from IPTG induced
cultures were cooled on ice and sedimented by centrifugation at
4,000.times. g for 20 minutes at 4.degree. C., and resuspended in
0.5 ml of cold phosphate-buffered saline (PBS). Cells were lysed by
sonication, and cell debris were sedimented by centrifugation at
10,000.times. g for 20 minutes. The resulting pellet was analyzed
for proteins by 10% SDS-PAGE, essentially as described in Harlow,
E. and Lane, D. Eds. in Antibodies, a laboratory manual. CSH
Laboratory press. New-York.
Experimental Results
[0218] The expression of recombinant heparanase in E. coli
BL21(DE3)pLysS cells containing the pRSEThpaS1 was analyzed by
SDS-PAGE followed by commassie blue staining for proteins.
Bacterial cells were fractionated and a protein of approximately 70
kDa, which is the expected size of the recombinant heparanase, was
observed in the insoluble fraction (FIG. 1, lanes 2-3). That band
did not appear when negative control cells transformed with pRSET
were employed (FIG. 1, lane 1).
[0219] The identification of the recombinant heparanase expressed
in E. coli was confirmed by a Western blot (data not shown) using a
rabbit anti-heparanase polyclonal antibody (disclosed in U.S.
patent application Ser. No. 09/071,739), followed by ECL detection
(Amersham, UK).
[0220] As compared to known quantities of co-size separated and
stained BSA, the estimated yield of the heparanase recombinant
protein under the conditions described was about 0.2 mg/ml of
culture (not shown). The protein was found in the insoluble
fraction (inclusion bodies) and had no enzymatic activity, as was
determined by the soluble .sup.35S-ECM degradation assay (not
shown), however, the recombinant heparanase protein expressed in E.
coli could provide a source for large quantities of heparanase.
[0221] It will be appreciated that solubillization and refolding of
recombinant proteins expressed in E. coli are well known in the art
(see, for example, for insulin, 70; others are reviewed in 71) and
these procedures should be applied in order to obtain a functional
protein having heparanase activity.
[0222] The expression of the recombinant heparanase in bacterial
cells is thus demonstrated in this Example. It will be further
appreciated that changes in protein length and/or amino acid
composition might affect the efficiency of expression, correct
folding and the potential yield of functional enzyme.
Example 2
Expression of Recombinant Human Heparanase in Yeast
Experimental Methods
[0223] Construction of Expression Vectors for Expression in
Yeast:
[0224] Two expression vectors were constructed for the expression
of hpa in Pichia pastoris. The first vector, designated
pPIC3.5K-Sheparanase (FIG. 2) contains nucleotides 63-1694 of the
hpa sequence (SEQ ID NO:1) cloned into the expression vector
pPIC3.5K (Invitrogen, CA) using a multistep procedure as
follows.
[0225] A pair of primers: HPU-6641-5'-AGGAATTCACCATGCTGCT
GCGCTCGAAGCCTGCG-3' (SEQ ID NO:6) and HPL-209
5'-GAGTAGCAATTGCTCCTGGTAG-3- ' (SEQ ID NO:7) were used in PCR
amplification to introduce an EcoRI site just upstream to the
predicted methionine. PCR conditions were: denaturation -94.degree.
C., 40 seconds; annealing -50.degree. C., 80 seconds; and
elongation -72.degree. C., 180 seconds, total of 30 cycles.
[0226] The resulting PCR product was digested with EcoRi and BamHI
and cloned into the EcoRI-BamHI sites of the vector phpa2
(described in U.S. patent application Ser. No. 08/922,170). The hpa
coding region was then removed as an EcoRI-NotI fragment and cloned
into the EcoRi-NotI sites of the expression vector pPIC3.5K to
generate the vector pPIC3.5K-Sheparanase (FIG. 2).
[0227] The second vector, designated pPIC9K-PP2 (FIG. 3), includes
the hpa coding region except for the predicted signal sequence
(N-terminal 36 amino acids, see SEQ ID NO:2). The hpa was cloned
in-frame to the .alpha.-factor prepro secretion signal in the
Pichia pastoris expression vector pPIC9K (Invitrogen, CA). A pair
of primers: HPU-559S, 5'-GTCTCGA GAAAAGACAGGACGTCGTGGACCTGGAC-3'
(SEQ ID NO:8) and HPL-209 (SEQ ID NO:7, described above) were used
in PCR amplification under the conditions described above.
[0228] The resulting PCR product was digested with XhoI and BamHIl
and inserted into the XhoI-BamHI sites of the vector phpa2 (U.S.
patent application Ser. No. 08/922,170).
[0229] Thereafter, the XhoI-NotI fragment containing the hpa
sequence was removed and cloned into an intermediate vector
harboring the SacI-NotI sites of pPIC9K.
[0230] The hpa was removed from the later vector as a SacI-NotI
fragment and cloned into the SacI-NotI sites of pPIC9K, thus
creating the vector pPIC9K-PP2 (FIG. 3).
[0231] Transformation and Screening:
[0232] Pichia pastoris strain SMD1 168 (his3, pep4) (Invitrogen,
CA) was used as a host for transformation. Transformation and
selection were carried out as described in the Pichia expression
Kit protocol (Invitrogen, CA). In all transformations the
expression vectors were linearized with SalI prior to their
introduction into the yeast cells.
[0233] Multiple copies integration clones were selected using G-418
(Boehringer Mannheim, Germany). Following transformation the top
agar layer containing the yeast cells was removed and re-suspended
in 10 ml of sterile water. Aliquots were removed and plated on YPD
plates (1% yeast extract, 2% peptone, 2% glucose) containing
increasing concentrations of G-418 (up to 4 mg/ml). Single isolates
were picked and streaked on YPD plates. G-418 resistance was then
further confirmed by streaking isolates on YPD-G-418 plates.
[0234] Expression Experiments:
[0235] Single colonies were inoculated into 6 ml BMGY--Buffered
Glycerol-complex Medium (1% yeast extract, 2% peptone, 100 mM
potassium phosphate pH 6.0, 1.34% yeast nitrogen base with ammonium
sulfate without amino acids, 4.times.10.sup.-5 biotin and 1%
glycerol) and incubated at 30.degree. C. at 250 RPM for 24 hours.
Cells were harvested using clinical centrifuge and re-suspended in
2.5 ml of BMMY--Buffered Methanol-complex Medium (The same as BMGY
except that 0.5% methanol replaces the 1% glycerol). Cells were
then incubated at 30.degree. C. at 250 RPM agitation for 48 hour.
Culture supernatants were separated on SDS-PAGE and
electrophoretically transferred to a nitrocellulose membrane using
the Hoeffer-Pharmacia apparatus, according to manufacturer
protocol. A rabbit anti-heparanase polyclonal antibody (disclosed
in U.S. patent application Ser. No. 09/071,739) was used as a
primary antibody in detection of heparanase. Horseradish
peroxidase-labeled anti-rabbit antibodies and ECL Western blotting
detection reagents (Amersham, UK) were used in subsequent detection
steps.
Experimental Results
[0236] Both pPIC3.5K-Sheparanase and pPIC9K-PP2 Pichia pastoris
transformants secreted a protein with a similar molecular weight of
about 70 kDa, as expected for heparanase. These results indicate
that the heparanase contains a signal sequence which efficiently
functions as a secretion signal in Pichia pastoris.
[0237] G-418 resistance was used to select isolates characterized
by multiple gene integration events. A faint heparanase band was
observed in the supernatant of pPIC3.5K-Sheparanase transformant
isolated without selection on G-418 (FIG. 4, lane 1), whereas no
band was observed in the corresponding position in pPIC3.5K
transformant, which served as negative control (FIG. 4, lane 2). A
profound increase in the secretion of heparanase was observed in
isolates resistance to 4 mg/ml of G-418 (FIG. 4, lanes 3-6).
Example 3
Expression and Secretion of Recombinant Human Heparanase in
Mammalian Cells
Experimental Methods
[0238] Construction of hpa DNA Expression Vectors:
[0239] A hpa gene fragment was cloned under the control of either
SV40 early promoter (pShpa, FIG. 20a) or the CMV promoter (pChpa,
FIG. 20e). One construct (pShpaCdhfr, FIG. 20b) also includes a
selection marker, the mouse dhfr gene.
[0240] Specifically, a 1740 bp hpa gene fragment encoding for a 543
amino acid protein was introduced into pSI (Promega, USA) or
pSI-Cdhfr vectors to yield vectors pShpa and pShpaCdhfr,
respectively (FIGS. 5a and 5b and 20a and 20b). In both cases the
gene was inserted under the SV40 early promoter regulation.
pShpaCdhfr also carries an expression unit of mouse dhfr gene under
the regulation of CMV promoter. Another plasmid, pcdhfr (FIG. 20f),
included expression unit of mouse dhfr gene under the regulation of
CMV promoter and served as control.
[0241] A vector designed pS1hpa (FIGS. 5c, 20c) was constructed by
ligating a truncated hpa gene fragment (nucleotides 169-1721 of SEQ
ID NO:1) to a heterologous signal peptide as follows. Preprotrypsin
(PPT) signal peptide (72) was generated by chemically synthesizing
two complementary oligonucleotides corresponding to the signal
peptide encoding DNA sequence, the first having a sequence
5'-AATTCACCATGTCTGCACTTCTGATCCTAGCTC TTGTTGGAGCTGCAGTTGCTCAGGAC-3'
(SEQ ID NO:9), whereas the second having a complementary sequence
5'-CCTGAGCAACTGCAGCTC CAACAAGAGCTAGGATCAGAAGTGCAGACATGGTG-3' (SEQ
ID NO:10). Annealing of the complementary oligonucleotides produced
the double strand sequence encoding to the PPT signal peptide
flanked by a sticky end of an EcoRI restriction site on the 5' end
thereof and a sticky end of an AatII restriction site on the 3' end
thereof. Following restriction by EcoRI and AatII of the pfasthpa
vector, a 145 bp fragment was removed, and replaced by the 52 bp
PPT DNA sequence to yield plasmid pS1hpa. The insert thereof was
cut out with EcoRI and DotI and ligated into the vector pSI.
[0242] A vector designed pS2hpa (FIGS. 5d and 20d) was constructed
by ligating a truncated hpa gene fragment (nucleotides 144-1721) to
the PPT signal peptide as follows. Preprotrypsin (PPT) signal
peptide (72) was generated by chemically synthesizing two
complementary oligonucleotides corresponding to the signal peptide
encoding DNA sequence, the first having a sequence
5'-AATTCACCATGTCTGCACTTCTGA TCCTAGCTCTTGTTGGAGCTGAGTTGC- -3' (SEQ
ID NO:11), whereas the second having a complementary sequence
5'-CGGCAACTGCA GCTCCAACAAGAGCTAGGATCAGAAGTGCAGACATGGTG-3' (SEQ ID
NO:12). Annealing of the complementary oligonucleotides produced
the double strand sequence encoding to the PPT signal peptide
flanked by a stick end of an EcoRI restriction site on the 5' end
thereof and a sticky end of a NarI restriction site on the 3' end
thereof.
[0243] Following restriction by EcoRI and NarI of pS1hpa plasmid, a
112 bp fragment was removed therefrom and replaced by the PPT DNA
sequence to give plasmid pS2hpa (FIGS. 5d, 20d).
[0244] Transfection of Vectors into Cells:
[0245] DNA constructs were introduced into animal cells using the
calcium-phosphate co-percipitaion technique essentially as
described in (73).
[0246] Selection for dhfr Expressing Stable Cellular Clones:
[0247] Following transfection, cells were incubated for 48 hours in
a non-selective growth medium (F12 medium supplemented with 10%
fetal calf serum). Then, the medium was changed to a selection
medium (DMEM supplemented with 10% dialyzed calf serum) and cells
were propagated to confluence at 37.degree. C., under 8% CO.sub.2
aeration. Methotrexate (MTX, 5000 nM) was added to the growth
selection medium and resistant cellular clones were isolated.
Alternatively, cells were transferred after transfection directly
to a selection medium containing MTX (100-1000 nM).
[0248] SDS Polyacrylamide Gel Electrophoresis and Western Blot
Analysis:
[0249] Denatured and reduced samples were loaded on ready made
gradient (4-20%) gels (Novex, USA) and separated under standard gel
running conditions (as described in Protein Electrophoresis
Application Guide, Hoeffer, U.S.A.). Transfer of proteins onto a
PVDF membrane was performed electrophoretically by a protein
transfer apparatus (Hoeffer-Pharmacia). Detection of specific
protein was accomplished by a rabbit anti-heparanase polyclonal
antibody (disclosed in U.S. patent application Ser. No. 09/071,739)
(.times.2000 dilution), followed by ECL detection (Amersham,
UK).
[0250] Determination of Heparanase Activity:
[0251] ECM-derived soluble HSPG assay was performed by incubating
cell extracts with solubillized .sup.35S-labeled ECM (18 hours,
37.degree. C.) in the presence of 20 mM phosphate buffer (pH 6.2),
and size fractionation of the hydrolyzed fraction of the ECM by gel
filtration on a Sepharose CL-6B column. Radiolabeling of
degradation fragments eluted at 0.5<Kav<0.8 (peak II) was
determined (61).
[0252] Alternatively, degradation of soluble high molecular weight
heparan sulfate or heparin molecules to smaller fragments was
detected by polyacrylamide gel electrophoresis analysis.
Polyacrylamide gels (7.5%) were loaded with 2.5 mg heparin that was
either untreated or incubated with heparanase containing cell
extracts or media. Staining by methylen blue (74) enabled detection
of the heparin molecules and its degradation products. The mobility
of the molecules on the gel reflects their size. Therefore,
activity of heparanase is reflected in a larger quantity of rapidly
migrating heparin degradation products.
[0253] Induction of Secretion:
[0254] CHO stable clones and untransfected CHO cells were induced
for secretion of proteins by either calcium ionophore calcimycin
(A23187) (Sigma) or phorbol 12-myristate 13-acetate (PMA, Sigma),
at different concentrations (0.01, 0.1 and 1.0 mg/ml), for various
incubation times (2, 8, 24, 48 hours). Induction was performed in
the absence of serum. Conditioned medium was collected with 10%
buffer citrate pH 5.6 and 200KIU/ml aprotinin (Protosol, Rad
Chemicals, Israel), centrifuged to remove floating cells, and kept
at -200.degree. C. The amount of secreted protein(s) was detected
by Western blot analysis, and its activity was determined by
.sup.35S-ECM degradation assay and soluble heparan sulfate
substrate hydrolysis assay. When necessary conditioned medium was
concentrated by ultrafiltration through a 10 kDa filter
(Millipore).
[0255] Large Scale Propagation of Animal Cells in a Spinner-Basket
Bioreactor:
[0256] The structure and mode of operation of the bioreactor is
described in detail in reference 75. A Spinner Basket bioreactor
(500 ml, New Brunswick Scientific) embedded with 10 grams of
Fibracel discs (Sterillin, U.K.) was inoculated with seeding
inoculum of 1.5.times.10.sup.8 cells of a stable clone of CHO cells
designated GGG.sub.11 that constitutively produces recombinant
heparanase. Propagation of cells was performed in a medium
containing 10% serum and cell proliferation was monitored by
measurement of glucose consumption.
[0257] Then growth medium was replaced with medium without serum,
suitable for the production of the recombinant protein. This medium
served as a source for recombinant heparanase for later
purification.
Experimental Results
[0258] Expression of hpa DNA in Animal Cells:
[0259] Expression of recombinant hpa gene products was detected in
a human kidney fibroblasts cell line (293), baby hamster kidney
cells (BHK21) and Chinese hamster ovary (CHO dhfr-) cells,
following transfection with the hpa gene (FIGS. 6a-b).
[0260] Analysis of recombinant heparanase by Western blotting
revealed two distinct specific protein products: a large protein of
about 70 kDa and a predominant protein of about 52 kDa (FIGS.
6a-b).
[0261] Transient expression of heparanase proteins was monitored
24-72 hours post transfection in various cell types.
[0262] Human fibroblasts (293 cell line) transfected with pShpa
(FIG. 5a) or pChpa constructs (FIG. 5e) exhibited heparanase
activity (FIG. 6a, lane 4, Table 1 below).
[0263] Transfection of CHO cells with the expression vector
pShpaCdhfr (FIG. 5b) and subsequent selection for MTX resistant
clones resulted in the isolation of numerous clones. These cellular
clones express hpa gene products in a constitutive and stable
manner (FIG. 6a, lanes 1-3).
[0264] Several CHO cellular clones have been particularly
productive in expressing hpa proteins, as determined by protein
blot analysis and by activity assays (FIG. 6a, FIG. 6b, lane 1, and
Table 1). Although the hpa DNA encodes for a large 543 amino acids
protein (expected molecular weight about 70 kDa) the results
clearly demonstrate the existence of two proteins, one of about 70
kDa and another of about 52 kDa. These observations are similar to
the results of the transient hpa gene expression in human 293 cells
(FIG. 6a, lane 4). Transient expression of pShpaCdhfr in CHO cells
revealed predominantly a 52 kDa heparanase protein (FIG. 6b, lane
2).
[0265] It has been previously shown that a 52 kDa protein with
heparanase activity was isolated from placenta (61) and from
platelets, (62). It is thus likely that the 70 kDa protein is
naturally processed in the host cell to yield the 52 kDa
protein.
[0266] Heparanase Secretion into the Growth Medium:
[0267] For large scale production and purification purposes,
secretion of the recombinant protein into the growth medium is
highly desirable. Therefore, expression vectors were constructed
(pS1hpa and pS2hpa, FIGS. 5c-d) that would drive translation of
heparanase attached to the PPT signal peptide.
[0268] Both pS1hpa and pS2hpa plasmids directed the expression of
protein product with heparanase activity in human 293 or CHO cells
(Table 1). The heparanase was not secreted to the medium in CHO
cells. However, transient expression of heparanase encoded by
pS1hpa and pS2hpa in human 293 cells resulted in the appearance of
a single size (about 65 kDa) heparanase protein (FIG. 7c, lanes
3-6).
1TABLE 1 Determination of Heparanase activity in transfected animal
cells transfected Heparanase Cell type DNA Activity Human 293 pChpa
+ (a) cells Human 293 pShpa + (b) cells Human 293 pS1hpa + (b)
cells Human 293 pS2pha + (b) cells CHO pShpaCdhfr + (a) Cell
extracts were assayed for heparanase activity using ECM-derived
soluble HSPG assay (a) or direct hydrolysis of soluble substrate
(b). Activity detected either in transiently expressing cells (293,
CHO) or stable cellular clones (CHO).
[0269] In order to induce secretion of the recombinant protein(s)
into the medium, stable clones and untransfected CHO cells were
induced with either calcium ionophore or PMA. The results show that
induction with 1 mg/ml calcium ionophore for 2 hours stimulates the
secretion of protein of about 52 kDa from stable clones but not
from untransfected cells (data not shown) or untreated stable
clones, while longer (24-48 hours) incubation time with 100 ng/ml
of calcium ionophore induces predominantly the secretion of protein
of about 70 kDa from stable clones (FIGS. 7a-b). The conditioned
medium obtained from the treated stable clone, which exhibited the
52 kDa protein, had strong heparanase activity in ECM-derived
soluble HSPG assay (FIGS. 8b-c), and in concentrated conditioned
medium, in the gel shift assay (FIG. 8a). The heparanase activity
in the conditioned medium from the treated stable clone, which
exhibited the 70 kDa, is lower than that of the 52 kDa fraction
(FIGS. 8d-g), since it was active when diluted eight fold while the
70 kDa protein failed to show activity in this dilution. It is thus
possible that the 52 kDa protein is the active form of a less
active pre heparanase of 70 kDa, which is naturally processed to
yield the mature-active 52 kDa heparanase.
[0270] Large Scale Production of Heparanase:
[0271] Large scale propagation of heparanase expressing cells was
set up in a 500 ml volume Spinner-Basket bioreactor to demonstrate
the ability to obtain a dense adherent cell culture for large scale
production of heparanase. Heparanase constitutively producing cell
line was propagated in the Spinner-Basket bioreactor and at the end
of the proliferation phase the medium was replaced with production
medium which has the same composition as the growth medium but
without serum. Cell proliferation and viability were constantly
monitored by daily measurements of glucose concentration in the
medium. Level of glucose was also the parameter used to determine
the frequency of medium refreshment in the bioreactor, as described
in reference 76. Results of a typical "batch run" that includes
proliferation and maintenance of heparanase producing cells in a
500 ml Spinner-Basket are shown in FIG. 9.
[0272] A "batch run" in a Spinner-Basket bioreactor can last about
four weeks, when serum is omitted from the culture medium. The
apparatus can be linearly enlarged to bioreactors of 5, 7 or 35
liters. Accordingly, larger amounts of Fibracel can be packed in
those vessels and accommodate, proportionally, larger numbers of
cells. The bioreactors can support cell growth for weeks, or even
months, depending on the nature of the cell line and the
composition of medium.
Example 4
Expression of Recombinant Heparanase in Virus Infected Insect
Cells
Experimental Methods
[0273] Cells:
[0274] High five and Sf21 insect cell lines were maintained as
monolayer cultures in SF900II-SFM medium (GibcoBRL).
[0275] Recombinant Baculovirus:
[0276] Recombinant virus containing the hpa gene was constructed
using the Bac to Bac system (GibcoBRL). The transfer vector
pFastBac (see U.S. patent application Ser. No. 08/922,180) was
digested with SalI and NotI and ligated with a 1.7 kb fragment of
phpa2 digested with XhoI and NotI. The resulting plasmid was
designated pFasthpa2. An identical plasmid designated pFasthpa4 was
prepared as a s duplicate and both independently served for further
experimentations. Recombinant bacmid was generated according to the
instructions of the manufacturer with pFasthpa2, pFasthpa4 and with
pFastBac. The latter served as a negative control. Recombinant
bacmid DNAs were transfected into Sf21 insect cells. Five days
after transfection recombinant viruses were harvested and used to
infect High five insect cells, 3.times.10.sup.6 cells in T-25
flasks. Cells were harvested 2-3 days after infection.
4.times.10.sup.6 cells were centrifuged and resuspended in a
reaction buffer containing 20 mM phosphate citrate buffer, 50 mM
NaCl. Cells underwent three cycles of freeze and thaw and lysates
were stored at -80.degree. C. Conditioned medium was stored at
4.degree. C.
Experimental Results
[0277] Degradation of Soluble ECM-Derived HSPG:
[0278] Monolayer cultures of High five cells were infected (72 h,
28.degree. C.) with recombinant bacoluvirus containing the pFasthpa
plasmid or with control virus containing an insert free plasmid.
The cells were harvested and lysed in heparanase reaction buffer by
three cycles of freezing and thawing. The cell lysates were then
incubated (18 h, 37.degree. C.) with sulfate labeled, ECM-derived
HSPG (peak I), followed by gel filtration analysis (Sepharose 6B)
of the reaction mixture.
[0279] As shown in FIG. 10, the substrate alone included almost
entirely high molecular weight (Mr) material eluted next to V.sub.O
(peak I, fractions 5-20, Kav<0.35). A similar elution pattern
was obtained when the HSPG substrate was incubated with lysates of
cells that were infected with control virus. In contrast,
incubation of the HSPG substrate with lysates of cells infected
with the hpa containing virus resulted in a complete conversion of
the high Mr substrate into low Mr labeled degradation fragments
(peak II, fractions 22-35, 0.5<Kav<0.75).
[0280] Fragments eluted in peak II were shown to be degradation
products of heparan sulfate, as they were (i) 5- to 6-fold smaller
than intact heparan sulfate side chains (Kav approx. 0.33) released
from ECM by treatment with either alkaline borohydride or papain;
and (ii) resistant to further digestion with papain or
chondroitinase ABC, and susceptible to deamination by nitrous acid.
Similar results (not shown) were obtained with Sf21 cells. Again,
heparanase activity was detected in cells infected with the hpa
containing virus (pFhpa), but not with control virus (pF). This
result was obtained with two independently generated recombinant
viruses. Lysates of control not infected High five cells failed to
degrade the HSPG substrate.
[0281] In subsequent experiments, the labeled HSPG substrate was
incubated with medium conditioned by infected High five or Sf21
cells.
[0282] As shown in FIGS. 11a-b, heparanase activity, reflected by
the conversion of the high Mr peak I substrate into the low Mr peak
II which represents HS degradation fragments, was found in the
growth medium of cells infected with the pFhpa2 or pFhpa4 viruses,
but not with the control pF1 or pF2 viruses. No heparanase activity
was detected in the growth medium of control non-infected High five
or Sf21 cells.
[0283] The medium of cells infected with the pFhpa4 virus was
passed through a 50 kDa cut off membrane to obtain a crude
estimation of the molecular weight of the recombinant heparanase
enzyme. As demonstrated in FIG. 12, all the enzymatic activity was
retained in the upper compartment and there was no activity in the
flow through (<50 kDa) material. This result is consistent with
the expected molecular weight of the hpa gene product.
[0284] In order to further characterize the hpa product the
competition effect of heparin, additional substrate of heparanase
was examined.
[0285] As demonstrated in FIGS. 13a-b, conversion of the peak I
substrate into peak II HS degradation fragments was completely
abolished in the presence of heparin.
[0286] Altogether, these results indicate that the heparanase
enzyme is expressed in an active form by insect cells infected with
Baculovirus containing the newly identified human hpa gene.
[0287] Degradation of HSPG in Intact ECM:
[0288] Next, the ability of intact infected insect cells to degrade
HS in intact, naturally produced ECM was investigated. For this
purpose, High five or Sf21 cells were seeded on metabolically
sulfate labeled ECM followed by infection (48 h, 28.degree. C.)
with either the pFhpa4 or control pF2 viruses. The pH of the medium
was then adjusted to pH 6.2-6.4 and the cells further incubated
with the labeled ECM for another 48 h at 28.degree. C. or 24 h at
37.degree. C. Sulfate labeled material released into the incubation
medium was analyzed by gel filtration on Sepharose 6B.
[0289] As shown in FIGS. 14a-b and 15a-b, incubation of the ECM
with I o cells infected with the control pF2 virus resulted in a
constant release of labeled material that consisted almost entirely
(>90%) of high Mr fragments (peak I) eluted with or next to
V.sub.O. It was previously shown that a proteolytic activity
residing in the ECM itself and/or expressed by cells is responsible
for release of the high Mr material. This nearly intact HSPG
provides a soluble substrate for subsequent degradation by
heparanase, as also indicated by the relatively large amount of
peak I material accumulating when the heparanase enzyme is
inhibited by heparin (FIG. 17). On the other hand, incubation of
the labeled ECM with cells infected with the pFhpa4 virus resulted
in release of 60-70% of the ECM-associated radioactivity in the
form of low Mr sulfate-labeled fragments (peak II,
0.5<Kav<0.75), regardless of whether the infected cells were
incubated with the ECM at 28.degree. C. or 37.degree. C. Control
intact non-infected Sf21 or High five cells failed to degrade the
ECM HS side chains.
[0290] In subsequent experiments, as demonstrated in FIGS. 16a-b,
High five and Sf21 cells were infected (96 h, 28.degree. C.) with
pFhpa4 or control pF1 viruses and the growth medium incubated with
sulfate-labeled ECM. Low Mr HS degradation fragments were released
from the ECM only upon incubation with medium conditioned by pFhpa4
infected cells. As shown in FIG. 17, production of these fragments
was abolished in the presence of heparin, due to its competitory
nature. No heparanase activity was detected in the growth medium of
control, non-infected cells. These results indicate that the
heparanase enzyme expressed by cells infected with the pFhpa4 virus
is capable of degrading HS when complexed to other macromolecular
constituents (i.e. fibronectin, laminin, collagen) of a naturally
produced intact ECM, in a manner similar to that reported for
highly metastatic tumor cells or activated cells of the immune
system.
[0291] Thus, insect cells of several origins (such as Sf21 from
Spodoptera frugiperda and High five from Trichoplusia ni) may be
infected productively with baculovirus. Insect cells are infected
with recombinant baculovirus in which viral DNA sequences have been
replaced with DNA sequences coding for a protein of interest. The
protein of interest is expressed during the very late phase of
infection. A major advantage of the baculovirus expression system
is that it can be used for expressing large amounts of recombinant
protein compared to other popular expression systems in eukaryotes
(e.g., expression in CHO cells). Another advantage of the system is
that insect cells have most of the protein processing capabilities
of higher eukaryotic cells. Thus, proteins produced in the
recombinant baculovirus-infected cells can undergo co-and post
translational processing yielding proteins which are similar to the
natural protein.
[0292] Scaling up the process of culturing and infecting insect
cells with baculovirus is required for the production of
recombinant protein of choice, in milligram and up to gram
quantities. These quantities may be required for both research or
commercial use. Scaling up the process involves a variety of
fields, such as medium development, metabolic studies, protein
purification and quantification.
[0293] Several problems are inherent to this system and effect the
process of scaling up. Upon infection, insect cells become
increasingly fragile and sensitive to the physiochemical
environment of the culture. One of the primary goals of the
bioengineer is to oxygenate large scale, high-density culture
sufficiently, at low shearing rates. Although oxygen uptake rates
of insect cells are similar to mammalian cell lines, it was found
that after infection oxygen uptake rates doubles. An optimization
process, aimed for setting-up of bioreactor parameters is required,
for supplying oxygen to the cells without damaging them.
[0294] The spinner Bellco, Cat. 1965-56001 was used for scaling up
as described. This is a double-wall type spinner. Temperature was
controlled by water circulated from a 12 liter water bath (Fried
Electric, Model TEP1) equipped with a heater and a thermostat. The
spinner was aerated with both air, using an aquarium pump (Rena
301) and oxygen. An oxygen cylinder (medical grade) was connected
to the spinner through a two stage regulator set to a pressure of 2
psi. Both air and oxygen were connected to the spinner through a
T-connector equipped with valves that enabled a control over the
flow rates of air and of oxygen. A tubing for delivering air mixed
with oxygen was connected to the sparger of the spinner through a
0.2.mu.-size filter. The sparger used was of an open type,
releasing air-oxygen mixture through an orifice of 3 mm inner
diameter. The stirring function was provided by a low-RPM magnetic
stirrer (LH, type 20, LH fermentation Co.), placed beneath the
spinner.
[0295] High five and Sf21 cells were used alternatively for large
scale production of heparanase. Cell culture was gradually built up
to 1.2.times.10.sup.10 cells. Eight shake flasks of 500 ml-size
were used for culturing cells to 3.times.10.sup.6 cells/ml. Cells
were cultured with protein-free medium (Insect-Xpress, Bio
Whittaker). 1.5 liters of the above culture was used for seeding a
6 liters-size spinner. At the time of seeding, culture was diluted
to 3 liters with fresh medium. Air was sparged into the culture at
0.5 liters/min. Stirring rate was 50 RPM and temperature was set to
28.degree. C. Two days after seeding, culture volume was doubled
again, from 3 liters to 6 liters. Cell density was adjusted at that
time to 1.times.10.sup.6 cells/ml. At that time pure oxygen was
sparged at 1.5 liters/min in addition to the sparging of air (at
0.5 liters/minute).
[0296] Infection of the culture took place one day after doubling
the culture volume from 3 liters to 6 liters, as described. Cells
were counted and infected with the heparanase-coding recombinant
virus pFhpa2 at a multiplicity of infection (MOI) of 0.1 or 1.0.
The infected culture was maintained for approximately 72 hours
under conditions set for 6 liters-size culture: Oxygen 1.5
liters/min, air 0.5 liters/min, temperature 28.degree. C.,
agitation at 50 RPM.
[0297] Viability of cells in culture was tested every 4 hours,
starting from 62 hours after virus infection and on. Viability of
cells was determined by staining cells with Trypan Blue dye. The
culture was harvested when viability reached 70-80%. Cells and cell
debris were removed by centrifugation (IEC B-22M, Rotor Cat. 878,
20 min. at 4.degree.]C. at 7,000 RPM). Supernatants were filtered
through 0.2.mu. size cartridge (Millipore, Cat. KV0304HB3). Virus
and small-size debris were removed with a 300 kda-size cross-flow
cartridge (Millipore, Cat. CDUF006LM). Heparanase was concentrated
from filtrate obtained from the 300 kda-size cartridge with 10 kDa
size cross-flow cartridge (Millipore, Cat. SK1P003W4). The final
concentrated solution had a volume of between 0.5 liters and 1
liters. Heparanase was purified from the concentrated solution on
HPLC. Table 2 below summarizes the results of two large scale
heparanase production by insect cells experiments.
2TABLE 2 Harvest Volume time Cell heparnase of post viability at in
Cells MOI culture infection harvest harvest No. used used (L)
(hours) (%) (mg/ml) 30 Sf21 0.1 4.5 78 76 0.44 31 Hi-5 0.1 6.0 75
76 0.16
Example 5
Purification of Recombinant Heparanase
Experimental Methods and Results
[0298] Methods and Results:
[0299] Baculovirus infected insect cells (1 or 5 liter of High five
cell suspension) were harvested by centrifugation. The supernatant
was passed through 0.2 micron filter (Millipore), then filtered
through 300K cartridge (Millipore). The <300 kDa retentate
(about 300 ml) was washed by further filtration with 2 volumes of
phosphate buffered saline (PBS). The <300 kDa filtrate was then
concentrated by 10K cellulose cartridge (Millipore). The >10 kDa
retentate was diluted three fold with 10 mM phosphate buffer pH 6.8
to prepare for applying the crude enzyme preparation onto a
Source-S column (Pharmacia).
[0300] The diluted >10 kDa retentate was subjected to a Source-S
column (2.5.times.10 cm) pre equilibrated with 10 mM phosphate
buffer pH 6.8, 50 mM NaCl). Most of the contaminating proteins did
not bind to the column while heparanase bound tightly. Heparanase
activity was eluted by a linear gradient of 0.05 M NaCl--1 M NaCl
in phosphate buffer pH 6.8 and fractions of 5 ml were
collected.
[0301] The fractions having the highest activity in degrading
sulfate labeled ECM were combined. The 0.4 M NaCl fractions were
about 90% pure and exhibited the highest activity (FIG. 18, lane
9). A rabbit anti-heparanase polyclonal antibody detected the
purified enzyme in Western blot--ECL analysis (FIG. 19, lane
9).
[0302] These results demonstrate a powerful single step
purification of recombinant heparanase from culture supernatants.
Obviously, other purification methods, such as affinity
purification using, for example, solid support bound heparanase
substrates, heparanase inhibitors or anti-heparanase antibodies,
size exclusion, hydrophobic interactions, etc. can be additionally
employed.
Example 6
Purification of Heparanase and Production of Highly Active
Heparanase Species by Proteolytic Processing
Experimental Methods
[0303] Construction of hpa DNA expression vectors, transfection
thereof into cells, selection for dhfr expressing stable cellular
clones, induction of secretion and SDS polyacrylamide gel
electrophoresis and Western blot analyses were all performed as
described hereinabove under Example 3.
[0304] Heparanase Activity Using DMB Assay:
[0305] For each sample, 100 .mu.l heparin sepharose (50% suspension
in 1.times. buffer A--containing 20 mM Phosphate citrate buffer pH
5.4, 1 mM CaCl.sub.2 and 1 mM NaCl) were incubated in 0.5 ml
eppendorf tube for 17 hours with a tested enzyme preparation. At
the end of the incubation period, samples were centrifuged for 2
minutes at 1000 rpm and the supernatants were analyzed for sulfated
polyanions (heparin) using the colorimetric dimethylmethylene blue
assay as follows.
[0306] Supernatants (100 .mu.l) were transferred to plastic
cuvettes and diluted to 0.5 ml with PBS supplemented with 1% BSA.
1,9-Dimethylmethylene blue (32 mg dissolved in 5 ml ethanol and
diluted to 1 liter with formate buffer) (0.5 ml) was added to each
cuvette. Absorbency at 530 nm was determined using a
spectrophotometer (Cary 100, Varian). For each sample a control, to
which the enzyme was added at the end of the incubation period, was
included. For further details, see U.S. patent Ser. No. 09/113,168,
which is incorporated by reference as if fully set forth
herein.
[0307] Heparanase Activity Using the Tetrazolium Assay:
[0308] Heparanase activity was determined in reactions containing
buffer A and 50 .mu.g heparan sulfate in a final volume of 100
.mu.l. Reactions were performed in a 96 well microtiterplate at
37.degree. C. for 17 hours. Reaction were thereafter stopped by the
addition of 100 pl tetrazolium blue reagent (0.1% tetrazolium blue
in 0.1 M NaOH) to each well. Color was developed following
incubation at 60.degree. C. for 40 minutes. Color intensity was
quantitatively determined at 580 nm using a microtiterplate reader
(Dynatech). For each sample a control, to which the enzyme was
added at the end of the incubation period, was included. A glucose
standard curve of 1-15 .mu.g glucose was included in each assay.
Heparanase activity was calculated as O.D. of the sample containing
the substrate minus the O.D of the control sample. The result was
converted to .mu.g glucose equivalent. One unit is defined as .mu.g
glucose equivalent produced per minute. For further details, see
U.S. patent Ser. No. 09/113,168, which is incorporated by reference
as if fully set forth herein.
[0309] Production of Rabbit Anti-Native Heparanase Polyclonal
Antibodies:
[0310] Rabbits were immunized in three two weeks intervals with 200
mg of purified human recombinant heparanase protein produced in
baculovirus infected Sf2l insect cells (see Examples 4-5 above)
emulsified with an equal volume of complete Freund's adjuvant. Ten
days after the third immunization rabbits were bled and serum was
examined for reactivity with recombinant heparanase. Four weeks
after bleeding another boost was injected and 10 days later blood
was collected.
[0311] Purification of Heparanase from Mammalian Cell Extract Using
Ion Exchange Chromatography:
[0312] 2TT1 CHO cells (2.times.10.sup.8 cells stably transfected
with pShpaCdhfr, FIG. 20b) were extracted in 2.5 ml of 10 mM
phosphate citrate buffer, pH 5.4. The extract was centrifuged at
2,750.times. g for 5 minutes and the supernatant was collected for
heparanase enzyme purification using cation exchange chromatography
as follows. An HPLC column (mono-S HR 5/5, Pharmacia Biotech) was
equilibrated with 20 mM sodium phosphate buffer, pH 6.8, and the
supernatant was loaded thereon. Proteins were eluted from the
column using a linear gradient of 0 to 1 M sodium chloride in 20 mM
sodium phosphate buffer, pH 6.8. The gradient was carried out in 20
column volumes at a flow rate of one ml per minute. Elution of
proteins was monitored at 214 nm (FIG. 23a) and fractions of 1 ml
each were collected. An aliquot from each fraction was analyzed for
heparanase activity using the DMB assay and for immunoreactivity
using a mouse anti-heparanase monoclonal antibody (see U.S. patent
Ser. No. 09/071,739, which is incorporated herein by reference).
Most of the heparanase was eluted in fractions 19-20.
[0313] Preparation of an Affinity Column with Anti-Native
Heparanase Antibodies:
[0314] An affinity column was prepared using the Immunopure Protein
G IgG Orientation Kit (Pierce). To this end, 17 mg of the above
described rabbit anti-native heparanase polyclonal antibody,
purified on protein G sepharose, were bound to a column containing
2 ml Immunopure immobilized protein G. The antibody was cross
linked to the protein G with DMP. Unreacted imidate groups were
blocked and the column was equilibrated with 20 mM phosphate
buffer, pH 6.8.
[0315] Purification of Heparanase Using the Affinity Column:
[0316] 0.5.times.10.sup.8 2TT1 CHO cells were suspended in 2.5 ml
of 20 mM phosphate citrate buffer, pH 5.4. Cells were frozen in
liquid nitrogen and subsequently thawed at 37.degree. C. Freezing
and thawing were repeated two more times. The extract was then
centrifuged for 15 minutes at 4000 g and the resulting supernatant
was loaded onto the affinity column and was incubated, to allow
binding of the enzyme to the column, at 4.degree. C. for 17 hours
under head-over-tail shaking. Thereafter, unbound proteins were
washed until absorbency at 280 nm reached zero. Proteins were
eluted from the column with 0.1 M glycine HCl buffer, pH 3.5. 900
.mu.l fractions were collected into eppendorf tubes each containing
100 .mu.l of 1 M phosphate buffer, pH 8. The presence of heparanase
in the eluted fractions was determined by Western blotting
following gradient 4-20% SDS-PAGE of 20 .mu.l samples using
anti-heparanase monoclonal antibody (see U.S. patent Ser. No.
09/071,739). Heparanase activity was determined in 30 .mu.l samples
using the above described DMB assay.
[0317] Construction of Heparanase Expression Vectors with a Unique
Protease Cleavage Sequence:
[0318] Expression vectors for the production of a heparanase
protein species carrying a unique proteolytic cleavage site were
designed and constructed. Two independent sites, just upstream of
amino acids 120 or 158 (SEQ ID NO:2), both are peaking on the
hydropathy plot, as calculated by the Kyte-Doolittle method for
calculating hydrophilicity, using the Wisconsin University GCG DNA
analysis software (FIG. 29a), were selected for insertion of either
one of two protease recognition and cleavage sequences within the
hpa cDNA sequence to yield two heparanase species designated herein
as pre-p56' and pre-p52', which, following digestion with their
respective protease, yield truncated proteins designated herein
p52' and p56', respectively. A first sequence included 4 amino
acids (Ile-Glu-Gly-Arg.dwnarw., SEQ ID NO:13) which constitute a
factor Xa recognition and cleavage sequence. An alternative,
second, sequence included 5 amino acids
(Asp-Asp-Asp-Asp-Lys.dwnarw., SEQ ID NO:14) which constitute a
enterokinase recognition and cleavage sequence. These sequences do
not appear in the natural enzyme (SEQ ID NO:2).
[0319] To this end, the following PCR primers were constructed:
52-Xa -5'-CCATCGATAGAAGGACGAAAAAAGTTCAAGAACAGCA CCTAC-3' (SEQ ID
NO:15); 52x-Cla--5'-GGATCGATTGGTAGTGT TCTCGGAGTAG-3' (SEQ ID
NO:16); 56-Xa--5'-GGATCGATAG AAGGACGATCTCAAGTCAACCAGGATATT-3' (SEQ
ID NO:17); 56x-Cla--5'-CCATCGATGCCCAGTAACTTCTCTCTTCAAAG-3' (SEQ ID
NO:18); hpl 967--5'-TCAGATGCAAGCAGCAACTTTGGC-3' (SEQ ID NO:19); hpu
685--5'-GAGCAGCCAGGTGAGCCCAAGAT-3' (SEQ ID NO:20); 52-EK
5'-CCATCGATGACGACGACAAGAAAAAGTTCA AGAACAGCACCTAC-3' (SEQ ID NO:21);
52e-Cla--5'-GGATCGATCTGGTAGTGTTCTCGGAGTAG-3' (SEQ ID NO:22);
56-EK--5'-GGATCGATGACGACGACAAGTCTCAAGTCAACC AGGATATTTG-3' (SEQ ID
NO:23); and 56e-Cla--5'-CCATCG ATTTGGGAGTAACTTCTCTCTTCAAAG-3' (SEQ
ID NO:24).
[0320] The following constructs were prepared (FIG. 29b):
[0321] (i) Construction of pre-p52'-Xa hpa in pFast: A first PCR
reaction was performed with a pFasthpa2 template and with primers
52-Xa and hpl 967. The resulted 1180 bp fragment was digested with
ClaI and AfII and a 220 bp fragment was isolated. A second PCR
reaction was performed with a pFasthpa2 template and with primers
52x-Cla and hpu 685. The resulting 500 bp fragment was digested
with ClaI and AatII and a 370 bp fragment was isolated. The
ClaI-AflII 220 bp and the ClaI-AatII 370 bp fragments were ligated
to a 5,900 AatII-AfII fragment of the pFasthpa2 plasmid.
[0322] (ii) Construction of pre-p56'-Xa hpa in pFast: A first PCR
reaction was performed with a pFasthpa2 template and with primers
56-Xa and hpl 967. The resulted 1290 bp fragment was digested with
ClaI and AfII and a 340 bp fragment was isolated. A second PCR
reaction was performed with a pFasthpa2 template and with primers
56x-Cla and hpu 685. The resulting 380 bp fragment was digested
with ClaI and AatII and a 250 bp fragment was isolated. The
ClaI-AfII 340 bp and the ClaI-AatII 250 bp fragments were ligated
to a 5,900 AatII-AJIlI fragment of the pFasthpa2 plasmid.
[0323] (iii) Construction of pre-p52'-Enterokinase hpa in pFast: A
first PCR reaction was performed with a pFasthpa2 template and with
primers 52-EK and hpl 967. The resulted 1180 bp fragment was
digested with ClaI and AfII and a 220 bp fragment was isolated. A
second PCR reaction was performed with a pFasthpa2 template and
with primers 52e-Cla and hpu 685. The resulting 500 bp fragment was
digested with ClaI and AatII and a 370 bp fragment was isolated.
The ClaI-AfII 220 bp and the ClaI-AatII 370 bp fragments were
ligated to a 5,900 AatII-AflII fragment of the pFasthpa2
plasmid.
[0324] (iv) Construction of pre-p56'-Enterokinase hpa in pFast: A
first PCR reaction was performed with a pFasthpa2 template and with
primers 56-EK and hpl 967. The resulted 1290 bp fragment was
digested with ClaI and AfII and a 340 bp fragment was isolated. A
second PCR reaction was performed with a pFasthpa2 template and
with primers 56e-Cla and hpu 685. The resulting 380 bp fragment was
digested with ClaI and AatII and a 250 bp fragment was isolated.
The ClaI-AfII 340 bp and the ClaI-AatII 250 bp fragments were
ligated to a 5,900 AatII-AflII fragment of the pFasthpa2
plasmid.
[0325] Construction of Plasmids for Expression of Heparanase with
Protease Digestion Sequence:
[0326] Each one of the four constructs (i to iv) described
hereinabove includes an AatII-AfII fragment which includes a factor
Xa or enterokinase recognition and cleavage sequence positioned at
one of the described alternative sites, i.e., upstream amino acids
120 or 158 (SEQ ID NO:2). The hpa constructs described in FIGS.
5a-e and 20 a-e, as well as the pFasthpa constructs, each includes
a single AatII site and a single AfII site within the hpa cDNA
sequence, thus enabling the insertion by replacement of the 220 or
340 AatII-AfII fragments as desired.
Experimental Results
[0327] Expression of hpa DNA in Animal Cells:
[0328] As already shown and discussed under Example 3 above, in
order to drive transient or stable expression of the hpa gene in
animal cells, the hpa gene was cloned into expression vectors,
where transcription is regulated by promoters of viral origin
(SV40, CMV) to ensure efficient transcription (FIGS. 5a-e). All
vectors were suitable for transient expression of hpa in animal
cells, but only vectors that include an expression cassette for the
mouse dhfr gene (FIGS. 5b and 20f, the latter serves as a negative
control) could be subjected to selection by mrthotrexate (MTX).
Selection enables the establishment of cell lines that
constitutively produce high levels of recombinant heparanase.
[0329] Cell lines of different origins have been transfected and
expressed human heparanase gene: Transient expression of
recombinant heparanase was detected in a human kidney fibroblasts
cell line 293 (FIG. 6a), baby hamster kidney cells (BHK21; FIG.
21a) and Chinese hamster ovary cells (CHO; FIG. 6b). Stable
expression of heparanase in CHO cells is shown in FIGS. 6a-b.
[0330] Transfection of CHO cells with the expression vector
pShpaCdhfr io (FIG. 5b) or co-transfection with pS1hpa and pcdhfr
(FIGS. 5c and 20f), followed by selection for MTX resistant clones
resulted in the isolation of numerous clones. These cellular clones
express hpa gene products in a constitutive and stable manner (FIG.
6a, lanes 1-3).
[0331] Analysis of expression of recombinant heparanase in
mammalian cells revealed two distinct specific protein products: a
large protein of about 70 kDa (which is referred to herein as p70)
and a predominant protein of about 50 kDa, which is referred to
herein as p52 (FIGS. 6a, 21a).
[0332] Although the hpa DNA encodes a large 543 amino acids protein
(expected molecular weight about 61 kDa), the results clearly
demonstrate the existence of two proteins. These observations are
similar to the results of the transient hpa gene expression in
human 293 cells (FIG. 6a, lane 4).
[0333] BHK21 cells, transiently transfected with pS1hpa (FIG. 5c)
express predominantly the p52 form of recombinant heparanase (FIG.
21a, lane 1 marked by an arrow). Stable CHO clones express
predominantly the p52 protein (FIG. 6b, lane 2).
[0334] The presence of both p70 and p52 heparanase was detected in
all cells that expressed the hpa gene, although the relative
concentrations of the proteins varied between different cell
types.
[0335] Cells transfected with pS1hpa (FIG. 5c) expressed p52 (FIG.
21a) indicating that the replacement of the putative heparanase
signal peptide by the PPT signal sequence did not affect the
expression and processing of the protein.
[0336] All cell extracts exhibited high heparanase activity
following the introduction of the hpa gene. Human. 293 cells
transfected with pShpa (FIG. 5e) exhibited high heparanase activity
(FIG. 21b).
[0337] It has been previously shown that a 52 kDa protein with
heparanase activity was isolated from placenta (61) an platelets
(62).
[0338] It is thus concluded that the p70 protein is a preheparanase
that is naturally processed in the host cell to yield the p52
protein.
[0339] Heparanase Secretion into the Growth Medium:
[0340] For large scale production and purification purposes,
secretion of the recombinant protein into the growth medium is
highly desirable. Therefore, expression vectors were constructed
(pS1hpa and pS2hpa, FIGS. 5c-d) to direct translation of heparanase
attached to the PPT signal peptide, a secretion signal peptide.
[0341] Both pS1hpa and pS2hpa plasmids directed the expression of
protein product with heparanase activity in human 293 or CHO cells
(FIGS. 7c, 22a-b). Transient expression of heparanase from pS1hpa
and pS2hpa resulted in the appearance of a single size (about 70
kDa) heparanase protein in the medium (FIG. 7c, lanes 3-6), similar
to the larger form of recombinant heparanase detected in the
cells.
[0342] CHO cells, stably transfected with either pShpaCdhfr (2TT1
clones) or pS1hpa (SIPPT clones) were further subeloned to yield
stable clones which maintain their genetic and cellular
characteristics stability in the absence of MTX selection. To this
end, the limiting dilution procedure was employed, in which cells
were cloned under non-selective conditions and clones exhibiting
the above stability were selected for further analysis. 2TT1 and
S1PPT clones under (clones 2TT1 and S1PPT-p) or after (clones
2TT1-2, 2TT1-8, S1PPT-4 and S1PPT-8) selection with high MTX
yielded stable clones exhibiting moderate (clones 2TT1 (FIG. 22b),
2TT1-2, 2TT1-8) or high (clones S1PPT-p, S1PPT-4, S1PPT-8 (FIG.
22a)) constitutive secretion of heparanase into the growth medium.
The secreted protein was of about 70 kDa, similar to p70, the
larger heparanase form found within the cells (FIGS. 22a-b). Only
when a large amount of p70 protein are found in the medium, a
residual amount of the smaller heparanase form, p52, could be
detected (FIG. 22a, lane 4).
[0343] In the conditioned medium containing heparanase, some
heparanase activity could be detected, although not as high as the
activity measured in the respective cell extracts which, as
determined immunologically, have comparable heparanase
concentrations. Some improvement in secretion could be detected by
calcium ionophore treatment, but the effect was transient (FIG.
22a, lane 4).
[0344] The Purification of Recombinant Heparanase from 2TT1 CHO
Cells by Ion Exchange Chromatography:
[0345] Clone 2TT1-8 was used for large scale production of
heparanase. In this cell line, the p52 form of heparanase is
predominantly expressed within the cells. The cells are grown
adherent to the tissue culture flask surface and were harvested
when the cell culture reaches confluency.
[0346] Purification of a non-abundant protein from cells is a
challenging task, where only an carefully designed and accurately
discriminating protocol enables purification. See U.S. Pat. No.
5,362,641 and references 61 and 62 describing the purification of
heparanase from placenta and platelets.
[0347] Here, a cation exchange chromatography procedure was
selected for purification based on successful use thereof in the
purification of insect cells produced recombinant heparanase, as
described in Example 5 hereinabove, Separation of the total protein
content of 2TT1-8 cell extract on a mono-S cation exchange column
is shown in FIG. 23a. The vast majority of cellular proteins were
eluted from the column prior to the elution of heparanase (FIG.
23b). It is important to note that the p52 and the p70 were
co-eluted under these conditions. Furthermore, a tight correlation
was found between the presence of heparanase, as detected
immunologically (FIG. 23b), and its activity, as measured by the
DMB (FIG. 23c) and the tetrazolium (FIG. 23d) activity assays.
[0348] Thus, using the above described purification protocol, one
obtains ample amounts of highly active and purified heparanase
which is highly suitable for use in a high throughput screening
assay for heparanase activity, e.g., in the presence of candidate
heparanase inhibitors, for example, combinatorial inhibitor
libraries. Further details relating to a heparanase high throughput
assay are provided in U.S. patent application Ser. No. 09/113,168,
which is incorporated herein by reference.
[0349] The Purification of Heparanase by an Anti-Heparanse Affinity
Column:
[0350] Partially purified, active recombinant heparanse produced in
SF21 insect cells infected with a baculovirus containing the hpa
cDNA, was used to immunize rabbits for the production of polyclonal
antibodies against the native recombinant heparanase protein. This
antibody was thereafter purified and was used to construct a
heparanase affinity column.
[0351] Cellular extract of CHO 2TT1-8 cells was loaded on the
column for affinity separation. FIG. 24a-b clearly show that
heparanase was specifically and efficiently bound to the affinity
column. Moreover, high salt elution of the bound heparanase from
the column was efficient and the activity of the eluted heparanase
(FIG. 24b) was tightly correlated with the presence of the
recombinant enzyme (FIG. 24a). Thus, using an affinity column as
herein described, one can obtain a highly purified and highly
active recombinant or natural heparanase in single step
purification, which can be used in pharmaceutical applications.
Furthermore, combining the Mono-S and affinity columns into a two
step purification procedure, will ensure even better results in
terms of both purification and yield.
[0352] In addition, the tetrazolium assay is based on the detection
of free reducing sugar ends. As such it requires heparanase
preparations devoid of such reducing ends. Heparanase purified
using the above described affinity column is devoid of such
reducing ends, and is therefore highly applicable for the
tetrazolium activity assay.
[0353] Proteolytic Processing of Heparanase by Protease from Insect
Cells:
[0354] Production of human recombinant heparanase in insect cells
(Sf21), via baculovirus infection, and the subsequent purification
of that protein are described in U.S. patent Ser. Nos. 08/922,170;
09/071,618; 09/109,386; and in PCT/US98/17954, all of which are
incorporated herein by reference.
[0355] Briefly, conditioned medium of Sf21 cells that were infected
with recombinant baculovirus, secrete heparanase to the medium.
This heparanase is a glycosylated protein with an apparent
molecular weight of 70 kDa. The size of that protein is similar to
the p70 produced by mammalian cells, and it possesses limited
heparanase activity. This heparanase protein is referred to herein
as p70-bac heparanase.
[0356] Purification of p70-bac heparanase from insect cells
conditioned medium involved sequential filtration steps and a
cation exchange column (Source-S). Fractions that contain
predominantly p70-bac heparanase protein are collected. This
purification protocol and results are described hereinabove.
[0357] The effect of different pH values on the activity and
intactness of p70-bac heparanase was examined in an attempt to
establish a pH optimum for heparanase activity. It was found that
exposure of p70-bac heparanase to pH 4.0 for one week at 4.degree.
C. resulted in significant (seven fold) increase in activity (FIG.
25b). This activation was protease dependent as is evident form the
inhibition of activation caused by a protease inhibitors cocktail
(FIG. 25b).
[0358] The fate of the p70-bac heparanase following exposure to
acidic pH was uncovered by Western-blot analysis (FIG. 25a).
Following exposure to pH 4, p70-bac heparanase was converted into a
lower molecular weight form, of about 56 kDa, which is referred to
herein as p56 (FIG. 25a, lane C). Proteolysis was inhibited in the
presence of protease inhibitors (FIG. 25a, lane B).
[0359] This is the first record demonstrating (i) in vitro
proteolytic processing of recombinant heparanase, (ii) associated
with a significant increase in heparanase activity.
[0360] To further characterize the protease(s) involved in
processing and activation of p70-bac heparanase, a collection of
individual protease inhibitors was employed (FIGS. 25c-d). The
inhibitors antipain, E-64, leupeptin and chemostatin were most
effective in preventing the activation of p70-bac heparanase at low
pH. The effect was due to inhibition of the proteolytic processing
of the p70-bac heparanase as is evident from the Western blot
analysis of FIG. 25c. Antipain and leupeptin are known to inhibit
serine and cysteine proteases, while E-64 inhibits only cysteine
proteases. These results therefore indicate that a cysteine
protease(s) present in the conditioned medium of insect cells are
responsible for the activation of p70-bac heparanase, by processing
the enzyme into a lower and more active p56 molecular weight
form.
[0361] N-terminal sequencing of gel separated and PVDF transferred
p56 heparanase revealed the sequence Ser-Gln-Val-Asn-Gln (SEQ ID
NO:25), which corresponds to a new heparanase species that starts
at Ser 120 of the full length enzyme (SEQ ID NO:2).
[0362] Proteolytic Processing of Heparanase by Trypsin and
Cathepsin L:
[0363] The activation of p70-bac heparanase by protease(s) from
insect cells conditioned medium could be reproduced by mild
digestion with trypsin (FIGS. 26a-b). Trypsin, 1.5 to 500 units per
10 .mu.g p70-bac heparanase, gradually activated the protein,
reaching maximal activation of five-fold already at 15 units
trypsin (FIG. 26a). Activation of p70-bac heparanase correlated
with the expected cleavage of a portion of the p70-bac heparanase
into smaller heparanase species, of about 56 kDa (FIG. 26b).
Smaller fragments of heparanase were also obtained by
trypsinization (FIG. 26b, lanes 2-3).
[0364] Similarly, recombinant heparanase processing and activation
occurred when mild trypsin digestion was employed on a crude
conditioned medium of CHO cells that secrete mammalian p70
heparanase (FIG. 27). Activation was dose dependent.
[0365] Processing and activation of recombinant CHO produced and
secreted heparanase (p70) was also obtained by mild treatment with
Cathepsin L, which is a known cysteine protease (FIGS. 28a-b).
Processing by this protease resulted in several digestion products,
of about 56, 34 and 21 kDa (FIG. 28b, lane 2).
[0366] It is shown herein that proteolytic digestion of recombinant
heparanase from a variety of sources and by a variety of proteases
results in (i) processing of the enzyme into a lower molecular
weigh species; and (ii) increased catalytic activity. Processing
and activation of heparanase in a similar fashion is anticipated to
take place in vivo as well and therefore in vivo inhibition of
proteases can be used to indirectly inhibit heparanase processing
and activation.
[0367] Design of Expression Vectors to Express Heparanase Precursor
Species Adapted for in vitro Activation by Proteases:
[0368] The p52 heparanase protein (as characterized in CHO, 293 and
BHK21 cells, placental and platelets heparanase) and the p56
heparanase protein (as characterized after processing of the
p70-bac heparanase) are presently the forms of heparanase that
exhibit the highest enzymatic activity. It is shown herein that
these heparanase species are the result of proteolytic cleavages of
heparanase. As was determined by solid phase microsequencing the
cleavage site of p70-bac heparanase is effected between amino acids
119 and 120 (SEQ ID NO:2, see above) within the first peak of
hydrophilicity (FIG. 29a, peak I). The second peak of
hydrophilicity (FIG. 29a, peak II) is expected to contain the
cleavage site yielding the p52 heparanase species. This is not
surprising, considering the fact that these regions, are positioned
at the surface of the heparanase molecule and are thus susceptible
to proteolysis.
[0369] FIG. 29c demonstrates the steps undertaken in constructing
four basic nucleic acid constructs harboring a unique protease
recognition and cleavage sequence of factor
Xa--Ile-Glu-Gly-Arg.dwnarw.--or of
enterokinase--Asp-Asp-Asp-Asp-Lys.dwnarw. downstream amino acids
119 or 157. AatII-AfII restriction fragments derived from these
four basic constructs can be used to replace a corresponding region
in any of the hpa constructs described herein (FIGS. 5a-e) and for
that effect, any other construct harboring a hpa derived sequence.
FIG. 29b shows the modified heparanase species (pre-p56' and
pre-p52') that contain these unique protease recognition and
cleavage sequences (shaded regions) which enable proteolytic
processing by the respective proteases to obtain homogeneously
processed and highly active heparanase species (p56' and p52',
respectively).
[0370] The above described constructs are highly suitable for
expression of heparanase in any expression system which is
characterized by secretion of the recombinant heparanase to the
growth medium. Such a precursor enzyme can be readily and precisely
processed into a mature active form of heparanase--p56' or
p52'.
[0371] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
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Sequence CWU 1
1
25 1 1721 DNA Homo sapiens 1 ctagagcttt cgactctccg ctgcgcggca
gctggcgggg ggagcagcca ggtgagccca 60 agatgctgct gcgctcgaag
cctgcgctgc cgccgccgct gatgctgctg ctcctggggc 120 cgctgggtcc
cctctcccct ggcgccctgc cccgacctgc gcaagcacag gacgtcgtgg 180
acctggactt cttcacccag gagccgctgc acctggtgag cccctcgttc ctgtccgtca
240 ccattgacgc caacctggcc acggacccgc ggttcctcat cctcctgggt
tctccaaagc 300 ttcgtacctt ggccagaggc ttgtctcctg cgtacctgag
gtttggtggc accaagacag 360 acttcctaat tttcgatccc aagaaggaat
caacctttga agagagaagt tactggcaat 420 ctcaagtcaa ccaggatatt
tgcaaatatg gatccatccc tcctgatgtg gaggagaagt 480 tacggttgga
atggccctac caggagcaat tgctactccg agaacactac cagaaaaagt 540
tcaagaacag cacctactca agaagctctg tagatgtgct atacactttt gcaaactgct
600 caggactgga cttgatcttt ggcctaaatg cgttattaag aacagcagat
ttgcagtgga 660 acagttctaa tgctcagttg ctcctggact actgctcttc
caaggggtat aacatttctt 720 gggaactagg caatgaacct aacagtttcc
ttaagaaggc tgatattttc atcaatgggt 780 cgcagttagg agaagattat
attcaattgc ataaacttct aagaaagtcc accttcaaaa 840 atgcaaaact
ctatggtcct gatgttggtc agcctcgaag aaagacggct aagatgctga 900
agagcttcct gaaggctggt ggagaagtga ttgattcagt tacatggcat cactactatt
960 tgaatggacg gactgctacc agggaagatt ttctaaaccc tgatgtattg
gacattttta 1020 tttcatctgt gcaaaaagtt ttccaggtgg ttgagagcac
caggcctggc aagaaggtct 1080 ggttaggaga aacaagctct gcatatggag
gcggagcgcc cttgctatcc gacacctttg 1140 cagctggctt tatgtggctg
gataaattgg gcctgtcagc ccgaatggga atagaagtgg 1200 tgatgaggca
agtattcttt ggagcaggaa actaccattt agtggatgaa aacttcgatc 1260
ctttacctga ttattggcta tctcttctgt tcaagaaatt ggtgggcacc aaggtgttaa
1320 tggcaagcgt gcaaggttca aagagaagga agcttcgagt ataccttcat
tgcacaaaca 1380 ctgacaatcc aaggtataaa gaaggagatt taactctgta
tgccataaac ctccataacg 1440 tcaccaagta cttgcggtta ccctatcctt
tttctaacaa gcaagtggat aaataccttc 1500 taagaccttt gggacctcat
ggattacttt ccaaatctgt ccaactcaat ggtctaactc 1560 taaagatggt
ggatgatcaa accttgccac ctttaatgga aaaacctctc cggccaggaa 1620
gttcactggg cttgccagct ttctcatata gtttttttgt gataagaaat gccaaagttg
1680 ctgcttgcat ctgaaaataa aatatactag tcctgacact g 1721 2 543 PRT
Homo sapiens 2 Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu
Met Leu Leu 1 5 10 15 Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly
Ala Leu Pro Arg Pro 20 25 30 Ala Gln Ala Gln Asp Val Val Asp Leu
Asp Phe Phe Thr Gln Glu Pro 35 40 45 Leu His Leu Val Ser Pro Ser
Phe Leu Ser Val Thr Ile Asp Ala Asn 50 55 60 Leu Ala Thr Asp Pro
Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu 65 70 75 80 Arg Thr Leu
Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly 85 90 95 Thr
Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe 100 105
110 Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys
115 120 125 Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu
Glu Trp 130 135 140 Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr
Gln Lys Lys Phe 145 150 155 160 Lys Asn Ser Thr Tyr Ser Arg Ser Ser
Val Asp Val Leu Tyr Thr Phe 165 170 175 Ala Asn Cys Ser Gly Leu Asp
Leu Ile Phe Gly Leu Asn Ala Leu Leu 180 185 190 Arg Thr Ala Asp Leu
Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu 195 200 205 Asp Tyr Cys
Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn 210 215 220 Glu
Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser 225 230
235 240 Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys
Ser 245 250 255 Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly
Gln Pro Arg 260 265 270 Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu
Lys Ala Gly Gly Glu 275 280 285 Val Ile Asp Ser Val Thr Trp His His
Tyr Tyr Leu Asn Gly Arg Thr 290 295 300 Ala Thr Arg Glu Asp Phe Leu
Asn Pro Asp Val Leu Asp Ile Phe Ile 305 310 315 320 Ser Ser Val Gln
Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly 325 330 335 Lys Lys
Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala 340 345 350
Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys 355
360 365 Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln
Val 370 375 380 Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn
Phe Asp Pro 385 390 395 400 Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe
Lys Lys Leu Val Gly Thr 405 410 415 Lys Val Leu Met Ala Ser Val Gln
Gly Ser Lys Arg Arg Lys Leu Arg 420 425 430 Val Tyr Leu His Cys Thr
Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly 435 440 445 Asp Leu Thr Leu
Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr Leu 450 455 460 Arg Leu
Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu 465 470 475
480 Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn
485 490 495 Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro
Leu Met 500 505 510 Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu
Pro Ala Phe Ser 515 520 525 Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys
Val Ala Ala Cys Ile 530 535 540 3 1721 DNA Homo sapiens CDS
(63)..(1691) 3 ctagagcttt cgactctccg ctgcgcggca gctggcgggg
ggagcagcca ggtgagccca 60 ag atg ctg ctg cgc tcg aag cct gcg ctg ccg
ccg ccg ctg atg ctg 107 Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro
Pro Leu Met Leu 1 5 10 15 ctg ctc ctg ggg ccg ctg ggt ccc ctc tcc
cct ggc gcc ctg ccc cga 155 Leu Leu Leu Gly Pro Leu Gly Pro Leu Ser
Pro Gly Ala Leu Pro Arg 20 25 30 cct gcg caa gca cag gac gtc gtg
gac ctg gac ttc ttc acc cag gag 203 Pro Ala Gln Ala Gln Asp Val Val
Asp Leu Asp Phe Phe Thr Gln Glu 35 40 45 ccg ctg cac ctg gtg agc
ccc tcg ttc ctg tcc gtc acc att gac gcc 251 Pro Leu His Leu Val Ser
Pro Ser Phe Leu Ser Val Thr Ile Asp Ala 50 55 60 aac ctg gcc acg
gac ccg cgg ttc ctc atc ctc ctg ggt tct cca aag 299 Asn Leu Ala Thr
Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys 65 70 75 ctt cgt
acc ttg gcc aga ggc ttg tct cct gcg tac ctg agg ttt ggt 347 Leu Arg
Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly 80 85 90 95
ggc acc aag aca gac ttc cta att ttc gat ccc aag aag gaa tca acc 395
Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr 100
105 110 ttt gaa gag aga agt tac tgg caa tct caa gtc aac cag gat att
tgc 443 Phe Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile
Cys 115 120 125 aaa tat gga tcc atc cct cct gat gtg gag gag aag tta
cgg ttg gaa 491 Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu
Arg Leu Glu 130 135 140 tgg ccc tac cag gag caa ttg cta ctc cga gaa
cac tac cag aaa aag 539 Trp Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu
His Tyr Gln Lys Lys 145 150 155 ttc aag aac agc acc tac tca aga agc
tct gta gat gtg cta tac act 587 Phe Lys Asn Ser Thr Tyr Ser Arg Ser
Ser Val Asp Val Leu Tyr Thr 160 165 170 175 ttt gca aac tgc tca gga
ctg gac ttg atc ttt ggc cta aat gcg tta 635 Phe Ala Asn Cys Ser Gly
Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu 180 185 190 tta aga aca gca
gat ttg cag tgg aac agt tct aat gct cag ttg ctc 683 Leu Arg Thr Ala
Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu 195 200 205 ctg gac
tac tgc tct tcc aag ggg tat aac att tct tgg gaa cta ggc 731 Leu Asp
Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly 210 215 220
aat gaa cct aac agt ttc ctt aag aag gct gat att ttc atc aat ggg 779
Asn Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly 225
230 235 tcg cag tta gga gaa gat tat att caa ttg cat aaa ctt cta aga
aag 827 Ser Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg
Lys 240 245 250 255 tcc acc ttc aaa aat gca aaa ctc tat ggt cct gat
gtt ggt cag cct 875 Ser Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp
Val Gly Gln Pro 260 265 270 cga aga aag acg gct aag atg ctg aag agc
ttc ctg aag gct ggt gga 923 Arg Arg Lys Thr Ala Lys Met Leu Lys Ser
Phe Leu Lys Ala Gly Gly 275 280 285 gaa gtg att gat tca gtt aca tgg
cat cac tac tat ttg aat gga cgg 971 Glu Val Ile Asp Ser Val Thr Trp
His His Tyr Tyr Leu Asn Gly Arg 290 295 300 act gct acc agg gaa gat
ttt cta aac cct gat gta ttg gac att ttt 1019 Thr Ala Thr Arg Glu
Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe 305 310 315 att tca tct
gtg caa aaa gtt ttc cag gtg gtt gag agc acc agg cct 1067 Ile Ser
Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro 320 325 330
335 ggc aag aag gtc tgg tta gga gaa aca agc tct gca tat gga ggc gga
1115 Gly Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly
Gly 340 345 350 gcg ccc ttg cta tcc gac acc ttt gca gct ggc ttt atg
tgg ctg gat 1163 Ala Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe
Met Trp Leu Asp 355 360 365 aaa ttg ggc ctg tca gcc cga atg gga ata
gaa gtg gtg atg agg caa 1211 Lys Leu Gly Leu Ser Ala Arg Met Gly
Ile Glu Val Val Met Arg Gln 370 375 380 gta ttc ttt gga gca gga aac
tac cat tta gtg gat gaa aac ttc gat 1259 Val Phe Phe Gly Ala Gly
Asn Tyr His Leu Val Asp Glu Asn Phe Asp 385 390 395 cct tta cct gat
tat tgg cta tct ctt ctg ttc aag aaa ttg gtg ggc 1307 Pro Leu Pro
Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly 400 405 410 415
acc aag gtg tta atg gca agc gtg caa ggt tca aag aga agg aag ctt
1355 Thr Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys
Leu 420 425 430 cga gta tac ctt cat tgc aca aac act gac aat cca agg
tat aaa gaa 1403 Arg Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro
Arg Tyr Lys Glu 435 440 445 gga gat tta act ctg tat gcc ata aac ctc
cat aac gtc acc aag tac 1451 Gly Asp Leu Thr Leu Tyr Ala Ile Asn
Leu His Asn Val Thr Lys Tyr 450 455 460 ttg cgg tta ccc tat cct ttt
tct aac aag caa gtg gat aaa tac ctt 1499 Leu Arg Leu Pro Tyr Pro
Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu 465 470 475 cta aga cct ttg
gga cct cat gga tta ctt tcc aaa tct gtc caa ctc 1547 Leu Arg Pro
Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu 480 485 490 495
aat ggt cta act cta aag atg gtg gat gat caa acc ttg cca cct tta
1595 Asn Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro
Leu 500 505 510 atg gaa aaa cct ctc cgg cca gga agt tca ctg ggc ttg
cca gct ttc 1643 Met Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly
Leu Pro Ala Phe 515 520 525 tca tat agt ttt ttt gtg ata aga aat gcc
aaa gtt gct gct tgc atc 1691 Ser Tyr Ser Phe Phe Val Ile Arg Asn
Ala Lys Val Ala Ala Cys Ile 530 535 540 tgaaaataaa atatactagt
cctgacactg 1721 4 26 DNA Artificial sequence Single strand DNA
oligonucleotide 4 cgcatatgca ggacgtcgtg gacctg 26 5 24 DNA
Artificial sequence Single strand DNA oligonucleotide 5 tatgatcctc
tagtacttct cgac 24 6 35 DNA Artificial sequence Single strand DNA
oligonucleotide 6 aggaattcac catgctgctg cgctcgaagc ctgcg 35 7 22
DNA Artificial sequence Single strand DNA oligonucleotide 7
gagtagcaat tgctcctggt ag 22 8 35 DNA Artificial sequence Single
strand DNA oligonucleotide 8 gtctcgagaa aagacaggac gtcgtggacc tggac
35 9 59 DNA Artificial sequence Single strand DNA oligonucleotide 9
aattcaccat gtctgcactt ctgatcctag ctcttgttgg agctgcagtt gctcaggac 59
10 53 DNA Artificial sequence Single strand DNA oligonucleotide 10
cctgagcaac tgcagctcca acaagagcta ggatcagaag tgcagacatg gtg 53 11 52
DNA Artificial sequence Single strand DNA oligonucleotide 11
aattcaccat gtctgcactt ctgatcctag ctcttgttgg agctgcagtt gc 52 12 50
DNA Artificial sequence Single strand DNA oligonucleotide 12
cggcaactgc agctccaaca agagctagga tcagaagtgc agacatggtg 50 13 4 PRT
Artificial sequence Factor Xa protease recognition and cleavage
site 13 Ile Glu Gly Arg 1 14 5 PRT Artificial sequence Enterokinase
recognition and cleavage site 14 Asp Asp Asp Asp Lys 1 5 15 42 DNA
Artificial sequence Single strand DNA oligonucleotide 15 ccatcgatag
aaggacgaaa aaagttcaag aacagcacct ac 42 16 28 DNA Artificial
sequence Single strand DNA oligonucleotide 16 ggatcgattg gtagtgttct
cggagtag 28 17 39 DNA Artificial sequence Single strand DNA
oligonucleotide 17 ggatcgatag aaggacgatc tcaagtcaac caggatatt 39 18
32 DNA Artificial sequence Single strand DNA oligonucleotide 18
ccatcgatgc ccagtaactt ctctcttcaa ag 32 19 24 DNA Artificial
sequence Single strand DNA oligonucleotide 19 tcagatgcaa gcagcaactt
tggc 24 20 23 DNA Artificial sequence Single strand DNA
oligonucleotide 20 gagcagccag gtgagcccaa gat 23 21 44 DNA
Artificial sequence Single strand DNA oligonucleotide 21 ccatcgatga
cgacgacaag aaaaagttca agaacagcac ctac 44 22 29 DNA Artificial
sequence Single strand DNA oligonucleotide 22 ggatcgatct ggtagtgttc
tcggagtag 29 23 43 DNA Artificial sequence Single strand DNA
oligonucleotide 23 ggatcgatga cgacgacaag tctcaagtca accaggatat ttg
43 24 33 DNA Artificial sequence Single strand DNA oligonucleotide
24 ccatcgattt gggagtaact tctctcttca aag 33 25 5 PRT Artificial
sequence Sequencing product corresponding with the homo sapiens
heparanase protein 25 Ser Gln Val Asn Gln 1 5
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