Title: Methods for preparing
low molecular weight heparin with modified heparinase III
United States Patent: 7,390,633
Issued: June 24, 2008
Inventors: Liu; Dongfang
(Yorktown Heights, NY), Pojasek; Kevin (Cambridge, MA), Shriver; Zachary
(Boston, MA), Holley; Kristine (Boston, MA), El-Shabrawi; Yosuf (Graz,
AT), Venkataraman; Ganesh (Bedford, MA), Sasisekharan; Ram (Bedford, MA)
Institute of Technology (Cambridge, MA)
Appl. No.: 11/406,214
Filed: April 18, 2006
George Washington University's Healthcare MBA
The invention relates to heparinase III
and mutants thereof. Modified forms of heparinase III having reduced
enzymatic activity which are useful for a variety of purposes, including
sequencing of heparin-like glycosaminoglycans (HLGAGs), removing active
heparan sulfate from a solution, inhibition of angiogenesis, etc. have
been discovered according to the invention. The invention in other aspects
relates to methods of treating cancer and inhibiting tumor cell growth
and/or metastasis using heparinase III, or products produced by enzymatic
cleavage by heparinase III of HLGAGs.
Description of the
The invention in some aspects
relates to heparinase III, modified forms thereof and uses thereof. The
invention arose from several scientific findings which expand the field of
heparinase biology. In particular the invention is based in part on the
discovery of new modified forms of heparinase that have varying enzymatic
activity and produce differing product profiles. The invention is also based
on the finding that native heparinase III, modified forms of heparinase III,
and modified forms of heparinase II having heparinase III like activity are
useful for the treatment and prevention of tumor cell growth and metastasis.
The present invention provides a series of new modified heparinase III
molecules. In particular, based upon a detailed structural and functional
characterization of heparinase III, new heparinases with altered stability,
activity and specificity are provided. The modified heparinases of the
invention have many in vivo, in vitro and ex vivo utilities. For instance,
they have great value in generating low molecular weight HLGAGs, heparan
sulfate, or heparan sulfate fragments for clinical use. Additionally they
can be used to neutralize the function of heparan sulfate containing HLGAGs
or they can be used to identify the sequence of HLGAGs. Other uses are
Heparinase III is unique in that it is the only member of the heparinase
family that recognizes and cleaves heparan sulfate as its only substrate.
Heparinase III is also unique among its heparin-degrading family members in
that it contains no cysteines in its primary amino acid sequence (Su, H.,
Blain, F., Musil, R. A., Zimmermann, J. J., Gu, K., and Bennett, D. C.
(1996) Appl. Environ. Micro. 62, 2723-34 and Godavarti, R., Davis, M.,
Venkataraman, G., Cooney, C. L., Langer, R., and Sasisekharan, R. (1996)
Biochem. and Biophys. Res. Comm. 225, 751-58). Heparinase III, however, does
contain thirteen histidines of which one or several might be involved in the
activity of the enzyme. Through a combination of chemical modification,
peptide mapping, and site-directed mutagenesis studies, the role of
histidines in the catalytic activity of heparinase III has been identified,
according to the invention.
The nucleotide and amino acid sequences of heparinase III are provided in
SEQ ID NO: 1 and SEQ ID NO: 2. The sequence of heparinase III has been
reported in Su, H., Blain, F., Musil, R. A., Zimmermann, J. J., Gu, K., and
Bennett, D. C. (1996) Appl. Environ. Micro. 62, 2723-34. and Godavarti, R.,
Davis, M., Venkataraman, G., Cooney, C. L., Langer, R., and Sasisekharan, R.
(1996) Biochem. and Biophys. Res. Comm. 225, 751-58, U.S. Pat. Nos.
5,919,693 and 5,681,733, and is listed in Accession number I71365. These
sequences have provided the first insight into the primary structure of the
native heparinase III of F. heparinum.
The present disclosure provides additional information about the secondary
and tertiary structure of the heparinase III, as well as, information
relating to the functional roles of the various regions of the enzyme. This
information is based upon detailed biochemical mapping of the important
sites within the enzyme and characterization of these sites through kinetic
studies, characterization of mutants created by site-directed mutagenesis,
etc. The result is a detailed picture of the primary, secondary, and
tertiary structures of heparinase III and the functional roles of various
regions of the enzyme as well as the functions of specific mutants thereof.
The invention is based on several scientific findings. It was discovered
according to the invention that various amino acid residues within
heparinase III are essential to the catalytic function of these enzymes and
can be modified to alter the enzymatic activity of these compounds. It was
also discovered that other amino acid residues are absolutely critical to
the function of heparinase III and if they are substituted or modified the
activity of these compounds is lost completely. In particular, it has been
shown according to the invention through a combination of chemical
modification, peptide mapping, and site-directed mutagenesis experiments
that two histidines, histidine 295 and histidine 510, are critical for the
enzymatic degradation of HLGAGs by heparinase III.
As shown in the Examples section, DEPC was used in the first step of the
analysis of heparinase III. DEPC is extremely useful in elucidating the role
of histidines in enzymatic function. Care has to be taken, though, to ensure
that DEPC doesn't modify other nucleophilic amino acids such as tyrosine,
lysine or cysteine (Godavarti, R., Cooney, C. L., Langer, R., and
Sasisekharan, R. (1996) Biochemistry 35, 6846-52 and Shriver, Z., Hu, Y.,
and Sasisekharan, R. (1998) J. Biol. Chem. 273, 10160-67). In the case of
heparinase III, there are no cysteine residues in the primary amino acid
sequence, eliminating this amino acid as a potential confounding factor in
the chemical modification studies. Also, no decrease in the absorbance at
278 nm was observed after heparinase III was incubated with DEPC, indicating
that tyrosine residues were not modified. An increase in the inactivation
kinetics without a change in the order of the reaction was observed from pH
6.0-7.5 upon DEPC treatment. Furthermore, the DEPC modification was 90%
reversible upon incubation with 300 mM hydroxylamine. Above pH 8.0, the
inactivation kinetics were no longer first order for DEPC and the
modification could not be reversed by hydroxylamine, indicating that
residues other than histidines (i.e. lysines) were being modified at those
pHs. However, at neutral pH, the data indicates that DEPC specifically
modifies the histidine residues of heparinase III.
Consistent with the observation that DEPC is modifying a histidine residue,
there was an increase in the absorbance at 240 nm as a function of time.
This is indicative of formation of an N-carbethoxyhistidyl derivative, the
product of a reaction between DEPC and a histidine residue. Over the course
of ten minutes, 1.8 histidine residues were modified and the enzymatic
activity was decreased by 90%. Also, pre-incubation with heparan sulfate
resulted in lower inactivation kinetics of heparinase III by DEPC. These
data indicated that DEPC specifically modified a critical histidine residue
proximate to the substrate binding/active site of heparinase III,
inactivating the enzyme.
An apparent discrepancy arose from these results in that the reaction of
DEPC with heparinase III follows pseudo-first order kinetics, yet two
histidines appeared to be independently modified, suggesting that two
surface accessible histidines react with DEPC at identical rates. It could
be the case that either one or both of the modified residues is responsible
for inactivating the enzyme. Site-directed mutagenesis experiments were
performed to determine if two histidines were essential for heparinase III's
catalytic activity. The results from the site-directed mutagenesis
experiments confirmed and expanded upon the chemical modification data in
that surface accessible histidines are critical for heparinase III activity.
These results identify histidine 295 and histidine 510 as the primary
histidines involved in the degradation of HLGAGs by heparinase III. When
these residues are replaced with alanines, the enzyme loses all activity
towards its substrate. None of the other histidine residues when mutated to
alanine show a complete loss of activity. The results from the peptide
mapping studies confirm the importance of the surface accessibility of
The loss of activity with the H295A and H510A enzymes can be explained in
several ways. It may be that these histidines are necessary for proper
folding of heparinase III. However, the CD spectrum of H295A, H510A, and
recombinant heparinase III were nearly identical, strongly indicating that
this is not the case. It is more likely that histidine 295 and histidine 510
play a direct role in the binding of HLGAGs to the enzyme or that histidine
295 and histidine 510 are critical active site residues directly involved in
the catalytic degradation of HLGAGs. Modified heparinase III molecules
having a change in amino acid at His 295 or 510 can be useful for a variety
of purposes, e.g., as a competitive inhibitor to functional heparinase III.
The studies described in the Examples section also identified several
heparinase III mutants which had altered levels of activity but which were
still active. These mutants include heparinase III molecules having the
following residues mutated or substituted: His36, His105, His110, His139,
His152, His225, His234, His241, His424, His469, and His539. Thus, the
present invention provides for novel modified heparinases rationally
designed on the basis of the sequence of the heparinase III of F. heparinum
and the structural and functional characterizations disclosed herein.
In the description herein, reference is made to the amino acid residues and
residue positions of native heparinase III disclosed in SEQ ID NO 2. In
particular, residues and residue positions are referred to as "corresponding
to" a particular residue or residue position of heparinase III. As will be
obvious to one of ordinary skill in the art, these positions are relative
and, therefore, insertions or deletions of one or more residues would have
the effect of altering the numbering of downstream residues. In particular,
N-terminal insertions or deletions would alter the numbering of all
subsequent residues. Therefore, as used herein, a residue in a recombinant
modified heparinase will be referred to as "corresponding to" a residue of
the full heparinase III if, using standard sequence comparison programs,
they would be aligned. Many such sequence alignment programs are now
available to one of ordinary skill in the art and their use in sequence
comparisons has become standard. As used herein, this convention of
referring to the positions of residues of the recombinant modified
heparinases by their corresponding heparinase III residues shall extend not
only to embodiments including N-terminal insertions or deletions but also to
internal insertions or deletions (e.g., insertions or deletions in "loop"
In addition, in the description herein, certain substitutions of one amino
acid residue for another in a recombinant modified heparinase are referred
to as "conservative substitutions." As used herein, a "conservative amino
acid substitution" or "conservative substitution" refers to an amino acid
substitution in which the substituted amino acid residue is of similar
charge as the replaced residue and is of similar or smaller size than the
replaced residue. Conservative substitutions of amino acids include
substitutions made amongst amino acids within the following groups: (a) the
small non-polar amino acids, A, M, I, L, and V; (b) the small polar amino
acids, G, S, T and C; (c) the amido amino acids, Q and N; (d) the aromatic
amino acids, F, Y and W; (e) the basic amino acids, K, R and H; and (f) the
acidic amino acids, E and D. Substitutions which are charge neutral and
which replace a residue with a smaller residue may also be considered
"conservative substitutions" even if the residues are in different groups
(e.g., replacement of phenylalanine with the smaller isoleucine). The term
"conservative amino acid substitution" also refers to the use of amino acid
analogs or variants.
Methods for making amino acid substitutions, additions or deletions are well
known in the art and are described in detail in the Examples below. The
terms "conservative substitution", "non-conservative substitutions",
"non-polar amino acids", "polar amino acids", and "acidic amino acids" are
all used consistently with the prior art terminology. Each of these terms is
well-known in the art and has been extensively described in numerous
publications, including standard biochemistry text books, such as
"Biochemistry" by Geoffrey Zubay, Addison-Wesley Publishing Co., 1986
edition, which describes conservative and non-conservative substitutions,
and properties of amino acids which lead to their definition as polar,
non-polar or acidic.
Even when it is difficult to predict the exact effect of a substitution in
advance of doing so, one skilled in the art will appreciate that the effect
can be evaluated by routine screening assays, preferably the biological
assays described herein. Modifications of peptide properties including
thermal stability, hydrophobicity, susceptibility to proteolytic degradation
or the tendency-to aggregate with carriers or into multimers are assayed by
methods well known to the ordinarily skilled artisan. For additional
detailed description of protein chemistry and structure, see Schulz, G. E.
et al., Principles of Protein Structure, Springer-Verlag, New York, 1979,
and Creighton, T. E., Proteins: Structure and Molecular Principles, W. H.
Freeman & Co., San Francisco, 1984.
Additionally, some of the amino acid substitutions are non-conservative
substitutions. In certain embodiments where the substitution is remote from
the active or binding sites, the non-conservative substitutions are easily
tolerated provided that they preserve the tertiary structure characteristic
of native heparinase, thereby preserving the active and binding sites.
Non-conservative substitutions, such as between, rather than within, the
above groups (or two other amino acid groups not shown above), which will
differ more significantly in their effect on maintaining (a) the structure
of the peptide backbone in the area of the substitution (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the
In one aspect, the invention is a substantially pure heparinase which is a
modified heparinase III having a modified heparinase III k.sub.cat value,
wherein the modified heparinase III k.sub.cat value is at least 10%
different than a native heparinase III k.sub.cat value. In a preferred
embodiment, the modified heparinase III k.sub.cat value is at least 20%
different than a native heparinase III k.sub.cat value. In another preferred
embodiment the modified heparinase III k.sub.cat value is at least 50%
different than a native heparinase III k.sub.cat value. A "modified
heparinase III k.sub.cat value" as used herein is a measurement of the
catalytic activity of the modified heparinase III enzyme with respect to a
heparan sulfate-like glycosaminoglycan substrate.
The k.sub.cat value may be determined using any enzymatic activity assay
which is useful for assessing the activity of a heparinase enzyme, such as
the assays set forth in the Examples below. Several such assays are
well-known in the art. For instance, an assay for measuring k.sub.cat is
described in (Ernst, S. E., Venkataraman, G., Winkler, S., Godavarti, R.,
Langer, R., Cooney, C. and Sasisekharan. R. (1996) Biochem. J. 315, 589-597.
The "native heparinase III k.sub.cat value" is the measure of enzymatic
activity of the native heparinase III.
The modified heparinase may have a reduced enzymatic activity with respect
to HLGAGs. A "reduced enzymatic activity" is assessed by comparing the
k.sub.cat value of the modified heparinase with that of native heparinase.
Preferably the k.sub.cat value of the modified heparinase III will be less
than or equal to 75% of the native heparinase III k.sub.cat value. A
modified heparinase having reduced enzymatic activity with respect to HLGAGs
is one which has modifications in the residues essential for catalytic
activity. For instance, mutation of His.sup.110 or His.sup.241 causes the
heparinase III to have a reduced enzymatic activity. A modified heparinase
III which has a increased enzymatic activity is one which has altered
residues which produce an enzyme with greater enzymatic activity. For
instance, mutation of His.sup.139 produces modified heparinase III molecules
having increased enzymatic activity. Additionally, when His.sup.225 is
mutated in heparinase III, a modified heparinase III is produced which
displays nearly the same enzymatic activity as native heparinase III. These
enzymes are also useful.
As used herein, with respect to heparinases, the term "substantially pure"
means that the heparinases are essentially free of other substances with
which they may be found in nature or in vivo systems to an extent practical
and appropriate for their intended use. In particular, the heparinases are
sufficiently free from other biological constituents of their hosts cells so
as to be useful in, for example, producing pharmaceutical preparations or
sequencing. Because the heparinases of the invention may be admixed with a
pharmaceutically acceptable carrier in a pharmaceutical preparation, the
heparinase may comprise only a small percentage by weight of the
preparation. The heparinase is nonetheless substantially pure in that it has
been substantially separated from the substances with which it may be
associated in living systems.
Based on the disclosure provided herein, those of ordinary skill in the art
will be able to identify other modified heparinase III molecules having
altered enzymatic activity with respect to the native heparinase III
In another aspect, the invention is a substantially pure heparinase which is
a modified heparinase III having a modified product profile, wherein the
modified product profile of the modified heparinase III is at least 10%
different than a native product profile of a native heparinase III.
Preferably it is at least 20% or even at least 50%. A "modified product
profile" as used herein is a set of degradation products produced by a
modified heparinase which differ from the degradation products which are
produced by a native heparinase under identical enzymatic conditions. The
difference in the product profile may be due to the presence of different
enzymatic products or simply in the number of enzymatic products formed by
the modified heparinase compared to the native heparinase, or a combination
of the two. For instance, the formation of different enzymatic products by a
modified heparinase as opposed to the native heparinase, would constitute a
modified product profile. Additionally, the production of the same types of
enzymatic products but in a lesser or greater amount by the modified
heparinase as opposed to the native heparinase, would also constitute a
modified product profile.
The product profile produced by a modified heparinase or a native heparinase
may be determined by any method known in the art for examining the type or
quantity of degradation product produced by heparinase. One preferred method
for determining the type and quantity of product is described in Rhomberg,
A. J. et al., PNAS, v. 95, p. 4176-4181 (April 1998), which is hereby
incorporated in its entirety by reference. The method disclosed in the
Rhomberg reference utilizes a combination of mass spectrometry and capillary
electrophoretic techniques to identify the enzymatic products produced by
heparinase. The Rhomberg study utilizes heparinase to degrade HLGAGs to
produce HLGAG oligosaccharides. MALDI (Matrix-Assisted Laser Desorption
Ionization) mass spectrometry can be used for the identification and
semiquantitative measurement of substrates, enzymes, and end products in the
enzymatic reaction. The capillary electrophoresis technique separates the
products to resolve even small differences amongst the products and is
applied in combination with mass spectrometry to quantitate the products
produced. Capillary electrophoresis may even resolve the difference between
a disaccharide and its semicarbazone derivative. Detailed methods for
sequencing polysaccharides and other polymers are disclosed in co-pending
U.S. patent applications Ser. Nos. 09/557,997 and 09/558,137, both filed on
Apr. 24, 2000 and having common inventorship. The entire contents of both
applications are hereby incorporated by reference.
Briefly, the method is performed by enzymatic digestion, followed by mass
spectrometry and capillary electrophoresis. The enzymatic assays can be
performed in a variety of manners, as long as the assays are performed
identically on the modified heparinase and the native heparinase, so that
the results may be compared. In the example described in the Rhomberg
reference, enzymatic reactions are performed by adding 1 mL of enzyme
solution to 5 mL of substrate solution. The digestion is then carried out at
room temperature (22.degree. C.), and the reaction is stopped at various
time points by removing 0.5 mL of the reaction mixture and adding it to 4.5
mL of a MALDI matrix solution, such as caffeic acid (approximately 12 mg/mL)
and 70% acetonitrile/water. The reaction mixture is then subjected to MALDI
mass spectrometry. The MALDI surface is prepared by the method of Xiang and
Beavis (Xiang and Beavis (1994) Rapid. Commun. Mass. Spectrom. 8, 199-204).
A two-fold lower access of basic peptide (Arg/Gly).sub.15 is premixed with
matrix before being added to the oligosaccharide solution. A 1 mL aliquot of
sample/matrix mixture containing 1-3 picomoles of oligosaccharide is
deposited on the surface. After crystallization occurs (typically within 60
seconds), excess liquid is rinsed off with water. MALDI mass spectrometry
spectra is then acquired in the linear mode by using a PerSeptive Biosystems
(Framingham, Mass.) Voyager Elite reflectron time-of-flight instrument
fitted with a 337 nanometer nitrogen laser. Delayed extraction is used to
increase resolution (22 kV, grid at 93%, guidewire at 0.15%, pulse delay 150
ns, low mass gate at 1,000, 128 shots averaged). Mass spectra are calibrated
externally by using the signals for proteinated (Arg/Gly).sub.15 and its
complex with the oligosaccharide.
Capillary electrophoresis is then performed on a Hewlett-Packard.sup.3D CE
unit by using uncoated fused silica capillaries (internal diameter 75
micrometers, outer diameter 363 micrometers, 1.sub.det 72.1 cm, and
1.sub.tot 85 cm). Analytes are monitored by using UV detection at 230 nm and
an extended light path cell (Hewlett-Packard). The electrolyte is a solution
of 10 mL dextran sulfate and 50 millimolar Tris/phosphoric acid (pH2.5).
Dextran sulfate is used to suppress nonspecific interactions of the heparin
oligosaccharides with a silica wall. Separations are carried out at 30 kV
with the anode at the detector side (reversed polarity). A mixture of a
1/5-naphtalenedisulfonic acid and 2-naphtalenesulfonic acid (10 micromolar
each) is used as an internal standard.
Other methods for assessing the product profile may also be utilized. For
instance, other methods include methods which rely on parameters such as
viscosity (Jandik, K. A., Gu, K. and Linhardt, R. J., (1994), Glycobiology,
4:284-296) or total UV absorbance (Ernst, S. et al., (1996), Biochem. J.,
315:589-597) or mass spectrometry or capillary electrophoresis alone.
The modified heparinases of the invention may be used for any of the same
purposes as native heparinase III. For instance, the modified heparinase III
molecules can be used to specifically cleave a HLGAG by contacting the HLGAG
substrate with one of the modified heparinases of the invention. The
invention is useful in a variety of in vitro, in vivo and ex vivo methods in
which it is useful to cleave HLGAGs.
The modified heparinase III may be used, for instance, in a method for
inhibiting angiogenesis. In this method an effective amount for inhibiting
angiogenesis of the heparinase III is administered to a subject in need of
treatment thereof. Angiogenesis as used herein is the inappropriate
formation of new blood vessels. "Angiogenesis" often occurs in tumors when
endothelial cells secrete a group of growth factors that are mitogenic for
endothelium causing the elongation and proliferation of endothelial cells
which results in a generation of new blood vessels. Several of the
angiogenic mitogens are heparin or heparan sulfate binding peptides which
are related to endothelial cell growth factors.
The modified heparinases are also useful for treating or preventing cancer
cell growth or metastasis. This aspect of the invention is discussed in more
detail below, with respect to both native and modified heparinase III.
The modified heparinases are also useful for inhibiting neovascularization
associated with disease such as eye disease. Neovascularization, or
angiogenesis, is the growth and development of new arteries. It is critical
to the normal development of the vascular system, including injury-repair.
There are, however, conditions characterized by abnormal neovascularization,
including diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis,
and certain cancers. For example, diabetic retinopathy is a leading cause of
blindness. There are two types of diabetic retinopathy, simple and
proliferative. Proliferative retinopathy is characterized by
neovascularization and scarring. About one-half of those patients with
proliferative retinopathy progress to blindness within about five years.
Another example of abnormal neovascularization is that associated with solid
tumors. It is now established that unrestricted growth of tumors is
dependant upon angiogenesis, and that induction of angiogenesis by
liberation of angiogenic factors can be an important step in carcinogenesis.
For example, basic fibroblast growth factor (bFGF) is liberated by several
cancer cells and plays a crucial role in cancer angiogenesis. As used
herein, an angiogenic condition means a disease or undesirable medical
condition having a pathology including neovascularization. Such diseases or
conditions include diabetic retinopathy, neovascular glaucoma and rheumatoid
arthritis (non-cancer angiogenic conditions). Cancer angiogenic conditions
are solid tumors and cancers or tumors otherwise associated with
neovascularization such as hemangioendotheliomas, hemangiomas and Kaposi's
Proliferation of endothelial and vascular smooth muscle cells is the main
feature of neovascularization. Thus the modified heparinase III of the
invention is useful for preventing proliferation and, therefore, inhibiting
or arresting altogether the progression of the angiogenic condition which
depends in whole or in part upon such neovascularization.
Neovascularization and angiogenesis are also important in a number of other
pathological processes, including arthritis, psoriasis, diabetic
retinopathy, chronic inflammation, scleroderma, hemangioma, retrolental
fibroplasia and abnormal capillary proliferation in hemophiliac joints,
prolonged menstruation and bleeding, and other disorders of the female
reproductive system (J. Folkman, Nature Medicine, Vol 1, p. 27-31, (1995);
J. W. Miller, et al., J. Pathol., Vol. 145, pp. 574-584 (1994); A. P. Adamid,
et al., Amer. J. Ophthal., Vol. 118, pp. 445-450 (1994); K. Takahashi, at
al., J. Clin. Invest., Vol. 93, pp. 2357-2364 (1994); D. J. Peacock, et al.,
J. Exp. Med., Vol. 175, pp. 1135-1138 (1992); B. J. Nickoloff, et al., Amer.
J. Pathol., Vol. 44, pp. 820-828 (1994); J. Folkman, Steroid Hormones and
Uterine Bleeding, N. J. Alexander and C. d'Arcangues, Eds., American
Association for the Advancement of Science Press, Washington, D.C., U.S.A.,
pp. 144-158 (1992)). Thus, in another embodiment, the modified heparinase is
administered to treat diseases such as psoriasis. Psoriasis is a common
dermatological disease caused by chronic inflammation.
The H295A and H510A modified heparinases are also useful according to the
invention as inhibitors of heparinase III activity. These modified
heparinases have a minimum one base pair modification from native heparinase
but have no enzymatic activity. Thus, modified heparinases having a H295A or
H510A modification can be used as competitive inhibitors of native or
functional modified forms of heparinase III. These compounds are useful any
time it is desirable to block heparinase III activity, e.g., when cell
proliferation and migration is desirable or to block the activity of
heparinase III in a solution.
The modified heparinases of the invention are also useful as tools for
sequencing HLGAGs. Detailed methods for sequencing polysaccharides and other
polymers are disclosed in co-pending U.S. patent applications Ser. Nos.
09/557,997 and 09/558,137, both filed on Apr. 24, 2000 and having common
inventorship. These methods utilize tools such as heparinases in the
sequencing process. The modified heparinase III of the invention is useful
as such a tool.
The modified heparinases of the invention may also be used to remove active
HLGAGs from a HLGAG containing fluid. A HLGAG containing fluid is contacted
with the modified heparinase III of the invention to degrade the HLGAG. The
method is particularly useful for the ex vivo removal of HLGAGs from blood.
In one embodiment of the invention the modified heparinase is immobilized on
a solid support as is conventional in the art. The solid support containing
the immobilized modified heparinase may be used in extracorporeal medical
devices (e.g. hemodialyzer, pump-oxygenator) for systemic heparinization to
prevent the blood in the device from clotting. The support membrane
containing immobilized heparinase III is positioned at the end of the device
to neutralize the HLGAG before the blood is returned to the body.
In another aspect, the invention is an immobilized substantially pure
heparinase of the invention. The heparinase may be immobilized to any type
of support but if the support is to be used in vivo or ex vivo it is desired
that the support is sterile and biocompatible. A biocompatible support is
one which would not cause an immune or other type of damaging reaction when
used in a subject. The heparinase may be immobilized by any method known in
the art. Many methods are known for immobilizing proteins to supports.
The heparinase III is, in some embodiments, immobilized on a solid support.
A "solid support" as used herein refers to any solid material to which a
protein can be immobilized. Solid supports, for example, include but are not
limited to membranes, e.g., natural and modified celluloses such as
nitrocellulose or nylon, Sepharose, Agarose, glass, polystyrene,
polypropylene, polyethylene, dextran, amylases, polyacrylamides,
polyvinylidene difluoride, other agaroses, and magnetite, including magnetic
beads. The carrier can be totally insoluble or partially soluble and may
have any possible structural configuration. Thus, the support may be
spherical, as in a bead, or cylindrical, as in the inside surface of a test
tube or microplate well, or the external surface of a rod. Alternatively,
the surface may be flat such as a sheet, test strip, bottom surface of a
microplate well, etc.
The modified heparinase III molecules are also useful for generating LMWHs
which have many therapeutic utilities. The modified heparinase III molecules
and LMWH can be used for the treatment of any type of condition in which
LMWH therapy has been identified as a useful therapy, e.g., preventing
coagulation, preventing psoriasis.
Thus, the modified heparinase molecules are useful for treating or
preventing disorders associated with coagulation. A "disease associated with
coagulation" as used herein refers to a condition characterized by local
inflammation resulting from an interruption in the blood supply to a tissue
due to a blockage of the blood vessel responsible for supplying blood to the
tissue such as is seen for myocardial or cerebral infarction. A cerebral
ischemic attack or cerebral ischemia is a form of ischemic condition in
which the blood supply to the brain is blocked. This interruption in the
blood supply to the brain may result from a variety of causes, including an
intrinsic blockage or occlusion of the blood vessel itself, a remotely
originated source of occlusion, decreased perfusion pressure or increased
blood viscosity resulting in inadequate cerebral blood flow, or a ruptured
blood vessel in the subarachnoid space or intracerebral tissue.
The methods of the invention are useful also for treating cerebral ischemia.
Cerebral ischemia may result in either transient or permanent deficits and
the seriousness of the neurological damage in a patient who has experienced
cerebral ischemia depends on the intensity and duration of the ischemic
event. A transient ischemic attack is one in which the blood flow to the
brain is interrupted only briefly and causes temporary neurological
deficits, which often are clear in less than 24 hours. Symptoms of TIA
include numbness or weakness of face or limbs, loss of the ability to speak
clearly and/or to understand the speech of others, a loss of vision or
dimness of vision, and a feeling of dizziness. Permanent cerebral ischemic
attacks, also called stroke, are caused by a longer interruption in blood
flow to the brain resulting from either a thromboembolism. A stroke causes a
loss of neurons typically resulting in a neurologic deficit that may improve
but that does not entirely resolve. Thromboembolic stroke is due to the
occlusion of an extracranial or intracranial blood vessel by a thrombus or
embolus. Because it is often difficult to discern whether a stroke is caused
by a thrombosis or an embolism, the term "thromboembolism" is used to cover
strokes caused by either of these mechanisms.
The methods of the invention in some embodiments are directed to the
treatment of acute thromboembolic stroke using modified heparinase III or
the LMWHs generated therewith. An acute stroke is a medical syndrome
involving neurological injury resulting from an ischemic event, which is an
interruption in the blood supply to the brain.
An effective amount of a modified heparinase III or the LMWHs generated
therewith alone or in combination with another therapeutic for the treatment
of stroke is that amount sufficient to reduce in vivo brain injury resulting
from the stroke. A reduction of brain injury is any prevention of injury to
the brain which otherwise would have occurred in a subject experiencing a
thromboembolic stroke absent the treatment of the invention. Several
physiological parameters may be used to assess reduction of brain injury,
including smaller infarct size, improved regional cerebral blood flow, and
decreased intracranial pressure, for example, as compared to pretreatment
patient parameters, untreated stroke patients or stroke patients treated
with thrombolytic agents alone.
The modified heparinase III or the LMWHs generated therewith may be used
alone or in combination with a therapeutic agent for treating a disease
associated with coagulation. Examples of therapeutics useful in the
treatment of diseases associated with coagulation include anticoagulation
agents, antiplatelet agents, and thrombolytic agents.
Anticoagulation agents prevent the coagulation of blood components and thus
prevent clot formation. Anticoagulants include, but are not limited to,
heparin, warfarin, coumadin, dicumarol, phenprocoumon, acenocoumarol, ethyl
biscoumacetate, and indandione derivatives.
Antiplatelet agents inhibit platelet aggregation and are often used to
prevent thromboembolic stroke in patients who have experienced a transient
ischemic attack or stroke. Antiplatelet agents include, but are not limited
to, aspirin, thienopyridine derivatives such as ticlopodine and clopidogrel,
dipyridamole and sulfinpyrazone, as well as RGD mimetics and also
antithrombin agents such as, but not limited to, hirudin.
Thrombolytic agents lyse clots which cause the thromboembolic stroke.
Thrombolytic agents have been used in the treatment of acute venous
thromboembolism and pulmonary emboli and are well known in the art (e.g. see
Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S (1995);
Holmes, et al, J Am Coll Cardiol; v.25 (7 suppl), p. 10S-17S(1995)).
Thrombolytic agents include, but are not limited to, plasminogen,
a.sub.2-antiplasmin, streptokinase, antistreplase, tissue plasminogen
activator (tPA), and urokinase. "tPA" as used herein includes native tPA and
recombinant tPA, as well as modified forms of tPA that retain the enzymatic
or fibrinolytic activities of native tPA. The enzymatic activity of tPA can
be measured by assessing the ability of the molecule to convert plasminogen
to plasmin. The fibrinolytic activity of tPA may be determined by any in
vitro clot lysis activity known in the art, such as the purified clot lysis
assay described by Carlson, et. al., Anal. Biochem. 168, 428-435 (1988) and
its modified form described by Bennett, W. F. Et al., 1991, Supra, the
entire contents of which are hereby incorporated by reference.
The invention also relates to the discovery that heparinase III, modified
forms thereof, modified forms of heparinase II and degradation products of
heparinases (HLGAG fragments) actually are useful for treating and
preventing cancer cell proliferation and metastasis. Thus, according to
another aspect of the invention, there is provided methods for treating
subjects having or at risk of having cancer.
Heparinases degrade HLGAGs, which are linear polysaccharides characterized
by a disaccharide-repeat unit of a uronic acid [.alpha.-L-iduronic acid (I)
or .beta.-D-glucuronic acid (G)] linked 1,4 to .alpha.-D-hexosamine (H).
HLGAGs are the most acidic, heterogeneous and information dense biopolymer
found in nature due to the highly variable chemical modification of the
disaccharide repeat unit--primarily in the form of sulfation at the N--, 3O
and 6O positions of H, and the 2O of the uronic acids. Critically, HLGAGs
(along with collagen) are key components of the cell surface-extracellular
matrix (ECM) interface. While collagen-like proteins provide the necessary
extracellular scaffold for cells to attach and form tissues, the complex
polysaccharides fill the space created by the scaffold and act as a
molecular sponge by specifically binding and regulating the biological
activities of numerous signaling molecules like growth factors, cytokines
etc. It has recently been recognized that cells synthesize distinct HLGAG
sequences and decorate themselves with these sequences, using the
extraordinary information content present in the sequences to bind
specifically to many signaling molecules and thereby regulate various
Tumor metastasis involves the spread of tumor cells primarily via the
vasculature following the disassembly of tumor cell-ECM interactions through
the degradation of the ECM, and tumor cell extravasation through the
capillary bed. Recent evidence has suggested that collagen (and related
proteins), enzymes (collagenases and others) that degrade the proteinaceous
component of the ECM may play roles in the regulation of tumor angiogenesis
or tumor cell invasion of the ECM. However, the chemical heterogeneity of
complex polysaccharides and lack of effective tools, has seriously limited
investigations into the roles of HLGAGs in tumor growth and metastasis.
Interestingly, however, in parallel with collagen and the proteases, it has
been hypothesized that HLGAG degrading enzymes (heparinases) assist in the
breakdown of ECM to promote tumor growth, angiogenesis and metastasis. Other
evidence such as the recent cloning of tumor heparinase genes has led to the
paradigm that, the expression of HLGAG degrading enzymes represents a
`switch` from a primary tumor to a metastatic disease state.
In surprising contrast to the findings of the prior art, it has now been
discovered according to the invention that not only is the prior art
incorrect in stating that HLGAG degrading enzymes may contribute to tumor
growth and metastasis, but in fact that certain HLGAG degrading enzymes and
HLGAG fragments (including LMWH compositions generated by heparinase III),
actually, are very effective in inhibiting cancer cell growth and
metastasis. In particular, it has been discovered that heparinases having
similar functional activity to native heparinase III prevent in vivo tumor
growth and metastasis. It has also been discovered that the enzymatic
products of heparinase III (HLGAG fragments and LMWH) are useful for
preventing tumor growth and metastasis.
The Examples section provides in vitro and in vivo data demonstrating the
effectiveness of the heparinases in preventing tumor growth and metastasis.
Using two different animal models of cancer, B16BL6 and LLC, strikingly
similar data was obtained, indicating an important role for HLGAGs in tumor
growth and metastasis. The data also demonstrated the differential effects
of heparinases I and III, and the HLGAG fragments generated by these
heparinases on physiological processes. Heparinase I was unable to prevent
cancer cell proliferation or metastasis, indicating that the effects are
specific to heparinase III and functional variants thereof. These results
are consistent with the unique specificities of heparinases, and hence the
distinct oligosaccharide products they generate. Additionally, the data
demonstrated that HLGAG fragments for one cell type were able to influence
effects on another cell type, strongly indicating the involvement of
specific sequences of HLGAG in modulating effects on tumor growth and
Thus, the invention includes methods for treating or preventing tumor
formation and/or metastasis by administering to a subject a heparinase III
molecule (native or modified) and/or therapeutic HLGAG fragments (including
The heparinases useful in this aspect of the invention include native
heparinase III, modified heparinase III and modified heparinases having the
functional activity of heparinase III. "Native heparinase III" as used
herein refers to the naturally occurring heparinase III molecule in an
isolated form. The sequence of the naturally occurring molecule from F.
heparinum is provided as SEQ ID NO.: 1 (nucleic acid sequence) and 2 (amino
acid sequence), and has been extensively described in art including in
issued patents. An isolated molecule is a molecule that is substantially
pure and is free of other substances with which it is ordinarily found in
nature or in vivo systems to an extent practical and appropriate for its
intended use. In particular, the molecular species are sufficiently pure and
are sufficiently free from other biological constituents of host cells so as
to be useful in, for example, producing pharmaceutical preparations or
sequencing if the molecular species is a nucleic acid, peptide, or
polysaccharide. Because an isolated molecular species of the invention may
be admixed with a pharmaceutically-acceptable carrier in a pharmaceutical
preparation, the molecular species may comprise only a small percentage by
weight of the preparation. The molecular species is nonetheless
substantially pure in that it has been substantially separated from the
substances with which it may be associated in living systems.
A "modified heparinase III" as used herein is any heparinase III molecule
which has at least one mutation, deletion or substitution, compared to
native heparinase III but which retains the ability to enzymatically cleave
heparan sulfate. These include the particular modified heparinases described
herein as well as any other modified heparinase having the appropriate
function. These can be identified by those of ordinary skill in the art
using the methods described above or in the examples section. For instance,
the modified heparinase III may have a simple conservative substitution
within a region of the molecule which is not critical for enzymatic activity
or folding and thus which has no effect on the ability of the heparinase to
cleave the substrate. Additionally, substitutions such as the histidine
substitutions described herein which influence the enzymatic activity or
product profile of the heparinase but which still retain some enzymatic
activity are also useful for this aspect of the invention because they are
still able to cleave heparan sulfate. The two histidine mutations (His 295
and His 510) which lost all activity, however, are not useful in this aspect
of the invention. (These two mutants have other utilities, such as
The term "modified heparinases having functional activity of heparinase III"
as used herein refers to heparinases other than heparinase III which have
been modified such that they are enzymatically active towards heparan
sulfate but only have minimal or no activity towards heparin. For instance,
mutation of Cys.sup.348 of heparinase II, a residue which is involved in
heparin binding, causes the heparinase II to have a reduced enzymatic
activity with respect to heparin. This modification produces a modified
heparinase II which becomes exclusively a heparan sulfate degrading enzyme.
Additionally, when histidine 440 is mutated in heparinase III, a modified
heparinase III is produced which has reduced enzymatic activity with respect
to heparin but which displays nearly the same enzymatic activity as native
heparinase III when heparan sulfate is used as the substrate. Mutation of
histidines 451, 238, and 579 of heparinase II produces modified heparinase
II molecules having reduced enzymatic activity with respect to heparan
sulfate. Thus modified heparinase II molecules in which the Cys.sup.348 or
His.sup.440 is mutated are "modified heparinases having functional activity
of heparinase III" according to the invention, whereas heparinases in which
histidines 451, 238, or 579 have been mutated are not within this class of
The invention also contemplates the use of therapeutic HLGAGs for the
treatment and prevention of tumor cell proliferation and metastasis. A
therapeutic HLGAG fragment as used herein refers to a molecule or molecules
which are pieces or fragments of an HLGAG that have been identified through
the use of the native heparinase III, modified heparinase III and modified
heparinases having the functional activity of heparinase III described
above. HLGAG fragments also include low molecular weight heparins (LMWHs).
The compositional analysis of some therapeutic HLGAGs is described below in
the Examples section.
The invention also encompasses screening assays for identifying therapeutic
HLGAG fragments for the treatment of a tumor and for preventing metastasis.
The assays are accomplished by treating a tumor or isolated tumor cells with
heparinase III, native or modified and isolating the resultant HLGAG
fragments. Surprisingly, these HLGAG fragments have therapeutic activity in
the prevention of tumor cell proliferation and metastasis. As described in
more detail in the Examples section, these HLGAG fragments are useful as
therapeutic agents for the treatment of the tumor cells from which they were
generated as well as other tumors. Thus the invention encompasses
individualized therapies, in which a tumor or portion of a tumor is isolated
from a subject and used to prepare the therapeutic HLGAG fragments. These
therapeutic fragments can be re-administered to the subject to protect the
subject from further tumor cell proliferation or metastasis or from the
initiation of metastasis if the tumor is not yet metastatic. Alternatively
the fragments can be used in a different subject having the same type or
tumor or a different type of tumor.
The term "therapeutic HLGAG fragment" as used herein refers to an HLGAG
which has therapeutic activity in that it prevents the proliferation and/or
metastasis of a tumor cell. Such compounds can be generated using heparinase
III to produce therapeutic fragments or they can be synthesized de novo.
Putative HLGAG fragments can be tested for therapeutic activity using any of
the assays described herein or known in the art. Thus the therapeutic HLGAG
fragment may be a synthetic HLGAG fragment generated based on the sequence
of the HLGAG fragment identified when the tumor is contacted with heparinase
III, or having minor variations which do not interfere with the activity of
the compound. Alternatively the therapeutic HLGAG fragment may be an
isolated HLGAG fragment produced when the tumor is contacted with heparinase
The invention is useful for treating and/or preventing tumor cell
proliferation or metastasis in a subject. The terms "prevent" and
"preventing" as used herein refer to inhibiting completely or partially the
proliferation or metastasis of a cancer or tumor cell, as well as inhibiting
any increase in the proliferation or metastasis of a cancer or tumor cell.
A "subject having a cancer" is a subject that has detectable cancerous
cells. The cancer may be a malignant or non-malignant cancer. Cancers or
tumors include but are not limited to biliary tract cancer; brain cancer;
breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial
cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms;
lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell);
melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer;
prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer;
thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.
A "subject at risk of having a cancer" as used herein is a subject who has a
high probability of developing cancer. These subjects include, for instance,
subjects having a genetic abnormality, the presence of which has been
demonstrated to have a correlative relation to a higher likelihood of
developing a cancer and subjects exposed to cancer causing agents such as
tobacco, asbestos, or other chemical toxins, or a subject who has previously
been treated for cancer and is in apparent remission. When a subject at risk
of developing a cancer is treated with a heparinase III the subject may be
able to kill the cancer cells as they develop.
Effective amounts of the native heparinase III, modified heparinases, or
therapeutic HLGAGs of the invention are administered to subjects in need of
such treatment. Effective amounts are those amounts which will result in a
desired reduction in cellular proliferation or metastasis without causing
other medically unacceptable side effects. Such amounts can be determined
with no more than routine experimentation. It is believed that doses ranging
from 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode
of administration, will be effective. The absolute amount will depend upon a
variety of factors (including whether the administration is in conjunction
with other methods of treatment, the number of doses and individual patient
parameters including age, physical condition, size and weight) and can be
determined with routine experimentation. It is preferred generally that a
maximum dose be used, that is, the highest safe dose according to sound
medical judgment. The mode of administration may be any medically acceptable
mode including oral, subcutaneous, intravenous, etc.
In some aspects of the invention the effective amount of heparinase III is
that amount effective to prevent invasion of a tumor cell across a barrier.
The invasion and h6metastasis of cancer is a complex process which involves
changes in cell adhesion properties which allow a transformed cell to invade
and migrate through the extracellular matrix (ECM) and acquire
anchorage-independent growth properties. Liotta, L. A., et al., Cell
64:327-336 (1991). Some of these changes occur at focal adhesions, which are
cell/ECM contact points containing membrane-associated, cytoskeletal, and
intracellular signaling molecules. Metastatic disease occurs when the
disseminated foci of tumor cells seed a tissue which supports their growth
and propagation, and this secondary spread of tumor cells is responsible for
the morbidity and mortality associated with the majority of cancers. Thus
the term "metastasis" as used herein refers to the invasion and migration of
tumor cells away from the primary tumor site.
The barrier for the tumor cells may be an artificial barrier in vitro or a
natural barrier in vivo. In vitro barriers include e but are not limited to
extracellular matrix coated membranes, such as Matrigel. Thus the heparinase
compositions can be tested for their ability to inhibit tumor cell invasion
in a Matrigel invasion assay system as described in detail by Parish, C. R.,
et al., "A Basement-Membrane Permeability Assay which Correlates with the
Metastatic Potential of Tumour Cells," Int. J. Cancer (1992) 52:378-383.
Matrigel is a reconstituted basement membrane containing type IV collagen,
laminin, heparan sulfate proteoglycans such as perlecan, which bind to and
localize bFGF, vitronectin as well as transforming growth factor-.beta. (TGF-.beta.),
urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA),
and the serpin known as plasminogen activator inhibitor type 1 (PAI-1).
Other in vitro and in vivo assays for metastasis have been described in the
prior art, see, e.g., U.S. Pat. No. 5,935,850, issued on Aug. 10, 1999,
which is incorporated by reference. An in vivo barrier refers to a cellular
barrier present in the body of a subject.
In general, when administered for therapeutic purposes, the formulations of
the invention are applied in pharmaceutically acceptable solutions. Such
preparations may routinely contain pharmaceutically acceptable
concentrations of salt, buffering agents, preservatives, compatible
carriers, adjuvants, and optionally other therapeutic ingredients.
The compositions of the invention may be administered per se (neat) or in
the form of a pharmaceutically acceptable salt. When used in medicine the
salts should be pharmaceutically acceptable, but non-pharmaceutically
acceptable salts may conveniently be used to prepare pharmaceutically
acceptable salts thereof and are not excluded from the scope of the
invention. Such pharmacologically and pharmaceutically acceptable salts
include, but are not limited to, those prepared from the following acids:
hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic,
salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic,
malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also,
pharmaceutically acceptable salts can be prepared as alkaline metal or
alkaline earth salts, such as sodium, potassium or calcium salts of the
carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric
acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and
phosphoric acid and a salt (0.8-2% W/V). Suitable preservatives include
benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V);
parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).
The present invention provides pharmaceutical compositions, for medical use,
which comprise native heparinase III, modified heparinases of the invention,
or therapeutic HLGAG fragments together with one or more pharmaceutically
acceptable carriers and optionally other therapeutic ingredients. The term
"pharmaceutically-acceptable carrier" as used herein, and described more
fully below, means one or more compatible solid or liquid filler, dilutants
or encapsulating substances which are suitable for administration to a human
or other animal. In the present invention, the term "carrier" denotes an
organic or inorganic ingredient, natural or synthetic, with which the active
ingredient is combined to facilitate the application. The components of the
pharmaceutical compositions also are capable of being commingled with the
modified heparinases of the present invention, and with each other, in a
manner such that there is no interaction which would substantially impair
the desired pharmaceutical efficiency.
A variety of administration routes are available. The particular mode
selected will depend, of course, upon the particular modified heparinase
selected, the particular condition being treated and the dosage required for
therapeutic efficacy. The methods of this invention, generally speaking, may
be practiced using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of an immune response
without causing clinically unacceptable adverse effects. A preferred mode of
administration is a parenteral route. The term "parenteral" includes
subcutaneous injections, intravenous, intramuscular, intraperitoneal, intra
sternal injection or infusion techniques. Other modes of administration
include oral, mucosal, rectal, vaginal, sublingual, intranasal,
intratracheal, inhalation, ocular, transdermal, etc.
For oral administration, the compounds can be formulated readily by
combining the active compound(s) with pharmaceutically acceptable carriers
well known in the art. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a subject
to be treated. Pharmaceutical preparations for oral use can be obtained as
solid excipient, optionally grinding a resulting mixture, and processing the
mixture of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat starch,
rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added,
such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a
salt thereof such as sodium alginate. Optionally the oral formulations may
also be formulated in saline or buffers for neutralizing internal acid
conditions or may be administered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for identification or to characterize different combinations
of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit
capsules made of gelatin, as well as soft, sealed capsules made of gelatin
and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can
contain the active ingredients in admixture with filler such as lactose,
binders such as starches, and/or lubricants such as talc or magnesium
stearate and, optionally, stabilizers. In soft capsules, the active
compounds may be dissolved or suspended in suitable liquids, such as fatty
oils, liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. Microspheres formulated for oral administration
may also be used. Such microspheres have been well defined in the art. All
formulations for oral administration should be in dosages suitable for such
For buccal administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present invention may be conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebulizer, with the use of a
suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a pressurized aerosol the dosage unit may be determined by providing a
valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin
for use in an inhaler or insufflator may be formulated containing a powder
mix of the compound and a suitable powder base such as lactose or starch.
The compounds, when it is desirable to deliver them systemically, may be
formulated for parenteral administration by injection, e.g., by bolus
injection or continuous infusion. Formulations for injection may be
presented in unit dosage form, e.g., in ampoules or in multi-dose
containers, with an added preservative. The compositions may take such forms
as suspensions, solutions or emulsions in oily or aqueous vehicles, and may
contain formulatory agents such as suspending, stabilizing and/or dispersing
Pharmaceutical formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form. Additionally,
suspensions of the active compounds may be prepared as appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include
fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate or triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension, such as
sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents which increase
the solubility of the compounds to allow for the preparation of highly
Alternatively, the active compounds may be in powder form for constitution
with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal or vaginal compositions such
as suppositories or retention enemas, e.g., containing conventional
suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also
be formulated as a depot preparation. Such long acting formulations may be
formulated with suitable polymeric or hydrophobic materials (for example as
an emulsion in an acceptable oil) or ion exchange resins, or as sparingly
soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel
phase carriers or excipients. Examples of such carriers or excipients
include but are not limited to calcium carbonate, calcium phosphate, various
sugars, starches, cellulose derivatives, gelatin, and polymers such as
Suitable liquid or solid pharmaceutical preparation forms are, for example,
aqueous or saline solutions for inhalation, microencapsulated, encochleated,
coated onto microscopic gold particles, contained in liposomes, nebulized,
aerosols, pellets for implantation into the skin, or dried onto a sharp
object to be scratched into the skin. The pharmaceutical compositions also
include granules, powders, tablets, coated tablets, (micro)capsules,
suppositories, syrups, emulsions, suspensions, creams, drops or preparations
with protracted release of active compounds, in whose preparation excipients
and additives and/or auxiliaries such as disintegrants, binders, coating
agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers
are customarily used as described above. The pharmaceutical compositions are
suitable for use in a variety of drug delivery systems. For a brief review
of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which
is incorporated herein by reference.
The compositions may conveniently be presented in unit dosage form and may
be prepared by any of the methods well known in the art of pharmacy. All
methods include the step of bringing the active modified heparinase into
association with a carrier which constitutes one or more accessory
ingredients. In general, the compositions are prepared by uniformly and
intimately bringing the polymer into association with a liquid carrier, a
finely divided solid carrier, or both, and then, if necessary, shaping the
product. The polymer may be stored lyophilized.
Other delivery systems can include time-release, delayed release or
sustained release delivery systems. Such systems can avoid repeated
administrations of the heparinases of the invention, increasing convenience
to the subject and the physician. Many types of release delivery systems are
available and known to those of ordinary skill in the art. They include
polymer based systems such as polylactic and polyglycolic acid,
polyanhydrides and polycaprolactone; nonpolymer systems that are lipids
including sterols such as cholesterol, cholesterol esters and fatty acids or
neutral fats such as mono-, di and triglycerides; hydrogel release systems;
silastic systems; peptide based systems; wax coatings, compressed tablets
using conventional binders and excipients, partially fused implants and the
like. Specific examples include, but are not limited to: (a) erosional
systems in which the polysaccharide is contained in a form within a matrix,
found in U.S. Pat. Nos. 4,452,775 (Kent); U.S. Pat. No. 4,667,014 (Nestor et
al.); and U.S. Pat. Nos. 4,748,034 and 5,239,660 (Leonard) and (b)
diffusional systems in which an active component permeates at a controlled
rate through a polymer, found in U.S. Pat. No. 3,832,253 (Higuchi et al.)
and U.S. Pat. No. 3,854,480 (Zaffaroni). In addition, a pump-based hardware
delivery system can be used, some of which are adapted for implantation.
A subject is any human or non-human vertebrate, e.g., dog, cat, horse, cow,
When administered to a patient undergoing cancer treatment, the heparinase
III compounds may be administered in cocktails containing other anti-cancer
agents. The compounds may also be administered in cocktails containing
agents that treat the side-effects of radiation therapy, such as
anti-emetics, radiation protectants, etc.
Anti-cancer drugs that can be co-administered with the compounds of the
invention include, but are not limited to Acivicin; Aclarubicin; Acodazole
Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine;
Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole;
Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin;
Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide
Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine;
Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;
Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil;
Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide;
Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride;
Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone;
Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene
Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine
Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin
Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine;
Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate;
Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;
Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin
Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin
Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon
Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a;
Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide
Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol
Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;
Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;
Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;
Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;
Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;
Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel;
Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide;
Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane;
Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride;
Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide;
Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;
Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;
Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan Sodium;
Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone;
Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine;
Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine
Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole
Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine
Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine
Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate;
Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin;
The heparinase III compounds may also be linked to a targeting molecule. A
targeting molecule is any molecule or compound which is specific for a
particular cell or tissue and which can be used to direct the heparinase III
to the cell or tissue. Preferably the targeting molecule is a molecule which
specifically interacts with a cancer cell or a tumor. For instance, the
targeting molecule may be a protein or other type of molecule that
recognizes and specifically interacts with a tumor antigen.
Tumor-antigens include Melan-A/M.quadrature.ART-1, Dipeptidyl peptidase IV (DPPIV),
adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal
associated antigen (CRC)--C017-1A/GA733, Carcinoembryonic Antigen (CEA) and
its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific
Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3,
prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain,
MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,
MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,
MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,
MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g.,
GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9),
BAGE, RAGE, LAGE-B, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family,
HER2/neu, p2lras, RCAS1, .alpha.-fetoprotein, E-cadherin, .alpha.-catenin,
.beta.-catenin and .gamma.-catenin, p120ctn, gp100.sup.Pmel117, PRAME,
NY-ESO-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1,
SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatous polyposis coli protein (APC),
fodrin, P1A, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides,
viral products such as human papilloma virus proteins, Smad family of tumor
antigens, lmp-1, EBV-encoded nuclear antigen (EBNA)-1, and c-erbB-2.
Examples of tumor antigens which bind to either or both MHC class I and MHC
class II molecules, see the following references: Coulie, Stem Cells
13:393-403, 1995; Traversari et al., J. Exp. Med. 176:1453-1457, 1992; Chaux
et al., J. Immunol. 163:2928-2936, 1999; Fujie et al., Int. J. Cancer
80:169-172, 1999; Tanzarella et al., Cancer Res. 59:2668-2674, 1999; van der
Bruggen et al., Eur. J. Immunol. 24:2134-2140, 1994; Chaux et al., J. Exp.
Med. 189:767-778, 1999; Kawashima et al, Hum. Immunol. 59:1-14, 1998; Tahara
et al., Clin. Cancer Res. 5:2236-2241, 1999; Gaugler et al., J. Exp. Med.
179:921-930, 1994; van der Bruggen et al., Eur. J. Immunol. 24:3038-3043,
1994; Tanaka et al., Cancer Res. 57:4465-4468, 1997; Oiso et al., Int. J.
Cancer 81:387-394, 1999; Herman et al., Immunogenetics 43:377-383, 1996;
Manici et al., J. Exp. Med. 189:871-876, 1999; Duffour et al., Eur. J.
Immunol. 29:3329-3337, 1999; Zorn et al., Eur. J. Immunol. 29:602-607, 1999;
Huang et al., J. Immunol.162:6849-6854, 1999; Boel et al., Immunity
2:167-175, 1995; Van den Eynde et al., J. Exp. Med. 182:689-698, 1995; De
Backer et al., Cancer Res. 59:3157-3165, 1999; Jager et al., J. Exp. Med.
187:265-270, 1998; Wang et al., J. Immunol. 161:3596-3606, 1998; Aarnoudse
et al., Int. J. Cancer 82:442-448, 1999; Guilloux et al., J. Exp. Med.
183:1173-1183, 1996; Lupetti et al., J. Exp. Med. 188:1005-1016, 1998;
Wolfel et al., Eur. J. Immunol. 24:759-764, 1994; Skipper et al., J. Exp.
Med. 183:527-534, 1996; Kang et al., J. Immunol. 155:1343-1348, 1995; Morel
et al., Int. J. Cancer 83:755-759, 1999; Brichard et al., Eur. J. Immunol.
26:224-230, 1996; Kittlesen et al., J. Immunol. 160:2099-2106, 1998;
Kawakami et al., J. Immunol. 161:6985-6992, 1998; Topalian et al., J. Exp.
Med. 183:1965-1971, 1996; Kobayashi et al., Cancer Research 58:296-301,
1998; Kawakami et al., J. Immunol. 154:3961-3968, 1995; Tsai et al., J.
Immunol. 158:1796-1802, 1997; Cox et al., Science 264:716-719, 1994;
Kawakami et al., Proc. Natl. Acad. Sci. USA 91:6458-6462, 1994; Skipper et
al., J. Immunol. 157:5027-5033, 1996; Robbins et al., J. Immunol.
159:303-308, 1997; Castelli et al, J. Immunol. 162:1739-1748, 1999; Kawakami
et al., J. Exp. Med. 180:347-352, 1994; Castelli et al., J. Exp. Med.
181:363-368, 1995; Schneider et al., Int. J. Cancer 75:451-458, 1998; Wang
et al., J. Exp. Med. 183:1131-1140, 1996; Wang et al., J. Exp. Med.
184:2207-2216, 1996; Parkhurst et al., Cancer Research 58:4895-4901, 1998;
Tsang et al., J. Natl Cancer Inst 87:982-990, 1995; Correale et al., J. Natl
Cancer Inst 89:293-300, 1997; Coulie et al., Proc. Natl. Acad. Sci. USA
92:7976-7980, 1995; Wolfel et al., Science 269:1281-1284, 1995; Robbins et
al., J. Exp. Med. 183:1185-1192, 1996; Brandle et al., J. Exp. Med.
183:2501-2508, 1996; ten Bosch et al., Blood 88:3522-3527, 1996; Mandruzzato
et al., J. Exp. Med. 186:785-793, 1997; Gueguen et al., J. Immunol.
160:6188-6194, 1998; Gjertsen et al., Int. J. Cancer 72:784-790, 1997;
Gaudin et al., J. Immunol. 162:1730-1738, 1999; Chiari et al., Cancer Res.
59:5785-5792, 1999; Hogan et al., Cancer Res. 58:5144-5150, 1998; Pieper et
al., J. Exp. Med. 189:757-765, 1999; Wang et al., Science 284:1351-1354,
1999; Fisk et al., J. Exp. Med. 181:2109-2117, 1995; Brossart et al., Cancer
Res. 58:732-736, 1998; Ropke et al., Proc. Natl. Acad. Sci. USA
93:14704-14707, 1996; Ikeda et al., Immunity 6:199-208, 1997; Ronsin et al.,
J. Immunol. 163:483-490, 1999; Vonderheide et al., Immunity 10:673-679,1999.
These antigens as well as others are disclosed in PCT Application
One of ordinary skill in the art, in light of the present disclosure, is
enabled to produce substantially pure preparations of any of the native or
modified heparinases by standard technology, including recombinant
technology, direct synthesis, mutagenesis, etc. For instance, using
recombinant technology one may substitute appropriate codons in SEQ ID NO: 1
to produce the desired amino acid substitutions by standard site-directed
mutagenesis techniques. Obviously, one may also use any sequence which
differs from SEQ ID NO: 1 only due to the degeneracy of the genetic code as
the starting point for site directed mutagenesis. The mutated nucleic acid
sequence may then be ligated into an appropriate expression vector and
expressed in a host such as F. heparinum or E. coli. The resultant modified
heparinase may then be purified by techniques well known in the art,
including those disclosed below and in Sasisekharan, et al. (1993). As used
herein, the term "substantially pure" means that the proteins are
essentially free of other substances to an extent practical and appropriate
for their intended use. In particular, the proteins are sufficiently pure
and are sufficiently free from other biological constituents of their hosts
cells so as to be useful in, for example, protein sequencing, or producing
In another set of embodiments an isolated nucleic acid encoding the
substantially pure modified heparinase of the invention is provided. As used
herein with respect to nucleic acids, the term "isolated" means: (i)
amplified in vitro by, for example, polymerase chain reaction (PCR); (ii)
recombinantly produced by cloning; (iii) purified, as by cleavage and gel
separation; or (iv) synthesized by, for example, chemical synthesis. An
isolated nucleic acid is one which is readily manipulable by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence contained in a
vector in which 5' and 3' restriction sites are known or for which
polymerase chain reaction (PCR) primer sequences have been disclosed is
considered isolated but a nucleic acid sequence existing in its native state
in its natural host is not. An isolated nucleic acid may be substantially
purified, but need not be. For example, a nucleic acid that is isolated
within a cloning or expression vector is not pure in that it may comprise
only a tiny percentage of the material in the cell in which it resides. Such
a nucleic acid is isolated, however, as the term is used herein because it
is readily manipulable by standard techniques known to those of ordinary
skill in the art.
As used herein, a coding sequence and regulatory sequences are said to be "operably
joined" when they are covalently linked in such a way as to place the
expression or transcription of the coding sequence under the influence or
control of the regulatory sequences. If it is desired that the coding
sequences be translated into a functional protein the coding sequences are
operably joined to regulatory sequences. Two DNA sequences are said to be
operably joined if induction of a promoter in the 5' regulatory sequences
results in the transcription of the coding sequence and if the nature of the
linkage between the two DNA sequences does not (1) result in the
introduction of a frame-shift mutation, (2) interfere with the ability of
the promoter region to direct the transcription of the coding sequences, or
(3) interfere with the ability of the corresponding RNA transcript to be
translated into a protein. Thus, a promoter region would be operably joined
to a coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting transcript might
be translated into the desired protein or polypeptide.
The precise nature of the regulatory sequences needed for gene expression
may vary between species or cell types, but shall in general include, as
necessary, 5' non-transcribing and 5' non-translating sequences involved
with initiation of transcription and translation respectively, such as a
TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5'
non-transcribing regulatory sequences will include a promoter region which
includes a promoter sequence for transcriptional control of the operably
joined gene. Promoters may be constitutive or inducible. Regulatory
sequences may also include enhancer sequences or upstream activator
sequences, as desired.
As used herein, a "vector" may be any of a number of nucleic acids into
which a desired sequence may be inserted by restriction and ligation for
transport between different genetic environments or for expression in a host
cell. Vectors are typically composed of DNA although RNA vectors are also
available. Vectors include, but are not limited to, plasmids and phagemids.
A cloning vector is one which is able to replicate in a host cell, and which
is further characterized by one or more endonuclease restriction sites at
which the vector may be cut in a determinable fashion and into which a
desired DNA sequence may be ligated such that the new recombinant vector
retains its ability to replicate in the host cell. In the case of plasmids,
replication of the desired sequence may occur many times as the plasmid
increases in copy number within the host bacterium, or just a single time
per host as the host reproduces by mitosis. In the case of phage,
replication may occur actively during a lytic phase or passively during a
lysogenic phase. An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it is
operably joined to regulatory sequences and may be expressed as an RNA
transcript. Vectors may further contain one or more marker sequences
suitable for use in the identification of cells which have or have not been
transformed or transfected with the vector. Markers include, for example,
genes encoding proteins which increase or decrease either resistance or
sensitivity to antibiotics or other compounds, genes which encode enzymes
whose activities are detectable by standard assays known in the art (e.g.,
.beta.-galactosidase or alkaline phosphatase), and genes which visibly
affect the phenotype of transformed or transfected cells, hosts, colonies or
plaques. Preferred vectors are those capable of autonomous replication and
expression of the structural gene products present in the DNA segments to
which they are operably joined.
As used herein, the term "stringent conditions" refers to parameters known
to those skilled in the art. One example of stringent conditions is
hybridization at 65.degree. C. in hybridization buffer (3.5.times.SSC, 0.02%
Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin (BSA), 25 mM
NaH.sub.2PO.sub.4 (pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium
chloride/0.15M sodium citrate, pH7; SDS is sodium dodecylsulphate; and EDTA
is ethylene diamine tetra acetic acid. There are other conditions, reagents,
and so forth which can be used, which result in the same degree of
stringency. A skilled artisan will be familiar with such conditions, and
thus they are not given here. The skilled artisan also is familiar with the
methodology for screening cells for expression of such molecules, which then
are routinely isolated, followed by isolation of the pertinent nucleic acid.
Thus, homologs and alleles of the substantially pure modified heparinases of
the invention, as well as nucleic acids encoding the same, may be obtained
routinely, and the invention is not intended to be limited to the specific
For prokaryotic systems, plasmid vectors that contain replication sites and
control sequences derived from a species compatible with the host may be
used. Examples of suitable plasmid vectors include pBR322, pUC18, pUC19 and
the like; suitable phage or bacteriophage vectors include .lamda.gt10,
.lamda.gt11 and the like; and suitable virus vectors include pMAM-neo, pKRC
and the like. Preferably, the selected vector of the present invention has
the capacity to autonomously replicate in the selected host cell. Useful
prokaryotic hosts include bacteria such as E. coli, Flavobacterium heparinum,
Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like.
To express the substantially pure modified heparinases of the invention in a
prokaryotic cell, it is necessary to operably join the nucleic acid sequence
of a substantially pure modified heparinase of the invention to a functional
prokaryotic promoter. Such promoter may be either constitutive or, more
preferably, regulatable (i.e., inducible or derepressible). Examples of
constitutive promoters include the int promoter of bacteriophage .lamda.,
the bla promoter of the .beta.-lactamase gene sequence of pBR322, and the
CAT promoter of the chloramphenicol acetyl transferase gene sequence of
pPR325, and the like. Examples of inducible prokaryotic promoters include
the major right and left promoters of bacteriophage .lamda. (P.sub.L and
P.sub.R), the trp, recA, lacZ, lacI, and gal promoters of E. coli, the
.alpha.-amylase (Ulmanen et al., J. Bacteriol. 162:176-182 (1985)) and the
.xi.-28-specific promoters of B. subtilis (Gilman et al., Gene sequence
32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan,
In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)),
and Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478
Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol. 1:277-282
(1987)); Cenatiempo (Biochimie 68:505-516 (1986)); and Gottesman (Ann. Rev.
Genet. 18:415-442 (1984)).
Proper expression in a prokaryotic cell also requires the presence of a
ribosome binding site upstream of the encoding sequence. Such ribosome
binding sites are disclosed, for example, by Gold et al. (Ann. Rev.
Microbiol. 35:365-404 (1981)).
Because prokaryotic cells will not produce the modified heparinases of the
invention with normal eukaryotic glycosylation, expression of the modified
heparinases of the invention of the invention by eukaryotic hosts is
possible when glycosylation is desired. Preferred eukaryotic hosts include,
for example, yeast, fungi, insect cells, and mammalian cells, either in vivo
or in tissue culture. Mammalian cells which may be useful as hosts include
HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of
lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8,
and their derivatives. Preferred mammalian host cells include SP2/0 and
J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide
better capacities for correct post-translational processing. Embryonic cells
and mature cells of a transplantable organ also are useful according to some
aspects of the invention.
In addition, plant cells are also available as hosts, and control sequences
compatible with plant cells are available, such as the nopaline synthase
promoter and polyadenylation signal sequences.
Another preferred host is an insect cell, for example in Drosophila larvae.
Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter
can be used (Rubin, Science 240:1453-1459 (1988)). Alternatively,
baculovirus vectors can be engineered to express large amounts of the
modified heparinases of the invention in insects cells (Jasny, Science
238:1653 (1987); Miller et al., In: Genetic Engineering (1986), Setlow, J.
K., et al., eds., Plenum, Vol. 8, pp. 277-297).
Any of a series of yeast gene sequence expression systems which incorporate
promoter and termination elements from the genes coding for glycolytic
enzymes and which are produced in large quantities when the yeast are grown
in media rich in glucose may also be utilized. Known glycolytic gene
sequences can also provide very efficient transcriptional control signals.
Yeast provide substantial advantages in that they can also carry out
post-translational peptide modifications. A number of recombinant DNA
strategies exist which utilize strong promoter sequences and high copy
number plasmids which can be utilized for production of the desired proteins
in yeast. Yeast recognize leader sequences on cloned mammalian gene sequence
products and secrete peptides bearing leader sequences (i.e., pre-peptides).
A wide variety of transcriptional and translational regulatory sequences may
be employed, depending upon the nature of the host. The transcriptional and
translational regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, simian virus, or the like, where the
regulatory signals are associated with a particular gene sequence which has
a high level of expression. Alternatively, promoters from mammalian
expression products, such as actin, collagen, myosin, and the like, may be
employed. Transcriptional initiation regulatory signals may be selected
which allow for repression or activation, so that expression of the gene
sequences can be modulated. Of interest are regulatory signals which are
temperature-sensitive so that by varying the temperature, expression can be
repressed or initiated, or which are subject to chemical (such as
As discussed above, expression of the modified heparinases of the invention
in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such
regions will, in general, include a promoter region sufficient to direct the
initiation of RNA synthesis. Preferred eukaryotic promoters include, for
example, the promoter of the mouse metallothionein I gene sequence (Hamer et
al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus
(McKnight, Cell 31:355-365 (1982)); the SV40 early promoter (Benoist et al.,
Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter
(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silveret
al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).
As is widely known, translation of eukaryotic mRNA is initiated at the codon
which encodes the first methionine. For this reason, it is preferable to
ensure that the linkage between a eukaryotic promoter and a DNA sequence
which encodes the modified heparinases of the invention does not contain any
intervening codons which are capable of encoding a methionine (i.e., AUG).
The presence of such codons results either in the formation of a fusion
protein (if the AUG codon is in the same reading frame as the modified
heparinases of the invention coding sequence) or a frame-shift mutation (if
the AUG codon is not in the same reading frame as the modified heparinases
of the invention coding sequence).
In one embodiment, a vector is employed which is capable of integrating the
desired gene sequences into the host cell chromosome. Cells which have
stably integrated the introduced DNA into their chromosomes can be selected
by also introducing one or more markers which allow for selection of host
cells which contain the expression vector. The marker may, for example,
provide for prototrophy to an auxotrophic host or may confer biocide
resistance to, e.g., antibiotics, heavy metals, or the like. The selectable
marker gene sequence can either be directly linked to the DNA gene sequences
to be expressed, or introduced into the same cell by co-transfection.
Additional elements may also be needed for optimal synthesis of the modified
heparinases of the invention mRNA. These elements may include splice
signals, as well as transcription promoters, enhancers, and termination
signals. cDNA expression vectors incorporating such elements include those
described by Okayama, Molec. Cell. Biol. 3:280 (1983).
In a preferred embodiment, the introduced sequence will be incorporated into
a plasmid or viral vector capable of autonomous replication in the recipient
host. Any of a wide variety of vectors may be employed for this purpose.
Factors of importance in selecting a particular plasmid or viral vector
include: the ease with which recipient cells that contain the vector may be
recognized and selected from those recipient cells which do not contain the
vector; the number of copies of the vector which are desired in a particular
host; and whether it is desirable to be able to "shuttle" the vector between
host cells of different species. Preferred prokaryotic vectors include
plasmids such as those capable of replication in E. coli (such as, for
example, pBR322, ColE1, pSC101, pACYC 184, and .pi.VX. Such plasmids are,
for example, disclosed by Sambrook, et al. (Molecular Cloning: A Laboratory
Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring
Harbor Laboratory, 1989)). Bacillus plasmids include pC194, pC221, pT127,
and the like. Such plasmids are disclosed by Gryczan (In: The Molecular
Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable
Streptomyces plasmids include pIJ101 (Kendall et al., J. Bacteriol.
169:4177-4183 (1987)), and streptomyces bacteriophages such as .phi.C31 (Chater
et al., In: Sixth International Symposium on Actinomycetales Biology,
Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids
are reviewed by John et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki
(Jpn. J. Bacteriol. 33:729-742 (1978)).
Preferred eukaryotic plasmids include, for example, BPV, EBV, SV40, 2-micron
circle, and the like, or their derivatives. Such plasmids are well known in
the art (Botstein et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, In:
The Molecular Biology of the Yeast Saccharomyces: Life Cycle and
Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p.
445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon et al., J. Clin.
Hematol. Oncol. 10:39-48 (1980); Maniatis, In: Cell Biology. A Comprehensive
Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608
(1980)). Other preferred eukaryotic vectors are viral vectors. For example,
and not by way of limitation, the pox virus, herpes virus, adenovirus and
various retroviruses may be employed. The viral vectors may include either
DNA or RNA viruses to cause expression of the insert DNA or insert RNA.
Additionally, DNA or RNA encoding the modified heparinases of the invention
polypeptides may be directly injected into cells or may be impelled through
cell membranes after being adhered to microparticles.
Once the vector or DNA sequence containing the construct(s) has been
prepared for expression, the DNA construct(s) may be introduced into an
appropriate host cell by any of a variety of suitable means, i.e.,
transformation, transfection, conjugation, protoplast fusion,
electroporation, calcium phosphate-precipitation, direct microinjection, and
the like. After the introduction of the vector, recipient cells are grown in
a selective medium, which selects for the growth of vector-containing cells.
Expression of the cloned gene sequence(s) results in the production of the
modified heparinases of the invention. This can take place in the
transformed cells as such, or following the induction of these cells to
differentiate (for example, by administration of bromodeoxyuracil to
neuroblastoma cells or the like).
Claim 1 of 11 Claims
1. A method for preparing low molecular
weight heparin (LMWH), comprising: contacting sample comprising a linear
polysaccharide with a disaccharide repeat unit of a uronic acid
[.alpha.-L-iduronic acid (I) or .beta.-D-glucuronic acid (G)] linked 1, 4
to .alpha.-D-hexosamine (H) with a protein comprising a modified
heparinase III to produce LMWH, wherein the modified heparinase III has
the amino acid sequence of the mature peptide of SEQ ID NO: 2, wherein at
least one histidine residue selected from the group consisting of His36,
His105, His110, His139, His152, His225, His234, His241, His424, His469,
and His539 has been substituted with a residue selected from the group
consisting of alanine, serine, tyrosine, threonine, and lysine.
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