Note: Descriptions are shown in the official language in which they were submitted.
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RECOMBINANT a-L-IDURONIDASE, METHODS FOR PRODUCING AND
PURIFYING THE SAME AND METHODS FOR TREATING
DISEASES CAUSED BY DEFICIENCIES THEREOF
FIELD OF THE INVENTION
The present invention is in 'the field of molecular biology, enzymology,
biochemistry and
clinical medicine. In particular, the present invention provides a recombinant
a-L-iduronidase,
methods to produce and purify this enzyme as well as methods to treat certain
genetic disorders
including a-L-iduronidase deficiency and mucopolysaccharidosis I (MPS I).
BACKGROUND OF THE INVENTION
Carbohydrates play a number of important roles in the functioning of living
organisms. In
addition to their metabolic roles, carbohydrates are structural components of
the human body
covalently attached to numerous other entities such as proteins and lipids
(called glycoconjugates).
For example, human connective tissues and cell membranes comprise proteins,
carbohydrates and a
proteoglycan matrix. The carbohydrate portion of this proteoglycan matrix
provides important
properties to the body's structure.
A genetic deficiency of the carbohydrate-cleaving, lysosomal enzyme a-L-
iduronidase causes
a lysosomal storage disorder known as mucopolysaccharidosis I (MPS I)
(Neufeld, E. F., and
Muenzer, J. (1989). 'The mucopoly,saccharidoses in "The Metabolic Basis of
Inherited Disease"
(Scriver, C.R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), pp. 1565-
1587, MeGraw-Hill, New
York). In a severe form, MPS I is commonly known as Hurler syndrome and is
associated with
multiple problems such as mental retardation, clouding of the cornea,
coarsened facial features,
cardiac disease, respiratory disease, liver and spleen enlargement, hernias,
and joint stiffness.
Patients suffering, from Hurler syndrome usually die before age 10. In an
intermediate form known
as Hurler-Scheie syndrome, mental function is generally not severely affected,
but physical problems
may lead to death by the teens or twenties. Scheie syndrome is the mildest
form of MPS I. It is
compatible with a normal life span, but joint stiffness, corneal clouding and
heart valve disease cause
significant problems.
The frequency of MPS I is estimated to be 1:100,000 according to a British
Columbia survey
of all newborns (Lowry et al., Human Genetics 85:389-390 (1990)) and 1:70,000
according to an Irish
study (Nelson, Human Genetics 101:355-358 (1990)). There appears to be no
ethnic predilection for
this disease. It is likely that worldwide the disease is underdiagnosed either
because the patient dies
of a complication before the diagnosis is made or because the milder forms of
the syndrome may be
CA 02328518 2000-11-10
WO 99/58691 PCT/US99/10102
mistaken for arthritis or missed entirely. Effective newborn screening for MPS
I would likely find
some previously undetected patients.
Except for bone marrow transplantation, there are no significant therapies
available for
MPS I. Bone marrow transplants can be effective in treating some of the
symptoms of the disorder
but have high morbidity and mortality in MPS I and often are not available to
patients because of a
lack of suitable donors. An alternative therapy available to all affected
patients would provide an
important breakthrough in treating and managing this disease.
Enzyme replacement therapy has long been considered a potential therapy for
MPS I
following the discovery that a-L-iduronidase can correct the enzymatic defect
in Hurler cells in
culture. In this corrective process, tlhe enzyme containing a mannose-6-
phosphate residue is taken up
into cells through receptor-mediated. endocytosis and transported to the
lysosomes where it clears the
stored substrates, heparan sulfate and dermatan sulfate. Application of this
therapy to humans has
previously not been possible due to inadequate sources of a-L-iduronidase in
tissues. The enzyme
replacement concept was first effectively applied to Gaucher patients in a
modified placental
glucocerebrosidase. The delivery and effective uptake of glucocerebrosidase in
Gaucher patients
demonstrated that an enzyme could be taken up in vivo in sufficient quantities
to provide effective
therapy.
For a-L-iduronidase enzyme therapy in MPS I, a recombinant source of enzyme
has been
needed in order to obtain therapeutically sufficient supplies of the enzyme.
The mammalian enzyme
was cloned in 1990 (Stoltzfus et al., J. Biol. Chem. 267:6570-6575 (1992), and
the human enzyme
was cloned in 1991 (Moskowitz et a!l., FASEB J 6:A77 (1992)).
DESCRIPTION OF THE FIGURES
FIGURE 1 represents the nucleotide and deduced amino acid sequences of cDNA
encoding
a-L-iduronidase. Nucleotides 1 through 6200 are provided. Amino acids are
provided starting with
the first methionine in the open reading frame.
FIGURE 2 represents the results from an SDS-PAGE run of eluate obtained
according to the
procedure set forth in Example 1. Lane 1 is blank. Lane 2 contained high
molecular weight
standards. Lane 3 is a blank. Lane 4 contained bovine serum albumin in a
concentration of 50 wg.
Lanes S through 10 represent eluate containing recombinantly produced human a-
L-iduronidase in
amounts of 1 pg, 2 pg, S ug, 5 pg, ~~ p,g and 5 fig, respectively.
FIGURE 3 reveals the urinary GAG levels in 16 MPS I patients in relation to
normal excretion
values. There is a wide range of urine GAG values in untreated MPS I patients.
A greater than SO%
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reduction in excretion of undegraded GAGs following therapy with recombinant a-
L-iduronidase is a
valid means to measure an individualf's response to therapy.
FIGURE 4 demonstrates leukocyte iduronidase activity before and after enzyme
therapy in
MPS I patients.
FIGURE 5 demonstrates the buccal iduronidase activity before and after enzyme
therapy.
FIGURE 6 demonstrates in three patients that a substantial shrinkage of liver
and spleen
together with significant clinical improvement in joint and soft tissue
storage was associated with a
greater than 65% reduction in undegraded GAG after only 8 weeks of treatment
with recombinant
enzyme.
FIGURE 7 demonstrates that; there is substantial normalization of livers and
spleens in
patients treated with recombinant enzyme after only 12 weeks of therapy.
FIGURE 8 demonstrates a precipitous drop in urinary GAG excretion over 22
weeks of therapy
with recombinant enzyme in 6 patients.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention features a method to produce a-L-
iduronidase in amounts
which enable using the enzyme therapeutically. In a broad embodiment, the
method comprises the
step of transfecting a cDNA encoding for all or a part of an a-L-iduronidase
into a cell suitable for the
expression thereof. In some embodiments, a cDNA encoding for a complete a-L-
iduronidase is used,
preferably a human a-L-iduronidase. However, in other embodiments, a cDNA
encoding for a
biologically active fragment or mutant thereof may be used. Specifically, one
or more amino acid
substitutions may be made while preserving or enhancing the biological
activity of the enzyme. In
other preferred embodiments, an expression vector is used to transfer the cDNA
into a suitable cell or
cell line for expression thereof. In one particularly preferred embodiment,
the cDNA is transfected
into a Chinese hamster ovary cell to create cell line 2.131. W yet other
preferred embodiments, the
production procedure features one or more of the following characteristics
which have demonstrated
particularly high production levels: (a) the pH of the cell growth culture may
be lowered to about 6.5
to 7.0, preferably to about 6.7-6.8 during the production process, (b) about
2/3 to 3/4 of the medium
may be changed approximately every 12 hours, (c) oxygen saturation may be
optimized at about 80%
using intermittent pure oxygen sparging, (d) microcarriers with about 10%
serum initially may be
used to produce cell mass followed by a rapid washout shift to protein-free
medium for production,
(e) a protein-free or low protein medium such as a JRH Biosciences PF-CHO
product may be
optimized to include supplemental amounts of one or more ingredients selected
from the group
consisting of glutamate, aspartate, glycine, ribonucleosides and
deoxyribonucleosides, (f) a perfusion
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wand such as a Bellco perfusion wand rnay be used in a frequent batch-feed
process rather than a
standard intended perfusion process, and (g) a mild sodium butyrate induction
process may be used to
induceinereased a-L-iduronidase expression.
In a second aspect, the present invention provides a transfected cell line
which features the
ability to produce a-L-iduronidase in amounts which enable using the enzyme
therapeutically. In
preferred embodiments, the present invention features a recombinant Chinese
hamster ovary cell line
such as the 2.131 cell line that stabl:y and reliably produces amounts of a-L-
iduronidase which enable
using the enzyme therapeutically. In some preferred embodiments, the cell line
may contain at least
about 10 copies of a an expression construct. In even more preferred
embodiments, the cell line
expresses recombinant a-L-iduronidase in amounts of at least about 20-40
micrograms per 10' cells
per day.
In a third aspect, the present invention provides novel vectors suitable to
produce
a-L-iduronidase in amounts which enable using the enzyme therapeutically. In
preferred
embodiments, the present invention features an expression vector comprising a
cytomegalovirus
promoter/enhancer element, a 5' intron consisting of a murine Ca intron, a
cDNA encoding all or a
fragment or mutant of an a-L-iduronidase, and a 3' bovine growth hormone
polyadenylation site.
Aiso, preferably the cDNA encoding all or a fragment or mutant of an a-L-
iduronidase is about
2.2 kb in length. This expression vc;ctor rnay be transfected at, for example,
a 50 to 1 ratio with any
appropriate common selection vector such as, for example, pSV2NE0, to enhance
multiple copy
insertions. Alternatively, gene amplification may be used to induce multiple
copy insertions.
In a fourth aspect, the present invention provides novel a-L-iduronidase
produced in
accordance with the methods of the present invention and thereby present in
amounts which enable
using the enzyme therapeutically. 'lChe specific activity of the a-L-
iduronidase according to the
present invention is in excess of 200,000 units per milligram protein.
Preferably, it is in excess of
about 240,000 units per milligram protein. The molecular weight of the a-L-
iduronidase of the
present invention is about 82,000 daltons, about 70,000 daltons being amino
acid and about 12,000
daltons being carbohydrates.
In a fifth aspect, the presemt invention features a novel method to purify a-L-
iduronidase.
According to a first embodiment, a cell mass rnay be grown in about 10% serum
containing medium
followed by a switch to a modified protein-free production medium without any
significant adaptation
to produce a high specific activity starting material for purification.
Preferably, a
concentration/diafiltration scheme is employed that allows for the removal of
exogenous materials
that may be required for recombinant production of the same such as, for
example, Pluronics F-68, a
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commonly used surfactant for protecsting cells from sparging damage. Such
exogenous materials
should normally be separated from t:he crude bulk to prevent fouling of the
columns. In another
prefenred embodiment, a first ~olurnn load is acidified to minimize the
competitive inhibition effect of
uronic acids found in protein-free medium formulations. Also preferably, a
heparin, phenyl and
sizing column purification scheme is used to produce pure enzyme using
automatable steps and
validatable media. In another preferred embodiment, the heparin and phenyl
column steps are used to
eliminate less desirable a-L-iduronidase that is nicked or degraded. In
another preferred
embodiment, an acid pH treatment step is used to inactivate potential viruses
without harming the
enzyme.
In a sixth aspect, the present. invention features novel methods of treating
diseases caused all
or in part by a deficiency in a-L-iduronidase. In one embodiment, this method
features administering
a recombinant a-L-iduronidase or a biologically active fragment or mutant
thereof alone or in
combination with a pharmaceutically suitable carrier. In other embodiments,
this method features
transferring a nucleic acid encoding all or a part of an a-L-iduronidase into
one or more host cells in
vivo. Preferred embodiments include optimizing the dosage to the needs of the
organism to be
treated, preferably mammals or humans, to effectively ameliorate the disease
symptoms. In prefer ed
embodiments, the disease is mucopolysaccharidosis 1 (MPS I), Hurler syndrome,
Hurler-Scheie
syndrome or Scheie syndrome.
In a seventh aspect, the present invention features novel pharmaceutical
compositions
comprising a-L-iduronidase useful i:or treating a disease caused all or in
part by a deficiency in a-L-
iduronidase. Such compositions may be suitable for administration in a number
of ways such as
parenteral, topical, intranasal, inhalation or oral administration. Within the
scope of this aspect are
embodiments featuring nucleic acid sequences encoding all or a part of an a-L-
iduronidase which
may be administered in vivo into cells affected with an a-L-iduronidase
deficiency.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention features a method to produce a-L-
iduronidase in amounts
which enable using the enzyme therapeutically. In general, the method features
transforming a
suitable cell line with the cDNA encoding for all of a-L-iduronidase or a
biologically active fragment
or mutant thereof. Those of skill in the art may prepare expression constructs
other than those
expressly described herein for optimal production of a-L-iduronidase in
suitable cell lines transfected
therewith. Moreover, skilled artisans may easily design fragments of cDNA
encoding biologically
active fragments and mutants of the naturally occurring a-L-iduronidase which
possess the same or
similar biological activity to the naturally occurring full-length enzyme.
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To create a recombinant source for a-L-iduronidase, a large series of
expression vectors may
be constructed and tested for expression of a a-L-iduronidase cDNA. Based on
transient transfection
experiments as well as stable transfections, an expression construct may be
identified that provides
particularly high level expression. v1 one embodiment of the present
invention, a Chinese hamster
cell line 2.131 developed by transfection of the a-L-iduronidase expression
construct and selection
for a high expression clone provides particularly high level expression. Such
a Chinese hamster cell
line according to this embodiment of the present invention may secrete about
5,000 to 7,000 fold
more a-L-iduronidase than normal. The a-L-iduronidase produced thereby may be
properly
processed, taken up into cells with high affinity and is corrective for a-L-
iduronidase deficient cells
such as those from patients suffering; from Hurler's Syndrome.
The method for producing a-L-iduronidase in amounts that enable using the
enzyme
therapeutically features a production process specifically designed to produce
the
enzyme in high quantities. According to preferred embodiments of such a
process, microcarriers are
used as a low cost scalable surface on which to grow adherent cells.
According to other preferred embodiments of the method for producing a-L-
iduronidase
according to the present invention, a culture system is optimized. In a first
embodiment, the culture
pH is lowered to about 6.S to 7.0, preferably to about 6.7-6.8 during the
production process. One
advantage of such a pH is to enhance accumulation of lysosornal enzymes that
are more stable at
acidic pH. In a second embodiment., about 2/3 to 3/4 of the medium is changed
approximately every
12 hours. One advantage of this procedure is to enhance the secretion rate of
recombinant a-L-
iduronidase and capture more active enzyme. In a third embodiment, oxygen
saturation is optimized
at about 80% using intermittent pure: oxygen sparging rather than continuous
sparging. In a fourth
embodiment, cytodex 2 microcarriers with about 10% serum initially are used to
produce a cell mass
followed by a rapid washout shift to a protein-free medium for production. In
a fifth embodiment, a
growth medium such as a JRH Biosciences PF-CHO product rnay be optimized to
include
supplemental amounts of one or more ingredients selected from the group
consisting of glutamate,
aspartate, glycine, ribonucleosides and deoxyribonucleosides. In a sixth
embodiment, a perfusion
wand such as a Bellco perfusion wand may be used in a frequent batch-feed
process rather than a
standard intended perfusion process. In a seventh embodiment, a mild sodium
butyrate induction
process may be used to induce increased a-L-iduronidase expression without a
substantial effect on
the carbohydrate processing and cellular uptake of the enzyme. Such an
induction process may
provide about a two-fold increase in production without significantly altering
post-translational
processing.
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Particularly preferred embodiments of the method for producing a-L-iduronidase
according to the present invention feature one, more than one or all of the
optimizations
described herein. The production method of the present invention may therefore
provide a production
culture process having the following features:
1. A microcarrier based culture using Cytodex 2 beads or an equivalent thereof
is
preferably used in large scale culture; flasks with overhead wand stirnng
using a Bellco perfusion
wand or an equivalent thereof. Attachment to these beads may be achieved by
culture in a 10% fetal
bovine serum in DME/F12 1:1 medium modified with ingredients including
ribonucleosides,
deoxyribonucleosides, pyruvate, non-essential amino acids, and HEPES and at a
pH of about 6.7-6.9.
After about 3 days in this medium, a washout procedure is begun in which
protein-free medium
replaces approximately 2/3 of the medium approximately every 12 hours for a
total of about 3-4
washes. Subsequently and throughout the entire remaining culture period, the
cells are cultivated in
protein-free medium.
2. The culture conditions are preferably maintained at a dissolved oxygen of
80% of air
saturation at a pH of about 6.7 and at a temperature of about 37° C.
This may be achieved using a
control tower, service unit and appropriate probes such as those produced by
Wheaton. However,
skilled artisans will readily appreciate that this can easily be achieved by
equivalent control systems
produced by other manufacturers. An air saturation of about 80% results in
improved a-L-
iduronidase secretion over 40% and 60% air saturation. However 90% air
saturation does not provide
significantly enhanced secretion over 80% air saturation. The dissolved oxygen
may be supplied by
intermittent pure oxygen sparging using a 5 micron stainless steel sparger or
equivalent thereof. A
pH of about 6.7 is optimal for the accumulation of the a-L-iduronidase enzyme.
The enzyme is
particularly unstable at pH's above about 7Ø Below a pH of about 6.7, the
secretion rate may
decrease, particularly below a pH of about 6.5. The culture is therefore
maintained optimally between
a pH of about 6.6 to 6.8 .
3: The production culture medium may be a modified form of the commercially
available proprietary medium from YRH Biosciences called Excell PF CHO. This
medium supports
levels of secretion equivalent to that of serum using a cell line such as the
2.131 cell line. It may be
preferably modified to include an acidic pH of about 6.7 (+/- 0. 1), and it
may be buffered with
HEPES at 7.5 mM. The medium may contain 0.05 to 0.1% of Pluronics F-68 (BASF),
a non-ionic
surfactant or an equivalent thereof vrrhich features the advantage of
protecting cells from shear forces
associated with sparging. The medium may further contain a proprietary
supplement that proves to
be important in increasing the productivity of the medium over other protein-
free mediums that are
presently available. Those skilled in the art will readily understand that the
choice of culture medium
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may be optimized continually according to particular commercial embodiments
available at particular
points in time. Such changes encompass no more than routine experimentation
and are intended to be
within the scope of the present invention.
4. The production medium may be analyzed using an amino acid analyzer
comparing
spent medium with starting medium. Such analyses have demonstrated that the
2.131 cell line
depletes a standard PF CHO medium of glycine, glutamate and aspartate to a
level of around 10% of
the starting concentration. Supplementation of these amino acids to higher
levels may result in
enhanced culture density and productivity that may lead to a 2-3 fold higher
production than at
baseline. Skilled artisans will appreciate that other cell lines within the
scope of the present invention
may be equally useful for producing a-L-iduronidase according to the present
method. Hence, more
or less supplemental nutrients may be required to optimize the medium. Such
optimizations are
intended to be within the scope of the present invention and may be practiced
without undue
experimentation.
The medium may beg supplemented with ribonucleosides and deoxyribonucleosides
to
support the dihydrofolate reductase deficient cell line 2.131. Skilled
artisans will appreciate that
other cell lines within the scope of the present invention may be equally
useful for producing a-L-
iduronidase according to the present method. Hence, more or less
ribonucleosides and
deoxyribonucleosides may be required to optimize the medium. Such
optimizations are intended
within the scope of the present invention and may be practiced without undue
experimentation.
6. After reaching confluence at about 3-4 days of culture, approximately 2/3
of the
medium may be changed out approximately every 12 hours. The change out of
medium may be
accomplished using, for instance, a Bellco perfusion wand which is a stirring
device with a hollow
center and screen filter at its tip. By pumping out medium through the hollow
interior of the wand
through the 40 micron screen. The microcarrie;rs with the 2.131 cell mass are
separated from
supernatant containing the enzyme.
'The rapid and frequent turnover of the medium has been shown by productivity
studies to result in improved overall collection of enzyme from the cell
culture. Less frequent
changes result in less total accumulation of enzyme. Studies of the secretion
rate of the enzyme
during a 12 hour culture cycle demonstrate that the cells are actively
secreting enzyme for the
majority of the culture period. More frequent changes are unlikely to yield
substantially more
enzyme. The method of this embodiment has proven to be superior to perfusion
culture and far
superior to strict batch culture or daily or every other day batch/feed
strategies. Using the every
approximately 12 hour change, the cells may be maintained in excellent
condition with high degrees
of viability and a high level of productivity.
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8. Production of a-L-iduronidase may be enhanced by the use of sodium butyrate
induction of gene expression. Systematic studies of a 2.131 cell line
demonstrated that about 2 mM
butyrate can be applied and result in about a two-fold or greater induction of
enzyme production with
minimal effects on carbohydrate processing. Lower levels of butyrate have not
been shown to induce
as well, and substantially higher levels may result in higher induction but
declining affinity of the
produced enzyme for cells from patients suffering from a-L-iduronidase
deficiency. The results
suggest that two-fold or greater induction results in less processing of the
carbohydrates and less
phosphate addition to the enzyme as well as increasing toxicity. One
particularly preferred method
uses 2 mM butyrate addition every 4.$ hours to the culture system. This
embodiment results in about
a two-fold induction of enzyme production using this method without
significant effect on the uptake
affinity of the enzyme, (K-uptake of less than 30 U/ml or 2.0 mM). Using
embodiments of the
present method featuring all of the above modifications and induction, a 1 S
liter culture system may
produce approximately 25 mg per liter of culture per day, or more at peak
culturing density.
In a second aspect, the present invention provides a transfected cell line
which possesses the
unique ability to produce a-L-iduronidase in amounts which enable using the
enzyme therapeutically.
In preferred embodiments, the present invention features a recombinant Chinese
hamster ovary cell
line such as the 2.131 cell line that stably and reliably produces amounts of
a-L-iduronidase. In
preferred embodiments, the cell line may contain at least about 10 copies of
an expression construct
comprising a CMV promoter, a Ca intron, a human a-L-iduronidase cDNA, and a
bovine growth
hormone polyadenylation sequence. In even more preferred embodiments, the cell
line expresses a-
L-iduronidase at amounts of at least about 20-40 micrograms per 10' cells per
day in a properly
processed, high uptake form appropriate for enzyme replacement therapy.
According, to preferred
embodiments of this aspect of the irmention, the transfected cell line adapted
to produce a-L-
iduronidase in amounts which enable using the enzyme therapeutically possesses
one or more of the
following features:
The cell line of preferred embodiments is derived from a parent cell line
wherein the
cells are passaged in culture until they have acquired a smaller size and more
rapid growth rate and
until they readily attach to substrates.
2. The cell line of preferred embodiments is transfected with an expression
vector
containing the 2 and 3, a human cDNA of about 2.2 kb in length, and a 3'
bovine growth hormone
cytomegalovirus promoter/enhance:r element, a 5' intron consisting of the
marine Ca intron between
exons polyadenylation site. This expression vector may be transfected at, for
example, a 50 to 1 ratio
with any appropriate common selection vector such as pSV2NE0. The selection
vector pSV2NE0 in
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turn confers 6418 resistance on successfully transfected cells. In
particularly preferred embodiments,
a ratio of about SO to 1 is used since this ratio enhances the acquisition of
multiple copy number
inserts. According to one embodiment wherein the Chinese hamster ovary cell
line 2.131 is provided,
there are approximately 10 copies of the expression vector for a-L-
iduronidase. Such a cell line has
demonstrated the ability to produce large quantities of human a-L-iduronidase
(minimum 20
micrograms per 10 million cells per day). Particularly preferred embodiments
such as the 2.131 cell
line possess the ability to produce properly processed enzyme that contains N-
linked oligosaccharides
containing high mannose chains modified with phosphate at the 6 position in
sufficient quantity to
produce an enzyme with high affinity (K-uptake of less than 3 nM).
3. The enzyme producc;d from the cell lines of the present invention such as a
Chinese
hamster ovary cell line 2.131 is rapidly assimilated into cells, eliminates
glycosaminoglycan storage
and has a half life of about 5 days in cells from patients suffering from a-L-
iduronidase deficiency.
4. The cell line of preferred embodiments such as a 2.131 cell line adapts to
large scale
culture and stably produces human a-L-iduronidase under these conditions. The
cells of preferred
embodiments are able to grow and secrete a-L-iduronidase at the acid pH of
about 6.6 to 6.8 at which
enhanced accumulation of a-L-iduronidase can occur.
5. Particularly preferred embodiments of the cell line according to the
invention, such as
a 2.131 cell line are able to secrete human a-L-iduronidase at levels
exceeding 2,000 units per ml (8
micrograms per ml) twice per day using a specially formulated protein-free
medium.
In a third aspect, the present invention provides novel vectors suitable to
produce a-L-
iduronidase in amounts which enable using the enzyme therapeutically. The
production of adequate
quantities of recombinant a-L-iduronidase is a critical prerequisite for
studies on the structure of the
enzyme as well as for enzyme replacement therapy. The cell lines according to
the present invention
permit the production of significant quantities of recombinant a-L-iduronidase
that is appropriately
processed for uptake. Overexpression in Chinese hamster ovary (CHO) cells has
been described for
three other lysosomal enzymes, a-galactosidase (Ioannou et al., J Cell. Biol.
119:1137-1150
(1992)), iduronate 2-sulfatase (Bielicki et al., Biochem. J. 289: 241-246
(1993)), and N-
acetylgalactosamine 4 -sulfatase (Amson et al., Biochem. J. 284:789-794
(1992)), using, a variety of
promoters and, in one case, amplification. The present invention features a
dihydrofolate reductase-
deficient CHO cell line, but according to preferred embodiments of the
invention amplification is
unnecessary. Additionally, the present invention provides a high level of
expression of the human a-
L-iduronidase using the CMV immediate early gene promoter/enhancer.
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The present invention features in preferred embodiments an expression vector
comprising a
cytomegalovirus promoter/enhancer element, a 5' intron consisting of the
marine Ca intron derived
from the marine long chain immunoglobulin Ca gene between exons 2 and 3, a
human cDNA of
about 2.2 kb in length, and a 3' bovine growth hormone polyadenylation site.
This expression vector
may be transfected at, for example, a 50 to 1 ratio with any appropriate
common selection vector such
as, for example, pSV2NE0. The selection vector such as pSV2NE0 in turn confers
6418 resistance
on successfully transfected cells. In particularly preferred embodiments, a
ratio of about 50 to 1
expression vector to selection vector is used since this ratio enhances the
acquisition of multiple copy
number inserts. According to one embodiment wherein the Chinese hamster ovary
cell line 2.131 is
provided, there are approximately 10 copies of the expression vector for a-L-
iduronidase. Such an
expression construct has demonstrated the ability to produce large quantities
of human a-L-
iduronidase (minimum 20 micrograms per 10 million cells per day) in a suitable
cell line such as, for
example, a Chinese hamster ovary cell line 2.131.
In a fourth aspect, the present invention provides novel a-L-iduronidase
produced in
accordance with the methods of the present invention and thereby present in
amounts. that enable
using the enzyme therapeutically. The methods of the present invention produce
a substantially pure
a-L-iduronidase that is properly processed and in high uptake form appropriate
for enzyme
replacement therapy and that is effective in therapy in vivo.
The specific activity of the ec-L-iduronidase according to the present
invention is in excess of
about 200,000 units per milligram protein. Preferably, it is in excess of
about 240,000 units per
milligram protein. The molecular weight of the full length a-L-iduronidase of
the present invention
is about 82,000 daltons comprising about 70,000 daltons of amino acids arid
12,000 daltons of
carbohydrates. The recombinant en;.yme of the present invention is endocytosed
even more
efficiently than has been previously reported for a partially purified
preparation of urinary enzyme.
The recombinant enzyme according to the present invention is effective in
reducing the accumulation
of radioactive S-labeled GAG in a-l:: iduronidase-deficient fibroblasts,
indicating that it is transported
to lysosomes, the site of GAG storage. The remarkably low concentration of a-L-
iduronidase needed
for such correction (half maximal correction at 0.7 pM) may be very important
for the success of
enzyme replacement therapy.
The human cDNA of a-L-iduronidase predicts a protein of 653 amino acids and an
expected
molecular weight of 70,000 daltons after signal peptide cleavage. Amino acid
sequencing reveals
alanine 26 at the N-terminus giving an expected protein of 629 amino acids.
Human recombinant a-
L-iduronidase has a Histidine at position 8 of the mature protein. The
predicted protein sequence
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comprises six potential N-linked oligosaccharide modificatian sites. All of
these may be modified in
the recombinant protein. The third and sixth sites have been demonstrated to
contain one or more
mannose 6-phosphate residues responsible for high affinity uptake into cells.
The following peptide
corresponds to Amino Acids 26-45 of Human Recombinant a-L-iduronidase with an
N-terminus
alanine and the following sequence:
ala-glu-aia-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-leu-arg-
arg
The overexpression of the a-L-iduronidase of the present invention does not
result in
generalized secretion of other iysosomal enzymes that are dependent on mannose-
6-P targeting. The
secreted recombinant a-L-iduronidase is similar to normal secreted enzyme in
many respects. Its
molecular size, found in various determinations to be 77, 82, 84, and 89 kDa,
is comparable to 87
kDa, found for urinary corrective factor (Barton et al., J. Biol. Chem. 246:
7773-7779 (1971)), and to
76 kDa and 82 kDa, found for enzyme secreted by cultured human fibroblasts
(Myerowitz et al., J.
Biol. Chem. 256: 3044-3048 (1991); Taylor et al., Biochem. J274:263-268
(1991)). The differences
within and between the studies are attributed to imprecision of the
measurements. The pattern of
intracellular processing of the recombinant enzyme-a slow decrease in
molecular size and the
eventual appearance of an additional band smaller by 9 kDa is the same as for
the human fibroblast
enzyme. This faster band arises by proteolytic cleavage of 80 N-terminal amino
acids .
In a fifth aspect, the present invention features a novel method to purify a-L-
iduronidase. In
preferred embodiments, the present invention features a method to purify
recombinant a-L-
iduronidase that has been optimized to produce a rapid and efficient
purification with validatible
chromatography resins and easy load, wash and elute operation. The method of
purifying a-L-
iduronidase of the present invention involves a series of column
chromatography steps which allow
the high yield purification of enzyme from protein-free production medium.
According to a first embodiment, the cell mass is grown in about 10 % serum
containing
medium followed by a switch to a modified protein-free production medium
without any significant
adaptation to produce a high specific activity starting material for
purification. In a second
embodiment, a concentration/diafiltcvation scheme is employed that allows for
the removal of such
exogenous materials as Pluronics F-~68 from the crude bulk to prevent fouling
of columns. In a third
embodiment, a first column load is acidified to minimize the competitive
inhibition effect of such
compounds as uronic acids found in protein-free medium formulations. In a
fourth embodiment, a
heparin, phenyl and sizing column purification scheme is used to produce pure
enzyme using
automatable steps. In a fifth embodiment, the heparin and phenyl column steps
are used to eliminate
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less desirable a-L-iduronidase that is nicked or degraded. In a sixth
embodiment, an acid pH
treatment step is used to inactivate potential viruses without harming the
enzyme.
Particularly preferred embodiments of the method for purifying a-L-iduronidase
according to
the present invention feature more than one or all of the optimizations
according to the following
particular embodiments. The purification method of the present invention may
therefore provide a
purified a-L-iduronidase having the .characteristics described herein.
Concentration/diafiltration: Crude supernatant is processed with a hollow
fiber
concentrator (A/G Technologies, 30K cutoff) to reduce fluid volume by about
75% and is then
diafiltrated with a heparin load buffer ( 10 mM NaPO,, pH 5.3, NaCI 200 mM).
The diafiltration is an
important step that eliminates undesirable compounds such as Pluronics F-68
from the supernatant, a
surfactant needed in many cell cultures of the present invention that can foul
columns. The
diafiltration may also partly remove competitor inhibitors that may prevent
binding to the heparin
column. These inhibitors may be found in PF-CHO medium and are believed to be
uronic acids
derived from a soybean hydrolysate present in this particular medium.
2. Heparin column: The load may be adjusted to a pH of about S.0 before
loading on
Heparin Sepharose CL-6B. Other types of heparin columns such as a heparin FF
(Pharmacia) have
different linkages and do not bind a-L-iduronidase as efficiently. A lower pH
neutralizes uronic
acids to some extent which lessens their competitive effect. Without the
diafiltration and pH
adjustment, heparin columns cannot be run using PF-CHO medium without having
substantial
enzyme flowthrough. The column nnay be washed with a pH of about 5.3 buffer
and then eluted in
0.6 M NaCI. The narrow range of binding and elution salt concentration leads
to an efficient
purification step and enzyme that is often greater than 90% pure after one
step.
Phenyl column: A Phenyl-Sepharose BP (Pharmacia) may be used in the next step.
The heparin eluate may be adjusted to about 1.5 M NaCI and loaded on the
column. The choice of
resin is important as is the salt concentration in ensuring that the enzyme
binds completely (no flow
through) and yet elutes easily and completely with about 0.15 M NaCI. The
eluate obtained is nearly
pure a-L-iduronidase.
4. A pH inactivation may be performed to provide a robust step for the removal
of
potential viruses. The phenyl pool i,s adjusted to a pH of about 3.3 using
Citrate pH 3.0 and held at
room temperature for about 4 hours. The enzyme may then be neutralized.
Embodiments featuring
this step have been shown to eliminate viruses at a minimum of about 5 log
units. The step does not
substantially inactivate or affect the: enzyme activity.
5. The enzyme may then be concentrated and injected onto a Sephacryl S-200
column
and the peak of enzyme collected.
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Enzyme purified in this manner has been shown to contain mannose-6-phosphate
residues of
sufficient quantity at positions 3 and 6 of the N-linked sugars to give the
enzyme uptake affinity of
less than 30 units per ml (less than 2 :nM) enzyme. The enzyme is
substantially corrective for
glycosamino glycan storage disorders and has a half life inside cells of
approximately S days.
In a sixth aspect, the present invention features novel methods of treating
diseases caused all
or in part by a deficiency in a-L-iduronidase. Recombinant a-L-iduronidase
provides enzyme
replacement therapy in a canine model of MPS 1. This canine model is deficient
in a-L-iduronidase
due to a genetic mutation and is similar to human MPS 1. Purified, properly
processed a-L-
iduronidase was administered intravenously to 11 dogs. In those dogs treated
with weekly doses of
25,000 to 125,000 units per kg for 3, 6 or 13 months, the enzyme was taken up
in a variety of tissues
and decreased the lysosomal storage in many tissues. The long term treatment
of the disease was
associated with clinical improvement in demeanor, joint stiffness, coat and
growth. Higher doses of
therapy ( 125,000 units per kg per week) result in better efficacy and
including normalization of
urinary GAG excretion in addition to more rapid clinical improvement in
demeanor, joint stiffness
and coat.
Enzyme therapy at even small doses of 25,000 units (0.1 mg/kglwk) resulted in
significant
enzyme distribution to some tissues and decreases in GAG storage. If continued
for over 1 year,
significant clinical effects of the therapy were evident in terms of activity,
mobility, growth and
overall health. The therapy at this dose did not improve other tissues that
are important sites for
disease in this entity such as cartilage and brain. Higher doses of 125,000
units (0.5 mg/kg) given 5
times over two weeks demonstrate that improved tissue penetration can be
achieved, and a therapeutic
effect at the tissue level was accomplished in as little as 2 weeks. Studies
at this increased dose haven
been preformed in two dogs. These :MPS I dogs show significant clinical
improvement and
substantial decreases in urinary GAC~ excretion into the normal range. Other
than an immune reaction
controlled by altered administration techniques, the enzyme therapy has not
shown significant clinical
or biochemical toxicity. Enzyme therapy at this higher weekly dose is
effective at improving some
clinical features of MPS I and decreasing storage without significant
toxicity.
In a seventh aspect, the present invention features novel pharmaceutical
compositions
comprising human a-L-iduronidase useful for treating a deficiency in a-L-
iduronidase. The
recombinant enzyme may be administered in a number of ways such as parenteral,
topical, intranasal,
inhalation or oral administration. Another aspect of the invention is to
provide for the administration
of the enzyme by formulating it with a pharmaceutically-acceptable carrier
which may be solid, semi-
solid or liquid or an ingestable .capsule. Examples of pharmaceutical
compositions include tablets,
drops such as nasal drops, compositions for topical application such as
ointments, jellies, creams and
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suspensions, aerosols for inhalation, nasal spray, and liposomes. Usually the
recombinant enzyme
comprises between 0.05 and 99% or between 0.5 and 99% by weight of the
composition, for example,
between 0.5-and 20% for compositions intended for injection and between 0.1
and 50% for
compositions intended for oral administration.
To produce pharmaceutical compositions in this form of dosage units for oral
application
containing a therapeutic enzyme, the enzyme may be mixed with a solid,
pulverulent carnet, for
example lactose, saccharose, sorbitol, mannitol, a starch such as potato
starch, corn starch,
amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or
gelatine and also may
include lubricants such as magnesium or calcium stearate or a Carbowax or
other polyethylene glycol
waxes and compressed to form tablets or cores for dragees. If dragees are
required, the cores may be
coated for example with concentrated sugar solutions which may contain gum
arabic, talc and/or
titanium dioxide, or alternatively with a film forming agent dissolved in
easily volatile organic
solvents or mixtures of organic solvents. Dyestuffs can be added to these
coatings, for example, to
distinguish between different contents. of active substance. For the
composition of soft gelatine
capsules consisting of gelatine and, far example, glycerol as a plasticizer,
or similar closed capsules,
the active substance may be admixed with a Carbowax~ or a suitable oil as
e.g., sesame oil, olive oil,
or arachis oil. Hard gelatine capsules may contain granulates of the active
substance with solid,
pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches
such as potato starch,
corn starch or amylopectin, cellulose derivatives or gelatine, and may also
include magnesium
stearate or stearic acid as lubricants.
Therapeutic enzymes of the subject invention may also be administered
parenterally such as
by subcutaneous, intramuscular or intravenous injection or by sustained
release subcutaneous implant.
In subcutaneous, intramuscular and intravenous injection, the therapeutic
enzyme (the active
ingredient) may be dissolved or dispersed in a liquid carrier vehicle. For
parenteral administration,
the active material may be suitably admixed with an acceptable vehicle,
preferably of the vegetable
oil variety such as peanut oil, cottonseed oil and the like. Other parenteral
vehicles such as organic
compositions using solketal, glycerol., formal, and aqueous parenteral
formulations may also be used.
For parenteral application by injection, compositions may comprise an aqueous
solution of a
water soluble pharmaceutically acceptable salt of the active acids according
to the invention,
desirably in a concentration of 0.5-10%, and optionally also a stabilizing
agent and/or buffer
substances in aqueous solution. Dosage units of the solution may
advantageously be enclosed in
ampules.
When therapeutic enzymes are administered in the form of a subcutaneous
implant, the
compound is suspended or dissolved in a slowly dispersed material known to
those skilled in the art,
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or administered in a device which slowly releases the active material through
the use of a constant
driving force such as an osmotic pump.. In such cases, administration over an
extended period of time
is possible. .
For topical application, the pharmaceutical compositions are suitably in the
form of an
ointment, gel, suspension, cream or the like. The amount of active substance
may vary, for example,
between 0.05- 20% by weight of the active substance. Such pharmaceutical
compositions for topical
application may be prepared in known manner by mixing the active substance
with known carrier
materials such as isopropanol, glycerol, paraffin, stearyl alcohol,
polyethylene glycol, etc. The
pharmaceutically acceptable Garner may also include a known chemical
absorption promoter.
Examples of absorption promoters are., e.g., dimethylacetamide (U.S. Patent
No. 3,472,931), trichloro
ethanol or trifluoroethanol (U.S. Patent No. 3,$91,757), certain alcohols and
mixtures thereof (British
Patent No. 1,001,949). A carrier material for topical application to unbroken
skin is also described in
the British patent specification No. 1,464,975, which discloses a carrier
material consisting of a
solvent comprising 40-70% (v/v) isopropanol and 0-60% (v/v) glycerol, the
balance, if any, being an
inert constituent of a diluent not exceeding 40% of the total volume of
solvent.
The dosage at which the therapeutic enzyme containing pharmaceutical
compositions are
administered may vary within a wide range and will depend on various factors
such as, for example,
the severity of the disease, the age of the patient, etc., and may have to be
individually adjusted. As a
possible range for the amount of therapeutic enzyme which may be administered
per day be
mentioned from about 0.1 mg to about: 2000 mg or from about. 1 mg to about
2000 mg.
The pharmaceutical compositions containing the therapeutic enzyme may suitably
be
formulated so that they provide doses within these ranges either as single
dosage units or as multiple
dosage units. In addition to containing a therapeutic enzyme (or therapeutic
enzymes), the subject
formulations may contain one or more substrates or cofactors for the reaction
catalyzed by the
therapeutic enzyme in the compositions. Therapeutic enzyme containing
compositions may also
contain more than one therapeutic enzyme.
The recombinant enzyme employed in the subject methods and compositions may
also be
administered by means of transforming patient cells with nucleic acids
encoding the recombinant a-
L-iduronidase. The nucleic acid sequc;nce so encoding may be incorporated into
a vector for
transformation into cells of the subject to be treated. Preferred embodiments
of such vectors are
described herein. The vector may be designed so as to integrate into the
chromosomes of the subject,
e.g., retroviral vectors, or to replicate autonomously in the host cells.
Vectors containing encoding a-
L-iduronidase nucleotide sequences may be designed so as to provide for
continuous or regulated
expression of the enzyme. Additionally, the genetic vector encoding the enzyme
may be designed so
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as to stably integrate into the cell genome or to only be present transiently.
The general methodology
of conventional genetic therapy may lie applied to polynucleotide sequences
encoding a-L-
iduronidase.. Reviews of conventional genetic therapy techniques can be found
in Friedman, Science
244:1275-1281 (1989}; Ledley, J. Inherit. Metab. Dis. 13:587-616 (1990}; and
Tolstoshev et al., Curr
Opinions Biotech. 1:55-61 (1990).
A particularly prefenred method of administering the recombinant enzyme is
intravenously.
A particularly preferred composition comprises recombinant a-L-iduronidase,
normal saline,
phosphate buffer to maintain the pH at about 5.8 and human albumin. These
ingredients may be
provided in the following amounts:
a-L-iduronidase 0.05-0.2 mg/mL or 12,500-50,000 units per mL
Sodium chloride solution 150 mM in an 1V bag, 50-250 cc total volume
Sodium phosphate buffer 10-50 mM, pH S.8
Human albumin 1 mg/mL
The invention having been described, the following examples are offered to
illustrate the
subject invention by way of illustration, not by way of limitation.
EXAMPLE 1
Producing Recombinant lduronidase
Standard techniques such as those described by Sambrook et al. (1987)
"Molecular Cloning:
A Laboratory Manual", 2°d ed., Cold .Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. may be
used to clone cDNA encoding human a-L-iduronidase. The human a-L-iduronidase
cDNA
previously cloned was subcloned into PRCCMV (InVitrogeri) as a HindIII-XbaI
fragment from a
bluescript KS subclone. An intron cassette derived from the rnurine
immunoglobulin Cot intron
between exons 2 and 3 was constructed using PCR amplification of bases 788-
1372 (Tucker et al.,
Proc. Natl. Acad. Sci. USA 78: 7684-7688 (1991) of clone pRIR14.5 (Kakkis et
al., Nucleic Acids
Res. 16:7796 (1988)). The cassette included 136 by of the 3' end of exon 2 and
242 by of the 5' end of
exon 3, which would remain in the properly spliced cDNA. No ATG sequences are
present in the
coding, region of the intron cassette. The intron cassette was cloned into the
HindIII site S' of the a-
L-iduronidase cDNA. The neo gene was deleted by digestion with Xhol followed
by recircularizing
the vector to make pCMVhldu.
One vial of the master cell bank is thawed and placed in three T150 flasks in
DME/F12 plus
supplements plus 10% FBS and 500 1 lg/ml 6418. After 3-4 days, the cells are
passaged using
trypsin-EDTA to 6 high capacity roller bottles in the same medium. The
innoculum of 2 x 109 cells is
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added to a Wheaton microcarrier flask: containing 60 grams of Cytodex 2
microcarriers, and
DME/F12 plus supplements, 10% FBS and 500 11g/ml of 6418 at a final volume of
13 liters. The
flask is stirred by a Bellco overhead dove with a Perfusion wand stirrer. The
culture is monitored by
temperature, DO and pH probes and controlled using the Wheaton mini-pilot
plant control system
with a PC interface (BioPro software). The parameters are controlled at the
set points, 37° C, 80% air
saturation, and pH 6.7, using a heating;- blanket, oxygen sparger and base
pump. The culture is
incubated for 3-4 days at which time the culture is coming out of log phase
growth at 1-3 x 106 cells
per ml. Thereafter, at 12 hour intervals, the medium is changed with PF-CHO
medium (with custom
modifications, JRH Biosciences). The; first 2 collections are set aside as
"washout". The third
collection is the beginning of the production run. Sodium butyrate at final 2
mM is added every 48
hours to induce an increase in iduronidase expression. Production continues
with medium changes of
liters every 12 hours and the collections filtered through a 1 micron filter
to eliminate free cells
and debris. The culture is monitored for temperature, pH and DO on a
continuous basis. Twice daily,
the culture is sampled before the medium change and assayed for cell condition
and microorganisms
by phase contrast microscopy, glucose: content using a portable glucometer,
iduronidase activity using
a fluorescent substrate assay. Cell mass is assayed several times during the
run using a total cellular
protein assay. By the middle of the nm, cell mass reaches 10' cells per ml.
Collected production
medium containing iduronidase is then concentrated five fold using an A/G
Technology hollow fiber
molecular filter with a 30,000 molecular weight cutoff. The concentrate is
then diafiltrated with a
minimum three fold volume of 0.2 M NaCI in 10 mM NaP04, pH 5. 8 over a period
of 8 hours. This
step removes Pluronics F68 and uronic acids from the concentrate. These
molecules can inhibit
function of the Heparin column. The concentrate is adjusted to pH 5.0,
filtered through 1.0 and 0.2
micron filters and then loaded on a Heparin-Sepharose CL-6B column. The column
is washed with
10 column volumes of 0.2 M NaCl, 10 mM NaP04, pH 5.3), and the enzyme eluted
with 0.6 MI,
lOmMNaPO,,pH 5.8. The eluate is adjusted to 1.5 M NaCI, filtered through a 1
micron filter and
loaded on a Phenyl-Sepharose HP column. The column is washed with 10 column
volumes of 1.5 M
NaCI, 10 mM NaPO,, pH 5.8 and the enzyme eluted with 0. l.5 M NaCI, 10 mM
NaP04, pH 5.8.
Viral inactivation is performed by acidifying the enzyme fraction to pH 3.3
using 1 M citric
acid pH 2.9 and incubating the enzyme at pH 3.3 at room temperature for 4
hours and readjusting the
pH to 5.8 using 1 M phosphate buffer. This step has been demonstrated to
remove 5 logs or better of
a retrovirus in spiking experiments. 'Ilie inactivated enzyme is filtered
through a 0.2 p filter,
concentrated on an A/G Technologies hollow fiber concentrator apparatus
(30,000 molecular weight
cutoff) and injected in cycles on a Sephacryl S200 gel filtration column and
the peaks collected. The
pooled peaks are filtered through a 0.2 p filter, formulated to 0.1 M NaPO,,
pH 5.8 and vialed.
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A set of studies may be performed to assess the quality, purity, potency of
the enzyme.
Results of an SDS-PAGE analysis of the eluate is provided in Figure 2.
One recombinant human a-L-iduronidase obtained from this procedure
demonstrates a
potency of 100,000 units per milliliter and has a total protein concentration
of 0.313 mg/ml.
EXAMPLE 2
Recombinant a-L-Iduronidase Therapy is Efficacious
Short-term intravenous administration of purified human recombinant a-L-
iduronidase to 9
MPS I dogs and 6 MPS I cats has shown significant uptake o:f enzyme in a
variety of tissues with an
estimated 50% or more recovery in tissues 24 hours after a single dose.
Although liver and spleen
take up the largest amount of enzyme, and have the best pathologic
improvement, improvements in
pathology and glycosaminoglycan content has been observed in many, but not all
tissues. In
particular, the cartilage, brain and heart valve did not have significant
improvement. Clinical
improvement was observed in a single dog on long-term treatment for 13 months,
but other studies
have been limited to 6 months or less. All dogs, and most cats, that received
recombinant human
enzyme developed antibodies to the human product. The IgCi antibodies are of
the complement
activating type (probable canine IgG equivalent). This phenomena is also
observed in at least 13% of
alglucerase-treated Gaucher patients. Proteinuria has been observed in one dog
which may be related
to immune complex disease. No othE,r effects of the antibodies have been
observed in the other
treated animals. Specific toxicity was not observed and clinical laboratory
studies (complete blood
counts, electrolytes, BLJN/creatinine, liver enzymes, urinalysis) have been
otherwise normal.
Enzyme therapy at even small doses of 25,000 units (0.1 mg/kg/wk) resulted in
significant
enzyme distribution to some tissues and decreases in GAG storage. If continued
for over 1 year,
significant clinical effects of the therapy were evident in ternns of
activity, mobility, growth and
overall health. The therapy at this dose did not improve other tissues that
are important sites for
disease in this entity such as cartilage and brain. Higher doses of 125,000
units (0.5 mg/kg) given 5
times over two weeks demonstrate that improved tissue penetration can be
achieved, and a therapeutic
effect at the tissue level was accomplished in as little as 2 weeks. Studies
at this increased dose are
ongoing in two dogs for six months to date. These MPS I dogs showed
significant clinical
improvement and substantial decreases in urinary GAG excretion into the normal
range. Other than
an immune reaction controlled by altered administration techniques, the enzyme
therapy has not
shown significant clinical or biochemical toxicity. Enzyme therapy at this
higher weekly dose is
effective at improving some clinical features of MPS I and decreasing storage
without significant
toxicity.
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The results of these various studies in MPS I dogs and one study in MPS I cats
shows that
human recombinant a-L-iduronidase is safe. These same results also provide a
significant rationale
that this recombinant enzyme should be effective in treating a-L-iduronidase
deficiency.
EXAMPLE 3
Recombinant a-L-Iduronidase Therapy in EfFcacious in Humans
The human cDNA of a-L-iduronidase predicts a protein of 653 amino acids and an
expected
molecular weight of 70,000 daltons after signal peptide cleavage. Amino acid
sequencing reveals
alanine 26 at the N-terminus giving an expected protein of 629 amino acids.
Human recombinant a-
L-iduronidase has a Histidine at position 8 of the mature protein. The
predicted protein sequence
comprises six potential N-linked oligosaccharide modification sites. All of
these sites are modified in
the recombinant protein. The third anal sixth sites have been demonstrated to
contain one or more
mannose 6-phosphate residues responsible for high affinity uptake into cells.
This peptide corresponds to Amino Acids 26-45 of Human Recombinant a-L-
iduronidase
with an N-terminus alanine and the following sequence:
ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-leu-arg-
arg
The recombinant enzyme has an apparent molecular weight of 82,000 daltons on
SDS-PAGE
due to carbohydrate modifications. Purified human recombinant a-L-iduronidase
has been sequenced
by the UCLA Protein Sequencing facility. It is preferred to administer the
recombinant enzyme
intravenously. Human recombinant a-L-iduronidase was supplied in 10 mL
polypropylene vials at a
concentration of 0.05-0.2 mg/ml (12,500-50,000 units per mL). The final dosage
form of the enzyme
includes human recombinant a-L-iduronidase, normal saline, phosphate buffer at
pH 5.8 and human
albumin at 1 mglml. These are prepared in a bag of normal saline.
Component Composition
a-L-iduronidase 0.05-0.2 mg/mL or 12,500-50,000 units per mL
Sodium chloride solution 150 mM in an 1V bag, 50-250 cc total volume
Sodium phosphate buffer 10-50 mM, pH 5.8
Human albumin 1 mg/mL
Human patients manifesting a clinical phenotype of MPS-I disorder with an a-L-
iduronidase
level of less than 1 % of normal in leukocytes and fibroblasts were included
in the study. All patients
manifested some clinical evidence of visceral and soft tissue accumulation of
glycosaminoglycans
with varying degrees of functional irr~pairment. Efficacy was determined by
measuring the
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percentage reduction in urinary GAG excretion over time. FIGURE 3 reveals the
urinary GAG levels
in 16 MPS-I patients in relation to normal excretion values. There is a wide
range of urine GAG
values in untreated MPS-I patients. A greater than 50% reduction in excretion
of undegraded GAGS
following therapy with recombinant a-L-iduronidase is a valid means to measure
an individual's
response to therapy. FIGURE 4 demonstrates leukocyte iduronidase activity
before and after enzyme
therapy in MPS I patients. The buccal iduronidase activity before and after
enzyme therapy is
depicted in FIGURE 5. FIGURE 6 demonstrates in three patients that a
substantial shrinkage of liver
and spleen together with significant clinical improvement in joint and soft
tissue storage was
associated with a greater than 65% reduction in undegraded GAG after only 8
weeks of treatment
with recombinant enzyme. FIGURE T demonstrates that there is substantial
normalization of livers
and spleens in patients treated with rec;ombinant enzyme after only 12 weeks
of therapy with
recombinant enzyme. FIGURE 8 demonstrates a precipitous drop in urinary GAG
excretion over 22
weeks of therapy with recombinant enzyme in 11 patients. Clinical assessment
of liver and spleen
size has been the most widely accepted means for evaluating successful bone
marrow transplant
treatment in MPS-I patients (Hoogerbrugge et al., Lancet 345:1398 (1995)).
Such measurements are
highly correlated with a decreased visceral storage of GAGS in MPS-I patients.
Although the invention has been described with reference to the presently
preferred
embodiments, it should be understood that various modifications can be made
without departing from
the spirit of the invention. Accordingly, the invention is limited only by the
following claims.
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