U.S. patent application number 09/993038 was filed with the patent office on 2002-10-10 for methods for producing and purifying recombinant alpha-l-iduronidase.
Invention is credited to Chan, Wai-Pan, Chen, Lin, Fitzpatrick, Paul A., Henstrand, John M., Kakkis, Emil D., Qin, Minmin, Starr, Christopher M., Wendt, Dan J., Zecherle, Gary N..
Application Number | 20020146802 09/993038 |
Document ID | / |
Family ID | 24857160 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020146802 |
Kind Code |
A1 |
Qin, Minmin ; et
al. |
October 10, 2002 |
Methods for producing and purifying recombinant
alpha-L-iduronidase
Abstract
The present invention provides a recombinant human
.alpha.-L-iduronidase and biologically active fragments and muteins
thereof with a purity greater than 99%. The present invention
further provides large-scale methods to produce and purify
commercial grade recombinant human .alpha.-L-iduronidase enzyme
thereof.
Inventors: |
Qin, Minmin; (Pleasanton,
CA) ; Chan, Wai-Pan; (Castro Valley, CA) ;
Chen, Lin; (San Francisco, CA) ; Fitzpatrick, Paul
A.; (Albany, CA) ; Henstrand, John M.; (Davis,
CA) ; Wendt, Dan J.; (Walnut Creek, CA) ;
Zecherle, Gary N.; (Novato, CA) ; Starr, Christopher
M.; (Sonoma, CA) ; Kakkis, Emil D.; (Novato,
CA) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE, LLP
BOX 34
301 RAVENSWOOD AVE.
MENLO PARK
CA
94025
US
|
Family ID: |
24857160 |
Appl. No.: |
09/993038 |
Filed: |
November 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09993038 |
Nov 13, 2001 |
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09711202 |
Nov 9, 2000 |
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09711202 |
Nov 9, 2000 |
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09439923 |
Nov 12, 1999 |
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Current U.S.
Class: |
435/200 ;
435/320.1; 435/325; 435/455; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12Y 402/02004 20130101;
A61P 19/00 20180101; A61P 3/00 20180101; C12Y 302/01014 20130101;
A61K 38/00 20130101; C12N 9/88 20130101; A61K 48/00 20130101; A61P
43/00 20180101; C12Y 302/01076 20130101; C12N 9/2402 20130101; C12Y
302/01075 20130101 |
Class at
Publication: |
435/200 ;
435/69.1; 435/455; 435/325; 435/320.1; 536/23.2 |
International
Class: |
C12N 009/24; C07H
021/04; C12P 021/02; C12N 005/06; C12N 015/87 |
Claims
What is claimed is:
1. A recombinant .alpha.-L-iduronidase enzyme or biologically
active fragments or mutein thereof with a purity of greater than
99%.
2. The recombinant .alpha.-L-iduronidase enzyme or biologically
active fragments or mutein thereof of claim 1 with a specific
activity greater than about 240,000 units per milligram
protein.
3. A method of producing human .alpha.-L-iduronidase of claim 1,
comprising the steps of: (a) preparing a seed train of cells
transformed with nucleic acids encoding for inoculation into a
bioreactor; (b) preparing a mixture containing macroporous
microcarriers by washing and autoclaving said microcarriers in
phosphate buffered saline, combining said microcarriers with growth
medium and fetal bovine serum, and pumping said microcarrier
mixture into said bioreactor; (c) inoculating and incubating said
cells in said bioreactor under control of pH, dissolved oxygen and
perfusion; and (d) harvesting cells when cell density reaches about
10.sup.6.
4. A method of preparing seed train of said cells of claim 3 for
mass production, comprising: (a) washing and resuspending an
aliquot of working cell bank CHO cells 2.131 in culture medium
containing protein-free medium with supplementation of 7.6 mg/L
thymidine, 13.6 mg/L hypoxanthine, 375 .mu.g/mL G418 and 5% fetal
bovine serum; (b) incubating said cell suspension for two to three
days at 37.degree. C. and 5% carbon dioxide in three 225 cm-flasks;
(c) splitting said cell suspension by adding the cells sequentially
to one 1-liter spinner flask, two 3-liter flasks, and four 8-liter
flasks; (d) rotating said cell suspension at 50 revolutions per
minute, followed by increasing inoculum volume by incubating and
subculturing cells to a final cell density of about
2.0.times.10.sup.5 to 2.5.times.10.sup.5.
5. A method of purifying of .alpha.-L-iduronidase of claim 1 to
greater than about 99% purity, comprising the steps of: (a)
harvesting and filtering fluid obtained from a culture of cells
transformed with nucleic acids encoding said human
.alpha.-L-iduronidase; (b) adjusting the pH of the fluid to an
acidic pH, followed by filtration through a 0.2 micron to 0.54
micron filter; (c) passing the fluid through a blue sepharose FF
column to capture said human recombinant .alpha.-L-iduronidase; (d)
passing the fluid through a copper chelating sepharose column to
remove contaminating CHO proteins; (e) passing the fluid through a
phenyl sepharose column to reduce residual leached Cibacron blue
dye and copper ions carried over from previous columns; and (f)
concentrating and diafiltering the purified
.alpha.-L-iduronidase.
6. The method of claim 5, wherein said blue sepharose FF column is
used to purify said human .alpha.-L-iduronidase seven to ten
fold.
7. The method of claim 5, wherein said method comprises using 10%
glycerol in all buffers to increase the quantitative recovery of
said human .alpha.-L-iduronidase.
Description
[0001] This application claims priority to U.S. application Ser.
No. 09/711,202, filed Nov. 9, 2000, which is a continuation-in-part
of U.S. patent application Ser. No. 09/439,923, filed Nov. 12,
1999.
FIELD OF THE INVENTION
[0002] The present invention is in the field of molecular biology,
enzymology, biochemistry and clinical medicine. In particular, the
present invention provides a human recombinant
.alpha.-L-iduronidase, methods of large-scale production and
purification of commercial grade human recombinant
.alpha.-L-iduronidase enzyme, and methods to treat certain genetic
disorders including .alpha.-L-iduronidase deficiency and
mucopolysaccharidosis I (MPS I).
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] A genetic deficiency of the carbohydrate-cleaving, lysosomal
enzyme .alpha.-L-iduronidase causes a lysosomal storage disorder
known as mucopolysaccharidosis I (MPS I) (Neufeld and Muenzer, pp.
1565-1587, in The Metabolic Basis of Inherited Disease, Eds., C. R.
Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, McGraw-Hill, New
York (1989)) 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.
[0005] 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 mistaken for
arthritis or missed entirely. Effective newborn screening for MPS I
would likely find some previously undetected patients.
[0006] Except for a few patients which qualify for bone marrow
transplantation, there are no significant therapies available for
all MPS I patients. Hobbs, et al. (Lancet 2: 709-712 (1981)) first
reported that bone marrow transplantation successfully treated a
Hurler patient. Since that time, clinical studies at several
transplant centers have shown improvement in physical disease and
slowing or stabilizing of developmental decline if performed early.
(Whitley, et al, Am. J Med. Genet. 46: 209-218 (1993); Vellodi, et
al., Arch. Dis. Child. 76: 92-99 (1997); Peters, et al., Blood 91:
2601-2608 (1998); Guffon, et al., J Pediatrics 133: 119-125 (1998))
However, the significant morbidity and mortality, and the need for
matched donor marrow, limits the utility of bone marrow
transplants. An alternative therapy available to all affected
patients, would provide an important breakthrough in treating and
managing this disease.
[0007] Enzyme replacement therapy has been considered a potential
therapy for MPS I following the discovery that
.alpha.-L-iduronidase can correct the enzymatic defect in Hurler
cells in culture, but the development of human therapy has been
technically unfeasible until now. In the corrective process, the
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
.alpha.-L-iduronidase in tissues.
[0008] For .alpha.-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 cDNA for the
canine enzyme was cloned in 1991 (Stoltzfus, et al., J. Biol. Chem.
267:6570-6575 (1992) and for the human enzyme in the same year.
(Scott, et al., Proc. Natl. Acad. Sci. U.S.A. 88:9695-9699 (1991),
Moskowitz, et al., FASEB J 6:A77 (1992)). Following the cloning of
cDNA for .alpha.-L-iduronidase, the production of adequate
quantities of recombinant .alpha.-L-iduronidase allowed the study
of enzyme replacement therapy in canine MPS I. (Kakkis, et al.,
Protein Expr. Purif. 5: 225-232 (1994)) Enzyme replacement studies
in the canine MPS I model demonstrated that
intravenously-administered recombinant .alpha.-L-iduronidase
distributed widely and reduced lysosomal storage from many tissues.
(Shull, et al., Proc. Natl. Acad. Sci. U.S.A. 91: 12937-12941
(1994); Kakkis, et al., Biochem. Mol. Med. 58: 156-167 (1996))
BRIEF SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention features a method to
mass produce human recombinant .alpha.-L-iduronidase in large scale
amounts with appropriate purity to enable large scale production
for long term patient use of the enzyme therapy. In a broad
embodiment, the method comprises the step of transfecting a cDNA
encoding for all or part of an .alpha.-L-iduronidase into a cell
suitable for the expression thereof. In some embodiments, a cDNA
encoding for a complete .alpha.-L-iduronidase is used, preferably a
human .alpha.-L-iduronidase. However, in other embodiments, a cDNA
encoding for a biologically active fragment or mutein 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. In 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 6.8-7.0 during the production process, (b)
as many as 2 to 3.5 culture volumes of the medium may be changed
during each 24-hour period by continuous perfusion, (c) oxygen
saturation may be optimized to about 40% but may be as high as 80%,
(d) macroporous cellulose microcarriers with about 5% serum in the
medium 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 stirred tank suspension culture may be perfused in a continuous
process to produce iduronidase.
[0010] In a second aspect, the present invention provides a
transfected cell line which features the ability to produce
.alpha.-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
.alpha.-L-iduronidase which enable using the enzyme
therapeutically. In some preferred embodiments, the cell line may
contain more than 1 copy of an expression construct. In even more
preferred embodiments, the cell line expresses recombinant
.alpha.-L-iduronidase in amounts of at least 20 micrograms per
10.sup.7 cells per day.
[0011] In a third aspect, the present invention provides novel
vectors suitable to produce
[0012] .alpha.-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 C.alpha. intron, a cDNA encoding all or a fragment or
mutein of an .alpha.-L-iduronidase, and a 3' bovine growth hormone
polyadenylation site. Also, preferably the cDNA encoding all or a
fragment or mutein .alpha.-L-iduronidase is about 2.2 kb in length.
This expression vector may be transfected at, for example, a 50 to
1 ratio with any appropriate common selection vector such as
pSV2NEO, to enhance multiple copy insertions. Alternatively, gene
amplification may be used to induce multiple copy insertions.
[0013] In a fourth aspect, the present invention provides novel
.alpha.-L-iduronidase produced in accordance with the methods of
the present invention and thereby present in amounts which enable
using the enzyme therapeutically. The specific activity of the
.alpha.-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 .alpha.-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.
[0014] In a fifth aspect, the present invention features a novel
method to purify .alpha.-L-iduronidase. According to a first
embodiment, a cell mass may be grown in about 5% 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 one
preferred embodiment, a three step column chromatography may be
used to purify the enzyme. Such a three step column chromatography
may include using a blue sepharose FF, a Cu++ chelating sepharose
chromatography and a phenyl sepharose HP chromatography. In another
preferred embodiment, an acid pH treatment step is used to
inactivate potential viruses without harming the enzyme.
Concanavalin A-Sepharose, Heparin-Sepharose and Sephacryl 200
columns are removed and Blue-Sepharose and copper chelating columns
added to increase the capacity of the large scale purification
process, to reduce undesirable leachables inappropriate for long
term patient use, and to improve the purity of the product.
[0015] In a sixth aspect, the present invention features novel
methods of treating diseases caused all or in part by a deficiency
in .alpha.-L-iduronidase. In one embodiment, this method features
administering a recombinant .alpha.-L-iduronidase or a biologically
active fragment or mutein therof 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 .alpha.-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 preferred
embodiments, the disease is Mucopolysaccharidosis I (MPS I), Hurler
syndrome, Hurler-Scheie syndrome or Scheie syndrome.
[0016] In a seventh aspect, the present invention features novel
pharmaceutical compositions comprising .alpha.-L-iduronidase useful
for treating a disease caused all or in part by a deficiency in
.alpha.-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 .alpha.-L-iduronidase which may be
administered in vivo into cells affected with an
.alpha.-L-iduronidase deficiency.
DESCRIPTION OF THE FIGURES
[0017] FIG. 1 represents the nucleotide and deduced amino acid
sequences of cDNA encoding .alpha.-L-iduronidase (SEQ ID NOS:1 and
2). Nucleotides 1 through 6200 are provided. Amino acids are
provided starting with the first methionine in the open reading
frame.
[0018] FIG. 2 represents the results from SDS-PAGE runs of eluate
obtained according to the procedures as described below. The top
panel shows the SDS-PAGE results of purified .alpha.-L-iduronidase
(3 micrograms) and contaminants from the production/purification
scheme disclosed in Kakkis, et al., Protein Expr. Purif. 5: 225-232
(1994). In the bottom panel, SDS-PAGE results of purified
.alpha.-L-iduronidase with contaminants from an unpublished prior
production/purification process (U.S. patent application Ser. Nos.
09/078,209 and 09/170,977) referred to as the Carson method in
Lanes 2 (7.5 microgram .alpha.-L-iduronidase) and Lane 3 (5.0
microgram .alpha.-L-iduronidase) are compared to that of the
production/purification process of the present invention referred
to as the Galli Process (Lane 4 5 micrograms
.alpha.-L-iduronidase). Lane 1 contains the molecular weight
marker. FIG. 2 shows that the Galli production/purification method
of the present invention yields a highly purified
.alpha.-L-iduronidase product with fewer contaminants in comparison
with prior production/purification schemes.
[0019] FIG. 3 demonstrates the .alpha.-iduronidase production level
over a 30-day period, during which time cells are switched at day 5
from a serum--containing medium to a serum-free medium.
.alpha.-Iduronidase production was characterized by: (1) absence of
a need for adaptation when cells are switched from serum-containing
to serum-free medium at 100200 (top and bottom panels) with an
uninterrupted increase in productivity (top panel); (2) a high
level of production in excess of 4 mg per liter (1000 per mL) in a
protein-free medium (bottom panel); and (3) a boost in
.alpha.-iduronidase production with butyrate induction events
(bottom panel).
[0020] FIG. 4 demonstrates a decrease in liver volume during enzyme
therapy in MPS I patients.
[0021] FIG. 5 demonstrates urinary GAG excretion during enzyme
therapy.
[0022] FIG. 6 demonstrates elbow and knee extension in HAC002
during enzyme therapy.
[0023] FIG. 7 demonstrates shoulder flexion to 104 weeks in four
patients with the most restriction during enzyme therapy.
[0024] FIG. 8 demonstrates improvement in sleep apnea before and
after six weeks of therapy.
[0025] FIG. 9 demonstrates the improvement in apneas and hypopneas
during sleep with enzyme therapy in each individual patient.
[0026] FIG. 10 demonstrates the improvement in pulmonary function
tests before and after 12 and 52 weeks of enzyme therapy in one
patient.
[0027] FIG. 11 demonstrates increased height growth velocity with
enzyme therapy.
[0028] FIG. 12 shows the degree of contamination by Chinese Hamster
Ovary Protein (CHOP) and degree of purity of .alpha.-L-iduronidase,
produced by (1) the Carson method, an unpublished prior
production/purification process (U.S. patent application Ser. Nos.
09/078,209 and 09/170,977 and (2) the Galli method, the
production/purification process of the present invention. Thus,
FIG. 12 shows that .alpha.-L-iduronidase produced and purified by
the Galli method has a higher degree of purity and lower degree of
CHOP contamination in comparison to that of the Carson method.
[0029] FIG. 13 shows a comparison of .alpha.-L-iduronidase produced
by the Galli method versus the Carson method. On the left side of
the Figure, results from a Western Blot show that the Galli
material (left side, column 2) comprise fewer contaminating protein
bands (between 48 kDa and 17 kDa) in comparison with the Carson
material (left side, column 3). On the right side of the Figure,
results from an SDS-PAGE silver stain show the absence of a band at
the 62 kDa in the Galli material (column 2) in comparison to the
presence of such a band in the Carson material (column 3).
DETAILED DESCRIPTION OF THE INVENTION
[0030] In one aspect, the present invention features a method to
produce .alpha.-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
.alpha.-L-iduronidase or a biologically active fragment or mutein
thereof. Those of skill in the art may prepare expression
constructs other than those expressly described herein for optimal
production of .alpha.-L-iduronidase in suitable cell lines
transfected therewith. Moreover, skilled artisans may easily design
fragments of cDNA encoding biologically active fragments and
muteins naturally occurring .alpha.-L-iduronidase which possess the
same or similar biological activity to the naturally occurring
full-length enzyme.
[0031] To create a recombinant source for .alpha.-L-iduronidase, a
large series of expression vectors may be constructed and tested
for expression of a .alpha.-L-iduronidase cDNA. Based on transient
transfection experiments, as well as stable transfections, an
expression construct may be identified that provides a particularly
high level of expression. In one embodiment of the present
invention, a Chinese hamster cell line 2.131 developed by
transfection of the .alpha.-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 .alpha.-L-iduronidase than normal. The
.alpha.-L-iduronidase produced thereby may be properly processed,
taken up into cells with high affinity and is corrective for
.alpha.-L-iduronidase deficient cells, such as those from patients
suffering from Hurler's Syndrome.
[0032] The method for producing .alpha.-L-iduronidase in amounts
that enable using the enzyme therapeutically features a production
process specifically designed to mass produce commercial grade
enzyme, wherein the quality of the enzyme has been deemed
acceptable for administration to humans by regulatory authorities
of various countries. The large scale production of commercial
grade enzyme necessitates modifications of the cell culture scale,
microcarrier systems, and purification scheme. In preferred
embodiments, the cell culture scale is increased from 45 liters to
110 liters or more, with a change to continuous perfusion. The
increase in scale is necessary to produce sufficient material for
potential large scale production for long term patient use.
According to preferred embodiments of such a process, microcarriers
are used as a low cost scalable surface on which to grow adherent
cells. In particularly preferred embodiments, such microcarriers
are macroporous and are specifically composed of modified
carbohydrates such as cellulose, e.g., Cytopore beads manufactured
by Pharmacia. Macroporous cellulose microcarriers allow improved
cell attachment and provide a larger surface area for attachment,
which is expected to yield an increased cell density during the
culture process. Higher cell densities are expected to increase
productivity. In preferred embodiments, heparin-Sepharose and
Sephacryl 200 columns are replaced with Blue-Sepharose and Copper
chelating columns to increase the capacity of the large scale
purification process and to improve the purity of the product. In a
particularly preferred embodiment, the copper chelating column is
used to reduce Chinese hamster ovary cell protein contaminants to
very low levels appropriate for large scale distribution. Using
embodiments of the present method featuring modifications and
induction described below, approximately 15 mg per liter of culture
per day, or more at peak culturing density can be produced starting
with a 110 liter culture system.
[0033] According to other preferred embodiments of the method for
producing .alpha.-L-iduronidase according to the present invention,
a culture system is optimized. In a first embodiment, the culture
pH is lowered to about 6.5 to 7.0, preferably to about 6.7-7.0
during the production process. One advantage of such a pH is to
enhance accumulation of lysosomal enzymes that are more stable at
acidic pH. In a second embodiment, as many as 2 to 3.5 culture
volumes of the medium may be changed during each 24-hour period by
continuous perfusion. One advantage of this procedure is to enhance
the secretion rate of recombinant .alpha.-L-iduronidase and to
capture more active enzyme. In a third embodiment, oxygen
saturation is optimized at about 40%. In a fourth embodiment,
macroporous microcarriers with about 5% serum initially in the
medium, are used to produce a cell mass followed by a rapid washout
shift to a protein-free medium for production (FIG. 3). In a fifth
embodiment, a protein-free growth medium, such as a JRHI
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. In a sixth embodiment, as many as 2 to
3.5 culture volumes of the medium may be changed during each
24-hour period by continuous perfusion. Such an induction process
may provide about a two-fold increase in production without
significantly altering post-translational processing.
[0034] Particularly preferred embodiments of the method for
producing .alpha.-L-iduronidase according to the present invention
feature one, more than one, or all of the optimizations described
herein and may be employed as described in more detail below. The
production method of the present invention may, therefore, provide
a production culture process having the following features:
[0035] 1. A microcarrier based culture using macroporous
microcarrier beads made of modified cellulose or an equivalent
thereof is preferably used in large scale culture flasks with
overhead stirring or an equivalent thereof. Attachment of cells to
these beads may be achieved by culture in a 5% fetal bovine serum
may be added to DME/F12 1:1 or a protein-free medium modified with
ingredients including ribonucleosides, deoxyribonucleosides,
pyruvate, non-essential amino acids, and HEPES. After about 3-6
days in this medium, a washout procedure is begun in which
protein-free medium replaces the serum-containing medium at an
increasing perfusion rate dependent on the glucose content and
culture condition. Subsequently, and throughout the entire
remaining culture period, the cells are cultivated in a
protein-free medium. The use of a protein-free medium in enzyme
production is beneficial in reducing the exposure risk of bovine
spongiform encephalopathy (BSE) and other infectious biologic
agents such as viruses to patients being treated with the enzyme,
wherein the risk of BSE or other harmful agents is dependent on the
amount of potential serum exposure. In prior published studies, the
carriers used to grow the cells were bovine gelatin microcarriers,
used at 1 gram per liter or 100 times the product concentration.
Leaching of 1% of the gelatin protein from the microcarriers would
represent a relative 100% contamination and thereby contribute to
the risk of BSE. Thus, new carriers are either dextran or
cellulose-based and consist of carbohydrates, and not
animal-derived materials.
[0036] FIG. 3 shows that the cells are grown to a density in 5%
serum containing medium and then switched without any adaptation to
a protein-free medium. FIG. 3 specifically shows that: 1) Cells
survive and continue to produce iduronidase when shifted without
adaptation. In contrast, other studies would suggest that
adaptation to a protein-free medium is necessary. In the method of
the present invention, enzyme production continues at levels
comparable to serum containing medium. 2) .alpha.-L-Iduronidase
produced in a protein-free medium retains a level of production in
excess of 4 mg per liter or 1,000 units per ml. 3)
.alpha.-L-Iduronidase produced in a protein-free medium has high
uptake indicating that the shift in medium and, hence, a shift in
carbohydrates being fed to cells, does not adversely affect the
high uptake character of the enzyme. Eight lots of
.alpha.-L-iduronidase have been produced and released in this
manner with an uptake half maximal value of less than 2 nM in all
lots.
[0037] 2. The culture conditions are preferably maintained at a
dissolved oxygen of 40% of air saturation at a pH of about 6.8-7.0
and at a temperature of about 35-37.degree. C. This may be achieved
using a control unit, monitoring unit and appropriate probes such
as those produced by Applikon.RTM. or Mettler.RTM.. 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 40% results in improved
.alpha.-L-iduronidase secretion though up to 80%% air saturation
may be used. However, further increases in oxygen to, for example,
90% air saturation, do not provide significantly enhanced secretion
over 80% air saturation. The dissolved oxygen may be supplied by
intermittent or continuous oxygen sparging using a 5 micron
stainless steel or larger opening sparger, or equivalent thereof. A
pH of about 6.8-7.0 is optimal for the accumulation of the
.alpha.-L-iduronidase enzyme. The enzyme is particularly unstable
at pHs above about 7.0. 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 6.8-7.0.
[0038] 3. The production culture medium may be a modified form of
the commercially available proprietary medium from JRH 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 6.8-7.0 (.+-.0.1), and buffered with HEPES at 7.5 mM or 15
mM. The medium may contain 0.05 to 0.1% of Pluronics F-68 (BASF), a
non-ionic surfactant or an equivalent thereof which features the
advantage of protecting cells from shear forces associated with
sparging. The medium may further contain a proprietary supplement
that is important in increasing the productivity of the medium over
other protein-free media that are presently available. Those
skilled in the art will readily understand that the choice of
culture medium 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.
[0039] 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 .alpha.-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.
[0040] 5. The medium may be supplemented with the four
ribonucleosides and four deoxyribonucleosides each at about 10
mg/liter 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 .alpha.-L-iduronidase according to the present method.
Hence, more or less ribonucleosides and deoxyribonucleosides may be
required to optimize the medium, and alternative sources of purines
and pyrmidines for nucleic acid synthesis may be used such as
hypoxanthine and thymidine. Such optimizations are intended within
the scope of the present invention and may be practiced without
undue experimentation.
[0041] 6. After reaching confluence at about 3-6 days of culture,
an increasing rate of continuous perfusion is initiated. A change
of medium may be accomplished, for example, using a slant feed tube
constructed and positioned to allow the uptake of medium without
removal of the microcarriers even while the culture is stirred. By
pumping out medium through the slant feed tube, microcarriers
settle within the body of the tube inside the culture and are not
removed from the culture during the change on medium. In this
manner, the microcarriers with the cell mass are separated from
supernatant containing the enzyme.
[0042] 7. 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 turnover of medium
results in less total production of enzyme on a daily basis. Using
the perfusion of 2-3.5 culture volumes per day, the cells may be
maintained in excellent condition with high degrees of viability
and a high level of productivity.
[0043] 8. Production of .alpha.-L-iduronidase may be enhanced by
the use of sodium butyrate induction of gene expression (FIG. 3).
Twenty lots of .alpha.-L-iduronidase were produced using butyrate
induction at 2 nM concentration with 2/3 washout every 12 hours
after induction and reinduction every 48 hours for a 21-day
production period. In FIG. 3, the vertical arrows at the bottom
indicate butyrate induction events. Each induction triggered a
boost in .alpha.-L-iduronidase concentration in the medium.
[0044] 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 .alpha.-L-iduronidase
deficiency. Butyrate induction performed in vitro at 2 mM for 24
hours or 5 mM, a more commonly used concentration resulted in
uptakes in excess of 3 nM or 40 U/ml, or an average of three times
the value observed in production lots. In addition, commonly used
times of 24 hours or more and concentration of 5 mM were toxic to
.alpha.-L-iduronidase producing cells and resulted in detachment
and loss of cell mass.
[0045] 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. With respect to
carbohydrate processing and the addition of phosphate groups, the
importance of mannose-6-phosphate in enzyme replacement therapy is
demonstrated by the observations that removal of the phosphate of
two lysosomal enzymes, glucosidase and galactosamine 4-sulfatase
leads to decreased uptake (Van der Ploeg, et al., J. Clin. Invest.
87: 513-518 (1991); Crawley, et al., J. Clin. Invest. 97: 1864-1873
(1996)). In addition, enzyme with low phosphate (Van Hove, et al.,
Proc. Natl. Acad. Sci. USA 93: 65-70 (1996) requires 1,000 units
per ml for uptake experiments (nearly 100 times used for
iduronidase) and effective doses in animal models require 14 mg/kg,
or 28 times the dose used with high phosphate containing
iduronidase (Kikuchi, et al., J. Clin. Invest. 101: 827-833
(1998)).
[0046] One particularly preferred aspect of the invention method
uses 2 mM butyrate addition every 48 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).
[0047] In a second aspect, the present invention provides a
transfected cell line, which possesses the unique ability to
produce .alpha.-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 .alpha.-L-iduronidase. In preferred embodiments, the
cell line may contain more than 1 copy of an expression construct
comprising a CMV promoter, a C.alpha. intron, a human
.alpha.-L-iduronidase cDNA, and a bovine growth hormone
polyadenylation sequence. In even more preferred embodiments, the
cell line expresses .alpha.-L-iduronidase at amounts of at least
about 20-40 micrograms per 10.sup.7 cells per day in a properly
processed, high uptake form appropriate for enzyme replacement
therapy. According to preferred embodiments of this aspect of the
invention, the transfected cell line adapted to produce
.alpha.-L-iduronidase in amounts which enable using the enzyme
therapeutically, possesses one or more of the following
features:
[0048] 1. 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.
[0049] 2. The cell line of preferred embodiments is transfected
with an expression vector containing the cytomegalovirus
promoter/enhancer element, a 5' intron consisting of the murine
C.alpha. intron 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
pSV2NEO. The selection vector pSV2NEO in turn confers G418
resistance on successfully transfected cells. In particularly
preferred embodiments, a ratio of about 50 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 is at least 1 copy of the expression
vector for .alpha.-L-iduronidase. Such a cell line has demonstrated
the ability to produce large quantities of human
.alpha.-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).
[0050] 3. The enzyme produced 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 .alpha.-L-iduronidase deficiency.
[0051] 4. The cell line of preferred embodiments such as a 2.131
cell line adapts to large scale culture and stably produces human
.alpha.-L-iduronidase under these conditions. The cells of
preferred embodiments are able to grow and secrete
.alpha.-L-iduronidase at the acid pH of about 6.6 to 7.0 at which
enhanced accumulation of .alpha.-L-iduronidase can occur.
[0052] 5. Particularly preferred embodiments of the cell line
according to the invention, such as a 2.131 cell line are able to
secrete human .alpha.-L-iduronidase at levels exceeding 2,000 units
per ml (8 micrograms per ml) harvested twice per day or exceeding
15 mg per liter of culture per day using a specially formulated
protein-free medium.
[0053] In a third aspect, the present invention provides novel
vectors suitable to produce .alpha.-L-iduronidase in amounts which
enable using the enzyme therapeutically. The production of adequate
quantities of recombinant .alpha.-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 .alpha.-L-iduronidase that is appropriately
processed for uptake. Overexpression in Chinese hamster ovary (CHO)
cells has been described for three other lysosomal enzymes,
.alpha.-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 .alpha.-L-iduronidase using the
CMV immediate early gene promoter/enhancer.
[0054] The present invention features in preferred embodiments, an
expression vector comprising a cytomegalovirus promoter/enhancer
element, a 5' intron consisting of the murine C.alpha. intron
derived from the murine long chain immunoglobulin C.alpha. 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 pSV2NEO. The selection
vector such as pSV2NEO in turn confers G418 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
.alpha.-L-iduronidase. Such an expression construct has
demonstrated the ability to produce large quantities of human
.alpha.-L-iduronidase (minimum 20 micrograms per 10 million cells
per day) in a suitable cell line such as a Chinese hamster ovary
cell line 2.131.
[0055] In a fourth aspect, the present invention provides novel
.alpha.-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 .alpha.-L-iduronidase that
is properly processed and in high uptake form, appropriate for
enzyme replacement therapy and effective in therapy in vivo.
[0056] The specific activity of the ac-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 using the original assay methods for
activity and protein concentration. A novel validated assay for the
same enzyme with units expressed as micromoles per min demonstrates
an activity of 100 units/ml (range of 70-130) and a protein
concentration by absorbance at 280 nM of 0.7 mg/ml (0.6-0.8) with
an average specific activity of 143 units per mg. The molecular
weight of the full length .alpha.-L-iduronidase of the present
invention is about 82,000 daltons comprising about 70,000 daltons
of amino acids and 12,000 daltons of carbohydrates. The recombinant
enzyme 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
.alpha.-L-iduronidase-deficient fibroblasts, indicating that it is
transported to lysosomes, the site of GAG storage. The remarkably
low concentration of .alpha.-L-iduronidase needed for such
correction (half-maximal correction at 0.7 pM) may be very
important for the success of enzyme replacement therapy.
[0057] The human cDNA of .alpha.-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 .alpha.-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 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 .alpha.-L-iduronidase
with an N-terminus alanine and the following sequence:
[0058]
ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-
-leu-arg-arg (art of SEQ ID NO:2)
[0059] The overexpression of the .alpha.-L-iduronidase of the
present invention does not result in generalized secretion of other
lysosomal enzymes that are dependent on mannose-6-P targeting. The
secreted recombinant .alpha.-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.
J 274:263-268 (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.
[0060] In a fifth aspect, the present invention features a novel
method to purify .alpha.-L-iduronidase. The U.S. Food and Drug
Administration has issued recommendations for assembling chemical
and technological data currently considered appropriate for an
enzyme preparation, including guidelines regarding the purity of
enzyme preparations (Enzyme Preparations: Chemistry Recommendations
for Food Additive and GRAS [Generally Recommended As Safe]
Affirmation Petitions, Version 1.1, Jan. 23, 1993; U.S. Food and
Drug Administration, Center For Food Safety and Applied Nutrition,
Office of Premarket Approval, Chemistry Review Branch). Various
studies have shown that impurities, such as anticomplement
activity, in protein preparations, including immunoglobulin
preparations, may be associated with the development of allergic
and anaphylactic reactions (Lundblad, et al., Rev. Infect. Dis. 8
(Suppl. 4):S382-90 (1986); Scheiermann and Kuwert, Dev. Biol.
Stand. 44:165-171(1979)). Furthermore, impurities may be associated
with unwanted biological activities and interference with desired
therapeutic effects. Thus, enhanced purity of protein preparations
would contribute to greater efficacy of the therapeutic protein
(Ueshima, et al., J. Clin. Hosp. Pharm. 10(2): 193-202 (1985);
Ehrlich, et al., Clin. Chem. 34(9): 1681-8 (1988)).
[0061] The relationship between enzyme purity and immunogenicity is
demonstrated in Studies 1 (Example 5) and 2 (Example 6). Two types
of immune reactions, urticaria and complement activation (indicated
by laboratory analysis), were documented during enzyme infusion and
may be associated with enzyme therapy. In the Phase I study
(Example 5), the purity of recombinant human .alpha.-L-iduronidase
was between 96% to 98%. In the Phase III study (Example 6),
recombinant human .alpha.-L-iduronidase was purified to greater
than 99%. FIGS. 12 and 13 compare the degree of contamination by
the other proteins, such as Chinese Hamster Ovary Protein, and the
purity of the recombinant human .alpha.-L-iduronidase produced by
the previous Carson and current Galli methods. The results show
that the recombinant human .alpha.-L-iduronidase purified according
to the Galli method has fewer protein contaminants than enzyme
produced by the Carson method. In the Phase I study using enyme
purified to 96-98%, five patients developed urticaria and evidence
of complement activation was observed in four patients. In the
Phase III study using enzyme purified to greater than 99%, none of
the enzyme-treated patients developed urticaria. Although all
enzyme--treated patients seroconverted in immunogenicity testing
for IgG, seroconversion did not result in increased
infusion-associated reactions or other adverse events. In patients
tested for IgE, results were negative. The relationship between
purity and immunogenicity is even more evident in the animal
studies described in Example 3, wherein the purity of the
recombinant human .alpha.-L-iduronidase was equal or less than or
about 95%. In the animal studies, all MPS I dogs and most MPS I
cats receiving enzyme treatment developed antibodies, including IgG
antibodies of the complement-acrivating type, a phenomenon observed
in 13% of alglucerase-treated Gaucher patients. One MPS I dog also
developed proteinuria thought to be related to immune complex
disease. These studies suggest that an increased level of enzyme
purity is associated with a lower frequency of immune-related
adverse side effects and hence with greater safety and efficacy of
enzyme therapy.
[0062] In preferred embodiments, the present invention features a
method to purify recombinant .alpha.-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 .alpha.-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. Specifically, Concanavalin A-Sepharose,
Heparin-Sepharose and Sephacryl 200 columns were replaced with
Blue-Sepharose and Copper chelating columns to increase the
capacity of a large-scale purification process, to reduce
leachables and to improve the purity of the product. Concanavalin A
lectin is often used to bind enzyme in an initial purification step
in the prior published study, and is a protein lectin derived from
plants. Concanavalin A is known to leach from columns and
contaminate lysosomal enzyme preparations. Such leaching could
cause activation of T cells in treated patients and hence is deemed
inappropriate for human administration (Furbish, et al., Proc.
Natl. Acad. Sci. USA 74: 3560-3563 (1977)). Thus, the use of
Concanavalin A is avoided in the present purification scheme. In a
prior study, the human liver .alpha.-L-iduronidase could not be
recovered from phenyl columns without high concentrations of
detergent (1% Triton X100) denaturation. Hence, a phenyl column was
not used in a published purification scheme of this enzyme
(Clements, et al., Eur. J. Biochem. 152: 21-28 (1985). The
endogenous human liver enzyme is highly modified within the
lysosomes by hydrolases which remove sialic acid and phosphate
residues and proteases which nick the enzyme. In contrast, the
overexpression of recombinant .alpha.-L-iduronidase causes 50% of
the enzyme to be secreted rather than transported to the lysosome
(Zhao, et al., J. Biol Chem. 272: 22758-22765 (1997). Hence,
recombinant iduronidase will have a full array of sialic acid and
phosphate residues, which lead to a higher degree of water
solubility and lower affinity to the phenyl column. The increased
hydrophilicity allows the enzyme to be eluted under non-denaturing
conditions using the low salt solutions of around 150-700 mM NaCl.
This feature of the recombinant enzyme allows it to be purified in
large scale without the use of detergents.
[0063] Recombinant .alpha.-L-iduronidase over-expressed in a
Chinese Hamster Ovary (CHO) cell line, has been purified to near
homogeneity following a 3-step column chromatography process. The
first column involves an affinity chromatography step using Blue
Sepharose 6 FF. The Blue Sepharose 6 FF eluate is then further
purified by another affinity chromatography step using Cu.sup.++
Chelating Sepharose FF. The final polish of the highly purified
enzyme is achieved by hydrophobic interaction chromatography using
Phenyl Sepharose High Performance (HP). The over-all yield ranges
from 45 to 55 percent and the purity of the final product is
>99%. The process is robust, reproducible, and scalable for
large-scale manufacturing. The purified enzyme has been
characterized with respect to its enzymatic activity using a
fluorescence-based substrate, and its functional uptake by
fibroblast cells. The enzyme has also been characterized for
substrate specificity, carbohydrate profiles, and isoelectric
focusing (IEF) profiles.
[0064] Particularly preferred embodiments of the method for
purifying .alpha.-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
.alpha.-L-iduronidase having the characteristics described
herein.
1 Outline of the .alpha.-L-Iduronidase Purification Process 1
.dwnarw. 2 .dwnarw. 3 .dwnarw. 4 .dwnarw. 5 .dwnarw. 6
[0065] 1. pH Adjustment/Filtration: The pH of filtered harvest
fluid (HF) is adjusted to 5.3 with 1 M H.sub.3PO.sub.4 and then
filtered through a 0.45.mu. filter (e.g. Sartoclean,
Sartorius).
[0066] 2. Blue Sepharose FF chromatography: This affinity
chromatography step serves to capture iduronidase to reduce the
volume and to purify iduronidase by approximately seven to ten
fold.
2 Loading capacity: 4 mg/ml (total protein per ml of resin)
Equilibration buffer: 10 mM NaPO.sub.4, pH 5.3 Wash buffer: 400 mM
NaCl, 10 mM NaPO.sub.4, pH 5.3 Elution buffer: 0.8 M NaCl, 10 mM
NaPO.sub.4, pH 5.3 Regeneration buffer: 2 M NaCl, 10 mM NaPO.sub.4,
pH 5.3 Fold of purification: 7-10 Yield: 70-85%
[0067] 3. Cu.sup.++ Chelating Sepharose FF chromatography: The
Cu.sup.++ Chelating affinity chromatography step is very effective
for removing some contaminating CHO proteins. The inclusion of 10%
glycerol in all the buffers seems to be crucial for the
quantitative recovery of iduronidase.
3 Loading capacity: 2 mg/ml Equilibration buffer: 1 M NaCl, 25 mM
NaAc, pH 6.0, 10% Glycerol Wash buffer: 1 M NaCl, 25 mM NaAc, pH
4.0, 10% Glycerol Elution buffer: 1 M NaCl, 25 mM NaAc, pH 3.7, 10%
Glycerol Regeneration buffer: 1 M NaCl, 50 mM EDTA, pH 8.0 Fold of
purification: 2-5 Yield: 80%
[0068] 4. Phenyl Sephrose HP chromatography: Phenyl Sephrose is
used as the last step to further purify the product as well as to
reduce residual leached Cibacron blue dye and Cu.sup.++ ion carried
over from previous columns.
4 Loading capacity: 1 mg/ml Equilibration buffer: 2 M NaCl, 10 mM
NaPO.sub.4, pH 5.7 Wash buffer: 1.5 M NaCl, 10 mM NaPO.sub.4, pH
5.7 Elution buffer: 0.7 M NaCl, 10 mM NaPO.sub.4, pH 5.7
Regeneration buffer: 0 M NaCl, 10 mM NaPO.sub.4, pH 5.7 Fold of
purification: 1.5 Yield: 90%
[0069] 5. Ultrafiltration (UF)/Diafiltration (DF)/Final
formulation: The purified iduronidase is concentrated and
diafiltered to a final concentration of 1 mg/ml in formulation
buffer (150 mM NaCl, 100 mM NaPO.sub.4, pH 5.8) using a tangential
flow filtration (TFF) system (e.g. Sartocon Slice from Sartorius).
The enzyme is then sterilized by filtering through a 0.2-micron
filter (e.g., cellulose acetate or polysulfone) and filled into
sterile vials.
[0070] 6. Characterization of Purified Iduronidase: Analysis of
enzyme purity using SDS-PAGE stained with Coomassie Blue or Silver
and Western blot analysis. Analysis of enzymatic activity using
4MU-sulfate as substrate. Analysis of functional uptake using
fibroblast cell assay. Analysis of carbohydrates by FACE. Analysis
of IEF profiles.
[0071] 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 glycosaminoglycan storage disorders
caused by iduronidase deficiency and has a half-life inside cells
of approximately 5 days.
[0072] Prior .alpha.-L-iduronidase purification schemes (Kakkis, et
al, Protein Expr. Purif 5: 225-232 (1994); Kakkis, et al., Biochem.
Mol. Med. 58: 156-167 (1996); U.S. patent application Ser. Nos.
09/078,209 and 09/170,977) produced degrees of purity between 90%
and less than 99%, which is not optimal for long-term human
administration (See FIG. 12). (These and all other U.S. patents
herein are specifically incorporated herein by reference in their
entirety.) Treatment with human recombinant .alpha.-L-iduronidase
with a minimum purity of 97% was associated with some clinical
reactions, specifically hives in 5 patients, and complement
activation in 4 patients. All patients demonstrated a reaction to a
protein that is a trace contaminant to the .alpha.-L-iduronidase.
(FIG. 2) Because this protein exists in both the final product and
in the serum-free blank CHO cell line supernatant, the extraneous
protein most likely originates from the CHO cell. The common
proteins that appear to be activating the clinical allergic
response are approximately 60 kDaltons and 50 kDaltons
respectively, which are too small to be recombinant human
iduronidase. Four patients developed an immune reaction to
.alpha.-L-iduronidase at least transiently as well as to the
Chinese hamster ovary cell host proteins. It is clear that even
though the enzyme used to treat patients is highly purified, the
degree of purification is important in reducing the immune response
to contaminants. FIG. 2 (SDS-PAGE), FIG. 12 (CHOP assay), and FIG.
13 (Western Blot, Silver Stain) demonstrate that
.alpha.-L-iduronidase produced and purified by the
production/purification scheme of the present invention has a
higher degree of purity and lower degree of CHOP contamination in
comparison to that of prior methods of production/purification.
Thus, a greater than 97% purity is adequate for patient use, higher
levels of purity are desirable and preferable. As shown in FIG. 12,
the optimized purification scheme described above achieves a degree
of purity that is greater than 99% and importantly reduces Chinese
hamster ovary cell host proteins to less than 1 percent, as
determined by the Chinese Hamster Ovary Protein (CHOP) assay.
[0073] In a sixth aspect, the present invention features novel
methods of treating diseases caused all or in part by a deficiency
in .alpha.-L-iduronidase. Recombinant .alpha.-L-iduronidase
provides enzyme replacement therapy in a canine model of MPS 1.
This canine model is deficient in .alpha.-L-iduronidase due to a
genetic mutation and is similar to human MPS 1. Purified, properly
processed .alpha.-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 0.5, 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, including normalization of urinary
GAG excretion in addition to more rapid clinical improvement in
demeanor, joint stiffness and coat.
[0074] 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,
some clinical effects were evident in terms of increased activity,
size and overall appearance of 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 have been completed in two
dogs for 15 months. These MPS I dogs are showing significant
clinical improvement and substantial decreases in urinary GAG
excretion into the near 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.
[0075] In a seventh aspect, the present invention features novel
pharmaceutical compositions comprising human .alpha.-L-iduronidase
useful for treating a deficiency in .alpha.-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, 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 suspensions, aerosols for inhalation, nasal
spray, and liposomes. Usually the recombinant enzyme comprises
between 0.01 and 99% or between 0.01 and 99% by weight of the
composition, for example, between 0.01 and 20% for compositions
intended for injection and between 0.1 and 50% for compositions
intended for oral administration.
[0076] 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 carrier, 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 gelatin and also may include
lubricants such as magnesium or calcium stearate or a Carbowax.RTM.
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 gelatin
capsules consisting of gelatin and, for example, glycerol as a
plasticizer, or similar closed capsules, the active substance may
be admixed with a Carbowax.RTM. or a suitable oil, e.g., sesame
oil, olive oil, or arachis oil. Hard gelatin 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
gelatin, and may also include magnesium stearate or stearic acid as
lubricants.
[0077] 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.
[0078] 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 0.01-10%, and optionally also a
stabilizing agent and/or buffer substances in aqueous solution.
Dosage units of the solution may advantageously be enclosed in
ampules.
[0079] 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, 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.
[0080] 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 carrier
may also include a known chemical absorption promoter. Examples of
absorption promoters are, e.g., dimethylacetamide (U.S. Pat. No.
3,472,931), trichloro ethanol or trifluoroethanol (U.S. Pat. No.
3,891,757), 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.
[0081] 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 the severity of
the disease, the age of the patient, etc., and may have to be
individually adjusted. A possible range for the amount of
therapeutic enzyme which may be administered per day is about 0.1
mg to about 2000 mg or about 1 mg to about 2000 mg.
[0082] 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 enzymes
containing compositions may also contain more than one therapeutic
enzyme.
[0083] 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
.alpha.-L-iduronidase. The nucleic acid sequence so encoded 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 .alpha.-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 as to stably integrate into the cell genome or to only
be present transiently. The general methodology of conventional
genetic therapy may be applied to polynucleotide sequences encoding
.alpha.-L-iduronidase. Conventional genetic therapy techniques have
been extensively reviewed. (Friedman, Science 244:1275-1281(1989);
Ledley, J. Inherit. Metab. Dis. 13:587-616 (1990); Tososhev, et
al., Curr Opinions Biotech. 1:55-61 (1990)).
[0084] A particularly preferred method of administering the
recombinant enzyme is intravenously. A particularly preferred
composition comprises recombinant .alpha.-L-iduronidase, normal
saline, phosphate buffer to maintain the pH at about 5.8 and human
albumin. These ingredients maybe provided in the following
amounts:
5 .alpha.-L-iduronidase 0.05-0.2 mg/mL or 12,500-50,000 units per
mL Sodium chloride solution 150 mM in an IV bag, 50-250 cc total
volume Sodium phosphate buffer 10-50 mM, pH 5.8 Human albumin 1
mg/mL
[0085]
6 Composition of Recombinant Human .alpha.-L-Iduronidase (rhIDU,
Aldurazyme .TM.) Drug Product Composition Name of Ingredient
Concentration per vial Function rhIDU 100 U/mL 3.07 mg Active
ingredient (Range 80-150 U/mL) NaCl 150 mM 46.5 mg Tonicity
Modifier Sodium Phosphate 92 mM 67.3 mg Buffer monobasic Sodium
Phosphate 8 mM 11.3 mg Buffer dibasic Polysorbate 80 10 .mu.g/mL
0.05 mg Stabilizer
[0086] The proposed commercial formulation for Aldurazyme.TM. is
100 Units/mL (approximately 0.58 mg/mL) for recombinant human
.alpha.-L-lduronidase (rhIDQ), 100 mM sodium phosphate, 150 mM
sodium chloride, and 10 .mu.M/mL polysorbate 80, pH of 5.8. The
Phase I study formula was identical to the Phase III study formula
and proposed commercial formulation with the exception that
polysorbate 80 was added as a stabilizer in the Phase III and
commercial formula. This commercial formulation was also used in
Good Laboratory Practice (GLP) toxicology studies.
[0087] Polysorbate 80, at a concentration of 10 .mu.M/mL was added
to the formulation to act as a stabilizer. The change was
implemented when the rhIDU production process was scaled up and
prompted by the observation of a fine precipitate in the vialed
drug product and coincided with the change from polypropylene vials
to glass vials. Formulation studies have demonstrated that
polysorbate-20 (10 .mu.M/mL) and polysorbate-80 (5 .mu.M/mL) both
minimized the formation of precipitates in vialed Aldurazyme.TM.
even after forced agitation. The concentration of polysorbate-20 or
polysorbate-80 needed to minimize the formation of precipitates was
5 .mu.M/mL for polysorbate-80 and 10 .mu.M/mL for polysorbate-20.
Preliminary data demonstrated that Aldurazyme.TM., when formulated
with polysorbate 80 at 10 .mu.M/mL, retained activity when stored
at 2-8.degree. C. Polysorbate 80 was chosen over polysorbate 20
because it performed slightly better in preventing precipitate
formation and it is more commonly used in marketed pharmaceutical
product formulations. Polysorbate is known to be effective against
agitation-induced aggregation of proteins, and a review of the
literature regarding the use of polysorbate 80 in chronic
intravenous therapies found the proposed level to be included in
Aldurazyme.TM. (10 .mu.M/mL) to be well below that used in other
pharmaceutical formulations (Bam, et al., J. Pharm. Sci.
87(12):1554-9 (1998); Kreilgaard, et al., J. Pharm. Sci.
87(12):1597-603 (1998)). The safety and efficacy of the commercial
formulation were assessed in Good Laboratory Practice (GLP)
toxicology studies as well as Phase III study.
[0088] 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 .alpha.-L-lduronidase
[0089] Standard techniques such as those described by Sambrook, et
al (Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1987)) maybe
used to clone cDNA encoding human .alpha.-L-iduronidase. The human
.alpha.-L-iduronidase cDNA previously cloned was subcloned into
PRCCMV (In Vitrogen) as a HindIII-XbaI fragment from a bluescript
KS subclone. An intron cassette derived from the murine
immunoglobulin C.alpha. 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 bp of the 3' end of exon 2 and 242 bp 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 5'
of the .alpha.-L-iduronidase cDNA. The neo gene was deleted by
digestion with Xhol followed by recircularizing the vector to make
pCMVhldu.
[0090] One vial of the working cell bank is thawed and placed in
three T225 flasks in DME/F12 or PF-CHO plus supplements, plus 5%
FBS and 500 .mu.g/ml G418. After 2-5 days, the cells are passaged
using trypsin-EDTA to a 1-liter spinner flask in the same medium
for 2-5 days. The cells are then transferred to two 3-liter spinner
flasks for 2-5 days, followed by four 8-liter spinner flasks for
2-5 days. The inoculum from the 8-liter spinner flasks is added to
two 110-liter Applikon.RTM. stirred tank bioreactors with an 80-90
liter working volume. Macroporous cellulose microcarriers are added
at 2 grams per liter (160 grams), with PF-CHO or DME/F12 plus
supplements, 5% FBS and 500 .mu.g/ml of G418 at a final volume of
80-90 liters. The flask is stirred by an overhead drive with a
marine impeller. The culture is monitored for agitation speed,
temperature, DO and pH probes and controlled the Applikon(.RTM.
control system with a PC interface. The parameters are controlled
at the set points or range, 35-37.degree. C. depending on culture
conditions, 40% air saturation, and pH 6.95, using a heating
blanket, oxygen sparger and base pump. The culture is incubated for
3-5 days at which time the culture is emerging from the log phase
growth at 1-3.times.10.sup.6 cells per ml. Thereafter, perfusion is
initiated at an increasing rate with PF-CHO medium (with custom
modifications, JRH Biosciences). The first four days of collection
(range of 3-5 days) are set aside as "washout." The collection
thereafter is the beginning of the production run. Production
continues with medium changes of as much as 2-3.5 culture volumes
per day for 20-36 days. The culture may be extended for 40 days or
longer. The culture is monitored for temperature, pH and DO on a
continuous basis. The purification of the enzyme proceeds as
described above. Collected production medium containing iduronidase
is then acidified to pH 5.3, filtered through a 0.2-micron filter
and purified using Blue-Sepharose chromatography. The purified
enzyme from multiple rounds of Blue-Sepharose chromatography are
then pooled and applied to a copper chelating column and eluted
with glycerol in the buffer at a pH of 3.7. The enzyme is held at
the acidic pH to inactivate potential viruses. The copper column
eluate is then adjusted to pH 5.7 and 2 M NaCl and loaded on the
phenyl Sepharose column. The enzyme is eluted at 0.7 M NaCl. The
eluate is concentrated and diafiltered into a formulation buffer of
150 mM NaCl, 100 mM NaPO4, pH5.8. The enzyme is filtered through a
40 nM filter to remove potential viruses and the filtrate adjusted
to 0.001% polysorbate 80. The formulated enzyme is sterilely bulk
filled into sterile polyethylene containers. The bulk enzyme is
then filtered and filled into 5 cc Type 1 glass vials appropriate
for injectable pharmaceuticals, stoppered and capped.
EXAMPLE 2
[0091] For bioreactors using single cell suspensions, the seed
train is prepared as described above in EXAMPLE 1. Using a single
cell suspension simplifies bioreactor preparation and inoculation.
The bioreactor is inoculated with cells in DMEM/F12 medium (25% of
reactor volume) and JRH 325 modified (25% of reactor volume).
Medium equal to 50% of the working reactor volume is added over 48
hours. Perfusion (and harvest) is started when cell density reaches
1.0 e.sup.6 and the perfusion medium is the same as described
above.
EXAMPLE 3
[0092] Short-term intravenous administration of purified human
recombinant .alpha.-L-iduronidase to 9 MPS I dogs and 6 MPS I cats
has shown significant uptake of an 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 enzymes, 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 IgG 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 other
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.
[0093] 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 terms
of activity, size and overall appearance of 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 are showing
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.
[0094] The results of these various studies in MPS I dogs and one
study in MPS I cats show that human recombinant
.alpha.-L-iduronidase is safe. Although these same results provide
significant rationale that this recombinant enzyme should be
effective in treating .alpha.-L-iduronidase deficiency, they do not
predict the clinical benefits or the potential immunological risks
of enzyme therapy in humans.
EXAMPLE 4
[0095] The human cDNA of .alpha.-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 .alpha.-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 and sixth sites have been
demonstrated to contain one or more mannose 6-phosphate residues
responsible for high affinity uptake into cells.
[0096] This peptide corresponds to Amnino Acids 26-45 of Human
Recombinant .alpha.-L-iduronidase with an N-terminus alanine and
the following sequence:
[0097]
ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-
-leu-arg-arg (part of SEQ ID NO:2)
[0098] The recombinant enzyme has an apparent molecular weight of
82,000 daltons on SDS-PAGE due to carbohydrate modifications.
Purified human recombinant .alpha.-L-iduronidase has been sequenced
by the UCLA Protein Sequencing facility. It is preferred to
administer the recombinant enzyme intravenously. Human recombinant
.alpha.-L-iduronidase was supplied for the clinical trial in 10 mL
polypropylene vials at a concentration of 100,000-200,000 units per
mL. The final dosage form of the enzyme used in the clinical trial
includes human recombinant .alpha.-L-iduronidase, normal saline,
and 100 mM phosphate buffer at pH 5.8. These are prepared in a bag
of normal saline. Polysorbate 80 at a final concentration of 0.001%
was added to the formulation to stabilize the protein against
shear, thereby avoiding precipitation in the final product
vials.
7 Final Vial Formulation Currently in Use Component Composition
.alpha.-L-iduronidase Target to 0.7 mg/mL or 100 (new) units per mL
Sodium chloride solution 150 mM Sodium phosphate buffer 100 mM, pH
5.8 Polysorbate 80 0.001%
[0099]
8 Final Dosage Form Used in the Treatment of Patients Component
Composition .alpha.-L-iduronidase product 5-12 fold dilution of
vial concentration Sodium chloride solution 50 mM Sodium phosphate
buffer 100-250 cc bag IV Human albumin 1 mg/ml
EXAMPLE 5
[0100] Phase I Study--Effects of Intravenous Administration of
.alpha.-L-Iduronidase in Patients with Mucopolysaccharidosis I (52
weeks)
[0101] Based on studies of cloning of cDNA encoding
.alpha.-L-iduronidase (Scott, et al., Proc. Natl. Acad. Sci. USA
88: 9695-99 (1991); Stoltzfus, et al., J. Biol. Chem. 267: 6570-75
(1992)) and animal studies showing effects of .alpha.-L-iduronidase
to reduce lysosomal storage in many tissues (Shull, et al., Proc.
Natl. Acad. Sci. USA 91: 12937-41 (1994); Kakkis, et al., Biochem.
Mol. Med. 58: 156-67 (1996)), a 52-week study was conducted to
assess the safety and clinical efficacy of intravenous
administration of highly purified .alpha.-L-iduronidase in ten
patients with mucopolysaccharidosis I (MPS I).
[0102] Recombinant human .alpha.-L-iduronidase was produced and
purified to greater than 97-99%. Patients demonstrated typical
clinical manifestations of the disorder and diagnosis was confirmed
by biochemical determination of .alpha.-L-iduronidase deficiency in
leukocytes.
[0103] Patients were given recombinant human .alpha.-L-iduronidase
(diluted in normal saline with 0.1% human serum albumin)
intravenously at a dose of 125,000 units per kg (using original
assay and unit definition); 3,000 units per kg were given over the
first hour, and 61,000 units per kg in each of the following two
hours. The dose of 125,000 units per kg is equivalent to 100 SI
units per kg using the new assay. The infusions were prolonged up
to 4-6 hours in patients who had hypersensitivity reactions.
[0104] At baseline and at 6, 12, 26 and 52 weeks depending on the
evaluation, the patients underwent examinations including history,
physical examinations by specialists, echocardiography, EKG, MRI,
polysomnography (weeks 0 and 26), skeletal survey (weeks 0, 26,
52), range of motion measurements, corneal photographs, and skin
biopsy (week 0) to set up fibroblast cultures for enzyme
determination and genotyping. Range of motion measurements were
performed with a goniometer and the maximum active (patient
initiated) range was recorded for each motion. Shoulder flexion is
movement of the elbow anteriorly from the side of the body and
elbow and knee extension represent straightening of the joint.
Degrees of restriction represent the difference between the normal
maximum range of motion for age and the measured value.
Polysomnography was performed according to American Thoracic
Society guidelines and apneic events (cessation of oro-nasal
airflow for 10 seconds or more), hypopneic events (decreased
oro-nasal airflow of 50% or more with desaturation of 2% or more,
or evidence of arousal), minutes below 89% oxygen saturation and
total sleep time recorded among the standard measurements required.
From these data an apnea/hypopnea index was calculated by dividing
the total number of apneic and hypopneic events by the number of
hours of sleep. Biochemical studies included measurement of enzyme
activity in leukocytes and brushings of buccal mucosal, urinary
glycosaminoglycan levels, and tests for serum antibodies to
recombinant human .alpha.-L-iduronidase (ELISA and Western blot).
Organ volumes were determined by analysis of MRI digital image data
using Advantage Windows workstation software from General Electric.
The organ volume was measured in milliliters and was converted to
weight assuming a density of 1 gram per ml. Urinary
glycosaminoglycan excretion was assayed by an adaptation of a
published method. Western blots and ELISA assays for antibodies to
recombinant human .alpha.-L-iduronidase were performed by standard
methods. Uronic acids and N-sulfate of urinary glycosaminoglycans
were analyzed by the orcinol, carbazole and MBTH methods, and by
electrophoretic separations.
[0105] All patients received weekly infusions of recombinant human
.alpha.-L-iduronidase administered for 52 weeks. The mean activity
of .alpha.-L-iduronidase in leukocytes was 0.04 units per mg before
treatment and when measured on average 7 days after an infusion
(i.e. immediately before the next infusion), 4.98 units per mg, or
15.0 percent of normal. Enzyme activity was not detectable in
buccal brushings prior to treatment, but 7 days after infusions it
reached a level of 1 percent of normal.
[0106] Liver volume decreased by 19 to 37 percent from baseline in
9 patients and 5 percent in one patient at 52 weeks; the mean
decrease was 25.0 percent (n=10, P<0.001). By 26 weeks, liver
size was normal for body weight and age in 8 patients (FIG. 1). In
2 patients (patients 6 and 9) with the largest relative liver size
at baseline, liver size was close to normal at 52 weeks (3.2 and
3.3 percent of body weight, respectively). Spleen size decreased in
8 patients by 13 to 42 percent from baseline (mean decrease of 20
percent in 10 patients, P<0.001).
[0107] Urinary glycosaminoglycan excretion declined rapidly by 3 to
4 weeks and by 8-12 weeks had fallen by 60-80 percent of baseline.
At 52 weeks, the mean reduction was 63 percent (range 53-74;
p<0.001). Eight of ten patients had a 75 percent or greater
reduction of the baseline amount of urinary glycosaminoglycan in
excess of the upper limit of normal for age. The results were
confirmed by assay of uronic acids and N-sulfate (a test specific
for heparan sulfate). Electrophoresis studies of urine detected a
significant reduction in heparan sulfate and dermatan sulfate
excretion but some excess dermatan sulfate excretion persisted in
all patients.
[0108] The mean height increased 6.0 cm (5.2 percent) in the 6
prepubertal patients (Table 2) and their mean height growth
velocity increased from 2.8 cm/yr to 5.2 cm/yr during treatment
(P=0.011). For all 10 patients, mean body weight increased 3.2 kg
(8.8 percent) and the mean increase was 4.2 kg (17.1 percent) for
the 6 prepubertal patients (Table 2). In these 6 patients, the mean
pretreatment weight growth velocity increased from 1.7 kg per year
to 3.8 kg per year during treatment (P=0.04).
[0109] Shoulder flexion (moving the elbow anteriorly) increased in
6 of the 8 subjects evaluated at baseline with a mean improvement
for the right and left shoulders of 28.degree. and 26.degree.,
respectively (P<0.002; FIG. 2). Elbow extension and knee
extension increased by a mean of 7.0.degree. (P<0.03) and 3.20
(P=0.10), respectively, in the 10 patients (FIG. 2).
[0110] Analysis of the improvement in individual patients revealed
that the most restricted joints had the greatest improvement. For
example at baseline, patients 5, 9 and 10 could not flex their
shoulders (move the elbow anteriorly) beyond 100.degree., which
increased 21.degree. to 51.degree. after treatment. Similarly,
patients 2 and 9 had a substantial increase in knee extension. The
changes in range of motion were accompanied by patient-reported
increases in physical activities such as being able to wash their
hair, hold a hamburger normally, hang from monkey bars, and play
sports better.
[0111] Seven patients had a decrease in apnea and hypopnea events
from 155 to 60 per night upon treatment (a 61 percent decrease)
with a change in mean apnea/hypopnea index (total number of events
per hour) from 2.1 to 1.0. Three patients had clinically
significant sleep apnea and all three improved during treatment. In
patient 2, the apnea/hypopnea index decreased from 4.5 at baseline
to 0.4 at 26 weeks and total time of oxygen desaturation decreased
from 48 minutes to 1 minute per night. Patient 6 required nightly
continuous positive airway pressure therapy before treatment due to
severe desaturation (61 minutes below 89 percent saturation with
continuous positive airway pressure in 368 minutes of sleep), but
by 52 weeks, the patient tolerated the sleep study without CPAP and
desaturated below 89 percent for only 8 minutes during 332 minutes
of sleep. Patient 9 had an apnea hypopnea index of 9.5 which
decreased to 4.0 by 26 weeks. Patient 8 worsened with an apnea
hypopnea index of 0.1 increasing to 3.1 at 26 weeks and 9.3 at 52
weeks for unclear reasons. Eight of ten patients or their families
reported improved breathing, and 5 of 7 noted quieter nighttime
breathing, improved sleep quality and decreased daytime
somnolence.
[0112] New York Heart Association functional classification was
determined by serial patient interviews. All 10 patients reported
improvement by one or two classes but there was no significant
objective data from echocardiographic studies to verify direct
cardiac benefit. The improved functional scores may reflect
improvements in other aspects of MPS I disease rather than cardiac
function. Comparing baseline to 52 weeks of treatment,
echocardiography demonstrated decreased tricuspid regurgitation or
pulmonic regurgitation in 4 patients but two patients (patients 2
and 7) had worsening mitral regurgitation. At baseline, patient 6
had atrial flutter and clinical signs of cardiac failure including
dyspnea at rest and peripheral edema. By 12 weeks, he had normal
sinus rhythm with first degree block and his dyspnea at rest and
pitting edema resolved.
[0113] All 10 patients reported a lack of endurance and limitations
of daily activities before treatment but exercise tolerance was not
formally tested. During treatment, all patients improved and by 26
weeks, many were able to walk more, run and play sports. Patients
3, 4 and 5 reported the resolution of severe incapacitating
headaches after treatment for 6-12 weeks.
[0114] Several patients reported decreased photophobia or
conjunctival irritation. Visual acuity improved in one patient
(20/1000 to 20/200 in one eye) and modestly in 2 others.
[0115] The results of this study indicate that intravenous
administration of the highly purified recombinant human
.alpha.-L-iduronidase of the present invention results in clinical
and biochemical improvement in patients with Mucopolysaccharidosis
I. The normalization of liver size and near normalization of
urinary glycosaminoglycan excretion is consistent with data from
studies in dogs with Mucopolysaccharidosis I, which demonstrated
clearance of storage in the liver and decreased urinary
glycosaminoglycan excretion in as little as 2 weeks.
[0116] Hypersensitivity reactions to the infusions of recombinant
human .alpha.-L-iduronidase were less severe than predicted from
studies in dogs. Though important in some patients, recurrent
urticaria was manageable with premedication and adjustments in
infusion rate. Antibodies specific to .alpha.-L-iduronidase were
detected in 4 patients with usually subclinical complement
activation, and both the antibodies and complement activation
declined with time. Similar IgG-mediated immune responses have been
previously noted in patients with Gaucher disease treat with
glucocerebrosidase, although the events were more frequent in our
patients. Mucopolysaccharidosis I patients with a null genotype may
have a greater immune response than in these 10 patients, none of
whom has a null.
[0117] Thus, recombinant human .alpha.-L-iduronidase can reduce
lysosomal storage and ameliorates some aspects of clinical disease
of Mucopolysaccharidosis I.
EXAMPLE 6
[0118] Phase III Study--Effects of Intravenous Administration of
.alpha.-L-Iduronidase in Patients with Mucopolysaccharidosis 1 (26
weeks)
[0119] A multi-national, multi-center, double-blind, randomized,
placebo-controlled study was conducted to further assess the safety
and clinical efficacy of intravenous administration of highly
purified .alpha.-L-Iduronidase in 45 MPS I patients.
[0120] Recombinant human .alpha.-L-Iduronidase was purified to
greater than 99%. The patients were characterized by age of at
least five years old, less than 10 percent of normal enzyme
activity, a baseline forced vital capacity (FVC) reflecting
pulmonary function of 80% or less of percent predicted normal, and
a capability of standing for 6 minutes and walking at least 5
meters. Of the 45 patients, 22 patients were treated with highly
purified .alpha.-L-Iduronidase and the remaining 23 received a
placebo. Patients were administered human .alpha.-L-Iduronidase
intravenously at a dose of 100 units per kilogram via a 4-hour
intravenous infusion each week for 26 weeks.
[0121] Efficacy Endpoints
[0122] Patients were assessed by measuring primary efficacy
endpoints, the change from baseline to week 26 in the % FVC and a
six-minute walk distance using the Wilcoxon Rank Sum Test. Patients
were further assessed by secondary efficacy endpoints including
apnea/hypoxia index (sleep study), liver organ volume
(hepatomegaly), disability score index (Child Health Assessment
Questionnaire/Health Assessment Questionnaire, CHAQ/HAQ), and
shoulder flexion reflecting joint range of motion. These endpoints
were measured as a change in baseline to week 26 by the Analysis of
Variance test. Patients were also assessed by measuring tertiary
efficacy endpoints, including urinary glucosaminoglycan (GAG)
levels, totally respiratory event index (sleep study), pain scale
(CHAQ), shoulder extension, knee extension and flexion, quality of
life (50-question Child Health Questionnaire Physical Functioning,
CHQ PF 50; 87-question Child Health Questionnaire directed to the
child with questions combined to create concepts, CHQ CF87;
36-question Short Form Health Status Survey, SF-36), growth in
prepubertal only, visual acuity, echocardiogram, force expriatory
volume (FEVI), and investigator global assessment. The investigator
global assessment comprises a series of seven categories in which
the investigator is providing an assessment as to how each patient
is improving during the trial.
[0123] Safety Endpoints
[0124] Safety was assessed by measurement of the frequency of
adverse events, serious adverse events, and infusion-associated
reactions, immunogenicity testing, and measurement of other safety
parameters by physical examinations, testing of vital signs,
brain/cranio-cervical junction magnetic resonance imaging (MRI, and
standard laboratory evaluations.
Results
[0125] Efficacy Endpoints
[0126] With respect to primary efficacy endpoints, a statistically
significant difference (p=0.028) was seen in the change in %
predicted FVC (see Table I). A close to statistically significant
difference (p=0.066) was noted in the change in 6-minute walk
(Table II).
[0127] Although there was no significant overall difference
observed in the sleep apnea/hypopnea index, a reduction of events
was observed in enzyme-treated patients with clinically significant
disease (n=6/9, p=0.011). Consistent with the prior study, there
was a significant reduction in liver volume (p=0.001) and hence
improvement in occurrence of hepatomegaly. There were no
significant differences in CHAQ/HAQ Diability Index or Joint Range
of Motion, although there was a trend towards improvement in more
severe patients. There was a statistically significant rapid
reduction in urinary GAG (p<0.001). Trends in favor of enzyme
treatment were noted in measurements of right shoulder extension,
left knee flexion, and LVDS (Left Venticle Internal Dimension at
End-Systole in cm) as measured by echocardiography.
9TABLE I Percent Predicted Change From Baseline Intent To Treat
Population Difference Baseline (% Week 26 (% from Predicted)
Predicted) Change Placebo .alpha.-L-Iduronidase 48.4 .+-. 14.85
50.2 .+-. 17.10 1.8 .+-. 7.70 4.5 (Aldurazyme .TM.) p = 0.028 n =
22 Placebo 54.2 .+-. 16.00 51.5 .+-. 13.13 -2.7 .+-. 7.12 n =
23
[0128]
10TABLE II Six-Minute Walk Change from Baseline Intent To Treat
Population Difference Baseline Week 26 from (m) (m) Change Placebo
.alpha.-L-Idur- 319.0 .+-. 131.41 338.8 .+-. 127.06 19.7 .+-. 68.56
38.1 onidase p = 0.066 (Aldura- zyme .TM.) n = 22 Placebo 366.7
.+-. 113.68 348.3 .+-. 128.81 -18.3 .+-. 67.49 n = 23
[0129] Comparison with Phase I Study
[0130] The results from measurement of secondary and tertiary
endpoints were consistent with that of the Phase I study. For
example, in both studies there was a significant reduction in liver
volume (p=0.001). Liver volume recovered to nomal in almost 60% of
enzyme-treated patients in the Phase III study at the end of 26
weeks. Similarly, in the Phase I study, liver size was normal for
body weight and age in eight of ten patients by 26 weeks. In both
studies, there was a reduction in sleep apnea/hypopnea events. As
described above, in the Phase In study, a reduction in events
(p=0.011) was observed in six of nine enzyme-treated patients with
clinically significant disease. Similarly, seven of ten patients in
the Phase I study showed a decrease in apnea and hypopnea events
from 155 to 60 per night upon treatment with a reduction in the
mean apnea/hypopnea index. There was also an improvement in the
joint range of motion of more severe patients treated with the
enzyme. Urinary GAG excretion rapidly declined in enzyme-treated
patients in both studies. In the Phase III study, there was a
statistically significant rapid reduction in urinary GAG levels
(p<0.001). Similarly, in the Phase I study, urinary GAG
excretion declined rapidly by three to four weeks and by eight to
twelve weeks had fallen by 60-80% of baseline. Thus, there appeared
to a strong correclations in the secondary and tertiary efficacy
endpoints of Phase I and III studies.
[0131] The results show that .alpha.-L-Iduronidase appears to be
safe and well-tolerated. The types of adverse events were similar
between days of infusion and non-infusion days. The frequency of
infusion--associated reactions was similar between placbo and
enzyme-treated groups. With respect to immunogenicity testing of
IgG, all 22 patients in the enzyme-reated group seroconverted with
a mean time to seroconversion of 41 days. Seroconversion did not
result in increased infusion-associated reactions or other adverse
events. Among three patients tested for IgE, including one patient
from the placebo group and two enyme-treated patients, all IgE
tests were negative. There were no clinically significant changes
in observations from physical examination, vital, and
brain/cranio--cervical junction MRI from baseline to week 26.
Standard laboratory evaluations showed: (1) no significant
laboratory chnges indicating a negative treatment effect; (2) a
significant increase in platelet counts in enzyme--treated
patients; and (3) improvement in liver enzyme abnormalities in
enyme-treated patients.
[0132] Summary
[0133] In the Phase I studies i) all patients developed antibodies
to the treatment with all 10 to contaminating proteins and 4 to IDU
itself; ii) 5 patients (50%) had clinical manifestations of an
allergic response of which the most common urticaria (hives); and
iii) several of these reactions were classified as serious adverse
events (SAEs) (meaning they required medical intervention) related
to treatment with .alpha.-L-Iduronidase.
[0134] In the Phase III study, i) all patients developed antibodies
to the treatment but it is not yet known whether these were to CP
or IDU itself; ii) clinical manifestations of an allergic response
were mild in all patients and were comparable between the placebo
and .alpha.-L-Iduronidase treated groups; iii) there were no SAEs
considered related to treatment with x-L-Iduronidase; and iv) there
was no urticaria reported.
[0135] In summary, the efficacy data gathered in the MPS I dog
studies and the two human clinical trials tells a consistent story
of improvement in disease symptoms. The safety profile of the
product improved significantly in the Phase im versus the Phase I.
This corroborates the theory that the material of increased purity
used in the Phase III trial is an improvment over the material used
in the Phase I trial.
[0136] The invention, and the manner and process of making and
using it, are now described in such full, clear, concise and exact
terms as to enable any person skilled in the art to which it
pertains, to make and use the same. It is to be understood that the
foregoing describes preferred embodiments of the present invention
and that modifications may be made therein without departing from
the spirit or scope of the present invention as set forth in the
claims. To particularly point out and distinctly claim the subject
matter regarded as invention, the following claims conclude this
specification.
Sequence CWU 1
1
2 1 6200 DNA Homo sapiens CDS (1558)...(3510) 1 gacggatcgg
gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca
gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc
agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt
ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
840 ctgcttaact ggcttatcga aattaatacg actcactata gggagaccca
agcttcgcag 900 aattcctgcg gctgctacag tgtgtccagc gtcctgcctg
gctgtgctga gcgctggaac 960 agtggcgcat cattcaagtg cacagttacc
catcctgagt ctggcacctt aactggcaca 1020 attgccaaag tcacaggtga
gctcagatgc ataccaggac attgtatgac gttccctgct 1080 cacatgcctg
ctttcttcct ataatacaga tgctcaacta actgctcatg tccttatatc 1140
acagagggaa attggagcta tctgaggaac tgcccagaag ggaagggcag aggggtcttg
1200 ctctccttgt ctgagccata actcttcttt ctaccttcca gtgaacacct
tcccacccca 1260 ggtccacctg ctaccgccgc cgtcggagga gctggccctg
aatgagctct tgtccctgac 1320 atgcctggtg cgagctttca accctaaaga
agtgctggtg cgatggctgc atggaaatga 1380 ggagctgtcc ccagaaagct
acctagtgtt tgagccccta aaggagccag gcgagggagc 1440 caccacctac
ctggtgacaa gcgtgttgcg tgtatcagct gaaagcttga tatcgaattc 1500
cggaggcgga accggcagtg cagcccgaag ccccgcagtc cccgagcacg cgtggcc atg
1560 Met 1 cgt ccc ctg cgc ccc cgc gcc gcg ctg ctg gcg ctc ctg gcc
tcg ctc 1608 Arg Pro Leu Arg Pro Arg Ala Ala Leu Leu Ala Leu Leu
Ala Ser Leu 5 10 15 ctg gcc gcg ccc ccg gtg gcc ccg gcc gag gcc ccg
cac ctg gtg cat 1656 Leu Ala Ala Pro Pro Val Ala Pro Ala Glu Ala
Pro His Leu Val His 20 25 30 gtg gac gcg gcc cgc gcg ctg tgg ccc
ctg cgg cgc ttc tgg agg agc 1704 Val Asp Ala Ala Arg Ala Leu Trp
Pro Leu Arg Arg Phe Trp Arg Ser 35 40 45 aca ggc ttc tgc ccc ccg
ctg cca cac agc cag gct gac cag tac gtg 1752 Thr Gly Phe Cys Pro
Pro Leu Pro His Ser Gln Ala Asp Gln Tyr Val 50 55 60 65 ctc agc tgg
gac cag cag ctc aac ctc gcc tat gtg ggc gcc gtc cct 1800 Leu Ser
Trp Asp Gln Gln Leu Asn Leu Ala Tyr Val Gly Ala Val Pro 70 75 80
cac cgc ggc atc aag cag gtc cgg acc cac tgg ctg ctg gag ctt gtc
1848 His Arg Gly Ile Lys Gln Val Arg Thr His Trp Leu Leu Glu Leu
Val 85 90 95 acc acc agg ggg tcc act gga cgg ggc ctg agc tac aac
ttc acc cac 1896 Thr Thr Arg Gly Ser Thr Gly Arg Gly Leu Ser Tyr
Asn Phe Thr His 100 105 110 ctg gac ggg tac ctg gac ctt ctc agg gag
aac cag ctc ggg ttt gag 1944 Leu Asp Gly Tyr Leu Asp Leu Leu Arg
Glu Asn Gln Leu Gly Phe Glu 115 120 125 ctg atg ggc agc gcc tcg ggc
cac ttc act gac ttt gag gac aag cag 1992 Leu Met Gly Ser Ala Ser
Gly His Phe Thr Asp Phe Glu Asp Lys Gln 130 135 140 145 cag gtg ttt
gag tgg aag gac ttg gtc tcc agc ctg gcc agg aga tac 2040 Gln Val
Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala Arg Arg Tyr 150 155 160
atc ggt agg tac gga ctg gcg cat gtt tcc aag tgg aac ttc gag acg
2088 Ile Gly Arg Tyr Gly Leu Ala His Val Ser Lys Trp Asn Phe Glu
Thr 165 170 175 tgg aat gag cca gac cac cac gac ttt gac aac gtc tcc
atg acc atg 2136 Trp Asn Glu Pro Asp His His Asp Phe Asp Asn Val
Ser Met Thr Met 180 185 190 caa ggc ttc ctg aac tac tac gat gcc tgc
tcg gag ggt ctg cgc gcc 2184 Gln Gly Phe Leu Asn Tyr Tyr Asp Ala
Cys Ser Glu Gly Leu Arg Ala 195 200 205 gcc agc ccc gcc ctg cgg ctg
gga ggc ccc ggc gac tcc ttc cac acc 2232 Ala Ser Pro Ala Leu Arg
Leu Gly Gly Pro Gly Asp Ser Phe His Thr 210 215 220 225 cca ccg cga
tcc ccg ctg agc tgg ggc ctc ctg cgc cac tgc cac gac 2280 Pro Pro
Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His Cys His Asp 230 235 240
ggt acc aac ttc ttc act ggg gag gcg ggc gtg cgg ctg gac tac atc
2328 Gly Thr Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu Asp Tyr
Ile 245 250 255 tcc ctc cac agg aag ggt gcg cgc agc tcc atc tcc atc
ctg gag cag 2376 Ser Leu His Arg Lys Gly Ala Arg Ser Ser Ile Ser
Ile Leu Glu Gln 260 265 270 gag aag gtc gtc gcg cag cag atc cgg cag
ctc ttc ccc aag ttc gcg 2424 Glu Lys Val Val Ala Gln Gln Ile Arg
Gln Leu Phe Pro Lys Phe Ala 275 280 285 gac acc ccc att tac aac gac
gag gcg gac ccg ctg gtg ggc tgg tcc 2472 Asp Thr Pro Ile Tyr Asn
Asp Glu Ala Asp Pro Leu Val Gly Trp Ser 290 295 300 305 ctg cca cag
ccg tgg agg gcg gac gtg acc tac gcg gcc atg gtg gtg 2520 Leu Pro
Gln Pro Trp Arg Ala Asp Val Thr Tyr Ala Ala Met Val Val 310 315 320
aag gtc atc gcg cag cat cag aac ctg cta ctg gcc aac acc acc tcc
2568 Lys Val Ile Ala Gln His Gln Asn Leu Leu Leu Ala Asn Thr Thr
Ser 325 330 335 gcc ttc ccc tac gcg ctc ctg agc aac gac aat gcc ttc
ctg agc tac 2616 Ala Phe Pro Tyr Ala Leu Leu Ser Asn Asp Asn Ala
Phe Leu Ser Tyr 340 345 350 cac ccg cac ccc ttc gcg cag cgc acg ctc
acc gcg cgc ttc cag gtc 2664 His Pro His Pro Phe Ala Gln Arg Thr
Leu Thr Ala Arg Phe Gln Val 355 360 365 aac aac acc cgc ccg ccg cac
gtg cag ctg ttg cgc aag ccg gtg ctc 2712 Asn Asn Thr Arg Pro Pro
His Val Gln Leu Leu Arg Lys Pro Val Leu 370 375 380 385 acg gcc atg
ggg ctg ctg gcg ctg ctg gat gag gag cag ctc tgg gcc 2760 Thr Ala
Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln Leu Trp Ala 390 395 400
gaa gtg tcg cag gcc ggg acc gtc ctg gac agc aac cac acg gtg ggc
2808 Glu Val Ser Gln Ala Gly Thr Val Leu Asp Ser Asn His Thr Val
Gly 405 410 415 gtc ctg gcc agc gcc cac cgc ccc cag ggc ccg gcc gac
gcc tgg cgc 2856 Val Leu Ala Ser Ala His Arg Pro Gln Gly Pro Ala
Asp Ala Trp Arg 420 425 430 gcc gcg gtg ctg atc tac gcg agc gac gac
acc cgc gcc cac ccc aac 2904 Ala Ala Val Leu Ile Tyr Ala Ser Asp
Asp Thr Arg Ala His Pro Asn 435 440 445 cgc agc gtc gcg gtg acc ctg
cgg ctg cgc ggg gtg ccc ccc ggc ccg 2952 Arg Ser Val Ala Val Thr
Leu Arg Leu Arg Gly Val Pro Pro Gly Pro 450 455 460 465 ggc ctg gtc
tac gtc acg cgc tac ctg gac aac ggg ctc tgc agc ccc 3000 Gly Leu
Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu Cys Ser Pro 470 475 480
gac ggc gag tgg cgg cgc ctg ggc cgg ccc gtc ttc ccc acg gca gag
3048 Asp Gly Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro Thr Ala
Glu 485 490 495 cag ttc cgg cgc tag cgc gcg gct gag gac ccg gtg gcc
gcg gcg ccc 3096 Gln Phe Arg Arg * Arg Ala Ala Glu Asp Pro Val Ala
Ala Ala Pro 500 505 510 cgc ccc tta ccc gcc ggc ggc cgc ctg agg ctg
cgc ccc gcg ctg cgg 3144 Arg Pro Leu Pro Ala Gly Gly Arg Leu Arg
Leu Arg Pro Ala Leu Arg 515 520 525 ctg ccg tcg ctt ttg ctg gtg cac
gtg tgt gcg cgc ccc gag aag ccg 3192 Leu Pro Ser Leu Leu Leu Val
His Val Cys Ala Arg Pro Glu Lys Pro 530 535 540 ccc ggg cag gtc acg
cgg ctc cgc gcc ctg ccc ctg acc caa ggg cag 3240 Pro Gly Gln Val
Thr Arg Leu Arg Ala Leu Pro Leu Thr Gln Gly Gln 545 550 555 560 ctg
gtt ctg gtc tgg tcg gat gaa cac gtg ggc tcc aag tgc ctg tgg 3288
Leu Val Leu Val Trp Ser Asp Glu His Val Gly Ser Lys Cys Leu Trp 565
570 575 aca tac gag atc cag ttc tct cag gac ggt aag gcg tac acc ccg
gtc 3336 Thr Tyr Glu Ile Gln Phe Ser Gln Asp Gly Lys Ala Tyr Thr
Pro Val 580 585 590 agc agg aag cca tcg acc ttc aac ctc ttt gtg ttc
agc cca gac aca 3384 Ser Arg Lys Pro Ser Thr Phe Asn Leu Phe Val
Phe Ser Pro Asp Thr 595 600 605 ggt gct gtc tct ggc tcc tac cga gtt
cga gcc ctg gac tac tgg gcc 3432 Gly Ala Val Ser Gly Ser Tyr Arg
Val Arg Ala Leu Asp Tyr Trp Ala 610 615 620 cga cca ggc ccc ttc tcg
gac cct gtg ccg tac ctg gag gtc cct gtg 3480 Arg Pro Gly Pro Phe
Ser Asp Pro Val Pro Tyr Leu Glu Val Pro Val 625 630 635 640 cca aga
ggg ccc cca tcc ccg ggc aat cca tgagcctgtg ctgagcccca 3530 Pro Arg
Gly Pro Pro Ser Pro Gly Asn Pro 645 650 gtgggttgca cctccaccgg
cagtcagcga gctggggctg cactgtgccc atgctgccct 3590 cccatcaccc
cctttgcaat atatttttat attttaaaaa aaaaaaaaaa aaaaaaaaaa 3650
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag aattcctgca
3710 gcccggggga tccactagtt ctagagggcc cgtttaaacc cgctgatcag
cctcgactgt 3770 gccttctagt tgccagccat ctgttgtttg cccctccccc
gtgccttcct tgaccctgga 3830 aggtgccact cccactgtcc tttcctaata
aaatgaggaa attgcatcgc attgtctgag 3890 taggtgtcat tctattctgg
ggggtggggt ggggcaggac agcaaggggg aggattggga 3950 agacaatagc
aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 4010
cagctggggc tcgagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
4070 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
agcctggggt 4130 gcctaatgag tgagctaact cacattaatt gcgttgcgct
cactgcccgc tttccagtcg 4190 ggaaacctgt cgtgccagct gcattaatga
atcggccaac gcgcggggag aggcggtttg 4250 cgtattgggc gctcttccgc
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 4310 cggcgagcgg
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 4370
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
4430 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
aaatcgacgc 4490 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
accaggcgtt tccccctgga 4550 agctccctcg tgcgctctcc tgttccgacc
ctgccgctta ccggatacct gtccgccttt 4610 ctcccttcgg gaagcgtggc
gctttctcaa tgctcacgct gtaggtatct cagttcggtg 4670 taggtcgttc
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4730
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
4790 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
tacagagttc 4850 ttgaagtggt ggcctaacta cggctacact agaaggacag
tatttggtat ctgcgctctg 4910 ctgaagccag ttaccttcgg aaaaagagtt
ggtagctctt gatccggcaa acaaaccacc 4970 gctggtagcg gtggtttttt
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 5030 caagaagatc
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 5090
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
5150 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
cagttaccaa 5210 tgcttaatca gtgaggcacc tatctcagcg atctgtctat
ttcgttcatc catagttgcc 5270 tgactccccg tcgtgtagat aactacgata
cgggagggct taccatctgg ccccagtgct 5330 gcaatgatac cgcgagaccc
acgctcaccg gctccagatt tatcagcaat aaaccagcca 5390 gccggaaggg
ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 5450
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
5510 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
attcagctcc 5570 ggttcccaac gatcaaggcg agttacatga tcccccatgt
tgtgcaaaaa agcggttagc 5630 tccttcggtc ctccgatcgt tgtcagaagt
aagttggccg cagtgttatc actcatggtt 5690 atggcagcac tgcataattc
tcttactgtc atgccatccg taagatgctt ttctgtgact 5750 ggtgagtact
caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 5810
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
5870 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
atccagttcg 5930 atgtaaccca ctcgtgcacc caactgatct tcagcatctt
ttactttcac cagcgtttct 5990 gggtgagcaa aaacaggaag gcaaaatgcc
gcaaaaaagg gaataagggc gacacggaaa 6050 tgttgaatac tcatactctt
cctttttcaa tattattgaa gcatttatca gggttattgt 6110 ctcatgagcg
gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 6170
acatttcccc gaaaagtgcc acctgacgtc 6200 2 650 PRT Homo sapiens 2 Met
Arg Pro Leu Arg Pro Arg Ala Ala Leu Leu Ala Leu Leu Ala Ser 1 5 10
15 Leu Leu Ala Ala Pro Pro Val Ala Pro Ala Glu Ala Pro His Leu Val
20 25 30 His Val Asp Ala Ala Arg Ala Leu Trp Pro Leu Arg Arg Phe
Trp Arg 35 40 45 Ser Thr Gly Phe Cys Pro Pro Leu Pro His Ser Gln
Ala Asp Gln Tyr 50 55 60 Val Leu Ser Trp Asp Gln Gln Leu Asn Leu
Ala Tyr Val Gly Ala Val 65 70 75 80 Pro His Arg Gly Ile Lys Gln Val
Arg Thr His Trp Leu Leu Glu Leu 85 90 95 Val Thr Thr Arg Gly Ser
Thr Gly Arg Gly Leu Ser Tyr Asn Phe Thr 100 105 110 His Leu Asp Gly
Tyr Leu Asp Leu Leu Arg Glu Asn Gln Leu Gly Phe 115 120 125 Glu Leu
Met Gly Ser Ala Ser Gly His Phe Thr Asp Phe Glu Asp Lys 130 135 140
Gln Gln Val Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala Arg Arg 145
150 155 160 Tyr Ile Gly Arg Tyr Gly Leu Ala His Val Ser Lys Trp Asn
Phe Glu 165 170 175 Thr Trp Asn Glu Pro Asp His His Asp Phe Asp Asn
Val Ser Met Thr 180 185 190 Met Gln Gly Phe Leu Asn Tyr Tyr Asp Ala
Cys Ser Glu Gly Leu Arg 195 200 205 Ala Ala Ser Pro Ala Leu Arg Leu
Gly Gly Pro Gly Asp Ser Phe His 210 215 220 Thr Pro Pro Arg Ser Pro
Leu Ser Trp Gly Leu Leu Arg His Cys His 225 230 235 240 Asp Gly Thr
Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu Asp Tyr 245 250 255 Ile
Ser Leu His Arg Lys Gly Ala Arg Ser Ser Ile Ser Ile Leu Glu 260 265
270 Gln Glu Lys Val Val Ala Gln Gln Ile Arg Gln Leu Phe Pro Lys Phe
275 280 285 Ala Asp Thr Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu Val
Gly Trp 290 295 300 Ser Leu Pro Gln Pro Trp Arg Ala Asp Val Thr Tyr
Ala Ala Met Val 305 310 315 320 Val Lys Val Ile Ala Gln His Gln Asn
Leu Leu Leu Ala Asn Thr Thr 325 330 335 Ser Ala Phe Pro Tyr Ala Leu
Leu Ser Asn Asp Asn Ala Phe Leu Ser 340 345 350 Tyr His Pro His Pro
Phe Ala Gln Arg Thr Leu Thr Ala Arg Phe Gln 355 360 365 Val Asn Asn
Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys Pro Val 370 375 380 Leu
Thr Ala Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln Leu Trp 385 390
395 400 Ala Glu Val Ser Gln Ala Gly Thr Val Leu Asp Ser Asn His Thr
Val 405 410 415 Gly Val Leu Ala Ser Ala His Arg Pro Gln Gly Pro Ala
Asp Ala Trp 420 425 430 Arg Ala Ala Val Leu Ile Tyr Ala Ser Asp Asp
Thr Arg Ala His Pro 435 440 445 Asn Arg Ser Val Ala Val Thr Leu Arg
Leu Arg Gly Val Pro Pro Gly 450 455 460 Pro Gly Leu Val Tyr Val Thr
Arg Tyr Leu Asp Asn Gly Leu Cys Ser 465 470 475 480 Pro Asp Gly Glu
Trp Arg Arg Leu Gly Arg Pro Val Phe Pro Thr Ala 485 490 495 Glu Gln
Phe Arg Arg Arg Ala Ala Glu Asp Pro Val Ala Ala Ala Pro 500 505 510
Arg Pro Leu Pro Ala Gly Gly Arg Leu Arg Leu Arg Pro Ala Leu Arg 515
520 525 Leu Pro Ser Leu Leu Leu Val His Val Cys Ala Arg Pro Glu Lys
Pro 530 535 540 Pro Gly Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr
Gln Gly Gln 545 550 555 560 Leu Val Leu Val Trp Ser Asp Glu His Val
Gly Ser Lys Cys Leu Trp 565 570 575 Thr Tyr Glu Ile Gln Phe Ser Gln
Asp Gly Lys Ala Tyr Thr Pro Val 580 585 590 Ser Arg Lys Pro Ser Thr
Phe Asn Leu Phe Val Phe Ser Pro Asp Thr 595 600 605 Gly Ala Val Ser
Gly Ser Tyr Arg Val Arg Ala Leu Asp Tyr Trp Ala 610 615 620 Arg Pro
Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu Glu Val Pro Val 625 630 635
640 Pro Arg Gly Pro Pro Ser Pro Gly Asn Pro 645 650
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