U.S. patent application number 17/524628 was filed with the patent office on 2022-06-30 for sialylated glycoprotein compositons and uses thereof.
The applicant listed for this patent is Ultragenyx Pharmaceutical Inc.. Invention is credited to Jeff GRUBB, Steven JUNGLES, Emil D. KAKKIS, Gabrielle MORRIS, Michael VELLARD.
Application Number | 20220202915 17/524628 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220202915 |
Kind Code |
A1 |
JUNGLES; Steven ; et
al. |
June 30, 2022 |
SIALYLATED GLYCOPROTEIN COMPOSITONS AND USES THEREOF
Abstract
The present application relates to sialylated glycoprotein
compositions and methods of their use in treating various
conditions and disorders.
Inventors: |
JUNGLES; Steven;
(Naperville, IL) ; MORRIS; Gabrielle; (Novato,
CA) ; GRUBB; Jeff; (Petaluma, CA) ; VELLARD;
Michael; (San Rafael, CA) ; KAKKIS; Emil D.;
(San Rafael, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ultragenyx Pharmaceutical Inc. |
Novato |
CA |
US |
|
|
Appl. No.: |
17/524628 |
Filed: |
November 11, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16738938 |
Jan 9, 2020 |
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17524628 |
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15943526 |
Apr 2, 2018 |
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16738938 |
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15607257 |
May 26, 2017 |
9937243 |
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15943526 |
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15251701 |
Aug 30, 2016 |
9687532 |
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15607257 |
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14639171 |
Mar 5, 2015 |
9457067 |
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15251701 |
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62114313 |
Feb 10, 2015 |
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61948421 |
Mar 5, 2014 |
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International
Class: |
A61K 38/47 20060101
A61K038/47; C12N 9/24 20060101 C12N009/24; A61K 45/06 20060101
A61K045/06; C12N 9/38 20060101 C12N009/38; A61K 9/00 20060101
A61K009/00 |
Claims
1.-27. (canceled)
28. A method for treating mucopolysaccharidosis type 7 in a subject
in need thereof, comprising administering to the subject a
composition comprising a recombinant .beta.-glucuronidase, wherein
the recombinant .beta.-glucuronidase is administered every other
week by continuous intravenous infusion at a dose of 1 mg/kg to 8
mg/kg.
29. The method of claim 28, wherein the subject is a human.
30. The method of claim 28, wherein the recombinant
.beta.-glucuronidase is administered at a dose of 2 mg/kg to 6
mg/kg.
31. The method of claim 28, wherein the recombinant
.beta.-glucuronidase is administered at a dose of 4 mg/kg.
32. The method of claim 28, wherein the recombinant
.beta.-glucuronidase is administered concurrently with or following
antihistamine therapy.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 16/738,938, filed Jan. 9, 2020, which is a
Continuation of U.S. patent application Ser. No. 15/943,526, filed
Apr. 2, 2018, which is a Continuation of Ser. No. 15/607,257, filed
May 26, 2017, (now, U.S. Pat. No. 9,937,243, issued, Apr. 10,
2018), which is a Continuation of U.S. patent application Ser. No.
15/251,701, filed Aug. 30, 2016, (now U.S. Pat. No. 9,687,532,
issued Jun. 27, 2017), which is a Continuation of U.S. patent
application Ser. No. 14/639,171, filed Mar. 5, 2015, (now U.S. Pat.
No. 9,457,067, issued Oct. 4, 2016), which claims priority to U.S.
Provisional Application Ser. No. 61/948,421, filed Mar. 5, 2014,
and U.S. Provisional Application Ser. No. 62/114,313, filed Feb.
10, 2015, each of which is herein incorporated by reference in
their entireties for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to sialylated glycoprotein
compositions and methods of their use in treating various
conditions and disorders.
DESCRIPTION OF TEXT FILE SUBMITTED ELECTRONICALLY
[0003] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
ULPI_020_08US_SeqList_ST25.txt, date recorded: Nov. 1, 2021, file
size: 5.88 kilobytes).
BACKGROUND OF THE INVENTION
[0004] The number of commercially-available therapeutic proteins
has increased dramatically in recent years and most of these
proteins are glycoproteins. The presence of sialic acid in a
glycoprotein can positively affect absorption, serum half-life, and
clearance from the serum, as well as the physical, chemical and
immunogenic properties of the respective glycoprotein. In certain
circumstances, it may therefore be desirable to increase the sialic
acid content of a glycoprotein intended for use in pharmacologic
applications.
SUMMARY OF THE INVENTION
[0005] The present invention is based, in part, on the discovery
that recombinant glycoprotein produced from mammalian cells through
the use of serum/protein free media improves sialylation of the
recombinant glycoprotein, e.g., without reducing the M6P content of
the recombinant glycoprotein.
[0006] In some embodiments of the present invention, a composition
comprises a recombinant glycoprotein having a sialic acid content
greater than 0.05 mol/mol of the recombinant glycoprotein. In some
embodiments, a composition comprises a recombinant glycoprotein
having a sialic acid content greater than 0.1 mol/mol of the
recombinant glycoprotein. In some embodiments, a composition
comprises a recombinant glycoprotein having a sialic acid content
greater than 0.5 mol/mol of the recombinant glycoprotein. In some
embodiments, a composition comprises a recombinant glycoprotein
having a sialic acid content greater than 0.7 mol/mol of the
recombinant glycoprotein. In some embodiments, a composition
comprises a recombinant glycoprotein having a sialic acid content
greater than 1 mol/mol of the recombinant glycoprotein. In some
embodiments, a composition comprises a recombinant glycoprotein
having a sialic acid content greater than 1.5 mol/mol of the
recombinant glycoprotein. In some embodiments, a composition
comprises a recombinant glycoprotein having a sialic acid content
greater than 2 mol/mol of the recombinant glycoprotein. In some
embodiments, a composition comprises a recombinant glycoprotein
having a sialic acid content greater than 5 mol/mol of the
recombinant glycoprotein. In some embodiments, a composition
comprises a recombinant glycoprotein having a sialic acid content
greater than 10 mol/mol of the recombinant glycoprotein. In some
embodiments, a composition comprises a recombinant glycoprotein
having a sialic acid content greater than 20 mol/mol of the
recombinant glycoprotein.
[0007] In some embodiments, the present invention provides a
composition comprising a recombinant glycoprotein, wherein at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of galactose
residues of the recombinant glycoprotein are sialylated.
[0008] In some embodiments, the present invention provides a
composition comprising a recombinant glycoprotein, wherein the
recombinant glycoprotein is human .beta.-glucuronidase and has a
sialylation content of at least 0.7 mol/mol of the recombinant
glycoprotein.
[0009] In some other embodiments, the present invention provides a
composition comprising a recombinant glycoprotein, wherein the
recombinant glycoprotein is human .beta.-glucuronidase and has a
sialylation content of at least 0.7 mol/mol of the recombinant
glycoprotein and a high level of mannose-6-phosphate (M6P)
moieties.
[0010] In one embodiment, the present invention provides a
composition comprising a recombinant glycoprotein, wherein the
recombinant glycoprotein is human .beta.-glucuronidase and has a
sialylation content of at least 0.7 mol/mol of the recombinant
glycoprotein and a high level of mannose-6-phosphate (M6P) moieties
of at least 10 mol % of the total glycan of the recombinant
glycoprotein.
[0011] In another embodiment, the present invention provides a
composition comprising a recombinant glycoprotein, wherein the
recombinant glycoprotein is human .beta.-glucuronidase and has a
sialylation content of at least 0.7 mol/mol of the recombinant
glycoprotein and a high level of mannose-6-phosphate (M6P)
moieties, e.g., K uptake is at its 60%, 70%, 80%, 90% or maximum
such as from about 1 nM to about 3 nM when tested in human
fibroblast cells (MPSI).
[0012] In yet another embodiment, the present invention provides a
composition comprising a recombinant glycoprotein, wherein the
recombinant glycoprotein is human .beta.-glucuronidase and has a
sialylation content of at least 0.7 mol/mol of the recombinant
glycoprotein and a high level of mannose-6-phosphate (M6P)
moieties, e.g., half-maximal uptake in human fibroblast cells such
as concentrations at which the glycoprotein (e.g., human
.beta.-glucuronidase) reaches 50% of maximal uptake is about no
more than 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM.
[0013] In some embodiments, the present invention provides a
preparation of a population of a recombinant glycoprotein, wherein
at least 50 percent of the population is sialylated. In some
embodiments, the present invention provides a preparation of a
population of a recombinant glycoprotein, wherein at least 60
percent of the population is sialylated. In some embodiments, the
present invention provides a preparation of a population of a
recombinant glycoprotein, wherein at least 70 percent of the
population is sialylated. In some embodiments, the present
invention provides a preparation of a population of a recombinant
glycoprotein, wherein at least 80 percent of the population is
sialylated. In some embodiments, the present invention provides a
preparation of a population of a recombinant glycoprotein, wherein
at least 90 percent of the population is sialylated.
[0014] Also provided is a method of making a
composition/preparation according to the present invention. In some
embodiments, the method comprises expressing the recombinant
glycoprotein in a cell culture with a serum or protein free media.
In some embodiments, a protein-free, chemically defined media may
be used to grow cells. In some embodiments, the media do not
include an effective amount of a sugar selected from galactose,
fructose, n-acetyl-mannosamine, mannose and combinations thereof.
For example, the effective amount of a sugar is greater than 0.01
mM, 0.05 mM, or 0.1 mM.
[0015] In some embodiments, a cell culture comprises a mammalian
cell. Exemplary mammalian cells include but are not limited to
Chinese Hamster Ovary (CHO), HeLa, VERO, BHK, Cos, MDCK, 293, 3T3,
myeloma (e.g. NSO, NSI), or WI38 cells. In a specific embodiment,
the mammalian cells are Chinese Hamster Ovary (CHO) cells.
[0016] In some other embodiments, a cell culture comprises a plant
cell. Exemplary plant cells include but are not limited to carrot
cells or any other plant cell based cell culture, e.g., developed
for recombinant protein production.
[0017] Further provided is a method of treating lysosomal storage
disorder (LSD) comprising administering to an individual in need of
such treatment a therapeutically effective amount of the
composition/preparation as described herein. In an exemplary
embodiment, the composition/preparation comprises recombinant human
.beta.-glucuronidase. In a further exemplary embodiment, the LSD is
mucopolysaccharidosis type 7 (i.e., MPS 7, MPS VII, or Sly
Syndrome). In some embodiments, the recombinant human
.beta.-glucuronidases provided herein have increased sialic acid
content and are particularly useful in treating a LSD, e.g., MPS
7.
[0018] In some embodiments, the present invention provides a method
for treating a lysosomal storage disorder (LSD) in a subject,
comprising administering a regimen of the composition/preparation
as described herein, wherein the administration provides a
statistically significant therapeutic effect for the treatment of
the LSD. In an exemplary embodiment, the composition/preparation
comprises recombinant human .beta.-glucuronidase. In a further
exemplary embodiment, the LSD is MPS 7.
Definitions
[0019] As used herein, the term "effective" (e.g., "an effective
amount") means adequate to accomplish a desired, expected, or
intended result. An effective amount can be a therapeutically
effective amount. A "therapeutically effective amount" refers to
the amount of an active ingredient that, when administered to a
subject, is sufficient to effect such treatment of a particular
disease or condition. The "therapeutically effective amount" will
vary depending on, e.g., the disease or condition, the severity of
the disease or condition, and the age, weight, etc., of the subject
to be treated.
[0020] In general, "treating" or "treatment" of any condition,
disease or disorder refers, in some embodiments, to ameliorating
the condition, disease or disorder (i.e., arresting or reducing the
development of the disease or at least one of the clinical symptoms
thereof). In some embodiments "treating" or "treatment" refers to
ameliorating at least one physical parameter, which may not be
discernible by the subject. In some embodiments, "treating" or
"treatment" refers to inhibiting the condition, disease or
disorder, either physically, (e.g., stabilization of a discernible
symptom), physiologically, (e.g., stabilization of a physical
parameter) or both. In some embodiments, "treating" or "treatment"
refers to delaying the onset of a condition, disease, or
disorder.
[0021] Following long-standing patent law convention, the terms
"a", "an", and "the" refer to "one or more" when used in this
application, including the claims. The use of the term "or" in the
claims is used to mean "and/or" unless explicitly indicated to
refer to alternatives only or the alternatives are mutually
exclusive. It is specifically contemplated that any listing of
items using the term "or" means that any of those listed items may
also be specifically excluded from the related embodiment.
[0022] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0023] A glycoprotein, as used herein, is a protein that has been
modified by the addition of one or more carbohydrates, including,
especially, the addition of one or more sugar residues.
[0024] As used herein, "GUS" refers to .beta.-glucuronidase, an
exemplary glycoprotein in accordance with the present
invention.
[0025] Sialylation, as used herein, is the addition of a sialic
acid residue to a protein, which may be a glycoprotein. The term
sialic acid, as used herein, encompasses a family of sugars
containing 9 or more carbon atoms, including a carboxyl group. A
generic structure encompassing all known natural forms of sialic
acid is shown below.
##STR00001##
[0026] R1 groups at various positions on a single molecule can be
the same as or different from each other. R1 can be a hydrogen or
an acetyl, lactyl, methyl, sulfate, phosphate, anhydro, sialic
acid, fucose, glucose, or galactose group. R2 can be an N-acetyl,
N-glycolyl, amino, hydroxyl, N-glycolyl-O-acetyl, or
N-glycolyl-O-methyl group. R3 represents the preceding sugar
residue in an oligosaccharide to which sialic acid is attached in
the context of a glycoprotein. R3 can be galactose (connected at
its 3, 4, or 5 position), N-acetyl-galactose (connected at its 6
position), N-acetyl-glucose (connected at its 4 or 6 position),
sialic acid (connected at its 8 or 9 position), or
5-N-glycolyl-neuraminic acid. Essentials of Glycobiology, Ch. 15,
Varki et al., eds., Cold Spring Harbor Laboratory Press, New York
(1999). More than 40 forms of sialic acid have been found in
nature. Essentials of Glycobiology, Ch. 15, Varki et al., eds.,
Cold Spring Harbor Laboratory Press, New York (1999). A common form
of sialic acid is N-acetylneuraminic acid (NANA), in which R1 is a
hydrogen at all positions and R2 is an N-acetyl group.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a plot comparing pharmacokinetics of recombinant
human .beta.-glucuronidases, GUS CR01 vs. GUS Lot 43/44 in rats via
a two-stage infusion. The data show that the infusion with the
higher sialylated CR01 results in less rapid clearance and a higher
mean concentration during the infusion, which increases the total
exposure to the enzyme, potentially enhancing its penetration of
tissues that are more difficult to treat.
[0028] FIG. 2 is a plot showing only the post infusion clearance
phase of GUS CR01 vs. GUS Lot 43/44 which was used to calculate the
t.sub.1/2 values. These differences in the rate of clearance are
sufficient to result in higher serum levels of enzymes as shown in
FIG. 1.
[0029] FIG. 3 is a series of plots showing tissue GUS levels for
the pharmacokinetics study of GUS CR01 vs. GUS Lot 43/44. The
tissue delivery and uptake of human .beta.-glucuronidases is shown
to be enhanced as the total uptake of sialylated human
.beta.-glucuronidases (CR01) is higher in all tissues than the
lower sialylated enzyme (43-44). When the endogenous glucuronidase
activity is substracted, the effective delivery of therapeutic
human .beta.-glucuronidases is increased 2 fold to almost 10 fold,
an exceptional and surprising finding.
[0030] FIG. 4 is a plot showing the measurement of urinary
glycosaminoglycan (uGAG) levels over 36 weeks in three subjects
treated with recombinant human .beta.-glucuronidase (rhGUS). A
rapid and sustained dose-dependent reduction in uGAG levels was
observed in subjects treated with rhGUS.
[0031] FIG. 5 illustrates the mean reduction in urinary
glycosaminoglycan (uGAG) levels at the end of each dosing interval
during a 36 week evaluation of three subjects treated with rhGUS. A
4 mg/kg QOW dose resulted in the greatest reduction in uGAG
levels.
[0032] FIG. 6 is a plot showing the measurement of serum
glycosaminoglycan (GAG) levels over 36 weeks in three subjects
treated with rhGUS. Each subject demonstrated at least a 25%
reduction in serum GAG levels at the end of the 36-week treatment
schedule.
[0033] FIG. 7 is a plot showing the measurement of liver size in
subjects treated with rhGUS. A significant reduction in
hepatomegaly resulting from the 36 week treatment protocol was
observed.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention is based, in part, on the discovery
that recombinant glycoprotein produced from mammalian cells through
the use of serum/protein free media improves sialylation of the
recombinant glycoprotein, and additionally levels of
mannose-6-phosphate moieties of the recombinant glycoprotein.
Sialylated Glycoprotein Compositions
[0035] Compositions as described herein comprise one or more
glycoproteins that have a high level or increased sialic acid
content.
[0036] Sialic acids represent a family of aminosugars with
9-carbons with over 50 members derived from N-acetyleuraminic acid.
Sialic acid is only one component out of several monosaccharides
building glycans of glycoproteins, but has an outstanding impact on
the quality and stability of any therapeutic glycoproteins for
several reasons: (I) terminal galactose residues are one of the
major factors determining the serum half-life of glycoproteins. The
serum half-life is regulated by the expression of liver
asialo-glycoprotein receptors. These receptors bind nonsialylated
glycoproteins and bound asialo-glycoproteins are removed from the
serum by endocytosis. As a consequence, expression of terminal
sialic acid on galactose residues prevents serum glycoproteins from
degradation; (II) sialic acids are important for masking antigenic
determinants or epitopes. It is known that the receptors of the
immune system (T- and B-cell receptors) often prefer nonsialylated
structures. Therefore, the possibility of the generation of
antibodies against the therapeutic glycoproteins correlates with
the degree of its sialylation; (III) negatively charged sialic
acids influence protein-specific parameters such as the thermal
stability, the resistance to proteolytic degradation or its
solubility (Bork et al., Increasing the Sialylation of Therapeutic
Glycoproteins: The potential of the Sialic Acid Biosynthetic
Pathway, Journal of Pharmaceutical Sciences, Vol. 98, No. 10,
October 2009).
[0037] In one aspect, the invention provides compositions
comprising a recombinant glycoprotein having a sialic acid content
greater than 0.05 mol/mol, 0.1 mol/mol, 0.5 mol/mol, 0.7 mol/mol, 1
mol/mol, 1.5 mol/mol, 2 mol/mol, 5 mol/mol, 10 mol/mol or 20
mol/mol of the recombinant glycoprotein. In some embodiments, the
invention provides compositions comprising a recombinant
glycoprotein having a sialic acid content greater than 0.5 mol/mol
of the recombinant glycoprotein. In additional embodiments, the
invention provides compositions comprising a recombinant
glycoprotein having a sialic acid content greater than 0.7 mol/mol
of the recombinant glycoprotein. In certain additional embodiments,
the invention provides compositions comprising a recombinant
glycoprotein having a sialic acid content greater than 1 mol/mol of
the recombinant glycoprotein.
[0038] In certain embodiments, the recombinant glycoprotein is a
recombinant form of human .beta.-glucuronidase, an enzyme
responsible for catalyzing the hydrolysis of .beta.-D-glucuronic
acid residues from the non-reducing end of mucopolysaccharides. In
some embodiments, the recombinant human .beta.-glucuronidase
(rhGUS) has a sialic acid content greater than 0.1 mol/mol, 0.5
mol/mol, 0.7 mol/mol, 1 mol/mol, 1.5 mol/mol, 2 mol/mol, or 5
mol/mol of the rhGUS. In one exemplary embodiment, the recombinant
human .beta.-glucuronidase (rhGUS) has a sialic acid content
greater than 0.7 mol/mol of the rhGUS. In another exemplary
embodiment, the recombinant human .beta.-glucuronidase (rhGUS) has
a sialic acid content greater than 1.0 mol/mol of the rhGUS. In yet
another exemplary embodiment, the recombinant human
.beta.-glucuronidase (rhGUS) has a sialic acid content of about 1.2
mol/mol of the rhGUS.
[0039] In some embodiments, the recombinant human
.beta.-glucuronidase (rhGUS) has a sialic acid content of about 0.5
mol/mol to about 2.0 mol/mol of the rhGUS. In one embodiment, the
recombinant human .beta.-glucuronidase (rhGUS) has a sialic acid
content of about 0.6 mol/mol to about 1.5 mol/mol of the rhGUS. In
another embodiment, the recombinant human .beta.-glucuronidase
(rhGUS) has a sialic acid content of about 0.7 mol/mol to about 1.4
mol/mol of the rhGUS. In an exemplary embodiment, the recombinant
human .beta.-glucuronidase (rhGUS) has a sialic acid content of
about 0.8 mol/mol to about 1.3 mol/mol of the rhGUS. In another
exemplary embodiment, the recombinant human .beta.-glucuronidase
(rhGUS) has a sialic acid content of about 1.0 mol/mol to about 1.2
mol/mol of the rhGUS.
[0040] In some embodiments, the composition of the present
invention includes a recombinant glycoprotein having at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of sites suitable for
sialic acid linkage sialylated. In general, galactose is the site
suitable for sialic acid linkage or sialylation. In certain
embodiments, a composition comprises a recombinant glycoprotein,
wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of
the galactose residues of the recombinant glycoprotein are
sialylated. In some embodiments, the composition comprises a
recombinant glycoprotein, wherein at least 50% of the galactose
residues of the recombinant glycoprotein are sialylated. In
additional embodiments, the composition comprises a recombinant
glycoprotein, wherein at least 60% of the galactose residues of the
recombinant glycoprotein are sialylated. In certain additional
embodiments, the composition comprises a recombinant glycoprotein,
wherein at least 70% of the galactose residues of the recombinant
glycoprotein are sialylated.
[0041] In certain embodiments, the recombinant glycoprotein is a
recombinant human .beta.-glucuronidase (rhGUS). In some
embodiments, at least 40%, 50%, 60%, 70%, or 80% of the galactose
residues of the recombinant human .beta.-glucuronidase (rhGUS) are
sialylated. In one exemplary embodiment, at least 50% of the
galactose residues of the rhGUS are sialylated. In another
exemplary embodiment, at least 60% of the galactose residues of the
rhGUS are sialylated. In yet another exemplary embodiment, at least
70% of the galactose residues of the rhGUS are sialylated. In yet
another exemplary embodiment, about 70%, 71%, 72%, 73%, 74%, or
about 75% of the galactose residues of the rhGUS are
sialylated.
[0042] In some embodiments, at least about 40% to at least about
90% of the galactose residues of the recombinant human
.beta.-glucuronidase (rhGUS) are sialylated. In one embodiment, at
least about 50% to at least about 80% of the galactose residues of
the recombinant human .beta.-glucuronidase (rhGUS) are sialylated.
In another embodiment, at least about 60% to at least about 80% of
the galactose residues of the recombinant human
.beta.-glucuronidase (rhGUS) are sialylated. In an exemplary
embodiment, at least about 65% to at least about 75% of the
galactose residues of the recombinant human .beta.-glucuronidase
(rhGUS) are sialylated.
[0043] In another aspect, the invention provides compositions
comprising a recombinant glycoprotein having a high level of sialic
acid content as well as a high level of mannose-6-phosphate (M6P)
moieties. As used herein, M6P moieties include any
mannose-6-phosphate capable of binding to or being recognized by
M6P receptors including without any limitation mono-phosphorylated
and bis-phosphorylated mannose-6-phosphate. In one embodiment, M6P
moieties include any M6P binding to cation-independent M6P receptor
(CI-MPR). In another embodiment, M6P moieties include any M6P
binding to cation-dependent M6P receptor (CD-MRP). In yet another
embodiment, M6P moieties include any bis-phosphorylated M6P.
[0044] According to the present invention, a high level of
mannose-6-phosphate moieties can include any level of M6P moieties
that is considered high by one skilled in the art, e.g., measured
using any suitable means known to or later developed by one skilled
in the art. In one embodiment, a high level of M6P moieties of a
recombinant glycoprotein includes M6P moiety levels of at least 10
mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol % or 15 mol % of the
total glycan of the recombinant glycoprotein. For example, the
recombinant glycoprotein can have a high level of M6P as determined
by the percentage of M6P peak area over total glycan peak area,
e.g., at least 10%, 11%, 12%, 13%, 14% or 15%. In some embodiments,
the recombinant glycoprotein is a recombinant human
.beta.-glucuronidase (rhGUS) and comprises M6P moiety levels of at
least 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol % or 15 mol %
of the total glycan of the rhGUS. In an exemplary embodiment, the
rhGUS comprises M6P moiety levels of about 13% to about 15%.
[0045] In another embodiment, a high level of M6P moieties of a
recombinant glycoprotein includes a high level of uptake of the
recombinant glycoprotein by human cells, e.g., high affinity uptake
amount by human fibroblast cells. For example, the recombinant
glycoprotein can have a M6P dependent K uptake of no more than 1
nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM,
1.9 nM, 2 nM, 2.1 nM, 2.2 nM, 2.3 nM, 2.4 nM, 2.5 nM, 2.6 nM, 2.7
nM, 2.8 nM, 2.9 nM, 3 nM, 4 nM, or 5 nM by any suitable human
cells, e.g., human fibroblast cells. In some embodiments, the
recombinant glycoprotein is a recombinant human
.beta.-glucuronidase (rhGUS) and has a M6P dependent K uptake of
less than 5 nM, less than 4 nM, less than 3 nM, or less than 2 nM.
In an exemplary embodiment, the rhGUS has a M6P dependent K uptake
of about 1.2 nM to about 1.8 nM.
[0046] In yet another embodiment, a high level of M6P moieties in a
recombinant glycoprotein includes lower concentrations required to
achieve maximum uptake of the recombinant glycoprotein by human
cells, e.g., lower half-maximum concentration. For example, the
recombinant glycoprotein can achieve maximum uptake by human cells,
e.g., fibroblast cells at concentrations less than 10 nM, 9 nM, 8
nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM or 1 nM. In some
embodiments, the recombinant glycoprotein is a recombinant human
.beta.-glucuronidase (rhGUS) can achieve maximum uptake by human
cells, e.g., fibroblast cells at concentrations of less than 5 nM,
less than 4 nM, less than 3 nM, or less than 2 nM. In an exemplary
embodiment, the rhGUS can achieve maximum uptake by human cells,
e.g., fibroblast cells at concentrations of about 1.2 nM to about
1.8 nM.
[0047] In still another embodiment, a high level of M6P moieties in
a recombinant glycoprotein includes one or more levels of M6P
moieties corresponding to levels of M6P moieties associated with
natural sialylation content of the recombinant glycoprotein, e.g.,
sialylation content of the recombinant glycoprotein prior to any
means for enhancement such as using the methods disclosed in the
present application.
[0048] According to the present invention, in some embodiments the
recombinant glycoprotein, e.g., a recombinant human
.beta.-glucuronidase or any other lysosomal enzyme, has a
sialylation content of at least 1 mol/mol and a high level of M6P
moieties of at least 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol
% or 15 mol % of the total glycan of the recombinant glycoprotein.
In one embodiment, the recombinant glycoprotein, e.g., a
recombinant human .beta.-glucuronidase or any other lysosomal
enzyme, has a sialylation content of at least 1 mol/mol and a high
level of M6P moieties with an uptake of at least 1 nM, 1.1 nM, 1.2
nM, 1.3 nM, 1.4 nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM or 2 nM
by human cells, e.g., human fibroblast cells. In another
embodiment, the recombinant glycoprotein, e.g., a recombinant human
.beta.-glucuronidase or any other lysosomal enzyme has a
sialylation content of at least 1 mol/mol and a high level of M6P
moieties with maximum uptake by human cells, e.g., human fibroblast
cells at less than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM,
2 nM, or 1 nM of the recombinant glycoprotein.
[0049] It yet another aspect, the invention provides a composition
comprising a population of recombinant glycoproteins, wherein at
least 50%, 60%, 70%, 80%, or 90% of the population is sialylated.
In one embodiment, at least 50%, 60%, 70%, 80% or 90% of the
population is recombinant glycoprotein in accordance to the present
invention, e.g., with respect to sialylation content and M6P
level.
[0050] The recombinant glycoprotein provided by the present
invention can be any glycoprotein. Exemplary recombinant
glycoproteins include those comprising amino acid sequences
identical to or substantially similar to all or part of one of the
following proteins: a Flt3 ligand (as described in WO 94/28391), a
CD40 ligand (as described in U.S. Pat. No. 6,087,329),
erythropoietin, thrombopoietin, calcitonin, leptin, IL-2,
angiopoietin-2 (as described by Maisonpierre et al. (1997), Science
277(5322):55-60, incorporated herein by reference), Fas ligand,
ligand for receptor activator of NF-kappa B (RANKL, as described in
WO 01/36637), tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL, as described in WO 97/01633),
thymic stroma-derived lymphopoietin, granulocyte colony stimulating
factor, granulocyte-macrophage colony stimulating factor (GM-CSF,
as described in Australian Patent No. 588819), mast cell growth
factor, stem cell growth factor (described in e.g. U.S. Pat. No.
6,204,363, incorporated herein by reference), epidermal growth
factor, keratinocyte growth factor, megakaryote growth and
development factor, RANTES, growth hormone, insulin,
insulinotropin, insulin-like growth factors, parathyroid hormone,
interferons including a interferons, y interferons, and consensus
interferons (such as those described in U.S. Pat. Nos. 4,695,623
and 4,897,471, both of which are incorporated herein by reference),
nerve growth factor, brain-derived neurotrophic factor,
synaptotagmin-like proteins (SLP 1-5), neurotrophin-3, glucagon,
interleukins 1 through 18, colony stimulating factors,
lymphotoxin-13, tumor necrosis factor (TNF), leukemia inhibitory
factor, oncostatin-M, and various ligands for cell surface
molecules ELK and Hek (such as the ligands for eph-related kinases
or LERKS). Descriptions of proteins that can be produced according
to the inventive methods may be found in, for example, Human
Cytokines: Handbook for Basic and Clinical Research, Vol. II
(Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge, Mass.,
1998); Growth Factors: A Practical Approach (McKay and Leigh, eds.,
Oxford University Press Inc., New York, 1993); and The Cytokine
Handbook (A. W. Thompson, ed., Academic Press, San Diego, Calif.,
1991), all of which are incorporated herein by reference.
[0051] The recombinant glycoproteins of the present invention can
include any lysosomal enzyme, especially any enzyme useful for
enzyme replacement therapy (ERT). Examples of such enzymes include,
without any limitation, acid alpha-glucosidase, acid
beta-glucosidase or glucocerebrosidase, alpha-Galactosidase A, acid
beta-galactosidase, beta-Hexosaminidase A, beta-Hexosaminidase B,
acid sphingomyelinase, galactocerebrosidase, acid ceramidase,
arylsulfatase, alpha-L-Iduronidase, Iduronate-2-sulfatase, heparan
N-sulfatase, alpha-N-Acetylglucosaminidase, Acetyl-CoA,
alpha-glucosaminide N-acetyltransferase,
N-Acetylglucosamine-6-sulfate sulfatase,
N-Acetylgalactosamine-6-sulfate sulfatase, Acid beta-galactosidase,
Arylsulfatase B, acid alpha-mannosidase, acid beta-mannosidase,
acid alpha-L-fucosidase, sialidase, and
alpha-N-acetylgalactosaminidase.
[0052] In certain exemplary embodiments, the recombinant
glycoprotein of the present invention is recombinant human
.beta.-glucuronidase (rhGUS). Human .beta.-glucuronidase is a
glycoprotein which contains up to 16 oligosaccharides per molecule
including a variety of chains that are of the high mannose, complex
and hybrid types.
[0053] Also described herein are isolated or purified glycoprotein
polypeptides. For example, disclosed herein are isolated or
purified rhGUS polypeptides. The disclosed isolated or purified
rhGUS polypeptides can be used in one or more of the compositions
or methods disclosed herein.
[0054] The rhGUS polypeptides can include the rhGUS peptide
sequence as well as fragments thereof, natural variants thereof,
and unnatural variants thereof. The rhGUS sequence is provided in
SEQ ID NO: 1. Disclosed herein are isolated or purified
polypeptides that consist of SEQ ID NO: 1. Also disclosed herein
are isolated or purified polypeptides that comprise SEQ ID NO: 1,
as well as fragments thereof. Fragments may be at least about 10,
20, 50, 100, 200, 300, 400, or 500, or more contiguous amino acids.
Also disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
encoding the amino acid sequence of SEQ ID NO: 1.
[0055] In some embodiments, the rhGUS polypeptide has a sialic acid
content greater than 0.1 mol/mol, 0.5 mol/mol, 0.7 mol/mol, 1
mol/mol, 1.5 mol/mol, 2 mol/mol, or 5 mol/mol of the rhGUS
polypeptide. In some embodiments, the rhGUS polypeptide has a
sialic acid content of about 0.5 mol/mol to about 2.0 mol/mol of
the rhGUS polypeptide. In one embodiment, the rhGUS polypeptide has
a sialic acid content of about 0.6 mol/mol to about 1.5 mol/mol of
the rhGUS polypeptide. In another embodiment, the rhGUS polypeptide
has a sialic acid content of about 0.7 mol/mol to about 1.4 mol/mol
of the rhGUS polypeptide. In an exemplary embodiment, the rhGUS
polypeptide has a sialic acid content of about 0.8 mol/mol to about
1.3 mol/mol of the rhGUS polypeptide. In another exemplary
embodiment, the rhGUS has a sialic acid content of about 1.0
mol/mol to about 1.2 mol/mol of the rhGUS polypeptide.
[0056] In additional embodiments, at least 40%, 50%, 60%, 70%, or
80% of the galactose residues of the rhGUS polypeptide are
sialylated. In an exemplary embodiment, at least about 65% to at
least about 75% of the galactose residues of the rhGUS polypeptide
are sialylated.
[0057] As described herein, the glycoproteins of the invention may
be produced recombinantly. A polynucleotide encoding a recombinant
glycoprotein of the invention can be introduced into a recombinant
expression vector. In an exemplary embodiment, the recombinant
glycoprotein is rhGUS. Accordingly, the application also relates to
a recombinant expression vector comprising a polynucleotide
encoding rhGUS. In one embodiment, the rhGUS protein produced by
the recombinant expression vector consists of or comprises SEQ ID
NO: 1.
[0058] As is understood in the art, recombinant vectors can be
expressed in a suitable host cell system using techniques well
known in the art. Accordingly, the application also relates to a
host cell comprising a polynucleotide encoding rhGUS. In one
embodiment, the rhGUS protein produced by the host cell consists of
or comprises SEQ ID NO: 1. Suitable host cells for expressing the
rhGUS protein of the present invention can include any cell line
that can glycosylate proteins, preferably a mammalian cell line
that has been genetically engineered to express a protein. For
example, Chinese hamster ovary (CHO), HeLa, VERO, BHK, Cos, MDCK,
293, 3T3, myeloma (e.g. NSO, NSI), or WI38 cells may be used. In an
exemplary embodiment, the cells used to produce the recombinant
glycoprotein are Chinese Hamster Ovary (CHO) cells.
[0059] It yet another aspect, the invention provides a formulation
comprising one or more glycoproteins that have a high level or
increased sialic acid content. Formulations in general include
liquid forms (solutions) such as, but not limited to reconstituted
lyophilizates, and solid forms such as, but not limited to
lyophilized forms, gels, microencapsulated particles and pastes.
The formulations in accordance with some embodiments of the present
invention can be combinations of liquid formulations,
lyophilizates, and liquid solutions prepared from reconstituted
lyophilizates used in combination with gel, particles, or
pastes.
[0060] In some embodiments, the formulation is a solution including
an aqueous buffer and the recombinant glycoprotein. The buffer may
include Sodium Phosphate (Na--Pt), histidine arginine
glycylglycine, tartaric acid, malic acid, lactic acid, aspartic
acid, succinic acid or combination thereof. In an exemplary
embodiment, the buffer includes Na-Pi and histidine. In another
embodiment, the buffer includes na-Pi, histidine and arginine.
[0061] In some embodiments, the buffer further includes one or more
other ingredients such as, but not limited to sodium chloride
(NaCl), polyxyethylene (Tween-20), potassium chloride, and sorbitol
(e.g., D-sorbitol). In an exemplary embodiment, the buffer includes
Na-Pi, histidine, NaCl and Tween 20.
[0062] As is well appreciated in the art, the stability of proteins
may be dependent upon the pH and/or the ionic strength of a
formulation. According to some embodiments of the present
invention, the pH of the formulation is about 9.0 to about 5.0, for
example, about 7.5 to about 6.0. In some embodiments, the pH is
about 9.0, about 8.0, about 7.5, about 7.0, about 6.5, about 6.0,
about 5.5 or about 5.0. It was the present invention that first
recognized that lower pH of a formulation would improve the
stability of the recombinant glycoprotein. For example, Table 6 in
Example 2 demonstrates the improved stability as measured by the
percentage of tetramers when the pH was changed to 6.0 from
7.5.
Methods of Production
[0063] In yet another aspect, the present invention provides a
method for increasing the sialylation of a glycoprotein and
additionally the M6P level of a glycoprotein produced by a cell
culture with a serum or protein free media.
[0064] In general, culture media can be divided into several
subsets based on the level of defined media. For example, a culture
media can be: 1) Serum-containing media (commonly 10-20% Fetal
Bovine Serum (FBS)); 2) Reduced-serum media (commonly 1-5% FBS); 3)
Serum-free media (synonymous with defined media); 4) Protein-free
media (no protein but contains undefined peptides from plant
hydrolysates); 5) Chemically-defined media (with only recombinant
proteins and/or hormones); 6) Protein-free, chemically defined
media (contains only low molecular weight constituents, but can
contain synthetic peptides/hormones); and 7) Peptide-free,
protein-free chemically defined media (contains only low molecular
weight constituents).
[0065] In some embodiments of the present invention, a
reduced-serum media may be used to grow cells for the expression of
glycoproteins. In some embodiments of the present invention, a
serum-free media may be used to grow cells for the expression of
glycoproteins. In some embodiments, a protein-free media may be
used to grow cells for the expression of glycoproteins. In some
embodiments, a chemically defined media may be used to grow cells
for the expression of glycoproteins. In some embodiments, a
protein-free, chemically defined media may be used to grow cells as
demonstrated in Example 1. Further in some other embodiments, a
peptide-free, protein-free chemically defined media is used to grow
cells for the expression of glycoproteins.
[0066] As is well understood in the art, serum-free media may
contain undefined animal-derived products such as serum albumin
(purified from blood), hydrolysates, growth factors, hormones,
carrier proteins, and attachment factors. These undefined
animal-derived products will contain complex contaminants, such as
the lipid content of albumin. In contrast, chemically defined media
is defined as all of the components being identified and having
their exact concentrations known. In some embodiments, a chemically
defined medium is entirely free of animal-derived components. In
some embodiments, a chemically defined medium excludes FBS, bovine
serum albumin (BSA), human serum albumin (HAS) or combinations
thereof. To achieve this, chemically defined media is commonly
supplemented with recombinant versions of albumin and growth
factors, usually derived from rice or E. coli, or synthetic
chemical such as the polymer polyvinyl alcohol which can reproduce
some of the functions of BSA/HSA.
[0067] In some embodiments, the protein free medium described
herein does not contain any proteins or components of biological
origin. The absence of proteins in the medium eliminates the risk
from contamination with blood borne or other pathogens or non-human
proteins. In addition, such protein free media are usually
completely defined as to identity and quantity of all of its
ingredients, which may provide unrivalled product consistency,
superior product quality control profile and better product
stability than protein-containing media.
[0068] In some embodiments, the medium used herein does not include
an effective amount of a sugar selected from galactose, fructose,
n-acetyl-mannosamine, mannose and combinations thereof. For
example, the effective amount of a sugar is greater than 0.01 mM,
0.05 mM, or 0.1 mM.
[0069] In some embodiments, the present invention provides a method
for culturing mammalian cells comprising growing in culture a
mammalian cell to produce a protein, e.g., a glycoprotein, in a
serum or protein free media. Suitable cells for practicing the
present invention include any cell line that can glycosylate
proteins, preferably a mammalian cell line that has been
genetically engineered to express a protein. In some embodiments,
cells are homogenous cell lines. Numerous suitable cell lines are
known in the art. For example, Chinese hamster ovary (CHO), HeLa,
VERO, BHK, Cos, MDCK, 293, 3T3, myeloma (e.g. NSO, NSI), or WI38
cells may be used. In an exemplary embodiment, the cells used to
produce the recombinant glycoprotein are Chinese Hamster Ovary
(CHO) cells.
[0070] In accordance with some embodiments of the present
invention, particularly useful cells are CHO cells, which are
widely used for the production of recombinant proteins, e.g.
cytokines, clotting factors, and antibodies (Brasel et al. (1996),
Blood 88: 2004-2012; Kaufman et al. (1988), J. Biol Chem
263:6352-6362; McKinnon et al. (1991), J Mol Endocrinol 6: 231-239;
Wood et al. (1990), J. Immunol 145: 3011-3016). A dihydrofolate
reductase (DHFR)-deficient mutant cell line (Urlaub et al. (1980),
Proc. Natl. Acad. Sci. USA 77:4216-4220), such as DXB11 or DG-44,
is useful because the efficient DHFR selectable and amplifiable
gene expression system allows high level recombinant protein
expression in these cells (Kaufman (1990), Meth. Enzymol. 185:
527-566). In addition, these cells are easy to manipulate as
adherent or suspension cultures and exhibit relatively good genetic
stability. CHO cells and recombinant proteins expressed in them
have been extensively characterized and have been approved for use
in clinical commercial manufacturing by regulatory agencies.
[0071] In some embodiments, cells are grown in a fed batch mode. A
fed-batch process is defined as an operational technique where one
or more nutrients (substrates) are added to a culture medium to
increase growth and achieve a high cell density in a bioreactor.
Generally, adding nutrients in a controlled manner has a positive
effect on the culture's growth rate and production. In some
embodiments, a cell concentration greater than 10.sup.6 cells/mL,
10.sup.7 cells/mL, 2.times.10.sup.7 cells/mL, 5.times.10.sup.7
cells/mL, or 10.sup.8 cells/mL in bioreactors can be achieved. In
some embodiments, bioreactors used in the fed batch mode have a
volume of at least 10 L, 20 L, SOL, 80 L, 100 L, 250 L, 500 L or
1000 L. Cells can be grown either in suspension or adherent
cultures. In an exemplary embodiment, cells are grown in
suspension. Mammalian cells are preferred, and in a particular
exemplary embodiment, the mammalian cells are Chinese hamster ovary
cells.
Therapeutic Treatment
[0072] In yet another aspect, the invention provides methods of
treating a condition or disorder comprising administering to an
individual in need of such treatment a therapeutically effective
amount of the composition/preparation as described herein.
[0073] Compositions/preparations as described herein can be used
alone or together with any therapeutic agents/compositions for
various purposes, such as in the treatment methods described
herein. In this regard, the compositions/preparations can be
pharmaceutically acceptable.
[0074] In some embodiments, the condition or disorder requiring
treatment is associated with an enzyme deficiency. Enzyme
deficiencies in cellular compartments such as the golgi, the
endoplasmic reticulum, and the lysosome cause a wide variety of
human diseases. For example, lysyl hydroxylase, an enzyme normally
in the lumen of the endoplasmic reticulum, is required for proper
processing of collagen; absence of the enzyme causes Ehlers-Danlos
syndrome type VI, a serious connective tissue disorder. GnT II,
normally found in the golgi, is required for normal glycosylation
of proteins; absence of GnT II leads to defects in brain
development.
[0075] In an exemplary embodiment, the condition or disorder
associated with an enzyme deficiency is a lysosomal storage
disorder (LSD). More than forty lysosomal storage diseases (LSDs)
are caused, directly or indirectly, by the absence of one or more
proteins in the lysosome. LSDs arise from abnormal metabolism of
various substrates, including glycosphingolipids, glycogen,
mucopolysaccharides and glycoproteins. The metabolism of the
substrates normally occurs in the lysosome and the process is
regulated in a stepwise process by various degradative enzymes.
Therefore, a deficiency in any one enzyme activity can perturb the
entire process and result in the accumulation of particular
substrates. Listed below are a number of lysosomal storage
disorders and the corresponding defective enzymes: [0076] Pompe
disease: Acid alpha-glucosidase [0077] Gaucher disease: Acid
beta-glucosidase or glucocerebrosidase [0078] Fabry disease:
alpha-Galactosidase A [0079] GMI-gangliosidosis: Acid
beta-galactosidase [0080] Tay-Sachs disease: beta-Hexosaminidase A
[0081] Sandhoff disease: beta-Hexosaminidase B [0082] Niemann-Pick
disease: Acid sphingomyelinase [0083] Krabbe disease:
Galactocerebrosidase [0084] Farber disease: Acid ceramidase [0085]
Metachromatic leukodystrophy: Arylsulfatase A [0086] Hurler-Scheie
disease: alpha-L-Iduronidase [0087] Hunter disease:
lduronate-2-sulfatase [0088] Sanfilippo disease A: Heparan
N-sulfatase [0089] Sanfilippo disease B:
alpha-N-Acetylglucosaminidase [0090] Sanfilippo disease C:
Acetyl-CoA: alpha-glucosaminide N-acetyltransferase [0091]
Sanfilippo disease D: N-Acetylglucosamine-6-sulfate sulfatase
[0092] Morquio disease A: N-Acetylgalactosamine-6-sulfate sulfatase
[0093] Morquio disease B: Acid beta-galactosidase [0094]
Maroteaux-Lamy disease: Arylsulfatase B [0095] Sly disease:
beta-Glucuronidase [0096] alpha-Mannosidosis: Acid
alpha-mannosidase [0097] beta-Mannosidosis: Acid beta-mannosidase
[0098] Fucosidosis: Acid alpha-L-fucosidase [0099] Sialidosis:
Sialidase [0100] Schindler-Kanzaki disease:
alpha-N-acetylgalactosaminidase
[0101] In certain exemplary embodiments, the present invention
provides a method of treating a LSD comprising administering to an
individual in need of such treatment a therapeutically effective
amount of the composition/preparation as described herein. In one
exemplary embodiment, the composition/preparation comprises
recombinant human .beta.-glucuronidase. In another exemplary
embodiment, the LSD is mucopolysaccharidosis type 7 (i.e., MPS 7,
MPS VII, or Sly Syndrome), a disorder resulting from the deficiency
of .beta.-glucuronidase. In some embodiments, the recombinant human
.beta.-glucuronidase harbors an increased sialic acid content and
is particularly useful in treating a LSD, e.g., MPS 7.
[0102] In some embodiments, the present invention provides a method
for treating a condition or disorder in a subject, comprising
administering a regimen of a composition/preparation as described
herein, wherein the administration provides a statistically
significant therapeutic effect for the treatment of the condition
or disorder. In some embodiments, the subject is human. In some
embodiments, the composition/preparation comprises a recombinant
glycoprotein that harbors an increased sialic acid content. In an
exemplary embodiment, the recombinant glycoprotein has a
sialylation content of at least 0.7 mol/mol of the recombinant
glycoprotein. In another exemplary embodiment, the recombinant
glycoprotein has a sialylation content of at least 1 mol/mol of the
recombinant glycoprotein. In some embodiments, the condition or
disorder is associated with an enzyme deficiency. In an exemplary
embodiment, the condition or disorder associated with an enzyme
deficiency is a lysosomal storage disorder (LSD).
[0103] Accordingly, the present invention provides a method for
treating a lysosomal storage disorder (LSD) in a subject,
comprising administering a regimen of the composition/preparation
as described herein, wherein the administration provides a
statistically significant therapeutic effect for the treatment of
the LSD. In an exemplary embodiment, the composition/preparation
comprises a recombinant human .beta.-glucuronidase that harbors an
increased sialic acid content. In a further exemplary embodiment,
the LSD is mucopolysaccharidosis type 7 (i.e., MPS 7, MPS VII, or
Sly Syndrome).
[0104] According to the present invention, treatment of the LSD
includes any form of treating the LSD, e.g., reducing any symptom
of the LSD, reducing the severity of any symptom of the LSD,
shortening the duration of one or more symptoms of the LSD,
treating or inhibiting any cause or condition associated with the
LSD, or reducing any clinical criteria or measurement of the degree
or condition of the LSD.
[0105] According to the present invention, the recombinant human
.beta.-glucuronidase of the present invention is administered in a
regimen for the treatment of a LSD. In one embodiment, the LSD is
MPS 7. Such regimen includes dosage per administration, per day,
per every two weeks, as well as number of doses per treatment
cycle, or combinations thereof.
[0106] In general, the recombinant human .beta.-glucuronidase
(rhGUS) of the present invention can be administered at a dosage of
from about 0.1 mg to 20 mg, 0.2 mg to 15 mg, 0.5 to 12 mg, 1 mg to
10 mg. 1.5 mg to 8 mg, 2 mg to 6 mg per kg. In some embodiments,
the rhGUS is administered at a dosage of about 0.1 mg, 0.2 mg, 0.5
mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11
mg, or about 12 mg per kg. In an exemplary embodiment, the rhGUS is
administered at a dosage of about 4 mg per kg. Dosages may be
adjusted for the condition of each patient as well as other drugs
taken by the patient.
[0107] In some embodiments, such dosage is administered hourly,
daily, weekly (i.e., QW), every two weeks (i.e., QOW), or
monthly.
[0108] In some embodiments, rhGUS is administered hourly, about
every 1 to 24 hours, 1 to 20 hours, 1 to 16 hours, 1 to 12 hours, 1
to 8 hours, 1 to 6 hours, 1 to 4 hours, 1 to 2 hours or every hour.
In some embodiments, rhGUS is administered about every 2, 3, 4, 5,
or 6 hours, or is administered about every 10 minutes, 15 minutes,
30 minutes, 45 minutes or 60 minutes.
[0109] In some embodiments, rhGUS may be administered by continuous
infusion. In some embodiments, rhGUS may be administered to the
patient for treatment periods of at least about 2, 4, 6, 10, 12
hours, or longer, which may improve effectiveness in some
embodiments. In some embodiments, rhGUS is administered by
continuous infusion for 1 to 24 hours, 1 to 20 hours, 1 to 16
hours, 1 to 12 hours 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1
to 4 hours to 1 to 2 hours. In some embodiments, rhGUS is
administered by continues infusions for about 10 minutes, 15
minutes, 30 minutes, 45 minutes or 60 minutes. In some embodiments,
rhGUS is administered by continuous infusion for about 1 hour, 2
hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10
hours, 12 hours, 24 hours or more. In an exemplary embodiment,
rhGUS is administered by continuous infusion for about 4 hours. In
some embodiments, the continuous infusion periods are separated by
periods of non-infusion (i.e., periods where no rhGUS is
administered). The infusion may be carried out by any suitable
means, such as by minipump.
[0110] In some embodiments, rhGUS is administered about every 1 to
30 days, every 1 to 25 days, every 1 to 20 days, every 1 to 14
days, every 1 to 10 days, every 1 to 5 days or daily.
[0111] In some embodiments, rhGUS is administered for about 1 to 12
weeks, about 1 to 24 weeks, about 1 to 36 weeks, about 1 to 48
weeks, about 1 to 60 weeks, or about 1 to 72 weeks. In some
embodiments, the rhGUS is administered for about 1 month, 4 months,
8 months, 12 months, 16 months, 20 months, or more. In some
embodiments, the rhGUS is administered for about 1 year, 2 years, 5
years, 10 years, or more. In some embodiments, the rhGUS is
administered permanently (i.e., long-term use).
[0112] The rhGUS may be provided in lyophilized form, and
reconstituted with sterile (e.g., aqueous) diluent prior to
administration. The rhGUS may be administered by any effective
route, including by subcutaneous injection, intramuscular
injection, intravenous injection or infusion, and orally. In
certain exemplary embodiments, the rhGUS is administered by
intravenous infusion. Generally, the scheduled dose of rhGUS may be
administered as a single dose (e.g., injection), or may be spaced
out over the course of 24 hours or less, for example, by continuous
infusion or repeated injection of subdose, or the like or as
described extensively herein. In one embodiment, the scheduled dose
of rhGUS may be administered as a single injection or as multiple
injections.
[0113] In one embodiment, the patient receives approximately every
other weekly (i.e., QOW) administration of rhGUS, at a dose between
about 0.5 and 12 mg (e.g., about 1, 2, 4, 8, or 12 mg) per kg to
reduce the severity of the LSD. In an exemplary embodiment, the
patient receives approximately every other weekly administration of
rhGUS at a dose of about 4 mg per kg. The regimen may continue in
some embodiments for 12, 24, 36, 48, or 60 weeks, or permanently
(i.e., long-term use).
[0114] According to the present invention, the rhGUS used in
methods of the present invention can be administered either alone
or in combination with a standard of care for the LSD, or as part
of treatment regimen involving the standard of care for the LSD. In
some embodiments, patients may be administered prophylactic
antihistamine prior to each infusion of rhGUS. In additional
embodiments, patients may be administered an antipyretic medication
(e.g., ibuprofen or acetaminophen) prior to each infusion of
rhGUS.
[0115] According to some embodiments of the present invention,
administration of rhGUS provides a statistically significant
therapeutic effect. In one embodiment, the statistically
significant therapeutic effect is determined based on one or more
standards or criteria provided by one or more regulatory agencies
in the United States, e.g., FDA or other countries. In another
embodiment, the statistically significant therapeutic effect is
determined based on results obtained from regulatory agency
approved clinical trial set up and/or procedure.
[0116] In some embodiments, the statistically significant
therapeutic effect is determined based on a randomized,
placebo-controlled, blind-start, single-crossover clinical trial
set up. In some embodiments, the statistically significant
therapeutic effect is determined based on data from a clinical
trial design whereby subjects are randomized to one of 4 groups,
each representing a different treatment sequence at different
pre-defined time points in a blinded manner. In some embodiments,
the statistically significant therapeutic effect is determined
based on data from a patient population of at least 4, 6, 8, 10, or
12 subjects. In an exemplary embodiment, the statistically
significant therapeutic effect is determined based on data from a
patient population of 12 subjects.
[0117] In some embodiments, the statistically significant
therapeutic effect is determined based on a study involving 12
subjects randomized 1:1:1:1 to one of four treatment sequence
groups to either start treatment with 4 mg/kg rhGUS every other
week (i.e., QOW), or placebo and cross over to 4 mg/kg rhGUS QOW at
different, pre-defined time points. In some embodiments, the
statistically significant therapeutic effect is determined based on
a study in subjects dosed with either 4 mg/kg rhGUS or placebo QOW
for 48 weeks.
[0118] In some embodiments, the statistically significant
therapeutic effect is determined based on a study wherein rhGUS is
administered QOW by slow IV infusion over a period of approximately
4 hours. In some embodiments, patients are pre-medicated with
prophylactic antihistamine (e.g., cetirizine or loratadine) prior
to each infusion of rhGUS.
[0119] In some embodiments, the statistically significant
therapeutic effect is determined by measuring urinary
glycosaminoglycan (uGAG) levels as the primary endpoint. Extensive
research conducted over the last 20 years on MPS disorders provides
significant relevant scientific data that allows for the
qualification of uGAG levels as a biomarker that is reasonably
likely to predict clinical benefit. The disease process and
mechanism of action for the rhGUS in MPS 7 are well understood and
data from other similar MPS disorders with comparable enzyme
replacement therapies (ERTs) have established that uGAG is a direct
pathophysiological and readily measured marker of the MPS disease
process and uGAG is a reasonable predictor of treatment effect and
clinical benefit in MPS disorders. In an exemplary embodiment, the
statistically significant therapeutic effect is determined based on
the determination of uGAG levels a clinical study involving 12
subjects who have been treated with 4 mg/kg rhGUS or placebo QOW
over a 48-week period.
[0120] In some embodiments, the statistically significant
therapeutic effect is determined using secondary efficacy measures
(i.e., secondary endpoints) such as a multi-domain responder index
and an evaluation of individualized clinical response.
[0121] In some embodiments, the statistically significant
therapeutic effect is determined using a multi-domain responder
index, which combines independent multi-domain analyses to assure
that the broader basis for efficacy can be assessed without the
complexity of trying to construct qualified composite endpoints. In
some embodiments, the multi-domain responder index provides an
assessment of rhGUS efficacy across a broad spectrum of clinical
characteristics commonly observed in MPS 7 patients.
[0122] In some embodiments, the statistically significant
therapeutic effect is determined by evaluating individualized
clinical response (ICR). This is a measure of each subject's
response to treatment that is selected based on the relevance of
the outcome measure to concerns that the subject/parent/caregiver
has reported, the subject's ability to complete clinical outcome
assessment reliably, and the extent of impairment for that
individual. Use of an ICR enables evaluation of the clinical
benefit of rhGUS by assessing change in a prespecified
individualized clinical outcome that is deemed most relevant for
each subject and then determining an overall response rate for the
study population. In some embodiments, the secondary efficacy
measures (i.e., secondary endpoints) may include the evaluation of
treatment subjects for signs and symptoms of MPSI that interfere
most with the subject's daily life (i.e., clinical problem
evaluation). In some embodiments, the evaluation may include
testing of pulmonary function, testing of walking distance, testing
of shoulder flexion range of motion, and testing of fine motor
function.
[0123] In some embodiments, the statistically significant
therapeutic effect is determined based on data with an alpha value
of less than or equal to about 0.05, 0.04, 0.03, 0.02 or 0.01. In
some embodiments, the statistically significant therapeutic effect
is determined based on data with a confidence interval greater than
or equal to 95%, 96%, 97%, 98% or 99%. In some embodiments, the
statistically significant therapeutic effect is determined based on
data with a p value of less than or equal to about 0.05, 0.04,
0.03, 0.02 or 0.01. In some embodiments, the statistically
significant therapeutic effect is determined on approval of Phase
III clinical trial of the compositions and methods provided by the
present invention, e.g., by FDA in the US.
[0124] In general, statistical analysis can include any suitable
method permitted by a regulatory agency, e.g., FDA in the US or
China or any other country. In some embodiments, statistical
analysis includes non-stratified analysis, log-rank analysis, e.g.,
from Kaplan-Meier, Jacobson-Truax, Gulliken-Lord-Novick,
Edwards-Nunnally, Hageman-Arrindel and Hierarchical Linear Modeling
(HLM) and Cox regression analysis.
[0125] In some embodiments, lysosomal storage biomarkers can be
used for predicting treatment response and/or determining treatment
efficacy. In some embodiments, urinary glycosaminoglycan (uGAG)
levels can be measured and a reduction in uGAG levels employed as
an indicator of positive treatment response. In some embodiments,
elevated levels of the uGAG biomarker, which later decrease upon
administration of rhGUS, is predictive of treatment response. In
some embodiments, this information can be employed in determining a
treatment regimen (as described herein) for the treatment of a
lysosomal storage disorder (e.g., MPS 7) using rhGUS. As such, the
present invention provides methods for determining a treatment
regimen which includes detecting a decrease in the level of a LSD
biomarker in a biological sample from a subject treated with rhGUS
and determining a treatment regimen of the rhGUS based on a
decrease in the level of one or more one or more LSD biomarkers in
a biological sample. In some embodiments, the LSD biomarker is
uGAG. In some embodiments, a decreased or reduced level of uGAG is
indicative of treatment response and/or treatment efficacy of
treatment with rhGUS. In some embodiments, reduction of uGAG levels
to a predetermined standard level is indicative of better treatment
prognosis with rhGUS.
EXAMPLES
Example 1: Production and Quantitation of Total Sialic Acid
[0126] The recombinant human .beta.-glucuronidase (rhGUS) produced
in accordance with the present invention was labeled GUS CR01. The
recombinant protein is produced from Chinese Hamster Ovary (CHO)
cells that have been engineered to express and secrete the enzyme
into the culture medium using a bioreactor culture system.
[0127] Previous batches of .beta.-glucuronidase (labeled as GUS Lot
43/44) have been produced using the same cell line by a process in
which the cells are grown attached to microcarriers in a continuous
perfusion system. Cells are generally expanded in growth media
containing Fetal Bovine Serum (FBS). Afterward, FBS is washed out
and replaced with media containing hydrolysates and supernatant was
harvested in perfusion mode.
[0128] In contrast to the previously reported methods, GUS CR01 was
produced in a culture system in which the cells are grown in
suspension in a fed batch mode. Another difference is GUS CR01 was
cultured only in chemically defined protein free medium as opposed
to serum containing medium used previously.
[0129] A method for quantitation of total sialic acid in GUS was
developed at the Rentchler Biotechnologie (RB) Quality Control
department for release-testing of the GUS drug substance. In this
method, sialic acid residues are released from the rhGUS glycan
structures with acid hydrolysis. The released sialic acid is then
labeled with OPD (O-phenylenediamine dihydrochloride) and analyzed
by Reversed-Phase HPLC analysis (RP-HPLC). To date, Lot 43/44 and
six RB-produced lots of rhGUS have been analyzed at RB for total
sialic acid. These results (Table 1) are consistent with the result
seen at GlycoSolutions.
TABLE-US-00001 TABLE 1 Results for Total Sialic Acid Analysis of
GUS Sialic Acid (mol/mol Lot GUS Production Method GUS monomer) Lot
43/44 Previously Reported 0.04 PR01 In accordance with the present
invention 1.0 PR02 In accordance with the present invention 0.7
CR01 In accordance with the present invention 1.1 GMP1 In
accordance with the present invention 1.2 GMP2 In accordance with
the present invention 1.2 GMP3 In accordance with the present
invention 1.2
Example 2: Pharmacokinetics in Male Sprague Dawley Rats
[0130] In this Example, recombinant human .beta.-glucuronidases
produced in accordance with the present invention were compared to
ones produced as previously reported in the art. The objective of
this study was to evaluate the pharmacokinetics and tissue
distribution of recombinant human .beta.-glucuronidases
administered intravenously as a single two hour infusion in male
Sprague Dawley rats. The rate of infusion was 1/3 of the total
volume for the first hour followed by 2/3 of the total volume in
the second hour. This dosing regimen was designed to simulate the
dosing regimen to be used in the patients.
Materials and Methods
[0131] Test Articles and Infusions
[0132] Three test articles were used in this study: 0.9% sodium
chloride as the non-enzyme control, GUS CR01 and GUS Lot 43/44.
Complete specifications for the test articles can be found in Table
2.
TABLE-US-00002 TABLE 2 Test Articles Test Article 1 Name: 0.9%
Sodium Chloride for Injection, USP (Saline) Source: Baxter
Healthcare (Marion, NC) Physical Properties: Clear liquid
Identifier/Lot Number: C883827 Sterility Status: Sterile Storage
Conditions: Room temperature Expiration Date April 2014 Test
Article 2 Name: GUS CR01 Source: Ultragenyx Pharmaceutical Inc.
(Novato, CA) Quantity: ~32.5 mL Concentration: 2.0 mg/mL GUS
Activity units/ml 10.75 Munits/ml Specific Activity units/mg 5.35
Munits/mg Physical Properties: Clear liquid Identifier/Lot Number:
CR01 Storage Conditions: 2 to 8.degree. C. Expiration Date: Not
provided Test Article 3 Name: GUS Lot 43/44 Source: Ultragenyx
Pharmaceutical Inc. (Novato, CA) Quantity: ~25 mL Concentration:
2.5 mg/mL* (2.18 mg/ml) GUS Activity units/ml 11.4 Munits/ml
Specific Activity units/mg 5.23 Munits/mg Physical Properties:
Clear liquid Identifier/Lot Number: 43/44 Storage Conditions: -60
to -80.degree. C. Expiration Date: Not provided
[0133] The test articles were infused into male Sprague-Dawley rats
at a dose of -2 mg/Kg body weight during a single infusion
consisting of two 1 hour phases. One third of the dose was infused
over the first hour and two thirds of the dose was infused during
the second hour (Table 3).
TABLE-US-00003 TABLE 3 Rat Group Numbers, Dose and Infusion Rates
Dose Dose Total Actual Test Dose Concentration rate Dose Dose*
Group # Article Gender n Route (mg/mL) (mL/min) (mg/kg) (mg/kg) 1
Saline M 5 iv n/a 1.sup.st hr: 0.99 n/a n/a 2.sup.nd hour: 1.98 2
GUS lot: M 5 iv 0.203 1.sup.st hr: 0.94 ~2.sup.1 ~2.sup.1 CR01
2.sup.nd hour: 1.87 3 GUS lot: M 5 iv 0.253 1.sup.st hr: 0.78
~2.sup.1 ~1.7.sup.1 43/44 2.sup.nd hour: 1.57 .sup.1Dose was based
on the average body weight of all five rats in each group. *The
dose for GUS Lot 43/44 was originally based on a protein value of
2.5 mg/ml determined by BCA assay. If the protein concentration was
based on absorbance at OD 280 and an extinction coefficient of
2.12, the actual dose was 84.8% .times. 2 = 1.7 mg/Kg.
[0134] Blood samples were taken from each rat pre-treatment and
then at intervals during the slow infusion, fast infusion and post
infusion phases by the schedule outlined in Table 4. Blood was
allowed to clot, serum was separated and stored frozen at
-80.degree. C. pending shipment on dry ice for analysis.
TABLE-US-00004 TABLE 4 Infusion and Bleed Schedule Stage Nominal
Interval Predose Slow Infusion 2 min post start of infusion 10 min
post start of infusion 30 min post start of infusion 60 min post
start of infusion Fast Infusion 120 min post start of infusion Post
Infusion 2 min (post end of infusion) 10 min (post end of infusion)
30 min (post end of infusion) 60 min (post end of infusion) 120 min
(post end of infusion) 240 min (post end of infusion) 480 min (post
end of infusion) 24 hr (post end of infusion)
GUS Activity in Serum
[0135] .beta.-glucuronidase activity was determined as follows: 25
.mu.L of serum diluted 1:4 to 1:300 in 0.1 M sodium acetate, pH
4.8, and 1 mg/mL crystalline BSA was mixed with 50 .mu.L of 10 mM
4-MU-.beta.-D-glucuronide substrate in 0.1 M sodium acetate, pH
4.8, 1 mg/mL crystalline BSA. All solutions were pre-warmed to
37.degree. C. mixed then incubated at 37.degree. C. for 30 minutes.
The assays were stopped by the addition of 200 .mu.L glycine
carbonate, pH 10.5, and read on a Molecular Devices M2' plate
reader at excitation/emission wavelengths of 366/446 nm. Activity
was expressed as 1 unit=1 nmole 4 MU released/mL/hr at 37.degree.
C.
GUS Activity in Tissues
[0136] Tissues were collected at necropsy and placed in cryovials,
snap frozen in liquid nitrogen, and stored at -80.degree. C.
pending shipment on dry ice for analysis. The distribution of GUS
activity in the tissues was assessed as follows. Whole or partial
tissue specimens were thawed and combined with 10 to 20 volumes of
25 mM Tris, 140 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, pH
7.2. Tissue homogenates were prepared using a Kinematica Polytron
homogenizer for 30 seconds on ice; the resultant homogenates were
freeze/thawed once (at -80.degree. C.) followed by sonication for
20 seconds with cooling on ice. A 25 .mu.L total volume of each
homogenate was assayed for .beta.-glucuronidase using 4
MU-.beta.-glucuronide as described previously. Protein
concentration of the homogenates was determined by the
bicinchoninic acid method. Tissue .beta.-glucuronidase levels were
expressed as nmoles of 4 MU hydrolyzed/hr/mg protein.
Results and Discussion
[0137] Pharmacokinetics of GUS CR01 vs. GUS Lot 43/44 in the
Plasma
[0138] FIG. 1 shows the .beta.-glucuronidase activity in the sera
of rats from each infusion group during the slow infusion stage,
the fast infusion stage and the post infusion stage. The curve for
Group 1 infused with saline only, indicates the low endogenous
level of rat (3-glucuronidase that is present in the sera of these
rats. The endogenous level has been subtracted from the values in
the other two plots from the rats infused with enzyme.
[0139] The plots for both GUS CR01 and GUS Lot 43/44 show a time
dependent increase in enzyme activity levels that reach a
steady-state level by the end of the slow infusion period then
increases again concomitant with the start of the fast infusion
period. However in contrast, GUS CR01 reaches a level in the serum
2-fold higher at the end of the slow infusion and 3-fold higher at
the end of the fast infusion period compared to GUS Lot 43/44. It
can also be seen in FIG. 1 that the rapid clearance of both enzymes
from the serum after the infusions cease which is characteristic
for lysosomal enzymes in general.
[0140] In FIG. 2, we present the post infusion clearance phase for
both enzymes from which were calculated the t.sub.1/2 values. GUS
Lot 43/44 is cleared from the circulation with a 1.sup.st phase
t.sub.1/2 of 4.50 minutes. In contrast, GUS CR01 is cleared at the
slightly slower t.sub.1/2 of 5.30 minutes. Raw clearance data was
analyzed by a different method to re-calculate the t.sub.1/2 values
(FIGS. 4 A and B). The Cmax (14800 for GUS CR01 vs 4300 for GUS lot
43/44) and the AUC-t (18700 vs 5580) are showing also a 3 fold
higher for GUS (Table 5). The t.sub.1/2 was calculated very
differently as only the second phase was taken into account. For
GUS CR01, the 2.sup.nd phase t.sub.1/2 is 1.1 hr and 0.967 hr for
GUS lot 43/44 (Table 5).
TABLE-US-00005 TABLE 5 Clearance Vz Cmax Tmax AUCO-t AUCO-inf
Half-life (min*Units/mL)/ (Units/mL)/ Group Animal (Units/mL) (h)
(h*Units/mL) (h*Units/mL) (h) mg/kg) mg/kg) Grp 2 6-10 CR01 2-6
12900 0 15300 15500 2.05 2.19E-06 0.000389 2-7 14200 0 16900 16900
0.794 2.01E-06 0.000138 2-8 14200 0 18300 18400 0.926 1.84E-06
0.000148 2-9 16700 0 22800 23000 1.03 1.47E-06 0.000132 2-10 16000
0 20300 20300 0.67 1.66E-06 9.65E-05 Mean 14800 0 18700 18800 1.1
1.84E-06 0.000181 CV % 10.4 15.7 15.7 50.5 15.3 65.4 Geometric Mean
14800 18500 18600 1.01 1.82E-06 0.000159 Grp 3 11-15 43/44 3-11
4640 0 5380 5380 0.677 6.26E-06 0.000367 3-12 7010 0.03 6940 6950
0.832 4.84E-06 0.000349 3-13 4870 0 6220 6250 1.02 5.38E-06
0.000476 3-14 2740 0.03 5420 5430 1.39 6.20E-06 0.000747 3-15 2270
0 3970 3980 0.913 8.45E-06 0.000668 Mean 4300 0.0133 5580 5600
0.967 6.23E-06 0.000521 CV % 44 136.9 19.9 19.8 27.8 22.1 34.4
Geometric Mean 3970 5490 5510 0.939 6.11E-06 0.000497
[0141] Tissue Distribution of GUS CR01 vs. GUS Lot 43/44
[0142] In addition to clearance of the two enzymes, we assessed the
tissue distribution of GUS CR01 compared to GUS Lot 43/44 in liver,
spleen, heart, kidney, brain and lung. Tissue extracts prepared
from each of these tissues were assayed for .beta.-glucuronidase
and protein as described in the methods. The results of the assays
were expressed as units of .beta.-glucuronidase activity/mg of
tissue protein. The summary of these assays can be seen in FIG. 4.
In each of the graphs of this figure, the total enzyme levels
including the endogenous rat .beta.-glucuronidase is shown on the
left side. On the right side of each graph the average endogenous
enzyme level has been subtracted from the total enzyme level. The
average endogenous .beta.-glucuronidase level from each tissue was
calculated using values from all five rats from saline infused
Group 1.
[0143] In each tissue, the level of GUS in rats infused with either
GUS CR01 or GUS Lot 43/44 is higher than the saline infused rats.
When the endogenous GUS levels are subtracted, it becomes apparent
that rats infused with GUS CR01 contain GUS levels that are at
least two times greater than rats infused with GUS Lot 43/44.
[0144] This study was designed to assess the .beta.-glucuronidase
pharmacokinetic and tissue distribution properties of GUS CR01 and
GUS Lot 43/44. The current study determined that GUS CR01 was
cleared from the circulation with a 1st phase t.sub.1/2 of 5.30
minutes, compared with a faster t.sub.1/2 of 4.50 minutes for GUS
Lot 43/44. The second phase finis also a bit bigger for GUS
CR01.
[0145] More significant differences between the 2 enzymes were
demonstrated for Cmax, AUC-t and tissue distribution. The maximum
concentration (Cmax) of .beta.-glucuronidase activity in the serum
at the end of the two hour infusion period for GUS CR01 was 14,829
units/ml, 4.2 times the concentration of 3537 units/ml attained by
GUS Lot 43/44. This increase in Cmax could be explained by the
accumulation of the slower clearing enzyme to a higher
concentration in the blood during the infusion period. AUC-t was
also highly increased (more than 3 time) with GUS CR01 vs. GUS Lot
43/44 as represented in FIG. 1.
[0146] Last but not least, we observed that in all of the tissues
tested that GUS CR01 was delivered to tissues at levels up to two
times or greater than that of GUS Lot 43/44. Changing the clearance
characteristics of a lysosomal enzyme from the circulation is known
to have an effect on tissue distribution. It is conceivable that
slowing the t.sub.1/2 of GUS CR01 in the circulation could allow
for a more efficient distribution of the enzyme to selected
tissues. More importantly the changes in tissue distribution seem
to correlate very well with the changes of Cmax and AUC-t. As can
be seen in FIG. 3, the error bars are quite large, reflecting a
large range of values obtained in the individual rats of each study
group. Repeat .beta.-glucuronidase assays in triplicate on selected
tissues confirmed the original values leading us to believe that
the wide range of values seen in these rats were real.
[0147] Previously, analysis of GUS CR01 has shown that whereas the
level of mannose 6 phosphate and most other properties are quite
similar between the 2 enzymes, the sialic acid content of GUS CR01
is 28 times that of GUS Lot 43/44 (1.1 mol/mol GUS monomer vs. 0.04
mol/mol GUS monomer). See Table 6.
TABLE-US-00006 TABLE 6 Characteristics of GUS CR01 and GUS Lot
43/44 GUS GUS Characteristic/Assay Units CR01 Lot 43/44 GMP1 GMP2
GMP3* Titer mg/L ~400 ~50 ~400 ~400 ~400 pH -log [H.sup.+] 7.4 7.5
7.4 7.6 6.0 Purity (Reducing SDS-PAGE) % 99.2 >95.0 99.2 98.8
99.3 Tetramer (SE-HPLC) % 97.7 99.0 98.4 98.6 99.1 Molecular Weight
Tetramer Daltons 290,249 300,000 300,000 300,000 300,000 Mass
Extinction Coefficient (mg/mL).sup.-1cm.sup.-1 2.08 -- 2.0 2.0 2.1
Charge Heterogeneity (IEF) pH Range comparable 6.6-7.7 comparable
comparable comparable M6P N-Glycan Analysis mol-% 14.2 comparable
14 14 12 (Sum of Peaks 15-17) Sialic Acid Content (moles/mole
monomer) 1.1 0.04 1.2 1.2 1.2 Specific Activity (MU/mg) 3.6 3.70
3.9 3.7 3.5 Cellular Uptake Kuptake nM 1.2-1.7 1.4 1.8 1.6 1.4
Half-life in MPS7 Fibroblasts Days 0-21 d 20.0 18.9 NA NA NA 5-21 d
21.6 20.5 *Monosaccharide analysis indicated that 71% of the
galactose residues on GMP3 GUS are sialylated.
[0148] By putting together the data it is becoming quite apparent
that the better tissue distribution of GUS CR01 demonstrated here
is due to its increase in sialic acid content as sialic acid is
well known to slow glycoproteins clearance from the circulation by
mannose receptors located in the endothelial cells in the interior
walls of the blood vessels. The combination of high sialic acid
levels and high affinity mannose-6-phosphate moieties provides an
optimal combination for reducing tissue uptake via other
carbohydrate receptors due to the high sialic acid content,
assuring higher concentrations in circulation and then achieving
excellent tissue uptake in the target tissues due to the high
affinity mannose-6-phosphate levels.
Example 3: Treatment of MPS VII Using Enzyme Replacement
Therapy
[0149] The purpose of this example is to demonstrate that enzyme
replacement therapy for mucopolysaccharidosis type VII (i.e., MPS
VII; Sly's Syndrome) using recombinantly produced human
.beta.-glucuronidase (rhGUS) reduces lysosomal storage in a 36-week
clinical study.
[0150] In this example, three subjects diagnosed with MPS VII were
administered rhGUS with increased sialic acid content. Dosing was
performed according to the following 36-week schedule: [0151] Weeks
1-12: 2 mg/kg every other week; [0152] Weeks 13-20: 1 mg/kg every
other week; [0153] Weeks 21-28: 4 mg/kg every other week; and
[0154] Weeks 29-36: 2 mg/kg every other week.
[0155] The safety and efficacy of rhGUS was assessed during the
36-week treatment schedule. The rhGUS compound appeared to be safe
and well tolerated. Importantly, no serious adverse events were
observed up to 36 weeks and there were no drug-related or
hypersensitivity infusion-associated reactions in any of the three
subjects.
[0156] To measure efficacy, urinary and serum levels of
glycosaminoglycans (GAGs) were first evaluated, as lysosomal
accumulation of GAGs is a hallmark of GUS deficiency. A rapid and
sustained dose-dependent reduction in urinary glycosaminoglycan
(uGAG) was observed in subjects treated with rhGUS. See FIG. 4. The
mean reduction in uGAG at the end of each dosing interval is shown
in FIG. 5 and illustrates that a 4 mg/kg dose resulted in the
greatest reduction of uGAG levels.
[0157] A progressive reduction in serum glycosaminoglycan (GAG) was
also seen in all three subjects treated with rhGUS. Notably, each
subject demonstrated at least a 25% reduction in serum GAG levels
at the end of the 36-week treatment schedule. See FIG. 6.
[0158] Lastly, liver size was evaluated in subjects treated with
rhGUS, as enlarged liver size is frequently observed in patients
suffering from MPS VII. There was a significant reduction in
hepatomegaly resulting from the 36-week treatment protocol. See
FIG. 7.
[0159] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the present application belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present application, representative methods and materials are
herein described.
[0160] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
[0161] The disclosures, including the claims, figures and/or
drawings, of each and every patent, patent application, and
publication cited herein are hereby incorporated herein by
reference in their entireties. In the case of any conflict between
a cited reference and this specification, the specification shall
control. In describing embodiments of the present application,
specific terminology is employed for the sake of clarity. However,
the invention is not intended to be limited to the specific
terminology so selected. Nothing in this specification should be
considered as limiting the scope of the present invention. All
examples presented are representative and non-limiting. The
above-described embodiments may be modified or varied, without
departing from the invention, as appreciated by those skilled in
the art in light of the above teachings.
Sequence CWU 1
1
11629PRTArtificial Sequencerecombinant human beta-glucuronidase
1Leu Gln Gly Gly Met Leu Tyr Pro Gln Glu Ser Pro Ser Arg Glu Cys1 5
10 15Lys Glu Leu Asp Gly Leu Trp Ser Phe Arg Ala Asp Phe Ser Asp
Asn 20 25 30Arg Arg Arg Gly Phe Glu Glu Gln Trp Tyr Arg Arg Pro Leu
Trp Glu 35 40 45Ser Gly Pro Thr Val Asp Met Pro Val Pro Ser Ser Phe
Asn Asp Ile 50 55 60Ser Gln Asp Trp Arg Leu Arg His Phe Val Gly Trp
Val Trp Tyr Glu65 70 75 80Arg Glu Val Ile Leu Pro Glu Arg Trp Thr
Gln Asp Leu Arg Thr Arg 85 90 95Val Val Leu Arg Ile Gly Ser Ala His
Ser Tyr Ala Ile Val Trp Val 100 105 110Asn Gly Val Asp Thr Leu Glu
His Glu Gly Gly Tyr Leu Pro Phe Glu 115 120 125Ala Asp Ile Ser Asn
Leu Val Gln Val Gly Pro Leu Pro Ser Arg Leu 130 135 140Arg Ile Thr
Ile Ala Ile Asn Asn Thr Leu Thr Pro Thr Thr Leu Pro145 150 155
160Pro Gly Thr Ile Gln Tyr Leu Thr Asp Thr Ser Lys Tyr Pro Lys Gly
165 170 175Tyr Phe Val Gln Asn Thr Tyr Phe Asp Phe Phe Asn Tyr Ala
Gly Leu 180 185 190Gln Arg Ser Val Leu Leu Tyr Thr Thr Pro Thr Thr
Tyr Ile Asp Asp 195 200 205Ile Thr Val Thr Thr Ser Val Glu Gln Asp
Ser Gly Leu Val Asn Tyr 210 215 220Gln Ile Ser Val Lys Gly Ser Asn
Leu Phe Lys Leu Glu Val Arg Leu225 230 235 240Leu Asp Ala Glu Asn
Lys Val Val Ala Asn Gly Thr Gly Thr Gln Gly 245 250 255Gln Leu Lys
Val Pro Gly Val Ser Leu Trp Trp Pro Tyr Leu Met His 260 265 270Glu
Arg Pro Ala Tyr Leu Tyr Ser Leu Glu Val Gln Leu Thr Ala Gln 275 280
285Thr Ser Leu Gly Pro Val Ser Asp Phe Tyr Thr Leu Pro Val Gly Ile
290 295 300Arg Thr Val Ala Val Thr Lys Ser Gln Phe Leu Ile Asn Gly
Lys Pro305 310 315 320Phe Tyr Phe His Gly Val Asn Lys His Glu Asp
Ala Asp Ile Arg Gly 325 330 335Lys Gly Phe Asp Trp Pro Leu Leu Val
Lys Asp Phe Asn Leu Leu Arg 340 345 350Trp Leu Gly Ala Asn Ala Phe
Arg Thr Ser His Tyr Pro Tyr Ala Glu 355 360 365Glu Val Met Gln Met
Cys Asp Arg Tyr Gly Ile Val Val Ile Asp Glu 370 375 380Cys Pro Gly
Val Gly Leu Ala Leu Pro Gln Phe Phe Asn Asn Val Ser385 390 395
400Leu His His His Met Gln Val Met Glu Glu Val Val Arg Arg Asp Lys
405 410 415Asn His Pro Ala Val Val Met Trp Ser Val Ala Asn Glu Pro
Ala Ser 420 425 430His Leu Glu Ser Ala Gly Tyr Tyr Leu Lys Met Val
Ile Ala His Thr 435 440 445Lys Ser Leu Asp Pro Ser Arg Pro Val Thr
Phe Val Ser Asn Ser Asn 450 455 460Tyr Ala Ala Asp Lys Gly Ala Pro
Tyr Val Asp Val Ile Cys Leu Asn465 470 475 480Ser Tyr Tyr Ser Trp
Tyr His Asp Tyr Gly His Leu Glu Leu Ile Gln 485 490 495Leu Gln Leu
Ala Thr Gln Phe Glu Asn Trp Tyr Lys Lys Tyr Gln Lys 500 505 510Pro
Ile Ile Gln Ser Glu Tyr Gly Ala Glu Thr Ile Ala Gly Phe His 515 520
525Gln Asp Pro Pro Leu Met Phe Thr Glu Glu Tyr Gln Lys Ser Leu Leu
530 535 540Glu Gln Tyr His Leu Gly Leu Asp Gln Lys Arg Arg Lys Tyr
Val Val545 550 555 560Gly Glu Leu Ile Trp Asn Phe Ala Asp Phe Met
Thr Glu Gln Ser Pro 565 570 575Thr Arg Val Leu Gly Asn Lys Lys Gly
Ile Phe Thr Arg Gln Arg Gln 580 585 590Pro Lys Ser Ala Ala Phe Leu
Leu Arg Glu Arg Tyr Trp Lys Ile Ala 595 600 605Asn Glu Thr Arg Tyr
Pro His Ser Val Ala Lys Ser Gln Cys Leu Glu 610 615 620Asn Ser Leu
Phe Thr625
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