U.S. patent application number 13/306336 was filed with the patent office on 2012-06-14 for peg-urate oxidase conjugates and use thereof.
This patent application is currently assigned to Duke University. Invention is credited to Michael S. Hershfield, Susan J. Kelly, Mark G.P. Saifer, Merry R. Sherman, L. David Williams.
Application Number | 20120149083 13/306336 |
Document ID | / |
Family ID | 22818799 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120149083 |
Kind Code |
A1 |
Williams; L. David ; et
al. |
June 14, 2012 |
PEG-Urate Oxidase Conjugates and Use Thereof
Abstract
A naturally occurring or recombinant urate oxidase (uricase)
covalently coupled to poly(ethylene glycol) or poly(ethylene oxide)
(both referred to as PEG), wherein an average of 2 to 10 strands of
PEG are conjugated to each uricase subunit and the PEG has an
average molecular weight between about 5 kDa and 100 kDa. The
resulting PEG-uricase conjugates are substantially non-immunogenic
and retain at least 75% of the uricolytic activity of the
unmodified enzyme.
Inventors: |
Williams; L. David;
(Fremont, CA) ; Hershfield; Michael S.; (Durham,
NC) ; Kelly; Susan J.; (Chapel Hill, NC) ;
Saifer; Mark G.P.; (San Carlos, CA) ; Sherman; Merry
R.; (San Carlos, CA) |
Assignee: |
Duke University
Durham
NC
Mountain View Pharmaceuticals, Inc.
Menlo Park
CA
|
Family ID: |
22818799 |
Appl. No.: |
13/306336 |
Filed: |
November 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12769570 |
Apr 28, 2010 |
8067553 |
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13306336 |
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09839946 |
Apr 19, 2001 |
7723089 |
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12769570 |
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09370084 |
Aug 6, 1999 |
6576235 |
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09839946 |
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60219318 |
Aug 6, 1998 |
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Current U.S.
Class: |
435/188 ;
435/191 |
Current CPC
Class: |
C12N 9/0093 20130101;
C12N 9/0046 20130101; C12N 9/96 20130101; A61P 3/00 20180101; C12Y
107/03003 20130101; A61P 13/12 20180101; A61P 19/06 20180101; C12N
9/0048 20130101; A61K 38/00 20130101; A61K 47/60 20170801 |
Class at
Publication: |
435/188 ;
435/191 |
International
Class: |
C12N 9/96 20060101
C12N009/96; C12N 9/06 20060101 C12N009/06 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0002] A portion of the research described in this application was
made with support from Grant DK48529 from the National Institutes
of Health. Accordingly, the U.S. government may have certain rights
in this invention.
Claims
1-41. (canceled)
42. An isolated tetrameric uricase produced by a method comprising
applying a solution of uricase to at least one separation column at
a pH of 9 to 10.5; and recovering from said column one or more
fractions that contain isolated tetrameric uricase, wherein said
one or more fractions are substantially free of uricase
aggregates.
43. The isolated tetrameric uricase of claim 42, wherein the
uricase is a mammalian uricase or a recombinant form thereof.
44. The isolated tetrameric uricase of claim 42, wherein the
uricase is a fungal or microbial uricase or a recombinant form
thereof.
45. The isolated tetrameric uricase of claim 44, wherein the fungal
or microbial uricase is isolated from Aspergillus flavus,
Arthrobacter globiformis, or Candida utilis, or is a recombinant
enzyme having substantially the sequence of one of those
uricases.
46. The isolated tetrameric uricase of claim 42, wherein the
uricase is an invertebrate uricase or a recombinant form
thereof.
47. The isolated tetrameric uricase of claim 46, wherein the
invertebrate uricase is isolated from Drosophila melanogaster or
Drosophila pseudoobscura, or is a recombinant enzyme having
substantially the sequence of one of those uricases.
48. The isolated tetrameric uricase of claim 42, wherein the
uricase is a plant uricase or a recombinant form thereof.
49. The isolated tetrameric uricase of claim 48, wherein the plant
uricase is isolated from root nodules of Glycine max or is a
recombinant enzyme having substantially the sequence of that
uricase.
50. The isolated tetrameric uricase of claim 42, each subunit of
the uricase being subsequently covalently linked to an average of 2
to 12 strands of PEG to form a PEG-uricase conjugate, wherein each
molecule of PEG has a molecular weight of 5 kDa to 100 kDa.
51. The isolated tetrameric uricase of claim 50, wherein the PEG
has an average molecular weight of 10 kDa to 60 kDa.
52. The isolated tetrameric uricase of claim 51, wherein the PEG
has an average molecular weight of 20 kDa to 40 kDa.
53. The isolated tetrameric unease of claim 50, wherein the average
number of covalently coupled strands of PEG is 2 to 10 strands per
uricase subunit.
54. The isolated tetrameric uricase of claim 53, wherein the
average number of covalently coupled strands of PEG is 3 to 8
strands per uricase subunit.
55. The isolated tetrameric uricase of claim 54, wherein the
average number of covalently coupled strands of PEG is 4 to 6
strands per uricase subunit.
56. The isolated tetrameric uricase of claim 50, wherein the
strands of PEG are covalently coupled to uricase via linkages
selected from the group consisting of urethane linkages, secondary
amine linkages, and amide linkages.
57. The isolated tetrameric uricase of claim 50, wherein the PEG is
linear.
58. The isolated tetrameric uricase of claim 50, wherein the PEG is
branched.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/769,570, filed Apr. 28, 2010, now U.S. Pat. No. 8,067,553,
which is a divisional of U.S. application Ser. No. 09/839,946,
filed Apr. 19, 2001, now U.S. Pat. No. 7,723,089, which is a
divisional of U.S. application Ser. No. 09/370,084, filed Aug. 6,
1999, now U.S. Pat. No. 6,576,235, which claims the benefit of U.S.
Provisional Application No. 60/219,318, filed Aug. 6, 1998; each of
which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to chemical modification of
proteins to prolong their circulating lifetimes and reduce their
immunogenicity. More specifically, the invention relates to
conjugation of polyethylene glycols) or polyethylene oxides) to
urate oxidases, which substantially eliminates urate oxidase
immunogenicity without compromising its uricolytic activity.
BACKGROUND OF THE INVENTION
[0004] Statements contained in this background section do not
constitute an admission of prior art, but instead reflect the
inventors' own subjective comments on and interpretations of the
state of the art at the time the invention was made. These
interpretations may include personal, heretofore undisclosed,
insights of the inventors, which insights were not themselves part
of the prior art.
[0005] Urate oxidases (uricases; E.C. 1.7.3.3) are enzymes that
catalyze the oxidation of uric acid to a more soluble product,
allantoin, a purine metabolite that is more readily excreted.
Humans do not produce enzymatically active uricase, as a result of
several mutations in the gene for uricase acquired during the
evolution of higher primates. Wu, X, et al., (1992) J Mol Evol
34:78-84. As a consequence, in susceptible individuals, excessive
concentrations of uric acid in the blood (hyperuricemia) and in the
urine (hyperuricosuria) can lead to painful arthritis (gout),
disfiguring urate deposits (tophi) and renal failure. In some
affected individuals, available drugs such as allopurinol (an
inhibitor of uric acid synthesis) produce treatment-limiting
adverse effects or do not relieve these conditions adequately.
Hande, K R, et al., (1984) Am J Med 76:47-56; Fam, A G, (1990)
Bailliere's Clin Rheumatol 4:177-192. Injections of uricase can
decrease hyperuricemia and hyperuricosuria, at least transiently.
Since uricase is a foreign protein in humans, however, even the
first injection of the unmodified protein from Aspergillus flavus
has induced anaphylactic reactions in several percent of treated
patients (Pui, C-H, et al., (1997) Leukemia 11:1813-1816), and
immunologic responses limit its utility for chronic or intermittent
treatment. Donadio, D, et al., (1981) Nouv Presse Meed 10:711-712;
Leaustic, M, et al., (1983) Rev Rhum Mal Osteoartic 50:553-554.
[0006] The sub-optimal performance of available treatments for
hyperuricemia has been recognized for several decades. Kissel, P,
et al., (1968) Nature 217:72-74. Similarly, the possibility that
certain groups of patients with severe gout might benefit from a
safe and effective form of injectable uricase has been recognized
for many years. Davis, F F, et al., (1978) in G B Braun, et al.,
(Eds.) Enzyme Engineering, Vol. 3 (pp. 169-173) New York, Plenum
Press; Nishimura, H, et al., (1979) Enzyme 24:261-264; Nishimura,
H, et al., (1981) Enzyme 26:49-53; Davis, S, et al., (1981) Lancet
2(8241)281-283; Abuchowski, A, et al., (1981) J Pharmacol Exp Ther
219:352-354; Chen, RH-L, et al., (1981) Biochim Biophys Acta
660:293-298; Chua, C C, et al., (1988) Ann Int Med 109:114-117;
Greenberg, M L, et al., (1989) Anal Biochem 176:290-293. Uricases
derived from animal organs are nearly insoluble in solvents that
are compatible with safe administration by injection. U.S. Pat. No.
3,616,231. Certain uricases derived from plants or from
microorganisms are more soluble in medically acceptable solvents.
However, injection of the microbial enzymes quickly induces
immunological responses that can lead to life-threatening allergic
reactions or to inactivation and/or accelerated clearance of the
uricase from the circulation. Donadio, et al., (1981); Leaustic, et
al., (1983). Enzymes based on the deduced amino acid sequences of
uricases from mammals, including pig and baboon, or from insects,
such as, for example, Drosophila melanogaster or Drosophila
pseudoobscura (Wallrath, L L, et al., (1990) Mol Cell Biol
10:5114-5127), have not been suitable candidates for clinical use,
due to problems of immunogenicity and insolubility at physiological
pH.
[0007] Covalent modification of proteins with poly(ethylene glycol)
or poly(ethylene oxide) (both referred to as PEG), has been used to
increase protein half-life and reduce immunogenicity. U.S. Pat.
Nos. 4,179,337, 4,766,106, and 4,847,325; Saifer, M G P, et al.,
(1994) Adv Exp Med Biol 366:377-387. The coupling of PEG of high
molecular weight to produce conjugates with prolonged circulating
lifetimes and/or decreased immunogenicity, while conserving
functional activity, was previously demonstrated for another
enzyme, superoxide dismutase (Somack, R, et al., (1991) Free Rad
Res Commun 12-13:553-562; U.S. Pat. Nos. 5,283,317 and 5,468,478)
and for other types of proteins, e.g., cytokines (Saifer, M G P, et
al., (1997) Polym Preprints 38:576-577; Sherman, M R, et al.,
(1997) in J M Harris, et al., (Eds.), Poly(ethylene glycol)
Chemistry and Biological Applications. ACS Symposium Series 680
(pp. 155-169) Washington, D.C.: American Chemical Society).
Conjugates of uricase with polymers other than PEG have also been
described. U.S. Pat. No. 4,460,683.
[0008] In nearly all of the reported attempts to PEGylate uricase
(i.e. to covalently couple PEG to uricase), the PEG was attached
primarily to amino groups, including the amino-terminal residue and
the available lysine residues. In the uricases commonly used, the
total number of lysines in each of the four identical subunits is
between 25 (Aspergillus flavus (U.S. Pat. No. 5,382,518)) and 29
(pig (Wu, X, et al., (1989) Proc Natl Acad Sci USA 86:9412-9416)).
Some of the lysines are unavailable for PEGylation in the native
conformation of the enzyme. The most common approach to reducing
the immunogenicity of uricase has been to couple large numbers of
strands of low molecular weight PEG. This has invariably resulted
in large decreases in the enzymatic activity of the resultant
conjugates.
[0009] Previous investigators have used injected uricase to
catalyze the conversion of uric acid to allantoin in vivo. See Pui,
et al., (1997). This is the basis for the use in France and Italy
of uricase from the fungus Aspergillus flavus (Uricozyme.RTM.) to
prevent or temporarily correct the hyperuricemia associated with
cytotoxic therapy for hematologic malignancies and to transiently
reduce severe hyperuricemia in patients with gout. Potaux, L, et
al., (1975) Nouv Presse Med 4:1109-1112; Legoux, R, et al., (1992)
J Biol Chem 267:8565-8570; U.S. Pat. Nos. 5,382,518 and 5,541,098.
Because of its short circulating lifetime, Uricozyme.RTM. requires
daily injections. Furthermore, it is not well suited for long-term
therapy because of its immunogenicity.
[0010] A single intravenous injection of a preparation of Candida
utilis uricase coupled to 5 kDa PEG reduced serum urate to
undetectable levels in five human subjects whose average
pre-injection serum urate concentration was 6.2 mg/dL, which is
within the normal range. Davis, et al., (1981). The subjects were
given an additional injection four weeks later, but their responses
were not reported. No antibodies to uricase were detected following
the second (and last) injection, using a relatively insensitive gel
diffusion assay. This reference reported no results from chronic or
subchronic treatments of human patients or experimental
animals.
[0011] A preparation of uricase from Arthrobacter protoformiae
coupled to 5 kDa PEG was used to temporarily control hyperuricemia
in a single patient with lymphoma whose pre-injection serum urate
concentration was 15 mg/dL. Chua, et al., (1988). Because of the
critical condition of the patient and the short duration of
treatment (four injections during 14 days), it was not possible to
evaluate the long-term efficacy or safety of the conjugate.
[0012] In this application, the term "immunogenicity" refers to the
induction of an immune response by an injected preparation of
PEG-modified or unmodified uricase (the antigen), while
"antigenicity" refers to the reaction of an antigen with
preexisting antibodies. Collectively, antigenicity and
immunogenicity are referred to as "immunoreactivity." In previous
studies of PEG-uricase, immunoreactivity was assessed by a variety
of methods, including: 1) the reaction in vitro of PEG-uricase with
preformed antibodies; 2) measurements of induced antibody
synthesis; and 3) accelerated clearance rates after repeated
injections.
[0013] Previous attempts to eliminate the immunogenicity of
uricases from several sources by coupling various numbers of
strands of PEG through various linkers have met with limited
success. PEG-uricases were first disclosed by F F Davis and by Y
Inada and their colleagues. Davis, et al., (1978); U.S. Pat. No.
4,179,337; Nishimura, et al., (1979); Japanese Patents 55-99189 and
62-55079. The conjugate disclosed in the '337 patent was
synthesized by reacting uricase of unspecified origin with a
2,000-fold molar excess of 750 dalton PEG, indicating that a large
number of polymer molecules was likely to have been attached to
each uricase subunit. The '337 patent discloses the coupling of
either PEG or poly(propylene glycol) with molecular weights of 500
to 20,000 daltons, preferably about 500 to 5,000 daltons, to
provide active, water-soluble, non-immunogenic conjugates of
various polypeptide hormones and enzymes including oxidoreductases,
of which uricase is one of three examples. In addition, the '337
patent emphasizes the coupling of 10 to 100 polymer strands per
molecule of enzyme, and the retention of at least 40% of enzymatic
activity. No test results were reported for the extent of coupling
of PEG to the available amino groups of uricase, the residual
specific uricolytic activity, or the immunoreactivity of the
conjugate.
[0014] Data from 13 citations relating to PEGylation of uricase are
summarized in Table 1. Some of these results are also presented
graphically in FIGS. 1A-2B. Seven of these publications describe
significant decreases in uricolytic activity measured in vitro
caused by coupling various numbers of strands of PEG to uricase
from Candida utilis. Coupling a large number of strands of 5 kDa
PEG to porcine liver uricase gave similar results, as described in
both the Chen publication and a symposium report by the same group.
Chen, et al., (1981); Davis, et al., (1978).
[0015] Among the studies summarized in Table 1, the
immunoreactivity of uricase was reported to be decreased by
PEGylation in seven of them and eliminated in five of them. In
three of the latter five studies, the elimination of
immunoreactivity was associated with profound decreases in
uricolytic activity--to at most 15%, 28%, or 45% of the initial
activity. Nishimura, et al., (1979) (15% activity); Chen, et al.,
(1981) (28% activity); Nishimura, et al., (1981) (45% activity). In
the fourth report, PEG was reported to be coupled to 61% of the
available lysine residues, but the residual specific activity was
not stated. Abuchowski, et al., (1981). However, a research team
that included two of the same scientists and used the same methods
reported elsewhere that this extent of coupling left residual
activity of only 23-28%. Chen, et al., (1981). The 1981
publications of Abuchowski et al., and Chen et al., indicate that
to reduce the immunogenicity of uricase substantially, PEG must be
coupled to approximately 60% of the available lysine residues
(Table 1). The fifth publication in which the immunoreactivity of
uricase was reported to have been eliminated does not disclose the
extent of PEG coupling, the residual uricolytic activity, or the
nature of the PEG-protein linkage. Veronese, F M, et al., (1997) in
J M Harris, et al., (Eds.), Poly(ethylene glycol) Chemistry and
Biological Applications. ACS Symposium Series 680 (pp. 182-192)
Washington, D.C.: American Chemical Society.
[0016] Conjugation of PEG to a smaller fraction of the lysine
residues in uricase reduced but did not eliminate its
immunoreactivity in experimental animals. Tsuji, J, et al., (1985)
Int J Immunopharmacol 7:725-730 (28-45% of the amino groups
coupled); Yasuda, Y, et al., (1990) Chem Pharm Bull 38:2053-2056
(38% of the amino groups coupled). The residual uricolytic
activities of the corresponding adducts ranged from <33% (Tsuji,
et al.) to 60% (Yasuda, et al.) of their initial values. Tsuji, et
al., synthesized PEG-uricase conjugates with 7.5 kDa and 10 kDa
PEGs, in addition to 5 kDa PEG. All of the resultant conjugates
were somewhat immunogenic and antigenic, while displaying markedly
reduced enzymatic activities (Table 1; FIGS. 1A-1B).
[0017] A PEGylated preparation of uricase from Candida utilis that
was safely administered twice to each of five humans was reported
to have retained only 11% of its initial activity. Davis, et al.,
(1981). Several years later, PEG-modified uricase from Arthrobacter
protoformiae was administered four times to one patient with
advanced lymphoma and severe hyperuricemia. Chua, et al., (1988).
While the residual activity of that enzyme preparation was not
measured, Chua, et al., demonstrated the absence of anti-uricase
antibodies in the patient's serum 26 days after the first
PEG-uricase injection, using an enzyme-linked immunosorbent assay
(ELISA).
[0018] As summarized in Table 1, previous studies of PEGylated
uricase show that catalytic activity is markedly depressed by
coupling a sufficient number of strands of PEG to decrease its
immunoreactivity substantially. Furthermore, most previous
preparations of PEG-uricase were synthesized using PEG activated
with cyanuric chloride, a triazine derivative
(2,4,6-trichloro-1,3,5-triazine) that has been shown to introduce
new antigenic determinants and to induce the formation of
antibodies in rabbits. Tsuji, et al., (1985).
TABLE-US-00001 TABLE 1 Characteristics of PEG-Uricases from
Previous Studies Molecular Percent of Residual Antigenicity or
Source of Coupling Weight of Lysines with Uricolytic Immunogenicity
Uricase Linkage PEG (kDa) PEG Attached Activity (%) Comments
Reference Not Azide 0.7 (diol) Not Not Not U.S. Pat. No. reported
reported reported reported 4,179,337 Candida Triazine 5 % of "98":
Antigenicity with rabbit Nishimura, utilis (Cyanuric serum (% of
that of the et al., 1979 chloride) unmodified enzyme) 20 31 70% 26
21 6% 43 15 0 48 5 0 Candida PEG.sub.2 2 .times. 5 22 87 86%
Nishimura, utilis triazine 25 70 49% et al., 1981 36 45 0 46 31 0
50 27 0 Candida Triazine 5 71 11 Five men tolerated two Davis,
utilis injections in 30 days. et al., 1981 Candida Triazine 5 49
Not Similar immunogenicity in Abuchowski, utilis according reported
birds to native uricase et al., 1981 to Chen 61 Not Immunogenicity
negative et al., 1981 reported Porcine Triazine 5 37 60 Accelerated
clearance in mice Chen, liver 47 45 '' et al., 1981 58 28 Constant
Clearance (half-life ca. 8 hours) Candida Triazine 5 57 23 Constant
Clearance utilis (half life ca. 8 hours) Candida Triazine 5 35 Not
PEG decreased the Savoca, KV, utilis according reported
immunogenicity in rabbits. et al., (1984) to Chen, 70 Not PEG
decreased the Int Arch Allergy et al., 1981 reported immunogenicity
in rabbits. Appl Immunol 75: 58-67 Candida Triazine 5 Not Not
PEG-uricase was given Nishida, Y, utilis reported reported orally
to chickens in et al., (1984) liposomes (once). J Pharm Pharmacol
36: 354-355 Candida Triazine 5 44 9.4 Immunogenicity was reduced,
Tsuji, utilis 7.5 45 7.8 but positive in rabbits. et al., 1985 10
28 32 (Antibodies are not to 37 11 uricase; they cross react 41 3
with PEG-superoxide dismutase.) 45 7.3 Antigenicity tested with
guinea pig antibodies was reduced. Arthrobacter Not 5 Not Not No
antibodies were detected by Chua, protoformiae reported reported
reported ELISA 26 days after the first et al., 1988 of four
PEG-uricase injections. Candida PEG.sub.2 2 .times. 5 10 90 Not
reported Yasuda, utilis triazine 12 89 Not reported et al., 1990 15
80 Not reported 21 70 Not reported 38 60 Antigenicity tested with
rabbit serum was reduced by 75%. Candida PEG.sub.2 2 .times. 5 22
68 Single injection. PEG increased Fujita, utilis triazine the
half-life from ca. 1 h to et al., 1991 ca. 8 h in mice. PEG blocked
clearance by liver, spleen and kidney (24-h study duration). Not
PEG Not Not Not Immunogenicity in mice was Veronese, reported
PEG.sub.2 reported reported reported decreased by 98% (PEG) et al.,
1997 Linkage Reported to Not or 100% (PEG.sub.2). not stated be the
same reported as for PEG
[0019] Japanese Patent No. 3-148298 to A Sano, et al., discloses
modified proteins, including uricase, derivatized with PEG having a
molecular weight of 1-12 kDa that show reduced antigenicity and
"improved prolonged" action, and methods of making such derivatized
peptides. However, there are no disclosures regarding strand
counts, enzyme assays, biological tests or the meaning of "improved
prolonged." Japanese Patents 55-99189 and 62-55079, both to Y
Inada, disclose uricase conjugates prepared with PEG-triazine or
bis-PEG-triazine (denoted as PEG.sub.2 in Table 1), respectively.
See Nishimura, et al., (1979 and 1981). In the first type of
conjugate, the molecular weights of the PEGs were 2 kDa and 5 kDa,
while in the second, only 5 kDa PEG was used. Nishimura, et al.,
(1979) reported the recovery of 15% of the uricolytic activity
after modification of 43% of the available lysines with linear 5
kDa PEG, while Nishimura, et al., (1981) reported the recovery of
31% or 45% of the uricolytic activity after modification of 46% or
36% of the lysines, respectively, with PEG.sub.2.
SUMMARY OF THE INVENTION
[0020] In one aspect of this embodiment, the uricase may be
tetrameric. The strands of PEG may be covalently linked to uricase
via urethane (carbamate) linkages, secondary amine linkages, and/or
amide linkages. When the uricase is a recombinant form of any of
the uricases mentioned herein, the recombinant form may have
substantially the sequence of the naturally occurring form.
[0021] Other embodiments of the present invention are a method for
isolating a tetrameric form of uricase from a solution containing
multiple forms of uricase and the product of that method.
Initially, the solution may contain tetrameric uricase and uricase
aggregates. The method may include the steps of: applying the
solution to at least one separation column at a pH between about 9
and 10.5, such as, for example, 10.2; recovering fractions of the
eluate and identifying those that may contain isolated tetrameric
uricase, wherein the fractions are substantially free of uricase
aggregates; and pooling the fractions of the isolated tetrameric
uricase. The separation column may be based on ion exchange, size
exclusion, or any other effective separation property. The method
may also include analysis of the fractions to determine the
presence of tetrameric uricase and/or the absence of uricase
aggregates. For example, such analysis may include high performance
liquid chromatography (HPLC), other chromatographic methods, light
scattering, centrifugation and/or electrophoresis. In one aspect of
this embodiment, the purified tetrameric uricase may contain less
than about 10% uricase aggregates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A shows the retention of activity by PEGylated uricase
from Candida utilis as a function of the number of strands of PEG
coupled per subunit.
[0023] FIG. 1B shows the retention of activity by PEGylated uricase
from Candida utilis as a function of the total mass of PEG coupled
per subunit.
[0024] FIG. 2A shows the retention of activity by PEGylated uricase
from porcine liver as a function of the number of strands of PEG
coupled per subunit.
[0025] FIG. 2B shows the retention of activity by PEGylated uricase
from porcine liver as a function of the total mass of PEG coupled
per subunit.
[0026] FIG. 3A shows the retention of activity by PEGylated
pig-baboon chimeric (PBC) uricase as a function of the number of
strands coupled per subunit.
[0027] FIG. 3B shows the retention of activity by PEGylated PBC
uricase as a function of the total mass of PEG coupled per
subunit.
[0028] FIG. 4A shows the retention of activity by PEGylated uricase
from Aspergillus flavus as a function of the number of strands of
PEG coupled per subunit.
[0029] FIG. 4B shows the retention of activity by PEGylated uricase
from Aspergillus flavus as a function of the total mass of PEG
coupled per subunit.
[0030] FIG. 5A shows the retention of activity by PEGylated
recombinant soybean root nodule uricase as a function of the number
of strands of PEG coupled per subunit.
[0031] FIG. 5B shows the retention of activity by PEGylated
recombinant soybean root nodule uricase as a function of the total
mass of PEG coupled per subunit.
[0032] FIG. 6 shows the deduced amino acid sequences of pig-baboon
chimeric uricase (PBC uricase), PBC uricase that is truncated at
both the amino and carboxyl terminals (PBC-NT-CT) and porcine
uricase containing the mutations R291K and T301S (PKS uricase),
compared with the porcine (SEQ ID NO:1) and baboon (SEQ ID NO:2)
sequences.
[0033] FIG. 7 shows the activity of uricase in mouse serum 24 h
after each of four or five intraperitoneal injections of
PEG-modified PBC uricase, relative to the value 24 h after the
first injection.
[0034] FIG. 8 shows the inverse relationship between the activity
of injected PEG-modified PBC uricase in the serum of a
uricase-deficient mouse and the concentrations of uric acid in the
serum and urine.
[0035] FIG. 9 shows the decreased severity of a urine-concentrating
defect in uricase-deficient (uox -/-) mice that were treated with
PEG-modified PBC uricase.
[0036] FIG. 10 shows the decreased severity of nephrogenic diabetes
insipidus in uricase-deficient (uox -/-) mice that were treated
with PEG-modified PBC uricase.
[0037] FIG. 11 shows the decreased severity of uric acid-induced
nephropathy, as visualized by magnetic resonance microscopy, in
uricase-deficient (uox -/-) mice that were treated with
PEG-modified PBC uricase.
[0038] FIG. 12 shows the accelerated clearance from the circulation
of BALB/c mice of injected PBC uricase octamer, compared with the
tetramer, when both were coupled to 5-6 strands of 10 kDa PEG per
subunit.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] Purified preparations of naturally occurring and recombinant
uricases usually contain a mixture of aggregates of the enzyme, in
addition to the tetrameric (140 kDa) form. The percentage of each
uricase preparation that is in the tetrameric form generally varies
from approximately 20% to 90%. Despite evidence that unPEGylated
aggregates of several other proteins are highly immunogenic (see,
e.g., Moore, W V, et al., (1980) J Clin Endocrinol Metab
51:691-697), previous studies of PEG-uricase do not describe any
efforts to limit the content of aggregates, suggesting that the
potential immunogenicity of the PEG-modified aggregates was not
considered. On the basis of the observations of the present
inventors, it appears likely that such aggregates were present in
the enzyme preparations used for previous syntheses of PEG-uricase.
Their presence may have rendered the task of preparing
non-immunogenic conjugates more difficult. It also appears that the
large losses of uricolytic activity observed in previous efforts to
PEGylate uricase were related to the large number of strands of low
molecular weight PEG that were coupled. On the other hand, the
methods of uricase purification and PEGylation described herein
permit the covalent attachment of as many as 10 strands of PEG per
subunit while retaining more than 75% of the uricolytic activity,
at least for certain uricases, e.g., pig-baboon chimeric uricase
and the enzyme from A. flavus (see FIGS. 3A and 4A).
[0040] In another preferred embodiment, substantially all
aggregates of the tetrameric form of the enzyme may be removed by
ion-exchange or size-exclusion chromatography at a pH between about
9 and 10.5, preferably 10.2, prior to PEG conjugation of the
resulting substantially tetrameric preparation of uricase. The
molecular weight of the uricase in each fraction from the
preparative column may be monitored by any size-dependent
analytical technique, including, for example, HPLC, conventional
size-exclusion chromatography, centrifugation, light scattering,
capillary electrophoresis or gel electrophoresis in a
non-denaturing buffer. For tetrameric uricase isolated using
size-exclusion chromatography, fractions containing only the 140
kDa form of the enzyme may be pooled and used for conjugation to
PEG. For tetrameric uricase isolated using ion-exchange
chromatography, fractions from the ion-exchange column may be
analyzed with respect to size to determine which fractions contain
substantial amounts of the tetrameric form without detectable
aggregates. Of the uricase thus pooled, at least 90% may be in the
tetrameric form; the undesirable aggregates may thus constitute as
little as about 10%, 5%, 2%, or less, of the total isolated
uricase.
[0041] The results presented herein indicate that, even when
extensively PEGylated, forms of PBC uricase larger than the
tetramer are highly immunogenic in mice (FIG. 12). Furthermore, in
mice that had been injected once with PEG conjugates of uricase
aggregates, the uricolytic activity in subsequent injections of
either PEGylated tetramers or PEGylated aggregates was cleared
rapidly from the circulation. In contrast, conjugates prepared from
uricase containing less than 5% aggregates could be reinjected many
times without any acceleration of their clearance rates (FIG. 7)
and without the detectable formation of antibodies, as measured by
a sensitive enzyme-linked immunoassay. The use of highly purified
tetrameric uricase further distinguishes the improved conjugates of
the present invention from the PEG-uricase preparations described
previously. In contrast, the presence of a significant proportion
(e.g., >10%) of aggregates in the uricase preparations used by
some previous investigators may have led them to couple large
numbers of strands of PEG in efforts to suppress the
immunogenicity. Consequently, the enzymatic activity of the
resultant conjugates was decreased substantially.
[0042] In one embodiment of the invention, uricase may be
conjugated via a biologically stable, nontoxic, covalent linkage to
a relatively small number of strands of PEG. Such linkages may
include urethane (carbamate) linkages, secondary amine linkages,
and amide linkages. Various activated PEGs suitable for such
conjugation are available commercially from Shearwater Polymers,
Huntsville, Ala.
[0043] For example, urethane linkages to unease may be formed by
incubating unease in the presence of the succinimidyl carbonate
(SC) or 4-nitrophenyl carbonate (NPC) derivative of PEG; SC-PEG may
be synthesized using the procedure described in U.S. Pat. No.
5,612,460, which is hereby incorporated by reference. NPC-PEG may
be synthesized by reacting PEG with 4-nitrophenyl chloroformate
according to methods described in Veronese, F M, et al., (1985)
Appl Biochem Biotechnol 11:141-'152, and in U.S. Pat. No.
5,286,637, which is hereby incorporated by reference. The methods
described in the '637 patent are adapted to PEGs of higher
molecular weight by adjusting the concentrations of the reactants
to maintain similar stoichiometry. An alternative method of
synthesis of NPC-PEG is described by Buettner, W, et al., East
German Patent Specification DD 479 486 A1.
[0044] Amide linkages to unease may be obtained using an
N-hydroxysuccinimide ester of a carboxylic acid derivative of PEG
(Shearwater Polymers). Secondary amine linkages may be formed using
2,2,2-trifluoroethanesulfonyl PEG (tresyl PEG; Shearwater Polymers)
or by reductive alkylation using PEG aldehyde (Shearwater Polymers)
and sodium cyanoborohydride.
[0045] There are several factors that may affect the choice of the
optimal molecular weight and number of strands of PEG for coupling
to a given form of unease. In general, the reduction or elimination
of immunogenicity without substantial loss of uricolytic activity
may require the coupling of relatively more strands of PEG of lower
molecular weight, compared to relatively fewer strands of PEG of
higher molecular weight. For example, either 6 strands of 20 kDa
PEG per subunit or 4 strands of 30 kDa PEG per subunit might be
optimally effective. Likewise, each different form of uricase may
have a different optimum with respect to both the size and number
of strands. See FIGS. 1A-5B.
[0046] When PEG conjugates of PBC uricase were prepared from the
purified tetrameric form of the enzyme (four 35 kDa subunits), they
displayed profoundly reduced immunogenicity in mice (FIG. 7), in
contrast to the moderate immunogenicity of PEG conjugates of larger
forms of the enzyme (e.g. octamers of the 35 kDa subunit; see FIG.
12), and the very high immunogenicity of the unmodified enzyme.
Repeated injections of uricase-deficient mice with PEG-uricase of
the present invention eliminated their hyperuricemia for more than
2 months and protected the structure and function of their kidneys
against uric acid-related damage (FIGS. 8-11).
[0047] The following examples, which are not to be construed as
limiting the invention in any way, illustrate the various aspects
disclosed above. These examples describe PEG-uricases prepared by
coupling activated (i.e., electrophilic) PEG derivatives of several
sizes and compositions with naturally occurring porcine, fungal or
bacterial uricases, or with recombinant soybean, porcine or
pig-baboon chimeric uricases. Results of activity, solubility,
stability, pharmacokinetic, pharmacodynamic and immunological
studies are included. The data in FIGS. 8-11 provide evidence of
the ability of PEG-modified PBC uricase of this invention to
correct hyperuricemia and hyperuricosuria and to preserve renal
structure and function in an animal model in which hyperuricemia
and hyperuricosuria occur and cause serious renal damage. Wu, X, et
al., (1994) Proc Natl Acad Sci USA 91:742-746. These examples
provide guidance to one with ordinary skill in the art for
producing substantially non-immunogenic conjugates of uricase that
retain at least about 75% of the uricolytic activity of the
unmodified enzyme.
Example 1
Purification of the Tetrameric Form of Uricase
[0048] The tetrameric form of uricase (molecular weight ca. 140
kDa) was purified from a solution of porcine liver uricase by
preparative size-exclusion or ion-exchange chromatography, followed
by analytical size-exclusion chromatography. Porcine liver uricase
was obtained from Sigma-Aldrich, St. Louis, Mo., catalog No. U2350
or U3377; or Boehringer Mannheim, Indianapolis, Ind.
[0049] Preparative and analytical size-exclusion chromatography
were performed at pH 10-10.5, preferably 10.2, in 10 mM sodium
carbonate buffer containing 0.1 M NaCl on Superdex 200 columns that
had been previously calibrated with proteins of known molecular
weight. Superdex was obtained from Amersham Pharmacia, Piscataway,
N.J. Any buffer may be used that is capable of maintaining the
desired pH and that is compatible with the chemistry to be used for
subsequent PEG coupling. Such buffers are well known in the art.
The ultraviolet absorbance of the eluate from the preparative
column was monitored at 280 nm, and uricase-containing portions of
the eluate corresponding to the molecular weight of the desired
tetrameric form, but free of higher molecular weight species, were
collected for use in synthesizing substantially non-immunogenic
PEG-uricase as described in Example 2. Alternatively, tetrameric
forms of uricase can be isolated using other size-exclusion media
such as, for example, Superose 12 (Amersham Pharmacia) or any other
medium that is compatible with mildly alkaline solutions and that
has an appropriate size fractionation range. Such media are readily
available and are well known in the art.
[0050] Ion-exchange chromatography was performed at pH 10-10.5,
preferably 10.2, on Mono Q columns (Amersham Pharmacia, Piscataway,
N.J.) that had been equilibrated with 0.1 M sodium carbonate
buffer. Any buffer that is compatible with the chemistry of PEG
coupling and that is capable of maintaining the desired pH may be
used at sufficiently low ionic strength to permit the adsorption of
uricase to the column. Such buffers are well known in the art. The
ultraviolet absorbance of the eluate was monitored at 280 nm during
elution of the uricase from the ion-exchange resin by increasing
the ionic strength of the applied buffer solution, e.g. by a linear
gradient of 0 to 0.5 M NaCl in the sodium carbonate buffer.
Size-exclusion HPLC was then used to identify the fractions of the
eluate containing the desired tetrameric form of uricase, without
detectable aggregates, for the synthesis of substantially
non-immunogenic PEG-uricase. Alternatively, the tetrameric form of
uricase can be isolated using other ion-exchange media, such as
Q-Sepharose (Amersham Pharmacia) or any other medium that is
compatible with mildly alkaline solutions. Such media are readily
available and are well known in the art.
[0051] Uricase activity was assayed using a modification of
standard methods. See, e.g., Fridovich (1965); Nishimura, et al.,
(1979). Solutions of uric acid were prepared fresh daily in 50 mM
sodium borate buffer, pH 9.2, to provide final concentrations in
the assay of 6-150 .mu.M. Uricase preparations were diluted in this
borate buffer containing bovine serum albumin (Sigma-Aldrich, St.
Louis, Mo., catalog No. A-7030), so that the final concentration of
albumin in the assay was 0.1 mg/mL. After mixing various dilutions
of the enzyme with the substrate in the wells of a microtiter plate
in a microplate reader, the rate of disappearance of uric acid at
25.degree. C. was monitored at 292 nm every 4 seconds for 3
minutes. From samples in which between 10% and 40% of the substrate
was consumed within 3 minutes, at least 20 data points were used to
calculate the maximal rate of decrease in the absorbance per
minute. One international unit (IU) of uricase activity is defined
as the amount of enzyme that consumes one micromole of uric acid
per minute; specific activities are expressed as IU/mg protein.
Some of the data for relative uricase activities in FIGS. 1A-5B
were obtained using 100 .mu.M uric acid in the assay. Other results
for the velocity at 100 .mu.M uric acid (V.sub.100) were calculated
from the values of the Michaelis constant (K.sub.M) and the maximal
velocity (V.sub.max) for the respective enzyme preparations, using
the formula:
V.sub.100=100.times.V.sub.max/(K.sub.M+100)
[0052] where K.sub.M is expressed in micromolar units.
Example 2
PEG Coupling to Tetrameric Porcine Uricase
[0053] To a solution of tetrameric uricase in 0.1 M sodium
carbonate buffer, pH 10.2, 10-200 moles of an activated derivative
of monomethoxyPEG, e.g., the 4-nitrophenyl carbonate (NPC-PEG), of
various sizes (5 kDa to 30 kDa) were added for each mole of uricase
subunit (molecular weight 35 kDa). These and other suitable
activated PEGs are available from Shearwater Polymers. Instructions
for coupling these PEGs to proteins are given in the catalog of
Shearwater Polymers, on the Internet at www.swpolymers.com, and in
J M Harris, et al., (Eds.) (1997) Poly(ethylene glycol) Chemistry
and Biological Applications. ACS Symposium Series 680, Washington,
D.C.: American Chemical Society. The coupling reaction was allowed
to proceed at 0-8.degree. C. until the extent of PEG coupling no
longer changed significantly with time. Unreacted PEG was then
removed from the reaction product by chromatography and/or
ultrafiltration
[0054] The number of strands of PEG coupled per subunit of uricase
was determined by an adaptation of the methods described by
Kunitani, M, et al., (1991) J Chromatogr 588:125-137; Saifer, et
al., (1997) and Sherman, et al., (1997). Briefly, aliquots of the
PEGylation reaction mixtures or fractions from the preparative
ion-exchange or size-exclusion columns were characterized by
analytical size-exclusion HPLC on a TSK 5,000 PW.sub.XL column at
room temperature in 10 mM sodium carbonate buffer, pH 10.2,
containing 0.1 M NaCl. The HPLC column was obtained from TosoHaas,
Montgomeryville, Pa. Proteins and PEGs were monitored by
ultraviolet absorbance and refractive index detectors. The amount
of protein in the conjugate was calculated from the ultraviolet
absorbance relative to that of the appropriate unmodified uricase
standard. The amount of PEG in the conjugate was then calculated
from the area of the refractive index peak, corrected for the
contribution of the protein to refractive index, relative to the
area of the refractive index peak of the appropriate PEG
standard.
[0055] FIG. 2A shows the retention of activity by PEGylated porcine
liver uricase as a function of the number of strands of PEG coupled
per subunit. Data of the present inventors (.DELTA., .quadrature.)
are compared with those of Chen, et al., (1981). The data point
within a large circle denotes a conjugate reported to be
non-immunoreactive by Chen, et al., (1981). As shown in FIG. 2A,
conjugates of tetrameric porcine uricase with up to 6 strands of 30
kDa PEG per subunit or up to 7 strands of 5 kDa PEG per subunit
retained at least 75% of the activity of the unmodified enzyme. The
apparent increase in specific activity with an increasing number of
strands of 5 kDa or 30 kDa PEG (up to about 4 strands per subunit)
may reflect the relative insolubility or instability of the
unmodified enzyme compared to the conjugates. As shown in FIG. 2B,
conjugates of porcine uricase with an average of more than 3
strands of 30 kDa PEG per subunit contain a greater mass of PEG
than was found sufficient to preclude immunoreactivity by Chen, et
al., (1981).
Example 3
Properties of PEG Conjugates of Tetrameric Recombinant PBC
Uricase
[0056] Recombinant pig-baboon chimeric (PBC) uricase cDNA was
subcloned into the pET3d expression vector (Novagen, Madison, Wis.)
and the resultant plasmid construct was transformed into and
expressed in a strain of Escherichia coli BL21(DE3)pLysS (Novagen).
These procedures were carried out using methods well known in the
art of molecular biology. See Erlich (1989); Sambrook, et al.,
(1989); Ausubel, F, et al., (Eds.), (1997) Short Protocols in
Molecular Biology. New York: John Wiley & Sons.
[0057] FIG. 6 shows the deduced amino acid sequence of PBC uricase
(amino acids 1-225 of SEQ ID NO: 1 and amino acids 226-304 of SEQ
ID NO: 2), compared with the porcine (SEQ ID NO: 1) and baboon (SEQ
ID NO: 2) sequences. Residues in the baboon sequence that differ
from those in the porcine sequence are shown in bold type. The
porcine and baboon sequences were first determined by Wu, et al.,
(1989) and were confirmed by the present inventors. SEQ ID NO. 1 is
identical to Accession Number p16164 of GenBank, except for the
absence of the initial methionyl residue in the GenBank sequence.
SEQ ID NO. 2 is identical to Accession Number p25689 of GenBank,
except for the absence of the initial methionyl residue and a
change from histidine to threonine at residue 153 in the GenBank
sequence (residue 154 in FIG. 6).
[0058] The tetrameric form of PBC uricase was isolated and coupled
to PEGs of various molecular weights as described in Examples 1 and
2. Conjugates prepared with 5 kDa, 10 kDa, 19 kDa or 30 kDa PEG
contained up to 10 strands of PEG per subunit. Those prepared with
PEGs of at least 10 kDa retained more than 95% of the initial
specific activity of the recombinant uricase (FIGS. 3A-3B).
[0059] The following properties of a conjugate of tetrameric PBC
uricase with approximately 6 strands of 10 kDa PEG per subunit are
illustrated in the indicated figures: the lack of immunogenicity
(FIG. 7) and the efficacy in uricase-deficient mice in 1)
correcting hyperuricemia and hyperuricosuria (FIG. 8); 2)
decreasing the severity of a urine-concentrating defect (FIG. 9),
and 3) decreasing the severity of nephrogenic diabetes insipidus
(FIG. 10). In addition, this PEG-uricase case decreased the
severity of uric acid-related renal damage, as visualized by
magnetic resonance microscopy (FIG. 11).
[0060] FIG. 7 shows the activity of PBC uricase in mouse serum 24 h
after each of four or five intraperitoneal injections of
PEG-uricase, relative to the value 24 h after the first injection.
PEG conjugates were prepared from three different preparations of
PBC uricase using two different techniques for PEG activation. One
preparation ( ) was tested in uricase-deficient (uox -/-) mice; the
other two (.DELTA., .box-solid.) were tested in normal BALB/c mice.
The most immunoreactive preparation (.DELTA.) was prepared from
purified PBC uricase containing an unknown quantity of uricase
aggregates coupled to an average of 7 strands of 5 kDa PEG per
subunit, using the succinimidyl carbonate derivative of PEG
(SC-PEG). Zalipsky, U.S. Pat. No. 5,612,460, hereby incorporated by
reference. The moderately immunoreactive preparation (.box-solid.)
was prepared by coupling a PBC uricase preparation containing 11%
aggregates to an average of 2 strands of 19 kDa PEG per subunit,
using a 4-nitrophenyl carbonate derivative of PEG (NPC-PEG).
Sherman, et al., (1997). The least immunoreactive conjugate ( ) was
prepared by coupling an average of 6 strands of 10 kDa NPC-PEG per
subunit to a preparation of PBC uricase containing <5%
aggregated uricase.
[0061] FIG. 8 shows the inverse relationship between the
concentrations of uric acid in the serum and urine and the activity
of injected PEG-uricase in the serum of a uricase-deficient (uox
-/-) mouse. Injections at zero time and after 72 h contained 0.43
IU of PBC uricase conjugated to an average of 6 strands of 10 kDa
PEG per enzyme subunit.
[0062] FIG. 9 shows that treatment of uricase-deficient mice with
PEG-modified PBC uricase decreased the severity of a
urine-concentrating defect. The mean and standard deviation of data
for urine osmolality are shown for two mice containing one copy of
the normal murine uricase gene (uox +/-), six untreated homozygous
uricase-deficient mice (uox -/-) and six homozygous
uricase-deficient mice that were injected ten times between the
third and 72nd day of life with either 95 or 190 mIU of
PEG-uricase. Mice of each genetic background either had received
water ad libitum (solid bars) or had been deprived of water for 12
h (hatched bars) prior to collection of their urine.
[0063] FIG. 10 shows that treatment of uricase-deficient mice with
PEG-modified PBC uricase decreased the severity of nephrogenic
diabetes insipidus, characterized by abnormally high consumption of
water and abnormally high urine output. The genetic backgrounds of
the mice and treatment protocol were the same as in FIG. 9. The
mean and standard deviation of the daily water consumption (solid
bars) and urine output (hatched bars) are shown for three groups of
six mice.
[0064] FIG. 11 shows that treatment of uricase-deficient mice with
PEG-modified PBC uricase decreased the severity of uric
acid-induced nephropathy, as visualized by magnetic resonance
microscopy. The genetic backgrounds of the three groups of mice and
the treatment protocol were the same as in FIGS. 9 and 10. Magnetic
resonance microscopy was performed at the Center for in vivo
Microscopy, Duke University Medical Center, Durham, N.C.
[0065] In addition to the results summarized in FIGS. 8-11, it was
demonstrated that the uric acid levels in the urine of all
uricase-deficient mice decreased dramatically after treatment with
PEG-modified PBC uricase. Finally, FIG. 12 shows that, unlike the
PEG-modified tetrameric form of PBC uricase, the octameric form
(molecular weight=280 kDa), even when extensively PEGylated, is
immunogenic in mice. This property is reflected in the accelerated
clearance of the PEG-modified octamer within 5 days after a single
intraperitoneal injection. The same mice were re-injected with the
same dose of the same PEG-uricase preparations on days 8 and 15.
Twenty-four hours after the second and third injections, uricolytic
activity was undetectable in the sera of mice injected with the
PEGylated octamer, but was readily detected in the sera of those
injected with the PEGylated tetramer. These findings, in
combination with the accelerated clearance of the PEGylated octamer
observed after the first injection (FIG. 12), support the utility
of removing all forms of uricase larger than the tetramer prior to
PEGylation of the enzyme.
Example 4
PEG Conjugation of Uricase from Candida utilis
[0066] Uricase from Candida utilis was obtained from either
Sigma-Aldrich (St. Louis, Mo.; catalog No. U1878) or Worthington
Biochemical Corporation (Freehold, N.J.; catalog No. URYW).
Proceeding as described in Examples 1 and 2, the tetrameric form
was isolated and PEG conjugates were synthesized with 5 kDa, 10 kDa
or 30 kDa PEG (FIGS. 1A-1B). FIG. 1A shows the retention of
activity by PEGylated uricase from Candida utilis as a function of
the number of strands of PEG coupled per subunit. Data of the
present inventors (.DELTA., , .quadrature.) are compared with those
of Nishimura, et al., (1979); Nishimura, et al., (1981); Chen, et
al., (1981); Davis, et al., (1981); Tsuji, et al., (1985); Yasuda,
et al., (1990), and Fujita, et al., (1991). Data points within
large circles denote conjugates reported to be non-antigenic by
Nishimura, et al., (1979 or 1981) or non-immunoreactive by Chen, et
al., (1981).
[0067] FIG. 1B shows the retention of activity by PEGylated uricase
from Candida utilis as a function of the total mass of PEG coupled
per subunit. Data of the present inventors (.DELTA., ,
.quadrature.) are compared with those of the same reports as in
FIG. 1A. Data points within large circles have the same meaning as
in FIG. 1A.
[0068] As shown in FIGS. 1A and 1B, conjugates with an average of
up to 6 strands of 5 kDa or 30 kDa PEG or 9 strands of 10 kDa PEG
per subunit retained at least 75% of the activity of the unmodified
enzyme. The apparent increase in specific activity as an increasing
number of strands of 30 kDa PEG is attached (up to 5 or 6 strands
per subunit) may reflect the relative insolubility or instability
of the unmodified enzyme compared to the conjugates.
Example 5
PEG Conjugation of Uricase Aspergillus flavus
[0069] Uricase from Aspergillus flavus was obtained from Sanofi
Winthrop (Gentilly Cedex, France). Proceeding as described in
Example 2, conjugates with PEGs of various molecular weights were
synthesized (FIGS. 4A-4B). Conjugates prepared by coupling the
enzyme from A. flavus with an average of up to 12 strands of 5 kDa
PEG or up to 7 strands of 30 kDa PEG per subunit retained at least
75% of the initial specific activity of this fungal uricase.
Example 6
PEG Conjugation of Soybean Uricase
[0070] Recombinant uricase from soybean root nodule (also called
nodulin 35) was prepared and purified as described by Kahn and
Tipton (Kahn, K, et al., (1997) Biochemistry 36:4731-4738), and was
provided by Dr. Tipton (University of Missouri, Columbia, Mo.).
Proceeding as described in Examples 1 and 2, the tetrameric form
was isolated and conjugates were prepared with PEGs of various
molecular weights (FIGS. 5A-5B). In contrast to uricase from
Candida utilis (FIG. 1A), porcine uricase (FIG. 2A), pig-baboon
chimeric uricase (FIG. 3A) and uricase from Aspergillus flavus
(FIG. 4A), the soybean enzyme tolerated coupling of only
approximately 2 strands of 5 kDa or 30 kDa PEG per subunit with
retention of at least 75% of the initial uricolytic activity.
Example 7
PEG Conjugation of Uricase from Arthrobacter globiformis
[0071] Uricase from Arthrobacter globiformis was obtained from
Sigma-Aldrich (catalog No. U7128). See Japanese Patent 9-154581.
Proceeding as described in Examples 1 and 2, the tetrameric form
was isolated and conjugates with 5 kDa and 30 kDa PEG were
prepared. While conjugates with an average of more than 3 strands
of 5 kDa PEG per subunit retained less than 60% of the initial
specific activity, conjugates with an average of approximately 2
strands of 30 kDa PEG per subunit retained at least 85% of the
initial specific activity.
Example 8
PEG Conjugation of Amino-Truncated Porcine and PBC Uricases
[0072] Recombinant porcine and PBC uricases from which the first
six amino acids at the amino terminal are deleted are expressed in
and purified from E. coli by standard techniques, as described in
Example 3. Proceeding as described in Examples 1 and 2, PEG
conjugates of the amino-truncated uricases are synthesized to
produce substantially non-immunogenic conjugates that retain at
least 75% of the initial specific activity.
Example 9
PEG Conjugation of Porcine and PBC Uricases Truncated at the
Carboxyl Terminal or Both the Amino and Carboxyl Terminals
[0073] Recombinant porcine and PBC uricases from which the last
three amino acids at the carboxyl terminal are deleted are
expressed in and purified from E. coli by standard techniques, as
described in Example 3. This carboxyl-terminal deletion may enhance
the solubility of the unmodified enzymes, since it removes the
peroxisomal-targeting signal. See Miura, et al., (1994). Proceeding
as described in Examples 1 and 2, PEG conjugates of the
carboxyl-truncated uricases are synthesized to produce
substantially non-immunogenic conjugates that retain at least 75%
of the initial specific activity. The sequence of recombinant PBC
uricase truncated by six residues at the amino terminal and by
three residues at the carboxyl terminal (PBC-NT-CT) is shown in
FIG. 6. This uricase is expressed, purified and PEGylated as
described in Examples 1, 2 and 3 to produce substantially
non-immunogenic conjugates that retain at least 75% of the initial
specific activity.
Example 10
PEG Conjugation of Porcine Uricase Mutants Containing an Increased
Number of PEG Attachment Sites
[0074] Recombinant porcine uricases are prepared as described in
Example 3, in which the potential number of sites of PEG attachment
is increased by replacing one or more arginine residues with
lysine. See Hershfield, M S, et al., (1991) Proc Natl Acad Sci USA
88:7185-7189. The amino acid sequence of one example of such a
mutant (PKS uricase), in which the arginine at residue 291 is
replaced by lysine and the threonine at residue 301 is replaced by
serine, is shown in FIG. 6. Proceeding as described in Examples 1
and 2, PEG is conjugated to this uricase to produce substantially
non-immunogenic conjugates that retain at least 75% of the initial
specific activity of the recombinant uricase.
Example 11
PEG Conjugation of a Recombinant Baboon Uricase Mutant
[0075] Using standard methods of molecular biology, as in Example
3, recombinant baboon uricase is constructed having an amino acid
substitution (histidine for tyrosine) at position 97 (we baboon
sequence in FIG. 6). Proceeding as described in Examples and 2, PEG
conjugates of the tetrameric form of the recombinant baboon uricase
mutant are synthesized to produce conjugates of substantially
reduced immunogenicity that retain at least 75% of the initial
specific activity of the recombinant uricase.
Example 12
Immunogenicity of PEG Conjugates from Candida utilis, Aspergillus
flavus, and Arthrobacter globiformis
[0076] Uricase from Candida utilis, Aspergillus flavus, and
Arthrobacter globiformis are obtained as described in Examples 4,
5, and 7, respectively. Proceeding as described in Examples 1 and
2, PEG conjugates are synthesized with 5 kDa, 10 kDa, 20 kDa or 30
kDa PEG. The immunogenicity of these conjugates is substantially
reduced or eliminated.
Sequence CWU 1
1
31304PRTSus scrofa 1Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp
Glu Val Glu Phe1 5 10 15Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys
Val Leu His Ile Gln 20 25 30Arg Asp Gly Lys Tyr His Ser Ile Lys Glu
Val Ala Thr Ser Val Gln 35 40 45Leu Thr Leu Ser Ser Lys Lys Asp Tyr
Leu His Gly Asp Asn Ser Asp 50 55 60Val Ile Pro Thr Asp Thr Ile Lys
Asn Thr Val Asn Val Leu Ala Lys65 70 75 80Phe Lys Gly Ile Lys Ser
Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95His Phe Leu Ser Ser
Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110Glu Glu Val
Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125His
Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135
140Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp
Leu145 150 155 160Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly
Phe Ile Lys Asp 165 170 175Gln Phe Thr Thr Leu Pro Glu Val Lys Asp
Arg Cys Phe Ala Thr Gln 180 185 190Val Tyr Cys Lys Trp Arg Tyr His
Gln Gly Arg Asp Val Asp Phe Glu 195 200 205Ala Thr Trp Asp Thr Val
Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220Pro Tyr Asp Lys
Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr225 230 235 240Asp
Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu Ile Glu Asp Met 245 250
255Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys
260 265 270Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp
Asn Pro 275 280 285Tyr Gly Arg Ile Thr Gly Thr Val Lys Arg Lys Leu
Thr Ser Arg Leu 290 295 3002304PRTPapio hamadryas 2Met Ala Asp Tyr
His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe1 5 10 15Val Arg Thr
Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln 20 25 30Arg Asp
Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45Leu
Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55
60Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys65
70 75 80Phe Lys Gly Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys
Glu 85 90 95Tyr Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val
Tyr Val 100 105 110Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly
Val Lys His Val 115 120 125His Ala Phe Ile His Thr Pro Thr Gly Thr
His Phe Cys Glu Val Glu 130 135 140Gln Leu Arg Ser Gly Pro Pro Val
Ile His Ser Gly Ile Lys Asp Leu145 150 155 160Lys Val Leu Lys Thr
Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175Gln Phe Thr
Thr Lys Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190Val
Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu 195 200
205Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly
210 215 220Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr
Leu Tyr225 230 235 240Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro
Glu Ile Glu Asp Met 245 250 255Glu Ile Ser Leu Pro Asn Ile His Tyr
Phe Asn Ile Asp Met Ser Lys 260 265 270Met Gly Leu Ile Asn Lys Glu
Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285Tyr Gly Lys Ile Thr
Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295
3003304PRTMutant combination of Sus scrofa & Papio hamadryas
3Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe1 5
10 15Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile
Gln 20 25 30Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser
Val Gln 35 40 45Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp
Asn Ser Asp 50 55 60Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn
Val Leu Ala Lys65 70 75 80Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe
Ala Val Thr Ile Cys Glu 85 90 95His Phe Leu Ser Ser Phe Lys His Val
Ile Arg Ala Gln Val Tyr Val 100 105 110Glu Glu Val Pro Trp Lys Arg
Phe Glu Lys Asn Gly Val Lys His Val 115 120 125His Ala Phe Ile Tyr
Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140Gln Ile Arg
Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu145 150 155
160Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala
Thr Gln 180 185 190Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp
Val Asp Phe Glu 195 200 205Ala Thr Trp Asp Thr Val Arg Ser Ile Val
Leu Gln Lys Phe Ala Gly 210 215 220Pro Tyr Asp Lys Gly Glu Tyr Ser
Pro Ser Val Gln Lys Thr Leu Tyr225 230 235 240Asp Ile Gln Val Leu
Thr Leu Gly Gln Val Pro Glu Ile Glu Asp Met 245 250 255Glu Ile Ser
Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys 260 265 270Met
Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280
285Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu
290 295 300
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References