U.S. patent application number 10/928370 was filed with the patent office on 2005-01-20 for aggregate-free protein compositions and methods of preparing same.
This patent application is currently assigned to Mountain View Pharmaceuticals, Inc.. Invention is credited to Saifer, Mark G.P., Sherman, Merry R., Williams, L. David.
Application Number | 20050014240 10/928370 |
Document ID | / |
Family ID | 22818799 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050014240 |
Kind Code |
A1 |
Sherman, Merry R. ; et
al. |
January 20, 2005 |
Aggregate-free protein compositions and methods of preparing
same
Abstract
A naturally occurring or recombinant protein, especially a
mutein of porcine urate oxidase (uricase), that is essentially free
of large aggregates can be rendered substantially non-immunogenic
by conjugation with a sufficiently small number of strands of
polymer such that the bioactivity of the protein is essentially
retained in the conjugate. Such conjugates are unusually well
suited for treatment of chronic conditions because they are less
likely to induce the formation of antibodies and/or accelerated
clearance than are similar conjugates prepared from protein
preparations containing traces of large aggregates.
Inventors: |
Sherman, Merry R.; (San
Carlos, CA) ; Saifer, Mark G.P.; (San Carlos, CA)
; Williams, L. David; (Fremont, CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Mountain View Pharmaceuticals,
Inc.
Menlo Park
CA
|
Family ID: |
22818799 |
Appl. No.: |
10/928370 |
Filed: |
August 30, 2004 |
Related U.S. Patent Documents
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Patent Number |
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10928370 |
Aug 30, 2004 |
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09501730 |
Feb 10, 2000 |
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6783965 |
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10928370 |
Aug 30, 2004 |
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09839946 |
Apr 19, 2001 |
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09839946 |
Apr 19, 2001 |
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09370084 |
Aug 6, 1999 |
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6576235 |
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60219318 |
Aug 6, 1998 |
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Current U.S.
Class: |
435/191 ;
435/320.1; 435/348; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61K 47/60 20170801;
C12N 9/0093 20130101; A61P 3/00 20180101; C12N 9/96 20130101; C12N
9/0048 20130101; A61P 13/12 20180101; C12Y 107/03003 20130101; A61K
38/00 20130101; C12N 9/0046 20130101; A61P 19/06 20180101 |
Class at
Publication: |
435/191 ;
435/069.1; 435/320.1; 435/348; 536/023.2 |
International
Class: |
C07H 021/04; C12N
005/06; C12N 009/06 |
Goverment Interests
[0001] A portion of the research described in this application was
made with support from the U.S.-Israel Binational Industrial
Research and Development Foundation. Accordingly, the U.S.
Government may have certain rights in the invention.
Claims
1-33. (cancelled).
34. A conjugate comprising a therapeutic protein covalently linked
to poly(ethylene glycol) or poly(ethylene oxide), wherein the
protein in said conjugate is substantially free of aggregates.
35. The conjugate of claim 34, wherein said protein is selected
from the group consisting of a naturally occurring protein, a
recombinant protein and a mutant protein that has substantially the
structure of a naturally occurring protein.
36. The conjugate of claim 34, wherein said protein is a growth
factor or a growth hormone.
37. The conjugate of claim 34, wherein said protein is an
interferon.
38. The conjugate of claim 37, wherein said interferon is
interferon-alpha.
39. The conjugate of claim 34, wherein said protein is an
Immunoglobulin G (IgG).
40. The conjugate of claim 34, wherein said poly(ethylene glycol)
is monomethoxy poly(ethylene glycol).
41. The conjugate of claim 34, wherein said protein is conjugated
to said poly(ethylene glycol) or poly(ethylene oxide) via a linkage
selected from the group consisting of a urethane (carbamate)
linkage, a secondary amine linkage and an amide linkage.
42. The conjugate of claim 34, wherein said poly(ethylene glycol)
or poly(ethylene oxide) has a molecular weight of between about 10
kDa and about 60 kDa.
43. The conjugate of claim 34, wherein the average number of
strands of said poly(ethylene glycol) or poly(ethylene oxide) that
are covalently linked to said protein is between 2 and 12.
44. The conjugate of claim 34, wherein said poly(ethylene glycol)
or poly(ethylene oxide) is linear.
45. The conjugate of claim 34, wherein said poly(ethylene glycol)
or poly(ethylene oxide) is branched.
46. A method for preparing a protein composition that is
substantially free of aggregates of said protein, comprising: (a)
preparing a solution of a protein in a suitable solvent; (b)
separating said protein solution by a separation process, whereby a
plurality of fractions of said protein is obtained; (c) assessing
the level of aggregation of said protein during or following said
separation; and (d) separating the fractions of said protein that
contain substantially aggregated protein from those fractions that
are substantially free of aggregates of said protein.
47. The method of claim 46, wherein the aggregation of said protein
in said individual fractions is assessed by measuring the light
scattering of each of said fractions.
48. The method of claim 46, wherein said separation process is
selected from the group consisting of ion-exchange chromatography,
size-exclusion chromatography and ultrafiltration.
49. The method of claim 46, wherein said protein is selected from
the group consisting of a naturally occurring protein, a
recombinant protein and a mutant protein that has substantially the
structure of a naturally occurring protein.
50. The method of claim 46, wherein said protein is a growth factor
or a growth hormone.
51. The method of claim 46, wherein said protein is an
interferon.
52. The method of claim 51, wherein said interferon is
interferon-alpha.
53. The method of claim 46, wherein said protein is an
Immunoglobulin G (IgG).
54. A method for preparing a conjugate of a therapeutic protein
having reduced immunogenicity, comprising: (a) preparing a protein
that is substantially free of aggregates of said protein according
to the method of claim 46; and (b) covalently linking said protein
to one or more poly(ethylene glycol) or poly(ethylene oxide)
molecules.
55. The method of claim 54, wherein said poly(ethylene glycol) is
monomethoxy poly(ethylene glycol).
56. The method of claim 54, wherein said protein is conjugated to
said poly(ethylene glycol) or poly(ethylene oxide) via a linkage
selected from the group consisting of a urethane (carbamate)
linkage, a secondary amine linkage and an amide linkage.
57. The method of claim 54, wherein said poly(ethylene glycol) or
poly(ethylene oxide) has a molecular weight of between about 10 kDa
and about 60 kDa.
58. The method of claim 54, wherein the average number of strands
of said poly(ethylene glycol) or poly(ethylene oxide) that are
covalently linked to said protein is between 2 and 12.
59. The method of claim 54, wherein said poly(ethylene glycol) or
poly(ethylene oxide) is linear.
60. The method of claim 54, wherein said poly(ethylene glycol) or
poly(ethylene oxide) is branched.
61. A pharmaceutical composition comprising the conjugate of claim
34 and one or more pharmaceutically acceptable excipients.
62. The pharmaceutical composition of claim 61, wherein said
composition is stabilized by lyophilization and dissolves upon
reconstitution to provide a solution suitable for parenteral
administration.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to purification and chemical
modification of proteins to prolong their circulating lifetimes and
reduce their immunogenicity. More specifically, the invention
relates to the removal of aggregates larger than octamers from
urate oxidases (uricases) prior to conjugation of poly(ethylene
glycols) or poly(ethylene oxides). This substantially eliminates
uricase immunogenicity without compromising its uricolytic
activity.
[0004] 2. Description of the Related Art
[0005] 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.
[0006] 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; Fain, A G, (1990)
Baillire'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 Med 10:711-712;
Leaustic, M, et al., (1983) Rev Rhum Mal Osteoartic 50:553-554.
[0007] U.S. patent application Ser. No. 09/370,084 and published
International Application No. PCT/US99/17514, the entire contents
of which are incorporated herein by reference, disclose poly
(ethylene glycol)-urate oxidase (PEG-uricase) that retains at least
about 75% of the uricolytic activity of unconjugated uricase and
has substantially reduced immunogenicity. In one such purified
uricase, each subunit is covalently linked to an average of 2 to 10
strands of PEG, wherein each molecule of PEG may have a molecular
weight between about 5 kDa and 100 kDa.
[0008] The aggregation of proteins is known to increase their
immunogenicity. This understanding has contributed to the
development of methods for intentionally aggregating proteins by
treatments such as thermal denaturation and cross-linking by
exposure to glutaraldehyde prior to use in the preparation of
vaccines or for immunization of animals to produce antisera.
[0009] Unintentional aggregation of proteins has also beers
recognized as contributing to immunization or sensitization during
clinical use of therapeutic proteins, e.g. for human gamma globulin
(Henney et al. (1968) N. Engl. J. Med. 278:2244-2246) and for human
growth hormone (Moore et al. (1980) J. Clin. Endocrinol. Metab.
51:691-697). The contribution of aggregates to the immunogenicity
of human interferon alpha has been demonstrated in BALB/c mice
(Braun et al. (1997) Pharm. Res. 14:1472-1478) and an enzyme-linked
immunosorbent assay (ELISA) has been developed for their
measurement (Braun et al. (1997) Pharm. Res. 14:1394-1400).
[0010] In contrast to the known effects of aggregation on the
immunogenicity of proteins, there are not reports of the effect of
aggregation on the immunogenicity of proteins conjugated to
poly(alkylene glycols) such as PEG. There is a need for
poly(alkylene glycol)-uricase conjugates that substantially
eliminates uricase immunogenicity without compromising its
uricolytic activity. The present invention provide such
compositions.
SUMMARY OF THE INVENTION
[0011] Conjugation of proteins with poly(alkylene glycols),
especially PEG, produces conjugates with reduced immunogenicity and
increased persistence in the bloodstream. In attempting to produce
substantially non-immunogenic conjugates of uricase that retain
substantially all of the uricolytic activity of the unmodified
uricase preparation, it was discovered that traces of large
aggregates of uricase in the starting material were surprisingly
effective at provoking both antibody formation and accelerated
clearance from the circulation, both of which are deleterious,
after repeated infections of PEG conjugates prepared from uricase
containing such aggregates. Surprisingly, the present inventors
found that the increased immunogenicity and accelerated clearance
were not due to the presence of well-defined, moderate-sized
aggregates of the uricase subunit that are larger than the native
tetramer, e.g. aggregates containing eight subunits (octamers). The
octameric form of uricase is present at sufficiently high
concentrations in most preparations of uricase to be delectable by
its absorbance of UV light, e.g. at 214 nm or 276 nm, or by its
contribution to the refractive index or other measurements of
protein concentration. Nevertheless, the octamers themselves were
found to contribute minimally to the immunogenicity and accelerated
clearance of PEG-uricase conjugates, in contrast with the much
smaller quantities of the much larger aggregates that are
undetectable by UV absorbance under the conditions tested but are
readily detected by static (Raleigh) or dynamic light scattering.
Therefore, the removal of such traces of very large aggregates
prior to conjugation with PEG was found to decrease the
immunogenicity and the accelerated clearance of the resultant
PEG-uricase conjugates to a surprising extent.
[0012] One embodiment of the present invention is purified urate
oxidase (uricase) substantially free of aggregates larger than
octamers. Preferably, the uricase is mammalian uricase. More
preferably, the uricase is porcine liver, bovine liver or ovine
liver uricase. In one aspect of this preferred embodiment, the
uricase is recombinant. In another aspect of this preferred
embodiment, the uricase has substantially the sequence of porcine,
bovine, ovine or baboon liver uricase. Advantageously, the uricase
is chimeric. Preferably, the uricase is PKS uricase. In another
aspect of this preferred embodiment, the uricase has substantially
the sequence of baboon liver uricase in which tyrosine 97 has been
replace by histidine. Preferably, the uricase comprises an amino
terminus and a carboxy terminus, and wherein the uricase is
truncated at one terminus or both termini. Advantageously, the
uricase is a fungal or microbial uricase. Preferably, the fungal or
microbial uricase is isolated from Aspergillus flavus, Arthrobacter
globiformis, Bacillus sp. or Candida utilis, or is a recombinant
enzyme having substantially the sequence of one of said uricases.
Alternatively, the uricase is an invertebrate uricase. Preferably,
the invertebrate uricase is isolated from Drosophila melanogaster
or Drosophila pseudoobscura, or is a recombinant enzyme having
substantially the sequence of one of said uricases. In another
aspect of this preferred embodiment, the uricase is a plant
uricase. Preferably, the plant uricase is isolated from root
nodules of Glycine max or is a recombinant enzyme having
substantially the sequence of the uricase.
[0013] In one aspect of this preferred embodiment, the uricase
described above is conjugated to poly(ethylene glycol) or
poly(ethylene oxide), under conditions such that the uricase in the
conjugate is substantially free of aggregates larger than octamers.
Preferably, the uricase is conjugated to poly(ethylene glycol) or
poly(ethylene oxide) via a urethane (carbamate), secondary amine or
amide linkage. In one aspect of this preferred embodiment, the
poly(ethylene glycol) is monomethoxy poly(ethylene glycol). In
another aspect of this preferred embodiment, the poly(ethylene
glycol) or poly(ethylene oxide) has a molecular weight between
about 5 kDa and 30 kDa. Preferably, the poly(ethylene glycol) or
poly(ethylene oxide) has a molecular weight between about 10 kDa
and 20 kDa. Advantageously, the average number of strands of said
poly(ethylene glycol) or poly(ethylene oxide) is between about 2
and 12 per uricase subunit. More advantageously, the average number
of strands of said poly(ethylene glycol) or poly(ethylene oxide) is
between about 6 and 10 per uricase subunit. Most advantageously,
the average number of strands of said poly(ethylene glycol) or
poly(ethylene oxide) is between about 7 and 9 per uricase subunit.
Preferably, the poly(ethylene glycol) or poly(ethylene oxide) is
linear. Alternatively, the poly(ethylene glycol) or polyethylene
oxide) is branched.
[0014] The present invention also provides a pharmaceutical
composition for lowering uric acid levels in a body fluid or
tissue, comprising the uricase conjugate described above and a
pharmaceutically acceptable carrier. Preferably, the composition is
stabilized by lyophilization and dissolves upon reconstitution to
provide solutions suitable for parenteral administration.
[0015] Another embodiment of the invention is a method for
purifying a uricase having reduced immunogenicity, comprising the
step of separating uricase aggregates larger than octamers in
uricase fractions, and excluding such aggregates from the purified
uricase. Preferably, the separating step comprises the step of
detecting aggregates larger than octamers from at least a portion
of the uricase fractions and excluding the fractions containing the
aggregates. Advantageously, the detecting step comprises
measurement of light scattering.
[0016] The present invention also provides isolated uricase
prepared by the method described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 illustrates uricase activity, total protein and salt
concentrations in fractions from a Pharmacia Biotech Mono Q
(1.times.10 cm) anion exchange column. Uricase activity was
measured at room temperature by monitoring the decrease in
absorbance at 292 nm of 100 EM uric acid in 200 mM sodium borate,
pH 9.2. Total protein was determined from the area under the curve
of the absorbance peak of uricase in size-exclusion HPLC analyses.
Salt concentrations were calculated from the conductivities at room
temperature using a standard curve for NaCl in the same buffer.
[0018] FIG. 2 illustrates size-exclusion HPLC analysis on a
Phamacia Superdex 200 column (1.times.30 cm) of the load and
selected fractions from a preparative Mono Q chromatography of
porcine uricase containing the mutations R29:K and T301S (PKS
uricase) showing data obtained by a light-scattering detector at
90.degree. C. (upper curves) and by absorbance at 276 nm (lower
curves). The different signal strengths of the tetrameric.
octameric and more highly aggregated forms of uricase in the
unfractionated sample (load) and the various fractions are evident.
The load was diluted 1/5 with Mono Q column buffer, fraction 5 was
diluted 1/3 and fraction 6 was diluted {fraction (1/9)}. Fractions
5 and 6 were combined to form the "low salt pool."
[0019] FIG. 3 illustrates size-exclusion analyses of fractions from
the Mono Q column in FIG. 1, showing data obtained by a
light-scattering detector at 90.degree. and by absorbance at 276
run, as in FIG. 2. The fractions shown in this figure where used to
form the "high salt pool", from which PEG conjugates were prepared
and injected into BALB/c mice. The resultant serum activities and
immunologic responses in BALB/c mice are shown in FIGS. 5 and
6.
[0020] FIG. 4 illustrates octamer content, determined by absorbance
at 276 nm and by light scattering at 90.degree., calculated from
the data in FIGS. 2 and 3, of unfractionated PKS uricase and of
selected fractions from the preparative MonoQ column chromatography
of PKS uricase (FIG. 1).
[0021] FIG. 5 illustrates UV assays, as in FIG. 1, of uricase
activity after a 4-hour incubation at 37.degree. C., in sera drawn
24 hours after each of six weekly injections of 6.times.10-kDa PEG
conjugates of PKS uricase or of pools from Mono Q column
fractions.
[0022] FIG. 6 illustrates ELISA analyses of IgG antibody formation
against PEG conjugates of PKS uricase and against PEG conjugates of
the pools of fractions from the Mono Q column shown in FIG. 1, in
sera drawn 24 hours after each of six weekly injections of female
BALB/c mice with 0.2 mg of uricase protein per 20 grams of body
weight. For each mouse, data from bleedings 24 hours after the
first through sixth injections are shown from left to right. The
assay conditions are described in Example 6. Data for the eight
mice in each group were arranged in order of increasing immune
response, from left to right.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] Previous studies have shown that when a significant
reduction in the immunogenicity and/or antigenicity of uricase is
achieved by conjugation with PEG (PEGylation), it is invariably
associated with a substantial loss of uricolyic activity. The
present invention includes the observation that traces of
aggregates of urate oxidases larger than octamers substantially
contribute to immunogenicity and the induction of accelerated
clearance of PEG-uricase conjugates. This discovery is most likely
applicable to proteins other than uricases, including interferons
and growth factors.
[0024] The safety, convenience and cost-effectiveness of
biopharmaceuticals are all adversely impacted by decreases in their
potencies and the resultant need to increase the administered dose.
Thus, there is a reed for a safe and effective alternative means
for lowering elevated levels of uric acid in body fluids, including
blood and urine. The present invention provides a method for
producing uricase that excludes uricase aggregates larger than
octamers for use in the synthesis of PEG-uricase. This PEG-uricase
retains all or nearly all of the uricolytic activity of the
unmodified enzyme. The present invention also provides purified
uricase substantially free of aggregates larger than octamers. The
term "substantially free" indicates that the purified uricase
comprises no more than about 2%, ard preferably no more than about
1% of aggregates larger than octamers.
[0025] The present invention pros ides a method for purifying
uricase such that aggregates larger then octamers are excluded from
the purified preparation Because these larger aggregates are highly
immunogenic, their presence in the purified uricase preparation is
undesirable. The method involves monitoring column fractions by
light scattering rather than or in addition to ultraviolet
absorbance at 280 nm, because the aggregates may be too dilute to
be detected by ultraviolet absorbance. The purified uricase is then
conjugated to water-soluble polymers, preferably poly(ethylene
glycols) or poly(ethylene oxides) as described in copending U.S.
application Ser. No. 09/370,084, the entire contents of which are
incorporated herein by reference
[0026] The removal of aggregated uricase from a preparation
consisting predominantly of tetrameric uricase can be accomplished
by any of the methods know to those skilled in the art, including
size-exclusion chromatography, ion-exchange chromatography,
ultrafiltration through a microporous membrane and centrifugation
including ultracentrifugation. The separation method may include
separation and analysis of fractions and the rejection or exclusion
of those fractions containing excessive quantities of large
aggregates. The resultant uricase preparation is better suited for
the synthesis of substantially non-immunogenic conjugates of
uricase than is the unfractionated uricase. For chronic
administration, it is important that PEG conjugates of proteins,
e.g. PEG-uricase, have low immunogenicity and do not provoke
progressively more rapid clearance from the bloodstream after
repeated doses.
[0027] The invention also provides pharmaceutical compositions of
the polymer-uricase conjugates. These conjugates are substantially
non-immunogenic and retain at least 75%, preferably 85 to, and more
preferably 950/o or more of the uricolytic activity of the
unmodified enzyme. Uricases suitable for conjugation to
water-soluble polymers include naturally occurring urate oxidases
isolated from bacteria, fungi and the tissues of plants and
animals, both vertebrates and invertebrates, as well as recombinant
foes of uricase, including mutated, hybrid, and/or truncated
enzymatically active variant of uricase. Water-soluble polymers
suitable for use in the present invention include linear and
branched poly(ethylene glycols) or poly(ethylene oxides), all
commonly known as PEGs. Examples of branched PEG are the subject of
U.S. Pat. No. 5,643,575. One preferred example of linear PEG is
monomethoxyPEG, of the general structure
CH.sub.3O--(CH.sub.2CH.sub.2O).s- ub.nH, where n varies from about
100 to about 2,300.
[0028] One embodiment of the present invention is a conjugate of
urate oxicase (uricase) that retains at least about 75% of the
uricolytic activity of unconjugated uricase and has substantially
reduced immunogenicity. The uricase of this aspect of the invention
may be recombinant. Whether recombinant or not, the uricase may be
of mammalian origin. In one aspect of this embodiment, the uricase
may be porcine, bovine or ovine liver uricase. In another aspect of
this embodiment, the uricase mat be chimeric. The chimeric uricase
may contain portions of porcine liver and/or baboon liver uricase.
For example, the chimeric uricase may be porcine uricase containing
the mutations R291K and T301S (PKS uricase). Alternatively the
uricase may be baboon liver uricase in which tyrosine 97 has been
replaced by histidine, whereby the specific activity of the uricase
may be increased by at least about 60%. The uricase of the
invention, whatever the origin, may also be in a form that is
truncated, either at the amino terminal, or at the carboxyl
terminal, or at both terminals. Likewise, the uricase may be fungal
or microbial uricase. In one aspect of this embodiment, the fungal
or microbial uricase may be a naturally occurring or recombinant
form of uricase from Aspergillus flavus, Arthrobacter globiforimis,
Bacillus sp. or Candida utilis. Alternatively, the uricase may be
an invertebrate uricase, such as, for example, a naturally
occurring or recombinant form of uricase from Drosophila
melanogaster or Drosophila pseudoobscura. The uricase of the
invention may also be a plant uricase, for example, a naturally
occurring or recombinant form of uricase from soybean root nodule
(Glycine max). The PEG may have an average molecular weight between
about 5 kDa and 100 kDa; preferably the PEG may have an average
molecular weight between about 8 kDa and 60 kDa; more preferably,
the PEG may have an average molecular weight between about 10 kDa
and about 40 kDa, such as, for example, 10 to 20 kDa. The average
number of covalently coupled strands of PEG may be 2 to 12 strands
per uricase subunit; preferably, the average number of covalently
coupled strands may be 6 to 10 per subunit; more preferably, the
average number of strands of PEG may be 7 to 9 per subunit. 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.
[0029] One preferred mammalian uricase is recombinant pig-baboon
chimeric uricase, composed of portions of the sequences of pig
liver and baboon liver uricase, both of which were first determined
by Wu, et al, (1989). One example of such a chimeric uricase
contains the first 288 amino acids from the porcine sequence (SEQ
ID NO: 1) and the last 16 amino acids from the baboon sequence (SEQ
ID NO: 2). Since the latter sequence differs from the porcine
sequence at only two positions, having a lysine (K) in place of
arginine at residue 291 and a serine (S), in place of threonine at
residue 301, this mutant is referred to as pig-K-S or PKS uricase
(SEQ ID NO: 3). PKS uricase has one more lysine residue and, hence,
one more potential site of PEGylation than either the porcine or
baboon sequence.
[0030] The cDNAs for various mammalian uricases, including PKS
uricase, were subcloned and the optimal conditions were determined
for expression in E. coli, using standard methods. See Erlich, H A,
(Ed.) (1989) PCR Technology. Principles and Applications for DNA
Amplification. New York: Stockton Press; Sambrook, J, et al.,
(1989) Molecular Cloning. A Laboratory Manual, Second Edition. Cold
Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. The
recombinant uricases were extracted, purified and their stability
and activity were assessed using a modification of standard assays.
See Fridovich, I, (1965) J Biol Chem 240:2491-2494; Nishimura, et
al., (1979), and Examples 1 and 5.
[0031] 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 mine linkages, and
amide linkages. Various activated PEGs suitable for such
conjugation acre available commercially from Shearwater Polymers,
Huntsville, Ala.
[0032] For example, urethane linkages to uricase may be formed by
incubating uricase in the presence of the succinimidyl carbonate
(SC) or p-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 p-nitrophenyl chloroformate
according to methods described in Veronese, FM, 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 Buttner, W, et al. East German Patent Specification DD
279 486 A1.
[0033] Amide linkages to uricase 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.
[0034] In conjugates containing PEG with a molecular weight of 10
kDa, the maximum number of strands of PEG that were coupled per
subunit, while retaining at least 75% of the uricolytic activity of
the unmodified enzyme, was about 12 strands for mammalian uricases
(e.g. PKS uricase, a mutein of porcine uricase; see assay
conditions in Example 5). The latter extent of PEGylation
corresponds to about 40% of the total amino groups. In one
embodiment of the invention, the average number of strands of PEG
coupled per uricase subunit is between about 2 and 12. In a
preferred embodiment, the average number of strands of PEG coupled
per uricase subunit is between about 6 and 10. In a more preferred
embodiment, the average number of covalently linked strands of PEG
per uricase subunit is between about 7 and 9. In another
embodiment, the molecular weight of PEG used for the coupling
reaction is between about 5 kDa and 30 kDa, preferably between
about 10 kDa and 20 kDa.
[0035] 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 uricase. 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. Likewise, each
different form of uricase may have a different optimum with respect
to both the size and number of strands. The optimal number of
strands of PEG and PEG molecular weight can be readily determined
using the methods described herein.
[0036] When PEG conjugates of mammalian uricase were prepared from
the purified tetrameric and octameric forms of the enzyme
(containing four or eight subunits of approximately 35 kDa), they
displayed profoundly reduced immunogenicity in mice, in contrast to
the moderate immunogenicity of PEG conjugates of uricase
preparations containing large aggregates (see FIG. 6) and the very
high immunogenicity of the unmodified enzyme.
[0037] Purified preparations of naturally occurring and recombinant
uricases usually contain a mixture of very large aggregates of the
enzyme, in addition to the tetrameric (140-kDa) and the octameric
(280-kDa) forms. The percentage of each uricase preparation that is
in either the tetrameric or octameric form generally varies from
about 20% to 95% (see FIGS. 2-4). Despite evidence that unPEGylated
aggregates of several other proteins are highly immunogenic (see,
e.g., Moore, V et al., (1980) J Clit, Endocrinol Metab 51:691-697),
precious studies of PEG-uricase do not describe an) 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 12 strands of PEG per
subunit while retaining more than 75% of the uricolytic activity,
at least for certain uricases, e.g., PKS uricase (a mutein of
porcine uricase) and the enzyme from thermophilic Bacillus sp.
[0038] In another preferred embodiment, substantially all large
aggregates of the enzyme may be removed by ion-exchange
chromatography (FIGS. 1-3) or size-exclusion chromatography at a pH
between about 9 and 10.5, preferably 10.2, prior to conjugation of
the resulting substantially aggregate-free preparation of uricase
to PEG. 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 aggregate-free uricase isolated using
size-exclusion chromatography, fractions containing only the
140-kDa and 280-kDa forms of the enzyme may be pooled and used for
conjugation to PEG. For tetrameric plus octameric 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 and
octameric forms without the large aggregates detected by light
scattering. In the purified product, the undesirable large
aggregates may thus constitute as little as about 1%, or less, of
the total uricase.
[0039] The results presented herein indicate that, even when
extensively PEGylated, forms of PKS uricase larger than the octamer
provoke accelerated clearance (FIG. 5) and are somewhat immunogenic
in mice (FIG. 6). In contrast, conjugates prepared from uricase
that is essentially free of large aggregates (detectable by light
scattering) could be reinjected at least six times at one-week
intervals with much less evidence of accelerated clearance rates
(FIG. 5) and without the detectable formation of antibodies, as
measured by a sensitive enzyme-linked immunoassay (FIG. 6). The use
of highly purified tetrameric or octameric uricase further
distinguishes the improved conjugates of the present invention from
the PEG-uricase preparations described previously. In contrast, the
presence of a significant content of large 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.
[0040] The PEG-uricase conjugates of the present invention are
useful for lowering the levels of uric acid in the body fluids and
tissues of mammals, preferably humans, and can thus be used for
treatment of elevated uric acid levels associated with conditions
including gout, tophi, renal insufficiency, organ transplantation
and malignant disease. PEG-uricase conjugates may be injected into
a mammal having excessive uric acid levels by any of a number of
routes, including intravenous, subcutaneous, intradermal,
intramuscular and intraperitoneal routes. Alternatively, they may
be aerosolized and inhaled. See Patton, JS, (1996) Adv Drug
Delivery Rev 19:3-36 and U.S. Pat. No. 5,458,135. The effective
dose of PEG-uricase of the present invention will depend on the
level of uric acid and the size of the individual. In one
embodiment of this aspect of the invention. PEG-uricase is
administered in a pharmaceutically acceptable excipient or diluent
in an amount ranging from about 10 .mu.g to about 1 g. In a
preferred embodiment, the amount administered is between about 100
.mu.g and 500 mg. More preferably, the conjugated uricase is
administered in an amount between 1 mg and 100 mg, such as, for
example, 5 mg, 20 mg or 50 mg. Masses given for dosage amounts of
the embodiments refer to the amount of protein in the
conjugate.
[0041] Pharmaceutical formulations containing PEG-uricase can be
prepared by conventional techniques, e.g., as described in Gennaro,
A R (Ed.) (1990) Remington's Pharmaceutical Sciences, 18th Edition,
Easton, Pa.: Mack Publishing Co. Suitable excipients for the
preparation of injectable solutions include, for example, phosphate
buffered saline, lactated Ringer's solution, water, polyols and
glycerol. Pharmaceutical compositions for parenteral injection
comprise pharmaceutically acceptable sterile aqueous or non-aqueous
liquids, dispersions, suspensions, or emulsions as well as sterile
powders for reconstitution into sterile injectable solutions or
dispersions just prior to use. These formulations may contain
additional components, such as, for example, preservatives,
solubilizers, stabilizers, % Netting agents, emulsifiers, buffers,
antioxidants and diluents.
[0042] PEG-uricase may also be provided as controlled-release
compositions for implantation into an individual to continually
control elevated uric acid levels in body fluids. For example,
polylactic acid, polyglycolic acid, regenerated collagen,
poly-L-lysine, sodium alginate, gellan gum, chitosan, agarose,
multi lamellar liposomes and many other conventional depot
formulations comprise bioerodible or biodegradable materials that
can be formulated with biologically active compositions. These
materials, when implanted or injected, gradually break down and
release the active material to the surrounding tissue. For example,
one method of encapsulating PEG-uricase comprises the method
disclosed in U.S. Pat. No. 5,653,974, which is hereby incorporated
by reference. The use of bioerodible, biodegradable and other depot
formulations is expressly contemplated in the present invention.
The use of infusion pumps and matrix entrapment systems for
delivery of PEG-uricase is also within the scope of the present
invention. PEG-uricase may also advantageously be enclosed in
micelles or liposomes. Liposome encapsulation technology is well
known in the art. See, e.g., Lasic, D, et al., (Eds.) (1995)
Stealth Lip osomes. Boca Raton, Fla.: CRC Press.
[0043] The PEG-uricase pharmaceutical compositions of the invention
will decrease the need for hemodialysis in patients at high risk of
urate-induced renal failure, e.g., organ transplant recipients (see
Venkataseshan, VS, et al., (1990) Nephron 56:317-321) and patients
with some malignant diseases. In patients with large accumulations
of crystalline urate (tophi), such pharmaceutical compositions will
improve the quality of life more rapidly than currently available
treatments.
[0044] 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 PEG (e.g., the p-nitrophenyl carbonate
derivative) to a mutein of porcine uricases. These examples provide
guidance to one of 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
and are well suited for chronic administration.
EXAMPLE 1
Preparative Ion-Exchange Chromatography of Uricase
[0045] Preparative ion-exchange chromatography was performed on a
Fast Protein Liquid Chromatography (FPLC) apparatus (Amersham
Pharmacia, Piscataway, N.J.). The Mono Q column (1.times.10 cm,
Amersham Pharmacia) was eluted with a gradient of 50 mM sodium
carbonate, pH 10.3, 0.1 M NaCl (Buffer A) to 50 mM sodium
carbonate, pH 10.3, 0.6 M NaCl (Buffer B) at a flow rate of 0.5
ml/min except that the sample was loaded at a lower flow-rate. This
technique was used to fractionate 25 mL of a solution of PKS
uricase (pH 10.3). PKS uricase was obtained from Bio-Technology
General Limited (Rehovot, Israel). The latter is recombinant
porcine uricase in which one residue of lysine (K) and one residue
of serine (S) have replaced one residue of arginine and one residue
of threonine, respectively, in the parental porcine sequence (Lee
et al. (1988) Science 239:1288-1291; Wu et al. (1989) Proc. Natl.
Acad. Sci. U.S.A. 86::9412-9416). After the sample was loaded, the
column was washed with 100 mL of Buffer A. The peak of uricase
began to elute at the end of a 31-mL linear gradient of 0 to 26%
Buffer B. Most of the uricase was eluted isocratically by 7 mL of
buffer containing 26% Buffer B. The remainder of the recovered
uricase was eluted by a linear 89-mL gradient of 26% to 100% buffer
B. Fractions of 4 mL or 6 mL were collected. Aliquots of Fractions
#4-11 were assayed for uricase, total protein and NaCl
concentration (FIG. 1) and were analyzed by size-exclusion high
performance liquid chromatography (HPLC) as described in Example 2
(FIGS. 2 and 3). The remaining portions of Fractions #5-10 were
coupled to PEG, as described in Example 3. Based on the results of
the analyses in Example 2, the PEG conjugates of Fractions #5 and 6
were combined as the "Low-Salt Pool" and the PEG conjugates of
Fractions #7-10 were combined as the "High-Salt Pool., as indicated
in FIG. 1.
EXAMPLE 2
Size-Exclusion Chromatography of Uricase Monitored by Light
Scattering and Ultraviolet Absorbance
[0046] Size-exclusion HPLC was performed at room temperature on a
Superdex 200 column (1.times.30 cm, Amersham Pharmacia Biotech) on
unfractionated PKS uricase and on selected fractions from the
preparative Mono Q chromatography of PKS uricase of Example 1. The
eluate from the absorbance monitor (UV 2000) of the Thermo
Separations HPLC (Sunnyvale, Calif.) was analyzed by light
scattering at 90.degree. to the incident light, using a MiniDawn
detector from Wyatt Technologies (Santa Barbara, Calif.).
[0047] The results shown in FIGS. 2-4 illustrate the resolution
among the tetramer, octamer and larger aggregates of the uricase
subunit and the different proportions of the signals detected from
these forms of uricase in the various samples. Unlike the
absorbance signal, which is directly proportional to the
concentration, the light scattering signal is proportional to the
product of the concentration times the size of the light scattering
unit. The resultant sensitivity of the light scattering detector to
very small amounts of highly aggregated uricase revealed the
presence of the largest aggregates, which are eluted at or near the
void volume (approximately 7 mL).
EXAMPLE 3
Synthesis of PEG-Uricase Conjugates
[0048] Unfractionated PKS uricase (from Bio-Technology General
Limited) and the uricase in fractions from the Mono Q column of
Example 1 were coupled to 10-kDa PEG using the p-nitrophenyl
carbonate derivative of PEG (NPC-PEG) obtained from Shearwater
Polymers (Huntsville, Ala.). The preparation of NPC-PEG from PEG
using phenylchloroformates has been described in several reports
(e.g. Veronese, F M. et al., (1985) Appl Biochem Biotechnol
11:141-152; Kito, M, et al., (1996) J Clin Biochem Nutr 21:101-111)
and NPC-PEG has been used for the synthesis of PEG-protein
conjugates by previous investigators including the present
inventors (e.g. Veronese et al., supra; Sherman, M R, et al., in J
M Harris, et al., (Eds.) Poly(ethylene glycol) Chemisny and
Biological Applications. ACS Symposium Series 680 (pp. 155-176)
Washington, D.C.: American Chemical Society). The number of strands
of 10-kDa PEG coupled to each subunit of uricase was determined to
be six by the method described by Kunitani, M, et al., (1991) J
Chromatogr 588:125-137.
EXAMPLE 4
In Vivo Serum Persistence and Immunogenicity of Uricase and
PEG-Uricase
[0049] PEG conjugates of recombinant mammalian uricases, prepared
according to the method of Example 3, were adjusted to 1 mg
protein/mL in phosphate-buffered saline (PBS), pH 7.4, for
injection. Samples were frozen and stored until analyzed or
injected. Samples were warmed to 37.degree. C. for up to 1 hour
prior to injection into groups of eight BALB/c female mice. The
groups of mice had mean weights in the range of 18-22 g at the
start of the studies.
[0050] The weights of all mice were monitored and evidence of
adverse reactions to the injections or other evidence of ill health
was recorded. Twenty-four hours after each of six weekly
injections, the animals were anesthetized with ketamine and 100-200
.mu.L of blood was obtained retro-orbitally, except at sacrifice
(exsanguination), when a larger volume was collected. Serum was
prepared from blood that had clotted for between 4 and 32 hours at
2-8.degree. C. Sera were stored at -20.degree. C. Sera were
analyzed for uricolytic activity as described in Example 5 and
analyzed for antibodies against uricases as described in Example
6.
EXAMPLE 5
Uricolytic Activity Assays of PEG-Uricase in Sera from Mice
Injected with PEG-Uricase
[0051] An activity assay based on ultraviolet light absorbance (UV
assay) was performed with 100 .mu.M uric acid as the substrate in
200 mM sodium borate, pH 9.2, in a microplate adaptation of the
method of I. Fridovich (J Biol Chem. (1965) 240:2491-2494). The
decrease in absorbance at 292 nm was monitored for 15 minutes at
room temperature in a 96-well plate with a UV-transparent bottom
(Costar, Corning, NY), using a SpectraMAX 250 microplate reader
from Molecular Devices (Sunnyvale, Calif.). The data were analyzed
by finding the maximum slope (in milli-absorbance units per minute)
of absorbance measurements made during the interval Awhile between
10 and 40% of the substrate was oxidized. Results obtained faith
this assay are illustrated in FIGS. 1 and 5.
[0052] The mean half-life in sera of mice injected for the first
time with PKS uricase coupled to six strands of 10-kDa PEG per
subunit (6.times.10-kDa PEG PKS) was 29.+-.4 hours, based on data
from sera obtained 24 and 72 hours after the injection.
[0053] In separate experiments, it was established that the
detectable uricolytic activity in the sera of mice injected with
PEG-uricase declines during storage at -20.degree. C. and that
maximal recovery of this activity is obtained by a 4-hour
incubation at 37' prior to assay. FIG. 5 shows that the recovery of
uricolytic activity after repeated weekly injections of
6.times.10-kDa PEG PKS uricase was greatest when the enzyme was
purified by Mono Q column chromatography, as in Example 1, prior to
PEGylation according to the method of Example 3. Recovery was
highest after the injection of conjugates prepared from the
high-salt eluate pool of Example 1 (see FIG. 1), which has the
smallest content of the very large aggregates (see the light
scattering profiles of Fractions 7-10 in FIG. 3). Intermediate
recovery was obtained with conjugates prepared from the low-salt
eluate pool from the Mono Q column of Example 1, and the poorest
recovery was obtained with conjugates made from unfractionated PKS
uricase, which has the highest content of very large aggregates
(see FIG. 2). The same order of relative activities recovered in
sera after repeated injections (high salt pool>low salt
pool>unfractionated uricase) was observed regardless of whether
the UV assay described above or a colorimetric assay adapted from
P. Fossati et al. (J. Clin Chem (1980) 26:227-231), was used and
regardless of whether the sera were incubated at 37.degree. C.
before they were assayed.
EXAMPLE 6
Enzyme-Linked Immunosorbent Assay (ELISA) of Sera from Mice
Injected with PEG-Uricase
[0054] Non-competitive ELISA analyses were performed with porcine
uricase bound to 96-well Immulon 2 plates (Dynex Technologies, from
VWR Scientific, San Francisco, Calif.). The primary antisera were
from mice injected with uricase or 6.times.10-kDa PEG conjugates
prepared according to the method of Example 3. The secondary
antibody was goat anti-mouse IgG coupled to horseradish peroxidase
(Calbiochem-Novabiochem #401 253, La Jolla, Calif.) and the
substrate was o-phenylenediamine dihydrochloride (Sigma P-9187, St.
Louis, Mo.), as described by B. Porstmann et al. (J. Clin. Chem.
Clin. Biochem. (1981)19:435-440).
[0055] FIG. 6 illustrates the results of the non-competitive ELISA
analyses. The results demonstrate that the 6.times.10-kDa a PEG PKS
uricase synthesized according to the method of Example 3 from the
high-salt eluate from the Mono Q column of Example 1 (shown in FIG.
1) did not produce detectable immune responses in any of the eight
mice that received weekly injections for six weeks. A few mice
injected with conjugates prepared from unfractionated PKS uricase
according to the method of Example 3 showed low but detectable
immune responses. The highest incidence of immune responses was in
mice injected with conjugates prepared according to the method of
Example 3 from the tow-salt eluate pool from the Mono Q column of
Example 1.
[0056] Without the benefit of the light scattering detector for the
size-exclusion HPLC analyses, as described in Example 2, it would
not have been apparent that the presence of the largest aggregates,
not of the octameric form of uricase, is associated with
progressively decreased recovery of PEG-uricase conjugates after
repeated injections, as observed in Example 5 (FIG. 5) and with an
increase in immunogenicity in BALB/c mice, as observed in Example 6
(FIG. 6). These results have important implications for the
specifications of the uricase used as a starting material for the
production of PEG-uricase for clinical use.
[0057] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit and scope of that which is described and claimed.
Sequence CWU 1
1
3 1 304 PRT Sus scrofa 1 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys
Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp
Met Ile Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His
Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser
Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val Ile
Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85
90 95 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr
Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val
Lys His Val 115 120 125 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His
Phe Cys Glu Val Glu 130 135 140 Gln Ile Arg Asn Gly Pro Pro Val Ile
His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr
Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr
Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr
Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205
Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210
215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu
Tyr 225 230 235 240 Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu
Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Leu
Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu
Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Arg Ile Thr Gly
Thr Val Lys Arg Lys Leu Thr Ser Arg Leu 290 295 300 2 304 PRT Papio
hamadryas 2 Met Ala Asp Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu
Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val
Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu
Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp
Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Ile Ile Pro Thr Asp Thr
Ile Lys Asn Thr Val His Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile
Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu 85 90 95 Tyr Phe
Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val 100 105 110
Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val 115
120 125 His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val
Glu 130 135 140 Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile
Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe
Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Lys Pro Glu Val
Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg
Tyr His Gln Cys Arg Asp Val Asp Phe Glu 195 200 205 Ala Thr Trp Gly
Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly 210 215 220 Pro Tyr
Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235
240 Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met
245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met
Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro
Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg
Lys Leu Ser Ser Arg Leu 290 295 300 3 304 PRT Chimera of Sus scrofa
and Papio hamadryas 3 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn
Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met
Ile Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser
Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser
Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val Ile Pro
Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 Phe
Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90
95 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val
100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys
His Val 115 120 125 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe
Cys Glu Val Glu 130 135 140 Gln Ile Arg Asn Gly Pro Pro Val Ile His
Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln
Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Leu
Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys
Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Ala
Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215
220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240 Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu Ile
Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn
Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val
Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr
Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300
* * * * *