U.S. patent application number 16/951764 was filed with the patent office on 2021-08-19 for uricase sequences and methods of treatment.
The applicant listed for this patent is MedImmune, LLC. Invention is credited to Manuel BACA, Andrew C. NYBORG.
Application Number | 20210254024 16/951764 |
Document ID | / |
Family ID | 1000005541209 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210254024 |
Kind Code |
A1 |
BACA; Manuel ; et
al. |
August 19, 2021 |
URICASE SEQUENCES AND METHODS OF TREATMENT
Abstract
Described are improved uricase sequences having beneficial
effects and methods of treating patients suffering from
hyperuricemia.
Inventors: |
BACA; Manuel; (Gaithersburg,
MD) ; NYBORG; Andrew C.; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune, LLC |
Gaithersburg |
MD |
US |
|
|
Family ID: |
1000005541209 |
Appl. No.: |
16/951764 |
Filed: |
November 18, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16167765 |
Oct 23, 2018 |
10883087 |
|
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16951764 |
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15573993 |
Nov 14, 2017 |
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PCT/US2016/032415 |
May 13, 2016 |
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16167765 |
|
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62162280 |
May 15, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/0048 20130101;
A61K 9/0019 20130101; A61P 13/02 20180101; A61K 38/00 20130101;
C12Y 107/03003 20130101 |
International
Class: |
C12N 9/06 20060101
C12N009/06; A61P 13/02 20060101 A61P013/02; A61K 9/00 20060101
A61K009/00 |
Claims
1-12. (canceled)
13. A method of reducing serum uric acid levels in a patient in
need thereof comprising administering a uricase, wherein the
uricase comprises at least one monomer comprising: (a) two
engineered cysteine residues; (b) two polyethylene glycol (PEG)
molecules, wherein each PEG is conjugated to each unique engineered
cysteine residue; (c) a deletion of a threonine residue at position
2, wherein the position numbering is relative to the amino acid
sequence of SEQ ID NO:27; and (d) a truncation of the C-terminus,
wherein the truncation removes an endogenous C-terminal cysteine
residue, and wherein the uricase retains enzymatic activity.
14. The method of claim 13, wherein the patient has gout or tumor
lysis syndrome.
15. The method of claim 14, wherein the patient has chronic
refractory gout and/or tophaceous gout.
16. The method of claim 13, wherein the uricase is administered
subcutaneously or intravenously.
17-25. (canceled)
26. A method of reducing serum uric acid levels in a patient in
need thereof, comprising administering a pharmaceutical composition
comprising the uricase as recited in claim 13.
27. The method of claim 26, wherein the patient has chronic
refractory gout, tophaceous gout, and/or tumor lysis syndrome.
28. The method of claim 26, wherein the uricase is administered
subcutaneously or intravenously.
29. (canceled)
30. A method of reducing uric acid levels in a patient in need
thereof comprising administering a uricase comprising SEQ ID NO:1
covalently linked to a polyethylene glycol (PEG) molecule at each
cysteine residue, wherein each PEG is maleimide-functionalized
PEG-10 with a molecular weight of about 10 kDa.
31. The method of claim 13, wherein the two unique engineered
cysteine residues are selected from the following amino acid
positions: 11C, 33C, 119C, 120C, 142C, 196C, 238C, 286C, and 289C,
wherein the position numbering is relative to the amino acid
sequence of SEQ ID NO:27.
32. The method of claim 31, wherein the two unique engineered
cysteine residues are located at amino acid positions 11C and
33C.
33. The method of claim 13, wherein the uricase does not comprise
an RGD motif.
34. The method of claim 13, wherein the uricase comprises the amino
acid sequence of SEQ ID NO:1.
35. The method of claim 13, wherein the PEG molecules each have a
molecular weight of about 10 kDa.
36. The method of claim 35, wherein the PEG is
maleimide-functionalized PEG-10.
37. The method of claim 13, wherein the uricase comprises a
homotetramer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 16/167,765, filed Oct. 23, 2018, which is a
continuation of U.S. patent application Ser. No. 15/573,993, filed
Nov. 14, 2017, which is a U.S. National Stage application of
International Application No. PCT/US2016/032415, filed May 13,
2016, which claims the benefit of U.S. Provisional Patent
Application No. 62/162,280, filed May 15, 2015, the disclosures of
which are incorporated by reference herein in their entireties,
including drawings and sequence listings.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 103,701 Byte
ASCII (Text) file named "UCASE-100WO1SequenceListing.TXT," created
on May 12, 2016.
BACKGROUND OF THE INVENTION
[0003] A functional uricase can be found in a wide range of
organisms, such as archaea, bacteria, and eukaryotes. However, in
humans and some primates uricase is not expressed. The lack of
uricase expression in humans has resulted in higher systemic uric
acid levels, and in some cases, hyperuricemia conditions such as
gout and tumor lysis syndrome.
[0004] Gout affects more than 8 million Americans and is a painful
and debilitating inflammatory arthritis defined as serum uric acid
levels exceeding uric acid solubility in body fluids. The damage
caused by gout can result in chronic pain, functional impairment at
work and at home, and compromised health-related quality of life
(see, e.g., Wertheimer, et al., Curr Ther Res Clin Exp., 75: 1-4
(2013)).
[0005] Tumor lysis syndrome (TLS) usually occurs in patients with
bulky, rapidly-proliferating, and treatment-responsive tumors. TLS
is a potentially lethal complication of anticancer treatment that
arises when large numbers of cancer cells are killed quickly and
release breakdown products that lead to a sharp increase in
systemic uric acid.
[0006] A variety of mechanisms of action exist for controlling
hyperuricemia, such as inhibitors of xanthine oxidase (enzyme that
converts xanthine to uric acid), uricosuric drugs (molecules that
inhibit URAT1), and uricase treatment.
[0007] There are two clinically approved uricases, Krystexxa.RTM.
and Elitek.RTM.. Krystexxa.RTM. (pegloticase) is a PEGylated
uricase approved for the treatment of chronic gout in adult
patients refractory to conventional therapy. Krystexxa.RTM. is a
chimeric protein of the pig and baboon uricase sequence that is
hyper-PEGylated (.about.440 kDa PEG per tetramer). Krystexxa.RTM.
is administered by an intravenous (IV) infusion over a 2 hour
period. During phase 3 clinical trials, 26% of patients experienced
infusion reactions and 6.5% of patients had reactions characterized
as anaphylaxis (Baraf et al., Arthritis Res Ther., 15(5):R137
(2013) and Strand et al., J Rheumatol., 39(7): 1450-1457 (2012).
Krystexxa.RTM. contains a black box warning for anaphylaxis and
infusion reactions (see Krystexxa.RTM. prescribing information). As
a result, patients are typically pretreated with antihistamines or
corticosteroids prior to the IV infusion and then monitored
post-infusion. Pretreatment, IV-infusion and post-infusion
monitoring takes about 6-8 hours in an IV clinic.
[0008] Elitek.RTM. (rasburicase) is a modified recombinant
Aspergillus flavus uricase that is indicated for initial management
of plasma uric acid levels in pediatric and adult patients with
leukemia, lymphoma, and solid tumor malignancies who are receiving
anti-cancer therapy expected to result in tumor lysis and
subsequent elevation of plasma uric acid. Elitek.RTM. has a
half-life of 16-21 hours in humans and must be dosed daily via IV
infusion. Similar to Krystexxa.RTM., Elitek.RTM. also has a
black-box warning for anaphylaxis and hemolysis (especially in
patients with a G6PD deficiency). Dosing frequency (daily), route
of administration (IV), immunogenicity, and cost make Elitek.RTM.
an unlikely option for chronic gout treatment.
[0009] In view of the foregoing, there is a need in the art to
develop safer, more convenient, and less immunogenic options for
treating hyperuricemia. The invention described herein fulfills
this need.
SUMMARY
[0010] To overcome the significant and known side-effects of prior
art treatments, the potential of a plurality of uricase sequences
has been evaluated and specific and meaningful improvements in
those sequences have been made to arrive at improved uricases that
are safer, less immunogenic, and more convenient than existing
therapies.
[0011] In some aspects, a number of different uricase sequences are
encompassed herein. A uricase may comprise an amino acid sequence
that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or 99% identical to any one of SEQ ID NOS: 1-34, wherein the
sequence is not any one of SEQ ID NOS: 27-33.
[0012] In some embodiments, the uricase is at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1
or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NO: 2.
[0013] In some embodiments, the uricase is a sequence that differs
from any one of SEQ ID NOS: 1-34 by from about 1 to about 35 amino
acids (e.g., by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34 or 35 amino acids). For example, the uricase may
differ from SEQ ID NO: 1 or SEQ ID NO: 2 by from about 1 to about
35 amino acids.
[0014] In some aspects, the uricase is a sequence that differs from
any one of SEQ ID NOS: 1-34 by from about 1 to about 14 amino acids
(e.g., by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14
amino acids). For example, the uricase may differ from SEQ ID NO: 1
or SEQ ID NO: 2 by from about 1 to about 14 amino acids. In certain
aspects, the uricase is SEQ ID NO: 1 or SEQ ID NO: 2. In certain
embodiments, the uricase is any one of SEQ ID NOS: 3-26 or 34.
[0015] In accordance with the description, methods of treatment are
also provided for hyperuricemia, gout (including various forms of
gout), and tumor lysis syndrome.
[0016] Additional objects and advantages will be set forth in part
in the description which follows, and in part will be obvious from
the description, or may be learned by practice. The objects and
advantages will be realized and attained by means of the elements
and combinations particularly pointed out in the appended
claims.
[0017] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the claims.
[0018] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate one (several)
embodiment(s) and together with the description, serve to explain
the principles described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 depicts an SDS-PAGE analysis of the soluble (S) and
insoluble (P) proteins present in the cell lysates of E. coli cells
expressing various uricases.
[0020] FIGS. 2A-B depict the differential scanning calorimetry
stability for Deinococcus geothermalis uricase (FIG. 2A) and
Deinococcus radiodurans uricase (FIG. 2B).
[0021] FIG. 3 is a line graph that depicts the results of uricase
activity assays at a variety of substrate (UA) concentrations. The
solid lines depict a Michaelis-Menten kinetic fit.
[0022] FIG. 4 is a line graph that depicts the adhesion of M21
cells to immobilized fibronectin, Fab9mCys, or uricase
variants.
[0023] FIG. 5 is a line graph that depicts the results of uricase
activity assays at a variety of substrate (UA) concentrations. The
solid and dashed lines depict a Michaelis-Menten kinetic fit.
[0024] FIG. 6 is a bar graph that depicts the HLA-DRB1 frequencies
in the study (donor) population as compared to those found in the
Caucasian population.
[0025] FIGS. 7A-C provide individual donor data for ex vivo
immunogenicity assessments. FIG. 7A is a scatter plot that depicts
the stimulation index of the buffer (negative control) as compared
to KLH (positive control). FIG. 7B is a scatter plot that depicts
the stimulation index of the buffer (negative control) as compared
to the uricase that was tested. FIG. 7C is a bar graph that depicts
the mean stimulation index (SI) for the buffer, KLH, and
uricase.
[0026] FIGS. 8A-B depict the analysis of various N-terminal uricase
truncations. FIG. 8A is an SDS-PAGE analysis of 3 N-terminal
truncated uricase variants (V1, V2, and V3), as compared to SGD
uricase. FIG. 8B is a line graph that depicts the results of
uricase activity assays at a variety of substrate (UA)
concentrations. The solid lines depict a Michaelis-Menten kinetic
fit.
[0027] FIGS. 9A-B are line graphs that depict the results of
uricase activity assays at a variety of substrate (UA)
concentrations. The solid lines depict a Michaelis-Menten kinetic
fit. FIG. 9A depicts the results of uricase activity assays done in
the presence of DTT for di-Cys and tri-Cys uricases (no PEG). FIG.
9B depicts the results of uricase activity assays for di-PEGylated
and tri-PEGylated uricases.
[0028] FIGS. 10A-D depict the analysis of di-pegylated uricase.
FIG. 10A shows the three dimensional solvent accessible sites
within the tetrameric crystal structure of Arthrobacter globiformis
uricase (PDB accession code: 2YZB). FIG. 10B is an SDS-PAGE
analysis of non-pegylated and di-pegylated uricase. FIG. 10C is a
reverse-phase chromatography analysis of purified di-pegylated
uricase. FIG. 10D is a line graph that depicts the results of
uricase activity assays at a variety of substrate (UA)
concentrations. The solid and dashed lines depict a
Michaelis-Menten kinetic fit.
[0029] FIGS. 11A-B are graphs that depict pharmacokinetic data for
PEGylated uricase. FIG. 11A depicts rat pharmacokinetic data for
di-PEGylated and tri-PEGylated uricase. FIG. 11B depicts dog
pharmacokinetic data for di-PEGylated uricase.
[0030] FIGS. 12A-C depict the ex vivo human serum-based analysis of
di-PEGylated uricase activity and stability. FIG. 12A show the
comparison of di-PEGylated uricase activity and Krystexxa.RTM. in
50% human serum at 37.degree. C. FIG. 12B depicts the activity of
di-PEGylated uricase that has been incubated in human serum at
37.degree. C. for various lengths of time. FIG. 12C depicts the
activity of di-PEGylated uricase in response to repeated doses of
UA.
[0031] FIG. 13 depicts RP-HPLC analysis of pegylation efficiency at
various incubation times. RP-HPLC was used to measure PEG
conjugation, which results in well-resolved peaks that correspond
to species with different degrees of conjugation.
[0032] FIGS. 14A-B depict response surface plots demonstrating the
effect of reagent concentration on overall PEGylation efficiency
for time points from 10 minutes to 2 hours. FIG. 14A illustrates
the data based on an analysis of "fully PEGylated subunit" (i.e. 3
out of 3 functionalized conjugation sites per monomer) as directly
obtained from the RP-HPLC assay trace. FIG. 14B illustrates the
data analysis when the overall derivatization is computed based on
equation (1).
DESCRIPTION OF THE SEQUENCES
[0033] Table 1 provides a listing of certain sequences referenced
herein.
TABLE-US-00001 TABLE 1 Description of the Sequences SEQ ID
Description Sequences NO Modified Arthrobacter
MATAETSTGCKVVLGQNQYGKAEVRLVKVTRCTARHEIQDLNVTSQLSGDFEAAHTAGDNAHVVA 1
globiformis Uricase,
TDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSE
modified N-terminus,
VRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVE
SGD, 2-Cys, C-terminal
VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFG
truncation (SGD V1 C2) QDNPNEVFYAADRPYGLIEATIQREGSRAD Modified
Arthrobacter
MATAETSTGCKVVLGQNQYGKAEVRLVKVTRCTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVA 2
globiformis Uricase,
TDTQKNTVYAFARDGFATTEEFLLRLGKHFTFGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSE
modified N-terminus,
VRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVE
RGD, 2-Cys, C-terminal
VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFG
truncation (RGD V1 C2) QDNPNEVFYAADRPYGLIEATIQREGSRAD Modified
Arthrobacter
MATAETSTGCKVVLGQNQYGKAEVRLVKVTRCTARHEIQDLNVTSQLXaa.sub.1Xaa.sub.2Xaa.sub.-
3FEAAHTA 3 globiformis Uricase,
GDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDH
modified N-terminus,
AFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSA
RGD variants, 2-Cys, C-
RWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHF
terminal truncation LVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRAD (RGD
variants of V1 C2) wherein Xaa.sub.1 is either R or any natural
amino acid except C; Xaa.sub.2 is either G or any natural amino
acid except C Xaa.sub.3 is either D or any natural amino acid
except C. Genus sequence, with
MXaa.sub.1ATAETSTGXaa.sub.2KVVLGQNQYGKAEVRLVKVTRXaa.sub.3TARHEIQDLNVTSQLX-
aa.sub.4GDFEA 4 optional N-terminal
AHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
modification, 4 possible
Xaa.sub.5DHDHAFSRNKSEVRTAVLEISGXaa.sub.6EQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQ-
ETT cysteines, R/SGD,
DRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEI
optionally with or
KMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADhpiwsniagf without
C-terminal wherein truncation in lowercase Xaa.sub.1 is either
present or absent, and if present is T; letters Xaa.sub.2 is either
T or C; Xaa.sub.3 is either N or C; Xaa.sub.4 is either R or S;
Xaa.sub.5 is either N or C; Xaa.sub.6 is either S or C; and wherein
from at least one, two, three, or four cysteines are included in
the sequence and wherein one or more lowercase amino acids in the
C-terminus (hpiwsniagf) are optional. Genus sequence, with
MATAETSTGXaa.sub.1KVVLGQNQYGKAEVRLVKVTRXaa.sub.2TARHEIQDLNVTSQLSGDFEAAHTA-
GDN 5 modified N-terminus,
AHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRIXaa.sub.3DHDH
with 4 possible cysteines,
AFSRNKSEVRTAVLEISGXaa.sub.4EQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATD
SGD, optionally with or
VSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNK
without C-terminal
HHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADhpiwsniagf truncation in
lowercase wherein letters Xaa.sub.1 is either T or C; Xaa.sub.2 is
either N or C; Xaa.sub.3 is either N or C; Xaa.sub.4 is either S or
C; and wherein from at least one, two, three, or four cysteines are
included in the sequence and wherein one or more lowercase amino
acids in the C-terminus (hpiwsniagf) are optional. Genus sequence,
with
MXaa.sub.1KVVLGQNQYGKAEVRLVKVTRXaa.sub.2TARHEIQDLNVTSQLSGDFEAAHTAGDNAHVVA-
TDT 6 truncated N-terminus, 4
QKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRIXaa.sub.3DHDHAFSRNKSE
possible cysteines, SGD,
VRTAVLEISGXaa.sub.4EQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYN
optionally with or
TVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQ
without C-terminal PFGQDNPNEVFYAADRPYGLIEATIQREGSRADhpiwsniagf
truncation in lowercase wherein letters Xaa.sub.1 is either T or C;
Xaa.sub.2 is either N or C; Xaa.sub.3 is either N or C; Xaa.sub.4
is either S or C; and wherein from at least one, two, three, or
four cysteines are included in the sequence and wherein one or more
lowercase amino acids in the C-terminus (hpiwsniagf) are optional.
Genus sequence, with
MXaa.sub.1ATAETSTGXaa.sub.2KVVLGQNQYGKAEVRLVKVTRXaa.sub.3TARHEIQDLNVTSQLX-
aa.sub.4GDFEA 7 optional N-terminal
AHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
modification, 9 possible
Xaa.sub.5Xaa.sub.6HDHAFSRNKSEVRTAVLEISGXaa.sub.7EQAIVAGIEGLTVLKSTGSEFHGFP-
RDKYTTLQ cysteines, R/SGD,
ETTDRILATDVSARWRYNTVXaa.sub.8VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETH
optionally with or
Xaa.sub.9EIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQRXaa.sub.10GSXaa.s-
ub.11ADhp without C-terminal iwsniagf truncation in lowercase
wherein letters Xaa.sub.1 is either present or absent, and if
present is T; Xaa.sub.2 is either T or C; Xaa.sub.3 is either N or
C; Xaa.sub.4 is either R or S; Xaa.sub.5 is either N or C;
Xaa.sub.6 is either D or C; Xaa.sub.7 is either S or C; Xaa.sub.8
is either E or C; Xaa.sub.9 is either P or C; Xaa.sub.10 is either
E or C; Xaan is either R or C; and wherein from at least one, two,
three, or four cysteines are included in the sequence and wherein
one or more lowercase amino acids in the C-terminus (hpiwsniagf)
are optional. Genus sequence, with
MXaa.sub.1ATAETSTGXaa.sub.2KVVLGQNQYGKAEVRLVKVTRXaa.sub.3TARHEIQDLNVTSQLX-
aa.sub.4GDFEA 8 optional N-terminal
AHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
modification, 9 possible
Xaa.sub.5Xaa.sub.6HDHAFSRNKSEVRTAVLEISGXaa.sub.7EQAIVAGIEGLTVLKSTGSEFHGFP-
RDKYTTLQ cysteines, XGD,
ETTDRILATDVSARWRYNTVXaa.sub.8VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETH
optionally with or
Xaa.sub.9EIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQRXaa.sub.10GSXaa.s-
ub.11ADhp without C-terminal iwsniagf truncation in lowercase
wherein letters Xaa.sub.1 is either present or absent, and if
present is T; Xaa.sub.2 is either T or C; Xaa.sub.3 is either N or
C; Xaa.sub.4 is any naturally occurring amino acid except C;
Xaa.sub.5 is either N or C; Xaa.sub.6 is either D or C; Xaa.sub.7
is either S or C; Xaa.sub.8 is either E or C; Xaa.sub.9 is either P
or C; Xaa.sub.10 is either E or C; Xaa.sub.11 is either R or C; and
wherein from at least one, two, three, or four cysteines are
included in the sequence and wherein one or more lowercase amino
acids in the C-terminus (hpiwsniagf) are optional. Genus
sequence,with N-
MXaa.sub.1KVVLGQNQYGKAEVRLVKVTRXaa.sub.2TARHEIQDLNVTSQLXaa.sub.3GDFEAAHTA-
GDNAHVVA 9 terminal truncation, 9
TDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI possible
cysteines, XGD,
Xaa.sub.4Xaa.sub.5HDHAFSRNKSEVRTAVLEISGXaa.sub.6EQAIVAGIEGLTVLKSTGSEFHGFP-
RDKYTTLQ optionally with or
ETTDRILATDVSARWRYNTVXaa.sub.7VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETH
without C-terminal
Xaa.sub.8EIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQRXaa.sub.9GSXaa.su-
b.10ADhp truncation in lowercase iwsniagf letters wherein Xaa.sub.1
is either T or C; Xaa.sub.2 is either N or C; Xaa.sub.3 is any
naturally occurring amino acid except C; Xaa.sub.4 is either N or
C; Xaa.sub.5 is either D or C; Xaa.sub.6 is either S or C;
Xaa.sub.7 is either E or C; Xaa.sub.8 is either P or C; Xaa.sub.9
is either E or C; Xaa.sub.10 is either R or C; and wherein from at
least one, two, three, or four cysteines are included in the
sequence and wherein one or more lowercase amino acids in the
C-terminus (hpiwsniagf) are optional. Genus sequence, with
MXaa.sub.1ATAETSTGXaa.sub.2KVVLGQNQYGKAEVRLVKVTRXaa.sub.3TARHEIQDLNVTSQLX-
aa.sub.4GDFEA 10 optional N-terminal
AHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
modification, 9 possible
Xaa.sub.5Xaa.sub.6HDHAFSRNKSEVRTAVLEISGXaa.sub.7EQAIVAGIEGLTVLKSTGSEFHGFP-
RDKYTTLQ conjugation sites, XGD,
ETTDRILATDVSARWRYNTVXaa.sub.8VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETH
optionally with or
Xaa.sub.9EIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQRXaa.sub.10GSXaa.s-
ub.11ADhp without C-terminal iwsniagf truncation in lowercase
wherein letters Xaa.sub.1 is either present or absent, and if
present is T; Xaa.sub.2 is either T or any natural or unnatural
amino acid used for site-specific conjugation; Xaa.sub.3 is either
N or any natural or unnatural amino acid used for site-specific
conjugation; Xaa.sub.4 is any naturally occurring amino acid except
C; Xaa.sub.5 is either N or any natural or unnatural amino acid
used for site-specific conjugation; Xaa.sub.6 is either D or any
natural or unnatural amino acid used for site-specific conjugation;
Xaa.sub.7 is either S or any natural or unnatural amino acid used
for site-specific conjugation; Xaa.sub.8 is either E or any natural
or unnatural amino acid used for site-specific conjugation;
Xaa.sub.9 is either P or any natural or unnatural amino acid used
for site-specific conjugation; Xaa.sub.10 is either E or any
natural or unnatural amino acid used for site-specific conjugation;
Xaa.sub.11 is either R or any natural or unnatural amino acid used
for site-specific conjugation; and wherein from at least one, two,
three, or four cysteines are included in the sequence and wherein
one or more lowercase amino acids in the C-terminus (hpiwsniagf)
are optional. Genus sequence, with N-
MXaa.sub.1KVVLGQNQYGKAEVRLVKVTRXaa.sub.2TARHEIQDLNVTSQLXaa.sub.3GDFEAAHTA-
GDNAHVVA 11 terminal truncation, 9
TDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI possible
conjugation
Xaa.sub.4Xaa.sub.5HDHAFSRNKSEVRTAVLEISGXaa.sub.6EQAIVAGIEGLTVLKSTGSEFHGFP-
RDKYTTLQ sites, XGD, optionally
ETTDRILATDVSARWRYNTVXaa.sub.7VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETH
with or without C-
Xaa.sub.8EIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQRXaa.sub.9GSXaa.su-
b.10ADhp terminal truncation in iwsniagf lowercase letters wherein
Xaa.sub.1 is either T or any natural or unnatural amino acid used
for site-specific conjugation; Xaa.sub.2 is either N or any natural
or unnatural amino acid used for
site-specific conjugation; Xaa.sub.3 is any naturally occurring
amino acid except C; Xaa.sub.4 is either N or any natural or
unnatural amino acid used for site-specific conjugation; Xaa.sub.5
is either D or any natural or unnatural amino acid used for
site-specific conjugation; Xaa.sub.6 is either S or any natural or
unnatural amino acid used for site-specific conjugation; Xaa.sub.7
is either E or any natural or unnatural amino acid used for
site-specific conjugation; Xaa.sub.8 is either P or any natural or
unnatural amino acid used for site-specific conjugation; Xaa.sub.9
is either E or any natural or unnatural amino acid used for
site-specific conjugation; Xaa.sub.10 is either R or any natural or
unnatural amino acid used for site-specific conjugation; and
wherein from at least one, two, three, or four cysteines are
included in the sequence and wherein one or more lowercase amino
acids in the C-terminus (hpiwsniagf) are optional. Modified
Arthrobacter
mgshhhhhhgarqTATAETSTGCKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLSGDFE
12 globiformis Uricase C1
AAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
construct (T11C
NDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILA
mutation, SGD, optional
TDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLP
N-terminal His tag and NKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRAD
optional short linker, both in lowercase (first Uricase residue
corresponds to Thr2)) Modified Arthrobacter
MATAETSTGCKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLSGDFEAAHTAGDNAHVVA
13 globiformis Uricase C1
TDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSE
construct (variant 1), with
VRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVE
tag eliminated, deletion of
VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFG
Thr2 (to avoid partial N- QDNPNEVFYAADRPYGLIEATIQREGSRAD term Met
cleavage) and Cys at position 11 (in another embodiment, SGD may be
RGD) Modified Arthrobacter
METSTGCKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLSGDFEAAHTAGDNAHVVATDT
14 globiformis Uricase C1
QKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRT
construct (variant 2 - N-
AVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDF
term truncation) with tag
DAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDN
eliminated, deletion of PNEVFYAADRPYGLIEATIQREGSRAD Thr2-Ala5 and
Cys at position 11, expect complete retention of N- term Met (in
another embodiment, SGD may be RGD) Modified Arthrobacter
MGCKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLSGDFEAAHTAGDNAHVVATDTQKNT
15 globiformis Uricase C1
VYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLE
construct (variant 3 - N-
ISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVY
term truncation) with tag
ASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEV
eliminated, deletion of FYAADRPYGLIEATIQREGSRAD Thr2-Thr9, Cys at
position 11, expect processing of N-term met (in another
embodiment, SGD may be RGD) Modified Arthrobacter
mgshhhhhhgarqTATAETSTGCKVVLGQNQYGKAEVRLVKVTRCTARHEIQDLNVTSQLSGDFE
16 globiformis Uricase with
AAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
SGD, and PEGylation
NDHDHAFSRNKSEVRTAVLEISGCEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILA
available sites at T11C,
TDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLP
N33C, S142C, optional NKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRAD
N-terminal His tag and optional short linker, both in lowercase (in
another embodiment an additional PEGylation site may optionally be
placed at N119C (not shown here and/or SGD may be RGD) Modified
Arthrobacter
mgshhhhhhgarqTATAETSTGCKVVLGQNQYGKAEVRLVKVTRCTARHEIQDLNVTSQLSGDFE
17 globiformis Uricase, NH2-
AAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
terminal truncated, SGD,
NDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILA
PEGylation available
TDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLP
sites at T11C and N33C NKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRAD
2-Cys (SGD His C2) with optional N-terminal His tag and optional
short linker, both in lowercase (in another embodiment, SGD may be
RGD) Modified Arthrobacter
MATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLSGDFEAAHTAGDNAHVVA
18 globiformis Uricase (C-term
TDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSE
truncation with SGD)(in
VRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVE
another embodiment,
VDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFG
SGD may be RGD) QDNPNEVFYAADRPYGLIEATIQREGSRAD Modified
Arthrobacter
ATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLSGDFEAAHTAGDNAHVVAT
19 globiformis Uricase
DTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEV
(processed form - Met
RTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEV
cleaved at N-term., SGD,
DFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQ
and C-term truncation) DNPNEVFYAADRPYGLIEATIQREGSRAD (in another
embodiment, SGD may be RGD) Modified Arthrobacter
mgshhhhhhgarqTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLSGDFE
20 globiformis Uricase
AAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
(contains optional N-
NDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILA
terminal His tag and
TDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLP
optional short linker, NKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRAD
both in lowercase; contains SGD instead of RGD)(C-term truncation
with his tag and SGD) Modified Arthrobacter
mgshhhhhhgarqTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFE
21 globiformis Uricase
AAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRI
(contains optional N-
NDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILA
terminal His tag and
TDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLP
optional short linker, NKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRAD
both in lowercase)(C- term truncation with his tag) Modified
Arthrobacter
MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV
22 globiformis Uricase (0
ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS
cysteines)(truncated the
EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTV
C-terminal 11 amino
EVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPF
acids to eliminate the GQDNPNEVFYAADRPYGLIEATIQREGSRAD Cys)(C-term
truncation) (in another embodiment, RGD may be SGD) Modified
Arthrobacter
MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLXaa.sub.1Xaa.sub.2Xaa.sub-
.3FEAAHT 23 globiformis Uricase (0
AGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHD
cysteines)(RGD variants,
HAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVS
truncated the C-terminal
ARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHH
11 amino acids to FLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRAD eliminate
the Cys)(C- wherein term truncation)(in Xaa.sub.1 is either R or
any natural amino acid except C; another embodiment, Xaa.sub.2 is
either G or any natural amino acid except C RGD may be SGD)
Xaa.sub.3 is either D or any natural amino acid except C. Modified
Arthrobacter
MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV
24 globiformis Uricase (0
ATDTQKNTVYAFARDGFATTEEFLLRLGKHETEGEDWVTGGRWAAQQFFWDRINDHDHAFSRNKS
cysteines)(truncated the
EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTV
C-terminal aa to eliminate
EVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPF
the cysteine) GQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGF Modified
Arthrobacter
MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLXaa.sub.1Xaa.sub.2Xaa.sub-
.3FEAAHT 25 globiformis Uricase (0
AGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHD
cysteines)(RGD variants,
HAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVS
truncated the C-terminal
ARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHH
aa to eliminate the
FLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGF cysteine) wherein
Xaa.sub.1 is either R or any natural amino acid except C; Xaa.sub.2
is either G or any natural amino acid except C Xaa.sub.3 is either
D or any natural amino acid except C. Modified Arthrobacter
MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLXaa.sub.1Xaa.sub.2Xaa.sub-
.3FEAAHT 26 globiformis Uricase (RGD
AGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHD
variants)
HAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDV- S
(contains the C-terminal
ARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHH
11 amino acids) FLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC
wherein Xaa.sub.1 is either R or any natural amino acid; Xaa.sub.2
is either G or any natural amino acid Xaa.sub.3 is either D or any
natural amino acid. Arthrobacter globiformis
MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV
27 Uricase (wt)
ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS
(contains the C-terminal
EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTV
11 amino acids)(in
EVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPF
another embodiment, GQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC RGD
may be SGD) Deinococcus geothermalis
MTQTQQNQQPKVKARLGANNYGKAEVNLMKVKRDSERHEIRELQVRVALIGDFAAAHEQGDNTDL
28 Uricase 1 cysteine, gram-
LATDTVRNTIYGLAKEGFQASPEAFGKELISHFVTTGPKVTGGFMEFTEYLWERIQVGGEGHNHA
positive, thermophilic
FVRQMPQRTGRVESEDGKTFKITSGLQNLYVLKTTESGWANYLLNERFTTLPETHERLMASFVTA
radiophile
KWEYNEDQVDYDDVWPRVYRQLQETFTDHYSPSLQRTLFLMGQAVLTRCPEMSRIWLQMPNKH- HL
QYNLERFGLDNNLEIFHVDPEPYGLMEAWVERA Deinococcus radiodurans
MMTGTQQPGTQPKVKVRLGENNYGKAEVQLMKIKRGTPRHELREAKVRVAMYGDFGAAHSEGDNT
29 Uricase (2 cysteines), is
DLVATDTVRNTVYGLAKEGFESSIEEFGKELLTHFVKVGPRVTGGFAEFTEHLWERVQTPAQPQG
an extremophilic
HDHAFVRQMPKRTARVETQDGRRFTVTSGIEELYVLKTTESGWENYLLDERFTTLPETHDRVMAT
bacterium FVTAKWEYAVESCDYDAVWERVYRQIQHTFTDHYSPSLQRTLYLM
GEAVLSVCPEISRIWFQMPNKHHLVYNLGRFGLENNNEILHVDPEPYGLMEAWVERAE
Granulicella tundricola
MAELTDAKFEIVANRYGKSKVRLLKVTRAEGRSDVHEWTVQVLLRGDFETAHTVGDNSKIVTTDT
30 Uricase (1 cysteine)
MKNTVYSLARWSSATTMEEFAEELIEHLLRRNEQVSSVRVHIEAALWKRLTVDGKEHPDTFMRGS
NEVQTATVEQARAGEKKFIAGFANLQLLKTANSAFSGFQRDELTTLPETRDRVFGTAVDAKWTYS
GPVEFAAMRKAAREVMLKVFADHMSESVQHTLYAMADAALEAVAEITEIELAMPNKHCLLVDLSK
FGQDNPNQIFVPTDEPHGYIEARVRRK Acidic Bacteria Solibacter
MERFASGWKQNYYGKGDVIVYRLNRDGVVPQGCCPVFGANVKMLLYGDAFWPTYTTGDNTNLVAT
31 usitatus Uricase (6
DSMKNFIQRETCNFTGYDLESYCDFLARKFMATYPHTAGIQLSARQAPYSGVAEGKVAFAPSGPD
cysteines)
VATACVELRRNGEALESVEASSGIHGFRLLRLGGSAFQGFLRDQYTTLPDIHNRPLHMWLDLE- WH
YIAPEAALTGGEVTAQVRRLVHEGFHSFESGSIQQVIYQLGTKMLADIPTISEVRLEANNRTWDT
IVEQGDRLGVYTDARPPYGCLGLTLRR Terriglobus saanensis
MAKLIDSRYGKARVRVMKLDRSQPQHQLLEWTVRVLLEGDFETAHTVGDNSNILPTDTMKNTVYS
32 Acidobacterium Uricase
RAKESKAETPEEFAIELAEFLLGRNPQVHTVEVKIETAMWKRLVVDGKPHGSSFMRGSDELGTVL
(only 2 cysteines and
HHATRETKTMVCGVENMVILKSQNSSFEGYIQDDLTTLKPTADRLFATAMTADWDYTDGGSAFAA
short 280 aa)
RREAIREAMLKAFAEHDSKSVQQTLYAMAEAAMAAVPAVNRVHMVMPNKHCLLVDLKHFGQENNN
EIFVPTEDPHGYIEATVVRE Kyrpidia tusciae Uricase
MIMTGTMTSGTDQRTMYYGKGDVWVYRSYAKPLRGLGQIPESAFAGRPNVIFGMNVQMAVEGEAF
33
LPSFTEGDNSMVVATDSMKNFILRQAGAFEGATAEGFLEFVAGKFLEKYAHVSGVRLFGRQIPFD
ELPVPEQEGFRPGELVFRYSMNEYPTAFVAVRRGPEGPVVVEHAGGVAGLKLIKIKGSSFYGYIH
DEYTTLPEAQDRPLFIYLYIKWKYEHPEDFRAEHPERYVAAEQVRDIAHTVFHELTSPSIQNLIY
HIGRRVLTRFPQLLEVSFEANNRTWETVLEEVEDLAGKRAEAKVYTEPRPPYGFQGFTVTRKDLE E
Consensus Uricase
MTATAETSTGTKIVLGQNQYGKAEVRVVKITRDGDTHHIKDLNVSVALSGDMDAVHLSGDNANVL
34 sequence from alignment
PTDTQKNTVYAFAKEHGIGSAEQFGIRLARHFVTSQEPIHGARIRIEEYAWERIETSHDHSFVRK
GQETRTAQITYDGDWEVVSGLKDLTVLNSTGSEFWGYVKDKYTTLPETYDRILATDVSARWRYNW
TDDQPMPDWDKSYEQVRKHLLEAFAETYSLSLQQTLYQMGSRVLEARPEIDEIRFSLPNKHHFLV
DLEPFGLDNDNEVYFAADRPYGLIEATVLRDGAEPRIPVDMTNL
DETAILED DESCRIPTION OF THE INVENTION
[0034] Urate oxidase (Uricase EC 1.7.3.3, uox) is a homotetrameric
enzyme composed of four identical 34 KDa subunits. The enzyme is
responsible for the initial step that begins a series of reactions
that convert uric acid to a more soluble and easily excreted
product, allantoin. In short, uricase catalyzes the reaction of
uric acid (UA) with 02 and H.sub.2O to form 5-hydroxy-isourate
(HIU) and the release of H.sub.2O.sub.2. HIU is an unstable product
that undergoes non-enzymatic hydrolysis to
2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and then
decarboxylates spontaneously to form racemic allantoin. In species
that contains a functional uricase, two additional enzymes are
expressed (HIU hydrolase and OHCU decarboxylase) which catalyze
these reaction more quickly to generate (s)-allantoin. A functional
uricase can be found in a wide range of organisms: archaea,
bacteria, and eukaryotes. However, in humans and some primates
uricase is not expressed. The lack of uricase expression is
attributed to three genetic mutations: a nonsense mutation at codon
33 (impacting orangutans, gorillas, chimpanzees, and humans),
another nonsense mutation at codon 187 (impacting chimpanzees and
humans) and a mutation at the splice acceptor site in intron 2
(impacting chimpanzees and humans). A number of hypotheses have
been proposed to explain the evolutionary elimination of uricase
activity and commensurate increase in UA levels. These include the
idea that an increase in UA levels (powerful antioxidant and
scavenger of oxygen radical) led to a decrease in oxygen free
radical associated disease (cancer) and an increase in lifespan.
Additionally, the fact that UA structurally resembles neuro
stimulants such as caffeine and theobromine has led to the
speculation that increased UA levels may have led to an
intellectual/cognitive jump. Lastly it has been suggested that an
increase in uric acid led to and helped maintain blood pressure
levels required by hominids while consuming a very low salt
vegetarian diet (1-2 million years ago). Regardless of the
evolutionary advantage that may have resulted, the lack of uricase
expression in humans has resulted in higher systemic UA levels and
in some cases hyperuricemia conditions such as gout and tumor lysis
syndrome.
[0035] Gout affects more than 8 million Americans and is a painful
and debilitating inflammatory arthritis defined as serum UA levels
exceeding UA solubility in body fluids. Serum UA levels higher than
6.8 mg/dL can result in UA crystal formation in tissues, provoking
an acute inflammatory response. Acute gouty arthritic attacks
(flares) and chronic inflammation that deposits UA crystals in
fibrous tissues are painful and debilitating. The damage caused by
gout can result in chronic pain, functional impairment at work and
at home, and compromised health-related quality of life
(Wertheimer, et al., supra).
[0036] Tumor lysis syndrome (TLS) usually occurs in patients with
bulky, rapidly-proliferating, and treatment-responsive tumors. TLS
is a potentially lethal complication of anticancer treatment that
arises when large numbers of cancer cells are killed quickly and
release breakdown products. Nucleic acid purines are metabolized to
UA leading to a sharp increase in systemic UA. In severe cases, UA
crystals form in the renal tubules causing UA nephropathy (acute
renal failure). TLS has been reported across a broad range of tumor
types (Ikeda, et al., Drugs, Diseases & Procedures, Medscape
(Dec. 3, 2014)).
[0037] A variety of mechanisms of action exist for controlling
hyperuricemia. Inhibitors of xanthine oxidase (enzyme that converts
xanthine to UA) have been clinically prescribed since the 1960s.
The most common of these, Allopurinol, is used by more the 2
million gout patients in the US. However, many patients continue to
have higher than acceptable UA levels suggesting that hyperuricemia
is not just a UA production problem. More recent studies have shown
that UA levels in patients can also be controlled by inhibiting
URAT1, an enzyme responsible for UA recycling. Uricosuric drugs
(molecules that inhibit URAT1) act on the proximal tubules in the
kidneys, where they interfere with the absorption of UA from the
kidney back into the blood. Uricosuric drugs, such as Benzbromarone
and Lesinurad, promote excretion of UA. Lastly, it has been shown
that uricase treatment rapidly reduces UA levels in the peripheral
blood stream by oxidizing UA to a more soluble product, allantoin.
There are two clinically approved uricases, Krystexxa.RTM. and
Elitek.RTM..
[0038] Krystexxa.RTM. (pegloticase) is a PEGylated uricase approved
for the treatment of chronic gout in adult patients refractory to
conventional therapy. Krystexxa.RTM. is a chimeric protein of the
pig and baboon uricase sequence that is hyper-PEGylated (.about.440
kDa PEG per tetramer). Krystexxa.RTM. is administered by an
intravenous (IV) infusion over a 2 hour period. During phase 3
clinical trials, 26% of patients experienced infusion reactions and
6.5% of patients had reactions characterized as anaphylaxis (Baraf
et al., Arthritis Res Ther., 15(5):R137 (2013) and Strand et al., J
Rheumatol., 39(7): 1450-1457 (2012). Krystexxa.RTM. contains a
black box warning for anaphylaxis and infusion reactions (see
Krystexxa.RTM. prescribing information). As a result, patients are
typically pretreated with antihistamines or corticosteroids prior
to the IV infusion and then monitored post-infusion. Pretreatment,
IV-infusion and post-infusion monitoring takes about 6-8 hours in
an IV clinic. Treatment frequency is once every two weeks. In phase
3 clinical trials, a high percentage of patients developed
anti-drug antibodies (.about.92%) and approximately 40% of patients
experienced a positive primary endpoint (reduction in UA levels
below 6 mg/dl for 6 months). In spite of the infusion reactions,
anti-drug response, and inconvenient dosing schedule, dramatic
results have been observed in clinical trials and case studies
demonstrating the reduction or resolution of tophi (uric acid
crystal deposits). Digital photos of patients with tophaceous gout
(hands or feet) before and after multiple Krystexxa.RTM. treatments
have demonstrated the potential for a uricase in resolving tophi
and UA burden.
[0039] Elitek.RTM. (rasburicase) is a modified recombinant
Aspergillus flavus uricase that is indicated for initial management
of plasma uric acid levels in pediatric and adult patients with
leukemia, lymphoma, and solid tumor malignancies who are receiving
anti-cancer therapy expected to result in tumor lysis and
subsequent elevation of plasma uric acid. Elitek.RTM. has a
half-life of 16-21 hours in humans and must be dosed daily via IV
infusion. Similar to Krystexxa.RTM., Elitek.RTM. also has a
black-box warning for anaphylaxis and hemolysis (especially in
patients with a G6PD deficiency). Dosing frequency (daily), route
of administration (IV), immunogenicity, and cost make Elitek.RTM.
an unlikely option for chronic gout treatment.
[0040] In view of the foregoing, there is a need in the art to
develop improved uricases that are safer, more convenient, and less
immunogenic than the uricases that are currently available. The
invention described herein fulfills this need.
I. Improved Uricase Sequences
[0041] In some aspects, a number of different uricase sequences are
encompassed herein. The uricase described herein may comprise an
amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS:
1-34, wherein the sequence is not any one of SEQ ID NOS: 27-33. In
one embodiment, the uricase has the amino acid sequence of any one
of SEQ ID NOS: 1-26 or 34. In another embodiment, the uricase
comprises an amino acid sequence that is at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of
SEQ ID NOS: 1-34, wherein the uricase sequence is not a naturally
occurring uricase sequence.
[0042] In some embodiments, the uricase is at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1
or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NO: 2.
[0043] In some embodiments, the uricase is a sequence that differs
from any one of SEQ ID NOS: 1-34 by from about 1 to about 35 amino
acids (e.g., by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34 or 35 amino acids). For example, the uricase may
differ from SEQ ID NO: 1 or SEQ ID NO: 2 by from about 1 to about
35 amino acids.
[0044] In some aspects, the uricase is a sequence that differs from
any one of SEQ ID NOS: 1-34 by from about 1 to about 14 amino acids
(e.g., by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14
amino acids). For example, the uricase may differ from SEQ ID NO: 1
or SEQ ID NO: 2 by from about 1 to about 14 amino acids. In certain
aspects, the uricase is SEQ ID NO 1 or SEQ ID NO: 2. In certain
embodiments, the uricase is any one of SEQ ID NOs: 3-26 or 34. The
uricase may "differ from" any one of SEQ ID NOs: 1-34 by comprising
an addition, deletion, or substitution in the amino acid sequence.
Methods for preparing amino acid additions, deletions, and
substitutions are well known in the art.
[0045] In some embodiments, the uricase comprises a truncation at
the N- and/or C-terminus, wherein the truncated uricase retains
enzymatic activity. In one embodiment, from about 1-15 (e.g., about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids
are truncated from the N-terminus. In another embodiment, from
about 1-20 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20) amino acids are truncated from the
C-terminus. In yet another embodiment, from about 1-15 (e.g., about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids
are truncated from the N-terminus and from about 1-20 (e.g., about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20) amino acids are truncated from the C-terminus. In one
embodiment, the uricase is a uricase of any one of SEQ ID NOs:
27-34, wherein the uricase comprises a truncation at the N- and/or
C-terminus, as described above, wherein the truncated uricase
retains enzymatic activity. In a further embodiment, the
aforementioned truncated uricase contains from about 1 to about 14
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14)
additional amino acid changes (e.g., additions, deletions, or
substitutions). Methods for assaying enzymatic activity of a
uricase are known in the art (e.g., product formation and substrate
depletion assays), and any suitable method known in the art can be
used to measure the enzymatic activity of the uricases described
herein.
[0046] In some embodiments, the uricase is at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs:
27-33. In some aspects, the uricase differs from any one of SEQ ID
NOs: 27-33 by from about 1 to about 35 amino acids (e.g., by about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35
amino acids).
[0047] In one embodiment, the uricase is at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to NCBI
Accession Number D0VWQ1, WP_011525965, WP_010887803,
WP_013581210.1, WP_011682147, WP_013569963, or ADG06709. In some
aspects, the uricase differs from any one of NCBI Accession Number
D0VWQ1, WP_011525965, WP_010887803, WP_013581210.1, WP_011682147,
WP_013569963, or ADG06709 by from about 1 to about 35 amino acids
(e.g., by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34 or 35 amino acids).
[0048] In some embodiments, the uricase is about 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27. In
some aspects, the uricase differs from SEQ ID NO: 27 by from about
1 to about 35 amino acids (e.g., by about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids).
[0049] In some embodiments, the uricase is at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
28. In some aspects, the uricase differs from SEQ ID NO: 28 by from
about 1 to about 35 amino acids (e.g., by about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids).
[0050] It is well understood in the art that processing of
expressed proteins can result in the cleavage of the N-terminal
methionine residue, a co-translational modification that can occur
in both prokaryotic and eukaryotic hosts (Sherman, et al.,
Bioessays, 3: 27-31 (1985)). This processing, which is
enzymatically effected by methionine aminopeptidase, is dependent
upon the identity of the amino acid residue adjacent to the amino
terminus. Methionine is efficiently removed from proteins when the
second residue is glycine or an amino acid with a small side chain
such as alanine (Hirel et al., Proc. Natl. Acad. Sci. U.S.A., 86:
8247-8251 (1989) and Huang et al., Biochemistry, 26: 8242-8246
(1987)). However, N-terminal methionine is not cleaved when an
amino acid with a large side chain is the adjacent residue.
Variable degrees of cleavage may occur when the second residue is
an intermediate sized amino acid such as threonine or asparagine
(Hirel et al., supra). Thus, in some embodiments, the methionine at
position 1 of the uricase is cleaved, such that the processed form
of the uricase does not contain a methionine at position 1. In
another embodiment, the uricase retains the methionine at position
1. In order to prevent cleavage of the methionine at position 1,
the uricase may comprise one or more amino acid substitutions or
deletions following the N-terminal methionine. Such substitutions
or deletions would be designed to result in a large amino acid
being at the second position within a sequence. Examples of large
amino acids are glutamine, glutamic acid, phenylalanine,
methionine, lysine, tyrosine, tryptophan and arginine. For example,
in some aspects, the uricase may not comprise a threonine at
position 2 and position 2 is either deleted or substituted, wherein
the numbering is relative to SEQ ID NO: 27. In some embodiments,
the uricase sequence has been modified to comprise an alanine or
other small amino acid at position 2 (i.e., the amino acid next to
the N-terminal methionine). Examples of small amino acids are
glycine, alanine, serine, proline, threonine, valine and cysteine,
but preference is given to the smallest of these (glycine and
alanine) to limit the possibility of partial processing (Hirel et
al., supra).
[0051] In some embodiments, the uricase sequence is conjugated or
recombinantly fused to a synthetic or biosynthetic polymer in order
to extend the half-life of the protein and/or to mitigate
immunogenicity. Exemplary polymers that may be used in the
invention are polyethylene glycol (PEG), polymers of
phosphorylcholine (see, e.g., US Patent Application Publication
2013/0034517), polymers of repeating peptides such as "PAS" or
"X-TEN" sequences (see, e.g., Schlapschy et al., Protein Eng. Des.
Sel. 26: 489-501 (2013), Schellenberger et al., Nat. Biotechnol.,
27: 1186-1190 (2009), and Podust et al., Protein Eng. Des. Sel.,
26: 743-753 (2013)), or carbohydrate-based polymers such as
heparosan (see, e.g., International Patent Application Publication
WO 2014/060397) or hydroxyethyl starch (see, e.g., EP 2270036). In
other embodiments, the uricase sequence may be recombinantly fused
to polypeptides that prolong the circulation half-life by reducing
the rate of renal clearance. Such fusion partners are well
understood in the art, and include agents that directly bind the
neonatal Fc receptor (FcRn) in a pH dependent manner (e.g., Fc
region of immunoglobulins or serum albumin), or alternatively bind
to a naturally-occurring FcRn-binding moiety (e.g., polypeptides
that bind to serum albumin). In another embodiment, the uricase
sequence may be conjugated or recombinantly fused to one or more
repeats of a C-terminal peptide fragment derived from the beta
subunit of human chorionic gonadotropin (see, e.g., U.S. Pat. No.
6,225,449).
[0052] In some embodiments, a synthetic or biosynthetic polymer is
conjugated to the N- and/or C-terminus of the uricase in order to
extend the half-life of the protein and/or to mitigate
immunogenicity.
[0053] In some embodiments, the uricase sequence is modified to
create 1-6 (e.g., 1, 2, 3, 4, 5, or 6) surface accessible sites for
conjugation. For example, in some embodiments, the uricase sequence
is modified to contain 1, 2, 3, 4, 5, or 6 surface accessible
cysteine residues to which a polymer (e.g., PEG) may be conjugated.
In some embodiments, a naturally-occurring uricase sequence that
does not contain any cysteines or contains only a few cysteines
provides a beneficial starting sequence, so that cysteines can be
inserted into the appropriate, surface-accessible locations. In
other embodiments, the uricase sequence is modified to contain 1,
2, 3, 4, 5, or 6 surface accessible non-naturally occurring amino
acids to which a polymer may be conjugated.
[0054] In some embodiments, a naturally-occurring uricase sequence
is modified to mutate some or all of the existing cysteines
(through deletion and/or substitution) with alternative amino
acids. In some embodiments, new cysteines are introduced at desired
locations (through addition and/or substitution), to enable
site-specific conjugation of polymers or polypeptides that can
modify pharmacokinetic behavior. In one embodiment, selection of an
appropriate amino acid for Cys-substitution is guided by alignment
of the uricase of interest to other uricase sequences in order to
determine the natural amino acid diversity at the equivalent
position across all uricases. A non-cysteine amino acid is then
selected based on its prevalence at the position of interest within
other uricases. In another embodiment, selection of an appropriate
amino acid for Cys-substitution is guided by analysis of the
crystal structure for that particular uricase in order to determine
which amino acid residues are surface accessible. One or more
surface accessible amino acids are then selected and modified to
cysteine. In some instances, the cysteines in the final modified
uricase sequence are located in surface accessible positions so
that the cysteines may be PEGylated.
[0055] In some embodiments, the uricase comprises from about 1 to
about 6 cysteines, specifically about 1, 2, 3, 4, 5, or 6
cysteines. In one embodiment, the uricase comprises about 2
cysteines.
[0056] In certain embodiments, the uricase comprises a PEG moiety
attached to the cysteine residue(s). Control over the number and
placement of cysteine residues allows for control over the number
of PEG attachment sites, and optimal properties of the resultant
conjugate including biophysical attributes and enzymatic
activity.
[0057] Polyethylene glycol (PEG) is a polyether compound with the
structure H--(O--CH.sub.2--CH.sub.2).sub.n--OH. The PEG reagents
most typically used for protein conjugation are monomethoxy
poly(ethylene glycol) derivatives, having the structure
CH.sub.3--O--(CH.sub.2--CH.sub.2--O).sub.n--X, wherein X contains a
linear linker and reactive functional group (linear PEG). In some
cases, X may contain a branching element, such that the PEG reagent
contains one reactive functional group and more than one PEG
polymer chain (branched PEG) or more than one reactive functional
group and PEG polymer chains (forked PEG). PEG reagents may include
about 5, 10, 20, 40, 60 and 80 kDa of total PEG polymer.
[0058] In some embodiments, thiol-reactive PEGs may be used to
react with the thiol group on at least one cysteine. For example,
PEG-maleimide may be used, as well as PEG-orthopyridyl-disulphide,
PEG-vinylsulphone, and PEG-iodoacetamide. In other embodiments,
thiol-reactive PEGs may have a linear or branched structure with a
single thiol-reactive moiety, or may have a forked structure with
two or more reactive groups per PEG molecule.
[0059] A variety of approaches, thus, are known in the art and any
suitable method known in the art may be used to PEGylate the
cysteine(s) in the uricase.
[0060] In some embodiments, the uricase comprises a cysteine in at
least one of the following positions: 11C, 33C, 119C, and 142C,
wherein the position numbering is relative to SEQ ID NO: 27.
[0061] In one embodiment, the uricase comprises a cysteine in at
least one of the following positions: 11C, 33C, 119C, 120C, 142C,
196C, 238C, 286C, and 289C wherein the position numbering is
relative to SEQ ID NO: 27.
[0062] As a major family of cell adhesion receptors, integrins are
known play a key role in cell-cell and cell-extracellular matrix
interactions. The tripeptide Arg-Gly-Asp (RGD) within fibronectin
has been shown to mediate cell adhesion through integrin binding.
Synthetic peptides containing an RGD motif have been generated
specifically to target alpha(v)-integrin for internalization by
integrin-dependent endocytosis as a potential cancer therapeutic.
Putatively, an integrin binding motif (RGD) could be problematic
for a therapeutic that is expected to function in the peripheral
blood stream. Thus, in certain aspects of the invention, the
uricase does not comprise an RGD sequence.
[0063] Methods for mutating amino acids are well-known in the art,
and such methods can be used to mutate one or more of the RGD amino
acids to any other naturally occurring amino acid. In one
embodiment, the arginine in the RGD motif is mutated to a serine,
such that the uricase contains an SGD instead of an RGD. In another
embodiment, the arginine, the glycine, and/or the aspartic acid in
the RGD motif is mutated to any other naturally occurring amino
acid, such that the uricase does not contain an RGD motif. In one
embodiment, a number of uricase amino acid sequences are aligned
using methods known in the art to determine the most highly
conserved residue at the amino acid positions where an RGD motif is
present, and one or more amino acids present in the RGD motif are
mutated to the amino acid residue that is most conserved at that
particular amino acid position. For example, if the G and the D of
the RGD motif are highly conserved, only the R would be mutated to
the amino acid residue that is most highly conserved at that
particular position (e.g., serine).
[0064] Methods for preparing nucleotide sequences encoding the
uricase amino acid sequences disclosed herein are well-known in the
art, such that one of ordinary skill in the art can readily prepare
a nucleic acid sequence encoding the uricase amino acid sequences
disclosed herein. Thus, in one embodiment, the invention comprises
a nucleic acid sequence encoding the uricase amino acid sequence
disclosed herein. Suitable expression vectors are known and
available in the art, such that the invention also encompasses a
vector comprising a nucleic acid sequence encoding the uricase
amino acid sequence disclosed herein. In yet another embodiment,
the invention encompasses a cell line comprising the expression
vector. The cell line can be a eukaryotic or a prokaryotic cell
line. In a preferred embodiment, the cell line is a prokaryotic
cell line, such as E. coli, corynebacterium, or Pseudomonas
fluorescens. In another embodiment, the cell line is a eukaryotic
cell line such as Saccharomyces cerevisiae, insect cells, etc.
Mammalian cell lines such as Chinese hamster ovary (CHO) cells may
also be used.
[0065] In one embodiment, the invention encompasses a composition
comprising the uricase described herein. In one aspect, the uricase
in the composition forms a tetramer. In some aspects, at least 93%
(e.g., 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%) of the
uricase monomers present in the composition are mono-pegylated
(e.g., one PEG moiety is present on each monomer). In some aspects,
at least 93% (e.g., 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%)
of the uricase monomers present in the composition are di-pegylated
(e.g., two PEG moieties are present on each monomer). In some
aspects, at least 93% (e.g., 93%, 94%, 95%, 96%, 97%, 98%, 99% or
even 100%) of the uricase monomers present in the composition are
tri-pegylated (e.g., three PEG moieties are present on each
monomer).
[0066] In another embodiment, the invention encompasses a
statistical model for determining the PEGylation efficiency of an
oligomeric protein, such as a tetramer of a uricase protein
described herein. The invention also provides a statistical measure
for deriving the overall functionalization of an oligomeric protein
from the data obtained from readily accessible assays that cause
non-covalently associated subunits to dissociate, as described in
Example 14 herein.
[0067] In another embodiment, the invention encompasses a
statistical approach based on a multinomial distribution that
allows the computation of overall protein conjugation for
oligomeric proteins when the size and nature of the protein or the
biophysical properties of the conjugate do not allow analysis under
native conditions.
II. Methods of Treatment
[0068] In some aspects, the invention encompasses a method of
treating a hyperuricemic patient comprising administering any of
the uricases described herein, and thereby reducing levels of uric
acid and/or UA crystal burden. The patient may have any number of
conditions resulting in hyperuricemia. For example, the patient may
have gout, such as, but not limited to chronic refractory gout,
tophaceous gout and/or high UA burden. As another example, the
patient may have or be at risk for tumor lysis syndrome.
[0069] In some aspects of the method, the uricase may be
administered subcutaneously. In other aspects, it may be
administered intravenously or intramuscularly.
[0070] For certain treatment methods, the patient may have a serum
UA level higher than 6.8 mg/dL before treatment and a serum UA
level lower than 6.8 mg/dL after treatment.
[0071] In some embodiments, the uricase or the method of treatment
is not associated with anaphylaxis. In one embodiment, the uricase
or the method of treatment is non-immunogenic.
EXAMPLES
Example 1. Selection of Uricase Enzyme
[0072] More than 200 uricase sequences from publicly-available
databases were aligned, including mammalian, plant, microbial, etc.
uricases. Candidate uricases with sequences available in the
databases were selected using proprietary criteria that included
(but not limited to): favorable biological properties (such as
expression in E. coli, neutral pH solubility, neutral pH activity),
low sequence identity or similarity to other sequences (diversity),
low endogenous Cys content, and organisms having interesting
properties suggesting that its uricase would have favorable
properties (extremophile, thermophile, acidophile, etc).
[0073] After this process, the following 7 candidate sequences were
chosen for further investigation: Arthrobacter globiformis uricase
(SEQ ID NO: 27), Deinococcus geothermalis uricase (SEQ ID NO: 28),
Deinococcus radiodurans uricase (SEQ ID NO: 29), Granulicella
tundricola uricase (SEQ ID NO: 30), Solibacter usitatus uricase
(SEQ ID NO: 31), Terriglobus saanensis uricase (SEQ ID NO: 32) and
Kyrpidia tusciae uricase (SEQ ID NO: 33). Additionally, as an
8.sup.th sequence, a consensus uricase sequence was also devised
from the alignment of many uricase sequences. The consensus
sequence is represented by SEQ ID NO: 34. As shown in Table 2
below, there is a significant amount of diversity between the 8
sequences that were selected.
TABLE-US-00002 TABLE 2 Uricase Sequence Identity Comparison
Arthrobactor Deinococcus Deinococcus Granulicella Solibacter
Terriglobus Kyrpidia Consensus globiformis geothermalis radiodurans
tundricola usitatus saanensis tusciae Consensus 100 61.7 44.4 44.1
36.7 28.0 39.4 28.5 Arthrobactor 100 41.2 43.4 42.7 29.2 38.4 27.3
globiformis Deinococcus 100 75.1 36.0 23.4 34.5 22.2 geothermalis
Deinococcus 100 36.3 23.1 37.0 21.6 radiodurans Granulicella 100
26.4 58.4 27.1 tundricola Solibacter 100 21.5 43.7 usitatus
Terriglobus 100 25.6 saanensis Kyrpidia 100 tusciae
Example 2. Screening Paradigm
[0074] An initial screening paradigm was used to identify
candidates for further optimization. The 8 uricase sequences
described in Example 1 were cloned with an amino terminal His tag
and expressed in E. coli. Each uricase construct was evaluated for
expression level and in particular, soluble expression. Uricase
expressing E. coli were lysed and soluble material was separated
from insoluble (pellet) material. The lysates were separated by
SDS-PAGE and the proteins were visualized by Coomassie blue
staining. As shown in FIG. 1, most uricases were present at high
level in the insoluble (P) material. The pig-baboon chimera appears
to express almost entirely in the pellet (P) (insoluble) fraction
(FIG. 1, Lane 9). Cytosolic soluble (S) expression was considered a
favorable property. The 8 uricases were then purified from the E.
coli cell lysates by Ni-affinity chromatography. Protein yield was
determined by measuring the absorbance at 280 nm. Protein size was
verified by mass spectrometry and tetramer formation was confirmed
by size exclusion chromatography and light-scattering detection
(see Table 3 below).
TABLE-US-00003 TABLE 3 Mass Spec and SEC-LS Analysis Pre- Measure
Measured dicted Mass Theoretical SEC-LS Mass Monomer Tetramer
Tetramer Tetramer (kDa) (kDa) (kDa) (kDa) Formation Arthrobactor
33.88 33.88 135.52 135.20 globiformis Deinococcus 35.19 35.19
140.76 136.30 geothermalis Terriglobus 32.69 32.69 130.76 126.80
saanensis Consensus 35.83 35.83 143.32 141.30 Deinococcus 35.58
35.58 142.32 140.40 radiodurans Granulicella 33.60 33.60 134.40
128.00 tundricola Kyrpidia 38.24 38.24 152.96 147.20 tusciae
Solibacter 33.24 33.24 132.96 137.70 usitatus
[0075] Three uricases were eliminated from further evaluation based
on unfavorable expression, solubility or purification yields,
namely, Solibacter usitatus, Kyrpidia tusciae, and Granulicella
tundricola.
[0076] Differential scanning calorimetry (DSC) measurements were
performed to assess thermal stability (see Table 4 below). Two
transitions were observed for each uricase. Terriglobus saanensis
and Deinococcus radiodurans exhibited a thermal transition (TM1)
that was lower than desired and, as a result, these two uricases
were eliminated from the pool of candidates. FIGS. 2A and 2B show
two examples of the DSC results (Deinococcus geothermalis uricase
(FIG. 2A) and Deinococcus radiodurans uricase (FIG. 2B)).
TABLE-US-00004 TABLE 4 Differential scanning calorimetry stability
TM1 (.degree. C.) TM2 (.degree. C.) Arthrobactor globiformis 47.5
73.0 Deinococcus geothermalis 55.0 63.0 Terriglobus saanensis 42.0
90.0 Consensus 56.0 69.0 Deinococcus radiodurans 32.0 54.0
[0077] Five uricases (SEQ ID NOs: 27, 28, 29, 32, and 34) were
evaluated for neutral pH solubility characteristics and activity in
terms of product formation (H.sub.2O.sub.2) at pH 9.0 and 7.4. In
the product formation assay, allantoin formation is proportional to
H.sub.2O.sub.2 formation, which is linked to colorimetric
horseradish peroxidase-catalyzed, colorimetric reaction. The
appearance of hydrogen peroxide can be measured by an increase in
absorbance at 540 nm. Product formation assays were proportional to
substrate depletion assays in terms of uricase activity. However,
product formation assay do not allow for continuous monitoring of
enzyme activity over time. The substrate depletion assays were much
better for assessing kinetic parameters like V max and Km.
[0078] Substrate depletion (UA) is another common method for
assessing uricase activity. In the substrate depletion assay,
uricase, UA, and phosphate buffer were incubated for 1 hour at the
stated temperature (typically 30.degree. C.). Uricase was then
diluted to 1 .mu.g/mL and combined with a curve of UA (400 .mu.M
diluted down 1:1.6 to 23.8 .mu.M) in 0.1M phosphate buffer (PB), pH
7.4. In some assays 1 mM DTT was added to the assay. The Molecular
Devices reader temperature was set to the 30.degree. C. Absorbance
measurements at 292 nm were captured every 20 seconds for a period
of 10 minutes. The rate of UA degradation was calculated by SoftMax
Pro software. V max and Km were calculated for these uricases (see
Table 5 below). Data are shown in FIG. 3. Each curve represents
4320 specific activity data points.
TABLE-US-00005 TABLE 5 Vmax, Km and kcat/Km Uricase Buffer pH Vmax
Km Krystexxa PB 7.4 5.68 116.3 Arthrobacter globiformis PB 7.4
10.80 109.7 Deinococcus geothermalis PB 7.4 5.75 55.73 Terriglobus
saanensis PB 7.4 5.48 76.18 Consensus PB 7.4 4.09 31.76 Deinococcus
radiodurans PB 7.4 5.20 83.06
[0079] Based on kinetic parameters, two uricases were selected for
further study, Deinococcus geothermalis, which had a 2 fold
improved V max relative to Krystexxa.RTM. (10.8 versus 5.7), and
Arthrobacter globiformis, which had a 2 fold better Km relative to
Krystexxa.RTM. (55.7 versus 116.3). Both uricases had about a 2
fold better kcat/Km relative to Krystexxa.RTM.. Lastly, although
the consensus uricase exhibited favorable kinetics, the consensus
sequence is 61.7% identical to Arthrobacter globiformis whereas
Deinococcus geothermalis and Arthrobacter globiformis are only
41.2% identical to each other, suggesting a greater diversity
between these two. The high degree of diversity was deemed
advantageous, and therefore, the Deinococcus geothermalis and
Arthrobacter globiformis uricases were selected for further
investigation.
Example 3. Dose Modeling Suggests that Kcat is the Most Important
Kinetic Parameter
[0080] Gout and tumor lysis syndrome patients typically have
saturating levels of UA (>6.8 mg/dl, 408 .mu.M). Therefore, it
was hypothesized that V max (kcat) is the most important kinetic
parameter for a therapeutic uricase. Dose models were generated
based on an improvement in kcat (Arthrobacter Globiformis) or Km
(Deinococcus geothermalis). Although the modeling predicted an
improved km (Deinococcus geothermalis) would provide no advantage
in dosing amount or frequency, the modeling predicted that an
improved kcat (Arthrobacter globiformis) would provide an advantage
in terms of dosing amount and frequency. Thus, the results of the
dose modeling suggest that kcat is the most important kinetic
parameter for a therapeutic uricase.
Example 4. Immunogenicity Based on Overlapping Peptide Analyses
[0081] Since immunogenicity has proven to be a problem in the
clinic for currently available uricases, both Arthrobacter
Globiformis and Deinococcus geothermalis uricases were screened for
putative T-cell immunogenicity by EpiScreen.TM. analysis. The
sequences of both uricase enzymes were analyzed using overlapping
peptides for the presence of CD4+ T cell epitopes (EpiScreen.TM. T
cell epitope mapping analysis). A total of 93 overlapping 15mer
peptides spanning the sequence of uricase Arthrobacter Globiformis
and 94 for Deinococcus geothermalis were tested against a cohort of
54 healthy donors screened to represent a cross section of HLA-DRB1
haplotypes. CD4+ T cell responses against individual peptides were
measured using .sup.3H thymidine incorporation proliferation assays
and the results were used to compile a T cell epitope map of the
two uricase sequences. A putative T-cell epitope was considered if
3 or more donor samples elicited a CD4 stimulation index score
greater than 2.00 in the assay. A total of five putative T-cell
epitopes were identified in the Arthrobacter globiformis sequence.
In this case, no peptides elicited a T-cell response in greater
than 4 donors samples (<10%). In addition, stimulation index
magnitude for each positive peptide was relatively low suggesting
that the peptides may not be strong T-cell epitopes. Overlapping
peptide T-cell analysis of Deinococcus geothermalis suggested the
existence of six putative epitopes. Some of these peptides elicited
a positive T-cell response in greater than 10% of the donors
screened and the magnitude of the response (stimulation index) was
greater.
[0082] Based on these results and the improved V max for
Arthrobacter globiformis it was determined to further optimize this
sequence.
Example 5. Sequence Evolution for Arthrobacter globiformis
Uricase
[0083] A. Initial Sequence Evolution
[0084] SEQ ID NO: 22 was modified to add an N-terminal His tag and
short linker, and to truncate the C-terminal 11 amino acids in
order to eliminate the C-terminal Cys, resulting in SEQ ID NO:
21.
[0085] B. Changing RGD to SGD
[0086] As a major family of cell adhesion receptors integrins are
known play a key role in cell-cell and cell-extracellular matrix
interactions. The tripeptide RGD within fibronectin has been shown
to mediate cell adhesion through the RGD motif. Putatively, an
integrin binding motif (RGD) could be problematic for a therapeutic
that is expected to function in the peripheral blood stream. SEQ ID
NO: 21 and SEQ ID NO: 22 both contain an RGD motif. An M21 tumor
cell adhesion assay was conducted to determine if the RGD is
surface accessible. M21 cells were used because they express
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins.
An RGD-containing fibronectin substrate, PBS (negative control), or
test article was coated on an ELISA plate at 0-100 ug/ml overnight
in PBS. Fluorescently labeled (calcein-AM) M21 cells were incubated
for 1 hour at 37.degree. C. on coated plates. Unbound cells were
washed away and bound cells were measured by total fluorescence.
Fab9mCys is an IgG that contains an RGD within the CDR-H3 loop and
serves as a positive control along with fibronectin. Results are
shown in FIG. 4 illustrating that RGD containing Arthrobacter
Globiformis uricase binds the M21 cells. These data suggest that
the RGD in Arthrobacter Globiformis uricase is surface
accessible.
[0087] Using a database of greater than 200 aligned uricases it was
determined that the Glycine (G) and Aspartate (D) in the RGD motif
were highly conserved residues across the aligned uricases.
However, the Arginine (R) was not a highly conserved position and
the consensus residue at this position is a Serine (S). Therefore,
site-directed mutagenesis was used to replace the R in the RGD
motif with an S, thus making the RGD an SGD. This modification
removes the potential integrin binding motif, thereby generating a
uricase having SEQ ID NO: 20, wherein the His and linker tag on the
N-terminus of the sequence as shown is optional.
[0088] C. Evaluating SGD Modification
[0089] The RGD to SGD mutation was evaluated for its impact on the
expression, solubility, purification yield, etc. Although, the SGD
mutation appears to have decreased the soluble expression a bit,
culture conditions can be optimized to improve soluble
expression.
[0090] The RGD to SGD mutation showed a marked reduction in the
integrin binding assay (see FIG. 4). Both uricases were evaluated
for activity at pH 7.4. The SGD mutation appears to have comparable
activity (see FIG. 5).
Example 6. Assessing Immunogenicity of Modified Arthrobacter
globiformis Uricase
[0091] A. LONZA Immunogenicity Assay (Epibase.RTM.)
[0092] Although Arthrobacter globiformis uricase (SEQ ID NO: 22)
had 5 putative T-cell epitopes based on the EpiScreen assay, none
of these elicited a strong response in greater than 10% of the
donor samples. Additionally, overlapping synthetic peptide T-cell
epitope assays are known to over predict MHC-class 2 epitopes. This
is likely due to the fact that not all potential peptide variants
will exist within an endogenous endosomal degradation process of
the protein therapeutic. As a result, modified Arthrobacter
globiformis (SEQ ID NO: 18) was screened as a holoprotein in the
Epibase.RTM. immunogenicity assay. The Epibase.RTM. assay is a
human PBMC T-cell immunogenicity assay used to assess
"immunogenicity risk." Although this assay cannot necessarily
predict clinical immunogenicity, it can be used to identify "high
risk" and "low risk" proteins based on the number of responders and
the overall response magnitude (Stimulation index). In this assay,
PBMC samples from 202 normal donors were used to screen the T-cell
immunogenicity of a uricase candidate relative to a negative
control (buffer) and a positive control (KLH). Here, 202 donors
were selected to represent HLA-DRB1 frequencies in the Caucasian
population (see FIG. 6). PBMC from frozen stocks were thawed and
added to a 96 well plate at a density of 3.times.10.sup.5 cells per
well. Test articles were added to media at 30 ug/ml (Buffer, KLH,
SEQ ID NO: 18). Each test condition was carried out in 8-plicate
(n=8). PBMC were incubated for 7 days. On day 7, PBMCs were labeled
for surface CD3.sup.+ and CD4.sup.+ markers. Proliferating
CD4.sup.+ T-cells were identified by flow cytometry. Stimulation
indices (SI) values describes the ratio of proliferating
CD3.sup.+CD4.sup.+ T-cell in antigen treated versus untreated
wells. SI values >2 are considered positive which is supported
by p-value <0.05. Population immunogenicity analysis was also
determined by calculating the magnitude of the T-cell response for
the entire population.
[0093] The results were as follows: negative control--0/202 donor
samples (0%) responded with a mean population SI=1.0; uricase
candidate--1/202 donor samples (0.5%) responded with a mean
population SI=1.03; positive control--181/202 donors (91%)
responded with a mean SI=4.2. Individual donor data is shown in
FIGS. 7A-C. FIG. 7A shows that the buffer (negative control)
stimulation index is 1.0 and the KLH (positive control) had a 91%
response (SI>2). The KLH mean total SI=4.2. FIG. 7B shows the
uricase candidate in comparison to the buffer control. A response
rate of 4% or less in this assay is considered to be "low risk" and
historical screening of other potential clinical compounds has
produced rates in the range of 20-25% immunogenicity. FIG. 7C shows
that the buffer stimulation index is 1.0, the KLH stimulation index
is 4.2, and the uricase candidate stimulation index is 1.03.
Although this assay cannot necessarily predict clinical
immunogenicity, it can be used to assess risk of immunogenicity and
these data suggest that the uricase evaluated is "low risk" for
clinical immunogenicity. Considering that the uricase protein
sequence that was tested is microbial in origin (Arthrobacter
globiformis), this is quite a surprising finding.
Example 7. His Tag Removal
[0094] The N-terminal His tag was added to the N-terminus of the
uricase sequences in order to provide an efficient method of
purifying the uricase proteins that were generated (i.e.,
Ni-affinity purification). While the His tag provides advantages
during discovery, especially in the area of purification, it is
desirable to remove the His tag before preparation of a drug
product.
Example 8. Optimizing the N-Terminus
[0095] It is well-known that when proteins are express in E. coli
the N-terminal methionine can be removed by Met aminopeptidase
depending on the second residue following the methionine. If a
small residue is in the second position, cleavage typically occurs.
If a bulky residue is in the second position, no cleavage occurs.
Whereas, if the second residue is neither bulky nor particularly
small the Met aminopeptidase may function to cleave some Met but
not all generating a heterogenous drug substance. Three N-terminal
variants were generated and analyzed for expression, solubility,
methionine cleavage, and activity in order to determine the optimum
N-terminal sequence.
[0096] The starting sequence for this process was SEQ ID NO: 12 and
three variants were created. SEQ ID NO: 13 (variant 1) has a
deletion of Thr2. SEQ ID NO: 14 (variant 2) has a deletion of
Thr2-Ala5, and it was expected that the N-terminal Met would be
retained. SEQ ID NO: 15 (variant 3) has a deletion of Thr2-Thr9,
and it was expected that the N-terminal Met would be processed.
[0097] The three N-terminal uricase variants were cloned, expressed
in E. coli, and purified. Each construct was expressed in the
soluble fraction similar to the His-tagged constructs. Due to the
lack of a His tag, a purification procedure was worked out for
these constructs. In short, this included a Q ion-exchange
chromatography step (Buffer A: PBS, pH 7.8, 5 mM DTT; Buffer B:
10.times.PBS, pH7.2) followed by size exclusion chromatography
(SEC). Fractions from the SEC were run on SDS-PAGE and the proteins
were visualized by Coomassie blue staining. Fractions containing
high levels of uricase were combined for further analyses. FIG. 8A
shows the Coomassie Blue stained SDS-PAGE from the purified
preparations relative to His tagged construct (labeled SGD).
Variants 1 and 3 were found to have the N-terminal methionine
processed (removed), whereas variant 2 was found to have the
N-terminal methionine retained. All processing appeared
uniform.
[0098] FIG. 8B shows the activity of V1, V2 and V3. Variant 1 and 2
had considerably better activity than variant 3. Variant 1 was
selected for further development.
Example 9. Specific PEGylation
[0099] Modification of therapeutic proteins with polyethylene
glycol (PEG) can be performed as either random attachment to
selected protein residues (e.g. lysine side chains), or
site-specific to a unique predetermined site. The latter approach
has the advantage that the conjugation chemistry can be better
controlled and manufactured consistently, yielding a highly
homogenous PEGylated product with defined bioactivity. Among
methods for site-specific attachment, the most widely used approach
is coupling to unpaired cysteine residues, and this generally
involves the introduction of one or more free cysteine residues
into the protein sequence. Sites for Cys introduction can be
carefully selected to avoid any negative impact on bioactivity or
biophysical properties of the conjugate product following
modification with PEG.
[0100] The Arthrobacter globiformis uricase sequence described in
SEQ ID NO: 27 is particularly well suited for cysteine-based
site-specific modification as it contains only one native
C-terminal Cys residue. The C-terminal region was truncated (SEQ ID
NOs: 18 and 20) so the protein contains no Cys. Thus, modification
of this protein by Cys-reactive reagents is easily confined to
sites where Cys residues have been introduced. To select potential
sites for Cys residue introduction in the Arthrobacter globiformis
uricase sequence, the following criteria were taken into
consideration [0101] i. Sites must be on a solvent-exposed surface
of the protein to ensure efficient reaction with a thio-reactive
PEG reagent; [0102] ii. Sites must be not be close to the enzyme
active sites to avoid the risk of impacting activity; and [0103]
iii. Sites must not be in close proximity to each other so that
PEGylation of one site does not sterically hinder PEGylation of
other sites.
[0104] Moreover, given the tetrameric nature of uricase, intra- and
inter-subunit distances ideally should be considered in the case of
ii. and iii.
[0105] In order to compute parameters relevant to these
considerations, a three-dimensional structure of the Arthrobacter
globiformis uricase was used. A limited number of different uricase
structures have been reported, and one of these is the crystal
structure of Arthrobacter globiformis uricase bound to uric acid
substrate (PDB accession code: 2YZB) (see FIG. 10A). The atomic
coordinates for this structure were used to compute the following
set of parameters: [0106] i. Solvent accessible area surface area
for each amino acid residue within this uricase and [0107] ii.
Atomic distances between each side chain C.alpha. atom and the C5
atom of the uric acid substrate (C.alpha.-C5 distance).
[0108] To identify preferred positions within the Arthrobacter
globiformis uricase for substitution with cysteine, the following
criteria were initially set. First, residues were identified with
total solvent accessible surface area >100 .ANG..sup.2 and which
with a C.alpha.-C5 distance >25 .ANG. (i.e. to each C5 in the 4
uric acid molecules bound to the uricase tetramer). Second, as a
further restriction, for any given uricase residue, these criteria
had to be met in all four subunits. Of the 287 amino acid residues
in each uricase subunit, only 9 satisfied these criteria. These
were Thr11; Asn33; Asn119; Asp120; Ser142; Glu196; Pro238; Glu286
and Arg289. The third criteria were then considered by calculating
the matrix of atomic distances between pairs of C.alpha. atoms
within this set of residues across the tetrameric structure (see
Table 6). From this analysis, Thr11, Asn33, Glu196 and Asn119 were
selected as preferred residues for substitution with cysteine, as
their C.alpha. atoms across the tetramer are well separated
(.gtoreq.19.5 .ANG. for all pairs).
[0109] Table 6 below is a matrix showing atomic distances (in
.ANG.) between selected C.alpha. atoms in uricase structure 2YZB.
Subunits are by a letter, i.e. -A, -B, -C, -D. Due to the highly
symmetric nature of the tetramer, the set of distances below
suffices to characterize all distance pairs across the tetramer
(example: T11-A to T11-B distance is equivalent to T11-C to T11-D;
T11-A to T11-C is equivalent to T11-B to T11-D; T11-A to T11-D is
equivalent to T11-B to T11-C).
TABLE-US-00006 TABLE 6 Matrix showing atomic distances (in .ANG.)
T11-A N33-A N119-A D120-A S142-A E196-A P238-A E286-A R289-A T11-A
0 62.2 67.2 66.4 35.5 47.3 48.6 43.1 35.4 N33-A 0 20.7 17.5 35.4
27.4 35.9 46.3 43.6 N119-A 0 3.8 45.8 35 37.6 46.6 46.5 D120-A 0
43.3 32.2 35.5 45.1 44.9 S142-A 0 15 21.8 24.7 17.3 E196-A 0 11.5
21.1 18.7 P238-A 0 11.9 14.5 E286-A 0 9 R289-A 0 T11-B 68.2 72 62.3
65 79.1 79 79.8 80.1 76.8 N33-B 71.3 52.9 35.1 37.3 59.8 49.5 43.2
45 49.3 N119-B 61.3 34.7 22 22.3 41.5 29.5 24.9 30.8 34.3 D120-B
64.1 36.9 22.3 23.1 45.3 33.3 28.5 34.1 37.8 S142-B 79.8 60.6 42.8
46.5 76.4 69.6 68.3 71.2 71.5 E196-B 79.2 49.6 29.8 33.6 68.8 59.5
58.2 63 64.3 P238-B 80.2 43 24.8 28.5 67.2 58 58.9 65.4 65.7 E286-B
80.9 44.8 30.6 33.9 70.3 62.9 65.4 71.9 70.8 R289-B 77.7 49.2 34.4
37.8 70.7 64.3 66 71.1 69.9 T11-C 83.5 42.7 27.2 30.5 69.9 61.2
63.5 70.7 70.3 N33-C 43.5 54 58.5 58.7 53.7 60.1 66.1 67.6 60.3
N119-C 28 58.6 59.7 60.1 48.2 55.5 58.3 56.3 49.5 D120-C 31.2 58.8
60.1 60.6 50.3 57.5 60.7 59.3 52.4 S142-C 71.2 53.6 47.5 49.7 71.1
69.5 73.7 78.1 74.1 E196-C 61.9 60.8 56.1 58 69.9 71.2 74.8 76.8
71.9 P238-C 63.8 66.4 58.7 61.1 73.9 74.5 76.4 77.3 73.3 E286-C
70.9 67.9 56.7 59.6 78 76.5 77.2 78.4 75.7 R289-C 70.7 60.8 49.9
52.8 74.1 71.8 73.4 75.9 73.1 T11-D 50.9 44.3 43.4 41.8 28.6 20.4
9.1 8.2 16 N33-D 44.2 74.8 66.8 68.4 63.8 66.4 62.9 57.1 54.8
N119-D 44 66.6 60.6 62.3 61.9 65.1 65.1 62.5 58.2 D120-D 42.4 68.4
62.4 64 61.7 65.3 64.8 61.6 57.3 S142-D 27.5 63.6 61.9 61.6 38.5
43.2 37.8 27.9 25.6 E196-D 19.5 66.5 65.4 65.5 43.9 51 48.5 40.8
36.1 P238-D 8.5 63 65.4 65 38.9 48.7 48.7 42.5 35.7 E286-D 6.3 57.5
63.1 62 29.4 41.3 42.8 37.5 29.5 R289-D 13.8 55.1 58.8 57.8 26.7
36.4 35.9 29.4 22.1
Example 10. Cysteine Containing Variants of Uricase for
Site-Specific PEGylation
[0110] A number of different combinations of 1, 2, 3, and 4 Cys
residues per uricase monomer were generated. These were analyzed
for expression, solubility, purity, and activity both before and
after PEGylation. Due to the solvent exposed nature of the Cys,
these constructs tend to aggregate (disulfide bond) unless they are
kept under reducing conditions. This necessitates that a reducing
agent (DTT or other) be present during purification and assay
procedures. Once the Cys has been PEGylated, reducing agent is no
longer necessary. All tested permutations of Cys containing
constructs could be expressed, purified and demonstrated good
activity both before and after PEGylation.
[0111] FIG. 10A shows the three dimensional solvent accessible
sites within the tetrameric crystal structure of Arthrobacter
globiformis uricase (PDB accession code: 2YZB) (1). Each uricase
monomer subunit of the tetrameric enzyme is shown, and residues
selected for substitution with cysteine (T11, N33, 5142) are
identified. These side chains are highly surface exposed, distant
from each other, and distant from each active site within the
tetramer. Two Cys containing variants (T11C, N33C (SEQ ID NO: 17)
and T11C, N33C, S142C (SEQ ID NO: 16)) were analyzed for
expression, solubility, purity, and activity both before and after
PEGylation. FIG. 9A shows non-Cys (SEQ ID NO: 20), di-Cys (T11C,
N33C) (SEQ ID NO: 17) and tri-Cys (T11C, N33C, and S142C) (SEQ ID
NO: 16) uricase activity. All assays are run in the presence of DTT
to eliminate the potential for disulfide bonding.
Example 11. Optimizing PEGylation
[0112] Long-term suppression of UA by uricase requires that the
molecule be modified in some fashion to extend half-life.
Commercially available rasburicase, which is not PEGylated and
contains no conjugate half-life extending properties, has a
half-life in humans of 16-21 hours requiring daily IV dosing for
tumor lysis syndrome (Ueng et al 2005). PEGylation has been
employed to extend the half-life of a number of uricases
preclinically. Krystexxa.RTM. is a hyper-PEGylated uricase that
contains .about.44.times.10 kDa PEG molecules (.about.440 kDa of
total PEG per tetramer) conjugated to the surface of active
tetramer. Based on the literature, during the early development of
Krystexxa.RTM. it was hypothesized that PEG would effectively mask
the uricase, which is a foreign protein, and make it less
immunogenic (Hershfield et al, 2010 PNAS). Preclinical studies were
performed to maximize the amount of PEG on the surface of the
uricase while retaining enzymatic activity. 44.times.10 kDa PEG per
tetramer was found to be the maximum amount of PEG that could be
conjugated to the uricase and retain enzymatic activity. The PEG
conjugation was achieved by randomly PEGylating primary amines.
Clinical data from the Krystexxa.RTM. trials demonstrate that
.about.90% of patients develop and anti-Krystexxa.RTM. drug
response (Lipsky et al 2014 Arthritis Research & therapy). A
large percentage of the anti-drug response appears to be to PEG and
not the protein. Most compelling, is the fact that antibodies from
patients that developed an anti-drug (PEG) response bind to
non-uricase PEGylated proteins demonstrating that the response is
against PEG and not the protein or protein-PEG interface. Prior to
these trials, conventional wisdom was that the PEG motif of a
PEGylated therapeutic was unlikely to be immunogenic. The vast
majority of PEGylated therapeutics contain only enough PEG to
extend half-life and are not "hyper-PEGylated" like Krystexxa.RTM..
One hypothesis is that the amount of PEG on Krystexxa.RTM. has led
to the anti-PEG immunogenicity associate with the drug. The
Krystexxa.RTM. tetramer is about 136 kDa with approximately 440 kDa
of PEG conjugated to the surface. This leads to a .about.576 kDa
molecule, a size that is unprecedented for PEGylated therapeutics.
As a result, a limited number of Cys residues for site specific
PEGylation were engineered, as described in Example 10.
[0113] Uricases with either two (di-cys) or three (tri-cys)
cysteines were generated initially with a His tag for purification
ease. FIG. 9A shows that these di-cys and tri-cys uricases retain
uricase enzymatic activity.
[0114] Di-PEGylation reaction conditions were optimized by varying
time, pH, phosphate concentration, NaCl, protein, PEG and TCEP.
Higher PEG concentrations were shown to improve PEGylation
efficiency and higher TCEP concentration decreased PEGylation
efficiency slightly. Other variables had very little effect on
PEGylation efficiency. Table 7 below shows the variables that were
tested to optimize PEGylation and the optimum conditions
achieved.
TABLE-US-00007 TABLE 7 Optimization of PEGylation Factor Range
studied Effect Optimum pH 6.5-7.5 Little effect pH 7 Phosphate
conc. 20 mM-100 mM Little effect 60 mM Protein conc. 1-5 mg/ml
Little effect 1-3 mg/ml PEG conc. 50-1000 uM Pronounced 700-900 uM
effect TCEP conc 0-500 uM Clear effect No TCEP Reaction time 10
mins-4 hrs Reaction complete at 4.degree. C. after 1.75 hrs
[0115] FIG. 9B shows that the di-PEGylated and tri-PEGylated
uricases retain uricase enzymatic activity.
[0116] Analysis of di-PEGylated material by SDS-PAGE (FIG. 10B)
confirmed that most of the protein was uniformly conjugated with
PEG. Reverse phase chromatography analysis of the di-PEGylated
material suggested that 92.6% was di-PEGylated, 4.4% mono-PEGylated
and a small amount of material over-PEGylated (.about.3%) was
observed (FIG. 10C). PEGylation did not appear to impact the
enzymatic activity. Di-PEGylated material showed a similar rate of
UA oxidation compared to the non-PEGylated enzyme (FIG. 10D).
Comparable results were obtained for the tri-PEGylated material
(data not shown). These methods of analysis disrupt the quaternary
structure of the uricase, but as a homo-tetramer the predominant
native state product for the di-PEG uricase would be expected to
have 8.times.10 kDa PEG chains, while the tri-PEG uricase would be
expected to have 12.times.10 kDa PEG chains.
[0117] Based on the optimized PEGylation conditions identified (see
Table 7), non-his tagged di-PEG and triPEG uricases were generated,
purified and analyzed for PEGylation efficiency and enzyme
activity. These PEGylated molecules were further analyzed in vivo
PK studies.
Example 12. In Vivo PK for Di-PEG and Tri-PEG Uricases
[0118] Two PEGylated uricases (di-PEG T11C, N33C and tri-PEG T11C,
N33C, and S142C) were evaluated in a rat study. A rat study was
chosen because there was precedence in testing PK of PEGylated
uricases in rats (Zhang et al, 2012 International Journal of
Pharmaceutics). SEQ ID NOs: 16 and 17 were used in this assay. In
vivo pharmacokinetics were determined in rat for both di-PEGylated
and tri-PEGylated uricases. 4 rats in each group were dosed IV at 5
mg/kg and 10 samples were collected (Day -1), 0.5, 2, 4, 8, 24, 48,
72, 96 and terminal at 144 hours post injection. Whole blood was
collected in serum separator tubes and frozen. Serum was analyzed
for residual uricase activity and data were fit to a titration
curve. The enzymatic specific activity of the uricase that went
into the rats (predose) and the uricase that was measured from
serum (postdose) was comparable suggesting activity was retained
during the in vivo study. FIG. 11A demonstrates that di and
tri-PEGylated uricases have substantially longer half-lives than
the non-PEGylated uricase. Non-PEGylated uricase had a half-life of
2-3 hours in this study (FIG. 11A, triangles). Both di- and
tri-pegylated uricases exhibit mono-phasic profiles. The
half-lives, volume of distribution (Vd) and clearance rate for each
uricase are shown in Table 8 below. Coefficient of variation is
expressed as a percentage within the parentheses.
TABLE-US-00008 TABLE 8 Rat PK for Di-PEGylated and Tri-PEGylated
uricase Half-Life Vd Clearance (hr) (L/kg) (L/hr/kg) Di-PEGylated
22.8 (7.4) 0.03 (7.2) 0.00096 (8.1) Tri-PEGylated 29.9 (12.1) 0.03
(25) 0.00077 (12.8)
[0119] The results shown in Table 8 indicate that di-PEG and
tri-PEG showed very similar pharmacokinetic profiles with a slight
advantage of tri-PEG over di-PEG. However, di-PEG was considered to
be slightly more desirable with respect to manufacturing and
analysis, and therefore, the di-PEG T11C, N33C was selected for
further testing. With substantially less PEG than Krystexxa.RTM.,
the di-PEGylated uricase may be advantageous from an immunogenicity
standpoint.
[0120] The pharmacokinetic behavior of Krystexxa.RTM. also was
evaluated in the same rat PK study. Unlike the di- and
tri-PEGylated uricases, Krystexxa.RTM. did not exhibit monophasic
elimination, but a complex profile in which Krystexxa.RTM. was
rapidly eliminated in the first 2 hours followed by a more gradual
elimination profile (FIG. 11A, squares). The Krystexxa.RTM.
elimination profile is distinctly different from that of the di and
tri-PEGylated uricases.
[0121] The PK of di-PEGylated uricase administered SC was studied
in canines. A canine study was chosen because there was precedence
for testing PK of PEGylated uricase in canines
(Pegloticase/Krystexxa.RTM. FDA BLA No. 125293, section 2.6.5.4.1).
Canines were dosed SC at 3 and 10 mg/kg and serum or blood samples
were collected at various time points and analyzed for uricase
activity (PK) or uric acid (PD). The enzymatic specific activity of
the uricase that went into the canines (predose) and the uricase
that was measured from serum (postdose) was comparable suggesting
activity was retained during the in vivo study. FIG. 11B
demonstrates that di-PEGylated uricase delivered to canines via SC
route of administration had a half-life of 1.81.+-.0.31 days for 3
mg/kg (n=3) and 1.82.+-.0.22 days for 10 mg/kg (n=3). A substantial
reduction (.about.85%) in UA levels was observed and appears to be
proportional to serum uricase levels (FIG. 11B). Blood UA levels
returned to normal as the uricase levels were depleted.
Example 13. Ex Vivo Evaluation of Activity and Stability
[0122] Di-PEG uricase (SEQ ID NO: 1) activity was evaluated in 50%
human serum (whole blood is .about.50% serum) at 37.degree. C. to
mimic the complex in vivo matrix environment and temperature. The
assay was performed as follows: UA, phosphate buffer, and serum
were warmed to 37.degree. C. All reaction steps were done at
37.degree. C. Uricase was diluted to 8 ug/mL in serum for 20-30
minutes to deplete endogenous uric acid in the human serum sample.
An equal volume of a titration of UA in phosphate buffer was then
added and the reaction was stopped at 0, 1, 2, 4 or 6 minutes using
50% percholoric acid. Perchloric acid has been shown to precipitate
protein but does not precipitate the UA (Sakuma et al, 1987
Clinical Chemistry and Stove et al, 2007 Clinical Chemistry). The
precipitate was pelleted and 100 uL of the supernatant was
transferred to a UV plate. Absorbance was measured at 292 nM. Rate
was calculated by plotting the slope of the 4 time-points at each
UA concentration using SoftMax Pro software. FIG. 12A show the
comparison of di-PEGylated uricase activity and Krystexxa.RTM. in
50% human serum at 37.degree. C.
[0123] A human serum based stability assay was performed as well.
Di-PEGylated uricase was incubated in 50% human serum for 0, 0.5,
1, 2, 4, or 24 hours at 37.degree. C. and then assayed for
activity. Di-PEG uricase retains activity at each point suggesting
that the protein is stable in 50% human serum for at least 24 hours
and retains its UA oxidase activity. FIG. 12B shows the results
from the serum stability experiment.
[0124] Lastly, the activity of di-PEGylated uricase was explored
with repeat doses (recharge) of UA at 37.degree. C. In short, UA
(100 .mu.M) and di-PEG uricase (1 .mu.g/ml) were combined in a 100
.mu.L volume (UV transparent 96 well plate) at 37.degree. C.
Absorbance at 292 nm was monitored for 10 minutes. 2.5 .mu.L of
2000 uM UA was added to the 100 .mu.L wells ("recharge") and the
absorbance at 292 nm was monitored for an additional 10 min. The
process was repeated and activity (slope of UA depletion) remained
relatively constant for each "recharge". FIG. 12C shows the results
from this study.
Example 14. PEG Conjugation Efficiency Determination
[0125] When multiple conjugation sites are present in a
biomolecule, conjugation reactions frequently lead to a
heterogeneous mixture of products that are characterized by varying
degrees of functionalization and/or different sites of
modification. This is generally the case for first generation,
non-specific coupling chemistries, such as protein conjugations
targeting .epsilon.-amino groups of lysine residues. However, even
when a site-specific conjugation approach is chosen, for example in
the frequently employed approach of targeting engineered cysteine
residues, the reaction might not go to completion, e.g. due to
steric constraints. This likewise results in a distribution of
conjugated proteins with varying degree of derivatization. As
bioactivities can vary significantly with the degree of
modification, the final product needs to be thoroughly
characterized in terms of modification to ensure a well-defined and
consistently manufactured bioconjugate.
[0126] Several analytical approaches can be employed to
characterize the overall derivatization of bioconjugates, including
mass spectrometry or HPLC-based methods. However, for the important
class of bioconjugations that involve the attachment of polymers
like poly(ethylene glycol) (PEG) to proteins, most of these
techniques become challenging for conjugates containing multiple
attached polymers. Due to the size and charge distribution of
polymer and protein, as well as polydispersity of the PEG, mass
spectrometry approaches based on electrospray ionization (ESI) are
generally not feasible, and MALDI MS frequently results in a broad
continuous mass spectrum. For smaller PEGylated proteins and/or in
the case of a low number of conjugation sites (N<3), HPLC-based
techniques under native conditions (based on size-exclusion or ion
exchange) may still provide sufficient resolution to distinguish
individual species. However, these techniques are generally not
feasible or not sufficiently resolved in the case of larger
proteins with multiple conjugation sites. The heavily hydrated PEG
polymer imparts a large hydrodynamic radius on the protein
conjugates which prevents SEC-based separation of sufficient
resolution, and the shielding of surface charges weakens
electrostatic interactions with IEX resins. In this case, Reversed
Phase (RP) HPLC is frequently the method of choice for accurate
reaction monitoring and product characterization. However, in the
case of oligomeric proteins, this technique generally leads to a
dissociation of subunits and provides only a description of the
monomeric unit which affords a partial understanding of molecule
functionalization that can be misleading for process optimization
efforts. To accurately quantify the true conjugation status of an
oligomeric biotherapeutic for process optimization and product
characterization, an understanding of the relationship between the
extent of modification at the monomer level and the resulting
overall derivatization at the quaternary level must be derived.
Experimental Design
[0127] Maleimide-functionalized PEG-10 (10 kDa, Sunbright MA-100)
was obtained from NOF. All buffer components and reagents were
purchased from Sigma (St Louis, Mo.) or Avantor Performance
Materials (Center Valley, Pa.). PEGylation reactions of tri-cys
uricase were performed in sodium phosphate buffer, pH 7.0.
PEGylation reactions were quenched after selected timepoints by the
addition of DTT to a final concentration of 10 mM and analyzed by
analytical reverse-phase high performance liquid chromatography RP
HPLC (RP-HPLC) using a YMC-Pack Protein-RP column (250.times.2.0
mm, S-5 .mu.m) from YMC America (Allentown, Pa., USA) with an
Agilent HPLC1200 system. Mobile phase A was 0.1% TFA in water and
mobile phase B consisted of 0.1% TFA in acetonitrile. The sample
was eluted with a linear gradient of increasing mobile phase B at a
flow rate of 0.4 ml/min. Elution profiles were monitored by UV
absorbance at 280 nm.
[0128] A two-step Box-Behnken design was employed with the goal to
maximize protein PEGylation. The concentration of protein (1-3
mg/ml), PEG-10 (0.5-1 mM) and reducing agent TCEP (0-0.5 mM) were
varied for di-cys and tri-cys uricases containing 8 or 12
conjugation sites per tetramer, respectively, while pH, salt and
phosphate buffer ion concentration were kept at fixed values. Data
were analyzed after selected time points ranging from 10 minutes to
4 hours. All second order effects as well as time were treated as
categorical variables, yielding a design of 64 experiments for
screening studies for each protein variant in round 1 and 60
additional experiments per protein variant for the second round of
optimization studies. Data analysis was performed with the software
JMP 10.
[0129] Statistical Model:
[0130] A statistical measure for deriving the overall
derivatization of an oligomeric protein from data obtained from
readily accessible assays (like Reverse Phase (RP) HPLC) that cause
non-covalently associated subunits to dissociate was generated. For
a protein (or biomolecule) containing n subunits, which has m
potential conjugation sites in each subunit. Let p.sub.i, with i=0,
. . . , m, be the experimentally observed proportion of the
subunits that have i conjugated sites, then .SIGMA..sub.i=0.sup.m
p.sub.i=1. The probability, q.sub.j, of observing an oligomeric
protein with j total conjugated sites can be summarized using the
following multinomial probability table.
TABLE-US-00009 TABLE 9 Total Conjugated Sites Probability 0 q.sub.0
= p.sub.0.sup.n 1 q.sub.1 = np.sub.0.sup.n-1p.sub.1 2 q 2 = ( 2 n )
.times. p 1 2 .times. p 0 n - 2 + np 2 .times. p 0 n - 1
##EQU00001## . . . . . . J q j = 0 .times. k 0 + 1 .times. k 1 + +
mk m = j .times. ( k 0 n ) .times. ( k 1 n - k 0 ) .times. .times.
.times. .times. ( k m - 1 k m - 1 + k m ) .times. i = 0 m .times. p
i k i ##EQU00002## . . . . . . n .times. m q.sub.n.times.m =
p.sub.m.sup.n
[0131] where i=0, . . . , m, are the number of subunits that have
exactly i conjugated sites and the ensemble of these subunits has
total j conjugated sites and
.SIGMA..sub.j=0.sup.n.times.mq.sub.j=1. The mean overall
derivatization of the molecule is then readily written as:
Derivatization.sub.overall=1q.sub.1+2q.sub.2+ . . . +jq.sub.j+ . .
. +n.times.m.times.q.sub.n.times.m (Eq. 1)
[0132] Normalizing this value to the total number of available
conjugation sites (n.times.m) yields the conjugation
efficiency.
[0133] For the biochemically and pharmaceutically important classes
of dimeric, trimeric, and tetrameric proteins, calculations are
detailed below.
TABLE-US-00010 TABLE 10 Calculation of overall derivatization for a
dimeric protein (n = 2) with m = 2 or 3 conjugation sites per
subunit 1. Input of experimentally observed conjugation per subunit
into table in Excel as follows: A B C D E 1 P.sub.0 P.sub.1 P.sub.2
P.sub.3 Sum Check 2 =SUM(A2:D2)* 2. Calculations of multinomial
probabilities and overall derivatization Set up Excel table as
follows: A B C D E 9 Total Probabilities q.sub.j Expected Overall
Derivatization Conjugation Conjugated Conjugations Efficiency (%)
Sites j (from 0, 1 . . . (nxm)) 10 0 =A2{circumflex over ( )}2
=A10*B10 =SUM(C10:C16) =D10*100/(nxm)*** 11 1 =2*B2*A2 =A11*B11 12
2 =B2{circumflex over ( )}2 + 2*C2*A2 =A12*B12 13 3 =2*C2*B2 +
2*D2*A2 =A13*B13 14 4 =C2{circumflex over ( )}2 + 2*D2*B2 =A14*B14
15 5 =2*D2*C2 =A15*B15 16 6 =D2{circumflex over ( )}2 =A16*B16 17
Sum Check =SUM(B10:B16)** p0, p1, . . . p3: Experimentally observed
proportions of subunits with 0, 1, . . . 3 conjugated molecules.
For m = 2 conjugation sites per subunit, complete fields A2, B2,
C2; for 3 conjugation sites per subunit, complete fields A2, B2,
C2, D2. *E2 = Sum Check = SUM(A2:D2). Total proportions need to add
up to value of 1, e.g. 100%. If the protein has only m = 2
conjugation sites per subunit, fields B15 and B16 will not be
populated. **Field B17 (SumCheck) needs to be = 1 ***(nxm), the
total number of conjugation sites, needs to be entered in numerical
form here.
TABLE-US-00011 TABLE 11 Calculation of overall derivatization for a
trimeric protein (n = 3) with m = 2 or 3 conjugation sites per
subunit 1. Input of experimentally observed conjugation per subunit
into table in Excel as follows: A B C D E 1 P.sub.0 P.sub.1 P.sub.2
P.sub.3 Sum Check 2 =SUM(A2:D2)* 2. Calculations of multinomial
probabilities and overall derivatization Set up Excel table as
follows: A B C D E 9 Total Probabilities q.sub.j Expected
Conjugations Overall Derivatization Conjugation Conjugated
Efficiency (%) Sites j (from 0, 1 . . . (nxm)) 10 0 =A2{circumflex
over ( )}3 =A10*B10 =SUM(C10:C19) =D10*100/(nxm)*** 11 1
=3*B2*A2{circumflex over ( )}2 =A11*B11 12 2 =3*B2{circumflex over
( )}2*A2 + 3*C2*A2{circumflex over ( )}2 =A12*B12 13 3
=B2{circumflex over ( )}3 + 6*C2*B2*A2 + 3*D2*A2{circumflex over (
)}2 =A13*B13 14 4 =3*C2{circumflex over ( )}2*A2 + 6*D2*B2*A2 +
3*C2*B2{circumflex over ( )}2 =A14*B14 15 5 =3*D2*B2{circumflex
over ( )}2 + 6*D2*C2*A2 + 3*B2*C2{circumflex over ( )}2 =A15*B15 16
6 =3*D2{circumflex over ( )}2*A2 + 6*B2*C2*D2 + C2{circumflex over
( )}3 =A16*B16 17 7 =3*D2{circumflex over ( )}2*B2 +
3*D2*C2{circumflex over ( )}2 =A17*B17 18 8 =3*D2{circumflex over (
)}2*C2 =A18*B18 19 9 =D2{circumflex over ( )}3 =A19*B19 20 Sum
Check =SUM(B10:B19)** p0, p1, . . . p3: Experimentally observed
proportions of subunits with 0, 1, . . . 3 conjugated molecules.
For m = 2 conjugation sites per subunit, complete fields A2, B2,
C2; for 3 conjugation sites per subunit, complete fields A2, B2,
C2, D2. *E2 = Sum Check = SUM(A2:D2). Total proportions need to add
up to value of 1, e.g. 100%. If the protein has only m = 2
conjugation sites per subunit, fields B17-B19 will not be
populated. **Field B20 (SumCheck) sum needs to be = 1 ***(nxm), the
total number of conjugation sites, needs to be entered in numerical
form here.
TABLE-US-00012 TABLE 12 Calculation of overall derivatization for a
tetrameric protein (n = 4) with m = 2 or 3 conjugation sites per
subunit 1. Input of experimentally observed conjugation per subunit
into table in Excel as follows: A B C D E 1 P.sub.0 P.sub.1 P.sub.2
P.sub.3 Sum Check 2 =SUM(A2:D2)* 2. Calculations of multinomial
probabilities and overall derivatization Set up Excel table as
follows: A B C D E 9 Total Probabilities q.sub.j Expected Overall
Conjugation Conjug. Conjugations Derivatization Efficiency (%)
Sites j 10 0 =A2{circumflex over ( )}4 =A10*B10 =SUM(C10:C22)
=D10*100/(nxm)*** 11 1 =4*B2*A2{circumflex over ( )}3 =A11*B11 12 2
=6*B2{circumflex over ( )}2*A2{circumflex over ( )}2 +
4*C2*A2{circumflex over ( )}3 =A12*B12 13 3 =4*D2*A2{circumflex
over ( )}3 + 4*B2{circumflex over ( )}3*A2 + 12*B2*C2*A2{circumflex
over ( )}2 =A13*B13 14 4 =6*C2{circumflex over ( )}2*A2{circumflex
over ( )}2 + B2{circumflex over ( )}4 + 12*D2*B2*A2{circumflex over
( )}2 + =A14*B14 12*C2*B2{circumflex over ( )}2*A2 15 5
=4*C2*B2{circumflex over ( )}3 + 12*D2*C2*A2{circumflex over ( )}2
+ 12*D2*B2{circumflex over ( )}2*A2 + =A15*B15 12*C2{circumflex
over ( )}2*B2*A2 16 6 =6*C2{circumflex over ( )}2*B2{circumflex
over ( )}2 + 24*A2*B2*C2*D2 + 4*D2*B2{circumflex over ( )}3 +
=A16*B16 6*D2{circumflex over ( )}2*A2{circumflex over ( )}2 +
4*C2{circumflex over ( )}3*A2 17 7 =12*D2*C2{circumflex over (
)}2*A2 + 12*D2{circumflex over ( )}2*B2*A2 + 4*C2{circumflex over (
)}3*B2 + =A17*B17 12*D2*C2*B2{circumflex over ( )}2 18 8
=C2{circumflex over ( )}4 + 12*D2*B2*C2{circumflex over ( )}2 +
12*D2{circumflex over ( )}2*C2*A2 + =A18*B18 6*D2{circumflex over (
)}2*B2{circumflex over ( )}2 19 9 =4*C2{circumflex over ( )}3*D2 +
4*D2{circumflex over ( )}3*A2 + 12*D2{circumflex over ( )}2*C2*B2
=A19*B19 20 10 =4*D2{circumflex over ( )}3*B2 + 6*D2{circumflex
over ( )}2*C2{circumflex over ( )}2 =A20*B20 21 11 =4*D2{circumflex
over ( )}3*C2 =A21*B21 22 12 =D2{circumflex over ( )}4 =A22*B22 23
Sum =SUM(B10:B22) Check p0, p1, . . . p3: Experimentally observed
proportions of subunits with 0, 1, . . . 3 conjugated molecules.
For m = 2 conjugation sites per subunit, complete fields A2, B2,
C2; for 3 conjugation sites per subunit, complete fields A2, B2,
C2, D2. *E2 = Sum Check = SUM(A2:D2). Total proportions need to add
up to value of 1, e.g. 100%. If the protein has only m = 2
conjugation sites per subunit, fields B19-B22 will not be
populated. ** Field B23 (SumCheck) sum needs to be = 1 ***(nxm),
the total number of conjugation sites, needs to be entered in
numerical form here.
[0134] The data above describes a statistical measure for deriving
the overall functionalization of an oligomeric protein from the
data obtained from readily accessible assays that cause
non-covalently associated subunits to dissociate. The data above
illustrates this method using the conjugation of a homo-tetrameric
uricase protein with poly(ethylene glycol) (PEG) as a model system.
The covalent modification of therapeutic proteins with PEG is now a
well-established approach to increase the half-life in vivo, reduce
immunogenicity, improve solubility and reduce susceptibility to
proteolytic degradation. However, the method is equally applicable
to other bioconjugation processes of oligomers which result in
partial functionalization at a fixed number of conjugation
sites.
[0135] The tetrameric uricase protein (n=4) studied here has a
total mass >100 kDa. A fixed number of conjugation sites was
introduced by engineering free cysteine residues that allow
PEGylation with maleimide-functionalized PEG. The following
discussion centers on the protein variant with m=3 conjugation
sites per subunit, resulting in a total number of 12 possible
conjugation sites for the tetramer. As described above, due to the
size of the protein and the number of conjugation sites, all
analytical tools that characterize the bioconjugate product with
sufficient resolution are based on techniques that dissociate the
non-covalently bound oligomer into individual subunits. FIG. 13
illustrates the reaction analysis over time using RP HPLC, which
results in well-resolved peaks that correspond to species with
different degree of conjugation that can readily be quantitated by
integration. The assay outputs are, therefore, the relative amounts
of monomer with different degrees of functionalization: the
proportions p0, p1, p2, p3, of subunits containing 0, 1, 2, or 3
attached PEG chains. From these values, the individual
probabilities q0, q1, q2, q3, . . . q12, are calculated for the
tetrameric protein with 0, 1, 2, 3, . . . 12 attached PEG chains
according to the above multinomial probability table as:
q j = 0 .times. k 0 + 1 .times. k 1 + + 3 .times. k 3 = j .times. (
k 0 4 ) .times. ( k 1 4 - k 0 ) .times. ... .times. ( k 3 .times.
.times. 1 k 3 .times. .times. 1 + k 3 ) .times. i = 0 3 .times. p i
k i ##EQU00003##
For this example, equation 1 then becomes:
Derivatization.sub.overall=1q.sub.1+2q.sub.2+3q.sub.3+ . . .
+12q.sub.12.
Table 13 exemplifies this analysis and lists the relative amounts
of differently PEGylated subunits as derived from RP HPLC analysis,
together with the computed overall derivatization calculated from
equation (1), and the PEGylation efficiency.
TABLE-US-00013 TABLE 13 Data analysis for the experiment
illustrated in FIG. 13. Computed Overall Conjugation Overall RP
HPLC Assay Result derivatization Overall UnPEGylated Subunit +
Subunit + Subunit + according to equ. 1 conjugation Experimental
subunit 1 PEG 2 PEGs 3 PEGs (total conjugated efficiency Timepoint
[p.sub.0, %] [p.sub.1, %] [p.sub.2, %] [p.sub.3, %] sites) in %* 10
minutes 1.3 15.9 53.9 28.9 8.4 70.1 1 hour 0.7 7.1 39.6 52.6 9.8
81.4 2 hours 0.4 5.3 32.1 62.2 10.3 84.4 4 hours 0 4.0 25.3 70.7
10.7 88.9 *This protein has a total of 12 possible conjugation
sites.
[0136] The data illustrate that the computed overall derivatization
is a valuable tool for process developers that allows immediately
gauging the overall protein modification. For example, after the 10
minute time point the chosen reaction condition yields 28.9% of
subunits with 3 (out of 3) functionalized conjugation sites.
However, accounting for the fact that the remaining subunits are
partially conjugated (15.9% with 1 PEG chain, 53.9% with 2 PEG
chains) and feeding these data into the multinomial distribution
according to equation 1 lets one immediately realize that the mean
overall derivatization of the protein is indeed 8.4 out of 12 total
conjugation sites, amounting to 70.1%.
[0137] To further illustrate the value of considering the overall
derivatization of a molecule for process optimization, this value
was employed as a response parameter for a Design-of-Experiment
(DOE) approach with the goal to optimize reaction conditions that
yielded maximum protein PEGylation. These results were compared to
data analysis when the experimental output of conjugation for
individual subunits was chosen. As described in the experimental
section, a Box-Behnken design was employed. FIGS. 14A and 14B
exemplify the response surface plots for the first round of
studies, demonstrating the effect of reagent concentration on
overall PEGylation efficiency for time points from 10 minutes to 2
hours. Whereas FIG. 14A illustrates the data based on an analysis
of "fully PEGylated subunit" (i.e. 3 out of 3 functionalized
conjugation sites per monomer) as directly obtained from the RP
HPLC assay trace, FIG. 14B illustrates the data analysis when the
overall derivatization is computed based on equation (1). Both
panels show that PEGylation efficiency is increased with increasing
concentrations of PEG. However, conclusions are different for the
two ways of analysis: If, for example, a conjugation efficiency of
>90% is the target, the analysis based on FIG. 14A might lead
process developers to add at least 2-fold higher concentration of
PEG and incubate for longer reaction times compared to the analysis
based on overall derivatization (FIG. 14B). This apparent
difference becomes more pronounced the more conjugation sites are
present per subunit. Choosing only the maximum conjugation per
subunit as response parameter instead of overall computed
derivatization does not provide the true picture of the
functionalization for an oligomeric molecule and might be
misleading. Considering the high cost of PEG and other conjugation
reagents, process optimization based on overall derivatization can
result in significant cost-of-goods (as well as time) savings.
[0138] This example provides an applicable statistical approach
based on a multinomial distribution that allows the computation of
overall protein conjugation for oligomeric proteins when the size
and nature of the protein or the biophysical properties of the
conjugate do not allow analysis under native conditions. The
quantitative description of overall molecule derivatization
computed according to equation (1) will support both process
optimization efforts as well as the accurate characterization of
the conjugate for regulatory filings, and hopefully aid in the
successful translation of novel bioconjugates to the clinic.
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EQUIVALENTS
[0176] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
embodiments. The foregoing description and Examples detail certain
embodiments and describes the best mode contemplated by the
inventors. It will be appreciated, however, that no matter how
detailed the foregoing may appear in text, the embodiment may be
practiced in many ways and should be construed in accordance with
the appended claims and any equivalents thereof.
[0177] As used herein, the term about refers to a numeric value,
including, for example, whole numbers, fractions, and percentages,
whether or not explicitly indicated. The term about generally
refers to a range of numerical values (e.g., +/-5-10% of the
recited range) that one of ordinary skill in the art would consider
equivalent to the recited value (e.g., having the same function or
result). When terms such as at least and about precede a list of
numerical values or ranges, the terms modify all of the values or
ranges provided in the list. In some instances, the term about may
include numerical values that are rounded to the nearest
significant figure.
Sequence CWU 1
1
341290PRTArtificial SequenceModified Arthrobacter globiformis
Uricase, modified N-terminus, SGD, 2-Cys, C-terminal truncation
(SGD V1 C2) 1Met Ala Thr Ala Glu Thr Ser Thr Gly Cys Lys Val Val
Leu Gly Gln1 5 10 15Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys
Val Thr Arg Cys 20 25 30Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val
Thr Ser Gln Leu Ser 35 40 45Gly Asp Phe Glu Ala Ala His Thr Ala Gly
Asp Asn Ala His Val Val 50 55 60Ala Thr Asp Thr Gln Lys Asn Thr Val
Tyr Ala Phe Ala Arg Asp Gly65 70 75 80Phe Ala Thr Thr Glu Glu Phe
Leu Leu Arg Leu Gly Lys His Phe Thr 85 90 95Glu Gly Phe Asp Trp Val
Thr Gly Gly Arg Trp Ala Ala Gln Gln Phe 100 105 110Phe Trp Asp Arg
Ile Asn Asp His Asp His Ala Phe Ser Arg Asn Lys 115 120 125Ser Glu
Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln Ala 130 135
140Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly
Ser145 150 155 160Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr
Leu Gln Glu Thr 165 170 175Thr Asp Arg Ile Leu Ala Thr Asp Val Ser
Ala Arg Trp Arg Tyr Asn 180 185 190Thr Val Glu Val Asp Phe Asp Ala
Val Tyr Ala Ser Val Arg Gly Leu 195 200 205Leu Leu Lys Ala Phe Ala
Glu Thr His Ser Leu Ala Leu Gln Gln Thr 210 215 220Met Tyr Glu Met
Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile Asp225 230 235 240Glu
Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp Leu 245 250
255Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp
260 265 270Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly
Ser Arg 275 280 285Ala Asp 2902290PRTArtificial SequenceModified
Arthrobacter globiformis Uricase, modified N-terminus, RGD, 2-Cys,
C-terminal truncation (RGD V1 C2) 2Met Ala Thr Ala Glu Thr Ser Thr
Gly Cys Lys Val Val Leu Gly Gln1 5 10 15Asn Gln Tyr Gly Lys Ala Glu
Val Arg Leu Val Lys Val Thr Arg Cys 20 25 30Thr Ala Arg His Glu Ile
Gln Asp Leu Asn Val Thr Ser Gln Leu Arg 35 40 45Gly Asp Phe Glu Ala
Ala His Thr Ala Gly Asp Asn Ala His Val Val 50 55 60Ala Thr Asp Thr
Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp Gly65 70 75 80Phe Ala
Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe Thr 85 90 95Glu
Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln Phe 100 105
110Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn Lys
115 120 125Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu
Gln Ala 130 135 140Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys
Ser Thr Gly Ser145 150 155 160Glu Phe His Gly Phe Pro Arg Asp Lys
Tyr Thr Thr Leu Gln Glu Thr 165 170 175Thr Asp Arg Ile Leu Ala Thr
Asp Val Ser Ala Arg Trp Arg Tyr Asn 180 185 190Thr Val Glu Val Asp
Phe Asp Ala Val Tyr Ala Ser Val Arg Gly Leu 195 200 205Leu Leu Lys
Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln Thr 210 215 220Met
Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile Asp225 230
235 240Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp
Leu 245 250 255Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr
Ala Ala Asp 260 265 270Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln
Arg Glu Gly Ser Arg 275 280 285Ala Asp 2903290PRTArtificial
SequenceModified Arthrobacter globiformis Uricase, modified
N-terminus, RGD variants, 2-Cys, C-terminal truncation (RGD
variants of V1 C2)MISC_FEATURE(48)..(48)X is either R or any
natural amino acid except CMISC_FEATURE(49)..(49)X is either G or
any natural amino acid except CMISC_FEATURE(50)..(50)X is either D
or any natural amino acid except C 3Met Ala Thr Ala Glu Thr Ser Thr
Gly Cys Lys Val Val Leu Gly Gln1 5 10 15Asn Gln Tyr Gly Lys Ala Glu
Val Arg Leu Val Lys Val Thr Arg Cys 20 25 30Thr Ala Arg His Glu Ile
Gln Asp Leu Asn Val Thr Ser Gln Leu Xaa 35 40 45Xaa Xaa Phe Glu Ala
Ala His Thr Ala Gly Asp Asn Ala His Val Val 50 55 60Ala Thr Asp Thr
Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp Gly65 70 75 80Phe Ala
Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe Thr 85 90 95Glu
Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln Phe 100 105
110Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn Lys
115 120 125Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu
Gln Ala 130 135 140Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys
Ser Thr Gly Ser145 150 155 160Glu Phe His Gly Phe Pro Arg Asp Lys
Tyr Thr Thr Leu Gln Glu Thr 165 170 175Thr Asp Arg Ile Leu Ala Thr
Asp Val Ser Ala Arg Trp Arg Tyr Asn 180 185 190Thr Val Glu Val Asp
Phe Asp Ala Val Tyr Ala Ser Val Arg Gly Leu 195 200 205Leu Leu Lys
Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln Thr 210 215 220Met
Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile Asp225 230
235 240Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp
Leu 245 250 255Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr
Ala Ala Asp 260 265 270Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln
Arg Glu Gly Ser Arg 275 280 285Ala Asp 2904301PRTArtificial
SequenceGenus sequence, with optional N-terminal modification, 4
possible cysteines, R/SGD, optionally with or without C-terminal
truncationMISC_FEATURE(1)..(301)From at least one, two, three, or
four cysteines are included in the sequenceMISC_FEATURE(2)..(2)X is
either present or absent, and if present is
TMISC_FEATURE(11)..(11)X is either T or CMISC_FEATURE(33)..(33)X is
either N or CMISC_FEATURE(49)..(49)X is either R or
SMISC_FEATURE(119)..(119)X is either N or
CMISC_FEATURE(142)..(142)X is either S or
CMISC_FEATURE(292)..(301)One or more amino acids in the C-terminus
are optional 4Met Xaa Ala Thr Ala Glu Thr Ser Thr Gly Xaa Lys Val
Val Leu Gly1 5 10 15Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val
Lys Val Thr Arg 20 25 30Xaa Thr Ala Arg His Glu Ile Gln Asp Leu Asn
Val Thr Ser Gln Leu 35 40 45Xaa Gly Asp Phe Glu Ala Ala His Thr Ala
Gly Asp Asn Ala His Val 50 55 60Val Ala Thr Asp Thr Gln Lys Asn Thr
Val Tyr Ala Phe Ala Arg Asp65 70 75 80Gly Phe Ala Thr Thr Glu Glu
Phe Leu Leu Arg Leu Gly Lys His Phe 85 90 95Thr Glu Gly Phe Asp Trp
Val Thr Gly Gly Arg Trp Ala Ala Gln Gln 100 105 110Phe Phe Trp Asp
Arg Ile Xaa Asp His Asp His Ala Phe Ser Arg Asn 115 120 125Lys Ser
Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Xaa Glu Gln 130 135
140Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr
Gly145 150 155 160Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr
Thr Leu Gln Glu 165 170 175Thr Thr Asp Arg Ile Leu Ala Thr Asp Val
Ser Ala Arg Trp Arg Tyr 180 185 190Asn Thr Val Glu Val Asp Phe Asp
Ala Val Tyr Ala Ser Val Arg Gly 195 200 205Leu Leu Leu Lys Ala Phe
Ala Glu Thr His Ser Leu Ala Leu Gln Gln 210 215 220Thr Met Tyr Glu
Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile225 230 235 240Asp
Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp 245 250
255Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala
260 265 270Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu
Gly Ser 275 280 285Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly
Phe 290 295 3005300PRTArtificial SequenceGenus sequence, with
modified N-terminus, with 4 possible cysteines, SGD, optionally
with or without C-terminal truncationMISC_FEATURE(1)..(300)From at
least one, two, three, or four cysteines are included in the
sequenceMISC_FEATURE(10)..(10)X is either T or
CMISC_FEATURE(32)..(32)X is either N or CMISC_FEATURE(118)..(118)X
is either N or CMISC_FEATURE(141)..(141)X is either S or
CMISC_FEATURE(291)..(300)One or more amino acids in the C-terminus
are optional 5Met Ala Thr Ala Glu Thr Ser Thr Gly Xaa Lys Val Val
Leu Gly Gln1 5 10 15Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys
Val Thr Arg Xaa 20 25 30Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val
Thr Ser Gln Leu Ser 35 40 45Gly Asp Phe Glu Ala Ala His Thr Ala Gly
Asp Asn Ala His Val Val 50 55 60Ala Thr Asp Thr Gln Lys Asn Thr Val
Tyr Ala Phe Ala Arg Asp Gly65 70 75 80Phe Ala Thr Thr Glu Glu Phe
Leu Leu Arg Leu Gly Lys His Phe Thr 85 90 95Glu Gly Phe Asp Trp Val
Thr Gly Gly Arg Trp Ala Ala Gln Gln Phe 100 105 110Phe Trp Asp Arg
Ile Xaa Asp His Asp His Ala Phe Ser Arg Asn Lys 115 120 125Ser Glu
Val Arg Thr Ala Val Leu Glu Ile Ser Gly Xaa Glu Gln Ala 130 135
140Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly
Ser145 150 155 160Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr
Leu Gln Glu Thr 165 170 175Thr Asp Arg Ile Leu Ala Thr Asp Val Ser
Ala Arg Trp Arg Tyr Asn 180 185 190Thr Val Glu Val Asp Phe Asp Ala
Val Tyr Ala Ser Val Arg Gly Leu 195 200 205Leu Leu Lys Ala Phe Ala
Glu Thr His Ser Leu Ala Leu Gln Gln Thr 210 215 220Met Tyr Glu Met
Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile Asp225 230 235 240Glu
Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp Leu 245 250
255Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp
260 265 270Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly
Ser Arg 275 280 285Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe
290 295 3006292PRTArtificial SequenceGenus sequence, with truncated
N-terminus, 4 possible cysteines, SGD, optionally with or without
C-terminal truncationMISC_FEATURE(1)..(292)From at least one, two,
three, or four cysteines are included in the
sequenceMISC_FEATURE(2)..(2)X is either T or
CMISC_FEATURE(24)..(24)X is either N or CMISC_FEATURE(110)..(110)X
is either N or CMISC_FEATURE(133)..(133)X is either S or
CMISC_FEATURE(283)..(292)One or more amino acids in the C-terminus
are optional 6Met Xaa Lys Val Val Leu Gly Gln Asn Gln Tyr Gly Lys
Ala Glu Val1 5 10 15Arg Leu Val Lys Val Thr Arg Xaa Thr Ala Arg His
Glu Ile Gln Asp 20 25 30Leu Asn Val Thr Ser Gln Leu Ser Gly Asp Phe
Glu Ala Ala His Thr 35 40 45Ala Gly Asp Asn Ala His Val Val Ala Thr
Asp Thr Gln Lys Asn Thr 50 55 60Val Tyr Ala Phe Ala Arg Asp Gly Phe
Ala Thr Thr Glu Glu Phe Leu65 70 75 80Leu Arg Leu Gly Lys His Phe
Thr Glu Gly Phe Asp Trp Val Thr Gly 85 90 95Gly Arg Trp Ala Ala Gln
Gln Phe Phe Trp Asp Arg Ile Xaa Asp His 100 105 110Asp His Ala Phe
Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val Leu 115 120 125Glu Ile
Ser Gly Xaa Glu Gln Ala Ile Val Ala Gly Ile Glu Gly Leu 130 135
140Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His Gly Phe Pro Arg
Asp145 150 155 160Lys Tyr Thr Thr Leu Gln Glu Thr Thr Asp Arg Ile
Leu Ala Thr Asp 165 170 175Val Ser Ala Arg Trp Arg Tyr Asn Thr Val
Glu Val Asp Phe Asp Ala 180 185 190Val Tyr Ala Ser Val Arg Gly Leu
Leu Leu Lys Ala Phe Ala Glu Thr 195 200 205His Ser Leu Ala Leu Gln
Gln Thr Met Tyr Glu Met Gly Arg Ala Val 210 215 220Ile Glu Thr His
Pro Glu Ile Asp Glu Ile Lys Met Ser Leu Pro Asn225 230 235 240Lys
His His Phe Leu Val Asp Leu Gln Pro Phe Gly Gln Asp Asn Pro 245 250
255Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly Leu Ile Glu Ala
260 265 270Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp His Pro Ile Trp
Ser Asn 275 280 285Ile Ala Gly Phe 2907301PRTArtificial
SequenceGenus sequence, with optional N-terminal modification, 9
possible cysteines, R/SGD, optionally with or without C-terminal
truncationMISC_FEATURE(1)..(301)From at least one, two, three, or
four cysteines are included in the sequenceMISC_FEATURE(2)..(2)X is
either present or absent, and if present is
TMISC_FEATURE(11)..(11)X is either T or CMISC_FEATURE(33)..(33)X is
either N or CMISC_FEATURE(49)..(49)X is either R or
SMISC_FEATURE(119)..(119)X is either N or
CMISC_FEATURE(120)..(120)X is either D or
CMISC_FEATURE(142)..(142)X is either S or
CMISC_FEATURE(196)..(196)X is either E or
CMISC_FEATURE(238)..(238)X is either P or
CMISC_FEATURE(286)..(286)X is either E or
CMISC_FEATURE(289)..(289)X is either R or
CMISC_FEATURE(292)..(301)One or more amino acids in the C-terminus
are optional 7Met Xaa Ala Thr Ala Glu Thr Ser Thr Gly Xaa Lys Val
Val Leu Gly1 5 10 15Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val
Lys Val Thr Arg 20 25 30Xaa Thr Ala Arg His Glu Ile Gln Asp Leu Asn
Val Thr Ser Gln Leu 35 40 45Xaa Gly Asp Phe Glu Ala Ala His Thr Ala
Gly Asp Asn Ala His Val 50 55 60Val Ala Thr Asp Thr Gln Lys Asn Thr
Val Tyr Ala Phe Ala Arg Asp65 70 75 80Gly Phe Ala Thr Thr Glu Glu
Phe Leu Leu Arg Leu Gly Lys His Phe 85 90 95Thr Glu Gly Phe Asp Trp
Val Thr Gly Gly Arg Trp Ala Ala Gln Gln 100 105 110Phe Phe Trp Asp
Arg Ile Xaa Xaa His Asp His Ala Phe Ser Arg Asn 115 120 125Lys Ser
Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Xaa Glu Gln 130 135
140Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr
Gly145 150 155 160Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr
Thr Leu Gln Glu 165 170 175Thr Thr Asp Arg Ile Leu Ala Thr Asp Val
Ser Ala Arg Trp Arg Tyr 180 185 190Asn Thr Val Xaa Val Asp Phe Asp
Ala Val Tyr Ala Ser Val Arg Gly 195 200 205Leu Leu Leu Lys Ala Phe
Ala Glu Thr His Ser Leu Ala Leu Gln Gln 210 215 220Thr Met Tyr Glu
Met Gly Arg Ala Val Ile Glu Thr His Xaa Glu Ile225 230 235 240Asp
Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp 245
250 255Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala
Ala 260 265 270Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg
Xaa Gly Ser 275 280 285Xaa Ala Asp His Pro Ile Trp Ser Asn Ile Ala
Gly Phe 290 295 3008301PRTArtificial SequenceGenus sequence, with
optional N-terminal modification, 9 possible cysteines, XGD,
optionally with or without C-terminal
truncationMISC_FEATURE(1)..(301)From at least one, two, three, or
four cysteines are included in the sequenceMISC_FEATURE(2)..(2)X is
either present or absent, and if present is
TMISC_FEATURE(11)..(11)X is either T or CMISC_FEATURE(33)..(33)X is
either N or CMISC_FEATURE(49)..(49)X is any naturally occurring
amino acid except CMISC_FEATURE(119)..(119)X is either N or
CMISC_FEATURE(120)..(120)X is either D or
CMISC_FEATURE(142)..(142)X is either S or
CMISC_FEATURE(196)..(196)X is either E or
CMISC_FEATURE(238)..(238)X is either P or
CMISC_FEATURE(286)..(286)X is either E or
CMISC_FEATURE(289)..(289)X is either R or
CMISC_FEATURE(292)..(301)One or more amino acids in the C-terminus
are optional 8Met Xaa Ala Thr Ala Glu Thr Ser Thr Gly Xaa Lys Val
Val Leu Gly1 5 10 15Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val
Lys Val Thr Arg 20 25 30Xaa Thr Ala Arg His Glu Ile Gln Asp Leu Asn
Val Thr Ser Gln Leu 35 40 45Xaa Gly Asp Phe Glu Ala Ala His Thr Ala
Gly Asp Asn Ala His Val 50 55 60Val Ala Thr Asp Thr Gln Lys Asn Thr
Val Tyr Ala Phe Ala Arg Asp65 70 75 80Gly Phe Ala Thr Thr Glu Glu
Phe Leu Leu Arg Leu Gly Lys His Phe 85 90 95Thr Glu Gly Phe Asp Trp
Val Thr Gly Gly Arg Trp Ala Ala Gln Gln 100 105 110Phe Phe Trp Asp
Arg Ile Xaa Xaa His Asp His Ala Phe Ser Arg Asn 115 120 125Lys Ser
Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Xaa Glu Gln 130 135
140Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr
Gly145 150 155 160Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr
Thr Leu Gln Glu 165 170 175Thr Thr Asp Arg Ile Leu Ala Thr Asp Val
Ser Ala Arg Trp Arg Tyr 180 185 190Asn Thr Val Xaa Val Asp Phe Asp
Ala Val Tyr Ala Ser Val Arg Gly 195 200 205Leu Leu Leu Lys Ala Phe
Ala Glu Thr His Ser Leu Ala Leu Gln Gln 210 215 220Thr Met Tyr Glu
Met Gly Arg Ala Val Ile Glu Thr His Xaa Glu Ile225 230 235 240Asp
Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp 245 250
255Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala
260 265 270Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Xaa
Gly Ser 275 280 285Xaa Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly
Phe 290 295 3009292PRTArtificial SequenceGenus sequence, with
N-terminal truncation, 9 possible cysteines, XGD, optionally with
or without C-terminal truncationMISC_FEATURE(1)..(292)From at least
one, two, three, or four cysteines are included in the
sequenceMISC_FEATURE(2)..(2)X is either T or
CMISC_FEATURE(24)..(24)X is either N or CMISC_FEATURE(40)..(40)X is
any naturally occurring amino acid except
CMISC_FEATURE(110)..(110)X is either N or
CMISC_FEATURE(111)..(111)X is either D or
CMISC_FEATURE(133)..(133)X is either S or
CMISC_FEATURE(187)..(187)X is either E or
CMISC_FEATURE(229)..(229)X is either P or
CMISC_FEATURE(277)..(277)X is either E or
CMISC_FEATURE(280)..(280)X is either R or
CMISC_FEATURE(283)..(292)One or more amino acids in the C-terminus
are optional 9Met Xaa Lys Val Val Leu Gly Gln Asn Gln Tyr Gly Lys
Ala Glu Val1 5 10 15Arg Leu Val Lys Val Thr Arg Xaa Thr Ala Arg His
Glu Ile Gln Asp 20 25 30Leu Asn Val Thr Ser Gln Leu Xaa Gly Asp Phe
Glu Ala Ala His Thr 35 40 45Ala Gly Asp Asn Ala His Val Val Ala Thr
Asp Thr Gln Lys Asn Thr 50 55 60Val Tyr Ala Phe Ala Arg Asp Gly Phe
Ala Thr Thr Glu Glu Phe Leu65 70 75 80Leu Arg Leu Gly Lys His Phe
Thr Glu Gly Phe Asp Trp Val Thr Gly 85 90 95Gly Arg Trp Ala Ala Gln
Gln Phe Phe Trp Asp Arg Ile Xaa Xaa His 100 105 110Asp His Ala Phe
Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val Leu 115 120 125Glu Ile
Ser Gly Xaa Glu Gln Ala Ile Val Ala Gly Ile Glu Gly Leu 130 135
140Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His Gly Phe Pro Arg
Asp145 150 155 160Lys Tyr Thr Thr Leu Gln Glu Thr Thr Asp Arg Ile
Leu Ala Thr Asp 165 170 175Val Ser Ala Arg Trp Arg Tyr Asn Thr Val
Xaa Val Asp Phe Asp Ala 180 185 190Val Tyr Ala Ser Val Arg Gly Leu
Leu Leu Lys Ala Phe Ala Glu Thr 195 200 205His Ser Leu Ala Leu Gln
Gln Thr Met Tyr Glu Met Gly Arg Ala Val 210 215 220Ile Glu Thr His
Xaa Glu Ile Asp Glu Ile Lys Met Ser Leu Pro Asn225 230 235 240Lys
His His Phe Leu Val Asp Leu Gln Pro Phe Gly Gln Asp Asn Pro 245 250
255Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly Leu Ile Glu Ala
260 265 270Thr Ile Gln Arg Xaa Gly Ser Xaa Ala Asp His Pro Ile Trp
Ser Asn 275 280 285Ile Ala Gly Phe 29010301PRTArtificial
SequenceGenus sequence, with optional N-terminal modification, 9
possible conjugation sites, XGD, optionally with or without
C-terminal truncationMISC_FEATURE(1)..(301)From at least one, two,
three, or four cysteines are included in the
sequenceMISC_FEATURE(2)..(2)X is either present or absent, and if
present is TMISC_FEATURE(11)..(11)X is either T or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(33)..(33)X is either N or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(49)..(49)X is any naturally occurring amino
acid except CMISC_FEATURE(119)..(119)X is either N or any natural
or unnatural amino acid used for site-specific
conjugationMISC_FEATURE(120)..(120)X is either D or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(142)..(142)X is either S or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(196)..(196)X is either E or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(238)..(238)X is either P or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(286)..(286)X is either E or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(289)..(289)X is either R or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(292)..(301)One or more amino acids in the
C-terminus are optional 10Met Xaa Ala Thr Ala Glu Thr Ser Thr Gly
Xaa Lys Val Val Leu Gly1 5 10 15Gln Asn Gln Tyr Gly Lys Ala Glu Val
Arg Leu Val Lys Val Thr Arg 20 25 30Xaa Thr Ala Arg His Glu Ile Gln
Asp Leu Asn Val Thr Ser Gln Leu 35 40 45Xaa Gly Asp Phe Glu Ala Ala
His Thr Ala Gly Asp Asn Ala His Val 50 55 60Val Ala Thr Asp Thr Gln
Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp65 70 75 80Gly Phe Ala Thr
Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe 85 90 95Thr Glu Gly
Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln 100 105 110Phe
Phe Trp Asp Arg Ile Xaa Xaa His Asp His Ala Phe Ser Arg Asn 115 120
125Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Xaa Glu Gln
130 135 140Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser
Thr Gly145 150 155 160Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr
Thr Thr Leu Gln Glu 165 170 175Thr Thr Asp Arg Ile Leu Ala Thr Asp
Val Ser Ala Arg Trp Arg Tyr 180 185 190Asn Thr Val Xaa Val Asp Phe
Asp Ala Val Tyr Ala Ser Val Arg Gly 195 200 205Leu Leu Leu Lys Ala
Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln 210 215 220Thr Met Tyr
Glu Met Gly Arg Ala Val Ile Glu Thr His Xaa Glu Ile225 230 235
240Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp
245 250 255Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr
Ala Ala 260 265 270Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln
Arg Xaa Gly Ser 275 280 285Xaa Ala Asp His Pro Ile Trp Ser Asn Ile
Ala Gly Phe 290 295 30011292PRTArtificial SequenceGenus sequence,
with N-terminal truncation, 9 possible conjugation sites, XGD,
optionally with or without C-terminal
truncationMISC_FEATURE(1)..(292)From at least one, two, three, or
four cysteines are included in the sequenceMISC_FEATURE(2)..(2)X is
either T or any natural or unnatural amino acid used for
site-specific conjugationMISC_FEATURE(24)..(24)X is either N or any
natural or unnatural amino acid used for site-specific
conjugationMISC_FEATURE(40)..(40)X is any naturally occurring amino
acid except CMISC_FEATURE(110)..(110)X is either N or any natural
or unnatural amino acid used for site-specific
conjugationMISC_FEATURE(111)..(111)X is either D or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(133)..(133)X is either S or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(187)..(187)X is either E or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(229)..(229)X is either P or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(277)..(277)X is either E or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(280)..(280)X is either R or any natural or
unnatural amino acid used for site-specific
conjugationMISC_FEATURE(283)..(292)One or more amino acids in the
C-terminus are optional 11Met Xaa Lys Val Val Leu Gly Gln Asn Gln
Tyr Gly Lys Ala Glu Val1 5 10 15Arg Leu Val Lys Val Thr Arg Xaa Thr
Ala Arg His Glu Ile Gln Asp 20 25 30Leu Asn Val Thr Ser Gln Leu Xaa
Gly Asp Phe Glu Ala Ala His Thr 35 40 45Ala Gly Asp Asn Ala His Val
Val Ala Thr Asp Thr Gln Lys Asn Thr 50 55 60Val Tyr Ala Phe Ala Arg
Asp Gly Phe Ala Thr Thr Glu Glu Phe Leu65 70 75 80Leu Arg Leu Gly
Lys His Phe Thr Glu Gly Phe Asp Trp Val Thr Gly 85 90 95Gly Arg Trp
Ala Ala Gln Gln Phe Phe Trp Asp Arg Ile Xaa Xaa His 100 105 110Asp
His Ala Phe Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val Leu 115 120
125Glu Ile Ser Gly Xaa Glu Gln Ala Ile Val Ala Gly Ile Glu Gly Leu
130 135 140Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His Gly Phe Pro
Arg Asp145 150 155 160Lys Tyr Thr Thr Leu Gln Glu Thr Thr Asp Arg
Ile Leu Ala Thr Asp 165 170 175Val Ser Ala Arg Trp Arg Tyr Asn Thr
Val Xaa Val Asp Phe Asp Ala 180 185 190Val Tyr Ala Ser Val Arg Gly
Leu Leu Leu Lys Ala Phe Ala Glu Thr 195 200 205His Ser Leu Ala Leu
Gln Gln Thr Met Tyr Glu Met Gly Arg Ala Val 210 215 220Ile Glu Thr
His Xaa Glu Ile Asp Glu Ile Lys Met Ser Leu Pro Asn225 230 235
240Lys His His Phe Leu Val Asp Leu Gln Pro Phe Gly Gln Asp Asn Pro
245 250 255Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly Leu Ile
Glu Ala 260 265 270Thr Ile Gln Arg Xaa Gly Ser Xaa Ala Asp His Pro
Ile Trp Ser Asn 275 280 285Ile Ala Gly Phe 29012303PRTArtificial
SequenceModified Arthrobacter globiformis Uricase C1 construct
(T11C mutation, SGD, optional N-terminal His tag and optional short
linker (first Uricase residue corresponds to
Thr2))MISC_FEATURE(1)..(13)Optional N-terminal His tag and optional
linker 12Met Gly Ser His His His His His His Gly Ala Arg Gln Thr
Ala Thr1 5 10 15Ala Glu Thr Ser Thr Gly Cys Lys Val Val Leu Gly Gln
Asn Gln Tyr 20 25 30Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg
Asn Thr Ala Arg 35 40 45His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln
Leu Ser Gly Asp Phe 50 55 60Glu Ala Ala His Thr Ala Gly Asp Asn Ala
His Val Val Ala Thr Asp65 70 75 80Thr Gln Lys Asn Thr Val Tyr Ala
Phe Ala Arg Asp Gly Phe Ala Thr 85 90 95Thr Glu Glu Phe Leu Leu Arg
Leu Gly Lys His Phe Thr Glu Gly Phe 100 105 110Asp Trp Val Thr Gly
Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp 115 120 125Arg Ile Asn
Asp His Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val 130 135 140Arg
Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala145 150
155 160Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly Ser Glu Phe
His 165 170 175Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu Thr
Thr Asp Arg 180 185 190Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg
Tyr Asn Thr Val Glu 195 200 205Val Asp Phe Asp Ala Val Tyr Ala Ser
Val Arg Gly Leu Leu Leu Lys 210 215 220Ala Phe Ala Glu Thr His Ser
Leu Ala Leu Gln Gln Thr Met Tyr Glu225 230 235 240Met Gly Arg Ala
Val Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys 245 250 255Met Ser
Leu Pro Asn Lys His His Phe Leu Val Asp Leu Gln Pro Phe 260 265
270Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr
275 280 285Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser Arg Ala
Asp 290 295 30013290PRTArtificial SequenceModified Arthrobacter
globiformis Uricase C1 construct (variant 1), with tag eliminated,
deletion of Thr2 (to avoid partial N-term Met cleavage) and Cys at
position 11 13Met Ala Thr Ala Glu Thr Ser Thr Gly Cys Lys Val Val
Leu Gly Gln1 5 10 15Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys
Val Thr Arg Asn 20 25 30Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val
Thr Ser Gln Leu Ser 35 40 45Gly Asp Phe Glu Ala Ala His Thr Ala Gly
Asp Asn Ala His Val Val 50 55 60Ala Thr Asp Thr Gln Lys Asn Thr Val
Tyr Ala Phe Ala Arg Asp Gly65 70 75 80Phe Ala Thr Thr Glu Glu Phe
Leu Leu Arg Leu Gly Lys His Phe Thr 85 90 95Glu Gly Phe Asp Trp Val
Thr Gly Gly Arg Trp Ala Ala Gln Gln Phe 100 105 110Phe Trp Asp Arg
Ile Asn Asp His Asp His Ala Phe Ser Arg Asn Lys 115 120 125Ser Glu
Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln Ala 130 135
140Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys
Ser Thr Gly Ser145 150 155 160Glu Phe His Gly Phe Pro Arg Asp Lys
Tyr Thr Thr Leu Gln Glu Thr 165 170 175Thr Asp Arg Ile Leu Ala Thr
Asp Val Ser Ala Arg Trp Arg Tyr Asn 180 185 190Thr Val Glu Val Asp
Phe Asp Ala Val Tyr Ala Ser Val Arg Gly Leu 195 200 205Leu Leu Lys
Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln Thr 210 215 220Met
Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile Asp225 230
235 240Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp
Leu 245 250 255Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr
Ala Ala Asp 260 265 270Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln
Arg Glu Gly Ser Arg 275 280 285Ala Asp 29014287PRTArtificial
SequenceModified Arthrobacter globiformis Uricase C1 construct
(variant 2 - N-term truncation) with tag eliminated, deletion of
Thr2-Ala5 and Cys at position 11, expect complete retention of
N-term Met 14Met Glu Thr Ser Thr Gly Cys Lys Val Val Leu Gly Gln
Asn Gln Tyr1 5 10 15Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg
Asn Thr Ala Arg 20 25 30His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln
Leu Ser Gly Asp Phe 35 40 45Glu Ala Ala His Thr Ala Gly Asp Asn Ala
His Val Val Ala Thr Asp 50 55 60Thr Gln Lys Asn Thr Val Tyr Ala Phe
Ala Arg Asp Gly Phe Ala Thr65 70 75 80Thr Glu Glu Phe Leu Leu Arg
Leu Gly Lys His Phe Thr Glu Gly Phe 85 90 95Asp Trp Val Thr Gly Gly
Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp 100 105 110Arg Ile Asn Asp
His Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val 115 120 125Arg Thr
Ala Val Leu Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala 130 135
140Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly Ser Glu Phe
His145 150 155 160Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu
Thr Thr Asp Arg 165 170 175Ile Leu Ala Thr Asp Val Ser Ala Arg Trp
Arg Tyr Asn Thr Val Glu 180 185 190Val Asp Phe Asp Ala Val Tyr Ala
Ser Val Arg Gly Leu Leu Leu Lys 195 200 205Ala Phe Ala Glu Thr His
Ser Leu Ala Leu Gln Gln Thr Met Tyr Glu 210 215 220Met Gly Arg Ala
Val Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys225 230 235 240Met
Ser Leu Pro Asn Lys His His Phe Leu Val Asp Leu Gln Pro Phe 245 250
255Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr
260 265 270Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser Arg Ala
Asp 275 280 28515283PRTArtificial SequenceModified Arthrobacter
globiformis Uricase C1 construct (variant 3 - N-term truncation)
with tag eliminated, deletion of Thr2-Thr9, Cys at position 11,
expect processing of N-term met 15Met Gly Cys Lys Val Val Leu Gly
Gln Asn Gln Tyr Gly Lys Ala Glu1 5 10 15Val Arg Leu Val Lys Val Thr
Arg Asn Thr Ala Arg His Glu Ile Gln 20 25 30Asp Leu Asn Val Thr Ser
Gln Leu Ser Gly Asp Phe Glu Ala Ala His 35 40 45Thr Ala Gly Asp Asn
Ala His Val Val Ala Thr Asp Thr Gln Lys Asn 50 55 60Thr Val Tyr Ala
Phe Ala Arg Asp Gly Phe Ala Thr Thr Glu Glu Phe65 70 75 80Leu Leu
Arg Leu Gly Lys His Phe Thr Glu Gly Phe Asp Trp Val Thr 85 90 95Gly
Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp Arg Ile Asn Asp 100 105
110His Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val
115 120 125Leu Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala Gly Ile
Glu Gly 130 135 140Leu Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His
Gly Phe Pro Arg145 150 155 160Asp Lys Tyr Thr Thr Leu Gln Glu Thr
Thr Asp Arg Ile Leu Ala Thr 165 170 175Asp Val Ser Ala Arg Trp Arg
Tyr Asn Thr Val Glu Val Asp Phe Asp 180 185 190Ala Val Tyr Ala Ser
Val Arg Gly Leu Leu Leu Lys Ala Phe Ala Glu 195 200 205Thr His Ser
Leu Ala Leu Gln Gln Thr Met Tyr Glu Met Gly Arg Ala 210 215 220Val
Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys Met Ser Leu Pro225 230
235 240Asn Lys His His Phe Leu Val Asp Leu Gln Pro Phe Gly Gln Asp
Asn 245 250 255Pro Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly
Leu Ile Glu 260 265 270Ala Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp
275 28016303PRTArtificial SequenceModified Arthrobacter globiformis
Uricase with SGD, and PEGylation available sites at T11C, N33C,
S142C, optional N-terminal His tag and optional short
linkerMISC_FEATURE(1)..(13)Optional N-terminal His tag and optional
linker 16Met Gly Ser His His His His His His Gly Ala Arg Gln Thr
Ala Thr1 5 10 15Ala Glu Thr Ser Thr Gly Cys Lys Val Val Leu Gly Gln
Asn Gln Tyr 20 25 30Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg
Cys Thr Ala Arg 35 40 45His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln
Leu Ser Gly Asp Phe 50 55 60Glu Ala Ala His Thr Ala Gly Asp Asn Ala
His Val Val Ala Thr Asp65 70 75 80Thr Gln Lys Asn Thr Val Tyr Ala
Phe Ala Arg Asp Gly Phe Ala Thr 85 90 95Thr Glu Glu Phe Leu Leu Arg
Leu Gly Lys His Phe Thr Glu Gly Phe 100 105 110Asp Trp Val Thr Gly
Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp 115 120 125Arg Ile Asn
Asp His Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val 130 135 140Arg
Thr Ala Val Leu Glu Ile Ser Gly Cys Glu Gln Ala Ile Val Ala145 150
155 160Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly Ser Glu Phe
His 165 170 175Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu Thr
Thr Asp Arg 180 185 190Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg
Tyr Asn Thr Val Glu 195 200 205Val Asp Phe Asp Ala Val Tyr Ala Ser
Val Arg Gly Leu Leu Leu Lys 210 215 220Ala Phe Ala Glu Thr His Ser
Leu Ala Leu Gln Gln Thr Met Tyr Glu225 230 235 240Met Gly Arg Ala
Val Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys 245 250 255Met Ser
Leu Pro Asn Lys His His Phe Leu Val Asp Leu Gln Pro Phe 260 265
270Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr
275 280 285Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser Arg Ala
Asp 290 295 30017303PRTArtificial SequenceModified Arthrobacter
globiformis Uricase, NH2-terminal truncated, SGD, PEGylation
available sites at T11C and N33C 2-Cys (SGD His C2) with optional
N-terminal His tag and optional short
linkerMISC_FEATURE(1)..(13)Optional N-terminal His tag and optional
linker 17Met Gly Ser His His His His His His Gly Ala Arg Gln Thr
Ala Thr1 5 10 15Ala Glu Thr Ser Thr Gly Cys Lys Val Val Leu Gly Gln
Asn Gln Tyr 20 25 30Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg
Cys Thr Ala Arg 35 40 45His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln
Leu Ser Gly Asp Phe 50 55 60Glu Ala Ala His Thr Ala Gly Asp Asn Ala
His Val Val Ala Thr Asp65 70 75 80Thr Gln Lys Asn Thr Val Tyr Ala
Phe Ala Arg Asp Gly Phe Ala Thr 85 90 95Thr Glu Glu Phe Leu Leu Arg
Leu Gly Lys His Phe Thr Glu Gly Phe 100 105 110Asp Trp Val Thr Gly
Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp 115 120 125Arg Ile Asn
Asp His Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val 130 135 140Arg
Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala145 150
155 160Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly Ser Glu Phe
His 165 170 175Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu Thr
Thr Asp Arg 180 185 190Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg
Tyr Asn Thr Val Glu 195 200 205Val Asp Phe Asp Ala Val Tyr Ala Ser
Val Arg Gly Leu Leu Leu Lys 210 215 220Ala Phe Ala Glu Thr His Ser
Leu Ala Leu Gln Gln Thr Met Tyr Glu225 230 235 240Met Gly Arg Ala
Val Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys 245 250 255Met Ser
Leu Pro Asn Lys His His Phe Leu Val Asp Leu Gln Pro Phe 260 265
270Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr
275 280 285Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser Arg Ala
Asp 290 295 30018290PRTArtificial SequenceModified Arthrobacter
globiformis Uricase (C-term truncation with SGD) 18Met Ala Thr Ala
Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly Gln1 5 10 15Asn Gln Tyr
Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg Asn 20 25 30Thr Ala
Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu Ser 35 40 45Gly
Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val Val 50 55
60Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp Gly65
70 75 80Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe
Thr 85 90 95Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln
Gln Phe 100 105 110Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe
Ser Arg Asn Lys 115 120 125Ser Glu Val Arg Thr Ala Val Leu Glu Ile
Ser Gly Ser Glu Gln Ala 130 135 140Ile Val Ala Gly Ile Glu Gly Leu
Thr Val Leu Lys Ser Thr Gly Ser145 150 155 160Glu Phe His Gly Phe
Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu Thr 165 170 175Thr Asp Arg
Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr Asn 180 185 190Thr
Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly Leu 195 200
205Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln Thr
210 215 220Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu
Ile Asp225 230 235 240Glu Ile Lys Met Ser Leu Pro Asn Lys His His
Phe Leu Val Asp Leu 245 250 255Gln Pro Phe Gly Gln Asp Asn Pro Asn
Glu Val Phe Tyr Ala Ala Asp 260 265 270Arg Pro Tyr Gly Leu Ile Glu
Ala Thr Ile Gln Arg Glu Gly Ser Arg 275 280 285Ala Asp
29019289PRTArtificial SequenceModified Arthrobacter globiformis
Uricase (processed form - Met cleaved at N-term., SGD, and C-term
truncation) 19Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu
Gly Gln Asn1 5 10 15Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val
Thr Arg Asn Thr 20 25 30Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr
Ser Gln Leu Ser Gly 35 40 45Asp Phe Glu Ala Ala His Thr Ala Gly Asp
Asn Ala His Val Val Ala 50 55 60Thr Asp Thr Gln Lys Asn Thr Val Tyr
Ala Phe Ala Arg Asp Gly Phe65 70 75 80Ala Thr Thr Glu Glu Phe Leu
Leu Arg Leu Gly Lys His Phe Thr Glu 85 90 95Gly Phe Asp Trp Val Thr
Gly Gly Arg Trp Ala Ala Gln Gln Phe Phe 100 105 110Trp Asp Arg Ile
Asn Asp His Asp His Ala Phe Ser Arg Asn Lys Ser 115 120 125Glu Val
Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln Ala Ile 130 135
140Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly Ser
Glu145 150 155 160Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu
Gln Glu Thr Thr 165 170 175Asp Arg Ile Leu Ala Thr Asp Val Ser Ala
Arg Trp Arg Tyr Asn Thr 180 185 190Val Glu Val Asp Phe Asp Ala Val
Tyr Ala Ser Val Arg Gly Leu Leu 195 200 205Leu Lys Ala Phe Ala Glu
Thr His Ser Leu Ala Leu Gln Gln Thr Met 210 215 220Tyr Glu Met Gly
Arg Ala Val Ile Glu Thr His Pro Glu Ile Asp Glu225 230 235 240Ile
Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp Leu Gln 245 250
255Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp Arg
260 265 270Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser
Arg Ala 275 280 285Asp20303PRTArtificial SequenceModified
Arthrobacter globiformis Uricase (contains optional N-terminal His
tag and optional short linker, contains SGD instead of RGD) (C-term
truncation with his tag and SGD)MISC_FEATURE(1)..(13)Optional
N-terminal His tag and optional linker 20Met Gly Ser His His His
His His His Gly Ala Arg Gln Thr Ala Thr1 5 10 15Ala Glu Thr Ser Thr
Gly Thr Lys Val Val Leu Gly Gln Asn Gln Tyr 20 25 30Gly Lys Ala Glu
Val Arg Leu Val Lys Val Thr Arg Asn Thr Ala Arg 35 40 45His Glu Ile
Gln Asp Leu Asn Val Thr Ser Gln Leu Ser Gly Asp Phe 50 55 60Glu Ala
Ala His Thr Ala Gly Asp Asn Ala His Val Val Ala Thr Asp65 70 75
80Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp Gly Phe Ala Thr
85 90 95Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe Thr Glu Gly
Phe 100 105 110Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln Phe
Phe Trp Asp 115 120 125Arg Ile Asn Asp His Asp His Ala Phe Ser Arg
Asn Lys Ser Glu Val 130 135 140Arg Thr Ala Val Leu Glu Ile Ser Gly
Ser Glu Gln Ala Ile Val Ala145 150 155 160Gly Ile Glu Gly Leu Thr
Val Leu Lys Ser Thr Gly Ser Glu Phe His 165 170 175Gly Phe Pro Arg
Asp Lys Tyr Thr Thr Leu Gln Glu Thr Thr Asp Arg 180 185 190Ile Leu
Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr Asn Thr Val Glu 195 200
205Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly Leu Leu Leu Lys
210 215 220Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln Thr Met
Tyr Glu225 230 235 240Met Gly Arg Ala Val Ile Glu Thr His Pro Glu
Ile Asp Glu Ile Lys 245 250 255Met Ser Leu Pro Asn Lys His His Phe
Leu Val Asp Leu Gln Pro Phe 260 265 270Gly Gln Asp Asn Pro Asn Glu
Val Phe Tyr Ala Ala Asp Arg Pro Tyr 275 280 285Gly Leu Ile Glu Ala
Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp 290 295
30021303PRTArtificial SequenceModified Arthrobacter globiformis
Uricase (contains optional N-terminal His tag and optional short
linker) (C-term truncation with his
tag)MISC_FEATURE(1)..(13)Optional N-terminal His tag and optional
linker 21Met Gly Ser His His His His His His Gly Ala Arg Gln Thr
Ala Thr1 5 10 15Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly Gln
Asn Gln Tyr
20 25 30Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg Asn Thr Ala
Arg 35 40 45His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu Arg Gly
Asp Phe 50 55 60Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val Val
Ala Thr Asp65 70 75 80Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg
Asp Gly Phe Ala Thr 85 90 95Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys
His Phe Thr Glu Gly Phe 100 105 110Asp Trp Val Thr Gly Gly Arg Trp
Ala Ala Gln Gln Phe Phe Trp Asp 115 120 125Arg Ile Asn Asp His Asp
His Ala Phe Ser Arg Asn Lys Ser Glu Val 130 135 140Arg Thr Ala Val
Leu Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala145 150 155 160Gly
Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His 165 170
175Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu Thr Thr Asp Arg
180 185 190Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr Asn Thr
Val Glu 195 200 205Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly
Leu Leu Leu Lys 210 215 220Ala Phe Ala Glu Thr His Ser Leu Ala Leu
Gln Gln Thr Met Tyr Glu225 230 235 240Met Gly Arg Ala Val Ile Glu
Thr His Pro Glu Ile Asp Glu Ile Lys 245 250 255Met Ser Leu Pro Asn
Lys His His Phe Leu Val Asp Leu Gln Pro Phe 260 265 270Gly Gln Asp
Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr 275 280 285Gly
Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp 290 295
30022291PRTArtificial SequenceModified Arthrobacter globiformis
Uricase (0 cysteines) (truncated the C-terminal 11 amino acids to
eliminate the Cys) (C-term truncation) 22Met Thr Ala Thr Ala Glu
Thr Ser Thr Gly Thr Lys Val Val Leu Gly1 5 10 15Gln Asn Gln Tyr Gly
Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg 20 25 30Asn Thr Ala Arg
His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu 35 40 45Arg Gly Asp
Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val 50 55 60Val Ala
Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp65 70 75
80Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe
85 90 95Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln
Gln 100 105 110Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe
Ser Arg Asn 115 120 125Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile
Ser Gly Ser Glu Gln 130 135 140Ala Ile Val Ala Gly Ile Glu Gly Leu
Thr Val Leu Lys Ser Thr Gly145 150 155 160Ser Glu Phe His Gly Phe
Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu 165 170 175Thr Thr Asp Arg
Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr 180 185 190Asn Thr
Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly 195 200
205Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln
210 215 220Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro
Glu Ile225 230 235 240Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His
His Phe Leu Val Asp 245 250 255Leu Gln Pro Phe Gly Gln Asp Asn Pro
Asn Glu Val Phe Tyr Ala Ala 260 265 270Asp Arg Pro Tyr Gly Leu Ile
Glu Ala Thr Ile Gln Arg Glu Gly Ser 275 280 285Arg Ala Asp
29023291PRTArtificial SequenceModified Arthrobacter globiformis
Uricase (0 cysteines) (RGD variants, truncated the C-terminal 11
amino acids to eliminate the Cys) (C-term
truncation)MISC_FEATURE(49)..(49)X is either R or any natural amino
acid except CMISC_FEATURE(50)..(50)X is either G or any natural
amino acid except CMISC_FEATURE(51)..(51)X is either D or any
natural amino acid except C 23Met Thr Ala Thr Ala Glu Thr Ser Thr
Gly Thr Lys Val Val Leu Gly1 5 10 15Gln Asn Gln Tyr Gly Lys Ala Glu
Val Arg Leu Val Lys Val Thr Arg 20 25 30Asn Thr Ala Arg His Glu Ile
Gln Asp Leu Asn Val Thr Ser Gln Leu 35 40 45Xaa Xaa Xaa Phe Glu Ala
Ala His Thr Ala Gly Asp Asn Ala His Val 50 55 60Val Ala Thr Asp Thr
Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp65 70 75 80Gly Phe Ala
Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe 85 90 95Thr Glu
Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln 100 105
110Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn
115 120 125Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser
Glu Gln 130 135 140Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu
Lys Ser Thr Gly145 150 155 160Ser Glu Phe His Gly Phe Pro Arg Asp
Lys Tyr Thr Thr Leu Gln Glu 165 170 175Thr Thr Asp Arg Ile Leu Ala
Thr Asp Val Ser Ala Arg Trp Arg Tyr 180 185 190Asn Thr Val Glu Val
Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly 195 200 205Leu Leu Leu
Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln 210 215 220Thr
Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile225 230
235 240Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val
Asp 245 250 255Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe
Tyr Ala Ala 260 265 270Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile
Gln Arg Glu Gly Ser 275 280 285Arg Ala Asp 29024301PRTArtificial
SequenceModified Arthrobacter globiformis Uricase (0 cysteines)
(truncated the C-terminal aa to eliminate the cysteine) 24Met Thr
Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly1 5 10 15Gln
Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg 20 25
30Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu
35 40 45Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His
Val 50 55 60Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala
Arg Asp65 70 75 80Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu
Gly Lys His Phe 85 90 95Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg
Trp Ala Ala Gln Gln 100 105 110Phe Phe Trp Asp Arg Ile Asn Asp His
Asp His Ala Phe Ser Arg Asn 115 120 125Lys Ser Glu Val Arg Thr Ala
Val Leu Glu Ile Ser Gly Ser Glu Gln 130 135 140Ala Ile Val Ala Gly
Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly145 150 155 160Ser Glu
Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu 165 170
175Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr
180 185 190Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val
Arg Gly 195 200 205Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu
Ala Leu Gln Gln 210 215 220Thr Met Tyr Glu Met Gly Arg Ala Val Ile
Glu Thr His Pro Glu Ile225 230 235 240Asp Glu Ile Lys Met Ser Leu
Pro Asn Lys His His Phe Leu Val Asp 245 250 255Leu Gln Pro Phe Gly
Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala 260 265 270Asp Arg Pro
Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser 275 280 285Arg
Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe 290 295
30025301PRTArtificial SequenceModified Arthrobacter globiformis
Uricase (0 cysteines) (RGD variants, truncated the C-terminal aa to
eliminate the cysteine)MISC_FEATURE(49)..(49)X is either R or any
natural amino acid except CMISC_FEATURE(50)..(50)X is either G or
any natural amino acid except CMISC_FEATURE(51)..(51)X is either D
or any natural amino acid except C 25Met Thr Ala Thr Ala Glu Thr
Ser Thr Gly Thr Lys Val Val Leu Gly1 5 10 15Gln Asn Gln Tyr Gly Lys
Ala Glu Val Arg Leu Val Lys Val Thr Arg 20 25 30Asn Thr Ala Arg His
Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu 35 40 45Xaa Xaa Xaa Phe
Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val 50 55 60Val Ala Thr
Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp65 70 75 80Gly
Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe 85 90
95Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln
100 105 110Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser
Arg Asn 115 120 125Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser
Gly Ser Glu Gln 130 135 140Ala Ile Val Ala Gly Ile Glu Gly Leu Thr
Val Leu Lys Ser Thr Gly145 150 155 160Ser Glu Phe His Gly Phe Pro
Arg Asp Lys Tyr Thr Thr Leu Gln Glu 165 170 175Thr Thr Asp Arg Ile
Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr 180 185 190Asn Thr Val
Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly 195 200 205Leu
Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln 210 215
220Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu
Ile225 230 235 240Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His
Phe Leu Val Asp 245 250 255Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn
Glu Val Phe Tyr Ala Ala 260 265 270Asp Arg Pro Tyr Gly Leu Ile Glu
Ala Thr Ile Gln Arg Glu Gly Ser 275 280 285Arg Ala Asp His Pro Ile
Trp Ser Asn Ile Ala Gly Phe 290 295 30026302PRTArtificial
SequenceModified Arthrobacter globiformis Uricase (RGD variants)
(contains the C-terminal 11 amino acids)MISC_FEATURE(49)..(49)X is
either R or any natural amino acidMISC_FEATURE(50)..(50)X is either
G or any natural amino acidMISC_FEATURE(51)..(51)X is either D or
any natural amino acid 26Met Thr Ala Thr Ala Glu Thr Ser Thr Gly
Thr Lys Val Val Leu Gly1 5 10 15Gln Asn Gln Tyr Gly Lys Ala Glu Val
Arg Leu Val Lys Val Thr Arg 20 25 30Asn Thr Ala Arg His Glu Ile Gln
Asp Leu Asn Val Thr Ser Gln Leu 35 40 45Xaa Xaa Xaa Phe Glu Ala Ala
His Thr Ala Gly Asp Asn Ala His Val 50 55 60Val Ala Thr Asp Thr Gln
Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp65 70 75 80Gly Phe Ala Thr
Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe 85 90 95Thr Glu Gly
Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln 100 105 110Phe
Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn 115 120
125Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln
130 135 140Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser
Thr Gly145 150 155 160Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr
Thr Thr Leu Gln Glu 165 170 175Thr Thr Asp Arg Ile Leu Ala Thr Asp
Val Ser Ala Arg Trp Arg Tyr 180 185 190Asn Thr Val Glu Val Asp Phe
Asp Ala Val Tyr Ala Ser Val Arg Gly 195 200 205Leu Leu Leu Lys Ala
Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln 210 215 220Thr Met Tyr
Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile225 230 235
240Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp
245 250 255Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr
Ala Ala 260 265 270Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln
Arg Glu Gly Ser 275 280 285Arg Ala Asp His Pro Ile Trp Ser Asn Ile
Ala Gly Phe Cys 290 295 30027302PRTArthrobacter globiformis 27Met
Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly1 5 10
15Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg
20 25 30Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln
Leu 35 40 45Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala
His Val 50 55 60Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe
Ala Arg Asp65 70 75 80Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg
Leu Gly Lys His Phe 85 90 95Thr Glu Gly Phe Asp Trp Val Thr Gly Gly
Arg Trp Ala Ala Gln Gln 100 105 110Phe Phe Trp Asp Arg Ile Asn Asp
His Asp His Ala Phe Ser Arg Asn 115 120 125Lys Ser Glu Val Arg Thr
Ala Val Leu Glu Ile Ser Gly Ser Glu Gln 130 135 140Ala Ile Val Ala
Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly145 150 155 160Ser
Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu 165 170
175Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr
180 185 190Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val
Arg Gly 195 200 205Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu
Ala Leu Gln Gln 210 215 220Thr Met Tyr Glu Met Gly Arg Ala Val Ile
Glu Thr His Pro Glu Ile225 230 235 240Asp Glu Ile Lys Met Ser Leu
Pro Asn Lys His His Phe Leu Val Asp 245 250 255Leu Gln Pro Phe Gly
Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala 260 265 270Asp Arg Pro
Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser 275 280 285Arg
Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys 290 295
30028293PRTDeinococcus geothermalis 28Met Thr Gln Thr Gln Gln Asn
Gln Gln Pro Lys Val Lys Ala Arg Leu1 5 10 15Gly Ala Asn Asn Tyr Gly
Lys Ala Glu Val Asn Leu Met Lys Val Lys 20 25 30Arg Asp Ser Glu Arg
His Glu Ile Arg Glu Leu Gln Val Arg Val Ala 35 40 45Leu Ile Gly Asp
Phe Ala Ala Ala His Glu Gln Gly Asp Asn Thr Asp 50 55 60Leu Leu Ala
Thr Asp Thr Val Arg Asn Thr Ile Tyr Gly Leu Ala Lys65 70 75 80Glu
Gly Phe Gln Ala Ser Pro Glu Ala Phe Gly Lys Glu Leu Ile Ser 85 90
95His Phe Val Thr Thr Gly Pro Lys Val Thr Gly Gly Phe Met Glu Phe
100 105 110Thr Glu Tyr Leu Trp Glu Arg Ile Gln Val Gly Gly Glu Gly
His Asn 115 120 125His Ala Phe Val Arg Gln Met Pro Gln Arg Thr Gly
Arg Val Glu Ser 130 135 140Glu Asp Gly Lys Thr Phe Lys Ile Thr Ser
Gly Leu Gln Asn Leu Tyr145 150 155 160Val Leu Lys Thr Thr Glu Ser
Gly Trp Ala Asn Tyr Leu Leu Asn Glu 165 170 175Arg Phe Thr Thr Leu
Pro Glu Thr His Glu Arg Leu Met Ala Ser Phe 180 185 190Val Thr Ala
Lys Trp Glu Tyr Asn Glu Asp Gln Val Asp Tyr
Asp Asp 195 200 205Val Trp Pro Arg Val Tyr Arg Gln Leu Gln Glu Thr
Phe Thr Asp His 210 215 220Tyr Ser Pro Ser Leu Gln Arg Thr Leu Phe
Leu Met Gly Gln Ala Val225 230 235 240Leu Thr Arg Cys Pro Glu Met
Ser Arg Ile Trp Leu Gln Met Pro Asn 245 250 255Lys His His Leu Gln
Tyr Asn Leu Glu Arg Phe Gly Leu Asp Asn Asn 260 265 270Leu Glu Ile
Phe His Val Asp Pro Glu Pro Tyr Gly Leu Met Glu Ala 275 280 285Trp
Val Glu Arg Ala 29029298PRTDeinococcus radiodurans 29Met Met Thr
Gly Thr Gln Gln Pro Gly Thr Gln Pro Lys Val Lys Val1 5 10 15Arg Leu
Gly Glu Asn Asn Tyr Gly Lys Ala Glu Val Gln Leu Met Lys 20 25 30Ile
Lys Arg Gly Thr Pro Arg His Glu Leu Arg Glu Ala Lys Val Arg 35 40
45Val Ala Met Tyr Gly Asp Phe Gly Ala Ala His Ser Glu Gly Asp Asn
50 55 60Thr Asp Leu Val Ala Thr Asp Thr Val Arg Asn Thr Val Tyr Gly
Leu65 70 75 80Ala Lys Glu Gly Phe Glu Ser Ser Ile Glu Glu Phe Gly
Lys Glu Leu 85 90 95Leu Thr His Phe Val Lys Val Gly Pro Arg Val Thr
Gly Gly Phe Ala 100 105 110Glu Phe Thr Glu His Leu Trp Glu Arg Val
Gln Thr Pro Ala Gln Pro 115 120 125Gln Gly His Asp His Ala Phe Val
Arg Gln Met Pro Lys Arg Thr Ala 130 135 140Arg Val Glu Thr Gln Asp
Gly Arg Arg Phe Thr Val Thr Ser Gly Ile145 150 155 160Glu Glu Leu
Tyr Val Leu Lys Thr Thr Glu Ser Gly Trp Glu Asn Tyr 165 170 175Leu
Leu Asp Glu Arg Phe Thr Thr Leu Pro Glu Thr His Asp Arg Val 180 185
190Met Ala Thr Phe Val Thr Ala Lys Trp Glu Tyr Ala Val Glu Ser Cys
195 200 205Asp Tyr Asp Ala Val Trp Glu Arg Val Tyr Arg Gln Ile Gln
His Thr 210 215 220Phe Thr Asp His Tyr Ser Pro Ser Leu Gln Arg Thr
Leu Tyr Leu Met225 230 235 240Gly Glu Ala Val Leu Ser Val Cys Pro
Glu Ile Ser Arg Ile Trp Phe 245 250 255Gln Met Pro Asn Lys His His
Leu Val Tyr Asn Leu Gly Arg Phe Gly 260 265 270Leu Glu Asn Asn Asn
Glu Ile Leu His Val Asp Pro Glu Pro Tyr Gly 275 280 285Leu Met Glu
Ala Trp Val Glu Arg Ala Glu 290 29530287PRTGranulicella tundricola
30Met Ala Glu Leu Thr Asp Ala Lys Phe Glu Ile Val Ala Asn Arg Tyr1
5 10 15Gly Lys Ser Lys Val Arg Leu Leu Lys Val Thr Arg Ala Glu Gly
Arg 20 25 30Ser Asp Val His Glu Trp Thr Val Gln Val Leu Leu Arg Gly
Asp Phe 35 40 45Glu Thr Ala His Thr Val Gly Asp Asn Ser Lys Ile Val
Thr Thr Asp 50 55 60Thr Met Lys Asn Thr Val Tyr Ser Leu Ala Arg Trp
Ser Ser Ala Thr65 70 75 80Thr Met Glu Glu Phe Ala Glu Glu Leu Ile
Glu His Leu Leu Arg Arg 85 90 95Asn Glu Gln Val Ser Ser Val Arg Val
His Ile Glu Ala Ala Leu Trp 100 105 110Lys Arg Leu Thr Val Asp Gly
Lys Glu His Pro Asp Thr Phe Met Arg 115 120 125Gly Ser Asn Glu Val
Gln Thr Ala Thr Val Glu Gln Ala Arg Ala Gly 130 135 140Glu Lys Lys
Phe Ile Ala Gly Phe Ala Asn Leu Gln Leu Leu Lys Thr145 150 155
160Ala Asn Ser Ala Phe Ser Gly Phe Gln Arg Asp Glu Leu Thr Thr Leu
165 170 175Pro Glu Thr Arg Asp Arg Val Phe Gly Thr Ala Val Asp Ala
Lys Trp 180 185 190Thr Tyr Ser Gly Pro Val Glu Phe Ala Ala Met Arg
Lys Ala Ala Arg 195 200 205Glu Val Met Leu Lys Val Phe Ala Asp His
Met Ser Glu Ser Val Gln 210 215 220His Thr Leu Tyr Ala Met Ala Asp
Ala Ala Leu Glu Ala Val Ala Glu225 230 235 240Ile Thr Glu Ile Glu
Leu Ala Met Pro Asn Lys His Cys Leu Leu Val 245 250 255Asp Leu Ser
Lys Phe Gly Gln Asp Asn Pro Asn Gln Ile Phe Val Pro 260 265 270Thr
Asp Glu Pro His Gly Tyr Ile Glu Ala Arg Val Arg Arg Lys 275 280
28531287PRTSolibacter usitatus 31Met Glu Arg Phe Ala Ser Gly Trp
Lys Gln Asn Tyr Tyr Gly Lys Gly1 5 10 15Asp Val Ile Val Tyr Arg Leu
Asn Arg Asp Gly Val Val Pro Gln Gly 20 25 30Cys Cys Pro Val Phe Gly
Ala Asn Val Lys Met Leu Leu Tyr Gly Asp 35 40 45Ala Phe Trp Pro Thr
Tyr Thr Thr Gly Asp Asn Thr Asn Leu Val Ala 50 55 60Thr Asp Ser Met
Lys Asn Phe Ile Gln Arg Glu Thr Cys Asn Phe Thr65 70 75 80Gly Tyr
Asp Leu Glu Ser Tyr Cys Asp Phe Leu Ala Arg Lys Phe Met 85 90 95Ala
Thr Tyr Pro His Thr Ala Gly Ile Gln Leu Ser Ala Arg Gln Ala 100 105
110Pro Tyr Ser Gly Val Ala Glu Gly Lys Val Ala Phe Ala Pro Ser Gly
115 120 125Pro Asp Val Ala Thr Ala Cys Val Glu Leu Arg Arg Asn Gly
Glu Ala 130 135 140Leu Glu Ser Val Glu Ala Ser Ser Gly Ile His Gly
Phe Arg Leu Leu145 150 155 160Arg Leu Gly Gly Ser Ala Phe Gln Gly
Phe Leu Arg Asp Gln Tyr Thr 165 170 175Thr Leu Pro Asp Ile His Asn
Arg Pro Leu His Met Trp Leu Asp Leu 180 185 190Glu Trp His Tyr Ile
Ala Pro Glu Ala Ala Leu Thr Gly Gly Glu Val 195 200 205Thr Ala Gln
Val Arg Arg Leu Val His Glu Gly Phe His Ser Phe Glu 210 215 220Ser
Gly Ser Ile Gln Gln Val Ile Tyr Gln Leu Gly Thr Lys Met Leu225 230
235 240Ala Asp Ile Pro Thr Ile Ser Glu Val Arg Leu Glu Ala Asn Asn
Arg 245 250 255Thr Trp Asp Thr Ile Val Glu Gln Gly Asp Arg Leu Gly
Val Tyr Thr 260 265 270Asp Ala Arg Pro Pro Tyr Gly Cys Leu Gly Leu
Thr Leu Arg Arg 275 280 28532280PRTTerriglobus saanensis 32Met Ala
Lys Leu Ile Asp Ser Arg Tyr Gly Lys Ala Arg Val Arg Val1 5 10 15Met
Lys Leu Asp Arg Ser Gln Pro Gln His Gln Leu Leu Glu Trp Thr 20 25
30Val Arg Val Leu Leu Glu Gly Asp Phe Glu Thr Ala His Thr Val Gly
35 40 45Asp Asn Ser Asn Ile Leu Pro Thr Asp Thr Met Lys Asn Thr Val
Tyr 50 55 60Ser Arg Ala Lys Glu Ser Lys Ala Glu Thr Pro Glu Glu Phe
Ala Ile65 70 75 80Glu Leu Ala Glu Phe Leu Leu Gly Arg Asn Pro Gln
Val His Thr Val 85 90 95Glu Val Lys Ile Glu Thr Ala Met Trp Lys Arg
Leu Val Val Asp Gly 100 105 110Lys Pro His Gly Ser Ser Phe Met Arg
Gly Ser Asp Glu Leu Gly Thr 115 120 125Val Leu His His Ala Thr Arg
Glu Thr Lys Thr Met Val Cys Gly Val 130 135 140Glu Asn Met Val Ile
Leu Lys Ser Gln Asn Ser Ser Phe Glu Gly Tyr145 150 155 160Ile Gln
Asp Asp Leu Thr Thr Leu Lys Pro Thr Ala Asp Arg Leu Phe 165 170
175Ala Thr Ala Met Thr Ala Asp Trp Asp Tyr Thr Asp Gly Gly Ser Ala
180 185 190Phe Ala Ala Arg Arg Glu Ala Ile Arg Glu Ala Met Leu Lys
Ala Phe 195 200 205Ala Glu His Asp Ser Lys Ser Val Gln Gln Thr Leu
Tyr Ala Met Ala 210 215 220Glu Ala Ala Met Ala Ala Val Pro Ala Val
Asn Arg Val His Met Val225 230 235 240Met Pro Asn Lys His Cys Leu
Leu Val Asp Leu Lys His Phe Gly Gln 245 250 255Glu Asn Asn Asn Glu
Ile Phe Val Pro Thr Glu Asp Pro His Gly Tyr 260 265 270Ile Glu Ala
Thr Val Val Arg Glu 275 28033326PRTKyrpidia tusciae 33Met Ile Met
Thr Gly Thr Met Thr Ser Gly Thr Asp Gln Arg Thr Met1 5 10 15Tyr Tyr
Gly Lys Gly Asp Val Trp Val Tyr Arg Ser Tyr Ala Lys Pro 20 25 30Leu
Arg Gly Leu Gly Gln Ile Pro Glu Ser Ala Phe Ala Gly Arg Pro 35 40
45Asn Val Ile Phe Gly Met Asn Val Gln Met Ala Val Glu Gly Glu Ala
50 55 60Phe Leu Pro Ser Phe Thr Glu Gly Asp Asn Ser Met Val Val Ala
Thr65 70 75 80Asp Ser Met Lys Asn Phe Ile Leu Arg Gln Ala Gly Ala
Phe Glu Gly 85 90 95Ala Thr Ala Glu Gly Phe Leu Glu Phe Val Ala Gly
Lys Phe Leu Glu 100 105 110Lys Tyr Ala His Val Ser Gly Val Arg Leu
Phe Gly Arg Gln Ile Pro 115 120 125Phe Asp Glu Leu Pro Val Pro Glu
Gln Glu Gly Phe Arg Pro Gly Glu 130 135 140Leu Val Phe Arg Tyr Ser
Met Asn Glu Tyr Pro Thr Ala Phe Val Ala145 150 155 160Val Arg Arg
Gly Pro Glu Gly Pro Val Val Val Glu His Ala Gly Gly 165 170 175Val
Ala Gly Leu Lys Leu Ile Lys Ile Lys Gly Ser Ser Phe Tyr Gly 180 185
190Tyr Ile His Asp Glu Tyr Thr Thr Leu Pro Glu Ala Gln Asp Arg Pro
195 200 205Leu Phe Ile Tyr Leu Tyr Ile Lys Trp Lys Tyr Glu His Pro
Glu Asp 210 215 220Phe Arg Ala Glu His Pro Glu Arg Tyr Val Ala Ala
Glu Gln Val Arg225 230 235 240Asp Ile Ala His Thr Val Phe His Glu
Leu Thr Ser Pro Ser Ile Gln 245 250 255Asn Leu Ile Tyr His Ile Gly
Arg Arg Val Leu Thr Arg Phe Pro Gln 260 265 270Leu Leu Glu Val Ser
Phe Glu Ala Asn Asn Arg Thr Trp Glu Thr Val 275 280 285Leu Glu Glu
Val Glu Asp Leu Ala Gly Lys Arg Ala Glu Ala Lys Val 290 295 300Tyr
Thr Glu Pro Arg Pro Pro Tyr Gly Phe Gln Gly Phe Thr Val Thr305 310
315 320Arg Lys Asp Leu Glu Glu 32534304PRTArtificial
SequenceConsensus Uricase sequence from alignment 34Met Thr Ala Thr
Ala Glu Thr Ser Thr Gly Thr Lys Ile Val Leu Gly1 5 10 15Gln Asn Gln
Tyr Gly Lys Ala Glu Val Arg Val Val Lys Ile Thr Arg 20 25 30Asp Gly
Asp Thr His His Ile Lys Asp Leu Asn Val Ser Val Ala Leu 35 40 45Ser
Gly Asp Met Asp Ala Val His Leu Ser Gly Asp Asn Ala Asn Val 50 55
60Leu Pro Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Lys Glu65
70 75 80His Gly Ile Gly Ser Ala Glu Gln Phe Gly Ile Arg Leu Ala Arg
His 85 90 95Phe Val Thr Ser Gln Glu Pro Ile His Gly Ala Arg Ile Arg
Ile Glu 100 105 110Glu Tyr Ala Trp Glu Arg Ile Glu Thr Ser His Asp
His Ser Phe Val 115 120 125Arg Lys Gly Gln Glu Thr Arg Thr Ala Gln
Ile Thr Tyr Asp Gly Asp 130 135 140Trp Glu Val Val Ser Gly Leu Lys
Asp Leu Thr Val Leu Asn Ser Thr145 150 155 160Gly Ser Glu Phe Trp
Gly Tyr Val Lys Asp Lys Tyr Thr Thr Leu Pro 165 170 175Glu Thr Tyr
Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg 180 185 190Tyr
Asn Trp Thr Asp Asp Gln Pro Met Pro Asp Trp Asp Lys Ser Tyr 195 200
205Glu Gln Val Arg Lys His Leu Leu Glu Ala Phe Ala Glu Thr Tyr Ser
210 215 220Leu Ser Leu Gln Gln Thr Leu Tyr Gln Met Gly Ser Arg Val
Leu Glu225 230 235 240Ala Arg Pro Glu Ile Asp Glu Ile Arg Phe Ser
Leu Pro Asn Lys His 245 250 255His Phe Leu Val Asp Leu Glu Pro Phe
Gly Leu Asp Asn Asp Asn Glu 260 265 270Val Tyr Phe Ala Ala Asp Arg
Pro Tyr Gly Leu Ile Glu Ala Thr Val 275 280 285Leu Arg Asp Gly Ala
Glu Pro Arg Ile Pro Val Asp Met Thr Asn Leu 290 295 300
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