U.S. patent application number 10/645723 was filed with the patent office on 2004-12-23 for novel mutated form of arginine deiminase.
This patent application is currently assigned to Phoenix Pharmacologics, Inc.. Invention is credited to Clark, Mike A., Ensor, Charles Mark, Holtsberg, Frederick Wayne.
Application Number | 20040258675 10/645723 |
Document ID | / |
Family ID | 24254954 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040258675 |
Kind Code |
A1 |
Ensor, Charles Mark ; et
al. |
December 23, 2004 |
Novel mutated form of arginine deiminase
Abstract
The present invention discloses arginine deiminase that is
genetically modified for more efficient manufacturing and
processing. The present invention discloses recombinant DNA
molecules and vectors and other therapeutic and pharmaceutical
compositions. The present invention also discloses methods for
preparing modified arginine deiminase as well as methods of
treating cancer and other disease states using modified arginine
deiminase.
Inventors: |
Ensor, Charles Mark;
(Lexington, KY) ; Holtsberg, Frederick Wayne;
(Nicholasville, KY) ; Clark, Mike A.; (Big Pine,
FL) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
Phoenix Pharmacologics,
Inc.
|
Family ID: |
24254954 |
Appl. No.: |
10/645723 |
Filed: |
August 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10645723 |
Aug 21, 2003 |
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09564559 |
May 4, 2000 |
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6635462 |
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09564559 |
May 4, 2000 |
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09023809 |
Feb 13, 1998 |
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6183738 |
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60046200 |
May 12, 1997 |
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Current U.S.
Class: |
424/94.4 ;
435/191 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 38/00 20130101; A61P 35/04 20180101; C12Y 305/03006 20130101;
C12N 9/96 20130101; C12N 9/14 20130101; A61P 35/00 20180101; Y02A
50/401 20180101; A61P 35/02 20180101; C12N 9/78 20130101; A61P
33/00 20180101; A61K 47/60 20170801 |
Class at
Publication: |
424/094.4 ;
435/191 |
International
Class: |
A61K 038/44; C12N
009/06 |
Claims
1-70. (canceled).
71. An arginine deiminase comprising arginine deiminase isolated
from Mycoplasma hominis that has been modified by elimination of at
least one pegylation site at or adjacent to the catalytic region of
the polypeptide.
72. The arginine deiminase of claim 71 wherein the arginine
deiminase has been modified by deletion or substitution of at least
one amino acid.
73. The arginine deiminase of claim 71 covalently bound to
polyethylene glycol.
74. The arginine deiminase of claim 73 wherein the arginine
deiminase is covalently bound to polyethylene glycol via a linking
group.
75. A composition comprising the arginine deiminase of any one of
claims 71 to 74 and at least one carrier, diluent, or excipient.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to mutations of arginine
deiminase for more efficient production and processing and thus
improved treatment of cancer and other disease states.
BACKGROUND OF THE INVENTION
[0002] It is presently believed that the amino acid arginine may
play an important role in mediating certain disease states. For
example, it has been reported that tumors such as melanomas,
hepatomas, sarcomas and leukemia require arginine for growth.
Sugimura et al., Melanoma Res. 2: 191-196 (1992); Takaku et al.,
Int. J. Cancer 51:244-249 (1992); Miyazaki et al, Cancer Res.
50:4522-4527 (1990); J. B. Jones, "The Effect of Arginine Deiminase
on Murine Leukemic Lymphoblasts," Ph.D. Dissertation, The
University of Oklahoma, pages 1 -165 (1981). Malignant melanoma
(stage 3) and hepatoma are fatal diseases which kill most patients
within one year of diagnosis. In the United States, approximately
16,000 people die from these diseases annually. The incidence of
melanoma is rapidly increasing in the United States and is even
higher in other countries, such as Australia. The incidence of
hepatoma, in parts of the world where hepatitis is endemic, is even
greater. For example, hepatoma is one of the leading forms of
cancer in Japan and Taiwan.
[0003] It has also been reported that many protozoa require
arginine for growth and thus arginine may play an important role in
many parasitic diseases. van Wagtendonk et al., "Nitrogen
Metabolism in Protozoa", in Comparative Biochemistry of Nitrogen
Metabolism, pages 1-56 (J. W. Campbell ed. 1970). Arginine (derived
from the circulation) has also been shown to be a source of nitrous
oxide which can play an important role in mediating septic shock.
Chang et al., Am. J Physiol. 274:H342-H348 (1998); McDonald et al.,
J. Biol. Chem. 272:31213-31216 (1997). Effective treatments for
these diseases are urgently needed.
[0004] It has been reported that enzymes which degrade
non-essential amino acids, such as arginine, may be an effective
means of controlling some forms of cancer. For example, arginine
deiminase isolated from Pseudomonas pudita was described by J. B.
Jones, "The Effect of Arginine Deiminase on Murine Leukemic
Lymphoblasts," Ph.D. Dissertation, The University of Oklahoma,
pages 1-165 (1981). Because arginine deiminase catalyzes the
conversion of arginine into citrulline, thus helping to eliminate
arginine from the circulation of animals, it is believed that
arginine deiminase can be used as an effective therapy for cancer
and other disease states where arginine plays a role. Although
arginine deiminase is not produced in mammals, it is found in a
variety of bacteria, fungi and mycoplasma. Arginine deiminase can
be thus be isolated from those organisms which produce it or, in
the alternative, the enzyme may be produced using recombinant DNA
technology. Misawa et al., J. Biotechnol. 36: 145-155 (1994).
[0005] Certain disadvantages have come to be associated with the
isolation of arginine deiminase from organisms. Although effective
in killing tumor cells in vitro, arginine deiminase isolated from
Pseudomonas pudita failed to exhibit efficacy in vivo because it
had little enzyme activity at a neutral pH and was rapidly cleared
from the circulation of experimental animals. Arginine deiminase
derived from Mycoplasma arginini is described, for example, by
Takaku et al, Int. J. Cancer, 51:244-249 (1992), and U.S. Pat. No.
5,474,928, the disclosures of which are hereby incorporated by
reference herein in their entirety. A problem associated with the
therapeutic use of such a heterologous protein is its antigenicity.
The chemical modification of arginine deiminase from Mycoplasma
arginini, via a cyanuric chloride linking group, with polyethylene
glycol was described by Takaku et al., Int. J. Cancer Res.
84:1195-1200 (1993). The modified protein was toxic when
metabolized due to the release of cyanide from the cyanuric
chloride linking group.
[0006] The production of arginine deiminase via recombinant DNA
techniques also provides for certain disadvantages. For example,
arginine deiminase produced in Escheria coli is enzymatically
inactive and thus must be denatured and then properly renatured in
order for it to become enzymatically active. The usual method for
renaturing arginine deiminase produced in E. coli is to isolate the
inactive enzyme, dissolve it in guanidium hydrochloride and
renature it by rapid dilution into low ionic strength buffer. This
last step requires very large volumes of buffer thus making the
manufacture of arginine deiminase both expensive and time
consuming. However, recombinant technology does have certain
advantages. For example, organisms more amenable to fermentation
can be used as hosts. Additionally, these fermentation hosts are
generally much less pathogenic and larger amounts of arginine
deiminase can be obtained. It has been shown the E. coli may
produce large amounts of Mycoplasma arginine deiminase.
[0007] Another problem associated with arginine deiminase is that
the enzyme is highly antigenic and thus rapidly cleared from
circulation. Accordingly, arginine deiminase must be properly
formulated before being used as a therapeutic agent. For purposes
of the present invention, the term formulation can be defined as
the chemical modification of any arginine deiminase for purposes of
reducing antigenicity of the enzyme. For example, it has been shown
that the formulation of several proteins including arginine
deiminase with polyethylene glycol, i.e. pegylation, can reduce the
antigenicity of the protein and greatly increase its circulating
half-life. Unfortunately, the formulation of arginine deiminase
with polyethylene glycol often inactivates the enzyme.
[0008] There is a need for methods and compounds which address
these problems associated with the prior art. The present invention
is directed to these, as well as other, important ends.
SUMMARY OF THE INVENTION
[0009] The present invention relates to a modified arginine
deiminase enzyme capable of more efficient production and
processing.
[0010] The present invention further relates to a recombinant DNA
molecule which encodes modified arginine deiminase.
[0011] In related aspects, the present invention further relates to
recombinant vectors which comprise a nucleotide sequence that
encodes modified arginine deiminase and to host cells comprising
such vectors.
[0012] The present invention further relates to methods for
preparing the modified arginine deiminase enzyme of the invention.
These methods comprise growing a host cell transformed with the
recombinant DNA molecule of this invention in a suitable culture
medium.
[0013] In other aspects, this invention to methods of treating
cancer as well as treating and/or inhibiting the metastasis of
tumor cells. The invention also relates to methods of treating
parasitic disease, septic shock and other disease states.
[0014] These and other aspects of the present invention will be
elucidated in the following detailed description of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 depicts the amino acid sequence of arginine deiminase
cloned from wild-type Mycoplasma hominus.
[0016] FIG. 2 depicts the amino acid sequence of modified arginine
deiminase from Mycoplasma hominus in accordance with preferred
embodiments of the present invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0017] The present invention is based on the discovery that
modifications of one or more of the naturally occurring amino acid
residues of arginine deiminase from Mycoplasma hominus can provide
for an enzyme that is more easily renatured and formulated thereby
improving existing techniques for the manufacture of arginine
deiminase and therapeutic compositions comprising the same. The
therapeutic compositions of the invention may comprise recombinant
DNA molecules, recombinant vectors including plasmids, transformed
host cells and other pharmaceutical compositions. The therapeutic
compositions may also comprise biocompatible carriers or diluents
as known to those skilled in the art. The therapeutic compositions
of the invention are easily made sterile and are non-pyrogenic.
Such improved techniques and compositions are necessary for the
effective treatment of cancer and other disease states.
[0018] As used herein, the term "melanoma" may be a malignant or
benign tumor arising from the melanocytic system of the skin and
other organs, including the oral cavity, esophagus, anal canal,
vagina, leptomeninges, and/or the conjunctivae or eye. The term
"melanoma" includes, for example, acral-lentiginous melanoma,
amelanotic melanoma, benign juvenile melanoma, lentigo maligna
melanoma, malignant melanoma, nodular melanoma, subungual melanoma
and superficial spreading melanoma.
[0019] "Hepatoma" may be a malignant or benign tumor of the liver,
including, for example, hepatocellular carcinoma.
[0020] "Patient" refers to an animal, preferably a mammal, more
preferably a human.
[0021] "Biocompatible" refers to materials or compounds which are
generally not injurious to biological functions and which will not
result in any degree of unacceptable toxicity, including allergenic
and disease states.
[0022] "Polyethylene glycol" or "PEG" refers to mixtures of
condensation polymers of ethylene oxide and water, in a branched or
straight chain, represented by the general formula
H(OCH.sub.2CH.sub.2).sub.nOH, wherein n is at least 4. It is
preferred that the polyethylene glycol is a straight chain.
Generally, increasing the molecular weight of the polyethylene
glycol decreases the immunogenicity of arginine deiminase. The
polyethylene glycol may be used in conjunction with arginine
deiminase, and, optionally, a biocompatible linking group, to treat
cancer, including, for example, melanomas, hepatomas and sarcomas,
preferably melanomas.
[0023] Normal cells do not require arginine for growth because they
can synthesize arginine from citrulline in a two step process
catalyzed by arginosuccinate synthase and arginosuccinate lyase. In
contrast, melanomas, hepatomas and some sarcomas do not express
arginosuccinate synthase; therefore, they are auxotrophic for
arginine, i.e., they require arginine for growth. This metabolic
difference may be capitalized upon to develop a safe and effective
therapy to treat these forms of cancer. Arginine deiminase
catalyzes the conversion of arginine to citrulline, and may be used
to eliminate arginine. Thus, arginine deiminase may be utilized as
a treatment for melanomas, hepatomas, some sarcomas and other
disease states.
[0024] The amino acid sequences of arginine deiminase from the
Mycoplasma hominus gene is disclosed by FIGS. 1 or 2. Chemical and
genetic modification of the arginine deiminase enzyme can affect
its biological activities. For example, it has been shown that
arginine deiminase is typically antigenic and rapidly cleared from
circulation in a patient. However, it has also been shown that the
formulation of arginine deiminase with polyethylene glycol reduces
the antigenicity and increases the circulating half-life of the
enzyme. Abuchowski et al., Cancer Biochem. Biophys. 7:175-186
(1984); Abuchowski et al., J. Biol. Chem. 252:3582-3586 (1977). In
particular, arginine deiminase can be covalently modified with
polyethylene glycol. Arginine deiminase covalently modified with
polyethylene glycol (with or without a linking group) may be
hereinafter referred to as "ADI-PEG." In U.S. patent application
Ser. No. 09/023,809, Clark describes improved modifications of
arginine deiminase with polyethylene glycol, the disclosure of
which is hereby incorporated by reference herein in its entirety.
When compared to native arginine deiminase, ADI-PEG retains most of
its enzymatic activity, is far less antigenic, has a greatly
extended circulating half-life, and is much more efficacious in the
treatment of tumors. For purposes of the invention, the
modification of any arginine deiminase with polyethylene glycol may
be referred to as pegylation.
[0025] It is necessary, however, to modify arginine deiminase with
the maximum amount of polyethylene glycol in order to decrease
antigenicity. For example, arginine deiminase can be formulated
with the maximum amount of succinimidyl succinate polyethylene
glycol (SS-PEG). The SS-PEG attaches to primary amines on proteins
(the n-terminus and lysines) during the pegylation process.
Unfortunately, it has been shown that the pegylation process can
inactivate arginine deiminase if allowed to go to completion. While
not meant to limit the present invention in any way, it is
presently believed that arginine deiminase is inactivated because
polyethylene glycol attaches to sites on the enzyme which interfere
with the enzymes ability to catalyze a reaction. Thus, it has
traditionally been necessary to carefully determine the exact ratio
of SS-PEG to enzyme, concentration of reactant and time of reaction
in order to prevent inactivation while decreasing antigenicity
(i.e., optimal pegylation). Additionally, it becomes increasingly
difficult to control pegylation reactions as manufacturing scale
increases, and often it is necessary to inactivate the SS-PEG prior
to removal of excess polyethylene glycol.
[0026] It has also been shown that arginine deiminase produced by
recombinant technology, in particular arginine deiminase produced
in Escheria coli cells, is initially inactive. It has thus been
necessary to activate the recombinant arginine deiminase by
denaturing and then properly renaturing the enzyme. Typically, the
inactive enzyme has been isolated, dissolved in guanidium
hydrochloride, and then renatured via rapid dilution with low ionic
strength buffer. However, large volumes of buffer are required,
thus making the production of recombinant arginine deiminase both
expensive and time-consuming.
[0027] It has now been discovered that certain modifications of
arginine deiminase can facilitate both the renaturation and
formulation (i.e., pegylation) of the enzyme thereby improving
manufacturing processes. The present invention is based on the
unexpected discovery that certain amino acid changes in arginine
deiminase provide for excellent results in the recombinant
production and formulation of the enzyme. The production of
arginine deiminase has traditionally been expensive and time
consuming due to difficulties associated with recombinant
production and formulation. The present invention thus discloses a
modified arginine deiminase that provides for improved renaturation
and formulation processes while retaining the ability to convert
arginine to citrulline. For purposes of the present invention,
modified arginine deiminase can be defined as arginine deiminase
having one or more amino acid substitutions. It is to be understood
that modified arginine deiminase can include arginine deiminase
having a single amino acid substitution or a plurality of amino
acid substitutions. The invention also discloses a DNA sequence
which encodes for the modified arginine deiminase of the invention.
The modified arginine deiminase of the invention can provide for
excellent results in the treatment of certain types of cancer,
inhibiting the metastasis of cancer, and treating other disease
states.
[0028] It is to be understood that preferred embodiments of the
invention are based on the discovery that certain pegylation sites
associated with arginine deiminase may be located at or adjacent
the catalytic region of the enzyme. For purposes of the present
invention, the phrase "pegylation site" may be defined as any site
or position of arginine deiminase that may be covalently modified
with polyethylene glycol. A "pegylation site" can be considered
located at or adjacent the catalytic region of the enzyme where
pegylation of the site results in a significant reduction in
catalytic activity of the enzyme. The pegylation of such sites has
traditionally resulted in the inactivation of the enzyme. For
example, arginine deiminase from Mycoplasma hominus has a lysine at
the 112 position which is believed to be at or adjacent the
catalytic region of the enzyme. The attachment of polyethylene
glycol to this lysine at the 112 position may inactivate the
enzyme. In addition, arginine deiminase from Mycoplasma hominus has
a cysteine at the 397 position which is believed to be at or
adjacent the catalytic region of the enzyme. The importance of this
cysteine has been shown as it has now been discovered that amino
acid substitutions for cysteine at the 397 position may inactivate
the enzyme. In particular, it has been shown that substituting
alanine, histidine, arginine, serine, lysine or tyrosine for
cysteine at the 397 position results in a loss of all detectable
enzyme activity. Arginine deiminase from Mycoplasma hominus also
has three lysines located near this conserved cysteine, in
particular Lys.sup.374, Lys.sup.405 and LyS.sup.408. The attachment
of polyethylene glycol to Lys.sup.374, Lys.sup.405, Lys.sup.408 or
combinations thereof is believed to inactivate the enzyme.
[0029] It is to be understood that arginine deiminase derived from
other organisms may also have pegylation sites corresponding to 112
position of arginine deiminase from Mycoplasma hominus. For
example, arginine deiminase from Steptococcus pyrogenes has lysine
at the 104 position, arginine deiminase from Mycoplasma pneumoniae
has lysine at the 106 position, and arginine deiminase from Qiardia
intestinalis has lysine at the 114 position. In addition, arginine
deiminase from some organisms may have lysines corresponding to the
same general location as the 112 position of arginine deiminase
from Mycoplasma hominus. The location of lysine in arginine
deiminase from such organisms may be indicated as follows:
1TABLE 1 Pegylation sites of arginine deiminase from various
organisms Position of lysine in arginine Organisms producing
arginine deiminase deiminase Mycoplasma hominus 112 Mycoplasma
arginini 111 Clostridium perfringens 105 Bacillus licheniformis 97,
108 Borrelia burgdorferi 102, 111 Borrelia afzellii 101
Enterococcus faecalis 102, 110 Streptococcus pyogenes 104
Steptococcus pneumoniae 103 Lactobacillus sake 97, 106 Qiardia
intestinalis 114, 116
[0030] It is presently believed that the attachment of polyethylene
glycol to such lysines or combinations thereof may inactivate the
enzyme. It is presently believed that amino acid substitutions at
such lysines may result in a protein that loses less of its
enzymatic activity upon pegylation.
[0031] The present invention thus provides for certain amino acid
substitutions in the polypeptide chain of arginine deiminase. These
amino acid substitutions provide for modified arginine deiminase
that loses less activity upon pegylation; i.e. upon pegylation, the
reduction of enzyme activity following pegylation in the modified
arginine deiminases is less than the reduction of enzyme activity
following pegylation in the unmodified arginine deiminases. By
eliminating pegylation sites at or adjacent to the catalytic region
of enzyme, optimal pegylation can be achieved without the
traditional loss of activity. As discussed above, arginine
deiminase from certain organisms have pegylation sites located at
various positions on the peptide chain. While not limiting the
present invention, it is presently believed that arginine deiminase
may have the amino acid lysine located at or adjacent to the
catalytic region of the enzyme and that pegylation of these sites
may inactivate the enzyme. By eliminating at least one of these
pegylation sites, pegylation can be achieved and more enzyme
activity retained. In accordance with the invention, it is
preferred that lysine is substituted with glutamic acid, valine,
aspartic acid, alanine, isoleucine, leucine or combinations
thereof. More preferred is that lysine is substituted with glutamic
acid. In one embodiment of the invention, it is preferred that
modified arginine deiminase from Mycoplasma hominus has an amino
acid substitution at Lys.sup.112, Lys.sup.374, Lys405, Lys.sup.408
or combinations thereof. Preferably, modified arginine deiminase
from Mycoplasma hominus has an amino acid substitution Lys.sup.112
preferred is that modified arginine deiminase from Mycoplasma
hominus has lysine at position 112 substituted with glutamic
acid.
[0032] It is to be understood that other preferred embodiments of
the invention are based on the discovery that certain structural
characteristics of arginine deiminase may prevent or interfere with
the proper and rapid renaturation of arginine deiminase when
produced via recombinant technology. In particular, these
structural characteristics hinder or prevent the enzyme from
assuming an active conformation during recombinant production. For
purposes of the present invention, the phrase "active conformation"
may be defined as a three-dimensional structure that allows for
enzymatic activity by unmodified or modified arginine deiminase.
The active conformation may, in particular, be necessary for
catalyzing the conversion of arginine into citrulline. The phrase
"structural characteristic" may be defined as any trait, quality or
property of the polypeptide chain resulting from a particular amino
acid or combination of amino acids. For instance, arginine
deiminase may contain an amino acid that results in a bend or kink
in the normal peptide chain and thus hinders the enzyme from
assuming an active conformation during renaturation of the enzyme.
In particular, arginine deiminase from Mycoplasma hominus has a
proline at the 210 position that may result in a bend or kink in
the peptide chain, making it more difficult to renature the enzyme
during recombinant production. It is to be understood that arginine
deiminase derived from other organisms may also have sites
corresponding to the 210 position of arginine deiminase from
Mycoplasma hominus.
[0033] The present invention thus again provides for certain amino
acid substitutions in the polypeptide chain of arginine deiminase.
Such amino acid substitutions can eliminate the problematic
structural characteristics in the peptide chain of arginine
deiminase. Such amino acid substitutions provide for improved
renaturation of the modified arginine deiminase. These amino acid
substitutions make possible rapid renaturing of modified arginine
deiminase using reduced amounts of buffer. These amino acid
substitutions may also provide for increased yields of renatured
modified arginine deiminase. In one embodiment of the invention, it
is preferred that the modified arginine deiminase have a single
amino acid substitution at Pro.sup.210. As mentioned above,
arginine deiminase derived from Mycoplasma hominus has the amino
acid proline located at the 210 position. While not limiting the
present invention, it is presently believed that the presence of
the amino acid proline at position 210 results in a bend or kink in
the normal polypeptide chain that increases the difficulty of
renaturing (i.e., refolding) arginine deiminase. Substitutions for
proline at position 210 make possible the rapid renaturation of
modified arginine deiminase using reduced amounts of buffer.
Substitutions for proline at position 210 may also provide for
increased yields of renatured modified arginine deiminase. In a
preferred embodiment, the proline at position 210 is substituted
with serine. It is to be understood that in accordance with this
aspect of the invention, other substitutions at position 210 may be
made. Examples of preferred substitutions include Pro.sup.210to
Thr.sup.210, Pro.sup.210to Arg.sup.210, Pro.sup.210to Asn.sup.210,
Pro.sup.210to Gln.sup.210 or Pro.sup.210 to Met.sup.210. By
eliminating those structural characteristics associated with the
amino acid of position 210 of the wild-type arginine deiminase,
proper refolding of the enzyme can be achieved.
[0034] In another embodiment of the invention, it is preferred that
the modified arginine deiminase have multiple amino acid
substitutions. The modified arginine deiminase may have at least
one amino acid substitution eliminating pegylation sites at or
adjacent a catalytic region of the enzyme. The modified arginine
deiminase may also have at least one amino acid substitution
eliminating those structural characteristics that interfere with
the renaturation of the enzyme. The amino acid substitutions may
thus provide for a modified arginine deiminase of the invention.
The amino acid substitutions may provide for the pegylation of
modified arginine deiminase without a loss of enzymatic activity.
The amino acid substitutions may provide for a modified arginine
deiminase that can be rapidly renatured using reduced amounts of
buffer. The amino acid substitutions may also provide for increased
yields of renatured modified arginine deiminase. In a preferred
embodiment, the modified arginine deiminase derived from Mycoplasma
hominus includes the proline at position 210 substituted with
serine and the lysine at position 112 substituted with glutamic
acid. As discussed above, however, it is to be understood that the
modified arginine deiminase may include other preferred
substitutions.
[0035] It is preferred that the modified arginine deiminase of the
invention is derived from Mycoplasma hominus. In accordance with
the invention, however, it will be understood by those skilled in
the art that modified arginine deiminase may be derived from other
organisms. For example, as discussed above, modified arginine
deiminase of the invention can be derived from Steptococcus
pyrogenes, Mycoplasma pneumoniae, Qiardia intestinalis, Mycoplasma
hominus, Mycoplasma arginini, Clostridium perfringens, Bacillus
licheniformis, Borrelia burgdorferi, Borrelia afzellii,
Enterococcus faecalis, Streptococcus pyogenes, Steptococcus
pneumoniae, Lactobacillus sake, Qiardia intestinalis. It is to be
understood that modified arginine deiminase may have one or more
amino acid substitutions, in accordance with the various aspects of
the invention.
[0036] It is to be understood that in certain embodiments of the
present invention, modified arginine deiminase can be formulated
with polyethylene glycol. The present invention provides for the
attachment of polyethylene glycol to modified arginine deiminase
for increasing circulation half-life, without the traditional
inactivation of the enzyme. Traditionally, selection of the
attachment site of polyethylene glycol on arginine deiminase was
determined by the role of each of the sites within the active
domain of the protein, as would be known to the skilled artisan. In
accordance with the present invention, polyethylene glycol may be
attached to a primary amine of the modified arginine deiminase. The
present invention provides for the attachment of polyethylene
glycol to the primary amines of arginine deiminase without
substantial loss of enzymatic activity. For example, modified
arginine deiminase from Mycoplasma hominus contains lysine amino
acids that may be modified with polyethylene glycol. In other
words, these lysines are all possible points at which modified
arginine deiminase can be attached to polyethylene glycol. It is to
be understood, however, that polyethylene glycol may also be
attached to other sites on modified arginine deiminase, as would be
apparent to those skilled in the art. Increasing the number of
polyethylene glycol units on modified arginine deiminase increases
the circulating half life of the enzyme without the traditional
decrease in specific activity of the enzyme.
[0037] The linking group used to covalently attach polyethylene
glycol to modified arginine deiminase may be any biocompatible
linking group. As discussed above, "biocompatible" indicates that
the compound or group is non-toxic and may be utilized in vitro or
in vivo without causing injury, sickness, disease or death.
Polyethylene glycol can be bonded to the linking group, for
example, via an ether bond, an ester bond, a thiol bond or an amide
bond. Suitable biocompatible linking groups include, for example,
an ester group, an amide group, an imide group, a carbamate group,
a carboxyl group, a hydroxyl group, a carbohydrate, a maleimide
group (including, for example, succinimidyl succinate (SS),
succinimidyl propionate (SPA), succinimidyl carboxymethylate (SCM),
succinimidyl succinamide (SSA) or N-hydroxy succinimide (NHS)), an
epoxide group, an oxycarbonylimidazole group (including, for
example, carbonyldimidazole (CDI)), a nitro phenyl group
(including, for example, nitrophenyl carbonate (NPC) or
trichlorophenyl carbonate (TPC)), a trysylate group, an aldehyde
group, an isocyanate group, a vinylsulfone group, a tyrosine group,
a cysteine group, a histidine group or a primary amine. Preferably,
the biocompatible linking group is an ester group and/or a
maleimide group. More preferably, the linking group is SS, SPA,
SCM, SSA or NHS; with SS, SPA or NHS being more preferred, and with
SS or SPA being most preferred.
[0038] Alternatively, polyethylene glycol may be coupled directly
to modified arginine deiminase (i.e., without a linking group)
through an amino group, a sulfhydral group, a hydroxyl group or a
carboxyl group. Polyethylene glycol may be covalently bonded to
modified arginine deiminase via a biocompatible linking group,
using methods known in the art, as described, for example, by Park
et al, Cancer Res., 33:3-14 (1973); and Zaplipsky and Lee,
Polyethylene Glycol Chemistry: Biotechnical and Biomedical
Applications, J. M. Harris, ed., Plenum Press, N.Y., Chapter 21
(1992), the disclosures of which are hereby incorporated by
reference herein in their entirety.
[0039] The present invention also relates to recombinant DNA
molecules encoding the modified arginine deiminase of the
invention. It is preferred that the DNA molecules comprising the
recombinant DNA molecules of this invention are derived from any
Mycoplasma hominus strain using known techniques, e.g., isolating
the gene from a gene bank, making complementary or cDNAs from a
mRNA template or via the polymerase chain reaction(see, U.S. Pat.
No.4,800,159) or from isolates of clinical specimens.
Alternatively, such recombinant DNA molecules may be synthesized by
standard DNA synthesis techniques. Various Mycoplasma hominus
strains are also publicly available from commercial depositories,
e.g., from the American Type Culture Collection (ATCC), Bethesda,
Md., U.S.A.
[0040] The DNA molecules of this invention may comprise additional
DNA sequences including, for example, a regulatory element, one or
more selectable markers, and sequences that code for replication
and maintenance functions. The regulatory region typically contains
a promoter found upstream from the coding sequence of this
invention, which functions in the binding of RNA polymerase and in
the initiation of RNA transcription. In other words, the regulatory
element or region can be operatively linked to the coding sequence
of this invention. It will be appreciated by one of skill in the
art that the selection of regulatory regions will depend upon the
host cell employed.
[0041] The invention also relates to recombinant vectors,
particularly recombinant plasmids that comprise sequences encoding
the modified arginine deiminase of the invention.
[0042] Another aspect of this invention is a host cell transformed
with the recombinant DNA molecule of this invention. Such host cell
is capable of growth in a suitable culture medium and expressing
the coding sequence of the invention. Such host cell can be
prepared by methods of this invention, e.g., by transforming a
desired host cell with the plasmid of this invention. Such
transformation can be accomplished by utilization of conventional
transformation techniques. Furthermore, the recombinant DNA
molecule of this invention can be integrated into the host cell's
genome by conventional techniques, e.g., homologous recombination.
Those host cells suitable for use in the present invention include,
but are not limited to, mammalian cells, insect cells, yeast and
other bacteria cells, e.g., Streptomyces, Bacillus and Salmonella.
It is preferred that the host cells of this invention include those
belonging to the species E. coli. This invention and the product
thereof is not limited to any specific host cell. It is preferred
that the modified arginine deiminase is produced by the transformed
host cell of this invention, but such enzyme can be prepared by
conventional peptide synthesis techniques.
[0043] The present invention also relates to methods of producing
the enzyme encoded by the recombinant DNA molecule of this
invention which comprises culturing the transformed host of the
invention in an appropriate culture media and the isolation of such
enzyme. For purposes of the present invention, the phrase
"appropriate culture media" can be defined as that media which
facilitates such host in expressing recombinant DNA molecules
encoding the modified arginine deiminase of the invention. It will
be appreciated by those skilled in the art that the appropriate
culture media will depend upon the host cell used. The isolation of
the enzyme so produced can be accomplished from a culture lysate of
the host, or if appropriate, directly from the host's culture
medium, and such isolation is carried out by conventional protein
isolation techniques.
[0044] In a preferred embodiment, the DNA coding sequence of the
modified arginine deiminase of the invention is expressed in a
transformed Mycoplasma hominus host cell. Preferably the Mycoplasma
hominus cell is deficient in arginine deiminase and thus requires
complementation. In such systems, sequences that encode the
modified arginine deiminase are typically located on a vector. Such
vectors contain sufficient amount of bacterial DNA to propagate the
vector in E. coli or some other suitable host. Such vector also
contains a sufficient amount of Mycoplasma hominus DNA flanking the
enzyme coding sequence so as to permit recombination between a
Mycoplasma hominus host deficient in the arginine deiminase gene
and the heterologous modified arginine deiminase gene. It is to be
understood by those skilled in the art that it is not essential to
use a Mycoplasma hominus host deficient in the modified arginine
deiminase gene, but that the absence of the gene in the host prior
to recombination will facilitate the screening and isolation of
recombinant hosts which have incorporated the gene of interest. The
recombinant Mycoplasma hominus arising from such homologous
recombination can then selected by standard techniques as known to
those skilled in the art.
[0045] The invention also encompasses methods for the treatment of
cancer and/or other disease states comprising the administration of
a therapeutically effective amount of one of the compounds of the
present invention. A therapeutically effective amount of one of the
compounds of the present invention is an amount that is effective
in reducing the incidence of the disease state. Where the disease
state is cancer, a therapeutically effective amount of one of the
compounds of the present invention can be an amount that is
effective to inhibit tumor growth. Generally, treatment is
initiated with small dosages which can be increased by small
increments until the optimum effect under the circumstances is
achieved. Generally, a therapeutic dosage of compounds of the
present invention may be from about 1 to about 200 mg/kg twice a
week to about once every two weeks. For example, the dosage may be
about 1 mg/kg once a week as a 2 ml intravenous injection to about
20 mg/kg once every 3 days. PEG-ADI may be mixed with a phosphate
buffered saline solution, or any other appropriate solution known
to those skilled in the art, prior to injection. The PEG-ADI
formulation may be administered as a solid (lyophalate) or as a
liquid formulation, as desired.
[0046] The methods of the present invention can involve either in
vitro or in vivo applications. In the case of in vitro
applications, including cell culture applications, the compounds
described herein can be added to the cells in cultures and then
incubated. The compounds of the present invention may also be used
to facilitate the production of monoclonal and/or polyclonal
antibodies, using antibody production techniques well known in the
art. The monoclonal and/or polyclonal antibodies can then be used
in a wide variety of diagnostic applications, as would be apparent
to one skilled in the art.
[0047] The in vivo means of administration of the compounds of the
present invention will vary depending upon the intended
application. As one skilled in the art will recognize,
administration of the PEG-ADI composition of the present invention
can be carried out, for example, orally, intranasally,
intraperitoneally, parenterally, intravenously, intralymphatically,
intratumorly, intramuscularly, interstitially, intra-arterially,
subcutaneously, intraocularly, intrasynovial, transepithelial, and
transdermally.
EXAMPLES
[0048] The invention is further demonstrated in the following
examples, which are for purposes of illustration, and are not
intended to limit the scope of the present invention.
Example 1
Cloning and Site-directed Mutagenisis of ADI
[0049] Cultures of Mycoplasma hominus (ATCC 23114) were obtained
rom the American Type Culture, Bethesda, Md. DNA was extracted and
the gene coding for arginine deiminase isolated by the polymerase
chain reaction.
[0050] The arginine deiminase gene was subcloned into pGEM-t and
the E. coli strain JM101. Site directed mutagenesis was performed
using the Altered Sites II kit (Promega, Madison, Wis.).
[0051] Modified arginine deiminase was expressed in JM101 cells as
previously described by Takaku et al., supra. The modified arginine
deiminase included glutamic acid at the 112 position and serine at
the 210 position. The amino acid sequence of modified arginine
deiminase from Mycoplasma hominus is described in FIG. 2.
Example 2
Renaturation and Purification of Enzymatically Active ADI
[0052] The modified arginine deiminase was isolated and purified as
previously described by Takaku et al., supra However, several
improvements were observed.
2TABLE 2 Renaturation of Arginine Deiminase Dilution Compound Time
(.degree. C.) Ratio Yield Arginine Deiminase 90 hours (15.degree.
C.) 1:200 70 mg/L Modified Arginine 6-12 hours (room temp.) 1:50
500 mg/L Deiminase
[0053] As indicated by Table 2 above, renaturation of the modified
arginine deiminase was completed at room temperature in about 6 to
12 hours using a 1:50 dilution ratio of guanidium hydrochloride
inclusion bodies in buffer. In contrast, Takaku et al. reported
that renaturation required 90 hours at 15.degree. C. using a 1:200
dilution ratio. In addition, the yield of modified arginine
deiminase was routinely about 500 mg per liter of fermentation
whereas Takaku et al. reported a yields of approximately 70 mg per
liter of fermentation.
Example 3
Formulation of Modified ADI with PEG
[0054] Modified arginine deiminase was formulated using SS-PEG as
previously described. The pegylation process was allowed to go
unchecked for over 4 hours without the modified arginine deiminase
becoming inactivated. In addition, it was not necessary to quench
the pegylation process through the addition of glycine. With
reference to Table 3, approximately 70-80% of enzymatic activity of
the modified arginine deiminase was retained.
3TABLE 3 Specific Enzyme Activity (IU/mg protein) Without
Pegylation Pegylated wild type ADI 20-21 5-8 modified ADI 20-21
12-16
[0055] This indicates that the modified arginine deiminase allows
for more consistent formulations. In addition, the modified
arginine deiminase allows for scaling up of the manufacturing
process as compared to the wild-type arginine deiminase.
[0056] Each of the patents, patent applications and publications
described herein are hereby incorporated by reference in their
entirety.
[0057] Various modifications of the invention, in addition to those
described herein, will be apparent to one skilled in the art in
view of the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
[0058] It is to be understood that the specification of the present
application hereby incorporates by reference the claims and their
disclosures in their entirety.
Sequence CWU 1
1
10 1 409 PRT Mycoplasma hominis 1 Met Ser Val Phe Asp Ser Lys Phe
Asn Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Thr
Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr
Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu
Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys Ile 50 55 60
Met Lys Asp Arg Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala 65
70 75 80 Glu Thr Tyr Asp Leu Ala Ser Lys Ala Ala Lys Glu Glu Phe
Ile Glu 85 90 95 Thr Phe Leu Glu Glu Thr Val Pro Val Leu Thr Glu
Ala Asn Lys Lys 100 105 110 Ala Val Arg Ala Phe Leu Leu Ser Lys Pro
Thr His Glu Met Val Glu 115 120 125 Phe Met Met Ser Gly Ile Thr Lys
Tyr Glu Leu Gly Val Glu Ser Glu 130 135 140 Asn Glu Leu Ile Val Asp
Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp 145 150 155 160 Pro Phe Ala
Ser Val Gly Asn Gly Val Thr Ile His Phe Met Arg Tyr 165 170 175 Ile
Val Arg Arg Arg Glu Thr Leu Phe Ala Arg Phe Val Phe Arg Asn 180 185
190 His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met Lys
195 200 205 Met Pro Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu
Thr Leu 210 215 220 Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr
Ile Thr Leu Leu 225 230 235 240 Ala Lys Asn Ile Lys Ala Asn Lys Glu
Val Glu Phe Lys Arg Ile Val 245 250 255 Ala Ile Asn Val Pro Lys Trp
Thr Asn Leu Met His Leu Asp Thr Trp 260 265 270 Leu Thr Met Leu Asp
Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala Asn 275 280 285 Asp Val Phe
Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala Glu 290 295 300 Pro
Gln Pro Gln Leu Asn Gly Leu Pro Leu Asp Lys Leu Leu Ala Ser 305 310
315 320 Ile Ile Asn Lys Glu Pro Val Leu Ile Pro Ile Gly Gly Ala Gly
Ala 325 330 335 Thr Glu Met Glu Ile Ala Arg Glu Thr Asn Phe Asp Gly
Thr Asn Tyr 340 345 350 Leu Ala Ile Lys Pro Gly Leu Val Ile Gly Tyr
Asp Arg Asn Glu Lys 355 360 365 Thr Asn Ala Ala Leu Lys Ala Ala Gly
Ile Thr Val Leu Pro Phe His 370 375 380 Gly Asn Gln Leu Ser Leu Gly
Met Gly Asn Ala Arg Cys Met Ser Met 385 390 395 400 Pro Leu Ser Arg
Lys Asp Val Lys Trp 405 2 409 PRT Mycoplasma hominis 2 Met Ser Val
Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile
Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25
30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile
35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val
Lys Ile 50 55 60 Met Lys Asp Arg Gly Ile Asn Val Val Glu Leu Thr
Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Lys Ala Ala
Lys Glu Glu Phe Ile Glu 85 90 95 Thr Phe Leu Glu Glu Thr Val Pro
Val Leu Thr Glu Ala Asn Lys Glu 100 105 110 Ala Val Arg Ala Phe Leu
Leu Ser Lys Pro Thr His Glu Met Val Glu 115 120 125 Phe Met Met Ser
Gly Ile Thr Lys Tyr Glu Leu Gly Val Glu Ser Glu 130 135 140 Asn Glu
Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp 145 150 155
160 Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Phe Met Arg Tyr
165 170 175 Ile Val Arg Arg Arg Glu Thr Leu Phe Ala Arg Phe Val Phe
Arg Asn 180 185 190 His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp
Pro Ala Met Lys 195 200 205 Met Pro Ile Glu Gly Gly Asp Val Phe Ile
Tyr Asn Asn Glu Thr Leu 210 215 220 Val Val Gly Val Ser Glu Arg Thr
Asp Leu Asp Thr Ile Thr Leu Leu 225 230 235 240 Ala Lys Asn Ile Lys
Ala Asn Lys Glu Val Glu Phe Lys Arg Ile Val 245 250 255 Ala Ile Asn
Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr Trp 260 265 270 Leu
Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala Asn 275 280
285 Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala Glu
290 295 300 Pro Gln Pro Gln Leu Asn Gly Leu Pro Leu Asp Lys Leu Leu
Ala Ser 305 310 315 320 Ile Ile Asn Lys Glu Pro Val Leu Ile Pro Ile
Gly Gly Ala Gly Ala 325 330 335 Thr Glu Met Glu Ile Ala Arg Glu Thr
Asn Phe Asp Gly Thr Asn Tyr 340 345 350 Leu Ala Ile Lys Pro Gly Leu
Val Ile Gly Tyr Asp Arg Asn Glu Lys 355 360 365 Thr Asn Ala Ala Leu
Lys Ala Ala Gly Ile Thr Val Leu Pro Phe His 370 375 380 Gly Asn Gln
Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met 385 390 395 400
Pro Leu Ser Arg Lys Asp Val Lys Trp 405 3 409 PRT Mycoplasma
hominis 3 Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr
Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro
Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu
Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys
Glu His Gln Ser Phe Val Lys Ile 50 55 60 Met Lys Asp Arg Gly Ile
Asn Val Val Glu Leu Thr Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp
Leu Ala Ser Lys Ala Ala Lys Glu Glu Phe Ile Glu 85 90 95 Thr Phe
Leu Glu Glu Thr Val Pro Val Leu Thr Glu Ala Asn Lys Lys 100 105 110
Ala Val Arg Ala Phe Leu Leu Ser Lys Pro Thr His Glu Met Val Glu 115
120 125 Phe Met Met Ser Gly Ile Thr Lys Tyr Glu Leu Gly Val Glu Ser
Glu 130 135 140 Asn Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe
Thr Arg Asp 145 150 155 160 Pro Phe Ala Ser Val Gly Asn Gly Val Thr
Ile His Phe Met Arg Tyr 165 170 175 Ile Val Arg Arg Arg Glu Thr Leu
Phe Ala Arg Phe Val Phe Arg Asn 180 185 190 His Pro Lys Leu Val Lys
Thr Pro Trp Tyr Tyr Asp Pro Ala Met Lys 195 200 205 Met Ser Ile Glu
Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu Thr Leu 210 215 220 Val Val
Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu Leu 225 230 235
240 Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg Ile Val
245 250 255 Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp
Thr Trp 260 265 270 Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser
Pro Ile Ala Asn 275 280 285 Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu
Val Asn Gly Gly Ala Glu 290 295 300 Pro Gln Pro Gln Leu Asn Gly Leu
Pro Leu Asp Lys Leu Leu Ala Ser 305 310 315 320 Ile Ile Asn Lys Glu
Pro Val Leu Ile Pro Ile Gly Gly Ala Gly Ala 325 330 335 Thr Glu Met
Glu Ile Ala Arg Glu Thr Asn Phe Asp Gly Thr Asn Tyr 340 345 350 Leu
Ala Ile Lys Pro Gly Leu Val Ile Gly Tyr Asp Arg Asn Glu Lys 355 360
365 Thr Asn Ala Ala Leu Lys Ala Ala Gly Ile Thr Val Leu Pro Phe His
370 375 380 Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met
Ser Met 385 390 395 400 Pro Leu Ser Arg Lys Asp Val Lys Trp 405 4
409 PRT Mycoplasma hominis 4 Met Ser Val Phe Asp Ser Lys Phe Asn
Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Thr Val
Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro
Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser
His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys Ile 50 55 60 Met
Lys Asp Arg Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala 65 70
75 80 Glu Thr Tyr Asp Leu Ala Ser Lys Ala Ala Lys Glu Glu Phe Ile
Glu 85 90 95 Thr Phe Leu Glu Glu Thr Val Pro Val Leu Thr Glu Ala
Asn Lys Glu 100 105 110 Ala Val Arg Ala Phe Leu Leu Ser Lys Pro Thr
His Glu Met Val Glu 115 120 125 Phe Met Met Ser Gly Ile Thr Lys Tyr
Glu Leu Gly Val Glu Ser Glu 130 135 140 Asn Glu Leu Ile Val Asp Pro
Met Pro Asn Leu Tyr Phe Thr Arg Asp 145 150 155 160 Pro Phe Ala Ser
Val Gly Asn Gly Val Thr Ile His Phe Met Arg Tyr 165 170 175 Ile Val
Arg Arg Arg Glu Thr Leu Phe Ala Arg Phe Val Phe Arg Asn 180 185 190
His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met Lys 195
200 205 Met Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu Thr
Leu 210 215 220 Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile
Thr Leu Leu 225 230 235 240 Ala Lys Asn Ile Lys Ala Asn Lys Glu Val
Glu Phe Lys Arg Ile Val 245 250 255 Ala Ile Asn Val Pro Lys Trp Thr
Asn Leu Met His Leu Asp Thr Trp 260 265 270 Leu Thr Met Leu Asp Lys
Asn Lys Phe Leu Tyr Ser Pro Ile Ala Asn 275 280 285 Asp Val Phe Lys
Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala Glu 290 295 300 Pro Gln
Pro Gln Leu Asn Gly Leu Pro Leu Asp Lys Leu Leu Ala Ser 305 310 315
320 Ile Ile Asn Lys Glu Pro Val Leu Ile Pro Ile Gly Gly Ala Gly Ala
325 330 335 Thr Glu Met Glu Ile Ala Arg Glu Thr Asn Phe Asp Gly Thr
Asn Tyr 340 345 350 Leu Ala Ile Lys Pro Gly Leu Val Ile Gly Tyr Asp
Arg Asn Glu Lys 355 360 365 Thr Asn Ala Ala Leu Lys Ala Ala Gly Ile
Thr Val Leu Pro Phe His 370 375 380 Gly Asn Gln Leu Ser Leu Gly Met
Gly Asn Ala Arg Cys Met Ser Met 385 390 395 400 Pro Leu Ser Arg Lys
Asp Val Lys Trp 405 5 409 PRT Mycoplasma arginini 5 Met Ser Val Phe
Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly
Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30
Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35
40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala
Glu 50 55 60 Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Ile Asp
Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys
Asp Lys Leu Ile Glu 85 90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val
Leu Ser Glu Glu His Lys Val 100 105 110 Val Val Arg Asn Phe Leu Lys
Ala Lys Lys Thr Ser Arg Lys Leu Val 115 120 125 Glu Ile Met Met Ala
Gly Ile Thr Lys Tyr Asp Leu Gly Ile Gly Ala 130 135 140 Asp His Glu
Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160
Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165
170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe
Ser 180 185 190 Asn His Pro Lys Leu Ile Asn Thr Pro Trp Tyr Tyr Asp
Pro Ser Leu 195 200 205 Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile
Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr
Asp Leu Gln Thr Val Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Val
Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn
Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu
Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285
Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290
295 300 Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu
Gln 305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile
Ala Gly Glu Gly 325 330 335 Ala Ser Gln Met Glu Ile Glu Arg Glu Thr
His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile Arg Pro Gly Val
Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu
Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380 His Gly Asn Gln
Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met
Pro Leu Ser Arg Lys Asp Val Lys 405 6 409 PRT Mycoplasma arginini 6
Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5
10 15 Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu
Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe
Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Lys
Gln Phe Val Ala Glu 50 55 60 Leu Lys Ala Asn Asp Ile Asn Val Val
Glu Leu Ile Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser
Gln Glu Ala Lys Asp Lys Leu Ile Glu 85 90 95 Glu Phe Leu Glu Asp
Ser Glu Pro Val Leu Ser Glu Glu His Glu Val 100 105 110 Val Val Arg
Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Lys Leu Val 115 120 125 Glu
Ile Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Gly Ala 130 135
140 Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg
145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His
Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser
Arg Phe Val Phe Ser 180 185 190 Asn His Pro Lys Leu Ile Asn Thr Pro
Trp Tyr Tyr Asp Pro Ser Leu 195 200 205 Lys Leu Ser Ile Glu Gly Gly
Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val
Ser Glu Arg Thr Asp Leu Gln Thr Val Thr Leu 225 230 235 240 Leu Ala
Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255
Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260
265 270 Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile
Ala 275 280 285 Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn
Gly Gly Ala 290 295 300 Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu
Glu Gly Leu Leu Gln 305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Val
Leu Ile Pro Ile Ala Gly Glu Gly 325 330 335 Ala Ser Gln Met Glu Ile
Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile
Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365 Lys Thr
Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380
His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser
385
390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys 405 7 409 PRT
Mycoplasma arthritidis 7 Met Ser Val Phe Asp Ser Lys Phe Lys Gly
Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Ser Val Leu
Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala
Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His
Asp Ala Arg Lys Glu Gln Ser Gln Phe Val Ala Ile 50 55 60 Leu Lys
Ala Asn Asp Ile Asn Val Val Glu Thr Ile Asp Leu Val Ala 65 70 75 80
Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Arg Lys Ile Glu 85
90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Ala His Lys
Lys 100 105 110 Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg
Lys Leu Val 115 120 125 Glu Leu Met Met Ala Gly Ile Thr Lys Tyr Asp
Leu Gly Val Glu Ala 130 135 140 Asp His Glu Leu Ile Val Asp Pro Met
Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val
Gly Asn Gly Val Thr Ile His Phe Met Arg 165 170 175 Tyr Lys Val Arg
Arg Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Arg 180 185 190 Asn His
Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala Met 195 200 205
Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210
215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Val Thr
Leu 225 230 235 240 Leu Ala Lys Asn Leu Val Ala Asn Lys Glu Cys Glu
Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn
Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asn
Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Val Phe Lys Phe
Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300 Glu Pro Gln Pro
Val Glu Asn Gly Leu Pro Leu Glu Lys Leu Leu Gln 305 310 315 320 Ser
Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu Gly 325 330
335 Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn
340 345 350 Tyr Ile Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg
Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Lys Ala Ala Gly Ile Lys
Val Leu Pro Phe 370 375 380 His Gly Asn Gln Leu Ser Leu Gly Met Gly
Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp
Val Lys 405 8 409 PRT Mycoplasma arthritidis 8 Met Ser Val Phe Asp
Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu
Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp
Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40
45 Leu Glu Ser His Asp Ala Arg Lys Glu Gln Ser Gln Phe Val Ala Ile
50 55 60 Leu Lys Ala Asn Asp Ile Asn Val Val Glu Thr Ile Asp Leu
Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp
Arg Lys Ile Glu 85 90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu
Ser Glu Ala His Glu Glu 100 105 110 Val Val Arg Asn Phe Leu Lys Ala
Lys Lys Thr Ser Arg Lys Leu Val 115 120 125 Glu Leu Met Met Ala Gly
Ile Thr Lys Tyr Asp Leu Gly Val Glu Ala 130 135 140 Asp His Glu Leu
Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp
Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Phe Met Arg 165 170
175 Tyr Lys Val Arg Arg Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Arg
180 185 190 Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro
Ala Met 195 200 205 Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr
Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp
Leu Asp Thr Val Thr Leu 225 230 235 240 Leu Ala Lys Asn Leu Val Ala
Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val
Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr
Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn
Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295
300 Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Lys Leu Leu Gln
305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala
Gly Glu Gly 325 330 335 Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His
Phe Asp Gly Thr Asn 340 345 350 Tyr Ile Ala Ile Arg Pro Gly Val Val
Ile Gly Tyr Ser Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Lys
Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380 His Gly Asn Gln Leu
Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro
Leu Ser Arg Lys Asp Val Lys 405 9 409 PRT Mycoplasma arthritidis 9
Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5
10 15 Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu
Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe
Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu Gln Ser
Gln Phe Val Ala Ile 50 55 60 Leu Lys Ala Asn Asp Ile Asn Val Val
Glu Thr Ile Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser
Gln Glu Ala Lys Asp Arg Lys Ile Glu 85 90 95 Glu Phe Leu Glu Asp
Ser Glu Pro Val Leu Ser Glu Ala His Glu Lys 100 105 110 Val Val Arg
Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Lys Leu Val 115 120 125 Glu
Leu Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Val Glu Ala 130 135
140 Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg
145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His
Phe Met Arg 165 170 175 Tyr Lys Val Arg Arg Arg Glu Thr Leu Phe Ser
Arg Phe Val Phe Arg 180 185 190 Asn His Pro Lys Leu Val Asn Thr Pro
Trp Tyr Tyr Asp Pro Ala Met 195 200 205 Lys Leu Ser Ile Glu Gly Gly
Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val
Ser Glu Arg Thr Asp Leu Asp Thr Val Thr Leu 225 230 235 240 Leu Ala
Lys Asn Leu Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255
Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260
265 270 Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile
Ala 275 280 285 Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn
Gly Gly Ala 290 295 300 Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu
Glu Lys Leu Leu Gln 305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Val
Leu Ile Pro Ile Ala Gly Glu Gly 325 330 335 Ala Ser Gln Met Glu Ile
Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Ile Ala Ile
Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365 Lys Thr
Asn Ala Ala Leu Lys Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380
His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385
390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys 405 10 409 PRT
Mycoplasma arthritidis 10 Met Ser Val Phe Asp Ser Lys Phe Lys Gly
Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Ser Val Leu
Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala
Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His
Asp Ala Arg Lys Glu Gln Ser Gln Phe Val Ala Ile 50 55 60 Leu Lys
Ala Asn Asp Ile Asn Val Val Glu Thr Ile Asp Leu Val Ala 65 70 75 80
Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Arg Lys Ile Glu 85
90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Ala His Lys
Glu 100 105 110 Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg
Lys Leu Val 115 120 125 Glu Leu Met Met Ala Gly Ile Thr Lys Tyr Asp
Leu Gly Val Glu Ala 130 135 140 Asp His Glu Leu Ile Val Asp Pro Met
Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val
Gly Asn Gly Val Thr Ile His Phe Met Arg 165 170 175 Tyr Lys Val Arg
Arg Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Arg 180 185 190 Asn His
Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala Met 195 200 205
Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210
215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Val Thr
Leu 225 230 235 240 Leu Ala Lys Asn Leu Val Ala Asn Lys Glu Cys Glu
Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn
Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asn
Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Val Phe Lys Phe
Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300 Glu Pro Gln Pro
Val Glu Asn Gly Leu Pro Leu Glu Lys Leu Leu Gln 305 310 315 320 Ser
Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu Gly 325 330
335 Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn
340 345 350 Tyr Ile Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg
Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Lys Ala Ala Gly Ile Lys
Val Leu Pro Phe 370 375 380 His Gly Asn Gln Leu Ser Leu Gly Met Gly
Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp
Val Lys 405
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