Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MODIFIED ARGININE DEIMINASE
Related A~~lications
This application is a continuation in part application of U.S. Patent
Application Serial No. 091023,809, allowed, which claims priority to U.S.
Provisional
Patent Application Serial No. 60/046,200, filed on May 12, 1997.
Field of the Invention
The present invention is directed to arginine deiminase modified with
polyethylene glycol, to methods for treating cancer, and to methods for
treating andlor
inhibiting metastasis.
Background of the Invention
Malignant melanoma (stage 3) and hepatoma are fatal diseases which bill
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.
Effective
treatments for these diseases are urgently needed.
Selective deprivation of essential amino acids has been used to treat some
forms of cancer. The best known example is the use of L-asparaginase to lower
levels of
asparagine as a treatment for acute lymphoblastic leukemia. The L-asparaginase
most
frequently used is isolated from E. coli. However, clinical use of this enzyme
is
compromised by its inherent antigenicity and short circulating half life, as
described by
~.K. Park, et al, Ahticafacef° Res., 1:373-376 (1981). Covalent
modification of E. coli L-
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asparaginase with polyethylene glycol reduces its antigenicity and prolongs
its circulating
half life, as described, for example, by Park, A~Zticahce~ Res., supra; Y.
Kamisalci et al, J.
Pharmacol. Exp. Tlaer., 216:410-414 (I98I); and Y. Kamisaki et al, Gahya.,
73:47-474
(1982). Although there has been a great deal of effort to identify other
essential amino
acid degrading enzymes for the treatment of cancer, none have been approved,
primarily
because deprivation of essential amino acids, by definition, results in
numerous, and
severe, side effects.
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, axginine deiminase (ADI) isolated from Pseudomofaas 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). Although
effective in
killing tumor cells in vitro, ADI isolated from P. 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. Patent No. 5,474,928, the disclosures of which are hereby incorporated by
reference
herein in their entirety. However, a problem associated with the therapeutic
use of such a
heterologous protein is its antigenicity. The chemical modification of
arginine deiminase
from Mycoplasma argihini, via a cyanuric chloride linking group, with
polyethylene
glycol was described by Takaku et al., Jpn. J. Cancer Res., 84:1195-1200
(1993).
However, the modified protein was toxic when metabolized due to the release of
cyanide
from the cyanuric chloride linl~ing group.
There is a need for compositions which degrade non-essential amino acids
and which do not have the problems associated with the prior art. The present
invention is
directed to these, as well as other, important ends.
Summary of the Invention
The present invention is directed to arginine deiminase modified with
polyethylene glycol. In a preferred embodiment, the arginine deiminase is
modified with
polyethylene glycol, having a total weight average molecular weight of about
1,000 to
about 50,000, directly or through a biocompatible linking group.
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Another embodiment of the invention is directed to methods of treating
cancer, including, for example, sarcomas, hepatomas and melanomas. The
invention is
also directed to methods of treating and/or inhibiting the metastasis of tumor
cells.
These and other aspects of the present invention will be elucidated in the
following detailed description of the invention.
Brief Description of the Drawi~s
Figure 1 depicts the amino acid sequences of arginine deiminase cloned
from Mycoplasma arginini (the top amino acid sequence SEQ ID NO: 1, identified
as
ADIPROT), Mycoplasma arthritides (the middle amino acid sequence SEQ ID NO: 2,
identified as ARTADIPRO), and Mycoplasma hominus (the bottom amino acid
sequence
SEQ ID NO: 3, identified as HOMADIPRO).
Figures 2A and 2B are graphs showing the effect of a single dose of native
arginine deiminase and arginine deiminase modified with polyethylene glycol
(e.g.,
molecular weight 5,000) on serum arginine levels and serum citrulline levels
in mice.
Figure 3 is a graph showing the effects on serum arginine levels when
PEG10,000 is covalently bonded to ADI via various linking groups.
Figure 4 is a graph showing the effect that the linking group and the
molecular weight of the polyethylene glycol have on citrulline production in
mice injected
with a single dose of PEG-ADI.
Figures 5A and SB are graphs showing the dose response that ADI-SS-
PEG5,000 had on serum arginine and citrulline levels. Figures 5C and SD are
graphs
showing the dose response that ADI-SS-PEG20,000 had on serum arginine and
citrulline
levels.
Figure 6 is a graph showing the antigenicity of native ADI, ADI-SS-
PEG5,000, and ADI-SS-PEG20,000.
Figure 7 is a graph showing the effect that treatments with ADI-SS-
PEG5,000, ADI-SS-PEG12,000 or ADI-SS-PEG20,000 had on tumor size in mice which
were inj ected with SK-mel 2 human melanoma cells.
Figure 8 is a graph showing the effect that treatments with ADI-
PEG20,000 had on tumor size in mice which were injected with SK-mel 28, SK-mel
2 or
M24-met human melanoma cells.
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Figure 9 is a graph showing the effect that treatments with ADI-PEG5,000,
ADI-PEG12,000 or ADI-PEG20,000 had on the survival of mice which were inj
ected with
human hepatoma SK-Hepl cells.
Figure 10 depicts the amino acid sequences of arginine deiminase cloned
from Steptococcus pyogenes (the top amino acid sequence SEQ ID NO: 6,
identified as
STRADIPYR) and Steptococcus pneunaoniae(the bottom amino acid sequence SEQ ID
NO: 7, identified as STRADIPNE).
Figure 11 depicts the amino acid sequences of arginine deiminase cloned
from Bo~y~elia bu~gdo~fe~i (the top amino acid sequence SEQ ID NO: 8,
identified as
BORADIBUR) and Borrelia afzelii (the bottom amino acid sequence SEQ ID NO: 9,
identified as BORADIAFZ).
Figure 12 depicts the amino acid sequence of Qia~diu intestinalis (the top
amino acid sequence SEQ ID NO: 10, identified as QIAADI1NT), Clostridium
peyf~ingens
(the middle amino acid sequence SEQ ID NO: 11, identified as CLOADIPER) and
Bacillus lichenifof°mis (the bottom amino acid sequence SEQ ID NO: 12,
identified as
BACADILIC).
Figure 13 depicts the amino acid sequence of Ente~ococcus faecalis (the
top amino acid sequence SEQ ID NO: 13, identified as ENTADIFAE) and
Lactobacillus
sake (the bottom amino acid sequence SEQ ID NO: 14, identified as LACADISAK).
Detailed Description of the Invention
Normal cells do not require arginine for growth, since they can synthesize
arginine from citrulline in a two step process catalyzed by argininosuccinate
synthase and
argininosuccinate lyase. In contrast, melanomas, hepatomas and some sarcomas
do not
express arginosuccinate synthase; therefore, they are auxotrophic for
arginine. 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 and some sarcomas.
Native arginine deiminase may be found in microorgaiusms and is antigenic
and rapidly cleared from circulation in a patient. These problems may be
overcome by
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covalently modifying arginine deiminase with polyethylene glycol (PEG).
Arginine
deiminase covalently modified with polyethylene glycol (with or without a
linking group)
may be hereinafter referred to as "ADI-PEG." 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.
"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(OCHZCHZ)"OH, wherein n is at least 4. "Polyethylene glycol"
or
"PEG" is used in combination with a numeric suffix to indicate the approximate
weight
average molecular weight thereof. For example, PEG5,000 refers to polyethylene
glycol
having a total weight average molecular weight of about 5,000; PEG12,000
refers to
polyethylene glycol having a total weight average molecular weight of about
12,000; and
PEG20,000 refers to polyethylene glycol having a total weight average
molecular weight
of about 20,000.
"Melanoma" may be a malignant or benign tumor arising from the
melanocytic system of the shin and other organs, including the oral cavity,
esophagus, anal
caaial, 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.
"Hepatoma" may be a malignant or benign tumor of the liver, including, for
example, hepatocellular carcinoma.
"Patient" refers to an animal, preferably a mammal, more preferably a
human.
"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.
Throughout the present disclosure, the following abbreviations may be
used: PEG, polyethylene glycol; ADI, arginine deiminase; SS, succinimidyl
succinate;
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SSA, succinimidyl succinamide; SPA, succinimidyl propionate; and NHS, N-
hydroxy-
succinimide.
The present invention is based on the unexpected discovery that ADI
modified with polyethylene glycol provides excellent results in treating
certain types of
cancer and inhibiting the metastasis of cancer. ADI may be covalently bonded
to
polyethylene glycol with or without a linking group, although a preferred
embodiment
utilizes a linl~ing group.
In the present invention, the arginine deiminase gene may be derived,
cloned or produced from any source, including, for example, microorganisms,
recombinant
bioteclmology or any combination thereof. For example, argiune deiminase may
be
cloned from microorganisms of the genera Mycoplasma, Clostf°idium,
Bacillus, Bo~~elia,
Enterococcus, Streptococcus, Lactobacillus, Qia~dia. It is preferred that
arginine
deiminase is cloned from Mycoplasma pneumoniae, Mycoplasma IZOminus,
Mycoplasma
of ginirai, Steptococcus pyogenes, Steptococcus pneumoniae, Bor~elia
bu~gdorfe~i, Bo~relia
afzelii, Qiay~dia intestinalis, Clostridium pelf °ihget~s, Bacillus
liclaeszifo~mis, Ente~ococcus
faecalis, Lactobacillus sake, or any combination thereof. In particular, the
arginine
deiminase used in the present invention may have one or more of the amino acid
sequences
depicted in Figures 1 and 10-13.
In certain embodiments of the present invention, it is preferred that arginine
deiminase is cloned from microorganisms of the genus Mycoplasma. More
preferably, the
arginine deiminase is cloned from Mycoplasma a~gihini, Mycoplasnaa laominus,
Mycoplasma a~tlz~itides, or any combination thereof. In particular, the
arginine deiminase
used in the present invention may have one or more of the amino acid sequences
depicted
in Figure 1.
hi one embodiment of the present invention, the polyethylene glycol (PEG)
has a total weight average molecular weight of about 1,000 to about 50,000;
more
preferably from about 3,000 to about 40,000, more preferably from about 5,000
to about
30,000; more preferably from about 8,000 to about 30,000; more preferably from
about
11,000 to about 30,000; even more preferably from about 12,000 to about
28,000; still
more preferably from about 16,000 to about 24,000; even more preferably from
about
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18,000 to about 22,000; even more preferably from about 19,000 to about
21,000, and
most preferably about 20,000. Generally, polyethylene glycol with a molecular
weight of
30,000 or more is difficult to dissolve, and yields of the formulated product
are greatly
reduced. The polyethylene glycol may be a branched or straight chain,
preferably a
straight chain. Generally, increasing the molecular weight of the polyethylene
glycol
decreases the immunogenicity of the ADI. The polyethylene glycol having a
molecular
weight described in this embodiment may be used in conjunction with ADI, and,
optionally, a biocompatible linlcing group, to treat cancer, including, for
example,
melanomas, hepatomas and sarcomas, preferably melanomas.
In another embodiment of the present invention, the polyethylene glycol
has a total weight average molecular weight of about 1,000 to about 50,000;
preferably
about 3,000 to about 30,000; more preferably from about 3,000 to about 20,000;
more
preferably from about 4,000 to about 12,000; still more preferably from about
4,000 to
about 10,000; even more preferably from about 4,000 to about 8,000; still more
preferably
from about 4,000 to about 6,000; with about 5,000 being most preferred. The
polyethylene
glycol may be a branched or straight chain, preferably a straight chain. The
polyethylene
glycol having a molecular weight described in this embodiment may be used in
conjunction with ADI, and optionally, a biocompatible linl~ing group, to treat
cancer,
including, for example, melanomas, hepatomas and sarcomas, preferably
hepatomas.
The linking group used to covalently attach ADI to PEG may be any
biocompatible linking group. As discussed above, "biocompatible" indicates
that the
compound or group is non-toxic and may be utilized ih vitro or in vivo without
causing
injury, sicl~ness, disease or death. PEG 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
linlcing 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
succinimide 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,
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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
linl~ing group
is an ester group and/or a succinimide group. More preferably, the lii~lcing
group is SS,
SPA, SCM, SSA or NHS; with SS, SPA or NHS being more preferred, and with SS or
SPA being most preferred.
Alternatively, ADI may be coupled directly to PEG (i.e., without a linking
group) through an amino group, a sulflzydral group, a hydroxyl group or a
carboxyl group.
ADI may be covalently bonded to PEG, via a biocompatible linking group,
using methods laiown in the art, as described, for example, by Parlc et al,
Anticahee~ Res.,
1: 373-376 (1981); and Zaplipsky and Lee, Polyethylene Glycol Chemistry:
Biotechnical
and Biomedical Applications, J.M. Harris, ed., Plenum Press, NY, Chapter 21
(1992), the
disclosures of which are hereby incorporated by reference herein in their
entirety.
The attachment of PEG to ADI increases the circulating half life of ADI.
Generally, PEG is attached to a primary amine of ADI. Selection of the
attachment site of
polyethylene glycol on the axginine deiminase is determined by the role of
each of the sites
within the active domain of the protein, as would be known to the skilled
artisan. PEG
may be attached to the primary amines of arginine deiminase without
substantial loss of
enzymatic activity. For example, ADI cloned from Mycoplasma argiraini,
Mycoplasma
a~th~itides amd Mycoplasma hominus has about 17 lysines that may be modified
by this
procedure. In other words, the 17 lysines are all possible points at which ADI
can be
attached to PEG via a biocompatible linking group, such as SS, SPA, SCM, SSA
and/or
NHS. PEG may also be attached to other sites on ADI, as would be apparent to
one skilled
in the art in view of the present disclosure.
From 1 to about 30 PEG molecules may be covalently bonded to ADI.
Preferably, ADI is modified with about 7 to about 15 PEG molecules, more
preferably
from about 9 to about 12 PEG molecules. In other words, about 30% to about 70%
of the
primary amino groups in arginine deiminase are modified with PEG, preferably
about 40%
to about 60%, more preferably about 45% to about 55%, and most preferably
about 50% of
the primary amino groups in arginine deiminase are modified with PEG. When PEG
is
covalently bonded to the end terminus of ADI, preferably only 1 PEG molecule
is utilized.
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Increasing the number of PEG units on ADI increases the circulating half life
of the
enzyme. However, increasing the number of PEG units on ADI decreases the
specific
activity of the enzyme. Thus, a balance needs to be achieved between the two,
as would
be apparent to one sleilled in the art in view of the present disclosure.
In the present invention, a common feature of the most preferred
biocompatible linking groups is that they attach to a primary amine of
arginine deiminase
via a maleimide group. Once coupled with arginine deiminase, SS-PEG has aaz
ester
linkage next to the PEG, which may render this site sensitive to serum
esterase, which may
release PEG from ADI in the body. SPA-PEG and PEG2-NHS do not have an ester
linkage, so they are not sensitive to serum esterase.
In the present invention, the particular linking groups do not appear to
influence the circulating half life of PEG-ADI or its specific enzyme
activity. However, it
is critical to use a biocompatible linking group in the present invention. PEG
which is
attached to the protein may be either a single chain, as with SS-PEG, SPA-PEG
and SC-
PEG, or a branched chain of PEG may be used, as with PEG2-NHS. The structural
formulas of the preferred linking groups in the present invention are set
forth below.
SS-PEG:
SPA-PEG:
PEG
O
O C CH2CH2 C O N
SS O
PEG
O
O CH2CH2 C O N
SPA
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PEG2-NHS
PE~
O
C O N
PE~
O
NHS
A therapeutically effective amount of one of the compounds of the present
invention is 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/lcg twice a week
to about
once every two weeks. For example, the dosage may be about 1 mglkg once a week
as a 2
ml intravenous injection to about 20 mg/kg once every 3 days. The optimum
dosage with
ADI-SS-PEG5,000 may be about twice a week, while the optimum dosage with ADI-
SS-
PEG20,000 may be from about once a weelc to about once every two weeks. 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 (lyophilate) or as a liquid formulation, as desired.
The methods of the present invention can involve either iya vitro or ih 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
vaxiety of diagnostic applications, as would be apparent to one skilled in the
art.
The ih. vivo means of administration of the compounds of the present
invention will vary depending upon the intended application. As one spilled in
the art will
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recognize, administration of the PEG-ADI composition of the present invention
can be
carried out, for example, orally, intxanasally, intraperitoneally,
parenterally, intravenously,
intralymphatically, intratumorly, intramuscularly, interstitially, infra-
arterially,
subcutaneously, intraocularly, intrasynovial, transepithelial, and
transdermally.
Examples
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: P~oductio~z of Recombiuaut ADI
Cultures of Mycoplaszzza a~gitzitzi (ATCC 23243), Mycoplasma hozninus
(ATCC 23114) and Mycoplaszzza arth~itides (ATCC 23192) were obtained from the
American Type Culture Collection, Rockville, Maryland.
Arginine deiminase was cloned from Mycoplasma a>"ginini, Mycoplasma
hoznizzus and Mycoplaszna a~th~itides and expressed in E. coli as previously
described by
S. Misawa et al, J. Biotechnology, 36:145-155 (1994), the disclosure of which
is hereby
incorporated herein by reference in its entirety. The amino acid sequences of
arginine
deiminase from each of the above species is set forth in Figure 1. The top
amino acid
sequence, identified as A:DIPROT, is from Mycoplasma aYginini; the middle
amino acid
sequence, identified as ARTADIPRO, is from Mycoplasma a>"th>~itides; and the
bottom
amino acid sequence, identified as HOMADIPRO, is from Mycoplasma lzomizzzrs.
Each of
the amino acid sequences are more than 96% conserved. Characterization, by
methods
l~nown to those spilled in the art, of each of the proteins with respect to
specific enzyme
activity, Kn" Vmax and pH optima revealed that they were biochemically
indistinguishable
from each other. The pH optima was determined using a citrate buffer (pH 5-
6.5), a
phosphate buffer (pH 6.5-7.5) and a borate buffer (pH 7.5-8.5). The I~m and
Vm~ were
determined by incubating the enzyme with various concentrations of argiiune
and
quantifying citrulline production. The K", for the various enzymes was about
0.02 to 0.06
~,M and the V",~ was about 15-20 ~,mol/min/mg, the values of which are within
standard
error of each other.
The arginine deiminase genes were amplified by polymerase chain reaction
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using the following primer pair derived from the published sequence of M.
a~gihihi, as
described, for example, by T. Olmo et al, Infect. Irnmun., 58:3788-3795
(1990), the
disclosure of which is hereby incorporated by reference herein in its
entirety:
SEQ ID NO: 4, 5'-GGGATCCATGTCTGTATTTGACAGT-3'
SEQ ID NO: 5, 5'-TGA.A.AGCTTTTACTACCACTTAACATCTTTACG-3'
The polymerase chain reaction products were cloned as a Bam H1-Hind III
fragment into
expression plasmid pQEl6. DNA sequence analysis indicated that the fragment
derived
from M. arginini by PCR had the same sequence for the arginine deiminase gene
as
described by Ohno et al, Infect. Immun., sups°a. The five TGA codons in
the ADI gene
which encode tryptophan in Mycoplasma were changed to TGG codons by
oligonucleotide-directed mutagenesis prior to gene expression in E. coli, as
taught, for
example, by J.R. Sayers et al, Biotechraiques, 13:592-596 (1992). Recombinant
ADI was
expressed in inclusion bodies at levels of 10% of total cell protein.
The proteins from each of the above three species of Mycoplasma have
approximately 95% homology and are readily purified by column chromatography.
Approximately 200 mg of pure protein may be isolated from 1 liter of
fermentation broth.
Recombinant ADI is stable for about 2 weeks at 37°C and for at least 8
months when
stored at 4°C. As determined by methods known to those skilled in the
art, the proteins
had a high affinity for arginine (0.04 ~,M), and a physiological pH optima of
about 7.2 to
about 7.4.
Example 2: Rezzatu~atiofz and Purification of Recozzzbiuaut ~iDI
ADI protein was renatured, with minor modifications, as described by
Misawa et al, J. Biotechnology, 36:145-155 (1994), the disclosure of which is
hereby
incorporated herein by reference in its entirety. 100 g of cell paste was
resuspended in 800
ml of 10 mM KZP04 pH 7.0, 1 mM EDTA (buffer 1) and the cells were disrupted by
two
passes in a Microfluidizer (Microfluidics Corporation, Newton, MA). Triton X-
100 was
added to achieve a final concentration of 4% (v/v). The homogenate was stirred
for 30
min at 4°C, then centrifuged for 30 min at 13,000 g. The pellet was
collected and
resuspended in one liter of buffer 1 containing 0.5% Triton X-100. The
solution was
diafiltered against 5 volumes of denaturation buffer (50 mM Tris HCI, pH 8.5,
10 mM
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DTT) using hollow-fiber cartridges with 100 kD retention rating (Microgon
Inc., Lagtma
Hills, CA). Guanidine HCl was added to achieve a final concentration of 6 M
and the
solution was stirred for 1 S min at 4°C. The solution was diluted 100-
fold into refolding
buffer 1, 10 rmn I~ZP04, pH 7.0 and stirred for 48 hours at 1S°C,
particulates were
S removed by centrifugation at 15,000 x g.
The resulting supernatant was concentrated on a Q Sepharose Fast Flow
(Pharmacia Inc., Piscataway, NJ) column preequilabrated in refolding buffer.
ADI was
eluted using refolding buffer containing 0.2 M NaCI. The purification
procedure yielded
ADI protein, which was >9S% pure as estimated by SDS-PAGE analysis. 8 g of
pure
renatured ADI protein was produced from 1 kg of cell paste wluch corresponds
to 200 mg
purified ADI per liter of fermentation.
ADI activity was determined by micro-modification of the method
described by Oginsky et al, Meth. Ehzymol., (1957) 3:639-642. 10 ~1 samples in
0.1 m
NazP04, pH 7.0 (BUN assay buffer) were placed in a 96 well microliter plate,
40 ~l of O.f
1 S mM arginine in BUN assay buffer was added, and the plate was covered and
incubated at
37 ° C for 1 S minutes. 20 ~.1 of complete BLTN reagent (Sigma
Diagnostics) was added and
the plate was incubated for 10 minutes at 100°C. The plate was then
cooled to 22°C and
analyzed at 490 nm by a microliter plate reader (Molecular Devices, Inc). 1.0
ICT is the
amount of enzyme which converts 1 mole of L-axginine to L-citrulline per
minute.
Protein concentrations were determined using Pierce Coomassie Blue Protein
Assay
Reagent (Pierce Co., Rockford, IL) with bovine serum albumin as a standaxd.
The enzyme activity of the purified ADI preparations was 17-2S IU/mg.
Example 3: Attaclasneht of PEG to ADI
PEG was covalently bonded to ADI in a 100 mM phosphate buffer, pH 7.4.
2S Briefly, ADI in phosphate buffer was mixed with a 100 molar excess of PEG.
The
reaction was stirred at room temperature for 1 hour, then the mixture was
extensively
dialized to remove unincorporated PEG.
A first experiment was performed where the effect of the licking group
used in the PEG-ADI compositions was evaluated. PEG and ADI were covalently
bonded
via four different linking groups: an ester group or maleimide group,
including SS, SSA,
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SPA and SSPA, where the PEG had a total weight average molecular weight of
5,000,
10,000, 12,000, 20,000, 30,000 and 40,000; an epoxy group, PEG-epoxy, where
the PEG
had a total weight average molecular weight of 5,000; and a branched PEG
group, PEG2-
NHS, where the PEG had a total weight average molecular weight of 10,000,
20,000 and
S 40,000.
S.0 ILT of the resulting compositions were injected into mice (S mice in each
group). To determine the serum levels of arginine, the mice were bled from the
retro
orbital plexus (100 u1). Immediately following collection an equal volume of
SO% (w/v) of
trichloroacetic acid was added. The precipitate was removed by centrifugation
(13,000 x g
for 30 minutes) and the supernatant removed and stored frozen at -70°C.
The samples were
then analyzed using an automated amino acid analyzer and reagents from Beckman
Instruments using protocols supplied by the manufacturer. The limits of
sensitivity for
arginine by this method was approximately 2-6 ~M and the reproducibility of
measurements within about 8%. The amount of serum arginine was determined by
amino
1 S acid analysis. As can be seen from the results in Figure 3, the linking
group covalently
bonding the PEG and ADI did not have an appreciable effect on the ability of
ADI to
reduce serum argiune ih vivo. In other words, the linking group may not be
critical to the
results of the experiment, except that a non-toxic linking group must be used
for ih vivo
applications.
A second experiment was performed wherein the effect of the linking group
and molecular weight of PEG on serum citrulline levels ih vivo was evaluated.
Mice (S in
each group) were given various compositions of ADI and PEG-ADI in an amount of
S.0
ILT. To determine the serum levels of citrulline, the mice were bled from the
retro orbital
plexus (100 u1). Tm_m__ediately following collection an equal volume of SO%
(w/v) of
2S trichloroacetic acid was added. The precipitate was removed by
centrifugation (13,000 x g
for 30 minutes) and the supernatant removed and stored frozen at -70°C.
The samples were
then analyzed using an automated amino acid analyzer and reagents from
Becl~nan
Instruments using protocols supplied by the manufacturer. The limits of
sensitivity for
citrulline by this method was approximately 2-6 ~,M and the reproducibility of
measurements within about 8%. The amount of citrulline was determined, and the
area
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under the curve approximated and expressed as ~mol days.
In Figure 4, the open circles indicate the amount of citrulline produced by
native ADI, the filled circles are ADI-SC-PEG, the open squares are ADI-SS-
PEG, the
open triangles are ADI-SPA-PEG, and the filled triangles are branched chain
PEG-NHS-
PEGZ. The results in Figure 4 demonstrate that the molecular weight of the PEG
determines the effectiveness of the PEG-ADI composition. The effectiveness of
the PEG-
ADI compositions is not necessarily based on the method or means of attachment
of the
PEG to ADI, except that a biocompatible linking group must be used for in vivo
applications.
The results in Figure 4 also demonstrate that the optimal molecular weight
of PEG is 20,000. Although PEG30,000 appears to be superior to PEG20,000 in
terms of
its phanmacodynamics, PEG30,000 is less soluble, which makes it more difficult
to work
with. The yields, which were based on the recovery of enzyme activity, were
about 90%
for PEG5,000 and PEG12,000; about 85% for PEG20,000 and about 40% for
PEG30,000.
Therefore, PEG20,000 is the best compromise between yield and circulating half
life, as
determined by citrulline production.
In a third experiment, the dose response of serum arginine depletion and the
production of citrulline with ADI-SS-PEG5,000 and ADI-SS-PEG20,000 was
determined.
Mice (5 in each group) were given a single injection of 0.05 ILJ, 0.5 ICT or
5.0 ILJ of either
ADI-SS-PEG5,000 or ADI-SS-PEG20,000. At indicated times, serum was collected,
as
described above, and an amino acid analysis was performed to quantify serum
arginine
(Figures 5A and SC) and serum citrulline (Figures SB and SD). Both
formulations induced
a dose dependent decrease in serum arginine and an increase in serum
citrulline. However,
the effects induced by ADI-SS-PEG20,000 were more pronounced and of longer
duration
than the effects induced by ADI-SS-PEG5,000.
Example 4: Selectivity of ADl Mediated Cytotoxicity
The selectivity of arginine deiminase mediated cytotoxicity was
demonstrated using a number of human tumors. Specifically, human tumors were
tested if2
vitYO for sensitivity to ADI-SS-PEG5,000 (50 ng/ml). Viability of cultures was
determined after 7 days. For a culture to be defined as "inhibited," greater
than 95% of the
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cells must take up Trypan blue dye. A host of normal cells were also tested,
including
endothelial cells, smooth muscle cells, epithelial cells and fibroblasts, and
none were
inhibited by ADI-SS-PEG5,000. Although arginine deiminase has no appreciable
toxicity
towards normal, and most tumor cells, ADI-SS-PEG5,000 greatly inhibited all
hiunan
melanomas and hepatomas that were commercially available from the ATCC, MSKCC
and Europe.
Table 1: Specificity of Arginine Deiminase Cytotoxicity
Tumor Type Number of Tumors Tumors inhibited
Tested (%)
Brain 16 0
. Colon 34 0
Bladder 3 0
Breast 12 0
Kidney 5 0
S arcoma 11 64
Hepatoma 17 100
Melanoma 37 100
In a parallel set of experiments, mRNA was isolated from the tumors.
Northern blot analyses, using the human axgininosuccinate synthase cDNA probe,
indicated complete concordance between the sensitivity to arginine deiminase
treatment
and an inability to express argininosuccinate synthase. This data suggests
that ADI
toxicity results from an inability to induce argininosuccinate synthase.
Therefore, these
cells cannot synthesize arginine from citrulline, and are unable to synthesize
the proteins
necessary for growth.
Example 5: Circulatisag Half Life
Balb C mice (5 in each group) were injected intravenously with a single 5.0
ICT dose of either native arginine deiminase or various formulations of
arginine deiminase
modified with polyethylene glycol, as indicated in Figures 2A and 2B. To
determine the
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serum levels of arginine and citrulline, the mice were bled from the retro
orbital plexus
(100 u1). Immediately following collection an equal volume of 50% (w/v) of
trichloro-
acetic acid was added. The precipitate was removed by centrifugation (13,000 x
g for 30
minutes) and the supernatant removed and stored frozen at -70°C. The
samples were then
analyzed using an automated amino acid analyzer and reagents from Beckman
Instruments
using protocols supplied by the manufacturer. The limits of sensitivity for
arginine by this
method was approximately 6 pM and the reproducibility of measurements within
about
8%.
A dose dependent decrease in serum arginine levels, as shown by the solid
circles in Figure 2A, and a rise in serum citrulline, as shown by the open
triangles in
Figure 2B, were detected from the single dose administration of native ADI
(filled circles)
or ADI-SS-PEG (open triangles). However, the decrease in senun arginine and
rise in
serum citrulline was short lived, and soon returned to normal. The half life
of arginine
depletion is summarized in the Table below.
Table 2: Half Life of Serum Arginine Depletion
Compound Half Life in
Days
Native ADI 1
ADI-SS-PEG5,000 5
ADI-SS-PEG12,000 15
ADI-SS-PEG20,000 20
ADI-SS-PEG30,000 22
This experiment demonstrates that normal cells and tissues are able to
convert the citrulline baclc into arginine intracellularly while melanomas and
hepatomas
cannot because they laclc argininosuccinate synthetase.
Example 6: A~atigehicity of PEG modified ADI
To determine the antigenicity of native ADI, ADI-SS-PEG5,000, and ADI-
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_I8_
SS-PEG20,000, the procedures described in, for example, Park, Ahtica~zcer
Res., supra,
and Kamisalci, J. Plaarmacol. Exp. Ther., supra, were followed.. Briefly, Balb
C mice (5
in each group) were intravenously injected weekly for 12 weeks with
approximately O.S IU
(100 ~g of protein) of native ADI, ADI-SS-PEG5,000 or ADI-SS-PEG20,000. The
animals were bled (0.05 ml) from the retro orbital plexus at the beginning of
the
experiment and at weeks 4, 8 and 12. The serum was isolated and stored at -
70°C. The
titers of anti-ADI IgG were determined by ELISA. 50 ~,g of ADI was added to
each well
of a 96 well micro-titer plate and was incubated at room temperature for 4
hours. The
plates were rinsed with PBS and then coated with bovine serum albumin (1
mg/ml) to
bloclc nonspecific protein binding sites, and stored over night at 4°C.
The next day serum
from the mice was diluted and added to the wells. After 1 hour the plates were
rinsed with
PBS and rabbit anti-mouse IgG coupled to peroxidase was added to the wells.
The plates
were incubated for 30 min and then the resulting UV absorbance was measured
using a
micro-titer plate reader. The titer was defined as the highest dilution of the
serum which
resulted in a two-fold increase from background absorbance (approximately 0.50
OD).
The results are shown in Figure 6. The open circles represent the data
obtained from animals inj ected with native ADI, which was very anta.genic.
The filled
circles represent the data obtained from the animals injected with ADI-SS-
PEG5,000,
while the open triangles represent the data obtained from the animals injected
with ADI-
SS-PEG20,000. As can be seen from Figure 6, ADI-SS-PEG5,000 and ADI-SS-
PEG20,000 are significantly less antigenic than native ADI. For example, as
few as 4
injections of native ADI resulted in a titer of about 10~, while 4 injections
of any of the
PEG-ADI formulations failed to produce any measurable antibody. However, after
8
injections, the ADI-PEG5,000 had a titer of about 10z, while ADI-PEG20,000 did
not
induce this much of an immune response until after 12 injections. The results
demonstrate
that attaching PEG to ADI blunts the immune response to the protein.
Example 7: Tuyzzor I>zhibitiou of Ilumau Melauonzas
The effect of PEG-ADI on the growth of human melanoma (SK-Mel 28) in
nude mice was determined. Nude mice (5 in each group) were injected with 10~
SK-mel 2
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human melanoma cells which were allowed to grow until the tumors reached a
diameter of
about 3-5 mm. The mice were left untreated (open circles) or were treated once
a week for
8 weeks with 5.0 IU of ADI-SS-PEG5,000 (filled triangles), ADI-SS-PEG12,000
(open
triangles) or ADI-SS-PEG20,000 (filled circles). The tumor size was measured
weekly,
and the mean diameter of the tumors is presented in Figure 7.
Figure 8 shows the effectiveness of ADI-SS-PEG20,000 on three human
melanomas (SK-mel 2, SK-mel 28, M24-met) grown ih vivo in nude mice. Nude mice
(5
in each group) were injected with 10~ SK-mel 2, SK-mel 28 or M24-met human
melanoma
cells. The tumors were allowed to grow until they were approximately 3-5 mm in
diameter. Thereafter, the animals were injected once a week with 5.01TJ of ADI-
SS-
PEG20,000. The results are shown in Figure 8, and show that PEG-ADI inhibited
tumor
growth and that eventually the tumors began to regress and disappear. Because
the tumors
did not have argininosuccinate synthatase, they were unable to synthesize
proteins
(because ADI eliminated arginine and the tumors could not make it) so that the
cells
"starved to death."
Since M24-met human melanoma is highly metastatic, the animals injected
with M24-met human melanoma cells were sacrificed after 4 weeks of treatment
and the
number of metastases in the lungs of the animals was determined. The control
animals had
an average of 32 metastases, while the animals treated with ADI-SS-PEG20,000
did not
have any metastases. The results appear to indicate that ADI-SS-PEG20,000 not
only
inhibited the growth of the primary melanoma tumor, but also inhibited the
formation of
metastases.
It is of interest to note that in over 200 animals tested, the average number
of metastases in the control group was 49 ~ 18, while only a single metastasis
was
observed in 1 treated animal.
Example 8: Tumor Iulzibitiozz of Human Hepatoszzas
The ability of PEG-ADI to inhibit the growth of a human hepatoma ira vivo
was tested. Nude mice (5 in each group) were injected with 10~ human hepatoma
SK-
Hepl cells. The tumors were allowed to grow for two weeks and then the animals
were
treated once a weelc with 5.0 IU of SS-PEG5,000-ADI (solid circles), SS-
PEG12,000-ADI
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(solid triangles) or SS-PEG20,000-ADI (open triangles). The results are set
forth in Figure
9. The untreated animals (open circles) all died within 3 weelcs. In contrast,
animals
treated with ADI had a far longer life expectancy, as can be seen from Figure
9. AlI the
surviving mice were euthanized after 6 months, and necropsy indicated that
they were free
of tumors.
Surprisingly, PEG5,000-ADI is most effective in inhibiting hepatoma
growth in vivo. The exact mechaiusm by which this occurs is unknown. Without
being
bound to any theory of the invention worles, it appears that proteins
formulated with SS-
PEG5,000-ADI become sequestered in the liver. Larger molecular weights of PEG
do not,
which may be due to the uniqueness of the hepatic endothelium and the spaces
(fenestrae)
being of such a size that larger molecular weights of PEG-ADI conjugates are
excluded.
Example 9: Application to Humans
PEG5,000-ADI and PEG20,000-ADI were incubated ex vivo with normal
human serum and the effects on arginine concentration was determined by amino
acid
analysis, where the enzyme was found to be fully active and capable of
degrading all the
detectable arginine with the same kinetics as in the experiments involving
mice. The
reaction was conducted at a volume of 0.1 ml in a time of 1 hour at 37
° C.
Additionally, the levels of arginine and citrulline in human serum are
identical with that
found in mice. PEG-proteins circulate longer in humans than they do in mice.
For
example, the circulating half life of PEG conjugated adenosine deiminase,
asparaginase,
glucocerbrocidase, uricase, hemoglobulin and superoxide dismutase all have a
circulating
half life that is 5 to 10 times longer than the same formulations in mice.
What this has
meant in the past is that the human dose is most often 1/5 to 1/10 of that
used in mice.
Accordingly, PEG-ADI should circulate even longer in humans than it does in
mice.
Each of the patents, patent applications and publications described herein
are hereby incorporated by reference herein in their entirety.
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.
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SEQUENCE LISTING
<110> Phoenix Pharmacologics, Inc.
<120> Modified Arginine Deiminase
<130> PHOE0064
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<141>
<150> 09/023,809
<151> 1998-02-13
<150> 09/723,546
<151> 2000-11-28
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Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Lys Leu Val
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Ser Ile I1e Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly G1u Gly
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7
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Cys Gly Gly Asp Asn Leu Val Ala Ala Gly Arg G1u Gln Trp Asn Asp
340 345 350
Gly Ser Asn Thr Leu Thr Ile Ala Pro Gly Val Va1 Val Val Tyr Asn
355 360 365
Arg Asn Thr Tle Thr Asn Ala Ile Leu Glu Ser Lys Gly Leu Lys Leu
370 375 380
Ile Lys Ile His Gly Ser G1u Leu Va1 Arg Gly Arg Gly Gly Pro Arg
385 390 395 400
Cys Met Ser Met Pro Phe Glu Arg Glu Asp Ile
405 410
<210> 7
<211> 409
<2l2> PRT
<213> Steptococcus pneumoniae
8
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<400> 7
Met Ser Ser His Pro Ile Gln Val Phe Ser Glu Ile Gly Lys Leu Lys
1 5 10 15
Lys Val Met Leu His Arg Pro Gly Lys Glu Leu Glu Asn Leu Leu Pro
20 25 30
Asp Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Asp
35 40 45
Ala Gln Lys Glu His Asp Ala Phe Ala Gln Ala Leu Arg Asp Glu Gly
50 55 60
Ile Glu Val Leu Tyr Leu Glu G1n Leu Ala Ala Glu Ser Leu Thr Ser
65 70 75 80
Pro Glu Ile Arg Asp Gln Phe Ile Glu Glu Tyr Leu Asp Glu Ala Asn
85 90 95
Ile Arg Asp Arg Gln Thr Lys Val Ala Tle Arg Glu Leu Leu His Gly
100 105 110
Ile Lys Asp Asn Gln Glu Leu Val Glu Lys Thr Met Ala Gly Ile G1n
115 120 125
Lys Val Glu Leu Pro Glu Ile Pro Asp Glu Ala Lys Asp Leu Thr Asp
130 135 140
Leu Val Glu Ser Glu Tyr Pro Phe Ala I1e Asp Pro Met Pro Asn Leu
145 150 155 160
Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile G1y Asn Ala Val Ser Leu
165 170 175
Asn His Met Phe A1a Asp Thr Arg Asn Arg Glu Thr Leu Tyr Gly Lys
180 185 190
Tyr Ile Phe Lys Tyr His Pro Ile Tyr Gly Gly Lys Val Asp Leu Val
195 200 205
Tyr Asn Arg Glu Glu Asp Thr Arg Ile Glu Gly G1y Asp Glu Leu Val
210 215 220
Leu Ser Lys Asp Val Leu Ala Va1 Gly Ile Ser Gln Arg Thr Asp Ala
225 230 235 240
A1a Ser Ile Glu Lys Leu Leu Val Asn 2le Phe Lys Lys Asn Val Gly
245 250 255
9
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Phe Lys Lys Val Leu Ala Phe Glu Phe Ala Asn Asn Arg Lys Phe Met
260 265 270
His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe Thr I1e
275 280 285
His Pro Glu Ile Glu Gly Asp Leu His Val Tyr Ser Val Thr Tyr Glu
290 295 300
Asn Glu Lys Leu Lys Ile Val Glu Glu Lys Gly Asp Leu Ala Glu Leu
305 310 315 320
Leu Ala Gln Asn Leu Gly Val Glu Lys Val His Leu Ile Arg Cys Gly
325 330 335
Gly Gly Asn Ile Va1 Ala Ala Ala Arg Glu Gln Trp Asn Asp Gly Ser
340 345 350
Asn Thr Leu Thr Ile Ala Pro Gly Val Val Val Val Tyr Asp Arg Asn
355 360 365
Thr Val Thr Asn Lys Ile Leu Glu Glu Tyr Gly Leu Arg Leu Ile Lys
370 375 380
Ile Arg Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg Cys Met
385 390 395 400
Ser Met Pro Phe Glu Arg Glu Glu Val
405
<210> 8
<211> 410
<212> PRT
<213> Borrelia burgdorferi
<400> 8
Met G1u Glu Glu Tyr Leu Asn Pro Tle Asn I1e Phe Ser Glu Ile Gly
1 5 10 15
Arg Leu Lys Lys Val Leu Leu His Arg Pro Gly Glu Glu Leu Glu Asn
20 25 30
Leu Thr Pro Leu Ile Met Lys Asn Phe Leu Phe Asp Asp Ile Pro Tyr
35 40 45
Leu Lys Val Ala Arg Gln Glu His Glu Val Phe Val Asn Ile Leu Lys
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50 55 60
Asp Asn Ser Val Glu Ile Glu Tyr Val Glu Asp Leu Val Ser Glu Val
65 70 ~ 75 80
Leu Ala Ser Ser Val Ala Leu Lys Asn Lys Phe Ile Ser Gln Phe Ile
85 90 95
Leu Glu Ala Glu Ile Lys Thr Asp Gly Val Ile Asn Ile Leu Lys Asp
100 l05 110
Tyr Phe Ser Asn Leu Thr Val Asp Asn Met Val Ser Lys Met Ile Ser
l15 120 125
Gly Val Ala Arg Glu Glu Leu Lys Asp Cys Glu Phe Ser Leu Asp Asp
130 135 140
Trp Val Asn Gly Ser Ser Leu Phe Val Ile Asp Pro Met Pro Asn Val
145 150 155 160
Leu Phe Thr Arg Asp Pro Phe Ala Ser Ile Gly Asn Gly I1e Thr Ile
165 170 175
Asn Lys Met Tyr Thr Lys Va1 Arg Arg Arg Glu Thr Ile Phe Ala Glu
180 185 190
Tyr Ile Phe Lys Tyr His Ser Ala Tyr Lys Glu Asn Val Pro Ile Trp
l95 200 205
Phe Asn Arg Trp Glu Glu Thr Ser Leu Glu Gly Gly Asp Glu Phe Val
210 215 220
Leu Asn Lys Asp Leu Leu Val Ile Gly Ile Ser Glu Arg Thr G1u Ala
225 230 235 240
Gly Ser Va1 Glu Lys Leu Ala Ala Ser Leu Phe Lys Asn Lys Ala Pro
245 250 255
Phe Ser Thr Ile Leu Ala Phe Lys Ile Pro Lys Asn Arg Ala Tyr Met
260 265 270
His Leu Asp Thr Val Phe Thr Gln Ile Asp Tyr Ser Val Phe Thr Ser
275 280 285
Phe Thr Ser Asp Asp Met Tyr Phe Ser Ile Tyr Val Leu Thr Tyr Asn
290 295 300
Ser Asn Ser Asn Lys Ile Asn Ile Lys Lys Glu Lys Ala Lys Leu Lys
11
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305 310 315 320
Asp Val Leu Ser Phe Tyr Leu Gly Arg Lys Ile Asp Ile Ile Lys Cys
325 330 335
Ala Gly Gly Asp Leu Ile His Gly Ala Arg Glu Gln Trp Asn Asp Gly
340 345 350
Ala Asn Val Leu Ala Tle Ala Pro Gly Glu Val Ile Ala Tyr Ser Arg
355 360 365
Asn His Val Thr Asn Lys Leu Phe Glu Glu Asn Gly Ile Lys Val His
370 375 380
Arg Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys
385 390 395 400
Met Ser Met Ser Leu Va1 Arg Glu Asp Ile
405 410
<210> 9
<211> 409
<212> PRT
<213> Borrelia afzelii
<400> 9
Met Glu Glu Tyr Leu Asn Pro Ile Asn Ile Phe Ser Glu Ile Gly Arg
1 5 10 15
Leu Lys Lys Va1 Leu Leu His Arg Pro Gly Glu Glu Leu Glu Asn Leu
20 25 - 30
Thr Pro Phe Ile Met Lys Asn Phe Leu Phe Asp Asp Ile Pro Tyr Leu
35 40 45
Glu Va1 Ala Arg Gln Glu His Glu Val Phe Ala Ser Ile Leu Lys Asn
50 55 60
Asn Leu Val Glu Ile Glu Tyr Ile Glu Asp Leu Ile Ser Glu Val Leu
65 70 75 80
Val Ser Ser Val Ala Leu Glu Asn Lys Phe Tle Ser Gln Phe Ile Leu
85 90 95
G1u Ala Glu Ile Lys Thr Asp Phe Thr Ile Asn Leu Leu Lys Asp Tyr
100 105 110
12
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Phe Ser Ser Leu Thr Ile Asp Asn Met Ile Ser Lys Met Ile Ser Gly
115 120 125
Val Val Thr Glu Glu Leu Lys Asn Tyr Thr Ser Ser Leu Asp Asp Leu
130 135 140
Val Asn Gly Ala Asn Leu Phe Ile Ile Asp Pro Met Pro Asn Val Leu
145 150 155 l60
Phe Thr Arg Asp Pro Phe Ala Ser Ile Gly Asn Gly Val Thr Ile Asn
165 170 175
Lys Met Phe Thr Lys Val Arg Gln Arg Glu Thr Ile Phe Ala Glu Tyr
180 185 190
Ile Phe Lys Tyr His Pro Val Tyr Lys Glu Asn Val Pro Ile Trp Leu
195 200 205
Asn Arg Trp Glu Glu Ala Ser Leu Glu Gly Gly Asp Glu Leu Val Leu
210 215 220
Asn Lys Gly Leu Leu Val Ile Gly Ile Ser Glu Arg Thr Glu Ala Lys
225 230 235 240
Ser Val Glu Lys Leu A1a Ile Ser Leu Phe Lys Asn Lys Thr Ser Phe
245 250 255
Asp Thr Ile Leu Ala Phe Gln Tle Pro Lys Asn Arg Ser Tyr Met His
260 265 270
Leu Asp Thr Val Phe Thr Gln Ile Asp Tyr Ser Val Phe Thr Ser Phe
275 280 285
Thr Ser Asp Asp Met Tyr Phe Ser Ile Tyr Val Leu Thr Tyr Asn Pro
290 295 300
Ser Ser Ser Lys Ile His Ile Lys Lys Glu Lys Ala Arg Ile Lys Asp
305 310 315 320
Val Leu Ser Phe Tyr Leu Gly Arg Lys Ile Asp I1e Ile Lys Cys Ala
325 330 335
Gly Gly Asp Leu Ile His Gly Ala Arg Glu Gln Trp Asn Asp Gly Ala
340 345 350
Asn Va1 Leu Ala Ile Ala Pro G1y Glu Ile Ile Ala Tyr Ser Arg Asn
355 360 365
13
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His Val Thr Asn Lys Leu Phe Glu Glu Asn Gly I1e Lys Val His Arg
370 375 380
Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met
385 390 395 400
Ser Met Pro Leu Ile Arg Glu Asp Ile
405
<210> 10
<211> 580
<212> PRT
<213> Qiardia intestinalis
<400> 10
Met Thr Asp Phe Ser Lys Asp Lys Glu Lys Leu Ala Gln Ala Thr Gln
1 5 10 15
Gly Gly Glu Asn G1u Arg Ala Glu Ile Val Val Val His Leu Pro Gln
20 25 30
Gly Thr Ser Phe Leu Thr Ser Leu Asn Pro Glu Gly Asn Leu Leu Glu
35 40 45
Glu Pro Ile Cys Pro Asp Glu Leu Arg Arg Asp His Glu Gly Phe Gln
50 55 60
Ala Val Leu Lys Glu Lys G1y Cys Arg Val Tyr Met Pro Tyr Asp Val
65 70 75 80
Leu Ser Glu A1a Ser Pro Ala Glu Arg Glu Val Leu Met Asp Gln Ala
85 90 95
Met Ala Ser Leu Lys Tyr Glu Leu His Ala Thr Gly Ala Arg Ile Thr
100 l05 110
Pro hys Met Lys Tyr Cys Val Ser Asp Glu Tyr Lys Arg Lys Val Leu
115 120 125
Ser Ala Leu Ser Thr Arg Asn Leu Val Asp Val Ile Leu Ser Glu Pro
130 135 140
Val Ile His Leu Ala Pro Gly Va1 Arg Asn Thr Ala Leu Val Thr Asn
145 150 155 160
Ser Val Glu Ile His Asp Ser Asn Asn Met Val Phe Met Arg Asp Gln
165 170 175
14
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Gln Ile Thr Thr Arg Arg Gly Ile Val Met Gly Gln Phe Gln Ala Pro
180 185 190
Gln Arg Arg Arg Glu Gln Val Leu Ala Leu Ile Phe Trp Lys Arg Leu
195 200 205
Gly Ala Arg Val Val Gly Asp Cys Arg Glu Gly Gly Pro His Cys Met
210 215 220
Leu Glu Gly Gly Asp Phe Val Pro Val Ser Pro Gly Leu Ala Met Met
225 230 235 240
Gly Val Gly Leu Arg Ser Thr Tyr Val Gly Ala Gln Tyr Leu Met Ser
245 250 255
Lys Asp Leu Leu Gly Thr Arg Arg Phe Ala Val Val Lys Asp Cys Phe
260 265 270
Asp Gln His Gln Asp Arg Met His Leu Asp Cys Thr Phe Ser Val Leu
275 280 285
His Asp Lys Leu Val Val Leu Asp Asp Tyr Ile Cys Ser Gly Met Gly
290 295 300
Leu Arg Tyr Val Asp Glu Trp Ile Asp Val Gly Ala Asp Ala Val Lys
305 310 315 320
Lys Ala Lys Ser Ser Ala Va1 Thr Cys Gly Asn Tyr Val Leu Ala Lys
325 330 335
A1a Asn Val Glu Phe Gln Gln Trp Leu Ser Glu Asn Gly Tyr Thr Ile
340 345 350
Val Arg I1e Pro His Glu Tyr Gln Leu Ala Tyr Gly Cys Asn Asn Leu
355 360 365
Asn Leu Gly Asn Asn Cys Val Leu Ser Val His Gln Pro Thr Val Asp
370 375 380
Phe Ile Lys Ala Asp Pro Ala Tyr Ile Ser Tyr Cys Lys Ser Asn Asn
385 390 395 400
Leu Pro Asn Gly Leu Asp Leu Val Tyr Val Pro Phe Arg Gly Ile Thr
405 410 415
Arg Met Tyr Gly Ser Leu His Cys Ala Ser Gln Val Val Tyr Arg Thr
420 425 430
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Pro Leu Ala Pro Ala Ala Val Lys Ala Cys Glu Gln Glu Gly Asp Gly
435 440 445
Ile Ala Ala Ile Tyr Glu Lys Asn Gly Glu Pro Val Asp Ala Ala Gly
450 455 460
Lys Lys Phe Asp Cys Val Ile Tyr Ile Pro Ser Ser Val Asp Asp Leu
465 470 475 480
Ile Asp Gly Leu Lys Ile Asn Leu Arg Asp Asp Ala Ala Pro Ser Arg
485 490 495
Glu Ile Ile Ala Asp Ala Tyr Gly Leu Tyr Gln Lys Leu Val Ser Glu
500 505 510
Gly Arg Val Pro Tyr Ile Thr Trp Arg Met Pro Ser Met Pro Va1 Val
515 520 525
Ser Leu Lys Gly Ala Ala Lys Ala Gly Ser Leu Lys Ala Val Leu Asp
530 535 540
Lys Ile Pro Gln Leu Thr Pro Phe Thr Pro Lys Ala Val Glu Gly Ala
545 550 555 560
Pro Ala Ala Tyr Thr Arg Tyr Leu Gly Leu Glu Gln Ala Asp Ile Cys
565 570 575
Val Asp I1e Lys
580
<210> 11
<211> 413
<212> PRT
<213> Clostridium perfringens
<400> 11
Met Arg Asp Asp Arg Ala Leu Asn Val Thr Ser Glu Ile Gly Arg Leu
1 5 10 15
Lys Thr Val Leu Leu His Arg Pro Gly Glu Glu Ile Glu Asn Leu Thr
20 25 30
Pro Asp Leu Leu Asp Arg Leu Leu Phe Asp Asp I1e Pro Tyr Leu Lys
35 40 45
Val Ala Arg Glu Glu His Asp Ala Phe Ala Gln Thr Leu Arg Glu Ala
16
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50 55 60
Gly Val Glu Val Leu Tyr Leu Glu Val Leu Ala Ala Glu Ala Ile Glu
65 70 75 80
Thr Ser Asp Glu Val Lys Gln Gln Phe Ile Ser Glu Phe Ile Asp Glu
85 90 95
Ala Gly Val Glu Ser Glu Arg Leu Lys Glu Ala Leu Ile Glu Tyr Phe
100 105 110
Asn Ser Phe Ser Asp Asn Lys Ala Met Val Asp Lys Met Met Ala Gly
l15 120 125
Val Arg Lys Glu G1u Leu Lys Asp Tyr His Arg Glu Ser Leu Tyr Asp
130 135 140
Gln Val Asn Asn Val Tyr Pro Phe Val Cys Asp Pro Met Pro Asn Leu
145 150 155 160
Tyr Phe Thr Arg Glu Pro Phe Ala Thr Ile Gly His Gly Ile Thr Leu
165 170 175
Asn His Met Arg Thr Asp Thr Arg Asn Arg Glu Thr Ile Phe A1a Lys
180 185 190
Tyr Ile Phe Arg His His Pro Arg Phe G1u Gly Lys Asp Ile Pro Phe
195 200 205
Trp Phe Asn Arg Asn Asp Lys Thr Ser Leu Glu Gly Gly Asp Glu Leu
210 215 220
Ile Leu Ser Lys Glu Ile Leu Ala Val Gly Ile Ser Gln Arg Thr Asp
225 230 235 240
Ser Ala Ser Val G1u Lys Leu Ala Lys Lys Leu Leu Tyr Tyr Pro Asp
245 250 255
Thr Ser Phe Lys Thr Val Leu Ala Phe Lys Ile Pro Val Ser Arg Ala
260 265 270
Phe Met His Leu Asp Thr Val Phe Thr Gln Val Asp Tyr Asp Lys Phe
275 280 285
Thr Val His Pro Gly Ile Val Gly Pro Leu Glu Val Tyr Ala Leu Thr
290 295 300
Lys Asp Pro Glu Asn Asp Gly Gln Leu Leu Val Thr Glu Glu Val Asp
17
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305 310 315 320
Thr Leu Glu Asn Ile Leu Lys Lys Tyr Leu Asp Arg Asp Ile Lys Leu
325 330 335
Ile Lys Cys Gly Gly Gly Asp Glu Ile Ile Ala Ala Arg G1u Gln Trp
340 345 350
Asn Asp Gly Ser Asn Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val
355 360 365
Tyr Ser Arg Asn Tyr Val Thr Asn Glu Ile Leu Glu Lys Glu Gly Ile
370 375 380
Lys Leu His Val Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly
385 390 395 400
Pro Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp Leu
405 410
<210> 12
<211> 413
<212> PRT
<213> Bacillus licheniformis
<400> 12
Met Ile Met Thr Thr Pro I1e His Va1 Tyr Ser Glu Ile Gly Pro Leu
1 5 10 15
Lys Thr Val Met Leu Lys Arg Pro G1y Arg Glu Leu Glu Asn Leu Thr
20 25 30
Pro Glu Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Pro
35 40 45
Ala Val Gln Lys Glu His Asp Gln Phe Ala Glu Thr Leu Lys Gln Gln
50 55 60
Gly Ala Glu Val Leu Tyr Leu Glu Lys Leu Thr Ala Glu Ala Leu Asp
65 70 75 80
Asp Ala Leu Val Arg Glu Gln Phe Ile Asp Glu Leu Leu Thr Glu Ser
85 90 95
Lys Ala Asp Ile Asn Gly Ala Tyr Asp Arg Leu Lys Glu Phe Leu Leu
100 105 110
18
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Thr Phe Asp Ala Asp Ser Met Val Glu Gln Val Met Ser Gly Ile Arg
115 120 125
Lys Asn Glu Leu Glu Arg Glu Lys Lys Ser His Leu His Glu Leu Met
130 135 140
Glu Asp His Tyr Pro Phe Tyr Leu Asp Pro Met Pro Asn Leu Tyr Phe
145 150 155 160
Thr Arg Asp Pro Ala Ala Ala Ile Gly Ser Gly Leu Thr Ile Asn Lys
165 170 175
Met Lys Glu Pro Ala Arg Arg Arg Glu Ser Leu Phe Met Arg Tyr Ile
180 185 190
Ile Asn His His Pro Arg Phe Lys Gly His Glu Ile Pro Val Trp Leu
195 200 205
Asp Arg Asp Phe Lys Phe Asn Ile Glu Gly Gly Asp Glu Leu Val Leu
210 215 220
Asn Glu Glu Thr Val Ala Ile Gly Val Ser Glu Arg Thr Thr Ala Gln
225 230 235 240
Ala Ile Glu Arg Leu Val Arg Asn Leu Phe Gln Arg Gln Ser Arg Ile
245 250 255
Arg Arg Val Leu Ala Val Glu Ile Pro Lys Ser Arg Ala Phe Met His
260 265 270
Leu Asp Thr Val Phe Thr Met Val Asp Arg Asp Gln Phe Thr 21e His
275 280 285
Pro Ala Ile G1n Gly Pro Glu Gly Asp Met Arg Ile Phe Val Leu Glu
290 295 300
Arg Gly Lys Thr A1a Asp Glu Ile His Thr Thr Glu Glu His Asn Leu
305 310 315 320
Pro Glu Val Leu Lys Arg Thr Leu Gly Leu Ser Asp Val Asn Leu Ile
325 330 335
Phe Cys Gly Gly Gly Asp Glu Ile Ala Ser Ala Arg Glu Gln Trp Asn
340 345 350
Asp G1y Ser Asn Thr Leu Ala Ile Ala Pro Gly Val Val Val Thr Tyr
355 360 365
19
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Asp Arg Asn Tyr Ile Ser Asn Glu Cys Leu Arg Glu Gln G1y Ile Lys
370 375 380
Val Ile Glu Ile Pro Ser Gly Glu Leu Ser Arg Gly Arg Gly Gly Pro
385 390 395 400
Arg Cys Met Ser Met Pro Leu Tyr Arg Glu Asp Val Lys
405 410
<210> 13
<211> 408
<212> PRT
<213> Enterococcus faecalis
<400> 13
Met Ser His Pro Tle Asn Val Phe Ser Glu Ile Gly Lys Leu Lys Thr
1 5 10 15
Val Met Leu His Arg Pro Gly Lys Glu Leu Glu Asn Leu Met Pro Asp
20 25 30
Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Lys Ala
35 40 45
Gln Ala Glu His Asp Ala Phe A1a Glu Leu Leu Arg Ser Lys Asp Ile
50 55 60
Glu Va1 Val Tyr Leu G1u Asp Leu Ala Ala Glu Ala Leu Ile Asn Glu
65 70 75 80
Glu Val Arg Arg Gln Phe Ile Asp Gln Phe Leu Glu Glu Ala Asn Ile
85 90 95
Arg Ser Glu Ser Ala Lys Glu Lys Val Arg Glu Leu Met Leu Glu Ile
100 105 110
Asp Asp Asn Glu G1u Leu Tle G1n Lys Ala 21e Ala Gly Ile Gln Lys
115 120 125
Gln Glu Leu Pro Lys Tyr Glu Gln Glu Phe Leu Thr Asp Met Val Glu
130 135 140
A1a Asp Tyr Pro Phe Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr
145 150 155 160
Arg Asp Asn Phe Ala Thr Met Gly His Gly Tle Ser Leu Asn His Met
165 170 175
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Tyr Ser Val Thr Arg Gln Arg Glu Thr Ile Phe Gly Gln Tyr Ile Phe
180 185 190
Asp Tyr His Pro Arg Phe Ala Gly Lys Glu Val Pro Arg Val Tyr Asp
l95 200 205
Arg Ser Glu Ser Thr Arg Ile Glu Gly Gly Asp Glu Leu Ile Leu Ser
210 215 220
Lys G1u Val Val Ala Ile Gly Ile Ser Gln Arg Thr Asp Ala Ala Ser
225 230 235 240
Ile Glu Lys Ile Ala Arg Asn Ile Phe Glu Gln Lys Leu Gly Phe Lys
245 250 255
Asn I1e Leu Ala Phe Asp Ile Gly Glu His Arg Lys Phe Met His Leu
260 265 270
Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Ile His Pro
275 280 285
Glu I1e Glu Gly Gly Leu Val Val Tyr Ser Ile Thr Glu Lys Ala Asp
290 295 300
Gly Asp Ile Gln Ile Thr Lys Glu Lys Asp Thr Leu Asp Asn Ile Leu
305 310 315 320
Cys Lys Tyr Leu His Leu Asp Asn Val Gln Leu Ile Arg Cys Gly Ala
325 330 335
Gly Asn Leu Thr Ala Ala Ala Arg Glu Gln Trp Asn Asp Gly Ser Asn
340 345 350
Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asp Arg Asn Thr
355 360 365
Ile Thr Asn Lys Ala Leu Glu Glu Ala Gly Val Lys Leu Asn Tyr Ile
370 375 380
Pro Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg Cys Met Ser
385 390 395 400
Met Pro Leu Tyr Arg Glu Asp Leu
405
<210> 14
21
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<211> 409
<212> PRT
<213> Lactobacillus sake
<400> 14
Met Thr Ser Pro Ile His Val Asn Ser Glu Ile Gly Lys Leu Lys Thr
1 5 10 15
Val Leu Leu Lys Arg Pro Gly Lys Glu Val Glu Asn Ile Thr Pro Asp
20 25 30
Ile Met Tyr Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu Pro Thr Ile
35 40 45
Gln Lys Glu His Asp Gln Phe Ala Gln Thr Leu Arg Asp Asn Gly Val
50 55 60
Glu Val Leu Tyr Leu Glu Asn Leu Ala Ala Glu Ala Ile Asp Ala Gly
65 70 75 80
Asp Val Lys Glu Ala Phe Leu Asp Lys Met Leu Asn Glu Ser His Ile
85 90 95
Lys Ser Pro Gln Val Gln Ala Ala Leu Lys Asp Tyr Leu Ile Ser Met
100 105 110
Ala Thr Leu Asp Met Val Glu Lys Ile Met Ala G1y Val Arg Thr Asn
115 120 125
Glu Ile Asp Ile Lys Ser Lys Ala Leu Ile Asp Val Ser Ala Asp Asp
130 135 140
Asp Tyr Pro Phe Tyr Met Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg
145 150 155 160
Asp Pro Ala Ala Ser Met Gly Asp Gly Leu Thr Ile Asn Lys Met Thr
165 170 175
Phe Glu Ala Arg Gln Arg Glu Ser Met Phe Met Glu Val Ile Met Gln
180 185 190
His His Pro Arg Phe A1a Asn Gln Gly Ala Gln Val Trp Arg Asp Arg
195 200 205
Asp His Ile Asp Arg Met Glu Gly G1y Asp Glu Leu Ile Leu Ser Asp
210 215 220
Lys Val Leu Ala Ile Gly Ile Ser Gln Arg Thr Ser Ala Gln Ser Ile
22
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225 230 235 240
Glu Glu Leu Ala Lys Val Leu Phe Ala Asn His Ser Gly Phe Glu Lys
245 250 255
Ile Leu Ala Tle Lys Ile Pro His Lys His Ala Met Met His Leu Asp
260 265 270
Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Ile His Pro Gly
275 280 285
Ile Gln Gly Ala Gly Gly Met Val Asp Thr Tyr Ile Leu Glu Pro Gly
290 295 300
Asn Asn Asp Glu I1e Lys Ile Thr His Gln Thr Asp Leu Glu Lys Val
305 310 315 320
Leu Arg Asp Ala Leu Glu Val Pro Glu Leu Thr Leu Ile Pro Cys Gly
325 330 335
' Gly Gly Asp Ala Val Val Ala Pro Arg Glu Gln Trp Asn Asp Gly Ser
340 345 350
Asn Thr Leu Ala Ile A1a Pro Gly Val Val Val Thr Tyr Asp Arg Asn
355 360 365
Tyr Val Ser Asn Glu Asn Leu Arg Gln Tyr Gly Ile Lys Val Ile Glu
370 375 380
Val Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met
385 390 395 400
Ser Met Pro Leu Val Arg Arg Lys Thr
405
23
