U.S. patent application number 11/689166 was filed with the patent office on 2007-07-26 for methods for inhibiting viral replication in vivo.
This patent application is currently assigned to Phoenix Pharmacologics, Inc.. Invention is credited to Mike Clark.
Application Number | 20070172469 11/689166 |
Document ID | / |
Family ID | 32326546 |
Filed Date | 2007-07-26 |
United States Patent
Application |
20070172469 |
Kind Code |
A1 |
Clark; Mike |
July 26, 2007 |
Methods For Inhibiting Viral Replication In Vivo
Abstract
The present invention is directed to methods of modulating viral
replication in vivo comprising administering to an individual a
therapeutically or prophylactically effective amount of a
composition comprising arginine deiminase modified with
polyethylene glycol, to methods of concurrently modulating viral
replication and treating cancer, and to methods of modulating
nitric oxide levels in a patient, among others.
Inventors: |
Clark; Mike; (Lexington,
KY) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR
2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
Phoenix Pharmacologics,
Inc.
|
Family ID: |
32326546 |
Appl. No.: |
11/689166 |
Filed: |
March 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10674666 |
Sep 29, 2003 |
7204980 |
|
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11689166 |
Mar 21, 2007 |
|
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60427497 |
Nov 18, 2002 |
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Current U.S.
Class: |
424/94.5 ;
424/85.7; 514/220; 514/263.31; 514/43; 514/47; 514/49 |
Current CPC
Class: |
A61P 31/14 20180101;
A61K 38/50 20130101; C12Q 1/18 20130101; A61K 47/60 20170801; A61K
45/06 20130101; Y02A 50/393 20180101; G01N 2333/978 20130101; A61P
35/04 20180101; Y02A 50/30 20180101; A61P 1/16 20180101; A61P 35/00
20180101; A61P 31/12 20180101; Y02A 50/465 20180101; A61P 43/00
20180101; A61K 38/50 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/094.5 ;
424/085.7; 514/043; 514/047; 514/049; 514/220; 514/263.31 |
International
Class: |
A61K 38/51 20060101
A61K038/51; A61K 38/21 20060101 A61K038/21; A61K 31/7076 20060101
A61K031/7076; A61K 31/7072 20060101 A61K031/7072; A61K 31/7056
20060101 A61K031/7056; A61K 31/551 20060101 A61K031/551; A61K
31/522 20060101 A61K031/522 |
Claims
1. A method of inhibiting replication of human immunodeficiency
virus in an individual comprising administering to said individual
a composition comprising an arginine deiminase bonded to
polyethylene glycol in an amount effective to inhibit human
immunodeficiency virus replication in said individual.
2. The method of claim 1 further comprising the step of
administering to said individual one or more compounds selected
from the group consisting of antibiotics, anti-virals, antifungals,
and anti-protozoan drugs.
3. The method of claim 1 further comprising the step of
administering to said individual one or more other anti-viral
compounds.
4. The method of claim 3 wherein said anti-viral compounds are one
or more of azidovudine (AZT), didanosine (dideoxyinosine, ddl),
d4T, zalcitabine (dideoxycytosine, ddC), nevirapine, lamivudine
(epivir, 3TC), saquinavir (Invirase), ritonavir (Norvir), indinavir
(Crixivan), delavirdine (Rescriptor), pegylated (PEG)
interferon-.alpha. (IFN), or ribavirin.
5. The method of claim 1 wherein said composition is administered
intramuscularly, intradermally, or intraperitoneally.
6. The method of claim 1 wherein said composition comprising an
arginine deiminase bonded to polyethylene glycol is effective at a
concentration of less than about 1 mM to inhibit human
immunodeficiency virus replication by at least 50% in greater than
50% of cells in an assay to measure human immunodeficiency virus
replication.
7. The method of claim 1 wherein the amount of arginine deiminase
bonded to polyethylene glycol effective to inhibit human
immunodeficiency virus replication is between about 40 IU/m and
about 160 IU/m.sup.2 per week.
8. The method of claim 1 wherein the amount of arginine deiminase
bonded to polyethylene glycol effective to inhibit human
immunodeficiency virus replication is about 160 IU/m2 per week.
9. The method of claim 1 wherein the amount of arginine deiminase
bonded to polyethylene glycol effective to inhibit human
immunodeficiency virus replication lowers plasma arginine levels to
less than 5 .mu.M.
10. The method of claim 1 wherein the arginine deiminase is
covalently bonded via a linking group to polyethylene glycol,
wherein each of said polyethylene glycol molecules has a molecular
weight of about 10,000 to about 30,000.
11. The method of claim 1 wherein each of said polyethylene glycol
molecules has a molecular weight of about 20,000.
12. The method of claim 10 wherein the linking group is selected
from the group consisting of a succinimide group, an amide group,
an imide group, a carbamate group, an ester group, an epoxy group,
a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine
group, a cysteine group, and a histidine group, and combinations
thereof.
13. The method of claim 10 wherein the linking group is
succinimidyl succinate.
14. The method of claim 1 wherein from about 7 to about 15
polyethylene glycol molecules are bonded to arginine deiminase.
15. The method of claim 1 wherein from about 9 to about 12 said
polyethylene glycol molecules are bonded to arginine deiminase.
16. The method of claim 1 wherein said arginine deiminase is
derived from a microorganism of the genus Mycoplasma.
17. The method of claim 16 wherein said microorganism is selected
from the group consisting of Mycoplasma arginini, Mycoplasma
hominus, Mycoplasma arthritides and combinations thereof.
18. The method of claim 1 wherein the arginine deiminase has an
amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
13, 14, 15, 16, 17, 18, 19, 20 or 21.
19. The method of claim 1 wherein the arginine deiminase has an
amino acid sequence of SEQ ID NO: 1 or 4.
20-22. (canceled)
23. A method of treating an individual who is suspected of having
been exposed to human immunodeficiency virus comprising the step of
administering to said individual an amount of a composition
comprising an arginine deiminase bonded to polyethylene glycol
effective to inhibit human immunodeficiency virus replication in
said individual.
24. A method of inhibiting human immunodeficiency virus replication
in an individual at risk for one or more viruses comprising
administering to said individual an amount of a composition
comprising an arginine deiminase bonded to polyethylene glycol
effective to inhibit human immunodeficiency virus replication in
said individual.
25. A method of inhibiting human immunodeficiency virus replication
in an individual who has been identified as having been infected
with one or more viruses comprising administering to said
individual an amount of a composition comprising an arginine
deiminase bonded to polyethylene glycol effective to inhibit human
immunodeficiency virus replication in said individual.
26-51. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority of Application Ser.
No. 60/427,497, filed Nov. 18, 2002, which is hereby incorporated
by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to methods for inhibiting
viral replication, to methods for treating cancer, to methods for
treating and/or inhibiting metastasis, and to methods for
concurrently inhibiting viral replication and treating cancer or
treating and/or inhibiting metastasis, and others.
BACKGROUND OF THE INVENTION
[0003] Viral infections are among the leading causes of death with
millions of deaths each year being directly attributable to several
viruses including hepatitis and HIV.
[0004] Hepatitis is a disease of the human liver. It is manifested
with inflammation of the liver and is usually caused by viral
infections. Several viruses such as hepatitis A, B, C, D, E and G
are known to cause viral hepatitis. Among them, HBV and HCV are the
most serious.
[0005] Hepatitis C virus (HCV) is pandemic with more than 170
million persons worldwide infected. Among viral diseases, it is 5
times more widespread that human immunodeficiency virus type 1
(HIV-1), and approximately 10,000 Americans will die this year from
cirrhosis and hepatocellular carcinoma (HCC) resultant from chronic
HCV infection (Sun C A, Wu D M, Lin C C, L U S N, You S L, Wang L
Y, Wu M H, Chen C J. 2003. Incidence and cofactors of hepatitis C
virus-related hepatocellular carcinoma: a prospective study of
12,008 men in Taiwan. Am J Epidemiol 157:674-682; Herrine S K.
2002. Approach to the patient with chronic hepatitis C virus
infection. Ann Intern Med 136: 747-757; Hoofnagle J H. 2002. Course
and outcome of hepatitis C. Hepatology 36:S21-S29; Lauer G M,
Walker B D. 2001 Hepatitis C virus infection. N Engl J Med
345:41-52; Liang T J, Rehermann B, Seeff L B, Hoofnagle J H. 2001.
Pathogenesis, natural history, treatment, and prevention of
hepatitis C. Ann Intern Med 132:296-305). Furthermore, the
prevalence of HCV continues to increase in the USA, Western Europe
and Asia despite the institution of blood donor screening programs.
Progression to chronic disease occurs in most HCV infected
patients. In addition, HCV causes HCC in 1-4% annually of all
chronically infected individuals. Moreover, HCC can occur even in
those without cirrhosis (Shiratori Y, Shiina S, Teratani T, Imamura
M, Obi S, Sato S, Koike Y, Yoshida H, Omata M. 2003. Interferon
therapy after tumor ablation improves prognosis in patients with
hepatocellular carcinoma associated with hepatitis C virus. Ann Int
Med 138:299-306; Smith M W, Yue Z N, Geiss G K, Sadovnikova N Y,
Carter V S, Boix L, Lazaro C A, Rosenberg G B, Bumgarner R E,
Fausto N, Bruix J, Katze M G. 2003. Identification of novel tumor
markers in hepatitis C virus-associated hepatocellular carcinoma.
Cancer Res 63:859-864; Yoshizawa H. 2002. hepatocellular carcinoma
associated with hepatitis C virus infection in Japan: projection to
other countries in the foreseeable future. Oncology 62 (Suppl
1):8-17; Colombo M. 1999. Natural history and pathogenesis of
hepatitis C virus related hepatocellular carcinoma. J Hepatology 31
(Suppl 1):25-30). Given the current prevalence of HCV infection
among persons 30 to 50 years of age, the incidence and mortality
rates of HCC are estimated to double in the United States over the
next 10 to 20 years (E1-Serag H B. 2002. Hepatocellular carcinoma
and hepatitis C in the United States. Hepatology 36:S74-S83). It is
estimated that there are 500 million people infected with it
worldwide. No effective immunization is currently available, and
hepatitis C can only be controlled by other preventive measures
such as improvement in hygiene and sanitary conditions and
interrupting the route of transmission.
[0006] Today, there is no effective therapy for HCC except surgical
resection (Ryder S D. 2003. Guidelines for the diagnosis and
treatment of hepatocellular carcinoma (HCC) in adults. Gut 52
(Suppl III):iii1-iii8; E1-Serag H B. 2002. Hepatocellular carcinoma
and hepatitis C in the United States. Hepatology 36:S74-S83;
E1-Serag H B. 2001. Global epidemiology of hepatocellular
carcinoma. Clin Liver Dis 5:87-107; DiMaio M, DeMaio E, Perrone F,
Pegnata S, Daniele B. 2002. Hepatocellular carcinoma: systemic
treatments. J Clin Gastroenterol 35 (Suppl. 2):S109-S114; Curley S
A, Izzo F, Ellis L M, Vauthey J N, Vallone P. 2000. Radiofrequency
ablation of hepatocellular cancer in 110 patients with cirrhosis.
Ann Surg 232:381-391; Watkins K T, Curley Sa. 2000. Liver and bile
ducts. In Clinical Oncology, 2.sup.nd ed. Editors M D Abeloff, J O
Armitage, A S Lichter, J E Niederhuber. New York: Churchill
Livingstone, pp. 1681-1748). However, only <5% of HCC patients
are surgical candidates and only .about.1% actually undergo
resection. Even among those resected, recurrence of HCC is common,
especially in those infected with HCV.
[0007] Amino acid deprivation therapy is an effective means for the
treatment of some cancers. Although normal cells do not require
arginine, many cancer cell lines are auxotrophic for this amino
acid. Thus, cancers, including but not limited to HCC, may be
selectively killed by arginine deprivation therapy (Ensor C M,
Holtsberg F W, Bomalaski J S, Clark M A. 2002. Pegylated arginine
deiminase (ADI-SS PEG .sub.20,000 mw) inhibits human melanomas and
hepatocellular carcinomas in vitro and in vivo. Cancer Res
62:5443-5440; Takaku, H, Misawa, S, Hayashi H and Miyazaki K.
(1993). Chemical modification by polyethylene glycol of the
anti-tumor enzyme arginine deiminase from Mycoplasma arginini. Jpn.
J. Cancer Res. 84:1195-1200; Takaku H, Takase M, Abe S, Hayashi H
and Miyazaki K. (1992). In vivo anti-tumor activity of arginine
deiminase purified from Mycoplasma arginini. Int. J. Cancer
51:244-249; Sugimura K, Ohno T, Kussyama T, Azuma I. 1992. High
sensitivity of human melanoma cell lines to the growth inhibitory
activity of Mycoplasma arginini deiminase in vitro. Melanoma Res.
2:191-196). High sensitivity of human melanoma cell lines to the
growth inhibitory activity of Mycoplasma arginini deiminase in
vitro. Melanoma Res. 2:191-196). This therapy is well tolerated as
arginine is not an essential amino acid in humans (Rose W C. 1949.
Amino acid requirements of man. Fed Proc 8:546-452, Snyderman, S.,
E., Boyer, A., and L. E. Holt 1959. The arginine requirement of the
infant. J. Dis. Child. 97:192 and for review see Rodgers Q R. 1994.
Species variation in arginine requirements. In Proceedings from a
Symposium Honoring Willard J. Visek--from Ammonia to Cancer and
Gene Expression. Special Publication 86-April 1994, Agriculture
Experiment Station, University of Illinois, 211 Mumford Hall,
Urbana, Ill. 61801, pp. 9-21, as it can be synthesized from
citrulline. ADI converts extracellular arginine into citrulline
which may be taken up by normal cells and converted into arginine
intracellularly but not by cancer cells, especially HCC cells,
because they lack the rate limiting enzyme argininosuccinate
synthetase (Ensor C M, Holtsberg F W, Bomalaski J S, Clark M A.
2002. Pegylated arginine deiminase (ADI-SS PEG .sub.20,000 mw)
inhibits human melanomas and hepatocellular carcinomas in vitro and
in vivo. Cancer Res 62:5443-5440). This inability to express
argininosuccinate synthetase has recently been confirmed by others
(Shen L J, Lin W C, Beloussow K, Shen W C. 2003. Resistance to the
anti-proliferative activity of recombinant arginine deiminase in
cell culture correlates with the endogenous enzyme,
argininosuccinate synthetase. Cancer Lett 191:165-170) We have
extended this study of argininosuccinate synthetase deficiency to
other tumors (Dillon B J, Prieto V G, Curley S A, Ensor C M,
Holtsberg F W, Bomalaski J S, Clark M A. 2003. The method incidence
and distribution of argininosuccinate synthetase deficiency in
human cancers: a method for identifying cancers sensitive to
arginine deprivation. Cancer (in press). Preliminary results from
human clinical testing of ADI-SS PEG 20,000 mw indicates this
therapy to be both safe and effective as an anti-cancer
treatment.
[0008] Hepatitis B virus infection can lead to a wide spectrum of
liver injury. Moreover, chronic hepatitis B infection has been
linked to the subsequent development of hepatocellular carcinoma, a
major cause of death. Current prevention of HBV infection is a
hepatitis B vaccination which is safe and effective. However,
vaccination is not effective in treating those already infected
(i.e., carriers and patients).
[0009] Acquired immune deficiency syndrome (AIDS) is a fatal
disease, reported cases of which have increased dramatically within
the past several years. The AIDS virus was first identified in
1983. It has been known by several names and acronyms. It is the
third known T-lymphotropic virus (HTLV-III), and it has the
capacity to replicate within cells of the immune system, causing
profound cell destruction. The AIDS virus is a retrovirus, a virus
that uses reverse transcriptase during replication. This particular
retrovirus is also known as lymphadenopathy-associated virus (LAV),
AIDS-related virus (ARV) and, most recently, as human
immunodeficiency virus (LIV). Two distinct families of HIV have
been described to date, namely HIV-1 and HIV-2. The acronym "HIV"
is used herein to refer to human immunodeficiency viruses
generically.
[0010] Herpes simplex virus (HSV) types 1 and 2 are persistent
viruses that commonly infect humans; they cause a variety of
troubling human diseases. HSV type 1 causes oral "fever blisters"
(recurrent herpes labialis), and HSV type 2 causes genital herpes,
which has become a major venereal disease in many parts of the
world. No fully satisfactory treatment for genital herpes currently
exists. In addition, although it is uncommon, HSV can also cause
encephalitis, a life-threatening infection of the brain. (The Merck
Manual, Holvey, Ed., 1972; Whitley, Herpes Simplex Viruses, In:
Virology, 2nd Ed., Raven Press (1990)). A most serious HSV-caused
disorder is dendritic keratitis, an eye infection that produces a
branched lesion of the cornea, which can in turn lead to permanent
scarring and loss of vision. Ocular infections with HSV are a major
cause of blindness. HSV is also a virus which is difficult, if not
impossible to cure.
[0011] Anti-Viral Therapies
[0012] There are several problems with current anti-viral
therapies. First, there are relatively few effective antiviral
drugs. Many of the existing anti-virals cause adverse or
undesirable side-effects. Most effective therapies (such as
vaccination) are highly specific for only a single strain of virus.
Frequently the virus undergoes mutation such that it becomes
resistant to either the drug or vaccine.
[0013] Many of the current treatments for viral infections revolve
around interferon-.alpha. (IFN-.alpha.). It is believed that
IFN-.alpha. binds to cellular receptors and initiates an
intracellular response that includes enzymes involved in protein
synthesis. This ultimately leads to the anti-viral
activity/response. However, data from various clinical trials have
shown that approximately 40% of patients treated with IFN-.alpha.
initially responded to the therapy, but 70% of these relapsed after
the treatment ended. (Damen, M., and Bresters, D., in H. W. (ed.):
Curr. Stud. Hematol. Blood Transf., Darger Publishers 1998, Basel.)
Overall, the long-term therapeutic effect and response was observed
in only 10 to 30% of the patients. (Houghton, M., in Fields, B. N.
et al., Fields Virology, Raven Publishers 1996, Philadelphia). In
addition many side effects were observed such as severe flu,
fatigue, muscle and head aches, even depression, weight loss and
diarrhea. (Damen, M., and Bresters, D., in H. W. (ed.): Curr. Stud.
Hematol. Blood Transf., Darger Publishers 1998, Basel.)
[0014] HCV therapy
[0015] The current standard therapy for HCV infection is pegylated
(PEG) interferon-.alpha. (IFN) and ribavirin. Although this therapy
can result in sustained anti-viral response, significant numbers of
patients do not respond to this therapy or are excluded from this
treatment (Falck-Ytter Y, Kale H, Mullen K D, Sarbah S A, Sorescu
L, McCullough A J. 2002. Surprisingly small effect of antiviral
treatment in patients with hepatitis C. Ann Intern Med 136:288-292;
Fried M W. 2002. Side effects of therapy of hepatitis C and their
management. Hepatology 36:S237-S244; Fried M W, Shiffman M L, Reddy
K R, Smith C, Marinos G, Goncales F L Jr, Haussinger K, Diago M,
Carosi G, Dhumeaux K, Craxi A, Lin A, Hoffman J, Yu J. 2002.
Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus
infection. N Engl J Med 347:975-982.; Herrine S K. 2002. Approach
to the patient with chronic hepatitis C virus infection. Ann Intern
Med 136:747-757; Lauer G M, Walker B D. 2001. Hepatitis C virus
infection. N Engl J Med 345:41-52; Liang T J, Rehermann B, Seeff L
B, Hoofnagle J H. 2001. Pathogenesis, natural history, treatment
and prevention of hepatitis C. Ann Intern Med 132:296-305; Manns M
P, McHutchinson J G, Gordon S C, Rustgi V K, Shiffman M, Reindollar
R, Goodman Z D, Koury K, Ling M-H, Albrecht J K. 2001.
Peginterferon alfa-2b plus ribavirin compared with interferon
alfa-2b plus ribavirin for initial treatment of chronic hepatitis
C: a randomized trial. Lancet 358:958-965). For example, recent
studies of PEG-IFN .alpha.-2a (Pegasys.sup.TN) plus ribavirin, and
PEG-IFN .alpha.-2b (Pegintron.sup.TN) plus ribavirin demonstrate
that .about.56% of studied patients had a sustained viral response
(Dantzler T D, Lawitz E J. 2003. Treatment of chronic hepatitis C
in nonresponders to previous therapy. Curr Gastroenterol Rep
5:78-85; Masci P, Bukowski R M, Patten P A, Osborn B L, Borden E C.
2003. New and modified interferon alfas: preclinical and clinical
data. Curr Oncol Rep 5:108-113; Chandler G, Sulkowski M S, Jenckes
M W, Torbenson M S, Herlong H F, Bass E B, Gebo K A. 2002.
Treatment of chronic hepatitis C: a systematic review. Hepatology
36:S135-S144; DiBisceglie A M, Hoofnagle J H. 2002. Optimal therapy
of hepatitis C. Hepatology 36:S121-127; Fried M W. 2002. Side
effects of therapy of hepatitis C and their management. Hepatology
36:S237-S244; Lindsay K L. 2002. Introduction to therapy of
hepatitis C. Hepatology 36:S1 14-S 120. Lopez-Guerrero J A,
Carrasco L. 1998. Effect of nitric oxide on poliovirus infection of
two human cell lines. J Virol 72:2538-2540; Wedemeyer H, Wiegand J,
Cornberg M, Manns M P.; Polyethylene glycol-interferon: Current
status in hepatitis C virus therapy, J Gastroenterol Hepatol.
December 2002; 17 Suppl 3:S344-S350; Manns M P, McHutchinson J G,
Gordon S C, Rustgi V K, Shiffman M, Reindollar R, Goodman Z D,
Koury K, Ling M-H, Albrecht J K. 2001. Peginterferon alfa-2b plus
ribavirin compared with interferon alfa-2b plus ribavirin for
initial treatment of chronic hepatitis C: a randomized trial.
Lancet 358:958-965). However, for HCV genotypes 1a and 1b, the most
common genotypes in the USA and western Europe, the response was
only .about.46%. HCV genotypes 2 and 3 had a better response (76%
-82%). Furthermore, this response rate of 50% is only for patients
studied in clinical trials; it does not represent the entire
patient population and is, therefore, biased ((Dantzler T D, Lawitz
E J. 2003. Treatment of chronic hepatitis C in nonresponders to
previous therapy. Curr Gastroenterol Rep 5:78-85; Masci P, Bukowski
R M, Patten P A, Osborn B L, Borden E C. 2003. New and modified
interferon alfas: preclinical and clinical data. Curr Oncol Rep
5:108-113; Chandler G, Sulkowski M S, Jenckes M W, Torbenson M S,
Herlong H F, Bass E B, Gebo K A. 2002. Treatment of chronic
hepatitis C: a systematic review. Hepatology 36:S135-S144;
DiBisceglie A M, Hoofnagle J H. 2002. Optimal therapy of hepatitis
C. Hepatology 36:S121-127; Fried M W. 2002. Side effects of therapy
of hepatitis C and their management. Hepatology 36:S237-S244; Fried
M W, Shiffman M L, Reddy K R, Smith C, Marinos G, Goncales F L Jr,
Haussinger K, Diago M, Carosi G, Dhumeaux K, Craxi A, Lin A,
Hoffman J, Yu J. 2002. Peginterferon alfa-2a plus ribavirin for
chronic hepatitis C virus infection. N Engl J Med 347:975-982;
Lindsay K L. 2002. Introduction to therapy of hepatitis C.
Hepatology 36:S114-S120. Lopez-Guerrero J A, Carrasco L. 1998.
Effect of nitric oxide on poliovirus infection of two human cell
lines. J Virol 72:2538-2540; Wedemeyer 2002, Manns M P,
McHutchinson J G, Gordon S C, Rustgi V K, Shiffman M, Reindollar R,
Goodman Z D, Koury K, Ling M-H, Albrecht J K. 2001. Peginterferon
alfa-2b plus ribavirin compared with interferon alfa-2b plus
ribavirin for initial treatment of chronic hepatitis C: a
randomized trial. Lancet 358:958-965). For example, a large study
in the USA excluded 404 out of 1337 (or .about.30%) of potential
patients due to selection criteria (McHutchinson J G, Gordon S C,
Schiff E R, Shiffman M L, Lee W M, Rustgi V K, et al. 1998.
Interferon alfa-2b alone or in combination with ribavirin as
initial treatment for chronic hepatitis C. Hepatitis Interventional
Therapy Group. N Engl J Med 339:1485-1492). Other large studies
often fail to describe their screening criteria or the percentage
of patients enrolled. A recent study performed in the USA by a
large teaching hospital noted that 72% of all HCV patients were not
treated with IFN for reasons such as medical or psychiatric
contraindications, ongoing substance or alcohol abuse, failure to
adhere to evaluation procedures, normal liver enzymes or even
patient preference of no treatment (Falck-Ytter Y, Kale H, Mullen K
D, Sarbah S A, Sorescu L, McCullough A J. 2002. Surprisingly small
effect of antiviral treatment in patients with hepatitis C. Ann
Intern Med 136:288-292). Similar results have been confirmed by
others (Diamond C, Lee J H. 2002. Use of antiviral therapy in
patients with hepatitis C. Annals Intern Med 137:1012). Thus a
significant portion of the HCV infected population does not receive
current "best standard of care" treatment due to a variety of
medical or psychiatric contraindications. Even in studies using the
"best" patients in the USA and western Europe, only .about.50%
achieve sustained viral response.
[0016] IFN-.alpha. also has significant side effects which occur
with approximately the same frequency in both the PEG and non PEG
formulated versions (Masci P, Bukowski R M, Patten P A, Osborn B L,
Borden E C. 2003. New and modified interferon alfas: preclinical
and clinical data. Curr Oncol Rep 5:108-113; Fried M W. 2002. Side
effects of therapy of hepatitis C and their management. Hepatology
36:S237-S244; Wedemeyer 2002, Herrine S K. 2002. Approach to the
patient with chronic hepatitis C virus infection. Ann Intern Med
136:747-757; Lauer G M, Walker B D. 2001. Hepatitis C virus
infection. N Engl J Med 345:41-52; Liang T J, Rehermann B, Seeff L
B, Hoofnagle J H. 2001. Pathogenesis, natural history, treatment,
and prevention of hepatitis C. Ann Intern Med 132:296-305). These
side effects include an influenza-like illness with fever, chills,
myalgias and malaise in up to 82% of patients studied, with
neuropsychiatric complications such as depression, irritability and
depression and anxiety in .about.20% of patients. Bone marrow
suppression with granulocytopenia, anemia or thrombocytopenia
occurs in .about.5%, as does alopecia. These side effects are
frequently so severe that further treatment with IFN alpha is
discontinued, thus further limiting the utility of IFN therapy.
Therefore, new treatments for HCV are needed.
[0017] HIV therapy
[0018] Several drugs have been approved for treatment of HIV,
including azidovudine (AZT), didanosine (dideoxyinosine, ddI), d4T,
zalcitabine (dideoxycytosine, ddC), nevirapine, lamivudine (epivir,
3TC), saquinavir (Invirase), ritonavir (Norvir), indinavir
(Crixivan), and delavirdine (Rescriptor). See M. I. Johnston &
D. F. Hoth, Science, 260(5112), 1286-1293 (1993) and D. D. Richman,
Science, 272(5270), 1886-1888 (1996). An alternative treatment for
HCV has been ribavirin. Ribavirin is an anti-viral with a broad
range of target viral activities. Ribavirin is a guanosine analogue
harboring a modified base
(1-.beta.-D-ribo-furanosyl-1,2,4-trizole-3-carboxamide), and has
been proposed to inhibit the cellular enzyme inosine monophosphate
dehydrogenase, resulting in a decrease of guanosine triphosphate.
Damen, M., and Bresters, D., in H. W. (ed.): Curr. Stud. Hematol.
Blood Transf., Darger Publishers 1998, Basel. However, ribavirin
will cause side effects. Christie, J. M. and Chapman, R. W., Hosp
Med. 60, 357 (1999). In particular ribavirin accumulates in the
erythrocytes of patients and can cause hemolytic anemia.
[0019] An AIDS vaccine (Salk's vaccine) has been tested and several
proteins which are chemokines from CD8 have been discovered to act
as HIV suppressors. In addition to the above synthetic nucleoside
analogs, proteins, and antibodies, several plants and substances
derived from plants have been found to have in vitro anti-HIV
activity. However, HIV virus is not easily destroyed nor is there a
good mechanism for keeping the host cells from replicating the
virus.
[0020] In vitro Use of Arginine Deprivation
[0021] Many studies over the last 30 years have demonstrated that
extracellular arginine is required for viral replication in vitro.
Historically this has been accomplished by making tissue culture
media deficient in arginine and dialyzing the serum used as a
supplement in order to achieve arginine free medium. Using this
methodology to achieve arginine deprivation results in inhibition
of replication of a large number of diverse families of viruses
including: adeno virus (Rouse H C, Bonifas V H, Schlesinger R W.
1963. Dependence of adenovirus replication on arginine and
inhibition of plaque formation by pleuropneumonia-like organisms.
Virology 20:357-365), herpes virus (Tankersley R W Jr. 1964. Amino
acid requirements of herpes simplex virus in human cells. J
Bacteriol 87:608-613), SV 40 (Goldblum N, Ravid Z, Becker Y.
1968.Effect of withdrawal of arginine and other amino acids on the
synthesis of tumour and viral antigens of SV40 virus. J Gen Virol
3:143-146), cytomegalovirus (Minamishima Y, Benyesh-Melnick M.
1969. Arginine-dependent events in cytomegalovirus infection.
Bacteriol Proc 170:334-339), respiratory syncytial virus (Levine S,
Buthala D, Hamilton R D. 1971. Late stage synchronization of
respiratory syncytial virus replication. Virology 45:390-400),
polyoma virus (Winters A L, Consigli R A, Rogers O R 1972. A
non-functional arginine biosynthetic pathway in polyoma-infected
mouse embryo cells. Biochem Biophys Res Comm 47:1045-1051),
Newcastle disease virus (Ilnuma M, Maemo K, Matsumoto T. 1973.
Studies on the assembly of Newcastle disease virus: an
arginine-dependent step in virus replication. Virology 51:205-215),
measles virus (Romano N, Scarlata G. 1973. Amino acid requirements
of measles virus in HeLa cells. Arch Gesamte Virus Forschung
43:359-366), influenza (Lisok T P, Sominina A A. 1977. Improved
methods of influenza virus propagation. I. Enhancement of virus
reproduction in cell cultures. Acta Virol 21:234-240), and perhaps
even more relevant, vaccinia virus (Holterman O A. 1969. Amino acid
requirements for the propagation of vaccinia virus in Earle's L
cells. J Gen Virol 4:585-591, Singer S H, Fitzgerald E A, Barile M
F, Kirschstein R L. 1970. Effect of mycoplasmas on vaccinia virus
growth: requirement of arginine. Proc Soc Exp Biol Med
133:1439-1442, Obert G, Tripier F, Guir J. 1971. Arginine
requirement for late mRNA transcription of vaccinia virus in KB
cells. Biochem Biophys Res Comm 44:362-367, Archard L C, Williamson
J D. 1971. The effect of arginine deprivation on the replication of
vaccinia virus. J Gen Virol 12:249-258.) and rabbit pox virus
(Cooke B C, Williamson J D. 1973. Enhanced utilization of
citrulline in rabbit pox virus-infected mouse sarcoma 180 cells. J
Gen Virol 21:339-348). Vaccinia virus is the prototypical member of
the Orthopoxvirus genera that includes smallpox (variola virus).
Inhibition of viral replication is observed in vitro, even though
protein synthesis and replication of infected cells is not
affected.
[0022] Enzymes which degrade arginine are known and include
arginine deiminase (ADI). However, 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, H, Misawa, S, Hayashi H and
Miyazaki K. (1993). Chemical modification by polyethylene glycol of
the anti-tumor enzyme arginine deiminase from Mycoplasma arginini.
Jpn. J. Cancer Res. 84:1195-1200. However, the modified protein was
toxic when metabolized due to the release of cyanide from the
cyanuric chloride linking group.
[0023] There is a need for methods for inhibiting viral replication
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
[0024] The present invention is directed to methods of modulating
viral replication comprising administering to a patient arginine
deiminase bonded to polyethylene glycol. The present invention is
also directed to methods of concurrently modulating viral
replication and treating cancer, including, for example, sarcomas,
hepatomas and melanomas. The present invention is also directed to
methods of determining the susceptibility of an individual to
arginine deprivation therapy for a viral infection, methods for
improving liver function, and the like. These and other aspects of
the present invention will be elucidated in the following detailed
description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Overview
[0026] The present invention is based on the unexpected discovery
that ADI modified with polyethylene glycol inhibits viral
replication. ADI may be covalently bonded to polyethylene glycol
with or without a linking group, although some embodiments utilize
a linking group. PEG-20,000, for example, exhibits useful enzymatic
activity levels, antigenicity, circulating half-life, efficacy, and
relative ease of manufacture.
[0027] The mechanism by which lowering of extracellular arginine
inhibits viral replication is not known. Herbivores such as human
and mice (unlike carnivores which have an absolute requirement for
arginine) (for review see Rodgers Q R. 1994. Species variation in
arginine requirements. In Proceedings from a Symposium Honoring
Willard J. Visek--from Ammonia to Cancer and Gene Expression.
Special Publication 86-April 1994, Agriculture Experiment Station,
University of Illinois, 211 Mumford Hall, Urbana, Ill. 61801, pp.
9-21) and most cells do not require arginine for growth as it may
be synthesized from citrulline using two intracellular enzymes
(argininosuccinate synthase and argininosuccinate lyase). Thus
elimination of extracellular arginine dose not affect intracellular
levels of arginine provided citrulline is available to the cells.
As viral replication is an intracellular process, it is unexpected
that a decrease in extracellular arginine could inhibit viral
replication.
[0028] Although not wishing to be bound by theory, one possible
mechanism by which lowering of extracellular arginine may inhibit
viral replication is by inhibiting nitric oxide synthesis. Nitric
oxide is synthesized from extracellular arginine, thus elimination
of this arginine pool effectively inhibits the production of this
important metabolite. Although nitric oxide is thought to be
protective against some virus infections (Akaike T, Maeda H. 2000.
Nitric oxide and virus infection. Immunology 101:300-308),
inhibition of nitric oxide synthesis has been shown to block the
replication of lymphocytic choriomeningitis virus (Campbell I L
Samimi A, Chiang C S. 1994. Expression of the inducible nitric
oxide synthase. Correlation with neuropathology and clinical
features in mice with lymphocytic choriomeningitis. J Immunol
153:3622-3629) and HIV (Blond D, Raoul H, LeGrand R, Dormont D.
2000. Nitric oxide synthesis enhances human immunodeficiency virus
replication in primary human macrophages. J Virol 74:8904-8912).
Inhibition of nitric oxide synthesis has also been shown to protect
animals from the lethal effects of influenza (Akaike T, Noguchi Y,
Ijiri S, Setoguchi K, Suga M, Zheng Y M, Dietzschold B, Maeda H.
1996. Pathogenesis of influenza virus-induced pneumonia:
involvement of both nitric oxide and oxygen radicals. Proc Natl
Acad Sci USA 93:2448-2453; Karupiah G, Chen J-H, Mahalingam S,
Nathan C F, MacMicking J D. 1998. Rapid interferon
.gamma.-dependent clearance of influenza A virus and protection
from consolidating pneumonitis in nitric oxide synthase 2-deficient
mice. J Exp Med 188:1541-1546), polio virus (Lopez-Guerrero J A,
Carrasco L. 1998. Effect of nitric oxide on poliovirus infection of
two human cell lines. J Virol 72:2538-2540), rabies virus (Ubol S,
Sukwattanapan C, Maneerat Y. 2001. Inducible nitric oxide synthase
delays death of rabies virus-infected mice. J Med Microbiol
50:238-42) and flavivirus (Kreil T R, Eibl M M. 1996. Nitric oxide
and viral infection: no antiviral activity against a flavivirus in
vitro, and evidence for contribution to pathogenesis in
experimental infection in vivo. Virology 219:304-306). However,
these previously used nitric oxide synthesis inhibitors have been
limited by their toxicities (liver failure, seizure and death) in
both animals and humans. Thus it is not clear that inhibition of
viral replication resulting from elimination of arginine from the
culture media (a process which clearly eliminates nitric oxide
production) is the only mechanism by which inhibition of viral
replication occurs. This stimulation/inhibition duality of nitric
oxide and virus infection is also observed with nitric oxide in
other pathological events (Colasanti M, Suzuki H. 2000. The dual
personality of NO. Trends Pharm Sci 21:249-252). Thus inhibition of
nitric oxide should not be expected to abrogate all sequella of an
infectious event (Bogdan C. 2001. Nitric oxide and the immune
system. Nature Immunology 2:907-916). However, unlike the nitric
oxide synthesis inhibitors used in the past, ADI-PEG 20 appears to
be safe and effective in inhibiting production of nitric oxide and
can be used to help elucidate the role of this biomediator in
protection against viral infection.
[0029] Definitions
[0030] Throughout the present disclosure, the following
abbreviations may be used: PEG, polyethylene glycol; ADI, arginine
deiminase; SS, succinimidyl succinate; SSA, succinimidyl
succinamide; SPA, succinimidyl propionate; and NHS,
N-hydroxy-succinimide.
[0031] ADI covalently modified with polyethylene glycol (with or
without a linking group) may be hereinafter referred to as
"ADI-PEG", or "PEG-ADI".
[0032] "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(OCH2CH2).sub.nOH, 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, PEG-5,000 (PEG5) refers to polyethylene glycol molecules
having an average molecular weight of about 5,000; PEG-12,000
(PEG12) refers to polyethylene glycol molecules having an average
molecular weight of about 12,000; and PEG-20,000 (PEG20) refers to
polyethylene glycol molecules having an average molecular weight of
about 20,000.
[0033] As used herein, the term "individual" refers to an animal,
in some embodiments a mammal, and in some embodiments a human. The
term "individual" includes biological samples taken from such
animals.
[0034] As used herein, the term "viral disease" refers to diseases
and disorders caused by a virus. Viral diseases include without
limitation viruses that infect animals or mammals, including
humans. Human viruses include viruses from the following viral
families: Pox, Herpes, Adeno, Papova, Parvo, Hepadna, Picoma,
Calici, Astro, Toga, Flavi, Corona, Paramyxo, Orthomyxo, Bunya,
Arena, Rhabdo, Filo, Borna, Reo, and Retro.
[0035] Examples of viruses and associated diseases that may be
treated by the present invention include without limitation:
variola (smallpox); herpesviruses, such as herpes simplex virus
(cold sores), varicella-zoster (chicken pox, shingles),
Epstein-Barr virus (mononucleosis, Burkitt's lymphoma), KSHV
(Kaposi's sarcoma), and cytomegalovirus (blindness); adenoviruses;
hepatitis (A/B/C); polioviruses, rhinociruses, rubella, yellow
fever, West Nile virus, dengue, equine encephalitis, respiratory
syncytial virus (RSV), parainfluenza virus, and tobacco mosaic
virus.
[0036] In some embodiments the virus is one or more of HIV,
influenza, polio viruses, herpes simplex, hepatitis B, hepatitis C
and other viral strains of hepatitis, Kaposi's sarcoma,
rhinoviruses, West Nile virus, smallpox, and vaccinia, among
others.
[0037] As used herein, "modulation" means either an increase
(stimulation) or a decrease (inhibition) in the expression of a
gene. In some embodiments of the present invention, inhibition is
the form of modulation of gene expression.
[0038] As used herein, the term "inhibit" refers to a reduction or
decrease in a quality or quantity, compared to a baseline. For
example, in the context of the present invention, inhibition of
viral replication refers to a decrease in viral replication as
compared to baseline. In some embodiments there is a reduction of
about 30%, about 40%, about 50%, about 60%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 95%, about 99%, and about
100%. Those of ordinary skill in the art can readily determine
whether or not viral replication has been inhibited and to what
extent.
[0039] As used herein, the term "about" refers to .+-.20%, .+-.15%,
.+-.10%, or .+-.5% of the value.
[0040] As used herein, the term "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.
[0041] "Circulating half life" refers to the period of time, after
injection of the modified ADI into a patient, until a quantity of
the ADI has been cleared to levels one half of the original peak
serum level. Circulating half-life may be determined in any
relevant species, including humans or mice.
[0042] As used herein, the terms "covalently bonded", "bonded" and
"coupled" are used interchangeably and refer to a covalent bond
linking ADI to the PEG molecule, either directly or through a
linker.
[0043] As used herein, the term "therapeutically effective amount"
is meant an amount of a compound of the present invention effective
to yield the desired therapeutic response. The specific
therapeutically effective amount will, obviously, vary with such
factors as the particular condition being treated, the physical
condition of the patient, the type of mammal or animal being
treated, the duration of the treatment, the nature of concurrent
therapy (if any), and the specific formulations employed and the
structure of the compounds or its derivatives. In the context of
improving liver function, the term "therapeutically effective
amount" refers to an amount of arginine deiminase bonded to
polyethylene glycol that improves liver function. In some
embodiments the therapeutically effective amount is effective to
improve the Child-Pugh scale or the Mayo End-stage Liver Disease
(MELD) score of the individual. In some embodiments the
therapeutically effective amount is effective to improve liver
function based on comparison of markers of liver function
including, without limitation, billrubin levels, creatine levels,
and international normalized ratio.
[0044] As used herein, the term "an amount effective to inhibit
viral replication" refers to the amount of a compound comprising
ADI covalently bonded via a linking group to polyethylene glycol
administered to an individual that results in a reduced level of
viral replication and thus a reduced amount of detectable virus in
the individual, i.e., a reduction in viral titer or viral load. To
determine an amount effective to inhibit viral replication, the
individual's viral load can be determined prior to treatment with a
compound of the present invention and then subsequent to treatment.
The level of viral replication can be quantified by any number of
routine methodologies including, for example: quantifying the
actual number of viral particles in a sample prior to and
subsequent to compound administration, and quantifying the level of
one or more viral antigen present in a sample prior to and
subsequent to compound administration. In some embodiments "an
amount effective to inhibit viral replication" is the amount
necessary to decrease plasma arginine concentrations below about 5
.mu.M. Methods of measuring plasma arginine concentrations are well
known in the art.
[0045] Assays for viral replication also provide one with the
ability to determine the efficacy of viral inhibitors and are well
known to those skilled in the art. Such assays may be conducted in
vivo or in vitro. HCV is known to occur in chimpanzees where the
infection closely resembles that seen in humans. There have also
been reports of experimental infection in tupaias, closely related
to the primates, and in immunodeficient mice. (Xie, Z. C. et al.,
Virology, 244, 513 (1998); Schinazi, R. F. et al., Antiviral Chem.
Chemother. 10, 99, (1999)).
[0046] The inhibition of viral replication contributes to a
reduction in the severity of the viral infection or of the symptoms
of the viral infection.
[0047] As used herein, the term "prophylactically effective amount"
is meant an amount of a compound of the present invention effective
to yield the desired prophylactic response. The specific
prophylactically effective amount will, obviously, vary with such
factors as the particular virus, the physical condition of the
patient, the type of mammal or animal being treated, the duration
of the treatment, the nature of concurrent therapy (if any), and
the specific formulations employed and the structure of the
compounds or its derivatives
[0048] As used herein "combination therapy" means that the
individual in need of treatment is given another drug for the
disease in conjunction with PEG-ADI. This combination therapy can
be sequential therapy where the individual is treated first with
one or more drugs and then the other, or two or more drugs are
given simultaneously.
[0049] As used herein, the phrase "arginine deprivation therapy"
refers to a treatment regimen that involves the use of an agent
that reduces, minimizes, or abolishes arginine levels in the
patient. Arginine deprivation therapy is often performed using ADI.
Arginine deprivation therapy and agents used in arginine
deprivation therapy are described in detail in allowed U.S.
application Ser. No. 09/023,809, filed Feb. 13, 1998, now U.S. Pat.
No. 6,183,738, issued Feb. 6, 2001; and pending application U.S.
Ser. No. 09/504,280, filed Feb. 15, 2000, each of which is hereby
incorporated by reference in its entirety.
[0050] As used herein, the term "an individual suspected of having
been exposed to one or more viruses" refers to an individual who
has not been diagnosed as being positive for one or more viruses
but who could possibly have been exposed to one or more viruses due
to a recent high risk activity or activity that likely put them in
contact with the viruses. For example, an individual suspected of
having been exposed to HIV refers to an individual that has been
stuck with a needle that has been in contact with either a sample
that contains HIV or HIV infected individual. Examples of such
samples include, without limitation, laboratory or research samples
or samples of blood, semen, bodily secretions, and the like from
patients. Individuals suspected of being exposed to HCV include
individuals that have received blood transfusions with blood of
unknown quality. The blood that is being transfused may have not
been tested or the test results indicating that the blood does not
contain HCV are not reliable or are doubted. In some embodiments,
an individual suspected of being infected with a virus includes
individuals who have been exposed to the virus through another
individual including, for example, through sexual intercourse,
contact with bodily fluids of another individual, sharing of
hypodermic needles, and the like. The individual from which the
virus originated may or may not have been tested for the presence
and/or absence of the virus. The term "an individual suspected of
having been exposed to one or more viruses" also includes
individuals who have been diagnosed as being positive for one virus
but are also infected with at least one further virus. For example,
often those infected with HIV are also positive for one or more
forms of hepatitis. Such an individual may be classified as being
at "high-risk" for one or more viruses.
[0051] As used herein, the term "selectively inhibit" refers
selective inhibition of viral replication and is, in some
embodiments, the ratio of CC.sub.50/EC.sub.50% of viral mRNA
levels. An SI>10 is considered to reflect a selective inhibition
of viral replication.
[0052] As used herein, the term "sample" refers to biological
material from a patient. The sample assayed by the present
invention is not limited to any particular type. Samples include,
as non-limiting examples, single cells, multiple cells, tissues,
tumors, biological fluids, biological molecules, or supernatants or
extracts of any of the foregoing. Examples include tissue removed
for biopsy, tissue removed during resection, blood, urine, lymph
tissue, lymph fluid, cerebrospinal fluid, mucous, and stool
samples. The sample used will vary based on the assay format, the
detection method and the nature of the tumors, tissues, cells or
extracts to be assayed. Methods for preparing samples are well
known in the art and can be readily adapted in order to obtain a
sample that is compatible with the method utilized.
[0053] ADI
[0054] Arginine deiminase catalyzes the conversion of arginine to
citrulline, and may be used to eliminate arginine. In the present
invention, the arginine deiminase gene may be derived, cloned or
produced from any source, including, for example, microorganisms,
recombinant biotechnology or any combination thereof. Arginine
deiminase may be cloned from microorganisms of the genus
Mycoplasma. In some embodiments, the arginine deiminase is cloned
from Mycoplasma arginini, Mycoplasma hominus, Mycoplasma
arthritides, or any combination thereof. In some embodiments, the
arginine deiminase used in the present invention may have one or
more of the amino acid sequences of SEQ ID NOS: 1-10 and 13-21.
[0055] Native arginine deiminase may be found in microorganisms and
is antigenic and rapidly cleared from circulation in a patient.
These problems may be overcome by 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.
[0056] 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 (SEQ ID NO:5) is described, for
example, by Takaku H, Takase M, Abe S, Hayashi H and Miyazaki K.
(1992). In vivo anti-tumor activity of arginine deiminase purified
from Mycoplasma arginini. Int. J. Cancer 51:244-249, 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, H, Misawa, S, Hayashi H and
Miyazaki K. (1993). Chemical modification by polyethylene glycol of
the anti-tumor enzyme arginine deiminase from Mycoplasma arginini.
Jpn. J. Cancer Res. 84:1195-1200. The modified protein was toxic
when metabolized due to the release of cyanide from the cyanuric
chloride linking group.
[0057] The production of arginine deiminase via recombinant DNA
techniques also provides for certain disadvantages. For example,
arginine deiminase produced in Escherichia 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 guanidinium 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.
[0058] 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 from Mycoplasma hominus (SEQ ID
NO:1), Mycoplasma arginini (SEQ ID NO:5), and Mycoplasma
arthritides (SEQ ID NO:7) 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.
[0059] 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 Streptococcus 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:
TABLE-US-00001 TABLE 1 Pegylation sites of arginine deiminase from
various organisms Position of lysine Organisms producing arginine
deiminase in arginine deiminase Mycoplasma hominus (SEQ ID NO: 1)
112 Mycoplasma arginini (SEQ ID NO: 5) 111 Clostridium
perfringens(SEQ ID NO: 18) 105 Bacillus licheniformis(SEQ ID NO:
19) 97, 108 Borrelia burgdorferi(SEQ ID NO: 15) 102, 111 Borrelia
afzellii (SEQ ID NO: 16) 101 Enterococcus faecalis(SEQ ID NO: 20)
102, 110 Streptococcus pyogenes(SEQ ID NO: 13) 104 Streptococcus
pneumoniae(SEQ ID NO: 14) 103 Lactobacillus sake (SEQ ID NO: 21)
97, 106 Qiardia intestinalis(SEQ ID NO: 17) 114, 116
[0060] 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.
[0061] In some embodiments the present invention 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, in some embodiments lysine is substituted with glutamic
acid, valine, aspartic acid, alanine, isoleucine, leucine or
combinations thereof. In some embodiments lysine is substituted
with glutamic acid. In some embodiments of the invention, modified
arginine deiminase from Mycoplasma hominus has an amino acid
substitution at Lys.sup.112, Lys.sup.374, Lys.sup.405, Lys.sup.408
or combinations or subcombinations thereof. In some embodiments
modified arginine deiminase from Mycoplasma hominus has an amino
acid substitution Lys.sup.112 to Glu.sup.112, Lys.sup.374 to
Glu.sup.374, Lys.sup.405 to Glu.sup.405, Lys.sup.408 to Glu.sup.408
or combinations thereof. In some embodiments modified arginine
deiminase from Mycoplasma hominus has lysine at position 112
substituted with glutamic acid (SEQ ID NO:2).
[0062] The present invention thus 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 some embodiments of the invention, the modified
arginine deiminase has 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 may 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 some embodiments, the proline at
position 210 is substituted with serine (SEQ ID NO:3). It is to be
understood that in accordance with this aspect of the invention,
other substitutions at position 210 may be made. Examples of
substitutions include Pro.sup.210 to Thr210, Pro.sup.210 to
Arg.sup.210, Pro.sup.210 to Asn.sup.210, Pro.sup.210 to 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.
[0063] In some embodiments of the invention, the modified arginine
deiminase has 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 some embodiments, 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 (SEQ ID NO:4). As
discussed above, however, it is to be understood that the modified
arginine deiminase may include other substitutions. In some
embodiments, conservative substitutions may be made at positions
112 and/or 210 of the wild-type arginine deiminase.
[0064] 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. In some embodiments the amino acid sequence of
modified arginine deiminase from Mycoplasma hominus is a sequence
of SEQ ID NO:4.
[0065] In some embodiments arginine deiminase is derived from
Mycoplasma hominus, Mycoplasma pneumoniae, Mycoplasma arginini,
Qiardia intestinalis, Clostridium perfringens, Bacillus
licheniformis, Borrelia burgdorferi, Borrelia afzellii,
Enterococcus faecalis, Streptococcus pyogenes, Streptococcus
pneumoniae, Lactobacillus sake or Qiardia intestinalis arginine
deiminase.
[0066] In some embodiments arginine deiminase is derived from
Mycoplasma hominus arginine deiminase (SEQ ID NO:1). In some
embodiments, the arginine deiminase comprises at the substitution
or deletion of at least one proline residue as compared to SEQ ID
NO:1. In some embodiments, the substitution or deletion of at least
one proline residue comprises substitution or deletion of the
proline residue at or corresponding to residue 210 of SEQ ID NO:1.
In some embodiments, the substitution or deletion of at least one
proline residue comprises substitution of the proline residue at or
corresponding to residue 210 of SEQ ID NO:1 with Ser, Thr, Arg,
Asn, Gln, or Met. In some embodiments, the substitution or deletion
of at least one proline residue comprises substitution of the
proline residue at or corresponding to residue 210 of SEQ ID NO:1
with Ser.
[0067] In some embodiments of the present invention the arginine
deiminase is modified and comprises at least one amino acid
substitution or deletion wherein the modified arginine deiminase
has a reduced number of pegylation sites at or adjacent to a
catalytic region, as compared to SEQ ID NO:1. In some embodiments,
the substitution or deletion of at least one lysine residue
comprises the substitution or deletion of at least one lysine
residue at or corresponding to residues 112, 374, 405 or 408 of SEQ
ID NO:1. In some embodiments, the substitution or deletion of at
least one lysine residue comprises the substitution of at least one
lysine residue at or corresponding to residues 112, 374, 405 or 408
of SEQ ID NO:1 with Glu, Val, Asp, Ala, Ile or Leu. In some
embodiments, the substitution or deletion of at least one lysine
residue comprises substitution of the lysine residue at or
corresponding to residue 112 of SEQ ID NO:1 with Glu, Val, Asp,
Ala, Ile or Leu. In some embodiments, the substitution or deletion
of at least one lysine residue comprises substitution of the lysine
residue at or corresponding to residue 112 of SEQ ID NO:1 with Glu.
In some embodiments, the modified arginine deiminase comprises the
further substitution or deletion of at least one proline
residue.
[0068] In some embodiments, the substitution or deletion of at
least one proline residue comprises substitution of the proline
residue at or corresponding to residue 210 of SEQ ID NO:1 with Ser,
Thr, Arg, Asn, Gln, or Met.
[0069] In some embodiments the arginine deiminase comprises
arginine deiminase modified to be free of at least one pegylation
site at or adjacent to a catalytic region as compared to SEQ ID
NO:1, wherein said modified arginine deiminase comprises at least
one amino acid substitution or deletion at or corresponding to
residues 112, 374, 405, or 408 of SEQ ID NO:1. In some embodiments
the at least one amino acid substitution or deletion comprises
substitution of the lysine residue at or corresponding to residue 1
12 of SEQ ID NO:1 with Glu, Val, Asp, Ala, Ile or Leu. In some
embodiments the at least one amino acid substitution or deletion
further comprises substitution or deletion of at least one proline
residue. In some embodiments the substitution or deletion of at
least one proline residue comprises substitution or deletion of the
proline residue at or corresponding to residue 210 of SEQ ID NO:1.
In some embodiments the substitution or deletion of at least one
proline residue comprises substitution of the proline residue at or
corresponding to residue 210 of SEQ ID NO:l with Ser, Thr, Arg,
Asn, Gln, or Met.
[0070] In some embodiments the arginine deiminase from Mycoplasma
hominus comprises a substitution of lysine at residue 112 of SEQ ID
NO:1 with glutamic acid (SEQ ID NO:2). In some embodiments the
arginine deiminase from Mycoplasma hominus comprises a substitution
of proline at residue 210 of SEQ ID NO:1 with serine (SEQ ID NO:3).
In some embodiments the arginine deiminase from Mycoplasma hominus
comprises a substitution of lysine at residue 112 of SEQ ID NO:1
with glutamic acid and a substitution of proline at residue 210 of
SEQ ID NO:1 with serine (SEQ ID NO:4). In some embodiments arginine
deiminase from Mycoplasma arginini comprises a substitution of
lysine at residue 111 of SEQ ID NO:5 with glutamic acid (SEQ ID
NO:6). In some embodiments the arginine deiminase from Mycoplasma
arthritides comprises substitutions of lysine at residues 111 and
112 of SEQ ID NO:7 with glutamic acid (SEQ ID NO:8). In some
embodiments the arginine deiminase from Mycoplasma arthritides
comprises a substitution of lysine at residue 111 of SEQ ID NO:7
with glutamic acid (SEQ ID NO:9). In some embodiments the arginine
deiminase from Mycoplasma arthritides comprises a substitution of
lysine at residue 112 of SEQ ID NO:7 with glutamic acid (SEQ ID
NO:10).
[0071] Such modifications and/or substitutions as well as
nucleotide and polypeptide sequences are described in U.S. Pat. No.
6,183,738, issued Feb. 6, 2001, and co-pending application Ser. No.
09/564,559, filed May 4, 2000, each of which is hereby incorporated
by reference in its entirety.
[0072] Polyethylene Glycol
[0073] There are many polyethylene glycols available that differ in
their molecular weight and linking group. These PEGs can have
varying effects on the antigencity, immunogenicity and circulating
half-life of a protein (Zalipsky, S. and Lee, C. Polyethylene
Glycol Chemistry: Biotechnical and Biomedical Applications. Pp.
347-370, Plenum Press, New York, 1992; Monfardini, C., et. al.
bioconjugate Chem. 6, 62-69, 1995; Delgado C; Francis G E; Fisher
D. The uses and properties of PEG-linked proteins. Crit. Rev. Ther.
Drug Carrier Sys., 9:249-304, 1992.)
[0074] In some embodiments of the present invention, each
polyethylene glycol molecule has an average molecular weight of
about 10,000 to about 50,000; from about 12,000 to about 40,000,
from about 15,000 to about 30,000; and 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.
[0075] The polyethylene glycol may be a branched or straight chain.
In some embodiments the polyethylene glycol is a straight chain.
Increasing the molecular weight of the polyethylene glycol
generally tends to decrease the immunogenicity of the ADI. The
polyethylene glycols having the molecular weights described in the
present invention may be used in conjunction with ADI, and,
optionally, a biocompatible linking group, to treat viral
diseases.
[0076] Pegylation
[0077] ADI may be covalently bonded to PEG via a biocompatible
linking group, using methods known in the art, as described, for
example, by Park et al, Anticancer 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.
[0078] The linking group used to covalently attach PEG to ADI may
be any compatible linking group. In some embodiments the linking
group is a 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. 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 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 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, 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. In some
embodiments the linking group is an ester group and/or a
succinimide group. In some embodiments, the linking group is SS,
SPA, SCM, SSA or NHS.
[0079] 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, if a linking group is used, in
some embodiments it is important to use a biocompatible linking
group. The 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.
[0080] Alternatively, ADI may be coupled directly to PEG (i.e.,
without a linking group) through an amino group, a sulfhydral
group, a hydroxyl group or a carboxyl group. In some embodiments,
PEG is coupled to lysine residues on ADI.
[0081] ADI-PEG
[0082] The attachment of PEG to ADI increases the circulating
half-life of ADI. The number of PEG molecules on ADI appears to be
related to the circulating half-life of the enzyme, while the
amount of retained enzymatic activity appears related to the
average molecular weight of the PEG used. Increasing the number of
PEG units on ADI decreases the enzymatic activity of the enzyme.
Also, it is known that some PEG formulations are difficult to
produce and yield relatively low amounts of product. Thus, to
achieve an efficacious product, a balance needs to be achieved
among circulating half-life, antigenicity, efficiency of
production, and enzymatic activity.
[0083] Generally, PEG is attached to a primary amine of ADI.
Selection of the attachment site of polyethylene glycol on the
arginine 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 arginini, Mycoplasma
arthritides and 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.
[0084] From 1 to about 30 PEG molecules may be covalently bonded to
ADI. In some embodiments ADI is modified with about 7 to about 15
PEG molecules, 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, about 40% to about 60%,
about 45% to about 55%, and about 50% of the primary amino groups
in arginine deiminase are modified with PEG. In some embodiments
when PEG is covalently bonded to the end terminus of ADI, only 1
PEG molecule is utilized. 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, in some embodiments a balance needs
to be achieved between the two, as would be apparent to one skilled
in the art in view of the present disclosure.
[0085] In the present invention, in some embodiments the linking
groups attach to a primary amine of arginine deiminase via a
maleimide group. Once coupled with arginine deiminase, SS-PEG has
an 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.
[0086] The structural formulas of certain linking groups useful in
the present invention are set forth below. ##STR1##
[0087] Methods of Treatment
[0088] In some embodiments, the present invention provides methods
of inhibiting viral replication in an individual comprising
administering to said individual a therapeutically or
prophylactically effective amount of a compound comprising ADI
covalently bonded via a linking group to polyethylene glycol,
wherein each polyethylene glycol molecule has an average molecular
weight of from about 10,000 to about 30,000. In some embodiments
ADI is modified with polyethylene glycol molecules, each molecule
having an average molecular weight of about 20,000. In some
embodiments the linking group is selected from the group consisting
of a succinimide group, an amide group, an imide group, a carbamate
group, an ester group, an epoxy group, a carboxyl group, a hydroxyl
group, a carbohydrate, a tyrosine group, a cysteine group, a
histidine group and combinations thereof. In some embodiments the
linking group is succinimidyl succinate. In some embodiments from
about 7 to about 15 polyethylene glycol molecules are bonded to
arginine deiminase. In some embodiments from about 9 to about 12
polyethylene glycol molecules are bonded to arginine deiminase. In
some embodiments the arginine deiminase is derived from a
microorganism of the genus Mycoplasma. In some embodiments the
arginine deiminase is derived from Mycoplasma arginini, Mycoplasma
hominus, Mycoplasma arthritides and combinations thereof. In some
embodiments the virus is HCV. In some embodiments the methods
further comprise the step of administering a therapeutically
effective amount of an additional anti-viral agent prior to,
simultaneously with, or following administration of the arginine
deiminase.
[0089] A therapeutically effective amount of one of the compounds
of the present invention is an amount that is effective to inhibit
viral replication. 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. The compounds can be administered in one dose, continuously
or intermittently throughout the course of treatment. ADI-PEG maybe
administered several times each day, once a day, once a week, or
once every two weeks.
[0090] In some embodiments, ADI-PEG is administered in a weekly
dose of at least about 40 IU/m.sup.2, at least about 80 IU/m.sup.2,
at least about 160 IU/m.sup.2, or at least about 200 IU/m.sup.2. In
some embodiments the dose administered lowers plasma levels of
arginine to less than about 10, .mu.M, 5 .mu.M, 1 .mu.M, or 100 nM.
In some embodiments, ADI-PEG20 is administered in a weekly dose of
about 160 IU/m.sup.2 resulting in a plasma level in the patient of
less than about 5 .mu.M.
[0091] The present invention provides methods of inhibiting
replication of one or more viruses in an individual comprising
administering a therapeutically or prophylactically effective
amount of an arginine deiminase bonded to polyethylene glycol to
said individual. In some embodiments the virus is a human virus. In
some embodiments the virus is HCV. In some embodiments, the
individual is infected with two or more different viruses. In some
embodiments the two or more viruses are HIV and HCV. In some
embodiments the presence and and/or identity of an infecting virus
is unknown at or before the time of administration. In some
embodiments the methods further comprise the step of administering
a therapeutically effective amount of an additional anti-viral
agent prior to, simultaneously with, or following administration of
the arginine deiminase.
[0092] The present invention also provides methods for treating an
individual suspected of having been exposed to one or more viruses
comprising administering a therapeutically or prophylactically
effective amount of an arginine deiminase bonded to polyethylene
glycol to said individual. As discussed above, some individuals who
have not been diagnosed as being infected with one or more viruses
are put in circumstances where it is possible that they could have
possibly been exposed to the virus. The treatment of individuals
suspected of being exposed to one or more viruses may also include
the administration of additional therapeutics as described above.
The course of prophylactic treatment may be performed in
conjunction with periodic monitoring for indications of viral
infection. In some embodiments, following commencement of treatment
according to the present invention the individual is diagnosed as
being positive for one or more viruses.
[0093] In some embodiments the present invention provides methods
of inhibiting viral replication in an individual at risk for one or
more viruses. The methods comprise administering to the individual
an amount of a composition comprising an arginine deiminase bonded
to polyethylene glycol effective to inhibit viral replication.
[0094] In some embodiments the present invention provides methods
of inhibiting viral replication in an individual who has been
identified as having been infected with a viral infection. The
methods comprise administering to the individual an amount of a
composition comprising an arginine deiminase bonded to polyethylene
glycol effective to inhibit viral replication.
[0095] In some embodiments the composition comprising an arginine
deiminase bonded to polyethylene glycol is effective at a
concentration of less than 0.1 mM to inhibit viral replication by
at least 50% in greater than 50% of cells in an in vitro assay to
measure viral replication. In some embodiments the composition
comprising an arginine deiminase bonded to polyethylene glycol is
effective at a concentration of less than 0.05 mM to inhibit viral
replication by at least 50% in greater than 50% of cells in an in
vitro assay to measure viral replication. In some embodiments the
composition comprising an arginine deiminase bonded to polyethylene
glycol is effective at a concentration of less than 0.01 mM to
inhibit viral replication by at least 50% in greater than 50% of
cells in an in vitro assay to measure viral replication.
[0096] In some embodiments the present invention provides methods
of concurrently treating a tumor and inhibiting replication of one
or more viruses in an individual. The method comprises
administering a therapeutically or prophylactically effective
amount of an arginine deiminase covalently bonded via a linking
group to polyethylene glycol to the individual. In some embodiments
the tumor is selected from the group consisting of melanoma,
sarcoma, and hepatoma. In some embodiments the tumor is hepatoma
and the virus is HCV. In some embodiments, the presence and/or
identity of the tumor is unknown at the time of treatment. In some
embodiments the presence and/or identity of the virus is unknown at
the time of treatment. In some embodiments the methods further
comprise administering a therapeutically effective amount of an
additional anti-viral agent prior to, simultaneously with, or
following administration of the arginine deiminase.
[0097] In some embodiments the present invention provides methods
for modulating nitric oxide levels in an individual comprising
administering a therapeutically or prophylactically effective
amount of an arginine deiminase bonded to polyethylene glycol to
said individual. In some embodiments, modulation is inhibition of
nitric oxide levels. In some embodiments the methods further
comprise administering a therapeutically or prophylactically
effective amount of an additional anti-viral agent prior to,
simultaneously with, or following administration of the arginine
deiminase. In some embodiments the individual has been identified
as having been infected with one or more viruses.
[0098] In some embodiments the present invention provides methods
to determine the sensitivity of viral replication to modulating
levels of arginine contacting a sample with a composition
comprising arginine deiminase bonded to polyethylene glycol and
measuring levels of viral RNA or products of viral RNA. Methods of
measuring levels of viral RNA or products thereof are well known to
those of ordinary skill in the art.
[0099] In some embodiments the present invention provides methods
of selectively inhibiting viral replication in an individual
infected with one or more viruses. The methods comprise
administering a therapeutically or prophylactically effective
amount of a composition comprising an arginine deiminase bonded to
polyethylene glycol to the individual. In some embodiments the
virus is HCV. In some embodiments the SI is above 10, above 15,
above 20, or above 25.
[0100] In some embodiments the present invention provides methods
for improving liver function in an individual comprising
administering a therapeutically or prophylactically effective
amount of a composition comprising arginine deiminase bonded to
polyethylene glycol to said individual.
[0101] Those of skill in the art are readily able to determine the
quality of liver function. In some embodiments, the relative
quantity of one or more markers is compared between a healthy
patient and a patient with a liver disease or disorder.
[0102] In some embodiments, liver function is assessed using the
Child-Pugh scale or the Mayo End-stage Liver Disease (MELD) score.
The Child-Pugh scale of grading liver function uses several factors
to predict mortality in liver disease. Factors considered in the
Child Pugh scale include billrubin levels, creatine levels,
international normalized ratio (INR; also known as prothrombin time
(measure of blood's ability to clot)), presence of ascites in the
abdomen, and grade of encephalopathy. Grades are assigned to levels
of increasing abnormality of liver function; the grade "A" reflects
a Child-Pugh score of 5-6 points and indicates the lowest level of
liver abnormality. The grade "B" reflects a Child-Pugh score of 7-9
points and indicates an intermediate level of liver abnormality.
The grade "C" reflects a Child-Pugh score of 10-15 points and
indicates the highest level of liver abnormality. The MELD scale of
grading liver function considers billrubin levels, creatine levels,
and international normalized ratio.
[0103] In some embodiments the liver function of the individual
prior to administration of the arginine deiminase bonded to
polyethylene glycol is Child-Pugh level A, level B, or level C.
[0104] In some embodiments the present invention provides methods
for identifying an individual identified as having one or more
viral infections as susceptible to arginine deprivation therapy.
The methods comprise obtaining a viral sample from the individual
and comparing viral replication in the sample in the presence and
absence of a composition comprising arginine deiminase bonded to
polyethylene glycol under conditions suitable for viral
replication. In some embodiments an inhibition of viral replication
of at least 40%, at least 50%, or at least 80% in the sample
contacted with ADI-PEG is indicative of an individual who is a
candidate for arginine deprivation therapy and an inhibition of
viral replication by ADI-PEG of less than 40%, less than 30%, or
less than 20% is indicative of an individual who is not a candidate
for arginine deprivation therapy.
[0105] In some embodiments, the present invention provides methods
for treating one or more viral infections in an individual. The
methods comprise determining if the individual is a candidate for
arginine deprivation therapy as described above and treating the
individual with arginine deprivation therapy if the individual is a
candidate for arginine deprivation therapy and treating the
individual with conventional antiviral treatment if the individual
is not a candidate for arginine deprivation therapy.
[0106] Methods of determining the most effective means and dosage
of administration are well known to those of skill in the art. In
some embodiments twice weekly dosing over a period of at least
several weeks is used. Often the anti-viral compounds will be
administered for extended periods of time and may be administered
for the lifetime of the individual. Methods of determining the most
effective means and dosage of administration are well known to
those of skill in the art. Single or multiple administrations can
be carried out with one dose level and pattern being selected by
the administrator.
[0107] The dosage administered will, of course, vary depending upon
known factors, such as the pharmacodynamic characteristics of the
particular agent and its mode and route of administration; the age,
health and/or weight of the individual; the nature and extent of
the symptoms; the kind of concurrent treatment; the frequency of
treatment; the symptoms exhibited by the individual, and the effect
desired.
[0108] Symptoms or criteria for response to anti-viral treatment
center around the level of viral replication in the case of most
viral infections. Tests for viral circulating viral RNA levels and
changes therein are standard and can be applied in cells and
animals, including humans. In human patients, tests for liver
activities may be performed. One exemplary test is the ALT (Serum
Glutamic Pyruvic Transaminase) test. ALT is an enzyme found
primarily in the liver but also to a lesser degree, the heart and
other tissues, and is useful in diagnosing liver function. The
normal adult range for humans is from 0 to about 48 U/L with an
optimal adult reading of about 24 U/L. Improvement in one or more
of these criteria signals an effective dosage or treatment.
[0109] The compounds may be administered in admixture with suitable
pharmaceutical diluents, extenders, excipients, or carriers
(collectively referred to herein as a pharmaceutically acceptable
carrier) selected with respect to the intended form of
administration and as consistent with conventional pharmaceutical
practices. For example, in some embodiments ADI-PEG 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
ADI-PEG formulation may be administered as a solid (lyophilate) or
as a liquid formulation, as desired.
[0110] The compositions of the present invention are formulated
according to the mode of administration to be used. In cases where
pharmaceutical compositions are injectable pharmaceutical
compositions, they are sterile, pyrogen free and particulate free.
In some embodiments the compositions are isotonic formulations. In
some embodiments additives for isotonicity can include one or more
of sodium chloride, dextrose, mannitol, sorbitol and lactose. In
some embodiments, the compositions are provided as isotonic
solutions such as phosphate buffered saline. Stabilizers for the
compositions include gelatin and albumin in some embodiments.
[0111] The present invention also provides methods of treating a
broad spectrum of genetically diverse viruses in a patient
comprising administering to the individual a therapeutically
effective amount of a compound comprising ADI covalently bonded via
a linking group to polyethylene glycol.
[0112] Combination Therapy
[0113] The compounds of the present invention may additionally be
combined with other antiviral compounds to provide a combination
therapy. Any known anti-viral may be combined with the compositions
of the present invention, as long as the combination does not
eliminate the antiviral activity of the compound of ADI-PEG. In the
case of HIV a combination therapy of ADI-PEG with AZT, TC-3 or
protease inhibitors may be more effective than either agent
individually. In the case of hepatitis, a combination of ADI-PEG
with one or more of cyclovir, famciclovir or valacyclovir,
ribavirin, interferon or beta globulin is administered as a
combination therapy. For herpes, a recombinant alpha interferon can
be used as a combination therapy with ADI-PEG.
[0114] Other anti-viral agents suitable for use in combination
therapy are known to the art-skilled and include, without
limitation, one or more of AZT (zidovudine, Retrovir), ddI
(didanosine, Videx), 3TC (lamivudine, Epivir), d4T (stavudine,
Zerit), abacavir (Ziagen), ddC (zalcitabine, Hivid), nevirapine
(Viramune), Delavirdine (Rescriptor), indinavir (Crixivan),
ritonavir (Norvir), nelfinavir (Viracept), saquinavir,
lopinavir/ritonavir (Kaletra), Amprenavir (Agenerase) Atazanavir,
tipranavir, fusion inhibitor T-20, Interleukin-2, hydroxyurea,
AR177 (Zintevir), fomivirsen sodium (Vitravene), GEM 132, GEM 91,
GEM 92, AMD 3100, n-docosanol (1-docosanol), PRO2000, T-1249, T-20,
arbidol, SP-303 (Virend), hypericin (VIMRxyn), MDL 28574, SC-48334,
ADA, imiquimod (Aldera), ISIS 5320, resiquimod, adefovir dipivoxil
(Preveon), DAPD, emtricitabine (Coviracil), entecavir, lamivudine
(Zeffix, Epivir-HBV, Heptovir, Heptodin), amantadine (Symmetrel),
oseltamivir (Tamiflu), pirodavir, pleconaril (VP-63843), ribavirin
(Virazid/Virazide/Virazole), rimantadine (Flumadine), WIN 54954,
zanamivir (Relenza), foscarnet (Foscavir), maribavir, ABT-378,
atevirdine mesylate, calanolide A, capravirine, efavirenz
(Sustiva), emivirine (Coactinon), GW420 867X (aka HBY 1293), HBY
097, L-697,661, loviride, MIV-150, PETT-5, R165335-TMC125,
talviraline, tivirapine, trovirdine, acyclovir (Zovirax), brivudin
(Helpin, Zostrex), cidofovir (Vistide (i.v.); Forvade (topical)),
cyclic HPMPC, famciclovir (Famvir), fiacitabine, fialuridine,
ganciclovir (Cymvene/Cytovene), GW-273175X, idoxuridine (Herpid,
Kerecid/Herplex Liquifilm, Idoxene, Virudox, Iduridin, Stoxil),
lobucavir, netivudine (Zonavir), penciclovir (Vectavir/Denavir),
sorivudine (Usevir), trifluridine (Viroptic), valaciclovir
(Valtrex; Zelitrex), valomaciclovir stearate (MIV-606), vidarabine
(Vira-A), 935U83, abacavir (Ziagen/Trizivir), adefovir, adefovir
dipivoxil (Preveon), alovudine, AzdU, CS-92, DAPD, didanosine
(Videx), dOTC, emtricitabine (Coviracil), fozivudine tidoxil,
lamivudine (Epivir/Combivir/Trizivir), lobucavir, lodenosine,
stavudine (Zerit), tenofovir (Viread), tenofovir disoproxil
fumarate, zalcitabine (Hivid), zidovudine (Retrovir), A-77003,
AG7088, amprenavir (Agenerase), BMS-232632, delavirdine
(Rescriptor), DMP-323, DMP-450, GW 433 908, indinavir (Crixivan),
KNI-272, lasinavir, lopinavir (Kaletra), Mozenavir, nelfinavir
(Viracept), PD178390, ritonavir (Norvir), RPI 312, saquinavir
(Invirase/Fortovase), SC-52151, SDZ PRI 053, tipranavir, U-103017,
U-96988, Hydroxyurea (Hydrea), AGI549, foscarnet (Foscavir), LiGLA,
Aciclovir--Valaciclovir, Famciclovir, Idoxuridine, Ganciclovir,
Foscarnet, Cidofovir, and Adefovir, enfuvirtide, Valcyte,
clevudine, thymalfasin, IL-12, among others.
[0115] Combination therapy can be sequential, that is the treatment
with one agent first and then the second agent, or it can be
treatment with both agents at the same time. The sequential therapy
can be within a reasonable time after the completion of the first
therapy before beginning the second therapy. The treatment with
both agents at the same time can be in the same daily dose or in
separate doses. For example in some embodiments treatment with one
agent occurs on day 1 and with the other on day 2. The exact
regimen will depend on the disease being treated, the severity of
the infection and the response to the treatment.
[0116] 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 ADI-PEG composition of the present invention
can be carried out, for example, by inhalation or suppository or to
mucosal tissue such as by lavage to vaginal, rectal, urethral,
buccal and sublingual tissue, orally, topically, intranasally,
intraperitoneally, parenterally, intravenously, intralymphatically,
intratumorly, intramuscularly, interstitially, intra-arterially,
subcutaneously, intraoccularly, intrasynovial, transepithelial, and
transdermally. The compounds of the present invention can be
administered in oral dosage forms as tablets, capsules, pills,
powders, granules, elixirs, tinctures, suspensions, syrups, and
emulsions. The compounds may also be administered in intravenous
(bolus or infusion), intraperitoneal, subcutaneous, or
intramuscular form, all using dosage forms well known to those of
ordinary skill in the pharmaceutical arts.
EXAMPLES
[0117] 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
Production of Recombinant ADI
[0118] Cultures of Mycoplasma arginini (ATCC 23243), Mycoplasma
hominus (ATCC 23114) and Mycoplasma arthritides (ATCC 23192) were
obtained from the American Type Culture Collection, Rockville,
Md.
[0119] Arginine deiminase was cloned from Mycoplasma arginini,
Mycoplasma hominus and Mycoplasma arthritides 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. Characterization, by methods
known to those skilled in the art, of each of the proteins with
respect to specific enzyme activity, K.sub.m, V.sub.max 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 K.sub.m and V.sub.max were determined by incubating
the enzyme with various concentrations of arginine and quantifying
citrulline production. The K.sub.m for the various enzymes was
about 0.02 to 0.06 .mu.M and the V.sub.max was about 15-20
[mol/min/mg, the values of which are within standard error of each
other.
[0120] The arginine deiminase genes were amplified by polymerase
chain reaction using the following primer pair derived from the
published sequence of M. arginini, as described, for example, by T.
Ohno et al, Infect. Immun., 58:3788-3795 (1990), the disclosure of
which is hereby incorporated by reference herein in its entirety:
TABLE-US-00002 (SEQ ID NO:11) 5'-GCAATCGATGTGTATTTGACAGT-3' (SEQ ID
NO:12) 5'-TGAGGATCCTTACTACCACTTAACATCTTTACG-3'
[0121] The polymerase chain reaction product was cloned as a Bam
H1-Hind III fragment into expression plasmid pQE16. DNA sequence
analysis indicated this fragment had the same sequence for the
arginine deiminase gene as described by Ohno et al, Infect. Immun.,
supra. 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, Biotechniques,
13:592-596 (1992). Recombinant ADI was expressed in inclusion
bodies at levels of 10% of total cell protein.
[0122] The proteins from each of the above three species of
Mycoplasma have approximately 95% homology and are readily purified
by column chromatography. Approximately 1.2 g of pure protein may
be isolated from 1 liter of fermentation broth. Recombinant ADI is
stable for about 2 weeks at 37.degree. C. and for at least 8 months
when stored at 4.degree. C. As determined by methods known to those
skilled in the art, the proteins had a high affinity for arginine
(0.04 .mu.M), and a physiological pH optima of about 7.2 to about
7.4.
Example 2
Renaturation and Purification of Recombinant ADI
[0123] 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 K2PO4 pH 7.0, 1 mM EDTA (buffer 1) and the cells were disrupted
by two passes in a Microfluidizer (Microfluidics Corporation,
Newton, Mass.). Triton X-100 was added to achieve a final
concentration of 4% (v/v). The homogenate was stirred for 30 min at
4.degree. 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 HCl, pH 8.5, 10 mM DTT) using
hollow-fiber cartridges with 100 kD retention rating (Microgon
Inc., Laguna Hills, Calif.). Guanidine HCl was added to achieve a
final concentration of 6 M and the solution was stirred for 15 min
at 4.degree. C. The solution was diluted 100-fold into refolding
buffer 1, 10 mm K.sub.2PO.sub.4, pH 7.0 and stirred for 48 hours at
15.degree. C., particulates were removed by centrifugation at
15,000.times.g.
[0124] The resulting supernatant was concentrated on a Q Sepharose
Past Flow (Pharmacia Inc., Piscataway, N.J.) column preequilabrated
in refolding buffer. ADI was eluted using refolding buffer
containing 0.2 M NaCl. The purification procedure yielded ADI
protein, which was >95% pure as estimated by SDS-PAGE analysis.
Eight g of pure renatured ADI protein was produced from 1 kg of
cell paste which corresponds to 200 mg purified ADI per liter of
fermentation.
[0125] ADI activity was determined by micro-modification of the
method described by Oginsky et al, Meth. Enzymol., (1957)
3:639-642. Ten [t samples in 0.1 M Na.sub.2PO.sub.4, pH 7.0 (BLN
assay buffer) were placed in a 96 well microliter plate, 40 .mu.l
of 0.5 mM arginine in BUN assay buffer was added, and the plate was
covered and incubated at 37.degree. C. for 15 minutes. Twenty .mu.l
of complete BUN reagent (Sigma Diagnostics) was added and the plate
was incubated for 10 minutes at 100.degree. C. The plate was then
cooled to 22.degree. C. and analyzed at 490 nm by a microliter
plate reader (Molecular Devices, Inc). One IU is the amount of
enzyme which converts 1 .mu.mole of L-arginine to L-citrulline per
minute. Protein concentrations were determined using Pierce
Coomassie Blue Protein Assay Reagent (Pierce Co., Rockford, Ill.)
with bovine serum albumin as a standard. The enzyme activity of
the-purified ADI preparations was 17-25 IU/mg.
Example 3
Attachment of PEG to ADI
[0126] PEG was covalently bonded to ADI in a 100 mM phosphate
buffer, pH 7.4. 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 dialysed
to remove unincorporated PEG.
[0127] A first experiment was performed where the effect of the
linking group used in the PEG-ADI compositions was evaluated.
PEG10,000 and ADI were covalently bonded via four different linking
groups: an ester group or maleimide group, including SS, SSA, SPA
and SSPA, where each PEG molecule had an average molecular weight
of 5,000, 10,000, 12,000, 20,000, 30,000 and 40,000; an epoxy
group, PEG-epoxy, where each PEG molecule had an average molecular
weight of 5,000; and a branched PEG group, PEG2-NHS, where each PEG
molecule had an average molecular weight of 10,000, 20,000 and
40,000.
[0128] Five IU of the resulting compositions were injected into
mice (5 mice in each group). To determine the serum levels of
arginine, the mice were bled from the retro orbital plexus (100
.mu.l). Immediately following collection an equal volume of 50%
(w/v) of trichloroacetic acid was added. The precipitate was
removed by centrifugation (13,000.times.g for 30 minutes) and the
supernatant removed and stored frozen at -70.degree. 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 citrulline by this
method was approximately 2-6 .mu.M and the reproducibility of
measurements within about 8%. The amount of serum arginine was
determined by amino acid analysis. The linking group covalently
bonding the PEG and ADI did not have an appreciable effect on the
ability of ADI to reduce serum arginine in vivo.
[0129] A second experiment was performed wherein the effect of the
linking group and molecular weight of PEG on serum citrulline
levels in vivo was evaluated. Mice (5 in each group) were given
various compositions of ADI and PEG-ADI in an amount of 5.0 IU. To
determine the serum levels of citrulline, the mice were bled from
the retro orbital plexus (100 .mu.l). Immediately following
collection an equal volume of 50% (w/v) of trichloroacetic acid was
added. The precipitate was removed by centrifugation
(13,000.times.g for 30 minutes) and the supernatant removed and
stored frozen at -70.degree. 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 citrulline by this method was
approximately 2-6 .mu.M and the reproducibility of measurements
within about 8%. The amount of citrulline was determined, and the
area under the curve approximated and expressed as .mu.mol
days.
[0130] The results demonstrate that the molecular weight of the PEG
determines the effectiveness of the PEG-ADI composition. The
effectiveness of the PEG-ADI compositions does not appear to be
based on the method or means of attachment of the PEG to ADI.
[0131] The results demonstrate that the optimal molecular weight of
PEG is about 20,000. Although PEG30,000 appears to be superior to
PEG20,000 in terms of its pharmacodynamics, 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, in some embodiments PEG20,000 appears to
be a good compromise between yield and circulating half life, as
determined by citrulline production.
[0132] 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 IU, 0.5 IU or 5.0 IU 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 and serum citrulline. 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
Circulating Half-Life
[0133] Balb C mice (5 in each group) were injected intravenously
with a single 5.0 IU does of either native arginine deiminase or
various formulations of arginine deiminase modified with
polyethylene glycol. To determine the serum levels of arginine and
citrulline, the mice were bled from the retro orbital plexus (100
.mu.l). Immediately following collection an equal volume of 50%
(w/v) of trichloro-acetic acid was added. The precipitate was
removed by centrifugation (13,000.times.g for 30 minutes) and the
supernatant removed and stored frozen at -70.degree. 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%.
[0134] A dose dependent decrease in serum arginine levels and a
rise in serum citrulline were detected from the single dose
administration of native ADI or ADI-SS-PEG. However, the decrease
in serum arginine and rise in serum citrulline was short lived, and
soon returned to normal. The half-life of arginine depletion is
summarized in Table 2 below. TABLE-US-00003 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
Example 5
Antigenicity of PEG modified ADI
[0135] To determine the antigenicity of native ADI,
ADI-SS-PEG5,000, and ADI-SS-PEG20,000, the procedures described in,
for example, Park, Anticancer Res., supra, and Kamisaki, J.
Pharmacol. Exp. Ther., supra, were followed. Briefly, Balb C mice
(5 in each group) were intravenously injected weekly for 12 weeks
with approximately 0.5 IU (100 .mu.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.degree. C. The titers of anti-ADI IgG were determined
by ELISA. Fifty .mu.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 block nonspecific protein binding sites,
and stored over night at 4.degree. 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).
[0136] 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.sup.6, 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 10.sup.2, 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 6
Application to Humans
[0137] 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.degree. C.
[0138] 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.
Example 7
[0139] The antiviral activity of ADI-PEG20 was tested in a stably
HCV RNA replicating cell line AVA5 derived by transfection of a
human hepatoblastome cell line Huh7 (Blight et al., Efficient
Initiation of HCV RNA Replication in Cell Culture, Science 2000
290: 1972-1974).
[0140] In vitro Replication Assay
[0141] A stable HCV RNA replicating cell line AVA5 derived by
transfection of a human hepatoblastoma cell line Huh7 was used.
Dividing cultures of AVA5 cells were treated once daily for three
days (media was changed with each addition of compound) with 4
concentrations of test compound (3 cultures per concentration). A
total of 6 untreated control cultures, and triplicate cultures
treated with 10, 3, and 1 IU/ml .alpha.-interferon (active
antiviral with no cytotoxicity), and 100, 10 and 1 uM ribavirin (no
antiviral activity and cytotoxic) served as controls. HCV RNA and
cellular .beta.-actin RNA levels were assessed 24 hours after the
last dose of compound using dot blot hybridization. .beta.-actin
RNA levels were used to normalize the amount of cellular RNA in
each sample. Toxicity analyses were performed on separate plates
from those used for the antiviral assays. Cells for the toxicity
analyses were cultured and treated with test compounds with the
same schedule and under identical culture conditions as used for
the antiviral evaluations. Each compound was tested at 4
concentrations, each in triplicate cultures. Uptake of neutral red
dye was used to determine the relative level of toxicity 24 hours
following the last treatment. The absorbance of internalized dye at
510 nm (A.sub.510) was used for the quantitative analysis. Values
in test cultures were compared to 9 cultures of untreated cells
maintained on the same plate as the test cultures. The 50% and 90%
effective antiviral concentrations (EC.sub.50,EC.sub.90) and the
50% cytotoxic concentrations (CC.sub.50) were calculated and used
to generate Selectivity Indexes (CC.sub.50/EC.sub.50). An S.I. of
10 or greater is considered to be a selective antiviral effect.
[0142] Antiviral activity of ADI-PEG20
[0143] A single dose of ADI-PEG20 (0.01 IU/ml) was added to
dividing cultures of these cells when they are at 50% confluence.
As a control alpha interferon (10 IU/ml) and ribavirin (100 .mu.M)
were used as positive controls. After 3 days of treatment RNA was
isolated from the cultures using standard laboratory techniques and
assayed using dot blots. The amount of HCV mRNA was determined and
compared to the mRNA for actin (which is used as a control). The
amount of drug (ADI, alpha interferon or raboviron) required to
inhibit 50% of the control levels of HCV mRNA is determined. Any
dose of drug that causes a 50% inhibition of actin mRNA is
considered to have nonspecific inhibitory activity. The results
obtained from this experiment are shown below. TABLE-US-00004 Drug
% inhibition of HCV mRNA % inhibition of actin ADI-PEG20 86% 12%
alpha interferon 92% 11% ribavirin 25% 98%
These data demonstrate that ADI-PEG inhibits HCV viral replication
in vitro nearly as well as alpha interferon and much greater than
ribavirin.
Example 8
[0144] Dividing cultures of AVA5 cells were treated with various
concentrations of PEG-ADI (or in control experiments alpha
interferon or ribavirin) for 3 days. HCV mRNA levels were assayed
as above. Cell viability was determined using neutral red. The
concentrations which inhibit 50% (IC.sub.50) and 90% (IC.sub.90) of
HCV mRNA levels were determined. The concentration of drug which
kills 50% of the cells (CC.sub.50) was also determined. The
CC.sub.50/EC.sub.50 is calculated to determine the selectivity
index (SI). An SI>10 is considered to be a selective inhibition
of the viral replication. The results are shown below.
TABLE-US-00005 Drug CC.sub.50 IC.sub.50 IC.sub.90 SI ADI-PEG20
0.335 IU/ml 0.27 IU/ml 0.188 IU/ml 12 alpha >10000 IU/ml 2.1
IU/ml 9.0 IU/ml >4762 interferon ribavirin 74 .mu.M >10 .mu.M
>10 .mu.M NA
These data confirm that ADI-PEG20 inhibits HCV replication and that
this drug is selective.
Example 9
Antiviral Activity and NO Synthesis in Tumor Patients
[0145] ADI-PEG 20 was tested for anti-tumor activity in patients
with hepatocellular cancer also chronically infected with HCV.
Viral titers of HCV in the plasma of these patients using standard
clinical assays developed by Hoffman La Roche were also determined.
Plasma was obtained prior to treatment with ADI-PEG 20. The
patients were injected with 160 IU/m.sup.2 of ADI-PEG 20 once a
week for 3 weeks. One week following the third injection with
ADI-PEG 20, plasma was isolated from the patients and again assayed
for HCV titer using the same assay. The results from this
experiment are shown below. TABLE-US-00006 Patient Number HCV titer
Pretreatment HCV titer Post treatment 1 614,836 485,900 2 1,255,542
254,729 3 328,134 97,535 4 1,466,460 63,902 5 1,187,730 485,190
[0146] These data demonstrate that ADI-PEG treatment of humans
chronically infected with HCV results in significantly lower titers
of HCV in their plasma. Moreover as alpha interferon is only
effective in .about.50% of these patients and it frequently
requires 3-6 months of treatment to achieved a 50% reduction in HCV
titers, it appears that ADI-PEG 20 is much more effective in this
regard.
[0147] ADI-SS PEG 20,000 mw was tested in a Phase 2 study of
individuals with inoperable HCC according to Richard Simon
statistical design for rapid optimal two-stage Phase 2 testing
(Simon R. 1989. Optimal two-stage designs for phase II clinical
trials. Control Clin Trials 10:1-10; Simon R M, Steinberg S M,
Hamilton M, Hildesheim A, Khleif S, Kwak L W, Mackall C L, Schlom
J, Topalian S L, Berzofsky J A. 2001. Clinical trial designs for
the early clinical development of therapeutic cancer vaccines. J
Clin Oncol 19:1848-1854.). This testing was performed under
approval by the Italian Health Ministry at the Pascale National
Cancer Institute in Naples, Italy and with the approval of the
local institutional review board. All subjects were provided
informed consent according to the Declaration of Helsinki. A total
of 18 individuals with inoperable HCC were enrolled in this study
who were chronically infected with HCV (Izzo submitted). During
this study 3 died from progressive disease and failed to receive
all 3 cycles of treatment and thus were excluded form further
analysis. All remaining 15 subjects received 3 cycles (each
consisting of 4 once a week injections) of ADI-SS PEG 20,000 mw at
the Optimum Biological Dose. The Optimum Biological Dose was
defined as that amount of ADI-SS PEG 20,000 mw which lowered plasma
arginine from a resting level of .about.130 .mu.M to below the
level of detection (<2 .mu.M) for at least 7 days (.about.160
IU/m.sup.2).
[0148] The action this therapy had on the tumors was assessed by CT
scans once every 4 weeks. Response was defined as either
Progressive disease (PD), stable disease (SD), partial response
(PR) or complete response (CR) according to standard National
Cancer Institute (NCI) criteria. The results from this testing
indicated that in the 15 subjects with HCC and HCV the following
responses were seen: TABLE-US-00007 Status of Disease Number of
Subjects Complete Response (CR) 2 Partial Response (PR) 7 Stable
Disease (SD) 10
[0149] None of the subjects had received any systemic anti-tumor
treatment (or anti-viral treatment) either prior to or during this
study. Clinical laboratory testing was performed at least twice a
week during the study and plasma samples were collected once a week
and archived frozen at -70.degree. C. It was these frozen archived
plasma samples that were later tested for HCV.
[0150] Assay for HCV Titers and Serotyping of Human Plasma
Samples
[0151] HCV viral titers were determined in the hospital infectious
disease clinical laboratory using a standard clinical
polymerase-chain-reaction (P CR) assay, Cobas Amplicor HCV Monitor
Test, version 2.0; Roche Diagnostics (Germer 1999). The genotype
was similarly determined. Viral titers were determined on plasma
samples collected prior to ADI-SS PEG 20,000 mw treatment and after
12 weeks of therapy.
[0152] NO Synthesis
[0153] Treatment with ADI-SS PEG 20,000 mw results in a dose
dependent decrease in plasma arginine and concomitant decrease in
NO synthesis (data not shown). Although this treatment
significantly decreased NO levels, there was no measurable effect
of this treatment on blood pressure or heart rate.
[0154] The following Table 3 lists the effect of ADI-SS PEG 20,000
mw on Hepatitis C Titers and Liver Function Tests. TABLE-US-00008
TABLE 3 Effect of ADI-SS PEG 20,000 mw on Hepatitis C Titers and
Liver Function Tests. Patient HCV Titer HCV Titer Titer % Sero ALT
ALT AST AST Bilirubin Bilirubin Number Response pre Rx post Rx
Decrease Type Pre Rx Post Rx Pre Rx Post Rx Total Pre Rx Total Post
Rx 1 PR 614,836 <6000 >99 1b 271 227 245 182 1.16 0.92 2 SD
1,466,400 63,902 96 1b 104 115 101 98 0.69 0.33 3 SD 269,000 28,200
90 1b 118 81 76 71 1.32 0.60 4 SD 1,187,730 40,200 97 1b 153 110
145 80 3.4 0.45 5 PR 614836 40,200 93 1b 87 122 85 108 1.8 0.45 6
CR 328,134 173,000 47 1b 63 66 51 47 0.94 0.45 7 CR <6000
<6000 -- 1b 57 67 65 74 1.72 0.45 8 SD 676,000 120,000 82 1b 57
67 65 74 1.72 0.45 9 SD <6000 <6000 -- 1b 63 25 37 14 1.73
0.60 10 PR 1,950,000 921,000 53 1b 68 58 69 47 1.70 0.73 11 SD
386,000 331,000 14 1b 89 153 95 151 2.83 3.1 12 PR 2,830,000
3,390,000 increase 1b 77 65 85 83 0.72 0.83 13 SD 689,000 1,010,000
increase 2c 66 54 68 47 1.66 0.99 14 SD 351,000 690,000 increase 2c
115 137 111 152 3.13 3.1 15 SD 801,000 1,210,000 increase 2c 79 87
85 76 1.68 1.80 Note All post RX values are after 3 cycles at the
OBD.
[0155] Each of the patents, Genbank accession numbers, patent
applications and publications described herein are hereby
incorporated by reference herein in their entirety.
[0156] 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.
Sequence CWU 1
1
21 1 409 PRT Mycoplasma hominus 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 hominus 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
hominus 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 hominus 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 5 410 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 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 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 Trp 405 410 6 410 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 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 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
Trp 405 410 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 Leu 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 Ile 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 Ile 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 Leu 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 Ile 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 Ile 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 Leu 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 Ile 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 Ile 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 Leu 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 Ile 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 Ile 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 11 23 DNA Artificial Sequence
oligonucleotide 11 gcaatcgatg tgtatttgac agt 23 12 33 DNA
Artificial Sequence oligonucleotide 12 tgaggatcct tactaccact
taacatcttt acg 33 13 411 PRT Streptococcus pyogenes 13 Met Thr Ala
Gln Thr Pro Ile His Val Tyr Ser Glu Ile Gly Lys Leu 1 5 10 15 Lys
Lys Val Leu Leu His Arg Pro Gly Lys Glu Ile Glu Asn Leu Met 20 25
30 Pro Asp Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu
35 40 45 Asp Ala Gln Lys Glu His Asp Ala Phe Ala Gln Ala Leu Arg
Asp Glu 50 55 60 Gly Ile Glu Val Leu Tyr Leu Glu Thr Leu Ala Ala
Glu Ser Leu Val 65 70 75 80 Thr Pro Glu Ile Arg Glu Ala Phe Ile Asp
Glu Tyr Leu Ser Glu Ala 85 90 95 Asn Ile Arg Gly Arg Ala Thr Lys
Lys Ala Ile Arg Glu Leu Leu Met 100 105 110 Ala Ile Glu Asp Asn Gln
Glu Leu Ile Glu Lys Thr Met Ala Gly Val 115 120 125 Gln Lys Ser Glu
Leu Pro Glu Ile Pro Ala Ser Glu Lys Gly Leu Thr 130 135 140 Asp Leu
Val Glu Ser Asn Tyr Pro Phe Ala Ile Asp Pro Met Pro Asn 145 150 155
160 Leu Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Val Ser
165 170 175 Leu Asn His Met Phe Ser Glu Thr Arg Asn Arg Glu Thr Leu
Tyr Gly 180 185 190 Lys Tyr Ile Phe Thr His His Pro Ile Tyr Gly Gly
Gly Lys Val Pro 195 200 205 Met Val Tyr Asp Arg Asn Glu Thr Thr Arg
Ile Glu Gly Gly Asp Glu 210 215 220 Leu Val Leu Ser Lys Asp Val Leu
Ala Val Gly Ile Ser Gln Arg Thr 225 230 235 240 Asp Ala Ala Ser Ile
Glu Lys Leu Leu Val Asn Ile Phe Lys Gln Asn 245 250 255 Leu Gly Phe
Lys Lys Val Leu Ala Phe Glu Phe Ala Asn Asn Arg Lys 260 265 270 Phe
Met His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe 275 280
285 Thr Ile His Pro Glu Ile Glu Gly Asp Leu Arg Val Tyr Ser Val Thr
290 295 300 Tyr Asp Asn Glu Glu Leu His Ile Val Glu Glu Lys Gly Asp
Leu Ala 305 310 315 320 Glu Leu Leu Ala Ala Asn Leu Gly Val Glu Lys
Val Asp Leu Ile Arg 325 330 335 Cys Gly Gly Asp Asn Leu Val Ala Ala
Gly Arg Glu Gln Trp Asn Asp 340 345 350 Gly Ser Asn Thr Leu Thr Ile
Ala Pro Gly Val Val Val Val Tyr Asn 355 360 365 Arg Asn Thr Ile Thr
Asn Ala Ile Leu Glu Ser Lys Gly Leu Lys Leu 370 375 380 Ile Lys Ile
His Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg 385 390 395 400
Cys Met Ser Met Pro Phe Glu Arg Glu Asp Ile 405 410 14 409 PRT
Streptococcus pneumoniae 14 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 Gln 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 Ile Arg Glu Leu Leu His
Gly 100 105 110 Ile Lys Asp Asn Gln Glu Leu Val Glu Lys Thr Met Ala
Gly Ile Gln 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
Ile Asp Pro Met Pro Asn Leu 145 150 155 160 Tyr Phe Thr Arg Asp Pro
Phe Ala Thr Ile Gly Asn Ala Val Ser Leu 165 170 175 Asn His Met Phe
Ala 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 Gly Asp Glu Leu Val 210
215 220 Leu Ser Lys Asp Val Leu Ala Val Gly Ile Ser Gln Arg Thr Asp
Ala 225 230 235 240 Ala Ser Ile Glu Lys Leu Leu Val Asn Ile Phe Lys
Lys Asn Val Gly 245 250 255 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 Ile 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 Val 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 15 410 PRT Borrelia burgdorferi 15 Met Glu Glu
Glu Tyr Leu Asn Pro Ile Asn Ile 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 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 100 105 110 Tyr Phe Ser Asn Leu Thr
Val Asp Asn Met Val Ser Lys Met Ile Ser 115 120 125 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 Ile Thr Ile
165 170 175 Asn Lys Met Tyr Thr Lys Val 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 195 200 205 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 Glu Ala 225 230 235 240 Gly Ser Val 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 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 Ile 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 Val Arg Glu Asp Ile 405 410 16 409 PRT Borellia
afzellii 16 Met Glu Glu Tyr Leu Asn Pro Ile Asn Ile Phe Ser Glu Ile
Gly Arg 1 5 10 15 Leu Lys Lys Val 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 Val 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 Ile Ser Gln Phe Ile Leu 85 90 95 Glu Ala
Glu Ile Lys Thr Asp Phe Thr Ile Asn Leu Leu Lys Asp Tyr 100 105 110
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 145 150 155 160 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 Ala Ile Ser Leu Phe Lys Asn Lys Thr Ser Phe
245 250 255 Asp Thr Ile Leu Ala Phe Gln Ile 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 Ile 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
Val Leu Ala Ile Ala Pro Gly Glu Ile Ile Ala Tyr Ser Arg Asn 355 360
365 His Val Thr Asn Lys Leu Phe Glu Glu Asn Gly Ile 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 17
580 PRT Giardia intestinalis 17 Met Thr Asp Phe Ser Lys Asp Lys Glu
Lys Leu Ala Gln Ala Thr Gln 1 5 10 15 Gly Gly Glu Asn Glu 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 Gly Cys Arg Val Tyr Met Pro Tyr Asp Val 65 70
75 80 Leu Ser Glu Ala 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 100 105 110 Pro Lys 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
Val 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 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 Val Thr Cys Gly Asn Tyr Val Leu Ala Lys
325 330 335 Ala Asn Val Glu Phe Gln Gln Trp Leu Ser Glu Asn Gly Tyr
Thr Ile 340 345 350 Val Arg Ile 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 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 Val 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 Ile Lys 580 18 413 PRT Clostridium perfringens 18
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 Ile Pro
Tyr Leu Lys 35 40 45 Val Ala Arg Glu Glu His Asp Ala Phe Ala Gln
Thr Leu Arg Glu Ala 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 115 120 125 Val
Arg Lys Glu Glu 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 Ala Lys 180 185 190 Tyr Ile Phe Arg His His Pro Arg Phe
Glu 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 Glu 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 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 Glu 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 19 413 PRT Bacillus licheniformis 19 Met Ile Met Thr Thr Pro
Ile His Val Tyr Ser Glu Ile Gly Pro Leu 1 5 10 15 Lys Thr Val Met
Leu Lys Arg Pro Gly 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 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 Ile His 275 280 285 Pro Ala
Ile Gln Gly Pro Glu Gly Asp Met Arg Ile Phe Val Leu Glu 290 295 300
Arg Gly Lys Thr Ala 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 Gly Ser Asn Thr Leu Ala Ile Ala Pro
Gly Val Val Val Thr Tyr 355 360 365 Asp Arg Asn Tyr Ile Ser Asn Glu
Cys Leu Arg Glu Gln Gly 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 20 408 PRT
Enterococcus faecalis 20 Met Ser His Pro Ile 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 Ala Glu Leu Leu Arg Ser Lys Asp Ile 50 55 60 Glu Val
Val Tyr Leu Glu 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 Glu Leu Ile Gln Lys Ala Ile 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 Ala 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 Ile Ser Leu Asn His Met 165 170 175 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 195 200 205 Arg
Ser Glu Ser Thr Arg Ile Glu Gly Gly Asp Glu Leu Ile Leu Ser 210 215
220 Lys Glu 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 Ile 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 Ile 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 21 409 PRT Lactobacillus sake 21 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 Gly 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 Ala Asn Gln Gly Ala Gln Val Trp Arg Asp Arg
195 200 205 Asp His Ile Asp Arg Met Glu Gly Gly 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 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 Ile 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 Ile 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 Ala 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
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