CA2430077A1 - Modified arginine deiminase - Google Patents

Abstract

The present invention is directed to arginine deiminase modified with polythylene glycol, to methods of treating cancer, and to methods of treating and(or inhibiting metastasis.

Description

MODIFIED ARGININE DEIMINASE
Related A~~lications This application is a continuation in part application of U.S. Patent Application Serial No. 091023,809, allowed, which claims priority to U.S.
Provisional Patent Application Serial No. 60/046,200, filed on May 12, 1997.
Field of the Invention The present invention is directed to arginine deiminase modified with polyethylene glycol, to methods for treating cancer, and to methods for treating andlor inhibiting metastasis.
Background of the Invention Malignant melanoma (stage 3) and hepatoma are fatal diseases which bill most patients within one year of diagnosis. In the United States, approximately 16,000 people die from these diseases annually. The incidence of melanoma is rapidly increasing in the United States and is even higher in other countries, such as Australia.
The incidence of hepatoma, in parts of the world where hepatitis is endemic, is even greater. For example, hepatoma is one of the leading forms of cancer in Japan and Taiwan.
Effective treatments for these diseases are urgently needed.
Selective deprivation of essential amino acids has been used to treat some forms of cancer. The best known example is the use of L-asparaginase to lower levels of asparagine as a treatment for acute lymphoblastic leukemia. The L-asparaginase most frequently used is isolated from E. coli. However, clinical use of this enzyme is compromised by its inherent antigenicity and short circulating half life, as described by ~.K. Park, et al, Ahticafacef° Res., 1:373-376 (1981). Covalent modification of E. coli L-asparaginase with polyethylene glycol reduces its antigenicity and prolongs its circulating half life, as described, for example, by Park, A~Zticahce~ Res., supra; Y.
Kamisalci et al, J.
Pharmacol. Exp. Tlaer., 216:410-414 (I98I); and Y. Kamisaki et al, Gahya., 73:47-474 (1982). Although there has been a great deal of effort to identify other essential amino acid degrading enzymes for the treatment of cancer, none have been approved, primarily because deprivation of essential amino acids, by definition, results in numerous, and severe, side effects.
It has been reported that enzymes which degrade non-essential amino acids, such as arginine, may be an effective means of controlling some forms of cancer. For example, axginine deiminase (ADI) isolated from Pseudomofaas pudita was described by J.B. Jones, "The Effect of Arginine Deiminase on Murine Leukemic Lymphoblasts," Ph.D.
Dissertation, The University of Oklahoma, pages 1-165 (1981). Although effective in killing tumor cells in vitro, ADI isolated from P. pudita failed to exhibit efficacy in vivo because it had little enzyme activity at a neutral pH and was rapidly cleared from the circulation of experimental animals. Arginine deiminase derived from Mycoplasma arginini is described, for example, by Takaku et al, Int. J. Cancer, 51:244-249 (1992), and U.S. Patent No. 5,474,928, the disclosures of which are hereby incorporated by reference herein in their entirety. However, a problem associated with the therapeutic use of such a heterologous protein is its antigenicity. The chemical modification of arginine deiminase from Mycoplasma argihini, via a cyanuric chloride linking group, with polyethylene glycol was described by Takaku et al., Jpn. J. Cancer Res., 84:1195-1200 (1993).
However, the modified protein was toxic when metabolized due to the release of cyanide from the cyanuric chloride linl~ing group.
There is a need for compositions which degrade non-essential amino acids and which do not have the problems associated with the prior art. The present invention is directed to these, as well as other, important ends.
Summary of the Invention The present invention is directed to arginine deiminase modified with polyethylene glycol. In a preferred embodiment, the arginine deiminase is modified with polyethylene glycol, having a total weight average molecular weight of about 1,000 to about 50,000, directly or through a biocompatible linking group.

_3_ Another embodiment of the invention is directed to methods of treating cancer, including, for example, sarcomas, hepatomas and melanomas. The invention is also directed to methods of treating and/or inhibiting the metastasis of tumor cells.
These and other aspects of the present invention will be elucidated in the following detailed description of the invention.
Brief Description of the Drawi~s Figure 1 depicts the amino acid sequences of arginine deiminase cloned from Mycoplasma arginini (the top amino acid sequence SEQ ID NO: 1, identified as ADIPROT), Mycoplasma arthritides (the middle amino acid sequence SEQ ID NO: 2, identified as ARTADIPRO), and Mycoplasma hominus (the bottom amino acid sequence SEQ ID NO: 3, identified as HOMADIPRO).
Figures 2A and 2B are graphs showing the effect of a single dose of native arginine deiminase and arginine deiminase modified with polyethylene glycol (e.g., molecular weight 5,000) on serum arginine levels and serum citrulline levels in mice.
Figure 3 is a graph showing the effects on serum arginine levels when PEG10,000 is covalently bonded to ADI via various linking groups.
Figure 4 is a graph showing the effect that the linking group and the molecular weight of the polyethylene glycol have on citrulline production in mice injected with a single dose of PEG-ADI.
Figures 5A and SB are graphs showing the dose response that ADI-SS-PEG5,000 had on serum arginine and citrulline levels. Figures 5C and SD are graphs showing the dose response that ADI-SS-PEG20,000 had on serum arginine and citrulline levels.
Figure 6 is a graph showing the antigenicity of native ADI, ADI-SS-PEG5,000, and ADI-SS-PEG20,000.
Figure 7 is a graph showing the effect that treatments with ADI-SS-PEG5,000, ADI-SS-PEG12,000 or ADI-SS-PEG20,000 had on tumor size in mice which were inj ected with SK-mel 2 human melanoma cells.
Figure 8 is a graph showing the effect that treatments with ADI-PEG20,000 had on tumor size in mice which were injected with SK-mel 28, SK-mel 2 or M24-met human melanoma cells.

Figure 9 is a graph showing the effect that treatments with ADI-PEG5,000, ADI-PEG12,000 or ADI-PEG20,000 had on the survival of mice which were inj ected with human hepatoma SK-Hepl cells.
Figure 10 depicts the amino acid sequences of arginine deiminase cloned from Steptococcus pyogenes (the top amino acid sequence SEQ ID NO: 6, identified as STRADIPYR) and Steptococcus pneunaoniae(the bottom amino acid sequence SEQ ID
NO: 7, identified as STRADIPNE).
Figure 11 depicts the amino acid sequences of arginine deiminase cloned from Bo~y~elia bu~gdo~fe~i (the top amino acid sequence SEQ ID NO: 8, identified as BORADIBUR) and Borrelia afzelii (the bottom amino acid sequence SEQ ID NO: 9, identified as BORADIAFZ).
Figure 12 depicts the amino acid sequence of Qia~diu intestinalis (the top amino acid sequence SEQ ID NO: 10, identified as QIAADI1NT), Clostridium peyf~ingens (the middle amino acid sequence SEQ ID NO: 11, identified as CLOADIPER) and Bacillus lichenifof°mis (the bottom amino acid sequence SEQ ID NO: 12, identified as BACADILIC).
Figure 13 depicts the amino acid sequence of Ente~ococcus faecalis (the top amino acid sequence SEQ ID NO: 13, identified as ENTADIFAE) and Lactobacillus sake (the bottom amino acid sequence SEQ ID NO: 14, identified as LACADISAK).
Detailed Description of the Invention Normal cells do not require arginine for growth, since they can synthesize arginine from citrulline in a two step process catalyzed by argininosuccinate synthase and argininosuccinate lyase. In contrast, melanomas, hepatomas and some sarcomas do not express arginosuccinate synthase; therefore, they are auxotrophic for arginine. This metabolic difference may be capitalized upon to develop a safe and effective therapy to treat these forms of cancer. Arginine deiminase catalyzes the conversion of arginine to citrulline, and may be used to eliminate arginine. Thus, arginine deiminase may be utilized as a treatment for melanomas, hepatomas and some sarcomas.
Native arginine deiminase may be found in microorgaiusms and is antigenic and rapidly cleared from circulation in a patient. These problems may be overcome by covalently modifying arginine deiminase with polyethylene glycol (PEG).
Arginine deiminase covalently modified with polyethylene glycol (with or without a linking group) may be hereinafter referred to as "ADI-PEG." When compared to native arginine deiminase, ADI-PEG retains most of its enzymatic activity, is far less antigenic, has a greatly extended circulating half life, and is much more efficacious in the treatment of tumors.
"Polyethylene glycol" or "PEG" refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCHZCHZ)"OH, wherein n is at least 4. "Polyethylene glycol"
or "PEG" is used in combination with a numeric suffix to indicate the approximate weight average molecular weight thereof. For example, PEG5,000 refers to polyethylene glycol having a total weight average molecular weight of about 5,000; PEG12,000 refers to polyethylene glycol having a total weight average molecular weight of about 12,000; and PEG20,000 refers to polyethylene glycol having a total weight average molecular weight of about 20,000.
"Melanoma" may be a malignant or benign tumor arising from the melanocytic system of the shin and other organs, including the oral cavity, esophagus, anal caaial, vagina, leptomeninges, and/or the conjunctivae or eye. The term "melanoma"
includes, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma and superficial spreading melanoma.
"Hepatoma" may be a malignant or benign tumor of the liver, including, for example, hepatocellular carcinoma.
"Patient" refers to an animal, preferably a mammal, more preferably a human.
"Biocompatible" refers to materials or compounds which are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic and disease states.
Throughout the present disclosure, the following abbreviations may be used: PEG, polyethylene glycol; ADI, arginine deiminase; SS, succinimidyl succinate;

SSA, succinimidyl succinamide; SPA, succinimidyl propionate; and NHS, N-hydroxy-succinimide.
The present invention is based on the unexpected discovery that ADI
modified with polyethylene glycol provides excellent results in treating certain types of cancer and inhibiting the metastasis of cancer. ADI may be covalently bonded to polyethylene glycol with or without a linking group, although a preferred embodiment utilizes a linl~ing group.
In the present invention, the arginine deiminase gene may be derived, cloned or produced from any source, including, for example, microorganisms, recombinant bioteclmology or any combination thereof. For example, argiune deiminase may be cloned from microorganisms of the genera Mycoplasma, Clostf°idium, Bacillus, Bo~~elia, Enterococcus, Streptococcus, Lactobacillus, Qia~dia. It is preferred that arginine deiminase is cloned from Mycoplasma pneumoniae, Mycoplasma IZOminus, Mycoplasma of ginirai, Steptococcus pyogenes, Steptococcus pneumoniae, Bor~elia bu~gdorfe~i, Bo~relia afzelii, Qiay~dia intestinalis, Clostridium pelf °ihget~s, Bacillus liclaeszifo~mis, Ente~ococcus faecalis, Lactobacillus sake, or any combination thereof. In particular, the arginine deiminase used in the present invention may have one or more of the amino acid sequences depicted in Figures 1 and 10-13.
In certain embodiments of the present invention, it is preferred that arginine deiminase is cloned from microorganisms of the genus Mycoplasma. More preferably, the arginine deiminase is cloned from Mycoplasma a~gihini, Mycoplasnaa laominus, Mycoplasma a~tlz~itides, or any combination thereof. In particular, the arginine deiminase used in the present invention may have one or more of the amino acid sequences depicted in Figure 1.
hi one embodiment of the present invention, the polyethylene glycol (PEG) has a total weight average molecular weight of about 1,000 to about 50,000;
more preferably from about 3,000 to about 40,000, more preferably from about 5,000 to about 30,000; more preferably from about 8,000 to about 30,000; more preferably from about 11,000 to about 30,000; even more preferably from about 12,000 to about 28,000; still more preferably from about 16,000 to about 24,000; even more preferably from about _7_ 18,000 to about 22,000; even more preferably from about 19,000 to about 21,000, and most preferably about 20,000. Generally, polyethylene glycol with a molecular weight of 30,000 or more is difficult to dissolve, and yields of the formulated product are greatly reduced. The polyethylene glycol may be a branched or straight chain, preferably a straight chain. Generally, increasing the molecular weight of the polyethylene glycol decreases the immunogenicity of the ADI. The polyethylene glycol having a molecular weight described in this embodiment may be used in conjunction with ADI, and, optionally, a biocompatible linlcing group, to treat cancer, including, for example, melanomas, hepatomas and sarcomas, preferably melanomas.
In another embodiment of the present invention, the polyethylene glycol has a total weight average molecular weight of about 1,000 to about 50,000;
preferably about 3,000 to about 30,000; more preferably from about 3,000 to about 20,000;
more preferably from about 4,000 to about 12,000; still more preferably from about 4,000 to about 10,000; even more preferably from about 4,000 to about 8,000; still more preferably from about 4,000 to about 6,000; with about 5,000 being most preferred. The polyethylene glycol may be a branched or straight chain, preferably a straight chain. The polyethylene glycol having a molecular weight described in this embodiment may be used in conjunction with ADI, and optionally, a biocompatible linl~ing group, to treat cancer, including, for example, melanomas, hepatomas and sarcomas, preferably hepatomas.
The linking group used to covalently attach ADI to PEG may be any biocompatible linking group. As discussed above, "biocompatible" indicates that the compound or group is non-toxic and may be utilized ih vitro or in vivo without causing injury, sicl~ness, disease or death. PEG can be bonded to the linking group, for example, via an ether bond, an ester bond, a thiol bond or an amide bond. Suitable biocompatible linlcing groups include, for example, an ester group, an amide group, an imide group, a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, a succinimide group (including, for example, succinimidyl succinate (SS), succinimidyl propionate (SPA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) or N-hydroxy succinimide (NHS)), an epoxide group, an oxycarbonylimidazole group (including, for example, carbonyldimidazole (CDI)), a nitro phenyl group (including, for example, _g_ nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), a trysylate group, an aldehyde group, an isocyanate group, a vinylsulfone group, a tyrosine group, a cysteine group, a histidine group or a primary amine. Preferably, the biocompatible linl~ing group is an ester group and/or a succinimide group. More preferably, the lii~lcing group is SS, SPA, SCM, SSA or NHS; with SS, SPA or NHS being more preferred, and with SS or SPA being most preferred.
Alternatively, ADI may be coupled directly to PEG (i.e., without a linking group) through an amino group, a sulflzydral group, a hydroxyl group or a carboxyl group.
ADI may be covalently bonded to PEG, via a biocompatible linking group, using methods laiown in the art, as described, for example, by Parlc et al, Anticahee~ Res., 1: 373-376 (1981); and Zaplipsky and Lee, Polyethylene Glycol Chemistry:
Biotechnical and Biomedical Applications, J.M. Harris, ed., Plenum Press, NY, Chapter 21 (1992), the disclosures of which are hereby incorporated by reference herein in their entirety.
The attachment of PEG to ADI increases the circulating half life of ADI.
Generally, PEG is attached to a primary amine of ADI. Selection of the attachment site of polyethylene glycol on the axginine deiminase is determined by the role of each of the sites within the active domain of the protein, as would be known to the skilled artisan. PEG
may be attached to the primary amines of arginine deiminase without substantial loss of enzymatic activity. For example, ADI cloned from Mycoplasma argiraini, Mycoplasma a~th~itides amd Mycoplasma hominus has about 17 lysines that may be modified by this procedure. In other words, the 17 lysines are all possible points at which ADI
can be attached to PEG via a biocompatible linking group, such as SS, SPA, SCM, SSA
and/or NHS. PEG may also be attached to other sites on ADI, as would be apparent to one skilled in the art in view of the present disclosure.
From 1 to about 30 PEG molecules may be covalently bonded to ADI.
Preferably, ADI is modified with about 7 to about 15 PEG molecules, more preferably from about 9 to about 12 PEG molecules. In other words, about 30% to about 70%
of the primary amino groups in arginine deiminase are modified with PEG, preferably about 40%
to about 60%, more preferably about 45% to about 55%, and most preferably about 50% of the primary amino groups in arginine deiminase are modified with PEG. When PEG
is covalently bonded to the end terminus of ADI, preferably only 1 PEG molecule is utilized.

Increasing the number of PEG units on ADI increases the circulating half life of the enzyme. However, increasing the number of PEG units on ADI decreases the specific activity of the enzyme. Thus, a balance needs to be achieved between the two, as would be apparent to one sleilled in the art in view of the present disclosure.
In the present invention, a common feature of the most preferred biocompatible linking groups is that they attach to a primary amine of arginine deiminase via a maleimide group. Once coupled with arginine deiminase, SS-PEG has aaz ester linkage next to the PEG, which may render this site sensitive to serum esterase, which may release PEG from ADI in the body. SPA-PEG and PEG2-NHS do not have an ester linkage, so they are not sensitive to serum esterase.
In the present invention, the particular linking groups do not appear to influence the circulating half life of PEG-ADI or its specific enzyme activity. However, it is critical to use a biocompatible linking group in the present invention. PEG
which is attached to the protein may be either a single chain, as with SS-PEG, SPA-PEG
and SC-PEG, or a branched chain of PEG may be used, as with PEG2-NHS. The structural formulas of the preferred linking groups in the present invention are set forth below.
SS-PEG:
SPA-PEG:
PEG
O

SS O
PEG
O

SPA

PE~
O
C O N
PE~
O
NHS
A therapeutically effective amount of one of the compounds of the present invention is an amount that is effective to inhibit tumor growth. Generally, treatment is initiated with small dosages which can be increased by small increments until the optimum effect under the circumstances is achieved. Generally, a therapeutic dosage of compounds of the present invention may be from about 1 to about 200 mg/lcg twice a week to about once every two weeks. For example, the dosage may be about 1 mglkg once a week as a 2 ml intravenous injection to about 20 mg/kg once every 3 days. The optimum dosage with ADI-SS-PEG5,000 may be about twice a week, while the optimum dosage with ADI-SS-PEG20,000 may be from about once a weelc to about once every two weeks. PEG-ADI
may be mixed with a phosphate buffered saline solution, or any other appropriate solution known to those skilled in the art, prior to injection. The PEG-ADI formulation may be administered as a solid (lyophilate) or as a liquid formulation, as desired.
The methods of the present invention can involve either iya vitro or ih vivo applications. In the case of in vitro applications, including cell culture applications, the compounds described herein can be added to the cells in cultures and then incubated. The compounds of the present invention may also be used to facilitate the production of monoclonal and/or polyclonal antibodies, using antibody production techniques well known in the art. The monoclonal and/or polyclonal antibodies can then be used in a wide vaxiety of diagnostic applications, as would be apparent to one skilled in the art.
The ih. vivo means of administration of the compounds of the present invention will vary depending upon the intended application. As one spilled in the art will recognize, administration of the PEG-ADI composition of the present invention can be carried out, for example, orally, intxanasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorly, intramuscularly, interstitially, infra-arterially, subcutaneously, intraocularly, intrasynovial, transepithelial, and transdermally.
Examples The invention is further demonstrated in the following examples, which are for purposes of illustration, and are not intended to limit the scope of the present invention.
Example 1: P~oductio~z of Recombiuaut ADI
Cultures of Mycoplaszzza a~gitzitzi (ATCC 23243), Mycoplasma hozninus (ATCC 23114) and Mycoplaszzza arth~itides (ATCC 23192) were obtained from the American Type Culture Collection, Rockville, Maryland.
Arginine deiminase was cloned from Mycoplasma a>"ginini, Mycoplasma hoznizzus and Mycoplaszna a~th~itides and expressed in E. coli as previously described by S. Misawa et al, J. Biotechnology, 36:145-155 (1994), the disclosure of which is hereby incorporated herein by reference in its entirety. The amino acid sequences of arginine deiminase from each of the above species is set forth in Figure 1. The top amino acid sequence, identified as A:DIPROT, is from Mycoplasma aYginini; the middle amino acid sequence, identified as ARTADIPRO, is from Mycoplasma a>"th>~itides; and the bottom amino acid sequence, identified as HOMADIPRO, is from Mycoplasma lzomizzzrs.
Each of the amino acid sequences are more than 96% conserved. Characterization, by methods l~nown to those spilled in the art, of each of the proteins with respect to specific enzyme activity, Kn" Vmax and pH optima revealed that they were biochemically indistinguishable from each other. The pH optima was determined using a citrate buffer (pH 5-6.5), a phosphate buffer (pH 6.5-7.5) and a borate buffer (pH 7.5-8.5). The I~m and Vm~ were determined by incubating the enzyme with various concentrations of argiiune and quantifying citrulline production. The K", for the various enzymes was about 0.02 to 0.06 ~,M and the V",~ was about 15-20 ~,mol/min/mg, the values of which are within standard error of each other.
The arginine deiminase genes were amplified by polymerase chain reaction -m-using the following primer pair derived from the published sequence of M.
a~gihihi, as described, for example, by T. Olmo et al, Infect. Irnmun., 58:3788-3795 (1990), the disclosure of which is hereby incorporated by reference herein in its entirety:
SEQ ID NO: 4, 5'-GGGATCCATGTCTGTATTTGACAGT-3' SEQ ID NO: 5, 5'-TGA.A.AGCTTTTACTACCACTTAACATCTTTACG-3' The polymerase chain reaction products were cloned as a Bam H1-Hind III
fragment into expression plasmid pQEl6. DNA sequence analysis indicated that the fragment derived from M. arginini by PCR had the same sequence for the arginine deiminase gene as described by Ohno et al, Infect. Immun., sups°a. The five TGA codons in the ADI gene which encode tryptophan in Mycoplasma were changed to TGG codons by oligonucleotide-directed mutagenesis prior to gene expression in E. coli, as taught, for example, by J.R. Sayers et al, Biotechraiques, 13:592-596 (1992). Recombinant ADI was expressed in inclusion bodies at levels of 10% of total cell protein.
The proteins from each of the above three species of Mycoplasma have approximately 95% homology and are readily purified by column chromatography.
Approximately 200 mg of pure protein may be isolated from 1 liter of fermentation broth.
Recombinant ADI is stable for about 2 weeks at 37°C and for at least 8 months when stored at 4°C. As determined by methods known to those skilled in the art, the proteins had a high affinity for arginine (0.04 ~,M), and a physiological pH optima of about 7.2 to about 7.4.
Example 2: Rezzatu~atiofz and Purification of Recozzzbiuaut ~iDI
ADI protein was renatured, with minor modifications, as described by Misawa et al, J. Biotechnology, 36:145-155 (1994), the disclosure of which is hereby incorporated herein by reference in its entirety. 100 g of cell paste was resuspended in 800 ml of 10 mM KZP04 pH 7.0, 1 mM EDTA (buffer 1) and the cells were disrupted by two passes in a Microfluidizer (Microfluidics Corporation, Newton, MA). Triton X-100 was added to achieve a final concentration of 4% (v/v). The homogenate was stirred for 30 min at 4°C, then centrifuged for 30 min at 13,000 g. The pellet was collected and resuspended in one liter of buffer 1 containing 0.5% Triton X-100. The solution was diafiltered against 5 volumes of denaturation buffer (50 mM Tris HCI, pH 8.5, 10 mM

DTT) using hollow-fiber cartridges with 100 kD retention rating (Microgon Inc., Lagtma Hills, CA). Guanidine HCl was added to achieve a final concentration of 6 M
and the solution was stirred for 1 S min at 4°C. The solution was diluted 100-fold into refolding buffer 1, 10 rmn I~ZP04, pH 7.0 and stirred for 48 hours at 1S°C, particulates were S removed by centrifugation at 15,000 x g.
The resulting supernatant was concentrated on a Q Sepharose Fast Flow (Pharmacia Inc., Piscataway, NJ) column preequilabrated in refolding buffer.
ADI was eluted using refolding buffer containing 0.2 M NaCI. The purification procedure yielded ADI protein, which was >9S% pure as estimated by SDS-PAGE analysis. 8 g of pure renatured ADI protein was produced from 1 kg of cell paste wluch corresponds to 200 mg purified ADI per liter of fermentation.
ADI activity was determined by micro-modification of the method described by Oginsky et al, Meth. Ehzymol., (1957) 3:639-642. 10 ~1 samples in 0.1 m NazP04, pH 7.0 (BUN assay buffer) were placed in a 96 well microliter plate, 40 ~l of O.f 1 S mM arginine in BUN assay buffer was added, and the plate was covered and incubated at 37 ° C for 1 S minutes. 20 ~.1 of complete BLTN reagent (Sigma Diagnostics) was added and the plate was incubated for 10 minutes at 100°C. The plate was then cooled to 22°C and analyzed at 490 nm by a microliter plate reader (Molecular Devices, Inc). 1.0 ICT is the amount of enzyme which converts 1 mole of L-axginine to L-citrulline per minute.
Protein concentrations were determined using Pierce Coomassie Blue Protein Assay Reagent (Pierce Co., Rockford, IL) with bovine serum albumin as a standaxd.
The enzyme activity of the purified ADI preparations was 17-2S IU/mg.
Example 3: Attaclasneht of PEG to ADI
PEG was covalently bonded to ADI in a 100 mM phosphate buffer, pH 7.4.
2S Briefly, ADI in phosphate buffer was mixed with a 100 molar excess of PEG.
The reaction was stirred at room temperature for 1 hour, then the mixture was extensively dialized to remove unincorporated PEG.
A first experiment was performed where the effect of the licking group used in the PEG-ADI compositions was evaluated. PEG and ADI were covalently bonded via four different linking groups: an ester group or maleimide group, including SS, SSA, SPA and SSPA, where the PEG had a total weight average molecular weight of 5,000, 10,000, 12,000, 20,000, 30,000 and 40,000; an epoxy group, PEG-epoxy, where the PEG
had a total weight average molecular weight of 5,000; and a branched PEG
group, PEG2-NHS, where the PEG had a total weight average molecular weight of 10,000, 20,000 and S 40,000.
S.0 ILT of the resulting compositions were injected into mice (S mice in each group). To determine the serum levels of arginine, the mice were bled from the retro orbital plexus (100 u1). Immediately following collection an equal volume of SO% (w/v) of trichloroacetic acid was added. The precipitate was removed by centrifugation (13,000 x g for 30 minutes) and the supernatant removed and stored frozen at -70°C.
The samples were then analyzed using an automated amino acid analyzer and reagents from Beckman Instruments using protocols supplied by the manufacturer. The limits of sensitivity for arginine by this method was approximately 2-6 ~M and the reproducibility of measurements within about 8%. The amount of serum arginine was determined by amino 1 S acid analysis. As can be seen from the results in Figure 3, the linking group covalently bonding the PEG and ADI did not have an appreciable effect on the ability of ADI to reduce serum argiune ih vivo. In other words, the linking group may not be critical to the results of the experiment, except that a non-toxic linking group must be used for ih vivo applications.
A second experiment was performed wherein the effect of the linking group and molecular weight of PEG on serum citrulline levels ih vivo was evaluated.
Mice (S in each group) were given various compositions of ADI and PEG-ADI in an amount of S.0 ILT. To determine the serum levels of citrulline, the mice were bled from the retro orbital plexus (100 u1). Tm_m__ediately following collection an equal volume of SO%
(w/v) of 2S trichloroacetic acid was added. The precipitate was removed by centrifugation (13,000 x g for 30 minutes) and the supernatant removed and stored frozen at -70°C.
The samples were then analyzed using an automated amino acid analyzer and reagents from Becl~nan Instruments using protocols supplied by the manufacturer. The limits of sensitivity for citrulline by this method was approximately 2-6 ~,M and the reproducibility of measurements within about 8%. The amount of citrulline was determined, and the area under the curve approximated and expressed as ~mol days.
In Figure 4, the open circles indicate the amount of citrulline produced by native ADI, the filled circles are ADI-SC-PEG, the open squares are ADI-SS-PEG, the open triangles are ADI-SPA-PEG, and the filled triangles are branched chain PEG-NHS-PEGZ. The results in Figure 4 demonstrate that the molecular weight of the PEG
determines the effectiveness of the PEG-ADI composition. The effectiveness of the PEG-ADI compositions is not necessarily based on the method or means of attachment of the PEG to ADI, except that a biocompatible linking group must be used for in vivo applications.
The results in Figure 4 also demonstrate that the optimal molecular weight of PEG is 20,000. Although PEG30,000 appears to be superior to PEG20,000 in terms of its phanmacodynamics, PEG30,000 is less soluble, which makes it more difficult to work with. The yields, which were based on the recovery of enzyme activity, were about 90%
for PEG5,000 and PEG12,000; about 85% for PEG20,000 and about 40% for PEG30,000.
Therefore, PEG20,000 is the best compromise between yield and circulating half life, as determined by citrulline production.
In a third experiment, the dose response of serum arginine depletion and the production of citrulline with ADI-SS-PEG5,000 and ADI-SS-PEG20,000 was determined.
Mice (5 in each group) were given a single injection of 0.05 ILJ, 0.5 ICT or 5.0 ILJ of either ADI-SS-PEG5,000 or ADI-SS-PEG20,000. At indicated times, serum was collected, as described above, and an amino acid analysis was performed to quantify serum arginine (Figures 5A and SC) and serum citrulline (Figures SB and SD). Both formulations induced a dose dependent decrease in serum arginine and an increase in serum citrulline. However, the effects induced by ADI-SS-PEG20,000 were more pronounced and of longer duration than the effects induced by ADI-SS-PEG5,000.
Example 4: Selectivity of ADl Mediated Cytotoxicity The selectivity of arginine deiminase mediated cytotoxicity was demonstrated using a number of human tumors. Specifically, human tumors were tested if2 vitYO for sensitivity to ADI-SS-PEG5,000 (50 ng/ml). Viability of cultures was determined after 7 days. For a culture to be defined as "inhibited," greater than 95% of the cells must take up Trypan blue dye. A host of normal cells were also tested, including endothelial cells, smooth muscle cells, epithelial cells and fibroblasts, and none were inhibited by ADI-SS-PEG5,000. Although arginine deiminase has no appreciable toxicity towards normal, and most tumor cells, ADI-SS-PEG5,000 greatly inhibited all hiunan melanomas and hepatomas that were commercially available from the ATCC, MSKCC
and Europe.
Table 1: Specificity of Arginine Deiminase Cytotoxicity Tumor Type Number of Tumors Tumors inhibited Tested (%) Brain 16 0 . Colon 34 0 Bladder 3 0 Breast 12 0 Kidney 5 0 S arcoma 11 64 Hepatoma 17 100 Melanoma 37 100 In a parallel set of experiments, mRNA was isolated from the tumors.
Northern blot analyses, using the human axgininosuccinate synthase cDNA probe, indicated complete concordance between the sensitivity to arginine deiminase treatment and an inability to express argininosuccinate synthase. This data suggests that ADI
toxicity results from an inability to induce argininosuccinate synthase.
Therefore, these cells cannot synthesize arginine from citrulline, and are unable to synthesize the proteins necessary for growth.
Example 5: Circulatisag Half Life Balb C mice (5 in each group) were injected intravenously with a single 5.0 ICT dose of either native arginine deiminase or various formulations of arginine deiminase modified with polyethylene glycol, as indicated in Figures 2A and 2B. To determine the serum levels of arginine and citrulline, the mice were bled from the retro orbital plexus (100 u1). Immediately following collection an equal volume of 50% (w/v) of trichloro-acetic acid was added. The precipitate was removed by centrifugation (13,000 x g for 30 minutes) and the supernatant removed and stored frozen at -70°C. The samples were then analyzed using an automated amino acid analyzer and reagents from Beckman Instruments using protocols supplied by the manufacturer. The limits of sensitivity for arginine by this method was approximately 6 pM and the reproducibility of measurements within about 8%.
A dose dependent decrease in serum arginine levels, as shown by the solid circles in Figure 2A, and a rise in serum citrulline, as shown by the open triangles in Figure 2B, were detected from the single dose administration of native ADI
(filled circles) or ADI-SS-PEG (open triangles). However, the decrease in senun arginine and rise in serum citrulline was short lived, and soon returned to normal. The half life of arginine depletion is summarized in the Table below.
Table 2: Half Life of Serum Arginine Depletion Compound Half Life in Days Native ADI 1 ADI-SS-PEG5,000 5 ADI-SS-PEG12,000 15 ADI-SS-PEG20,000 20 ADI-SS-PEG30,000 22 This experiment demonstrates that normal cells and tissues are able to convert the citrulline baclc into arginine intracellularly while melanomas and hepatomas cannot because they laclc argininosuccinate synthetase.
Example 6: A~atigehicity of PEG modified ADI
To determine the antigenicity of native ADI, ADI-SS-PEG5,000, and ADI-_I8_ SS-PEG20,000, the procedures described in, for example, Park, Ahtica~zcer Res., supra, and Kamisalci, J. Plaarmacol. Exp. Ther., supra, were followed.. Briefly, Balb C mice (5 in each group) were intravenously injected weekly for 12 weeks with approximately O.S IU
(100 ~g of protein) of native ADI, ADI-SS-PEG5,000 or ADI-SS-PEG20,000. The animals were bled (0.05 ml) from the retro orbital plexus at the beginning of the experiment and at weeks 4, 8 and 12. The serum was isolated and stored at -70°C. The titers of anti-ADI IgG were determined by ELISA. 50 ~,g of ADI was added to each well of a 96 well micro-titer plate and was incubated at room temperature for 4 hours. The plates were rinsed with PBS and then coated with bovine serum albumin (1 mg/ml) to bloclc nonspecific protein binding sites, and stored over night at 4°C.
The next day serum from the mice was diluted and added to the wells. After 1 hour the plates were rinsed with PBS and rabbit anti-mouse IgG coupled to peroxidase was added to the wells.
The plates were incubated for 30 min and then the resulting UV absorbance was measured using a micro-titer plate reader. The titer was defined as the highest dilution of the serum which resulted in a two-fold increase from background absorbance (approximately 0.50 OD).
The results are shown in Figure 6. The open circles represent the data obtained from animals inj ected with native ADI, which was very anta.genic.
The filled circles represent the data obtained from the animals injected with ADI-SS-PEG5,000, while the open triangles represent the data obtained from the animals injected with ADI-SS-PEG20,000. As can be seen from Figure 6, ADI-SS-PEG5,000 and ADI-SS-PEG20,000 are significantly less antigenic than native ADI. For example, as few as 4 injections of native ADI resulted in a titer of about 10~, while 4 injections of any of the PEG-ADI formulations failed to produce any measurable antibody. However, after injections, the ADI-PEG5,000 had a titer of about 10z, while ADI-PEG20,000 did not induce this much of an immune response until after 12 injections. The results demonstrate that attaching PEG to ADI blunts the immune response to the protein.
Example 7: Tuyzzor I>zhibitiou of Ilumau Melauonzas The effect of PEG-ADI on the growth of human melanoma (SK-Mel 28) in nude mice was determined. Nude mice (5 in each group) were injected with 10~
SK-mel 2 human melanoma cells which were allowed to grow until the tumors reached a diameter of about 3-5 mm. The mice were left untreated (open circles) or were treated once a week for 8 weeks with 5.0 IU of ADI-SS-PEG5,000 (filled triangles), ADI-SS-PEG12,000 (open triangles) or ADI-SS-PEG20,000 (filled circles). The tumor size was measured weekly, and the mean diameter of the tumors is presented in Figure 7.
Figure 8 shows the effectiveness of ADI-SS-PEG20,000 on three human melanomas (SK-mel 2, SK-mel 28, M24-met) grown ih vivo in nude mice. Nude mice (5 in each group) were injected with 10~ SK-mel 2, SK-mel 28 or M24-met human melanoma cells. The tumors were allowed to grow until they were approximately 3-5 mm in diameter. Thereafter, the animals were injected once a week with 5.01TJ of ADI-SS-PEG20,000. The results are shown in Figure 8, and show that PEG-ADI inhibited tumor growth and that eventually the tumors began to regress and disappear. Because the tumors did not have argininosuccinate synthatase, they were unable to synthesize proteins (because ADI eliminated arginine and the tumors could not make it) so that the cells "starved to death."
Since M24-met human melanoma is highly metastatic, the animals injected with M24-met human melanoma cells were sacrificed after 4 weeks of treatment and the number of metastases in the lungs of the animals was determined. The control animals had an average of 32 metastases, while the animals treated with ADI-SS-PEG20,000 did not have any metastases. The results appear to indicate that ADI-SS-PEG20,000 not only inhibited the growth of the primary melanoma tumor, but also inhibited the formation of metastases.
It is of interest to note that in over 200 animals tested, the average number of metastases in the control group was 49 ~ 18, while only a single metastasis was observed in 1 treated animal.
Example 8: Tumor Iulzibitiozz of Human Hepatoszzas The ability of PEG-ADI to inhibit the growth of a human hepatoma ira vivo was tested. Nude mice (5 in each group) were injected with 10~ human hepatoma SK-Hepl cells. The tumors were allowed to grow for two weeks and then the animals were treated once a weelc with 5.0 IU of SS-PEG5,000-ADI (solid circles), SS-PEG12,000-ADI

(solid triangles) or SS-PEG20,000-ADI (open triangles). The results are set forth in Figure 9. The untreated animals (open circles) all died within 3 weelcs. In contrast, animals treated with ADI had a far longer life expectancy, as can be seen from Figure 9. AlI the surviving mice were euthanized after 6 months, and necropsy indicated that they were free of tumors.
Surprisingly, PEG5,000-ADI is most effective in inhibiting hepatoma growth in vivo. The exact mechaiusm by which this occurs is unknown. Without being bound to any theory of the invention worles, it appears that proteins formulated with SS-PEG5,000-ADI become sequestered in the liver. Larger molecular weights of PEG
do not, which may be due to the uniqueness of the hepatic endothelium and the spaces (fenestrae) being of such a size that larger molecular weights of PEG-ADI conjugates are excluded.
Example 9: Application to Humans PEG5,000-ADI and PEG20,000-ADI were incubated ex vivo with normal human serum and the effects on arginine concentration was determined by amino acid analysis, where the enzyme was found to be fully active and capable of degrading all the detectable arginine with the same kinetics as in the experiments involving mice. The reaction was conducted at a volume of 0.1 ml in a time of 1 hour at 37 ° C.
Additionally, the levels of arginine and citrulline in human serum are identical with that found in mice. PEG-proteins circulate longer in humans than they do in mice.
For example, the circulating half life of PEG conjugated adenosine deiminase, asparaginase, glucocerbrocidase, uricase, hemoglobulin and superoxide dismutase all have a circulating half life that is 5 to 10 times longer than the same formulations in mice.
What this has meant in the past is that the human dose is most often 1/5 to 1/10 of that used in mice.
Accordingly, PEG-ADI should circulate even longer in humans than it does in mice.
Each of the patents, patent applications and publications described herein are hereby incorporated by reference herein in their entirety.
Various modifications of the invention, in addition to those described herein, will be apparent to one skilled in the art in view of the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

SEQUENCE LISTING
<110> Phoenix Pharmacologics, Inc.
<120> Modified Arginine Deiminase <130> PHOE0064 <l40>
<141>
<150> 09/023,809 <151> 1998-02-13 <150> 09/723,546 <151> 2000-11-28 <160> 14 <170> PatentIn Ver. 2.1 <210>1 <211>410 <212>PRT

<213>Mycoplasma arginini <400> 1 Met Ser Val Phe Asp Ser Lys Phe Lys Gly I1e His Val Tyr Ser Glu Ile G1y Glu Leu Glu Ser Val Leu Val His G1u Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Ile Asp Leu Val Ala Glu Thr Tyr Asp Leu Ala Ser G1n Glu Ala Lys Asp Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Val Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Lys Leu Val Glu I1e Met Met Ala G1y Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg Tyr Lys Val Arg G1n Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser Asn His Pro Lys Leu Ile Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Va1 Thr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln Ser Ile I1e Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly G1u Gly Ala Ser G1n Met Glu Ile G1u Arg Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met Pro Leu Ser Arg Lys Asp Val Lys Trp <210> 2 <211> 410 <212> PRT
<213> Mycoplasma arthritidis <400> 2 Met Ser Val Phe Asp Ser Lys Phe Lys G1y Tle His Val Tyr Ser Glu Ile Gly Glu Leu Glu Ser Va1 Leu Val His Glu Pro Gly Arg Glu Tle Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu Gln Ser Gln Phe Val Ala Ile Leu Lys Ala Asn Asp Ile Asn Val Val Glu Thr Ile Asp Leu Va1 Ala Glu Thr Tyr Asp Leu Ala Ser G1n Glu Ala Lys Asp Arg Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Ala His Lys Lys Val Va1 Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Lys Leu Val Glu Leu Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Val Glu Ala 130 l35 140 Asp His Glu Leu I1e Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Phe Met Arg Tyr Lys Val Arg Arg Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Arg Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala Met Lys Leu Ser Tle Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Val Thr Leu Leu Ala Lys Asn Leu Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro I1e Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Lys Leu Leu Gln Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn Tyr Ile Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu Lys Thr Asn Ala Ala Leu Lys Ala Ala G1y Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met Pro Leu Ser Arg Lys Asp Val Lys Trp <210> 3 <211> 409 <212> PRT
<213> Mycoplasma hominis <400> 3 Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg G1u Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala I1e Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys Ile Met Lys Asp Arg Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala Glu Thr Tyr Asp Leu Ala Ser Lys Ala Ala Lys Glu Glu Phe Ile Glu Thr Phe Leu Glu Glu Thr Val Pro Val Leu Thr Glu Ala Asn Lys Lys Ala Va1 Arg Ala Phe Leu Leu Ser Lys Pro Thr His Glu Met Val Glu Phe Met Met Ser Gly Ile Thr Lys Tyr Glu Leu Gly Val Glu Ser G1u Asn Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Phe Met Arg Tyr Ile Val Arg Arg Arg Glu Thr Leu Phe Ala Arg Phe Va1 Phe Arg Asn His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met Lys Met Pro Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu Thr Leu 210 2l5 220 Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly A1a Glu Pro Gln Pro Gln Leu Asn Gly Leu Pro Leu Asp Lys Leu Leu Ala Ser Ile Ile Asn Lys Glu Pro Val Leu Ile Pro Ile Gly Gly A1a Gly Ala Thr Glu Met Glu Ile Ala Arg Glu Thr Asn Phe Asp Gly Thr Asn Tyr Leu Ala Ile Lys Pro Gly Leu Val Ile Gly Tyr Asp Arg Asn Glu Lys Thr Asn Ala Ala Leu Lys Ala Ala Gly Ile Thr Val Leu Pro Phe His G1y Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met Pro Leu Ser Arg Lys Asp Val Lys Trp <210> 4 <211> 23 <212> DNA
<213> Mycoplasma arginini <400> 4 gcaatcgatg tgtatttgac agt 23 <210> 5 <211> 33 <212> DNA

<213> Mycoplasma arginini <400> 5 tgaggatcct tactaccact taacatcttt acg 33 <210> 6 <211> 411 <212> PRT
<213> Steptococcus pyogenes <400> 6 Met Thr Ala Gln Thr Pro Tle His Val Tyr Ser Glu Ile Gly Lys Leu Lys Lys Val Leu Leu His Arg Pro Gly Lys Glu Ile Glu Asn Leu Met Pro Asp Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Asp Ala Gln Lys Glu His Asp Ala Phe Ala Gln Ala Leu Arg Asp G1u G1y Tle Glu Val Leu Tyr Leu Glu Thr Leu Ala Ala G1u Ser Leu Val Thr Pro Glu Ile Arg Glu A1a Phe Ile Asp Glu Tyr Leu Ser Glu Ala Asn Tle Arg Gly Arg Ala Thr Lys Lys Ala Ile Arg Glu Leu Leu Met Ala Tle Glu Asp Asn Gln Glu Leu Ile Glu Lys Thr Met Ala Gly Val 115 l20 l25 Gln Lys Ser G1u Leu Pro Glu Ile Pro A1a Ser Glu Lys Gly Leu Thr l30 135 140 Asp Leu Val Glu Ser Asn Tyr Pro Phe Ala Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile G1y Thr G1y Val Sex Leu Asn His Met Phe Ser Glu Thr Arg Asn Arg Glu Thr Leu Tyr Gly 180 l85 190 Lys Tyr Ile Phe Thr His His Pro Ile Tyr Gly Gly Gly Lys Val Pro Met Val Tyr Asp Arg Asn Glu Thr Thr Arg Ile Glu Gly Gly Asp G1u Leu Val Leu Ser Lys Asp Val Leu Ala Val Gly Ile Ser Gln Arg Thr Asp Ala Ala Ser Ile Glu Lys Leu Leu Val Asn Ile Phe Lys Gln Asn Leu Gly Phe Lys Lys Val Leu Ala Phe Glu Phe Ala Asn Asn Arg Lys Phe Met His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe Thr Ile His Pro Glu Ile Glu Gly Asp Leu Arg Val Tyr Ser Val Thr Tyr Asp Asn G1u Glu Leu His Ile Val Glu Glu Lys Gly Asp Leu Ala Glu Leu Leu A1a Ala Asn Leu Gly Val Glu Lys Val Asp Leu Ile Arg Cys Gly Gly Asp Asn Leu Val Ala Ala Gly Arg G1u Gln Trp Asn Asp Gly Ser Asn Thr Leu Thr Ile Ala Pro Gly Val Va1 Val Val Tyr Asn Arg Asn Thr Tle Thr Asn Ala Ile Leu Glu Ser Lys Gly Leu Lys Leu Ile Lys Ile His Gly Ser G1u Leu Va1 Arg Gly Arg Gly Gly Pro Arg Cys Met Ser Met Pro Phe Glu Arg Glu Asp Ile <210> 7 <211> 409 <2l2> PRT
<213> Steptococcus pneumoniae <400> 7 Met Ser Ser His Pro Ile Gln Val Phe Ser Glu Ile Gly Lys Leu Lys Lys Val Met Leu His Arg Pro Gly Lys Glu Leu Glu Asn Leu Leu Pro Asp Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Asp Ala Gln Lys Glu His Asp Ala Phe Ala Gln Ala Leu Arg Asp Glu Gly Ile Glu Val Leu Tyr Leu Glu G1n Leu Ala Ala Glu Ser Leu Thr Ser Pro Glu Ile Arg Asp Gln Phe Ile Glu Glu Tyr Leu Asp Glu Ala Asn Ile Arg Asp Arg Gln Thr Lys Val Ala Tle Arg Glu Leu Leu His Gly Ile Lys Asp Asn Gln Glu Leu Val Glu Lys Thr Met Ala Gly Ile G1n Lys Val Glu Leu Pro Glu Ile Pro Asp Glu Ala Lys Asp Leu Thr Asp Leu Val Glu Ser Glu Tyr Pro Phe Ala I1e Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile G1y Asn Ala Val Ser Leu Asn His Met Phe A1a Asp Thr Arg Asn Arg Glu Thr Leu Tyr Gly Lys Tyr Ile Phe Lys Tyr His Pro Ile Tyr Gly Gly Lys Val Asp Leu Val Tyr Asn Arg Glu Glu Asp Thr Arg Ile Glu Gly G1y Asp Glu Leu Val Leu Ser Lys Asp Val Leu Ala Va1 Gly Ile Ser Gln Arg Thr Asp Ala A1a Ser Ile Glu Lys Leu Leu Val Asn 2le Phe Lys Lys Asn Val Gly Phe Lys Lys Val Leu Ala Phe Glu Phe Ala Asn Asn Arg Lys Phe Met His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe Thr I1e His Pro Glu Ile Glu Gly Asp Leu His Val Tyr Ser Val Thr Tyr Glu Asn Glu Lys Leu Lys Ile Val Glu Glu Lys Gly Asp Leu Ala Glu Leu Leu Ala Gln Asn Leu Gly Val Glu Lys Val His Leu Ile Arg Cys Gly Gly Gly Asn Ile Va1 Ala Ala Ala Arg Glu Gln Trp Asn Asp Gly Ser Asn Thr Leu Thr Ile Ala Pro Gly Val Val Val Val Tyr Asp Arg Asn Thr Val Thr Asn Lys Ile Leu Glu Glu Tyr Gly Leu Arg Leu Ile Lys Ile Arg Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg Cys Met Ser Met Pro Phe Glu Arg Glu Glu Val <210> 8 <211> 410 <212> PRT
<213> Borrelia burgdorferi <400> 8 Met G1u Glu Glu Tyr Leu Asn Pro Tle Asn I1e Phe Ser Glu Ile Gly Arg Leu Lys Lys Val Leu Leu His Arg Pro Gly Glu Glu Leu Glu Asn Leu Thr Pro Leu Ile Met Lys Asn Phe Leu Phe Asp Asp Ile Pro Tyr Leu Lys Val Ala Arg Gln Glu His Glu Val Phe Val Asn Ile Leu Lys 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 Leu Glu Ala Glu Ile Lys Thr Asp Gly Val Ile Asn Ile Leu Lys Asp 100 l05 110 Tyr Phe Ser Asn Leu Thr Val Asp Asn Met Val Ser Lys Met Ile Ser l15 120 125 Gly Val Ala Arg Glu Glu Leu Lys Asp Cys Glu Phe Ser Leu Asp Asp Trp Val Asn Gly Ser Ser Leu Phe Val Ile Asp Pro Met Pro Asn Val Leu Phe Thr Arg Asp Pro Phe Ala Ser Ile Gly Asn Gly I1e Thr Ile Asn Lys Met Tyr Thr Lys Va1 Arg Arg Arg Glu Thr Ile Phe Ala Glu Tyr Ile Phe Lys Tyr His Ser Ala Tyr Lys Glu Asn Val Pro Ile Trp l95 200 205 Phe Asn Arg Trp Glu Glu Thr Ser Leu Glu Gly Gly Asp Glu Phe Val Leu Asn Lys Asp Leu Leu Val Ile Gly Ile Ser Glu Arg Thr G1u Ala Gly Ser Va1 Glu Lys Leu Ala Ala Ser Leu Phe Lys Asn Lys Ala Pro Phe Ser Thr Ile Leu Ala Phe Lys Ile Pro Lys Asn Arg Ala Tyr Met His Leu Asp Thr Val Phe Thr Gln Ile Asp Tyr Ser Val Phe Thr Ser Phe Thr Ser Asp Asp Met Tyr Phe Ser Ile Tyr Val Leu Thr Tyr Asn Ser Asn Ser Asn Lys Ile Asn Ile Lys Lys Glu Lys Ala Lys Leu Lys Asp Val Leu Ser Phe Tyr Leu Gly Arg Lys Ile Asp Ile Ile Lys Cys Ala Gly Gly Asp Leu Ile His Gly Ala Arg Glu Gln Trp Asn Asp Gly Ala Asn Val Leu Ala Tle Ala Pro Gly Glu Val Ile Ala Tyr Ser Arg Asn His Val Thr Asn Lys Leu Phe Glu Glu Asn Gly Ile Lys Val His Arg Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met Ser Met Ser Leu Va1 Arg Glu Asp Ile <210> 9 <211> 409 <212> PRT
<213> Borrelia afzelii <400> 9 Met Glu Glu Tyr Leu Asn Pro Ile Asn Ile Phe Ser Glu Ile Gly Arg Leu Lys Lys Va1 Leu Leu His Arg Pro Gly Glu Glu Leu Glu Asn Leu Thr Pro Phe Ile Met Lys Asn Phe Leu Phe Asp Asp Ile Pro Tyr Leu Glu Va1 Ala Arg Gln Glu His Glu Val Phe Ala Ser Ile Leu Lys Asn Asn Leu Val Glu Ile Glu Tyr Ile Glu Asp Leu Ile Ser Glu Val Leu Val Ser Ser Val Ala Leu Glu Asn Lys Phe Tle Ser Gln Phe Ile Leu G1u Ala Glu Ile Lys Thr Asp Phe Thr Ile Asn Leu Leu Lys Asp Tyr Phe Ser Ser Leu Thr Ile Asp Asn Met Ile Ser Lys Met Ile Ser Gly Val Val Thr Glu Glu Leu Lys Asn Tyr Thr Ser Ser Leu Asp Asp Leu Val Asn Gly Ala Asn Leu Phe Ile Ile Asp Pro Met Pro Asn Val Leu 145 150 155 l60 Phe Thr Arg Asp Pro Phe Ala Ser Ile Gly Asn Gly Val Thr Ile Asn Lys Met Phe Thr Lys Val Arg Gln Arg Glu Thr Ile Phe Ala Glu Tyr Ile Phe Lys Tyr His Pro Val Tyr Lys Glu Asn Val Pro Ile Trp Leu Asn Arg Trp Glu Glu Ala Ser Leu Glu Gly Gly Asp Glu Leu Val Leu Asn Lys Gly Leu Leu Val Ile Gly Ile Ser Glu Arg Thr Glu Ala Lys Ser Val Glu Lys Leu A1a Ile Ser Leu Phe Lys Asn Lys Thr Ser Phe Asp Thr Ile Leu Ala Phe Gln Tle Pro Lys Asn Arg Ser Tyr Met His Leu Asp Thr Val Phe Thr Gln Ile Asp Tyr Ser Val Phe Thr Ser Phe Thr Ser Asp Asp Met Tyr Phe Ser Ile Tyr Val Leu Thr Tyr Asn Pro Ser Ser Ser Lys Ile His Ile Lys Lys Glu Lys Ala Arg Ile Lys Asp Val Leu Ser Phe Tyr Leu Gly Arg Lys Ile Asp I1e Ile Lys Cys Ala Gly Gly Asp Leu Ile His Gly Ala Arg Glu Gln Trp Asn Asp Gly Ala Asn Va1 Leu Ala Ile Ala Pro G1y Glu Ile Ile Ala Tyr Ser Arg Asn His Val Thr Asn Lys Leu Phe Glu Glu Asn Gly I1e Lys Val His Arg Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp Ile <210> 10 <211> 580 <212> PRT
<213> Qiardia intestinalis <400> 10 Met Thr Asp Phe Ser Lys Asp Lys Glu Lys Leu Ala Gln Ala Thr Gln Gly Gly Glu Asn G1u Arg Ala Glu Ile Val Val Val His Leu Pro Gln Gly Thr Ser Phe Leu Thr Ser Leu Asn Pro Glu Gly Asn Leu Leu Glu Glu Pro Ile Cys Pro Asp Glu Leu Arg Arg Asp His Glu Gly Phe Gln Ala Val Leu Lys Glu Lys G1y Cys Arg Val Tyr Met Pro Tyr Asp Val Leu Ser Glu A1a Ser Pro Ala Glu Arg Glu Val Leu Met Asp Gln Ala Met Ala Ser Leu Lys Tyr Glu Leu His Ala Thr Gly Ala Arg Ile Thr 100 l05 110 Pro hys Met Lys Tyr Cys Val Ser Asp Glu Tyr Lys Arg Lys Val Leu Ser Ala Leu Ser Thr Arg Asn Leu Val Asp Val Ile Leu Ser Glu Pro Val Ile His Leu Ala Pro Gly Va1 Arg Asn Thr Ala Leu Val Thr Asn Ser Val Glu Ile His Asp Ser Asn Asn Met Val Phe Met Arg Asp Gln Gln Ile Thr Thr Arg Arg Gly Ile Val Met Gly Gln Phe Gln Ala Pro Gln Arg Arg Arg Glu Gln Val Leu Ala Leu Ile Phe Trp Lys Arg Leu Gly Ala Arg Val Val Gly Asp Cys Arg Glu Gly Gly Pro His Cys Met Leu Glu Gly Gly Asp Phe Val Pro Val Ser Pro Gly Leu Ala Met Met Gly Val Gly Leu Arg Ser Thr Tyr Val Gly Ala Gln Tyr Leu Met Ser Lys Asp Leu Leu Gly Thr Arg Arg Phe Ala Val Val Lys Asp Cys Phe Asp Gln His Gln Asp Arg Met His Leu Asp Cys Thr Phe Ser Val Leu His Asp Lys Leu Val Val Leu Asp Asp Tyr Ile Cys Ser Gly Met Gly Leu Arg Tyr Val Asp Glu Trp Ile Asp Val Gly Ala Asp Ala Val Lys Lys Ala Lys Ser Ser Ala Va1 Thr Cys Gly Asn Tyr Val Leu Ala Lys A1a Asn Val Glu Phe Gln Gln Trp Leu Ser Glu Asn Gly Tyr Thr Ile Val Arg I1e Pro His Glu Tyr Gln Leu Ala Tyr Gly Cys Asn Asn Leu Asn Leu Gly Asn Asn Cys Val Leu Ser Val His Gln Pro Thr Val Asp Phe Ile Lys Ala Asp Pro Ala Tyr Ile Ser Tyr Cys Lys Ser Asn Asn Leu Pro Asn Gly Leu Asp Leu Val Tyr Val Pro Phe Arg Gly Ile Thr Arg Met Tyr Gly Ser Leu His Cys Ala Ser Gln Val Val Tyr Arg Thr Pro Leu Ala Pro Ala Ala Val Lys Ala Cys Glu Gln Glu Gly Asp Gly Ile Ala Ala Ile Tyr Glu Lys Asn Gly Glu Pro Val Asp Ala Ala Gly Lys Lys Phe Asp Cys Val Ile Tyr Ile Pro Ser Ser Val Asp Asp Leu Ile Asp Gly Leu Lys Ile Asn Leu Arg Asp Asp Ala Ala Pro Ser Arg Glu Ile Ile Ala Asp Ala Tyr Gly Leu Tyr Gln Lys Leu Val Ser Glu Gly Arg Val Pro Tyr Ile Thr Trp Arg Met Pro Ser Met Pro Va1 Val Ser Leu Lys Gly Ala Ala Lys Ala Gly Ser Leu Lys Ala Val Leu Asp Lys Ile Pro Gln Leu Thr Pro Phe Thr Pro Lys Ala Val Glu Gly Ala Pro Ala Ala Tyr Thr Arg Tyr Leu Gly Leu Glu Gln Ala Asp Ile Cys Val Asp I1e Lys <210> 11 <211> 413 <212> PRT
<213> Clostridium perfringens <400> 11 Met Arg Asp Asp Arg Ala Leu Asn Val Thr Ser Glu Ile Gly Arg Leu Lys Thr Val Leu Leu His Arg Pro Gly Glu Glu Ile Glu Asn Leu Thr Pro Asp Leu Leu Asp Arg Leu Leu Phe Asp Asp I1e Pro Tyr Leu Lys Val Ala Arg Glu Glu His Asp Ala Phe Ala Gln Thr Leu Arg Glu Ala Gly Val Glu Val Leu Tyr Leu Glu Val Leu Ala Ala Glu Ala Ile Glu Thr Ser Asp Glu Val Lys Gln Gln Phe Ile Ser Glu Phe Ile Asp Glu Ala Gly Val Glu Ser Glu Arg Leu Lys Glu Ala Leu Ile Glu Tyr Phe Asn Ser Phe Ser Asp Asn Lys Ala Met Val Asp Lys Met Met Ala Gly l15 120 125 Val Arg Lys Glu G1u Leu Lys Asp Tyr His Arg Glu Ser Leu Tyr Asp Gln Val Asn Asn Val Tyr Pro Phe Val Cys Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Glu Pro Phe Ala Thr Ile Gly His Gly Ile Thr Leu Asn His Met Arg Thr Asp Thr Arg Asn Arg Glu Thr Ile Phe A1a Lys Tyr Ile Phe Arg His His Pro Arg Phe G1u Gly Lys Asp Ile Pro Phe Trp Phe Asn Arg Asn Asp Lys Thr Ser Leu Glu Gly Gly Asp Glu Leu Ile Leu Ser Lys Glu Ile Leu Ala Val Gly Ile Ser Gln Arg Thr Asp Ser Ala Ser Val G1u Lys Leu Ala Lys Lys Leu Leu Tyr Tyr Pro Asp Thr Ser Phe Lys Thr Val Leu Ala Phe Lys Ile Pro Val Ser Arg Ala Phe Met His Leu Asp Thr Val Phe Thr Gln Val Asp Tyr Asp Lys Phe Thr Val His Pro Gly Ile Val Gly Pro Leu Glu Val Tyr Ala Leu Thr Lys Asp Pro Glu Asn Asp Gly Gln Leu Leu Val Thr Glu Glu Val Asp Thr Leu Glu Asn Ile Leu Lys Lys Tyr Leu Asp Arg Asp Ile Lys Leu Ile Lys Cys Gly Gly Gly Asp Glu Ile Ile Ala Ala Arg G1u Gln Trp Asn Asp Gly Ser Asn Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Ser Arg Asn Tyr Val Thr Asn Glu Ile Leu Glu Lys Glu Gly Ile Lys Leu His Val Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp Leu <210> 12 <211> 413 <212> PRT
<213> Bacillus licheniformis <400> 12 Met Ile Met Thr Thr Pro I1e His Va1 Tyr Ser Glu Ile Gly Pro Leu Lys Thr Val Met Leu Lys Arg Pro G1y Arg Glu Leu Glu Asn Leu Thr Pro Glu Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Pro Ala Val Gln Lys Glu His Asp Gln Phe Ala Glu Thr Leu Lys Gln Gln Gly Ala Glu Val Leu Tyr Leu Glu Lys Leu Thr Ala Glu Ala Leu Asp Asp Ala Leu Val Arg Glu Gln Phe Ile Asp Glu Leu Leu Thr Glu Ser Lys Ala Asp Ile Asn Gly Ala Tyr Asp Arg Leu Lys Glu Phe Leu Leu Thr Phe Asp Ala Asp Ser Met Val Glu Gln Val Met Ser Gly Ile Arg Lys Asn Glu Leu Glu Arg Glu Lys Lys Ser His Leu His Glu Leu Met Glu Asp His Tyr Pro Phe Tyr Leu Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Ala Ala Ala Ile Gly Ser Gly Leu Thr Ile Asn Lys Met Lys Glu Pro Ala Arg Arg Arg Glu Ser Leu Phe Met Arg Tyr Ile Ile Asn His His Pro Arg Phe Lys Gly His Glu Ile Pro Val Trp Leu Asp Arg Asp Phe Lys Phe Asn Ile Glu Gly Gly Asp Glu Leu Val Leu Asn Glu Glu Thr Val Ala Ile Gly Val Ser Glu Arg Thr Thr Ala Gln Ala Ile Glu Arg Leu Val Arg Asn Leu Phe Gln Arg Gln Ser Arg Ile Arg Arg Val Leu Ala Val Glu Ile Pro Lys Ser Arg Ala Phe Met His Leu Asp Thr Val Phe Thr Met Val Asp Arg Asp Gln Phe Thr 21e His Pro Ala Ile G1n Gly Pro Glu Gly Asp Met Arg Ile Phe Val Leu Glu Arg Gly Lys Thr A1a Asp Glu Ile His Thr Thr Glu Glu His Asn Leu Pro Glu Val Leu Lys Arg Thr Leu Gly Leu Ser Asp Val Asn Leu Ile Phe Cys Gly Gly Gly Asp Glu Ile Ala Ser Ala Arg Glu Gln Trp Asn Asp G1y Ser Asn Thr Leu Ala Ile Ala Pro Gly Val Val Val Thr Tyr Asp Arg Asn Tyr Ile Ser Asn Glu Cys Leu Arg Glu Gln G1y Ile Lys Val Ile Glu Ile Pro Ser Gly Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met Ser Met Pro Leu Tyr Arg Glu Asp Val Lys <210> 13 <211> 408 <212> PRT
<213> Enterococcus faecalis <400> 13 Met Ser His Pro Tle Asn Val Phe Ser Glu Ile Gly Lys Leu Lys Thr Val Met Leu His Arg Pro Gly Lys Glu Leu Glu Asn Leu Met Pro Asp Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Lys Ala Gln Ala Glu His Asp Ala Phe A1a Glu Leu Leu Arg Ser Lys Asp Ile Glu Va1 Val Tyr Leu G1u Asp Leu Ala Ala Glu Ala Leu Ile Asn Glu Glu Val Arg Arg Gln Phe Ile Asp Gln Phe Leu Glu Glu Ala Asn Ile Arg Ser Glu Ser Ala Lys Glu Lys Val Arg Glu Leu Met Leu Glu Ile Asp Asp Asn Glu G1u Leu Tle G1n Lys Ala 21e Ala Gly Ile Gln Lys Gln Glu Leu Pro Lys Tyr Glu Gln Glu Phe Leu Thr Asp Met Val Glu A1a Asp Tyr Pro Phe Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Asn Phe Ala Thr Met Gly His Gly Tle Ser Leu Asn His Met Tyr Ser Val Thr Arg Gln Arg Glu Thr Ile Phe Gly Gln Tyr Ile Phe Asp Tyr His Pro Arg Phe Ala Gly Lys Glu Val Pro Arg Val Tyr Asp l95 200 205 Arg Ser Glu Ser Thr Arg Ile Glu Gly Gly Asp Glu Leu Ile Leu Ser Lys G1u Val Val Ala Ile Gly Ile Ser Gln Arg Thr Asp Ala Ala Ser Ile Glu Lys Ile Ala Arg Asn Ile Phe Glu Gln Lys Leu Gly Phe Lys Asn I1e Leu Ala Phe Asp Ile Gly Glu His Arg Lys Phe Met His Leu Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Ile His Pro Glu I1e Glu Gly Gly Leu Val Val Tyr Ser Ile Thr Glu Lys Ala Asp Gly Asp Ile Gln Ile Thr Lys Glu Lys Asp Thr Leu Asp Asn Ile Leu Cys Lys Tyr Leu His Leu Asp Asn Val Gln Leu Ile Arg Cys Gly Ala Gly Asn Leu Thr Ala Ala Ala Arg Glu Gln Trp Asn Asp Gly Ser Asn Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asp Arg Asn Thr Ile Thr Asn Lys Ala Leu Glu Glu Ala Gly Val Lys Leu Asn Tyr Ile Pro Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg Cys Met Ser Met Pro Leu Tyr Arg Glu Asp Leu <210> 14 <211> 409 <212> PRT
<213> Lactobacillus sake <400> 14 Met Thr Ser Pro Ile His Val Asn Ser Glu Ile Gly Lys Leu Lys Thr Val Leu Leu Lys Arg Pro Gly Lys Glu Val Glu Asn Ile Thr Pro Asp Ile Met Tyr Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu Pro Thr Ile Gln Lys Glu His Asp Gln Phe Ala Gln Thr Leu Arg Asp Asn Gly Val Glu Val Leu Tyr Leu Glu Asn Leu Ala Ala Glu Ala Ile Asp Ala Gly Asp Val Lys Glu Ala Phe Leu Asp Lys Met Leu Asn Glu Ser His Ile Lys Ser Pro Gln Val Gln Ala Ala Leu Lys Asp Tyr Leu Ile Ser Met Ala Thr Leu Asp Met Val Glu Lys Ile Met Ala G1y Val Arg Thr Asn Glu Ile Asp Ile Lys Ser Lys Ala Leu Ile Asp Val Ser Ala Asp Asp Asp Tyr Pro Phe Tyr Met Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Ala Ala Ser Met Gly Asp Gly Leu Thr Ile Asn Lys Met Thr Phe Glu Ala Arg Gln Arg Glu Ser Met Phe Met Glu Val Ile Met Gln His His Pro Arg Phe A1a Asn Gln Gly Ala Gln Val Trp Arg Asp Arg Asp His Ile Asp Arg Met Glu Gly G1y Asp Glu Leu Ile Leu Ser Asp Lys Val Leu Ala Ile Gly Ile Ser Gln Arg Thr Ser Ala Gln Ser Ile Glu Glu Leu Ala Lys Val Leu Phe Ala Asn His Ser Gly Phe Glu Lys Ile Leu Ala Tle Lys Ile Pro His Lys His Ala Met Met His Leu Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Ile His Pro Gly Ile Gln Gly Ala Gly Gly Met Val Asp Thr Tyr Ile Leu Glu Pro Gly Asn Asn Asp Glu I1e Lys Ile Thr His Gln Thr Asp Leu Glu Lys Val Leu Arg Asp Ala Leu Glu Val Pro Glu Leu Thr Leu Ile Pro Cys Gly ' Gly Gly Asp Ala Val Val Ala Pro Arg Glu Gln Trp Asn Asp Gly Ser Asn Thr Leu Ala Ile A1a Pro Gly Val Val Val Thr Tyr Asp Arg Asn Tyr Val Ser Asn Glu Asn Leu Arg Gln Tyr Gly Ile Lys Val Ile Glu Val Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met Ser Met Pro Leu Val Arg Arg Lys Thr

Claims (37)

What is claimed is:

1. A compound comprising arginine deiminase covalently bonded via a linking group to polyethylene glycol, wherein the polyethylene glycol has a total weight average molecular weight of from about 1,000 to about 40,000, and 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, a histidine group and combinations thereof.

2. The compound of claim 1, wherein said linking group is a succinimide group.

3. The compound of claim 2, wherein said succinimide group is succinimidyl succinate, succinimidyl propionate, succinimidyl carboxymethylate, succinimidyl succinamide, N-hydroxy succinimide or combinations thereof.

4. The compound of claim 3, wherein said succinimide group is succinimidyl succinate, succinimidyl propionate or combinations thereof.

5. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Mycoplasma.

6. The compound of claim 5, wherein said microorganism is selected from the group consisting of Mycoplasma arginini, Mycoplasma hominus, Mycoplasma arthritides and combinations thereof.

7. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Steptococcus.

8. The compound of claim 7, wherein said microorganism is selected from the group consisting of Steptococcus pyogenes, Steptococeus pneumoniae and combinations thereof.

9. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Borrelia.

10. The compound of claim 9, wherein said microorganism is selected from the group consisting of Borrelia burgdorferi, Borrelia afzelii, and combinations thereof.

11. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Qiardia.

12. The compound of claim 11, wherein said microorganism is Qiardia intestinalis.

13. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Clostridium.

14. The compound of claim 13, wherein said microorganism is Clostridium perfringens.

15. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Enterococcus.

16. The compound of claim 15, wherein said microorganism is Enterococcus faecalis.

17. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Lactobacillus.

18. The compound of claim 17, wherein said microorganism is Lactobacillus sake.

19. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Bacillus.

20. The compound of claim 19, wherein said microorganism is Bacillus licheniformis.

21. The compound of claim 1, wherein said microorganism is selected from the group consisting of Mycoplasma pneumoniae, Mycoplasma hominus, Mycoplasma arginini, Steptococcus pyogenes, Steptococcus pneumoniae, Borrelia burgdorferi, Borrelia afzelii, Qiardia intestinalis, Clostridium perfingens, Bacillus licheniformis, Enterococcus faecalis, Lactobacillus sake, and combinations thereof.

22. The compound of claim 1, wherein said arginine deiminase is covalently bonded to about 7 to about 15 polyethylene glycol molecules.

23. The compound of claim 22, wherein said arginine deiminase is covalently bonded to about 9 to about 12 polyethylene glycol molecules.

24. The compound of claim 1, wherein said polyethylene glycol has a total weight average molecular weight of from about 10,000 to about 30,000.

25. A method of enhancing the circulating half life of arginine deiminase comprising modifying said arginine deiminase by covalently bonding said arginine deiminase via a linking group to polyethylene glycol, wherein the polyethylene glycol has a total weight average molecular weight of from about 1,000 to about 40,000, and 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, a histidine group and combinations thereof.

26. A method of enhancing the tumoricidal activity of arginine deiminase comprising modifying said arginine deiminase by covalently bonding said arginine deiminase via a linking group to polyethylene glycol, wherein the polyethylene glycol has a total weight average molecular weight of from about 1,000 to about 40,000, and 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, a histidine group and combinations thereof.

27. A method of treating a tumor in a patient comprising administering to said patient the compound of Claim 1.

28. The method of claim 27, wherein said tumor is a melanoma.

29. The method of claim 28, wherein said polyethylene glycol has a total weight average molecular weight of about 20,000

30. The method of claim 28, wherein said linking group is a succinimide group.

31. The method of claim 30, wherein said succinimide group is, succinimidyl succinate, succinimidyl propionate, succinimidyl carboxymethylate, succinimidyl succinamide, N-hydroxy succinimide or combinations thereof.

32. The method of claim 27, wherein said tumor is a hepatoma.

33. The method of claim 32, wherein said polyethylene glycol has a total weight average molecular weight of about 5,000

34. The method of claim 32, wherein said linking group is a succinimide group.

35. The method of claim 34, wherein said succinimide group is succinimidyl succinate, succinimidyl propionate, succinimidyl carboxymethylate, succiiumidyl succinamide, N-hydroxy succinimide or combinations thereof.

36. The method of claim 27, wherein said tumor is a sarcoma.

37. A method of treating and inhibiting metastases in a patient comprising administering to said patient the compound of claim 1.