Arginine Deiminase With Reduced Cross-reactivity Toward Adi - Peg 20 Antibodies For Cancer Treatment

Abstract

The present invention relates generally to isolated to arginine deiminase (ADI) proteins that have reduced cross-reactivity with anti-ADI-PEG 20 antibodies as compared to ADI-PEG 20, but which can have functional characteristics comparable to or better than ADI-PEG 20, compositions comprising the ADI proteins, and related methods of treating arginine-dependent diseases or related diseases such as cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Application No. 61/790,833, filed Mar. 15, 2013, which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is POLA_003_01US_ST25.txt. The text file is about 114 KB, was created on Mar. 14, 2014, and is being submitted electronically via EFS-Web.

BACKGROUND

Technical Field

[0003] The present invention relates generally to arginine deiminase (ADI) proteins, including ADI proteins having reduced cross-reactivity with ADI-PEG 20 antibodies. Such ADI proteins are useful for treating arginine-dependent or related diseases such as cancer.

Description of the Related Art

[0004] Amino acid deprivation therapy can be an effective treatment of some forms of cancer. To date, there is one known clinical example relevant to this approach which utilizes asparaginase to lower circulating levels of asparagine and inhibit protein synthesis. This treatment is particularly effective for acute lymphoblastic leukemia (Avramis 2005, Viera Pinheiro 2004). Acute lymphoblastic leukemia cells require the amino acid asparagine for growth and proliferation. In contrast, most normal human cells are capable of synthesizing asparagine and are unaffected by asparagine depletion. Therefore, decreasing serum asparagine with asparaginase can selectively kill the cancer cells without harming the normal cells, tissues, and host. An E. coli derived form of asparaginase has been approved for human use. However, asparaginase is found only in microbes; which makes it highly immunogenic in humans and also has a short serum half-life following injection (Avramis 2005). To make asparaginase a more effective drug, these drawbacks were minimized by formulating the E. coli derived asparaginase with polyethylene glycol (PEG) to reduce the immunogenicity of this enzyme and the associated allergic reactions. In addition, PEG greatly prolongs the circulating half-life of asparaginase, which reduces both the frequency of treatment and the total cost of the therapy. PEG formulated asparaginase is approved for use and is marketed under the trade name Oncaspar.RTM. (Oncaspar.RTM. 2011, Avramis 2005, Viera Pinheiro 2004, Fu 2007, Zeidan 2008).

[0005] Arginine is another non-essential amino acid for humans and mice (for review see Rogers 1994). In humans, arginine can be synthesized from citrulline in two steps via the Krebs (urea) cycle enzymes argininosuccinate synthetase (ASS, L-citrulline:L-aspartate ligase [AMP-forming], EC 6.3.4.5) and argininosuccinate lyase (ASL, L-argininosuccinate arginine-lyase, EC 4.3.2.) (Haines 2011, Wu 2009, Morris 2006, Husson 2003, Tapiero 2002, Rogers 1994). ASS catalyzes the conversion of citrulline and aspartic acid to argininosuccinate, which is then converted to arginine and fumaric acid by ASL. An arginine deficient diet in humans does not evoke hyperammonemia, orotic aciduria, nor alter the rate of whole body nitric oxide (NO) synthesis in adult humans (Tapiero 2002, Castillo 1995, Rogers 1994, Carey 1987, Barbul 1986, Snyderman 1959, Rose 1949). Although preterm infants appear to require arginine (Wu 2004), arginine levels do not correlate with age among infants, children and young adults (Lucke 2007). In 1992, Takaku and Sugimura separately reported that human melanomas and hepatocellular carcinoma (HCC) cell lines appear to require arginine for growth. Other studies showed that pegylated ADI was effective for the treatment of melanomas and hepatomas with few adverse effects.

[0006] ADI-PEG 20 treatment requires multiple doses over a period of time. After a number of treatments, anti-ADI-PEG 20 antibodies can develop that may limit its continued effectiveness. Therefore, there is a need in the art for ADI that has reduced cross-reactivity to anti-ADI-PEG20 antibodies for use in treatment in order to improve and extend the efficacy of arginine depletion therapy. The present invention provides this and other advantages for the treatment of cancers.

[0007] References: Avramis V I, Panosyan E H. 2005. Clin Pharmacokinet 44:367-393; Barbul A. 1986. J Parenteral Enteral Nutr 10:227-238; Carey G P, et al. 1987. J Nutr 117:1734-1739; Castillo L, et al. 1995. Am J Physiol 268 (Endocrinol Metab 31):E360-367; Fu C H, Sakamoto K M. 2007. Expert Opin Pharmacother 8:1977-1984; Haines R J, et al. 2011. Int J Biochem Mol Biol 2:8-23; Husson A, et al. 2003. Eur J Biochem 270:1887-1899; Lucke T, et al. 2007. Clin Chem Lab Med 45:1525-1530; Morris S M Jr. 2006. Am J Clin Nutr 83(Suppl):5985-5125; Rogers Q R. 1994. 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; Tapiero H, et al. 2002. Biomed Pharmacother 56:439-445, 2002; Viera Pinheiro J P, Boos J. 2004. Br J Haematol 125: 117-127; Wu G, et al. 2009. Amino Acids 37:153-168; Wu G, et al. 2004. J Nutr Biochem 15:442-451; Zeidan A, et al. 2008. Expert Opin Biol Ther 9:111-119).

BRIEF SUMMARY

[0008] One aspect of the present invention provides an isolated arginine deiminase, wherein the isolated arginine deiminase has reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies. Also included are therapeutic or pharmaceutical compositions comprising an isolated arginine deiminase or a fragment thereof having ADI activity, and a pharmaceutically-acceptable carrier. In certain embodiments, the composition is sterile and/or substantially free of pyrogens such as endotoxins. In one embodiment, the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies is not from M. hominis. In another embodiment, the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies is from an organism listed in Table 1. In certain embodiments the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies has one or more properties comparable to or better than those of ADI-PEG 20. In this regard, the one or more properties includes, but is not limited to, Kcat, Km, pH optimum, stability, in vivo proteolytic stability, or no requirement for ions or cofactors that are not already present in blood, or any combination thereof. In one embodiment, the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies, has at least 20 surface residue changes as compared to M. hominis arginine deiminase. In another embodiment, the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies has between 20 and 135 surface residue changes, between 40 and 100 surface residue changes, between 30 and 60 surface residue changes, between 80 and 100 surface residues changes, or between 100 and 120 surface residues changes, as compared to M. hominis arginine deiminase.

[0009] In another embodiment, the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies is from M. arginini, M. arthritidis, M. phocicerebrale, M. gateae, M. phocidae, M. columbinum, M. iowae, M. crocodyli, M. alligatoris, H. orenii, or M. bovis. Illustrative arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies comprise the amino acid sequence set forth in any one of SEQ ID NOs:2-32.

[0010] In another embodiment, the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies has been modified to remove at least one pegylation site. In another embodiment of the arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies, at least one lysine residue has been modified by an amino acid substitution. In this regard, in certain embodiments, at least 5 lysine residues, at least 10 lysine residues, or at least 20 lysine residues have been modified by an amino acid substitution.

[0011] In another embodiment, the arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies is covalently bonded via a linker to a PEG molecule. In this regard, the arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies may be covalently bonded to one or more PEG molecule, such as to about 1 to about 10 or about 2 to about 8 PEG molecules. The PEG molecules may be straight chain or branch chain PEG molecules and may have a total weight average molecular weight of from about 1,000 to about 40,000, or a total weight average molecular weight of from about 10,000 to about 30,000. In those embodiments where the PEG is covalently bonded to the ADIr of the present invention, via a linker, the linker may comprise a succinyl 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, a methylene group, or any combinations thereof. In one embodiment, the source of the succinyl group is succinimidyl succinate.

[0012] Another aspect of the present invention provides a polynucleotide encoding an isolated arginine deiminase described herein, vectors comprising the polynucleotide, and isolated host cells comprising the vectors.

[0013] An additional aspect of the present invention provides a composition comprising the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies as described herein and a physiologically acceptable carrier. In certain embodiments, the compositions further comprise a chemotherapeutic agent. Exemplary themotherapeutic agents include, but are not limited to, docetaxel, carboplatin, cyclophosphamide, gemcitabine, cisplatin, sorafenib, sunitinib, and everolimus.

[0014] Another aspect of the present invention provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies as described herein and a physiologically acceptable carrier, thereby treating, ameliorating the symptoms of, or inhibiting the progression of the cancer. In certain embodiments, the patient in need thereof has been determined to have anti-ADI-PEG 20 antibodies. In another embodiment, the cancer is selected from the group consisting of hepatocellular carcinoma, melanoma including metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, and stomach cancer.

[0015] Another aspect of the invention provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising ADI-PEG 20, and after a period of time, administering to the patient a composition comprising the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies as described herein and a physiologically acceptable carrier, thereby treating, ameliorating the symptoms of, or inhibiting the progression of the cancer. In this regard, the period of time may be determined, for example, by detecting a predetermined level of anti-ADI-PEG 20 antibodies in the patient and/or measuring or otherwise observing ADI activity in the patient, wherein the composition comprising the isolated arginine deiminase having reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies is administered following detection of the predetermined level of said anti-ADI-PEG 20 antibodies and/or measurement or observation of a predetermined level of ADI activity in the patient.

[0016] Also included are isolated arginine deiminase proteins described herein for use in the preparation or manufacture of a medicament for treating, ameliorating the symptoms of, or inhibiting the progression of a cancer.

DETAILED DESCRIPTION

[0017] Embodiments of the present invention relate to selected ADI enzymes, which in some embodiments are engineered to have a small number of surface lysine residues, and conjugated with PEG through a stable linker. The selected ADI enzymes are chosen from a large number of ADI enzymes, from different organisms, based on their beneficial properties. These properties include the ability of the enzyme to establish and maintain low arginine concentrations in human blood through ADI conversion of arginine to citrulline and ammonia. In addition, the selected ADI molecules have reduced cross-reactivity toward anti-ADI-PEG 20 antibodies as compared to ADI-PEG 20, such antibodies possibly resulting from a patient's previous treatment with ADI-PEG 20.

[0018] In certain embodiments, the enzymes in this invention are pegylated to provide protection against renal clearance and proteolysis, as well as reduced immunogenicity or antigenicity. To increase the effectiveness of the pegylation, modifications to the enzymes may be engineered to reduce the number of surface lysine residues and therefore limit the number of available PEG attachment sites. This provides more complete and uniform pegylation at the remaining lysine attachment residues.

[0019] The PEG linker selected to attach methoxy-PEG to ADI is chosen to provide a chemically stable linkage. It is expected this will increase the molecule's bioactive lifetime. A chemically stable linker will also eliminate hydrolysis and reduce an immune response that might occur to a de-pegylated linker attached to the enzyme surface.

[0020] These cumulative specifications result in one or more molecules that effectively remove arginine from a patient's blood and are not neutralized or cleared by anti-ADI-PEG 20 antibodies from previous arginine depletion therapy. The molecules are pegylated so as to delay neutralization and clearance due to their own immunogenicity. These factors will permit their use instead of ADI-PEG 20 or in addition to ADI-PEG 20 (e.g., as a follow-on drug) to extend arginine depletion therapy and therefore increase effectiveness of arginine depletion treatment as an anti-cancer therapeutic.

[0021] Normal cells do not require arginine for growth, since they can synthesize arginine from citrulline in a two step process catalyzed by ASS and ASL. In contrast, certain cancers do not express ASS. Certain cancers do not express ASL, and other cancers may have diminished expression of, or may not express ASS and/or ASL. Therefore, these cancers are auxotrophic for arginine. This metabolic difference may be capitalized upon to develop a safe and effective therapy to treat these forms of cancer. ADI catalyzes the conversion of arginine to citrulline via the arginine dihydrolase pathway, and may thus be used to eliminate arginine.

[0022] The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Protein Science, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

[0023] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

[0024] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0025] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0026] By "statistically significant," it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.

[0027] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0028] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0029] "Patient" or "subject" refers to an animal, in certain embodiments a mammal, and in a specific embodiment, a human.

[0030] "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.

[0031] The term "reference sequence" refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.

[0032] Throughout the present disclosure, the following abbreviations may be used: PEG, polyethylene glycol; ADI, arginine deiminase; SS, succinimidyl succinate; SSA, succinimidyl succinimide; SPA, succinimidyl propionate; NHS, N-hydroxy-succinimide; ASS1 or ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase.

[0033] In the present invention, a polynucleotide encoding ADI may be derived, cloned, isolated, synthesized or produced from any source, including, for example, microorganisms, recombinant biotechnology or any combination thereof. For example, arginine deiminase may be cloned from microorganisms of the genera Mycoplasma, Clostridium, Bacillus, Borrelia, Enterococcus, Streptococcus, Lactobacillus, and/or Giardia. In certain embodiments, arginine deiminase is cloned from Mycoplasma arthritidis, Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma arginini, Steptococcus pyogenes, Steptococcus pneumoniae, Borrelia burgdorferi, Borrelia afzelii, Giardia intestinalis, Clostridium perfringens, Bacillus licheniformis, Enterococcus faecalis, Lactobacillus sake, or any combination thereof. In other embodiments, the arginine deiminase is cloned from a species listed in Table 1. In particular, the ADI used in the present invention may comprise the amino acid sequence of any one of SEQ ID NOs: 1-32, or a variant or fragment or extension thereof having ADI activity (e.g., able to metabolize arginine into citrulline and ammonia). Some of the sequences provided in the sequence listing do not represent full-length ADI protein sequences. Thus, in certain embodiments, additional amino acid residues can be added to either end of the sequences provided herein to make a full-length protein having ADI activity. The specific amino acids to be added can be determined by the skilled person based on alignments of known ADI sequences. Such ADI molecules can be synthesized using known techniques. Illustrative "extended" ADI(r) are provided, for example, in SEQ ID NOs:26-32.

[0034] In certain embodiments, the ADI enzymes as described herein are compared to the benchmark ADI-PEG 20 molecule derived from M. hominis. As used herein, "ADI-PEG 20" refers to the ADI molecule known in the art and described for example in U.S. Pat. Nos. 6,183,738; 6,635,462; Ascierto P A, et al. (2005) Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies. J Clin Oncol 23(30): 7660-7668; Izzo F, et al. (2004) Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I/II studies. J Clin Oncol 22(10): 1815-1822; Holtsberg F W, et al. (2002), Poly(ethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties. J Control Release 80(1-3): 259-271; Kelly et al., (2012) British Journal of Cancer 106, 324-332. As would be recognized by the skilled artisan, this molecule is a pegylated (PEG 20,000) ADI enzyme derived from M. hominis, and has two substitutions (K112E; P210S) relative to the wild type M. hominis ADI enzyme.

[0035] The arginine deiminase enzymes as described herein were screened from a large number of ADI enzymes and have a reduced level of reactivity with anti-ADI-PEG 20 antibodies from patients. Anti-ADI-PEG 20 antibodies can appear in subjects treated with ADI-PEG 20 and can be measured using known methodologies. Reactivity to anti-ADI-PEG 20 antibodies can be determined for example using ELISA or other similar assays known to the skilled artisan.

[0036] In this regard, ADI-PEG 20 can be used as a comparison to assess cross-reactivity level to patient anti-ADI-PEG 20 antibodies. A cross-reactivity level that is statistically significantly lower than that of ADI-PEG 20 to patient anti-ADI-PEG 20 antibodies may be useful herein. In certain embodiments, the arginine deiminase enzymes as described herein have low or no cross-reactivity to anti-ADI-PEG 20 antibodies. In another embodiment, any reduction in reactivity to anti-ADI-PEG 20 antibodies as compared to reactivity with ADI-PEG 20 can be beneficial as such an ADI enzyme would improve treatment options for patients in need of arginine depletion therapy. Thus, the arginine deiminase enzymes as described herein have reduced cross-reactivity to patient anti-ADI-PEG 20 antibodies as compared to ADI-PEG 20 reactivity to such antibodies.

[0037] "ADIr" is used herein to refer to an ADI enzyme of the present invention having reduced cross-reactivity to anti-ADI-PEG 20 antibodies as compared to ADI-PEG 20 reactivity to such antibodies. "ADIr" nomenclature is used to distinguish the molecules identified herein from ADI and ADI-PEG 20 as known in the art.

[0038] The ADIr enzymes of the invention have characteristics or properties comparable to or better than those of ADI-PEG 20, in order to reduce and maintain low blood arginine levels for effective cancer treatment. Such properties include Kcat, Km, pH optimum, stability, in vivo proteolytic stability and lack of requirement for ions or cofactors not already present in the blood, or any combination thereof. In certain embodiments, an ADIr as described herein has properties that are about or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, than comparable properties of ADI-PEG 20. In other embodiments an ADIr described herein has properties that are about or at least about 100%, 105%, 110%, 120%, 140%, 150%, 160%, 180%, 200%, 220%, 240%, 250%, 260%, 280%, 300%, 320%, 340, 350%, 360%, 400%, 420%, 450%, 460%, 500%, 520%, 550% or higher than the specific property of ADI-PEG 20 being compared.

[0039] Thus, in certain embodiments, an ADIr has a Kcat that is about or at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the Kcat of ADI-PEG 20, or better. In certain embodiments, an ADIr has a Kcat that is about or at least about 100%, 105%, 110%, 120%, 125%, 140%, 150%, 160%, 180%, 200%, 220%, 240%, 250%, 260%, 280%, 300%, 320%, 340, 350%, 360%, 400%, 420%, 450%, 460%, 500%, 520%, 550% or higher, times that of the ADI-PEG 20 Kcat. In certain embodiments, the Kcat of the ADIr enzymes described herein, or compositions comprising same, is about 0.5 sec.sup.-1 to about 15 sec.sup.-1, and in a further embodiment, is from about 1 sec.sup.-1 to about 12 sec.sup.-1, about 1 sec.sup.-1 to about 10 sec.sup.-1, about 1.5 sec.sup.-1 to about 9 sec.sup.-1, about 2 sec.sup.-1 to about 8 sec.sup.-1 or about 2.5 sec.sup.-1 to about 7 sec.sup.-1. In certain embodiments, the ADIr or ADIr-PEG in a composition has a Kcat of about 2.5 sec.sup.-1 to about 7.5 sec.sup.-1. In some embodiments, the ADIr or ADIr-PEG in a composition has a Kcat of about 2.5 sec.sup.-1, about 3 sec.sup.-1, about 3.5 sec.sup.-1, about 4 sec.sup.-1, about 4.5 sec.sup.-1, about 5 sec.sup.-1, about 5.5 sec.sup.-1, about 6 sec.sup.-1, about 6.5 sec.sup.-1, about 7 sec.sup.-1, about 7.2 sec.sup.-1, about 7.5 sec.sup.-1, about 8 sec.sup.-1, about 10 sec.sup.-1, about 15 sec.sup.-1, about 20 sec.sup.-1, about 25 sec.sup.-1, about 30 sec.sup.-1, about 35 sec.sup.-1, about 40 sec.sup.-1, about 45 sec.sup.-1, about 50 sec.sup.-1, about 55 sec.sup.-1, about 60 sec.sup.-1, about 65 sec.sup.-1, about 70 sec.sup.-1, about 75 sec.sup.-1, about 80 sec.sup.-1, about 85 sec.sup.-1, about 90 sec.sup.-1, about 95 sec.sup.-1, or about 100 sec.sup.-1.

[0040] Thus, in certain embodiments, an ADIr has a Km that is about or at least about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the Km of ADI-PEG 20, or better. Thus, in certain embodiments, an ADIr has a Km that is about or at least about 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 180%, 200%, 220%, 240%, or 250% that of the Km of ADI-PEG 20. In one embodiment, an ADIr, or a pegylated formulation thereof, has a Km of from about 0.5 .mu.M to about 50 .mu.M, or about 1.6 .mu.M to about 48 .mu.M, or about 0.5 .mu.M to about 15 .mu.M, and in a further embodiment, is from about 1 .mu.M to about 12 .mu.M, about 1 .mu.M to about 10 .mu.M, about 1.5 .mu.M to about 9 .mu.M, about 1.5 .mu.M to about 8 .mu.M or about 1.5 .mu.M to about 7 .mu.M. In certain embodiments, the ADIr or ADIr-PEG in a composition has a Km of about 1.5 .mu.M to about 6.5 .mu.M. In some embodiments, the ADIr or pegylated formulation thereof has a Km of about 1.5 .mu.M, about 1.6 .mu.M, about 2 .mu.M, about 2.5 .mu.M, about 3 .mu.M, about 3.5 .mu.M, about 4 .mu.M, about 4.5 .mu.M, about 5 .mu.M, about 5.5 .mu.M, about 6 .mu.M, about 6.5 .mu.M, about 7 .mu.M, about 8 .mu.M, about 9 .mu.M, about 10 .mu.M, about 12 .mu.M, about 14 .mu.M, about 15 .mu.M, about 16 .mu.M, about 18 .mu.M about 20 .mu.M, about 22 .mu.M, about 24 .mu.M, about 25 .mu.M, about 26 .mu.M, about 28 .mu.M, about 30 .mu.M, about 32 .mu.M, about 34 .mu.M, about 35 .mu.M, about 36 .mu.M, about 38 .mu.M, about 40 .mu.M, about 42 .mu.M, about 44 .mu.M, about 45 .mu.M, about 46 .mu.M, about 48 .mu.M, or about 50 .mu.M.

[0041] In certain embodiments, an ADIr functions at a pH close to the physiological pH of human blood. Thus, in one embodiment, an ADIr functions at a pH of about 4 to about 10.8, or about 6 to about 8, or about 6.5 to about 7.5. In one embodiment, an ADIr has good enzyme activity at about pH 7.4.

[0042] In certain embodiments, an ADIr has stability during long term storage and temperature and proteolytic stability during treatment in the human body. In further embodiments, an ADIr does not require ions or cofactors for activity that are not already present in blood.

[0043] In certain embodiments, an ADIr described herein generally has an amino acid sequence sufficiently different from M. hominis so that there are surface residue changes which will reduce or eliminate antigenic sites for anti-ADI-PEG 20 antibodies. In one embodiment, there will be no cross reactivity between the selected ADIr molecule and existing anti-ADI-PEG 20 antibodies in a subject, and a completely new immune response will be generated in a subject rather than a maturation of the existing response to M. hominis ADI. Thus, in one embodiment, an ADIr as described herein has from 20%-85% sequence identity to M. hominis ADI as set forth in SEQ ID NO:1. In certain embodiments, an ADIr as described herein has even lower percent sequence identity to M. hominis ADI, such as 10% or 15% identity. In another embodiment, an ADIr as described herein has 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% or even 83% identity to M. hominis ADI, and still has reduced cross-reactivity toward anti-ADI-PEG 20 antibodies.

[0044] In one embodiment, an ADIr as described herein has from about 25-140 surface residue changes as compared to M. hominis ADI. Surface residues can be identified from the crystal structure of M. hominis ADI and surface residues for ADI from other organisms can be determined by sequence homology. An ADIr as described herein may have about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or about 140 surface residue changes as compared to M. hominis ADI (see SEQ ID NO:1).

[0045] In another embodiment, an ADIr as described herein has from about 25-140 residue changes as compared to M. hominis ADI. Such residue changes need not only be of surface amino acid residues. Such residue changes (or additions or deletions) can be at either end of the molecule or may be at any residue of the ADI, such that the modified ADI has the desired ADI activity as described herein. Residues to be changed can be identified from the crystal structure of M. hominis ADI and residues for ADI from other organisms can be determined by sequence homology. An ADIr as described herein may have about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or about 140 amino acid residue changes as compared to M. hominis ADI (see SEQ ID NO:1).

[0046] From a large number of ADI enzymes, Table 1 lists 24 ADIr enzymes with their sequence percent identity relative to M. hominis ADI. From the literature, M. hominis, M. arginini, and M. arthritidis ADI amino acid sequences are closely related and these enzymes have good catalytic properties. More recently, additional ADI enzymes have been discovered that have sequences closely related to these three. More distantly related Mycoplasma ADI enzymes have been identified, although less is known about them. And even more distantly related ADI enzymes from bacterial and other sources exist.

[0047] In certain embodiments, the ADIr enzymes identified herein from a number of selected species, have surface lysine residues (in certain embodiments, up to 30 or more). However, in certain embodiments an ADIr enzyme may have many fewer surface lysine residues, such as just 2 lysine residues as in the case of Mycobacterium bovis ADI, or even no lysine residues (see e.g., ADI from Mycobacterium sp. MCS; GenBank No. ABG10381). Therefore, the ADIr enzymes identified herein that have reduced cross-reactivity with anti-ADI-PEG 20 antibodies, have about 0, 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or more surface lysine residues.

[0048] The terms "polypeptide," "protein" and "peptide" are used interchangeably and mean a polymer of amino acids not limited to any particular length. The terms do not exclude modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms "polypeptide" or "protein" means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass the ADIr proteins of the present disclosure, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of the ADIr proteins. In certain embodiments, the polypeptide is a "recombinant" polypeptide, produced by recombinant cell that comprises one or more recombinant DNA molecules, which are typically made of of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell.

[0049] The term "isolated protein" referred to herein means that a subject protein (1) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a protein with which the "isolated protein" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0050] The term "variant" includes a polypeptide that differs from a reference polypeptide specifically disclosed herein (e.g., SEQ ID NOS:1-32) by one or more substitutions, deletions, additions and/or insertions. Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.

[0051] In many instances, a biologically active variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.

[0052] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility.

[0053] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within .+-.2 is preferred, those within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

[0054] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within .+-.2 is preferred, those within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

[0055] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0056] Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0057] A variant may also, or alternatively, contain non-conservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide.

[0058] In general, variants will display about or at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to a reference polypeptide sequence (e.g., SEQ ID NOS:1-32). Moreover, sequences differing from the native or parent sequences by the addition (e.g., C-terminal addition, N-terminal addition, both), deletion, truncation, insertion, or substitution of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids but which retain the properties or activities of a parent or reference polypeptide sequence are contemplated.

[0059] In some embodiments, variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other embodiments, variant polypeptides differ from a reference sequence by about or at least 0.5% or 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.).

[0060] The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion or substitution of a naturally-occurring or recombinantly-produced polypeptide. In certain embodiments, a polypeptide fragment can comprise an amino acid chain at least 5 to about 400 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, or 400 amino acids long. Particularly useful polypeptide fragments include functional domains, including the catalytic ADI domains of the ADIr described herein. In the case of an ADIr, useful fragments include, but are not limited to, the catalytic domain and the .alpha.-helical domain.

[0061] Many activated PEGs used for conjugation to ADI covalently bond to lysine residues. There are usually many fewer PEG molecules attached to ADI than there are lysine residues. Both the number and distribution of attachments can be heterogeneous from molecule to molecule. Any particular lysine residue will be modified in only a small fraction of the ADI molecules. This site modification heterogeneity and low PEG occupancy can result in problems with both drug characterization and the effectiveness of PEG shielding at antigenic sites. Therefore, in certain embodiments, the selected ADIr enzymes as described herein, are modified by lysine replacement with other residue types to reduce the number of lysine residues. This produces a more uniformly pegylated protein and increases the PEG occupancy at the remaining lysine residues. Specific lysine residues chosen to be changed to other residues will be selected in order to preserve enzyme activity. This more uniform pegylation is expected to provide increased protection against proteolysis in blood and increased shielding of antigenic sites from patient antibodies.

[0062] In certain embodiments, the ADIr of the present disclosure may be modified as described in U.S. Pat. No. 6,635,462. In particular, modifications of one or more of the naturally occurring amino acid residues of an ADIr can provide for an enzyme that is more easily renatured and formulated thereby improving the manufacture of ADIr and therapeutic compositions comprising the same. In one embodiment, the ADIr of the present disclosure is modified to remove one or more lysine residues (e.g., the lysine can be substituted with another amino acid or analogues thereof, or a non-natural amino acid). In particular, in one embodiment, the ADIr is modified to be free of the lysine at a position equivalent to 112, 374, 405 or 408 of SEQ ID NO:1 (M. hominis ADI), or a combination of one or more of these positions. In a further embodiment, the ADIr is modified to be free of one or more lysines, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more lysine residues, should they be present, can be substituted with another amino acid or analogues thereof, or a nonnatural amino acid. In one embodiment, an ADIr has 5 lysines substituted, for example, at an equivalent position to position 7, 88, 137, 209, and 380 of SEQ ID NO: 1. In another embodiment, an ADIr has 10 lysines substituted, for example, at positions equivalent to positions 7, 9, 59, 88, 115, 116, 137, 178, 209, and 380 of SEQ ID NO: 1. In yet another embodiment, an ADIr has 15 lysines substituted, for example, at positions equivalent to positions 7, 9, 59, 66, 88, 91, 93, 115, 116, 137, 141, 178, 209, 279, and at position 380 of SEQ ID NO: 1. In one embodiment, an ADIr comprises 21 lysines substituted, for example, at positions equivalent to positions 7, 9, 56, 59, 66, 88, 91, 93, 96, 115, 116, 137, 141, 178, 209, 254, 279, 325, 326, 380, and 406 of SEQ ID NO: 1.

[0063] A native ADIr may be found in microorganisms and is immunogenic and rapidly cleared from circulation in a patient. These problems may be overcome by modifying an ADIr. Thus, the present disclosure provides ADIr modified by a modifying agent, including, but not limited to macromolecule polymers, proteins, peptides, polysaccharides, or other compounds. Arginine deiminase as described herein and the modifying agent may be linked by either covalent bonds or non-covalent interaction to form a stable conjugate or a stable composition to achieve a desired effect. In certain embodiments, the modified ADIr retains the biological activity of an unmodified ADIr and has a longer half life in vivo and lower antigenicity than the unmodified, ADIr. In certain embodiments, the modified ADIr retains at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the biological activity of unmodified ADIr. Generally, the modified ADIr retains biological activity sufficient for therapeutic use.

[0064] In one embodiment, a modifying agent can be a polymer or a protein or a fragment thereof that is biocompatible and can increase the half life of ADIr in blood. The modifying agent can be either chemically coupled to ADIr or where applicable, linked to the ADIr via fusion protein expression.

[0065] Macromolecule polymers may include a non-peptide macromolecule polymer, which in certain embodiments, may have its own bioactivity. Suitable polymers include, but are not limited to, polyenol compounds, polyether compounds, polyvinylpyrrolidone, poly amino acids, copolymer of divinyl ether and maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, polysaccharide, polyoxyethylated polyol, heparin or its fragment, poly-alkyl-ethylene glycol and its derivatives, copolymers of poly-alkyl-ethylene glycol and its derivatives, poly(vinyl ethyl ether), a,P-Poly[(2-hydroxyethyl)-DL-aspartamide], polycarboxylates, poly oxyethylene-oxymethylenes, polyacryloyl morpholines, copolymer of amino compounds and oxyolefin, poly hyaluronic acid, polyoxiranes, copolymer of ethanedioic acid and malonic acid, poly (1,3-dioxolane), ethylene and maleic hydrazide copolymer, poly sialic acid, cyclodextrin, etc. In certain embodiments, the polymer is polyethylene glycol.

[0066] The polyenol compounds as used herein include, but are not limited to, polyethylene glycol (including monomethoxy polyethylene glycol, monohydroxyl polyethylene glycol), polyvinyl alcohol, polyallyl alcohol, polybutenol and the like, and their derivatives, such as lipids.

[0067] The polyether compounds include, but are not limited to poly alkylene glycol (HO((CH2).sub.xO).sub.nH), polypropylene glycol, polyoxyrehylene (HO((CH.sub.2).sub.2O).sub.nH), polyvinyl alcohol ((CH.sub.2CHOH).sub.n).

[0068] Poly amino acids include, but are not limited to, polymers of one type of amino acid or copolymers of two or more types of amino acids, for example, polyalanine or polylysine, or block co-polymers thereof.

[0069] Polysaccharides include but are not limited to, glucosan and its derivatives, for example dextran sulfate, cellulose and its derivatives (including methyl cellulose and carboxymethyl cellulose), starch and its derivatives, polysucrose, etc.

[0070] In one specific embodiment of the present invention, ADIr is modified by coupling with proteins or peptides, wherein one or more proteins or peptides are directly or indirectly linked to ADIr. The proteins can either be naturally existing proteins or their fragments, including but not limited to naturally existing human serum proteins or their fragments, such as thyroxine-binding protein, transthyretin, a1-acid glycoprotein, transferrin, fibrinogen, immunoglobulin, Ig Fc regions, albumin, and fragments thereof. By "fragment" is meant any portion of a protein that is smaller than the whole protein but which retains the desired function of the protein. The ADIr as described herein may be directly or indirectly linked to a protein via a covalent bond. Direct linking means that one amino acid of ADIr is directly linked to one amino acid of the modifying protein, via a peptide bond or a disulfide bridge. Indirect linking refers to the linkages between a ADIr and a modifying protein, via originally existing chemical groups therebetween or specific chemical groups added through biological or chemical means, or the combination of the above-mentioned linkages.

[0071] In one particular embodiment, ADIr is modified by covalent attachment with PEG. ADIr covalently modified with PEG (with or without a linker) may be hereinafter referred to as "ADIr-PEG." When compared to unmodified ADIr, ADIr-PEG retains most of its enzymatic activity, is far less immunogenic or antigenic, has a greatly extended circulating half-life, and is much more efficacious in the treatment of tumors.

[0072] "Polyethylene glycol" or "PEG" refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH.sub.2CH.sub.2)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, PEG5,000 refers to PEG having a total weight average molecular weight of about 5,000; PEG12,000 refers to PEG having a total weight average molecular weight of about 12,000; and PEG20,000 refers to PEG having a total weight average molecular weight of about 20,000.

[0073] In one embodiment of the present invention, the PEG has a total weight average molecular weight of about 1,000 to about 50,000; in one embodiment from about 3,000 to about 40,000, and in another embodiment from about 5,000 to about 30,000; in certain embodiments from about 8,000 to about 30,000; in other embodiments from about 11,000 to about 30,000; in additional embodiments, from about 12,000 to about 28,000; in still other embodiments, from about 16,000 to about 24,000; and in other embodiments, about 18,000 to about 22,000; in another embodiment, from 19,000 to about 21,000, and in one embodiment, the PEG has a total weight average molecular weight of about 20,000. Generally, PEG with a molecular weight of 30,000 or more is difficult to dissolve, and yields of the formulated product may be reduced. The PEG may be a branched or straight chain. Generally, increasing the molecular weight of the PEG decreases the immunogenicity of the ADIr. The PEG having a molecular weight described in this embodiment may be used in conjunction with ADIr, and, optionally, a biocompatible linker, to treat cancer, including, for example, acute myeloid leukemia, such as relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, stomach cancer and esophageal cancer.

[0074] In another embodiment of the present invention, the PEG has a total weight average molecular weight of about 1,000 to about 50,000; in certain embodiments about 3,000 to about 30,000; in other embodiments from about 3,000 to about 20,000; in one embodiment from about 4,000 to about 12,000; in still other embodiments from about 4,000 to about 10,000; in additional embodiments from about 4,000 to about 8,000; still further embodiments from about 4,000 to about 6,000; and about 5,000 in another embodiment. The PEG may be a branched or straight chain, and in certain embodiments is a straight chain. The PEG having a molecular weight described in this embodiment may be used in conjunction with ADIr, and optionally, a biocompatible linker, to treat graft versus host disease (GVHD) or cancer.

[0075] While ADIr-PEG is the illustrative modified ADIr described herein, as would be recognized by the skilled person ADIr may be modified with other polymers or appropriate molecules for the desired effect, in particular reducing antigenicity and increasing serum half-life.

[0076] ADIr may be covalently bonded to a modifying agent, such as PEG, with or without a linker, although a preferred embodiment utilizes a linker.

[0077] The linker used to covalently attach ADIr to a modifying agent, e.g. PEG, may be any biocompatible linker. 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. A modifying agent, such as PEG, can be bonded to the linker, for example, via an ether bond, a thiol bond, or an amide bond. The linker group includes, for example, a succinyl 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, a methylene group, and combinations thereof. In one embodiment, the source of the biocompatible linker is succinimidyl succinate (SS). Other suitable sources of linker may include an oxycarbonylimidazole group (including, for example, carbonylimidazole (CDI), a nitro phenyl group (including, for example, nitrophenyl carbonate (NCP) or trichlorophenyl carbonate (TCP)), a trysylate group, an aldehyde group, an isocyanate group, a vinylsulfone group, or a primary amine. In another embodiment, the linker is derived from SS, SPA, SCM, or NHS; in certain embodiments, SS, SPA, or NHS are used, and in other embodiments, SS or SPA are used. Thus, in certain embodiments, potential linkers can be formed from methoxy-PEG succinimidyl succinate (SS), methoxy-PEG succinimidyl glutarate (SG), methoxy-PEG succinimidyl carbonate (SC), methoxy-PEG succinimidyl carboxymethyl ester (SCM), methoxy-PEG2 N-hydroxy succinimide (NHS), methoxy-PEG succinimidyl butanoate (SBA), methoxy-PEG succinimidyl propionate (SPA), methoxy-PEG succinimidyl glutaramide, and methoxy-PEG succinimidyl succinamide.

[0078] Alternatively, ADIr may be coupled directly to a modifying agent, such as PEG (i.e., without a linker) through an amino group, a sulfhydryl group, a hydroxyl group or a carboxyl group.

[0079] ADIr may be covalently bonded to PEG, via a biocompatible linker, 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.

[0080] The attachment of PEG to ADIr increases the circulating half-life of ADIr. Generally, PEG is attached to a primary amine of ADIr. Selection of the attachment site of PEG, or other modifying agent, on the ADIr 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 ADIr without substantial loss of enzymatic activity. For example, the lysine residues present in ADIr are all possible points at which ADIr as described herein can be attached to PEG via a biocompatible linker, such as SS, SPA, SCM, SSA and/or NHS. PEG may also be attached to other sites on ADIr, as would be apparent to one skilled in the art in view of the present disclosure.

[0081] From 1 to about 30 PEG molecules may be covalently bonded to ADIr. In certain embodiments, ADIr is modified with one PEG molecule. In other embodiments, ADIr is modified with more than one PEG molecule. In one embodiment, ADIr is modified with about 1 to about 10, or from about 7 to about 15 PEG molecules, and in one embodiment from about 2 to about 8 or about 9 to about 12 PEG molecules. In another embodiment, the ADIr is modified with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG molecules. In one specific embodiment, ADIr is modified with 4.5-5.5 PEG molecules per ADIr. In another embodiment, ADIr is modified with 5.+-.1.5 PEG molecules.

[0082] In another embodiment, about 15% to about 70% of the primary amino groups in ADIr are modified with PEG, in one embodiment about 20% to about 65%, about 25% to about 60%, or in certain embodiments about 30% to about 55%, or 45% to about 50%, and in other embodiments about 50% of the primary amino groups in arginine deiminase are modified with PEG. When PEG is covalently bonded to the end terminus of ADIr, it may be desirable to have only 1 PEG molecule utilized. Increasing the number of PEG units on ADIr increases the circulating half life of the enzyme. However, increasing the number of PEG units on ADIr decreases the specific activity of the enzyme. Thus, 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.

[0083] In the present invention, a common feature of biocompatible linkers is that they attach to a primary amine of arginine deiminase via a succinimide group. Once coupled with ADIr, SS-PEG has an ester linkage next to the PEG, which may render this site sensitive to serum esterase, which may release PEG from ADIr in the body. SPA-PEG and PEG2-NHS do not have an ester linkage, so they are not sensitive to serum esterase.

[0084] In certain embodiments, a biocompatible linker is used in the present invention. PEG which is attached to the protein may be either a straight chain, as with SS-PEG, SPA-PEG and SC-PEG, or a branched chain of PEG may be used, as with PEG2-NHS.

[0085] In certain embodiments, pegylation sites associated with ADIr located at or adjacent to the catalytic region of the enzyme are modified. For purposes of the present invention, the phrase "pegylation site" may be defined as any site or position of ADI or a ADIr that may be covalently modified with polyethylene glycol. A "pegylation site" can be considered located at or adjacent the catalytic region of the enzyme where pegylation of the site results in a significant reduction in catalytic activity of the enzyme. The pegylation of such sites has traditionally resulted in the inactivation of the enzyme. For example, ADI from Mycoplasma hominis has a lysine at the 112 position which can be considered to be at or adjacent the catalytic region of the enzyme. The attachment of PEG to this lysine at the 112 position can inactivate the enzyme. In addition, ADI from Mycoplasma hominis has a cysteine at the 397 position which can be considered to be at or adjacent the catalytic region of the enzyme. The amino acid substitutions for cysteine at the 397 position can inactivate the enzyme. In particular, substituting alanine, histidine, arginine, serine, lysine or tyrosine for cysteine at the 397 position can result in a loss of all detectable enzyme activity. ADI from Mycoplasma hominis also has three lysines located near this conserved cysteine, in particular Lys374, Lys405 and Lys408. The attachment of PEG to Lys374, Lys405, Lys408 or combinations thereof can inactivate the enzyme.

[0086] It is to be understood that ADIr derived from other organisms may also have pegylation sites corresponding to 112 position of ADI from Mycoplasma hominis. In addition, ADI from some organisms may have lysines corresponding to the same general location as the 112 position of ADI from Mycoplasma hominis. The location of lysine in ADI from such organisms are known to the skilled person and are described in U.S. Pat. No. 6,635,462.

[0087] Thus, in one embodiment, the present invention provides for certain amino acid substitutions in the polypeptide chain of ADIr. These amino acid substitutions provide for modified ADIr that loses less activity when modified by a modifying agent, e.g., upon pegylation. By eliminating pegylation sites, or other known modification sites, at or adjacent to the catalytic region of enzyme, optimal modification, e.g., pegylation, can be achieved without the loss of activity.

[0088] It is to be understood that other embodiments of the invention are based on the understanding that certain structural characteristics of arginine deiminase may prevent or interfere with the proper and rapid renaturation when produced via recombinant technology. In particular, these structural characteristics hinder or prevent the enzyme from assuming an active conformation during recombinant production. For purposes of the present invention, the phrase "active conformation" may be defined as a three-dimensional structure that allows for enzymatic activity by unmodified or modified arginine deiminase. The active conformation may, in particular, be necessary for catalyzing the conversion of arginine into citrulline. The phrase "structural characteristic" may be defined as any trait, quality or property of the polypeptide chain resulting from a particular amino acid or combination of amino acids. For instance, arginine deiminase may contain an amino acid that results in a bend or kink in the normal peptide chain and thus hinders the enzyme from assuming an active conformation during renaturation of the enzyme. In particular, arginine deiminase from Mycoplasma hominis has a proline at the 210 position that may result in a bend or kink in the peptide chain, making it more difficult to renature the enzyme during recombinant production. It is to be understood that arginine deiminase derived from other organisms may also have sites corresponding to the 210 position of arginine deiminase from Mycoplasma hominis.

[0089] The present invention thus again provides for certain amino acid substitutions in the polypeptide chain of wild type arginine deiminases. 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 deiminases using reduced amounts of buffer. These amino acid substitutions may also provide for increased yields of renatured modified arginine deiminase. In one embodiment of the invention, the modified arginine deiminase has an amino acid substitution at P210 or the equivalent residue. As mentioned above, arginine deiminase derived from Mycoplasma hominis has the amino acid proline located at the 210 position. While not limiting the present invention, it is presently believed that the presence of the amino acid proline at position 210 results in a bend or kink in the normal polypeptide chain that increases the difficulty of renaturing (i.e., refolding) arginine deiminase. Substitutions for proline at position 210 make possible the rapid renaturation of modified arginine deiminase using reduced amounts of buffer. Substitutions for proline at position 210 may also provide for increased yields of renatured modified arginine deiminase. In one embodiment, the proline at position 210 is substituted with serine. It is to be understood that in accordance with this aspect of the invention, other substitutions at position 210 may be made. Examples of other substitutions include Pro210 to Thr210, Pro210 to Arg210, Pro210 to Asn210, Pro210 to Gln210 or Pro210 to Met210. 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.

[0090] The methods of the present invention can involve either in vitro or in vivo applications. In the case of in vitro applications, including cell culture applications, the compounds described herein can be added to the cells in cultures and then incubated. The compounds of the present invention may also be used to facilitate the production of monoclonal and/or polyclonal antibodies, using antibody production techniques well known in the art. The monoclonal and/or polyclonal antibodies can then be used in a wide variety of diagnostic applications, as would be apparent to one skilled in the art.

[0091] The in vivo means of administration of the compounds of the present invention will vary depending upon the intended application. Administration of the ADIr compositions described herein, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions can be prepared by combining ADIr, e.g., ADIr-PEG, ADIr-PEG 20, with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmaceutically active ingredients (including other anti-cancer agents as described elsewhere herein) and/or suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition. Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical. Modes of administration depend upon the nature of the condition to be treated or prevented. Thus, ADIr-PEG, e.g., ADIr-PEG 20, may be administered orally, intranasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorly, intramuscularly, interstitially, intra-arterially, subcutaneously, intraocularly, intrasynovial, transepithelial, and transdermally. An amount that, following administration, reduces, inhibits, prevents or delays the progression and/or metastasis of a cancer is considered effective. In certain embodiment, the ADIr compositions herein increase median survival time of patients by a statistically significant amount. In one embodiment, the ADIr treatments described herein increase median survival time of a patient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certain embodiments, ADIr treatments increase median survival time of a patient by 1 year, 2 years, 3 years, or longer. In one embodiment, the ADIr treatments described herein increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. In certain embodiments, the ADIr treatments described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.

[0092] In certain embodiments, the amount administered is sufficient to result in tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 10%, 20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered (e.g., decreased with statistical significance) scan dimensions. In certain embodiments, the amount administered is sufficient to result in stable disease. In other embodiments, the amount administered is sufficient to result in clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician.

[0093] In certain embodiments the amount administered is sufficient to inhibit NO synthesis, inhibit angiogenesis, and or is sufficient to induce apoptosis in tumor cells or any combination thereof. NO synthesis, angiogenesis and apoptosis may be measured using methods known in the art, see, e.g., Current Protocols in Immunology or Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. (2009 and updates thereto); Ausubel et al., Short Protocols in Molecular Biology, 3.sup.rd ed., Wiley & Sons, 1995; and other like references. In one particular embodiment the amount administered inhibits NO synthesis and inhibits the growth of melanoma and complements, adds to, or synergizes with other chemotherapies as described herein, such as cisplatin. Accordingly, one embodiment of the present disclosure provides a method of treating melanoma by administering ADIr-PEG 20 in combination with cisplatin, wherein the treatment depletes endogenous nitric oxide (NO).

[0094] The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.

[0095] The ADIr compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions may also be administered in combination with antibiotics.

[0096] The ADIr compositions may also be administered alone or in combination with ADI-PEG 20 therapy. In certain embodiments, the ADIr as described herein are used in patients who have been treated with ADI-PEG 20 and who have developed anti-ADI-PEG 20 antibodies. Such patients no longer benefit from ADI-PEG 20 treatment as the enzyme is neutralized by the antibodies. Thus, in certain embodiments, the invention provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising ADI-PEG 20, and after a period of time, administering to the patient a composition comprising an ADIr as described herein, thereby treating, ameliorating the symptoms of, or inhibiting the progression of the cancer.

[0097] In one embodiment of the method, the period of time is determined by detecting a predetermined level of anti-ADI-PEG 20 antibodies in the patient, wherein the composition comprising an ADIr is administered following detection of the predetermined level of said anti-ADI-PEG 20 antibodies. In certain embodiments, threshold level(s) or predetermined levels of anti-ADI-PEG 20 antibodies in patients to be treated with ADI-PEG 20 and the ADIr of the present invention can be established. A "predetermined threshold level" (also referred to as "predetermined level" or "predetermined cut-off value"), or sometimes referred to as a predetermined cut off, of anti-ADI-PEG 20 antibodies may be established using methods known in the art, for example, using Receiver Operator Characteristic curves or "ROC" curves. In one embodiment, even very low levels of anti-ADI-PEG 20 antibodies is deemed sufficient to warrant switching treatment from ADI-PEG 20 to an ADIr-PEG of the present invention. In certain embodiments, an appropriate level of anti-ADI-PEG 20 that will determine when to terminate ADI-PEG 20 treatment and begin treatment with an ADIr-PEG of the present invention can be determined by the skilled clinician.

[0098] In some embodiments, the period of time is determined by detecting or otherwise observing ADI activity in the patient, wherein the composition is administered following detection or observation of a predetermined level of ADI activity. In particular embodiments, the composition is administered following detection or observation of a reduced level of ADI activity in the patient. ADI activity can be measured directly, for example, by assaying a biological sample for at least one indicator of ADI activity, or indirectly, for example, by observing the desired or intended effect of the ADI-PEG 20 treatment. In certain embodiments, an appropriate level of ADI activity that will determine when to terminate ADI-PEG 20 treatment and begin treatment with an ADIr-PEG of the present invention can be determined by the skilled clinician.

[0099] Typical routes of administering these and related pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

[0100] Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described ADIr composition in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of an ADIr-PEG of the present disclosure, such as ADIr-PEG 20, for treatment of a disease or condition of interest in accordance with teachings herein. In certain embodiments, the pharmaceutical or therapeutic compositions are sterile and/or pyrogen-free.

[0101] A pharmaceutical composition may be in the form of a solid or liquid. In one embodiment, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, anoral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is generally either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0102] As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0103] The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[0104] The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, in certain embodiments, physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0105] A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of ADIr as herein disclosed, such as ADIr-PEG 20, such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of ADIr in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of ADIr-PEG. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of ADIr-PEG prior to dilution.

[0106] The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

[0107] The pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The pharmaceutical composition in solid or liquid form may include an agent that binds to ADIr-PEG and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome. The pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.

[0108] The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a composition that comprises ADIr-PEG as described herein and optionally, one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the ADIr-PEG composition so as to facilitate dissolution or homogeneous suspension of the ADIr-PEG in the aqueous delivery system.

[0109] The compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound (e.g., ADIr-PEG) employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.

[0110] 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/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. In a further embodiment, the dose may be from about 50 IU/m.sup.2 to about 700 IU/m.sup.2, administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks. In certain embodiments, the dose may be about 50 IU/m.sup.2, 60 IU/m.sup.2, 70 IU/m.sup.2, 80 IU/m.sup.2, 90 IU/m.sup.2, 100 IU/m.sup.2, 110 IU/m.sup.2, 120 IU/m.sup.2, 130 IU/m.sup.2, 140 IU/m.sup.2 150 IU/m.sup.2, 160 IU/m.sup.2, 170 IU/m.sup.2, 180 IU/m.sup.2, 190 IU/m.sup.2, 200 IU/m.sup.2, 210 IU/m.sup.2, 220 IU/m.sup.2, 230 IU/m.sup.2, 240 IU/m.sup.2, 250 IU/m.sup.2, 260 IU/m.sup.2, 270 IU/m.sup.2, 280 IU/m.sup.2, 290 IU/m.sup.2, 300 IU/m.sup.2, 310 IU/m.sup.2, about 320 IU/m.sup.2, about 330 IU/m.sup.2, 340 IU/m.sup.2 about 350 IU/m.sup.2 360 IU/m.sup.2, 370 IU/m.sup.2, 380 IU/m.sup.2, 390 IU/m.sup.2, 400 IU/m.sup.2, 410 IU/m.sup.2, 420 IU/m.sup.2, 430 IU/m.sup.2, 440 IU/m.sup.2, 450 IU/m.sup.2, 500 IU/m.sup.2, 550 IU/m.sup.2, 600 IU/m.sup.2, 620 IU/m.sup.2, 630 IU/m.sup.2, 640 IU/m.sup.2, 650 IU/m.sup.2, 660 IU/m.sup.2, 670 IU/m.sup.2, 680 IU/m.sup.2, 690 IU/m.sup.2, or about 700 IU/m.sup.2 administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks. In certain embodiments, the dose may be modified as desired by the skilled clinician.

[0111] The optimum dosage with ADIr-SS-PEG5,000 may be about twice a week, while the optimum dosage with ADIr-SS-PEG20,000 may be from about once a week to about once every two weeks. In certain embodiments, the optimum dosage with ADIr-SS-PEG20,000 may be about twice a week.

[0112] ADIr-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. In one embodiment, a liquid composition comprising ADIr-PEG comprises about 10 to about 12 mg of ADIr, about 20 to about 40 mg of polyethylene glycol, 1.27 mg+5% monobasic sodium phosphate, USP; about 3 mg+5% dibasic sodium phosphate, USP; 7.6 mg+5% sodium chloride, USP; at a pH of about 6.6 to about 7; in an appropriate amount of water for injection (e.g., about 1 ml or about 2 ml). In one embodiment, a liquid composition comprising an ADIr-PEG comprises histidine--HCl, and in certain embodiments, the composition buffer is from about 0.0035 M Histidine-HCl to about 0.35 M Histidine-HCl. In one particular embodiment, the composition is formulated in a buffer comprising 0.035 M Histidine-HCl at pH 6.8 with 0.13 M sodium chloride. In another embodiment, the composition is formulated in a buffer comprising 0.02 M sodium phosphate buffer at pH 6.8 with 0.13 M sodium chloride.

[0113] In one embodiment, a composition comprising ADIr or ADIr-PEG has a pH of about 5 to about 9, about 6 to about 8, or about 6.5 to about 7.5. In some embodiments, the composition comprising ADIr has a pH of about 6.8.+-.1.0.

[0114] In one embodiment, free PEG in a composition comprising ADIr-PEG is between 1-10%, and in a further embodiment, is less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the total PEG. In certain embodiments, the unmodified ADIr in a composition comprising ADIr-PEG is less than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or less than 0.1%. Generally, compositions comprising ADIr-PEG have total impurities less than or equal to about 4%, 3%, 2%, 1.5%, 1% or 0.5%. In one embodiment, the endotoxin limit meets the requirements stated in USP, i.e., <50 EU/mL.

[0115] In one embodiment, the free sulfhydryl in a composition comprising ADIr or ADIr-PEG is greater than about 90%. In some embodiments, the free sulfhydryl in a composition comprising ADIr or ADIr-PEG is about 91%, about 92%, about 93%, about 94% or about 95%, about 96% about 97%, about 98% about 99% or more.

[0116] In one embodiment, the ADIr or ADIr-PEG in a composition has a Km of from about 0.1 .mu.M or 0.5 .mu.M to about 15 .mu.M, and in a further embodiment, is from about 1 .mu.M to about 12 .mu.M, about 1 .mu.M to about 10 .mu.M, about 1.5 .mu.M to about 9 .mu.M, about 1.5 .mu.M to about 8 .mu.M or about 1.5 .mu.M to about 7 .mu.M. In certain embodiments, the ADIr or ADIr-PEG in a composition has a Km of about 1.0 .mu.M to about 10 .mu.M or about 1.5 .mu.M to about 6.5 .mu.M. In some embodiments, the ADIr or ADIr-PEG in a composition has a Km of about, at least about, or less than about 0.1 .mu.M, about 0.5 .mu.M, about 1.0 .mu.M, about 1.5 .mu.M, about 2 .mu.M, about 2.5 .mu.M, about 3 .mu.M, about 3.5 .mu.M, about 4 .mu.M, about 4.5 .mu.M, about 5 .mu.M, about 5.5 .mu.M, about 6 .mu.M, about 6.5 .mu.M, or about 7 .mu.M, or about 8 .mu.M, or about 9 .mu.M, or about 10 .mu.M.

[0117] In one embodiment, the ADIr or ADIr-PEG in a composition has a Kcat of from about 0.5 sec.sup.-1 to about 80 sec.sup.-1, or about 0.5 sec.sup.-1 to about 70 sec.sup.-1, or about 0.5 sec.sup.-1 to about 60 sec.sup.-1, or about 0.5 sec.sup.-1 to about 50 sec.sup.-1, or about 0.5 sec.sup.-1 to about 40 sec.sup.-1, or about 0.5 sec.sup.-1 to about 30 sec.sup.-1, or about 0.5 sec.sup.-1 to about 20 sec.sup.-1, or about 0.5 sec.sup.-1 to about 15 sec.sup.-1, and in a further embodiment, is from about 0.5 sec.sup.-1 to about 80 sec.sup.-1, or about 1 sec.sup.-1 to about 80 sec.sup.-1, or about 5 sec.sup.-1 to about 80 sec.sup.-1, or about 10 sec.sup.-1 to about 80 sec.sup.-1, or about 20 sec.sup.-1 to about 80 sec.sup.-1, or about 30 sec.sup.-1 to about 80 sec.sup.-1, or about 40 sec.sup.-1 to about 80 sec.sup.-1, or about 50 sec.sup.-1 to about 80 sec.sup.-1, or about 60 sec.sup.-1 to about 80 sec.sup.-1, or about 70 sec.sup.-1 to about 80 sec.sup.-1, or about 1 sec.sup.-1 to about 12 sec.sup.-1, about 1 sec.sup.-1 to about 10 sec.sup.-1, about 1.5 sec.sup.-1 to about 9 sec.sup.-1, about 2 sec.sup.-1 to about 8 sec.sup.-1 or about 2.5 sec.sup.-1 to about 7 sec.sup.-1. In certain embodiments, the ADIr or ADIr-PEG in a composition has a Kcat of about 2.5 sec.sup.-1 to about 7.5 sec.sup.-1. In some embodiments, the ADIr or ADIr-PEG in a composition has a Kcat of about or at least about 2.5 sec.sup.-1, about 3 sec.sup.-1, about 3.5 sec.sup.-1, about 4 sec.sup.-1, about 4.5 sec.sup.-1, about 5 sec.sup.-1, about 5.5 sec.sup.-1, about 6 sec.sup.-1, about 6.5 sec.sup.-1, about 7 sec.sup.-1, about 7.5 sec.sup.-1 or about 8 sec.sup.-1, about 10 sec.sup.-1, about 15 sec.sup.-1, about 20 sec.sup.-1, about 25 sec.sup.-1, about 30 sec.sup.-1, about 35 sec.sup.-1, about 40 sec.sup.-1, about 45 sec.sup.-1, about 50 sec.sup.-1, about 55 sec.sup.-1, about 60 sec.sup.-1, about 65 sec.sup.-1, about 70 sec.sup.-1, about 75 sec.sup.-1, about 80 sec.sup.-1, about 85 sec.sup.-1, about 90 sec.sup.-1, about 95 sec.sup.-1, or about 100 sec.sup.-1.

[0118] In one embodiment, the ADIr or ADIr-PEG in a composition has a conductivity (also referred to in the art as specific conductance) of about 5 mS/cm to about 20 mS/cm, and in further embodiments, from about 5 mS/cm to about 15 mS/cm, about 7 mS/cm to about 15 mS/cm, about 9 mS/cm to about 15 mS/cm or about 10 mS/cm to about 15 mS/cm. In some embodiments, the ADIr or ADIr-PEG in a composition has a conductivity of about 9 mS/cm, about 10 mS/cm, about 11 mS/cm, about 12 mS/cm or about 13 mS/cm, about 14 mS/cm or about 15 mS/cm. In certain embodiments, the ADIr or ADIr-PEG in a composition has a conductivity of about 13 mS/cm.+-.1.0 mS/cm.

[0119] In one embodiment, the ADIr or ADIr-PEG in a composition has an osmolality of about 50 mOsm/kg to about 500 mOsm/kg, about 100 mOsm/kg to about 400 mOsm/kg, about 150 mOsm/kg to about 350 mOsm/kg, about 200 mOsm/kg to about 350 mOsm/kg or about 250 mOsm/kg to about 350 mOsm/kg. In certain embodiments, the ADIr or ADIr-PEG in a composition has an osmolality of about 300.+-.30 mOsm/kg.

[0120] In one embodiment, the protein concentration is about 11.0.+-.1.0 mg/mL. In certain embodiments, the protein concentration is between about 8 and about 15 mg/mL. In another embodiment, the protein concentration is about 8, 9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 15 mg/mL.

[0121] In one embodiment, the specific enzyme activity is between about 5.0 and 90 IU/mg or between about 5 and 55 IU/mg, where 1 IU is defined as the amount of enzyme that converts one .mu.mol of arginine into one .mu.mol of citrulline and 1 .mu.mol of ammonia in one minute at 37.degree. C. and the potency is 100.+-.20 IU/mL. In another embodiment, the specific enzyme activity is about 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 35, 40, 45, 50, 55, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100.+-.2.0 IU/mg. In one particular embodiment, the specific enzyme activity is 9.+-.2.0 IU/mg.

[0122] Compositions comprising ADIr-PEG of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents, including ADI-PEG 20. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising ADIr-PEG (e.g., ADIr-PEG 20) of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, ADIr-PEG as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Similarly, ADIr-PEG as described herein and the other active agent can be administered to the patient together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations, by the same or different routes (e.g., one by injection, one by oral). Where separate dosage formulations are used, the compositions comprising ADIr-PEG and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.

[0123] Thus, in certain embodiments, also contemplated is the administration of the ADIr compositions of this disclosure in combination with one or more other therapeutic agents. Such therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer or GVHD. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics, autophagy inhibitors, or other active and ancillary agents.

[0124] In certain embodiments, the ADIr compositions disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE.RTM., Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin.TM. (bexarotene), Panretin.TM. (alitretinoin); ONTAK.TM. (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin. Further chemotherapeutic agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib, bevacizumab, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, trastuzumab, vandetanib, vemurafenib, and sunitinib; sirolimus (rapamycin), everolimus and other mTOR inhibitors. Pharmaceutically acceptable salts, acids or derivatives of any of the above are also contemplated for use herein.

[0125] In certain embodiments, the ADIr compositions disclosed herein may be administered in conjunction with any number of autophagy inhibitors. In some preferred embodiments, the autophagy inhibitor is selected from the group consisting of: chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil.TM.), bafilomycin A1, 5-amino-4-imidazole carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1, analogues of cAMP, and drugs which elevate cAMP levels, adenosine, N6-mercaptopurine riboside, wortmannin, and vinblastine. In addition, antisense or siRNA that inhibits expression of proteins essential for autophagy, such as for example ATGS, may also be used.

[0126] In one embodiment, the combination of ADIr-PEG with one or more therapeutic agents acts complementary, additively, or synergistically. In this regard, complementary or synergizing agents are described herein, which include a therapeutic agent (e.g., chemotherapeutic agent, autophagy inhibitor, mTOR inhibitor, or any other therapeutic agent used for the treatment of cancer, GVHD, or inflammatory bowel disease as described herein) that is capable of acting complementary or synergistically with ADIr-PEG as provided herein, where such complementarity or synergy manifests as a detectable effect that is greater (i.e., in a statistically significant manner relative to an appropriate control condition) in magnitude than the effect that can be detected when the chemotherapeutic agent is present but the ADIr-PEG composition is absent, and/or when the ADIr-PEG is present but the chemotherapeutic agent is absent. Methods for measuring synergy and complementarity are known in the art (see e.g., Cancer Res Jan. 15, 2010 70; 440).

[0127] The compositions comprising ADIr, and optionally other therapeutic agents, as described herein may be used in therapeutic methods for treating cancer and methods for preventing metastasis of a cancer. Thus, the present invention provides for methods for treating, ameliorating the symptoms of, or inhibiting the progression of or prevention of a variety of different cancers. In another embodiment, the present disclosure provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of GVHD. In particular the present disclosure provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of a cancer or GVHD in a patient comprising administering to the patient a therapeutically effective amount of ADIr composition as described herein, optionally, following treatment with ADI-PEG 20, particularly where a patient develops anti-ADI-PEG 20 antibodies, thereby treating, ameliorating the symptoms of, or inhibiting the progression of the cancer or GVHD. Thus, the ADIr compositions described herein may be administered to an individual afflicted with inflammatory bowel disease (e.g., Crohn's disease; ulcerative colitis), GVHD or a cancer, including, but not limited to hepatocellular carcinoma, leukemia (e.g. acute myeloid leukemia and relapsed acute myeloid leukemia), melanoma including metastatic melanoma, sarcomas (including, but not limited to, metastatic sarcomas, uterine leiomyosarcoma), pancreatic cancer, prostate cancer (such as, but not limited to, hormone refractory prostate cancer), mesothelioma, lymphatic leukemia, chronic myelogenous leukemia, lymphoma, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer (including, but not limited to, gastric adenocarcinoma), glioma, glioblastoma multi-form, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), kidney cancer (including but not limited to renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers (including, but not limited to, squamous cell carcinoma of the head and neck; cancer of the tongue), cervical cancer, testicular cancer, gallbladder, cholangiocarcinoma, and stomach cancer.

[0128] In another embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer in a patient comprising administering to the patient a composition comprising ADIr, and optionally one or more other therapeutic agents, as described herein, wherein the cancer is deficient in ASS, ASL, or both. In this regard, ASS or ASL deficiency may be a reduction in expression as measured by mRNA expression or protein expression, or may be a reduction in protein activity, and generally comprises a statistically significant reduction in expression or activity as determined by the skilled person. Reduced ASS or ASL expression or activity may be a reduction in expression or activity of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more, as compared to expression or activity in an appropriate control sample known to be cancer free. In certain embodiments, ASS or ASL expression or activity is reduced by at least twofold as compared to expression or activity in a non-cancer control sample.

[0129] In certain embodiments, the reduced expression or activity of ASS or ASL results from methylation of the ASS or ASL promoter or inhibition of the ASS or ASL promoter. In another embodiment the reduction in expression or activity of ASS or ASL results from a DNA mutation (e.g., one or more point mutations, small deletions, insertions, and the like) or a chromosomal abnormality resulting in deletion of the gene. In one embodiment, the cancer is ASS or ASL negative, meaning no expression or activity is observed.

[0130] Reduction in ASS or ASL expression or activity may be measured using any methods known in the art, such as but not limited to, quantitative PCR, immunohistochemistry, enzyme activity assays (e.g., assay to measure conversion of citrulline into argininosuccinate or conversion of argininosuccinate into arginine and fumarate), and the like.

[0131] Thus, the present invention provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of a cancer in a patient comprising administering to the patient a composition comprising ADIr as described herein, wherein the cancer exhibits reduced expression or activity of ASS or ASL, or both, wherein the cancer includes, but is not limited to hepatocellular carcinoma, leukemia (e.g. acute myeloid leukemia and relapsed acute myeloid leukemia), melanoma including metastatic melanoma, sarcomas (including, but not limited to, metastatic sarcomas, uterine leiomyosarcoma), pancreatic cancer, prostate cancer (such as, but not limited to, hormone refractory prostate cancer), mesothelioma, lymphatic leukemia, chronic myelogenous leukemia, lymphoma, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer (including, but not limited to, gastric adenocarcinoma), glioma, glioblastoma multi-form, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), kidney cancer (including but not limited to renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers (including, but not limited to, squamous cell carcinoma of the head and neck; cancer of the tongue), cervical cancer, testicular cancer, gallbladder, cholangiocarcinoma, and stomach cancer.

[0132] Various studies in the literature have shown that ASS is deficient in the following tumors: acute myelogenous leukemia (AML), bladder, breast, colorectal, gastric, glioblastoma, HCC, lymphoma, melanoma, mesothelioma, non-small cell lung, ovarian, pancreatic, prostate, renal, sarcoma, and small cell lung. Accordingly, treatment of these ASS-deficient cancers is specifically contemplated herein, with ADIr-PEG alone or in combination with other treatments, including treatment first with ADI-PEG 20.

[0133] The present invention further provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of cancer in a patient comprising administering to the patient a composition comprising ADIr as described herein (e.g; ADIr-PEG and in particular ADIr-PEG 20), in combination with an autophagy inhibitor. In one embodiment, the present invention provides methods for treating cancer in a patient comprising administering to the patient a therapeutically effective amount of a composition comprising ADIr as described herein in combination with autophagy inhibitor wherein the cancer is pancreatic cancer or small cell lung cancer.

[0134] In certain embodiments, the present invention provides methods of treatment where administration of the compositions comprising ADIr described herein depletes arginine in the plasma for at least one month, 2 months, 3 months, 4 months, 5 months, 6 months or longer. In another embodiment, the present invention provides methods of treatment where administration of the compositions comprising ADIr described herein depletes arginine in the plasma for at least one month, 2 months, 3 months, 4 months, 5 months, 6 months or longer after terminating treatment with ADI-PEG 20 following detection of anti-ADI-PEG 20 antibodies.

EXAMPLES

Example 1

Screening and Selection of ADI Enzymes that have Low Cross-Reactivity with Patient Anti-ADI-PEG 20 Antibodies

[0135] This example describes the screening and selection of ADI enzymes that have low cross-reactivity with patient anti-ADI-PEG 20 antibodies.

[0136] From a large number of ADI enzymes, Table 1 lists 24 ADI enzymes selected for their sequence percent identity relative to M. hominis ADI. From the literature, M. hominis, M. arginini, and M. arthritidis ADI amino acid sequences are closely related and these enzymes have good catalytic properties. More recently, additional ADI enzymes have been discovered that have sequences closely related to these three. More distantly related Mycoplasma ADI enzymes have been identified, although less is known about them. Even more distantly related ADI enzymes from bacterial and other sources exist.

TABLE-US-00001 TABLE 1 Selected ADI Sequences with Varying Degrees of Similarity to M. hominis ADI SEQUENCE PERCENT ACCESSION SEQ ORGANISM IDENTITY NUMBER ID NO: Mycoplasma hominis 100.0% gi | 728876 1 Mycoplasma phocicerebrale 82.1% gi | 154184333 2 Mycoplasma arginini 82.1% gi | 728875 3 Mycoplasma arthritidis 80.4% gi | 238692486 4 Mycoplasma orale 77.8% gi | 2170494 5 Mycoplasma gateae 76.8% gi | 148361415 6 Mycoplasma phocidae 75.3% gi | 154184335 7 Mycoplasma columbinum 58.2% gi | 343491689 8 Mycoplasma iowae 55.2% gi | 350546321 9 Mycoplasma crocodyli 52.3% gi | 291600396 10 Mycoplasma fermentans 52.0% gi | 238809916 11 Mycoplasma penetrans 51.7% gi | 26554060 12 Mycoplasma gallisepticum 51.5% gi | 31544533 13 Mycoplasma alligatoris 50.8% gi | 292552899 14 Mycoplasma pneumoniae 50.7% gi | 440453687 15 Mycoplasma mobile 47.3% gi | 47458387 16 Streptococcus pyogenes 37.7% gi | 15675444 17 Enterococcus faecalis 37.1% gi | 60389809 18 Mycoplasma capricolum 36.6% gi | 83319656 19 Halothermothrix orenii 34.8% gi | 254803235 20 Staphylococcus aureus 33.8% gi | 123549453 21 Pseudomonas plecoglossicida 28.7% gi | 154183755 22 Pseudomonas putida 27.5% gi | 431801013 23 Pseudomonas aeruginosa 27.0% gi | 452183609 24 Mycobacterium bovis 26.8% gi | 378770764 25

[0137] Several of the protein sequences available in the public databases may not have been full-length ADI sequences. In those cases, the publicly available sequences were extended where needed to make full-length ADI based on known ADI sequences. In certain cases, the ADI proteins were modified elsewhere (e.g., C251S substitution). These synthesized ADI sequences are provided in SEQ ID NOs:26-32 and correspond to the extended and/or modified ADI sequences of Mycoplasma arginini (C251S), Mycoplasma arthritidis (C251S), Mycoplasma phocicerebrale, Mycoplasma gateae, Mycoplasma phocidae, H. orenii, and Mycobacterium bovis.

[0138] A number of ADI enzymes from a variety of organisms were characterized to determine which enzymes would be expected to remove and maintain low concentrations of arginine in patient blood, even in the presence of anti-ADI-PEG 20 antibodies. Table 2 (below) lists a selected subset of ADI enzymes from Table 1 that were studied. As detailed below, the data from these studies show that ADI from a number of species that are closely related to M. hominis, based on sequence identity, have sufficiently good enzyme catalytic properties and reduced cross-reactivity with anti-ADI-PEG 20 antibodies.

[0139] ADI Preparation. Recombinant ADI enzymes were cloned, expressed, and purified for testing according to standard protocols, as described, for example, in Gallego et al., PLOS One, 7(10):e47886, 2012; Monstadt and Holldorf, Biochem. J. 273:739-745, 1990; Joo Noh et al., Molecules and Cells. 13:137-143, 2002; and Sugimura et al., Infection and Immunity. 58:2510-2515, 1990.

[0140] Human Anti-ADI-PEG20 Antibody Purification. Anti-ADI-PEG20 antibody was purified from plasma samples of patients who had received ADI-PEG20 during a clinical study. A total of 60 ml of plasma was pooled from 8 different patients that had reached high titer (titer>/=4) against ADI-PEG20 as determined by an ELISA assay. A two-step purification was used, a Protein "A" chromatography (GE Healthcare) followed by an ADI affinity chromatography. .about.20 mg of purified antibody was obtained and stored at -80.degree. C. in aliquots until needed.

[0141] ADI Enzyme Assays. Arginine deiminase (ADI) catalyzes the conversion of L-arginine to L-citrulline and ammonia. The amount of L-citrulline can be detected by a colorimetric endpoint assay (see, for example, Knipp and Vasak, Analytical Biochem. 286:257-264, 2000) and compared to a standard curve of known amounts of L-citrulline in order to calculate the specific activity of ADI expressed as IU/mg of protein. One IU of enzyme activity is defined as the amount of enzyme that produces 1 .mu.mol of citrulline per minute at the pH and temperature being tested. Standard assay conditions were performed at 37.degree. C. in Physiological HEPES Buffer (PHB) 50 mM HEPES, 160 mM NaCl pH 7.4 (Lang and Zander, Clin Chem Lab Med. 37:563-571, 1999) plus 0.1% BSA. All samples and standards were run in duplicate or triplicate where conditions permitted.

[0142] Km and Kcat values were determined by using a variation of the activity assay described above. As with the activity assay, all reactions were run at 37.degree. C. in PHB plus 0.1% BSA. Enzyme concentration, reaction time, and substrate concentration range were adjusted for each of the ADI or ADIr constructs to account for their differences in activity. In general, 2 nM enzyme, 5 minute reaction time, and a 0-160 .mu.M arginine was used as starting conditions. When optimizing the conditions, particular attention was paid towards the amount of substrate consumed as a percentage of total substrate added to the reaction. The lower limit of detection is 1 .mu.M of citrulline with the lower limit of quantitation being 2 .mu.M. A citrulline standard curve was run on every plate and used to quantify the citrulline produced by the enzymatic reaction.

[0143] Activity assays were also performed to assess enzymatic activity in the presence of anti-ADI-PEG20 (antibody neutralization profiles). These assays were performed as described above and in the presence of 800 nM, 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 0 nM of anti-ADI-PEG20 antibodies.

[0144] Calculations. The citrulline concentration (.mu.M) produced in each reaction well was calculated and averaged using the citrulline standard curve. The velocity of each reaction was then calculated in .mu.M/min/50 nM ADI. Specific activity (IU/mg or .mu.mols product/min/mg ADI) was calculated by multiplying this value by the "IU" factor (IU factor was calculated from the molecular weight of the ADI and the reaction volume). The results are summarized in Table 2 below.

TABLE-US-00002 TABLE 2 Selected ADI Sequences and Properties NUMBER OF SEQUENCE SPECIFIC SURFACE REDUCED AB PERCENT ACTIVITY RESIDUE CROSS- ORGANISM IDENTITY (IU/MG)*** CHANGES* REACTIVITY**** Mycoplasma hominis 100.0 + 0 -- Mycoplasma phocicerebrale 82.1 + 33 Y Mycoplasma arginini 82.1 + 50 Y Mycoplasma arthritidis 80.4 + 53 Y Mycoplasma gateae 76.8 + 48 Y Mycoplasma phocidae 75.3 + 51 Y Mycoplasma columbinum 58.2 + 93 Y Mycoplasma iowae 55.2 - 107 ND Mycoplasma crocodyli 52.3 - 107 ND Mycoplasma gallisepticum 51.5 - 108 ND Mycoplasma alligatoris 50.8 + 111 Y Mycoplasma mobile 47.3 ND 111 ND Halothermothrix orenii 34.8 - 122 ND Mycobacterium bovis 26.8 - 132 ND *Surface residues were identified from the crystal structure of M. hominis ADI and surface residues for ADI from other organisms was determined by sequence homology. ** ND: Not Determined. ***"+" indicates specific enzymatic activity of .gtoreq.8 IU/mg under physiological conditions and in the absence of anti-ADI-PEG20 antibodies. ****"Y" indicates reduced anti-ADI-PEG20 cross-reactivity relative to M. hominis ADI, as measured by specific enzymatic activity in the presence of anti-ADI-PEG20 antibodies.

[0145] These data show that ADI enzymes that are highly homologous to M. hominis ADI (about 50-100 percent identity) maintained excellent catalytic activity. They also showed reduced affinity toward patient anti-ADI-PEG 20 antibodies, as measured by enzyme activity in the presence of anti-ADI-PEG 20 antibodies, for example, relative to that of Mycoplasma hominis. Accordingly, these ADI enzymes may have therapeutic utility for use in therapy for the treatment of cancer, either alone or following ADI-PEG 20 treatment, to extend and/or increase the effectiveness of arginine depletion therapy.

[0146] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments.

[0147] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Sequence CWU 1

1

321409PRTMycoplasma hominis 1Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu1 5 10 15Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys Ile 50 55 60Met Lys Asp Arg Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala65 70 75 80Glu Thr Tyr Asp Leu Ala Ser Lys Ala Ala Lys Glu Glu Phe Ile Glu 85 90 95Thr Phe Leu Glu Glu Thr Val Pro Val Leu Thr Glu Ala Asn Lys Lys 100 105 110Ala Val Arg Ala Phe Leu Leu Ser Lys Pro Thr His Glu Met Val Glu 115 120 125Phe Met Met Ser Gly Ile Thr Lys Tyr Glu Leu Gly Val Glu Ser Glu 130 135 140Asn Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp145 150 155 160Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Phe Met Arg Tyr 165 170 175Ile Val Arg Arg Arg Glu Thr Leu Phe Ala Arg Phe Val Phe Arg Asn 180 185 190His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met Lys 195 200 205Met Pro Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu Thr Leu 210 215 220Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu Leu225 230 235 240Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg Ile Val 245 250 255Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr Trp 260 265 270Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala Asn 275 280 285Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala Glu 290 295 300Pro Gln Pro Gln Leu Asn Gly Leu Pro Leu Asp Lys Leu Leu Ala Ser305 310 315 320Ile Ile Asn Lys Glu Pro Val Leu Ile Pro Ile Gly Gly Ala Gly Ala 325 330 335Thr Glu Met Glu Ile Ala Arg Glu Thr Asn Phe Asp Gly Thr Asn Tyr 340 345 350Leu Ala Ile Lys Pro Gly Leu Val Ile Gly Tyr Asp Arg Asn Glu Lys 355 360 365Thr Asn Ala Ala Leu Lys Ala Ala Gly Ile Thr Val Leu Pro Phe His 370 375 380Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met385 390 395 400Pro Leu Ser Arg Lys Asp Val Lys Trp 4052392PRTMycoplasma phocicerebrale 2Ile His Val Tyr Ser Glu Ile Gly Glu Leu Glu Thr Val Leu Val His1 5 10 15Glu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu 20 25 30Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His 35 40 45Gln Ser Phe Val Lys Gln Leu Lys Asp Asn Gly Ile Asn Val Val Glu 50 55 60Leu Thr Asp Leu Val Ala Glu Thr Phe Asp Leu Ala Ser Lys Glu Glu65 70 75 80Gln Glu Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu 85 90 95Ser Glu Ala His Lys Thr Ala Val Arg Lys Phe Leu Thr Ser Arg Lys 100 105 110Ser Thr Arg Glu Met Val Glu Phe Met Met Ala Gly Ile Thr Lys Tyr 115 120 125Asp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro 130 135 140Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val145 150 155 160Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe 165 170 175Ser Arg Phe Val Phe Ser Asn His Pro Lys Leu Val Lys Thr Pro Trp 180 185 190Tyr Tyr Asp Pro Ala Met Lys Met Ser Ile Glu Gly Gly Asp Val Phe 195 200 205Ile Tyr Asn Asn Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp 210 215 220Leu Glu Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu225 230 235 240Val Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn 245 250 255Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe 260 265 270Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp 275 280 285Leu Val Asn Gly Gly Ala Glu Pro Gln Pro Lys Glu Asn Gly Leu Pro 290 295 300Leu Glu Gly Leu Leu Gln Ser Ile Ile Asn Lys Lys Pro Val Leu Ile305 310 315 320Pro Ile Ala Gly Asn Asn Ala Ser His Ile Asp Ile Glu Arg Glu Thr 325 330 335His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Lys Pro Gly Val Val Ile 340 345 350Gly Tyr Ala Arg Asn Glu Lys Thr Asn Ala Ala Leu Ala Ala Ala Gly 355 360 365Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly 370 375 380Asn Ala Arg Cys Met Ser Met Pro385 3903410PRTMycoplasma arginini 3Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu1 5 10 15Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu 50 55 60Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Ile Asp Leu Val Ala65 70 75 80Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu 85 90 95Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Val 100 105 110Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Glu Leu Val 115 120 125Glu Ile Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser 180 185 190Asn His Pro Lys Leu Ile Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu 195 200 205Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr Leu225 230 235 240Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln305 310 315 320Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu Gly 325 330 335Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser385 390 395 400Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 4104409PRTMycoplasma arthritidis 4Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu1 5 10 15Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu Ile 20 25 30Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Ala Glu 50 55 60Leu Lys Lys Arg Gly Ile Asn Val Val Glu Leu Val Asp Leu Ile Val65 70 75 80Glu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Lys Glu Lys Leu Leu Glu 85 90 95Glu Phe Leu Asp Asp Ser Val Pro Val Leu Ser Asp Glu His Arg Ala 100 105 110Ala Val Lys Lys Phe Leu Gln Ser Gln Lys Ser Thr Arg Ser Leu Val 115 120 125Glu Tyr Met Ile Ala Gly Ile Thr Lys His Asp Leu Lys Ile Glu Ser 130 135 140Asp Leu Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser 180 185 190Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala Glu 195 200 205Gly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr Leu225 230 235 240Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp 290 295 300Ala Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Glu Asp Leu Leu Lys305 310 315 320Ser Ile Ile Gly Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Ala Gly 325 330 335Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350Tyr Leu Ala Val Ala Pro Gly Ile Val Ile Gly Tyr Ala Arg Asn Glu 355 360 365Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Thr Val Leu Pro Phe 370 375 380Arg Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser385 390 395 400Met Pro Leu Ser Arg Lys Asp Val Lys 4055408PRTMycoplasma oralemisc_feature(201)..(201)Xaa can be any naturally occurring amino acidmisc_feature(263)..(263)Xaa can be any naturally occurring amino acidmisc_feature(272)..(272)Xaa can be any naturally occurring amino acidmisc_feature(294)..(294)Xaa can be any naturally occurring amino acid 5Ser Val Phe Ser Asp Lys Phe Asn Gly Ile His Val Tyr Ser Glu Ile1 5 10 15Gly Asp Leu Glu Ser Val Leu Val His Glu Pro Gly Lys Glu Ile Asp 20 25 30Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu 35 40 45Glu Ser Thr Asp Ala Arg Lys Glu His Lys Glu Phe Val Glu Ile Leu 50 55 60Lys Lys Gln Gly Ile Asn Val Val Glu Leu Val Asp Leu Val Val Glu65 70 75 80Thr Tyr Asn Leu Val Asp Lys Lys Thr Gln Glu Lys Leu Leu Lys Asp 85 90 95Phe Leu Asp Asp Ser Glu Pro Val Leu Ser Pro Glu His Arg Lys Ala 100 105 110Val Glu Lys Phe Leu Lys Ser Leu Lys Ser Thr Lys Glu Leu Ile Gln 115 120 125Tyr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys Ala Asp 130 135 140Lys Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp145 150 155 160Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg Tyr 165 170 175Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Lys Phe Ile Phe Thr Asn 180 185 190His Pro Lys Leu Val Lys Thr Pro Xaa Tyr Tyr Asp Pro Ala Met Lys 195 200 205Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr Leu 210 215 220Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile Thr Leu Leu225 230 235 240Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile Val 245 250 255Ala Ile Asn Val Pro Lys Xaa Thr Asn Leu Met His Leu Asp Thr Xaa 260 265 270Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala Asn 275 280 285Asp Val Phe Lys Phe Xaa Asp Tyr Asp Leu Val Asn Gly Gly Ser Asn 290 295 300Pro Glu Pro Val Val Asn Gly Leu Pro Leu Asp Lys Leu Leu Glu Ser305 310 315 320Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Lys Gly Ala 325 330 335Thr Glu Ile Glu Thr Ala Val Glu Thr His Phe Asp Gly Thr Asn Tyr 340 345 350Leu Ala Ile Lys Pro Gly Val Val Val Gly Tyr Ser Arg Asn Val Lys 355 360 365Thr Asn Ala Ala Leu Glu Ala Asn Gly Ile Lys Val Leu Pro Phe Lys 370 375 380Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met385 390 395 400Pro Leu Ser Arg Lys Asp Val Lys 4056391PRTMycoplasma gateae 6Ile His Val Tyr Ser Glu Ile Gly Glu Leu Glu Ser Val Leu Val His1 5 10 15Glu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu 20 25 30Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His 35 40 45Lys Leu Phe Val Ser Glu Leu Lys Ala Asn Asp Ile Asn Val Val Glu 50 55 60Leu Thr Asp Leu Val Thr Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala65 70 75 80Lys Asp Asn Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu 85 90 95Thr Glu Glu Leu Lys Ser Val Val Arg Thr Tyr Leu Lys Ser Ile Lys 100 105 110Ser Thr Arg Glu Leu Ile Gln Met Met Met Ala Gly Ile Thr Lys Tyr 115 120 125Asp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro 130 135 140Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val145 150 155 160Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe 165 170 175Ser Arg Phe Val Phe Ser Asn His Pro Lys Leu Val Asn Thr Pro Trp 180 185 190Tyr Tyr Asp Pro Ser Leu Lys Leu Ser Ile Glu Gly Gly Asp Val Phe 195 200 205Ile Tyr Asn Asn Asn Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp 210 215 220Leu Glu Thr Val Thr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys Glu225 230 235 240Cys Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn 245 250 255Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe 260 265 270Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp 275 280 285Leu Val Asn Gly Gly Glu Glu Pro Gln Pro Val Glu Asn Gly Leu Pro 290 295 300Leu Glu Gly Leu Leu Glu Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile305 310 315 320Pro Ile Ala Gly Glu Gly Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr 325 330 335His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val Ile 340 345 350Gly Tyr Ser Arg Asn Glu Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly 355 360 365Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly 370 375

380Asn Ala Arg Cys Met Ser Met385 3907392PRTMycoplasma phocidae 7Ile His Val Tyr Ser Glu Ile Gly Glu Leu Gln Thr Val Leu Val His1 5 10 15Glu Pro Gly Arg Glu Ile Glu Tyr Ile Thr Pro Ala Arg Leu Asp Glu 20 25 30Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His 35 40 45Gln Glu Phe Val Ala Glu Leu Lys Lys Asn Asn Ile Asn Val Val Glu 50 55 60Leu Thr Asp Leu Val Ser Glu Thr Tyr Asp Met Val Ser Lys Glu Lys65 70 75 80Gln Glu Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu 85 90 95Ser Glu Glu His Lys Gly Leu Val Arg Lys Phe Leu Lys Ser Leu Lys 100 105 110Ser Ser Lys Glu Leu Ile Gln Tyr Met Met Ala Gly Ile Thr Lys His 115 120 125Asp Leu Asn Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro 130 135 140Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val145 150 155 160Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe 165 170 175Ser Arg Phe Ile Phe Ala Asn His Pro Lys Leu Met Asn Thr Pro Leu 180 185 190Tyr Tyr Asn Pro Asp Met Lys Leu Ser Ile Glu Gly Gly Asp Val Phe 195 200 205Val Tyr Asn Asn Glu Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp 210 215 220Leu Asp Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu225 230 235 240Arg Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn 245 250 255Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe 260 265 270Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp 275 280 285Leu Val Asn Gly Gly Asp Glu Pro Gln Pro Lys Val Asn Gly Leu Pro 290 295 300Leu Glu Lys Leu Leu Glu Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile305 310 315 320Pro Ile Ala Gly Thr Ser Ala Ser Asn Ile Asp Val Glu Arg Glu Thr 325 330 335His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Ala Pro Gly Val Val Ile 340 345 350Gly Tyr Ser Arg Asn Val Lys Thr Asn Glu Ala Leu Glu Ala Ala Gly 355 360 365Ile Lys Val Leu Pro Phe Lys Gly Asn Gln Leu Ser Leu Gly Met Gly 370 375 380Asn Ala Arg Cys Met Ser Met Pro385 3908401PRTMycoplasma columbinum 8Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu Val1 5 10 15Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg 20 25 30Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala Ile 35 40 45Lys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile Lys 50 55 60Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His Ala65 70 75 80Thr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala 85 90 95Glu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr Val 100 105 110Leu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met Ala 115 120 125Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu Val 130 135 140Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser145 150 155 160Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys 165 170 175Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp Tyr 180 185 190Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile Glu 195 200 205Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val 210 215 220Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys Ile225 230 235 240Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val 245 250 255Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val 260 265 270Asp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys 275 280 285Val Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr Asn 290 295 300Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro Val305 310 315 320Leu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp Ile 325 330 335Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val 340 345 350Val Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg Ala 355 360 365Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly 370 375 380Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val385 390 395 400Lys9407PRTMycoplasma iowae 9Met Gly Asn Asn Ile Pro Lys Lys Ile Asn Val Phe Ser Glu Ile Gly1 5 10 15Asn Leu Lys Arg Val Leu Val His Thr Pro Gly Lys Glu Ile Glu Tyr 20 25 30Val Thr Pro Gln Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Asp 35 40 45Pro Val Arg Ala Arg Glu Glu His Lys Glu Phe Ile Lys Ile Leu Glu 50 55 60Ser Gln Gly Val Glu Val Val Gln Leu Val Asp Leu Thr Ala Glu Thr65 70 75 80Tyr Asp Val Ala Glu Ser Gln Ala Lys Glu Asn Phe Ile Gln Lys Trp 85 90 95Leu Asp Glu Ser Leu Pro Lys Leu Thr Asp Glu Asn Arg Asn Lys Val 100 105 110Tyr Ser Leu Leu Lys Ser Leu Glu Lys Asp Pro Lys Glu Met Ile Arg 115 120 125Lys Met Met Ser Gly Val Leu Ala Ser Glu Ile Gly Val Lys Ser Asp 130 135 140Val Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp145 150 155 160Pro Phe Ala Ser Val Gly Asn Gly Ile Thr Leu His Arg Met Phe Arg 165 170 175Pro Thr Arg Arg Arg Glu Thr Ile Phe Ala Asp Phe Ile Phe Ser Asn 180 185 190His Pro Glu Tyr Lys Ser Thr Gln Lys Tyr Tyr Glu Arg Glu Asp Lys 195 200 205Phe Ser Leu Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Lys Thr Leu 210 215 220Val Val Gly Val Ser Glu Arg Thr Glu Lys Gly Ala Ile Lys Ala Leu225 230 235 240Ala Lys Ala Val Gln Asn Asn Ser Asn Met Ser Phe Glu Lys Ile Tyr 245 250 255Ala Ile Asn Val Pro Lys Met Ser Asn Leu Met His Leu Asp Thr Trp 260 265 270Leu Thr Met Leu Asp Thr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met 275 280 285Gly Val Leu Lys Ile Trp Glu Ile Asp Leu Ser Asp Lys Ser Leu Lys 290 295 300Trp Lys Glu Ile Arg Asp Ser Leu Asp His Phe Leu Ser Thr Ile Ile305 310 315 320Gly Lys Lys Ala Ile Thr Val Pro Val Ala Gly Lys Asp Ala Met Gln 325 330 335Phe Glu Ile Asp Ile Glu Thr His Phe Asp Ala Thr Asn Phe Ile Ala 340 345 350Val Ala Pro Gly Val Val Ile Gly Tyr Asp Arg Asn Lys Lys Thr Asn 355 360 365Glu Ala Leu Lys Glu Ala Gly Ile Lys Val Leu Ser Trp Asn Gly Asp 370 375 380Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Thr Met Pro Leu385 390 395 400Tyr Arg Glu Glu Leu Lys Lys 40510402PRTMycoplasma crocodyli 10Met Asn Lys Ile Asn Val Tyr Ser Glu Val Gly Lys Leu Lys Glu Val1 5 10 15Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg 20 25 30Leu Glu Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Ser Ala Ile 35 40 45Glu Glu His Lys Arg Phe Leu Lys Ile Leu Glu Asp Asn Asn Ile Lys 50 55 60Val Ile Gln Leu Asp Gln Leu Val Ala Asp Thr Tyr Glu Leu Val Asn65 70 75 80Pro Ser Val Arg Asp Ala Phe Ile Glu Lys Trp Leu Asn Glu Ser Glu 85 90 95Pro Lys Leu Asp Lys Lys Leu Arg Glu Lys Val Lys Glu Tyr Leu Leu 100 105 110His Thr Gln Lys Thr Val Gly Thr Lys Arg Met Val Arg Ile Met Met 115 120 125Ala Gly Val Asp Arg Val Glu Leu Gly Val Glu Leu Asp Arg Gln Leu 130 135 140Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala145 150 155 160Ser Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg 165 170 175Lys Arg Glu Thr Ile Phe Ser Glu Phe Ile Phe Glu Asn His Pro Asp 180 185 190Tyr Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile 195 200 205Glu Gly Gly Asp Val Phe Ile Tyr Asn Arg Thr Thr Leu Val Ile Gly 210 215 220Ile Ser Glu Arg Thr Asn Lys Asp Ala Leu Leu Thr Ile Ala Asn Asn225 230 235 240Ile Lys Ser Asn Lys Glu Ser Lys Phe Glu Arg Ile Val Ala Val Asn 245 250 255Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met 260 265 270Val Asp His Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Lys Thr Leu 275 280 285Lys Phe Trp Thr Ile Asp Leu Thr Lys Pro Ile Lys Met Val Glu Leu 290 295 300Glu Glu Ser Leu Ser Asp Met Ile Glu Thr Ile Ile Gly Lys Lys Pro305 310 315 320Val Leu Ile Pro Ile Ala Gly His Asp Ala Ser Pro Leu Asp Val Asp 325 330 335Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly 340 345 350Val Val Val Gly Tyr Ser Arg Asn Lys Leu Thr Glu Lys Ala Leu Thr 355 360 365Lys Ala Gly Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu 370 375 380Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp385 390 395 400Ile Lys11430PRTMycoplasma fermentans 11Met Gln Ile Ile Ala Lys Ile Asp Leu Leu Thr Asn Met Leu Ile Phe1 5 10 15Met Lys Ile Tyr Phe Ile Gly Arg Leu Ile Met Lys Lys Ile Asn Val 20 25 30Tyr Ser Glu Tyr Gly Lys Leu Lys Glu Val Leu Val His Thr Pro Gly 35 40 45Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg Leu Asp Glu Leu Leu Phe 50 55 60Ser Ala Ile Leu Glu Pro Asp Ser Ala Ile Ala Glu His Lys Arg Phe65 70 75 80Val Gln Leu Leu Lys Asp Asn Gly Ile Lys Val Ile Gln Leu Asp Glu 85 90 95Leu Phe Ala Lys Thr Phe Asp Leu Val Ser Glu Ser Val Lys Gln Ser 100 105 110Leu Ile Glu Arg Trp Leu Asp Glu Cys Glu Pro Lys Leu Asp Ala Thr 115 120 125Leu Arg Ala Lys Val Lys Glu Tyr Ile Leu Glu Leu Lys Ala Lys Ser 130 135 140Ser Lys Lys Met Val Arg Val Met Met Ala Gly Ile Asp Lys Lys Glu145 150 155 160Leu Gly Ile Glu Leu Asp Arg Asp Leu Val Val Asp Pro Met Pro Asn 165 170 175Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Ile Ser 180 185 190Leu His His Met Lys Tyr Val Thr Arg Gln Arg Glu Thr Ile Phe Ser 195 200 205Glu Phe Ile Phe Asp Asn Asn Leu Asp Tyr Asn Thr Val Pro Arg Trp 210 215 220Phe Asp Arg Lys Asp Glu Gly Arg Ile Glu Gly Gly Asp Val Phe Ile225 230 235 240Tyr Ser Ala Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asn Lys 245 250 255Glu Ala Ile Asn Val Met Ala Arg Lys Ile Ala Ala Asp Lys Glu Val 260 265 270Lys Phe Lys Arg Ile Tyr Ala Ile Asn Val Pro Pro Met Pro Asn Leu 275 280 285Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu 290 295 300Tyr Ser Pro Asn Met Leu Ser Val Leu Lys Val Trp Arg Ile Asp Leu305 310 315 320Asn Asp Pro Asp Phe Val Trp His Glu Ile Glu Gly Ser Leu Glu Glu 325 330 335Ile Leu Glu Gln Ile Ile Gly Met Lys Pro Ile Leu Ile Pro Ile Ala 340 345 350Gly Lys Gly Ala Ser Gln Leu Asp Ile Asp Ile Glu Thr His Phe Asp 355 360 365Gly Thr Asn Tyr Leu Thr Ile Ala Pro Ser Val Val Val Gly Tyr Ser 370 375 380Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala Ala Lys Val Lys Val385 390 395 400Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ala Arg 405 410 415Cys Met Ser Met Pro Leu Ile Arg Glu Asp Ile Lys Lys Lys 420 425 43012452PRTMycoplasma penetrans 12Met Val Ile Thr Ile Ala Leu Asn Ile Leu Asn Lys Ile Tyr Phe Lys1 5 10 15Pro Gln Asn Arg Ser Ile Leu Lys Leu Tyr Arg Leu Pro Ser Leu Cys 20 25 30Thr Gln Ile Ser Ile Phe Ile Gly Gly Lys Met Ser Ser Ile Asp Lys 35 40 45Asn Ser Leu Gly Asn Gly Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu 50 55 60Lys Glu Val Leu Val His Thr Pro Gly Asp Glu Ile Arg Tyr Thr Ala65 70 75 80Pro Ser Arg Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Lys Ala Asp 85 90 95Thr Ala Ile Glu Glu His Lys Gly Phe Val Lys Ile Leu Gln Asn Asn 100 105 110Gly Ile Lys Val Ile Gln Leu Cys Asp Leu Val Ala Glu Thr Tyr Glu 115 120 125Leu Cys Ser Lys Glu Val Arg Asn Ser Phe Ile Glu Gln Tyr Leu Asp 130 135 140Glu Ala Leu Pro Val Leu Lys Lys Glu Ile Arg Pro Val Val Lys Asp145 150 155 160Tyr Leu Leu Ser Phe Pro Thr Val Gln Met Val Arg Lys Met Met Ser 165 170 175Gly Ile Leu Ala Asn Glu Leu Asn Ile Lys Gln Asp Asn Pro Leu Ile 180 185 190Ile Asp Gly Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser 195 200 205Met Gly Asn Gly Val Ser Ile Asn Cys Met Lys Tyr Pro Thr Arg Lys 210 215 220Arg Glu Val Ile Phe Ser Arg Phe Val Phe Thr Asn Asn Pro Lys Tyr225 230 235 240Lys Asn Thr Pro Arg Tyr Phe Asp Ile Val Gly Asn Asn Gly Thr Ile 245 250 255Glu Gly Gly Asp Ile Phe Ile Tyr Asn Ser Lys Thr Leu Val Ile Gly 260 265 270Asn Ser Glu Arg Thr Asn Phe Ala Ala Ile Glu Ser Val Ala Lys Asn 275 280 285Ile Gln Ala Asn Lys Asp Cys Thr Phe Glu Arg Ile Val Val Ile Asn 290 295 300Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met305 310 315 320Leu Asp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Asn Val Leu 325 330 335Lys Ile Trp Glu Ile Asp Leu Asn Val Lys Pro Val Lys Phe Val Glu 340 345 350Lys Lys Gly Thr Leu Glu Glu Val Leu Tyr Ser Ile Ile Asp Lys Lys 355 360 365Pro Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Asn Gln Leu Asp Ile 370 375 380Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro385 390 395 400Gly Val Val Val Gly Tyr Glu Arg Asn Glu Lys Thr Gln Lys Ala

Leu 405 410 415Val Glu Ala Gly Ile Lys Val Leu Ser Phe Asn Gly Ser Gln Leu Ser 420 425 430Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu 435 440 445Asn Leu Lys Lys 45013408PRTMycoplasma gallisepticum 13Met Arg Arg Lys Phe Met Phe Asn Lys Ile Arg Val Tyr Ser Glu Ile1 5 10 15Gly Lys Leu Arg Lys Val Leu Val His Thr Pro Gly Lys Glu Leu Asp 20 25 30Tyr Val Thr Pro Gln Arg Leu Asp Glu Leu Leu Phe Ser Ser Leu Leu 35 40 45Asn Pro Ile Lys Ala Arg Gln Glu His Glu Thr Phe Ile Lys Leu Leu 50 55 60Glu Asp His Asp Val Glu Cys Val Gln Leu Ser Thr Leu Thr Ala Gln65 70 75 80Thr Phe Gln Ala Val Asn Ser Lys Ile Gln Glu Glu Phe Ile Asn Arg 85 90 95Trp Leu Asp Glu Cys Leu Pro Val Leu Ser Glu Ile Asn Arg Leu Lys 100 105 110Val Tyr Asp Tyr Leu Lys Ser Leu Ala Thr Asn Pro Gln Val Met Ile 115 120 125Arg Lys Met Met Ser Gly Ile Leu Ala Lys Glu Val Gly Ile Gln Ser 130 135 140Glu Val Glu Leu Val Ala Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Phe Ala Ser Ile Gly Lys Gly Ile Thr Leu His Ser Met Phe 165 170 175His Pro Thr Arg Lys Arg Glu Thr Ile Phe Ala Asp Phe Ile Phe Ser 180 185 190His His Pro Glu Tyr Lys Asn Ala Pro Lys Tyr Tyr Ser Arg Glu Asp 195 200 205Lys Tyr Ser Ile Glu Gly Gly Asp Leu Phe Val Tyr Asp Asp Lys Thr 210 215 220Leu Val Ile Gly Val Ser Glu Arg Thr Glu Lys Lys Ala Ile Gln Ser225 230 235 240Leu Ala Glu Lys Leu Arg Gln Asn Asp Glu Thr Ser Phe Glu Lys Ile 245 250 255Tyr Ala Ile Asn Val Pro Lys Met Ser Asn Leu Met His Leu Asp Thr 260 265 270Trp Leu Thr Met Leu Asp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met 275 280 285Met Gly Val Leu Lys Ile Trp Glu Ile Asp Leu Ile His Pro Thr Leu 290 295 300Ile Trp Arg Glu Leu Asn Glu Ser Leu Glu Gly Phe Leu Ser Met Val305 310 315 320Ile Gly Lys Lys Ala Thr Leu Ile Pro Val Ala Gly Glu Asp Ser Thr 325 330 335Gln Ile Glu Ile Asp Val Glu Thr Asn Phe Asp Ala Thr Asn Phe Leu 340 345 350Val Ile Gln Pro Gly Val Val Val Gly Tyr Asp Arg Asn Tyr Lys Thr 355 360 365Asn Gln Ala Leu Arg Asp Ala Gly Val Lys Val Ile Ser Trp Asn Gly 370 375 380Asp Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro385 390 395 400Leu Tyr Arg Asp Pro Ile Lys Lys 40514402PRTMycoplasma alligatoris 14Met Ser Lys Ile Asn Val Tyr Ser Glu Val Gly Arg Leu Lys Glu Val1 5 10 15Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Thr Arg 20 25 30Leu Glu Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Thr Ala Ile 35 40 45Glu Glu His Lys Arg Phe Leu Asn Val Leu Glu Lys Asn Gly Ile Lys 50 55 60Ala Ile Gln Leu Asp Glu Leu Val Ala Gln Thr Tyr Asp Gln Val Asp65 70 75 80Gln Lys Ile Lys Asp Glu Phe Ile Asp Gln Trp Leu Gln Glu Ala Lys 85 90 95Pro Val Leu Asn Asp Gln Leu Lys Lys Leu Val Lys Asn Tyr Leu Leu 100 105 110Lys Ser Gln Lys Glu Phe Ser Thr Lys Lys Met Val Arg Ile Met Met 115 120 125Ala Gly Ile Asp Lys Lys Glu Ile Asn Ile Asp Leu Asp Arg Asp Leu 130 135 140Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala145 150 155 160Ser Val Gly Asn Gly Ile Ser Leu His Asn Met Lys Tyr Gln Thr Arg 165 170 175Lys Arg Glu Thr Ile Phe Ala Gln Phe Ile Phe Lys Tyr Asn Lys Asp 180 185 190Tyr Lys Thr Thr Pro His Trp Phe Asp Arg Phe Asp His Gly Ser Ile 195 200 205Glu Gly Gly Asp Val Phe Val Tyr Thr Lys Asp Thr Leu Val Ile Gly 210 215 220Ile Ser Glu Arg Thr Thr Lys Glu Ala Val Leu Asn Ile Ala Lys Lys225 230 235 240Ile Lys Ala Asn Thr Asp Ser Lys Phe Lys Lys Ile Val Ala Ile Asn 245 250 255Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Ile Thr Met 260 265 270Val Asp His Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Lys Ser Leu 275 280 285Lys Phe Trp Leu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Leu 290 295 300Glu Glu Ser Leu Ser Asn Met Leu Glu Ala Ile Ile Gly Lys Lys Pro305 310 315 320Ile Leu Ile Pro Ile Ala Gly Lys Asn Ala Ser Gln Leu Asp Ile Asp 325 330 335Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly 340 345 350Val Val Val Gly Tyr Ser Arg Asn Lys Leu Thr Gln Lys Ala Leu Glu 355 360 365Asp Ala Gly Val Lys Val Leu Ser Phe Asp Gly Asn Gln Leu Ser Leu 370 375 380Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp385 390 395 400Ile Lys15438PRTMycoplasma pneumoniae 15Met Ser Lys Lys Gln Leu Val Lys Thr Asp Gly His Asn Gln Leu Asp1 5 10 15Gln Pro Asn Thr Lys Ala Leu Gln Leu Lys Lys Lys Gln Phe Asn Ser 20 25 30Gly Val Arg Val Thr Ser Glu Ile Ser Phe Leu Arg Glu Val Ile Ala 35 40 45His His Pro Gly Ile Glu Thr Glu Arg Val Ile Asp Asn Gln Thr Phe 50 55 60Gly Ser Ala Met Tyr Leu Glu Arg Ala Gln Lys Glu His Gln Leu Phe65 70 75 80Ile Lys Ile Leu Arg Gln His Gly Thr Lys Val His Tyr Leu Gln Asp 85 90 95Leu Leu Leu Glu Ala Leu Ser Ala Ala Asp Pro Asn Val Arg Gln Asp 100 105 110Phe Ile Lys Asn Phe Leu Leu Glu Ser Gly Ile Lys Ser Val Ser Thr 115 120 125Phe Glu Ala Cys Leu Asn Phe Phe Arg Ser Leu Asp Ser Leu Val Asp 130 135 140Val Ile Lys Val Met Phe Gly Gly Ile Lys Val Ser Asp Val Pro Pro145 150 155 160Ile Thr Pro Gln Arg Phe Ala Asp Ile His Val Ser Asn Ser Pro Phe 165 170 175Leu Ile Lys Pro Leu Ser Phe Ser Leu Tyr Pro His Lys Phe Phe Asn 180 185 190Thr Leu Gly Thr Gly Val Ala Leu Phe Val Thr Asn Asp Ser Glu Leu 195 200 205Lys Arg His Ser Leu Val Tyr Glu Tyr Ile Met Arg Phe His Pro Arg 210 215 220Phe Asp Gly Val Lys Leu Tyr Thr Asn Arg Asp Phe Lys Asn Cys Leu225 230 235 240Ile Asn Ser Ser Asp Ile Ile Gln Ile Ser Asn Glu Ile Leu Leu Ile 245 250 255Gly Ile Ser His Asp Thr Asp Val Leu Gly Ile Glu Ser Leu Ala Arg 260 265 270Asn Leu Leu Ser Asp His Thr Asn Pro Ile Lys Gln Ile Ile Ala Ile 275 280 285Asn Ile His Lys Phe Gly Ala Lys Thr Asn Leu Asn Lys Leu Ile Ala 290 295 300Met Val Asp Val Asp Lys Phe Ile Ile Ala Arg Lys Val Leu Gln Ala305 310 315 320Thr Glu Ile Phe Glu Leu Thr Ala Thr Ala Gln Arg Asp Val Asp Gly 325 330 335Leu Ala Gln Ile Lys Phe Lys Pro Leu Lys Phe Asn Phe Gly Glu Ile 340 345 350Ile Glu Ala Ile Ile Asp Lys Gln Pro Arg Phe Val Ile Ile Gly Gly 355 360 365Gly Asp Glu Val Ala Glu Arg Lys Glu Leu Leu Asp Cys Gly Met Gly 370 375 380Val Leu Asn Leu Ser Pro Gly Glu Ile Val Val Phe Asp Arg Asn His385 390 395 400Tyr Thr Asn Asn Leu Leu Asn Glu Leu Gly Leu Ile Ile His Lys Ile 405 410 415Pro Ala Ser Glu Leu Ser Arg Gly Pro Ser Gly Pro Leu Glu Met Val 420 425 430Cys Ser Leu Trp Arg Glu 43516409PRTMycoplasma mobile 16Met Lys Asp Thr Lys Asp Ile Ile Asn Val Phe Ser Glu Ile Gly Glu1 5 10 15Leu Lys Lys Val Leu Ile His Thr Pro Gly Asn Glu Leu Lys Tyr Val 20 25 30Ser Pro Tyr Arg Leu Asp Glu Leu Leu Phe Ser Asn Val Leu Glu Trp 35 40 45Arg Glu Ala Lys Lys Glu His Asn Glu Phe Ile Gln Lys Leu Lys Ser 50 55 60Glu Gly Val Glu Pro Val Glu Leu Thr Asp Leu Val Ala Glu Ser Phe65 70 75 80Glu Glu Ser Ser Ile Lys Val Lys Asn Asp Phe Ile Arg Gln Tyr Leu 85 90 95Asp Glu Ala Thr Pro Ile Leu Asp Gly Leu Thr Lys Gln Lys Leu Leu 100 105 110Pro Phe Phe Leu Asp Ile Lys His Ser Thr Arg Lys Thr Ile Glu Leu 115 120 125Met Met Ser Gly Ile Thr Gln Lys Asp Ile Ser Ile Ser His Ile Glu 130 135 140Arg Glu Leu Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Ser Arg Asp145 150 155 160Asn Phe Ile Ser Ile Gly Asn Ser Val Ile Ile Ser Asn Met Lys Tyr 165 170 175Lys Thr Arg Lys Arg Glu Thr Ile Phe Thr Asp Phe Ile Phe Lys Asn 180 185 190His Pro Leu Tyr Lys Lys Val Asn Met Ala Phe Glu Arg Lys Asp Leu 195 200 205Asn Asn Gln Ile Ser Ile Ile Glu Gly Gly Asp Val Leu Val Tyr Ser 210 215 220Lys Glu Ile Leu Ile Ile Gly Ile Ser Glu Arg Thr Thr Met Ser Ala225 230 235 240Ile Leu Glu Leu Ala Glu Asn Phe Lys Lys Thr Lys Arg Ser Phe Lys 245 250 255Lys Ile Tyr Gly Val Glu Val Pro Lys Met Lys Asn Leu Met His Leu 260 265 270Asp Thr Trp Leu Thr Met Ile Asp Tyr Asp Lys Phe Ile Tyr Ser Pro 275 280 285Asn Val Leu Thr Asp Leu Lys Phe Trp Glu Ile Asn Leu Asp Tyr Glu 290 295 300Lys Ile Ser Ser Lys Glu Leu His Ala Ser Leu Ser Glu Phe Leu Lys305 310 315 320Leu Ile Ile Gly Lys Asp Pro Ile Leu Ile Pro Ile Gly Gly Lys Gly 325 330 335Ala Ser Gln Ile Thr Ile Asp Ile Glu Thr Asn Phe Val Ala Ala Asn 340 345 350Tyr Leu Val Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Tyr 355 360 365Glu Thr Gln Lys Ala Leu Glu Gly His Gly Val Lys Val Ile Ala Phe 370 375 380Glu Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ser Arg Cys Met Ser385 390 395 400Met Pro Leu Ile Arg Ser Asn Leu Lys 40517411PRTStreptococcus pyogenes 17Met Thr Ala Gln Thr Pro Ile His Val Tyr Ser Glu Ile Gly Lys Leu1 5 10 15Lys Lys Val Leu Leu His Arg Pro Gly Lys Glu Ile Glu Asn Leu Met 20 25 30Pro Asp Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu 35 40 45Asp Ala Gln Lys Glu His Asp Ala Phe Ala Gln Ala Leu Arg Asp Glu 50 55 60Gly Ile Glu Val Leu Tyr Leu Glu Thr Leu Ala Ala Glu Ser Leu Val65 70 75 80Thr Pro Glu Ile Arg Glu Ala Phe Ile Asp Glu Tyr Leu Ser Glu Ala 85 90 95Asn Ile Arg Gly Arg Ala Thr Lys Lys Ala Ile Arg Glu Leu Leu Met 100 105 110Ala Ile Glu Asp Asn Gln Glu Leu Ile Glu Lys Thr Met Ala Gly Val 115 120 125Gln Lys Ser Glu Leu Pro Glu Ile Pro Ala Ser Glu Lys Gly Leu Thr 130 135 140Asp Leu Val Glu Ser Asn Tyr Pro Phe Ala Ile Asp Pro Met Pro Asn145 150 155 160Leu Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Val Ser 165 170 175Leu Asn His Met Phe Ser Glu Thr Arg Asn Arg Glu Thr Leu Tyr Gly 180 185 190Lys Tyr Ile Phe Thr His His Pro Ile Tyr Gly Gly Gly Lys Val Pro 195 200 205Met Val Tyr Asp Arg Asn Glu Thr Thr Arg Ile Glu Gly Gly Asp Glu 210 215 220Leu Val Leu Ser Lys Asp Val Leu Ala Val Gly Ile Ser Gln Arg Thr225 230 235 240Asp Ala Ala Ser Ile Glu Lys Leu Leu Val Asn Ile Phe Lys Gln Asn 245 250 255Leu Gly Phe Lys Lys Val Leu Ala Phe Glu Phe Ala Asn Asn Arg Lys 260 265 270Phe Met His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe 275 280 285Thr Ile His Pro Glu Ile Glu Gly Asp Leu Arg Val Tyr Ser Val Thr 290 295 300Tyr Asp Asn Glu Glu Leu His Ile Val Glu Glu Lys Gly Asp Leu Ala305 310 315 320Glu Leu Leu Ala Ala Asn Leu Gly Val Glu Lys Val Asp Leu Ile Arg 325 330 335Cys Gly Gly Asp Asn Leu Val Ala Ala Gly Arg Glu Gln Trp Asn Asp 340 345 350Gly Ser Asn Thr Leu Thr Ile Ala Pro Gly Val Val Val Val Tyr Asn 355 360 365Arg Asn Thr Ile Thr Asn Ala Ile Leu Glu Ser Lys Gly Leu Lys Leu 370 375 380Ile Lys Ile His Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg385 390 395 400Cys Met Ser Met Pro Phe Glu Arg Glu Asp Ile 405 41018408PRTEnterococcus faecalis 18Met Ser His Pro Ile Asn Val Phe Ser Glu Ile Gly Lys Leu Lys Thr1 5 10 15Val Met Leu His Arg Pro Gly Lys Glu Leu Glu Asn Leu Met Pro Asp 20 25 30Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Lys Ala 35 40 45Gln Ala Glu His Asp Ala Phe Ala Glu Leu Leu Arg Ser Lys Asp Ile 50 55 60Glu Val Val Tyr Leu Glu Asp Leu Ala Ala Glu Ala Leu Ile Asn Glu65 70 75 80Glu Val Arg Arg Gln Phe Ile Asp Gln Phe Leu Glu Glu Ala Asn Ile 85 90 95Arg Ser Glu Ser Ala Lys Glu Lys Val Arg Glu Leu Met Leu Glu Ile 100 105 110Asp Asp Asn Glu Glu Leu Ile Gln Lys Ala Ile Ala Gly Ile Gln Lys 115 120 125Gln Glu Leu Pro Lys Tyr Glu Gln Glu Phe Leu Thr Asp Met Val Glu 130 135 140Ala Asp Tyr Pro Phe Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr145 150 155 160Arg Asp Asn Phe Ala Thr Met Gly His Gly Ile Ser Leu Asn His Met 165 170 175Tyr Ser Val Thr Arg Gln Arg Glu Thr Ile Phe Gly Gln Tyr Ile Phe 180 185 190Asp Tyr His Pro Arg Phe Ala Gly Lys Glu Val Pro Arg Val Tyr Asp 195 200 205Arg Ser Glu Ser Thr Arg Ile Glu Gly Gly Asp Glu Leu Ile Leu Ser 210 215 220Lys Glu Val Val Ala Ile Gly Ile Ser Gln Arg Thr Asp Ala Ala Ser225 230 235 240Ile Glu Lys Ile Ala Arg Asn Ile Phe Glu Gln Lys Leu Gly Phe Lys 245 250 255Asn Ile Leu Ala Phe Asp Ile Gly Glu His Arg Lys Phe Met His Leu 260 265 270Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Ile His Pro 275 280 285Glu Ile Glu Gly Gly Leu Val Val Tyr Ser Ile Thr Glu Lys Ala Asp 290 295 300Gly Asp Ile Gln Ile Thr Lys Glu Lys Asp Thr Leu Asp Asn Ile Leu305 310 315 320Cys Lys Tyr Leu His Leu Asp Asn Val Gln Leu Ile Arg Cys Gly Ala 325 330 335Gly Asn Leu Thr Ala

Ala Ala Arg Glu Gln Trp Asn Asp Gly Ser Asn 340 345 350Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asp Arg Asn Thr 355 360 365Ile Thr Asn Lys Ala Leu Glu Glu Ala Gly Val Lys Leu Asn Tyr Ile 370 375 380Pro Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg Cys Met Ser385 390 395 400Met Pro Leu Tyr Arg Glu Asp Leu 40519403PRTMycoplasma capricolum 19Met Glu Lys Lys Ile Asn Val Phe Ser Glu Ile Gly Thr Leu Lys Thr1 5 10 15Val Leu Val His Arg Pro Gly Asp Glu Ile Glu Asn Leu Thr Pro Glu 20 25 30Leu Leu Glu Arg Leu Leu Phe Asp Asp Val Pro Phe Lys Asp Val Ala 35 40 45Val Lys Glu His Asp Ala Phe Thr Lys Ile Met Arg Asp Asn Gly Val 50 55 60Glu Val Leu Tyr Ile Glu Lys Leu Ala Ala Glu Thr Leu Asp Gln His65 70 75 80Pro Asp Leu Arg Glu Lys Phe Ile Asp Gln Phe Ile Ser Glu Ala Asn 85 90 95Ile Glu Asp Lys Tyr Lys Glu Lys Tyr Arg Asp Phe Ile Ser Ser Leu 100 105 110Asp Asn Tyr Arg Met Ile Lys Lys Met Ile Ala Gly Thr Lys Lys Leu 115 120 125Glu Leu Gly Ile Asp Glu Gly Tyr Lys Ala Tyr Pro Phe Ile Ala Asp 130 135 140Pro Leu Pro Asn Val Leu Phe Gln Arg Asp Pro Phe Ser Ser Val Gly145 150 155 160Phe Gly Ile Thr Met Asn Arg Met Trp Ser Val Thr Arg Asn Arg Glu 165 170 175Thr Ile Phe Pro Asp Leu Val Phe Lys His His Asn Arg Phe Ala Asn 180 185 190Gln Val Pro Tyr Tyr Tyr Glu Arg Asp Trp Lys Glu Glu Thr Ile Glu 195 200 205Gly Gly Asp Ile Leu Val Leu Asn Lys Glu Thr Leu Ile Ile Gly Val 210 215 220Thr Gln Arg Thr Thr Leu Lys Ala Ile Glu Lys Phe Ser Glu Arg Leu225 230 235 240Phe Asn Asp Pro Glu Ser Ser Tyr Ser Lys Val Ile Ala Leu Asp Leu 245 250 255Pro Lys Ser Arg Ala Phe Met His Leu Asp Thr Val Phe Thr Asn Ile 260 265 270Asp Tyr Asp Lys Phe Ile Ala His Pro Leu Ile Phe Asp Cys Ile Asp 275 280 285Glu Phe Lys Ile Tyr Glu Val Ser Lys Gln Gly Thr Lys Glu Val Lys 290 295 300Lys Thr Leu Ile Glu Leu Leu Ser Asp Ala Ala Gly Arg Glu Val Gln305 310 315 320Ile Ile Arg Cys Gly Gly Asn Asp Val Val Gly Ala Ser Arg Glu Gln 325 330 335Trp Asn Asp Gly Thr Asn Val Val Ala Leu Arg Pro Gly Lys Val Ile 340 345 350Ala Tyr Glu Arg Asn Trp Ile Thr Ile Asp Leu Leu Arg Lys Ala Gly 355 360 365Val Glu Val Leu Thr Ile Ala Ser Ser Glu Leu Ser Arg Gly Arg Gly 370 375 380Gly Pro Arg Cys Met Thr Met Pro Leu Trp Arg Glu Asp Leu Gln Glu385 390 395 400Ile Lys Arg20410PRTHalothermothrix orenii 20Met Phe Lys Lys Ser Pro Leu Asn Val Thr Ser Glu Ile Gly Lys Leu1 5 10 15Lys Lys Val Leu Leu His Arg Pro Gly His Glu Ile Glu Asn Leu Thr 20 25 30Pro Asp Leu Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu Lys 35 40 45Val Ala Gln Glu Glu His Asp Ala Phe Ala Gln Thr Leu Arg Asp Asn 50 55 60Gly Val Glu Val Leu Tyr Leu His Glu Leu Ala Ala Glu Ala Ile Gln65 70 75 80Glu Asp Glu Ile Arg Lys Lys Phe Ile Glu Gln Phe Leu Asp Glu Ala 85 90 95Gly Val Ile Gly Lys Gly Ala Arg Gln Val Leu Lys Glu Tyr Phe Ala 100 105 110Asp Met Asp Asn Glu Thr Leu Ile Arg Lys Met Met Ala Gly Val Arg 115 120 125Lys Lys Glu Ile Pro Ala Ile Glu Lys Val Ala Ser Leu Asn Asp Met 130 135 140Val Glu Glu Asp Tyr Pro Phe Val Leu Asp Pro Met Pro Asn Leu Tyr145 150 155 160Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Ile Thr Leu Asn 165 170 175His Met Arg Thr Glu Thr Arg Asn Arg Glu Val Ile Phe Ala Glu Tyr 180 185 190Ile Phe Ser Tyr His Pro Asp Phe Lys Asp Thr Glu Ile Pro Phe Trp 195 200 205Phe Asp Arg Asn Glu Thr Thr Ser Ile Glu Gly Gly Asp Glu Leu Ile 210 215 220Leu Ser Asp Lys Val Leu Ala Met Gly Ile Ser Glu Arg Thr Asp Ala225 230 235 240Ala Ser Ile Glu Lys Val Ala Arg Asn Ile Phe Thr Asp Gly Gln Pro 245 250 255Phe Glu Thr Ile Leu Ala Phe Lys Ile Pro Glu Lys Arg Ala Phe Met 260 265 270His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe Thr Ile 275 280 285His Ala Glu Ile Glu Gly Pro Leu Lys Val Tyr Ser Ile Thr Lys Gly 290 295 300Asp Asn Asp Glu Leu Lys Ile Asp Glu Glu Lys Ala Thr Leu Glu Asp305 310 315 320Thr Leu Lys Lys Tyr Leu Gly Leu Asp Glu Val Thr Leu Ile Arg Cys 325 330 335Ala Gly Gly Asp Tyr Ile Asp Ala Gly Arg Glu Gln Trp Asn Asp Gly 340 345 350Ser Asn Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asn Arg 355 360 365Asn His Thr Thr Asn Arg Leu Leu Glu Glu His Gly Ile Lys Leu His 370 375 380Val Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys385 390 395 400Met Ser Met Pro Leu Val Arg Glu Asp Ile 405 41021411PRTStaphylococcus aureus 21Met Thr Asp Gly Pro Ile Lys Val Asn Ser Glu Ile Gly Ala Leu Lys1 5 10 15Thr Val Leu Leu Lys Arg Pro Gly Lys Glu Leu Glu Asn Leu Val Pro 20 25 30Asp Tyr Leu Asp Gly Leu Leu Phe Asp Asp Ile Pro Tyr Leu Glu Val 35 40 45Ala Gln Lys Glu His Asp His Phe Ala Gln Val Leu Arg Glu Glu Gly 50 55 60Val Glu Val Leu Tyr Leu Glu Lys Leu Ala Ala Glu Ser Ile Glu Asn65 70 75 80Pro Gln Val Arg Ser Glu Phe Ile Asp Asp Val Leu Ala Glu Ser Lys 85 90 95Lys Thr Ile Leu Gly His Glu Glu Glu Ile Lys Ala Leu Phe Ala Thr 100 105 110Leu Ser Asn Gln Glu Leu Val Asp Lys Ile Met Ser Gly Val Arg Lys 115 120 125Glu Glu Ile Asn Pro Lys Cys Thr His Leu Val Glu Tyr Met Asp Asp 130 135 140Lys Tyr Pro Phe Tyr Leu Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Gln Ala Ser Ile Gly His Gly Ile Thr Ile Asn Arg Met Phe 165 170 175Trp Arg Ala Arg Arg Arg Glu Ser Ile Phe Ile Gln Tyr Ile Val Lys 180 185 190His His Pro Arg Phe Lys Asp Ala Asn Ile Pro Ile Trp Leu Asp Arg 195 200 205Asp Cys Pro Phe Asn Ile Glu Gly Gly Asp Glu Leu Val Leu Ser Lys 210 215 220Asp Val Leu Ala Ile Gly Val Ser Glu Arg Thr Ser Ala Gln Ala Ile225 230 235 240Glu Lys Leu Ala Arg Arg Ile Phe Glu Asn Pro Gln Ala Thr Phe Lys 245 250 255Lys Val Val Ala Ile Glu Ile Pro Thr Ser Arg Thr Phe Met His Leu 260 265 270Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Met His Ser 275 280 285Ala Ile Leu Lys Ala Glu Gly Asn Met Asn Ile Phe Ile Ile Glu Tyr 290 295 300Asp Asp Val Asn Lys Asp Ile Ala Ile Lys Gln Ser Ser His Leu Lys305 310 315 320Asp Thr Leu Glu Asp Val Leu Gly Ile Asp Asp Ile Gln Phe Ile Pro 325 330 335Thr Gly Asn Gly Asp Val Ile Asp Gly Ala Arg Glu Gln Trp Asn Asp 340 345 350Gly Ser Asn Thr Leu Cys Ile Arg Pro Gly Val Val Val Thr Tyr Asp 355 360 365Arg Asn Tyr Val Ser Asn Asp Leu Leu Arg Gln Lys Gly Ile Lys Val 370 375 380Ile Glu Ile Ser Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg385 390 395 400Cys Met Ser Gln Pro Leu Phe Arg Glu Asp Ile 405 41022417PRTPseudomonas plecoglossicida 22Met Ser Ala Glu Lys Gln Lys Tyr Gly Val His Ser Glu Ala Gly Lys1 5 10 15Leu Arg Lys Val Met Val Cys Ala Pro Gly Leu Ala His Lys Arg Leu 20 25 30Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val 35 40 45Asp Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu 50 55 60Arg Gly Val Asp Val Leu Glu Met His Asn Leu Leu Thr Asp Ile Val65 70 75 80Gln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Pro 85 90 95Asp Thr Val Gly Val Gly Leu Thr Asn Glu Val Arg Ser Trp Leu Glu 100 105 110Gly Gln Glu Pro Arg His Leu Ala Glu Phe Leu Ile Gly Gly Val Ala 115 120 125Gly Gln Asp Leu Pro Glu Ser Glu Gly Ala Ser Val Val Lys Met Tyr 130 135 140Asn Asp Tyr Leu Gly His Ser Ser Phe Ile Leu Pro Pro Leu Pro Asn145 150 155 160Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr 165 170 175Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr 180 185 190Thr Ala Ile Tyr Lys Phe His Pro Glu Phe Thr Lys Ala Asp Phe Gln 195 200 205Val Trp Tyr Gly Asp Pro Asp Gln Glu His Gly Gln Ala Thr Leu Glu 210 215 220Gly Gly Asp Val Met Pro Ile Gly Lys Gly Ile Val Leu Ile Gly Met225 230 235 240Gly Glu Arg Thr Ser Arg Gln Ala Ile Gly Gln Leu Ala Gln Asn Leu 245 250 255Phe Ala Lys Gly Ala Val Glu Gln Val Ile Val Ala Gly Leu Pro Lys 260 265 270Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg 275 280 285Asp Leu Val Thr Val Phe Pro Glu Val Val Arg Glu Ile Val Pro Phe 290 295 300Ile Ile Arg Pro Asp Glu Ser Lys Pro Tyr Gly Met Asp Val Arg Arg305 310 315 320Glu Asn Lys Ser Phe Ile Glu Val Val Gly Glu Gln Leu Gly Val Lys 325 330 335Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg Glu 340 345 350Gln Trp Asp Asp Gly Asn Asn Val Val Ala Leu Glu Pro Gly Val Val 355 360 365Ile Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys Ala 370 375 380Gly Ile Glu Val Ile Thr Ile Ser Ala Gly Glu Leu Gly Arg Gly Arg385 390 395 400Gly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile Asn 405 410 415Tyr23417PRTPseudomonas putida 23Met Ser Ala Glu Lys Gln Lys Tyr Gly Val His Ser Glu Ala Gly Lys1 5 10 15Leu Arg Lys Val Met Val Cys Ser Pro Gly Leu Ala His Lys Arg Leu 20 25 30Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val 35 40 45Asp Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu 50 55 60Arg Gly Val Asp Val Leu Glu Met His Asn Leu Leu Thr Asp Ile Val65 70 75 80Gln Gln Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Ser 85 90 95Asp Thr Val Gly Val Gly Leu Thr Asn Glu Val Arg Ser Trp Leu Glu 100 105 110Gly Leu Glu Pro Arg His Leu Ala Glu Phe Leu Ile Gly Gly Val Ala 115 120 125Gly Gln Asp Leu Pro Glu Ser Glu Gly Ala Ser Val Val Lys Met Tyr 130 135 140Asn Asp Tyr Leu Gly His Ser Ser Phe Ile Leu Pro Pro Leu Pro Asn145 150 155 160Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr 165 170 175Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr 180 185 190Thr Ala Ile Tyr Lys Phe His Lys Glu Phe Thr Gly Ala Asp Phe Gln 195 200 205Val Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Asn Ala Thr Leu Glu 210 215 220Gly Gly Asp Val Met Pro Ile Gly Lys Gly Ile Val Leu Ile Gly Met225 230 235 240Gly Glu Arg Thr Ser Arg Gln Ala Ile Gly Gln Leu Ala Gln Asn Leu 245 250 255Phe Ala Lys Gly Ala Val Glu Lys Val Ile Val Ala Gly Leu Pro Lys 260 265 270Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg 275 280 285Asp Leu Val Thr Val Phe Pro Glu Val Val Lys Glu Ile Val Pro Phe 290 295 300Ile Ile Arg Pro Asp Glu Ser Lys Pro Tyr Gly Met Asp Val Arg Arg305 310 315 320Glu Asn Lys Ser Phe Ile Glu Val Val Gly Glu Gln Leu Gly Val Lys 325 330 335Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg Glu 340 345 350Gln Trp Asp Asp Gly Asn Asn Val Val Ala Met Glu Pro Gly Val Val 355 360 365Ile Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys Ala 370 375 380Gly Ile Glu Val Ile Thr Ile Ser Ala Gly Glu Leu Gly Arg Gly Arg385 390 395 400Gly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile Asp 405 410 415Tyr24418PRTPseudomonas aeruginosa 24Met Ser Thr Glu Lys Thr Lys Leu Gly Val His Ser Glu Ala Gly Lys1 5 10 15Leu Arg Lys Val Met Val Cys Ser Pro Gly Leu Ala His Gln Arg Leu 20 25 30Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val 35 40 45Asn Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu 50 55 60Arg Gly Ile Asp Val Leu Glu Met His Asn Leu Leu Thr Glu Thr Ile65 70 75 80Gln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Ala 85 90 95Asp Ser Val Gly Leu Gly Leu Thr Ser Glu Leu Arg Ser Trp Leu Glu 100 105 110Ser Leu Glu Pro Arg Lys Leu Ala Glu Tyr Leu Ile Gly Gly Val Ala 115 120 125Ala Asp Asp Leu Pro Ala Ser Glu Gly Ala Asn Ile Leu Lys Met Tyr 130 135 140Arg Glu Tyr Leu Gly His Ser Ser Phe Leu Leu Pro Pro Leu Pro Asn145 150 155 160Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr 165 170 175Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr 180 185 190Thr Ala Ile Tyr Lys Phe His Pro Glu Phe Ala Asn Ala Glu Phe Glu 195 200 205Ile Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Ser Ser Thr Leu Glu 210 215 220Gly Gly Asp Val Met Pro Ile Gly Asn Gly Val Val Leu Ile Gly Met225 230 235 240Gly Glu Arg Ser Ser Arg Gln Ala Ile Gly Gln Val Ala Gln Ser Leu 245 250 255Phe Ala Lys Gly Ala Ala Glu Arg Val Ile Val Ala Gly Leu Pro Lys 260 265 270Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg 275 280 285Asp Leu Val Thr Val Phe Pro Glu Val Val Lys Glu Ile Val Pro Phe 290 295 300Ser Leu Arg Pro Asp Ala Ser Ser Pro Tyr Gly Met Ser Ile Arg Arg305 310 315 320Glu Glu

Lys Thr Phe Leu Glu Val Val Ala Glu Ser Leu Gly Leu Lys 325 330 335Lys Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg 340 345 350Glu Gln Trp Asp Asp Gly Asn Asn Val Val Cys Leu Glu Pro Gly Val 355 360 365Val Val Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys 370 375 380Ala Gly Val Glu Val Ile Thr Ile Ser Ala Ser Glu Leu Gly Arg Gly385 390 395 400Arg Gly Gly Gly His Cys Met Thr Cys Pro Ile Ile Arg Asp Pro Ile 405 410 415Asp Tyr25402PRTMycobacterium bovis 25Met Gly Val Glu Leu Gly Ser Asn Ser Glu Val Gly Ala Leu Arg Val1 5 10 15Val Ile Leu His Arg Pro Gly Ala Glu Leu Arg Arg Leu Thr Pro Arg 20 25 30Asn Thr Asp Gln Leu Leu Phe Asp Gly Leu Pro Trp Val Ser Arg Ala 35 40 45Gln Asp Glu His Asp Glu Phe Ala Glu Leu Leu Ala Ser Arg Gly Ala 50 55 60Glu Val Leu Leu Leu Ser Asp Leu Leu Thr Glu Ala Leu His His Ser65 70 75 80Gly Ala Ala Arg Met Gln Gly Ile Ala Ala Ala Val Asp Ala Pro Arg 85 90 95Leu Gly Leu Pro Leu Ala Gln Glu Leu Ser Ala Tyr Leu Arg Ser Leu 100 105 110Asp Pro Gly Arg Leu Ala His Val Leu Thr Ala Gly Met Thr Phe Asn 115 120 125Glu Leu Pro Ser Asp Thr Arg Thr Asp Val Ser Leu Val Leu Arg Met 130 135 140His His Gly Gly Asp Phe Val Ile Glu Pro Leu Pro Asn Leu Val Phe145 150 155 160Thr Arg Asp Ser Ser Ile Trp Ile Gly Pro Arg Val Val Ile Pro Ser 165 170 175Leu Ala Leu Arg Ala Arg Val Arg Glu Ala Ser Leu Thr Asp Leu Ile 180 185 190Tyr Ala His His Pro Arg Phe Thr Gly Val Arg Arg Ala Tyr Glu Ser 195 200 205Arg Thr Ala Pro Val Glu Gly Gly Asp Val Leu Leu Leu Ala Pro Gly 210 215 220Val Val Ala Val Gly Val Gly Glu Arg Thr Thr Pro Ala Gly Ala Glu225 230 235 240Ala Leu Ala Arg Ser Leu Phe Asp Asp Asp Leu Ala His Thr Val Leu 245 250 255Ala Val Pro Ile Ala Gln Gln Arg Ala Gln Met His Leu Asp Thr Val 260 265 270Cys Thr Met Val Asp Thr Asp Thr Met Val Met Tyr Ala Asn Val Val 275 280 285Asp Thr Leu Glu Ala Phe Thr Ile Gln Arg Thr Pro Asp Gly Val Thr 290 295 300Ile Gly Asp Ala Ala Pro Phe Ala Glu Ala Ala Ala Lys Ala Met Gly305 310 315 320Ile Asp Lys Leu Arg Val Ile His Thr Gly Met Asp Pro Val Val Ala 325 330 335Glu Arg Glu Gln Trp Asp Asp Gly Asn Asn Thr Leu Ala Leu Ala Pro 340 345 350Gly Val Val Val Ala Tyr Glu Arg Asn Val Gln Thr Asn Ala Arg Leu 355 360 365Gln Asp Ala Gly Ile Glu Val Leu Thr Ile Ala Gly Ser Glu Leu Gly 370 375 380Thr Gly Arg Gly Gly Pro Arg Cys Met Ser Cys Pro Ala Ala Arg Asp385 390 395 400Pro Leu26410PRTArtificial SequenceMycoplasma arginini arginine deiminase with flanking synthesized residues added 26Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu1 5 10 15Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu 50 55 60Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Ile Asp Leu Val Ala65 70 75 80Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu 85 90 95Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Val 100 105 110Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Glu Leu Val 115 120 125Glu Ile Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser 180 185 190Asn His Pro Lys Leu Ile Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu 195 200 205Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr Leu225 230 235 240Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Ser Glu Phe Lys Arg Ile 245 250 255Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln305 310 315 320Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu Gly 325 330 335Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser385 390 395 400Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 41027410PRTArtificial Sequencemycoplasma arthritidis arginine deiminase with flanking synthesized residues added 27Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu1 5 10 15Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu Ile 20 25 30Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Ala Glu 50 55 60Leu Lys Lys Arg Gly Ile Asn Val Val Glu Leu Val Asp Leu Ile Val65 70 75 80Glu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Lys Glu Lys Leu Leu Glu 85 90 95Glu Phe Leu Asp Asp Ser Val Pro Val Leu Ser Asp Glu His Arg Ala 100 105 110Thr Val Lys Lys Phe Leu Gln Ser Gln Lys Ser Thr Arg Ser Leu Val 115 120 125Glu Tyr Met Ile Ala Gly Ile Thr Lys His Asp Leu Lys Ile Glu Ser 130 135 140Asp Leu Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser 180 185 190Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala Glu 195 200 205Gly Leu Thr Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr Leu225 230 235 240Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Ser Glu Phe Lys Arg Ile 245 250 255Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp 290 295 300Ala Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Glu Asp Leu Leu Lys305 310 315 320Ser Ile Ile Gly Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Ala Gly 325 330 335Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350Tyr Leu Ala Val Ala Pro Gly Ile Val Ile Gly Tyr Ala Arg Asn Glu 355 360 365Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Thr Val Leu Pro Phe 370 375 380Arg Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser385 390 395 400Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 41028410PRTArtificial Sequencemycoplasma phocicerebrale arginine deiminase with flanking synthesized residues added 28Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu1 5 10 15Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys Gln 50 55 60Leu Lys Asp Asn Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala65 70 75 80Glu Thr Phe Asp Leu Ala Ser Lys Glu Glu Gln Glu Lys Leu Ile Glu 85 90 95Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Ala His Lys Thr 100 105 110Ala Val Arg Lys Phe Leu Thr Ser Arg Lys Ser Thr Arg Glu Met Val 115 120 125Glu Phe Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser 180 185 190Asn His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met 195 200 205Lys Met Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile Thr Leu225 230 235 240Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg Ile 245 250 255Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300Glu Pro Gln Pro Lys Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln305 310 315 320Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Asn Asn 325 330 335Ala Ser His Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350Tyr Leu Ala Ile Lys Pro Gly Val Val Ile Gly Tyr Ala Arg Asn Glu 355 360 365Lys Thr Asn Ala Ala Leu Ala Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser385 390 395 400Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 41029410PRTArtificial Sequencemycoplasma gateae arginine deiminase with flanking synthesized residues added 29Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu1 5 10 15Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Leu Phe Val Ser Glu 50 55 60Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val Thr65 70 75 80Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Asn Leu Ile Glu 85 90 95Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Thr Glu Glu Leu Lys Ser 100 105 110Val Val Arg Thr Tyr Leu Lys Ser Ile Lys Ser Thr Arg Glu Leu Ile 115 120 125Gln Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser 180 185 190Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu 195 200 205Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asn Thr 210 215 220Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Val Thr Leu225 230 235 240Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Glu 290 295 300Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Glu305 310 315 320Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu Gly 325 330 335Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser385 390 395 400Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 41030410PRTArtificial Sequencemycoplasma phocidae arginine deiminase with flanking synthesized residues added 30Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu1 5 10 15Ile Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30Glu Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Glu Phe Val Ala Glu 50 55 60Leu Lys Lys Asn Asn Ile Asn Val Val Glu Leu Thr Asp Leu Val Ser65 70 75 80Glu Thr Tyr Asp Met Val Ser Lys Glu Lys Gln Glu Lys Leu Ile Glu 85 90 95Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Gly 100 105 110Leu Val Arg Lys Phe Leu Lys Ser Leu Lys Ser Ser Lys Glu Leu Ile 115 120 125Gln Tyr Met Met Ala Gly Ile Thr Lys His Asp Leu Asn Ile Glu Ala 130 135 140Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg145 150 155 160Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Ala 180 185 190Asn His Pro Lys Leu Met Asn Thr Pro Leu Tyr Tyr Asn Pro Asp Met 195 200 205Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Asn Glu Thr 210 215 220Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu225 230 235 240Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Arg Glu Phe Lys Arg Ile

245 250 255Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp 290 295 300Glu Pro Gln Pro Lys Val Asn Gly Leu Pro Leu Glu Lys Leu Leu Glu305 310 315 320Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Thr Ser 325 330 335Ala Ser Asn Ile Asp Val Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350Tyr Leu Ala Ile Ala Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Val 355 360 365Lys Thr Asn Glu Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380Lys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser385 390 395 400Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 41031411PRTArtificial Sequencemycoplasma H.orenii arginine deiminase with flanking synthesized residues added 31Met Ser Phe Lys Lys Ser Pro Leu Asn Val Thr Ser Glu Ile Gly Lys1 5 10 15Leu Lys Lys Val Leu Leu His Arg Pro Gly His Glu Ile Glu Asn Leu 20 25 30Thr Pro Asp Leu Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu 35 40 45Lys Val Ala Gln Glu Glu His Asp Ala Phe Ala Gln Thr Leu Arg Asp 50 55 60Asn Gly Val Glu Val Leu Tyr Leu His Glu Leu Ala Ala Glu Ala Ile65 70 75 80Gln Glu Asp Glu Ile Arg Lys Lys Phe Ile Glu Gln Phe Leu Asp Glu 85 90 95Ala Gly Val Ile Gly Lys Gly Ala Arg Gln Val Leu Lys Glu Tyr Phe 100 105 110Ala Asp Met Asp Asn Glu Thr Leu Ile Arg Lys Met Met Ala Gly Val 115 120 125Arg Lys Lys Glu Ile Pro Ala Ile Glu Lys Val Ala Ser Leu Asn Asp 130 135 140Met Val Glu Glu Asp Tyr Pro Phe Val Leu Asp Pro Met Pro Asn Leu145 150 155 160Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Ile Thr Leu 165 170 175Asn His Met Arg Thr Glu Thr Arg Asn Arg Glu Val Ile Phe Ala Glu 180 185 190Tyr Ile Phe Ser Tyr His Pro Asp Phe Lys Asp Thr Glu Ile Pro Phe 195 200 205Trp Phe Asp Arg Asn Glu Thr Thr Ser Ile Glu Gly Gly Asp Glu Leu 210 215 220Ile Leu Ser Asp Lys Val Leu Ala Met Gly Ile Ser Glu Arg Thr Asp225 230 235 240Ala Ala Ser Ile Glu Lys Val Ala Arg Asn Ile Phe Thr Asp Gly Gln 245 250 255Pro Phe Glu Thr Ile Leu Ala Phe Lys Ile Pro Glu Lys Arg Ala Phe 260 265 270Met His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe Thr 275 280 285Ile His Ala Glu Ile Glu Gly Pro Leu Lys Val Tyr Ser Ile Thr Lys 290 295 300Gly Asp Asn Asp Glu Leu Lys Ile Asp Glu Glu Lys Ala Thr Leu Glu305 310 315 320Asp Thr Leu Lys Lys Tyr Leu Gly Leu Asp Glu Val Thr Leu Ile Arg 325 330 335Cys Ala Gly Gly Asp Tyr Ile Asp Ala Gly Arg Glu Gln Trp Asn Asp 340 345 350Gly Ser Asn Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asn 355 360 365Arg Asn His Thr Thr Asn Arg Leu Leu Glu Glu His Gly Ile Lys Leu 370 375 380His Val Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg385 390 395 400Cys Met Ser Met Pro Leu Val Arg Glu Asp Ile 405 41032404PRTArtificial Sequencemycobacterium bovis arginine deiminase with flanking synthesized residues added 32Met Ser Val Gly Val Glu Leu Gly Ser Asn Ser Glu Val Gly Ala Leu1 5 10 15Arg Val Val Ile Leu His Arg Pro Gly Ala Glu Leu Arg Arg Leu Thr 20 25 30Pro Arg Asn Thr Asp Gln Leu Leu Phe Asp Gly Leu Pro Trp Val Ser 35 40 45Arg Ala Gln Asp Glu His Asp Glu Phe Ala Glu Leu Leu Ala Ser Arg 50 55 60Gly Ala Glu Val Leu Leu Leu Ser Asp Leu Leu Thr Glu Ala Leu His65 70 75 80His Ser Gly Ala Ala Arg Met Gln Gly Ile Ala Ala Ala Val Asp Ala 85 90 95Pro Arg Leu Gly Leu Pro Leu Ala Gln Glu Leu Ser Ala Tyr Leu Arg 100 105 110Ser Leu Asp Pro Gly Arg Leu Ala His Val Leu Thr Ala Gly Met Thr 115 120 125Phe Asn Glu Leu Pro Ser Asp Thr Arg Thr Asp Val Ser Leu Val Leu 130 135 140Arg Met His His Gly Gly Asp Phe Val Ile Glu Pro Leu Pro Asn Leu145 150 155 160Val Phe Thr Arg Asp Ser Ser Ile Trp Ile Gly Pro Arg Val Val Ile 165 170 175Pro Ser Leu Ala Leu Arg Ala Arg Val Arg Glu Ala Ser Leu Thr Asp 180 185 190Leu Ile Tyr Ala His His Pro Arg Phe Thr Gly Val Arg Arg Ala Tyr 195 200 205Glu Ser Arg Thr Ala Pro Val Glu Gly Gly Asp Val Leu Leu Leu Ala 210 215 220Pro Gly Val Val Ala Val Gly Val Gly Glu Arg Thr Thr Pro Ala Gly225 230 235 240Ala Glu Ala Leu Ala Arg Ser Leu Phe Asp Asp Asp Leu Ala His Thr 245 250 255Val Leu Ala Val Pro Ile Ala Gln Gln Arg Ala Gln Met His Leu Asp 260 265 270Thr Val Cys Thr Met Val Asp Thr Asp Thr Met Val Met Tyr Ala Asn 275 280 285Val Val Asp Thr Leu Glu Ala Phe Thr Ile Gln Arg Thr Pro Asp Gly 290 295 300Val Thr Ile Gly Asp Ala Ala Pro Phe Ala Glu Ala Ala Ala Lys Ala305 310 315 320Met Gly Ile Asp Lys Leu Arg Val Ile His Thr Gly Met Asp Pro Val 325 330 335Val Ala Glu Arg Glu Gln Trp Asp Asp Gly Asn Asn Thr Leu Ala Leu 340 345 350Ala Pro Gly Val Val Val Ala Tyr Glu Arg Asn Val Gln Thr Asn Ala 355 360 365Arg Leu Gln Asp Ala Gly Ile Glu Val Leu Thr Ile Ala Gly Ser Glu 370 375 380Leu Gly Thr Gly Arg Gly Gly Pro Arg Cys Met Ser Cys Pro Ala Ala385 390 395 400Arg Asp Pro Leu

Claims

1.-28. (canceled)

29. An isolated arginine deiminase, or a fragment thereof having ADI activity, wherein the isolated arginine deiminase has reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies.

30. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase is not from M. hominis.

31. The isolated arginine deiminase of claim 30 wherein the isolated arginine deiminase is from an organism listed in Table 1.

32. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase has one or more properties comparable to or better than those of ADI-PEG 20.

33. The isolated arginine deiminase of claim 32 wherein the one or more properties is Keat, Km, pH optimum, stability, in vivo proteolytic stability, or no requirement for ions or cofactors that are not already present in blood, or any combination thereof.

34. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase has at least 20 surface residue changes as compared to M. hominis arginine deiminase.

35. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase has between 20 and 135 surface residue changes as compared to M. hominis arginine deiminase.

36. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase has between 40 and 100 surface residue changes as compared to M. hominis arginine deiminase.

37. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase has between 30 and 60 surface residue changes as compared to M. hominis arginine deiminase.

38. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase has between 80 and 100 surface residues changes as compared to M. hominis arginine deiminase.

39. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase has between 100 and 120 surface residues changes as compared to M. hominis arginine deiminase.

40. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase is from M. arginini, M. arthritidis, M. phocicerebrale, M. gateae, M. phocidae, M. columbinum, M. iowae, M. crocodyli, M. alligatoris, H. orenii, or M. bovis.

41. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase comprises the amino acid sequence set forth in any one of SEQ ID NOs:2-32.

42. The isolated arginine deiminase of claim 29 wherein the isolated arginine deiminase has been modified to remove at least one pegylation site.

43. The isolated arginine deiminase of claim 29 wherein at least one lysine residue has been modified by an amino acid substitution.

44. The isolated arginine deiminase of claim 43 wherein at least 5 lysine residues have been modified by an amino acid substitution.

45. The isolated arginine deiminase of claim 43 wherein at least 10 lysine residues have been modified by an amino acid substitution.

46. The isolated arginine deiminase of claim 43 wherein at least 15 lysine residues have been modified by an amino acid substitution.

47. The isolated arginine deiminase of claim 43 wherein at least 20 lysine residues have been modified by an amino acid substitution.

48. The isolated arginine deiminase of claim 29 wherein the arginine deiminase is covalently bonded via a linker to a PEG molecule.

49. The isolated arginine deiminase of claim 48 wherein the arginine deiminase is covalently bonded to more than one PEG molecule.

50. The isolated arginine deiminase of claim 48 wherein the arginine deiminase is covalently bonded to about 1 to about 10 PEG molecules.

51. The isolated arginine deiminase of claim 48 wherein the arginine deiminase is covalently bonded to about 2 to about 8 PEG molecules.

52. The isolated arginine deiminase of claim 48 wherein the PEG molecules are straight chain or branch chain PEG molecules.

53. The isolated arginine deiminase of claim 48 wherein the PEG has a total weight average molecular weight of from about 1,000 to about 40,000.

54. The isolated arginine deiminase of claim 48 wherein the PEG has a total weight average molecular weight of from about 10,000 to about 30,000.

55. The isolated arginine deiminase of claim 48 wherein the linker is a succinyl 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, a methylene group, or any combinations thereof.

56. The isolated arginine deiminase of claim 55 wherein the source of the succinyl group is succinimidyl succinate.

57. A polynucleotide encoding the isolated arginine deiminase of claim 29.

58. A vector comprising the polynucleotide of claim 57.

59. An isolated host cell comprising the vector of claim 58.

60.-68. (canceled)