Arginine deiminase with reduced cross-reactivity to ADI-PEG20 for cancer treatment

Abstract

The present invention generally relates to an isolated arginine deiminase (ADI) protein that has reduced cross-reactivity with an anti-ADI-PEG 20 antibody compared to ADI-PEG 20, but may have functional characteristics similar to or superior to those of ADI-PEG 20; compositions comprising the ADI protein, and related methods for treating arginine-dependent diseases or related diseases such as cancer.

Description

[0001] This application is a divisional application of the invention application with the application date of September 16, 2015, the Chinese application number of 201580057712.X, and the invention name of "Arginine deiminase with reduced cross-reactivity to ADI-PEG 20 antibody for cancer treatment".

[0002] Cross-reference to related applications

[0003] This application claims the priority of U.S. Application No. 62 / 051,182 filed on September 16, 2014 under 35 U.S.C.§119(e), which is incorporated herein by reference in its entirety.

[0004] Statement regarding the Sequence Listing

[0005] The Sequence Listing related to this application is provided in text format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the Sequence Listing is POLA_005_01WO_SeqList_ST25.txt. This file is approximately 97 KB, was created on September 15, 2015, and was submitted electronically via EFS-Web. Background Technical Field

[0006] The present invention generally relates to arginine deiminase (ADI) proteins, including ADI proteins with reduced cross-reactivity to ADI-PEG20 antibodies. Such ADI proteins can be used to treat arginine-dependent or related diseases such as cancer.

[0007] Description of the Related Art

[0008] Amino acid deprivation therapy can be an effective treatment for certain forms of cancer. To date, there is one known clinical example related to this method, which uses asparaginase to reduce the circulating levels of asparagine and inhibit protein synthesis. This treatment is particularly effective against 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 able to synthesize asparagine and are not affected by asparagine depletion. Thus, reducing serum asparagine with asparaginase can selectively kill cancer cells without damaging normal cells, tissues, and the host. The Escherichia coli (E. coli)-derived form of asparaginase has been approved for human use. However, asparaginase is only found in microorganisms; this makes it highly immunogenic in humans and also has a short serum half-life after injection (Avramis 2005). To make asparaginase a more effective drug, these disadvantages are minimized by formulating the E. coli-derived asparaginase with polyethylene glycol (PEG) to reduce the immunogenicity of this enzyme and related allergic reactions. In addition, PEG greatly extends the circulating half-life of asparaginase, thus reducing both the frequency of treatment and the total cost of the therapy. PEG-formulated asparaginase is approved for use and is sold under the trade name ( 2011, Avramis 2005, Viera Pinheiro 2004, Fu 2007, Zeidan 2008).

[0009] Arginine is another non-essential amino acid for humans and mice (for a review see Rogers 1994). In the human body, arginine can be synthesized from citrulline in two steps via the Krebs (urea) cycle enzymes argininosuccinate synthetase (ASS, L-citrulline:L-aspartate ligase [forming AMP], 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 aspartate into argininosuccinate, which is then converted into arginine and fumarate by ASL. An arginine-deficient diet does not cause hyperammonemia or orotic aciduria in humans and also does not alter the rate of systemic nitric oxide (NO) synthesis in adults (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 are not age-related among infants, children, and young adults (Lücke 2007). In 1992, Takaku and Sugimura independently reported that human melanoma and hepatocellular carcinoma (HCC) cell lines appeared to require arginine for growth. Other studies have shown that PEGylated ADI is effective in treating melanoma and hepatoma with few adverse effects.

[0010] ADI-PEG 20 treatment requires multiple doses over a period of time. After many treatments, anti-ADI-PEG 20 antibodies can be produced that can limit its lasting effectiveness. Therefore, there is a need in the art for an ADI with reduced cross-reactivity with anti-ADI-PEG 20 antibodies for use in therapy to improve and extend the efficacy of arginine depletion therapy. The present invention provides this advantage and other advantages regarding the treatment of cancer.

[0011] References: Avramis VI, Panosyan EH. 2005. Clin Pharmacokinet 44:367-393; Barbul A. 1986. J Parenteral Enteral Nutr 10:227-238; Carey GP et al. 1987. J Nutr 117:1734-1739; Castillo L et al. 1995. Am J Physiol 268(Endocrinol Metab 31):E360-367; Fu CH, Sakamoto KM. 2007. Expert Opin Pharmacother 8:1977-1984; Haines RJ et al. 2011. Int J Biochem Mol Biol 2:8-23; Husson A et al. 2003. Eur J Biochem 270:1887-1899; Lücke T et al. 2007. Clin Chem Lab Med 45:1525-1530; Morris SM Jr. 2006. Am J Clin Nutr 83(Suppl):598S-512S; Rogers QR. 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, IL 61801, pp. 9-21; Tapiero H et al. 2002. Biomed Pharmacother 56:439-445, 2002; Viera Pinheiro JP, 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). Summary of the Invention

[0012] Certain embodiments relate to an isolated arginine deiminase, wherein the isolated arginine deiminase has reduced cross-reactivity with a patient anti-ADI-PEG 20 antibody. Also included is a therapeutic or pharmaceutical composition comprising the isolated arginine deiminase or an ADI-active fragment thereof and a pharmaceutically acceptable carrier. In certain embodiments, the composition is sterile and / or substantially pyrogen-free, such as endotoxin-free. In some embodiments, the isolated arginine deiminase having reduced cross-reactivity with a patient anti-ADI-PEG 20 antibody is not from Mycoplasma hominis. In some embodiments, the isolated arginine deiminase having reduced cross-reactivity with a patient anti-ADI-PEG 20 antibody is from an organism listed in Table 1.

[0013] In certain embodiments, the isolated arginine deiminase having reduced cross-reactivity with a patient anti-ADI-PEG 20 antibody has one or more properties similar to or superior to ADI-PEG 20. In this regard, the one or more properties include, but are not limited to, Kcat, Km, optimal pH, stability, in vivo proteolytic stability, or the absence of a need for ions or cofactors not already present in the blood or any combination thereof. In some embodiments, the isolated arginine deiminase having reduced cross-reactivity with a patient anti-ADI-PEG 20 antibody has at least 5, 10, 15, or 20 surface residue changes compared to Mycoplasma hominis arginine deiminase. In certain embodiments, the isolated arginine deiminase having reduced cross-reactivity with a patient anti-ADI-PEG 20 antibody has from about 20 to 135 surface residue changes, from about 40 to 100 surface residue changes, from about 30 to 60 surface residue changes, from about 80 to 100 surface residue changes, or from about 100 to 120 surface residue changes compared to Mycoplasma hominis arginine deiminase.

[0014] In certain embodiments, the isolated arginine deiminase having reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody is from Mycoplasma salivarium, Mycoplasma spumans, Mycoplasma canadense, Mycoplasma auris, Mycoplasma hyosynoviae, Mycoplasma cloacale, Mycoplasma anseris, Mycoplasma alkalescens, Mycoplasma orale, Mycoplasma iners, Mycoplasma meleagridis, Mycoplasma alvi, Mycoplasma penetrans, Mycoplasma gallinarum, Mycoplasma pirum, Mycoplasma primatum, Mycoplasma fermentans, Mycoplasma lipofaciens, Mycoplasma felifaucium, Mycoplasma imitans, Mycoplasma opalescens, Mycoplasma moatsii, Mycoplasma elephantis, Mycoplasma pneumoniae, Mycoplasma testudinis, Mycoplasma sp. CAG:877 or Mycoplasma sp. CAG:472. Exemplary arginine deiminases having reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody include any one or more of the amino acid sequences set forth in SEQ ID NO:2 to SEQ ID NO:28.

[0015] In some embodiments, an isolated arginine deiminase having reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody has been modified to remove at least one pegylation site. In certain embodiments of an arginine deiminase having reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody, at least one lysine residue has been modified by amino acid substitution. In this regard, in certain embodiments, at least about 5 lysine residues, at least about 10 lysine residues, or at least about 20 lysine residues have been modified by amino acid substitution.

[0016] In some embodiments, an arginine deiminase having reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody is covalently bonded to a PEG molecule via a linker. In this regard, an arginine deiminase having reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody can be covalently bonded to one or more PEG molecules, such as covalently bonded to about 1 to about 10 or about 2 to about 8 PEG molecules. The PEG molecule can be a linear or branched PEG molecule and the total weight average molecular weight can be about 1,000 to about 40,000 or the total weight average molecular weight is about 10,000 to about 30,000. In some embodiments, where the PEG is covalently bonded to an ADIr as described herein via a linker, the linker can 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 combination thereof. In a specific embodiment, the source of the succinyl group is succinimidyl succinate.

[0017] Also included are polynucleotides encoding the isolated arginine deiminases described herein, vectors comprising the polynucleotides, and isolated host cells comprising the vectors.

[0018] Certain embodiments relate to compositions comprising an isolated arginine deiminase having reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody as described herein and a physiologically acceptable carrier. In certain embodiments, the composition further comprises a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, docetaxel, carboplatin, cyclophosphamide, gemcitabine, cisplatin, sorafenib, sunitinib, and everolimus.

[0019] Also included are methods of treating cancer, alleviating its symptoms or inhibiting its progression, which comprise administering to a patient in need thereof a therapeutically effective amount of a composition comprising an isolated arginine deiminase having reduced cross-reactivity with the patient's anti-ADI-PEG20 antibody as described herein and a physiologically acceptable carrier, thereby treating cancer, alleviating its symptoms or inhibiting its progression. In certain embodiments, the patient in need has been determined to have anti-ADI-PEG 20 antibody. In some embodiments, 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), renal cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, and gastric cancer.

[0020] Some embodiments include methods of treating cancer, alleviating its symptoms or inhibiting its progression, which comprise 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 isolated arginine deiminase having reduced cross-reactivity with the patient's anti-ADI-PEG20 antibody as described herein and a physiologically acceptable carrier, thereby treating cancer, alleviating its symptoms or inhibiting its progression. The period of time can be determined, for example, by detecting a predetermined level of anti-ADI-PEG 20 antibody in the patient and / or measuring or otherwise observing the ADI activity in the patient, wherein the composition comprising the isolated arginine deiminase having reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody is administered after detecting the predetermined level of the anti-ADI-PEG 20 antibody in the patient and / or measuring or observing a predetermined level of ADI activity.

[0021] Also included is the isolated arginine deiminase protein described herein for use in the preparation or manufacture of a medicament for treating cancer, alleviating its symptoms or inhibiting its progression.

[0022] The present invention also relates to the following:

[0023] 1. A therapeutic composition comprising an isolated arginine deiminase or a fragment thereof having ADI activity and a pharmaceutically acceptable carrier, wherein the isolated arginine deiminase has reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody.

[0024] 2. The therapeutic composition according to item 1, wherein the isolated arginine deiminase is not from Mycoplasma hominis.

[0025] 3. The therapeutic composition according to item 1 or 2, wherein the isolated arginine deiminase is from an organism listed in Table 1.

[0026] 4. The therapeutic composition according to any one of items 1 to 3, wherein the isolated arginine deiminase has one or more properties similar to or superior to those of ADI-PEG 20.

[0027] 5. The therapeutic composition according to item 4, wherein the one or more properties are Kcat, Km, optimal pH value, stability, in vivo proteolytic stability, or do not require ions or cofactors not already present in the blood, or any combination thereof.

[0028] 6. The therapeutic composition according to any one of items 1 to 5, wherein the isolated arginine deiminase has at least 20 surface residue changes compared to Mycoplasma hominis arginine deiminase.

[0029] 7. The therapeutic composition according to item 6, wherein the isolated arginine deiminase has 20 to 135 surface residue changes compared to Mycoplasma hominis arginine deiminase.

[0030] 8. The therapeutic composition according to item 6, wherein the isolated arginine deiminase has 40 to 100 surface residue changes compared to Mycoplasma hominis arginine deiminase.

[0031] 9. The therapeutic composition according to item 6, wherein the isolated arginine deiminase has 30 to 60 surface residue changes compared to Mycoplasma hominis arginine deiminase.

[0032] 10. The therapeutic composition according to item 6, wherein the isolated arginine deiminase has 80 to 100 surface residue changes compared to Mycoplasma hominis arginine deiminase.

[0033] 11. The therapeutic composition according to item 6, wherein the isolated arginine deiminase has 100 to 120 surface residue changes compared to Mycoplasma hominis arginine deiminase.

[0034] 12. The therapeutic composition according to any one of items 1 to 11, wherein the isolated arginine deiminase is from Mycoplasma salivarium, Mycoplasma spumans, Mycoplasma canadense, Mycoplasma auris, Mycoplasma hyosynoviae, Mycoplasma cloacale, Mycoplasma anseris, Mycoplasma alcaligenes, Mycoplasma orale, Mycoplasma inertum, Mycoplasma meleagridis, Mycoplasma mellis, Mycoplasma penetrans, Mycoplasma gallisepticum, Mycoplasma pirum, Mycoplasma primatum, Mycoplasma fermentans, Mycoplasma lipophilum, Mycoplasma tracheale, Mycoplasma imitans, Mycoplasma lactucae, Mycoplasma moatsii, Mycoplasma elephantis, Mycoplasma pneumoniae, Mycoplasma testudineum, Mycoplasma sp. CAG:877 or Mycoplasma sp. CAG:472.

[0035] 13. The therapeutic composition according to any one of items 1 to 12, wherein the isolated arginine deiminase comprises the amino acid sequence set forth in any one of SEQ ID NO:2 to SEQ ID NO:28.

[0036] 14. The therapeutic composition according to any one of items 1 to 13, wherein the isolated arginine deiminase has been modified to remove at least one polyethylene glycolylation site.

[0037] 15. The therapeutic composition according to any one of items 1 to 14, wherein at least one lysine residue has been modified by amino acid substitution.

[0038] 16. The therapeutic composition according to item 15, wherein at least 5 lysine residues have been modified by amino acid substitution.

[0039] 17. The therapeutic composition according to item 15, wherein at least 10 lysine residues have been modified by amino acid substitution.

[0040] 18. The therapeutic composition according to item 15, wherein at least 15 lysine residues have been modified by amino acid substitution.

[0041] 19. The therapeutic composition according to item 15, wherein at least 20 lysine residues have been modified by amino acid substitution.

[0042] 20. The therapeutic composition according to any one of items 1 to 19, wherein the arginine deiminase is covalently bonded to a PEG molecule via a linker.

[0043] 21. The therapeutic composition according to item 20, wherein the arginine deiminase is covalently bonded to more than one PEG molecule.

[0044] 22. The therapeutic composition according to item 20, wherein the arginine deiminase is covalently bonded to about 1 to about 10 PEG molecules.

[0045] 23. The therapeutic composition according to item 20, wherein the arginine deiminase is covalently bound to about 2 to about 8 PEG molecules.

[0046] 24. The therapeutic composition according to item 20, wherein the PEG molecules are linear or branched PEG molecules.

[0047] 25. The therapeutic composition according to item 20, wherein the total weight average molecular weight of the PEG is about 1,000 to about 40,000.

[0048] 26. The therapeutic composition according to item 20, wherein the total weight average molecular weight of the PEG is about 10,000 to about 30,000.

[0049] 27. The isolated arginine deiminase according to any one of items 20 to 26, wherein the linker is succinyl, amide, imide, carbamate, ester, epoxy, carboxyl, hydroxyl, carbohydrate, tyrosine group, cysteine group, histidine group, methylene or any combination thereof.

[0050] 28. The therapeutic composition according to item 27, wherein the source of the succinyl group is succinimidyl succinate.

[0051] 29. An isolated arginine deiminase or a fragment thereof having ADI activity, wherein the isolated arginine deiminase has a reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody.

[0052] 30. The isolated arginine deiminase according to item 29, wherein the isolated arginine deiminase is not from Mycoplasma hominis.

[0053] 31. The isolated arginine deiminase according to item 29 or 30, wherein the isolated arginine deiminase is from an organism listed in Table 1.

[0054] 32. The isolated arginine deiminase according to any one of items 29 to 31, wherein the isolated arginine deiminase has one or more properties similar to or superior to ADI-PEG20.

[0055] 33. The isolated arginine deiminase according to item 32, wherein the one or more properties are Kcat, Km, optimal pH, stability, in vivo proteolytic stability, or do not require ions or cofactors not already present in the blood or any combination thereof.

[0056] 34. The isolated arginine deiminase according to any one of items 29 to 33, wherein the isolated arginine deiminase has at least 20 surface residue changes compared to Mycoplasma hominis arginine deiminase.

[0057] 35. The isolated arginine deiminase as described in item 34, wherein the isolated arginine deiminase has 20 to 135 surface residue changes compared with Mycoplasma hominis arginine deiminase.

[0058] 36. The isolated arginine deiminase as described in item 34, wherein the isolated arginine deiminase has 40 to 100 surface residue changes compared with Mycoplasma hominis arginine deiminase.

[0059] 37. The isolated arginine deiminase as described in item 34, wherein the isolated arginine deiminase has 30 to 60 surface residue changes compared with Mycoplasma hominis arginine deiminase.

[0060] 38. The isolated arginine deiminase as described in item 34, wherein the isolated arginine deiminase has 80 to 100 surface residue changes compared with Mycoplasma hominis arginine deiminase.

[0061] 39. The isolated arginine deiminase as described in item 34, wherein the isolated arginine deiminase has 100 to 120 surface residue changes compared with Mycoplasma hominis arginine deiminase.

[0062] 40. The isolated arginine deiminase as described in any one of items 29 to 39, wherein the isolated arginine deiminase is from Mycoplasma salivarium, Mycoplasma spumans, Mycoplasma canadense, Mycoplasma auris, Mycoplasma hyosynoviae, Mycoplasma cloacale, Mycoplasma anseris, Mycoplasma alcaligenes, Mycoplasma orale, Mycoplasma iners, Mycoplasma meleagridis, Mycoplasma mellis, Mycoplasma penetrans, Mycoplasma gallisepticum, Mycoplasma pirum, Mycoplasma primatum, Mycoplasma fermentans, Mycoplasma lipophilum, Mycoplasma tracheale, Mycoplasma imitans, Mycoplasma lactucae, Mycoplasma moatsii, Mycoplasma elephantis, Mycoplasma pneumoniae, Mycoplasma testudineum, Mycoplasma sp. CAG:877 or Mycoplasma sp. CAG:472.

[0063] 41. The isolated arginine deiminase as described in any one of items 29 to 40, wherein the isolated arginine deiminase comprises the amino acid sequence set forth in any one of SEQ ID NO:2 to SEQ ID NO:28.

[0064] 42. The isolated arginine deiminase as described in any one of items 29 to 41, wherein the isolated arginine deiminase has been modified to remove at least one polyethylene glycolylation site.

[0065] 43. The isolated arginine deiminase as described in any one of items 29 to 42, wherein at least one lysine residue has been modified by amino acid substitution.

[0066] 44. The isolated arginine deiminase as described in item 43, wherein at least 5 lysine residues have been modified by amino acid substitution.

[0067] 45. The isolated arginine deiminase as described in item 43, wherein at least 10 lysine residues have been modified by amino acid substitution.

[0068] 46. The isolated arginine deiminase as described in item 43, wherein at least 15 lysine residues have been modified by amino acid substitution.

[0069] 47. The isolated arginine deiminase as described in item 43, wherein at least 20 lysine residues have been modified by amino acid substitution.

[0070] 48. The isolated arginine deiminase as described in any one of items 29 to 47, wherein the arginine deiminase is covalently bonded to a PEG molecule via a linker.

[0071] 49. The isolated arginine deiminase as described in item 48, wherein the arginine deiminase is covalently bonded to more than one PEG molecule.

[0072] 50. The isolated arginine deiminase as described in item 48 or 49, wherein the arginine deiminase is covalently bonded to about 1 to about 10 PEG molecules.

[0073] 51. The isolated arginine deiminase as described in item 50, wherein the arginine deiminase is covalently bonded to about 2 to about 8 PEG molecules.

[0074] 52. The isolated arginine deiminase as described in any one of items 48 to 51, wherein the PEG molecule is a linear or branched PEG molecule.

[0075] 53. The isolated arginine deiminase as described in any one of items 48 to 52, wherein the total weight average molecular weight of the PEG is about 1,000 to about 40,000.

[0076] 54. The isolated arginine deiminase as described in item 53, wherein the total weight average molecular weight of the PEG is about 10,000 to about 30,000.

[0077] 55. The isolated arginine deiminase as described in any one of items 48 to 54, 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 combination thereof.

[0078] 56. The isolated arginine deiminase as described in item 55, wherein the source of the succinyl group is succinimidyl succinate.

[0079] 57. A polynucleotide encoding the isolated arginine deiminase as described in any one of the preceding items.

[0080] 58. A vector comprising the polynucleotide as described in item 57.

[0081] 59. An isolated host cell comprising the vector as described in item 58.

[0082] 60. The therapeutic composition as described in any one of the preceding items, further comprising a chemotherapeutic agent.

[0083] 61. The therapeutic composition as described in item 60, wherein the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, cyclophosphamide, gemcitabine, cisplatin, sorafenib, sunitinib, and everolimus.

[0084] 62. A method for treating cancer, alleviating its symptoms, or inhibiting its progression, the method comprising administering to a patient in need a therapeutically effective amount of the therapeutic composition or isolated arginine deiminase as described in any one of the preceding items, thereby treating the cancer, alleviating its symptoms, or inhibiting its progression.

[0085] 63. The method as described in item 62, wherein the patient in need has been determined to have anti-ADI-PEG 20 antibodies.

[0086] 64. The method as described in item 62 or 63, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myeloid leukemia, recurrent acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), renal cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, and gastric cancer.

[0087] 65. A method for treating cancer, alleviating its symptoms, or inhibiting its progression, the method comprising administering to a patient in need a therapeutically effective amount of a composition comprising ADI-PEG 20, and after a period of time, administering to the patient the therapeutic composition or arginine deiminase as described in any one of the preceding items, thereby treating the cancer, alleviating its symptoms, or inhibiting its progression.

[0088] 66. The method according to item 65, wherein the time period is determined by detecting a predetermined level of anti-ADI-PEG 20 antibody in the patient, and wherein the therapeutic composition is administered after detection of the predetermined level of the anti-ADI-PEG 20 antibody.

[0089] 67. The method according to item 65, wherein the time period is determined by detecting ADI activity in the patient, and wherein the therapeutic composition is administered after detection of a predetermined or reduced level of ADI activity.

[0090] 68. Use of a therapeutic composition or isolated arginine deiminase as described in any of the preceding items for the manufacture of a medicament for treating cancer, alleviating its symptoms or inhibiting its progression. Brief Description of the Drawings

[0091] Figure 1A-1D Illustrate various cysteine-reactive PEG molecules that can be conjugated to the ADIr enzyme described herein. Detailed Description of the Invention

[0092] Embodiments of the present invention relate to selected ADI enzymes that are conjugated to PEG via a linker such as a stable linker in some embodiments. In some embodiments, the ADI enzyme is engineered or selected to have a reduced number of surface lysine residues relative to, for example, a wild-type sequence or a reference sequence (see, for example, Table 1). 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 form and maintain a low arginine concentration in human blood through the conversion of arginine to citrulline and ammonia by ADI. In some embodiments, the selected ADI molecule has reduced cross-reactivity with anti-ADI-PEG 20 antibodies compared to ADI-PEG 20, such antibodies that may be generated due to prior treatment of the patient with ADI-PEG 20.

[0093] In certain embodiments, the ADI enzyme is pegylated to provide protection against renal clearance and proteolysis, as well as reduced immunogenicity or antigenicity. To increase the effectiveness of pegylation, the enzyme can be modified to engineer a reduced number of surface lysine residues and thus limit the number of available PEG attachment sites. In some cases, reducing the number of lysine residues provides more complete and uniform pegylation at the remaining lysine attachment residues.

[0094] In some embodiments, the PEG linker selected to attach methoxy-PEG to ADI provides a chemically stable bond. It is expected that the stable linker will increase the biological activity lifetime of the molecule. The chemically stable linker will also eliminate hydrolysis and reduce the immune response that may occur with a de-pegylated linker attached to the enzyme surface.

[0095] 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 a previous arginine depletion therapy. The molecules are pegylated to delay neutralization and clearance due to their immunogenicity. These factors will allow their use as an alternative to ADI-PEG 20 or in addition to ADI-PEG 20 (e.g., as a successor drug) to extend arginine depletion therapy and thus increase the effectiveness of arginine depletion therapy as an anti-cancer therapeutic agent.

[0096] Normal cells do not require arginine for growth because it can be synthesized from citrulline in a two-step process by ASS and ASL. In contrast, certain cancers do not express ASS. Certain cancers do not express ASL, while other cancers may have reduced expression of ASS and / or ASL, or may not express ASS and / or ASL. Thus, these cancers are auxotrophic for arginine. This metabolic difference can be exploited to develop safe and effective therapies to treat these forms of cancer. ADI catalyzes the conversion of arginine to citrulline via the arginine dihydrolase pathway and can thus be used to deplete arginine.

[0097] Unless specifically indicated to the contrary, the practice of the invention will employ 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 illustrative purposes. Such techniques are well explained in the literature. See, for example, 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 ed., 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Volumes I and II (D. Glover ed.); Oligonucleotide Synthesis (N. Gait ed., 1984); Nucleic Acid Hybridization (B. Hames and S. Higgins ed., 1985); Transcription and Translation (B. Hames and S. Higgins ed., 1984); Animal Cell Culture (R. Freshney ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar literature.

[0098] Standard techniques are available for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. Unless otherwise specifically defined, the nomenclature used in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as well as the experimental procedures and techniques of molecular biology, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly employed in the art. Standard techniques can be used in recombinant techniques, molecular biosynthesis, microbiological synthesis, chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients.

[0099] Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents.

[0100] "About" means that a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0101] As used herein, the term "amino acid" includes naturally occurring amino acids and non-naturally occurring amino acids as well as amino acid analogs and mimetics. For example, naturally occurring amino acids include the 20 (L)-amino acids used during protein biosynthesis as well as other amino acids such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, norleucine, norvaline, p-fluorophenylalanine, ethionine, and their analogs, which are known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functional similarity properties such as the charge and charge-spacing characteristics of a reference amino acid. For example, an organic structure mimicking arginine (Arg or R) will have positively charged moieties positioned at a similar molecular spacing and with the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures that maintain the optimal spacing and charge interactions of the amino acid or amino acid functional group. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0102] 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 the 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.

[0103] "Biocompatible" means that a material or compound is generally not harmful to biological functions and will not produce any degree of unacceptable toxicity, including allergenicity and disease conditions.

[0104] The terms "endotoxin-free" and "substantially endotoxin-free" generally refer to compositions, solvents, and / or containers that contain at most trace amounts (e.g., amounts that have no harmful physiological effects on a subject clinically) of endotoxin and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain bacteria, typically Gram-negative bacteria, although endotoxins can be found in Gram-positive bacteria such as Listeria monocytogenes. The most common endotoxin is lipopolysaccharide (LPS) or lipooligosaccharide (LOS) found in the outer membrane of various Gram-negative bacteria, and it represents a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in the human body can cause fever, decreased blood pressure, and activation of inflammation and coagulation, as well as other harmful physiological effects.

[0105] Thus, in drug preparation, it is often necessary to remove most or all trace amounts of endotoxin from drug products and / or drug containers because even small amounts can cause harmful effects in the human body. A pyrogen oven can be used for this purpose because typically temperatures above 300 °C are required to decompose most endotoxins. For example, based on the primary packaging material such as syringes or vials, a combination of a glass transition temperature of 250 °C and a hold time of 30 minutes is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods of removing endotoxin are covered, including, for example, chromatography and filtration methods as described herein and known in the art. Also included are methods of producing polypeptides in eukaryotic cells such as mammalian cells and separating them from the eukaryotic cells to reduce (if not eliminate) the risk of endotoxin being present in the compositions of the present invention. Methods of producing polypeptides in serum-free cells and separating them from the serum-free cells are included.

[0106] Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus Amebocyte Lysate assay using blood from the horseshoe crab is a highly sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of the Limulus lysate, which is attributed to a powerful enzymatic cascade that potentiates this reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, the endotoxin level can be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10 EU / ml. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1 - 10 EU.

[0107] The "half-life" of a polypeptide can refer to the time it takes for the pharmacological, physiological, or other activity of the polypeptide to be reduced by half relative to its activity at the time of administration to the serum or tissue of an organism or relative to any other specified time point. "Half-life" can also refer to the time it takes for the amount or concentration of the polypeptide to be reduced by half relative to the starting amount administered to the serum or tissue of an organism or relative to any other specified time point. The half-life can be measured in serum and / or any one or more selected tissues.

[0108] "Homology" refers to the percentage of amino acids that are identical or constitute conservative substitutions. Homology can be determined using sequence comparison programs such as GAP (Deveraux et al., Nucleic Acids Research. 12, 387 - 395, 1984), which is incorporated herein by reference. In this way, sequences that are similar or substantially different in length from those listed herein can be compared by inserting gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0109] The terms "modulate" and "alter" include "increase", "elevate" or "stimulate" and "decrease" or "reduce" typically in a statistically significant or physiologically significant amount or degree relative to a control. The amount of "increase", "stimulate" or "elevate" is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) the amount produced by no composition (e.g., absence of a reagent) or a control composition (including all integers and ranges therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.). The amount of "decrease" or "reduce" is typically a "statistically significant" amount and can include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% decrease in the amount produced by no composition (e.g., absence of a reagent) or a control composition (including all integers and ranges therebetween). Examples of comparing values and "statistically significant" amounts are described herein.

[0110] "Patient" or "subject" refers to an animal, in certain embodiments a mammal, and in a specific embodiment a human.

[0111] In certain embodiments, the "purity" of any given reagent (e.g., ADIr, ADIr-PEG) in a composition can be specified. For example, a reagent contained in certain compositions can be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure as measured by, for example and without limitation, high performance liquid chromatography (HPLC) (a well-known form of column chromatography often used in biochemistry and analytical chemistry to separate, identify and quantify compounds), including all decimals and ranges therebetween.

[0112] The term "reference sequence" generally refers to a nucleic acid encoding a sequence or an amino acid sequence being compared to another sequence. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name and those described in tables and sequence listings.

[0113] The term "sequence identity" or, for example, "a sequence that is 50% identical to" refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a comparison window. Thus, the "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window to determine the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present in both sequences, to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity.

[0114] Terms used to describe sequence relationships between two or more polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" can be at least 12 but often 15 to 18 and frequently at least 25 monomer units in length, including nucleotide and amino acid residues. Since two polypeptides can each contain (1) sequences similar between the two polypeptides (i.e., only a portion of the complete polypeptide sequence) and (2) sequences that diverge between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing the sequences of the two polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6, usually about 50 to about 100, more usually about 100 to about 150 consecutive positions, where the two sequences are compared after the best alignment of the sequence with a reference sequence having the same number of consecutive positions. For the best alignment of two sequences, the comparison window can include about 20% or less additions or deletions (i.e., gaps) as compared to the reference sequence (which does not contain additions or deletions). The best alignment of sequences for aligning the comparison window can be conducted by a computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and by any of a variety of methods selected to produce the best alignment (i.e., the highest percentage homology over the comparison window). Reference can also be made to programs of the BLAST family, such as those disclosed by Altschul et al. (Nucl. Acids Res. 25:3389, 1997). Details of sequence analysis discussions can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994 - 1998, Chapter 15).

[0115] The calculation of sequence similarity or sequence identity between sequences (these terms are used interchangeably herein) can be carried out as follows. To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment and non-homologous sequences can be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90% or 100% of the length of the reference sequence. Then, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0116] The percent identity between two sequences varies with the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0117] The comparison of sequences and the determination of the percent identity between two sequences can be achieved using mathematical algorithms. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (J. Mol. Biol. 48:444-453, 1970), which has been incorporated into the GAP program in the GCG software package, using the Blossum62 matrix or the PAM250 matrix, and the gap weight is 16, 14, 12, 10, 8, 6 or 4 and the length weight is 1, 2, 3, 4, 5 or 6. In some embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, with its NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. Another exemplary set of parameters (and unless otherwise specified, should be used by the user) includes the Blossum 62 scoring matrix, where the gap penalty is 12, the gap extension penalty is 4 and the frameshift gap penalty is 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:11-17, 1989), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, the diagonals length penalty is 12 and the gap penalty is 4.

[0118] The term "solubility" refers to the property of the ADIr enzyme provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration by the mass of solute per unit volume of solvent (grams of solute per kilogram of solvent, g / dL (100 mL), mg / ml, etc.), molarity, molality, mole fraction, or other similar descriptions of concentration. The maximum equilibrium amount of solute that can be dissolved in a unit amount of solvent is the solubility of the solute in the solvent under specified conditions, including temperature, pressure, pH, and solvent properties. In certain embodiments, solubility is measured at physiological pH or other pH values, such as at pH 5.0, pH 6.0, pH 7.0, pH 7.2, pH 7.4, pH 7.6, pH 7.8, or pH 8.0. In certain embodiments, solubility is measured in water or a physiological buffer (such as PBS or NaCl) (in the presence or absence of NaP) or other buffers / compositions described herein. In a specific embodiment, solubility is measured at a relatively low pH value (such as pH 6.0) and relatively high salt (such as 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (such as about 20, 21, 22, 23, 24, 25 °C) or about body temperature (37 °C). In certain embodiments, the solubility of the ADIr enzyme is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg / ml at room temperature or at 37 °C.

[0119] "Substantially" or "essentially" means almost entirely or completely, such as 95%, 96%, 97%, 98%, 99%, or greater of a given amount.

[0120] "Statistically significant" means that the result is not likely to occur by chance. Statistical significance can be determined by any method known in the art. Common measures of significance include the p-value, which is the frequency or probability that the observed event would occur if the null hypothesis were true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In a simple case, the significance level is defined as a p-value of 0.05 or less.

[0121] Unless otherwise expressly stated, the various embodiments in this specification, with necessary modifications, apply to each other embodiment.

[0122] Throughout the present disclosure, the following abbreviations may be used: PEG, polyethylene glycol; ADI, arginine deiminase; SS, succinimidyl succinate; SSA, succinimidyl succinamate; SPA, succinimidyl propionate; NHS, N-hydroxysuccinimide; ASS1 or ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase.

[0123] The polynucleotide encoding the ADI enzyme 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 a microorganism of the genus Mycoplasma. In certain embodiments, the arginine deiminase is cloned from Mycoplasma salivarium, Mycoplasma spumans, Mycoplasma canadense, Mycoplasma auris, Mycoplasma hyorhinis, Mycoplasma cloacae, Mycoplasma anseris, Mycoplasma alcaligenes, Mycoplasma orale, Mycoplasma inertum, Mycoplasma meleagridis, Mycoplasma mellis, Mycoplasma penetrans, Mycoplasma gallinaceum, Mycoplasma pirum, Mycoplasma primatum, Mycoplasma fermentans, Mycoplasma lipophilum, Mycoplasma tracheale, Mycoplasma imitans, Mycoplasma lactucae, Mycoplasma moatsii, Mycoplasma elephantis, Mycoplasma pneumoniae, Mycoplasma testudineum, a Mycoplasma sp. CAG:877 or a Mycoplasma sp. CAG:472 or any combination thereof. In some embodiments, the arginine deiminase is cloned from a species listed in Table 1. In a particular embodiment, the ADI comprises the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:28 or a variant or fragment or extension thereof having ADI activity (e.g., capable of metabolizing arginine into citrulline and ammonia). Such ADI enzymes may be prepared or synthesized using known techniques.

[0124] In certain embodiments, the ADI enzymes described herein are compared to a benchmark ADI-PEG20 molecule derived from Mycoplasma hominis. As used herein, "ADI-PEG 20" refers to an ADI molecule known in the art and described, for example, in U.S. Patent Nos. 6,183,738 and 6,635,462; see also Ascierto 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 FW 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 will be appreciated by those skilled in the art, this molecule is a polyethylene glycolylated (PEG 20,000) ADI enzyme derived from Mycoplasma hominis and has two substitutions (K112E; P210S) relative to the wild-type Mycoplasma hominis ADI enzyme.

[0125] The arginine deiminases described herein are screened from a large number of ADI enzymes and are believed to have a reduced level of reactivity with anti-ADI-PEG20 antibodies of a patient and / or have other beneficial properties. Anti-ADI-PEG 20 antibodies can occur in subjects treated with ADI-PEG 20 and can be measured using known methods. Reactivity with anti-ADI-PEG 20 antibodies can be determined, for example, using ELISA or other similar assays known to those skilled in the art.

[0126] In this regard, ADI-PEG 20 can be used as a comparator to evaluate the level of cross-reactivity with patient anti-ADI-PEG 20 antibodies. Cross-reactivity levels that are statistically significantly lower compared to the level of cross-reactivity of ADI-PEG 20 with patient anti-ADI-PEG 20 antibodies are applicable herein. In certain embodiments, the arginine deiminases as described herein have low or no cross-reactivity with anti-ADI-PEG 20 antibodies. In certain embodiments, any decrease in reactivity with anti-ADI-PEG 20 antibodies compared to reactivity with ADI-PEG 20 can be beneficial, such that the ADI enzyme would improve treatment options for patients in need of arginine depletion therapy. Thus, in some embodiments, the arginine deiminases as described herein have a reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies compared to the reactivity of ADI-PEG 20 with such antibodies.

[0127] "ADIr" is used herein to refer to an ADI enzyme of the invention that has a reduced cross-reactivity with anti-ADI-PEG 20 antibodies compared to the reactivity of ADI-PEG 20 with such antibodies. The name "ADIr" is used to distinguish the molecules identified herein from ADI and ADI-PEG 20 as known in the art. Examples of ADIr enzymes include SEQ ID NO:2 to SEQ ID NO:28 and variants whose amino acid sequences are different from SEQ ID NO:1 or ADI-PEG 20.

[0128] In some embodiments, the ADIr enzyme of the present invention has properties or characteristics similar to or superior to those of ADI-PEG 20 to reduce and maintain low blood arginine levels for effective cancer treatment. Examples of such properties include Kcat, Km, optimal pH, stability, in vivo proteolytic stability, and the lack of need for ions or cofactors not already present in the blood, or any combination thereof. In certain embodiments, the properties of ADIr as described herein 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 compared to the similar properties of ADI-PEG 20. In some embodiments, the properties of ADIr as described herein 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 compared to the specific properties of ADI-PEG 20 being compared.

[0129] Thus, in certain embodiments, the Kcat of ADIr 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% or better than the Kcat of ADI-PEG 20. In certain embodiments, the Kcat of ADIr 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 than the Kcat of ADI-PEG 20. In certain embodiments, the Kcat of the ADIr enzyme or a composition comprising the same as described herein is about 0.5 seconds -1 to about 15 seconds -1 、about 1 second -1 to about 12 seconds -1 、about 1 second -1 to about 10 seconds-1 、 About 1.5 seconds -1 to about 9 seconds -1 、 About 2 seconds -1 to about 8 seconds -1 or about 2.5 seconds -1 to about 7 seconds -1 。 In certain embodiments, the kcat of ADIr or ADIr-PEG in the composition is about 2.5 seconds -1 to about 7.5 seconds -1 。 In some embodiments, the kcat of ADIr or ADIr-PEG in the composition is about 2.5 seconds -1 、 About 3 seconds -1 、 About 3.5 seconds -1 、 About 4 seconds -1 、 About 4.5 seconds -1 、 About 5 seconds -1 、 About 5.5 seconds -1 、 About 6 seconds -1 、 About 6.5 seconds -1 、 About 7 seconds -1 、 About 7.2 seconds -1 、 About 7.5 seconds -1 、 About 8 seconds -1 、 About 10 seconds -1 、 About 15 seconds -1 、 About 20 seconds -1 、 About 25 seconds -1 、 About 30 seconds -1 、 About 35 seconds -1 、 About 40 seconds -1 、 About 45 seconds -1 、 About 50 seconds -1 、 About 55 seconds -1 、 About 60 seconds -1 、 About 65 seconds -1 、 About 70 seconds -1 、 About 75 seconds -1 、 About 80 seconds -1 、 About 85 seconds -1 、 About 90 seconds -1 、 About 95 seconds -1 or about 100 seconds -1 。

[0130] In certain embodiments, the Km of ADIr 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% or better than the Km of ADI-PEG 20. In certain embodiments, the Km of ADIr is about or at least about 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 180%, 200%, 220%, 240% or 250% of the Km of ADI-PEG 20. In certain embodiments, the Km of ADIr or its pegylated formulation is from about 0.5 μM to about 50 μM or from about 1.6 μM to about 48 μM or from about 0.5 μM to about 15 μM, from about 1 μM to about 12 μM, from about 1 μM to about 10 μM, from about 1.5 μM to about 9 μM, from about 1.5 μM to about 8 μM or from about 1.5 μM to about 7 μM. In certain embodiments, the Km of ADIr or ADIr-PEG in the composition is from about 1.5 μM to about 6.5 μM. In some embodiments, the Km of ADIr or its pegylated formulation is about 1.5 μM, about 1.6 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 22 μM, about 24 μM, about 25 μM, about 26 μM, about 28 μM, about 30 μM, about 32 μM, about 34 μM, about 35 μM, about 36 μM, about 38 μM, about 40 μM, about 42 μM, about 44 μM, about 45 μM, about 46 μM, about 48 μM or about 50 μM.

[0131] In certain embodiments, the ADIr functional group is at a pH value close to the physiological pH value of human blood. Thus, in some embodiments, the ADIr functional group is at a pH value from about 4 to about 10.8 or from about 6 to about 8 or from about 6.5 to about 7.5. In certain embodiments, ADIr has good enzyme activity at about pH 7.4.

[0132] In certain embodiments, ADIr has stability during long-term storage and has temperature and proteolytic stability during processing in the human body. In some embodiments, ADIr does not require ions or cofactors that are not already present in the blood to acquire activity.

[0133] In certain embodiments, the ADIrs described herein generally have an amino acid sequence that is quite different from that of Mycoplasma hominis, such that there are surface residue changes that will reduce or eliminate the antigenic sites of anti-ADI-PEG 20 antibodies. In some embodiments, there will be no cross-reactivity (e.g., no statistically significant cross-reactivity) between the selected ADIr molecule and existing anti-ADI-PEG 20 antibodies in the subject, and a completely new immune response will be generated in the subject rather than a maturation of the existing response to Mycoplasma hominis ADI. Thus, in some embodiments, the ADIrs described herein have 20%-85% sequence identity with Mycoplasma hominis ADI as set forth in SEQ ID NO:1. In certain embodiments, the ADIrs described herein have an even lower percentage of sequence identity with Mycoplasma hominis ADI, such as 10% or 15% identity. In certain embodiments, the ADIrs described herein have 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 with Mycoplasma hominis ADI and still have reduced cross-reactivity with anti-ADI-PEG 20 antibodies.

[0134] In certain embodiments, the ADIr as described herein has about 10 - 140, 15 - 140, or 25 - 140 surface residue changes compared to Mycoplasma hominis ADI. The surface residues can be identified from the crystal structure of Mycoplasma hominis ADI, and the surface residues of ADI from other organisms can be determined by sequence homology. The ADIr as described herein can have about 10, 15, 20, 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 compared to Mycoplasma hominis ADI (see SEQ ID NO:1).

[0135] In some embodiments, the ADIr as described herein has about 10 - 140, 15 - 140, or 25 - 140 surface residue changes compared to Mycoplasma hominis ADI. Such residue changes require not only surface amino acid residues. Such residue changes (or additions or deletions) can be at either end of the molecule or at any residue of the ADI such that the modified ADI has the desired ADI activity as described herein. The residues to be altered can be identified from the crystal structure of Mycoplasma hominis ADI, and the residues of ADI from other organisms can be determined by sequence homology. The ADIr as described herein can have about 10, 15, 20, 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 compared to Mycoplasma hominis ADI (see SEQ ID NO:1).

[0136] From a large number of ADI enzymes, Table 1 lists 27 ADIr enzymes and their percent sequence identity relative to Mycoplasma hominis ADI.

[0137] In certain embodiments, the ADIr enzymes identified herein from a number of selected species have a defined number of surface lysine residues (in certain embodiments, up to 30 or more, for example). Some of the ADIr enzymes identified herein with reduced cross-reactivity with anti-ADI-PEG 20 antibody have about, at least about, or no more than 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, 59, or 60 surface lysine residues, including all ranges therebetween.

[0138] The terms "polypeptide", "protein", and "peptide" are used interchangeably and mean an amino acid polymer of any specific length is not limited. The term "enzyme" includes polypeptide or protein catalysts and can be used interchangeably with protein, polypeptide, or peptide with respect to ADIr. The term includes modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more chains of amino acids, wherein each chain contains amino acids covalently linked by peptide bonds, and wherein the polypeptide or protein can contain multiple chains linked together non-covalently and / or covalently by peptide bonds, having the sequence of a native protein (i.e., a protein produced by a naturally occurring and specifically non-recombinant cell or a genetically engineered or recombinant cell), and including a molecule having the amino acid sequence of a native protein or a molecule having deletions, additions, and / or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically cover the ADIr enzymes / proteins described herein, or sequences having deletions, additions, and / or substitutions of one or more amino acids of the ADIr protein. In certain embodiments, the polypeptide is a "recombinant" polypeptide produced by a recombinant cell, the recombinant cell containing one or more recombinant DNA molecules typically composed of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would otherwise not be present in the cell.

[0139] As used herein, the term "isolated protein" means a target protein that (1) is free of at least some other proteins that would typically be found with it in nature, (2) is substantially free of other proteins from the same source (e.g., from the same species), (3) is expressed by cells from different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials that associate with it in nature, (5) does not associate (by covalent or non-covalent interactions) with portions of proteins that associate with the "isolated protein" in nature, (6) is operably associated (by covalent or non-covalent interactions) with polypeptides that do not associate with it in nature, or (7) does not exist in nature. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNAs that may have a synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0140] The term "variant" includes polypeptides that differ from a reference polypeptide specifically disclosed herein (e.g., SEQ ID NOs: 1 to SEQ ID NO: 28) by one or more substitutions, deletions, additions, and / or insertions. Variant polypeptides are biologically active, i.e., they continue to possess the enzymatic or binding activity of the reference polypeptide. Such variants can be produced, for example, by genetic polymorphisms and / or from human manipulation.

[0141] In many cases, biologically active variants will contain one or more conservative substitutions. A "conservative substitution" is the replacement of one amino acid with another amino acid having similar properties such that a person skilled in the art of peptide chemistry would expect the secondary structure and hydrophilic nature of the polypeptide to remain substantially unchanged. As described above, modifications can be made in the structures of the polynucleotides and polypeptides described herein and still obtain a functional molecule encoding a variant or derivative polypeptide having the desired properties.

[0142] For example, certain amino acids can replace other amino acids in the structure of a protein without a significant loss of the ability to interact and bind to structures such as the antigen-binding region of an antibody or a binding site on a substrate molecule. Since the interaction capabilities and properties of a protein define its biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence and, of course, its underlying DNA coding sequence, and still obtain a protein with similar properties. Accordingly, various changes are expected to be made in the peptide sequences of the disclosed compositions or the corresponding DNA sequences encoding such peptides without a significant loss of their efficacy.

[0143] In making such changes, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilic amino acid index in conferring protein interaction biological functions is generally understood in the art (Kyte and Doolittle, 1982, which is incorporated herein by reference). It is recognized that the relative hydrophilic nature of the amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens and the like. Hydrophilicity indices have been assigned to the amino acids based on their hydrophobicity and charge characteristics (Kyte and 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 can be replaced by other amino acids having similar hydrophilicity indices or fractions and still produce a protein having similar biological activity, i.e., still obtain a biologically functionally equivalent protein. In making such changes, amino acid substitutions within ±2 of the hydrophilicity index are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0144] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, specifically incorporated herein by reference in its entirety, states that the greatest local average hydrophilicity of a protein as controlled by the hydrophilicity of its neighboring amino acids correlates with the biological properties 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 will be appreciated that an amino acid may 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, 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 particularly preferred.

[0145] As outlined above, therefore, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account various of the foregoing characteristics are well known to those skilled in the art, and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0146] Amino acid substitutions may be further made based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or 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 and 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 exhibit 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.

[0147] Alternatively, the variant may also contain non-conservative changes. In a preferred embodiment, the variant polypeptide differs from the native sequence by the substitution, deletion, or addition of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids or even 1 amino acid. The variant may also (or) be modified by, for example, the deletion or addition of amino acids that have a minimal impact on the immunogenicity, secondary structure, enzymatic activity, and / or hydrophilicity of the polypeptide.

[0148] Generally, the variant will exhibit 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 the reference polypeptide sequence (e.g., SEQ ID NO:1 to SEQ ID NO:28). In addition, sequences that differ from the native or parental sequence 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 retain the properties or activities of the parental or reference polypeptide sequence are encompassed.

[0149] In some embodiments, the variant polypeptide has at least one but less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residues different from the reference sequence. In some embodiments, the variant polypeptide has about or at least 0.5% or 1% but less than 20%, 15%, 10% or 5% of the residues different from the reference sequence. (If such a comparison requires alignment, the sequences should be aligned to obtain maximum similarity. Sequences that are "circled" due to deletions or insertions or mismatches are considered differences).

[0150] In some embodiments, the length of the ADIr will be from about 300 to about 500 amino acids, including all integers and ranges therebetween. In a specific embodiment, the length of the ADIr will be about 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495 or 500 amino acids, including all integers and ranges therebetween.

[0151] The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxy-terminal deletion, and / or an internal deletion or substitution in a naturally occurring or recombinantly produced polypeptide. In certain embodiments, the polypeptide fragment can comprise an amino acid chain that is at least 5 to about 400 amino acids in length. It is understood that in certain embodiments, the fragment is 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 in length. Particularly useful polypeptide fragments include functional domains, including the catalytic ADI domain of ADIr described herein. In the case of ADIr, useful fragments include, but are not limited to, the catalytic domain and the alpha-helical domain.

[0152] Many of the activated PEGs used for conjugation to ADI covalently bind to lysine residues. There are generally many fewer PEG molecules attached to ADI compared to lysine residues. The number and distribution of the linkages from molecule to molecule can both be non-uniform. Any particular lysine residue will be modified in only a small fraction of the ADI molecules. This site modification non-uniformity and low PEG occupancy can create problems with drug characterization and the effectiveness of PEG shielding at antigenic sites. Thus, in certain embodiments, selected ADIr enzymes as described herein are modified by lysine replacement with other residue types to reduce the number of lysine residues. This results in a more uniformly polyethylene glycolated protein and increases the PEG occupancy at the remaining lysine residues. The specific lysine residues chosen to be changed to other residues will be selected to retain enzyme activity. This more uniform polyethylene glycolation is expected to provide increased protection against proteolysis in the blood and increased sequestration against anti-site and patient antibodies.

[0153] In certain embodiments, the ADIr enzyme is modified as described in U.S. Patent No. 6,635,462. Specifically, modification of one or more of the naturally occurring amino acid residues of ADIr can provide an enzyme that is more amenable to refolding and formulation, thereby improving the manufacture of ADIr and therapeutic compositions containing it. In some embodiments, the ADIr enzyme is modified to remove one or more lysine residues (e.g., lysine can be replaced by another amino acid or an analogue or non-natural amino acid thereof). Specifically, in some embodiments, the ADIr enzyme is modified to be free of lysine at positions equivalent to 112, 374, 405, or 408 of SEQ ID NO:1 (Mycoplasma hominis ADI) or a combination of one or more of these positions. In some embodiments, the ADIr enzyme is modified to be free of one or more lysine residues, e.g., 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 (if present) can be replaced by another amino acid or an analogue or non-natural amino acid thereof. In a specific embodiment, the ADIr enzyme has 5 substituted lysine residues at positions equivalent to positions 7, 88, 137, 209, and 380 of SEQ ID NO:1, for example. In some embodiments, the ADIr enzyme has 10 substituted lysine residues at positions equivalent to positions 7, 9, 59, 88, 115, 116, 137, 178, 209, and 380 of SEQ ID NO:1, for example. In certain embodiments, the ADIr enzyme has 15 substituted lysine residues at positions equivalent to positions 7, 9, 59, 66, 88, 91, 93, 115, 116, 137, 141, 178, 209, 279, and position 380 of SEQ ID NO:1, for example. In some embodiments, the ADIr enzyme has 21 substituted lysine residues 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, for example.

[0154] In some cases, native ADIr can be found in microorganisms and is thus immunogenic and rapidly cleared from the circulation in patients. These problems can be overcome by modifying the ADIr to produce a "modified ADIr" enzyme. Accordingly, certain embodiments include an ADIr enzyme comprising a "modifier", examples of which include but are not limited to macromolecular polymers, proteins, peptides, polysaccharides, and other compounds. The ADIr enzyme and the modifier can be linked by covalent bonds or non-covalent interactions to form a stable conjugate or stable composition to achieve the desired effect. In certain embodiments, the modified ADIr retains the biological activity of the corresponding unmodified ADIr (e.g., having the same or similar sequence) and has a longer in vivo half-life and lower antigenicity compared to the corresponding 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 the corresponding unmodified ADIr. Generally, the modified ADIr retains sufficient biological activity for therapeutic use.

[0155] In some embodiments, the modifier can be a biocompatible polymer or protein or a fragment thereof and can increase the half-life of the ADIr in the blood. The modifier can be chemically conjugated to the ADIr or, where applicable, linked to the ADIr via fusion protein expression.

[0156] Macromolecular polymers can include non-peptide macromolecular polymers, which in certain embodiments can have their own biological activity. Suitable polymers include but are not limited to polyvinyl alcohol compounds, polyether compounds, polyvinylpyrrolidone, polyamino acids, copolymers of divinyl ether and maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, polysaccharides, polyoxyethylated polyols, heparin or fragments thereof, polyalkyl-ethylene glycols and their derivatives, copolymers of polyalkyl-ethylene glycols and their derivatives, poly(vinyl ethyl ether), α,β-poly[(2-hydroxyethyl)-DL-asparagine], polycarboxylates, poly(ethylene oxide-formaldehyde), polyacrylmorpholine, copolymers of amino compounds and alkylene oxides, polyhyaluronic acid, poly(ethylene oxide), copolymers of oxalic acid and malonic acid, poly(1,3-dioxolane), copolymers of ethylene and maleic hydrazide, polysialic acid, cyclodextrin, etc. In certain embodiments, the polymer is polyethylene glycol.

[0157] As used herein, polyvinyl alcohol compounds include but are not limited to polyethylene glycol (including monomethoxy polyethylene glycol, monohydroxy polyethylene glycol), polyvinyl alcohol, polyallyl alcohol, polybutenol and the like, and derivatives thereof, such as lipids.

[0158] Polyether compounds include but are not limited to polyalkylene glycols (HO((CH2) x O)n H), polypropylene glycol, polyethylene oxide (HO((CH2)2O) n H), polyvinyl alcohol ((CH2CHOH) n ).

[0159] Polyamino acids include, but are not limited to, polymers of one type of amino acid or copolymers of two or more types of amino acids, such as polyalanine or polylysine or their block copolymers.

[0160] Polysaccharides include, but are not limited to, dextrans and their derivatives, such as dextran sulfate; cellulose and its derivatives (including methylcellulose and carboxymethylcellulose); starch and its derivatives; polysucrose, etc.

[0161] In a specific embodiment, ADIr is modified by coupling with a protein or peptide, wherein one or more proteins or peptides are directly or indirectly linked to ADIr. The protein can be a naturally occurring protein or a fragment thereof, including but not limited to naturally occurring human serum proteins or fragments thereof, such as thyroxine-binding protein, transthyretin, a1-acid glycoprotein, transferrin, fibrinogen, immunoglobulin, Ig Fc region, albumin and fragments thereof. "Fragment" means any portion of a protein that is less than the whole protein but retains the desired protein function. The ADIr as described herein can be directly or indirectly linked to the protein via a covalent bond. Direct linkage means that an amino acid of ADIr is directly linked to an amino acid of the modified protein via a peptide bond or a disulfide bond. Indirect linkage refers to the linkage between ADIr and the modified protein via a chemical group that initially exists between them or a specific chemical group added by biological or chemical means or a combination of the linkages mentioned above.

[0162] In a particular embodiment, ADIr is modified by covalently linking to one or more PEG molecules. ADIr covalently modified with PEG (in the presence or absence of a linker) may hereinafter be referred to as "ADIr-PEG". When compared to unmodified ADIr, ADIr-PEG retains most of its enzyme activity, has much lower immunogenicity or antigenicity, has a greatly extended circulatory half-life, and is more effective in tumor therapy.

[0163] "Polyethylene glycol" or "PEG" refers to a mixture of cold condensates of ethylene oxide and water in branched or straight-chain form, which is represented by the general formula H(OCH2CH2) nOH represents, where n is at least 4. "Polyethylene glycol" or "PEG" is used in combination with a numerical suffix to indicate its approximate weight average molecular weight. For example, PEG5,000 refers to PEG with a total weight average molecular weight of approximately 5,000; PEG12,000 refers to PEG with a total weight average molecular weight of approximately 12,000; and PEG20,000 refers to PEG with a total weight average molecular weight of approximately 20,000.

[0164] In some embodiments, the total weight average molecular weight of the PEG is from about 1,000 to about 50,000; from about 3,000 to about 40,000; from about 5,000 to about 30,000; from about 8,000 to about 30,000; from about 11,000 to about 30,000; from about 12,000 to about 28,000; from about 16,000 to about 24,000; from about 18,000 to about 22,000; or from about 19,000 to about 21,000. In some embodiments, the total weight average molecular weight of the PEG is from about 1,000 to about 50,000; from about 3,000 to about 30,000; from about 3,000 to about 20,000; from about 4,000 to about 12,000; from about 4,000 to about 10,000; from about 4,000 to about 8,000; from about 4,000 to about 6,000; or about 5,000. In a specific embodiment, the total weight average molecular weight of the PEG is about 20,000. Generally, PEG with a molecular weight of 30,000 or greater is difficult to dissolve, and the yield of the formulated product can be reduced. The PEG can be branched or linear. Generally, an increase in the molecular weight of the PEG reduces the immunogenicity of the ADIr. The PEG can be branched or linear, and in certain embodiments is linear. PEGs having the molecular weights described herein can be used in combination with ADIr and an optionally present biocompatible linker to treat graft-versus-host disease (GVHD) or cancer, including, for example, hepatocellular carcinoma, 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), renal cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, gastric cancer or esophageal cancer, and other cancers described herein.

[0165] Certain embodiments use thiol, sulfhydryl or cysteine - reactive PEGs. In some embodiments, the thiol, sulfhydryl or cysteine - reactive PEGs are linked to one or more naturally - occurring cysteine residues, one or more introduced cysteine residues (e.g., substituting one or more wild - type residues with cysteine residues, inserting one or more cysteine residues), or any combination thereof (see, e.g., Doherty et al., Bioconjug Chem. 16:1291 - 98, 2005). In certain embodiments, some of the wild - type ADI cysteine residues may first be substituted with another amino acid to prevent the PEG polymer from linking to the wild - type cysteine, e.g., to prevent the PEG from disrupting a bioactivity that is otherwise required. Some embodiments use one or more non - natural cysteine derivatives (e.g., homocysteine) in place of cysteine.

[0166] Non - limiting examples of thiol, sulfhydryl or cysteine - reactive PEGs include methoxy PEG maleimide (M - PEG - MAL) (e.g., MW 2000, MW 5000, MW 10000, MW 20000, MW 30000, MW 40000). M - PEG - MAL reacts with the thiol groups on the cysteine side chains in proteins and peptides to produce stable 3 - thiosuccinimide ether bonds. This reaction is highly selective and can be carried out under mild conditions at about pH 5.0 - 6.5 in the presence of other functional groups. Specific examples of commercially available thiol, sulfhydryl or cysteine - reactive PEG molecules are shown in Figure 1A-1D . Thus, in certain embodiments, the ADIr enzyme is conjugated to any one or more of the thiol, sulfhydryl or cysteine - reactive PEG molecules described herein.

[0167] ADIr can be covalently bonded to a modifier, such as PEG, in the presence or absence of a linker, although preferred embodiments use a linker. ADIr can be covalently bonded to PEG via a biocompatible linker using methods known in the art as described, for example, in 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 incorporated herein by reference. In some cases, ADIr can be directly coupled (i.e., without using a linker) to a modifier such as PEG through, for example, an amino, sulfhydryl, hydroxyl, carboxyl or other group.

[0168] The linker for covalently linking an ADIr to a modifier (e.g., PEG) can be any biocompatible linker. As discussed above, "biocompatible" indicates that the compound or group is non-toxic and can be used in vitro or in vivo without causing injury, disease, illness, or death. A modifier such as PEG can be bonded to the linker, for example, via an ether bond, a thiol bond, an amide bond, or other bonds.

[0169] In some embodiments, the total chain length of a suitable linker can be about 1 - 100 atoms, 1 - 80 atoms, 1 - 60 atoms, 1 - 40 atoms, 1 - 30 atoms, 1 - 20 atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms, such as where the atoms in the chain include C, S, N, P, and / or O. In certain embodiments, the linker is optionally present, e.g., a PEG-conjugated ADIr enzyme does not contain a linker. In some cases, 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. Specific examples of stable linkers include succinimide, propionic acid, carboxymethoxide linkage, ether, carbamate, amide, amine, urea, imide, aliphatic C-C bond, and thioether. In certain embodiments, the biocompatible linker is a succinimidyl succinate (SS) group.

[0170] Other suitable linkers include an oxycarbonyl imidazole group (including, for example, carbonyl imidazole (CDI)), a nitrophenyl group (including, for example, nitrophenyl carbonate (NCP) or trichlorophenyl carbonate (TCP)), a trysylate group, an aldehyde group, an isocyanate group, a vinyl sulfone group, or a primary amine. In certain embodiments, the linker is derived from SS, SPA, SCM, or NHS; in certain embodiments, SS, SPA, or NHS is used, and in some embodiments, SS or SPA is used. Thus, in certain embodiments, possible linkers can be formed from: methoxy-PEG succinimidyl succinate (SS), methoxy-PEG succinimidyl glutarate (SG), methoxy-PEG succinimidyl carbonate (SC), methoxy-PEG succinimidyl carboxymethyl ether (SCM), methoxy-PEG2 N-hydroxysuccinimide (NHS), methoxy-PEG succinimidyl butyrate (SBA), methoxy-PEG succinimidyl propionate (SPA), methoxy-PEG succinimidyl glutaric amide, and / or methoxy-PEG succinimidyl succinimide.

[0171] Other examples of linkers include, but are not limited to, one or more of the following: -O-, -NH-, -S-, -C(O)-, C(O)-NH, NH-C(O)-NH, O-C(O)-NH, -C(S)-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -O-CH2-, -CH2-O-, -O-CH2-CH2-, -CH2-O-CH2-, -CH2-CH2-O-, -O-CH2-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, -CH2-CH2-CH2-O-, -O-CH2-CH2-CH2-CH2-, -CH2-O-CH2-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-CH2-O-CH2-, -CH2-CH2-CH2-CH2-O-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-CH2-, -CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -NH-C(O)-CH2-, -CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, -NH-C(O)-CH2-CH2-, -CH2-NH-C(O)-CH2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -O-C(O)-NH-CH2-, -O-C(O)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH2-NH-CH2-, -C(O)-CH2-, -C(O)-CH2-CH2-, -CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-C(O)-,-CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-, a divalent cycloalkyl group, -N(R6)-, where R6 is H or an organic group selected from the group consisting of: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl. Additionally, any of the linker moieties described herein may further comprise an ethylene oxide oligomer chain comprising from 1 to 20 ethylene oxide monomer units [i.e., -(CH2CH2O)-, 1-20 -]. That is, the ethylene oxide oligomer chain may be present before or after the linker and optionally between any two atoms of a linker moiety comprising two or more atoms. Additionally, if the oligomer is adjacent to a polymer fragment, the oligomer chain will not be considered part of the linker moiety, but merely represents an extension of the polymer fragment.

[0172] Specific exemplary PEG molecules and linkers are described in Table A below.

[0173]

[0174]

[0175] In certain embodiments, the ADIr enzyme comprises one or more PEG molecules and / or linkers as described herein (e.g., in Table A).

[0176] PEGylation of ADIr increases the circulatory half-life of ADIr. Generally, PEG is attached to the primary amine of ADIr. As will be known to those skilled in the art, the choice of the site of attachment of PEG or other modifiers to ADIr is determined by the effect of individual sites within the active domain of the protein. PEG can be attached to the primary amine of ADIr without a substantial loss of enzyme activity. For example, all of the lysine residues present in ADIr are potential 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. As will be apparent to those skilled in the art in light of the present disclosure, PEG can also be attached to other sites on ADIr.

[0177] From 1 to about 30 PEG molecules can be covalently bonded to ADIr. In certain embodiments, ADIr is modified with one PEG molecule (i.e., contains one PEG molecule). In some embodiments, ADIr is modified with more than one PEG molecule. In certain embodiments, ADIr is modified with about 1 to about 10 or about 7 to about 15 PEG molecules or about 2 to about 8 or about 9 to about 12 PEG molecules. In some embodiments, ADIr is modified with about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG molecules. In specific embodiments, ADIr is modified with 4.5 - 5.5 PEG molecules per ADIr. In some embodiments, ADIr is modified with 5 ± 1.5 PEG molecules.

[0178] In certain embodiments, about 15% to about 70% of the primary amines in ADIr are modified with PEG. In some embodiments, about 20% to about 65%, about 25% to about 60%, or in certain embodiments, about 30% to about 55% or 45% to about 50%, or in some embodiments, about 50% of the primary amines in arginine deiminase are modified with PEG. When PEG is covalently bonded to the end of ADIr, it may be necessary to use only 1 PEG molecule. An increase in the number of PEG units on ADIr increases the circulatory half-life of the enzyme. However, an increase in the number of PEG units on ADIr decreases the specific activity of the enzyme. Thus, as will be apparent to those skilled in the art in view of the present disclosure, a balance needs to be achieved between the two.

[0179] In some embodiments, a common feature of the biocompatible linker is that it is attached to the primary amine of arginine deiminase via a succinimidyl group. Once coupled to ADIr, SS-PEG has an ester bond next to the PEG, which can confer serum esterase sensitivity at this site, and the serum esterase can release PEG from ADIr in vivo. SPA-PEG and PEG2-NHS do not have an ester bond, so they are not sensitive to serum esterase.

[0180] The PEG linked to the protein can be linear, as in the case of SS-PEG, SPA-PEG, and SC-PEG, or branched PEG can be used, as in the case of PEG2-NHS.

[0181] In certain embodiments, the PEGylation sites associated with ADIr that are located at or near the catalytic region of the enzyme are modified. In certain embodiments, the phrase "PEGylation site" is defined as any site or position in ADI or ADIr that can be covalently modified with polyethylene glycol. The "PEGylation site" can be considered to be located at or near the catalytic region of the enzyme, where PEGylation of the site results in a significant decrease in the catalytic activity of the enzyme. PEGylation of such sites has traditionally caused inactivation of the enzyme. For example, ADI from Mycoplasma hominis has a lysine at position 112, which can be considered to be located at or near the catalytic region of the enzyme. PEGylation of this lysine at position 112 can inactivate the enzyme. In addition, ADI from Mycoplasma hominis has a cysteine at position 397, which can be considered to be located at or near the catalytic region of the enzyme. Amino acid substitution of the cysteine at position 397 can inactivate the enzyme. Specifically, substitution of the cysteine at position 397 with alanine, histidine, arginine, serine, lysine, or tyrosine can result in the loss of all detectable enzyme activity. ADI from Mycoplasma hominis has three lysine residues that are located in proximity to this conserved cysteine, specifically Lys374, Lys405, and Lys408. PEGylation of Lys374, Lys405, Lys408, or a combination thereof can inactivate the enzyme.

[0182] It should be understood that ADIr derived from other organisms may also have PEGylation sites corresponding to position 112 of ADI from Mycoplasma hominis. In addition, ADI from some organisms may have lysine residues at positions corresponding to the general location of position 112 of ADI from Mycoplasma hominis. The positions of the lysines in ADI from such organisms are known to those of skill in the art and are described in U.S. Patent No. 6,635,462.

[0183] Accordingly, some embodiments provide certain amino acid substitutions in the polypeptide chain of ADIr. These amino acid substitutions provide a modified ADIr that loses less activity when modified by a modifier such as after PEGylation. Optimal modification (e.g., PEGylation) can be achieved without loss of activity by eliminating the PEGylation sites or other known modification sites that are located at or near the catalytic region of the enzyme.

[0184] In some embodiments, for example, as described above, amino acid substitutions are made using unnatural amino acids conjugated to PEG or other modifiers (see, e.g., de Graaf et al., Bioconjug Chem. 20:1281-95, 2009). Accordingly, certain embodiments include ADIr enzymes conjugated to one or more PEGs via one or more unnatural amino acids. In some embodiments, the unnatural amino acid comprises a side chain having a functional group selected from the group consisting of: alkyl, aryl, aryl halide, vinyl halide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl, keto, azide, hydroxyl, hydrazine, cyano, halo, acylhydrazide, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boric acid, boronate ester, borane, phenylboronic acid, thiol, seleno, sulfonyl, borate, boronate, phosphate, phosphonyl, phosphine, heterocycle-, pyridyl, naphthyl, benzophenone, constrained ring (such as cyclooctyne), thioester, ketene, imine, aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid, α-ketocarboxylic acid, α or β unsaturated acid and amide, glyoxylamide and organosilyl group. In some embodiments, the unnatural amino acid is selected from the group consisting of: p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, homocysteine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, β-O-GlcNAc-L-serine, tri-O-acetyl-GalNAc-α-threonine, α-GalNAc-L-threonine, L-dopa, fluorophenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonylserine, phosphonyltyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine and isopropyl-L-phenylalanine.

[0185] Although ADIr-PEG is an illustrative modified ADIr described herein, as will be appreciated by those skilled in the art, ADIr can be modified with other polymers or suitable molecules to achieve the desired effects, particularly reducing antigenicity and increasing serum half-life.

[0186] It should be understood that some embodiments are based on the understanding that when prepared via recombinant techniques, certain structural features of arginine deiminase can prevent or impede proper and rapid refolding. Specifically, these structural features impede or prevent the enzyme from assuming an active conformation during recombinant preparation. In some embodiments, the term "active conformation" is defined as the three-dimensional structure that permits enzymatic activity produced by an unmodified or modified arginine deiminase. The active conformation may be particularly necessary for catalyzing the conversion of arginine to citrulline. The term "structural feature" may be defined as any characteristic, quality, or property of a polypeptide chain produced by a particular amino acid or combination of amino acids. For example, arginine deiminase may contain amino acids that produce a bend or kink in the normal peptide chain and thus impede the enzyme from assuming an active conformation during refolding of the enzyme. Specifically, arginine deiminase from Mycoplasma hominis has a proline at position 210, which may produce a bend or kink in the peptide chain, making it more difficult for the enzyme to refold during recombinant preparation. It should be understood that arginine deiminases derived from other organisms may also have a site corresponding to position 210 of arginine deiminase from Mycoplasma hominis.

[0187] Accordingly, some embodiments are provided regarding certain amino acid substitutions in the polypeptide chain of wild-type arginine deiminase. Examples include substitutions that eliminate problem structural features in the peptide chain of arginine deiminase. Also included are substitutions that provide improved refolding of the modified arginine deiminase. These amino acid substitutions make it possible to rapidly refold the modified arginine deiminase using a reduced amount of buffer. These amino acid substitutions may also provide a refolded modified arginine deiminase with increased yield. In some embodiments, the modified arginine deiminase has an amino acid substitution at P210 or an equivalent residue. As mentioned above, arginine deiminase derived from Mycoplasma hominis has the amino acid proline at position 210. While not limiting the invention, it is believed that the presence of the amino acid proline at position 210 produces a bend or kink in the normal polypeptide chain, thereby increasing the difficulty of refolding (i.e., refolding) certain arginine deiminases. Substituting the proline at position 210 makes it possible to rapidly refold the modified arginine deiminase using a reduced amount of buffer. Substituting the proline at position 210 (or an equivalent residue) may also provide a refolded modified arginine deiminase with increased yield. In some embodiments, the proline at position 210 (or an equivalent residue) is substituted with serine. Non-limiting 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 features associated with the amino acid at position 210 of the wild-type arginine deiminase, optimal refolding of the enzyme can be achieved.

[0188] The methods provided herein may involve in vitro or in vivo applications. In the case of in vitro applications (including cell culture applications), the compounds described herein may be added to cells in a culture and then incubated. The compounds described herein may also be used to facilitate the preparation of monoclonal and / or polyclonal antibodies using antibody preparation techniques well known in the art. As will be readily apparent to those skilled in the art, the monoclonal and / or polyclonal antibodies may then be used in a variety of diagnostic applications.

[0189] The in vivo means of administering the compounds or compositions described herein will vary depending on the intended application. Administration of the ADIr compositions described herein, in pure form or in a suitable pharmaceutical composition, may be effected by any of the accepted modes of drug delivery for providing similar effects. The pharmaceutical composition may be prepared by combining an ADIr (e.g., ADIr-PEG, ADIr-PEG 20) with a suitable 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. Additionally, 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 in the composition.

[0190] Administration may be achieved by a variety of different routes including: oral, parenteral, intranasal, intravenous, intradermal, subcutaneous, or topical. The mode of administration depends on the nature of the condition to be treated or prevented. Thus, the ADIr enzyme (e.g., ADIr-PEG, ADIr-PEG 20) may be administered orally, intranasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorally, intramuscularly, interstitially, intraarterially, subcutaneously, intraocularly, intrasynovially, trans-epithelially, and transdermally. After administration, an amount that alleviates, inhibits, prevents cancer, or delays its progression and / or metastasis is considered an effective amount. In certain embodiments, the ADIr compositions herein increase the median survival time of a patient by a statistically significant amount. In some embodiments, the ADIr treatment described herein increases the 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, the ADIr treatment increases the median survival time of a patient by 1 year, 2 years, 3 years, or longer. In some embodiments, the ADIr treatment described herein increases 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 treatment described herein increases progression-free survival by 1 year, 2 years, 3 years, or longer.

[0191] In certain embodiments, the amount administered is sufficient to cause tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor, such as at least 10%, 20%, 30%, 40%, 50%, or greater decrease in tumor mass; or as indicated by a change in scan size (e.g., a statistically significant decrease). In certain embodiments, the amount administered is sufficient to cause disease stabilization. In certain embodiments, the amount administered is sufficient to produce a clinically relevant reduction in symptoms having specific disease signs known to a skilled clinician.

[0192] In certain embodiments, the amount administered is sufficient to inhibit NO synthesis, inhibit angiogenesis, and / or sufficient to induce tumor cell apoptosis or any combination thereof. NO synthesis, angiogenesis, and apoptosis can be measured using methods known in the art, see, for example, Current Protocols in Immunology or Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. (2009 and updates); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; and other similar references. In some embodiments, the amount administered inhibits NO synthesis and inhibits melanoma growth, and complements, increases, or synergistically enhances other chemotherapeutic agents as described herein, such as cisplatin. Thus, some embodiments provide a method of treating melanoma by administering ADIr-PEG20 in combination with cisplatin, wherein the treatment depletes endogenous nitric oxide (NO).

[0193] The precise dosage and duration of treatment vary with the disease being treated and can be determined empirically using known assays or by testing the composition in model systems known in the art and extrapolating therefrom. Controlled clinical trials can also be conducted. The dosage can also vary with the severity of the condition to be alleviated. The pharmaceutical compositions are generally formulated and administered to provide a therapeutically effective action while minimizing unwanted side effects. The compositions can be administered as a single dose, or can be divided into a number of smaller doses to be administered at multiple time intervals. For any particular subject, the specific dosing regimen can be adjusted over time according to individual needs.

[0194] ADIr (e.g., ADIr-PEG) compositions can be administered alone or in combination with other known cancer treatments such as: radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions can also be administered in combination with antibiotics.

[0195] The ADIr composition can also be administered alone or in combination with ADI-PEG 20 therapy. In certain embodiments, the ADIr as described herein is for use in patients who have received ADI-PEG 20 therapy and have developed anti-ADI-PEG 20 antibodies. Such patients no longer benefit from ADI-PEG 20 treatment because the enzyme is neutralized by the antibodies. Thus, in certain embodiments, the present invention provides a method of treating cancer, alleviating its symptoms or inhibiting its progression, which comprises 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 ADIr as described herein, thereby treating cancer, alleviating its symptoms or inhibiting its progression.

[0196] In some embodiments 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 ADIr is administered after the detection of the predetermined level of the anti-ADI-PEG 20 antibodies. In certain embodiments, a threshold level or a predetermined level of anti-ADI-PEG 20 antibodies in a patient to be treated with ADI-PEG 20 and the ADIr enzyme as described herein can be determined. The "predetermined threshold level" (also referred to as the "predetermined level" or "predetermined cut-off value") of anti-ADI-PEG 20 antibodies, or sometimes referred to as the predetermined cut-off value, can be determined using methods known in the art, such as using Receiver Operator Characteristic curves or "ROC" curves. In some embodiments, even very low levels of anti-ADI-PEG 20 antibodies are considered sufficient to be a basis for switching the treatment from ADI-PEG 20 to ADIr-PEG as described herein. In certain embodiments, the appropriate level of anti-ADI-PEG 20 for determining when to end ADI-PEG 20 treatment and start treatment with the ADIr composition as described herein can be determined by a skilled clinician.

[0197] In some embodiments, the period of time is determined by detecting or otherwise observing ADI activity in the patient, wherein the composition is administered after the detection or observation of a predetermined level of ADI activity. In certain embodiments, the composition is administered after the detection or observation of a reduced level of ADI activity in the patient. ADI activity can be measured directly, for example, by assaying at least one ADI activity indicator of a biological sample, or indirectly, for example, by observing the desired or expected effects of ADI-PEG 20 treatment. In certain embodiments, the appropriate level of ADI activity for determining when to end ADI-PEG 20 treatment and start treatment with ADIr-PEG as described herein can be determined by a skilled clinician.

[0198] Thus, typical routes of administration of these and related pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques.

[0199] Certain pharmaceutical compositions are formulated such that the active ingredient(s) contained therein are bioavailable after administration of the composition to a patient. The composition to 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 the container of an ADIr composition in the form of an aerosol as described herein may contain multiple dosage units. The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, 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 as described herein, such as ADIr-PEG 20, for treating the relevant diseases or conditions according to the teachings herein. In certain embodiments, the pharmaceutical or therapeutic composition is sterile and / or pyrogen-free.

[0200] Pharmaceutical compositions can be in solid or liquid form. In some embodiments, the carrier is particulate, such that the composition is in the form of, for example, a tablet or powder. The carrier can be liquid, and the composition is, for example, an oral oil, an injectable liquid, or an aerosol, which is suitable for, for example, inhalational administration. When intended for oral administration, pharmaceutical compositions are generally in solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered solid or liquid herein.

[0201] As a solid composition for oral administration, the pharmaceutical composition can be formulated as a powder, granule, compressed tablet, pill, capsule, chewing gum, dry film, or the like. Such solid compositions 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, ethylcellulose, microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginic acid, sodium alginate, Primogel, corn starch, and the like; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silica; sweetening agents, such as sucrose or saccharin; flavoring agents, such as peppermint, methyl salicylate, or orange flavor; and coloring agents. When the pharmaceutical composition is in the form of a capsule (e.g., a gelatin capsule), it may contain a liquid carrier such as polyethylene glycol or an oil in addition to the above types of materials.

[0202] The pharmaceutical composition can be in liquid form, such as an elixir, syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral administration or for delivery by injection. When intended for oral administration, preferably the composition contains one or more of the following in addition to the compound of the present invention: sweetening agents, preservatives, coloring agents / colorants, and flavoring agents. In the composition intended to be administered by injection, one or more of the following can be included: surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents.

[0203] The liquid pharmaceutical composition (whether it is a solution, suspension or other similar form) can include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution (physiological saline in certain embodiments), Ringer's solution, isotonic sodium chloride, non-volatile oils such as synthetic glycerol monoesters or glycerol diesters that can act as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for adjusting tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0204] The liquid pharmaceutical composition intended for parenteral or oral administration should contain an amount of ADIr (such as ADIr-PEG, ADIr-PEG 20) as disclosed herein such that a suitable dose will be obtained. Typically, this amount is at least 0.01% of ADIr contained in the composition. When intended for oral administration, this amount can vary between 0.1% and about 70% by weight of the composition. Some oral pharmaceutical compositions contain between about 4% and about 75% of ADIr. In certain embodiments, the pharmaceutical composition and formulation are prepared such that the parenteral dose unit contains between 0.01% and 10% by weight of ADIr before dilution.

[0205] A pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, an emulsion, an ointment or a gel matrix. For example, the matrix may comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and ethanol, and emulsifying and stabilizing agents. Thickeners may be present in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device. A pharmaceutical composition may be intended for rectal administration in the form of, for example, a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous matrix as a suitable non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0206] A pharmaceutical composition may include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell surrounding the active ingredient. The materials forming the coating shell are typically inert and may be selected from, for example, sugars, shellac and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. A pharmaceutical composition in solid or liquid form may include a reagent that binds to an ADIr (such as ADIr-PEG) and thereby aids in the delivery of the compound. Suitable reagents that may function in this capacity include monoclonal or polyclonal antibodies, one or more proteins or liposomes. A pharmaceutical composition may consist essentially of dosage units that can be administered in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from systems with colloidal properties to systems consisting of pressurized packages. Delivery may be effected by a liquefied or compressed gas or by a suitable pump system for dispensing the active ingredient. An aerosol may be delivered in the form of a single-phase, two-phase or three-phase system to deliver the active ingredient. The delivery of an aerosol includes the necessary container, activator, valve, sub-container and the like, which together may form a kit. One of ordinary skill in the art can determine the preferred aerosol without undue experimentation.

[0207] A pharmaceutical composition may be prepared by methods well known in the pharmaceutical art. For example, a pharmaceutical composition intended for administration by injection may be prepared by combining a composition comprising an ADIr (such as ADIr-PEG) as described herein and optionally one or more of salts, buffers and / or stabilizers with sterile distilled water to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. A surfactant is a compound that non-covalently interacts with the ADIr (such as ADIr-PEG) composition to facilitate the dissolution or homogeneous suspension of the ADIr (such as ADIr-PEG) in an aqueous delivery system.

[0208] The composition can be administered in a therapeutically effective amount, which will vary depending on a variety of factors including: the activity of the specific compound used (e.g., ADIr-PEG); the metabolic stability and duration of action of the compound; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; drug combinations; the severity of the particular disease or condition; and the subject being treated.

[0209] A therapeutically effective amount of the compounds described herein is an amount effective to inhibit tumor growth. Generally, treatment is initiated at a low dose, which can be increased in small increments until the optimal effect in the situation is achieved. Generally, the therapeutic dose of the compound can be about 1 to about 200 mg / kg twice a week to about once every two weeks. For example, the dose can be about 1 mg / kg once a week to about 20 mg / kg once every 3 days in the form of a 2 ml intravenous injection. In some embodiments, the dose can be administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks at about 50 IU / m 2 to about 700 IU / m 2 . In certain embodiments, the dose can be administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks at about 50 IU / m 2 , 60 IU / m 2 , 70 IU / m 2 , 80 IU / m 2 , 90 IU / m 2 , 100 IU / m 2 , 110 IU / m 2 , 120 IU / m 2 , 130 IU / m 2 , 140 IU / m 2 , 150 IU / m 2 , 160 IU / m 2 , 170 IU / m 2 , 180 IU / m 2 , 190 IU / m 2 , 200 IU / m 2 , 210 IU / m 2 , 220 IU / m 2 , 230 IU / m 2 , 240 IU / m 2 , 250 IU / m 2 , 260 IU / m 2 , 270 IU / m 2 , 280 IU / m 2 , 290 IU / m 2 , 300 IU / m 2 , 310 IU / m 2, about 320 IU / m 2 , about 330 IU / m 2 , 340 IU / m 2 , about 350 IU / m 2 , 360 IU / m 2 , 370 IU / m 2 , 380 IU / m 2 , 390 IU / m 2 , 400 IU / m 2 , 410 IU / m 2 , 420 IU / m 2 , 430 IU / m 2 , 440 IU / m 2 , 450 IU / m 2 , 500 IU / m 2 , 550 IU / m 2 , 600 IU / m 2 , 620 IU / m 2 , 630 IU / m 2 , 640 IU / m 2 , 650 IU / m 2 , 660 IU / m 2 , 670 IU / m 2 , 680 IU / m 2 , 690 IU / m 2 or about 700 IU / m 2 . In certain embodiments, the dosage can be modified by a skilled clinician as needed.

[0210] In some cases, the optimal dosage in the case of ADIr-SS-PEG5,000 can be about twice a week, while the optimal dosage in the case of ADIr-SS-PEG20,000 can be from about once a week to about once every two weeks. In certain embodiments, the optimal dosage in the case of ADIr-SS-PEG20,000 can be about twice a week.

[0211] ADIr (such as ADIr-PEG) can be mixed with phosphate buffered saline solution or any other suitable solution known to those skilled in the art before injection. In some embodiments, the liquid composition containing ADIr-PEG contains about 10 to about 12 mg of ADIr; about 20 to about 40 mg of polyethylene glycol; about 1.27 mg ± 5% sodium dihydrogen phosphate, USP; about 3 mg ± 5% disodium hydrogen phosphate, USP; about 7.6 mg ± 5% sodium chloride, USP; at a pH of about 6.6 to about 7; contained in a suitable amount of water for injection (such as about 1 ml or about 2 ml).

[0212] In some embodiments, the liquid composition containing ADIr-PEG contains 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 containing 0.035 M histidine-HCl at pH 6.8 in the presence of 0.13 M sodium chloride. In certain embodiments, the composition is formulated in a buffer containing 0.02 M sodium phosphate buffer at pH 6.8 in the presence of 0.13 M sodium chloride. In some embodiments, the liquid composition containing ADIr-PEG contains from about 10 to about 12 mg of ADIr; from about 20 to about 40 mg of polyethylene glycol; about 5.4 mg ± 5% histidine, USP; about 7.6 mg ± 5% sodium chloride, USP; at a pH value of from about 6.6 to about 7; contained in a suitable amount of water for injection (e.g., about 1 ml or about 2 ml).

[0213] In some embodiments, the pH value of the composition containing ADIr (such as ADIr-PEG) is from about 5 to about 9, from about 6 to about 8, or from about 6.5 to about 7.5. In some embodiments, the pH value of the composition containing ADIr is about 6.8 ± 1.0.

[0214] In some embodiments, the free PEG in the composition containing ADIr (such as ADIr-PEG) is between 1-10%. In some embodiments, it 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 the composition containing ADIr (such as 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, the total impurities of the composition containing ADIr-PEG are less than or equal to about 4%, 3%, 2%, 1.5%, 1%, or 0.5%. In some embodiments, the endotoxin limit meets the requirements stated in the USP, i.e., ≤50 EU / mL.

[0215] In some embodiments, the free sulfhydryl groups in the composition containing ADIr (such as ADIr-PEG) are greater than about 90%. In some embodiments, the free sulfhydryl groups in the composition containing ADIr or ADIr-PEG are about 91%, about 92%, about 93%, about 94%, or about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0216] In some embodiments, the Km of the ADIr (e.g., ADIr-PEG) in the composition is from about 0.1 μM or 0.5 μM to about 15 μM, or from about 1 μM to about 12 μM, from about 1 μM to about 10 μM, from about 1.5 μM to about 9 μM, from about 1.5 μM to about 8 μM, or from about 1.5 μM to about 7 μM. In certain embodiments, the Km of the ADIr (e.g., ADIr-PEG) in the composition is from about 1.0 μM to about 10 μM or from about 1.5 μM to about 6.5 μM. In some embodiments, the Km of the ADIr or ADIr-PEG in the composition is about, at least about, or less than about 0.1 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, or 7 μM or 8 μM or 9 μM or 10 μM.

[0217] In some embodiments, the Kcat of the ADIr (e.g., ADIr-PEG) in the composition is about 0.5 seconds -1 to about 80 seconds -1 or about 0.5 seconds -1 to about 70 seconds -1 or about 0.5 seconds -1 to about 60 seconds -1 or about 0.5 seconds -1 to about 50 seconds -1 or about 0.5 seconds -1 to about 40 seconds -1 or about 0.5 seconds -1 to about 30 seconds -1 or about 0.5 seconds -1 to about 20 seconds -1 or about 0.5 seconds -1 to about 15 seconds -1 , or is about 0.5 seconds -1 to about 80 seconds -1 or about 1 second -1 to about 80 seconds -1 or about 5 seconds -1 to about 80 seconds -1 or about 10 seconds -1 to about 80 seconds -1 or about 20 seconds -1 to about 80 seconds -1 or about 30 seconds -1 to about 80 seconds -1 or about 40 seconds -1 to about 80 seconds -1 or about 50 seconds -1 to about 80 seconds -1 or about 60 seconds -1 to about 80 seconds-1 or about 70 seconds -1 to about 80 seconds -1 or about 1 second -1 to about 12 seconds -1 about 1 second -1 to about 10 seconds -1 about 1.5 seconds -1 to about 9 seconds -1 about 2 seconds -1 to about 8 seconds -1 or about 2.5 seconds -1 to about 7 seconds -1 In certain embodiments, the Kcat of ADIr (e.g., ADIr-PEG) in the composition is about 2.5 seconds -1 to about 7.5 seconds -1 In some embodiments, the Kcat of ADIr or ADIr-PEG in the composition is about or at least about 2.5 seconds -1 about 3 seconds -1 about 3.5 seconds -1 about 4 seconds -1 about 4.5 seconds -1 about 5 seconds -1 about 5.5 seconds -1 about 6 seconds -1 about 6.5 seconds -1 about 7 seconds -1 about 7.5 seconds -1 or about 8 seconds -1 about 10 seconds -1 about 15 seconds -1 about 20 seconds -1 about 25 seconds -1 about 30 seconds -1 about 35 seconds -1 about 40 seconds -1 about 45 seconds -1 about 50 seconds -1 about 55 seconds -1 about 60 seconds -1 about 65 seconds -1 about 70 seconds -1 about 75 seconds -1 about 80 seconds -1 about 85 seconds -1 about 90 seconds -1 about 95 seconds -1 or about 100 seconds -1 .

[0218] In some embodiments, the conductivity (also known as specific conductance in the art) of the ADIr (e.g., ADIr-PEG) in the composition is from about 5 mS / cm to about 20 mS / cm or from about 5 mS / cm to about 15 mS / cm, from about 7 mS / cm to about 15 mS / cm, from about 9 mS / cm to about 15 mS / cm, or from about 10 mS / cm to about 15 mS / cm. In some embodiments, the conductivity of the ADIr (e.g., ADIr-PEG) in the composition is 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 conductivity of the ADIr (e.g., ADIr-PEG) in the composition is about 13 mS / cm ± 1.0 mS / cm.

[0219] In some embodiments, the osmotic pressure of the ADIr (e.g., ADIr-PEG) in the composition is from about 50 mOsm / kg to about 500 mOsm / kg, from about 100 mOsm / kg to about 400 mOsm / kg, from about 150 mOsm / kg to about 350 mOsm / kg, from about 200 mOsm / kg to about 350 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg. In certain embodiments, the osmotic pressure of the ADIr (e.g., ADIr-PEG) in the composition is about 300 ± 30 mOsm / kg.

[0220] In some embodiments, 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 certain embodiments, the protein concentration is about 8, 9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 15 mg / mL.

[0221] In some embodiments, the enzyme specific 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 1 μmol of arginine to 1 μmol of citrulline and 1 μmol of ammonia in one minute at 37 °C and the titer is 100 ± 20 IU / ml. In certain embodiments, the enzyme specific 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 enzyme specific activity is 9 ± 2.0 IU / mg.

[0222] Compositions comprising an ADIr described herein (e.g., ADIr-PEG) can be administered simultaneously with, prior to, or following the administration of one or more other therapeutic agents including ADI-PEG 20. Such combination therapies can include the administration of a single pharmaceutical dosage formulation containing a compound of the invention and one or more other active agents, as well as the administration of a composition comprising the ADIr described herein (e.g., ADIr-PEG) and each active agent contained in its own separate pharmaceutical dosage formulation. For example, the ADIr (e.g., ADIr-PEG) and the other active agent can be administered to a patient together in a single oral dosage composition such as a tablet or capsule, or the respective agents can be administered in separate oral dosage formulations. Similarly, the ADIr (e.g., ADIr-PEG) and the other active agent can be administered to a patient together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or the respective agents can be administered in separate parenteral dosage formulations by the same or different routes (e.g., one by injection and one by oral). In the case of using separate dosage formulations, the compositions comprising the ADIr (e.g., ADIr-PEG) and one or more other active agents can be administered substantially simultaneously (i.e., in parallel) or separately at staggered times (i.e., sequentially) and in any order; combination therapy is to be understood as including all such regimens.

[0223] Thus, in certain embodiments, co - administration of the ADIr compositions of the present disclosure with one or more other therapeutic agents is also contemplated. Such therapeutic agents may be accepted in the art as standard treatments for specific disease conditions such as specific cancers or GVHD as described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti - inflammatory agents, chemotherapeutic agents, radiotherapy agents, autophagy inhibitors or other active agents and adjuvants.

[0224] In certain embodiments, the ADIr (e.g., ADIr - PEG) compositions disclosed herein may be co - administered in combination with a 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; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dimethylfolic acid, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as cyclosine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone;Anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamideglycoside; 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; podophyllic acid; 2-ethylhydrazine; procarbazine; PSK; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichloroethylamine; carbamate; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoid, such as paclitaxel)(; Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel( 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 RFS2000; Difluoromethylornithine (DMFO); Retinoic acid derivatives such as Targretin TM (Bexarotene), Panretin TM (Alitretinoin); ONTAK TMDenileukin diftitox; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, 4(5)-imidazoles that inhibit aromatase, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin. Other 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 foregoing are also contemplated for use herein.

[0225] In certain embodiments, the ADIr (e.g., ADIr-PEG) compositions disclosed herein can be administered in combination with a number of autophagy inhibitors. In some preferred embodiments, the autophagy inhibitors are selected from the group consisting of: chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil.TM.), bafilomycin A1, 5-amino-4-imidazolecarboxamide ribonucleoside (AICAR), okadaic acid, autophagy-inhibiting marine toxins (which inhibit type 2A or type 1 protein phosphatases), analogs of cAMP and drugs that increase cAMP levels, adenosine, N6-mercaptopurine ribonucleoside, wortmannin, and vinblastine. In addition, antisense or siRNA that inhibits the expression of proteins (such as ATG5) necessary for autophagy can also be used.

[0226] In some embodiments, the combination of ADIr (e.g., ADIr-PEG) with one or more therapeutic agents acts complementarily, additively, or synergistically. In this regard, complementary or synergistic enhancers are described herein, which include therapeutic agents (e.g., chemotherapeutic agents, autophagy inhibitors, mTOR inhibitors, or any other therapeutic agent for treating cancer, GVHD, or inflammatory bowel disease as described herein) that can act complementarily or synergistically with the ADIr-PEG provided herein, where such complementary or synergistic action is manifested as a detectable action with a greater magnitude (i.e., in a statistically significant manner relative to an appropriate control situation) compared to the detectable action that can be detected when the chemotherapeutic agent is present but the ADIr (e.g., ADIr-PEG) composition is absent and / or when the ADIr (e.g., 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 January 15, 2010 70; 440).

[0227] Compositions comprising an ADIr (e.g., ADIr-PEG) and optionally one or more other therapeutic agents as described herein can be used in methods of treatment for treating cancer and methods for preventing cancer metastasis. Thus, some embodiments include methods for treating a variety of different cancers, alleviating their symptoms, inhibiting their progression, or preventing them. Some embodiments include methods for treating GVHD, alleviating its symptoms, or inhibiting its progression. Particular embodiments include methods for treating a patient's cancer or GVHD, alleviating its symptoms, or inhibiting its progression, which include, optionally after treatment with ADI-PEG 20, particularly in the case where the patient develops anti-ADI-PEG 20 antibodies, administering to the patient a therapeutically effective amount of an ADIr composition as described herein, thereby treating the cancer or GVHD, alleviating its symptoms, or inhibiting its progression. Thus, the ADIr compositions described herein can be administered to an individual having an 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), sarcoma (including but not limited to metastatic sarcoma, uterine leiomyosarcoma), pancreatic cancer, prostate cancer (such as but not limited to hormone-refractory prostate cancer), mesothelioma, lymphocytic 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 multiforme, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), renal cancer (including but not limited to renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer (including but not limited to head and neck squamous cell carcinoma; tongue cancer), cervical cancer, testicular cancer, gallbladder cancer, cholangiocarcinoma, and gastric cancer.

[0228] Also included are methods for treating a patient's cancer, alleviating its symptoms, or inhibiting its progression, which include administering to the patient a composition comprising an ADIr (e.g., ADIr-PEG) and optionally one or more other therapeutic agents as described herein, wherein the cancer lacks ASS, ASL, or both. In this regard, the lack of ASS or ASL can be a reduced expression as measured by mRNA expression or protein expression, or can be a reduced protein activity, and generally comprises a statistically significant reduction in expression or activity as determined by a person skilled in the art. The reduced ASS or ASL expression or activity can 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 the expression or activity in a suitable control sample known not to have cancer. In certain embodiments, the ASS or ASL expression or activity is reduced by at least two-fold as compared to the expression or activity in a non-cancer control sample.

[0229] In certain embodiments, the reduced expression or activity of ASS or ASL results from methylation of an ASS or ASL promoter or inhibition of an ASS or ASL promoter. In some embodiments, the reduced 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 aberration that results in gene deletion. In some embodiments, the cancer is ASS- or ASL-negative, meaning that no expression or activity is observed.

[0230] Reduced ASS or ASL expression or activity can be measured using any method known in the art such as, but not limited to, the following: quantitative PCR, immunohistochemistry, enzyme activity assays (e.g., assays for measuring the conversion of citrulline to argininosuccinate or the conversion of argininosuccinate to arginine and fumarate), and the like.

[0231] Accordingly, certain embodiments include methods for treating a patient's cancer, alleviating its symptoms, or inhibiting its progression, which include administering to the patient a composition comprising an ADIr (e.g., ADIr-PEG) as described herein, wherein the cancer exhibits reduced expression or activity of ASS or ASL or both, and 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), sarcoma (including, but not limited to, metastatic sarcoma, uterine leiomyosarcoma), pancreatic cancer, prostate cancer (such as, but not limited to, hormone-refractory prostate cancer), mesothelioma, lymphocytic 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 multiforme, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), renal cancer (including, but not limited to, renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer (including, but not limited to, head and neck squamous cell carcinoma; tongue cancer), cervical cancer, testicular cancer, gallbladder cancer, cholangiocarcinoma, and gastric cancer.

[0232] Various studies in the literature have shown a lack of ASS in the following tumors: acute myeloid leukemia (AML), bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, HCC, lymphoma, melanoma, mesothelioma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, and small cell lung cancer. Accordingly, the treatment of these ASS-deficient cancers is specifically covered herein using ADIr-PEG alone or in combination with other treatments (including treatment first with ADI-PEG 20).

[0233] Also included are methods for treating a patient's cancer, alleviating its symptoms, or inhibiting its progression, which include administering to the patient, in combination with an autophagy inhibitor, a composition comprising an ADIr (e.g., ADIr-PEG, ADIr-PEG 20) as described herein. Some embodiments include methods for treating a patient's cancer, which include administering to the patient, in combination with an autophagy inhibitor, a therapeutically effective amount of a composition comprising an ADIr as described herein, wherein the cancer is pancreatic cancer or small cell lung cancer.

[0234] Some embodiments include methods of treatment wherein administration of the composition comprising an ADIr as described herein depletes arginine in the plasma for at least one month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. Certain embodiments include methods of treatment wherein, after termination of treatment with ADI-PEG 20 and after detection of anti-ADI-PEG 20 antibodies, administration of the composition comprising an ADIr as described herein depletes arginine in the plasma for at least one month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. Examples

[0235] Example 1

[0236] Screening and selection of ADI enzymes with low cross-reactivity to patient anti-ADI-PEG 20 antibodies

[0237] This example describes the screening and selection of ADI enzymes with low cross-reactivity to patient anti-ADI-PEG 20 antibodies.

[0238] From a large number of ADI enzymes, Table 1 lists 27 ADI enzymes selected with respect to the percentage of sequence identity to Mycoplasma hominis ADI.

[0239]

[0240]

[0241] Many ADI enzymes from various organisms were characterized to determine which enzymes are expected to remove arginine from the patient's blood and maintain low concentrations even in the presence of anti-ADI-PEG 20 antibodies. As detailed below, these studies showed that ADIs from many species closely related to Mycoplasma hominis based on sequence identity have sufficiently good enzyme catalytic properties measured at various temperatures and pH values, and also show reduced cross-reactivity with anti-ADI-PEG 20 antibodies.

[0242] ADI preparation. The recombinant ADI enzyme was cloned, expressed, and purified for testing according to standard protocols as described, for example, in the following references: 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.

[0243] Purification of human anti-ADI-PEG20 antibody . The anti-ADI-PEG20 antibody was purified from plasma samples of patients who had received ADI-PEG20 during clinical studies. For example, a total of 60 ml of plasma was pooled from 8 different patients who had achieved a high titer (titer > / = 4) against ADI-PEG20 as determined by ELISA assay. Purification was performed using a two-step method, with protein "A" chromatography (GE Healthcare) followed by ADI affinity chromatography. Approximately 20 mg of purified antibody was obtained and stored in aliquots at -80 °C until needed.

[0244] ADI enzyme assay . 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 to calculate the ADI specific activity expressed as IU per milligram of protein. One IU of enzyme activity is defined as the amount of enzyme that produces 1 μmol of citrulline per minute at the pH and temperature being tested. Standard assay conditions are performed at 37 °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. Whenever conditions permit, all samples and standards are run in duplicate or triplicate.

[0245] The Km and Kcat values were determined by using a modified form of the activity assay described above. As in the case of the activity assay (or unless otherwise indicated, all reactions were carried out at 37 °C in PHB plus 0.1% BSA). The enzyme concentration, reaction time, and substrate concentration range were adjusted for each of the ADI or ADIr constructs to account for their activity differences. Generally, 2 nM enzyme, a 5-minute reaction time, and 0 - 160 μM arginine were used as starting conditions. When optimizing conditions, special attention was paid to the amount of substrate consumed in the form of a percentage of the total substrate added to the reaction. Typically, the lower limit of detection was approximately 1 μM citrulline, and the lower limit of quantification was approximately 2 μM. A citrulline standard curve assay was performed for each plate and used to quantify the citrulline produced by the enzymatic reaction.

[0246] Activity assays were also performed to evaluate the enzyme activity (antibody neutralization characteristics) in the presence of anti-ADI-PEG20. These assays were carried out as described above and in the presence of 640 nM, 320 nM, 160 nM, 80 nM, 40 nM, 20 nM, 10 nM, and 0 nM anti-ADI-PEG20 antibodies.

[0247] Calculation . The concentration of citrulline (μM) produced in each reaction was calculated using the citrulline standard curve and averaged. Then, the rate of each reaction was calculated as μM / min / 50 nM ADI. The specific activity (IU / mg or micromoles of product per minute per milligram of ADI) was calculated by multiplying this value by the “IU” factor (the IU factor was calculated from the molecular weight of ADI and the reaction volume).

[0248] The data are shown in Table 2 - 7 below.

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] These data in particular show that native and pegylated ADI enzymes highly homologous to Mycoplasma hominis ADI (about 50%-100% identity) maintain excellent catalytic activity (Table 2-4), including under different pH values (Table 6) and temperature limitations (Table 7). As measured by (increased) enzyme activity in the presence of anti-ADI-PEG 20 antibody (Table 4-5), for example relative to Mycoplasma hominis, it also shows reduced affinity for patient anti-ADI-PEG 20 antibody. Thus, these ADI enzymes may have therapeutic efficacy when used in therapies for treating cancer alone or after ADI-PEG 20 treatment, to extend and / or increase the effectiveness of arginine depletion therapy.

[0260] The various embodiments described above may be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned and / or listed in the data tables used herein are incorporated herein by reference in their entirety. Aspects of the embodiments may be modified as necessary to use the concepts of the various patents, applications, and publications to provide other embodiments.

[0261] These and other changes may 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 as limiting the claims to the specific embodiments disclosed in this specification and the claims, but should be understood to include all possible embodiments and the full scope of equivalents to such claims. Thus, the claims are not limited by the present disclosure. Sequence Listing <110> Ruihua Pharmaceutical Group Showalter, Richard E. Almassy, Robert Thomson, James A. Sisson, Wes Shia, Wei-Jong Chen, Li-Chang Lee, Yang <120> Arginine Deiminase with Reduced Cross-Reactivity to ADI-PEG 20 Antibody for Cancer Treatment <130> POLA-005 / 01WO <150> US 62,051,182 <151> 2014-09-16 <160> 28 <170> PatentIn Version 3.5 <210> 1 <211> 409 <212> PRT <213> Mycoplasma hominis <400> 1 Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys Ile 50 55 60 Met Lys Asp Arg Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Lys Ala Ala Lys Glu Glu Phe Ile Glu 85 90 95 Thr Phe Leu Glu Glu Thr Val Pro Val Leu Thr Glu Ala Asn Lys Lys 100 105 110 Ala Val Arg Ala Phe Leu Leu Ser Lys Pro Thr His Glu Met Val Glu 115 120 125 Phe Met Met Ser Gly Ile Thr Lys Tyr Glu Leu Gly Val Glu Ser Glu 130 135 140 Asn Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp 145 150 155 160 Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Phe Met Arg Tyr 165 170 175 Ile Val Arg Arg Arg Glu Thr Leu Phe Ala Arg Phe Val Phe Arg Asn 180 185 190 His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met Lys 195 200 205 Met Pro Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu Thr Leu 210 215 220 Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu Leu 225 230 235 240 Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg Ile Val 245 250 255 Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr Trp 260 265 270 Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala Asn 275 280 285 Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala Glu 290 295 300 Pro Gln Pro Gln Leu Asn Gly Leu Pro Leu Asp Lys Leu Leu Ala Ser 305 310 315 320 Ile Ile Asn Lys Glu Pro Val Leu Ile Pro Ile Gly Gly Ala Gly Ala 325 330 335 Thr Glu Met Glu Ile Ala Arg Glu Thr Asn Phe Asp Gly Thr Asn Tyr 340 345 350 Leu Ala Ile Lys Pro Gly Leu Val Ile Gly Tyr Asp Arg Asn Glu Lys 355 360 365 Thr Asn Ala Ala Leu Lys Ala Ala Gly Ile Thr Val Leu Pro Phe His 370 375 380 Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met 385 390 395 400 Pro Leu Ser Arg Lys Asp Val Lys Trp 405 <210> 2 <211> 410 <212> PRT <213> Mycoplasma salivarium <400> 2 Met Ser Val Phe Ser Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Glu Phe Val Ala Thr 50 55 60 Leu Lys Lys Glu Lys Ile Asn Val Val Glu Leu Thr Asp Leu Val Thr 65 70 75 80 Glu Thr Tyr Asp Leu Val Asp Gln Lys Thr Lys Asp Lys Leu Ile Asp 85 90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Thr Ala Glu Leu Lys Ala 100 105 110 Thr Val Lys Lys Phe Leu Lys Ser Phe Lys Glu Thr Arg Lys Leu Ile 115 120 125 Glu Val Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys Ala 130 135 140 Asp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Asn 180 185 190 Asn His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met 195 200 205 Lys Met Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Ile Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300 Asn Pro Gln Pro Lys Asp Asn Gly Leu Pro Leu Asp Lys Leu Leu Lys 305 310 315 320 Ser Ile Ile Gly Lys Glu Pro Val Leu Ile Pro Ile Ala Gly His His 325 330 335 Ala Thr Glu Ile Glu Val Ala Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ala Arg Asn Glu 355 360 365 Lys Thr Asn Glu Ala Leu Lys Asp Ala Gly Ile Thr Val Leu Pro Phe 370 375 380 Lys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 410 <210> 3 <211> 411 <212> PRT <213> Mycoplasma pneumoniae <400> 3 Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gly Phe Val Ala Glu 50 55 60 Leu Lys Lys Gln Asn Val Asn Val Ile Glu Leu Thr Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Glu Leu Ala Ser Lys Glu Ala Gln Ala Lys Leu Ile Glu 85 90 95 Asp Phe Ile Glu Asp Ser Glu Pro Val Leu Asn Ala Glu Glu Ala Gln 100 105 110 Ala Val Arg Lys Phe Leu Ser Glu Arg Lys Ser Thr Arg Glu Met Val 115 120 125 Glu Tyr Met Met Ser Gly Leu Thr Lys Tyr Glu Leu Gly Leu Glu Ser 130 135 140 Ala Asp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr 145 150 155 160 Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met 165 170 175 Lys Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ala Lys Phe Val Phe 180 185 190 Ser Asn His Pro Lys Leu Val Asn Thr Pro Arg Tyr Tyr Asp Pro Ser 195 200 205 Met Lys Leu Pro Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu 210 215 220 Thr Leu Val Val Gly Cys Ser Glu Arg Thr Glu Leu Glu Thr Ile Thr 225 230 235 240 Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg 245 250 255 Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp 260 265 270 Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile 275 280 285 Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly 290 295 300 Glu Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Glu Leu Leu 305 310 315 320 Ala Ser Ile Ile Asn Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Glu 325 330 335 Gly Ala Thr His Ile Asp Val Glu Arg Glu Thr His Phe Asp Gly Thr 340 345 350 Asn Tyr Leu Ala Ile Ala Pro Ala Leu Ile Ile Gly Tyr Ser Arg Asn 355 360 365 Glu Lys Thr Asn Ala Ala Leu Glu Lys Ala Gly Ile Thr Val Leu Pro 370 375 380 Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met 385 390 395 400 Ser Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 410 <210> 4 <211> 410 <212> PRT <213> Mycoplasma ear <400> 4 Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Lys Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Glu Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu 50 55 60 Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Val Ser Gln Glu Leu Lys Asp Lys Leu Ile Glu 85 90 95 Glu Phe Leu Asp Asp Ser Tyr Pro Val Leu Thr Glu Glu His Lys Lys 100 105 110 Ala Val Arg Ser Phe Leu Lys Ser Arg Ser Ser Thr Arg Glu Leu Ile 115 120 125 Glu Tyr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140 Glu Gly Asp Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Asp 180 185 190 Asn His Pro Lys Leu Val Asn Thr Pro Arg Tyr Tyr Asp Pro Ser Leu 195 200 205 Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Met Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Val Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro His Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Tyr Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300 Glu Pro Gln Pro Val Val Asn Glu Leu Pro Leu Asp Lys Leu Leu Glu 305 310 315 320 Ser Ile Ile His Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu Gly 325 330 335 Ala Ser Gln Ile Asp Leu Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Val Leu Arg Pro Gly Val Val Val Gly Tyr Ala Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Glu Ala Val Gly Ile Lys Val Leu Pro Phe 370 375 380 Tyr Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ser Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 410 <210> 5 <211> 410 <212> PRT <213> Mycoplasma hyosynoviae <400> 5 Met Ser Val Phe Asn Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Asp Leu Glu Ser Val Leu Val His Glu Pro Gly Lys Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser Asn Asp Ala Arg Lys Glu His Lys Glu Phe Val Glu Ile 50 55 60 Leu Lys Lys Glu Gly Val Asn Val Val Glu Leu Val Asp Leu Ile Ala 65 70 75 80 Glu Thr Ile Asp Leu Val Asp Ala Lys Lys Lys Glu Ala Leu Ile Asp 85 90 95 Glu Tyr Ile Glu Asp Ser Glu Pro Val Val Asp Ala Lys Val Lys Pro 100 105 110 Leu Val Lys Lys Leu Leu Leu Gly Ile Lys Asp Thr Lys Glu Leu Val 115 120 125 Lys Leu Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Glu Ile Glu Ser 130 135 140 Glu Lys Glu Leu Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Arg 180 185 190 Asn His Pro Lys Leu Thr Ser Thr Pro Trp Tyr Tyr Asp Pro Ala Met 195 200 205 Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Ile Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ser 290 295 300 Glu Pro Gln Pro Lys Asp Asn Gly Leu Pro Leu Glu Lys Leu Leu Glu 305 310 315 320 Ser Ile Ile Gly Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Cys Cys 325 330 335 Ala Ser Asp Ile Glu Ile Ala Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile Lys Pro Gly Val Val Ile Gly Tyr Ala Arg Asn Glu 355 360 365 Lys Thr Asn Lys Ala Leu Glu Lys Ala Gly Ile Lys Val Leu Pro Phe 370 375 380 Lys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 410 <210> 6 <211> 409 <212> PRT <213> Mycoplasma cloacae <400> 6 Met Ser Val Phe Asp Lys Arg Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Lys Ile 50 55 60 Leu Glu Ser Gln Gly Ile Asn Val Val Glu Leu Thr Asp Leu Ile Ala 65 70 75 80 Glu Thr Tyr Glu Leu Ala Ser Glu Glu Ala Lys Asp Asn Leu Ile Glu 85 90 95 Glu Phe Leu Asp Glu Ser Glu Pro Val Leu Ser Glu Glu His Arg Ile 100 105 110 Leu Val Arg Asn Phe Leu Lys Gly Ile Thr Lys Thr Lys Glu Leu Val 115 120 125 Lys Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140 Asp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Glu 180 185 190 Asn His Pro Lys Leu Val Ser Thr Pro Ile Tyr Tyr His Pro Ser Gln 195 200 205 Gly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Lys Ala Asn Glu Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp 290 295 300 Glu Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Asn Glu Leu Leu Ala 305 310 315 320 Ser Ile Ile Gly Glu Glu Pro Val Leu Val Pro Ile Ala Gly Glu Gly 325 330 335 Ala Ser Lys Met Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile Ala Pro Gly Val Val Val Gly Tyr Ser Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Glu Lys Ala Gly Ile Lys Val Leu Pro Phe 370 375 380 Lys Gly His Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Tyr Arg Lys Asp Val Lys 405 <210> 7 <211> 412 <212> PRT <213> Mycoplasma alcaligenes <400> 7 Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly His Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala Met 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu 50 55 60 Leu Lys Ala Asn Asn Val Asn Val Ile Glu Leu Thr Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu 85 90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu Asn Lys Ile 100 105 110 Ala Val Arg Asp Phe Leu Lys Ser Arg Lys Thr Thr Arg Glu Leu Ile 115 120 125 Glu Val Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys Asn 130 135 140 Cys Lys Cys Gln Asp Leu Val Val Asp Pro Met Pro Asn Leu Tyr Phe 145 150 155 160 Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Ile Thr Ile His Tyr 165 170 175 Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile 180 185 190 Phe Ala Asn His Pro Lys Leu Val Asn Thr Pro Ile Tyr Tyr His Pro 195 200 205 Ser Leu Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn 210 215 220 Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile 225 230 235 240 Thr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys 245 250 255 Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu 260 265 270 Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro 275 280 285 Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly 290 295 300 Gly Ala Glu Pro Lys Pro Val Glu Asn Gly Ser Ser Leu Glu Ala Ile 305 310 315 320 Leu Glu Ser Ile Ile His Lys Lys Pro Ile Leu Ile Pro Ile Gly Gly 325 330 335 Asp Ser Ala Ser Gln Ile Glu Val Glu Arg Glu Thr His Phe Asp Gly 340 345 350 Thr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg 355 360 365 Asn Val Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Ile 370 375 380 Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys 385 390 395 400 Met Ser Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 410 <210> 8 <211> 410 <212> PRT <213> Mycoplasma orale <400> 8 Methionine, Serine, Valine, Phenylalanine, Serine, Aspartic acid, Lysine, Phenylalanine, Asparagine, Glycine, Isoleucine, Histidine, Valine, Tyrosine, Serine, Glutamic acid 1 5 10 15 Isoleucine, Glycine, Aspartic acid, Leucine, Glutamic acid, Serine, Valine, Leucine, Valine, Histidine, Glutamic acid, Proline, Glycine, Lysine, Glutamic acid, Isoleucine 20 25 30 Aspartic acid, Tyrosine, Isoleucine, Threonine, Proline, Alanine, Arginine, Leucine, Aspartic acid, Glutamic acid, Leucine, Leucine, Phenylalanine, Serine, Alanine, Isoleucine 35 40 45 Leucine, Glutamic acid, Serine, Threonine, Aspartic acid, Alanine, Arginine, Lysine, Glutamic acid, Histidine, Lysine, Glutamic acid, Phenylalanine, Valine, Glutamic acid, Isoleucine 50 55 60 Leucine, Lysine, Lysine, Glutamine, Glycine, Isoleucine, Asparagine, Valine, Valine, Glutamic acid, Leucine, Valine, Aspartic acid, Leucine, Valine, Valine 65 70 75 80 Glutamic acid, Threonine, Tyrosine, Asparagine, Leucine, Valine, Aspartic acid, Lysine, Lysine, Threonine, Glutamine, Glutamic acid, Lysine, Leucine, Leucine, Lysine 85 90 95 Aspartic acid, Phenylalanine, Leucine, Aspartic acid, Aspartic acid, Serine, Glutamic acid, Proline, Valine, Leucine, Serine, Proline, Glutamic acid, Histidine, Arginine, Lysine 100 105 110 Alanine, Valine, Glutamic acid, Lysine, Phenylalanine, Leucine, Lysine, Serine, Leucine, Lysine, Serine, Threonine, Lysine, Glutamic acid, Leucine, Isoleucine 115 120 125 Glutamine, Tyrosine, Methionine, Methionine, Alanine, Glycine, Isoleucine, Threonine, Lysine, Tyrosine, Aspartic acid, Leucine, Glycine, Isoleucine, Lysine, Alanine 130 135 140 Aspartic acid, Lysine, Glutamic acid, Leucine, Isoleucine, Valine, Aspartic acid, Proline, Methionine, Proline, Asparagine, Leucine, Tyrosine, Phenylalanine, Threonine, Arginine 145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Lys Phe Ile Phe Thr 180 185 190 Asn His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met 195 200 205 Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ser 290 295 300 Asn Pro Glu Pro Val Val Asn Gly Leu Pro Leu Asp Lys Leu Leu Glu 305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Lys Gly 325 330 335 Ala Thr Glu Ile Glu Thr Ala Val Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile Lys Pro Gly Val Val Val Gly Tyr Ser Arg Asn Val 355 360 365 Lys Thr Asn Ala Ala Leu Glu Ala Asn Gly Ile Lys Val Leu Pro Phe 370 375 380 Lys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 410 <210> 9 <211> 401 <212> PRT <213> Mycoplasma inertum <400> 9 Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val 1 5 10 15 Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg 20 25 30 Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala Ile 35 40 45 Gln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile Lys 50 55 60 Thr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr Ala 65 70 75 80 Ser Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala 85 90 95 Thr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr Ile 100 105 110 Leu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met Ala 115 120 125 Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu Ile 130 135 140 Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser 145 150 155 160 Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys 165 170 175 Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr 180 185 190 Lys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile Glu 195 200 205 Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val 210 215 220 Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile 225 230 235 240 Gln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn Val 245 250 255 Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val 260 265 270 Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys 275 280 285 Val Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr Asn 290 295 300 Lys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile 305 310 315 320 Leu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp Ile 325 330 335 Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val 340 345 350 Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg Ala 355 360 365 Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly 370 375 380 Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val 385 390 395 400 Lys <210> 10 <211> 401 <212> PRT <213> Mycoplasma gallisepticum <400> 10 Met Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys Val 1 5 10 15 Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg 20 25 30 Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala Ile 35 40 45 Glu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile Gln 50 55 60 Ala Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala 65 70 75 80 Thr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala 85 90 95 Thr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr Ile 100 105 110 Leu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met Ala 115 120 125 Gly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu Ile 130 135 140 Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser 145 150 155 160 Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys 165 170 175 Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu Tyr 180 185 190 Lys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile Glu 195 200 205 Gly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly Val 210 215 220 Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile 225 230 235 240 Gln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn Val 245 250 255 Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val 260 265 270 Asp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu Lys 275 280 285 Ile Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser Asn 290 295 300 Lys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile 305 310 315 320 Leu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp Ile 325 330 335 Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val 340 345 350 Val Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala 355 360 365 Ala Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly 370 375 380 Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val 385 390 395 400 Lys <210> 11 <211> 405 <212> PRT <213> Mycoplasma pirum <400> 11 Met Asn Ser Asn Gln Lys Gly Ile His Val Tyr Ser Glu Ile Gly Lys 1 5 10 15 Leu Lys Glu Val Leu Val His Arg Pro Gly Arg Glu Leu Asp Phe Leu 20 25 30 Asp Pro Thr Arg Leu Asp Glu Leu Leu Phe Ala Ala Thr Leu Glu Ala 35 40 45 Glu Thr Ala Arg Leu Glu His Asp Asn Phe Thr Asn Ala Leu Lys Asn 50 55 60 Gln Gly Val Thr Val Ile Glu Leu Ala Asp Leu Val Ala Gln Thr Tyr 65 70 75 80 Ser Ser Ser Thr Pro Thr Ile Lys Ala Ala Phe Ile Asn Lys Tyr Leu 85 90 95 Asp Glu Ala Thr Pro Ala Leu Thr Thr Lys Leu Arg Thr Leu Val Lys 100 105 110 Asp Phe Leu Thr Lys Gln Lys Ser Val Arg Lys Met Val Asp Tyr Met 115 120 125 Ile Gly Gly Ile Leu Ser Thr Asp Leu Asn Ile Lys Gly Lys Pro Glu 130 135 140 Leu Ile Val Glu Pro Met Pro Asn Ala Tyr Phe Thr His Asp Pro Phe 145 150 155 160 Ala Ser Val Gly Asn Gly Val Thr Leu His Tyr Met Lys His Asn Val 165 170 175 Arg Arg Arg Glu Val Leu Phe Ser Glu Phe Ile Phe Asn Asn Asn Glu 180 185 190 Arg Phe Gln Asn Thr Pro Arg Tyr Ile Val Pro Thr Lys Gly Leu Asp 195 200 205 Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Lys Asn Thr Leu Val Val 210 215 220 Gly Val Ser Glu Arg Thr Lys Met Val Thr Ile Lys Glu Leu Ala Lys 225 230 235 240 Asn Ile Leu Lys Asn Lys Glu Cys Leu Phe Lys Lys Ile Tyr Ala Ile 245 250 255 Asn Val Pro Lys Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr 260 265 270 Met Leu Asp His Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val 275 280 285 Leu Lys Ile Trp Glu Ile Asp Ile Ser Ser Gly Lys Ser Ile Ser Ser 290 295 300 Pro Lys Glu Leu Asn Met Asp Leu Ser Lys Ala Leu Ser Ile Ile Ile 305 310 315 320 Gly Lys Lys Pro Ile Leu Ile Pro Val Ala Gly Glu Asn Ala Ser Gln 325 330 335 Ile Asp Ile Asn Ile Glu Thr Asn Phe Asp Ala Thr Asn Tyr Leu Val 340 345 350 Thr Gln Pro Gly Val Val Val Gly Tyr Ser Arg Asn Lys Lys Thr Glu 355 360 365 Ala Ala Leu Ile Lys Ala Gly Ile Glu Val Ile Pro Phe Gln Gly Asn 370 375 380 Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu 385 390 395 400 Ile Arg Glu Asp Val 405 <210> 12 <211> 404 <212> PRT <213> Mycoplasma primatum <400> 12 Met Ser Lys Ser Lys Ile Asn Val Tyr Ser Glu Tyr Gly Asn Leu Lys 1 5 10 15 Glu Val Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Thr Pro 20 25 30 Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Lys Ser 35 40 45 Ala Ile Ala Glu His Lys Ser Phe Cys Gln Ile Leu Lys Asp Asn Lys 50 55 60 Val Lys Ala Ile Gln Leu Asp Glu Leu Val Ala Ala Thr Tyr Lys Gly 65 70 75 80 Val Ser Glu Ser Val Gln Asn Ser Phe Val Glu Arg Trp Leu Asp Glu 85 90 95 Cys Glu Pro Lys Leu Glu Asn Asn Val Arg Pro Ile Val Lys Glu Tyr 100 105 110 Leu Leu Lys Ala Ala Glu Gln Ser Val Lys Lys Met Ile Arg Ile Met 115 120 125 Met Ala Gly Ile Asp Lys Arg Glu Ile Gly Val Glu Ser Glu Val Asp 130 135 140 Phe Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe 145 150 155 160 Ala Ser Val Gly Asn Gly Ile Thr Leu His His Met Lys Tyr Val Val 165 170 175 Arg Gln Arg Glu Thr Leu Phe Ser Glu Phe Ile Phe Asp Asn His Pro 180 185 190 Asp Tyr Lys Phe Val Pro Arg Tyr Phe Asp Arg Asp Asp Glu Gly Lys 195 200 205 Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Ser Lys Thr Leu Val Val 210 215 220 Gly Ile Ser Glu Arg Thr Asn Lys Asp Ala Ile Arg Ile Val Ala Lys 225 230 235 240 Lys Ile Gln Ala Asn Ala Asp Ala Lys Phe Glu Lys Ile Phe Ala Ile 245 250 255 Asn Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr 260 265 270 Met Leu Asp Ser Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val 275 280 285 Leu Lys Val Trp Glu Ile Asn Leu Asp Asp Pro Ala Leu Glu Trp Lys 290 295 300 Glu Ile Ser Gly Ser Leu Glu Glu Ile Leu Thr Tyr Ile Ile Gly Lys 305 310 315 320 Lys Pro Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Ser Gln Phe Glu 325 330 335 Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Ala 340 345 350 Pro Ser Val Val Ile Gly Tyr Ser Arg Asn Glu Leu Thr Glu Lys Ala 355 360 365 Leu Lys Lys Ala Gly Val Lys Val Leu Ser Leu Asp Gly Asn Gln Leu 370 375 380 Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg 385 390 395 400 Glu Asp Val Lys <210> 13 <211> 401 <212> PRT <213> Mycoplasma lipofaciens <400> 13 Met Ser Lys Ile Asn Val Tyr Ser Glu Val Gly Val Leu Lys Glu Val 1 5 10 15 Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Val Ala Pro Ser Arg 20 25 30 Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Gln Asp Ala Ile 35 40 45 Ala Glu His Lys Arg Phe Ile Lys Ile Leu Glu Asp Asn Asn Ile Lys 50 55 60 Val Ile Gln Leu Asp Glu Leu Val Ser Glu Thr Trp Glu Lys Ala Thr 65 70 75 80 Ala Glu Gln Arg Asp Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala Glu 85 90 95 Pro Val Leu Asp Ala Lys Leu Arg Glu Thr Val Lys Lys Tyr Leu Leu 100 105 110 Ser Leu Asn Pro Val Lys Lys Met Val Arg Thr Met Met Ala Gly Ile 115 120 125 Asp Lys Lys Glu Leu Lys Ile Glu Leu Asp Arg Asp Leu Val Val Asp 130 135 140 Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Ala Gly 145 150 155 160 Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys Arg Glu 165 170 175 Thr Ile Phe Ala Glu Phe Ile Phe Asn Ile His Pro Asp Tyr Lys Thr 180 185 190 Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile Glu Gly Gly 195 200 205 Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Leu Gly Val Ser Glu 210 215 220 Arg Thr Asn Lys Asp Ala Val Met Thr Ile Ala Lys His Ile Gln Ser 225 230 235 240 Asn Glu Gln Ala Lys Phe Lys Lys Leu Val Ala Ile Asn Val Pro Pro 245 250 255 Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val Asp His 260 265 270 Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys Ile Trp 275 280 285 Glu Ile Asp Leu Thr Pro Gly Lys Glu Ile Glu Met Val Glu Ser Thr 290 295 300 Lys Ser Leu Ser Asp Met Leu Glu Ser Ile Ile Gly Lys Lys Pro Val 305 310 315 320 Leu Ile Pro Ile Ala Gly Lys Asp Ala Ser Gln Leu Asp Ile Asp Ile 325 330 335 Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Arg Pro Gly Val 340 345 350 Val Val Gly Tyr Ser Arg Asn Cys Leu Thr Glu Gln Ala Leu Lys Asp 355 360 365 Ala Gly Val Thr Val Leu Ser Phe Asp Gly Asn Gln Leu Ser Leu Gly 370 375 380 Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Ile 385 390 395 400 Lys <210> 14 <211> 405 <212> PRT <213> Mycoplasma tracheae <400> 14 Met Asn Lys Ile Asn Val Tyr Ser Glu Ile Gly Lys Leu Lys Glu Val 1 5 10 15 Leu Val His Thr Pro Gly Asn Glu Ile Arg Arg Ile Ser Pro Ser Arg 20 25 30 Leu Asp Glu Leu Leu Phe Ser Ala Leu Leu Glu Pro Asn Phe Ala Ala 35 40 45 Lys Glu His Thr Ala Phe Cys Glu Ile Leu Lys Glu Asn Gly Ile Lys 50 55 60 Ala Ile Gln Leu Val Asp Leu Val Ser Asp Thr Trp Arg Ile Ala Ser 65 70 75 80 Glu Lys Ala Lys Thr Glu Phe Ile Glu Arg Trp Leu Asp Glu Cys Glu 85 90 95 Pro Lys Leu Asp Ser Asn Leu Arg Glu Ile Val Arg Lys His Ile Tyr 100 105 110 Ala Ile Glu Lys Arg Ser Val Lys Arg Met Val Lys Thr Met Met Ala 115 120 125 Gly Ile Glu Arg Arg Glu Leu Pro Val Thr Ser Lys Glu Val Ala Arg 130 135 140 Glu Leu Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro 145 150 155 160 Phe Ala Ser Val Gly Asn Gly Ile Ser Leu His His Met Lys Tyr Val 165 170 175 Thr Arg Gln Arg Glu Thr Ile Phe Ala Glu Phe Val Phe Gly Asn His 180 185 190 Pro Asp Tyr Ile Asp Thr Pro Arg Trp Phe Asp Arg Ser Asp Asp Gly 195 200 205 Arg Ile Glu Gly Gly Asp Val Phe Ile Tyr Gly Ser Lys Thr Leu Val 210 215 220 Ile Gly Val Ser Glu Arg Thr Asn Lys Glu Ala Ile Lys Val Met Ala 225 230 235 240 Lys Lys Ile Gln Ala Asn Lys Glu Ala Thr Phe Glu Lys Ile Tyr Ala 245 250 255 Ile Asn Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu 260 265 270 Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ala 275 280 285 Val Leu Gln Val Trp Glu Ile Asp Leu Lys Asp Pro Glu Leu Thr Trp 290 295 300 His Glu Leu Ser Gly Ser Leu Glu Glu Ile Leu His Lys Ile Ile Gly 305 310 315 320 Arg Lys Pro Ile Leu Ile Pro Ile Ala Gly His Gly Ala Gln Gln Ile 325 330 335 Asp Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Ala Ile 340 345 350 Ala Pro Gly Val Val Val Gly Tyr Asn Arg Asn Val Leu Thr Glu Arg 355 360 365 Ala Leu Lys Lys Ala Gly Ile Lys Val Leu Ser Phe Glu Gly Asn Gln 370 375 380 Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile 385 390 395 400 Arg Glu Asn Leu Lys 405 <210> 15 <211> 404 <212> PRT <213> Mycoplasma imitans <400> 15 Met Phe Asn Lys Ile Lys Val Tyr Ser Glu Ile Gly Arg Leu Arg Lys 1 5 10 15 Val Leu Val His Thr Pro Gly Lys Glu Leu Glu Tyr Val Thr Pro Gln 20 25 30 Arg Leu Asp Glu Leu Leu Phe Ser Ser Leu Leu Asn Pro Val Lys Ala 35 40 45 Arg Gln Glu His Glu Ala Phe Ile Lys Ile Leu Gln Asp Gln Gly Val 50 55 60 Glu Cys Val Gln Leu Thr Thr Leu Thr Ala Gln Thr Phe Gln Ser Ala 65 70 75 80 Thr Ser Glu Val Lys Glu Lys Phe Ile Asn Arg Trp Leu Asp Glu Cys 85 90 95 Leu Pro Lys Leu Ser Asp Asp Asn Arg Ile Lys Val Tyr Ala Tyr Leu 100 105 110 Lys Asp Leu Ser Ser Asp Pro Glu Val Met Ile Arg Lys Met Met Ser 115 120 125 Gly Ile Leu Ala Lys Glu Val Asn Val Gln Ser Asp Val Glu Leu Ile 130 135 140 Ala Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser 145 150 155 160 Ile Gly Lys Gly Val Thr Leu His Ser Met Phe His Pro Thr Arg Lys 165 170 175 Arg Glu Thr Ile Phe Ala Asp Phe Val Phe Ser His His Pro Glu Tyr 180 185 190 Lys Gln Thr Pro Lys Tyr Tyr Ser Arg Leu Asn Glu Tyr Ser Ile Glu 195 200 205 Gly Gly Asp Leu Phe Val Tyr Asp Asp Lys Thr Leu Val Ile Gly Val 210 215 220 Ser Glu Arg Thr Glu Lys Lys Ala Ile Gln Phe Leu Ala Glu Lys Leu 225 230 235 240 Arg Glu Asn Tyr Glu Thr Thr Phe Glu Lys Ile Tyr Ala Ile Asn Val 245 250 255 Pro Lys Met Ser Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Leu 260 265 270 Asp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Gly Val Leu Lys 275 280 285 Ile Trp Glu Ile Asp Leu Thr His Glu Gln Leu Ser Trp Arg Glu Leu 290 295 300 Asn Glu Ser Leu Glu Glu Phe Leu Ser Met Val Ile Gly Lys Lys Ala 305 310 315 320 Thr Thr Ile Pro Val Ala Gly Glu Asp Ser Thr Gln Ile Glu Ile Asp 325 330 335 Val Glu Thr Asn Phe Asp Ala Thr Asn Phe Leu Val Ile Gln Pro Gly 340 345 350 Val Val Val Gly Tyr Asp Arg Asn Tyr Lys Thr Asn Gln Ala Leu Val 355 360 365 Asn Ala Gly Ile Lys Val Leu Ser Trp Asn Gly Asp Gln Leu Ser Leu 370 375 380 Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Tyr Arg Asp Pro 385 390 395 400 Ile Lys Lys Gly <210> 16 <211> 401 <212> PRT <213> Mycoplasma lactucae <400> 16 Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Thr Leu Lys Glu Val 1 5 10 15 Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Val Ala Pro Ala Arg 20 25 30 Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn His Ala Ile 35 40 45 Ala Glu His Lys Ala Phe Ile Lys Ile Leu Glu Asp Asn Gly Ile Lys 50 55 60 Val Ile Gln Leu Asp Glu Leu Val Val Gln Thr Trp Asn Gln Val Asp 65 70 75 80 Glu Ala Thr Arg Lys Ala Phe Val Thr Lys Trp Leu Asp Glu Cys Glu 85 90 95 Pro Lys Leu Glu Ser Asn Val Arg Val Glu Val Glu Lys Tyr Ile Tyr 100 105 110 Ser Leu Ala Lys Glu Pro Lys Lys Met Val Arg Thr Met Met Ala Gly 115 120 125 Ile Ser Lys Glu Glu Leu Pro Leu Asn Val Asn Arg Pro Leu Val Val 130 135 140 Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val 145 150 155 160 Gly Thr Gly Ile Ser Leu His His Met Lys Tyr Val Thr Arg Gln Arg 165 170 175 Glu Thr Ile Phe Ala Gln Phe Val Phe Asp Asn His Lys Asp Tyr Asn 180 185 190 Thr Val Pro Arg Trp Phe Asp Asn Lys Asp Gln Gly Arg Ile Glu Gly 195 200 205 Gly Asp Val Phe Ile Tyr Asn Thr Lys Thr Leu Val Ile Gly Val Ser 210 215 220 Glu Arg Thr Asp Lys Asp Ala Ile Lys Ile Met Ala Lys Lys Ile Gln 225 230 235 240 Ala Asp Lys Asn Cys Lys Phe Glu Lys Ile Phe Ala Ile Asn Val Pro 245 250 255 Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val Asp 260 265 270 Arg Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys Val 275 280 285 Trp Glu Ile Asp Leu Lys Asp Ala Ser Leu Ala Trp Lys Glu Ile Glu 290 295 300 Gly Ser Leu Ser Gln Ile Leu Glu Lys Ile Ile Gly Glu Lys Pro Ile 305 310 315 320 Leu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp Ile 325 330 335 Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val 340 345 350 Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Gln Ala Leu Lys Ala 355 360 365 Ala Gly Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly 370 375 380 Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp Leu 385 390 395 400 Lys <210> 17 <211> 431 <212> PRT <213> Mycoplasma mobile <400> 17 Met Gly Ile Lys Lys Ile Met Lys Lys Asn Ala Ile Asn Val Tyr Ser 1 5 10 15 Glu Ile Gly Lys Leu Lys Lys Val Leu Val His Arg Pro Gly Asp Glu 20 25 30 Leu Lys Tyr Val Thr Pro Gln Arg Met Asp Glu Leu Leu Met Ser Ala 35 40 45 Ile Ile Glu Leu Glu Gln Ala Lys Glu Glu His Asp Ala Phe Thr Lys 50 55 60 Ile Leu Arg Asp Asn Gly Val Glu Val Ile Glu Leu Ala Asp Leu Thr 65 70 75 80 Ala Glu Met Tyr Asp Ser Leu Thr Pro Ser Glu Lys Asp Ala Phe Leu 85 90 95 Asn Gln Trp Val Lys Glu Ala Ser Trp Gly Lys Lys Ser Ser Ile Asp 100 105 110 Ala Leu Lys Ile Lys Lys Asn Leu Ser Lys Lys Val Phe Asp Tyr Val 115 120 125 Lys Ser Ile Lys Pro Thr Arg Lys Met Ile Asp Lys Leu Met Ala Gly 130 135 140 Val Leu Leu Ser Glu Ile Gly Glu Lys Ser Ile Ile Leu Asn Lys Asp 145 150 155 160 Lys Lys Asn Glu Met Val Ile Asp Leu Val Val Asp Pro Met Pro Asn 165 170 175 Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Ile Thr 180 185 190 Leu His Asn Met Lys Tyr Pro Thr Arg Lys Arg Glu Thr Ile Phe Ala 195 200 205 Gln Trp Ile Phe Asn Lys His Pro Glu Tyr Lys Asp Val Pro Gln Phe 210 215 220 Ile Ser Lys Arg Asp Gly Lys Glu Thr Ile Glu Gly Gly Asp Val Phe 225 230 235 240 Ile Tyr Thr Lys Asp Val Leu Ala Ile Gly Val Ser Glu Arg Thr Asn 245 250 255 Met Glu Ala Ile Leu Arg Ile Ala Thr Asn Ile Lys Lys Asp Lys Asn 260 265 270 Cys Glu Phe Lys Lys Ile Val Ala Ile Asn Val Pro Pro Met Gly Asn 275 280 285 Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Leu Phe 290 295 300 Leu Tyr Ser Gly Asn Ile Lys Ser Ala Leu Lys Val Trp Glu Ile Asp 305 310 315 320 Leu Thr Lys Pro Ile Thr Pro Lys Ser Pro Lys Leu Ser Thr Ala Lys 325 330 335 Leu Ala Asp Ile Leu Ala Lys Ile Val Gly Lys Lys Val Arg Met Ile 340 345 350 Pro Ile Gly Gly Lys Asp Gly Asn Gln Met Asp Ile Asp Ile Glu Thr 355 360 365 His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Ala Pro Gly Val Val Val 370 375 380 Gly Tyr His Arg Asn Arg Lys Thr Gln Lys Ala Leu Glu Glu Ala Gly 385 390 395 400 Val Lys Val Leu Ala Phe Gln Gly Asn Gln Leu Ser Leu Gly Met Gly 405 410 415 Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Glu Val Lys 420 425 430 <210> 18 <211> 399 <212> PRT <213> Mycoplasma elephantis <400> 18 Methionine Serine Glutamine Isoleucine Asparagine Valine Phenylalanine Serine Glutamic acid Isoleucine Glycine Glutamine Leucine Lysine Glutamic acid Valine 1 5 10 15 Leucine Valine Histidine Threonine Proline Glycine Aspartic acid Glutamic acid Isoleucine Arginine Arginine Isoleucine Serine Proline Lysine Arginine 20 25 30 Tyrosine Asparagine Glutamic acid Leucine Leucine Phenylalanine Serine Alanine Isoleucine Leucine Glutamic acid Alanine Aspartic acid Valine Alanine Isoleucine 35 40 45 Lysine Glutamic acid Histidine Lysine Serine Phenylalanine Valine Lysine Isoleucine Leucine Glutamic acid Glutamic acid Asparagine Asparagine Valine Lysine 50 55 60 Valine Isoleucine Glutamine Leucine Lysine Aspartic acid Isoleucine Leucine Leucine Glutamic acid Threonine Tryptophan Asparagine Isoleucine Cysteine Serine 65 70 75 80 Lysine Glutamic acid Alanine Lysine Asparagine Isoleucine Phenylalanine Isoleucine Asparagine Lysine Tryptophan Isoleucine Glutamic acid Glutamic acid Alanine Glutamine 85 90 95 Proline Valine Isoleucine Histidine Serine Serine Serine Leucine Lysine Glutamic acid Lysine Isoleucine Lysine Leucine Phenylalanine Leucine 100 105 110 Lysine Serine Lysine Threonine Proline Leucine Glutamic acid Isoleucine Isoleucine Aspartic acid Isoleucine Methionine Methionine Lysine Glycine Isoleucine 115 120 125 Leucine Lysine Glutamine Glutamic acid Leucine Glycine Isoleucine Glutamic acid Tyrosine Lysine Histidine Glutamic acid Leucine Isoleucine Isoleucine Aspartic acid 130 135 140 Proline Methionine Proline Asparagine Leucine Tyrosine Phenylalanine Threonine Arginine Aspartic acid Proline Phenylalanine Threonine Serine Methionine Glycine 145 150 155 160 Ser Gly Ile Thr Ile Asn Asn Met Lys Tyr Gln Thr Arg Lys Arg Glu 165 170 175 Thr Ile Phe Ser Glu Phe Ile Phe Asn Asn His Pro Lys Tyr Lys Asn 180 185 190 Thr Pro Arg Trp Phe Asp Arg Phe Asp Ser Gly Asn Ile Glu Gly Gly 195 200 205 Asp Leu Phe Val Tyr Thr Lys Glu Thr Ile Val Val Gly Val Ser Glu 210 215 220 Arg Thr Lys Lys Lys Ala Ile Leu Lys Ile Ala Lys Asn Ile Gln Glu 225 230 235 240 Asn Asn Asn Ser Phe Lys Lys Ile Val Val Ile Lys Val Pro Ile Met 245 250 255 Gln Asn Leu Met His Leu Asp Thr Trp Ile Val Met Val Asp Phe Asp 260 265 270 Lys Phe Ile Tyr Ser Pro Asn Val Thr Lys Ser Leu Lys Phe Trp Glu 275 280 285 Ile Asp Leu Thr Lys Lys Pro Lys Phe Ile Gln Leu Lys Asn Glu Thr 290 295 300 Leu Glu Asp Val Leu Tyr Arg Val Ile Gly Lys Lys Pro Ile Leu Ile 305 310 315 320 Pro Val Ala Gly Glu Asn Ala Asn Gln Ile Asp Ile Asp Val Glu Thr 325 330 335 His Phe Asp Ala Thr Asn Tyr Leu Thr Ile Arg Pro Gly Val Val Val 340 345 350 Gly Tyr Ser Arg Asn Lys Lys Thr Glu Glu Ala Leu Ile Asn Ala Gly 355 360 365 Val Lys Val Tyr Ala Phe Glu Gly Asn Gln Leu Ser Leu Gly Met Gly 370 375 380 Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp Ile Ile 385 390 395 <210> 19 <211> 399 <212> PRT <213> Mycoplasma testudineum <400> 19 Met Lys Asn Ile Asn Val Tyr Ser Glu Val Gly Lys Leu Lys Glu Val 1 5 10 15 Val Val His Thr Pro Gly Glu Glu Leu His Asn Val Ala Pro Ser Arg 20 25 30 Leu Gln Glu Leu Leu Thr Ser Ala Val Leu Glu Pro Glu Val Ala Arg 35 40 45 Lys Glu His Leu Lys Phe Ile Lys Ile Leu Asn Asp Tyr Gly Val Lys 50 55 60 Val Ile Gln Ile Val Asp Leu Ile Thr Glu Thr Tyr Glu Ala Val Asp 65 70 75 80 Ser Asn Lys Lys Glu Ala Phe Ile Asn Asn Trp Leu Asp Asn Ser Val 85 90 95 Pro Lys Leu Thr Asp Lys Asn Arg Met Ile Leu Arg Asn Tyr Leu Thr 100 105 110 Gln Phe Ser Thr Lys Ala Met Ile Arg Lys Met Ile Ser Gly Ile Arg 115 120 125 Ala Lys Glu Leu Asn Leu Lys Thr Pro Ser Ala Leu Leu Val Asp Pro 130 135 140 Met Pro Asn Leu Cys Phe Ala Arg Asp Thr Phe Ala Cys Val Gly Ser 145 150 155 160 Ala Ile Ser Leu Ser Thr Met Lys His Pro Thr Arg Arg Arg Glu Ala 165 170 175 Leu Leu Thr Glu Phe Ile Phe Gln Asn His Pro Lys Tyr Lys Asp Val 180 185 190 Ile Lys Tyr Phe Asp Ser Lys Asn Ser Lys Ala Thr Ile Glu Gly Gly 195 200 205 Asp Ile Phe Val Tyr Asn Pro Lys Thr Leu Val Val Gly Asn Ser Glu 210 215 220 Arg Thr Asn Met Gln Ala Cys Leu Leu Leu Ala Lys Lys Ile Gln Ser 225 230 235 240 Asn Pro Asn Asn Lys Phe Glu Lys Ile Val Ile Val Asn Val Pro Pro 245 250 255 Leu Pro His Leu Met His Leu Asp Thr Trp Leu Thr Met Val Asp Tyr 260 265 270 Asp Lys Phe Ile Tyr Ser Pro Asn Ile Leu His Thr Leu Lys Phe Trp 275 280 285 Val Ile Asp Leu Lys Lys Arg Lys Leu Glu Ala Val Glu Lys His Asn 290 295 300 Thr Leu Lys Ala Met Leu Arg Met Ile Ile Lys Lys Glu Pro Ile Leu 305 310 315 320 Ile Pro Val Gly Asp Val Gly Ala Asp Gln Leu Asp Ile Asp Leu Glu 325 330 335 Thr His Phe Asp Ala Thr Asn Tyr Leu Ala Leu Ala Pro Gly Val Val 340 345 350 Val Gly Tyr Asp Arg Asn Ile Lys Thr Gln Arg Ala Leu Glu Lys Ala 355 360 365 Gly Val Lys Val Leu Ser Phe Ser Gly Asn Gln Leu Ser Leu Ala Met 370 375 380 Gly Ser Ala Arg Cys Leu Ser Met Pro Leu Ile Arg Glu Glu Asn 385 390 395 <210> 20 <211> 410 <212> PRT <213> Mycoplasma canadense <400> 20 Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ser Glu 50 55 60 Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu 85 90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Ala 100 105 110 Ile Val Arg Lys Tyr Leu Lys Gly Ile Gln Pro Thr Arg Lys Leu Ile 115 120 125 Glu Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140 Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser 180 185 190 Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu 195 200 205 Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ser 290 295 300 Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Glu 305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu Gly 325 330 335 Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380 His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys Trp 405 410 <210> 21 <211> 409 <212> PRT <213> Mycoplasma anseris <400> 21 Met Ser Val Phe Asp Lys Arg Phe Lys Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Ala Glu His Lys Lys Phe Val Ala Thr 50 55 60 Leu Lys Glu Gln Gly Ile Asn Thr Val Glu Leu Thr Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Arg Asp Asn Leu Leu Glu 85 90 95 Glu Phe Leu Asp Asp Ser Ala Pro Val Leu Ser Glu Glu His Lys Glu 100 105 110 Ile Val Arg Thr Tyr Leu Lys Gly Ile Lys Gly Thr Arg Lys Leu Ile 115 120 125 Glu Thr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140 Glu Gln Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Ser 180 185 190 Asn His Pro Gln Leu Val Asn Thr Pro Trp Tyr Tyr Asn Pro Ala Glu 195 200 205 Gly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Lys Ala Asn Glu Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Thr Asn Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp 290 295 300 Glu Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Asn Glu Leu Leu Lys 305 310 315 320 Ser Ile Ile Gly Glu Glu Pro Ile Leu Ile Pro Ile Ala Gly Asp Gly 325 330 335 Ala Thr Gln Ile Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile Ala Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380 Lys Gly His Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Tyr Arg Lys Asp Val Lys 405 <210> 22 <211> 401 <212> PRT <213> Mycoplasma gallisepticum <400> 22 Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val 1 5 10 15 Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg 20 25 30 Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala Ile 35 40 45 Lys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile Lys 50 55 60 Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala 65 70 75 80 Thr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu Ala 85 90 95 Thr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr Ile 100 105 110 Thr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met Ala 115 120 125 Gly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu Ile 130 135 140 Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser 145 150 155 160 Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys 165 170 175 Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr 180 185 190 Lys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile Glu 195 200 205 Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val 210 215 220 Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His Ile 225 230 235 240 Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val 245 250 255 Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val 260 265 270 Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys 275 280 285 Ile Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr Ser 290 295 300 Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile 305 310 315 320 Leu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp Ile 325 330 335 Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val 340 345 350 Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys Ala 355 360 365 Ala Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu Gly 370 375 380 Met Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val 385 390 395 400 Lys <210> 23 <211> 404 <212> PRT <213> Mycoplasma mellis <400> 23 Met Ser Ile Lys Glu Asn Gly Ile His Val Tyr Ser Glu Ile Gly Lys 1 5 10 15 Leu Arg Asp Val Leu Val His Arg Pro Gly Arg Glu Leu Asn Phe Leu 20 25 30 Asp Pro Ser Arg Leu Asp Glu Leu Leu Phe Ala Ala Thr Leu Glu Pro 35 40 45 Glu Thr Ala Arg Leu Glu His Asp Asn Phe Thr Thr Val Leu Lys Asn 50 55 60 Gln Gly Val Asn Val Ile Glu Leu Ala Asp Leu Val Ser Gln Thr Tyr 65 70 75 80 Ser Lys Val Asp Ser Lys Val Lys Lys Glu Phe Ile Asp Gln Tyr Leu 85 90 95 Asn Glu Ala Thr Pro Lys Leu Thr Ser Glu Leu Ser Lys Lys Val Tyr 100 105 110 Asp Phe Leu Thr Lys Gln Lys Ser Asn Arg Glu Met Val Asp Phe Met 115 120 125 Met Gly Gly Ile Leu Ser Ser Asp Leu Asn Ile Lys Gly Gln Pro Tyr 130 135 140 Leu Ile Val Glu Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe 145 150 155 160 Ala Ser Val Gly Asn Gly Ala Thr Ile His Trp Met Lys His Asn Val 165 170 175 Arg Arg Arg Glu Val Leu Phe Ala Asn Phe Ile Phe Lys Tyr Asn Glu 180 185 190 Arg Phe Gln Asn Thr Pro Lys Tyr Ile Thr Pro Thr Lys Gly Leu Asp 195 200 205 Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Lys Lys Thr Leu Val Val 210 215 220 Gly Val Ser Glu Arg Thr Lys Met Glu Thr Ile Lys Glu Leu Ala Lys 225 230 235 240 Asn Ile Ser Lys Asn Lys Glu Cys Thr Phe Thr Lys Ile Tyr Ala Ile 245 250 255 Asn Val Pro Lys Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr 260 265 270 Met Leu Asp Tyr Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val 275 280 285 Leu Lys Val Trp Glu Ile Asn Ile Ser Asn Asn Lys Val Ser Ala Pro 290 295 300 Lys Glu Leu Asn Val Asn Leu Glu Lys Ala Leu Ser Met Ile Ile Gly 305 310 315 320 Lys Lys Pro Ile Leu Ile Pro Val Ala Gly Ala Asn Ala Ser Gln Ile 325 330 335 Asp Ile Asn Ile Glu Thr Asn Phe Asp Ala Thr Asn Tyr Leu Val Ile 340 345 350 Glu Pro Gly Val Val Val Gly Tyr Ser Arg Asn Lys Lys Thr Glu Glu 355 360 365 Ala Leu Val Lys Ala Gly Ile Lys Val Leu Pro Phe His Gly Asn Gln 370 375 380 Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Tyr 385 390 395 400 Arg Glu Asp Val <210> 24 <211> 410 <212> PRT <213> Mycoplasma penetrans <400> 24 Met Ser Ser Ile Asp Lys Asn Ser Leu Gly Asn Gly Ile Asn Val Tyr 1 5 10 15 Ser Glu Ile Gly Glu Leu Lys Glu Val Leu Val His Thr Pro Gly Asp 20 25 30 Glu Ile Arg Tyr Thr Ala Pro Ser Arg Leu Glu Glu Leu Leu Phe Ser 35 40 45 Ala Val Leu Lys Ala Asp Thr Ala Ile Glu Glu His Lys Gly Phe Val 50 55 60 Lys Ile Leu Gln Asn Asn Gly Ile Lys Val Ile Gln Leu Cys Asp Leu 65 70 75 80 Val Ala Glu Thr Tyr Glu Leu Cys Ser Lys Glu Val Arg Asn Ser Phe 85 90 95 Ile Glu Gln Tyr Leu Asp Glu Ala Leu Pro Val Leu Lys Lys Glu Ile 100 105 110 Arg Pro Val Val Lys Asp Tyr Leu Leu Ser Phe Pro Thr Val Gln Met 115 120 125 Val Arg Lys Met Met Ser Gly Ile Leu Ala Asn Glu Leu Asn Ile Lys 130 135 140 Gln Asp Asn Pro Leu Ile Ile Asp Gly Met Pro Asn Leu Tyr Phe Thr 145 150 155 160 Arg Asp Pro Phe Ala Ser Met Gly Asn Gly Val Ser Ile Asn Cys Met 165 170 175 Lys Tyr Pro Thr Arg Lys Arg Glu Val Ile Phe Ser Arg Phe Val Phe 180 185 190 Thr Asn Asn Pro Lys Tyr Lys Asn Thr Pro Arg Tyr Phe Asp Ile Val 195 200 205 Gly Asn Asn Gly Thr Ile Glu Gly Gly Asp Ile Phe Ile Tyr Asn Ser 210 215 220 Lys Thr Leu Val Ile Gly Asn Ser Glu Arg Thr Asn Phe Ala Ala Ile 225 230 235 240 Glu Ser Val Ala Lys Asn Ile Gln Ala Asn Lys Asp Cys Thr Phe Glu 245 250 255 Arg Ile Val Val Ile Asn Val Pro Pro Met Pro Asn Leu Met His Leu 260 265 270 Asp Thr Trp Leu Thr Met Leu Asp Tyr Asp Lys Phe Leu Tyr Ser Pro 275 280 285 Asn Met Met Asn Val Leu Lys Ile Trp Glu Ile Asp Leu Asn Val Lys 290 295 300 Pro Val Lys Phe Val Glu Lys Lys Gly Thr Leu Glu Glu Val Leu Tyr 305 310 315 320 Ser Ile Ile Asp Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Lys Gly 325 330 335 Ala Asn Gln Leu Asp Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Thr Ile Ala Pro Gly Val Val Val Gly Tyr Glu Arg Asn Glu 355 360 365 Lys Thr Gln Lys Ala Leu Val Glu Ala Gly Ile Lys Val Leu Ser Phe 370 375 380 Asn Gly Ser Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ile Arg Glu Asn Leu Lys Lys 405 410 <210> 25 <211> 404 <212> PRT <213> Mycoplasma fermentans <400> 25 Met Lys Lys Ile Asn Val Tyr Ser Glu Tyr Gly Lys Leu Lys Glu Val 1 5 10 15 Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg 20 25 30 Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Ser Ala Ile 35 40 45 Ala Glu His Lys Arg Phe Val Gln Leu Leu Lys Asp Asn Gly Ile Lys 50 55 60 Val Ile Gln Leu Asp Glu Leu Phe Ala Lys Thr Phe Asp Leu Val Ser 65 70 75 80 Glu Ser Val Lys Gln Ser Phe Ile Glu Arg Trp Leu Asp Glu Cys Glu 85 90 95 Pro Lys Leu Asp Ala Thr Leu Arg Ala Lys Val Lys Glu Tyr Ile Leu 100 105 110 Glu Leu Lys Ala Lys Ser Ser Lys Lys Met Val Arg Val Met Met Ala 115 120 125 Gly Ile Asp Lys Lys Glu Leu Gly Ile Glu Leu Asp Arg Asp Leu Val 130 135 140 Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser 145 150 155 160 Val Gly Asn Gly Ile Ser Leu His His Met Lys Tyr Val Thr Arg Gln 165 170 175 Arg Glu Thr Ile Phe Ser Glu Phe Ile Phe Asp Asn Asn Leu Asp Tyr 180 185 190 Asn Thr Val Pro Arg Trp Phe Asp Arg Lys Asp Glu Gly Arg Ile Glu 195 200 205 Gly Gly Asp Val Phe Ile Tyr Ser Ala Asp Thr Leu Val Val Gly Val 210 215 220 Ser Glu Arg Thr Asn Lys Glu Ala Ile Asn Val Met Ala Arg Lys Ile 225 230 235 240 Ala Ala Asp Lys Glu Val Lys Phe Lys Arg Ile Tyr Ala Ile Asn Val 245 250 255 Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Leu 260 265 270 Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys 275 280 285 Val Trp Arg Ile Asp Leu Asn Asp Pro Asp Phe Val Trp His Glu Ile 290 295 300 Glu Gly Ser Leu Glu Glu Ile Leu Glu Gln Ile Ile Gly Met Lys Pro 305 310 315 320 Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Ser Gln Leu Asp Ile Asp 325 330 335 Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Ser 340 345 350 Val Val Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys 355 360 365 Ala Ala Lys Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu 370 375 380 Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp 385 390 395 400 Ile Lys Lys Lys <210> 26 <211> 404 <212> PRT <213> Mycoplasma pneumoniae <400> 26 Met Lys Tyr Asn Ile Asn Val His Ser Glu Ile Gly Gln Leu Gln Thr 1 5 10 15 Val Leu Val His Thr Pro Gly Asn Glu Ile Arg Arg Ile Ser Pro Arg 20 25 30 Arg Leu Asp Asp Leu Leu Phe Ser Ala Val Ile Glu Pro Asp Thr Ala 35 40 45 Ile Gln Glu His Gln Thr Phe Cys Gln Leu Leu Gln Glu Gln Asn Ile 50 55 60 Glu Val Val Gln Leu Thr Asp Leu Thr Ala Thr Thr Phe Asp Lys Ala 65 70 75 80 Asn Ala Thr Ala Gln Asn Gln Phe Ile Glu Thr Trp Leu Asp Gln Ala 85 90 95 Glu Pro Lys Leu Thr Pro Glu His Arg Lys Val Ala Lys Gln Tyr Leu 100 105 110 Leu Glu Gln Lys Ala Lys Ser Thr Leu Ser Met Val Arg Ser Met Met 115 120 125 Gly Gly Ile Asp Lys Arg Lys Val Ala Ala Ala Asn Thr Ile Asn Gly 130 135 140 Asp Phe Leu Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro 145 150 155 160 Phe Ala Ser Ile Gly His Gly Ile Ser Ile Asn Arg Met Lys Tyr Leu 165 170 175 Thr Arg Arg Arg Glu Thr Leu Phe Ala Ser Phe Ile Phe Ala Asn His 180 185 190 Pro Ile Ile Ala Ala Arg Lys Phe Tyr Phe Lys Pro Ile Asp Met Gly 195 200 205 Thr Ile Glu Gly Gly Asp Ile Phe Val Tyr Asp Gln Gln Thr Val Val 210 215 220 Met Gly Leu Ser Glu Arg Thr Thr Glu Ala Ala Ile Asn Val Leu Ala 225 230 235 240 Lys Lys Ile Gln Gln Asp Ser Ser Thr Ser Phe Lys Arg Ile Phe Val 245 250 255 Ile Asn Val Pro Gln Leu Pro Asn Leu Met His Leu Asp Thr Trp Leu 260 265 270 Thr Met Leu Asp Arg Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ala 275 280 285 Val Leu Lys Ala Trp Arg Ile Asp Phe Thr Asp Pro Ala Leu Lys Trp 290 295 300 Asn Glu Ile Ala Gly Asp Leu Ser Thr Ile Leu His Thr Ile Ile Gly 305 310 315 320 Gln Lys Pro Met Leu Ile Pro Ile Ala Gly Ala Asp Ala Asn Gln Thr 325 330 335 Glu Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile 340 345 350 Ala Pro Ser Val Val Val Gly Tyr Ala Arg Asn Lys Leu Thr His Gln 355 360 365 Thr Leu Glu Ala Ala Gly Val Lys Val Ile Ala Phe Lys Gly Asn Gln 370 375 380 Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val 385 390 395 400 Arg Lys Pro Leu <210> 27 <211> 414 <212> PRT <213> Mycoplasma sp. <400> 27 Met Glu Lys Ile His Val Thr Ser Glu Ile Gly Pro Leu Lys Lys Val 1 5 10 15 Leu Leu His Arg Pro Gly Asn Glu Leu Leu Asn Leu Thr Pro Asp Thr 20 25 30 Leu Ser Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu Pro Asp Ala Ile 35 40 45 Lys Glu His Asp Glu Phe Ala Asp Ala Leu Arg Ala Asn Gly Val Glu 50 55 60 Val Val Tyr Leu Glu Asn Leu Met Ala Asp Val Leu Asp Leu Ser Asp 65 70 75 80 Glu Ile Arg Asp Lys Phe Ile Lys Gln Phe Ile Tyr Glu Ala Gly Ile 85 90 95 Arg Thr Pro Lys Tyr Lys Tyr Leu Val Phe Asp Tyr Leu Asp Gln Ile 100 105 110 Thr Asn Ser Lys Lys Leu Val Leu Lys Thr Met Glu Gly Ile Gln Ile 115 120 125 Ser Asp Ile Pro Arg Arg Lys Arg Glu Ile Glu Lys Ser Leu Val Asp 130 135 140 Leu Ile Glu Thr Glu Asp Glu Phe Ile Ala Asp Pro Met Pro Asn Leu 145 150 155 160 Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Glu Gly Ile Ser Leu 165 170 175 Asn Lys Met Tyr Ser Val Thr Arg Asn Arg Glu Thr Ile Tyr Ala Glu 180 185 190 Tyr Ile Phe Lys Tyr His Pro Asp Tyr Lys Asp Gln Ala Arg Leu Tyr 195 200 205 Tyr Asp Arg Tyr Asn Pro Tyr His Ile Glu Gly Gly Asp Val Leu Asn 210 215 220 Ile Asn Asp His Val Leu Ala Ile Gly Ile Ser Gln Arg Thr Thr Ala 225 230 235 240 Glu Ala Ile Asp Gln Ile Ala Lys Asn Leu Phe Lys Asp Pro Glu Cys 245 250 255 Lys Ile Asp Thr Ile Leu Ala Phe Asn Ile Pro Glu Ser Arg Ala Phe 260 265 270 Met His Leu Asp Thr Val Phe Thr Gln Val Asp Tyr Asp Lys Phe Thr 275 280 285 Tyr His Pro Gly Ile Met Gly Thr Leu Gln Val Phe Glu Ile Thr Glu 290 295 300 Gly Asp Asp Pro Asn Ser Asp Glu Asp Leu Thr Val Thr Glu Ile Asn 305 310 315 320 Ala Pro Leu Glu Glu Ile Leu Thr Lys Tyr Val Gly Arg Lys Val Thr 325 330 335 Leu Ile Pro Cys Ala Gly Gly Asp Lys Val Ser Ala Glu Arg Glu Gln 340 345 350 Trp Asn Asp Gly Ser Asn Thr Leu Cys Ile Ala Pro Gly Val Val Val 355 360 365 Val Tyr Asp Arg Asn Asn Leu Thr Asn Ala Val Leu Arg Ser Tyr Gly 370 375 380 Leu Lys Val Ile Glu Ile His Gly Ala Glu Leu Ser Arg Gly Arg Gly 385 390 395 400 Gly Pro Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Ile 405 410 <210> 28 <211> 408 <212> PRT <213> Mycoplasma sp. <400> 28 Met His Val Thr Ser Glu Ile Lys Lys Leu Lys Lys Val Leu Val His 1 5 10 15 Arg Pro Gly Lys Glu Leu Leu Asn Leu Thr Pro Asp Thr Leu Gly Arg 20 25 30 Leu Leu Phe Asp Asp Ile Pro Tyr Leu Lys Asp Ala Ile Leu Glu His 35 40 45 Asp Glu Phe Cys Gln Ile Leu Arg Asp Asn Asp Val Glu Val Val Tyr 50 55 60 Leu Glu Asp Leu Met Ala Glu Thr Leu Asp Glu Asn Pro Gln Val Lys 65 70 75 80 Pro Ser Phe Ile Arg Gln Phe Ile Tyr Glu Ala Gly Val Arg Thr Pro 85 90 95 Lys Tyr Lys Asp Leu Leu Phe Asp Tyr Leu Met Ser Tyr Thr Asn Asn 100 105 110 Lys Glu Leu Val Leu Lys Thr Met Glu Gly Ile Lys Val Ser Glu Val 115 120 125 His Arg Asn Lys Gln Asp Ser Glu Tyr Ser Leu Val Asp Gln Ile Ser 130 135 140 Glu Glu Thr Lys Phe Leu Ala Glu Pro Met Pro Asn Leu Tyr Phe Thr 145 150 155 160 Arg Asp Pro Phe Ala Ser Val Gly Asp Gly Ile Ile Leu Asn Lys Met 165 170 175 His Ser Val Thr Arg Ser Arg Glu Thr Ile Tyr Ala Tyr Tyr Ile Phe 180 185 190 Asn Tyr His Pro Asp Tyr Met Asp Lys Val Pro Lys Tyr Tyr Asp Arg 195 200 205 Glu Asn Pro Phe Ser Ile Glu Gly Gly Asp Val Leu Asn Leu Asn Glu 210 215 220 His Thr Leu Ala Ile Gly Ile Ser Gln Arg Thr Ser Ala Glu Ala Ile 225 230 235 240 Asp Leu Val Ala Lys Asn Met Phe Asn Asp Glu Lys Cys Asn Ile Asp 245 250 255 Thr Ile Leu Ala Phe Lys Ile Pro Glu Cys Arg Ala Phe Met His Leu 260 265 270 Asp Thr Val Phe Thr Gln Ile Asp Ile Asp Lys Phe Thr Tyr His Pro 275 280 285 Gly Ile Met Asp Thr Leu Glu Val Phe Glu Ile Thr Lys Asn Glu Asp 290 295 300 Asp Leu Asp Glu Val Arg Val Ile Lys Lys Glu Gly Ser Leu Glu Asn 305 310 315 320 Ile Leu Glu Glu Tyr Leu Gly Ile Asp Ile Thr Leu Ile Pro Cys Ala 325 330 335 Gly Gly Asp Lys Ile Ala Ser Glu Arg Glu Gln Trp Asn Asp Gly Thr 340 345 350 Asn Thr Leu Cys Ile Ala Pro Gly Val Val Val Val Tyr Asn Arg Asn 355 360 365 Asn Ile Thr Asn Glu Val Leu Arg Glu Lys Gly Ile Lys Val Ile Glu 370 375 380 Met Asn Ser Ala Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met 385 390 395 400 Ser Met Pro Leu Glu Arg Glu Asp 405

Claims

1. A therapeutic composition comprising a purified arginine deiminase as shown in SEQ ID NO:22 and a pharmaceutically acceptable carrier, wherein the purified arginine deiminase has reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody.

2. The therapeutic composition according to claim 1, wherein the arginine deiminase is covalently bonded to a PEG molecule via a linker.

3. The therapeutic composition according to claim 2, wherein the arginine deiminase is covalently bonded to more than one PEG molecule.

4. The therapeutic composition according to claim 2, wherein the arginine deiminase is covalently bonded to 1 to 10 PEG molecules.

5. The therapeutic composition according to claim 2, wherein the arginine deiminase is covalently bonded to 2 to 8 PEG molecules.

6. The therapeutic composition according to claim 2, wherein the PEG molecule is a linear or branched PEG molecule.

7. The therapeutic composition according to claim 2, wherein the total weight average molecular weight of the PEG is from 1,000 to 40,000.

8. The therapeutic composition according to claim 2, wherein the total weight average molecular weight of the PEG is from 10,000 to 30,000.

9. The therapeutic composition according to claim 2, 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 combination thereof.

10. The therapeutic composition according to claim 9, wherein the source of the succinyl group is succinimidyl succinate.

11. The therapeutic composition according to claim 1, further comprising a chemotherapeutic agent.

12. The therapeutic composition according to claim 11, wherein the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, cyclophosphamide, gemcitabine, cisplatin, sorafenib, sunitinib and everolimus.

13. A purified arginine deiminase as shown in SEQ ID NO:22, wherein the purified arginine deiminase has reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody.

14. A polynucleotide encoding the purified arginine deiminase according to claim 13.

15. A vector comprising the polynucleotide according to claim 14.

16. An isolated host cell comprising the vector according to claim 15.

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