Combination cancer immunotherapy using arginine depleting agents

Abstract

Arginine depletion agents such as ADI-PEG are provided for use in combination with cancer immunotherapy, such as immune checkpoint regulators and T cell adoptive immunotherapy to treat various cancers. Related methods, compositions, patient care kits, and cell cultures are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Patent Application No. 62 / 515,807, filed June 6, 2017, and U.S. Patent Application No. 62 / 358,479, filed July 5, 2016, pursuant to 35 U.S. SC §119(e), each of which is incorporated herein by reference in its entirety.

[0003] Declaration of sequence list

[0004] The sequence list related to this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence list is named POLA_006_02WO_ST25.txt. This text file is 196KB in size, was created on July 5, 2017, and submitted electronically via EFS-Web. Background Technology Technical Field

[0005] Embodiments of this disclosure particularly relate to the use of arginine-depleting agents such as ADI-PEG in combination with cancer immunotherapies, such as immune checkpoint modulators and T-cell adoptive immunotherapy, to treat various cancers.

[0006] Related technical specifications

[0007] Arginine is used in many metabolic pathways, including those involved in regulating immune responses. It is a substrate of arginase and iNOS, enzymes that play important roles in the regulation of immune cells (see, for example, Peranzoni et al., Immunobiology, 2007; 212:795-812; Rodriguez and Ochoa, Immunol Rev, 2008; 222:180-91; Rath et al., Front Immunol, October 27, 2014; 5:532; Delage et al., International Journal of Cancer, June 15, 2010; 126(12):2762-72; Bronte and Zanovello et al., Nature Review: Immunology, August 2005; 5(8):641-54; Raber et al., Immunological Research, Immunol Rev, 2005; 5(8):641-54). Invest), 2012; 41(6-7):614-34; and Albina et al., Journal of Experimental Medicine, March 1, 1989; 169(3):1021-9). T cells require arginine for proliferation, TCR expression and memory development (ibid.).

[0008] Although arginine starvation has been shown to inhibit the proliferation and cell cycle progression of activated T cells in vitro, this effect can be reversed by adding citrulline (see, for example, Bansal et al., JPEN: Journal of Parenteral and Enteral Nutrition, 2004; 28, 423-430; Fletcher et al., Cancer Research, 75, 275–83 (2015); and Rodriguez et al., Blood, 2007; 109: 1568–73). Similarly, delayed loss of CD3 and T cell inhibition can be induced in vitro by macrophages expressing arginase I instead of those producing NOS2 (converting arginine to citrulline and NO) (see, for example, Rodriguez et al., J Immunol, 1 Aug 2003; 171(3): 1232-9).

[0009] Arginine deficiency induces increased transcription and stability of RNA encoding a multi-amino acid transport system, including CAT-1, which increases the transport of cationic amino acids into the cell (see, for example, Gazzola et al., *Biochim Biophys Acta*, 1972; 266:407-421; Hyatt et al., *J Biol Chem*, 1997; 272:19951-19957; Aulak et al., *J Biol Chem*, 1996; 271:29799-29806). Under low arginine conditions, T cells increase citrulline uptake and upregulate ASS1 expression, which converts citrulline to arginine (see, for example, Bansal et al., 2004; Fletcher et al., 2015; and Tarasenko et al., *J Leukoc Biol*, Feb. 2015; 97(2):273-8). Therefore, although the conversion of arginine to ornithine by arginase can lead to immunosuppression in the tumor microenvironment (as above), the conversion of arginine to citrulline may have significant benefits.

[0010] PEGylated arginine deiminoase (ADI-PEG 20) depletes the external supply of arginine by converting arginine to citrulline and ammonia. Clinically, ADI-PEG 20 is being investigated for tumors lacking argininosuccinate synthase-1 (ASS1), a key enzyme involved in the conversion of citrulline to arginine. ADI-PEG 20 has been well-tolerated and has shown promise in clinical studies (see, for example, Qiu et al., Cancer Lett, August 1, 2015; 364(1):1-7; Phillips et al., Cancer Research and Treatment, December 2013; 45(4):251-62; Feun et al., Curr Pharm Des, 2008; 14(11):1049-57; Feun and Savarage, Expert Opin Investig Drugs, July 2006; 15(7):815-22; Feun et al., Curr Opin Clin Nutr Metab Care, January 2015; 18(1):78-82). The antitumor properties of ADI-PEG 20 have been extensively studied; however, its effects on immune cells such as T cells remain largely unknown. This provides an opportunity to explore new immunotherapies for arginine depletion. Summary of the Invention

[0011] Embodiments of this disclosure relate to the discovery that arginine depletion agents (which convert arginine to citrulline) possess unexpected immunomodulatory properties. For example, the exemplary arginine depletion agent ADI-PEG 20 has been shown to reduce the depletion of immunosuppressive regulatory T cells (Treg cells) from alleviated effector T cells (Teff cells) in vivo and to induce or increase T cell infiltration in non-immunogenic tumors in vivo. As a specific example, ADI treatment during T cell stimulation enhances effector T cell activation (CD69 expression), prevents the upregulation of immunosuppressive receptors PD-1 and CTLA-4 on T cells, thereby reducing T cell depletion, and reduces the upregulation of PD-L1 on T cells after stimulation.

[0012] These undesirable properties suggest that arginine depletion agents, such as ADI-PEG 20, can be combined with other cancer immunotherapies to improve those therapies. Specific examples of cancer immunotherapies benefiting from combination with arginine depletion agents include PD-1 inhibitors, PD-L1 inhibitors, and T-cell adoptive immunotherapy.

[0013] Therefore, some embodiments include a method of treating cancer in a subject in need, the method comprising administering to the subject:

[0014] (a) an arginine-depleting agent that converts arginine to citrulline; and

[0015] (b) Cancer immunotherapy agents.

[0016] In some embodiments, the arginine depleting agent of (a) is an ADI polypeptide. In some embodiments, the ADI polypeptide comprises, consists of, or is substantially composed of sequences selected from Table A1, including variants and fragments that convert arginine to citrulline. In some embodiments, the variants comprise sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% equivalent to sequences from Table A1 and convert arginine to citrulline.

[0017] In some embodiments, the ADI polypeptide is covalently bonded to at least one PEG molecule via an optional linker group. In some embodiments, the ADI polypeptide is covalently bonded to more than one PEG molecule. In specific embodiments, the ADI polypeptide is covalently bonded to about 1 to about 10 PEG molecules. In some embodiments, the ADI polypeptide is covalently bonded to about 2 to about 8 PEG molecules. In some embodiments, the PEG molecules are linear or branched PEG molecules.

[0018] In some embodiments, the PEG has a total average molecular weight of about 1,000 to about 40,000 Daltons. In some embodiments, the PEG has a total average molecular weight of about 10,000 to about 30,000 Daltons. In a particular embodiment, the PEG has a total average molecular weight of about 20,000 Daltons.

[0019] In some embodiments, the linking group 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 group, a tyrosine group, a cysteine ​​group, a histidine group, a methylene group, or any combination thereof. In some embodiments, the succinyl group is derived from succinimide succinate.

[0020] In some embodiments, the arginine depleting agent is polyethylene glycol-modified arginine deiminase (ADI-PEG). In a particular exemplary embodiment, the ADI-PEG is ADI-PEG 20.

[0021] In some embodiments, the cancer immunotherapy agent of (b) is selected from one or more of immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies. In some embodiments, the immune checkpoint modulator is a peptide, optionally an antibody or its antigen-binding fragment or ligand or small molecule.

[0022] In some embodiments, the immune checkpoint modulator comprises:

[0023] (i) Antagonists of inhibitory immune checkpoint molecules; or

[0024] (ii) Agonists that stimulate immune checkpoint molecules.

[0025] In some embodiments, the immune checkpoint modulator specifically binds to the immune checkpoint molecule.

[0026] In a particular embodiment, the inhibitory immune checkpoint molecule is selected from one or more of the following: programmed death-ligand 1 (PD-L1), programmed death-1 (PD-1), programmed death-ligand 2 (PD-L2), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), T-cell activation V-domain Ig repressor (VISTA), B and T lymphocyte attenuator (BTLA), CD160, herpesvirus entry mediator (HVEM), and T-cell immune receptor having Ig and ITIM domains (TIGIT).

[0027] In some embodiments, the antagonist is a PD-L1 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to it, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and wherein the cancer is optionally selected from one or more of colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma. In some embodiments, the antagonist is a PD-1 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to it, nivolumab, pembrolizumab, PDR001, and pidilizumab. In some embodiments, the PD-1 antagonist is nivolumab, and the cancer is optionally selected from one or more of Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer. In some embodiments, the PD-1 antagonist is pembrolizumab, and the cancer is optionally selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma.

[0028] In some embodiments, the antagonist is a PD-L2 antagonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules that specifically bind to it.

[0029] In some embodiments, the antagonist is a CTLA-4 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to it, ipilimumab, and tremelimumab. In specific embodiments, the cancer is selected from one or more of melanoma, prostate cancer, lung cancer, and bladder cancer.

[0030] In some embodiments, the antagonist is an IDO antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to it, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, and wherein the cancer is optionally selected from metastatic breast cancer and brain cancer, optionally one or more of glioblastoma multiforme, glioma, gliosarcoma, or malignant brain tumor.

[0031] In some embodiments, the antagonist is a TDO antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to it, 680C91, and LM10.

[0032] In some embodiments, the antagonist is a TIM-3 antagonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules that specifically bind to it.

[0033] In some embodiments, the antagonist is a LAG-3 antagonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules that specifically bind to it and BMS-986016.

[0034] In some embodiments, the antagonist is a VISTA antagonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules that specifically bind to it.

[0035] In some embodiments, the antagonist is a BTLA antagonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules that specifically bind to it.

[0036] In some embodiments, the antagonist is a CD160 antagonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules that specifically bind to it.

[0037] In some embodiments, the antagonist is an HVEM antagonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules that specifically bind to it.

[0038] In some embodiments, the antagonist is a TIGIT antagonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules that specifically bind to it.

[0039] In some embodiments, the stimulating immune checkpoint molecule is selected from one or more of OX40, CD40, glucocorticoid-induced TNFR family-associated gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0040] In some embodiments, the agonist is an OX40 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to it, OX86, Fc-OX40L, and GSK3174998.

[0041] In some embodiments, the agonist is a CD40 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, CP-870, 893, dacetuzumab, Chi Lob7 / 4, ADC-1013, and rhCD40L, and the cancer is optionally selected from one or more of melanoma, pancreatic cancer, mesothelioma, and hematologic malignancies, and optionally lymphomas such as non-Hodgkin's lymphoma.

[0042] In some embodiments, the agonist is a GITR agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to it, INCAGN01876, DTA-1, and MEDI1873.

[0043] In some embodiments, the agonist is a CD137 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to it, utomilumab, and 4-1BB ligand.

[0044] In a particular embodiment, the agonist is a CD27 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, varlilumab, and CDX-1127 (1F5).

[0045] In some embodiments, the agonist is a CD28 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to it and TAB08.

[0046] In some embodiments, the agonist is a CD226 agonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to it.

[0047] In some embodiments, the agonist is an HVEM agonist, optionally selected from one or more of antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to it.

[0048] In some embodiments, (b) the cancer immunotherapy agent is one or more cancer vaccines selected from Oncophage, optionally Gardasil or Cervarix human papillomavirus HPV vaccines, optionally Engerix-B, Recombivax HB or Twinrix hepatitis B vaccines, and sipuleucel-T (Provenge), or includes one or more cancer antigens selected from: human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial cells Long-term factors VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tendinin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA) -IX), Carcinoembryonic antigen (CEA), Guanylate cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activating protein α (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B) Or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), liver regeneration phosphatase 3 (PRL-3), prostate acid phosphatase, Lewis-Y antigen, GD2 (disialiac ganglioside expressed on neuroectodermal tumors), phosphatidylinositol polysaccharide-3 (GPC3), and mesothelin.

[0049] In some embodiments, (b) the cancer immunotherapy agent is an oncolytic virus selected from one or more of the following: talimogene laherparepvec (T-VEC), Coxsackievirus A21 (CAVATAK). TM ), Oncorine(H101), pelareorep Seneca Valley virus (NTX-010), Seneca virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0050] In some embodiments, (b) the cancer immunotherapy agent is a cytokine selected from one or more of the following: interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21 and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0051] In some embodiments, the cancer immunotherapy agent of (b) is a cell-based immunotherapy comprising cancer antigen-specific T cells, optionally ex vivo derived T cells. In some embodiments, the cancer immunotherapy agent of cancer antigen-specific T cells is selected from one or more of chimeric antigen receptor (CAR) modified T cells and T cell receptor (TCR) modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0052] In some embodiments, (b) the cancer immunotherapy agent is selected from one or more of IDO antagonists, CD20 antagonists, B-Raf antagonists, IL-2, GM-CSF and oncolytic viruses.

[0053] In some embodiments, the IDO antagonist is indomod, and / or the CD20 agonist is rituximab, and / or the B-Raf antagonist is vemurafenib, and / or the oncolytic virus is talimogene laherparepvec (T-VEC) or Coxsackievirus A21 (CAVATAK). TM ).

[0054] In some embodiments, the arginine-depleting agent of (a) and the cancer immunotherapy agent of (b) are administered individually. In some embodiments, the arginine-depleting agent of (a) and the cancer immunotherapy agent of (b) are administered together as part of the same composition.

[0055] In some embodiments, the cancer is selected from one or more of the following: hepatocellular carcinoma, melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myeloid leukemia, lymphoma, liver cancer, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, and stomach cancer.

[0056] It also includes methods for adoptive T-cell immunotherapy for treating cancer in subjects in need, comprising:

[0057] (a) Incubate in vitro derived T cells (i) with arginine depletion agent or (ii) in arginine-free medium supplemented with citrulline.

[0058] (b) Administering ex vivo derived T cells to the subject.

[0059] In some embodiments, the ex vivo derived T cells of (a) or (b) include cancer antigen-specific T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR) modified T cells and T cell receptor (TCR) modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0060] It also includes methods for increasing T cell activation and / or regulating T cell (Treg) downregulation in vivo or in vitro, comprising (a) incubating T cells with an arginine depletion agent, (b) incubating T cells in an arginine-free medium supplemented with citrulline, or both (a) and (b).

[0061] In some embodiments, the arginine depleting agent is an ADI polypeptide. In some embodiments, the ADI polypeptide comprises, consists of, or is substantially composed of sequences selected from Table A1, including variants and fragments thereof having ADI activity. In some embodiments, the variants comprise sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% equivalent to sequences from Table A1.

[0062] In some embodiments, the ADI polypeptide is covalently bonded to at least one PEG molecule via an optional linker group. In some embodiments, the ADI polypeptide is covalently bonded to more than one PEG molecule. In some embodiments, the ADI polypeptide is covalently bonded to about 1 to about 10 PEG molecules. In some embodiments, the ADI polypeptide is covalently bonded to about 2 to about 8 PEG molecules. In some embodiments, the PEG molecules are linear or branched PEG molecules. In some embodiments, the PEG has a total average molecular weight of about 1,000 to about 40,000. In some embodiments, the PEG has a total average molecular weight of about 10,000 to about 30,000. In a particular embodiment, the PEG has a total average molecular weight of about 20,000.

[0063] In some embodiments, the linking group 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 group, a tyrosine group, a cysteine ​​group, a histidine group, a methylene group, or any combination thereof. In some embodiments, the succinyl group is derived from succinimide succinate.

[0064] In some embodiments, the arginine depleting agent is polyethylene glycol-modified arginine deiminase (ADI-PEG). In a particular exemplary embodiment, the ADI-PEG is ADI-PEG 20.

[0065] Some embodiments include a patient care kit, which comprises:

[0066] (a) an arginine-depleting agent that converts arginine to citrulline; and

[0067] (b) Cancer immunotherapy agents.

[0068] In some embodiments, (a) and (b) are in separate compositions. In some embodiments, (a) and (b) are in the same composition. Attached Figure Description

[0069] Figures 1A to 1C illustrate how ADI-PEG 20 enhances anti-CD3 / CD28-induced T cell activation. PBMCs were stimulated with anti-CD3 / CD28 Dynabeads in or without ADI-PEG 20 or mutADI-PEG 20. The percentage of CD69+ cells among all T cells (A), CD4+ T cells (B), and CD8+ T cells (Figure 1C) was determined by flow cytometry at 24, 48, and 72 hours.

[0070] Figures 2A to 2C illustrate the blockade of anti-CD3 / CD28-induced PD-1 upregulation on T cells by ADI-PEG 20. PBMCs were stimulated with anti-CD3 / CD28 Dynabeads in or without ADI-PEG 20 or mutADI-PEG 20. The percentage of PD-1+ cells in all T cells (A), CD4+ T cells (B), and CD8+ T cells (C) was determined by flow cytometry at 24, 48, and 72 hours.

[0071] Figures 3A to 3C illustrate how ADI-PEG 20 mitigates anti-CD3 / CD28-induced PD-L1 upregulation on T cells. PBMCs were stimulated with anti-CD3 / CD28 Dynabeads in or without ADI-PEG 20 or mutADI-PEG 20. The percentage of PD-L1+ cells in all T cells (A), CD4+ T cells (B), and CD8+ T cells (C) was determined by flow cytometry at 24, 48, and 72 hours.

[0072] Figures 4A to 4C illustrate the inhibition of anti-CD3 / CD28-induced CTLA-4 upregulation on T cells by ADI-PEG 20. PBMCs were stimulated with anti-CD3 / CD28 Dynabeads in or without ADI-PEG 20 or mutADI-PEG 20. The percentage of CTLA-4+ cells in all T cells (A), CD4+ T cells (B), and CD8+ T cells (C) was determined by flow cytometry at 24, 48, and 72 hours.

[0073] Figures 5A to 5C illustrate how ADI-PEG 20 reversed anti-CD3 / CD28-induced Treg accumulation. PBMCs were stimulated with anti-CD3 / CD28 Dynabeads in or without ADI-PEG 20 or mutADI-PEG 20. The percentage of Tregs in CD4+ T cells was determined by flow cytometry at 24, 48, and 72 hours. Treg cells were identified by labeling with CD3+CD4+CD25+FoxP3+ (A), CD3+CD4+CD25+FoxP3+PD-L1+ (B), or CD3+CD4+CD25+FoxP3+CTLA-4+ (C).

[0074] Figure 6A The effect of ADI-PEG 20 on the body weight of C57BL / 6 mice implanted with B16-F10 syngeneic tumors and treated with a loading agent (PBS; control) versus ADI-PEG 20 was demonstrated. Figure 6BThe effect of ADI-PEG 20 on dynamic tumor volume in C57BL / 6 mice implanted with B16-F10 syngeneic tumors and treated with a load (PBS; control) compared to those treated with ADI-PEG 20.

[0075] Figure 7 This study illustrates T-cell infiltration in B16-F10 tumors induced by ADI-PEG 20 treatment in C57BL / 6 mice. The number of T cells detected in tumor sections from ADI-PEG 20-treated mice is shown compared to the control group treated with the load cell.

[0076] Figure 8 The body weights of female Balb / c mice carrying CT26-established tumors are shown after administration of ADI-PEG20 and / or anti-PD-L1 mAb. Data points represent group mean body weights. Error bars represent the standard error (SEM) of the mean.

[0077] Figure 9 The tumor volume trajectory (tumor growth curve) of female BALB / c mice with CT26-infused tumors is shown. Data points represent group means, and error bars represent the standard error (SEM) of the means.

[0078] Figure 10 Survival curves are shown for female BALB / c mice with CT26-infused tumors after administration of ADI-PEG20 and / or anti-PD-L1 mAb. Survival curves were observed when the tumor reached 3000 mm. 3 At that time, each animal was executed.

[0079] Figure 11 The body weights of female C57BL / 6J mice carrying B16F10-derived tumors are shown after administration of ADI-PEG 20 and / or anti-PD-1 mAb. Data points represent group mean body weights. Error bars represent the standard error (SEM) of the mean.

[0080] Figure 12 Tumor volume is shown on day 13 in female C57BL / 6J mice with tumors established by B16F10 after administration of ADI-PEG20 and / or anti-PD-1 mAb. Detailed Implementation

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods, materials, compositions, reagents, or cells similar or equivalent to those described herein may be used to practice or test the subject matter of this disclosure, preferred methods and materials are described. All publications and references cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety as if each individual publication or reference were specifically and individually indicated as fully elucidated and incorporated herein by reference. Any patent application claiming priority to this application is also incorporated herein by reference in its entirety as described above regarding the publications and references.

[0082] Unless otherwise stated to the contrary, the practice of this disclosure will employ conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the scope 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 and Sons, New York (NY), (2009); Ausubel et al., *Short Protocols in Molecular Biology*, 3rd edition, Wiley and Sons, 1995; Sambrook and Russell, *Molecular Cloning: A Laboratory Manual*, 3rd edition, 2001; Maniatis et al., *Molecular Cloning: A Laboratory Manual*, (1982); *DNA Cloning: A Practical Approach*, Volumes I and II (edited by D. Glover); *Oligonucleotide Synthesis* (edited by N. Gait, 1984); *Nucleic Acid Hybridization*. Acid Hybridization (edited by B. Hames and S. Higgins, 1985); Transcription and Translation (edited by B. Hames and S. Higgins, 1984); Animal Cell Culture (edited by R. Freshney, 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0083] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions, or as commonly practiced in the art or as described herein. These and related techniques and procedures can generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. Unless specifically defined, the nomenclature and laboratory procedures and techniques used in conjunction with those described herein in molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry are those well known and commonly used in the art. Standard techniques can be used for recombinant technologies, molecular biology, microbiology, chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0084] For the purposes of this disclosure, the following terms are defined as follows.

[0085] This article uses the articles “a” and “an” to refer to one or more (i.e., at least one) grammatical objects of the articles mentioned. By way of example, “element” means one element or more elements.

[0086] "Approximately" means that a quantity, level, value, number, frequency, percentage, size, size, total, weight, or length varies relative to a reference quantity, level, value, number, frequency, percentage, size, total, weight, or length by as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0087] An "antagonist" is a biological structure or chemical agent that interferes with or otherwise reduces the physiological effects of another drug or molecule. In some cases, antagonists specifically bind to other drugs or molecules. This includes both full-acting and partial antagonists.

[0088] An "agonist" is a biological structure or chemical agent that increases or enhances the physiological effects of another drug or molecule. In some cases, agonists specifically bind to other drugs or molecules. This includes both full-acting and partial agonists.

[0089] As used herein, the term "amino acid" is intended to refer to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimics. For example, naturally occurring amino acids include 20(L)-amino acids used during protein biosynthesis, as well as others such as 4-hydroxyproline, hydroxylysine, desmodium, isodesmodium, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, leucine, valine, p-fluorophenylalanine, ethionine, etc., known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and moieties on the amino acid, or derivatization of the amino acid. Amino acid mimics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristics of the reference amino acid. For example, an organic structure mimicking arginine (Arg or R) would have a positively charged moiety located in a similar molecular space and having the same mobility of the e-amino group of the side chain as the naturally occurring Arg amino acid. Mimics also include constrained structures to maintain optimal spacing and charge interactions between the amino acid or amino acid functional groups. Those skilled in the art know or can identify which amino acid analogs and amino acid mimics have equivalent structures and functions.

[0090] "Biocompatible" refers to materials or compounds that are generally harmless to biological functions and do not cause any degree of unacceptable toxicity, including allergens and disease states.

[0091] A “coding sequence” refers to any nucleic acid sequence that contributes to the encoding of the polypeptide product of a gene. Conversely, the term “non-coding sequence” refers to any nucleic acid sequence that does not directly contribute to the encoding of the polypeptide product of a gene.

[0092] Throughout this disclosure, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of the stated steps or elements or a set of steps or elements, but not to exclude any other steps or elements or a set of steps or elements.

[0093] The phrase "consisting of..." means encompassing and limited to anything that follows the phrase "consisting of...". Therefore, the phrase "consisting of..." indicates that the listed element is necessary or mandatory, and other elements may not be present. The phrase "substantially constitutes..." means encompassing any element listed following this phrase, and is limited to other elements that do not interfere with or contribute to the activity or effect specified for the listed element in this disclosure. Therefore, the phrase "substantially constitutes..." indicates that the listed element is necessary or mandatory, but other elements are optional and may be present or absent depending on whether they substantially affect the activity or effect of the listed element.

[0094] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to endotoxins present in at most trace amounts (e.g., amounts that have no clinically adverse physiological effects on the subject), and preferably in amounts that are undetectable. Endotoxins are toxins associated with certain microorganisms, such as bacteria (typically Gram-negative bacteria), although they may also be found in Gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipoolipoproteins (LOS) found in the outer membranes of various Gram-negative bacteria, representing the central pathogenic characteristic of these bacteria's ability to cause disease. Small amounts of endotoxins in the human body can produce fever, lower blood pressure, activate inflammation and coagulation, and other adverse physiological effects.

[0095] Therefore, in pharmaceutical manufacturing, it is often necessary to remove most or all trace amounts of endotoxins from pharmaceutical products and / or containers, as even small amounts can have adverse effects on humans. Pyrogen-free ovens can be used for this purpose, as temperatures exceeding 300°C are typically required to decompose most endotoxins. For example, based on primary packaging materials such as syringes or vials, a glass temperature of 250°C and a holding time of 30 minutes are often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for endotoxin removal are also considered, including, for example, chromatography and filtration methods as described herein and known in the art.

[0096] Endotoxins can be detected using conventional techniques known in the art. For example, the horseshoe crab blood-based lipopolysaccharide (LPS) assay is a highly sensitive method for detecting the presence of endotoxins. In this test, very low levels of LPS can induce detectable coagulation of the horseshoe crab lysate due to the amplification of the reaction by a strong enzymatic cascade. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be essentially endotoxin-free, endotoxin levels can be below approximately 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 / mg of the active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1 to 10 EU.

[0097] The "half-life" of a polypeptide can refer to the time it takes for the polypeptide to lose half of its pharmacological, physiological, or other activity relative to the amount or concentration of the polypeptide when applied to the serum or tissue of a organism, or relative to any other defined time point. The "half-life" can also refer to the time it takes for the amount or concentration of the polypeptide to decrease by half when applied to the serum or tissue of a organism, or relative to any other defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0098] The terms “modification” and “change” include “increase,” “enhancement,” or “stimulation,” as well as “decrease” or “reduction,” generally of a statistically or physiologically significant amount or degree relative to a control. An “increased,” “stimulated,” or “enhanced” amount is typically a “statistically significant” amount and may 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) (inclusive of all integers and ranges between them, e.g., 1.5, 1.6, 1.7, 1.8, etc.) in the amount produced by a non-composite (e.g., no drug) or control composition. A “reduction” or “decrease” is typically a “statistically significant” amount and can include reductions of 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% (inclusive of all integers and ranges in between) in the amount produced by a non-composite (e.g., no-drug) or control composition. Examples of comparative and “statistically significant” amounts are described herein.

[0099] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably and refer to polymers of amino acids not limited to any particular length. The term “enzyme” includes a polypeptide or protein catalyst and, in the context of ADI, is used interchangeably with protein, polypeptide, or peptide. The term includes modifications such as myristylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of a signal sequence. The terms “polypeptide” or “protein” refer to one or more amino acid chains, each chain comprising amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein may comprise multiple chains non-covalently and / or covalently linked by peptide bonds and having a native protein sequence, i.e., proteins produced by naturally occurring and, particularly, non-recombinant cells, or genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a native protein, or molecules having one or more amino acids with a natural sequence but with deletions, additions, and / or substitutions. The terms “polypeptide” and “protein” specifically include the ADI enzyme / protein described herein, or sequences having one or more amino acids with deletions, additions, and / or substitutions of ADI proteins. In some embodiments, the polypeptide is a “recombinant” polypeptide produced by recombinant cells comprising one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that cannot be found otherwise in the cell.

[0100] The term “isolated” polypeptide or protein as used herein refers to a test protein that: (1) is free from at least some other proteins, typically found in nature using said at least some other proteins; (2) is substantially free from other proteins of the same origin, e.g., of the same species; (3) is expressed by cells of a different species; (4) has been isolated from at least about 50% of polynucleotides, lipids, carbohydrates, or other substances naturally associated with them; (5) is not associated with any protein portion naturally associated with the “isolated protein” (through covalent or non-covalent interactions); (6) is operatively associated with any polypeptide not naturally associated with it (through covalent or non-covalent interactions); or (7) is not present in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or may have a synthetic origin, or any combination thereof. In some embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that would be found in its natural environment and would interfere with its use (therapeutic, diagnostic, preventative, research, or otherwise).

[0101] In some embodiments, the “purity” of any given reagent in the composition (e.g., ADI, ADI-PEG) can be specifically defined. For example, some compositions may include reagents with a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, encompassing all decimals and ranges therebetween, measured, for example and by no means limited, by high-performance liquid chromatography (HPLC), a well-known form of column chromatography commonly used in biochemistry and analytical chemistry for the separation, identification, and quantification of compounds.

[0102] The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence that is 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 the tables and sequence listings.

[0103] As used herein, the term "sequence identity" or, for example, "a sequence that is 50% identical to" refers to the degree of sequence consistency within a comparison window, either on a nucleotide-by-nucleotide or amino acid-by-amino acid basis. Therefore, the "sequence identity percentage" can be calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions in the two sequences where 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 found, thus obtaining 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 obtain the sequence identity percentage. The optimal alignment of sequences used for the comparison window can be achieved through a computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in version 7.0 of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin, USA), or by examining the optimal alignment produced by any of the various selected methods (i.e., the one that results in the highest percentage of homology within the comparison window). Reference can also be made to the family of BLAST programs, such as those disclosed, for example, by Altschul et al., Nucleic Acids Res, 25:3389, 1997.

[0104] The term "solubility" refers to the property of a pharmaceutical agent (e.g., ADI, ADI-PEG) provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as concentration and is the mass of solute per unit volume of solvent (grams of solute per kilogram of solvent, g / dL (100 mL), mg / ml, etc.), molar concentration, mass molar concentration, mole fraction, or other similar concentration descriptions. The maximum equilibrium amount of solute that can dissolve each unit volume of solvent is the solubility of said solute in said solvent under specific conditions including temperature, pressure, pH, and the properties of the solvent. In some embodiments, solubility is measured at physiological pH or other pH values, such as pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5 to pH 8). In some embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In certain embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt concentrations (e.g., 500 mM NaCl and 10 mM NaP). In some embodiments, solubility is measured in biological fluids (solvents) such as blood or serum. In some embodiments, the temperature may be approximately room temperature (e.g., approximately 20°C, 21°C, 22°C, 23°C, 24°C, 25°C) or approximately body temperature (37°C). In some embodiments, the agent has a solubility of 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, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37°C.

[0105] The terms “subject” or “subject in need” or “patient” or “patient in need” include mammalian subjects such as the test subject.

[0106] "Substantially" or "essentially" means almost entirely or completely, for example, 95%, 96%, 97%, 98%, 99%, or higher of a given quantity.

[0107] "Statistically significant" means that the result is unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Commonly used significance measures include the p-value, which is the frequency or probability of the observed event if the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In a simple example, the significance level is defined as a p-value of 0.05 or less.

[0108] "Therapeutic response" refers to the improvement of symptoms (whether or not) based on the administration of one or more therapeutic agents.

[0109] As used herein, “treatment” for a subject (e.g., a mammal, such as a human) or cell is any type of intervention intended to alter the natural processes of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be administered preventively or after the onset of a pathological event or exposure to a pathogen. It also includes “preventive” treatment, which may be aimed at reducing the rate of progression of the treated disease or condition, delaying the onset of said disease or condition, or reducing the severity of its onset. “Treatment” or “prevention” does not necessarily imply the complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

[0110] The term "wild type" refers to the gene or gene product (e.g., polypeptide) that is most frequently observed in a population and is therefore arbitrarily referred to as the "normal" or "wild type" form of a gene.

[0111] Unless otherwise expressly stated, each embodiment in this specification is applicable to all other embodiments with necessary modifications.

[0112] Throughout this disclosure, the following abbreviations may be used: PEG, polyethylene glycol; ADI, arginine deiminase; SS, succinimide succinate; SSA, succinimide succinimide; SPA, succinimide propionate; NHS, N-hydroxy-succinimide; ASS1 or ASS, arginine succinate synthase.

[0113] Arginine depletion agent

[0114] Some embodiments employ one or more arginine depletion agents. In certain cases, arginine depletion agents convert arginine into citrulline (and ammonia), thereby depleting or reducing the supply of arginine available to cells such as cancer cells and / or T cells.

[0115] Some exemplary arginine depleting agents contain an arginine deiminase (ADI) enzyme or polypeptide. In some embodiments, the ADI polypeptide is derived from Mycoplasma hominis, Mycoplasma argininae, Mycoplasma arthritis, Mycoplasma phocomelia, Mycoplasma felis, Mycoplasma sepium, Mycoplasma pigeonis, Mycoplasma Iowa, Mycoplasma crocodilia, Mycoplasma alligatoris, Haemophilus ostreatus, or Mycoplasma bovis. In some embodiments, the ADI polypeptide is derived from Mycoplasma salivariae, Mycoplasma foamii, Mycoplasma cantonensis, Mycoplasma ototrichum, Mycoplasma synovitis, Mycoplasma cloacae, Mycoplasma gossypii, Mycoplasma albicans, Mycoplasma stomatum, Mycoplasma inertia, Mycoplasma turkeyii, Mycoplasma honeycomb, Mycoplasma penetratingum, Mycoplasma chickenii, Mycoplasma pearii, Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma leonhinum, Mycoplasma trachea, Mycoplasma mimicrysipelas, Mycoplasma milky white, Mycoplasma mousnezoffii, Mycoplasma elephantii, Mycoplasma pneumoniae, Mycoplasma turkeyii, Mycoplasma genus CAG:877, or Mycoplasma genus CAG:472.

[0116] Illustrative ADI peptides are provided in Table A1 below.

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] Therefore, in some embodiments, the ADI polypeptide comprises, is composed of, or is substantially composed of exemplary sequences from Table A1 (SEQ ID NO: 1-56), or variants or fragments thereof having ADI activity.

[0133] Some embodiments contain variants of the reference ADI polypeptide sequence described herein, whether described by name or by reference tables or sequence identifiers (e.g., Table A1, SEQ ID NO: 1-56). A “variant” sequence refers to a polypeptide or polynucleotide sequence that differs from the reference sequence in one or more substitutions, deletions (e.g., truncation), additions, and / or insertions. Thus, some variants contain fragments of the reference sequence described herein. The variant polypeptides possess biological activity, i.e., they retain the enzymatic or binding activity of the reference polypeptide. Such variants can be generated, for example, by genetic polymorphism and / or artificial manipulation.

[0134] In many cases, bioactive variants will contain one or more conserved substitutions. A “conserved substitution” is a substitution of one amino acid with another having similar properties, such that those skilled in the art of peptide chemistry expect the secondary structure and hydrophilic properties of the polypeptide to remain substantially unchanged. As described above, the structures of the polynucleotides and polypeptides of this disclosure can be modified, and functional molecules encoding variants or derived polypeptides with the desired characteristics are still obtained. When it is desired to change the amino acid sequence of a polypeptide to produce an equivalent or even a modified variant or portion of the polypeptide described herein, those skilled in the art typically change one or more codons encoding the DNA sequence.

[0135] For example, certain amino acids can replace other amino acids in a protein structure without significantly losing their ability to interact with structures such as antigen-binding regions of antibodies or binding sites on substrate molecules. Since the interacting abilities and properties of proteins define their biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence, and of course, in its basic DNA coding sequence, and thus obtain protein properties similar to those of the protein. Therefore, it is anticipated that various changes can be made to the peptide sequence of the disclosed composition or the corresponding DNA sequence encoding the peptide without significantly losing its utility.

[0136] When making such changes, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilic amino acid index in conferring biological functions of protein-protein interactions is well known in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is understood that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydrophilicity index based on its 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 substituted with other amino acids having similar hydrophilic indices or fractions, and still produce proteins with similar biological activities, i.e., proteins with equivalent biological functions. When making such a change, it is preferable to replace amino acids with a hydrophilicity index within ±2, particularly preferably within ±1, and even more preferably within ±0.5.

[0137] It should also be understood in the art that similar amino acids can be effectively substituted based on hydrophilicity. U.S. Patent 4,554,101 (incorporated herein by reference in its entirety) states that the maximum local average hydrophilicity of a protein, governed by the hydrophilicity of its adjacent amino acids, is related to the biological properties of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values ​​are specified for 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 should be understood that an amino acid can be substituted for another amino acid with a similar hydrophilicity value, and biologically equivalent, especially immunologically equivalent, proteins can still be obtained. In such a change, substitution of amino acids with a hydrophilicity value within ±2 is preferred, particularly within ±1, and even more particularly preferred within ±0.5.

[0138] As mentioned above, amino acid substitution is therefore generally based on the relative similarity of the substituents in the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various 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.

[0139] Amino acid substitutions can be further made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties 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 without polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; and serine, phenylalanine, and tyrosine. Other amino acid groups that can represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0140] Variants may also or alternatively contain non-conserved variations. In preferred embodiments, the variant peptide differs from the native or reference sequence in that it has been substituted, deleted, or added to fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified, for example, by the deletion or addition of amino acids that have the least impact on the peptide's immunogenicity, secondary structure, enzymatic activity, and / or hydrophilic properties.

[0141] In some embodiments, the polypeptide sequence is about, at least about, or more than about 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 in length. 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 700, 710, 720, 730, 740, 750, 760 770, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more (inclusive of all integers in between) consecutive amino acids, and may include all or part of a reference sequence (see, for example, sequence listing).

[0142] In some embodiments, the polypeptide sequence consists of about or no more than about 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, 5 0, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 40 0, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770 It consists of 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more (inclusive of all integers in between) consecutive amino acids, and may include all or part of a reference sequence (see, for example, sequence listing).

[0143] In some embodiments, the polypeptide sequence is approximately 10 to 1000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 1000, 20 to 900, 20 to 800, 20 to 700, 20 to 600, 20 to 500, 20 to 400, 20 to 300, 20 to 200, 20 to 100, 20 to 50, 20 to 40, 20 to 30, 50 to 1000, 50 to 900, 50 to 800, 5 0 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 100 to 1000, 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 1000, 200 to 900, 200 to 800, 200 to 700, 200 to 600, 200 to 500, 200 to 400 or 200 to 300 (inclusive) consecutive amino acids, and may include all or part of the reference sequence. In some embodiments, the C-terminal or N-terminal region of any reference polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more (inclusive) amino acids. Or approximately 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more (inclusive of all integers therein, e.g., 101, 102, 103, 104, 105) amino acids, provided that the truncated polypeptide retains the binding properties and / or activity of the reference polypeptide. Typically, the bioactive fragment has not less than approximately 1%, approximately 5%, approximately 10%, approximately 25%, or approximately 50% of the activity of the bioactive reference polypeptide from which it is derived.

[0144] Typically, variants will show at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity, sequence identity, or sequence homology to the reference polypeptide sequence. Furthermore, the sequence differs from the native or parental sequence in that it includes additions (e.g., C-terminal addition, N-terminal addition, both C-terminal and N-terminal addition), deletions, truncations, insertions, or substitutions (e.g., conserved substitutions) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2 6, 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, or 100 (inclusive of all integers and ranges) amino acids, but retaining the characteristics or activity of the parent or reference polypeptide sequence.

[0145] In some embodiments, the variant peptide differs from the reference sequence by at least one but fewer than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3, or 2 amino acid residues. In some embodiments, the variant peptide differs from the reference sequence by at least 1% but less than 20%, 15%, 10%, or 5% of residues. (If this comparison requires alignment, the sequences should be aligned to obtain the maximum similarity. Deleted or inserted or mismatched "loop" sequences are considered differences.)

[0146] The calculation of sequence similarity or sequence identity between sequences (these terms are used interchangeably herein) is performed as follows. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, sequences are aligned for optimal alignment purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In some embodiments, the sequence length aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a 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.

[0147] The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0148] Sequence comparison and determination of percentage identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences can be determined using the Needleman and Fhunsch (J. Mol. Biol., 48:444-453, 1970) algorithm in the GAP program, which is already incorporated in the GCG software package, using a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percentage identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (unless otherwise specified, the parameters to be used) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5.

[0149] The percentage of identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Computer Applications in the Biological Sciences (Cabios), 4:11-17, 1989), which has been incorporated in the ALIGN program (version 2.0), using the PAM120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4.

[0150] The nucleic acid and protein sequences described herein can be used as "query sequences" to search public databases, thereby, for example, identifying other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, *Journal of Molecular Biology*, 215:403-10). A BLAST nucleotide search can be performed using the XBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed using the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. For vacancy-based alignments for comparative purposes, vacancy-based BLAST can be used as described in Altschul et al. (*Nucleic Acids Res*, 25:3389-3402, 1997). When using the BLAST and Gapped BLAST procedures, the default parameters of each procedure (e.g., XBLAST and NBLAST) can be used.

[0151] In some embodiments, as described above, BLAST alignment tools can be used to evaluate polynucleotides and / or peptides. Local alignment consists of only one pair of sequence segments, with one segment from each sequence segment being compared. Modifications to the Smith-Waterman or Sellers algorithm will find all segment pairs, known as high-score fragment pairs (HSPs), whose scores cannot be improved by expansion or pruning. The results of BLAST alignment include statistical measures to indicate the likelihood that a BLAST score could be expected by chance.

[0152] The raw score S is calculated from the number of gaps and substitutions associated with each aligned sequence, where a higher similarity score indicates a more significant alignment. The substitution score is given by a lookup table (see PAM, BLOSUM).

[0153] The vacancy cost is typically calculated as the sum of G (vacancy opening penalty) and L (vacancy extension penalty). For a vacancy of length n, the vacancy cost is G + Ln. The choice of vacancy costs G and L is empirical, but conventionally, a high value for G (10 to 15), such as 11, and a low value for L (1 to 2), such as 1, are chosen.

[0154] The bit score S' is derived from the original alignment score S, which already takes into account the statistical properties of the scoring system used. The bit score is standardized relative to the scoring system, so it can be used to compare alignment scores from different retrievals. The terms "bit score" and "similarity score" are used interchangeably. The bit score indicates the degree of alignment quality; a higher score indicates a better alignment.

[0155] The E-value, or expected value, describes the probability that sequences with similar scores will coincidentally appear in a database. The E-value or expected value is a prediction of the number of distinct alignments with scores equal to or better than S that will coincidentally appear in a database retrieval. The smaller the E-value, the more significant the alignment. For example, an E-value of e... -117 The alignment implies that sequences with similar scores are unlikely to appear by chance. Furthermore, the expected score requirement for aligning random amino acid pairs is negative; otherwise, long alignments will tend to have high scores regardless of their relevance to the aligned region. Additionally, the BLAST algorithm uses appropriate substitution matrices, nucleotides, or amino acids, and for gap alignments, it uses gap generation and extension penalties. For example, BLAST alignments and peptide sequence comparisons typically use a BLOSUM62 matrix, a gap presence penalty of 11, and a gap extension penalty of 1.

[0156] In some embodiments, sequence similarity scores are reported from BLAST analysis using a BLOSUM62 matrix, a void presence penalty of 11, and a void extension penalty of 1.

[0157] In specific embodiments, the sequence identity / similarity scores provided herein refer to values ​​obtained using GAP version 10 (GCG, Accelrys, San Diego, California) with the following parameters: percentage identity and percentage similarity of nucleotide sequences using GAP weight 50 and length weight 3 and the nwsgapdna.cmp scoring matrix; and percentage identity and percentage similarity of amino acid sequences using GAP weight 8 and length weight 2 and the BLOSUM62 scoring matrix (Henikoff and Henikoff, Proceedings of the National Academy of Sciences (PNASUSA), 89:10915-10919, 1992). GAP uses the algorithm of Needleman and Wunsch (Journal of Molecular Biology, 48:443-453, 1970) to find alignments between two complete sequences that maximize the number of matches and minimize the number of gaps.

[0158] In certain embodiments, the variant polypeptide includes an amino acid sequence (see, for example, a sequence listing) that can be optimally aligned with a reference polypeptide sequence to produce at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 5 60, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more (inclusive of all integers and ranges) of BLAST bit scores or sequence similarity scores, where BLAST alignment uses a BLOSUM62 matrix, a gap presence penalty of 11, and a gap extension penalty of 1.

[0159] As described above, reference peptides can be modified in various ways, including amino acid substitution, deletion, truncation, addition, and insertion. Methods of such manipulation are well known in the art. For example, amino acid sequence variants of the reference peptide can be prepared by mutation in DNA. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Kunkel, (Proceedings of the National Academy of Sciences, 82:488-492, 1985); Kunkel et al., (Methods in Enzymol, 154:367-382, 1987); U.S. Patent No. 4,873,192; Watson, JD et al., (Molecular Biology of the Gene, 4th Edition, Benjamin Cummings, Menlo Park, California, 1987); and references cited herein. Guidelines on appropriate amino acid substitutions that do not affect the biological activity of the protein in question can be found in Dayhoff et al., (1978), Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington, D.C.).

[0160] Methods for screening gene products from combinatorial libraries prepared through such modification, and methods for screening cDNA libraries containing gene products with selected properties, are known in the art. These methods are suitable for rapid screening of gene libraries generated by combinatorial mutagenesis of a reference peptide. As an example, recursive total mutagenesis (REM)—a technique that enhances the frequency of functional mutants in a library—can be used in combination with screening assays to identify peptide variants (Arkin and Yourvan, *Proceedings of the National Academy of Sciences*, 89:7811-7815, 1992; Delgrave et al., *Protein Engineering*, 6:327-331, 1993).

[0161] In some cases, naturally occurring ADIs can be derived from microorganisms and therefore can be immunogenic and / or rapidly cleared from the patient's circulation. Such problems can be overcome by modifying ADIs to produce "modified ADI" enzymes. Therefore, some embodiments comprise ADI enzymes including "modifiers," examples of which include, but are not limited to, macromolecular polymers, proteins, peptides, polysaccharides, and other compounds. ADI enzymes and modifiers can be linked by covalent or non-covalent interactions to form stable conjugates or stable compositions to achieve the desired effect. In some embodiments, the modified ADI retains the biological activity of the corresponding unmodified ADI (e.g., the same or similar sequence) and has a longer in vivo half-life and lower antigenicity than the corresponding unmodified ADI. In some embodiments, the modified ADI 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 ADI. Typically, the modified ADI retains sufficient biological activity for therapeutic use.

[0162] In some embodiments, the modifier may be a biocompatible polymer or protein or fragment thereof, and may increase the half-life of ADI in the blood. The modifier may be chemically coupled to ADI or, where applicable, linked to ADI via fusion protein expression.

[0163] The polymer may include non-peptide polymers, which in some embodiments may possess their own biological activity. Suitable polymers include, but are not limited to, polyenol compounds, polyether compounds, polyvinylpyrrolidone, polyamino acids, copolymers of divinyl ether and maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, polysaccharides, polyoxyethyl polyols, heparin or fragments thereof, polyalkylethylene glycol and its derivatives, copolymers of polyalkylethylene glycol and its derivatives, poly(vinyl ethyl ether), α,P-poly[(2-hydroxyethyl)-DL-asparagine], polycarboxylates, polyoxyethylene-polyoxymethylene, polyacryloylmorpholine, copolymers of amino compounds and oxyolefins, polyhyaluronic acid, polyepoxides, copolymers of oxalic acid and malonic acid, copolymers of poly(1,3-dioxolane)ethylene and maleic hydrazine, polysialic acid, cyclodextrin, etc. In some embodiments, the polymer is polyethylene glycol.

[0164] Polyene alcohol compounds used in this article include, but are not limited to, polyethylene glycol (including monomethoxy polyethylene glycol and monohydroxy polyethylene glycol), polyvinyl alcohol, polyacryl alcohol, polybutenol, and their derivatives, such as lipids.

[0165] 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 ).

[0166] Polyamino acids include, but are not limited to, polymers of one amino acid or copolymers of two or more amino acids, such as polyalanine or polylysine, or block copolymers thereof.

[0167] Polysaccharides include, but are not limited to, dextran and its derivatives, such as sulfated dextran, cellulose and its derivatives (including methylcellulose and carboxymethylcellulose), starch and its derivatives, sucrose, etc.

[0168] In certain embodiments, the ADI is modified by coupling with a protein or peptide, wherein one or more proteins or peptides are directly or indirectly linked to the ADI. 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, α1-acid glycoprotein, transferrin, fibrinogen, immunoglobulins, IgFc regions, albumin, and fragments thereof. A “fragment” refers to any part of a protein that is smaller than the whole protein but retains the desired function of the protein. The ADI, as described herein, can be linked to a protein directly or indirectly via covalent bonds. A direct linkage refers to the direct linkage of one amino acid of the ADI to one amino acid of the modified protein via a peptide bond or disulfide bond. An indirect linkage refers to a linkage between the ADI and the modified protein via a chemical group originally present therein or a specific chemical group added by biological or chemical methods, or a combination of the above linkages.

[0169] In certain embodiments, ADI is modified by covalently linking it to one or more PEG molecules. ADI covalently modified with PEG (with or without a linker group) is hereinafter referred to as "ADI-PEG". Compared to unmodified ADI, ADI-PEG retains most of its enzymatic activity, has less immunogenicity or antigenicity, a significantly prolonged circulating half-life, and is more effective in cancer treatment.

[0170] "Polyethylene glycol" or "PEG" refers to a mixture of branched or linear ethylene oxide and water condensation polymers, with the general formula H(OCH2CH2). n OH indicates that 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.

[0171] In some embodiments, the total weight-average molecular weight of PEG is about 1,000 to about 50,000; about 3,000 to about 40,000; about 5,000 to about 30,000; about 8,000 to about 30,000; about 11,000 to about 30,000; about 12,000 to about 28,000; about 16,000 to about 24,000; about 18,000 to about 22,000; or about 19,000 to about 21,000. In some embodiments, the total weight-average molecular weight of PEG is from about 1,000 to about 50,000; about 3,000 to about 30,000; about 3,000 to about 20,000; about 4,000 to about 12,000; about 4,000 to about 10,000; about 4,000 to about 8,000; about 4,000 to about 6,000; or about 5,000. In some embodiments, the total weight-average molecular weight of PEG is about 20,000. Typically, PEG with a molecular weight of 30,000 or more is difficult to dissolve, and the yield of the formulated product may be reduced. PEG can be branched or linear. Generally, increasing the molecular weight of PEG reduces the immunogenicity of ADI. PEG can be branched or linear, and in some embodiments is linear. PEG having the molecular weights described herein can be used in combination with ADI, and optionally, in combination with a biocompatible linker.

[0172] Some embodiments use thiol, thiol, or cysteine-reactive PEG. In some embodiments, the thiol, thiol, or cysteine-reactive PEG is linked to one or more naturally occurring cysteine ​​residues, one or more introduced cysteine ​​residues (e.g., replacing one or more wild-type residues with (multiple) cysteine ​​residues), inserted with one or more cysteine ​​residues, or any combination thereof (see, for example, Doherty et al., Bioconjug Chemistry, 16:1291-98, 2005). In some embodiments, certain wild-type ADI cysteine ​​residues may first be substituted with another amino acid to prevent the PEG polymer from linking to wild-type cysteine, for example, to prevent PEG from disrupting other desired biological activities. Some embodiments use one or more non-natural cysteine ​​derivatives (e.g., homocysteine) instead of cysteine.

[0173] Non-limiting examples of thiol, thiol, or cysteine-reactive PEGs include methoxyPEG maleimide (M-PEG-MAL) (e.g., MW 2000, MW 5000, MW 10000, MW 20000, MW 30000, MW 40000). M-PEG-MAL reacts with thiol groups on the cysteine ​​side chains of proteins and peptides to produce stable 3-thiosuccinimide ether bonds. This reaction is highly selective and can be carried out under mild conditions from about pH 5.0 to pH 6.5 in the presence of other functional groups. Specific examples of commercially available thiol, thiol, or cysteine-reactive PEG molecules are shown in Figures 1A through 1D. Thus, in some embodiments, the ADI enzyme is conjugated with any one or more of the thiol, thiol, or cysteine-reactive PEG molecules described herein.

[0174] Although preferred embodiments use linker groups, ADI can be covalently bonded to modifiers such as PEG, with or without linker groups. ADI can be covalently bonded to PEG using methods known in the art via biocompatible linker groups, for example, Park et al., *Anticancer Res.*, 1:373-376 (1981); and Zaplipsky and Lee, *Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications*, edited by J.M. Harris, Plenicom Press, New York, Chapter 21 (1992), the disclosures of which are incorporated herein by reference in their entirety. In some cases, ADI can be directly (i.e., without linker groups), for example, coupled to modifiers such as PEG via amino, thiol, hydroxyl, carboxyl, or other groups.

[0175] The linker group used to covalently link ADI to a modifier (such as PEG) can be any biocompatible linker group. As mentioned above, "biocompatible" means that the compound or group is non-toxic and can be used in vitro or in vivo without causing damage, disease, illness, or death. Modifiers such as PEG can be bonded to the linker group, for example, via ether bonds, thiol bonds, amide bonds, or other bonds.

[0176] In some embodiments, a suitable linker group may have, for example, a total chain length of about 1 to 100 atoms, 1 to 80 atoms, 1 to 60 atoms, 1 to 40 atoms, 1 to 30 atoms, 1 to 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, wherein the atoms in the chain include C, S, N, P, and / or O. In some embodiments, the linker group is optional; for example, PEG-conjugated ADI enzymes do not include a linker group. In some cases, the linker group comprises, for example, succinyl, amide, imide, carbamate, ester, epoxy, carboxyl, hydroxyl, carbohydrate, tyrosine, cysteine, histidine, methylene, or combinations thereof. Specific examples of stable linking groups include succinimide, propionic acid, carboxymethyl linkage, ether, carbamate, amide, amine, carbamate, imide, aliphatic C-link, and thioether. In some embodiments, the biocompatible linking group is a succinimide succinate (SS) group.

[0177] Other suitable linking groups include oxycarbonylimidazolium (containing, for example, carbonylimidazolium (CDI)), nitrophenyl (containing, for example, nitrophenyl carbonate (NCP) or trichlorophenyl carbonate (TCP)), toluenesulfonate, aldehyde, isocyanate, vinyl sulfone, or primary amine. In some embodiments, the linking group is derived from SS, SPA, SCM, or NHS; in some embodiments, SS, SPA, or NHS is used, and in some embodiments, SS or SPA is used. Therefore, in some embodiments, the potential linking group may be formed from methoxy-PEG succinimide succinate (SS), methoxy-PEG succinimide glutarate (SG), methoxy-PEG succinimide carbonate (SC), methoxy-PEG succinimide carboxymethyl ester (SCM), methoxy-PEG2N-hydroxysuccinimide (NHS), methoxy-PEG succinimide butyrate (SBA), methoxy-PEG succinimide propionate (SPA), methoxy-PEG succinimide glutaramide, and / or methoxy-PEG succinimide succinimide.

[0178] Additional examples of linking groups 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—O—, —O—CH2—CH2—, —CH2—O—CH2—CH2—O—, —O—CH2—CH2—CH2—, —CH2—CH2—O—CH2—CH2—O—CH2—CH2—O—, —O—CH2—CH2—CH2—C H2—,—CH2—O—CH2—CH2—CH2—,—CH2—CH2—O—CH2—CH2—,—CH2—C H2—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—C H2—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—、divalent cycloalkyl、—N(R6)—、R6 is an organogroup selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl groups. Additionally, any linking group portion described herein may further comprise an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e., -(CH2CH2O]]. 1-20 That is, ethylene oxide oligomer chains may appear before or after the linking group, and optionally between any two atoms of the linking group portion comprising two or more atoms. Furthermore, if the oligomer is adjacent to a polymer chain segment and represents only an extension of that segment, the oligomer chain will not be considered part of the linking group portion.

[0179] Specific exemplary PEG molecules and linking groups are described in Table A2 below.

[0180]

[0181] In some embodiments, the ADI enzyme includes one or more PEG molecules and / or linker groups as described herein (e.g., in Table A2).

[0182] The binding of PEG to ADI increases the cyclic half-life of ADI. Typically, PEG is bound to the primary amine of ADI. As is known to those skilled in the art, the choice of binding sites for PEG or other modifiers on ADI is determined by the role of each site within the protein's active domain. PEG can bind to the primary amine of ADI without substantially losing enzyme activity. For example, lysine residues present in ADI are all possible sites where, as described herein, ADI can be bound to PEG via biocompatible binding groups such as SS, SPA, SCM, SSA, and / or NHS. As will be apparent to those skilled in the art, PEG can also bind to other sites on ADI.

[0183] One to about 30 PEG molecules can be covalently bonded to ADI. In some embodiments, ADI is modified with (i.e., including) one PEG molecule. In some embodiments, ADI is modified with more than one PEG molecule. In particular embodiments, ADI 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, ADI is modified with about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG molecules. In particular embodiments, ADI is modified with 4.5 to 5.5 PEG molecules per ADI. In some embodiments, ADI is modified with 5 ± 1.5 PEG molecules.

[0184] In some embodiments, about 15% to about 70% of the primary amino groups in the ADI are modified with PEG; in some embodiments, about 20% to about 65%, about 25% to about 60%, or in some embodiments, about 30% to about 55%, or 45% to about 50%, or in some embodiments, about 50% of the primary amino groups in the arginine deiminase are modified with PEG. When PEG is covalently bonded to the end of the ADI, it is desirable to use only one PEG molecule. Increasing the number of PEG units on the ADI increases the enzyme's cycle half-life. However, increasing the number of PEG units on the ADI decreases the enzyme's specific activity. Therefore, given this disclosure, it will be apparent to those skilled in the art that a balance needs to be struck between the two.

[0185] In some embodiments, a common feature of the biocompatible linker groups is that they are linked to the primary amine of arginine deiminase via a succinimide group. Once coupled to ADI, SS-PEG has an ester bond next to PEG, which can sensitize the site to serum esterases, which can release PEG from ADI in vivo. SPA-PEG and PEG2-NHS do not have ester bonds and are therefore insensitive to serum esterases.

[0186] Similar to SS-PEG, SPA-PEG, and SC-PEG, the PEG linked to the protein can be a straight chain, or, like PEG2-NHS, it can use branched PEG chains.

[0187] In some embodiments, the polyethylene glycol (PEG) esterification site associated with the ADI located at or near the catalytic region of the enzyme is modified. In some embodiments, the phrase "PEG esterification site" is defined as any site or location of the ADI that can be covalently modified with polyethylene glycol. A "PEG esterification site" can be considered to be located at or near the catalytic region of the enzyme, wherein PEGylation of the site significantly reduces the catalytic activity of the enzyme. PEGylation of such sites conventionally leads to enzyme inactivation. For example, the ADI from Mycoplasma hominis has a lysine residue at position 112, which can be considered to be at or near the catalytic region of the enzyme. The connection of PEG to the lysine residue at position 112 can inactivate the enzyme. Furthermore, the ADI from Mycoplasma hominis has a cysteine ​​residue at position 397, which can be considered to be at or near the catalytic region of the enzyme. Amino acid substitution of the cysteine ​​residue at position 397 can inactivate the enzyme. Specifically, replacing cysteine ​​at position 397 with alanine, histidine, arginine, serine, lysine, or tyrosine results in the loss of all detectable enzyme activity. ADI from Mycoplasma hominis also contains three lysine residues near this conserved cysteine, specifically Lys374, Lys405, and Lys408. Linking PEG with Lys374, Lys405, Lys408, or a combination thereof, can inactivate the enzyme.

[0188] It should be understood that ADIs derived from other organisms may also have a polyethylene glycolation site corresponding to position 112 of the ADI from Mycoplasma hominis. Furthermore, ADIs from certain organisms may have lysine residues corresponding to the same general position as position 112 of the ADI from Mycoplasma hominis. The positions of lysine residues in ADIs from these organisms are known to those skilled in the art and are described in U.S. Patent No. 6,635,462.

[0189] Therefore, some embodiments provide substitutions of certain amino acids in the polypeptide chain of the ADI. These amino acid substitutions provide a modified ADI that loses less activity when modified with a modifier, such as PEGylation. Optimal modification, such as PEGylation, can be achieved without loss of activity by eliminating the PEGylation site or other known modification sites at or near the catalytic region of the enzyme.

[0190] In some embodiments, for example, as described above, amino acid substitution is performed using non-natural amino acids conjugated with PEG or other modifiers (see, for example, de Graaf et al., Bioconjugated Chemistry, 20:1281-95, 2009). Thus, some embodiments comprise an ADI enzyme conjugated with one or more non-natural amino acids and one or more PEGs. In some embodiments, non-natural amino acids include side chains having functional groups selected from the following groups: alkyl, aryl, aryl halogen, vinyl halogen, alkyl halogen, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl, ketone, azide, hydroxyl, hydrazine, cyano, halogen, acylhydrazine, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boric acid, borate ester, borane, phenylboronic acid, thiol, selenyl, sulfonyl, borate ester, borate, phosphonium, phosphonic carboxyl, phosphine, heterocyclic, aziridine, naphthyl, benzophenone, restricted rings such as cyclooctyne, thioester, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid, α-ketocarboxylic acid, α or β unsaturated acid and amide, acetaldehyde amide and organosilicon alkyl groups. In some embodiments, the non-natural amino acid is selected from groups 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, levodopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonyltyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

[0191] While ADI-PEG is an exemplary modified ADI described herein, as those skilled in the art will recognize, ADI can be modified with other polymers or appropriate molecules to achieve the desired effects, particularly reducing antigenicity and increasing serum half-life.

[0192] It should be understood that some embodiments are based on the understanding that certain structural features of arginine deiminases can hinder or interfere with proper and rapid renaturation when generated via recombination technology. Specifically, these structural features prevent or inhibit the enzyme from presenting its active conformation during recombination production. In some embodiments, the term "active conformation" is defined as the three-dimensional structure that allows for enzymatic activity by unmodified or modified arginine deiminases. In particular, the active conformation may be necessary for catalyzing the conversion of arginine to citrulline. The term "structural feature" can be defined as any characteristic, quality, or property of a polypeptide chain resulting from a particular amino acid or combination of amino acids. For example, arginine deiminases may contain amino acids that cause normal peptide chain bending or kinking, thereby preventing the enzyme from presenting its active conformation during enzyme renaturation. In particular, arginine deiminases derived from Mycoplasma hominis have a proline at position 210, which may cause bending or kinking in the peptide chain, making it more difficult for the enzyme to reform during recombination production. It should be understood that arginine deiminases derived from other organisms may also have a site corresponding to position 210 of arginine deiminases derived from Mycoplasma hominis.

[0193] Therefore, some embodiments provide certain amino acid substitutions in the polypeptide chain of wild-type arginine deiminases. Examples include substitutions that eliminate problematic structural features in the polypeptide chain of arginine deiminases. Substitutions that provide improved refolding for modified arginine deiminases are also included. These amino acid substitutions enable rapid refolding of modified arginine deiminases using reduced amounts of buffer. These amino acid substitutions can also provide increased yields for refolding modified arginine deiminases. In some embodiments, the modified arginine deiminase has an amino acid substitution or equivalent residue at position P210. As mentioned above, arginine deiminases derived from Mycoplasma hominis have the amino acid proline at position 210. While not limiting, it is believed that the presence of the amino acid proline at position 210 causes bending or kinking in the normal polypeptide chain, which increases the difficulty of refolding (i.e., refolding) some arginine deiminases. The proline substitution at position 210 enables rapid refolding of modified arginine deiminases using reduced amounts of buffer. Substitution (or equivalent residues) of proline at position 210 can also provide improved yields for renatured arginine deiminases. In some embodiments, proline (or equivalent residues) at position 210 is substituted with serine. Other non-limiting examples of substitutions include Pro210 to Thr210, Pro210 to Arg210, Pro210 to Asn210, Pro210 to Gln210, or Pro210 to Met210. Optimal enzyme refolding can be achieved by eliminating those structural features associated with the amino acid at position 210 of the wild-type arginine deiminase.

[0194] In a specific embodiment, the modified ADI is ADI-PEG 20. ADI-PEG 20 refers to the ADI molecule described in the following, for example, U.S. Patent No. 6,183,738; U.S. Patent No. 6,635,462; Ascierto PA 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. Studies), *Journal of Clinical Oncology*, 22(10):1815–1822; Holtsberg FW et al., (2002), Poly(ethylene glycol)(PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties, *Journal of Controlled Release*, 80(1–3):259–271; and Kelly et al., (2012), *British Journal of Cancer*, 106, 324–332. As will be appreciated by those skilled in the art, ADI-PEG20 is a PEGylated ADI enzyme derived from Mycoplasma hominis (5.5 ± 1.0 bonded PEG 20,000) with two substitutions (K112E; P210S) relative to the wild-type Mycoplasma hominis ADI sequence.

[0195] Those skilled in the art will understand that the various arginine depletion agents described herein can be combined with any one or more of the various cancer immunotherapy agents described herein and used in accordance with any one or more of the methods or compositions described herein.

[0196] Immunotherapy agents

[0197] Some embodiments employ one or more cancer immunotherapeutic agents. In some instances, the immunotherapeutic agent modulates the subject's immune response, for example, to increase or maintain a cancer-related or cancer-specific immune response, thereby resulting in increased immune cell suppression or a reduction in cancer cells. Exemplary immunotherapeutic agents comprise peptides, such as antibodies and their antigen-binding fragments, ligands, and small peptides and mixtures thereof. Immunotherapeutic agents also comprise small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapy agents, or other polynucleotide-based agents, including viral agents such as oncolytic viruses, and other agents known in the art. Thus, in some embodiments, the cancer immunotherapeutic agent is selected from one or more of immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies.

[0198] In some embodiments, cancer immunotherapeutic agents are immune checkpoint modulators. Specific examples include “antagonists” of one or more inhibitory immune checkpoint molecules and “agonists” of one or more stimulating immune checkpoint molecules. Typically, immune checkpoint molecules are components of enhancing (co-stimulatory) or de-stimulating signals of the immune system, and targeting these components has therapeutic potential against cancer because cancer cells can disrupt the natural function of immune checkpoint molecules (see, for example, Sharma and Allison, Science, 348:56-61, 2015; Topalian et al., Cancer Cell, 27:450-461, 2015; Pardoll, Nature Reviews Cancer, 12:252-264, 2012). In some embodiments, immune checkpoint modulators (e.g., antagonists, agonists) “bind” or “specifically bind” to one or more immune checkpoint molecules as described herein.

[0199] In certain embodiments, the immune checkpoint modulator is a polypeptide or peptide. The terms "peptide" and "polypeptide" are used interchangeably herein; however, in some instances, the term "peptide" may refer to a shorter polypeptide, such as a polypeptide consisting of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, encompassing all integers and ranges therebetween (e.g., 5 to 10, 8 to 12, 10 to 15). Polypeptides and peptides may consist of naturally occurring and / or non-naturally occurring amino acids as described herein. Antibodies are also included as polypeptides.

[0200] The binding properties of peptides can be quantified using methods known in the art (see Davies et al., *Annual Rev. Biochem.*, 59:439-473, 1990). In some embodiments, the peptide specifically binds to target molecules, such as immune checkpoint molecules or their epitopes, with an equilibrium dissociation constant of about ≤10⁻⁷ M to about 10⁻⁸ M. In some embodiments, the equilibrium dissociation constant is about ≤10⁻⁹ M to ≤10⁻¹⁰ M. In some illustrative embodiments, the peptide has a specific binding affinity to the target described herein of about, at least about, or less than about 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, etc. Affinity (Kd) at M, 7nM, 8nM, 9nM, 10nM, 11nM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, 20nM, 21nM, 22nM, 23nM, 24nM, 25nM, 26nM, 27nM, 28nM, 29nM, 30nM, 40nM, or 50nM.

[0201] In some embodiments, the immune checkpoint modulating peptide is an antibody or "its antigen-binding fragment". The antibody or antigen-binding fragment can be of virtually any type. As is well known in the art, an antibody is an immunoglobulin molecule, such as an immune checkpoint molecule, capable of specifically binding to a target through at least one epitope recognition site located in the variable region of an immunoglobulin molecule.

[0202] As used herein, the term "antibody" includes not only complete polyclonal or monoclonal antibodies, but also their fragments (such as dAb, Fab, Fab', F(ab')2, Fv), single chains (ScFv), their synthetic variants, naturally occurring variants, fusion proteins including antibody portions having antigen-binding fragments possessing desired specificity, humanized antibodies, chimeric antibodies, and any other modified conformation of immunoglobulin molecules including antigen-binding sites or fragments (epitope recognition sites) with desired specificity. Certain characteristics and properties of antibodies (and their antigen-binding fragments) are described in more detail herein.

[0203] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of the heavy and / or light chains of an immunoglobulin that binds to the antigen of interest. In this respect, the antigen-binding fragment of an antibody described herein may include one, two, three, four, five, or all six CDRs of the VH and VL sequences of an antibody that binds to a target molecule.

[0204] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binder such as an antibody and can also be used in animals to produce antibodies that can bind to epitopes of that antigen. An antigen may have one or more epitopes.

[0205] The term "epitope" encompasses any determinant, preferably a polypeptide determinant, capable of specifically binding to immunoglobulins or T-cell receptors. An epitope is a region of an antigen that an antibody binds to. In some embodiments, the epitope determinant comprises chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics. Epitopes can be continuous or discontinuous with respect to the primary structure of the antigen.

[0206] A molecule, such as a peptide or antibody, is said to exhibit “specific binding” or “preferential binding” if it reacts or associates with a specific cell or substance more frequently, more rapidly, for a longer duration, and / or with a greater affinity than it reacts or associates with a substitute cell or substance. An antibody is said to “specifically bind” or “wire-bind” a target if it binds to a target with greater affinity, easier binding, and / or longer duration than it binds to other substances, for example, by a statistically significant amount. For example, an antibody that specifically or preferentially binds to a specific epitope is an antibody that binds to that specific epitope with greater affinity, easier binding, and / or longer duration than it binds to other epitopes. It should also be understood from this definition that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, “specific binding” or “preferential binding” does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, the mention of binding refers to preferential binding.

[0207] Immune binding generally refers to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen, such as, in an illustrative rather than restrictive manner, due to electrostatic, ionic, hydrophilic and / or hydrophobic attractive or repulsive forces, steric forces, hydrogen bonding, van der Waals forces, and other interactions, which give the immunoglobulin specificity for the antigen. The strength or affinity of an immune-binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, where a smaller Kd indicates a greater affinity. The immune-binding properties of a selected peptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, where those rates depend on the concentration of the complex's conjugate, the affinity of the interaction, and geometric parameters that also affect the rates in both directions. Thus, the "association rate constant" (Kon) and the "dissociation rate constant" (Koff) can be determined by calculating the concentrations of association and dissociation and the actual rates. Rates with Koff / Kon eliminate all parameters unrelated to affinity and are therefore equal to the dissociation constant Kd.

[0208] Antibodies can be prepared using any of a variety of techniques known to those skilled in the art. See, for example, Harlow and Lane, *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, 1988. Monoclonal antibodies specific to peptides of interest can be prepared, for example, using Kohler and Milstein, *Eur. J. Immunol.*, 6:511-519, 1976, and modifications thereof. Methods for expressing human antibodies using transgenic animals such as mice are also included. See, for example, Neuberger et al., *Nature Biotechnology*, 14:826, 1996; Lonberg et al., *Handbook of Experimental Pharmacology*, 113:49-101, 1994; and Lonberg et al., *Internal Review of Immunology*, 13:65-93, 1995. Specific examples include those from... of Platform (see, for example, U.S. Patent No. 6,596,541).

[0209] Antibodies can also be generated or identified using phage display libraries or yeast display libraries (see, for example, U.S. Patent No. 7,244,592; Chao et al., *Nature Protocols*, 1:755-768, 2006). Non-limiting examples of usable libraries include cloned or synthetic libraries, such as the Human Combinatorial Antibody Library (HuCAL), in which the structural diversity of the human antibody profile is represented by genes encoding seven heavy chain and seven light chain variable regions. Combinations of these genes produce 49 frames in the master library. Large-scale human antibody profiles can be replicated by superimposing highly variable gene cassettes (CDRs = complementarity-determining regions) onto these frames. Human libraries designed with human donor-derived fragments encoding light chain variable regions, heavy chain CDR-3, synthetic DNA encoding diversity in heavy chain CDR-1, and synthetic DNA encoding diversity in heavy chain CDR-2 are also included. Other libraries suitable for use will be apparent to those skilled in the art.

[0210] In some embodiments, antibodies and their antigen-binding fragments as described herein comprise heavy and light chain CDR groups, respectively inserted between heavy and light chain framework region (FR) groups, which provide support for the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR group" refers to three hypervariable regions of the V region of the heavy or light chain. These regions are designated "CDR1," "CDR2," and "CDR3," respectively, starting from the N-terminus of the heavy or light chain. Thus, the antigen-binding site comprises six CDRs, including CDR groups from each of the V regions of the heavy and light chains. Peptides comprising a single CDR (e.g., CDR1, CDR2, or CDR3) are referred to herein as "molecular recognition units." Crystallographic analysis of various antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0211] As used herein, the term "FR group" refers to the four flanking amino acid sequences of the CDR within the CDR group that frames the V region of the heavy or light chain. Some FR residues can contact the bound antigen; however, the FRs are primarily responsible for folding the V region into the antigen-binding site, specifically the FR residues directly adjacent to the CDR. Within the FR, certain amino acid collection and certain structural features are highly conserved. In this respect, all V region sequences contain an internal disulfide ring with approximately 90 amino acid residues. When the V region folds into the binding site, the CDR is presented as a prominent loop motif forming the antigen-binding surface. It is generally recognized that there are conserved structural regions of the FR that shape the folding shape of the CDR ring into certain "canonical" structures—regardless of the exact CDR amino acid sequence. Furthermore, certain FR residues are known to participate in non-covalent contacts that stabilize the interaction between the antibody heavy and light chains.

[0212] The structure and location of immunoglobulin variable domains can be determined by referring to Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, 1987, and its updates.

[0213] It also includes “monoclonal” antibodies, which refers to a homogeneous group of antibodies, wherein a monoclonal antibody includes the amino acids (naturally and non-naturally present) involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, targeting a single epitope. The term “monoclonal antibody” includes not only complete and full-length monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab')2, Fv), single chains (ScFv), their variants, fusion proteins including antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified conformation of immunoglobulin molecules including antigen-binding fragments (epitope recognition sites) with the desired specificity and the ability to bind epitopes. It is not intended to limit the source of antibodies or the manner in which they are formed (e.g., via hybridoma, phage selection, recombinant expression, transgenic animals). The term includes whole immunoglobulins as well as the fragments described above under the definition of “antibody.”

[0214] Papain, a proteolytic enzyme, preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each comprise a covalent heterodimer containing an intact antigen-binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including an F(ab')2 fragment comprising two antigen-binding sites. Fv fragments for use according to certain embodiments of the invention can be produced by preferential proteolytic cleavage of IgM and, in small amounts, IgG or IgA immunoglobulin molecules. However, more generally, Fv fragments are derived using recombinant techniques known in the art. Fv fragments comprise a non-covalent VH::VL heterodimer containing antigen-binding sites that retain the ability to recognize and bind to many native antibody molecules. See Inbar et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS USA.), 69:2659-2662, 1972; Hochman et al., Biochem., 15:2706-2710, 1976; and Ehrlich et al., Biochem., 19:4091-4096, 1980.

[0215] In some embodiments, single-chain Fv or scFV antibodies are envisioned. For example, k-body (Ill et al., *Protein Engineering*, 10:949-57, 1997); mini-body (Martin et al., *Journal of the European Society for Molecular Biology*, 13:5305-9, 1994); dimorph (Holliger et al., *Proceedings of the National Academy of Sciences*, 90:6444-8, 1993); or Janusins ​​(Traunecker et al., *Journal of the European Society for Molecular Biology*, 10:3655-59, 1991; and Traunecker et al., *Int.J. Cancer Suppl.*, 7:51-52, 1992) can be prepared using standard molecular biology techniques following the teachings of this application regarding the selection of antibodies with desired specificity.

[0216] Single-chain Fv (sFv) polypeptides are covalently linked VH::VL heterodimers expressed via gene fusion containing VH and VL encoding genes linked by peptide-encoding linker groups. (Huston et al., Proceedings of the National Academy of Sciences, 85(16):5879-5883, 1988). Various methods have been described to identify the chemical structures used to convert naturally polymerized but chemically separated light and heavy polypeptide chains from the antibody V region into sFv molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Patent No. 4,946,778 to Ladner et al.

[0217] In some embodiments, antibodies as described herein are in the form of "dimers". A dimer is a multimer of polypeptides, each polypeptide comprising a first domain and a second domain, the first domain comprising a binding domain for the immunoglobulin light chain and the second domain comprising a binding domain for the immunoglobulin heavy chain, the two domains being linked (e.g., via a peptide linker) but not associating with each other to form an antigen-binding site: an antigen-binding site is formed by associating the first domain of one polypeptide within the dimer with the second domain of another polypeptide within the dimer (WO94 / 13804). The dAb fragment of the antibody consists of a VH domain (Ward et al., *Nature*, 341:544-546, 1989). Dimers and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO94 / 13804; and Holliger et al., *Proceedings of the National Academy of Sciences*, 90:6444-6448, 1993)).

[0218] It also includes miniatures of scFvs linked to the CH3 domain (see Hu et al., Cancer Research, 56:3055-3061, 1996). See also Ward et al., Nature, 341:544-546, 1989; Bird et al., Science, 242:423-426, 1988; Huston et al., Proceedings of the National Academy of Sciences, 85:5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; and Reiter et al., Nature Biotech, 14:1239-1245, 1996.

[0219] When using bispecific antibodies, these antibodies can be conventional bispecific antibodies that can be manufactured by various methods (Holliger and Winter, Current Opinion Biotechnol., 4:446-449, 1993), such as those prepared chemically or derived from hybridoma cells, or can be any of the bispecific antibody fragments mentioned above. Dimers and scFvs without Fc regions can be constructed using only the variable domain, potentially reducing the impact of anti-idiotype reactions.

[0220] Bispecific dimers can also be particularly useful as a contrast to intact bispecific antibodies because they can be readily constructed and expressed in *E. coli*. Phage display (WO 94 / 13804) can be used to easily select dimers (and many other peptides, such as antibody fragments) with suitable binding specificity from a library. If one arm of the dimer remains constant, for example, with specificity against antigen X, a library with variations in the other arm can be generated and antibodies with suitable specificity selected. Intact bispecific antibodies can be produced using a knocks-in-holes engineering method (knobs-into-holes et al., *Protein Engineering*, 9:616-621, 1996).

[0221] In some embodiments, the antibodies described herein can be used as... Provided in a specific format. This refers to IgG4 antibodies with the hinge region removed (see GenMab Utrecht, Netherlands; also see, for example, US20090226421). This antibody technology produces stable, smaller antibody forms with a longer therapeutic window than currently expected for smaller antibody forms. IgG4 antibodies are considered inert and therefore do not interact with the immune system. Intact human IgG4 antibodies can be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments with different stability properties compared to the corresponding intact IgG4 (GenMab, Utrecht). Dividing the IgG4 molecule allows leaving only one region in place that can bind homologous antigens (e.g., disease targets). Up, and Therefore, it binds monovalently to only one site on the target cell. For certain cancer cell surface antigens, this monovalent binding may not stimulate cancer cells to grow as we would see with bivalent antibodies that have the same antigen specificity, and therefore... Technology can provide treatment options for some types of cancer that may be difficult to treat, in order to treat conventional antibodies. When treating certain forms of cancer, The small size may have significant advantages, allowing for better distribution of molecules in larger solid tumors and potentially increasing therapeutic efficacy.

[0222] In some embodiments, the antibodies provided herein may be in the form of nanobodies. Minibodies are encoded by a single gene and are efficiently produced in virtually all prokaryotic and eukaryotic hosts, such as *Escherichia coli* (see U.S. Patent No. 6,765,087), molds (e.g., *Aspergillus* or *Trichoderma*), and yeasts (e.g., *Saccharomyces*, *Kluyveromyces*, *Hansenula*, or *Pichia pastoris* (see U.S. Patent No. 6,838,254). The production process is scalable, and multiple kilograms of nanobodies have been produced. Nanobodies can be formulated into ready-to-use solutions with long shelf lives. A monoclonal method (see WO 06 / 079372) is a patented method for producing nanobodies against a desired target based on automated, high-throughput selection of B cells.

[0223] In some embodiments, the antibody or its antigen-binding fragment is humanized. These embodiments relate to chimeric molecules typically prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and a residual immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise an intact variable domain fused to a constant domain or a CDR grafted only to an appropriate frame region within the variable domain. The epitope binding site may be wild-type or modified by substitution of one or more amino acids. This eliminates the constant region of the immunogen in the human individual, but the possibility of an immune response to a foreign variable region remains (LoBuglio et al., Proceedings of the National Academy of Sciences, 86:4220-4224, 1989; Queen et al., Proceedings of the National Academy of Sciences, 86:10029-10033, 1988; Riechmann et al., Nature, 332:323-327, 1988). Illustrative methods for humanized antibodies include those described in U.S. Patent No. 7,462,697.

[0224] Another approach focuses not only on providing human-derived constant regions but also on modifying variable regions to reshape them as closely as possible to human form. Variable regions of both heavy and light chains are known to contain three complementary determinant regions (CDRs), which vary in response to the epitope in question and determine binding capacity, and are side-mounted by four frame regions (FRs), which are relatively conserved in a given species and presumably provide scaffolding for the CDRs. When preparing non-human antibodies for a specific epitope, the variable regions can be “reshaped” or “humanized” by grafting CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this method to various antibodies has been reported by the following: Sato et al., Cancer Research, 53:851-856, 1993; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988; Kettleborough et al., Protein Engineering, 4:773-3783, 1991; Maeda et al., Human Antibody Hybridoma. Hybridoma, 2:124-134, 1991; Gorman et al., Proceedings of the National Academy of Sciences, 88:4181-4185, 1991; Tempest et al., Bio / Technology, 9:266-271, 1991; Co et al., Proceedings of the National Academy of Sciences, 88:2869-2873, 1991; Carter et al., Proceedings of the National Academy of Sciences, 89:4285-4289, 1992; and Co et al., Journal of Immunology, 148:1149-1154, 1992. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) modified with respect to the original antibody, also referred to as "derived from" one or more CDRs from the original antibody.

[0225] In some embodiments, the antibody may be a chimeric antibody. In this regard, a chimeric antibody comprises an antigen-binding fragment operatively linked to or otherwise fused to a heterologous Fc portion of a different antibody. In some embodiments, the heterologous Fc domain is human. In other embodiments, the heterologous Fc domain may be derived from different Ig classes derived from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may include one or more CH2 and CH3 domains from different Ig classes. As described above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody may include only one or more of the CDRs of the antibodies described herein (e.g., one, two, three, four, five, or six CDRs of the antibodies described herein), or may include the entire variable domain (VL, VH, or both).

[0226] In some embodiments, the agent is or includes a "ligand" of an immune checkpoint molecule, such as a natural ligand. A "ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) for biological purposes and comprises a "protein ligand" that typically generates a signal by binding to a site on a target molecule or target protein. Thus, some agents are natural protein ligands that bind to immune checkpoint molecules and generate a signal. "Modified ligands" are also included, such as protein ligands fused to a pharmacokinetic modifier, for example, an Fc domain derived from an immunoglobulin.

[0227] In some embodiments, the agent is a "small molecule," which refers to an organic compound that is synthetically or biologically derived (biomolecule) but is not typically a polymer. Organic compounds refer to a broad class of chemical compounds whose molecules contain carbon, typically excluding chemical compounds containing only carbonates, simple carbon oxides, or cyanides. "Biomolecule" generally refers to organic molecules produced by living organisms, including large polymers (biopolymers) such as peptides, polysaccharides, and nucleic acids, as well as small molecules such as primary and secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerides, vitamins, and hormones. "Polymer" generally refers to a large molecule or macromolecule comprising repeating structural units typically linked by covalent chemical bonds.

[0228] In some embodiments, the small molecules have a molecular weight of about or less than about 1,000 Daltons to 2,000 Daltons, typically between about 300 Daltons and 700 Daltons, and include about or less than about 50 Daltons, 100 Daltons, 150 Daltons, 200 Daltons, 250 Daltons, 300 Daltons, 350 Daltons, 400 Daltons, 450 Daltons, 500 Daltons, 550 Daltons, 500 Daltons, 650 Daltons, 600 Daltons, 750 Daltons, 700 Daltons, 850 Daltons, 800 Daltons, 950 Daltons, 1,000 Daltons, or 2,000 Daltons.

[0229] Certain small molecules may possess the “specific binding” property described herein for peptides, such as antibodies. For example, in some embodiments, small molecules specifically bind to targets with an affinity (Kd) of about, at least about, or less than about the following values, such as immune checkpoint molecules: 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 11nM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, 20nM, 21nM, 22nM, 23nM, 24nM, 25nM, 26nM, 27nM, 28nM, 29nM, 30nM, 40nM, or 50nM.

[0230] In some embodiments, an immune checkpoint modulator is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include: programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), programmed death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), T-cell activation V-domain Ig repressor (VISTA), B and T lymphocyte attenuator (BTLA), CD160, and a T-cell immune receptor having Ig and ITIM domains (TIGIT).

[0231] In some embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, whose targeting has been shown to be the restoration of immune function in the tumor environment (see, for example, Phillips et al., *International Journal of Immunology*, 27:39-46, 2015). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and expressed on T cells and progenitor B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 acts as a suppressive immune checkpoint molecule, for example, by reducing or preventing T cell activation, thereby reducing autoimmune diseases and increasing self-tolerance. The inhibitory effect of PD-1 is accomplished at least in part through a dual mechanism of promoting apoptosis of antigen-specific T cells in lymph nodes and reducing apoptosis of regulatory T cells (suppressive T cells). Some examples of PD-1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1 and reduce its immunosuppressive activity, such as its downstream signaling pathways or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, and pidilizumab, as well as their antigen-binding fragments.

[0232] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As described above, PD-L1 is one of the natural ligands of the PD-1 receptor. General examples of PD-L1 antagonists or inhibitors include one or more of those that specifically bind to PD-L1 and reduce its immunosuppressive activity, such as antibodies or antigen-binding fragments or small molecules that bind to the PD-1 receptor. Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avermab (MSB0010718C), and duvalumab (MEDI4736) and their antigen-binding fragments.

[0233] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As described above, PD-L2 is one of the natural ligands of the PD-1 receptor. General examples of PD-L2 antagonists or inhibitors include one or more antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L2 and reduce its immunosuppressive activity, such as those that bind to the PD-1 receptor.

[0234] In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (differentiation cluster 152), is a protein receptor that acts as a suppressive immune checkpoint molecule, for example, by transmitting an inhibitory signal to a T cell when the T cell binds to CD80 or CD86 on the surface of an antigen-presenting cell. General examples of CTLA-4 antagonists or inhibitors comprise antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Specific examples comprise the antibodies ipilimumab and trimemumab, and their antigen-binding fragments. It is believed that at least some of the activity of ipilimumab is mediated by antibody-dependent cell-mediated cytotoxic (ADCC) killing of Tregs, which inhibit CTLA-4. Therefore, the ability of ADI to downregulate the inhibitory factor Tregs is parallel to the ability of certain CTLA-4 antagonists or inhibitors, suggesting that ADI may be useful not only in the same case as a CTLA-4 inhibitor (e.g., ipilimumab) but also in combination with said CTLA-4 inhibitors.

[0235] In some embodiments, the agent is an IDO antagonist or inhibitor or a TDO antagonist or inhibitor. IDOs and TDOs are enzymes that catabolize tryptophan and have immunosuppressive properties. For example, IDOs are known to inhibit T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. General examples of IDO and TDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDOs or TDOs (see, for example, Platten et al., Frontiers in Immunology, 5:673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indomethacin (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norhalman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadorestans (see, for example, Sheridan, Nature Biotechnology, 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, for example, Pilotte et al., Proceedings of the National Academy of Sciences, 109:2497-2502, 2012).

[0236] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by triggering cell death upon interaction with its ligand, galactagogue-9. TIM-3 contributes to a suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (see, for example, Li et al., *Acta Oncol.*, 54:1706-13, 2015). General examples of TIM-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3 and reduce or inhibit its immunosuppressive activity.

[0237] In some embodiments, the agent is a LAG-3 antagonist or inhibitor. Lymphocyte activation gene-3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1 (see, for example, Workman and Vignali, *European Journal of Immunology*, 33:970-9, 2003; and Workman et al., *Journal of Immunology*, 172:5450–5, 2004), and has been reported for its Treg-suppressive function (see, for example, Huang et al., *Immunity*, 21:503-13, 2004). LAG3 also maintains CD8+ T cells in a tolerant state and, in combination with PD-1, maintains CD8 T cell depletion. General examples of LAG-3 antagonists or inhibitors include one or more antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and inhibit its immunosuppressive activity. Specific examples include antibody BMS-986016 and its antigen-binding fragment.

[0238] In some embodiments, the agent is a VISTA antagonist or inhibitor. V-domain Ig repressor of T cell activation (VISTA) is primarily expressed on hematopoietic cells and is a suppressive immune checkpoint regulator that inhibits T cell activation, induces Foxp3 expression, and is highly expressed within the tumor microenvironment, where it suppresses anti-tumor T cell responses (see, for example, Lines et al., Cancer Research, 74:1924-32, 2014). Typical examples of VISTA antagonists or inhibitors include one or more antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA and reduce its immunosuppressive activity.

[0239] In some embodiments, the agent is a BTLA antagonist or inhibitor. B and T lymphocyte attenuator (BTLA; CD272) expression is induced during T cell activation, and it suppresses T cells by interacting with tumor necrosis family receptors (TNF-R) and the B7 cell surface receptor family. BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as a herpesvirus entry mediator (HVEM). The BTLA-HVEM complex negatively regulates T cell immune responses, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, for example, Derré et al., *Journal of Clinical Investigation (J ClinInvest)*, 120:157–67, 2009). General examples of BTLA antagonists or inhibitors comprise one or more antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce its immunosuppressive activity.

[0240] In some embodiments, the agent is an HVEM antagonist or inhibitor, such as an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD160. General examples of HVEM antagonists or inhibitors include one or more antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM, optionally reduce HVEM / BTLA and / or HVEM / CD160 interactions, and thereby reduce the immunosuppressive activity of HVEM.

[0241] In some embodiments, the agent is a CD160 antagonist or inhibitor, such as an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. General examples of CD160 antagonists or inhibitors include one or more antibodies or antigen-binding fragments or small molecules that specifically bind to CD160, optionally reduce CD160 / HVEM interaction, and thereby reduce or inhibit their immunosuppressive activity.

[0242] In some embodiments, the agent is a TIGIT antagonist or inhibitor. T-cell Ig and ITIM domains (TIGIT) are found on the surface of various lymphocytes and act as co-inhibitory receptors for inhibiting antitumor immunity, for example, through Tregs (Kurtulus et al., *Journal of Clinical Investigation*, 125:4053-4062, 2015). Typical examples of TIGIT antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT and reduce its immunosuppressive activity (see, for example, Johnston et al., *Cancer Cell*, 26:923-37, 2014).

[0243] In some embodiments, the immune checkpoint modulator is an agonist of one or more stimulating immune checkpoint molecules. Exemplary stimulating immune checkpoint molecules include OX40, CD40, glucocorticoid-induced TNFR family-associated gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0244] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the expansion of effector T cells and memory T cells and inhibits the differentiation and activity of regulatory T cells (see, for example, Croft et al., *Immunological Reviews*, 229:173–91, 2009). Its ligand is OX40L (CD252). Because OX40 signaling affects T cell activation and survival, it plays an important role in initiating antitumor immune responses in lymph nodes and maintaining antitumor immune responses in the tumor microenvironment. General examples of OX40 agonists include one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and increase its immunostimulatory activity. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (mouse OX4 agonist), and MEDI6383 (OX40 agonist) and their antigen-binding fragments.

[0245] In some embodiments, the agent is a CD40 agonist. CD40 is expressed on antigen-presenting cells (APCs) and some malignancies. Its ligand is CD40L (CD154). On APCs, this linkage leads to upregulation of co-stimulatory molecules, potentially bypassing the need for T-cell help in anti-tumor immune responses. CD40 agonist therapy plays an important role in APC maturation and its migration from tumors to lymph nodes, resulting in enhanced antigen presentation and T-cell activation. Anti-CD40 agonist antibodies generate substantial responses and durable anti-cancer immunity in animal models, an effect at least partially mediated by cytotoxic T cells (see, for example, Johnson et al., *Clinical Cancer Research*, 21:1321-1328, 2015; and Vonderheide and Glennie, *Clinical Cancer Research*, 19:1035-43, 2013). General examples of CD40 agonists comprise one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD40 and increase its immunostimulatory activity. Specific examples include CP-870, 893, dasizumab, Chi Lob7 / 4, ADC-1013, CD40L, rhCD40L, and their antigen-binding fragments.

[0246] In some embodiments, the agent is a GITR agonist. Glucocorticoid-induced TNFR family-associated genes (GITRs) increase T cell proliferation, inhibit the suppressive activity of Tregs, and prolong the survival of T effector cells. GITR agonists have been shown to promote antitumor responses by depriving Treg profile stability (see, for example, Schaer et al., Cancer Immunol Res., 1:320–31, 2013). These different mechanisms demonstrate the important role of GITRs in initiating immune responses in lymph nodes and in maintaining immune responses in tumor tissues. The ligand is GITRL. General examples of GITR agonists include one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITRs and increase their immunostimulatory activity. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and their antigen-binding fragments.

[0247] In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. General examples of CD137 agonists comprise one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and increase its immunostimulatory activity. Specific examples comprise a CD137 (or 4-1BB) ligand (see, for example, Shao and Schwarz, *Journal of Leukocyte Biology*, 89:21-9, 2011) and the antibody utomomab, which comprises its antigen-binding fragment.

[0248] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific amplification of naive T cells and contributes to the long-term maintenance of T cell memory and T cell immunity. Its ligand is CD70. Targeting of human CD27 with agonist antibodies stimulates T cell activation and anti-tumor immunity (see, for example, Thomas et al., *Oncoimmunology*, 2014; 3:e27255.doi:10.4161 / onci.27255; and He et al., *Journal of Immunology*, 191:4174-83, 2013). General examples of CD27 agonists comprise one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27 and increase its immunostimulatory activity. Specific examples include CD70 and the antibodies valilumab and CDX-1127(1F5), which contain their antigen-binding fragments.

[0249] In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed CD4+ T cells and some CD8+ T cells. Its ligands include CD80 and CD86, and its stimulation increases T cell proliferation. General examples of CD28 agonists include one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and increase its immunostimulatory activity. Specific examples include CD80, CD86, antibody TAB08, and its antigen-binding fragment.

[0250] In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and unlike TIGIT, binding to CD226 enhances T cell activity (see, for example, Kurtulus et al., *Journal of Clinical Research*, 125:4053-4062, 2015; Bottino et al., *Journal of Experimental Medicine*, 1984:557-567, 2003; and Tahara-Hanaoka et al., *International Journal of Immunology*, 16:533-538, 2004). General examples of CD226 agonists include one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD226 and increase its immunostimulatory activity (e.g., CD112, CD155).

[0251] In some embodiments, the agent is an HVEM agonist. Herpesvirus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF receptor superfamily. HVEM is found on a variety of cells including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T cells and is downregulated upon activation. HVEM signaling has been shown to play an important role in the early stages of T cell activation and during the expansion of tumor-specific lymphocyte populations in lymph nodes. General examples of HVEM agonists include one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM and increase its immunostimulatory activity.

[0252] In some embodiments, the cancer immunotherapy agent is a cancer vaccine. The efficacy of a cancer vaccine is limited by the activity of Tregs in the subject, and the ability of arginine-depleting agents such as ADI to reduce Tregs (among other properties described herein) can be used to enhance the efficacy of any type of cancer vaccine. Exemplary cancer vaccines include oncophage, human papillomavirus (HPV) vaccines such as Gardasil or Cervarix, hepatitis B vaccines such as Engerix-B, Recombivax HB, or Twinrix, and sipuleucel-T (Provinci). In some embodiments, the cancer vaccine includes or utilizes one or more cancer antigens or cancer-associated antigens. Exemplary cancer antigens include, but are not limited to, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, and C. D40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tendinin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclase C, NY-ESO-1, p5 3. Survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein α (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family Member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), liver regeneration phosphatase 3 (PRL-3), prostate acid phosphatase, Lewis-Y antigen, GD2 (bisialidylganglioside expressed on neuroectodermal tumors), phosphatidylinositol polysaccharide-3 (GPC3), and mesothelin.

[0253] In some embodiments, the cancer immunotherapy agent is an oncolytic virus. Oncolytic viruses are viruses that preferentially infect and kill cancer cells. They include naturally occurring and artificially created or engineered oncolytic viruses. Similar to the above, the efficacy of oncolytic viruses is limited by the activity of Tregs in the subject, and the ability of arginine-depleting agents such as ADI to reduce Tregs (among other properties described herein) can be used to enhance the efficacy of any type of oncolytic virus. Most oncolytic viruses are engineered for tumor selectivity, although naturally occurring examples exist such as reovirus and SVV-001 Seneca Valley virus. General examples of oncolytic viruses include VSV, poliovirus, reovirus, Seneca virus, and RIGVIR, as well as their engineered versions. Non-limiting examples of oncolytic viruses include herpes simplex virus (HSV) and its engineered versions, talimogene laherparepvec (T-VEC), and Coxsackievirus A21 (CAVATAK). TM ), Oncorine(H101), pelareorep Seneca Valley virus (NTX-010), Seneca virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS and DNX-2401, etc.

[0254] In some embodiments, the cancer immunotherapy agent is a cytokine. Exemplary cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0255] In some embodiments, the cancer immunotherapy agent is a cell-based immunotherapy, such as T-cell-based adoptive immunotherapy. In some embodiments, the cell-based immunotherapy includes cancer antigen-specific T cells, optionally ex vivo derived T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells. In a particular embodiment, the CAR-modified T cells target CD-19 (see, for example, Maude et al., Hematology, 125:4017-4023, 2015).

[0256] In specific embodiments, the cancer immunotherapy agent is selected from one or more of IDO antagonists, CD20 antagonists, β-Raf antagonists, IL-2, GM-CSF, and oncolytic viruses. In specific embodiments, the IDO antagonist is indomod, and / or the CD20 agonist is rituximab, and / or the β-Raf antagonist is vemurafenib, and / or the oncolytic virus is talimogenelaherparepvec (T-VEC) or Coxsackievirus A21 (CAVATAK). TM ).

[0257] Those skilled in the art will understand that the various cancer immunotherapy agents described herein can be combined with any one or more of the various arginine depletion agents described herein and used in accordance with any one or more of the methods or compositions described herein.

[0258] How to use

[0259] As described above, embodiments of this disclosure relate to the discovery that arginine depletion agents possess unexpected immunomodulatory properties. For example, the exemplary arginine depletion agent ADI-PEG 20 has been shown in vitro to reduce immunosuppressive regulatory T cells (Treg cells), which would otherwise minimize the magnitude and duration of inflammatory responses, particularly antitumor responses. It has also been shown to neutralize the depletion of effector T cells (Teff cells) in vitro and increase T cell tumor infiltration in vivo. Therefore, arginine depletion agents such as ADI can be used to enhance antitumor T cell responses, for example, by enhancing T cell initiation and transport to the tumor microenvironment, and thereby boosting other cancer immunotherapies.

[0260] Therefore, some embodiments include methods for treating and alleviating symptoms of cancer or inhibiting its progression in subjects in need, including administering to the subject (a) an arginine-depleting agent that converts arginine to citrulline; and (b) a cancer immunotherapy agent. Arginine-depleting agents and cancer immunotherapy agents, in combination thereof, are described elsewhere herein.

[0261] In some cases, (a) the arginine depleting agent and (b) the cancer immunotherapy agent are administered separately, for example, in separate compositions and at different times. In some embodiments, (a) the arginine depleting agent and (b) the cancer immunotherapy agent are administered simultaneously as part of the same composition.

[0262] In some embodiments, administration of an arginine-depleting agent (e.g., ADI, ADI-PEG, ADI-PEG 20), for example, in the tumor microenvironment of a subject, reduces immunosuppressive regulatory T cells (Treg cells). In some cases, relative to a control, such as a control composition (without the agent) or a control time point, such as an earlier time point, Tregs are reduced by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more.

[0263] In some embodiments, administration of an arginine depletion agent (e.g., ADI, ADI-PEG, ADI-PEG 20), for example, in the tumor microenvironment of a subject, reduces or mitigates the depletion of effector T cells (Teff cells). In some cases, relative to a control, such as a control composition (without the agent) or a control time point, such as an earlier time point, Teff cell depletion is reduced by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more.

[0264] In some embodiments, administration of an arginine-depleting agent (e.g., ADI, ADI-PEG, ADI-PEG 20) increases tumor infiltration of T cells. In some cases, relative to a control, such as a control composition (without the agent) or a control time point, such as an earlier time point, tumor infiltration of T cells increases by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. Tumor infiltration can be measured according to conventional techniques in the art (see, for example, Example 2).

[0265] In some cases, administration of arginine-depleting agents (e.g., ADI, ADI-PEG, ADI-PEG 20) enhances effector T cell activation (CD69 expression) and / or reduces the upregulation of immunosuppressive receptors PD-1 and CTLA-4 on T cells. In some cases, relative to a control, such as a control composition (without the agent) or a control time point, such as an earlier time point, CD69 expression on effector T cells is increased by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. In some cases, relative to a control, such as a control composition (without the agent) or a control time point, such as an earlier time point, PD-1 expression on T cells is decreased by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. In some cases, relative to a control, such as a control composition (without the drug) or a control time point, such as an earlier time point, CTLA-4 expression on T cells is reduced by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more.

[0266] In some embodiments, the combination therapies described herein increase median survival by 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 weeks, or longer. In some embodiments, the combination therapies described herein increase median survival by 1 year, 2 years, 3 years, or longer. In some embodiments, the combination therapies described herein increase progression-free survival by 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or longer. In some embodiments, the combination therapies described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.

[0267] In some embodiments, the administered composition is sufficient to induce tumor regression, as indicated by a statistically significant reduction in tumor survival, for example, a reduction in tumor mass of at least 10%, 20%, 30%, 40%, 50%, or more, or as indicated by a change in scan size (e.g., a statistically significant reduction). In some embodiments, the administered combination is sufficient to stabilize the disease. In some embodiments, the administered combination is sufficient to alleviate the clinical relevance of symptoms of a specific disease indication known to a skilled clinician. In some embodiments, the arginine depletion agent increases, complements, or otherwise enhances the antitumor and / or immunostimulatory activity of the cancer immunotherapy agent relative to the cancer immunotherapy agent alone.

[0268] The methods and compositions described herein can be used to treat any type of cancer. In some embodiments, the cancer is selected from one or more of the following: hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myeloid leukemia, lymphoma, liver cancer, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma (e.g., astrocytoma, oligodendroglioma, ependymoma, or choroid plexus papilloma), glioblastoma multiforme (e.g., giant cell glioma or gliosarcoma), meningioma, pituitary adenoma, vestibular schwannoma, primary central nervous system lymphoma, primitive neuroectodermal tumor (neuroblastoma), non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, and gastric cancer.

[0269] Certain combinations contain arginine-depleting agents and PD-L1 antagonists or inhibitors, such as atezolizumab (MPDL3280A), avermab (MSB0010718C), and durvalumab (MEDI4736), for the treatment of one or more cancers selected from colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma.

[0270] Some specific combinations contain arginine-depleting agents and PD-1 antagonists, such as nivolumab, for the treatment of one or more cancers selected from Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer.

[0271] Specific combinations include arginine-depleting agents and PD-1 antagonists, such as pembrolizumab, for the treatment of one or more cancers selected from melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma.

[0272] Certain combinations contain arginine-depleting agents and CTLA-4 antagonists, such as ipilimumab and trimemumab, for the treatment of one or more cancers selected from melanoma, prostate cancer, lung cancer, and bladder cancer.

[0273] Some specific combinations contain arginine-depleting agents and IDO antagonists, such as indomod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norhalman, 9H-pyrido[3,4-b]indole), rosmarinic acid, or epaccaldostane, for the treatment of one or more cancers selected from metastatic breast cancer and brain cancer, optionally glioblastoma multiforme, glioma, gliosarcoma, or malignant brain tumors.

[0274] Certain combinations contain arginine-depleting agents and the cytokine INF-α for the treatment of melanoma, Kaposi's sarcoma, and blood cancers. Combinations also include arginine-depleting agents and IL-2 (such as adeleukin) for the treatment of metastatic renal cell carcinoma or metastatic melanoma.

[0275] Some specific combinations involve arginine-depleting agents and T-cell-based adoptive immunotherapy, such as CAR-modified T cells targeting CD19, for the treatment of blood cancers such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell tumors (see, for example, Maude et al., 2015, ibid.; Lorentzen and Straten, Scand J Immunol, 82:307-19, 2015; and Ramos et al., Cancer J, 20:112–118, 2014).

[0276] In some embodiments, as described above, administration of arginine-depleting agents (e.g., ADI, ADI-PEG, ADI-PEG20) reduces the upregulation of the immunosuppressive receptor CTLA-4 on T cells and downregulates Tregs. Without being limited by any single theory, this activity is at least partially parallel to the anti-CTLA4 monoclonal antibody ipilimumab. The antitumor activity of arginine depletion agents is therefore important. Thus, arginine depletion agents should be found for therapeutic use in clinical settings or in combinations similar to those identified against ipilimumab. Specific examples include administration of arginine depletion agents in combination with one or more IDO antagonists, CD20 antagonists, B-Raf antagonists, IL-2, GM-CSF, and oncolytic viruses. In specific embodiments, the IDO antagonist is indomod, the CD20 agonist is rituximab, the B-Raf antagonist is vemurafenib, and / or the oncolytic virus is talimogene laherparepvec (T-VEC) or Coxsackievirus A21 (CAVATAK). TM ).

[0277] Cancer treatments can be combined with other therapies. For example, the combination therapies described herein can be administered to subjects before, during, or after other therapeutic interventions, and include symptomatic care, radiation therapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic care includes the administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment, and vomiting, and the administration of anticonvulsants to reduce seizures. Radiation therapy includes whole-brain irradiation, fractionated radiation therapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with conventional surgery.

[0278] Methods for identifying subjects who have one or more of the diseases or conditions described herein are known in the art.

[0279] For in vivo use, such as for treating human diseases or for testing, the pharmaceutical agents described herein are typically incorporated into one or more pharmaceutical or therapeutic compositions prior to administration. In some cases, the pharmaceutical or therapeutic composition comprises a combination of one or more pharmaceutical agents described herein with a physiologically acceptable carrier or excipient.

[0280] To prepare a pharmaceutical composition, one or more effective or desired pharmaceutical agents are mixed with any drug carrier or excipient known to those skilled in the art to suit a particular agent and / or route of administration. The drug carrier may be liquid, semi-liquid, or solid. Solutions or suspensions for parenteral, intradermal, subcutaneous, or topical application may contain, for example, sterile diluents (such as water), saline solutions (e.g., phosphate-buffered saline; PBS), fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents (such as benzyl alcohol and methylparaben); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates, and phosphates). If administered intravenously (e.g., via IV infusion), a suitable carrier comprises physiological saline or phosphate-buffered saline (PBS), and a solution containing thickeners and solubilizers such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0281] The pharmaceutical agents described herein may be administered, either in their pure form or in a suitable pharmaceutical composition, via any acceptable method of pharmaceutical administration for similar purposes. Pharmaceutical compositions may be prepared by mixing a composition containing the pharmaceutical agent with a suitable physiologically acceptable carrier, diluent, or excipient, and may be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Furthermore, other active pharmaceutical ingredients (including other small molecules described elsewhere herein) and / or suitable excipients, such as salts, buffers, and stabilizers, may be present in the composition, but are not necessarily required to be present.

[0282] Administration can be achieved through various routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or local administration. The preferred route of administration depends on the nature of the condition to be treated or prevented. Specific embodiments include administration via intravenous infusion.

[0283] The carrier may contain, for example, pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or mammals exposed to them at the doses and concentrations used. Physiologically acceptable carriers are typically aqueous pH buffer solutions. Examples of physiologically acceptable carriers include buffers of phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or polysorbate 20 (TWEEN). TM Polyethylene glycol (PEG) and poloxamer (PLURONICS) TM Nonionic surfactants such as )

[0284] In some embodiments, one or more pharmaceutical agents may be encapsulated, for example, by coagulation techniques or by interfacial polymerization, in prepared microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Oslo A., (1980). The particles or liposomes may further include other therapeutic or diagnostic agents.

[0285] The precise dosage and duration of treatment are functions of the disease being treated and can be determined empirically using known testing protocols or inferred by testing the composition in model systems known in the art. Controlled clinical trials can also be conducted. The dosage may also vary depending on the severity of the condition to be alleviated. Pharmaceutical compositions are typically formulated and administered to achieve a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered once or divided into many smaller doses administered at intervals. For any given subject, a specific dosage regimen can be adjusted over time according to individual needs.

[0286] Therefore, typical routes of administration of these and related pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal administration. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. Pharmaceutical compositions according to certain embodiments of the invention are formulated such that the active ingredient contained therein is bioavailable when administered to a patient. Compositions administered to a subject or patient may be in the form of one or more dose units, wherein, for example, tablets may be single dose units, and containers of the aerosol form of the pharmaceutical agent described herein may contain multiple dose units. Practical methods for preparing such dosage forms are known or obvious to those skilled in the art; see, for example, *The Science and Practice of Pharmacy*, 20th edition (Philadelphia College of Medical Sciences, 2000). Compositions to be administered typically contain a therapeutically effective amount of the pharmaceutical agent described herein for the treatment of a disease or condition of concern.

[0287] The pharmaceutical composition may be in solid or liquid form. In one embodiment, the carrier is granular, and therefore the composition is, for example, in tablet or powder form. The carrier may be liquid, and the composition is, for example, an oral oil, an injectable liquid, or an aerosol, which may be used, for example, for inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension, and gel forms are included in the solid or liquid forms described herein.

[0288] As a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, sheets, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginate, sodium alginate, sodium carboxymethyl starch, corn starch, etc.; lubricants such as magnesium stearate or hydrogenated castor oil; glidants such as colloidal silica; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavorings; and colorants. When the pharmaceutical composition is in capsule form, for example, gelatin capsules, in addition to the substances of the types described above, the pharmaceutical composition may also contain liquid carriers such as polyethylene glycol or oils.

[0289] The pharmaceutical composition may be in liquid form, such as an elixir, syrup, solution, emulsion, or suspension. As two examples, the liquid may be for oral administration or for administration by injection. When for oral administration, in addition to the compounds of the present invention, preferred compositions may contain one or more sweeteners, preservatives, dyes / colorants, and flavor enhancers. Compositions intended for administration by injection may contain one or more of surfactants, preservatives, humectants, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0290] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following adjuvants: such as water for injection, saline solution, preferably physiological saline, Ringer's solution, sterile diluents of isotonic sodium chloride, such as synthetic monoglycerides or diglycerides of glycerol, polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as a solvent or suspension medium; 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 acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0291] Liquid pharmaceutical compositions intended for parenteral or oral administration should contain an amount of the drug to obtain a suitable dosage. Typically, this amount is at least 0.01% of the drug of interest in the composition. When intended for oral administration, this amount can vary from 0.1% to about 70% by weight of the composition. Some oral pharmaceutical compositions contain about 4% to about 75% of the drug of interest. In some embodiments, pharmaceutical compositions and formulations according to the invention are prepared such that the parenteral dose unit contains 0.01% to 10% by weight of the drug of interest before dilution.

[0292] The pharmaceutical composition may be intended for topical application, in which case the carrier may suitably comprise a solution, emulsion, cream, or gel matrix. For example, the matrix may include one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. A thickener may be present in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device.

[0293] The pharmaceutical composition can be used, for example, in the form of a suppository for rectal administration, which dissolves in the rectum and releases the drug. Compositions for rectal administration may contain an oily matrix as a suitable, non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0294] Pharmaceutical compositions can comprise a variety of materials that alter the physical form of solid or liquid dosage units. For example, a composition may contain a material that forms a coating around the active ingredient. The coating material is typically inert and can be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. Pharmaceutical compositions in solid or liquid form may contain components that bind the drug, thereby facilitating the delivery of the compound. Suitable components that can function in this way include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0295] Pharmaceutical compositions can consist essentially of dosage units and can be administered as aerosols. The term aerosol is used to refer to a variety of systems, ranging from colloidal to systems consisting of pressurized packaging. Delivery can be made by liquefying or compressing gases or by a suitable pump system dispensing the active ingredient. Aerosols can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which together can form a kit. Preferred aerosols can be determined by those skilled in the art without extensive experimentation.

[0296] The compositions described herein can be prepared using a carrier that protects the pharmaceutical agent from rapid elimination from the body, such as a timed-release formulation or a coating. Such carriers include controlled-release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, polyorthoester, polylactic acid, and other substances known to those skilled in the art.

[0297] Pharmaceutical compositions can be prepared using methods well-known in the pharmaceutical industry. For example, a pharmaceutical composition for administration by injection may include one or more salts, buffers, and / or stabilizers, and form a solution together with sterile distilled water. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that interact nonvalently with a pharmaceutical agent to promote the dissolution or homogeneous suspension of the agent in an aqueous delivery system.

[0298] The composition may be administered at a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the specific compound used; the metabolic stability and duration of action of the compound; the patient's age, weight, general health, sex, and diet; the route and timing of administration; the excretion rate; the combination of drugs; the severity of the specific disease or condition; and the treatment the subject is receiving. In some cases, the therapeutically effective daily dose (for a 70 kg mammal) is from about 0.001 mg / kg (i.e., about 0.07 mg) to about 100 mg / kg (i.e., about 7.0 g); preferably, the therapeutically effective dose (for a 70 kg mammal) is from about 0.01 mg / kg (i.e., about 0.7 mg) to about 50 mg / kg (i.e., about 3.5 g); more preferably, the therapeutically effective dose (for a 70 kg mammal) is from about 1 mg / kg (i.e., about 70 mg) to about 25 mg / kg (i.e., about 1.75 g).

[0299] In some embodiments, the composition comprising ADI (e.g., ADI-PEG, ADI-PEG 20) has a pH of about 5 to about 9, about 6 to about 8, or about 6.5 to about 7.5. In some embodiments, the composition comprising ADI (e.g., ADI-PEG, ADI-PEG 20) has a pH of about 6.8 ± 1.0.

[0300] In some embodiments, the free PEG in the composition comprising an ADI (e.g., ADI-PEG, ADI-PEG 20) is 1% to 10%. In some embodiments, the free PEG 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 some embodiments, the unmodified ADI in the composition comprising ADI-PEG (e.g., ADI-PEG 20) 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%. Typically, the composition comprising an ADI (e.g., ADI-PEG, ADI-PEG 20) has less than or equal to about 4%, 3%, 2%, 1.5%, 1%, or 0.5% of total impurities. In some embodiments, the endotoxin limit meets the requirements stated in the USP, i.e., ≤50 EU / mL.

[0301] In some embodiments, the composition comprising ADI (e.g., ADI-PEG, ADI-PEG 20) contains more than about 90% free thiol groups. In some embodiments, the composition comprising ADI (e.g., ADI-PEG, ADI-PEG 20) contains about 91%, about 92%, about 93%, about 94%, or about 95%, about 96%, about 97%, about 98%, about 99%, or more free thiol groups.

[0302] In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a Km of about 0.1 μM or 0.5 μM to about 15 μM, or about 1 μM to about 12 μM, about 1 μM to about 10 μM, about 1.5 μM to about 9 μM, about 1.5 μM to about 8 μM, or about 1.5 μM to about 7 μM. In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a Km of about 1.0 μM to about 10 μM or about 1.5 μM to about 6.5 μM. In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a Km of about, at least about, or less than about 0.1 μM, about 0.5 μM, about 1.0 μM, about 1.5 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, or about 7 μM, or about 8 μM, or about 9 μM, or about 10 μM.

[0303] In some embodiments, the ADI in the composition (e.g., ADI-PEG, ADI-PEG 20) has a time of approximately 0.5 seconds. -1 About 80 seconds -1 or about 0.5 seconds -1 About 70 seconds -1 or about 0.5 seconds -1 About 60 seconds -1 or about 0.5 seconds -1 About 50 seconds -1 or about 0.5 seconds -1 About 40 seconds -1 or about 0.5 seconds -1 About 30 seconds -1 or about 0.5 seconds -1 About 20 seconds -1 or about 0.5 seconds -1 About 15 seconds -1 or from 0.5 seconds -1 About 80 seconds -1 or about 1 second -1 About 80 seconds -1 or about 5 seconds -1 About 80 seconds -1 or about 10 seconds -1 About 80 seconds -1 or about 20 seconds -1 About 80 seconds -1 or about 30 seconds -1 About 80 seconds -1 or about 40 seconds -1 About 80 seconds-1 or about 50 seconds -1 About 80 seconds -1 or about 60 seconds -1 About 80 seconds -1 or about 70 seconds -1 About 80 seconds -1 or about 1 second -1 Approximately 12 seconds -1 Approximately 1 second -1 About 10 seconds -1 Approximately 1.5 seconds -1 Approximately 9 seconds -1 Approximately 2 seconds -1 About 8 seconds -1 or about 2.5 seconds -1 About 7 seconds -1 The Kcat. In some embodiments, the ADI in the composition (e.g., ADI-PEG, ADI-PEG 20) has a Kcat of about 2.5 seconds. -1 Approximately 7.5 seconds -1 Kcat. In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a Kcat of about or at least about 2.5 seconds. -1 Approximately 3 seconds -1 Approximately 3.5 seconds -1 Approximately 4 seconds -1 Approximately 4.5 seconds -1 Approximately 5 seconds -1 Approximately 5.5 seconds -1 Approximately 6 seconds -1 Approximately 6.5 seconds -1 Approximately 7 seconds -1 Approximately 7.5 seconds -1 or about 8 seconds -1 Approximately 10 seconds -1 Approximately 15 seconds -1 Approximately 20 seconds -1 Approximately 25 seconds -1 Approximately 30 seconds -1 Approximately 35 seconds -1 Approximately 40 seconds -1 Approximately 45 seconds -1 Approximately 50 seconds -1 Approximately 55 seconds -1 Approximately 60 seconds -1 Approximately 65 seconds -1 Approximately 70 seconds -1 Approximately 75 seconds -1 Approximately 80 seconds -1 Approximately 85 seconds -1 Approximately 90 seconds -1 Approximately 95 seconds -1 or about 100 seconds -1 Kcat.

[0304] In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a conductivity (also referred to in the art as electrical conductivity) of about 5 mS / cm to about 20 mS / cm, or about 5 mS / cm to about 15 mS / cm, about 7 mS / cm to about 15 mS / cm, about 9 mS / cm to about 15 mS / cm, or about 10 mS / cm to about 15 mS / cm. In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a conductivity of about 9 mS / cm, about 10 mS / cm, about 11 mS / cm, about 12 mS / cm, about 13 mS / cm, about 14 mS / cm, or about 15 mS / cm. In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a conductivity of about 13 mS / cm ± 1.0 mS / cm.

[0305] In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a permeability of about 50 mOsm / kg to about 500 mOsm / kg, about 100 mOsm / kg to about 400 mOsm / kg, about 150 mOsm / kg to about 350 mOsm / kg, about 200 mOsm / kg to about 350 mOsm / kg, or about 250 mOsm / kg to about 350 mOsm / kg. In some embodiments, the ADI (e.g., ADI-PEG, ADI-PEG 20) in the composition has a permeability of about 300 ± 30 mOsm / kg.

[0306] In some embodiments, the protein concentration is about 8 to about 15 mg / mL. In some 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.

[0307] In some embodiments, the specific activity of the ADI (e.g., ADI-PEG, ADI-PEG 20) is about 5.0 to 90 IU / mg or about 5 to 55 IU / mg, wherein 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 potency is 100 ± 20 IU / mL. In some embodiments, the 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.

[0308] The combination therapies described herein may comprise the administration of a single drug dosage form comprising an arginine depleting agent and an immunotherapy agent (optionally having one or more additional active agents), or the administration of a composition comprising an arginine depleting agent and a cancer immunotherapy agent in its own separate drug dosage form. For example, the arginine depleting agent and cancer immunotherapy agent described herein may be administered to a patient together in a single oral dose composition such as tablets or capsules, or each agent may be administered in a separate oral dosage form. Similarly, the arginine depleting agent and cancer immunotherapy agent described herein may be administered to a patient together in a single parenteral dose composition, such as in saline solution or other physiologically acceptable solutions, or each agent may be administered in a separate parenteral dosage form. As another example, for cell-based therapies, the arginine depleting agent may be mixed with cells prior to administration, administered as part of a separate composition, or administered with the composition. When using separate dose formulations, the compositions may be administered substantially simultaneously, i.e., concurrently, or separately and alternately, i.e., sequentially and in any order; combination therapy should be understood to include all of these regimens.

[0309] This also includes in vitro or ex vivo uses of arginine depletion agents and related embodiments described herein. Typically, arginine depletion can be used, for example, to increase in vitro T cell activation and / or Treg downreg in tissue culture studies or as a primer for T cell-based immunotherapies. Therefore, some embodiments relate to methods for increasing T cell activation and / or Treg downreg in vitro or ex vivo, including incubating or contacting T cells with one or more arginine depletion agents, as described elsewhere herein. Methods for increasing T cell activation and / or Treg downreg in vitro or ex vivo also include incubating T cells in an arginine-free medium supplemented with citrulline. In some embodiments, the T cells are primary T cells, for example, isolated or obtained from a subject or patient.

[0310] Specific embodiments relate to a method of adoptive T-cell immunotherapy for treating cancer in a subject of need, comprising (a) incubating ex vivo-derived T cells (i) with an arginine-depleted ADI-PEG20 agent or (ii) in an arginine-free medium supplemented with citrulline; and (b) administering the ex vivo-derived T cells to the subject. See, for example, June, *Journal of Clinical Investigation*, 117:1466-1476, 2007; and Rosenberg and Restifo, *Science*, 348:62-68, 2015, for descriptions of adoptive T-cell immunotherapy. In some cases, the ex vivo-derived T cells are obtained from the subject to be treated. In some embodiments, arginine depletion enhances the efficacy of the adopted T-cell transfer.

[0311] Exemplary arginine depletion agents are described elsewhere in this document. Arginine-free culture media and citrulline supplements are commercially available (see, for example, Thermo Fisher Scientific). In some cases, arginine-free culture media comprise about 0.01 to 100 mM, or about, at least about, or no more than about 0.01, 0.05, 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, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 mM of citrulline.

[0312] Similarly, exemplary cancers treated with adoptive T-cell immunotherapy combined with arginine depletion include hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myeloid leukemia, lymphoma, liver cancer, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, and gastric cancer. Gliomas (e.g., astrocytomas, oligodendrogliomas, ependymomas, or choroid plexus papillomas), glioblastomas multiforme (e.g., giant cell gliomas or gliosarcomas), meningiomas, pituitary adenomas, vestibular schwannomas, primary central nervous system lymphomas, primitive neuroectodermal tumors (neuroblastomas), non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, and gastric cancer.

[0313] In some embodiments, T cells (e.g., ex vivo derived T cells) include cancer antigen-specific T cells. Cancer antigen-specific T cells can be expanded, enriched, modified, or otherwise prepared before, during, and / or after exposure to arginine depletion (e.g., contact with an arginine depletion agent, incubation in an arginine-free medium supplemented with citrulline). In certain cases, the cancer to be treated is bound to a cancer antigen; that is, the cancer antigen-specific T cells target or enrich at least one known antigen associated with the cancer to be treated. In some embodiments, the cancer antigen is selected from CD19, human Her2 / neu, Her1 / EGF receptor (EGFR, Her3, A33 antigen, B7H3, CD5, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, etc. CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tendinin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folic acid receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein α ( FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein The protein disulfide isomerase (PDI), liver regeneration phosphatase-3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (bisialidylganglioside expressed on neuroectodermal tumors), phosphatidylinositol polysaccharide-3 (GPC3), and mesothelin are selected from one or more of the following: protein disulfide isomerase (PDI), liver regeneration phosphatase-3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialotetrahexosylganglioside expressed on neuroectodermal tumors), phosphatidylinositol polysaccharide-3 (GPC3), and mesothelin. In some cases, cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR) modified T cells (e.g., targeting cancer antigens), T cell receptor (TCR) modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0314] It also includes a patient care kit comprising (a) one or more arginine-depleting agents that convert arginine to citrulline; and one or more cancer immunotherapy agents. In some kits, (a) and (b) are in separate compositions. In some kits, (a) and (b) are in the same composition.

[0315] The kit described herein may also contain one or more additional therapeutic agents or other components suitable for or intended for use in the therapeutic indication or for intended diagnostic applications. The kit described herein may also contain one or more syringes or other components required or desired to facilitate the intended delivery modality (e.g., stents, implantable reservoirs, etc.).

[0316] All publications, patent applications and granted patents cited in this specification are incorporated herein by reference as if each individual publication, patent application or granted patent were specifically and individually indicated to be incorporated by reference.

[0317] Although the invention has been described in detail by way of illustration and example for the purpose of clarity, it should be readily apparent to those skilled in the art, inspired by the teachings of this invention, that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration rather than limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially similar results.

[0318] Example

[0319] Example 1

[0320] ADI-PEG 20 regulates stimulated T cells rather than resting T cells.

[0321] The effects of ADI PEG-20 on T cells were tested, as described below.

[0322] reagents ADI PEG-20 is a recombinant protein cloned from Mycoplasma hominis, produced in E. coli and PEGylated with 20,000 MW PEG (see, for example, Feun and Savaraj, Research Drug Expert Review, July 2006; 15(7):815-22; Holtsberg et al., Journal of Controlled Release, April 23, 2002; 80(1-3):259-71; and Bomalaski et al., Preclinica (2003), Vol. 1, pp. 284-293). An inactivated ADI PEG-20 mutant (mutADI) was generated using C397A substitution.

[0323] Frozen healthy donor PBMCs were purchased from AllCells. PBMCs from two different donors were tested in three replicate experiments. PBMCs were thawed, washed, and allowed to stand overnight (18 hours). Cells were collected the following day, resuspended in AIM-V medium supplemented with Glutamax, and counted. The cell concentration was adjusted to 2 x 10⁶ cells / mL. 6 2 mL of cells were added to each well of a 6-well plate. PBMCs were treated with 0, 0.8, 4, or 20 nM ADI-PEG 20, with or without CD3 / CD28Dynabeads or PHA. CD3 / CD28Dynabeads were prepared according to the manufacturer's instructions. Cells were analyzed after 24, 48, and 72 hours of treatment.

[0324] Flow cytometry analysis Dynabeads were removed from the sample prior to flow cytometry analysis. Cells were blocked in human Fc blockade, stained with a live / dead fixative near-IR staining agent, and then stained with an appropriate antibody solution mixture. For effector T cell (Teff) analysis, the antibody mixture included: CD3-PE-Cy7, CD8-FITC, CD4-PerCP-Cy5.5, CD69-APC, and CD274-PE, CD279-PE, or CD152-PE. For regulatory T cell (Treg) analysis, the antibody mixture included CD3-PE-Cy7, CD4-PerCP-Cy5.5, CD25-APC, and CD127-PE, CD274-PE, CD279-PE, or CD152-PE. Immediately after washing the surface-stained samples, samples stained with the Teff antibody mixture were analyzed by flow cytometry. Simultaneously, samples stained with the Treg antibody mixture were permeabilized using a human Foxp3 buffer kit and intracellularly stained with FoxP3-FITC antibody. 50,000 events were acquired for each sample on a Guava EasyCyte 8HT (Millipore). Data acquisition and analysis were performed using InCyte software (Millipore). Monochromatic fluorophore controls were generated using an AbC total antibody compensation bead kit and an ArC amine reactive bead kit, and spectral overlap compensation was performed on the cytometer.

[0325] Gating strategy.Single-cell gates were determined using FSC-H versus FSC-A curves. Fluorescence minus one (FMO) controls were used to establish other analytical gates. Single-cell gating was performed on live cells using a viable / dead immobilizable near-IR staining agent, and further analysis of single-cell and live cells was conducted to determine the presence of CD3T cell markers. CD4+ and CD8+ gates were established in CD3+ live and single-cell populations, and each of these populations was analyzed to determine the presence of other markers CD69, CD274, CD279, and CD152. For Treg analysis, single and viable CD3+CD4+ cells were analyzed to determine the presence of markers CD25, FoxP3, CD127, CD274, CD279, and CD152.

[0326] Experiment Overview. PBMCs were left to stand overnight and treated for 24, 48, or 72 hours with 0, 0.8, 4, or 20 nM ADI-PEG 20, with or without CD3 / CD28 microbeads or PHA. To control for any potential effects of PEG 20 and to verify that the observed therapeutic outcomes of ADI-PEG 20 were mediated by ADI activity, the effect of a 20 nM inactivated ADI mutant (mutADI) was evaluated as a control (ADI with C397A substitution PEGylated with 20K PEG).

[0327] After incubation, cells were collected, stained to express surface markers, and analyzed using flow cytometry and immunocellular phenotyping. Three independent experiments were performed using PBMCs from two different donors, and the results were consistent across experimental replicates. Representative data are shown in Figures 1 through 5.

[0328] Although it had no effect on T cells under static conditions, treatment with ADI-PEG 20 during stimulation with anti-CD3 / CD28 microbeads (Figures 1-5) or PHA (data not shown) resulted in T cell modulation, as detailed below. Under any experimental conditions, non-functional mutADI-PEG 20 did not modulate T cells.

[0329] Results: ADI-PEG 20 promoted stimulation-induced T cell activation, manifested as CD69 expression. CD69 upregulation on T cells was enhanced in a concentration-dependent manner by ADI-PEG 20 24 hours after stimulation. Furthermore, at the 48-hour and 72-hour time points, although CD69 levels declined in the presence of ADI-PEG 20 (as expected), stimulated T cells maintained high CD69 levels in the presence of ADI-PEG 20.

[0330] A 0.8 nM dose of ADI-PEG 20 showed no significant effect at the 24 and 48 hour time points, and induced a modest increase in CD69 expression at 72 hours, which must have required sufficient time to convert the critical amount of arginine to citrulline. Both 4 nM and 20 nM doses significantly enhanced CD69 expression (inducing this expression in approximately 80% of T cells), with the 20 nM dose being slightly more effective than the 4 nM dose; 100 nM of ADI-PEG 20 had the same effect as 20 nM (data not shown).

[0331] On CD4+ and CD8+ T cells, ADI-PEG 20 dose-related enhancement of surface CD69 was significant. Representative data are shown in Figure 1.

[0332] ADI-PEG 20 also slowed T cell exhaustion. Treatment with 4 nM or 20 nM ADI-PEG 20 prevented the induction of PD-1 on T cells at 48 and 72 hours post-stimulation (see Figure 2). PD-1 levels in T cells remained low during PBMC stimulation in the presence of ADI-PEG 20, similar to levels at rest.

[0333] Analysis of CTLA-4 expression on T cell subsets revealed that 4 nM and 20 nM doses of ADI-PEG 20 significantly reduced the upregulation of CTLA-4 on CD4+ and CD8+ cells after stimulation, while 20 nM mutADI-PEG 20 or 0.8 nM ADI-PEG 20 had no significant effect on CTLA-4 levels (see Figure 3).

[0334] Following stimulation with anti-CD3 / CD28 beads (or PHA) containing surface PD-L1, detectable on almost all T cells at 48 hours, PD-L1 surface levels on T cells were significantly upregulated. The presence of 4 nM or 20 nM (instead of 0.8 nM) ADI-PEG 20 somewhat mitigated this induction, but the percentage of PD-L1+ T cells remained significantly higher than in the unstimulated control. The ADI-PEG 20-mediated reduction in PD-L1+ activated T cells was more pronounced in CD8+ cells compared to CD4+ cells (see Figure 4).

[0335] ADI-PEG 20 downregulated regulatory T cells (Tregs). CD3 / CD28 microbead stimulation induced CD3+CD4+CD25+FoxP3+ Tregs, and ADI-PEG 20 reversed this effect in a concentration-related manner (see Figure 5). 4 nM and 20 nM doses of ADI-PEG 20 significantly reduced Treg levels at all three test time points, with the 20 nM dose producing a larger effect and reducing Treg levels to those found in unstimulated PBMCs.

[0336] Similar to the T cell activation data (CD69 levels), 0.8 nM ADI-PEG 20 had no effect on Treg cells at the 24 and 48 hour time points, but regulated them at 72 hour. In PBMC samples stimulated with anti-CD3 / CD28 microbeads and treated with 0.8 nM ADI-PEG 20, the percentage of Treg cells in CD4+ cells decreased approximately two-fold from the 48 to 72 hour time points, while in untreated or mutADI-PEG 20-treated controls, the percentage decreased only slightly. This suggests that ADI-PEG 20 not only prevents the accumulation of Treg cells but also reverses it.

[0337] Further analysis of Treg cells determined their expression of the PD-L1 and CTLA-4 markers. The dose-related effects of ADI-PEG 20 on Treg cells expressing PD-L1+ (mostly Treg cells) or CTLA-4+ were similar to those observed using all Treg cells (CD3+CD4+CD25+FoxP3+). These results were similar regardless of whether gating to CD127- cells was included (almost all CD3+CD4+CD25+FoxP3+ were CD127-, data not shown).

[0338] Example 2

[0339] ADI-PEG 20 induced T cell infiltration and showed significant inhibitory effects in a B16-F10 syngeneic mouse melanoma model.

[0340] To evaluate the efficacy and effect of ADI-PEG 20 on T cell activity in vivo, C57BL / 6 mice were implanted with B16-F10 melanoma cells (5 × 10⁻⁶). 5 One B16-F10 cell was subcutaneously implanted into the right side of a C57BL / 6 mouse in 0.1 ml PBS and the mouse was treated with the experimental agent.

[0341] To achieve therapeutic efficacy, the animals were randomly divided into three groups of eight mice each (n=8). Treatment began on the first day after tumor inoculation. Each mouse in group 1 received 0.5 ml of the carrier (PBS) via intraperitoneal injection every 3 days for a total of 4 times. Each mouse in group 2 received 18 mg / m² via intramuscular injection at a dose of 40 μl every 6 days for a total of 2 times. 2 ADI-PEG20. In group 3, each mouse received 36 mg / ml via intraperitoneal injection at a dose of 0.25 ml, administered four times every three days. 2 ADI-PEG20. Measure tumor size three times weekly until the average tumor volume in each group reaches 3500 mm. 3 Up to 4000mm 3 And use the following formula in mm 3 Tumor volume was expressed in units of: V = 0.536a × b², where a and b are the major and minor axes of the tumor, respectively. Weight was measured and recorded three times per week during this period. Results are expressed as mean ± SEM, as previously described. Figure 6A The effect of ADI-PEG 20 on the body weight of inoculated mice was shown. Figure 6B The effect of ADI-PEG20 on dynamic tumor volume was shown. These results indicate that ADI-PEG20 significantly reduced tumor volume over time.

[0342] For tumor T-cell infiltration analysis, animals were randomly divided into two groups: a control group of 4 mice and an ADI-PEG20 treatment group of 6 mice. Treatment was administered on days 1 and 7 after tumor implantation. Control animals were injected intramuscularly with PBS, while mice in the experimental group were injected intramuscularly with 36 mg / m². 2 ADI-PEG 20, both in 40 μl volumes.

[0343] Animals were euthanized on day 14 after tumor implantation, and the tumors were removed, preserved in 4% paraformaldehyde solution for at least 24 hours, and embedded in paraffin for immunohistochemical (IHC) analysis. Tumor sections were stained with anti-CD3 mAb to examine for the presence of T cells. Three sections of the tumor from each animal were stained, and the level of T cell infiltration was similar between sections derived from the same tumor.

[0344] Of the six animals treated with ADI-PEG 20, five had a high number of T cells present in their tumors (see [link to article]). Figure 7 This indicates that ADI-PEG 20 induces tumor infiltration into non-immunogenic B16-F10 tumors via T cells. Tumor sections from only one ADI-PEG 20-treated animal showed very few infiltrating T cells, similar to the levels observed in tumor sections from untreated control mice.

[0345] Example 3

[0346] Antitumor efficacy of ADI-PEG20 and mouse-specific PD-1 and PD-L1 antibodies in syngeneic mouse models.

[0347] The efficacy of ADI-PEG combined with PD-1 and PD-L1 antibodies was tested in two different mouse colorectal cancer models.

[0348] CT26 mouse colorectal cancer model In 0.1 ml PBS, CT26 tumor cells (2 x 10⁻⁶) were subcutaneously injected into the right side of Balb / c mice. 5 This allows for tumor development. Animals were randomly assigned to groups based on tumor size, with the average tumor size reaching 81 mm. 3 Treatment will begin at that time to conduct efficacy studies.

[0349] ADI-PEG20 was administered via intramuscular injection (im) and mouse-specific anti-PD-L1 antibody was administered via intraperitoneal injection (ip).

[0350] Figure 9 Table E1 (below) shows the effects of ADI-PEG20, PD-L1 inhibitors, and combination therapies on tumor volume in tumor-bearing mice.

[0351]

[0352] On day 11 post-treatment, the mean tumor volume in the control group reached 1,347 mm. 3 Treatment with BioXcell-mPD-L1 at a dose of 10 mg / kg showed significant antitumor activity, with an average tumor size of 1,033 mm. 3 (T / C value = 77%, TGI value = 25%, and p = 0.889, relative to the load cell group). Treatment with 12 mg / kg ADI-PEG20 showed moderate antitumor activity; mean tumor size was 667 mm. 3(T / C value = 50%, TGI value = 54%, and p = 0.113, relative to the load cell group). The combination of ADI-PEG20 and BioXcell-mPD-L1 also showed significant antitumor activity; the average tumor size was 469 mm. 3 (T / C value = 35%); TGI value = 69%, and p = 0.018).

[0353] Figure 10 Table E2 (below) shows the effects of ADI-PEG20, PD-L1 inhibitors, and combination therapy on the survival of tumor-bearing mice.

[0354]

[0355] Life expectancy in control mice treated with PBS ranged from 14 to 21 days, with a mean time to survival (MST) of 15.5 days. Treatment with ADI-PEG20 (12 mg / kg) resulted in an MST of 29.5 days, representing 90% ILS compared to the control group (p = 0.003). Treatment with the same dose of BioXcell-mPD-L1 (10 mg / kg) resulted in an MST of 27.5 days (ILS = 77%, p = 0.052). The combination of ADI-PEG20 and BioXcell-mPD-L1 presented an MST of 32 days (ILS = 106%, p = 0.0002).

[0356] B16F10 mouse model of colorectal cancer B16F10 tumor cells (2 x 10⁻⁵ cells) were subcutaneously inoculated on the right side of female C57BL / 6J mice in 0.1 ml PBS (containing 0.05 ml of matrix gel). 5 This is to support tumor development. Animals were randomly assigned to groups based on body weight, and treatment began on the first day after cell inoculation.

[0357] ADI-PEG20 was administered via intramuscular injection (im) and mouse-specific anti-PD-1 antibody was administered via intraperitoneal injection (ip).

[0358] Figure 12The effects of ADI-PEG20, PD-1 inhibitors, and combination therapy on the survival of tumor-bearing mice were shown. On day 13 after treatment initiation, the mean tumor size in the control group reached 2,157 mm3. Mice treated with anti-PD-1 mAb (10 mg / kg) reached a mean tumor size of 1,078 mm3 (T / C = 50%; TGI = 50%, p < 0.001, relative to the load group). Treatment with 12 mg / kg of ADI-PEG20 produced antitumor activity; the mean tumor size was 1,466 mm3 (T / C = 68%; TGI = 32%, p = 0.003, relative to the load group). The combination of ADI-PEG20 and anti-PD-1 mAb produced significant antitumor activity; the mean tumor size was 1,002 mm3 (T / C = 46%; TGI = 54%, p < 0.001). sequence list <110> Ruihua Pharmaceutical Group E. Brin He Wei <120> Combination cancer immunotherapy using arginine depletion agents <130> POLA-006 / 02WO <150> US 62 / 358,479 <151> 2016-07-05 <150> US 62 / 515,807 <151> 2017-06-06 <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 409 <212> PRT <213> Human mycoplasma <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> 409 <212> PRT <213> artificial sequence <220> <223> Mutant <400> 2 Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu 1 5 10 15 Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys Ile 50 55 60 Met Lys Asp Arg Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Lys Ala Ala Lys Glu Glu Phe Ile Glu 85 90 95 Thr Phe Leu Glu Glu Thr Val Pro Val Leu Thr Glu Ala Asn Lys Glu 100 105 110 Ala Val Arg Ala Phe Leu Leu Ser Lys Pro Thr His Glu Met Val Glu 115 120 125 Phe Met Met Ser Gly Ile Thr Lys Tyr Glu Leu Gly Val Glu Ser Glu 130 135 140 Asn Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp 145 150 155 160 Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Phe Met Arg Tyr 165 170 175 Ile Val Arg Arg Arg Glu Thr Leu Phe Ala Arg Phe Val Phe Arg Asn 180 185 190 His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met Lys 195 200 205 Met Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu Thr Leu 210 215 220 Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu Leu 225 230 235 240 Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg Ile Val 245 250 255 Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr Trp 260 265 270 Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala Asn 275 280 285 Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala Glu 290 295 300 Pro Gln Pro Gln Leu Asn Gly Leu Pro Leu Asp Lys Leu Leu Ala Ser 305 310 315 320 Ile Ile Asn Lys Glu Pro Val Leu Ile Pro Ile Gly Gly Ala Gly Ala 325 330 335 Thr Glu Met Glu Ile Ala Arg Glu Thr Asn Phe Asp Gly Thr Asn Tyr 340 345 350 Leu Ala Ile Lys Pro Gly Leu Val Ile Gly Tyr Asp Arg Asn Glu Lys 355 360 365 Thr Asn Ala Ala Leu Lys Ala Ala Gly Ile Thr Val Leu Pro Phe His 370 375 380 Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met 385 390 395 400 Pro Leu Ser Arg Lys Asp Val Lys Trp 405 <210> 3 <211> 392 <212> PRT <213> Mycoplasma phocicerebrale <400> 3 Ile His Val Tyr Ser Glu Ile Gly Glu Leu Glu Thr Val Leu Val His 1 5 10 15 Glu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu 20 25 30 Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His 35 40 45 Gln Ser Phe Val Lys Gln Leu Lys Asp Asn Gly Ile Asn Val Val Glu 50 55 60 Leu Thr Asp Leu Val Ala Glu Thr Phe Asp Leu Ala Ser Lys Glu Glu 65 70 75 80 Gln Glu Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu 85 90 95 Ser Glu Ala His Lys Thr Ala Val Arg Lys Phe Leu Thr Ser Arg Lys 100 105 110 Ser Thr Arg Glu Met Val Glu Phe Met Met Ala Gly Ile Thr Lys Tyr 115 120 125 Asp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro 130 135 140 Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val 145 150 155 160 Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe 165 170 175 Ser Arg Phe Val Phe Ser Asn His Pro Lys Leu Val Lys Thr Pro Trp 180 185 190 Tyr Tyr Asp Pro Ala Met Lys Met Ser Ile Glu Gly Gly Asp Val Phe 195 200 205 Ile Tyr Asn Asn Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp 210 215 220 Leu Glu Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu 225 230 235 240 Val Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn 245 250 255 Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe 260 265 270 Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp 275 280 285 Leu Val Asn Gly Gly Ala Glu Pro Gln Pro Lys Glu Asn Gly Leu Pro 290 295 300 Leu Glu Gly Leu Leu Gln Ser Ile Ile Asn Lys Lys Pro Val Leu Ile 305 310 315 320 Pro Ile Ala Gly Asn Asn Ala Ser His Ile Asp Ile Glu Arg Glu Thr 325 330 335 His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Lys Pro Gly Val Val Ile 340 345 350 Gly Tyr Ala Arg Asn Glu Lys Thr Asn Ala Ala Leu Ala Ala Ala Gly 355 360 365 Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly 370 375 380 Asn Ala Arg Cys Met Ser Met Pro 385 390 <210> 4 <211> 410 <212> PRT <213> Mycoplasma arginini <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 Ser Val Leu Val His Glu Pro Gly Arg Glu Ile 20 25 30 Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile 35 40 45 Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu 50 55 60 Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Ile Asp Leu Val Ala 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu 85 90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Val 100 105 110 Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Glu Leu Val 115 120 125 Glu Ile Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala 130 135 140 Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg 165 170 175 Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser 180 185 190 Asn His Pro Lys Leu Ile Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu 195 200 205 Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr 210 215 220 Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300 Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln 305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu Gly 325 330 335 Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe 370 375 380 His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys Trp<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Lys Glu Lys Leu Leu Glu 85 90 95 Glu Phe Leu Asp Asp Ser Val Pro Val Leu Ser Asp Glu His Arg Ala 100 105 110 Ala Val Lys Lys Phe Leu Gln Ser Gln Lys Ser Thr Arg Ser Leu Val 115 120 125 Glu Tyr Met Ile Ala Gly Ile Thr Lys His Asp Leu Lys Ile Glu Ser 130 135 140 Asp Leu 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 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 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 Asp 290 295 300 Ala Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Glu Asp Leu Leu Lys 305 310 315 320 Ser Ile Ile Gly Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Ala Gly 325 330 335 Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Val Ala Pro Gly Ile Val Ile Gly Tyr Ala Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Thr Val Leu Pro Phe 370 375 380 Arg Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys 405 <210> 6 <211> 408 <212> PRT <213> Mycoplasma orale <220> <221> misc_feature <222> (201)..(201) <223> Xaa can be any naturally occurring amino acid. <220> <221> misc_feature <222> (263)..(263) <223> Xaa can be any naturally occurring amino acid. <220> <221> misc_feature <222> (272)..(272) <223> Xaa can be any naturally occurring amino acid. <220> <221> misc_feature <222> (294)..(294) <223> Xaa can be any naturally occurring amino acid. <400> 6 Ser Val Phe Ser Asp Lys Phe Asn Gly Ile His Val Tyr Ser Glu Ile 1 5 10 15 Gly Asp Leu Glu Ser Val Leu Val His Glu Pro Gly Lys Glu Ile Asp 20 25 30 Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu 35 40 45 Glu Ser Thr Asp Ala Arg Lys Glu His Lys Glu Phe Val Glu Ile Leu 50 55 60 Lys Lys Gln Gly Ile Asn Val Val Glu Leu Val Asp Leu Val Val Glu 65 70 75 80 Thr Tyr Asn Leu Val Asp Lys Lys Thr Gln Glu Lys Leu Leu Lys Asp 85 90 95 Phe Leu Asp Asp Ser Glu Pro Val Leu Ser Pro Glu His Arg Lys Ala 100 105 110 Val Glu Lys Phe Leu Lys Ser Leu Lys Ser Thr Lys Glu Leu Ile Gln 115 120 125 Tyr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys Ala Asp 130 135 140 Lys 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 Tyr Met Arg Tyr 165 170 175 Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Lys Phe Ile Phe Thr Asn 180 185 190 His Pro Lys Leu Val Lys Thr Pro Xaa Tyr Tyr Asp Pro Ala Met Lys 195 200 205 Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr Leu 210 215 220 Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile Thr Leu Leu 225 230 235 240 Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile Val 245 250 255 Ala Ile Asn Val Pro Lys Xaa Thr Asn Leu Met His Leu Asp Thr Xaa 260 265 270 Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala Asn 275 280 285 Asp Val Phe Lys Phe Xaa Asp Tyr Asp Leu Val Asn Gly Gly Ser Asn 290 295 300 Pro Glu Pro Val Val Asn Gly Leu Pro Leu Asp Lys Leu Leu Glu Ser 305 310 315 320 Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Lys Gly Ala 325 330 335 Thr Glu Ile Glu Thr Ala Val Glu Thr His Phe Asp Gly Thr Asn Tyr 340 345 350 Leu Ala Ile Lys Pro Gly Val Val Val Gly Tyr Ser Arg Asn Val Lys 355 360 365 Thr Asn Ala Ala Leu Glu Ala Asn Gly Ile Lys Val Leu Pro Phe Lys 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 405 <210> 7 <211> 391 <212> PRT <213> Mycoplasma gateae <400> 7 Ile His Val Tyr Ser Glu Ile Gly Glu Leu Glu Ser Val Leu Val His 1 5 10 15 Glu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu 20 25 30 Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His 35 40 45 Lys Leu Phe Val Ser Glu Leu Lys Ala Asn Asp Ile Asn Val Val Glu 50 55 60 Leu Thr Asp Leu Val Thr Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala 65 70 75 80 Lys Asp Asn Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu 85 90 95 Thr Glu Glu Leu Lys Ser Val Val Arg Thr Tyr Leu Lys Ser Ile Lys 100 105 110 Ser Thr Arg Glu Leu Ile Gln Met Met Met Ala Gly Ile Thr Lys Tyr 115 120 125 Asp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro 130 135 140 Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val 145 150 155 160 Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe 165 170 175 Ser Arg Phe Val Phe Ser Asn His Pro Lys Leu Val Asn Thr Pro Trp 180 185 190 Tyr Tyr Asp Pro Ser Leu Lys Leu Ser Ile Glu Gly Gly Asp Val Phe 195 200 205 Ile Tyr Asn Asn Asn Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp 210 215 220 Leu Glu Thr Val Thr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys Glu 225 230 235 240 Cys Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn 245 250 255 Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe 260 265 270 Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp 275 280 285 Leu Val Asn Gly Gly Glu Glu Pro Gln Pro Val Glu Asn Gly Leu Pro 290 295 300 Leu Glu Gly Leu Leu Glu Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile 305 310 315 320 Pro Ile Ala Gly Glu Gly Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr 325 330 335 His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val Ile 340 345 350 Gly Tyr Ser Arg Asn Glu Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly 355 360 365 Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly 370 375 380 Asn Ala Arg Cys Met Ser Met 385 390 <210> 8 <211> 392 <212> PRT <213> Mycoplasma phocidae <400> 8 Ile His Val Tyr Ser Glu Ile Gly Glu Leu Gln Thr Val Leu Val His 1 5 10 15 Glu Pro Gly Arg Glu Ile Glu Tyr Ile Thr Pro Ala Arg Leu Asp Glu 20 25 30 Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His 35 40 45 Gln Glu Phe Val Ala Glu Leu Lys Lys Asn Asn Ile Asn Val Val Glu 50 55 60 Leu Thr Asp Leu Val Ser Glu Thr Tyr Asp Met Val Ser Lys Glu Lys 65 70 75 80 Gln Glu Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu 85 90 95 Ser Glu Glu His Lys Gly Leu Val Arg Lys Phe Leu Lys Ser Leu Lys 100 105 110 Ser Ser Lys Glu Leu Ile Gln Tyr Met Met Ala Gly Ile Thr Lys His 115 120 125 Asp Leu Asn Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro 130 135 140 Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val 145 150 155 160 Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe 165 170 175 Ser Arg Phe Ile Phe Ala Asn His Pro Lys Leu Met Asn Thr Pro Leu 180 185 190 Tyr Tyr Asn Pro Asp Met Lys Leu Ser Ile Glu Gly Gly Asp Val Phe 195 200 205 Val Tyr Asn Asn Glu Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp 210 215 220 Leu Asp Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu 225 230 235 240 Arg Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn 245 250 255 Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe 260 265 270 Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp 275 280 285 Leu Val Asn Gly Gly Asp Glu Pro Gln Pro Lys Val Asn Gly Leu Pro 290 295 300 Leu Glu Lys Leu Leu Glu Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile 305 310 315 320 Pro Ile Ala Gly Thr Ser Ala Ser Asn Ile Asp Val Glu Arg Glu Thr 325 330 335 His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Ala Pro Gly Val Val Ile 340 345 350 Gly Tyr Ser Arg Asn Val Lys Thr Asn Glu Ala Leu Glu Ala Ala Gly 355 360 365 Ile Lys Val Leu Pro Phe Lys Gly Asn Gln Leu Ser Leu Gly Met Gly 370 375 380 Asn Ala Arg Cys Met Ser Met Pro 385 390 <210> 9<00013​​​​​​​​​​​​​​​​​​​​Lys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile Lys 50 55 60 Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His Ala 65 70 75 80 Thr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala 85 90 95 Glu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr Val 100 105 110 Leu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met Ala 115 120 125 Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu 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 Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys 165 170 175 Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp Tyr 180 185 190 Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu 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 Lys Lys 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 Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys 275 280 285 Val Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr Asn 290 295 300 Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro Val 305 310 315 320 Leu Ile Pro Ile Ala Gly Lys Gly 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 Ile 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> 407 <212> PRT <213> Mycoplasma iowae [[ID=第十九]]<400> 10 Met Gly Asn Asn Ile Pro Lys Lys Ile Asn Val Phe Ser Glu Ile Gly 1 5 10 15 Asn Leu Lys Arg Val Leu Val His Thr Pro Gly Lys Glu Ile Glu Tyr 20 25 30 Val Thr Pro Gln Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Asp 35 40 45 Pro Val Arg Ala Arg Glu Glu His Lys Glu Phe Ile Lys Ile Leu Glu 50 55 60 Ser Gln Gly Val Glu Val Val Gln Leu Val Asp Leu Thr Ala Glu Thr 65 70 75 80 Tyr Asp Val Ala Glu Ser Gln Ala Lys Glu Asn Phe Ile Gln Lys Trp 85 90 95 Leu Asp Glu Ser Leu Pro Lys Leu Thr Asp Glu Asn Arg Asn Lys Val 100 105 110 Tyr Ser Leu Leu Lys Ser Leu Glu Lys Asp Pro Lys Glu Met Ile Arg 115 120 125 Lys Met Met Ser Gly Val Leu Ala Ser Glu Ile Gly Val Lys Ser Asp 130 135 140 Val 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 Ile Thr Leu His Arg Met Phe Arg 165 170 175 Pro Thr Arg Arg Arg Glu Thr Ile Phe Ala Asp Phe Ile Phe Ser Asn 180 185 190 His Pro Glu Tyr Lys Ser Thr Gln Lys Tyr Tyr Glu Arg Glu Asp Lys 195 200 205 Phe Ser Leu Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Lys Thr Leu 210 215 220 Val Val Gly Val Ser Glu Arg Thr Glu Lys Gly Ala Ile Lys Ala Leu 225 230 235 240 Ala Lys Ala Val Gln Asn Asn Ser Asn Met Ser Phe Glu Lys Ile Tyr 245 250 255 Ala Ile Asn Val Pro Lys Met Ser Asn Leu Met His Leu Asp Thr Trp 260 265 270 Leu Thr Met Leu Asp Thr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met 275 280 285 Gly Val Leu Lys Ile Trp Glu Ile Asp Leu Ser Asp Lys Ser Leu Lys 290 295 300 Trp Lys Glu Ile Arg Asp Ser Leu Asp His Phe Leu Ser Thr Ile Ile 305 310 315 320 Gly Lys Lys Ala Ile Thr Val Pro Val Ala Gly Lys Asp Ala Met Gln 325 330 335 Phe Glu Ile Asp Ile Glu Thr His Phe Asp Ala Thr Asn Phe Ile Ala 340 345 350 Val Ala Pro Gly Val Val Ile Gly Tyr Asp Arg Asn Lys Lys Thr Asn 355 360 365 Glu Ala Leu Lys Glu Ala Gly Ile Lys Val Leu Ser Trp Asn Gly Asp 370 375 380 Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Thr Met Pro Leu 385 390 395 400 Tyr Arg Glu Glu Leu Lys Lys 405 <210> 11 <211> 402 <212> PRT <213> Mycoplasma crocodyli <400> 11 Met Asn Lys Ile Asn Val Tyr Ser Glu Val Gly Lys 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 Glu Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Ser Ala Ile 35 40 45 Glu Glu His Lys Arg Phe Leu Lys Ile Leu Glu Asp Asn Asn Ile Lys 50 55 60 Val Ile Gln Leu Asp Gln Leu Val Ala Asp Thr Tyr Glu Leu Val Asn 65 70 75 80 Pro Ser Val Arg Asp Ala Phe Ile Glu Lys Trp Leu Asn Glu Ser Glu 85 90 95 Pro Lys Leu Asp Lys Lys Leu Arg Glu Lys Val Lys Glu Tyr Leu Leu 100 105 110 His Thr Gln Lys Thr Val Gly Thr Lys Arg Met Val Arg Ile Met Met 115 120 125 Ala Gly Val Asp Arg Val Glu Leu Gly Val Glu Leu Asp Arg Gln Leu 130 135 140 Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala 145 150 155 160 Ser Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg 165 170 175 Lys Arg Glu Thr Ile Phe Ser Glu Phe Ile Phe Glu Asn His Pro Asp 180 185 190 Tyr Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile 195 200 205 Glu Gly Gly Asp Val Phe Ile Tyr Asn Arg Thr Thr Leu Val Ile Gly 210 215 220 Ile Ser Glu Arg Thr Asn Lys Asp Ala Leu Leu Thr Ile Ala Asn Asn 225 230 235 240 Ile Lys Ser Asn Lys Glu Ser Lys Phe Glu Arg Ile Val Ala Val Asn 245 250 255 Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met 260 265 270 Val Asp His Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Lys Thr Leu 275 280 285 Lys Phe Trp Thr Ile Asp Leu Thr Lys Pro Ile Lys Met Val Glu Leu 290 295 300 Glu Glu Ser Leu Ser Asp Met Ile Glu Thr Ile Ile Gly Lys Lys Pro 305 310 315 320 Val Leu Ile Pro Ile Ala Gly His Asp Ala Ser Pro Leu Asp Val Asp 325 330 335 Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly 340 345 350 Val Val Val Gly Tyr Ser Arg Asn Lys Leu Thr Glu Lys Ala Leu Thr 355 360 365 Lys Ala Gly 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 Val Arg Glu Asp 385 390 395 400 Ile Lys <210> 十二 <211> 430 <212> PRT <213> Mycoplasma fermentans <400> 12 Met Gln Ile Ile Ala Lys Ile Asp Leu Leu Thr Asn Met Leu Ile Phe 1 5 10 15 Met Lys Ile Tyr Phe Ile Gly Arg Leu Ile Met Lys Lys Ile Asn Val 20 25 30 Tyr Ser Glu Tyr Gly Lys Leu Lys Glu Val Leu Val His Thr Pro Gly 35 40 45 Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg Leu Asp Glu Leu Leu Phe 50 55 60 Ser Ala Ile Leu Glu Pro Asp Ser Ala Ile Ala Glu His Lys Arg Phe 65 70 75 80 Val Gln Leu Leu Lys Asp Asn Gly Ile Lys Val Ile Gln Leu Asp Glu 85 90 95 Leu Phe Ala Lys Thr Phe Asp Leu Val Ser Glu Ser Val Lys Gln Ser 100 105 110 Leu Ile Glu Arg Trp Leu Asp Glu Cys Glu Pro Lys Leu Asp Ala Thr 115 120 125 Leu Arg Ala Lys Val Lys Glu Tyr Ile Leu Glu Leu Lys Ala Lys Ser 130 135 140 Ser Lys Lys Met Val Arg Val Met Met Ala Gly Ile Asp Lys Lys Glu 145 150 155 160 Leu Gly Ile Glu Leu Asp Arg 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 Ser 180 185 190 Leu His His Met Lys Tyr Val Thr Arg Gln Arg Glu Thr Ile Phe Ser 195 200 205 Glu Phe Ile Phe Asp Asn Asn Leu Asp Tyr Asn Thr Val Pro Arg Trp 210 215 220 Phe Asp Arg Lys Asp Glu Gly Arg Ile Glu Gly Gly Asp Val Phe Ile 225 230 235 240 Tyr Ser Ala Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asn Lys 245 250 255 Glu Ala Ile Asn Val Met Ala Arg Lys Ile Ala Ala Asp Lys Glu Val 260 265 270 Lys Phe Lys Arg Ile Tyr Ala Ile Asn Val Pro Pro Met Pro Asn Leu 275 280 285 Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu 290 295 300 Tyr Ser Pro Asn Met Leu Ser Val Leu Lys Val Trp Arg Ile Asp Leu 305 310 315 320 Asn Asp Pro Asp Phe Val Trp His Glu Ile Glu Gly Ser Leu Glu Glu 325 330 335 Ile Leu Glu Gln Ile Ile Gly Met Lys Pro Ile Leu Ile Pro Ile Ala 340 345 350 Gly Lys Gly Ala Ser Gln Leu Asp Ile Asp Ile Glu Thr His Phe Asp 355 360 365 Gly Thr Asn Tyr Leu Thr Ile Ala Pro Ser Val Val Val Gly Tyr Ser 370 375 380 Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala Ala Lys Val Lys Val 385 390 395 400 Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ala Arg 405 410 415 Cys Met Ser Met Pro Leu Ile Arg Glu Asp Ile Lys Lys Lys 420 425 430 <210> 13 <211> 452<000​​​​​​​​​​​​​​​​​​​ 50 55 60 Lys Glu Val Leu Val His Thr Pro Gly Asp Glu Ile Arg Tyr Thr Ala 65 70 75 80 Pro Ser Arg Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Lys Ala Asp 85 90 95 Thr Ala Ile Glu Glu His Lys Gly Phe Val Lys Ile Leu Gln Asn Asn 100 105 110 Gly Ile Lys Val Ile Gln Leu Cys Asp Leu Val Ala Glu Thr Tyr Glu 115 120 125 Leu Cys Ser Lys Glu Val Arg Asn Ser Phe Ile Glu Gln Tyr Leu Asp 130 135 140 Glu Ala Leu Pro Val Leu Lys Lys Glu Ile Arg Pro Val Val Lys Asp 145 150 155 160 Tyr Leu Leu Ser Phe Pro Thr Val Gln Met Val Arg Lys Met Met Ser 165 170 175 Gly Ile Leu Ala Asn Glu Leu Asn Ile Lys Gln Asp Asn Pro Leu Ile 180 185 190 Ile Asp Gly Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser 195 200 205 Met Gly Asn Gly Val Ser Ile Asn Cys Met Lys Tyr Pro Thr Arg Lys 210 215 220 Arg Glu Val Ile Phe Ser Arg Phe Val Phe Thr Asn Asn Pro Lys Tyr 225 230 235 240 Lys Asn Thr Pro Arg Tyr Phe Asp Ile Val Gly Asn Asn Gly Thr Ile 245 250 255 Glu Gly Gly Asp Ile Phe Ile Tyr Asn Ser Lys Thr Leu Val Ile Gly 260 265 270 Asn Ser Glu Arg Thr Asn Phe Ala Ala Ile Glu Ser Val Ala Lys Asn 275 280 285 Ile Gln Ala Asn Lys Asp Cys Thr Phe Glu Arg Ile Val Val Ile Asn 290 295 300 Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met 305 310 315 320 Leu Asp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Asn Val Leu 325 330 335 Lys Ile Trp Glu Ile Asp Leu Asn Val Lys Pro Val Lys Phe Val Glu 340 345 350 Lys Lys Gly Thr Leu Glu Glu Val Leu Tyr Ser Ile Ile Asp Lys Lys 355 360 365 Pro Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Asn Gln Leu Asp Ile 370 375 380 Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro 385 390 395 400 Gly Val Val Val Gly Tyr Glu Arg Asn Glu Lys Thr Gln Lys Ala Leu 405 410 415 Val Glu Ala Gly Ile Lys Val Leu Ser Phe Asn Gly Ser Gln Leu Ser 420 425 430 Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu 435 440 445 Asn Leu Lys Lys 450 <210> 14 <211> 403 <212> PRT <213> Mycoplasma gallisepticum <400> 14 Met Phe Asn Lys Ile Arg Val Tyr Ser Glu Ile Gly Lys Leu Arg Lys 1 5 10 15 Val Leu Val His Thr Pro Gly Lys Glu Leu Asp Tyr Val Thr Pro Gln 020 25 30 Arg Leu Asp Glu Leu Leu Phe Ser Ser Leu Leu Asn Pro Ile Lys Ala 35 40 45 Arg Gln Glu His Glu Thr Phe Ile Lys Leu Leu Glu Asp His Asp Val 50 55 60 Glu Cys Val Gln Leu Ser Thr Leu Thr Ala Gln Thr Phe Gln Ala Val 65 70 75 80 Asn Ser Lys Ile Gln Glu Glu Phe Ile Asn Arg Trp Leu Asp Glu Cys 85 90 95 Leu Pro Val Leu Ser Glu Ile Asn Arg Leu Lys Val Tyr Asp Tyr Leu 100 105 110 Lys Ser Leu Ala Thr Asn Pro Gln Val Met Ile Arg Lys Met Met Ser 115 120 125 Gly Ile Leu Ala Lys Glu Val Gly Ile Gln Ser Glu Val Glu Leu Val 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 Ile Thr Leu His Ser Met Phe His Pro Thr Arg Lys 165 170 175 Arg Glu Thr Ile Phe Ala Asp Phe Ile Phe Ser His His Pro Glu Tyr 180 185 190 Lys Asn Ala Pro Lys Tyr Tyr Ser Arg Glu Asp Lys 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 Ser Leu Ala Glu Lys Leu 225 230 235 240 Arg Gln Asn Asp Glu Thr Ser 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 Ile His Pro Thr Leu Ile Trp Arg Glu Leu 290 295 300 Asn Glu Ser Leu Glu Gly Phe Leu Ser Met Val Ile Gly Lys Lys Ala 305 310 315 320 Thr Leu 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 Arg 355 360 365 Asp Ala Gly Val Lys Val Ile 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 <210> 15 <211> 402 <212> PRT <213> Mycoplasma alligatoris <400> 15 Met Ser Lys Ile Asn Val Tyr Ser Glu Val Gly Arg Leu Lys Glu Val 1 5 10 15 Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Thr Arg 20 25 30 Leu Glu Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Thr Ala Ile 35 40 45 Glu Glu His Lys Arg Phe Leu Asn Val Leu Glu Lys Asn Gly Ile Lys 50 55 60 Ala Ile Gln Leu Asp Glu Leu Val Ala Gln Thr Tyr Asp Gln Val Asp 65 70 75 80 Gln Lys Ile Lys Asp Glu Phe Ile Asp Gln Trp Leu Gln Glu Ala Lys 85 90 95 Pro Val Leu Asn Asp Gln Leu Lys Lys Leu Val Lys Asn Tyr Leu Leu 100 105 110 Lys Ser Gln Lys Glu Phe Ser Thr Lys Lys Met Val Arg Ile Met Met 115 120 125 Ala Gly Ile Asp Lys Lys Glu Ile Asn Ile Asp Leu Asp Arg Asp Leu 130 135 140 Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala 145 150 155 160 Ser Val Gly Asn Gly Ile Ser Leu His Asn Met Lys Tyr Gln Thr Arg 165 170 175 Lys Arg Glu Thr Ile Phe Ala Gln Phe Ile Phe Lys Tyr Asn Lys Asp 180 185 190 Tyr Lys Thr Thr Pro His Trp Phe Asp Arg Phe Asp His Gly Ser Ile 195 200 205 Glu Gly Gly Asp Val Phe Val Tyr Thr Lys Asp Thr Leu Val Ile Gly 210 215 220 Ile Ser Glu Arg Thr Thr Lys Glu Ala Val Leu Asn Ile Ala Lys Lys 225 230 235 240 Ile Lys Ala Asn Thr Asp Ser Lys Phe Lys Lys Ile Val Ala Ile Asn 245 250 255 Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Ile Thr Met 260 265 270 Val Asp His Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Lys Ser Leu 275 280 285 Lys Phe Trp Leu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Leu 290 295 300 Glu Glu Ser Leu Ser Asn Met Leu Glu Ala Ile Ile Gly Lys Lys Pro 305 310 315 320 Ile Leu Ile Pro Ile Ala Gly Lys Asn 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 Gly 340 345 350 Val Val Val Gly Tyr Ser Arg Asn Lys Leu Thr Gln Lys Ala Leu Glu 355 360 365 Asp Ala Gly Val Lys Val Leu Ser Phe Asp Gly Asn Gln Leu Ser Leu 370 375 380 Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp 385 390 395 400 Ile Lys <210> 16 <211> 438 <212> PRT <213> Mycoplasma pneumoniae <400> 16 Met Ser Lys Lys Gln Leu Val Lys Thr Asp Gly His Asn Gln Leu Asp 1 5 10 15 Gln Pro Asn Thr Lys Ala Leu Gln Leu Lys Lys Lys Gln Phe Asn Ser 20 25 30 Gly Val Arg Val Thr Ser Glu Ile Ser Phe Leu Arg Glu Val Ile Ala 35 40 45 His His Pro Gly Ile Glu Thr Glu Arg Val Ile Asp Asn Gln Thr Phe 50 55 60 Gly Ser Ala Met Tyr Leu Glu Arg Ala Gln Lys Glu His Gln Leu Phe 65 70 75 80 Ile Lys Ile Leu Arg Gln His Gly Thr Lys Val His Tyr Leu Gln Asp 85 90 95 Leu Leu Leu Glu Ala Leu Ser Ala Ala Asp Pro Asn Val Arg Gln Asp 100 105 110 Phe Ile Lys Asn Phe Leu Leu Glu Ser Gly Ile Lys Ser Val Ser Thr 115 120 125 Phe Glu Ala Cys Leu Asn Phe Phe Arg Ser Leu Asp Ser Leu Val Asp 130 135 140 Val Ile Lys Val Met Phe Gly Gly Ile Lys Val Ser Asp Val Pro Pro 145 150 155 160 Ile Thr Pro Gln Arg Phe Ala Asp Ile His Val Ser Asn Ser Pro Phe 165 170 175 Leu Ile Lys Pro Leu Ser Phe Ser Leu Tyr Pro His Lys Phe Phe Asn 180 185 190 Thr Leu Gly Thr Gly Val Ala Leu Phe Val Thr Asn Asp Ser Glu Leu 195 200 205 Lys Arg His Ser Leu Val Tyr Glu Tyr Ile Met Arg Phe His Pro Arg 210 215 220 Phe Asp Gly Val Lys Leu Tyr Thr Asn Arg Asp Phe Lys Asn Cys Leu 225 230 235 240 Ile Asn Ser Ser Asp Ile Ile Gln Ile Ser Asn Glu Ile Leu Leu Ile 245 250 255 Gly Ile Ser His Asp Thr Asp Val Leu Gly Ile Glu Ser Leu Ala Arg 260 265 270 Asn Leu Leu Ser Asp His Thr Asn Pro Ile Lys Gln Ile Ile Ala Ile 275 280 285 Asn Ile His Lys Phe Gly Ala Lys Thr Asn Leu Asn Lys Leu Ile Ala 290 295 300 Met Val Asp Val Asp Lys Phe Ile Ile Ala Arg Lys Val Leu Gln Ala 305 310 315 320 Thr Glu Ile Phe Glu Leu Thr Ala Thr Ala Gln Arg Asp Val Asp Gly 325 330 335 Leu Ala Gln Ile Lys Phe Lys Pro Leu Lys Phe Asn Phe Gly Glu Ile 340 345 350 Ile Glu Ala Ile Ile Asp Lys Gln Pro Arg Phe Val Ile Ile Gly Gly [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Met Lys Asp Thr Lys Asp Ile Ile Asn Val Phe Ser Glu Ile Gly Glu 1 5 10 15 Leu Lys Lys Val Leu Ile His Thr Pro Gly Asn Glu Leu Lys Tyr Val 20 25 30 Ser Pro Tyr Arg Leu Asp Glu Leu Leu Phe Ser Asn Val Leu Glu Trp 35 40 45 Arg Glu Ala Lys Lys Glu His Asn Glu Phe Ile Gln Lys Leu Lys Ser 50 55 60 Glu Gly Val Glu Pro Val Glu Leu Thr Asp Leu Val Ala Glu Ser Phe 65 70 75 80 Glu Glu Ser Ser Ile Lys Val Lys Asn Asp Phe Ile Arg Gln Tyr Leu 85 90 95 Asp Glu Ala Thr Pro Ile Leu Asp Gly Leu Thr Lys Gln Lys Leu Leu 100 105 110 Pro Phe Phe Leu Asp Ile Lys His Ser Thr Arg Lys Thr Ile Glu Leu 115 120 125 Met Met Ser Gly Ile Thr Gln Lys Asp Ile Ser Ile Ser His Ile Glu 130 135 140 Arg Glu Leu Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Ser Arg Asp 145 150 155 160 Asn Phe Ile Ser Ile Gly Asn Ser Val Ile Ile Ser Asn Met Lys Tyr 165 170 175 Lys Thr Arg Lys Arg Glu Thr Ile Phe Thr Asp Phe Ile Phe Lys Asn 180 185 190 His Pro Leu Tyr Lys Lys Val Asn Met Ala Phe Glu Arg Lys Asp Leu 195 200 205 Asn Asn Gln Ile Ser Ile Ile Glu Gly Gly Asp Val Leu Val Tyr Ser 210 215 220 Lys Glu Ile Leu Ile Ile Gly Ile Ser Glu Arg Thr Thr Met Ser Ala 225 230 235 240 Ile Leu Glu Leu Ala Glu Asn Phe Lys Lys Thr Lys Arg Ser Phe Lys 245 250 255 Lys Ile Tyr Gly Val Glu Val Pro Lys Met Lys Asn Leu Met His Leu 260 265 270 Asp Thr Trp Leu Thr Met Ile Asp Tyr Asp Lys Phe Ile Tyr Ser Pro 275 280 285 Asn Val Leu Thr Asp Leu Lys Phe Trp Glu Ile Asn Leu Asp Tyr Glu 290 295 300 Lys Ile Ser Ser Lys Glu Leu His Ala Ser Leu Ser Glu Phe Leu Lys 305 310 315 320 Leu Ile Ile Gly Lys Asp Pro Ile Leu Ile Pro Ile Gly Gly Lys Gly 325 330 335 Ala Ser Gln Ile Thr Ile Asp Ile Glu Thr Asn Phe Val Ala Ala Asn 340 345 350 Tyr Leu Val Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Tyr 355 360 365 Glu Thr Gln Lys Ala Leu Glu Gly His Gly Val Lys Val Ile Ala Phe 370 375 380 Glu Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ser Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ile Arg Ser Asn Leu Lys 405 <210> 18<000188​​​​​​​​​​​​​​​​​​​ Asp Ala Gln Lys Glu His Asp Ala Phe Ala Gln Ala Leu Arg Asp Glu 50 55 60 Gly Ile Glu Val Leu Tyr Leu Glu Thr Leu Ala Ala Glu Ser Leu Val 65 70 75 80 Thr Pro Glu Ile Arg Glu Ala Phe Ile Asp Glu Tyr Leu Ser Glu Ala 85 90 95 Asn Ile Arg Gly Arg Ala Thr Lys Lys Ala Ile Arg Glu Leu Leu Met 100 105 110 Ala Ile Glu Asp Asn Gln Glu Leu Ile Glu Lys Thr Met Ala Gly Val 115 120 125 Gln Lys Ser Glu Leu Pro Glu Ile Pro Ala Ser Glu Lys Gly Leu Thr 130 135 140 Asp Leu Val Glu Ser Asn Tyr Pro Phe Ala Ile Asp Pro Met Pro Asn 145 150 155 160 Leu Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Val Ser 165 170 175 Leu Asn His Met Phe Ser Glu Thr Arg Asn Arg Glu Thr Leu Tyr Gly 180 185 190 Lys Tyr Ile Phe Thr His His Pro Ile Tyr Gly Gly Gly Lys Val Pro 195 200 205 Met Val Tyr Asp Arg Asn Glu Thr Thr Arg Ile Glu Gly Gly Asp Glu 210 215 220 Leu Val Leu Ser Lys Asp Val Leu Ala Val Gly Ile Ser Gln Arg Thr 225 230 235 240 Asp Ala Ala Ser Ile Glu Lys Leu Leu Val Asn Ile Phe Lys Gln Asn 245 250 255 Leu Gly Phe Lys Lys Val Leu Ala Phe Glu Phe Ala Asn Asn Arg Lys 260 265 270 Phe Met His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe 275 280 285 Thr Ile His Pro Glu Ile Glu Gly Asp Leu Arg Val Tyr Ser Val Thr 290 295 300 Tyr Asp Asn Glu Glu Leu His Ile Val Glu Glu Lys Gly Asp Leu Ala 305 310 315 320 Glu Leu Leu Ala Ala Asn Leu Gly Val Glu Lys Val Asp Leu Ile Arg 325 330 335 Cys Gly Gly Asp Asn Leu Val Ala Ala Gly Arg Glu Gln Trp Asn Asp 340 345 350 Gly Ser Asn Thr Leu Thr Ile Ala Pro Gly Val Val Val Val Tyr Asn 355 360 365 Arg Asn Thr Ile Thr Asn Ala Ile Leu Glu Ser Lys Gly Leu Lys Leu '370 375 380 Ile Lys Ile His Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg 385 390 395 400 Cys Met Ser Met Pro Phe Glu Arg Glu Asp Ile 405 410 <210> 19 <211> 408 <212> PRT <213> Enterococcus faecalis <400> 19 Met Ser His Pro Ile Asn Val Phe Ser Glu Ile Gly Lys Leu Lys Thr 1 5 10 15 Val Met Leu His Arg Pro Gly Lys Glu Leu Glu Asn Leu Met Pro Asp 20 25 30 Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Lys Ala 35 40 45 Gln Ala Glu His Asp Ala Phe Ala Glu Leu Leu Arg Ser Lys Asp Ile 50 55 6 Glu Val Val Tyr Leu Glu Asp Leu Ala Ala Glu Ala Leu Ile Asn Glu 65 70 75 80 Glu Val Arg Arg Gln Phe Ile Asp Gln Phe Leu Glu Glu Ala Asn Ile 85 90 95 Arg Ser Glu Ser Ala Lys Glu Lys Val Arg Glu Leu Met Leu Glu Ile 100 105 110 Asp Asp Asn Glu Glu Leu Ile Gln Lys Ala Ile Ala Gly Ile Gln Lys 115 120 125 Gln Glu Leu Pro Lys Tyr Glu Gln Glu Phe Leu Thr Asp Met Val Glu 130 135 140 Ala Asp Tyr Pro Phe Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr 145 150 155 160 Arg Asp Asn Phe Ala Thr Met Gly His Gly Ile Ser Leu Asn His Met 165 170 175 Tyr Ser Val Thr Arg Gln Arg Glu Thr Ile Phe Gly Gln Tyr Ile Phe 180 185 190 Asp Tyr His Pro Arg Phe Ala Gly Lys Glu Val Pro Arg Val Tyr Asp 195 200 205 Arg Ser Glu Ser Thr Arg Ile Glu Gly Gly Asp Glu Leu Ile Leu Ser 210 215 220 Lys Glu Val Val Ala Ile Gly Ile Ser Gln Arg Thr Asp Ala Ala Ser 225 230 235 240 Ile Glu Lys Ile Ala Arg Asn Ile Phe Glu Gln Lys Leu Gly Phe Lys 245 250 255 Asn Ile Leu Ala Phe Asp Ile Gly Glu His Arg Lys Phe Met His Leu 260 265 270 Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Ile His Pro 275 280 285 Glu Ile Glu Gly Gly Leu Val Val Tyr Ser Ile Thr Glu Lys Ala Asp 290 295 300 Gly Asp Ile Gln Ile Thr Lys Glu Lys Asp Thr Leu Asp Asn Ile Leu 305 310 315 320 Cys Lys Tyr Leu His Leu Asp Asn Val Gln Leu Ile Arg Cys Gly Ala 325 330 335 Gly Asn Leu Thr Ala Ala Ala Arg Glu Gln Trp Asn Asp Gly Ser Asn 340 345 350 Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asp Arg Asn Thr 355 360 365 Ile Thr Asn Lys Ala Leu Glu Glu Ala Gly Val Lys Leu Asn Tyr Ile 370 375 380 Pro Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg Cys Met Ser 385 390 395 400 Met Pro Leu Tyr Arg Glu Asp Leu 405 <210> 20 <211> 403 <212> PRT <213> Mycoplasma capricolum <400> 20 Met Glu Lys Lys Ile Asn Val Phe Ser Glu Ile Gly Thr Leu Lys Thr 1 5 10 15 Val Leu Val His Arg Pro Gly Asp Glu Ile Glu Asn Leu Thr Pro Glu 20 25 30 Leu Leu Glu Arg Leu Leu Phe Asp Asp Val Pro Phe Lys Asp Val Ala 35 40 45 Val Lys Glu His Asp Ala Phe Thr Lys Ile Met Arg Asp Asn Gly Val 50 55 60 Glu Val Leu Tyr Ile Glu Lys Leu Ala Ala Glu Thr Leu Asp Gln His 65 70 75 80 Pro Asp Leu Arg Glu Lys Phe Ile Asp Gln Phe Ile Ser Glu Ala Asn 85 90 95 Ile Glu Asp Lys Tyr Lys Glu Lys Tyr Arg Asp Phe Ile Ser Ser Leu 100 105 110 Asp Asn Tyr Arg Met Ile Lys Lys Met Ile Ala Gly Thr Lys Lys Leu 115 120 125 Glu Leu Gly Ile Asp Glu Gly Tyr Lys Ala Tyr Pro Phe Ile Ala Asp 130 135 140 Pro Leu Pro Asn Val Leu Phe Gln Arg Asp Pro Phe Ser Ser Val Gly 145 150 155 160 Phe Gly Ile Thr Met Asn Arg Met Trp Ser Val Thr Arg Asn Arg Glu 165 170 175 Thr Ile Phe Pro Asp Leu Val Phe Lys His His Asn Arg Phe Ala Asn 180 185 190 Gln Val Pro Tyr Tyr Tyr Glu Arg Asp Trp Lys Glu Glu Thr Ile Glu 195 200 205 Gly Gly Asp Ile Leu Val Leu Asn Lys Glu Thr Leu Ile Ile Gly Val 210 215 220 Thr Gln Arg Thr Thr Leu Lys Ala Ile Glu Lys Phe Ser Glu Arg Leu 225 230 235 240 Phe Asn Asp Pro Glu Ser Ser Tyr Ser Lys Val Ile Ala Leu Asp Leu 245 250 255 Pro Lys Ser Arg Ala Phe Met His Leu Asp Thr Val Phe Thr Asn Ile 260 265 270 Asp Tyr Asp Lys Phe Ile Ala His Pro Leu Ile Phe Asp Cys Ile Asp 275 280 285 Glu Phe Lys Ile Tyr Glu Val Ser Lys Gln Gly Thr Lys Glu Val Lys 290 295 300 Lys Thr Leu Ile Glu Leu Leu Ser Asp Ala Ala Gly Arg Glu Val Gln 305 310 315 320 Ile Ile Arg Cys Gly Gly Asn Asp Val Val Gly Ala Ser Arg Glu Gln 325 330 335 Trp Asn Asp Gly Thr Asn Val Val Ala Leu Arg Pro Gly Lys Val Ile 340 345 350 Ala Tyr Glu Arg Asn Trp Ile Thr Ile Asp Leu Leu Arg Lys Ala Gly 355 360 365 Val Glu Val Leu Thr Ile Ala Ser Ser Glu Leu Ser Arg Gly Arg Gly 370 375 380 Gly Pro Arg Cys Met Thr Met Pro Leu Trp Arg Glu Asp Leu Gln Glu 385 390 395 400 Ile Lys Arg <210> 21 <211> 410 <212> PRT <213> Halothermothrix orenii <400> 21 Met Phe Lys Lys Ser Pro Leu Asn Val Thr Ser Glu Ile Gly Lys Leu 1 5 10 15 Lys Lys Val Leu Leu His Arg Pro Gly His Glu Ile Glu Asn Leu Thr 20 25 30 Pro Asp Leu Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu Lys 35 40 45 Val Ala Gln Glu Glu His Asp Ala Phe Ala Gln Thr Leu Arg Asp Asn 50 55 60 Gly Val Glu Val Leu Tyr Leu His Glu Leu Ala Ala Glu Ala Ile Gln 65 70 75 80 Glu Asp Glu Ile Arg Lys Lys Phe Ile Glu Gln Phe Leu Asp Glu Ala 85 90 95 Gly Val Ile Gly Lys Gly Ala Arg Gln Val Leu Lys Glu Tyr Phe Ala 100 105 110 Asp Met Asp Asn Glu Thr Leu Ile Arg Lys Met Met Ala Gly Val Arg 115 120 125 Lys Lys Glu Ile Pro Ala Ile Glu Lys Val Ala Ser Leu Asn Asp Met 130 135 140 Val Glu Glu Asp Tyr Pro Phe Val Leu Asp Pro Met Pro Asn Leu Tyr 145 150 155 160 Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Ile Thr Leu Asn 165 170 175 His Met Arg Thr Glu Thr Arg Asn Arg Glu Val Ile Phe Ala Glu Tyr 180 185 190 Ile Phe Ser Tyr His Pro Asp Phe Lys Asp Thr Glu Ile Pro Phe Trp 195 200 205 Phe Asp Arg Asn Glu Thr Thr Ser Ile Glu Gly Gly Asp Glu Leu Ile 210 215 220 Leu Ser Asp Lys Val Leu Ala Met Gly Ile Ser Glu Arg Thr Asp Ala 225 230 235 240 Ala Ser Ile Glu Lys Val Ala Arg Asn Ile Phe Thr Asp Gly Gln Pro 245 250 255 Phe Glu Thr Ile Leu Ala Phe Lys Ile Pro Glu Lys Arg Ala Phe Met 260 265 270 His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe Thr Ile 275 280 285 His Ala Glu Ile Glu Gly Pro Leu Lys Val Tyr Ser Ile Thr Lys Gly 290 295 300 Asp Asn Asp Glu Leu Lys Ile Asp Glu Glu Lys Ala Thr Leu Glu Asp 305 310 315 320 Thr Leu Lys Lys Tyr Leu Gly Leu Asp Glu Val Thr Leu Ile Arg Cys 325 330 335 Ala Gly Gly Asp Tyr Ile Asp Ala Gly Arg Glu Gln Trp Asn Asp Gly 340 345 350 Ser Asn Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asn Arg 355 360 365 Asn His Thr Thr Asn Arg Leu Leu Glu Glu His Gly Ile Lys Leu His 370 375 380 Val Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys 385 390 395 400 Met Ser Met Pro Leu Val Arg Glu Asp Ile 405 410 <210> 22 <211> 411 <212> PRT <213> Staphylococcus aureus <400> 22 Met Thr Asp Gly Pro Ile Lys Val Asn Ser Glu Ile Gly Ala Leu Lys 1 5 10 15 Thr Val Leu Leu Lys Arg Pro Gly Lys Glu Leu Glu Asn Leu Val Pro 20 25 30 Asp Tyr Leu Asp Gly Leu Leu Phe Asp Asp Ile Pro Tyr Leu Glu Val 35 40 45 Ala Gln Lys Glu His Asp His Phe Ala Gln Val Leu Arg Glu Glu Gly 50 55 60 Val Glu Val Leu Tyr Leu Glu Lys Leu Ala Ala Glu Ser Ile Glu Asn 65 70 75 80 Pro Gln Val Arg Ser Glu Phe Ile Asp Asp Val Leu Ala Glu Ser Lys 85 90 95 Lys Thr Ile Leu Gly His Glu Glu Glu Ile Lys Ala Leu Phe Ala Thr 100 105 110 Leu Ser Asn Gln Glu Leu Val Asp Lys Ile Met Ser Gly Val Arg Lys 115 120 125 Glu Glu Ile Asn Pro Lys Cys Thr His Leu Val Glu Tyr Met Asp Asp 130 135 140 Lys Tyr Pro Phe Tyr Leu Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg 145 150 155 160 Asp Pro Gln Ala Ser Ile Gly His Gly Ile Thr Ile Asn Arg Met Phe 165 170 175 Trp Arg Ala Arg Arg Arg Glu Ser Ile Phe Ile Gln Tyr Ile Val Lys 180 185 190 His His Pro Arg Phe Lys Asp Ala Asn Ile Pro Ile Trp Leu Asp Arg 195 200 205 Asp Cys Pro Phe Asn Ile Glu Gly Gly Asp Glu Leu Val Leu Ser Lys 210 215 220 Asp Val Leu Ala Ile Gly Val Ser Glu Arg Thr Ser Ala Gln Ala Ile 225 230 235 240 Glu Lys Leu Ala Arg Arg Ile Phe Glu Asn Pro Gln Ala Thr Phe Lys 245 250 255 Lys Val Val Ala Ile Glu Ile Pro Thr Ser Arg Thr Phe Met His Leu 260 265 270 Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Met His Ser 275 280 285 Ala Ile Leu Lys Ala Glu Gly Asn Met Asn Ile Phe Ile Ile Glu Tyr 290 295 300 Asp Asp Val Asn Lys Asp Ile Ala Ile Lys Gln Ser Ser His Leu Lys 305 310 315 320 Asp Thr Leu Glu Asp Val Leu Gly Ile Asp Asp Ile Gln Phe Ile Pro 325 330 335 Thr Gly Asn Gly Asp Val Ile Asp Gly Ala Arg Glu Gln Trp Asn Asp 340 345 350 Gly Ser Asn Thr Leu Cys Ile Arg Pro Gly Val Val Val Thr Tyr Asp 355 360 365 Arg Asn Tyr Val Ser Asn Asp Leu Leu Arg Gln Lys Gly Ile Lys Val 370 375 380 Ile Glu Ile Ser Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg 385 390 395 400 Cys Met Ser Gln Pro Leu Phe Arg Glu Asp Ile 405 410 <210> 23 <211> 417 <212> PRT <213> Pseudomonas plecoglossicida <400> 23 Met Ser Ala Glu Lys Gln Lys Tyr Gly Val His Ser Glu Ala Gly Lys 1 5 10 15 Leu Arg Lys Val Met Val Cys Ala Pro Gly Leu Ala His Lys Arg Leu 20 25 30 Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val 35 40 45 Asp Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu 50 55 60 Arg Gly Val Asp Val Leu Glu Met His Asn Leu Leu Thr Asp Ile Val 65 70 75 80 Gln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Pro 85 90 95 Asp Thr Val Gly Val Gly Leu Thr Asn Glu Val Arg Ser Trp Leu Glu 100 105 110 Gly Gln Glu Pro Arg His Leu Ala Glu Phe Leu Ile Gly Gly Val Ala 115 120 125 Gly Gln Asp Leu Pro Glu Ser Glu Gly Ala Ser Val Val Lys Met Tyr 130 135 140 Asn Asp Tyr Leu Gly His Ser Ser Phe Ile Leu Pro Pro Leu Pro Asn 145 150 155 160 Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr 165 170 175 Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr 180 185 190 Thr Ala Ile Tyr Lys Phe His Pro Glu Phe Thr Lys Ala Asp Phe Gln 195 200 205 Val Trp Tyr Gly Asp Pro Asp Gln Glu His Gly Gln Ala Thr Leu Glu 210 215 220 Gly Gly Asp Val Met Pro Ile Gly Lys Gly Ile Val Leu Ile Gly Met 225 230 235 240 Gly Glu Arg Thr Ser Arg Gln Ala Ile Gly Gln Leu Ala Gln Asn Leu 245 250 255 Phe Ala Lys Gly Ala Val Glu Gln Val Ile Val Ala Gly Leu Pro Lys 260 265 270 Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg 275 280 285 Asp Leu Val Thr Val Phe Pro Glu Val Val Arg Glu Ile Val Pro Phe 290 295 300 Ile Ile Arg Pro Asp Glu Ser Lys Pro Tyr Gly Met Asp Val Arg Arg 305 310 315 320 Glu Asn Lys Ser Phe Ile Glu Val Val Gly Glu Gln Leu Gly Val Lys 325 330 335 Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg Glu 340 345 350 Gln Trp Asp Asp Gly Asn Asn Val Val Ala Leu Glu Pro Gly Val Val 355 360 365 Ile Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys Ala 370 375 380 Gly Ile Glu Val Ile Thr Ile Ser Ala Gly Glu Leu Gly Arg Gly Arg 385 390 395 400 Gly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile Asn 405 410 415 Tyr <210> 24 <211> 417 <212> PRT <213> Pseudomonas putida <400> 24 Met Ser Ala Glu Lys Gln Lys Tyr Gly Val His Ser Glu Ala Gly Lys 1 5 10 15 Leu Arg Lys Val Met Val Cys Ala Pro Gly Leu Ala His Lys Arg Leu 020 25 30 Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val 35 40 45 Asp Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu 50 55 60 Arg Gly Val Asp Val Leu Glu Met His Asn Leu Leu Thr Asp Ile Val 65 70 75 80 Gln Asn Lys Asp Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Pro 85 90 95 Asp Thr Val Gly Val Gly Leu Thr Asn Glu Val Arg Ser Trp Leu Glu 100 105 110 Gly Leu Glu Pro Arg His Leu Ala Glu Phe Leu Ile Gly Gly Val Ala ​​​Asn Asp Tyr Leu Gly His Ser Ser Phe Ile Leu Pro Pro Leu Pro Asn 145 150 155 160 Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr 165 170 175 Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr 180 185 190 Thr Ala Ile Tyr Lys Phe His Pro Gln Phe Thr Gly Ala Asp Phe Gln 195 200 205 Val Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Asn Ala Thr Leu Glu 210 215 220 Gly Gly Asp Val Met Pro Ile Gly Lys Gly Ile Val Leu Ile Gly Met 225 230 235 240 Gly Glu Arg Thr Ser Arg Gln Ala Ile Gly Gln Leu Ala Gln Asn Leu 245 250 255 Phe Ala Lys Gly Ala Val Glu Lys Val Ile Val Ala Gly Leu Pro Lys 260 265 270 Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg 275 280 285 Asp Leu Val Thr Ile Phe Pro Glu Val Val Lys Glu Ile Val Pro Phe 290 295 300 Ile Ile Arg Pro Asp Glu Ser Lys Pro Tyr Gly Met Asp Val Arg Arg 305 310 315 320 Glu Asn Lys Ser Phe Ile Glu Val Val Gly Glu Gln Leu Gly Val Lys 325 330 335 Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg Glu 340 345 350 Gln Trp Asp Asp Gly Asn Asn Val Val Ala Val Glu Pro Gly Val Val 355 360 365 Ile Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys Ala 370 375 380 Gly Ile Glu Val Ile Thr Ile Ser Ala Gly Glu Leu Gly Arg Gly Arg 385 390 395 400 Gly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile Asp 405 410 415 Tyr[[ID=3​​​​​​​​​​​​​​Leu Arg Lys Val Met Val Cys Ser Pro Gly Leu Ala His Gln Arg Leu 20 25 30 Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val 35 40 45 Asn Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu 50 55 60 Arg Gly Ile Asp Val Leu Glu Met His Asn Leu Leu Thr Glu Thr Ile 65 70 75 80 Gln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Ala 85 90 95 Asp Ser Val Gly Leu Gly Leu Thr Ser Glu Leu Arg Ser Trp Leu Glu 100 105 110 Ser Leu Glu Pro Arg Lys Leu Ala Glu Tyr Leu Ile Gly Gly Val Ala 115 120 125 Ala Asp Asp Leu Pro Ala Ser Glu Gly Ala Asn Ile Leu Lys Met Tyr 130 135 140 Arg Glu Tyr Leu Gly His Ser Ser Phe Leu Leu Pro Pro Leu Pro Asn 145 150 155 160 Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr 165 170 175 Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr 180 185 190 Thr Ala Ile Tyr Lys Phe His Pro Glu Phe Ala Asn Ala Glu Phe Glu 195 200 205 Ile Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Ser Ser Thr Leu Glu 210 215 220 Gly Gly Asp Val Met Pro Ile Gly Asn Gly Val Val Leu Ile Gly Met 225 230 235 240 Gly Glu Arg Ser Ser Arg Gln Ala Ile Gly Gln Val Ala Gln Ser Leu 245 250 255 Phe Ala Lys Gly Ala Ala Glu Arg Val Ile Val Ala Gly Leu Pro Lys 260 265 270 Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg 275 280 285 Asp Leu Val Thr Val Phe Pro Glu Val Val Lys Glu Ile Val Pro Phe 290 295 300 Ser Leu Arg Pro Asp Ala Ser Ser Pro Tyr Gly Met Ser Ile Arg Arg 305 310 315 320 Glu Glu Lys Thr Phe Leu Glu Val Val Ala Glu Ser Leu Gly Leu Lys 325 330 335 Lys Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg 340 345 350 Glu Gln Trp Asp Asp Gly Asn Asn Val Val Cys Leu Glu Pro Gly Val 355 360 365 Val Val Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys 370 375 380 Ala Gly Val Glu Val Ile Thr Ile Ser Ala Ser Glu Leu Gly Arg Gly 385 390 395 400 Arg Gly Gly Gly His Cys Met Thr Cys Pro Ile Ile Arg Asp Pro Ile 405 410 415 Asp Tyr <210> 26 <211> 402 <212> PRT <213> Binding to Mycobacterium tuberculosis complex <400> 26 Met Gly Val Glu Leu Gly Ser Asn Ser Glu Val Gly Ala Leu Arg Val 1 5 10 15 Val Ile Leu His Arg Pro Gly Ala Glu Leu Arg Arg Leu Thr Pro Arg 20 25 30 Asn Thr Asp Gln Leu Leu Phe Asp Gly Leu Pro Trp Val Ser Arg Ala 35 40 45 Gln Asp Glu His Asp Glu Phe Ala Glu Leu Leu Ala Ser Arg Gly Ala 50 55 60 Glu Val Leu Leu Leu Ser Asp Leu Leu Thr Glu Ala Leu His His Ser 65 70 75 80 Gly Ala Ala Arg Met Gln Gly Ile Ala Ala Ala Val Asp Ala Pro Arg 85 90 95 Leu Gly Leu Pro Leu Ala Gln Glu Leu Ser Ala Tyr Leu Arg Ser Leu 100 105 110 Asp Pro Gly Arg Leu Ala His Val Leu Thr Ala Gly Met Thr Phe Asn 115 120 125 Glu Leu Pro Ser Asp Thr Arg Thr Asp Val Ser Leu Val Leu Arg Met 130 135 140 His His Gly Gly Asp Phe Val Ile Glu Pro Leu Pro Asn Leu Val Phe 145 150 155 160 Thr Arg Asp Ser Ser Ile Trp Ile Gly Pro Arg Val Val Ile Pro Ser 165 170 175 Leu Ala Leu Arg Ala Arg Val Arg Glu Ala Ser Leu Thr Asp Leu Ile 180 185 190 Tyr Ala His His Pro Arg Phe Thr Gly Val Arg Arg Ala Tyr Glu Ser 195 200 205 Arg Thr Ala Pro Val Glu Gly Gly Asp Val Leu Leu Leu Ala Pro Gly 210 215 220 Val Val Ala Val Gly Val Gly Glu Arg Thr Thr Pro Ala Gly Ala Glu 225 230 235 240 Ala Leu Ala Arg Ser Leu Phe Asp Asp Asp Leu Ala His Thr Val Leu 245 250 255 Ala Val Pro Ile Ala Gln Gln Arg Ala Gln Met His Leu Asp Thr Val 260 265 270 Cys Thr Met Val Asp Thr Asp Thr Met Val Met Tyr Ala Asn Val Val 275 280 285 Asp Thr Leu Glu Ala Phe Thr Ile Gln Arg Thr Pro Asp Gly Val Thr 290 295 300 Ile Gly Asp Ala Ala Pro Phe Ala Glu Ala Ala Ala Lys Ala Met Gly 305 310 315 320 Ile Asp Lys Leu Arg Val Ile His Thr Gly Met Asp Pro Val Val Ala 325 330 335 Glu Arg Glu Gln Trp Asp Asp Gly Asn Asn Thr Leu Ala Leu Ala Pro 340 345 350 Gly Val Val Val Ala Tyr Glu Arg Asn Val Gln Thr Asn Ala Arg Leu 355 360 365 Gln Asp Ala Gly Ile Glu Val Leu Thr Ile Ala Gly Ser Glu Leu Gly 370 375 380 Thr Gly Arg Gly Gly Pro Arg Cys Met Ser Cys Pro Ala Ala Arg Asp 385 390 395 400 Pro Leu <210> 27 <211> 409 <212> PRT <213> Mycoplasma arthritidis <400> 27 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 Lys 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 Ala Glu 50 55 60 Leu Lys Lys Arg Gly Ile Asn Val Val Glu Leu Val Asp Leu Ile Val 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Lys Glu Lys Leu Leu Glu 85 90 95 Glu Phe Leu Asp Asp Ser Val Pro Val Leu Ser Asp Glu His Arg Ala 100 105 110 Ala Val Lys Lys Phe Leu Gln Ser Gln Lys Ser Thr Arg Ser Leu Val 115 120 125 Glu Tyr Met Ile Ala Gly Ile Thr Lys His Asp Leu Lys Ile Glu Ser 130 135 140 Asp Leu 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 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 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 Asp 290 295 300 Ala Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Glu Asp Leu Leu Lys 305 310 315 320 Ser Ile Ile Gly Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Ala Gly 325 330 335 Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn 340 345 350 Tyr Leu Ala Val Ala Pro Gly Ile Val Ile Gly Tyr Ala Arg Asn Glu 355 360 365 Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Thr Val Leu Pro Phe 370 375 380 Arg Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser 385 390 395 400 Met Pro Leu Ser Arg Lys Asp Val Lys 405 <210> 28 <211> 410 <212> PRT <213> Artificial Sequence <220> <223> Artificial full - length Mycoplasma phocicerebrale <400> 28 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 ​​​​​​​​​​​​​​​​​​​​​​​​​​Glu Phe 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 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 Glu Thr Ile Thr Leu 225 230 235 240 Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg Ile 245 250 255 Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr 260 265 270 Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala 275 280 285 Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala 290 295 300 Glu Pro Gln Pro Lys Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln 305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Asn Asn 325 330 335 Ala Ser His Ile Asp Ile Glu 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 Ala Ala Leu Ala 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> 29 <211> 410 <212> PRT <213> Artificial sequence <220> <223> Artificial full-length Mycoplasma gateae <400> 29 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 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 Leu Phe Val Ser Glu 50 55 60 Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val Thr 65 70 75 80 Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Asn Leu Ile Glu 85 90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Thr Glu Glu Leu Lys Ser 100 105 110 Val Val Arg Thr Tyr Leu Lys Ser Ile Lys Ser Thr Arg Glu Leu Ile 115 120 125 Gln 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 Ile Tyr Asn Asn Asn Thr 210 215 220 Leu Val Val 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 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 Glu 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 Ile Asp 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> 30 <211> 410 <212> PRT <213> Artificial Sequence <220> <223> Artificial full-length Mycoplasma phocidae[[ID=�6]] <400> 30 Met Ser Val Phe Asp Ser Lys Phe Asn 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<〇〇〇2581>20 25 30 Glu 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 Glu Phe Val Ala Glu 50 55 60 Leu Lys Lys Asn Asn Ile Asn Val Val Glu Leu Thr Asp Leu Val Ser 65 70 75 80 Glu Thr Tyr Asp Met Val Ser Lys Glu Lys Gln Glu Lys Leu Ile Glu 85 90 95 Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Gly 100 105 110 Leu Val Arg Lys Phe Leu Lys Ser Leu Lys Ser Ser Lys Glu Leu Ile 115 120 125 Gln Tyr Met Met Ala Gly Ile Thr Lys His Asp Leu Asn 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 Ile Phe Ala 180 185 190 Asn His Pro Lys Leu Met Asn Thr Pro Leu Tyr Tyr Asn Pro Asp Met 195 200 205 Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Asn Glu 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 Arg 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 Asp 290 295 300 Glu Pro Gln Pro Lys Val Asn Gly Leu Pro Leu Glu Lys Leu Leu Glu 305 310 315 320 Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Thr Ser 325 330 335 Ala Ser Asn Ile Asp Val 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 Val 355 360 365 Lys Thr Asn Glu Ala Leu Glu Ala 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> 31 <211> 410 <212> PRT <213> Mycoplasma salivarium <400> 31 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> 32 <211> 411 <212> PRT <213> Mycoplasma spumans <400> 32 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​​​​​​​​​​​​​​​​​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<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 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 Light Light Light 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 [[ID=1十四]]<210> 35 <211> 409 <212> PRT <213> Mycoplasma cloacale <400> 35 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<00​​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> 36 <211> 412 <212> PRT <213> Mycoplasma alkalescens <400> 36 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<00​​​​​​​​​​​​​​​​​​​ 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> 38 <211> 401 <212> PRT <213> Mycoplasma gallinarum <400> 38 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> 39 <211> 405 <212> PRT <213> Mycoplasma pirum <400> 39 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> 40 <211> 404 <212> PRT <213> Mycoplasma primatum <400> 40 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> 41 <211> 401 <212> PRT <213> Mycoplasma lipofaciens <400> 41 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> 42 <211> 405 <212> PRT <213> Mycoplasma felifaucium <400> 42 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> 43 <211> 404 <212> PRT <213> Mycoplasma imitans <400> 43 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> 44 <211> 401 <212> PRT <213> Mycoplasma opalescens <400> 44 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> 45 <211> 425<00034२4><212> PRT <213> Mycoplasma moatsii <400> 45 Met Lys Lys Asn Ala Ile Asn Val Tyr Ser Glu Ile Gly Lys Leu Lys 1 5 10 1५ Lys Val Leu Val His Arg Pro Gly Asp Glu Leu Lys Tyr Val Thr Pro 20 25 30 Gln Arg Met Asp Glu Leu Leu Met Ser Ala Ile Ile Glu Leu Glu Gln<00034३2>[ 35 40 45 Ala Lys Glu Glu His Asp Ala Phe Thr Lys Ile Leu Arg Asp Asn Gly 5० 55 60 Val Glu Val Ile Glu Leu Ala Asp Leu Thr Ala Glu Met Tyr Asp Ser 65 70 75 80 It should be noted that there may be some errors in the original text where "२" appears in the tag <00034२4>, which should probably be a normal number or character. This translation is done based on the best understanding of the provided content.Leu Thr Pro Ser Glu Lys Asp Ala Phe Leu Asn Gln Trp Val Lys Glu 85 90 95 Ala Ser Trp Gly Lys Lys Ser Ser Ile Asp Ala Leu Lys Ile Lys Lys 100 105 110 Asn Leu Ser Lys Lys Val Phe Asp Tyr Val Lys Ser Ile Lys Pro Thr 115 120 125 Arg Lys Met Ile Asp Lys Leu Met Ala Gly Val Leu Leu Ser Glu Ile 130 135 140 Gly Glu Lys Ser Ile Ile Leu Asn Lys Asp Lys Lys Asn Glu Met Val 145 150 155 160 Ile Asp Leu Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp 165 170 175 Pro Phe Ala Ser Val Gly Asn Gly Ile Thr Leu His Asn Met Lys Tyr 180 185 190 Pro Thr Arg Lys Arg Glu Thr Ile Phe Ala Gln Trp Ile Phe Asn Lys 195 200 205 His Pro Glu Tyr Lys Asp Val Pro Gln Phe Ile Ser Lys Arg Asp Gly 210 215 220 Lys Glu Thr Ile Glu Gly Gly Asp Val Phe Ile Tyr Thr Lys Asp Val 225 230 235 240 Leu Ala Ile Gly Val Ser Glu Arg Thr Asn Met Glu Ala Ile Leu Arg 245 250 255 Ile Ala Thr Asn Ile Lys Lys Asp Lys Asn Cys Glu Phe Lys Lys Ile 260 265 270 Val Ala Ile Asn Val Pro Pro Met Gly Asn Leu Met His Leu Asp Thr 275 280 285 Trp Leu Thr Met Leu Asp Lys Asp Leu Phe Leu Tyr Ser Gly Asn Ile 290 295 300 Lys Ser Ala Leu Lys Val Trp Glu Ile Asp Leu Thr Lys Pro Ile Thr 305 310 315 320 Pro Lys Ser Pro Lys Leu Ser Thr Ala Lys Leu Ala Asp Ile Leu Ala 325 330 335 Lys Ile Val Gly Lys Lys Val Arg Met Ile Pro Ile Gly Gly Lys Asp 340 345 350 Gly Asn Gln Met Asp Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn 355 360 365 Tyr Leu Ala Ile Ala Pro Gly Val Val Val Gly Tyr His Arg Asn Arg 370 375 380 Lys Thr Gln Lys Ala Leu Glu Glu Ala Gly Val Lys Val Leu Ala Phe 385 390 395 400 Gln Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser 405 410 415 Met Pro Leu Val Arg Glu Glu Val Lys 420 425 <210> 46 <211> 399 <212> PRT <213> Mycoplasma elephantis <400> 46 Met Ser Gln Ile Asn Val Phe Ser Glu Ile Gly Gln Leu Lys Glu Val 1 5 10 15 Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Lys Arg 20 25 30 Tyr Asn Glu Leu Leu Phe Ser Ala Ile Leu Glu Ala Asp Val Ala Ile 35 40 45 Lys Glu His Lys Ser Phe Val Lys Ile Leu Glu Glu Asn Asn Val Lys 50 55 60 Val Ile Gln Leu Lys Asp Ile Leu Leu Glu Thr Trp Asn Ile Cys Ser<​​​​​​​​100 105 110 Lys Ser Lys Thr Pro Leu Glu Ile Ile Asp Ile Met Met Lys Gly Ile 115 120 125 Leu Lys Gln Glu Leu Gly Ile Glu Tyr Lys His Glu Leu Ile Ile Asp 130 135 140 Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Thr Ser Met Gly 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 ...

Claims

1. Use of a combination of (a) an arginine depleting agent and (b) a cancer immunotherapeutic agent in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the cancer immunotherapeutic agent is a PD-1 antagonist or a PD-L1 antagonist, wherein the subject's cancer is a cancer targeted by immunotherapy treatment, wherein the arginine depleting agent is ADI-PEG, the ADI is from Mycoplasma hominis, the PEG has an overall average molecular weight of 20,000 Daltons, the cancer is colorectal cancer, and The PD-1 antagonist is a PD-1 antibody, and the PD-L1 antagonist is a PD-L1 antibody.

2. The use according to claim 1, wherein the PD-L1 antagonist is selected from one or more of atezolizumab (MPDL3280A), avelumab (MSB0010718C) and durvalumab (MEDI4736).

3. The use according to claim 1, wherein the PD-1 antagonist is selected from one or more of nivolumab, pembrolizumab, PDR001 and pidilizumab.

4. The use according to claim 3, wherein the PD-1 antagonist is nivolumab.

5. The use according to claim 3, wherein the PD-1 antagonist is pembrolizumab.

6. The use according to any one of claims 1 to 5, wherein (a) and (b) are administered separately.

7. Use according to any one of claims 1 to 5, wherein (a) and (b) are administered together as part of the same composition.

8. A patient care kit comprising: (a) Arginine depleting agents; as well as (b) PD-1 antagonist or PD-L1 antagonist, wherein the arginine depleting agent is ADI-PEG, the ADI is from Mycoplasma hominis, the PEG has a total average molecular weight of 20,000 Daltons, and The PD-1 antagonist is a PD-1 antibody, and the PD-L1 antagonist is a PD-L1 antibody.

9. The patient care kit of claim 8, wherein (a) and (b) are in separate compositions.

10. The patient care kit of claim 8, wherein (a) and (b) are in the same composition.

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