Modified arginine deiminase

Abstract

The present invention provides modified arginine deiminase (ADI) proteins comprising ADI proteins comprising one or more substitutions that increase inclusion bodies that exhibit insoluble and refoldable properties in bacteria. Also provided are methods of producing the modified ADI proteins, compositions comprising the ADI proteins, and related methods of treating arginine-dependent and related diseases, such as cancer.

Description

Technical Field

[0001] Broadly speaking, the present invention relates to modified arginine deiminase (ADI) proteins (including ADI proteins containing one or more substitutions that increase expression as insoluble and refoldable inclusion bodies in bacteria), methods for producing such modified ADI proteins, compositions comprising such ADI proteins, and related methods for treating arginine-dependent and related diseases such as cancer. Background Art

[0002] Compared with other diseases, arginine depletion therapy can be an effective treatment for certain forms of cancer. For example, polyethylene glycolylated arginine deiminase (ADI-PEG) can be used to deplete its blood supply by converting arginine into citrulline and ammonia. ADI-PEG 20 is an exemplary ADI-PEG that is being studied clinically for tumors lacking the key enzyme argininosuccinate synthetase-1 (ASS1) involved in the conversion of citrulline to arginine. ADI-PEG 20 has been well tolerated and shown potential in clinical studies (see, for example, Qiu et al., Cancer Lett. 2015 Aug 1; 364(1):1-7; Phillips et al., Cancer Res Treat. 2013 Dec; 45(4):251-62; Feun et al., Curr Pharm Des. 2008; 14(11):1049-57; Feun and Savaraj, Expert Opin Investig Drugs. 2006 Jul; 15(7):815-22; Feun et al., Curr Opin Clin Nutr Metab Care. 2015 Jan; 18(1):78-82).

[0003] Soluble, active, high-level expression of the protein of interest is generally the ultimate goal of biotechnology. However, soluble expression of ADI proteins presents unique problems. Since some ADIs are soluble, highly active, and have a low Km for the substrate (<10 uM), these enzymes are capable of depleting the intracellular arginine of the host cells in which they are expressed. Escherichia coli (E. coli) can convert citrulline back to arginine via argininosuccinate synthetase and argininosuccinate lyase, but this competing process places excessive stress on the host cells, as evidenced by low cell density and low-level protein expression. Additionally, constant arginine turnover reduces the tRNA pool of arginine, and thereby limits the amount of overexpressed ADI protein in the host cells.

[0004] Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble precipitates called inclusion bodies (see, e.g., Taylor et al., Bio / Technology 4, 553, 1986). Over the years, a great deal of research has been carried out to try to convert insoluble expression into soluble expression in order to functionally characterize the protein of interest up to commercial scale production for industrial, biotechnological, or pharmaceutical applications (see, e.g., Misawa and Kumagai, Biopolymers 51:297 - 307, 1999). That is, when all other methods fail, the inclusion bodies themselves can be used to attempt to denature and refold the aggregated protein into a properly folded functional state. This process can be extremely inefficient, difficult, and time - and resource - expensive. Each protein is unique and requires an appropriate combination of denaturants, dilution ratios, dilution rates, dialysis conditions, solid - phase carriers, incubation temperatures, pH, salt concentrations, incubation times, excipients (such as glycerol, diols), solvents, arginine, detergents, oxidizing and reducing agents, cofactors, etc. to achieve proper protein refolding (see the above references and Middleberg, Trends in Biotech. 20:437 - 443, 2002).

[0005] Given these challenges, there is a need to increase the conversion of soluble ADI protein into insoluble / refoldable inclusion bodies, and thereby minimize the stress on host cells from soluble ADI expression and optimize the recombinant production of ADI protein. Such an approach would allow for the production of ADI protein at the scale required for drug development and enhance the commercial potential of ADI protein as a therapeutic enzyme. Summary of the Invention

[0006] Certain embodiments include an isolated arginine deiminase (ADI) that comprises, consists of, or consists essentially of an amino acid sequence that is at least 90, 95, 96, 97, 98, 99, or 100% identical to an amino acid sequence selected from Tables A1.1 and A1.2 (excluding SEQ ID NO:1).

[0007] In some embodiments, the isolated ADI is recombinantly expressed (or can be expressed) as an insoluble and refoldable inclusion body in a bacterial host cell, optionally Escherichia coli. In some embodiments, at least about 10 - 100% or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the ADI is recombinantly expressed (or can be expressed) as an insoluble and refoldable inclusion body in a bacterial host cell.

[0008] In some embodiments, the isolated ADI has ADI activity under physiological conditions of temperature, salinity, and pH as appropriate. In some embodiments, the isolated ADI has at least about 50, 60, 70, 80, 90, 100, 110, or 120% ADI activity under comparable physiological conditions relative to the isolated ADI consisting of SEQ ID NO:1 (wild-type Mycoplasma columbinum).

[0009] Certain isolated ADIs include an amino acid sequence that is at least 90, 95, 96, 97, 98, 99, or 100% identical to, consists of, or consists essentially of an amino acid sequence selected from SEQ ID NOs: 2-178, which amino acid sequence retains one or more lysine substitutions selected from K2G, K13E, K63N, K82S, K90T, K90V, K101D, K106L, K108R, K108A, K131R, K170R, K175R, K192V, K192C, K216N, K216V, K229L, K237N, K238N, K240V, K243T, K246E, K248R, K249R, K273R, K275A, K287A, K287Q, K287C, K295A, K295I, K304L, K317R, K326A, and K400A (relative to SEQ ID NO:1). Certain isolated ADIs retain 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, 26, 27, 28, or 29 lysine substitutions selected from K2G, K13E, K63N, K82S, K90T, K90V, K101D, K106L, K108R, K108A, K131R, K170R, K175R, K192V, K192C, K216N, K216V, K229L, K237N, K238N, K240V, K243T, K246E, K248R, K249R, K273R, K275A, K287Q, K287C, K287A, K295A, K295I, K304L, K317R, K326A, and K400A (relative to SEQ ID NO:1), such as all or some of the lysine substitutions indicated for the selected sequences in Table A2 and Table 3.

[0010] In some embodiments, the isolated ADI is covalently bonded via a linker to at least one PEG molecule. In some embodiments, the isolated ADI is covalently bonded to from about 1 to about 10 PEG molecules. In some embodiments, the isolated ADI is covalently bonded to from about 2 to about 8 PEG molecules. In some embodiments, the PEG molecule is a linear or branched PEG molecule. In some embodiments, the PEG has a total weight average molecular weight of from about 1,000 to about 40,000, optionally from about 2,000 to about 20,000, optionally from about 2,000 to about 10,000, optionally about 5,000.

[0011] In some embodiments, the linker is a succinyl group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, a histidine group, a methylene group, or any combination thereof. In some embodiments, the source of the succinyl group is methoxy-PEG succinimidyl carboxymethyl ester (SCM) or N-hydroxysuccinimide (NHS).

[0012] Also included are compositions (including therapeutic compositions) containing the isolated arginine deiminase (ADI) described herein and a pharmaceutically acceptable carrier.

[0013] In some embodiments, the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% (protein-based or weight-weight-based) and is substantially free of aggregates. Certain compositions are substantially endotoxin-free.

[0014] Also included are methods of treating, ameliorating the symptoms of cancer, or inhibiting cancer progression in a subject in need thereof, which comprise administering to the subject a therapeutic composition or isolated ADI as described herein.

[0015] In some embodiments, the cancer is one or more selected from the following: hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myelogenous leukemia, relapsed acute myelogenous leukemia, B cell malignancies, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma (e.g., astrocytoma, oligodendroglioma, ependymoma, or choroid plexus papilloma), glioblastoma multiforme (e.g., giant cell glioblastoma or gliosarcoma), meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), non-small cell lung cancer (NSCLC), renal cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, and gastric cancer. In some embodiments, the cancer exhibits decreased argininosuccinate synthetase-1 expression.

[0016] Certain embodiments include an isolated polynucleotide encoding one or more of the isolated arginine deiminases (ADIs) described herein or a vector comprising the polynucleotide. Some polynucleotides or vectors include at least one AAG codon encoding a lysine residue, such as the K317 residue. Some polynucleotides or vectors include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 AAG codons encoding lysine residues.

[0017] Also included are recombinant bacterial host cells containing the nucleotides or vectors described herein, such as Escherichia coli.

[0018] Some embodiments include a method of recombinantly producing an isolated arginine deiminase (ADI), which includes

[0019] (a) expressing ADI in a recombinant bacterial host cell described herein, wherein the ADI is expressed in the host cell as an insoluble and refoldable inclusion body;

[0020] (b) removing the insoluble inclusion body from the host cell;

[0021] (c) purifying the ADI from the insoluble inclusion body;

[0022] (d) refolding the ADI in a refolding buffer; and

[0023] (e) purifying the ADI from the refolding buffer,

[0024] thereby producing an isolated ADI

[0025] In some embodiments, at least about 10 - 100% or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the ADI is expressed in the bacterial host cell as an insoluble and refoldable inclusion body. Certain embodiments further include measuring the ADI activity of the isolated ADI under physiological conditions of optionally temperature, salinity, and pH, wherein the isolated ADI has ADI activity under physiological conditions.

[0026] In some embodiments, the isolated ADI has at least about 50, 60, 70, 80, 90, 100, 110, or 120% of the ADI activity of the isolated ADI consisting of SEQ ID NO:1 (wild-type Mycoplasma gallisepticum) under comparable physiological conditions.

[0027] Certain embodiments further include preparing a therapeutic composition comprising isolated ADI, for example, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% (protein-based or weight-by-weight), and / or wherein the composition is substantially free of aggregates and substantially free of endotoxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] None. DETAILED DESCRIPTION

[0029] 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 invention belongs. Although any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this invention, the preferred methods and materials are described. The entire contents of all publications and references (including but not limited to patents and patent applications) cited in this specification are incorporated herein by reference as if each individual publication or reference were specifically and individually indicated to be incorporated by reference in its entirety. The entire contents of any patent application for which priority is claimed in this application are also incorporated herein by reference in the manner described above for publications and references.

[0030] Unless otherwise indicated specifically, the practice of the present invention will employ conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques that are well known to those of ordinary skill in the art. Many of these methods are described below for illustrative purposes. These techniques are fully explained in the literature. See, for example, Current Protocols in Protein Science, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Volumes I & II (D. Glover ed.); Oligonucleotide Synthesis (N. Gait ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins eds., 1985); Transcription and Translation (B. Hames & S. Higgins eds., 1984); Animal Cell Culture (R. Freshney ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0031] For recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation, standard techniques (e.g., electroporation, lipofection) can be used. 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 various general and more specific references, which are cited and discussed throughout this specification. Unless otherwise provided with specific definitions, the nomenclature and laboratory procedures and techniques utilized in connection with the molecular biology, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those that are well known and commonly used in the art.

[0032] Recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment can use standard techniques.

[0033] For the purposes of the present invention, the following terms are defined hereinbelow.

[0034] As used herein, the articles “a” and “an” refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, “a component” means one component or more than one component.

[0035] “About” means that a quantity, degree, value, number, frequency, percentage, size, dimension, amount, weight or length varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference quantity, degree, value, number, frequency, percentage, size, dimension, amount, weight or length.

[0036] As used herein, the term “amino acid” is intended to refer to natural and unnatural amino acids, as well as amino acid analogs and mimetics. Natural amino acids include the 20 (L)-amino acids used during protein biosynthesis, as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine. Unnatural amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like known to those skilled in the art. Amino acid analogs include modified forms of natural and unnatural amino acids. Such modifications can include, for example, substituting or replacing chemical groups and moieties on the amino acid or derivatizing the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functional similar properties (such as charge and charge-spacing characteristics) of a reference amino acid. For example, an organic structure mimicking arginine (Arg or R) has a positively charged moiety located in a similar molecular space and having the same mobility as the side-chain e-amino group of the natural Arg amino acid. Mimetics also include constrained structures to maintain optimal spacing and charge interactions of the amino acid or amino acid functional groups. Those skilled in the art know or can determine the structures that constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0037] “Biocompatible” means a material or compound that is generally not harmful to biological functions and does not produce any degree of unacceptable toxicity (including sensitization and disease states).

[0038] “Coding sequence” means any nucleic acid sequence that contributes to encoding the polypeptide product of a gene. In contrast, the term “non-coding sequence” means any nucleic acid sequence that does not directly contribute to encoding the polypeptide product of a gene.

[0039] Throughout the present invention, unless the context requires otherwise, the word "comprise", "comprises" and "comprising" shall be understood to mean including the stated step or element or steps or element groups, but not excluding any other step or element or steps or element groups.

[0040] "Consisting of" means including (and limited to) whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that no other elements may be present. "Consisting essentially of" means including any of the elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or function specified for the listed elements in the present invention. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present, depending on whether such other elements significantly affect the activity or function of the listed elements.

[0041] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents and / or containers containing up to trace amounts (e.g., amounts that do not have clinically adverse physiological effects on a subject) of endotoxin and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms (e.g., bacteria, typically gram-negative bacteria), but endotoxins can be found in gram-positive bacteria (e.g., Listeria monocytogenes). The most prevalent endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in the outer membranes of various gram-negative bacteria, which represent a major pathogenicity feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can produce fever, decreased blood pressure, and inflammation and activation of blood clotting, as well as other adverse physiological effects.

[0042] Thus, in pharmaceutical production, it is generally desirable to remove most or all trace amounts of endotoxin from drugs and / or drug containers, since even very small amounts can cause adverse effects in humans. Depyrogenation ovens can be used for this purpose, since temperatures in excess of 300 °C are generally required to decompose most endotoxins. For example, based on the primary packaging material (e.g., syringes or vials), a combination of a glass temperature of 250 °C and a holding time of 30 minutes is generally sufficient to achieve a 3-log reduction in endotoxin content. Other methods for removing endotoxin are also covered, including (e.g.) chromatography and filtration methods as described herein and known in the art.

[0043] Conventional techniques known in the art can be used to detect endotoxins. For example, the Limulus amebocyte lysate assay (which utilizes blood from the horseshoe crab) is a highly sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of the Limulus lysate due to the presence of a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, the endotoxin content can be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10 EU / mg of the active compound. Generally, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1 - 10 EU.

[0044] The "half-life" of a polypeptide can refer to the time required for the polypeptide to lose half of its pharmacological, physiological or other activity relative to its activity when administered to the serum or tissue of an organism or relative to any other specified time point. The "half-life" can also refer to the time required for the amount or concentration of the polypeptide to decrease by half from its starting amount in the serum or tissue of an organism relative to the amount or concentration when administered to the serum or tissue of an organism or relative to any other specified time point. The half-life can be measured in serum and / or any one or more selected tissues.

[0045] The terms "modulate" and "alter" include generally "increase", "enhance" or "stimulate" as well as "decrease" or "reduce" relative to a control by a statistically significant or physiologically significant amount or degree. An "increase", "stimulate" or "enhance" amount is generally a "statistically significant" amount and can include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and ranges therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) compared to the amount produced by a composition-free (e.g., in the absence of an agent) or control composition. A "decrease" or "reduce" amount is generally a "statistically significant" amount and can include a decrease 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% (including all integers and ranges therebetween) compared to the amount produced by a composition-free (e.g., in the absence of an agent) or control composition. Examples of the comparison and "statistically significant" amounts are set forth herein.

[0046] The terms "polypeptide", "protein", and "peptide" are used interchangeably and refer to amino acid polymers of any specific length. The term "enzyme" includes polypeptide or protein catalysts, and ADI can be used interchangeably with protein, polypeptide, or peptide. These terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term "polypeptide" or "protein" refers to one or more amino acid chains, where each chain includes amino acids covalently linked by peptide bonds, and where the polypeptide or protein can include multiple chains non-covalently and / or covalently linked together and having the sequence of a native protein (i.e., a protein produced by natural and specifically non-recombinant cells or genetically engineered or recombinant cells), and includes molecules having the amino acid sequence of a native protein or molecules having deletions, additions, and / or substitutions of one or more amino acids from the native sequence. The terms "polypeptide" and "protein" specifically encompass the ADI enzyme / protein described herein or sequences having deletions, additions, and / or substitutions of one or more amino acids from the ADI protein. In certain embodiments, the polypeptide is a "recombinant" polypeptide produced by recombinant cells comprising one or more recombinant DNA molecules, typically made from a combination of heterologous polynucleotide sequences or polynucleotide sequences not originally found in the cell.

[0047] The term "isolated" polypeptide or protein as referred to herein means that the target protein (1) is free of at least some of the other proteins normally found associated with it in nature, (2) is substantially free of other proteins from the same source (e.g., from the same species), (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other substances with which it is associated in nature, (5) is not associated (by covalent or non-covalent interactions) with the protein moieties with which the "isolated protein" is associated in nature, (6) is operably associated (by covalent or non-covalent interactions) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. This isolated protein can be encoded by genomic DNA, cDNA, mRNA, or other RNA, can be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in its native environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or other uses).

[0048] In certain embodiments, the “purity” of any given agent (e.g., ADI or PEGylated ADI) in a composition may be specifically defined. For example, certain compositions may include an agent that is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% pure (e.g., based on the protein, including all decimals and ranges therebetween), as measured (e.g.,) by high performance liquid chromatography (HPLC), a well-known column chromatography format commonly used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0049] The term “reference sequence” generally refers to a nucleic acid coding sequence or an amino acid sequence to which another sequence is compared. All polypeptide and polynucleotide sequences set forth herein are included as reference sequences, including those set forth by name and those set forth in the tables and sequence listings.

[0050] As used herein, the term “sequence identity” (or e.g., including “sequence 50% identical”) refers to the degree of identity of a sequence based on each nucleotide or based on each amino acid in a comparison window. Thus, the “percent sequence identity” can be calculated by: comparing two optimally aligned sequences in a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity. The optimal alignment of sequences for comparison windows can be conducted by computerized implementation algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis, USA) or by inspection of the best alignment generated using any of a variety of methods selected (i.e., yielding the highest percent homology in the comparison window). Reference may also be made to the BLAST program listings, as disclosed, e.g., by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0051] The term "solubility" refers to the property of the agents provided herein (e.g., ADI or PEGylated ADI) to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration in the following forms: mass of solute / volume of unit solvent (g solute / kg solvent, g / dL (100 mL), mg / ml, etc.), molarity, molality, mole fraction, or other similar concentration presentation forms. The maximum equilibrium amount of solute that can be dissolved in each amount of solvent is the solubility of the solute in that solvent under specified conditions (including temperature, pressure, pH, and solvent properties). In certain embodiments, solubility is measured at physiological pH or other pH values (e.g., at 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 - 8)). In certain embodiments, solubility is measured in water or a physiological buffer (e.g., PBS or NaCl (with or without NaP)). In a specific embodiment, solubility is measured at a relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO4). In certain embodiments, solubility is measured in a biological fluid (solvent) (e.g., blood or serum). In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25 °C) or about body temperature (37 °C). In certain 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 at 37 °C.

[0052] "Subject" or "subject in need" or "patient" or "patient in need" includes mammalian subjects, such as human subjects.

[0053] "Substantially" or "essentially" means almost entirely or completely, e.g., 95%, 96%, 97%, 98%, 99%, or higher of a given quantity.

[0054] "Statistically significant" means that the result is not likely to occur by chance. Statistical significance can be determined by any method known in the art. Common measures of significance include the p-value, which is the frequency or probability of the observed event occurring when the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In a simple case, the significance level is defined at a p-value of 0.05 or less.

[0055] "Treatment response" refers to symptom improvement (whether continuous or not) based on the administration of one or more therapeutic agents.

[0056] As used herein, "treating" a subject (e.g., a mammal, such as a human) or "processing" a cell is any type of intervention used to attempt to alter a natural process in an individual or cell. Treating includes (but is not limited to) administering a pharmaceutical composition and can be implemented prophylactically, or after the onset of a pathological event; or contact with a pathogen. Also included is "prophylactic" treatment, which can involve reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

[0057] The term "wild type" refers to the gene or gene product (e.g., polypeptide) most commonly observed in a population and thus the "normal" or "wild type" form of any arbitrarily designed gene.

[0058] Unless otherwise explicitly stated, each embodiment in this specification applies to every other embodiment.

[0059] Throughout the present invention, the following abbreviations may be used: PEG, polyethylene glycol; ADI, arginine deiminase; SS, succinimidyl succinate (succinic acid succinimide ester); SSA, succinimidyl succinate; SPA, succinimidyl propionate (propionic acid succinimide ester); NHS, N-hydroxy-succinimide; ASS-1, argininosuccinate synthetase-1 (argininosuccinate synthetase-1)

[0060] Modified arginine deiminase

[0061] Certain embodiments are directed to an isolated arginine deiminase ("ADI" or "ADI protein", including its variants / fragments and pegylated forms) derived from wild-type ADI of Mycoplasma gallisepticum, which is modified to increase insolubility and refoldable inclusion bodies expressed in bacteria. In some cases, the ADI protein is modified to include one or more amino acid substitutions of solvent-accessible residues. In some cases, the ADI protein is modified to include one or more substitutions of wild-type lysine residues. In some cases, the coding sequence of the ADI protein is modified to include one or more non-preferred codons encoding lysine residues, such as the AAG codon instead of the preferred AAA codon.

[0062] In some embodiments, the ADI protein is recombinantly expressed (or expressible) in bacterial host cells as insoluble and refoldable inclusion bodies. For example, in some embodiments, at least about 10-100% or 50-100% or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the ADI protein is recombinantly expressed (or expressible) in bacterial host cells as insoluble and refoldable inclusion bodies. "Inclusion bodies" generally refer to nuclear or cytoplasmic aggregates of stable substances, usually proteins, and usually contain very little host protein, ribosomal components or DNA / RNA fragments. Inclusion bodies can be found in the cytoplasm and periplasmic space during high-level expression of heterologous proteins in bacteria such as Escherichia coli as dense electron-refracting particles that aggregate proteins.

[0063] Inclusion bodies have a higher density than many cellular components and can thus be easily separated by high-speed centrifugation after cell lysis. Although inclusion bodies are dense particles, they are highly hydrated and have a porous architecture (see, for example, Sing and Panda, Journal of Bioscience and Bioengineering. 99:303-310, 2005). In certain embodiments, the inclusion bodies consist essentially of overexpressed ADI protein. In a particular embodiment, the bacterial host cell is Escherichia coli.

[0064] In certain embodiments, as described above, the ADI protein has "ADI activity", i.e., the ability to convert or metabolize arginine into citrulline and ammonia. ADI or "arginine deiminase" activity can be measured according to conventional techniques in the art. For example, the amount of L-citrulline can be detected by a colorimetric endpoint assay (see, for example, Knipp and Vasak, Analytical Biochem. 286:257-264, 2000) and compared to a standard curve of known amounts of L-citrulline to calculate the specific activity of ADI, which can be expressed in (for example) IU / mg protein. In some embodiments, one IU of ADI enzyme activity is defined as the amount of enzyme that produces 1 μmol of citrulline per minute at the tested pH and temperature. In some embodiments, the isolated ADI protein has ADI activity under physiological conditions (e.g., under physiological conditions of temperature, salinity (e.g., a solution of one or more salts substantially isotonic with tissue fluid or blood), and pH, e.g., about 37 °C and about pH 7.2-7.6 or about pH 7.4). In certain embodiments, relative to the ADI protein consisting of SEQ ID NO:1 (wild-type Mycoplasma gallisepticum), the ADI proteins described herein have at least about 50, 60, 70, 80, 90, 100, 110 or 120% of the ADI activity under comparable physiological conditions.

[0065] The amino acid sequences of the exemplary modifications of ADI are provided in Tables A1.1 - A1.2 below and do not include SEQ ID NO:1. The % ADI activity relative to the wild - type ADI from Mycoplasma gallisepticum (SEQ ID NO:1) is also indicated in Table A1.1, as described in the examples.

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Table A1.2

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] Thus, in some embodiments, the ADI protein comprises, consists of, or consists essentially of an amino acid sequence selected from Tables A1.1 and A1.2 (e.g., SEQ ID NOs: 2-178), and does not include SEQ ID NO: 1. Also included are variants and / or fragments thereof having ADI activity. For example, in certain embodiments, the ADI protein comprises, consists of, or consists essentially of an amino acid sequence that is at least 90, 95, 96, 97, 98, 99, or 100% identical to a reference amino acid sequence selected from Tables A1.1 and A1.2 (e.g., SEQ ID NOs: 2-178), and does not include SEQ ID NO: 1.

[0094] A "variant" sequence is a polypeptide or polynucleotide sequence that differs from a reference sequence by one or more substitutions, deletions (e.g., truncations), additions, and / or insertions. Certain variants thus include fragments of the reference sequences set forth herein. Variant polypeptides have biological activity, i.e., they continue to possess the enzymatic or binding activity of the reference polypeptide. Such variants can be derived, for example, from genetic polymorphisms and / or from human manipulation.

[0095] In certain embodiments, variants and / or fragments of ADI in Table A1.1 or Table A1.2 retain one or more lysine substitutions selected from K2G, K13E, K63N, K82S, K90T, K90V, K101D, K106L, K108R, K108A, K131R, K170R, K175R, K192V, K192C, K216N, K216V, K229L, K237N, K238N, K240V, K243T, K246E, K248R, K249R, K273R, K275A, K287Q, K287C, K295A, K304L, K317R, K326A, and K400A (relative to SEQ ID NO:1). In some embodiments, variants and / or fragments of ADI in Table A1 retain 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, 26, 27, 28, or 29 lysine substitutions selected from K2G, K13E, K63N, K82S, K90T, K90V, K101D, K106L, K108R, K108A, K131R, K170R, K175R, K192V, K192C, K216N, K216V, K229L, K237N, K238N, K240V, K243T, K246E, K248R, K249R, K273R, K275A, K287Q, K287C, K287A, K295A, K295I, K304L, K317R, K326A, and K400A (relative to SEQ ID NO:1).

[0096] In certain embodiments, variants and / or fragments of ADI in Tables A1.1 - A1.2 retain all or part of the lysine substitutions specified in Tables A2 and A3 below (as indicated for that particular sequence).

[0097]

[0098]

[0099]

[0100] Table A3. Modified Protein Amino Acid Substitutions and Measured Activity

[0101]

[0102]

[0103] * Use AAG codon instead of preferred AAA.

[0104] ND not measured

[0105] K246E enhances the enzyme activity by about 5%

[0106] K106L, K170R, K175R, K229L, K275A, K317R each reduce the enzyme activity by 5 - 20%.

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] In some cases, variants include one or more "conservative" changes or substitutions. A "conservative substitution" is the replacement of one amino acid by another amino acid having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophilic properties of the polypeptide to be substantially unchanged. As described above, modifications can be made in the structures of the polynucleotides and polypeptides of the present invention and still obtain functional molecules encoding variant or derivative polypeptides having the desired characteristics. When it is desired to alter the amino acid sequence of a polypeptide to produce an equivalent or even improved variant or portion of the polypeptides described herein, one skilled in the art typically alters one or more codons of the encoding DNA sequence.

[0157] For example, certain amino acids in a protein structure can be replaced by other amino acids without significantly losing the ability to bind to structural interactions such as the antigen-binding region of an antibody or a binding site on a substrate molecule. Since the interaction ability and properties of a protein define the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, its underlying DNA coding sequence, and still obtain a protein having similar properties. With this in mind, various changes can be made in the peptide sequences of the disclosed compositions or the corresponding DNA sequences encoding such peptides without significantly losing their utility.

[0158] In making such changes, the hydrophilicity-hydrophobicity index of the amino acids can be considered. It is generally understood in the art that the hydrophilicity-hydrophobicity amino acid index is important in conferring interactive biological functions to proteins (Kyte & Doolittle, 1982, which is incorporated herein by reference). It is believed that the relative hydrophilicity-hydrophobicity characteristics of the amino acids contribute to the secondary structure of the resulting protein, and the secondary structure in turn defines the interaction of the protein with other molecules (such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like). Each amino acid has been assigned a hydrophilicity-hydrophobicity index based on its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982). The values are as follows: 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids can be replaced by other amino acids having similar hydrophilicity-hydrophobicity indices or scores and still produce a protein having similar biological activity, i.e., still obtain a biologically functionally equivalent protein. In making such changes, the hydrophilicity-hydrophobicity index of the amino acid substitution is preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0159] It is also understood in the art that substitutions similar to amino acids can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, the entire content of which is specifically incorporated herein by reference, states that the maximum local average hydrophilicity of a protein (as determined by the hydrophilicity of its neighboring amino acids) is related to the biological properties of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilic values are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It should be understood that one amino acid can substitute for another with a similar hydrophilic value and still obtain a bioequivalent and particularly immunoequivalent protein. Among such changes, substitutions of amino acids with hydrophilic values within ±2 are preferred, those within ±1 are more preferred, and those within ±0.5 are even more preferred.

[0160] As outlined above, therefore, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions considering 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.

[0161] Amino acid substitutions can further be made based on the similarity of the residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic characteristics. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids containing uncharged polar head groups and having similar hydrophilic values include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, 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.

[0162] Variants may alternatively or additionally contain non-conservative changes. In some embodiments, the variant polypeptide differs from the native or reference sequence by substitution, deletion, or addition of about or fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids or even 1 amino acid. The variant may also (or alternatively) be modified by, for example, deleting or adding amino acids that have minimal impact on the immunogenicity, secondary structure, enzymatic activity, and / or hydrophilic or hydrophobic properties of the polypeptide.

[0163] In certain embodiments, the length of the polypeptide sequence is about, at least about, or at most 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, 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, 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 contiguous amino acids (including all integers therebetween), and may include all or a portion of the reference sequence (see, e.g., Tables A1.1 and A1.2, Sequence Listing).

[0164] 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, 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, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids (including all integers therebetween), and may include all or a portion of a reference sequence (see, e.g., Tables A1 and A1.2, Sequence Listing).

[0165] In certain embodiments, the polypeptide sequence contains about 10 - 1000, 10 - 900, 10 - 800, 10 - 700, 10 - 600, 10 - 500, 10 - 400, 10 - 300, 10 - 200, 10 - 100, 10 - 50, 10 - 40, 10 - 30, 10 - 20, 20 - 1000, 20 - 900, 20 - 800, 20 - 700, 20 - 600, 20 - 500, 20 - 400, 20 - 300, 20 - 200, 20 - 100, 20 - 50, 20 - 40, 20 - 30, 50 - 1000, 50 - 900, 50 - 800, 50 - 700, 50 - 600, 50 - 500, 50 - 400, 50 - 300, 50 - 200, 50 - 100, 100 - 1000, 100 - 900, 100 - 800, 100 - 700, 100 - 600, 100 - 500, 100 - 400, 100 - 300, 100 - 200, 200 - 1000, 200 - 900, 200 - 800, 200 - 700, 200 - 600, 200 - 500, 200 - 400 or 200 - 300 contiguous amino acids (including all ranges therebetween), and includes all or a portion of a reference sequence (see, e.g., Table A1, Sequence Listing). In certain 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 amino acids or about 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 amino acids (including all integers and ranges therebetween, e.g., 101, 102, 103, 104, 105), provided that the truncated polypeptide retains the binding properties and / or activity of the reference polypeptide (see, e.g., Tables A1.1 and A1.2, Sequence Listing). Generally, a bioactive fragment has an activity of no less than about 1%, about 5%, about 10%, about 25% or about 50% of the bioactive reference polypeptide from which it is derived.

[0166] In general, variants will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to a reference polypeptide sequence (see, e.g., Tables A1 and A1.2, Sequence Listing). Additionally, sequences that differ from a native or parental sequence by addition (e.g., C-terminal addition, N-terminal addition, both), deletion, truncation, insertion, or substitution (e.g., conservative substitution) 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids (including all integers and ranges therebetween) but retain the properties or activities of the parental or reference polypeptide sequence are considered (see, e.g., Tables A1.1 and A1.2, Sequence Listing).

[0167] In some embodiments, the variant polypeptide differs from the reference sequence by at least one but less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3, or 2 amino acid residues. In certain embodiments, the variant polypeptide differs from the reference sequence by at least 1% but less than 20%, 15%, 10%, or 5% of the residues. (If such a comparison requires alignment, the sequences should be aligned for maximum similarity. "Gapped" sequences from deletions or insertions or mismatches can be considered differences.)

[0168] Sequence similarity or sequence identity between sequences is calculated as described below (these terms are used interchangeably herein). To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of the reference sequence aligned for representative purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. Then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0169] The percent identity between two sequences varies with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap introduced to achieve the best alignment of the two sequences.

[0170] Sequence comparisons between two sequences and determination of percent homology can be accomplished using a mathematical algorithm. In a preferred embodiment, percent homology between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970)) algorithm in the GAP program incorporated into the GCG software package, using a Blossum 62 matrix or PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, percent homology between two nucleotide sequences is determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The particularly preferred set of parameters (and those to be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0171] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:11-17, 1989) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0172] The nucleic acid and protein sequences described herein can be used as "query sequences" to perform a search of public databases to, for example, identify 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, J. Mol. Biol, 215:403-10). A BLAST nucleotide search can be performed using the NBLAST program (score = 100, wordlength = 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, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. For purposes of comparison, to obtain gapped alignments, gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25:3389-3402, 1997). When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0173] In some embodiments, as described above, BLAST alignment tools can be used to evaluate polynucleotides and / or polypeptides. A local alignment consists of only a pair of sequence segments, each segment from each of the sequences being compared. Modified forms of the Smith-Waterman or Sellers algorithms will find all segment pairs that cannot be improved in score by extension or trimming, called high-scoring segment pairs (HSPs). The results of a BLAST alignment include statistical measures that indicate the likelihood that the BLAST score could be expected by chance alone.

[0174] The raw score S is calculated from the gaps and substitutions associated with each aligned sequence, where a higher similarity score indicates a more significant alignment. Substitution scores are given by a lookup table (see PAM, BLOSUM).

[0175] The gap score is typically calculated as the sum of G (gap opening penalty) and L (gap extension penalty). For a gap of length n, the gap cost is G + Ln. The choice of gap costs G and L is empirical, but typically a high G value (10 - 15, e.g., 11) and a low L value (1 - 2, e.g., 1) are chosen.

[0176] The bit score S' is derived from the raw alignment score S, where the statistical nature of the scoring system used has been taken into account. The bit score is normalized with respect to the scoring system so that it can be used to compare alignment scores from different searches. The terms "bit score" and "similarity score" are used interchangeably. The bit score can indicate how well the alignment is; the higher the score, the better the alignment.

[0177] The E-value or expected value describes the likelihood that a sequence with a similar score would occur by chance in the database. It can predict the number of different alignments with scores equal to or better than S that would be expected to occur by chance in a database search. The smaller the E-value, the more significant the alignment. For example, an alignment with an E-value of e-117 means that it is extremely unlikely that a sequence with a similar score would occur by chance alone. Additionally, the expected score for aligning random amino acid pairs is negative, otherwise long alignments would tend to have high scores independent of whether the aligned segments are related or not. Additionally, the BLAST algorithm uses an appropriate substitution matrix, nucleotides or amino acids, and gap alignment uses gap creation and extension penalties. For example, BLAST alignments and comparisons of polypeptide sequences are typically performed using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0178] In some embodiments, sequence similarity scores are reported from BLAST analyses performed using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0179] In one particular embodiment, the sequence identity / similarity scores provided herein refer to the values obtained using GAP version 10 (GCG, Accelrys, San Diego, Calif.) with the following parameters: percent identity and percent similarity of nucleotide sequences using a gap weight of 50 and a length weight of 3 and the nwsgapdna.cmp scoring matrix; percent identity and percent similarity of amino acid sequences using a gap weight of 8 and a length weight of 2 and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992). GAP uses the algorithm of Needman and Wunsch (J Mol Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

[0180] In certain embodiments, variant polypeptides include amino acid sequences that can be optimally aligned with a reference polypeptide sequence (see, e.g., Tables A1.1 and A1.2, Sequence Listing) to generate a BLAST bit score or sequence similarity score of 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, 560, 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 higher (including all integers and ranges therebetween), wherein the BLAST alignment uses the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0181] As described above, reference polypeptides can be modified in various ways, including amino acid substitution, deletion, truncation, addition, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutagenesis of the DNA. Methods of mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Kunkel (PNAS USA. 82:488-492, 1985); Kunkel et al., (Methods in Enzymol. 154:367-382, 1987); U.S. Patent No. 4,873,192; Watson, J.D. et al., ("Molecular Biology of the Gene", Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidelines for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

[0182] Methods for screening gene products of combinatorial libraries prepared by such modifications and for screening gene products in cDNA libraries having selected properties are known in the art. Such methods are suitable for rapid screening of gene libraries generated by combinatorial mutagenesis of a reference polypeptide. As an example, recursive ensemble mutagenesis (REM) is a technique for enhancing the frequency of functional mutants in a library, which can be used in combination with screening assays to identify polypeptide variants (Arkin and Yourvan, PNAS USA 89:7811-7815, 1992; Delgrave et al., Protein Engineering. 6:327-331, 1993).

[0183] In some embodiments, the isolated ADI is covalently bonded to at least one PEG molecule via an optional linker. In some cases, such molecules may be referred to as "ADI-PEG"; however, as used herein the term "ADI" includes "ADI-PEG". "Polyethylene glycol" or "PEG" refers to a mixture of condensation polymers of ethylene oxide and water, which are branched or linear and have the general formula H(OCH 2 CH 2 ) nOH represents, where n is at least 4. "Polyethylene glycol" or "PEG" combined with a numerical suffix is used to indicate its approximate weight-average molecular weight. For example, PEG5,000 refers to PEG with a total weight-average molecular weight of about 5,000; PEG12,000 refers to PEG with a total weight-average molecular weight of about 12,000; and PEG20,000 refers to PEG with a total weight-average molecular weight of about 20,000.

[0184] In some embodiments, the PEG has a total weight-average molecular weight of 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 PEG has a total weight-average molecular weight of 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 specific embodiments, the PEG has a total weight-average molecular weight of about 5,000 or about 20,000. Generally, PEG with a molecular weight of 30,000 or higher is difficult to dissolve and the yield of the formulated product may be reduced. The PEG can be branched or linear. Generally, increasing the molecular weight of the PEG can reduce the immunogenicity of the ADI. The PEG can be branched or linear, and in certain embodiments is linear. PEGs having the molecular weights described herein can be used in combination with ADI and, optionally, a biocompatible linker.

[0185] Certain embodiments employ thiol, sulfhydryl, or cysteine-reactive PEG. In some embodiments, the thiol, sulfhydryl, or cysteine-reactive PEG is attached to one or more native cysteine residues, one or more introduced cysteine residues (e.g., using cysteine residues to replace one or more wild-type residues), one or more inserted cysteine residues, or any combination thereof (see, e.g., Doherty et al., Bioconjug Chem. 16:1291-98, 2005). In certain embodiments, certain wild-type ADI cysteine residues can first be replaced with another amino acid to prevent attachment of the PEG polymer to the wild-type cysteine, e.g., to prevent the PEG from disrupting the originally desired biological activity. Some embodiments employ one or more non-natural cysteine derivatives (e.g., homocysteine) in place of cysteine.

[0186] Non-limiting examples of thiol, sulfhydryl or cysteine reactive PEGs include methoxy PEG maleimide (M-PEG-MAL) (e.g., MW 2000, MW 5000, MW 10000, MW 20000, MW 30000, MW 40000). M-PEG-MAL reacts with the thiol groups on the cysteine side chains in proteins and peptides to form stable 3-thiosuccinimidyl ether linkages. This reaction is highly selective and can occur under mild conditions in the presence of other functional groups at about pH 5.0 - 6.5. Thus, in certain embodiments, the ADI enzyme is conjugated to any one or more of the thiol, sulfhydryl or cysteine reactive PEG molecules described herein.

[0187] ADI can be covalently bonded to a modifier (e.g., PEG) with or without a linker, but a preferred embodiment utilizes a linker. ADI can be covalently bonded to PEG via a biocompatible linker using methods known in the art, such as (e.g.) those described in: Park et al., Anticancer Res., 1:373 - 376 (1981); and Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J.M. Harris ed., Plenum Press, NY, chapter 21 1992), the disclosures of which are incorporated herein by reference in their entirety. In some cases, ADI can be coupled directly (i.e., without a linker) to a modifier (e.g., PEG) via, for example, an amino group, a sulfhydryl group, a hydroxyl group, a carboxyl group or other groups.

[0188] The linker used to covalently attach ADI to a modifier (e.g., PEG) can be any biocompatible linker. As discussed above, "biocompatible" indicates that the compound or group is non-toxic and can be used in vitro or in vivo without causing injury, morbidity, disease or death. The modifier (e.g., PEG) can be bonded to the linker via, for example, an ether bond, a thiol bond, an amide bond or other bonds.

[0189] In some embodiments, a suitable linker may have a total chain length of about 1-100 atoms, 1-80 atoms, 1-60 atoms, 1-40 atoms, 1-30 atoms, 1-20 atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms, for example, where the atoms in the chain include C, S, N, P, and / or O. In certain embodiments, the linker is optional, for example, the PEG-conjugated ADI enzyme does not include a linker. In some cases, the linker group includes, for example, a succinyl group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, a histidine group, a methylene group, and combinations thereof. Specific examples of stable linkers include succinimide, propionic acid, carboxymethylated linkages, ethers, carbamates, amides, amines, carbonamides, imides, aliphatic C-C bonds, and thioethers. In certain embodiments, the biocompatible linker is a methoxy-PEG succinimidyl carboxymethyl ester (SCM) or an N-hydroxysuccinimide (NHS) group.

[0190] Other suitable linkers include an oxycarbonylimidazolyl group (including, for example, carbonyldiimidazole (CDI)), a nitrophenyl group (including, for example, nitrophenyl carbonate (NCP) or trichlorophenyl carbonate (TCP)), a trifluoroethylsulfonate group, an aldehyde group, an isocyanate group, a vinylsulfone group, or a primary amine. In certain embodiments, the linker is derived from SS, SPA, SCM, or NHS; in certain embodiments, SS, SPA, or NHS is used, and in some embodiments, SS or SPA is used. Thus, in certain embodiments, the possible linker may be formed from methoxy-PEG succinimidyl succinate (SS), methoxy-PEG glutaric acid succinimidyl ester (SG), methoxy-PEG carbonic acid succinimidyl ester (SC), methoxy-PEG succinimidyl carboxymethyl ester (SCM), methoxy-PEG2 N-hydroxysuccinimide (NHS), methoxy-PEG butyric acid succinimidyl ester (SBA), methoxy-PEG succinimidyl propionate (SPA), methoxy-PEG succinimidyl glutaramide, and / or methoxy-PEG succinimidyl succinimide.

[0191] Other examples of linkers include, but are not limited to, one or more of the following: -O-, -NH-, -S-, -C(O)-, C(O)-NH, NH-C(O)-NH, O-C(O)-NH, -C(S)-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -O-CH2-, -CH2-O-, -O-CH2-CH2-, -CH2-O-CH2-, -CH2-CH2-O-, -O-CH2-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, -CH2-CH2-CH2-O-, -O-CH2-CH2-CH2-CH2-, -CH2-O-CH2-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-CH2-O-CH2-, -CH2-CH2-CH2-CH2-O-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-CH2-, -CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -NH-C(O)-CH2-, -CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, -NH-C(O)-CH2-CH2-, -CH2-NH-C(O)-CH2-CH2, -CH2-CH2-NH-C(O)-CH2-CH2, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -O-C(O)-NH-CH2-, -O-C(O)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH2-NH-CH2-, -C(O)-CH2-, -C(O)-CH2-CH2-, -CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-C(O)-,-CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-, divalent cycloalkyl, -N(R6)-, where R6 is H or an organic group selected from the group consisting of: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

[0192] In addition, any linker moiety described herein may further comprise an ethylene oxide oligomer chain containing from 1 to 20 ethylene oxide monomer units [i.e., -(CH 2 CH 2 O) 1-20 -]. That is, the ethylene oxide oligomer chain may occur before or after the linker and, optionally, between any two atoms of a linker moiety comprising two or more atoms. Additionally, if the oligomer is adjacent to a polymer segment, the oligomer chain is not considered part of the linker moiety and represents only an extension of the polymer segment.

[0193] In certain embodiments, the ADI enzyme comprises one or more PEG molecules and / or linkers as described herein. In certain embodiments, the linker is a water-labile linker.

[0194] Attachment of PEG to ADI increases the circulatory half-life of ADI. Generally, PEG is attached to a primary amine of ADI. The choice of the attachment site of PEG or other modifiers on ADI depends on the role of each site within the active domain of the protein, as is known to those skilled in the art. PEG can be attached to a primary amine of ADI without substantial loss of enzymatic activity. For example, the lysine residues present in ADI are all possible points at which ADI can be attached to PEG via a biocompatible linker (such as SS, SPA, SCM, SSA, and / or NHS) as described herein. PEG can also be attached to other sites on ADI, as will be appreciated by those skilled in the art in accordance with the present invention.

[0195] One to about 30 PEG molecules can be covalently bonded to ADI. In certain embodiments, ADI is modified by (i.e., includes) one PEG molecule. In some embodiments, ADI is modified by more than one PEG molecule. In certain embodiments, ADI is modified by 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 by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG molecules. In a specific embodiment, ADI is modified by 4.5 - 5.5 PEG molecules (per ADI). In some embodiments, ADI is modified by 5 ± 1.5 PEG molecules. In some embodiments, isolated ADI is covalently bonded to about 1 to about 10 PEG molecules. In some embodiments, isolated ADI is covalently bonded to about 2 to about 8 PEG molecules.

[0196] In certain embodiments, about 15% to about 70% of the primary amino groups in ADI are modified with PEG. For example, in some embodiments, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, or about 20% to about 25% of the primary amino groups in arginine deiminase are modified with PEG. In a specific embodiment, about 20% of the primary amino groups in arginine deiminase are modified with PEG. When PEG is covalently bonded to the terminus of ADI, it may be desirable to use only 1 PEG molecule.

[0197] Increasing the number of PEG units on ADI can increase the circulatory half-life of the enzyme. However, increasing the number of PEG units on ADI can decrease the specific activity of the enzyme. Thus, a balance needs to be achieved between the two, as will be appreciated by those skilled in the art in light of the present invention.

[0198] In some embodiments, a common feature of biocompatible linkers is that they are attached to the primary amine of arginine deiminase via a succinimidyl group. After coupling with ADI, SS-PEG has an ester linkage adjacent to the PEG, which can render this site sensitive to serum esterases that can release PEG from ADI in vivo. SPA-PEG and PEG2-NHS do not have an ester linkage and are thus insensitive to serum esterases.

[0199] The PEG attached to the protein can be linear (such as SS-PEG, SPA-PEG, and SC-PEG), or branched PEG (such as PEG2-NHS) can be used.

[0200] In some embodiments, for example, as described above, amino acid substitutions are used to conjugate non-natural amino acids to PEG or other modifiers (see, e.g., de Graaf et al., Bioconjug Chem. 20:1281-95, 2009). Accordingly, certain embodiments include ADIr enzymes conjugated to one or more PEGs via one or more non-natural amino acids. In some embodiments, the non-natural amino acid comprises a side chain having a functional group selected from the group consisting of alkyl, aryl, aryl halide, vinyl halide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl, keto, azide, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boronic acid, boronate ester, borane, phenylboronic acid, thiol, seleno, sulfonyl, borate, boronate, phosphate, phosphonyl, phosphine, heterocyclic-, pyridyl, naphthyl, benzophenone, constrained ring (such as cyclooctyne), thioester, ketene, imine, aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid, α-ketocarboxylic acid, α or β unsaturated acid and amide, glyoxylamide and organosilyl group. In some embodiments, the non-natural amino acids are selected from the group consisting of p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, homocysteine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, β-O-GlcNAc-L-serine, tri-O-acetyl-GalNAc-α-threonine, α-GalNAc-L-threonine, levodopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonylserine, phosphonyltyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine and isopropyl-L-phenylalanine.

[0201] Although ADI-PEG is an illustrative modification of ADI set forth herein, those skilled in the art will recognize that other polymers or suitable molecules can be used to modify ADI to achieve the desired effects, particularly reducing antigenicity and increasing serum half-life.

[0202] Compositions and Methods of Use

[0203] Certain embodiments include therapeutic compositions containing the ADI proteins set forth herein and methods of using them for arginine depletion therapy, including treating various cancers.

[0204] For example, certain embodiments include compositions containing the isolated ADI described herein and a pharmaceutically acceptable carrier, such as a therapeutic or pharmaceutical composition. Certain compositions are substantially pure, based on protein or by weight. For example, based on protein or weight - weight, certain compositions have a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% and are substantially free of aggregates (e.g., less than about 10, 9, 8, 7, 6 or 5% aggregates). Certain compositions are substantially free of endotoxins, as described herein.

[0205] Compositions can be prepared by methods well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a composition comprising the isolated ADI described herein and optionally one or more buffers or excipients with sterile distilled water, as appropriate, to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension. A surfactant is a compound that interacts non - covalently with the ADI in the composition to facilitate the dissolution or homogeneous suspension of the ADI in an aqueous delivery system.

[0206] Some embodiments include pharmaceutically acceptable buffers, such as buffers selected from one or more of histidine, sodium citrate, glycyl - glycine, sodium phosphate, Tris, and lysine. In certain embodiments, the concentration of the buffer is from about 0.10 mM to about 200 mM or about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 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, 100, 105, 110, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 mM (including all integers and ranges therebetween). In certain embodiments, the buffer is at about 1 mM to about 50 mM or about 10 mM to about 30 mM or about 15 mM to about 25 mM or about 20 mM or about 10 mM.

[0207] Some embodiments include pharmaceutically acceptable excipients, such as one or more excipients selected from cryoprotectants, lyoprotectants, stabilizers, bulking agents, tonicity modifiers, surfactants, pharmaceutical plasticizers, chelating agents, and any combination of the foregoing agents.

[0208] In some embodiments, the cryoprotectant is present in an amount of from about 0.001% to about 20% (wt%), including all integers and ranges therebetween. In some embodiments, the cryoprotectant is one or more selected from sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, and any combination of the foregoing agents.

[0209] In some embodiments, the lyoprotectant is present in an amount of from about 0.001% to about 20% (wt%), including all integers and ranges therebetween. In some embodiments, the lyoprotectant is one or more selected from sucrose, trehalose, mannitol, sorbitol, glycerol, and any combination of the foregoing agents.

[0210] In some embodiments, the stabilizer is present in an amount of from about 0.001% to about 20% (wt%), including all integers and ranges therebetween. In certain embodiments, the stabilizer is one or more selected from sucrose, mannitol, lactose, trehalose, maltose, sorbitol, gelatin, albumin, and any combination of the foregoing agents.

[0211] In some embodiments, the bulking agent is present in an amount of from about 0.001% to about 20% (wt%), including all integers and ranges therebetween. In certain embodiments, the bulking agent is one or more selected from mannitol, sorbitol, lactose, glucose, sucrose, glycine, albumin, dextran 40.

[0212] In certain embodiments, the tonicity modifier is present in an amount of from about 0.001% to about 20% (wt%), including all integers and ranges therebetween. In specific embodiments, the tonicity modifier is one or more selected from sodium chloride, sucrose, mannitol, and any combination of the foregoing agents.

[0213] Certain compositions include one or more pharmaceutically acceptable excipients selected from the group consisting of sucrose, trehalose, dextran, mannitol, proline, glycine, surfactants, pharmaceutical plasticizers, chelating agents, and any combination of the foregoing agents.

[0214] Certain compositions include chelating agents, such as ethylenediaminetetraacetic acid (EDTA). In some embodiments, the chelating agent is present in an amount of from about 0.001% to about 1% (wt%) or about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0% (including all integers and ranges therebetween).

[0215] Certain compositions are at a pharmaceutically acceptable pH. For example, in some embodiments, the pharmaceutically acceptable pH is from about 5.0 to about 8.0 (±0.01 to ±0.1) or about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0 (±0.01 to ±0.1) (including all integers and ranges therebetween).

[0216] In some embodiments, ADI has ADI activity at a pH close to the physiological pH of human blood. Thus, in some embodiments, ADI has ADI activity at a pH of from about 4 to about 10.8 or from about 6 to about 8 or from about 6.5 to about 7.5. In some embodiments, ADI has good ADI enzyme activity at about pH 7.4.

[0217] In some embodiments, ADI has stability during long-term storage and has temperature and proteolytic stability during treatment of a human body. In some embodiments, ADI does not require active ions or cofactors that are not already present in the blood.

[0218] In some embodiments, the isolated ADI or a composition comprising the same has an osmotic pressure of about 50 mOsm / kg to about 500 mOsm / kg or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495 or about 500 mOsm / kg.

[0219] In some embodiments, the ADI enzyme specific activity of the composition is between about 5.0 IU / mg and 120 IU / mg, where 1 IU is defined as the amount of enzyme that converts 1 μmol of arginine into 1 μmol of citrulline and 1 μmol of ammonia in one minute at 37 °C and the efficacy is 100 ± 20 IU / mL. In certain embodiments, the enzyme specific activity is about 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 35, 40, 45, 50, 55, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 IU / mg.

[0220] In certain embodiments, the ADI protein concentration is between about 5 - 20 mg / mL, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg / mL.

[0221] In a specific embodiment, the composition has one or more of the following purity determination values: less than about 1 EU endotoxin / mg protein, less than about 100 ng host cell protein / mg protein, less than about 10 pg host cell DNA / mg protein, and / or greater than about 95% unimodal purity (by SEC HPLC).

[0222] Also included is a method of treating, ameliorating cancer symptoms or inhibiting cancer progression in a subject in need thereof, which comprises administering to the subject a composition comprising at least one isolated ADI as described herein.

[0223] The methods and compositions described herein can be used to treat any type of cancer. In some embodiments, the cancer is one or more selected from the following: hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myelogenous leukemia, recurrent acute myelogenous leukemia, B cell malignancies, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma (such as astrocytoma, oligodendroglioma, ependymoma or choroid plexus papilloma), glioblastoma multiforme (such as giant cell glioblastoma or gliosarcoma), meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), non-small cell lung cancer (NSCLC), renal cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, and gastric cancer.

[0224] In some embodiments, the cancer exhibits reduced argininosuccinate synthetase-1 (ASS-1) expression and / or activity, or alternatively lacks argininosuccinate synthetase-1. In some cases, the reduced ASS-1 expression or activity is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more relative to the expression and / or activity in a suitable control sample (such as normal cells or tissues). In certain embodiments, the ASS or ASL expression or activity is reduced by at least two-fold relative to the expression or activity in the control sample. The reduction in ASS-1 expression or activity can be measured according to conventional techniques in the art, including (for example) quantitative PCR, immunohistochemical techniques, enzyme activity assays (such as ADI activity assays for measuring the conversion of citrulline to argininosuccinate or argininosuccinate to arginine and fumarate), and the like.

[0225] Administration can be achieved by a variety of different routes. The mode of administration depends on the nature of the condition to be treated or prevented. For example, ADI can be administered orally, intranasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorally, intramuscularly, interstitially, intraarterially, subcutaneously, intravitreally, intrasynovially, trans-epithelially, and / or transdermally. Specific embodiments include administration by IV infusion. An amount that reduces, inhibits, prevents, or delays the growth, progression, and / or metastasis of cancer after administration can be considered effective.

[0226] In some embodiments, the methods or compositions described herein increase the median survival time of a patient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or more. In certain embodiments, the methods or compositions described herein increase the median survival time of a patient by 1 year, 2 years, 3 years, or more. In some embodiments, the methods or compositions described herein increase the progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more. In certain embodiments, the methods or compositions described herein increase the progression-free survival by 1 year, 2 years, 3 years, or more.

[0227] In certain embodiments, the administered composition achieves tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor (e.g., a decrease in tumor mass of at least 10%, 20%, 30%, 40%, 50%, or more) or by a change in scan size (e.g., a statistically significant decrease). In certain embodiments, the administered composition is sufficient to achieve stable disease. In certain embodiments, the administered composition is sufficient to stabilize or clinically relevantly reduce the symptoms of a particular disease indication known to a skilled clinician.

[0228] The methods or compositions for treating cancer can be combined with other treatment modalities. For example, the compositions described herein can be administered to a subject before, during, or after other therapeutic interventions, including symptomatic care, radiation therapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic care includes administering corticosteroids to reduce cerebral edema, headache, cognitive dysfunction, and vomiting, and administering 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 traditional surgery.

[0229] Methods for identifying a subject having one or more of the diseases or conditions described herein are known in the art.

[0230] The precise dosage and duration of treatment vary with the disease being treated and can be determined empirically using known testing protocols or by testing the composition in model systems known in the art and extrapolating therefrom. Controlled clinical trials can also be conducted. The dosage can also vary with the severity of the condition to be alleviated. Pharmaceutical compositions are generally formulated and administered to impart a therapeutically useful effect while minimizing undesirable side effects. The composition can be administered as a single dose, or it can be divided into a number of smaller doses and administered at intervals over time. For any particular subject, the specific dosage regimen can be adjusted over time according to individual need.

[0231] In some embodiments, a therapeutically effective amount or therapeutic dose of the compositions described herein is an amount effective to reduce or stabilize tumor growth. In some cases, treatment is initiated at a low dose and the dose is increased in small increments until the optimal effect in those cases is achieved.

[0232] In some embodiments, the dose is administered from about once daily to about once every two or three weeks. For example, in certain embodiments, the dose is administered at the following frequencies: about once every 1, 2, 3, 4, 5, 6, or 7 days or about once a week or about twice a week or about three times a week or about once every two or three weeks.

[0233] In some embodiments, the dose is from about 0.1 mg / kg to about 20 mg / kg or to about 10 mg / kg or to about 5 mg / kg or to about 3 mg / kg. In some embodiments, the dose is about 0.10 mg / kg, 0.15 mg / kg, 0.20 mg / kg, 0.25 mg / kg, 0.30 mg / kg, 0.35 mg / kg, 0.40 mg / kg, 0.45 mg / kg, 0.50 mg / kg, 0.55 mg / kg, 0.60 mg / kg, 0.65 mg / kg, 0.70 mg / kg, 0.75 mg / kg, 0.80 mg / kg, 0.85 mg / kg, 0.90 mg / kg, 0.95 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg or 20 mg / kg (including all integers and ranges therebetween). In a specific embodiment, the dose is from about 1 mg / kg (in the form of a 2 ml intravenous injection) once a week to about 20 mg / kg once every 3 days.

[0234] In some embodiments, the dose is about 50 IU / m 2 to about 1000 IU / m 2 . In certain embodiments, the dose is about 50 IU / m 2 , 60 IU / m 2 , 70 IU / m 2 , 80 IU / m 2 , 90 IU / m 2 , 100 IU / m 2 , 110 IU / m 2 , 120 IU / m 2 , 130 IU / m 2 , 140 IU / m 2 , 150 IU / m 2 , 160 IU / m 2 , 170 IU / m 2 , 180 IU / m 2 , 190 IU / m 2 , 200 IU / m 2, 210 IU / m 2 , 220 IU / m 2 , 230 IU / m 2 , 240 IU / m 2 , 250 IU / m 2 , 260 IU / m 2 , 270 IU / m 2 , 280 IU / m 2 , 290 IU / m 2 , 300 IU / m 2 , 310 IU / m 2 , approximately 320 IU / m 2 , approximately 330 IU / m 2 , 340 IU / m 2 , approximately 350 IU / m 2 , 360 IU / m 2 , 370 IU / m 2 , 380 IU / m 2 , 390 IU / m 2 , 400 IU / m 2 , 410 IU / m 2 , 420 IU / m 2 , 430 IU / m 2 , 440 IU / m 2 , 450 IU / m 2 , 500 IU / m 2 , 550 IU / m 2 , 600 IU / m 2 , 620 IU / m 2 , 630 IU / m 2 , 640 IU / m 2 , 650 IU / m 2 , 660 IU / m 2 , 670 IU / m 2 , 680 IU / m 2 , 690 IU / m 2 , 700 IU / m 2 , 710 IU / m 2 , 720 IU / m 2 , 730 IU / m 2 , 740 IU / m 2 , 750 IU / m 2 , 760 IU / m 2 , 770 IU / m 2 , 780 IU / m 2 , 790 IU / m 2 , 800 IU / m 2 , 810 IU / m2 , 820 IU / m 2 , 830 IU / m 2 , 840 IU / m 2 , 850 IU / m 2 , 860 IU / m 2 , 870 IU / m 2 , 880 IU / m 2 , 890 IU / m 2 , 900 IU / m 2 , 910 IU / m 2 , 920 IU / m 2 , 930 IU / m 2 , 940 IU / m 2 , 950 IU / m 2 , 960 IU / m 2 , 970 IU / m 2 , 980 IU / m 2 , 990 IU / m 2 or about 1000 IU / m 2 (including all integers and ranges therebetween).

[0235] Also included are patient care kits that include one or more of the compositions or isolated ADIs described herein. Some kits also include one or more pharmaceutically acceptable diluents or solvents, such as water (e.g., sterile water). In some embodiments, the composition or ADI is stored in a vial, cartridge, dual-chamber syringe, and / or prefilled mixing system.

[0236] The kits herein may also include one or more other therapeutic agents or other components suitable for or desired for the indicated indication or desired diagnostic application. The kits herein may also include one or more syringes or other components (e.g., stents, implantable reservoirs, etc.) needed or desired to facilitate the intended delivery mode.

[0237] Polynucleotides, expression vectors, and host cells

[0238] Certain embodiments relate to polynucleotides encoding ADI proteins modified as described herein. Accordingly, certain embodiments include polynucleotides encoding any one or more individual ADI polypeptides in Tables A1.1 and A1.2 (excluding SEQ ID NO:1), including variants and / or fragments thereof. For example, certain polynucleotides encode ADI proteins comprising, consisting of, or consisting essentially of an amino acid sequence that is at least 90, 95, 96, 97, 98, 99, or 100% identical to a reference amino acid sequence selected from Tables A1.1 and A1.2 (e.g., SEQ ID NOs: 2-178), excluding SEQ ID NO:1.

[0239] In some embodiments, the polynucleotide includes at least one AAG codon (instead of the preferred AAA codon) encoding a lysine residue, such as the AAG codon encoding the K317 residue. Some polynucleotides include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 AAG codons (instead of the preferred AAA codons) encoding lysine residues.

[0240] Among other uses, these and related embodiments are particularly useful for recombinantly producing ADI proteins in host cells. Those skilled in the art will appreciate that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides may have minimal homology to the nucleotide sequences of any natural gene. However, polynucleotides that vary due to codon usage are specifically contemplated, such as polynucleotides optimized for human, yeast, or bacterial codon selection.

[0241] As recognized by those skilled in the art, the polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be a DNA (genomic, cDNA, or synthetic) or RNA molecule. The polynucleotide can include a native sequence (i.e., an endogenous sequence encoding the ADI protein) or can include a variant or biological functional equivalent of such a sequence. Polynucleotide variants can contain one or more of the substitutions, additions, deletions, and / or insertions described herein, such that preferably the activity of the polypeptide is not substantially diminished relative to the unmodified polypeptide.

[0242] Other coding or non-coding sequences may (but need not) be present within the polynucleotide, and the polynucleotide may (but need not) be linked to other molecules and / or vector materials. Thus, regardless of the length of the coding sequence itself, the polynucleotide can be combined with other DNA or RNA sequences (such as promoters, enhancers, polyadenylation signals, other restriction enzyme sites, multiple cloning sites, other coding segments, and the like), such that its overall length can vary significantly.

[0243] The polynucleotide sequence may also be a polynucleotide sequence of a metagenome, cDNA, RNA, and synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide may be ligated to a genomic or cDNA sequence encoding a polypeptide, and then the DNA or RNA sequence may be modified at the site by homologous recombination according to well-known procedures by inserting a synthetic oligonucleotide encoding the desired amino acid sequence or preferably by PCR using appropriate oligonucleotides to generate the desired sequence. In some embodiments, a signal sequence may be included in front of the coding sequence. This sequence encodes a signal peptide located at the N-terminus of the coding sequence, which communicates with the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the culture medium. Generally, the signal peptide is cleaved off by the cell before the protein leaves the host cell. Signal peptides can be found in various proteins in prokaryotes and eukaryotes.

[0244] One or more polynucleotides may encode the ADI proteins described herein. In addition, the polynucleotide sequence may be manipulated for various reasons. Examples include (but are not limited to) incorporating preferred codons to enhance the expression of the polynucleotide in various organisms (see generally Nakamura et al., Nuc. Acid. Res. 28:292, 2000).

[0245] Also included are expression vectors containing the polynucleotide and host cells containing the polynucleotide and / or the expression vector. The ADI protein can be produced by expressing the DNA or RNA sequence encoding the polypeptide in a suitable host cell by well-known techniques. The term "host cell" is used to mean a cell that has been introduced or is capable of being introduced with a nucleic acid sequence encoding one or more polypeptides described herein and further expresses or is capable of expressing the polypeptide of interest (e.g., a polynucleotide encoding any polypeptide described herein).

[0246] The term includes the progeny of the parental cell, regardless of whether the morphology or genetic composition of the progeny is the same as that of the original parental cell, as long as the selected gene is present. Host cells may be selected for certain characteristics, such as expressing formylglycine-generating enzyme (FGE) to convert cysteine or serine residues within a sulfatase motif into formylglycine (FGly) residues; or expressing an aminoacyl-tRNA synthetase that can incorporate unnatural amino acids into a polypeptide (including unnatural amino acids having azide side chains, alkyne side chains, or other desired side chains) to facilitate chemical conjugation or modification.

[0247] In some cases, the polynucleotide or expression vector includes other non-coding sequences. For example, "control elements" or "regulatory sequences" present in the expression vector are non-translated regions in the vector that interact with host cell proteins to effect transcription and translation, including enhancers, promoters, 5' and 3' non-translated regions. The strength and specificity of these elements can vary. Depending on the vector and host utilized, any number of suitable transcriptional and translational elements can be used, including constitutive and inducible promoters. For example, when cloned into a bacterial system, an inducible promoter such as the hybrid lacZ promoter of pBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like can be used.

[0248] A variety of expression vector / host systems are known and can be used to contain and express polynucleotide sequences. These systems include (but are not limited to) microorganisms such as bacteria transformed with an expression vector (e.g., a recombinant phage, plasmid or cosmid DNA expression vector). Certain embodiments thus include an expression vector containing a polynucleotide sequence encoding a polypeptide (e.g., an ADI protein) as described herein. Also included are host cells containing the polynucleotide and / or expression vector.

[0249] Certain embodiments employ an E. coli-based expression system (see, e.g., Structural Genomics, Consortium et al., Nature Methods. 5:135-146, 2008). These and related embodiments can optionally utilize ligation-independent cloning (LIC) to generate a suitable expression vector. In a specific embodiment, protein expression can be controlled by T7 RNA polymerase (e.g., the pET vector series) or a modified pET vector with an alternative promoter (including, e.g., the TAC promoter). These and related embodiments can utilize the expression host strain BL21(DE3) (a lysogen of BL21 that supports T7-mediated expression and lacks the lon and ompT proteases) to improve target protein stability. Also included are expression host strains carrying a plasmid encoding tRNAs rarely used in E. coli, such as ROSETTA TM(DE3) and Rosetta2(DE3) strains. In some embodiments, other E. coli strains that can reduce the degree of post-translational modification during fermentation can be utilized, including other E. coli K-12 strains such as W3110 (F-λ-IN(rrnD-rrnE)1rph-1) and UT5600 (F, araC14, leuB6(Am), secA206(aziR), lacY1, proC14, tsx67,.(ompTfepC)266, entA403, glnX44(AS), A-, trpE38, rfbC1, rpsL109(strR), xylA5, mtl-1, thiE1). Reagents sold under the trademarks nucleases and protein extraction reagents can also be used to improve cell lysis and sample handling. For cell culture, autoinduction media can improve the efficiency of many expression systems, including high-throughput expression systems. Without the addition of artificial inducers such as IPTG, such media (e.g., OVERNIGHT EXPRESS TM Autoinduction System) gradually induce protein expression via metabolic shift.

[0250] Certain embodiments employ hexahistidine tags (e.g., those sold under the trademark Fusion) and are subsequently purified by immobilized metal affinity chromatography (IMAC) or related techniques. However, in certain aspects, clinical-grade proteins can be isolated from E. coli inclusion bodies with or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006).

[0251] Also included are methods for recombinantly producing ADI proteins as described herein. In some embodiments, a polynucleotide encoding an ADI protein is directly introduced into a host cell, and the cells are cultured under conditions sufficient to induce expression of the encoded protein. Standard techniques well known to those skilled in the art can be used in combination with the polypeptide and nucleic acid sequences provided herein to prepare the polypeptide sequences of the present invention.

[0252] Accordingly, in certain embodiments, there are provided recombinant host cells comprising a polynucleotide or a fusion polynucleotide encoding an ADI protein as described herein. The ADI protein can be expressed in the host cell by culturing the recombinant host cell containing the polynucleotide under appropriate conditions. After production by expression, the ADI protein can be isolated and / or purified using any suitable technique and then used as needed. For example, some methods include: (a) expressing ADI in a recombinant bacterial host cell, where ADI is expressed in the host cell as an insoluble and refoldable inclusion body; (b) removing the insoluble inclusion body from the host cell; (c) purifying ADI from the insoluble inclusion body; (d) refolding ADI in a refolding buffer; and (e) purifying ADI from the refolding buffer, thereby producing isolated ADI (see Examples).

[0253] In some embodiments, at least about 40, 50, 60, 70, 80, or 90% of the ADI is expressed in the bacterial host cell as an insoluble and refoldable inclusion body. In certain embodiments, the host cell is Escherichia coli.

[0254] The ADI protein produced by the recombinant host cell can be purified and characterized according to various techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (such as the AKTA and Bio-Rad FPLC systems), high performance liquid chromatography (HPLC) (such as the Beckman and Waters HPLC). Exemplary chemical assays for purification include ion exchange chromatography (such as Q, S), size exclusion chromatography, salt gradient, affinity purification (such as Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reverse phase, ceramic ion exchange chromatography, and hydrophobic interaction columns (HIC). See also Examples.

[0255] Also included is evaluating or measuring the ADI activity of the isolated ADI under physiological conditions of temperature and pH as appropriate, where the isolated ADI has ADI activity under physiological conditions. In some embodiments, the isolated ADI has at least about 50, 60, 70, 80, 90, 100, 110, or 120% of the ADI activity of the isolated ADI consisting of SEQ ID NO:1 (wild-type Mycoplasma gallisepticum) under comparable physiological conditions.

[0256] Certain aspects further include preparing a composition comprising the isolated ADI, for example, where the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% based on protein or weight-weight, and where the composition is substantially free of aggregates and substantially free of endotoxin.

[0257] All publications, patent applications, and issued patents cited in this specification are hereby incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually incorporated by reference.

[0258] Although the foregoing invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will readily appreciate that certain changes and modifications may be made to the invention without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration and not limitation. Those skilled in the art will readily identify various non-critical parameters that may be changed or modified to obtain substantially similar results.

[0259] Embodiments

[0260] Embodiment 1

[0261] Modification of ADI from Mycoplasma columbinasale

[0262] Mutants of ADI from Mycoplasma columbinasale were generated to identify those that increase the expression of the protein as insoluble and refoldable inclusion bodies in Escherichia coli. ADI from Mycoplasma columbinasale (M.col ADI) is a 400 amino acid sequence in which the amino-terminal methionine is removed during expression in Escherichia coli. This protein has 35 lysine residues in its sequence. Attachment of polyethylene glycol (PEG) molecules to the protein surface typically uses amine chemistry that links PEG to lysine and amino-terminal amines. Thus, first, an attempt was made to reduce the number of surface lysines to reduce the heterogeneity of PEGylation when using these techniques. To achieve this goal, the three-dimensional X-ray crystal structure of M.col ADI was solved to 2.4 Å and used to identify lysine residues among the 35 that are solvent-exposed and likely to participate in PEGylation, and residues that are buried in the structure or participate in protein-protein interactions in the fully assembled homohexamer. Six lysines were identified as non-solvent accessible.

[0263] The amino acid sequence of M.col ADI was then used to search the GenBank database using the BLASTP program at blast.ncbi.nlm.nih.gov. Thirty-seven Mycoplasma ADI enzymes with amino acid identities between 88% - 53% were selected and assembled into a multiple sequence alignment using AlignX from the VectorNTI 11.5.1 (Invitrogen) software package. Each of the remaining 29 lysines in M.col ADI was compared to the 37 Mycoplasma ADIs and any substitutions were listed.

[0264] Based on this analysis, a number of mutants were prepared. The sequence and activity information for each mutant discussed herein are summarized in Tables A1.1 - A1.2, A2, and A3.

[0265] First, mutations were introduced for all 29 lysines using the substitution pairs described in the multiple sequence alignment. However, two lysines are absolutely conserved in all sequences and alanine residues were thus selected for substitution at these positions. This mutant protein (McolM1) appeared as an insoluble inclusion body. The inclusion body was extracted, refolded, purified, and then tested for activity. McolM1 retained only about 17% of the wild - type enzyme activity. This low activity may result from improper folding of the mutant enzyme due to the large number of introduced mutations. Alternatively, certain lysine residues may be required to achieve the proper structure / function of the protein.

[0266] To identify lysine mutants that contribute to the insoluble phenotype and maintain most of the wild - type activity, the mutations were divided into two groups to generate McolM2 and McolM3. These constructs were also insoluble and were refolded, purified, and tested. McolM2 and McolM3 retained <80% of the wild - type activity.

[0267] McolM2 was further divided into McolM4 and McolM5, and McolM3 was divided into McolM6 and McolM7. McolM4 and McolM7 appeared as soluble enzymes and retained >80% of the wild - type activity. McolM5 and McolM6 were insoluble and retained <80% of the activity. Then the mutants from McolM4 and McolM7 were combined into McolM8, which appeared as a soluble enzyme and retained >80% of the wild - type activity.

[0268] The mutants from McolM5 and McolM6 were isolated and added to the McolM8 mutant group to generate McolM9, McolM10, McolM11, and McolM12. All four mutants were insoluble, refolded, purified, and tested for activity, where McolM9 and McolM10 had >80% activity and McolM11 and McolM12 had <80% activity. The McolM9 mutation showed a first set of lysine mutations that allowed for a high level of insoluble expression as inclusion bodies and enabled refolding into an active enzyme with >80% wild - type activity.

[0269] Since the only difference between McolM8 (soluble) and McolM9 (insoluble) is the addition of three mutants (K175R, K246E, K317R), each of these three mutations in McolM9 was converted back to lysine one at a time to generate each pairwise combination and each single mutant (see McolM29 - McolM34). All 6 mutants were expressed as insoluble inclusion bodies and refolded, with most having an activity > 80%. McolM33 was selected as the backbone sequence and then the lysine mutants were selectively mutated back to lysine starting from the terminal carboxyl and amino regions of the protein. McolM59 - McolM63 have 2, 3, 4, 5, and 6 lysine mutants replaced with wild - type lysine, respectively. McolM59 and McolM60 were insoluble, refolded, purified, tested, and found to have > 80% wild - type activity. McolM61 had an intermediate phenotype and only about 50% of the protein was expressed as insoluble inclusion bodies. The insoluble fraction was refolded, purified, tested, and found to have > 80% wild - type activity. McolM62 and McolM63 were soluble and not tested.

[0270] Other factors that can affect solubility were also tested. For example, the codon usage for back - translating from an amino acid sequence to a DNA sequence for protein expression can affect the protein expression levels in E. coli. Generally, the more the protein sequence utilizes the preferred codons of E. coli, the higher the protein expression (e.g., see Gouy and Gautier, Nucleic Acids Res. 10:7055 - 74, 1982). Tests were thus conducted to determine whether codon usage can affect the solubility of M.col ADI expressed in E. coli by changing the codon bias, as measured by the codon adaptation index (CAI) (e.g., see Sharp et al., Nucleic Acids Res. 15:1281 - 1295, 1987). CAI is expressed as a value from 1.0 to < 1, where 1.0 represents a perfect match between the codon bias of the organism used as the expression host and the gene of interest. Unexpectedly, a single change of one lysine codon (K317) from the preferred AAA to the non - preferred AAG in McolM8 caused the resulting construct (McolM46) to be insoluble and refoldable.

[0271] PEGylation - related mutagenesis strategies were also tested. For example, in addition to the random PEGylation strategy on amines, using cysteine to replace solvent - accessible residues can provide a mechanism for site - specific PEGylation on the molecule (e.g., see Dozier and Distefano, Int J Mol Sci.

[0272] (16:25831 - 25864, 2015). Due to the homotetrameric structure of M. col ADI, a single cysteine mutation allows up to 6 PEG molecules to attach to the functional enzyme. Mutating cysteine to many solvent-accessible residues on the wild-type M. col ADI sequence, the most effective ones are K192C and K287C, which have little (if any) impact on the structure / function of ADI. These mutations were carried out in the form of single mutants and double mutants (McolM35, McolM36, and McolM39, respectively) in the soluble background of McolM8. All three presented as insoluble / refoldable inclusion bodies and were able to be PEGylated with 20 kDa maleimide PEG by specifically modifying the solvent-accessible cysteine residues. These results demonstrate that the current set of lysine mutations in the McolM8 soluble construct can become insoluble / refoldable by simply changing the lysine-substituted amino acids to cysteine rather than adding additional lysine mutations.

[0273] In summary, these results demonstrate that modified ADIs from Mycoplasma columbinas can be generated by substitution and / or changing codon usage to selected lysine residues, and these ADIs not only form insoluble and refoldable inclusion bodies in E. coli but also retain significant ADI activity under physiological conditions.

[0274] Materials and Methods:

[0275] Molecular Biology: Unless otherwise indicated, buffer salts and general reagents were purchased from Sigma - Aldrich. The amino acid sequences of Mycoplasma columbinas arginine deiminase (Sequence ID: EGV00288 and mutant constructs of this sequence) were back-translated to DNA sequences using Vector NTI advance 11.5.1 software (Invitrogen) and the E. coli standard codon preference table. A unique NdeI restriction site was included at the 5' end encoding the start methionine and a unique XhoI site was added to the 3' end, thus forming a fusion with a hexahistidine tag encoded by the vector.

[0276] This DNA sequence was synthesized by Invitrogen and supplied in its standard vector.

[0277] The vector was restricted using NdeI and XhoI enzymes (New England Biolabs) and visualized on a 1% agarose E-gel (Invitrogen). The 1.2 kb NdeI-XhoI DNA fragment containing Mycoplasma gallisepticum arginine deiminase (M. col ADI) was excised from the gel under blue light of the Safe-Imager 2.0 (Invitrogen) gene and purified using the QIAquick Gel Extraction Kit (Qiagen). The expression vector pET21a (Novagen) digested with XhoI-NdeI was prepared in the same manner. The vector and the insert DNA were ligated with the Quick Ligation Kit (New England Biolabs) and transformed into chemically competent Mach1-T1 cells (Invitrogen), and selection was carried out on LB agar using 100 μg / ml ampicillin (Teknova). A single colony was inoculated into 5 ml of LB medium supplemented with 100 μg / ml ampicillin (GIBCO) and grown to an OD600 of approximately 1.

[0278] One ml aliquots with a final glycerol concentration of 20% were snap-frozen in liquid nitrogen for storage and subsequent use. Plasmid DNA was prepared from the remaining culture using the QIAprep Spin Miniprep Kit (Qiagen). The inserted sequence was verified by automated dideoxy chain termination sequencing (Retrogen, San Diego CA). The verified sequence was then transformed into the expression host BL21(DE3) (Invitrogen) and frozen stocks were prepared as mentioned above. The mutant residue was site-directed mutagenized back to its wild-type lysine using the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, San Diego CA) following the manufacturer's protocol.

[0279] All mutations were sequence-verified over the entire length of the gene sequence to rule out any secondary defects that might have been introduced by the mutagenesis procedure. The selected insoluble mutant of M. col ADI was also mutated to remove the carboxy-terminal hexahistidine tag by inserting a stop codon (TAG) precisely before the XhoI restriction site using site-directed mutagenesis, thus confirming the insoluble / refoldable activity phenotype.

[0280] Expression and purification: The expression of wild-type and mutant proteins of Mycoplasma columbinasale ADI in each construct was basically the same. Generally, a frozen glycerol stock of the expression host was inoculated into 5 ml of LB medium containing 100 μg / ml ampicillin and grown overnight at 37 °C. In a baffled Erlenmeyer flask, a 1:1000 dilution was prepared from this fermentation culture in 1 liter of Terrific Broth autoinduction medium (see, for example, Studier, Protein Expression and Purif. 41:207-234, 2005) containing 100 μg / ml ampicillin. The cells were grown overnight in an orbital shaker at 37 °C and 250 rpm. Cell pellets were harvested by centrifugation at 10,000 rpm for 20 minutes and then stored at -80 °C for future purification.

[0281] Mycoplasma columbinasale ADI wild type: Cell pellets with expressed ADI were resuspended in 20 mM NaPO 4 (pH 8.5) and 20 mM imidazole at a ratio of 5 ml of buffer per gram of cell slurry. The cells were then lysed using an M110L microfluidics homogenizer (Microfluidics, Westwood, MA). Insoluble materials were removed by centrifugation at 10,000 rpm. The supernatant was loaded onto a 50 ml NiNTA Superflow (Qiagen) column equilibrated with 20 mM NaPO 4 (pH 8.5) and 20 mM imidazole using an AKTA FPLC (GE Amersham Pharmacia). The column was washed to baseline with 20 mM NaPO 4 (pH 8.5) and 20 mM imidazole and then the bound protein was eluted using a step gradient of 20 mM NaPO 4 (pH 8.5) and 500 mM imidazole.

[0282] The eluted protein was then bound to a 50 ml Q-Sepharose Fast Flow (GE Amersham Pharmacia) column and washed to baseline with 20 mM NaPO 4 (pH 8.5). The bound protein was eluted using a linear gradient of 20 mM NaPO 4 (pH 8.5) to 20 mM NaPO 4 (pH 8.5) and 1 M NaCl over 10 column volumes. The peak fractions were pooled, brought to 1 M using ammonium sulfate, and then loaded onto a 50 ml high performance phenyl sepharose (GE Amersham Pharmacia) column. Using 20 mM NaPO4 (pH 8.5), 1 M ammonium sulfate was used to wash the column to baseline, and then 20 mM NaPO 4 (pH 8.5), 1 M ammonium sulfate to 20 mM NaPO 4 A linear gradient of pH 8.5 was used to elute the bound protein over 10 column volumes.

[0283] The peak fractions were pooled and concentrated to approximately 20 ml or less in an Amicon stirred cell concentrator with a 10 kDa MWCO membrane (EMD Millipore, Billerica MA), sterile filtered through a 25 mm 0.2 micron syringe filter (Pall Life Sciences), and then loaded onto a 500 ml Superdex 200 size exclusion column (GE Amersham Pharmacia). The column was run in 10 mM HEPES pH 7.5 150 mM NaCl buffer at 1 ml / min. The peak fractions were pooled and concentrated to approximately 1 mg / ml or higher, and stored at -80 °C.

[0284] Mycoplasma gallisepticum ADI insoluble mutant; the insoluble mutant was purified in the same manner as the wild-type protein with the following differences. After lysing the cells in 20 mM NaPO 4 (pH 8.5) buffer, the pellet formed by centrifugation at 10,000 rpm was resuspended in several hundred ml of 20 mM NaPO 4 (pH 8.5) wash buffer and centrifuged again. This washing step was repeated several times until a clear supernatant was formed.

[0285] The insoluble inclusion bodies in the pellet could then be refolded using the methods described in the literature (e.g., see U.S. Patent No. 6,132,713; Misawa et al., J Biotechnol. 36:145-55, 1994; and Noh et al., Molecules and Cells. 13:137-143, 2002). The refolded active ADI was then purified from the refolding buffer starting from the Q-Sepharose Fast Flow step and following all subsequent steps as performed with the wild-type protein.

[0286] Protein purity evaluation: Generally, protein purity is evaluated during the purification process by the chromatographic behavior of the protein, followed by polyacrylamide gel electrophoresis (PAGE), and then by Coomassie blue protein staining and gel densitometry analysis. Additionally, as a final test, after protein PEGylation, the average PEG number and purity are determined using reverse-phase liquid chromatography (RPLC). The purity value of the protein purified using the described method is >95%.

[0287] Activity analysis: ADI catalyzes the conversion of L-arginine to L-citrulline and ammonia. The amount of L-citrulline can be detected by a colorimetric endpoint assay (Knipp and Vasak, Anal Biochem. 286:257-64, 2000) and compared to a standard curve of known amounts of L-citrulline to calculate the specific activity of ADI (expressed as IU / mg protein). One IU of enzyme activity is defined as the amount of enzyme that produces 1 μmol of citrulline per minute at the tested pH and temperature. Standard condition assays are performed at 37°C in physiological HEPES buffer (PHB), 50 mM HEPES, 160 mM NaCl (pH 7.4) (Clin Chem Lab Med. 37:563-71, 1999) + 0.1% BSA. Whenever conditions permit, all samples and standards are run in duplicate or triplicate.

[0288] The Km and Kcat values are determined by using a variation of the above activity analysis. For the activity assays (or unless otherwise indicated), all reactions are run at 37°C in PHB + 0.1% BSA. The enzyme concentration, reaction time, and substrate concentration range for each ADI construct are adjusted to compensate for their activity differences. Generally, 2 nM enzyme, a 5-minute reaction time, and 0-160 μM arginine are used as starting conditions. When optimizing conditions, particular attention is paid to the amount of substrate consumed and expressed as a percentage of the total substrate added to the reaction. Typically, the lower limit of detection is approximately 1 μM citrulline and the lower limit of quantification is approximately 2 μM. A citrulline standard curve is run on each plate and used to quantify the citrulline produced by the enzymatic reaction.

[0289] Calculation: Calculate the concentration of citrulline (μM) produced in each reaction well and average using the citrulline standard curve. Then calculate the rate of each reaction in the form of μM / min / 50 nM ADI. Calculate the specific activity (IU / mg or μmol product / min / mg ADI) by multiplying this value by the "IU" factor (the IU factor is calculated from the molecular weight of ADI and the reaction volume).

[0290] Example 2

[0291] Modification of ADI from Mycoplasma columbinasale

[0292] Generate other mutants of ADI from Mycoplasma columbinasale to identify those that increase the expression of the protein as insoluble and refoldable inclusion bodies in E. coli. The sequence and activity information for each mutant discussed herein is summarized in Tables A1.1 - A1.2, Table A2, and Table A3. Use the method described above in Example 2 to test other mutants of ADI.

[0293] The mutant ADIs M1 - M56, as described above, identified a subgroup of lysine mutations that conferred the original desired phenotype (e.g., insoluble, refoldable proteins retaining ADI activity). M34 was found to have the most desired phenotype. Data generated from the study of mutant ADIs M59 - M86 identified a subgroup of substitutions found to be important for the desired phenotype. These mutants were identified as K90T, K108R, K192V, K216N, K240V, K287Q, and K295A. The first 6 substitutions were found to be more important, while the last substitution, K295A, was found to be less important. Thus, these 7 mutants were found to be important for conferring the desired phenotype and are represented by M86.

[0294] Other lysine codon changes were also included in the study and were found to have varying importance. Mutants M94 - M184 were studied, which changed each of the 7 identified mutants to other amino acids and used M34 as the background for these changes. Of the 20 amino acids available for substitution at each lysine position, 13 amino acids were retained, and lysine (wild - type), the original mutation (according to the site change), proline (causing a structural change), cysteine (which can form disulfide bonds with other molecules, cysteine used for site - specific PEGylation as needed, M35, M36, and M39), the large hydrophobic residues tryptophan, phenylalanine, and tyrosine (lysine is on the surface of the protein, and large hydrophobic side chains are not suitable on the surface of the protein and can cause aggregation) were not used. This left 13 other amino acids (M94 - M184) that could be substituted at each of the 7 lysine positions. The insoluble expression of ADI from Mycoplasma columbinasale of each was evaluated, and some from each position were selected for refolding, purification, and comparison of activity to the wild - type enzyme.

[0295] Seven variations were selected and incorporated into a single mutant, M188. This mutant did not have the appropriate enzyme activity, so each position was reverted back to wild-type lysine and designated as M189 - M195. No single variation produced the desired phenotype, so each individual mutation was added in the minimal amount required to obtain the desired phenotype. The K246E substitution was added because it was able to increase the enzyme activity by approximately 5%. M196 to M122 show the results of sequential addition. M214 had the desired phenotype and required only three mutations in addition to the K246E substitution, namely K90V, K287A, and K295I. M216 and M218 also had the desired phenotype, but each had four mutations in addition to the K246E substitution, where M218 shared the same three mutations with M214 and M216 shared only two of the three with M214. Although the data presented herein show many active mutant ADI proteins with the desired properties, other combinations of mutations may produce the same phenotype and can be identified using the methods disclosed herein.

Claims

1. An isolated arginine deiminase (ADI) that comprises, consists of, or consists essentially of an amino acid sequence that is at least 90, 95, 96, 97, 98, 99, or 100% identical to an amino acid sequence selected from Tables A1.1 and A1.2, and does not include SEQ ID NO:

1.

2. The isolated ADI according to claim 1, wherein, the isolated ADI is recombinantly expressed (or is capable of being expressed) in a bacterial host cell, optionally Escherichia coli (E. coli), as insoluble and refoldable inclusion bodies.

3. The isolated ADI according to claim 2, wherein, at least about 10 - 100% or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the ADI is recombinantly expressed (or is capable of being expressed) in the bacterial host cell as insoluble and refoldable inclusion bodies.

4. The isolated ADI according to any one of claims 1 to 3, wherein, the isolated ADI has ADI activity under physiological conditions, optionally temperature, salinity, and pH.

5. The isolated ADI according to claim 4, which has at least about 50, 60, 70, 80, 90, 100, 110, or 120% ADI activity relative to the isolated ADI consisting of SEQ ID NO:1 (wild-type Mycoplasma columbinum) under comparable physiological conditions.

6. The isolated ADI according to any one of claims 1 to 5, which comprises, consists of, or consists essentially of an amino acid sequence that is at least 90, 95, 96, 97, 98, 99, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 2 - 178, and the amino acid sequence retains one or more lysine substitutions selected from K2G, K13E, K63N, K82S, K90T, K90V, K101D, K106L, K108R, K108A, K131R, K170R, K175R, K192V, K192C, K216N, K216V, K229L, K237N, K238N, K240V, K243T, K246E, K248R, K249R, K273R, K275A, K287Q, K287C, K287A, K295A, K295I, K304L, K317R, K326A, and K400A (relative to SEQ ID NO:1).

7. The isolated ADI as claimed in claim 6, which retains 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, 26, 27, 28, or 29 lysine substitutions selected from K2G, K13E, K63N, K82S, K90T, K90V, K101D, K106L, K108R, K108A, K131R, K170R, K175R, K192V, K192C, K216N, K216V, K229L, K237N, K238N, K240V, K243T, K246E, K248R, K249R, K273R, K275A, K287Q, K287C, K287A, K295A, K295I, K304L, K317R, K326A, and K400A (relative to SEQ INO:1), optionally all or some of the lysine substitutions indicated for the selected sequences in Tables A2 and A3.

8. The isolated ADI as claimed in any one of claims 1 to 7, wherein, the isolated ADI is covalently bonded via a linker to at least one PEG molecule.

9. The isolated ADI as claimed in claim 8, wherein, the isolated ADI is covalently bonded to about 1 to about 10 PEG molecules.

10. The isolated ADI as claimed in claim 8, wherein, the isolated ADI is covalently bonded to about 2 to about 8 PEG molecules.

11. The isolated ADI as claimed in any one of claims 8 to 10, wherein, the PEG molecule is a linear or branched PEG molecule.

12. The isolated ADI as claimed in any one of claims 8 to 11, wherein, the PEG has a total weight average molecular weight of about 1,000 to about 40,000, optionally about 2,000 to about 20,000, optionally about 2,000 to about 10,000, optionally about 5,000.

13. The isolated ADI as claimed in any one of claims 8 to 12, wherein, the linker is a succinyl group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, a histidine group, a methylene group, or any combination thereof.

14. The arginine deiminase as claimed in claim 13, wherein, the source of the succinyl group is methoxy-PEG succinimidyl carboxymethyl ester (SCM) or N-hydroxy-succinimide (NHS).

15. A therapeutic composition comprising the isolated arginine deiminase (ADI) as claimed in any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. The therapeutic composition as claimed in claim 15, wherein, The composition is protein-based or has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% weight-by-weight and is substantially free of aggregates.

17. The therapeutic composition according to claim 15 or 16, which is substantially free of endotoxins.

18. A method of treating, ameliorating cancer symptoms or inhibiting cancer progression in an individual in need thereof, which comprises administering to the individual a therapeutic composition according to any one of claims 15 to 17.

19. The method according to claim 18, wherein, the cancer is selected from one or more of hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myeloid leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, B cell malignancies, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma (such as astrocytoma, oligodendroglioma, ependymoma or choroid plexus papilloma), glioblastoma multiforme (such as giant cell glioblastoma or gliosarcoma), meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), non-small cell lung cancer (NSCLC), renal cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer and gastric cancer.

20. The method according to claim 18 or 19, wherein, the cancer exhibits decreased spermidine synthase-1 expression.

21. A polynucleotide encoding an isolated arginine deiminase (ADI) according to any one of claims 1 to 7 or a vector comprising the polynucleotide.

22. The polynucleotide or vector according to claim 21, which comprises at least one AAG codon encoding a lysine residue, optionally the K317 residue.

23. The polynucleotide or vector according to claim 22, which comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 AAG codons encoding lysine residues.

24. A recombinant bacterial host cell, optionally Escherichia coli, which comprises the polynucleotide or vector according to any one of claims 21 to 23.

25. A method for recombinantly producing an isolated arginine deiminase (ADI), which comprises (a) expressing the ADI in a recombinant bacterial host cell according to claim 23, wherein, the ADI is expressed in the host cell as an insoluble and refoldable inclusion body; (b) removing the insoluble inclusion body from the host cell; (c) purifying the ADI from the insoluble inclusion body; (d) refolding the ADI in a refolding buffer; and (e) purifying the ADI from the refolding buffer, thereby producing an isolated ADI.

26. The method according to claim 25, wherein, At least about 10 - 100% or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of said ADI is present as insoluble and refoldable inclusion bodies in said bacterial host cell.

27. The method according to claim 26, further comprising measuring the ADI activity of said isolated ADI under physiological conditions, optionally temperature, salinity and pH, characterized in that, said isolated ADI has ADI activity under physiological conditions.

28. The method according to claim 27, characterized in that, relative to the isolated ADI (wild-type Mycoplasma gallisepticum) consisting of SEQ ID NO:1, said isolated ADI has at least about 50, 60, 70, 80, 90, 100, 110 or 120% ADI activity under comparable physiological conditions.

29. The method according to any one of claims 25 to 28, further comprising preparing a therapeutic composition comprising said isolated ADI, characterized in that, said composition has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% based on protein or weight - weight, and characterized in that said composition is substantially free of aggregates and substantially free of endotoxins.

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