TWI676683B - Arginine deiminase with reduced cross-reactivity toward adi-peg 20 antibodies for cancer treatment - Google Patents

Abstract

The present invention relates generally to isolated arginine deiminase (ADI) protein (which has lower cross-reactivity with anti-ADI-PEG 20 antibody than ADI-PEG 20, but its functional characteristics are similar to ADI-PEG 20 is similar or better than this), compositions containing such ADI proteins, and related methods for treating arginine-dependent or related diseases such as cancer.

Description

Arginine deiminase with reduced cross-reactivity to ADI-PEG 20 antibody for cancer treatment Cross-reference to related applications

This application claims priority from US Provisional Application No. 61 / 790,833, filed March 15, 2013, which is incorporated herein by reference in its entirety.

Statement about sequence listing

The sequence listings related to this application are provided in text format rather than paper copies, and are hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is POLA_003_01TW_ST25.txt. The text file is about 117KB and was created on March 12, 2014.

The present invention relates generally to arginine deiminase (ADI) proteins, including ADI proteins with reduced cross-reactivity with ADI-PEG 20 antibodies. These ADI proteins are suitable for the treatment of arginine-dependent or related diseases, such as cancer.

Amino acid removal therapy can be an effective treatment for some forms of cancer. To date, there is a known clinical example related to this method, which uses asparaginase to reduce the amount of asparagine circulating and inhibit protein synthesis. This treatment is particularly effective for acute lymphoblastic leukemia (Avramis 2005, Viera Pinheiro 2004). Acute lymphoblastic leukemia cells require asparagine for growth and proliferation. In contrast, most normal human cells are able to synthesize asparagine and are not affected by asparagine depletion. Therefore, reducing asparagine with asparaginase can selectively kill cancer cells without damaging normal cells, tissues, and hosts. Approved derived from Escherichia coli (E. coli) of asparagine in the form of an enzyme for human use. However, asparaginase is only found in microorganisms; this makes it highly immunogenic in humans and also has a short serum half-life after injection (Avramis 2005). In order to make asparaginase a more effective drug, the asparaginase derived from E. coli is formulated with polyethylene glycol (PEG) to reduce the enzyme's immunogenicity and related allergic reactions. Minimize these disadvantages. In addition, PEG greatly prolongs the circulating half-life of asparaginase, which reduces the frequency of treatment and the total cost of treatment. Asparaginase formulated with PEG is approved for use and sold under the trade name Oncaspar® (Oncaspar® 2011, Avramis 2005, Viera Pinheiro 2004, Fu 2007, Zeidan 2008).

Arginine is another non-essential amino acid used in humans and mice (for a review see Rogers 1994). In humans, arginine can be used in two steps with the help of the Krebs (urea) cycle enzyme spermine succinate synthetase (ASS, L-citrulline: L-aspartic acid) Conjugating enzyme [AMP-forming], EC 6.3.4.5) and spermine succinate lyase (ASL, L-spermine succinate lysine lyase, EC 4.3.2.) Are synthesized from citrulline (Haines 2011, Wu 2009, Morris 2006, Husson 2003, Tapiero 2002, Rogers 1994). ASS catalyzes the conversion of citrulnic acid and aspartic acid to spermine succinate, which is subsequently converted to spermine and fumarate by ASL. Human arginine-deficient diet does not induce hyperammonemia, orotic aciduria, nor does it alter the rate of nitric oxide (NO) synthesis in adults (Tapiero 2002, Castillo 1995, Rogers 1994, Carey 1987, Barbul 1986 , Snyderman 1959, Rose 1949). Although it seems that arginine is needed in premature babies (Wu 2004), the amount of arginine is not related to the age of the infant, child, and young people (Lücke 2007). In 1992, Takaku and Sugimura reported separately that human melanoma and hepatocellular carcinoma (HCC) cell lines appear to require arginine for growth. Other studies show Glycolated ADI is effective and has few adverse effects on the treatment of melanoma and liver tumors.

ADI-PEG 20 treatment requires multiple doses over time. After multiple treatments, anti-ADI-PEG 20 antibodies may appear that limit the continued effectiveness of the treatment. Therefore, there is a need in the art for ADI with reduced cross-reactivity to anti-ADI-PEG20 antibodies for treatment in order to improve and prolong the efficacy of arginine depletion therapy. The present invention provides this and other advantages for treating cancer.

References: Avramis VI, Panosyan EH. 2005. Clin Pharmacokinet 44: 367-393; Barbul A. 1986. J Parenteral Enteral Nutr 10: 227-238; Carey GP et al. 1987. J Nutr 117: 1734-1739; Castillo L 1995. Am J Physiol 268 (Endocrinology Metabolism 31): E360-367; Fu CH, Sakamoto KM. 2007. Expert Opin Pharmacother 8: 1977-1984; Haines RJ et al. 2011. Int J Biochem Mol Biol 2: 8 -23; Husson A et al. 2003. Eur J Biochem 270: 1887-1899; Lücke T et al. 2007. Clin Chem Lab Med 45: 1525-1530; Morris SM Jr. 2006. Am J Clin Nutr 83 (Supplement): 598S -512S; Rogers QR. 1994. In Proceedings from a Symposium Honoring Willard J. Visek-from Ammonia to Cancer and Gene Expression. Special Publication 86-April 1994, Agriculture Experiment Station, University of Illinois, 211 Mumford Hall, Urbana , IL 61801, pages 9-21; Tapiero H et al. 2002. Biomed Pharmacother 56: 439-445, 2002; Viera Pinheiro JP, Boos J. 2004. Br J Haematol 125: 117-127; Wu G et al. 2009. Amino Acids 37: 153-168; Wu G et al. 2004. J Nutr Bioche m 15: 442-451; Zeidan A et al. 2008. Expert Opin Biol Ther 9: 111-119).

One aspect of the present invention provides an isolated arginine deiminase, wherein the isolated arginine deiminase has reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody. Also included are therapeutic or pharmaceutical compositions that include isolated arginine deiminase or a fragment thereof having ADI activity, and a pharmaceutically acceptable carrier. In certain embodiments, the composition is sterile and / or substantially free of pyrogens such as endotoxins. In one embodiment, the isolated arginine deiminase with reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody is not derived from M. hominis . In another embodiment, the isolated arginine deiminase with reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody is from an organism listed in Table 1. In certain embodiments, one or more characteristics of the isolated arginine deiminase with reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody are similar to or better than those of ADI-PEG 20 better. In this regard, the one or more characteristics include, but are not limited to, Kcat, Km, optimal pH, stability, proteolytic stability in vivo, or the need for ions or cofactors not present in the blood, or Any combination. In one embodiment, the isolated arginine deiminase with reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody has at least 20 surfaces compared to the human mold arginine deiminase Residue changes. In another embodiment, the isolated arginine deiminase with reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody has a value between 20 and 20 compared to human mold arginine deiminase And 135 surface residue changes, between 40 and 100 surface residue changes, between 30 and 60 surface residue changes, between 80 and 100 surface residue changes Base changes, or surface residue changes between 100 and 120.

In another embodiment, the isolated arginine deiminase with reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody is from M. arginini , arthritis ( M.arthritidis ), M.phocicerebrale , M.gateae , M.phocidae , M.columbinum , Aia M.iowae , M.crocodyli , M.alligatoris , H.orenii , or Mycobacterium bovis M.bovis ). Illustrative reduction in cross-reactivity with patient anti-ADI-PEG 20 antibodies includes the amino acid sequence set forth in any one of SEQ ID NOs: 2-32.

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

In another embodiment, the arginine deiminase with reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody is covalently bound to the PEG molecule via a linker. In this regard, the reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody can covalently bind to one or more PEG molecules, such as to about 1 to about 10 or about 2 to about 8 PEG molecules. The PEG molecules may be linear or branched PEG molecules, and the total weight average molecular weight may be about 1,000 to about 40,000, or the total weight average molecular weight may be about 10,000 to about 30,000. In those embodiments in which the PEG is covalently bound to the ADir of the present invention by means of a linker, the linker may include succinimide, amido, amido, carbamate, ester, Epoxy, carboxyl, hydroxyl, carbohydrate, tyrosine, cysteine, histamine, methylene, or any combination thereof. In one embodiment, the source of the succinyl group is succinimide succinate.

Another aspect of the present invention provides a polynucleotide encoding the isolated arginine deiminase described herein, a vector comprising the polynucleotide, and an isolated host cell comprising the vectors .

Another aspect of the present invention provides a composition comprising an isolated arginine deiminase with reduced cross-reactivity with a patient's anti-ADI-PEG 20 antibody as described herein, and a physiologically Acceptable vehicle. In certain embodiments, the compositions further comprise a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, polyenes Docetaxel, carboplatin, cyclophosphamide, gemcitabine, cisplatin, sorafenib, sunitinib, and everolimus (everolimus).

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

Another aspect of the present invention provides a method for treating cancer, improving the symptoms of cancer, or inhibiting the progression of cancer, which comprises administering to a patient in need thereof a therapeutically effective amount of a composition comprising ADI-PEG 20, and After that time, the patient is administered a composition comprising an isolated arginine deiminase with reduced cross-reactivity with the patient's anti-ADI-PEG 20 antibody, as described herein, and a physiologically acceptable carrier To treat the cancer, improve the symptoms of the cancer, or suppress the progression of the cancer. In this regard, the period of time can be determined by detecting a predetermined amount of anti-ADI-PEG 20 antibody in the patient and / or measuring or otherwise observing the ADI activity in the patient, which includes -An isolated arginine deiminase composition with reduced cross-reactivity of PEG 20 antibodies is detecting the predetermined content of the anti-ADI-PEG 20 antibodies and / or measuring or observing the Patients are administered after a predetermined degree of ADI activity.

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

Embodiments of the present invention pertain to selected ADI enzymes, which in some embodiments are engineered to have a small number of surface lysine residues and are conjugated to PEG via a stable linker. The selected ADI enzyme is selected from a large number of ADI enzymes from different organisms based on its beneficial properties. These properties include the ability of the enzyme to establish and maintain low spermine concentrations in human blood by converting arginine to citrulline and ammonia via ADI. In addition, the cross-reactivity of selected ADI molecules against ADI-PEG 20 antibodies is reduced compared to ADI-PEG 20, and these antibodies may be generated by patients previously treated with ADI-PEG 20.

In certain embodiments, the enzymes of the present invention are pegylated to provide protection against renal clearance and proteolysis, and to reduce immunogenicity or antigenicity. To increase the effectiveness of pegylation, modifications to the enzyme can be engineered to reduce the number of lysine residues on the surface and therefore limit the number of PEG attachment sites available. This provides more complete and uniform pegylation at the residual lysine linking residues.

PEG linkers selected to link methoxy-PEG to ADI are selected to provide chemically stable linkages. This is expected to increase the biologically active life of the molecule. Chemically stable linkers will also eliminate hydrolysis and reduce the immune response that can occur on dePEGylated linkers attached to the enzyme surface.

These accumulations account for the production of one or more molecules that effectively remove arginine from the patient's blood without being offset or eliminated by anti-ADI-PEG 20 antibodies from previous arginine depletion therapy. These molecules are pegylated to delay offset and clearance due to their autoimmunity. These factors will allow its use in place of or in addition to ADI-PEG 20 (e.g., as a follow-up drug) to prolong arginine depletion therapy and therefore increase the effectiveness of arginine depletion therapy as an anticancer treatment .

Arginine is not required for normal cell growth because it can be synthesized from citrulline in a two-step process catalyzed by ASS and ASL. In contrast, some cancers do not show ASS. Some cancers do not show ASL, while other cancers may reduce the performance of ASS and / or ASL or may not show ASS and / or ASL. Therefore, these cancers are auxotrophic to arginine. This can take advantage of metabolic differences to develop safe and effective therapies for treating these forms of cancer. ADI catalyzes the conversion of arginine to citrulline via the arginine dihydrolase pathway and can therefore be used to eliminate arginine.

Unless specifically directed to the contrary, the practice of the present invention will employ conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology within the skill of this technology, many of which are for illustrative purposes It is described below. These techniques are explained fully 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, NY (2009); Ausubel et al., Short Protocols in Molecular Biology , 3rd Edition, Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach , vols . I and II (eds. D. Glover) ); Oligonucleotide Synthesis (edited by N. Gait, 1984); Nucleic Acid Hybridization (edited by B. Hames and S. Higgins, 1985); Transcription and Translation (edited by B. Hames and S. Higgins, 1984); Animal Cell Culture (R (Freshney, 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

As used in this specification and the scope of the attached patent application, unless the content Make sure that otherwise, the singular forms "a / a", "an" and "the" include plural references.

Throughout this specification, unless otherwise stated herein, the words "comprise" or variations such as "comprises" or "comprising" shall be understood to imply the inclusion of stated elements, or Integer, or element or group of integers, but does not exclude any other element, or integer, or group of elements or integers.

"About" means quantity, degree, value, number, frequency, percentage, size, size, amount, weight or length, quantity, degree, value, number, frequency, percentage, size, size, amount, weight, relative to the reference Or length changes such as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

"Statistically significant" means that the results are unlikely to happen by chance. Statistical significance can be determined by any method known in the art. A common measure of significance includes the p-value, which is the frequency or probability that the observed event will occur if the null hypothesis is true. If the p-value obtained is less than a significant level, the null hypothesis is rejected. In simple cases, a significant level is defined as a p-value of 0.05 or less.

Unless explicitly stated otherwise, the embodiments in this specification are applied to every other embodiment with necessary modifications.

Recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, liposome transfection) can use standard techniques. Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly accomplished in the technique or as described herein. These and related techniques and procedures may generally be performed according to well-known methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. Unless specific definitions are provided, the nomenclature used in molecular biology, analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described in this article, and the laboratory procedures and techniques of these disciplines are well known in the technology And they are often used. Standard technology can be used in recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis Analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

"Patient" or "individual" means an animal, in some embodiments a mammal, and in a particular embodiment, a human.

"Biocompatibility" means that the material or compound is generally not harmful to biological functions and does not produce any degree of unacceptable toxicity (including allergenicity and disease conditions).

The term "reference sequence" generally refers to a nucleic acid coding or amino acid sequence against which another sequence is compared. Included in the form of reference sequences are all polypeptide and polynucleotide sequences described herein, including their sequences described by name and their sequences described in tables and sequence listings.

Throughout the present invention, the following abbreviations may be used: PEG, polyethylene glycol; ADI, arginine deiminase; SS, succinimide succinate; SSA, succinimide Dimethylimide; SPA, succinimide propionate; NHS, N-hydroxysuccinimide; ASS1 or ASS, spermine succinate synthase; ASL, spermine succinate lyase .

In the present invention, the polynucleotide encoding the ADI may be derived, cloned, isolated, synthesized, or produced from any source including, for example, microorganisms, recombinant biotechnology, or any combination thereof. For example, enzymes optionally arginine to colonize the imine from the genus Mycoplasma (Mycoplasma), Clostridium (Clostridium), Bacillus (Bacillus), Borrelia (of Borrelia), Enterococcus (of Enterococcus) Microorganisms of the genus Streptococcus , Lactobacillus and / or Giardia . In some embodiments, the arginine deiminase is selected from the group consisting of Mycoplasma arthritidis , Mycoplasma pneumoniae , Mycoplasma hominis , and Arginine Mycoplasma arginini , Steptococcus pyogenes , Steptococcus pneumoniae , Borrelia burgdorferi , Borrelia afzelii , Giardia intestinalis ), Clostridium perfringens , Bacillus licheniformis , Enterococcus faecalis , Lactobacillus sake , or any combination thereof. In other embodiments, the arginine deiminase is selected from the species listed in Table 1. In detail, the ADI used in the present invention may include the amino acid sequence of any one of SEQ ID NOs: 1-32 or have ADI activity (for example, it can metabolize arginine to citrulline and ammonia) Variants or fragments or extensions. Some of the sequences provided in the Sequence Listing do not represent the full-length ADI protein sequence. Therefore, in certain embodiments, additional amino acid residues can be added at either terminus of the sequences provided herein to form a full-length protein with ADI activity. The particular amino acid to be added can be determined by the skilled person based on an alignment of known ADI sequences. The ADI molecules can be synthesized using known techniques. For example, an illustrative "extended" ADI (r) is provided in SEQ ID NOs: 26-32.

In certain embodiments, an ADI enzyme as described herein is compared to a benchmark ADI-PEG 20 molecule derived from a human mold. As used herein, "ADI-PEG 20" refers to known in the art and described in, for example, US6183738; US6635462; Ascierto PA et al. (2005) Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies J Clin Oncol 23 (30): 7660-7668; Izzo F et al. (2004) Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I / II studies. J Clin Oncol 22 (10): 1815-1222 Holtsberg FW et al. (2002), Poly (ethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties. J Control Release 80 (1-3): 259-271; Kelly et al., (2012 ) ADI molecule in British Journal of Cancer 106, 324-332. As will be recognized by those skilled in the art, this molecule is a polyethylene glycol (PEG 20,000) -derived ADI enzyme derived from human mold, and has two substitutions relative to wild-type human mold mold ADI enzyme ( K112E; P210S).

Arginine deiminase as described herein was screened from a large number of ADI enzymes, and the degree of reactivity of these enzymes with anti-ADI-PEG 20 antibodies from patients was reduced. Anti-ADI-PEG 20 antibodies can be found in individuals treated with ADI-PEG 20 and can be measured using known methods. Reactivity to anti-ADI-PEG 20 antibodies can be determined, for example, using an ELISA or other similar analysis known to those skilled in the art.

In this regard, ADI-PEG 20 can be used as a comparison to assess the degree of cross-reactivity to a patient's anti-ADI-PEG 20 antibody. A degree of cross-reactivity that is statistically significantly lower than the degree of cross-reactivity of ADI-PEG 20 to a patient's anti-ADI-PEG 20 antibody may be applicable herein. In certain embodiments, the arginine deiminase as described herein is less cross-reactive or non-reactive to the anti-ADI-PEG 20 antibody. In another embodiment, any decrease in the reactivity of the anti-ADI-PEG 20 antibody compared to the reactivity using ADI-PEG 20 may be beneficial because this ADI enzyme will improve the patient's need for arginine depletion therapy. Treatment options. Therefore, the cross-reactivity of arginine deiminase to patients' anti-ADI-PEG 20 antibodies as described herein is reduced compared to the reactivity of ADI-PEG 20 to these antibodies.

As used herein, "ADIr" refers to the ADI enzyme of the present invention whose cross-reactivity against anti-ADI-PEG 20 antibodies is reduced compared to the reactivity of ADI-PEG 20 to these antibodies. The "ADIr" nomenclature is used to distinguish molecules identified herein from ADI and ADI-PEG 20 as known in the art.

The features or characteristics of the ADir enzyme of the present invention are similar to or better than those of ADI-PEG 20, thereby reducing and maintaining a low blood spermine level for effective cancer treatment. These characteristics include Kcat, Km, optimal pH, stability, proteolytic stability in vivo, and the absence of ions or cofactors that are not present in the blood, or any combination thereof. In certain embodiments, the characteristics of an ADDi as described herein are about or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% of similar characteristics of ADI-PEG 20. , 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In other embodiments, the characteristics of the ADDi described herein are about or at least about 100%, 105%, 110%, 120%, 140%, 150%, 160%, 180 of the specific characteristics of the ADI-PEG 20 being compared. %, 200%, 220%, 240%, 250%, 260%, 280%, 300%, 320%, 340%, 350%, 360%, 400%, 420%, 450%, 46%, 500%, 520%, 550% or higher.

Therefore, in certain embodiments, the Kcat of ADir is about or at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% of the Kcat of ADI-PEG 20. , 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or better. In certain embodiments, the Kcat of ADir is about or at least about 100%, 105%, 110%, 120%, 125%, 140%, 150%, 160%, 180%, 200%, 220%, 240%, 250%, 260%, 280%, 300%, 320%, 340%, 350%, 360%, 400%, 420%, 450%, 46%, 500%, 520%, 550% Or higher. In certain embodiments, as described herein ADIr the enzyme or composition comprising such Kcat ADIr enzyme is from about 0.5 seconds to about 15 ^-1 sec ^-1, and in another embodiment from about 1 sec ^-1 To about 12 seconds ^-1 , about 1 second ^-1 to about 10 seconds ^-1 , about 1.5 seconds ^-1 to about 9 seconds ^-1 , about 2 seconds ^-1 to about 8 seconds ^-1 , or about 2.5 seconds ^-1 to About 7 seconds ^-1 . In certain embodiments, the composition of the ADIR or Kcat ADIr-PEG is from about 2.5 to about 7.5 sec ^-1 sec ^-1. In some embodiments, the Kcat of ADir or ADIR-PEG in the composition is about 2.5 sec ^-1 , about 3 sec ^-1 , about 3.5 sec ^-1 , about 4 sec ^-1 , about 4.5 sec ^-1 , about 5 sec ^-1, of about 5.5 ^sec-1, to about 6 sec ^-1, 6.5 sec ^-1 sec ^-1 to about 7, about 7.2 sec ^-1, of about 7.5 sec ^{-1, -1} about 8 seconds, about 10 seconds ^{- 1} , about 15 seconds ^-1 , about 20 seconds ^-1 , about 25 seconds ^-1 , about 30 seconds ^-1 , about 35 seconds ^-1 , about 40 seconds ^-1 , about 45 seconds ^-1 , about 50 seconds ^-1 , About 55 sec ^-1 , about 60 sec ^-1 , about 65 sec ^-1 , about 70 sec ^-1 , about 75 sec ^-1 , about 80 sec ^-1 , about 85 sec ^-1 , about 90 sec ^-1 , about 95 Seconds ^-1 , or about 100 seconds ^-1 .

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

In some embodiments, the ADIR functions at a pH value close to the physiological pH value of human blood. Therefore, in one embodiment, the ADIR functions at a pH of about 4 to about 10.8, or about 6 to about 8, or about 6.5 to about 7.5. In one embodiment, ADIR has good enzyme activity at about pH 7.4.

In certain embodiments, ADir is stable during long-term storage, and temperature and proteolytically stable during human treatment. In other embodiments, the activity of ADir does not require ions or cofactors that are not present in the blood.

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

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

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

From a large number of ADI enzymes, Table 1 lists 24 kinds of ADir enzymes and their percent sequence identity with respect to human mold mold ADI. According to the literature, the ADI amino acid sequences of human mold, arginine, and arthritis ADI are closely related, and these enzymes have good catalytic properties. Recently, additional ADI enzymes with sequences closely related to these three enzymes have been discovered. Far more relevant mycoplasma ADI enzymes have been identified, although less is known about them. And there are even farther related ADI enzymes from bacteria and other sources.

In certain embodiments, the ADDi enzymes identified herein from multiple selected species have surface lysine residues (in some embodiments up to 30 or more). However, in some embodiments, the ADIR enzyme may have much less surface lysine residues, such as only 2 lysine residues in the case of Mycobacterium bovis ADI, or even none Lysine residues (see, eg, ADI from Mycobacterium sp. MCS; GenBank number ABG10381). Therefore, the ADir enzymes with reduced cross-reactivity with anti-ADI-PEG 20 antibodies identified herein have about 0, 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or more surface ionizing amino acid residues.

The terms "polypeptide", "protein" and "peptide" are used interchangeably and are meant to be not limited to amino acid polymers of any particular length. The term does not exclude modifications, such as myristate, sulfate, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more amino acid chains, wherein each chain comprises an amino acid covalently linked by a peptide bond, and wherein the polypeptide or protein may include a plurality of non- Covalent and / or covalently linked chains (such chains have natural proteins (i.e., proteins produced by naturally occurring and specifically non-recombinant cells) or genetically engineered or recombinant cells Sequence), and contains molecules with amino acid sequences of natural proteins, or molecules with deletions, additions and / or substitutions to one or more amino acids of natural sequences. The terms "polypeptide" and "protein" specifically encompass the ADIr proteins of the invention, or sequences having deletions, additions and / or substitutions to one or more amino acids of these ADIr proteins. In certain embodiments, the polypeptide is a "recombinant" polypeptide produced by a recombinant cell comprising one or more recombinant DNA molecules, which are typically made of heterologous cells that will not be found in the cell Polynucleotide sequences or combinations of polynucleotide sequences.

The term "isolated protein" as referred to herein means that an individual protein (1) is free of at least some other proteins that would typically be found in nature with it, and (2) is substantially free of the same source (e.g., from Other proteins of the same species), (3) expressed by cells from different species, (4) separated from at least about 50% of polynucleotides, lipids, carbohydrates or other materials associated with them in nature, (5) Not related to the part of the protein related to the "isolated protein" in nature (by covalent or non-covalent) (Interactions), (6) operably related (by covalent or non-covalent interactions) to a polypeptide with which it is not related in nature, or (7) does not occur in nature. This isolated protein may be encoded by genomic DNA, cDNA, mRNA or other RNA, may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free of proteins or polypeptides or other contaminants that are found in its natural environment and will interfere with its use (therapeutic, diagnostic, prophylactic, research or other).

The term "variant" includes polypeptides that differ from a reference polypeptide (eg, SEQ ID NOs: 1-32) specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. The variant polypeptide has biological activity, that is, it continues to have the enzymatic or binding activity of the reference polypeptide. Such variants may be produced, for example, by genetic polymorphisms and / or by human manipulation.

In many cases, the biologically active variant will contain one or more conservative substitutions. A "conservative substitution" is one in which one amino acid is replaced by another amino acid having similar characteristics, so those skilled in peptide chemistry can expect that the secondary structure and hydrophilic properties of the polypeptide will not substantially change. As described above, modifications can be made in the structure of the polynucleotides and polypeptides of the present invention, while still obtaining functional molecules encoding variants or derived polypeptides with desired characteristics.

For example, in a protein structure, certain amino acids can be replaced by other amino acids without significantly losing their ability to interact with structures such as the antigen-binding region of an antibody or a binding site on a receptor molecule. Because the biological functional activity of a protein is defined by its interaction capabilities and properties, certain amino acid sequence substitutions can be made in the protein sequence and, of course, its basic DNA coding sequence, while still obtaining a protein with similar characteristics. It is therefore expected that various changes may be made in the peptide sequence of the disclosed composition or the corresponding DNA sequence encoding such peptides without significant loss of its utility.

In making these changes, the hydrophilic index of amino acids can be considered. The importance of amino acid hydrophilicity index in conferring protein-interacting biological functions is generally understood in related arts (Kyte and Doolittle, 1982, incorporated herein by reference). It is recognized that the relatively hydrophilic nature of amino acids contributes to the secondary structure of the resulting protein, which subsequently defines the protein Interaction with other molecules (such as enzymes, substrates, receptors, DNA, antibodies, antigens and their analogs). Each amino acid has been assigned a hydrophilic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3) ); Proline (-1.6); histamine (-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 substituted with other amino acids having similar hydrophilic indices or scores, while still producing proteins with similar biological activity, that is, still obtaining biologically functionally equivalent proteins. In making these changes, substitution with an amino acid having a hydropathic index within ± 2 is preferred, substitution with their amino acid having a hydropathic index within ± 1 is particularly preferred, and using a hydropathic index within ± 0.5 Substitution of their amino acids is even more preferred.

It should also be understood in the art that substitutions similar to amino acids can be performed efficiently based on hydrophilicity. US Patent 4,554,101, specifically incorporated herein by reference in its entirety, states that the maximum local average hydrophilicity of a protein (as controlled by the hydrophilicity of its adjacent amino acid) is related to the biological properties of the protein. As detailed in US Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arginine (+3.0); Lysine (+3.0); Aspartic acid (+ 3.0 ± 1); Bran Glycine (+ 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 may be substituted with another amino acid having a similar hydrophilicity value, while still obtaining a biologically equivalent and especially immunologically equivalent protein. Among these changes, substitution with an amino acid having a hydrophilicity value within ± 2 is preferred, substitution with their amino acid having a hydrophilicity index within ± 1 is particularly preferred, and using a hydrophilicity index between Substitution of their amino acids within ± 0.5 is even more preferred.

As outlined above, amino acid substitutions are therefore generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and similar forms. Exemplary substitutions that take into account the foregoing features are familiar to those skilled in the art and include: spermine and lysine; glutamic and aspartic acid; serine and threonine; bran Phenylamine and Asparagine; and Valine, Leucine, and Isoleucine.

Amino acid substitutions can be further performed based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphoteric similarity of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and those without anionic head groups with similar hydrophilicity values Amino acids 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.

Variants can also (or alternatively) contain non-conservative changes. In a preferred embodiment, the variant polypeptide differs from the natural sequence by less than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids or even 1 amino acid. Replace, delete or add. Variants can also (or alternatively) be modified by, for example, deletions or additions of amino acids that have minimal impact on the immunogenicity, secondary structure, enzymatic activity, and / or hydrophilic properties of the polypeptide.

Overall, the variant will show about or at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75% of the reference polypeptide sequence (e.g., SEQ ID NO: 1-32). , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% for similarity or sequence identity or sequence homology. In addition, the coverage differs from natural or parental sequences by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, Addition of 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids (e.g. C-terminal addition, N-terminal addition Plus, both), deletions, truncations, insertions, or substitutions but that retain the identity or activity of the parent or reference polypeptide sequence.

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 other embodiments, the variant polypeptide differs from the reference sequence by about or at least 0.5% or 1% but less than 20%, 15%, 10%, or 5% residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. "Loop" bursts or mismatches from deletions or insertions are considered differences.)

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

Many activated PEGs for conjugation to ADI are covalently bound to lysine residues. The number of PEG molecules attached to ADI is usually much less than the number of lysine residues. Both the number and distribution of linkages may be non-uniform between molecules. Any particular lysine residue will only be modified in a small portion of the ADI molecule. This site modification heterogeneity and low PEG occupancy can lead to problems with drug characterization and the effectiveness of PEG shielding at the antigenic site. Therefore, in certain embodiments, the selected ADir enzyme as described herein is modified by replacing the lysine with other residue types to reduce the number of lysine residues. This produces a more uniform polyethylene glycol Protein, and increase the PEG occupancy at the residual lysine residues. Specific lysine residues selected to become other residues will be selected to maintain enzyme activity. It is expected that this more homogeneous PEGylation results in increased protection against proteolysis in blood and increased screening of antigenic sites by antibodies from patients.

In some embodiments, the ADIR of the present invention can be modified as described in US Patent No. 6,635,462. In particular, modifications to one or more of the naturally occurring amino acid residues of the ADir can provide enzymes that are easier to refold and deploy, thereby improving the production of the ADir and therapeutic compositions comprising the ADir. In one embodiment, the ADIR of the present invention is modified to remove one or more lysine residues (e.g., the lysine can be replaced with another amino acid or its analog, or an unnatural amino acid ). In particular, in one embodiment, the ADIR is modified to be at a position corresponding to 112, 374, 405, or 408 of SEQ ID NO: 1 (human mold mycoplasma ADI) or a combination of one or more of these positions It does not contain lysine. In another embodiment, the ADIR is modified to be free of one or more lysines, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21 or more lysine residues, if present, may be substituted with another amino acid or its analog, or an unnatural amino acid. In one embodiment, the ADIR has, for example, substituted 5 lysines at positions corresponding to positions 7, 88, 137, 209, and 380 of SEQ ID NO: 1. In another embodiment, the ADIR has, for example, substituted 10 lysines at positions corresponding to positions 7, 9, 59, 88, 115, 116, 137, 178, 209, and 380 of SEQ ID NO: 1 . In yet another embodiment, the ADIR is, for example, at a position corresponding to positions 7, 9, 59, 66, 88, 91, 93, 115, 116, 137, 141, 178, 209, 279 of SEQ ID NO: 1 And has 15 substituted lysines at position 380 of SEQ ID NO: 1. In one embodiment, the ADIR is, for example, at positions 7, 9, 56, 59, 66, 88, 91, 93, 96, 115, 116, 137, 141, 178, 209, 254, SEQ ID NO: 1, Positions 279, 325, 326, 380 and 406 contain 21 substituted lysines.

Natural ADir is found in microorganisms, is immunogenic and circulates quickly from patients Quickly clear. These problems can be solved by modifying the ADir. Therefore, the present invention provides ADir modified by a modifier, which includes, but is not limited to, a macromolecular polymer, protein, peptide, polysaccharide, or other compound. Arginine deiminase and modifiers as described herein can be linked by covalent bonds or non-covalent interactions to form a stable conjugate or a stable composition to achieve the desired effect. In certain embodiments, the modified ADir retains the biological activity of the unmodified ADir and has a longer half-life and less antigenicity in vivo than the unmodified ADir. In certain embodiments, the modified ADir retains at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% of the biological activity of the unmodified ADir %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. In general, the modified ADir retains biological activity sufficient for therapeutic use.

In one embodiment, the modifying agent can be a polymer or protein or a fragment thereof that is biocompatible and can increase the half-life of ADir in the blood. Modifiers can be chemically coupled to the ADir or, where applicable, linked to the ADir by means of fusion protein expression.

The macromolecular polymer may include a non-peptide macromolecular polymer, which in some embodiments may have its own biological activity. Suitable polymers include, but are not limited to, polyenol compounds, polyether compounds, polyvinylpyrrolidone, polyamino acids, copolymers of divinyl ether and maleic anhydride, N- (2-hydroxypropyl ) -Methacrylamide, polysaccharides, polyoxyethylated polyols, heparin or fragments thereof, polyalkylene glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives, poly ( Vinyl ether), α, β-poly [(2-hydroxyethyl) -DL-asparagine], polycarboxylates, polyoxyethylene-formaldehyde, polypropylene morpholine, amine compounds and olefin oxides Copolymers, polyhyaluronic acid, polyethylene oxide, copolymers of oxalic acid and malonic acid, poly (1,3-dioxolane), copolymers of ethylene and maleic acid , Polysialic acid, cyclodextrin and so on. In certain embodiments, the polymer is polyethylene glycol.

Polyenol compounds as used herein include, but are not limited to, polyethylene glycol (including monomethoxy polyethylene glycol, monohydroxy polyethylene glycol), polyvinyl alcohol, polyallyl alcohol, polybutenol And their analogs, and their derivatives (such as lipids).

Polyether compounds include, but are not limited to, polyalkylene glycols (HO ((CH2) _x O) _n H), polypropylene glycol, polyoxyethylene (HO ((CH ₂ ) ₂ O) _n H), polyvinyl alcohol ( (CH ₂ CHOH) _n ).

The polyamino acid includes, but is not limited to, a polymer of an amino acid or a copolymer of two or more amino acids, such as a polyamino acid or a polyamino acid, or a block copolymer thereof.

Polysaccharides include (but are not limited to) polydextrose and its derivatives (such as dextran sulfate), cellulose and its derivatives (including methyl cellulose and carboxymethyl cellulose), starch and its derivatives, polysucrose, etc. .

In a specific embodiment of the present invention, the AD Ir is modified by coupling with a protein or peptide, wherein one or more proteins or peptides are directly or indirectly connected to the AD Ir. The protein may be a naturally occurring protein or a fragment thereof, including (but not limited to) naturally occurring human serum proteins or fragments thereof, such as thyroxine binding protein, thyroxin, a1-acid glycoprotein, transferrin, fibrin Progeny, immunoglobulin, Ig Fc region, albumin, and fragments thereof. A "fragment" means any part of a protein that is smaller than the entire protein but retains the desired function of the protein. An ADDi as described herein can be directly or indirectly attached to a protein by means of a covalent bond. Direct linking means that an amino acid of ADir is directly connected to an amino acid of the modified protein by means of a peptide bond or a disulfide bridge. Indirect connection refers to the connection between ADir and the protein for modification by means of a chemical group that previously exists therebetween or a specific chemical group added by biological or chemical means, or a combination of the above.

In a particular embodiment, the AD Ir is modified by covalent attachment to PEG. An ADIr covalently modified with PEG (in the presence or absence of a linker) may hereinafter be referred to as "ADIr-PEG". When compared to unmodified ADir, ADir-PEG retains most of its enzymatic activity, is much less immunogenic or antigenic, has a significantly longer circulating half-life, and is much more effective in treating tumors.

"Polyethylene glycol" or "PEG" refers to a mixture of condensation polymers of ethylene oxide and water (in the form of branched or linear chains), which is represented by the general formula H (OCH ₂ CH ₂ ) nOH, where n is At least 4. "Polyethylene glycol" or "PEG" is used in combination with a suffix 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.

In one embodiment of the invention, the total weight average molecular weight of PEG is from about 1,000 to about 50,000; in one embodiment, from about 3,000 to about 40,000; and in another embodiment, from about 5,000 to about 30,000; About 8,000 to about 30,000 in some embodiments; about 11,000 to about 30,000 in other embodiments; about 12,000 to about 28,000 in other embodiments; about 16,000 to about 24,000 in still other embodiments; and In other embodiments it is about 18,000 to about 22,000; in another embodiment it is 19,000 to about 21,000, and in one embodiment the total weight average molecular weight of PEG is about 20,000. In general, PEG with a molecular weight of 30,000 or more is difficult to dissolve, and the yield of the formulated product can be reduced. PEG can be branched or linear. In general, increasing the molecular weight of PEG decreases the immunogenicity of ADir. PEG having a molecular weight as described in this example can be used in combination with ADir and optionally biocompatible linkers to treat cancer, including, for example, acute myeloid leukemia (such as recurrent acute myeloid leukemia), breast cancer, ovarian cancer, Colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, gastric cancer And esophageal cancer.

In another embodiment of the invention, the total weight average molecular weight of PEG is from about 1,000 to about 50,000; in some embodiments from about 3,000 to about 30,000; in other embodiments from about 3,000 to about 20,000; About 4,000 to about 12,000 in embodiments; about 4,000 to about 10,000 in still other embodiments; about 4,000 to about 8,000 in other embodiments; about 4,000 to about 6,000 in still other embodiments; and In another embodiment it is about 5,000. PEG may be branched or linear, and in some embodiments is linear. PEG having the molecular weight described in this example can be used in combination with ADir and optionally biocompatible linkers to treat graft-versus-host disease (GVHD) Or cancer.

Although ADir-PEG is an illustrative modified ADir described herein, as will be recognized by the skilled person, ADIR can be modified by other polymers or appropriate molecules to achieve the desired effect, especially reducing antigenicity and increasing serum half-life.

ADir can covalently bind to a modifier (such as PEG) in the presence or absence of a linker, although a preferred embodiment utilizes a linker.

The linker used to covalently link the ADDi to a modifier (eg, PEG) can be any biocompatible linker. As discussed above, "biocompatible" indicates that a compound or group is non-toxic and can be utilized in vitro or in vivo without causing damage, sickness, disease, or death. Modifiers, such as PEG, can be bound to a linker, for example, by means of an ether bond, a thiol bond, or a amide bond. Linker groups include, for example, succinimidyl, amido, amido, urethane, ester, epoxy, carboxyl, hydroxyl, carbohydrate, tyrosine, cysteine, Histamine, methylene, and combinations thereof. In one embodiment, the source of the biocompatible linker is succinate succinate (SS). Other suitable linker sources may include oxycarbonylimidazolyl (including, for example, carbonylimidazole (CDI)), nitrophenyl (including, for example, nitrophenyl carbonate (NCP) or trichlorophenyl carbonate (TCP)), Fluorethionate, aldehyde, isocyanate, vinylfluorenyl, or primary amine. In another embodiment, the linker is derived from SS, SPA, SCM, or NHS; in some embodiments, SS, SPA, or NHS is used, while in other embodiments, SS or SPA is used. Therefore, in certain embodiments, a potential linker may be formed by: methoxy-PEG succinate succinate (SS), methoxy-PEG succinate succinate (SG), methoxy-PEG succinimide carbonate (SC), methoxy-PEG succinimide acetate (SCM), methoxy-PEG2 N-hydroxybutyl Dimethyimine (NHS), Methoxy-PEG butyrate butyrate (SBA), Methoxy-PEG butyrate butyrate propionate (SPA), Methoxy-PEG butyrate Imidinoglutaridine and methoxy-PEG succinimide succinimide.

Alternatively, the ADDi can be directly coupled to a modifier (such as PEG) via an amine, sulfhydryl, hydroxyl, or carboxyl group (ie, without a linker).

The methods known in the art can be used to covalently bind ADir to PEG by means of biocompatible linkers, as described, for example, by Park et al., Anticancer Res., 1: 373-376 (1981 ); And Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, edited by JM Harris, Plenum Press, NY, Chapter 21 (1992), the disclosure of which is hereby incorporated by reference in its entirety.

Linking PEG to the AD Ir increases the circulating half-life of the AD Ir. In general, PEG is attached to the primary amine of ADir. As should be known to those skilled in the art, the selection of the attachment site of PEG or other modifiers on the ADIR is determined by the action of each of the sites within the active domain of the protein. PEG can be attached to the primary amine of ADir without substantial loss of enzymatic activity. For example, the lysine residues present in the ADir are all that the ADir can be linked to PEG by means of biocompatible linkers such as SS, SPA, SCM, SSA, and / or NHS as described herein Maybe. As will be apparent to those skilled in the art in view of the present invention, PEG can also be linked to other sites on the ADIR.

1 to about 30 PEG molecules can be covalently bound to the AD Ir. In certain embodiments, the ADIR is modified by a PEG molecule. In other embodiments, the AD Ir is modified by more than one PEG molecule. In one embodiment, the ADIR is modified by about 1 to about 10 or about 7 to about 15 PEG molecules, and in one embodiment, is modified by about 2 to about 8 or about 9 to about 12 PEG molecules. In another embodiment, the ADIR is modified by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG molecules. In a particular embodiment, the AD Ir is modified by 4.5-5.5 PEG molecules per AD Ir. In another embodiment, the ADIR is modified by 5 ± 1.5 PEG molecules.

In another embodiment, about 15% to about 70% of the primary amine groups in ADir are modified by PEG. In one embodiment, about 20% to about 65%, and about 25% to arginine deiminase. About 60%, or in some embodiments, about 30% to about 55%, or 45% to about 50%, and in other embodiments, about 50% of the primary amine groups are modified by PEG. When PEG covalently binds to the end of the ADIR, it may be necessary to utilize only 1 PEG molecule. Increasing the number of PEG units on the ADir increases the circulating half-life of the enzyme. However, increasing the number of PEG units on the ADir decreases the specific activity of the enzyme. Therefore, as will be apparent to those skilled in the art in view of the present invention, a balance needs to be struck between the two.

A common feature of the biocompatible linker in the present invention is that it is linked to the primary amine of arginine deiminase by means of a succinimide group. Once coupled with ADir, SS-PEG has an ester bond immediately adjacent to PEG, which can make this site sensitive to serum esterase, which can release PEG from ADir in the body. SPA-PEG and PEG2-NHS do not have ester bonds, so they are not sensitive to serum esterase.

In certain embodiments, a biocompatible linker is used in the present invention. The PEG attached to the protein may be a straight chain, such as SS-PEG, SPA-PEG, and SC-PEG, or a branched chain PEG, such as PEG2-NHS.

In certain embodiments, the ADir-associated pegylation site at or adjacent to the catalytic region of the enzyme is modified. For the purposes of the present invention, the phrase "pegylated site" can be defined as any site or position of ADI or ADir that can be covalently modified by polyethylene glycol. A "pegylation site" can be considered to be located at or adjacent to the catalytic region of an enzyme, where PEGylation of the site results in a significant reduction in the catalytic activity of the enzyme. Pegylation of these sites has traditionally led to enzyme inactivation. For example, ADI from human molds has an lysine at position 112, which can be considered to be at or adjacent to the catalytic region of the enzyme. Attachment of PEG to this lysine at position 112 can render the enzyme inactive. In addition, ADI from human mycelia has a cysteine at position 397, which can be regarded as being at or adjacent to the catalytic region of the enzyme. Amino acid substitution of cysteine at position 397 can render the enzyme inactive. In particular, replacing cysteine at position 397 with alanine, histamine, arginine, serine, lysine, or tyrosine can cause Loss of all detectable enzyme activity. ADI from human mycelia also has three lysines located close to this conserved cysteine, specifically Lys374, Lys405 and Lys408. Linking PEG to Lys374, Lys405, Lys408, or a combination thereof can render the enzyme inactive.

It should be understood that ADIr derived from other organisms may also have a pegylation site corresponding to position 112 of ADI from human mold. In addition, the ADI from some organisms may have an lysine corresponding to the same general position as the 112 position of the ADI from human mold. The position of lysine in ADI from such organisms is known to the skilled person and is described in US Patent No. 6,635,462.

Thus, in one embodiment, the invention provides certain amino acid substitutions in the polypeptide chain of ADir. These amino acid substitutions provide a modified ADir with less loss of activity when modified by a modifier (e.g., after pegylation). By eliminating PEGylation sites or other known modification sites at or adjacent to the catalytic region of the enzyme, optimal modifications (eg, pegylation) can be achieved without loss of activity.

It should be understood that other embodiments of the present invention are based on the understanding that certain structural features of arginine deiminase can prevent or interfere with proper and rapid renaturation when produced by means of recombinant technology. In particular, these structural features prevent or prevent the enzyme from adopting an active configuration during recombinant production. For the purposes of the present invention, the phrase "active configuration" may be defined as a three-dimensional structure that allows the enzymatic activity of an unmodified or modified arginine deiminase. The active configuration may be particularly necessary to catalyze the conversion of arginine to citrulline. The phrase "structural characteristic" can be defined as any trait, quality, or property of a polypeptide chain produced by a particular amino acid or combination of amino acids. For example, arginine deiminase may contain amino acids that cause bending or kinking in normal peptide chains and thus prevent the enzyme from adopting an active configuration during enzymatic renaturation. In detail, the arginine deiminase from human mold fungus has a proline at position 210, which can cause bending or kinking in the peptide chain, making the enzyme complex during recombinant production Sex is more difficult. Understand Arginine deiminase derived from other organisms may also have a site corresponding to the 210 position of arginine deiminase from human mold.

The present invention thus provides certain amino acid substitutions in the polypeptide chain of wild-type arginine deiminase. These amino acid substitutions can eliminate problematic structural features in the peptide chain of arginine deiminase. These amino acid substitutions provide renaturation improvements of the modified arginine deiminase. These amino acid substitutions make it possible to use a reduced amount of buffering agent for rapid renaturation of the modified arginine deiminase. These amino acid substitutions can also increase the yield of renatured modified arginine deiminase. In one embodiment of the invention, the modified arginine deiminase has an amino acid substitution at P210 or an equivalent residue. As mentioned above, the arginine deiminase derived from human mold fungi has the amino acid proline at position 210. Although the present invention is not limited, the presence of the amino acid proline at position 210 currently causes bending or kinking in the normal polypeptide chain, and the increase in bending or kinking refolds arginine deiminase (i.e. (Folding). Substitution of proline at position 210 makes it possible to use a reduced amount of buffering agent for rapid renaturation of modified arginine deiminase. Substitution of proline at position 210 can also increase the yield of renatured modified sperine deiminase. In one embodiment, the proline at position 210 is substituted with serine. It should be understood that according to this aspect of the invention, other substitutions may be made at position 210. Examples of other substitutions include Pro210 for Thr210, Pro210 for Arg210, Pro210 for Asn210, Pro210 for Gln210, or Pro210 for Met210. By eliminating the structural features associated with the amino acid at position 210 of the wild-type sperine deiminase, proper refolding of the enzyme can be achieved.

The method of the invention may involve in vitro or in vivo application. In the case of in vitro applications (including cell culture applications), the compounds described herein can be added to cells in culture and subsequently grown. The compounds of the present invention can also be used to promote the production of single and / or multiple antibodies using antibody production techniques well known in the art. As will be apparent to those skilled in the art, single and / or multiple strain antibodies can subsequently be used in a variety of diagnostic applications.

Means of in vivo administration of a compound of the present invention will vary depending on the intended application. Administration of the ADDi composition described herein in pure form or in the form of a suitable pharmaceutical composition can be performed by any of the accepted modes of administration for agents that serve a similar purpose. Pharmaceutical compositions can be prepared by combining ADir (e.g., ADir-PEG, ADDi-PEG 20) with an appropriate physiologically acceptable carrier, diluent or excipient, and can be formulated as a solid, semi-solid, Preparations in liquid or gaseous form, such as lozenges, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. In addition, other pharmaceutically active ingredients (including other anticancer agents as described elsewhere herein) and / or suitable excipients (such as salts, buffers, and stabilizers) may (but need not) be present in the combination Inside. Administration can be achieved by a number of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or local. The mode of administration depends on the nature of the condition to be treated or prevented. Therefore, ADir-PEG (such as ADir-PEG 20) can be administered orally, intranasally, intraperitoneally, parenterally, intravenously, intralymphally, intratumorally, intramuscularly, Intramembrane, transepithelial and transdermal administration. After administration, the amount that slows, inhibits, prevents or delays the progression of cancer and / or cancer metastasis is considered effective. In certain embodiments, the ADDi compositions herein increase a patient's median survival time by a statistically significant amount. In one embodiment, the ADir treatment described herein increases the median survival time of a patient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks or longer. In certain embodiments, ADir treatment increases the median survival time of a patient by one, two, three, or more years. In one embodiment, the ADDi treatment described herein increases survival without disease progression 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 ADDi treatment described herein increases survival without disease progression by one, two, three or more years.

In certain embodiments, such as by a statistically significant reduction in the amount of live tumors (e.g., a decrease in tumor mass by at least 10%, 20%, 30%, 40%, 50% or more) or by a change in scan size ( (E.g., a statistically significant reduction) indicates that the amount administered is sufficient to cause tumor regression. in In some embodiments, the amount administered is sufficient to cause stable disease. In other embodiments, the amount administered is sufficient to cause a clinically relevant reduction in the symptoms indicated by a particular disease known to the skilled clinician.

In certain embodiments, the amount administered is sufficient to inhibit NO synthesis, inhibit angiogenesis, and / or sufficient to induce apoptosis in tumor cells, or any combination thereof. NO synthesis, angiogenesis, and apoptosis can be measured using methods known in the art, see, for example, Current Protocols in Immunology or Current Protocols in Molecular Biology , John Wiley & Sons, New York, NY (2009 and the latest); Ausubel et al., Short Protocols in Molecular Biology , 3rd Edition, Wiley & Sons, 1995; and other similar references. In a specific embodiment, the administered amount inhibits NO synthesis and inhibits the growth of melanoma and complement, enhancing or synergistically enhancing other chemotherapy (such as cisplatin) as described herein. Accordingly, one embodiment of the present invention provides a method for treating melanoma by administering ADir-PEG 20 in combination with cisplatin, wherein the treatment depletes endogenous nitrogen oxide (NO).

The precise dose and duration of treatment will vary with the disease being treated, and can be determined empirically using known testing protocols, or by testing and inferring the composition in a model system known in the art. Controlled clinical trials are also possible. The dose may also vary with the severity of the condition to be alleviated. Pharmaceutical compositions are generally formulated and administered to exert therapeutically applicable effects while minimizing unwanted side effects. The composition may be administered at once, or may be divided into multiple smaller doses for administration at various time intervals. For any particular individual, specific dosage regimens can be adjusted over time according to individual needs.

ADir compositions can be administered alone or in combination with other known cancer treatments such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, and the like. The composition can also be administered in combination with antibiotics.

The ADIr composition can also be administered alone or in combination with an ADI-PEG 20 therapy. In certain embodiments, an ADIr as described herein is used that has been treated with ADI-PEG 20 and has developed anti- Patients with ADI-PEG 20 antibody. These patients no longer benefit from ADI-PEG 20 treatment because the enzyme is offset by antibodies. Therefore, in certain embodiments, the present invention provides a method for treating cancer, improving the symptoms of cancer, or inhibiting the progression of cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising ADI-PEG 20 And, after a period of time, administering to the patient a composition comprising an ADDi as described herein, thereby treating the cancer, ameliorating the symptoms of the cancer, or inhibiting the progression of the cancer.

In one embodiment of the method, the period of time is determined by detecting a predetermined amount of an anti-ADI-PEG 20 antibody in a patient, wherein the composition containing the ADir is detecting the anti-ADI-PEG 20 antibody in the patient. After a predetermined amount is administered. In certain embodiments, a threshold or predetermined amount of an anti-ADI-PEG 20 antibody in a patient to be treated with ADI-PEG 20 and an ADIr of the invention may be established. Methods known in the art (e.g., using a Receiver Operator Characteristic curve or an `` ROC '' curve) can be used to establish the `` predetermined threshold content '' of anti-ADI-PEG 20 antibodies (also known as `` scheduled Content "or" predetermined cut-off value ") or sometimes called a pre-determined cut-off. In one embodiment, it is believed that even very low levels of anti-ADI-PEG 20 antibodies are sufficient to ensure conversion of the treatment from ADI-PEG 20 to the ADIr-PEG of the present invention. In some embodiments, the appropriate content of the anti-ADI-PEG 20 that will decide when to terminate ADI-PEG 20 treatment and begin treatment with the ADIr-PEG of the present invention can be determined by a skilled clinician.

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

Typical routes for administration of these and related pharmaceutical compositions therefore include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, intrabuccal, transrectal, transvaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

The method of formulating a pharmaceutical composition according to some embodiments of the present invention should be such that after the composition is administered to a patient, the active ingredients contained therein can be bioavailable. The composition to be administered to an individual or patient may be in the form of one or more dosage units, where, for example, a lozenge may be a single dosage unit, and a container of the ADIR composition in the form of an aerosol described herein may hold multiple doses unit. The actual method of preparing these dosage forms is known to those skilled in the art or knows well; for example, see Remington: The Science and Practice of Pharmacy , 20th Edition (Philadelphia College of Pharmacy and Science, 2000). According to the teachings herein, the composition to be administered will, in any event, contain a therapeutically effective amount of the ADir-PEG of the present invention (such as ADir-PEG 20) for the treatment of a disease or condition of interest. In certain embodiments, the pharmaceutical or therapeutic composition is sterile and / or pyrogen-free.

The pharmaceutical composition may be in a solid or liquid form. In one embodiment, the carrier (cluster) is granular so that the composition is in the form of, for example, a lozenge or powder. The carrier (group) can be a liquid, such that the composition is, for example, an oral oil, an injection, or an aerosol suitable for, for example, inhalation administration. When intended for oral administration, pharmaceutical compositions are generally in solid or liquid form, with semi-solid, semi-liquid, suspension and gel forms being included in the forms considered herein as solid or liquid.

When used as a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewable tablets, powder tablets or the like. The solid composition typically contains one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or paste Disintegrating agents, such as alginic acid, sodium alginate, starch starch (primogel), corn starch, and the like; lubricants, such as magnesium stearate or hydrogenated vegetable oil (Sterotex); slip agents, such as colloid Silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or citrus flavoring; and coloring agents. When the pharmaceutical composition is in the form of a capsule (eg, a gelatin capsule), it may contain a liquid carrier such as polyethylene glycol or oil in addition to the above types of materials.

The pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. For two examples, liquids can be used for oral administration or for delivery by injection. When intended for oral administration, the preferred composition contains one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer in addition to the compound of the present invention. In the composition intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffering agent, a stabilizer, and an isotonicity agent may be included.

Liquid pharmaceutical compositions, whether in solution, suspension, or other similar forms, can include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solutions (in some embodiments, physiological saline) , Ringer's solution, isotonic sodium chloride, non-volatile oils (such as synthetic mono- or diglycerides that can act as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol, or other solvents; anti- Bacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetate, citrate or phosphate; and Tonic agents, such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

Liquid pharmaceutical compositions intended for parenteral or oral administration should contain an amount of ADir (such as ADir-PEG 20) as disclosed herein, such that a suitable dosage is obtained. Typically, this amount is at least 0.01% ADIr in the composition. When intended for oral administration, this amount may vary between 0.1% and about 70% by weight of the composition. Certain oral pharmaceutical compositions contain between ADir-PEG between about 4% and about 75%. In certain embodiments, the pharmaceutical compositions and formulations of the present invention are prepared such that parenteral dosage units contain between AD and 10% by weight ADir-PEG before dilution.

The pharmaceutical composition is intended for topical administration, in which case the downloading agent may suitably comprise a solution, emulsion, ointment or gel base. For example, the matrix may include one or more of the following: paraffin fat, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (such as water and alcohol), and emulsifiers and stabilizers. Thickeners may be present in pharmaceutical compositions for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device. The pharmaceutical composition is intended for rectal administration in the form of, for example, a suppository, which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

A pharmaceutical composition can include a variety of materials that alter the physical form of a solid or liquid dosage unit. For example, the composition may include a material forming a coating shell surrounding the active ingredient. The materials forming the coating shell are typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be enclosed in a gelatin capsule. A pharmaceutical composition in solid or liquid form may include a medicament bound to AD Ir-PEG and thereby helping to deliver the compound. Suitable agents that can serve this purpose include single or multiple strains of antibodies, one or more proteins, or a liposome. A pharmaceutical composition may consist essentially of dosage units which can be administered in the form of an aerosol. The term aerosol is used to refer to various systems ranging from their systems with colloidal properties to systems consisting of pressurized packages. Delivery can be performed by liquefying or compressing the gas or by a suitable pump system that dispenses the active ingredient. Aerosols can be delivered in a single-phase, two-phase or three-phase system to deliver the active ingredient. Aerosol delivery includes essential containers, activators, valves, sub-containers, and the like, which together form a kit. Those skilled in the art can generally determine the preferred aerosol without undue experimentation.

Pharmaceutical compositions can be prepared by methods well known in the pharmaceutical arts. For example, a pharmaceutical composition intended to be administered by injection may be a composition that will include one or more of ADIr-PEG and optionally a salt, buffer, and / or stabilizer as described herein It is prepared by combining with sterile distilled water to form a solution. A surfactant may be added to promote the formation of a homogeneous solution or suspension. A surfactant is a compound that non-covalently interacts with an ADDi-PEG composition to promote dissolution or uniform suspension of the ADDi-PEG in an aqueous delivery system.

The composition can be administered in a therapeutically effective amount, which will vary depending on a number of factors including: the activity of the particular compound employed (e.g., ADIR-PEG); the metabolic stability and duration of action of the compound; the patient Age, weight, general health, gender, and diet; mode and timing of administration; rate of excretion; combination of drugs; severity of specific illness or condition; and individuals undergoing therapy.

A therapeutically effective amount of one of the compounds of the present invention is an amount effective for inhibiting tumor growth. Generally speaking, starting with smaller doses, these doses can be increased in smaller increments until the best results in these cases are achieved. In general, a therapeutic dose of a compound of the present invention can be from twice a week to about once every two weeks, each time from about 1 to about 200 mg / kg. For example, the dose may be about 1 mg / kg once a week to about 20 mg / kg once every 3 days in the form of a 2 ml intravenous injection. In another embodiment, the dose may be about every 3 days, about once a week, twice a week, or about once to about administration and about 50IU / m ² to about 700IU / m ² every 2 weeks. In certain embodiments, the dose may be about every 3 days, about once a week, twice a week, or about once administered about every two weeks with about ^{50IU / m 2, 60IU / m} 2, 70IU / m 2, 80IU / m ² , 90IU / m ² , 100IU / m ² , 110IU / m ² , 120IU / m ² , 130IU / m ² , 140IU / m ² , 150IU / m ² , 160IU / m ² , 170IU / m ² , 180IU / m ² , 190IU / m ² , 200IU / m ² , 210IU / m ² , 220IU / m ² , 230IU / m ² , 240IU / m ² , 250IU / m ² , 260IU / m ² , 270IU / m ² , ^{280IU / m 2, 290IU / m} 2, 300IU / m 2, 310IU / m 2, about 320IU / m ^2, about ^{330IU / m 2, 340IU / m} 2, about ^{350IU / m 2, 360IU / m} 2, 370IU / m ² , 380IU / m ² , 390IU / m ² , 400IU / m ² , 410IU / m ² , 420IU / m ² , 430IU / m ² , 440IU / m ² , 450IU / m ² , 500IU / m ² , 550IU / ^{m 2, 600IU / m 2,} 620IU / m 2, 630IU / m 2, 640IU / m 2, 650IU / m 2, 660IU / m 2, 670IU / m 2, 680IU / m 2, 690IU / m 2 , or about 700IU / m ² . In certain embodiments, the dosage can be changed as needed by a skilled clinician.

The optimal dose of ADIr-SS-PEG5,000 may be about twice a week, and the optimal dose of ADIr-SS-PEG20,000 may be about once a week to about once every two weeks. In certain embodiments, the optimal dose of ADDi-SS-PEG20,000 may be about twice a week.

ADIr-PEG can be mixed with a phosphate buffered saline solution or any other suitable solution known to those skilled in the art prior to injection. In one embodiment, the liquid composition comprising ADir-PEG comprises about 10 to about 12 mg of ADIr, about 20 to about 40 mg of polyethylene glycol, 1.27 mg + 5 in an appropriate amount of water for injection (e.g., about 1 ml or about 2 ml). % Sodium dihydrogen phosphate, USP; about 3 mg + 5% disodium hydrogen phosphate, USP; 7.6 mg + 5% sodium chloride, USP; pH value is about 6.6 to about 7. In one embodiment, the liquid composition comprising ADir-PEG comprises histidine-HCl, and in certain embodiments, the composition buffer is from about 0.0035M histidine-HCl to about 0.35M histidine- HCl. In a specific embodiment, the composition is formulated in a buffer comprising 0.035M histidine-HCl (pH 6.8) and 0.13M sodium chloride. In another embodiment, the composition is formulated in a buffer comprising 0.02M sodium phosphate buffer (pH 6.8) and 0.13M sodium chloride.

In one embodiment, the pH of the composition comprising ADir or ADir-PEG is about 5 to about 9, about 6 to about 8, or about 6.5 to about 7.5. In some embodiments, the pH of the composition comprising ADir is about 6.8 ± 1.0.

50EU/mL。 In one embodiment, the free PEG in the composition containing ADDi-PEG is between 1% and 10%, and in another embodiment is less than 7% of the total PEG, less than 6%, less than 5%, and less than 4%, less than 3%, less than 2% or less than 1%. In certain embodiments, the unmodified ADIr in a composition comprising ADIr-PEG is less than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or Less than 0.1%. Generally, the total impurities of the composition containing ADDi-PEG are less than or equal to about 4%, 3%, 2%, 1.5%, 1%, or 0.5%. In one embodiment, the endotoxin limit meets the needs stated in the USP, that is,

50EU / mL.

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

In one embodiment, the Km of ADir or ADIR-PEG in the composition is about 0.1 μM or 0.5 μM to about 15 μM, and in another embodiment, about 1 μM to about 12 μM, about 1 μM to about 10 μM, about 1.5 μM to about 9 μM, about 1.5 μM to about 8 μM, or about 1.5 μM to about 7 μM. In certain embodiments, the Km of ADir or ADIR-PEG in the composition is from about 1.0 μM to about 10 μM, or from about 1.5 μM to about 6.5 μM. In some embodiments, the Km of ADir or ADIR-PEG in the composition is about, at least about, or less than about 0.1 μM, about 0.5 μM, about 1.0 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, About 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, or about 7 μM, or about 8 μM, or about 9 μM, or about 10 μM.

In one embodiment, the composition of the ADIR or Kcat ADIr-PEG from about 0.5 to about 80 sec ^-1 sec ^-1, or about 0.5 to about 70 sec ^-1 sec ^-1, or about 0.5 sec ^-1 to about 60 sec ^-1, or about 0.5 to about 50 sec ^-1 sec ^-1, or about 0.5 to about 40 sec ^-1 sec ^-1, or about 0.5 to about 30 ^{sec-1 sec-1,} or about 0.5 seconds ^{- 1} sec ^-1 to about 20, or about 0.5 to about 15 ^{sec-1 sec-1,} and in another embodiment from about 0.5 to about 80 sec ^-1 sec ^-1, or from about 1 to about 80 ^sec-1 Sec ^-1 , or about 5 sec ^-1 to about 80 sec ^-1 , or about 10 sec ^-1 to about 80 sec ^-1 , or about 20 sec ^-1 to about 80 sec ^-1 , or about 30 sec ^-1 to about 80 sec ^-1, or about 40 to about 80 sec ^-1 sec ^-1, or about 50 seconds to about 80 ^-1 sec ^-1, or about 60 to about 80 sec ^-1 sec ^-1, or about 70 seconds ^{- 1} sec ^-1 to about 80, or about 1 ^{sec-1 sec-1} to about 12, from about 1 to about 10 sec ^-1 sec ^-1, 1.5 sec ^-1 sec ^-1 to about 9, from about 2 sec ^-1 To about 8 seconds ^-1 , or about 2.5 seconds ^-1 to about 7 seconds ^-1 . In certain embodiments, the composition of the ADIR or Kcat ADIr-PEG is from about 2.5 to about 7.5 sec ^-1 sec ^-1. In some embodiments, the Kcat of ADir or ADDi-PEG in the composition is about 2.5 seconds ^-1 , about 3 seconds ^-1 , about 3.5 seconds ^-1 , about 4 seconds ^-1 , about 4.5 seconds ^-1 , about 5 seconds ^-1 , about 5.5 seconds ^-1 , about 6 seconds ^-1 , about 6.5 seconds ^-1 , about 7 seconds ^-1 , about 7.5 seconds ^-1 or about 8 seconds ^-1 , about 10 seconds ^-1 , and about 15 seconds ^-1 About 20 seconds ^-1 , about 25 seconds ^-1 , about 30 seconds ^-1 , about 35 seconds ^-1 , about 40 seconds ^-1 , about 45 seconds ^-1 , about 50 seconds ^-1 , about 55 seconds ^-1 , about 60 seconds ^-1 , about 65 seconds ^-1 , about 70 seconds ^-1 , about 75 seconds ^-1 , about 80 seconds ^-1 , about 85 seconds ^-1 , about 90 seconds ^-1 , about 95 seconds ^-1 , or about 100 Seconds ^-1 .

In one embodiment, the conductivity (also referred to as the specific conductivity in this technique) of ADir or ADDi-PEG in the composition is about 5 mS / cm to about 20 mS / cm, and in other embodiments about 5 mS / cm to about 15 mS / cm, about 7 mS / cm to about 15 mS / cm, about 9 mS / cm to about 15 mS / cm, or about 10 mS / cm to about 15 mS / cm. In some embodiments, the conductivity of the ADIR or ADDi-PEG in the composition is about 9 mS / cm, about 10 mS / cm, about 11 mS / cm, about 12 mS / cm or about 13 mS / cm, about 14 mS / cm, or about 15mS / cm. In certain embodiments, the conductivity of the ADir or ADir-PEG in the composition is about 13 mS / cm ± 1.0 mS / cm.

In one embodiment, the weight molar osmolarity of the ADIR or ADDi-PEG in the composition is about 50 mOsm / kg to about 500 mOsm / kg, about 100 mOsm / kg to about 400 mOsm / kg, about 150 mOsm / kg to about 350 mOsm / kg, about 200 mOsm / kg to about 350 mOsm / kg, or about 250 mOsm / kg to about 350 mOsm / kg. In certain embodiments, the weight mole osmolality of the ADIR or ADIR-PEG in the composition is about 300 ± 30 mOsm / kg.

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

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

The ADir-PEG-containing composition of the present invention may also be administered at the same time as, or before, one or more other therapeutic agents, including ADI-PEG 20. The combination therapy may include the administration of a single pharmaceutical dosage formulation containing a compound of the present invention and one or more additional active agents, and the administration of the ADir-PEG-containing (such as Composition of ADIr-PEG 20) and each active agent. For example, ADir-PEG and other active agents as described herein may be administered to a patient together in the form of a single oral dose composition, such as a lozenge or capsule, or each agent may be administered as a separate oral dose formulation versus. Similarly, ADIR-PEG and other active agents as described herein may be administered to a patient together in the form of a single parenteral dosage composition, such as in a saline solution or other physiologically acceptable solution, or each agent may be borrowed. They are administered by the same or different routes (for example, one by injection, one by mouth) as separate parenteral dosage formulations. Where individual dosage formulations are used, compositions comprising ADir-PEG and one or more additional active agents may be administered at substantially the same time (i.e., simultaneously), or at separately staggered times (i.e., sequentially And in any order); combination therapy is understood to include all such regimens.

Accordingly, in certain embodiments, it is also contemplated to administer the ADir composition of the invention in combination with one or more additional therapeutic agents. Such therapeutic agents are acceptable in the art as standard treatments for specific disease conditions, such as specific cancers or GVHD, as described herein. All Exemplary therapeutic agents covered include cytokines, growth factors, steroids, NSAID, DMARD, anti-inflammatory agents, chemotherapeutics, radiation therapy agents, autophagy inhibitors or other active and adjuvant agents.

In certain embodiments, the ADDi compositions disclosed herein can be administered in combination with a variety of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ^™ ); alkyl sulfonates such as busulfan, improsulfan, and Piposulfan; aziridine, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimine and methyl melamine , Including altretamine, triethylenemelamine, triyleneethylphosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; Nitrogen mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, nitrogen Mustard (mechlorethamine), nitrogen mustard oxide hydrochloride, melphalan, novembichin, cholesterol p-phenylacetate nitrogen mustard (phenesterine), prednimustine (tredifosin), tromethamine ( trofosfamide), uracil mustard; Nitrosurea, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ramo Ranimustine; antibiotics such as aclacinomysin, actinomycin, autramycin, azaserine, bleomycin, actinomycetes C (cactinomycin), calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, actinomycin D (dactinomycin) Daunorubicin, detorubicin, 6-diazo-5-sideoxy-L-n-leucine, doxorubicin, epirubicin ), Esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, Olivomycin, peplomycin, potfiromycin, puromycin, quinine Quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, netazita Zinostatin, Zorubicin; Antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs, such as denopterin, methylamine Methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, Thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine ( cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, flexor Dromostanolone propionate, epitiostanol, mepitiosta ne), testolactone; anti-adrenal, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; Aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene Edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; eytoglu (etoglucid); gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; Nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazine; procarbazine ( procarbazine); PSK.RTM .; razoxane; sizofiran; germanium snail (spirogermanium); tenuazonic acid; triaziquone; 2,2 ', 2 "-trichlorotriethylamine;urethan;vindesine;Dacarbazine;mannomustine;miobronitol;mitoactol;piperobromine;piobroman;gacytosine; arabinoside ) ("Ara-C");cyclophosphamide;thiotepas; taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE® , Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; thiopurine; methotrexate; platinum analogs, such as Cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone ); Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin ^TM (bexarotene), Panretin ^TM (alitretinoin); ONTAK ^TM ( Devilukin diftitox); esperamicin; capecitabine; and a pharmaceutically acceptable salt, acid or derivative of any of the above. Also included in this definition are anti-hormonal agents, such as anti-estrogens, which function to regulate or inhibit the effects of hormones on tumors, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4 ( 5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and Anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin. Other chemotherapeutic agents include sorafenib and other protein kinase inhibitors, such as afatinib, axitinib, bevacizumab, cetuximab ( cetuximab), crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, latin Lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegastat Pegaptanib, ranibizumab, ruxolitinib, trastuzumab, vandetanib, vemurafenib and suni Sunitinib; sirolimus (rapamycin), everolimus and other mTOR inhibitors. A pharmaceutically acceptable salt, acid or derivative of any of the above is also encompassed for use herein.

In certain embodiments, the ADDi compositions disclosed herein can be administered in combination with a variety of autophagy inhibitors. In some preferred embodiments, the autophagy inhibitor is selected from the group consisting of chloroquinine, 3-methyladenine, hydroxychloroquine (Plaquenil.TM.), Bava Bafilomycin A1, 5-amino-4-imidazolidine riboside (AICAR), okadaic acid, autophagy-inhibiting algae toxin (which inhibits type 2A or type 1 proteins Phosphatase), cAMP analogs, and drugs that increase cAMP content, adenosine, N6-mercaptopurine riboside, wortmannin, and vinblastine. Alternatively, it can be used to inhibit autophagy Antisense RNA or siRNA expressing essential proteins, such as ATG5.

In one embodiment, the combination of ADIR-PEG and one or more therapeutic agents acts complementary, additively, or synergistically. In this regard, described herein are complementary or synergistic agents that include therapeutic agents (e.g., chemotherapeutic agents, autophagy inhibitors, mTOR inhibitors, or For treating cancer, GVHD, or any other therapeutic agent as described herein for inflammatory bowel disease), wherein the complementary or synergistic effect is shown as a detectable effect in a magnitude ratio in the presence of a chemotherapeutic agent but without the ADir-PEG The effect detectable is greater in the composition and / or in the presence of ADir-PEG but in the absence of a chemotherapeutic agent (ie, in a statistically significant manner relative to appropriately controlled conditions). Methods for measuring synergy and complementarity are known in the art (see, for example, Cancer Res January 15, 2010; 70 ; 440).

Compositions including ADir and optionally other therapeutic agents as described herein can be used in therapeutic methods for treating cancer and methods for preventing cancer metastasis. Accordingly, the present invention provides methods for treating a variety of different cancers, improving their symptoms, or suppressing their progress or preventing them. In another embodiment, the present invention provides a method for treating GVHD, ameliorating symptoms of GVHD, or inhibiting the progression of GVHD. In particular, the present invention provides methods for treating cancer, GVHD, ameliorating symptoms, or inhibiting its progression in a patient, the methods comprising optionally treating with ADI-PEG 20 (especially in patients with anti-ADI- In the case of a PEG 20 antibody), the patient is administered a therapeutically effective amount of an ADDi composition as described herein, thereby treating the cancer or GVHD, improving its symptoms, or inhibiting its progression. Thus, the ADir compositions described herein can be administered to individuals suffering from inflammatory bowel disease (such as Crohn's disease; ulcerative colitis), GVHD, or cancer including, but not limited to, hepatocytes Cancer, leukemia (e.g., acute myeloid leukemia and recurrent acute myeloid leukemia), melanoma (including metastatic melanoma), sarcoma (including (but not limited to) metastatic sarcoma, uterine leiomyosarcoma), pancreatic cancer, prostate cancer ( (Such as, but not limited to, hormone-refractory prostate cancer), mesothelioma, and lymphocytic white blood Disease, chronic myelogenous leukemia, lymphoma, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer (including (but not limited to) gastric adenocarcinoma), glioma, pleomorphic glioblastoma Tumor, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), kidney cancer (including (but not limited to) renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer (including (But not limited to) squamous cell carcinoma of the head and neck; tongue cancer), cervical cancer, testicular cancer, gallbladder, bile duct cancer, and gastric cancer.

In another embodiment, the present invention provides a method of treating cancer, ameliorating symptoms, or inhibiting its progression in a patient, the method comprising administering to the patient one or more of including ADir and optionally as described herein Compositions of other therapeutic agents, wherein the cancer lacks ASS, ASL, or both. In this regard, ASS or ASL deficiency may be a decrease in performance as measured by mRNA or protein performance, or may be a decrease in protein activity, and generally includes a statistically significant as determined by a skilled person Reduced performance or activity. A decrease in ASS or ASL performance or activity can be about a 5%, 10%, 15%, 20%, 25%, 30%, 35% decrease in performance or activity compared to the performance or activity in a suitable control sample known to be cancer-free. %, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more. In certain embodiments, the ASS or ASL performance or activity is reduced by at least one-half compared to the performance or activity in a non-cancer control sample.

In certain embodiments, the reduction in the performance or activity of ASS or ASL results from methylation of the ASS or ASL promoter or inhibition of the ASS or ASL promoter. In another embodiment, the reduction in the performance or activity of ASS or ASL is caused by a DNA mutation (such as one or more point mutations, small deletions, insertions, and the like) or a chromosomal abnormality that results in a gene deletion. In one embodiment, the cancer is ASS or ASL negative, meaning that no performance or activity is observed.

The decrease in ASS or ASL performance or activity can be measured using any method known in the art, such as (but not limited to) quantitative PCR, immunohistochemistry, enzyme activity analysis (e.g., measuring the conversion of citrulline to spermine Conversion of succinate or spermine succinate to Analysis of the conversion of arginine and fumarate) and its method.

Accordingly, the invention provides methods for treating cancer, ameliorating symptoms, or inhibiting its progression in a patient, the methods comprising administering to the patient a composition comprising an ADDi as described herein, wherein the cancer exhibits ASS or Reduced performance or activity of ASL or both, where the cancer includes, but is not limited to, hepatocellular carcinoma, leukemia (e.g., acute myeloid leukemia and recurrent acute myeloid leukemia), melanoma (including metastatic melanoma), and sarcoma (including (But not limited to) metastatic sarcoma, leiomyosarcoma of the uterus), pancreatic cancer, prostate cancer (such as (but not limited to) hormone-refractory prostate cancer), mesothelioma, lymphocytic leukemia, chronic myeloid leukemia, lymphoma, Small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer (including, but not limited to, gastric adenocarcinoma), glioma, glioblastoma multiforme, retinoblastoma, neuroblastoma , Non-small cell lung cancer (NSCLC), kidney cancer (including (but not limited to) renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer (including (but not limited to) head And squamous cell carcinoma of the neck; tongue cancer), cervical cancer, testicular cancer, gallbladder, bile duct cancer, and stomach cancer.

Various studies in the literature have shown the lack of ASS in the following tumors: acute myeloid leukemia (AML), bladder tumors, breast tumors, colorectal tumors, gastric tumors, glioblastoma, HCC, lymphoma, Melanoma, mesothelioma, non-small cell lung tumor, ovarian tumor, pancreatic tumor, prostate tumor, kidney tumor, sarcoma and small cell lung tumor. Accordingly, the use of AD Ir-PEG to treat such ASS-deficient cancers, alone or in combination with other treatments, including first treatment with ADI-PEG 20, is specifically covered herein.

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

In certain embodiments, the invention provides a method of treatment, wherein administration of a composition comprising an ADDi as described herein depletes arginine in the plasma for at least one month, two months, three months, and four months , 5 months, 6 months or longer. In another embodiment, the present invention provides a method of treatment, wherein administering a composition comprising an ADIr as described herein after the detection of an anti-ADI-PEG 20 antibody terminates treatment with ADI-PEG 20 Arginine is depleted for at least one month, two months, three months, four months, five months, six months or more.

Examples Example 1

Screening and selection of ADI enzymes with low cross-reactivity with patients' anti-ADI-PEG20 antibodies

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

A large number of ADI enzymes are listed. Table 1 lists the 24 ADI enzymes selected for the percent sequence identity with respect to human AMI. According to the literature, the ADI amino acid sequences of human mold, arginine, and arthritis ADI are closely related, and these enzymes have good catalytic properties. More recently, additional ADI enzymes have been found with sequences closely related to these three enzymes. Far more relevant mycoplasma ADI enzymes have been identified, although less is known about them. There are even more distantly related ADI enzymes from bacteria and other sources.

Certain protein sequences available in public databases may not necessarily be full-length ADI sequences. In those cases, publicly available sequences are extended based on known ADI sequences where full-length ADI is required. In some cases, the ADI protein is modified elsewhere (eg, C251S substitution). These synthetic ADI sequences are provided in SEQ ID NOs: 26-32 and correspond to the expanded and / or modified ADI sequences of: Arginine mold (C251S), Arthritis mold (C251S), Cerebral seal mycelium, Mycelium feline, Mycelium seal, Halobacterium australis and Mycobacterium bovis.

A variety of ADI enzymes from a variety of organisms are characterized to determine which enzymes are expected (even in the presence of anti-ADI-PEG 20 antibodies) to remove and maintain low spermine levels in a patient's blood. Table 2 (below) lists the subset of ADI enzymes selected from Table 1 studied. As detailed below It is stated that the data from these studies show that ADI from multiple species, which is closely related to human mold plasmodium based on sequence identity, has sufficiently good enzymatic properties and reduced cross-reactivity with anti-ADI-PEG 20 antibodies .

ADI preparation. Colony, expression and purification of recombinant ADI enzymes for testing according to standard protocols as described, for example, in Gallego et al., PLOS One, 7 (10): e47886, 2012; Monstadt and Holldorf, Biochem. J. 273: 739-745, 1990; Joo Noh et al., Molecules and Cells. 13: 137-143, 2002; and Sugimura et al., Infection and Immunity. 58: 2510-2515, 1990.

Human anti-ADI-PEG20 antibody purification. Anti-ADI-PEG20 antibodies were purified from plasma samples of patients who had received ADI-PEG20 during clinical studies. A total of 60 ml of plasma from 8 different patients who had reached a high titer (titer> / = 4) for ADI-PEG20 as determined by ELISA analysis was pooled. Using two-step purification, protein "A" chromatography (GE Healthcare) followed by ADI affinity chromatography. About 20 mg of purified antibody was obtained and stored in aliquots at -80 ° C until needed.

ADI enzyme analysis. Arginine deiminase (ADI) catalyzes the conversion of L-arginine to L-citrulline and ammonia. The colorimetric endpoint analysis (see, eg, Knipp and Vasak, Analytical Biochem. 286: 257-264, 2000) can be used to detect the amount of L-citrulline and a standard curve with known amounts of L-citrulline The specific activity of ADI was calculated by comparison (expressed as IU / mg protein). 1IU enzyme activity is defined as the amount of enzyme that produces 1 μmol of citrulline per minute at the pH and temperature tested. Standard analysis conditions were performed at 37 ° C in physiological HEPES buffer (PHB) (50 mM HEPES, 160 mM NaCl, pH 7.4) (Lang and Zander, Clin Chem Lab Med. 37: 563-571, 1999) plus 0.1% BSA. All samples and standards were performed in duplicate or in triplicate if conditions permit.

The Km and Kcat values were determined by using a variation of the above-mentioned activity analysis. As with the activity analysis, all reactions were performed at 37 ° C in PHB plus 0.1% BSA. Considering differences in activity, the enzyme concentration, reaction time, and substrate concentration were adjusted for each of the ADI or ADir constructs Degree range. Overall, 2nM enzyme, 5 minute reaction time, and 0-160 μM arginine were used as the starting conditions. When optimizing these conditions, particular attention is paid to the percentage of substrates consumed relative to the total substrates added to the reaction. The lower detection limit is 1 μM citrulamic acid and the lower limit of quantification is 2 μM. The citrulline standard curve relates to each plate and is used to quantify the citrulline produced by the enzymatic reaction.

An activity analysis was also performed to assess enzyme activity in the presence of anti-ADI-PEG20 (antibody offset curve). These analyses were performed as described above and in the presence of 800nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, and 0nM anti-ADI-PEG20 antibodies.

Calculation. The citrulamic acid concentration (μM) produced in each reaction well was calculated and averaged using a citrulline standard curve. The speed of each reaction was then calculated in μM / min / 50nM ADI. The specific activity (IU / mg or micromolar product / minute / mg ADI) was calculated by multiplying this value by the "IU" factor (the IU factor was calculated from the molecular weight and reaction volume of ADI). The results are summarized in Table 2 below.

These data show that ADI enzymes, which are highly homologous (approximately 50% to 100% identical) to AMI of human mold, maintain excellent catalytic activity. It has also been shown that such enzymes have a reduced affinity for anti-ADI-PEG 20 antibodies to patients, as measured by enzyme activity in the presence of anti-ADI-PEG 20 antibodies, such as the affinity for human mold mold. Accordingly, these ADI enzymes may have therapeutic utility for therapies used to treat cancer alone or after treatment with ADI-PEG 20 to prolong and / or increase the effectiveness of arginine depletion therapy.

The various embodiments described above may be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in this application information form are cited in their entirety. Ways are incorporated herein. Aspects of the embodiments may be modified as necessary to use the concepts of various patents, applications, and publications to provide yet other embodiments.

These and other changes can be made to the embodiments in light of the above embodiments. In general, in the following patent application scope, the terms used should not be interpreted to limit the scope of patent application to the specific embodiments disclosed in this specification and the scope of patent application, but should be interpreted to include all possible embodiments and the application The patent scope has the right to claim the full scope of equivalents. Accordingly, the scope of patent application is not limited by the present invention.

<110> Cayman Islands Shangpraris Group

<120> Arginine deiminase with reduced cross-reactivity to ADI-PEG 20 antibody for cancer treatment

<130> POLA-003 / 011W

<150> US 61 / 790,833

<151> 2013-03-15

<160> 32

<170> PatentIn version 3.5

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Claims (18)

A therapeutic composition comprising a pegylated arginine deiminase (ADI) and a pharmaceutically acceptable carrier, wherein the pegylated ADI is covalently bound to The PEGylated ADI comprises the amino acid sequence shown in any one of SEQ ID NO: 8, 2 or 6 on at least one PEG molecule, or is shown to be identical to any of SEQ ID NO: 8, 2 or 6 One is an amino acid sequence with at least 95% sequence identity and arginine deiminase activity, and wherein the cross-reactivity between the PEGylated ADI and the patient's anti-ADI-PEG 20 antibody has been reduced. The therapeutic composition of claim 1, wherein the pegylated ADI has been modified to remove at least one pegylation site. The therapeutic composition of claim 1 or 2, wherein at least one of the lysine residues has been modified with an amino acid substitution. The therapeutic composition of claim 3, wherein: (a) at least 5 lysine residues have been modified by amino acid substitutions; (b) at least 10 lysine residues have been modified by amino acid substitutions; c) at least 15 lysine residues have been modified by amino acid substitution; or (d) at least 20 lysine residues have been modified by amino acid substitution. The therapeutic composition of claim 1 or 2, wherein: (a) the ADI is covalently bound to more than one PEG molecule; (b) the ADI is covalently bound to about 1 to about 10 PEG molecules; or (c ) The ADI is covalently bound to about 2 to about 8 PEG molecules. The therapeutic composition of claim 5, wherein the PEG molecules are linear or branched PEG molecules. The therapeutic composition of claim 1, wherein the total weight average molecular weight of the PEG is 1,000 to 40,000. The therapeutic composition of claim 1, wherein the total weight average molecular weight of the PEG is from 10,000 to 30,000. The therapeutic composition according to claim 1, wherein the linker is succinimide, amido, amido, amino formate, ester, epoxy, carboxyl, hydroxyl, carbohydrate, tyrosine Group, cysteine group, histidine group, methylene group, or any combination thereof. The therapeutic composition according to claim 9, wherein the source of the succinyl group is succinimide succinate. The therapeutic composition of claim 1 or 2, further comprising a chemotherapeutic agent. The therapeutic composition according to claim 11, wherein the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, cyclophosphamide, gemcitabine, gemcitabine, Cisplatin, sorafenib, sunitinib and everolimus. A use of a therapeutic composition according to any one of claims 1 to 12 for preparing a medicament for treating cancer, improving the symptoms of cancer, or inhibiting the progress of cancer in a patient in need. For the use of claim 13, wherein the patient in need has been determined to have an anti-ADI-PEG 20 antibody. The use according to claim 13 or 14, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymph Globular leukemia, chronic myeloid leukemia, lymphoma, liver tumor, sarcoma, leukemia, acute myeloid leukemia, recurrent acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, polymorphism Glioblastoma, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, and stomach cancer. A use of a therapeutic composition according to any one of claims 1 to 12, which is a medicament for treating cancer, improving the symptoms of cancer, or suppressing the progression of cancer in a patient in need, and is characterized in that the composition The patient is administered after a period of administration of a composition comprising ADI-PEG 20. For the purpose of claim 16, wherein the period of time is determined by detecting a predetermined content of the anti-ADI-PEG 20 antibody in the patient, which is characterized in that the therapeutic composition is detecting the anti-ADI-PEG 20 antibody. This predetermined content is administered afterwards. The use of claim 16, wherein the period of time is determined by detecting ADI activity in the patient, and is characterized in that the therapeutic composition is administered after detecting a predetermined or reduced degree of ADI activity.