Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/38—Albumins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA

Definitions

• the present invention relates generally to the fields of biology and medicine, and more specifically to methods for treating acute myeloid leukemia (AML) . Still more specifically, the present invention relates to methods for treating AML subtypes using arginine-depleting agents and the use of arginine-depleting agents in the manufacture of medicaments for the treatment of AML.
• AML acute myeloid leukemia
• AML Acute myeloid leukemia
• AML also known as acute myelogenous leukemia
• AML is a genetically heterogeneous aggressive cancer in which the accumulation of genetic alterations results in uncontrolled clonal proliferation of myeloid progenitor cells in the bone marrow and blood. More severe cases involve the infiltration of organs by the abnormal cells.
• AML is one of the most common acute leukemias in adults and children, accounting for approximately 80%of adult cases and approximately 20%of childhood leukemia diagnoses. Most cases of AML occur in adults, with the average age of diagnosis being 68 years. The five-year survival rate for people over the age of 20 diagnosed with AML is only 25%.
• AML is a heterogeneous disease which is classified into several subtypes.
• the FAB classification system is the one most commonly used and is the one referred to herein.
• Most people diagnosed with AML have one of nine different FAB subtypes of AML (M0, M1, M2, M3, M4, M4 eos, M5, M6 &M7) .
• M0, M1, M2, M3, M4, M4 eos, M5, M6 &M7 The prognosis of a case of AML is often dependent, inter alia, on the FAB AML subtype.
• Chemotherapy is currently the main mode of treatment for AML and includes two main phases: induction and consolidation.
• Induction therapy aims for complete remission of the cancer.
• Consolidation is a term given to post-remission therapy.
• Patients may die within a few days of the commencement of treatment due to treatment-related mortality. The major reason patients are not cured is resistance to treatment, which often manifests as a relapse from remission.
• there is no current standard of care for adult relapsed or refractory AML and the prognosis in such patients is generally poor.
• AML remains a challenging illness and a need exists for new therapeutic approaches for the treatment of this aggressive cancer.
• the present invention provides methods for treating acute myeloid leukemia (AML) using arginine-depleting agents and the use of arginine-depleting agents in the manufacture of medicaments for the treatment of AML.
• AML acute myeloid leukemia
• the inventors of the present invention have surprisingly found that certain FAB AML subtypes respond markedly better to arginine depletion than others.
• the methods and uses described herein may therefore be useful for targeted treatment of AML based on FAB AML subtype.
• the present invention provides a method for treating acute myeloid leukemia (AML) in a subject in need thereof, said method comprising administering a therapeutically effective amount of an arginine-depleting agent to the subject, wherein the AML is of the French-American-British (FAB) subtype M0 (undifferentiated acute myeloblastic leukemia) , M2 (acute myeloblastic leukemia with maturation) , M4 (acute myeloblastic leukemia with maturation) , M4 eos (acute myelomonocytic leukemia with eosinophilia) , M5 (acute monocytic leukemia) , M6 (acute erythroid leukemia) or M7 (acute megakaryoblastic leukemia) .
• FAB French-American-British
• the arginine-depleting agent comprises an arginine catabolic enzyme.
• the arginine catabolic enzyme is an arginine deiminase, arginase, arginine decarboxylase or arginine 2-monooxygenase.
• the arginine-depleting agent is a synthetic arginine-depleting agent.
• the arginine-depleting agent comprises human serum albumin, an albumin binding domain, an Fe region of an immunoglobulin, a polyethylene glycol (PEG) group, human transferrin, XTEN, a proline-alanine-serine polymer (PAS) , an elastin-like polypeptide (ELP) , a homo-amino-acid polymer (HAP) , artificial gelatin-like protein (GLK) , a carboxy-terminal peptide (CTP) , or a combination thereof.
• the arginine-depleting agent comprises human serum albumin, an albumin binding domain, or a combination thereof.
• the arginine-depleting agent comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%or 99%sequence identity to SEQ ID NO: 1.
• the arginine-depleting agent comprises or consists of an amino acid sequence as defined in SEQ ID NO: 1.
• the AML is of the FAB subtype M4 or M7.
• the AML is of the FAB subtype M7 and the arginine-depleting agent comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%or 99%sequence identity to SEQ ID NO: 1.
• the AML is of the FAB subtype M7 and the arginine-depleting agent comprises or consists of an amino acid sequence as defined in SEQ ID NO: 1.
• the AML is auxotrophic for arginine.
• the arginine-depleting agent is administered intramuscularly, intravenously, subcutaneously or orally.
• the arginine-depleting agent is administered intravenously.
• the subject is human.
• the present invention provides the use of an arginine-depleting agent for the manufacture of a medicament for the treatment of AML in a subject in need thereof, wherein the AML is of the French-American-British (FAB) subtype M0 (undifferentiated acute myeloblastic leukemia) , M2 (acute myeloblastic leukemia with maturation) , M4 (acute myeloblastic leukemia with maturation) , M4 eos (acute myelomonocytic leukemia with eosinophilia) , M5 (acute monocytic leukemia) , M6 (acute erythroid leukemia) or M7 (acute megakaryoblastic leukemia) .
• FAB French-American-British
• the present invention provides an arginine-depleting agent for use in treating AML in a subject in need thereof, wherein the AML is of the French-American-British (FAB) subtype M0 (undifferentiated acute myeloblastic leukemia) , M2 (acute myeloblastic leukemia with maturation) , M4 (acute myeloblastic leukemia with maturation) , M4 eos (acute myelomonocytic leukemia with eosinophilia) , M5 (acute monocytic leukemia) , M6 (acute erythroid leukemia) or M7 (acute megakaryoblastic leukemia) .
• FAB French-American-British
• the arginine-depleting agent comprises an arginine catabolic enzyme.
• the arginine catabolic enzyme is an arginine deiminase, arginase, arginine decarboxylase or arginine 2-monooxygenase.
• the arginine-depleting agent is a synthetic arginine-depleting agent.
• the arginine-depleting agent comprises human serum albumin, an albumin binding domain, an Fe region of an immunoglobulin, a PEG group, human transferrin, XTEN, a proline-alanine-serine polymer (PAS) , an elastin-like polypeptide (ELP) , a homo-amino-acid polymer (HAP) , artificial gelatin-like protein (GLK) , a carboxy-terminal peptide (CTP) , or a combination thereof.
• PAS proline-alanine-serine polymer
• ELP elastin-like polypeptide
• HAP homo-amino-acid polymer
• GLK artificial gelatin-like protein
• CTP carboxy-terminal peptide
• the arginine-depleting agent comprises human serum albumin, an albumin binding domain, or a combination thereof.
• the AML is auxotrophic for arginine.
• the medicament is formulated for intramuscular, intravenous, subcutaneous or oral administration.
• the medicament is formulated for intravenous administration.
• the arginine-depleting agent is administered to the subject intramuscularly, intravenously, subcutaneously or orally.
• the arginine-depleting agent is administered to the subject intravenously.
• the subject is human.
• the term “comprising” means “including” , in a non-exhaustive sense. Variations of the word “comprising” , such as “comprise” and “comprises” have correspondingly varied meanings.
• a plurality means more than one.
• a plurality may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or more, and any numerical value derivable therein, and any range derivable therein.
• the term “between” when used in reference to a range of numerical values encompasses the numerical values at each endpoint of the range.
• arginine-depleting agent refers to an arginine-depleting agent which has been produced by artificial chemical reactions.
• the terms “treat” , “treating” , “treatment” , and the like refer to reducing or ameliorating a disorder/disease and/or symptoms associated therewith. It will be appreciated, although not precluded, that treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.
• the term “catabolism” or “catabolic” refers to a chemical reaction in which a molecule decomposes into other, e.g., smaller, molecules.
• arginine catabolic enzyme includes any enzyme capable of reacting with arginine thereby transforming it into other molecules, such as ornithine, citrulline, and agmatine.
• the term "subject” refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, and rodents.
• FAB subtype As used herein, the terms “FAB subtype” , “FAB AML subtype” and “FAB AML” refer to a subtype of AML as classified by the French-American-British (FAB) classification system.
• the FAB system divides AML into nine subtypes, M0, M1, M2, M3, M4, M4 eos, M5, M6 and M7, based on the type of cell the leukemia develops from and how mature the cells are.
• a percentage of “sequence identity” will be understood to arise from a comparison of two sequences in which they are aligned to give a maximum correlation between the sequences. This may include inserting “gaps” in either one or both sequences to enhance the degree of alignment. The percentage of sequence identity may then be determined over the length of each of the sequences being compared.
• a nucleotide sequence “subject sequence” ) having at least 95% “sequence identity” with another nucleotide sequence ( “query sequence” ) is intended to mean that the subject sequence is identical to the query sequence except that the subject sequence may include up to five nucleotide alterations per 100 nucleotides of the query sequence.
• nucleotide sequence of at least 95%sequence identity to a query sequence up to 5% (i.e. 5 in 100) of the nucleotides in the subject sequence may be inserted or substituted with another nucleotide or deleted.
• an AML which is “auxotrophic for arginine” refers to an AML which is unable to synthesize arginine.
• Figure 1 provides a schematic of the relationship between argininosuccinate synthetase (ASS) and the urea cycle.
• Figure 2 is an image of a gel showing expression levels of autophagic (LC3-II, BECLIN-1 and phospho-AMPK ⁇ ) and apoptotic (PARP-1) markers in a pancreatic cell line (Mia PaCa-2) after NEI-01 treatment.
• autophagic LC3-II, BECLIN-1 and phospho-AMPK ⁇
• PARP-1 apoptotic marker
• Figure 3 is an image of a gel showing expression levels of autophagic (LC3-II, p62, phospho-AMPK- ⁇ , AMPK- ⁇ ) and apoptotic (Caspase-9) markers in an AML cell line (HL-60) after NEI-01 treatment.
• autophagic LC3-II, p62, phospho-AMPK- ⁇ , AMPK- ⁇
• Caspase-9 apoptotic markers in an AML cell line (HL-60) after NEI-01 treatment.
• Figure 4 provides Kaplan-Meier survival curves of mice with AML in a C1498 (M4) syngeneic AML model.
• Figure 5 provides images A-D of the tumour burden monitored and quantified using in vivo bioluminescence imaging in a C1498 (M4) syngeneic AML model.
• Figure 6 provides a graph of the tumour burden monitored and quantified using in vivo bioluminescence imaging in a C1498 (M4) syngeneic AML model.
• Figure 7 provides graphs showing the inhibition of tumour growth following repeated NEI-01 treatments in a KG-1-Derived Acute Myeloid Leukaemia (FAB AML M0) Xenograft Model.
• Figure 7A shows the change in average tumour volume in 4 weeks.
• Figure 7B shows the change in average T/C%over 4 weeks. Tumour volume was measured every 3 days. By day 28, a 39%reduction was observed in the treatment group.
• Statistics were calculated using RM two-way ANOVA, followed by Sidak’s multiple comparison for post-hoc analysis. **indicates a p-value of less than 0.01. ***indicates a p-value of less than 0.001.
• Figure 8 provides a graph showing bioluminescence signals in mice transplanted with HL-60-gfphi-Luc+ AML cells. In vivo BLI was performed twice a week and the changes in BLI intensity were plotted. Data are expressed as mean ⁇ SEM.
• Figure 9 provides graphs which show the inhibition of tumour growth following repeated NEI-01 treatments in a P31/FUJ-Derived Acute Myeloid Leukaemia (FAB AML M5) Xenograft Model.
• Figure 9A shows the change in average tumour volume in 4 weeks.
• Figure 9B shows the change in average T/C%over 4 weeks.
• Statistics were calculated using RM two-way ANOVA, followed by Sidak’s multiple comparison for post-hoc analysis. *indicates a p-value of less than 0.05. **indicates a p-value of less than 0.01. ***indicates a p-value of less than 0.001.
• Figure 9C shows the difference in tumour weight between control and treated groups.
• Figure 10 provides graphs which show the inhibition of tumour growth following repeated NEI-01 treatments in a MKPL-1-Derived Acute Myeloid Leukaemia (FAB AML M7) Xenograft Model.
• Figure 10A shows the change in average tumor volume in 3 weeks.
• Figure 10B provides the change in average T/C%over 3 weeks.
• Figure 10C shows the tumor weight after the termination of the study.
• Statistics were calculated using RM two-way ANOVA, followed by Sidak’s multiple comparison for post-hoc analysis. *indicates a p-value of less than 0.05. ***indicates a p-value of less than 0.001.
• Figure 11 provides a graph of growth curves of tumour burden (presented by %of hCD45+ cells) in peripheral blood. Data is expressed as mean ⁇ SEM.
• Figure 12 is a graph of Kaplan-Meirer survival curves of mice in an AM8096 model.
• Figure 13 provides graphs showing the in vivo response of Jurkat cells to NEI-01.
• Figure 13A shows the change in tumour volume (%) in 4 weeks.
• Figure 13B shows the change in average T/C%in 4 weeks. Tumour volume was measured twice a week. Data are expressed as mean ⁇ SEM. A two-tailed student T-test was used. *indicates a p value of equal to or less than 0.05.
• Figure 14 provides graphs of the mean plasma concentration of NEI-01 in (A) male mice and (B) female mice for a Repeated Dose Study on Day 1 and Day 22.
• AML Acute myeloid leukemia
• the present invention provides methods for treating AML using arginine-depleting agents.
• the methods provided herein may reduce or ameliorate the disease and/or symptoms associated therewith.
• the methods may or may not completely eliminate said disease and/or symptoms.
• the arginine-depleting agents described herein may also be used for the manufacture of medicaments for the treatment of AML, which may reduce or ameliorate the disease and/or symptoms associated therewith, and may or may not completely eliminate said disease and/or symptoms.
• AML is a heterogeneous disease which is classified into several subtypes.
• the FAB classification system is the one most commonly used and is the one referred to herein.
• Most people diagnosed with AML have one of nine different FAB subtypes of AML (M0, M1, M2, M3, M4, M4 eos, M5, M6 &M7) .
• M0, M1, M2, M3, M4, M4 eos, M5, M6 &M7 The prognosis of a case of AML is often dependent, inter alia, on the AML subtype.
• the FAB classification system divides AML into subtypes M0 to M7 based on the type of cell the leukemia develops from and how mature the cells are, as outlined in Table 1:
• the present invention provides methods for treating AML and the use of arginine-depleting agents for the manufacture of a medicament for the treatment of AML of any subtype.
• the invention provides methods, and the use of arginine-depleting agents for the manufacture of medicaments for treating FAB AML M0.
• the methods and medicaments of the present invention may also be used to treat FAB AML M2.
• the invention provides methods and medicaments for treating FAB AML M4.
• the invention provides methods and medicaments for treating FAB AML M4 eos.
• the invention provides methods and medicaments for treating FAB AML M5. Methods and medicaments for treating FAB AML M6 are also provided herein.
• the present invention also provides methods and medicaments for treating FAB AML M7.
• methods and medicaments provided herein are presented in the context of treating AML subtypes as defined by the FAB classification system, the skilled person will understand that they may be used to treat cases of AML classified using any other system or method of classification.
• the FAB AML classification system was established in 1976 and is well known in the art. A person skilled in the art can easily identify the FAB AML subtype of a sample using, for example, histochemical staining and microscopy.
• the AML sample used may be obtained, for example, from peripheral blood, a bone marrow aspirate or a biopsy.
• a detailed description of each FAB AML subtype, including images to aid identification, is provided in Charles A. Schiffer, MD and Richard M. Stone, MD (2003) in “Holland-Frei Cancer Medicine, 6th edition” , Kufe DW, Pollock RE, Weichselbaum RR, et al. (eds. ) Hamilton (ON) , 1983.
• Arginine is required for a variety of metabolic pathways. Many tumours are auxotrophic for arginine due to low levels or the absence of argininosuccinate synthetase (ASS) and/or ornithine transcarbamoylase (OTC) , which are required for arginine synthesis. In most cases of AML, the cells are deficient in ASS1, the gene encoding ASS in humans. It would be easy for a person skilled in the art to determine whether the cells from an AML sample were deficient in one or both of the aforementioned enzymes using well-known methods such as Western Blot, ELISA SDS-PAGE or immunoprecipitation.
• Some embodiments of the present invention provide methods for treating AML in a subject comprising administering a therapeutically effective amount of an arginine-depleting agent to the subject. Further embodiments provide the use of arginine-depleting agents for the manufacture of a medicament for the treatment of AML in a subject in need thereof.
• the subject may be any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, and rodents.
• An arginine-depleting agent used in the treatment methods and medicaments described herein may be any arginine-depleting agent known in the art to be capable of reducing plasma and/or cellular levels of arginine in a subject.
• the arginine-depleting agent may, for example, be a small molecule or protein.
• the arginine-depleting agent comprises an arginine catabolic enzyme.
• arginine catabolic enzymes include arginase, arginine deiminase, arginine decarboxylase and arginine 2-monooxygenase.
• the arginase may be any arginase known in the art, such as those produced by bacteria, fungi, fish, humans, bovines, swine, rabbits, rodents, primates, sheep and goats.
• arginases include Bacillus caldovelox arginase, Thermus thermophilus arginase, Capra hircus arginase I, Heterocephalus glaber arginase I, Bos taurus arginase I, Sus scrofa arginase I, Plecoglossus altivelis arginase I, Salmo salar arginase I, Oncorhynchus mykiss arginase I, Osmerus mordax arginase I, Hyriopsis cumingii arginase I, Rattus norvegicus arginase I, Mus musculus arginase I, Homo sapiens (human) arginase I, Pan troglodytes ar
• arginases from Bacillus methanolicus Bacillus sp. NRRL B-14911, Planococcus donghaensis, Paenibacillus dendritiformis, Desmospora sp., Methylobacter tundripaludum, Stenotrophomonas sp., Microbacterium laevaniformans, Porphyromonas uenonis, Agrobacterium sp., Octadecabacter arcticus, Agrobacterium tumefaciens, Anoxybacillus flavithermus, Bacillus pumilus, Geobacillus thermoglucosidasius, Geobacillus thermoglucosidans, Brevibacillus laterosporus, Desulfotomaculum ruminis, Geobacillus kaustophilus, Geobacillus thermoleovorans, Geobacillus thermodenitrificans, Staphylococcus aureus, Halophilic archa
• An arginine deiminase used in the methods and medicaments of the present invention may be any arginine deiminase known in the art, such as those produced from Mycoplasma, Lactococcus, Pseudomonas, Steptococcus, Escherichia, Mycobacterium or Bacillus microorganisms.
• Exemplary arginine deiminases include, but are not limited, to those produced by Mycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis, Clostridium perfringens, Bacillus licheniformis, Borrelia burgdorferi, Borrelia afzellii, Enterococcus faecalis, Lactococcus lactis, Bacillus cereus, Streptococcus pyogenes, Steptococcus pneumoniae, Lactobacillus sake, Giardia intestinalis, Mycobacterium tuberculosis, Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas aeruginosa, and the like.
• the arginine decarboxylase may be any arginine decarboxylase known in the art, such as those produced by Escherichia coli., Salmonella typhimurium, Chlamydophila pneumoniae, Methanocaldococcus jannaschii, Paramecium bursaria Chlorella virus 1, Vibrio vulnificus YJ016, Campylobacter jejuni subsp., Trypanosoma cruzi, Sulfolobus solfataricus, Bacillus licheniformis, Bacillus cereus, Carica papaya, Nicotianatobacum, Glycine max, Lotus coniculata, Vibrio vulnificus, Vibrio cholerae, Mus musculus, Thermotoga, Rattus norvegzcus, Homo sapiens, Bos taurus, Susscrofa, Thermus thermophiles, Thermus parvatiensis, Thermus aquaticus, Thermus thermophilus
• An arginine 2-monooxygenase used in the methods and medicaments of the present invention may be any arginine 2-monooxygenase known in the art, such as those produced from Arthrobacter globiformis IFO 12137, Arthrobacter simplex IFO 12069, Brevibacterium helvolum IFO 12073, Helicobacter cinaedi CCUG 18818, Streptomyces griseus, and the like.
• the arginine-depleting agents of the present invention may comprise naturally occurring and/or synthetic products.
• the arginine-depleting agents comprise naturally occurring arginine catabolic enzymes.
• the arginine-depleting agents comprise synthetic arginine catabolic enzymes.
• the arginine-depleting agents may comprise full proteins and/or functional fragments and/or variants thereof.
• Arginine decarboxylases, arginine deiminases, arginine 2-monooxygenases, arginases and other arginine-depleting agents used in the methods and uses may be modified to improve their pharmacokinetic properties, such as by fusion of proteins and/or functional fragments and/or variants thereof with human serum albumin, an albumin binding domain, an Fe region of an immunoglobulin, a polyethylene glycol (PEG) group, or a combination thereof.
• PEG polyethylene glycol
• the increase in half-life results in a reduction of the frequency of administration of the arginine-depleting agent required to achieve the same outcome.
• One or more of the aforementioned modifications to the arginine-depleting agents may reduce immunogenicity, which may help to avoid adverse effects.
• arginine catabolic enzymes may be engineered to include specific sites on the enzyme where, for example, PEG can be selectively attached.
• the selected PEGylation sites may be located at a site removed from the active site of the enzyme, and/or may be generally exposed to solvent to allow reaction with PEGylation reagents.
• PEGylation reagents include, but are not limited to mPEG-ALD (methoxypolyethylene glycol-propionaldehyde) ; mPEG-MAL (methoxypolyethylene glycol-maleimide) ; mPEG-NHS (methoxypolyethylene glycol-N-hydroxy-succinimide) ; mPEG-SPA (methoxypolyethylene glycol-succinimidyl propionate) ; and mPEG-CN (methoxypolyethylene glycol-cyanuric chloride) .
• mPEG-ALD methoxypolyethylene glycol-propionaldehyde
• MAL methoxypolyethylene glycol-maleimide
• mPEG-NHS methoxypolyethylene glycol-N-hydroxy-succinimide
• mPEG-SPA methoxypolyethylene glycol-succinimidyl propionate
• mPEG-CN methoxypoly
• the PEG group has a molecular weight of about 5,000 to about 20,000 amu, about 5,000 to about 15,000 amu, about 5,000 to about 12,000 amu, about 7,000 to about 12,000 amu, or about 7,000 to about 10,000 amu. In certain embodiments, the PEG group has a molecular weight of about 2,000 amu to 10,000 amu. In some embodiments, the PEG group is PEG4,000, PEG5,000, PEG6,000, or PEG7,000.
• the PEG group may be covalently attached directly to the enzyme or attached via a linker.
• the enzyme is covalently attached via a propionic acid linker to PEG.
• Arginine catabolic enzymes may be fused to proteins with an inherently long serum half-life, which may result in more desirable pharmacokinetic profiles.
• the arginine-depleting agents of the present invention may comprise an antibody Fc domain and/or serum albumin.
• the arginine-depleting agents may comprise arginine catabolic enzymes genetically fused to an antibody Fc domain and/or serum albumin.
• the Fe region of an immunoglobulin is from human immunoglobulin, for example, human IgG.
• the enzymes may be fused to an albumin binding domain.
• the enzymes may be fused to human transferrin.
• the arginine-depleting agents comprise arginine catabolic enzymes fused to non-structured polypeptides. Fusion of the enzymes to non-structured polypeptides may increase the overall size and/or hydrodynamic radius of the agents.
• arginine catabolic enzymes are fused to any one or more of XTEN, which is a recombinant PEG mimetic (XTENylation) , PAS, which is a proline-alanine-serine polymer (PASylation) , ELP, which is an elastin-like polypeptide (ELPylation) , HAP, which is a homo-amino-acid polymer (HAPylation) , and artificial gelatin-like protein (GLK) .
• XTEN which is a recombinant PEG mimetic
• PAS which is a proline-alanine-serine polymer
• ELP which is an elastin-like polypeptide (ELPylation)
• HAP which is a homo-amino-acid polymer (HAPylation)
• GLK artificial gelatin-like protein
• the arginine catabolic enzymes used in some embodiments of the invention may be fused to anionic polypeptides, which may increase the negative charge of the agents. Enzymes may be fused to a carboxy-terminal peptide (CTP) .
• CTP carboxy-terminal peptide
• suitable CTP fusion is the genetic fusion of the CTP from the human chorionic gonadotropin (CG) ⁇ chain.
• Arginine catabolic enzymes may be linked to serum albumin via non-covalent interactions with serum albumin, which may also extend the half-life of the agents.
• an albumin-binding moiety is either conjugated or genetically fused to the therapeutic enzyme.
• moieties can be used, including, but not limited to (i) molecules with intrinsic affinity for albumin; (ii) peptides, antibody fragments, alternative scaffolds, and small chemicals generated and selected to exhibit albumin binding activity.
• fusion proteins were first used in the 1980s and are created by the fusion of two or more genes which each encode a separate protein.
• a variety of methods for the synthesis of fusion proteins are well known in the art. See, for example, Yu et al., Biotechnology Advances, 2015; 33: 155-164, which provides a review of the most common approaches currently used for the design and construction of synthetic fusion proteins. Strohl, Biodrugs, 2015; 29 (4) : 215-239, provides another detailed review of fusion proteins and outlines the advantages and disadvantages of both fusion methods and different types of fusion protein.
• the arginine-depleting agent comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to SEQ ID NO: 1.
• the arginine-depleting agent comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%99%or 100%sequence identity to SEQ ID NO: 1.
• the arginine-depleting agent comprises or consists of an amino acid sequence as defined in SEQ ID NO: 1.
• BLAST program is freely accessible at https: //blast. ncbi. nlm. nih. gov/Blast. cgi.
• Other non-limiting examples include the Clustal (http: //www. clustal. org/) and FASTA (Pearson (1990) , Methods Enzymol. 83, 63-98; Pearson and Lipman (1988) , Proc. Natl. Acad. Sci. U.S.A 85, 2444-2448. ) programs. These and other programs can be used to identify sequences which are at least to some level identical to a given input sequence. Additionally or alternatively, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux et al.
• the arginine-depleting agents described herein may be prepared as pharmaceutical compositions containing a therapeutically effective amount of an arginine-depleting agent described herein as an active ingredient in a pharmaceutically acceptable carrier.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered.
• vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• 0.9%saline and 0.3%glycine can be used. These solutions may be sterile and generally free of particulate matter.
• compositions may be sterilized by conventional, well-known sterilization techniques (e.g., filtration) .
• the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and colouring agents, etc.
• concentration of the arginine-depleting agent in such pharmaceutical formulation can vary widely and may be selected based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.
• the concentration of plasma arginine in the subject needed to observe a therapeutic effect may vary based on numerous factors, including the condition of the subject and/or the type and severity of the AML and/or diet composition.
• the selection of the target plasma arginine levels is well within the skill of a person of ordinary skill in the art.
• the duration of treatment is more than 1 week, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 5 weeks, more than 6 weeks, more than 7 weeks, more than 8 weeks, more than 9 weeks, more than 10 weeks, more than 11 weeks, more than 12 weeks, more than 24 weeks, more than 28 weeks, more than 32 weeks, more than 36 weeks, more than 40 weeks, more than 44 weeks, more than 48 weeks, more than 52 weeks, or more than 56 weeks.
• the mode of administration of the arginine-depleting agents is intravenous.
• the mode of administration for therapeutic use of the the arginine-depleting agents described herein may be any suitable route that delivers the agents to the subject, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous and/or subcutaneous; pulmonary; transmucosal (e.g., oral, intranasal, intravaginal and/or rectal) ; using a formulation in a tablet, capsule, solution, suspension, powder, gel and/or particle; and contained in a syringe, an implanted device, osmotic pump, cartridge and/or micropump; or other means appreciated by the skilled artisan, as well known in the art.
• Example 1 Cytotoxicity of NEI-01 in a range of cancer cell lines.
• NEI-01 is a recombinant albumin-binding arginine deiminase which can convert arginine to citrulline &ammonia, and inhibits growth in various ASS-deficient cancers by depleting arginine ( Figure 1) . Cytotoxicity assay results demonstrated that NEI-01 depleted arginine and inhibited cancer cell growth (especially in the case of ASS1 deficient cancer cell lines) in a range of different cancer cell lines (Table 2) .
• Example 2 Apoptosis and autophagy. NEI-01 treatment of pancreatic cancer cell line Mia PaCa-2.
• Mia PaCa-2 cells were treated with designated concentrations of NEI-01 with or without choloquine (CQ) .
• CQ choloquine
• Example 3 NEI-01 treatment of AML cell line HL-60.
• ASS1-deficient HL-60 AML cells were treated with NEI-01 and CQ.
• expression levels of autophagic markers LC3-II, p62, phospho-AMPK ⁇ and AMPK ⁇ increased upon NEI-01 treatment, suggesting autophagy plays a role in NEI-01-induced cell death.
• expressions levels of apoptotic marker Caspase-9 decreased following NEI-01 treatment, demonstrating the activation of apoptotic pathways.
• FAB French-American-British
• WHO World Health Organization
• Example 4 Anticancer activity of NEI-01 in the C1498 Syngeneic Acute Myeloid Leukemia (FAB AML M4) Model.
• Murine argininosuccinate synthase (ASS1) -deficient C1498 cells co-labeled with luciferase and green fluorescent protein (GFP) were intravenously (i.v. ) transplanted into C57BL/6 mice to establish a syngeneic AML model.
• the mice were randomly divided into 4 groups. Details of the 4 groups and their corresponding treatment regimens are provided in Table 4.
• Table 4 Groups and treatment regimens for C1498 Syngeneic Acute Myeloid Leukaemia (FAB AML M4) Model study.
• mice in Group 3 (treated with NEI-01 280 U/kg once a week) and all of the mice in Group 4 (treated with NEI-01 280 U/kg twice a week) survived until the end of the experiment.
• treatment with NEI-01 significantly reduced the total leukemia burden in addition to slowing down disease progression ( Figures 5 and 6) . This anticancer activity of NEI-01 was exhibited in a dose-dependence manner.
• Example 5 Anticancer activity of NEI-01 in a KG-1-Derived Acute Myeloid Leukaemia (FAB AML M0) Xenograft Model.
• mice Human argininosuccinate synthase (ASS1) -deficient M0-subtype acute myeloid leukemia KG-1 cells were subcutaneously injected into immunodeficient BALB/c nude mice. When the tumour volume reached 180 mm 3 , the mice were intravenously (i.v. ) treated with buffer MHT or NEI-01 (280 U/kg) once a week. Details of the study groups and their corresponding treatment regimens are provided in Table 5.
• Table 5 Groups and treatment regimens for KG-1-Derived Acute Myeloid Leukaemia (FAB AML M0) Xenograft Model study.
• tumour weights were 3.41 ⁇ 0.53g in the control group and 2.03 ⁇ 0.47g in the NEI-01 (280 U/kg once a week) treatment group. There was a 40.38%decrease in tumour weight.
• Example 6 Anticancer activity of NEI-01 in a HL-60 Derived Acute Myeloid Leukemia (FAB AML M2) Orthotopic Model.
• mice Human argininosuccinate synthase (ASS1) -deficient M2-subtype acute myeloid leukemia HL-60 cells co-labeled with luciferase and green fluorescent protein (GFP) were intravenously (i.v. ) transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to establish an orthotopic AML model.
• the mice were randomly divided into 3 groups. Details of the study groups and their corresponding treatment regimens are provided in Table 6.
• Table 6 Groups and treatment regimens for HL-60 Derived Acute Myeloid Leukemia (FAB AML M2) Orthotopic Model study.
• Leukemia cells (HL-60-gfphi-Luc+ cells) were tracked and the total leukemia burden was quantified by in vivo BLI.
• the disease progression was determined by the changes in BLI intensity. The results are shown in Figure 8. An aggressive disease progression was found with a strong signal evident throughout the AML mice. This progression was significantly inhibited when the mice were treated with NEI-01 either once a week (p ⁇ 0.05, from Day 4 to Day 25) or twice a week (p ⁇ 0.01, whole treatment period) .
• Example 7 Anticancer activity of NEI-01 in a P31/FUJ-Derived Acute Myeloid Leukemia (FAB AML M5) Xenograft Model.
• mice Human argininosuccinate synthase (ASS1) -deficient M5-subtype acute myeloid leukemia P31/FUJ cells were subcutaneously inoculated in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice.
• NOD/SCID mice nonobese diabetic/severe combined immunodeficiency mice.
• the tumour volume reached 200 mm 3
• the mice were intravenously (i.v. ) treated with buffer MHT or NEI-01 (280 U/kg) once a week.
• the tumour volume was measured every 3-4 days.
• Table 7 Groups and treatment regimens for P31/FUJ-Derived Acute Myeloid Leukemia (FAB AML M5) Xenograft Model study.
• Figure 9 shows that NEI-01 treatment significantly reduced the tumour volume as well as the tumour weight, resulting in a 51.27%reduction in tumour volume by Day 28. A final T/C ratio reached 51.14%.
• the tumour weights were 1.08 ⁇ 0.98 g in the control group and 0.78 ⁇ 0.08g in NEI-01 (280 U/kg once a week) treatment group (p ⁇ 0.05) .
• Example 8 Anticancer activity of NEI-01 in a MKPL-1-Derived Acute Myeloid Leukemia (FAB AML M7) Xenograft Model.
• mice Human argininosuccinate synthase (ASS1) -deficient M7-subtype acute myeloid leukemia MKPL-1 cells were subcutaneously inoculated into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice.
• NOD/SCID nonobese diabetic/severe combined immunodeficiency mice.
• the tumour size reached 120 mm 3
• the mice were intravenously (i.v. ) treated with buffer MHT or NEI-01 (280 U/kg) once a week.
• the tumour volume was measured every 2-3 days.
• Table 8 Groups and treatment regimens for MKPL-1-Derived Acute Myeloid Leukemia (FAB AML M7) Xenograft Model study.
• Figure 10 shows that NEI-01 treatment significantly reduced the tumour volume as well as the tumour weight, resulting in a 99%reduction in tumour volume by Day 22.
• Tumour weights were 8.05 ⁇ 0.056 g in the control group and 0.15 ⁇ 0.05 g in the NEI-01 (280 U/kg once a week) treatment group. A final T/C ratio reached at 99%.
• Example 9 In vivo Efficacy Study of NEI-01 in the Treatment of a Patient-Derived AM5512 Acute Myeloid Leukemia (FAB AML M7) Model.
• PDX Patient-derived xenograft
• FAB AML M7 Acute Myeloid Leukemia Model
• mice Human AM5512 cells were intravenously (i.v. ) inoculated into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the tumour burden in peripheral blood was ⁇ 1.33%, the mice were randomly divided into 3 groups: group 1 (Vehicle) , group 2 (NEI-01, 140 U/kg) and group 3 (NEI-01, 280 U/kg) as outlined in Table 9.
• Example 10 In vivo Efficacy Study of NEI-01 in the Treatment of a Patient-Derived AM8096 Acute Myeloid Leukemia (FAB AML M2) Model.
• mice Human AM8096 cells were intravenously (i.v. ) inoculated into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the tumour burden in peripheral blood was ⁇ 1.5%, the mice were randomly divided into 3 groups: group 1 (Vehicle) , group 2 (NEI-01, 140 U/kg) and group 3 (NEI-01, 280 U/kg) as outlined in Table 10.
• Table 10 Groups and treatment regimens for Patient-Derived AM8096 Acute Myeloid Leukemia (FAB AML M2) Model study.
• Table 11 Median survival days and life-span (ILS) for each group in the AM8096 model.
• Example 11 Anticancer Activity of NEI-01 in Jurkat-Derived Leukemia Cancer Xenograft Model.
• T-ALL acute lymphoblastic leukemia
• Drug-induced amino acid deprivation is one strategy that has been successfully used in the treatment of acute lymphoblastic leukemia, where asparaginase is an important part of induction chemotherapy.
• Arginine as a precursor for initiation of a variety of metabolic pathways, has been confirmed to have a modulatory effect on tumourigenesis.
• Arginine deprivation has been demonstrated as a promising therapeutic approach against arginine-auxotrophic tumours which lack argininosuccinate synthase (ASS1) , a limiting enzyme to synthesize arginine from citrulline.
• ASS1 argininosuccinate synthase
• This study aimed to evaluate the anticancer activity of the arginine-depriving enzyme, NEI-01 in a T-ALL Jurkat xenograft model.
• mice Human ASS1-deficient T-ALL Jurkat cells were subcutaneously inoculated into immunodeficient BALB/c nude mice. When the tumour volume reached around 40 mm 3 , the mice were randomly divided into two groups: a control and NEI-01 treatment group, as outlined in Table 12. The mice were intraperitoneally (i.p. ) administered with PBS or NEI-01 (5 U per mouse, ⁇ 280U/kg) twice a week for 4 weeks. The tumour volume was measured twice a week.
• Table 12 Groups and treatment regimens for Jurkat-Derived Leukemia Cancer Xenograft Model study.
• Example 12 Determination of NEI-01 in Mice Plasma from Repeated Dose Study.
• NEI-01 was administered to ICR mice by intravenously once per week for 4 weeks at dosages of 160, 280 and 560U/kg.
• Blood samples were taken on Day 1 and Day 22 at pre-dose (-1) , 0.25, 6, 24, 48 and 72 h post-dose for all groups on Day 1, pre-dose (-1) for all groups on Day 8 (Week 2) , pre-dose (-1) for all groups on Day 15 (Week 3) , pre-dose (-1) , 0.25, 6, 24, 48 and 72 h post-dose on Day 22 (Week 4) and before sacrificing the mice on Day 29 (Week 5) .
• 5 animals/group/sex/time point and plasma concentrations were quantified (Figure 14) .
• Table 13 Pharmacokinetic parameters and measurements for Day 1 Mice for NEI-01 treatment groups.
• AUC 0-72h the area under the curve in a plot of drug concentration versus time from time of drug administration (time “0” to time “72h” )
• AUC 0-72h the area under the curve in a plot of drug concentration versus time from time of drug administration (time “0” to time “72h” )
• T max was 0.25h post-dose in both males and females on Day 1.
• the T 1/2 was between 21.04 and 36.58 hours.
• Systemic exposure (as measured by AUC 0-72 ) to NEI-01 increased with dose in a proportional manner in males and females on both Day 1 and Day 22.
• the AUC 0-72 was similar in males and females on Day 1 (5.82x10 6 ng. h/ml ⁇ 6.04x10 6 ng. h/ml) and Day 22 (5.97x10 6 ng.h/ml ⁇ 6.27x10 6 ng. h/ml) at 560 U/kg.
• C max results were similar to AUC 0-72 in that results were similar for males and females on both Day 1 and Day 22. There was also no significant difference in body weight between males and female treatment groups.
• the objective of the presently claimed invention is to provide alternative methods for treating AML.

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