CN115697320A - Methods of treating AML subtypes using arginine depleting agents - Google Patents
Methods of treating AML subtypes using arginine depleting agents Download PDFInfo
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- A61K38/00—Medicinal preparations containing peptides
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- 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
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- C07K14/76—Albumins
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Abstract
The present invention provides a method for treating Acute Myeloid Leukemia (AML) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an arginine depleting agent, wherein the AML is french-usa-british (FAB) subtype M0 (undifferentiated acute myeloid leukemia), M2 (acute mature myeloid leukemia), M4eos (acute myelomonocytic leukemia with eosinophilia), M5 (acute monocytic leukemia), M6 (acute erythroleukemia) or M7 (acute megakaryocytic leukemia). The invention also provides a medicament comprising an arginine depleting agent for treating AML in a subject in need thereof, wherein the AML is of french-american-british (FAB) subtype M0 (undifferentiated acute myeloid leukemia), M2 (acute mature myeloid myeloblastic leukemia), M4 (acute mature myeloid leukemia), M4eos (acute myelomonocytic leukemia with eosinophilia), M5 (acute monocytic leukemia), M6 (acute erythroleukemia) or M7 (acute megakaryocytic leukemia).
Description
Technical Field
The present invention relates generally to the fields of biology and medicine, and more particularly to methods for treating Acute Myeloid Leukemia (AML). Still more particularly, the invention relates to methods of treating AML subtypes using arginine depleting agents, and the use of arginine depleting agents in the manufacture of medicaments for treating AML.
Background
The following discussion of the background of the invention is merely provided to assist the reader in understanding the invention and is not intended to describe or constitute background to the invention.
Acute Myeloid Leukemia (AML), also known as acute myelogenous leukemia (acute myelogenous leukemia), is a genetically heterogeneous, aggressive cancer in which the accumulation of genetic changes leads to uncontrolled, replicative proliferation of myeloid progenitor cells in the bone marrow and blood. A more serious case involves abnormal cellular infiltration of the organ. AML is one of the most common acute leukemias in adults and children, with leukemia diagnosis accounting for about 80% of adult cases and 20% of childhood cases. Most cases of AML occur in adults with a mean diagnostic age of 68 years. The five-year survival rate for people over 20 years of age diagnosed with AML is only 25%.
AML is a heterogeneous disease, which is classified into several subtypes. There are two major classification systems for the AML subtype-the french-american-british (FAB) system and the World Health Organization (WHO) classification system. The FAB classification system is the most commonly used one, and is also referred to herein. Most people diagnosed with AML have one of 9 different FAB AML subtypes (M0, M1, M2, M3, M4eos, M5, M6 and M7). The prognosis of AML cases in particular often depends on the FAB AML subtype.
Despite technological advances and new insights into the disease, the overall survival rate of patients diagnosed with AML has tended to stagnate and people continue to die largely of the disease. Chemotherapy is currently the primary model for treating AML and comprises two major phases: induction and consolidation (consolidation). Induction therapy aims at complete remission of the cancer. Consolidation is the term for administering post-remission therapy. Within the days of treatment initiation, patients may die due to the mortality associated with treatment. The main reason for the inability of patients to cure is resistance to treatment, which is often manifested as relapse after remission. However, there is currently no standard of care for relapsed or refractory AML in adults, and the prognosis for such patients is generally poor.
AML remains a challenging disease and there is a need for new therapeutic approaches for treating this aggressive cancer.
Disclosure of Invention
The present invention provides methods of treating Acute Myeloid Leukemia (AML) using an arginine depleting agent, and the use of an arginine depleting agent in the manufacture of a medicament for treating AML.
The inventors of the present invention surprisingly found that a specific FAB AML subtype responds significantly better to arginine elimination than others. Thus, the methods and uses described herein are useful for AML targeted therapies based on the FAB AML subtype.
In a first aspect, the invention provides a method for treating Acute Myeloid Leukemia (AML) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an arginine depleting agent, wherein the AML is french-usa-british (FAB) subtype M0 (undifferentiated acute myeloid leukemia), M2 (acute mature myeloid leukemia), M4eos (acute myeloid monocytic leukemia with eosinophilia), M5 (acute monocytic leukemia), M6 (acute erythroleukemia), or M7 (acute megakaryocytic leukemia).
In one embodiment of the first aspect, the arginine depleting agent comprises an arginine catabolic enzyme.
In one embodiment of the first aspect, the arginine catabolic enzyme is arginine deiminase, arginase, arginine decarboxylase, or arginine 2-monooxygenase.
In one embodiment of the first aspect, the arginine depleting agent is a synthetic arginine depleting agent.
In one embodiment of the first aspect, 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), an artificial gelatin-like protein (GLK), a carboxy-terminal peptide (CTP), or a combination thereof.
In one embodiment of the first aspect, the arginine depleting agent comprises human serum albumin, an albumin binding domain, or a combination thereof.
In one embodiment of the first aspect, the arginine depleting agent comprises an amino acid sequence substantially identical to SEQ ID NO:1 or consists of an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity.
In one embodiment of the first aspect, the arginine depleting agent comprises the amino acid sequence set forth in SEQ ID NO:1 or consists thereof.
In one embodiment of the first aspect, the AML is a FAB subtype M4 or M7.
In one embodiment of the first aspect, the AML is FAB subtype M7 and the arginine depleting agent comprises an amino acid sequence identical to SEQ ID NO:1 or consists of an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity.
In one embodiment of the first aspect, the AML is FAB subtype M7 and the arginine depleting agent comprises the amino acid sequence set forth in SEQ ID NO:1 or consists thereof.
In one embodiment of the first aspect, AML is arginine auxotrophic.
In one embodiment of the first aspect, the arginine depleting agent is administered intramuscularly, intravenously, subcutaneously, or orally.
In one embodiment of the first aspect, the arginine depleting agent is administered intravenously.
In one embodiment of the first aspect, the subject is a human.
In a second aspect, the invention provides the use of an arginine depleting agent for the manufacture of a medicament for treating AML in a subject in need thereof, wherein the AML is of the french-american-british (FAB) subtype M0 (undifferentiated acute myeloid leukemia), M2 (acute mature myeloid leukemia), M4eos (acute myelomonocytic leukemia with eosinophilia), M5 (acute monocytic leukemia), M6 (acute erythroleukemia) or M7 (acute megakaryocytic leukemia).
In a third aspect, the present invention provides an arginine depleting agent for treating AML in a subject in need thereof, wherein the AML is french-american-united kingdom (FAB) subtype M0 (undifferentiated acute myeloid leukemia), M2 (acute mature myeloid leukemia), M4eos (acute myelomonocytic leukemia with eosinophilia), M5 (acute monocytic leukemia), M6 (acute erythroleukemia) or M7 (acute megakaryocytic leukemia).
In one embodiment of the second or third aspect, the arginine depleting agent comprises an arginine catabolic enzyme.
In one embodiment of the second or third aspect, the arginine catabolic enzyme is arginine deiminase, arginase, arginine decarboxylase, or arginine 2-monooxygenase.
In one embodiment of the second or third aspect, the arginine depleting agent is a synthetic arginine depleting agent.
In one embodiment of the second or third aspect, 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), an artificial gelatin-like protein (GLK), a carboxyl-terminal peptide (CTP), or a combination thereof.
In one embodiment of the second or third aspect, the arginine depleting agent comprises human serum albumin, an albumin binding domain, or a combination thereof.
In one embodiment of the second aspect, AML is arginine auxotrophic.
In one embodiment of the second aspect, the medicament is formulated for intramuscular, intravenous, subcutaneous or oral administration.
In one embodiment of the second aspect, the medicament is formulated for intravenous administration.
In one embodiment of the third aspect, the arginine depleting agent is administered to the subject intramuscularly, intravenously, subcutaneously, or orally.
In one embodiment of the third aspect, the arginine depleting agent is administered to the subject intravenously.
In one embodiment of the second or third aspect, the subject is a human.
Drawings
The above and other aspects and embodiments of the present disclosure will become apparent from the following description of the present disclosure when taken in conjunction with the accompanying drawings, wherein:
FIG. 1 provides a schematic representation of the relationship between argininosuccinate synthetase (ASS) and the urea cycle.
FIG. 2 shows a gel diagram of the expression levels of autophagy (LC 3-II, BECLIN-1 and phospho-AMPK. Alpha.) and apoptosis (PARP-1) markers in pancreatic cell strains (Mia PaCa-2) after NEI-01 treatment.
FIG. 3 shows a gel diagram of the expression levels of markers of autophagy (LC 3-II, p62, phospho-AMPK-alpha, AMPK-alpha) and apoptosis (Caspase-9) in an AML cell line (HL-60) after NEI-01 treatment.
Fig. 4 provides Kaplan-Meier survival curves for mice with AML in the C1498 (M4) homologous AML model.
Fig. 5 provides graphs a-D of tumor burden monitored and quantified using in vivo bioluminescence imaging in the C1498 (M4) homologous AML model.
Fig. 6 provides a graph of tumor burden monitored and quantified using in vivo bioluminescence imaging in the C1498 (M4) homologous AML model.
FIG. 7 provides a graph showing tumor growth inhibition following repeated NEI-01 treatment in a KG-1 derived acute myeloid leukemia (FAB AML M0) xenograft model. Figure 7A shows the change in mean tumor volume within four weeks. FIG. 7B shows the change in mean T/C% over four weeks. Tumor volumes were measured every 3 days. By day 28, a 39% reduction was observed in the treated groups. Statistical data were calculated using RM two-factor variance analysis (two-way ANOVA), followed by multiple comparisons using Sidak for post hoc analysis. * Denotes a p value of less than 0.01.* Denotes a p value of less than 0.001.
FIG. 8 provides a graph showing bioluminescence signals in mice engrafted with HL-60-gfp hi-Luc + AML cells. In vivo BLI was performed twice weekly and changes in BLI intensity were plotted. Data are presented as mean ± SEM.
FIG. 9 provides a graph showing tumor growth inhibition following repeated NEI-01 treatment in a P31/FUJ-derived acute myeloid leukemia (FAB AML M5) xenograft model. Figure 9A shows the change in mean tumor volume within four weeks. FIG. 9B shows the change in mean T/C% over four weeks. Statistical data were calculated using RM two-factor variance analysis (two-way ANOVA), followed by multiple comparisons using Sidak for post hoc analysis. * Indicating a p value of less than 0.05.* Denotes a p value of less than 0.01.* Indicates p value less than 0.001. Fig. 9C shows the difference in tumor weight between the control group and the treated group.
FIG. 10 provides a graph showing tumor growth inhibition following repeated NEI-01 treatment in a MKPL-1 derived acute myeloid leukemia (FAB AML M7) xenograft model. Figure 10A shows the change in mean tumor volume over three weeks. FIG. 10B provides the change in mean T/C% over three weeks. Fig. 10C shows tumor weight after the end of the study. Statistical data were calculated using RM two-factor variance analysis (two-way ANOVA), followed by multiple comparisons using Sidak for post hoc analysis. * Indicating a p value of less than 0.05.* Denotes a p value of less than 0.001.
Figure 11 provides a growth graph of tumor burden (presented by the percentage of hCD45+ cells) in peripheral blood. Data are presented as mean ± SEM.
FIG. 12 is a Kaplan-Meirer survival plot for mice in the AM8096 model.
FIG. 13 provides an in vivo response graph showing Jurkat cells to NEI-01. Fig. 13A shows the change (%) in tumor volume within four weeks. FIG. 13B shows the variation of the mean T/C% over four weeks. Tumor volume was measured twice weekly. Data are presented as mean ± SEM. Two-tailed student's T-test was used. * Indicating a p-value equal to or less than 0.05.
FIG. 14 provides a graph of mean plasma concentrations of NEI-01 in (A) male mice and (B) female mice subjected to a repeat dose study on days 1 and 22.
Detailed Description
Definition of
Certain terms used herein shall have the meanings set forth below.
As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.
As used herein, the term "comprising" means "including" in a non-exhaustive sense. Variations of the word "comprising", such as "comprising" and "comprises", have a meaning that is varied accordingly.
The term "plurality", as used herein, means more than one. In a particular specific aspect or embodiment, 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.
As used herein, the term "between" when referring to a range of values encompasses the value at each end of the range.
As used herein, the term "synthetic" when used to describe a product refers to a product produced by a human agent as opposed to a naturally occurring product. For example, a "synthetic" arginine depleting agent refers to an arginine depleting agent that has been produced through an artificial chemical reaction.
As used herein, the terms "treat", "treating", "treat") "and like terms refer to the alleviation or amelioration of the conditions/diseases and/or symptoms associated therewith. It will be understood that, although not exclusively, treatment of a disorder or condition need not completely eliminate the disorder, condition or symptom associated therewith.
As used herein, the term "catabolism" or "catabolic" refers to a chemical reaction in which a molecule breaks down into other molecules, e.g., smaller molecules. For example, the term "arginine catabolic enzyme" includes any enzyme that is capable of reacting with arginine thereby converting it into other molecules, such as ornithine, citrulline, and agmatine (agmatine).
As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, canines, felines, and rodents.
As used herein, the terms "FAB subtype", "FAB AML subtype" and "FAB AML" refer to a subtype of AML classified by the french-american-british (FAB) classification system. The FAB system classifies AML into 9 subtypes, M0, M1, M2, M3, M4eos, M5, M6 and M7, based on the cell type from which leukemia develops and the degree of maturity of the cell.
As used herein, a percentage of "sequence identity" will be understood to result from a comparison of two sequences in which they are aligned to give the greatest correlation between the sequences. This may include inserting "gaps" in one or both sequences to enhance the degree of alignment. The percentage of sequence identity can then be determined over the length of each sequence compared. For example, 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 variations relative to every 100 nucleotides of the query sequence. In other words, in order to obtain a nucleotide sequence having at least 95% sequence identity with respect to the query sequence, at most 5% (i.e., 5 out of 100) of the nucleotides in the subject sequence may be inserted or replaced with another nucleotide or deleted.
As used herein, the term "auxotrophic," when used to describe cancer, refers to a cancer that is incapable of synthesizing one or more specific substances required for growth and/or metabolism. For example, AML "auxotrophic for arginine" refers to AML that is unable to synthesize arginine.
Acute Myeloid Leukemia (AML) is one of the most common acute leukemias in adults and children. Previous methods for treating AML sometimes resulted in treatment-related mortality. With initial success of treatment, recurrence after remission is common. It is important to develop new treatments for this aggressive cancer.
The present invention provides methods of treating AML using arginine depleting agents. The methods provided herein can reduce or ameliorate the diseases and/or symptoms associated therewith. The method may eliminate the disease and/or symptoms or may not completely eliminate the disease and/or symptoms. The arginine depleting agents described herein may also be used in the manufacture of a medicament for treating AML, which may reduce or ameliorate a disease and/or symptom associated therewith, and which may or may not completely eliminate the disease and/or symptom.
FAB AML subtype
AML is a heterogeneous disease, which is classified into several subtypes. There are two major classification systems for the AML subtype-the french-us-british (FAB) system and the World Health Organization (WHO) classification system. The FAB classification system is the most commonly used one, and is also referred to herein. Most people diagnosed with AML have one of 9 different FAB AML subtypes (M0, M1, M2, M3, M4eos, M5, M6 and M7). The prognosis of AML cases in particular often depends on the AML subtype.
The FAB classification system classifies AML into subtypes M0 to M7 based on the cell type from which leukemia develops and the degree of maturity of the cells, as shown in table 1:
table 1: FAB AML subtypes and descriptions
The present invention provides methods for treating AML and the use of an arginine depleting agent in the manufacture of a medicament for treating AML of any subtype. In some embodiments, the invention provides methods for treating FAB AML M0 and the use of an arginine depleting agent in the manufacture of a medicament for treating FAB AML M0. The methods and agents of the invention may also be used to treat FAB AML M2. In other embodiments, the invention provides methods and agents for treating FAB AML M4. In further embodiments, the invention provides methods and medicaments for treating FAB AML M4 eos. In yet further embodiments, the invention provides methods and agents for treating FAB AML M5. Methods and agents for treating FAB AML M6 are also provided herein. The invention also provides methods and agents for treating FAB AML M7. Although the methods and agents provided herein are presented in the context of treating AML subtypes defined by the FAB classification system, the skilled artisan will appreciate that they can be used to treat AML cases classified using any other classification system or method.
The FAB AML classification system was established in 1976 and is well known in the art. One of ordinary skill in the art to which the invention pertains can readily identify the FAB AML subtype of a specimen using, for example, histochemical staining and microscopy. The AML samples used may be obtained, for example, from peripheral blood, bone marrow aspirate or biopsy. The medical doctors Charles a. Schiffer and Richard m.stone (2003) provide detailed descriptions of various FAB AML subtypes in "Holland-free Cancer Medicine, 6 th edition," including maps to aid identification, kufe DW, pollock RE, weichselbaum RR, et al. (eds.) Hamilton (ON), 1983.
Arginine is required for various metabolic pathways. Many tumors are arginine auxotrophic due to the low degree or absence of argininosuccinate synthetase (ASS) and/or ornithine carbamoyltransferase (OTC) required for arginine synthesis. In most cases of AML, ASS1, the gene encoding ASS in humans, is absent from the cells. It will be readily apparent to one of ordinary skill in the art to which the invention pertains whether cells from AML samples lack one or both of the aforementioned enzymes, using well-known methods such as western blotting, ELISA, SDS-PAGE, or immunoprecipitation.
Some embodiments of the invention provide methods for treating AML in a subject comprising administering to the subject a therapeutically effective amount of an arginine depleting agent. A further embodiment provides the use of an arginine depleting agent for the manufacture of a medicament for treating AML in a subject in need thereof. The subject can be any animal (e.g., a mammal), including but not limited to humans, non-human primates, canines, felines, and rodents.
Arginine depleting agents
The arginine depleting agent used in the methods and medicaments described herein may be any arginine depleting agent known in the art capable of reducing the level of arginine in the plasma and/or cells of a subject. The arginine depleting agent may be, for example, a small molecule or a protein.
In some embodiments, the arginine depleting agent comprises an arginine catabolic enzyme. Non-limiting examples of 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, human, bovine, porcine, rabbit, rodent, primate, ovine, and caprine. Non-limiting examples of arginases include Bacillus caldolox arginase (Bacillus caldolox arginase), thermus thermophilus arginase (Thermus thermophilus arginase), goat arginase I (Capra hirginase I), naked mole arginase I (Heterococcus glaber arginase I), bovine arginase I (Bos taurus arginase I), porcine arginase I (Sus scrofa arginase I), carnius sativus arginase I (Plectoglomus altis arginase I), atlantic salmon arginase I (Salmo sara arginase I), rainbow arginase I (Salmonella arginase I), salmonella arginase I (Salmonella arginase II), salmonella arginase I (Morinase II), salmonella arginase I (Homo arginase II), salmonella arginase II (Homo arginase I), salmonella arginase II (Homo arginase II), salmonella arginase I (Homo arginase II), arginase I (Homo arginase I), arginase I (Homo arginase II (Homo, phaffia bacteria arginase (Delftia arginase), bacillus coagulans arginase (Bacillus coagulans arginase), phototrophic Hersches arginase (Hoeflea phototrophic arginase), and photosynthetic Rose Bengal arginase (Rosefilexus castenthizii arginase). Other examples include those from Bacillus methanolicus (Bacillus methanolicus), NRRL B-14911 Bacillus (Bacillus sp. NRRL B-14911), rhodococcus eastern sea (Planococcus donghalensis), paenibacillus arborescens (Paenibacillus Dendritiformis), streptomyces (Desmospora sp), methylobacter juniperi (Methylobacter tundiformis), methylobacter stenotrophicus (Stenotrophorus sp), microbacterium laeviformis (Microbacterium laeviformis), porphyromonas winogradskyi (Porphyromonas) and Agrobacterium (Agrobacterium sp), octadecbacter (Octadecabacter arcticum), agrobacterium tumefaciens (Agrobacterium tumefaciens), thermomyces aerobicas (Anaxybacter), bacillus pumilus (Bacillus pumilus) and Bacillus pumilus (Bacillus pumilus) as Bacillus thermophilus (Geobacillus thermogluconoides), geobacillus thermogluconans, brevibacillus laterosporus (Brevibacillus laterosporus), enterobacter ruminants (Desutomacculus ruminis), geobacillus thermophilus (Geobacillus kautophilus), geobacillus thermoacidophilus (Geobacillus thermoacidophilus), geobacillus thermodenitrificans (Geobacillus thermonitrificans), staphylococcus aureus (Staphyloccocus aureus), archaeoglobus halophilus DL31 (Halophilicaceae DL 31), halioticus bisporus halodurans (Halopilaginoensis), alcaligenes malabaricum (Natalimenta magadiii), plasmodium falciparum (Plasmodium), helicobacter pylori (Helicobacter pylori), and the like.
The arginine deiminase used in the methods and medicaments of the present invention may be any arginine deiminase known in the art, such as arginine deiminase produced by Mycoplasma (Mycoplasma), lactococcus (Lactococcus), pseudomonas (Pseudomonas), streptococcus (Steptococcus), escherichia (Escherichia), mycobacterium (Mycobacterium), or Bacillus microorganisms (Bacillus micorganisms). Exemplary arginine deiminases include, but are not limited to, those produced by Mycobacterium of human origin (Mycoplasma hominis), mycoplasma arginini (Mycoplasma arginini), mycoplasma arthritides (Mycoplasma arthritides), clostridium perfringens (Clostridium perfringens), bacillus licheniformis (Bacillus licheniformis), borrelia burgdorferi, borrelia afzellii, enterococcus faecalis (Enterococcus faecalis), lactococcus lactis (Lactobacillus lactis), cactus (Bacillus cereus), streptococcus pyogenes (Streptococcus pyelonogus), streptococcus pneumoniae (Streptococcus neoniae), lactobacillus sake (Lactobacillus saxiella sarmentosum), giardia intestinalis (Giardia intestinalis), mycobacterium tuberculosis (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida), and the like.
Arginine decarboxylase may be any arginine decarboxylase known in the art, such as produced by Escherichia coli (Escherichia coli), salmonella typhimurium (Salmonella typhimurium), chlamydomonas pneumoniae (Klamydophylla pneumoniae), methanococcus jannaschii (Methanococcus jannaschii), paraphora viridis virus I (Paracoccus burlara virus 1), vibrio vulnificus YJ016 (Vibrio vulgaris YJ 016), campylobacter jejuni (Campylobacter jejuni subsp), trypanosoma cruzi (Trypanosoma cruzi), sulfolobus solfataricus (Sulfolobus solfataricus), bacillus licheniformis (Bacillus licheniformis), vibrio cerivis (Vibrio cerivirus), carica papaya (Carica), thermococcus thermophilus (Thermoascus), thermoascus aurantiacus (Thermoascus), thermoascus sp), thermoascus sp (Thermoascus, thermoascus purpurea, thermoascus sp).
The 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 produced from Arthrobacter globiformis ifoo 12137 (Arthrobacter globiformis ifoo 12137), arthrobacter simplex IFO 12069 (Arthrobacter simplex IFO 12069), brevibacterium flavum IFO 12073 (Brevibacterium helvolvulum IFO 12073), helicobacter homochrosis ccu 18818 (Helicobacter cinacaldus CCUG 18818), streptomyces griseus (Streptomyces griseus) and similar species.
The arginine depleting agents of the present invention may comprise naturally occurring and/or synthetic products. In some embodiments of the invention, the arginine depleting agent comprises a naturally occurring arginine catabolic enzyme. In other embodiments, the arginine depleting agent comprises a synthetic arginine catabolic enzyme.
The arginine depleting agent may comprise the intact protein and/or functional fragments and/or variants thereof. Arginine decarboxylase, arginine deiminase, arginine 2-monooxygenase, arginase, and other arginine depleting agents used in the methods and uses may be modified to improve their pharmacokinetic properties, such as by fusing the protein and/or functional fragments and/or variants thereof to human serum albumin, albumin binding domains, fe regions of immunoglobulins, polyethylene glycol (PEG) groups, or combinations thereof. In some embodiments of the invention, one or more of the foregoing modifications increase the half-life of the arginine depleting agent. In further embodiments, the increase in half-life results in a decrease in the frequency of administration of arginine depleting agents required to achieve the same result. One or more of the foregoing modifications to arginine depleting agents may reduce immunogenicity, which may help avoid adverse effects.
In some embodiments of the invention, the arginine catabolic enzyme may be engineered to include specific sites on the enzyme, e.g., PEG may be selectively attached. The selected pegylation site may be located at a site removed from the active site of the enzyme, and/or may generally be exposed to a solvent to allow reaction with the pegylation reagent.
Any pegylation reagent known in the art can be used to covalently attach PEG to the arginine catabolic enzyme described herein. Exemplary PEGylation reagents include, but are not limited to, mPEG-ALD (methoxy polyethylene glycol-propionaldehyde), mPEG-MAL (methoxy polyethylene glycol-maleimide), mPEG-NHS (methoxy polyethylene glycol-N-hydroxysuccinimide), mPEG-SPA (methoxy polyethylene glycol-succinimidyl propionate), and mPEG-CN (methoxy polyethylene glycol-cyanuric chloride).
In some embodiments, the PEG group has a molecular weight of about 5,000 to about 20,000amu, about 5,000 to about 15,000amu, about 5,000 to about 12,000amu, about 7,000 to about 12,000amu, or about 7,000 to about 10,000amu. In particular embodiments, the PEG group has a molecular weight of about 2,000amu to 10,000amu. 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 through a linker. In particular embodiments, the enzyme is covalently attached to the PEG via a propionic acid linker.
Arginine catabolic enzymes may be fused to proteins with inherently long serum half-lives, which may result in a more desirable pharmacokinetic profile. The arginine depleting agents of the present invention may comprise an antibody Fc domain and/or serum albumin. The arginine depleting agent may comprise an arginine catabolic enzyme genetically fused to an antibody Fc domain and/or serum albumin. In some embodiments, the Fe region of the immunoglobulin is from a human immunoglobulin, such as a human IgG. In some embodiments, the enzyme may be fused to an albumin domain. In a further embodiment, the enzyme may be fused to human transferrin.
In a further embodiment of the invention, the arginine depleting agent comprises an arginine catabolic enzyme fused to a nonstructural polypeptide. The fusion of the enzyme to the unstructured polypeptide may increase the overall size and/or hydrodynamic radius of the agent. In some embodiments of the invention, the arginine catabolic enzyme is fused to any one or more of XTEN (which is a recombinant PEG mimetic (XTENylation), PAS (proline-alanine-serine polymer (PASylation)), ELP (which is an elastin-like polypeptide (ELPylation)), HAP (which is a high amino acid polymer (haphylation)), and artificial gelatin-like protein (GLK).
The arginine catabolic enzyme used in some embodiments of the present invention may be fused to an anionic polypeptide, which may increase the negative charge of the agent. The enzyme may be fused to a Carboxy Terminal Peptide (CTP). One suitable non-limiting embodiment of the CTP fusion is the genetic fusion of CTPs from the beta chain of human Chorionic Gonadotropin (CG).
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 agent. In one non-limiting example of an embodiment of the invention, the albumin binding moiety is conjugated or genetically fused to a therapeutic enzyme. Many types of moieties can be used, including but not limited to (i) molecules with internal affinity for albumin; (ii) Peptides, antibody fragments, alternative architectures and small chemicals produced and selected to exhibit albumin binding activity.
Recombinant fusion proteins were first used in the 1980 s and were produced through the fusion of two or more genes, each encoding a separate protein. Methods for the synthesis of various fusion proteins are well known in the art. See, e.g., yu et al, biotechnology Advances,2015;33:155-164, which provides a review of the most common methods currently used for designing and constructing synthetic fusion proteins. Strohl, biodrugs,2015;29 (4): 215-239 which provides another detailed review of the fusion protein and outlines the advantages and disadvantages of the fusion method and the different types of fusion proteins.
In some embodiments of the invention, the arginine depleting agent comprises or consists of an amino acid sequence having an amino acid sequence identical to SEQ ID NO:1 at least 75%, 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
In further embodiments, the arginine depleting agent comprises or consists of an amino acid sequence having an amino acid sequence identical to SEQ ID NO:1 at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In yet a further embodiment, the arginine depleting agent comprises the amino acid sequence set forth in SEQ ID NO:1 or consists of an amino acid sequence as defined in 1.
Methods for assessing the degree of homology and identity between sequences are well known in the art. For example, a mathematical algorithm can be used to calculate the percentage of sequence identity between two sequences. Non-limiting examples of suitable mathematical algorithms are described in the publication by Karlin and co-workers (1993, PNAS USA, 90. This algorithm is integrated into the BLAST (basic local alignment search tool) program family (see also Altschul et al, (1990), J.mol.biol.215, 403-410, or Altschul et al, (1997), nucleic Acids Res,25, 3389-3402), available via the National Center for Biotechnology Information (NCBI) Web site homepage (https:// www.ncbi.nlm.nih.gov). BLAST programs are freely available at https:// BLAST. 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. A85, 2444-2448.) programs. These programs and others can be used to identify sequences that are at least to some extent identical to a given input sequence. Additionally or alternatively, the percentage of Sequence identity between two polypeptide sequences can be determined using the programs Devereux et al 1984, nucleic Acids Res.,387-395, such as the programs GAP and BESTFIT, available in the Wisconsin Sequence Analysis Package (Wisconsin Sequence Analysis Package) version 9.1. BESTFIT uses the local homology algorithm of Smith and Waterman (1981, J.mol.biol.147, 195-197) and identifies the best single region of similarity between the two sequences. When reference is made herein to an amino acid sequence that shares a specified percentage of sequence identity with a reference amino acid sequence, differences between the sequences may result in partial or complete retention of amino acid substitutions. In such cases, the sequence recognized by the substitution with one or more retained amino acids may retain substantially or completely the same biological activity as the reference sequence.
Administration of arginine depleting agents
For therapeutic use, the arginine depleting agents described herein may be prepared as pharmaceutical compositions containing a therapeutically effective amount of the arginine depleting agent described herein as the active ingredient in a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle that carries the active compound to be administered. Such vehicles may 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. As a non-limiting example, 0.9% saline and 0.3% glycine may be used. These solutions may be sterile and generally free of particulate matter. They may be sterilized by conventional well-known sterilization techniques, such as filtration. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizing agents, thickening agents, lubricating agents, coloring agents, and the like. The concentration of arginine depleting agent in such pharmaceutical formulations can vary widely and can be selected based on the desired dosage, liquid volume, viscosity, etc., depending on the particular administration model selected. Suitable vehicles and formulations are described, for example, in Remington: the Science and Practice of Pharmacy,21st edition, troy, D.B.ed., lipinctt Williams and Wilkins, philadelphia, pa.2006, part 5, pharmaceutical Manufacturing pp 691-1092, see in particular pp.958-989.
The concentration of plasma arginine in a subject whose therapeutic effect is to be observed may vary based on a number of factors, including the condition of the subject and/or the type and severity of AML and/or dietary composition. The choice of the target plasma arginine level is well within the skill of one of ordinary skill in the art to which the invention pertains.
Determination of the duration of treatment, e.g., the duration of time that the plasma arginine concentration in the subject is maintained in a depleted state, is well within the skill of one of ordinary skill in the art to which this invention pertains. In particular embodiments, 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 4 weeks, more than 48 weeks, more than 52 weeks, or more than 56 weeks.
In the methods and uses of the present invention, there is no limitation as to the application of the administration model of the arginine depleting agent. In some embodiments of the invention, the administration model of the arginine depleting agent is intravenous. The administration model for therapeutic use of the arginine depleting agents described herein may be any suitable route of delivering the agent to a subject, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous, and/or subcutaneous administration; pulmonary administration; transmucosal (transmucosal) administration (e.g., oral, intranasal, intravaginal, and/or rectal); dosage forms using tablets, capsules, solutions, suspensions, powders, gels and/or granules; and in syringes, implant devices, osmotic pumps, syringes, and/or micropumps; or other devices known to those of ordinary skill in the art to which the invention pertains.
Those skilled in the art to which the invention pertains will appreciate that numerous changes and/or modifications may be made to the invention disclosed in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Examples
The invention will now be described with reference to the following specific examples, which should not be construed as limiting in any way.
Example 1: cytotoxicity of NEI-01 in a range of cancer cell lines.
Exogenous arginine is essential for the growth of certain argininosuccinate synthetase (ASS) deficient cancers. NEI-01 is a recombinant albumin-binding arginine deiminase that can convert arginine to citrulline and ammonia and inhibit the growth of various ASS-deficient cancers by consuming arginine (fig. 1). The results of the cytotoxicity experiments demonstrated that NEI-01 consumed arginine and inhibited the growth of cancer cells in a range of different cancer cell lines (especially in ASS 1-deficient cancer cell lines) (table 2).
TABLE 2 cytotoxicity test results against different cancer cell lines after treatment with NEI-01.
The results of cytotoxicity assays against different AML cell lines after treatment with NEI-01 are provided in table 3.
TABLE 3 cytotoxicity test results against different cancer cell lines after treatment with NEI-01.
Example 2: apoptosis and autophagy.
NEI-01 treated the pancreatic cancer cell line Mia PaCa-2.
To demonstrate the arginine deprivation-mediated decrease in cell viability caused by autophagic cell death, mia PaCa-2 cells were treated with the indicated concentration of NEI-01 with or without Chloroquine (CQ). At the indicated time points, cells were harvested and immunoblotted with antibodies against several markers of autophagy and apoptosis.
As shown in FIG. 2, the expression levels of autophagy markers LC3-II, BECLIN-1 and phosphorylated AMPK α increased when NEI-01 treatment was performed, suggesting that autophagy plays a role in NEI-01-induced cell death. In contrast, when NEI-01 treatment was performed, the expression level of the apoptosis marker PARP-1 was decreased, demonstrating activation of the apoptotic pathway. These results show that apoptosis and autophagy play a role in arginine deprivation mediated cell death mechanisms.
Example 3 NEI-01 treatment of AML cell line HL-60.
To further demonstrate the arginine deprivation-mediated decrease in cell viability caused by autophagic cell death, ASS 1-deficient HL-60AML cells were treated with NEI-01 and CQ. As shown in FIG. 3, when NEI-01 treatment was performed, the expression levels of autophagy markers LC3-II, p62, phospho-AMPK α and AMPK α were increased, suggesting that autophagy plays a role in NEI-01-induced cell death. Conversely, following NEI-01 treatment, expression levels of the apoptotic marker Caspase-9 were reduced, demonstrating activation of the apoptotic pathway. These results show that apoptosis and autophagy play a role in arginine deprivation mediated cell death mechanisms.
Examples 4 to 10: effect of NEI-01 on AML subtypes
There are two major classification systems for identifying AML subtypes-the french-united states-british (FAB) system and the World Health Organization (WHO) classification system. The FAB system is the most commonly used one, and is also referred to herein. According to the FAB system, most people diagnosed with AML have one of 9 different AML types (subtypes): m0, M1, M2, M3, M4eos, M5, M6 and M7.
Example 4: anti-cancer activity of NEI-01 in a C1498 homologous acute myeloid leukemia (FAB AML M4) model.
This study was aimed at assessing the anti-cancer activity of arginine deprivation enzyme NEI-01 in the C1498 homologous AML (FAB AML M4) model.
Murine argininosuccinate synthetase (ASS 1) -deficient C1498 cells co-labeled with luciferase and Green Fluorescent Protein (GFP) were transplanted intravenously (i.v.) into C57BL/6 mice to establish a homogeneous AML model. Mice were randomly divided into 4 groups. Table 4 provides 4 sets of detailed information and their corresponding processing schemes.
Table 4: group and treatment protocol for C1498 homogeneous acute myeloid leukemia (FAB AML M4) model study.
The results show that treatment with NEI-01 significantly prolonged the overall survival of mice with AML subtype M4 compared to PBS treated controls (figure 4). Median Survival Day (MSD) was extended from 24 days in group 1 (control) to 29 days in group 2 (treatment with NEI-01 140U/kg once a week, p =0.0058 versus control).
In addition, more than 60% of the mice in group 3 (treated with NEI-01 280U/kg once a week) and all the mice in group 4 (treated with NEI-01 280U/kg twice a week) survived to the end of the experiment. Median survival for group 3 and group 4 was >31 days (group 3 vs control, p =0.0003; group 4 vs control, p < 0.0001). Consistent with the overall survival results observed, treatment with NEI-01 significantly reduced the overall leukemic burden in addition to slowing disease progression (fig. 5 and 6). This anticancer activity of NEI-01 is presented in a dose-dependent manner.
This study demonstrated the potential anticancer activity of NEI-01 in the C1498 homologous AML M4 model.
Example 5: anti-cancer activity of NEI-01 in KG-1 derived acute myeloid leukemia (FAB AML M0) xenograft model.
In this study, the anticancer effect of NEI-01 was evaluated in a murine xenograft model.
Human argininosuccinate synthetase (ASS 1) -deficient M0 subtype acute myeloid leukemia KG-1 cells were injected subcutaneously into immunodeficient BALB/c nude mice. When the tumor volume reaches 180mm 3 Once a week mice were treated intravenously (i.v.) with either buffer MHT or NEI-01 (280U/kg). Table 5 provides detailed information of the study groups and their corresponding treatment protocols.
Table 5: study cohort and treatment protocol for KG-1 derived acute myeloid leukemia (FAB AML M0) xenograft model.
Tumor volumes were measured every 3 days. After four weeks, mice were sacrificed (n = 9). The xenograft tumors were then dissected and weighed individually.
The results show that NEI-01 treatment significantly reduced tumor volume (fig. 7). By day 28, a 39% reduction was observed. The final T/C ratio reached 60.6%. At the end of the study, the tumor weight in the control group was 3.41. + -. 0.53g, and the tumor weight in the NEI-01 (280U/kg once a week) treated group was 2.03. + -. 0.47g. Tumor weight was reduced by 40.38%.
This study demonstrated potent anticancer activity of NEI-01 in murine AML M0 KG-1 xenografts.
Example 6: anti-cancer activity of NEI-01 in an in situ model of HL-60 derived acute myeloid leukemia (FAB AML M2).
In this study, NEI-01 was evaluated for anti-cancer activity in a murine orthotopic AML model.
Human argininosuccinate synthetase (ASS 1) deficient cells of M2 subtype acute myeloid leukemia HL-60 co-labeled with luciferase and Green Fluorescent Protein (GFP) were transplanted intravenously (iv) into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice to establish orthotopic AML model. Mice were randomly divided into 3 groups. Table 6 provides detailed information of the study groups and their corresponding treatment protocols.
Table 6: groups and treatment protocol for in situ model study of HL-60 derived acute myeloid leukemia (FAB AML M2).
Leukemic cells (HL-60-gfp hi-Luc + cells) were followed and the overall leukemic burden quantified by BLI in vivo. Disease progression is determined by changes in BLI intensity. The results are shown in fig. 8. Aggressive disease progression was found from strong signal evidence throughout AML mice. This progression was significantly inhibited when mice were treated with NEI-01 once a week (p <0.05, from day 4 to day 25) or twice a week (p <0.01, throughout the treatment period).
The results demonstrate that treatment with NEI-01 effectively depletes arginine from mouse plasma, resulting in inhibition of disease progression and a reduction in tumor burden in hematopoietic tissues (including bone marrow and spleen). Disease progression was significantly inhibited when mice were treated with NEI-01 once a week (p <0.05, from day 4 to day 25) or twice a week (p <0.01, throughout the treatment period). Particularly potent activity was observed in bone marrow and spleen; when mice were treated twice a week with 280U/kg NEI-01, tumor burden was significantly reduced in bone marrow (p < 0.0001) and spleen (p < 0.005).
This study demonstrates the potential anticancer activity of NEI-01 in the HL-60 in situ AML M2 model.
Example 7: anti-cancer activity of NEI-01 in P31/FUJ derived acute myeloid leukemia (FAB AML M5) xenograft model.
In this study, the anti-cancer activity of NEI-01 was evaluated in a murine xenograft model.
Human argininosuccinate synthetase (ASS 1) deficient M5 subtype acute myeloid leukemia P31/FUJ cells were subcutaneously inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the tumor volume reached 200mm3, mice were treated intravenously (i.v.) once a week with buffer MHT or NEI-01 (280U/kg). Tumor volumes were measured every 3-4 days. After four weeks, mice were sacrificed (n = 10) and xenografted tumors were dissected and weighed individually. Table 7 provides detailed information of the study groups and their corresponding treatment protocols.
Table 7: group and treatment protocol for P31/FUJ derived acute myeloid leukemia (FAB AML M5) xenograft model study.
Figure 9 shows that NEI-01 treatment significantly reduced tumor volume as well as tumor weight, resulting in a 51.27% reduction in tumor volume by day 28. The final T/C ratio reached 51.14%. At the end of the study, the tumor weight in the control group was 1.08 ± 0.98g, and the tumor weight in the NEI-01 (280U/kg once a week) treated group was 0.78 ± 0.08g (p < 0.05).
This study demonstrates potent anticancer activity of NEI-01 in murine AML M5P 31/FUJ xenografts.
Example 8: anti-cancer activity of NEI-01 in a MKPL-1 derived acute myeloid leukemia (FAB AML M7) xenograft model.
In this study, the anti-cancer effect of NEI-01 was evaluated in a murine xenograft model.
Human argininosuccinate synthetase (ASS 1) -deficient M7 subtype acute myeloid leukemia MKPL-1 cells were subcutaneously inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the tumor volume reaches 120mm 3 Once a week mice were treated intravenously (i.v.) with either buffer MHT or NEI-01 (280U/kg). Tumor volumes were measured every 2-3 days. Three weeks later, mice were sacrificed (n = 9) and xenograft tumors were dissected and weighed individually. Table 8 provides detailed information of the study groups and their corresponding treatment protocols.
Table 8: group and treatment protocol for MKPL-1 derived acute myeloid leukemia (FAB AML M7) xenograft model study.
Figure 10 shows that NEI-01 treatment significantly reduced tumor volume as well as tumor weight, resulting in a 99% reduction in tumor volume by day 22. Tumor weights were 8.05. + -. 0.056g in the control group and 0.15. + -. 0.05g in the NEI-01 (280U/kg once a week) treated group. The final T/C ratio reached 99%.
This study demonstrated potent anticancer activity of NEI-01 in murine AML M7 MKPL-1 xenografts.
Example 9: in vivo efficacy studies of NEI-01 in the treatment of a patient-derived AM5512 acute myeloid leukemia (FAB AML M7) model.
Patient-derived xenografts (PDX) provide the most metastatic preclinical model for efficacy screening in cancer drug development. PDX models are derived directly from patient tumors and have never been adapted to in vitro growth, reflecting the heterogeneity and diversity of human patient populations. In this study, the anticancer effect of NEI-01 was evaluated in a patient-derived AM5512 (FAB AML M7) acute myeloid leukemia model.
Human AM5512 cells were inoculated intravenously (i.v.) into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the tumor burden in peripheral blood was about 1.33%, mice were randomized into 3 groups: group 1 (vehicle), group 2 (NEI-01, 140U/kg) and group 3 (NEI-01, 280U/kg) as described in Table 9.
Table 9 group and treatment protocol for patient derived AM5512 acute myeloid leukemia (FAB AML M7) model study.
Treatment with NEI-01 (280U/kg or 140U/kg once a week) significantly inhibited the growth of tumor burden in peripheral blood after the 3 rd NEI-01 dose (FIG. 11). At the end of the study (one week after the 4 th NEI-01 dose), a significant reduction in tumor burden was observed in peripheral blood and hematopoietic tissues (including spleen, liver and bone marrow) after treatment with NEI-01 (280U/kg or 140U/kg once a week) (p <0.05 compared to vehicle group). These anti-leukemic effects are presented in a dose-dependent manner.
This study provides strong evidence that supports the potential anti-leukemic effect of NEI-01 in the AM5512 (M7) PDX model.
Example 10: in vivo efficacy studies of NEI-01 in the treatment of a patient-derived AM8096 acute myeloid leukemia (FAB AML M2) model.
In this study, the anticancer effect of NEI-01 was evaluated in a patient-derived AM8096 acute myeloid leukemia model.
Human AM8096 cells were inoculated intravenously (i.v.) into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the tumor burden in peripheral blood was about 1.5%, mice were randomized into 3 groups: group 1 (vehicle), group 2 (NEI-01, 140U/kg) and group 3 (NEI-01, 280U/kg) as described in Table 10.
Table 10: cohort and treatment protocol for patient-derived AM8096 acute myeloid leukemia (FAB AML M2) model study.
The results show that once a week treatment with NEI-01 slightly extended the median number of survival days from 12 days in the vehicle control group to 14.5 days in the NEI-01 (140U/kg) treatment group and 16.5 days in the NEI-01 (280U/kg) treatment group (fig. 12 and table 11).
The increase In Lifespan (ILS) of NEI-01 treated mice (140U/kg or 280U/kg) was 20.8% and 37.5%, respectively, when compared to the vehicle control group (Table 11). Exciting was that one mouse in the NEI-01 (280U/kg) treatment group survived to 41 days after the initial treatment. These data suggest that NEI-01 has an anti-leukemic effect in at least some AM8096 model populations.
Table 11: median survival days and lifespan (ILS) for each group in the AM8096 model.
Example 11: anti-cancer activity of NEI-01 in a Jurkat derived leukemia cancer xenograft model.
The T cell immunophenotype of acute lymphocytic leukemia (T-ALL) accounts for approximately 15% to 25% of acute leukemias in adults and children. With the benefit of rapid technological advances and emerging understanding, treatment of T-ALL has advanced significantly. However, a significant number of patients remain at high risk for relapse, and few patients survive when the disease recurs. Thus, new therapeutic approaches are urgently needed.
Drug-induced amino acid deprivation is a strategy that has been successfully used in the treatment of acute lymphocytic leukemia, where asparaginase is an important part of induction chemotherapy. Arginine, as a precursor to the initiation of various metabolic pathways, has been shown to have a regulatory effect on tumor formation. Arginine deprivation has proven to be a promising treatment for arginine auxotrophic tumors lacking argininosuccinate synthase (ASS 1), a restriction enzyme that synthesizes arginine from citrulline. This study was aimed at assessing the anti-cancer activity of arginine deprivation enzyme NEI-01 in a T-ALL Jurkat xenograft model.
Human ASS 1-deficient T-ALL Jurkat cells were inoculated subcutaneously into immunodeficient BALB/c nude mice. When the tumor volume reaches 40mm 3 When nearby, mice were randomly divided into two groups: control and NEI-01 treatment groups as described in table 12. Mice were administered either PBS or NEI-01 intraperitoneally (i.p.) 5U per mouse, approximately 280U/kg twice a week for four weeks. Tumor volume was measured twice a week.
Table 12: groups and treatment protocols for Jurkat-derived leukemia cancer xenograft model studies.
The results show that treatment with NEI-01 (5U per mouse, approximately 280U/kg) twice a week significantly inhibited (p.ltoreq.0.05) tumor growth when compared to the control group on day 28 (FIG. 13).
These data provide support for the potential anticancer activity of NEI-01 in the Jurkat-derived leukemia subcutaneous xenograft model.
Example 12: NEI-01 in the plasma of mice was determined from repeated dose studies.
NEI-01 was administered intravenously to ICR mice at doses of 160, 280 and 560U/kg once weekly for four weeks. Blood samples were taken for all groups on days 1 and 22, pre-dose (-1 h), 0.25, 6, 24, 48 and 72h post-dose; on day 8 (week two), blood samples were taken prior to dosing (-1 h) for all groups; on day 15 (week three), blood samples were taken prior to dosing (-1 h) for all groups; blood samples were taken on day 22 (week four), pre-dose (-1 h), 0.25, 6, 24, 48 and 72h post-dose; and blood samples were taken prior to sacrifice of the mice on day 29 (week 5). Quantification was performed on 5 animals/group/sex/time point and plasma concentrations (figure 14).
The parameters and results obtained for the pharmacokinetic assessment of NEI-01 for the treatment groups are presented in table 13 (day 1) and table 14 (day 22). Plasma concentrations of all NEI-01 in the vehicle control group were below the limit of quantitation. Thus, vehicle control group data are not presented in the table.
Table 13: pharmacokinetic parameters and measurements for day 1 mice of the NEI-01 treatment group.
a C max : maximum NEI-01 concentration
b T max :C max Time of occurrence
c T 1/2 : half life, C max Time required for half reduction
d AUC 0-72h : from the time of drug administration (time "0" to time "72"), the area under the curve in the drug concentration versus time plot
TABLE 14 pharmacokinetic parameters and measurements for day 22 mice of NEI-01 treatment group.
a C max : maximum NEI-01 concentration
b T max :C max Time of occurrence
c T 1/2 : half life, C max Time required for half reduction
d AUC 0-72h : from the time of drug administration (time "0" to time "72"), the area under the curve in the drug concentration versus time plot
Following intravenous administration of NEI-01 to mice, systemic exposure to NEI-01 was observed, with T in males and females on day 1 max Mean value of (d) was 0.25h after dosing. T1/2 is between 21.04 hours and 36.58 hours.
Systemic exposure to NEI-01 (measured by AUC 0-72) increased in a dose-proportional manner in males and females on days 1 and 22. At 560U/kg, day 1 (5.82x10) 6 ng.h/ml-6.04x106ng.h/ml) and day 22 (5.97x10) 6 ng.h/ml~6.27x10 6 ng.h/ml), AUC0-72 were similar in males and females. Likewise, cmax results were similar to AUC0-72, where male and female results were similar on days 1 and 22. There was also no significant difference in body weight between the male and female treatment groups.
Industrial applicability
It is an object of the presently claimed invention to provide an alternative method of treating AML.
Sequence listing
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Ile Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu Lys Leu Ser Ile Glu
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Claims (25)
1. A method for treating Acute Myeloid Leukemia (AML) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an arginine depleting agent, wherein the AML is subtype M0 (undifferentiated acute myeloid leukemia), M2 (acute mature myeloid leukemia), M4eos (acute myelomonocytic leukemia with eosinophilia), M5 (acute monocytic leukemia), M6 (acute erythroleukemia) or M7 (acute megakaryocytic leukemia).
2. The method of claim 1, wherein the arginine depleting agent comprises an arginine catabolic enzyme.
3. The method of claim 2, wherein the arginine catabolic enzyme is arginine deiminase, arginase, arginine decarboxylase, or arginine 2-monooxygenase.
4. The method of claim 1, wherein the arginine depleting agent is a synthetic arginine depleting agent.
5. The method of claim 1, wherein 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, proline-alanine-serine Polymer (PAS), an elastin-like polypeptide (ELP), a high amino acid polymer (HAP), an artificial gelatin-like protein (GLK), a carboxyl-terminal peptide (CTP), or a combination thereof.
6. The method of claim 1, wherein the arginine depleting agent comprises human serum albumin, an albumin binding domain, or a combination thereof.
7. The method of claim 1, wherein the arginine depleting agent comprises an amino acid sequence substantially identical to SEQ ID NO:1 or consists of an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity.
8. The method of claim 1, wherein the arginine depleting agent comprises the amino acid sequence of SEQ ID NO:1 or consists of said amino acid sequence.
9. The method according to claim 1, wherein the AML is FAB subtype M4 or M7.
10. The method of claim 9, wherein the arginine depleting agent comprises an amino acid sequence substantially identical to SEQ ID NO:1 or consists of an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity.
11. The method of claim 9, wherein the arginine depleting agent comprises the amino acid sequence of SEQ ID NO:1 or consists of said amino acid sequence.
12. The method of claim 1, wherein the AML is arginine auxotrophic.
13. The method of claim 1, wherein the arginine depleting agent is administered intramuscularly, intravenously, subcutaneously, or orally.
14. The method of claim 1, wherein the arginine depleting agent is administered intravenously.
15. The method of claim 1, wherein the subject is a human.
16. An arginine depleting agent for use in the treatment of AML in a subject in need thereof, wherein said AML is of the french-american-british (FAB) subtype M0 (undifferentiated acute myeloid leukemia), M2 (acute mature myeloid leukemia), M4eoS (acute myelomonocytic leukemia with eosinophilia), M5 (acute monocytic leukemia), M6 (acute erythroleukemia) or M7 (acute megakaryocytic leukemia).
17. The arginine depleting agent according to claim 16, wherein the arginine depleting agent comprises an arginine catabolic enzyme.
18. The arginine depleting agent according to claim 17, wherein the arginine catabolic enzyme is arginine deiminase, arginase, arginine decarboxylase, or arginine 2-monooxygenase.
19. The arginine depleting agent according to claim 16, wherein the arginine depleting agent is a synthetic arginine depleting agent.
20. The arginine depleting agent of claim 16, wherein 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), an artificial gelatin-like protein (GLK), a carboxyl-terminal peptide (CTP), or a combination thereof.
21. The arginine depleting agent of claim 16, wherein the arginine depleting agent comprises human serum albumin, an albumin binding domain, or a combination thereof.
22. The arginine depleting agent according to claim 16, wherein the AML is arginine auxotrophic.
23. The arginine depleting agent according to claim 16, wherein the arginine depleting agent is administered intramuscularly, intravenously, subcutaneously, or orally.
24. The arginine depleting agent according to claim 16, wherein the arginine depleting agent is administered intravenously.
25. The arginine depleting agent according to claim 16, wherein the subject is a human.
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