Treatment using asparaginase

Abstract

To provide a therapy of a disease by using L-asparaginase.SOLUTION: A therapy of a disease that can be treated by depletion of L-asparagine in a patient includes administering a composite of protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol (PEG) by an effective dose, where a molecular weight of polyethylene glycol is approximately 5000 Da or less, and protein is L-asparaginase derived from the genus Erwinia.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a complex of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol, more particularly to a complex in which the polyethylene glycol has a molecular weight of about 5000 Da or less, and more particularly to a complex in which the protein is an L-asparaginase derived from the genus Erwinia, and to its use in therapy. [Background technology]

[0002] Proteins possessing L-asparagine aminohydrolase activity, commonly known as L-asparaginases, have been used for many years in the treatment of acute lymphoblastic leukemia (ALL) in children with success. ALL is the most common childhood malignancy (Avramis and Panosyan, (2005) 44:367-393).

[0003] L-asparaginase has also been used to treat Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum sarcoma, and melanoma (Kotzia (2007) J. Biotechnol. 127, 657-669). The antitumor activity of L-asparaginase is thought to be due to the lack or low ability of L-asparagine synthesis in certain malignant cells (Kotzia (2007) J. Biotechnol. 127, 657-669). These malignant cells depend on extracellular supply of L-asparagine. However, the L-asparaginase enzyme catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia, thereby depleting the circulating pool of L-asparagine and killing tumor cells that cannot synthesize proteins without L-asparagine (Kotzia (2007) J. Biotechnol. 127, 657-669).

[0004] L-asparaginase derived from E. coli was the first enzyme drug used to treat ALL and is marketed in the United States as Elspar® and in Europe as Kidrolase® and L-asparaginase Medac®. L-asparaginase has also been isolated from other microorganisms; for example, the L-asparaginase protein from Erwinia chrysanthemi is named chrysanthanspase and is marketed as Erwinase® (Wriston (1985) Meth. Enzymol. 113, 608-618; Goward (1992) Bioseparation 2, 335-341). L-asparaginases from other species of the genus Erwinia have also been identified, including, for example, Erwinia chrysanthemi 3937 (Genbank registration number AAS67028), Erwinia chrysanthemi NCPPB 1125 (Genbank registration number CAA31239), Erwinia carotovora (Genbank registration number AAP92666), and Erwinia carotovora subsp. astroseptica (Genbank registration number AAS67027). These Erwinia chrysanthemi L-asparaginases share approximately 91-98% amino acid sequence identity with each other, while Erwinia carotovora L-asparaginase shares approximately 75-77% amino acid sequence identity with Erwinia chrysanthemi L-asparaginase (Kotzia (2007) J. Biotechnol. 127 657-669).

[0005] Currently available L-asparaginase preparations offer neither alternative nor complementary therapies, particularly for the treatment of ALL, characterized by high catalytic activity, significantly improved pharmacological and pharmacokinetic properties, and reduced immunogenicity.

[0006] In one embodiment, the problem that the present invention seeks to solve is to provide an L-asparaginase formulation having: high in vitro physiological activity, stable PEG-protein binding, a long in vivo half-life, significantly reduced immunogenicity, which is supported, for example, by the reduction or elimination of antibody responses to the L-asparaginase formulation after repeated administration, and usefulness as a first-line treatment, for example, as a second-line treatment for patients who have developed sensitivity to treatment using E. coli-derived L-asparaginase.

[0007] This problem remains unresolved with known L-asparaginase complexes that either exhibit significant cross-reactivity with modified L-asparaginase formulations (Wang (2003) Leukemia 17, 1583-1588, which is incorporated herein by reference in its entirety) or have significantly reduced in vitro activity (Kuchumova (2007) Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 1, 230-232, which is incorporated herein by reference in its entirety). This problem is solved according to the present invention by providing a complex of Erwinia L-asparaginase with a hydrophilic polymer, more particularly with polyethylene glycol having a molecular weight of 5000 Da or less, a method for preparing such a complex, and the use of the complex. [Overview of the Initiative]

[0008] The present invention encompasses a method for treating diseases treatable by L-asparagine depletion in patients, the method comprising administering an effective amount of a complex of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol (PEG), wherein the polyethylene glycol has a molecular weight of about 5000 Da or less, and the protein is L-asparaginase derived from the genus Erwinia. In some embodiments, the L-asparaginase has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acids of SEQ ID NO: 1. In some embodiments, the complex contains L-asparaginase derived from the genus Erwinia that has 100% sequence identity with respect to the amino acids of SEQ ID NO: 1. In some embodiments, the PEG has a molecular weight of about 5000 Da, 4000 Da, 3000 Da, 2500 Da, or 2000 Da. In some embodiments, the complex has at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% in vitro activity compared to L-asparaginase that is not complexed with PEG. In some embodiments, the complex has at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times stronger L-asparaginine depletion activity than L-asparaginase that is not complexed with PEG. In some embodiments, the complex drastically reduces plasma levels of L-asparagine to undetectable levels for at least about 12, 24, 48, 96, 108, or 120 hours. In some embodiments, the complex has a longer in vivo circulating half-life than L-asparaginase that does not form a complex with PEG. In some embodiments, the complex has a longer t1 / 2 than PEG-asparagase administered at an equivalent protein dose.In some embodiments, the complex has a t1 / 2 of at least about 58 to about 65 hours at a dose of about 50 μg / kg based on protein content after IV administration to mice, and a t1 / 2 of at least about 34 to about 40 hours at a dose of about 10 μg / kg based on protein content. In some embodiments, the complex has a concentration of about 10,000 to about 15,000 IU / m³. 2 (Approximately 20-30 mg of protein / m³) 2 At doses in the range of ), it has a t1 / 2 of at least about 100 to about 200 hours. In some embodiments, the complex has a larger area under the curve (AUC) than L-asparaginase that is not complexed with PEG. In some embodiments, the average AUC of the complex is at least about 3 times greater than that of PEG-asparaginase at an equivalent protein dose. In some embodiments, PEG is covalently bonded to one or more amino groups of L-asparaginase. In some embodiments, PEG is covalently bonded to one or more amino groups by amide bonds. In some embodiments, PEG is covalently bonded to at least about 40% to about 100% of the accessible amino groups or at least about 40% to about 90% of the total amino groups.

[0009] The method of the present invention involves the use of a complex having the following formula: Asp-[NH-CO-(CH2) x -CO-NH-PEG] n In the formula, Asp is L-asparaginase, NH is one or more lysine residues and / or N-terminal NH groups of Asp, PEG is a polyethylene glycol moiety, n is a number representing at least about 40% to about 100% of the accessible amino groups of Asp, and x is an integer in the range of about 1 to about 8, more specifically, about 2 to about 5. In certain embodiments, PEG is monomethoxy-polyethylene glycol (mPEG).

[0010] The method of the present invention involves the use of an L-asparaginase complex comprising one or more peptides, each peptide independently of peptide R N -(P / A)-RC where (P / A) is an amino acid sequence consisting of only proline and alanine amino acid residues, where R N is a protecting group bonded to the N-terminal amino group of the amino acid sequence, where R C is an amino acid residue bonded to the C-terminal carboxy group of the amino acid sequence via its amino group, and each peptide forms a complex with L-asparaginase via an amide bond formed from the carboxy group of the C-terminal amino acid residue R C of the peptide and the free amino group of L-asparaginase, and where at least one of the free amino groups to which the peptide is bonded is not the N-terminal α-amino group of L-asparaginase.

[0011] The method of the present invention encompasses the use of the complex for cancer treatment. In some embodiments, the cancer is selected from the group consisting of lymphoma, large cell immunoblastic lymphoma, non-Hodgkin lymphoma, diffuse large B cell lymphoma, NK lymphoma, Hodgkin's disease, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute T cell leukemia, acute myeloid leukemia (AML), biphenotypic B-cell myelomonocytic leukemia, and chronic lymphocytic leukemia.

[0012] In some embodiments, the disease is renal cell carcinoma Tumor , renal cell adenocarcinoma, glioblastoma including glioblastoma multiforme and astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma Tumor , squamous cell carcinoma Tumor , large cell lung carcinoma Tumor and small cell lung carcinoma Tumor including lung cancer Tumor , endometrial carcinoma Tumor , ovarian adenocarcinoma, ovarian teratocarcinoma, cervical adenocarcinoma, breast cancer Tumor , breast carcinoma, intraductal carcinoma of breast Tumor , pancreatic adenocarcinoma, pancreatic ductal carcinoma Tumor , colon cancer Tumor , colorectal adenocarcinoma, colorectal adenocarcinoma, transitional epithelial carcinoma of bladder Tumor , bladder papilloma, prostate cancer Tumor , osteosarcoma, squamous cell carcinoma of bone Tumor prostate cancer Tumor The group is selected from the following: , and thyroid cancer. Depending on the embodiment, the complex is administered in a dose of approximately 5 U / kg body weight to approximately 50 U / kg body weight.

[0013] Depending on the embodiment, the composite may have a concentration of approximately 100 to 15,000 IU / m³. 2 It is administered in doses within the range of [specify dose range]. Depending on the embodiment, administration is intravenous or intramuscular, once, twice, or three times per week. Depending on the embodiment, the complex is administered as monotherapy. Depending on the embodiment, the complex is administered as part of combination therapy. Depending on the embodiment, the complex is administered as part of combination therapy using Oncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactricib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839. Depending on the embodiment, the patient receiving treatment has previously experienced hypersensitivity to E. coli asparaginase or its PEGylated counterpart, or to Erwinia asparaginase. Depending on the embodiment, the patient receiving treatment has experienced disease relapse, particularly relapse after treatment with E. coli asparaginase or its PEGylated counterpart. [Brief explanation of the drawing]

[0014] [Figure 1] In vivo experimental data showing the combined use of peg chrysanthase with other compounds are presented. [Figure 2] In vivo experimental data showing the combined use of peg chrysanthase with other compounds are presented. [Figure 3] The dose-response curve for monotherapy cases is shown. [Figure 4] The dose-response curve for a mixture with an inert agent is shown. [Figure 5] Comparative data between single-component and mixed-component examples are shown. [Figure 6]A dose-centered plot is shown to indicate which drug combinations are synergistic. [Figure 7] CNS cell line data is shown. [Figure 8] This demonstrates the IC50 effect of PEG chrysanthase. [Figure 9] This demonstrates the IC50 effect of PEG chrysanthase. [Figure 10] This study demonstrates in vitro sensitivity of PEG chrysanthase in leukemia and lymphoma cell lines. [Modes for carrying out the invention]

[0015] Bacterial L-asparaginases have the potential for high immunogenicity and antigenicity, frequently inducing adverse reactions ranging from mild allergic reactions to anaphylactic shock in sensitized patients (Wang (2003) Leukemia 17, 1583-1588). E. coli L-asparaginase is particularly immunogenic, and the presence of anti-asparaginase antibodies against E. coli L-asparaginase after IV or im administration has been reported to reach as high as 78% in adults and 70% in children (Wang (2003) Leukemia 17, 1583-1588).

[0016] L-asparaginases from Escherichia coli and Erwinia chrysanthemi have different pharmacokinetic properties and each possesses a unique immunogenicity profile (Klug Albertsen (2001) Brit. J. Haematol. 115, 983-990). Furthermore, it has been shown that antibodies generated after treatment with E. coli-derived L-asparaginase do not cross-react with L-asparaginases from the Erwinia genus (Wang (2003) Leukemia 17, 1583-1588). Therefore, L-asparaginase derived from Erwinia crisantaspase has been used as a second-line treatment for ALL in patients who respond to E. coli L-asparaginase (Duval (2002) Blood 15, 2734-2739; Avramis (2005) Clin. Pharmacokinet. 44, 367-393).

[0017] Another attempt to reduce the immunogenicity associated with the administration of microbial L-asparaginase has led to the development of E. coli species L-asparaginase modified with methoxy-polyethylene glycol (mPEG). This method, commonly known as "PEGylation," has been shown to alter the immunogenic properties of the protein (Abuchowski (1977) J. Biol. Chem. 252, 3578-3581). This so-called mPEG-L-asparaginase, or peg-asparaginase, is marketed as Oncaspar® and was first approved in the United States as a second-line treatment in 1994, and has been approved as a first-line treatment for ALL in children and adults since 2006. Oncaspar® has a long in vivo half-life and reduced immunogenicity / antigenicity.

[0018] Oncaspar® is an E. coli species L-asparaginase modified with 5 kDa mPEG-succinimidyl succinate (SS-PEG) at multiple lysine residues (U.S. Patent No. 4,179,337). SS-PEG is a first-generation PEG reagent with an unstable ester bond that is sensitive to enzymatic hydrolysis or weakly alkaline pH values ​​(U.S. Patent No. 4,670,417). These properties reduce stability both in vitro and in vivo, potentially compromising drug safety.

[0019] Furthermore, it has been demonstrated that antibodies generated against L-asparaginase derived from E. coli may cross-react with Oncaspar® (Wang (2003) Leukemia 17, 1583-1588). Even if these antibodies are not neutralizing antibodies, this finding clearly demonstrates a high possibility of cross-hypersensitivity or cross-inactivation in vivo. In fact, one report indicated that 30-41% of children administered peg-asparaginase experienced allergic reactions (Wang (2003) Leukemia 17, 1583-1588).

[0020] In addition to overtly manifested allergic reactions, a recent issue of "asymptomatic hypersensitivity" has been reported, in which patients develop anti-asparaginase antibodies without showing any clinical evidence of a hypersensitivity reaction (Wang (2003) Leukemia 17, 1583-1588). This reaction may lead to the formation of neutralizing antibodies against E. coli L-asparaginase and peg-asparaginase. However, because these patients do not show overt signs of hypersensitivity, they are not switched to Erwinia L-asparaginase, resulting in a shorter effective treatment period (Holcenberg (2004) Pediatr. Hematol. Oncol. 26, 273-274).

[0021] Treatment with Erwinia chrysanthemi L-asparaginase is often used in cases of hypersensitivity to E. coli-derived L-asparaginase. However, it has been observed that 30-50% of patients administered Erwinia L-asparaginase become antibody-positive (Avramis (2005) Clin. Pharmacokinet. 44, 367-393). Furthermore, because Erwinia chrysanthemi L-asparaginase has a significantly shorter elimination half-life than E. coli L-asparaginase, it must be administered more frequently (Avramis (2005) Clin. Pharmacokinet. 44, 367-393). In a study by Avramis, Erwinia asparaginases had an inferior pharmacokinetic profile (Avramis (2007) J. Pediatr. Hematol. Oncol. 29, 239-247). Therefore, E. coli L-asparaginase and pegasparagase were preferred as first-line treatments for ALL over Erwinia L-asparaginases.

[0022] Over many years, PEGylation and commercialization have been successfully achieved for numerous biopharmaceuticals. To couple PEG with proteins, PEG must be activated at its OH terminus. The activating group is selected based on the available reactive groups of the protein to be PEGylated. For proteins, the most important amino acids are lysine, cysteine, glutamic acid, aspartic acid, C-terminal carboxylic acid, and N-terminal amino group. Given the wide range of reactive groups in proteins, almost all peptide chemical reactions have been applied to activate the PEG moiety. Examples of these PEG activation reagents include activated carbonate esters, such as p-nitrophenyl carbonate and succinimidyl carbonate, and activated esters, such as succinimidyl ester. Furthermore, aldehydes and maleimides have been developed for site-specific coupling (Harris (2002) Adv. Drug Del. Rev. 54, 459-476). The availability of diverse chemical methods for PEG modification indicates that each new development of PEGylated proteins can be a case-specific research project. Not only chemical reactions, but also the molecular weight of the PEG bound to the protein significantly influences the pharmaceutical properties of PEGylated proteins. In most cases, it is expected that the pharmaceutical properties will improve as the molecular weight of PEG increases (Sherman (2008) Adv. Drug Del. Rev. 60, 59-68; Holtsberg (2002) Journal of Controlled Release 80, 259-271). For example, Holtsberg et al. found that when PEG was complexed with arginine deaminase, another amino acid-degrading enzyme isolated from microbial raw materials, the pharmacokinetic and pharmacodynamic functions of the enzyme increased as the molecular weight of the bound PEG increased from 5,000 Da to 20,000 Da (Holtsberg (2002) Journal of Controlled Release 80, 259-271).

[0023] However, in many cases, PEGylated biologics exhibit significantly reduced activity compared to unmodified biologics (Fishburn (2008) J. Pharm. Sci., 1-17). In the case of L-asparaginase from Erwinia carotovora, PEGylation was observed to reduce its in vitro activity to approximately 57% (Kuchumova (2007) Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 1, 230-232). L-asparaginase from Erwinia carotovora has only about 75% homology to L-asparaginase (chrysanthanspase) from Erwinia chrysanthemi. For Oncaspar®, its in vitro activity is also known to be about 50% compared to unmodified E. coli L-asparaginase.

[0024] Described herein is a PEGylated L-asparaginase derived from the genus Erwinia that possesses improved pharmacological properties compared to unmodified L-asparaginase protein and to E. coli-derived PEG-asparaginase preparations. The PEGylated L-asparaginase complex described herein, for example, Erwinia chrysanthemi L-asparaginase PEGylated with PEG of molecular weight 5000 Da, acts as a therapeutic agent, particularly in patients who exhibit hypersensitivity (e.g., allergic reactions or asymptomatic hypersensitivity) to treatment with E. coli-derived L-asparaginase, PEGylated L-asparaginase, or unmodified L-asparaginase derived from the genus Erwinia. The PEGylated L-asparaginase complex described herein is also useful as a therapeutic agent for patients with disease relapses, such as relapses of ALL, and patients who have previously been treated with other forms of asparaginase, such as E. coli-derived L-asparaginase or PEGylated L-asparaginase.

[0025] As described in detail herein, the complex of the present invention unexpectedly exhibits superior properties compared to known L-asparaginase preparations, such as peg-asparaginase. For example, unmodified L-asparaginase (chrysanthapase) derived from Erwinia chrysanthemi has a significantly shorter half-life than unmodified L-asparaginase derived from E. coli (Avramis (2005) Clin. Pharmacokinet. 44, 367-393, which is incorporated herein by reference in its entirety). The PEGylated complex of the present invention has a longer half-life than PEGylated L-asparaginase derived from E. coli at an equivalent protein dose.

[0026] definition Unless otherwise explicitly defined, terms used herein shall be understood in accordance with their common meanings in the art.

[0027] As used herein, the term “including” means “including, but not limited to,” and when used in the singular form, it includes the plural form unless otherwise specified in the context, and vice versa.

[0028] As used herein, the term “disease treatable by asparagine depletion” refers to a condition or disorder in which cells involved in or contributing to the condition or disorder exhibit either a lack or reduced capacity to synthesize L-asparagine. The depletion or loss of L-asparagine may be partial or substantially complete (e.g., at levels undetectable using methods and apparatus known in the art).

[0029] As used herein, the term “therapeutically effective dose” refers to the amount of protein (e.g., asparaginase or a complex thereof) required to produce the desired therapeutic effect.

[0030] As used herein, the term “sequence identity” is used synonymously with “homology,” and therefore may have the same meaning where appropriate.

[0031] The terms “simultaneous administration,” “administered simultaneously,” “administered in combination with,” “administered in combination with,” “simultaneously,” and “simultaneously occurring,” as used herein, encompass the administration of two or more active pharmaceutical ingredients to a human subject such that both active pharmaceutical ingredients and / or their metabolites are present in the human subject at the same time. Simultaneous administration includes simultaneous administration in separate compositions, administration in separate compositions at different time points, or administration in a single composition containing two or more active pharmaceutical ingredients. Simultaneous administration in separate compositions and administration in a single composition containing both agents are also encompassed in the methods of the present invention.

[0032] L-asparaginase protein The protein according to the present invention is an enzyme having L-asparagine aminohydrolase activity, so-called L-asparaginase.

[0033] Many L-asparaginase proteins have been identified in this field and isolated from microorganisms by known methods (see, for example, Savitri (2003) Indian J. Biotechnol 2, 184-194, which is incorporated herein by reference in its entirety). The most widely used commercially available L-asparaginases are derived from E. coli or Erwinia chrysanthemi, which have less than 50% structural homology to each other. Within the Erwinia species, typically 75-77% sequence identity has been reported between enzymes from Erwinia chrysanthemi and Erwinia carotovora, and approximately 90% sequence identity has been found between different subspecies of Erwinia chrysanthemi (Kotzia GA, Labrou E, Journal of Biotechnology (2007) 127:657-669, which is incorporated herein by reference in its entirety). Some representative Erwinia L-asparaginases are shown in Table 1, for example: [Table 1]

[0034] The sequences and GenBank registrations of Erwinia L-asparaginases in Table 1 are incorporated herein by reference. L-asparaginases suitable for therapeutic use are those isolated from E. coli and Erwinia species, particularly Erwinia chrysanthemi.

[0035] L-asparaginase can be a naturally occurring enzyme isolated from microorganisms. L-asparaginase can also be produced by recombinant enzyme techniques in producing microorganisms such as E. coli. For example, the protein used in the complex of the present invention can be a protein produced by E. coli from a recombinant E. coli strain producing a protein of the Erwinia species, specifically E. coli from an Erwinia chrysanthemi strain.

[0036] Enzymes can be identified by their specific activity. That is, this definition encompasses all polypeptides that possess a defined specific activity and are also present in other organisms, more specifically, other microorganisms. Enzymes with similar activity can often be identified by grouping them into certain families defined as PFAMs or COGs. PFAMs (Protein Family Database by Sequence Comparison and Hidden Markov Models; pfam.sanfferac.ukl) represent a vast collection of protein sequence comparisons. Each PFAM allows for the visualization of multiple sequence comparisons, display of protein domains, evaluation of inter-organism distribution, access to other databases, and visualization of known protein structures. COGs (Clusters of Homologous Molecular Species of Proteins; vv-ww.nebi.nlm.nih.gov / COG / ) are obtained by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenetic lineages. Each COG is defined from at least three lineages, which allows for the identification of previously conserved domains.

[0037] Means for identifying homologous sequences and identifying their homology or sequence identity percentages are well known to those skilled in the art, and such means include, in particular, the BLAST program. The BLAST program can be used from the following website, blast.ncbi.olo.nih.gov / Blast.cgi, where initial parameters are shown. The resulting sequences can then be utilized (e.g., aligned) in programs such as CLUSTALW (www.ebi.ac.uk / Tools / clustalw2 / index.html) or MULTALIN (bioinfo.genotoul.fr / multalin / multalin.html) using the initial parameters shown on those websites. Using the references provided in GenBank for known genes, those skilled in the art can identify equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc. This common method is advantageously performed using consensus sequences, which can be identified by comparing their sequences with those of genes from other microorganisms and designing denaturing probes to clone the corresponding genes in other organisms. Such common methods in molecular biology are well known to those skilled in the art and are described, for example, in Sambrook (2012) Molecular Cloning: A Laboratory Manual, 4th ed. Cold Spring Harbor Lab Press).

[0038] In fact, those skilled in the art will know how to select and design homologous proteins that substantially retain L-asparaginase activity. Typically, the Nessler assay is used to measure L-asparaginase activity according to the method described by Mashburn and Wriston (Mashburn (1963) Biochem. Biophys. Res. Comm. 12, 50, which is incorporated herein by reference in its entirety).

[0039] In certain embodiments of the complex of the present invention, the L-asparaginase protein has at least about 80% homology or sequence identity with respect to the protein containing the sequence of SEQ ID NO: 1, more specifically, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 2095%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity with respect to the protein containing the sequence of SEQ ID NO: 1. SEQ ID NO: 1 is as follows: ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSA RYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0040] The phrase "contains the sequence of SEQ ID NO: 1" means that the amino acid sequence of the protein is not strictly limited to SEQ ID NO: 1 and may contain additional amino acids.

[0041] In certain embodiments, the protein is L-asparaginase from Erwinia chrysanthemi having the sequence of SEQ ID NO: 1. In other embodiments, the L-asparaginase is derived from Erwinia chrysanthemi NCPPB1066 (Genbank registration number CAA32884, which is incorporated herein by reference in its entirety, with or without the presence of a signal peptide and / or leader sequence).

[0042] The protein fragment of SEQ ID NO: 1 is also included in the definition of the protein used in the complex of the present invention. The term "fragment of SEQ ID NO: 1" means that the polypeptide sequence may have fewer amino acids than SEQ ID NO: 1, but still has enough amino acids to confer L-aminohydrolase activity.

[0043] It is well known in the art that polypeptides can be modified while retaining their enzymatic activity by substitution, insertion, deletion, and / or addition of one or more amino acids. For example, it is common to substitute one amino acid at a given position with a chemically equivalent amino acid that does not affect the functional properties of the protein. A substitution can be defined as an exchange within one of the following groups: Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly, Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln, Polar, positively charged residues: His, Arg, Lys, Large aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys, Large aromatic residues: Phe, Tyr, Trp.

[0044] In other words, substitutions involving one negatively charged residue to another (for example, glutamate to aspartate) or one positively charged residue to another (lysine to arginine) can be expected to yield functionally equivalent products.

[0045] The modification sites of amino acids in the amino acid sequence and the number of amino acids to be modified are not particularly limited. Those skilled in the art will know which modifications can be introduced without affecting the activity of the protein. For example, modifications at the N-terminal or C-terminal region of a protein can be expected not to alter the protein's activity under certain conditions. In particular, asparaginases have been thoroughly characterized, especially in terms of their sequence, structure, and the residues that form the active catalytic site. This provides guidance on residues that can be modified without affecting the enzyme's activity. All known bacterial L-asparaginases share a common structural characteristic: they are all homotetramers with four active sites between the N-terminal and C-terminal domains of two adjacent monomers (Aghaipour (2001) Biochemistry 40, 5655-5664, which is incorporated herein by reference in its entirety). These all exhibit a high degree of similarity in their tertiary and quaternary structures (Papageorgiou (2008) FEBSJ. 275, 4306-4316, which is incorporated herein by reference in its entirety). The sequence of the catalytic site of L-asparaginase is highly conserved among Erwinia chrysanthemi, Erwinia carotovora, and E. coli species L-asparaginase II (Papageorgiou (2008) FEBSJ. 275, 4306-4316). The flexible loop of the active site contains amino acid residues 14-33, and structural analysis has shown that Thr 15 , Thr 95 , Ser 62 , Glu 63 Asp 96 , and Ala 120It has been shown that it contacts the ligand (Papageorgiou (2008) FEBSJ. 275, 4306-4316). Aghaipour et al. performed a detailed analysis of the four active sites of Erwinia chrysanthemi L-asparaginase by high-resolution crystal structure analysis of the enzyme conjugated with the substrate (Aghaipour (2001) Biochemistry 40, 5655-5664). Kotzia et al. sequenced L-asparaginases from multiple species and subspecies of the genus Erwinia and reported that despite having only about 75-77% identity between their proteins in Erwinia chrysanthemi and Erwinia carotovora, they still possess L-asparaginase activity (Kotzia (2007) J. Biotechnol. 127, 657-669, which is incorporated herein by reference in its entirety). Moola et al. performed epitope mapping on 3937 species of Erwinia chrysanthemi L-asparaginase and found that this L-asparaginase retained its enzymatic activity even after various antigenic sequence mutations were made to reduce the immunogenicity of the asparaginase (Moola (1994) Biochem. J. 302, 921-927, which is incorporated herein by reference in its entirety). Each of the above references is incorporated herein by reference in its entirety. Considering the extensive characterizations that have been performed on L-asparaginase, those skilled in the art can determine how to prepare fragments and / or perform sequence substitutions while retaining enzymatic activity.

[0046] Polymers used in the composite The polymer is selected from the group consisting of non-toxic, water-soluble polymers, such as polysaccharides, such as hydroxyethyl starch; polyamino acids, such as polylysine; polyesters, such as polylactic acid; and polyalkylene oxides, such as polyethylene glycol (PEG).

[0047] Polyethylene glycol (PEG) or mono-methoxy-polyethylene glycol (mPEG) is well known in the art and includes linear and branched polymers. Examples of several polymers, in particular PEG, are provided below, each of which is incorporated herein by whole reference as is: U.S. Patent Nos. 5,672,662, 4,179,337, 5,252,714, U.S. Patent Publication No. 2003 / 0114647, U.S. Patent Nos. 6,113,906, 7,419,600, 9,920,311, and PCT Publication No. WO2004 / 083258.

[0048] The quality of such polymers is characterized by the polydispersity index (PDI). PDI reflects the molecular weight distribution in a given polymer sample and is calculated by dividing the weight-average molecular weight by the number-average molecular weight. PDI indicates the distribution of individual molecular weights within a batch of polymers. PDI is always greater than 1, but as the polymer chain approaches an ideal Gaussian distribution (=monodisperse), PDI also approaches 1.

[0049] Polyethylene glycols have a molecular weight in the range of about 500 Da to about 9,000 Da. More specifically, polyethylene glycols (e.g., mPEG) have molecular weights selected from the group consisting of polyethylene glycols of 2,000 Da, 2,500 Da, 3,000 Da, 3,500 Da, 4,000 Da, 4,500 Da, and 5,000 Da. In certain embodiments, polyethylene glycols (e.g., mPEG) have a molecular weight of 5,000 Da.

[0050] Method for preparing the complex Subsequently, to couple the polymer with a protein having L-asparagine aminohydrolase activity, the polymer portion has an activated functional group that reacts suitably with amino groups in the protein. In one embodiment, the present invention relates to a method for preparing a complex, which comprises mixing a certain amount of polyethylene glycol (PEG) and a certain amount of L-asparaginase in a buffer solution for a time sufficient to allow PEG and L-asparaginase to covalently bond. In certain embodiments, the L-asparaginase is derived from the Erwinia species, more particularly from Erwinia chrysanthemi, and more specifically, an L-asparaginase comprising the sequence of SEQ ID NO: 1. In one embodiment, PEG is monomethoxy-polyethylene glycol (mPEG).

[0051] In one embodiment, the reaction between polyethylene glycol and L-asparaginase is carried out in a buffer solution. In some of the specific embodiments, the pH of the buffer solution is in the range of about 7.0 to about 9.0. The optimal pH is in the range of about 7.5 to about 8.5, for example, pH values ​​of about 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 158.5. In certain embodiments, the L-asparaginase is derived from the species Erwinia, more specifically from Erwinia chrysanthemi, and more specifically, is an L-asparaginase containing the sequence of SEQ ID NO: 1.

[0052] Furthermore, PEGylation of L-asparaginase is carried out at protein concentrations of approximately 0.5 to 25 mg / mL, more specifically, approximately 2 to 20 mg / mL, and in particular, approximately 3 to 15 mg / mL. For example, protein concentrations are approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL. In certain embodiments, PEGylation of L-asparaginase at these protein concentrations is for Erwinia species, more specifically for Erwinia chrysanthemi, and more specifically for L-asparaginase containing the sequence of Sequence ID No. 1.

[0053] At high protein concentrations exceeding 2 mg / mL, the PEGylation reaction proceeds rapidly in less than 2 hours. Furthermore, the molar excess ratio of the polymer to the amino groups of L-asparaginase is less than approximately 20:1. For example, molar excess ratios are approximately 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7.5:1, 7:1, 6.5:1, 6:1, 5.5:1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.5:1, or less than 1:1. In certain embodiments, the molar excess ratio is less than approximately 10:1, and in more detailed embodiments, the molar excess ratio is less than approximately 8:1. In certain embodiments, the L-asparaginase is derived from the Erwinia species, more specifically from Erwinia chrysanthemi, and more specifically, is an L-asparaginase containing the sequence of SEQ ID NO: 1.

[0054] The number of PEG moieties that can be coupled to a protein depends on the number of free amino groups, and furthermore, on the number of amino groups accessible in the PEGylation reaction. In certain embodiments, the degree of PEGylation (i.e., the number of PEG moieties coupled to the amino groups of L-asparaginase) is in the range of about 10% to about 100% of the free and / or accessible amino groups (e.g., about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). 100% PEGylation of accessible amino groups (e.g., lysine residues and / or N-terminus of a protein) is also referred to herein as "maximum PEGylation". One method for identifying the modified amino groups in the mPEG-r-chrysanthase complex (the degree of PEGylation) is described by Habeeb (AFSA Habeeb, "Determination of free amino groups in proteins by trinitrobenzensulfonic acid", Anal. Biochem. 14 (1966), p. 328, which is incorporated herein by reference in its entirety). In one embodiment, the PEG moiety is coupled to one or more amino groups of L-asparaginase (the amino groups include lysine residues and / or the N-terminus). In certain embodiments, the degree of PEGylation is in the range of about 10% to about 100% of all amino groups or accessible amino groups (e.g., lysine residues and / or N-terminus), for example, about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total amino groups (e.g., lysine residues and / or N-terminus) are coupled with the PEG moiety.In another specific embodiment, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66% of accessible amino groups (e.g., lysine residues and / or N-terminus) 67%, 68%, 70%, 71%, 72%, 7%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the PEG moiety couples with the PEG moiety. In certain embodiments, 40-55% or 100% of the accessible amino groups (e.g., lysine residues and / or N-terminus) couple with the PEG moiety. In some embodiments, the PEG moiety couples with L-asparaginase by covalent bond. In certain embodiments, the L-asparaginase is derived from the Erwinia species, more specifically from Erwinia chrysanthemi, and more specifically, is an L-asparaginase containing the sequence of SEQ ID NO: 1.

[0055] In one embodiment, the composite of the present invention can be represented by the following formula Asp-[NH-CO-(CH2) x -CO-NH-PEG] n In the formula, Asp is the L-asparaginase protein, NH is the lysine residue and / or the N-terminal NH group of the protein chain, PEG is the polyethylene glycol moiety, n is the number of at least 40% to about 100% of the accessible amino groups in the protein (e.g., the lysine residue and / or N-terminus), all of which are defined in the above and below examples, and x is an integer in the range of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), preferably 2 to 5 (e.g., 2, 3, 4, 5). In certain embodiments, the L-asparaginase is derived from the species Erwinia, more specifically from Erwinia chrysanthemi, and more specifically, is the L-asparaginase comprising the sequence of Sequence ID No. 1.

[0056] Other PEGylation methods that can be used to form the composite of the present invention are presented, for example, in U.S. Patent Nos. 4,179,337, 5,766,897, U.S. Patent Application Publication No. 2002 / 0065397A1, and U.S. Patent Application Publication No. 2009 / 0054590A1, which are each incorporated herein by reference in their entirety.

[0057] Specific embodiments include proteins having substantial L-asparagine aminohydrolase activity and polyethylene glycol, which are selected from the following group of complexes: (A) The protein has a structure that is at least 90% homologous to L-asparaginase from Erwinia chrysanthemi as disclosed in Sequence ID No. 1, the polyethylene glycol has a molecular weight of approximately 5000 Da, and the polyethylene glycol moiety is covalently bonded to the protein by amide bonds, with approximately 100% of the accessible amino groups (e.g., lysine residues and / or N-terminus), or approximately 80-90% of the total amino groups (e.g., lysine residues and / or N-terminus), specifically about 84%, being bonded to the polyethylene glycol moiety. (B) The protein has a structure that is at least 90% homologous to L-asparaginase from Erwinia chrysanthemi as disclosed in Sequence ID No. 1, the polyethylene glycol has a molecular weight of approximately 5000 Da, and the polyethylene glycol moiety is covalently bonded to the protein by amide bonds, with approximately 40% to 45%, more specifically about 43%, or about 36% of all amino groups (e.g., lysine residues and / or N-terminus) being bonded to the polyethylene glycol moiety. (C) The protein has a structure that is at least 90% homologous to L-asparaginase from Erwinia chrysanthemi as disclosed in Sequence ID No. 1, the polyethylene glycol has a molecular weight of approximately 2000 Da, and the polyethylene glycol moiety is covalently bonded to the protein by amide bonds, with approximately 100% of the accessible amino groups (e.g., lysine residues and / or N-terminus), or approximately 80-90% of the total amino groups (e.g., lysine residues and / or N-terminus), specifically about 84%, being bonded to the polyethylene glycol moiety. (D) The protein has a structure that is at least 90% homologous to L-asparaginase from Erwinia chrysanthemi as disclosed in Sequence ID No. 1, the polyethylene glycol has a molecular weight of about 2000 Da, and the polyethylene glycol moiety is covalently bonded to the protein by amide bonds, with about 50% to about 60%, more specifically about 55%, or about 47% of all amino groups (e.g., lysine residues and / or N-terminus) being bonded to the polyethylene glycol moiety.

[0058] L-asparaginase-PEG complex The complex of the present invention has certain advantages and unexpected properties compared to unmodified L-asparaginase, more specifically compared to unmodified Erwinia genus L-asparaginase, more specifically compared to unmodified L-asparaginase derived from Erwinia chrysanthemi, and more specifically compared to unmodified L-asparaginase having the sequence of SEQ ID NO: 1.

[0059] In some embodiments, the method of the present invention comprises a complex that, when administered at a dose of 5 U / kg body weight (bw) or 10 μg / kg (based on protein content), lowers plasma L-asparagine and glutamine levels for at least about 12, 24, 48, 72, 96, or 120 hours. In other embodiments, the complex of the present invention, when administered at a dose of 25 U / kg bw or 50 μg / kg (based on protein content), lowers plasma L-asparagine levels to undetectable levels for at least about 12, 24, 48, 72, 96, 120, or 144 hours. In other embodiments, the complex of the present invention, when administered at a dose of 50 U / kg bw or 100 μg / kg (based on protein content), lowers plasma L-asparagine levels for at least about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours. In another embodiment, the composite of the present invention has a concentration of about 100 to about 15,000 IU / m³. 2 (approximately 1-30 mg protein / m³) 2 When administered in doses within the range of ), it reduces plasma L-asparagine levels to undetectable levels for a period of at least about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours. In certain embodiments, the complex comprises L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, and more specifically, L-asparaginase comprising the sequence of SEQ ID NO: 1. In detailed embodiments, the complex comprises PEG (e.g., mPEG) with a molecular weight of about 5000 Da or less. In more detailed embodiments, at least about 40% to about 100% of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated.

[0060] In one embodiment, the complex has a molPEG / mol monomer ratio of about 4.5 to about 8.5, more particularly about 6.5, a specific activity of about 450 to about 550 U / mg, more particularly about 501 U / mg, and a relative activity of about 75% to about 85%, more particularly about 81%, compared to the corresponding unmodified L-asparaginase. In certain embodiments, the complex having these properties comprises an L-asparaginase derived from the Erwinia species, more particularly from Erwinia chrysanthemi, more particularly an L-asparaginase containing the sequence of SEQ ID NO: 1, wherein about 40-55% of the accessible amino groups (e.g., lysine residues and / or the N-terminus) are PEGylated with 5000 Da mPEG.

[0061] In one embodiment, the complex has a molPEG / mol monomer ratio of about 12.0 to about 18.0, more specifically about 15.1, a specific activity of about 450 to about 550 U / mg, more specifically about 483 U / mg, and a relative activity of about 75% to about 85%, more specifically about 78%, compared to the corresponding unmodified L-asparaginase. In a particular embodiment, the complex having these properties comprises an L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, more specifically an L-asparaginase containing the sequence of SEQ ID NO: 1, wherein about 100% of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated with 5000 Da mPEG.

[0062] In one embodiment, the complex has a molPEG / mol monomer ratio of about 5.0 to about 9.0, more specifically about 7.0, a specific activity of about 450 to about 550 U / mg, more specifically about 501 U / mg, and a relative activity of about 80 to about 90%, more specifically about 87%, compared to the corresponding unmodified L-asparaginase. In a particular embodiment, the complex having these properties comprises an L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, more specifically an L-asparaginase containing the sequence of SEQ ID NO: 1, wherein about 40 to 55% of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated with 10,000 Da mPEG.

[0063] In one embodiment, the complex has a molPEG / mol monomer ratio of about 11.0 to about 17.0, more specifically about 14.1, a specific activity of about 450 to about 550 U / mg, more specifically about 541 U / mg, and a relative activity of about 80 to about 90%, more specifically about 87%, compared to the corresponding unmodified L-asparaginase. In a particular embodiment, the complex having these properties comprises an L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, more specifically an L-asparaginase containing the sequence of SEQ ID NO: 1, wherein about 100% of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated with 10,000 Da mPEG.

[0064] In one embodiment, the complex has a molPEG / mol monomer ratio of about 6.5 to about 10.5, more particularly about 8.5, a specific activity of about 450 to about 550 U / mg, more particularly about 524 U / mg, and a relative activity of about 80 to about 90%, more particularly about 84%, compared to the corresponding unmodified L-asparaginase. In certain embodiments, the complex having these properties comprises an L-asparaginase derived from the Erwinia species, more particularly from Erwinia chrysanthemi, more particularly an L-asparaginase containing the sequence of SEQ ID NO: 1, wherein about 40 to 55% of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated with 2,000 Da mPEG.

[0065] In one embodiment, the complex has a molPEG / mol monomer ratio of about 12.5 to about 18.5, more specifically about 15.5, a specific activity of about 450 to about 550 U / mg, more specifically about 515 U / mg, and a relative activity of about 80 to about 90%, more specifically about 83%, compared to the corresponding unmodified L-asparaginase. In a particular embodiment, the complex having these properties comprises an L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, more specifically an L-asparaginase containing the sequence of SEQ ID NO: 1, wherein about 100% of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated with 2,000 Da mPEG.

[0066] In other embodiments, the complex of the present invention exhibits at least a 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold increase in potency after a single injection compared to the corresponding unmodified L-asparaginase. In certain embodiments, the complex having these properties comprises an L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, and more specifically, an L-asparaginase containing the sequence of SEQ ID NO: 1. In certain embodiments, the complex comprises a PEG (e.g., mPEG) with a molecular weight of about 5000 Da or less. In more specific embodiments, at least about 40% to about 100% of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated.

[0067] In one embodiment, the complex of the present invention, when measured as described in PCT Publication WO2011003886, has the following single-dose pharmacokinetic profile, more specifically, the complex comprises mPEG with a molecular weight of 2000 Da or less, and L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, and more specifically, L-asparaginase containing the sequence of SEQ ID NO: 1. A max :Approx. 150U / L~Approx. 250U / L, T Amax Approximately 4 to 8 hours, or more specifically, about 6 hours. d Amax Approximately 220 hours to 250 hours, more precisely, approximately 238.5 hours (over 0, approximately 90 minutes to 240 hours). AUC: Approximately 12000 to 30000, and t1 / 2: Approximately 50 to 90 hours. In one embodiment, the complex of the present invention has the following single-dose pharmacokinetic profile, more specifically, the complex comprises mPEG with a molecular weight of 5000 Da or less, and L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, and more specifically L-asparaginase containing the sequence of SEQ ID NO: 1 A max:Approx. 18U / L~Approx. 250U / L, T Amax Approximately 1 hour to approximately 50 hours. d Amax Approximately 90 hours to 250 hours, more precisely, approximately 238.5 hours (over 0, approximately 90 minutes to 240 hours). AUC: Approximately 500 to 35000, and t1 / 2: Approximately 30 hours to 120 hours. In one embodiment, the complex of the present invention, with a protein content equivalent to that of peg asparagase, results in a similar level of L-asparagine depletion after a single dose, over a period of time (e.g., 24, 48, or 72 hours). In certain embodiments, the complex comprises an L-asparagase derived from the Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly an L-asparagase containing the sequence of SEQ ID NO: 1. In certain embodiments, the complex comprises a PEG (e.g., mPEG) with a molecular weight of 5000 Da or less. In more specific embodiments, at least about 40% to about 100%, more particularly about 40% to 55% or 100%, of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated.

[0068] In one embodiment, the complex of the present invention has a longer t1 / 2 than pegasparagase administered at an equivalent protein dose. In a particular embodiment, the complex has a t1 / 2 of at least about 50, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, or 65 hours at a dose of about 50 μg / kg (based on protein content). In another particular embodiment, the complex has a t1 / 2 of at least about 30, 32, 34, 36, 37, 38, 39, or 40 hours at a dose of about 10 μg / kg (based on protein content). In yet another particular embodiment, the complex has a t1 / 2 of about 100 to about 15,000 IU / m³ 2 (approximately 1-30 mg protein / m³) 2 The dose range has a t1 / 2 of at least about 100 to about 200 hours.

[0069] In one embodiment, the complex of the present invention has an average AUC that is at least about 2, 3, 4, or 5 times greater than that of pegasparagase at an equivalent protein dose.

[0070] In one embodiment, the conjugate of the present invention does not induce any significant antibody response for a certain period of time after a single dose, for example, for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or more than 12 weeks. In a particular embodiment, the conjugate of the present invention does not induce any significant antibody response for at least 8 weeks. In one example, "does not induce any significant antibody response" means that the subject to whom the conjugate is administered is identified as antibody-negative within the parameter range recognized in the art. Antibody levels can be measured by methods known in the art, such as ELISA or surface plasmon resonance (SPR-Biacore) assays (Zalewska-Szewczyk (2009) Clin. Exp. Med. 9, 113-116; Avramis (2009) AntiCancer Research 29, 299-302, which are respectively incorporated herein by whole reference). The complexes of the present invention may possess these properties in any combination.

[0071] PAS-modified L-asparaginase Depending on the embodiment, the method of the present invention may include an L-asparaginase complex comprising one or more peptides, each peptide independently of peptide R N -(P / A)-R C In the formula, (P / A) is an amino acid sequence consisting only of proline and alanine amino acid residues, and in the formula, R N R is a protecting group bonded to the N-terminal amino group of an amino acid sequence, and in the formula, C R is an amino acid residue that is attached to the C-terminal carboxyl group of the amino acid sequence via its own amino group, and each peptide is the C-terminal amino acid residue R of the peptide. CThe peptide forms a complex with L-asparaginase via an amide bond formed from the carboxyl group of the peptide and the free amino group of L-asparaginase, and at least one of the free amino groups to which the peptide is bound is not the N-terminal α-amino group of L-asparaginase. These molecules are also known as PAS derivatives of L-asparaginase and are referred to herein as complexes.

[0072] The monomers of modified L-asparaginase proteins have approximately 350, 400, 450, 500 amino acids to approximately 550, 600, 650, 700, or 750 amino acids after modification. In an additional embodiment, the modified L-asparaginase proteins have approximately 350 to approximately 750 amino acids, or approximately 500 to approximately 750 amino acids.

[0073] Each peptide contained in the modified L-asparaginase protein as described herein is independently peptide R N -(P / A)-R C Therefore, with respect to each peptide contained in the modified L-asparaginase protein as described herein, the N-terminal protecting group R N , amino acid sequence (P / A), and C-terminal amino acid residue R C Each of these is independently selected from its own meaning. Therefore, two or more peptides contained in a modified L-asparaginase protein may be the same or different from one another. In one embodiment, all peptides contained in a modified L-asparaginase protein are the same.

[0074] The chemically bonded portion (P / A) in the modified L-asparaginase protein is peptide R N -(P / A)-R CAlthough included in this, this portion is an amino acid sequence that can consist of a total of 10 to 100 or more proline and alanine amino acid residues, a total of 15 to 60 proline and alanine amino acid residues, a total of 15 to 45 proline and alanine amino acid residues, for example, a total of 20 to about 40 proline and alanine amino acid residues, for example, 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, or 45 proline and alanine amino acid residues. In a preferred embodiment, the above amino acid sequence consists of about 20 proline and alanine amino acid residues. In another preferred embodiment, the above amino acid sequence consists of about 40 proline and alanine amino acid residues. Peptide R N -(P / A)-R C In this case, the ratio of the number of proline residues in (P / A) to the number of amino acid residues in the portion (P / A) is preferably ≥10% and ≤70%, more preferably ≥20% and ≤50%, and even more preferably ≥25% and ≤40%. Therefore, it is preferable that 10% to 70% of the total number of amino acid residues in (P / A) are proline residues, more preferably 20% to 50% of the total number of amino acid residues in (P / A) are proline residues, and even more preferably 25% to 40% (e.g., 25%, 30%, 35%, or 40%) of the total number of amino acid residues in (P / A) are proline residues. Furthermore, it is preferable that (P / A) does not contain any consecutive proline residues (i.e., (P / A) does not contain any sub-sequence PP). In a preferred embodiment, (P / A) is the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 2). In another preferred embodiment, (P / A) is the amino acid sequence AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 3).

[0075] Peptide R N -(P / A)-R C The base R NThis may be a protecting group attached to the N-terminal amino group of the amino acid sequence (P / A), more specifically, to the N-terminal α-amino group. N It is preferable that this is pyroglutamoyl or acetyl.

[0076] Peptide R N -(P / A)-R C The base R C R is an amino acid residue bonded to the C-terminal carboxyl group of (P / A) via its own amino group, and this group has at least two carbon atoms between its own amino group and its own carboxyl group. Naturally, C At least two carbon atoms between the amino group and the carboxyl group are R C This can provide a gap of at least two carbon atoms between the amino group and the carboxyl group (for example, R C ω-amino-C 3-15 This is the case with alkanates, for example, ε-aminohexanoic acid. C It is preferably ε-aminohexanoic acid.

[0077]

[0001] In one embodiment, the peptide is Pga-AAPAAPAPAAPAAPAPAPAAPA-Ahx-COOH (SEQ ID NO: 4) or Pga-AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQ ID NO: 5). The term "Pga" is an abbreviation for "pyroglutamoyl" or "pyroglutamic acid". The term "Ahx" is an abbreviation for "ε-aminohexanoic acid".

[0078] In the modified L-asparaginase protein as described herein, each peptide R N -(P / A)-R C This is the C-terminal amino acid residue R of the peptide. CIt can form a complex with L-asparaginase via an amide bond formed from the carboxyl group of L-asparaginase and the free amino group of L-asparaginase. The free amino group of L-asparaginase can be, for example, the N-terminal α-amino group or a side-chain amino group of L-asparaginase (e.g., the ε-amino group of a lysine residue contained in L-asparaginase). If L-asparaginase is composed of multiple subunits, for example, if L-asparaginase is a tetramer, there may be multiple N-terminal α-amino groups (i.e., one in each subunit). In one embodiment, peptides as defined herein in sections 9-13 (e.g., peptides 9, 11, 12, or 13) can be chemically bonded to L-asparaginase (e.g., to each subunit / monomer of L-asparaginase).

[0079] Accordingly, in one embodiment, at least one of the free amino groups to which the peptide is chemically bound is not the N-terminal α-amino group of L-asparaginase (i.e., it is different from the N-terminal α-amino group). Therefore, it is preferable that at least one of the free amino groups to which the peptide is bound is a side-chain amino group of L-asparaginase, and it is particularly preferable that at least one of the free amino groups to which the peptide is bound is an ε-amino group of a lysine residue of L-asparaginase.

[0080] Furthermore, it is preferable that the free amino groups to which the peptide binds are selected from the ε-amino groups of any lysine residue(s) of L-asparaginase, the N-terminal α-amino groups of L-asparaginase or any subunit(s) of L-asparaginase, and any combination thereof. It is particularly preferable that one of the free amino groups to which the peptide binds is an N-terminal α-amino group, and the other one(s) of the free amino groups to which the peptide binds is an ε-amino group of a lysine residue of L-asparaginase. Alternatively, it is preferable that the free amino groups to which the peptide binds are each ε-amino groups of a lysine residue of L-asparaginase.

[0081] A modified L-asparaginase protein as described herein consists of L-asparaginase and one or more peptides as defined herein. A corresponding modified L-asparaginase protein may consist, for example, of one L-asparaginase and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 (or more) peptides, each peptide bound to L-asparaginase. L-asparaginase may be, for example, a monomeric protein or a protein composed of multiple subunits, such as a tetramer. If L-asparaginase is a monomeric protein, the corresponding modified L-asparaginase protein may consist, for example, of one monomeric L-asparaginase and 9 to 13 (or more) peptides (e.g., 8, 9, 10, 11, 12, or 13), each peptide bound to a monomeric L-asparaginase. An example of the amino acid sequence of monomeric L-asparaginase is shown in Sequence ID No. 1. If L-asparaginase is a protein composed of multiple subunits, for example, four subunits (i.e., the above L-asparaginase is a tetramer), the corresponding modified L-asparaginase protein may consist, for example, of four L-asparaginase subunits and 9 to 13 (or more) peptides (e.g., 9, 10, 11, 12, or 13) as defined above, each peptide bound to a subunit of L-asparaginase. An example of the amino acid sequence of a subunit of L-asparaginase is shown in Sequence ID No. 1. Similarly, if L-asparaginase is a protein composed of multiple subunits, for example four subunits (i.e., if the above L-asparaginase is a tetramer), the corresponding modified L-asparaginase protein may consist, for example, of four L-asparaginase subunits and 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 (or more) peptides, each peptide bound to the L-asparaginase tetramer.In one embodiment, the present invention relates to L-asparaginase and modified L-asparaginase proteins having a plurality of chemically bonded peptide sequences. In a further embodiment, the length of the peptide sequences is about 10 to about 100, about 15 to about 60, or about 20 to about 40.

[0082] Peptides consisting solely of proline and alanine amino acid residues can covalently bind to one or more amino acids of the L-asparaginase, such as lysine residues and / or N-terminal residues, and / or peptides consisting solely of proline and alanine amino acid residues may covalently bind to at least about 40, 50, 60, 70, 80, or 90% to about 60, 70, 80, 90, or 100% of the accessible amino groups, including the amino groups of the lysine residues and / or N-terminal residues, on the surface of the L-asparaginase. For example, there are about 11 to 12 accessible lysine residues per L-asparaginase, and about 8 to 12 lysine residues may bind to peptides consisting solely of proline and alanine amino acid residues. In a further embodiment, a peptide consisting solely of proline and alanine amino acid residues is covalently bonded to approximately 20, 30, 40, 50, or 60% to approximately 30, 40, 50, 60, 70, 80, or 90% of the total lysine residues of the L-asparaginase. In a further embodiment, a peptide consisting solely of proline and alanine amino acid residues is covalently bonded to the L-asparaginase via a linker. An example of a linker is disclosed in U.S. Patent Application No. 2015 / 0037359, which is incorporated herein by reference in its entirety.

[0083] In one embodiment, the complex is a fusion protein comprising L-asparaginase and a polypeptide consisting solely of proline and alanine amino acid residues having a length of about 200 to about 400. In other words, the polypeptide may consist of about 200 to about 400 proline and alanine amino acid residues. In one embodiment, the polypeptide consists of a total of about 200 (e.g., 201) proline and alanine amino acid residues (i.e., having a length of about 200 (e.g., 201) proline and alanine amino acid residues), or the polypeptide consists of a total of about 400 (e.g., 401) proline and alanine amino acid residues (i.e., having a length of about 400 (e.g., 401) proline and alanine amino acid residues). In some preferred embodiments, the polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 6 or 7. In some embodiments, each monomer of the fusion protein has about 350, 400, 450, 500 amino acids to about 550, 600, 650, 700, 750, or 1,000 amino acids, including the monomer and P / A amino acid sequences. In further embodiments, the modified protein has about 350 to about 800 amino acids or about 500 to about 750 amino acids. For example, the polypeptide includes the peptide prepared in U.S. Patent No. 9,221,882. In some embodiments, the L-asparaginase is derived from the species Erwinia, more particularly from Erwinia chrysanthemi, and more specifically, is an L-asparaginase comprising the sequence of Sequence ID No. 1 as described herein.

[0084] In a further embodiment, the L-asparaginase disclosed herein can be produced using a (recombinant) vector comprising a nucleotide sequence encoding a modified L-asparaginase protein comprising L-asparaginase and a polypeptide, wherein the polypeptide consists only of proline and alanine amino acid residues, and preferably the modified protein is a fusion protein as described herein, and the vector can express the modified protein (e.g., a fusion protein). In a further embodiment, the present invention also relates to a host comprising the (recombinant) vector described herein. Possible hosts include bacteria, actinomycetes, fungi, algae, and other microorganisms, such as yeasts, e.g., Saccharomyces cerevisiae and Pichia Pistoris, as well as yeasts, e.g., Saccharomyces cerevisiae and Pichia Pistoris, e.g., bacteria, actinomycetes, fungi, algae, and other microorganisms, e.g., Escherichia coli, Bacillus species, Pseudomonas fluorescens, Corynebacterium glutamicum, and bacterial hosts of the following genera: Serratia, Proteus, Acinetobacter, and Alcaligenes. Other hosts are also known to those skilled in the art, including Nocardiopsis alba expressing an asparaginase mutant lacking glutaminase activity, and those disclosed in Savitri et al. (2003) Indian Journal of Biotechnology, 2, 184-194, which are incorporated herein by reference in their entirety.

[0085] Treatment methods and usage The complex of the present invention can be used to treat diseases treatable by asparagine and / or glutamine depletion. For example, the complex is useful in treating acute lymphoblastic leukemia (ALL) in both adults and children, as well as in the manufacture of pharmaceuticals for the treatment of other conditions in which asparagine and / or glutamine depletion is expected to have a beneficial effect. Such conditions include, but are not limited to, malignant tumors or cancers, such as hematological malignancies, lymphomas, large immunoblastic lymphomas, non-Hodgkin lymphomas, diffuse large B-cell lymphomas, NK lymphomas, Hodgkin's disease, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute T-cell leukemia, acute myeloid leukemia (AML), double phenotype B-cell myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanoma. Depending on the embodiment, the disease may be acute myeloid leukemia or diffuse large B-cell lymphoma. As a malignant tumor or cancer, renal cell carcinoma. Tumor , renal cell adenocarcinoma, glioblastoma including glioblastoma multiforme and astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma Tumor , squamous cell carcinoma Tumor , lung large cell carcinoma Tumor and small cell lung cancer Tumor Lung cancer including Tumor , endometrial cancer Tumor Ovarian adenocarcinoma, ovarian malformation carcinoma, cervical adenocarcinoma, breast cancer Tumor , breast cancer, ductal carcinoma Tumor Pancreatic adenocarcinoma, pancreatic ductal carcinoma Tumor , colon cancer Tumor , colon adenocarcinoma, colorectal adenocarcinoma, bladder transitional cell carcinoma Tumor Bladder papilloma, prostate cancer Tumor Osteosarcoma, epithelioid carcinoma of bone Tumor prostate cancer Tumor This includes, but is not limited to, thyroid cancer.

[0086] Representative non-malignant hematological disorders that respond to asparagine and / or glutamine depletion include immune-mediated hematological disorders, such as infections caused by HIV infection (i.e., AIDS). Non-hematological disorders associated with asparagine and / or glutamine dependence include autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and collagen vascular diseases. Other autoimmune diseases include osteoarthritis, Isaacs syndrome, psoriasis, insulin-dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), primary biliary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease. Cells suspected of causing the disease can be tested for asparagine and / or glutamine dependence in any suitable in vitro or in vivo assay, for example, in an in vitro assay in which the growth medium does not contain asparagine and / or glutamine. That is, in one embodiment, the present invention relates to a method for treating a treatable disease in a patient, the method comprising administering to the patient an effective amount of the complex of the present invention. In another embodiment, the complex of the invention is administered co-administered with another active pharmaceutical ingredient. In some embodiments, the complex of the invention is administered co-administered with Oncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactricib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839. In a particular embodiment, the disease is ALL. In certain embodiments, a complex used to treat diseases treatable by asparagine and / or glutamine depletion comprises an L-asparaginase derived from the Erwinia species, more particularly from Erwinia chrysanthemi, and more specifically an L-asparaginase comprising the sequence of SEQ ID NO: 1 as described herein.

[0087] In one embodiment, treatment with the complex of the invention would be carried out as a first-line treatment. In another embodiment, treatment with the complex of the invention would be carried out as a second-line treatment in patients who have developed objective signs of hypersensitivity, including allergy or “asymptomatic hypersensitivity,” to other asparaginase preparations, particularly to unmodified Escherichia-coli-derived L-asparaginase or its PEGylated variant (peguasparagase), specifically in patients with ALL. A non-limiting example of objective signs of allergy or hypersensitivity is being “antibody-positive” in tests for the asparaginase enzyme. In a particular embodiment, the complex of the invention would be used as a second-line treatment after treatment with pegasparagase. In a more specific embodiment, the complex used as a second-line treatment would contain L-asparaginase derived from the Erwinia species, more specifically from Erwinia chrysanthemi, and more specifically, L-asparaginase containing the sequence of SEQ ID NO: 1. In a more specific embodiment, the complex further comprises PEG (e.g., mPEG) with a molecular weight of about 5000 Da or less, more specifically about 5000 Da. In an even more specific embodiment, at least about 40% to about 100%, more specifically about 40% to 55% or 100%, of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated.

[0088] In another embodiment, the present invention relates to a treatment for acute lymphoblastic leukemia, comprising administering a therapeutically effective amount of the complex of the present invention to a patient in need of treatment. In another embodiment, the present invention relates to a treatment for acute myeloid leukemia, comprising administering a therapeutically effective amount of the complex of the present invention to a patient in need of treatment in combination with daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactricib, bortezomib, and / or carfilzomib. In another embodiment, the present invention relates to a treatment for acute myeloid leukemia, comprising administering a therapeutically effective amount of the complex of the present invention to a patient in need of treatment in combination with venetoclax. In another embodiment, the present invention relates to a treatment for diffuse large B-cell lymphoma, comprising administering a therapeutically effective amount of the complex of the present invention to a patient in need of treatment in combination with ABT-737, venetoclax, carfilzomib, vincristine, and / or prednisolone. In another embodiment, the present invention relates to a treatment for diffuse large B-cell lymphoma, comprising administering a therapeutically effective amount of the complex of the present invention to a patient in need of treatment in combination with vincristine.

[0089] In another embodiment, the composite described herein has a concentration of approximately 1500 IU / m³ 2 ~Approx. 15,000IU / m 2 Typically, approximately 10,000 to 15,000 IU / m³ 2 (Approximately 20-30 mg protein / m³) 2The complex of the present invention will be administered in doses ranging from approximately twice a week to approximately once a month, typically on a schedule ranging from once a week to once every other week, either as a monotherapy (e.g., monotherapy) or as part of a combination of chemotherapy agents, including but not limited to glucocorticoids, corticosteroids, anticancer compounds, or other agonists, such as methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracyclines. For example, a patient with ALL will be administered the complex of the present invention as a component of a multi-agent chemotherapy regimen during a chemotherapy period including induction therapy, consolidation or strengthening therapy, and maintenance therapy. In specific examples, the complex will not be administered with an asparagine synthetase inhibitor (e.g., as described in U.S. Patent No. 9,920,311, which is incorporated herein by reference in its entirety). In another specific example, the complex will not be administered with an asparagine synthetase inhibitor but will be administered with other chemotherapy agents. The conjugate can be administered as part of a multi-agent chemotherapy regimen, either before, after, or concurrently with other compounds.

[0090] In certain embodiments, the method involves administering the complex of the present invention in amounts ranging from about 1 U / kg to about 25 U / kg (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 U / kg) or equivalent amounts 20 (e.g., based on protein content). In more specific embodiments, the complex is administered in amounts selected from the group consisting of about 5, about 10, and about 25 U / kg. In another specific embodiment, the complex is administered in amounts of about 1,000 IU / m³ 2 ~Approx. 20,000IU / m 2 (For example, 1,000 IU / m 2 , 2,000 IU / m 2 3,000 IU / m 2 , 4,000 IU / m 2 5,000 IU / m 2 6,000 IU / m 2 7,000 IU / m 28,000 IU / m 2 9,000 IU / m 2 , 10,000 IU / m 2 , 11,000 IU / m 2 , 12,000 IU / m 2 , 13,000 IU / m 2 , 14,000 IU / m 2 , 15,000 IU / m 2 , 16,000 IU / m 2 , 17,000 IU / m 2 , 18,000 IU / m 2 , 19,000 IU / m 2 , or 20,000 IU / m 2 ) are administered in doses within the range of ). In another specific embodiment, the complex is administered in single doses for a period of about 3 to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) in doses that reduce L-asparagine and / or glutamine to undetectable levels using methods and apparatus known in the art.

[0091] In another embodiment, the method includes administering the complex of the present invention to a patient that induces a lower immunogenic response than uncomplexed L-asparaginase. In another embodiment, the method includes administering the complex of the present invention having a longer post-single-dose in vivo circulating half-life than uncomplexed L-asparaginase. In one embodiment, the method includes administering the complex having a longer t1 / 2 than pegasparagase administered at an equivalent protein dose. In a particular embodiment, the method includes administering the complex having a t1 / 2 of at least about 50, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, or 65 hours at a dose of about 50 μg / kg (based on protein content). In another specific embodiment, the method includes administering the complex having a t1 / 2 of at least about 30, 32, 34, 36, 37, 37, 39, or 40 hours at a dose of about 10 μg / kg (based on protein content). In another specific embodiment, the method produces approximately 10,000 to approximately 15,000 IU / IU / m³ 2 (Approximately 20-30 mg protein / IU / m³)2 The method includes administering a complex having a t1 / 2 of at least about 100 to about 200 hours at a dose in the range of ). In one embodiment, the method includes administering a complex having an average AUC at least about 2, 3, 4, or 5 times greater than pegasparagase administered at an equivalent protein dose.

[0092] The relapse rate in ALL patients after treatment with L-asparaginase remains high, with early relapses occurring in approximately 10–25% of pediatric ALL patients (e.g., sometimes during the maintenance period 30–36 months after induction) (Avramis (2005) Clin. Pharmacokinet. 44, 367–393). If relapse occurs in patients treated with E. coli-derived L-asparaginase, subsequent treatment with E. coli preparations may result in a "vaccination" effect, thereby increasing the immunogenicity of E. coli preparations during subsequent administrations. In one embodiment, the complex of the present invention can be used to treat patients with relapsing ALL who have been previously treated with other asparaginase preparations, specifically with E. coli-derived asparaginase.

[0093] Depending on the embodiment, the therapeutic use and methods of the present invention may involve administering an L-asparaginase complex having the properties or combinations of properties described above in this specification (for example, in the chapter titled L-asparaginase PEG complex or PAS-modified L-asparaginase) or below in this specification.

[0094] Compositions, formulations, and routes of administration The present invention also includes pharmaceutical compositions comprising the complex of the present invention. In certain embodiments, the pharmaceutical composition is contained in a vial as a lyophilized powder intended to be reconstituted in a solvent, regardless of what the bacterial raw material used in its manufacture was, such as currently available natural L-asparaginase (Kidrolase®, Elspar®, Erwinase®). In another embodiment, the pharmaceutical composition may further include, as a “ready-to-use” liquid, pegasparagase (Oncaspar®), which allows for appropriate handling and administration, for example, via intramuscular, intravenous (infusion and / or bolus), intraventricular (icv), or subcutaneous routes. In further embodiments, the pharmaceutical composition comprises the complex of the present invention in combination with Oncaspar®, daunorubicin, cytarabine, ABT-737, venetoclax, dactricib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839.

[0095] The complex of the present invention, including compositions containing the complex (e.g., pharmaceutical compositions), can be administered to patients using standard techniques. The techniques and formulations can be found in Remington's Pharmaceutical Sciences (2013) 22nd ed., Mack Publishing, which are incorporated herein by reference.

[0096] The appropriate dosage form depends, in part, on the method of use or route of delivery, such as orally, transdermally, transmucosally, or by injection (parenteral). Such a dosage form must allow the therapeutic agent to reach the target cells and, in any case, to have the desired therapeutic effect. For example, a pharmaceutical composition injected into the bloodstream is preferably soluble.

[0097] The complexes and / or pharmaceutical compositions according to the present invention can be formulated as pharmaceutically acceptable salts and complexes. A pharmaceutically acceptable salt is a salt that exists in a non-toxic manner at the dose and concentration in which it is administered. Formulations of such salts can facilitate the medical use of a compound by modifying its physical properties without preventing the compound from exerting its physiological effects. Useful modifications of physical properties include promoting transmucosal administration by lowering the melting point, and promoting administration by increasing the drug concentration through increased solubility. Pharmaceutically acceptable salts of asparaginase can also exist as complexes, as is recognized in the art.

[0098] Pharmaceutically acceptable salts include acid addition salts, such as those containing sulfate ions, hydrochloride ions, fumarate ions, maleate ions, phosphate ions, sulfamate ions, acetate ions, citrate ions, lactate ions, tartrate ions, methanesulfonate ions, ethanesulfonate ions, benzenesulfonate ions, p-toluenesulfonate ions, cyclohexylsulfamate ions, and quinic acid ions. Pharmaceutically acceptable salts can be obtained from acids, and examples of acids include hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumarate, and quinic acid.

[0099] In the presence of acidic functional groups, such as carboxylic acids or phenols, pharmaceutically acceptable salts may include base addition salts, such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc. See, for example, Remington's Pharmaceutical Sciences (previously mentioned). Such salts can be prepared using appropriate bases.

[0100] Pharmaceutically acceptable carriers and / or excipients can also be incorporated into the pharmaceutical compositions according to the present invention to facilitate the administration of specific asparaginases. Examples of carriers suitable for use in carrying out the present invention include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or various starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and physiologically compatible solvents. Examples of physiologically compatible solvents include water for injection (WFI), physiological saline, and sterile glucose solutions.

[0101] The pharmaceutical composition according to the present invention can be administered by various routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration. For systemic administration, oral administration is preferred. For oral administration, for example, the compound can be incorporated into conventional oral dosage forms, such as capsules, tablets, and liquid formulations, such as syrups, elixirs, and concentrated drops.

[0102] Alternatively, injection (parenteral administration), such as intramuscular, intravenous, intraperitoneal, and subcutaneous injections, may be used. In the case of injection, the pharmaceutical composition is formulated in a liquid form, preferably in a physiologically compatible buffer or solution, such as saline, Hanks' solution, or Ringer's solution. The compound may also be formulated in solid form and redissolved or suspended immediately before use. For example, a lyophilized complex can be produced. In a specific embodiment, the complex is administered intramuscularly. In another specific embodiment, the complex is administered intravenously.

[0103] Systemic administration can also be achieved by transmucosal or transdermal means. In the case of transmucosal or transdermal administration, a suitable penetration enhancer is used in the formulation for the barrier to be permeated. Such penetration enhancers are well known in the art and include, for example, bile salts and fusidic acid derivatives in the case of transmucosal administration. Also, surfactants may be used to facilitate penetration. Transmucosal administration can be, for example, via nasal spray, inhaler (for delivery to the lungs), rectal suppository, or vaginal suppository. In the case of topical administration, the compound can be formulated into an ointment, salve, gel, or cream as is well known in the art.

[0104] The amount of the complex to be delivered depends on many factors such as IC 50 、EC 50 、the biological half-life of the compound, the age, size, weight, and physical condition of the patient, and the disease or disorder to be treated, etc. The importance of these and other factors to be considered is well known to those skilled in the art. Generally, the amount of the complex to be administered is in the range of about 10 international units per square meter of the patient's body surface area (IU / m 2 ) to 50,000 IU / m 2 , and the dosage range is preferably about 1,000 IU / m 2 to about 15,000 IU / m 2 , more preferably in the range of about 6,000 IU / m 2 to about 15,000 IU / m 2 , and the range of about 10,000 to about 15,000 IU / m 2 (about 20 - 30 mg protein / m 2 ) is particularly suitable for treating malignant blood diseases such as leukemia. Typically, these dosages are administered via intramuscular or intravenous injection at intervals of 3 times a week to about once a month during the treatment process, typically once a week or once every other week. Of course, other dosages and / or treatment regimens can be employed as determined by the attending physician.

[0105] The present invention is further illustrated by the following additional examples, which should not be considered limiting. Those skilled in the art will see that, in light of this disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or equivalent results can still be obtained. [Examples]

[0106] The subject matter of U.S. Patent No. 9,920,311, including the examples disclosing methods for producing and testing PEGylated asparaginase, is incorporated herein by reference. The mPEG-r-chrysanthase complex used in the following examples was prepared as described in U.S. Patent No. 9,920,311.

[0107] Example 1 The mPEG-r-chrysanthase complex (Peg chrysanthase) was tested in various cell lines in two steps, as shown below.

[0108] Cell preparation. All cell lines were licensed from the American Cell Culture and Cell Line Preservation Center (ATCC) in Manassas, Virginia (US). Master cell banks and working cell banks (MCB and WCB) were prepared by subculturing and freezing cells in ATCC-recommended medium according to the ATCC-recommended protocol (www.atcc.org).

[0109] Compound preparation. The test compounds were prepared as stock solutions using DMSO or aqueous buffer as appropriate, and then sequentially diluted to obtain a dilution series.

[0110] Cell proliferation assay. Cell proliferation was evaluated using a commercially available fluorescence assay with ATP as the endpoint.

[0111] Control. Signal at t = 0. 45 μl of cells were dispensed onto parallel plates and incubated at 37 °C in a humidified atmosphere of 5% CO2. After 24 hours, 5 μl of DMSO-containing Hepes buffer and 25 μl of ATPlite 1Step™ solution were mixed, and fluorescence was measured after a 10-minute incubation (= fluorescence at t = 0).

[0112] Reference compound. The IC of the reference compound doxorubicin 50 is measured in a separate plate. For IC 50 , there is a tendency. If IC 50 is out of specification (a deviation of 0.32 - 3.16 times from the past average), the assay is invalid.

[0113] Cell growth control. The doubling time of cells for all cell lines is calculated from the growth signals of untreated cells at t = 0 hours and t = end. If the doubling time is out of specification (a deviation of 0.5 - 2.0 times from the past average), the assay is invalid.

[0114] Maximum signal. For each cell line, the maximum fluorescence was recorded at t = end after incubation without compound in the presence of 0.4% DMSO (= fluorescence 未処置,t=end ).

[0115] Drug sensitivity. Between the "modified" and "wild-type" groups of cell lines 10 logIC 50The differences were analyzed in three ways. First, the drug sensitivity of individual cell lines for the top 18 most frequent gene alterations was visualized using a waterfall plot. Second, a larger subset (38 in total) of the most commonly occurring and well-understood oncogenes was analyzed using Type II Anova analysis in the statistical program R. The results are displayed in a volcano plot. Third, a complete set of 114 oncogenes was analyzed using a two-tailed equal-variance t-test in R. The p-values ​​from the Anova and t-tests were subjected to the Benjamini-Hochberg multiple comparison correction method. Only gene associations with a false-find rate of less than 20% were considered significant. The Type II Anova analysis for the 38 oncogenes is a different test from the equal-variance t-test for the 114 oncogenes, which means that the significance of the association may differ. For further information on the Oncolines™ Act, see: www.ntrc.nl / services / oncolinestm.

[0116] I C 50 This was calculated using nonlinear regression with IDBS XLfit. The growth percentage (%-growth) from incubation to t=end was calculated as follows: 100% × (fluorescence) t=end / fluorescence 未処置,t=end ). This can be expressed using a four-coefficient logistic curve. 10 The logarithmic compound concentration (conc) was applied as follows: %-growth = minimum value + (maximum value - minimum value) / (1 + 10 (logIC50-conc)*hill) In the formula, hill is the Hill coefficient, and the minimum and maximum values ​​are the asymptotic minimum and maximum cell proliferation values ​​that the compound allows in that assay. [Table 2]

[0117] NCI60 parameters. LD 50 That is, the concentration at which 50% of cells die is fluorescence t=end = 1 / 2 × fluorescence t=0h This is the concentration. GI 50That is, the concentration at which proliferation is inhibited by 50% is the concentration at which cell proliferation is halved. This is the concentration associated with the following signal: ((fluorescence 未処置,t=end -fluorescence t=0 ) / 2) + fluorescence t=0 .

[0118] Curve fitting. The curves automatically calculated by the software were manually adjusted according to the following protocol: If the calculated curve had a minimum value lower than 0, the minimum value of the curve was fixed at 0%. If the software calculated a value lower than -6, the slope was fixed at -6. If the F-test value for fit quality was >1.5, or if the compound was inert (maximum effect <20%), the curve was invalid, and in those cases, the curve was removed from the graph. If the curve exhibited biphasic characteristics, that curve was the strongest IC. 50 The fitting was performed. If, by chance, it appeared to be a technical error, the concentration point was excluded. This is always shown in the dose-dependent graph. If the dose-response curve was fully identified for more than 85%, the maximum effect was calculated as 100% (signal from untreated cells) - minimum value of the curve. The dose-response curve is considered 100% complete when the data point at the highest concentration reaches the minimum value of the curve. If the completeness was less than 85%, the maximum effect was calculated as 100% - mean of minimum signal. If the minimum value of the curve was fixed at 0%, the maximum effect was always calculated as 100% - growth inhibition at the highest concentration.

[0119] Volcano plot. The volcano plot in Figure 8 shows how gene transformation is statistically associated with shifts in compound sensitivity in 38 key genes. 10 LogIC 50 (Assuming measured by IC). The P-value (y-axis of the volcano plot) is IC 50 This indicates the confidence level of genetic association for mutations in specific genes that involve shifts. 50The shift coefficient is shown on the x-axis. The area of ​​the circle is proportional to the number of mutations in the cell panel (each mutation is present at least 3 times). To calculate significance, the p-value is subjected to the Benjamin-Hochberg multiple comparison test correction, and only gene associations with a false-find rate of less than 20% are shown in gray. The relevant cutoff p-value (0.059) is shown by a horizontal line. If there is no significant association, neither the gray circle nor the horizontal line is drawn.

[0120] T-test results. For the 98 validated cancer driver genes, since their variants also occur in patients, the presence of "wild-type" and "mutant" forms of the gene in the cell line indicates a significant IC of the test compound. 50 We tested whether it was related to the shift. 50 The "Shift" column is, 10 LogIC 50 This shows the difference. Negative IC 50 A shift indicates that the compound is stronger in cell lines carrying the "mutant" gene. The "p-value" column shows the results of a two-sided t-test. To calculate significance, the p-values ​​were subjected to the Benjamin-Hochberg multiple comparison correction method. Only gene associations with a false-find rate of less than 20% are highlighted ("adjusted p-value" column). If there is no significant association, there are no gray cells in the table below. [Table 3]

[0121] The special volcano plot in Figure 9 is related to the sensitivity of compounds to the presence of hotspot mutations in cancer. 10 LogIC 50 (Assuming measured by [method]). This provides greater attention to clinically relevant cancer driver mutations in comparison to previous analyses. Hotspot mutations were derived from statistical analysis of mutation repetition patterns and copy number changes in patients through separate studies. The axes and statistical analyses are identical to those in the Volcato plot in Figure 8. The significance cutoff p-level is 0.32.

[0122] Example 3: Synergistic activity of Peg chrysanthanspase and Oncaspar®. Effect 20 In the SynergyScreen™ experiment, a low, fixed concentration equivalent to a 20% inhibition of cell proliferation is used to identify the effect of this compound on the activity of other anticancer drugs. This concentration is determined using the dose-response curve of the single compound. The concentration is on the x-axis and corresponds to the 80% survival rate in the untreated group on the y-axis. [Table 4]

[0123] mPEG-r-crisanthase complex (PEG crisanthase, see the first table below) or Oncaspar® (see the second table below) were tested with other agonists typically used in the standard of care (SOC) for AML or DLBCL. Increased efficacy was observed in AML when used in combination with daunorubicin, cytarabine, ABT-737, venetoclax, dactricib, bortezomib, and carfilzomib. Furthermore, increased efficacy was observed in DLBCL when used in combination with vincristine, prednisolone, ABT-737, venetoclax, everolimus, dactricib, bortezomib, carfilzomib, and CB-839. See the table below. Gray highlighting indicates synergistic activity. Light gray highlighting indicates one experiment, and dark gray highlighting indicates two experiments. [Table 5]

[0124] Example 3: The mPEG-r-chrysanthase complex (Peg chrysanthase) was tested in vivo with cytarabine and daunorubicin. Each group of five mice received either mPEG-r-chrysanthase (PegC) as monotherapy (5 & 50 IU / kg), or in combination with the state-of-care drug cytarabine (50 mg / kg once daily for 5 days, followed by a 2-day rest period, repeated twice) and daunorubicin (1 mg / kg weekly for 2 weeks). These doses were well-tolerated. See Figure 1. Group 1 was the PBS control group, Group 3 was the PegC group, Group 11 was the daunorubicin + PegC group, and Group 13 was the daunorubicin group. A decrease of approximately 10% in mean relative body weight was attributed to daunorubicin.

[0125] Example 4: This example was carried out in the same manner as Example 1, except that the mPEG-r-chrysanthase complex (Peg chrysanthase) was tested in combination with other compounds. Figure 2 shows that Peg chrysanthase enhances the effects of cytarabine, venetoclax, and ABT-737, exhibiting a synergistic effect.

[0126] Example 5: The mPEG-r-chrysanthase complex (Peg chrysanthase) was tested in combination with ABT-737 on the HL-60 cell line.

[0127] Plate preparation. The stock solutions of the mixture and individual compounds were diluted with DMSO or 0.9% sodium chloride to generate a 7-point dose-response dilution series. After further 31.6-fold dilution with 20 mM sterile Hepes buffer pH 7.4 (reference compound) or medium (PEG chrysanthanspase), 5 μl of PEG chrysanthanspase solution and 5 μl of the reference compound were added in pairs to 40 μl of cells pre-seeded in a 384-well assay plate. The final DMSO concentration during incubation was 0.4% in all wells. The final assay concentrations for the individual compounds were determined by their IC50 levels. 50 10 to 0.01 times (IC 50 The range was 10 times and 0.01 times the equivalent.

[0128] Cell proliferation assay. Assay-prepared storage cells were thawed and diluted in appropriate medium and dispensed into 384-well plates. The cell concentrations ranged from 800 to 3200 cells per well in 45 μl of medium, depending on the cell line used: DB: 800 cells per well; RL: 1000 cells per well; MV-4-11: 1600 cells per well; KG-1, HL-60, and HT: 3200 cells per well. The cell density was pre-optimized for each cell line used. The plate margins were filled with phosphate-buffered saline. Seeded cells were incubated at 37°C in a humidified atmosphere of 5% CO2. After 24 hours, 5 μl of PEG chrysanthase solution and 5 μl of reference compound were added, and the plates were incubated for an additional 72 hours. After 72 hours, the plate was cooled to room temperature in 30 minutes, 25 μl of ATPlite 1Step™ (PerkinElmer) solution was added to each well, and the mixture was then shaken for 2 minutes. After incubation in the dark at room temperature for 5 minutes, fluorescence was recorded using an Envision multimode reader (PerkinElmer).

[0129] Control: Signal at t=0. 40 μl of cells were dispensed in four sets into parallel plates and incubated at 37°C in a humidified atmosphere of 5% CO2. After 24 hours, the plates were cooled to room temperature in 30 minutes. 5 μl of DMSO-containing Hepes buffer, 5 μl of 0.9% sodium chloride medium, and 25 μl of ATPlite 1Step™ solution were added and mixed for 2 minutes. After 10 minutes of incubation, fluorescence was measured in the dark (=fluorescence). t=0 ).

[0130] Cell proliferation control. Cell doubling time for all cell lines is calculated from the t=0 time and t=end proliferation signals of untreated cells. If the doubling time deviates from the specifications (deviation of 0.5 to 2.0 times from the past average), the assay is considered invalid.

[0131] Maximum signal. In each 384-well plate, maximum fluorescence was recorded after 72 hours of incubation in the presence of 0.4% DMSO, without the compound. All equivalent wells (typically 14) were averaged. This average is defined as follows: fluorescence 未処置,t=72h . Dose-response curve. Accurate monotherapy informed consent is necessary for combination therapy analysis. 50 This is necessary. For each monotherapy agent, its dose-response signal was fitted using a 4-coefficient logistic curve with XL-fit 5 (IDBS software): Fluorescence = Minimum value + (Maximum value - Minimum value) / (1 + 10) (log I C 50 -log[cpd])·hill) ) [cpd] is the concentration of the compound tested. hill is the Hill coefficient. The minimum and maximum values ​​are the asymptotic minimum and maximum of the curve. Identifying the Combination Index (CI). CI is one of the most widely used metrics for quantitatively expressing synergistic effects. CI evaluates the concentration required to achieve a fixed effect. A CI less than 1 indicates synergy. A CI less than 0.3 indicates strong synergy. For example, a CI of 0.1 indicates that the combination requires only 1 / 10 times the concentration expected from the single-agent data to achieve the same level of effect. For instance, if a potent compound and a less potent compound are combined with a CI of 0.1, the effective concentration of the potent compound is improved tenfold by the less potent compound.

[0132] CI is defined in relation to a specific percentage of cell viability (V), where V is the signal relative to the unexposed control: V = 100% × fluorescence 処置,t=72h / fluorescence 未処置,t=72h Next, the concentrations required to achieve a specific cell viability percentage V when the two compounds, cpd1 and cpd2, are used in combination are compared to the concentrations required when each compound is used alone: CI (100-V) =[cpd1] V / I C (100-V),cpd1 +[cpd2] V / I C (100-V),cpd2 For example, [cpd1] 50 This represents the CPD1 concentration in the mixture that gives a 50% survival rate. 50,cpd1 This is a standalone IC for cpd1. 50 This represents CI, which, by convention, is labeled with %-effect, so CI 75 This represents the CI at a 25% survival rate. Curve shift analysis. This analysis provides a visual confirmation of synergistic effects. 1 The concentrations of the mixture of compounds 1 and 2 (cpd1 and cpd2), as well as the individual compounds, are measured in IC. 50 In equivalent terms (IC 50 Expressed in units: [mix]=[cpd1] / IC 50,cpd1 +[cpd2] / IC 50,cpd2 The dose-response signals were fitted using a 4-coefficient logistic curve with XL-fit 5 (IDBS software): Fluorescence = Minimum value + (Maximum value - Minimum value) / (1 + 10) (logX-log[mix])·hill) ) In the equation, hill is the Hill coefficient, and X is the inflection point of the curve. The minimum and maximum values ​​are the asymptotic minimum and maximum of the curve. [mix] is IC 50 Because it is expressed in equivalent units, the curves for the single agents will overlap, and their inflection points will be where the value is 1. The IC used in the calculation... 50 The values ​​were measured in parallel for each individual agent.

[0133] In mixtures where no synergistic effect exists, the curve will overlap with that of the single agent. In mixtures where a synergistic effect exists, the curve will overlap with that of the single agent. 50 The equivalent weight will shift to the left towards the lower end: the mixture will appear to be stronger than expected based on its individual components. This is a good indicator of synergy.

[0134] Isobologram. An isobologram is a dose-centered plot that reveals whether a combination of drugs is synergistic. This is defined by a specific efficacy level, usually 75%. If the monotherapy curve does not reach this efficacy level, the isobologram level is set to 50%, 30%, 25%, or 20%. If the monotherapy does not reach 20% efficacy, the isobologram is not drawn. On the axis, the calculated dose of a single compound that gives a predetermined growth effect is plotted. A straight line connects the two points (additive action line). For drug combinations, the concentration of the individual components at the point is plotted on the isobologram, and which dilution gives the predetermined growth effect is calculated. In the case of additive drug effects, the drug combination will be close to the additive action line. In the case of synergistic or antagonistic effects, the points will be below or above the additive action line, respectively.

[0135] Experiment using an inactive agent. In a specific agreed-upon case, a synergistic experiment is performed in the presence of an "inactive" agent. An "inactive" agent is a compound that does not yield a dose-response curve as a single agent at the tested concentration. The experiment is carried out as described above, except that the "inactive" agent is added to each well of the experiment at a fixed concentration. Since the "inactive" agent alone does not show an effect, its contribution to CI is negligible. Therefore, the CI value is based on the reaction of the activating agent. Curve shifts in the mixture are identified by comparison with other activating agents. Isobolograms are not calculated. The dose-response curves for each single agent are shown in Figure 3. ABT-737 has an IC 50 While the IC50 is 835nM and the maximum efficacy is 67%, PEG chrysanthase has an IC50 ratio of 835nM. 50 The concentration was 0.15 nM, and the maximum efficacy was 88%.

[0136] Curve shift analysis: The x-axis of the single-component curves (gray and dark gray) and mixture curves (red, orange, and pink) is the IC of the single-component curve. 50 Based on IC 50 The values ​​were converted to equivalents. These converted values ​​were then compared to the dose-response curve of the mixture shown in Figure 4.

[0137] I C 50 For the dose-response curves of the mixture at the baseline, all curves were superimposed and the shift was recorded. A mixture curve shifted to the left compared to the single-component curves (gray and dark gray) indicates a synergistic effect, while a shift to the right indicates an antagonistic effect (see Figure 5 and the table below). IC of the mixture compared to the single agent 50 shift [Table 6]

[0138] The results of using peg chrysanthanspase and ABT-737 in combination are shown below. CI values ​​and ED calculated from the mixture data. 75 This corresponds to a 25% survival rate. A typical value is the average CI at a 50% survival rate for the three mixtures, which is shown in the summary. [Table 7-1] [Table 7-2]

[0139] Using the combination data, an isobologram was created as shown in Figure 6. An isobologram is a dose-centered plot that reveals whether the combination of drugs is synergistic. In the case of synergy, the combination point lies below the additive line. The concentration of peg chrysanthanaspase is shown in IU / mL. The additive line (dark gray) shows the combination at concentrations that are expected to theoretically produce an additive effect. Combinations of drugs are plotted as red, pink, and orange points. In summary, as shown below, a potent synergistic effect was observed between peg chrysanthanaspase and ABT-737 in the HL-60 cell line. [Table 8]

[0140] Example 6: This example was carried out in the same manner as Example 5, except that the synergistic effect with additional anticancer drugs was tested in different cell types, as shown below. [Table 9]

[0141] Example 7: This example was carried out in the same manner as Example 1, except that the activity of the mPEG-r-chrysanthase complex against CNS cell lines was tested. The CNS cell lines included, for example, glioblastoma, medulloblastoma, glioblastoma multiforme, and astrocytoma. The results are shown in Figure 7. Additional experiments were performed using different cell lines. The results are shown in Figure 10.

[0142] Example 8: Following the method described in Example 1, the mPEG-r-chrysanthase complex (Peg chrysanthase) was tested in combination with additional compounds against AML (acute myeloid leukemia) and DLBCL (diffuse large B-cell lymphoma) cell lines. The results are shown below. KG-1, HL-60, and MV4-11 are AML cell lines, and DB, HT, and RL are DLBCL cell lines. The data for the combination of peg chrysanthase and venetoclax showed a potent synergistic effect in AML cell lines. [Table 10]

[0143] Example 9 Following the method described in Example 1, Pas-modified chrysanthaspase complexes were tested in multiple cell lines, comparing them with PEGylated (PEG-chrysanthaspase) and non-PEGylated (Erwinase) chrysanthaspsases, along with E. coli-derived L-asparaginase (Oncaspar). PA-20 and PA-40 are Pas-modified chrysanthaspase complexes produced in Corynebacterium or Pseudomonas expression systems, and PA-200 is a Pas-modified fusion protein produced in a Pseudomonas expression system. The PA-20, PA-40, PA-200, and PA-400 structures are those of Sequence IDs 2, 3, 6, and 7. The results are shown below. CCRF-CEM, MOLT-4, and RS4:11 are all AML cell lines, Jurkat E6-1 is an acute T-cell leukemia cell line, HL-60 is an acute promyelocytic leukemia cell line, MV4-11 is a dual-phenotype B-cell myelomonocytic leukemia cell line, THP-1 is an AML cell line, RL is a non-Hodgkin lymphoma cell line, and H9 is a lymphoma cell line. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5]

[0144] Those skilled in the art will recognize, or can verify by common practice, many equivalents of the specific embodiments of the present invention described herein. Such equivalents shall be covered by the following claims.

Claims

1. A composition for treating glioblastoma or medulloblastoma in a patient, A complex comprising L-asparaginase protein derived from the genus Erwinia containing SEQ ID NO: 1 and polyethylene glycol (PEG), wherein the molecular weight of the polyethylene glycol is 5000 Da or less, and containing an effective amount of the complex. The glioblastoma or medulloblastoma includes cells having a copy number mutation in the CDKN2A gene. composition.

2. The composition according to claim 1, wherein the L-asparaginase complex exhibits at least 10 times higher activity against glioblastoma or medulloblastoma compared to Escherichia coli asparaginase.

3. A composition for treating acute promyelocytic leukemia in patients, It contains an effective amount of a complex of a protein having L-asparagine aminohydrolase activity and polyethylene glycol (PEG), The polyethylene glycol has a molecular weight of 5000 Da or less. The protein in question is an L-asparaginase derived from the genus Erwinia containing SEQ ID NO:

1. The aforementioned complex is administered as part of combination therapy with ABT-737. composition.

4. A composition for treating diseases treatable by L-asparagine depletion in patients, It contains an effective amount of a complex of a protein having L-asparagine aminohydrolase activity and polyethylene glycol (PEG), The polyethylene glycol has a molecular weight of 5000 Da or less. The protein is an L-asparaginase derived from the genus Erwinia, and has at least 95% sequence identity with the amino acids of SEQ ID NO:

1. The aforementioned disease is a cancer selected from glioblastoma, neuroblastoma, and medulloblastoma. The cancer includes cells having mutations in genes selected from NRAS, PTEN, ERBB2, CDKN2A, or combinations thereof. composition.

5. The composition according to claim 4, wherein the L-asparaginase has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid of SEQ ID NO:

1.

6. The composition according to claim 5, wherein the complex comprises an L-asparaginase derived from the genus Erwinia having 100% sequence identity with the amino acid of SEQ ID NO:

1.

7. The composition according to any one of claims 4 to 6, wherein the PEG has a molecular weight of 5000 Da, 4000 Da, 3000 Da, 2500 Da, or 2000 Da.

8. i) The complex has at least 60% of the in vitro activity compared to the L-asparaginase when it is not complexed with PEG; ii) The complex has at least 10 times stronger L-asparagine depletion activity than the L-asparaginase when it is not complexed with PEG; iii) The complex depletes plasma L-asparagine levels to undetectable levels for at least 12 hours; iv) The complex has a longer in vivo circulating half-life than the L-asparaginase when it is not complexed with PEG; v) The complex has a longer t1 / 2 than pegasparagase administered at an equivalent protein dose; vi) The complex has a t1 / 2 of at least 58 to 65 hours at a dose of 50 μg / kg based on protein content, and a t1 / 2 of at least 34 to 40 hours at a dose of 10 μg / kg based on protein content, after administration iv in mice; vii) The composite has a concentration of 10,000 to 15,000 IU / m³ ２ (20 to 30 mg protein / m²) ２ ) having a dose in the range of t1 / 2 of at least 100 to 200 hours; viiii) The complex has a larger area under the curve (AUC) than the L-asparaginase when it is not complexed with PEG; and / or ix) The complex has an average AUC at an equivalent protein dose that is at least three times greater than that of pegasparagase. The composition according to any one of claims 4 to 7.

9. i) The PEG is covalently bonded to one or more amino groups of the L-asparaginase; ii) The PEG is covalently bonded to one or more amino groups by an amide bond; iii) The PEG is covalently bonded to at least 40% to 100% of the accessible amino groups. iv) The composite is given by the following formula: Asp-[NH-CO-(CH) ２ ) ｘ -CO-NH-PEG] ｎ The formula comprises, where Asp is the L-asparaginase, NH is one or more of the lysine residues and / or N-terminal NH groups of Asp, PEG is a polyethylene glycol moiety, n is the number of polyethylene glycol moieties coupled to Asp, and x is an integer in the range of 1 to 8; v) The composite is given by the following formula: Asp-[NH-CO-(CH) ２ ) ｘ -CO-NH-PEG] ｎ The formula comprises, where Asp is the L-asparaginase, NH is one or more of the lysine residues and / or N-terminal NH groups of Asp, PEG is a polyethylene glycol moiety, n is the number of polyethylene glycol moieties coupled to Asp, and x is an integer in the range of 2 to 5; and / or vi) The PEG is monomethoxy-polyethylene glycol (mPEG), The composition according to any one of claims 4 to 8.

10. The aforementioned complex, i) In amounts ranging from 5 U / kg body weight to 50 U / kg body weight; ii) at a dosage in the range of 10,000 to 15,000 IU / m ２ ; iii) By intravenous administration; iv) By intramuscular administration; and / or v) Once a week, twice a week, or three times a week, A composition according to any one of claims 4 to 9, which is administered.

11. The composition according to any one of claims 4 to 10, wherein the complex is administered as part of monotherapy or combination therapy.

12. The composition according to claim 11, wherein the combination therapy comprises Oncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactricib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839.

13. The composition according to any one of claims 4 to 12, wherein the disease is a cancer comprising cells having mutations in the NRAS gene, the PTEN gene, and / or the ERBB2 gene.

14. The composition according to any one of claims 4 to 13, wherein the disease is a cancer containing cells having a mutation in the CDKN2A gene, and the mutation is a copy number mutation.

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