Therapy 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 conjugates of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol, particularly where the polyethylene glycol has a molecular weight of about 5000 Da or less and particularly where the protein is L-asparaginase from the genus Erwinia, and to uses thereof in therapy. [Background technology]

[0002] Proteins with L-asparagine aminohydrolase activity, commonly known as L-asparaginase, have been used successfully for many years in the treatment of childhood acute lymphoblastic leukemia (ALL), 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 cell sarcoma, and melanosarcoma (Kotzia (2007) J. Biotechnol. 127, 657-669). The antitumor activity of L-asparaginase is likely due to the lack or reduced ability of certain malignant cells to synthesize L-asparagine (Kotzia (2007) J. Biotechnol. 127, 657-669). These malignant cells are dependent on an exogenous 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 perform protein synthesis without L-asparagine (Kotzia (2007) J. Biotechnol. 127, 657-669).

[0004] L-asparaginase from E. coli was the first enzyme drug used to treat ALL and is sold in the United States as Elspar® and in Europe as Kidrolase® and L-asparaginase Medic®. L-asparaginase has also been isolated from other microorganisms; for example, the L-asparaginase protein from Erwinia chrysanthemi has been named crisantaspase and is commercially available 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 Erwinia chrysanthemi 3937 (Genbank Accession No. AAS67028), Erwinia chrysanthemi NCPPB 1125 (Genbank Accession No. CAA31239), Erwinia carotovora (Genbank Accession No. AAP92666), and Erwinia carotovora subsp. astroseptica (Genbank Accession No. AAS67027). These Erwinia chrysanthemi L-asparaginases share about 91-98% amino acid sequence identity with each other, while Erwinia carotovora L-asparaginase shares about 75-77% amino acid sequence identity with Erwinia chrysanthemi L-asparaginase (Kotzia (2007) J. Biotechnol. 127 657-669).

[0005] Currently available L-asparaginase preparations do not offer an alternative or complementary therapy characterized by high catalytic activity and significantly improved pharmacological and pharmacokinetic properties, as well as reduced immunogenicity, particularly for the treatment of ALL.

[0006] In one aspect, the problem to be solved by the present invention is to provide an L-asparaginase formulation that has: high in vitro bioactivity, stable PEG-protein conjugation, long in vivo half-life, greatly reduced immunogenicity, as evidenced, for example, by reduction or elimination of antibody responses to the L-asparaginase formulation after repeated administration, and utility as a second line treatment for patients who have developed sensitivity to first line treatments, e.g., treatments using E. coli derived L-asparaginase.

[0007] This problem has not been solved by known L-asparaginase conjugates, which either have significant cross-reactivity with modified L-asparaginase preparations (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 conjugate of Erwinia sp. 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 conjugate, and the use of the conjugate. Summary of the Invention

[0008] The present invention encompasses a method of treating a disease treatable by L-asparagine depletion in a patient, comprising administering an effective amount of a conjugate 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 an L-asparaginase 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 to the amino acids of SEQ ID NO:1. In some embodiments, the conjugate comprises an L-asparaginase from the genus Erwinia having 100% sequence identity 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 conjugates have an in vitro activity of 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% of that of L-asparaginase not conjugated to PEG. In some embodiments, the conjugates have an L-asparagine depletion activity that is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more potent than L-asparaginase not conjugated to PEG. In some embodiments, the conjugate depletes plasma levels of L-asparagine to undetectable levels for at least about 12, 24, 48, 96, 108, or 120 hours. In some embodiments, the conjugate has a longer in vivo circulatory half-life than L-asparaginase not conjugated to PEG. In some embodiments, the conjugate has a longer t1 / 2 than pegaspargase administered at an equivalent protein dose.In some embodiments, the conjugate has a t of at least about 58 to about 65 hours at a dose of about 50 μg / kg of protein content, and at least about 34 to about 40 hours at a dose of about 10 μg / kg of protein content, after iv administration to mice. In some embodiments, the conjugate has a t of about 10,000 to about 15,000 IU / m. 2 (Approximately 20-30 mg of protein / m 2 ) has a t1 / 2 of at least about 100 to about 200 hours. In some embodiments, the complex has an area under the curve (AUC) greater than L-asparaginase not complexed with PEG. In some embodiments, the complex has a mean AUC at least about 3-fold greater than PEG asparaginase at an equivalent protein dose. In some embodiments, PEG is covalently attached to one or more amino groups of L-asparaginase. In some embodiments, PEG is covalently attached to one or more amino groups by an amide bond. In some embodiments, PEG is covalently attached 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 methods of the present invention involve the use of a conjugate having the formula: Asp-[NH-CO-(CH2) x -CO-NH-PEG] n where Asp is 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 a number representing at least about 40% to about 100% of the accessible amino groups of Asp, and x is an integer ranging from about 1 to about 8, more particularly, from about 2 to about 5. In certain embodiments, PEG is monomethoxy-polyethylene glycol (mPEG).

[0010] The methods of the invention encompass the use of an L-asparaginase complex that includes one or more peptide(s), each peptide being independently selected from the group consisting of peptide R N -(P / A)-RC where (P / A) is an amino acid sequence consisting of only proline and alanine amino acid residues, and where R N is a protecting group attached to the N-terminal amino group of the amino acid sequence, where R C is an amino acid residue linked via its amino group to the C-terminal carboxyl group of the amino acid sequence, and each peptide is C and a free amino group of L-asparaginase, and wherein at least one of the free amino groups to which the peptide is attached is not the N-terminal α-amino group of L-asparaginase.

[0011] The methods of the invention encompass the use of the conjugates for the treatment of cancer. In some embodiments, the cancer is selected from the group consisting of lymphoma, large cell immunoblastic lymphoma, non-Hodgkin's 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 selected from the group consisting of renal cell carcinoma, renal cell adenocarcinoma, glioblastoma, including glioblastoma multiforme and astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung cancer, including large cell carcinoma and small cell lung carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian teratocarcinoma, cervical adenocarcinoma, breast cancer, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon cancer, colon adenocarcinoma, colorectal adenocarcinoma, transitional cell carcinoma of the bladder, bladder papilloma, prostate cancer, osteosarcoma, epithelioid carcinoma of bone, prostate cancer, and thyroid cancer. In some embodiments, the conjugate is administered in an amount of about 5 U / kg body weight to about 50 U / kg body weight.

[0013] In some embodiments, the complex has a concentration of about 100 to about 15,000 IU / m 2In some embodiments, the administration is intravenous or intramuscular, once a week, twice a week, or three times a week. In some embodiments, the conjugate is administered as a monotherapy. In some embodiments, the conjugate is administered as part of a combination therapy. In some embodiments, the conjugate is administered as part of a combination therapy with Oncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactolisib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839. In some embodiments, the patient being treated has previously developed hypersensitivity to E. coli species asparaginase or a PEGylated version thereof, or to Erwinia species asparaginase. In some embodiments, the patient being treated has previously experienced disease recurrence, particularly after treatment with E. coli species asparaginase or a PEGylated version thereof. [Brief description of the drawings]

[0014] [Figure 1] In vivo experimental data on peg crisantaspase in combination with other compounds is shown. [Diagram 2] In vivo experimental data on peg crisantaspase in combination with other compounds is shown. [Diagram 3] The dose-response curve for single agent cases is shown. [Figure 4] 1 shows dose-response curves for example mixtures with inactive agents. [Diagram 5] Comparative data between single agent examples and mixture examples is shown. [Figure 6] 1 shows dose center plots indicating which drug combinations are synergistic. [Figure 7] CNS cell line data is shown. [Figure 8] The IC50 effect of peg crisantaspase is shown. [Figure 9] The IC50 effect of peg crisantaspase is shown. [Figure 10]FIG. 1 shows the in vitro sensitivity of PEG-crisantaspase in leukemia and lymphoma cell lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] L-asparaginase of bacterial origin has high immunogenic and antigenic potential and frequently induces adverse reactions ranging from mild allergic reactions to anaphylactic shock in sensitized patients (Wang (2003) Leukemia 17, 1583-1588). E. coli species L-asparaginase is particularly immunogenic, and the presence of anti-asparaginase antibodies against E. coli species L-asparaginase after iv or im administration has been reported to be as high as 78% in adults and 70% in children (Wang (2003) Leukemia 17, 1583-1588).

[0016] L-asparaginase from Escherichia coli and Erwinia chrysanthemi have different pharmacokinetic properties and each has its own immunogenicity profile (Klug Albertsen (2001) Brit. J. Haematol. 115, 983-990). Furthermore, it has been shown that antibodies generated after treatment with L-asparaginase from E. coli do not cross-react with L-asparaginase from Erwinia species (Wang (2003) Leukemia 17, 1583-1588). Therefore, L-asparaginase 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] In another attempt to reduce the immunogenicity associated with the administration of microbial L-asparaginase, E. coli species L-asparaginase modified with methoxy-polyethylene glycol (mPEG) has been developed. This method is commonly known as "PEGylation" and has been shown to modify the immunogenic properties of proteins (Abuchowski (1977) J. Biol. Chem. 252, 3578-3581). This so-called mPEG-L-asparaginase, or pegaspargase, is marketed as Oncaspar®, which was first approved in the United States in 1994 as a second-line treatment and since 2006 as a first-line treatment for pediatric and adult ALL. Oncaspar® has a long in vivo half-life and reduced immunogenicity / antigenicity.

[0018] Oncaspar® is an E. coli 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 labile ester bonds that are sensitive to enzymatic hydrolysis or mildly 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 was demonstrated that antibodies developed against L-asparaginase from E. coli may cross-react with Oncaspar® (Wang (2003) Leukemia 17, 1583-1588). Even if these antibodies were not neutralizing, this finding clearly demonstrated a high possibility of cross-hypersensitivity or cross-inactivation in vivo. Indeed, in one report, allergic reactions occurred in 30-41% of children administered pegaspargase (Wang (2003) Leukemia 17, 1583-1588).

[0020] Besides overt allergic reactions, the problem of "asymptomatic hypersensitivity" has recently been reported, where patients develop anti-asparaginase antibodies without any clinical evidence of a hypersensitivity reaction (Wang (2003) Leukemia 17, 1583-1588). This reaction may result in the formation of neutralizing antibodies against E. coli species L-asparaginase and pegaspargase. However, in these patients, the lack of overt signs of hypersensitivity prevents a switch to Erwinia species L-asparaginase, and as a result, these patients receive a shorter period of effective treatment (Holcenberg (2004) Pediatr. Hematol. Oncol. 26, 273-274).

[0021] Erwinia chrysanthemi species L-asparaginase therapy is often used in the event of hypersensitivity to E. coli-derived L-asparaginase. However, as many as 30-50% of patients administered Erwinia species L-asparaginase have been observed to become seropositive (Avramis (2005) Clin. Pharmacokinet. 44, 367-393). Moreover, Erwinia chrysanthemi species L-asparaginase has a significantly shorter elimination half-life than E. coli species L-asparaginase and must therefore be administered more frequently (Avramis (2005) Clin. Pharmacokinet. 44, 367-393). In a study by Avramis, Erwinia asparaginase had an inferior pharmacokinetic profile (Avramis (2007) J. Pediatr. Hematol. Oncol. 29, 239-247). Therefore, E. coli L-asparaginase and pegaspargase were preferred over Erwinia L-asparaginase as first-line treatment for ALL.

[0022] Over the years, numerous biologics have been successfully PEGylated and commercialized. To couple PEG to a protein, PEG must be activated at its OH-terminus. The activation 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 on proteins, almost all peptide chemical reactions have been applied to activate the PEG moiety. Examples of such activated PEG reagents include activated carbonates, e.g., p-nitrophenyl carbonate, succinimidyl carbonate, and active esters, e.g., succinimidyl esters, and aldehydes and maleimides have been developed for site-specific coupling (Harris (2002) Adv. Drug Del. Rev. 54, 459-476). The availability of a wide variety of chemical approaches for PEG modification means that each new development of a PEGylated protein is a case-by-case study. Not only the chemical reaction, but also the molecular weight of PEG attached to a protein has a significant effect on the pharmaceutical properties of PEGylated proteins. In most cases, the higher the molecular weight of PEG, the better the improvement in pharmaceutical properties is expected (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 conjugated to arginine deaminase, another amino acid degrading enzyme isolated from a microbial source, the pharmacokinetic and pharmacodynamic functions of the enzyme increased as the molecular weight of the attached PEG increased from 5000 Da to 20,000 Da (Holtsberg (2002) Journal of Controlled Release 80, 259-271).

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

[0024] Described herein is a PEGylated L-asparaginase from Erwinia sp., which has improved pharmacological properties when compared to unmodified L-asparaginase protein and when compared to E. coli-derived PEG asparaginase preparations. The PEGylated L-asparaginase conjugates described herein, for example Erwinia chrysanthemi sp. L-asparaginase PEGylated with a molecular weight of 5000 Da, serve as therapeutic agents, particularly for use in patients who exhibit hypersensitivity (e.g., allergic reaction or asymptomatic hypersensitivity) to treatment with E. coli-derived L-asparaginase or PEGylated L-asparaginase or Erwinia sp.-derived unmodified L-asparaginase. The PEGylated L-asparaginase conjugates described herein are also useful as therapeutic agents for use in patients with disease relapse, for example, relapsed ALL, and in patients who have previously been treated with another form of asparaginase, for example, E. coli-derived L-asparaginase or PEGylated L-asparaginase.

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

[0026] definition Unless expressly defined otherwise, terms used herein are to be understood according to their ordinary meaning in the art.

[0027] As used herein, the term "including" means "including but not limited to," and terms used in the singular include the plural and vice versa, unless the context specifically dictates otherwise.

[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 ability 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 equipment known in the art).

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

[0030] As used herein, the term "sequence identity" is used synonymously with "homology," and as such can have the same meaning, where appropriate.

[0031] The terms "co-administration," "co-administer," "administered in combination with," "administered in combination with," "simultaneously," and "concurrently," as used herein, encompass 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. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in one composition in which two or more active pharmaceutical ingredients are present. Co-administration in separate compositions and administration in one composition in which both agents are present are also encompassed by the methods of the 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 the art 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 from E. coli or Erwinia chrysanthemi, which have less than 50% structural homology with each other. Within Erwinia species, typically 75-77% sequence identity has been reported between the enzymes from Erwinia chrysanthemi and Erwinia carotovora, and about 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 include, for example, those provided in Table 1: [Table 1]

[0034] The sequences and GenBank entries of the Erwinia sp. L-asparaginases in Table 1 are incorporated herein by reference. Preferred L-asparaginases for use in therapy are those isolated from E. coli and Erwinia sp., in particular Erwinia chrysanthemi.

[0035] L-asparaginase can be a natural enzyme isolated from a microorganism. L-asparaginase can also be produced by recombinant enzyme techniques in a production microorganism, such as E. coli. By way of example, the protein used in the complex of the present invention can be a protein from Erwinia species, specifically Erwinia chrysanthemi, produced in a recombinant E. coli production strain.

[0036] Enzymes are identifiable by their specific activity; that is, this definition includes all polypeptides that have a defined specific activity and are also present in other organisms, more specifically other microorganisms. Enzymes with similar activities can often be identified by grouping them into certain families, defined as PFAMs or COGs. PFAMs (Protein Families Database by Sequence Comparisons and Hidden Markov Models; pfam.sanfferac.ukl) represent a large collection of protein sequence comparisons. Each PFAM allows the visualization of multiple sequence comparisons, display of protein domains, evaluation of distribution among organisms, access to other databases, and visualization of known protein structures. COGs (Clusters of Protein Orthologs; vv-ww.nebi.nlm.nih.gov / COG / ) were derived 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 the identification of previously conserved domains.

[0037] The means of identifying homologous sequences and their homology or sequence identity percentage are well known to those skilled in the art, and include, in particular, the BLAST program. The BLAST program can be used at the following website: blast.ncbi.olo.nih.gov / Blast.cgi, with the default parameters shown on the website. The obtained sequences can then be utilized (e.g. aligned) in, for example, the following programs: CLUSTALW (www.ebi.ac.uk / Tools / clustalw2 / index.html) or MULTALIN (bioinfo.genotoul.fr / multalin / multalin.html), with the default parameters shown on the websites. Using the references provided in GenBank for known genes, those skilled in the art can identify equivalent genes in other organisms, bacterial strains, yeast, fungi, mammals, plants, etc. This routine is advantageously carried out using consensus sequences, which can be identified by sequence comparison with genes from other microorganisms and designing degenerate probes to clone the corresponding genes in other organisms. Such routine methods of 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] Indeed, one of skill in the art would 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 to a protein comprising 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 to a protein comprising the sequence of SEQ ID NO: 1. SEQ ID NO: 1 is as follows: ADKLPNIVILATGGTIAAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSA RYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0040] The term "comprising 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, but may have additional amino acids.

[0041] In a particular embodiment, the protein is an Erwinia chrysanthemi L-asparaginase having the sequence of SEQ ID NO: 1. In another embodiment, the L-asparaginase is derived from Erwinia chrysanthemi NCPPB1066 (Genbank Accession No. CAA32884, which is incorporated herein by reference in its entirety), with or without a signal peptide and / or leader sequence.

[0042] Fragments of the protein of SEQ ID NO: 1 are also encompassed in the definition of the protein used in the conjugates of the 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 have sufficient amino acids to confer L-aminohydrolase activity.

[0043] It is well known in the art that a polypeptide can be modified by substitution, insertion, deletion and / or addition of one or more amino acids while retaining its enzymatic activity. For example, at a given position, it is common to replace an amino acid 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, non-polar 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] That is, exchanges that result in the substitution of one negatively charged residue for another (e.g., glutamic acid to aspartic acid) or one positively charged residue for another (lysine to arginine) can be expected to result in functionally equivalent products.

[0045] The position of the amino acid modification 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 the modifications that can be introduced without affecting the activity of the protein. For example, modifications at the N- or C-terminal portion of the protein can be expected not to change the activity of the protein under certain conditions. In particular, asparaginase has been thoroughly characterized, especially with respect to the sequence, structure, and residues that form the active catalytic site. This provides guidance as to the residues that can be modified without affecting the activity of the enzyme. All known L-asparaginases from bacterial sources share common structural characteristics: they are all homotetramers, with four active sites between the N- and C-terminal domains of two adjacent monomers (Aghaipour (2001) Biochemistry 40, 5655-5664, which is incorporated herein by reference in its entirety). They all share 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 includes amino acid residues 14-33, and structural analysis has revealed that Thr 15 , Thr 95 , Ser 62 , Glu 63 , Asp 96 , and Ala 120It has been shown that the 4 active sites of Erwinia chrysanthemi L-asparaginase are in contact with the ligand (Papageorgiou (2008) FEBSJ. 275, 4306-4316). Aghaipour et al. performed detailed analysis of the four active sites of Erwinia chrysanthemi L-asparaginase by high-resolution crystal structure analysis of the enzyme complexed with a substrate (Aghaipour (2001) Biochemistry 40, 5655-5664). Kotzia et al. sequenced L-asparaginases from multiple species and subspecies of the Erwinia genus and reported that they still have L-asparaginase activity, even though the proteins between Erwinia chrysanthemi and Erwinia carotovora are only about 75-77% identical (Kotzia (2007) J. Biotechnol. 127, 657-669, which is incorporated herein by reference in its entirety). Moola et al. conducted an epitope mapping study of Erwinia chrysanthemi 3937 L-asparaginase and found that even after various antigenic sequence mutations were made to reduce the immunogenicity of asparaginase, the L-asparaginase was able to retain its enzymatic activity (Moola (1994) Biochem. J. 302, 921-927, which is incorporated herein by reference in its entirety). Each of the above documents is incorporated herein by reference in its entirety. Given the extensive characterization that has been performed on L-asparaginase, one skilled in the art can determine how to make fragments and / or sequence substitutions while retaining enzymatic activity.

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

[0047] Polyethylene glycol (PEG) or mono-methoxy-polyethylene glycol (mPEG) are well known in the art and include linear and branched polymers. Examples of some polymers, specifically PEG, are provided below, each of which is incorporated herein by reference in its entirety: U.S. Patent Nos. 5,672,662, 4,179,337, 5,252,714, U.S. Patent Application 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 distribution of molecular weights 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 in a batch of polymer. PDI always has a value greater than 1, but the closer the polymer chains are to an ideal Gaussian distribution (=monodisperse), the closer the PDI is to 1.

[0049] The polyethylene glycol advantageously has a molecular weight in the range of about 500 Da to about 9,000 Da. More particularly, the polyethylene glycol (e.g., mPEG) has a molecular weight selected from the group consisting of 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da, and 5000 Da polyethylene glycol. In a particular embodiment, the polyethylene glycol (e.g., mPEG) has a molecular weight of 5000 Da.

[0050] Preparation of the Complex To subsequently couple the polymer to a protein having L-asparagine aminohydrolase activity, the polymer moiety has an activated functional group suitable for reacting with an amino group in the protein. In one aspect, the invention relates to a method for making a conjugate, the method comprising mixing an amount of polyethylene glycol (PEG) with an amount of L-asparaginase in a buffered solution for a sufficient length of time for covalently binding the PEG and the L-asparaginase. In a particular embodiment, the L-asparaginase is from an Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly an L-asparaginase comprising the sequence of SEQ ID NO: 1. In one embodiment, the PEG is monomethoxy-polyethylene glycol (mPEG).

[0051] In one embodiment, the reaction of polyethylene glycol with L-asparaginase is carried out in a buffer solution. In some specific embodiments, the pH value of the buffer solution is in the range of about 7.0 to about 9.0. The optimal pH value is in the range of about 7.5 to about 8.5, for example, a pH value 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 a specific embodiment, the L-asparaginase is from Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly is an L-asparaginase comprising the sequence of SEQ ID NO:1.

[0052] Furthermore, PEGylation of L-asparaginase is carried out at a protein concentration of about 0.5 to about 25 mg / mL, more particularly about 2 to about 20 mg / mL, and particularly about 3 to about 15 mg / mL. For example, the protein concentration is about 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 particularly for Erwinia chrysanthemi, and more particularly for L-asparaginase comprising the sequence of SEQ ID NO:1.

[0053] At high protein concentrations of more than 2 mg / mL, the PEGylation reaction proceeds quickly, in less than 2 hours. Furthermore, the molar excess ratio of polymer to amino groups of L-asparaginase is applied at less than about 20:1. For example, the molar excess ratio is less than about 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 1:1. In certain embodiments, the molar excess ratio is less than about 10:1, and in more specific embodiments, the molar excess ratio is less than about 8:1. In a particular embodiment, the L-asparaginase is from an Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly an L-asparaginase comprising 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 further 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 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 the N-terminus of the protein) is also referred to herein as "maximum PEGylation." One method for identifying modified amino groups in the mPEG-r-crisantaspase conjugate (degree of PEGylation) is that 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 (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 the N-terminus), e.g., 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 all amino groups (e.g., lysine residues and / or the N-terminus) are coupled to a PEG moiety.In another particular 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%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... , 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% are coupled to a PEG moiety. In certain embodiments, 40-55% or 100% of the accessible amino groups (e.g., lysine residues and / or the N-terminus) are coupled to a PEG moiety. In some embodiments, the PEG moiety is covalently coupled to the L-asparaginase. In a particular embodiment, the L-asparaginase is from an Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly an L-asparaginase comprising the sequence of SEQ ID NO:1.

[0055] In one embodiment, the conjugate of the invention can be represented by the formula: Asp-[NH-CO-(CH2) x -CO-NH-PEG] n where Asp is L-asparaginase protein, NH is the NH group of the lysine residues and / or the N-terminus of the protein chain, PEG is a polyethylene glycol moiety, n is a number representing at least 40% to about 100% of the accessible amino groups in the protein (e.g., lysine residues and / or the N-terminus), all as defined above and in the Examples below, and x is an integer ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), preferably 2 to 5 (e.g., 2, 3, 4, 5). In a particular embodiment, the L-asparaginase is from an Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly an L-asparaginase comprising the sequence of SEQ ID NO:1.

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

[0057] A specific embodiment comprises a protein having substantial L-asparagine aminohydrolase activity and a polyethylene glycol, which is selected from the group of conjugates: (A) Protein has a structure at least 90% homologous to L-asparaginase derived from Erwinia chrysanthemi as disclosed in SEQ ID NO:1, polyethylene glycol has a molecular weight of about 5000 Da, the protein and the polyethylene glycol moiety are covalently bonded to the protein by an amide bond, and about 100% of accessible amino groups (e.g., lysine residues and / or the N-terminus) or about 80-90%, specifically about 84%, of all amino groups (e.g., lysine residues and / or the N-terminus) are bound to the polyethylene glycol moiety. (B) The protein has a structure at least 90% homologous to L-asparaginase from Erwinia chrysanthemi as disclosed in SEQ ID NO:1, the polyethylene glycol has a molecular weight of about 5000 Da, the protein and the polyethylene glycol moiety are covalently bonded to the protein by an amide bond, and about 40% to about 45% of the accessible amino groups (e.g., lysine residues and / or the N-terminus), more specifically about 43%, or about 36% of the total amino groups (e.g., lysine residues and / or the N-terminus) are bound to the polyethylene glycol moiety. (C) The protein has a structure at least 90% homologous to L-asparaginase derived from Erwinia chrysanthemi as disclosed in SEQ ID NO:1, the polyethylene glycol has a molecular weight of about 2000 Da, the protein and the polyethylene glycol moiety are covalently bonded to the protein by an amide bond, and about 100% of the accessible amino groups (e.g., lysine residues and / or the N-terminus) or about 80-90%, specifically about 84%, of all amino groups (e.g., lysine residues and / or the N-terminus) are bound to the polyethylene glycol moiety. (D) The protein has a structure at least 90% homologous to L-asparaginase from Erwinia chrysanthemi as disclosed in SEQ ID NO:1, the polyethylene glycol has a molecular weight of about 2000 Da, the protein and the polyethylene glycol moiety are covalently bonded to the protein by an amide bond, and about 50% to about 60%, more specifically about 55%, or about 47% of the total amino groups (e.g., lysine residues and / or N-terminus) are bound to the polyethylene glycol moiety.

[0058] L-Asparaginase-PEG Conjugate The conjugates of the present invention have certain advantages and unexpected properties compared to unmodified L-asparaginase, in particular compared to unmodified Erwinia sp. L-asparaginase, more particularly compared to unmodified L-asparaginase from Erwinia chrysanthemi, and more particularly compared to unmodified L-asparaginase having the sequence of SEQ ID NO:1.

[0059] In some embodiments, the methods of the invention include complexes that reduce plasma L-asparagine and glutamine levels for a period of at least about 12, 24, 48, 72, 96, or 120 hours when administered at a dose of 5 U / kg body weight (bw) or 10 μg / kg (protein content basis). In other embodiments, complexes of the invention reduce plasma L-asparagine levels to undetectable levels for a period of at least about 12, 24, 48, 72, 96, 120, or 144 hours when administered at a dose of 25 U / kg bw or 50 μg / kg (protein content basis). In other embodiments, complexes of the invention reduce plasma L-asparagine levels for a period of at least about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours when administered at a dose of 50 U / kg bw or 100 μg / kg (protein content basis). In another embodiment, the complex of the present invention has a concentration of about 100 to about 15,000 IU / m 2 (Approximately 1-30 mg protein / m 2 ), 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 particular embodiments, the complex comprises an L-asparaginase from an Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly comprising an L-asparaginase comprising the sequence of SEQ ID NO:1. In particular embodiments, the complex comprises a PEG (e.g., mPEG) having a molecular weight of about 5000 Da or less. In more particular embodiments, at least about 40% to about 100% of accessible amino groups (e.g., lysine residues and / or the N-terminus) are PEGylated.

[0060] In one embodiment, the conjugate has a mol PEG / mol monomer ratio of about 4.5 to about 8.5, particularly about 6.5, a specific activity of about 450 to about 550 U / mg, particularly about 501 U / mg, and a relative activity of about 75% to about 85%, particularly about 81%, compared to the corresponding unmodified L-asparaginase. In a particular embodiment, a conjugate having these properties comprises an L-asparaginase from an Erwinia species, more particularly from Erwinia chrysanthemi, more particularly an L-asparaginase comprising the sequence of SEQ ID NO: 1, in which 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 conjugate has a mol PEG / mol monomer ratio of about 12.0 to about 18.0, particularly about 15.1, a specific activity of about 450 to about 550 U / mg, particularly about 483 U / mg, and a relative activity of about 75% to about 85%, particularly about 78%, compared to the corresponding unmodified L-asparaginase. In a particular embodiment, a conjugate having these properties comprises an L-asparaginase from an Erwinia species, more particularly from Erwinia chrysanthemi, more particularly an L-asparaginase comprising the sequence of SEQ ID NO: 1, in which about 100% of the accessible amino groups (e.g., lysine residues and / or the N-terminus) are PEGylated with 5000 Da mPEG.

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

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

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

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

[0066] In other embodiments, the conjugates of the invention have at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold increased potency after a single injection compared to the corresponding unmodified L-asparaginase. In certain embodiments, a conjugate having these properties comprises an L-asparaginase from an Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly an L-asparaginase comprising the sequence of SEQ ID NO: 1. In certain embodiments, the conjugate comprises a PEG (e.g., mPEG) having 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 the N-terminus) are PEGylated.

[0067] In one embodiment, the conjugate of the invention has a single dose pharmacokinetic profile, as measured as described in PCT Publication No. WO2011003886, as follows, specifically, the conjugate comprises mPEG having a molecular weight of 2000 Da or less, and an L-asparaginase from an Erwinia species, more specifically, from Erwinia chrysanthemi, more specifically, an L-asparaginase comprising the sequence of SEQ ID NO:1: A max :Approx. 150U / L~Approx. 250U / L, T Amax : About 4 hours to about 8 hours, specifically about 6 hours, d Amax : About 220 hours to about 250 hours, specifically, about 238.5 hours (over 0, about 90 minutes to about 240 hours), AUC: about 12,000 to about 30,000, and t1 / 2: Approximately 50 hours to approximately 90 hours. In one embodiment, the conjugate of the invention has a single dose pharmacokinetic profile as follows, specifically, the conjugate comprises mPEG with a molecular weight of 5000 Da or less, and L-asparaginase from Erwinia species, more specifically from Erwinia chrysanthemi, more specifically comprising the sequence of SEQ ID NO:1: A max:Approx. 18U / L~Approx. 250U / L, T Amax : About 1 hour to about 50 hours, d Amax : About 90 hours to about 250 hours, specifically, about 238.5 hours (over 0, about 90 minutes to about 240 hours), AUC: about 500 to about 35,000, and t1 / 2: Approximately 30 hours to approximately 120 hours. In one embodiment, the conjugates of the invention provide a similar level of L-asparagine depletion over a period of time (e.g., 24, 48, or 72 hours) after a single dose compared to an equivalent amount of protein of pegaspargase. In a particular embodiment, the conjugates comprise an L-asparaginase from an Erwinia species, more particularly from Erwinia chrysanthemi, more particularly comprising the sequence of SEQ ID NO: 1. In a particular embodiment, the conjugates comprise a PEG (e.g., mPEG) having a molecular weight of 5000 Da or less. In a more particular embodiment, at least about 40% to about 100%, more particularly about 40-55% or 100%, of the accessible amino groups (e.g., lysine residues and / or N-terminus) are PEGylated.

[0068] In one embodiment, the complexes of the invention have a longer t1 / 2 than pegaspargase administered at an equivalent protein dose. In a specific embodiment, the complexes have 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 (protein content basis). In another specific embodiment, the complexes have 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 (protein content basis). In another specific embodiment, the complexes have a t1 / 2 of at least about 100 to about 15,000 IU / m 2 (Approximately 1-30 mg protein / m 2 ) has a t1 / 2 of at least about 100 to about 200 hours.

[0069] In one embodiment, a conjugate of the invention has a mean AUC that is at least about 2, 3, 4, or 5 times greater than pegaspargase at an equivalent protein dose.

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

[0071] PAS-L-asparaginase In some embodiments, the methods of the invention include an L-asparaginase complex that includes one or more peptide(s), each peptide being independently selected from the group consisting of peptide R and peptide B. N -(P / A)-R C where (P / A) is an amino acid sequence consisting of only proline and alanine amino acid residues, and where R N is a protecting group attached to the N-terminal amino group of the amino acid sequence, where R C is an amino acid residue linked via its amino group to the C-terminal carboxyl group of the amino acid sequence, and each peptide is Cand a free amino group of L-asparaginase, and at least one of the free amino groups to which the peptide is attached is not the N-terminal α-amino group of L-asparaginase. These molecules are also known as PAS derivatives of L-asparaginase, and are also referred to herein as complexes.

[0072] The modified L-asparaginase protein monomer has from about 350, 400, 450, 500 amino acids to about 550, 600, 650, 700, or 750 amino acids after modification. In additional embodiments, the modified L-asparaginase protein has from about 350 to about 750 amino acids, or from about 500 to about 750 amino acids.

[0073] Each peptide contained in the modified L-asparaginase protein as described herein is independently designated peptide R N -(P / A)-R C Thus, for each peptide contained in the modified L-asparaginase protein as described herein, an N-terminal protecting group R N , amino acid sequence (P / A), and C-terminal amino acid residue R C are each independently selected from their respective meanings. Thus, two or more peptides contained in the modified L-asparaginase protein may be the same or different from each other. In one embodiment, all peptides contained in the modified L-asparaginase protein are the same.

[0074] The portion of the modified L-asparaginase protein (P / A) that is chemically bonded to peptide R N -(P / A)-R CThe 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 amino acid sequence consists of about 20 proline and alanine amino acid residues. In another preferred embodiment, the amino acid sequence consists of about 40 proline and alanine amino acid residues. Peptide R N -(P / A)-R C In the above, the ratio of the number of proline residues contained in (P / A) to the number of amino acid residues contained 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 contained 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 contained in (P / A) are proline residues. Moreover, it is preferable that (P / A) does not contain any consecutive proline residues (i.e., (P / A) does not contain any partial 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 Group R NR may be a protecting group attached to the N-terminal amino group of the amino acid sequence (P / A), specifically the N-terminal α-amino group. N is preferably pyroglutamoyl or acetyl.

[0076] Peptide R N -(P / A)-R C Group R C is an amino acid residue linked through its amino group to the C-terminal carboxy group of (P / A), which has at least two carbon atoms between its amino group and its carboxy group. C At least two carbon atoms between the amino group and the carboxy group of R C can provide a spacing of at least two carbon atoms between the amino and carboxy groups of R C ω-amino-C 3-15 This is the case for alkanoic acids, e.g., ε-aminohexanoic acid). C is preferably ε-aminohexanoic acid.

[0077] In one embodiment, the peptide is Pga-AAPAAPAPAAPAAPAPAAPA-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 is the C-terminal amino acid residue R of the peptide Cand 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). When L-asparaginase is composed of multiple subunits, for example, when L-asparaginase is a tetramer, there can be multiple N-terminal α-amino groups (i.e., one for each subunit). In one embodiment, 9-13 peptides as defined herein (e.g., 9, 11, 12, or 13 peptides) can be chemically conjugated to L-asparaginase (e.g., each subunit / monomer of L-asparaginase).

[0079] In accordance with the above, 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., different from the N-terminal α-amino group). Thus, it is preferred 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 preferred that at least one of the free amino groups to which the peptide is bound is the ε-amino group of a lysine residue of L-asparaginase.

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

[0081] The modified L-asparaginase protein as described herein is composed of L-asparaginase and one or more peptides as defined herein.The corresponding modified L-asparaginase protein can be composed of, for example, 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 of which is bound to the L-asparaginase.The L-asparaginase can be, for example, a monomeric protein or a protein composed of multiple subunits, for example, a tetramer. When L-asparaginase is a monomeric protein, the corresponding modified L-asparaginase protein can be, for example, composed of one monomeric L-asparaginase and 9 to 13 (or more), (e.g., 8, 9, 10, 11, 12, or 13) peptides, each of which is bound to the monomeric L-asparaginase. An example of the amino acid sequence of a monomeric L-asparaginase is shown in SEQ ID NO: 1. When L-asparaginase is a protein composed of multiple subunits, for example, four subunits (i.e., when the above L-asparaginase is a tetramer), the corresponding modified L-asparaginase protein can be, for example, composed of four L-asparaginase subunits and 9 to 13 (or more), (e.g., 9, 10, 11, 12, or 13) peptides as defined above, each of which is bound to each subunit of L-asparaginase. An example of the amino acid sequence of an L-asparaginase subunit is shown in SEQ ID NO: 1. Similarly, when the L-asparaginase is a protein composed of multiple subunits, for example, four subunits (i.e., when the L-asparaginase is a tetramer), the corresponding modified L-asparaginase protein can consist of, for example, 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 of which is associated with the L-asparaginase tetramer.In one embodiment, the present invention relates to an L-asparaginase and a modified L-asparaginase protein 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] A peptide consisting of only proline and alanine amino acid residues can be covalently bonded to one or more amino acids of the L-asparaginase, such as lysine residues and / or the N-terminal residue, and / or the peptide consisting of only proline and alanine amino acid residues may be covalently bonded 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 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 lysines may be bonded to a peptide consisting of only proline and alanine amino acid residues. In a further aspect, the peptide consisting of only proline and alanine amino acid residues is covalently bonded to about 20, 30, 40, 50, or 60% to about 30, 40, 50, 60, 70, 80, or 90% of the total lysine residues of the L-asparaginase. In a further embodiment, the peptide consisting of only proline and alanine amino acid residues is covalently bonded to the L-asparaginase via a linker. Examples of linkers include the linkers disclosed in U.S. Patent Application No. 2015 / 0037359, the entirety of which is incorporated herein by reference.

[0083] In one aspect, the complex is a fusion protein comprising an L-asparaginase and a polypeptide consisting of only proline and alanine amino acid residues having a length of about 200 to about 400 proline and alanine amino acid residues. In other words, the polypeptide may consist of about 200 to about 400 proline and alanine amino acid residues. In one aspect, the polypeptide consists of a total of about 200 (e.g., 201) proline and alanine amino acid residues (i.e., has 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., has a length of about 400 (e.g., 401) proline and alanine amino acid residues). In some preferred embodiments, the polypeptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 6 or 7. In some embodiments, each monomer of the fusion protein has from 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 from about 350 to about 800 amino acids or from about 500 to about 750 amino acids. For example, the polypeptide comprises a peptide prepared in U.S. Pat. No. 9,221,882. In some embodiments, the L-asparaginase is from an Erwinia species, more particularly from Erwinia chrysanthemi, and more particularly an L-asparaginase comprising the sequence of SEQ ID NO: 1 as described herein.

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

[0085] Treatment and Use The complexes of the present invention can be used to treat diseases treatable by depletion of asparagine and / or glutamine. For example, the complexes are useful for treating or manufacturing medicaments for treating acute lymphoblastic leukemia (ALL) in both adults and children, as well as other conditions in which depletion of asparagine and / or glutamine is expected to have a beneficial effect. Such conditions include, but are not limited to, malignancies or cancers, such as hematological malignancies, lymphomas, large cell immunoblastic lymphomas, non-Hodgkin's 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), biphenotypic B-cell myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma, and melanosarcoma. In some embodiments, the disease may be acute myeloid leukemia or diffuse large B-cell lymphoma. Malignant tumors or cancers include, but are not limited to, renal cell carcinoma, renal cell adenocarcinoma, glioblastoma including glioblastoma multiforme and astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung cancer including large cell carcinoma and small cell lung carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian teratocarcinoma, cervical adenocarcinoma, breast cancer, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon cancer, colon adenocarcinoma, colorectal adenocarcinoma, transitional cell carcinoma of the bladder, bladder papilloma, prostate cancer, osteosarcoma, epithelioid carcinoma of bone, prostate cancer, and thyroid cancer.

[0086] Representative non-malignant hematological diseases that respond to asparagine and / or glutamine depletion include immune system-mediated hematological diseases, such as infectious diseases caused by HIV infection (i.e., AIDS). Non-hematological diseases associated with asparagine and / or glutamine dependency include autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus (SLE), collagen vascular disease, etc. 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 dependency in any suitable in vitro or in vivo assay, for example, in vitro assays in which the growth medium does not contain asparagine and / or glutamine. Thus, in one aspect, the invention relates to a method of treating a treatable disease in a patient, comprising administering to the patient an effective amount of a conjugate of the invention. In another aspect, the conjugate of the invention is co-administered with another active pharmaceutical ingredient. In some embodiments, the conjugate of the invention is co-administered with Oncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactolisib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839. In a particular embodiment, the disease is ALL. In certain embodiments, the conjugate used to treat a disease treatable by asparagine and / or glutamine depletion comprises an L-asparaginase from an Erwinia species, more particularly from Erwinia chrysanthemi, more particularly comprising the sequence of SEQ ID NO:1 as described herein.

[0087] In one embodiment, treatment with the inventive complex will be performed as a first-line treatment. In another embodiment, treatment with the inventive complex will be performed as a second-line treatment in patients, particularly in patients with ALL, who have developed objective signs of allergy or hypersensitivity, including "asymptomatic hypersensitivity", to other asparaginase preparations, particularly to native Escherichia coli-derived L-asparaginase or its PEGylated variant (pegaspargase). Non-limiting examples of objective signs of allergy or hypersensitivity include "antibody positive" tests for the asparaginase enzyme. In a particular embodiment, the inventive complex will be used in a second-line treatment after treatment with pegaspargase. In a more specific embodiment, the complex used in the second-line treatment comprises an L-asparaginase from Erwinia species, more particularly from Erwinia chrysanthemi, more particularly comprising the sequence of SEQ ID NO:1. In more specific embodiments, the conjugate further comprises PEG (e.g., mPEG) having a molecular weight of about 5000 Da or less, more specifically about 5000 Da. In even more specific embodiments, at least about 40% to about 100%, more specifically about 40-55% or 100%, of the accessible amino groups (e.g., lysine residues and / or the N-terminus) are PEGylated.

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

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

[0090] In a specific embodiment, the method comprises administering a complex of the invention at 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 an equivalent amount thereof (e.g., on a protein content basis). In a more specific embodiment, the complex is administered in an amount selected from the group consisting of about 5, about 10, and about 25 U / kg. In another specific embodiment, the complex is administered at about 1,000 IU / m 2 ~About 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 2, 8,000 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 In another specific embodiment, the complex is administered at a dose that depletes L-asparagine and / or glutamine to undetectable levels using methods and devices known in the art for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) in a single dose.

[0091] In another embodiment, the method includes administering a conjugate of the invention that induces a lower immunogenic response in a patient than unconjugated L-asparaginase. In another embodiment, the method includes administering a conjugate of the invention that has a longer in vivo circulatory half-life after a single dose than unconjugated L-asparaginase. In one embodiment, the method includes administering a conjugate that has a longer t1 / 2 than pegasparagase administered at an equivalent protein dose. In a specific embodiment, the method includes administering a conjugate that 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 (protein content basis). In another specific embodiment, the method includes administering a conjugate that has 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 (protein content basis). In another specific embodiment, the method provides a dose of about 10,000 to about 15,000 IU / IU / m 2 (Approximately 20-30 mg protein / IU / m2 ) having a t1 / 2 of at least about 100 to about 200 hours. In one embodiment, the method comprises administering a conjugate having a mean AUC that is at least about 2, 3, 4, or 5 times greater than pegaspargase administered at an equivalent protein dose.

[0092] Relapses in ALL patients following treatment with L-asparaginase remain frequent, with early relapses occurring in approximately 10-25% of pediatric ALL patients (e.g., in some cases during the maintenance period 30-36 months after induction) (Avramis (2005) Clin. Pharmacokinet. 44, 367-393). If relapse occurs in a patient treated with E. coli derived L-asparaginase, subsequent treatment with E. coli preparations may result in a "vaccination" effect, whereby the E. coli preparation becomes more immunogenic during subsequent administrations. In one embodiment, the conjugates of the present invention can be used in the treatment of patients with relapsed ALL who have been previously treated with other asparaginase preparations, particularly with E. coli derived asparaginase.

[0093] In some embodiments, the therapeutic uses and methods of the present invention comprise administering an L-asparaginase conjugate having the properties or combination of properties described herein above (e.g., in the sections entitled L-asparaginase PEG conjugates or PASylated L-asparaginase) or herein below.

[0094] Compositions, Formulations, and Routes of Administration The present invention also includes pharmaceutical compositions comprising the conjugates of the present invention. In certain embodiments, the pharmaceutical compositions are packaged in vials as lyophilized powders intended to be reconstituted with a solvent, regardless of the bacterial source used for its production, such as currently available native L-asparaginase (Kidrolase®, Elspar®, Erwinase®). In another embodiment, the pharmaceutical composition may further include pegaspargase, etc., as a "ready-to-use" liquid formulation (Oncaspar®), which further allows for proper handling and administration via, for example, intramuscular, intravenous (infusion and / or bolus), intracerebroventricular (icv), subcutaneous routes. In a further embodiment, the pharmaceutical composition comprises the conjugates of the present invention in combination with Oncaspar®, daunorubicin, cytarabine, ABT-737, venetoclax, dactolisib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839.

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

[0096] Appropriate dosage forms depend, in part, on the use or route of introduction, e.g., oral, transdermal, transmucosal, or by injection (parenteral). Such dosage forms must allow the therapeutic agent to reach target cells or, in any event, have the desired therapeutic effect. For example, pharmaceutical compositions injected into the bloodstream are preferably soluble.

[0097] The conjugates and / or pharmaceutical compositions according to the invention can be formulated as their pharma- ceutically acceptable salts and complexes. Pharmaceutically acceptable salts are salts that exist in a non-toxic manner in the amounts and concentrations at which they are administered. Such salt formulations can facilitate medical use by modifying the physical properties of the compounds without preventing them from exerting their physiological effects. Useful modifications of physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administration at higher drug concentrations. Pharmaceutically acceptable salts of asparaginase can also exist as complexes, as recognized in the art.

[0098] Pharmaceutically acceptable salts include acid addition salts, such as those containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinate. Pharmaceutically acceptable salts can be obtained from acids, including 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, fumaric acid, and quinic acid.

[0099] When an acidic functional group, such as a carboxylic acid or phenol, is present, pharma- ceutically acceptable salts also include base addition salts, such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamines, and zinc. See, for example, Remington's Pharmaceutical Sciences, supra. Such salts can be prepared using the appropriate base.

[0100] Pharmaceutically acceptable carriers and / or excipients can also be incorporated into the pharmaceutical compositions of the present invention to facilitate administration of the specific asparaginase. 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 glycols, and physiologically compatible solvents. Examples of physiologically compatible solvents include water for injection (WFI), saline, and sterile solutions of glucose.

[0101] The pharmaceutical composition according to the present invention can be administered by various routes, such as 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 formulated into conventional oral dosage forms, such as capsules, tablets, and liquid preparations, such as syrups, elixirs, and concentrated drops.

[0102] Alternatively, injection (parenteral administration), such as intramuscular, intravenous, intraperitoneal, and subcutaneous injection, may be used. For injection, the pharmaceutical composition is formulated in a liquid agent, preferably in a physiologically compatible buffer or solution, such as physiological saline, Hank's solution, or Ringer's solution. The compound may also be formulated in solid form and redissolved or suspended immediately prior to use. For example, the complex may be prepared in lyophilized form. 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. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are well known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. Surfactants may also be used to enhance penetration. Transmucosal administration can be, for example, through nasal sprays, inhalers (for delivery to the lungs), rectal suppositories, or vaginal suppositories. For topical administration, the compound can be formulated into ointments, salves, gels, or creams, as is well known in the art.

[0104] The amount of complex delivered depends on many factors, e.g., IC 50 , E.C. 50 The amount of conjugate administered will depend on the biological half-life of the compound, the age, size, weight, and physical condition of the patient, and the disease or disorder being treated. The importance of these and other factors to be considered is well known to those of skill in the art. Generally, the amount of conjugate administered will be about 10 International Units per square meter (IU / m2) of the surface area of ​​the patient's body. 2 )~50,000IU / m 2 and the dosage range is approximately 1,000 IU / m 2 ~Approx. 15,000IU / m 2 is preferred, about 6,000 IU / m 2 ~Approx. 15,000IU / m 2 More preferably, the range is about 10,000 to about 15,000 IU / m 2 (Approximately 20-30 mg protein / m 2 ) are particularly suitable for treating malignant hematological diseases, such as leukemia. Typically, these dosages are administered via intramuscular or intravenous injection at intervals of 3 times per week to about once per month, typically once per week or once every other week, during the course of treatment. 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 construed as limiting: Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLES

[0106] The subject matter of U.S. Patent No. 9,920,311 is incorporated herein by reference, including the Examples which disclose how to make and test PEGylated asparaginase. The mPEG-r-crisantaspase conjugate used in the following Examples was prepared as described in U.S. Patent No. 9,920,311.

[0107] Example 1 The mPEG-r-crisantaspase conjugate (Peg crisantaspase) was tested against various cell lines in two stages as shown below.

[0108] Cell preparation. All cell lines were licensed from the American Type Culture Collection (ATCC), Manassas, Virginia (US). Master and working cell banks (MCB and WCB) were prepared by subculturing and freezing in ATCC recommended media according to the ATCC recommended protocols (www.atcc.org).

[0109] Compound Preparation. Test compounds were prepared as stock solutions and serially diluted using DMSO or aqueous buffer as appropriate to obtain a dilution series.

[0110] Cell proliferation assay. Cell proliferation was assessed using a commercially available fluorescent assay using ATP as the endpoint.

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

[0112] Reference compound. IC of reference compound doxorubicin 50 is measured in a separate plate. 50 There is a tendency for IC 50 If falls outside of specification (deviations of 0.32-3.16 fold from the historical average), the assay is invalid.

[0113] Cell proliferation controls. Cell doubling times for all cell lines are calculated from the proliferation signals at t=0 and t=end of untreated cells. If the doubling time falls outside of specification (0.5-2.0 fold deviation from the historical average), the assay is invalid.

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

[0115] Drug sensitivity. Between “modified” and “wild-type” cell lines. 10 logIC 50The differences were analyzed in three ways. First, the drug sensitivity of individual cell lines for the top 18 frequent genetic alterations was visualized in a waterfall plot. Second, a larger subset of the most commonly occurring and best understood cancer genes (38 in total) was analyzed with a Type II Anova analysis in the statistical program R. Results are displayed in a volcano plot. Third, the complete set of 114 cancer genes was analyzed with a two-tailed equal variance t-test in R. P values ​​from the anova and t-tests were subjected to the Benjamini-Hochberg multiple comparison test correction method. Only genetic associations with a false discovery rate below 20% are considered significant. The Type II Anova analysis for the 38 cancer genes is a different test than the equal variance t-test for the 114 cancer genes, which means that the significance of the associations may be different. For more information about the Oncolines™ method, see: www.ntrc.nl / services / oncolinestm.

[0116] I C 50 was calculated by nonlinear regression using IDBS XLfit. After incubation, the percentage of growth (%-growth) until t=end was calculated as follows: 100%×(fluorescence t=end / fluorescence 未処置,t=end ) This can be expressed as follows using a four-coefficient logistic curve: 10 Fitted to log compound concentration (conc): %-growth = min + (max - min) / (1 + 10 (logIC50-conc)*hill) ), where hill is the Hill coefficient and min and max are the asymptotic minimum and maximum cell proliferation values ​​tolerated by the compound in that assay. [Table 2]

[0117] NCI60 parameters.LD 50 , i.e., the concentration at which 50% of the cells die is t=end = 1 / 2 x fluorescence t=0h This is the concentration at which GI 50i.e., the concentration at which proliferation is inhibited by 50%, is the concentration at which cell proliferation is half-maximal. 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. Curves calculated automatically by the software were adjusted manually according to the following protocol: If the calculated curve had a minimum below 0, the minimum of the curve was fixed at 0%. If the software calculated a value below -6, the slope was fixed at -6. If the F-test value for the quality of the fit was >1.5 or if the compound was inactive (maximal effect <20%), the curve was invalid and removed from the graph. If the curve had a biphasic character, it was determined that the most potent IC 50 Fitting was performed with . Concentration points were excluded if, by chance, it seemed to be a technical error. This was always shown in the dose-response graph. If the dose-response curve was more than 85% perfectly specified, the Max effect was calculated as 100% (signal of untreated cells) - minimum of the curve. A dose-response curve is considered to be 100% complete if the data point at the highest concentration reaches the minimum of the curve. If the completeness was less than 85%, the Max effect was calculated as 100% - mean of the minimum signal. If the minimum of the curve was fixed at 0%, the Max effect was always calculated as 100% - growth inhibition at the highest concentration.

[0119] Volcano plot. The volcano plot in Figure 8 shows how genetic transformation is statistically associated with shifts in compound sensitivity in the 38 key genes ( 10 logIC 50 P values ​​(y-axis of the volcano plot) represent the IC 50 Indicates the confidence level for the genetic association of a variant in a particular gene with a shift. IC 50The coefficient of shift 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-fold). To calculate significance, P values ​​were subjected to the Benjamin-Hochberg multiple comparison test correction method, and only genetic associations with a false discovery rate below 20% are shown in grey. The relevant cutoff P value (0.059) is indicated by a horizontal line. If there is no significant association, neither the grey circle nor the horizontal line is drawn.

[0120] T-test results. For the 98 validated cancer driver genes, whose mutant forms also occur in patients, the presence of the “wild-type” and “mutant” forms of the genes in the cell lines correlated with significant IC of the test compounds. 50 We tested whether it was related to the IC shift. 50 The "Shift" column is 10 logIC 50 The difference between the negative IC 50 A shift indicates that the compound is more potent in cell lines carrying the "mutated" gene. The "p-value" column shows the results of a two-tailed t-test. To calculate significance, the p-values ​​were subjected to the Benjamin-Hochberg multiple comparison test correction method. Only genetic associations with a false discovery rate below 20% are highlighted (column "adjusted p-value"). If there is no significant association, there are no grey cells in the table below. [Table 3]

[0121] The special volcano plot in Figure 9 relates compound sensitivity to the presence of cancer hotspot mutations ( 10 logIC 50 As measured by . This provides a higher focus on clinically relevant cancer driver mutations in comparison to previous analyses. Hotspot mutations were derived from statistical analysis of repeat patterns and copy number changes of mutations in patients across separate studies. Axes and statistical analysis are the same as Volcate plot in Figure 8. The cutoff p-level for significance is 0.32.

[0122] Example 3: Synergistic activity of Peg crisantaspase and Oncaspar®. Effect 20 To determine the effect of the compounds on the activity of other anti-cancer drugs in SynergyScreen™ experiments, a low, fixed concentration is used that corresponds to a concentration at which cell proliferation is inhibited by 20%. This concentration is determined using a dose-response curve of a single compound. The concentration is the value on the x-axis that corresponds to 80% untreated viability on the y-axis. [Table 4]

[0123] mPEG-r-crisantaspase conjugate (Peg crisantaspase, see first table below) or Oncaspar® (see second table below) were tested with other agents typically used in standard of care (SOC) for AML or DLBCL. Increased efficacy was seen in AML when combined with daunorubicin, cytarabine, ABT-737, venetoclax, dactolisib, bortezomib, and carfilzomib. Additionally, increased efficacy was seen in DLBCL when combined with vincristine, prednisolone, ABT-737, venetoclax, everolimus, dactolisib, bortezomib, carfilzomib, and CB-839. See table below. Grey coloring indicates synergistic activity. Light grey coloring indicates one experiment, dark grey coloring indicates two experiments. [Table 5]

[0124] Example 3: mPEG-r-crisantaspase conjugate (PegCrisantapase) was tested in vivo with cytarabine and daunorubicin. Groups of 5 mice were each administered mPEG-r-crisantaspase (PegC) as a single agent (5 & 50 IU / kg) and in combination with the SOC drugs cytarabine (50 mg / kg once daily for 5 days followed by 2 days off for 2 cycles) and daunorubicin (1 mg / kg weekly for 2 weeks). These doses were well tolerated. See Figure 1. Group 1 is the PBS control group, group 3 is the PegC group, group 11 is the daunorubicin + PegC group, and group 13 is the daunorubicin group. Approximately 10% decrease in mean relative body weight was due to daunorubicin.

[0125] Example 4: This example was performed in a similar manner to Example 1, except that mPEG-r-crisantaspase conjugate (Peg crisantaspase) was tested in combination with other compounds. Figure 2 shows that Peg crisantaspase enhances the effects of cytarabine, venetoclax, and ABT-737, demonstrating synergy.

[0126] Example 5: mPEG-r-crisantaspase conjugate (Peg crisantaspase) was tested in combination with ABT-737 against the HL-60 cell line.

[0127] Plate preparation. Stock solutions of mixtures and single agents were diluted in DMSO or 0.9% sodium chloride to generate a 7-point dose-response dilution series. After a further 31.6-fold dilution in 20 mM sterile Hepes buffer pH 7.4 (reference compound) or vehicle (peg-crisantaspase), 5 μl of peg-crisantaspase solution and 5 μl of reference compound were added in duplicate 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. Final assay concentrations were determined for the single agents to be 0.01% or lower than their IC 50 10 to 0.01 times (IC 50 The concentrations ranged from 10 and 0.01 equivalents of 10 and 0.01, respectively.

[0128] Cell proliferation assay. The assay stock cells were thawed and diluted in the appropriate medium and dispensed into 384-well plates. The concentration was 800-3200 cells in 45 μl of medium per well 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 margins of the plate were filled with phosphate-buffered saline. The seeded cells were incubated at 37°C in a humidified atmosphere of 5% CO2. After 24 h, 5 μl of peg-crisantaspase solution and 5 μl of reference compound were added and the plates were further incubated for an additional 72 h. After 72 hours, the plates were cooled to room temperature for 30 minutes and 25 μl of ATPlite 1Step™ (PerkinElmer) solution was added to each well, followed by shaking for 2 minutes. After 5 minutes of incubation at room temperature in the dark, fluorescence was recorded on an Envision multimode reader (PerkinElmer).

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

[0130] Cell proliferation controls. Cell doubling times for all cell lines are calculated from the t=0 and t=end proliferation signals of untreated cells. If the doubling time is out of specification (0.5-2.0 fold deviation from the historical average) the assay is invalid.

[0131] Maximum signal. For each 384-well plate, the maximum fluorescence was recorded after 72 hours of incubation in the presence of 0.4% DMSO and without compound. All equivalent wells (usually 14) were averaged. This average is defined as: fluorescence 未処置,t=72h . Dose-response curves. Accurate single-agent IC for combination analysis 50 For each single agent, the dose-response signal was fitted with a four-parameter logistic curve using XL-fit 5 (IDBS Software): Fluorescence = Min + (Max - Min) / (1 + 10 (log I C 50 -log[cpd])·hill) ) [cpd] is the compound concentration tested. hill is the Hill coefficient. min and max are the asymptotic minimum and maximum of the curve. Identification of Combination Index (CI). CI is one of the most widely used quantitative indications of synergy. 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 a 10-fold lower concentration than would be expected from the single agent data to achieve the same level of effect. For example, 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 by 10-fold by the less potent compound.

[0132] CI is defined with respect to a certain cell viability percentage (V), where V is the signal relative to the unexposed control: V = 100% x fluorescence. 処置,t=72h / fluorescence 未処置,t=72h The concentrations of the two compounds, cpd1 and cpd2, required in combination to reach a certain percentage of cell viability V are then compared to the concentrations required for the single agents: CI (100-V) =[cpd1] V / I C (100-V),cpd1 +[cpd2] V / I C (100-V),cpd2 For example, [cpd1] 50 IC represents the concentration of CPD1 in the mixture that gives 50% survival. 50,cpd1 is IC of cpd1 alone 50 CI is, by convention, labeled by %-effect, so CI 75 represents the CI at 25% survival rate. Curve shift analysis. This analysis provides visual confirmation of synergy. 1 The concentrations of the mixture of compounds 1 and 2 (cpd1 and cpd2) and the single agents were determined based on the IC 50 In terms of equivalent (IC 50 Expressed in "units": [mix]=[cpd1] / IC 50,cpd1 +[cpd2] / IC 50,cpd2 The dose-response signal was fitted with a four-parameter logistic curve using XL-fit 5 (IDBS Software): Fluorescence = Min + (Max - Min) / (1 + 10 (logX-log[mix])·hill) ) where hill is the Hill coefficient and X is the inflection point of the curve. The minimum and maximum are the asymptotic minimum and maximum of the curve. [mix] is the IC 50 Since they are expressed in terms of equivalents, the curves for the single agents will overlap and their inflection point will be at a value of 1. The IC used in the calculation 50 Values ​​are from parallel determinations of single agents.

[0133] For mixtures where synergy is not present, the curves will overlap with those of the single agents. For mixtures where synergy is present, the curves will be closer to the IC 50 The equivalence will shift to the left toward lower equivalence: the mixture will appear to be more potent than would be expected based on the individual components. This is a good indication of synergy.

[0134] Isobolograms. Isobolograms are dose-centered plots that reveal whether drug combinations are synergistic. This is defined at a certain efficacy level, usually 75%. If the single agent curves do not reach this efficacy level, the isobologram level is set to 50%, 30%, 25%, or 20%. If the single agents do not reach 20% efficacy, no isobologram is drawn. On the axis, the calculated doses of the single compounds that give the predefined growth effect are plotted. Both points are connected by a straight line (additivity line). For drug combinations, it is calculated which dilution gives the predefined growth effect and the concentrations of the individual components at that point are plotted on the isobologram. In case of additive drug effects, the drug combination will be close to the additivity line. In case of synergy or antagonism, the points will be below or above the additivity line, respectively.

[0135] Experiments with inactive agents. In certain cases, synergy experiments are performed in the presence of an "inactive" agent. An "inactive" agent is a compound that does not give a dose-response curve as a single agent at the concentrations tested. The experiments are performed as described above, except that the "inactive" agent is added at a fixed concentration to each well of the experiment. Since the "inactive" agent alone has no effect, its contribution to the CI is negligible. Therefore, the CI value is based on the response of the active agent. The curve shift of the mixture is determined relative to the other, active agent. Isobolograms are not calculated. The dose-response curves for the single agents are shown in Figure 3. ABT-737 had an IC 50 835 nM and maximum efficacy of 67%, whereas PEG-crisantaspase has an IC 50 was 0.15 nM, and the maximum efficacy was 88%.

[0136] Curve shift analysis: The x-axis of the single agent curves (gray and dark gray) and mixture curves (red, orange, and pink) are plotted as the IC 50 Based on the IC 50 The conversions were then compared to the dose-response curves of the mixtures as shown in FIG.

[0137] I C 50 For the dose-response curves of the mixture at baseline, all curves were superimposed and the shifts recorded. A leftward shift of the mixture curve compared to the single agent curves (gray and dark grey) indicates synergy, whereas a rightward shift indicates antagonism (see FIG. 5 and table below). IC of the mixture compared to single agents 50 shift [Table 6]

[0138] The results using the combination of peg-crisantaspase and ABT-737 are shown below. CI values ​​calculated from mixture data, ED 75 corresponds to 25% viability. Representative values ​​are the mean CI at 50% viability for the three mixtures and are shown in the summary. [Table 7-1] [Table 7-2]

[0139] The combination data was used to generate an isobologram, shown in FIG. 6. An isobologram is a dose-centered plot that reveals whether a drug combination is synergistic. In the case of synergy, the combination points fall directly below the additivity line. The concentrations of peg-crisantaspase are shown in IU / mL. The additivity line (dark grey) indicates the concentration combination that would give theoretical additivity. Drug combinations are plotted as red, pink, and orange points. In summary, strong synergy was observed between peg-crisantaspase and ABT-737 in the HL-60 cell line, as shown below. [Table 8]

[0140] Example 6: This example was carried out in a similar manner to Example 5, except that synergy with additional anti-cancer drugs was tested in different cell types, as shown below. [Table 9]

[0141] Example 7: This example was performed in a similar manner to Example 1, except that mPEG-r-crisantaspase conjugates were tested for activity against CNS cell lines. CNS cell lines included, for example, glioblastoma, medulloblastoma, glioblastoma multiforme, and astrocytoma. The results are shown in Figure 7. Additional experiments with different cell lines were performed. The results are shown in Figure 10.

[0142] Example 8: Following the methods described in Example 1, mPEG-r-crisantaspase conjugate (Peg crisantaspase) 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. Data for the combination of peg crisantaspase and venetoclax showed strong synergy in AML cell lines. [Table 10]

[0143] Example 9 Following the method described in Example 1, the Pas-conjugated conjugates of crisantaspase were tested in multiple cell lines against PEGylated (PEG-crisantaspase) and non-PEGylated (Erwinase) forms of crisantaspase, together with L-asparaginase from E. coli (Oncaspar). PA-20 and PA-40 are Pas-conjugated crisantaspase conjugates produced in Corynebacterium or Pseudomonas expression systems, and PA-200 is a Pas-conjugated fusion protein produced in a Pseudomonas expression system. The PA-20, PA-40, PA-200, and PA-400 constructs are SEQ ID NOs: 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 biphenotypic B-cell myelomonocytic leukemia cell line, THP-1 is an AML cell line, RL is a non-Hodgkin's 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 be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A composition for treating glioblastoma or medulloblastoma in a patient, comprising: containing an effective amount of an L-asparaginase protein derived from the genus Erwinia comprising SEQ ID NO: 1; The glioblastoma or medulloblastoma comprises cells having a copy number mutation of the CDKN2A gene. composition.

2. 2. The composition of claim 1, wherein the L-asparaginase exhibits at least 10-fold greater activity against the glioblastoma or medulloblastoma compared to E. coli asparaginase.

3. 1. A composition for treating acute promyelocytic leukemia in a patient, comprising: containing an effective amount of a conjugate of a protein having L-asparagine aminohydrolase activity and polyethylene glycol (PEG); The molecular weight of the polyethylene glycol is 5000 Da or less, the protein is an L-asparaginase derived from the genus Erwinia comprising SEQ ID NO: 1; the conjugate is administered as part of a combination therapy with ABT-737; composition.

4. 1. A composition for treating colon cancer in a patient, comprising: containing an effective amount of a conjugate of a protein having L-asparagine aminohydrolase activity and polyethylene glycol (PEG); The molecular weight of the polyethylene glycol is 5000 Da or less, The protein is an L-asparaginase from the genus Erwinia and has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

1. composition.

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

1.

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

1.

7. 7. The composition of claim 4, wherein the PEG has a molecular weight of 5000 Da, 4000 Da, 3000 Da, 2500 Da, or 2000 Da.

8. i) the conjugate has at least 60% of the in vitro activity of the L-asparaginase when not conjugated with PEG; ii) the conjugate has an L-asparagine-depleting activity that is at least 10 times more potent than the L-asparaginase when not conjugated to PEG; iii) the complex depletes plasma L-asparagine levels to undetectable levels for at least 12 hours; iv) the conjugate has a longer in vivo circulatory half-life than the L-asparaginase when not conjugated to PEG; v) the conjugate has a longer t½ than pegaspargase administered at an equivalent protein dose; vi) the conjugate has a t of at least 58 to 65 hours at a dose of 50 μg / kg of protein content and a t of at least 34 to 40 hours at a dose of 10 μg / kg of protein content after iv administration in mice; vii) the complex has a concentration of 10,000 to 15,000 IU / m ２ (20 to 30 mg protein / m ２ ) has a t of at least 100 to 200 hours at doses in the range of viii) the conjugate has a greater area under the curve (AUC) than the L-asparaginase when not conjugated to PEG; and / or ix) the conjugate has a mean AUC at least 3 times greater than pegaspargase at an equivalent protein dose; The composition according to any one of claims 4 to 7.

9. i) the PEG is covalently attached to one or more amino groups of the L-asparaginase; ii) the PEG is covalently attached to the one or more amino groups via an amide bond; iii) the PEG is covalently bonded to at least 40% to 100% of the accessible amino groups, or at least 40% to 90% of the total amino groups; iv) the conjugate has the following formula: Asp-[NH-CO-(CH ２ ) ｘ -CO-NH-PEG] ｎ wherein Asp is said L-asparaginase, NH is one or more of the lysine residues and / or N-terminal NH groups of said Asp, PEG is a polyethylene glycol moiety, n is a number representing at least 40% to 100% of the accessible amino groups of said Asp, and x is an integer ranging from 1 to 8; v) the conjugate has the following formula: Asp-[NH-CO-(CH ２ ) ｘ -CO-NH-PEG] ｎ wherein Asp is said L-asparaginase, NH is one or more of the lysine residues and / or N-terminal NH groups of said Asp, PEG is a polyethylene glycol moiety, n is a number representing at least 40% to 100% of the accessible amino groups of said Asp, and x is an integer ranging from 2 to 5; and / or vi) the PEG is monomethoxy-polyethylene glycol (mPEG); 9. The composition of any one of claims 4 to 8.

10. 10. The composition of any one of claims 4 to 9, wherein the colon cancer is colon carcinoma, colon adenocarcinoma, or colorectal adenocarcinoma.

11. The composition of claim 10 , wherein the colon cancer is colon adenocarcinoma.

12. The complex is i) in an amount of 5 U / kg body weight to 50 U / kg body weight; ii) 10,000 to 15,000 IU / m ２ At doses ranging from; iii) via intravenous administration; iv) intramuscularly; and / or v) once a week, twice a week, or three times a week; The composition of any one of claims 4 to 11, wherein the composition is administered intravenously.

13. 13. The composition of any one of claims 4 to 12, wherein the conjugate is administered as a monotherapy or as part of a combination therapy.

14. 14. The composition of claim 13, wherein the combination therapy comprises Oncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactolisib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839.

15. 15. The composition of any one of claims 4 to 14, wherein the colon cancer comprises cells with mutations in the NRAS gene, the PTEN gene, the ERBB2 gene, and / or the CDKN2A gene.

16. The composition of claim 15, wherein the mutation in the CDKN2A gene is a copy number mutation.

17. 1. A composition for treating a disease treatable by L-asparagine depletion in a patient, comprising: containing an effective amount of a conjugate of a protein having L-asparagine aminohydrolase activity and polyethylene glycol (PEG); The molecular weight of the polyethylene glycol is 5000 Da or less, the protein is an L-asparaginase from the genus Erwinia and has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1; the disease is a cancer selected from glioblastoma, neuroblastoma, medulloblastoma, breast carcinoma, breast adenocarcinoma, ductal carcinoma, colon carcinoma, colon adenocarcinoma, or colorectal adenocarcinoma; the cancer comprises cells with a mutation in a gene selected from NRAS, PTEN, ERBB2, CDKN2A, or a combination thereof; composition.

18. 18. The composition of claim 17, wherein the L-asparaginase has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

1.

19. 19. The composition of claim 18, wherein the complex comprises an L-asparaginase from the genus Erwinia having 100% sequence identity with the amino acid sequence of SEQ ID NO:

1.

20. 20. The composition of any one of claims 17 to 19, wherein the PEG has a molecular weight of 5000 Da, 4000 Da, 3000 Da, 2500 Da, or 2000 Da.

21. i) the conjugate has at least 60% of the in vitro activity of the L-asparaginase when not conjugated with PEG; ii) the conjugate has an L-asparagine-depleting activity that is at least 10 times more potent than the L-asparaginase when not conjugated to PEG; iii) the complex depletes plasma L-asparagine levels to undetectable levels for at least 12 hours; iv) the conjugate has a longer in vivo circulatory half-life than the L-asparaginase when not conjugated to PEG; v) the conjugate has a longer t½ than pegaspargase administered at an equivalent protein dose; vi) the conjugate has a t of at least 58 to 65 hours at a dose of 50 μg / kg of protein content and a t of at least 34 to 40 hours at a dose of 10 μg / kg of protein content after iv administration in mice; vii) the complex has a concentration of 10,000 to 15,000 IU / m ２ (20 to 30 mg protein / m ２ ) has a t of at least 100 to 200 hours at doses in the range of viii) the conjugate has a greater area under the curve (AUC) than the L-asparaginase when not conjugated to PEG; and / or ix) the conjugate has a mean AUC at least 3 times greater than pegaspargase at an equivalent protein dose; 21. The composition of any one of claims 17 to 20.

22. i) the PEG is covalently attached to one or more amino groups of the L-asparaginase; ii) the PEG is covalently attached to the one or more amino groups via an amide bond; iii) the PEG is covalently bonded to at least 40% to 100% of the accessible amino groups, or at least 40% to 90% of the total amino groups; iv) the conjugate has the following formula: Asp-[NH-CO-(CH ２ ) ｘ -CO-NH-PEG] ｎ wherein Asp is said L-asparaginase, NH is one or more of the lysine residues and / or N-terminal NH groups of said Asp, PEG is a polyethylene glycol moiety, n is a number representing at least 40% to 100% of the accessible amino groups of said Asp, and x is an integer ranging from 1 to 8; v) the conjugate has the following formula: Asp-[NH-CO-(CH ２ ) ｘ -CO-NH-PEG] ｎ wherein Asp is said L-asparaginase, NH is one or more of the lysine residues and / or N-terminal NH groups of said Asp, PEG is a polyethylene glycol moiety, n is a number representing at least 40% to 100% of the accessible amino groups of said Asp, and x is an integer ranging from 2 to 5; and / or vi) the PEG is monomethoxy-polyethylene glycol (mPEG); 22. The composition of any one of claims 17 to 21.

23. 23. The composition of any one of claims 17 to 22, wherein the disease is selected from glioblastoma, neuroblastoma, or medulloblastoma.

24. 23. The composition of any one of claims 17 to 22, wherein the disease is selected from breast carcinoma, breast adenocarcinoma, or breast ductal carcinoma.

25. 23. The composition of any one of claims 17 to 22, wherein the disease is colon carcinoma, colon adenocarcinoma, or colorectal adenocarcinoma.

26. The complex is i) in an amount of 5 U / kg body weight to 50 U / kg body weight; ii) 10,000 to 15,000 IU / m ２ At doses ranging from; iii) via intravenous administration; iv) intramuscularly; and / or v) once a week, twice a week, or three times a week; 26. The composition of any one of claims 17 to 25, wherein the composition is administered intravenously.

27. 27. The composition of any one of claims 17 to 26, wherein the conjugate is administered as a monotherapy or as part of a combination therapy.

28. 28. The composition of claim 27, wherein the combination therapy comprises Oncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactolisib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839.

29. 29. The composition of any one of claims 17 to 28, wherein the disease is cancer containing cells with mutations in the NRAS gene, the PTEN gene, and / or the ERBB2 gene.

30. A composition described in any one of claims 17 to 29, wherein the disease is cancer containing cells having a mutation in the CDKN2A gene, and the mutation is a copy number mutation.

31. 1. A composition for treating cancer in a patient, comprising: containing an effective amount of a conjugate of a protein having L-asparagine aminohydrolase activity and polyethylene glycol (PEG); The molecular weight of the polyethylene glycol is 5000 Da or less, the protein is an L-asparaginase from the genus Erwinia and has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1; the conjugate is administered as part of a combination therapy with ABT-737, venetoclax, everolimus, and / or CB-839; the cancer is selected from lymphoma, large cell immunoblastic lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, NK lymphoma, Hodgkin's disease, leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, renal cell carcinoma, renal cell adenocarcinoma, glioblastoma, neuroblastoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung carcinoma including large cell lung carcinoma and small cell lung carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian teratocarcinoma, cervical adenocarcinoma, breast carcinoma, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon carcinoma, colorectal adenocarcinoma, transitional cell carcinoma of the bladder, bladder papilloma, prostate carcinoma, osteosarcoma, epidermoid carcinoma of bone, or thyroid cancer; composition.

32. 32. The composition of claim 31, wherein the L-asparaginase has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

1.

33. 33. The composition of claim 32, wherein the complex comprises an L-asparaginase from the genus Erwinia having 100% sequence identity with the amino acid sequence of SEQ ID NO:

1.

34. 34. The composition of any one of claims 31 to 33, wherein the PEG has a molecular weight of 5000 Da, 4000 Da, 3000 Da, 2500 Da, or 2000 Da.

35. i) the conjugate has at least 60% of the in vitro activity of the L-asparaginase when not conjugated with PEG; ii) the conjugate has an L-asparagine-depleting activity that is at least 10 times more potent than the L-asparaginase when not conjugated to PEG; iii) the complex depletes plasma L-asparagine levels to undetectable levels for at least 12 hours; iv) the conjugate has a longer in vivo circulatory half-life than the L-asparaginase when not conjugated to PEG; v) the conjugate has a longer t½ than pegaspargase administered at an equivalent protein dose; vi) the conjugate has a t of at least 58 to 65 hours at a dose of 50 μg / kg of protein content and a t of at least 34 to 40 hours at a dose of 10 μg / kg of protein content after iv administration in mice; vii) the complex has a concentration of 10,000 to 15,000 IU / m ２ (20 to 30 mg protein / m ２ ) has a t of at least 100 to 200 hours at doses in the range of viii) the conjugate has a greater area under the curve (AUC) than the L-asparaginase when not conjugated to PEG; and / or ix) the conjugate has a mean AUC at least 3 times greater than pegaspargase at an equivalent protein dose; 35. The composition of any one of claims 31 to 34.

36. i) the PEG is covalently attached to one or more amino groups of the L-asparaginase; ii) the PEG is covalently attached to the one or more amino groups via an amide bond; iii) the PEG is covalently bonded to at least 40% to 100% of the accessible amino groups, or at least 40% to 90% of the total amino groups; iv) the conjugate has the following formula: Asp-[NH-CO-(CH ２ ) ｘ -CO-NH-PEG] ｎ wherein Asp is said L-asparaginase, NH is one or more of the lysine residues and / or N-terminal NH groups of said Asp, PEG is a polyethylene glycol moiety, n is a number representing at least 40% to 100% of the accessible amino groups of said Asp, and x is an integer ranging from 1 to 8; v) the conjugate has the following formula: Asp-[NH-CO-(CH ２ ) ｘ -CO-NH-PEG] ｎ wherein Asp is said L-asparaginase, NH is one or more of the lysine residues and / or N-terminal NH groups of said Asp, PEG is a polyethylene glycol moiety, n is a number representing at least 40% to 100% of the accessible amino groups of said Asp, and x is an integer ranging from 2 to 5; and / or vi) the PEG is monomethoxy-polyethylene glycol (mPEG); 36. The composition of any one of claims 31 to 35.

37. 37. The composition of any one of claims 31 to 36, wherein the cancer is glioblastoma, neuroblastoma, or medulloblastoma.

38. 37. The composition of any one of claims 31 to 36, wherein the cancer is leukemia.

39. 39. The composition of claim 38, wherein the leukemia is selected from acute monocytic leukemia, acute T-cell leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), biphenotypic B-cell myelomonocytic leukemia, or chronic lymphocytic leukemia.

40. The cancer is i) breast carcinoma, adenocarcinoma, or ductal carcinoma; or ii) colon carcinoma, colon adenocarcinoma, or colorectal adenocarcinoma 37. The composition of any one of claims 31 to 36, selected from:

41. The complex is i) in an amount of 5 U / kg body weight to 50 U / kg body weight; ii) 10,000 to 15,000 IU / m ２ At doses ranging from; iii) via intravenous administration; iv) intramuscularly; and / or v) once a week, twice a week, or three times a week; 41. The composition of any one of claims 31 to 40, wherein the composition is administered intravenously.

42. 42. The composition of any one of claims 37 to 41, wherein the conjugate is administered as a monotherapy or as part of a combination therapy.

43. 43. The composition of claim 42, wherein the combination therapy further comprises Oncaspar®, daunorubicin, cytarabine, Vyxeos®, dactolisib, bortezomib, carfilzomib, vincristine, and / or prednisolone.

44. 44. The composition of any one of claims 31 to 43, wherein the cancer comprises cells with mutations in the NRAS gene, the PTEN gene, the ERBB2 gene, and / or the CDKN2A gene.

45. 45. The composition of claim 44, wherein the mutation in the CDKN2A gene is a copy number mutation.

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