Modified L-asparaginase

Abstract

The present invention relates to a modified L-asparaginase. [Solution] The present invention relates to modified proteins that are combinations of (i) L-asparaginase and (ii) one or more (poly)peptides, where the (poly)peptides consist solely of proline and alanine amino acid residues. The modified proteins can be formed in several ways, including by expressing the modified proteins as chemical conjugates or fusion proteins between L-asparaginase and the (poly)peptides. Also provided herein are nucleic acids encoding the modified proteins, vectors and / or host cells containing them, and processes for their production. Compositions containing the modified proteins and their use in medicine, particularly in the treatment of cancer, are disclosed. In another aspect of the invention, the L-asparaginase can be derived from Erwinia and / or has at least 85% identity to the amino acid sequence of SEQ ID NO:1. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention relates to modified proteins that are combinations of (i) L-asparaginase and (ii) one or more (poly)peptides, where the (poly)peptides consist solely of proline and alanine amino acid residues. The modified proteins can be formed in several ways, including by expressing the modified proteins as chemical conjugates or fusion proteins between L-asparaginase and the (poly)peptides. Also provided herein are nucleic acids encoding the modified proteins, vectors and / or host cells containing the same, and processes for their production. Compositions containing the modified proteins and their use in medicine, particularly in the treatment of cancer, are disclosed. In another aspect of the invention, the L-asparaginase is derived from Erwinia and / or has at least 85% identity to the amino acid sequence of SEQ ID NO:1. [Background technology]

[0002] A protein with L-asparagine aminohydrolase activity, commonly known as L-asparaginase, has been used successfully for many years in the treatment of acute lymphoblastic leukemia (ALL) in children, the most common childhood malignancy (Avramis, (2005), Clin. Pharmacokinet. 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 thought to result from the inability or reduced ability of certain malignant cells to synthesize L-asparagine (ibid.). These malignant cells depend on an extracellular supply of L-asparagine. However, the L-asparaginase enzyme catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia, thereby depleting the circulating pool of L-asparagine and killing tumor cells that cannot perform protein synthesis without it (ibid.).

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

[0005] L-asparaginase of bacterial origin has high immunogenic and antigenic potential, often causing adverse reactions ranging from mild allergic reactions to anaphylactic shock in sensitized patients (Wang, (2003), Leukemia, 17, 1583-1588). E. coli L-asparaginase is particularly immunogenic, with reports of the presence of anti-asparaginase antibodies against E. coli L-asparaginase reaching as high as 78% in adults and 70% in children after intravenous or intramuscular administration (ibid.).

[0006] L-asparaginase derived from Escherichia coli and Erwinia chrysanthemi differ in their pharmacokinetic properties and have distinct immunogenicity profiles (Klug Albertsen, (2001), Brit. J. Haematol. 115, 983-990). Furthermore, it has been shown that antibodies generated after treatment with L-asparaginase derived from E. coli do not cross-react with L-asparaginase derived from Erwinia (Wang, (2003), Leukemia, 17, 1583-1588). Therefore, L-asparaginase derived from Erwinia (crisantaspase) has been used as a second-line treatment for ALL in patients who respond to L-asparaginase derived from E. coli (Duval, (2002), Blood, 15, 2734-2739; Avramis, (2005), Clin. Pharmacokinet. 44, 367-393).

[0007] Another attempt to reduce the immunogenicity associated with the administration of microbial L-asparaginase has involved the development of E. coli L-asparaginase modified with methoxy-polyethylene glycol (mPEG). This so-called mPEG-L-asparaginase, or pegaspargase commercially available as Oncaspar® (Enzon Inc.), was first approved in the United States for second-line treatment of ALL in 1994 and has been approved for first-line therapy of ALL in children and adults since 2006.

[0008] Oncaspar® is an L-asparaginase from E. coli modified at multiple lysine residues with 5 kDa mPEG-succinimidyl succinate (SS-PEG) (U.S. Patent No. 4,179,337). SS-PEG is a first-generation PEG reagent that contains labile ester bonds that are susceptible to enzymatic hydrolysis or at slightly alkaline pH values ​​(U.S. Patent No. 4,670,417). These properties may reduce stability both in vitro and in vivo, compromising the safety of the drug.

[0009] Furthermore, it has been shown that antibodies developed against L-asparaginase from E. coli may cross-react with Oncaspar® (Wang, (2003), Leukemia, 17, 1583-1588). Although these were not neutralizing antibodies, these findings clearly indicated a high likelihood of cross-hypersensitivity or cross-inactivation in vivo. Indeed, in one report, 30-41% of children treated with pegaspargase had an allergic reaction (ibid.).

[0010] In addition to apparent allergic reactions, the problem of "silent hypersensitivity" has recently been reported, whereby patients develop anti-asparaginase antibodies without clinical evidence of a hypersensitivity reaction (Wang, (2003), Leukemia, 17, 1583-1588). This reaction can result in the formation of neutralizing antibodies against E. coli L-asparaginase and pegaspargase, but because of the lack of outward signs of hypersensitivity, these patients are not transitioned to Erwinia L-asparaginase, and therefore they receive effective treatment for a shorter period of time (Holcenberg, (2004), J. Pediatr. Hematol. Oncol. 26, 273-274).

[0011] Treatment with Erwinia chrysanthemi L-asparaginase is often used in cases of hypersensitivity to E. coli-derived L-asparaginase. However, as many as 30-50% of patients treated with Erwinia L-asparaginase have been observed to be seropositive (Avramis, (2005), Clin. Pharmacokinet. 44, 367-393). Furthermore, Erwinia chrysanthemi L-asparaginase has a shorter elimination half-life than E. coli L-asparaginase, so it must be administered more frequently (ibid.). In a study by Avramis et al., Erwinia asparaginase was associated with inferior pharmacokinetic properties (Avramis, (2007), J. Pediatr. Hematol. Oncol. 29, 239-247). Therefore, E. coli L-asparaginase and pegaspargase have become the preferred first-line therapy for ALL compared with Erwinia L-asparaginase.

[0012] Many biopharmaceuticals have been successfully PEGylated and have been commercially available for many years. However, in many cases, PEGylated biopharmaceuticals exhibit significantly reduced activity compared to unmodified biopharmaceuticals. In the case of L-asparaginase from Erwinia carotovora, PEGylation was observed to reduce its in vitro activity to approximately 57% (Kuchumova, (2007), Biochemistry, (Moscow), Supplement Series, B: Biomedical Chemistry, 1, 230-232). L-asparaginase from Erwinia carotovora shares only about 75% homology with L-asparaginase (crisantaspase) from Erwinia chrysanthemi. It is also known that Oncaspar® exhibits approximately 50% in vitro activity compared to unmodified L-asparaginase from E. coli.

[0013] Therefore, the technical problem underlying the present invention is to provide means and methods for treating cancer, such as, for example, leukemia or non-Hodgkin's lymphoma, that avoid the limitations and shortcomings of prior art therapies, particularly some pegylated asparaginases.

[0014] This technical problem is solved by providing the embodiments characterized in the claims. Summary of the Invention

[0015] In one aspect, the present invention relates to a modified protein comprising (i) an L-asparaginase and (ii) one or more (poly)peptides, wherein the (poly)peptides consist solely of proline and alanine amino acid residues. In a preferred aspect, the present invention relates to a modified protein comprising (i) an L-asparaginase having at least 85% identity to the amino acid sequence of SEQ ID NO: 1 and (ii) one or more (poly)peptides, wherein the (poly)peptides consist solely of proline and alanine amino acid residues.

[0016] The present invention relates, inter alia, to the following items: 1. A modified protein comprising (i) L-asparaginase and (ii) one or more (poly)peptides, wherein the (poly)peptides consist solely of proline and alanine amino acid residues.

[0017] 2. The modified protein according to item 1, wherein the L-asparaginase has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.

[0018] 3. The modified protein according to item 1 or 2, wherein the L-asparaginase has the amino acid sequence of SEQ ID NO: 1.

[0019] 4. The modified protein according to any one of items 1 to 3, wherein the modified protein has higher asparaginase activity or glutaminase activity than that of unmodified L-asparaginase.

[0020] 5. The modified protein according to any one of items 1 to 4, wherein the modified protein has an L-asparagine-depleting activity that is at least about 20% higher than that of unmodified L-asparaginase.

[0021] 6. The modified protein according to any one of items 1 to 5, wherein the L-asparaginase is a tetramer.

[0022] 7. The modified protein according to any one of items 1 to 6, which is a modified protein of the L-asparaginase and a polypeptide, wherein the polypeptide consists only of proline and alanine amino acid residues.

[0023] 8. The modified protein according to item 7, wherein the polypeptide consists of about 100 to 600 amino acid residues of proline and alanine, particularly about 200 to 400 amino acid residues of proline and alanine.

[0024] 9. The modified protein according to item 7, wherein the polypeptide consists of a total of about 200 proline and alanine amino acid residues, or a total of about 400 proline and alanine amino acid residues.

[0025] 10. The modified protein according to any one of items 7 to 9, wherein the proline residues constitute more than about 10% and less than about 70% of the polypeptide.

[0026] 11. The modified protein according to any one of items 7 to 10, wherein the polypeptide comprises a plurality of amino acid repeats, the repeats consisting of proline and alanine residues, and wherein no more than six consecutive amino acid residues are identical.

[0027] 12. The polypeptide has the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or the sequence of the above sequence as a whole or a part thereof. circular permutation type 12. The modified protein according to any one of items 7 to 11, comprising or consisting of a multimer(s).

[0028] 13. (a) the polypeptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 7 or 9; (b) the polypeptide comprises or consists of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 8 or 10. 13. The modified protein according to any one of items 7 to 12.

[0029] 14. (a) the modified protein comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 11 or 13; (b) the modified protein comprises or consists of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 12 or 14. 14. The modified protein according to any one of items 7 to 13.

[0030] 15. The modified protein according to any one of items 7 to 14, wherein the polypeptide is a random coil polypeptide.

[0031] 16. The modified protein according to any one of items 7 to 15, wherein the modified protein is a fusion protein of L-asparaginase and a polypeptide.

[0032] 17. L-asparaginase and peptide R N -(P / A)-R C and a modified protein with one or more peptides, (P / A) is an amino acid sequence consisting of only proline and alanine amino acid residues, and R N is a protecting group linked to the N-terminal amino group of the amino acid sequence, R C is an amino acid residue bound to the C-terminal carboxyl group of the amino acid sequence via its amino group, Each peptide is composed of the C-terminal amino acid residue R Cand the free amino group of the L-asparaginase, 7. The modified protein according to any one of items 1 to 6, wherein at least one of the free amino groups to which the peptide is conjugated is not the N-terminal α-amino group of the L-asparaginase.

[0033] 18. The modified protein according to item 17, wherein the amino acid sequence consists of a total of 15 to 45 proline and alanine amino acid residues.

[0034] 19. The modified protein according to item 17 or 18, wherein the amino acid sequence consists of 20 proline and alanine amino acid residues.

[0035] 20. The modified protein according to item 17 or 18, wherein the amino acid sequence consists of 40 proline and alanine amino acid residues.

[0036] 21. The modified protein according to any one of items 17 to 20, wherein the proline residues constitute more than about 10% and about 70% of the amino acid sequence.

[0037] 22. The modified protein according to any one of items 17 to 21, wherein the amino acid sequence is AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 15).

[0038] 23.R N is pyroglutamoyl or acetyl, and / or R C 23. The modified protein according to any one of items 17 to 22, wherein the amino acid is ε-aminohexanoic acid.

[0039] 24. The modified protein according to any one of items 17 to 23, wherein a peptide contained in the modified protein adopts a random coil structure.

[0040] 25. The modified protein according to any one of items 17 to 24, wherein all of the peptides contained in the modified protein are identical.

[0041] 26. The modified protein according to any one of items 17 to 25, wherein at least one of the free amino groups to which the peptide is conjugated is the ε-amino group of a lysine residue of the L-asparaginase.

[0042] 27. The modified protein according to any one of items 17 to 26, wherein the free amino group to which the peptide is conjugated is selected from the group comprising the ε-amino group(s) of any lysine residue(s) of the L-asparaginase and the N-terminal α-amino group(s) of the L-asparaginase.

[0043] 28. The modified protein according to any one of items 17 to 27, wherein the L-asparaginase is composed of four subunits and 9 to 13 peptides as defined in any one of items 15 to 24 are conjugated to each subunit of the L-asparaginase.

[0044] 29. The modified protein according to any one of items 1 to 28, wherein said polypeptide or peptide mediates a reduction in the immunogenicity of said modified protein.

[0045] 30. A nucleic acid encoding a modified protein according to any one of items 1 to 16.

[0046] 31. The nucleic acid is (a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12 or 14; (b) a nucleic acid comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence as defined in (a), and (c) a nucleic acid that is degenerate as a result of the genetic code for a nucleotide sequence as defined in (a) or (b). 31. The nucleic acid according to item 30, selected from the group consisting of:

[0047] 32. A vector comprising the nucleic acid according to item 30 or 31.

[0048] 33. A host cell comprising a nucleic acid according to item 30 or 31 or a vector according to item 32.

[0049] 34. The host cell according to item 33, wherein the host cell is selected from the group consisting of Pseudomonas fluorescens and Corynebacterium glutamicum.

[0050] 35. A process for the preparation of a modified protein according to any one of items 1 to 16, 29 or a nucleic acid according to item 30 or 31.

[0051] 36. The process according to item 35, comprising culturing a host cell according to item 33 or 34 and isolating the modified protein from the culture or from the cell.

[0052] 37. A process for preparing a protein as defined in any one of items 17 to 29, comprising: The process comprises: (a) reacting a compound of formula R N -(P / A)-R C-act (In the formula, R C-act is R C is the carboxy-activated form of R C and (P / A) are as defined for the modified protein to be prepared, and R N is a protecting group linked to the N-terminal amino group of (P / A)) is coupled with L-asparaginase to form a peptide having a structure similar to that of the L-asparaginase. N wherein is a protecting group to obtain a modified protein.

[0053] 38. The amino acid residue R in the activated peptide C-act38. The process according to item 37, wherein the activated carboxy group is an activated ester group.

[0054] 39. A composition comprising a modified protein according to any one of items 1 to 29 or a modified protein prepared by the process according to any one of items 35 to 38.

[0055] 40. The composition according to item 39, which is a pharmaceutical composition optionally further comprising pharmaceutically acceptable carrier(s) or excipient(s).

[0056] 41. A modified protein according to any one of items 1 to 29 or a modified protein prepared by the process according to any one of items 35 to 38, or a composition according to item 39 or 40, for use as a medicament.

[0057] 42. A modified protein according to any one of items 1 to 29 or a modified protein prepared by a process according to any one of items 35 to 38, or a composition according to item 39 or 40, for use in treating a disease in a patient, for example a disease that can be treated by depletion of L-asparagine.

[0058] 43. A method for treating a disease treatable by L-asparagine depletion in a patient, the method comprising administering to the patient an effective amount of the modified protein of any one of items 1 to 29, or the modified protein prepared by the process of any one of items 35 to 38, or the composition of item 39 or 40.

[0059] 44. The modified protein for use according to item 42, the composition for use according to item 42, or the method according to item 43, wherein the disease treatable by depletion of L-asparagine is cancer.

[0060] 45. A modified protein according to any one of items 1 to 29, a modified protein prepared by the process according to any one of items 35 to 38, or a composition according to item 39 or 40 for use in the treatment of cancer.

[0061] 46. ​​A method for treating cancer, comprising administering to a subject a modified protein according to any one of items 1 to 29, a modified protein prepared by the process according to any one of items 35 to 38, or a composition according to item 39 or 40.

[0062] 47. The modified protein for use according to item 44 or 45, or the composition for use according to item 44 or 45, wherein the cancer is a non-solid cancer, or the method according to item 44 or 46, wherein the cancer is a non-solid cancer.

[0063] 48. The modified protein for use according to item 47, or the composition for use according to item 47, wherein the non-solid cancer is leukemia or non-Hodgkin's lymphoma, or the method according to item 47, wherein the non-solid cancer is leukemia or non-Hodgkin's lymphoma.

[0064] 49. The modified protein for use according to item 48 or the composition for use according to item 48, wherein the leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML), or the method according to item 48, wherein the leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

[0065] 50. The modified protein for use according to any one of items 42, 44, 45 and 47 to 49, or the composition for use according to any one of items 42, 44, 45 and 47 to 49, or the method according to any one of items 43, 44 and 46 to 49, wherein the modified protein causes a lower immunogenic response in the patient compared to unmodified L-asparaginase.

[0066] 51. The modified protein for use according to any one of items 42, 44, 45 and 47 to 50, or the composition for use according to any one of items 42, 44, 45 and 47 to 50, or the method according to any one of items 43, 44 and 46 to 50, wherein the modified protein has a longer circulating half-life in vivo after a single administration compared to unmodified L-asparaginase.

[0067] 52. The modified protein for use according to any one of items 42, 44, 45, and 47 to 51, or the composition for use according to any one of items 42, 44, 45, and 47 to 51, or the method according to any one of items 43, 44, and 46 to 51, wherein the modified protein has a greater AUC value after a single administration compared to unmodified L-asparaginase.

[0068] 53. The modified protein for use according to any one of items 42, 44, 45 and 47 to 52, or the composition for use according to any one of items 42, 44, 45 and 47 to 52, or the method according to any one of items 43, 44 and 46 to 52, wherein the patient has a history of hypersensitivity to E. coli L-asparaginase or a pegylated form thereof.

[0069] 54. The modified protein for use according to any one of items 42, 44, 45 and 47 to 53, or the composition for use according to any one of items 42, 44, 45 and 47 to 53, or the method according to any one of items 43, 44 and 46 to 53, wherein the patient has a history of hypersensitivity to Erwinia L-asparaginase.

[0070] 55. The modified protein for use according to any one of items 42, 44, 45 and 47 to 54, or the composition for use according to any one of items 42, 44, 45 and 47 to 54, or the method according to any one of items 43, 44 and 46 to 54, wherein the treatment comprises intravenous administration of the modified protein. [Brief explanation of the drawings]

[0071] [Figure 1] Figure 1 shows the chemistry of conjugation of crisantaspase with an N-terminally protected P / A peptide via the amino group. (A) and (B) show the chemical structures of P / A peptides containing 20 or 40 Pro / Ala residues (respectively) obtained by solid-phase peptide synthesis (SEQ ID NOs: 16 and 17, amino acid sequences shown in SEQ ID NOs: 5 and 15). To prevent peptide polymerization during chemical activation of the C-terminus, the N-terminus was protected with a pyroglutamyl (Pga) residue. Aminohexanoic acid (Ahx) was incorporated into the C-terminus of the peptide as a linker. (C) The N-terminally protected P / A peptide was activated at the C-terminus with the benzotriazole derivative O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) in the presence of the non-nucleophilic base N,N-diisopropylethylamine (DIPEA, Hunig's base) and DMSO as the solvent. The amino groups of crisantaspase (the ε-amino groups of lysine residues or the α-amino group at the N-terminus) are then derivatized with the P / A peptide via the formation of a peptide or isopeptide bond using an HOBt active ester of the peptide, releasing free hydroxybenzotriazole (HOBt). This coupling step is performed in an aqueous solution (e.g., PBS buffer) containing 30% or less organic solvent. The modified P / A-crisantaspase protein may be purified from the remaining P / A peptide / coupling reagent by dialysis and / or chromatography (e.g., ion exchange chromatography). [Figure 2]Figure 1 shows the optimization of the coupling ratio of crisantaspase / Pga-P / A(20)-Ahx. As described in Example 1, recombinant crisantaspase produced in E. coli was conjugated with the Pga-P / A#1(20)-Ahx peptide (SEQ ID NOs: 16 and 17; the amino acid sequences are shown in SEQ ID NOs: 5 and 15). The peptide-to-protein ratio was varied between 3.5 mg and 10 mg of P / A peptide per 1 mg of crisantaspase. Seven micrograms of crisantaspase from each coupling reaction was loaded onto the gel. Additionally, a mix of coupling reactions at ratios of 0.3 to 10 mg of peptide per mg of protein was applied as a size standard ("Std"). The number of coupled P / A peptides can be determined by counting the bands in the ladder starting from unconjugated crisantaspase, as indicated on the right. Lane "kDa": Pierce™ unstained protein molecular weight marker (Thermo Fisher Scientific). [Figure 3]Figure 1 shows the purification of the crisantaspase / Pga-P / A(40)-Ahx peptide coupling product via anion exchange chromatography. As described in Example 2, recombinant crisantaspase produced in E. coli was conjugated to the Pga-P / A(40)-Ahx peptide (Figure 1B) (SEQ ID NO: 17; the amino acid sequence is shown in SEQ ID NO: 15). After dialysis against AIX running buffer (25 mM boric acid / NaOH, pH 9.0, 1 mM EDTA), anion exchange chromatography was performed on an 85 mL Source™ 15Q column (A). By applying a NaCl gradient, the enzymatically modified protein eluted in a single sharp peak, as revealed by a UV trace at 280 nm. Separation of the remaining uncoupled peptide and other non-proteinaceous by-products of the chemical conjugate, which lack UV absorption at 280 nm, was monitored by a UV trace at 225 nm. (B) SDS-PAGE analysis of the modified crisantaspase / Pga-P / A(40)-Ahx protein after purification by anion exchange chromatography (lane 1). The coupling reaction mix at a ratio of 0.3–10 mg of peptide per mg of protein was applied to lane 2 to allow determination of the number of coupled P / A peptides per crisantaspase monomer. A PageRuler™ Plus prestained marker (Thermo Fisher Scientific) was applied to lane "M." [Figure 4]Figure 1 shows the purification of the crisantaspase / Pga-P / A(20)-Ahx peptide coupling product via anion exchange chromatography. Recombinant crisantaspase produced in E. coli was conjugated to the Pga-P / A(20)-Ahx peptide (Figure 1A) (SEQ ID NO: 16; the amino acid sequence is shown in SEQ ID NO: 5) as described in Example 3. After dialysis against AIX running buffer (25 mM boric acid / NaOH, pH 9.0, 1 mM EDTA), anion exchange chromatography was performed on an 85 mL Source™ 15Q column (A). By applying a NaCl gradient, the enzymatically modified protein eluted in a single sharp peak, as revealed by a UV trace at 280 nm. Separation of the remaining uncoupled peptide and other non-proteinaceous by-products of the chemical conjugate, which lack UV absorption at 280 nm, was revealed by a UV trace at 225 nm. (B) SDS-PAGE analysis of the modified crisantaspase / Pga-P / A(20)-Ahx protein after purification by anion exchange chromatography (lane 1). The coupling reaction mix at a ratio of 0.3–10 mg of peptide per mg of protein was applied to lane 2 to allow determination of the number of coupled P / A peptides per crisantaspase monomer. A PageRuler™ Plus prestained marker (Thermo Fisher Scientific) was applied to lane "M." [Figure 5]Figure 1 shows the cloning of expression vectors for the production of PAS-ylated crisantaspase in E. coli. (A) Plasmid maps of (A) pASK75-SapI-crisantaspase (SEQ ID NO: 4) and (B) its derivative pASK75-PA400-crisantaspase (SEQ ID NO: 14) after seamless insertion into the gene cassette of PA#1c / 1b(400) (SEQ ID NO: 10) via two oppositely oriented SapI restriction sites. The structural gene of the biologically / pharmacologically active (pre)protein PA#1(400)-crisantaspase (SEQ ID NO: 13), which contains the coding region of the bacterial Enx signal sequence (SPEnx) as well as the low-repetitive nucleotide sequence encoding the PA#1 polypeptide with 401 amino acid residues and the structural gene of crisantaspase, is cloned under the transcriptional control of the tet promoter / operator (tetp / o). The plasmid backbone outside the expression cassette, flanked by XbaI and HindIII restriction sites, is identical to that of the gene expression vector pASK75 (Skerra, (1994), Gene, 151:131-135). The plasmid for expression of crisantaspase fused to PA#1(200) (SEQ ID NO: 11) was cloned in the same way using the PA#1b(200) gene cassette (SEQ ID NO: 12). [Figure 6]Figure 1 shows SDS-PAGE analysis of recombinant crisantaspase genetically fused to PA200 or PA400. (A) 10% SDS-PAGE analysis of the mature PA#1(400)-crisantaspase fusion protein (SEQ ID NO: 13) after periplasmic extraction (PPE), ammonium sulfate precipitation (ASP), and anion exchange chromatography (AEX). (B) The gel shows a 5 μg sample of purified mature PA#1(200)-crisantaspase (lane 1) (SEQ ID NO: 11) or PA#1(400)-crisantaspase (lane 2) (SEQ ID NO: 13). The size of the marker protein (M) is indicated on the left. The PA#1(200)-crisantaspase and PA#1(400)-crisantaspase fusion proteins appear as single homogenous bands with apparent molecular sizes of approximately 105 kDa (lane 1) and 200 kDa (lane 2), respectively. Due to insufficient SDS binding, PA fusion proteins generally exhibit sizes significantly larger than, for example, the calculated mass of 51 kDa for the PA#1(200)-crisantaspase monomer or the calculated mass of 67 kDa for the PA#1(400)-crisantaspase monomer (Schlapschy, (2013), Protein Eng. Des. Sel. 26:489-501). [Figure 7]Size-exclusion chromatography of PAS-modified crisantaspase variants. (A) Overlay of the elution profiles of unmodified crisantaspase, crisantaspase chemically conjugated to either Pga-P / A(20)-Ahx or Pga-P / A(40)-Ahx (described in Examples 3 and 2, respectively), and recombinant crisantaspase genetically fused to the PA#1(200) (SEQ ID NO: 7) or PA#1(400) (SEQ ID NO: 9) polypeptides (described in Example 5). 150 μL of purified protein at a concentration of 1 mg / mL was applied to a Superdex™ S200 10 / 300GL column equilibrated with PBS buffer. Absorbance at 280 nm was monitored, and peaks at each chromatographic setting were normalized to 100%. (B) Calibration curve for the chromatogram from (A) using a Superdex S200 10 / 300GL column. The logarithm of the molecular weights of marker proteins (ovalbumin: 43.0 kDa, bovine serum albumin: 66.3 kDa, alcohol dehydrogenase: 150 kDa, β-amylase: 200 kDa, apoferritin: 440 kDa) was plotted against their elution volumes (black circles) and fitted with a straight line. From the observed elution volumes of tetrameric crisantaspase, its PA#1 peptide-modified protein, and its recombinant PA#1 fusion protein (black squares), their apparent molecular sizes were determined as follows: Crisantaspase: 105 kDa (true mass 140 kDa), crisantaspase / Pga-P / A(20)-Ahx-modified protein: 531 kDa (true mass 228 kDa), crisantaspase / Pga-P / A(40)-Ahx-modified protein: 820 kDa (true mass 284 kDa), PA200-crisantaspase: 595 kDa (true mass 205 kDa), PA400-crisantaspase: 1087 kDa (true mass 269 kDa). These data indicate that both chemically conjugated P / A peptides and genetic fusions with the PA#1 polypeptide confer greatly expanded hydrodynamic volumes. [Figure 8]Figure 4 shows ESI-MS analysis of PAS-modified crisantaspase mutants. (A) The raw m / z spectrum obtained by electrospray ionization mass spectrometry (ESI-MS) of the purified crisantaspase / Pga-P / A(20)-Ahx-modified protein prepared as described in Example 3 was deconvoluted to obtain the mass spectrum. (B) The observed mass species could be unambiguously assigned to crisantaspase conjugated with peptides 9 to 14 (see Table 3). However, a major peak was observed only for the protein species with peptides 10 to 13, which corresponds to the determination of the peptide coupling ratio by SDS-PAGE (see Figure 4B). (C) and (E) show the raw m / z spectra of the PA200-crisantaspase and PA400-crisantaspase fusion proteins prepared in Example 5. The deconvoluted mass spectra (D) and (F) showed masses of 51164.75 Da and 67199.17 Da, respectively, which correspond almost perfectly to the calculated mass of 51163.58 Da. [Figure 9] Mean (±SD) plasma concentration versus time profile following a single IV bolus administration to male CD-1 mice. Figure shows plasma asparaginase activity of PA·crisantaspase conjugate following a single IV bolus administration to male mice. DETAILED DESCRIPTION OF THE INVENTION

[0072] In one aspect, the present invention relates to a modified protein comprising (i) a recombinant L-asparaginase having at least 85% identity to the amino acid sequence of SEQ ID NO: 1 and (ii) one or more (poly)peptides, wherein the (poly)peptides consist solely of proline and alanine amino acid residues. The explanations and definitions provided herein for terms such as "modified protein," "L-asparaginase," "(poly)peptide," etc., apply mutatis mutandis. The term "recombinant L-asparaginase," as used herein, refers to a recombinant form of L-asparaginase having at least 85% identity to the amino acid sequence of a native Erwinia L-asparaginase. The term "recombinant" can refer to a recombinantly produced L-asparaginase, e.g., an L-asparaginase produced in a host cell containing a nucleic acid encoding the L-asparaginase.

[0073] The modified proteins also exhibit improved plasma half-lives and therefore a prolonged duration of action compared to unconjugated L-asparaginase, allowing for a reduced administration frequency and therefore a reduced burden of side effects. The present invention also provides processes for preparing the modified proteins described herein.

[0074] In certain embodiments, the present invention relates to a modified protein comprising (i) an L-asparaginase having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence of SEQ ID NO: 1, and (ii) one or more (poly)peptides, wherein the (poly)peptides consist exclusively of proline and alanine amino acid residues. The term "consisting exclusively of proline and alanine amino acid residues" means that at least one proline residue and at least one alanine residue must be present, i.e., both at least one proline residue and at least one alanine residue must be present. In a preferred embodiment, the present invention relates to a modified protein comprising (i) a recombinant L-asparaginase having the amino acid sequence of SEQ ID NO: 1, and (ii) one or more (poly)peptides, wherein the (poly)peptides consist exclusively of proline and alanine amino acid residues. In one embodiment, the L-asparaginase is a tetramer (i.e., an L-asparaginase composed of four subunits or monomers). An exemplary subunit or monomer has the amino acid sequence of SEQ ID NO:1.

[0075] In one aspect, the (poly)peptide (i.e., polypeptide or peptide) mediates a reduction in the immunogenicity of a modified protein described herein, e.g., a reduction in the immunogenicity of the modified protein compared to unconjugated L-asparaginase.

[0076] As shown in the accompanying Examples, the PA#1(200)-crisantaspase protein had 109% and the PA#1(400)-crisantaspase protein had 118% of the enzymatic activity of unmodified crisantaspase (see Example 5). This demonstrates that fusion of asparaginase to a polypeptide as described herein does not affect enzymatic activity. Surprisingly, activity even increased with the length of the PA-polypeptide.

[0077] More generally, the modified proteins provided herein have the same or substantially the same enzymatic activity as unmodified asparaginase. Enzymatic activity may be assessed by a Nessler assay. Details of the Nessler assay are provided in the accompanying Examples and / or are disclosed in the prior art, e.g., Mashburn, (1963), Biochem. Biophys. Res. Commun. 12, 50 (incorporated herein by reference in its entirety). Thus, in one aspect, the modified proteins provided herein have the same or substantially the same enzymatic activity as unmodified asparaginase, as assessed by a Nessler assay. The term "unmodified asparaginase," as used herein, refers to native asparaginase, i.e., asparaginase that has not been modified by fusion / conjugation with a (poly)peptide as defined herein.

[0078] For example, an "unmodified asparaginase" is an L-asparaginase having at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, an "unmodified asparaginase" is an L-asparaginase having the amino acid sequence of SEQ ID NO: 1.

[0079] In some aspects, the modified proteins provided herein have higher enzymatic activity than that of unmodified L-asparaginase. Enzymatic activity may be assessed, for example, by a Nessler assay. Details of the Nessler assay are provided in the accompanying Examples and / or are disclosed in the prior art, e.g., Mashburn, (1963), Biochem. Biophys. Res. Commun. 12, 50 (incorporated herein by reference in its entirety). Thus, in one aspect, the modified proteins provided herein have higher enzymatic activity than that of unmodified L-asparaginase, as assessed by a Nessler assay. The term "unmodified asparaginase," as used herein, refers to native asparaginase, i.e., asparaginase that has not been modified by fusion / conjugation with a (poly)peptide as defined herein. For example, an "unmodified asparaginase" is an L-asparaginase having at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the "unmodified asparaginase" is an L-asparaginase having the amino acid sequence of SEQ ID NO: 1. For example, the modified protein has an enzymatic activity that is at least 5% and / or up to 30% (e.g., at least 10%, 15%, 20%, 25%, or more) higher than that of L-asparaginase, particularly unmodified asparaginase, as assessed, particularly by a Nessler assay. The above description particularly applies to, but is not limited to, the fusion proteins provided herein (e.g., modified proteins of L-asparaginase and a polypeptide, where the polypeptide consists solely of proline and alanine amino acid residues).

[0080] In some aspects, the modified protein has higher asparaginase or glutaminase activity than that of unmodified L-asparaginase. For example, the modified protein can have at least 5% and / or up to 30% (e.g., at least 10%, 15%, 20%, 25%, or more) higher asparaginase or glutaminase activity than that of L-asparaginase, particularly unmodified L-asparaginase, particularly as assessed by a Nessler assay. In some embodiments, the asparaginase or glutaminase activity can be measured by a Nessler assay. The rate of asparagine hydrolysis can be determined by measuring the ammonia released, and the amount of ammonia released from the modified proteins disclosed herein can be compared to the amount of ammonia released from L-asparaginase or unmodified L-asparaginase. In additional embodiments, the modified protein has an L-asparagine-depleting activity that is greater than that of unmodified L-asparaginase. For example, the modified protein has an L-asparagine-depleting activity that is at least 5% and / or up to 30% (e.g., at least 10%, 15%, 20%, 25%, (or more)) greater than that of L-asparaginase, particularly unmodified L-asparaginase, as assessed particularly by a Nessler assay. The present invention also relates to pharmaceutical compositions comprising the modified proteins, and to the modified proteins or pharmaceutical compositions for use in therapy, as a drug, or for use in medicine.

[0081] Generally, modified proteins can be obtained by chemical coupling or genetic fusion (in the case of conjugates with another protein or peptide). The term "fusion protein," as used herein, primarily refers to a modified protein comprising (i) L-asparaginase and (ii) one or more polypeptides, where the polypeptides consist solely of proline and alanine amino acid residues. In this context, the polypeptides can consist of about 200 to about 400 proline and alanine amino acid residues. Exemplary amino acid sequences of such polypeptides are set forth in SEQ ID NOs: 7 and 9.

[0082] If the modified protein is obtained by chemical coupling, it comprises (i) L-asparaginase and (ii) one or more peptides, the peptides consisting solely of proline and alanine amino acid residues. In this context, the peptides can consist of a total of 10 to 100 proline and alanine amino acid residues, about 15 to about 60 proline and alanine amino acid residues, about 15 to about 45 proline and alanine amino acid residues, for example, about 20 to about 40, e.g., 20 proline and alanine amino acid residues, or 40 proline and alanine amino acid residues. Exemplary amino acid sequences of such peptides are AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or AAPAAPAPAAPAAPAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 15).

[0083] As used herein, the term "modified protein" can be used interchangeably with the term "conjugate," particularly if it refers to a modified protein obtained by chemical coupling or as a fusion protein, i.e., it primarily comprises (i) L-asparaginase and (ii) one or more (poly)peptides, where the (poly)peptides consist solely of proline and alanine amino acid residues. Similarly, the terms "unmodified" and "unconjugated" can be used interchangeably herein.

[0084] The present invention also provides a compound of formula (a) R N -(P / A)-R C-act The activated peptide is coupled with L-asparaginase to produce a peptide containing L-asparaginase and R N The present invention also relates to a process for preparing a modified protein, comprising obtaining the modified protein with a peptide in which R C-act is R C is the carboxy-activated form of R C and (P / A) are as defined in the modified protein to be prepared, and R N is a protecting group linked to the N-terminal amino group of (P / A).

[0085] The accompanying examples (see Example 1, Table 1) demonstrate that modified proteins can be prepared using various mass ratios of activated peptide to asparaginase. For example, mass ratios of 10:1 (activated peptide:asparaginase), 7.5:1, 5:1, or 3.5:1 can be used. The highest (enzymatic) activity of the modified protein was observed when a ratio of 5:1 or less was used (see Example 1, Table 2). Therefore, in the processes described herein above, it may be advantageous to use a mass ratio of activated peptide:asparaginase of 5:1 or less, e.g., 5:1, 4:1, 3.5:1, or 3:1. The term "mass ratio," as used herein, refers to the ratio of the molecular weights of an activated peptide, as defined herein, to an asparaginase, as defined herein (e.g., an asparaginase as set forth in SEQ ID NO:1 and a protein having at least 85% identity to SEQ ID NO:1). "Molecular weight" is typically expressed herein using the scientific unit Dalton (Da). It is well known that the molecular weight unit of asparaginase or peptides as denoted herein in Daltons (Da) is an alternative name for the unified atomic mass unit (u). Thus, for example, a molecular weight of 500 Da is equivalent to 500 g / mol. The term "kDa" (kilodalton) refers to 1000 Da.

[0086] The molecular weight of an asparaginase or peptide can be determined using methods known in the art, such as mass spectroscopy (e.g., electrospray ionization mass spectrometry, ESI-MS, or matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS)), gel electrophoresis (e.g., polyacrylamide gel electrophoresis with sodium dodecyl sulfate, SDS-PAGE), hydrodynamic methods (e.g., gel filtration / size-exclusion chromatography, SEC, or gradient sedimentation), or dynamic (DLS) or static light scattering (e.g., multi-angle light scattering, MALS), or the molecular weight of an asparaginase or peptide can be calculated from the known amino acid sequence of the asparaginase or peptide (and known post-translational modifications, if present). Preferably, the molecular weight of an asparaginase or peptide is determined using mass spectrometry.

[0087] The present invention also relates to processes for preparing modified proteins or nucleic acids encoding modified proteins. In some embodiments, the process comprises producing L-asparaginase in a host selected from the group including yeasts such as Saccharomyces cerevisiae and Pichia pistoris, as well as bacteria, actinomycetes, fungi, algae, and other microorganisms, including Escherichia coli, Bacillus sp., Pseudomonas fluorescens, Corynebacterium glutamicum, and bacterial hosts of the following genera: Serratia, Proteus, Acinetobacter, and Alcaligenes. Other hosts are known to those skilled in the art, including Nocardiopsis alba (Meena, et al. (2014), Bioprocess Biosyst. Eng. October 2014, Article, incorporated herein by reference in its entirety), which expresses a mutant of asparaginase lacking glutaminase activity, and those disclosed in Savitri, et al. (2003), Indian Journal of Biotechnology, 2, 184-194, incorporated herein by reference in its entirety.

[0088] The modified protein can be a fusion protein comprising (i) an L-asparaginase having at least 85% identity to the amino acid sequence of SEQ ID NO: 1 and (ii) one or more polypeptides, wherein the polypeptides consist solely of proline and alanine amino acid residues.

[0089] In a polypeptide consisting only of proline and alanine amino acid residues, proline residues may constitute more than about 10% but less than about 70% of the polypeptide. Therefore, proline residues preferably account for 10% to 70% of the total number of amino acid residues in the polypeptide, more preferably 20% to 50% of the total number of amino acid residues contained in the polypeptide, and even more preferably 30% to 40% (e.g., 30%, 35%, or 40%) of the total number of amino acid residues contained in the polypeptide.

[0090] The polypeptide may comprise a plurality of amino acid repeats, the repeats consisting of proline and alanine residues, wherein no more than six consecutive amino acid residues are identical. In particular, the polypeptide may comprise the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or any of the sequences thereof, as whole or in part. circular permutation type or may comprise or consist of multimer(s).

[0091] Preferably, the polypeptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 7 or 9, or the polypeptide comprises or consists of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 8 or 10. Preferred herein are modified proteins that (a) comprise or consist of an amino acid sequence as set forth in SEQ ID NO: 11 or 13, or (b) comprise or consist of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 12 or 14. In one embodiment, the polypeptide is a random coil polypeptide.

[0092] In some embodiments, the modified protein, e.g., a fusion protein, has asparaginase or glutaminase activity that is higher than that of unconjugated L-asparaginase. For example, the modified protein can have asparaginase or glutaminase activity that is at least 5% and / or up to 30% (e.g., at least 10%, 15%, 20%, 25%, (or more)) higher than that of unmodified L-asparaginase, particularly as assessed by a Nessler assay. In further embodiments, the L-asparaginase in the modified protein, e.g., fusion protein, is directly covalently attached to the terminal residue of the polypeptide via an amine bond, and / or the fusion protein is recombinantly produced. In preferred embodiments, the modified protein, e.g., fusion protein, includes a linker between the L-asparaginase and the polypeptide. An exemplary linker may be an alanine amino acid residue. The present invention also relates to pharmaceutical compositions comprising the modified proteins, e.g., fusion proteins, or their use in therapy, or for use as a drug, or for use in medicine.

[0093] The present invention also relates to nucleic acids encoding modified proteins, particularly fusion proteins, as defined herein. Preferably, the nucleic acid is selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleotide sequence having SEQ ID NO: 12 or 14, (b) a nucleic acid molecule comprising a nucleotide sequence having at least 85% identity to a nucleotide sequence as defined in (a), and (c) a nucleic acid molecule that is degenerate as a result of the genetic code for a nucleotide sequence as defined in (a).

[0094] One aspect of the present invention further relates to a process for preparing a modified protein as defined herein or a nucleic acid as defined herein. The process may comprise culturing a host cell as defined herein and isolating the modified protein from the culture or the cell. The process for preparing a modified protein, particularly a fusion protein, as defined herein may comprise culturing a host cell transformed with or comprising a vector comprising a nucleic acid encoding the modified protein, particularly a fusion protein, under conditions that result in expression of the modified protein, particularly the fusion protein. In some aspects, the host cell is selected from the group listed above.

[0095] The present invention further relates to a method for treating a disease treatable by L-asparagine depletion in a patient, the method comprising administering to the patient an effective amount of a modified protein, e.g., a fusion protein, as defined herein. The disease treatable by L-asparagine depletion can be cancer. The modified protein as defined herein can elicit a lower immunogenic response in a patient compared to unconjugated L-asparaginase, can have a longer circulating half-life in vivo after a single administration compared to unconjugated L-asparaginase, and / or can have a larger AUC value after a single administration compared to L-asparaginase, particularly unconjugated L-asparaginase.

[0096] The problem solved by the present invention can be viewed as providing an L-asparaginase preparation that has high biological activity in vitro, stable protein / modifier binding, a long half-life in vivo, significantly reduced immunogenicity as evidenced, for example, by a decline or disappearance of antibody responses to the L-asparaginase preparation following repeated administration, and / or utility, for example, as a second-line treatment for patients who have developed sensitivity to first-line treatment with L-asparaginase not derived from E. coli.

[0097] This problem is solved according to the present invention by the embodiments characterized in the claims, in particular by providing a modified protein comprising L-asparaginase and a modifier, i.e., (ii) one or more (poly)peptides, wherein the (poly)peptides consist exclusively of proline and alanine amino acid residues, and by providing methods for preparing and using the same.

[0098] In one aspect, described herein are modified L-asparaginases that have improved pharmacological properties compared to the unmodified L-asparaginase protein.

[0099] The term "modified L-asparaginase," as used herein, refers to a modified protein comprising (i) an L-asparaginase and (ii) one or more (poly)peptides, wherein the (poly)peptides consist solely of proline and alanine amino acid residues, as defined and described herein. In one aspect of the invention, the L-asparaginase is derived from Erwinia and has at least 85% identity to SEQ ID NO:1.

[0100] The modified L-asparaginases described herein, e.g., L-asparaginase conjugated or fused to one or more (poly)peptides, where the (poly)peptide consists solely of proline and alanine amino acid residues, are particularly useful as therapeutic agents for use in patients who exhibit hypersensitivity (e.g., allergic reaction or asymptomatic hypersensitivity) to treatment with Erwinia and / or E. coli-derived L-asparaginase or pegylated L-asparaginase, or unmodified Erwinia-derived L-asparaginase. The modified L-asparaginases described herein are also useful as therapeutic agents for use in patients who have a disease relapse, e.g., a relapse of ALL, and who have previously been treated with another form of asparaginase.

[0101] Erwinia chrysanthemi (also known as Pectobacterium chrysanthemi) has been renamed Dickeya chrysanthemi. Thus, the terms Erwinia chrysanthemi, Pectobacterium chrysanthemi, and Dickeya chrysanthemi are used interchangeably herein.

[0102] Unless otherwise defined, terms used herein are to be understood according to their ordinary meaning in the art.

[0103] As used herein, unless the context necessarily dictates otherwise, the term "including" means "including, without limitation" and terms used in the singular shall include the plural and vice versa.

[0104] As used herein, the terms "comprising", "including", "having", or grammatical variations thereof, should be interpreted as specifying the stated features, integers, steps or components, but not excluding the addition of one or more additional features, integers, steps, components or groups thereof. The terms "comprising" / "including" / "having" encompass "consisting of" and "essentially consisting of". Thus, whenever the terms "comprising" / "including" / "having" are used herein, they can be replaced by "consisting essentially of" or, preferably, "consisting of".

[0105] The terms "comprising" / "including" / "having" mean that optional additional components (or similar features, integers, steps, etc.) may be present.

[0106] "Consisting of" means that no further components (or similar features, integers, steps, etc.) can be present.

[0107] As used herein, "consisting essentially of" or grammatical variations thereof should be construed to specify the stated features, integers, steps, or components, but does not preclude the addition of one or more additional features, integers, steps, components, or groups thereof, so long as such additional features, integers, steps, components, or groups thereof do not materially alter the basic and novel characteristics of the claimed product, composition, apparatus, or method, etc.

[0108] Thus, "consisting essentially of" means that certain additional components (or similar features, integers, steps, etc.) can be present, i.e., those that do not materially affect the essential characteristics of the product, composition, device, or method. In other words, the term "consisting essentially of" (which may be used interchangeably herein with the term "comprising substantially") permits the presence of other components in a product, composition, device, or method in addition to the essential components (or similar features, integers, steps, etc.), provided that the essential characteristics of the product, composition, device, or method are not materially affected by the presence of the other components.

[0109] As used herein, the term "about" refers to ±10%, unless otherwise indicated herein.

[0110] As used herein, "a" or "an" may mean one or more.

[0111] As used herein, the term "disease treatable by asparagine depletion" refers to a condition or disease in which the cells involved in or causing the condition or disease lack or have a reduced ability to synthesize L-asparagine. The depletion or loss of L-asparagine can be partial or can be substantially complete (e.g., to levels undetectable using methods and devices known in the art).

[0112] As used herein, the term "therapeutically effective amount" refers to the amount of protein (e.g., asparaginase or modified protein thereof) needed to produce a desired therapeutic effect.

[0113] As used herein, the term "L-asparaginase" refers to an enzyme with L-asparagine aminohydrolase activity. The enzymatic activity of L-asparaginase can include deamidation of asparagine to aspartic acid and ammonia, as well as deamidation of glutamine to glutamic acid and ammonia. Asparaginases are typically composed of four monomers (although some have been reported with five or six). Each monomer can be about 32,000 to about 36,000 daltons.

[0114] Numerous L-asparaginase proteins isolated by known methods from microorganisms have been identified in the art (see, e.g., Savitri and Azmi, Indian J. Biotechnol. 2, (2003), 184-194, which is incorporated herein by reference in its entirety). The most widely used and commercially available L-asparaginases are derived from E. coli or Erwinia chrysanthemi, both of which share less than 50% structural homology.

[0115] The following relates to "L-asparaginase" for use in accordance with the present invention. Among Erwinia species, 75-77% sequence identity is typically reported between enzymes from Erwinia chrysanthemi and Erwinia carotovora, and approximately 90% sequence identity has been found between different subspecies of Erwinia chrysanthemi (Kotzia, (2007), Journal of Biotechnology, 127, 657-669, incorporated herein by reference in its entirety). Some representative Erwinia L-asparaginases include those provided in Table 1 below, which discloses the percent sequence identity to Erwinia chrysanthemi NCPPB1066, for example. [Table A]

[0116] The Erwinia L-asparaginase sequences and GenBank entries in Table 1 are incorporated herein by reference. Exemplary L-asparaginases for use in therapeutic methods are those isolated from E. coli and from Erwinia, specifically Erwinia chrysanthemi.

[0117] L-asparaginase may be a naturally occurring enzyme isolated from a microorganism. It can also be produced by recombinant enzyme technology using microorganisms such as E. coli. For example, the protein used in the modified protein of the present invention can be a recombinant protein produced in an E. coli strain, preferably a protein derived from an Erwinia species, particularly Erwinia chrysanthemi, produced in a recombinant E. coli strain.

[0118] Enzymes can be identified by their specific activity. This definition therefore includes all polypeptides with a defined specific activity that are also present in other organisms, particularly other microorganisms. Enzymes with similar activities can often be identified by grouping them into specific families, defined as PFAMs or COGs. PFAMs (Protein Family Database of Sequence Comparisons and Hidden Markov Models, pfam.sanfferac.ukl) represent a large collection of protein sequence comparisons. Each PFAM allows for visualization of multiple sequence comparisons, identification of protein domains, assessment of distribution among organisms, access to other databases, and visualization of known protein structures. COGs (Clusters of Orthologous Groups of Proteins, vv-ww.nebi.nlm.nih.gov / COG / ) are derived by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenetic strains. Each COG is defined from at least three strains, allowing for the identification of prior conserved domains.

[0119] Means for determining the percentage of sequence identity are well known to those skilled in the art, and include, in particular, the BLAST program, which can be used at the website blast.ncbi.olo.nih.gov / Blast.cgi with the default parameters set forth therein. The obtained sequences can then be utilized (e.g., aligned) using, for example, the program CLUSTALW (ebi.ac.uk / Tools / clustalw2 / index.html) with the default parameters. Using the references provided in GenBank for known genes, those skilled in the art can determine equivalent genes in other organisms, bacterial strains, yeast, fungi, mammals, plants, etc. This routine is advantageously performed using consensus sequences, which can be determined by performing sequence comparisons with genes from other microorganisms and designing degenerate probes to clone the corresponding genes in other organisms.

[0120] Those skilled in the art will understand how to select and design proteins that substantially retain L-asparaginase activity. One approach to measuring L-asparaginase activity is the Nessler assay as described by Mashburn, (1963), Biochem. Biophys. Res. Commun. 12, 50 (incorporated herein by reference in its entirety).

[0121] In particular embodiments of the modified proteins of the present invention, the L-asparaginase has at least about 85% homology or sequence identity to the amino acid sequence of SEQ ID NO: 1, and more particularly at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to the amino acid sequence of SEQ ID NO: 1 as set forth in the accompanying sequence listing. The terms "homology" and "sequence identity" are used interchangeably herein.

[0122] The term "comprising the sequence of SEQ ID NO: 1" (e.g., if the L-asparaginase has 100% homology or sequence identity to the amino acid sequence of SEQ ID NO: 1) means that the amino acid sequence of the asparaginase may not be strictly limited to SEQ ID NO: 1, but may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional amino acids. In other words, if the L-asparaginase used herein has 100% homology or sequence identity to the amino acid sequence of SEQ ID NO: 1, the L-asparaginase comprises or consists of the amino acid sequence of SEQ ID NO: 1. The term "comprising" in this context means that the amino acid sequence of the L-asparaginase of SEQ ID NO: 1 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional amino acids.

[0123] In a particular embodiment, the protein is an Erwinia chrysantherni L-asparaginase comprising or consisting of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the L-asparaginase is derived from Erwinia chrysantherni NCPPB1066 (GenBank Accession No. CAA32884, which is incorporated herein by reference in its entirety), with or without a signal peptide and / or leader sequence.

[0124] Fragments of L-asparaginase, preferably the L-asparaginase of SEQ ID NO: 1, are also included within the definition of L-asparaginase for use in the modified proteins of the present invention. The term "fragment of asparaginase" (e.g., a fragment of the asparaginase of SEQ ID NO: 1) means that the sequence of the asparaginase may contain fewer amino acids than in the asparaginases exemplified herein (e.g., the asparaginase of SEQ ID NO: 1), but may still contain sufficient amino acids to confer L-asparaginase activity. For example, a "fragment of asparaginase" is / consists of at least about 150 or 200 contiguous amino acids (e.g., about 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, 325, 326 contiguous amino acids) of one of the asparaginases exemplified herein (e.g., the asparaginase of SEQ ID NO: 1), and / or the fragment is / consists of up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 326 contiguous amino acids) from the N-terminus of said asparaginase (e.g., the asparaginase of SEQ ID NO: 1). and / or the fragment has up to 75 or 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95 or 100) amino acids deleted from the C-terminus of the asparaginase exemplified herein (e.g., the asparaginase of SEQ ID NO: 1), and / or has amino acids deleted from both the N-terminus and C-terminus of the asparaginase exemplified herein (e.g., the asparaginase of SEQ ID NO: 1), and the total number of deleted amino acids can be up to 125 or 150 amino acids.

[0125] It is well known in the art that polypeptides can be modified by substitution, insertion, deletion, and / or addition of one or more amino acids while retaining their enzymatic activity. The term "one or more amino acids" in this context can refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. For example, substitution of one amino acid at a given position with a chemically equivalent amino acid that does not affect the functional properties of the protein is common. Substitutions may be defined as exchanges 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, non-polar residues: Met, Leu, Ile, Val, Cys Large aromatic residues: Phe, Tyr, Trp

[0126] Thus, changes that result in the substitution of one negatively charged residue for another negatively charged residue (e.g., substitution of glutamic acid for aspartic acid) or one positively charged residue for another positively charged residue (e.g., substitution of lysine for arginine) can be expected to result in functionally equivalent products.

[0127] 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. A skilled artisan will recognize modifications that can be introduced without affecting the activity of a protein. For example, modifications in the N- or C-terminal portions of a protein may be expected not to alter the activity of the protein under certain circumstances. With regard to asparaginase, extensive characterization has been carried out, particularly with regard to the sequence, structure, and residues that form the particularly active catalytic site. This provides guidance regarding residues that can be modified without affecting the activity of the enzyme. All known L-asparaginases derived from bacterial sources share common structural features. All are homotetramers with four active sites located between the N- and C-terminal domains of two adjacent monomers (Aghaipour, (2001), Biochemistry, 40, 5655-5664, the entire contents of which are incorporated herein by reference). All of them share a high degree of similarity in their tertiary and quaternary structures (Papageorgiou, (2008), FEBS J. 275, 4306-4316, incorporated herein by reference in its entirety). The catalytic site sequence of L-asparaginase is highly conserved among Erwinia chrysanthemi, Erwinia carotovora, and E. coli L-asparaginase (II) (ibid.). The flexible loop in the active site contains amino acid residues 14-33, and structural analysis indicates that Thr15, Thr95, Ser62, G1u63, Asp96, and A1a120 contact the ligand (ibid.). Aghaipour et al. have performed a detailed analysis of the four active sites of L-asparaginase from Erwinia chrysanthemi by examining the high-resolution crystal structure of the enzyme complexed with its substrate (Aghaipour, (2001), Biochemistry, 40, 5655-5664).Kotzia et al. provided sequences of L-asparaginases from several species and subspecies of Erwinia, and although the proteins share only about 75-77% identity between Erwinia chrysanthemi and Erwinia carotovora, each still possesses L-asparaginase activity (Kotzia, (2007), J. Biotechnol. 127, 657-669). Moola et al. performed epitope mapping studies on Erwinia chrysanthemi 3937 L-asparaginase and were able to retain enzymatic activity even after mutating various antigenic sequences in an attempt to reduce the immunogenicity of asparaginase (Moola, (1994), Biochem. J. 302, 921-927). Given the extensive characterization that has been performed on L-asparaginase, those skilled in the art can determine how to create fragments and / or perform sequence substitutions while retaining enzymatic activity.

[0128] As used herein, the term "about," as used to modify, for example, dimensions, volumes, amounts, concentrations, process temperatures, process times, yields, flow rates, pressures, and similar values ​​and ranges of components in a composition, refers to variations in quantity that may occur through typical measuring and handling procedures used to make a compound, composition, concentrate, or use formulation, through inadvertent errors in these procedures, through differences in the manufacture, source, or purity of starting materials or components used to carry out the method, and similar considerations. The term "about" also encompasses, for example, amounts that vary with aging of a particular initial concentration or mixture of a composition, formulation, or cell culture, and amounts that vary with mixing or processing of a particular initial concentration or mixture of a composition or formulation. Whether modified by the term "about," the claims appended hereto include equivalents to these quantities. The term "about" may also refer to a range of values ​​similar to a stated reference value. In certain embodiments, the term "about" refers to a range of values ​​that are within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percent of a stated reference value.

[0129] In the context of the present invention, it is surprising to note that the specific C-terminal amino acid residue (R C It has been found that chemical conjugation to L-asparaginase via a peptide having a particularly high coupling ratio of the peptide per molecule of asparaginase makes it possible to provide L-asparaginase-modified proteins with significantly reduced immunogenicity and improved plasma half-life. This novel technique can also be applied to L-asparaginase without compromising its catalytic activity, which has further been found to greatly enhance the therapeutic value of the corresponding modified proteins described herein.

[0130] In one aspect, described herein is a modified protein comprising: (i) an L-asparaginase having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence of SEQ ID NO:1; and (ii) one or more peptides, wherein the peptides consist solely of proline and alanine amino acid residues.

[0131] In a preferred embodiment, the modified protein comprises L-asparaginase and peptide R N -(P / A)-R C wherein (P / A) is an amino acid sequence consisting of only proline and alanine amino acid residues, and R N is a protecting group linked to the N-terminal amino group of the amino acid sequence, and R C is an amino acid residue that is bonded via its amino group to the C-terminal carboxyl group of the amino acid sequence, and each peptide is composed of the C-terminal amino acid residue R C and a free amino group of the L-asparaginase, and at least one of the free amino groups to which the peptide is conjugated is not the N-terminal α-amino group of the L-asparaginase.

[0132] In some embodiments, the modified protein monomers have, after modification, from about 350, 400, 450, 500 amino acids to about 550, 600, 650, 700, or 750 amino acids. In additional embodiments, the modified proteins have from about 350 to about 750 amino acids, or from about 500 to about 750 amino acids.

[0133] Each peptide contained in a modified protein as described herein is independently referred to as peptide R N -(P / A)-R C Thus, for each of the peptides included in the modified proteins described herein, an N-terminal protecting group, R N , the amino acid sequence (P / A), and the C-terminal amino acid residue R C are each independently selected from their respective meanings. Thus, two or more peptides contained in the modified protein may be the same, or they may be different from each other. In one embodiment, all of the peptides contained in the modified protein are the same.

[0134] Furthermore, peptides contained in the modified protein preferably adopt a random coil conformation, especially when the modified protein is in an aqueous environment (e.g., aqueous solution or aqueous buffer). The presence of random coil conformation can be determined using methods known in the art, especially by spectroscopic techniques such as, for example, circular dichroism spectroscopy (CD).

[0135] Peptide R N -(P / A)-R CThe moiety (P / A) in the chemically conjugated modified protein included in the formula (I) can be an amino acid sequence consisting of a total of 10 to 100 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 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 20 proline and alanine amino acid residues. In another preferred embodiment, the amino acid sequence consists of 40 proline and alanine amino acid residues. Peptide R N -(P / A)-R C In the case of (P / A), the ratio of the number of proline residues contained in the portion (P / A) to the total number of amino acid residues contained in (P / A) is preferably 10% or more and 70% or less, more preferably 20% or more and 50% or less, and even more preferably 25% or more and 40% or less. Therefore, it is preferred that 10% to 70% of the total number of amino acid residues in (P / A) are proline residues, more preferably 20% to 50% of the total number of amino acid residues in (P / A) are proline residues, and even more preferably 25% to 40% (e.g., 25%, 30%, 35%, or 40%) of the total number of amino acid residues in (P / A) are proline residues. Furthermore, it is preferred that (P / A) does not contain consecutive proline residues (i.e., it does not contain the subsequence PP). In a preferred embodiment, (P / A) has the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5). In another preferred embodiment, (P / A) is the amino acid sequence AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 15).

[0136] Peptide R N -(P / A)-R C R in NThe group may be a protecting group linked to the N-terminal amino group, particularly the N-terminal alpha amino group, of the amino acid sequence (P / A). N is preferably pyroglutamoyl or acetyl.

[0137] Peptide R N -(P / A)-R C R in C The R group is an amino acid residue that is linked through its amino group to the C-terminal carboxy group of (P / A) and contains 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 may provide a distance of at least two carbon atoms between the amino and carboxy groups of (e.g., R C ω-amino-C 3-15 It will be understood that the R C is preferably ω-aminohexanoic acid.

[0138] In one embodiment, the peptide is Pga-AAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQ ID NO: 16) or Pga-AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQ ID NO: 17). The term "Pga" is an abbreviation for "pyroglutamoyl" or "pyroglutamic acid." The term "Ahx" is an abbreviation for "ω-aminohexanoic acid."

[0139] As also demonstrated in the accompanying examples, the C-terminal amino acid residue R as defined herein, including in particular ω-aminohexanoic acid, C The use of makes it possible to provide the modified protein with an advantageously high coupling ratio of peptides consisting only of proline and alanine amino acid residues per molecule of asparaginase, and therefore makes it possible to provide the modified protein with advantageously reduced immunogenicity and advantageously increased plasma half-life.

[0140] In modified proteins as described herein, each peptide R N -(P / A)-R C is the C-terminal amino acid residue R of the peptide C The peptide can be conjugated to L-asparaginase via an amide bond formed between the carboxy group of the peptide and a free amino group of L-asparaginase. The free amino group of L-asparaginase may be, for example, the N-terminal α-amino group of L-asparaginase or a side chain amino group (e.g., the ε-amino group of a lysine residue contained in L-asparaginase). If L-asparaginase is composed of multiple subunits, for example, if L-asparaginase is a tetramer, there may be multiple N-terminal α-amino groups (i.e., one for each subunit). In one embodiment, a 9-13 peptide (e.g., 9, 11, 12, or 13 peptides) as defined herein can be chemically conjugated to L-asparaginase (e.g., to each subunit / monomer of L-asparaginase).

[0141] In accordance with the above, in one embodiment, at least one of the free amino groups to which the peptide is chemically conjugated is not (i.e., different from) the N-terminal α-amino group of L-asparaginase. Thus, it is preferred that at least one of the free amino groups to which the peptide is conjugated 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 conjugated is the ε-amino group of a lysine residue of L-asparaginase.

[0142] Furthermore, the free amino groups to which the peptide is conjugated are preferably selected from the ε-amino group(s) of the lysine residue(s) of L-asparaginase, the N-terminal α-amino group(s) of L-asparaginase or any subunit(s) of L-asparaginase, and combinations thereof. It is particularly preferred that one of the free amino groups to which the peptide is conjugated is the N-terminal α-amino group, while the other(s) of the free amino groups to which the peptide is conjugated are each the ε-amino group of a lysine residue of L-asparaginase. Alternatively, it is preferred that each of the free amino groups to which the peptide is conjugated is the ε-amino group of a lysine residue of L-asparaginase.

[0143] The modified proteins described herein are composed of L-asparaginase, as defined herein, and one or more peptides. The corresponding modified proteins may, for example, consist of an 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 conjugated to the L-asparaginase. L-asparaginase may, for example, be a monomeric protein or a protein composed of multiple subunits, e.g., a tetramer. If L-asparaginase is a monomeric protein, the corresponding modified protein may, for example, consist of a monomeric L-asparaginase and 9 to 13 (or more) (e.g., 9, 11, 12, or 13) peptides, each conjugated to the monomeric L-asparaginase. An exemplary amino acid sequence of a monomeric L-asparaginase is set forth in SEQ ID NO: 1. If L-asparaginase is a protein composed of multiple subunits, e.g., four subunits (i.e., if the L-asparaginase is a tetramer), the corresponding modified protein may consist of, for example, four L-asparaginase subunits and 9 to 13 (or more) (e.g., 9, 11, 12, or 13) peptides, as defined herein, each conjugated to a respective L-asparaginase subunit. An exemplary amino acid sequence of an L-asparaginase subunit is set forth in SEQ ID NO: 1. Similarly, if L-asparaginase is a protein composed of multiple subunits, e.g., four subunits (i.e., if the L-asparaginase is a tetramer), the corresponding modified 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 conjugated to the L-asparaginase tetramer.In one embodiment, the present invention relates to a modified protein comprising L-asparaginase and multiple chemically linked peptide sequences. In a further embodiment, the peptide sequences are about 10 to about 100, about 15 to about 60, or about 20 to about 40 in length.

[0144] A peptide consisting only of proline and alanine amino acid residues may be covalently attached to one or more amino acids of the L-asparaginase, e.g., lysine residues and / or the N-terminal residue, and / or a peptide consisting only of proline and alanine amino acid residues may be covalently attached to at least about 40, 50, 60, 70, 80, or 90% to about 60, 70, 80, 90, or 100% of the accessible amino groups on the surface of the L-asparaginase, including the amino groups of the lysine residues and / or the N-terminal residue. For example, there may be about 11-12 accessible lysine residues per L-asparaginase, and about 9-12 lysines that may be conjugated to a peptide consisting only of proline and alanine amino acid residues. In a further aspect, the peptide consisting of only proline and alanine amino acid residues is covalently attached 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 attached to the L-asparaginase via a linker. Exemplary linkers include those disclosed in U.S. Patent Application Publication No. 2015 / 0037359, which is incorporated herein by reference in its entirety.

[0145] Additionally, the modified protein may have a half-life of at least about 5, 10, 12, 15, 24, 36, 48, 60, 72, 84, or 96 hours at a dose of about 25 μg protein / kg, and / or a longer circulating half-life in vivo compared to unmodified L-asparaginase.Furthermore, the modified protein may have a larger area under the plasma drug concentration-time curve (AUC) compared to L-asparaginase.

[0146] The modified protein of the present invention can be prepared using methods known in the art, in particular, it can be prepared using the processes described below and / or according to or analogously to the procedures described in the Examples.

[0147] The present invention further relates to a process for preparing a modified protein as defined herein, said process comprising: (a) a compound of formula R N -(P / A)-R C-act The activated peptide is coupled with L-asparaginase to produce a peptide containing L-asparaginase and R N is a protecting group, to obtain a modified protein with a peptide, C-act is R C is the carboxy-activated form of R C and (P / A) are as defined for the modified protein to be prepared, and R N is a protecting group linked to the N-terminal amino group of (P / A).

[0148] Carboxy-activated C-terminal amino acid residue R contained in the activated peptide C-act is an amino acid residue R as described and defined herein for a peptide. C R may be C The carboxy group of the amino acid residue R in the activated peptide is preferably in the form of an activated carboxy group. C-act The activated carboxy group of is an active ester group.

[0149] R C-act is an activated ester group, it is preferably an activated ester group: [ka] is selected from one of the following.

[0150] A particularly preferred active ester group is the 1-hydroxybenzotriazole (HOBt) active ester group. C-act The activated carboxy group of the formula: [ka] (HOBt active ester group) group.

[0151] The process further comprises, before step (a), C and (P / A) are as defined for the modified protein to be prepared, and R N is a protecting group of formula R linked to the N-terminal amino group of (P / A) N -(P / A)-R C The peptide may comprise the further step of converting the peptide to an activated P / A peptide.

[0152] For example, R C-act The step of converting the peptide into an activated peptide to obtain an activated peptide having a 1-hydroxybenzotriazole active ester group as the activated carboxy group can be carried out by reacting the peptide with a salt of a phosphonium ester, uronium ester, or imonium ester of 1-hydroxybenzotriazole (HOBt) in the presence of a base. The salt of the phosphonium, uronium, or imonium derivative of HOBt is preferably O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU).

[0153] The coupling step (a) and any preceding steps of converting the peptide into an activated peptide can be carried out as described, for example, in El-Faham, et al., 2011, Chem. Rev. 111(11), 6557-6602; Montalbetti, et al., 2005, Tetrahedron, 61(46), 10827-10852; Klose, et al., 1999, Chem. Commun. 18, 1847-1848; Valeur, et al., 2007; Carpino, et al., 1995, J. Am. Chem. Soc. 117(19), 5401-5402; Valeur, et al., 2009, Chem. Soc. Rev., 38(2), 606-631, or Hermanson, 2013, Bioconjugate techniques. Third edition. Academic This can be accomplished using peptide coupling or amide bond formation procedures described in the literature, either in the US or elsewhere in the press. Suitable reagents and reaction conditions for such procedures are further described in the aforementioned literature and in the further references cited therein. Additional descriptions can be found in U.S. Patent Nos. 8,563,521, 9,260,494, and 9,221,882, all of which are incorporated herein by reference in their entireties.

[0154] A protecting group R, as required in optional step (b), N Procedures for removing the protecting group R are well known in the art and are described, for example, in Wuts, et al., 2012, Greene's Protective Groups in Organic Synthesis. Fourth Edition. John Wiley & Sons and / or Isidro-Llobet, et al., 2009, Chem. Rev. 109(6), 2455-2504. Thus, optional step (b) can be carried out by, for example, removing the corresponding protecting group R N The method can be carried out as described for

[0155] In some aspects, the present invention relates to a modified protein comprising (i) an L-asparaginase having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence of SEQ ID NO: 1, and (ii) a polypeptide, wherein the polypeptide consists solely of proline and alanine amino acid residues. In one aspect, the modified protein is a fusion protein. The polypeptide consisting solely of proline and alanine amino acid residues may have 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 preferred aspects, 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 (e.g., 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: 7 or 9, or the polypeptide comprises or consists of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 8 or 10. In some aspects, preferably the modified protein is a fusion protein, and the modified protein comprises monomers and the amino acid sequence of P / A, each monomer having from about 350, 400, 450, 500 amino acids to about 550, 600, 650, 700, 750, or 1000 amino acids. In additional embodiments, the modified protein has from about 350 to about 800 amino acids or from about 500 to about 750 amino acids.

[0156] For example, polypeptides include peptides prepared in US Pat. No. 9,221,882.

[0157] In preferred embodiments, the modified protein (a) comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 11 or 13, or (b) comprises or consists of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 12 or 14. As used herein, modified proteins are contemplated to include (a) a protein having an amino acid sequence as set forth in SEQ ID NO: 11 or 13, (b) a protein as defined in (a) in which 1 to 65 amino acids in asparaginase have been deleted, inserted, added, or substituted, (c) a protein encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 12 or 14, (d) a protein having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (c), (e) a protein having at least 85% identity to any one of the proteins in (a) through (d), and (f) a protein having an amino acid sequence encoded by a nucleic acid that is degenerate as a result of the genetic code for the nucleotide sequence of a nucleic acid as defined in (c) or (d).

[0158] The modified protein as defined herein may be composed of four subunits, the subunits being selected from the group consisting of: (a) a protein having an amino acid sequence as set forth in SEQ ID NO:1; (b) a protein as defined in (a) in which 1 to 65 amino acids have been deleted, inserted, added, or substituted in asparaginase; (c) a protein encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO:2; (d) a protein having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (c); (e) a protein having at least 85% identity to any one of the proteins in (a)-(d); and (f) a protein having an amino acid sequence encoded by a nucleic acid that is degenerate as a result of the genetic code for the nucleotide sequence of a nucleic acid as defined in (c) or (d).

[0159] Specifically, if the modified protein is a modified protein of L-asparaginase and a polypeptide, the present invention relates to a nucleic acid encoding the modified protein as defined herein, wherein the polypeptide consists only of proline and alanine amino acid residues. In a preferred embodiment, the modified protein is a fusion protein. In a preferred embodiment, the nucleic acid is selected from the group consisting of: (a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12 or 14; (b) a nucleic acid comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence as defined in (a); and (c) a nucleic acid that is degenerate as a result of the genetic code for the nucleotide sequence as defined in (a).

[0160] In a further aspect, the present invention relates to a nucleotide sequence encoding a fusion protein, comprising a nucleotide sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO: 12 or 14. While the encoded polypeptide comprises a repetitive amino acid sequence that may form a random coil, the encoding nucleic acid preferably comprises a less repetitive nucleotide sequence. In other words, the nucleic acid can comprise a nucleotide sequence encoding a PA-rich polypeptide, wherein the encoding nucleotide sequence comprises nucleotide repeats having a maximum length of 14, 15, 16, 17, about 20, about 25, about 30, about 35, about 40, about 45, about 50, or about 55 nucleotides. Less repetitive nucleic acids as disclosed herein can be advantageous compared to more repetitive nucleic acid molecules. In particular, the genetic stability of the less repetitive nucleic acid molecules used herein can be improved.

[0161] In some embodiments, the nucleotide sequence encodes any of the modified proteins comprising L-asparaginase and a polypeptide, where the polypeptide consists only of proline and alanine amino acid residues, preferably a fusion protein as described herein, except that one or more amino acids are added, deleted, inserted, or substituted, provided that the fusion protein having this amino acid sequence has L-asparaginase activity.

[0162] In an additional aspect, the present invention relates to a (recombinant) vector comprising a nucleotide sequence encoding a modified protein comprising an L-asparaginase and a polypeptide, wherein the polypeptide consists solely of proline and alanine amino acid residues, preferably a fusion protein as described herein, wherein the vector is 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. The host may be, for example, a bacterium, actinomycete, fungus, algae, or other microorganism, including yeasts such as Saccharomyces cerevisiae and Pichia pistoris, as well as Escherichia coli, Bacillus sp., Pseudomonas fluorescens, Corynebacterium glutamicum, and bacterial hosts of the following genera: Serratia, Proteus, Acinetobacter, and Alcaligenes. Other hosts are known to those skilled in the art and include Nocardiopsis alba, which expresses a mutant of asparaginase lacking glutaminase activity (Meena, et al. (2014), Bioprocess Biosyst. Eng. October 2014, Article, incorporated herein by reference in its entirety), and those disclosed in Savitri, et al. (2003), Indian Journal of Biotechnology, 2, 184-194, incorporated herein by reference in its entirety.

[0163] The present invention relates to a vector comprising a nucleic acid as described herein above, i.e., a nucleic acid encoding a modified protein as defined herein, in particular a nucleic acid encoding a modified protein, e.g., a fusion protein, of L-asparaginase and a polypeptide, wherein the polypeptide consists only of proline and alanine amino acid residues. In a preferred embodiment, the nucleic acid is selected from the group consisting of: (a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12 or 14; (b) a nucleic acid comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence as defined in (a); and (c) a nucleic acid that is degenerate as a result of the genetic code for the nucleotide sequence as defined in (a).

[0164] The present invention relates to a host cell comprising a nucleic acid as defined herein or comprising a vector as defined herein. Exemplary hosts are listed above.

[0165] The present invention further relates to a process for preparing a modified protein, preferably a fusion protein, as described herein, or a process for preparing a nucleic acid encoding same. The process can include culturing a host cell as defined herein and isolating said modified protein from the culture or said cell. The process can include culturing a host cell (e.g., a host cell transformed with or containing a nucleic acid and / or vector comprising a nucleotide sequence encoding the modified protein, preferably a fusion protein) under conditions that result in expression of the modified protein, preferably a fusion protein. Exemplary hosts are listed above.

[0166] Many suitable vectors are known to those skilled in the art of molecular biology. The choice of suitable vector depends on the desired function, including plasmids, cosmids, viruses, bacteriophages, and other vectors conventionally used in genetic engineering.

[0167] Various plasmids can be constructed using methods well known to those skilled in the art. See, for example, the techniques described in Sambrook, (2012), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Typical plasmid vectors include, for example, pQE-12, the pUC series of plasmids, pBluescript (Stratagene), the pET series of expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE, the pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, and pTACT1. Exemplary vectors suitable for expression in mammalian cells include the E-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), and pEAK-10 (Edge). Non-limiting examples of suitable plasmid vectors for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen).

[0168] Generally, a vector can contain one or more origins of replication (ori) and inheritance for cloning and expression, one or more markers for selection in a host, e.g., antibiotic resistance, and one or more expression cassettes. Examples of suitable origins of replication include, for example, full-length ColE1, truncated forms thereof, such as those present in pUC plasmids, SV40 virus, and M13 phage replication origins. Non-limiting examples of selectable markers include ampicillin, chloramphenicol, tetracycline, kanamycin, dhfr, gpt, neomycin, hygromycin, blasticidin, or geneticin. Additionally, the vector comprises a regulatory sequence operably linked to the nucleotide sequence or nucleic acid molecule defined herein.

[0169] The coding sequence(s) contained in the vector, e.g., the nucleotide sequence encoding a polypeptide, can be ligated to transcriptional regulatory sequence(s) and / or other amino acid-encoding sequences using established methods. Such regulatory sequences are well known to those of skill in the art and include, but are not limited to, regulatory sequences ensuring transcription initiation, internal ribosome entry sites (IRES), and, optionally, regulatory sequences ensuring transcription termination and transcript stabilization. Non-limiting examples of such regulatory sequences ensuring transcription initiation include promoters, translation initiation codons, enhancers, insulators, and / or regulatory sequences ensuring transcription termination. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleic acid sequences encoding secretion signals, or signal sequences capable of directing expressed proteins to a cellular compartment or culture medium, depending on the expression system used.

[0170] Examples of suitable promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV (Rous sarcoma virus) promoter, the lacZ promoter, the chicken β-actin promoter, the CAG promoter (a combination of the chicken β-actin promoter and the cytomegalovirus immediate-early enhancer), the human elongation factor 1α promoter, the AOX1 promoter, the GAL1 promoter, the CaM kinase promoter, the lac promoter, the trp promoter, or the tac promoter, the lacUV5 promoter, the T7 or T5 promoter, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) polyhedron promoter, or the globin intron in mammalian and other animal cells. An example of an enhancer is, for example, the SV40 enhancer. Additional non-limiting examples of regulatory sequences / sequences ensuring transcription termination include the SV40 polyA site, the tk polyA site, or the AcMNPV polyhedron polyadenylation signal.

[0171] Additionally, depending on the expression system, a leader sequence may be added to the coding sequence of the nucleic acids provided herein that can direct the polypeptide to a cellular compartment or secrete it into the medium. The leader sequence(s) are constructed in frame with translation initiation and termination sequences, and preferably, the leader sequence is capable of directing secretion of the translated protein or a portion thereof into the periplasm or extracellular medium. Suitable leader sequences are, for example, the signal sequences of BAP (bacterial alkaline phosphatase), CTB (cholera toxin subunit B), DsbA, ENX, OmpA, PhoA, stII, OmpT, PelB, Tat (twin-arginine translocation) in E. coli, and the signal sequences of bovine growth hormone, human chymotrypsinogen, human factor VIII, human Ig-kappa, human insulin, human interleukin-2, luciferase from Metrida or Vargula, human trypsinogen-2, inulinase from Kluyveromyces marxianus, mating factor alpha-1 from Saccharomyces cerevisiae, melittin, human azurocidin and analogs in eukaryotic cells.

[0172] The vector may also contain additional expressible nucleic acid sequences encoding one or more chaperones to facilitate correct protein folding.

[0173] In some aspects, a vector of the invention is an expression vector, which is capable of directing the replication and expression of a nucleic acid molecule of the invention, e.g., a nucleic acid comprising a nucleotide sequence encoding a polypeptide and a nucleotide sequence encoding an asparaginase.

[0174] Nucleic acid molecules and / or vectors such as those described herein above may be designed for introduction into cells by, for example, non-chemical methods (electroporation, sonoporation, phototransfection, gene electrotransfer, hydrodynamic delivery upon contacting cells with a nucleic acid molecule of the invention, or spontaneous transformation), chemical-based methods (calcium phosphate, DMSO, PEG, liposomes, DEAE-dextran, polyethyleneimine, nucleofection, etc.), particle-based methods (gene guns, magnetofection, imparefection), phage or phagemid vector-based methods, and viral methods. For example, expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpesvirus, Semliki Forest virus, or bovine papillomavirus may be used to deliver nucleic acid molecules to targeted cell populations.

[0175] The present invention also relates to host cells or non-human hosts transformed with the vectors or nucleic acids described herein. It will be appreciated that the term "host cells or non-human hosts transformed with a vector" refers to host cells or non-human hosts comprising a vector or nucleic acid as described herein. Host cells for the expression of polypeptides are well known in the art and include eukaryotic cells as well as prokaryotic cells. Appropriate culture media and conditions for the host cells described above are known in the art.

[0176] "Culturing a host or host cell" includes expression of a modified protein, including a fusion protein as defined herein, and / or a polypeptide and / or asparaginase as defined herein in a host or host cell.

[0177] Methods for isolating modified proteins and / or polypeptides and / or asparaginase as defined herein include, but are not limited to, purification steps such as affinity chromatography (preferably using a fusion tag such as Strep-tag II or His6-tag), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high-pressure liquid chromatography (HPLC), reverse-phase HPLC, ammonium sulfate precipitation, or immunoprecipitation. These methods are well known in the art and are generally described, for example, in Scopes, (1994), Protein Purification—Principles and Practice, Springer. Such methods provide substantially pure polypeptides. The pure polypeptides preferably have at least about 90-95% homogeneity (at the protein level), more preferably at least about 98-99% homogeneity. Most preferably, these pure polypeptides are suitable for pharmaceutical uses / applications.

[0178] It is contemplated that a modified protein comprising an L-asparaginase and a polypeptide can be prepared by expressing a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide and a nucleic acid sequence encoding the asparaginase. The expressed modified protein can be isolated. Alternatively, the modified protein can be prepared by culturing / producing a host comprising a nucleotide sequence or nucleic acid sequence encoding the polypeptide consisting solely of proline and alanine. Thus, the nucleic acid is expressed in the host. The produced polypeptide can be isolated. The produced polypeptide can be conjugated to the asparaginase, for example, via a peptide or non-peptide bond.

[0179] The modified proteins described herein can be used to treat diseases treatable by asparagine depletion. The disease treatable by asparagine depletion is preferably cancer, such as, for example, a non-solid cancer. Preferably, the non-solid cancer is leukemia or non-Hodgkin's lymphoma. The leukemia is preferably acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). For example, the modified proteins are useful in the treatment of, or in the manufacture of a medicament for use in the treatment of, acute lymphoblastic leukemia (ALL) in both adults and children or acute myeloid leukemia (AML) in both adults and children. The use of the modified proteins described herein in the treatment of other conditions in which asparagine depletion is expected to have a beneficial effect is also contemplated. Such conditions include, but are not limited to, malignant tumors or cancers, including, but not limited to, hematologic malignancies, NK lymphoma, pancreatic cancer, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma, melanosarcoma, and diffuse large B-cell lymphoma (DLBCL). The cancer may be a solid cancer, such as lung cancer or breast cancer. Exemplary non-malignant hematologic disorders responsive to asparagine depletion include immune system-mediated hematologic disorders, such as infectious diseases caused by HIV infection (i.e., AIDS). Non-hematologic disorders associated with asparagine dependency include autoimmune diseases, such as rheumatoid arthritis, SLE, autoimmune, collagen-associated vascular diseases, and the like. Other autoimmune diseases include osteoarthritis, Issac's syndrome, psoriasis, insulin-dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus, 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 disease can be tested for asparagine dependence in a suitable in vitro or in vivo assay, e.g., an in vitro assay in which the growth medium lacks asparagine.

[0180] The present invention further relates to methods for treating a disease treatable by L-asparagine depletion in a patient, the method comprising administering to the patient an effective amount of the modified protein. In some preferred aspects, the disease treatable by L-asparagine depletion is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or non-Hodgkin's lymphoma. In some aspects, the disease treatable by L-asparagine depletion is cancer, including, but not limited to, NK lymphoma and pancreatic cancer. In additional embodiments, the modified proteins described herein elicit a less immunogenic response in the patient compared to the modified protein L-asparaginase.

[0181] In some embodiments, the modified proteins described above have a longer circulating half-life in vivo after a single administration compared to the unmodified L-asparaginase of the modified protein. The modified proteins described herein can reduce plasma L-asparagine levels for 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). The modified proteins described herein can reduce plasma L-asparagine levels to undetectable levels for 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). The modified proteins described herein, when administered at a dose of 50 U / kg (bw) or 100 μg / kg (protein content basis), can reduce plasma L-asparagine levels for at least about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours. The modified proteins described herein can reduce plasma L-asparagine levels by about 10,000 to about 15,000 IU / m 2 (approximately 20-30 mg of protein / m 2), can reduce plasma L-asparagine levels to undetectable levels for at least about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours.

[0182] The modified proteins described herein can produce similar levels of L-asparagine depletion for a period of time (eg, 24, 48, or 72 hours) after a single administration.

[0183] The modified proteins described herein have a longer t than unmodified L-asparaginase administered at an equivalent protein dose. 1 / 2 The modified proteins described above can have a greater AUC value (e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater) after a single administration compared to the unmodified L-asparaginase protein.

[0184] In some embodiments, the modified proteins described herein do not elicit a significant antibody response for a specified period of time, e.g., about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or more, following administration of a single dose. For example, the modified protein does not elicit a significant antibody response for at least 8 weeks. In one example, "does not elicit a significant antibody response" means that subjects receiving the modified protein are identified as antibody-negative within art-recognized parameters. Antibody levels can be determined by methods known in the art, such as ELISA or surface plasmon resonance 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 modified proteins may have any combination of these properties.

[0185] In some embodiments, treatment with the modified proteins described herein is administered as first-line therapy. In other embodiments, treatment with the modified proteins is administered as second-line therapy in patients, particularly patients with ALL, who have developed objective signs of allergy or hypersensitivity, including "silent hypersensitivity," to other asparaginase preparations, particularly native Escherichia coli L-asparaginase or its pegylated variant (pegaspargase). Non-limiting examples of objective signs of allergy or hypersensitivity include testing for "antibody positivity" to the asparaginase enzyme. In specific embodiments, the modified proteins are used as second-line therapy after treatment with pegaspargase. Patients may have a history of hypersensitivity to E. coli L-asparaginase and / or may have a history of hypersensitivity to Erwinia L-asparaginase. The hypersensitivity may be selected from the group consisting of allergic reaction, anaphylactic shock, and asymptomatic hypersensitivity.

[0186] The incidence of relapse in ALL patients following treatment with L-asparaginase remains high, with approximately 10-25% of pediatric ALL patients experiencing early relapse (e.g., some during the maintenance phase at 30-36 months after induction therapy) (Avramis, (2005), Clin. Pharmacokinet. 44, 367-393). When patients treated with E. coli-derived L-asparaginase relapse, subsequent treatment with E. coli preparations can result in a "vaccination" effect, whereby the E. coli preparation becomes more immunogenic during subsequent administrations. The modified proteins described herein may be used in methods to treat patients with relapsed ALL who have been previously treated with other asparaginase preparations, particularly those previously treated with E. coli-derived L-asparaginase. Disease relapse may occur following treatment with E. coli L-asparaginase or its pegylated forms.

[0187] In another aspect, the present invention is directed to a method of treating acute lymphoblastic leukemia, comprising administering to a patient in need of treatment a therapeutically effective amount of the modified protein described above. In a specific aspect, the amount is about 1500 IU / m on a schedule ranging from about twice a week to about once a month, usually once a week to once every two weeks. 2 ~Approx. 15,000IU / m 2 , usually about 10,000~15,000IU / m 2 (approximately 20-30 mg of protein / m 2 ) is administered. The modified proteins described above may be administered as a single agent (monotherapy) or as part of a combination with other chemotherapeutic agents, including, but not limited to, glucocorticoids, corticosteroids, anti-cancer compounds, or other agents, including, but not limited to, methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracyclines. By way of example, an ALL patient would be administered the modified proteins described above as a component of multi-agent chemotherapy over three chemotherapy phases, including induction, adjuvant or consolidation, and maintenance. In a specific example, the modified proteins described above are not administered in conjunction with an asparagine synthase inhibitor (e.g., as described in WO 2007 / 103290, the entire contents of which are incorporated herein by reference). In another example, the modified proteins described above are not administered in conjunction with an asparagine synthase inhibitor, but are administered in conjunction with other chemotherapeutic agents. The modified proteins described above can be administered before, after, or simultaneously with other compounds as part of a multi-drug chemotherapy regimen.

[0188] In specific embodiments, the method involves administering an amount of 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 (e.g., on a protein content basis) of a modified protein as described above. The amount of modified protein delivered will depend on a number of factors, including the IC 50 , E.C. 50 The amount of modified protein administered will depend on the patient's biological half-life, 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. In certain embodiments, the amount of modified protein administered is about 10 International Units per square meter (IU / m) of the patient's body surface area. 2 ) to 50,000 IU / m 2 In additional embodiments, the modified protein 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 modified protein is administered in an amount selected from the group consisting of about 1,000 IU / m 2 to approximately 20,000 IU / m 2 (e.g., 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 modified protein described above is administered in a single dose for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) at a dose that depletes L-asparagine to undetectable levels using methods and devices known in the art.

[0189] The modified protein may be administered at a dose that depletes L-asparagine to undetectable levels for about 3 to about 10 days, about 5 to 20 days, about 1 to 15 days, or about 2 to 30 days. The modified protein may be administered at a dose that depletes L-asparagine to undetectable levels for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. The modified protein may be administered intravenously or intramuscularly. In additional embodiments, the modified protein may be administered once or twice per week, less than once per week, or as monotherapy.

[0190] The present invention relates to a composition comprising a modified protein as defined herein or a modified protein prepared by a process as described herein. The composition may be a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier(s) or excipient(s).

[0191] The present invention also relates to pharmaceutical compositions comprising the modified proteins described above. In a specific embodiment, pharmaceutical compositions, such as currently available natural L-asparaginase (e.g., Kidrolase®, Elspar®, Erwinase®), regardless of the bacterial source used for its production, are contained in vials as lyophilized powders to be reconstituted with solvent. In another embodiment, pharmaceutical compositions, such as pegaspargase (Oncaspar®), are "ready-to-use" solutions that allow for appropriate handling, e.g., administration via intramuscular, intravenous (infusion and / or bolus), intracerebroventricular (icv), or subcutaneous routes.

[0192] The modified protein, including compositions containing it (e.g., pharmaceutical compositions), can be administered to patients using standard techniques. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, 22nd ed., Pharmaceutical Press, (2012). The appropriate dosage form depends, in part, on the use or route of administration, e.g., oral, transdermal, transmucosal, or parenteral (by injection). Such dosage forms should enable the therapeutic agent to reach target cells or otherwise have the desired therapeutic effect. For example, pharmaceutical compositions injected into the bloodstream are preferably soluble. Pharmaceutical compositions of the present invention can be formulated as pharmaceutically acceptable salts and complexes thereof. Pharmaceutically acceptable salts are non-toxic salts present in the amounts and concentrations at which they are administered. Such salt formulations can facilitate pharmaceutical use by altering the physical characteristics of a compound without preventing it from exerting its physiological effect. Useful changes in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate the administration of high drug concentrations. Pharmaceutically acceptable salts of modified proteins as described herein may exist as complexes, as will be appreciated by those skilled in the art. Pharmaceutically acceptable salts include, for example, 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.Pharmaceutically acceptable salts also include base addition salts containing, for example, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamines, and zinc salts when an acidic functional group, such as a carboxylic acid or phenol, is present. See, for example, Remington's Pharmaceutical Sciences, supra. Such salts can be prepared using the appropriate corresponding base. Pharmaceutically acceptable carriers and / or excipients can also be incorporated into the pharmaceutical compositions of the present invention to facilitate administration of a particular asparaginase. Examples of carriers suitable for use in the practice of the present invention include calcium carbonate, calcium phosphate, various sugars, such as lactose, glucose, or sucrose, or various starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (EFI), saline, and dextrose. The pharmaceutical compositions of the present invention can be administered by various routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration. For systemic administration, oral administration is preferred. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms, such as capsules, tablets, and liquid preparations, such as syrups, elixirs, and concentrated drops. Alternatively, injections (parenteral administration), such as intramuscular, intravenous, intraperitoneal, and subcutaneous injections, can be used. For injections, the pharmaceutical compositions are formulated in liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline, Hank's solution, or Ringer's solution. In addition, the compounds can be formulated in solid form and redissolved or suspended immediately before use. For example, lyophilized forms of the modified proteins can be produced. In a specific embodiment, the modified proteins are administered intramuscularly. In a preferred specific embodiment, the modified proteins are administered intravenously.

[0193] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are well known in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, surfactants may be used to enhance penetration. Transmucosal administration may be via, for example, nasal sprays, inhalers (for pulmonary delivery), rectal suppositories, or vaginal suppositories. For topical administration, the compounds can be formulated into ointments, salves, gels, or creams, as is well known in the art.

[0194] In one aspect, the invention also relates to the use of a modified protein as described herein in therapy. The use may be for treating a disease treatable by L-asparagine depletion, as described above for the methods of treating a disease treatable by L-asparagine depletion. In one aspect, the invention relates to a modified protein as described herein or a modified protein prepared by a process as described herein, or a composition comprising a modified protein as described herein, for use as a medicament / for use in therapy / for use in medicine.

[0195] In one aspect, the invention relates to a modified protein as described herein, or a modified protein prepared by a process as described herein, or a composition comprising a modified protein as described herein, for use in treating a disease treatable by L-asparagine depletion in a patient. The invention also relates to the use of a modified protein as described herein, or a modified protein prepared by a process as described herein, or a composition comprising a modified protein as described herein, in the preparation of a medicament for treating a disease treatable by L-asparagine depletion in a patient. The invention also relates to a method of treating a disease treatable by L-asparagine depletion in a patient, the method comprising administering to the patient an effective amount of a modified protein as described herein, a modified protein prepared by a process as described herein, or a composition as described herein. Preferably, the disease treatable by L-asparagine depletion is cancer.

[0196] In a preferred aspect, the invention relates to a modified protein as described herein or a modified protein prepared by a process as described herein, or a composition comprising a modified protein as described herein, for use in the treatment of cancer. The invention also relates to the use of a modified protein as described herein or a modified protein prepared by a process as described herein, or a composition comprising a modified protein as described herein, in the preparation of a medicament for treating cancer. The invention also relates to a method of treating cancer comprising administering to a subject a modified protein as described herein or a modified protein prepared by a process as described herein, or a composition as described herein.

[0197] As used herein, the subject to be treated is preferably a mammal, particularly a human.

[0198] The cancer may be a non-solid cancer, such as leukemia or non-Hodgkin's lymphoma. Preferably, the leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

[0199] The modified protein may elicit a less immunogenic response in patients compared to unconjugated L-asparaginase. The modified protein may have a longer circulating half-life in vivo after a single dose compared to unconjugated L-asparaginase. The modified protein may have a larger AUC value after a single dose compared to unconjugated L-asparaginase. The patient may have a history of hypersensitivity to E. coli L-asparaginase or its pegylated form.

[0200] The present invention is further illustrated with reference to the following non-limiting figures and examples. [Example]

[0201] The following examples illustrate the invention.

[0202] Example 1: Optimization of coupling ratio for the preparation of pyroglutamoyl-P / A(20)-aminohexanoyl-crisantaspase 4.38 mg of Pga-P / A#1(20)-Ahx peptide (Figure 1A, TFA salt, 98% purity, PSL Peptide Specialty Laboratories, Heidelberg, Germany) (SEQ ID NO: 16, amino acid sequence shown in SEQ ID NO: 5) was dissolved in 66.3 μL of DMSO. Chemical activation of the P / A peptide via its terminal carboxylic acid group was initiated by adding 23.7 μL of a 500 mM TBTU (CAS#125700-67-6; Iris Biotech, Marktredwitz, Germany) solution in DMAO and 2.7 μL of DIPEA to the peptide solution and vortexing (see Figure 1C). In this setup, the peptide concentration was 25.8 mM, and the molar ratio between DIPEA, TBTU, and Pga-P / A#1(20)-Ahx was 5:5:1. After 10 minutes of incubation at 25°C, the mixture was diluted in Eppendorf tubes according to Table 1.

[0203] A solution of Dickeya chrysanthemi L-asparaginase (crisantaspase, SEQ ID NO: 1, recombinantly produced in E. coli (lot RE-LAP-P57D) at a concentration of 2 mg / mL was prepared in phosphate-buffered saline (PBS: 115 mM NaCl, 4 mM KH2PO4, and 16 mM Na2HPO4, pH 7.4) and pipetted into each Eppendorf tube according to the volumes stated in Table 1. After mixing by repeated pipetting and vortexing, the coupling reaction was carried out for 30 min at 25 °C. The reaction was stopped by adding glycine (pH 8.0 adjusted with Tris base) to a final concentration of 250 mM.

[0204] [Table 1]

[0205] SDS-PAGE analysis of the modified proteins is shown in Figure 2. Each individual band corresponds to a protein modified with a different protein, each with one coupled P / A peptide. Additional application of the coupling reaction mix at ratios of 0.3–10 mg of peptide per mg of protein allowed counting of bands in a continuous ladder starting from the unconjugated protein, allowing for accurate determination of the number of coupled P / A peptides. Band intensities were quantified densitometrically using Quant v12 software (TotalLab, Newcastle upon Tyne, UK), and the arithmetic mean number of coupled peptides per crisantaspase, weighted for band intensity, was calculated (see Table 2). A concentration of 3.5 mg of P / A peptide per mg of crisantaspase resulted in coupling ratios ranging from 9–12 P / A peptides per crisantaspase monomer (mean value: 10.4). Increasing the applied mass ratio to 10 mg P / A peptide per mg crisantaspase only resulted in a slight increase, resulting in a coupling ratio of 10-13 P / A peptide per mg crisantaspase (mean value: 12.0), indicating saturation of accessible amino groups.

[0206] The modified proteins were purified by anion exchange chromatography on a MonoQ HR5 / 5 column (GE Healthcare) using 25 mM Na-borate, pH 9.0, 1 mM EDTA as the running buffer and a 0-1 M NaCl gradient to elute the proteins. The L-asparaginase aminohydrolase activity of each crisantaspase-modified protein was determined by reaction of the ammonia liberated via L-asparagine enzymatic activity with Nessler's reagent. Briefly, 50 μL of the enzyme solution was mixed with 20 mM L-asparagine in 100 mM sodium borate buffer, pH 8.6, containing 0.015% (w / v) bovine serum albumin and incubated at 37°C for 15 min. The reaction was stopped by the addition of 200 μL of Nessler's reagent (Sigma-Aldrich). The absorbance of this solution was measured at 450 nm. Activity was calculated from a calibration curve obtained using ammonium sulfate as a reference. The results are summarized in Table 2.

[0207] [Table 2]

[0208] Example 2: Preparation of pyroglutamoyl-P / A(40)-aminohexanoyl-crisantaspase 28 mg of pyroglutamoyl-P / A#1(40)-Ahx peptide (SEQ ID NO: 17, amino acid sequence shown in SEQ ID NO: 15, Figure 1B, TFA salt, 98% purity, Almac Group, Craigavon, UK) was dissolved in 1324 μL of anhydrous DMSO (99.9%, Sigma-Aldrich, Taufkirchen, Germany). To achieve chemical activation of the P / A peptide via its terminal carboxylic acid group, 162 μL of a 500 mM TBTU (CAS#125700-67-6, Iris Biotech, Marktredwitz, Germany) DMSO solution was added, mixed, and then 14 μL of DIPEA (99.5%, biotechnology grade, Sigma-Aldrich) was added. The entire mixture was briefly vortexed and incubated at 25 °C for 20 min (see Figure 1C). In this setup, the peptide concentration was 5.41 mM, and the molar ratio between DIPEA, TBTU, and Pga-P / A#1(40)-Ahx was 10:10:1.

[0209] 3.5 mL of ice-cold crisantaspase solution (SEQ ID NO: 1) (2 mg / mL in PBS) was mixed with 1.5 mL of activated peptide solution (resulting in a 5:1 mass ratio of Pga-P / A#1(40)-Ahx to crisantaspase) and incubated at room temperature for 30 min to allow coupling. The solution was dialyzed against 5 L of AEX running buffer (25 mM Na-borate, pH 9.0, 1 mM EDTA) using regenerated cellulose membrane dialysis tubing (MWCO 50 kDa, Spectrum Laboratories, Los Angeles, CA) and subjected to anion exchange chromatography on a HiScale™ 16 / 40 column (GE Healthcare) packed with Source™ 15Q resin. The column was equilibrated with AEX running buffer, and the modified proteins were eluted using a segmented gradient of NaCl from 0 to 150 mM in 1 column volume and from 150 to 1000 mM in 0.25 column volumes (Figure 3A).

[0210] Applying the eluate to SDS-PAGE in parallel with a ladder obtained from a mixture of coupling reactions at ratios of 0.3–10 mg of peptide per mg of protein allowed the determination of coupling ratios of 9–11 PA peptides per crisantaspase monomer (mean value: 10.0) (Figure 3B). The enzymatic activity of the crisantaspase / PA(40)-modified protein, determined using the Nessler assay described in Example 1, was 78.2% of the activity of unmodified crisantaspase assayed in the same way.

[0211] Example 3: Preparation of pyroglutamoyl-P / A(20)-aminohexanoyl-crisantaspase 21 mg of pyroglutamoyl-P / A#1(20)-Ahx peptide (SEQ ID NO: 5, Figure 1A, TFA salt, 98% purity, PSL Peptide Specialty Laboratories, Heidelberg, Germany) was dissolved in 1376 μL of anhydrous DMSO (99.9%, Sigma-Aldrich, Taufkirchen, Germany). To achieve chemical activation of the P / A peptide via its terminal carboxylic acid group, 114 μL of a DMSO solution of 500 mM TBTU (CAS#125700-67-6, purchased from Iris Biotech, Marktredwitz, Germany) was added. After mixing, 10 μL of DIPEA (99.5%, biotechnology grade, Sigma-Aldrich) was added. The entire mixture was briefly vortexed and incubated at 25 °C for 20 min (Figure 1C). In this setup, the peptide concentration was 7.58 mM, and the molar ratio between DIPEA, TBTU, and Pga-P / A#1(20)-Ahx was 5:5:1.

[0212] 3.5 mL of ice-cold crisantaspase solution (SEQ ID NO: 1) (2 mg / mL in PBS) was mixed with 1.5 mL of activated peptide solution to yield a 5:1 mass ratio between Pga-P / A#1(40)-Ahx and crisantaspase and incubated at room temperature for 30 min to allow coupling. The solution was dialyzed against 5 L of AEX running buffer (25 mM Na-borate, pH 9.0, 1 mM EDTA) using regenerated cellulose membrane dialysis tubing (MWCO 50 kDa, Spectrum Laboratories, Los Angeles, CA) and subjected to anion exchange chromatography on a HiScale™ 16 / 40 column (GE Healthcare) packed with Source™ 15Q resin. The column was equilibrated with AEX running buffer, and the modified proteins were eluted using a discontinuous gradient of NaCl from 0 to 150 mM in 1 column volume and from 150 to 1000 mM in 0.25 column volumes (Figure 4A).

[0213] Applying the eluate to SDS-PAGE in parallel with a ladder obtained from a mixture of coupling reactions at ratios of 0.3–10 mg of peptide per mg of protein allowed the determination of coupling ratios of 10–13 PA peptides per crisantaspase monomer (average value: 11.9) (Figure 4B). The enzymatic activity of the crisantaspase / PA(20)-modified protein, determined using the Nessler assay described in Example 1, was 91.2% of the activity of unmodified crisantaspase assayed in the same way.

[0214] Example 4: Cloning of expression plasmids for periplasmic production of crisantaspase N-terminally fused to P / A sequences of various lengths A synthetic DNA fragment encoding the mature amino acid sequence of Dickeya chrysanthemi L-asparaginase (UniProt IDP06608) was obtained from a gene synthesis supplier (Thermo Fisher Scientific, Regensburg, Germany). This gene fragment (SEQ ID NO: 4) contained an XbaI restriction site followed by a ribosome binding site, a nucleotide sequence encoding the Enx signal peptide, followed by a GCC-alanine codon, a first SpaI recognition sequence GCTCTTC on the non-coding strand, an 11-nucleotide spacer, a second SpaI restriction sequence in the reverse-complementary orientation to its recognition sequence GCTCTTC on the coding strand, followed by a GCC-alanine codon directly linked to the coding sequence for mature L-asparaginase, and finally a HindIII restriction site.

[0215] This gene fragment was cloned into pASk75 via the flanking restriction sites XbaI and HindIII according to standard procedures (Sambrook, (2012), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press). The resulting plasmid (Figure 5A) was digested with SpaI, leading to the release of a small (30 bp) DNA insert containing a SpaI recognition site and a truncated vector backbone with compatible 5'-GCC / 5'-GGC cohesive ends immediately preceding the encoded mature N-terminus of L-asparaginase, ideally suited for the insertion of less repetitive nucleic acid molecules encoding proline / alanine-rich amino acid repeats. After isolation of the vector fragment using the Promega Wizard Gel Extraction Kit (Promega, Mannheim, Germany) and dephosphorylation with the heat-sensitive alkaline phosphatase FastAP (Thermo Fisher Scientific, Waltham, MA), both according to the manufacturer's instructions, it was ligated via EarI restriction digestion to the PA#1b(200) gene cassette excised from pXL2-PA#1b(200) (SEQ ID NO: 8) or the PA#1c / 1b(400) gene cassette excised from pXL2-PA#1c / 1b(400) (SEQ ID NO: 10). The resulting plasmids (SEQ ID NOs: 12 and 14) (Figure 5B) allow bacterial expression (after in vivo processing of the Enx signal peptide for periplasmic secretion in E. coli) of a fusion protein consisting of a proline / alanine-rich amino acid repeat sequence (SEQ ID NOs: 11 and 13) fused to the biologically active protein crisantaspase.

[0216] Example 5: Bacterial production and purification of fusion proteins between either the PA#1(200) or PA#1(400) sequence and crisantaspase Both PA#1(200)-crisantaspase and PA#1(400)-crisantaspase fusion proteins (calculated masses: 51 kDa and 67 kDa, respectively) were produced in E. coli W3110 harboring the expression plasmids pASK75-PA200-crisantaspase or pASK75-PA400-crisantaspase (Figure 5B) from Example 4 at 25°C using an 8 L benchtop fermentor with synthetic glucose mineral medium supplemented with 100 mg / L ampicillin, according to published procedures (Schiweck, (1995), Proteins, 23:561-565). Expression of the recombinant genes was confirmed by the CD400-mediated fusion of the PA#1(200)-crisantaspase and PA#1(400)-crisantaspase strains. 550 Once the OD reached 40, the cells were induced by adding 500 μg / L of anhydrotetracycline (Skerra, (1994), loc. cit.). After a 2.5-h incubation period, the cells were harvested by centrifugation and resuspended in ice-cold periplasmic fractionation buffer (500 mM sucrose, 1 mM EDTA, 200 mM boric acid / NaOH, pH 8.0, 2 ml / L) and OD reached 40. 550 After adding 15 mM EDTA and 250 μg / mL lysozyme, the cell suspension was incubated on ice for 20 minutes, centrifuged several times, and the clear supernatant containing the recombinant protein was collected.

[0217] The periplasmic extracts were dialyzed twice each against 15 L of PBS containing 1 mM EDTA for at least 6 h at 4 °C, filtered using a 0.2 μm cellulose nitrate membrane (GE Healthcare), and precipitated by adding ammonium sulfate (European Pharmacopoeia grade, Applichem, Darmstadt, Germany) to 25% saturation at 25 °C. After centrifugation, the supernatant was removed, and the precipitate was resuspended in AEX running buffer (25 mM Na-borate, pH 9.0, 1 mM EDTA) and dialyzed against 5 L of AEX running buffer for at least 6 h at 4 °C. The dialyzed protein solution was clarified from remaining insoluble material by centrifugation and subjected to subtractive anion exchange chromatography using an 85 mL HiScale™ column (GE Healthcare, Freiburg, Germany) packed with Source 15Q resin and connected to an Äkta™ purifier system (GE Healthcare, Freiburg, Germany) equilibrated with AEX running buffer. The column flow-through fraction containing the pure protein (see Figures 6A and 6B) was dialyzed twice against 5 L of PBS.

[0218] Homogeneous protein preparations without signs of aggregation were obtained with final yields of 128 mg for PA#1(200)-crisantaspase and 48 mg for PA#1(400)-crisantaspase from one 8 L fermentor each. Protein concentrations were 19370 M. -1 cm -1The activity of the fusion proteins was determined by measuring the absorbance at 280 nm using a calculated extinction coefficient of 0.05 (Gill, (1989), Anal. Biochem. 182:319-326). The enzymatic activity of the fusion proteins was determined using the Nessler assay described in Example 1. In this setting, the PA#1(200)-crisantaspase fusion protein had 109% of the enzymatic activity, and PA#1(400)-crisantaspase had 118% of the enzymatic activity of unmodified crisantaspase assayed in the same manner. This demonstrates that N-terminal fusion of crisantaspase with P / A polypeptides up to a length of at least 401 amino acids does not affect enzymatic activity.

[0219] Example 6: Determination of hydrodynamic volume for both genetically and chemically PAS-ylated crisantaspase by analytical gel filtration Size exclusion chromatography (SEC) was performed on a Superdex™ S200 ascending 10 / 300GL column (GE Healthcare Europe, Freiburg, Germany) at a flow rate of 0.5 mL / min using an Akta™ Purifier 10 system (GE Healthcare) with PBS (115 mM NaCl, 4 mM KH2PO4, 16 mM Na2HPO4, pH 7.4) as running buffer. Using a disposable regenerated cellulose ultrafiltration device (MWCO 10 kDa; Merck-Millipore, Darmstadt, Germany), recombinant crisantaspase genetically fused to the PA#1(200) or PA#1(400) polypeptide (described in Example 5) and crisantaspase chemically conjugated to the Pga-P / A(40)-Ahx peptide (described in Example 2) or the Pga-P / A(20)-Ahx peptide (described in Example 3) were adjusted to a concentration of 1 mg / mL in PBS. 150 μL samples of the concentrated PAS-conjugated and unconjugated enzymes were applied to the column, and the chromatographic traces were overlaid (Figure 7A). All five proteins eluted as a single homogenous peak.

[0220] For column calibration (Figure 7B), an appropriate mixture of the following globular proteins (Sigma, Deisenhofen, Germany) was applied in PBS at protein concentrations between 0.5 mg / mL and 1.0 mg / mL: cytochrome c: 12.4 kDa, ovalbumin: 43.0 kDa, bovine serum albumin: 66.3 kDa, alcohol dehydrogenase: 150 kDa, β-amylase: 200 kDa, apoferritin: 440 kDa, and thyroglobulin: 660 kDa.

[0221] As a result, both the recombinant PA fusion protein and the chemically conjugated enzyme preparation exhibited significantly larger sizes than the corresponding globular proteins of the same molecular weight. This molecular weight / hydrodynamic volume disparity became even larger with increasing size of the P / A (poly)peptide moiety. The apparent size increase for PA(200)-crisantaspase was 5.1-fold compared to unfused crisantaspase, whereas the true mass was only 1.5-fold larger. The apparent size increase for PA(400)-crisantaspase compared to unfused crisantaspase was 10.4-fold, whereas the true mass was only 1.9-fold larger. This observation clearly demonstrates the greatly increased hydrodynamic volume conferred to the biologically active crisantaspase enzyme by the Pro / Ala polypeptide segment of the present invention.

[0222] Example 7: ESI-MS analysis of chemically or genetically PAS-modified crisantaspase Two hundred fifty microliters of purified, chemically modified crisantaspase protein with Pga-P / A(20)-Ahx from Example 3, and 250 μL of recombinant PA200 and PA400 fusion proteins from Example 5, all at a concentration of 1 mg / mL, were applied to a 1 mL Resource™ RPC column (GE Healthcare, Freiburg, Germany) connected to an Akta™ Purifier System using 2% v / v acetonitrile and 1% v / v formic acid as the running buffer. Proteins were eluted using an acetonitrile gradient from 2% v / v acetonitrile and 1% v / v formic acid over 20 column volumes to 80% v / v acetonitrile and 0.1% v / v formic acid. Eluted proteins were directly analyzed via ESI mass spectrometry on a maXis™ microTOF instrument (Bruker Daltonik, Bremen, Germany) using positive ion mode. The raw m / z spectrum of the crisantaspase / Pga-P / A(20)-Ahx chemically modified protein is shown in Figure 8A. The masses revealed by the deconvoluted mass spectrum (Figure 8B) are given in Table 3. The distribution of masses is consistent with the coupling ratios determined by SDS-PAGE analysis as described in Example 2.

[0223] The raw m / z spectrum of the recombinant PA#1(200)-crisantaspase (SEQ ID NO: 11) fusion protein is shown in Figure 8C. The deconvoluted mass spectrum revealed a mass of 51164.75 Da (Figure 8D), which is essentially consistent with the calculated mass of this protein (51163.58 Da). The raw m / z spectrum of the recombinant PA#1(400)-crisantaspase fusion protein (SEQ ID NO: 13) is shown in Figure 8E. The deconvoluted spectrum (Figure 8F) revealed a mass of 67199.17 Da, which is essentially consistent with the calculated mass of this protein (67201.99 Da). This clearly demonstrates that intact crisantaspase enzymes genetically fused to either PA200 or PA400 can be produced in E. coli in a highly homogeneous form.

[0224] [Table 3]

[0225] Example 8: Asparaginase activity The enzyme activity of PAS-modified L-asparaginase was determined by catalyzing the conversion of L-asparagine to L-aspartic acid. This reaction liberates one mole of ammonia per mole of L-asparagine converted. The released ammonia was detected using Nessler's reagent. In the presence of Nessler's reagent, ammonia forms a water-soluble yellow complex that can be quantified by absorbance measurement at 450 nm (Mashburn, et al. (1963), Biochem. Biophys. Res. Commun. 12, 50). One unit of L-asparaginase enzyme activity (International Unit or IU) is defined as the amount of enzyme that catalyzes the conversion of 1 μmol of L-asparagine per minute. The specific activity (IU / mg) of a sample was determined by dividing the L-asparaginase activity value expressed in IU / mL by the protein concentration expressed in mg / mL. The mass of the protein monomer containing the PAS-modified sequence was measured.

[0226] Measurement of L-asparaginase activity is based on an endpoint assay in which samples are diluted to a series of final enzyme concentrations, which are then incubated at 37°C for 15 minutes under saturating L-asparagine concentrations. The reaction is stopped by the addition of Nessler's reagent, and the amount of ammonia produced by the reaction is extrapolated from a calibration curve constructed from known amounts of ammonium sulfate used as a standard. A plot of enzyme concentration versus ammonia is then made for each sample, and the slope of the curve is divided by the reaction time to obtain the specific activity in IU / mg. Specific activity is reported as IU / mg and is reported to the nearest whole number.

[0227] For each modified or fusion protein, the initial test results are shown in the table below. [Table 4]

[0228] Example 9: Pharmacokinetics The pharmacokinetic profile of recombinant crisantaspase expressed in E. coli as a PASylated fusion protein (PA-200) or chemically conjugated to the PA-peptide (PA-20) was characterized following administration of a single intravenous bolus dose to CD-1 mice, a model of healthy mice.

[0229] All animals received a single intravenous (IV) bolus (10 mL / kg) via the lateral tail vein based on body weights obtained prior to dosing. Individual doses were calculated based on the most recent body weight to provide the appropriate dose. Day 1 of dosing was based on body weights on study day 0. All animals were observed twice daily, once in the morning and once in the afternoon, for mortality, abnormalities, and signs of pain or distress.

[0230] Mice were administered PAS-modified asparaginase as a single IV dose of 25 IU / kg body weight. Groups of mice were dosed at 25 IU / kg body weight, and plasma samples were collected at scheduled times up to 10 days (240 hours) following administration. Asparaginase activity in mouse plasma was measured using a qualified biochemical assay as described in the previous example. Average plasma asparaginase activity (n=4) versus time data was plotted (Figure 1) and pharmacokinetic analysis was performed.

[0231] Blood samples were collected prior to dosing and approximately 6 hours, 24 hours (Day 1), 48 hours (Day 2), 51 hours (Day 2), 54 hours (Day 2), 60 hours (Day 2), 96 hours (Day 4), 168 hours (Day 7), and 240 hours (Day 10) after dosing. A tail snip (tail-tip snip) blood collection method was used. For the initial blood collection, an approximately 1-2 mm snip was made at the distal end of the tail. All subsequent blood collections were taken from the same site by removing the eschar and stroking the tail to stimulate blood flow. Approximately 100 μL of blood per time point was collected into chilled K3EDTA (Minivette) collection tubes. The blood was transferred to tubes suitable for centrifugation. For plasma isolation, all samples were centrifuged at 3,000 × g for approximately 10 minutes within approximately 20 minutes of collection in a refrigerated centrifuge set to maintain approximately 4°C. Following centrifugation, the maximum amount of plasma was collected (targeting 30 μL) and placed into plastic vials, which were stored at −65° C. to −85° C. until testing.

[0232] Asparaginase activity was measured as the concentration of asparaginase in plasma samples as previously described (Allas, et al. (2009), Blood, 114, 2033). 2 The concentration versus time profile (t 1 / 2 , CL and V ss Reporting of parameters dependent on adequate characterization of the terminal phase of the pharmacokinetic (t) study was performed. Pharmacokinetic data were imported into Phoenix WinNonlin v6.4 (Certara / Pharsight) for analysis. Plasma asparaginase activity versus time data was analyzed using a non-compartmental method with fractional sampling in an IV bolus model. Activity values ​​below the limit of quantitation of the assay (10 U / L) were set to zero for group mean calculations. Nominal dose levels and sampling times were used in the calculations. t 1 / 2 The estimated values ​​for PA-20 crisantaspase were 50.2 hours and PA-200 crisantaspase were 17.9 hours.

[0233] The present invention refers to the following nucleotide and amino acid sequences:

[0234] Some sequences provided herein are available in the NCBI database and can be retrieved at www.ncbi.nlm.nih.gov / sites / entrez?db=gene. These sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods for using variants of the homologous and concise sequences provided herein. Preferably, such "variants" are genetic variants.

[0235] SEQ ID NO:1: Amino acid sequence of L-asparaginase from Dickeya chrysanthemi ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSA RYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0236] SEQ ID NO:2: Nucleotide sequence encoding L-asparaginase from Dickeya chrysanthemi GCAGATAAACTGCCGAATATTGTTATTCTGGCAACCGGTGGCACCATTGCAGGTAGCGCAGCAACCGGCACCCAAACCACAGGTTATAAAGCCGGTGCACTGGGTGTTGATACCCTGATTAATGCAGTTCCGGAAGTTAAAAAACTGGCCAATGTGAAAGGTGAACAGTTTAGCAATATGGCCAGCGAAAATATGACCGGTGATGTTGTTCTGAAACTGAGCCAGCGTGTTAATGAACTGCTGGCACGTGATGATGTTGATGGTGTGGTTATTACCCATGGCACCGATACCGTTGAAGAAAGCGCCTATTTTCTGCATCTGACCGTGAAAAGCGATAAACCGGTTGTTTTTGTTGCAGCAATGCGTCCGGCAACCGCAATTAGCGCAGATGGTCCGATGAATCTGCTGGAAGCAGTTCGTGTTGCCGGTGATAAACAGAGCCGTGGTCGTGGTGTTATGGTTGTTCTGAATGATCGTATTGGTAGCGCACGCTATATTACCAAAACCAATGCAAGCACCCTGGATACCTTTAAAGCCAATGAAGAAGGTTATCTGGGCGTTATTATTGGCAATCGCATTTATTATCAGAATCGCATTGATAAACTGCATACCACCCGTAGCGTTTTTGATGTTCGTGGTCTGACCAGCCTGCCGAAAGTTGATATTCTGTATGGCTATCAGGATGATCCGGAATATCTGTATGATGCAGCCATTCAGCATGGTGTTAAAGGTATTGTGTATGCAGGTATGGGTGCAGGTAGCGTTAGCGTTCGTGGTATTGCAGGTATGCGTAAAGCAATGGAAAAAGGCGTTGTTGTTATTCGTAGCACCCGTACCGGTAATGGTATTGTTCCGCCGGATGAAGAACTGCCGGGTCTGGTTAGCGATAGCCTGAATCCGGCACATGCACGTATTCTGCTGATGCTGGCACTGACCCGTACCAGCGATCCGAAAGTGATTCAGGAATATTTTCATACCTAT

[0237] SEQ ID NO:3: Amino acid sequence of L-asparaginase from Dickeya chrysanthemi Signal peptide: 1-28; removed during cloning: 29-39; 40-366: asparaginase [ka]

[0238] SEQ ID NO:4 Nucleotide sequence encoding L-asparaginase from Dickeya chrysanthemi (synthetic) Mature asparaginase encoded by bases 160 to 1140 (bold). Thus, the nucleotide sequence encoding L-asparaginase spans nucleotides 160 to 1140. [ka]

[0239] SEQ ID NO:5: Amino acid sequence of PA(20) peptide AAPAAPAPAAPAAPAPAAPA

[0240] SEQ ID NO:6: Nucleotide sequence encoding the PA(20) peptide GCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCC

[0241] SEQ ID NO:7: Amino acid sequence of PA(200)-polypeptide AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAA

[0242] Accession number 8: Nucleotide sequence encoding the PA(200)-polypeptide GCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCCGCCGCTCCTGCGGCACCTGCGCCCGCCGCGCCGGCAGCGCCGGCACCGGCAGCTCCGGCGGCCGCGCCTGCAGCTCCTGCACCGGCGGCTCCAGCAGCCCCGGCGCCGGCCGCACCTGCGGCGGCGCCCGCGGCGCCTGCACCCGCAGCGCCTGCGGCACCGGCCCCAGCAGCCCCTGCCGCCGCACCGGCTGCGCCTGCCCCAGCGGCCCCCGCTGCCCCGGCCCCGGCGGCTCCAGCCGCAGCGCCTGCCGCCCCAGCGCCCGCAGCACCGGCGGCACCAGCTCCGGCGGCGCCGGCGGCGGCTCCGGCAGCTCCGGCCCCTGCTGCGCCGGCTGCGCCGGCTCCGGCGGCCCCTGCGGCGGCTCCGGCCGCACCTGCACCTGCCGCGCCGGCTGCTCCGGCCCCGGCTGCCCCAGCAGCGGCACCAGCAGCGCCTGCTCCTGCGGCGCCTGCAGCTCCGGCGCCGGCAGCCCCGGCCGCCGCACCCGCGGCTCCAGCCCCCGCCGCTCCAGCAGCCCCCGCGCCAGCTGCACCTGCTGCC

[0243] Accession number 9: Amino acid sequence of the PA(400)-polypeptide AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAA

[0244] SEQ ID NO:10: Nucleotide sequence encoding PA(400)-polypeptide

[0245] SEQ ID NO:11: Amino acid sequence of asparaginase-PA(200)-fusion protein AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPA APAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASE NMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYY QNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0246] SEQ ID NO:12: Nucleotide sequence encoding asparaginase-PA(200)-fusion protein (XbaI / HindIII) The mature fusion protein (SEQ ID NO: 11) is encoded by bases 127 to 1710 (bold). Thus, the nucleotide sequence encoding the fusion protein can span nucleotides 127 to 1710 of SEQ ID NO: 12. Consequently, the term "a modified protein comprising or consisting of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 12" as used herein can be more narrowly defined as "a modified protein comprising or consisting of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 12 from positions 127 to 1710." [ka]

[0247] SEQ ID NO: 13: Amino acid sequence of asparaginase-PA(400)-fusion protein AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAA PAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPA APAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDK QSRRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0248] SEQ ID NO: 14: Nucleotide sequence encoding asparaginase-PA(400)-fusion protein (XbaI / HindIII) The mature fusion protein (SEQ ID NO: 13) is encoded by bases 127 to 2184 (bold). Thus, the nucleotide sequence encoding the fusion protein can span nucleotides 127 to 2184 of SEQ ID NO: 14. Consequently, the term "a modified protein comprising or consisting of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 14" as used herein can be more narrowly defined as "a modified protein comprising or consisting of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 14 from positions 127 to 2184." [ka] JPEG0007808424000012.jpg153108 JPEG0007808424000013.jpg49108

[0249] SEQ ID NO: 15: Amino acid sequence of PA(40) peptide AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA

[0250] SEQ ID NO: 16: Modified PA(20) peptide Pga-AAPAAPAPAAPAAPAPAAPA-Ahx-COOH

[0251] SEQ ID NO: 17: Modified PA(40) peptide Pga-AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA-Ahx-COOH

[0252] All references cited herein are incorporated by reference in their entirety. Having now fully described the invention, it will be understood by those skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters, and the like, without affecting the spirit or scope of the invention and its embodiments. List of References Carpino&El-Faham, 1995 Carpino, L.A. and El-Faham, A. (1995), Tetramethylfluoroformamidinium hexafluorophosphate: a rapid-acting peptide coupling reagent for solution and solid phase peptide synthesis. J. Am. Chem. Soc. 117(19), 5401 - 5402. El-Faham, et al., 2011 El-Faham, A. & Albericio, F. (2011), peptide coupling reagents, more than a letter soup. Chem. Rev. 111(11), 6557 - 6602. Hermanson, 2013 Hermanson, G.T. (2013), Bioconjugate techniques. Third edition. Academic press Isidro-Llobet, 2009 Isidro-Llobet, A., Alvarez, M. & Albericio, F. (2009), Amino acid-protecting groups. Chem. Rev. 109(6), 2455 - 2504. Klose, et al., 1999 Klose, J., Bienert, M., Mollenkopf, C., Wehle, D., Zhang, C.-W., Carpino, L.A. & Henklein, P. (1999), 2-Propanephosphonic acid anhydride (T3P)-mediated segment coupling and head-to-tail cyclization of sterically hindered peptides. Chem. Commun. 18, 1847 - 1848 Montalbetti, et al., 2005 Montalbetti,C.A.&Falque,V.(2005),Amide bond formation and peptide coupling.Tetrahedron,61(46),10827-10852 Valeur,et al.,2007 Valeur,E.&Bradley,M.(2009),Amide bond formation:beyond the myth of coupling reagents.Chem.Soc.Rev.,38(2),606-631 Valeur,et al.,2009 Valeur,E.&Bradley,M.(2009),Amide bond formation:beyond the myth of coupling reagents.Chem.Soc.Rev.,38(2),606-631 Wuts,2012 Wuts,P.G.&Greene,T.W.(2012),Greene’s Protective Groups in Organic Synthesis.Fourth Edition.John Wiley & Sons.

Claims

1. A modified protein having L-asparaginase activity, wherein the modified protein is a tetramer, each monomer of the tetramer comprising (i) an L-asparaginase having the amino acid sequence of SEQ ID NO: 1 and (ii) one or more polypeptides, each of the polypeptides consisting exclusively of about 200 to about 400 proline and alanine amino acid residues, and wherein the average coupling ratio of the polypeptides per monomer is less than 12; The modified protein, wherein the monomer of the tetramer is a fusion protein of the L-asparaginase and the polypeptide.

2. The modified protein of claim 1, wherein the polypeptide comprises the amino acid sequence AAPAAPAPAAAPAPAPAAPA (SEQ ID NO: 5) or a circularly permuted or multimerized form of said sequence as the sequence of SEQ ID NO: 5 or as part of the sequence of SEQ ID NO:

5.

3. (a) the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 9, or (b) the polypeptide comprises or consists of an amino acid sequence encoded by a nucleic acid having the nucleotide sequence of SEQ ID NO: 8 or 10; A modified protein according to claim 1 or 2.

4. (a) the modified protein comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 13; (b) the modified protein comprises or consists of an amino acid sequence encoded by a nucleic acid having the nucleotide sequence of SEQ ID NO: 12 or 14; The modified protein according to any one of claims 1 to 3.

5. The modified protein according to any one of claims 1 to 4, wherein said polypeptide mediates a reduction in the immunogenicity of said modified protein.

6. A nucleic acid encoding the modified protein according to any one of claims 1 to 4.

7. The nucleic acid (a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12 or 14, and (b) a nucleic acid according to claim 6, which is selected from the group consisting of nucleic acids that are degenerate as a result of the genetic code for the nucleotide sequence of SEQ ID NO: 12 or 14.

8. A vector comprising the nucleic acid of claim 6 or 7.

9. A host cell comprising a nucleic acid according to claim 6 or 7 or a vector according to claim 8.

10. 10. A process for the preparation of the modified protein of any one of claims 1 to 5, said process comprising culturing a host cell of claim 9 and isolating the modified protein from the culture or from the cell.

11. 11. A pharmaceutical composition comprising a modified protein according to any one of claims 1 to 5 or said modified protein prepared by the process of claim 10, optionally further comprising a pharmaceutically acceptable carrier(s) or excipient(s).

12. A modified protein according to any one of claims 1 to 5 or said modified protein prepared by the process according to claim 10 or a composition according to claim 11 for use in the treatment of a disease.

13. 13. The modified protein of claim 12 or the composition of claim 12, wherein the disease is treatable by depletion of L-asparagine and is cancer; or A modified protein according to any one of claims 1 to 5, said modified protein prepared by the process according to claim 10 or the composition according to claim 12 for the treatment of cancer.

14. 14. The modified protein of claim 13 or the composition of claim 13, wherein the cancer is a non-solid cancer.

15. 15. The modified protein of claim 14 or the composition of claim 14, wherein the non-solid cancer is leukemia or non-Hodgkin's lymphoma.

16. 16. The modified protein of claim 15 or the composition of claim 15, wherein the non-solid cancer is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

17. The modified protein of any one of claims 13 to 16 or the composition of claims 13 to 16, wherein the modified protein induces a reduced immunogenic response in patients compared to unmodified L-asparaginase.

Images