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

[Technology Field]

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

[0002] Proteins with L-asparagine aminohydrolase activity, commonly known as L-asparaginase, have been successfully used for many years in the treatment of acute lymphoblastic leukemia (ALL) in children. ALL is 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 sarcoma, and melanoma (Kotzia, (2007), J. Biotechnol. 127, 657-669). The antitumor activity of L-asparaginase is thought to be due to 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 synthesize proteins without L-asparagine (ibid.).

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

[0005] Bacterial L-asparaginases possess high immunogenicity 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, and there are reports of anti-asparaginase antibodies against E. coli L-asparaginase reaching levels as high as 78% in adults and 70% in children after intravenous or intramuscular administration (ibid.).

[0006] L-asparaginases 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, antibodies generated after treatment with E. coli-derived L-asparaginase have been shown not to cross-react with Erwinia-derived L-asparaginase (Wang, (2003), Leukemia, 17, 1583-1588). Therefore, Erwinia-derived L-asparaginase (chrysanthanspase) is used as a second-line treatment for ALL in patients who respond to E. coli L-asparaginase (Duval, (2002), Blood, 15, 2734-2739; Avramis, (2005), Clin. Pharmacokinet. 44, 367-393).

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

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

[0009] Furthermore, it has been shown that antibodies generated against L-asparaginase derived 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 possibility of cross-hypersensitivity or cross-inactivation in vivo. In fact, one report indicated that 30-41% of children treated with pegaspar gauze had allergic reactions (ibid.).

[0010] In addition to apparent allergic reactions, the problem of "asymptomatic hypersensitivity" has recently been reported, in which patients develop anti-asparaginase antibodies without showing clinical evidence of a hypersensitivity reaction (Wang, (2003), Leukemia, 17, 1583-1588). This reaction can lead to the formation of neutralizing antibodies against E. coli's L-asparaginase and pegaspargase, but because there are no apparent signs of hypersensitivity, these patients do not progress to Erwinia's L-asparaginase, and therefore the effective treatment they receive is even shorter in duration (Holcenberg, (2004), J. Pediatr. Hematol. Oncol. 26, 273-274).

[0011] Treatment with Erwinia chrysanthemi L-asparaginase is often used when there is hypersensitivity to L-asparaginase derived from E. coli. However, it has been observed that as many as 30–50% of patients receiving Erwinia L-asparaginase treatment are antibody-positive (Avramis, (2005), Clin. Pharmacokinet. 44, 367-393). Furthermore, Erwinia chrysanthemi L-asparaginase has a shorter elimination half-life than E. coli L-asparaginase, requiring more frequent administration (ibid.). A study by Avramis et al. showed that Erwinia asparaginase had inferior pharmacokinetic properties (Avramis, (2007), J. Pediatr. Hematol. Oncol. 29, 239-247). Therefore, E. coli L-asparaginase and pegaspargase are preferred first-line treatments for ALL compared to Erwinia L-asparaginase.

[0012] Numerous 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 derived 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 derived from Erwinia carotovora has only about 75% homology to L-asparaginase (chrysanthanspase) from Erwinia chrysanthemi. For Oncaspar®, its in vitro activity is also known to be approximately 50% of that of unmodified L-asparaginase from E. coli.

[0013] Therefore, the underlying technical problem of the present invention is to provide means and methods for treating cancers such as leukemia or non-Hodgkin lymphoma, for example, while avoiding the limitations and shortcomings of prior art treatments, particularly those using some pegylated asparaginases.

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

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

[0016] This invention relates, in particular, to the following items. 1. A modified protein comprising (i) L-asparaginase and (ii) one or more (poly)peptides, wherein the (poly)peptides consist only 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 with 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 described in any one of items 1 to 3, wherein the modified protein has higher asparaginase activity or glutaminase activity than unmodified L-asparaginase.

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

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

[0022] 7. A modified protein comprising the L-asparaginase and polypeptide, wherein the polypeptide consists only of proline and alanine amino acid residues, as described in any one of items 1 to 6.

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

[0024] 9. The modified protein described in item 7, wherein the polypeptide comprises a total of approximately 200 proline and alanine amino acid residues, or a total of approximately 400 proline and alanine amino acid residues.

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

[0026] 11. A 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 six or fewer consecutive amino acid residues are identical.

[0027] 12. A modified protein according to any one of items 7 to 11, wherein the polypeptide comprises or consists of the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or a circular permutation or polymer(s) of the sequence, in whole or in part.

[0028] 13. (a) The polypeptide comprises or consists of an amino acid sequence such as that shown 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 shown in SEQ ID NO: 8 or 10. A modified protein as described in any one of items 7 to 12.

[0029] 14. (a) The modified protein contains or consists of an amino acid sequence such as that shown 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 such as that shown in SEQ ID NO: 12 or 14. A modified protein as described in any one of items 7-13.

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

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

[0032] 17. L-asparaginase and peptide R N -(P / A)-R C It is a modified protein with one or more peptides, (P / A) is an amino acid sequence consisting only of proline and alanine amino acid residues, R N This is a protecting group that is attached to the N-terminal amino group of the aforementioned amino acid sequence. R C This is an amino acid residue that is bonded to the C-terminal carboxyl group of the aforementioned amino acid sequence via its amino group. Each peptide is the C-terminal amino acid residue R of the peptide. CIt is conjugated to L-asparaginase via an amide bond formed by the carboxyl group of and the free amino group of the L-asparaginase, 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 described in item 17, the amino acid sequence comprising a total of 15 to 45 proline and alanine amino acid residues.

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

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

[0036] 21. A 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. A 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 A modified protein as described in any one of items 17-22, wherein the modified protein is ε-aminohexanoic acid.

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

[0040] 25. A 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 the lysine residue of the L-asparaginase.

[0042] 27. A modified protein according to any one of items 17 to 26, selected from the group comprising the free amino groups to which the peptide is conjugated, including the ε-amino groups of any lysine residue(s) of the L-asparaginase and the N-terminal α-amino groups(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. A modified protein according to any one of items 1 to 28, wherein the polypeptide or peptide mediates the reduction of the immunogenicity of the modified protein.

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

[0046] 31. The nucleic acid is (a) Nucleic acids containing the nucleotide sequence of sequence number 12 or 14, (b) A nucleic acid comprising a nucleotide sequence having at least 85% identity with a nucleotide sequence as defined in (a), and (c) Nucleic acids that are degenerate as a result of the genetic code for nucleotide sequences as defined in (a) or (b). 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 the nucleic acid according to item 30 or 31 or the 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 the modified protein according to any one of items 1 to 16, 29 or the nucleic acid according to item 30 or 31.

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

[0052] 37. A process for preparing the protein defined in any one of items 17 to 29, wherein the process comprises (a) the formula R N -(P / A)-R C-act (wherein R C-act is the carboxy-activated form of R C , 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)) of the activated peptide is coupled with L-asparaginase to obtain a modified protein of the L-asparaginase and a peptide wherein R N is a protecting group.

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

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

[0055] 40. The composition described in item 39, which is a pharmaceutical composition comprising optionally further pharmaceutically acceptable carriers or excipients.

[0056] 41. Modified proteins as described in any one of items 1 to 29 or modified proteins prepared by the process described in any one of items 35 to 38, or compositions as described in item 39 or 40, for use as a drug.

[0057] 42. Modified proteins as described in any one of items 1 to 29 or modified proteins prepared by a process as described in any one of items 35 to 38, or compositions as described in item 39 or 40, for use in the treatment of a disease in a patient, for example, a disease that can be treated by L-asparagine depletion.

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

[0059] 44. The modified protein for use described in item 42, the composition for use described in item 42, or the method described in item 43, wherein the disease that can be treated by L-asparagine depletion is cancer.

[0060] 45. Modified proteins as described in any one of items 1 to 29 for use in the treatment of cancer, modified proteins prepared by the process described in any one of items 35 to 38, or compositions as described in item 39 or 40.

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

[0062] 47. A modified protein for use as described in item 44 or 45, or a composition for use as described in item 44 or 45, wherein the cancer is a non-solid tumor, or the method described in item 44 or 46, wherein the cancer is a non-solid tumor.

[0063] 48. The modified protein for use described in item 47, or the composition for use described in item 47, wherein the non-solid tumor is leukemia or non-Hodgkin lymphoma, or the method described in item 47, wherein the non-solid tumor is leukemia or non-Hodgkin lymphoma.

[0064] 49. A modified protein for use as described in item 48, or a composition for use as described in item 48, wherein the leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML), or the method described in item 48, wherein the leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

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

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

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

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

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

[0070] 55. A modified protein for use as described in any one of items 42, 44, 45 and 47-54, wherein the treatment involves intravenous administration of the modified protein, or a composition for use as described in any one of items 42, 44, 45 and 47-54, or the method as described in any one of items 43, 44 and 46-54. [Brief explanation of the drawing]

[0071] [Figure 1] This figure shows the chemistry of conjugation of chrysanthaspsase with N-terminally protected P / A peptides via an amino group. (A) and (B) show the chemical structures of P / A peptides (SEQ ID NOs. 16 and 17, amino acid sequences shown in SEQ ID NOs. 5 and 15) obtained by solid-phase peptide synthesis, each containing 20 or 40 Pro / Ala residues, respectively. The N-terminus was protected with a pyroglutamyl (Pga) residue to avoid peptide polymerization during C-terminal chemical activation. Aminohexanoic acid (Ahx) was incorporated into the C-terminus of the peptide as a linker. (C) In the presence of the non-nucleophilic base N,N-diisopropylethylamine (DIPEA, Hug's base) and with DMSO as the solvent, the N-terminally protected P / A peptide was activated at the C-terminus with the benzotriazole derivative tetrafluoroborate O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium (TBTU). While releasing free hydroxybenzotriazole (HOBt), the amino group of the chrysanthaspase (ε-amino group of the lysine residue or α-amino group of the N-terminus) is subsequently derivatized by the P / A peptide via the formation of a peptide bond or isopeptide bond using the HOBt active ester of the peptide. This coupling step is carried out in an aqueous solution with an organic solvent content of 30% or less (e.g., PBS buffer). The modified protein of P / A-chrysanthaspase may be purified from the residual P / A peptide / coupling reagent by dialysis and / or chromatography (e.g., ion exchange chromatography). [Figure 2] This figure shows the optimization of the coupling ratio of chrysanthase / Pga-P / A(20)-Ahx. Recombinant chrysanthase produced in E. coli was conjugated with Pga-P / A#1(20)-Ahx peptides (SEQ ID NOs. 16 and 17, amino acid sequences shown in SEQ ID NOs. 5 and 15) as described in Example 1. The peptide-to-protein ratio was varied between 3.5 mg and 10 mg of P / A peptide per 1 mg of chrysanthase. 7 μg of chrysanthase from each coupling reaction was loaded onto the gel. Furthermore, a mix of coupling reactions with a peptide ratio of 0.3 to 10 mg 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 its ladder, starting from the unconjugated chrysanthase as indicated on the right. Lane "kDa": Pierce® unstained protein molecular weight marker (Thermo Fisher Scientific). [Figure 3] This figure shows the purification of the chrysanthaspase / Pga-P / A(40)-Ahx peptide coupling product via anion exchange chromatography. Recombinant chrysanthaspase produced in E. coli was conjugated with the Pga-P / A(40)-Ahx peptide (Figure 1B) (SEQ ID NO: 17, amino acid sequence shown in SEQ ID NO: 15) as described in Example 2. After dialysis with AIX running buffer (25 mM borate / NaOH, pH 9.0, 1 mM EDTA), anion exchange chromatography was performed on an 85 mL Source® 15Q column (A). By applying a NaCl concentration gradient, the enzyme-modified protein eluted as a single sharp peak, as revealed by UV tracing at 280 nm. Separation of the remaining uncoupled peptide and other non-proteinoid byproducts of the chemical conjugate lacking UV absorption at 280 nm was monitored by UV tracing at 225 nm. (B) SDS-PAGE analysis of modified chrysanthaspase / Pga-P / A(40)-Ahx protein after purification by anion exchange chromatography (lane 1). A mix of coupling reactions at a peptide ratio of 0.3–10 mg per mg of protein was applied to lane 2 to determine the number of coupled P / A peptides per chrysanthaspase monomer. A pre-stained marker for PageRuler® Plus (Thermo Fisher Scientific) was applied to lane "M". [Figure 4] This figure shows the purification of the chrysanthaspase / Pga-P / A(20)-Ahx peptide coupling product via anion exchange chromatography. Recombinant chrysanthaspase produced in E. coli was conjugated with the Pga-P / A(20)-Ahx peptide (Figure 1A) (SEQ ID NO: 16, amino acid sequence shown in SEQ ID NO: 5) as described in Example 3. After dialysis with AIX running buffer (25 mM borate / NaOH, pH 9.0, 1 mM EDTA), anion exchange chromatography was performed on an 85 mL Source® 15Q column (A). By applying a NaCl concentration gradient, the enzyme-modified protein eluted as a single sharp peak, as revealed by UV tracing at 280 nm. Separation of the remaining uncoupled peptide and other non-proteinogenic byproducts of the chemical conjugate lacking UV absorption at 280 nm was revealed by UV tracing at 225 nm. (B) SDS-PAGE analysis of modified chrysanthaspase / Pga-P / A(20)-Ahx protein after purification by anion exchange chromatography (lane 1). A mix of coupling reactions at a peptide ratio of 0.3–10 mg per mg of protein was applied to lane 2 to determine the number of coupled P / A peptides per chrysanthaspase monomer. A pre-stained marker for PageRuler® Plus (Thermo Fisher Scientific) was applied to lane "M". [Figure 5] This figure shows the cloning of an expression vector for the production of PAS-modified chrysanthaspas in E. coli. (A) This figure shows plasmid maps of (A) pASK75-SapI-chrysanthaspas (SEQ ID NO: 4) and (B) its derivative pASK75-PA400-chrysanthaspas (SEQ ID NO: 14) after seamless insertion of PA#1c / 1b(400) (SEQ ID NO: 10) into the gene cassette via two inversely oriented SapI restriction sites. The low repetition nucleotide sequence encoding the PA#1 polypeptide with 401 amino acid residues and the bacterial Enx signal sequence (SP) are shown as well as the chrysanthaspas structure gene. EnxThe structural gene of the biologically / pharmacologically active (pre)protein PA#1(400)-chrysanthaspase (SEQ ID NO: 13), which includes the coding region of ), has been modified with a tet promoter / operator (tet p / o Cloning was performed under the transcriptional control of ). The plasmid backbone outside the expression cassette, adjacent to the XbaI and HindIII restriction sites, is identical to that of the gene expression vector pASK75 (Skerra, (1994), Gene, 151:131-135). A plasmid for the expression of chrysanthaspase fused to PA#1(200) (SEQ ID NO: 11) was cloned using the same method with the PA#1b(200) gene cassette (SEQ ID NO: 12). [Figure 6] This figure shows the SDS-PAGE analysis of recombinant chrysanthaspas fused to PA200 or PA400. (A) Analysis of mature PA#1(400)-chrysanthaspas fusion protein (SEQ ID NO: 13) by 10% SDS-PAGE 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)-chrysanthaspas (lane 1) (SEQ ID NO: 11) or PA#1(400)-chrysanthaspas (lane 2) (SEQ ID NO: 13). The size of the marker protein (M) is shown on the left. The PA#1(200)-chrysanthaspas and PA#1(400)-chrysanthaspas fusion proteins appear as a single homogeneous band 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 a significantly larger size than, for example, the 51 kDa mass calculated for the PA#1(200)-chrysanthaspase monomer or the 67 kDa mass calculated for the PA#1(400)-chrysanthaspase monomer (Schlapschy, (2013), Protein Eng. Des. Sel. 26:489-501). [Figure 7] This figure shows size exclusion chromatography of PAS-modified chrysanthanaspases. (A) Superposition of elution characteristics of unmodified chrysanthanaspases, chrysanthanaspases chemically conjugated to either Pga-P / A(20)-Ahx or Pga-P / A(40)-Ahx (described in Examples 3 and 2, respectively), and recombinant chrysanthanaspases fused to the polypeptide PA#1(200) (SEQ ID NO: 7) or PA#1(400) (SEQ ID NO: 9) (described in Example 5). 150 μL of purified protein at a concentration of 1 mg / mL was subjected to a Superdex® S200 10 / 300GL column equilibrated in PBS buffer. Absorption at 280 nm was monitored, and peaks at each chromatographic setting were normalized to 100%. (B) Calibration curves for the chromatograms from (A) using the Superdex S200 10 / 300GL column. The logarithms 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) were plotted against their elution volumes (black circles), and a straight line was fitted to the plot. From the observed elution volumes of tetrameric chrysanthaspase, its PA#1 peptide-modified protein, and its recombinant PA#1 fusion protein (black square), their apparent molecular sizes were determined as follows. Chrysanthantapase: 105 kDa (true mass 140 kDa), Chrysanthantapase / Pga-P / A(20)-Ahx modified protein: 531 kDa (true mass 228 kDa), Chrysanthantapase / Pga-P / A(40)-Ahx modified protein: 820 kDa (true mass 284 kDa), PA200-chrysanthantapase: 595 kDa (true mass 205 kDa), PA400-chrysanthantapase: 1087 kDa (true mass 269 kDa). These data indicate that gene fusion with chemically conjugated P / A peptides and PA#1 polypeptides both confer significantly expanded hydrodynamic volume. [Figure 8] This figure shows the ESI-MS analysis of PAS-modified chrysanthaspases. (A) The raw m / z spectra obtained by electron-spray ionization mass spectrometry (ESI-MS) of purified chrysanthaspases / Pga-P / A(20)-Ahx modified proteins prepared as described in Example 3 were deconvoluted to obtain mass spectra (B). The observed mass species could be clearly assigned to chrysanthaspases conjugated with 9–14 peptides (see Table 3). However, the major peaks were observed only for protein species with 10–13 peptides, corresponding to the determination of peptide coupling ratios by SDS-PAGE (see Figure 4B). (C) and (E) show the raw m / z spectra of PA200-chrysanthaspases and PA400-chrysanthaspases 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] This figure shows the mean (±SD) plasma concentration over time following a single IV bolus administration to male CD-1 mice. The figure shows the plasma asparaginase activity of the PA-chrysanthanspase conjugate following a single IV bolus administration to male mice. [Modes for carrying out the invention]

[0072] In one embodiment, 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, the (poly)peptides consisting only of proline and alanine amino acid residues. The explanations and definitions given herein with respect to the terms “modified protein,” “L-asparaginase,” and “(poly)peptide” provided herein shall apply mutatis mutandis. When used herein, the term “recombinant L-asparaginase” refers to a recombinant form of L-asparaginase having at least 85% identity to the amino acid sequence of natural Erwinia L-asparaginase. The term “recombinant” may refer to L-asparaginase produced by recombinant means, for example, L-asparaginase produced in a host cell containing the nucleic acid encoding L-asparaginase.

[0073] The modified proteins exhibit an even more enhanced plasma half-life, resulting in an extended duration of action compared to each unconjugated L-asparaginase. This allows for a reduction in administration frequency and, consequently, a reduction in the burden of side effects. The present invention also provides a process for preparing modified proteins as 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 only of proline and alanine amino acid residues. The phrase "consisting only of proline and alanine amino acid residues" means that there must be at least one proline residue and at least one alanine residue, i.e., both at least one proline residue and at least one alanine residue must be present. In preferred embodiments, 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 only of proline and alanine amino acid residues. In one embodiment, L-asparaginase is a tetramer (i.e., L-asparaginase composed of four subunits or monomers). An example subunit or monomer has the amino acid sequence of SEQ ID NO: 1.

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

[0076] As shown in the attached examples, the PA#1(200)-chrysanthaspase protein exhibited 109% of the enzymatic activity and the PA#1(400)-chrysanthaspase protein exhibited 118% of the enzymatic activity compared to unmodified chrysanthaspase. See Example 5. This demonstrates that fusion with asparaginase polypeptides, as described herein, does not affect enzymatic activity. Surprisingly, the 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 (enzyme) activity as unmodified asparaginase. (Enzyme) activity may be evaluated by the Nessler assay. Details of the Nessler assay are provided in the accompanying examples and / or disclosed in the prior art, e.g., Mashburn, (1963), Biochem. Biophys. Res. Commun. 12, 50 (the whole of which is incorporated herein by reference). Thus, in one embodiment, the modified proteins provided herein, when evaluated by the Nessler assay, have the same or substantially the same (enzyme) activity as unmodified asparaginase. The term “unmodified asparaginase” as used herein refers to natural asparaginase, i.e., asparaginase that has not been modified by fusion / conjugate with a (poly)peptide as defined herein.

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

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

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

[0081] Generally, modified proteins can be obtained by chemical coupling or gene fusion (in the case of conjugation 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, the polypeptides consisting solely of proline and alanine amino acid residues. In this context, the polypeptides may consist of approximately 200 to 400 proline and alanine amino acid residues. An example amino acid sequence of such a polypeptide is shown in SEQ ID NOs. 7 or 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 only 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, for example, 20 proline and alanine amino acid residues, or 40 proline and alanine amino acid residues. An example amino acid sequence of such a peptide is AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 15).

[0083] In particular, if the term “modified protein” refers to a modified protein obtained by chemical coupling or as a fusion protein, that is, if it mainly comprises (i) L-asparaginase and (ii) one or more (poly)peptides, then the term “modified protein” may be used interchangeably with the term “conjugated” as used herein, where the (poly)peptide consists only of proline and alanine amino acid residues. Similarly, the terms “unmodified” and “unconjugated” may be used interchangeably as used herein.

[0084] The present invention also includes (a) formula R N -(P / A)-R C-act The activated peptide is coupled with L-asparaginase, and L-asparaginase and R N This also relates to a process for preparing modified proteins, including obtaining a modified protein with a peptide in which is a protecting group, where R C-act is R C It is a carboxyl-activated form of R C And (P / A) are as defined in the modified protein being prepared, R N (P / A) is a protecting group that is attached to the N-terminal amino group.

[0085] The attached examples (see Example 1, Table 1) demonstrate that modified proteins can be prepared using various mass ratios of activated peptide and asparaginase. For example, mass ratios of 10:1 (activated peptide:asparaginase), 7.5:1, 5:1, or 3.5:1 can be used. It has been observed that the (enzymatic) activity of the modified protein is highest when ratios of 5:1 or less are used (see Example 1, Table 2). Therefore, in the processes described herein above, it may be advantageous to use activated peptide:asparaginase mass ratios of 5:1 or less, for example, 5:1, 4:1, 3.5:1, or 3:1. When used herein, the term “mass ratio” refers to the ratio of the molecular weights of an activated peptide as defined herein and an asparaginase as defined herein (e.g., an asparaginase as shown in SEQ ID NO: 1 and a protein having at least 85% identity to SEQ ID NO: 1). “Molecular weight” is usually expressed herein using the scientific unit Dalton (Da). It is well known that the molecular weight unit of asparaginases or peptides as shown herein in Daltons (Da) is a substitute name for the unified atomic mass unit (u). Therefore, 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 asparaginase or peptide can be determined by methods known in the art, such as mass spectrometry (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 precipitation), or dynamic (DLS) or static light scattering (e.g., multi-angle light scattering, MALS), or the molecular weight of 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 asparaginase or peptide is determined by mass spectrometry.

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

[0088] The modified protein may be a fusion protein comprising (i) L-asparaginase having at least 85% identity with the amino acid sequence of SEQ ID NO: 1 and (ii) one or more polypeptides, the polypeptides consisting only 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, it is preferable that 10% to 70% of the total number of amino acid residues in the polypeptide are proline residues, more preferably 20% to 50% of the total number of amino acid residues in the polypeptide are proline residues, and even more preferably 30% to 40% (for example, 30%, 35%, or 40%) of the total number of amino acid residues in the polypeptide are proline residues.

[0090] The polypeptide may contain multiple amino acid repeats, each repeat consisting of proline and alanine residues, with six or fewer consecutive amino acid residues being identical. In particular, the polypeptide may contain, or consist of, the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or the entire sequence as a circular permutation or polymer(s) of the sequence, in whole or in part.

[0091] Preferably, the polypeptide comprises or consists of an amino acid sequence such as that shown 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 such as that shown in SEQ ID NO: 8 or 10. It is preferred herein that the modified protein comprises or consists of (a) an amino acid sequence such as that shown in SEQ ID NO: 11 or 13, or (b) an amino acid sequence encoded by a nucleic acid having a nucleotide sequence such as that shown in SEQ ID NO: 12 or 14. In one embodiment, the polypeptide is a random coil polypeptide.

[0092] In some embodiments, a modified protein, such as a fusion protein, has higher asparaginase activity or glutaminase activity than that of unconjugated L-asparaginase. For example, a modified protein can have at least 5% and / or up to 30% (e.g., at least 10%, 15%, 20%, 25%, (or more)) higher asparaginase activity or glutaminase activity than that of unmodified L-asparaginase, particularly when evaluated by the Nessler assay. In further embodiments, the L-asparaginase in the modified protein, such as a fusion protein, is covalently linked directly to a terminal residue of the polypeptide by an amine bond, and / or the fusion protein is produced recombinantly. In preferred embodiments, the modified protein, such as a fusion protein, includes a linker between the L-asparaginase and the polypeptide. An example linker may be an alanine amino acid residue. The present invention also relates to a pharmaceutical composition comprising a modified protein, for example, a fusion protein, or a pharmaceutical composition for use in a therapeutic or pharmacopoeia, or a pharmaceutical composition 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) nucleic acid molecules comprising a nucleotide sequence having sequence number 12 or 14, (b) nucleic acid molecules comprising a nucleotide sequence having at least 85% identity to a nucleotide sequence as defined in (a), and (c) nucleic acid molecules that are 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 modified proteins or nucleic acids as defined herein. The process may include culturing host cells as defined herein and isolating the modified proteins from the culture or from the cells. A process for preparing modified proteins, particularly fusion proteins, as defined herein may include culturing host cells transformed with a vector containing a modified protein, particularly a fusion protein, or host cells containing such a vector, under conditions that induce expression of the modified protein, particularly a fusion protein. In some aspects, the host cells are selected from the group listed above.

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

[0096] The problems to be solved by the present invention can be considered to be the provision of an L-asparaginase preparation that has high biological activity in vitro, stable binding of proteins and modifying factors, a long half-life in vivo, significantly reduced immunogenicity as evidenced by, for example, a decrease or disappearance of the antibody response to the L-asparaginase preparation following repeated administration, and / or usefulness 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 in accordance with the present invention by the embodiments characterized in the claims, in particular by providing L-asparaginase and a modified protein comprising a modifying factor, i.e., (ii) a (poly)peptide consisting only of proline and alanine amino acid residues, and by providing a method for preparing the same and a method for using the same.

[0098] In one embodiment, the herein describes a modified L-asparaginase having improved pharmacological properties compared to an unmodified L-asparaginase protein.

[0099] When used herein, the term “modified L-asparaginase” refers to a “modified protein comprising (i) L-asparaginase and (ii) one or more (poly)peptides, wherein the (poly)peptide consists only of proline and alanine amino acid residues.” In one aspect of the present invention, the L-asparaginase is derived from Erwinia and has at least 85% identity with SEQ ID NO: 1.

[0100] The modified L-asparaginases described herein, for example, L-asparaginases in which a (poly)peptide is conjugated or fused to one or more (poly)peptides consisting only of proline and alanine amino acid residues, are particularly useful as therapeutic agents for use in patients who are hypersensitive (e.g., allergic reactions or asymptomatic hypersensitivity) to treatment with L-asparaginase or pegylated L-asparaginase derived from Erwinia and / or E. coli, or with unmodified L-asparaginase derived from Erwinia. The modified L-asparaginases described herein are also useful as therapeutic agents for use in patients with disease relapses, for example, those with relapses of ALL and who have been previously treated with other forms of asparaginase.

[0101] Erwinia chrysanthemi (also known as Pectobacterium chrysanthemi) has been renamed Dickeya chrysanthemi. Therefore, the terms Erwinia chrysanthemi, Pectobacterium chrysanthemi, and Dickeya chrysanthemi are used interchangeably in this specification.

[0102] Unless otherwise defined, terms used herein shall be understood in accordance with their ordinary meanings in the art.

[0103] As used herein, unless the context necessarily otherwise determines, the term "inlucing" means "including, without limitation," and a term used singularly is to include plurals, and vice versa.

[0104] When used herein, the terms “comprising,” “including,” “having,” or their grammatical variations should be interpreted as identifying the described feature, complete, step, or component, but not as precluding the addition of one or more additional features, completes, steps, components, or groups thereof. The terms “comprising” / “including” / “having” encompass “consisting of” and “substantially consisting of.” Accordingly, whenever the terms “comprising” / “including” / “having” are used herein, they may be replaced by “substantially consisting of” or preferably “consisting of.”

[0105] The terms "comprising," "including," and "having" mean that any further components (or similar features, completes, steps, etc.) may exist.

[0106] "Consists of ~" means that no further components (or similar features, complete forms, steps, etc.) can exist.

[0107] When used herein, “substantially consisting of” or its grammatical variation should be interpreted as identifying the described feature, whole, step, or component, but not to the extent that the addition of one or more additional features, wholes, steps, components, or groups thereof does not materially alter the basic and novel features of the claimed product, composition, apparatus, or method, etc.

[0108] Therefore, “substantially consisting of” means that specific additional components (or similar features, completes, steps, etc.) that do not materially affect the essential characteristics of the product, composition, apparatus, or method may be present. In other words, the term “substantially consisting of” (which may be used herein interchangeably with the term “comprising substantially”) allows the presence of other components in the product, composition, apparatus, or method in addition to the essential components (or similar features, completes, steps, etc.), provided that the essential characteristics of the product, composition, apparatus, or method are not materially affected by the presence of the other components.

[0109] As used herein, the term “approximately” means ±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” means a condition or disease in which the cells involved in or causing a condition or disease lack or have reduced ability to synthesize L-asparagine. The depletion or loss of L-asparagine may be partial or substantially complete (for example, to a level undetectable by methods and apparatus known in the art).

[0112] As used herein, the term “therapeutably effective amount” refers to the amount of protein (e.g., asparaginase or a modified version thereof) required to produce the desired therapeutic effect.

[0113] As used herein, the term “L-asparaginase” refers to an enzyme possessing L-asparagine aminohydrolase activity. The enzymatic activity of L-asparaginase may include not only the deamidation of asparagine to aspartic acid and ammonia, but also the deamidation of glutamine to glutamic acid and ammonia. Asparaginase is typically composed of four monomers (although some have been reported to have five or six). Each monomer can be approximately 32,000 to 36,000 daltons.

[0114] Numerous L-asparaginase proteins isolated from microorganisms by known methods have been identified using this technique (see, for example, Savitri and Azmi, Indian J. Biotechnol. 2, (2003), 184-194, which are incorporated herein by reference in their 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 the "L-asparaginase" used in accordance with the present invention. Among Erwinia species, sequence identity of 75–77% is typically reported between enzymes derived 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, the whole of which is incorporated herein by reference). Some representative Erwinia L-asparaginases are provided in Table 1 below, which discloses the percentage of sequence identity to Erwinia chrysanthemi NCPPB1066. [Table A]

[0116] The sequences and GenBank registrations of Erwinia L-asparaginases in Table 1 are incorporated herein by reference. Examples of L-asparaginases used in therapeutics 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. They can also be produced by recombinant enzyme technology that creates microorganisms such as E. coli. For example, the protein used in the modified protein of the present invention may be a recombinant protein produced by the E. coli strain, preferably a protein derived from an Erwinia species, particularly Erwinia chrysanthemi, produced by the recombinant E. coli strain.

[0118] Enzymes can be identified by their specific activity. Therefore, this definition includes all polypeptides with defined specific activity that are also present in other organisms, and more specifically, other microorganisms. Enzymes with similar activity can often be identified by grouping them into specific families, defined as PFAMs or COGs. PFAMs (Database of Protein Families and Hidden Markov Models for Sequence Comparison, pfam.sanfferac.ukl) represent a large collection of protein sequence comparisons. Each PFAM allows for the visualization of multiple sequence comparisons, identification of protein domains, assessment of inter-organism distribution, 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 obtained by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenetic strains. Each COG is defined from at least three strains, enabling the identification of previously conserved domains.

[0119] Means for identifying the ratio of sequence identity are well known to those skilled in the art, including the BLAST program, which can be used from the website blast.ncbi.olo.nih.gov / Blast.cgi with initial setup parameters shown on the website. The obtained sequences can then be used (e.g., aligned) using, for example, the CLUSTALW program (ebi.ac.uk / Tools / clustalw2 / index.html) with initial setup parameters. Using the references given in GenBank for known genes, those skilled in the art can determine equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc. This routine work is advantageously carried out using consensus sequences that can be determined by performing sequence comparisons with genes from other microorganisms and by 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 (the whole is incorporated herein by reference).

[0121] In certain embodiments of the modified protein of the present invention, L-asparaginase has at least about 85% homology or sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and more specifically, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity with respect to the amino acid sequence of SEQ ID NO: 1 as described in the attached sequence listing. The terms “homology” and “sequence identity” are used interchangeably herein.

[0122] The term "contains the sequence of SEQ ID NO: 1" (for example, if 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 asparaginase does not have to be strictly limited to SEQ ID NO: 1 and may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional amino acids. In other words, if L-asparaginase as used herein has 100% homology or sequence identity to the amino acid sequence of SEQ ID NO: 1, then L-asparaginase contains or consists of the amino acid sequence of SEQ ID NO: 1. The term "contains" in this context means that the amino acid sequence of 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 certain embodiments, the protein is L-asparaginase of Erwinia chrysantherni containing or comprising the amino acid sequence of Sequence ID No. 1. In other embodiments, the L-asparaginase is derived from NCPPB1066 of Erwinia chrysantherni (GenBank acceptance number CAA32884, which is incorporated herein by reference in its entirety), with or without the presence of a signal peptide and / or a leader sequence.

[0124] L-asparaginase, preferably the L-asparaginase fragment of SEQ ID NO: 1, is also included within the definition of L-asparaginase as used in the modified proteins of the present invention. The term "asparaginase fragment" (e.g., the asparaginase fragment of SEQ ID NO: 1) means that the asparaginase sequence may contain fewer amino acids than in the asparaginase exemplified herein (e.g., the asparaginase of SEQ ID NO: 1), but may still contain enough amino acids to confer L-asparaginase activity. For example, “fragment of asparaginase” is a fragment consisting of at least about 150 or 200 adjacent amino acids (for example, 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 adjacent amino acids) of one of the asparaginases exemplified herein (for example, asparaginase of SEQ ID NO: 1) / and / or the fragment is the first 50 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25) from the N-terminus of the asparaginase exemplified herein (for example, asparaginase of SEQ ID NO: 1), A fragment is missing amino acids up to 30, 35, 40, 45, or 50, and / or the fragment is missing amino acids up to 75 or 100 (for example, up to 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) from the C-terminus of the asparaginase exemplified herein (for example, the asparaginase of SEQ ID NO: 1), and / or the fragment is missing amino acids at both the N-terminus and C-terminus of the asparaginase exemplified herein (for example, the asparaginase of SEQ ID NO: 1), and the total number of missing amino acids may be up to 125 or 150.

[0125] It is well known in this art that polypeptides can be modified by substitution, insertion, deletion, and / or addition of one or more amino acids while retaining their enzymatic activity. In this context, the term "one or more amino acids" can refer to one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids. For example, the 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. A substitution may be defined as an exchange within one of the following groups: • Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly • Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln • Polarly charged residues: His, Arg, Lys • Large aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys • Large aromatic residues: Phe, Tyr, Trp

[0126] Therefore, it can be expected that changes resulting from substituting one negatively charged residue with another negatively charged residue (e.g., glutamic acid with aspartic acid) or substituting one positively charged residue with another positively charged residue (e.g., lysine with arginine) will produce functionally equivalent products.

[0127] The position of amino acid modification in the amino acid sequence and the number of amino acids targeted for modification are not particularly limited. Skilled experts can recognize modifications that can be introduced without affecting protein activity. For example, modifications at the N-terminus or C-terminus of a protein may be expected not to alter protein activity under certain circumstances. With regard to asparaginases, many characterizations have been performed, particularly concerning the sequences, structures, and residues that form the active catalytic sites. This provides guidance on residues that can be modified without affecting enzyme activity. All known L-asparaginases of bacterial origin share common structural features. They are all homotetramers with four active sites between the N-terminal and C-terminal domains of two adjacent monomers (the whole is incorporated herein by reference Aghaipour, (2001), Biochemistry, 40, 5655-5664). All of them exhibit a high degree of similarity in their tertiary and quaternary structures (Papageorgiou, (2008), FEBS J.275, 4306-4316, the whole is incorporated herein by reference). The sequence of the catalytic site of L-asparaginase is highly conserved among the L-asparaginase(II) of Erwinia chrysanthemi, Erwinia carotovora, and E. coli (ibid.). The flexible loop of the active site contains amino acid residues 14-33, and structural analysis shows that Thr15, Thr95, Ser62, G1u63, Asp96, and A1a120 contact the ligand (ibid.). Aghaipour et al. have conducted 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-asparaginase derived from several species and subspecies of Erwinia, and despite the proteins having only about 75–77% identity between Erwinia chrysanthemi and Erwinia carotovora, they still retain L-asparaginase activity (Kotzia, (2007), J. Biotechnol. 127, 657–669). Moola et al. performed epitope mapping on Erwinia chrysanthemi3937 L-asparaginase and found that the enzyme activity was retained even after various antigenic sequence mutations in an attempt to reduce the immunogenicity of the asparaginase (Moola, (1994), Biochem. J. 302, 921–927). Considering the extensive characterization that has been performed on L-asparaginase, those skilled in the art can determine methods for creating fragments and / or performing sequence substitutions while retaining enzyme activity.

[0128] When used herein, the term “about” modifies, for example, the dimensions, volume, quantity, concentration, process temperature, process time, yield, flow rate, pressure, and similar values ​​of components in a composition, and their ranges, to mean quantities that may arise through typical measurement and handling procedures used to make a compound, composition, concentrate, or formulation for use, through careless errors in these procedures, through differences in the manufacture, source, or purity of the starting materials or components used to carry out the method, and variations in similar considerations. The term “about” also includes, for example, quantities that differ due to the degradation over time of a particular initial concentration or mixture of a composition, formulation, or cell culture, and quantities that differ by mixing or processing a particular initial concentration or mixture of a composition or formulation. Whether or not modified by the term “about,” the claims attached herein include equivalents to these quantities. The term “about” may also mean a range of values ​​similar to the reference values ​​further stated. In certain embodiments, “about” means a range of values ​​that fall within 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 percent of the reference values ​​stated.

[0129] In the context of the present invention, surprisingly, a specific C-terminal amino acid residue (R) of one or more peptides consisting only of proline and alanine amino acid residues. C Chemical conjugation to L-asparaginase via ) has been found to provide L-asparaginase-modified proteins with significantly reduced immunogenicity and improved plasma half-life, as it has a particularly high coupling ratio of the peptide per molecule of asparaginase. This novel technique can also be applied to L-asparaginase without impairing its catalytic activity, which has been found to greatly enhance the therapeutic value of the corresponding modified proteins described herein.

[0130] In one embodiment, the herein describes 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 with the amino acid sequence of SEQ ID NO: 1, and (ii) one or more peptides, the peptides consisting only of proline and alanine amino acid residues.

[0131] In a preferred embodiment, the modified protein is L-asparaginase, and each independently forms peptide R N -(P / A)-R C A modified protein with one or more peptides, where (P / A) is an amino acid sequence consisting only of proline and alanine amino acid residues, and R N R is a protecting group that is attached to the N-terminal amino group of an amino acid sequence. C R is an amino acid residue that is bonded to the C-terminal carboxyl group of the amino acid sequence via its amino group, and each peptide has a C-terminal amino acid residue R C The peptide is conjugated to L-asparaginase via an amide bond formed from the carboxyl group of the peptide and a free amino group of 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 L-asparaginase.

[0132] In some embodiments, the monomer of the modified protein has approximately 350, 400, 450, or 500 amino acids after modification, ranging from approximately 550, 600, 650, 700, or 750 amino acids. In additional embodiments, the modified protein has approximately 350 to approximately 750 amino acids, or approximately 500 to approximately 750 amino acids.

[0133] Each peptide contained in the modified proteins described herein is independently peptide R N -(P / A)-R C Therefore, for each of the peptides contained in the modified proteins described herein, the N-terminal protecting group R N , amino acid sequence (P / A), and the C-terminal amino acid residue R C Each of these is independently selected according to its own intention. Therefore, two or more peptides contained in the modified protein may be identical, or they may be different from one another. In one embodiment, all of the peptides contained in the modified protein are identical.

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

[0135] Peptide R N -(P / A)-R CThe portion (P / A) in the chemically conjugated modified protein contained therein is an amino acid sequence that can consist 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 Therefore, the ratio of the number of proline residues in a portion (P / A) to the total number of amino acid residues 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. Accordingly, it is preferable that 10% to 70% of the total number of amino acid residues in (P / A) are proline residues, more preferably 20% to 50% of the total number of amino acid residues in (P / A) are proline residues, and even more preferably 25% to 40% (for example, 25%, 30%, 35%, or 40%) of the total number of amino acid residues in (P / A) are proline residues. Furthermore, it is preferable that (P / A) does not contain consecutive proline residues (i.e., it does not contain the sub-sequence PP). In a preferred embodiment, (P / A) is 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 In R NThe group may be a protecting group linked to the N-terminal amino group of the amino acid sequence (P / A), particularly the N-terminal α-amino group. N It is preferable that it be pyroglutamoyl or acetyl.

[0137] Peptide R N -(P / A)-R C In R C The group is an amino acid residue that is bonded to the C-terminal carboxyl group of (P / A) via its amino group, and contains at least two carbon atoms between the amino group and its carboxyl group. C At least two carbon atoms between the amino group and the carboxyl group are R C The distance between at least two carbon atoms between the amino group and the carboxyl group may be provided (for example, R C ω-amino-C 3-15 Alkanates (for example, ω-aminohexanoic acid) will be understood. C It is preferable that it is ω-aminohexanoic acid.

[0138] In one embodiment, the peptide is Pga-AAPAAPAPAAPAAPAPAPAAPA-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 is evident in the attached examples, C-terminal amino acid residues R as defined herein, particularly ω-aminohexanoic acid C The use of asparaginase allows for a favorably high coupling ratio of peptides consisting only of proline and alanine amino acid residues per molecule to be provided to the modified protein, thereby allowing for a favorably reduced immunogenicity and a favorably improved plasma half-life to be provided to the modified protein.

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

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

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

[0143] Modified proteins as described herein consist of L-asparaginase as defined herein and one or more peptides. A corresponding modified protein may consist, for example, of one L-asparaginase and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 (or more) peptides conjugated to the L-asparaginase. The L-asparaginase may be, for example, a monomeric protein or a protein composed of multiple subunits, for example, a tetramer. If the L-asparaginase is a monomeric protein, the corresponding modified protein may consist, for example, of one monomeric L-asparaginase and 9 to 13 (or more) (for example, 9, 11, 12, or 13) peptides conjugated to the monomeric L-asparaginase. An example amino acid sequence of monomeric L-asparaginase is shown in SEQ ID NO: 1. If L-asparaginase is a protein composed of multiple subunits, for example, four subunits (i.e., if L-asparaginase is a tetramer), then the corresponding modified protein may consist, for example, of four L-asparaginase subunits and 9 to 13 (or more) peptides as defined herein (e.g., 9, 11, 12, or 13) conjugated to each L-asparaginase subunit. An example amino acid sequence of an L-asparaginase subunit is shown in Sequence ID No. 1. Similarly, if L-asparaginase is a protein composed of multiple subunits, for example, four subunits (i.e., if L-asparaginase is a tetramer), the corresponding modified protein may consist, for example, of four L-asparaginase subunits and 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 (or more) peptides conjugated to the L-asparaginase tetramer, respectively.In one embodiment, the present invention relates to a modified protein having L-asparaginase and a plurality of chemically linked peptide sequences. In a further embodiment, the length of the peptide sequences is about 10 to about 100, about 15 to about 60, or about 20 to about 40.

[0144] A peptide consisting solely of proline and alanine amino acid residues may be covalently bonded to one or more amino acids of the L-asparaginase, such as a lysine residue and / or an N-terminal residue, and / or the peptide consisting solely of proline and alanine amino acid residues may be covalently bonded to at least about 40, 50, 60, 70, 80, or 90% to about 60, 70, 80, 90, or 100% of the contactable amino groups, including the amino groups of the lysine residue and / or N-terminal residue, on the surface of the L-asparaginase. For example, there may be about 11 to 12 contactable lysine residues per L-asparaginase, and about 9 to 12 lysine residues that would be conjugated with a peptide consisting solely of proline and alanine amino acid residues. In a further embodiment, a peptide consisting solely of proline and alanine amino acid residues is covalently bonded to approximately 20, 30, 40, 50, or 60% to approximately 30, 40, 50, 60, 70, 80, or 90% of the total lysine residues of the L-asparaginase. In a further embodiment, the peptide consisting solely of proline and alanine amino acid residues is covalently bonded to the L-asparaginase via a linker. An example linker is disclosed in U.S. Patent Application Publication No. 2015 / 0037359, which is incorporated herein by reference in its entirety.

[0145] In addition, the modified protein may have a half-life of at least approximately 5, 10, 12, 15, 24, 36, 48, 60, 72, 84, or 96 hours at a dose of approximately 25 μg of 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 proteins according to the present invention can be prepared using methods known in the art. In particular, they can be prepared using the processes described below and / or by following the procedures described in the examples, or similarly.

[0147] The present invention further relates to a process for preparing a modified protein as defined herein, the process being (a) formula R N -(P / A)-R C-act The activated peptide is coupled with L-asparaginase, and L-asparaginase and R N This involves obtaining a modified protein with a peptide in which is a protecting group, where R C-act is R C It is a carboxyl-activated form of R C And (P / A) are as defined in the modified protein being prepared, and R N (P / A) is a protecting group that is attached to the N-terminal amino group.

[0148] The carboxyl-activated C-terminal amino acid residue R in the activated peptide C-act R is an amino acid residue as described and defined herein with respect to peptides. C It may be R C The carboxyl group is in the form of an activated carboxyl group. Preferably, the amino acid residue R in the activated peptide. C-act The activated carboxyl group is an active ester group.

[0149] R C-act If the activated carboxyl group is an active ester group, it is preferably one of the following active ester groups: [Formula 1] One of the following will be selected.

[0150] The ester group with particularly favorable activity is the 1-hydroxybenzotriazole (HOBt) active ester group. Therefore, R C-actThe activated carboxyl group is given by the following formula: [chemical 2] It is the group of the (HOBt active ester group).

[0151] The process further involves, before step (a), R C And (P / A) is as defined for the modified protein that is prepared, R N The protecting group R is a protecting group that is linked to the N-terminal amino group of (P / A). N -(P / A)-R C The process may include a further step of converting the peptide into an activated P / A peptide.

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

[0153] The coupling step (a) and any preceding step that converts the peptide to the activated peptide can be found in, for example, 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. The procedure can be carried out using the peptide coupling or amide bond formation procedures described in the literature in any of the press. Suitable reagents and reaction conditions for such procedures are further described in the aforementioned literature and the further references cited therein. Additional descriptions can be found in U.S. Patents 8,563,521, 9,260,494, and 9,221,882, all of which are incorporated herein by reference in their entirety.

[0154] Protecting group R as required in any step (b) N The procedure for removing the protecting group R is well known in the art, for example, as described in Wuts, et al., 2012, Greene, Protective Groups in Organic Synthesis. Fourth Edition. John Wiley & Sons and / or Isidro-Llobet, et al., 2009, Chem. Rev. 109(6), 2455-2504. Therefore, any step (b) can be replaced with the corresponding protecting group R as described in, for example, one of the aforementioned references. N It can be implemented as described.

[0155] In some embodiments, 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 with the amino acid sequence of SEQ ID NO: 1, and (ii) a polypeptide, the polypeptide consisting solely of proline and alanine amino acid residues. In one embodiment, 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 embodiments, the polypeptide comprises a total of about 200 (e.g., 201) proline and alanine amino acid residues (i.e., having a length of about 200 (e.g., 201) proline and alanine amino acid residues), or the polypeptide comprises a total of about 400 (e.g., 401) proline and alanine amino acid residues (e.g., having a length of about 400 (e.g., 401) proline and alanine amino acid residues). In some preferred embodiments, the polypeptide comprises or comprises an amino acid sequence such as that shown in SEQ ID NO: 7 or 9, or the polypeptide comprises or comprises an amino acid sequence encoded by a nucleic acid having a nucleotide sequence such as that shown in SEQ ID NO: 8 or 10. In some embodiments, the modified protein is preferably a fusion protein, and the modified protein comprises a monomer and a P / A amino acid sequence, with each monomer having about 350, 400, 450, 500 amino acids to about 550, 600, 650, 700, 750, or 1000 amino acids. In an additional embodiment, the modified protein has approximately 350 to approximately 800 amino acids or approximately 500 to approximately 750 amino acids.

[0156] For example, the polypeptide includes the peptide prepared in U.S. Patent No. 9,221,882.

[0157] In a preferred embodiment, the modified protein comprises (a) an amino acid sequence such as that shown in SEQ ID NO: 11 or 13, or (b) an amino acid sequence encoded by a nucleic acid having a nucleotide sequence such as that shown in SEQ ID NO: 12 or 14. In this specification, the modified protein is intended to include (a) a protein having an amino acid sequence such as that shown in SEQ ID NO: 11 or 13, (b) a protein as defined in (a) in which 1 to 65 amino acids are deleted, inserted, added or substituted by asparaginase, (c) a protein encoded by a nucleic acid having a nucleotide sequence such as that shown 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 with a 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) to (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] A modified protein as defined herein may consist of four subunits, the subunits being selected from the group consisting of (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1, (b) a protein as defined in (a) in which 1 to 65 amino acids are deleted, inserted, added or substituted by asparaginase, (c) a protein encoded by a nucleic acid having a nucleotide sequence as shown in SEQ ID NO: 2, (d) a protein having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a 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) to (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 polypeptide, the present invention relates to a nucleic acid encoding a 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) nucleic acids comprising the nucleotide sequence of SEQ ID NO: 12 or 14, (b) nucleic acids comprising a nucleotide sequence having at least 85% identity to a nucleotide sequence as defined in (a), and (c) nucleic acids that are degenerate as a result of the genetic code for a nucleotide sequence as defined in (a).

[0160] In a further embodiment, 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 NOs: 12 or 14. The encoded polypeptide comprises a repeating amino acid sequence which may form a random coil, but the encoding nucleic acid preferably comprises a less repeating nucleotide sequence. In other words, the nucleic acid may comprise a nucleotide sequence encoding a PA-rich polypeptide, wherein the coding 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 repeating nucleic acids, such as those disclosed herein, can be advantageous compared to more repeating nucleic acid molecules. In particular, the genetic stability of less repeating nucleic acid molecules used herein can be improved.

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

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

[0163] The present invention relates to a nucleic acid described herein, i.e., a nucleic acid encoding a modified protein as defined herein, in particular a vector comprising a nucleic acid encoding a modified protein of L-asparaginase and polypeptide, for example, a fusion protein, 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 defined in (a), and (c) a nucleic acid that is degenerate as a result of the genetic code for the nucleotide sequence defined in (a).

[0164] The present invention relates to a host cell comprising a nucleic acid as defined herein, or a vector as defined herein. Examples of 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 nucleic acid encoding such a protein. The process may include culturing host cells as defined herein and isolating the modified protein from the culture or from the cells. The process may also include culturing host cells (for example, host cells transformed with a nucleic acid and / or vector containing a nucleotide sequence encoding the modified protein, preferably a fusion protein, or host cells containing such a vector) under conditions that induce expression of the modified protein (preferably a fusion protein). Examples of hosts are listed above.

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

[0167] Various plasmids can be constructed using methods well known to those skilled in the art. For example, see the technique 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. Typical 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, 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 Examples of suitable plasmid vectors for Pichia pastoris include pTriEx-Hygro (Novagen) and pCINeo (Promega). Non-limited examples of plasmid vectors suitable for Pichia pastoris include plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen).

[0168] Generally, a vector may contain one or more replication origins (oris) and modes of inheritance for cloning and expression, one or more markers for selection in a host, such as antibiotic resistance, and one or more expression cassettes. Examples of suitable replication origins include, for example, full-length ColE1, its truncated form such as that present in pUC plasmids, and replication origins of the SV40 virus and M13 phage. Non-limiting examples of selectable markers include ampicillin, chloramphenicol, tetracycline, kanamycin, dhfr, gpt, neomycin, hygromycin, blasticidine, or geneticin. Furthermore, the vector may include a moduloable sequence that is operably linked to the nucleotide sequence or nucleic acid molecules as defined herein.

[0169] The coding sequence(s) contained in the vector, for example, the nucleotide sequence encoding a polypeptide, can be ligated to transcriptional regulatory sequences(s) and / or sequences encoding other amino acids using established methods. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure the initiation of transcription, internal ribosome entry sites (IRESs), and optionally, regulatory sequences that ensure the termination of transcription and the stabilization of the transcript. Non-limited examples of such regulatory sequences that ensure the initiation of transcription include promoters, translation start codons, enhancers, insulators, and / or regulatory sequences that ensure the termination of transcription. Further examples include Kozak sequences and intervening sequences where donor and acceptor sites for RNA splicing are adjacent, nucleic acid sequences encoding secretory signals, or signal sequences that, depending on the expression system used, can direct the expressed protein towards a cell compartment or culture medium.

[0170] Examples of suitable promoters, without limitation, include the cytomegalovirus (CMV) promoter, SV40 promoter, RSV (Rous sarcoma virus) promoter, lacZ promoter, chicken β-actin promoter, CAG promoter (a combination of the chicken β-actin promoter and the cytomegalovirus pre-initial enhancer), human elongation factor 1α promoter, AOX1 promoter, GAL1 promoter, CaM kinase promoter, lac promoter, trp promoter or tac promoter, lacUV5 promoter, T7 or T5 promoter, Autographa californica polynuclear polyhedron disease virus (AcMNPV) polyhedron promoter, or globin introns in mammalian and other animal cells. An example of an enhancer is, for example, the SV40 enhancer. Examples of additional regulatory sequences / sequences that ensure transcription termination include the SV40 polyA site, the tk polyA site, or the AcMNPV polyhedron polyadenylation signal.

[0171] Furthermore, depending on the expression system, a leader sequence may be added to the nucleic acid coding sequence provided herein, which can direct the polypeptide into a cellular compartment or secrete it into the culture medium. The leader sequence(s) are constructed in frame with the translation start and termination sequences, and preferably the leader sequence can direct the secretion of the translated protein or a portion thereof into the periplasm or extracellular culture medium. Suitable leader sequences include, 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 derived from Metrida or Vargula, human trypsinogen-2, inulinase derived from Kluyveromyces marxianus, fermentation factor alpha-1 derived from Saccharomyces cerevisiae, melittin, human azulocidine, and its analogues 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 embodiments, the vector of the present invention is an expression vector. The expression vector can direct the replication and expression of a nucleic acid molecule of the present invention, for example, a nucleic acid comprising a nucleotide sequence encoding a polypeptide and a nucleotide sequence encoding asparaginase.

[0174] Nucleic acid molecules and / or vectors as described herein above may be designed for introduction into cells by, for example, non-chemical methods (electroporation, sonoporation, optical translocation, electrotransfer of genes, hydrodynamic delivery or spontaneous transformation upon contact between the nucleic acid molecules of the present invention and cells), chemical-based methods (calcium phosphate, DMSO, PEG, liposomes, DEAE-dextran, polyethyleneimine, nucleofection, etc.), particle-based methods (gene gun, magnetofection, imparefection), phage or phagemid vector-based methods, and viral methods. For example, expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, Semliki Forest viruses, or bovine papillomavirus may be used for delivery of nucleic acid molecules to a target cell population.

[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 fully understood that the term “host cells or non-human hosts transformed with vectors” refers to host cells or non-human hosts containing vectors or nucleic acids as described herein. Host cells for polypeptide expression are well known in the art and include eukaryotic and prokaryotic cells. Appropriate culture media and culture conditions for the host cells described above are known in the art.

[0176] "Culturing a host or host cells" includes the expression of modified proteins, including fusion proteins as defined herein, and / or polypeptides and / or asparaginases as defined herein, in a host or host cells.

[0177] Methods for isolating modified proteins and / or polypeptides and / or asparaginases as defined herein include, but are not limited to, affinity chromatography (preferably using fusion tags 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 commonly described, for example, in Scopes, (1994), Protein Purification - Principles and Practice, Springer. Such methods provide substantially pure polypeptides. The pure polypeptides preferably have a homogeneity of at least about 90–95% (at the protein level), and more preferably at least about 98–99%. Most preferably, these pure polypeptides are suitable for pharmaceutical applications.

[0178] It is assumed that a modified protein containing L-asparaginase and polypeptide can be prepared by expressing a nucleic acid molecule containing a nucleotide sequence encoding the polypeptide and a nucleic acid sequence encoding asparaginase. The expressed modified protein can be isolated. Alternatively, the modified protein can be prepared by culturing / producing a host containing a nucleotide sequence or nucleic acid sequence encoding the polypeptide consisting only 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 asparaginase, for example, via a peptide bond or a non-peptide bond.

[0179] The modified proteins described herein can be used to treat diseases that can be treated by asparagine depletion. Diseases that can be treated by asparagine depletion are preferably cancers, such as non-solid tumors. Preferably, non-solid tumors are leukemia or non-Hodgkin lymphoma. Leukemia is preferably acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). For example, the modified proteins are useful in the treatment of acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) in both adults and children, or in the manufacture of drugs for use in treatment. The use of the modified proteins described herein is also intended in the treatment of other conditions in which asparagine depletion is expected to have a beneficial effect. Such conditions include, but are not limited to, malignant tumors or cancers, including, hematological malignancies, NK lymphoma, pancreatic cancer, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum sarcoma, melanoma, and diffuse large B-cell lymphoma (DLBCL). Cancers may also be solid tumors, such as lung cancer or breast cancer. Representative non-malignant hematological disorders that respond to asparagine depletion include immune-mediated hematological disorders, such as infectious diseases caused by HIV infection (i.e., AIDS). Non-hematological disorders associated with asparagine dependence include autoimmune diseases, such as rheumatoid arthritis, SLE, autoimmune diseases, collagen vascular diseases, etc. Other autoimmune diseases include osteoarthritis, Issaac 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 the disease can be examined for asparagine dependence using suitable in vitro or in vivo assays, such as in vitro assays in which the growth medium lacks asparagine.

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

[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. 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 for at least about 12, 24, 48, 72, 96, or 120 hours. When administered at a dose of 25 U / kg (bw) or 50 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. The modified proteins described herein, when administered at doses of 50 U / kg (bw) or 100 μg / kg (protein content basis), can lower plasma L-asparagine levels for at least approximately 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours. The modified proteins described herein can lower plasma L-asparagine levels from approximately 10,000 to approximately 15,000 IU / m³. 2 (Approximately 20-30 mg of protein / m³) 2When administered in doses up to 12 hours, it can reduce plasma L-asparagine levels to undetectable levels for at least approximately 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours.

[0182] The modified proteins described herein can induce levels similar to L-asparagine depletion for a period of time (e.g., 24, 48, or 72 hours) after a single dose.

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

[0184] In some embodiments, the modified proteins described herein do not produce a significant antibody response for a specific period after a single dose, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or longer. For example, the modified protein does not produce a significant antibody response for at least 8 weeks. In one example, "does not produce a significant antibody response" means that the subject accepting the modified protein is identified as antibody-negative within the parameters recognized in the art. The antibody level can be determined by methods known in the art, such as ELISA or surface plasmon resonance assay (each of which is incorporated herein by reference as a whole: Zalewska-Szewczyk, (2009), Clin. Exp. Med. 9, 113-116; Avramis, (2009), Anticancer Research, 29, 299-302). The modified protein may have any combination of these properties.

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

[0186] The incidence of relapse in ALL patients following L-asparaginase treatment remains high, with approximately 10–25% of pediatric ALL patients experiencing early relapses (e.g., some during the maintenance phase 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 may produce a "vaccination" effect, thereby increasing the immunogenicity of the E. coli preparation during subsequent administrations. The modified proteins described herein may also be used in methods to treat patients with relapsed ALL previously treated with other asparaginase preparations, particularly those previously treated with E. coli-derived L-asparaginase. Disease relapse may occur after treatment with E. coli L-asparaginase or its pegylated form.

[0187] In another embodiment, the present invention relates to a method for treating acute lymphoblastic leukemia, comprising administering a therapeutically effective amount of the modified protein described above to a patient in need of treatment. In a specific embodiment, the dose is approximately 1500 IU / m², typically administered once a week to once every two weeks, ranging from about twice a week to about once a month. 2 ~Approx. 15,000IU / m 2 , usually about 10,000 to about 15,000IU / m 2 (Approximately 20-30 mg of protein / m³) 2 Treatment is administered at doses of ). The modified proteins described above may be administered as monotherapy, or as part of a combination with chemotherapeutic agents, including but not limited to glucocorticoids, corticosteroids, and anticancer compounds, or with other agents, including but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracyclines. For example, an ALL patient may be administered the modified proteins described above as a component of multi-agent chemotherapy during three chemotherapy phases, including induction, adjuvant or intensification, and maintenance. In specific examples, the modified proteins described above are not administered with asparagine synthase inhibitors (for example, as described in WO2007 / 103290, which is incorporated herein by reference in whole). In another specific example, the modified proteins described above are not administered with asparagine synthase inhibitors but with other chemotherapeutic agents. The modified proteins described above can be administered before, after, or concurrently with other compounds as part of a multi-agent chemotherapy regimen.

[0188] In certain embodiments, the method comprises administering the modified protein described above in 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). The amount of modified protein to be delivered will depend on a number of factors, such as the IC 50 , EC 50 , 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 will be well known to those skilled in the art. In certain embodiments, the amount of modified protein administered is from about 10 International Units per square meter of body surface area of the patient (IU / m 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 ranges from about 1,000 IU / m 2 to about 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 ) is administered in doses. In another specific embodiment, the modified protein described above is administered as a single dose for a period of about 3 to 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) in a dose that depletes L-asparagine to an undetectable level using methods and apparatus known in the art.

[0189] The modified protein may be administered in doses that deplete L-asparagine to undetectable levels for approximately 3 to 10 days, approximately 5 to 20 days, approximately 1 to 15 days, or approximately 2 to 30 days. The modified protein may be administered in doses that deplete L-asparagine to undetectable levels for periods ranging from approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days to approximately 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 compositions comprising a modified protein as defined herein or a modified protein prepared by a process as described herein. The composition may optionally further comprise a pharmaceutically acceptable carrier(s) or excipient(s).

[0191] The present invention also relates to pharmaceutical compositions comprising the modified proteins described above. In specific embodiments, a pharmaceutical composition such as currently available natural L-asparaginases (e.g., Kidrolase®, Elspar®, Erwinase®), regardless of the bacterial source used in its manufacture, is contained in a vial as a lyophilized powder that is reconstituted with a solvent. In another embodiment, a pharmaceutical composition such as pegaspar gauze (Oncaspar®) is a “ready-to-use” solution that allows for appropriate handling, e.g., administration via intramuscular, intravenous (infusion and / or bolus), intraventricular (icv), or subcutaneous routes.

[0192] Modified proteins, including compositions containing them (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 preferred dosage form depends to some extent on the use or route of administration, e.g., oral, transdermal, transmucosal, or injectable (parenteral) routes. Such dosage forms should allow 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 according to the present invention can be formulated as pharmaceutically acceptable salts and complexes thereof. Pharmaceutically acceptable salts are non-toxic salts present at the amounts and concentrations in which they are administered. Formulations of such salts can facilitate pharmaceutical use by altering the physical properties of the compound without interfering with its physiological effects. Useful alterations to physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate the administration of high concentrations of the drug. The pharmaceutically acceptable salts of modified proteins as described herein may exist as complexes, as will be readily apparent to those skilled in the art. Examples of pharmaceutically acceptable salts include acid addition salts containing sulfates, hydrochlorides, fumarates, maleates, phosphates, sulfamates, acetates, citrates, lactates, tartrates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, cyclohexylsulfamates, and quinates. 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 acidic functional groups such as carboxylic acids or phenols, such as benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamines, and trioxides. See, for example, Remington's Pharmaceutical Sciences, above. Such salts can be prepared using appropriate corresponding bases. Pharmaceutically acceptable carriers and / or excipients can also be incorporated into pharmaceutical compositions according to the present invention to facilitate the administration of specific asparaginases. Examples of carriers suitable for use in 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 water for injection (EFI), physiological saline, and sterile solutions of dextrose. The pharmaceutical compositions according to the present invention can be administered by various routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (percutaneous), or transmucosal administration. For systemic administration, oral administration is preferred. For oral administration, for example, the compound can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid formulations such as syrups, elixirs, and concentrated drops. Alternatively, injection (parenteral administration), such as intramuscular, intravenous, intraperitoneal, and subcutaneous injections, may be used. For injection, the pharmaceutical composition is formulated in a liquid solution, preferably a physiologically compatible buffer or solution such as physiological saline, Hanks' solution, or Ringer's solution. In addition, the compound may be formulated in solid form and redissolved or suspended immediately before use. For example, a lyophilized form of the modified protein can be produced. In a specific embodiment, the modified protein is administered intramuscularly. In a preferred specific embodiment, the modified protein is administered intravenously.

[0193] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrating agent suitable for the barrier to be permeated is used in the formulation. Such penetrating agents 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 promote permeation. Transmucosal administration may be via, for example, an intranasal spray, an inhaler (for pulmonary delivery), a rectal suppository, or a vaginal suppository. For topical administration, the compound can be formulated into an ointment, plaster, gel, or cream, as is well known in the art.

[0194] In one aspect, the present invention also relates to the use of modified proteins as described herein in therapeutics. The use may be for the treatment of diseases treatable by L-asparagine depletion as described above, as a method for treating diseases treatable by L-asparagine depletion. In one aspect, the present invention relates to modified proteins as described herein for use as drugs / for use in therapeutics / for use in medicine, or modified proteins prepared by processes as described herein, or compositions comprising modified proteins as described herein.

[0195] In one embodiment, the present 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 a disease treatable by L-asparagine depletion in a patient. The present 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 drug for treating a disease treatable by L-asparagine depletion in a patient. The present invention also 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 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 embodiment, the present 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 present 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 drug for the treatment of cancer. The present invention also relates to a method for 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] In this specification, the target of treatment is preferably a mammal, particularly a human.

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

[0199] Modified proteins may elicit a less immunogenic response in patients compared to unconjugated L-asparaginase. Modified proteins may have a longer circulating half-life in the body after a single dose compared to unconjugated L-asparaginase. Modified proteins may have a larger AUC after a single dose compared to unconjugated L-asparaginase. Patients may have a history of hypersensitivity to E. coli L-asparaginase or its pegylated form.

[0200] The present invention will be further described with reference to the following non-limiting figures and embodiments. [Examples]

[0201] The following examples illustrate the present invention.

[0202] Example 1: Optimization of coupling ratio for the preparation of pyroglutamoyl-P / A(20)-aminohexanoyl-chrysanthanspase 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 DMAO solution of 500 mM TBTU (CAS #125700-67-6; Iris Biotech, Marktredwitz, Germany) and 2.7 μL of DIPEA to the peptide solution and vortexing (see Figure 1C). In this setting, 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 incubation at 25°C for 10 minutes, the mixture was diluted in Eppendorf tubes according to Table 1.

[0203] A solution of L-asparaginase (chrysanthapase, SEQ ID NO: 1, recombinant (lot RE-LAP-P57D) of Dickeya chrysanthemi produced in E. coli) 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 described in Table 1. After mixing by repeated pipetting and vortex stirring, the coupling reaction was carried out at 25°C for 30 minutes. 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] Figure 2 shows the SDS-PAGE analysis of the modified proteins. Each individual band corresponds to a protein modified with one coupled P / A peptide. The application of additional coupling reaction mixes with peptide ratios of 0.3–10 mg per mg of protein allowed for counting bands in a continuous ladder starting from the unconjugated protein, thus enabling accurate determination of the number of coupled P / A peptides. Band intensity was quantified using a densitometer with Quant v12 software (TotalLab, Newcastle upon Tyne, UK), and the arithmetic mean of the number of coupled peptides per chrysanthanspase weighted by band intensity was calculated (see Table 2). 3.5 mg of P / A peptide per mg of chrysanthanspase resulted in coupling ratios ranging from 9–12 P / A peptides per chrysanthanspase monomer (mean: 10.4). The fact that increasing the mass ratio to apply up to 10 mg of P / A peptide per mg of chrysanthanspase only resulted in a slight increase in the coupling ratio of 10–13 P / A peptides per chrysanthanspase (average: 12.0) indicates saturation of accessible amino groups.

[0206] Modified proteins were purified by anion exchange chromatography on a MonoQ HR5 / 5 column (GE Healthcare) using a concentration gradient of 25 mM Na-boric acid as the running buffer, pH 9.0, 1 mM EDTA, and 0–1 M NaCl for protein elution. The L-asparaginase aminohydrolase activity of each chrysanthasapase-modified protein was determined by the reaction of ammonia released via L-asparagine enzyme activity with Nessler reagent. Briefly, 50 μL of the enzyme solution was mixed with 100 mM sodium borate buffer containing 0.015% (w / v) bovine serum albumin and 20 mM L-asparagine at pH 8.6, and incubated at 37°C for 15 minutes. The reaction was stopped by adding 200 μL of Nessler reagent (Sigma-Aldrich). The absorbance of this solution was measured at 450 nm. Activity was calculated from a calibration curve obtained from 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-chrysanthase 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 DMSO solution of 500 mM TBTU (CAS #125700-67-6, Iris Biotech, Marktredwitz, Germany) was added and mixed, followed by 14 μL of DIPEA (99.5%, biotechnology grade, Sigma-Aldrich). The entire mixture was briefly vortexed and incubated at 25°C for 20 minutes (see Figure 1C). In this configuration, 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 chrysanthase solution (SEQ ID NO: 1) (2 mg / mL in PBS) was mixed with an activated peptide solution (1.5 mL) to create a 5:1 mass ratio of Pga-P / A#1(40)-Ahx to chrysanthase, and the mixture was incubated at room temperature for 30 minutes to achieve coupling. The solution was dialyzed against 5 L of AEX running buffer (25 mM Na-boric acid, pH 9.0, 1 mM EDTA) using a regenerated cellulose membrane dialysis tube (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 protein-modified proteins were eluted using a partitioned concentration gradient of NaCl ranging from 0–150 mM at 1 column volume and 150–1000 mM at 0.25 column volume (Figure 3A).

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

[0211] Example 3: Preparation of pyroglutamoyl-P / A(20)-aminohexanoyl-chrysanthase 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 DMSO solution of 500 mM TBTU (CAS #125700-67-6, purchased from Iris Biotech, Marktredwitz, Germany) was added and mixed, followed by 10 μL of DIPEA (99.5%, biotechnology grade, Sigma-Aldrich). The entire mixture was briefly vortexed and incubated at 25°C for 20 minutes (Figure 1C). In this configuration, the peptide concentration was 7.58 mM, and the molar ratios between DIPEA, TBTU, and Pga-P / A#1(20)-Ahx were 5:5:1.

[0212] 3.5 mL of ice-cold chrysanthase solution (SEQ ID NO: 1) (2 mg / mL in PBS) was mixed with activated peptide solution (1.5 mL) to create a 5:1 mass ratio between Pga-P / A#1(40)-Ahx and chrysanthase, and the mixture was incubated at room temperature for 30 minutes to achieve coupling. The solution was dialyzed against 5 L of AEX running buffer (25 mM Na-boric acid, pH 9.0, 1 mM EDTA) using a regenerated cellulose membrane dialysis tube (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 protein-modified proteins were eluted using a partitioned concentration gradient of NaCl ranging from 0–150 mM in one column volume and 150–1000 mM in 0.25 column volumes (Figure 4A).

[0213] Applying the eluate to SDS-PAGE in parallel with the ladder obtained from coupling reaction mixes at a ratio of 0.3–10 mg of peptide per mg of protein allowed for the determination of coupling ratios of 10–13 PA peptides per chrysanthaspase monomer (mean: 11.9) (Figure 4B). The enzymatic activity of chrysanthaspase / PA(20) modified proteins determined using the Nessler assay described in Example 1 was 91.2% of the activity of unmodified chrysanthaspase similarly assayed.

[0214] Example 4: Cloning of expression plasmids for periplasmic production of chrysanthasps with N-terminal fusion to P / A sequences of various lengths. A synthetic DNA fragment encoding the mature amino acid sequence (UniProt IDP06608) of Dickeya chrysanthemi L-asparaginase 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 on the coding strand in a direction inversely complementary to its recognition sequence GCTCTTC, followed by a GCC alanine codon directly linked to the mature L-asparaginase coding sequence, and finally a HindIII restriction site.

[0215] Following the standard procedure (Sambrook, (2012), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press), this gene fragment was cloned into pASk75 via adjacent restriction sites XbaI and HindIII. The resulting plasmid (Figure 5A) was digested with SpaI, which led to the release of a small (30 bp) DNA insertion fragment containing the SpaI recognition site and a cleaved vector backbone with a 5'-GCC / 5'-GGC adherent end fitted immediately before the mature N-terminus encoded by L-asparaginase, which is ideally suited for the insertion of low-repetition nucleic acid molecules encoding proline / alanine-rich amino acid repeat sequences. In both cases, vector fragments were isolated using the Promega Wizard gel extraction kit (Promega, Mannheim, Germany) according to the manufacturer's instructions, and dephosphorylated with the heat-sensitive alkaline phosphatase FastAP (Thermo Fisher Scientific, Waltham, MA). These fragments were then ligated via EarI restriction digestion to either 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 NO: 12 and SEQ ID NO: 14) (Figure 5B) enable bacterial expression of fusion proteins (SEQ ID NO: 11 and SEQ ID NO: 13) consisting of proline / alanine-rich amino acid repeat sequences fused to the biologically active protein chrysanthase (after in vivo processing of the Enx signal peptide during periplasmic secretion in E. coli).

[0216] Example 5: Production and purification of a fusion protein between either the PA#1(200) sequence or the PA#1(400) sequence and chrysanthanspase in bacteria. The fusion proteins of PA#1(200)-chrysanthase and PA#1(400)-chrysanthase (calculated masses: 51 kDa and 67 kDa, respectively) were both produced at 25°C in E. coli W3110 containing the expression plasmid pASK75-PA200-chrysanthase or pASK75-PA400-chrysanthase (Figure 5B) derived from Example 4, using an 8 L benchtop fermenter with synthetic glucose inorganic medium supplemented with 100 mg / L ampicillin, according to the published procedure (Schiweck, (1995), Proteins, 23:561-565). Recombinant gene expression was confirmed when the culture was CD 550 As soon as the concentration reached 40, induction was achieved by adding 500 μg / L of anhydrotetracycline (Skerra, (1994), loc. cit.). After an incubation period of 2.5 hours, cells were harvested by centrifugation and cooled in ice-cold periplasm fraction buffer (500 mM sucrose, 1 mM EDTA, 200 mM borate / NaOH, pH 8.0, 2 ml / L and OD). 550 The cells were resuspended in ) for 10 minutes. 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 recombinant protein was collected.

[0217] Periplasm extracts were dialyzed twice in 15 L of PBS containing 1 mM EDTA at 4°C for at least 6 hours, filtered through 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-boric acid, pH 9.0, 1 mM EDTA) and dialyzed in 5 L of AEX running buffer at 4°C for at least 6 hours. The dialyzed protein solution was clarified from the remaining insoluble material by centrifugation, packed with Source15Q resin, and connected to an Akta® purification system (GE Healthcare, Freiburg, Germany). It was subjected to subtractive anion exchange chromatography using an 85 mL HiScale® column (GE Healthcare, Freiburg, Germany) equilibrated with AEX running buffer. The column-pass fraction containing pure protein (see Figures 6A and 6B) was dialyzed twice with 5 L of PBS.

[0218] Homogeneous protein preparations showing no signs of aggregation were obtained from one 8L fermenter, with final yields of 128 mg for PA#1(200)-chrysanthaspase and 48 mg for PA#1(400)-chrysanthaspase. The protein concentration was 19370M. -1 cm -1The absorption at 280 nm was determined by measuring the absorption using the calculated extinction coefficient (Gill, (1989), Anal. Biochem. 182:319-326). The enzymatic activity of the fusion protein was determined using the Nessler assay described in Example 1. In this setting, the PA#1(200)-chrysanthaspas fusion protein had 109% of the enzymatic activity compared to similarly assayed unmodified chrysanthaspas, and the PA#1(400)-chrysanthaspas had 118% of the enzymatic activity. This demonstrates that N-terminal fusion of chrysanthaspas with P / A polypeptides up to at least 401 amino acids in length does not affect the enzymatic activity.

[0219] Example 6: Measurement of hydrodynamic volume of both genetically and chemically PAS-modified chrysanthanaspase by analytical gel filtration. Size exclusion chromatography (SEC) was performed using an Akta® purification system 10 (GE Healthcare) with PBS (115 mM NaCl, 4 mM KH2PO4, 16 mM Na2HPO4, pH 7.4) as a running buffer, at a flow rate of 0.5 mL / min on a Superdex® S200 elevation 10 / 300 GL column (GE Healthcare Europe, Freiburg, Germany). Using a disposable ultrafiltration apparatus made of regenerated cellulose (MWCO 10kDa; Merck-Millipore, Darmstadt, Germany), recombinant chrysanthasps fused to PA#1(200) or PA#1(400) polypeptide (described in Example 5) and chrysanthasps chemically conjugated with Pga-P / A(40)-Ahx peptide (described in Example 2) or 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 unPAS-conjugated enzymes were individually applied to columns, and the chromatographic traces were superimposed (Figure 7A). All five proteins eluted as a single homogeneous peak.

[0220] For column calibration (Figure 7B), a suitable 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 preparations were significantly larger in size than the corresponding globular protein of the same molecular weight. This molecular weight / hydrodynamic volume imbalance increased further as the size of the P / A (poly)peptide portion increased. For PA(200)-chrysanthaspas, the apparent size increase was 5.1 times compared to unfused chrysanthaspas, while the true mass was only 1.5 times larger. For PA(400)-chrysanthaspas, the apparent size increase was 10.4 times compared to unfused chrysanthaspas, while the true mass was only 1.9 times larger. This observation clearly demonstrates the significantly increased hydrodynamic volume conferred to biologically active chrysanthaspas enzymes by the Pro / Ala polypeptide segment according to the present invention.

[0222] Example 7: ESI-MS analysis of chemically or genetically PAS-modified chrysanthasps 250 μL of purified chemically modified chrysanthase protein with Pga-P / A(20)-Ahx from Example 3, and 250 μL each of recombinant PA200 fusion protein and PA400 fusion protein from Example 5, all at a concentration of 1 mg / mL, were subjected to a 1 mL Resource® RPC column (GE Healthcare, Freiburg, Germany) connected to an Akta® purification system, using 2% v / v acetonitrile and 1% v / v formic acid as running buffers. Proteins were eluted using an acetonitrile gradient from 2% v / v acetonitrile, 1% v / v formic acid to 80% v / v acetonitrile, 0.1% v / v formic acid over 20 column volumes. The eluted proteins were directly analyzed by ESI mass spectrometry using a maXis® microOTOF instrument (Bruker Daltonik, Bremen, Germany) in cation mode. The raw m / z spectra of the chrysanthanspase / Pga-P / A(20)-Ahx chemically modified protein are shown in Figure 8A. The masses revealed by the deconvolutional mass spectra (Figure 8B) are given in Table 3. The mass distribution is consistent with the coupling ratio determined by the SDS-PAGE analysis described in Example 2.

[0223] The raw m / z spectrum of recombinant PA#1(200)-chrysanthaspase (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 recombinant PA#1(400)-chrysanthaspase 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 chrysanthaspase enzymes 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 enzymatic activity of PAS-modified L-asparaginase was determined by its catalysis of the conversion of L-asparagine to L-aspartic acid. This reaction releases 1 mole of ammonia per mole of converted L-asparagine. 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 (International Unit or IU) of L-asparaginase enzymatic activity 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 is determined by dividing the value of L-asparaginase activity, expressed in IU / mL, by the protein concentration, expressed in mg / mL. The mass of protein monomers with PAS-modified sequences was measured.

[0226] Measurement of L-asparaginase activity is based on an endpoint assay in which the sample is diluted to a series of final enzyme concentrations, which are then incubated at 37°C for 15 minutes under saturated L-asparagine concentration. The reaction is stopped by the addition of Nessler reagent, and the amount of ammonia produced by the reaction is extrapolated from a calibration curve constructed from a known amount of ammonium sulfate used as a reference. A plot of enzyme concentration against 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. The specific activity is reported as IU / mg and rounded to the nearest integer.

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

[0228] Example 9: Pharmacokinetics The pharmacokinetic profiles of recombinant chrysanthasps expressed in E. coli as a PAS-modified fusion protein (PA-200) or chemically conjugated to a PA-peptide (PA-20) were characterized following administration of a single intravenous bolus dose to CD-1 mice. CD-1 mice are a model of healthy mice.

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

[0230] PAS-modified asparaginase was administered to mice as a single IV dose of 25 IU / kg body weight. Plasma samples were collected from the mouse group at planned 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. Mean plasma asparaginase activity (n=4) was plotted against time data (Figure 1), and pharmacokinetic analysis was performed.

[0231] Blood samples were collected prior to administration and approximately 6 hours (day 1), 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 administration. A tail amputation (tail end amputation) blood collection method was employed. For the initial blood collection, approximately 1-2 mm was amputated at the distal end of the tail. For subsequent blood collections, all scabs were removed and blood flow was promoted by stroking the tail to collect blood from the same site. Approximately 100 μL of blood per time point was collected in a cold K3 EDTA (Minivette) sample collection tube. The blood was transferred to a tube suitable for centrifugation. For plasma isolation, all samples were centrifuged at 3,000 × g for approximately 10 minutes in a refrigerated centrifuge set to maintain approximately 4°C, within approximately 20 minutes of sample collection. Following centrifugation, the maximum volume of plasma (targeting 30 μL) was collected and placed in a plastic vial. The plastic vial was stored at -65°C to -85°C until testing.

[0232] As previously described (Allas, et al. (2009), Blood, 114, 2033), asparaginase activity was measured in plasma samples as the concentration of asparaginase. 2 The concentration-time profile (t) is only valid if (the square of the correlation coefficient for linear regression used to estimate the terminal disappearance rate constant, λz) is greater than 0.8. 1 / 2 CL and V ss We reported parameters that depend on a thorough characterization of the terminal phase of the assay. Pharmacokinetic data were imported into Phoenix WinNonlinv6.4 (Certara / Pharsight) for analysis. Plasma asparaginase activity against time data was analyzed using a non-compartmental method with small sample collections in an IV bolus administration model. Activity values ​​below the assay's quantification limit (10 U / L) were set to zero in the calculation of the group mean. Nominal dose levels and sample collection times were used in the calculations. 1 / 2 The estimated time for PA-20 chrysanthanpases was 50.2 hours, and for PA-200 chrysanthanpases it was 17.9 hours.

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

[0234] Some of the sequences provided herein are available in the NCBI database and can be accessed from 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 homologous sequences and variants of concise sequences provided herein. Preferably, such “variants” are genetic variants.

[0235] Sequence ID 1: Amino acid sequence of L-asparaginase from Dickeya chrysanthemi ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSA RYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0236] Sequence ID 2: Nucleotide sequence encoding L-asparaginase from Dickeya chrysanthemi GCAGATAAACTGCCGAATATTGTTATTCTGGCAACCGGTGGCACCATTGCAGGTAGCGCAGCAACCGGCACCCAAACCACAGGTTATAAAGCCGGTGCACTGGGTGTTGATACCCTGATTAATGCAGTTCCGGAAGTTAAAAAACTGGCCAATGTGAAAGGTGAACAGTTTAGCAATATGGCCAGCGAAAATATGACCGGTGATGTTGTTCTGAAACTGAGCCAGCGTGTTAATGAACTGCTGGCACGTGATGATGTTGATGGTGTGGTTATTACCCATGGCACCGATACCGTTGAAGAAAGCGCCTATTTTCTGCATCTGACCGTGAAAAGCGATAAACCGGTTGTTTTTGTTGCAGCAATGCGTCCGGCAACCGCAATTAGCGCAGATGGTCCGATGAATCTGCTGGAAGCAGTTCGTGTTGCCGGTGATAAACAGAGCCGTGGTCGTGGTGTTATGGTTGTTCTGAATGATCGTATTGGTAGCGCACGCTATATTACCAAAACCAATGCAAGCACCCTGGATACCTTTAAAGCCAATGAAGAAGGTTATCTGGGCGTTATTATTGGCAATCGCATTTATTATCAGAATCGCATTGATAAACTGCATACCACCCGTAGCGTTTTTGATGTTCGTGGTCTGACCAGCCTGCCGAAAGTTGATATTCTGTATGGCTATCAGGATGATCCGGAATATCTGTATGATGCAGCCATTCAGCATGGTGTTAAAGGTATTGTGTATGCAGGTATGGGTGCAGGTAGCGTTAGCGTTCGTGGTATTGCAGGTATGCGTAAAGCAATGGAAAAAGGCGTTGTTGTTATTCGTAGCACCCGTACCGGTAATGGTATTGTTCCGCCGGATGAAGAACTGCCGGGTCTGGTTAGCGATAGCCTGAATCCGGCACATGCACGTATTCTGCTGATGCTGGCACTGACCCGTACCAGCGATCCGAAAGTGATTCAGGAATATTTTCATACCTAT

[0237] Accession number 3: Amino acid sequence of the L-asparaginase of Dickeya chrysanthemi Signal peptide: 1-28; removed during cloning: 29-39; 40-366: asparaginase [Chemical Formula 3]

[0238] Accession number 4 Nucleotide sequence (synthetic) encoding the L-asparaginase of Dickeya chrysanthemi Mature asparaginase encoded by bases 160-1140 (bold). Thus, the nucleotide sequence encoding L-asparaginase extends from nucleotide position 160 to 1140. [Chemical Formula 4]

[0239] Accession number 5: Amino acid sequence of the PA(20) peptide AAPAAPAPAAPAAPAPAAPA

[0240] Accession number 6: Nucleotide sequence encoding the PA(20) peptide GCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCC

[0241] Accession number 7: Amino acid sequence of the PA(200)-polypeptide AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAA

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

[0243] SEQ ID NO: 9 Amino acid sequence of PA(400)-polypeptide AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAA

[0244] Sequence ID 10: PA(400) - Nucleotide sequence encoding polypeptide

[0245] Sequence ID 11: Amino acid sequence of asparaginase-PA(200)-fusion protein AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPA APAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASE NMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYY QNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0246] Sequence ID 12: Nucleotide sequence encoding the asparaginase-PA(200)-fusion protein (XbaI / HindIII) A mature fusion protein (SEQ ID NO: 11) encoded by bases 127–1710 (bold). Therefore, the nucleotide sequence encoding the fusion protein can extend to nucleotides from position 127 to 1710 of SEQ ID NO: 12. Consequently, the term "modified protein comprising or consisting of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence such as that shown in SEQ ID NO: 12" as used herein can be more narrowly defined as "modified protein comprising or consisting of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence such as that shown in SEQ ID NO: 12." [C5]

[0247] Sequence ID 13: Amino acid sequence of asparaginase-PA(400)-fusion protein AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAA PAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPA APAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDK QSRRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0248] Sequence ID 14: Nucleotide sequence encoding the asparaginase-PA(400)-fusion protein (XbaI / HindIII) A mature fusion protein (SEQ ID NO: 13) encoded by bases 127–2184 (bold). Therefore, the nucleotide sequence encoding the fusion protein can extend to nucleotides from position 127 to 2184 of SEQ ID NO: 14. Consequently, the term "modified protein comprising or consisting of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence such as that shown in SEQ ID NO: 14" as used herein can be more narrowly defined as "modified protein comprising or consisting of an amino acid sequence encoded by a nucleic acid having a nucleotide sequence such as that shown in SEQ ID NO: 14." [C6]

[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 entirely by reference. Now that the present invention has been fully described, it will be understood by those skilled in the art that the present invention may be practiced within a broad and equivalent range of conditions, parameters, etc., 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. 1. A modified protein having L-asparaginase activity, wherein the modified protein is a tetramer, each monomer of the tetramer comprises (i) an L-asparaginase having the amino acid sequence of SEQ ID NO:1 and (ii) one or more polypeptides, wherein the polypeptides consist of only about 200 to about 400 proline and alanine amino acid residues, and the average coupling ratio of the polypeptides per monomer is less than 12.

2. The modified protein of claim 1, wherein the monomer of the tetramer is a fusion protein of the L-asparaginase and the polypeptide.

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

5.

4. (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 2 or 3.

5. (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 2 to 4.

6. A modified protein comprising a conjugate of L-asparaginase and one or more peptides, The L-asparaginase comprises the amino acid sequence of SEQ ID NO:1, Each of the one or more peptides is independently selected from peptide R N -(P / A)-R C and (P / A) is an amino acid sequence consisting of 20 to 40 proline and alanine amino acid residues only, R N is a protecting group linked to the N-terminal amino group of the amino acid sequence of (P / A), R C is an amino acid residue that is bonded via its amino group to the C-terminal carboxy group of the amino acid sequence of (P / A), Each of the one or more peptides comprises the C-terminal amino acid residue R C and a free amino group of the L-asparaginase, at least one of the free amino groups of the L-asparaginase to which the peptide is conjugated is not the N-terminal alpha amino group of the L-asparaginase; The average coupling ratio of the polypeptide per L-asparaginase is less than 12. Modified proteins.

7. R N is pyroglutamoyl or acetyl, and / or R C The modified protein of claim 6, wherein is ε-aminohexanoic acid.

8. 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; or 8. The modified protein of claim 6 or 7, wherein the free amino group to which the peptide is conjugated is selected from the group consisting of 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.

9. The modified protein of claim 8, wherein the L-asparaginase is composed of four subunits, and the peptides 9 to 13 of claims 6 or 7 are conjugated to each subunit of the L-asparaginase.

10. The modified protein according to any one of claims 1 to 9, wherein said polypeptide or peptide mediates a reduction in the immunogenicity of said modified protein.

11. A nucleic acid encoding a modified protein according to any one of claims 1 to 5.

12. The nucleic acid (a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12 or 14, and (b) the nucleic acid of claim 11 selected from the group consisting of nucleic acids that are degenerate as a result of the genetic code to the nucleotide sequence of SEQ ID NO: 12 or 14.

13. A vector comprising the nucleic acid of claim 11 or 12.

14. A host cell comprising a nucleic acid according to claim 11 or 12 or a vector according to claim 13.

15. A process for the preparation of the modified protein according to any one of claims 1 to 5 and 10, said process comprising culturing a host cell according to claim 14 and isolating the modified protein from the culture or from the cell.

16. A process for preparing the modified protein according to any one of claims 6 to 9, comprising the steps of: The process (a) Formula R N -(P / A)-R C-act The activated peptide of is coupled with the L-asparaginase to obtain R N obtaining a modified protein of the L-asparaginase and a peptide, In the formula, R C-act is R C and R is a carboxy-activated form of C and (P / A) are as defined for the modified protein to be prepared, R N is a protecting group linked to the N-terminal amino group of (P / A).

17. The amino acid residue R in the activated peptide C-act 17. The process of claim 16, wherein the activated carboxy group of is an active ester group.

18. A pharmaceutical composition comprising a modified protein according to any one of claims 1 to 10 or said modified protein prepared by the process according to any one of claims 15 to 17, optionally further comprising a pharma- ceutically acceptable carrier(s) or excipient(s).

19. A modified protein according to any one of claims 1 to 10 or said modified protein prepared by a process according to any one of claims 15 to 17 or a composition according to claim 18 for use in the treatment of a disease.

20. 20. The modified protein for use according to claim 19, wherein the disease can be treated by depletion of L-asparagine and is cancer, or the composition for use according to claim 19. The modified protein according to any one of claims 1 to 10, said modified protein prepared by the process according to any one of claims 15 to 17 or the composition according to claim 19 for the treatment of cancer.

21. 21. The modified protein for use according to claim 20 or the composition for use according to claim 20, wherein the cancer is a non-solid cancer.

22. 22. The modified protein for use according to claim 21 or the composition for use according to claim 21, wherein the non-solid cancer is leukemia or non-Hodgkin's lymphoma.

23. 23. The modified protein for use according to claim 22 or the composition for use according to claim 22, wherein the non-solid cancer is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

24. The modified protein for use according to any one of claims 20 to 23 or the composition for use according to claims 20 to 23, wherein the modified protein induces a lower immunogenic response in a patient compared to unmodified L-asparaginase.

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