Variant asparaginase polypeptides for medical use

Abstract

Various embodiments are provided relating to variant asparaginase polypeptides having enhanced stability, pharmacodynamics and / or reduced immunogenicity. The variant asparaginase polypeptides can be used as therapeutic agents in mammals including humans, canines, felines and equines.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 660,189, filed on April 19, 2018, which is incorporated herein by reference in its entirety for any purpose.

[0003] Sequence Listing

[0004] This application is submitted with a sequence table in electronic format. The sequence table is provided as a file named 2019-04-15_01157-0013-00PCT_Seq_List_ST25.txt, created on April 15, 2019, with a size of 42Kb. The information in the sequence table in electronic format is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to variant asparaginase polypeptides having reduced immunogenicity and enhanced stability and pharmacokinetics, and methods of producing and using the variant asparaginase polypeptides, e.g., for treating acute lymphoblastic leukemia in humans and lymphomas in companion animals. Background Art

[0006] The natural form of L-asparaginase (also referred to as asparaginase in this article) is a bacterial enzyme consisting of identical subunits (each subunit is about 35kDa), which catalyzes the conversion of L-asparagine into aspartic acid and ammonia. Escherichia coli (Escherichia coli or E.coli) asparaginase can be a homodimer or a homotetramer, while Erwinia chrysanthemi (Erwinia chrysanthemi or E.chrysanthemi) (also referred to as Dickeya dadantii, D.dadantii, Dickeya chrysanthemi, D.chrysanthemi, etc.) asparaginase is a tetramer. Asparaginase is used to treat patients with acute lymphoblastic leukemia (ALL) and dogs and cats with lymphoma. Some leukemia cells cannot synthesize asparagine and rely on circulating asparagine to survive. As a therapeutic agent, asparaginase can deprive such cells of circulating asparagine, thereby causing cell death. Normal cells are less affected by asparaginase because they are able to synthesize asparagine.

[0007] Unfortunately, hypersensitivity reactions to asparaginase are commonly reported adverse events and often require termination of asparaginase therapy. Native E. coli asparaginase can be immunogenic when introduced into humans in therapeutic amounts. Antibodies to asparaginase can increase the elimination rate, and neutralizing antibodies, if present, can limit the effectiveness of the enzyme. Summary of the invention

[0008] Embodiment 1. A variant E. chrysanthemi asparaginase polypeptide comprising at least one cysteine ​​substitution.

[0009] Embodiment 2. A variant E. chrysanthemi asparaginase polypeptide comprising at least one PEG-conjugable amino acid substitution, wherein the variant polypeptide is less immunogenic or antigenic when conjugated to PEG compared to the corresponding wild-type E. chrysanthemi asparaginase polypeptide.

[0010] Embodiment 3. A variant E. chrysanthemi asparaginase polypeptide comprising at least one PEG-conjugable amino acid substitution at an amino acid position at or spatially nearby an immunogenic or antigenic site of a corresponding wild-type E. chrysanthemi asparaginase polypeptide.

[0011] Embodiment 4. The variant E. chrysanthemi asparaginase polypeptide of any one of Embodiments 1 to 3, wherein the at least one cysteine ​​or the at least one PEG-conjugatable amino acid is surface exposed.

[0012] Embodiment 5. The variant E. chrysanthemi asparaginase polypeptide of Embodiment 3 or 4, wherein the immunogenic or antigenic site in the wild-type asparaginase polypeptide elicits an immune response in humans or companion animal species.

[0013] Embodiment 6. The variant E. chrysanthemi asparaginase polypeptide of Embodiment 5, wherein the companion animal species is canine, feline, or equine.

[0014] Embodiment 7. A variant E. chrysanthemi asparaginase polypeptide comprising at least one amino acid substitution, wherein the substituted amino acid is surface exposed.

[0015] Embodiment 8. The variant E. chrysanthemi asparaginase polypeptide of any of the preceding embodiments, wherein the variant polypeptide lacks a leader sequence.

[0016] Embodiment 9. A variant E. chrysanthemi asparaginase polypeptide according to any of the preceding embodiments, wherein 30% or more of the at least one cysteine, the at least one amino acid, or the at least one PEG-conjugable amino acid are surface exposed as determined by standard protein modeling software.

[0017] Embodiment 10. A variant E. chrysanthemi asparaginase polypeptide according to any of the preceding embodiments, wherein 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 95% or more of the at least one cysteine, the at least one amino acid or the at least one PEG-conjugable amino acid are surface exposed as determined by standard protein modeling software.

[0018] Embodiment 11. The variant E. chrysanthemi asparaginase polypeptide of any one of Embodiments 2 to 10, wherein the at least one PEG-conjugable amino acid or the at least one amino acid is cysteine, lysine, or a PEG-conjugable amino acid derivative.

[0019] Embodiment 12. A variant E. chrysanthemi asparaginase polypeptide according to any one of the preceding embodiments, wherein the at least one cysteine, the at least one amino acid, or the at least one PEG-conjugable amino acid is 35 angstroms from the immunogenic or antigenic site. or smaller, as determined by three-dimensional protein structure analysis.

[0020] Embodiment 13. A variant E. chrysanthemi asparaginase polypeptide according to any one of the preceding embodiments, wherein the at least one cysteine, the at least one amino acid, or the at least one PEG-conjugable amino acid is 500 μg / cm3 from the immunogenic or antigenic site. or smaller, or smaller, or smaller, or smaller, or smaller or or smaller, as determined by three-dimensional protein structure analysis.

[0021] Embodiment 14. A variant E. chrysanthemi asparaginase polypeptide according to any of the preceding embodiments, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a wild-type asparaginase amino acid sequence such as SEQ ID NO:2.

[0022] Embodiment 15. The variant E. chrysanthemi asparaginase polypeptide of any one of Embodiments 2 to 6 and 8 to 14, wherein the wild-type E. chrysanthemi asparaginase polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0023] Embodiment 16. The variant E. chrysanthemi asparaginase polypeptide of any one of Embodiments 3 to 6 and 8 to 15, wherein the immunogenic or antigenic site corresponds to amino acid position 41, 72, 265 or 288 of SEQ ID NO:2.

[0024] Embodiment 17. The variant E. chrysanthemi asparaginase polypeptide of any of the preceding embodiments, wherein the at least one cysteine, at least one amino acid substitution, or the at least one PEG-conjugable amino acid substitution is at a position corresponding to position 3, 4, 17, 26, 37, 38, 41, 42, 44, 45, 47, 48, 51, 53, 54, 55, 56, 59, 60, 68, 72, 79, 82, 83, 84, 85, 87, 110, 112, 123, 124, 125, 127, 180, 190, 191, 192, 198, 202, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235 6, 208, 210, 212, 213, 215, 216, 219, 231, 239, 240, 241, 243, 257, 260, 261, 264, 265, 267, 268, 269, 270, 280, 286, 287, 288, 289, 312, 313, 315, 316, 317, 318 and 322.

[0025] Embodiment 18. The variant E. chrysanthemi asparaginase polypeptide of any one of Embodiments 2 to 17, wherein the at least one PEG conjugable amino acid substitution or the at least one amino acid substitution is at a position corresponding to position 3, 4, 17, 26, 37, 38, 41, 42, 44, 45, 47, 48, 51, 53, 54, 55, 56, 59, 60, 68, 72, 79, 82, 83, 84, 85, 87, 110, 112, 123, 124, 125, 127, 180, 190, 191, 192, 198, 202, 205, 206, 207, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235 280, 286, 287, 288, 289, 312, 313, 315, 316, 317, 318 and 322.

[0026] Embodiment 19. The variant E. chrysanthemi asparaginase polypeptide of any one of Embodiments 2 to 18, wherein the at least one amino acid substitution or the at least one PEG conjugable amino acid substitution is in a residue corresponding to a residue selected from SEQ ID 261, 264, 267, 268, 270, 280, 286, 287, 288, 289, 312, 313, 315, 316, 317, and 322 of NO:2.

[0027] Embodiment 20. The variant E. chrysanthemi asparaginase polypeptide of any of the preceding embodiments, comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0028] Embodiment 21. The variant E. chrysanthemi asparaginase polypeptide according to any one of the preceding embodiments, wherein the variant E. chrysanthemi asparaginase polypeptide is modified, such as sialylated or pegylated.

[0029] Embodiment 22. A tetramer comprising the variant E. chrysanthemi asparaginase polypeptide according to any one of the preceding embodiments.

[0030] Embodiment 23. A variant asparaginase polypeptide comprising at least one amino acid substitution at an unpaired cysteine ​​residue of the corresponding wild-type asparaginase polypeptide, wherein the at least one amino acid substitution comprises any amino acid other than cysteine.

[0031] Embodiment 24. A variant asparaginase polypeptide according to embodiment 23, which comprises at least one cysteine ​​substitution at an amino acid position at or spatially nearby the immunogenic or antigenic site of the corresponding wild-type asparaginase polypeptide or comprises at least one pair of cysteine ​​substitutions at said amino acid position.

[0032] Embodiment 25. A variant asparaginase polypeptide comprising at least one cysteine ​​substitution or at least one pair of cysteine ​​substitutions at an amino acid position, wherein the variant polypeptide is less immunogenic or antigenic when conjugated to PEG compared to the corresponding wild-type asparaginase polypeptide.

[0033] Embodiment 26. A variant asparaginase polypeptide comprising at least one cysteine ​​substitution at an amino acid position at or spatially nearby an immunogenic or antigenic site of a corresponding wild-type asparaginase polypeptide or comprising at least one pair of cysteine ​​substitutions at said amino acid position.

[0034] Embodiment 27. The variant asparaginase polypeptide of any one of embodiments 24 to 26, wherein the at least one cysteine ​​is partially buried.

[0035] Embodiment 28. A variant asparaginase polypeptide comprising at least one cysteine ​​substitution, wherein the cysteine ​​is partially buried.

[0036] Embodiment 29. The variant asparaginase polypeptide of any one of embodiments 24 to 28, wherein the variant asparaginase polypeptide lacks a leader sequence.

[0037] Embodiment 30. The variant asparaginase polypeptide of any one of embodiments 24 to 29, wherein the immunogenic or antigenic site in the wild-type asparaginase polypeptide elicits an immune response in humans or companion animal species.

[0038] Embodiment 31. The variant asparaginase polypeptide of Embodiment 30, wherein the companion animal species is canine, feline, or equine.

[0039] Embodiment 32. The variant asparaginase polypeptide of any one of embodiments 24 to 31, wherein between 5% and 25% of the at least one cysteine ​​is surface exposed as determined by standard protein modeling software.

[0040] Embodiment 33. The variant asparaginase polypeptide of any one of Embodiments 24 to 32, wherein between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 20%, between 20% and 30%, or between 10% and 30% of the at least one cysteine ​​is surface exposed as determined by standard protein modeling software.

[0041] Embodiment 34. A variant asparaginase polypeptide according to any one of embodiments 24 to 33, wherein the at least one cysteine ​​or the at least one pair of cysteines is 35 angstroms from the immunogenic or antigenic site. or smaller, as determined by three-dimensional protein structure analysis.

[0042] Embodiment 35. The variant asparaginase polypeptide of any one of Embodiments 24 to 34, wherein the at least one cysteine ​​or the at least one pair of cysteines is 1.5 to 2.0 mM away from the immunogenic or antigenic site. or smaller, or smaller, or smaller, or smaller, or smaller or or smaller, as determined by three-dimensional protein structure analysis.

[0043] Embodiment 36. A variant asparaginase polypeptide according to any one of embodiments 23 to 35, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a wild-type asparaginase amino acid sequence such as SEQ ID NO: 2, SEQ ID NO: 11 or SEQ ID NO: 14.

[0044] Embodiment 37. The variant asparaginase polypeptide of any one of embodiments 23 to 35, wherein the wild-type asparaginase polypeptide is a wild-type E. coli asparaginase polypeptide.

[0045] Embodiment 38. The variant asparaginase polypeptide of any one of embodiments 23 to 37, wherein the wild-type asparaginase polypeptide comprises SEQ ID NO:2, SEQ ID NO:11 or SEQ ID NO:14.

[0046] Embodiment 39. The variant asparaginase polypeptide of any one of Embodiments 24 to 38, wherein the immunogenic or antigenic site corresponds to amino acid position 55, 56, 57, 58, 114, 115, 116, 117, 118, 119, 201, 202, 203, 204, 205, 206, 207, 252, 253, 254, 255, 256, 257 or 258 of SEQ ID NO: 11.

[0047] Embodiment 40. The variant asparaginase polypeptide of any one of Embodiments 24 to 39, wherein the at least one cysteine ​​is located at a position corresponding to one or more amino acid positions selected from 51, 52, 118, 119, 196, 206, 285 and 311 of SEQ ID NO: 11.

[0048] Embodiment 41. The variant asparaginase polypeptide of any one of embodiments 24 to 40, wherein each of the at least one pair of cysteines is selected from one or more C1-C2 paired cysteine ​​substitutions listed in Table 5.

[0049] Embodiment 42. The variant asparaginase polypeptide of any one of embodiments 24 to 41, wherein the at least one pair of cysteines is located at positions corresponding to amino acid positions 116 and 120, amino acid positions 196 and 200, or amino acid positions 225 and 252 of SEQ ID NO: 11.

[0050] Embodiment 43. The variant asparaginase polypeptide of any one of embodiments 23 to 42, comprising the amino acid sequence of SEQ ID NO:12.

[0051] Embodiment 44. The variant asparaginase polypeptide of any one of embodiments 23 to 24, 27, and 29 to 42, wherein the unpaired cysteine ​​residue is located at a position corresponding to amino acid position 45, 67 and / or 242 of SEQ ID NO: 14.

[0052] Embodiment 45. The variant asparaginase polypeptide of any one of embodiments 23 to 44, comprising the amino acid sequence of SEQ ID NO:15.

[0053] Embodiment 46. A tetramer comprising the variant asparaginase polypeptide according to any one of embodiments 23 to 45.

[0054] Embodiment 47. A dimer comprising the variant asparaginase polypeptide according to any one of embodiments 23 to 45.

[0055] Embodiment 48. The variant asparaginase polypeptide of any one of embodiments 1 to 47, wherein the variant asparaginase polypeptide is modified, such as sialylated or pegylated.

[0056] Embodiment 49. The variant asparaginase polypeptide of any one of embodiments 1 to 48, wherein the variant asparaginase polypeptide is thiol-PEGylated or amine-PEGylated.

[0057] Embodiment 50. An isolated nucleic acid encoding the variant E. chrysanthemi asparaginase polypeptide of any one of embodiments 1 to 20 and 23 to 45.

[0058] Embodiment 51. A vector comprising the nucleic acid according to embodiment 50.

[0059] Embodiment 52. A host cell comprising the vector according to embodiment 51.

[0060] Embodiment 53. A host cell according to embodiment 52, wherein the cell is a prokaryotic cell.

[0061] Embodiment 54. The host cell of Embodiment 53, wherein the prokaryotic cell is an Erwinia chrysanthemi cell, an Escherichia coli cell, or a Pseudomonas cell.

[0062] Embodiment 55. A host cell according to Embodiment 52, wherein the cell is a eukaryotic cell.

[0063] Embodiment 56. The host cell of Embodiment 55, wherein the eukaryotic cell is a yeast cell.

[0064] Embodiment 57. A method for producing a variant E. chrysanthemi asparaginase in E. coli cells, comprising culturing E. coli cells expressing a variant E. chrysanthemi asparaginase lacking a leader sequence.

[0065] Embodiment 58. A method of producing a variant asparaginase polypeptide comprising culturing the host cell of any one of embodiments 51 to 56.

[0066] Embodiment 59. The method of embodiment 57 or embodiment 58, wherein the variant asparaginase polypeptide is isolated from a cell lysate.

[0067] Embodiment 60. The method of any one of Embodiments 57 to 59, wherein the variant asparaginase polypeptide is isolated from the periplasm.

[0068] Embodiment 61. The method of any one of Embodiments 57 to 60, wherein the variant asparaginase polypeptide is separated by cation exchange column chromatography, anion exchange column chromatography, mixed mode column chromatography and / or hydrophobic interaction column chromatography.

[0069] Embodiment 62. The method of any one of Embodiments 57 to 61, wherein the variant asparaginase polypeptide is combined with a reducing agent.

[0070] Embodiment 63. A method according to embodiment 62, wherein the reducing agent is DTT.

[0071] Embodiment 64. The method of any one of Embodiments 62 to 63, wherein the variant asparaginase polypeptide is combined with a polyoxyethylene compound or a sialic acid compound.

[0072] Embodiment 65. A method according to embodiment 64, wherein the polyoxyethylene compound is α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy, polyoxyethylene or α-succinimidyloxyglutaryl-ω-methoxy, polyoxyethylene.

[0073] Embodiment 66. A pharmaceutical composition comprising the variant asparaginase polypeptide according to any one of embodiments 1 to 50 and a pharmaceutically acceptable carrier.

[0074] Embodiment 67. A method of delivering a variant asparaginase polypeptide to a subject, comprising administering the variant asparaginase polypeptide according to any one of embodiments 1 to 50 or the pharmaceutical composition according to embodiment 66 via an intramuscular route, an intraperitoneal route, an intravenous route, a subcutaneous route, or an intraarterial route.

[0075] Embodiment 68. A method of treating a subject suffering from lymphoma, comprising administering to the subject a therapeutically effective amount of a variant asparaginase polypeptide according to any one of embodiments 1 to 50 or a pharmaceutical composition according to embodiment 66.

[0076] Embodiment 69. A method of treating a subject having acute lymphoblastic leukemia, comprising administering to the subject a therapeutically effective amount of a variant asparaginase polypeptide according to any one of embodiments 1 to 50 or a pharmaceutical composition according to embodiment 66.

[0077] Embodiment 70. The method of any one of Embodiments 67 to 69, wherein the subject is a human or a companion animal species.

[0078] Embodiment 71. The method of Embodiment 70, wherein the companion animal species is canine, feline, or equine. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1A TABLE 4 is a table listing the wash and elution buffers used for cation exchange purification of variant E. chrysanthemi asparaginase (SEQ ID NO: 5) as described in Example 4.

[0080] Figure 1B is a non-reducing SDS-PAGE with Coomassie staining showing the variant E. chrysanthemi asparaginase (SEQ ID NO: 5) after expression and purification in E. coli cells.

[0081] Figure 2A Size exclusion chromatography results of variant E. chrysanthemi asparaginase (SEQ ID NO: 5) and protein standard markers are shown.

[0082] Figure 2B Elution times of size exclusion chromatography of variant E. chrysanthemi asparaginase (SEQ ID NO: 5) and protein standard markers are listed.

[0083] Figure 3 Shown is SDS-PAGE of un-PEGylated and thiol-PEGylated variant E. chrysanthemi asparaginase (SEQ ID NO: 5) under reducing (+DTT) and non-reducing (-DTT) conditions, at different reaction times (5 min and 60 min) and different reaction temperatures (room temperature and 37°C). Lane 1: unPEGylated, -DTT; Lane 2: unPEGylated, +DTT; Lane 3: thiol-PEGylated, reaction time 5 min, RT, -DTT; Lane 4: thiol-PEGylated, reaction time 5 min, RT, +DTT; Lane 5: thiol-PEGylated, reaction time 60 min, RT, -DTT; Lane 6: thiol-PEGylated, reaction time 60 min, RT, +DTT; Lane 7: thiol-PEGylated, reaction time 60 min, 37°C, -DTT; Lane 8: thiol-PEGylated, reaction time 60 min, 37°C, +DTT.

[0084] Figure 4 Shown is a variant E. coli asparaginase polypeptide of SEQ ID NO: 12. Additional cysteine ​​pairs were identified.

[0085] Sequence Description

[0086] Table 1 provides a listing of certain sequences cited herein.

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] DETAILED DESCRIPTION

[0094] PEGylation of biomolecules can increase circulation time, prevent inactivation by proteolysis, mask immunogenic or antigenic sites, and / or reduce immunogenicity. (pegaspargase, Shire) is an FDA-approved PEGylated asparaginase derived from Escherichia coli. Package insert, each asparaginase molecule has an average of 69-82 lysine-linked PEG molecules and is approximately 483-548 kDa. However, random PEGylation can result in heterogeneous products, and hypersensitivity to PEGylated asparaginase from E. coli can still occur.

[0095] The asparaginase isolated from Erwinia chrysanthemi was also studied. (Jazz Pharmaceuticals) is a natural asparaginase derived from Erwinia chrysanthemi that has been approved by the FDA as an alternative to pegaspargase and natural Escherichia coli asparaginase for patients who have developed a hypersensitivity reaction to E. coli-derived asparaginase. Package insert; Gervais, D. Validation of a 30-year-old process for the manufacture of L-asparaginase from Erwiniachrysanthemi. Bioprocess Biosyst Eng (2013) 36: 453-60. It is reported that See fiercepharma.com / manufacturing / jazz-releases-some-erwinaze-children-s-leukem ia-drug-has-been-shortage, dated February 26, 2018.

[0096] It is reported that pegylated recombinant Erwinia chrysanthemi-derived L-asparaginase is pegylated using random primary amines. The product has lower immunogenicity and improved efficacy. Chien, W., Pharmacology, immunogenicity, and efficacy of a novel pegylated recombinant Erwinia chrysanthemi-derived L-asparaginase. Invest New Drugs (2014) 32: 795-805. However, random pegylation can lead to heterogeneous products. Therefore, it is advantageous to incorporate PEG-conjugated amino acids into bacterial asparaginase in a site-specific manner, but it is not without problems.

[0097] Antigenic epitopes of Escherichia coli and Escherichia coli asparaginase have been identified and mapped. See Moola, ZB et al. Epitope mapping and antigenic modification of L-asparaginase. Biochem J. (1994) 302: 921-927 and Werner A. et al. Mapping of B-cell epitopes in E. coliasparaginase II, an enzyme used in leukemia treatment. Biol. Chem. (2005) 386: 535-540. However, the literature shows a lack of variant bacterial asparaginase products with site-specific rather than random PEGylation to shield immunogenic or antigenic sites (e.g., via site-specific cysteine ​​substitutions for thiol-specific conjugation). Recombinant expression of variant asparaginase polypeptides in the periplasm may be challenging. For example, while the oxidative environment of the periplasm is important for disulfide bond formation between paired cysteine ​​residues for proper protein folding, unpaired cysteine ​​residues are also oxidized, thereby hindering PEGylation techniques.

[0098] Due to immunogenicity issues, shortages of clinical asparaginase products, and inefficiencies in production methods, there is a need for asparaginase products with reduced immunogenicity, derived from alternative sources, and made using more efficient production methods. Variant asparaginase polypeptides and methods that address these issues are described herein.

[0099] The present disclosure relates to variant asparaginase polypeptides with reduced immunogenicity and enhanced stability and pharmacokinetics, and methods of producing and using the variant asparaginase polypeptides, e.g., for treating acute lymphoblastic leukemia in humans and lymphomas in companion animals. For the convenience of the reader, the following definitions of terms used herein are provided.

[0100] As used herein, "a or an" means "at least one" or "one or more" unless otherwise specified. As used herein, the term "or" means "and / or" unless otherwise specified. In the context of multiple dependent claims, when referencing other claims, the use of "or" refers only to those claims that are in the alternative.

[0101] Exemplary Asparaginase Polypeptides

[0102] Variant asparaginase polypeptides are provided, for example, variant asparaginase polypeptides comprising PEG-conjugatable amino acid substitutions (such as cysteine ​​and lysine substitutions) to reduce immunogenicity.

[0103] "Amino acid sequence" means the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. The definition encompasses both full-length proteins and fragments thereof. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of this disclosure, "polypeptide" refers to a protein that includes modifications (such as deletions, additions and substitutions) to a natural (or wild-type) sequence (usually conservative in nature), as long as the protein maintains the desired activity. These modifications may be intentional (such as by site-directed mutagenesis), or may be accidental (such as by mutations of the host producing the protein or due to errors caused by PCR amplification).

[0104] As used herein, "amino acid derivatives" refers to any amino acid, modified amino acid and / or amino acid analog, rather than one of the 20 common natural amino acids found in humans. Exemplary amino acid derivatives include natural amino acids not found in humans (e.g., selenocysteine ​​and pyrrolysine that may be found in some microorganisms) and non-natural amino acids. Exemplary amino acid derivatives include, but are not limited to, amino acid derivatives available from chemical product manufacturers and distributors (e.g., sigmaaldrich.com / chemistry / chemistry-products.html? TablePage=16274965 accessed on May 6, 2017, which is incorporated herein by reference). One or more amino acid derivatives can be incorporated into a polypeptide at a specific position using a translation system utilizing a host cell, an orthogonal aminoacyl-tRNA synthetase derived from a true bacterial synthetase, an orthogonal tRNA, and an amino acid derivative. For further description, see, e.g., U.S. Patent No. 9,624,485.

[0105] As used herein, "asparaginase" or "L-asparaginase" is a polypeptide comprising the whole or a fragment of asparaginase, including variant polypeptides, precursor polypeptides, mature polypeptides, tetramers, dimers, monomers or any other forms.

[0106] For example, "asparaginase" refers to asparaginase polypeptides from any source, unless otherwise indicated, including plants, mammals (e.g., porcine and rodents), and bacteria (e.g., Escherichia coli, Erwinia chrysanthemi, Aerobacter aerogenes, Aeromonas hydrophila, A. liquefaciens, A. salmonicida, A. sinuosa, Bacillus megaterium, megaterium), B. subtilis, Erwinia amylovora, E. araoideae, E. carotovora, E. dissolvens, E. freundii, Hydrogenomonas eutropha, H. pantotropha, Photobacterium fischeri, Proteus americanus, P. mirabilis, P. morganii, P. paramericanus, P. psudovaleriei, P. shingides, P. vulgaris, Psudomonas acidovorans acidovorans), Pseudomonas ammoniagenes, Pseudomonas asplenii, Pseudomonas aureofaciens, Pseudomonas caviae, Pseudomonas convexa, Pseudomonas dacunhae, Pseudomonas fluorescens, Pseudomonas curvatus, Pseudomonas lemonnieri, Pseudomonas pavonacea, Pseudomonas putida, Pseudomonas reptilivora, Pseudomonas spp., Pseudomonas stutzeri, Pseudomonas synxantha, Pseudomonas rotundus, Pseudomonas spp.taetrolens), Serratia marcescens, Xanthomonas campestrils, etc.; see Peterson RE and Ciegler A, L-asparaginase production by various bacteria. Applied Microbiol (1969) 17: 929-930. In some embodiments, the asparaginase comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

[0107] "Wild-type" refers to the unmutated form of a naturally occurring polypeptide or fragment thereof. A wild-type polypeptide can be produced recombinantly or naturally. A naturally occurring wild-type polypeptide is also referred to as a native polypeptide.

[0108] "Variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with a wild-type (or native) sequence polypeptide after alignment of the sequences and introduction of gaps (if necessary) to achieve the maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added, deleted, or are present at the N-terminus or C-terminus of the polypeptide.

[0109] In some embodiments, the variant has at least about 80% amino acid sequence identity, at least about 85% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 95% amino acid sequence identity, at least about 97% amino acid sequence identity, at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity to a wild-type (native) sequence polypeptide.

[0110] A "biologically active" entity or an entity having "biological activity" is an entity having any function related to or associated with a metabolic or physiological process, and / or having a structural function, a regulatory function, or a biochemical function of a naturally occurring molecule. A biologically active polypeptide or fragment thereof includes one that can participate in a biological reaction, including but not limited to an enzyme reaction, a ligand-receptor interaction, or an antigen-antibody binding. Biological activity can include improved desired activity or reduced undesired activity. An entity can demonstrate biological activity when it participates in a molecular interaction with another molecule, when it has therapeutic value for alleviating a disease condition, when it has prophylactic value for inducing an immune response, when it has diagnostic and / or prognostic value for determining the presence of a molecule.

[0111] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN or MEGALIGN. TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any parameters needed to achieve maximal alignment over the full length of the sequences being compared.

[0112] As used herein, "a site corresponding to amino acid position n" or "a position corresponding to amino acid position n," where n is any number or range of numbers, refers to one or more amino acid positions of a subject polypeptide that align with position n of a reference polypeptide after aligning the amino acid sequences of the subject and reference polypeptides and introducing gaps. Alignment for the purpose of determining whether a position of a subject polypeptide corresponds to position n of a reference polypeptide can be accomplished in various ways within the skill in the art, for example using publicly available computer software such as BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN. TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for comparison, including any parameters required to achieve maximum comparison over the full length of the two sequences being compared. In some embodiments, the subject polypeptide and the reference polypeptide have different lengths.

[0113] Amino acid substitutions may include, but are not limited to, replacing an amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into a protein of interest and the product may be screened for desired activity, such as retained / improved antigen binding or reduced immunogenicity.

[0114] Table 2

[0115]

[0116] Amino acids can be grouped according to common side chain properties:

[0117] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0118] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0119] (3) Acidic: Asp, Glu;

[0120] (4) Basic: His, Lys, Arg;

[0121] (5) Residues that affect chain orientation: Gly, Pro;

[0122] (6) Aromatic: Trp, Tyr, Phe.

[0123] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0124] In some embodiments, the variant asparaginase polypeptide comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:15.

[0125] A "PEG-conjugable" amino acid refers to an amino acid or amino acid derivative capable of associating with or covalently or non-covalently attaching to at least one PEG molecule.

[0126] In some embodiments, the variant asparaginase polypeptide comprises at least one PEG conjugated amino acid substitution. In some embodiments, the PEG conjugated amino acid comprises lysine, cysteine, histidine, arginine, aspartic acid, glutamine, serine, threonine, tyrosine or an amino acid derivative of any of these amino acids.

[0127] As used herein, "PEG" refers to a polyethylene glycol moiety.

[0128] As used herein, "PEGylated" refers to a polypeptide having one or more PEG moieties associated or covalently or non-covalently attached.

[0129] As used herein, "sialylated" refers to a polypeptide having one or more covalently attached sialic acid moieties.

[0130] A variety of methods have been developed for producing glycosylated and sialylated proteins. See, for example, Savinova et al. Applied Biochem & Microbiol. (2015) 51(8): 827-833.

[0131] In some embodiments, the variant asparaginase polypeptide is pegylated and / or sialylated. In some embodiments, the variant asparaginase polypeptide is pegylated and / or sialylated at cysteine, lysine, histidine, arginine, aspartic acid, glutamine, serine, threonine, tyrosine, or any of these amino acid derivatives (such as cysteine, lysine, histidine, arginine, aspartic acid, glutamine, serine, threonine, tyrosine amino acid derivatives).

[0132] "Surface exposed amino acids" refer to amino acid residues whose 30% or more of their surface is accessible to solvent as determined using any standard protein modeling software known in the art (e.g., Swiss model, MOE (Molecular Operating Environment), Rosetta, and Modeller).

[0133] In some embodiments, surface exposed amino acids include amino acids having 30% or greater surface exposure (or 30% or greater of their surface accessible to solvent) as determined by standard protein modeling software. In some embodiments, surface exposed amino acids include amino acids having 30% or greater, 35% or greater, 40% or greater, 45% or greater, 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater surface exposure.

[0134] "Partially buried amino acid" refers to an amino acid residue that has between 5% and 30% of its surface inaccessible to solvent as determined using any standard protein modeling software known in the art (e.g., Swiss model, MOE (Molecular Operating Environment), Rosetta, and Modeller).

[0135] In some embodiments, partially buried amino acids include amino acids having between 5% and 30% surface exposure (or between 5% and 30% of their surface accessible to solvent), as determined by standard protein modeling software. In some embodiments, surface exposed amino acids include amino acids having between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 20%, between 20% and 30%, or between 10% and 30% surface exposure.

[0136] As used herein, "immunogenic or antigenic site" refers to any amino acid or group of amino acids within a polypeptide that is involved in eliciting an immune response.

[0137] In some embodiments, the immunogenic or antigenic site is any amino acid within the epitope. In some embodiments, when the immunogenic or antigenic site or an amino acid that is spatially close to the immunogenic or antigenic site is conjugated to PEG, the immunogenic or antigenic site is less immunogenic or antigenic.

[0138] An amino acid that is "spatially close" to another amino acid refers to their three-dimensional proximity, as determined by three-dimensional protein modeling. If an amino acid is 35 angstroms from another amino acid, or smaller, or smaller, or smaller, or smaller, or smaller, or smaller or or smaller, then the two amino acids are likely to be close in space.

[0139] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate or lipid) where an antigen-binding molecule (e.g., an antibody, an antibody fragment or a scaffold protein containing an antibody binding region) binds to the target molecule. The epitope generally includes a chemically active surface grouping of molecules such as amino acids, polypeptides or sugar side chains, and has specific three-dimensional structural characteristics and specific charge characteristics. The epitope can be formed by continuous or juxtaposed non-continuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. The epitope formed by continuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) is usually retained when exposed to a denaturing solvent, while the epitope formed by tertiary folding is usually lost when treated with a denaturing solvent. The epitope may include, but is not limited to, at least 3, at least 5 or 8-10 residues (e.g., amino acids or nucleotides). In some examples, the epitope length is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues or less than 12 residues. If two antibodies show competitive binding to an antigen, they can bind to the same epitope within the antigen. In some embodiments, epitope can be identified by a certain minimum distance with the CDR residues on the antigen binding molecules. In some embodiments, epitope can be identified by the above distance, and further limited to those residues that participate in the bond (e.g., hydrogen bond) between the antibody residues and the antigen residues. Epitope can also be identified by various scans, such as alanine or arginine scanning can indicate one or more residues that the antigen binding molecules can interact with. Unless explicitly stated, a group of residues as an epitope does not exclude other residues as a part of the epitope of a specific antibody. On the contrary, the existence of such a group represents the minimum series (or species group) of an epitope. Therefore, in some embodiments, a group of residues identified as an epitope represents the minimum epitope associated with an antigen, rather than an exclusive list of residues of an epitope on an antigen.

[0140] In some embodiments, immunogenic or antigenic sites can be determined by epitope scanning methods known in the art, for example, according to the method described in Moola, ZB, Erwinia chrysanthemi L-asparaginase: epitopemapping and production of antigenically modified enzymes. Biochem J. (1994) 921-927.

[0141] In some embodiments, the immunogenic or antigenic site is an epitope corresponding to amino acid positions 37 to 40 or 205 to 212 of SEQ ID NO: 2. In some embodiments, the immunogenic or antigenic site corresponds to amino acid positions 37, 38, 39, 40, 41, 72, 205, 206, 207, 208, 209, 210, 211, 212, 265, or 288 of SEQ ID NO: 2. In some embodiments, the immunogenic or antigenic site corresponds to amino acid positions 55, 56, 57, 58, 114, 115, 116, 117, 118, 119, 201, 202, 203, 204, 205, 206, 207, 252, 253, 254, 255, 256, 257, or 258 of SEQ ID NO: 11.

[0142] In some embodiments, the variant asparaginase polypeptide comprises at least one PEG conjugable amino acid substitution or at least one cysteine ​​substitution at at least one position corresponding to a position selected from the group consisting of: position 3, 4, 17, 26, 37, 38, 41, 42, 44, 45, 47, 48, 51, 53, 54, 55, 56, 59, 60, 68, 72, 79, 82, 83, 84, 85, 87, 110, 112, 123, 124, 125, 127, 180, 190, 191, 192, 198, 202, 205, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235 , 206, 208, 210, 212, 213, 215, 216, 219, 231, 239, 240, 241, 243, 257, 260, 261, 264, 265, 267, 268, 269, 270, 280, 286, 287, 288, 289, 312, 313, 315, 316, 317, 318 and 322.

[0143] In some embodiments, the variant asparaginase polypeptide comprises at least one PEG conjugated amino acid substitution or at least one lysine substitution at at least one position corresponding to a position selected from: SEQ ID NO: 213, 215, 216, 231, 239, 240, 241, 257, 260, 261, 264, 267, 268, 270, 280, 286, 287, 288, 289, 312, 313, 315, 316, 317 and 322 of NO:2.

[0144] In some embodiments, the variant asparaginase polypeptide comprises at least one cysteine ​​located at at least one position corresponding to a position selected from 51, 52, 118, 119, 196, 206, 285, and 311 of SEQ ID NO: 11. In some embodiments, the variant asparaginase polypeptide comprises at least one pair of cysteines selected from any one or combination of one or more C1-C2 paired cysteine ​​substitutions listed in Table 5. For example, the variant asparaginase polypeptide may comprise one or more pairs of cysteines located at positions corresponding to amino acid positions 116 and 120, amino acid positions 196 and 200, or amino acid positions 225 and 252 of SEQ ID NO: 11.

[0145] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D Affinity can be measured by conventional methods known in the art such as, for example, immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI) or surface plasmon resonance apparatus.

[0146] The term "K D ”, “K d "," Kd "or "Kd value" are used interchangeably to refer to the equilibrium dissociation constant of an antibody-antigen interaction. In some embodiments, according to the supplier's instructions, the dissociation constant is determined using a method such as Biosensors such as the Pall ForteBio system (Pall ForteBio LLC, Fremont, CA) measure the K of antibodies using biolayer interferometry. d Briefly, biotinylated antigen was bound to the sensor tip and antibody association was monitored for 90 seconds and dissociation for 600 seconds. The buffer used for the dilution and binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer-only blank curve was subtracted to correct for any drift. The data were fit to a 2:1 binding model using ForteBio data analysis software to determine the association rate constant (k on ), dissociation rate constant (k off ) and K d The equilibrium dissociation constant (K d ) is calculated as k off / k on The term "kon" refers to the rate constant for the association of an antibody with an antigen, while the term "koff" refers to the rate constant for the dissociation of an antibody from an antibody / antigen complex.

[0147] "Surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example using BIAcore TM System (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26.

[0148] "Bio-layer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from an immobilized protein layer on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause shifts in the interference pattern that can be measured in real time. A non-limiting exemplary apparatus for bio-layer interferometry is System (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem. 377: 209-277.

[0149] "Reducing" or "inhibiting" means reducing, lowering or stagnating an activity, function or amount compared to a reference. In some embodiments, "reducing" or "inhibiting" means the ability to cause an overall reduction of 20% or more. In some embodiments, "reducing" or "inhibiting" means the ability to cause an overall reduction of 50% or more. In some embodiments, "reducing" or "inhibiting" means causing an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above amount is suppressed or reduced over a period of time relative to a control dose (such as a placebo) over the same time period. As used herein, "reference" refers to any sample, standard or level for comparison purposes. References can be obtained from healthy or non-diseased samples. In some examples, references are obtained from non-diseased or untreated samples of companion animals. In some examples, references are obtained from one or more healthy animals of a particular species rather than the animals being tested or treated.

[0150] As used herein, the term "significantly reduced" means a sufficiently high degree of reduction between a numerical value and a reference value such that one skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by the value. In some embodiments, a significantly reduced numerical value is reduced by greater than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% compared to a reference value.

[0151] Exemplary expression and production

[0152] Polynucleotide sequences are provided that encode all or part of an asparaginase polypeptide with or without a leader sequence.If a homologous leader sequence (ie, a leader sequence of a wild-type asparaginase) is not used in the construction of the nucleic acid molecule, another bacterial leader sequence may be used.

[0153] Typically, a nucleotide sequence encoding a polypeptide of interest (such as an asparaginase polypeptide described herein) is inserted into an expression vector suitable for expression in a host cell of choice.

[0154] The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate expression of a polypeptide of interest (such as, for example, a promoter or enhancer), or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0155] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary prokaryotic cells include Escherichia coli, Bacillus, and Pseudomonas. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NS0 cells, Host cells include cells (Crucell), 293 cells and CHO cells, and derivatives thereof such as 293-6E, DG44, CHO-S and CHO-K cells. Host cells include progeny of a single host cell, and due to natural, accidental or deliberate mutations, the progeny may not necessarily be identical to the original parent cell (in terms of morphology or genomic DNA complementary sequence). Host cells include cells transfected in vivo with one or more polynucleotides encoding one or more amino acid sequences provided herein.

[0156] As used herein, the term "isolated" refers to a molecule that has been separated from at least some components that are usually found or produced in nature. For example, when a polypeptide is separated from at least some components of the cell that produces it, it is called "isolated". When a polypeptide is secreted by a cell after expression, the supernatant containing the polypeptide is physically separated from the cell that produces it, and it is considered to be "isolated" the polypeptide. Similarly, when a polynucleotide is not a part of a larger polynucleotide that is usually found in nature (such as, for example, genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or when, for example, it is separated from at least some components of the cell that produces it in the case of an RNA polynucleotide, the polynucleotide is called "isolated". Therefore, the DNA polynucleotide contained in the vector in the host cell can be called "isolated". In some embodiments, a chromatogram is used, such as size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and CHT chromatography or mixed mode chromatography to purify asparaginase.

[0157] Exemplary Pharmaceutical Compositions

[0158] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in such form as to render the biological activity of one or more active ingredients effective, and that contains no other components that are unacceptably toxic to a subject to which the formulation would be administered.

[0159] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier conventional in the art, which is used together with the therapeutic agent that comprises the "pharmaceutical composition" for administration to a subject. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations used and are compatible with the other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for the formulation used. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin, canine or other animal albumin; buffers such as phosphate, citrate, tromethamine or HEPES buffer; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide or magnesium trisilicate; polyvinyl pyrrolidone, cellulose-based substances; polyethylene glycol; sucrose; mannitol; or amino acids including but not limited to arginine.

[0160] The pharmaceutical composition can be stored in a lyophilized form. Therefore, in some embodiments, the preparation method includes a lyophilization step. The lyophilized composition can then be reconstituted before being administered to the subject, usually as an aqueous composition suitable for parenteral administration. In other embodiments, particularly when the polypeptide is highly stable to thermal denaturation and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., stored as an aqueous composition, which can be administered to the subject directly or in the case of appropriate dilution. The lyophilized composition can be reconstituted with sterile water for injection (WFI). Antibacterial agents such as benzyl alcohol can be included. Therefore, the invention provides pharmaceutical compositions in solid or liquid form.

[0161] When administered, the pH of the pharmaceutical composition may be in the range of about pH 5 to about pH 8. If they are used for therapeutic purposes, the compositions of the present invention are sterile. Sterility can be achieved by any of several means known in the art, including filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane). Sterility may be maintained with or without the use of an antibacterial agent.

[0162] Exemplary Uses of Variant Asparaginase Polypeptides

[0163] Variant asparaginase polypeptides of the present invention or pharmaceutical compositions comprising the variant asparaginase polypeptides can be used to treat a variety of conditions, including cancers such as lymphomas or leukemias. In some embodiments, variant asparaginase polypeptides of the present invention or pharmaceutical compositions comprising the variant asparaginase polypeptides can be used to treat ALL in humans or lymphomas in companion animal species.

[0164] The term "companion animal species" refers to animals suitable as companions for humans. In some embodiments, companion animal species are small mammals such as canines, felines, dogs, cats, horses, rabbits, ferrets, guinea pigs, rodents, etc. In some embodiments, companion animal species are farm animals such as horses, cattle, pigs, etc.

[0165] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. As used herein, "treatment" includes any administration or application of a disease therapeutic agent in a mammal (including a companion animal). For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, any one or more of the following: alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, inhibiting the disease or disease progression, inhibiting or slowing the disease or disease progression, stagnating the development of the disease and alleviating (whether in part or in whole). "Treatment" also encompasses the pathological consequences of reducing proliferative diseases. The method provided herein takes into account any one or more of these treatment aspects. Consistent with the above, the term treatment does not require 100% removal of all aspects of the obstacle.

[0166] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary according to factors such as the type of disease to be treated, the disease state, the severity and course of the disease, the type of therapeutic objective, any previous treatment, clinical history, response to previous treatment, the judgment of the attending veterinarian, the age, sex and weight of the animal, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the animal. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the substance / molecule, agonist or antagonist. A therapeutically effective amount may be delivered in one or more administrations. A "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic or preventive result at the necessary dosage and for the necessary period of time.

[0167] In some embodiments, as described herein, the variant asparaginase polypeptide or pharmaceutical composition comprising the variant asparaginase polypeptide is administered parenterally, by subcutaneous administration, intravenous infusion or intramuscular injection. In some embodiments, the variant asparaginase polypeptide or pharmaceutical composition is administered by push injection or continuous infusion for a period of time. In some embodiments, the variant asparaginase polypeptide or pharmaceutical composition is administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal or inhalation route.

[0168] In some embodiments, the dose is administered once a week for at least two or three consecutive weeks, and in some embodiments, the treatment cycle is repeated two or more times, optionally interspersed with one or more weeks without treatment. In other embodiments, the therapeutically effective dose is administered once a day for two to five consecutive days, and in some embodiments, the treatment cycle is repeated two or more times, optionally interspersed with one or more days or one or more weeks without treatment.

[0169] Administration "in combination" with one or more other therapeutic agents includes simultaneous (concurrent) and continuous or sequential administration in any order. The term "concurrent" is used herein to refer to the administration of two or more therapeutic agents, wherein at least part of the administration overlaps in time or the administration of one therapeutic agent occurs in a very short time relative to the administration of another therapeutic agent. For example, the two or more therapeutic agents are administered at a time interval not exceeding about a specified number of minutes. The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents, wherein one or more other agents are continued to be administered after interrupting the administration of one or more medicaments, or wherein one or more other medicaments are administered before administering one or more medicaments. For example, the two or more therapeutic agents are administered at a time interval exceeding about a specified number of minutes. As used herein, the term "in combination with..." refers to administering another therapeutic mode in addition to a treatment mode. Therefore, "in combination with..." refers to administering another treatment mode before, during, or after administering a treatment mode to an animal.

[0170] The following examples illustrate specific aspects of the disclosure and are not intended to limit the disclosure in any way.

[0171] Example

[0172] Example 1

[0173] Expression of Asparaginase from Erwinia chrysanthemi in Escherichia coli

[0174] Natural Erwinia chrysanthemi asparaginase expressed in its natural host cells is usually processed and secreted into the periplasmic space or culture medium. The expression of Erwinia chrysanthemi asparaginase in Escherichia coli cells was studied. The nucleotide sequence encoding the natural Erwinia chrysanthemi asparaginase (SEQ ID NO: 1) containing a leader sequence was cloned into pET28a (+) (an Escherichia coli expression vector with a T7 promoter and a kanamycin resistance gene) (Novagen) between the Xba1 and HindIII sites. After the resulting plasmid was transformed into Escherichia coli BL21 (DE3) cells, the cells were incubated at 37°C until the optical density at 600nm reached approximately 1. Then 1mM IPTG was added to the culture medium, and the cells were incubated at 37°C for 2 hours to induce the expression of the Erwinia chrysanthemi asparaginase protein.

[0175] Cells are subjected to osmotic shock to release proteins from the periplasm. Centrifugal cells are separated and supernatant (also referred to as osmotic fraction). Cell pellets are resuspended and ultrasonicated to lyse cells. Centrifugal cells, and lysate (also referred to as soluble fraction) is separated from precipitation (also referred to as insoluble fraction). These three fractions (osmotic, soluble and insoluble fractions) are separated by SDS-PAGE and visualized using Coomassie staining.

[0176] Based on band intensity and molecular weight differences, the permeate fraction appears to contain very low processed asparaginase production, while most of the full-length asparaginase products are observed in the cell pellet (insoluble fraction). A small amount of unprocessed asparagine products are observed in the lysate fraction (soluble fraction). These results indicate that the periplasmic expression efficiency of the E. chrysanthemi asparaginase in E. coli is low, and most of the unprocessed asparaginase products are trapped in the cell in an insoluble form (e.g., aggregates, inclusion bodies, etc.), which may be due to the processing failure of the leader sequence.

[0177] Example 2

[0178] Intracellular expression of asparaginase from Erwinia chrysanthemi

[0179] The expression of the Erwinia chrysanthemi asparaginase lacking a leader sequence in Escherichia coli cells was also studied. The nucleotide sequence encoding the natural Erwinia chrysanthemi asparaginase lacking its leader sequence (SEQ ID NO:3) was cloned between the Xba1 and HindIII sites of pET28a (+) (Novagen), transformed into Escherichia coli BL21 (DE3) cells, and cultured in the presence of 1mM IPTG as described in Example 1. The cells were lysed by ultrasonic treatment, centrifuged, and the lysate (soluble fraction) and the precipitation (insoluble fraction) were separated by SDS-PAGE, and visualized using Coomassie staining. Unexpectedly, higher yields of soluble asparaginase expressed in the cells were observed in the lysate.

[0180] Example 3

[0181] Variant Erwinia chrysanthemi asparaginase polypeptides for site-directed thiol conjugation

[0182] Secretory proteins of Gram-negative bacteria often contain disulfide bonds, which are catalyzed by thiol-disulfide oxidoreductases in the periplasm at cysteine ​​residues. For example, native E. coli asparaginase has a disulfide bond between only two cysteine ​​residues in the protein. However, inspection of the primary amino acid sequence of E. chrysanthemi asparaginase revealed that the protein lacks cysteine ​​residues. This observation suggests that native E. chrysanthemi asparaginase lacks disulfide bonds, and that the oxidative environment of the periplasmic space may not be required to fold this protein.

[0183] Bacterial proteins are highly immunogenic to humans. PEGylation of bacterial proteins can shield immunogenic sites and reduce immunogenicity. In addition, PEGylation can prolong the in vivo half-life of proteins. (pegasparagase, Shire), E. coli-derived asparaginase is PEGylated at surface-exposed primary amines (e.g., lysine residues). Unfortunately, this nonspecific PEGylation may result in heterogeneous products. It may be advantageous to PEGylate at specific immunogenic sites rather than on the entire protein.

[0184] Since the natural Erwinia chrysanthemi asparaginase does not include cysteine ​​residues, variants comprising one or more cysteine ​​substitutions can be specifically pegylated at those positions. The introduction of one or more cysteine ​​residues at or near the immunogenicity or antigenicity sites of Erwinia chrysanthemi asparaginase was studied. The three-dimensional protein structure analysis of tetrameric Erwinia chrysanthemi asparaginase was used to identify surface-exposed amino acids at potential immunogenicity or antigenicity sites or at positions close in space, where pegylation can shield the sites without affecting one or more active sites. Potential immunogenicity or antigenicity sites include amino acid positions 37 to 41, 72, 205 to 212, 265 and 288 of the mature amino acid sequence (SEQ ID NO:2). Table 2 lists the amino acid positions of SEQ ID NO:2, at which cysteine ​​residues can be introduced to carry out site-specific pegylation to reduce immunogenicity.

[0185] Table 2.

[0186]

[0187]

[0188] Variant E. chrysanthemi asparaginase polypeptides can be prepared by introducing one or more cysteine ​​residues at any one or any combination of one or more amino acid positions listed in Table 2. For example, 2, 3, 4, 5, 6 or more cysteine ​​residues can be introduced into the E. chrysanthemi asparaginase.

[0189] For example, a variant precursor E. chrysanthemi asparaginase polypeptide can be prepared having three cysteine ​​amino acid substitutions at positions 41, 72, and 265 of the mature amino acid sequence (SEQ ID NO: 4) or four cysteine ​​amino acid substitutions at positions 41, 72, 265, and 288 of the mature amino acid sequence (SEQ ID NO: 7).

[0190] Example 4

[0191] Intracellular expression of variant Erwinia chrysanthemi asparaginase polypeptide

[0192] Although the oxidative environment of the periplasm catalyzes disulfide bonds between cysteine ​​residues, cysteine ​​thiol groups are protected from oxidation in the reducing environment of the cytoplasm. The intracellular expression of variant Erwinia chrysanthemi asparaginase polypeptides comprising cysteine ​​substitutions at immunogenic or antigenic sites was explored in Escherichia coli cells. The nucleotide sequence encoding the variant Erwinia chrysanthemi asparaginase was cloned into pET28a (+) (Novagen), and the variant Erwinia chrysanthemi asparaginase had an N-terminal poly-His tag replacing the leader sequence and had three cysteine ​​amino acid substitutions at positions 41, 72 and 265 of the mature amino acid sequence (SEQ ID NO: 5). After the resulting plasmid was transformed into Escherichia coli BL21 (DE3) cells, the cells were incubated at 37°C until the optical density at 600nm reached about 1. Then 1mM IPTG was added to the culture medium, and the cells were incubated at 37°C for 2 hours to induce the expression of the variant Erwinia chrysanthemi asparaginase protein.

[0193] The cells were lysed by sonication, centrifuged, and the variant E. chrysanthemi asparaginase (SEQ ID NO: 5) was purified from the lysate by affinity chromatography using Ni Sepharose excel histidine tag protein purification resin (GE Healthcare, catalog number 17371201), followed by cation exchange chromatography using SulfphopropylSepharose Fast Flow (SP FF) cation exchange chromatography resin (GE Healthcare, catalog number 17072901). The SP FF column was washed with 0.1 M sodium phosphate at pH 6 and then pH 7, and the protein was eluted with a solution of 0.1 M sodium phosphate (pH 7) and 1 M sodium chloride. See Figure 1A Purified proteins were separated using non-reducing SDS-PAGE and visualized using Coomassie staining ( Figure 1B ).

[0194] In addition, size exclusion chromatography was used to compare the hydrodynamic radius of the purified variant Erwinia chrysanthemi asparaginase (SEQ ID NO: 5) with a Novex Sharp prestained protein standard marker (ThermoFisher, catalog number LC5800), and to confirm that the purified protein was in tetrameric form. Using an Agilent 1100 chromatography system, the purified protein was loaded onto a Shodex KW803 column (8mm x 300mm) with a KW-G guard column at a constant flow rate of 0.5mL / min and a running buffer of 0.1M sodium phosphate (pH 6.6) and 1M sodium chloride. The eluted material was monitored by absorbance at a wavelength of 214nm. The purified variant Erwinia chrysanthemi asparaginase product (SEQ ID NO: 5) eluted at 16.5 minutes, consistent with the predicted molecular weight (about 117.4kDa) of the protein tetramer form in the solution. See Figure 2A and Figure 2B .

[0195] Example 5

[0196] Site-directed PEGylation of variant Erwinia chrysanthemi asparaginase polypeptides

[0197] Conjugation of purified variant E. chrysanthemi asparaginase (SEQ ID NO: 5) was performed according to the manufacturer's instructions using: (1) conjugation with α-succinimidyloxyglutaryl-ω-methoxy, polyoxyethylene (mw5kD; ME-050GS; NOF Corporation) and (2) random amine PEGylation with α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy, polyoxyethylene (mw10kD; ME-100MA; NOF Corporation) for thiol-specific PEGylation, or (3) two methods.

[0198] For thiol-specific PEGylation, different reaction times (5 min and 60 min) and different reaction temperatures (room temperature and 37°C) were tested. PEGylated proteins were separated by SDS-PAGE under reducing (with +DTT) and non-reducing (without DTT) conditions and visualized using Coomassie staining ( Figure 3 ). UnPEGylated proteins were exposed to reducing conditions ( Figure 3 , lane 2) under non-reducing conditions ( Figure 3 , lane 3) runs at approximately 35 kDa, indicating the absence of disulfide bonds in the purified variant E. chrysanthemi asparaginase (SEQ ID NO: 5). Figure 3 , lanes 4-9) run at a higher molecular weight consistent with protein PEGylation.

[0199] The asparaginase activity of variant Erwinia chrysanthemi asparaginase (SEQ ID NO: 5) conjugated via random amine PEGylation and / or thiol-specific PEGylation as described above was measured in duplicate using an asparaginase activity assay kit (BioVision; Catalog No. K754-100) according to the manufacturer's instructions. The results of the assay are provided in Table 3 below. The results show that after PEGylation, variant Erwinia chrysanthemi asparaginase (SEQ ID NO: 5) retains enzyme activity.

[0200] Table 3

[0201]

[0202] *, Each assay was performed in duplicate, except sample A which was performed once.

[0203] Example 6

[0204] Variant Erwinia chrysanthemi asparaginase polypeptides for site-directed amine conjugation

[0205] Surface exposed primary amines (e.g., lysine) can be conjugated with amine reactive PEG derivatives. The specificity of PEGylation may be low, and the product may be heterogeneous. Introducing one or more additional lysine residues at surface exposed positions can provide additional sites for amine reactive PEGylation. For example, variant Erwinia chrysanthemi asparaginase polypeptides can be prepared by replacing one or more lysine residues at or near potential immunogenic or antigenic sites (such as amino acid positions 41, 72, 265 and 288 of the mature amino acid sequence (SEQ ID NO:2). Table 4 lists exemplary surface exposed amino acid positions of Erwinia chrysanthemi asparaginase at amino acid positions 41, 72, 265 and 288 of the mature amino acid sequence (SEQ ID NO:2), at which positions lysine residues can be introduced to carry out site-specific PEGylation.

[0206] Table 4.

[0207]

[0208] Variant E. chrysanthemi asparaginase polypeptides can be prepared by introducing one or more lysine residues at any one or any combination of one or more amino acid positions listed in Table 4. For example, 2, 3, 4, 5, 6 or more lysine residues can be introduced into the E. chrysanthemi asparaginase.

[0209] For example, a variant precursor E. chrysanthemi asparaginase polypeptide can be prepared having a lysine amino acid substitution at position 41 (SEQ ID NO:8) or at positions 41 and 288 (SEQ ID NO:9) of the mature amino acid sequence.

[0210] Example 7

[0211] Variant Escherichia coli asparaginase polypeptides for site-directed amine conjugation

[0212] Escherichia coli L-asparaginase II (SEQ ID NO:10) is currently approved for the treatment of acute lymphoblastic leukemia. Unfortunately, immunological response is a significant adverse side effect. Site-specific PEGylation will help reduce immunological side effects. The natural E. coli asparaginase protein has a leader sequence and two cysteine ​​residues that form a single disulfide bond. It is believed that the protein is processed and secreted into the periplasmic space to form a disulfide bond. Unlike the Erwinia chrysanthemi asparaginase, the E. coli asparaginase may not be suitable for intracellular expression because the disulfide bond in the periplasm may be necessary for correct protein folding. The different methods for the site-specific PEGylation of E. coli asparaginase have been studied.

[0213] A method of introducing a specific pegylation site into E. coli asparaginase involves replacing unpaired cysteine ​​at a position partially embedded in or near a potential immunogenic or antigenic site. The thiol group of the unpaired cysteine ​​at the partially embedded site can be shielded from oxidation in the periplasm, but is sufficiently exposed for a conjugation reaction such as pegylation. The three-dimensional protein structure analysis of tetrameric E. coli L-asparaginase II is used to identify partially embedded amino acids at potential immunogenic or antigenic sites or at positions close in space, where pegylation can shield the site without affecting one or more active sites. For example, one or more unpaired cysteine ​​residues can be substituted at immunogenic or antigenic sites (such as amino acid positions 55-58, 114-119, 201-207 and 252-258 of the mature amino acid sequence (SEQ ID NO: 11)) or at partially embedded sites close in space. For example, a variant E. coli asparaginase polypeptide can be prepared by substituting one or more unpaired cysteine ​​residues at any one or more of the following amino acid positions based on the mature amino acid sequence (SEQ ID NO: 11): E51C, Q52C, S118C, T119C, K196C, S206C, D285C and T311C.

[0214] A second method for introducing specific PEGylation sites into E. coli asparaginase involves the introduction of one or more paired cysteine ​​residues at or near a potential immunogenic or antigenic site. The thiol groups of the substituted cysteine ​​pairs should not be oxidized in the periplasm, but rather the pairs form disulfide bonds. The disulfide bonds (including the original bonds) can then be reduced using a reducing agent such as DTT to allow conjugation. PEGylation or labeling can be achieved by rebridging the paired cysteines using various chemical methods such as thiol-yne coupling reactions. See Griebenow N. et al., Site-specific conjugation of peptides and proteins via rebridging of disulfide bonds using the thiol-yne coupling reaction. Bioconjug Chem (2016) 4: 911-917.

[0215] Three-dimensional protein structure analysis of tetrameric E. coli L-asparaginase II was used to identify locations for introducing paired cysteine ​​residues at or near potential immunogenic or antigenic sites where PEGylation can shield the site without affecting one or more of its active sites. Paired cysteines can be introduced within the same domain of the tetrameric protein or across domains. For example, Table 5 provides a list of combinations of paired cysteine ​​residues (C1 and C2) that can be introduced into tetrameric E. coli asparaginase within the same domain or across domains. For example, Figure 4 Shown is a hypothetical three-dimensional structure of a variant E. coli asparaginase polypeptide (SEQ ID NO: 12) with a cysteine ​​pair introduced at positions 8 and 32 of SEQ ID NO: 11. Additional cysteine ​​pairs are identified in the figure.

[0216] Table 5.

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225] A third method for introducing specific PEGylation sites into E. coli asparaginase involves introducing one or more additional lysine residues at surface exposed positions at or in close spatial proximity to potential immunogenic or antigenic sites, such as at or in close spatial proximity to amino acid positions 55-58, 114-119, 201-207, and 252-258 of the mature amino acid sequence (SEQ ID NO: 11). For example, variant E. coli asparaginase polypeptides having one or more of the following lysine substitutions can be prepared: S58K, R116K, and / or D240K.

[0226] Example 8

[0227] Variant Escherichia coli K-12 asparaginase polypeptide is an E. coli L-asparaginase that has been indicated for the treatment of ALL and has been prescribed by veterinarians for the treatment of lymphoma in dogs and cats. Derived from E. coli strain K-12, and has five cysteine ​​residues. The precursor amino acid sequence is provided as SEQ ID NO: 13, and the mature sequence is provided as SEQ ID NO: 14. Use tetramer The three-dimensional protein structure analysis of SEQ ID NO:14 is used to identify that the two cysteines at positions 67 and 200 may form a disulfide bond, while the remaining three cysteines at positions 45, 67 and 242 of SEQ ID NO:14 may be unpaired. In order to reduce the possibility of protein aggregation and / or reactivity with other molecules, one, two or all three of the unpaired cysteines may be replaced by other amino acids (such as serine). For example, a variant Escherichia coli asparaginase (SEQ ID NO:15) in which three unpaired cysteines are replaced by serine at amino acid positions 45, 67 and 242 of SEQ ID NO:14 can be prepared. Asparaginase polypeptides with one or more unpaired cysteine ​​residues (e.g., polypeptides with an amino acid sequence of SEQ ID NO:14 or SEQ ID NO:14 in which only one or two cysteine ​​residues are substituted) can be used for conjugation. Sequence Listing <110> Kindred Biosciences, Inc. <120> Variant asparaginase polypeptides for medical use <130> 01157-0013-00PCT <150> US 62 / 660,189 <151> 2018-04-19 <160> 15 <170> PatentIn Version 3.5 <210> 1 <211> 348 <212> PRT <213> Dickeya chrysanthemi <400> 1 Met Glu Arg Trp Phe Lys Ser Leu Phe Val Leu Val Leu Phe Phe Val 1 5 10 15 Phe Thr Ala Ser Ala Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala 20 25 30 Thr Gly Gly Thr Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr 35 40 45 Gly Tyr Lys Ala Gly Ala Leu Gly Val Asp Thr Leu Ile Asn Ala Val 50 55 60 Pro Glu Val Lys Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn 65 70 75 80 Met Ala Ser Glu Asn Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln 85 90 95 Arg Val Asn Glu Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile 100 105 110 Thr His Gly Thr Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu 115 120 125 Thr Val Lys Ser Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro 130 135 140 Ala Thr Ala Ile Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val 145 150 155 160 Arg Val Ala Gly Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val 165 170 175 Ile Asn Asp Arg Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala 180 185 190 Ser Thr Leu Asp Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val 195 200 205 Ile Ile Gly Asn Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His 210 215 220 Thr Thr Arg Ser Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys 225 230 235 240 Val Asp Ile Leu Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp 245 250 255 Ala Ala Ile Gln His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly 260 265 270 Ala Gly Ser Val Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu 275 280 285 Glu Lys Gly Val Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile 290 295 300 Val Pro Pro Asp Glu Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn 305 310 315 320 Pro Ala His Ala Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser 325 330 335 Asp Pro Lys Val Ile Gln Glu Tyr Phe His Thr Tyr 340 345 <210> 2 <211> 327 <212> PRT <213> Erwinia chrysanthemi <400> 2 Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr Ile 1 5 10 15 Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala Gly 20 25 30 Ala Leu Gly Val Asp Thr Leu Ile Asn Ala Val Pro Glu Val Lys Lys 35 40 45 Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu Asn 50 55 60 Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu Leu 65 70 75 80 Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr Asp 85 90 95 Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser Asp 100 105 110 Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile Ser 115 120 125 Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly Asp 130 135 140 Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg Ile 145 150 155 160 Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp Thr 165 170 175 Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn Arg 180 185 190 Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser Val 195 200 205 Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu Tyr 210 215 220 Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln His 225 230 235 240 Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val Ser 245 250 255 Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu Glu Lys Gly Val Val 260 265 270 Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp Glu 275 280 285 Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala Arg 290 295 300 Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val Ile 305 310 315 320 Gln Glu Tyr Phe His Thr Tyr 325 <210> 3 <211> 328 <212> PRT <213> Artificial sequence <220> <223> Variant Erwinia chrysanthemi asparaginase without leader sequence for intracellular expression <400> 3 Met Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr 1 5 10 15 Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala 20 25 30 Gly Ala Leu Gly Val Asp Thr Leu Ile Asn Ala Val Pro Glu Val Lys 35 40 45 Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu 50 55 60 Asn Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu 65 70 75 80 Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr 85 90 95 Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 100 105 110 Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 115 120 125 Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly 130 135 140 Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg 145 150 155 160 Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp 165 170 175 Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn 180 185 190 Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser 195 200 205 Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 210 215 220 Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln 225 230 235 240 His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val 245 250 255 Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu Glu Lys Gly Val 260 265 270 Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp 275 280 285 Glu Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala 290 295 300 Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val 305 310 315 320 Ile Gln Glu Tyr Phe His Thr Tyr 325 <210> 4 <211> 327 <212> PRT <213> artificial sequence <220> <223> Variant Erwinia chrysanthemi asparaginase mature sequence with three cysteine ​​substitutions <400> 4 Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr Ile 1 5 10 15 Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala Gly 20 25 30 Ala Leu Gly Val Asp Thr Leu Ile Cys Ala Val Pro Glu Val Lys Lys 35 40 45 Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu Asn 50 55 60 Met Thr Gly Asp Val Val Leu Cys Leu Ser Gln Arg Val Asn Glu Leu 65 70 75 80 Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr Asp 85 90 95 Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser Asp 100 105 110 Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile Ser 115 120 125 Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly Asp 130 135 140 Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg Ile 145 150 155 160 Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp Thr 165 170 175 Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn Arg 180 185 190 Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser Val 195 200 205 Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu Tyr 210 215 220 Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln His 225 230 235 240 Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val Ser 245 250 255 Val Arg Gly Ile Ala Gly Met Arg Cys Ala Leu Glu Lys Gly Val Val 260 265 270 Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp Glu 275 280 285 Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala Arg 290 295 300 Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val Ile 305 310 315 320 Gln Glu Tyr Phe His Thr Tyr 325 <210> 5 <211> 336 <212> PRT <213> Artificial sequence <220> <223> Variant Erwinia chrysanthemi asparaginase mature sequence with three cysteine ​​substitutions and an N-terminal poly-His tag <400> 5 Met Ala His His His His His His Ala Asp Lys Leu Pro Asn Ile 1 5 10 15 Val Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Ser Ala Ala Thr Gly 20 25 30 Thr Gln Thr Thr Gly Tyr Lys Ala Gly Ala Leu Gly Val Asp Thr Leu 35 40 45 Ile Cys Ala Val Pro Glu Val Lys Lys Leu Ala Asn Val Lys Gly Glu 50 55 60 Gln Phe Ser Asn Met Ala Ser Glu Asn Met Thr Gly Asp Val Val Leu 65 70 75 80 Cys Leu Ser Gln Arg Val Asn Glu Leu Leu Ala Arg Asp Asp Val Asp 85 90 95 Gly Val Val Ile Thr His Gly Thr Asp Thr Val Glu Glu Ser Ala Tyr 100 105 110 Phe Leu His Leu Thr Val Lys Ser Asp Lys Pro Val Val Phe Val Ala 115 120 125 Ala Met Arg Pro Ala Thr Ala Ile Ser Ala Asp Gly Pro Met Asn Leu 130 135 140 Leu Glu Ala Val Arg Val Ala Gly Asp Lys Gln Ser Arg Gly Arg Gly 145 150 155 160 Val Met Val Val Ile Asn Asp Arg Ile Gly Ser Ala Arg Tyr Ile Thr 165 170 175 Lys Thr Asn Ala Ser Thr Leu Asp Thr Phe Arg Ala Asn Glu Glu Gly 180 185 190 Tyr Leu Gly Val Ile Ile Gly Asn Arg Ile Tyr Tyr Gln Asn Arg Ile 195 200 205 Asp Lys Leu His Thr Thr Arg Ser Val Phe Asp Val Arg Gly Leu Thr 210 215 220 Ser Leu Pro Lys Val Asp Ile Leu Tyr Gly Tyr Gln Asp Asp Pro Glu 225 230 235 240 Tyr Leu Tyr Asp Ala Ala Ile Gln His Gly Val Lys Gly Ile Val Tyr 245 250 255 Ala Gly Met Gly Ala Gly Ser Val Ser Val Arg Gly Ile Ala Gly Met 260 265 270 Arg Cys Ala Leu Glu Lys Gly Val Val Val Met Arg Ser Thr Arg Thr 275 280 285 Gly Asn Gly Ile Val Pro Pro Asp Glu Glu Leu Pro Gly Leu Val Ser 290 295 300 Asp Ser Leu Asn Pro Ala His Ala Arg Ile Leu Leu Met Leu Ala Leu 305 310 315 320 Thr Arg Thr Ser Asp Pro Lys Val Ile Gln Glu Tyr Phe His Thr Tyr 325 330 335 <210> 6 <211> 336 <212> PRT <213> Artificial sequence <220> <223> Variant Erwinia chrysanthemi asparaginase sequence with N-terminal poly-His tag <400> 6 Met Ala His His His His His His Ala Asp Lys Leu Pro Asn Ile 1 5 10 15 Val Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Ser Ala Ala Thr Gly 20 25 30 Thr Gln Thr Thr Gly Tyr Lys Ala Gly Ala Leu Gly Val Asp Thr Leu 35 40 45 Ile Asn Ala Val Pro Glu Val Lys Lys Leu Ala Asn Val Lys Gly Glu 50 55 60 Gln Phe Ser Asn Met Ala Ser Glu Asn Met Thr Gly Asp Val Val Leu 65 70 75 80 Lys Leu Ser Gln Arg Val Asn Glu Leu Leu Ala Arg Asp Asp Val Asp 85 90 95 Gly Val Val Ile Thr His Gly Thr Asp Thr Val Glu Glu Ser Ala Tyr 100 105 110 Phe Leu His Leu Thr Val Lys Ser Asp Lys Pro Val Val Phe Val Ala 115 120 125 Ala Met Arg Pro Ala Thr Ala Ile Ser Ala Asp Gly Pro Met Asn Leu 130 135 140 Leu Glu Ala Val Arg Val Ala Gly Asp Lys Gln Ser Arg Gly Arg Gly 145 150 155 160 Val Met Val Val Ile Asn Asp Arg Ile Gly Ser Ala Arg Tyr Ile Thr 165 170 175 Lys Thr Asn Ala Ser Thr Leu Asp Thr Phe Arg Ala Asn Glu Glu Gly 180 185 190 Tyr Leu Gly Val Ile Ile Gly Asn Arg Ile Tyr Tyr Gln Asn Arg Ile 195 200 205 Asp Lys Leu His Thr Thr Arg Ser Val Phe Asp Val Arg Gly Leu Thr 210 215 220 Ser Leu Pro Lys Val Asp Ile Leu Tyr Gly Tyr Gln Asp Asp Pro Glu 225 230 235 240 Tyr Leu Tyr Asp Ala Ala Ile Gln His Gly Val Lys Gly Ile Val Tyr 245 250 255 Ala Gly Met Gly Ala Gly Ser Val Ser Val Arg Gly Ile Ala Gly Met 260 265 270 Arg Lys Ala Leu Glu Lys Gly Val Val Val Met Arg Ser Thr Arg Thr 275 280 285 Gly Asn Gly Ile Val Pro Pro Asp Glu Glu Leu Pro Gly Leu Val Ser 290 295 300 Asp Ser Leu Asn Pro Ala His Ala Arg Ile Leu Leu Met Leu Ala Leu 305 310 315 320 Thr Arg Thr Ser Asp Pro Lys Val Ile Gln Glu Tyr Phe His Thr Tyr 325 330 335 <210> 7 <211> 328 <212> PRT <213> Artificial sequence <220> <223> Variant Erwinia chrysanthemi asparaginase mature sequence with four cysteine ​​substitutions <400> 7 Met Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr 1 5 10 15 Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala 20 25 30 Gly Ala Leu Gly Val Asp Thr Leu Ile Cys Ala Val Pro Glu Val Lys 35 40 45 Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu 50 55 60 Asn Met Thr Gly Asp Val Val Leu Cys Leu Ser Gln Arg Val Asn Glu 65 70 75 80 Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr 85 90 95 Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 100 105 110 Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 115 120 125 Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly 130 135 140 Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg 145 150 155 160 Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp 165 170 175 Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn 180 185 190 Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser 195 200 205 Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 210 215 220 Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln 225 230 235 240 His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val 245 250 255 Ser Val Arg Gly Ile Ala Gly Met Arg Cys Ala Leu Glu Lys Gly Val 260 265 270 Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp 275 280 285 Cys Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala 290 295 300 Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val 305 310 315 320 Ile Gln Glu Tyr Phe His Thr Tyr 325 <210> 8 <211> 328 <212> PRT <213> Artificial sequence <220> <223> Variant Erwinia chrysanthemi asparaginase mature sequence with 1 lysine substitution <400> 8 Met Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr 1 5 10 15 Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala 20 25 30 Gly Ala Leu Gly Val Asp Thr Leu Ile Lys Ala Val Pro Glu Val Lys 35 40 45 Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu 50 55 60 Asn Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu 65 70 75 80 Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr 85 90 95 Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 100 105 110 Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 115 120 125 Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly 130 135 140 Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg 145 150 155 160 Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp 165 170 175 Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn 180 185 190 Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser 195 200 205 Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 210 215 220 Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln 225 230 235 240 His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val 245 250 255 Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu Glu Lys Gly Val 260 265 270 Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp 275 280 285 Glu Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala 290 295 300 Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val 305 310 315 320 Ile Gln Glu Tyr Phe His Thr Tyr 325 <210> 9 <211> 328 <212> PRT <213> Artificial sequence <220> <223> Variant Erwinia chrysanthemi asparaginase mature sequence with two lysine substitutions <400> 9 Met Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr 1 5 10 15 Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala 20 25 30 Gly Ala Leu Gly Val Asp Thr Leu Ile Lys Ala Val Pro Glu Val Lys 35 40 45 Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu 50 55 60 Asn Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu 65 70 75 80 Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr 85 90 95 Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 100 105 110 Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 115 120 125 Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly 130 135 140 Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg 145 150 155 160 Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp 165 170 175 Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn 180 185 190 Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser 195 200 205 Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 210 215 220 Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln 225 230 235 240 His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val 245 250 255 Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu Glu Lys Gly Val 260 265 270 Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp 275 280 285 Lys Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala 290 295 300 Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val 305 310 315 320 Ile Gln Glu Tyr Phe His Thr Tyr 325 <210> 10 <211> 348 <212> PRT <213> Escherichia coli <400> 10 Met Glu Phe Phe Lys Lys Thr Ala Leu Ala Ala Leu Val Met Gly Phe 1 5 10 15 Ser Gly Ala Ala Leu Ala Leu Pro Asn Ile Thr Ile Leu Ala Thr Gly 20 25 30 Gly Thr Ile Ala Gly Gly Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr 35 40 45 Val Gly Lys Val Gly Val Glu Asn Leu Val Asn Ala Val Pro Gln Leu 50 55 60 Lys Asp Ile Ala Asn Val Lys Gly Glu Gln Val Val Asn Ile Gly Ser 65 70 75 80 Gln Asp Met Asn Asp Asn Val Trp Leu Thr Leu Ala Lys Lys Ile Asn 85 90 95 Thr Asp Cys Asp Lys Thr Asp Gly Phe Val Ile Thr His Gly Thr Asp 100 105 110 Thr Met Glu Glu Thr Ala Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp 115 120 125 Lys Pro Val Val Met Val Gly Ala Met Arg Pro Ser Thr Ser Met Ser 130 135 140 Ala Asp Gly Pro Phe Asn Leu Tyr Asn Ala Val Val Thr Ala Ala Asp 145 150 155 160 Lys Ala Ser Ala Asn Arg Gly Val Leu Val Val Met Asn Asp Thr Val 165 170 175 Leu Asp Gly Arg Asp Val Thr Lys Thr Asn Thr Thr Asp Val Ala Thr 180 185 190 Phe Lys Ser Val Asn Tyr Gly Pro Leu Gly Tyr Ile His Asn Gly Lys 195 200 205 Ile Asp Tyr Gln Arg Thr Pro Ala Arg Lys His Thr Ser Asp Thr Pro 210 215 220 Phe Asp Val Ser Lys Leu Asn Glu Leu Pro Lys Val Gly Ile Val Tyr 225 230 235 240 Asn Tyr Ala Asn Ala Ser Asp Leu Pro Ala Lys Ala Leu Val Asp Ala 245 250 255 Gly Tyr Asp Gly Ile Val Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr 260 265 270 Lys Ser Val Phe Asp Thr Leu Ala Thr Ala Ala Lys Thr Gly Thr Ala 275 280 285 Val Val Arg Ser Ser Arg Val Pro Thr Gly Ala Thr Thr Gln Asp Ala 290 295 300 Glu Val Asp Asp Ala Lys Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn 305 310 315 320 Pro Gln Lys Ala Arg Val Leu Leu Gln Leu Ala Leu Thr Gln Thr Lys 325 330 335 Asp Pro Gln Gln Ile Gln Gln Ile Phe Asn Gln Tyr 340 345 <210> 11 <211> 326 <212> PRT <213> Escherichia coli <400> 11 Leu Pro Asn Ile Thr Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Gly 1 5 10 15 Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr Val Gly Lys Val Gly Val 20 25 30 Glu Asn Leu Val Asn Ala Val Pro Gln Leu Lys Asp Ile Ala Asn Val 35 40 45 Lys Gly Glu Gln Val Val Asn Ile Gly Ser Gln Asp Met Asn Asp Asn 50 55 60 Val Trp Leu Thr Leu Ala Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr 65 70 75 80 Asp Gly Phe Val Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala 85 90 95 Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val 100 105 110 Gly Ala Met Arg Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn 115 120 125 Leu Tyr Asn Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg 130 135 140 Gly Val Leu Val Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val 145 150 155 160 Thr Lys Thr Asn Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr 165 170 175 Gly Pro Leu Gly Tyr Ile His Asn Gly Lys Ile Asp Tyr Gln Arg Thr 180 185 190 Pro Ala Arg Lys His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu 195 200 205 Asn Glu Leu Pro Lys Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser 210 215 220 Asp Leu Pro Ala Lys Ala Leu Val Asp Ala Gly Tyr Asp Gly Ile Val 225 230 235 240 Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr Lys Ser Val Phe Asp Thr 245 250 255 Leu Ala Thr Ala Ala Lys Thr Gly Thr Ala Val Val Arg Ser Ser Arg 260 265 270 Val Pro Thr Gly Ala Thr Thr Gln Asp Ala Glu Val Asp Asp Ala Lys 275 280 285 Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn Pro Gln Lys Ala Arg Val 290 295 300 Leu Leu Gln Leu Ala Leu Thr Gln Thr Lys Asp Pro Gln Gln Ile Gln 305 310 315 320 Gln Ile Phe Asn Gln Tyr 325 <210> 12 <211> 326 <212> PRT <213> Artificial sequence <220> <223> Variant Escherichia coli asparaginase mature sequence with an additional cysteine ​​pair <400> 12 Leu Pro Asn Ile Thr Ile Leu Cys Thr Gly Gly Thr Ile Ala Gly Gly 1 5 10 15 Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr Val Gly Lys Val Gly Cys 20 25 30 Glu Asn Leu Val Asn Ala Val Pro Gln Leu Lys Asp Ile Ala Asn Val 35 40 45 Lys Gly Glu Gln Val Val Asn Ile Gly Ser Gln Asp Met Asn Asp Asn 50 55 60 Val Trp Leu Thr Leu Ala Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr 65 70 75 80 Asp Gly Phe Val Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala 85 90 95 Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val 100 105 110 Gly Ala Met Arg Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn 115 120 125 Leu Tyr Asn Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg 130 135 140 Gly Val Leu Val Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val 145 150 155 160 Thr Lys Thr Asn Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr 165 170 175 Gly Pro Leu Gly Tyr Ile His Asn Gly Lys Ile Asp Tyr Gln Arg Thr 180 185 190 Pro Ala Arg Lys His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu 195 200 205 Asn Glu Leu Pro Lys Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser 210 215 220 Asp Leu Pro Ala Lys Ala Leu Val Asp Ala Gly Tyr Asp Gly Ile Val 225 230 235 240 Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr Lys Ser Val Phe Asp Thr 245 250 255 Leu Ala Thr Ala Ala Lys Thr Gly Thr Ala Val Val Arg Ser Ser Arg 260 265 270 Val Pro Thr Gly Ala Thr Thr Gln Asp Ala Glu Val Asp Asp Ala Lys 275 280 285 Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn Pro Gln Lys Ala Arg Val 290 295 300 Leu Leu Gln Leu Ala Leu Thr Gln Thr Lys Asp Pro Gln Gln Ile Gln 305 310 315 320 Gln Ile Phe Asn Gln Tyr 325 <210> 13 <211> 321 <212> PRT <213> Escherichia coli <400> 13 Met Gly Lys Ala Val Ile Ala Ile His Gly Gly Ala Gly Ala Ile Ser 1 5 10 15 Arg Ala Gln Met Ser Leu Gln Gln Glu Leu Arg Tyr Ile Glu Ala Leu 20 25 30 Ser Ala Ile Val Glu Thr Gly Gln Lys Met Leu Glu Ala Gly Glu Ser 35 40 45 Ala Leu Asp Val Val Thr Glu Ala Val Arg Leu Leu Glu Glu Cys Pro 50 55 60 Leu Phe Asn Ala Gly Ile Gly Ala Val Phe Thr Arg Asp Glu Thr His 65 70 75 80 Glu Leu Asp Ala Cys Val Met Asp Gly Asn Thr Leu Lys Ala Gly Ala 85 90 95 Val Ala Gly Val Ser His Leu Arg Asn Pro Val Leu Ala Ala Arg Leu 100 105 110 Val Met Glu Gln Ser Pro His Val Met Met Ile Gly Glu Gly Ala Glu 115 120 125 Asn Phe Ala Phe Ala Arg Gly Met Glu Arg Val Ser Pro Glu Ile Phe 130 135 140 Ser Thr Ser Leu Arg Tyr Glu Gln Leu Leu Ala Ala Arg Lys Glu Gly 145 150 155 160 Ala Thr Val Leu Asp His Ser Gly Ala Pro Leu Asp Glu Lys Gln Lys 165 170 175 Met Gly Thr Val Gly Ala Val Ala Leu Asp Leu Asp Gly Asn Leu Ala 180 185 190 Ala Ala Thr Ser Thr Gly Gly Met Thr Asn Lys Leu Pro Gly Arg Val 195 200 205 Gly Asp Ser Pro Leu Val Gly Ala Gly Cys Tyr Ala Asn Asn Ala Ser 210 215 220 Val Ala Val Ser Cys Thr Gly Thr Gly Glu Val Phe Ile Arg Ala Leu 225 230 235 240 Ala Ala Tyr Asp Ile Ala Ala Leu Met Asp Tyr Gly Gly Leu Ser Leu 245 250 255 Ala Glu Ala Cys Glu Arg Val Val Met Glu Lys Leu Pro Ala Leu Gly 260 265 270 Gly Ser Gly Gly Leu Ile Ala Ile Asp His Glu Gly Asn Val Ala Leu 275 280 285 Pro Phe Asn Thr Glu Gly Met Tyr Arg Ala Trp Gly Tyr Ala Gly Asp 290 295 300 Thr Pro Thr Thr Gly Ile Tyr Arg Glu Lys Gly Asp Thr Val Ala Thr 305 310 315 320 Gln <210> 14 <211> 303 <212> PRT <213> Escherichia coli <400> 14 Gln Met Ser Leu Gln Gln Glu Leu Arg Tyr Ile Glu Ala Leu Ser Ala 1 5 10 15 Ile Val Glu Thr Gly Gln Lys Met Leu Glu Ala Gly Glu Ser Ala Leu 20 25 30 Asp Val Val Thr Glu Ala Val Arg Leu Leu Glu Glu Cys Pro Leu Phe 35 40 45 Asn Ala Gly Ile Gly Ala Val Phe Thr Arg Asp Glu Thr His Glu Leu 50 55 60 Asp Ala Cys Val Met Asp Gly Asn Thr Leu Lys Ala Gly Ala Val Ala 65 70 75 80 Gly Val Ser His Leu Arg Asn Pro Val Leu Ala Ala Arg Leu Val Met 85 90 95 Glu Gln Ser Pro His Val Met Met Ile Gly Glu Gly Ala Glu Asn Phe 100 105 110 Ala Phe Ala Arg Gly Met Glu Arg Val Ser Pro Glu Ile Phe Ser Thr 115 120 125 Ser Leu Arg Tyr Glu Gln Leu Leu Ala Ala Arg Lys Glu Gly Ala Thr 130 135 140 Val Leu Asp His Ser Gly Ala Pro Leu Asp Glu Lys Gln Lys Met Gly 145 150 155 160 Thr Val Gly Ala Val Ala Leu Asp Leu Asp Gly Asn Leu Ala Ala Ala 165 170 175 Thr Ser Thr Gly Gly Met Thr Asn Lys Leu Pro Gly Arg Val Gly Asp 180 185 190 Ser Pro Leu Val Gly Ala Gly Cys Tyr Ala Asn Asn Ala Ser Val Ala 195 200 205 Val Ser Cys Thr Gly Thr Gly Glu Val Phe Ile Arg Ala Leu Ala Ala 210 215 220 Tyr Asp Ile Ala Ala Leu Met Asp Tyr Gly Gly Leu Ser Leu Ala Glu 225 230 235 240 Ala Cys Glu Arg Val Val Met Glu Lys Leu Pro Ala Leu Gly Gly Ser 245 250 255 Gly Gly Leu Ile Ala Ile Asp His Glu Gly Asn Val Ala Leu Pro Phe 260 265 270 Asn Thr Glu Gly Met Tyr Arg Ala Trp Gly Tyr Ala Gly Asp Thr Pro 275 280 285 Thr Thr Gly Ile Tyr Arg Glu Lys Gly Asp Thr Val Ala Thr Gln 290 295 300 <210> 15 <211> 303 <212> PRT <213> Artificial sequence <220> <223> Variant Escherichia coli asparaginase mature sequence without unpaired cysteine <400> 15 Gln Met Ser Leu Gln Gln Glu Leu Arg Tyr Ile Glu Ala Leu Ser Ala 1 5 10 15 Ile Val Glu Thr Gly Gln Lys Met Leu Glu Ala Gly Glu Ser Ala Leu 20 25 30 Asp Val Val Thr Glu Ala Val Arg Leu Leu Glu Glu Ser Pro Leu Phe 35 40 45 Asn Ala Gly Ile Gly Ala Val Phe Thr Arg Asp Glu Thr His Glu Leu 50 55 60 Asp Ala Cys Val Met Asp Gly Asn Thr Leu Lys Ala Gly Ala Val Ala 65 70 75 80 Gly Val Ser His Leu Arg Asn Pro Val Leu Ala Ala Arg Leu Val Met 85 90 95 Glu Gln Ser Pro His Val Met Met Ile Gly Glu Gly Ala Glu Asn Phe 100 105 110 Ala Phe Ala Arg Gly Met Glu Arg Val Ser Pro Glu Ile Phe Ser Thr 115 120 125 Ser Leu Arg Tyr Glu Gln Leu Leu Ala Ala Arg Lys Glu Gly Ala Thr 130 135 140 Val Leu Asp His Ser Gly Ala Pro Leu Asp Glu Lys Gln Lys Met Gly 145 150 155 160 Thr Val Gly Ala Val Ala Leu Asp Leu Asp Gly Asn Leu Ala Ala Ala 165 170 175 Thr Ser Thr Gly Gly Met Thr Asn Lys Leu Pro Gly Arg Val Gly Asp 180 185 190 Ser Pro Leu Val Gly Ala Gly Cys Tyr Ala Asn Asn Ala Ser Val Ala 195 200 205 Val Ser Ser Thr Gly Thr Gly Glu Val Phe Ile Arg Ala Leu Ala Ala 210 215 220 Tyr Asp Ile Ala Ala Leu Met Asp Tyr Gly Gly Leu Ser Leu Ala Glu 225 230 235 240 Ala Ser Glu Arg Val Val Met Glu Lys Leu Pro Ala Leu Gly Gly Ser 245 250 255 Gly Gly Leu Ile Ala Ile Asp His Glu Gly Asn Val Ala Leu Pro Phe 260 265 270 Asn Thr Glu Gly Met Tyr Arg Ala Trp Gly Tyr Ala Gly Asp Thr Pro 275 280 285 Thr Thr Gly Ile Tyr Arg Glu Lys Gly Asp Thr Val Ala Thr Gln 290 295 300

Claims

1. An asparaginase polypeptide variant, which consists of the amino acid sequence of SEQ ID NO: 4 or 5.

2. The asparaginase polypeptide variant according to claim 1, wherein the variant is different from the corresponding wild-type Erwinia chrysanthemi ( E. chrysanthemi ) asparaginase polypeptide, the asparaginase polypeptide variant is less immunogenic or antigenic when conjugated to PEG.

3. The asparaginase polypeptide variant of claim 1, wherein 30% or more of the PEG-conjugable amino acids are surface exposed as determined by standard protein modeling software.

4. The asparaginase polypeptide variant of claim 1, wherein 35% or more of the at least one PEG-conjugable amino acid is surface exposed as determined by standard protein modeling software.

5. The asparaginase polypeptide variant according to claim 1, which consists of the amino acid sequence of SEQ ID NO:

5.

6. The asparaginase polypeptide variant according to claim 1, which consists of the amino acid sequence of SEQ ID NO:

4.

7. The asparaginase polypeptide variant according to claim 1, wherein the asparaginase polypeptide variant is sialylated or pegylated.

8. The asparaginase polypeptide variant according to claim 1, wherein the asparaginase polypeptide variant is pegylated.

9. The asparaginase polypeptide variant according to claim 1, wherein the asparaginase polypeptide variant is thiol-PEGylated.

10. A tetramer comprising the asparaginase polypeptide variant according to any one of claims 1 to 9.

11. An isolated nucleic acid encoding the asparaginase polypeptide variant according to any one of claims 1 to 9. A vector comprising the nucleic acid according to claim 11 .

13. A host cell comprising the vector according to claim 12, wherein the host cell does not comprise a plant cell.

14. The host cell of claim 13, wherein the cell is a prokaryotic cell.

15. The host cell of claim 14, wherein the prokaryotic cell is an Erwinia chrysanthemi cell, an Escherichia coli cell, or a Pseudomonas cell.

16. The host cell of claim 15, wherein the prokaryotic cell is an E. coli cell.

17. The host cell of claim 13, wherein the cell is a eukaryotic cell.

18. The host cell of claim 17, wherein the eukaryotic cell is a yeast cell.

19. A method for producing an asparaginase polypeptide variant, comprising culturing an Escherichia coli cell expressing the asparaginase polypeptide variant of claim 1.

20. A method for producing an asparaginase polypeptide variant, comprising culturing the host cell according to claim 13.

21. The method of claim 20, wherein the asparaginase polypeptide variant is isolated from a cell lysate.

22. The method of claim 20, wherein the asparaginase polypeptide variant is isolated from the periplasm.

23. The method of claim 20, wherein the asparaginase polypeptide variant is separated by cation exchange column chromatography, anion exchange column chromatography, mixed mode column chromatography and / or hydrophobic interaction column chromatography.

24. The method of claim 20, wherein the asparaginase polypeptide variant is combined with a reducing agent.

25. The method of claim 24, wherein the reducing agent is DTT.

26. The method of claim 24, wherein the asparaginase polypeptide variant is combined with a polyoxyethylene compound or a sialic acid compound.

27. The method according to claim 26, wherein the polyoxyethylene compound is α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy, polyoxyethylene or α-succinimidyloxyglutaryl-ω-methoxy, polyoxyethylene.

28. A pharmaceutical composition comprising the asparaginase polypeptide variant according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

29. A medicament comprising the asparaginase polypeptide variant according to any one of claims 1 to 9 for delivery to a subject via intramuscular route, intraperitoneal route, intravenous route or subcutaneous route.

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