Recombinant L-asparaginase

Abstract

This invention provides a method for treating diseases treatable by asparagine depletion in humans. [Solution] The method is 10 mg / m² 2 ~100mg / m 2 The method involves administering recombinant L-asparaginase to human subjects in a specified amount, wherein the recombinant L-asparaginase is a tetramer, the tetramer contains four monomers, and the monomers have an amino acid sequence containing a specific sequence.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to U.S. Provisional Application No. 62 / 926,201 filed on 25 October 2019 and U.S. Provisional Application No. 63 / 047,222 filed on 1 July 2020, which are incorporated herein by reference in their entirety.

[0002] This invention provides recombinant L-asparaginase and its use in therapy. [Background technology]

[0003] Proteins with L-asparagine aminohydrolase activity, commonly known as L-asparaginase, have been successfully used to treat a variety of potentially fatal diseases, including acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL). In these diseases, children constitute the majority of patients.

[0004] Bacterial L-asparaginases possess high immunogenicity and antigenicity. Currently available as first-line treatments are L-asparaginase and pegaspargase derived from E. coli. These products can cause adverse hypersensitivity reactions in patients, including allergic reactions, silent inactivation, and anaphylactic shock. Patients exhibiting hypersensitivity reactions to these products often have to discontinue administration, resulting in a poorer prognosis and survival rate. These patients have transitioned to Erwinaze® after experiencing hypersensitivity reactions. Erwinaze® has been reported to take up to nine months to prepare and has long suffered from supply problems (https: / / www.statnews.com / 2016 / 10 / 31 / cancer-drug-shortage / ). (See also https: / / www.drugs.com / drug-shortages / asparaginase-erwinia-chrysanthemi-482). The problem persists, and shortages of Erwinase® are common. (See also https: / / www.accessdata.fda.gov / scripts / drugshortages / dsp_ActiveIngredientDetails.cfm?AI=Asparaginase%20Erwinia%20Chrysanthemi%20(Erwinaze)&st=c). The FDA has issued a warning letter to manufacturers stating that "alterations of the raw material or cell line are likely to adversely affect the identity, potency, quality, purity, or potency of Erwinase®."

[0005] There is a need for immunogenically non-cross-reactive treatment options. Recombinant L-asparaginases that do not exhibit immunological cross-reactivity with E. coli-derived asparaginases (such as recombinant L-asparaginase recombinantly produced in Pseudomonas fluorescens) would address a critical medical need (as a component of multi-agent chemotherapy regimens) in patients with ALL / lymphoblastic lymphoma (LBL) by helping to ensure that asparaginase is available to patients who have developed hypersensitivity to E. coli-derived asparaginases. [Overview of the Initiative]

[0006] Accordingly, this disclosure provides a recombinant L-asparaginase that does not exhibit immunological cross-reactivity with asparaginase derived from E. coli.

[0007] In one embodiment, the disclosure relates to a method for treating a disease treatable by asparagine depletion in human subjects, wherein the method is approximately 10 mg / m² 2 ~about 100mg / m 2The present invention provides a method comprising administering recombinant L-asparaginase to a human subject in an amount, wherein the recombinant L-asparaginase is a tetramer, the tetramer has four monomers, and each monomer has an amino acid sequence containing SEQ ID NO: 1.

[0008] In some embodiments, recombinant L-asparaginase is not conjugated with a PEG moiety. In some embodiments, recombinant L-asparaginase is not conjugated with a peptide containing proline or alanine.

[0009] In some embodiments, human subjects have been previously treated with E. coli-derived asparaginase. In some further embodiments, the E. coli-derived asparaginase is conjugated to the PEG portion.

[0010] In some embodiments, human subjects have experienced silent inactivation of E. coli-derived asparaginase.

[0011] In some embodiments, human subjects have experienced allergic reactions to asparaginase derived from E. coli. In some embodiments, human subjects have experienced anaphylaxis to asparaginase derived from E. coli.

[0012] In some embodiments, the disease treatable by L-asparagine depletion is acute lymphoblastic leukemia (ALL). In some embodiments, the disease treatable by L-asparagine depletion is lymphoblastic lymphoma (LBL). In some embodiments, ALL or LBL is relapsed ALL or relapsed LBL.

[0013] In some embodiments, the recombinant L-asparaginase is administered three times a week. In some embodiments, the recombinant L-asparaginase is administered every other day for five consecutive days, followed by a drug holiday of two consecutive days. In some embodiments, the recombinant L-asparaginase is administered on Monday, Wednesday, and Friday of the same week. In some embodiments, the recombinant L-asparaginase is administered for two weeks.

[0014] In some embodiments, the recombinant L-asparaginase is administered intramuscularly. In some embodiments, the recombinant L-asparaginase is administered at about 25 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 15 mg / m 2 to about 45 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 27 mg / m 2 to about 37.5 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 25 mg / m 2 to about 80 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 37.5 mg / m 2 to about 80 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 37.5 mg / m 2 to about 65 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 25 mg / m 2 to about 37.5 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 30 mg / m 2 to about 75 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 35 mg / m 2 to about 70 mg / m 2 by intramuscular administration. In some embodiments, the recombinant L-asparaginase is administered at about 40 mg / m 2~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is [amount]. , about 40mg / m 2 ~about 75mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 45 mg / m². 2 ~about 70mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 50 mg / m². 2 ~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 55 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 40 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 55mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 47.5 mg / m². 2 ~about 50mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 35mg / m 2 It is administered intramuscularly in that amount.

[0015] In some embodiments, recombinant L-asparaginase is administered intravenously. In some embodiments, recombinant L-asparaginase is administered at approximately 37.5 mg / m². 2It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 27.5 mg / m². 2 ~about 47.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 21.5 mg / m². 2 ~about 38.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 37.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 100mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 80mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 35mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 95mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 35 mg / m². 2 ~about 90mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m². 2 ~about 85mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 80mg / m 2It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 50 mg / m². 2 ~about 75mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 55 mg / m². 2 ~about 70mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 60 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 60mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 35 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m². 2 ~about 50mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 42.5 mg / m². 2 ~about 57.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 75mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in that amount.

[0016] In some embodiments, the lowest serum asparaginase activity (NSAA) measured from serum samples derived from human subjects was 0.1 after administration and treatment with recombinant L-asparaginase. Equal to or greater than IU / mL.

[0017] In some embodiments, recombinant L-asparaginase is co-administered with one or more other chemotherapy agents as part of a multi-agent chemotherapy regimen.

[0018] In some embodiments, recombinant L-asparaginase exhibits aggregation of less than 6%. In some embodiments, recombinant L-asparaginase exhibits aggregation of less than 1%.

[0019] In some embodiments, recombinant L-asparaginase is not freeze-dried.

[0020] In another aspect, the Disclosure provides a method for treating a disease treatable by asparagine depletion in a human subject, the method comprising administering recombinant L-asparaginase to the human subject, wherein the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens, the recombinant L-asparaginase is a tetramer, the tetramer has four monomers, each monomer having an amino acid sequence including SEQ ID NO: 1.

[0021] In some embodiments, recombinant L-asparaginase is not conjugated with a PEG moiety. In some embodiments, recombinant L-asparaginase is not conjugated with a peptide containing proline or alanine.

[0022] In some embodiments, human subjects have been previously treated with E. coli-derived asparaginase. In some embodiments, E. coli-derived asparaginase is conjugated to the PEG portion.

[0023] In some embodiments, the human subject has experienced silent inactivation of E. coli-derived asparaginase.

[0024] In some embodiments, the human subject has experienced an allergic reaction to E. coli-derived asparaginase.

[0025] In some embodiments, the human subject has experienced anaphylaxis to E. coli-derived asparaginase.

[0026] In some embodiments, the disease treatable by L-asparagine depletion is acute lymphoblastic leukemia (ALL). In some embodiments, the disease treatable by L-asparagine depletion is lymphoblastic lymphoma (LBL). In some embodiments, the ALL or LBL is relapsed ALL or relapsed LBL.

[0027] In some embodiments, recombinant L-asparaginase is administered three times per week. In some embodiments, recombinant L-asparaginase is administered every other day for five consecutive days, followed by a two consecutive day drug holiday. In some embodiments, recombinant L-asparaginase is administered on Monday, Wednesday, and Friday of each week.

[0028] In some embodiments, recombinant L-asparaginase is administered for a period of two weeks.

[0029] In some embodiments, recombinant L-asparaginase is about 10 mg / m 2 to about 100 mg / m 2 is administered in an amount of

[0030] In some embodiments, recombinant L-asparaginase is administered intramuscularly. In some embodiments, recombinant L-asparaginase is about 25 mg / m 2is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 15 mg / m 2 to about 45 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 27 mg / m 2 to about 37.5 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 25 mg / m 2 to about 80 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 37.5 mg / m 2 to about 80 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 37.5 mg / m 2 to about 65 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 25 mg / m 2 to about 37.5 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 30 mg / m 2 to about 75 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 35 mg / m 2 to about 70 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 40 mg / m 2 to about 65 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 45 mg / m 2 to about 60 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 50 mg / m 2 to about 55 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 40 mg / m 2 to about 75 mg / m 2 is administered intramuscularly in an amount of. In some embodiments, the recombinant L-asparaginase is about 45 mg / m2 ~about 70mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 50 mg / m². 2 ~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 55 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 40 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 55mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 47.5 mg / m². 2 ~about 50mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 35mg / m 2 It is administered intramuscularly in that amount.

[0031] In some embodiments, recombinant L-asparaginase is administered intravenously. In some embodiments, recombinant L-asparaginase is administered at approximately 37.5 mg / m². 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 27.5 mg / m². 2 ~about 47.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 21.5 mg / m². 2 ~about 38.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 37.5mg / m 2It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 100mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 80mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 35mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 95mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 35 mg / m². 2 ~about 90mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m². 2 ~about 85mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 80mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 50 mg / m². 2 ~about 75mg / m 2 It is administered intravenously in an amount of 55 ml. In some embodiments, recombinant L-asparaginase is administered approximately 55 ml. g / m 2 ~about 70mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 60 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 60mg / m 2It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 35 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m². 2 ~about 50mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 42.5 mg / m². 2 ~about 57.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 75mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in that amount.

[0032] In some embodiments, the lowest serum asparaginase activity (NSAA) measured from serum samples derived from human subjects is equal to or greater than 0.1 IU / mL after treatment with recombinant L-asparaginase and administration.

[0033] In some embodiments, recombinant L-asparaginase is co-administered with one or more other chemotherapy agents as part of a multi-agent chemotherapy regimen.

[0034] In some embodiments, compositions containing recombinant L-asparaginase exhibit aggregation of less than 6% of the recombinant L-asparaginase protein. In some embodiments, compositions exhibit aggregation of less than 1% of the recombinant L-asparaginase protein.

[0035] In some embodiments, recombinant L-asparaginase is not freeze-dried, or the composition containing L-asparaginase is not a freeze-dried composition.

[0036] In another aspect, the Disclosure provides a method for treating a disease treatable by asparagine depletion in a human subject, the method comprising administering a composition comprising recombinant L-asparaginase to a human subject, wherein the recombinant L-asparaginase is a tetramer, the tetramer has four monomers, and the composition exhibits aggregation of less than 6% of the recombinant L-asparaginase protein.

[0037] In some embodiments, recombinant L-asparaginase is not conjugated with a PEG moiety. In some embodiments, recombinant L-asparaginase is not conjugated with a peptide containing proline or alanine.

[0038] In some embodiments, human subjects have been previously treated with E. coli-derived asparaginase. In some embodiments, E. coli-derived asparaginase is conjugated to the PEG portion.

[0039] In some embodiments, human subjects have experienced silent inactivation of E. coli-derived asparaginase. In some embodiments, human subjects have experienced allergic reactions to E. coli-derived asparaginase.

[0040] In some embodiments, human subjects have experienced anaphylaxis to asparaginase derived from E. coli.

[0041] In some embodiments, the disease treatable by L-asparagine depletion is acute lymphoblastic leukemia (ALL). In some embodiments, the disease treatable by L-asparagine depletion is lymphoblastic lymphoma (LBL). In some embodiments, ALL or LBL is relapsed ALL or relapsed LBL.

[0042] In some embodiments, recombinant L-asparaginase is administered three times a week. In some embodiments, recombinant L-asparaginase is administered every other day for five consecutive days, followed by a two-day rest period. In some embodiments, recombinant L-asparaginase is administered on Monday, Wednesday, and Friday of the same week.

[0043] In some embodiments, recombinant L-asparaginase is administered for two weeks.

[0044] In some embodiments, recombinant L-asparaginase is present at approximately 10 mg / m². 2 ~about 100mg / m 2 It is administered in that amount.

[0045] In some embodiments, recombinant L-asparaginase is administered intramuscularly. In some embodiments, recombinant L-asparaginase is administered at a dose of approximately 25 mg / m². 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 15 mg / m². 2 ~about 45mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 27 mg / m². 2 ~about 37.5mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 80mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 37.5 mg / m². 2 ~about 80mg / m 2It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 37.5 mg / m². 2 ~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 37.5mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 75mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 35 mg / m². 2 ~about 70mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 40 mg / m². 2 ~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 40 mg / m². 2 ~about 75mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 70mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 50 mg / m². 2 ~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 55 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 40 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m².2 ~about 55mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 47.5 mg / m². 2 ~about 50mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 35mg / m 2 It is administered intramuscularly in that amount.

[0046] In some embodiments, recombinant L-asparaginase is administered intravenously. In some embodiments, recombinant L-asparaginase is administered at approximately 37.5 mg / m². 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in the amount specified. In some embodiments, recombinant L-asparagus The enzyme concentration is approximately 27.5 mg / m². 2 ~about 47.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 21.5 mg / m². 2 ~about 38.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 37.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 100mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 25 mg / m². 2 ~about 80mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 35mg / m 2It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 95mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 35 mg / m². 2 ~about 90mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m². 2 ~about 85mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 80mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 50 mg / m². 2 ~about 75mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 55 mg / m². 2 ~about 70mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 60 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 60mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 35 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m². 2 ~about 50mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 42.5 mg / m². 2 ~about 57.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is administered at approximately 30 mg / m². 2 ~about 75mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 40 mg / m².2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, recombinant L-asparaginase is approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in that amount.

[0047] In some embodiments, the lowest serum asparaginase activity (NSAA) measured from serum samples derived from human subjects is equal to or greater than 0.1 IU / mL after administration and treatment with recombinant L-asparaginase.

[0048] In some embodiments, recombinant L-asparaginase is co-administered with one or more other chemotherapy agents as part of a multi-agent chemotherapy regimen.

[0049] In some embodiments, each monomer of the tetramer has an amino acid sequence containing SEQ ID NO: 1.

[0050] In some embodiments, recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens.

[0051] In some embodiments, the amount of aggregation shown by the composition containing recombinant L-asparaginase is less than 1%.

[0052] In some embodiments, recombinant L-asparaginase is not freeze-dried.

[0053] In some embodiments, the multi-drug chemotherapy regimen includes recombinant L-asparaginase and one additional chemotherapeutic agent.

[0054] In some embodiments, the multi-drug chemotherapy regimen includes recombinant L-asparaginase and two or more additional chemotherapeutic agents.

[0055] In one aspect, the present invention relates to a method for treating a disease in a human subject, wherein the method is approximately 10 mg / m² 2 ~about 100mg / m 2 The present invention provides a method comprising administering recombinant L-asparaginase to a human subject in an amount, wherein the recombinant L-asparaginase is a tetramer, the tetramer comprises four monomers, and each monomer has an amino acid sequence containing SEQ ID NO: 1.

[0056] In some embodiments, the disease is a member selected from sarcomas, breast cancer, metastatic breast cancer, liver cancer, gastric cancer, prostate cancer, colorectal cancer, and head and neck cancer.

[0057] In some embodiments, recombinant L-asparaginase is not conjugated with the PEG moiety.

[0058] In some embodiments, recombinant L-asparaginase is administered to human subjects as a substitute for long-acting asparaginase derived from E. coli.

[0059] In some embodiments, six doses of recombinant L-asparaginase are administered to human subjects instead of a single dose of long-acting asparaginase derived from E. coli.

[0060] In some embodiments, the long-acting asparaginase derived from E. coli is pegaspargase.

[0061] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle comprising a first dose, a second dose, and a third dose, the cycle being optionally repeatable, and the first, second, and third doses being administered at intervals of approximately 48 to 72 hours.

[0062] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle, which is optionally repeatable, and which comprises administration of recombinant L-asparaginase every other day for 5 consecutive days, followed by a 2 consecutive day rest period, with the first dose of the cycle being 25 mg / m². 2 Therefore, the second dose of that cycle is 25 mg / m². 2 Therefore, the third dose of that cycle was 37.5 mg / m². 2 This is followed by a two-day drug-free period.

[0063] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle, which is optionally repeatable, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days, followed by a two-day rest period, with the first dose of the cycle being 37.5 mg / m². 2 Therefore, the second dose of that cycle is 37.5 mg / m². 2 Therefore, the third dose of that cycle was 37.5 mg / m². 2 This is followed by a two-day drug-free period.

[0064] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle, which is optionally repeatable, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days, followed by a two-day rest period, with the first dose of the cycle being 37.5 mg / m². 2 This is the second time in that cycle. The dosage is 25 mg / m². 2 Therefore, the third dose of that cycle was 37.5 mg / m². 2 This is followed by a two-day drug-free period.

[0065] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle, which is optionally repeatable, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days, followed by a two-day rest period, with the first dose of the cycle being 37.5 mg / m². 2 Therefore, the second dose of that cycle is 25 mg / m². 2 Therefore, the third dose of that cycle is 25 mg / m². 2 This is followed by a two-day drug-free period.

[0066] In some embodiments, the first dose of the cycle is administered on Monday, the second dose of the cycle is administered on Wednesday, and the third dose of the cycle is administered on Friday.

[0067] The present invention may be best understood by the following modes for carrying out the invention, when read in conjunction with the accompanying drawings. The following drawings are included. [Brief explanation of the drawing]

[0068] [Figure 1] The results of the safety test for Example 2 are shown below. [Figure 2] The purity of recombinant L-asparaginase, recombinant chrysanthase derived from E. coli, and Erwinase®, as evaluated by the SEC-HPLC method outlined in Example 5, is shown. [Figure 3] The purity of recombinant L-asparaginase, recombinant chrysanthase derived from E. coli, and Erwinase®, as evaluated by the SEC-MALLS method outlined in Example 5, is shown. [Figure 4] The results obtained from the data analysis of the settling velocity AUC outlined in Example 5 are shown below. [Figure 5] This shows that the size profile of recombinant L-asparaginase was evaluated by SE-UHPLC according to the release method outlined in Example 6. [Figure 6]It shows that the high molecular weight species present in recombinant L-asparaginase were measured using SEC-MALLS outlined in Example 6. [Figure 7] It shows the normalized sedimentation coefficient distribution of recombinant L-asparaginase (commercial scale) outlined in Example 6. [Figure 8] It shows the normalized sedimentation coefficient distribution of Erwinase® (commercial scale) outlined in Example 6. [Figure 9] It shows the proportion of healthy subjects exhibiting SAA levels at 48 and 72 hours after administration according to Example 7. Abbreviations: ERW, Erwinia chrysanthemi asparaginase; IM, intramuscular; IV, intravenous; SAA, serum asparaginase activity. [Figure 10] It shows a summary of SAA-based PK in the study described in Example 7. Abbreviations: AUC0-inf, area under the curve extrapolated from time 0 to infinite time; AUC0-t, area under the curve from time 0 to the last time point at which SAA can be quantified; C48h, SAA at 48 hours; C72h, SAA at 72 hours; CL, clearance; Cmax, maximum SAA concentration; CV, coefficient of variation; ERW, Erwinia chrysanthemi asparaginase; IM, intramuscular; IV, intravenous; PK, pharmacokinetics; SAA, serum asparaginase activity; tmax, time point at which Cmax is observed; t1 / 2, half-life; V, volume of distribution. aFor IM administration, CL=CL / F (apparent clearance) and V=Vz / F (apparent volume of distribution). For IV administration, CL=CL and V=Vss (assessment of volume of distribution at steady state). bFor t1 / 2, AUC0-inf, CL, and V, n=5. N is the number of exposed healthy subjects. Mean values (CV%) are presented for all parameters except the tmax value, which is reported as median and range. [Figure 11] It shows TEAEs after treatment described in Example 7. Abbreviations: ERW, Erwinia chrysanthemi asparaginase; IM, intramuscular; IV, intravenous; TEAE, treatment-emergent adverse event. aPresented by preferred term using MedDRA terminology, version 22.0. Treatment-related TEAEs are presented in descending order of frequency. [Figure 12] Individual SAA-time profiles are shown. Abbreviations: ERW, *Erwinia chrysanthemi* asparaginase; IM, intramuscular; IV, intravenous; SAA, serum asparaginase activity. Further description can be found in Example 7. [Figure 13] Mean (95% CI) SAA-time profiles, as further described in Example 7, are shown. CI, confidence interval; IM, intramuscular; IV, intravenous; SAA, serum asparaginase activity. [Figure 14A] Mean SAA-time profiles for intramuscular injection of JZP-458 are shown. ERW, *Erwinia chrysanthemi* asparaginase; IM, intramuscular; SAC, serum asparaginase concentration. Note: lower limit of quantification (LLOQ) = 1.00 ng / mL. Values below the LLOQ were set to 0. [Figure 14B] Mean SAA-time profiles for intravenous injection of JZP-458 are shown. ERW, *Erwinia chrysanthemi* asparaginase; IV, intravenous; SAC, serum asparaginase concentration. Note: lower limit of quantification (LLOQ) = 1.00 ng / mL. Values below the LLOQ were set to 0. [Figure 14C] Individual asparaginase concentration-time profiles are shown. ERW, *Erwinia chrysanthemi* asparaginase; IM, intramuscular; IV, intravenous; SAC, serum asparaginase concentration. [Figure 14D] The correlation between SAC and SAA following intramuscular and intravenous administration of JZP-458 is shown. Abbreviations: IM, intramuscular; IV, intravenous; SAA, serum asparaginase activity; SAC, serum asparaginase concentration. Note: regression line equation: SAC = 1407.9 × SAA; Pearson correlation coefficient = 0.9779. Further description can be found in Example 7. [Figure 15]The mean SAA time profile and corresponding mean plasma L-asparagine levels are shown in the study of Example 7, which is further described below. IM: Intramuscular; IV: Intravenous; SAA: Serum asparaginase activity. Note: Lower limit of quantification (LLOQ): Asparaginase activity = 0.0250 IU / mL; L-asparagine = 0.0250 ug / mL. Values ​​below LLOQ were set to 0. [Figure 16] The baseline demographic characteristics of the study in Example 7 are shown. Abbreviations: ERW, Erwinia chrysanthemi asparaginase; IM, intramuscular; IV, intravenous; SD, standard deviation. a. Ethnicity was self-reported. Healthy subjects could identify as belonging to two or more ethnic groups. [Figure 17] The dose-proportionality evaluation of JZP-458 based on SAA, as further described in the study of Example 7, is shown. Abbreviations: AUC0-inf, area under the curve extrapolated from time 0 to infinite time; AUC0-t, area under the curve from time 0 to the last time point at which SAA quantification is possible; CI, confidence interval; Cmax, maximum SAA value; IM, intramuscular; IV, intravenous; PK, pharmacokinetics; SAA, serum asparaginase activity. The results are based on the following power model: ln(parameter) = intercept + slope × ln(dose). [Figure 18]The following is an overview of the PK based on SAC, which is further described in Example 7. Abbreviations: AUC0-inf, area under the curve extrapolated from time 0 to infinite time; AUC0-t, area under the curve from time 0 to the last time point when SAA can be quantified; CL, clearance; Cmax, maximum SAA value; CV, coefficient of variation; ERW, Erwinia chrysanthemi asparaginase; IM, intramuscular; IV, intravenous; PK, pharmacokinetics; t1 / 2, half-life; SAA, serum asparaginase activity; SAC, serum asparaginase concentration; tmax, time point when Cmax is observed; V, volume of distribution. a. For IM administration, CL = CL / F (apparent clearance) and V = Vz / F (apparent volume of distribution). For IV administration, CL = CL and V = Vss (assessment of volume of distribution at steady state). b. n=3 for AUC0-inf, CL, and V; n=5 for t1 / 2. N is the number of exposed healthy subjects. The mean (CV%) is shown for all parameters except the median and the tmax value, which is reported as a range. [Figure 19] This paper presents an evaluation of the dose-proportionality of JZP-458 based on SAC. Abbreviations: AUC0-inf, area under the curve extrapolated from time 0 to infinite time; AUC0-t, area under the curve from time 0 to the last time point at which SAA quantification is possible; CI, confidence interval; Cmax, maximum SAA value; IM, intramuscular; IV, intravenous; PK, pharmacokinetics; SAA, serum asparaginase activity; SAC, serum asparaginase concentration. The results are based on the following power model: ln(parameters) = intercept + slope × ln(dose). [Figure 20] The study design for Example 7 is shown. Abbreviations: ERW, Erwinia chrysanthemi asparaginase; IM, intramuscular; IV, intravenous; PK, pharmacokinetics. [Figure 21]The mean SAA time profile and corresponding mean plasma L-glutamine levels for all procedures further described in the study of Example 7 are shown. Abbreviations: IM, intramuscular; IV, intravenous; SAA, serum asparaginase activity. Note: Limit of quantification (LLOQ): Asparaginase activity = 0.0250 IU / mL; L-glutamine = 0.250 ug / mL. Values ​​less than LLOQ were set to 0. Abbreviations: IM, intramuscular; IV, intravenous; SAA, serum asparaginase activity. Note: Limit of quantification (LLOQ): Asparaginase activity = 0.0250 IU / mL; L-glutamine = 0.250 ug / mL. Values ​​less than LLOQ were set to 0. [Figure 22] The JZP-458 IM administration cohort (Example 8) in Part A is shown. The SDRC will evaluate safety and tolerability for participants in Cohort 1 to determine whether additional participants at different dose levels are needed or whether an appropriate JZP-458 IM dose level has been determined for advancing to an expanded cohort (Cohort 2). The SDRC will review NSAA and safety / tolerability data when 6 and 13 evaluable participants in each subcohort complete the first cycle. Enrollment will not be interrupted at any specific point for SDRC review. Abbreviations: IM = Intramuscular; IU = International Units; NSAA = Minimum Serum Asparaginase Activity; SDRC = Study Data Review Committee. [Figure 23] The JZP-458 administration cohort (Example 8) in Part B is shown. The SDRC will evaluate safety and tolerability for participants in Cohort 1 to determine if additional participants at different dose levels are needed. The SDRC will review NSAA and safety / tolerability data when six evaluable participants in each subcohort have completed the first cycle. Abbreviations: IU = International Units; IV = Intravenous; NSAA = Minimum Serum Asparaginase Activity; SDRC = Study Data Review Committee. [Figure 24] The results of SAA (IU / mL) in JZP-458 during the first cycle are summarized (evaluable participants) (Example 8). [Figure 25]This shows the proportion of patients with NSAA levels of 0.1 IU / mL or higher and 0.4 IU / mL or higher at the most recent 48 and 72 hours during the first cycle (evaluable participants) (Example 8). [Figure 26] The simulated median SAA profile of JZP-458, along with the 95% prediction interval, is shown on a semi-logarithmic scale (FMW administration schedule) (IM simulation model for Example 8). Abbreviations: IM = intramuscular; FMW = Friday, Monday, Wednesday; SAA = serum asparaginase activity. The centerline is the median. The bands (95% prediction interval) represent the 2.5 percentile (bottom) and 97.5 percentile (top). The x-axis is shown up to 336 hours with an additional offset. The plotted data include simulated observations up to 504 hours after the start of the cycle. [Figure 27] This shows the percentage of patients treated with JZP-458 who are expected to achieve the target SAA trough level (FMW dosing schedule) (IM simulation model for Example 8). [Figure 28] A summary of adverse events that occurred under treatment is shown (safety analysis population) (Example 8). [Figure 29] Adverse events occurring under Grade 3 or 4 treatment are shown (Safety Analysis Cohort 1a) (Example 8). [Figure 30] This shows adverse events that occurred under treatment of grade 3 or 4 (Safety Analysis Cohort 1a) (Example 8). Percentages were calculated using the number of participants in the safety analysis population as the denominator. Adverse events were coded to SOC and PT using MedDRA 22.1. The severity of AEs was recorded using CTCAE 5.0. TEAE was defined as any event that occurred on a date after the first dose of the study treatment up to the end of the study, or any ongoing event whose severity worsened on a date after the first dose of the study treatment up to the end of the study. Abbreviations: AE = Adverse Event; CTCAE = Common Terminology Criteria for Adverse Events; IM = Intramuscular Injection; PT = Basic Terminology; SOC = Organ-Specific Major Classification; TEAE = Adverse Event Occurring Under Treatment. [Modes for carrying out the invention]

[0069] Bacterial L-asparaginases possess high immunogenicity and antigenicity. These products can cause adverse hypersensitivity reactions in patients, including allergic reactions, silent inactivation, and anaphylactic shock. L-asparaginase is an enzyme with L-asparagine aminohydrolase activity. L-asparaginase enzyme activity can include not only the amidogenesis of asparagine to aspartic acid and ammonia, but also the amidogenesis of glutamine to glutamic acid and ammonia. L-asparaginases from E. coli and Erwinia chrysanthemi are commonly used to treat various diseases treated by asparagine depletion, including ALL and LBL. Healthy cells can produce asparagine, but some diseased cells cannot because they lack asparagine synthase. When L-asparaginase is administered to patients with the disease, it reduces the level of soluble asparagine, thereby starving diseased cells but not healthy cells, resulting in selective disease cell death. Antibodies produced after treatment with E. coli-derived L-asparaginase are found in Erwinia. It does not cross-react with L-asparaginase derived from chrysanthemi. L-asparaginase produced in Erwinia chrysanthemi requires a long manufacturing lead time (e.g., Erwinase®). The present invention provides a recombinant L-asparaginase that does not cross-react with L-asparaginase derived from E. coli, and offers improvements over L-asparaginase derived from E. coli and L-asparaginase derived from Erwinia chrysanthemi. These improvements are described herein.

[0070] I. Definition Unless explicitly defined otherwise, terms used herein shall be understood in accordance with their common meanings in the art.

[0071] As used herein, the term "disease treatable by asparagine depletion" refers to a condition or disorder, wherein cells involved in or causing the condition or disorder exhibit either a lack or a reduction in the ability to synthesize L-asparagine. Depletion or deficiency of L-asparagine can be partial or substantially complete (e.g., to an undetectable level using methods and equipment known in the art).

[0072] As used herein, the term "therapeutically effective amount" refers to the amount of a protein (e.g., asparaginase or a recombinant L-asparaginase thereof) required to produce a desired therapeutic effect.

[0073] The term "comprising the sequence of SEQ ID NO: 1" means that the amino acid sequence of the protein does not have to be strictly limited to SEQ ID NO: 1, and may contain additional amino acids.

[0074] The terms "subject" or "patient" refer to an animal, a mammal, or even a human patient.

[0075] The term "host cell or non-human host transformed with a vector" refers to a host cell or non-human host comprising a vector or nucleic acid described herein. Host cells for expression of polypeptides are well known in the art, and include prokaryotic cells as well as eukaryotic cells. Suitable culture media and conditions for the aforementioned host cells are known in the art.

[0076] "Culturing a host or host cell" includes expression of a protein in the host or host cell, including expression of a fusion protein as defined herein and / or a polypeptide as defined herein, and / or expression of asparaginase.

[0077] As used herein, the term “about” modifying, for example, size, volume, amount of components in a composition, concentration, process temperature, process time, yield, flow rate, pressure, and similar values ​​and their ranges, refers to variations in quantity that may occur, for example, due to typical measurement and processing procedures used for the manufacture of a compound, composition, concentrate, or formulation, due to careless errors in these procedures, due to differences in the manufacture, source, or purity of the starting materials or components used to carry out the method, and for similar reasons. The term “about” also includes different amounts resulting from the aging of a composition, formulation, or cell culture having a particular initial concentration or formulation, and different amounts resulting from mixing or processing a composition or formulation having a particular initial concentration or formulation. Whether modified by the term “about,” the claims attached herein include equivalent amounts of these quantities. Furthermore, the term “about” may refer to a range of values ​​similar to the specified reference value. In certain embodiments, the term "about" refers to a range of values ​​that are 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percent of the specified baseline value, or less.

[0078] As used herein, the terms “co-administration,” “co-administration,” “administered in combination,” “administered in combination,” “simultaneous,” and “concurrently” encompass the administration of two or more active pharmaceutical ingredients to a human subject such that both active pharmaceutical ingredients and / or their metabolites are present in the human subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration in separate compositions at different time points, or administration in a composition containing two or more active pharmaceutical ingredients. Simultaneous administration in separate compositions and administration in a composition containing both drugs are also encompassed in the methods of the present invention.

[0079] As used herein, the term “therapeutically effective dose” refers to the amount of protein (e.g., recombinant L-asparaginase or its conjugate) required to produce the desired therapeutic effect.

[0080] The terms "L-asparaginase derived from E. coli," "E. coli-derived L-asparaginase," "E. coli asparaginase," and "E. coli L-asparaginase" are used interchangeably to refer to the asparaginase naturally produced in E. coli.

[0081] The terms “L-asparaginase derived from the genus Erwinia,” “Erwinia genus asparaginase,” “Erwinia genus L-asparaginase,” “Erwinia genus asparaginase,” “L-asparaginase derived from the genus Erwinia,” and “asparaginase derived from the genus Erwinia” are used interchangeably herein to refer to asparaginase naturally produced in the genus Erwinia.

[0082] The terms “L-asparaginase from Erwinia chrysanthemi,” “Erwinia chrysanthemi L-asparaginase,” and “L-asparaginase from Erwinia chrysanthemi” are used interchangeably to refer to the asparaginase naturally produced in Erwinia chrysanthemi. Erwinia chrysanthemi (also known as Pectobacterium chrysanthemi) has been newly named Dickeya chrysanthemi. Therefore, the terms Erwinia chrysanthemi, Pectobacterium chrysanthemi, and Dickeya chrysanthemi are used interchangeably herein.

[0083] Erwinaze® (biological product application number 125359) is an Erwinia chrysanthemi type II L-asparaginase product commercially approved in the United States for the treatment of ALL in patients. Its active ingredient is Erwinia chrysanthemi type II L-asparaginase (see the Erwinaze® package insert incorporated herein by reference).

[0084] II. Recombinant L-asparaginase In one embodiment, the recombinant L-asparaginase provided herein is L-asparaginase. In a further embodiment, the recombinant L-asparaginase according to the present invention as described herein is an enzyme having L-asparagine aminohydrolase activity. The enzymatic activity of such recombinant L-asparaginase includes not only the amidodegradation of asparagine to aspartic acid and ammonia, but also the amidodegradation of glutamine to glutamic acid and ammonia.

[0085] In some embodiments, the recombinant L-asparaginase disclosed herein is active as a polymer. In some embodiments, the recombinant L-asparaginase is an active enzyme as a tetramer. The tetramer consists of four subunits (also known as monomers). In some embodiments, the recombinant L-asparaginase is a tetramer consisting of four identical 35kD subunits. In some embodiments, the recombinant L-asparaginase is a non-disulfide-bonded tetrameric therapeutic protein. In certain embodiments, each subunit or monomer of the polymer recombinant L-asparaginase contains the amino acid sequence of SEQ ID NO: 1. In certain embodiments, each subunit or monomer of the tetrameric recombinant L-asparaginase contains the amino acid sequence of SEQ ID NO: 1. In another embodiment, the L-asparaginase is derived from Erwinia chrysanthemi NCPPB 1066 (Genbank accession number CAA32884 (the whole thereof is incorporated herein by reference)) and may or may not have a signal peptide and / or a leader sequence.

[0086] In some embodiments, recombinant L-asparaginase is composed of multiple subunits, for example, four subunits or monomers (tetramers). In this case, the corresponding modified recombinant protein may consist of, for example, 1 to 20 (or more) peptides conjugated to each of the monomers of the tetramer. In some embodiments, recombinant L-asparaginase includes monomers and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 (or more) peptides conjugated to each of the L-asparaginase monomers. In certain embodiments, L-asparaginase is a polymer comprising multiple subunits or monomers, for example, a tetramer, where each monomer in the tetramer is conjugated to a peptide, thereby resulting in a tetramer containing four conjugated peptides, one for each monomer. In some embodiments, recombinant L- Asparaginase is a tetramer containing 1 to 4 peptides conjugated to each L-monomer. In some embodiments, recombinant L-asparaginase is a tetramer containing 4 to 20 peptides conjugated to each L-monomer. In some embodiments, recombinant L-asparaginase is a tetramer containing 6 to 18 peptides conjugated to each L-monomer. In some embodiments, recombinant L-asparaginase is a tetramer containing 6 to 18 peptides conjugated to each L-monomer. In some embodiments, recombinant L-asparaginase is a tetramer containing 10 to 15 peptides conjugated to each L-monomer.

[0087] In one embodiment, the present invention relates to recombinant L-asparaginase and a modified protein having a plurality of chemically linked peptide sequences. In a further embodiment, the length of the peptide sequence is about 10 to about 100, about 15 to about 60, or about 20 to about 40.

[0088] A recombinant L-asparaginase fragment, preferably the recombinant L-asparaginase fragment of SEQ ID NO: 1, may be useful in the inventions described herein. The term “recombinant L-asparaginase fragment” (e.g., the recombinant L-asparaginase fragment of SEQ ID NO: 1) means that the recombinant L-asparaginase sequence may contain fewer amino acids than the recombinant L-asparaginase exemplified herein (e.g., the recombinant L-asparaginase of SEQ ID NO: 1), but may still contain enough amino acids to confer L-aminohydrolase activity. For example, “recombinant L-asparaginase fragment” is a fragment consisting of at least about 150 or 200 consecutive amino acids (e.g., about 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, 325, 326 consecutive amino acids) of one of the recombinant L-asparaginases exemplified herein (e.g., recombinant L-asparaginase of SEQ ID NO: 1), and / or herein, the fragment is the first 50 amino acids from the N-terminus of the recombinant L-asparaginase exemplified herein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 The recombinant L-asparaginase has deletions of up to 15, 20, 25, 30, 35, 40, 45, or 50 amino acids, and / or has deletions of up to 75 or 100 amino acids from the C-terminus of the recombinant L-asparaginase exemplified herein (e.g., recombinant L-asparaginase of SEQ ID NO: 1) (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, or 100 amino acids), and / or has deletions of amino acids at both the N-terminus and C-terminus of the recombinant L-asparaginase exemplified herein (e.g., recombinant L-asparaginase of SEQ ID NO: 1), the total number of deleted amino acids may be up to 125 or 150 amino acids.

[0089] In fact, those skilled in the art will understand how to select and design homologous proteins that substantially retain L-asparaginase activity. Typically, the Nessler assay, following the method described by Mashburn, L., and Wriston, J. (1963) “Tumor Inhibitory Effect of L-Asparaginase,” Biochem Biophys Res Commun 12, 50 (which is incorporated herein by reference in its entirety) is used to determine L-asparaginase activity.

[0090] It is well known in the art that polypeptides can be modified by substitution, insertion, deletion, and / or addition of one or more amino acids while retaining their enzymatic activity. In this context, the term "one or more amino acids" can refer to one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids. For example, affecting the functional properties of the protein. Substitutions of one amino acid at a given position with a chemically equivalent amino acid that do not affect other amino acids are common. A substitution can be defined as an exchange within one of the following groups: Nonpolar or slightly polar small aliphatic residues: Ala, Ser, Thr, Pro, Gly, Negatively charged residues and their amides: Asp, Asn, Glu, Gln, Positively charged residues of polarity: His, Arg, Lys, Nonpolar large aliphatic residues: Met, Leu, Ile, Val, Cys, Large aromatic residues: Phe, Tyr, Trp.

[0091] Therefore, changes resulting from the substitution of one negatively charged residue with another (e.g., glutamate to aspartate) or one positively charged residue with another (lysine to arginine) can be expected to yield functionally equivalent products.

[0092] The position in the amino acid sequence where an amino acid is modified and the number of amino acids that can be modified are not particularly limited. Those skilled in the art can understand modifications that can be introduced without affecting the activity of the protein. For example, modifications in the N-terminal or C-terminal region of a protein can be expected not to alter the activity of the protein under certain conditions. In particular with respect to asparaginases, many characterizations have been made, particularly with respect to the sequence, structure, and residues that form the active catalytic site. This provides insights into residues that can be modified without affecting the activity of the enzyme. L-asparaginases from all known bacterial resources share common structural features. They are all homotetramers and have four active sites between the N-terminal and C-terminal domains of two adjacent monomers (Aghaipour (2001) Biochemistry 40, 5655-5664 (the whole of which is incorporated herein by reference)). They all exhibit a high degree of similarity in their tertiary and quaternary structures (Papageorgiou (2008) FEBS J.275, 4306-4316 (the whole is incorporated herein by reference)). The sequence of the catalytic site of L-asparaginase is highly conserved among L-asparaginase II of Erwinia chrysanthemi, Erwinia carotovora, and E. coli (ibid.). The flexible loop of the active site contains amino acid residues 14-33, and structural analysis has shown that Thr15, Thr95, Ser62, Glu63, Asp96, and Ala120 contact the ligand (ibid.). Aghaipour et al. performed a detailed analysis of the four active sites of L-asparaginase from Erwinia chrysanthemi by analyzing the high-resolution crystal structure of the enzyme that formed a complex with the substrate (Aghaipour (2001) Biochemistry 40, 5655-5664).Kotzia et al. have provided sequences of L-asparaginase from several species and subspecies of the genus Erwinia, and despite having only about 75–77% identity between Erwinia chrysanthemi and Erwinia carotovora, their proteins still retain L-asparaginase activity (Kotzia (2007) J. Biotechnol. 127, 657–669). Moola et al. performed epitope mapping of L-asparaginase from Erwinia chrysanthemi 3937 and found that enzyme activity could be retained even after various antigenic sequences were mutated to reduce the immunogenicity of the asparaginase (Moola (1994) Biochem. J. 302, 921–927). Considering the extensive characterization that has been carried out on L-asparaginase, those skilled in the art can determine how to prepare fragments and / or perform sequence substitutions while retaining enzyme activity. More specifically, a fragment of the protein of SEQ ID NO: 1 is also included within the definition of the protein used in the recombinant L-asparaginase of the present invention. The term “fragment of SEQ ID NO: 1” means that the polypeptide sequence may contain fewer amino acids than the full-length SEQ ID NO: 1, but may still retain enough protein to confer L-aminohydrolase activity. In some embodiments, the recombinant L-asparaginase contains a small amount of the protein including SEQ ID NO: 1. It has at least approximately 80% homology or identity. In some embodiments, recombinant L-asparaginase has sequence identity of at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 1. The terms “homology” and “sequence identity” are used interchangeably herein. The term “contains the sequence of SEQ ID NO: 1” (for example, if L-asparaginase has 100% homology or sequence identity to the amino acid sequence of SEQ ID NO: 1) means that the amino acid sequence of asparaginase is not strictly limited to SEQ ID NO: 1 and may contain one, two, three, four, five, six, seven, eight, nine, ten, or more additional amino acids.

[0093] Sequence ID 1 is as follows: ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALG VDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQ RVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPV VFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVV LNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYY QNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYD AAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIR STRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

[0094] This disclosure also relates to nucleic acids encoding recombinant L-asparaginase as described herein, and in particular to nucleic acids encoding Sequence ID No. 1 as defined herein.

[0095] A. Pegging In certain embodiments, the recombinant L-asparaginase of the present invention, as described herein, further comprises and / or is conjugated to a polymer. In some embodiments, the recombinant L-asparaginase described herein is conjugated with a polyethylene glycol (PEG) moiety. In other embodiments, the recombinant L-asparaginase is not conjugated with a PEG moiety.

[0096] The polymer is selected from the group of non-toxic, water-soluble polymers, e.g., polysaccharides, e.g., hydroxyethyl starch; polyamino acids, e.g., polylysine; polyesters, e.g., polylactic acid; and polyalkylene oxides, e.g., polyethylene glycol (PEG). Polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) is well known in the art and includes linear and branched polymers. Examples of several polymers (in particular PEG) are provided below (each of which is incorporated herein in whole by reference): U.S. Patent No. 5,672,662; U.S. Patent No. 4,179,337; U.S. Patent No. 5,252,714; U.S. Patent Application Publication No. 2003 / 0114647; U.S. Patent No. 6,113,906; U.S. Patent No. 7,419,600; U.S. Patent No. 9,920,311; PCT Publication No. WO2019 / 109018; and PCT Publication No. W02004 / 083258.

[0097] The quality of such polymers is characterized by their polydispersity index (PDI). PDI is... The PDI reflects the molecular weight distribution of a given polymer sample and is calculated by dividing the weight-average molecular weight by the number-average molecular weight. The PDI indicates the distribution of individual molecular weights within a batch of polymer. The PDI is always greater than 1, but as the polymer chain approaches an ideal Gaussian distribution (=monodisperse), the PDI approaches 1.

[0098] In one embodiment, the conjugate has the formula:Asp-[NH-CO-(CH2)x-CO-NH-PEG]n, where Asp is recombinant L-asparaginase, NH is one or more lysine residues and / or N-terminal NH groups of Asp, PEG is a polyethylene glycol moiety, n is a number representing at least 40% to about 100% of the accessible amino groups in Asp (e.g., lysine residues and / or N-terminus), and x is an integer in the range of about 1 to about 8, more specifically, about 2 to about 5. In certain embodiments, PEG is monomethoxypolyethylene glycol (mPEG).

[0099] B.PAS conversion In some embodiments, recombinant L-asparaginase is conjugated with a peptide containing proline or alanine. In other embodiments, recombinant chrysanthanspase is not conjugated with a peptide containing proline, alanine, or serine.

[0100] In some embodiments, recombinant L-asparaginase is a fusion protein comprising (i) recombinant L-asparaginase having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1, and (ii) one or more polypeptides, wherein the polypeptide consists only of proline and alanine amino acid residues.

[0101] In such a fusion protein, the proline residues in the polypeptide, which consist only of proline and alanine amino acid residues, may constitute more than 10% and less than 70% of the polypeptide. Therefore, in such a fusion protein, it is preferable that 10% to 70% of the total number of amino acid residues in the polypeptide are proline residues, more preferably 20% to 50% of the total number of amino acid residues in the polypeptide are proline residues, and even more preferably 30% to 40% (e.g., 30%, 35%, or 40%) of the total number of amino acid residues in the polypeptide are proline residues. The polypeptide may contain multiple amino acid repeats, where the repeats consist of proline and alanine residues, and six or fewer consecutive amino acid residues are identical. In particular, the polypeptide may contain or consist of the amino acid sequence AAPAAPAPAAPAAPAPAAPA, or a cyclically substituted version, or a polymer(s) of the sequence as the whole or as part of the sequence. In other embodiments, recombinant L-asparaginases are particularly lacking in such polypeptides; for example, recombinant L-asparaginases are not conjugated with polypeptides containing the above percentage or repeat of proline residues.

[0102] The present invention also relates to nucleic acids encoding recombinant L-asparaginases, and more particularly to fusion proteins as defined herein. In some embodiments, the nucleotide sequence is a sequence encoding either SEQ ID NO: 1 and a recombinant L-asparaginase comprising a polypeptide, wherein the polypeptide consists only of proline and alanine amino acid residues, and preferably the protein is a fusion protein as described herein, except that one or more amino acids are added, deleted, inserted, or substituted, provided that the fusion protein having this amino acid sequence retains L-asparaginase activity. In other embodiments, the nucleotide sequence is a sequence encoding any recombinant L-asparaginase comprising SEQ ID NO: 1, wherein the sequence is not conjugated to a polypeptide consisting only of proline and alanine amino acid residues (or is not part of a sequence encoding a fusion protein containing a polypeptide).

[0103] The recombinant L-asparaginases according to this disclosure may be prepared using methods known in the art, in particular the methods disclosed in U.S. Patent No. 10,174,302 and PCT Application No. W02019 / 109018, which are incorporated herein by reference for representative embodiments.

[0104] C. Composition containing recombinant L-asparaginase This disclosure also provides compositions comprising recombinant L-asparaginase. Such compositions may comprise recombinant L-asparaginase in combination with other elements (including, but not limited to, buffers, salts, and excipients). Such compositions may comprise vehicles for administering L-asparaginase to a target, including, for example, particles, powders, and encapsulations.

[0105] In some embodiments, the recombinant L-asparaginase described herein may be encapsulated. Encapsulation of asparaginase in red blood cells may, in some cases, help improve the therapeutic index (D. Schrijvers et al., Clin. Pharmacokinet. 2003, 42(9):779-791). For example, a method of encapsulation is described in EP1773452, which is incorporated herein by reference in its entirety for all teachings relating particularly to the encapsulation of L-asparaginase.

[0106] III. Functional aspects and other properties of recombinant L-asparaginase and its composition As is evident, the description herein regarding the functional aspects and other properties of recombinant L-asparaginase may also apply to compositions comprising the recombinant L-asparaginase of the present invention as disclosed herein.

[0107] In some embodiments, the recombinant L-asparaginase described herein may induce a weaker immunogenic response in patients compared to wild-type L-asparaginase. In some embodiments, the recombinant L-asparaginase described herein may have a higher AUC value after a single dose compared to natural L-asparaginase. These characteristics of the recombinant L-asparaginase described herein are beneficial for patients who are thought to have previously developed hypersensitivity to E. coli L-asparaginase or its PEGylated form. In some embodiments, the recombinant L-asparaginase described herein does not produce any significant antibody response for a specific period after a single dose, for example, more than about one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, or longer. In one example, "does not produce any significant antibody response" means that the subject being administered the recombinant L-asparaginase is confirmed to be antibody-negative and within the parameters recognized in the art. Antibody levels can be determined by methods known in the art, such as ELISA or surface plasmon resonance assays (Zalewska-Szewczyk (2009) Clin. Exp. Med. 9, 113-116; Avramis (2009) Anticancer Research 29, 299-302 (each of these is incorporated herein by reference in whole)).

[0108] The compositions comprising the recombinant L-asparaginase of the present invention exhibit reduced aggregation compared to those containing Erwinase® and L-asparaginase of Erwinia chrysanthemi recombinantly expressed in E. coli. In some embodiments, the compositions comprising the recombinant L-asparaginase described herein exhibit reduced aggregation compared to compositions containing other forms of L-asparaginase. For example, the process for producing the non-conjugate recombinant L-asparaginase of the present invention is Er This results in less aggregation compared to winase® and L-asparaginase of Erwinia chrysanthemi recombinantly expressed in E. coli. For example, a process for producing a batch of Erwinase® yields a product with approximately 6% aggregation (see Examples 5 and 6). A batch of the recombinant L-asparaginase of this disclosure typically has less than 1% aggregation (see Examples 5 and 6).

[0109] In some embodiments, the recombinant L-asparaginases of this disclosure have a higher purity than other L-asparaginases. In some embodiments, purity is measured by demonstrating the amount of aggregation of asparaginase in a given sample. The amount of aggregation can be demonstrated by various methods described in the art, including, but not limited to, size exclusion chromatography (SEC-HPLC), size exclusion ultrahigh performance liquid chromatography (SE-UHPLC), size exclusion chromatography-multiangle light scattering (SEC MALLS), and sedimentation velocity analysis ultracentrifugation (svAUC). In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.2%, 0.1%, or 0.01%. In some embodiments, the amount of aggregation observed in a composition containing recombinant L-asparaginase is between 1% and less than 10%. In some embodiments, the amount of aggregation observed in compositions containing recombinant L-asparaginase is less than 10%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 9%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 8%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 7%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 6%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 5%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 4%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 3%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 2%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 1%. In some embodiments, the amount of aggregation of recombinant L-asparaginase is less than 0.5%. In some embodiments, the amount of recombinant L-asparaginase aggregation is less than 0.25%. In some embodiments, the amount of recombinant L-asparaginase aggregation is less than 0.2%. In some embodiments, the amount of recombinant L-asparaginase aggregation is less than 0.1%.In some embodiments, the amount of recombinant L-asparaginase aggregation is less than 0.01%. In some embodiments, the amount of recombinant L-asparaginase aggregation is 0.01% to 10%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 0.01% to about 9%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 0.01% to about 8%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 0.01% to about 7%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 0.01% to about 6%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 0.1% to about 5%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 0.2% to about 4%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 0.25% to about 3%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 0.5% to about 2%. In some embodiments, the amount of recombinant L-asparaginase aggregation is about 1%. In some embodiments, the amount of recombinant L-asparaginase aggregation is 1%.

[0110] It is known to those skilled in the art that smaller amounts of aggregation generally result in products with lower immunogenicity. Immunogenicity is a significant factor in causing adverse events in clinical practice and is regulated by the Federal Drug Administration (FDA). See pp. 15-17 of Services, Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products, 2014, Quaternary Structure: Product Aggregates and Measurement of Aggregates, https: / / www.fda.gov / media / 85017 / download. Also see Ratanji et al; Immunogenicity of therapeutic proteins: Influence of Aggregation.Journal of Immunotoxicology,2014;11(2):99-109;Wang et al;Immunogenicity of Protein Aggregates- Concerns and Realities,International Journal of Pharmaceutics,2012,431(1-2):1-11;and Moussa See also et al, Immunogenicity of Therapeutic Protein Aggregates, Journal of Pharmaceutical Sciences, 2016;105(2):417-430.

[0111] In addition, protein aggregation is associated with enzyme activity because aggregation interferes with the enzyme's ability to function, and can also lead to a decrease in the total yield of active enzyme. Protein aggregation creates difficulties in manufacturing and development, thereby increasing the time it takes to deliver therapeutic drugs to patients and increasing costs. The recombinant chrysanthase of this disclosure exhibits less aggregation compared to other Erwinia chrysanthemi L-asparaginases recombinantly expressed in E. coli and L-asparaginases derived from Erwinia chrysanthemi. These aspects of recombinant L-asparaginase represent an improvement to the art.

[0112] The recombinant L-asparaginases of this disclosure may have any combination of the properties described in the above sections or any other properties described herein.

[0113] IV. Method for producing recombinant L-asparaginase In some embodiments, the recombinant chrysanthaspases disclosed herein are recombinantly produced in Pseudomonas fluorescens. In some embodiments, Pseudomonas fluorescens lacks the native L-asparaginase.

[0114] In some embodiments, the present disclosure provides a method for high-yield cytoplasmic production of soluble recombinant L-asparaginase, where the recombinant protein is produced in the periplasm at lower yields in its native host. In its native host, Erwinia chrysanthemi, L-asparaginase is produced in the periplasm. The present invention provides a method that enables the production of high levels of soluble and / or active recombinant L-asparaginase in the cytoplasm of host cells. In embodiments, the method provided herein generates high levels of soluble and / or active recombinant L-asparaginase in the cytoplasm of host cells of the orders Pseudomonadales, genus Pseudomonad, genus Pseudomonas, or Pseudomonas fluorescens.

[0115] A method that may be used to produce recombinant L-asparaginase is described, for example, in U.S. Publication No. 2019 / 0127742, which is incorporated herein by reference in its entirety for all purposes and in particular for all teachings relating to the production method for recombinant L-asparaginase.

[0116] A. Expression System The methods described herein, in some examples, involve expressing recombinant L-asparaginase from an expression construct in Pseudomonas host cells. In some embodiments, the expression construct is a plasmid. In some embodiments, the plasmid encoding the chrysanthaspase sequence includes a selection marker, and the host cells holding the plasmid are cultured under selection conditions. In some embodiments, the plasmid does not include a selection marker. In some embodiments, the expression construct is integrated into the host cell genome. In some embodiments, the expression construct encodes chrysanthaspase fused to a secretory signal that directs the chrysanthaspase to the periplasm. In some embodiments, the secretory signal is cleaved within the host cell. In some embodiments, the expression construct does not encode a secretory signal, and the chrysanthaspase is directed to the cytoplasm.

[0117] Methods for expressing heterologous proteins in host strains containing regulatory sequences (e.g., promoters, secretion leaders, and ribosome-binding sites) useful for the methods of the present invention, including Pseudomonas host strains, include, for example, U.S. Patent No. 7,618,799, “Bacterial leader sequences for increased expression”, U.S. Patent No. 7,985,564, “Expression systems with Sec-system secretion”, U.S. Patents No. 9,394,571 and 9,580,719, both titled “Method for Rapidly Screening Microbial Hosts to Identify Certain Strains with Improved Yield and / or Quality in the Expression of Heterologous Proteins”, and “Expression of U.S. Patent No. 9,453,251, “Mammalian Proteins in Pseudomonas fluorescens”, U.S. Patent No. 8,603,824, “Process for Improved Protein Expression by Strain Engineering”, and U.S. Patent No. 8,530,171, “High Level Expression of Recombinant Toxin Proteins” (each of which is incorporated herein by reference in its entirety). In embodiments, secretion leaders used in the context of the present invention are secretion leaders disclosed in any of U.S. Patents No. 7,618,799, No. 7,985,564, No. 9,394,571, No. 9,580,719, No. 9,453,251, No. 8,603,824, and No. 8,530,171. These patents also describe bacterial host strains useful for carrying out the methods herein, which have been engineered to overexpress folding modulators or have protease mutations introduced to increase heterologous protein expression.

[0118] B. Promoter The promoters used in accordance with the methods described herein may be constitutive or regulatory promoters. Common examples of useful regulatory promoters include those of the family derived from the lac promoter (i.e., the lacZ promoter), particularly the tac and trc promoters described in U.S. Patent No. 4,551,433, belonging to DeBoer, as well as the Ptac16, Ptac17, PtacII, PlacUV5, and T7lac promoters. In one embodiment, the promoter is not derived from a host cell bacterium. In certain embodiments, the promoter is derived from E. coli.

[0119] Inducible promoter sequences are used to modulate the expression of recombinant L-asparaginase according to the methods described herein. In embodiments, inducible promoters useful for the methods described herein include those of the family derived from the lac promoter (i.e., the lacZ promoter), particularly the tac and trc promoters described in U.S. Patent No. 4,551,433, belonging to DeBoer, as well as Ptac16, Ptac Examples include the 17, PtacII, PlacUV5, and T7lac promoters. In one embodiment, the promoter is not derived from the host cell bacterium. In certain embodiments, the promoter is derived from E. coli. In some embodiments, the lac promoter is used to regulate the expression of chrysanthase from a plasmid. In the case of lac promoter derivatives or family members, such as the tac promoter, the inducer is IPTG (isopropyl-β-D-1-thiogalactopyranoside, also known as "isopropylthiogalactoside"). In certain embodiments, IPTG is added to the culture to induce the expression of chrysanthase from the lac promoter in Pseudomonas host cells.

[0120] Common examples of non-lac promoters useful in expression systems according to the methods described herein include those specified in U.S. Patent Application Publication No. 2019 / 0127742, incorporated herein by reference, particularly those listed in Table 1.

[0121] See also, for example, J. Sanchez-Romero & V. De Lorenzo, 1999, Manual of Industrial Microbiology and Biotechnology (A. Demain & J. Davies, eds.) pp. 460-74 (ASM Press, Washington, DC); H. Schweizer, 2001, Current Opinion in Biotechnology, 12:439-445; R. Slater & R. Williams, 2000, Molecular Biology and Biotechnology (J. Walker & R. Rapley, eds.) pp. 125-54 (The Royal Society of Chemistry, Cambridge, UK), and L.-M. Guzman, et al., 1995, J. Bacteriol. 177(14):4121-4130, all of which are incorporated herein by reference. Promoter sequences that are native to selected bacterial host cells, such as the anthranilic or benzoic acid operon promoters of the genus Pseudomonas (Pant, Pben), may also be used to control the expression of transgenes encoding target polypeptides. Tandem promoters, in which two or more promoters are covalently linked to each other, whether their sequences are the same or different, or whether they originate from the same or different organisms, such as the Pant-Pben tandem promoter (promoter hybrid) or the Place-Plac tandem promoter, may also be used.

[0122] A regulatory promoter uses promoter-regulating proteins to control the transcription of a gene in which the promoter is part. Where a regulatory promoter is used herein, the corresponding promoter-regulating protein is also part of the expression system according to the method herein. Examples of promoter-regulating proteins include: activating proteins, e.g., E. coli catabolite activating protein, MalT protein; AraC family transcription activators; repressor proteins, e.g., E. coli Lad protein; and dual-function regulatory proteins, e.g., E. coli NagC protein. Numerous regulatory promoter / promoter-regulating protein pairs are known in the art. In one embodiment, expression constructs for a target protein(s) and a heterologous protein of interest are under the control of the same regulatory element.

[0123] Promoter regulatory proteins interact with effector compounds, which are compounds that reversibly or irreversibly associate with the regulatory protein to allow the protein to either release or bind to at least one DNA transcription regulatory region of a gene under the control of the promoter, thereby permitting or inhibiting the action of the transcriptase that initiates gene transcription. Effector compounds are inducers or corepressors. These compounds are classified into one of the following categories, and examples of these compounds include natural effector compounds and non-metabolic inducers. Numerous triads of regulatory promoter / promoter-regulating protein / effector compound are known in the art. In some cases, effector compounds are used through cell culture or fermentation, but in one embodiment in which a regulatory promoter is used, a suitable effector compound is added to the culture to directly or indirectly result in the expression of a desired gene(s) encoding a protein or polypeptide of interest after the host cell biomass has grown to a desired amount or density.

[0124] In embodiments where the lac family promoter is used, the lad gene may be present in the system. The lad gene, which is normally constitutively expressed, encodes the Lad protein, a Lac repressor protein, which binds to the lac operator of the lac family promoter. Therefore, when the lac family promoter is used, the lad gene may be included in the expression system and expressed. Useful promoter systems for the genus Pseudomonas are described in the literature, for example, U.S. Patent Application Publication 2008 / 0269070, which is cited above.

[0125] C. Other adjustment elements In some embodiments, the soluble recombinant L-asparaginase of this disclosure is present in the cytoplasm or periplasm of a cell at the time of production. Secretory leaders useful for targeting proteins, such as chrysanthaspsase, are described elsewhere in this specification, as well as in U.S. Patent Application Publication 2008 / 0193974, U.S. Patent Application Publication 2006 / 0008877, and U.S. Patent Application 12 / 610,207 cited above. In some embodiments, an expression construct encoding the recombinant L-asparaginase is provided, fused to a secretory leader that transports the recombinant L-asparaginase to the periplasm of a Pseudomonad or Pseudomonas cell. In some embodiments, the secretory leader is cleaved from the recombinant L-asparaginase. In some embodiments, the secretory leader promotes the production of soluble chrysanthaspsase.

[0126] In embodiments, the expression vector contains an optimal ribosome-binding sequence. To improve the production of heterologous cytoplasmic proteins that accumulate primarily as inclusion bodies due to excessively rapid translation rates, modulation of translation intensity by modifying the translation initiation region of the protein of interest may be used. Secretion of heterologous proteins into the periplasmic space of bacterial cells can also be enhanced not by maximizing the protein translation level, but by optimizing the translation rate to synchronize with the protein secretion rate. The translation initiation region is defined as a sequence extending from immediately upstream of the ribosome-binding site (RBS) to approximately 20 nucleotides downstream of the start codon (McCarthy et al. (1990) Trends in Genetics 6:78-85 (the entire text is incorporated herein by reference)). In prokaryotes, alternative RBS sequences may be used to optimize the translation level of heterologous proteins by providing a reduced translation rate compared to the translation level using the standard or consensus RBS sequence (AGGAGG) described by Shine and Dalgarno (Proc. Natl. Acad. Sci. USA 71:1342-1346, 1974). "Translation rate" or "translation efficiency" refers to the rate of mRNA translation into protein within a cell. In most prokaryotes, the Shine-Dalgarno sequence assists in the binding and positioning of the 30S ribosomal component to the mRNA start codon through interaction with the pyrimidine-rich region of 16S ribosomal RNA. The RBS (also referred to herein as the Shine-Dalgarno sequence) is located downstream of the start of mRNA transcription and upstream of the start of translation, typically 4–14 nucleotides upstream of the start codon, more typically 8–10 nucleotides upstream of the start codon. Due to the role of RBS sequences in translation, there is a direct causal relationship between translation efficiency and the efficiency (or strength) of RBS sequences.

[0127] In some embodiments, modification of the RBS sequence results in a decrease in the translation rate of a heterologous protein. This decrease in translation rate may correspond to an increase in the level of properly processed protein or polypeptide per gram of protein produced or per gram of host protein. The decrease in translation rate may also correlate with an increase in the level of recoverable protein or polypeptide produced per gram of recombinant protein or per gram of host cell protein. The decrease in translation rate may also correspond to any combination of increased expression, increased activity, increased solubility, or increased translocation (e.g., translocation to the periplasmic compartment or secretion into the extracellular space). In this embodiment, the term “increase” is an increase compared to the protein or polypeptide of interest that is produced, properly processed, soluble, and / or recoverable when expressed under the same or substantially the same conditions, where the nucleotide sequence encoding the polypeptide contains the standard RBS sequence. Similarly, the term “decrease” is a decrease compared to the translation rate of the protein or polypeptide of interest when the gene encoding the protein or polypeptide contains the standard RBS sequence. Translation speed may decrease by at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, at least approximately 75%, or more, or by at least approximately 1 / 2, approximately 1 / 3, approximately 1 / 4, approximately 1 / 5, approximately 1 / 6, approximately 1 / 7, or less.

[0128] In some embodiments, the RBS sequence variants described herein may be classified as resulting in high, medium, or low translation efficiency. In one embodiment, sequences are ranked according to their level of translational activity compared to the translational activity of a standard RBS sequence. High RBS sequences have about 60% to about 100% of the activity of the standard sequence. Medium RBS sequences have about 40% to about 60% of the activity of the standard sequence. Low RBS sequences have less than about 40% of the activity of the standard sequence.

[0129] Examples of RBS sequences produced by the methods described herein include those specified in U.S. Patent Application Publication No. 2019 / 0127742, incorporated herein by reference, particularly those listed in Table 2.

[0130] Expression constructs useful for carrying out the methods described herein include, in addition to a protein-coding sequence, the following regulatory elements operably linked thereto: a promoter, a ribosome-binding site (RBS), a transcriptional terminator, and translation start and stop signals. Useful RBSs can be obtained from any species useful as host cells in the expression system, for example, according to U.S. Patent Application Publication No. 2008 / 0269070 and U.S. Patent Application No. 12 / 610,207. Numerous specific and diverse consensus RBS, e.g., D. Frishman The following are publicly known, as described and cited in et al., Gene 234(2):257-65 (8 Jul. 1999); and BESuzek et al., Bioinformatics 17(12):1123-30 (December 2001). In addition, either natural or synthetic RBS can be used, for example, EP0207459 (synthetic RBS); as described in O. Ikehata et al., Eur.J. Biochem. 181(3):563-70 (1989). Further examples of methods, vectors, translation and transcription elements, and other elements useful for the methods described herein are described, for example, in U.S. Patent No. 5,055,294 belonging to Gilroy and U.S. Patent No. 5,128,130 belonging to Gilroy et al.; U.S. Patent No. 5,281,532 belonging to Rammler et al.; U.S. Patent No. 4,695,455 and 4,861,595 belonging to Barnes et al.; U.S. Patent No. 4,755,465 belonging to Gray et al.; and U.S. Patent No. 5,169,760 belonging to Wilcox.

[0131] D. host strain Bacterial hosts, including those of the genus Pseudomonad and closely related bacteria, are assumed to be used when carrying out the methods described herein. In certain embodiments, the Pseudomonad host cell is Pseudomonas fluorescens. In some embodiments, the host cell is a Pseudomonas fluorescens cell, which is deficient in native L-asparaginase.

[0132] Host cells and constructs useful for carrying out the methods described herein are identified or prepared using reagents and methods known in the art, as well as reagents and methods described in the literature, for example, U.S. Patent Application Publication 2009 / 0325230, “Protein Expression Systems” (which is incorporated herein by reference in its entirety). This publication describes the production of recombinant polypeptides by introducing a nucleic acid construct into trophic Pseudomonas fluorescens host cells containing a lacI gene insertion fragment in the chromosome. The nucleic acid construct comprises a nucleotide sequence encoding a recombinant polypeptide operably linked to a promoter that can direct nucleic acid expression in the host cell, and also comprises a nucleotide sequence encoding a trophicity selection marker. The trophicity selection marker is a polypeptide that restores the trophicity host cell to protrophicity. In embodiments, the cells are trophicity-dependent to proline, uracil, or a combination thereof. In embodiments, the host cells are derived from MB101 (ATCC deposit number PTA-7841). U.S. Patent Application Publication No. 2009 / 0325230, “Protein Expression Systems,” and Schneider, et al., 2005, “Auxotrophic markers pyrF and proC, in some cases, replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation,” Biotechnol. Progress 21(2):343-8 (both are incorporated herein by reference in their entirety) describe a uracil-dependent producing host strain constructed by deleting the pyrF gene from the MB101 strain. The pyrF gene was cloned from the MB214 strain (ATCC deposit number PTA-7840) to construct a plasmid to compensate for the pyrF deletion in order to restore protrophotrophy. In certain embodiments, a pyrF-proC dual trophotrophic selective marker system is used in P. fluorescens host cells.The pyrF-deficient producing host strains described herein are often used as a background for introducing other desired genomic alterations, including those described herein as useful for carrying out the methods herein.

[0133] In embodiments, host cells useful for the methods of the present invention are deficient in the expression of at least one protease, or overexpression of at least one folding modulator, or both. In embodiments, host cells are not deficient in the expression of proteases and do not overexpress folding modulators, and are therefore wild-type with respect to the expression of proteases and folding modulators. In any of these embodiments, host cells are additionally deficient in native L-asparaginase. In embodiments, the deficient in native L-asparaginase is caused by deleting or otherwise inactivating the native L-asparaginase gene using any suitable method known in the art. In embodiments, host cells are deficient in native type I L-asparaginase, native type II L-asparaginase, or both. In embodiments, host cells are wild-type with respect to the expression of proteases and folding modulators and are deficient in native type I L-asparaginase and native type II L-asparaginase. For example, host cells useful for the method of the present invention can be prepared from MB101 using methods known to those skilled in the art. In embodiments, the host cells are prepared from MB101 containing the native type I L-asparaginase gene and the native type II L-asparaginase gene. It is produced by deleting one or both of the offspring, or by otherwise inactivating them.

[0134] It will be understood by those skilled in the art that a producible host strain useful for the method of the present invention can be produced using commonly available host cells, e.g., P. fluorescens MB101, by inactivating the pyrF gene and / or the native type I L-asparaginase gene and / or the native type II L-asparaginase gene using, for example, one of a number of suitable methods known in the art and described in the literature. It will also be understood that a plasmid restoring protrophotrophy, e.g., a plasmid containing the pyrF gene from strain MB214, can be transformed into a useful strain using one of a number of suitable methods known in the art and described in the literature. In addition, in such strains, the protease can be inactivated using methods known in the art, and a folding modulator overexpression construct can be introduced.

[0135] In embodiments, the host cell belongs to the order Pseudomonadales. If the host cell belongs to the order Pseudomonadales, it may be a member of the family Pseudomonadaceae, which includes the genus Pseudomonas. Examples of γ-proteobacteria hosts include members of the species Escherichia coli and members of the species Pseudomonas fluorescens. Host cells of the order Pseudomonadales, family Pseudomonadaceae, or genus Pseudomonas can be identified by those skilled in the art and can be found in the literature (e.g., Bergey's Manual of Systematics of Archaea and It is described in "Bacteria" (online publication, 2015).

[0136] Other organisms of the genus Pseudomonas may also be useful. Examples of the genus Pseudomonad and its close relatives include Gram-negative proteobacteria subgroup 1, which includes proteobacteria belonging to families and / or genera described in Bergey's Manual of Systematics of Archaea and Bacteria (online publication, 2015). Table 3 shows organisms of these families and genera.

[0137] Examples of families and genera listed in the “Gram-Negative Aerobic Rods and Cocci” section (Bergey’s Manual of Systematics of Archaea and Bacteria (online publication, 2015)) include those specified in U.S. Patent Application Publication No. 2019 / 0127742, incorporated herein by reference, particularly those listed in Table 3.

[0138] The genus Pseudomonas and related bacteria are generally part of a group defined as “Gram(-) Proteobacteria subgroup 1” or “Gram-negative aerobic rods and cocci” (Bergey's Manual of Systematics of Archaea and Bacteria (online publication, 2015)). Host strains of the genus Pseudomonas are described in the literature, for example, U.S. Patent Application Publication No. 2006 / 0040352 cited above.

[0139] "Gram-negative proteobacteria subgroup 1" also includes proteobacteria that are classified under this item according to the criteria used for classification. This item includes groups that were previously classified under this section but are no longer classified under this section, as defined in Bergey's Manual of Systematics of Archaea and Bacteria (online publication, 2015), such as the genera Acidovax, Brevundimonas, Burkholderia, and Hydrogonia. This includes the genera Sphingomonas (and the derived genus Blastomonas), which was created by reorganizing organisms belonging to the genera nophaga, Oceanimonas, Ralstonia, Stenotrophomonas, and Xanthomonas (and organisms previously referred to as species of the genus Xanthomonas), as well as the genus Acidomonas, which was created by reorganizing organisms belonging to the genus Acetobacter. In addition, the hosts include Pseudomonas enalia (ATCC 14393) and Pseudomonas, which have been reclassified as Alteromonas haloplanktis, Alteromonas nigrifaciens, and Alteromonas putrefaciens, respectively. nigrifaciensi (ATCC 19375), and Pseudomonas Examples include cells of putrefaciens (ATCC 8071). Similarly, for example, Pseudomonas acidovorans (ATCC 15668) and Pseudomonas testosteroni (ATCC 11996) have since been reclassified as Comamonas acidovorans and Comamonas testosteroni, respectively, and Pseudomonas nigrifaciens (ATCC 19375) and Pseudomonas piscicida (ATCC 15057) have been reclassified as Pseudoalteromonas nigrifaciens and Pseudoalteromonas piscicida, respectively. "Gram-negative proteobacteria subgroup 1" also includes proteobacteria classified as belonging to one of the following families: Pseudomonadaceae, Azotobacteraceae (now often referred to as the "Azotobacter group" of Pseudomonadaceae), Rhizobiaceae, and Methylomonadaceae (now often referred to as the "Methylococcaceae"). As a result, in addition to the genera specified herein, further proteobacteria genera that fall within the scope of “Gram-negative proteobacteria subgroup 1” include: 1) Azotobacter group bacteria of the genus Azorhizophilus; 2) Pseudomonadaceae bacteria of the genera Cellvibrio, Oligella, and Teredinibacter; 3) Rhizobiaceae bacteria of the genera Chelatobacter, Ensifer, Liberibacter (also known as “Candidatus Liberibacter”), and Sinorhizobium; and 4) Methylococcaceae bacteria of the genera Methylobacter, Methylocaldum, Methylomicrobium, Methylosarcina, and Methylosphaera.

[0140] In some cases, the host cell is selected from "Gram-negative proteobacteria subgroup 16." "Gram-negative proteobacteria subgroup 16" is defined as the group of proteobacteria of the genus Pseudomonas (with the ATCC number or other deposit number of the exemplary strain(s) in parentheses): Pseudomonas abietaniphila(ATCC700689);Pseudomonas aeruginosa(ATCC10145);Pseudomonas alcaligenes(ATCC14909);Pseudomonas anguilliseptica(ATCC33660);Pseudomonas citronellolis(ATCC13674);Pseudomonas flavescens(ATCC51555);Pseudomonas mendocina(ATCC25411);Pseudomonas nitroreducens(ATCC33634);Pseudomonas oleovorans(ATCC8062);Pseudomonas pseudoakaligenes(ATCC17440);Pseudomonas resinovorans(ATCC14235);Pseudomonas straminea(ATCC33636);Pseudomonas agarici(AT CC25941);Pseudomonas alcaliphila;Pseudomonas alginovora;Pseudomonas andersonii;Pseudomonas asplenii(ATCC23835);Pseudomonas azelaica(ATCC27162);Pseudomonas beyerinckii(ATCC19372);Pseudomonas borealis;Pseudomonas boreopolis(ATCC33662);Pseudomonas brassicacearum;Pseudomonas butanovora(ATCC43655);Pseudomonas cellulosa(ATCC55703);Pseudomonas aurantiaca(ATCC33663);Pseudomonas chlororaphis(ATCC9446、ATCC13985、ATCC17418、ATCC17461);Pseudomonas fragi(ATCC4973);Pseudomonas lundensis(ATCC49968);Pseudomonas taetrolens(ATCC4683);Pseudomonas cissicola(ATCC33616);Pseudomonas coronafaciens;Pseudomonas diterpeniphila;Pseudomonas elongata(ATCC10144);Pseudomonas flectens(ATCC12775);Pseudomonas azotoformans;Pseudomonas brenneri;Pseudomonas cedrella;Pseudomonas corrugata(ATCC29736);Pseudomonas extremorientalis;Pseudomonas fluorescens(ATCC35858);Pseudomonas gessardii;Pseudomonas libanensis;Pseudomonas mandelii(ATCC700871);Pseudomonas marginalis(ATCC10844);Pseudomonas migulae;Pseudomonas mucidolens(ATCC4685);Pseudomonas orientalis;Pseudomonas rhodesiae;Pseudomonas synxantha(ATCC9890);Pseudomonas tolaasii(ATCC33618);Pseudomonas veronii(ATCC700474);Pseudomonas frederiksbergensis;Pseudomonas geniculata(ATCC19374);Pseudomonas gingeri;Pseudomonas graminis;Pseudomonas grimontii;Pseudomonas halodenitrificans;Pseudomonas halophila;Pseudomonas hibiscicola(ATCC19867);Pseudomonas huttiensis(ATCC14670);Pseudomonas; hydrogenovora;Pseudomonas jessenii(ATCC700870);Pseudomonas kilonensis;Pseudomonas lanceolata(ATCC14669);Pseudomonas lini;Pseudomonas marginata(ATCC25417);Pseudomonas mephitica(ATCC33665);Pseudomonas denitrificans(ATCC19244);Pseudomonas pertucinogena(ATCC190);Pseudomonas pictorum(ATCC23328);Pseudomonas psychrophila;Pseudomonas filva(ATCC31418);Pseudomonas monteilii(ATCC700476);Pseudomonas mosselii;Pseudomonas oryzihabitans(ATCC43272);Pseudomonas plecoglossicida(ATCC700383);Pseudomonas putida(ATCC12633);Pseudomonas reactans;Pseudomonas spinosa(ATCC14606);Pseudomonas balearica;Pseudomonas luteol a(ATCC43273);Pseudomonas stutzeri(ATCC17588);Pseudomonas amygdali(ATCC33614);Pseudomonas avellanae(ATCC700331);Pseudomonas caricapapayae(ATCC33615);Pseudomonas cichorii(ATCC10857);Pseudomonas ficuserectae(ATCC35104);Pseudomonas fuscovaginae;Pseudomonas meliae(ATCC33050);Pseudomonas syringae(ATCC19310);Pseudomonas viridiflava(ATCC13223);Pseudomonas thermocarboxydovorans(ATCC35961);Pseudomonas thermotolerans;Pseudomonas thivervalensis;Pseudomonas vancouverensis (ATCC700688); Pseudomonas wisconsinensis; and Pseudomonas xiamenensis. In one embodiment, the host cell for the expression of recombinant L-asparaginase is Pseudomonas fluorescens.

[0141] In some cases, the host cell is selected from "Gram-negative proteobacteria subgroup 17." "Gram-negative proteobacteria subgroup 17" is defined as a group of proteobacteria known in the art as "fluorescent pseudomonas," including, for example, the following Pseudomonas species: Pseudomonas azotoformans; Pseudomonas brenneri; Pseudomonas cedrella; Pseudomonas cedrina; Pseudomonas corrugata; Pseudomonas extremorientalis; Pseudomonas fluorescens; Pseudomonas gessardii; Pseudomonas libanensis; Pseudomonas mandelii; Pseudomonas marginalis; Pseudomonas migulae; Pseudomonas mucidolens; Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas synxantha; Pseudomonas tolaasii; and Pseudomonas veronii.

[0142] E. protease In embodiments, the methods provided herein involve using Pseudomonas host cells containing one or more mutations (e.g., partial or complete deletions) in one or more protease genes to produce recombinant L-asparaginase. In some embodiments, the mutations in the protease genes promote the production of recombinant L-asparaginase.

[0143] Examples of target protease genes include aminopeptidases; dipeptidases; dipeptidylpeptidases and tripeptidylpeptidases; peptidyldipeptidases; serine carboxypeptidases; metallocarboxypeptidases; cysteine ​​carboxypeptidases; omegapeptidases; serine proteinases; cysteine ​​proteinases; aspartate proteinases; metalloproteinases; or proteases classified as proteinases of unknown mechanisms.

[0144] Examples of aminopeptidases include cytosolic aminopeptidase (leucylaminopeptidase), membrane alanylaminopeptidase, cystinylaminopeptidase, tripeptide aminopeptidase, prolylaminopeptidase, arginylaminopeptidase, glutamylaminopeptidase, x-proaminopeptidase, and bacterial leucylaminopeptidase. Examples of peptidases include thermophilic aminopeptidases, Clostridium aminopeptidases, cytosolic alanyl aminopeptidases, lysyl aminopeptidases, x-trp aminopeptidases, tryptophanyl aminopeptidases, methionyl aminopeptidases, d-stereospecific aminopeptidases, and aminopeptidase ey. Examples of dipeptidases include x-his dipeptidase, x-arg dipeptidase, x-methyl-his dipeptidase, cys-gly dipeptidase, glu-glu dipeptidase, pro-x dipeptidase, x-pro dipeptidase, met-x dipeptidase, non-stereospecific dipeptidases, cytosolic nonspecific dipeptidases, membrane dipeptidases, and β-ala-his dipeptidases. Examples of dipeptidyl peptidases and tripeptidyl peptidases include dipeptidyl peptidase i, dipeptidyl peptidase ii, dipeptidyl peptidase iii, dipeptidyl-peptidase iv, dipeptidyl dipeptidase, tripeptidyl peptidase I, and tripeptidyl peptidase II. Examples of peptidyl dipeptidases include peptidyl dipeptidase a and peptidyl dipeptidase b. Examples of serine carboxypeptidases include lysosomal pro-x carboxypeptidase, serine D-ala-D-ala carboxypeptidase, carboxypeptidase C, and carboxypeptidase D. Examples of metallocarboxypeptidases include carboxypeptidase a, carboxypeptidase B, lysine (arginine) carboxypeptidase, gly-X carboxypeptidase, alanine carboxypeptidase, muramoyl pentapeptide carboxypeptidase, carboxypeptidase h, glutamate carboxypeptidase, carboxypeptidase M, muramoyl tetrapeptide carboxypeptidase, zinc d-ala-d-ala carboxypeptidase, carboxypeptidase A2, membrane pro-X carboxypeptidase, tubulinyl-tyr carboxypeptidase, and carboxypeptidase t.Examples of omega peptidases include acylaminoacyl peptidase, peptidylglycine amidase, pyroglutamyl peptidase I, β-aspartyl peptidase, pyroglutamyl peptidase II, n-formylmethionyl peptidase, pteroylpoly-[γ]-glutamate carboxypeptidase, γ-glu-X carboxypeptidase, and acylmuramoyl-ala peptidase. Examples of serine proteins include chymotrypsin, chymotrypsin c, metrizine, trypsin, thrombin, coagulation factor Xa, plasmin, enteropeptidase, acrosin, α-lysic protease, glutamyl endopeptidase, cathepsin G, coagulation factor viia, coagulation factor ixa, cucumisin, prolyl oligopeptidase, coagulation factor xia, brachiulin, plasma kallikrein, and tissue kallikrein. N, pancreatic elastase, leukocyte elastase, coagulation factor xiia, chymase, complement component c1r55, complement component c1s55, classical complement pathway c3 / c5 convertase, complement factor I, complement factor D, complement secondary pathway c3 / c5 convertase, cerevicin, hypodermin C, lysyl endopeptidase, endopeptidase la, γ-renin, benonbin ab, leucyl endopeptidase, tryptase, s Examples include ctellarin, kexin, subtilisin, orijen, endopeptidase k, thermomycoline, thermidase, endopeptidase SO, T-plasminogen activator, protein C, pancreatic endopeptidase E, pancreatic elastase II, IGA-specific serine endopeptidase, U-plasminogen activator, benonbin A, furin, myeloblastin, semenogenase, granzyme A or cytotoxic T lymphocyte proteinase 1, granzyme B or cytotoxic T lymphocyte proteinase 2, streptoglycin A, streptoglycin B, glutamyl endopeptidase II, oligopeptidase B, horseshoe crab coagulation factor c, horseshoe crab coagulation factor, horseshoe crab coagulation enzyme, onputin, repressor lexa, bacterial leader peptidase I, togavirin, and flavirin.Cysteine ​​proteins include cathepsin B, papain, ficin, chymopapain, asclepain, clostripine, streptopain, actinide, cathepsin 1, cathepsin H, calpain, cathepsin t, glycyl endopeptidase, tumor coagulation pro-factor, cathepsin S, picornaine 3C, picornaine 2A, calicaine, ananaine, and stem bromelay. Examples include flutebromelain, regmine, histrisine, and interleukin-1-β convertase. Examples of aspartic acid proteins include pepsin A, pepsin B, gastrixin, chymosin, cathepsin D, nepenthesin, renin, retropepsin, proopiomelanocortin-converting enzyme, aspergylopepsin I, aspergylopepsin II, penicillopepsin, lysopuspepsin, endothiapepsin, mucolopepsin, candidapepsin, saccharopepsin, rhodotorulapepsin, physalopepsin, acrocillindropepsin, polyporopepsin, pycnoporopepsin, citalidopepsin a, citalidopepsin b, xanthomonapepsin, cathepsin e, barrierpepsin, bacterial leader peptidase I, pseudomonapepsin, and plasmmepsin. Metalloproteinases include atrolysin a, microbial collagenase, leucolicin, interstitial collagenase, neprilysin, embellicin, IgA-specific metalloendopeptidase, procollagen N-endopeptidase, thymetoligopeptidase, neurolytic agents, stromelysin 1, mepurin A, procollagen C-endopeptidase, peptidyl-lys metalloendopeptidase, astacin, stromelysin 2, matrilysin, gelatinase, aeromonolisin, pseudolysin, thermolysin, basilicolicin, oleolisin, cocolicin, mycolicin, β-lytic metalloendopeptidase, peptidyl-asp metalloendopeptidase, and neutrophils. Examples include globular collagenase, gelatinase B, reisi manoricin, saccharolicin, autolysin, doiterolysin, ceralicin, atrolysin B, atrolysin C, atroxase, atrolysin E, atrolysin F, adamaricin, folyricin, lavaricin, bosolopacin, bosololicin, ohitolysin, trimeloricin I, trimeloricin II, mucrolysin, pyriricin, insulinin, O-sialycoprotein endopeptidase, russeriricin, mitochondrial intermediate peptidase, dactylicin, narijilicin, magnolicin, mepurin B, mitochondrial processing peptidase, macrophage elastase, coriolisin, and toxylysin.Examples of proteinases with unknown mechanisms include thermopsins and multicatalytic endopeptidase complexes.

[0145] Certain proteases possess both protease and chaperone-like activity. When these proteases negatively impact protein yield and / or quality, it is often useful to specifically delete their protease activity, and when their chaperone activity may positively impact protein yield and / or quality, they are overexpressed. Examples of these proteases, but not limited to, include: RXF04587.1(clpA), RXF08347.1, RXF04654.2(clpX), RXF04663.1, RXF01957.2(hslU), RXF01961.2(hslV), members of the Hsp100(Clp / Hsl) family; and RXF0534, a member of the peptidyl-prolyl cis-trans isomerase family. 5.2(ppiB); RXF04892.1 (aminohydrolase), a member of the metallopeptidase M20 family; RXF04693.1 (methionine aminopeptidase) and RXF03364.1 (methionine aminopeptidase), members of the metallopeptidase M24 family; and RXF01181.1 (signal peptidase), a member of the serine peptidase S26 signal peptidase I family.

[0146] In embodiments, a host strain useful for expressing recombinant L-asparaginase in the method of the present invention is a Pseudomonas host strain, e.g., P. fluorescens, that has a protease deficiency or inactivation (e.g., due to deletion, partial deletion, or knockout) and / or overexpresses a folding modulator from, for example, a plasmid or bacterial chromosome. In embodiments, the host strain is deficient in at least one protease selected from Lon, HslUV, DegP1, DegP2, Prc, AprA, DegP2 S219A, Prc1, and AprA. In this configuration, the host strain overexpresses a folding modulator selected from LepB, Tig, and DsbAC-Skp (i.e., a combination of DsbA, DsbC, and Skp; Skp is OmpH RXF4702.1, described as SEQ ID NO. 59 in U.S. Patent Application Publication 2019 / 0127742, where an example of the coding sequence is described as SEQ ID NO. 60). In a DsbAC-Skp overexpressing host, the folding modulators DsbA, DsbC, and Skp (SEQ ID NOs. 25 and 26 in U.S. Patent Application 9,394,571, and SEQ ID NO. 60 in U.S. Patent Application Publication 2019 / 0127742, respectively) can be expressed from an operon. In one embodiment, the host strain is deficient in at least one protease selected from Lon, HslUV, DegP1, DegP2, Prc, AprA, DegP2 S219A, Prc1, and AprA, and overexpresses a folding modulator selected from LepB, Tig, and DsbAC-Skp. In any of the above embodiments, the host strain expresses the nutritional requirement markers pyrF and proC, and is protease deficient and / or overexpresses a folding modulator. In another embodiment, the host strain expresses any other suitable select marker known in the art. In any of the above embodiments, the host strain's asparaginase, e.g., native type I and / or type II asparaginase, is inactivated.In the embodiment, the host cell line is a Pseudomonadales host cell lacking Lon and HslU / V; a Pseudomonadales host cell lacking Lon, DegP1, DegP2, Prc, and AprA; a Pseudomonadales host cell lacking Lon, DegP1, DegP2 S219A, Prc1, and AprA and overexpressing DsbAC-Skp; a Pseudomonadales host cell lacking AspG1 and / or AspG2; a Pseudomonadales host cell lacking AspG1 and / or AspG2 and overexpressing Tig; a Pseudomonadale lacking AspG1 and / or AspG2 and overexpressing LepB. s-order host cells; Pseudomonadales-order host cells lacking AspG1 and / or AspG2, and lacking Lon and HslU / V; host cells lacking AspG1 and / or AspG2, and lacking Lon, DegP1, DegP2, Prc, and AprA; or host cells lacking AspG1 and / or AspG2, Lon, DegP1, DegP2, Prc1, and AprA, and overexpressing DsbAC-Skp.

[0147] These and other proteases and folding modulators are known in the art and are described in the literature, e.g., U.S. Patent No. 8,603,824. For example, Table D of this patent describes Tig(tig, trigger factor, FKBP type ppiase (ec 5.2.1.8), RXF04655, UniProtKB-P0A850 (TIG_ECOLI)). "Method for Rapidly Screening Microbial Hosts to Identify Certain Strains with Improved Yield and / or Quality in the Expression of Heterologous WO2008 / 134461 and U.S. Patent No. 9,394,571, both titled “Proteins”, are incorporated herein by reference in their entirety. These patents describe Tig (RXF04655.2, including SEQ ID NO: 34), LepB (RXF01181.1, including SEQ ID NO: 56), DegP1 (RXF01250, including SEQ ID NO: 57), AprA (RXF04304.1, including SEQ ID NO: 86), Prc1 (RXF06586.1, including SEQ ID NO: 120), DegP2 (RXF07210.1, including SEQ ID NO: 124), Lon (RXF04653, including SEQ ID NO: 92); DsbA (RXF01002.1, including SEQ ID NO: 25), and DsbC (RXF03307.1, including SEQ ID NO: 26). These sequences, as well as the sequences of other proteases and folding modulators, are described in U.S. Patent No. 9,580,719 (the table of sequence numbers in columns 93-98). For example, U.S. Patent No. 9,580,719 lists the sequences The sequence is provided that codes for HslU (RXF01957.2) and HslV (RXF01961.2) as numbers 18 and 19.

[0148] F. Codon Optimization In one embodiment, the method described herein involves the expression of recombinant L-asparaginase from a construct optimized for the codon selectivity of a bacterial strain of interest. In this embodiment, the bacterial strain is a Pseudomonas host cell, e.g., Pseudomonas fluorescens. Methods for optimizing codons to improve expression in a bacterial host are known in the art and described in the literature. For example, codon optimization for expression in a Pseudomonas host strain is described, for example, in U.S. Patent Application Publication 2007 / 0292918, “Codon Optimization Method” (which is incorporated herein by reference in its entirety).

[0149] In heterologous expression systems, optimization steps can improve the host's ability to produce foreign proteins. Protein expression is governed by numerous factors, including those affecting transcription, mRNA processing, and translation stability and initiation. Polynucleotide optimization steps may include steps to improve the host's ability to produce foreign proteins and steps to help researchers efficiently design expression constructs. Optimization strategies may include, for example, modifications of the translation initiation region, modifications of mRNA structural elements, and the use of different codon biases. Methods for optimizing nucleic acid sequences to improve the expression of heterologous proteins in bacterial hosts are known in the art and described in the literature. For example, codon optimization for expression in Pseudomonas host strains is described, for example, in U.S. Patent Application Publication 2007 / 0292918, “Codon Optimization Method” (which is incorporated herein by reference in its entirety).

[0150] Optimization addresses one of numerous sequence features of heterologous genes. For example, rare codon-induced translational arrest often results in reduced heterologous protein expression. Rare codon-induced translational arrest involves the presence of codons in the polynucleotide of interest that are rarely used in the host organism and can negatively impact protein translation due to their scarcity in the available tRNA pool. One method to improve optimal translation in the host organism involves performing codon optimization, which may result in the removal of rare host codons from the synthetic polynucleotide sequence.

[0151] Alternative translation initiations can, in some cases, lead to a decrease in the expression of heterologous proteins. Alternative translation initiations involve synthetic polynucleotide sequences that unintentionally contain motifs that can function as ribosome-binding sites (RBS). These sites can, in some cases, lead to the initiation of translation of cleaved proteins from sites within the gene. One method to reduce the likelihood of producing cleaved proteins, which are often difficult to remove during purification, involves removing putative internal RBS sequences from optimized polynucleotide sequences.

[0152] Repeat-induced polymerase slippage often results in a decrease in the expression of heterologous proteins. Repeat-induced polymerase slippage involves nucleotide repeat sequences that have been shown to cause slippage or stuttering of DNA polymerase, sometimes resulting in frameshift mutations. Such repeats often also cause slippage of RNA polymerase. In organisms with a high G+C content tendency, more sophisticated repeats consisting of G or C nucleotide repeats may be present. Therefore, one method to reduce the likelihood of inducing RNA polymerase slippage involves modifying extended G or C nucleotide repeats.

[0153] Interfering secondary structures can, in some cases, lead to a decrease in the expression of heterologous proteins. This often involves sequestering the RBS sequence or start codon and is associated with decreased protein expression. Stem-loop structures are also frequently involved in transcriptional arrest and transcriptional decay. Optimized polynucleotide sequences typically have minimal secondary structures in the RBS and gene coding regions of the nucleotide sequence to enable improved transcription and translation.

[0154] Another feature that can sometimes affect the expression of heterologous proteins is the presence of restriction sites. Polynucleotide sequences are optimized by removing restriction sites that interfere with the subsequent subcloning of transcription units into host expression vectors.

[0155] For example, the optimization process often begins with identifying a desired amino acid sequence heterologously expressed by a host. From this amino acid sequence, a candidate polynucleotide or DNA is designed. During the design of the synthetic DNA sequence, codon usage is often compared to the codon usage of the host organism, and rare host codons are removed from the synthetic sequence. In addition, the candidate synthetic DNA sequence is sometimes modified to remove undesirable enzyme restriction sites and to add or remove any desired signal sequences, linkers, or untranslated regions. The synthetic DNA sequence is often analyzed for the presence of secondary structures that may interfere with the translation process, such as G / C repeats and stem-loop structures. Before the candidate DNA sequence is synthesized, the optimized sequence design is often checked to ensure that the sequence accurately codes for the desired amino acid sequence. Finally, the candidate DNA sequence is synthesized using DNA synthesis techniques, such as those known in the art.

[0156] In another embodiment of this specification, common codon usage frequencies in host organisms such as P. fluorescens are often used to optimize the expression of heterologous polynucleotide sequences. The percentage and distribution of codons that are rarely considered favorable for a particular amino acid in the host expression system are evaluated. Values ​​of 5% and 10% usage frequencies are often used as cutoff values ​​for determining rare codons. For example, the codons listed in Table 4 have calculated occurrence frequencies of less than 5% in the genome of P. fluorescens MB214 and are typically avoided in optimized genes expressed in the P. fluorescens host.

[0157] Examples of codons appearing at less than 5% in P. fluorescens by the methods described herein include those specified in U.S. Patent Application Publication No. 2019 / 0127742, incorporated herein by reference, particularly those listed in Table 4.

[0158] This disclosure intends to describe the use of any sequence encoding any recombinant L-asparaginase, which may include any sequence optimized for expression in Pseudomonas host cells used. Sequences intended for use may often be optimized to any desired degree, but may include, but are not limited to, optimizations to remove: codons occurring less than 5% in Pseudomonas host cells, codons occurring less than 10% in Pseudomonas host cells, rare codon-induced translational arrests, putative internal RBS sequences, extended G or C nucleotide repeats, interfering secondary structures, restriction sites, or combinations thereof.

[0159] Furthermore, any amino acid sequence of a secretion leader useful for carrying out the methods provided herein is encoded by any suitable nucleic acid sequence. Codon optimization for expression in E. coli can be found, for example, in Welch, et al., 2009, PLoS One, “Design Parameters to Control Synthetic Gene Expression in Escherichia coli,” 4(9):e7002, Ghane, et al., 2008, Krishna R. et al., (2008) Mol Biotechnology “Optimization of the AT-content of Codons Immediately This is described in "Downstream of the Initiation Codon and Evaluation of Culture Conditions for High-level Expression of Recombinant Human G-CSF in Escherichia coli," 38:221-232.

[0160] G. High-throughput screening In some embodiments, high-throughput screening is often performed to determine the optimal conditions for the expression of soluble recombinant L-asparaginase. Conditions that may be modified in the screening include, for example, the host cell, the genetic background of the host cell (e.g., deletion of different proteases), the type of promoter in the expression construct, the type of secretory leader fused to the encoded recombinant L-asparaginase, the growth temperature, the OD at induction when an inducible promoter is used, the amount of inducing factor added (e.g., the amount of IPTG used for induction when a lacZ promoter or its derivative is used), the duration of protein induction, the growth temperature after adding the inducing agent to the culture, the stirring rate of the culture, the selection method for plasmid maintenance, the culture volume in the container, and the cell lysis method.

[0161] In some embodiments, a library (or “array”) of host strains is provided, where each strain (or “population of host cells”) in the library is genetically modified to regulate the expression of one or more target genes in host cells. The “optimal host strain” or “optimal expression system” is often identified or selected by comparing it with other populations of host cells in the array that have different phenotypes, based on the quantity, quality, and / or location of the expressed protein of interest. Thus, the optimal host strain is a strain that produces the polypeptide of interest according to desired specifications. The desired specifications vary depending on the polypeptide produced, but may include the quality and / or quantity of the protein, protein folding, etc., regardless of whether the protein is sequestered (e.g., in inclusion bodies) or secreted. For example, an optimal host strain or optimal expression system yields a certain absolute level of yield or indicator strain, i.e., a certain level of yield compared to that produced by a strain used for comparison, characterized by the amount of soluble heterologous protein, the amount of recoverable heterologous protein, the amount of properly processed heterologous protein, the amount of properly folded heterologous protein, the amount of active heterologous protein, and / or the total amount of heterologous protein.

[0162] A method for screening microbial hosts to identify strains with improved yield and / or quality in the expression of heterologous proteins is described, for example, in U.S. Patent Application Publication No. 20080269070.

[0163] H. Conditions for bacterial growth The growth conditions useful for the methods described herein often include a temperature of about 4°C to about 42°C and a pH of about 5.7 to about 8.8. When expression constructs having the lacZ promoter or derivatives thereof are used, expression is often induced by adding IPTG to the culture at a final concentration of about 0.01 mM to about 1.0 mM.

[0164] The pH of the culture is maintained, if applicable, using pH buffers and methods known to those skilled in the art. pH control during cultivation is often achieved using aqueous ammonia. In some embodiments, the pH of the culture is about 5.7 to about 8.8. In certain embodiments, the pH is about 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8. In another embodiment, the pH is about 5.7 to 5.9. The ranges are 5.8-6.0, 5.9-6.1, 6.0-6.2, 6.1-6.3, 6.2-6.5, 6.4-6.7, 6.5-6.8, 6.6-6.9, 6.7-7.0, 6.8-7.1, 6.9-7.2, 7.0-7.3, 7.1-7.4, 7.2-7.5, 7.3-7.6, 7.4-7.7, 7.5-7.8, 7.6-7.9, 7.7-8.0, 7.8-8.1, 7.9-8.2, 8.0-8.3, 8.1-8.4, 8.2-8.5, 8.3-8.6, 8.4-8.7, or 8.5-8.8. In further other embodiments, the pH is approximately 5.7–6.0, 5.8–6.1, 5.9–6.2, 6.0–6.3, 6.1–6.4, or 6.2–6.5. In certain embodiments, the pH is approximately 5.7–6.25. In some embodiments, the pH is approximately 5.0–8.0.

[0165] In some embodiments, the growth temperature is maintained between approximately 4°C and approximately 42°C. In certain embodiments, the growth temperature is approximately 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C. In other embodiments, the growth temperature is approximately 25°C to 27°C, 25°C to 28°C, 25°C to 29°C, 25°C to 30°C, 25°C to 31°C, 25°C to 32°C, 25°C to 33°C, 26°C to 28°C, 26°C to 29°C, 26°C to 30°C, 26°C to 31°C, 26°C to 32°C, 27°C to 29°C, 27°C to 30°C, and 2 The temperature is maintained at approximately 7°C to 31°C, approximately 27°C to 32°C, approximately 26°C to 33°C, approximately 28°C to 30°C, approximately 28°C to 31°C, approximately 28°C to 32°C, approximately 29°C to 31°C, approximately 29°C to 32°C, approximately 29°C to 33°C, approximately 30°C to 32°C, approximately 30°C to 33°C, approximately 31°C to 33°C, approximately 31°C to 32°C, approximately 30°C to 33°C, or approximately 32°C to 33°C. In one embodiment, the growth temperature is maintained at approximately 22°C to 33°C. In another embodiment, the temperature is changed during culture. In certain embodiments, the temperature is maintained at approximately 30°C to approximately 32°C before an agent for inducing expression from a construct encoding a polypeptide or protein of interest is added to the culture, and then the temperature is lowered to approximately 25°C to approximately 27°C after the agent for inducing expression, such as IPTG, is added to the culture. In one embodiment, the temperature is maintained at approximately 30°C before an agent for inducing expression from a construct encoding a polypeptide or protein of interest is added to the culture, and then the temperature is lowered to approximately 25°C after the agent for inducing expression is added to the culture.

[0166] I. Induction In one embodiment, fermentation is used in a method for producing recombinant L-asparaginase. The expression system according to this disclosure is cultured in any fermentation manner. For example, batch fermentation, fed-batch fermentation, semi-continuous fermentation, and continuous fermentation manners may be used herein.

[0167] In some embodiments, the fermentation medium may be selected from nutrient-rich mediums, minimal mediums, and inorganic salt mediums. In other embodiments, minimal mediums or inorganic salt mediums are selected. In certain embodiments, inorganic salt mediums are selected.

[0168] Inorganic salt media consist of inorganic salts and a carbon source such as glucose, sucrose, or glycerol. Examples of inorganic salt media include M9 medium, Pseudomonas medium (ATCC179), and Davis·Mingioli medium (see BD Davis & ES Mingioli (1950) J.Bact. 60:17-28). Inorganic salts used to prepare inorganic salt media include, for example, potassium phosphate, ammonium sulfate or ammonium chloride, magnesium sulfate or magnesium chloride, and trace metals such as calcium chloride, iron, copper, and manganese. Examples include a selection from zinc borate and sulfate. Typically, organic nitrogen sources such as peptone, tryptone, amino acids, or yeast extracts are not included in inorganic salt media. Instead, an inorganic nitrogen source is used, which may be selected from, for example, ammonium salts, aqueous ammonia, and gaseous ammonia. Inorganic salt media typically contain glucose or glycerol as a carbon source. Compared to inorganic salt media, minimal media often contain inorganic salts and a carbon source, but are often supplemented with, for example, low levels of amino acids, vitamins, peptone, or other components. However, these are added at minimal levels. The media are often prepared using methods described in the art, for example, the method described in U.S. Patent Application Publication 2006 / 0040352, cited and incorporated by reference above. For details of culture procedures and inorganic salt media useful for the methods described herein, see Riesenberg, D et al., 1991, “High cell density cultivation of Escherichia coli This is described in "at controlled specific growth rate," J. Biotechnol. 20(l):17-27.

[0169] Fermentation can be carried out on any scale. The expression system according to this disclosure is useful for the expression of recombinant proteins on any scale. Therefore, for example, fermentation volumes on the microliter, milliliter, centiliter, and deciliter scales may be used, and in many cases, fermentation volumes of 1 liter or more are used.

[0170] In an embodiment, the fermentation volume is approximately 1 liter or more. In an embodiment, the fermentation volume is approximately 0.5 liters to approximately 100 liters. In an embodiment, the fermentation volume is approximately 0.5 liters, approximately 1 liter, approximately 2 liters, approximately 3 liters, approximately 4 liters, approximately 5 liters, approximately 6 liters, approximately 7 liters, approximately 8 liters, approximately 9 liters, or approximately 10 liters. In an embodiment, the fermentation volume is approximately 0.5 liters to approximately 2 liters, approximately 0.5 liters to approximately 5 liters, approximately 0.5 liters to approximately 10 liters, approximately 0.5 liters to approximately 25 liters, approximately 0.5 liters to approximately 50 liters, approximately 0.5 liters to approximately 75 liters, approximately 10 liters to approximately 25 liters, approximately 25 liters to approximately 50 liters, or approximately 50 liters to approximately 100 liters. In other embodiments, the fermentation volume is 5 liters or more, 10 liters or more, 15 liters or more, 20 liters or more, 25 liters or more, 50 liters or more, 75 liters or more, 100 liters or more, 200 liters or more, 500 liters or more, 1,000 liters or more, 2,000 liters or more, 5,000 liters or more, 10,000 liters or more, or 50,000 liters or more.

[0171] J. Fermentation Forms In one embodiment, fermentation is used in a method for producing recombinant L-asparaginase. The expression system according to this disclosure is cultured in any fermentation manner. For example, batch fermentation, fed-batch fermentation, semi-continuous fermentation, and continuous fermentation manners may be used herein.

[0172] In some embodiments, the fermentation medium may be selected from nutrient-rich mediums, minimal mediums, and inorganic salt mediums. In other embodiments, minimal mediums or inorganic salt mediums are selected. In certain embodiments, inorganic salt mediums are selected.

[0173] Inorganic salt media consist of inorganic salts and a carbon source such as glucose, sucrose, or glycerol. Examples of inorganic salt media include M9 medium, Pseudomonas medium (ATCC179), and Davis·Mingioli medium (see BD Davis & ES Mingioli (1950) J.Bact.60:17-28). Inorganic salts used to prepare inorganic salt media include, for example, potassium phosphate, ammonium sulfate or ammonium chloride, and magnesium sulfate. Examples include magnesium chloride or other minerals, and trace metals, such as borates and sulfates of calcium chloride, iron, copper, manganese, and zinc. Typically, organic nitrogen sources such as peptone, tryptone, amino acids, or yeast extracts are not included in inorganic salt media. Instead, inorganic nitrogen sources are used, which may be selected from, for example, ammonium salts, aqueous ammonia, and gaseous ammonia. Inorganic salt media typically contain glucose or glycerol as a carbon source. Compared to inorganic salt media, minimal media often contain inorganic salts and carbon sources, but are often supplemented with, for example, low levels of amino acids, vitamins, peptone, or other components. However, these are added at minimal levels. The media are often prepared using methods described in the art, for example, the method described in U.S. Patent Application Publication 2006 / 0040352, which is cited and incorporated by reference above. Details of the culture procedures and inorganic salt media useful for the methods described herein are described in Riesenberg, D et al., 1991, “High cell density cultivation of Escherichia coli at controlled specific growth rate,” J. Biotechnol. 20(l):17-27.

[0174] Fermentation can be carried out on any scale. The expression system according to this disclosure is useful for the expression of recombinant proteins on any scale. Therefore, for example, fermentation volumes on the microliter, milliliter, centiliter, and deciliter scales may be used, and in many cases, fermentation volumes of 1 liter or more are used.

[0175] In an embodiment, the fermentation volume is approximately 1 liter or more. In an embodiment, the fermentation volume is approximately 0.5 liters to approximately 100 liters. In an embodiment, the fermentation volume is approximately 0.5 liters, approximately 1 liter, approximately 2 liters, approximately 3 liters, approximately 4 liters, approximately 5 liters, approximately 6 liters, approximately 7 liters, approximately 8 liters, approximately 9 liters, or approximately 10 liters. In an embodiment, the fermentation volume is approximately 0.5 liters to approximately 2 liters, approximately 0.5 liters to approximately 5 liters, approximately 0.5 liters to approximately 10 liters, approximately 0.5 liters to approximately 25 liters, approximately 0.5 liters to approximately 50 liters, approximately 0.5 liters to approximately 75 liters, approximately 10 liters to approximately 25 liters, approximately 25 liters to approximately 50 liters, or approximately 50 liters to approximately 100 liters. In other embodiments, the fermentation volume is 5 liters or more, 10 liters or more, 15 liters or more, 20 liters or more, 25 liters or more, 50 liters or more, 75 liters or more, 100 liters or more, 200 liters or more, 500 liters or more, 1,000 liters or more, 2,000 liters or more, 5,000 liters or more, 10,000 liters or more, or 50,000 liters or more.

[0176] K. Protein Analysis In embodiments, recombinant L-asparaginase produced by the method provided herein is analyzed. Recombinant chrysanthase is optionally analyzed by, for example, biolayer interferometry, SDS-PAGE, Western blotting, far-western blotting, ELISA, absorbance, or mass spectrometry (e.g., tandem mass spectrometry).

[0177] In some embodiments, the concentration and / or amount of recombinant L-asparaginase produced is determined by, for example, Bradford assay, absorbance, Coomassi staining, mass spectrometry, etc.

[0178] The protein yields of the insoluble and soluble fractions described herein are often determined by methods known to those skilled in the art, such as capillary gel electrophoresis (CGE) and Western blotting. The soluble fraction is often evaluated using, for example, biolayer interferometry.

[0179] The recombinant L-asparaginase monomers of this disclosure can form active tetramers, for example, in cell lysates, in sonicated cell products, and upon further purification. After expression of recombinant L-asparaginase in a bacterial expression system, for example, an E. coli or Pseudomonas host strain, the recombinant protein can be purified using any suitable method known in the art, for example, to remove host cell proteins. Examples of purification methods include cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, high-performance liquid chromatography (HPLC), or combinations thereof and / or other known methods. Purification of asparaginase proteins is described in the literature, for example, U.S. Patent No. 5,310,670, “Method for the purification of Erwinia L-asparaginase” and U.S. Patent No. 8,323,948, “Asparaginases and uses thereof” (each incorporated herein in whole by reference). The type II asparaginase expressed in P. fluorescens exists as an active tetrameric asparaginase enzyme in sonicated materials.

[0180] In embodiments, a measurable characteristic (e.g., activity, size, length, or other characteristic indicating the presence of active and / or intact protein) of a certain amount of unpurified or purified asparaginase sample is compared to the same measurable characteristic of the same amount of asparaginase standard sample (e.g., commercially available asparaginase). It will be understood that the amount of asparaginase protein in the sample may be determined by any suitable assay known in the art for protein measurement, and the activity may be determined by any suitable assay, such as those described herein.

[0181] Useful measures of protein yield include, for example, the amount of recombinant protein per unit of culture volume (e.g., grams or milligrams of protein / liter of culture), the percentage or proportion of measured recombinant protein in the insoluble pellet obtained after lysis (e.g., amount of recombinant protein in the supernatant / amount of protein in the insoluble fraction), the percentage or proportion of soluble recombinant protein, the percentage or proportion of active protein (e.g., amount of active protein / amount of protein used in the assay), the percentage or proportion of total cellular protein (tcp), the amount of protein / cell, and the percentage of dry biomass.

[0182] In embodiments, the methods herein are used to obtain soluble recombinant L-asparaginase of this disclosure, for example monomers or tetramers, in yields of about 20% to about 90% total cell protein. In certain embodiments, the yield of soluble recombinant chrysanthaspasase is about 20% total cell protein, about 25% total cell protein, about 30% total cell protein, about 31% total cell protein, about 32% total cell protein, about 33% total cell protein, about 34% total cell protein, about 35% total cell protein, about 36% total cell protein, about 37% total cell protein, about 38% total cell protein, about 39% total cell protein, about 40% total cell protein, about 41% total cell protein, about 42% total cell protein, about 43% total cell protein, about 44% total cell protein, about 45% total cell protein, about 46% total cell protein The protein content is approximately 47% total cellular protein, 48% total cellular protein, 49% total cellular protein, 50% total cellular protein, 51% total cellular protein, 52% total cellular protein, 53% total cellular protein, 54% total cellular protein, 55% total cellular protein, 56% total cellular protein, 57% total cellular protein, 58% total cellular protein, 59% total cellular protein, 60% total cellular protein, 65% total cellular protein, 70% total cellular protein, 75% total cellular protein, 80% total cellular protein, 85% total cellular protein, or 90% total cellular protein. In some embodiments, the yield of soluble recombinant chrysanthase is approximately 20% to 25% total cellular protein. Quality, approximately 20% to 30% total cellular protein, approximately 20% to 35% total cellular protein, approximately 20% to 40% total cellular protein, approximately 20% to 45% total cellular protein, approximately 20% to 50% total cellular protein, approximately 20% to 55% total cellular protein, approximately 20% to 60% total cellular protein, approximately 20% to 65% total cellular protein, approximately 20% to 70% total cellular protein, approximately 20% to 75% total cellular protein, approximately 20% to 80% total cellular protein, approximately 20% to 85% total cellular protein, approximately 20% to 90% total cellular protein, approximately 25% to 90% total cellular protein, approximately 30% to 90% total cellular protein, approximately 35% to 90% total cellular protein, approximately 4 The yields are approximately 0% to 90% total cellular protein, approximately 45% to 90% total cellular protein, approximately 50% to 90% total cellular protein, approximately 55% to 90% total cellular protein, approximately 60% to 90% total cellular protein, approximately 65% ​​to 90% total cellular protein, approximately 70% to 90% total cellular protein, approximately 75% to 90% total cellular protein, approximately 80% to 90% total cellular protein, approximately 85% to 90% total cellular protein, approximately 20% to 40% total cellular protein, approximately 25% to 40% total cellular protein, approximately 35% to 40% total cellular protein, approximately 20% to 35% total cellular protein, approximately 20% to 30% total cellular protein, or approximately 20% to 25% total cellular protein. In some embodiments, the yield of soluble recombinant chrysanthanasase is approximately 20% to 40% total cellular protein.

[0183] In embodiments, the methods described herein are used to obtain soluble recombinant L-asparaginase of the present disclosure, for example, monomers or tetramers, in yields of about 1 gram to about 50 grams per liter. In certain embodiments, the yield of soluble recombinant L-asparaginase is approximately 1 gram per liter, approximately 2 grams per liter, approximately 3 grams per liter, approximately 4 grams per liter, approximately 5 grams per liter, approximately 6 grams per liter, approximately 7 grams per liter, approximately 8 grams per liter, approximately 9 grams per liter, approximately 10 grams per liter, approximately 11 grams per liter, approximately 12 grams per liter, approximately 13 grams per liter, approximately 14 grams per liter, approximately 15 grams per liter, approximately 16 grams per liter, approximately 17 grams per liter, approximately 18 grams per liter, approximately 19 grams per liter, approximately 20 grams per liter, approximately 21 grams per liter, approximately 22 grams per liter, approximately 23 grams per liter, approximately 24 grams per liter, approximately 25 grams per liter, approximately 26 grams per liter, approximately 27 grams per liter, and approximately 28 grams, approximately 30 grams per liter, approximately 35 grams per liter, approximately 40 grams per liter, approximately 45 grams per liter, approximately 50 grams per liter, approximately 1 gram to approximately 5 grams per liter, approximately 1 gram to approximately 10 grams per liter, approximately 10 grams to approximately 12 grams per liter, approximately 10 grams to approximately 13 grams per liter, approximately 10 grams to approximately 14 grams per liter, approximately 10 grams to approximately 15 grams per liter, approximately 10 grams to approximately 16 grams per liter, approximately 10 grams to approximately 17 grams per liter, approximately 10 grams to approximately 18 grams per liter, approximately 10 grams to approximately 19 grams per liter, approximately 10 grams to approximately 20 grams per liter, approximately 10 grams to approximately 21 grams per liter,Approximately 10 grams to 22 grams per liter, approximately 10 grams to 23 grams per liter, approximately 10 grams to 24 grams per liter, approximately 10 grams to 25 grams per liter, approximately 10 grams to 30 grams per liter, approximately 10 grams to 40 grams per liter, approximately 10 grams to 50 grams per liter, approximately 10 grams to 12 grams per liter, approximately 12 grams to 14 grams per liter, approximately 14 grams to 16 grams per liter, approximately 16 grams to 18 grams per liter, approximately 18 grams per liter and above. Approximately 20 grams per liter, approximately 20 grams to approximately 22 grams per liter, approximately 22 grams to approximately 24 grams per liter, approximately 23 grams to approximately 25 grams per liter, approximately 10 grams to approximately 25 grams per liter, approximately 11 grams to approximately 25 grams per liter, approximately 12 grams to approximately 25 grams per liter, approximately 13 grams to approximately 25 grams per liter, approximately 14 grams to approximately 25 grams per liter, approximately 15 grams to approximately 2 grams per liter 5 grams, approximately 16 to 25 grams per liter, approximately 17 to 25 grams per liter, approximately 18 to 25 grams per liter, approximately 19 to 25 grams per liter, approximately 20 to 25 grams per liter, approximately 21 to 25 grams per liter, approximately 22 to 25 grams per liter, approximately 23 to 25 grams per liter, or approximately 24 to 25 grams per liter. In the embodiment, the soluble recombinant protein yield is approximately 10 to 13 grams per liter, 12 to 14 grams per liter, 13 to 15 grams per liter, 14 to 16 grams per liter, 15 to 17 grams per liter, 16 to 18 grams per liter, 17 to 19 grams per liter, 18 to 20 grams per liter, 20 to 22 grams per liter, 22 to 24 grams per liter, or 23 to 25 grams per liter.In embodiments, the soluble recombinant protein yield is approximately 10 to 25 grams per liter, approximately 12 to 24 grams per liter, approximately 14 to 22 grams per liter, approximately 16 to 20 grams per liter, or approximately 18 to 20 grams per liter. In embodiments, the extracted protein yield is approximately 5 to 15 grams per liter, approximately 5 to 25 grams per liter, approximately 10 to 15 grams per liter, approximately 10 to 25 grams per liter, approximately 15 to 20 grams per liter, approximately 15 to 25 grams per liter, or approximately 18 to 25 grams per liter. In certain embodiments, the yield of soluble recombinant chrysanthase is approximately 10 grams to 25 grams per liter.

[0184] In embodiments, the amount of recombinant L-asparaginase of the Disclosure, e.g., monomers or tetramers, detected in the soluble fraction is about 10% to about 100% of the total amount of recombinant chrysanthaspas produced. In embodiments, this amount is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, or about 100% of the total amount of recombinant chrysanthaspas produced. In embodiments, this amount is approximately 10% to 20%, 20% to 50%, 25% to 50%, 25% to 50%, 25% to 95%, 30% to 50%, 30% to 40%, 30% to 60%, 30% to 70%, 35% to 50%, 35% to 70%, 35% to 75%, 35% to 95%, 40% to 50%, 40% to 95%, 50% to 75%, 50% to 95%, 70% to 95%, or 80% to 100% of the total amount of recombinant chrysanthaspase produced.

[0185] In some embodiments, the amount of soluble recombinant L-asparaginase of the present disclosure is in the culture. It is expressed as a percentage of the total soluble protein produced. Data expressed as recombinant asparaginase weight / cell culture volume at a given cell density can be converted to data expressed as a percentage of recombinant protein out of total cellular protein. For example, by knowing the amount of total cellular protein per unit volume of cell culture at a given cell density, it is within the capabilities of those skilled in the art to convert volume protein yield to % total cellular protein. This value can be determined if the percentage consisting of 1) cell weight / culture volume at a given cell density and 2) the amount of total protein out of the cell weight is known. For example, at OD550 of 1.0, the dry cell weight of E. coli has been reported to be 0.5 grams / liter ("Production of Heterologous Proteins from Recombinant DNA Escherichia coli in Bench Fermentors," Lin, NS, and Swartz, JR, 1992, METHODS: A Companion to Methods in Enzymology 4:159-168). Bacterial cells are composed of polysaccharides, lipids, nucleic acids, and proteins. While not limited to these, proteins are estimated to constitute 52.4% by weight of E. coli cells (Da Silva, NA, et al., 1986, “Theoretical Growth Yield Estimates for Recombinant Cells,” Biotechnology and Bioengineering, Vol. XXVIII: 741-746), and the protein content of E. coli is reported as 55% by dry cell weight (“Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology,”). Literature, including 1987, Ed. in Chief Frederick C. Neidhardt, Vol. 1, pp. 3-6, reports that E. coli cells are approximately 52.4–55% protein. Using the above measurements (i.e., 0.5 g / liter dry cell weight and 55% protein by cell weight), the total cellular protein per volume of cell culture at A550 with a ratio of 1.0 for E. coli is calculated to be 275 μg total cellular protein / ml / A550. For calculating total cellular protein per volume of cell culture based on wet cell weight, measurements by Glazyrina, et al. (incorporated herein by reference, Microbial Cell Factories 2010, 9:42), for example, that A600 with a ratio of 1.0 for E. coli resulted in a wet cell weight of 1.7 g / liter and a dry cell weight of 0.39 g / liter, can be used. For example, using this wet cell weight versus dry cell weight comparison and the protein as 55% dry cell weight as described above, the total cellular protein per volume of cell culture at A600 with a value of 1.0 for E. coli can be calculated as 215 μg total cellular protein / ml / A600. For Pseudomonas fluorescens, the total cellular protein per volume of cell culture at a given cell density is similar to that found in E. coli. P. fluorescens, like E. coli, is a Gram-negative rod. The dry cell weight of P. fluorescens ATCC 11150 is given in Edwards, et al., 1972, “Continuous Culture The amount is 0.5 grams / liter / A500, as reported in "of Pseudomonas fluorescens with Sodium Maleate as a Carbon Source," Biotechnology and Bioengineering, Vol. XIV, pp. 123-147. This is the same weight reported by Lin, et al. for E. coli at A550. The light scattering measurements obtained at 500 nm and 550 nm are expected to be approximately the same. For P. fluorescens HK44, the percentage of total protein content in the cell weight is, for example, Yarwood, et al., July 2002, "Noninvasive Quantitative Measurement of Bacterial Growth in Porous Media under Unsaturated-Flow Conditions," Applied According to *Environmental Microbiology* 68(7):3597-3605, the figure is 55%. This percentage is comparable to, or identical to, that given for *E. coli* in the aforementioned literature.

[0186] In embodiments, the amount of soluble recombinant L-asparaginase of the Disclosure produced, for example, monomers or tetramers, is about 0.1% to about 95% of the total soluble protein produced in the culture. In embodiments, this amount is more than about 0.1%, more than 0.5%, more than 1%, more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of the total soluble protein produced in the culture. In embodiments, this amount is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total soluble protein produced in the culture. In embodiments, this amount represents approximately 5% to 95%, 10% to 85%, 20% to 75%, 30% to 65%, 40% to 55%, 1% to 95%, 5% to 30%, 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, or 80% to 90% of the total soluble protein produced in the culture.

[0187] In embodiments, the amount of soluble recombinant L-asparaginase of the Disclosure produced, for example, monomers or tetramers, is about 0.1% to about 50% of the dry cell weight (DCW). In embodiments, this amount is more than about 0.1%, more than 0.5%, more than 1%, more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 45%, or more than 50% of the DCW. In embodiments, this amount is about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50% of the DCW. In this embodiment, this amount is approximately 5% to 50%, 10% to 40%, 20% to 30%, 1% to 20%, 5% to 25%, 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, or 40% to 50% of the total soluble protein produced in the culture.

[0188] In embodiments, the soluble proteins of the disclosure produced in the cytoplasm are described in terms of any of the following ways: the amount of recombinant protein per volume of culture (e.g., grams or milligrams of protein / liter of culture), the percentage or proportion of the measured recombinant protein in the insoluble pellet obtained after lysis (e.g., amount of recombinant protein in the supernatant / amount of protein in the insoluble fraction), the percentage or proportion of soluble recombinant protein, the percentage or proportion of active protein (e.g., amount of active protein / amount of protein used in the assay), the percentage or proportion of total cellular protein (tcp), the amount of protein / cell, and the percentage of dry biomass). The yield or amount of recombinant L-asparaginase is equivalent to or greater than the amount of soluble recombinant chrysantháspase produced in the periplasm obtained under similar or substantially similar conditions (conditions include, for example, host cells, genetic background of host cells (e.g., deletion of different proteases), type of promoter in the expression construct, growth temperature, OD at induction when an inducible promoter is used, amount of inducing factor added (e.g., amount of IPTG used for induction when a lacZ promoter or its derivative is used), duration of protein induction, growth temperature after adding the inducing agent to the culture, culture stirring rate, selection method for plasmid maintenance, culture volume in the container, and cell lysis method). In embodiments, the yield ratio of soluble recombinant L-asparaginase produced in the cytoplasm to soluble recombinant L-asparaginase produced in the periplasm obtained under similar or substantially similar conditions is about 1:1 (i.e., 1) to about 5:1 (i.e., 5). In embodiments, cytoplasmic production obtained under similar or substantially similar conditions The yield ratio of soluble recombinant L-asparaginase produced in the cytoplasm to soluble recombinant L-asparaginase produced in the periplasm is about 1 to about 5. In embodiments, the yield ratio of soluble recombinant L-asparaginase produced in the cytoplasm to soluble recombinant chrysanthase produced in the periplasm, obtained under similar or substantially similar conditions, is at least about 1. In embodiments, the yield ratio of soluble recombinant L-asparaginase produced in the cytoplasm to soluble recombinant L-asparaginase produced in the periplasm, obtained under similar or substantially similar conditions, is at most about 5. In embodiments, the yield ratios of soluble recombinant L-asparaginase produced in the cytoplasm versus soluble recombinant L-asparaginase produced in the periplasm, obtained under similar or substantially similar conditions, are approximately 1 to 1.25, 1 to 1.5, 1 to 1.75, 1 to 2, 1 to 2.5, 1 to 3, 1 to 3.5, 1 to 4, 1 to 4.5, 1 to 5, 1.25 to 1.5, 1.25 to 1.75, 1.25 to 2, 1.25 to 2.5, 1.25 to 3, 1.25 to 3.5, 1.25 to 4, 1.25 to 4.5, 1.25 to 5, 1.5 to 1.75, 1.5 to 2, and 1.5 to 2. 5, approximately 1.5 to approximately 3, approximately 1.5 to approximately 3.5, approximately 1.5 to approximately 4, approximately 1.5 to approximately 4.5, approximately 1.5 to approximately 5, approximately 1.75 to approximately 2, approximately 1.75 to approximately 2.5, approximately 1.75 to approximately 3, approximately 1.75 to approximately 3.5, approximately 1.75 to approximately 4, approximately 1.75 to approximately 4.5, approximately 1.75 to approximately 5, approximately 2 to approximately 2.5, approximately 2 to approximately 3, approximately 2 to approximately 3.5, approximately 2 to approximately 4, approximately 2 to approximately 4.5, approximately 2 to approximately 5, approximately 2.5 to approximately 3, approximately 2.5 to approximately 3.5, approximately 2.5 to approximately 4, approximately 2.5 to approximately 4.5, approximately 2.5 to approximately 5, approximately 3 to approximately 3.5, approximately 3 to approximately 4, approximately 3 to approximately 4.5, approximately 3 to approximately 5, approximately 3.5 to approximately 4, approximately 3.5 to approximately 4.5, approximately 3.5 to approximately 5, approximately 4 to approximately 4.5, approximately 4 to approximately 5, or approximately 4.5 to approximately 5. In embodiments, the yield ratio of soluble recombinant L-asparaginase produced in the cytoplasm versus soluble recombinant L-asparaginase produced in the periplasm, obtained under similar or substantially similar conditions, is approximately 1, approximately 1.25, approximately 1.5, approximately 1.75, approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, or approximately 5.

[0189] V. Treatment methods and methods for using recombinant chrysanthanspase protein A. Disease or disability The recombinant L-asparaginases of this disclosure may be used to treat diseases in human subjects, where the disease is treatable by asparagine depletion. In some embodiments, human subjects have experienced silent inactivation of E. coli-derived asparaginase prior to administration of recombinant L-asparaginase. In some embodiments, human subjects have experienced an allergic reaction to E. coli-derived asparaginase prior to administration of recombinant L-asparaginase. In some embodiments, human subjects have experienced anaphylaxis to E. coli-derived asparaginase prior to administration of recombinant L-asparaginase. Non-limiting examples of objective signs of allergy or hypersensitivity include testing positive for antibodies against the asparaginase enzyme.

[0190] In some embodiments, the recombinant L-asparaginase of the present disclosure is useful for the treatment of acute lymphoblastic leukemia (ALL) or for the manufacture of agents for use in the treatment of acute lymphoblastic leukemia (ALL). The relapse rate in ALL patients after treatment with L-asparaginase remains high, with approximately 10–25% of pediatric ALL patients experiencing early relapse (e.g., relapse may occur during the maintenance period 30–36 months after induction). If relapse occurs in patients treated with E. coli-derived L-asparaginase, subsequent treatment with E. coli formulations may produce a “vaccination” effect, thereby increasing the immunogenicity of the E. coli formulation in subsequent administrations. In one embodiment, the recombinant L-asparaginase of the present invention may be used in a method for treating patients with relapsing ALL who have been previously treated with other asparaginase formulations, particularly patients with relapsing ALL who have been previously treated with E. coli-derived asparaginase. In some embodiments, patients with relapsing ALL Recombinant L-asparaginase administered to patients is conjugated with a PEG moiety. In some embodiments, recombinant L-asparaginase administered to patients with recurrent ALL is not conjugated with a PEG moiety. In some embodiments, recombinant L-asparaginase administered to patients with recurrent ALL is conjugated with a peptide containing proline or alanine. In some embodiments, recombinant L-asparaginase administered to patients with recurrent ALL is not conjugated with a peptide containing proline or alanine.

[0191] In some embodiments, recombinant L-asparaginase of the present disclosure is useful for the treatment of lymphoblastic lymphoma (LBL) or for the manufacture of agents for use in the treatment of lymphoblastic lymphoma (LBL). In some embodiments, recombinant L-asparaginase administered to patients with relapsed LBL, as well as patients with ALL, is conjugated with a PEG moiety. In some embodiments, recombinant L-asparaginase administered to patients with relapsed LBL is not conjugated with a PEG moiety. In some embodiments, recombinant L-asparaginase administered to patients with relapsed LBL is conjugated with a peptide containing proline or alanine. In some embodiments, recombinant L-asparaginase administered to patients with relapsed LBL is not conjugated with a peptide containing proline or alanine.

[0192] Diseases or disorders for which recombinant L-asparaginase of this disclosure is useful in treatment include, but are not limited to, hematological malignancies, lymphomas, non-Hodgkin lymphomas, NK lymphomas, pancreatic cancers, Hodgkin's disease, immunoblastic large cell lymphomas, acute promyelocytic leukemia, acute myeloid leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute T-cell leukemia, acute myeloid leukemia (AML), diplasmic B-cell myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanoma, and diffuse large B-cell lymphoma (DLBCL). Other diseases or disorders for which recombinant L-asparaginase is useful in treatment include, but are not limited to, glioblastoma, including renal cell carcinoma, renal cell adenocarcinoma, glioblastoma multiforme and glioblastoma-astrocytoma, lung cancer, including medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung cancer, including large cell lung cancer and small cell lung cancer, endometrial cancer, ovarian adenocarcinoma, ovarian teratoma, cervical adenocarcinoma, breast cancer, mammary gland cancer, ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon cancer, colorectal adenocarcinoma, transitional cell carcinoma of the bladder, bladder papilloma, prostate cancer, osteosarcoma, epithelioid carcinoma of bone, prostate cancer, and thyroid cancer. Cancers may be solid tumors, such as lung cancer or breast cancer. Representative non-malignant hematological disorders that respond to asparagine depletion include, but are not limited to, immune-mediated hematological disorders, including infectious diseases such as those caused by HIV infection (i.e., AIDS). Non-hematological disorders associated with asparagine dependence include autoimmune diseases such as rheumatoid arthritis, collagen vascular disease, AIDS, osteoarthritis, Isaacs syndrome, psoriasis, insulin-dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus (SLE), rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), primary biliary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease. Cells suspected of causing disease can be tested for asparagine dependence in any suitable in vitro or in vivo assay, for example, in an in vitro assay in which the growth medium lacks asparagine.

[0193] Diseases or disorders for which the recombinant L-asparaginase of this disclosure is useful in treatment include sarcomas, breast cancer, metastatic breast cancer, liver cancer, gastric cancer, colorectal cancer, and head and neck cancers.

[0194] B. Methods for testing asparagine dependence Cells suspected of causing disease are tested in any suitable in vitro or in vivo assay, for example, in an in vitro assay where the growth medium is asparagine-free, in an asparagine-dependent manner. It can be tested for this. Accordingly, in some embodiments, the present disclosure relates to a method for treating a treatable disease in a patient, comprising administering an effective amount of the recombinant L-asparaginase of the present invention to the patient. In certain embodiments, the disease is ALL. In certain embodiments, the disease is LBL. In certain embodiments, the recombinant L-asparaginase used to treat a disease treatable by asparagine depletion comprises the sequence of SEQ ID NO: 1. In further embodiments, the recombinant L-asparaginase is not conjugated to a polymer such as PEG.

[0195] C. Method for evaluating the lowest serum asparaginase activity (NSAA) levels. Assays for measuring the lowest serum asparaginase activity (NSAA) in human subjects may be performed for evaluation of human subjects. In some embodiments, serum samples are taken from human subjects to evaluate NSAA. In some embodiments, whole blood samples are taken from subjects to evaluate NSAA. In some embodiments, the evaluation of NSAA is performed before recombinant L-asparaginase is administered to the patient. In some embodiments, the evaluation of NSAA is performed after recombinant L-asparaginase has been administered to the patient.

[0196] D. Treatment First-line treatment is the initial procedure administered for a disease. First-line treatment may be a monotherapy or a set of standard treatments.

[0197] Second-line treatment may be monotherapy or a set of standard treatments. Second-line treatment is administered after the initial treatment has failed, after its effectiveness has ceased (partially or completely), after the first treatment has caused unacceptable side effects, after the patient has decided to discontinue the initial treatment for any reason, or after a new treatment method has become available that may yield better results than the current treatment. In some embodiments, second-line treatment may be administered to human subjects in addition to first-line treatment for beneficial additive or synergistic results.

[0198] Additional elective therapies, including third, fourth, fifth, sixth, and any further elective therapies, are defined similarly to second-line therapies, except that both first and second-line therapies fail, their effects cease (partially or completely), they have unacceptable side effects, the patient decides to discontinue first and / or second-line therapies for any reason, or a new treatment becomes available that may produce better results than first and second-line therapies, or any combination of these reasons. Additional elective therapies may be monotherapy or a set of standard treatments. In some embodiments, additional elective therapies may be administered to human subjects in addition to first and / or second-line therapies for beneficial additive or synergistic results.

[0199] In some embodiments, treatment with recombinant L-asparaginase of the Disclosure is administered as first-line treatment. In other embodiments, treatment with recombinant L-asparaginase of the Disclosure is administered as second-line treatment in patients, particularly those with ALL and LBL, who have objective signs of allergy or hypersensitivity, including “silent inactivation” of other asparaginase preparations, in particular natural L-asparaginase derived from Escherichia coli or its PEGylated variant (pegaspargase). Non-limiting examples of objective signs of allergy or hypersensitivity include “antibody-positive” results for asparaginase enzymes on laboratory tests. Patients may have previously developed hypersensitivity to at least one E. coli-derived L-asparaginase and / or at least one Erwinia chrysanthemi-derived L-asparaginase. Hypersensitivity may be selected from the group consisting of allergic reactions, anaphylactic shock, and asymptomatic inactivation. In certain embodiments, recombinants of the present disclosure L-asparaginase is used as a second-line treatment after treatment with pegaspar gauze. In a more specific embodiment, the recombinant L-asparaginase of the present disclosure used as a second-line treatment comprises L-asparaginase produced in Pseudomonas fluorescens cells, more specifically, a tetramer-containing L-asparaginase, where each monomer or subunit contains the sequence of SEQ ID NO: 1.

[0200] In some embodiments, the recombinant L-asparaginase of this disclosure is used as a second-line treatment for patients who are hypersensitive to L-asparaginase derived from E. coli and / or who may have previously developed hypersensitivity to L-asparaginase derived from Erwinia chrysanthemi. In some embodiments, the recombinant L-asparaginase may be used as a second-line treatment for patients who are being administered long-acting asparaginase derived from E. coli. In some embodiments, six doses of recombinant L-asparaginase are administered to the patient instead of a single dose of long-acting asparaginase derived from E. coli. In some embodiments, the long-acting asparaginase derived from E. coli is pegaspargase. In some embodiments, treatment with the recombinant L-asparaginase of this disclosure is administered as a third-line treatment. In some embodiments, treatment with the recombinant L-asparaginase of this disclosure is administered as a fourth-line treatment. In some embodiments, treatment with recombinant L-asparaginase of the Disclosure is administered as a fifth-line treatment. In some embodiments, treatment with recombinant L-asparaginase of the Disclosure is administered as a sixth-line treatment. In some embodiments, treatment with recombinant L-asparaginase of the Disclosure is administered as maintenance therapy.

[0201] In some embodiments, treatment with recombinant L-asparaginase of the Disclosure is co-administered with a multi-agent chemotherapy regimen. In some embodiments, treatment with recombinant L-asparaginase of the Disclosure is co-administered with one or more other chemotherapy agents as part of a multi-agent chemotherapy regimen. In some embodiments, treating a patient with recombinant L-asparaginase of the Disclosure in addition to other agents helps ensure that asparaginase is available to patients who have developed hypersensitivity to E. coli-derived asparaginase. Examples of agents that may be part of a multi-agent chemotherapy regimen using recombinant L-asparaginase as disclosed herein include, but are not limited to, cytarabine, vincristine, daunorubicin, methotrexate, leucovorin, doxorubicin, anthracyclines, corticosteroids and glucocorticoids (including, but not limited to, prednisone, prednisolone, and / or dexamethasone), cyclophosphamide, 6-mercaptopurine, venetoclax, and etoposide. In some embodiments, the multi-agent chemotherapy regimen consists of recombinant L-asparaginase and one additional chemotherapeutic agent. In some embodiments, the multi-agent chemotherapy regimen consists of recombinant L-asparaginase and two or more additional chemotherapeutic agents.

[0202] As an example, patients with ALL are co-administered recombinant L-asparaginase of this disclosure with multi-agent chemotherapy during three chemotherapy phases, including the induction phase, the consolidation or intensification phase, and the maintenance phase. Specifically, recombinant L-asparaginase of this disclosure is co-administered with an asparagine synthase inhibitor (for example, as described in WO2007 / 103290, which is incorporated herein in its entirety by reference). In another specific example, recombinant L-asparaginase of this disclosure is not co-administered with an asparagine synthase inhibitor but is co-administered with other chemotherapeutic agents. In yet another specific example, recombinant L-asparaginase of this disclosure is co-administered with an asparagine synthase inhibitor and other chemotherapeutic agents. Recombinant L-asparaginase of this disclosure may be co-administered before, after, or concurrently with other compounds as part of a multi-agent chemotherapy regimen. In certain embodiments, the recombinant L-asparaginase of the present disclosure is a recombinant protein produced in Pseudomonas fluorescens, more specifically, a recombinant containing the sequence of SEQ ID NO: 1. Contains L-asparaginase.

[0203] VI. Compositions, Formulations, Routes of Administration, and Dosage A. Compositions, formulations, and routes of administration The present invention also includes pharmaceutical compositions comprising the recombinant L-asparaginase of the present disclosure. The pharmaceutical compositions may be administered to a patient using standard techniques. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, 22nd edition, Mack Publishing, 2015 (incorporated herein by reference).

[0204] The appropriate dosage form depends, in part, on the method of use or route of delivery, e.g., orally, transdermally, transmucosally, or by injection (parenterally). Such a dosage form must allow the therapeutic agent to reach target cells or otherwise exert the desired therapeutic effect. For example, a pharmaceutical composition injected into the bloodstream is preferably soluble. Pharmaceutical compositions according to this disclosure may be formulated as pharmaceutically acceptable salts and complexes thereof. A pharmaceutically acceptable salt is a non-toxic salt present at the amount and concentration in which they are administered. Formulations of such salts may facilitate medical use by altering the physical properties of the compound without interfering with the compound exerting its physiological effects. Useful alterations of physical properties include facilitating transmucosal administration by lowering the melting point and facilitating administration of higher concentrations of the drug by increasing solubility. The pharmaceutically acceptable salts of the modified proteins described herein may exist as complexes, as those skilled in the art will understand. Pharmaceutically acceptable salts include acid addition salts, such as sulfates, hydrochlorides, fumarates, maleates, phosphates, sulfamates, acetates, citrates, lactates, tartrates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, cyclohexylsulfamates, and quinates. Pharmaceutically acceptable salts can be obtained from acids, including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumarate, and quinic acid. When acidic functional groups, such as carboxylic acids or phenols, are present, pharmaceutically acceptable salts may include base addition salts containing, for example, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc. See, for example, Remington's Pharmaceutical Sciences (cited above). Such salts may be prepared using appropriate corresponding bases.To facilitate the administration of specific asparaginases, pharmaceutically acceptable carriers and / or excipients may also be incorporated into the pharmaceutical compositions according to the present invention. Examples of carriers suitable for use in the implementation of the present invention include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or various starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and physiologically compatible solvents. Examples of physiologically compatible solvents include water for injection (WFI), physiological saline, and sterile solutions of dextrose. The pharmaceutical compositions according to the present invention may be administered by various routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration. For oral administration, for example, the compound may be formulated into conventional oral dosage forms, such as capsules, tablets, and liquid formulations, such as syrups, elixirs, and concentrated drops. Alternatively, injection (parenteral administration), such as intramuscular, intravenous, intraperitoneal, and subcutaneous injections, may be used. For injection, the pharmaceutical composition is formulated in a liquid solution, preferably a physiologically compatible buffer or solution, such as saline, Hanks' solution, or Ringer's solution. In addition, the compound may be formulated in solid form and redissolved or suspended immediately before use. For example, recombinant L-asparaginase can be produced in lyophilized form. In certain embodiments, recombinant L-asparaginase is administered intramuscularly. In preferred specific embodiments, recombinant L-asparaginase is used. L-asparaginase is administered intravenously.

[0205] In preferred embodiments, the pharmaceutical composition is not freeze-dried. In further embodiments, the pharmaceutical composition is a solution. In some embodiments, the final freeze-drying step may induce stress and accelerate the degradation of the compound. In some embodiments, freeze-drying may increase aggregation. In some further preferred embodiments, the recombinant chrysanthanpasase is administered intramuscularly. In some further preferred embodiments, the recombinant chrysanthanpasase is administered intravenously. For each of these preferred embodiments of the dosage form, it will be understood that any form of recombinant L-asparaginase, including modified and unmodified forms, may be administered.

[0206] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrating agent suitable for the barrier to be penetrated is used in the formulation. Such penetrating agents are well known in the art, and examples of transmucosal administration include bile salts and fusidic acid derivatives. In addition, surfactants may be used to promote penetration. Transmucosal administration may be, for example, by nasal spray, intranasal spray, inhaler (delivery to the lungs), anal suppositories, or vaginal suppositories. For topical administration, the compound may be formulated into ointments, plasters, gels, or creams, as is well known in the art.

[0207] B. Administration In some embodiments, the dose is the amount administered to a human subject over a certain period of time and at a certain frequency. In some embodiments, the dose of recombinant L-asparaginase is administered to a human subject with hypersensitivity only when the hypersensitivity subsides.

[0208] In an exemplary embodiment, recombinant L-asparaginase is present in a dose of approximately 10 mg / m². 2 ~100mg / m 2 It is administered to human subjects in that amount.

[0209] In an exemplary embodiment, recombinant L-asparaginase is administered intramuscularly every other day for five consecutive days, followed by a two-day rest period, at a dose of approximately 25 mg / m². 2 That is the case.

[0210] In an exemplary embodiment, recombinant L-asparaginase is administered intravenously every other day for five consecutive days, followed by a two-day rest period, at a dose of approximately 37.5 mg / m². 2 That is the case.

[0211] In further exemplary embodiments, the recombinant L-asparaginase administered in such doses, according to any of the descriptions herein relating to administration, is not conjugated to a polymer such as a PEG moiety and / or to a peptide containing only alanine and / or proline residues.

[0212] 1.Dose The amount of recombinant L-asparaginase of this disclosure to be delivered depends on numerous factors, such as the compound's IC50, EC50, biological half-life, the patient's age, size, weight, and health status, as well as the disease or disorder being treated. The importance of these and other factors to be considered is well known to those skilled in the art. Generally, the amount of recombinant L-asparaginase of this disclosure is about 1 milligram (mg / m²) per square meter of the patient's body surface area. 2 ) ~1,000 / m 2 It is administered within a range, but is not limited to, approximately 10 mg / m² to treat diseases including ALL or LBL. 2 ~about 100mg / m 2 It is administered within the specified dosage range. Naturally, other dosages and / or treatment regimens may be used at the discretion of the attending physician.

[0213] In some embodiments, this method is used for approximately 10 mg / m² 2 ~about 100mg / m 2 The method includes administering recombinant L-asparaginase of the present disclosure in an amount of 10 mg / m². In some embodiments, the method is 10 mg / m². 2 ~100mg / m 2 This includes administering recombinant L-asparaginase of the Disclosure in an amount of about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 95, or about 95 mg / m². In some embodiments, recombinant L-asparaginase of the Disclosure is administered in an amount of about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 95, or about 95 mg / m². 2It is administered in an amount or equivalent thereto (for example, on a protein content basis). In more specific embodiments, the recombinant L-asparaginase of the present disclosure is administered in amounts of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, and about 100 mg / m². 2 It is administered in an amount selected from the group consisting of the following. In another specific embodiment, the recombinant L-asparaginase of the present disclosure is administered at approximately 1 mg / m². 2 More than about 10mg / m 2 More than about 15mg / m 2 More than about 20mg / m 2 More than about 25mg / m 2 More than about 30mg / m 2 Above, about 35mg / m 2 More than about 40mg / m 2 More than about 45mg / m 2 More than about 50mg / m 2 More than about 55mg / m 2 More than about 60mg / m 2 Above, about 65mg / m 2 More than about 70mg / m 2 Above, about 75mg / m 2 More than about 80mg / m 2 Above, about 85mg / m 2 Above, about 95mg / m 2 Above, about 95mg / m 2 More than about 100mg / m 2 More than about 200mg / m 2 Above, or approximately 300 mg / m² 2 It is administered in the above dose. In another specific embodiment, the recombinant L-asparaginase of the present disclosure is administered at approximately 300 mg / m². 2 Below about 200mg / m 2 Below, about 100mg / m 2 Below, approximately 95mg / m 2 Below, approximately 80mg / m 2 Below, about 75mg / m 2 Below about 70mg / m 2 Below about 65mg / m 2 Below about 60mg / m 2 Below, about 55mg / m 2 Below about 50mg / m 2 Below about 45mg / m2 Below about 40mg / m 2 Below, about 35mg / m 2 Below, about 30mg / m 2 Below about 25mg / m 2 Below, about 20mg / m 2 Below, about 15mg / m 2 Below, about 10mg / m 2 Below, about 5mg / m 2 The following, or approximately 1 mg / m² 2 It is administered in the following doses. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 12 mg / m². 2 ~about 90mg / m 2 It is administered in an amount of approximately 20 mg / m². In another exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in approximately 20 mg / m². 2 ~about 80mg / m 2 It is administered in an amount of approximately 25 mg / m². In another exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in approximately 25 mg / m². 2 ~about 70mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 25 mg / m². 2 ~about 80mg / m 2 It is administered in the amount of approximately 37.5 mg / m². In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered in approximately 37.5 mg / m². 2 ~about 80mg / m 2 It is administered in the amount of approximately 37.5 mg / m². In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered in approximately 37.5 mg / m². 2 ~about 65mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 25 mg / m². 2 ~about 37.5mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 25 mg / m². 2 ~about 100mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 25 mg / m². 2 ~about 65mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 25 mg / m². 2~about 80mg / m 2 It is administered in that amount.

[0214] In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 75mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 35 mg / m². 2 ~about 70mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 40 mg / m². 2 ~about 65mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered in that amount.

[0215] In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 40 mg / m². 2 ~about 75mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparagus [of the present disclosure] The amount of Nase is approximately 45 mg / m². 2 ~about 70mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 50 mg / m². 2 ~about 65mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of the present disclosure is administered at approximately 55 mg / m². 2 ~about 60mg / m 2 It is administered in that amount.

[0216] In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 40 mg / m². 2 ~about 60mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 45 mg / m². 2~about 55mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 47.5 mg / m². 2 ~about 50mg / m 2 It is administered in that amount.

[0217] In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 35mg / m 2 It is administered in that amount.

[0218] In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 95mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 35 mg / m². 2 ~about 90mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 40 mg / m². 2 ~about 85mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 45 mg / m². 2 ~about 80mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 50 mg / m². 2 ~about 75mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of the present disclosure is administered at approximately 55 mg / m². 2 ~about 70mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 60 mg / m². 2 ~about 65mg / m 2 It is administered in that amount.

[0219] In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 60mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 35 mg / m². 2 ~about 55mg / m2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 40 mg / m². 2 ~about 50mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 42.5 mg / m². 2 ~about 57.5mg / m 2 It is administered in that amount.

[0220] In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 75mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 35 mg / m². 2 ~about 70mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 40 mg / m². 2 ~about 65mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered in that amount.

[0221] In some embodiments, the recombinant L-asparaginase of the present disclosure is approximately 10 mg / m². 2 ~about 50mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 12.5 mg / m². 2 ~about 47.5mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 15 mg / m². 2 ~about 45mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 20 mg / m². 2 ~about 42.5mg / m 2It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 22.5 mg / m². 2 ~about 40mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 24 mg / m². 2 ~about 39mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 27 mg / m². 2 ~about 37.5mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 45mg / m 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 25 mg / m². 2 It is administered intramuscularly in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of the present disclosure is 25 mg / m². 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 25 mg / m². 2 ~about 80mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 37.5 mg / m². 2 ~about 80mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 37.5 mg / m². 2 ~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 25 mg / m². 2 ~about 37.5mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 75mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 35 mg / m². 2 ~about 70mg / m 2It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 40 mg / m². 2 ~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 40 mg / m². 2 ~about 75mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 45 mg / m². 2 ~about 70mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 50 mg / m². 2 ~about 65mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 55 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 40 mg / m². 2 ~about 60mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 45 mg / m². 2 ~about 55mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, recombinant L-asparaginase of the present disclosure is administered at approximately 47.5 mg / m². 2 ~about 50mg / m 2 It is administered intramuscularly in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 35mg / m 2 It is administered intramuscularly in that amount.

[0222] In some embodiments, the recombinant L-asparaginase of the present disclosure is approximately 10 mg / m².2 ~about 95mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 20 mg / m². 2 ~about 60mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 22.5 mg / m². 2 ~about 57.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 25 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 27.5 mg / m². 2 ~about 47.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 30 mg / m². 2 ~about 45mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 32.5 mg / m². 2 ~about 42.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 21.5 mg / m². 2 ~about 38.5mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is approximately 36 mg / m². 2 ~about 45mg / m 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 37.5 mg / m². 2 It is administered intravenously in the amount of [amount]. In some embodiments, the recombinant L-asparaginase of this disclosure is 37.5 mg / m². 2 It is administered intravenously in that amount.

[0223] In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 25 mg / m². 2 ~about 37.5 mg / m² 2It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 25 mg / m². 2 ~about 100mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 25 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 25 mg / m². 2 ~about 80mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 30 mg / m². 2 ~about 35mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 30 mg / m². 2 ~about 95mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 35 mg / m². 2 ~about 90mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 40 mg / m². 2 ~about 85mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 45 mg / m². 2 ~about 80mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 50 mg / m². 2 ~about 75mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 55 mg / m². 2 ~about 70mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 60 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 30 mg / m². 2 ~about 60mg / m 2It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 35 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 40 mg / m². 2 ~about 50mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 42.5 mg / m². 2 ~about 57.5mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 30 mg / m². 2 ~about 75mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is administered at approximately 35 mg / m². 2 ~about 70mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 40 mg / m². 2 ~about 65mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 45 mg / m². 2 ~about 60mg / m 2 It is administered intravenously in the amount of [amount]. In exemplary embodiments, the recombinant L-asparaginase of this disclosure is approximately 50 mg / m². 2 ~about 55mg / m 2 It is administered intravenously in that amount.

[0224] In another embodiment, the method includes administering recombinant L-asparaginase of the present disclosure, which induces a weaker immunogenic response in a patient compared to non-recombinant L-asparaginase.

[0225] 2. Dosage frequency In certain embodiments, the treatment is approximately 1 mg / m². 2 ~about 1000mg / m 2 Typically about 10 mg / m² 2 ~about 100mg / m 2In doses ranging from approximately three times a week to approximately once a month, typically on a schedule ranging from once a week to once every week, administered as a single agent (e.g., monotherapy) or as part of a combination of chemotherapeutic agents, including but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracyclines, glucocorticoids, corticosteroids, anticancer compounds, or other agents.

[0226] The recombinant L-asparaginases of this disclosure may be administered before, after, or concurrently with other compounds as part of a multi-agent chemotherapy regimen. In certain embodiments, the recombinant L-asparaginases of this disclosure include recombinant L-asparaginases comprising a protein recombinantly produced in Pseudomonas fluorescens, more specifically, a recombinant L-asparaginase comprising the sequence described in SEQ ID NO: 1. In some embodiments, the recombinant L-asparaginases of this disclosure -ze is administered as a single dose in a dose that depletes L-asparagine to undetectable levels for approximately 3 to 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) using methods and apparatus known in the art.

[0227] In some embodiments, the recombinant L-asparaginase of the Disclosure is administered three times a week. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered every other day for five consecutive days, followed by a two-day rest period. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered on Monday, Wednesday, and Friday of the same week.

[0228] In some embodiments, the recombinant L-asparaginase of the Disclosure is administered three times a week for at least 1 to 3 weeks. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered every other day for 5 consecutive days for about 1 to 3 weeks, followed by a 2-day rest period. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered on Mondays, Wednesdays, and Fridays of a week for about 1 to 3 weeks.

[0229] In some embodiments, the recombinant L-asparaginase of the Disclosure is administered three times a week for about two weeks. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered every other day for five consecutive days for about two weeks, followed by a two-day rest period. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered on Monday, Wednesday, and Friday of the same week for about two weeks.

[0230] In some embodiments, the recombinant L-asparaginase of the Disclosure is administered three times a week for two weeks. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered every other day for five consecutive days for two weeks, followed by a two-day rest period. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered on Monday, Wednesday, and Friday of the same week for two weeks.

[0231] In some embodiments, the recombinant L-asparaginase of the Disclosure is administered three times a week, and this continues until the patient's asparagine depletion eliminates treatable disease. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered every other day for five consecutive days, followed by a two-day rest period, and this continues until the patient's asparagine depletion eliminates treatable disease. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered on Monday, Wednesday, and Friday of the same week, and this continues until the patient's asparagine depletion eliminates treatable disease.

[0232] In some embodiments, the recombinant L-asparaginase of the Disclosure is administered three times a week, continuing until the patient decides to discontinue or postpone the treatment. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered every other day for five consecutive days, followed by a two-day rest period, continuing until the patient decides to discontinue or postpone the treatment. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered on Monday, Wednesday, and Friday of the same week, continuing until the patient decides to discontinue or postpone the treatment.

[0233] In some embodiments, the recombinant L-asparaginase of the Disclosure is administered approximately every 48 hours. In some embodiments, the recombinant L-asparaginase of the Disclosure is administered approximately every 72 hours.

[0234] In some embodiments, the recombinant L-asparaginases of the present disclosure may be hypersensitive to L-asparaginases derived from E. coli and / or may have previously developed hypersensitivity to L-asparaginases derived from Erwinia chrysanthemi. It is administered to patients as a second-line treatment.

[0235] In some embodiments, recombinant L-asparaginase is administered to human subjects in place of a long-acting asparaginase derived from E. coli. In some embodiments, six doses of recombinant L-asparaginase are administered to human subjects in place of a single dose of long-acting asparaginase derived from E. coli. In some embodiments, the long-acting asparaginase derived from E. coli is pegaspargase. In further embodiments, the six divided doses may be administered over a period of approximately two weeks.

[0236] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle comprising a first dose, a second dose, and a third dose, the cycle being optionally repeatable, and the first, second, and third doses being administered at intervals of approximately 48 to 72 hours.

[0237] In some embodiments, the dose may vary within a cycle.

[0238] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle, which is optionally repeatable, and which comprises administration of recombinant L-asparaginase every other day for 5 consecutive days, followed by a 2 consecutive day rest period, with the first dose of the cycle being 25 mg / m². 2 Therefore, the second dose of that cycle is 25 mg / m². 2 Therefore, the third dose of that cycle was 37.5 mg / m². 2 This is followed by a two-day drug-free period.

[0239] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle, which is optionally repeatable, and which comprises administration of recombinant L-asparaginase every other day for 5 consecutive days, followed by a 2 consecutive day rest period, with the first dose of the cycle being 37.5 mg / m². 2 Therefore, the second dose of that cycle was 37.5 mg / m². 2 Therefore, the third dose of that cycle was 37.5 mg / m². 2 This is followed by a two-day drug-free period.

[0240] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle, which is optionally repeatable, and which comprises administration of recombinant L-asparaginase every other day for 5 consecutive days, followed by a 2 consecutive day rest period, with the first dose of the cycle being 37.5 mg / m². 2 Therefore, the second dose of that cycle is 25 mg / m².2 Therefore, the third dose of that cycle was 37.5 mg / m². 2 This is followed by a two-day drug-free period.

[0241] In some embodiments, the recombinant L-asparaginase administration regimen comprises a cycle, which is optionally repeatable, and which comprises administration of recombinant L-asparaginase every other day for 5 consecutive days, followed by a 2 consecutive day rest period, with the first dose of the cycle being 37.5 mg / m². 2 Therefore, the second dose of that cycle is 25 mg / m². 2 Therefore, the third dose of that cycle is 25 mg / m². 2 This is followed by a two-day drug-free period. In some embodiments, the first dose of the cycle is administered on Monday, the second dose of the cycle is administered on Wednesday, and the third dose of the cycle is administered on Friday.

[0242] The administration regimen may encompass any number of cycles over any number of weeks, or any number of cycles up to any endpoint specified herein.

[0243] 3. Administration time In some embodiments, the administration time to human subjects is short, e.g., immediate or 1 second to 5 minutes, when delivered by intramuscular injection. In other embodiments, the administration time to human subjects is longer, for example, when delivered by intravenous injection. In some embodiments, the administration of recombinant L-asparaginase lasts from about 5 minutes to about 4 hours. In exemplary embodiments, the administration time to human subjects lasts from 90 minutes to about 150 minutes. In some embodiments, the administration time to human subjects lasts from about 2 hours. In some embodiments, the administration time to human subjects lasts from about 45 minutes to about 75 minutes. In some embodiments, the administration time to human subjects lasts from about 1 hour. In some embodiments, the administration time to human subjects lasts from 1 hour. [Examples]

[0244] Example 1: Test to evaluate toxicity and toxicological effects The toxicity and toxicological kinetics (TK) of recombinant L-asparaginase were investigated in rats using a 2-week daily intravenous infusion study under good laboratory standards (GLP). A 2-week recovery period was included to assess the reversibility or persistence of the effect and / or delayed toxicity. Considering that recombinant L-asparaginase is a biotechnology-derived product, the development of anti-drug antibodies (ADAs) was prospectively evaluated in all primary test animals to aid in interpreting the study results.

[0245] Recombinant L-asparaginase was administered to four groups of male and female Crl:CD(SD) rats (n=10 / sex / group) at doses of 0, 4.6, 15.2, and 45.6 mg / kg, respectively. Recovered animals were included in the control and high-dose groups (n=5 / sex / group). Toxicological evaluation animals (n=3 / sex for the control group and n=9 / sex / dose group) were also included. Recombinant L-asparaginase was administered intravenously over 2 hours (±10 minutes) at a rate of 5 mL / kg / hour via a catheter inserted into the femoral vein. The vehicle and control substances were 20 mM sodium phosphate, 50 mM sodium chloride, and 6.4% trehalose, with a pH of 7.0.

[0246] The sentinel group also received the highest dose before the start of the main trial, was observed for two days, and was euthanized on the third day without necropsy.

[0247] Mortality rates, clinical findings, body weight, changes in body weight, food intake, ocular findings, and clinicopathological and anatomical findings were recorded, along with toxicological analysis (assessed by serum asparaginase activity (SAA)) and anti-drug antibody analysis.

[0248] Toxicity and toxicological evaluation animals in groups 1 (control) to 3 (15.2 mg / kg) were administered for 14 days and survived until their scheduled necropsy. Administration in group 4 (45.6 mg / kg) was discontinued on day 6 due to adverse clinical findings; at this point, the animals were either necropsy according to their assigned group or transferred to a two-week recovery period. Recovered animals in group 1 were administered the vehicle once daily for 14 days, followed by a six-day recovery period, and were therefore euthanized on the same calendar day as the recovered animals in group 4.

[0249] SAA C max and AUC O-24 As assessed, exposure increased with increasing dose levels, from 4.6–45.6 mg / kg / day on day 1 and 4.6–15.2 mg / kg / day on day 14. max and AUC 0-24 The increase in values ​​was roughly dose-proportional, ranging from 4.6–15.2 and 15.2–45.6 mg / kg / day on day 1 and from 4.6–15.2 mg / kg / day on day 14. However, for 4.6–45.6 mg / kg / day on day 1, the AUC was different for a 10-fold increase in dose. 0-24 It increased 18-fold. SAA C max and AUC 0-24 There were no significant sex differences in the values. SAA accumulation was not observed in rats after repeated administration.

[0250] In high-dose animals euthanized early, clinical findings included piloerection, coat stiffness, redness of the skin on the feet, and clear oral secretions. Significant changes in hematological values ​​indicating decreased hematopoiesis were observed, including decreased reticulocyte, platelet, and white blood cell counts. These changes were found to be reversible in recovered animals. Increased blood glucose and serum urea nitrogen concentrations, as well as decreased protein and liver enzymes, suggested dehydration, prerenal azotemia, protein loss or intercompartmental migration, and downregulation of liver enzyme activity, respectively. Macroscopic findings were limited to localized redness of the mucosa in the glandular stomach of one female, which lacked microscopic correlation. Microscopic findings included decreased cellular integrity in the bone marrow of the femur, myocardial necrosis and myocardial hemorrhage, submucosal edema / abscess and epithelial hyperplasia / erosion in the non-glandular gastric / duodenal region, decreased red pulp, extramedullary hematopoiesis and lymphocytes in the spleen, decreased cortical lymphocytes in the thymus, and decreased pancreatic secretion. These changes were considered adverse. Increased spleen and thymic weight was observed in males and females at the time of sacrifice of recovered animals, but this was considered consistent with recovery. Microscopic evaluation of recovered animals was not performed.

[0251] A decrease in the dose-correlation of food intake and body weight was observed in animals administered at doses of 15.2 mg / kg / day or less, and piloerection was observed at 15.2 mg / kg / day. Changes in the dose-correlation of hematological and clinical chemistry values ​​were generally consistent with those observed in the high-dose group, although smaller changes were observed in most animals. These clinicopathological changes were not considered adverse. Microscopic findings in low- and medium-dose animals consisted of a slight to mild decrease in erythrocyte progenitor cells and an increase in myeloid progenitor cells, which were consistent with the hematological findings. A decrease in spleen weight correlated with a decrease in extramedullary hematopoiesis and red pulp, but a decrease in thymus weight did not show a microscopic correlation.

[0252] In conclusion, male and female Crl:CD(SD) rats were administered either a vehicle control or recombinant L-asparaginase at 4.6, 15.2, or 45.6 mg / kg / day intravenously once daily for 14 days. Adverse clinical findings requiring euthanasia and discontinuation of administration on day 6 of the administration phase, as well as decreased cellular integrity in the bone marrow of the femur, myocardial necrosis and myocardial hemorrhage in the heart, submucosal edema or abscess and epithelial hyperplasia in the non-glandular stomach, and gastric or duodenal erosion / ulcers, occurred in animals administered at 45.6 mg / kg / day. Non-adverse clinical findings, clinicopathological changes, and microscopic findings occurred in animals administered at 15.2 mg / kg / day or less. Therefore, the no-observed-adverse-effect level (NOAEL) is 15.2 mg / kg / day.

[0253] The highlights of this study include: male and female Crl:CD(SD) rats (n=10 / sex / group) with control and recovery groups (n=5 / sex / group) at high doses. Recombinant L-asparaginase (0, 4.6, 15.2, or 45.6 mg / kg / day) was administered daily by intravenous infusion over 2 hours for up to 14 days. In the high-dose group, euthanasia and discontinuation of administration were required on day 6 due to adverse clinical findings. Adverse findings, including decreased cytoplasmic density in the bone marrow of the femur, necrosis and hemorrhage in the heart, and erosion, edema, hyperplasia, abscess, and / or ulceration in the non-glandular gastric / duodenal region, occurred in the high-dose group. Non-adverse but qualitatively similar clinical findings, clinicopathological changes, and microscopic findings occurred in animals administered at doses of 15.2 mg / kg / day or less. The no-observed-adverse-effect level (NOAEL) was determined to be 15.2 mg / kg / day.

[0254] Example 2: Test to evaluate safety The subjects were between 18 and 55 years old and were in good health as determined by the clinical trial physician. In administration cohort 1, subjects received recombinant L-asparaginase (25 mg / m²) via either a 2-hour intravenous (IV) infusion (N=6) or intramuscular (IM) infusion (N=6). 2 They were randomized (1:1) to receive a single dose of ) to determine the need for another dosing cohort. To that end, the safety, tolerability, and pharmacokinetics of recombinant L-asparaginase were evaluated, and the subjects of administration cohort 2 received a dose of 37.5 mg / m². 2 IV (N=6) or 12.5 mg / m² 2 Participants were randomized (1:1) to receive a single dose of recombinant L-asparaginase via one of the IV (N=6) intensive care units (IM). Recombinant L-asparaginase was administered in the inpatient ward, and participants were discharged on day 5. Safety follow-up by telephone was conducted on days 6 and 30. The primary objective was to evaluate the safety and tolerability of recombinant L-asparaginase with IV and IM administration in each cohort. A secondary objective included characterizing the pharmacokinetics of recombinant L-asparaginase with IV and IM administration based on serum asparaginase activity (SAA).

[0255] The demographic characteristics (mean ± SD) of the 24 registered participants included: age (38.4 ± 8.30 years), weight (77.04 ± 10.00 kg), and body surface area (1.91 ± 0.15 m²). 2 ). In addition, 63% of the subjects were male, 97% were Hispanic / Latin American, 83% were white, and 17% were Black / African American. Both safety and pharmacokinetics were evaluated in this study. Regarding safety, 8 / 12 (67%) of subjects in administration cohort 1 experienced one or more adverse events (IV=4 subjects; IM=4 subjects). In administration cohort 2, 11 / 12 (92%) of subjects experienced one or more adverse events (IV=6 subjects; IM=5 subjects). No serious adverse events or grade 3 or higher adverse events were reported in any subject in either administration cohort. The most commonly observed treatment-dependent adverse event in two or more subjects in each administration cohort was nausea (see Figure 1 and Table 1). Dyspeptic malnutrition occurred at 12.5 mg / m² 2 The most common adverse event in subjects administered recombinant L-asparaginase via intravenous injection (IM) was [unclear] (see Figure 1 and Table 1). Pharmacokinetic evaluation was performed at 12.5 and 25 mg / m² when administered via IM. 2In the administration cohort, serum asparaginase activity levels of recombinant L-asparaginase reached 0.1 IU / mL or higher in 6 / 6 (100%) of the subjects at 48 and 72 hours after administration. After IV administration, 25 mg / m² 2 At this dose level, serum asparaginase activity levels reached 0.1 IU / mL or higher in 6 / 6 (100%) of subjects 48 hours after administration and 4 / 6 (67%) of subjects 72 hours after administration, while at 37.5 mg / m² 2 At this dose level, 6 / 6 (100%) of subjects reached 0.1 IU / mL or higher 48 and 72 hours after administration (see Figure 1 and Table 2).

[0256] In healthy adults, recombinant L-asparaginase administration was well-tolerated, no unexpected adverse events were observed, serum asparaginase activity was not reported, and no grade 3 or higher adverse events were observed. Serum asparaginase activity levels of 0.1 IU / mL or higher (a surrogate marker for asparagine depletion) were achieved at 48 hours in all human subjects who received recombinant L-asparaginase IM and IV. SAA levels of 0.1 IU / mL or higher were also achieved 72 hours after recombinant L-asparaginase administration, and 25 mg / m² was also observed. 2 This was achieved in all subjects except for two subjects in group IV. Based on the overall pharmacokinetic and safety data of this study, the recommended starting dose for Phase 2 / 3 is 25 mg / m² via the IM route with a Monday / Wednesday / Friday dosing schedule. 2 For the IV administration route, 37.5 mg / m² 2 That is the case.

[0257] Example 3: Development and simulation of a population pharmacokinetic (PK) model A population PK model for recombinant L-asparaginase was developed using aggregated serum asparaginase activity (SAA) data from a Phase 1 single-dose trial in healthy adult subjects (sbj), and the effects of endogenous covariates (body weight, body surface area, age, sex, and race) on PK parameters were evaluated. This population PK model was developed to determine the starting dose to be selected for a central Phase 2 / 3 trial of recombinant L-asparaginase. The starting dose was 25 mg / m². 2 [N=6] and 37.5 mg / m² 2 Intravenous (IV) infusion data for [N=6], and data for 12.5 mg / m² 2 [N=6] and 25 mg / m² 2 This includes intramuscular (IM) injection data from [N=6]. A total of 24 subjects were included in the model. Adult and pediatric SAA profiles (1000 sbj / population) were simulated using the developed model to investigate the possibility of achieving therapeutic target trough SAA levels of 0.1 IU / mL or higher based on different doses, schedules, and routes of administration.

[0258] The final model describing both the IV and IM pathways is a one-compartment model with linear elimination (IV) and mixed-order absorption (IM only), with body weight included as a non-proportional covariate to SAA clearance. Body size assessment measures (i.e., body weight and BSA (body surface area)) were identified as statistically significant covariates, accounting for 2.8% and 3.4% of the variability in recombinant L-asparaginase PK, respectively. Based on Phase 1 trial data and population PK modeling and simulation, the recommended starting dose for Phase 2 / 3 is 25 mg / m² for the IM administration route with a Monday / Wednesday / Friday dosing schedule. 2 For the IV administration route, 37.5 mg / m² 2 The recommended dose and schedule are expected to maintain a trough SAA level of 0.1 IU / mL or higher 72 hours after administration.

[0259] Example 4: Test to evaluate safety and efficacy This study is an open-label, multicenter, Phase 2 / 3 dose-confirming and pharmacokinetic study of recombinant L-asparaginase in patients with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) who exhibit hypersensitivity (allergic reaction or silent inactivation) to E. coli-derived asparaginase and whose treatment plan (Table 3) includes two or more doses of E. coli-derived asparaginase. The study is designed to evaluate the tolerability and efficacy of recombinant L-asparaginase, as measured by asparaginase activity, in patients who develop hypersensitivity to E. coli-derived asparaginase. In these patients, six doses of recombinant L-asparaginase will be used in place of each dose of long-acting E. coli-derived asparaginase. Individual patient treatment durations will vary depending on the number of E. coli-derived asparaginase doses remaining in the patient's original treatment plan.

[0260] This study consists of two consecutive parts: Part A, which determined the dose of recombinant L-asparaginase for intramuscular (IM) administration and confirmed its safety and efficacy; and Part B, which identified the optimal dose and schedule for intravenous (IV) infusion of recombinant L-asparaginase.

[0261] Part A of this trial has two IM cohorts. Cohort 1 is a repeated-dose / confirmatory cohort in which the final IM dose level will be selected. Cohort 2 is an extended cohort in which the efficacy and safety of the final IM dose level and schedule will be confirmed.

[0262] Part B will be conducted to determine the optimal dose of recombinant L-asparaginase intravenously for further testing in ALL / LBL patients as repeated doses.

[0263] As long as the patient achieves clinical efficacy, additional cycles of recombinant L-asparaginase will be administered based on each patient's original treatment plan.

[0264] Blood samples were collected at predetermined times to determine serum asparaginase activity levels. Patients were monitored for adverse events. Immunogenicity of recombinant L-asparaginase treatment was also evaluated.

[0265] The first objective is (1) the response rate in the first cycle of recombinant L-asparaginase IM administration (the response rate is defined as the response rate of 0.1 IU / mL or higher in the most recent 72 hours in the first cycle of treatment). (1) Determine the proportion of patients with the lowest level of serum asparaginase activity, and (2) evaluate the occurrence of adverse events (TEAEs) that occur under treatment.

[0266] The second objectives are to determine (1) the proportion of patients showing a minimum serum asparaginase activity level of 0.1 IU / mL or higher in the preceding 48 hours during the first cycle of recombinant L-asparaginase IM administration within a two-week timeframe, (2) the proportion of patients showing a minimum serum asparaginase activity level of 0.4 IU / mL or higher in the preceding 48 hours during the first cycle of recombinant L-asparaginase IM administration within a two-week timeframe, (3) the proportion of patients showing a minimum serum asparaginase level of 0.4 IU / mL or higher in the preceding 72 hours during the first cycle of recombinant L-asparaginase IM administration within a two-week timeframe, (4) the pharmacokinetic characterization of recombinant L-asparaginase IM infusion using a population pharmacokinetic approach and exposure-related correlations based on serum asparaginase activity in a timeframe up to 30 days after the last dose, and (5) the incidence of anti-drug antibody formation against recombinant L-asparaginase in a timeframe up to 30 days after the last dose.

[0267] The eligibility criteria include both males and females. Inclusion criteria include: (1) pediatric and adult patients with a diagnosis of ALL or LBL; (2) patients who have had an allergic reaction to E. coli-derived long-acting asparaginase or who have silent inactivation; (3) patients with one or more courses of E. coli-derived asparaginase remaining in their treatment plan; and (4) patients who, in the judgment of the investigator, must be completely recovered from a previous allergic reaction to E. coli-derived asparaginase. Exclusion criteria include: (1) patients who have previously received L-asparaginase derived from Erwinia chrysanthemi or recombinant L-asparaginase; (2) patients with recurrent ALL or LBL; (3) patients who are currently receiving another investigational drug and / or have been treated with an investigational medical device concurrently with recombinant L-asparaginase (within 48 hours) during one cycle of recombinant L-asparaginase; (4) patients with a history of grade 3 or higher pancreatitis; (5) patients with a history of asparaginase-related thrombosis requiring anticoagulation therapy, excluding grade 3 or higher bleeding events related to asparaginase or catheter-related thrombotic events.

[0268] Further trials are underway in an additional subcohort at 37.5 mg / m². 2 The treatment will be performed at an even higher dose, and each additional dose level(s) will not exceed a 50% increase from the previous dose level.

[0269] This trial allows registration for the evaluation of IV and IM formulations, and the involvement of the testing center is left to the sponsor's discretion.

[0270] Undetectable SAA levels may be based on the limit of quantification defined by an accredited laboratory authorized to perform this test under CLIA. CLIA-accredited laboratories utilize different limits of quantification depending on their assay method, and the results for determining undetectable SAA levels may vary between laboratories.

[0271] Serum asparaginase concentration is the same as the content of the PK in this test. Consistency between SAP (using serum asparaginase concentration) and the label on the laboratory tube (using the content of the PK).

[0272] In certain preferred embodiments, the time frame for the tests is 7 days for all clinical tests, including coagulation tests and clinical tests at the time of screening.

[0273] Example 5: Test to evaluate protein aggregation Quality testing was conducted on small batches of recombinant L-asparaginase produced on a laboratory scale. This testing consisted of an analytical evaluation of equivalence and homogeneity with the commercial product Erwinase® to confirm that the quality, safety, and potency of the recombinant L-asparaginase were equivalent to natural L-asparaginase (currently in clinical use) obtained from the spontaneous fermentation of Dickeya chrysanthemi, and were not adversely affected by the use of alternative expression systems and purification processes.

[0274] SEC-HPLC method: Purity was determined by size exclusion chromatography using SEC-HPLC with a Phenomenex BioSep SEC-s4000 column. Before injection, the column was conditioned with mobile phase (50 mM phosphate, 200 mM NaCl, pH 7.0) at 1 mL / min for 1 hour. BioRad gel filtration standards were diluted to 1 mg / mL in the mobile phase and injected in triple repeats after multiple injections of mobile phase blank to wash the column. The purity of recombinant L-asparaginase, recombinant chrysanthase from E. coli, and Erwinase® was evaluated (Figure 2).

[0275] The SEC-HPLC results show a main peak representing the tetramer and an aggregate peak. Different levels of HMW aggregates were present in all samples, with more aggregates in Erwinaze CAMR176 (6%) compared to recombinant L-asparaginase (0.3%).

[0276] Using the standard operating procedures of size exclusion chromatography-multi-angle light scattering (SEC MALLS): C.52.S1640 and Solvias, test solutions were prepared in double replicates for all samples. The samples were diluted to 10 mg / mL in a compounding buffer and mixed. At least 4 mL was prepared. The sample solution was filtered through a 0.2 μM syringe filter and directly into an HPLC vial. The first few drops were discarded. If the protein concentration was lower than 10 mg / mL, it was necessary to adjust the injection volume to allow for the injection of 750 μg. The following samples and reference substances were analyzed using this technique: all unstressed batches, and all batches stressed by 5 and 10 overnight freezes at -75°C and thawing at room temperature for 3 hours, respectively. The results are shown in Figure 3.

[0277] Based on the SEC MALLS results, the SEC-HPLC results were confirmed, and when compared to recombinant L-asparaginase (1%), the level of HMW aggregates was higher in Erwinaze® CAMR176 (6%).

[0278] Sedimentation velocity AUC (svAUC) method: Recombinant L-asparaginase and Erwinase® samples were diluted to 10 mg / mL in 20 mM sodium phosphate (pH 7.0) and dialyzed against the same buffer. The samples were then placed in an AN-60Ti analytical rotor and loaded into a Beckman Coulter ProteomeLab XL analytical ultracentrifuge. The data were analyzed using the c(s) function developed by Peter Schuck of the NIH and implemented in his analytical program SEDFIT (version 11.3). The resulting size distributions were graphed, and peaks were merged using OriginLab Origin® version 9.0. The results in Figure 4 demonstrate that recombinant L-asparaginase showed the highest homogeneity (based on the lowest aggregate content of 1%), while the Erwinase® sample was the least homogeneous (5% aggregates). In conclusion, recombinant L-asparaginase exhibits lower aggregate levels compared to Erwinase®, as can be measured by multiple methods with different detection principles.

[0279] Example 6: Test to evaluate protein aggregation on a commercial scale This quality test was performed on recombinant L-asparaginase in a production-scale batch manufactured at 1000 L (intended commercial scale). The test consisted of an analytical evaluation of equivalence and homogeneity with the commercial product Erwinase® to confirm that the equivalence and homogeneity testing of recombinant L-asparaginase in small-scale (SS) production in Example 5 was supported at production scale (1000 L). The SEC-MALLS method was also used to characterize the recombinant L-asparaginase.

[0280] A 1 mL aliquot of recombinant L-asparaginase was pipetteed into a 5 mL PETG bottle and stored at -20°C. The mixture consisted of 20 mM sodium phosphate, 50 mM sodium chloride, and 6.4% trehalose, with a pH of 7.0.

[0281] SE-UHPLC method: The purity of recombinant L-asparaginase DS samples is determined based on molecular size, i.e., hydrodynamic radius, using size exclusion ultra-high pressure liquid chromatography (SE-UPLC) with UV detection. Samples and standards are diluted to 2.5 mg / mL in the mobile phase (10 mM phosphate, 0.3 M arginine, 0.05% sodium azide) and injected into a Waters Acquity UPLC column (BEH 200 SEC, 1.7 μm, 4.6 x 30 mm). Recombinant L-asparaginase DS is eluted at a uniform concentration of 0.4 mL / min. The purity profile is characterized by UV detection at 280 nm. Purity is reported as an integrated % tetramer (main peak), % HMW, and % LMW. The SE-UPLC profile of recombinant L-asparaginase DS consists of a main peak identified as a tetrameric enzyme and an HMW peak identified as an octamer in the following SEC-MALLS analysis. Very low levels of low molecular weight (LMW) backstrap peaks are also present. The area ratio is shown in Figure 5.

[0282] Consistent with the small-scale data from Example 5, SEC-UPLC showed that recombinant L-asparaginase (0.2%) in the FS batch had significantly lower aggregate counts compared to Erwinase CAMR-174 (6%).

[0283] SEC-MALLS method: High molecular weight species present in two recombinant L-asparaginase samples (A and B) were measured using SEC-MALLS size exclusion chromatography (SEC-MALLS) combined with a multi-angle light scattering detector. Samples were diluted with a compound buffer and injected into a Superdex® 200 Increase column equipped with a Minidawn Treos MALLS detector. Figure 6 shows the UV peak area percentage. The results indicate that recombinant L-asparaginase exists primarily in tetrameric form, with the presence of low levels of octamers and higher-order aggregates. Lower levels of LMW species were also observed.

[0284] Recombinant L-asparaginase and Erwinaze® samples were diluted to 10 mg / mL in 20 mM sodium phosphate (pH 7.0) and dialyzed against the same buffer. The samples were then placed in an AN-60Ti analytical rotor and loaded into a Beckman Coulter ProteomeLab XL analytical ultracentrifuge. The data were analyzed using the c(s) function developed by Peter Schuck of the NIH and implemented in his analytical program, SEDFIT (version 11.3). The resulting size distributions were graphed, and peaks were merged using OriginLab Origin® version 9.0.

[0285] As shown in Figures 7 and 8, recombinant L-asparaginase is highly homogeneous, with 99.2% present as tetrameric enzymes, and low levels of octamers (0.61%) and higher-order aggregates (0.07%). This confirms the low aggregate levels observed by SE-UHPLC and SEC-MALLS methods. The Erwinase® batch was found to be more heterogeneous, with 93.6% identified as tetramers and higher-order aggregates. A certain level of HMW (6.4%) was present.

[0286] Example 7: Randomized Phase 1 study of the safety, tolerability, and pharmacokinetics of recombinant Erwinia asparaginase (JZP-458) in healthy adult subjects. JZP-458 is a recombinant Erwinia asparaginase produced using a novel Pseudomonas fluorescens expression platform that produces an enzyme that does not exhibit immunological cross-reactivity with E. coli-derived asparaginase. To evaluate the safety, tolerability, and pharmacokinetics of a single dose of JZP-458, a randomized, single-center, open-label phase 1 trial was conducted in healthy adult subjects, administered by intramuscular (IM) injection or intravenous (IV) infusion. Serum asparaginase activity (SAA) levels serve as a surrogate marker of asparagine depletion in determining the efficacy of asparaginase, with a minimum SAA level of 0.1 IU / mL or higher (NSAA) being the recognized threshold for demonstrating adequate asparagine depletion in clinical practice.

[0287] Methods: A randomized, single-center, open-label Phase 1 trial was conducted in the United States from November 19, 2018 to May 20, 2019. The trial was approved by the IntegReview Institutional Review Board in Austin, Texas, and conducted at QPS Miami Research Associates (Miami Clinical Research) in Miami, Florida, in accordance with the Declaration of Helsinki and GCP guidelines. All healthy subjects submitted signed informed consent prior to enrollment. Eligible subjects were men aged 18–55 years with a normal body mass index (i.e., 19.0–30.0 kg / m2), who were in good health as assessed by the investigator at screening and day 1-1, and who understood and could comply with the specific requirements of this trial. They were not pregnant and were not breastfeeding. The primary exclusion criteria from the trial included any medical history or presence of any disease, physical findings, clinical examination findings, or electrocardiographic findings that, in the judgment of the sponsor and / or the investigator, could interfere with the outcome or conduct of the trial, or pose a risk to healthy subjects. This included any condition that could interfere with the distribution, metabolism, or excretion of the drug.

[0288] Study Design: In this study, healthy adult subjects were screened for eligibility 2 to 28 days prior to administration (Figure 20). Eligible healthy subjects were admitted to the study center on day 1 for baseline assessment, then admitted to an inpatient ward, and received a single dose of the investigational drug on day 1. All subjects remained in the inpatient ward for pharmacokinetic (PK) and safety assessments until discharge on day 5. Telephone safety follow-up for adverse events (AEs) was conducted on days 6 and 30.

[0289] This trial was an open-label study enrolling a total of 30 healthy adult subjects. In this trial of JZP-458, the starting dose for Cohort 1 was 25 mg / m2, and dose selection for Cohort 2 was an adaptive design based on the safety, tolerability, and PK data of Cohort 1. The sentinel dosing approach for JZP-458 was applied to the first two subjects randomized to and administered in the trial only to JZP-458-administered Cohort 1. These two subjects were randomized to either IM or IV infusion of JZP-458 (one for each administration route). Dosage to the remaining subjects in the first cohort (JZP-458-administered Cohort 1 and Erwinaze-administered Cohort) was 1 week apart from the sentinel-administered subjects. Administration to the remaining subjects was permitted by protocol because the investigator and sponsor determined that the safety and tolerability were acceptable for the first two subjects (no grade 3 or higher adverse events related to the investigational drug).

[0290] The following 16 subjects were randomized to receive either JZP-458 or Erwinaze. Ten subjects were randomized to JZP-458 cohort 1, and six subjects were randomized to Erwinaze cohort. Within JZP-458 cohort 1 and Erwinaze cohort, subjects were randomized in a 1:1 ratio to either the IM or IV treatment group. This randomization scheme ensured that the first two randomized subjects received IM JZP-458 and IV JZP-458 according to a sentinel dosing approach, while all 18 subjects received IM JZP-458 and IV JZP-458 in a 2:2:1:1 ratio. This is equivalent to randomizing to JZP-458, IM Erwinaze, or IV Erwinaze.

[0291] To determine whether subjects needed to be enrolled in a separate cohort, JZP-458 Cohort 2, the investigators and sponsors evaluated safety, tolerability, and PK data for all subjects in JZP-458 Cohort 1. However, during SAP preparation, it was decided to conduct JZP-458 Cohort 2 and to evaluate the previously collected safety, tolerability, and PK data to determine the dose levels to be used in this cohort. In JZP-458 Cohort 2, 12 additional subjects were randomized in a 1:1 ratio to either the IM or IV treatment group.

[0292] Objectives: The primary objective was to evaluate the safety and tolerability of a single dose (IM or IV) of JZP-458 in healthy adult subjects. Safety and tolerability were assessed by the occurrence of therapeutic adverse events (TEAEs) and clinically significant changes in vital signs and laboratory tests. The secondary objective was to characterize the pharmacokinetic (PK) of a single dose (IM or IV) of JZP-458 in healthy adult subjects based on SAA data. Additional evaluations included measurement of serum asparaginase (SAC) levels with JZP-458, as well as measurement of L-asparagine and L-glutamine levels to assess the pharmacodynamic (PD) effects of JZP-458 in healthy adults.

[0293] Sampling and biochemical analysis for pharmacokinetic / pharmacodynamic evaluation: Sequential blood samples for PK / PD evaluation were collected from all healthy subjects at predetermined time points up to 96 hours after administration. For IM administration, samples were collected before administration and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours after administration. For IV administration, samples were collected before administration and at 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours after the start of infusion. Blood samples for PK analysis were collected in labeled 4 mL EDTA tubes and kept on ice until the samples were centrifuged into serum. Blood samples for PD analysis were collected in 4 mL EDTA tubes and kept on ice until the samples were centrifuged into plasma.

[0294] Biochemical analysis of PK samples was performed by Charles River Laboratories (Skokie, IL). PK samples were analyzed for SAA levels in the range of 0.025 IU / mL to 0.15 IU / mL using a validated enzyme activity assay with human serum. In addition, PK samples were also analyzed for SAC in the range of 1.0 ng / mL to 128 ng / mL using a validated ECLIA enzyme content assay with human serum. PD samples were analyzed by Syneos Health (Princeton, NJ) using validated liquid chromatography-tandem mass spectrometry (LC / MS / MS) for L-asparagine in the range of 0.025 μg / mL to 10.0 μg / mL and for L-glutamine in the range of 0.250 μg / mL to 100 μg / mL.

[0295] Pharmacokinetic and statistical analysis: The PK of JZP-458 was evaluated primarily based on SAA data. The following PK parameters were evaluated using non-compartmental analysis with Phoenix WinNonlin version 6.3: max =SAA highest value;C 48h =SAA value at 48 hours;C 72h=SAA value at 72 hours; t max =C max Arrival time; AUC 0-t = Area under the SAA-time curve from time 0 to the final point in time when SAA can be quantified; AUC 0-inf = Area under the SAA-time curve from time 0 to infinite time; CL = clearance; Vss = estimated volume of distribution at steady state after IV administration; V z / F = apparent volume of distribution after IM administration, and t 1 / 2 = Half-life of the disappearance during the terminal phase.

[0296] Continuous data were aggregated using descriptive statistics (n, mean, standard deviation, median, minimum, and maximum), while categorical data were aggregated using counts and percentages. Post-hoc analyses were performed to evaluate the relationship between SAA and serum asparaginase concentration (SAC). Correlation and linear regression analyses were performed for each investigational drug, across administration routes, and for each administration route. In the linear regression models, SAC was the dependent variable and SAA was the independent variable. Finally, formal hypothesis testing was not performed.

[0297] Results: The highest dose tested for each route of administration (i.e., 25 mg / m² for IM) 2 And IV: 37.5 mg / m² 2 In this study, JZP-458 resulted in serum asparaginase activity (SAA) levels of ≥0.1 IU / mL being achieved in 100% of healthy subjects 72 hours after administration. All dose levels of JZP-458 were well-tolerated, and no unexpected adverse events (AEs), serious AEs, or grade 3 or higher AEs were observed. Based on the pharmacokinetic and safety data from this study, the recommended starting dose of JZP-458 for central phase 2 / 3 trials is 25 mg / m2 for IM administration and 37.5 mg / m2 for IV administration, on a Monday / Wednesday / Friday administration schedule. 2 That is the case.

[0298] Baseline demographics: A total of 30 healthy adult subjects were enrolled and randomized in the study. Of the 30 enrolled subjects, all 30 completed the study, including the final scheduled telephone safety follow-up at day 30. Overall baseline demographics (mean ± SD) included a mean ± SD age of 38.4 ± 8.30 years, a body weight of 77.04 ± 9.998 kg, and a BSA of 1.91 ± 0.150 m² (Figure 16). In addition, 63% were male, 97% were Hispanic or Latino, 83% were white, and 17% were Black / African American.

[0299] Pharmacokinetic analysis: The PK analysis population consisted of all 30 healthy subjects (100%) enrolled in the study.

[0300] Serum asparaginase activity data: In this study, the starting dose for JZP-458 administration cohort 1 was 25 mg / m². 2 Individual SAA-time profiles were created for all treatment groups (Figure 12). Pre-administration SAA levels were below the limit of quantification for all subjects and all treatments. The number and percentage of subjects with SAA levels of 0.1 IU / mL or higher and 0.4 IU / mL or higher at 48 and 72 hours after administration are shown in Figure 9.

[0301] The dose level for Cohort 2 was determined based on the safety, tolerability, and PK SAA data from Cohort 1. No unexpected adverse events (AEs) were observed in Cohort 1, no serious AEs were reported, and no grade 3 or higher AEs were observed. The dose level for Cohort 1 was 25 mg / m². 2 The PK SAA data for JZP-458 is shown in Figure 12. 25 mg / m² 2 In IM administration, the SAA level 72 hours after administration was 0.1 IU / mL or higher in 6 out of 6 (100%) healthy subjects. This indicates that 25 mg / m² 2The administration of IM suggested that SAA levels of 0.1 IU / mL or higher would be maintained throughout the treatment period with a Monday / Wednesday / Friday administration schedule in the central Phase 2 / 3 trial. Therefore, in Cohort 2, the IM dose level was set to 12.5 mg / m². 2 The dose was reduced by 50%, and the dose-proportionality and safety profile at this dose were tested. Also, 25 mg / m² 2 In IV administration, the SAA level 72 hours after administration was 0.1 IU / mL or lower in only 4 out of 6 (67%) healthy subjects. It was above. This means 25 mg / m² 2 This suggests that IV administration was insufficient to maintain SAA levels above 0.1 IU / mL for 72 hours. Therefore, in Cohort 2, the IV dose level was increased to 37.5 mg / m². 2 It was increased by 50%.

[0302] After administration of JZP-458 IM, the SAA level was 12.5 mg / m². 2 and 25 mg / m² 2 At both dose levels, SAA levels reached 0.1 IU / mL or higher 48 and 72 hours after administration in 6 / 6 (100%) healthy subjects. After IV administration of JZP-458, SAA levels reached 25 mg / m². 2 At this dose level, levels reached 0.1 IU / mL or higher in 6 out of 6 (100%) healthy subjects 48 hours after administration and 4 out of 6 (67%) healthy subjects 72 hours after administration, while at 37.5 mg / m² 2 At this dose level, SAA levels reached ≥0.1 IU / mL in 6 / 6 (100%) healthy subjects at both 48 and 72 hours after administration. The data suggest that, at the same dose level, the IM administration route was able to maintain higher levels of SAA compared to IV. SAA data for healthy subjects administered ERW are also shown in Figures 12 and 9.

[0303] JZP-458 25 mg / m² 2 IM administration and 37.5 mg / m² 2For IV administration, mean and 95% confidence interval (CI) curves for SAA were also created based on observational data (N=6 each; Figure 13). The data showed that the lower limit of the 95% CI was 25 mg / m² for JZP-458. 2 IM administration and 37.5 mg / m² 2 Both IV administrations showed a dose level of 0.1 IU / mL or higher 72 hours after administration (the lower limit of the 95% CI was 0.31107 IU / mL and 0.09476 IU / mL for IM and IV, respectively). These data helped in recommending doses for the central phase 2 / 3 trial.

[0304] PK parameters were aggregated based on SAA for all treatment groups (Figure 10). When administered as an intramuscular dose (IM), JZP-458 was absorbed slowly based on SAA, at 12.5 mg / m². 2 and 25 mg / m² 2 t after administration of the dose max The median response times were 24 hours and 36 hours, respectively. 12.5 mg / m² 2 and 25 mg / m² 2 t after administration 1 / 2 The estimated mean times were 23.4 hours and 19.1 hours, respectively. When administered intravenously, the SAA level of JZP-458 was 25 mg / m². 2 and 37.5 mg / m² 2 After administration of the dose, t 11.5 hours and 12.6 hours, respectively. 1 / 2 The average value decreased. Generally, the t of JZP-458 after IM administration. 1 / 2 The elimination time was longer than that of IV infusion, due to the absorption rate-limiting elimination dynamics. Furthermore, the volume of distribution was almost the same as that of plasma volume. This suggests that JZP-458 was mainly localized to the central vascular compartment.

[0305] The dose-proportionality assessment based on SAA showed that exposure to JZP-458 increased with increasing dose based on SAA (Figure 17). For both IM and IV administrations, exposure to JZP-458 SAA (C maxThe increases in (and AUC) were approximately dose-proportional within the tested dose range. The bioavailability of JZP-458 via the IM administration route was also calculated and estimated to be 34.5%–36.8% for JZP-458 based on SAA data.

[0306] Serum asparaginase concentration data: Historically, asparaginase PK has been determined based on SAA data. However, SAA is not considered a true measure of PK, and therefore, enzyme content assays have been developed to measure JZP-458 asparaginase concentration in human serum.

[0307] Individual and mean SAC-time profiles were created for all treatment groups (Figures 14A-C). SAC-based PK parameters are summarized in Figure 18. When administered as an IM, JZP-458 is absorbed slowly based on SAC, and t max The median is 12.5 mg / m². 2 and 25 mg / m² 2 The duration was 30 hours for both doses. 1 / 2 The average value is 12.5 mg / m². 2 and 25 mg / m² 2 28.9 hours and 2 hours respectively for JZP-458 It was estimated to be 5.4 hours. After IV administration of JZP-458, the SAC level was 25 mg / m². 2 and 37.5 mg / m² 2 After administration of the dose, t 12.0 hours and 12.7 hours, respectively. 1 / 2 It decayed at the average value.

[0308] The dose-proportionality and bioavailability of JZP-458 were also evaluated based on SAC (Figure 19). Exposure to JZP-458 increased with increasing dose based on SAC. For both IM and IV administration, SAC(C max Based on AUC, the increase in exposure to JZP-458 was approximately dose-proportional within the tested dose range. In the IM administration route, the bioavailability of JZP-458 was estimated to be 37.7%–43.9% based on SAC data.

[0309] The relationship between SAA and SAC for JZP-458 was further investigated (Figure 14D). When evaluated across all routes of JZP-458 administration, a strong positive association was observed between SAA and SAC, with a correlation coefficient greater than 0.95. In addition, a linear regression model yielded the equation SAC = 1407.9 × SAA. These data suggest that for an SAA level of 0.1 IU / mL, the corresponding SAC is approximately 141 ng / mL in this healthy adult population.

[0310] Pharmacodynamic data: Asparaginase hydrolyzes the amino acid asparagine to aspartic acid and ammonia. Plasma levels of asparagine were monitored throughout the treatment period. The time course of mean plasma asparagine concentration versus mean SAA is shown in Figure 15. Baseline (pre-administration) mean plasma asparagine concentrations were similar in groups IM and IV. Individual asparagine concentrations ranged from 5.09 μg / mL to 13.8 μg / mL in all subjects. These values ​​are consistent with those reported in the literature. Following administration of JZP-458 (IM and IV), mean plasma asparagine levels rapidly decreased from pre-administration concentrations (Cohort 1: 8.62 μg / mL and 8.96 μg / mL for IM and IV, respectively; Cohort 2: 6.42 μg / mL and 5.89 μg / mL for IM and IV, respectively) to levels below the assay limit of quantification (LLOQ; 0.025 μg / mL) in both routes and remained undetectable until the final sample collection time 96 hours later. The data showed a direct correlation between SAA and the decrease in plasma asparagine levels. At all JZP-458 dose levels, plasma asparagine levels were completely depleted by JZP-458 administration. The highest dose of JZP-458 tested in this Phase 1 study with healthy subjects (i.e., 25 mg / m² for IM) was 25 mg / m². 2 And IV: 37.5 mg / m² 2 In this study, JZP-458 not only achieved SAA levels of 0.1 IU / mL or higher 72 hours after administration in 100% of healthy subjects in each pathway, but also resulted in complete depletion of plasma asparagine levels up to 96 hours after administration.

[0311] Asparaginase can hydrolyze glutamine to glutamic acid and ammonia, in addition to asparagine, albeit with very low efficiency. Plasma levels of glutamine were monitored for all treatments (Figure 17). Baseline (pre-administration) mean plasma glutamine concentrations were similar in the IM and IV groups. Individual pre-administration glutamine concentrations ranged from 60.4 μg / mL to 146 μg / mL in all subjects. These values ​​are also consistent with those reported in the literature. The data showed that mean plasma glutamine levels rapidly decreased for approximately 12 hours after IV administration of JZP-458 in cohorts 1 and 2, from pre-administration concentrations of 106.5 and 74.0 μg / mL, respectively, to levels below the assay LLOQ (0.25 μg / mL), and then glutamine levels recovered to nearly pre-administration levels at the final sample collection 96 hours after administration. In the IM pathway, mean plasma glutamine levels decay after IM administration of JZP-458, with the lowest glutamine level observed 36 hours after administration at 25 mg / m². 2 and 12.5 mg / m² 2 The glutamine levels decreased by 79% and 47% respectively at the time of administration, and then glutamine levels rose to the final sample 96 hours after administration. At the time of sampling, levels had recovered to levels similar to those before administration. Complete depletion of L-glutamine was not observed. Glutamine levels were moderately affected, only partially decreasing, and the data were more volatile than those observed with L-asparagine.

[0312] Safety and Tolerability: The safety profile observed for JZP-458 in this Phase 1 trial was consistent with that of other asparaginases. All dose levels of JZP-458 were well-tolerated, and no unexpected adverse events (AEs), serious AEs, or grade 3 or higher AEs were observed. The most common TEAE occurring in two or more healthy subjects in each administration cohort was nausea (Figure 11).

[0313] Discussion: JZP-458, a recombinant Erwinia asparaginase expected to be immune-free from E. coli-derived asparaginase, is being developed to make asparaginase therapy available to patients with ALL or LBL who have developed hypersensitivity to E. coli-derived asparaginase. In this randomized, single-center, open-label phase 1 trial, JZP-458 was administered at the highest dose tested for each route of administration (i.e., 25 mg / m² for IM). 2 And IV: 37.5 mg / m² 2 72 hours after administration, SAA levels of ≥0.1 IU / mL were achieved in 100% of healthy adult subjects in each route. The SAA levels observed in this study also demonstrated that JZP-458 can completely deplete plasma asparagine levels. This was confirmed by the asparagine concentrations measured in this study. At all JZP-458 dose levels, plasma asparagine levels were completely depleted by JZP-458 treatment in both the IM and IV administration routes. In addition, the safety profile of JZP-458 in this study was consistent with the profiles of other asparaginases. All dose levels of JZP-458 were well tolerable, with no unexpected adverse events (AEs), no serious AEs reported, and no grade 3 or higher AEs.

[0314] Based on accumulated PK and safety data, the recommended starting dose of JZP-458 in central Phase 2 / 3 is 25 mg / m² via the IM route with a Monday / Wednesday / Friday dosing schedule. 2 For the IV administration route, 37.5 mg / m² 2 In this Phase 1 trial in healthy adult subjects, these doses achieved SAA levels of 0.1 IU / mL or higher in 6 out of 6 (100%) healthy subjects 72 hours after administration. Furthermore, these doses are expected to maintain SAA levels of 0.1 IU / mL or higher throughout the treatment period in the central Phase 2 / 3 trial.

[0315] Completing asparaginase therapy is crucial for improving patient outcomes, as demonstrated in previous studies. In the Dana-Farber Cancer Institute ALL Consortium Protocol 91-01 trial, patients with asparaginase intolerance, defined as completing 25 weeks or less of the planned 30 weeks of asparaginase therapy, had a significantly lower 5-year EFS compared to patients who received 26 weeks or more of asparaginase therapy (73% vs. 90%, respectively; P<0.01). A recent Children's Oncology Group study demonstrated that standard-risk ALL patients with high-risk and late early responses who did not complete their prescribed asparaginase doses had a significantly inferior EFS compared to patients who received all prescribed asparaginase doses. Notably, patients who experienced hypersensitivity reactions and completed a course of treatment with Erwinia asparaginase substitutes showed a similar EFS to patients who completed a course of first-line asparaginase therapy. These trials suggest that patients who complete the prescribed asparaginase dose, regardless of whether it is a first-line or second-line asparaginase, have better outcomes than those who discontinue treatment early. These results indicate that patients who develop hypersensitivity to E. coli-derived asparaginase can complete their entire treatment cycle. The need for alternative asparaginase preparations to achieve this is emphasized.

[0316] Conclusion: In this Phase 1 trial, JZP-458 was the highest dose tested for each route of administration (i.e., 25 mg / m² for IM). 2 And IV: 37.5 mg / m² 2) 72 hours after administration, SAA levels of ≥0.1 IU / mL were achieved in 100% of healthy adult subjects in each route, resulting in complete asparagine depletion without unexpected AEs, SAEs, or grade 3 or higher AEs. Based on the PK and safety data accumulated in this study, the recommended starting dose of JZP-458 in Phase 2 / 3 is 25 mg / m² for the IM administration route with a Monday / Wednesday / Friday dosing schedule. 2 For the IV administration route, 37.5 mg / m² 2 Therefore, JZP-458 may be used as an alternative treatment for ALL / LBL patients who develop hypersensitivity to E. coli-derived asparaginase.

[0317] Example 8: Phase 2 / 3 trial of JZP-458 in ALL / LBL patients hypersensitive to E. coli-derived asparaginase. We planned an open-label, multicenter, dose-confirming, pharmacokinetic study of JZP-458 in participants (of any age) with ALL / LBL exhibiting hypersensitivity (allergic reaction or silent inactivation) to E. coli-derived asparaginase. This study was designed to evaluate the tolerability and efficacy of JZP-458, as measured by serum asparaginase activity, with additional supporting analyses for asparagine depletion and anti-drug antibody (ADA) levels. Six doses of JZP-458 were used in place of each dose of E. coli-derived long-acting asparaginase. A two-week consecutive treatment with JZP-458 was defined as one course.

[0318] Study Design: This study consists of two parts: Part A to determine the dose of JZP-458 in IM administration and to confirm its safety and efficacy; and Part B to define the optimal dose and schedule for IV infusion of JZP-458 (see Figures 22 and 23). Parts A and B may be considered in parallel. Part A (IM) examines multiple cohorts, with Cohort 1 determining the optimal dose and Cohort 2 increasing the number of participants treated with the optimal dose. Part B (IV) reflects Part A, and in both parts, a cycle of JZP-458 (six doses over two weeks) may begin on Monday, Wednesday, or Friday, and the number of cycles per participant depends on their individual treatment plan. In each part, a cycle of JZP-458 (six doses over two weeks) may begin on Monday, Wednesday, or Friday, and the number of cycles per participant depends on their individual treatment plan. The starting dose is 25 mg / m² via the IM administration route (cohort 1a). 2 The starting dose was selected based on the results of the Phase 1 trial.

[0319] Objective: The primary objective of the study is to determine the efficacy of IM administration of JZP-458, as measured by response (defined as an NSAA level of 0.1 IU / mL or higher in the most recent 72 hours during the first cycle) in Cohorts 1 and 2, and to evaluate the safety and tolerability of IM-administered JZP-458 in participants with ALL / LBL who exhibit hypersensitivity to E. coli-derived asparaginase. Additional objectives are as follows:

[0320] Second primary objective: To determine the efficacy of IM administration of JZP-458, as measured by response in Cohort 1 and Cohort 2 (defined as an NSAA level of 0.1 IU / mL or higher in the most recent 48 hours during the first cycle).

[0321] Second objective: 1) Response rate in Cohort 1 and Cohort 2 (0.4% in the first cycle) 1) Determine the efficacy of IM administration of JZP-458, as measured by (defined as NSAA levels of ≥ IU / mL in the most recent 48 and 72 hours); 2) Characterize the PK of IM infusion of JZP-458 using a PPK approach and investigate ER correlations using data from all participants at all dose levels and samples at all time points; and 3) Evaluate the immunogenicity of IM-administered JZP-458 after repeated administrations of JZP-458.

[0322] Exploratory Objectives (Part B of the Study): 1) To determine the efficacy of IV administration of JZP-458 as measured by response defined as NSAA levels of 0.1 IU / mL or higher in the most recent 48 hours and NSAA levels of 0.1 IU / mL or higher in the most recent 72 hours during the first cycle; 2) To determine the efficacy of IV administration of JZP-458 as measured by response defined as NSAA levels of 0.4 IU / mL or higher in the most recent 48 hours and NSAA levels of 0.4 IU / mL or higher in the most recent 72 hours during the first cycle; 3) To evaluate the safety and tolerability of IV-administered JZP-458 in participants with ALL / LBL who exhibit hypersensitivity to E. coli-derived asparaginase; 4) To characterize the PK of IV-administered JZP-458 using a population PK approach; and 5) To evaluate the immunogenicity of IV-administered JZP-458 after repeated administrations.

[0323] Preliminary Results: As of October 7, 2020, 31 participants have been enrolled in Cohort 1a and 56 participants in Cohort 1b of the central Phase 2 / 3 trial (JZP458-201). The first SDRC assessment of Cohort 1a, as defined in the protocol, included data from 6 evaluable participants, and the second SDRC assessment of Cohort 1a included cumulative data from 16 evaluable participants. Each SDRC recommendation is based on a review of all data available at that time. After evaluation of available efficacy data, 25 mg / m² 2 No unexpected safety events were observed at the IM dose level of 37.5 mg / m². 2Evaluation of IM dose levels was initiated (Cohort 1b). The first participant in Cohort 1b was enrolled on June 29, 2020. SDRC evaluation of Cohort 1b data was conducted on September 1, 2020, as a preliminary review of data up to the first seven participants enrolled in Cohort 1b. This review included all available SAA data, PPK modeling and simulation data, and safety data from the trial. The recommendation was to continue enrollment without change and to review the trial again, in accordance with SDRC policy, when data from at least 13 evaluable Cohort 1b participants were available. The available preliminary data for Cohorts 1a (N=31) and 1b (N=17) are summarized below.

[0324] Serum asparaginase activity level Serum samples were analyzed for SAA. SAA levels serve as a surrogate marker of efficacy and response. Biochemical analysis of SAA was performed by Charles River Laboratories (Skokie, IL) using a validated enzyme activity method with human serum and the JZP-458 reference standard (lot #RM-M-009; specific activity of 693 U / mg) in the range of 0.035 IU / mL to 0.210 IU / mL.

[0325] Evaluable participants in Cohort 1 were defined as those who received at least three IM doses of JZP-458 and had 72-hour NSAA levels collected within the protocol-defined sampling window (±2 hours) during the latter half of the first cycle. Of the 31 participants enrolled at 25 mg / m2, 26 were deemed evaluable for the primary efficacy objective of Cohort 1 according to the protocol. The reasons for participants being deemed ineligible were as follows: 1) Two participants had 72-hour PK samples collected outside the defined sampling window. 2) One participant did not have a 72-hour PK sample collected. 3) One participant withdrew informed consent and did not have a PK sample collected. None. 4) One participant had an SAA value (assessed by the biochemical analysis laboratory Charles River Laboratory) that was unassessable due to interference with the assay by increased lipids. This participant had grade 2 hypertriglyceridemia (318 mg / dL) at baseline and grade 4 hypertriglyceridemia (2679 mg / dL) during the first cycle (reported as TEAE; see Safety Summary below for details). This participant's triglycerides continued to decrease to baseline or below baseline in subsequent cycles of JZP-458, and most recently, the participant completed the fifth cycle with a triglyceride level of 234 mg / dL (grade 1).

[0326] Of the 17 participants included in the preliminary analysis of Cohort 1b (37.5 mg / m2), 16 participants were evaluable for the primary efficacy objective of Cohort 1 (one participant was excluded for the primary endpoint but included for other endpoints because they had a 72-hour PK sample collected outside the defined sampling frame). Individual preliminary SAA results at key time points are summarized in Figure 24. The observed preliminary SAA result was 25 mg / m2. 2 The results show that at the IM dose level of JZP-458 (N=26 evaluable participants), the mean SAA levels (95% CI) 72 and 48 hours after the most recent administration were 0.1560 (0.1144~0.1976) and 0.4504 (0.3637~0.5370), respectively (the median SAA levels [first quartile, Q1; third quartile, Q3] 72 and 48 hours after the most recent administration were 0.1345 [0.0886, 0.2178] and 0.4091 [0.2813, 0.6577], respectively). 37.5 mg / m² 2At the IM dose level of JZP-458 (N=16 evaluable participants), the mean (95% CI) SAA levels 72 and 48 hours after the most recent administration were 0.2605 (0.1326–0.3884) and 0.7146 (0.3976–1.0316), respectively (the median SAA levels [Q1, Q3] 72 and 48 hours after the most recent administration were 0.1732 [0.1157, 0.2849] and 0.6503 [0.3248, 0.8736], respectively).

[0327] Figure 25 shows the proportion of participants who showed NSAA levels of 0.1 IU / mL or higher and 0.4 IU / mL or higher 72 and 48 hours after the most recent administration. 25 mg / m² 2 (N=26 evaluable participants) and 37.5 mg / m² 2 In the IM dose levels of JZP-458 (N=16 evaluable participants), 65.4% and 81.3% of participants achieved an NSAA level of 0.1 IU / mL or higher in the most recent 72-hour assessment (primary endpoint), and 96.2% and 93.8% of participants achieved an NSAA level of 0.1 IU / mL or higher in the most recent 48-hour assessment (primary secondary endpoint).

[0328] Population pharmacokinetic modeling and simulation: A preliminary PPK analysis (secondary endpoint) was performed using SAA data (data received on September 28, 2020). This included 47 participants (25 mg / m²) from the JZP458-201 trial who received IM-administered JZP-458. 2 30 participants and 37.5 mg / m² 2 A total of 319 quantifiable SAA data points (from the first cycle only) from 17 participants were included in the development of the PPK model. The model was fitted to the SAA data to identify the structural model. Weight, height, body surface area, age, sex, race, ethnicity, disease, and disease subtype were tested as potential covariates for JZP-458 SAA clearance and volume. Only weight was found to be statistically significant and was included in the model.

[0329] The covariate models developed for JZP-458 included a one-compartment IM-specific model with linear vanishing and primary absorption, and a proportional residual error model, with body weight included as a non-proportional covariate to JZP-458 SAA clearance and volume. Model diagnostics showed good fitting based on predicted data versus observed data and predictive modeling methods.

[0330] To investigate the possibility of achieving therapeutic NSAA levels of 0.1 IU / mL or higher, a covariate model was used to simulate patient SAA profiles (N = 2000 subjects per dose level). The simulated hypothetical population was analyzed by Centers for Disease. The target population was created by re-extracting subjects from the National Health and Nutrition Examination Survey data in the control group by replacing demographic characteristics. The simulated population ranged from 2 to 85 years of age, with weights ranging from 8.9 to 174.6 kg (median 62.7 kg).

[0331] Both the Friday, Monday, Wednesday (FMW) and MWF dosing schedules were simulated with six doses per treatment cycle. This was because the FMW schedule represented the most conservative scenario, with NSAA levels in the first 72 hours following only one dose. The simulation data showed that the start date did not affect the simulated percentage of patients with NSAA levels of 0.1 IU / mL or higher at the most recent 48 or 72-hour point. The simulated profiles for the FMW dosing schedule, along with the median and 95% prediction interval, are shown in Figure 26. (25–80 mg / m²) 2 Figure 27 shows a summary table of simulated percentages and predicted mean SAA levels for patients achieving NSAA of 0.1 IU / mL or higher with IM doses within the specified range.

[0332] Based on PPK modeling and simulation, 37.5 mg / m² on the FMW schedule was determined. 2With IM administration of JZP-458, it is expected that JZP-458 will achieve an NSAA level of ≥0.1 IU / mL in the most recent 72 hours in 87% of patients (95% CI: 85.5%–88.5%) and an NSAA level of ≥0.1 IU / mL in the most recent 48 hours in 99.9% of patients (95% CI: 99.7%–100%). The simulated mean NSAA levels were 0.29 (95% prediction interval: 0.06–0.88) and 0.58 (95% prediction interval: 0.2–1.32) for the most recent 72 and 48 hours, respectively. The PPK model predicts 50 mg / m² 2 At this dose, 94.2% of patients are expected to achieve an NSAA level of 0.1 IU / mL or higher 72 hours after administration.

[0333] Preliminary results for asparagine depletion: Pharmacodynamic samples were validated by liquid chromatography-tandem mass spectrometry (Biomarker Partial Validation Report) by Syneos Health (Princeton, NJ). Asparagine concentrations were analyzed in the range of 0.025–10.0 μg / mL using Amendment 1, SN 0044). To ensure that the analytical measurements reflected in vivo asparagine levels, blood samples for the asparagine assay were placed in an ice bath immediately after collection, and the treated plasma was stored at -80°C within 30 minutes. Asparagine levels were measured to support the efficacy of JZP-458 during the treatment period. Mean baseline asparagine levels were 10.2557 μg / mL (95% CI: 9.2175–11.2938) in 30 participants treated in cohort 1a (25 mg / m2) and 10.2282 μg / mL (95% CI: 7.1852–13.2712) in 17 participants treated in cohort 1b (37.5 mg / m2). These values ​​are consistent with those reported in the literature (Tong 2014). In most participants, complete depletion of plasma asparagine was observed in most samples; that is, plasma asparagine levels rapidly decreased from pre-administration levels to below or near the assay limit of quantification (0.025 μg / mL) and persisted throughout the treatment period from the first cycle to the pre-administration period of the sixth cycle, when the last sample was taken. Four participants showed transient low-level increases in asparagine (three in cohort 1a and one in cohort 1b).

[0334] Preliminary safety results: In cohort 1a, a total of 31 participants received 25 mg / m². 2 of JZP-458 was administered via IM. In cohort 1b, the data showed a dose of 37.5 mg / m². 2 JZP-458 is available to 17 participants who received IM.

[0335] Figure 28 shows an overview of TEAEs in the treatment cohort. The majority of participants experienced at least one TEAE. JZP-458 IM 25 mg / m² 2In the cohort, the most frequent TEAEs were decreased neutrophil count (32.3% of participants, 10 / 31), decreased white blood cell count (25.8% of participants, 8 / 31), and anemia (22.6% of participants, 7 / 31) (Table 4, Cohort 1a). JZP-458 IM 37.5 mg / m² 2 In the cohort, the most frequent TEAEs were vomiting (35.3% of participants, 6 / 17) and nausea (23.5% of participants, 4 / 17) (Figure 27, Cohort 1b).

[0336] JZP-458 IM 25mg / m 2 In the cohort (N=31), grade 3 or 4 TEAEs were reported in 18 participants (58.1%), with the most common being neutropenia (29.0%), leukopenia (16.1%), and febrile neutropenia (19.4%) (Table 6). Overall, one participant reported an unexpected clinically significant non-hematological toxicity occurring under grade 3 or higher treatment (grade 4 hypertriglyceridemia considered to be related to the investigational drug) (Table 6 and List Table 1, Cohort 1a). In the JZP-458 IM 25 mg / m2 cohort, serious TEAEs were reported in 9 participants (29.0%) (Table 5). Serious TEAEs included near-syncope in one participant; rhinorrhea and febrile neutropenia in one participant; febrile neutropenia, dehydration, and fever in one participant; drug hypersensitivity and febrile neutropenia in one participant; febrile neutropenia in one participant; febrile neutropenia and stomatitis in one participant; exacerbation of oral mucositis in one participant; fever in one participant; and methemoglobinemia in one participant. All of these serious TEAEs, with the exception of the drug hypersensitivity and febrile neutropenia events in one participant, were not considered to be related to the investigational drug.

[0337] Overall, one participant (JZP-458 25 mg / m2) experienced a TEAE that led to discontinuation of the investigational drug. This participant (4 years old) showed a grade 2 allergic reaction (non-severe) to cefepime and sulfamethoxazole / trimethoprim on the fifth dose of the first cycle.

[0338] Both of these drugs were discontinued. Subsequently, one day after the third dose of the second cycle of JZP-458, the participant experienced a severe TEAE (grade 3) of drug hypersensitivity. The event resolved the following day, and the participant was discharged. This event was considered to be related to the investigational drug, leading to discontinuation of the investigational drug (Table 1, Cohort 1a). In the JZP-458 IM 37.5 mg / m2 cohort (N=17), six participants experienced grade 3 or 4 TEAEs, with febrile neutropenia (17.6%, 3 / 17) being the most common (Figure 30 and Table 1, Cohort 1b). Three participants in the JZP-458 IM 37.5 mg / m2 cohort experienced serious TEAEs (vomiting and abdominal pain in one participant [unrelated to the investigational drug]; drug hypersensitivity [reporter's terminology: allergic reaction to JZP-458; related to the investigational drug] and febrile neutropenia [related to the investigational drug] in one participant; and febrile neutropenia [related to the investigational drug; two events] in one participant) (Figure 28). No unexpected clinically significant non-hematological toxic events of grade 3 or higher occurred in these participants, and there were no TEAEs leading to study discontinuation (Figure 29, Cohort 1b).

[0339] Adverse events of interest related to asparaginase include allergic reactions, pancreatitis, and thrombosis (Stock 2011; Kearney 2009; Pieters 2011; Plourde 2014; Kloos 2020; Asparlas (Summary Basis of Approval 2018). At the data cutoff point for this preliminary safety analysis, only one participant (cohort 1a) experienced a grade 3 or higher allergic reaction / hypersensitivity (as described above), and no participants experienced a grade 3 or higher event of pancreatitis or thrombosis. No deaths were reported.

[0340] In summary, the observed TEAE profile was consistent with other asparaginases, at both dose levels of JZP-458 (25 and 37.5 mg / m²). 2 The study showed that the drug was well-tolerated by the participants.

[0341] Based on a detailed review of available trial data by experts inside and outside the SDRC, 37.5 mg / m² 2 The IM dose of JZP-458 has a favorable benefit-risk profile and is an appropriate dose for the JZP-458 approval application. This dose can ensure that patients complete the asparaginase treatment regimen and is consistent with FDA guidelines regarding benefit-risk and minimum effective administration.

[0342] Safety: Further safety studies are available for safety evaluation in Cohort 1a (25 mg / m²). 2 ) 31 people and Cohort 1b (37.5 mg / m²) 2 The program is planned to be conducted with a minimum of 82 participants, including 51 people from the group. 47 participants (25 mg / m²) 2 31 participants and 37.5 mg / m² 2 Preliminary data from 17 participants showed a dose of 37.5 mg / m². 2 The proposed dose was demonstrated to be safe and well tolerable. In a preliminary safety analysis, one participant (Cohort 1a, 25 mg / m²) was found to be safe. 2 Only one participant experienced a grade 3 or higher allergic reaction / hypersensitivity; no participants experienced a grade 3 or higher event of pancreatitis or thrombosis. A grade 3 serious adverse event (SAE) of drug hypersensitivity (related to the investigational drug) led to discontinuation of the investigational drug. No other participants discontinued the study due to a TEAE.

[0343] Efficacy Data: Criteria for early efficacy evaluation were based on a target SAA level of 0.1 IU / mL, as well as confirmation of asparagine depletion and consideration of clinically appropriate response rates in patients previously exposed to asparaginase. The observed preliminary SAA result was 25 mg / m². 2 The results show that at the IM dose level of JZP-458 (N=26 evaluable participants), the mean SAA levels (95% CI) 72 and 48 hours after the most recent administration were 0.1560 (0.1144~0.1976) and 0.4504 (0.3637~0.5370), respectively (the median SAA levels [Q1, Q3] 72 and 48 hours after the most recent administration were 0.1345 [0.0886, 0.2178] and 0.4091 [0.2813, 0.6577], respectively). 37.5 mg / m² 2 At the IM dose level of JZP-458 (N=16 evaluable participants), the mean (95% CI) SAA levels 72 and 48 hours after the most recent administration were 0.2605 (0.1326–0.3884) and 0.7146 (0.3976–1.0316), respectively (the median SAA levels [Q1, Q3] 72 and 48 hours after the most recent administration were 0.1732 [0.1157, 0.2849] and 0.6503 [0.3248, 0.8736], respectively).

[0344] 25 and 37.5 mg / m² 2 At the IM dose level of JZP-458, 65.4% and 81.3% of participants achieved an NSAA level of 0.1 IU / mL or higher in the most recent 72-hour assessment (primary endpoint), and 96.2% and 93.8% of participants achieved an NSAA level of 0.1 IU / mL or higher in the most recent 48-hour assessment (primary secondary endpoint). 37.5 mg / m² 2In (N=16), the percentage of participants achieving an NSAA level of 0.1 IU / mL or higher in the most recent 72-hour assessment did not meet the primary endpoint criterion (as defined in the statistical analysis plan [SAP]). However, this result demonstrates a clinically appropriate response rate consistent with the observed or predicted rates (80%–88%) published for patients treated with non-E. coli-based asparaginase after hypersensitivity reactions (Vrooman 2010; Panetta 2020; Salzer). (2013). 37.5 mg / m² 2 In the (N=16) study, 93.8% of participants achieved an NSAA level of 0.1 IU / mL or higher (the primary secondary endpoint) in the most recent 48-hour assessment. This percentage is expected to meet the predetermined success criteria outlined in the SAP for the IA study (N=51 participants are planned).

[0345] The efficacy of JZP-458 was further confirmed in the preliminary PPK results. The preliminary PPK results were consistent with the observed data and further supported the proposed dose selection of 37.5 mg / m2. Based on PPK modeling and simulation (secondary endpoint), 37.5 mg / m2 in the FMW schedule was selected. 2 With IM administration of JZP-458, JZP-458 is expected to achieve an NSAA level of 0.1 IU / mL or higher in the most recent 72 hours in 87% of patients (95% CI: 85.5%–88.5%) and an NSAA level of 0.1 IU / mL or higher in the most recent 48 hours in 99.9% of patients (95% CI: 99.7%–100%). The simulated mean NSAA levels were 0.29 (95% prediction interval: 0.06–0.88) and 0.58 (95% prediction interval: 0.2–1.32) for the most recent 72 and 48 hours, respectively. The simulation results showed that the start date did not affect the percentage of patients showing an NSAA level of 0.1 IU / mL or higher at the most recent 72 or 48-hour timeframe.

[0346] The PPK analysis robustly supports the observed data and provides a complete data package for BLA because this model uses available data for all patients, time points, and dose levels. The results of the PPK model can demonstrate the exposure-response characteristics of JZP-458 based on all available clinical and PK data. This model provides a reliable prediction of NSAA levels at meaningful time points because it is independent of classified data. This model demonstrates that the percentage of patients exhibiting NSAA levels of 0.1 IU / mL or higher 72 hours after IM administration of JZP-458 is consistent with the expected response in a patient population with hypersensitivity to E. coli-based asparaginase products. The modeled SAA value is 37.5 mg / m². 2 This suggests that IM administration is the appropriate dose in the MWF administration schedule.

[0347] JZP-458 37.5 mg / m² 2 As a further means of dose verification, the goal of complete asparagine depletion, measured by a validated method (Biomarker Partial Validation Report Amendment 1, SN 0044), was achieved at 25 and 37.5 mg / m². 2 Comparison of baseline asparagine levels versus trough asparagine levels throughout the administration period consistently demonstrated near-complete depletion of asparagine. Deviations from this pattern were small and transient. The effects of candidate doses, toxicity studies, number of injections, number of vials, and other factors were evaluated. Such studies compared the potential effects of higher doses on the increased risk of more severe adverse events.

[0348] While embodiments and uses of the present invention have been described in some detail with reference to figures and examples, it will be apparent to those skilled in the art that numerous further modifications are possible without departing from the inventive concept contained herein. All references cited herein are incorporated herein in their entirety.

Claims

1. A method for treating diseases treatable by asparagine depletion in humans, wherein the method is approximately 10 mg / m² ２ ~Approx. 100mg / m ２ The method comprises administering recombinant L-asparaginase to a human subject in an amount, wherein the recombinant L-asparaginase is a tetramer, the tetramer comprises four monomers, and each monomer has an amino acid sequence containing SEQ ID NO:

1.

2. The method according to claim 1, wherein the recombinant L-asparaginase is not conjugated with the PEG portion.

3. The method according to claim 1, wherein the recombinant L-asparaginase is not conjugated with a peptide containing proline or alanine.

4. The method according to claim 1, wherein the human subject has been previously treated with an asparaginase derived from E. coli.

5. The method according to claim 4, wherein the asparaginase derived from E. coli is conjugated to the PEG portion.

6. The method according to claim 4, wherein the human subject has previously experienced silent inactivation of asparaginase derived from E. coli.

7. The method according to claim 4, wherein the human subject has previously experienced an allergic reaction to asparaginase derived from E. coli.

8. The method according to claim 4, wherein the human subject has previously experienced anaphylaxis to asparaginase derived from E. coli.

9. The method according to claim 1 or 4, wherein the disease treatable by L-asparagine depletion is acute lymphoblastic leukemia (ALL).

10. The method according to claim 1 or 4, wherein the disease treatable by L-asparagine depletion is lymphoblastic lymphoma (LBL).

11. The method according to claim 9 or 10, wherein the ALL or LBL is recurrent ALL or recurrent LBL.

12. The method according to any one of claims 1 to 11, wherein the recombinant L-asparaginase is administered three times a week.

13. The method according to claim 12, wherein the recombinant L-asparaginase is administered every other day for five consecutive days, followed by a drug-free period of two consecutive days.

14. The method according to claim 12, wherein the recombinant L-asparaginase is administered on Monday, Wednesday, and Friday of the same week.

15. The method according to any one of claims 12 to 14, wherein the recombinant L-asparaginase is administered for two weeks.

16. The method according to any one of claims 1 to 15, wherein the recombinant L-asparaginase is administered intramuscularly.

17. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ The method according to claim 16, administered in an amount.

18. The recombinant L-asparaginase is present in approximately 15 mg / m². ２ ~Approx. 45mg / m ２ The method according to claim 16, administered in an amount.

19. The recombinant L-asparaginase is present in an amount of approximately 27 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 16, administered in an amount.

20. The method according to any one of claims 1 to 15, wherein the recombinant L-asparaginase is administered intravenously.

21. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ The method according to claim 20, administered in an amount.

22. The recombinant L-asparaginase is administered at a dose of about 25 mg / m ２ to about 55 mg / m ２ The method according to claim 20.

23. The recombinant L-asparaginase is present in approximately 27.5 mg / m². ２ ~About 47.5mg / m ２ The method according to claim 20, administered in an amount.

24. The recombinant L-asparaginase is present in approximately 21.5 mg / m². ２ ~Approx. 38.5mg / m ２ The method according to claim 20, administered in an amount.

25. The method according to any one of claims 1 to 24 and 103 to 139, wherein the lowest serum asparaginase activity (NSAA) measured from the serum sample derived from the human subject is equal to or greater than 0.1 IU / mL after administration and treatment with the recombinant L-asparaginase.

26. The method according to any one of claims 1 to 25 and 103 to 139, wherein the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a multi-agent chemotherapy regimen.

27. The method according to any one of claims 1 to 26 and 103 to 139, wherein the recombinant L-asparaginase exhibits aggregation of less than 6%.

28. The method according to any one of claims 1 to 26 and 103 to 139, wherein the recombinant L-asparaginase exhibits aggregation of less than 1%.

29. The method according to any one of claims 1 to 28 and 103 to 139, wherein the recombinant L-asparaginase is not freeze-dried.

30. A method for treating a disease treatable by asparagine depletion in a human subject, the method comprising administering recombinant L-asparaginase to the human subject, wherein the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens, the recombinant L-asparaginase is a tetramer, the tetramer has four monomers, and each monomer has an amino acid sequence including SEQ ID NO:

1.

31. The method according to claim 30, wherein the recombinant L-asparaginase is not conjugated with the PEG portion.

32. The method according to claim 30, wherein the recombinant L-asparaginase is not conjugated with a peptide containing proline or alanine.

33. The method according to claim 30, wherein the human subject has been previously treated with an asparaginase derived from E. coli.

34. The method according to claim 33, wherein the asparaginase derived from E. coli is conjugated to the PEG portion.

35. The method according to claim 33, wherein the human subject has previously experienced silent inactivation of asparaginase derived from E. coli.

36. The method according to claim 33, wherein the human subject has previously experienced an allergic reaction to asparaginase derived from E. coli.

37. The method according to claim 33, wherein the human subject has previously experienced anaphylaxis to asparaginase derived from E. coli.

38. The method according to claim 30 or 33, wherein the disease treatable by L-asparagine depletion is acute lymphoblastic leukemia (ALL).

39. The method according to claim 30 or 33, wherein the disease treatable by L-asparagine depletion is lymphoblastic lymphoma (LBL).

40. The method according to claim 38 or 39, wherein the ALL or LBL is recurrent ALL or recurrent LBL.

41. The method according to any one of claims 30 to 40, wherein the recombinant L-asparaginase is administered three times a week.

42. The method according to claim 41, wherein the recombinant L-asparaginase is administered every other day for five consecutive days, followed by a drug-free period of two consecutive days.

43. The method according to claim 41, wherein the recombinant L-asparaginase is administered on Monday, Wednesday, and Friday of the same week.

44. The method according to any one of claims 41 to 43, wherein the recombinant L-asparaginase is administered for two weeks.

45. The recombinant L-asparaginase is present in approximately 10 mg / m². ２ ~Approx. 100mg / m ２ The method according to any one of claims 30 to 44, administered in an amount.

46. The method according to any one of claims 30 to 45, wherein the recombinant L-asparaginase is administered intramuscularly.

47. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ The method according to claim 46, administered in an amount.

48. The recombinant L-asparaginase is present in approximately 15 mg / m². ２ ~Approx. 45mg / m ２ The method according to claim 46, administered in an amount.

49. The recombinant L-asparaginase is present in an amount of approximately 27 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 46, administered in an amount.

50. The method according to any one of claims 30 to 45, wherein the recombinant L-asparaginase is administered intravenously.

51. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ The method according to claim 50, administered in an amount.

52. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 55mg / m ２ The method according to claim 50, administered in an amount.

53. The recombinant L-asparaginase is present in approximately 27.5 mg / m². ２ ~About 47.5mg / m ２ The method according to claim 50, administered in an amount.

54. The recombinant L-asparaginase is present in approximately 21.5 mg / m². ２ ~Approx. 38.5mg / m ２ The method according to claim 50, administered in an amount.

55. The method according to any one of claims 30 to 54 and 140 to 176, wherein the lowest serum asparaginase activity (NSAA) measured from the serum sample derived from the human subject is equal to or greater than 0.1 IU / mL after administration and treatment with the recombinant L-asparaginase.

56. The method according to any one of claims 30 to 55 and 140 to 176, wherein the recombinant L-asparaginase is co-administered with one or more other chemotherapy agents as part of a multi-agent chemotherapy regimen.

57. The method according to any one of claims 30 to 56 and 140 to 176, wherein the recombinant L-asparaginase is contained in the composition, and the composition exhibits aggregation of less than 6% of the recombinant L-asparaginase protein.

58. The method according to any one of claims 30 to 56 and 140 to 176, wherein the recombinant L-asparaginase is contained in the composition, and the composition exhibits aggregation of less than 1% of the recombinant L-asparaginase protein.

59. The method according to any one of claims 30 to 58 and 140 to 176, wherein the recombinant L-asparaginase is not freeze-dried.

60. A method for treating a disease treatable by asparagine depletion in a human subject, the method comprising administering a composition containing recombinant L-asparaginase to the human subject, wherein the recombinant L-asparaginase is a tetramer, the tetramer has four monomers, and the composition exhibits aggregation of less than 6%.

61. Claim that the recombinant L-asparaginase is not conjugated with the PEG moiety. The method described in 60.

62. The method according to claim 60, wherein the recombinant L-asparaginase is not conjugated with a peptide containing proline or alanine.

63. The method according to claim 60, wherein the human subject has been previously treated with an asparaginase derived from E. coli.

64. The method according to claim 63, wherein the asparaginase derived from E. coli is conjugated to the PEG portion.

65. The method according to claim 63, wherein the human subject has previously experienced silent inactivation of asparaginase derived from E. coli.

66. The method according to claim 63, wherein the human subject has previously experienced an allergic reaction to asparaginase derived from E. coli.

67. The method according to claim 63, wherein the human subject has previously experienced anaphylaxis to asparaginase derived from E. coli.

68. The method according to claim 60 or 63, wherein the disease treatable by L-asparagine depletion is acute lymphoblastic leukemia (ALL).

69. The method according to claim 60 or 63, wherein the disease treatable by L-asparagine depletion is lymphoblastic lymphoma (LBL).

70. The method according to claim 68 or 69, wherein the ALL or LBL is recurrent ALL or recurrent LBL.

71. The method according to any one of claims 60 to 70, wherein the recombinant L-asparaginase is administered three times a week.

72. The method according to claim 71, wherein the recombinant L-asparaginase is administered every other day for five consecutive days, followed by a drug-free period of two consecutive days.

73. The method according to claim 71, wherein the recombinant L-asparaginase is administered on Monday, Wednesday, and Friday of the same week.

74. The method according to any one of claims 71 to 73, wherein the recombinant L-asparaginase is administered for two weeks.

75. The recombinant L-asparaginase is present in approximately 10 mg / m². ２ ~Approx. 100mg / m ２ The method according to any one of claims 60 to 74, administered in an amount.

76. The method according to any one of claims 60 to 75, wherein the recombinant L-asparaginase is administered intramuscularly.

77. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ The method according to claim 76, administered in an amount.

78. The recombinant L-asparaginase is present in approximately 15 mg / m². ２ ~Approx. 45mg / m ２ The method according to claim 76, administered in an amount.

79. The recombinant L-asparaginase is present in an amount of approximately 27 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 76, administered in an amount.

80. The method according to any one of claims 60 to 75, wherein the recombinant L-asparaginase is administered intravenously.

81. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ The method according to claim 80, administered in an amount.

82. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 55mg / m ２ The method according to claim 80, administered in an amount.

83. The recombinant L-asparaginase is present in approximately 27.5 mg / m². ２ ~About 47.5mg / m ２ The method according to claim 80, administered in an amount.

84. The recombinant L-asparaginase is present in approximately 21.5 mg / m². ２ ~Approx. 38.5mg / m ２ The method according to claim 80, administered in an amount.

85. The method according to any one of claims 60 to 84 and 177 to 213, wherein the lowest serum asparaginase activity (NSAA) measured from the serum sample derived from the human subject is equal to or greater than 0.1 IU / mL after administration and treatment with the recombinant L-asparaginase.

86. The method according to any one of claims 60 to 85 and 177 to 213, wherein the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a multi-agent chemotherapy regimen.

87. The method according to any one of claims 60 to 86 and 177 to 213, wherein each monomer of the tetramer has an amino acid sequence containing SEQ ID NO:

1.

88. The method according to any one of claims 60 to 87 and 177 to 213, wherein the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens.

89. The method according to any one of claims 60 to 88 and 177 to 213, wherein the amount of aggregation of the recombinant L-asparaginase is less than 1%.

90. The method according to any one of claims 60 to 89 and 177 to 213, wherein the recombinant L-asparaginase is not freeze-dried.

91. A method for treating a disease in human subjects, wherein the method is approximately 10 mg / m² ２ ~Approx. 100mg / m ２ The method comprises administering recombinant L-asparaginase to a human subject in an amount, wherein the recombinant L-asparaginase is a tetramer, the tetramer comprises four monomers, and each monomer has an amino acid sequence containing SEQ ID NO:

1.

92. The aforementioned diseases include sarcoma, breast cancer, metastatic breast cancer, liver cancer, gastric cancer, prostate cancer, colorectal cancer, and head cancer. The method according to claim 91, wherein the member is selected from cervical cancer.

93. The method according to claims 91 to 92, wherein the recombinant L-asparaginase is not conjugated with the PEG portion.

94. The method according to any one of the prior claims, wherein the recombinant L-asparaginase is administered to the human subject in place of the administration of a long-acting asparaginase derived from E. coli.

95. The method according to claim 94, wherein six doses of recombinant L-asparaginase are administered to the human subject in place of a single dose of the long-acting asparaginase derived from E. coli.

96. The method according to any one of claims 94 to 95, wherein the long-acting asparaginase derived from E. coli is pegaspar gauze.

97. The method according to any one of the prior claims, wherein the administration regimen of recombinant L-asparaginase comprises a cycle, the cycle comprises a first dose, a second dose, and a third dose, the cycle is optionally repeatable, and the first dose, second dose, and third dose are administered at intervals of approximately 48 to 72 hours.

98. The recombinant L-asparaginase administration regimen comprises a cycle, the cycle is optionally repeatable, the cycle comprises administration of recombinant L-asparaginase every other day for five consecutive days, followed by a two-day rest period, and the initial dose of the cycle is 25 mg / m². ２ Therefore, the second dose of the cycle is 25 mg / m². ２ Therefore, the third dose of the cycle was 37.5 mg / m². ２ The method according to any one of the prior claims, wherein a drug-free period of two consecutive days follows.

99. The recombinant L-asparaginase administration regimen comprises a cycle, the cycle is optionally repeatable, the cycle comprises administration of recombinant L-asparaginase every other day for five consecutive days, followed by a two-day rest period, and the initial dose of the cycle is 37.5 mg / m². ２ Therefore, the second dose of the cycle was 37.5 mg / m². ２ Therefore, the third dose of the cycle was 37.5 mg / m². ２ The method according to any one of claims 1 to 97, wherein a drug-free period of two consecutive days follows.

100. The recombinant L-asparaginase administration regimen comprises a cycle, the cycle is optionally repeatable, the cycle comprises administration of recombinant L-asparaginase every other day for five consecutive days, followed by a two-day rest period, and the initial dose of the cycle is 37.5 mg / m². ２ Therefore, the second dose of the cycle is 25 mg / m². ２ Therefore, the third dose of the cycle was 37.5 mg / m². ２ The method according to any one of claims 1 to 97, wherein a drug-free period of two consecutive days follows.

101. The recombinant L-asparaginase administration regimen comprises a cycle, the cycle is optionally repeatable, the cycle comprises administration of recombinant L-asparaginase every other day for five consecutive days, followed by a two-day rest period, and the initial dose of the cycle is 37.5 mg / m². ２ Therefore, the second dose of the cycle is 25 mg / m². ２ Therefore, the third dose of the cycle is 25 mg / m². ２ The method according to any one of claims 1 to 97, wherein a drug-free period of two consecutive days follows.

102. The first dose of the cycle is administered on Monday, and the second dose of the cycle is administered on Wednesday. The method according to any one of claims 97 to 101, wherein the dose is administered on a weekday, and the third dose of the cycle is administered on Friday.

103. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~about 80mg / m ２ The method according to claim 16, administered in an amount.

104. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ ~about 80mg / m ２ The method according to claim 16, administered in an amount.

105. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 16, administered in an amount.

106. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 16, administered in an amount.

107. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 75mg / m ２ The method according to claim 16, administered in an amount.

108. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 16, administered in an amount.

109. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 16, administered in an amount.

110. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 60mg / m ２ The method according to claim 16, administered in an amount.

111. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 55mg / m ２ The method according to claim 16, administered in an amount.

112. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 75mg / m ２ The method according to claim 16, administered in an amount.

113. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 16, administered in an amount.

114. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 16, administered in an amount.

115. The recombinant L-asparaginase is approximately 55 mg / m². ２ ~about 60mg / m ２ The method according to claim 16, administered in an amount.

116. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~about 60mg / m ２ The method according to claim 16, administered in an amount.

117. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~Approx. 55mg / m ２ The method according to claim 16, administered in an amount.

118. The recombinant L-asparaginase is present in approximately 47.5 mg / m². ２ ~Approx. 50mg / m ２ The method according to claim 16, administered in an amount.

119. The recombinant L-asparaginase is administered at a dose of about 30 mg / m ２ to about 35 mg / m ２ The method according to claim 16.

120. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 20, administered in an amount.

121. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 100mg / m ２ The method according to claim 20, administered in an amount.

122. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 20, administered in an amount.

123. The recombinant L-asparaginase is present at about 25 mg / m ２ to about 80 mg / m ２ when administered in an amount of according to claim 20. The method according to claim 20.

124. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 35mg / m ２ The method according to claim 20, administered in an amount.

125. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 95mg / m ２ The method according to claim 20, administered in an amount.

126. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 90mg / m ２ The method according to claim 20, administered in an amount.

127. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~about 85mg / m ２ The method according to claim 20, administered in an amount.

128. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 80mg / m ２ The method according to claim 20, administered in an amount.

129. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 75mg / m ２ The method according to claim 20, administered in an amount.

130. The recombinant L-asparaginase is approximately 55 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 20, administered in an amount.

131. The recombinant L-asparaginase is present at approximately 60 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 20, administered in an amount.

132. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~about 60mg / m ２ The method according to claim 20, administered in an amount.

133. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 55mg / m ２ The method according to claim 20, administered in an amount.

134. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 50mg / m ２ The method according to claim 20, administered in an amount.

135. The recombinant L-asparaginase is present in approximately 42.5 mg / m². ２ ~Approx. 57.5mg / m ２ of The method according to claim 20, administered in a certain quantity.

136. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 75mg / m ２ The method according to claim 20, administered in an amount.

137. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 20, administered in an amount.

138. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 60mg / m ２ The method according to claim 20, administered in an amount.

139. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 55mg / m ２ The method according to claim 20, administered in an amount.

140. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~about 80mg / m ２ The method according to claim 46, administered in an amount.

141. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ ~about 80mg / m ２ The method according to claim 46, administered in an amount.

142. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 46, administered in an amount.

143. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 46, administered in an amount.

144. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 75mg / m ２ The method according to claim 46, administered in an amount.

145. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 46, administered in an amount.

146. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 46, administered in an amount.

147. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 60mg / m ２ The method according to claim 46, administered in an amount.

148. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 55mg / m ２ The method according to claim 46, administered in an amount.

149. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 75mg / m ２ The method according to claim 46, administered in an amount.

150. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 46, administered in an amount.

151. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 46, administered in an amount.

152. The recombinant L-asparaginase is approximately 55 mg / m². ２ ~about 60mg / m ２ The method according to claim 46, administered in an amount.

153. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~about 60mg / m ２ The method according to claim 46, administered in an amount.

154. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~Approx. 55mg / m ２ The method according to claim 46, administered in an amount.

155. The recombinant L-asparaginase is present in approximately 47.5 mg / m². ２ ~Approx. 50mg / m ２ The method according to claim 46, administered in an amount.

156. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 35mg / m ２ The method according to claim 46, administered in an amount.

157. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 50, administered in an amount.

158. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 100mg / m ２ The method according to claim 50, administered in an amount.

159. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 50, administered in an amount.

160. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~about 80mg / m ２ The method according to claim 50, administered in an amount.

161. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 35mg / m ２ The method according to claim 50, administered in an amount.

162. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 95mg / m ２ The method according to claim 50, administered in an amount.

163. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 90mg / m ２ The method according to claim 50, administered in an amount.

164. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~about 85mg / m ２ The method according to claim 50, administered in an amount.

165. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 80mg / m ２ The method according to claim 50, administered in an amount.

166. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 75mg / m ２ The method according to claim 50, administered in an amount.

167. The recombinant L-asparaginase is approximately 55 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 50, administered in an amount.

168. The recombinant L-asparaginase is present at approximately 60 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 50, administered in an amount.

169. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~about 60mg / m ２ The method according to claim 50, administered in an amount.

170. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 55mg / m ２ The method according to claim 50, administered in an amount.

171. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 50mg / m ２ The method according to claim 50, administered in an amount.

172. The recombinant L-asparaginase is present in approximately 42.5 mg / m². ２ ~Approx. 57.5mg / m ２ The method according to claim 50, administered in an amount.

173. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 75mg / m ２ The method according to claim 50, administered in an amount.

174. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 50, administered in an amount.

175. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 60mg / m ２ The method according to claim 50, administered in an amount.

176. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 55mg / m ２ The method according to claim 50, administered in an amount.

177. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~about 80mg / m ２ The method according to claim 76, administered in an amount.

178. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ ~about 80mg / m ２ The method according to claim 76, administered in an amount.

179. The recombinant L-asparaginase is present in approximately 37.5 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 76, administered in an amount.

180. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 76, administered in an amount.

181. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 75mg / m ２ The method according to claim 76, administered in an amount.

182. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 76, administered in an amount.

183. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 76, administered in an amount.

184. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 60mg / m ２ The method according to claim 76, administered in an amount.

185. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 55mg / m ２ Administer in the amount provided. The method according to claim 76.

186. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 75mg / m ２ The method according to claim 76, administered in an amount.

187. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 76, administered in an amount.

188. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 76, administered in an amount.

189. The recombinant L-asparaginase is approximately 55 mg / m². ２ ~about 60mg / m ２ The method according to claim 76, administered in an amount.

190. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~about 60mg / m ２ The method according to claim 76, administered in an amount.

191. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~Approx. 55mg / m ２ The method according to claim 76, administered in an amount.

192. The recombinant L-asparaginase is present in approximately 47.5 mg / m². ２ ~Approx. 50mg / m ２ The method according to claim 76, administered in an amount.

193. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 35mg / m ２ The method according to claim 76, administered in an amount.

194. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 37.5mg / m ２ The method according to claim 80, administered in an amount.

195. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 100mg / m ２ The method according to claim 80, administered in an amount.

196. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 80, administered in an amount.

197. The recombinant L-asparaginase is present in approximately 25 mg / m². ２ ~about 80mg / m ２ The method according to claim 80, administered in an amount.

198. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 35mg / m ２ The method according to claim 80, administered in an amount.

199. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 95mg / m ２ The method according to claim 80, administered in an amount.

200. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 90mg / m ２ The method according to claim 80, administered in an amount.

201. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~about 85mg / m ２ The method according to claim 80, administered in an amount.

202. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 80mg / m ２ The method according to claim 80, administered in an amount.

203. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 75mg / m ２ The method according to claim 80, administered in an amount.

204. The recombinant L-asparaginase is approximately 55 mg / m². ２ ~Approx. 70mg / m ２ The method according to claim 80, administered in an amount.

205. The recombinant L-asparaginase is present at approximately 60 mg / m². ２ ~Approx. 65mg / m ２ The method according to claim 80, administered in an amount.

206. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~about 60mg / m ２ The method according to claim 80, administered in an amount.

207. The recombinant L-asparaginase is present in an amount of approximately 35 mg / m². ２ ~Approx. 55mg / m ２ The method according to claim 80, administered in an amount.

208. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 50mg / m ２ The method according to claim 80, administered in an amount.

209. The recombinant L-asparaginase is present in approximately 42.5 mg / m². ２ ~Approx. 57.5mg / m ２ The method according to claim 80, administered in an amount.

210. The recombinant L-asparaginase is present in an amount of approximately 30 mg / m². ２ ~Approx. 75mg / m ２ The method according to claim 80, administered in an amount.

211. The recombinant L-asparaginase is approximately 40 mg / m² ２ ~Approx. 65mg / m ２ The method according to claim 80, administered in an amount.

212. The recombinant L-asparaginase is present at approximately 45 mg / m². ２ ~about 60mg / m ２ The method according to claim 80, administered in an amount.

213. The recombinant L-asparaginase is approximately 50 mg / m² ２ ~Approx. 55mg / m ２ The method according to claim 80, administered in an amount.

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