Recombinant L-asparaginase dosage and use

Abstract

The present invention provides compositions and methods for treating a disease treatable by asparagine depletion in a human subject comprising administering to the human subject L-asparaginase.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 168,224, filed March 30, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created on March 30, 2022 is named 48929-4009_SL.txt and is 6,543 bytes in size.

[0003] Technical Field The present invention provides dosing regimens for L-asparaginase. [Background technology]

[0004] background A protein with L-asparagine aminohydrolase activity, commonly known as L-asparaginase, has been used successfully to treat a variety of potentially fatal diseases, including acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL), the majority of patients affected by these diseases are children.

[0005] L-asparaginase of bacterial origin has high potential immunogenicity and antigenicity. Currently on the market as first-line treatments are E. coli-derived L-asparaginase and pegaspargase. These products can cause adverse hypersensitivity reactions in patients, including allergic reactions, asymptomatic inactivation, and anaphylactic shock. Patients who develop hypersensitivity reactions to these products are often forced to discontinue treatment, resulting in poor prognosis and reduced survival. These patients who develop hypersensitivity reactions have been transitioned to Erwinaze®, which can reportedly take nine months to prepare and has long been plagued by supply issues (see https: / / www.statnews.com / 2016 / 10 / 31 / cancer-drug-shortage / and https: / / www.drugs.com / drug-shortages / asparaginase-erwinia-chrysanthemi-482). The problem continues to this day, with Erwinase® shortages becoming commonplace (see https: / / www.accessdata.fda.gov / scripts / drugshortages / dsp_ActiveIngredientDetails.cfm?AI=Asparaginase%20Erwinia%20Chrysanthemi%20(Erwinaze)&st=c). The FDA has issued warning letters to manufacturers stating that "any changes in source material or cell line are likely to adversely affect the identity, active ingredient content, quality, purity, or potency of Erwinase®."

[0006] There is a need for new and improved dosing regimens of L-asparaginase (such as recombinant L-asparaginase recombinantly produced in Pseudomonas fluorescens) that lack immunological cross-reactivity to E. coli-derived asparaginase. Such dosing regimens could be used as a first-line therapy or as an alternative to native E. coli-derived L-asparaginase and / or long-acting E. coli-derived L-asparaginase when human subjects develop hypersensitivity. Novel dosing regimens of recombinant L-asparaginase (as a component of a combination chemotherapy regimen) would address a significant medical need in ALL / lymphoblastic lymphoma (LBL) patients. Novel dosing regimens of L-asparaginase (including, but not limited to, dosing regimens that are components of combination chemotherapy regimens) will also address the significant medical needs of patients with colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), and acute myeloid leukemia (AML), particularly Wnt-negative CRC and R / R AML. Summary of the Invention

[0007] Thus, the present disclosure provides a recombinant L-asparaginase that does not have immunological cross-reactivity with asparaginase derived from E. coli.

[0008] In one aspect, the disclosure provides a method of treating cancer in a human subject in need of such treatment, comprising administering to the human subject an L-asparaginase, other than E. coli derived asparaginase, as a chronological dose set, wherein the chronological dose set comprises a first dose followed by a second dose, the first dose being about (i) 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and the second dose is (i) about 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and wherein the first dose is about 50 mg / m 2and wherein the administration interval between the first and second doses to the human subject is 3 days, and the first dose is about 25 mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 2 days, and the second dose is about 50 mg / m 2 and wherein said subject has not received any further administration of L-asparaginase for 3 days, and said second dose is about 25 mg / m 2 and no further administration of L-asparaginase to said subject occurs for 2 days when said L-asparaginase is about 25 mg / m 2 is administered intravenously and is approximately 50 mg / m 2 In some embodiments, the first dose is administered intramuscularly at a dose of about 50 mg / m 2 and said second dose is about 25 mg / m 2 and the chronological dose set further comprises a third dose after the second dose, the third dose being about 25 mg / m 2 wherein the administration interval between the second and third doses to the human subject is two days. In some embodiments, the first dose is administered on a Friday and the second dose is administered on a Monday. In some embodiments, the first dose is administered on a Friday, the second dose is administered on a Monday, and the third dose is administered on a Wednesday. In some embodiments, the first dose is about 25 mg / m 2 and said second dose is about 50 mg / m 2 and the chronological dose set further comprises a third dose after the second dose, the third dose being about 25 mg / m 2 and the administration interval between the second and third doses to the human subject is 3 days. In some embodiments, the first dose is administered on a Wednesday and the second dose is administered on a Friday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the first dose is about 25 mg / m 2 and said second dose is about 50 mg / m 2and said chronological dose set further comprises a third dose prior to said first dose, said third dose being about 25 mg / m 2 and the third dose is administered to the human subject two days prior to the first dose. In some embodiments, the first dose is administered on a Wednesday and the second dose is administered on a Friday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the method further comprises a third dose, where the first dose is administered on a Monday, the second dose is administered on a Wednesday, and the third dose is administered on a Friday. In some embodiments, the first dose is 25 mg / m 2 and said second dose is 25 mg / m 2 and said third dose is 50 mg / m 2In some embodiments, the method further comprises administering to the human subject a plurality of instances of the L-asparaginase of the chronological dose set, wherein each instance of the recombinant L-asparaginase of the chronological dose set is administered to the subject upon completion of a previous instance of the chronological dose set in the plurality of instances of the chronological dose set. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 100. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 15. In some embodiments, the E. coli derived asparaginase is naturally occurring. In some embodiments, the E. coli derived asparaginase is long acting. In some embodiments, the long acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult. In some embodiments, the human subject is a child. In some embodiments, the L-asparaginase exhibits less than 6% aggregation. In some embodiments, the L-asparaginase exhibits less than 1% aggregation. In some embodiments, the L-asparaginase is not lyophilized. In some embodiments, the L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the serum asparaginase activity in assay minimum (NSAA) as measured from a serum sample from the human subject is equal to or greater than 0.1 IU / mL after administration of the L-asparaginase post-treatment. In some embodiments, the L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0009] In another aspect, the disclosure provides a method of switching cancer treatment in a human subject in need of treatment, comprising administering to the human subject a series of six doses of L-asparaginase in place of each dose of long-acting E. coli derived asparaginase, wherein four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2 and the dosing interval between two doses of about 25 mg / m2 in said series is 7 days, the dosing interval between two doses of about 50 mg / m2 in said series is 2 days, and 2 and the dosing interval between the dose of about 25 mg / m 2 The dose is administered intravenously at approximately 50 mg / m 2and the L-asparaginase is not long-acting E. coli derived asparaginase. In some embodiments, each dose of the long-acting E. coli derived asparaginase in the multiple doses of long-acting E. coli derived asparaginase is separately replaced with an instance of the series of 6 doses of L-asparaginase. In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject has completed E. coli derived asparaginase therapy. In some embodiments, the human subject is an adult. In some embodiments, the human subject is a child. In some embodiments, the L-asparaginase exhibits less than 6% aggregation. In some embodiments, the L-asparaginase exhibits less than 1% aggregation. In some embodiments, the L-asparaginase is not lyophilized. In some embodiments, the L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the lowest serum asparaginase activity (NSAA) in an assay as measured from a serum sample from the human subject is 0.1 IU / mL or greater after administration of the L-asparaginase post-treatment. In some embodiments, the L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL). In some embodiments, the cancer is colorectal cancer (CRC). In some embodiments, the CRC is Wnt-negative CRC or Wnt-mutated CRC.In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the AML is R / R AML. In some embodiments, the cancer is DLBCL.

[0010] In another aspect, the disclosure provides a method of switching cancer treatment in a human subject in need thereof, comprising intravascularly administering to said human subject a series of seven doses of recombinant L-asparaginase in place of each dose of long-acting E. coli derived asparaginase, wherein each dose in said series of doses is about 25 mg / m 2and the administration interval between each dose in the series of doses is 2 days. In some embodiments, each dose of the long-acting E. coli derived asparaginase in the multiple doses of long-acting E. coli derived asparaginase is separately replaced with an instance of the series of 7 doses of recombinant L-asparaginase. In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 6% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 3% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the serum asparaginase activity minimum assay (NSAA) as measured from a serum sample from the human subject is equal to or greater than 0.1 IU / mL after administration with the recombinant L-asparaginase. In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0011] In another aspect, the disclosure provides a method of switching cancer treatment in a human subject in need thereof, comprising intravascularly administering to the human subject a series of six doses of L-asparaginase in place of each dose of long-acting E. coli derived asparaginase, wherein four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2 and the interval between two doses is 7 days, and the doses in the series are about 25 mg / m 2 and the interval between each dose is 2 days, and 2and a subsequent dose of L-asparaginase is not a long-acting E. coli derived asparaginase. In some embodiments, each dose of the long-acting E. coli derived asparaginase in the multiple doses of long-acting E. coli derived asparaginase is separately replaced with an instance of the series of 6 doses of L-asparaginase. In some embodiments, the long-acting E. coli derived asparaginase is a peg-asparaginase. In some embodiments, the L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject has completed E. coli derived asparaginase therapy. In some embodiments, the human subject is an adult. In some embodiments, the human subject is a child. In some embodiments, the L-asparaginase exhibits less than 6% aggregation. In some embodiments, the L-asparaginase exhibits less than 1% aggregation. In some embodiments, the L-asparaginase is not lyophilized. In some embodiments, the L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the lowest serum asparaginase activity (NSAA) in an assay as measured from a serum sample from the human subject is greater than or equal to 0.1 IU / mL after administration of the L-asparaginase post-treatment. In some embodiments, the L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0012] In another aspect, the present invention provides a method of treating cancer in a human subject in need of such treatment, comprising intravascularly administering to the human subject an L-asparaginase, other than E. coli derived asparaginase, as a set of chronological doses, wherein the set of chronological doses comprises a first dose followed by a second dose, the first dose being (i) about 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and the second dose is (i) about 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and wherein the first dose is about 50 mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 3 days, and the first dose is about 25 mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 2 days, and the second dose is about 50 mg / m 2 and the subject has not received any further administration of L-asparaginase for 3 days, and the second dose is about 25 mg / m 2 In some embodiments, the first dose is about 50 mg / m 2 and said second dose is about 25 mg / m 2 and the chronological dose set further comprises a third dose after the second dose, the third dose being about 25 mg / m 2 wherein the administration interval between the second and third doses to the human subject is two days. In some embodiments, the first dose is administered on a Friday and the second dose is administered on a Monday. In some embodiments, the first dose is administered on a Friday, the second dose is administered on a Monday, and the third dose is administered on a Wednesday. In some embodiments, the first dose is about 25 mg / m 2 and said second dose is about 50 mg / m 2 and the chronological dose set further comprises a third dose after the second dose, the third dose being about 25 mg / m 2and the administration interval between the second and third doses to the human subject is 3 days. In some embodiments, the first dose is administered on a Wednesday and the second dose is administered on a Friday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the first dose is about 25 mg / m 2 and said second dose is about 50 mg / m 2 and said chronological dose set further comprises a third dose prior to said first dose, said third dose being about 25 mg / m 2 and the third dose is administered to the human subject two days prior to the first dose. In some embodiments, the first dose is administered on a Wednesday and the second dose is administered on a Friday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the method further comprises a third dose, where the first dose is administered on a Monday, the second dose is administered on a Wednesday, and the third dose is administered on a Friday. In some embodiments, the first dose is 25 mg / m 2 and said second dose is 25 mg / m 2 and said third dose is 50 mg / m 2In some embodiments, the method further comprises administering a plurality of instances of the chronological dose set of L-asparaginase intravascularly to the human subject, wherein each instance of the recombinant L-asparaginase in the chronological dose set is administered to the subject upon completion of a previous instance of the chronological dose set in the plurality of instances of the chronological dose set. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 100. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 15. In some embodiments, the E. coli derived asparaginase is naturally occurring. In some embodiments, the E. coli derived asparaginase is long acting. In some embodiments, the long acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult. In some embodiments, the human subject is a child. In some embodiments, the L-asparaginase exhibits less than 6% aggregation. In some embodiments, the L-asparaginase exhibits less than 1% aggregation. In some embodiments, the L-asparaginase is not lyophilized. In some embodiments, the L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the serum asparaginase activity in assay minimum (NSAA) as measured from a serum sample from the human subject is equal to or greater than 0.1 IU / mL after administration of the L-asparaginase post-treatment. In some embodiments, the L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL). In some embodiments, the cancer is colorectal cancer (CRC). In some embodiments, the CRC is Wnt-negative CRC or Wnt-mutated CRC. In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the AML is R / R AML. In some embodiments, the cancer is DLBCL. [Brief description of the drawings]

[0013] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, in which: [Figure 1] 4 shows the results of a safety test in Example 2. [Diagram 2] FIG. 1 shows the purity of recombinant L-asparaginase (R-CRIS), recombinant crisantaspase from E. coli (E. coli RC), and Erwinase® as assessed by the SEC-HPLC method outlined in Example 5. [Diagram 3] FIG. 1 shows the purity of recombinant L-asparaginase (R-CRIS), recombinant crisantaspase from E. coli (E. coli RC), and Erwinase® as assessed by the SEC-MALLS method outlined in Example 5. [Figure 4] 1 shows the results obtained from the analysis of sedimentation velocity AUC data as outlined in Example 5. [Diagram 5] 1 shows the size profile of recombinant L-asparaginase assessed by SE-UHPLC following the release method outlined in Example 6. [Figure 6] 1 shows the measurement of high molecular weight species present in recombinant L-asparaginase using SEC-MALLS as outlined in Example 6. [Figure 7] FIG. 1 shows the normalized sedimentation coefficient distribution of recombinant L-asparaginase (commercial scale) as outlined in Example 6. [Figure 8]FIG. 1 shows the normalized sedimentation coefficient distribution of Erwinase® (commercial scale) as outlined in Example 6. [Figure 9] Figure 1 shows the percentage of healthy subjects who showed SAA levels after 48 and 72 doses according to Example 7. Abbreviations: ERW, Erwinia chrysanthemi asparaginase. IM, intramuscular. IV, intravenous. SAA, serum asparaginase activity. [Figure 10]

[0046] Figure 1 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 infinity. 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. CV, coefficient of variation. ERW, Erwinia chrysanthemiasparaginase. 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. a For IM treatment, CL=CL / F (apparent clearance) and V=Vz / F (apparent volume of distribution). For IV treatment, CL=CL and V=Vss (estimate of volume of distribution at steady state). b n=5 for t1 / 2, AUC0-inf, CL and V. N is the number of exposed healthy subjects. Mean values ​​(CV%) are shown for all parameters except tmax values, which are reported as median and range. [Figure 11] TEAEs following treatment as described in Example 7 are shown. Abbreviations: ERW, Erwinia chrysanthemiasparaginase. IM, intramuscular. IV, intravenous. TEAE, treatment-emergent adverse events. a Listed by preferred term using the MedDRA glossary, version 22.0. Treatment-related TEAEs are listed in descending order of frequency. [Figure 12] Individual SAA-time profiles are shown. Abbreviations: ERW, Erwinia chrysanthemiasparaginase. IM, intramuscular. IV, intravenous. SAA, serum asparaginase activity. Further explanation is given in Example 7. [Figure 13]Figure 1 shows SAA mean (95%CI)-time profiles as further described in Example 7. Abbreviations: CI, confidence interval. IM, intramuscular. IV, intravenous. SAA, serum asparaginase activity. [Figure 14] A) SAA mean-time profile for IM administration of JZP-458, a recombinant Erwinia asparaginase produced using a novel Pseudomonas fluorescens expression platform that generates an enzyme with no immunological cross-reactivity to E. coli-derived asparaginase. Abbreviations: ERW, Erwinia chrysanthemiasparaginase. IM, intramuscular. SAC, serum asparaginase concentration. Note: Lower limit of quantification (LLOQ) = 1.00 ng / mL. Values ​​below the LLOQ were set to 0. B) SAA mean-time profile for IV administration of JZP-458. ERW, Erwinia chrysanthemiasparaginase. IV, intravenous. SAC, serum asparaginase concentration. Note: Lower limit of quantification (LLOQ) = 1.00 ng / mL. Values ​​below the LLOQ were set to 0. C) Individual asparaginase concentration-time profile. ERW, Erwinia chrysanthemiasparaginase. IM, intramuscular. IV, intravenous. SAC, serum asparaginase concentration. D) Correlation between SAC and SAA with IM and IV administration of JZP-458. 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 explanation is provided in Example 7. [Figure 15] 1 shows the SAA mean versus time profile and corresponding mean plasma L-asparagine levels as further described in the study in Example 7. Abbreviations: IM, intramuscular. IV, intravenous. SAA, serum asparaginase activity. Note: For lower limit of quantification (LLOQ), asparaginase activity = 0.0250 IU / mL, L-asparagine = 0.0250 ug / mL. Values ​​below the LLOQ were set to 0. [Figure 16]

[0036] Figure 1 shows baseline demographics for the study in Example 7. Abbreviations: ERW, Erwinia chrysanthemiasparaginase. IM, intramuscular. IV, intravenous. SD, standard deviation. a Ethnicity was self-reported. Healthy subjects could self-identify as more than one ethnicity. [Figure 17] Figure 1 shows an assessment of dose proportionality of JZP-458 based on SAA as further described in the study in Example 7. Abbreviations: AUC0-inf, area under the curve extrapolated from time 0 to infinity. AUC0-t, area under the curve from time 0 to the last time point at which SAA can be quantified. CI, confidence interval. Cmax, maximum SAA value. IM, intramuscular. IV, intravenous. PK, pharmacokinetics. SAA, serum asparaginase activity. Results are based on a power model where ln(parameter)=intercept+slope×ln(dose). [Figure 18] Figure 1 shows a summary of PK based on SAC, which is further described in Example 7. Abbreviations: AUC0-inf, area under the curve extrapolated from time 0 to infinity. AUC0-t, area under the curve from time 0 to the last time point for which SAA is quantifiable. CL, clearance. Cmax, maximum SAA. CV, coefficient of variation. ERW, Erwinia chrysanthemiasparaginase. IM, intramuscular. IV, intravenous. PK, pharmacokinetics. t1 / 2, half-life. SAA, serum asparaginase activity. SAC, serum asparaginase concentration. tmax, time point at which Cmax is observed. V, volume of distribution. a For IM treatment, CL=CL / F (apparent clearance) and V=Vz / F (apparent volume of distribution). For IV treatment, CL=CL and V=Vss (estimate 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. Mean values ​​(CV%) are shown for all parameters except tmax values, which are reported as medians and ranges. [Figure 19]Figure 1 shows an assessment of dose proportionality of JZP-458 based on SAC. Abbreviations: AUC0-inf, area under the curve extrapolated from time 0 to infinity. AUC0-t, area under the curve from time 0 to the last time point at which SAA can be quantified. CI, confidence interval. Cmax, maximum SAA value. IM, intramuscular. IV, intravenous. PK, pharmacokinetics. SAA, serum asparaginase activity. SAC, serum asparaginase concentration. Results are based on a power model: ln(parameter) = intercept + slope x ln(dose). [Figure 20] Figure 1 shows the study design for the study in Example 7. Abbreviations: ERW, Erwinia chrysanthemiasparaginase. IM, intramuscular. IV, intravenous. PK, pharmacokinetics. [Figure 21] Figure 1 shows the SAA mean-time profile for all treatments and the corresponding mean plasma L-glutamine levels as further described in the study in Example 7. Abbreviations: IM, intramuscular. IV, intravenous. SAA, serum asparaginase activity. Note: for lower limit of quantification (LLOQ) asparaginase activity = 0.0250 IU / mL, L-glutamine = 0.250 ug / mL. Values ​​below the LLOQ were set to 0. Abbreviations: IM, intramuscular. IV, intravenous. SAA, serum asparaginase activity. Note: for lower limit of quantification (LLOQ) asparaginase activity = 0.0250 IU / mL, L-glutamine = 0.250 ug / mL. Values ​​below the LLOQ were set to 0. [Figure 22] Part A JZP-458 IM dosing cohort (Example 8) is shown. The SDRC will evaluate safety and tolerability issues 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 progression to the expansion cohort (Cohort 2). The SDRC will review NSAA and safety / tolerability data when 6 and 13 evaluable participants have completed the first course in each subcohort. Enrollment will not be stopped at any particular time point for SDRC review. Abbreviations: IM=intramuscular. IU=international units. NSAA=nadir serum asparaginase activity. SDRC=trial data review committee. [Figure 23]Part B JZP-458 dosing cohorts (Example 8) are shown. The SDRC will evaluate safety and tolerability issues 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 course. Abbreviations: IU = International Units. IV = intravenous. NSAA = nadir serum asparaginase activity. SDRC = Study Data Review Committee. [Figure 24] 1 shows a summary of SAA results (IU / mL) for JZP-458 in the first course (evaluable participants) (Example 8). [Diagram 25] 1 shows the percentage of patients with NSAA levels ≧0.1 IU / mL and ≧0.4 IU / mL at the last 48 and 72 hours in the first course (evaluable participants) (Example 8). [Figure 26] Simulated JZP-458 median SAA profile with 95% prediction interval - semi-log scale (FMW dosing schedule) (IM simulation model of Example 8). Abbreviations: IM = intramuscular. FMW = Friday, Monday, Wednesday. SAA = serum asparaginase activity. Center line is median. Bands (95% prediction interval) represent the 2.5th percentile (lower) and 97.5th percentile (upper). The x-axis is shown with an additional offset out to 336 hours, and plotted data includes simulated observations out to 504 hours after the start of the course. [Figure 27] 4 shows the percentage of patients treated with JZP-458 predicted to achieve target SAA trough levels (FMW dosing schedule) (IM simulation model of Example 8). [Figure 28] An overview of treatment-emergent adverse events is shown (safety analysis set) (Example 8). [Figure 29] Grade 3 or 4 treatment-emergent adverse events are shown (Safety Analysis Set Cohort 1a) (Example 8). [Diagram 30]Grade 3 or 4 treatment-emergent adverse events are shown (Safety Population Cohort 1a) (Example 8). Percentages were calculated using the number of participants in the safety population as the denominator. Adverse events were coded into SOC and PT using MedDRA 22.1. AE severity was recorded using CTCAE 5.0. TEAEs were defined as any event with onset on any date after the first dose of study treatment through the end of the study or any ongoing event worsening in severity on any date after the first dose of study treatment through the end of the study. Abbreviations: AE = adverse event. CTCAE = Common Terminology Criteria for Adverse Events. IM = intramuscular injection. PT = preferred term. SOC = system organ class. TEAE = treatment-emergent adverse event. [Diagram 31] Summary of treatment-emergent adverse events (data cutoff date: October 14, 2021) from Study JZP458-201 described in Example 9. Abbreviations: CTCAE = Common Terminology Criteria for Adverse Events. IM = intramuscular. MedDRA = International Medical Dictionary. MWF = Monday, Wednesday, Friday. PT = preferred term. SOC = system organ class. TEAE = treatment-emergent adverse event. (a) Doses were administered on a Monday, Wednesday, Friday schedule. (b) No participants died at the time of the initial analysis cutoff date (October 14, 2020). Three deaths were reported between October 15, 2020 and January 11, 2021. 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 MedDRA22.1. The severity of AEs was recorded using CTCAE 5.0. The severity of AEs was recorded using CTCAE 5.0. A TEAE was defined as any event that occurred on any date after the first dose of study treatment through the end of the study or any ongoing event that worsened in severity on any date after the first dose of study treatment through the end of the study. [Diagram 32]Summary of treatment-emergent adverse events in Study JZP458-201, i.e., Cohort 1c, discussed in Examples 10 and 11 (Safety Analysis Set; Data Cutoff Date: January 11, 2021). Abbreviations: CTCAE = Common Terminology Criteria for Adverse Events. IM = Intramuscular. MedDRA = International Dictionary of Clinical Terms. PT = Preferred Term. SOC = System Organ Class. TEAE = Treatment-Emergent Adverse Event. Percentages were calculated using the number of participants in the safety analysis set as the denominator. Adverse events were coded into SOC and PT using MedDRA22.1. The severity of AEs was recorded using CTCAE5.0. The severity of AEs was recorded using CTCAE5.0. A TEAE was defined as any event that occurred on the date after the first dose of study treatment through the end of the study or any ongoing event that worsened in severity on the date after the first dose of study treatment through the end of the study. [Figure 33A] Figure 33A, Figure 33B and Figure 33C show an update on Study JZP458-201 described in Example 12, namely demographics and baseline characteristics (safety population as of July 19, 2021). Abbreviations: ALL = acute lymphoblastic leukemia. B ALL = B-cell acute lymphoblastic leukemia. B LBL = B-cell lymphoblastic lymphoma. BSA = body surface area. IM = intramuscular. LBL = lymphoblastic lymphoma. T ALL = T-cell acute lymphoblastic leukemia. T-cell lymphoblastic lymphoma. T LBL = T-cell lymphoblastic lymphoma. Percentages were calculated using the number of participants in the safety population as the denominator. Time in months from diagnosis is calculated as the integer '[(study start date - diagnosis date + 1) / 365.25]*12'. Baseline values ​​are defined as the most recent non-missing value obtained prior to the start date and / or time of first dose of JZP-458 (study day 1), which is defined as the date of first dose of JZP-458. [Figure 33B] See legend to Figure 33A. [Figure 33C] See legend to Figure 33A. [Diagram 34]1 shows a summary of serum asparaginase activity results (IU / mL) for JZP-458 (intramuscular and intravenous) in Study JZP458-201, i.e., first cycle (efficacy analysis population as of July 19, 2020). Abbreviations: CI = confidence interval. IM = intramuscular. IV = intravenous. MW = Monday, Wednesday. MWF = Monday, Wednesday, Friday. Q1 = 1st quartile. Q3 = 3rd quartile. NSAA = nadir serum asparaginase activity. SAA = serum asparaginase activity. The efficacy analysis population included participants who received at least one dose of JZP-458 and had at least one 48-hour or 72-hour NSAA assessment collected within the protocol-defined sampling window (± 2 hours) for the first cycle. [Diagram 35] 1 shows an update on the correlation between serum asparaginase concentration and serum asparaginase activity (pharmacokinetic analysis population as of July 19, 2021) for intramuscular and intravenous JZP-458 administered in the first cycle of Study JZP458-201 described in Example 12. Abbreviations: IM = intramuscular. IV = intravenous. PK = pharmacokinetics. SAA = serum asparaginase activity. SAC = serum asparaginase concentration. [Figure 36A] Figures 36A and 36B show updated mean serum asparaginase activity versus time profiles with corresponding mean plasma L-asparagine levels from the first course of intramuscular and intravenous JZP-458 in Study JZP458-201 described in Example 12 (pharmacodynamic analysis population as of July 19, 2021) on linear and semi-log scales. [Figure 36B] See legend to Figure 36A. [Figure 37A]Figures 37A, 37B and 37C show updated mean serum asparaginase activity versus time profiles for the first course of intramuscular and intravenous JZP-458 on linear and semi-log scales (pharmacodynamic analysis population as of July 19, 2021). Abbreviations: FMW = Friday, Monday, Wednesday. IM = intramuscular. IV = intravenous. MWF = Monday, Wednesday, Friday. PD = pharmacodynamics. SAA = serum asparaginase activity. WFM = Wednesday, Friday, Monday. Lower limit of quantification (LLOQ) for SAA 0.0350 IU / mL, L-glutamine = 0.250 μg / mL. Values ​​below the LLOQ were set to 0. In the semi-log plots, only SAA is in semi-log scale. If the mean value is 0, then on a semi-log plot there will be missing values ​​because the scale is not set to 0. [Figure 37B] See legend to Figure 37A. [Figure 37C] See legend to Figure 37A. [Figure 38] 1 shows the dose proportionality of JZP-458 as described in Example 12. Abbreviations: AUC = area under the concentration-time curve. Cmax = maximum SAA. Cmax,6 = maximum SAA after the sixth dose of the first cycle. DP = formulation. [Figure 39] Figure 1 shows the accumulation of JZP-458 as described in Example 12. Abbreviations: Cmax = maximum SAA value. Cmax,6 = maximum SAA value after the sixth dose of the first cycle. [Diagram 40] FIG. 1 shows the hierarchical immunogenicity testing strategy outlined in Example 12. Abbreviations: ADA = anti-drug antibodies. NR = non-reactive. [Diagram 41] 1 shows the maximum number of courses by anti-drug antibody collection (data cutoff date: July 19, 2021) for study JZP458-201 described in Example 12. Abbreviations: ADA = anti-drug antibody. IM = intramuscular. IV = intravenous. MWF = Monday, Wednesday, Friday. [Figure 42A]Figures 42A and 42B show an overview of SAA levels in ADA+ participants in Study JZP458-201 described in Example 12, i.e., with at least one SAA level <0.1 IU / mL in the first cycle, as of July 19, 2021. Abbreviations: ADA = anti-drug antibodies. BLQ = below limit of quantification. FMW = Friday, Monday, Wednesday. h = hours. MWF = Monday, Wednesday, Friday. N / A = not available. SAA = serum asparaginase activity. [Figure 42B] See legend to Figure 42A. [Diagram 43] 1 shows a summary of the immunogenicity results described in Example 12. Abbreviations: ADA = anti-drug antibodies. IM = intramuscular. IV = intravenous. NAb = neutralizing antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Invention L-asparaginase from bacterial sources has high potential immunogenicity and antigenicity. These products can cause adverse hypersensitivity reactions in patients, including allergic reactions, asymptomatic inactivation, and anaphylactic shock. L-asparaginase is an enzyme with L-asparagine aminohydrolase activity. L-asparaginase enzyme activity can include the amidation of asparagine to aspartic acid and ammonia, as well as the amidation of glutamine to glutamic acid and ammonia. L-asparaginase from E. coli and Erwinia chrysanthemi is commonly used to treat a variety of diseases that are treated by asparagine depletion, including ALL and LBL. Healthy cells can produce asparagine, but some diseased cells cannot because they lack the asparagine synthase enzyme. When L-asparaginase is administered to a diseased patient, it reduces the level of soluble asparagine, thereby starving diseased cells but not healthy cells, resulting in selective diseased cell death. Antibodies made after treatment with E. coli-derived L-asparaginase do not cross-react with Erwinia chrysanthemi-derived L-asparaginase. L-asparaginase produced in Erwinia chrysanthemi requires long manufacturing lead times (e.g., Erwinase®). The present invention includes a novel and improved dosing regimen for L-asparaginase that does not cross-react with E. coli-derived L-asparaginase, providing improvements over E. coli-derived L-asparaginase and Erwinia chrysanthemi-derived L-asparaginase. These improvements are described herein.

[0015] I. Definition Unless expressly defined, terms used herein are to be understood according to their ordinary meaning in the art.

[0016] As used herein, the term "disease treatable by asparagine depletion" refers to a condition or disorder in which cells involved in or causing said condition or disorder exhibit either a lack or reduced ability to synthesize L-asparagine. The depletion or deficiency of L-asparagine can be partial or substantially complete (e.g., at levels undetectable using methods and devices known in the art).

[0017] As used herein, the term "therapeutically effective amount" refers to the amount of a protein (eg, asparaginase or a recombinant L-asparaginase thereof) needed to produce a desired therapeutic effect.

[0018] The term "comprising the sequence of SEQ ID NO:1" means that the amino acid sequence of the protein may not be strictly limited to SEQ ID NO:1, but may contain additional amino acids.

[0019] The term "subject" or "patient" intends an animal, mammal, or even a human patient.

[0020] The term "host cell transformed with a vector or non-human host" refers to a host cell or non-human host that contains a vector or nucleic acid described herein. Host cells for the expression of polypeptides are well known in the art and include prokaryotic and eukaryotic cells. Appropriate culture media and conditions for the above-mentioned host cells are known in the art.

[0021] "Culturing a host or host cell" includes expression of a protein in a host or host cell, including expression of a protein as a fusion protein as defined herein and / or a polypeptide as defined herein, and / or expression of asparaginase.

[0022] As used herein, the term "about" modifies, for example, size, volume, amount of components in a composition, concentration, process temperature, process time, yield, flow rate, pressure, and similar values ​​and ranges thereof, and refers to variations in quantity that may occur, for example, due to typical measuring and processing procedures used to manufacture a compound, composition, concentrate, or formulation used, due to inadvertent errors in these procedures, due to differences in manufacture, source, or purity of starting materials or components used to carry out the method, and for similar reasons. The term "about" also encompasses amounts that differ due to aging of a composition, formulation, or cell culture having a particular initial concentration or formulation, and amounts that differ due to mixing or processing a composition or formulation having a particular initial concentration or formulation. The claims appended hereto include equivalents of these amounts, whether modified by the term "about". In addition, the term "about" may refer to a range of values ​​that are comparable to the specified reference value. In certain embodiments, the term "about" refers to a range of values ​​that are within 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value.

[0023] As used herein, the terms "co-administration," "co-administering," "administered in combination with," "administering in combination with," "simultaneously," and "concurrently" encompass administration of two or more active pharmaceutical ingredients to a human subject such that both active pharmaceutical ingredients and / or their metabolites are present in the human subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are also encompassed by the methods of the invention.

[0024] As used herein, the term "therapeutically effective amount" refers to the amount of a protein (e.g., recombinant L-asparaginase or a conjugate thereof) needed to produce a desired therapeutic effect.

[0025] The terms "E. coli derived L-asparaginase," "E. coli derived L-asparaginase," "E. coli asparaginase," and "E. coli L-asparaginase" are used interchangeably to refer to asparaginase that is naturally produced in E. coli.

[0026] The terms "Erwinia sp.-derived L-asparaginase," "Erwinia sp. asparaginase," "Erwinia sp. L-asparaginase," "Erwinia sp. asparaginase," "Erwinia sp.-derived L-asparaginase" and "Erwinia sp.-derived asparaginase" are used interchangeably herein to refer to asparaginase that is naturally produced in the genus Erwinia.

[0027] The terms "L-asparaginase from Erwinia chrysanthemi", "Erwinia chrysanthemi L-asparaginase" and "L-asparaginase from Erwinia chrysanthemi" are used interchangeably to refer to asparaginase that is naturally produced in Erwinia chrysanthemi. Erwinia chrysanthemi (also known as Pectobacterium chrysanthemi) has been renamed Dickeya chrysanthemi. Thus, the terms Erwinia chrysanthemi, Pectobacterium chrysanthemi and Dickeya chrysanthemi are used interchangeably herein.

[0028] Erwinaze® (BLA number 125359) is an Erwinia chrysanthemi type II L-asparaginase product commercially approved for the treatment of ALL in patients in the United States. Its active ingredient is Erwinia chrysanthemi type II L-asparaginase (see Erwinaze® package insert, which is incorporated herein by reference).

[0029] The terms "homology" and "sequence identity" are used interchangeably herein. Percent amino acid sequence identity (%) for a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular (parent) sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. One specific program is the ALIGN-2 program, as outlined in U.S. Patent No. 20160244525, paragraphs

[0279] to

[0280] , which is incorporated herein by reference. The degree of identity between an amino acid sequence of the present invention ("invention sequence") and a parent amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences divided by the length of the "invention sequence" or the length of the parent sequence, whichever is shorter. The result is expressed as a percentage of identity. In some embodiments, two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

[0030] The term "two days" used in the context of administration refers to the time between a first dose and a second dose administered after the first dose. For example, if the first dose is administered on Monday of a given week, the second dose administered two days after the first dose will be administered on Wednesday of the given week. The term "two days" encompasses situations in which the first dose is administered at the same time as the second dose, and situations in which the first dose is administered at a different time than the second dose. Thus, in the above example in which the first dose is administered on Monday of a given week and the second dose is administered on Wednesday of the given week, the first dose can be administered in the early morning of Monday of the given week, and the second dose can be administered in the afternoon or evening of Wednesday of the given week without going beyond the scope of the term "two days". Conversely, the first dose can be administered in the afternoon or evening of Monday of the given week, and the second dose can be administered in the morning of Wednesday of the given week without going beyond the scope of the term "two days".

[0031] The term "three days" used in the context of administration refers to the time between a first dose and a second dose administered after the first dose. For example, if the first dose is administered on Friday of a given week, the second dose administered three days after the first dose will be administered on Monday of the given week. The term "three days" encompasses situations in which the first dose is administered at the same time as the second dose, and situations in which the first dose is administered at a different time than the second dose. Thus, in the above example in which the first dose is administered on Friday of a given week and the second dose is administered on Monday of the week immediately following the given week, the first dose can be administered early in the morning on Friday of the given week, and the second dose can be administered in the afternoon or evening on Monday of the week immediately following the given week, without going beyond the scope of the term "three days". Conversely, the first dose can be administered in the afternoon or evening on Friday of the given week, and the second dose can be administered in the morning on Monday of the week immediately following the given week, without going beyond the scope of the term "three days".

[0032] The term "7 days" as used in the context of administration refers to the time between a first dose and a second dose administered after the first dose. For example, if the first dose is administered on a Friday of a given week, then the second dose, administered 7 days after the first dose, will be administered on the Friday of the week immediately following the given week. The term "7 days" encompasses situations in which the first dose is administered at the same time as the second dose, and situations in which the first dose is administered at a different time than the second dose. Thus, in the above example in which the first dose is administered on a Friday of a given week and the second dose is administered on the Friday of the week immediately following the given week, the first dose can be administered early in the morning on the Friday of the given week, and the second dose can be administered in the afternoon or evening on the Friday of the week immediately following the given week, without exceeding a 7-day range. Conversely, the first dose can be administered in the afternoon or evening on the Friday of the given week, and the second dose can be administered in the morning on the Friday of the week immediately following the given week, without exceeding a 7-day range.

[0033] The term "about 48 hours" used in the context of administration refers to the time between a first dose and a second dose administered after the first dose. For example, if the first dose is administered on Monday of a given week, then the second dose administered about 48 hours after the first dose will be administered on Wednesday of the given week. The term "about 48 hours" encompasses situations in which the first dose is administered at the same time as the second dose, and situations in which the first dose is administered at a different time than the second dose. Thus, in the above example in which the first dose is administered on Monday of a given week and the second dose is administered on Wednesday of the same week, the first dose can be administered in the early morning of the Monday of the given week, and the second dose can be administered in the afternoon or evening of the Wednesday of the given week, without exceeding the scope of the term "about 48 hours". Conversely, the first dose can be administered in the afternoon or evening of the Monday of the given week, and the second dose can be administered in the morning of the Wednesday of the given week, without exceeding the scope of the term "about 48 hours".

[0034] The term "about 72 hours" used in the context of administration refers to the time between a first dose and a second dose administered after the first dose. For example, if the first dose is administered on Friday of a given week, the second dose administered about 72 hours after the first dose will be administered on Monday of the week immediately following the given week. The term "about 72 hours" encompasses the situation where the first dose is administered at the same time as the second dose, and the situation where the first dose is administered at a different time than the second dose. Thus, in the above example where the first dose is administered on Friday of a given week and the second dose is administered on Monday of the week immediately following the given week, the first dose can be administered in the early morning of the Friday of the given week, and the second dose can be administered in the afternoon or evening of the Monday of the week immediately following the given week, without exceeding the scope of the term "about 72 hours". Conversely, the first dose may be administered on the afternoon or evening of a Friday of the given week, and the second dose may be administered in the morning of the Monday of the week immediately following the given week, without exceeding the scope of the term "about 72 hours."

[0035] The term "about 168 hours" used in the context of administration refers to the time between a first dose and a second dose administered after the first dose. For example, if the first dose is administered on a Friday of a given week, then the second dose administered about 168 hours after the first dose will be administered on the Friday of the week immediately following the given week. The term "about 168 hours" encompasses situations in which the first dose is administered at the same time as the second dose, and situations in which the first dose is administered at a different time than the second dose. Thus, in the above example in which the first dose is administered on a Friday of a given week and the second dose is administered on the Friday of the week immediately following the given week, the first dose can be administered in the early morning of the Friday of the given week, and the second dose can be administered in the afternoon or evening of the Friday of the week immediately following the given week, without exceeding the scope of the term "about 168 hours". Conversely, the first dose may be administered in the early afternoon on the Friday of the given week, and the second dose may be administered in the morning on the Friday of the week immediately following the given week, without exceeding the range of "about 168 hours."

[0036] II. Recombinant L-Asparaginase In one embodiment, the recombinant L-asparaginase according to the present disclosure provided herein is an L-asparaginase. In a further embodiment, the recombinant L-asparaginase according to the present invention described herein is an enzyme having L-asparagine aminohydrolase activity. The enzyme activity of such recombinant L-asparaginase includes not only the amidation of asparagine to aspartic acid and ammonia, but also the amidation of glutamine to glutamic acid and ammonia.

[0037] In some embodiments, the recombinant L-asparaginase disclosed herein is active as a multimer. In some embodiments, the recombinant L-asparaginase is an active enzyme as a tetramer. A tetramer is composed of four subunits (also known as a monomer). In some embodiments, the recombinant L-asparaginase is a tetramer consisting of four identical 35 kD subunits. In some embodiments, the recombinant L-asparaginase is a non-disulfide-bonded tetrameric therapeutic protein. In certain embodiments, each of the subunits or monomers of the multimeric recombinant L-asparaginase comprises the amino acid sequence of SEQ ID NO:1. In certain embodiments, each of the subunits or monomers of the tetrameric recombinant L-asparaginase comprises the amino acid sequence of SEQ ID NO:1. In another embodiment, the L-asparaginase is derived from Erwinia chrysanthemi NCPPB 1066 (Genbank Accession No. CAA32884, incorporated herein by reference in its entirety), with or without a signal peptide and / or leader sequence.

[0038] In some embodiments, the recombinant L-asparaginase is composed of multiple subunits, e.g., four subunits or monomers (tetramer). The corresponding modified recombinant protein can then consist of, for example, 1 to 20 (or more) peptides conjugated to each of the monomers of the tetramer. In some embodiments, the recombinant L-asparaginase comprises a monomer 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, the L-asparaginase is a multimer, e.g., a tetramer, comprising multiple subunits or monomers, each of the monomers in the tetramer being conjugated to one peptide, resulting in a tetramer comprising four conjugated peptides, one for each monomer. In some embodiments, the recombinant L-asparaginase is a tetramer that includes 1-4 peptides conjugated to each of the L-monomers. In some embodiments, the recombinant L-asparaginase is a tetramer that includes 4-20 peptides conjugated to each of the L-monomers. In some embodiments, the recombinant L-asparaginase is a tetramer that includes 6-18 peptides conjugated to each of the L-monomers. In some embodiments, the recombinant L-asparaginase is a tetramer that includes 6-18 peptides conjugated to each of the L-monomers. In some embodiments, the recombinant L-asparaginase is a tetramer that includes 10-15 peptides conjugated to each of the L-monomers.

[0039] In one aspect, the present invention relates to recombinant L-asparaginase and modified proteins having multiple chemically linked peptide sequences. In a further aspect, the length of the peptide sequences is about 10 to about 100, about 15 to about 60, or about 20 to about 40.

[0040] Fragments of recombinant L-asparaginase, preferably those of SEQ ID NO: 1, may be useful in the invention described herein. The term "fragment of recombinant L-asparaginase" (e.g., those of SEQ ID NO: 1) means that the sequence of the recombinant L-asparaginase may contain fewer amino acids than those in the recombinant L-asparaginase exemplified herein (e.g., those of SEQ ID NO: 1), but may still contain sufficient amino acids to confer L-aminohydrolase activity. For example, a "fragment of recombinant L-asparaginase" is / consists 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 wherein the fragment is up to 50 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, 325, 326 consecutive amino acids) from the N-terminus of the recombinant L-asparaginase exemplified herein (e.g., recombinant L-asparaginase of SEQ ID NO:1). The recombinant L-asparaginase may have a deletion of up to 75 or 100 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, or 100 amino acids) from the C-terminus of the recombinant L-asparaginase exemplified herein (e.g., the recombinant L-asparaginase of SEQ ID NO:1), and / or have a deletion of amino acids at both the N-terminus and C-terminus of the recombinant L-asparaginase exemplified herein (e.g., the recombinant L-asparaginase of SEQ ID NO:1), and the total number of deleted amino acids may be up to 125 or 150 amino acids.

[0041] Indeed, one skilled in the art would understand how to select and design a homologous protein that substantially retains L-asparaginase activity. Typically, the Nessler assay according to the method described by Mashburn and Wriston (Mashburn, L., and Wriston, J. (1963) "Tumor Inhibitory Effect of L-Asparaginase," Biochem Biophys Res Commun 12, 50 (incorporated herein by reference in its entirety)) is used to determine L-asparaginase activity.

[0042] It is well known in the art that a polypeptide can be modified by substitution, insertion, deletion, and / or addition of one or more amino acids while retaining its enzymatic activity. In this context, the term "one or more amino acids" can refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. For example, it is common to replace one amino acid at a given position with a chemically equivalent amino acid, which does not affect the functional properties of the protein. Substitutions can be: The non-polar or slightly polar small aliphatic residues Ala, Ser, Thr, Pro, and Gly; Polar negatively charged residues and their amides Asp, Asn, Glu, Gln; The polar positively charged residues His, Arg, and Lys; The large non-polar aliphatic residues Met, Leu, Ile, Val, and Cys; Large aromatic residues Phe, Tyr, and Trp It can be defined as an exchange within one of the groups.

[0043] Thus, changes that result in the substitution of one negatively charged residue for another (e.g., glutamic acid for aspartic acid) or one positively charged residue for another (lysine for arginine) would be expected to result in functionally equivalent products.

[0044] The position in the amino acid sequence where the 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 the modifications that can be introduced without affecting the activity of the protein. For example, modifications in the N- or C-terminal part of the protein can be expected not to change the activity of the protein under certain conditions. For asparaginase in particular, much characterization has been done, especially regarding the sequence, structure, and residues that form the active catalytic site. This provides suggestions regarding the residues that can be modified without affecting the activity of the enzyme. All known L-asparaginases from bacterial sources share common structural features. They are all homotetramers, with four active sites between the N- and C-terminal domains of two adjacent monomers (Aghaipour (2001) Biochemistry 40, 5655-5664, incorporated herein by reference in its entirety). They all have a high degree of similarity in their tertiary and quaternary structures (Papageorgiou (2008) FEBS J. 275, 4306-4316, incorporated herein by reference in its entirety). The sequence of the catalytic site of L-asparaginase is highly conserved among Erwinia chrysanthemi, Erwinia carotovora, and E. coli L-asparaginase II (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. carried out detailed analysis of the four active sites of L-asparaginase from Erwinia chrysanthemi by high-resolution crystal structure analysis of the enzyme complexed with a substrate (Aghaipour (2001) Biochemistry 40, 5655-5664).Kotzia et al. provide sequences of L-asparaginase from several species and subspecies of the Erwinia genus, and although the proteins have only about 75-77% identity between Erwinia chrysanthemi and Erwinia carotovora, each of them still has L-asparaginase activity (Kotzia (2007) J. Biotechnol. 127, 657-669). Moola et al. performed epitope mapping studies of L-asparaginase from Erwinia chrysanthemi 3937, and were able to retain the enzyme activity even after mutating various antigenic sequences to reduce the immunogenicity of asparaginase (Moola (1994) Biochem. J. 302, 921-927). Considering the extensive characterization that has been performed on L-asparaginase, one skilled in the art can determine how to make fragments and / or sequence replacements while retaining the enzyme activity. More specifically, fragments of the protein of SEQ ID NO:1 are 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 sequence of the polypeptide may contain fewer amino acids than the full length SEQ ID NO:1, but may retain sufficient protein to confer L-aminohydrolase activity. In some embodiments, the recombinant L-asparaginase has at least about 80% homology or identity to a protein comprising SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase comprises at least about 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity sequence identity to the amino acid sequence of SEQ ID NO:1. The terms "homology" and "sequence identity" are used interchangeably herein. The term "comprising the sequence of SEQ ID NO:1" (e.g., where an L-asparaginase has 100% homology or sequence identity to the amino acid sequence of SEQ ID NO:1) means that the amino acid sequence of the asparaginase may not be strictly limited to SEQ ID NO:1, but may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional amino acids.

[0045] SEQ ID NO:1 is as follows: TIFF2024512784000001.tif61128

[0046] The present disclosure also relates to nucleic acids encoding the recombinant L-asparaginase described herein, in particular nucleic acids encoding SEQ ID NO:1 as defined herein.

[0047] A. PEGylation In certain aspects, 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 to a polyethylene glycol (PEG) moiety. In other embodiments, the recombinant L-asparaginase is not conjugated to a PEG moiety.

[0048] The polymer is selected from the group of non-toxic water-soluble polymers, such as polysaccharides, e.g., hydroxyethyl starch, polyamino acids, e.g., polylysine, polyesters, e.g., polylactic acid, and polyalkylene oxides, e.g., polyethylene glycol (PEG), or monomethoxypolyethylene glycol (mPEG), are well known in the art and include linear and branched chain polymers. Examples of some polymers, particularly PEG, are provided in U.S. Pat. No. 5,672,662, U.S. Pat. No. 4,179,337, U.S. Pat. No. 5,252,714, U.S. Patent Application Publication No. 2003 / 0114647, U.S. Pat. No. 6,113,906, U.S. Pat. No. 7,419,600, U.S. Pat. No. 9,920,311, PCT Publication No. WO2019 / 109018, and PCT Publication No. WO2004 / 083258, each of which is incorporated by reference in its entirety.

[0049] The quality of such polymers is characterized by the polydispersity index (PDI). 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. PDI indicates the distribution of individual molecular weights within a batch of polymer. PDI is always greater than 1, but as the polymer chains approach the ideal Gaussian distribution (=monodisperse), PDI approaches 1.

[0050] 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 of the NH groups of lysine residues and / or the N-terminus 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 the N-terminus), and x is an integer ranging from about 1 to about 8, more specifically, from about 2 to about 5. In certain embodiments, PEG is monomethoxypolyethylene glycol (mPEG).

[0051] B.PAS conversion In some embodiments, the recombinant L-asparaginase is conjugated to a peptide containing proline or alanine, while in other embodiments, the recombinant crisantaspase is not conjugated to a peptide containing proline, alanine, or serine.

[0052] In some embodiments, the recombinant L-asparaginase is a fusion protein comprising (i) a 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 polypeptide(s), wherein the polypeptide consists solely of proline and alanine amino acid residues.

[0053] In such fusion proteins, proline residues in a polypeptide consisting of only proline and alanine amino acid residues may constitute more than about 10% and less than about 70% of the polypeptide. Thus, in such fusion proteins, preferably 10% to 70% of the total number of amino acid residues in the polypeptide may be proline residues, more preferably 20% to 50% of the total number of amino acid residues contained 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 contained in the polypeptide are proline residues. The polypeptide may comprise a plurality of amino acid repeats, where the repeats consist of proline and alanine residues and no more than six consecutive amino acid residues are identical. In particular, the polypeptide may comprise or consist of the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 2), or a circularly permuted version, or a multimer(s) of the sequence as the whole or part of the sequence. In other embodiments, the recombinant L-asparaginase is specifically devoid of such polypeptides, e.g., the recombinant L-asparaginase is not conjugated to a polypeptide containing the above percentages or repeats of proline residues.

[0054] The present invention also relates to nucleic acids encoding recombinant L-asparaginase, in particular fusion proteins as defined herein. In some embodiments, the nucleotide sequence is a sequence encoding any recombinant L-asparaginase comprising SEQ ID NO:1 and a polypeptide, wherein said polypeptide consists of only 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 (or is not part of) a polypeptide consisting of only proline and alanine amino acid residues (or is not part of a sequence encoding a fusion protein containing the polypeptide).

[0055] Recombinant L-asparaginase according to the present disclosure can be prepared using methods known in the art, in particular, the methods disclosed in U.S. Pat. No. 10,174,302 and PCT Application No. WO2019 / 109018, which are incorporated by reference herein for representative embodiments.

[0056] C. Compositions Comprising Recombinant L-Asparaginase The present disclosure also provides compositions comprising recombinant L-asparaginase. Such compositions may include recombinant L-asparaginase in combination with other components, including but not limited to buffers, salts, and excipients. Such compositions may include a vehicle for administering L-asparaginase to a subject, including, for example, particles, powders, and encapsulations.

[0057] In some embodiments, the recombinant L-asparaginase described herein can be encapsulated. Encapsulation of asparaginase into erythrocytes can, in some cases, help improve therapeutic index (D. Schrijvers et al., Clin. Pharmacokinet. 2003, 42(9):779-791). For example, methods for encapsulation are described in EP1773452, which is incorporated herein by reference in its entirety for all teachings specifically related to the encapsulation of L-asparaginase.

[0058] III. Functional Aspects and Other Properties of Recombinant L-Asparaginase and Compositions Thereof As will be apparent, the descriptions herein regarding the functional aspects and other properties of recombinant L-asparaginase also apply to compositions comprising the recombinant L-asparaginase of the present disclosure.

[0059] In some aspects, 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 native L-asparaginase. These characteristics of the recombinant L-asparaginase described herein are beneficial for patients who may have previously developed hypersensitivity to E. coli L-asparaginase or its PEGylated form. In some embodiments, the recombinant L-asparaginase described herein does not elicit any significant antibody response for a certain period of time after administration of a single dose, for example, for more than about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or longer. In one example, "does not elicit any significant antibody response" means that the subject administered the recombinant L-asparaginase is confirmed to be antibody negative within 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 which is incorporated herein by reference in their entirety).

[0060] Compositions comprising the recombinant L-asparaginase of the present invention exhibit reduced aggregation compared to those containing Erwinase® and Erwinia chrysanthemi L-asparaginase recombinantly expressed in E. coli. In some embodiments, 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 unconjugated recombinant L-asparaginase of the present invention results in less aggregation compared to Erwinase® and Erwinia chrysanthemi L-asparaginase recombinantly expressed in E. coli. For example, the process for producing a batch of Erwinase® results in a product having about 6% aggregation (see Examples 5 and 6). Batches of recombinant L-asparaginase of the present disclosure typically have less than about 1% aggregation (see Examples 5 and 6).

[0061] In some embodiments, the recombinant L-asparaginase of the present disclosure has a higher purity than other L-asparaginases. In some embodiments, purity is measured by demonstrating the amount of aggregation in a given sample of asparaginase. The amount of aggregation can be demonstrated by a variety of methods described in the art, including, but not limited to, size exclusion chromatography (SEC-HPLC), size exclusion ultra-high performance liquid chromatography (SE-UHPLC), size exclusion chromatography-multi-angle light scattering (SEC MALLS), and sedimentation velocity analysis ultracentrifugation (svAUC). In some embodiments, the amount of aggregation of the 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 less than 1-10%. 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 aggregation of the recombinant L-asparaginase is less than 0.25%.In some embodiments, the amount of aggregation of the recombinant L-asparaginase is less than 0.2%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is less than 0.1%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is less than 0.01%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is 0.01% to 10%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 0.01% to about 9%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 0.01% to about 8%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 0.01% to about 7%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 0.01% to about 6%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 0.1% to about 5%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 0.2% to about 4%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 0.25% to about 3%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 0.5% to about 2%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is about 1%. In some embodiments, the amount of aggregation of the recombinant L-asparaginase is 1%.

[0062] It is known to those skilled in the art that a lower amount of aggregation generally results in a product that has lower immunogenicity. Immunogenicity is an important factor in causing adverse events in clinical practice and is regulated by the US Food and Drug Administration (FDA) (see US Department of Health and Human Services, Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products, 2014, Quaternary Structure: Product Aggregates and Measurement of Aggregates, p.15-17, https: / / www.fda.gov / media / 85017 / download; 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 et al, Immunogenicity of Therapeutic Protein Aggregates, Journal of Pharmaceutical Sciences, 2016; (see also 105(2):417-430).

[0063] In addition, protein aggregation can also be associated with enzyme activity, resulting in a reduction in the overall yield of active enzyme, as aggregation interferes with the enzyme's ability to function. Protein aggregation creates manufacturing and development challenges, thereby increasing the time and cost it takes to provide a therapeutic to a patient. The recombinant crisantaspase of the present disclosure exhibits less aggregation compared to other Erwinia chrysanthemi L-asparaginases and Erwinia chrysanthemi L-asparaginase recombinantly expressed in E. coli. These aspects of the recombinant L-asparaginase are an improvement to the art.

[0064] The recombinant L-asparaginase of the present disclosure can have any combination of the properties in the above sections or any other properties described herein.

[0065] IV. Methods for Producing Recombinant L-Asparaginase In some embodiments, the recombinant crisantaspase disclosed herein is recombinantly produced in Pseudomonas fluorescens, hi some embodiments, the Pseudomonas fluorescens is deficient in native L-asparaginase.

[0066] In some embodiments, the present disclosure provides methods for high-yield cytoplasmic production of soluble recombinant L-asparaginase, where the recombinant protein is produced in the periplasm at a lower yield than in its native host. In its native host, Erwinia chrysanthemi, L-asparaginase is produced in the periplasm. The present invention provides methods that allow for the production of high levels of soluble and / or active recombinant L-asparaginase in the cytoplasm of host cells. In embodiments, the methods provided herein produce high levels of soluble and / or active recombinant L-asparaginase in the cytoplasm of Pseudomonadales, Pseudomonad, Pseudomonas, or Pseudomonas fluorescens host cells.

[0067] Methods that can be used to produce recombinant L-asparaginase are described, for example, in U.S. Publication No. 2019 / 0127742, which is incorporated by reference in its entirety for all purposes, and in particular for all teachings related to production methods for recombinant L-asparaginase. Additional methods for isolating and developing L-asparaginase for use in the methods described herein include WO2011 / 003886A1, WO2018 / 234492A1, WO2019 / 109018, WO2021 / 078988, and PCT / US2021 / 032627, filed May 14, 2021, which are incorporated by reference in their entirety for all purposes, and in particular for all teachings related to production and development methods for L-asparaginase.

[0068] V. METHODS OF TREATMENT AND USE OF RECOMBINANT ASPARAGINASE PROTEINS A. Disease or Disorder The recombinant L-asparaginase of the present disclosure may be used to treat a disease in a human subject, where the disease is treatable by depletion of asparagine. In some embodiments, the recombinant L-asparaginase of the present disclosure may be used to treat cancer. In some embodiments, the human subject experiences asymptomatic inactivation of E. coli derived asparaginase prior to administration of the recombinant L-asparaginase. In some embodiments, the human subject experiences an allergic reaction to E. coli derived asparaginase prior to administration of the recombinant L-asparaginase. In some embodiments, the human subject experiences anaphylaxis to E. coli derived asparaginase prior to administration of the recombinant L-asparaginase. Non-limiting examples of objective signs of allergy or hypersensitivity include testing "antibody positive" to the asparaginase enzyme.

[0069] In some embodiments, the recombinant L-asparaginase of the present disclosure may be used in the treatment of cancer. In some embodiments, the recombinant L-asparaginase of the present disclosure is useful in the treatment of acute lymphoblastic leukemia (ALL) or in the manufacture of a medicament for use in the treatment of 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 a relapse occurs in a patient treated with E. coli-derived L-asparaginase, subsequent treatment with E. coli formulations may cause a "vaccination" effect, which makes the E. coli formulation more immunogenic in subsequent administrations. In one embodiment, the recombinant L-asparaginase of the present invention may be used in a method of treating patients with relapsed ALL who have been previously treated with other asparaginase formulations, particularly patients with relapsed ALL who have been previously treated with E. coli-derived asparaginase. In some embodiments, the recombinant L-asparaginase administered to the patient with relapsed ALL is conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to the patient with relapsed ALL is not conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to the patient with relapsed ALL is conjugated to a peptide containing proline or alanine. In some embodiments, the recombinant L-asparaginase administered to the patient with relapsed ALL is not conjugated to a peptide containing proline or alanine.

[0070] In some embodiments, the recombinant L-asparaginase of the present disclosure is useful for treating lymphoblastic lymphoma (LBL) or for manufacturing a medicament for use in treating LBL. As with patients with ALL, in some embodiments, the recombinant L-asparaginase administered to patients with recurrent LBL is conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to patients with recurrent LBL is not conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to patients with recurrent LBL is conjugated to a peptide containing proline or alanine. In some embodiments, the recombinant L-asparaginase administered to patients with recurrent LBL is not conjugated to a peptide containing proline or alanine.

[0071] In some embodiments, the recombinant L-asparaginase of the present disclosure is used to treat colorectal cancer (CRC) or to manufacture a medicament for use in treating CRC. In some embodiments, the recombinant L-asparaginase administered to the CRC patient is conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to the CRC patient is not conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to the CRC patient is conjugated to a peptide containing proline or alanine. In some embodiments, the recombinant L-asparaginase administered to the CRC patient is not conjugated to a peptide containing proline or alanine. In some embodiments, the CRC is a Wnt-negative CRC. In some embodiments, the CRC is a Wnt-mutated CRC.

[0072] In some embodiments, the recombinant L-asparaginase of the present disclosure is useful for treating acute myeloid leukemia (AML) or for manufacturing a medicament for use in treating AML. In some embodiments, the recombinant L-asparaginase administered to the AML patient is conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to the AML patient is not conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to the AML patient is conjugated to a peptide containing proline or alanine. In some embodiments, the recombinant L-asparaginase administered to the AML patient is not conjugated to a peptide containing proline or alanine. In some embodiments, the AML is relapsed / refractory acute myeloid leukemia (R / R AML).

[0073] In some embodiments, the recombinant L-asparaginase of the present disclosure is useful for treating diffuse large b-cell lymphoma (DLBCL) or for manufacturing a medicament for use in treating DLBCL. In some embodiments, the recombinant L-asparaginase administered to the DLBCL patient is conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to the DLBCL patient is not conjugated to a PEG moiety. In some embodiments, the recombinant L-asparaginase administered to the DLBCL patient is conjugated to a peptide containing proline or alanine. In some embodiments, the recombinant L-asparaginase administered to the DLBCL patient is not conjugated to a peptide containing proline or alanine.

[0074] Diseases or disorders for which the recombinant L-asparaginase of the present disclosure is useful in treating include, but are not limited to, malignancies or cancers, including, but not limited to, hematological malignancies, lymphoma, non-Hodgkin's lymphoma, NK lymphoma, pancreatic cancer, Hodgkin's disease, immunoblastic large cell lymphoma, acute promyelocytic leukemia, acute myeloid leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute T-cell leukemia, acute myeloid leukemia (AML), biplasmic B-cell myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma, and melanosarcoma, and diffuse large B-cell lymphoma (DLBCL). Other diseases or disorders for which recombinant L-asparaginase is useful in treating include, but are not limited to, renal cell carcinoma, renal cell adenocarcinoma, glioblastoma, including glioblastoma multiforme and glioblastoma-astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung cancer, including large cell lung carcinoma and small cell lung carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian teratocarcinoma, cervical adenocarcinoma, breast cancer, breast adenocarcinoma, ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon cancer, colon adenocarcinoma, colorectal adenocarcinoma, bladder transitional cell carcinoma, bladder papilloma, prostate cancer, osteosarcoma, epithelioid carcinoma of bone, prostate cancer, and thyroid cancer. The cancer may be a solid cancer, e.g., lung cancer or breast cancer. Exemplary non-malignant hematologic disorders that respond to asparagine depletion include immune system-mediated hematologic disorders, including, but not limited to, infectious diseases such as those caused by HIV infection (i.e., AIDS). Non-blood diseases associated with asparagine dependency 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 dependency in any suitable in vitro or in vivo assay, for example, in an in vitro assay in which the growth medium lacks asparagine.

[0075] Diseases or disorders for which the recombinant L-asparaginase of the present disclosure is useful in treating include sarcoma, breast cancer, metastatic breast cancer, liver cancer, gastric cancer, colorectal cancer, and head and neck cancer.

[0076] B. Methods for Testing Asparagine Dependence Cells suspected of causing the disease can be tested for asparagine dependency in any suitable in vitro or in vivo assay, for example, in an in vitro assay in which the growth medium lacks asparagine. Thus, in some embodiments, the present disclosure is directed to a method of treating a treatable disease in a patient, comprising administering to the patient an effective amount of a recombinant L-asparaginase of the present invention. 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.

[0077] C. Methods for assessing nadir serum asparaginase activity (NSAA) An assay for measuring serum asparaginase activity (NSAA) in a human subject can be performed to evaluate the human subject. In some embodiments, a serum sample is obtained from the human subject to evaluate the NSAA. In some embodiments, a whole blood sample is obtained from the subject to evaluate the NSAA. In some embodiments, the evaluation of the NSAA is performed before recombinant L-asparaginase is administered to the patient. In some embodiments, the evaluation of the NSAA is performed after recombinant L-asparaginase is administered to the patient.

[0078] D. Line of Treatment A first line treatment is the initial treatment given to a disease. A first line treatment can be a monotherapy or a course of standard treatment.

[0079] The second line treatment can be a monotherapy or a series of standard treatments. The second line treatment is a treatment administered after the first treatment has failed, lost its effectiveness (partially or completely), after the first treatment has unacceptable side effects, after the patient decides to discontinue the first treatment for any reason, or after a new treatment becomes available that may provide better results than the current treatment. In some embodiments, the second line treatment can be administered to a human subject in addition to the first line treatment for beneficial additive or synergistic results.

[0080] Additional selection therapy, including third, fourth, fifth, sixth and any further selection therapy, is defined similarly to second selection therapy, but in this case, both first and second selection therapy fail, lose their effect (partially or completely), have unacceptable side effects, the patient decides to discontinue first and / or second selection therapy for any reason, or a new therapy becomes available that may perform better than first and second selection therapy, or any combination of these reasons occurs. Additional selection therapy can be a monotherapy or a series of standard treatments. In some embodiments, additional selection therapy can be administered to human subjects in addition to first and / or second selection therapy for beneficial additive or synergistic results.

[0081] In some embodiments, treatment with the recombinant L-asparaginase of the present disclosure is administered as a first-line treatment. In other embodiments, treatment with the recombinant L-asparaginase of the present disclosure is administered as a second-line treatment in patients, particularly those with ALL and LBL, who have developed objective signs of allergy or hypersensitivity, including "asymptomatic inactivation," to other asparaginase preparations, particularly native Escherichia coli L-asparaginase or its PEGylated variants (pegaspargase). Non-limiting examples of objective signs of allergy or hypersensitivity include testing "antibody positive" to the asparaginase enzyme. The patient may have previously developed hypersensitivity to at least one E. coli L-asparaginase and / or may have previously developed hypersensitivity to at least one Erwinia chrysanthemi L-asparaginase. The hypersensitivity may be selected from the group consisting of an allergic reaction, anaphylactic shock, and asymptomatic inactivation. In certain embodiments, the recombinant L-asparaginase of the present disclosure is used in second line treatment following treatment with pegaspargase. In more specific embodiments, the recombinant L-asparaginase of the present disclosure used in second line treatment comprises an L-asparaginase produced in Pseudomonas fluorescens cells, more specifically an L-asparaginase that is a tetramer, where each monomer or subunit comprises the sequence of SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase of the present disclosure is used in second line treatment of patients who are hypersensitive to E. coli-derived L-asparaginase and / or may have previously developed hypersensitivity to Erwinia chrysanthemi-derived L-asparaginase. The E. coli-derived L-asparaginase may be a native or long-acting E. coli-derived L-asparaginase.Native E. coli L-asparaginase can be identified by any of the following examples, including but not limited to SEQ ID NO:3, Crastinin (Bayer), Elspar (Merck), Kidrolase (Rhone-Poulenc), and Leunase (Kyowa) (see also ASPG2_ECOLI, Uniprot Accession No. P00805, https: / / www.uniprot.org / uniprot / P00805).

[0082] SEQ ID NO:3 is as follows: TIFF2024512784000002.tif39153

[0083] In some embodiments, recombinant asparaginase is administered to CRC patients as a second line treatment. In some embodiments, recombinant asparaginase is administered to Wnt-negative CRC patients as a second line treatment. In some embodiments, recombinant asparaginase is administered to Wnt-mutated CRC patients as a second line treatment. In some embodiments, recombinant asparaginase is administered to AML patients as a second line treatment. In some embodiments, recombinant asparaginase is administered to relapsed / refractory AML patients as a second line treatment. In some embodiments, recombinant asparaginase is administered to DLBCL patients as a second line treatment.

[0084] In some embodiments, recombinant L-asparaginase may be used as a second line treatment for patients administered native E. coli derived asparaginase. In some embodiments, one dose of recombinant L-asparaginase is administered to the patient instead of one dose of native E. coli derived asparaginase. In some embodiments, recombinant L-asparaginase may be used as a second line treatment for patients administered long-acting E. coli derived asparaginase. In some embodiments, six doses of recombinant L-asparaginase are administered to the patient instead of one dose of long-acting E. coli derived asparaginase. In some embodiments, seven doses of recombinant L-asparaginase are administered to the patient instead of one dose of long-acting E. coli derived asparaginase. In some embodiments, the long-acting E. coli derived asparaginase is pegaspargase. In some embodiments, the long-acting E. coli derived asparaginase is calaspargase (see Liu, G., Space for Calaspargase? A New Asparaginase for Acute Lymphoblastic Leukemia Clin Cancer Res 2020;26:325-7). In some embodiments, treatment with a recombinant L-asparaginase of the present disclosure is administered as a third line treatment. In some embodiments, treatment with a recombinant L-asparaginase of the present disclosure is administered as a fourth line treatment. In some embodiments, treatment with a recombinant L-asparaginase of the present disclosure is administered as a fifth line treatment. In some embodiments, treatment with a recombinant L-asparaginase of the present disclosure is administered as a sixth line treatment. In some embodiments, treatment with a recombinant L-asparaginase of the present disclosure is administered as a maintenance treatment. In some embodiments, administering recombinant L-asparaginase to a patient in place of native or long-acting E. coli derived asparaginase can occur at any time within a treatment cycle with native or long-acting E. coli derived asparaginase. In some embodiments, a treatment cycle for native or long-acting E. coli derived asparaginase is 1 to 14 administrations.In some embodiments, the treatment cycle for native or long-acting E. coli derived asparaginase is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 administrations. In some embodiments, the treatment cycle for native or long-acting E. coli derived asparaginase is 2-12 administrations. In some embodiments, the treatment cycle for native or long-acting E. coli derived asparaginase is 4-10 administrations. In some embodiments, the treatment cycle for native or long-acting E. coli derived asparaginase is 4-10 administrations. In some embodiments, the treatment cycle for native or long-acting E. coli derived asparaginase is 4-8 administrations. In some embodiments, the treatment cycle for native or long-acting E. coli derived asparaginase is 4-6 administrations. In some embodiments, administration of recombinant L-asparaginase to a patient in place of native or long-acting E. coli derived asparaginase can occur at any point within a treatment cycle with native or long-acting E. coli derived asparaginase, depending on when the patient develops hypersensitivity.

[0085] In some embodiments, treatment with the recombinant L-asparaginase of the present disclosure is administered in conjunction with a multi-drug chemotherapy regimen. In some embodiments, treatment with the recombinant L-asparaginase of the present disclosure is co-administered with one or more other chemotherapeutic agents as part of a multi-drug chemotherapy regimen. In some embodiments, treating a patient with the recombinant L-asparaginase of the present 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-drug chemotherapy regimen using the recombinant L-asparaginase of the present disclosure 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 combination chemotherapy regimen is recombinant L-asparaginase and one additional chemotherapeutic agent. In some embodiments, the combination chemotherapy regimen is recombinant L-asparaginase and two or more additional chemotherapeutic agents.

[0086] As an example, a patient with ALL is co-administered with a recombinant L-asparaginase of the present disclosure with a combination chemotherapy during three chemotherapy phases, including induction, consolidation or intensification, and maintenance. In a specific example, the recombinant L-asparaginase of the present disclosure is co-administered with an asparagine synthetase inhibitor (e.g., as described in WO2007 / 103290, which is incorporated herein by reference in its entirety). In another example, the recombinant L-asparaginase of the present disclosure is not co-administered with an asparagine synthetase inhibitor, but is co-administered with other chemotherapeutic agents. In another example, the recombinant L-asparaginase of the present disclosure is co-administered with an asparagine synthetase inhibitor and other chemotherapeutic agents. The recombinant L-asparaginase of the present disclosure can be co-administered before, after, or simultaneously with other compounds as part of a combination chemotherapy regimen. In certain embodiments, the recombinant L-asparaginase of the present disclosure comprises a protein recombinantly produced in Pseudomonas fluorescens, more specifically, a recombinant L-asparaginase comprising the sequence of SEQ ID NO:1.

[0087] VI. Compositions, Formulations, Routes of Administration, and Administration 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 can be administered to patients using standard techniques. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, 22nd edition, Mack Publishing, 2015 (incorporated herein by reference). The embodiments disclosed herein can further be utilized in conjunction with or in addition to other regimens, for example, as described in WO2021 / 078988, filed October 23, 2020, and PCT / US2021 / 032627, filed May 14, 2021, each of which is incorporated herein by reference for all purposes, particularly for all teachings related to the administration and use of recombinant L-asparaginase.

[0088] The appropriate dosage form is determined, in part, by the method of use or route of introduction, e.g., injection (parenteral). Such dosage form must allow the therapeutic agent to reach target cells or otherwise exert the desired therapeutic effect. For example, pharmaceutical compositions injected into the bloodstream are preferably soluble. Pharmaceutical compositions according to the present disclosure may be formulated as their pharmaceutically acceptable salts and complexes. Pharmaceutically acceptable salts are non-toxic salts that are present in the amounts and concentrations to be administered. Such salt formulations may facilitate medical use by altering the physical properties of the compound without preventing the compound from exerting its physiological effect. Useful changes in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administration of higher concentrations of the drug. Pharmaceutically acceptable salts of the modified proteins described herein may also be present as complexes, as will be understood by those of skill in the art. Pharmaceutically acceptable salts include acid addition salts, such as those containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinate. Pharmaceutically acceptable salts may be obtained from acids, including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid. When an acidic functional group is present, such as a carboxylic acid or phenol, pharma- ceutically acceptable salts also include base addition salts containing, for example, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamines, and zinc. See, for example, Remington's Pharmaceutical Sciences, supra. Such salts can be prepared using the appropriate corresponding base.Pharmaceutically acceptable carriers and / or excipients may also be incorporated into pharmaceutical compositions according to the invention to facilitate administration of a particular asparaginase. Examples of suitable carriers for use in the practice of the invention include calcium carbonate, calcium phosphate, various sugars, such as lactose, glucose, or sucrose, or various starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline, and dextrose. Pharmaceutical compositions according to the invention may be administered by various routes, including intravenous (IV) and intramuscular (IM) administration. For injection, the pharmaceutical composition is formulated in a liquid solution, preferably in a physiologically compatible buffer or solution, such as saline, Hank's solution, or Ringer's solution. In addition, the compound may be formulated in solid form and redissolved or suspended immediately prior to use. For example, a lyophilized form of recombinant L-asparaginase may be produced. In certain embodiments, recombinant L-asparaginase is administered intramuscularly. In certain preferred embodiments, the recombinant L-asparaginase is administered intravenously.

[0089] In preferred embodiments, the pharmaceutical composition is not lyophilized. In further embodiments, the pharmaceutical composition is a solution. In some embodiments, the final lyophilization step may cause induced stress and accelerate degradation of the compound. In some embodiments, lyophilization may increase aggregation. In some further preferred embodiments, the recombinant crisantaspase is administered intramuscularly. In some further preferred embodiments, the recombinant crisantaspase is administered intravenously. For each of these preferred embodiments of dosage forms, it will be understood that any form of recombinant L-asparaginase may be administered, including modified and unmodified forms.

[0090] In some embodiments, recombinant L-asparaginase is administered intravenously to a human subject at certain times and intramuscularly to the same human subject at other certain times.

[0091] B. Administration In some embodiments, a dose is an amount administered to a human subject over a period of time and at a frequency, hi some embodiments, a dose of recombinant L-asparaginase is administered to a human subject with hypersensitivity only if the hypersensitivity subsides.

[0092] In an exemplary embodiment, the recombinant L-asparaginase is present at about 10 mg / m 2 ~100mg / m 2 is administered to a human subject in an amount of

[0093] In an exemplary embodiment, the recombinant L-asparaginase is present at about 25 mg / m 2 ~about 50mg / m 2 is administered to a human subject in an amount of

[0094] In an exemplary embodiment, recombinant L-asparaginase is administered intramuscularly every other day for five consecutive days, followed by a rest period of two consecutive days, where the amount is about 25 mg / m 2 It is.

[0095] In an exemplary embodiment, recombinant L-asparaginase is administered intravenously every other day for five consecutive days, followed by a drug holiday of two consecutive days, where the amount is about 50 mg / m 2 It is.

[0096] In further exemplary embodiments, the recombinant L-asparaginase administered in such doses according to any of the descriptions herein regarding administration is not conjugated to a polymer, such as a PEG moiety, and / or is not conjugated to a peptide containing only alanine and / or proline residues.

[0097] 1. Dosage The amount of recombinant L-asparaginase of the present disclosure to be delivered depends on a number of factors, such as the IC50, EC50, biological half-life of the compound, the age, size, weight, and health of the patient, and the disease or disorder being treated. The importance of these and other factors to be considered is well known to those of skill in the art. In general, the amount of recombinant L-asparaginase of the present disclosure is about 1 milligram per square meter (mg / m2) of the patient's body surface area. 2 )~1,000 / m 2 and about 10 mg / m for treating diseases including, but not limited to, ALL or LBL. 2 ~about 100mg / m 2 Of course, other dosages and / or treatment regimens may be used according to the judgment of the attending physician.

[0098] In some embodiments, the method comprises administering a dose of about 10 mg / m 2 ~about 100mg / m 2 In some embodiments, the method comprises administering a recombinant L-asparaginase of the present disclosure in an amount of 10 mg / m 2 ~100mg / m 2 In some embodiments, the 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 / m 2 or the equivalent thereof (e.g., based on protein content). In more specific embodiments, the recombinant L-asparaginase of the present disclosure is administered at about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, and about 100 mg / m 2 In another specific embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount selected from the group consisting of about 1 mg / m 2 More than about 10mg / m 2 More than about 15mg / m 2 More than about 20mg / m 2 More than about 25mg / m 2More 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 or more than 300 mg / m 2 In another specific embodiment, the recombinant L-asparaginase of the present disclosure is administered at a dose of about 300 mg / m 2 Below about 200mg / m 2 Below, about 100mg / m 2 Below, about 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 / m 2 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 Less than or equal to about 1 mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered at a dose of about 12 mg / m 2 ~about 90mg / m2 In another exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 20 mg / m 2 ~about 80mg / m 2 In another exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 80mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 37.5 mg / m 2 ~about 80mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 37.5 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 37.5mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 100mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 80mg / m 2 is administered in an amount of

[0099] In some embodiments, the method comprises administering a dose of about 25 mg / m 2 ~about 50mg / m 2 In some embodiments, the method comprises administering a recombinant L-asparaginase of the present disclosure in an amount of 25 mg / m 2 ~50mg / m 2In some embodiments, the recombinant L-asparaginase of the disclosure is administered in an amount of about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg / m 2 or the equivalent thereof (eg, based on protein content).

[0100] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 20 mg / m 2 ~about 30mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 22.5 mg / m 2 ~about 28.5mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 23 mg / m 2 ~about 27mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 24 mg / m 2 ~about 26mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 24.5 mg / m 2 ~about 25.5mg / m 2 is administered in an amount of

[0101] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 40 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 42.5 mg / m 2 ~about 58.5mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 43 mg / m 2 ~about 57mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 44 mg / m 2 ~about 56mg / m 2In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 55mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 46 mg / m 2 ~about 54mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 47.5 mg / m 2 ~about 52.5mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 48 mg / m 2 ~about 52mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 49 mg / m 2 ~about 51mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 49.5 mg / m 2 ~about 50.5mg / m 2 is administered in an amount of

[0102] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 30 mg / m 2 ~about 75mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 35 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 40 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 50 mg / m 2 ~about 55mg / m 2 is administered in an amount of

[0103] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 40 mg / m 2 ~about 75mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 50 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 55 mg / m 2 ~about 60mg / m 2 is administered in an amount of

[0104] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 40 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 55mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 47.5 mg / m 2 ~about 50mg / m 2 is administered in an amount of

[0105] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 30 mg / m 2 ~about 35mg / m 2 is administered in an amount of

[0106] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 30 mg / m 2 ~about 95mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 35 mg / m 2 ~about 90mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 40 mg / m 2 ~about 85mg / m 2In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 80mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 50 mg / m 2 ~about 75mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 55 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 60 mg / m 2 ~about 65mg / m 2 is administered in an amount of

[0107] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 30 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 35 mg / m 2 ~about 55mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 40 mg / m 2 ~about 50mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 42.5 mg / m 2 ~about 57.5mg / m 2 is administered in an amount of

[0108] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 30 mg / m 2 ~about 75mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 35 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 40 mg / m 2 ~about 65mg / m 2In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount of about 50 mg / m 2 ~about 55mg / m 2 is administered in an amount of

[0109] In some embodiments, the recombinant L-asparaginase of the present disclosure is at about 10 mg / m 2 ~about 50mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 12.5 mg / m 2 ~about 47.5mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 15 mg / m 2 ~about 45mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 20 mg / m 2 ~about 42.5mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 22.5 mg / m 2 ~about 40mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 24 mg / m 2 ~about 39mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 27 mg / m 2 ~about 37.5mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 30 mg / m 2 ~about 45mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 25 mg / m 2In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of 25 mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 25 mg / m 2 ~about 80mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 37.5 mg / m 2 ~about 80mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 37.5 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 25 mg / m 2 ~about 37.5mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 30 mg / m 2 ~about 75mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 35 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 40 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 45 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 50 mg / m 2 ~about 55mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 40 mg / m 2 ~about 75mg / m 2In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 45 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 50 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 55 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 40 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 45 mg / m 2 ~about 55mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 47.5 mg / m 2 ~about 50mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 30 mg / m 2 ~about 35mg / m 2 It is administered intramuscularly or intravenously in an amount of

[0110] In some embodiments, the recombinant L-asparaginase of the present disclosure is at about 10 mg / m 2 ~about 95mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 20 mg / m 2 ~about 60mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 22.5 mg / m 2 ~about 57.5mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 25 mg / m2 ~about 55mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 27.5 mg / m 2 ~about 47.5mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 30 mg / m 2 ~about 45mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 32.5 mg / m 2 ~about 42.5mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 21.5 mg / m 2 ~about 38.5mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 36 mg / m 2 ~about 45mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 37.5 mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 50 mg / m 2 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intravenously or intramuscularly in an amount of 50 mg / m 2 It is administered intravenously or intramuscularly in an amount of

[0111] In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is at about 25 mg / m 2 ~about 37.5mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 25 mg / m 2 ~about 100mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 25 mg / m 2~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 25 mg / m 2 ~about 80mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 30 mg / m 2 ~about 35mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 30 mg / m 2 ~about 95mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 35 mg / m 2 ~about 90mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 40 mg / m 2 ~about 85mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 45 mg / m 2 ~about 80mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 50 mg / m 2 ~about 75mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 55 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 60 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 30 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 35 mg / m 2 ~about 55mg / m 2In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 40 mg / m 2 ~about 50mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 42.5 mg / m 2 ~about 57.5mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 30 mg / m 2 ~about 75mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 35 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 40 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 45 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered intramuscularly or intravenously in an amount of about 50 mg / m 2 ~about 55mg / m 2 It is administered intramuscularly or intravenously in an amount of

[0112] In another embodiment, the method includes administering a recombinant L-asparaginase of the present disclosure that elicits a weaker immunogenic response in a patient as compared to non-recombinant L-asparaginase.

[0113] In some embodiments, the method comprises administering a series of seven doses of recombinant L-asparaginase intramuscularly or intravenously to a human subject, where each dose in the series is about 20 mg / m 2 and the administration interval between each dose in the series is 2 days.

[0114] In some embodiments, the method comprises administering a series of seven doses of recombinant L-asparaginase intramuscularly or intravenously to a human subject, wherein each dose in the series is about 22.5 mg / m 2 and the administration interval between each dose in the series is 2 days.

[0115] In some embodiments, the method comprises administering a series of seven doses of recombinant L-asparaginase intramuscularly or intravenously to a human subject, wherein each dose in the series is about 25 mg / m 2 and the administration interval between each dose in the series is 2 days.

[0116] In some embodiments, the method comprises administering a series of seven doses of recombinant L-asparaginase intramuscularly or intravenously to a human subject, wherein each dose in the series is about 27.5 mg / m 2 and the administration interval between each dose in the series is 2 days.

[0117] In some embodiments, the method comprises administering a series of seven doses of recombinant L-asparaginase intramuscularly or intravenously to a human subject, wherein each dose in the series is about 30 mg / m 2 and the administration interval between each dose in the series is 2 days.

[0118] In some embodiments, the method includes administering a series of six doses of L-asparaginase intramuscularly or intravascularly to a human subject, where four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2 The dosing interval between two doses in the series is 7 days, and the dose is about 25 mg / m 2 and the dosing interval between the dose of about 50 mg / m 2and the dosing interval between the dose of about 25 mg / m and the next dose is 3 days, and the L-asparaginase is not E. coli derived asparaginase. 2 The dose is administered intravenously and is approximately 50 mg / m 2 In some embodiments, the doses are administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0119] In some embodiments, the method includes administering a series of six doses of L-asparaginase intramuscularly or intravascularly to a human subject, where four of the six doses are about 22.5 mg / m 2 ~about 27.5mg / m 2 and two of the six doses are about 47.5 mg / m 2 ~about 52.5mg / m 2 is about 47.5 mg / m 2 ~about 52.5mg / m 2 The dosing interval between two doses in the series is 7 days, and the dose is about 22.5 mg / m 2 ~about 27.5mg / m 2 and the dosing interval between the dose of about 47.5 mg / m 2 ~about 52.5mg / m 2 and the dosing interval between the dose of about 22.5 mg / m and the next dose is 3 days, and the L-asparaginase is not E. coli derived asparaginase. 2 ~about 27.5mg / m 2 The dose is administered intravenously and is approximately 47.5 mg / m 2 ~about 52.5mg / m 2 In some embodiments, the doses are administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0120] In some embodiments, the method includes administering a series of six doses of L-asparaginase intramuscularly or intravascularly to a human subject, where four of the six doses are about 20 mg / m 2 ~about 30mg / m 2 and two of the six doses are about 45 mg / m 2 ~about 55mg / m 2 is about 45 mg / m 2 ~about 55mg / m 2 The dosing interval between two doses in the series is 7 days, and the dose is about 20 mg / m 2 ~about 30mg / m 2 and the dosing interval between the dose of about 45 mg / m 2 ~about 55mg / m 2 and the dosing interval between the dose of about 20 mg / m and the next dose is 3 days, and the L-asparaginase is not E. coli derived asparaginase. 2 ~about 30mg / m 2 The dose is administered intravenously and is approximately 45 mg / m 2 ~about 55mg / m 2 In some embodiments, the doses are administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0121] In some embodiments, the method comprises intramuscularly or intravenously administering to a human subject an L-asparaginase other than E. coli derived asparaginase as a set of chronological doses, wherein the set of chronological doses comprises a first dose followed by a second dose, the first dose being (i) about 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and the second dose is (i) about 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and wherein the first dose is about 50 mg / m 2and wherein the administration interval between the first and second doses to the human subject is 3 days, and the first dose is about 25 mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 2 days, and the second dose is about 50 mg / m 2 no further administration of L-asparaginase to the subject occurs for 3 days, and the second dose is about 25 mg / m 2 In some embodiments, when the L-asparaginase is about 25 mg / m, no further administration of L-asparaginase to the subject occurs for two days. 2 Any dose at 50 mg / m2 is administered intravenously. 2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0122] In some embodiments, the method includes intramuscularly or intravenously administering to a human subject an L-asparaginase other than E. coli derived asparaginase as a set of chronological doses, wherein the set of chronological doses includes a first dose followed by a second dose, the first dose being about (i) 47.5 mg / m of L-asparaginase. 2 ~about 52.5mg / m 2 and (ii) about 22.5 mg / m 2 ~about 27.5mg / m 2 and the second dose is (i) about 47.5 mg / m of L-asparaginase. 2 ~about 52.5mg / m 2 and (ii) about 22.5 mg / m 2 ~about 27.5mg / m 2 and wherein the first dose is about 47.5 mg / m 2 ~about 52.5mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 3 days, and the first dose is about 22.5 mg / m 2 ~about 27.5mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 2 days, and the second dose is about 47.5 mg / m2 ~about 52.5mg / m 2 no further administration of L-asparaginase to the subject occurs for 3 days, and the second dose is about 22.5 mg / m 2 ~about 27.5mg / m 2 In some embodiments, when the L-asparaginase is about 22.5 mg / m, no further administration of L-asparaginase to the subject occurs for two days. 2 ~about 27.5mg / m 2 Any dose at 47.5 mg / m2 is administered intravenously. 2 ~about 52.5mg / m 2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0123] In some embodiments, the method includes intramuscularly or intravenously administering to a human subject an L-asparaginase other than E. coli derived asparaginase as a set of chronological doses, wherein the set of chronological doses includes a first dose followed by a second dose, the first dose being about (i) 45 mg / m of L-asparaginase. 2 ~about 55mg / m 2 and (ii) about 20 mg / m 2 ~about 30mg / m 2 and the second dose is (i) about 45 mg / m of L-asparaginase. 2 ~about 55mg / m 2 and (ii) about 20 mg / m 2 ~about 30mg / m 2 and wherein the first dose is about 45 mg / m 2 ~about 55mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 3 days, and the first dose is about 20 mg / m 2 ~about 30mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 2 days, and the second dose is about 45 mg / m 2 ~about 55mg / m 2no further administration of L-asparaginase to the subject occurs for 3 days, and the second dose is about 20 mg / m 2 ~about 30mg / m 2 In some embodiments, when the L-asparaginase is about 20 mg / m, no further administration of L-asparaginase to the subject occurs for two days. 2 ~about 30mg / m 2 Any dose at 45 mg / m2 is administered intravenously. 2 ~about 55mg / m 2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0124] 2. Frequency of administration In certain embodiments, the treatment is at about 1 mg / m 2 ~about 1000mg / m 2 , typically about 10 mg / m 2 ~about 100mg / m 2 on a schedule ranging from about three times a week to about once a month, typically once a week or once every other week, as a single agent (e.g., monotherapy) or as part of a combination of chemotherapeutic agents including, but not limited to, glucocorticoids, corticosteroids, anti-cancer compounds, or other agents, including, but not limited to, methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracyclines.

[0125] The recombinant L-asparaginase of the present disclosure may be administered before, after, or simultaneously with other compounds as part of a combination chemotherapy regimen. In certain embodiments, the recombinant L-asparaginase of the present disclosure comprises a protein recombinantly produced in Pseudomonas fluorescens, more specifically, a recombinant L-asparaginase comprising the sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered in a single dose for about 3 to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) at a dose that depletes L-asparagine to undetectable levels using methods and devices known in the art.

[0126] In some embodiments, the recombinant L-asparaginase of the present disclosure is administered three times per week. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered every other day for five consecutive days, followed by a rest period of two consecutive days. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered on Monday, Wednesday, and Friday of the same week.

[0127] In some embodiments, the recombinant L-asparaginase of the disclosure is administered three times per week for at least one to three weeks. In some embodiments, the recombinant L-asparaginase of the disclosure is administered every other day for five consecutive days for about one to three weeks, followed by a rest period of two consecutive days. In some embodiments, the recombinant L-asparaginase of the disclosure is administered on Mondays, Wednesdays, and Fridays per week for about one to three weeks.

[0128] In some embodiments, the recombinant L-asparaginase of the disclosure is administered three times per 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 rest period of two consecutive days. 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.

[0129] In some embodiments, the recombinant L-asparaginase of the disclosure is administered three times per 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 rest period of two consecutive days. In some embodiments, the recombinant L-asparaginase of the disclosure is administered on Monday, Wednesday, and Friday of the same week for two weeks.

[0130] In some embodiments, the recombinant L-asparaginase of the present disclosure is administered three times a week, which continues until the patient is free of disease treatable by asparagine depletion. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered every other day for five consecutive days, followed by a rest period of two consecutive days, which continues until the patient is free of disease treatable by asparagine depletion. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered on Monday, Wednesday, and Friday of the same week, which continues until the patient is free of disease treatable by asparagine depletion.

[0131] In some embodiments, the recombinant L-asparaginase of the present disclosure is administered three times a week, which continues until the patient decides to terminate or postpone treatment. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered every other day for five consecutive days, followed by a rest period of two consecutive days, which continues until the patient decides to terminate or postpone treatment. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered on Monday, Wednesday, and Friday of the same week, which continues until the patient decides to terminate or postpone treatment.

[0132] In some embodiments, the recombinant L-asparaginase of the present disclosure is administered about every 48 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered every 40 to 58 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered about every 42 to 56 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered about every 44 to 52 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered about every 46 to 50 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered about every 72 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered every 64 to 80 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered about every 66 to 78 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered about every 68 to 76 hours. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered about every 70 to 74 hours.

[0133] In some embodiments, the recombinant L-asparaginase of the present disclosure is administered as a second line treatment to patients who are hypersensitive to E. coli derived L-asparaginase and / or who may have previously developed hypersensitivity to Erwinia chrysanthemi derived L-asparaginase.

[0134] In some embodiments, recombinant L-asparaginase is administered to a human subject in place of administration of E. coli derived long-acting asparaginase. In some embodiments, six doses of recombinant L-asparaginase are administered to a human subject in place of one dose of E. coli derived long-acting asparaginase. In some embodiments, seven doses of recombinant L-asparaginase are administered to a human subject in place of one dose of long-acting E. coli derived asparaginase. In some embodiments, the long-acting E. coli derived asparaginase is pegaspargase. In a further embodiment, six separate doses can be administered over a period of about two weeks. In another further embodiment, seven separate doses can be administered over a period of about two weeks.

[0135] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, wherein the cycle comprises a first dose, a second dose, and a third dose, which may be optionally repeated, and wherein the first dose, the second dose, and the third dose are administered about 48-72 hours apart.

[0136] In some embodiments, the dosage may vary within a cycle.

[0137] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, which may be optionally repeated, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days followed by a two consecutive day rest period, and the first dose of the cycle is 25 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle is 50 mg / m 2 followed by a drug-free period of 2 consecutive days.

[0138] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, which may be optionally repeated, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days followed by a two consecutive day rest period, and the first dose of the cycle is 25 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle was 37.5 mg / m 2 followed by a drug-free period of 2 consecutive days.

[0139] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, which may be optionally repeated, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days followed by a rest period of two consecutive days, and the first dose of the cycle is 37.5 mg / m 2 and the second dose of the cycle is 37.5 mg / m 2 and the third dose of the cycle was 37.5 mg / m 2 followed by a drug-free period of 2 consecutive days.

[0140] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, which may be optionally repeated, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days followed by a rest period of two consecutive days, and the first dose of the cycle is 37.5 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle was 37.5 mg / m 2 followed by a drug-free period of 2 consecutive days.

[0141] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, which may be optionally repeated, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days followed by a rest period of two consecutive days, and the first dose of the cycle is 37.5 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle is 25 mg / m 2 followed by a drug rest period of 2 consecutive days. In some embodiments, the first dose of the cycle is administered on a Monday, the second dose of the cycle is administered on a Wednesday, and the third dose of the cycle is administered on a Friday.

[0142] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, which may be optionally repeated, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days followed by a two consecutive day rest period, and the first dose of the cycle is 25 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle is 50 mg / m 2 followed by a drug rest period of 2 consecutive days. In some embodiments, the first dose of the cycle is administered on a Monday, the second dose of the cycle is administered on a Wednesday, and the third dose of the cycle is administered on a Friday.

[0143] In some embodiments, the disclosure provides a method for depleting asparaginase in a human subject to treat acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL), comprising administering about 25 mg / m L-asparaginase on Mondays, Wednesdays, and Fridays to a human subject for a total of 7 doses of L-asparaginase over a two week period. 2 The method comprises intramuscularly administering to a human subject an L-asparaginase comprising the compound of formula (I).

[0144] In some embodiments, the disclosure provides a method for depleting asparaginase in a human subject to treat cancer, including but not limited to acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), colorectal cancer (CRC), acute myeloid leukemia (AML), comprising administering to the human subject a total of six doses of L-asparaginase over a two week period, comprising: (a) administering about 25 mg / m2 L-asparaginase on Mondays and Wednesdays; 2 (b) administering to a human subject intramuscularly or intravenously about 50 mg / m 2 The method comprises intramuscularly or intravascularly administering to a human subject L-asparaginase of the formula:

[0145] The dosing regimen may include any number of cycles for any number of weeks or to any endpoint specified herein.

[0146] 3. Administration time In some embodiments, the administration time to a human subject is short, e.g., immediate or takes 1 second to 5 minutes, when delivered by intramuscular injection. In other embodiments, the administration time to a human subject is longer, e.g., when delivered by intravenous injection. In some embodiments, the administration of the recombinant L-asparaginase lasts from about 5 minutes to about 4 hours. In exemplary embodiments, the administration time to a human subject lasts from 90 minutes to about 150 minutes. In some embodiments, the administration time to a human subject lasts about 2 hours. In some embodiments, the administration time to a human subject lasts 2 hours. In some embodiments, the administration time to a human subject lasts from about 45 minutes to about 75 minutes. In some embodiments, the administration time to a human subject lasts about 1 hour. In some embodiments, the administration time to a human subject lasts 1 hour.

[0147] 4. Preferred Dosing Regimen In some embodiments, the disclosure provides a method of treating cancer in a human subject. In some embodiments, the disclosure provides a method of treating a human subject with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL). In some embodiments, the disclosure provides a method of treating a human subject with colorectal cancer (CRC). In some embodiments, the disclosure provides a method of treating a human subject with Wnt-negative colorectal cancer (CRC). In some embodiments, the disclosure provides a method of treating a human subject with Wnt-mutated colorectal cancer (CRC). In some embodiments, the disclosure provides a method of treating a human subject with acute myeloid leukemia (AML). In some embodiments, the disclosure provides a method of treating a human subject with diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disclosure provides a method of treating a human subject with relapsed / refractory AML (R / R AML). In some embodiments, recombinant L-asparaginase can be used as a first line treatment. In some embodiments, recombinant L-asparaginase can be used as a second line treatment. In some embodiments, recombinant L-asparaginase can be used as a third, fourth, or fifth line treatment.

[0148] In some embodiments, recombinant L-asparaginase may be used in place of native E. coli derived asparaginase. In some embodiments, one dose of recombinant L-asparaginase is administered to a human subject in place of one dose of native E. coli derived asparaginase. In some embodiments, recombinant L-asparaginase may be used in place of long-acting E. coli derived asparaginase. In some embodiments, six doses of recombinant L-asparaginase are administered to a human subject in place of one dose of long-acting E. coli derived asparaginase. In some embodiments, seven doses of recombinant L-asparaginase are administered to a human subject in place of one dose of long-acting E. coli derived asparaginase. In some embodiments, the long-acting E. coli derived asparaginase is pegaspargase. In some embodiments, treatment with the recombinant L-asparaginase of the present disclosure is administered as a maintenance treatment. In some embodiments, administration of recombinant L-asparaginase to a human subject in place of native or long-acting E. coli derived asparaginase can occur at any time during a treatment cycle of native or long-acting E. coli derived asparaginase. In some embodiments, a treatment cycle for native or long-acting E. coli derived asparaginase is 1-14 administrations. In some embodiments, a treatment cycle for native or long-acting E. coli derived asparaginase is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 administrations. In some embodiments, a treatment cycle for native or long-acting E. coli derived asparaginase is 2-12 administrations. In some embodiments, a treatment cycle for native or long-acting E. coli derived asparaginase is 4-10 administrations. In some embodiments, a treatment cycle for native or long-acting E. coli derived asparaginase is 4-10 administrations. In some embodiments, a treatment cycle for native or long-acting E. coli derived asparaginase is 4-8 doses. In some embodiments, a treatment cycle for native or long-acting E. coli derived asparaginase is 4-6 doses.In some embodiments, the recombinant L-asparaginase can be administered to a human subject in place of native or long-acting E. coli derived asparaginase at any time during the treatment cycle of native or long-acting E. coli derived asparaginase, depending on the time point at which the human subject develops hypersensitivity. Of course, other alternative treatment regimes may be adopted at the discretion of the attending physician. The amount of the recombinant L-asparaginase of the present disclosure may be administered in a mixed dosage manner, where the dosage varies within the administration regimen. In general, the amount of the recombinant L-asparaginase of the present disclosure in a mixed administration manner is about 25 mg / m to treat diseases, including but not limited to cancer. 2 ~about 50mg / m 2 In some embodiments, the dose is within the range of 25 mg / m 2 The dose was 50 mg / m 2 The dose is administered intramuscularly. The amount of recombinant L-asparaginase of the present disclosure may be administered in a mixed administration manner, where the administration method varies within the administration regimen. In some embodiments, the mixed administration manner includes intravenous and intramuscular administration. Of course, other dosages and / or treatment regimens may be employed at the discretion of the attending physician.

[0149] In some embodiments, the disclosure provides a method of switching cancer treatments for a human subject. In some embodiments, the disclosure provides a method of switching acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) treatments for a human subject. In some embodiments, the disclosure provides a method of switching colorectal cancer (CRC) treatments for a human subject. In some embodiments, the disclosure provides a method of switching Wnt-negative colorectal cancer (CRC) treatments for a human subject. In some embodiments, the disclosure provides a method of switching Wnt-mutated colorectal cancer (CRC) treatments for a human subject. In some embodiments, the disclosure provides a method of switching diffuse large B-cell lymphoma (DLBCL) treatments for a human subject. In some embodiments, the disclosure provides a method of switching acute myeloid leukemia (AML) treatments for a human subject. In some embodiments, the disclosure provides a method of switching relapsed / refractory AML (R / R AML) treatments for a human subject. In some embodiments, recombinant L-asparaginase can be used as a first line treatment. In some embodiments, recombinant L-asparaginase can be used as a second line treatment. In some embodiments, recombinant L-asparaginase may be used as a third, fourth, or fifth line treatment.

[0150] (a) Dose Regimen 1: 25 mg / m 2 7 doses at In some embodiments, the present invention provides a method of switching cancer therapy in a human subject in need of cancer treatment. In some embodiments, the present invention provides a method of switching acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) therapy in a human subject in need of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) treatment. In some embodiments, the present invention provides a method of switching colorectal cancer (CRC) therapy in a human subject in need of colorectal cancer (CRC) treatment. In some embodiments, the present invention provides a method of switching Wnt-negative colorectal cancer (CRC) therapy in a human subject in need of Wnt-negative colorectal cancer (CRC) treatment. In some embodiments, the present disclosure provides a method of switching Wnt-mutated colorectal cancer (CRC) therapy in a human subject in need of Wnt-mutated colorectal cancer (CRC) treatment. In some embodiments, the present disclosure provides a method of switching diffuse large B-cell lymphoma (DLBCL) therapy in a human subject. In some embodiments, the present invention provides methods of switching acute myeloid leukemia (AML) treatment in a human subject in need of AML treatment. In some embodiments, the present invention provides methods of switching relapsed / refractory (R / R) acute myeloid leukemia (AML) treatment in a human subject in need of AML treatment.

[0151] In some embodiments, the method includes administering a series of seven doses of recombinant L-asparaginase intramuscularly or intravenously to a human subject in place of each dose of long-acting E. coli derived asparaginase, wherein each dose in the series is about 25 mg / m 2 and the administration interval between each dose in the series is 2 days.

[0152] In some embodiments, the method includes administering a series of seven doses of recombinant L-asparaginase intramuscularly or intravenously to a human subject in place of each dose of long-acting E. coli derived asparaginase, wherein each dose in the series is about 25 mg / m2 of recombinant L-asparaginase, and the administration interval between each dose in the series is about 48 hours.

[0153] In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the recombinant L-asparaginase is a tetramer, and each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase is a tetramer, and each monomer of the tetramer has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the human subject exhibited hypersensitivity to E. coli derived asparaginase prior to switching. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 6% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the lowest serum asparaginase activity (NSAA) in an assay as measured from a serum sample from a human subject is 0.1 IU / mL or greater after administration with the recombinant L-asparaginase. In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0154] (b) Dose regimen 2: 25 mg / m 2 / 25mg / m 2 / 50mg / m 2 6 doses x 2 The amount of recombinant L-asparaginase of the present disclosure may be administered in a mixed dose manner, where the dosage varies within the administration regimen. Generally, the recombinant L-asparaginase of the present disclosure in a mixed administration manner is administered in an amount of about 25 mg / m 2 ~about 50mg / m 2 In some embodiments, the cancer being treated is ALL or LBL. In some embodiments, the cancer being treated is CRC. In some embodiments, the cancer being treated is Wnt-negative CRC. In some embodiments, the cancer being treated is Wnt-mutated CRC. In some embodiments, the cancer being treated is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer being treated is AML. In some embodiments, the cancer being treated is R / R AML. Of course, other dosages and / or treatment regimens may be employed at the discretion of the attending physician.

[0155] In some embodiments, the present disclosure provides a method of switching cancer therapy in a human subject in need of cancer treatment. In some embodiments, the present disclosure provides a method of switching acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) therapy in a human subject in need of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) therapy. In some embodiments, the present disclosure provides a method of switching colorectal cancer (CRC) therapy in a human subject in need of colorectal cancer (CRC) therapy. In some embodiments, the present disclosure provides a method of switching Wnt-negative colorectal cancer (CRC) therapy in a human subject in need of Wnt-negative colorectal cancer (CRC) therapy. In some embodiments, the present disclosure provides a method of switching Wnt-mutated colorectal cancer (CRC) therapy in a human subject in need of Wnt-mutated colorectal cancer (CRC) therapy. In some embodiments, the present disclosure provides a method of switching diffuse large B-cell lymphoma (DLBCL) therapy in a human subject. In some embodiments, the present disclosure provides a method of switching acute myeloid leukemia (AML) treatment in a human subject in need of AML treatment. In some embodiments, the present disclosure provides a method of switching relapsed / refractory acute myeloid leukemia (R / R AML) treatment in a human subject in need of R / R AML treatment.

[0156] In some embodiments, the method includes administering a series of six doses of L-asparaginase intramuscularly or intravenously to a human subject in place of each dose of E. coli derived asparaginase, where four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2 The dosing interval between two doses in the series is 7 days, and the dose is about 25 mg / m 2 and the dosing interval between the dose of about 50 mg / m 2and the administration interval between the doses is 3 days, and the L-asparaginase is not E. coli derived asparaginase.

[0157] In some embodiments, the method includes administering a series of six doses of L-asparaginase intramuscularly or intravenously to a human subject in place of each dose of E. coli derived asparaginase, where four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2 The dosing interval between two doses in the series is 7 days, and the dose is about 25 mg / m 2 and the dosing interval between the dose of about 50 mg / m 2 and the administration interval between the doses is 3 days, the L-asparaginase is not Escherichia coli derived asparaginase, and 2 The dose was 50 mg / m 2 The dose is administered intramuscularly.

[0158] In some embodiments, the method includes administering a series of six doses of L-asparaginase intramuscularly or intravenously to a human subject in place of each dose of E. coli derived asparaginase, where four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2 The dosing interval between two doses in the series is about 168 hours, and the doses in the series are about 25 mg / m 2 and the interval between doses of about 48 hours is about 50 mg / m 2 and the administration interval between said doses is about 72 hours, and said L-asparaginase is not E. coli derived asparaginase.

[0159] In some embodiments, the method includes administering a series of six doses of L-asparaginase intramuscularly or intravenously to a human subject in place of each dose of E. coli derived asparaginase, where four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2 The dosing interval between two doses in the series is about 168 hours, and the doses in the series are about 25 mg / m 2 and the interval between doses of about 48 hours is about 50 mg / m 2 and the administration interval between the doses is about 72 hours, the L-asparaginase is not E. coli derived asparaginase, and 2 The dose was 50 mg / m 2 The dose is administered intramuscularly.

[0160] In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 6% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the lowest serum asparaginase activity (NSAA) in an assay as measured from a serum sample from a human subject is 0.1 IU / mL or greater after administration with the recombinant L-asparaginase. In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0161] In some embodiments, the disclosure provides a method of switching cancer treatment in a human subject in need thereof, the method comprising administering a series of six doses of L-asparaginase intramuscularly or intravenously to the human subject in place of each dose of E. coli derived asparaginase, wherein four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2The administration interval between two doses in the series is 6 to 8 days, and the dose is about 25 mg / m 2 and the dosing interval between the dose of about 50 mg / m 2and the next dose is 3 days, and the L-asparaginase is not E. coli derived asparaginase. In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 6% aggregation of L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the lowest serum asparaginase activity (NSAA) in an assay as measured from a serum sample from a human subject is 0.1 IU / mL or greater after administration with the recombinant L-asparaginase. In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL). In some embodiments, the cancer is lymphoblastic lymphoma (LBL). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is Wnt-negative colorectal cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is Wnt-mutated colorectal cancer.In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the acute myeloid leukemia is relapsed / refractory AML.

[0162] In some embodiments, the disclosure provides a method of switching cancer treatment in a human subject in need thereof, the method comprising administering a series of six doses of L-asparaginase intramuscularly or intravenously to the human subject in place of each dose of E. coli derived asparaginase, wherein four of the six doses are about 25 mg / m 2 and two of the six doses are about 50 mg / m 2 is about 50 mg / m 2 The administration interval between two doses in the series is 6 to 8 days, and the dose is about 25 mg / m 2 and the dosing interval between the dose of about 50 mg / m 2 and the administration interval between the doses is 3 days, the L-asparaginase is not Escherichia coli derived asparaginase, and 2 The dose was 50 mg / m 2is administered intramuscularly. In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 6% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the lowest serum asparaginase activity (NSAA) in an assay as measured from a serum sample from a human subject is 0.1 IU / mL or greater after administration with the recombinant L-asparaginase. In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0163] (c) Dosing regimen 3: 50 / 25 dosing every 3 days The amount of recombinant L-asparaginase of the present disclosure may be administered in a mixed dosing manner, where the dosage and administration time vary within the dosing regimen. Generally, the amount of recombinant L-asparaginase of the present disclosure in a mixed dosing manner is about 25 mg / m2 to treat diseases including, but not limited to, ALL or LBL, CRC, AML, or DLBCL. 2 ~about 50mg / m2 In some embodiments, the CRC is a Wnt-negative CRC. In some embodiments, the CRC is a Wnt-mutated CRC. In some embodiments, the AML is an R / R AML. Of course, other dosages and / or treatment regimens may be employed at the discretion of the attending physician.

[0164] In some embodiments, the present disclosure provides a method of treating acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) in a human subject in need of such treatment.

[0165] In some embodiments, the method includes intramuscularly administering to a human subject an L-asparaginase other than E. coli derived asparaginase as a chronological dose set, wherein the chronological dose set includes a first dose followed by a second dose, the first dose being about (i) 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and the second dose is (i) about 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and wherein the first dose is about 50 mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 3 days, and the first dose is about 25 mg / m 2 and wherein the administration interval between the first and second doses to the human subject is 2 days, and the second dose is 50 mg / m 2 no further administration of L-asparaginase to the subject occurs for 3 days, and the second dose is 25 mg / m 2 no further administration of L-asparaginase to the subject occurs for two days.

[0166] In some embodiments, the first dose is about 50 mg / m 2 and the second dose is about 25 mg / m 2and the chronological dose set further comprises a third dose after the second dose, said third dose being about 25 mg / m 2 and the administration interval between the second and third doses to the human subject is two days. In some embodiments, the first dose is administered on a Friday and the second dose is administered on a Monday. In some further embodiments, the first dose is administered on a Friday, the second dose is administered on a Monday, and the third dose is administered on a Wednesday.

[0167] In some embodiments, the first dose is about 25 mg / m 2 and the second dose is about 50 mg / m 2 and the chronological dose set further comprises a third dose after the second dose, said third dose being about 25 mg / m 2 and the administration interval between the second and third doses for a human subject is 3 days. In some embodiments, the first dose is administered on a Wednesday and the second dose is administered on a Friday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. 2 Any dose at 50 mg / m2 is administered intravenously. 2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0168] In some embodiments, the first dose is about 25 mg / m 2 and the second dose is about 50 mg / m 2 and the chronological dose set further comprises a third dose prior to the first dose, said third dose being about 25 mg / m 2 and the third dose is administered to the human subject two days before the first dose. In some embodiments, the first dose is administered on a Wednesday and the second dose is administered on a Friday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the first dose is administered at about 25 mg / m 2 Any dose at 50 mg / m2 is administered intravenously.2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0169] In some embodiments, the method includes a first dose, a second dose, and a third dose, where the first dose is administered on Monday, the second dose is administered on Wednesday, and the third dose is administered on Friday. In some embodiments, the first dose is 25 mg / m 2 and the second dose was 25 mg / m 2 and the third dose was 50 mg / m 2 It is.

[0170] In some embodiments, the method further comprises administering a plurality of instances of L-asparaginase of the chronological dose set intramuscularly or intravascularly to a human subject, wherein each instance of the recombinant L-asparaginase of the chronological dose set is administered to the subject upon completion of a previous instance of the chronological dose set in the plurality of instances of the chronological dose set. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 100. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 80. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 60. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 50. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 40. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 30. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 20. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 15. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 14. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 13. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 12. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 11. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 10. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 9. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 8.In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 7. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 6. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 5. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 4. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 4 and 6.

[0171] In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase.

[0172] In some embodiments, the recombinant L-asparaginase is a tetramer and each monomer of the tetramer comprises SEQ ID NO: 1. In some embodiments, the recombinant L-asparaginase is a tetramer and each monomer of the tetramer has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1.

[0173] In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 6% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the serum minimal asparaginase activity (NSAA) in an assay as measured from a serum sample from the human subject is equal to or greater than 0.1 IU / mL after administration with the recombinant L-asparaginase. In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0174] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, which may be optionally repeated, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days followed by a two consecutive day rest period, and the first dose of the cycle is 25 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle is 50 mg / m 2 followed by a drug rest period of 2 consecutive days. In some embodiments, the first dose of the cycle is administered on a Monday, the second dose of the cycle is administered on a Wednesday, and the third dose of the cycle is administered on a Friday.

[0175] (d) Dosing Regimen 3: 50 / 25 doses administered approximately 72 hours apart The amount of recombinant L-asparaginase of the present disclosure may be administered in a mixed dosage manner, where the dosage amount and administration time vary within the administration regimen. Generally, the amount of recombinant L-asparaginase of the present disclosure in a mixed dosage manner is about 25 mg / m2 for treating diseases including, but not limited to, cancer. 2 ~about 50mg / m 2 The dosage range is within the range of 100 mg / kg / day. Some cancers treated using this dosage regimen include, but are not limited to, ALL or LBL, CRC, AML, or DLBCL. In some embodiments, the CRC is Wnt-negative CRC. In some embodiments, the CRC is Wnt-mutated CRC. In some embodiments, the AML is R / R AML. Of course, other dosages and / or treatment regimens may be employed at the discretion of the attending physician.

[0176] In some embodiments, the present disclosure provides a method of treating cancer in a human subject in need of cancer treatment. In some embodiments, the present disclosure provides a method of treating acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) in a human subject in need of LBL treatment. In some embodiments, the present disclosure provides a method of treating colorectal cancer (CRC) in a human subject in need of CRC treatment. In some embodiments, the present disclosure provides a method of treating Wnt-negative colorectal cancer (CRC) in a human subject in need of CRC treatment. In some embodiments, the present disclosure provides a method of treating Wnt-mutated colorectal cancer (CRC) in a human subject in need of CRC treatment. In some embodiments, the present disclosure provides a method of treating Wnt-mutated colorectal cancer (CRC) in a human subject in need of CRC treatment. In some embodiments, the present disclosure provides a method of treating diffuse large B-cell lymphoma (DLBCL). In some embodiments, the present disclosure provides a method of treating acute myeloid leukemia (AML) in a human subject in need of such treatment. In some embodiments, the present disclosure provides a method of treating relapsed / refractory acute myeloid leukemia (R / R AML) in a human subject in need of such treatment.

[0177] In some embodiments, the method comprises intramuscularly or intravenously administering to a human subject an L-asparaginase other than E. coli derived asparaginase as a set of chronological doses, wherein the set of chronological doses comprises a first dose followed by a second dose, the first dose being (i) about 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and the second dose is (i) about 50 mg / m of L-asparaginase. 2 and (ii) about 25 mg / m 2 and wherein the first dose is about 50 mg / m 2 wherein the administration interval between the first and second doses to the human subject is about 72 hours, and the first dose is about 25 mg / m2 wherein the administration interval between the first and second doses to the human subject is about 48 hours, and the second dose is about 50 mg / m 2 no further administration of L-asparaginase to the subject is administered for about 72 hours, and the second dose is about 25 mg / m 2 In some embodiments, no further administration of L-asparaginase to the subject occurs for about 48 hours. 2 Any dose at 50 mg / m2 is administered intravenously. 2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0178] In some embodiments, the first dose is about 50 mg / m 2 and the second dose is about 25 mg / m 2 and the chronological dose set further comprises a third dose after the second dose, said third dose being about 25 mg / m 2 and the administration interval between the second and third doses for a human subject is about 48 hours. In some embodiments, the first dose is administered on a Friday and the second dose is administered on a Monday. In some further embodiments, the first dose is administered on a Friday, the second dose is administered on a Monday, and the third dose is administered on a Wednesday. In some embodiments, the first dose is administered on a Friday, the second dose is administered on a Monday, and the third dose is administered on a Wednesday. 2 Any dose at 50 mg / m2 is administered intravenously. 2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0179] In some embodiments, the first dose is about 25 mg / m 2 and the second dose is about 50 mg / m 2 and the chronological dose set further comprises a third dose after the second dose, said third dose being about 25 mg / m 2and the administration interval between the second and third doses for a human subject is about 72 hours. In some embodiments, the first dose is administered on a Wednesday and the second dose is administered on a Friday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. 2 Any dose at 50 mg / m2 is administered intravenously. 2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0180] In some embodiments, the first dose is about 25 mg / m 2 and the second dose is about 50 mg / m 2 and the chronological dose set further comprises a third dose prior to the first dose, said third dose being about 25 mg / m 2 and the third dose is administered to the human subject about 48 hours before the first dose. In some embodiments, the first dose is administered on a Wednesday and the second dose is administered on a Friday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. In some embodiments, the first dose is administered on a Wednesday, the second dose is administered on a Friday, and the third dose is administered on a Monday. 2 Any dose at 50 mg / m2 is administered intravenously. 2 Any dose at is administered intramuscularly. In some embodiments, all doses are administered intramuscularly. In some embodiments, all doses are administered intravascularly.

[0181] In some embodiments, the method includes a first dose, a second dose, and a third dose, where the first dose is administered on Monday, the second dose is administered on Wednesday, and the third dose is administered on Friday. In some embodiments, the first dose is 25 mg / m 2 and the second dose was 25 mg / m 2 and the third dose was 50 mg / m 2 It is.

[0182] In some embodiments, the method further comprises administering a plurality of instances of L-asparaginase of the chronological dose set intramuscularly or intravascularly to a human subject, wherein each instance of the recombinant L-asparaginase of the chronological dose set is administered to the subject upon completion of a previous instance of the chronological dose set in the plurality of instances of the chronological dose set. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 100. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 80. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 60. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 50. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 40. In some embodiments, the plurality of instances of L-asparaginase of the chronological dose set is between 2 and 30. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 20. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 15. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 14. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 13. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 12. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 11. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 10. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 9. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 8.In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 7. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 6. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 5. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 2 and 4. In some embodiments, the plurality of instances of L-asparaginase in the chronological dose set is between 4 and 6.

[0183] In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase.

[0184] In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO: 1. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1.

[0185] In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 6% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition that exhibits less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the serum minimal asparaginase activity (NSAA) in an assay as measured from a serum sample from the human subject is equal to or greater than 0.1 IU / mL after administration with the recombinant L-asparaginase. In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0186] In some embodiments, the dosing regimen of recombinant L-asparaginase comprises a cycle, which may be optionally repeated, and which comprises administration of recombinant L-asparaginase every other day for five consecutive days followed by a rest period of about 48 hours, and the first dose of the cycle is 25 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle is 50 mg / m 2 followed by a drug holiday of about 48 hours. In some embodiments, the first dose of the cycle is administered on a Monday, the second dose of the cycle is administered on a Wednesday, and the third dose of the cycle is administered on a Friday.

[0187] (e) Alternative Preferred Dosing Regimens In some embodiments, the disclosure provides a method for depleting asparaginase in a human subject to treat cancer, comprising administering about 25 mg / m L-asparaginase to a human subject on Mondays, Wednesdays, and Fridays for a total of 7 doses of L-asparaginase over a two week period. 2The method includes intravascularly administering to a human subject an L-asparaginase of the formula: In some embodiments, the cancer being treated is ALL or LBL. In some embodiments, the cancer being treated is CRC. In some embodiments, the cancer being treated is Wnt-negative CRC. In some embodiments, the cancer being treated is Wnt-mutated CRC. In some embodiments, the cancer being treated is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer being treated is AML. In some embodiments, the cancer being treated is R / R AML. In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the recombinant L-asparaginase is a tetramer, and each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase is a tetramer, and each monomer of the tetramer has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition exhibiting less than 6% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition exhibiting less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the assay minimum serum asparaginase activity (NSAA) as measured from a serum sample from a human subject is 0.1 IU / mL or greater following treatment with recombinant L-asparaginase.In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen.

[0188] In some embodiments, the disclosure provides a method for depleting asparaginase in a human subject to treat cancer, comprising administering to the human subject a total of six doses of L-asparaginase over a two week period, comprising: (a) administering about 25 mg / m2 L-asparaginase on Mondays and Wednesdays; 2 of L-asparaginase to a human subject, and (b) administering about 50 mg / m 2The method includes intramuscularly administering to a human subject an L-asparaginase of the formula: In some embodiments, the cancer being treated is ALL or LBL. In some embodiments, the cancer being treated is CRC. In some embodiments, the cancer being treated is Wnt-negative CRC. In some embodiments, the cancer being treated is Wnt-mutated CRC. In some embodiments, the cancer being treated is AML. In some embodiments, the cancer being treated is R / R AML. In some embodiments, the cancer being treated is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the long-acting E. coli derived asparaginase is peg-asparaginase. In some embodiments, the recombinant L-asparaginase is a tetramer, wherein each monomer of the tetramer comprises SEQ ID NO:1. In some embodiments, the recombinant L-asparaginase is a tetramer, and each monomer of the tetramer has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the human subject has demonstrated hypersensitivity to E. coli derived asparaginase. In some embodiments, the human subject is an adult patient. In some embodiments, the human subject is a pediatric patient. In some embodiments, the recombinant L-asparaginase is administered in a composition exhibiting less than 6% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is administered in a composition exhibiting less than 1% aggregation of the L-asparaginase. In some embodiments, the recombinant L-asparaginase is not lyophilized. In some embodiments, the recombinant L-asparaginase is recombinantly produced in Pseudomonas fluorescens. In some embodiments, the assay minimum serum asparaginase activity (NSAA) as measured from a serum sample from a human subject is 0.1 IU / mL or greater following treatment with recombinant L-asparaginase.In some embodiments, the recombinant L-asparaginase is co-administered with one or more other chemotherapeutic agents as part of a combination chemotherapy regimen. EXAMPLES

[0189] Example 1 Studies to assess toxicity and toxicokinetics The toxicity and toxicokinetics (TK) of recombinant L-asparaginase was investigated in a 2-week daily intravenous infusion study in rats under good laboratory practice (GLP). A 2-week recovery period was included to evaluate reversibility or persistence of effects and / or delayed toxicity. Given that recombinant L-asparaginase is a biotechnology-derived product, the occurrence of anti-drug antibodies (ADA) was prospectively evaluated in all main study animals to aid in the interpretation of the study findings.

[0190] Four groups of male and female Crl:CD(SD) rats (n=10 / sex / group) were administered recombinant L-asparaginase at doses of 0, 4.6, 15.2, and 45.6 mg / kg, respectively. Recovery animals were included in the control and high dose groups (n=5 / sex / group). Toxicokinetic evaluation animals (n=3 / sex for control group, and n=9 / sex / dose group) were also included. Animals were administered recombinant L-asparaginase by intravenous infusion (±10 min) for 2 h at a rate of 5 mL / kg / h via a catheter inserted in the femoral vein. Vehicle and control substances were 20 mM sodium phosphate, 50 mM sodium chloride, and 6.4% trehalose, pH 7.0.

[0191] The sentinel group also received the highest dose before the start of the main study, were observed for two days, and were sacrificed on the third day without necropsy.

[0192] Mortality, clinical findings, body weight, body weight change, food consumption, ocular findings, and clinical and anatomic pathology were recorded, along with toxicokinetics (assessed by serum asparaginase activity (SAA)) and anti-drug antibody analyses.

[0193] Toxicity and toxicokinetic evaluation animals in Groups 1 (control) through 3 (15.2 mg / kg) were dosed for 14 days and survived until their scheduled necropsy. Dosing in Group 4 (45.6 mg / kg) was discontinued on Day 6 due to adverse clinical findings, at which point animals were either necropsied according to their assigned group or transferred to a 2-week recovery period. Group 1 recovery animals were dosed with vehicle once daily for 14 days, followed by a 6-day recovery period, and therefore sacrificed on the same calendar day as Group 4 recovery animals.

[0194] Exposure, as assessed by SAA Cmax and AUC0-24, increased with increasing dose levels from 4.6 to 45.6 mg / kg / day on day 1 and from 4.6 to 15.2 mg / kg / day on day 14. Increases in Cmax and AUC0-24 values ​​were approximately dose proportional from 4.6 to 15.2 and 15.2 to 45.6 mg / kg / day on day 1 and from 4.6 to 15.2 mg / kg / day on day 14, but from 4.6 to 45.6 mg / kg / day on day 1 there was an 18-fold increase in AUC0-24 for a 10-fold increase in dose. There were no significant sex differences in SAA Cmax and AUC0-24 values. No accumulation of SAA was observed in rats following repeated dosing.

[0195] In high-dose animals that were euthanized early, clinical findings included piloerection, bristly fur, reddened skin on the paws, and clear oral secretions. Significant hematological changes indicative of reduced hematopoiesis were noted, including decreased reticulocyte, platelet, and white blood cell counts. These changes were found to be reversible in recovered animals. Increases in blood glucose and blood urea nitrogen concentrations, and decreases in protein and liver enzymes suggested dehydration, prerenal azotemia, protein loss or intercompartmental shifts, and downregulation of liver enzyme activity, respectively. Gross findings were limited to focal red discoloration of the mucosa in the glandular stomach of one female, which lacked microscopic correlation. Microscopic findings included decreased cellularity in femoral bone marrow, myocardial necrosis and hemorrhage, submucosal edema / abscesses and epithelial hyperplasia / erosions in the non-glandular stomach / duodenum, decreased red pulp, extramedullary hematopoiesis, and lymphocytes in the spleen, decreased cortical lymphocytes in the thymus, and decreased secretions in the pancreas. These changes were considered adverse. Increases in spleen and thymus weights in males and females were noted at the time of sacrifice of recovered animals and were considered consistent with recovery. No microscopic evaluation was performed on recovered animals.

[0196] Dose-related decreases in food consumption and body weight were observed in animals dosed at or below 15.2 mg / kg / day, and piloerection was observed at 15.2 mg / kg / day. Dose-related changes in hematology and clinical chemistry parameters were generally consistent with those observed in the high-dose group, although smaller changes were observed in most animals. These clinical pathology changes were not considered adverse. Microscopic findings in low- and mid-dose animals consisted of a minimal to mild decrease in erythroid progenitors and an increase in myeloid progenitors, which were consistent with the hematological findings. The decrease in spleen weight correlated with the decrease in extramedullary hematopoiesis and red pulp, but the decrease in thymus weight lacked a microscopic correlation.

[0197] In conclusion, male and female Crl:CD(SD) rats were administered vehicle control material or recombinant L-asparaginase at 4.6, 15.2, or 45.6 mg / kg / day by intravenous infusion once daily for 14 days. Adverse clinical findings that necessitated sacrifice in moribund condition and discontinuation of treatment on day 6 of the treatment phase, as well as decreased cellularity in femoral bone marrow, myocardial necrosis and hemorrhage in the heart, submucosal edema or abscesses and epithelial hyperplasia in non-glandular stomach, and gastric or duodenal erosions / ulcers, occurred in animals administered 45.6 mg / kg / day. No adverse clinical findings, clinical pathological changes, or microscopic findings occurred in animals administered 15.2 mg / kg / day or less. Thus, the no observed adverse effect level (NOAEL) is 15.2 mg / kg / day.

[0198] The highlights of this study include male and female Crl:CD(SD) rats (n=10 / sex / group) with control and high-dose recovery groups (n=5 / sex / group). Recombinant L-asparaginase (0, 4.6, 15.2, or 45.6 mg / kg / day) was administered daily via 2-hour intravenous infusion for up to 14 days. Adverse clinical findings necessitated sacrifice and discontinuation of treatment on day 6 in the high-dose group. Adverse reductions in cellularity in femoral bone marrow, necrosis and hemorrhage in the heart, and erosions, edema, hyperplasia, abscesses, and / or ulcers in non-glandular stomach / duodenum occurred in the high-dose group. Non-adverse but qualitatively similar clinical findings, clinical pathological changes, and microscopic findings occurred in animals dosed at or below 15.2 mg / kg / day. The no observed adverse effect level (NOAEL) was determined to be 15.2 mg / kg / day.

[0199] Example 2 Tests to assess safety Subjects were 18-55 years old and in good health as determined by the investigator. In dosing cohort 1, subjects received recombinant L-asparaginase (25 mg / m2) by either a 2-hour intravenous (IV) infusion (N=6) or intramuscular (IM) infusion (N=6). 2After the safety, tolerability, and pharmacokinetics of recombinant L-asparaginase were evaluated to determine the need for separate dosing cohorts, subjects in dosing cohort 2 were randomized to receive a single dose of 37.5 mg / m 2 IV (N=6) or 12.5 mg / m 2 Patients were randomized (1:1) to receive a single dose of recombinant L-asparaginase either IV or IM (N=6). Recombinant L-asparaginase was administered in the inpatient ward, and subjects were discharged on day 5 with safety follow-up by telephone on days 6 and 30. The primary objective was to evaluate the safety and tolerability of recombinant L-asparaginase administered IV and IM in each cohort. Secondary objectives included characterizing the pharmacokinetics of recombinant L-asparaginase administered IV and IM based on serum asparaginase activity (SAA).

[0200] The demographic characteristics (mean ± SD) of the 24 subjects enrolled included age (38.4 ± 8.30 years), weight (77.04 ± 10.00 kg), and body surface area (1.91 ± 0.15 m 2 ) were included. Additionally, 63% of subjects were male, 97% ethnicity was 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%) subjects in dosing cohort 1 had 1 or more adverse events (IV=4 subjects, IM=4 subjects). In dosing cohort 2, 11 / 12 (92%) subjects had 1 or more adverse events (IV=6 subjects, IM=5 subjects). No serious adverse events or grade 3 or higher adverse events were reported for any subject in either dosing cohort. The most common treatment-emergent adverse event occurring in two or more subjects in each dosing cohort was nausea (see Figure 1, Table 1). Dyspepsia was reported at 12.5 mg / m 2 This was the most common adverse event in subjects receiving recombinant L-asparaginase IM (see Figure 1, Table 1). Pharmacokinetic evaluations were performed at doses of 12.5 and 25 mg / m when administered IM. 2Six out of six (100%) subjects in the cohort achieved serum asparaginase activity levels of 0.1 IU / mL or greater at 48 and 72 hours after administration of recombinant L-asparaginase. 2 At the dose level of 37.5 mg / m, serum asparaginase activity levels reached 0.1 IU / mL or higher in 6 / 6 (100%) subjects at 48 hours and in 4 / 6 (67%) subjects at 72 hours. 2 At this dose level, 6 / 6 (100%) subjects achieved 0.1 IU / mL or greater at 48 and 72 hours post-dose (see Figure 1, Table 2).

[0201] Administration of recombinant L-asparaginase was well tolerated in healthy adults, with no unexpected adverse events, no reported serum asparaginase activity, and no grade 3 or higher adverse events. Serum asparaginase activity levels of 0.1 IU / mL or greater (a surrogate marker of asparagine depletion) were achieved at 48 hours in all human subjects receiving recombinant L-asparaginase IM and IV. SAA levels of 0.1 IU / mL or greater at 72 hours after administration of recombinant L-asparaginase were also achieved in all subjects receiving 25 mg / m 2 This was achieved in all but two subjects in the IV group. Based on the overall pharmacokinetic and safety data from this study, the recommended starting dose for Phase 2 / 3 is 25 mg / m2 for the IM route on a Monday / Wednesday / Friday dosing schedule. 2 and 37.5 mg / m for the IV route of administration. 2 It is.

[0202] Example 3 Population Pharmacokinetic (PK) Model Development and Simulation A population PK model of recombinant L-asparaginase was developed using aggregated serum asparaginase activity (SAA) data from a Phase 1 single-dose study in healthy adult subjects (sbj) to evaluate the effect of endogenous covariates (weight, body surface area, age, sex, and race) on PK parameters. This population PK model was developed to inform the starting dose selection for a pivotal Phase 2 / 3 study of recombinant L-asparaginase. 2 [N=6] and 37.5 mg / m 2 Intravenous (IV) infusion data [N=6] and 12.5 mg / m 2 [N=6] and 25 mg / m 2 A total of 24 subjects were included in the model, with intramuscular (IM) injection data at [N=6].Adult and pediatric SAA profiles (1000sbj / population) were simulated using the developed model to explore the feasibility of achieving therapeutic target trough SAA levels ≥0.1IU / mL based on different doses, schedules, and routes of administration.

[0203] The final model describing both the IV and IM routes was a one-compartment model with linear elimination (IV) and mixed order absorption (IM only) with body weight included as a non-proportional covariate for SAA clearance. Body size 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 variance in recombinant L-asparaginase PK. Based on the Phase 1 study data and population PK modeling and simulations, the recommended starting dose in Phase 2 / 3 is 25 mg / m for the IM route on a Monday / Wednesday / Friday dosing schedule. 2 and 37.5 mg / m for the IV route of administration. 2 The recommended dose and schedule is expected to maintain trough SAA levels of 0.1 IU / mL or greater 72 hours after dosing.

[0204] Example 4. Studies to evaluate safety and efficacy This is an open-label, multicenter, Phase 2 / 3 dose-finding and pharmacokinetic study of recombinant L-asparaginase in patients with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) who have demonstrated hypersensitivity (allergic reaction or asymptomatic inactivation) to E. coli-derived asparaginase and have more than one dose of E. coli-derived asparaginase remaining on their treatment regimen (Table 3). 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 are substituted for each dose of E. coli-derived long-acting asparaginase. Individual patient treatment duration will vary depending on the number of doses of E. coli-derived asparaginase remaining on the patient's original treatment regimen.

[0205] This study consists of two consecutive parts: Part A, which determined the dose of recombinant L-asparaginase and confirmed its safety and efficacy for intramuscular (IM) administration, and Part B, which clarified the optimal dose and schedule for intravenous (IV) infusion of recombinant L-asparaginase.

[0206] Part A of this study has two IM cohorts: Cohort 1 is a repeat-dose / confirmatory cohort where the final IM dose level will be selected, and Cohort 2 is an expansion cohort to confirm the efficacy and safety of the final IM dose level and schedule.

[0207] Part B will be conducted to determine the optimal dose of IV administration of recombinant L-asparaginase for further testing in ALL / LBL patients as a repeat dose.

[0208] Additional courses of recombinant L-asparaginase will be administered based on each patient's original treatment plan as long as the patient is experiencing a clinical response.

[0209] Blood samples were taken at pre-determined time points to determine serum asparaginase activity levels. Patients were monitored for adverse events. The immunogenicity of recombinant L-asparaginase treatment was also evaluated.

[0210] The primary objectives were (1) to determine the response rate during the first cycle of IM recombinant L-asparaginase administration (response rate defined as the proportion of patients with a nadir level of serum asparaginase activity during the last 72 hours of ≥0.1 IU / mL during the first cycle of treatment) and (2) to evaluate the occurrence of treatment-emergent adverse events (TEAEs).

[0211] Secondary objectives were to determine (1) the proportion of patients with a 48-hour serum asparaginase activity nadir level of 0.1 IU / mL or greater during the first course of IM administration of recombinant L-asparaginase in a 2-week time frame; (2) the proportion of patients with a 48-hour serum asparaginase activity nadir level of 0.4 IU / mL or greater during the first course of IM administration of recombinant L-asparaginase in a 2-week time frame; (3) the proportion of patients with a 72-hour serum asparaginase activity nadir level of 0.4 IU / mL or greater during the first course of IM administration of recombinant L-asparaginase in a 2-week time frame; (4) characterize the pharmacokinetics of IM administered recombinant L-asparaginase using a population pharmacokinetic approach and exposure-related correlations based on serum asparaginase activity for a time frame up to 30 days after the last dose; and (5) the incidence of anti-drug antibody formation to recombinant L-asparaginase for a time frame up to 30 days after the last dose.

[0212] Eligibility criteria will allow both males and females. Inclusion criteria include: (1) pediatric and adult patients with a diagnosis of ALL or LBL, (2) patients who have developed an allergic reaction to or have asymptomatic inactivation of E. coli-derived long-acting asparaginase, (3) patients with at least one course of E. coli-derived asparaginase remaining in their treatment regimen, and (4) patients must have fully recovered from a previous allergic reaction to E. coli-derived asparaginase, as determined by the investigator. Exclusion criteria included: (1) patients who had previously received Erwinia chrysanthemi L-asparaginase or recombinant L-asparaginase; (2) patients with relapsed ALL or LBL; (3) patients currently receiving another investigational drug and / or being treated with an investigational device concurrently (within 48 hours) with recombinant L-asparaginase during one course of recombinant L-asparaginase; (4) patients with a history of grade 3 or higher pancreatitis; and (5) patients with a history of asparaginase-related grade 3 or higher bleeding events or asparaginase-related thrombosis requiring anticoagulant treatment, excluding catheter-related thrombotic events.

[0213] Further testing revealed a 37.5 mg / m 2 Above 100 mg / kg / day, higher doses are administered, with each additional dose level(s) not exceeding a 50% increase from the previous dose level.

[0214] The study will accept enrollment for evaluation of IV and IM formulations, and study center involvement will be at the sponsor's discretion.

[0215] An undetectable level of SAA may be based on the lower limit of quantification defined by a certified laboratory authorized to perform this test under CLIA. CLIA certified laboratories utilize different limits of quantification depending on their assay methodology, and results for determining an undetectable level of SAA may vary between laboratories.

[0216] Serum asparaginase concentration is the same as the PK content in this study. Consistency between SAP (which uses serum asparaginase concentration) and the label on the laboratory tube (which uses the PK content).

[0217] In certain preferred embodiments, the testing window is 7 days for all laboratory tests, including coagulation tests and laboratory tests at screening.

[0218] Example 5. Tests for assessing protein aggregation Quality testing was performed on small batches of recombinant L-asparaginase produced at laboratory scale, which consisted of an analytical evaluation of comparability and homogeneity with commercial Erwinase® to ensure that the quality, safety, and potency of the recombinant L-asparaginase was equivalent to native L-asparaginase from spontaneous fermentation of Dickeya chrysanthemi (currently in clinical use) and was not adversely affected by the use of alternative expression systems and purification processes.

[0219] SEC-HPLC method Purity determination by size exclusion chromatography was performed using SEC-HPLC using a Phenomenex BioSep SEC-s4000 column. The column was conditioned with mobile phase (50 mM phosphate, 200 mM NaCl, pH 7.0) at 1 mL / min for 1 hour prior to injection. BioRad gel filtration standards were diluted to 1 mg / mL in mobile phase and injected in triplicate after multiple injections of a mobile phase blank to wash the column. The purity of recombinant L-asparaginase, recombinant crisantaspase from E. coli, and Erwinase® was assessed (Figure 2).

[0220] The SEC-HPLC results show a main peak representing the tetramer and an aggregate peak. Different levels of HMW aggregates are present in all samples, with more aggregates present in Erwinaze CAMR176 (6%) when compared to recombinant L-asparaginase (0.3%).

[0221] Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC MALLS) Test solutions were prepared in duplicate for all samples using the C.52.S1640 and Solvias standard operating procedures. Samples were diluted to 10 mg / mL in formulation buffer and mixed. At least 4 mL was prepared. Sample solutions were filtered through a 0.2 μM syringe filter and directly into HPLC vials. The first few drops were discarded. If the protein concentration was lower than 10 mg / mL, it was necessary to adjust the injection volume so that 750 μg was injected. Samples and reference materials were analyzed by this technique: all unstressed batches, all batches stressed by 5 and 10 cycles, respectively, of freezing below -75°C overnight and thawing at room temperature for 3 hours. The results are shown in Figure 3.

[0222] The SEC MALLS results confirm the SEC-HPLC results and show higher levels of HMW aggregates in Erwinaze® CAMR176 (6%) when compared to recombinant L-asparaginase (1%).

[0223] Sedimentation velocity AUC (svAUC) method: 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. Samples were then placed in an AN-60Ti analytical rotor and loaded into an analytical ultracentrifuge, Beckman Coulter ProteomeLab XL. Data were analyzed using the c(s) function developed by Peter Schuck at NIH and implemented in his analysis program SEDFIT (version 11.3). The resulting size distributions were graphed and peaks integrated using OriginLab Origin® version 9.0. The results in Figure 4 demonstrate that recombinant L-asparaginase shows the highest homogeneity (based on the lowest aggregate content of 1%) and the Erwinase® sample is the least homogeneous (5% aggregates). In conclusion, recombinant L-asparaginase has lower aggregate levels compared to Erwinase® as measured by several methods with different detection principles.

[0224] Example 6. Studies to assess protein aggregation on a commercial scale This quality test was performed on a full scale batch of recombinant L-asparaginase produced at 1000 L (commercial scale). This test consisted of an analytical evaluation of the comparability to commercial Erwinase® to confirm that the small scale (SS) comparability test of recombinant L-asparaginase in Example 5 was supported at full scale (1000 L). This test also used SEC-MALLS methodology to characterize the recombinant L-asparaginase.

[0225] 1 mL aliquots of recombinant L-asparaginase were pipetted into 5 mL PETG bottles and stored at −20° C. The formulation was 20 mM sodium phosphate, 50 mM sodium chloride, 6.4% trehalose, pH 7.0.

[0226] SE-UHPLC methodSize-exclusion ultra-high pressure liquid chromatography (SE-UPLC) with UV detection is used to determine the purity of recombinant L-asparaginase DS samples based on molecular size, i.e., hydrodynamic radius. Samples and standards are diluted to 2.5 mg / mL in mobile phase (10 mM phosphate, 0.3 M arginine, 0.05% sodium azide) and injected onto a Waters Acquity UPLC column (BEH200 SEC, 1.7 μm, 4.6×30 mm). Recombinant L-asparaginase DS isocratically eluted at 0.4 mL / min. Purity profile is characterized by UV detection at 280 nm. Purity is reported as % tetramer (main peak), % HMW, and % LMW integrated. The SE-UPLC profile of recombinant L-asparaginase DS consists of a main peak identified as the tetrameric form of the enzyme and a HMW peak identified as the octamer in the following SEC-MALLS analysis. There is also a very low level of a low molecular weight (LMW) back shoulder peak. The area percentages are shown in Figure 5.

[0227] Consistent with the small scale data in Example 5, SEC-UPLC showed that the FS batch of recombinant L-asparaginase (0.2%) had significantly lower aggregate counts compared to Erwinase CAMR-174 (6%).

[0228] SEC-MALLS method The high molecular weight species present in two samples (A and B) of recombinant L-asparaginase were measured using SEC-MALLS size exclusion chromatography coupled with a multi-angle light scattering detector (SEC-MALLS). Samples were diluted with formulation buffer and injected onto a Superdex™ 200 Increase column equipped with a Minidawn Treos MALLS detector. Figure 6 reports the UV peak area percentages. The results show that recombinant L-asparaginase exists primarily in tetrameric form, with low levels of octamers and higher order aggregates. Lower levels of LMW species were also observed.

[0229] 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. Samples were then placed in an AN-60Ti analytical rotor and loaded into a Beckman Coulter ProteomeLab XL analytical ultracentrifuge. Data were analyzed using the c(s) function developed by Peter Schuck at NIH and implemented in his analysis program, SEDFIT (version 11.3). The resulting size distributions were graphed and peaks integrated using OriginLab Origin® version 9.0.

[0230] As shown in Figures 7 and 8, the recombinant L-asparaginase was highly homogeneous, with 99.2% of the enzyme present in tetrameric form, with low levels of octamers (0.61%) and higher order aggregates (0.07%). This confirms the low aggregate levels observed with the SE-UHPLC and SEC-MALLS methods. The Erwinase® batch was found to be more heterogeneous, with 93.6% identified as tetramers and higher levels of HMW species (6.4%).

[0231] Example 7. A randomized Phase 1 study of the safety, tolerability, and pharmacokinetics of Erwinia recombinant asparaginase (JZP-458) in healthy adult subjects. JZP-458 is an Erwinia recombinant asparaginase produced using a novel Pseudomonas fluorescens expression platform that produces an enzyme with no immunological cross-reactivity to E. coli-derived asparaginase. A randomized, single-center, open-label Phase 1 study was conducted to evaluate the safety, tolerability, and pharmacokinetics of a single dose of JZP-458 administered by intramuscular (IM) injection or intravenous (IV) infusion in healthy adult subjects. In determining the efficacy of asparaginase, serum asparaginase activity (SAA) levels serve as a surrogate marker of asparagine depletion, with a nadir SAA level (NSAA) of 0.1 IU / mL or greater being the accepted threshold for demonstrating adequate asparagine depletion in clinical practice.

[0232] Methods: A randomized, single-center, open-label Phase 1 study was conducted in the United States between November 19, 2018 and May 20, 2019. The study was approved by the IntegReview Institutional Review Board in Austin, Texas, and conducted at QPS Miami Research Associates (Miami Clinical Research), Miami, Florida, in accordance with the Declaration of Helsinki and GCP guidelines. All healthy subjects provided signed informed consent prior to enrollment. Eligible subjects were in good health as determined by the investigator at screening and day -1, able to understand and comply with the specific requirements of the study, had a normal body mass index (i.e., 19.0-30.0 kg / m 2 The subjects were men aged 18-55 years and non-pregnant, non-lactating women with any medical condition. Major exclusion criteria from the study included the history or presence of any disease, physical examination findings, laboratory tests, or electrocardiogram findings that, in the opinion of the sponsor and / or investigator, might interfere with the results or conduct of the study or pose a risk to healthy subjects. This included any condition that might interfere with the distribution, metabolism, or excretion of the drug.

[0233] Study Design: In this study, healthy adult subjects were screened for eligibility 2-28 days prior to dosing (Figure 20). Eligible healthy subjects were admitted to the study center on day -1 for baseline evaluation, then admitted to the inpatient ward and received a single dose of study drug on day 1. All subjects stayed in the inpatient ward for pharmacokinetic (PK) and safety evaluations until discharge on day 5. Telephone safety follow-ups for adverse events (AEs) were conducted on days 6 and 30.

[0234] This study was an open-label study enrolling a total of 30 healthy adult subjects. This study of JZP-458 used an adaptive design where the starting dose for cohort 1 was 25 mg / m2 and dose selection for cohort 2 was based on safety, tolerability, and PK data from cohort 1. A sentinel dosing approach for JZP-458 was applied to the first two subjects randomized to JZP-458 cohort 1 only and dosed in the study. These two subjects were randomized to either IM or IV infusion of JZP-458 (one for each route of administration). Dosing for the remaining subjects in the first cohorts (JZP-458 cohort 1 and Erwinaze cohort) was spaced 1 week apart from the sentinel subjects. Dosing of the remaining subjects was permitted by protocol as safety and tolerability were deemed acceptable (no grade 3 or higher study drug-related AEs) for the first two subjects by the investigator and sponsor.

[0235] The next 16 subjects were randomized to the JZP-458 and Erwinaze cohorts, 10 subjects were randomized to JZP-458 cohort 1, and 6 subjects were randomized to the Erwinaze cohort. Within JZP-458 cohort 1 and the Erwinaze cohort, subjects were randomized in a 1:1 ratio to IM or IV treatment groups. This randomization scheme equates to randomizing all 18 subjects in a 2:2:1:1 ratio to IM JZP-458, IV JZP-458, IM Erwinaze, or IV Erwinaze, with the first 2 randomized subjects receiving IM JZP-458 and IV JZP-458 following a sentinel dosing approach.

[0236] Safety, tolerability, and PK data were evaluated for all subjects in JZP-458 Cohort 1 by the investigators and sponsor to determine whether subjects should be enrolled in a separate cohort, JZP-458 Cohort 2. However, during SAP development, it was decided to conduct JZP-458 Cohort 2 and that previously collected safety, tolerability, and PK data would be evaluated to determine the dose level to be used in this cohort. In JZP-458 Cohort 2, 12 additional subjects were randomized 1:1 to either the IM or IV treatment arms.

[0237] 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 will be assessed by the occurrence of treatment-emergent adverse events (TEAEs) and clinically significant changes in vital signs and clinical laboratories. The secondary objective was to characterize the PK of a single dose (IM or IV) of JZP-458 in healthy adult subjects based on the SAA data. Additional evaluations included measurement of serum asparaginase concentration (SAC) with JZP-458, as well as L-asparagine and L-glutamine levels to evaluate the pharmacodynamic (PD) effects of JZP-458 in healthy adults.

[0238] Pharmacokinetic / Pharmacodynamic Sampling and Biochemistry Analyses: Serial blood samples for PK / PD evaluations were collected from all healthy subjects at pre-specified time points up to 96 hours post-dose. For IM dosing, samples were collected pre-dose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours post-dose. For IV dosing, samples were collected pre-dose and 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours post-infusion. Blood samples for PK analysis were collected in labeled 4 mL EDTA tubes and kept on ice until samples were centrifuged to serum. Blood samples for PD analysis were collected in 4 mL EDTA tubes and kept on ice until samples were centrifuged to plasma.

[0239] Biochemical analysis of PK samples was performed by Charles River Laboratories (Skokie, IL). PK samples were analyzed for SAA levels ranging from 0.025 IU / mL to 0.15 IU / mL using a validated enzyme activity method in human serum. In addition, PK samples were also analyzed for SAC ranging from 1.0 ng / mL to 128 ng / mL using a validated ECLIA enzyme content assay in human serum. PD samples were analyzed for L-asparagine and L-glutamine concentrations ranging from 0.025 μg / mL to 10.0 μg / mL for L-asparagine and 0.250 μg / mL to 100 μg / mL for L-glutamine using a validated liquid chromatography tandem mass spectrometry (LC / MS / MS) method by Syneos Health (Princeton, NJ).

[0240] Pharmacokinetic and statistical analysis: The PK of JZP-458 was evaluated primarily based on the SAA data. max = SAA maximum, 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 last time point for which SAA was quantifiable, AUC 0-inf = area under the SAA-time curve from time 0 to infinity, 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 The PK parameter terminal elimination half-life was assessed using noncompartmental analysis in Phoenix WinNonlin version 6.3.

[0241] Descriptive statistics (n, mean, standard deviation, median, minimum, and maximum) were used to summarize continuous data, and counts and percentages were used to summarize categorical data. 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 study drug and between and by route of administration. In linear regression modeling, SAC was the dependent variable and SAA was the independent variable. Finally, no formal hypothesis testing was performed.

[0242] Results: The highest dose tested for each route of administration (i.e., 25 mg / m for IM) 2 and 37.5 mg / m IV. 2 ), JZP-458 achieved serum asparaginase activity (SAA) levels of 0.1 IU / mL or greater at 72 hours post-dose in 100% of healthy subjects. All dose levels of JZP-458 were well tolerated with no unexpected adverse events (AEs), serious AEs, or grade ≥3 AEs. Based on the pharmacokinetic and safety data from this study, the recommended starting dose of JZP-458 for pivotal Phase 2 / 3 trials is 25 mg / m2 for IM and 37.5 mg / m2 for IV administration on a Monday / Wednesday / Friday dosing schedule. 2 It is.

[0243] Baseline Demographics: A total of 30 healthy adult subjects were enrolled and randomized in the study. Of the 30 subjects enrolled, all 30 completed the study, including the final scheduled telephone safety follow-up visit on day 30. Overall baseline demographics (mean ± SD) included mean ± SD age of 38.4 ± 8.30 years, weight of 77.04 ± 9.998 kg, and height of 1.91 ± 0.150 m. 2 16% were male, 97% were Hispanic or Latino in ethnicity, 83% were White, and 17% were Black / African American.

[0244] Pharmacokinetic Analysis: The PK analysis population consisted of all 30 healthy subjects (100%) enrolled in the study.

[0245] Serum asparaginase activity data: In this study, the starting dose of JZP-458 in cohort 1 was 25 mg / m 2 Individual SAA-time profiles were generated for all treatment groups (Figure 12). Pre-dose SAA values ​​were below the limit of quantification for all subjects and all treatments. The number and percentage of subjects with SAA levels ≥ 0.1 IU / mL and ≥ 0.4 IU / mL at 48 and 72 hours post-dose are shown in Figure 9.

[0246] The dose level for Cohort 2 was determined based on the safety, tolerability, and PK SAA data from Cohort 1. No unexpected AEs were observed in Cohort 1, no serious AEs were reported, and no grade ≥3 AEs were observed. 2 The PK SAA data for JZP-458 at 25 mg / m 2 In cohort 2, the IM dose level was 12.5 mg / m2, while in cohort 3, the SAA level was ≥ 0.1 IU / mL in 6 / 6 (100%) healthy subjects at 72 hours post-dose. 2 The dose was then reduced by 50% to 25 mg / m to study dose proportionality and the safety profile at this dose. 2With IV administration of 25 mg / m 2, SAA levels at 72 hours were 0.1 IU / mL or higher in only 4 / 6 (67%) healthy subjects. 2 The results suggest that IV administration of 37.5 mg / m IV was insufficient to maintain SAA levels at 0.1 IU / mL or higher for 72 hours. Therefore, in cohort 2, the IV dose level was reduced to 37.5 mg / m IV. 2 increased by 50%.

[0247] After IM administration of JZP-458, SAA levels were 12.5 mg / m 2 and 25 mg / m 2 At both dose levels, SAA levels reached 0.1 IU / mL or greater at 48 and 72 hours after dosing in 6 / 6 (100%) healthy subjects. After IV administration of JZP-458, SAA levels reached 0.1 IU / mL or greater at 25 mg / m 2 At the dose level of 37.5 mg / m2, 6 / 6 (100%) healthy subjects achieved >0.1 IU / mL at 48 hours and 4 / 6 (67%) healthy subjects achieved >0.1 IU / mL at 72 hours, whereas 37.5 mg / m2 2 At the dose level, SAA levels reached 0.1 IU / mL or higher in 6 / 6 (100%) healthy subjects at both 48 and 72 hours post-dose. At the same dose level, the IM route of administration was able to maintain higher levels of SAA when compared to IV. SAA data for healthy subjects receiving ERW are also shown in Figures 12 and 9.

[0248] JZP-458 at 25 mg / m 2 IM administration at 37.5 mg / m 2 Mean and 95% confidence interval (CI) curves for SAA were also generated based on the observed data for IV administration at 25 mg / m (N=6 for each, Figure 13). The data showed that the lower limit of the 95% CI was 0.01 for JZP-458 at 25 mg / m 2 IM administration at 37.5 mg / m 2 The study showed that both IM and IV doses reached ≥ 0.1 IU / mL at 72 hours after dosing (lower limits of 95% CI were 0.31107 IU / mL and 0.09476 IU / mL for IM and IV, respectively). These data helped to recommend doses for the pivotal Phase 2 / 3 study.

[0249] PK parameters were tabulated for all treatment groups based on the SAA (Figure 10). When administered IM, JZP-458 was absorbed slowly based on the SAA, with a maximum absorption rate of 12.5 mg / m 2 and 25 mg / m 2 The median tmax following administration of the 12.5 mg / m 2 and 25 mg / m 2 After administration of t 1 / 2 The mean values ​​were estimated to be 23.4 and 19.1 hours, respectively. When administered IV, the SAA levels of JZP-458 were 25 mg / m 2 and 37.5 mg / m 2 After administration of the doses, the t 1 / 2 In general, the t 1 / 2 The distribution volume was approximately the same as the plasma volume.

[0250] Assessment of dose proportionality based on the SAA showed that exposure to JZP-458 increased with increasing dose based on the SAA (Figure 17). For both IM and IV administration, exposure to JZP-458 SAA (C max The increases in AUC and AUC were approximately dose-proportional over the dose range tested. The bioavailability of JZP-458 via the IM route of administration was also calculated and estimated to be 34.5%-36.8% for JZP-458 based on the SAA data.

[0251] Serum Asparaginase Concentration Data: Historically, asparaginase PK has been determined based on SAA data. However, an enzyme content assay has been developed to measure JZP-458 asparaginase concentrations in human serum.

[0252] Individual and mean SAC-time profiles were generated for all treatment groups (Figures 14A-C). SAC-based PK parameters are summarized in Figure 18. When administered IM, JZP-458 was absorbed slowly based on the SAC, with a median tmax of 12.5 mg / m 2 and 25 mg / m 2 The time to administration was 30 hours for both doses. 1 / 2 The average value was 12.5 mg / m 2 and 25 mg / m 2 After IV administration of JZP-458, SAC levels were estimated to be 28.9 and 25.4 hours, respectively, at 25 mg / m 2 and 37.5 mg / m 2 and t of 12.0 and 12.7 hours, respectively, after administration of the doses 1 / 2 Attenuated on average.

[0253] 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 Increased exposure to JZP-458 based on AUC and AUC was approximately dose-proportional over the dose range tested. With the IM route of administration, the bioavailability of JZP-458 was estimated to be 37.7%-43.9% based on SAC data.

[0254] The relationship between SAA and SAC of JZP-458 was further investigated (Figure 14D). When evaluated by all administration routes of JZP-458, a strong positive association was observed between SAA and SAC, with a correlation coefficient of more than 0.95. In addition, the equation from the linear regression model was SAC = 1407.9 x SAA. These data suggest that when the SAA level is 0.1 IU / mL, the corresponding SAC is about 141 ng / mL in this healthy adult population.

[0255] Pharmacodynamic Data: Asparaginase hydrolyzes the amino acid asparagine to aspartic acid and ammonia. Plasma levels of asparagine were monitored throughout the treatment period. The mean SAA vs. mean plasma asparagine concentration over time is shown in Figure 15. Baseline (pre-dose) mean plasma asparagine concentrations were similar in the IM and IV groups. Individual asparagine concentrations ranged from 5.09 μg / mL to 13.8 μg / mL in all subjects. This is consistent with values ​​reported in the literature. After administration of JZP-458 (IM and IV), mean plasma asparagine levels rapidly declined from pre-dose concentrations (8.62 μg / mL and 8.96 μg / mL for IM and IV, respectively, in cohort 1 and 6.42 μg / mL and 5.89 μg / mL for IM and IV, respectively, in cohort 2) to levels below the lower limit of assay quantification (LLOQ, 0.025 μg / mL) by both routes and remained undetectable until the final sampling time point at 96 hours. The data showed that there was 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. At the highest dose of JZP-458 tested in this Phase 1 study in healthy subjects (i.e., 25 mg / m 2 and 37.5 mg / m IV. 2 ), JZP-458 not only achieved SAA levels of 0.1 IU / mL or greater at 72 hours after dosing in 100% of healthy subjects in each route, but also led to complete depletion of plasma asparagine levels by 96 hours after dosing.

[0256] In addition to asparagine, asparaginase can also hydrolyze glutamine to glutamic acid and ammonia, although with much less efficiency. Plasma levels of glutamine were monitored for all treatments (Figure 17). Baseline (pre-dose) mean plasma glutamine concentrations were similar in the IM and IV groups. Individual pre-dose glutamine concentrations ranged from 60.4 μg / mL to 146 μg / mL for all subjects. This value is also consistent with values ​​reported in the literature. The data showed that mean plasma glutamine levels rapidly declined from pre-dose concentrations of 106.5 and 74.0 μg / mL for cohort 1 and cohort 2, respectively, to levels below the assay LLOQ (0.25 μg / mL) for approximately 12 hours following IV administration of JZP-458, after which glutamine levels recovered to near pre-dose levels at the final sample collection time point 96 hours post-dose. With the IM route, mean plasma glutamine levels declined following IM administration of JZP-458, with the lowest glutamine levels observed 36 hours after administration at 25 mg / mL. 2 and 12.5 mg / m 2 There was a 79% and 47% glutamine decrease at 0.5 mg / kg / day and then glutamine levels recovered to similar levels as before dosing at the final sampling time 96 hours after dosing. Complete depletion of L-glutamine was not observed. Glutamine levels were only moderately affected and partially decreased, and the data were more variable than those observed with L-asparagine.

[0257] Safety and Tolerability: The safety profile observed for JZP-458 in this Phase 1 study was consistent with that of other asparaginases. JZP-458 at all dose levels was well tolerated with no unexpected, serious, or grade ≥3 AEs. The most common TEAE occurring in ≥2 healthy subjects in each dosing cohort was nausea (Figure 11).

[0258] Discussion: JZP-458, a recombinant Erwinia asparaginase that is expected to have no immune cross-reactivity to E. coli-derived asparaginase, is being developed to provide access to asparaginase therapy to ALL or LBL patients who have developed hypersensitivity to E. coli-derived asparaginase. In this randomized, single-center, open-label Phase 1 study, JZP-458 was administered at the highest dose tested for each route of administration (i.e., 25 mg / m for IM) and 10 mg / mL for IM. 2 and 37.5 mg / m IV. 2 ) 72 hours after administration, SAA levels of 0.1 IU / mL or greater were achieved in 100% of healthy adult subjects in each route. The SAA levels observed in this study also indicated that JZP-458 could 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 with both IM and IV administration routes. In addition, the safety profile of JZP-458 in this study was consistent with that of other asparaginases. JZP-458 at all dose levels was well tolerated, with no unexpected AEs, no serious AEs reported, and no grade 3 or higher AEs.

[0259] Based on accumulated PK and safety data, the recommended starting dose of JZP-458 in the central Phase 2 / 3 study is 25 mg / m IM on a Monday / Wednesday / Friday dosing schedule. 2 and 37.5 mg / m for the IV route of administration. 2 In this Phase 1 study in healthy adult subjects, these doses achieved SAA levels of 0.1 IU / mL or greater at 72 hours post-dose in 6 / 6 (100%) healthy subjects, and are expected to maintain SAA levels of 0.1 IU / mL or greater throughout the treatment period in pivotal Phase 2 / 3 studies.

[0260] Completing asparaginase therapy is important for improving patient outcomes, as shown in previous studies. In the Dana-Farber Cancer Institute ALL Consortium Protocol 91-01 trial, patients with asparaginase intolerance, defined as completion of 25 weeks or less of a total planned 30 weeks of asparaginase therapy, had a significantly lower 5-year EFS when compared with 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 high-risk and standard-risk ALL patients with late early responses who did not finish the prescribed asparaginase dose had a significantly inferior EFS compared with patients who received all prescribed asparaginase doses. Of note, patients with hypersensitivity reactions who completed a course of treatment with Erwinia asparaginase substitutes showed similar EFS to patients who completed a course of first-line asparaginase therapy. These studies suggest that patients who complete their prescribed dose of asparaginase, whether first- or second-line asparaginase, have better outcomes than patients who discontinue early. These results highlight the need for alternative asparaginase formulations to allow patients who develop hypersensitivity to E. coli-derived asparaginase to complete their full course of treatment.

[0261] Conclusion: In this Phase 1 study, JZP-458 was administered at the highest dose tested for each route of administration (i.e., 25 mg / m 2 and 37.5 mg / m IV. 2 ) achieved SAA levels of 0.1 IU / mL or greater in 100% of healthy adult subjects by each route, resulting in complete asparagine depletion without unexpected AEs, SAEs, or grade ≥3 AEs. Based on the PK and safety data accumulated from this study, the recommended starting dose of JZP-458 in Phase 2 / 3 is 25 mg / m2 for the IM route on a Monday / Wednesday / Friday dosing schedule. 2and 37.5 mg / m for the IV route of administration. 2 JZP-458 may be used as an alternative therapy for ALL / LBL patients who develop hypersensitivity to E. coli-derived asparaginase.

[0262] Example 8 Phase 2 / 3 Study of JZP-458 in ALL / LBL Patients with Hypersensitivity to E. coli-derived Asparaginase (Cohorts 1a and 1b) An open-label, multicenter, dose-finding, PK study of JZP-458 in participants (of any age) with ALL / LBL who demonstrate hypersensitivity (allergic reaction or asymptomatic inactivation) to E. coli-derived asparaginase was designed. The study was designed to evaluate the tolerability and efficacy of JZP-458, as measured by serum asparaginase activity, with additional supportive analyses of asparagine depletion and anti-drug antibody (ADA) levels. Six doses of JZP-458 will be substituted for each dose of E. coli-derived long-acting asparaginase. Two consecutive weeks of treatment with JZP-458 are defined as one course.

[0263] Study Design: This study consists of two parts, Part A to determine the dose of JZP-458 administered IM and to confirm safety and efficacy, and Part B to define the optimal dose and schedule of IV infusion of JZP-458 (see Figures 22 and 23). Parts A and B may be studied in parallel. Part A (IM) studies multiple cohorts, where in cohort 1 the optimal dose will be determined and in cohort 2 the number of participants treated with the optimal dose will be increased. Part B (IV) mirrors Part A, and in both parts, courses of JZP-458 (6 doses over 2 weeks) may be initiated on either Monday, Wednesday, or Friday, with the number of courses per participant depending on their individual treatment plan. In each part, courses of JZP-458 (6 doses over 2 weeks) may be initiated on either Monday, Wednesday, or Friday, with the number of courses per participant depending on their individual treatment plan. The starting dose was 25 mg / m via IM route (cohort 1a). 2The starting dose was selected based on the results of a phase 1 trial.

[0264] OBJECTIVES: The primary objectives of the study were to determine the efficacy of IM JZP-458 as measured by response (defined as a 72-hour NSAA level of ≥0.1 IU / mL in the first cycle) in cohorts 1 and 2, and to evaluate the safety and tolerability of IM JZP-458 in participants with ALL / LBL who exhibit hypersensitivity to E. coli-derived asparaginase. Additional objectives included the following:

[0265] Primary secondary objective: To determine the efficacy of IM administration of JZP-458 as measured by response (defined as a last 48-hour NSAA level of 0.1 IU / mL or greater in the first cycle) in cohort 1 and cohort 2.

[0266] Secondary objectives: 1) to determine the efficacy of IM administration of JZP-458 as measured by response (defined as last 48 hour and last 72 hour NSAA levels ≥ 0.4 IU / mL in the first cycle) in cohort 1 and cohort 2; 2) to characterize the PK of IM administered JZP-458 using a PPK approach and to explore ER correlations using data from samples from all participants and all time points at all dose levels; and 3) to evaluate the immunogenicity of IM administered JZP-458 following repeated dosing of JZP-458.

[0267] Exploratory Objectives (Part B of the Study): 1) to determine the efficacy of IV JZP-458 administered as measured by response defined as NSAA ≥ 0.1 IU / mL in the last 48 hours and NSAA ≥ 0.1 IU / mL in the last 72 hours during the first cycle; 2) to determine the efficacy of IV JZP-458 administered as measured by response defined as NSAA ≥ 0.4 IU / mL in the last 48 hours and NSAA ≥ 0.4 IU / mL in the last 72 hours during the first cycle; 3) to evaluate the safety and tolerability of IV JZP-458 in participants with ALL / LBL who demonstrate hypersensitivity to E. coli-derived asparaginase; 4) to characterize the PK of IV JZP-458 using a population PK approach; and 5) to evaluate the immunogenicity of IV JZP-458 following repeated dosing of JZP-458.

[0268] Preliminary Results: As of October 7, 2020, 31 participants had been enrolled in cohort 1a and 56 participants in cohort 1b of the pivotal Phase 2 / 3 study (JZP458-201). The first protocol-defined SDRC evaluation of cohort 1a included data from 6 evaluable participants, and the second SDRC evaluation of cohort 1a included cumulative data from 16 evaluable participants. Each SDRC recommendation was based on a review of all data available at the time. Following evaluation of the available efficacy data, 2 No unexpected safety events were observed at the IM dose level of 37.5 mg / m 2Evaluation of the IM dose level of 100 mg / kg / day has begun (Cohort 1b). The first participant in Cohort 1b was enrolled on June 29, 2020. SDRC evaluation of Cohort 1b data was performed on September 1, 2020 as a preliminary review of data up to the first 7 participants enrolled in Cohort 1b. This review included all available SAA data, PPK modeling and simulation data, and safety data from the study. The preliminary results supported continuing enrollment without change and subsequently reviewing the study again when data are available for at least 13 evaluable Cohort 1b participants, per SDRC policy. Available preliminary data for Cohorts 1a (N=31) and 1b (N=17) are summarized below.

[0269] Serum asparaginase activity levels: 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 in human serum and 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.

[0270] 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 second half of the first cycle. Of the 31 participants enrolled at 25 mg / m2, 26 participants were considered evaluable for the primary efficacy objective in Cohort 1 per protocol. Reasons for participants being considered non-evaluable were as follows: 1) 2 participants had 72-hour PK samples collected outside the defined sampling window; 2) 1 participant did not have a 72-hour PK sample collected; 3) 1 participant withdrew informed consent and a PK sample was not collected; and 4) 1 participant had an SAA value (assessed by bioanalytical laboratory Charles River Laboratory) that was not evaluable due to interference with the assay from elevated lipids. This participant had Grade 2 hypertriglyceridemia (318 mg / dL) at baseline and Grade 4 hypertriglyceridemia (2679 mg / dL) during the first course (reported as a TEAE, see Safety Summary below for more details). This participant's triglycerides continued to decrease to baseline or below baseline with subsequent courses of JZP-458, and most recently, the participant completed the fifth course with triglyceride levels decreasing to 234 mg / dL (Grade 1).

[0271] Of the 17 participants included in the exploratory analysis of Cohort 1b (37.5 mg / m2), 16 participants were evaluable for the primary efficacy objectives in Cohort 1 (one participant had a 72-hour PK sample taken outside the defined collection window and was therefore excluded for the primary endpoint but included for the other endpoints). Individual preliminary SAA results at key time points are summarized in Figure 24. Preliminary SAA results observed were: 2Results show that at the IM dose level of JZP-458 (N=26 evaluable participants), mean (95% CI) SAA levels 72 and 48 hours after the most recent dose were 0.1560 (0.1144-0.1976) and 0.4504 (0.3637-0.5370), respectively (median SAA levels [1st quartile, Q1; 3rd quartile, Q3] 72 and 48 hours after the most recent dose were 0.1345 [0.0886, 0.2178] and 0.4091 [0.2813, 0.6577], respectively). 2 At the IM dose level of JZP-458 (N=16 evaluable participants), mean SAA levels (95% CI) 72 and 48 hours after the most recent dose were 0.2605 (0.1326-0.3884) and 0.7146 (0.3976-1.0316), respectively (median SAA levels [Q1, Q3] 72 and 48 hours after the most recent dose were 0.1732 [0.1157, 0.2849] and 0.6503 [0.3248, 0.8736], respectively).

[0272] The percentage of participants with NSAA levels ≥0.1 IU / mL and ≥0.4 IU / mL 72 and 48 hours after most recent dose are shown in Figure 25. 2 (N = 26 evaluable participants) and 37.5 mg / m 2 At the IM dose level of JZP-458 (N=16 evaluable participants), 65.4% and 81.3% of participants, respectively, achieved NSAA levels of 0.1 IU / mL or greater at the last 72-hour assessment (primary endpoint), and 96.2% and 93.8% of participants, respectively, achieved NSAA levels of 0.1 IU / mL or greater at the last 48-hour assessment (key secondary endpoint).

[0273] Population Pharmacokinetic Modeling and Simulation: An exploratory PPK analysis (secondary endpoint) was performed using the SAA data (data received on September 28, 2020). Forty-seven participants in the JZP458-201 study who received JZP-458 IM (25 mg / m 2 Thirty participants and 37.5 mg / m 2A total of 319 quantifiable SAA data points (1st cycle only) from 17 participants (17 participants) were included in the development of the PPK model. The model was fitted to the SAA data to identify a 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.

[0274] The covariate models developed for JZP-458 were a one-compartment IM-only model with linear elimination and first-order absorption and a proportional residual error model, with body weight included as a nonproportional covariate for JZP-458 SAA clearance and volume. Model diagnostics showed good fitting based on predicted versus observed data and predictive modeling methods.

[0275] To examine the likelihood of achieving therapeutic NSAA levels of 0.1 IU / mL or greater, a covariate model was used to simulate patient SAA profiles (N=2000 subjects per dose level). A simulated hypothetical population was created by re-extracting subjects with replacement from demographic data from the Centers for Disease Control's National Health and Nutrition Examination Survey Data. The simulated population ranged in age from 2 to 85 years and in weight from 8.9 to 174.6 kg (median 62.7 kg).

[0276] Both Friday, Monday, Wednesday (FMW) and MWF dosing schedules were simulated with six doses per treatment course. Here, the FMW schedule was the focus of attention since it was the most conservative scenario with NSAAs in the first 72 hours after only one dose. 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 last 48 or 72 hours. The simulated profiles for the FMW dosing schedule are shown in Figure 26 along with the median and 95% prediction interval. 25-80 mg / m 2 A summary table of the simulated percentage of patients achieving NSAA ≥ 0.1 IU / mL and predicted mean SAA levels at IM doses ranging from 0.1 IU / mL to 0.2 IU / mL is shown in FIG.

[0277] Based on PPK modeling and simulation, 37.5 mg / m on the FMW schedule 2 With IM administration of JZP-458 at 50 mg / m, JZP-458 is predicted to achieve 72-hour NSAA levels of ≥0.1 IU / mL in 87% of patients (95% CI: 85.5%-88.5%) and 48-hour NSAA levels of ≥0.1 IU / mL 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) in the last 72 and 48 hours, respectively. PPK modeling suggested that at 50 mg / m 2 At this dose, 94.2% of patients would be predicted to achieve NSAA levels of 0.1 IU / mL or greater 72 hours after administration.

[0278] Preliminary results of asparagine depletion: Pharmacodynamic study samples were analyzed for asparagine concentrations in the range of 0.025-10.0 μg / mL by Syneos Health (Princeton, NJ) using a validated liquid chromatography tandem mass spectrometry method (Biomarker Partial Validation Report Amendment 1, SN0044). To ensure analytical measurements represent in vivo asparagine levels, blood samples for asparagine assays were placed in an ice bath immediately after collection and processed plasma was stored at -80°C for no more than 30 minutes. Asparagine levels were measured to support efficacy of JZP-458 during the dosing period. Baseline asparagine levels were measured in cohort 1a (25 mg / m 2 ) in 30 participants treated with cohort 1b (37.5 mg / m 2 In 17 participants treated with 10.2282 μg / mL (95% CI: 7.1852-13.2712), plasma asparagine was 10.2282 μg / mL (95% CI: 7.1852-13.2712). These values ​​are consistent with those reported in the literature (Tong 2014). In most participants, complete depletion of plasma asparagine was observed in the majority of samples, i.e., plasma asparagine levels rapidly decreased from pre-dose levels to levels below or near the lower limit of assay quantification (0.025 μg / mL) and persisted throughout the first course of treatment until before the sixth dose, when the final sample was taken. Four participants showed a transient low-level increase in asparagine (three in cohort 1a and one in cohort 1b).

[0279] Preliminary safety results: In cohort 1a, a total of 31 participants received 25 mg / m 2 In cohort 1b, data showed that patients received 37.5 mg / m 2 of JZP-458 was available in 17 participants who received IM.

[0280] A summary of TEAEs in treatment cohorts is shown in Figure 28. The majority of participants experienced at least one TEAE. JZP-458 IM 25 mg / m2 In this 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). 2 In this 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).

[0281] 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 grade 3 or 4 TEAEs being decreased neutrophil count (29.0%), decreased white blood cell count (16.1%), and febrile neutropenia (19.4%) (Table 6). Overall, grade 3 or higher treatment-emergent unexpected clinically significant non-hematological toxicity was reported in one participant (grade 4 hypertriglyceridemia deemed related to the study drug) (Table 6 and Table 1, Cohort 1a). JZP-458 IM 25 mg / m 2 In the cohort, serious TEAEs were reported in 9 participants (29.0%) (Table 5). Serious TEAEs included near syncope in 1 participant, rhinorrhea and febrile neutropenia in 1 participant, febrile neutropenia, dehydration, and fever in 1 participant, drug hypersensitivity and febrile neutropenia in 1 participant, febrile neutropenia, febrile neutropenia and stomatitis in 1 participant, worsening oral mucositis in 1 participant, pyrexia in 1 participant, and methemoglobinemia in 1 participant. All these serious TEAEs were not considered related to the study drug, except for the drug hypersensitivity and febrile neutropenia events in 1 participant.

[0282] Overall, one participant (JZP-458 25 mg / m 2) experienced a TEAE that led to discontinuation of the study drug. This participant (age 4) had a grade 2 allergic reaction (non-serious) to cefepime and sulfamethoxazole / trimethoprim on the day of the fifth dose of the first cycle.

[0283] Both of these medications were discontinued. Subsequently, 1 day after the third dose of the second cycle of JZP-458, the participant developed a serious TEAE (grade 3) of drug hypersensitivity. The event resolved the next day, and the participant was discharged. The event was considered related to the study drug and led to discontinuation of the study drug (Table 1, cohort 1a). JZP-458 IM 37.5 mg / m 2 In the cohort (N=17), 6 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). JZP-458 IM 37.5 mg / m 2 Three participants in the cohort experienced serious TEAEs (vomiting and abdominal pain [unrelated to study drug] in one participant, drug hypersensitivity [reporter's term: allergic reaction to JZP-458; study drug related] and febrile neutropenia [study drug related] in one participant, and febrile neutropenia [study drug related; 2 events] in one participant) (Figure 28). No grade 3 or higher treatment-emergent unexpected clinically significant non-hematologic toxic events were reported in these participants, and no TEAEs led to study discontinuation (Figure 29, cohort 1b).

[0284] Adverse events of interest for 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 time of data cutoff for this preliminary safety analysis, only one participant (cohort 1a) had a grade 3 or higher allergic reaction / hypersensitivity (as described above), and no participants had grade 3 or higher pancreatitis or thrombosis events. No deaths were reported.

[0285] In summary, the observed TEAE profile was consistent with other asparaginases and was consistent with both dose levels of JZP-458 (25 and 37.5 mg / m 2 ) was well tolerated in participants.

[0286] Based on a detailed review of available test data by experts both within and outside the SDRC, 2 "The IM dose of JZP-458 has a favorable benefit-risk profile and is an appropriate dose for regulatory submission of JZP-458. This dose would ensure that patients complete their asparaginase treatment regimen and is consistent with FDA guidelines regarding benefit-risk and minimum effective dosing."

[0287] Safety: Further safety studies are available for safety evaluation in cohort 1a (25 mg / m 2 ) and 31 patients in cohort 1b (37.5 mg / m 2 The study will involve a minimum of 82 participants, including 51 participants at 25 mg / m 2 31 participants and 37.5 mg / m 2 Preliminary data from a randomized controlled trial (17 participants) showed that 2 The study demonstrated that the proposed dose of 25 mg / m was safe and well tolerated. A preliminary safety analysis showed that 1 participant (cohort 1a, 25 mg / m 2Only one participant in the study had a grade 3 or higher allergic reaction / hypersensitivity event, and no participant had a grade 3 or higher pancreatitis or thrombotic event. A grade 3 serious adverse event (SAE) of drug hypersensitivity (related to the study drug) led to discontinuation of the study drug. No other participants were discontinuing the study due to TEAEs.

[0288] Efficacy Data: Criteria for early efficacy assessment were based on a target SAA value of 0.1 IU / mL, as well as confirmation of asparagine depletion and consideration of clinically relevant response rates in patients with previous exposure to asparaginase. Preliminary SAA results observed were 2 Results show that at the IM dose level of JZP-458 (N=26 evaluable participants), mean SAA levels (95% CI) 72 and 48 hours after the most recent dose were 0.1560 (0.1144 to 0.1976) and 0.4504 (0.3637 to 0.5370), respectively (median SAA levels [Q1, Q3] 72 and 48 hours after the most recent dose were 0.1345 [0.0886, 0.2178] and 0.4091 [0.2813, 0.6577], respectively). 2 At the IM dose level of JZP-458 (N=16 evaluable participants), mean SAA levels (95% CI) 72 and 48 hours after the most recent dose were 0.2605 (0.1326-0.3884) and 0.7146 (0.3976-1.0316), respectively (median SAA levels [Q1, Q3] 72 and 48 hours after the most recent dose were 0.1732 [0.1157, 0.2849] and 0.6503 [0.3248, 0.8736], respectively).

[0289] 25 and 37.5 mg / m 2 At the IM dose level of JZP-458 of 37.5 mg / m, 65.4% and 81.3% of participants, respectively, achieved NSAA levels of 0.1 IU / mL or greater at the last 72-hour assessment (primary endpoint), and 96.2% and 93.8% of participants, respectively, achieved NSAA levels of 0.1 IU / mL or greater at the last 48-hour assessment (key secondary endpoint). 2Although the percentage of participants achieving NSAA levels ≥ 0.1 IU / mL at the most recent 72-hour assessment (N=16) did not meet the primary endpoint criteria (defined in the statistical analysis plan [SAP]), the results demonstrate a clinically relevant response rate consistent with published observed or predicted rates (80%-88%) for patients receiving non-E. coli-based asparaginase for treatment after a hypersensitivity reaction (Vrooman 2010; Panetta 2020; Salzer 2013). 2 In (N=16), the percentage of participants who achieved NSAA levels ≥0.1 IU / mL (key secondary endpoint) at the last 48-hour assessment was 93.8%. This percentage is expected to meet the predefined success criteria outlined in the SAP for the IA (N=51 participants planned).

[0290] The efficacy of JZP-458 is further confirmed in preliminary PPK results. Preliminary PPK was consistent with observed data and was consistent with the proposed dose of 37.5 mg / m 2 The dose selection of 37.5 mg / m on the FMW schedule was further supported based on PPK modeling and simulation (secondary endpoints). 2 With IM administration of JZP-458, JZP-458 is predicted to achieve NSAA levels of ≥0.1 IU / mL in the last 72 hours in 87% of patients (95% CI: 85.5%-88.5%) and NSAA levels of ≥0.1 IU / mL in the last 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) in the last 72 and 48 hours, respectively. Simulation results showed that the start date did not affect the percentage of patients with NSAA levels of ≥0.1 IU / mL in the last 72 or 48 hours.

[0291] The PPK analysis robustly supports the observed data and provides a complete data package for the BLA as the model uses available data for all patients, time points, and dose levels. The results of the PPK model can represent the exposure-response characteristics of JZP-458 based on all available clinical and PK data. The model does not rely on categorized data and therefore provides reliable predictions of NSAA levels at relevant time points. The model demonstrates that the percentage of patients with NSAA levels ≥ 0.1 IU / mL 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 was 37.5 mg / m 2 These results suggest that IM administration of is an appropriate dose for the MWF dosing schedule.

[0292] JZP-458 at 37.5 mg / m 2 As a further measure of dose validation, the goal of complete asparagine depletion, as measured by a validated method (Biomarker Partial Validation Report Amendment 1, SN0044), was achieved at doses of 25 and 37.5 mg / m 2 Comparison of baseline asparagine levels versus asparagine levels measured at trough throughout the dosing period at 10 mg consistently demonstrated near-complete depletion of asparagine. Deviations from this pattern were small and transient. The effect of potential doses, toxicity studies, number of injections, number of vials, and other factors were evaluated. Such studies balanced the potential effect of higher doses versus increased risk of more severe adverse events.

[0293] Example 9 Comprehensive Clinical Data Package for JZP-458: Observed Efficacy, PD, Safety, and Immunogenicity Data for Cohorts 1a and 1b The following is a 25 / 25 / 50 mg / m dose given IM to MWF: 2 These are four key elements of data supporting the efficacy and safety of JZP-458:

[0294] 1. Observed efficacy data from cohorts 1a and 1b: 25 mg / m administered IM to MWF / WFM / FMW 2 For JZP-458 at 25 mg / m2 (N=29) and 37.5 mg / m2 (N=51), the percentage of participants achieving NSAA levels ≥0.1 IU / mL at the most recent 72-hour assessment during cycle 1 was 65.5% (95% CI: 48.2%, 82.8%) and 80.4% (95% CI: 69.5%, 91.3%), respectively (Initial Analyses Table 9.2.1.1). 2 (N=32) and 37.5 mg / m 2 For JZP-458 (N=53), the percentage of participants achieving NSAA levels ≥0.1 IU / mL at the most recent 48-hour assessment during cycle 1 was 96.9% (95%CI: 90.8%, 100%) and 98.1% (95%CI: 94.5%, 100.0%), respectively. The lower limit of the 95%CI was >90%, supporting the efficacy of 25 mg / m2 and 37.5 mg / m2 JZP-458 administered every 48 hours.

[0295] 2. PD data from cohorts 1a (N=32) and 1b (N=53): 25 and 37.5 mg / m 2 Administration of an IM dose of JZP-458 completely depleted plasma asparagine levels. Plasma asparagine levels rapidly declined from pre-dose levels to levels below or near the lower limit of assay quantification (0.025 μg / mL) and remained so throughout the first course of treatment until the sixth dose, when the final sample was taken. Four participants had a transient, low-level increase in plasma asparagine (three in cohort 1a and one in cohort 1b).

[0296] 3. Safety Data from Cohorts 1a and 1b (by cohort and overall): The overall safety profile of JZP-458 in ALL / LBL participants based on the combined safety data from Cohorts 1a and 1b (N=86) demonstrated that JZP-458 was well tolerated and that the safety profile of JZP-458 was predictable and consistent with the established safety profile of asparaginase.

[0297] 4. Adverse Events: Overall, 96.5% (83 / 86) of participants experienced one or more treatment-emergent adverse events (TEAEs) during the study (Figure 31). Overall, the most frequent TEAEs were anemia (37.2% [32 / 86] of participants), decreased neutrophil count (34.9% [30 / 86] of participants), and decreased platelet count (29.1% [25 / 86] of participants). The most frequent treatment-related TEAEs were decreased neutrophil count (18.6% [16 / 86] of participants), anemia (15.1% [13 / 86] of participants), and nausea (15.1% [13 / 86] of participants).

[0298] Example 10: Safety Study of Cohort 1c Cohort 1c (25 / 25 / 50 mg / m administered IM to MWF) 2 Preliminary safety (TEAE) data indicate that this dose and regimen of JZP-458 was well tolerated and consistent with the well-established safety profile of asparaginase products. Preliminary data indicate that in cohort 1c, 81.3% (13 / 16) of participants experienced one or more TEAEs (Figure 32). Overall, the most frequent TEAEs were anemia (25.0% [4 / 16] of participants), decreased platelet count (25.0% [4 / 16] of participants), and decreased lymphocyte count, decreased neutrophil count, and decreased white blood cell count (18.8% [3 / 16] of participants, respectively).

[0299] Preliminary data also indicate that no participants died in Cohort 1c. Four of 16 participants (25.0%) experienced an SAE. One participant experienced a treatment-related SAE of acute pancreatitis (grade 3) that led to discontinuation of study drug. This event began on study day 13 and resolved by study day 24. Preliminary data indicate that no other participants in Cohort 1c discontinued study drug due to TEAEs. Additionally, preliminary data from Cohort 1c (N=16) indicated that no TEAEs of allergic reactions (including hypersensitivity and anaphylaxis) or thrombosis occurred.

[0300] As of the data cutoff date, 2 of 16 participants (12.5%) in cohort 1c developed a TEAE of pancreatitis, both of which were assessed by the investigator as related to JZP-458. The first participant developed grade 2 pancreatitis 4 days after the sixth dose of the first cycle. This participant had grade 2 epigastric pain at baseline, no other symptoms, and an increase in lipase of 871 U / L (reference range 73-393 U / L). The amylase was reported by the investigator to be within normal limits (value not provided). The investigator reported that symptoms subsided without treatment, and lipase decreased to 300 U / L 8 days later. The investigator assessed the event to be grade 2 pancreatitis and related to JZP-458. No action was taken regarding JZP-458, and the participant began a second course of JZP-458 14 days after the onset of this event without further issues. Laboratory results at the start of the second course were lipase 218 U / L and amylase 64 (reference range 25-115 U / L).

[0301] The second participant developed grade 3 pancreatitis 1 day after completing the sixth dose of the first cycle. The participant presented to the emergency room with moderate abdominal pain (8 / 10) radiating to the left upper, middle, and lower quadrants. An IV injection of morphine was administered, which reduced pain to 7 / 10. Relevant laboratory results included lipase 549 U / L (reference range 73-393) and amylase 47 U / L (range 25-115 U / L). An x-ray revealed moderate constipation. COVID testing and urine culture were negative. The participant was admitted to the hospital for workup and treatment of pancreatitis. The event resolved approximately 10 days later, and the lipase result returned to range that same day (lipase 355 U / L). The investigator assessed the event to be related to JZP-458, and the participant discontinued treatment on study day 13 due to grade 3 pancreatitis.

[0302] A preliminary review of the clinical database was conducted for reports of pancreatitis in JZP458-201. Of the 161 currently enrolled participants, including 44 participants from cohort 1c, there were seven cases of pancreatitis reported across the cohorts. In addition to the two cohort 1c participants, five cases were reported in participants enrolled in cohort 1b. Of the seven reports, six were considered severe and one was non-severe. As noted above, two of the six severe reports of pancreatitis occurred in cohort 1b participants, who subsequently died (one participant developed sepsis and died, and the other developed aspiration pneumonia secondary to an esophageal tracheal fistula and died). The overall incidence of pancreatitis is within the range expected with asparaginase.

[0303] Summary: In this study, JZP458-201, three dose regimens were evaluated as having a favorable benefit-risk profile. Overall the data showed that 25 / 25 / 50 mg / m 2 administered IM in six separate doses on a MWF schedule to replace one course of long-acting E. coli asparaginase was not associated with a favorable benefit-risk profile. 2 The safety profile of JZP-458 (cohorts 1a, 1b and preliminary data from cohort 1c) is comparable to the established safety profile of asparaginase products.

[0304] Example 11 60-day Update: Efficacy PK and PD Data from Cohorts 1a, 1b, and 1c Available Efficacy, PK (SAA), and PD Data from Cohorts 1a (N=32) and 1b (N=53): Available data included data collected for Cohorts 1a (N=33) and 1b (N=53) through the data cutoff date and analyzed by the bioanalytical laboratory prior to the data extraction date.

[0305] Efficacy, PK (SAA), and PD data from cohort 1c (N=16): As of the data cutoff date, a total of 17 participants (including 1 untreated participant) had been enrolled in cohort 1c (25 / 25 / 50 mg / m administered IM to MWF). 2 For the 16 participants who received at least one dose of study drug, the median (range) duration of study was 4.75 (1.9-7.1) weeks, of which 2 participants had <2 weeks, 3 participants had 2-<4 weeks, 9 participants had 4-<6 weeks, and 2 participants had 6-<8 weeks. Of the 16 participants, 7 received 1 course of treatment and 9 received 2 courses of treatment.

[0306] Based on preliminary results, 25 / 25 / 50 mg / m administered IM to MWF 2 In JZP-458 (N=13), the percentage of participants who achieved NSAA levels of 0.1 IU / mL or greater at the most recent 72- and 48-hour assessments during the first cycle was 92.3% (12 / 13 participants) and 92.3% (12 / 13 participants), respectively. These results were consistent with the 25 / 25 / 50 mg / m IM administered MWF schedule. 2Supporting the efficacy of JZP-458 in patients with rheumatoid arthritis. One participant (age 12-<18 years) who did not achieve NSAA levels ≥0.1 IU / mL at the most recent 72- and 48-hour assessments in cycle 1 had a grade 3 allergic reaction to Oncaspar and received approximately 5% of the infusions when symptoms began. JZP-458 was first administered 7 days later. The participant's laboratory values ​​(including hemoglobin, bilirubin, triglycerides, and cholesterol) at screening and day 1 were not elevated. Two AEs have been reported to date for this participant: a sore throat (unrelated) and a mild (grade 1) injection site reaction (related to JZP-458) that occurred after the last dose (6th dose) of cycle 1. During the first cycle, this participant had NSAA levels >0.1 IU / mL (2.5 and 48 hours after first dose and 48 hours after second dose), which decreased to 0.0453 IU / mL 72 hours after the third dose (last 72-hour assessment), 0.1369 IU / mL 2.5 hours after the fourth dose, BLQ 48 hours after the fourth dose, and 0.0510 IU / mL 48 hours after the fifth dose (last 48-hour assessment). ADA data for this participant are pending.

[0307] Example 12: A 2 / 3 study of JZP-458 in ALL / LBL patients with hypersensitivity to E. coli-derived asparaginase This is an open-label, multicenter, dose-finding, PK study of JZP-458 in participants (of any age) with ALL / LBL who demonstrate hypersensitivity to E. coli-derived asparaginase (allergic reaction or asymptomatic inactivation). The study was designed to evaluate the tolerability and efficacy of JZP-458 as measured by SAA, with additional supportive analyses of asparagine depletion and ADA levels. Six doses of JZP-458 will be substituted for each dose of long-acting E. coli-derived asparaginase. Two consecutive weeks of treatment with JZP-458 are defined as one course. Courses of JZP-458 (six doses over two weeks) will begin on either Monday, Wednesday, or Friday, with the number of courses per participant determined by individual treatment plan.

[0308] The study consists of two parts: Part A, which will determine the dose of JZP-458 and confirm its safety and efficacy for IM administration, and Part B, which will identify the optimal dose and schedule for IV administration of JZP-458.

[0309] Part A: Cohort 1a received 25 mg / m MWF schedule over 2 weeks. 2 Data from cohort 1a showed that six evaluable participants received 25 mg / m IM on the MWF schedule. 2 The safety profile of Jazz at the 25 mg / m2 IM dose level was evaluated after participants enrolled in 37.5 mg / m2 IM on a MWF schedule and was evaluated again with cumulative data from 16 evaluable participants. Jazz was evaluated at the 37.5 mg / m2 IM dose level on a MWF schedule with an acceptable safety profile at the 25 mg / m2 IM dose level, as the percentage of participants with NSAA levels after the last 72 hours of dosing were below the protocol-specified threshold. 2 The decision was made to escalate to an IM dose of 10 mg / kg / day (cohort 1b).

[0310] Safety data, SAA data, PPK modeling, and simulation results were evaluated after the first 7 participants were enrolled in cohort 1b, and cumulatively again after 17 evaluable participants were enrolled in cohort 1b. Jazz was then administered at 37.5 mg / m2 on the MWF schedule without modification in cohort 1b. 2 It was decided to continue enrollment with treatment.

[0311] Based on PPK modeling and discussions with the FDA, cohort 1c will receive 25 mg / m 2 Dose and 50 mg / m administered on Friday 2 A preliminary PPK model using participant data from the first course of cohorts 1a (N=30) and 1b (N=32) suggested that the optimal dose to support the MWF dosing schedule was 25 / 25 / 50 mg / m administered in MWF for a total of 6 doses to replace each dose of long-acting E. coli asparaginase. 2As of July 19, 2021, a total of 51 participants had been enrolled in cohort 1c.

[0312] Part B Part B Cohort 1a (IV dose confirmation): Participants in this cohort will receive 6 doses of JZP458 IV. At least 6 evaluable participants were enrolled as a target. Evaluable participants are defined as those who received at least 3 doses of JZP458 IV and have 72-hour NSAA levels (obtained within ±2 hours) during the second half of the first cycle. Blood samples were collected for SAA level measurement and other PK / PD laboratory evaluations. Additional courses of IV JZP458 will be administered based on the participant's original treatment plan as long as the participant is achieving clinical benefit. Subsequent doses of long-acting E. coli-derived asparaginase scheduled in the participant's treatment plan that were not administered will be substituted with 6 doses of IV JZP458 equivalent to one cycle until the participant's asparaginase is completed.

[0313] After all required participants in one cohort have been enrolled and treated with six doses of JZP458, dose levels will be assessed and the need for additional participants / cohorts will be determined by the SDRC.

[0314] Part B (JZP458 IV Decision for Possible Additional Cohorts): If 6 of 6 evaluable participants in the initial IV dosing cohort have 72-hour NSAA levels ≥ 0.1 IU / mL and safety and tolerability are acceptable based on review by the SDRC, then the dose may be confirmed as the dose for further evaluation. If the dose cannot be confirmed, additional dosing cohorts may be tested as described in (Section 8.1 / Protocol Amendment 02 / Section 3.1.3).

[0315] This summary presents results from the secondary analysis population with a data cutoff date of July 19, 2021, which consisted of Part A (Cohort 1a [25 mg / m 2, N = 33], cohort 1b [37.5 mg / m administered IM on a MWF schedule 2 , N = 83], and cohort 1c [25 mg / m administered IM on Mondays and Wednesdays 2 and 50 mg / m2 administered IM on Fridays, N=51]), and Part B (25 mg / m2 administered IV on Mondays and Wednesdays, N=52). 2 and 50 mg / m IV on Friday. 2 , N=62).

[0316] The primary objectives of the study are to determine the efficacy of IM JZP-458 as measured by response (defined as a 72-hour NSAA level of ≥0.1 IU / mL in the first cycle) in cohort 1 and cohort 2 and to evaluate the safety and tolerability of IM JZP-458 in participants with ALL / LBL who demonstrate hypersensitivity to E. coli-derived asparaginase.

[0317] As of the data cutoff date of July 19, 2021, a total of 228 participants (Part A: 33 in cohort 1a [25 mg / m administered IM to MWF] 2 ], and 83 in cohort 1b [37.5 mg / m IM administered to MWF 2 ], and 51 in cohort 1c [25(MW) / 50(F)mg / m 2 Part B: 62 patients in cohort 1a [25(MW) / 50(F)mg / m administered IV 2 ]) were enrolled in the study. As of July 19, 2021, 28 (16.8%) participants in Part A and 39 (62.9%) participants in Part B remain on JZP-458 treatment in the study.

[0318] In Part A (IM), a total of 35 (21.0%) of 167 participants discontinued JZP-458 treatment (21 of 167 [12.6%] discontinued due to TEAEs, 8 of 167 [4.8%] discontinued at physician discretion, 3 of 167 [1.8%] discontinued due to progressive disease, 1 of 167 [0.6%] discontinued by parent or guardian, 1 of 167 [0.6%] discontinued due to participant withdrawal, and 1 of 167 [0.6%] discontinued due to "other"). As of the data cutoff date of July 19, 2021, a total of 3 [1.8%] of the 167 participants in Part A had died.

[0319] In Part B (IV), a total of 22 (35.5%) of 62 participants discontinued JZP-458 treatment (15 / 62 [24.2%] discontinued due to TEAEs, 4 / 62 [6.5%] discontinued at physician discretion, 1 / 62 [1.6%] discontinued due to recurrent disease, 1 / 62 [1.6%] discontinued due to participant withdrawal, and 1 / 62 [1.6%] discontinued due to "other"). As of the data cutoff date of July 19, 2021, there were no deaths in Part B.

[0320] Overall, in Part A (IM), the majority of participants were white (115 of 167 [68.9%]). The median age at enrollment was 10 years (range: 1-25 years). Of the 167 participants in the safety population, 44 (26.3%) were <6 years old, 57 (34.1%) were 6 to <12 years old, 45 (26.9%) were 12 to <18 years old, and 21 (12.6%) were 18 years old or older. A higher percentage of participants in the safety population were male (103 of 167 [61.7%]) than female (64 of 167 [38.3%]).

[0321] In Part B (IV), the majority of participants were white (43 of 61 [70.5%]). The median age at enrollment was 10 years (range: 1-24 years). Of the 61 participants in the safety population, 20 (32.8%) were <6 years old, 14 (23.0%) were 6 to <12 years old, 17 (27.9%) were 12 to <18 years old, and 10 (16.4%) were 18 years old or older. A higher percentage of participants in the safety population were male (36 of 61 [59.0%]) than female (25 of 61 [41.0%]).

[0322] Overall, in Part A (IM), B-cell ALL was reported in 124 of 167 participants [74.3%], T-cell ALL in 26 of 167 participants [15.6%], T-cell LBL in 16 of 167 participants [9.6%], and B-cell LBL in 1 of 167 participants (0.6%) as the primary disease state. Overall, time from primary diagnosis ranged from 0 to 3 months relative to study date for the majority of participants (122 of 167 [73.1%]). The previous asparaginase treatment for all participants (167 of 167) was Oncaspar, and the mean time from last asparaginase treatment was 24.3 days relative to study date (range 2 to 158 days).

[0323] Overall, in Part B (IV), B-cell ALL was reported in 51 of 61 participants [83.6%], T-cell ALL in 7 of 61 participants [11.5%], B-cell LBL in 2 of 61 participants (3.3%), and T-cell LBL in 1 of 61 participants [1.6%] as the primary disease status. Overall, time from primary diagnosis ranged from 0 to 3 months relative to study date for the majority of participants (47 of 61 [77.0%]). The previous asparaginase treatment for the majority of participants (60 of 61 [98.4%]) was Oncaspar, and the mean time from last asparaginase treatment was 24.9 days relative to study date (range 2 to 138 days). Demographic and baseline characteristics are shown in Figure 33.

[0324] Pharmacokinetics based on serum asparaginase activity: In Part A (IM), the PK analysis population was 2 32 participants (97.0%) in the MWF cohort, 37.5 mg / m 2 83 participants (100%) in the MWF cohort and 25(MW) / 50(F)mg / m 2 The cohort consisted of 51 participants (100%). In Part B (IV), the PK analysis population consisted of 25(MW) / 50(F)mg / m 2 The study consisted of 60 participants (96.8%) who received JZP458 IV at a dose of

[0325] The observed SAA results were 25 mg / m 2 MWF, 37.5 mg / m 2 MWF, and 25(MW) / 50(F)mg / mg 2 The results show that at the IM dose level of JZP-458, the mean and median SAA levels immediately preceding dosing at both 72 and 48 hours were 0.1 IU / mL or greater (FIG. 34).

[0326] Part A (IM) JZP-458 at 25 and 37.5 mg / m administered IM on a MWF schedule 2 At the dose level of 25(MW) / 50(F)mg / m, 64.3% (95%CI: 46.5%, 82.0%) and 90.8% (95%CI: 84.3%, 97.3%) of participants, respectively, achieved NSAA levels of 0.1 IU / mL or greater at the most recent 72-hour assessment during the first course of treatment. 2 When administered IM at the dose of 0.1 IU / mL or higher, 89.8% (95% CI: 81.3%, 98.3%) of participants achieved NSAA levels of 0.1 IU / mL or higher at the most recent 72-hour assessment during the first course of treatment.

[0327] JZP-458 at 25 and 37.5 mg / m administered IM on a MWF schedule 2At the dose level of 25(MW) / 50(F)mg / m, 96.9% (95%CI: 90.8%, 100.0%) and 98.8% (95%CI: 96.4%, 100.0%) of participants, respectively, achieved NSAA levels of 0.1 IU / mL or greater at the most recent 48-hour assessment during the first course of treatment. 2 When administered IM at the dose of 0.1 IU / mL or higher, 95.9% (95% CI: 90.4%, 100.0%) of participants achieved NSAA levels of 0.1 IU / mL or higher at the most recent 48-hour assessment during the first course of treatment.

[0328] Part B(IV) JZP-458 25(MW) / 50(F)mg / m administered IV on a MWF schedule 2 At dose level, 40.0% (95% CI: 26.4%, 53.6%) of participants achieved NSAA levels of 0.1 IU / mL or greater at the most recent 72-hour assessment during the first course of treatment, respectively.

[0329] 25(MW) / 50(F)mg / m 2 At the IV dose level of JZP-458 administered IV at a dose of , 89.8% (95% CI: 82.1%, 97.5%) of participants achieved NSAA levels of ≥0.1 IU / mL at the most recent 48-hour assessment during the first course of treatment.

[0330] Pharmacokinetics based on serum asparaginase concentration: The relationship between the SAA and SAC of JZP-458 was investigated in the JZP458-201 study.

[0331] Part A (IM): When JZP-458 was administered IM to participants with ALL / LBL, a strong positive association was observed between SAA and SAC, with pooled JZP458 IM 25 mg / m 2 MWF, IM 37.5mg / m 2 MWF and IM 25(MW) / 50(F)mg / m 2The correlation coefficient for was 0.9640 (Pearson correlation coefficient) (Figure 35). Furthermore, the equation from the linear regression model was SAC = 1254.7 x SAA (Figure 35). These data suggested that an SAA level of 0.1 IU / mL would correspond to a serum asparaginase concentration of approximately 125 ng / mL in this patient population.

[0332] Part B(IV) When JZP-458 was administered IV to participants with ALL / LBL, a strong positive association was observed between SAA and SAC, with a median age of 12 months after the first course of JZP-458 IV 25(MW) / 50(F)mg / m 2 The correlation coefficient for was 0.9263 (Pearson correlation coefficient) (Figure 35). Furthermore, the equation from the linear regression model was SAC = 1080.8 x SAA (Figure 35). These data suggested that an SAA level of 0.1 IU / mL would correspond to a serum asparaginase concentration of 108 ng / mL in this patient population.

[0333] Pharmacodynamic Results: The PD analysis population consisted of 166 participants (99.4%) enrolled in Part A (IM) of the study and 60 participants (96.8%) enrolled in Part B (IV) of the study. PD samples were analyzed for L-asparagine and L-glutamine concentrations in human plasma using a validated liquid chromatography tandem mass spectrometry method, with L-asparagine ranging from 0.025 to 10.0 μg / mL and L-glutamine ranging from 0.250 to 100 μg / mL.

[0334] Plasma L-asparagine depletion: Part A (IM): Complete depletion of plasma asparagine was achieved with 25 mg / m 2 MWF, 37.5 mg / m 2 MWF, and 25(MW) / 50(F)mg / m 2During the first cycle, mean predose L-asparagine concentrations ranged from 7.5865 to 11.3215 μg / mL at all levels and all schedules. Complete depletion of plasma asparagine was observed in the majority of participants. Figures 36A, 36B, and 36C show the effect of JZP-458 at 25 mg / m 2 MWF, 37.5 mg / m 2 MWF, and 25(MW) / 50(F)mg / m 2 The results show that after IM administration of 0.01 mg / mL, mean plasma asparagine levels rapidly declined from pre-dose levels to levels below or near the assay LLOQ (0.025 μg / mL). The decreased plasma asparagine levels persisted throughout the first course of treatment until before the sixth dose, when the final sample was taken. Four participants showed a transient, low-level increase in plasma asparagine (three in cohort 1a and one in cohort 1b).

[0335] Part B (IV): Complete depletion of plasma asparagine was achieved with JZP-458 at 25(MW) / 50(F)mg / m 2 During the first cycle, mean predose L-asparagine concentrations ranged from 8.2218 to 8.9348 μg / mL for all dosing schedules. Complete depletion of plasma asparagine was observed in the majority of participants. Figures 36A-36C show the effect of JZP-458 at 25(MW) / 50(F) mg / m 2 The results show that after IV administration of 0.01 mg / mL of 100 mg ...

[0336] Plasma L-glutamine depletion: Part A (IM): Plasma glutamine depletion was also achieved with 25 mg / m 2 MWF, 37.5 mg / m 2 MWF, and 25(MW) / 50(F)mg / m2 In the first course, mean pre-dose L-glutamine concentrations ranged from 88.3000 to 112.7875 μg / mL for all levels and all schedules. Figures 37A-37C show that mean L-glutamine levels were significantly higher after IM administration of JZP-458 at 25 mg / m 2 MWF, 37.5 mg / m 2 MWF, and 25(MW) / 50(F)mg / m 2 However, complete depletion of L-glutamine was not observed based on the profile means, and glutamine levels were only moderately affected.

[0337] Part B(IV): Plasma glutamine depletion was also ob...

Claims

1. A pharmaceutical comprising L-asparaginase for use in a method for treating cancer in a human subject, comprising: the method comprising administering a chronological dose set of L-asparaginase, the chronological dose set comprising a first dose, a second dose, and a third dose, to a human subject intravenously or intramuscularly on Monday, Wednesday, and Friday, respectively; The first dose is 25 mg / m ２ and the second dose is 25 mg / m ２ and said third dose is 50 mg / m 2 , and The L-asparaginase is not an E. coli derived asparaginase and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1; The said medicine.

2. The method further comprises administering to a human subject a plurality of instances of the time-ordered dose set of L-asparaginase; The pharmaceutical composition of claim 1, wherein each instance of the chronological dose set of L-asparaginase is administered to the subject upon completion of a previous instance of the chronological dose set in the multiple instances of the chronological dose set.

3. The method of claim 2, wherein the plurality of instances of the chronological dose set of L-asparaginase is between 2 and 100, or between 2 and 15.

4. A pharmaceutical comprising L-asparaginase for use in a method for treating cancer in a human subject, comprising: the method comprising administering to the human subject a series of six doses of L-asparaginase in place of each dose of E. coli derived asparaginase; Four of the six doses are 25 mg / m ２ and Two of the six doses are 50 mg / m ２ and 50 mg / m ２ the administration interval between the two doses is 7 days, 25 mg / m ２ and the administration interval between the dose of Of the series of doses, 50 mg / m ２ and the dosing interval between the dose is 3 days; and The L-asparaginase is not an asparaginase derived from Escherichia coli and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1; The said medicine.

5. 5. The method of claim 4, wherein each dose of the E. coli derived asparaginase in the multiple doses of the E. coli derived asparaginase is separately replaced with a series of six dose instances of the L-asparaginase.

6. The pharmaceutical described in any one of claims 1 to 5, wherein (a) the Escherichia coli-derived asparaginase is PEG-asparaginase, and (b) the L-asparaginase is a tetramer, and each monomer of the tetramer comprises an amino acid sequence at least 95% identical to SEQ ID NO:

1.

7. The pharmaceutical agent according to any one of claims 1 to 6, wherein the L-asparaginase is a tetramer, and each monomer of the tetramer comprises the amino acid sequence of SEQ ID NO:

1.

8. The L-asparaginase is (a) exhibiting less than 6% aggregation or less than 1% aggregation; (b) is not freeze-dried; (c) recombinantly produced in Pseudomonas fluorescens; and / or (d) is natural or long-acting; A pharmaceutical composition according to any one of claims 1 to 7.

9. A pharmaceutical agent according to any one of claims 1 to 8, used in combination with one or more other chemotherapy agents as part of a combination chemotherapy regimen.

10. 12. The method of claim 11, wherein the human subject (a) exhibited hypersensitivity to the E. coli-derived asparaginase, and / or (b) has completed E. coli-derived asparaginase therapy; A pharmaceutical composition according to any one of claims 1 to 9.

11. The pharmaceutical agent according to any one of claims 1 to 10, wherein the serum asparaginase activity nadir (NSAA) in an assay when measured from a serum sample from the human subject is 0.1 IU / mL or greater after administration of the L-asparaginase.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the cancer is acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL).

13. The cancer is (a) colorectal cancer (CRC); (b) acute myeloid leukemia (AML), or (c) Diffuse large B-cell lymphoma (DLBCL) The pharmaceutical composition according to any one of claims 1 to 12,

14. The pharmaceutical composition according to claim 13, wherein the CRC is Wnt-negative CRC or Wnt-mutated CRC.

15. The method of claim 13, wherein the AML is relapsed / refractory acute (R / R) AML.

16. The pharmaceutical composition of claim 1, wherein, in the method, each dose of the L-asparaginase is administered intramuscularly.

17. The method of claim 1, wherein each of the 25 mg / m 2 doses is administered intravascularly and each of the 50 mg / m 2 doses is administered intramuscularly.

18. The method of claim 1, wherein each dose of the L-asparaginase is administered intravenously.

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