Asparaginase erwinia chrysanthemi (recombinant)-rywn

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Rylaze

Sequence:

1ADKLPNIVIL ATGGTIAGSA ATGTQTTGYK AGALGVDTLI NAVPEVKKLA

51NVKGEQFSNM ASENMTGDVV LKLSQRVNEL LARDDVDGVV ITHGTDTVEE

101SAYFLHLTVK SDKPVVFVAA MRPATAISAD GPMNLLEAVR VAGDKQSRGR

151GVMVVLNDRI GSARYITKTN ASTLDTFKAN EEGYLGVIIG NRIYYQNRID

201KLHTTRSVFD VRGLTSLPKV DILYGYQDDP EYLYDAAIQH GVKGIVYAGM

251GAGSVSVRGI AGMRKAMEKG VVVIRSTRTG NGIVPPDEEL PGLVSDSLNP

301AHARILLMLA LTRTSDPKVI QEYFHTY

>Protein sequence for asparaginase (Erwinia chrysanthemi) monomer
ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNM
ASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAA
MRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKAN
EEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQH
GVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNP
AHARILLMLALTRTSDPKVIQEYFHTY

References:

Therapeutic Targets Database: TTD Biologic drug sequences in fasta format [Link]

Asparaginase erwinia chrysanthemi (recombinant)-rywn

JZP458-201

JZP458

CAS Registry Number 1349719-22-7

Protein Chemical FormulaC₁₅₄₆H₂₅₁₀N₄₃₂O₄₇₆S₉

Protein Average Weight 140000.0 Da

Rylaze, FDA APPROVED 6/30/2021, BLA 761179

L-Asparaginase (ec 3.5.1.1, L-asparagine amidohydrolase) erwinia chrysanthemi tetramer alpha4

Asparaginase (Dickeya chrysanthemi subunit)

Other Names

Asparaginase Erwinia chrysanthemi
Crisantaspase
Cristantaspase
Erwinase
Erwinaze

L-Asparagine amidohydrolase (Erwinia chrysanthemi subunit)

D733ET3F9O

1349719-22-7

Asparaginase erwinia chrysanthemi [USAN]

UNII-D733ET3F9O

L-Asparaginase (erwinia)

Erwinia asparaginase

L-Asparaginase, erwinia chrysanthemi

Asparaginase (erwinia chrysanthemi)

Erwinase

Asparaginase erwinia chrysanthemi

Erwinaze

Crisantaspase

Crisantaspase [INN]

L-Asparaginase (ec 3.5.1.1, L-asparagine amidohydrolase) erwinia chrysanthemi tetramer alpha4

Asparaginase erwinia sp. [MI]

Asparaginase erwinia chrysanthemi (recombinant) [USAN]

Asparaginase erwinia chrysanthemi (recombinant)

JZP-458

A hydrolase enzyme that converts L-asparagine and water to L-aspartate and NH3.

NCI: Asparaginase Erwinia chrysanthemi. An enzyme isolated from the bacterium Erwinia chrysanthemi (E. carotovora). Asparagine is critical to protein synthesis in leukemic cells, which cannot synthesize this amino acid due to the absence of the enzyme asparagine synthase. Asparaginase hydrolyzes L-asparagine to L-aspartic acid and ammonia, thereby depleting leukemic cells of asparagine and blocking protein synthesis and tumor cell proliferation, especially in the G1 phase of the cell cycle. This agent also induces apoptosis in tumor cells. The Erwinia-derived product is often used for those patients who have experienced a hypersensitivity reaction to the E. Coli formulation. (NCI Thesaurus)

Treatment of Acute Lymphoblastic Leukemia (ALL)
Antineoplastic Agents

10MG/0.5ML	INJECTABLE;INTRAMUSCULAR

Label (PDF)
Letter (PDF)

Label (PDF)

PATENT

WO 2011003633

https://patents.google.com/patent/WO2011003633A1/en

The present invention concerns a conjugate of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol, particularly wherein the polyethylene glycol has a molecular weight less than or equal to about 5000 Da, particularly a conjugate wherein the protein is a L-asparaginase from Erwinia, and its use in therapy.

Proteins with L-asparagine aminohydrolase activity, commonly known as L- asparaginases, have successfully been used for the treatment of Acute Lymphoblastic Leukemia

(ALL) in children for many years. ALL is the most common childhood malignancy (Avramis and Panosyan, Clin. Pharmacokinet. (2005) 44:367-393).

[0003] L-asparaginase has also been used to treat Hodgkin’s disease, acute myelocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).

The anti-tumor activity of L-asparaginase is believed to be due to the inability or reduced ability of certain malignant cells to synthesize L-asparagine (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669). These malignant cells rely on an extracellular supply of L-asparagine. However, the L-asparaginase enzyme catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia, thereby depleting circulating pools of L-asparagine and killing tumor cells which cannot perform protein synthesis without L-asparagine (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).

[0004] L-asparaginase from E. coli was the first enzyme drug used in ALL therapy and has been marketed as Elspar® in the USA or as Kidrolase® and L-asparaginase Medac® in Europe. L- asparaginases have also been isolated from other microorganisms, e.g., an L-asparaginase protein from Erwinia chrysanthemi, named crisantaspase, that has been marketed as Erwinase® (Wriston Jr., J.C. (1985) “L-asparaginase” Meth. Enzymol. 113, 608-618; Goward, CR. et al. (1992) “Rapid large scale preparation of recombinant Erwinia chrysanthemi L-asparaginase”, Bioseparation 2, 335-341). L-asparaginases from other species of Erwinia have also been identified, including, for example, Erwinia chrysanthemi 3937 (Genbank Accession

#AAS67028), Erwinia chrysanthemi NCPPB 1125 (Genbank Accession #CAA31239), Erwinia carotovora (Genbank Accession #AAP92666), and Erwinia carotovora subsp. Astroseptica (Genbank Accession #AAS67027). These Erwinia chrysanthemi L-asparaginases have about 91-98% amino acid sequence identity with each other, while the Erwinia carotovora L- asparaginases have approximately 75-77% amino acid sequence identity with the Erwinia chrysanthemi L-asparaginases (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).

[0005] L-asparaginases of bacterial origin have a high immunogenic and antigenic potential and frequently provoke adverse reactions ranging from mild allergic reaction to anaphylactic shock in sensitized patients (Wang, B. et al. (2003) “Evaluation of immunologic cross reaction of anti- asparaginase antibodies in acute lymphoblastic leukemia (ALL and lymphoma patients),

Leukemia 17, 1583-1588). E. coli L-asparaginase is particularly immunogenic, with reports of the presence of anti-asparaginase antibodies to E. coli L-asparaginase following i.v. or i.m. administration reaching as high as 78% in adults and 70% in children (Wang, B. et al. (2003) Leukemia 17, 1583-1588).

[0006] L-asparaginases from Escherichia coli and Erwinia chrysanthemi differ in their pharmacokinetic properties and have distinct immunogenic profiles, respectively (Klug Albertsen, B. et al. (2001) “Comparison of intramuscular therapy with Erwinia asparaginase and asparaginase Medac: pharmacokinetics, pharmacodynamics, formation of antibodies and influence on the coagulation system” Brit. J. Haematol. 115, 983-990). Furthermore, it has been shown that antibodies that developed after a treatment with L-asparaginase from E. coli do not cross react with L-Asparaginase from Erwinia (Wang, B. et al., Leukemia 17 (2003) 1583-1588). Thus, L-asparaginase from Erwinia (crisantaspase) has been used as a second line treatment of ALL in patients that react to E. coli L-asparaginase (Duval, M. et al. (2002) “Comparison of Escherichia co/z-asparaginase with £Vwzm^‘α-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment of

Cancer, Children’s Leukemia Group phase 3 trial” Blood 15, 2734-2739; Avramis and Panosyan,

Clin. Pharmacokinet. (2005) 44:367-393).

[0007] In another attempt to reduce immunogenicity associated with administration of microbial L-asparaginases, an E. coli L-asparaginase has been developed that is modified with methoxy- polyethyleneglycol (mPEG). This method is commonly known as “PEGylation” and has been shown to alter the immunological properties of proteins (Abuchowski, A. et al. (1977) “Alteration of Immunological Properties of Bovine Serum Albumin by Covalent Attachment of Polyethylene Glycol,” J.Biol.Chem. 252 (11), 3578-3581). This so-called mPEG-L- asparaginase, or pegaspargase, marketed as Oncaspar® (Enzon Inc., USA), was first approved in the U.S. for second line treatment of ALL in 1994, and has been approved for first- line therapy of ALL in children and adults since 2006. Oncaspar® has a prolonged in vivo half-life and a reduced immunogenicity/antigenicity.

[0008] Oncaspar® is E. coli L-asparaginase that has been modified at multiple lysine residues using 5 kDa mPEG-succinimidyl succinate (SS-PEG) (U.S. Patent No. 4,179,337). SS-PEG is a

PEG reagent of the first generation that contains an instable ester linkage that is sensitive to hydro lysis by enzymes or at slightly alkaline pH values (U.S. Patent No. 4,670,417; Makromol. Chem. 1986, 187, 1131-1144). These properties decrease both in vitro and in vivo stability and can impair drug safety.

[0009] Furthermore, it has been demonstrated that antibodies developed against L-asparaginase from E. coli will cross react with Oncaspar® (Wang, B. et al. (2003) “Evaluation of immunologic cross-reaction of anti-asparaginase antibodies in acute lymphoblastic leukemia (ALL and lymphoma patients),” Leukemia 17, 1583-1588). Even though these antibodies were not neutralizing, this finding clearly demonstrated the high potential for cross-hypersensitivity or cross-inactivation in vivo. Indeed, in one report 30-41% of children who received pegaspargase had an allergic reaction (Wang, B. et al. (2003) Leukemia 17, 1583-1588).

[0010] In addition to outward allergic reactions, the problem of “silent hypersensitivity” was recently reported, whereby patients develop anti-asparaginase antibodies without showing any clinical evidence of a hypersensitivity reaction (Wang, B. et al. (2003) Leukemia 17, 1583-1588). This reaction can result in the formation of neutralizing antibodies to E. coli L-asparaginase and pegaspargase; however, these patients are not switched to Erwinia L-asparaginase because there are not outward signs of hypersensitivity, and therefore they receive a shorter duration of effective treatment (Holcenberg, J., J. Pediatr. Hematol. Oncol. 26 (2004) 273-274).

[0011] Erwinia chrysanthemi L-asparaginase treatment is often used in the event of hypersensitivity to E. co/z-derived L-asparaginases. However, it has been observed that as many as 30-50% of patients receiving Erwinia L-asparaginase are antibody-positive (Avramis and

Panosyan, Clin. Pharmacokinet. (2005) 44:367-393). Moreover, because Erwinia chrysanthemi L-asparaginase has a significantly shorter elimination half-life than the E. coli L-asparaginases, it must be administered more frequently (Avramis and Panosyan, Clin. Pharmacokinet. (2005) 44:367-393). In a study by Avramis et al., Erwinia asparaginase was associated with inferior pharmacokinetic profiles (Avramis et al., J. Pediatr. Hematol. Oncol. 29 (2007) 239-247). E. coli L-asparaginase and pegaspargase therefore have been the preferred first-line therapies for ALL over Erwinia L-asparaginase.

[0012] Numerous biopharmaceuticals have successfully been PEGylated and marketed for many years. In order to couple PEG to a protein, the PEG has to be activated at its OH terminus. The activation group is chosen based on the available reactive group on the protein that will be

PEGylated. In the case of proteins, the most important amino acids are lysine, cysteine, glutamic acid, aspartic acid, C-terminal carboxylic acid and the N-terminal amino group. In view of the wide range of reactive groups in a protein nearly the entire peptide chemistry has been applied to activate the PEG moiety. Examples for this activated PEG-reagents are activated carbonates, e.g., p-nitrophenyl carbonate, succinimidyl carbonate; active esters, e.g., succinimidyl ester; and for site specific coupling aldehydes and maleimides have been developed (Harris, M., Adv. Drug – A –

DeI. Rev. 54 (2002), 459-476). The availability of various chemical methods for PEG modification shows that each new development of a PEGylated protein will be a case by case study. In addition to the chemistry the molecular weight of the PEG that is attached to the protein has a strong impact on the pharmaceutical properties of the PEGylated protein. In most cases it is expected that, the higher the molecular weight of the PEG, the better the improvement of the pharmaceutical properties (Sherman, M. R., Adv. Drug Del. Rev. 60 (2008), 59-68; Holtsberg, F. W., Journal of Controlled Release 80 (2002), 259-271). For example, Holtsberg et al. found that, when PEG was conjugated to arginine deaminase, another amino acid degrading enzyme isolated from a microbial source, pharmacokinetic and pharmacodynamic function of the enzyme increased as the size of the PEG attachment increased from a molecular weight of 5000

Da to 20,000 Da (Holtsberg, F.W., Journal of Controlled Release 80 (2002), 259-271).

[0013] However, in many cases, PEGylated biopharmaceuticals show significantly reduced activity compared to the unmodified biopharmaceutical (Fishburn, CS. (2008) Review “The Pharmacology of PEGylation: Balancing PD with PK to Generate Novel Therapeutics” J. Pharm. Sd., 1-17). In the case of L-asparaginase from Erwinia carotovora, it has been observed that PEGylation reduced its in vitro activity to approximately 57% (Kuchumova, A.V. et al. (2007) “Modification of Recombinant asparaginase from Erwinia carotovora with Polyethylene Glycol 5000” Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 1, 230-232). The L-asparaginase from Erwinia carotovora has only about 75% homology to the Erwinia chrysanthemi L-asparaginase (crisantaspase). For Oncaspar® it is also known that its in vitro activity is approximately 50% compared to the unmodified E. coli L-asparaginase.

[0014] The currently available L-asparaginase preparations do not provide alternative or complementary therapies— particularly therapies to treat ALL— that are characterized by high catalytic activity and significantly improved pharmacological and pharmacokinetic properties, as well as reduced immunogenicity.

L-asparaginase protein has at least about 80% homology or identity with the protein comprising the sequence of SEQ ID NO:1, more specifically at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with the protein comprising the sequence of SEQ ID NO:1. SEQ ID NO:1 is as follows:

ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGE QFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTV KSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSA RYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKV DILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVI

RSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY (SEQ ID NO:1) [0048] 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.

Examples

Example 1 : Preparation of Recombinant Crisantaspase [0100] The recombinant bacterial strain used to manufacture the naked recombinant Erwinia chrysanthemi L-asparaginase protein (also referred to herein as “r-crisantaspase”) was an E. coli BL21 strain with a deleted ansB gene (the gene encoding the endogenous E. coli type II L- asparaginase) to avoid potential contamination of the recombinant Erwinia chrysanthemi L- asparaginase with this enzyme. The deletion of the ansB gene relies on homologous recombination methods and phage transduction performed according to the three following steps:

1) a bacterial strain (NMI lOO) expressing a defective lambda phage which supplies functions that protect and recombine electroporated linear DNA substrate in the bacterial cell was transformed with a linear plasmid (kanamycin cassette) containing the kanamycin gene flanked by an FLP recognition target sequence (FRT). Recombination occurs to replace the ansB gene by the kanamycin cassette in the bacterial genome, resulting in a ΛansB strain; 2) phage transduction was used to integrate the integrated kanamycin cassette region from the ΛansB NMI lOO strain to the ansB locus in BL21 strain. This results in an E. coli BL21 strain with a deleted ansB gene and resistant to kanamycin; 3) this strain was transformed with a FLP -helper plasmid to remove the kanamycin gene by homologous recombination at the FRT sequence. The genome of the final strain (BL21 ΛansB strain) was sequenced, confirming full deletion of the endogenous ansB gene.

[0101] The E. co/z^‘-optimized DNA sequence encoding for the mature Erwinia chrysanthemi L- asparaginase fused with the ENX signal peptide from Bacillus subtilis was inserted into an expression vector. This vector allows expression of recombinant Erwinia chrysanthemi L- asparaginase under the control of hybrid T5/lac promoter induced by the addition of Isopropyl β- D-1-thiogalactopyranoside (IPTG) and confers resistance to kanamycin.

[0102] BL21 ΛansB strain was transformed with this expression vector. The transformed cells were used for production of the r-crisantaspase by feed batch glucose fermentation in Reisenberg medium. The induction of the cell was done 16h at 23°C with IPTG as inducer. After cell harvest and lysis by homogenization in 1OmM sodium phosphate buffer pH6 5mM EDTA (Buffer A), the protein solution was clarified by centrifugation twice at 1500Og, followed by 0.45μm and 0.22μm filtration steps. The recombinant Erwinia chrysanthemi L-asparaginase was next purified using a sequence of chromatography and concentration steps. Briefly, the theoretical isoelectric point of the Erwinia chrysanthemi L-asparaginase (7.23) permits the recombinant enzyme to adsorb to cation exchange resins at pH6. Thus, the recombinant enzyme was captured on a Capto S column (cation exchange chromatography) and eluted with salt gradient in Buffer A. Fractions containing the recombinant enzyme were pooled. The pooled solution was next purified on Capto MMC column (cation exchange chromatography) in Buffer A with salt gradient. . The eluted fractions containing Erwinia chrysanthemi L-asparaginase were pooled and concentrated before protein separation on Superdex 200pg size exclusion chromatography as polishing step. Fractions containing recombinant enzymes were pooled, concentrated, and diafiltered against 10OmM sodium phosphate buffer pH8. The purity of the final Erwinia chrysanthemi L-asparaginase preparation was evaluated by SDS-PAGE (Figure 1) and RP-HPLC and was at least 90%. The integrity of the recombinant enzyme was verified by

N-terminal sequencing and LC-MS. Enzyme activity was measured at 37°C using Nessler’s reagent. The specific activity of the purified recombinant Erwinia chrysanthemi L-asparaginase was around 600 U/mg. One unit of enzyme activity is defined as the amount of enzyme that liberates lμmol of ammonia from L-asparagine per minute at 37°C. Example 2: Preparation of 10 kDa mPEG-L- Asparaginase Conjugates

[0103] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer at pH 8.0, at a protein concentration between 2.5 and 4 mg/mL, in the presence of 150 mg/mL or 36 mg/mL 10 kDa mPEG-NHS, for 2 hours at 22°C. The resulting crude 10 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein-containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 10 kDa mPEG-L-asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as a reference, one corresponding to full PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N-terminus) residues being conjugated corresponding to PEGylation of 78% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (39% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 50% of accessible amino groups (e.g., lysine residues and/or the N-terminus)) . SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.

Example 3: Preparation of 5 kDa mPEG-L-Asparaginase Conjugates

[0104] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer at pH 8.0, at a protein concentration of 4 mg/mL, in the presence of 150 mg/mL or 22.5 mg/mL 5 kDa mPEG-NHS, for 2 hours at 22°C. The resulting crude 5 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein-containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 5 kDa mPEG-L- asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as a reference, one corresponding to full PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N-terminus) being conjugated corresponding to PEGylation of 84% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (36% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 43% of accessible amino groups (e.g., lysine residues and/or the N-terminus)). SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.

Example 4: Preparation of 2 kDa mPEG-L-Asparaginase Conjugates

[0105] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer pH 8.0 at a protein concentration of 4 mg/mL in the presence of

150 mg/mL or 22.5 mg/mL 2 kDa mPEG-NHS for 2 hours at 22°C. The resulting crude 2 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 2 kDa mPEG-L- asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as reference, one corresponding to maximum PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N- terminus) being conjugated corresponding to PEGylation of 86% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (47% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 55% of accessible amino groups {e.g., lysine residues and/or the N-terminus)). SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.

Example 5: Activity of mPEG-r-Crisantaspase Conjugates

[0106] L-asparaginase aminohydrolase activity of each conjugate described in the proceeding examples was determined by Nesslerization of ammonia that is liberated from L-asparagine by enzymatic activity. Briefly, 50μL of enzyme solution were mixed with 2OmM of L-asparagine in a 50 mM Sodium borate buffer pH 8.6 and incubated for 10 min at 37°C. The reaction was stopped by addition of 200μL of Nessler reagent. Absorbance of this solution was measured at 450 nm. The activity was calculated from a calibration curve that was obtained from Ammonia sulfate as reference. The results are summarized in Table 2, below:

Table 2: Activity of mPEG-r-crisantaspase conjugates

* the numbers “40%” and “100%” indicate an approximate degree of PEGylation of respectively 40-55% and 100% of accessible amino groups (see Examples 2-4, supra).

** the ratio mol PEG / mol monomer was extrapolated from data using TNBS assay, that makes the assumption that all amino groups from the protein (e.g., lysine residues and the N-terminus) are accessible.

[0107] Residual activity of mPEG-r-crisantaspase conjugates ranged between 483 and 543 Units/mg. This corresponds to 78-87% of L-asparagine aminohydrolase activity of the unmodified enzyme. Example 6: L-Asparagine-Depleting Effect of Unmodified Crisantaspase

PAPER

Biotechnology and Applied Biochemistry (2019), 66(3), 281-289. |

https://iubmb.onlinelibrary.wiley.com/doi/10.1002/bab.1723

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l-asparaginase (ASNase). The main disadvantages of crisantaspase are the short half-life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce immunogenicity but also improve plasma half-life. In this work, we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2-L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L⁻¹ H⁻¹, respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg⁻¹, 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol-N-hydroxysuccinimidyl (mPEG-NHS) at different pH values (6.5–9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with the lowest poly-PEGylation ratio (7%). PEG-crisantaspase was purified by size exclusion chromatography and presented a K_M value three times higher than crisantaspase (150 and 48.5 µM, respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL.

ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSN

MASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVV

FVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNAST

LDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEY

LYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEE

LPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

Figure S1 – Amino acid sequence of the enzyme crisantaspase without the signal peptide and with the lysines highlighted in red (Swiss-Prot/TrEMBL accession number: P06608|22-348 AA).

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As a component of a chemotherapy regimen to treat acute lymphoblastic leukemia and lymphoblastic lymphoma in patients who are allergic to E. coli-derived asparaginase products
Press Release

For Immediate Release:: June 30, 2021

FDA Approves Component of Treatment Regimen for Most Common Childhood Cancer

Alternative Has Been in Global Shortage Since 2016

Today, the U.S. Food and Drug Administration approved Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn) as a component of a chemotherapy regimen to treat acute lymphoblastic leukemia and lymphoblastic lymphoma in adult and pediatric patients who are allergic to the E. coli-derived asparaginase products used most commonly for treatment. The only other FDA-approved drug for such patients with allergic reactions has been in global shortage for years.

“It is extremely disconcerting to patients, families and providers when there is a lack of access to critical drugs for treatment of a life-threatening, but often curable cancer, due to supply issues,” said Gregory Reaman, M.D., associate director for pediatric oncology in the FDA’s Oncology Center of Excellence. “Today’s approval may provide a consistently sourced alternative to a pivotal component of potentially curative therapy for children and adults with this type of leukemia.”

Acute lymphoblastic leukemia occurs in approximately 5,700 patients annually, about half of whom are children. It is the most common type of childhood cancer. One component of the chemotherapy regimen is an enzyme called asparaginase that kills cancer cells by depriving them of substances needed to survive. An estimated 20% of patients are allergic to the standard E. coli-derived asparaginase and need an alternative their bodies can tolerate.

Rylaze’s efficacy was evaluated in a study of 102 patients who either had a hypersensitivity to E. coli-derived asparaginases or experienced silent inactivation. The main measurement was whether patients achieved and maintained a certain level of asparaginase activity. The study found that the recommended dosage would provide the target level of asparaginase activity in 94% of patients.

The most common side effects of Rylaze include hypersensitivity reactions, pancreatic toxicity, blood clots, hemorrhage and liver toxicity.

This review was conducted under Project Orbis, an initiative of the FDA Oncology Center of Excellence. Project Orbis provides a framework for concurrent submission and review of oncology drugs among international partners. For this review, FDA collaborated with Health Canada, where the application review is pending.

Rylaze received Fast Track and Orphan Drug designations for this indication. Fast Track is a process designed to facilitate the development and expedite the review of drugs to treat serious conditions and fulfill an unmet medical need. Orphan Drug designation provides incentives to assist and encourage drug development for rare diseases.

The FDA granted approval of Rylaze to Jazz Pharmaceuticals.

REF

https://www.accessdata.fda.gov/drugsatfda_docs/appletter/2021/761179Orig1s000ltr.pdf

https://www.prnewswire.com/news-releases/jazz-pharmaceuticals-announces-us-fda-approval-of-rylaze-asparaginase-erwinia-chrysanthemi-recombinant-rywn-for-the-treatment-of-acute-lymphoblastic-leukemia-or-lymphoblastic-lymphoma-301323782.html#:~:text=Jazz%20Pharmaceuticals%20Announces,details%20to%20follow

DUBLIN, June 30, 2021 /PRNewswire/ — Jazz Pharmaceuticals plc (Nasdaq: JAZZ) today announced the U.S. Food and Drug Administration (FDA) approval of Rylaze^™ (asparaginase erwinia chrysanthemi (recombinant)-rywn) for use as a component of a multi-agent chemotherapeutic regimen for the treatment of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) in pediatric and adult patients one month and older who have developed hypersensitivity to E. coli-derived asparaginase.¹ Rylaze is the only recombinant erwinia asparaginase manufactured product that maintains a clinically meaningful level of asparaginase activity throughout the entire duration of treatment, and it was developed by Jazz to address the needs of patients and healthcare providers with an innovative, high-quality erwinia-derived asparaginase with reliable supply.

“We are excited to bring this important new treatment to patients who are in critical need, and we are grateful to FDA for the approval of Rylaze based on its established safety and efficacy profile. We are pleased Rylaze was approved before the trial is complete and are diligently working to advance additional clinical trial data. We are committed to quickly engaging with FDA to evolve the Rylaze product profile with additional dosing options and an IV route of administration,” said Bruce Cozadd, chairman and CEO of Jazz Pharmaceuticals. “Thank you to our collaborators within the Children’s Oncology Group, the clinical trial investigators, patients and their families, and all of the other stakeholders who helped us achieve this significant milestone.”

Rylaze was granted orphan drug designation for the treatment of ALL/LBL by FDA in June 2021. The Biologics Licensing Application (BLA) approval followed review under the Real-Time Oncology Review (RTOR) program, an initiative of FDA’s Oncology Center of Excellence designed for efficient delivery of safe and effective cancer treatments to patients.

The company expects Rylaze will be commercially available in mid-July.

“The accelerated development and approval of Rylaze marks an important step in bringing a meaningful new treatment option for many ALL patients – most of whom are children – who cannot tolerate E. coli-derived asparaginase medicine,” said Dr. Luke Maese, assistant professor at the University of Utah, Primary Children’s Hospital and Huntsman Cancer Institute. “Before the approval of Rylaze, there was a significant need for an effective asparaginase medicine that would allow patients to start and complete their prescribed treatment program with confidence in supply.”

Recent data from a Children’s Oncology Group retrospective analysis of over 8,000 patients found that patients who did not receive a full course of asparaginase treatment due to associated toxicity had significantly lower survival outcomes – regardless of whether those patients were high risk or standard risk, slow early responders.²

About Study JZP458-201
The FDA approval of Rylaze, also known as JZP458, is based on clinical data from an ongoing pivotal Phase 2/3 single-arm, open-label, multicenter, dose confirmation study evaluating pediatric and adult patients with ALL or LBL who have had an allergic reaction to E. coli-derived asparaginases and have not previously received asparaginase erwinia chrysanthemi. The study was designed to assess the safety, tolerability and efficacy of JZP458. The determination of efficacy was measured by serum asparaginase activity (SAA) levels. The Phase 2/3 study is being conducted in two parts. The first part is investigating the intramuscular (IM) route of administration, including a Monday-Wednesday-Friday dosing schedule. The second part remains active to further confirm the dose and schedule for the intravenous (IV) route of administration.

The FDA approval of Rylaze was based on data from the first of three IM cohorts, which demonstrated the achievement and maintenance of nadir serum asparaginase activity (NSAA) greater than or equal to the level of 0.1 U/mL at 48 hours using IM doses of Rylaze 25 mg/m². The results of modeling and simulations showed that for a dosage of 25 mg/m² administered intramuscularly every 48 hours, the proportion of patients maintaining NSAA ≥ 0.1 U/mL at 48 hours after a dose of Rylaze was 93.6% (95% CI: 92.6%, 94.6%).¹

The most common adverse reactions (incidence >15%) were abnormal liver test, nausea, musculoskeletal pain, fatigue, infection, headache, pyrexia, drug hypersensitivity, febrile neutropenia, decreased appetite, stomatitis, bleeding and hyperglycemia. In patients treated with the Rylaze, a fatal adverse reaction (infection) occurred in one patient and serious adverse reactions occurred in 55% of patients. The most frequent serious adverse reactions (in ≥5% of patients) were febrile neutropenia, dehydration, pyrexia, stomatitis, diarrhea, drug hypersensitivity, infection, nausea and viral infection. Permanent discontinuation due to an adverse reaction occurred in 9% of patients who received Rylaze. Adverse reactions resulting in permanent discontinuation included hypersensitivity (6%) and infection (3%).¹

The company will continue to work with FDA and plans to submit additional data from a completed cohort of patients evaluating 25mg/m² IM given on Monday and Wednesday, and 50 mg/m² given on Friday in support of a M/W/F dosing schedule. Part 2 of the study is evaluating IV administration and is ongoing. The company also plans to submit these data for presentation at a future medical meeting.

Investor Webcast
The company will host an investor webcast on the Rylaze approval in July. Details will be announced separately.

About Rylaze^™ (asparaginase erwinia chrysanthemi (recombinant)-rywn)
Rylaze, also known as JZP458, is approved in the U.S. for use as a component of a multi-agent chemotherapeutic regimen for the treatment of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) in pediatric and adult patients one month and older who have developed hypersensitivity to E. coli-derived asparaginase. Rylaze has orphan drug designation for the treatment of ALL/LBL in the United States. Rylaze is a recombinant erwinia asparaginase that uses a novel Pseudomonas fluorescens expression platform. JZP458 was granted Fast Track designation by the U.S. Food and Drug Administration (FDA) in October 2019 for the treatment of this patient population. Rylaze was approved as part of the Real-Time Oncology Review program, an initiative of the FDA’s Oncology Center of Excellence designed for efficient delivery of safe and effective cancer treatments to patients.

The full U.S. Prescribing Information for Rylaze is available at: <http://pp.jazzpharma.com/pi/rylaze.en.USPI.pdf>

Important Safety Information

RYLAZE should not be given to people who have had:

Serious allergic reactions to RYLAZE
Serious swelling of the pancreas (stomach pain), serious blood clots, or serious bleeding during previous asparaginase treatment

RYLAZE may cause serious side effects, including:

Allergic reactions (a feeling of tightness in your throat, unusual swelling/redness in your throat and/or tongue, or trouble breathing), some of which may be life-threatening
Swelling of the pancreas (stomach pain)
Blood clots (may have a headache or pain in leg, arm, or chest)
Bleeding
Liver problems

Contact your doctor immediately if any of these side effects occur.

Some of the most common side effects with RYLAZE include: liver problems, nausea, bone and muscle pain, tiredness, infection, headache, fever, allergic reactions, fever with low white blood cell count, decreased appetite, mouth swelling (sometimes with sores), bleeding, and too much sugar in the blood.

RYLAZE can harm your unborn baby. Inform your doctor if you are pregnant, planning to become pregnant, or nursing. Females of reproductive potential should use effective contraception (other than oral contraceptives) during treatment and for 3 months following the final dose. Do not breastfeed while receiving RYLAZE and for 1 week after the final dose.

Tell your healthcare provider if there are any side effects that are bothersome or that do not go away.

These are not all the possible side effects of RYLAZE. For more information, ask your healthcare provider.

You are encouraged to report negative side effects of prescription drugs to the FDA. Visit www.fda.gov/medwatch, or call 1-800-FDA-1088 (1-800-332-1088).

About ALL
ALL is a cancer of the blood and bone marrow that can progress quickly if not treated.³ Leukemia is the most common cancer in children, and about three out of four of these cases are ALL.⁴ Although it is one of the most common cancers in children, ALL is among the most curable of the pediatric malignancies due to recent advancements in treatment.^5,6 Adults can also develop ALL, and about four of every 10 cases of ALL diagnosed are in adults.⁷ The American Cancer Society estimates that almost 6,000 new cases of ALL will be diagnosed in the United States in 2021.⁷ Asparaginase is a core component of multi-agent chemotherapeutic regimens in ALL.⁸ However, asparaginase treatments derived from E. coli are associated with the potential for development of hypersensitivity reactions.⁹

About Lymphoblastic Lymphoma
LBL is a rare, fast-growing, aggressive subtype of Non-Hodgkin’s lymphoma, most often seen in teenagers and young adults.⁸ LBL is a very aggressive lymphoma – also called high-grade lymphoma – which means the lymphoma grows quickly with early spread to different parts of the body.^10,11

About Jazz Pharmaceuticals plc
Jazz Pharmaceuticals plc (NASDAQ: JAZZ) is a global biopharmaceutical company whose purpose is to innovate to transform the lives of patients and their families. We are dedicated to developing life-changing medicines for people with serious diseases – often with limited or no therapeutic options. We have a diverse portfolio of marketed medicines and novel product candidates, from early- to late-stage development, in neuroscience and oncology. We actively explore new options for patients including novel compounds, small molecules and biologics, and through cannabinoid science and innovative delivery technologies. Jazz is headquartered in Dublin, Ireland and has employees around the globe, serving patients in nearly 75 countries. For more information, please visit www.jazzpharmaceuticals.com and follow @JazzPharma on Twitter.

About The Children’s Oncology Group (COG)
COG (childrensoncologygroup.org), a member of the NCI National Clinical Trials Network (NCTN), is the world’s largest organization devoted exclusively to childhood and adolescent cancer research. COG unites over 10,000 experts in childhood cancer at more than 200 leading children’s hospitals, universities, and cancer centers across North America, Australia, and New Zealand in the fight against childhood cancer. Today, more than 90% of the 14,000 children and adolescents diagnosed with cancer each year in the United States are cared for at COG member institutions. Research performed by COG institutions over the past 50 years has transformed childhood cancer from a virtually incurable disease to one with a combined 5-year survival rate of 80%. COG’s mission is to improve the cure rate and outcomes for all children with cancer.

Caution Concerning Forward-Looking Statements
This press release contains forward-looking statements, including, but not limited to, statements related to Jazz Pharmaceuticals’ belief in the potential of Rylaze to provide a reliable therapeutic option for adult and pediatric patients to maximize their chance for a cure, plans for a mid-July 2021 launch of Rylaze, the availability of a reliable supply of Rylaze and other statements that are not historical facts. These forward-looking statements are based on Jazz Pharmaceuticals’ current plans, objectives, estimates, expectations and intentions and inherently involve significant risks and uncertainties. Actual results and the timing of events could differ materially from those anticipated in such forward-looking statements as a result of these risks and uncertainties, which include, without limitation, effectively launching and commercializing new products; obtaining and maintaining adequate coverage and reimbursement for the company’s products; delays or problems in the supply or manufacture of the company’s products and other risks and uncertainties affecting the company, including those described from time to time under the caption “Risk Factors” and elsewhere in Jazz Pharmaceuticals’ Securities and Exchange Commission filings and reports (Commission File No. 001-33500), including Jazz Pharmaceuticals’ Annual Report on Form 10-K for the year ended December 31, 2020 and future filings and reports by Jazz Pharmaceuticals. Other risks and uncertainties of which Jazz Pharmaceuticals is not currently aware may also affect Jazz Pharmaceuticals’ forward-looking statements and may cause actual results and the timing of events to differ materially from those anticipated. The forward-looking statements herein are made only as of the date hereof or as of the dates indicated in the forward-looking statements, even if they are subsequently made available by Jazz Pharmaceuticals on its website or otherwise. Jazz Pharmaceuticals undertakes no obligation to update or supplement any forward-looking statements to reflect actual results, new information, future events, changes in its expectations or other circumstances that exist after the date as of which the forward-looking statements were made.

Jazz Media Contact:
Jacqueline Kirby
Vice President, Corporate Affairs
Jazz Pharmaceuticals plc
CorporateAffairsMediaInfo@jazzpharma.com
Ireland, +353 1 697 2141
U.S. +1 215 867 4910

Jazz Investor Contact:
Andrea N. Flynn, Ph.D.
Vice President, Head, Investor Relations
Jazz Pharmaceuticals plc
investorinfo@jazzpharma.com
Ireland, +353 1 634 3211

References

Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn) injection, for intramuscular use Prescribing Information. Palo Alto, CA: Jazz Pharmaceuticals, Inc.
Gupta S, Wang C, Raetz EA et al. Impact of Asparaginase Discontinuation on Outcome in Childhood Acute Lymphoblastic Leukemia: A Report From the Children’s Oncology Group. J Clin Oncol. 2020 Jun 10;38(17):1897-1905. doi: 10.1200/JCO.19.03024
National Cancer Institute. Adult Acute Lymphoblastic Leukemia Treatment (PDQ^®)–Patient Version. Available at www.cancer.gov/types/leukemia/patient/adult-all-treatment-pdq. Accessed June 29, 2021
American Cancer Society. Key Statistics for Childhood Leukemia. Available at https://www.cancer.org/cancer/leukemia-in-children/about/key-statistics.html. Accessed June 29, 2021.
American Cancer Society. Cancer Facts & Figures 2019. www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2019.html. Accessed June 29, 2021.
Pui C, Evans W. A 50-Year Journey to Cure Childhood Acute Lymphoblastic Leukemia. Seminars in Hematology. 2013;50(3), 185-196.
American Cancer Society. Key Statistics for Acute Lymphocytic Leukemia (ALL). Available at https://cancerstatisticscenter.cancer.org/?_ga=2.8163506.1018157754.1621008457-1989786785.1621008457#!/data-analysis/NewCaseEstimates. Accessed June 29, 2021.
Salzer W, Bostrom B, Messinger Y et al. 2018. Asparaginase activity levels and monitoring in patients with acute lymphoblastic leukemia. Leukemia & Lymphoma. 59:8, 1797-1806, DOI: 10.1080/10428194.2017.1386305.
Hijiya N, van der Sluis IM. Asparaginase-associated toxicity in children with acute lymphoblastic leukemia. Leuk Lymphoma. 2016;57(4):748–757. DOI: 10.3109/10428194.2015.1101098.
Leukemia Foundation. Lymphoblastic Lymphoma. Available at https://www.leukaemia.org.au/disease-information/lymphomas/non-hodgkin-lymphoma/other-non-hodgkin-lymphomas/lymphoblastic-lymphoma/. Accessed June 29, 2021.
Mayo Clinic. Acute Lymphocytic Leukemia Diagnosis. Available at https://www.mayoclinic.org/diseases-conditions/acute-lymphocytic-leukemia/diagnosis-treatment/drc-20369083. Accessed June 29, 2021.

SOURCE Jazz Pharmaceuticals plc

Asparaginase erwinia chrysanthemi (recombinant)-rywn

Sequence:

References:

Asparaginase erwinia chrysanthemi (recombinant)-rywn

Other Names

A hydrolase enzyme that converts L-asparagine and water to L-aspartate and NH3.

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