US20120100121A1 - Pegylated L-Asparaginase - Google Patents

Pegylated L-Asparaginase Download PDF

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US20120100121A1
US20120100121A1 US13/382,276 US201013382276A US2012100121A1 US 20120100121 A1 US20120100121 A1 US 20120100121A1 US 201013382276 A US201013382276 A US 201013382276A US 2012100121 A1 US2012100121 A1 US 2012100121A1
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asparaginase
conjugate
peg
protein
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Thierry Abribat
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Jazz Pharmaceuticals PLC
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Alize Pharma 2 SAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • L-asparaginases 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).
  • 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).
  • 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).
  • 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, C. R. et al.
  • 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).
  • 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).
  • 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).
  • 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.
  • L-asparaginase from Erwinia 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 coli -asparaginase with Erwinia -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).
  • an E. coli L-asparaginase has been developed that is modified with methoxy-polyethyleneglycol (mPEG).
  • mPEG methoxy-polyethyleneglycol
  • 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.
  • Oncaspar® is E. coli L-asparaginase that has been modified at multiple lysine residues using 5 kDa mPEG-succinimidyl succinate (SS-PEG) (U.S. Pat. No. 4,179,337).
  • SS-PEG is a PEG reagent of the first generation that contains an insatiable ester linkage that is sensitive to hydrolysis by enzymes or at slightly alkaline pH values (U.S. Pat. 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.
  • Erwinia chrysanthemi L-asparaginase treatment is often used in the event of hypersensitivity to E. coli -derived L-asparaginases.
  • Erwinia chrysanthemi L-asparaginase treatment is often used in the event of hypersensitivity to E. coli -derived L-asparaginases.
  • 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).
  • PEGylated and marketed Numerous biopharmaceuticals have successfully been PEGylated and marketed for many years.
  • the activation group is chosen based on the available reactive group on the protein that will be PEGylated.
  • the most important amino acids are lysine, cysteine, glutamic acid, aspartic acid, C-terminal carboxylic acid and the N-terminal amino group.
  • the entire peptide chemistry has been applied to activate the PEG moiety.
  • 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 Del. Rev. 54 (2002), 459-476).
  • activated carbonates e.g., p-nitrophenyl carbonate, succinimidyl carbonate
  • active esters e.g., succinimidyl ester
  • site specific coupling aldehydes and maleimides have been developed (Harris, M., Adv. Drug Del. 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.
  • the molecular weight of the PEG that is attached to the protein has a strong impact on the pharmaceutical properties of the PEG
  • 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.
  • the present invention is directed to a conjugate of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol, wherein the polyethylene glycol has a molecular weight less than or equal to about 5000 Da, particularly a conjugate where the protein is a L-asparaginase from Erwinia .
  • the conjugate comprises an L-asparaginase from Erwinia having at least 80% identity to the amino acid of SEQ ID NO:1 and polyethylene glycol (PEG), wherein the PEG has a molecular weight less than or equal to about 5000 Da.
  • the L-asparaginase has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid of SEQ ID NO:1.
  • the PEG has a molecular weight of about 5000 Da, 4000, Da, 3000 Da, 2500 Da, or 2000 Da.
  • the conjugate has an in vitro activity of at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the L-asparaginase when not conjugated to PEG.
  • the conjugate has an L-asparagine depletion activity at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more potent than the L-asparaginase when not conjugated to PEG.
  • the conjugate depletes plasma L-asparagine levels to an undetectable level for at least about 12, 24, 48, 96, 108, or 120 hours.
  • the conjugate has a longer in vivo circulating half life compared to the L-asparaginase when not conjugated to PEG.
  • the conjugate has a longer t 1/2 than pegaspargase (i.e., PEG-conjugated L-asparaginase from E. coli ) administered at an equivalent protein dose (e.g., measured in ⁇ g/kg).
  • the conjugate has a t 1/2 of at least about 58 to about 65 hours at a dose of about 50 ⁇ g/kg on a protein content basis, and a t 1/2 of at least about 34 to about 40 hours at a dose of about 10 ⁇ g/kg on a protein content basis, following iv administration in mice.
  • the conjugate has a t 1/2 of at least about 100 to about 200 hours at a dose ranging from about 10,000 to about 15,000 IU/m 2 (about 20-30 mg protein/m 2 ).
  • the conjugate has a greater area under the curve (AUC) compared to the L-asparaginase when not conjugated to PEG.
  • the conjugate has a mean AUC that is at least about 3 times greater than pegaspargase at an equivalent protein dose.
  • the PEG is covalently linked to one or more amino groups (wherein “amino groups” includes lysine residues and/or the N-terminus) of the L-asparaginase.
  • amino groups includes lysine residues and/or the N-terminus
  • the PEG is covalently linked to the one or more amino groups by an amide bond.
  • the PEG is covalently linked to at least from about 40% to about 100% of the accessible amino groups (e.g., lysine residues and/or the N-terminus of the protein) or at least from about 40% to about 90% of total amino groups (e.g., lysine residues and/or the N-terminus of the protein).
  • the conjugate has the formula:
  • Asp is the L-asparaginase
  • NH is one or more of the NH groups of the lysine residues and/or the N-terminus of the Asp
  • PEG is a polyethylene glycol moiety
  • n is a number that represents at least about 40% to about 100% of the accessible amino groups (e.g., lysine residues and/or the N-terminus) in the Asp
  • x is an integer ranging from about 1 to about 8, more specifically, from about 2 to about 5.
  • the PEG is monomethoxy-polyethylene glycol (mPEG).
  • the invention is directed to a method of making a conjugate comprising combining an amount of PEG with an amount of the L-asparaginase in a buffered solution for a time period sufficient to covalently link the PEG to the L-asparaginase.
  • the invention is directed to a pharmaceutical composition comprising the conjugate of the invention.
  • the invention is directed to a method of treating a disease treatable by L-asparagine depletion in a patient comprising administering an effective amount of the conjugate of the invention.
  • the disease is a cancer.
  • the cancer is ALL.
  • the conjugate is administered at an amount of about 5 U/kg body weight to about 50 U/kg body weight.
  • the conjugate is administered at a dose ranging from about 10,000 to about 15,000 IU/m 2 (about 20-30 mg protein/m 2 ).
  • the administration may be intravenous or intramuscular and may be less than once per week (e.g., once per month or once every other week), once per week, twice per week, or three times per week.
  • the conjugate is administered as monotherapy and, more specifically, without an asparagine synthetase inhibitor.
  • the conjugate is administered as part of a combination therapy (but in some embodiments, the combination therapy does not comprise an asparagine synthetase inhibitor).
  • the patient receiving treatment has had a previous hypersensitivity to an E. coli asparaginase or PEGylated form thereof or to an Erwinia asparaginase.
  • the patient receiving treatment has had a disease relapse, in particular a relapse that occurs after treatment with an E. coli asparaginase or PEGylated form thereof.
  • FIG. 1 SDS-polyacrylamide gel electrophoresis of purified recombinant Erwinia chrysanthemi L-asparaginase.
  • Purified recombinant Erwinia chrysanthemi L-asparaginase (r-crisantaspase) was analyzed on SDS-PAGE. Protein bands were stained with silver nitrate.
  • Lane 1 Molecular Weight Marker (116, 66.2, 45, 35, 25, 18.4, and 14.4 kDa)
  • lane 2 purified recombinant Erwinia chrysanthemi L-asparaginase (r-crisantaspase).
  • FIG. 2 SDS-PAGE analysis of mPEG-r-crisantaspase conjugates.
  • FIG. 3 Plasma L-asparagine levels following a single intravenous dose of Erwinase® (5 U/kg, 25 U/kg, 125 U/kg and 250 U/kg body weight).
  • FIG. 4 Plasma L-asparagine levels following a single intravenous injection of mPEG-r-crisantaspase conjugates compared to Erwinase® in mice.
  • the numbers “40%” and “100%” indicate an approximate degree of PEGylation of, respectively, about 40-55% (partially PEGylated) and about 100% (maximally PEGylated) of the accessible amino groups.
  • FIG. 5 Area under the curves (AUC) (residual enzymatic activity) calculated from L-asparaginase profiles following a single intravenous injection of mPEG-r-crisantaspase conjugates in mice.
  • AUC Area under the curves
  • FIG. 6 Plasma L-asparagine levels following a single intravenous dose in mice of 2 kDa-100% mPEG-r-crisantaspase (5 U/kg, 25 U/kg and 50 U/kg body weight) ( FIG. 6A ), 5 kDa-100% mPEG-r-crisantaspase (5 U/kg, 25 U/kg and 50 U/kg body weight) ( FIG. 6B ), or 2 kDa-100% mPEG-r-crisantaspase (5 U/kg), 5 kDa-100% mPEG-r-crisantaspase (5 U/kg), and pegaspargase (Oncaspar®) (1 U/kg) ( FIG. 6C ).
  • FIG. 7 Dose-effect Relationship of 2 kDa-100% PEGylated r-crisantaspase compared to 5 kDa-100% PEGylated r-crisantaspase.
  • FIG. 7A shows the residual enzymatic activity in plasma following a single intravenous dose of 2 kDa-100% PEGylated r-crisantaspase at 5 U/kg (10 ⁇ g/kg on a protein content basis), 25 U/kg, and 50 U/kg.
  • FIG. 7A shows the residual enzymatic activity in plasma following a single intravenous dose of 2 kDa-100% PEGylated r-crisantaspase at 5 U/kg (10 ⁇ g/kg on a protein content basis), 25 U/kg, and 50 U/kg.
  • 7B shows the residual enzymatic activity in plasma following a single intravenous dose of 5 kDa-100% PEGylated r-crisantaspase at 5 U/kg (10 ⁇ g/kg on a protein content basis), 25 U/kg, and 50 U/kg.
  • FIG. 8 Dose-effect relationship of 2 kDa-100% PEGylated r-crisantaspase compared to 5 kDa-100% PEGylated r-crisantaspase. AUCs of the residual enzymatic activity measured in mice after a single intravenous dose of 2 kDa-100% or 5 kDa-100% mPEG-conjugates. Overall, when compared at the same dose level, AUCs measured for the 5 kDa-100% mPEG-r-crisantaspase were higher than those observed for the 2-kDa-100% mPEG-r-crisantaspase. A difference of 31, 37, and 14% was observed at 5, 25, and 50 U/kg doses, respectively.
  • FIG. 9 Pharmacokinetics of mPEG-r-crisantaspase conjugates vs. pegaspargase (Oncaspar®) in mice.
  • FIG. 9A represents the residual enzymatic activity measured in mice after a single intravenous dose of 2 kDa-100% mPEG-r-crisantaspase, 5 kDa-100% mPEG-r-crisantaspase, or pegaspargase (Oncaspar®).
  • FIG. 9A represents the residual enzymatic activity measured in mice after a single intravenous dose of 2 kDa-100% mPEG-r-crisantaspase, 5 kDa-100% mPEG-r-crisantaspase, or pegaspargase (Oncaspar®).
  • FIG. 9A represents the residual enzymatic activity measured in mice after a single intravenous dose of 2 kDa-100% mPEG-r-crisantaspase,
  • 9B represents AUCs of the residual enzymatic activity measured in mice after a single intravenous dose of 2 kDa-100% mPEG-r-crisantaspase, 5 kDa-100% mPEG-r-crisantaspase, or pegaspargase (Oncaspar®).
  • FIG. 11 Serum levels of anti-conjugate specific antibodies after treatment with mPEG-r-crisantaspase maximally (100%) PEGylated conjugates.
  • FIG. 11B results presented as the percentage of animals with absorbance values >0.5 in the anti-conjugate ELISA.
  • the problem to be solved by the invention is to provide an L-asparaginase preparation with:
  • This problem is solved according to the present invention by providing a conjugate of Erwinia L-asparaginase with a hydrophilic polymer, more specifically, a polyethylene glycol with a molecular weight of 5000 Da or less, a method for preparing such a conjugate and the use of the conjugate.
  • Described herein is a PEGylated L-asparaginase from Erwinia with improved pharmacological properties as compared with the unmodified L-asparaginase protein, as well as compared to the pegaspargase preparation from E. coli .
  • the PEGylated L-asparaginase conjugate described herein e.g., Erwinia chrysanthemi L-asparaginase PEGylated with 5000 Da molecular weight PEG, serves as a therapeutic agent particularly for use in patients who show hypersensitivity (e.g., an allergic reaction or silent hypersensitivity) to treatment with L-asparaginase or PEGylated L-asparaginase from E.
  • the PEGylated L-asparaginase conjugate described herein is also useful as a therapeutic agent for use in patients who have had a disease relapse, e.g., a relapse of ALL, and have been previously treated with another form of asparaginase, e.g., with L-asparaginase or PEGylated L-asparaginase from E. coli.
  • the conjugate of the invention shows unexpectedly superior properties compared to known L-asparaginase preparations such as pegaspargase.
  • L-asparaginase preparations such as pegaspargase.
  • unmodified L-asparaginase from Erwinia chrysanthemi crisantaspase
  • has a significantly lower half-life than unmodified L-asparaginase from E. coli Avramis and Panosyan, Clin. Pharmacokinet . (2005) 44:367-393, incorporated herein by reference in its entirety.
  • the PEGylated conjugate of the invention has a half life that is greater than PEGylated L-asparaginase from E. coli at an equivalent protein dose.
  • disease treatable by depletion of asparagine refers to a condition or disorder wherein the cells involved in or responsible for the condition or disorder either lack or have a reduced ability to synthesize L-asparagine.
  • Depletion or deprivation of L-asparagine can be partial or substantially complete (e.g., to levels that are undetectable using methods and apparatus that are known in the art).
  • terapéuticaally effective amount refers to the amount of a protein (e.g., asparaginase or conjugate thereof), required to produce a desired therapeutic effect.
  • the protein according to the invention is an enzyme with L-asparagine aminohydrolase activity, namely an L-asparaginase.
  • L-asparaginase proteins have been identified in the art, isolated by known methods from microorganisms. (See, e.g., Savitri and Azmi, Indian J. Biotechnol 2 (2003) 184-194, incorporated herein by reference in its entirety). The most widely used and commercially available L-asparaginases are derived from E. coli or from Erwinia chrysanthemi , both of which share 50% or less structural homology.
  • Erwinia L-asparaginases include, for example, those provided in Table 1:
  • L-asparaginases used in therapy are L-asparaginase isolated from E. coli and from Erwinia , specifically, Erwinia chrysanthemi.
  • the L-asparaginases may be native enzymes isolated from the microorganisms. They can also be produced by recombinant enzyme technologies in producing microorganisms such as E. coli .
  • the protein used in the conjugate of the invention can be a protein form E. coli produced in a recombinant E. coli producing strain, of a protein from an Erwinia species, particularly Erwinia chrysanthemi , produced in a recombinant E. coli producing strain.
  • Enzymes can be identified by their specific activities. This definition thus includes all polypeptides that have the defined specific activity also present in other organisms, more particularly in other microorganisms. Often enzymes with similar activities can be identified by their grouping to certain families defined as PFAM or COG.
  • PFAM protein family database of alignments and hidden Markov models; http://pfam.sanger.ac.uk/) represents a large collection of protein sequence alignments. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures.
  • COGs Clusters of Orthologous Groups of proteins; http://www.ncbi.nlm.nih.gov/COG/) are obtained by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenetic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.
  • the means of identifying homologous sequences and their percentage homology and/or identity are well known to those skilled in the art, and include in particular the BLAST programs, which can be used from the website http://blast.ncbi.nlm.nih.gov/Blast.cgi with the default parameters indicated on that website.
  • the sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW (http://www.ebi.ac.uk/Tools/clustalw2/index.html) or MULTALIN (http://bioinfo.genotoul.fr/multalin/muitalin.html) with the default parameters indicated on those websites.
  • a Nessler assay is used for the determination of L-asparaginase activity according to a 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).
  • the 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:
  • amino-acid sequence of the protein may not be strictly limited to SEQ ID NO:1 but may contain additional amino-acids.
  • the protein is the L-asparaginase of Erwinia chrysanthemi having the sequence of SEQ ID NO: 1.
  • the L-asparaginase is from Erwinia chrysanthemi NCPPB 1066 (Genbank Accession No. CAA32884, incorporated herein by reference in its entirety), either with or without signal peptides and/or leader sequences.
  • Fragments of the protein of SEQ ID NO:1 are also comprised within the definition of the protein used in the conjugate of the invention.
  • the term “a fragment of SEQ ID NO:1” means that the sequence of the polypeptide may include less amino-acid than SEQ ID N01 but still enough amino-acids to confer L-aminohydrolase activity.
  • a polypeptide can be modified by substitution, insertion, deletion and/or addition of one or more amino-acids while retaining its enzymatic activity. For example, substitution of one amino-acid at a given position by a chemically equivalent amino-acid that does not affect the functional properties of a protein is common. Substitutions may be defined as exchanges within one of the following groups:
  • the positions where the amino-acids are modified and the number of amino-acids subject to modification in the amino-acid sequence are not particularly limited. The skilled artisan is able to recognize the modifications that can be introduced without affecting the activity of the protein. For example, modifications in the N- or C-terminal portion of a protein may be expected not to alter the activity of a protein under certain circumstances. With respect to asparaginases, in particular, much characterization has been done, particularly with respect to the sequences, structures, and the residues forming the active catalytic site. This provides guidance with respect to residues that can be modified without affecting the activity of the enzyme. All known L-asparaginases from bacterial sources have common structural features.
  • coli L-asparaginase II (Papageorgiou et al., FEBS J. 275 (2008) 4306-4316).
  • the active site flexible loop contains amino acid residues 14-33, and structural analysis show that Thr15, Thr95, Ser62, Glu63, Asp96, and Ala120 contact the ligand (Papageorgiou et al., FEBS J. 275 (2008) 4306-4316).
  • Aghaipour et al. have conducted a detailed analysis of the four active sites of Erwinia chrysanthemi L-asparaginase by examining high resolution crystal structures of the enzyme complexed with its substrates (Aghaipour et al., Biochemistry 40 (2001) 5655-5664).
  • Kotzia et al. provide sequences for L-asparaginases from several species and subspecies of Erwinia and, even though the proteins have only about 75-77% identity between Erwinia chrysanthemi and Erwinia carotovora , they each still have L-asparaginase activity (Kotzia et al., J. Biotechnol. 127 (2007) 657-669, incorporated herein by reference in its entirety). Moola et al.
  • Polymers are selected from the group of non-toxic water soluble polymers such as polysaccharides, e.g. hydroxyethyl starch, poly amino acids, e.g. poly lysine, polyester, e.g., polylactic acid, and poly alkylene oxides, e.g., polyethylene glycol (PEG).
  • non-toxic water soluble polymers such as polysaccharides, e.g. hydroxyethyl starch, poly amino acids, e.g. poly lysine, polyester, e.g., polylactic acid, and poly alkylene oxides, e.g., polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Polyethylene glycol (PEG) or mono-methoxy-polyethyleneglycol (mPEG) is well known in the art and comprises linear and branched polymers. Examples of some polymers, particularly PEG, are provided in the following, each of which is herein incorporated by reference in its entirety: U.S. Pat. No. 5,672,662; U.S. Pat. No. 4,179,337; U.S. Pat. No. 5,252,714; US Pat. Appl. Publ. No. 2003/0114647; U.S. Pat. No. 6,113,906; U.S. Pat. No. 7,419,600; and PCT Publ. No. WO2004/083258.
  • the quality of such polymers is characterized by the polydispersity index (PDI).
  • PDI polydispersity index
  • the PDI reflects the distribution of molecular weights in a given polymer sample and is calculated from the weight average molecular weight divided by the number average molecular weight. It indicates the distribution of individual molecular weights in a batch of polymers.
  • the polyethylene glycol has advantageously a molecular weight comprised within the range of about 500 Da to about 9,000 Da. More specifically, the polyethylene glycol (e.g, mPEG) has a molecular weight selected from the group consisting of polyethylene glycols of 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da, and 5000 Da. In a particular embodiment, the polyethylene glycol (e.g., mPEG) has a molecular weight of 5000 Da.
  • the polymer moiety contains an activated functionality that preferably reacts with amino groups in the protein.
  • the invention is directed to a method of making a conjugate, the method comprising combining an amount of polyethylene glycol (PEG) with an amount of L-asparaginase in a buffered solution for a time period sufficient to covalently link the PEG to the L-asparaginase.
  • PEG polyethylene glycol
  • the L-asparaginase is from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the PEG is monomethoxy-polyethylene glycol (mPEG).
  • the reaction between the polyethylene glycol and L-asparaginase is performed in a buffered solution.
  • the pH value of the buffer solution ranges between about 7.0 and about 9.0.
  • the most preferred pH value ranges between about 7.5 and about 8.5, e.g., a pH value of about 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.
  • the L-asparaginase is from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • PEGylation of L-asparaginase is performed at protein concentrations between about 0.5 and about 25 mg/mL, more specifically between about 2 and about 20 mg/mL and most specifically between about 3 and about 15 mg/mL.
  • the protein concentration is about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/mL.
  • the PEGylation of L-asparaginase at these protein concentrations is of Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the PEGylation reaction proceeds rapidly, within less than 2 hours.
  • a molar excess of polymer over amino groups in L-asparaginase of less than about 20:1 is applied.
  • the molar excess is less than about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7.5:1, 7:1, 6.5:1, 6:1, 5.5:1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.5:1, or 1:1.
  • the molar excess is less than about 10:1 and in a more specific embodiment, the molar excess is less than about 8:1.
  • the L-asparaginase is from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the number of PEG moieties which can be coupled to the protein will be subject to the number of free amino groups and, even more so, to which amino groups are accessible for a PEGylation reaction.
  • the degree of PEGylation i.e., the number of PEG moieties coupled to amino groups on the L-asparaginase
  • 100% PEGylation of accessible amino groups is also referred to herein as “maximally PEGylated.”
  • One method to determine the modified amino groups in mPEG-r-crisantaspase conjugates is a method described by Habeeb (A.F.S.A. Habeeb, “Determination of free amino groups in proteins by trinitrobenzensulfonic acid”, Anal. Biochem. 14 (1966), p. 328, incorporated herein by reference in its entirety).
  • the PEG moieties are coupled to one or more amino groups (wherein amino groups include lysine residues and/or the N-terminus) of the L-asparaginase.
  • the degree of PEGylation is within a range of from about 10% to about 100% of total or accessible amino groups (e.g., lysine residues and/or the N-terminus), e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
  • the accessible amino groups e.g., lysine residues and/or the N-terminus
  • the accessible amino groups e.g., lysine residues and/or the N-terminus
  • the PEG moieties are coupled to the L-asparaginase by a covalent linkage.
  • the L-asparaginase is from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate of the invention can be represented by the formula
  • Asp is a L-asparaginase protein
  • NH is the NH group of a lysine residue and/or the N-terminus of the protein chain
  • PEG is a polyethylene glycol moiety
  • n is a number of at least 40% to about 100% of the accessible amino groups (e.g., lysine residues and/or the N-terminus) in the protein, all being defined above and below in the examples
  • x is an integer ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), preferably 2 to 5 (e.g., 2, 3, 4, 5).
  • the L-asparaginase is from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • proteins having substantial L-Asparagine aminohydrolase activity and polyethylene glycol selected from the group of conjugates wherein:
  • Conjugates of the invention have certain advantageous and unexpected properties compared to unmodified L-asparaginases, particularly compared to unmodified Erwinia L-asparaginases, more particularly compared to unmodified L-asparaginase from Erwinia chrysanthemi , and more particularly compared to unmodified L-asparaginase having the sequence of SEQ ID NO:1.
  • the conjugate of the invention reduces plasma L-asparagine levels for a time period of at least about 12, 24, 48, 72, 96, or 120 hours when administered at a dose of 5 U/kg body weight (bw) or 10 ⁇ g/kg (protein content basis). In other embodiments, the conjugate of the invention reduces plasma L-asparagine levels to undetectable levels for a time period of at least about 12, 24, 48, 72, 96, 120, or 144 hours when administered at a dose of 25 U/kg bw or 50 ⁇ g/kg (protein content basis).
  • the conjugate of the invention reduces plasma L-asparagine levels for a time period of at least about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours when administered at a dose of 50 U/kg bw or 100 ⁇ g/kg (protein content basis).
  • the conjugate of the invention reduces plasma L-asparagine levels to undetectable levels for a time period of at least about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours when administered at a dose ranging from about 10,000 to about 15,000 IU/m 2 (about 20-30 mg protein/m 2 ).
  • the conjugate comprises L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da.
  • at least about 40% to about 100% of accessible amino groups are PEGylated.
  • the conjugate comprises a ratio of mol PEG/mol monomer of about 4.5 to about 8.5, particularly about 6.5; a specific activity of about 450 to about 550 U/mg, particularly about 501 U/mg; and a relative activity of about 75% to about 85%, particularly about 81% compared to the corresponding unmodified L-asparaginase.
  • the conjugate with these properties comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1, with PEGylation of approximately 40-55% accessible amino groups (e.g., lysine residues and/or the N-terminus) with 5000 Da mPEG.
  • the conjugate comprises a ratio of mol PEG/mol monomer of about 12.0 to about 18.0, particularly about 15.1; a specific activity of about 450 to about 550 U/mg, particularly about 483 U/mg; and a relative activity of about 75 to about 85%, particularly about 78% compared to the corresponding unmodified L-asparaginase.
  • the conjugate with these properties comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1, with PEGylation of approximately 100% accessible amino groups (e.g., lysine residues and/or the N-terminus) with 5000 Da mPEG.
  • the conjugate comprises a ratio of mol PEG/mol monomer of about 5.0 to about 9.0, particularly about 7.0; a specific activity of about 450 to about 550 U/mg, particularly about 501 U/mg; and a relative activity of about 80 to about 90%, particularly about 87% compared to the corresponding unmodified L-asparaginase.
  • the conjugate with these properties comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1, with PEGylation of approximately 40-55% accessible amino groups (e.g., lysine residues and/or the N-terminus) with 10,000 Da mPEG.
  • the conjugate comprises a ratio of mol PEG/mol monomer of about 11.0 to about 17.0, particularly about 14.1; a specific activity of about 450 to about 550 U/mg, particularly about 541 U/mg; and a relative activity of about 80 to about 90%, particularly about 87% compared to the corresponding unmodified L-asparaginase.
  • the conjugate with these properties comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1, with PEGylation of approximately 100% accessible amino groups (e.g., lysine residues and/or the N-terminus) with 10,000 Da mPEG.
  • the conjugate comprises a ratio of mol PEG/mol monomer of about 6.5 to about 10.5, particularly about 8.5; a specific activity of about 450 to about 550 U/mg, particularly about 524 U/mg; and a relative activity of about 80 to about 90%, particularly about 84% compared to the corresponding unmodified L-asparaginase.
  • the conjugate with these properties comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1, with PEGylation of approximately 40-55% accessible amino groups (e.g., lysine residues and/or the N-terminus) with 2,000 Da mPEG.
  • the conjugate comprises a ratio of mol PEG/mol monomer of about 12.5 to about 18.5, particularly about 15.5; a specific activity of about 450 to about 550 U/mg, particularly about 515 U/mg; and a relative activity of about 80 to about 90%, particularly about 83% compared to the corresponding unmodified L-asparaginase.
  • the conjugate with these properties comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1, with PEGylation of approximately 100% accessible amino groups (e.g., lysine residues and/or the N-terminus) with 2,000 Da mPEG.
  • the conjugate of the invention has an increased potency of at least about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times after a single injection compared to the corresponding unmodified L-asparaginase.
  • the conjugate with these properties comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da.
  • at least about 40% to about 100% of accessible amino groups are PEGylated.
  • the conjugate of the invention has a pharmacokinetic profile according to the following parameters:
  • the half-life time of the residual enzyme activity in plasma is derived from the following formula:
  • AUC Area under the curve
  • the conjugate of the invention has a single-dose pharmacokinetic profile according to the following, specifically wherein the conjugate comprises mPEG at molecular weight of less than or equal to 2000 Da and an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1:
  • the conjugate of the invention has a single-dose pharmacokinetic profile according to the following, specifically where the conjugate comprises mPEG at molecular weight of less than or equal to 5000 Da and an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1:
  • the conjugate of the invention results in a similar level of L-asparagine depletion over a period of time (e.g., 24, 48, or 72 hours) after a single dose compared to an equivalent quantity of protein of pegaspargase.
  • the conjugate comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da.
  • at least about 40% to about 100% of accessible amino groups are PEGylated, more particularly about 40-55% or 100%.
  • the conjugate of the invention has a longer t 1/2 than pegaspargase administered at an equivalent protein dose.
  • the conjugate has a t 1/2 of at least about 50, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, or 65 hours at a dose of about 50 ⁇ g/kg (protein content basis).
  • the conjugate has a t 1/2 of at least about 30, 32, 34, 36, 37, 38, 39, or 40 hours at a dose of about 10 ⁇ g/kg (protein content basis).
  • the conjugate has a t 1/2 of at least about 100 to about 200 hours at a dose ranging from about 10,000 to about 15,000 IU/m 2 (about 20-30 mg protein/m 2 ).
  • the conjugate of the invention has a mean AUC that is at least about 2, 3, 4 or 5 times greater than pegaspargase at an equivalent protein dose.
  • the conjugate of the invention does not raise any significant antibody response for a particular period of time after administration of a single dose, e.g, greater 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, etc. In a particular embodiment the conjugate of the invention does not raise any significant antibody response for at least 8 weeks.
  • “does not raise any significant antibody response” means that the subject receiving the conjugate is identified within art-recognized parameters as “antibody-negative.”
  • Antibody levels can be determined by methods known in the art, for example ELISA or surface plasmon resonance (SPR-Biacore) assays (Zalewska-Szewczyk et al., Clin. Exp. Med . (2009) 9:113-116; Avramis et al., Anticancer Research 29 (2009) 299-302, each of which is incorporated herein by reference in its entirety).
  • Conjugates of the invention may have any combination of these properties.
  • the conjugates of the invention can be used in the treatment of a disease treatable by depletion of asparagine.
  • the conjugate is useful in the treatment or the manufacture of a medicament for use in the treatment of acute lymphoblastic leukemia (ALL) in both adults and children, as well as other conditions where asparagine depletion is expected to have a useful effect.
  • ALL acute lymphoblastic leukemia
  • Such conditions include, but are not limited to the following: malignancies, or cancers, including but not limited to hematologic malignancies, non-Hodgkin's lymphoma, NK lymphoma, pancreatic cancer, Hodgkin's disease, acute myelocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma.
  • Representative non-malignant hematologic diseases which respond to asparagine depletion include immune system-mediated blood diseases, e.g., infectious diseases such as those caused by HIV infection (i.e., AIDS).
  • Non-hematologic diseases associated with asparagine dependence include autoimmune diseases, for example rheumatoid arthritis, SLE, autoimmune, collagen vascular diseases, AIDS, etc.
  • Other autoimmune diseases include osteoarthritis, Issac's syndrome, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus, rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), primary billiary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease.
  • autoimmune diseases for example rheumatoid arthritis, SLE, autoimmune, collagen vascular diseases, AIDS, etc.
  • Other autoimmune diseases include osteoarthritis, Issac's syndrome, psoriasis, insulin
  • the invention is directed to a method of treating a disease treatable in a patient, the method comprising administering to the patient an effective amount of a conjugate of the invention.
  • the disease is ALL.
  • the conjugate used in the treatment of a disease treatable by asparagine depletion comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da.
  • at least about 40% to about 100% of accessible amino groups are PEGylated, more particularly about 40-55% or 100%.
  • treatment with a conjugate of the invention will be administered as a first line therapy.
  • treatment with a conjugate of the invention will be administered as a second line therapy in patients, particularly patients with ALL, where objective signs of allergy or hypersensitivity, including “silent hypersensitivity,” have developed to other asparaginase preparations, in particular, the native Escherichia coli -derived L-asparaginase or its PEGylated variant (pegaspargase).
  • objective signs of allergy or hypersensitivity include testing “antibody positive” for an asparaginase enzyme.
  • the conjugate of the invention is used in second line therapy after treatment with pegaspargase.
  • the conjugate used in second line therapy comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate further comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da, more specifically about 5000 Da.
  • PEG e.g., mPEG
  • at least about 40% to about 100% of accessible amino groups are PEGylated, more particularly about 40-55% or 100%.
  • the invention is directed to a method for treating acute lymphoblastic leukemia comprising administering to a patient in need of the treatment a therapeutically effective amount of a conjugate of the invention.
  • treatment will be administered at a dose ranging from about 1500 IU/m 2 to about 15,000 IU/m 2 , typically about 10,000 to about 15,000 IU/m 2 (about 20-30 mg protein/m 2 ), at a schedule ranging from about twice a week to about once a month, typically once per week or once every other week, as a single agent (e.g., monotherapy) or as part of a combination of chemotherapy drugs, including, but not limited to glucocorticoids, corticosteroids, anticancer compounds or other agents, including, but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracycline.
  • the conjugate of the invention as a component of multi-agent chemotherapy during 3 chemotherapy phases including induction, consolidation or intensification, and maintenance.
  • the conjugate is not administered with an asparagine synthetase inhibitor (e.g., such as set forth in PCT Pub. No. WO 2007/103290, which is herein incorporated by reference in its entirety).
  • the conjugate is not administered with an asparagine synthetase inhibitor, but is administered with other chemotherapy drugs.
  • the conjugate can be administered before, after, or simultaneously with other compounds as part of a multi-agent chemotherapy regimen.
  • the conjugate comprises L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da.
  • at least about 40% to about 100% of accessible amino groups are PEGylated, more particularly about 40-55% or 100%.
  • the method comprises administering a conjugate of the invention at an amount of about 1 U/kg to about 25 U/kg (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 U/kg) or an equivalent amount thereof (e.g., on a protein content basis).
  • the conjugate is administered at an amount selected from the group consisting of about 5, about 10, and about 25 U/kg.
  • the conjugate is administered at a dose ranging from about 1,000 IU/m 2 to about 20,000 IU/m 2 (e.g., 1,000 IU/m 2 , 2,000 IU/m 2 , 3,000 IU/m 2 , 4,000 IU/m 2 , 5,000 IU/m 2 , 6,000 IU/m 2 7,000 IU/m 2 , 8,000 IU/m 2 , 9,000 IU/m 2 , 10,000 IU/m 2 , 11,000 IU/m 2 , 12,000 IU/m 2 13,000 IU/m 2 14,000 IU/m 2 , 15,000 IU/m 2 , 16,000 IU/m 2 , 17,000 IU/m 2 , 18,000 IU/m 2 , 19,000 IU/m 2 , or 20,000 IU/m 2 ).
  • 20,000 IU/m 2 e.g., 1,000 IU/m 2 , 2,000 IU/m 2 , 3,000 IU/m 2 , 4,000 IU/m
  • the conjugate is administered at a dose that depletes L-asparagine to undetectable levels using methods and apparatus known in the art for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) for a single dose.
  • the method comprises administering a conjugate of the invention that elicits a lower immunogenic response in a patient compared to an unconjugated L-asparaginase.
  • the method comprises administering a conjugate of the invention that has a longer in vivo circulating half-life after a single dose compared to the unconjugated L-asparaginase.
  • the method comprises administering a conjugate that has a longer t 112 than pegaspargase administered at an equivalent protein dose.
  • the method comprises administering a conjugate that has a t 1/2 of at least about 50, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, or 65 hours at a dose of about 50 ⁇ g/kg (protein content basis).
  • the method comprises administering a conjugate that has a t 1/2 of at least about 30, 32, 34, 36, 37, 37, 39, or 40 hours at a dose of about 10 ⁇ g/kg (protein content basis).
  • the method comprises administering a conjugate that has a t 1/2 of at least about 100 to about 200 hours at a dose ranging from about 10,000 to about 15,000 IU/m 2 (about 20-30 mg protein/m 2 ). In one embodiment, the method comprises administering a conjugate that has a mean AUC that is at least about 2, 3, 4 or 5 times greater than pegaspargase at an equivalent protein dose. In another embodiment, the method comprises administering a conjugate of the invention that has a greater AUC value after a single dose compared to the unconjugated L-asparaginase.
  • the conjugate comprises L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da.
  • at least about 40% to about 100% of accessible amino groups are PEGylated, more particularly about 40-55% or 100%.
  • the conjugate of the invention may be used in a method of treating patients with relapsed ALL who were previously treated with other asparaginase preparations, in particular those who were previously treated with E. coli -derived asparaginases.
  • the uses and methods of treatment of the invention comprise administering an L-asparaginase conjugate having properties or combinations of properties described herein above (e.g., in the section entitled “L-asparaginase PEG conjugates”) or herein below.
  • compositions Compositions, Formulations, and Routes of Administration
  • the invention also includes a pharmaceutical composition comprising a conjugate of the invention.
  • the pharmaceutical composition is contained in a vial as a lyophilized powder to be reconstituted with a solvent, such as currently available native L-asparaginases, whatever the bacterial source used for its production (Kidrolase®, Elspar®, Erwinase® . . . ).
  • the pharmaceutical composition is a “ready to use” solution, such as pegaspargase (Oncaspar®) enabling, further to an appropriate handling, an administration through, e.g., intramuscular, intravenous (infusion and/or bolus), intra-cerebroventricular (icy), sub-cutaneous routes.
  • Conjugates of the invention including compositions comprising conjugates of the invention (e.g., a pharmaceutical composition) can be administered to a patient using standard techniques. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa., 1990 (herein incorporated by reference).
  • Suitable dosage forms depend upon the use or the route of entry, for example, oral, transdermal, transmucosal, or by injection (parenteral). Such dosage forms should allow the therapeutic agent to reach a target cell or otherwise have the desired therapeutic effect.
  • pharmaceutical compositions injected into the blood stream preferably are soluble.
  • Conjugates and/or pharmaceutical compositions according to the invention can be formulated as pharmaceutically acceptable salts and complexes thereof.
  • Pharmaceutically acceptable salts are non-toxic salts present in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate pharmaceutical use by altering the physical characteristics of the compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administering higher concentrations of the drug.
  • the pharmaceutically acceptable salt of an asparaginase may be present as a complex, as those in the art will appreciate.
  • 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 can be obtained from acids, including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
  • acids including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
  • Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present.
  • basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present.
  • acidic functional groups such as carboxylic acid or phenol are present.
  • Pharmaceutically acceptable carriers and/or excipients can also be incorporated into a pharmaceutical composition according to the invention to facilitate administration of the particular asparaginase.
  • carriers suitable for use in the practice of the invention include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents.
  • physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution and dextrose.
  • compositions according to the invention can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration.
  • oral administration is preferred.
  • the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.
  • injection parenteral administration
  • pharmaceutical compositions are formulated in liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution.
  • the compounds may be formulated in solid form and redissolved or suspended immediately prior to use.
  • lyophilized forms of the conjugate can be produced.
  • the conjugate is administered intramuscularly.
  • the conjugate is administered intravenously.
  • Systemic administration can also be accomplished by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are well known in the art, and include, for example, for transmucosal administration, bile salts, and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration for example, may be through nasal sprays, inhalers (for pulmonary delivery), rectal suppositories, or vaginal suppositories.
  • compounds can be formulated into ointments, salves, gels, or creams, as is well known in the art.
  • the amounts of the conjugate to be delivered will depend on many factors, for example, the IC 50 , EC 50 , the biological half-life of the compound, the age, size, weight, and physical condition of the patient, and the disease or disorder to be treated. The importance of these and other factors to be considered are well known to those of ordinary skill in the art.
  • the amount of the conjugate to be administered will range from about 10 International Units per square meter of the surface area of the patient's body (IU/m 2 ) to 50,000 IU/m 2 , with a dosage range of about 1,000 IU/m 2 to about 15,000 IU/m 2 being preferred, and a range of about 6,000 IU/m 2 to about 15,000 IU/m 2 being more preferred, and a range of about 10,000 to about 15,000 IU/m 2 (about 20-30 mg protein/m 2 ) being particularly preferred to treat a malignant hematologic disease, e.g., leukemia.
  • a malignant hematologic disease e.g., leukemia.
  • these dosages are administered via intramuscular or intravenous injection at an interval of about 3 times weekly to about once per month, typically once per week or once every other week during the course of therapy.
  • other dosages and/or treatment regimens may be employed, as determined by the attending physician.
  • the conjugate and/or pharmaceutical composition or formulation to be administered as described herein comprises L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate comprises L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi , and more specifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.
  • the conjugate comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da.
  • at least about 40% to about 100% of amino groups are PEGylated.
  • 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 (NM1100) 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).
  • NM1100 bacterial strain
  • FRT FLP recognition target sequence
  • Recombination occurs to replace the ansB gene by the kanamycin cassette in the bacterial genome, resulting in a ⁇ ansB strain; 2) phage transduction was used to integrate the integrated kanamycin cassette region from the ⁇ ansB NM1100 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 ⁇ ansB strain) was sequenced, confirming full deletion of the endogenous ansB gene.
  • the E. coli - 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 ⁇ -D-1-thiogalactopyranoside (IPTG) and confers resistance to kanamycin.
  • IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
  • BL21 ⁇ ansB 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 16 h at 23° C. with IPTG as inducer.
  • the theoretical isoelectric point of the Erwinia chrysanthemi L-asparaginase permits the recombinant enzyme to adsorb to cation exchange resins at pH6.
  • 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 200 pg size exclusion chromatography as polishing step. Fractions containing recombinant enzymes were pooled, concentrated, and diafiltered against 100 mM sodium phosphate buffer pH8. The purity of the final Erwinia chrysanthemi L-asparaginase preparation was evaluated by SDS-PAGE ( FIG. 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 1 ⁇ mol of ammonia from L-asparagine per minute at 37° C.
  • 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 ⁇ kta 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 FIG. 2 .
  • the resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-
  • 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 ⁇ kta 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 FIG. 2 .
  • the resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-cri
  • 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 ⁇ kta 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 FIG. 2 .
  • the resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisant
  • 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, 50 ⁇ L of enzyme solution were mixed with 20 mM 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 200 ⁇ L 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:
  • 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.
  • Erwinase® The pharmacodynamic profile of Erwinase® was determined in B6D2F1-Hybrids (immune competent, females), Charles River Germany.
  • Erwinase® is a commercially available crisantaspase (L-asparaginase derived from Erwinia chrysanthemi ). Briefly, 2 animals per group received a single i.v. injection of 5, 25, 125, or 250 Units/kg bw Erwinase® At ⁇ 1 h pre-dose and at 6 h, 12 h, 24 h, and 48 h post-dose, plasma samples were collected from orbital sinus and analyzed for plasma levels of L-asparagine.
  • Plasma amino acid levels were determined with a PICO-TAG Amino Acid Analysis Kit (Waters). Briefly, plasma samples were deproteinised by methanol precipitation. Free amino acids in the supernatant were derivatised with phenylisothiocyanate and quantified by RP-HPLC.
  • the pharmacodynamic and pharmacokinetic profiles of 6 different mPEG-r-crisantaspase conjugates was determined in B6D2F1-Hybrids (immune competent, females), Charles River Germany The six conjugates tested differed in the molecular size of the PEG (2, 5 or 10 kDa) and in the degree of PEGylation (maximal vs. partial PEGylation). Unmodified crisantaspase (Erwinase®) was used as a reference. Briefly, 4 animals per group received a single i.v. injection of 5 Units/kg bw conjugate vs. 250 Units/kg bw Erwinase®.
  • plasma samples were collected from the orbital sinus of each animal and analyzed for plasma levels of L-asparagine and residual enzyme activity, respectively.
  • Plasma amino acid levels were determined with a PICO-TAG Amino Acid Analysis Kit (Waters). Briefly, plasma samples were deproteinised by methanol precipitation. Free amino acids in the supernatant were derivatised with phenylisothiocyanate and quantified by RP-HPLC.
  • Enzyme activity in plasma was determined by a chromogenic assay.
  • L-aspartic ⁇ -hydroxamate (AHA) was used as substrate.
  • the enzymes hydrolyzed AHA to L-Asp and hydroxylamine, which was determined at 710 nm after condensation with 8-hydroxyquinoline and oxidation to indooxine. (Analytical Biochemistry 309 (2002): 117-126, incorporated herein by reference in its entirety).
  • Enzymatic activity was consistent with L-asparagine depletion. As shown in FIG. 5 , the 5 kDa-100% conjugate exhibited the largest AUC, reflecting a longer half-life. Lower AUCs were observed with PEG-40% (partially PEGylated) vs. PEG-100% (maximally PEGylated) conjugates for the 2 kDa and 5 kDa candidates and no difference was observed for the 10 kDa candidates.
  • the pharmacodynamic profile of 2 mPEG-r-crisantaspase conjugates compared to pegaspargase (Oncaspar®) was determined in B6D2F1-Hybrids (immune competent, females), Charles River Germany The conjugates tested were the 2 kDa maximally (100%) PEGylated r-crisantaspase and the 5 kDa maximally (100%) PEGylated r-crisantaspase at 3 doses. Briefly, 8 animals per group received a single i.v. injection of 5, 25 or 50 Units/kg bw of the r-crisantaspase conjugates, corresponding to 10, 50 or 100 ⁇ g protein/kg.
  • Oncaspar® was tested at 1 Unit/kg, corresponding to 10 ⁇ g protein/kg.
  • 6 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 192 h and 240 h post-dose plasma samples were collected from orbital sinus and analyzed for plasma levels of L-asparagine.
  • Plasma amino acid levels were determined with a PICO-TAG Amino Acid Analysis Kit (Waters). Briefly, plasma samples were deproteinised by methanol precipitation. Free amino acids in the supernatant were derivatised with phenylisothiocyanate and quantified by RP-HPLC.
  • FIG. 6 The dose-related effects of the conjugates on plasma L-asparagine levels are shown in FIG. 6 .
  • both conjugates were highly efficient in depleting circulating L-asparagine.
  • total depletion was observed over 3, 6 and at least 10 days at the 5 U, 25 U and 50 U/kg doses, respectively.
  • total depletion was observed over 3, 10 and 10 days at the 5 U, 25 U and 50 U/kg doses, respectively.
  • the 5, 25 and 50 U/kg doses tested corresponded to 10, 50 and 100 ⁇ g/kg on a protein content basis, which is a very low amount of protein when compared to other available L-asparaginase preparations.
  • 250 U/kg Erwinase® corresponds to approximately 520 ⁇ g/kg
  • 1 U/kg Oncaspar® corresponds approximately to 10 ⁇ g/kg (protein content basis).
  • FIG. 6C shows that the administration of an equivalent quantity of protein (10 ⁇ g/kg) of either the 2 kDa-100% conjugate, the 5 kDa-100% conjugate or Oncaspar® resulted in a similar L-asparagine depletion over 72 hrs.
  • the pharmacokinetic profile of mPEG-r-crisantaspase conjugates was determined in B6D2F1-Hybrids (immune competent, females), Charles River Germany The conjugates tested were the 2 kDa maximally (100%) PEGylated r-crisantaspase and the 5 kDa maximally (100%) fully PEGylated r-crisantaspase at 3 doses. Unmodified crisantaspase (Erwinase®) at 250 U/kg and Oncaspar® at 1 U/kg were also tested as controls. Briefly, 8 animals per group received a single i.v.
  • Enzyme activity in plasma was determined by a chromogenic assay.
  • L-aspartic ⁇ -hydroxamate (AHA) was used as substrate.
  • the enzymes hydrolyzed AHA to L-Asp and hydroxylamine, which was determined at 710 nm after condensation with 8-hydroxyquinoline and oxidation to indooxine. ( Analytical Biochemistry 309 (2002): 117-126).
  • AUC areas under the curve
  • Table 5 summarizes pharmacokinetic and pharmacodynamic data gathered from several experiments, including those described in Examples 7-9 herein, showing that: 1) both the 2 kDa-100% and the 5 kDa-100% conjugates were highly potent in increasing potency and duration of action of crisantaspase, as shown by the marked differences observed compared to Erwinase®; 2) the 5 kDa-100% conjugate was longer-acting than both the 2 kDa-100% conjugate and Oncaspar®, as shown by a longer half-life observed at all doses tested.
  • the immunogenicity data showed that the 10 kDa-100% exhibited an unacceptable immunogenicity profile, a major drawback when considering administering the compound to patients who are allergic to E. coli L-asparaginase or have developed anti-L-asparaginase antibodies.
  • the 10 kDa-100% conjugate is really not suitable.
  • the 2 kDa-100% and the 5 kDa-100% are preferable, and the 5 kDa-100% conjugate is particularly preferable.
  • Immunogenicity of mPEG-r-crisantaspase conjugates was determined in B6D2F1-Hybrids (immune competent, females), Charles River Germany Animals were treated twice a week in weeks 1, 2, 3, 4, an 8 by i.v. injection of 250 U/kg bw for Erwinase® and 5 U/kg bw for all r-crisantaspase conjugates. Serum samples were collected at ⁇ 1 h pre-dose and after 1w, 2w, 4w, 6w and 8w from the orbital sinus. Anti-crisantaspase or anti-mPEG-r-crisantaspase antibody levels in serum were determined by ELISA. The results are summarized in FIGS. 10 and 11 .

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