WO2007083100A2 - Enzyme - Google Patents

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Publication number
WO2007083100A2
WO2007083100A2 PCT/GB2007/000124 GB2007000124W WO2007083100A2 WO 2007083100 A2 WO2007083100 A2 WO 2007083100A2 GB 2007000124 W GB2007000124 W GB 2007000124W WO 2007083100 A2 WO2007083100 A2 WO 2007083100A2
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Prior art keywords
asparaginase
aep
enzyme
coli
modified
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WO2007083100A3 (fr
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Vaskar Saha
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Cancer Research Technology Ltd
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Cancer Research Technology Ltd
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Priority claimed from GB0600862A external-priority patent/GB0600862D0/en
Priority claimed from GB0618149A external-priority patent/GB0618149D0/en
Application filed by Cancer Research Technology Ltd filed Critical Cancer Research Technology Ltd
Publication of WO2007083100A2 publication Critical patent/WO2007083100A2/fr
Publication of WO2007083100A3 publication Critical patent/WO2007083100A3/fr
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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)

Definitions

  • This invention relates to an enzyme that is suitable for therapeutic use.
  • ALL acute lymphoblastic leukaemia
  • Asparagine synthetase enzyme Most mammalian cells use an asparagine synthetase enzyme to make their own asparagine.
  • the enzyme takes aspartate and adds an amine thus forming asparagine.
  • some blood cells such as leukaemic blast cells instead rely on the blood for their supply of asparagine.
  • Asparaginase therapy in which recombinant purified L-asparaginase is administered to patients suffering from malignant haematological disease, takes advantage of this fact. When a large dose of this enzyme is introduced into the blood, it circulates and breaks-down asparagine, ultimately starving any cells that rely on the blood-borne supply (Capizzi et al, 1971).
  • E. coli Escherichia coli
  • Erwinia chi ⁇ santhemi also known as Erwinia carotovord
  • the amino acid sequences of the E. coli L-asparaginase and the Erwinia L-asparaginase are only 46% identical.
  • E. coli L-asparaginase has greater potency and a longer half-life than L-asparaginase from Erwinia ch ⁇ santhemi and hence is a better therapy (Albertsen et al, 2001).
  • L- asparaginases from both species are used therapeutically because the EEwinia L-asparaginase lacks cross-reactivity with E. coli enzyme.
  • E. coli L-asparaginase lacks cross-reactivity with E. coli enzyme.
  • patients that become resistant to E. coli L-asparaginase due to acquired immunity may still be treatable with Erwinia chrysanthemi L-asparaginase.
  • Erwinia L-asparaginase is commercially available as Erwinase from OPi Ltd., UK.
  • Native E. coli L- asparaginase is commercially available from Medac GmbH and from Merck & Co, USA.
  • US Patent No 6,251,388 describes an L-asparaginase enzyme from Wolinella succinogenes that is stated to be suitable for treating diseases that respond to L- asparaginase depletion.
  • the Wolinella L-asparaginase was predicted to lack cross- reactivity with E. coli L-asparaginase.
  • Pegaspargase In order to overcome resistance due to acquired immunity, polyethylene glycol (PEG) conjugation to L-asparaginase was developed to provide a form of the drug which is less likely to " cause the development of neutralizing antibodies.
  • Pegylated E. coli L-asparaginase is commercially available as Pegaspargase (Graham, 2003). Pegaspargase has been used to treat both paediatric and adult ALL, lymphoma, non-Hodgkin's lymphoma, AIDS related lymphoma, and advanced solid tumours (see, Section 5 "Clinical Trials" in Graham (2003), and references cited therein). Pegaspargase is commercially available from Enzon Pharmaceuticals as OncasparTM.
  • Lugthart et al (2005) also used micro-array technology to determine 45 genes associated with cross-resistance to the four anti-leukaemic drugs (including L-asparaginase) and 139 genes related to a phenotype of sensitive to vincristine but resistant to L-asparaginase. Nevertheless, these studies have not contributed to an improvement in the treatment of patients with ALL using prednisolone, vincristine, L-asparaginase and/or daunorubicin, or indeed any other therapeutic agent.
  • Chromosome 21 is a region of general genomic instability associated with acute leukaemias (Harewood et al, 2003), and some patients who are hyperdiploid for chromosome 21 demonstrate resistance to L-asparaginase therapy.
  • the inventor has identified a subset of patients who are hyperdiploid in only part of chromosome 21, and whose gene expression profile resembles that described for patients who demonstrate resistance to L-asparaginase therapy but appear resistant to vincristine (Lugthart et al, 2005). Genomic data from these patients was used to identify genes that may be involved in the development of, or propensity for, resistance to L-asparaginase.
  • LGMN human legumain
  • AEP asparaginyl endopeptidase
  • Asparaginyl endopeptidase (EC 3.4.22.34), also known as legumain, is a lysosomal cysteine protease that cleaves protein substrates on the C-terminal side of asparagine.
  • AEP has a strict specificity for cleavage of asparaginyl bonds at pH 5.8 (Chen et al, 1997) although it has also been shown to cleave aspartyl bonds slowly, especially under acidic conditions (Halfon et al (1998). This strict specificity is unusual among lysosomal enzymes.
  • AEP is also known to be up- regulated in a wide variety of solid tumours and its over-expression correlates with increased invasiveness (Liu et al, 2003). AEP was thus proposed for use as an enzymatic target for pro-drug therapy of solid tumours (Liu et al, 2003). AEP has also been shown to be involved in processing antigens for presentation within the class II MHC pathway (Manoury et al, 1998).
  • AEP expression is up-regulated and the protein is active in both leukaemic cell lines and in patient samples (see, Figures 3, 4 and 5).
  • the inventor has shown that AEP cleaves native E. coli L-asparaginase, thus contributing to resistance to this drug (see Figures 8 and 9 in Example X).
  • the inventor has also demonstrated that it is possible to modify the sequence of E. coli L-asparaginase so that it is not susceptible to cleavage by AEP ( Figure 10 in Example 3).
  • a modified E. coli L-asparaginase which is not susceptible to AEP cleavage will have a longer half-life in vivo than the native enzyme.
  • a modified E. coli L-asparaginase which is not susceptible to AEP cleavage will also be less allergenic than the native enzyme, since the AEP will no longer be able to cleave it and present it to T-cells.
  • the inventor has identified at least three potential AEP cleavage sites in E. coli L-asparaginase (see Figures 6 and 7), none of which are present in the Erwinia L-asparaginase. This is why the Erwinia L-asparaginase is not susceptible to AEP cleavage, and may explain why the Erwinia L-asparaginase can be substituted in patients with E. coli asparaginase hypersensitivity.
  • a first aspect of the invention thus provides a modified E, coli L-asparaginase II enzyme that is resistant to cleavage by human AEP.
  • the inactive prolegumain is processed to the active form of the AEP /legumain enzyme by autocatalytic activation (Chen et al, 2000) and/or by normal cellular processing (Li et al, 2003).
  • Methods for determining cleavage of a given polypeptide substrate by AEP/legumain are well known in the art and are provided, for example by Chen et al (1997).
  • Example 2 provides an example of a protocol for measuring cleavage of L-asparaginase by human AEP, and is suitable for determining the resistance of a modified E. coli L-asparaginase to cleavage by human AEP.
  • E. coli contains two L-asparaginase isoenzymes: L-asparaginase I, a low-affinity enzyme located in the cytoplasm, and L-asparaginase II, a high-affinity secreted enzyme.
  • E. coli L-asparaginase means the periplasmic form of the enzyme (type II), and not the cytoplasmic form of the enzyme (type I).
  • the periplasmic and cytoplasmic E. coli asparaginases only have about 30% amino acid identity.
  • E. coli L-asparaginase enzyme and by "native" E. coli L-asparaginase enzyme we mean the E. coli L-asparaginase II enzyme which has the amino acid sequence shown in Figure 7 (SEQ ID No: 1), and listed in Genbank Accession No P00805.
  • E. coli L-asparaginase enzyme that is resistant to cleavage by human AEP, we mean that the AEP is unable to cleave the enzyme at (the C- terminal side of) any one or more of the asparagine (Asn, N) or aspartate (Asp, D) residues within the L-asparaginase sequence (shown in Figures 6 and 7) that is normally cleaved by AEP.
  • the E. coli L-asparaginase enzyme is modified to be resistant to AEP cleavage by changing one or more of the Asn or Asp residues that is normally cleaved by AEP to another amino acid residue which is not susceptible to AEP cleavage.
  • a modified E. coli L-asparaginase enzyme that is resistant to cleavage by human AEP, we mean that the AEP is unable to cleave the L-asparaginase at (the C-terminal side of) a replacement residue which is present at any one or more of the positions in the native enzyme which is normally occupied by an Asn or Asp residue that is susceptible to AEP cleavage.
  • each replacement residue may, independently, be any of the naturally occurring amino acids which are encoded by DNA other than Asn or Asp, and is selected from alanine (Ala, A), arginine (Arg, R), cysteine (Cys, C), glutamine (GIn, Q), glutamate (GIu, E), glycine (GIy, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (VaI, V).
  • Figures 6 and 7 illustrate three potential AEP cleavage sites within E. coli L-asparaginase: Asn at position 46 (N46); Asn at position 165 (Nl 65) and Asp at position 146 (D146).
  • a preferred modified E. coli L-asparaginase enzyme that is resistant to cleavage by human AEP is one that the AEP is unable to cleave at the C-terminal side of any one or more of positions 46, 146 and 165 as shown in Figure 7 (SEQ ID No: 1).
  • the AEP is unable to cleave the asparaginase at the C-terminal side of residue 46, or at the C-terminal side of residue 165 or more preferably at the C-terminal side of both residues 46 and 165. Still more preferably, the AEP is unable to cleave the asparaginase at the C- terminal side of any of residues 46, 146 and 165.
  • the human AEP does not cleave the modified E: coli L-asparaginase enzyme at all, or does so at an undetectable level.
  • the Asn residue at position 46 of SEQ ID No: 1 (N46) of the modified E. coli L-asparaginase enzyme has been replaced with another amino acid residue. It is preferred if N46 has been replaced with a residue other than Asp, and preferably by a naturally occurring amino acid which is encoded by DNA. Suitable naturally occurring amino acids include Ala, Arg, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and VaI. It is more preferred if N46 has been replaced with a GIy residue. This is because GIy is present at the equivalent position in the Erwinia L-asparaginase.
  • the Asn residue at position 165 of SEQ ID No: 1 (Nl 65) of the modified E. coli L-asparaginase enzyme has been replaced with another amino acid residue. It is preferred if Nl 65 has been replaced with a residue other than Asp, and preferably by a naturally occurring amino acid which is encoded by DNA. Suitable naturally occurring amino acids include Ala, Arg, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and VaI. It is more preferred if Nl 65 has been replaced with a GIy residue. This is because GIy is present at the equivalent position in the Erwinia L-asparaginase.
  • both N46 and Nl 65 have been replaced with other naturally occurring amino acid residues which are encoded by DNA. It is preferred if both N46 and N165 have been replaced with residues other than Asp. It is more preferred if both N46 and Nl 65 have been replaced with GIy residues.
  • the Asp residue at position 146 of SEQ ID No: 1 is also replaced with another amino acid residue other than Asn, and preferably by a naturally occurring amino acid which is encoded by DNA.
  • Suitable naturally occurring amino acids include Ala, Arg, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tip, Tyr and VaI.
  • D146 is replaced with a GIy residue since GIy is present at the equivalent position in the Erwinia L- asparaginase.
  • Modified L-asparaginase enzymes thus include enzymes which comprise the mutations N46G, D146G and N165G; N46G and D146G; D146G arid N165G; and N46G and N165G.
  • L-asparaginase enzyme we do not necessarily mean that a "native" enzyme has been physically modified. Typically, the modified enzyme is made in its modified form.
  • the modified E. coli L- asparaginase is not limited to the amino acid sequence listed in Figure 7 (SEQ ID No: 1), modified at any of the Asp or Asn residues (especially at positions 46, 146 and 165) as described above, but includes naturally occurring variants of E. coli L- asparaginase II, that retain asparaginase activity, in which one or more of the Asp or Asn residues have been replaced with another amino acid.
  • the invention includes a modified enzyme that has at least 80% sequence identity, more preferably at least 85% or at least 90% sequence identity, and still more preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of native E. coli L-asparaginase (SEQ ID No: 1), and which is resistant to cleavage by AEP.
  • residues T34, Y47, S80, Till, Dl 12 and Kl 84 as numbered in SEQ ID No: 1, which define the active site of the enzyme remain unchanged.
  • residues T12, Y25, S58, T89, D90 and Kl 62 are equivalent to residues T12, Y25, S58, T89, D90 and Kl 62 numbered according to Kozak et al (2000) who studied a shorter variant of E. coli L-asparaginase II.
  • any one, two or all three of the residues R217, K218 and H219 as numbered in SEQ ID No: 1, which define an antigenic region of the enzyme, are replaced with less antigenic residues such as alanine.
  • These three residues are equivalent to residues Rl 95, Kl 96 and Hl 97 numbered according to Jianh.ua et al (2006) who studied the same shorter variant of E. coli L-asparaginase II as did Kozak et al (2000) supra.
  • the modified E. coli L-asparaginase may be a modified fragment of the full-length native E. coli L-asparaginase II polypeptide, which fragment retains asparaginase activity, in which one or more of the Asp or Asn residues have been replaced with another amino acid.
  • full- length native E. coli L-asparaginase II is 348 amino acid residues in length.
  • a 326 residue fragment of full-length native E. coli L-asparaginase II polypeptide that has asparaginase activity is described by Kozak et al (2000).
  • a suitable modified fragment is typically a contiguous portion of the native enzyme of at least 200 residues in length, more preferably at least 250 residues in length, and still more preferably at least 300, or at least 310, or at least 320, or at least 330, or at least 340 residues in length, or a sequence variant thereof.
  • any one or more of the Asp or Asn residues in the modified fragment may be replaced with another amino acid, it is preferred if one or more of the residues corresponding to N46, D146 and N165 of SEQ ID No: 1 have been replaced with another amino acid as described above.
  • the modified fragment preferably has at least 90% sequence identity with the corresponding portion of the native enzyme, and still more preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of the corresponding portion of the native E. coli L-asparaginase.
  • the modified fragment contains amino acid residues equivalent to T34, Y47, S80, Til l, Dl 12 and Kl 84 as numbered in SEQ ID No: 1, which define the active site of the enzyme.
  • the modified E. coli L-asparaginase enzyme that is resistant to cleavage by human AEP has the ability to hydrolyse L-asparagine to L-aspartate, i.e. it has asparaginase activity. This can be tested for and measured using methods well known in the art. Preferably, an immunoassay such as the Medac Asparaginase Activity Test (MAAT), commercially available form Medac , is used. Typically, the modified enzyme has at least 30% of the asparaginase activity of the native E. coli L-asparaginase enzyme.
  • MAAT Medac Asparaginase Activity Test
  • the modified enzyme has at least 50%, preferably at least 70%, more preferably at least 90%, and yet more preferably at least 99% of the asparaginase activity of the native enzyme. Most preferably, the modified enzyme has 100% or more of the asparaginase activity of the native E. coli L-asparaginase II enzyme.
  • the modified E. coli L-asparaginase may be glycosylated in order to prevent cleavage by human AEP.
  • This embodiment is less preferred because it is more complicated and expensive.
  • the modified enzyme is glycosylated at, or adjacent to, any one, any two, or all three of residues N46, D146 and Nl 65.
  • Methods for site-specific glycosylation are known in the art and are described, for example, by Zhang et al (2004).
  • the modified E. coli L-asparaginase of the first aspect of the invention has been pegylated.
  • Methods for pegylating an enzyme are well known in the art. Indeed, native E. coli L-asparaginase which has been pegylated is commercially available as Pegaspargase (Graham, 2003).
  • a second aspect of the invention provides a polynucleotide that encodes a modified E. coli L-asparaginase enzyme as defined above in the first aspect of the invention.
  • the invention also includes a vector comprising a polynucleotide that encodes a modified E. coli L-asparaginase enzyme as defined in the first aspect of the invention.
  • the vector is typically a plasmid vector or a viral vector.
  • the invention further includes a cell containing the polynucleotide or the vector.
  • the cell is typically a bacterial a yeast cell, or a cell from an insect or mammalian cell line.
  • a large number of suitable vectors and cell lines for cloning and expressing the modified E. coli L-asparaginase enzyme are very well known in the art.
  • a large number of gene therapy vectors suitable for administration to a patient for targeted expression of the modified E. coli L-asparaginase in the patient are well known in the art.
  • a third aspect of the invention provides a method of making a modified E. coli L-asparaginase enzyme as defined in the first aspect of the invention using recombinant DNA technology.
  • the method comprises providing a plurality of cells that contain a polynucleotide that encodes the modified E. coli L- asparaginase enzyme under conditions suitable for expression of the enzyme from the polynucleotide encoding it, and obtaining the enzyme thus produced.
  • the method further comprises isolating and/or purifying the enzyme thus obtained.
  • a fourth aspect of the invention provides a modified E. coli L-asparaginase enzyme as defined above in the first aspect of the invention for use in medicine.
  • a fifth aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a modified E. coli L-asparaginase as defined above in the first aspect of the invention, and a pharmaceutically acceptable excipient, solvent, diluent or carrier (including combinations thereof).
  • the carrier, diluent, solvent or excipient must be "acceptable” in the sense of being compatible with the active agents of the invention and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • Suitable excipients include mannitol and dextrose.
  • compositions may comprise as, or in addition to, the carrier, excipient, solvent or diluent any suitable binder, lubricant, suspending agent, coating agent, or solubilising agent. Preservatives, stabilisers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • the pharmaceutical composition is formulated for intravenous or intramuscular injection, or intravenous infusion.
  • the modified E. coli L-asparaginase may be in the form of a sterile lyophilised plug or powder that, once reconstituted with sterile water or saline solution, is suitable for intravenous or intramuscular injection.
  • a preferred formulation of the pharmaceutical composition is an isotonic sterile solution in phosphate buffered saline atpH 7-7.5.
  • a sixth aspect of the invention provides a kit of parts comprising a modified E. coli L-asparaginase enzyme as defined above in the first aspect of the invention, and at least one further therapeutic anticancer agent.
  • Suitable therapeutic anticancer agents include: alkylating agents including nitrogen mustards such as mechlorethamine (HN 2 ), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; emylenimines and methylmelamines such as hexamethyhnelamine, thiotepa; alkyl sulphonates such as busulphan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluor
  • Preferred therapeutic anticancer agents for use against leukaemia, include daunorubicin, steroids, vincristines, anthracyclines, nucleoside analogues, methotrexate, thiopurines and alkylating agents such as cyclophosphamide.
  • a seventh aspect of the invention provides a method of combating a disease or condition where asparagine depletion or deprivation would be therapeutically useful, the method comprising administering a modified E. coli L-asparaginase enzyme as defined above in the first aspect of the invention to an individual who has said disease or condition.
  • This aspect of the invention includes the use of a modified E. coli L-asparaginase enzyme as defined in the first aspect of the invention in the manufacture of a medicament for combating a disease or condition where asparagine depletion or deprivation would be therapeutically useful.
  • the disease or condition is one where asparagine depletion or deprivation in the blood would be therapeutically useful.
  • the invention includes treating a disease or condition that responds to asparagine depletion or deprivation.
  • a disease or condition which responds to asparagine depletion refers to a disorder wherein the cells responsible for the disease state either lack or have a reduced ability to synthesise asparagine. Depletion or deprivation of asparagine to such cells can be partial or substantially complete, so long as the desired therapeutic benefit is achieved. In certain embodiments, more than about 50% of asparagine in the serum is depleted, preferably greater than about 75%, with depletion of more than 95% being most preferably achieved.
  • diseases which respond to asparagine depletion or deprivation include certain malignant diseases, particularly malignant hematologic diseases, including lymphomas, leukaemias and myelomas.
  • malignant hematologic diseases including lymphomas, leukaemias and myelomas.
  • leukaemias treatable according to the invention include acute lymphoblastic leukaemia (ALL), acute non-lymphocytic leukaemias, B-cell and T-cell leukaemias, chronic leukaemias, and acute undifferentiated leukaemia.
  • 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-haematological diseases associated with asparagine dependence include autoimmune diseases, for example rheumatoid arthritis, SLE, autoimmune, collagen vascular diseases and AIDS (according to US 6,251,388).
  • 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 and AIDS (according to US 6,251,388).
  • Other autoimmune diseases include osteoarthritis, I
  • any disease the cells responsible for which cease proliferating, become senescent, undergo apoptosis or die in response to asparagine depletion may be treated using the modified asparaginase enzyme.
  • cells suspected of causing disease can be tested for asparagrne dependence in any suitable in vitro or in vivo assay, e.g., an in vitro assay wherein the growth medium lacks asparagrne.
  • the invention thus includes the use of a modified E. coli L-asparaginase enzyme as defined in the first aspect of the invention in the manufacture of a medicament for combating a neoplastic condition in an individual.
  • the neoplastic condition to be combated may be paediatric ALL, adult ALL, lymphoma, non-Hodgkin's lymphoma, AIDS related lymphoma, and advanced solid tumours.
  • the modified L-asparaginase of the invention is likely to be included in the induction, intensification and reinduction blocks for the treatment of childhood ALL and non-Hodgkin's lymphoma, and occasionally for intensification in acute myeloid leukaemia (AML).
  • AML acute myeloid leukaemia
  • the method may further comprise admir ⁇ stering at least one additional anticancer therapeutic agent to the individual.
  • the invention includes the use of a modified E. coli L-asparaginase enzyme as defined in the first aspect of the invention in the manufacture of a medicament for combating a neoplastic condition in an individual, wherein the individual is administered at least one additional anticancer therapeutic agent.
  • the invention includes the use of a modified E. coli L-asparaginase enzyme as defined in the first aspect of the invention and at least one additional anticancer therapeutic agent in the manufacture of a medicament for combating a neoplastic condition in an individual.
  • the invention also includes the use of at least one anticancer therapeutic agent (other than asparaginase) in the manufacture of a medicament for combating a neoplastic condition in an individual, wherein the individual is administered a modified E. coli L-asparaginase enzyme as defined in the first aspect of the invention.
  • Suitable therapeutic anticancer agents include those described above with respect to the kit of parts of the sixth aspect of the invention.
  • Preferred therapeutic anticancer agents include daunorubicin, steroids, vincristines, anthracyclines, nucleoside analogues, methotrexate, thiopurines and alkylating agents such as cyclophosphamide.
  • Suitable doses of the modified E. coli L-asparaginase to be administered typically range from 1,000 Units to 20,000 Units. (One unit of asparaginase splits one micromole of ammonia from L-asparagine in one minute under standard conditions.)
  • the dose of the modified E. coli L-asparaginase to be administered is sufficient to deplete at least 50% of the L-asparagine in the patient's serum, preferably at least 75%, and more preferably at least 90% or at least 95% of the L-asparagine in the patient's serum within 2 hours.
  • the frequency of administration will typically range from every day, to once every three weeks. In any event, the most appropriate treatment regimen for any particular patient will be determined by the physician.
  • An eighth aspect of the invention provides a method of identifying an L-asparaginase enzyme that may have advantageous properties for therapeutic use, the method comprising providing a test L-asparaginase, and determining whether and/or to what extent the L-asparaginase is resistant to cleavage by human AEP, wherein an L-asparaginase that is not cleaved by human AEP may have advantageous properties for therapeutic use.
  • the advantageous properties for therapeutic use include a longer half-life in vivo, and a reduced allergenicity, compared to native E. coli L-asparaginase.
  • the step of determining whether and/or to what extent the L-asparaginase is resistant to cleavage by human AEP, is typically performed in vitro.
  • test L-asparaginase is preferably a modified E. coli L-asparaginase as described above with respect to the first aspect of the invention.
  • test L-asparaginase may be from other species, and may be a modified Erwinia or Wolinella L-asparaginase.
  • a preferred embodiment of this aspect of the invention includes a method of identifying a modified E. coli L-asparaginase that has a longer half-life in vivo and/or a reduced allergenicity compared to native E. coli L-asparaginase, the method comprising providing a modified E. coli L-asparaginase, and determining whether and/or to what extent the modified L-asparaginase is resistant to cleavage by human AEP, wherein a modified E. coli L-asparaginase that is not cleaved by human AEP may have a longer half-life in vivo and/or a reduced allergenicity compared to native E. coli L-asparaginase.
  • a ninth aspect of the invention thus provides a kit of parts for an assay for determining the level of AEP in a suitable sample.
  • AEP protein levels may be determined using any of the well- known immunoassay techniques, such as an enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • Luminex or other bead-based assays for analysis of multiple biomarkers simultaneously in one sample may be used to ' detect AEP in the sample.
  • Western blotting may also be used but this is less-preferred.
  • the Mt comprises anti-AEP "capture” antibodies attached to a solid substrate, and anti-AEP "detecting” antibodies.
  • a kit can be used, for example, in a sandwich ELISA to determine the level of AEP protein in a suitable sample.
  • the kit is preferably for determining the level of human AEP in the sample, and the anti-AEP antibodies are preferably anti-human AEP antibodies.
  • Suitable anti-AEP antibodies can readily be obtained.
  • IgG2A antibodies (R&D Systems, Catalogue Numbers AF2199 and MAB2199, respectively) are commercially available.
  • suitable anti-AEP antibodies can be made using methods well known in the art, for example by immunising an animal with an appropriate AEP peptide and deriving antibodies therefrom, or by phage display technology.
  • the kit will also contain detectably-labelled secondary antibodies that specifically bind to the anti-AEP detecting antibodies.
  • the secondary antibody is specific for the mammalian species being tested, for example an anti-human antibody, as is well known in the art. Many such antibodies are commercially available.
  • the detectable-label in the conjugate is typically an enzyme, for example horseradish peroxidase.
  • the kit may further comprise a substrate for the enzyme.
  • detectable labels suitable for use on the secondary antibody include FITC, biotin and alkaline phosphatase, and RPE for luminex applications.
  • the kit may also comprise suitable wash buffers, instructions for carrying out the assay, and/or appropriate positive and negative controls.
  • the anti-AEP "capture” antibodies are typically coated on microtitre plates overnight at 4°C. Unbound antibody is washed off with a wash buffer such as phosphate buffered saline or Tris buffered saline. Serum or other samples are incubated on the plate, typically at 2O 0 C for between 1 and several hours, to allow AEP in the sample to bind to the antibodies. Unbound material is washed off, and the plates are incubated with the anti-AEP "detecting" antibodies, again typically at 2O 0 C for between 1 and several h-ours.
  • a wash buffer such as phosphate buffered saline or Tris buffered saline. Serum or other samples are incubated on the plate, typically at 2O 0 C for between 1 and several hours, to allow AEP in the sample to bind to the antibodies. Unbound material is washed off, and the plates are incubated with the anti-AEP "detecting" antibodies, again typically at 2O 0 C for
  • Unbound material is washed off, and plates are incubated with species specific enzyme- labelled (eg horseradish peroxidase) antibody, typically anti-IgG or IgM for serum samples, for 1 to several hours aL 20 0 C. Unbound antibody is washed off and plates are incubated with a substrate such as TMB for about 5 minutes, and the optical density measured in a photometer.
  • species specific enzyme- labelled antibody eg horseradish peroxidase
  • this ELISA is suitable for detecting AEP protein in a sample from other mammalian species.
  • conjugated and unconjugated secondary antibodies are readily available.
  • a variety of bovine, cat, dog, goat, guinea pig, hamster, horse, human, monkey, mouse, pig, rabbit, rat, and sheep secondary antibodies and conjugates are commercially available from Sigma-Aldrich and from Jackson Immunoresearch Laboratories, Inc.
  • the kit comprises AEP, or an antigenic fragment thereof, attached to a solid substrate, and anti-AEP "primary" antibodies.
  • AEP or an antigenic fragment thereof, attached to a solid substrate, and anti-AEP "primary" antibodies.
  • Such a kit can be used, for example, in a competitive ELISA to determine the level of AEP protein in a suitable sample.
  • the kit is preferably for determining the level of human AEP in the sample, and the AEP, or antigenic fragment thereof, attached to the solid substrate is preferably human AEP and the anti-AEP antibodies are preferably anti-human AEP antibodies, as described above.
  • an "antigenic fragment” of AEP we mean that the fragment can be bound by anti-AEP antibodies, and this can readily be determined by the skilled person using only standard techniques.
  • the AEP, or antigenic fragments thereof are made by expression of a suitable DNA construct encoding the protein using recombinant DNA technology.
  • suitable techniques for cloning, manipulation, modification and expression of nucleic acids, and purification of expressed proteins are well known in the art and are described for example in Sambrook et al (2001), incorporated herein by reference.
  • the kit will also contain detectably-labelled secondary antibodies that specifically bind to the anti-AEP primary antibodies.
  • This aspect of the invention thus includes both a solid substrate with human AEP attached thereto, and a solid substrate with anti-human AEP antibodies attached thereto.
  • the solid substrate is typically a microtitre plate.
  • a further embodiment of this aspect of the invention provides a kit of parts for an assay for determining the level of AEP polynucleotide in a suitable sample.
  • the kit may comprise a pair of amplification primers that can be used to amplify a given section of the AEP polynucleotide and, optionally, a probe that specifically binds to the AEP polynucleotide within the amplified section.
  • one or both of the amplification primers are fluorescently labeled.
  • DNA binding dyes such as SYBR Green I and BEBO, which become strongly fluorescent when bound to double stranded DNA, can be used.
  • SYBR Green I and BEBO which become strongly fluorescent when bound to double stranded DNA.
  • the assay is a TaqMan ® assay and the probe is fluorescently labeled with a reporter dye and a quenching dye.
  • the Taqman ® method is now well known in the art and has been described, for example, by Van der Velden et al (2003), Gabert et al (2005) and Branford et al (2006).
  • this method is based on the 5'-3' exonuclease activity of Taq DNA polymerase, which results in cleavage of fluorescent dye-labelled probes during PCR; the intensity of fluorescence is then measured by a detection system.
  • the probe is located between the two PCR primers and usually has a melting temperature around 10 0 C higher than that of the primers.
  • the probe has two fluorescent tags attached to it.
  • One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its emission spectra quenched due to the spatial proximity of a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA).
  • FAM 6-carboxyfluorescein
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • the ABI Prism 7700 is a laser-coupled spectrophotometer which is suitable for monitoring the fluorescence output of TaqMan ® performed in a microtitre plate format in real-time.
  • the Ct value is directly related to the amount of PCR product and therefore related to the initial amount of target DNA present in the PCR reaction.
  • Quantitative real-time TaqMan ® PCR has several advantages over the classical quantitative PCR system.
  • the use of fluorescent dye-labelled probes increases the sensitivity of the system by at several orders of magnitude and gives rise to a linear relationship between copy number and Ct values.
  • the liquid hybridisation step adds further specificity to the system.
  • the elimination of post- amplification steps increases reliability and reproducibility of the assay.
  • a major factor responsible for the accuracy of this method is the determination of Ct value within the logarithmic phase of the amplification reaction, instead of the endpoint determination used by conventional quantitative PCR system systems.
  • RQ relative quantitative RT- PCR using locked nucleic acid (LNA) technology with or without the Taqman approach (Dreier et al (2006); Kivinemi et al (2005)).
  • LNA locked nucleic acid
  • the kit also comprises amplification primers for a control polynucleotide and, when required for the particular technique used, a probe specific for the control polynucleotide.
  • control polynucleotide can be beta-2 -microglobulin (B2M).
  • B2M beta-2 -microglobulin
  • Other suitable controls include RPLPO, 18S, HMBS, UBC, PPIA, PGKl, IPO8, GUSB, POLR2A, TBP, ACTB, HPRT, TFRC, GAPDH and YWHAZ (Applied Biosystems, 2005) and ABL. B2M and GAPDH are preferred.
  • a patient with a high level of AEP should not be treated with an L- asparaginase that is susceptible to cleavage by AEP, such as native E. coli L- asparaginase.
  • Such a patient should be treated with an L-asparaginase that is resistant to cleavage by AEP such as Erwinia L-asparaginase or a modified E. coli L-asparaginase as defined in the first aspect of the invention.
  • a patient with a low level of AEP can be treated with any L-asparaginase, whether or not it is cleaved by AEP.
  • it is useful for a physician to be provided with a reference level of AEP above which is considered to be a high level of AEP.
  • a tenth aspect of the invention thus provides a method of assessing the optimal L- asparaginase for administration to a patient in need thereof, the method comprising: obtaining a suitable sample from a patient; determining the level of AEP in the sample; and comparing the level of AEP in the sample with a reference level of AEP.
  • the patient is a human patient.
  • a level of AEP in the sample higher than the reference level indicates that the patient should not be treated with an L-asparaginase that is cleaved by AEP.
  • the patient when the level of AEP in the sample is above the reference value, the patient is not treated with native E. coH L-asparaginase.
  • a level of AEP in the sample higher than the reference level indicates that the patient should be treated with an L-asparaginase that is resistant to cleavage by AEP.
  • the patient when the level of AEP in the sample is above the reference value, the patient is preferably treated with an Erwinia L-asparaginase or a modified E. coli L-asparaginase as defined in the first aspect of the invention.
  • determining the level of AEP in the sample may comprise determining the level of AEP protein in the sample, for example using an immunoassay. Additionally or alternatively, determining the level of AEP in the sample may comprise determining the level of AEP polynucleotide in the sample. It is preferred, if the AEP polynucleotide is a single stranded mRNA or a cDNA derived therefrom.
  • the reference level of AEP may be an absolute value of AEP protein or polynucleotide in the sample.
  • the reference value may be a relative value, i.e. a level of AEP protein or AEP polynucleotide relative to a control protein or polynucleotide.
  • One of the quantitative PCR methods known in the art may be used to measure levels of AEP polynucleotide and a control polynucleotide in a sample.
  • the ⁇ Ct method of comparison may be used to determine the relative level of AEP with respect to the control (see, Stalberg et al (2005) and Goff et al (2004)).
  • a level of AEP polynucleotide relative to beta-2 microglobulin (B2M) of 0.1 can be used. More preferably, a relative value of 0.15, and more preferably 0.2, or 0.3, or 0.4, or 0.5 or more in bone marrow cells can be used. This can be measured, for example, in mRNA extracted from bone marrow cells, using RT-PCR and the TaqMan methods which are well known in the art.
  • the reference level of AEP may depend upon the methodology used to detect AEP levels and the source of the sample.
  • the sample is a fluid sample, and suitable samples include whole blood, serum and plasma. It is particularly convenient if the sample is a plasma sample which may be prepared from a blood sample in a standard way (for example by collection in EDTA tubes followed by centrifugation).
  • AEP levels can also be measured in samples of, or containing, bone marrow cells, peripheral blood cells, bone marrow plasma and peripheral plasma.
  • This aspect thus includes the use of an assay for AEP levels to assess the optimal L-asparaginase for administration to a patient in need thereof.
  • the assay may comprise an immunoassay to measure AEP protein levels or may comprise a PCR- based assay to measure AEP polynucleotide levels. Many suitable assays are known in the art and discussed above.
  • This aspect of the invention includes categorizing a patient in need of treatment with L-asparaginase based on his or her AEP status.
  • a patient in need of treatment with L-asparaginase we mean a patient who has a disease or condition where asparagine depletion or deprivation would be therapeutically useful.
  • diseases and conditions are described above with respect to the seventh aspect of the invention, and includes neoplastic conditions such as both paediatric and adult ALL.
  • An eleventh aspect of the invention provides a method of treating a human patient who has a disease or condition where asparagine depletion or deprivation would be therapeutically useful, the method comprising: selecting the patient on the basis that the patient has a level of AEP above a reference level; and administering to the patient an L-asparaginase that is resistant to cleavage by AEP.
  • neoplastic conditions such as both paediatric and adult ALL.
  • the method may further comprise administering at least one additional therapeutic anticancer agent to the patient, such as those described above in the sixth aspect of the invention.
  • the patient may be one who is administered at least one additional therapeutic anticancer agent, such as those described above in the sixth aspect of the invention.
  • This aspect of the invention also provides the use of an L-asparaginase that is resistant to cleavage by AEP in the preparation of a medicament for treating a human patient with ALL, wherein the patient has been selected on the basis that the patient has a level of AEP above a reference level.
  • the medicament may further comprise at least one additional therapeutic anticancer agent, such as those described above in the sixth aspect of the invention.
  • the L-asparaginase that is resistant to cleavage by AEP is preferably Erwinia L- asparaginase or a modified E. coli L-asparaginase as defined in the first aspect of the invention.
  • preferences regarding methods for obtaining information on a patient's level of AEP, reference levels of AEP, additional therapeutic anticancer agents, doses and routes of administration of the L-asparaginase, and so on, are as described above.
  • Figure 1 Genomic analysis of DNA and cell suspension from patient 5989.
  • BAC aCGH results chromosome 21 is positioned horizontally, with the centromeric to telomeric positions running from left to right respectively.
  • Dye swap experiments are shown by the A (for the dup(21q)) and ⁇ (for normal controls) marked lines respectively. Double deviation of both these experiments from a normal value of 1.00 demonstrates loss or gain of DNA material. Deviation of the A and ® lines greater than 1.00 shows loss or gain of copy number respectively.
  • B Examples of the FISH confirmation of aCGH data: each numbered FISH probe is at the numbered position in (A).
  • Figure 2 Schematic diagram of SNP calls on chromosome 21 in patients with dup(21q).
  • FIG. 3 Box plot diagrams illustrating (A) LGMN expression and (B) expression of those genes within the CRA, compared to other ALL subtypes.
  • the y-axis represents the relative gene expression level of either LGMN (Panel A) or all those genes within the CRA (Panel B).
  • LGMN LGMN
  • CRA high hyperdiploid
  • the y-axis represents the relative gene expression level of either LGMN (Panel A) or all those genes within the CRA (Panel B).
  • Each box plot shows the distribution of expression levels from 25 th to the 75 th percentile.
  • the median is shown as a line across the box, where the + is the calculated mean expression level for the particular subtype.
  • the dotted line indicates the inner fence, and a far out value outside the outer fence is shown as *.
  • FIG. 4 Comparative expression of LGMN in childhood ALL (real-time quantitative PCR). On the x-axis are shown LGMN RNA levels normalised to B2M (house-keeping gene) in patients with ALL; normal karyotype (2 patients), t(12;21) (3 patients), Hyperdiploid (HD, 3 patients) and dup(21q) (5 patients). Expression in one normal (patient without leukaemia) and in one HRC57 sample (B-cell cell line, mature, non-malignant) are provided as controls.
  • Figure 5 Western blot illustrating active AEP/Legumain over-expression in dup21(q) patient sample.
  • HRC57 and Olga-1 are B-cell cell lines, mature, non-malignant.
  • REH is a t(12;21) cell line.
  • the primary anti-legumain antibody used was mouse monoclonal antibody 6EH, used at a dilution of 1 : 1000.
  • the secondary antibody was used at a dilution of 1 : 2000.
  • Figure 6 Schematic illustration of E. coli L-asparaginase II.
  • Figure 7 Amino acid and nucleic acid sequences of E. coli L-asparaginase II . Sequences provided are the amino acid sequence (SEQ ID No: 1) and nucleic acid sequence (SEQ ID No: 2) of E. coli L-asparaginase II. The three potential AEP/legumain cleavage sites are indicated with an arrow.
  • Figure 8 Cleavage of E. coli L-asparaginase by AEP.
  • E. coli L-asparaginase and the tetanus toxin C fragment (TTCF) were each incubated with and without recombinant human AEP.
  • the digestion products were separated on an SDS-PAGE gel and stained with Coomassie blue.
  • Lane 1 protein weight markers; lane 2: L-asparaginase with AEP; lane 3: L-asparaginase alone; lane 4: TTCF with AEP; lane 5: TTCF alone; and lane 6: AEP alone.
  • Lane 2 marked "Asp+AEP" shows cleavage products of the E. coli L-asparaginase by the AEP.
  • Figure 9 Cleavage of E. coli L-asparaginase by AEP.
  • E. coli L-asparaginase Medac and Kidrolase
  • Erwinia L-asparaginase Erwinase
  • Pegylated E. coli L- asparaginase Pegaspargase
  • Lane 1 protein weight markers
  • lanes 2-3 Medac L-asparaginase with and without AEP
  • lanes 4-5 Kidrolase L-asparaginase with and without AEP
  • lanes 6-7 Erwinase L-asparaginase with and without AEP
  • lanes 8-9 pegylated E. coli L-asparaginase with and without AEP
  • lane 10 Medac L-asparaginase alone.
  • the three preparations of E. coli L-asparaginase (Medac, Kidrolase and Pegaspargase) are each cleaved by AEP.
  • the Erwinase L- asparaginase is not cleaved by AEP.
  • Figure 10 Modified E. coli L-asparaginase is not cleaved by AEP.
  • E. coli L-asparaginase A laboratory preparation of wild-type E. coli L-asparaginase; E. coli L- asparaginase with N46G and D146G mutations; E. coli L-asparaginase with N46G, D146G and N165G mutations; and a wild-type E. coli L-asparaginase preparation from Medac were each incubated with and without AEP. The digestion products were separated on a 10% SDS-PAGE gel and stained with Coomassie blue. Lanes 1-2: wild-type E. coli L-asparaginase (ansBpRSETB) without (-) and with (+) AEP; lanes 3-4: E.
  • Figure 11 Sequence of Human AEP (SEQ ID No: 3).
  • Table 1 BAC Array CGH and FISH results for 13 ALL patients with duplicated chromosome 21
  • Example 1 Identification of Consistent Patterns of Genomic Amplification, Expression, and Chromosomal Instability in Acute Lymphoblastic Leukaemia with Duplication of 21q.
  • ALL B-lineage acute lymphoblastic leukaemia
  • FISH fluorescence in situ hybridisation
  • Dup(21q) has also been reported in patients with acute myeloid leukaemia (AML) (Baldus et al, 2004).
  • AML acute myeloid leukaemia
  • BAC aCGH BAC array-based comparative genomic hybridisation
  • Oligonucleotide expression analysis revealed that all significantly over-expressed genes were located within these amplicons, implying that the changes in gene expression were entirely related to alterations in copy number (Baldus et al, 2004).
  • Genomic copy number variation was assessed using a commercially available BAC aCGH system (Spectral Genomics, Genosystems, France).
  • the arrays comprise 2621 genomic clones positioned at approximately 1Mb intervals throughout the genome. Of these, 26 are located along 21q from position 15.1Mb (centromeric) to 46.9Mb (telomeric).
  • the position of genes and BAC clones were determined using the National Center for Biotechnology Information (NCBI) Map Viewer for Homo Sapiens, Build 35, version 1 (www.ncbi.nlm.nih.gov/mapview).
  • NCBI National Center for Biotechnology Information
  • oligonucleotide-based CGH (Oligo aCGH) arrays (NimbleGen, Wisconsin, USA), designed to tile through chromosome 21. Sequences (NCBI Build 35.1) were repeat-masked and oligonucleotides selected at a minimal spacing distance of 60 bp, from both the forward and reverse strands, resulting in approximately 45,000 features along the length of the chromosome. The arrays were synthesised as described previously (Singh-Gasson et al, 1999), and standard labeling, hybridisation and image capture was performed.
  • NimbleScan extraction software (NimbleGen, Wisconsin, USA), which allows automated grid alignment, extraction and generation of data files. Segmentation analysis of data sets indicated deletion and amplification breakpoints. Corrections for optical noise, background adjustments and normalisation were performed using Bioconductor as previously described (Gentleman et al, 2004). After a loss correction for probe GC content, the Iog2 ratios were averaged in windows ranging from 500-5000 bp to produce the final segmentations using SignalMap software (NimbleGen, Wisconsin, USA; Olshen et al, 2004).
  • the GeneChip mapping assay protocol (Affymetrix Inc., Santa Clara) was used to produce the 10,000 single nucleotide polymorphism (SNP) array results and is described elsewhere (Matsuzaki et al, 2004).
  • the protocol was adapted such that the purification of polymerase chain reaction (PCR) product was performed using the Ultrafree-MC filtration column (Millipore, Bill erica, MA).
  • Signal intensity data was analysed by the GeneChip DNA analysis software (GDAS), which uses a model algorithm to generate SNP calls (Kennedy et al, 2003).
  • GEP Global expression profiling
  • RNA was extracted with TRIzol (Invitxogen, Paisley, UK) followed by a second ethanol precipitation, prior to quality assessment using the Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany).
  • Fluorescently labeled cRNA probes were synthesised and hybridised to Affymetrix (Santa Clara, CA, USA) HG-U133A oligonucleotide arrays according to the manufacturers' instructions. The arrays were scanned on a GeneArray scanner (Agilent Technologies, Waldbronn, Germany), and the intensities of the fluorescent signals were captured and analysed with Affymetrix MAS 5.0 software. No scaling was applied.
  • GeneSpring 6.0 (Silicon Genetics, Redwood City, CA, USA) was used for raw data normalisation. Firstly, the data were normalised to the median per sample, using all genes not marked absent Each gene was then divided by the median of its measurements in all samples (i.e. across all arrays). If the median of the raw values was below 10, then each measurement for that gene was divided by 10. Signal intensities were log transformed for statistical analysis. Genes called absent in all samples were removed in order to exclude those with minimal variation over the experiments. Probe sets passing the filter were used to find statistically significant differentially expressed genes between the subgroups studied. Significance Analysis of Microarrays (SAM) was applied to the normalised and log transformed data.
  • SAM Significance Analysis of Microarrays
  • RT-RQPCR Real-Time quantitative RT-PCR
  • Genome- wide BAC aCGH showed genomic imbalances in all 10 patients with dup(21q) analysed in this study. Patterns of imbalance corresponding to over- and under-representation of specific regions of chromosome 21 were unique to each patient (Table 1). All BAC clones on chromosome 21 showed gain in at least one patient. These gains frequently involved clones between genomic positions 22.1 and 27.8Mb (clones RPl 1-64112 to RP11-90A12). The size of the amplified region varied considerably between patients, from 3-8.6 to 24.0-24.1Mb for patients 6783 and 6788 respectively. A representative aCGH result confirmed by FISH is shown in Figures 1 A & B .
  • tiling-path Oligo aCGH Using tiling-path Oligo aCGH, the extent of the CRA was further refined in five patients with dup(21q) to a region of 6.527-6.604Mb in size (between genomic positions 33.192 and 39.796Mb). Using oligo aCGH, the size of the CRD was refined to a 3.541Mb region.
  • FISH analysis confirmed the variation in copy number along 21 q in the cases analysed by BAC aCGH (Table 1 and Figure IB). The same CRA and CRD were identified. The high concordance between the two procedures indicated the accuracy of BAC aCGH in the determination of copy number changes, while FISH analysis provided precise quantification. Between three and eight additional copies of the clones within the CRA were demonstrated by FISH, indicating a 2.5- 5 fold gain. FISH data on copy number changes in a further three patients with dup(21q), provided further confirmation of the BAC aCGH results (data not included).
  • GEP Global gene expression profiling
  • the gene expression profiles of the eight patients were compared: to the full cohort of 85; to a subgroup of 21 patients with the ETV6-RUNX1 fusion; to a subgroup of 23 patients with HD+21.
  • the gene list was compiled in this manner, patients with dup(21q) exhibited a distinctive expression pattern.
  • the CRA was represented on the Affymetrix Ul 33 A GeneChip by 96 probe sets in total, including 40 well-characterised genes and six open reading frames. From a total of 768 probe sets within the CRA analysed for eight patients, 321 (42%) were present or marginal and up regulated. Of the 46 sequences, 13 were up regulated in at least 75% of patients.
  • the CRD was represented on the GeneChip by 83 probe sets, containing 33 genes, three open reading frames and 3 EST' s. From a total of 664 probe sets, 462 (70%) were absent. An absent flag was carried in 22 of the 39 gene sequences in at least 75% of patients.
  • Taqman ® Gene Expression Assays were used. The following genes were examined in cases with available cDNA: SODl, OLIG2, IFNAR2, ILlORB 5 ITSNl, CRYZLl, RUNXl, TTC3, ERG and ETS2. Affymetrix called OLIG2, IFNAR2, ITSNl and RUNXl absent and, in confirmation, no gene expression levels were detected by the Taqman assays. For all genes a strong correlation between the Affymetrix and Taqman data was identified.
  • FISH detection based solely on RUNXl copy number
  • interpretation may be misleading, particularly in the presence of a high hyperdiploid cell population with multiple copies of chromosome 21.
  • RUNXl copy number since the observed increase in RUNXl copy number was serendipitous, it may not be a causative mechanism. It is therefore important to characterise this abnormality to provide accurate diagnosis, particularly for ALL patients without any other high-risk clinical features.
  • RUNXl fusion and RUNXl are common findings in patients with t(12;21)
  • allelic imbalances correlate with changes in gene expression (Hyman et al, 2002). Consequently, this may result in expression variation of many genes unassociated with, but flanking genes important in cancer pathogenesis (Masayesva et al, 2004).
  • genomic and genotypic profiling we were unable to detect any consistent allelic imbalances outside the CRA and CRD. Thus, we could not correlate all gene expression changes to alteration at the genomic copy level. Not all genes within the amplicon were over-expressed and there was no linear correlation between the degree of amplification and expression. This may result from heterogeneity in amplification within the region or other regulatory mechanisms influencing gene expression, such as epigenetics and bio-feedback regulation.
  • Our data suggest that genomic amplification within the CRA resulted from allelic imbalance. Partial uniparental disomy (UPD) has recently been reported in patients with AML
  • RPS9 and MAFF under-expression
  • TCF4, F8A and TAF5 under-expression of a number of genes.
  • MAFF over-expression is known to correlate with steroid resistance and F8A down regulation with insensitivity to L-asparaginase.
  • AEP Over-expression of AEP may thus be a hitherto undiscovered mechanism of drug resistance, leading to therapeutic failure.
  • AEP has previously been proposed as a candidate for targeted therapy (Liu et al, 2003; Ekici et al, 2004) and potentially such an approach offers new treatment alternatives for this high-risk group.
  • this is completely different therapeutic approach to the modified E. coli asparaginase of the present invention.
  • Example 2A Cleavage of E. coli L-asparaginase by AEP
  • E. coli L-asparaginase and 4 ⁇ g of the tetanus toxin C fragment (TTCF) protein were each incubated with and without 0.4 ⁇ g of recombinant human AEP in 5OmM sodium citrate buffer pH 4.5 containing 5mM DTT for 3 hours at 37°C.
  • TTCF tetanus toxin C fragment
  • Asp+AEP shows cleavage products of the E. coli L-asparaginase corresponding to cleavage at the sites indicated in Figures 6 and 7 (N46, D 146 and Nl 65).
  • Example 2B Cleavage of commercially available L-asparaginases by AEP
  • E. coli L-asparaginase Medac from Medac UK and Kidrolase from OPi Pharmaceuticals
  • Erwinia L- asparaginase Erwinase ®
  • pegylated E. coli L-asparaginase Pegylated E. coli L-asparaginase (Pegaspargase from Medac UK) were each, incubated with and without 0.4 ⁇ g of recombinant human AEP in 5OmM sodium citrate buffer pH 4.5 containing 5mM DTT for 3 hours at 37 0 C.
  • the digestion products (20 ⁇ l) were separated on a 10% SDS- PAGE gel and stained with Coomassie blue. The results are shown in Figure 9.
  • coli L-asparaginase (Medac, Kidrolase and Pegaspargase) are each cleaved by AEP.
  • the Erwinase ® L-asparaginase is not cleaved by AEP.
  • Example 3 Modified E. coli L-asparaginase is Resistant to Cleavage by AEP
  • DNA from recombinant E. coli pKK233 containing ansB of E. coli was subcloned into the bacterial expression vector pRSETB (Invitrogen) using the Ncol and HindIll restriction enzyme sites. The obtained plasmid was verified by DNA sequencing and named ansBpRSETB. DNA from ansBpRSETB was then used as a template to generate two mutated versions of ansB using the PCR method of QuickChange Multi Site-Directed Mutagenesis (Stratagene). The following mutagenic primers were used:
  • N46G, D146G and N165G amino acid mutations The other mutated form named ansBpRSETBN46GDl 46G had N46G and D146G amino acid replacements.
  • DNA from all three forms of ansB were transformed into BL21(DE3)pLysS E. coli cells and transformants were selected for ampicillin and chloramphenicol resistance.
  • a single colony from each ansB form was grown overnight in SOB media containing 50 ⁇ g/ml ampicillin and 35 ⁇ g/ml chloramphenicol at 37 0 C, and this was used the next day to inoculate 170 ml of fresh SOB media containing 50 ⁇ g/ml ampicillin.
  • the culture was grown at 37 0 C to an OD 600 of 0.4 - 0.6 and then expression of the recombinant protein was induced by addition of ImM IPTG. After 3.5 hours, bacterial cells were harvested by centrifugation and the recombinant protein from each ansB form was purified using Qiagen Ni-NTA spin columns and concentrated using a Millipore Microcon YM- 10 filter unit.
  • E. coli L-asparaginase ansBpRSETB and Medac
  • AEP Neither of the mutated E. coli L- asparaginases are cleaved by AEP.
  • RT-PCR and the TaqMan ® methodology were used on mRNA extracted from bone marrow cells to determine cut-off levels for normal/low, intermediate and high levels of AEP.
  • the Taqman ready assay for AEP (probe 797894) Hs 00271599 - MlLGMN was used to test approximately 100 ALL patients along with 5 normal and 5 AML patients.
  • the ⁇ Ct method of comparison was used with beta-2 microglobulin (B2M) as a control.
  • AEP / B2M levels of 0 - 0.1 were considered to be normal/low, levels between 0.1 and 0.2 intermediate, and AEP levels above 0.2 were considered to be high.
  • Example 5 Treatment of a Patient with ALL Using a Modified E. coli L- asparaginase that is Resistant to Cleavage by AEP
  • a nine year old child presents to a physician with symptoms of paediatric ALL, which is subsequently diagnosed by the physician.
  • the patient is administered a modified E. coli L-asparaginase which has a polypeptide sequence identical to SEQ ID No: 1, but which has GIy residues at positions N46 and N165.
  • the patient is administered the asparaginase parenterally in place of native E. coli L- asparaginase in a standard treatment regime, and during subsequent reinduction blocks to maintain asparagine depletion during these blocks. Once remission is obtained, maintenance therapy is instituted without asparaginase.
  • Example 6 Treatment of a Patient with ALL Using a Modified E. coli L- asparaginase that is Resistant to Cleavage by AEP
  • a nine year old child presents to a physician with symptoms of paediatric ALL, which is subsequently diagnosed by the physician.
  • the patient is administered a modified E. coli L-asparaginase which has a polypeptide sequence identical to
  • SEQ ID No: 1 which has GIy residues at positions N46 and D146.
  • the patient is administered the asparaginase parenterally in place of native E. coli L- asparaginase in a standard treatment regime, and during subsequent reinduction blocks to maintain asparagine depletion during these blocks. Once remission is obtained, maintenance therapy is instituted without asparaginase.
  • Example 7 Treatment of a Patient with ALL Using a Modified E. coli L- asparaginase that is Resistant to Cleavage by AEP
  • a nine year old child presents to a physician with symptoms of paediatric ALL, which is subsequently diagnosed by the physician.
  • the patient is administered a modified E. coli L-asparaginase which has a polypeptide sequence identical to SEQ ID No: 1, but which has GIy residues at positions N46, D146 and N165.
  • the patient is administered the asparaginase parenterally in place of native E. coli L- asparaginase in a standard treatment regime, and during subsequent reinduction blocks to maintain asparagine depletion during these blocks. Once remission is obtained, maintenance therapy is instituted without asparaginase.

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Abstract

La présente invention porte sur une enzyme L-asparaginase E.coli modifiée qui est résistante au clivage par l'asparaginyl l'endopeptidase humaine (également appelée légumaïne). Les résidus d'aminoacides N46, D 146 et/ou N 165 ont été remplacés par un autre résidu d'aminoacide, de préférence par des résidus de glycine.
PCT/GB2007/000124 2006-01-17 2007-01-17 Enzyme Ceased WO2007083100A2 (fr)

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GB0600862A GB0600862D0 (en) 2006-01-17 2006-01-17 Enzyme
GB0600862.7 2006-01-17
GB0618149A GB0618149D0 (en) 2006-09-15 2006-09-15 Enzyme II
GB0618149.9 2006-09-15

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WO2007083100A3 WO2007083100A3 (fr) 2007-11-08

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012014228A1 (fr) 2010-07-28 2012-02-02 Abhishek Narain Singh Méthode pour supplanter l'activité du domaine allostérique d'une enzyme de façon à modifier sa rétroinhibition ou son activation
WO2012075173A3 (fr) * 2010-12-01 2013-01-03 Board Of Regents The University Of Texas System Compositions et procédé de désimmunisation de protéines
CN106556699A (zh) * 2015-09-30 2017-04-05 余笑虹 用于测定豆荚蛋白的血液水平的方法和组合物
WO2017059145A1 (fr) * 2015-09-30 2017-04-06 Xiaohong Yu Procédés et compositions de dosage des niveaux sanguins de la légumaïne
CN108226508A (zh) * 2017-02-16 2018-06-29 李伟 抗人Legumain蛋白的单克隆抗体、杂交瘤细胞株及其用途
CN110331157A (zh) * 2019-08-02 2019-10-15 湖北大学 一种aep环化酶在大肠杆菌中的融合表达方法、aep环化酶环化能力鉴定方法及其应用
CN110462035A (zh) * 2016-11-08 2019-11-15 帕维亚大学 高度稳定且抗蛋白酶的大肠杆菌天冬酰胺酶
EP4232575A1 (fr) * 2020-10-20 2023-08-30 Flagship Pioneering Innovations VI, LLC Protéines d'asparaginase

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1713928A2 (fr) * 2004-02-03 2006-10-25 Bayer HealthCare AG Outils de diagnostic et de traitement de maladies associees a la cysteine protease putative 1 (prsc1)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012014228A1 (fr) 2010-07-28 2012-02-02 Abhishek Narain Singh Méthode pour supplanter l'activité du domaine allostérique d'une enzyme de façon à modifier sa rétroinhibition ou son activation
WO2012075173A3 (fr) * 2010-12-01 2013-01-03 Board Of Regents The University Of Texas System Compositions et procédé de désimmunisation de protéines
CN106556699A (zh) * 2015-09-30 2017-04-05 余笑虹 用于测定豆荚蛋白的血液水平的方法和组合物
WO2017059145A1 (fr) * 2015-09-30 2017-04-06 Xiaohong Yu Procédés et compositions de dosage des niveaux sanguins de la légumaïne
US10048267B2 (en) 2015-09-30 2018-08-14 Ningbo Ziyuan Medical Devices Inc. Methods and compositions for assaying blood levels of legumain
CN110462035A (zh) * 2016-11-08 2019-11-15 帕维亚大学 高度稳定且抗蛋白酶的大肠杆菌天冬酰胺酶
CN108226508A (zh) * 2017-02-16 2018-06-29 李伟 抗人Legumain蛋白的单克隆抗体、杂交瘤细胞株及其用途
CN108226508B (zh) * 2017-02-16 2019-02-01 李伟 抗人Legumain蛋白的单克隆抗体、杂交瘤细胞株及其用途
CN110331157A (zh) * 2019-08-02 2019-10-15 湖北大学 一种aep环化酶在大肠杆菌中的融合表达方法、aep环化酶环化能力鉴定方法及其应用
EP4232575A1 (fr) * 2020-10-20 2023-08-30 Flagship Pioneering Innovations VI, LLC Protéines d'asparaginase

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