THERAPEUTIC CONJUGATE

Abstract

The invention provides conjugates comprising L-asparaginase and a water-soluble polymer for use in treating a disease treatable by L-asparagine depletion in a patient that has been previously administered E. coli derived L-asparaginase. The invention also provides methods of treatment, compositions comprising said conjugate, and methods of producing the conjugate.

Claims

1. A conjugate comprising L-asparaginase and a water-soluble polymer, for use in treating a disease treatable by L-asparagine depletion in a patient, wherein: (a) the L-asparaginase is from a source other than E. coli; (b) the L-asparaginase is expressed in a host cell other than E. coli; and (c) the patient has previously been administered E. coli-derived L-asparaginase.

2. The conjugate for use according to claim 1, wherein the L-asparaginase is a recombinant L-asparaginase.

3. A recombinant heterologously-expressed L-asparaginase for use in treating a disease treatable by L-asparagine depletion in a patient, wherein: (a) the L-asparaginase is from a source other than E. coli; (b) the heterologous host cell is a host cell other than E. coli; and (c) the patient has previously been administered E. coli-derived L-asparaginase.

4. The conjugate for use according to claim 1 or claim 2, or the recombinant heterologously-expressed L-asparaginase for use according to claim 3, wherein the patient has had a hypersensitivity to an E. coli-derived L-asparaginase.

5. The conjugate for use according to claim 4, or the recombinant heterologously-expressed L-asparaginase for use according to claim 4, wherein the hypersensitivity is selected from the group consisting of allergic reaction, anaphylactic shock, and silent inactivation.

6. The conjugate for use according to any one of claims 1 to 5, or the recombinant heterologously-expressed L-asparaginase for use according to any one of claims 1 to 5, wherein the E. coli-derived L-asparaginase is Oncaspar®.

7. The conjugate for use according to any one of claims 1 to 6, or the recombinant heterologously-expressed L-asparaginase for use according to any one of claims 1 to 6, wherein the L-asparaginase is Erwinia L-asparaginase.

8. The conjugate for use according to claim 7, or the recombinant heterologously-expressed L-asparaginase for use according to claim 7, wherein the Erwinia L-asparaginase is E. chrysanthemi L-asparaginase.

9. The conjugate for use according to claim 8, or the recombinant heterologously-expressed L-asparaginase for use according to claim 8, wherein the E. chrysanthemi L asparaginase has at least 80% identity to the amino acid sequence of SEQ ID NO: 2.

10. The conjugate for use according to any one of the preceding claims, wherein the water-soluble polymer comprises polyethylene glycol (PEG).

11. The conjugate for use according to claim 10, wherein the PEG has a molecular weight of less than about 10000 Da.

12. The conjugate for use according to claim 10 or claim 11, wherein the PEG has a molecular weight of less than about 5000 Da

13. The conjugate for use according to any one of claims 10 to 12, wherein the PEG has a molecular weight of less than about 4000 Da

14. The conjugate for use according to any one of claims 10 to 13, wherein the PEG has a molecular weight of less than about 3000 Da

15. The conjugate for use according to any one of claims 10 to 14, wherein the PEG has a molecular weight of less than about 2000 Da.

16. The conjugate for use according to any one of the preceding claims, wherein the water-soluble polymer is a PAS polymer.

17. The conjugate for use according to claim 16, wherein at least 80% of the amino acid residues in the PAS polymer consist of proline, alanine and serine.

18. The conjugate for use according to claim 16 or claim 17, wherein at least 90% of the amino acid residues in the PAS polymer consist of proline, alanine and serine.

19. The conjugate for use according to any one of claims 16 to 18, wherein at least 95% of the amino acid residues in the PAS polymer consist of proline, alanine and serine.

20. The conjugate for use according to any one of claims 16 to 19, wherein at least 97% of the amino acid residues in the PAS polymer consist of proline, alanine and serine.

21. The conjugate for use according to any one of claims 16 to 20, wherein at least 99% of the amino acid residues in the PAS polymer consist of proline, alanine and serine.

22. The conjugate for use according to any one of claims 16 to 21, wherein all of the amino acid residues in the PAS polymer consist of proline, alanine and serine.

23. The conjugate for use according to any one of claims 16 to 22, wherein the PAS polymer comprises at least 10 amino acid residues.

24. The conjugate for use according to any one of claims 16 to 23, wherein the PAS polymer comprises at least 15 amino acid residues.

25. The conjugate for use according to any one of claims 16 to 24, wherein the PAS polymer comprises at least 20 amino acid residues.

26. The conjugate for use according to any one of claims 16 to 25, wherein the PAS polymer comprises at least 25 amino acid residues.

27. The conjugate for use according to any one of claims 16 to 26, wherein the PAS polymer comprises at least 30 amino acid residues.

28. The conjugate for use according to any one of claims 16 to 27, wherein the PAS polymer comprises 10-60 amino acid residues.

29. The conjugate for use according to any one of claims 16 to 28, wherein the PAS polymer comprises 15-50 amino acid residues.

30. The conjugate for use according to any one of claims 16 to 29, wherein the PAS polymer comprises 20-40 amino acid residues.

31. The conjugate for use according to any one of claims 16 to 30, wherein the PAS polymer comprises 20-30 amino acid residues.

32. The conjugate for use according to any one of claims 16 to 31, wherein the PAS polymer additionally comprises a purification tag.

33. The conjugate for use according to claim 32, wherein the purification tag is a His.sub.6-tag.

34. The conjugate for use according to claim 32, wherein the purification tag is a Strep-tag.

35. The conjugate for use according to any one of the preceding claims, or the recombinant heterologously-expressed L-asparaginase for use according to any one of the preceding claims, wherein the host cell is of the genus Pseudomonas.

36. The conjugate for use according to claim 35, or the recombinant heterologously-expressed L-asparaginase for use according to claim 35, wherein the host cell is Pseudomonas aeruginosa.

37. The conjugate for use according to claim 35, or the recombinant heterologously-expressed L-asparaginase for use according to claim 35, wherein the host cell is Pseudomonas fluorescens.

38. The conjugate for use according to claim 35, or the recombinant heterologously-expressed L-asparaginase for use according to claim 35, wherein the host cell is Pseudomonas putida.

39. The conjugate for use according to any one of the preceding claims, or the recombinant heterologously-expressed L-asparaginase for use according to any one of the preceding claims, wherein the patient is 21 years old or younger.

40. The conjugate for use according to any one of the preceding claims, or the recombinant heterologously-expressed L-asparaginase for use according to any one of the preceding claims, wherein the conjugate, or the recombinant heterologously-expressed L-asparaginase is in lyophilised form.

41. The conjugate for use according to any one of the preceding claims, wherein the conjugate has an in vitro activity of at least 80% as compared to an equivalent conjugate that was expressed in E. coli.

42. The conjugate for use according to any one of the preceding claims, wherein the conjugate has an in vitro activity of at least 90% as compared to an equivalent conjugate that was expressed in E. coli.

43. The conjugate for use according to any one of the preceding claims, wherein the conjugate has an in vitro activity that is at least as high as an equivalent conjugate that was expressed in E. coli.

44. The conjugate for use according to any one of the preceding claims, or the recombinant heterologously-expressed L-asparaginase for use according to any one of the preceding claims, wherein said treatment comprises intravenous administration of the conjugate.

45. The conjugate for use according to any one of the preceding claims, or the recombinant heterologously-expressed L-asparaginase for use according to any one of the preceding claims, wherein said treatment comprises intramuscular administration of the conjugate.

46. The conjugate for use according to any one of the preceding claims, wherein said treatment comprises administration of the conjugate less than three times per week

47. The conjugate for use according to any one of claims 1 to 46, wherein said treatment comprises administration of the conjugate less than two times per week.

48. The conjugate for use according to any one of claims 1 to 47, wherein said treatment comprises administration of the conjugate less than one time per week.

49. The conjugate for use according to any one of the preceding claims, or the recombinant heterologously-expressed L-asparaginase for use according to any one of the preceding claims, wherein said disease treatable by L-asparagine depletion is a cancer.

50. The conjugate for use according to claim 49, or the recombinant heterologously-expressed L-asparaginase for use according to claim 49, wherein said cancer is selected from the group consisting of Acute Lymphoblastic Leukemia (ALL), non-Hodgkin's lymphoma, NK lymphoma, and pancreatic cancer.

51. The conjugate for use according to claim 49 or claim 50, or the recombinant heterologously-expressed L-asparaginase according to claim 49 or claim 50, wherein said cancer is ALL.

52. A composition comprising: (a) the conjugate for use according to any one of the preceding claims, and/or (b) the recombinant heterologously-expressed L-asparaginase for use according to any one of the preceding claims, and a pharmaceutically acceptable excipient.

53. A composition for use according to claim 52, wherein the composition comprises one or more lyoprotectant(s).

54. The composition for use according to claim 53, wherein the lyoprotectant(s) are selected from the list consisting of trehalose and sucrose.

55. The composition for use according to any one of claims 52 to 54, wherein the composition is in lyophilised form.

56. The composition for use according to any one of claims 52 to 54, wherein the composition is in solubilised form.

57. A method of treating a disease treatable by L-asparagine depletion in a patient that has been previously administered E. coli-derived L-asparaginase, said method comprising administering to said patient an effective amount of the conjugate or composition as defined in any one of the preceding claims.

58. A method of treating a disease treatable by L-asparagine depletion in a patient that has been previously administered E. coli-derived L-asparaginase, said method comprising administering to said patient an effective amount of the recombinant heterologously-expressed L-asparaginase or composition as defined in any one of the preceding claims.

59. The method according to claim 57 or claim 58, wherein the patient has had a hypersensitivity to an E. coli-derived L-asparaginase.

60. The method according to claim 59, wherein the hypersensitivity is selected from the group consisting of allergic reaction, anaphylactic shock, and silent inactivation.

61. The method according to any one of claims 57 to 60, wherein the patient has previously been administered Oncaspar®.

62. The method according to any one of claims 57 to 60, wherein the patient is 21 years old or younger.

63. The method according to any one of claims 57 to 62, wherein said disease treatable by L-asparagine depletion is a cancer.

64. The method according to claim 63, wherein said cancer is selected from the group consisting of Acute Lymphoblastic Leukemia (ALL), lymphosarcoma, non-Hodgkin's lymphoma, NK lymphoma, and pancreatic cancer.

65. The method according to claim 63 or claim 64, wherein said cancer is ALL.

66. The method according to any one of claims 57 to 65, wherein said conjugate or composition is administered intravenously.

67. The method according to any one of claims 57 to 66, wherein said conjugate or composition is administered intramuscularly.

68. The method according to any one of claims 57 to 67, wherein said conjugate or composition is administered less than three times per week.

69. The method according to any one of claims 57 to 68, wherein said conjugate or composition is administered less than two times per week.

70. The method according to any one of claims 57 to 69, wherein said conjugate or composition is administered less than one time per week.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0159] FIG. 1 Standard curve for the determination of E. coli HCPs in Oncaspar®. HCP concentration (ng/mL) is plotted on the X-axis against absorbance (A.sub.450) on the Y-axis. R.sup.2=0.9999.

EXAMPLES

Example 1: Detection of E. coli HCPs in Oncaspar®

[0160] A commercial vial of Oncaspar® was analysed for the presence of E. coli HCPs by enzyme-linked immunosorbent assay (ELISA) and by mass spectrometry. Both analyses confirmed the presence of E. coli HCPs in Oncaspar®.

(a) Detection of E. coli HCPs by ELISA

[0161] A commercial vial of Oncaspar® (3,750 IU) was reconstituted in water and analysed using anti-E. coli HCP ELISA. In the test kit, the ELISA plate was pre-coated with capture antibodies (anti-E. coli HCP antibodies). Following incubation of Oncaspar® with capture antibodies, detection antibodies (second anti-E. coli HCP antibodies, conjugated with biotin) were applied. Following incubation with detection antibodies, the plate was incubated with Streptavidin-Horseradish Peroxidase (HRP) conjugate, which binds to biotin-labelled antibody. “TMB” (3,3′,5,5″-tetramethylbenzidine) substrate was then added, which is converted by the captured HRP to a coloured product in proportion to the amount of HCP bound to the plate. Absorbance was measured at 450 nm.

[0162] A standard curve was generated to determine the relationship between the concentration of E. coli HCPs (ng/mL) and absorbance (405 nm). The standard curve (FIG. 1) had an R.sup.2 value of 0.9999, which signifies a very good ‘fit’ and a high degree of confidence in the data.

[0163] ELISA analysis determined that the vial of Oncaspar® contains ˜1.1 ng of E. coli HCPs.

[0164] A typical dose of Oncaspar® is 4500 IU, which corresponds to 1.2 vials of Oncaspar®. Accordingly, patients are administered ˜1.3 ng of E. coli HCPs every time they receive a dose of Oncaspar®.

(b) Detection of E. coli HCPs by Mass Spectrometry

[0165] Oncaspar® was also analysed using mass spectrometry (MS). Specifically, Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) was used to identify unknown HCPs in Oncaspar® by Independent Data Acquisition (‘IDA’) (Patel, V. J. et al. Journal of Proteome Research, (2009). 8: 3752-3759). The sample was digested using trypsin, ionised and mass to charge ratios (m/z) of the ions were determined. Results were searched against the Uniprot complete protein database to identify HCPs in the sample.

[0166] Five main peptides were identified by MS, which are summarised in Table 1:

TABLE-US-00007 Peptides Protein name Species (95%) L-asparaginase E. coli (strain K12) 690 L-asparaginase E. chrysanthemi 43 (strain 3937) Putative sulfatase AsIA E. coli (strain K12) 1 Uncharacterised protein YggE_OS E. coli (strain K12) 1 Protein UshA_OS E. coli (strain K12) 1

[0167] As expected, the most abundant peptide was identified as L-asparaginase from E. coli. The second most abundant peptide was identified as L-asparaginase from E. chrysanthemi. Without wishing to be bound by theory, the inventors believe that the peptides identified as L-asparaginase from E. chrysanthemi correspond to regions of E. coli L-asparaginase that have very high sequence identity to the native E. chrysanthemi L-asparaginase.

[0168] MS analysis also confirmed the presence of E. coli HCPs in Oncaspar®. These MS data further validate the results generated by ELISA. Of the E. coli HCPs in Oncaspar®, MS analysis identified (i) Putative sulfatase AsIA; (ii) Uncharacterised protein YggE_OS; and (iii) Protein UshA_OS.

Example 2: Identification of Highly Immunogenic E. coli HCPs in Oncaspar®

[0169] The E. coli HCPs identified in Oncaspar® were assessed for their binding affinity to major histocompatibility complex class II (MHC II), and the likelihood of being presented by any known MHC II receptor. This is critical in the development of a T-cell response and provides a strong indicator of immunogenicity.

[0170] The immunogenicity analysis was performed using the publicly-available “NetMHCIIpan” server. This server uses Artificial Neural Networks and is trained on a dataset of over 500,000 measurements of binding affinity, and eluted ligand mass spectrometry, covering the three MHC II isotypes HLA-DR, HLA-DQ, HLA-DP, as well as mouse molecules (H-2) (for details see Jensen et al. 2018, Immunology, 154. 394-406).

[0171] The immunogenicity results are shown in Table 2:

TABLE-US-00008 NetMHCIIpan 3.2 Number of No. of medium- plus E. coli HCP strong-binders strong- binders Putative sulfatase AsIA 45 1322 Uncharacterised protein 124 1920 YggE_OS Protein UshA_OS 66 789

[0172] Strong binders have <50 nM affinity across 25 HLA alleles covering >99% of the human population. Medium-plus-strong binders have <500 nM affinity across 25 HLA alleles covering >99% of the human population (Wang et al., 2010 BMC bioinformatics. 2010; 11:568).

[0173] As shown in Table 2, the E. coli HCPs identified in Oncaspar® by MS displayed extremely high numbers of strong- and medium-plus-strong binders. Each of the E. coli HCPs identified in Oncaspar® is therefore highly immunogenic.

[0174] Administration of E. coli HCPs immunologically pre-disposes patients to elicit a hypersensitive response to subsequently administered L-asparaginase that is derived from E. coli. The risk of a dangerous and undesirable immune reaction is increased when the HCPs are highly immunogenic.

Example 3: Production of a Conjugate Comprising E. chrysanthemi L-asparaginase and a PAS Polymer

[0175] Nucleic acid encoding E. chrysanthemi L-asparaginase and a PAS polymer is cloned into a pMMPc vector (GenBank accession number KC544266) under the control of a P.sub.c promoter. Cloning is confirmed by polymerase chain reaction analysis. Pseudomonas fluorescens strain MB214 is then transformed with the vector by electroporation.

[0176] Transformed Pseudomonas fluorescens is inoculated into broth and grown for 48 h, followed by centrifugation. A series of chromatography and concentration steps are performed. Sample purity is confirmed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. The amino acid sequence of the conjugate is confirmed by N-terminal protein sequencing and liquid chromatography—mass spectrometry.

[0177] L-asparaginase activity is confirmed in vitro by the Nesslerisation method at 37° C.

Example 4: Production of a Conjugate Comprising E. chrysanthemi L-asparaginase and 5000 Da PEG

[0178] Step A: Nucleic acid encoding E. chrysanthemi L-asparaginase is cloned into a pMMPc vector as described in Example 3. Pseudomonas fluorescens strain BM214 is then transformed with the vector and grown as per Example 3. Following inoculation and growth for 48 h, E. chrysanthemi L-asparaginase is then purified and concentrated as set out in Example 3.

[0179] Step B: Purified E. chrysanthemi L-asparaginase (5 mg/ml) is then mixed in the presence of 5000 Da functionalised mPEG (100 mg/ml) and sodium phosphate buffer (100 mM; pH 8.0) for 2.5 hours. The PEGylated E. chrysanthemi L-asparaginase is concentrated and purified, and L-asparaginase activity is confirmed in vitro by the Nesslerisation method at 37° C.

Example 5: In Vivo Half-Life Analysis of E. chrysanthemi L-asparaginase Conjugates

[0180] To assess the pharmacokinetic properties of the conjugates of Examples 3 and 4, the conjugates are administered intravenously to immune competent mice (“Group 1” and “Group 2”, respectively). As controls, “Group 3” mice are administered E. chrysanthemi L-asparaginase concentrated and purified in Example 3 Step A (i.e. not conjugated to PEG).

[0181] Blood is collected from the mice by retro-orbital bleeding. Bleedings are performed at 1 hr pre-administration, and at 6 h, 12 h, 18 h, 24 h, 36 h and 48 h post-administration. Residual L-asparaginase activity is assessed by the L-aspartic β-hydroxamate catalysis method, and in vivo t.sub.1/2 values are calculated.

[0182] Groups 1 and 2 display a significantly longer t.sub.1/2 than Group 3.

Example 6: Suitability for Use in Patients Who Have Developed Hypersensitivity to Oncaspar®

[0183] Patient #1: A male patient suffering from ALL experiences a hypersensitive reaction to his third dose of Oncaspar®. One week after this allergic reaction, he is intravenously administered conjugate prepared according to Example 3, at a dose of 750 IU/m.sup.2. The patient tolerates the conjugate of E. chrysanthemi L-asparaginase and PAS polymer, and his SAA levels are within therapeutic range (≥0.1 IU/ml) at 48 h after dosing.

[0184] Patient #2: A female patient suffering from ALL experiences an allergic reaction to her second dose of Oncaspar®. One week after this allergic reaction, she is intravenously administered conjugate prepared according to Example 3, at a dose of 750 IU/m.sup.2. The patient tolerates the conjugate of E. chrysanthemi L-asparaginase and PAS polymer, and her SAA levels are within therapeutic range at 48 h after dosing.