STABLE RECOMBINANT ADENOSINE DEAMINASE

• US 20090047271A1
• Filed: 04/18/2008
• Published: 02/19/2009
• Est. Priority Date: 04/20/2007
• Status: Active Grant

• Current Assignee: Unikeris Limited
• Original Assignee: Enzon Pharmaceuticals Inc. (Icahn Associates Holding LLC)
• Inventors: Youngster, Stephen K., Filpula, David R.
• Granted Patent: US 8,071,741 B2
• Time in Patent Office: Days
• US Class Current: 424/94.600
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• CPC Class Codes: C12Y 305/04014 Deoxycytidine deaminase (3.5.4.14)

Abstract

A mutein recombinant adenosine deaminase having any oxidizable cysteine residue replaced by a non-oxidizable amino acid residue is disclosed. Stabilized recombinant adenosine deaminase, polymer conjugates and methods of treatment using the same are also disclosed.

Claims

1. A recombinant adenosine deaminase, wherein an oxidizable amino acid residue expressed in the wild-type of the adenosine deaminase is replaced by a non-oxidizable amino acid residue.

2. The recombinant adenosine deaminase of claim 1, wherein the oxidizable amino acid residue is cysteine, methionine or tryptophan, and the non-oxidizable amino acid residue is selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tyrosine, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminobutyric acid, desmosine. 2,2′

-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethylglycine, n-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, n-methylglycine, sarcosine, n-methylisoleucine, 6-n-methyllysine, n-methylvaline, norvaline, norleucine and ornithine.

3. The recombinant adenosine deaminase of claim 1, wherein the non-oxidizable amino acid residue is selected from the group consisting of serine, alanine, asparagine, glutamine, glycine, isoleucine, leucine, phenylalanine, threonine, tyrosine, and valine.

4. The recombinant adenosine deaminase of claim 1, wherein the non-oxidizable amino acid residue is serine.

5. The recombinant adenosine deaminase of claim 2, wherein the oxidizable cysteine is located at about position 74 of the mature protein.

6. The recombinant adenosine deaminase of claim 5 that is a recombinant human adenosine deaminase or a recombinant bovine adenosine deaminase.

7. The recombinant adenosine deaminase of claim 6 that is translated from a DNA molecule according to SEQ ID NO:

2 or SEQ ID NO;

4.

8. The recombinant adenosine deaminase of claim 6 that comprises SEQ ID NO:

1 or SEQ ID NO;

3.

9. The recombinant adenosine deaminase of claim 8 that comprises SEQ ID NO:

1 with an amino acid substitution selected from the group consisting of Gln in place of Lys₁₉₈;

Ala in place of Thr₂₄₅;

Arg in place of Gly₃₅₁, and combinations thereof.

10. A polyalkylene oxide-adenosine deaminase conjugate, wherein the adenosine deaminase is the recombinant adenosine deaminase of claim 1.

11. The polyalkylene oxide-adenosine deaminase conjugate of claim 10, wherein the polyalkylene oxide is polyethylene glycol.

12. The polyalkylene oxide-adenosine deaminase conjugate of claim 11, wherein the polyethylene glycol is conjugated to the recombinant adenosine deaminase via a linker selected from the group consisting of succinimidyl carbonate, thiazolidine thione, urethane, and amide based linkers.

13. The polyalkylene oxide-adenosine deaminase conjugate of claim 11, wherein the polyethylene glycol is covalently attached to an epsilon amino group of a Lys of the recombinant adenosine deaminase.

14. The polyalkylene oxide-adenosine deaminase conjugate of claim 11, wherein the recombinant adenosine deaminase comprises one or more polyethylene glycol strands attached to epsilon amino groups of one or more Lys residues of the recombinant adenosine deaminase.

15. The polyalkylene oxide-adenosine deaminase conjugate of claim 11, wherein the recombinant adenosine deaminase comprises from about 11 to about 18 polyethylene glycol strands attached to epsilon amino groups of one or more Lys residues of the recombinant adenosine deaminase.

16. The polyalkylene oxide-adenosine deaminase conjugate of claim 11, wherein the polyethylene glycol is conjugated to the recombinant adenosine deaminase via a succinimidyl carbonate linker.

17. The polyalkylene oxide-adenosine deaminase conjugate of claim 10, wherein the polyethylene glycol has a molecular weight of from about 2,000 to about 100,000.

18. The polyalkylene oxide-adenosine deaminase conjugate of claim 10, wherein the polyethylene glycol has a molecular weight of from about 4,000 to about 45,000.

19. A method of treating an adenosine deaminase-mediated condition in mammals, comprising administering an effective amount of the recombinant adenosine deaminase of claim 1.

20. The method of claim 19, wherein the adenosine deaminase-mediated condition is severe combined immune disorder.

21. A process for purifying the recombinant adenosine deaminase of claim 10, comprising purifying the protein by ion exchange chromatography.

22. A process for purifying the recombinant adenosine deaminase of claim 10 that comprises SEQ ID NO:

1, comprising purifying the protein by hydrophobic interaction chromatography.

23. A recombinant adenosine deaminase produced by the process of claim 21.

24. A recombinant adenosine deaminase produced by the process of claim 22.

25. An isolated DNA that encodes a recombinant adenosine deaminase having the amino acid sequence SEQ ID NO:

1 or SEQ ID NO;

3.

26. The isolated DNA of claim 25, wherein the recombinant adenosine deaminase has an amino acid substitution selected from the group consisting of Gln in place of Lys₁₉₈;

Ala in place of Thr₂₄₅;

Arg in place of Gly₃₅₁, and combinations thereof.