ALKYLATED INTERLEUKIN-18 AND RELATED COMPOSITIONS, KITS, AND METHODS OF MAKING AND USING SAME

• US 20100166702A1
• Filed: 12/21/2009
• Published: 07/01/2010
• Est. Priority Date: 12/23/2008
• Status: Active Grant

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Zhao, Cheng, Brate, Elaine Marie, Rae, Tracey D.
• Granted Patent: US 8,168,165 B2
• Time in Patent Office: Days
• US Class Current: 424/85.200
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]

Abstract

Alkylated interleukin-18 (IL-18), compositions comprising alkylated IL-18, kits comprising such compositions, methods of alkylating IL-18, and methods of using compositions comprising alkylated IL-18.

Claims

1. An isolated or purified, alkylated interleukin-18 (IL-18), in which at least one free sulfhydryl of a cysteine residue has been alkylated.

2. The alkylated IL-18 of claim 1, in which the hydrogen of at least one free sulfhydryl of a cysteine residue has been replaced with carboxyamidomethyl.

3. The IL-18 of claim 2, which has a capping ratio of about 2.5 to about 4.0 as determined by mass spectrometry.

4. The alkylated IL-18 of claim 1, in which the hydrogen of at least one free sulfhydryl of a cysteine residue has been replaced with carboxymethyl.

5. The alkylated IL-18 of claim 4, which has a capping ratio of about 1.0 to about 4.0 as determined by mass spectrometry.

6. A composition comprising (i) an isolated or purified, alkylated IL-18, in which at least one free sulfhydryl of a cysteine residue has been alkylated, and (ii) at least one stabilizing protein.

7. The composition of claim 6, in which the hydrogen of at least one free sulfhydryl of a cysteine residue has been replaced with carboxyamidomethyl, and, optionally, in which the protein is bovine serum albumin (BSA).

8. The composition of claim 7, in which the alkylated IL-18 has a capping ratio of about 2.5 to about 4.0 as determined by mass spectrometry, and, optionally, in which the protein is BSA.

9. The composition of claim 6, in which the hydrogen of at least one free sulfhydryl of a cysteine residue has been replaced with carboxymethyl, and, optionally, in which the protein is BSA.

10. The composition of claim 9, in which the alkylated IL-18 has a capping ratio of about 1.0 to about 4.0 as determined by mass spectrometry, and, optionally, in which the protein is BSA.

11. The composition of claim 6, in which the protein is selected from the group consisting of BSA, human serum albumin (HSA), bovine γ

-globulin (BGG), Carnation®

Instant Nonfat Dry Milk, and any combination thereof.

12. The composition of claim 6, in which the protein is BSA.

13. The composition of claim 6, which comprises a known concentration of alkylated IL-18.

14. A series of calibrating compositions, which can be used in a method of determining the concentration of IL-18 in a sample, wherein each of the compositions in the series comprises (i) a known concentration of an isolated or purified, alkylated IL-18, in which at least one free sulfhydryl of a cysteine residue has been alkylated, and (ii) at least one stabilizing protein, wherein each of the compositions differs from the other compositions in the series by the concentration of alkylated IL-18.

15. The composition of claim 14, in which the hydrogen of at least one free sulfhydryl of a cysteine residue has been replaced with carboxyamidomethyl, and, optionally, in which the protein is BSA.

16. The composition of claim 15, in which the alkylated IL-18 has a capping ratio of about 2.5 to about 4.0 as determined by mass spectrometry, and, optionally, in which the protein is BSA.

17. The composition of claim 14, in which the hydrogen of at least one free sulfhydryl of a cysteine residue has been replaced with carboxymethyl, and, optionally, in which the protein is BSA.

18. The composition of claim 17, in which the alkylated IL-18 has a capping ratio of about 1.0 to about 4.0 as determined by mass spectrometry, and, optionally, in which the protein is BSA.

19. The composition of claim 14, in which the protein is selected from the group consisting of BSA, HSA, BGG, Carnation®

Instant Nonfat Dry Milk, and any combination thereof.

20. The composition of claim 14, in which the protein is BSA.

21. A kit for assaying a test sample for IL-18, which kit comprises at least one component for assaying the test sample for IL-18 and instructions for assaying the patient sample for IL-18, wherein the at least one component includes at least one composition comprising (i) a known amount of an isolated or purified, alkylated IL-18, in which at least one free sulfhydryl of a cysteine residue has been alkylated, and (ii) at least one stabilizing protein, wherein, if the kit comprises more than one composition, each of the compositions differs from the other compositions by the concentration of alkylated IL-18.

22. In a method of determining the presence, amount or concentration of IL-18 in a test sample by an immunoassay comprising comparing a signal generated by a detectably labeled antibody or a detectably labeled analyte as a direct or indirect indication of the concentration of IL-18 in the test sample to a signal generated as a direct or indirect indication of the concentration of IL-18 in a calibrator, which is optionally part of a series of calibrators in which each of the calibrators differs from the other calibrators in the series by the concentration of IL-18, the improvement comprising using a calibrator comprising (i) a known concentration of an isolated or purified, alkylated IL-18, in which at least one free sulfhydryl of a cysteine residue has been alkylated, and (ii) at least one stabilizing protein.

23. In the method of claim 22, in which the hydrogen of at least one free sulfhydryl of a cysteine residue has been replaced with carboxyamidomethyl, and, optionally, in which the protein is BSA.

24. In the method of claim 23, in which the alkylated IL-18 has a capping ratio of about 2.5 to about 4.0 as determined by mass spectrometry, and, optionally, in which the protein is BSA.

25. In the method of claim 22, in which the hydrogen of at least one free sulfhydryl of a cysteine residue has been replaced with carboxymethyl, and, optionally, in which the protein is BSA.

26. In the method of claim 25, in which the alkylated IL-18 has a capping ratio of about 1.0 to about 4.0 as determined by mass spectrometry, and, optionally, in which the protein is BSA.

27. In the method of claim 22, wherein the protein in (ii) is selected from the group consisting of BSA, HSA, BGG, Carnation®

Instant Nonfat Dry Milk, and any combination thereof.

28. In the method of claim 22, wherein the protein in (ii) is BSA.

29. A method of alkylating an isolated or purified IL-18, which method comprises contacting the IL-18 with an alkylating agent under conditions that promote alkylation of the free sulfhydryls of the cysteine residues of the IL-18, whereupon the IL-18 is alkylated.

30. The method of claim 29, wherein the alkylating agent is a sulfhydryl-reactive reagent comprising iodoacetic acid, and the conditions comprise a pH of about 7.2, a reaction time of about two hours, a temperature of about 22°

C. (+/−

2°

C.), and excess sulfhydryl-reactive reagent.

31. The method of claim 29, wherein the alkylating agent is a sulfhydryl-reactive reagent comprising iodoacetamide, and the conditions comprise a pH of about 7.2, a reaction time of about two hours, a temperature of about 22°

C. (+/−

2°

C.), the absence of light, and excess sulfhydryl-reactive reagent.

32. A pharmaceutical composition comprising (i) a therapeutically or prophylactically effective amount of an alkylated IL-18, (ii) a pharmaceutically acceptable carrier, diluent, and/or excipient, and (iii) optionally, another active agent and/or an adjuvant, wherein the pharmaceutical composition is optionally part of a kit comprising one or more containers in which the alkylated IL-18, another active agent and/or the adjuvant can be present in the same or different containers.

33. A method of treating a patient in therapeutic or prophylactic need of IL-18, which method comprises administering to the patient a pharmaceutical composition comprising (i) a therapeutically or prophylactically effective amount of an alkylated IL-18, (ii) a pharmaceutically acceptable carrier, diluent, and/or excipient, and (iii) optionally, another active agent and/or an adjuvant, whereupon the patient is treated.