Methods for reducing the mitogenicity of lectin compositions

• US 8,729,242 B2
• Filed: 01/27/2010
• Issued: 05/20/2014
• Est. Priority Date: 10/19/2005
• Status: Active Grant

• Current Assignee: SmartCells, Inc. (Merck & Co., Inc.)
• Original Assignee: SmartCells, Inc. (Merck & Co., Inc.)
• Inventors: Lancaster, Thomas M., Cai, Wenyi, Faucette, Ryan, Pecukonis, Matthew, Zion, Todd C.
• Primary Examiner(s): Mi, Qiuwen
• Application Number:
  US12/695,078
• Publication Number:
  US 20100130726A1
• Time in Patent Office: 1,574 Days
• Field of Search: None
• US Class Current: 530/396
• CPC Class Codes: A61K 38/25 Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 38/28 Insulins
• 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: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/6903 the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C08B 30/18 Dextrin , e.g. yellow canari, white dextrin, amylodextrin or maltodextrin; Methods of depolymerisation, e.g. by irradiation or mechanically
• CPC Class Codes: C08B 37/00 Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof cellulose D21; microbiological processes C12P
• CPC Class Codes: C08B 37/003 Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• CPC Class Codes: C08B 37/0069 Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• CPC Class Codes: C08B 37/0084 Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
• CPC Class Codes: Y10S 436/827 Lectins

Abstract

Methods for reducing the T-cell mitogenicity of lectin compositions are provided. In one aspect this is achieved by chemically modifying mitogenic lectin compositions under optimized conditions. Additionally or alternatively, the reduction in T-cell mitogenicity is achieved by removing unmodified subunits chemically modified mixtures. Modified lectin compositions with reduced T-cell mitogenicity are also provided as are uses of the inventive compositions.

Claims

1. A method of reducing the T-cell mitogenicity of a chemically modified lectin composition by affinity chromatography wherein the affinity chromatography is performed using beads in solution, wherein the method comprises:

(a) providing a mitogenic lectin composition that includes a plurality of mitogenic lectins and reacting the mitogenic lectin composition with one or more chemical compounds to produce a chemically modified lectin composition;

(b) contacting a chemically modified lectin composition with an immobilized ligand on the beads under conditions that allow at least a portion of the composition to bind the ligand;

(c) exposing the bound portion of the chemically modified lectin composition to glucose at a first concentration;

(d) exposing the remaining bound portion of the chemically modified lectin composition after step (c) to glucose at a second higher concentration; and

(e) recovering the portion of the chemically modified lectin composition that is released at the second higher glucose concentration, wherein the T-cell mitogenicity of the recovered portion of the chemically modified lectin composition is less than the T-cell mitogenicity of the original chemically modified lectin composition.

2. The method of claim 1, wherein in the step of reacting, the mitogenic lectin composition is reacted with a monovalent chemical compound.

3. The method of claim 1, wherein the mitogenic lectin composition includes native lectin subunits, and in the step of reacting, the mitogenic lectin composition is reacted with a multivalent chemical compound that covalently crosslinks native lectin subunits.

4. The method of claim 3, wherein in the step of reacting, the mitogenic lectin composition is further reacted with a monovalent chemical compound.

5. The method of claim 1, wherein the modified lectin composition includes a modified lectin that is multimeric and comprised of identical subunits in its native form.

6. The method of claim 5, wherein the modified lectin is tetrameric in its native form and the modified lectin composition comprises monomers, dimers, trimers, or tetramers of the modified lectin, or any mixture thereof.

7. The method of claim 1, wherein the modified lectin composition includes Concanavalin A.

8. The method of claim 2, wherein the monovalent chemical compound is a polymer.

9. The method of claim 4, wherein the monovalent chemical compound is a polymer.

10. The method of claim 8, wherein the polymer is a pegylation agent.

11. The method of claim 9, wherein the polymer is a pegylation agent.

12. The method of claim 1, wherein the ligand comprises a saccharide or polysaccharide.

13. The method of claim 1, wherein the ligand comprises glucose.

14. The method of claim 1, wherein the ligand comprises mannose.

15. The method of claim 1, wherein the ligand comprises a polysaccharide selected from the group consisting of dextran, glycogen, and mannan.

16. The method of claim 1, wherein the first glucose concentration is about 50 mg/dl.

17. The method of claim 1, wherein the second glucose concentration is about 800 mg/dl.

18. The method of claim 1, wherein the first glucose concentration is below physiologically relevant glucose levels.

19. The method of claim 18, wherein the first glucose concentration is below 400 mg/dl.

20. The method of claim 1, wherein the second glucose concentration is above physiologically relevant glucose levels.

21. The method of claim 20, wherein the second glucose concentration is above 400 mg/dl.

22. The method of claim 1 wherein the concentration of glucose is increased gradually from the first glucose concentration to the second glucose concentration.

23. The method of claim 1, wherein the concentration of glucose is increased stepwise from the first glucose concentration to the second glucose concentration.

24. The method of claim 1, wherein the affinity chromatography is performed using a column.