Recombinant lectins, binding-site modified lectins and uses thereof

• US 9,068,013 B2
• Filed: 07/22/2011
• Issued: 06/30/2015
• Est. Priority Date: 07/28/2010
• Status: Active Grant

• Current Assignee: SmartCells, Inc. (Merck & Co., Inc.)
• Original Assignee: Smart Cells Incorporated
• Inventors: Lancaster, Thomas M., Butters, Robert, Choodnovskiy, Naomi, Zion, Todd C.
• Primary Examiner(s): Rao, Manjunath
• Assistant Examiner(s): Moore, William W
• Application Number:
  US13/811,981
• Publication Number:
  US 20130324702A1
• Time in Patent Office: 1,439 Days
• Field of Search: 530/370
• US Class Current: 1/1
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/42 Lectins, e.g. concanavalin, phytohaemagglutinin

Abstract

In one aspect, the disclosure provides cross-linked materials that include multivalent lectins with at least two binding sites for glucose, wherein the lectins include at least one affinity ligand which is capable of competing with glucose for binding with at least one of said binding sites and is covalently linked to a cysteine residue of the lectins; and conjugates that include two or more separate affinity ligands bound to a conjugate framework, wherein the two or more affinity ligands compete with glucose for binding with the lectins at said binding sites and wherein conjugates are cross-linked within the material as a result of non-covalent interactions between lectins and affinity ligands on different conjugates. These materials are designed to release amounts of conjugate in response to desired concentrations of glucose. Depending on the end application, in various embodiments, the conjugates may also include a drug and/or a detectable label.

Claims

1. A cross-linked material comprising:

multivalent lectins with at least two binding sites for glucose, wherein the lectins include at least one affinity ligand which is capable of competing with glucose for binding with at least one of said binding sites and is covalently linked to a cysteine residue of the lectins wherein the affinity ligand includes a saccharide and a linker and the saccharide is covalently linked to the linker via an anomeric carbon; and

conjugates that include two or more separate affinity ligands, wherein the two or more affinity ligands compete with glucose for binding with the lectins at said binding sites and wherein conjugates are cross-linked within the material as a result of non-covalent interactions between lectins and affinity ligands on different conjugates.

2. The material of claim 1, wherein the conjugates further comprise a drug.

3. The material of claim 1, wherein the conjugates further comprise a detectable label.

4. The material of claim 1, wherein the lectins include only one covalently linked affinity ligand per lectin.

5. The material of claim 1, wherein the lectins include one covalently linked affinity ligand per glucose binding site.

6. The material of claim 1, wherein the lectins are recombinant Con A lectins that contain at least one cysteine residue introduced by site directed mutagenesis.

7. The material of claim 6, wherein the at least one cysteine residue substitutes an amino acid in the wild-type Con A sequence that forms part of a binding site for glucose.

8. The material of claim 6, wherein the at least one cysteine residue is at a location within the wild-type Con A sequence that is within 1-5 amino acids (as measured in the primary amino acid sequence) of an amino acid that forms part of a binding site for glucose.

9. The material of claim 6, wherein the at least one cysteine residue is at a location within the wild-type Con A sequence that it is separated by at least 1, 2, 3, 4 or 5 amino acids (as measured in the primary amino acid sequence) from any amino acid that forms part of a binding site for glucose.

10. The material of claim 6, wherein lectins have an amino acid sequence that is at least 90%, identical to the amino acid sequence of SEQ ID NO:

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 with the proviso that in each of these sequences each X is a codable amino acid and at least one X is a cysteine.

11. The material of claim 6, wherein lectins have an amino acid sequence that is at least 90%, identical to the amino acid sequence of SEQ ID NO:

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 where just one of the X positions is a cysteine and all other X positions are codable amino acids.

12. The material of claim 6, wherein the lectins have an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

17 with the proviso that at least one X is a cysteine.

13. The material of claim 1, wherein the affinity ligand covalently linked to the cysteine of the lectin includes a saccharide which is glucose.

14. The material of claim 1, wherein the affinity ligand covalently linked to the cysteine of the lectin includes a saccharide which is mannose.

15. The material of claim 2, wherein the drug is an insulin molecule.

16. The material of claim 1, wherein the conjugate has the general formula:

17. The material of claim 1, wherein the conjugate has the general formula: