Construction of a Multivalent SCFV Through Alkyne-Azide 1,3-Dipolar Cycloaddition

• US 20090234105A1
• Filed: 03/26/2007
• Published: 09/17/2009
• Est. Priority Date: 03/24/2006
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Du, Wenjun, Denardo, Sally, Gervay-Hague, Jacquelyn, Natarajan, Arutselvan
• Granted Patent: US 8,946,391 B2
• Time in Patent Office: Days
• US Class Current: 530/387.300
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/3015 Breast
• CPC Class Codes: C07K 16/3069 Reproductive system, e.g. ovaria, uterus, testes, prostate
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)

Abstract

The present invention provides for a practical, universal and efficient method to ligate two large macromolecules (e.g., proteins) using the alkyne-azide 1,3-dipolar cycloaddition reaction to produce a conjugated macromolecule, such as a multivalent scFv. The present invention also provides for conjugate macromolecules comprising a plurality of macromolecule components cross-linked through at least one linking group comprising at least one 1,2,3-triazole moiety, wherein at least 50 percent of the macromolecule components in the conjugate macromolecule has only one site available for cross-linking.

Claims

1. A method of preparing a multivalent scFv, said method comprising:

(a) contacting a first scFv component having from one to eight attached alkyne moieties with a second scFv component having an attached linking group with a terminal azide moiety under conditions sufficient for 1,2,3-triazole formation to occur; and

optionally

(b) attaching additional scFv components to the product of step (a) in a sequential manner by contacting the product of each step with another scFv component having an attached linking group with a terminal azide moiety under conditions sufficient for 1,2,3-triazole formation;

to produce said multivalent scFv.

2. A method in accordance with claim 1, wherein said multivalent scFv is a di-scFv.

3. A method in accordance with claim 1, wherein said first scFv component has from two to eight alkyne moieties present in a linking group.

4. A method in accordance with claim 3, wherein said first scFv component has from three to six alkyne moieties present in a linking group.

5. A method in accordance with claim 1, wherein said first scFv component has the formula:

6. A method in accordance with claim 1, wherein said second scFv component having an attached linking group with a terminal azide moiety has the formula:

7. A method in accordance with claim 2, wherein said di-scFv has the formula:

8. A method in accordance with claim 1, wherein said multivalent scFv is a tri-scFv.

9. A method in accordance with claim 1, wherein said multivalent scFv is a tetra-scFv.

10. A method in accordance with claim 4, wherein each linking group is attached to an scFv moiety through a thio ether linkage to a cysteine residue.

11. A method of covalently attaching two proteins to form a conjugate protein, said method comprising contacting a first protein component having from one to eight attached alkyne moieties with a second protein component having an attached linking group with a terminal azide moiety under conditions sufficient for 1,2,3-triazole formation to occur and produce said conjugate protein.

12. A method in accordance with claim 11, wherein said first protein component has from three to six attached alkyne moieties.

13. A method in accordance with claim 11, wherein said first protein component has the formula:

14. A method in accordance with claim 11, wherein said second protein component has the formula:

15. A method in accordance with claim 11, wherein said conjugate protein has the formula:

16. A conjugate protein comprising a plurality of protein components cross-linked through at least one linking group comprising at least one 1,2,3-triazole moiety, wherein at least 50 percent of the protein components in the conjugate protein has only one site available for cross-linking.

17. A conjugate protein in accordance with claim 16, wherein said conjugate protein is a multivalent scFv.

18. A multivalent scFv in accordance with claim 17, wherein the multivalent scFv is a di-scFv.

19. A multivalent scFv in accordance with claim 17, wherein the multivalent scFv is a tri-scFv.

20. A multivalent scFv in accordance with claim 17, wherein the multivalent scFv is a tetra-scFv.

21. A multivalent scFv in accordance with claim 18, wherein said multivalent scFv has the structure selected from the group consisting of:

22. A multivalent scFv in accordance with claim 21, wherein said scFv is an antibody fragment that binds to the MUC-1 mucin antigen expressed in breast cancer and prostate cancer cells.