Construction of a multivalent scFv through alkyne-azide 1,3-dipolar cycloaddition

• US 8,946,391 B2
• Filed: 03/26/2007
• Issued: 02/03/2015
• 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: Gervay-Hague, Jacquelyn, Du, Wenjun, Denardo, Sally, Natarajan, Arutselvan
• Primary Examiner(s): Reddig, Peter J
• Application Number:
  US12/294,227
• Publication Number:
  US 20090234105A1
• Time in Patent Office: 2,871 Days
• US Class Current: 530/387.3
• 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 the formula;

2. 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, wherein said second scFv component having an attached linking group with a terminal azide moiety has the formula;

3. A method of preparing a divalent scFv (“

di-scFv”

), said method comprising;

(a) contacting a first scFv component having the formula;

4. A method of covalently attaching two proteins to form a conjugate protein, said method comprising contacting a first protein component having the formula:

5. 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, wherein said second protein component has the formula:

6. A method of covalently attaching two proteins to form a conjugate protein, said method comprising contacting a first protein component having the formula: