Construction of a multivalent scFv through alkyne-azide 1,3-dipolar cycloaddition
AssigneeThe Regents of the University of California
InventorsJacquelyn Gervay-Hague, Wenjun Du, Sally Denardo, Arutselvan Natarajan
Life of this patent
Estimating the term…
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.
Claims6
A method of preparing a multivalent scFv, said method comprising:
- (a) contacting a first scFv component having the formula:
- 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.
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:
- 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.
A method of preparing a divalent scFv (“di-scFv”), said method comprising:
- (a) contacting a first scFv component having the formula:
- 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 di-scFv, wherein said di-scFv has the formula:
A method of covalently attaching two proteins to form a conjugate protein, said method comprising contacting a first protein component having the formula:
- 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.
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:
A method of covalently attaching two proteins to form a conjugate protein, said method comprising contacting a first protein component having the formula:
- 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 conjugate protein has the formula:
Dependencies are inferred from the claim text.