Bivalent binding molecules of 7 transmembrane G protein-coupled receptors
First Claim
1. A method of identifying a bivalent binding molecule of a 7TM G protein-coupled receptor wherein said bivalent binding molecule comprises a first and second aptamer to a first and second epitope of the same 7 transmembrane G protein-coupled receptor, said method comprising:
- a) identifying said first aptamer to said first epitope by the method comprising;
i) preparing a first candidate mixture of nucleic acids;
ii) contacting said first candidate mixture of nucleic acid with said first epitope, wherein nucleic acids having an increased affinity to said first epitope may be partitioned from the remainder of the first candidate mixture;
iii) partitioning said increased affinity nucleic acids from the remainder of the first candidate mixture; and
iv) amplifying said increased affinity nucleic acids, whereby said first aptamer to said first epitope may be identified;
b) identifying said second aptamer to said second epitope by the method comprising;
i) preparing a second candidate mixture of nucleic acids;
ii) contacting said second candidate mixture of nucleic acid with said second epitope, wherein nucleic acids having an increased affinity to said second epitope may be partitioned from the remainder of the second candidate mixture;
iii) partitioning said increased affinity nucleic acids from the remainder of the second candidate mixture; and
iv) amplifying said increased affinity nucleic acids, whereby said second aptamer to said second epitope may be identified; and
c) coupling said first aptamer to said second aptamer, whereby said bivalent binding molecule may be identified;
wherein said 7 transmembrane G protein-coupled receptor is selected from the group consisting of histamine, muscarinic, cholecystokinin, choriogonadotropin, corticotropin releasing factor, gastrin, neuropeptide Y, TSH and 5-HT 7 transmembrane G protein-coupled receptors.
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Abstract
Described herein are methods for identifying and preparing bivalent binding molecules to 7 transmembrane G protein-coupled receptors. The methods disclosed herein are based on the SELEX method for generating high affinity nucleic acid ligands. SELEX is an acronym for Systematic Evolution of Ligands by EXponential enrichment. The methods of this invention combine two or more binding domains to two or more different epitopes of the same 7 transmembrane G protein-coupled receptor. These bivalent binding molecules are useful as therapeutic and diagnostic agents.
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Citations
19 Claims
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1. A method of identifying a bivalent binding molecule of a 7TM G protein-coupled receptor wherein said bivalent binding molecule comprises a first and second aptamer to a first and second epitope of the same 7 transmembrane G protein-coupled receptor, said method comprising:
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a) identifying said first aptamer to said first epitope by the method comprising;
i) preparing a first candidate mixture of nucleic acids;
ii) contacting said first candidate mixture of nucleic acid with said first epitope, wherein nucleic acids having an increased affinity to said first epitope may be partitioned from the remainder of the first candidate mixture;
iii) partitioning said increased affinity nucleic acids from the remainder of the first candidate mixture; and
iv) amplifying said increased affinity nucleic acids, whereby said first aptamer to said first epitope may be identified;
b) identifying said second aptamer to said second epitope by the method comprising;
i) preparing a second candidate mixture of nucleic acids;
ii) contacting said second candidate mixture of nucleic acid with said second epitope, wherein nucleic acids having an increased affinity to said second epitope may be partitioned from the remainder of the second candidate mixture;
iii) partitioning said increased affinity nucleic acids from the remainder of the second candidate mixture; and
iv) amplifying said increased affinity nucleic acids, whereby said second aptamer to said second epitope may be identified; and
c) coupling said first aptamer to said second aptamer, whereby said bivalent binding molecule may be identified;
wherein said 7 transmembrane G protein-coupled receptor is selected from the group consisting of histamine, muscarinic, cholecystokinin, choriogonadotropin, corticotropin releasing factor, gastrin, neuropeptide Y, TSH and 5-HT 7 transmembrane G protein-coupled receptors. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method of identifying a bivalent binding molecule 7 transmembrane G protein-coupled receptor, wherein said bivalent binding molecule comprises an aptamer to a first epitope coupled to a non-aptamer binding domain which binds to a second epitope of the same receptor, said method comprising:
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a) preparing a blended candidate mixture of bivalent binding molecules comprising a candidate mixture of nucleic acid sequences coupled to a non-aptamer binding domain which binds to said second epitope of the receptor;
b) contacting said 7 transmembrane G protein-coupled receptor with said blended candidate mixture of bivalent binding molecules, wherein bivalent binding molecules having an aptamer of increased affinity to the first epitope of the 7 transmembrane G protein-coupled receptor relative to the blended candidate mixture may be partitioned from the remainder of the candidate mixture;
c) partitioning the increased affinity bivalent binding molecules from the remainder of the blended candidate mixture; and
d) amplifying the increased affinity bivalent binding molecules to yield an enriched mixture of bivalent binding molecules, whereby bivalent binding molecules to a 7 transmembrane G protein-coupled receptor may be identified, wherein said 7 transmembrane G protein-coupled receptor is selected from the group consisting of histamine, muscarinic, cholecystokinin, choriogonadotropin, corticotropin releasing factor, gastrin, neuropeptide Y, TSH and 5-HT 7 transmembrane G protein-coupled receptors. - View Dependent Claims (9, 10, 11, 12, 13)
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14. A method of identifying a bivalent binding molecule to 7 transmembrane G protein-coupled receptor, wherein said bivalent binding molecule comprises an aptamer to a first epitope on a first extracellular domain of said receptor coupled to a non-aptamer binding domain which binds to a second epitope on a second extracellular domain of the same 7TM G protein-coupled receptor, said method comprising:
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a) identifying an aptamer to said first epitope of said 7 transmembrane G protein-coupled receptor by the method comprising;
i) preparing a candidate mixture of nucleic acids;
ii) contacting said candidate mixture with said first epitope, wherein nucleic acids having an increased affinity to said first epitope relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;
iii) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and
iv) amplifying the increased affinity nucleic acids to yield an enriched mixture of nucleic acids, whereby an aptamer to said first epitope of said 7 transmembrane G protein-coupled receptor may be identified; and
b) coupling said aptamer to a non-aptamer binding domain which binds to said second epitope on the second extracellular domain of said 7 transmembrane G protein coupled receptor to yield a bivalent binding molecule, wherein said 7 transmembrane G protein-coupled receptor is selected from the group consisting of histamine, muscarinic, cholecystokinin, choriogonadotropin, corticotropin releasing factor, gastrin, neuropeptide Y, TSH and 5-HT 7 transmembrane G protein-coupled receptors. - View Dependent Claims (15, 16, 17, 18, 19)
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Specification