US5955358A
Expired / Lapsed
US
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Optimization of binding proteins
Assignee: Applied Molecular Evolution Inc
•
Filing: 1995-02-09
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Granted: 1999-09-21
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Expires: 2015-02-09
Abstract
Abstract The invention relates to methods for manipulating nucleic acids so as to optimize the binding characteristics of an encoded binding protein by providing two or more nucleic acids encoding binding proteins having at least one set of splicing sites, the set of splicing sites flanking opposite ends of one or more encoded binding domains; mixing the nucleic acids to produce a parent population of mixed nucleic acids encoding binding proteins; and randomly incorporating the binding domains between the nucleic acids through the set of splicing sites to produce a different population of nucleic acids encoding binding proteins wherein at least one binding protein is characterized by substantially different binding characteristics than a member of the parent population.
Inventors
- 👤 William D. Huse
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 11:32
CPC Classifications
C; C07; C07K; C07K16/00; C07K16/46; C07K16/461; C07K16/464; C07K2317/00; C07K2317/20; C07K2317/24; C07K2319/00
Claims Analysis
Total Claims: 33
Claim 1
Independent Claim
191 words
A method for manipulating nucleic acids so as to increase the binding of an encoded antibody to a particular ligand, said method comprising the steps of: (a) providing two or more nucleic acids encoding different antibodies all having selective binding for said particular ligand, said nucleic acids having at least one set of splicing sites, said set of splicing sites flanking opposite ends of one or more encoded CDRs, wherein the binding activity of at least one encoded antibody is known; (b) mixing said two or more nucleic acids to produce a parent population of mixed nucleic acids encoding said antibodies; (c) randomly incorporating said one or more encoded CDRs between said two or more nucleic acids through said set of splicing sites to produce a different population of nucleic acids than said parent population, wherein at least one antibody encoded by said different population is characterized by having increased binding to the particular ligand as compared to the antibody having the known binding activity; and (d) identifying at least one of the antibodies having increased binding to the particular ligand as compared to the antibody having the known binding activity.
Claim 2
Independent Claim
191 words
A method for manipulating nucleic acids so as to increase the binding of an encoded antibody to a particular ligand, said method comprising the steps of: (a) providing two or more nucleic acids encoding different antibodies all having selective binding for said particular ligand, said nucleic acids having at least one set of splicing sites, said set of splicing sites flanking opposite ends of one or more encoded CDRs, wherein the binding activity of at least one encoded antibody is known; (b) mixing said two or more nucleic acids to produce a parent population of mixed nucleic acids encoding said antibodies; (c) randomly incorporating said one or more encoded CDRs between said two or more nucleic acids through said set of splicing sites to produce a different population of nucleic acids than said parent population, wherein at least one antibody encoded by said different population is characterized by having increased binding to the particular ligand as compared to the antibody having the known binding activity; and (d) identifying at least one of the antibodies having increased binding to the particular ligand as compared to the antibody having the known binding activity.
Claim 3
Dependent Claim
20 words
The method of claim 1, wherein step (a) further comprises synthesis of at least one nucleic acid by enzymatic polymerization.
Claim 4
Dependent Claim
14 words
The method of claim 2, wherein said enzymatic polymerization is a polymerase chain reaction.
Claim 5
Dependent Claim
18 words
The method of claim 1, wherein step (a) further comprises chemical synthesis of at least one nucleic acid.
Claim 6
Dependent Claim
83 words
The method of claim 1, wherein step (c) further comprises: (c1) introducing at least one set of cleavage sites within said parent population of mixed nucleic acids at said splicing sites; (c2) cleaving said cleavage sites with a cleaving reagent to produce termini at each splicing site which are non-compatible for ligation and releasing said one or more encoded CDRs from said nucleic acids encoding said antibodies; and (c3) ligating said released one or more encoded CDRs to said nucleic acids encoding antibodies.
Claim 7
Dependent Claim
40 words
The method of claim 2, wherein step (c) further comprises annealing single strand regions of said splicing sites between said two or more nucleic acids, said splicing sites having sufficient nucleotide sequence identity between complementary strands to allow specific hybridization.
Claim 8
Dependent Claim
14 words
The method of claim 6, wherein said two or more nucleic acids are single-stranded.
Claim 9
Dependent Claim
39 words
The method of claim 4, wherein step (c) further comprises annealing single-stranded regions of said splicing sites between said two or more nucleic acids, said splicing sites having sufficient nucleotide sequence identity between complementary strands to allow specific hybridization.
Claim 10
Dependent Claim
14 words
The method of claim 8, wherein said two or more nucleic acids are single-stranded.
Claim 11
Dependent Claim
16 words
The method of claim 5, wherein said set of cleavage sites are restriction enzyme recognition sites.
Claim 12
Dependent Claim
25 words
The method of claim 10, wherein said restriction enzyme recognition sites are selected from the group consisting of Bst XI, Fok I and Dra III.
Claim 13
Dependent Claim
13 words
The method of claim 5, wherein said cleaving reagent is a restriction enzyme.
Claim 14
Dependent Claim
149 words
A population of nucleic acids encoding antibodies, said population of nucleic acids produced by a process, comprising the steps of: (a) providing two or more nucleic acids encoding different antibodies all having selective binding for said particular ligand, said nucleic acids having at least one set of splicing sites, said set of splicing sites flanking opposite ends of one or more encoded CDRs, wherein the binding activity of at least one encoded antibody is known; (b) mixing said two or more nucleic acids to produce a parent population of mixed nucleic acids encoding said antibodies; and (c) randomly incorporating said one or more encoded CDRs between said two or more nucleic acids through said set of splicing sites to produce a different population of nucleic acids than said parent population, wherein at least one antibody is characterized by different binding characteristics than the antibody having the known binding activity.
Claim 15
Dependent Claim
149 words
A population of nucleic acids encoding antibodies, said population of nucleic acids produced by a process, comprising the steps of: (a) providing two or more nucleic acids encoding different antibodies all having selective binding for said particular ligand, said nucleic acids having at least one set of splicing sites, said set of splicing sites flanking opposite ends of one or more encoded CDRs, wherein the binding activity of at least one encoded antibody is known; (b) mixing said two or more nucleic acids to produce a parent population of mixed nucleic acids encoding said antibodies; and (c) randomly incorporating said one or more encoded CDRs between said two or more nucleic acids through said set of splicing sites to produce a different population of nucleic acids than said parent population, wherein at least one antibody is characterized by different binding characteristics than the antibody having the known binding activity.
Claim 16
Dependent Claim
163 words
A population of vectors containing nucleic acids encoding antibodies, said population of vectors produced by a process, comprising the steps of: (a) providing two or more nucleic acids encoding different antibodies all having selective binding for said particular ligand, said nucleic acids having at least one set of splicing sites, said set of splicing sites flanking opposite ends of one or more encoded CDRs, wherein the binding activity of at least one encoded antibody is known; (b) mixing said two or more nucleic acids to produce a parent population of mixed nucleic acids encoding said antibodies; (c) randomly incorporating said one or more encoded CDRs between said two or more nucleic acids through said set of splicing sites to produce a different population of nucleic acids than said parent population, wherein at least one antibody is characterized by different binding characteristics than the antibody having the known binding activity; and (d) inserting said different population of nucleic acids encoding said antibodies into vectors.
Claim 17
Dependent Claim
163 words
A population of vectors containing nucleic acids encoding antibodies, said population of vectors produced by a process, comprising the steps of: (a) providing two or more nucleic acids encoding different antibodies all having selective binding for said particular ligand, said nucleic acids having at least one set of splicing sites, said set of splicing sites flanking opposite ends of one or more encoded CDRs, wherein the binding activity of at least one encoded antibody is known; (b) mixing said two or more nucleic acids to produce a parent population of mixed nucleic acids encoding said antibodies; (c) randomly incorporating said one or more encoded CDRs between said two or more nucleic acids through said set of splicing sites to produce a different population of nucleic acids than said parent population, wherein at least one antibody is characterized by different binding characteristics than the antibody having the known binding activity; and (d) inserting said different population of nucleic acids encoding said antibodies into vectors.
Claim 18
Dependent Claim
14 words
A population of host cells transformed with the population of vectors of claim 14.
Claim 19
Independent Claim
191 words
A method for manipulating nucleic acids so as to decrease the binding of an encoded antibody to a particular ligand, said method comprising the steps of: (a) providing two or more nucleic acids encoding different antibodies all having selective binding for said particular ligand, said nucleic acids having at least one set of splicing sites, said set of splicing sites flanking opposite ends of one or more encoded CDRs, wherein the binding activity of at least one encoded antibody is known; (b) mixing said two or more nucleic acids to produce a parent population of mixed nucleic acids encoding said antibodies; (c) randomly incorporating said one or more encoded CDRs between said two or more nucleic acids through said set of splicing sites to produce a different population of nucleic acids than said parent population, wherein at least one antibody encoded by said different population is characterized by having decreased binding to the particular ligand as compared to the antibody having the known binding activity; and (d) identifying at least one of the antibodies having decreased binding to the particular ligand as compared to the antibody having the known binding activity.
Claim 20
Independent Claim
191 words
A method for manipulating nucleic acids so as to decrease the binding of an encoded antibody to a particular ligand, said method comprising the steps of: (a) providing two or more nucleic acids encoding different antibodies all having selective binding for said particular ligand, said nucleic acids having at least one set of splicing sites, said set of splicing sites flanking opposite ends of one or more encoded CDRs, wherein the binding activity of at least one encoded antibody is known; (b) mixing said two or more nucleic acids to produce a parent population of mixed nucleic acids encoding said antibodies; (c) randomly incorporating said one or more encoded CDRs between said two or more nucleic acids through said set of splicing sites to produce a different population of nucleic acids than said parent population, wherein at least one antibody encoded by said different population is characterized by having decreased binding to the particular ligand as compared to the antibody having the known binding activity; and (d) identifying at least one of the antibodies having decreased binding to the particular ligand as compared to the antibody having the known binding activity.
Claim 21
Dependent Claim
20 words
The method of claim 16, wherein step (a) further comprises synthesis of at least one nucleic acid by enzymatic polymerization.
Claim 22
Dependent Claim
14 words
The method of claim 17, wherein said enzymatic polymerization is a polymerase chain reaction.
Claim 23
Dependent Claim
18 words
The method of claim 16, wherein step (a) further comprises chemical synthesis of at least one nucleic acid.
Claim 24
Dependent Claim
83 words
The method of claim 16, wherein step (c) further comprises: (c1) introducing at least one set of cleavage sites within said parent population of mixed nucleic acids at said splicing sites; (c2) cleaving said cleavage sites with a cleaving reagent to produce termini at each splicing site which are non-compatible for ligation and releasing said one or more encoded CDRs from said nucleic acids encoding said antibodies; and (c3) ligating said released one or more encoded CDRs to said nucleic acids encoding antibodies.
Claim 25
Dependent Claim
40 words
The method of claim 17, wherein step (c) further comprises annealing single strand regions of said splicing sites between said two or more nucleic acids, said splicing sites having sufficient nucleotide sequence identity between complementary strands to allow specific hybridization.
Claim 26
Dependent Claim
14 words
The method of claim 21, wherein said two or more nucleic acids are single-stranded.
Claim 27
Dependent Claim
39 words
The method of claim 19, wherein step (c) further comprises annealing single-stranded regions of said splicing sites between said two or more nucleic acids, said splicing sites having sufficient nucleotide sequence identity between complementary strands to allow specific hybridization.
Claim 28
Dependent Claim
14 words
The method of claim 23, wherein said two or more nucleic acids are single-stranded.
Claim 29
Dependent Claim
16 words
The method of claim 20, wherein said set of cleavage sites are restriction enzyme recognition sites.
Claim 30
Dependent Claim
25 words
The method of claim 25, wherein said restriction enzyme recognition sites are selected from the group consisting of Bst XI, Fok I and Dra III.
Claim 31
Dependent Claim
13 words
The method of claim 20, wherein said cleaving reagent is a restriction enzyme.
Claim 32
Dependent Claim
28 words
The method of claim 1, wherein steps (a) through (d) are repeated one or more times to further increase the binding of said antibody to said particular ligand.
Claim 33
Dependent Claim
29 words
The method of claim 16, wherein the steps (a) through (d) are repeated one or more times to further decrease the binding of said antibody to said particular ligand.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2002-09-20 | 2003-03-22 | 2003-09-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2006-09-20 | 2007-03-22 | 2007-09-21 | $3760.00 | Window Closed / Lapsed |
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