Polyvalent display libraries
First Claim
1. A method of producing a multivalent polypeptide display library, comprising:
- (a) providing a library of replicable genetic packages, wherein a member comprises a replicable genetic package capable of displaying from its outersurface a fusion protein comprising polypeptide to be screened and a tag, which fusion protein is encoded by a segment of a genome of the package, the polypeptide varying between library members, the number of copies of the fusion protein displayed per library member varying between library members, and the tag being the same in different library members; and
(b) contacting the library with a receptor having a specific affinity for the tag under conditions whereby library members displaying at least two copies of the fusion protein are preferentially bound to immobilized receptor by multivalent bonds between the receptor and the at least two copies of the tag;
(c) separating library members bound to the immobilized receptor from unbound library members to produce a sublibrary enriched relative to the library for members displaying at least two copies of the fusion protein.
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Abstract
The invention is directed to inter alia two related but self-sufficient improvements in conventional display methods. The first improvement provides methods of enriching conventional display libraries for members displaying more than one copy of a polypeptide prior to affinity screening of such libraries with a target of interest. These methods can achieve diverse populations in which the vast majority of members retaining full-length coding sequences encode polypeptides having specific affinity for the target. In a second aspect, the invention provides methods of subcloning nucleic acids encoding displayed polypeptides of enriched libraries from a display vector to an expression vector without the need for clonal isolation of individual members. These methods result in polyclonal libraries of antibodies and other polypeptides for use, e.g., as diagnostic or therapeutic reagents.
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Citations
19 Claims
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1. A method of producing a multivalent polypeptide display library, comprising:
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(a) providing a library of replicable genetic packages, wherein a member comprises a replicable genetic package capable of displaying from its outersurface a fusion protein comprising polypeptide to be screened and a tag, which fusion protein is encoded by a segment of a genome of the package, the polypeptide varying between library members, the number of copies of the fusion protein displayed per library member varying between library members, and the tag being the same in different library members; and (b) contacting the library with a receptor having a specific affinity for the tag under conditions whereby library members displaying at least two copies of the fusion protein are preferentially bound to immobilized receptor by multivalent bonds between the receptor and the at least two copies of the tag; (c) separating library members bound to the immobilized receptor from unbound library members to produce a sublibrary enriched relative to the library for members displaying at least two copies of the fusion protein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 19)
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11. A method of producing a multivalent Fab phage display library, comprising:
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providing a library of phage, wherein a library member comprises a phage capable of displaying from its outersurface a fusion protein comprising a phage coat protein, an antibody light or heavy chain variable domain, and a tag, wherein in at least some members, the antibody heavy or light chain is complexed with a partner antibody heavy or light chain variable domain chain, the complex forming a Fab fragment to be screened, wherein the fusion protein and/or the partner antibody heavy or light chain are encoded by segment(s) of the genome of the phage, and the number of copies of the fusion protein and the partner antibody chain displayed per phage vary between library members; contacting the library or a fraction thereof with a receptor having a specific affinity for the tag under conditions whereby library members displaying at least two copies of the fusion protein are preferentially bound to immobilized receptor by multivalent bonds between the receptor and the at least two copies of the tag; separating library members bound to the receptor from unbound library members to produce an sublibrary enriched relative to the library for members displaying at least two copies of the fusion protein. - View Dependent Claims (13, 14, 15, 16)
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12. A method of producing a multivalent Fab phage display library, comprising:
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providing a library of phage, wherein a library member comprises a phage capable of displaying from its outersurface a fusion protein comprising a phage coat protein, and an antibody light or heavy chain variable domain, wherein in at least some members, the antibody heavy or light chain is complexed with a partner antibody heavy or light chain variable domain chain fused to a tag, the complex forming a Fab fragment to be screened, wherein the fusion protein and/or the partner antibody heavy or light chain fused to the tag are encoded by segment(s) of the genome of the phage, and the number of copies of the fusion protein and the partner antibody chain displayed per phage vary between library members; contacting the library or a fraction thereof with a receptor having a specific affinity for the tag under conditions whereby library members displaying at least two copies of the partner antibody chain are preferentially bound to immobilized receptor by multivalent bonds between the immobilized receptor and the at least two copies of the tag; separating library members bound to the receptor from unbound library members to produce an sublibrary enriched relative to the library for members displaying at least two copies of the partner antibody chain.
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17. A method of producing a multivalent Fab phage display library, comprising:
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providing a library of phage, wherein a library member comprises a phage displaying from its outersurface a fusion protein comprising an antibody light or heavy chain variable domain and a tag, wherein in at least some members, the antibody heavy or light chain is complexed with a second fusion protein comprising a partner antibody heavy or light chain variable domain and a second tag, the complex forming a Fab fragment to be screened, wherein the fusion protein and/or the second fusion protein are encoded by segment(s) of the genome of the phage, the number of copies of the fusion protein displayed per phage varies between library members and the number of copies of the second fusion protein displayed per phage varies between library members; contacting the library or a fraction thereof with a receptor having a specific affinity for the tag under conditions whereby library members displaying at least two copies of the fusion protein are preferentially bound to immobilized receptor by formation of multivalent bonds between the receptor and the at least two copies of the tag; separating library members bound to the receptor from unbound library members to produce an sublibrary enriched relative to the library for members displaying at least two copies of the fusion protein; contacting the library or a second fraction thereof with a second receptor having a specific affinity for the second tag under conditions whereby library members displaying at least two copies of the second fusion protein are preferentially bound to the second receptor by multivalent bonds between the second receptor and the least two copies of the second tag; separating library members bound to the second receptor from unbound library members to produce a second sublibrary enriched relative to the library for members displaying at least two copies of the second fusion protein. - View Dependent Claims (18)
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Specification