Methods for producing members of specific binding pairs
First Claim
1. A specific binding pair member which is a single chain specific binding pair member comprising a first polypeptide chain component and a second polypeptide chain component and specific for a complementary specific binding pair member of interest, produced by a method which comprises:
- (I) introducing into host cells;
(i) first vectors comprising nucleic acid encoding a genetically diverse population of a first polypeptide chain component fused to a component of a secreted replicable genetic display package for display of said polypeptide chain component at the surface of replicable genetic display packages; and
(ii) second vectors comprising nucleic acid encoding a genetically diverse population of said second polypeptide chain component;
said first vectors being packaged in infectious replicable genetic display packages and their introduction into host cells being by infection into host cells harboring said second vectors;
or said second vectors being packaged in infectious replicable genetic display packages and their introducing into host cells being by infection into host cells harboring said first vectors;
(II) causing or allowing recombination between said first and second vectors within said host cells, the recombination being promoted by inclusion in said first and second vectors of sequences at which site-specific recombination occurs resulting in recombinant vectors each of which comprises nucleic acid encoding a said single chain specific binding pair member comprising a said first polypeptide chain component and a said second polypeptide chain component and an amino acid sequence encoded by a sequence provided by recombination between said sequences at which site-specific recombination occurs, and capable of being packaged into a replicable genetic display packages using said replicable genetic display package component;
(III) expressing said single chain specific binding pair members within the host cells to form a library of said single chain specific binding pair members displayed by replicable genetic display packages, whereby the genetic materials of each said replicable genetic display package encodes a single chain specific binding pair member displayed at its surface, (IV) selecting by binding with said complementary specific binding pair member of interest one or more single chain specific binding pair members specific for said complementary specific binding pair member of interest, each single chain specific binding pair member thus selected being associated in its respective replicable genetic display package with nucleic acid encoding that single chain specific binding pair member, (V) obtaining nucleic acid encoding a said single chain specific binding pair member from its replicable genetic display package displaying a single chain specific binding pair member selected in step (IV);
(V) producing, by expression of encoding nucleic acid in a recombinant host organism, a single chain specific binding pair member comprising a first polypeptide chain component and a second polypeptide chain component and an amino acid sequence encoded by a sequence provided by recombination between said sequences at which site-specific recombination occurs and specific for said complementary specific binding pair member of interest, which single chain specific binding pair comprises a polypeptide chain component which is as encoded by nucleic acid encoding a said polypeptide chain component of a specific binding pair member selected in step (IV) or is a derivative thereof by way of addition, deletion, substitution or insertion of one or more amino acids or by linkage of another molecule.
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Accused Products
Abstract
Methods, recombinant host cells and kits are disclosed for the production of members of specific binding pairs (sbp), e.g. antibodies, using display on the surface of secreted replicable genetic display packages (rgdps), e.g. filamentous phage. To produce a library of great diversity, recombination occurs between first and second vectors comprising nucleic acid encoding first and second polypeptide chains of sbp members respectively, thereby producing recombinant vectors each encoding both a fist and a second polypeptide chain component of an sbp member. The recombination may take place in vitro or intracellularly and may be site-specific, e.g. involving use of the loxP sequence and mutants thereof. Recombination may take place after prior screening or selecting for rgdps displaying sbp members which bind complementary sbp member of interest.
91 Citations
19 Claims
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1. A specific binding pair member which is a single chain specific binding pair member comprising a first polypeptide chain component and a second polypeptide chain component and specific for a complementary specific binding pair member of interest, produced by a method which comprises:
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(I) introducing into host cells;
(i) first vectors comprising nucleic acid encoding a genetically diverse population of a first polypeptide chain component fused to a component of a secreted replicable genetic display package for display of said polypeptide chain component at the surface of replicable genetic display packages; and
(ii) second vectors comprising nucleic acid encoding a genetically diverse population of said second polypeptide chain component;
said first vectors being packaged in infectious replicable genetic display packages and their introduction into host cells being by infection into host cells harboring said second vectors;
orsaid second vectors being packaged in infectious replicable genetic display packages and their introducing into host cells being by infection into host cells harboring said first vectors;
(II) causing or allowing recombination between said first and second vectors within said host cells, the recombination being promoted by inclusion in said first and second vectors of sequences at which site-specific recombination occurs resulting in recombinant vectors each of which comprises nucleic acid encoding a said single chain specific binding pair member comprising a said first polypeptide chain component and a said second polypeptide chain component and an amino acid sequence encoded by a sequence provided by recombination between said sequences at which site-specific recombination occurs, and capable of being packaged into a replicable genetic display packages using said replicable genetic display package component;
(III) expressing said single chain specific binding pair members within the host cells to form a library of said single chain specific binding pair members displayed by replicable genetic display packages, whereby the genetic materials of each said replicable genetic display package encodes a single chain specific binding pair member displayed at its surface, (IV) selecting by binding with said complementary specific binding pair member of interest one or more single chain specific binding pair members specific for said complementary specific binding pair member of interest, each single chain specific binding pair member thus selected being associated in its respective replicable genetic display package with nucleic acid encoding that single chain specific binding pair member, (V) obtaining nucleic acid encoding a said single chain specific binding pair member from its replicable genetic display package displaying a single chain specific binding pair member selected in step (IV);
(V) producing, by expression of encoding nucleic acid in a recombinant host organism, a single chain specific binding pair member comprising a first polypeptide chain component and a second polypeptide chain component and an amino acid sequence encoded by a sequence provided by recombination between said sequences at which site-specific recombination occurs and specific for said complementary specific binding pair member of interest, which single chain specific binding pair comprises a polypeptide chain component which is as encoded by nucleic acid encoding a said polypeptide chain component of a specific binding pair member selected in step (IV) or is a derivative thereof by way of addition, deletion, substitution or insertion of one or more amino acids or by linkage of another molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
(i) the repertoire of rearranged immunoglobulin genes of an animal immunized with a complementary sbp member;
(ii) the repertoire of rearranged immunoglobulin genes of an animal not immunized with a complementary sbp member;
(iii) a repertoire of an artificially rearranged immunoglobulin gene or genes;
(iv) a repertoire of an immunoglobulin homolog gene or genes;
(v) a repertoire of sequences derived from a germ-line immunoglobulin gene or genes;
(vi) a repertoire of an immunoglobulin gene or genes artificially mutated by the introduction of one or more point mutations; and
(vii) a mixture of any of (i), (ii), (iii), (iv), (v) and (vi).
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5. A specific binding pair member according to claim 1 wherein the replicable genetic display package is a bacteriophage, the host is a bacterium, and said component of the replicable genetic display package is a capsid protein for the bacteriophage.
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6. A specific binding pair member according to claim 5 wherein the phage is a filamentous phage.
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7. A specific binding pair member according to claim 6 wherein the phage is selected from the class I phages fd, M13, Ifl, Ike, ZJ/Z, Ff and the class II phages Xf, Pf1 and Pf3.
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8. A specific binding pair member according to claim 6 wherein the first polypeptide chain components are expressed as fusions with the gene III capsid protein of phage fd or its counterpart in another filamentous phage.
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9. A specific binding pair member according to claim 8 wherein the first polypeptide chain components are each inserted in the N-terminal region of the mature capsid protein downstream of a secretory leader peptide.
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10. A specific binding pair member according to claim 5 wherein the first polypeptide chain components are expressed as fusions with the gene III capsid protein of phage fd or its counterpart in another filamentous phage.
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11. A specific binding pair member according to claim 10 wherein the first polypeptide chain components are each inserted in the N-terminal region of the mature capsid protein downstream of a secretory leader peptide.
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12. A specific binding pair member according to claim 5 wherein the host is E. coli.
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13. A specific binding pair member according to claim 1, wherein said sequences at which site-specific recombination occurs are loxP sequences.
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14. A specific binding pair (sbp) member which is a single chain specific binding pair member specific for a counterpart specific binding pair member of interest, produced by a method which comprises:
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(i) causing or allowing intracellular recombination between (a) first vectors comprising nucleic acid encoding a population of a fusion of a first polypeptide chain component of a specific binding pair member and a component of a secreted replicable genetic display package and (b) second vectors comprising nucleic acid encoding a population of a second polypeptide chain component of a specific binding pair member, at least one of said populations being genetically diverse, the recombination between the vectors being at sequences at which site-specific recombination occurs and resulting in recombinant vectors each of which comprises nucleic acid encoding a single chain specific binding pair member comprising a said first polypeptide chain component, a said second polypeptide chain component, and an amino acid sequence encoded by a sequence provided by recombination between said sequences at which site-specific recombination occurs, which nucleic acid is capable of being packaged using said replicable genetic display package component; and
(ii) expressing said single chain specific binding pair members producing replicable genetic display packages which display at their surface said single chain specific binding pair members and which each comprise nucleic acid encoding a said single chain specific binding pair member (iii) selecting by binding with said counterpart specific binding pair member of interest one or more single chain specific binding pair members specific for said counterpart specific binding pair member of interest, each single chain specific binding pair member thus selected being associated in its respective replicable genetic display package with nucleic acid encoding that single chain specific binding pair member;
(iv) obtaining nucleic acid encoding a said single chain specific binding pair member from its replicable genetic display package displaying a specific binding pair member selected in step (v);
(v) producing, by expression of encoding nucleic acid in a recombinant host organism, a said single chain specific binding pair member comprising a first polypeptide chain component and a second polypeptide chain component and an amino acid sequence encoded by a sequence provided by recombination between said sequences at which site specific recombination occurs and specific for said complementary specific binding pair member of interest, which single chain specific binding pair comprises a polypeptide chain component which is as encoded by nucleic acid encoding a said polypeptide chain component of a specific binding pair member selected in step (v) or is a derivative thereof by way of addition, deletion, substitution or insertion of one or more amino acids or by linkage of another molecule. - View Dependent Claims (15, 16, 17, 18, 19)
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Specification