Methods for producing members of specific binding pairs
DCFirst Claim
1. A method of producing a member of a specific binding pair, which member is a single polypeptide chain comprising a binding domain of an antibody, the binding domain comprising a light chain variable region and a heavy chain variable region, from a nucleic acid library including nucleic acid encoding said specific binding pair member or a genetically diverse population of that type of specific binding pair member, which method comprises:
- expressing in recombinant host cells polypeptides encoded by said nucleic acid library fused to a component of a secreted filamentous phage, so that the phage displays said specific binding pair member in functional form at the surface of the phage, said nucleic acid library being contained within the host cells in a form that is packaged using said phage component, whereby the genetic material of a secreted filamentous phage displaying a specific binding pair member contains nucleic acid encoding said specific binding pair member.
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Abstract
A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.
1003 Citations
59 Claims
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1. A method of producing a member of a specific binding pair, which member is a single polypeptide chain comprising a binding domain of an antibody, the binding domain comprising a light chain variable region and a heavy chain variable region, from a nucleic acid library including nucleic acid encoding said specific binding pair member or a genetically diverse population of that type of specific binding pair member, which method comprises:
expressing in recombinant host cells polypeptides encoded by said nucleic acid library fused to a component of a secreted filamentous phage, so that the phage displays said specific binding pair member in functional form at the surface of the phage, said nucleic acid library being contained within the host cells in a form that is packaged using said phage component, whereby the genetic material of a secreted filamentous phage displaying a specific binding pair member contains nucleic acid encoding said specific binding pair member. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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2. A method of producing a member of a specific binding pair, which member is a single polypeptide chain comprising a binding domain of an antibody, the binding domain comprising a light chain variable region and a heavy chain variable region, which method comprises:
expressing in recombinant host cells nucleic acid encoding said specific binding pair member or a genetically diverse population of that type of specific binding pair member, wherein the or each said specific binding pair member is expressed as a fusion with a component of a secreted filamentous phage which displays said specific binding pair member at the surface of the phage, nucleic acid encoding said specific binding pair member being contained within the host cell in a form that is packaged using said phage component, whereby the genetic material of the phage displaying said specific binding pair member encodes said specific binding pair member, said host cell being a mutator strain which introduces genetic diversity into the specific binding pair member to produce said mixed population.
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3. A method of producing a member of a specific binding pair, which member is a single polypeptide chain comprising a binding domain of an antibody, the binding domain comprising a light chain variable region and a heavy chain variable region, which method comprises:
expressing in recombinant host cells nucleic acid encoding said specific binding pair member or a genetically diverse population of that type of specific binding pair member, wherein the or each specific binding pair member is expressed as a fusion with a component of a secreted filamentous phage which displays said specific binding pair member in functional form at the surface of the phage, nucleic acid encoding said specific binding pair member being contained within the host cell in a form that is packaged using said phage component, whereby the genetic material of the phage displaying a specific binding pair member encodes said specific binding pair member, said fusions being with phage capsid protein and the phage being formed with said fusions in the absence of said capsid protein expressed in wild-type form.
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4. A method of producing a member of a specific binding pair, which member is a single polypeptide chain comprising a binding domain of an antibody, the binding domain comprising a light chain variable region and a heavy chain variable region, which method comprises:
expressing in recombinant host cells nucleic acid encoding said specific binding pair member or a genetically diverse population of that type of specific binding pair member, wherein the or each specific binding pair member is expressed as a fusion with a component of a secreted replicable genetic display package which displays said specific binding pair member at the surface of the package, nucleic acid encoding said specific binding pair member being contained within the host cell in a form that is packaged using said replicable genetic display package component, whereby the genetic material of the replicable genetic display package displaying an specific binding pair member encodes said specific binding pair member, said specific binding pair member being expressed from a phagemid as a capsid fusion, and a helper phage, or a plasmid expressing complementing phage genes, it used along with said capsid fusions to package the phagemid nucleic acid. - View Dependent Claims (5)
- 21. Recombinant host cells harboring a library of nucleic acid fragments comprising fragments encoding a genetically diverse population of a type of member of a specific binding pair which member is a single polypeptide chain comprising a binding domain of an antibody, the binding domain comprising a light chain variable region and a heavy chain variable region, each specific binding pair member being expressed as a fusion with a surface component of a secretable filamentous phage, so that said specific binding pair members are displayed on the surface of the phage in functional form and the genetic material of the phage encode the associated displayed specific binding pair member.
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22. A phage having the form of a secreted replicable genetic display package displaying on its surface in functional form a member of a specific binding pair, which member is a single polypeptide chain comprising a binding domain of an antibody, the binding domain comprising a light chain variable region and a heavy chain variable region or a binding domain thereof.
Specification