Methods for producing members of specific binding pairs
View Patent ↗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.
1. A method of obtaining a member of a specific binding pair, the method comprising:
providing a library of in vitro mutagenized nucleic acid molecules from an existing antibody encoding nucleic acid molecule,
producing a library of filamentous bacteriophage particles displaying on the surface a population of specific binding pair members which comprise a binding domain of an immunoglobulin, each filamentous bacteriophage particle containing a nucleic acid molecule from the library of in vitro mutagenized nucleic acid molecules from an existing antibody encoding nucleic acid molecule,
contacting the library of filamentous bacteriophage particles with a desired epitope, and
separating filamentous bacteriophage particles displaying on the surface specific binding pair members comprising a binding domain which binds to said epitope, wherein the specific binding pair members are Fabs.
2. A method of producing a specific binding pair member, the method comprising:
(i) obtaining a nucleic acid molecule from a separated filamentous bacteriophage particle obtained by a method according to claim 1 ; and
(ii) producing by expression from the nucleic acid molecule obtained in step (i) the encoded specific binding pair member.
3. A method of producing a nucleic acid molecule encoding a specific binding pair member, the method comprising:
(i) obtaining a nucleic acid molecule from a separated filamentous bacteriophage particle obtained by a method according to claim 1 ; and
(ii) producing from the nucleic acid molecule obtained in step (i) a nucleic acid molecule which encodes a specific binding pair member.
4. A method of producing a nucleic acid molecule encoding a specific binding pair member, the method comprising:
(i) obtaining a nucleic acid molecule from a separated filamentous bacteriophage particle obtained by a method according to claim 1 ; and
(ii) producing from the nucleic acid molecule obtained in step (i) a nucleic acid molecule which encodes a derivative specific binding pair member, wherein said derivative specific binding pair member is produced by addition, deletion, substitution or insertion of one or more amino acids in, or by linkage of another molecule to, a specific binding pair member encoded by the nucleic acid molecule obtained in step (i).
5. A method of producing a specific binding pair member, the method comprising:
producing by expression from the encoding nucleic acid molecule obtained by a method according to claim 4 said derivative specific binding pair member.