Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions

摘要

A method of engineering immunoglobulins which each have one or more amino acid modifications in at least one structural loop region of such immunoglobulins, where the modified loop region specifically binds to an epitope of an antigen to which an unmodified immunoglobulin does not significantly bind.

主权项

1. A method for engineering an immunoglobulin comprising at least one altered structural loop region of a CH3 constant domain, said altered structural loop region comprising at least three amino acid modifications with respect to a wild type structural loop region of a CH3 constant domain of an immunoglobulin, comprising the steps of:
(i) providing a nucleic acid molecule encoding a CH3 domain, wherein the immunoglobulin comprises a CH3 constant domain comprising one or more structural loops regions; (ii) modifying the nucleic acid sequence encoding at least one of the structural loop regions of said CH3 domain; (iii) transferring the modified nucleic acid molecule into an expression system; (iv) expressing the modified immunoglobulin encoded by the nucleic acid sequence modified according to step (ii); wherein the modified structural loop region of said modified immunoglobulin comprises at least four amino acid modifications in a single non-CDR structural loop or at least three amino acid modifications in two or more non-CDR structural loops; (v) contacting the expressed modified immunoglobulin with an epitope; and (vi) determining whether the modified immunoglobulin specifically binds to said epitope, wherein said epitope is not specifically bound by a wild type structural loop region of a CH3 constant domain of an immunoglobulin, and wherein said altered structural loop region does not encompass an inserted peptide of predetermined sequence that specifically binds said epitope independently of its insertion into said structural loop region.