Patent ID: 7432063

Claim:
An in vivo method of affinity maturation by auto-inhibited reactivation to obtain a binding molecule that has an enhanced affinity for a target antigen relative to a reference antibody that specifically binds to the target antigen, the method comprising: (a) recombinantly altering a population of host cells by (i) introducing into the host cells a nucleic acid encoding a competitor antibody that can be secreted and that binds to the target antigen with the same specificity as the reference antibody; (ii) introducing into the host cells a nucleic acid encoding a reactivator complex that can be secreted and that comprises a reactivator molecule covalently linked to the target antigen; (iii) introducing into the host cells a library of genes, each of which encodes an auto-inhibited responder complex that can be secreted and that comprises a responder molecule covalently linked to an inhibitor and to a candidate binding molecule that is an antibody, wherein the responder molecule is an enzyme and the inhibitor is an inhibitor of the enzyme; (b) incubating the host cells under conditions in which the competitor antibody, the reactivator complex, and the auto-inhibited responder library are expressed and secreted, where the responder molecule is activated when a candidate binding molecule competes for binding with the competitor antibody and binds to the target antigen; whereupon the reactivator displaces the inhibitor from the responder complex; and (c) detecting a signal from the responder molecule that corresponds to a candidate binding molecule affinity for the target antigen that is greater than that of the reference antibody, thereby identifying a candidate binding molecule with an enhanced affinity for the target antigen.