INDUCIBLE MUTAGENESIS OF TARGET GENES

摘要

The present invention relates generally to mutagenesis of target genes that takes advantage of the natural mutagenic capabilities of B cells, and enhances those capabilities by bringing the process of diversification under control. The invention provides a method for rapidly and inducibly generating point mutations and other types of diversification in expressed genes, such as antibody genes. This method can be coupled with selection to identify B cell clones that produce, for example, antibodies of high affinity or specificity. The diversification process can be modulated, accelerated, halted, switched between methods of mutagenesis and the like. The modulation of diversification in accordance with the invention is both inducible and reversible. The invention provides a means of rapid and feasible development of a repertoire of variant immunoglobulins and other polypeptides.

主权项

1. A method of producing a repertoire of polypeptides having variant sequences of a polypeptide of interest, the method comprising:
(i) culturing a B cell modified to reversibly induce accelerated diversification of a target gene containing a coding region of the polypeptide of interest, in conditions that allow expression of a diversification factor, wherein said B cell comprises:
(a) a cis-regulatory element operably linked to the target gene, wherein the target gene comprises a promoter and a coding region; and(b) a fusion construct that encodes a tethering factor fused to a diversification factor, wherein the tethering factor specifically binds to the cis-regulatory element of (a) and the diversification factor reversibly induces accelerated diversification of the coding region in the target gene; (ii) maintaining the culture under conditions that permit proliferation of the B cell until a plurality of B cells comprising the desired repertoire of polypeptides having variant sequences of the polypeptide of interest is obtained.