Identification of antigen or ligand-specific binding proteins

摘要

The present invention discloses novel methods for the generation, expression and screening of diverse collections of binding proteins such as antibodies or fragments thereof in vertebrate host cells in vitro, for the identification and isolation of ligand- or antigen-specific binding proteins. The methods disclosed herein allow the expression of diverse collections of binding proteins from at least one vector construct, which optionally can give rise to collections of diverse binding proteins upon transfer and expression into vertebrate host cells in situ.

主权项

1. A method for the isolation and identification of at least one stably transduced vertebrate host cell that recombinantly expresses on its cell surface a full-length antibody specific for a desired antigen, the method comprising the steps of:
(a) transducing a first library of replication incompetent retroviral expression constructs encoding a plurality of full-length antibody heavy chain or light chain polypeptides into a population of vertebrate host cells such that a first library of stably transduced vertebrate host cells is generated, wherein each member of the first library of stably transduced vertebrate host cells comprises at least one retroviral expression construct from the first library of retroviral expression constructs stably integrated into the host cell genome, and wherein the vertebrate host cells are precursor B lymphocytes that express Igα and Igβ molecules and are deficient in at least one surrogate light chain component; (b) transducing the first library of stably transduced vertebrate host cells of step (a) with a second library of replication incompetent retroviral expression constructs encoding a plurality of full-length antibody heavy chain or light chain polypeptides such that a second library of stably transduced vertebrate host cells is generated, wherein (i) the first library of replication incompetent retroviral expression constructs encodes a plurality of full-length antibody heavy chain polypeptides and the second library of replication incompetent retroviral expression constructs encodes a plurality of full-length antibody light chain polypeptides, or (ii) the first library of replication incompetent retroviral expression constructs encodes a plurality of full-length antibody light chain polypeptides and the second library of replication incompetent retroviral expression constructs encodes a plurality of full-length antibody heavy chain polypeptides; and wherein each member of the second library of stably transduced vertebrate host cells comprises at least one replication incompetent retroviral expression construct from the first library of retroviral expression constructs stably integrated into the host cell genome and at least one replication incompetent retroviral expression construct from the second library of retroviral expression constructs stably integrated into the host cell genome; (c) allowing expression of the integrated retroviral expression constructs in the second library of stably transduced vertebrate host cells such that full-length antibodies are expressed on the cell surface of the second library of stably transduced vertebrate host cells; and (d) selecting from the second library of stably transduced vertebrate host cells at least one stably transduced vertebrate host cell expressing a full-length antibody specific for the desired antigen.