Patent ID: 8748353
Filing Date: 2014-06-10
Classification: C07K,C12N,C12Q,C40B,G01N

Abstract:
1. A method for the isolation and identification of at least one stably transduced vertebrate host cell clone that (i) comprises a stably integrated retroviral expression vector encoding a full-length antibody heavy chain polypeptide specific for a desired antigen, a stably integrated retroviral expression vector encoding a full-length antibody light chain polypeptide specific for the desired antigen, and a screening marker, and (ii) expresses on the cell surface a full-length antibody specific for the desired antigen that is encoded by the stably integrated retroviral expression vectors, 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 the retroviral transduction is performed at a multiplicity of infection of equal to or less than 0.1, wherein each member of the first library of stably transduced 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 Abelson-murine leukemia virus transformed precursor B lymphocytes that do not express lamda-5, VpreB1, and VpreB2 surrogate light chain genes; (b) transducing the first library of stably transduced 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 the first library of retroviral expression constructs encodes a plurality of full-length antibody heavy chain polypeptides and the second library of retroviral expression constructs encodes a plurality of full-length antibody light chain polypeptides, or the first library of retroviral expression constructs encodes a plurality of full-length antibody light chain polypeptides and the second library of retroviral expression constructs encodes a plurality of full-length antibody heavy chain polypeptides such that the first and the second library of retroviral expression constructs encode antibody heavy and light chain combinations, wherein the first or the second library of retroviral expression constructs further comprise at least one screening marker which is a fluorescent protein or cell surface marker, wherein the retroviral transduction is performed at a multiplicity of infection of equal to or less than 0.1, and wherein each member of the second library of stably transduced cells comprises at least one retroviral expression construct from the first library of retroviral expression constructs stably integrated into the host cell genome and at least one retroviral expression construct from the second library of retroviral expression constructs stably integrated into the host cell genome; (c) allowing expression of the first and second stably integrated retroviral expression constructs in the second library of stably transduced vertebrate host cells such that full-length antibodies are expressed on the 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 clone expressing an antibody specific for the desired antigen.