Identification of antigen- or ligand-specific binding proteins
AssigneeAgenus Inc.
InventorsUlf Grawunder, Jorn Stitz
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Abstract
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.
Claims18
A method of producing at least one stably transduced vertebrate host cell that recombinantly expresses on its cell surface a dimeric T cell receptor (TCR) 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 first TCR chain polypeptides or second TCR chain polypeptides into a population of vertebrate host cells that express low or substantially no endogenous TCR 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;
- (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 first TCR chain polypeptides or second TCR 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 first TCR chain polypeptides and the second library of replication incompetent retroviral expression constructs encodes a plurality of second TCR chain polypeptides, or (ii) the first library of replication incompetent retroviral expression constructs encodes a plurality of second TCR chain polypeptides and the second library of replication incompetent retroviral expression constructs encodes a plurality of first TCR 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 dimeric TCRs are expressed on the cell surface of the second library of stably transduced vertebrate host cells, wherein each expressed dimeric TCR comprises a first TCR chain polypeptide and a second TCR chain polypeptide;
- and
- (d) selecting from the second library of stably transduced vertebrate host cells at least one stably transduced vertebrate host cell expressing a dimeric TCR specific for the desired antigen.
Dependencies are inferred from the claim text.