| 摘要 |
The present invention discloses novel methods for the generation, expression and screening of diverse collections of binding proteins in vertebrate cells in vitro, for the identification and isolation of ligand- or antigen-specific binding proteins. I particular, the present invention relates to methods for the isolation and identification of at least one nucleotide sequence each encoding a binding protein or functional fragment thereof specific for a desired antigen or ligand. 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 cells in situ. Expression of the at least one binding protein in vertebrate cells is preferably mediated by retroviral transduction. Preferred binding proteins are full-length, fully human antibodies, and preferred cells for the realization of the invention are cells of the lymphocyte lineage. The methods disclosed herein include procedures allowing the isolation of cells displaying desired binding characteristics for a ligand or antigen of interest, and the isolation of genes encoding the at least one desired binding protein of interest. The preferred method of retroviral expression of binding proteins in vertebrate cells disclosed herein allows for stable and optionally clonal expression of binding proteins, which greatly facilitates the amplification, isolation, and cloning of binding protein encoding genes, in comparison to alternative, plasmid-based or non-integrating virus-based vertebrate expression systems known in the art. The disclosed methods allow for efficient generation of diverse collections of binding proteins in vitro by either a.) shuffling of at least one expression construct encoding at least one polypeptide chain of a multimeric binding protein (like e.g. a heavy chain of an antibody), with at least one expression construct encoding at least one matching polypeptide chain (like e.g. a light chain of an antibody) generating a functional multimeric binding protein(like e.g an antibody), or b.) by somatic recombination of V (variable), optionally D (diversity), and J (joining) gene segments encoding variable binding domains of immunoglobulins and immunoglobulin-like molecules contained in at least one expression vector upon transfer into vertebrate cells in situ, by the process known as V(D)J recombination, or c.) by somatic mutation of at least one expression vector encoding at least one binding protein upon transfer into vertebrate cells in situ, or (d) by any combination of procedures a.), b.), or c.) The preferred embodiments include retroviral transduction and expression of at least one binding protein on the surface of precursor lymphocytes. The method is therefore termed " Retro viral precursor lympho cyte display", or, in short, "Retrocyte Display". |
