# US 8,771,960 B2 · Method for producing protein libraries and for selecting proteins from said libraries > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,960 B2 - Document ID: US8771960B2 - Status: Granted - Kind: B2 - Application number: 10/491,653 - Filed: 2002-09-27 - Published: 2005-03-17 - Granted: 2014-07-08 - Earliest claimed: 2001-10-01 - Assignees: Deutsches Krebsforschungszentrum - Applicants: Frank Breitling; Gerhard Moldenhauer; Annemarie Poustka; Thorsten Kühlwein; Sandra Lüttgau - Inventors: Frank Breitling; Gerhard Moldenhauer; Annemarie Poustka; Thorsten Kühlwein; Sandra Lüttgau - Web page: https://patentgrep.com/patent/US8771960B2 ## CPC - C40B 40/02: Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors - C07K 16/30: from tumour cells - C07K 16/40: against enzymes - C07K 16/461: {Igs containing Ig-regions, -domains or -residues form different species} - C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display} - C07K 2317/31: multispecific - C07K 2317/55: Fab or Fab' - C07K 2317/622: Single chain antibody (scFv) - C07K 2319/035: containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The invention relates to a method for the production of a protein library, in particular an antibody library, which is highly diverse and for the selection of proteins, in particular antibodies, therefrom. ## Claims ### Claim 1 A method of producing a library of antibody-producing eukaryotic cells, the method comprising the steps of: (a) introducing specific FRT recombination signals into at least one chromosomal gene locus of a eukaryotic cell; (b) expanding the eukaryotic cell comprising the specific FRT recombination signals in the at least one gene locus; (c) introducing into the expanded eukaryotic cells of step (b) a plurality of nucleic acids comprising homologous sequences and encoding a plurality of antibodies; (d) introducing into the expanded eukaryotic cells of step (b) a nucleic acid encoding a Flp recombinase recognizing the specific FRT recombination signals; and (e) selecting eukaryotic cells expressing said plurality of antibodies, wherein the plurality of nucleic acids replace the at least one gene locus of the expanded eukaryotic cells, wherein a library of more than 102 different antibody-producing eukaryotic cells is produced, and wherein members of the library of antibody-producing eukaryotic cells express an antibody encoded by the plurality of nucleic acids and bound to the surface of the members of the library of antibody-producing eukaryotic cells, wherein said antibody-producing eukaryotic cells are mammalian cells or hybridoma cells. ### Claim 2 (depends on 1) The method according to claim 1, wherein step (a) comprises homologous recombination of transfected DNA with the at least one gene locus, and wherein the transfected DNA comprises the specific recombination signals and a region homologous to DNA sequences flanking at least one gene locus of the eukaryotic cell. ### Claim 3 (depends on 1) The method according to claim 1, wherein the plurality of nucleic acids comprises vH genes, vlambda genes or vkappa genes. ### Claim 4 (depends on 1) The method according to claim 1, wherein the mammalian cells are neoplastic lymphocytes or precursors thereof, leukemia cells or malignant lymphoma cells. ### Claim 5 (depends on 3) The method according to claim 3, wherein the vH genes, vlambda genes or vkappa genes are human genes. ### Claim 6 (depends on 3) The method according to claim 3, wherein the at least one gene locus is an antibody locus selected from the group consisting of a vH gene, a vlambda gene and a vkappa gene. ### Claim 7 (depends on 6) The method according to claim 6, wherein the antibody is a monoclonal human antibody. ### Claim 8 (depends on 7) The method according to claim 7, wherein the monoclonal human antibody is bound to the surface of the member of the protein-producing eukaryotic cells by differentially splicing the constant domains of IgG, IgM, IgA, IgD or IgE. ### Claim 9 (depends on 1) The method according to claim 1, wherein more than 103 different eukaryotic cells are obtained, each expressing a different protein. ### Claim 10 (depends on 2) The method according to claim 2, wherein the region homologous to DNA sequences flanking the at least one gene locus comprises at least 400 base pairs. ### Claim 11 (depends on 2) The method according to claim 2, wherein the region homologous to DNA sequences flanking the at least one gene locus comprises an IgG, IgM, IgA, IgD or IgE gene and an intron between the IgG, IgM, IgA, IgD or IgE gene and an M1 exon, and wherein the intron is shortened by more than 50 base pairs in the 5′ end direction before the M1-exon of an IgG, IgM, IgA, IgD or IgE gene when compared to a naturally occurring intron between an IgG, IgM, IgA, IgD or IgE gene and the M1 exon. ### Claim 12 (depends on 1) The method according to claim 1, wherein the library of antibody-producing eukaryotic cells comprises more than 106 cells. ### Claim 13 (depends on 2) The method according to claim 2, wherein the at least one gene locus is an antibody locus comprising an active vH gene, an active vlambda gene or an active vkappa gene. ### Claim 14 (depends on 8) The method according to claim 8, wherein the monoclonal human antibody is expressed by differentially splicing the constant domains of IgG. ### Claim 15 (depends on 1) The method according to claim 1, wherein the plurality of nucleic acids is obtained by error-prone PCR. ### Claim 16 (depends on 3) The method according to claim 3, wherein the plurality of nucleic acids is obtained by error-prone PCR. ### Claim 17 (depends on 2) The method according to claim 2, wherein the transfected DNA comprises a selectable marker. ### Claim 18 (depends on 1) The method according to claim 1, further comprising selecting the expanded eukaryotic cells by FACS. ### Claim 19 (depends on 1) The method according to claim 1, wherein the antibody is bound to the surface of the member of the library of antibody-producing eukaryotic cells by the immunoglobulin M1 domain. ### Claim 20 (depends on 1) The method according to claim 1, wherein the members of the library of antibody-producing eukaryotic cells do not express a resistance marker.