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Last Updated: October 21, 2019

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Claims for Patent: 8,771,960

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Summary for Patent: 8,771,960
Title:Method for producing protein libraries and for selecting proteins from said libraries
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.
Inventor(s): Breitling; Frank (Heidelberg, DE), Moldenhauer; Gerhard (Heidelberg, DE), Poustka; Annemarie (Heidelberg, DE), Kuhlwein; Thorsten (Viernheim, DE), Luttgau; Sandra (Dossenheim, DE)
Assignee: Deutsches Krebsforschungszentrum (Heidelburg, DE)
Application Number:10/491,653
Patent Claims: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 10.sup.2 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.

2. 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.

3. The method according to claim 1, wherein the plurality of nucleic acids comprises vH genes, vlambda genes or vkappa genes.

4. The method according to claim 1, wherein the mammalian cells are neoplastic lymphocytes or precursors thereof, leukemia cells or malignant lymphoma cells.

5. The method according to claim 3, wherein the vH genes, vlambda genes or vkappa genes are human genes.

6. 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.

7. The method according to claim 6, wherein the antibody is a monoclonal human antibody.

8. 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.

9. The method according to claim 1, wherein more than 10.sup.3 different eukaryotic cells are obtained, each expressing a different protein.

10. 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.

11. 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.

12. The method according to claim 1, wherein the library of antibody-producing eukaryotic cells comprises more than 10.sup.6 cells.

13. 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.

14. The method according to claim 8, wherein the monoclonal human antibody is expressed by differentially splicing the constant domains of IgG.

15. The method according to claim 1, wherein the plurality of nucleic acids is obtained by error-prone PCR.

16. The method according to claim 3, wherein the plurality of nucleic acids is obtained by error-prone PCR.

17. The method according to claim 2, wherein the transfected DNA comprises a selectable marker.

18. The method according to claim 1, further comprising selecting the expanded eukaryotic cells by FACS.

19. 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.

20. The method according to claim 1, wherein the members of the library of antibody-producing eukaryotic cells do not express a resistance marker.

Summary for Patent:   Start Trial

Foriegn Application Priority Data
Foreign Country Foreign Patent Number Foreign Patent Date
01123596Oct 1, 2001
PCT Information
PCT FiledSeptember 27, 2002PCT Application Number:PCT/EP02/10852
PCT Publication Date:April 10, 2003PCT Publication Number:WO03/029458

Details for Patent 8,771,960

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04   Start Trial Deutsches Krebsforschungszentrum (Heidelburg, DE) 2021-10-01 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04   Start Trial Deutsches Krebsforschungszentrum (Heidelburg, DE) 2021-10-01 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04   Start Trial Deutsches Krebsforschungszentrum (Heidelburg, DE) 2021-10-01 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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