Production of antibodies using Cre-mediated site-specific recombination

• US 20030013157A1
• Filed: 07/31/2002
• Published: 01/16/2003
• Est. Priority Date: 03/29/1995
• Status: Active Grant

• Current Assignee: Amgen Fremont Inc. (Amgen Inc.), Japan Tobacco Inc.
• Original Assignee: Amgen Fremont Inc. (Amgen Inc.)
• Inventors: Jakobovits, Aya, Zsebo, Krisztina M.
• Granted Patent: US 7,145,056 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

A method to produce a cell expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre-mediated site-specific recombination is disclosed. The method involves first transfecting an antibody-producing cell with a homology-targeting vector comprising a lox site and a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin loci of the genomic sequence which is to be converted to a modified region, so the first lox site is inserted into the genomic sequence via site-specific homologous recombination. Then the cell is transfected with a lox-targeting vector comprising a second lox site suitable for Cre-mediated recombination with the integrated lox site and a modifying sequence to convert the region of the immunoglobulin loci to the modified region. This conversion is performed by interacting the lox sites with Cre in vivo, so that the modifying sequence inserts into the genomic sequence via Cre-mediated site-specific recombination of the lox sites. Also disclosed are a form of the method used to produce a cell expressing a modified antibody molecule using Cre-mediated site-specific recombination, and antibody-producing cells obtainable by the disclosed methods. Class-switching modifications of human antibodies produced in murine hybridoma cells are exemplified.

Claims

1. A method to produce a cell expressing an antibody from a genomic sequence of said cell comprising a modified immunoglobulin locus, which method uses Cre-mediated site-specific recombination to modify said locus and comprises:

(a) transfecting an antibody-producing cell with a first homology-targeting vector comprising;

(i) a first lox site, and

(ii) a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin locus of said genomic sequence which is to be converted to a modified region, so that said first lox site is inserted into said genomic sequence via site-specific homologous recombination with genomic DNA in vivo;

(b) transfecting said cell with a lox-targeting vector comprising;

(i) a second lox site suitable for Cre-mediated recombination with said first lox site, and

(ii) a modifying sequence to convert said region of the immunoglobulin locus to said modified region;

(c) interacting the lox sites with Cre, so that said modifying sequence inserts into said genomic sequence via Cre-mediated site-specific recombination of said lox sites, thereby converting said region of the immunoglobulin locus to said modified region; and

(d) selecting a transfectant in which said region of the immunoglobulin locus is converted to said modified region and which transfectant produces said antibody molecule.

2. The method of claim 1 wherein said lox-targeting vector further comprises a selectable marker gene operably linked to control regions such that said marker gene is expressed in said cell,

so that in step (c), on interacting the lox sites with Cre, said marker gene inserts into said genomic sequence with said modifying sequence via Cre-mediated site-specific recombination of said lox sites, and

in step (d), said selecting for a transfectant comprises selecting for a transfectant expressing said marker gene.

3. The method of claim 1 further comprising

an additional step before step (b) which additional step comprises transfecting said cell with a second homology-targeting vector comprising:

(i) a third lox site suitable for Cre-mediated recombination with said first and second lox sites,

(ii) a first selectable marker gene operably linked to control regions such that said first marker gene is expressed in said cell, and

(iii) a targeting sequence homologous to a second DNA sequence adjacent to the region of the immunoglobulin locus of said genomic sequence which is to be converted, said region which is to be converted being flanked by said first and second DNA sequences, so that said third lox site and said first marker gene are inserted into said genomic sequence via site-specific homologous recombination with genomic DNA in vivo; and

wherein in step (b), said lox-targeting vector further comprises a second selectable marker gene operably linked to control regions such that said second marker gene is expressed in said cell,

so that in step (c), on interacting the lox sites with Cre, said second marker gene inserts into said genomic sequence with said modifying sequence via Cre-mediated site-specific recombination of said lox sites; and

in step (d), said selecting for a transfectant comprises selecting for a transfectant expressing said second marker gene.

4. The method of claim 3 wherein

in step (c), on interacting the lox sites with Cre, after said modifying sequence and second marker gene insert into said genomic sequence, said first marker gene and said region to be converted are deleted by Cre-mediated site-specific recombination;

and

in step (d) said selecting for a transfectant further comprises selecting for a transfectant not expressing said first marker gene.

5. The method of claim 1 wherein said modifying sequence comprises a regulatory nucleotide sequence which replaces all or a portion of the regulatory sequences of the immunoglobulin locus of said genomic sequence to provide modified expression of said immunoglobulin locus.

6. The method of claim 1 wherein said modifying sequence comprises a nucleotide sequence that encodes a translation product to replace all or a portion of either the constant region or the variable region of an antibody molecule to form a modified antibody molecule.

7. The method of claim 1 wherein said region of the immunoglobulin locus to be converted to a modified region comprises a constant region gene and said modifying sequence comprises a nucleotide sequence that encodes a translation product to replace all or a portion of the constant region of the antibody produced by said constant region gene with a modified constant region.

8. The method of claim 7 in which said constant region gene of said genomic sequence comprises a heavy chain gene.

9. The method of claim 7 wherein said constant region gene of said genomic sequence is a murine constant region gene and said modifying sequence comprises a human constant region gene or portion thereof.

10. The method of claim 7 wherein said constant region gene of said genomic sequence is a murine constant region gene and said modifying sequence comprises a human constant region gene or portion thereof.

11. The method of claim 7 wherein said constant region gene of said genomic sequence is a human constant region gene and said modifying sequence comprises a different human constant region gene or portion thereof.

12. The method of claim 11 wherein said constant region gene of said genomic sequence is a human C_μ

gene and said modifying sequence comprises a human C_γ

constant region gene.

13. The method of claim 7 wherein said replacement vector comprises a sequence encoding an enzyme, toxin, hormone, growth factor or linker.

14. The method of claim 1 wherein said cell expressing an antibody molecule is a cell of a lymphoid cell line.

15. The method of claim 14 wherein said lymphoid cell line is a murine hybridoma cell line.

16. The method of claim 15 wherein said murine hybridoma cell line is producing a human antibody by expression of a human immunoglobulin locus.

17. A cell expressing an antibody from a genomic sequence of said cell comprising a modified immunoglobulin locus wherein said cell is obtainable by the method of claim 1.

18. A method to produce an antibody from a cell expressing said antibody from a genomic sequence comprising a modified immunoglobulin locus wherein said cell is obtainable by the method of claim 1, which method comprises:

(a) incubating said cell under conditions such that said cell expresses said antibody, and

(b) recovering said antibody expressed by said cell.

20. A cell expressing an antibody molecule from a genomic sequence of said cell with a lox site adjacent to or integrated within a region of the immunoglobulin loci encoding said antibody molecule wherein said cell is obtainable by the method of claim 19.

19. A method to produce a cell expressing an antibody molecule from a genomic sequence of said cell with a lox site adjacent to or integrated within a region of an immunoglobulin locus encoding said antibody molecule, for use in modifying said region using Cre-mediated site-specific recombination, which method comprises:

(a) transfecting an antibody-producing cell with a first homology-targeting vector comprising;

(i) a first lox site, and

(ii) a targeting sequence homologous to a first DNA sequence adjacent to region of the immunoglobulin locus of said genomic sequence which is to be converted to a modified region, so that said first lox site is inserted into said genomic sequence via site-specific homologous recombination with genomic DNA in vivo; and

(b) selecting a transfectant with said lox site inserted into said genomic DNA adjacent to said region which is to be converted.

21. A method to modify a region of an immunoglobulin locus in a genomic sequence of a cell expressing an antibody molecule from said immunoglobulin locus wherein said genomic sequence comprises a first lox site adjacent to or integrated within a region of said immunoglobulin locus which is to be modified using Cre-mediated site-specific recombination, which method comprises:

(a) transfecting said cell with a lox-targeting vector comprising;

(i) a second lox site suitable for Cre-mediated recombination with said first lox site, and

(ii) a modifying sequence to convert said region of the immunoglobulin locus to said modified region;

(b) interacting the lox sites with Cre, so that said modifying sequence inserts into said genomic sequence via Cre-mediated site-specific recombination of said lox sites, thereby converting said region of the immunoglobulin locus to said modified region; and

(c) selecting a transfectant in which said region of the immunoglobulin locus is converted to said modified region and which produces said antibody molecule.

22. A cell expressing an antibody from a genomic sequence of said cell comprising a modified immunoglobulin locus wherein said cell is obtainable by the method of claim 21.

23. A method to produce an antibody from a cell expressing said antibody from a genomic sequence comprising a modified immunoglobulin locus wherein said cell is obtainable by the method of claim 21, which method comprises:

(a) incubating said cell under conditions such that said cell expresses said antibody, and

(b) recovering said antibody expressed by said cell.

25. An embryonic stem cell of claim 24 wherein said non-primate mammal is murine.

27. A transgenic non-primate mammal of claim 26 wherein said mammal is murine.

24. An embryonic stem cell of a non-primate mammal comprising a genome comprising:

at least a functional portion of a human heavy chain immunoglobulin locus or at least a functional portion of a human light chain immunoglobulin locus,

wherein a lox site is adjacent to or integrated within a region of said human heavy chain or light chain immunoglobulin locus.

26. A transgenic non-primate mammal comprising a genome comprising:

at least a functional portion of a human heavy chain immunoglobulin locus or at least a functional portion of a human light chain immunoglobulin locus,

wherein a lox site is adjacent to or integrated within a region of said human heavy chain or light chain immunoglobulin locus.