Production of antibodies using cre-mediated site-specific recombination

• US 6,458,592 B1
• Filed: 07/05/2000
• Issued: 10/01/2002
• Est. Priority Date: 03/29/1995
• Status: Expired due to Term

• Current Assignee: Amgen Fremont Inc. (Amgen Inc.)
• Original Assignee: Abgenix Inc (Amgen Inc.), Japan Tobacco Inc.
• Inventors: Jakobovits, Aya, Zsebo, Krisztina M.
• Primary Examiner(s): WEHBE, ANNE MARIE SABRINA
• Application Number:
  US09/610,259
• Time in Patent Office: 818 Days
• Field of Search: 435/320.1, 435/325, 435/326, 435/69.1, 435/69.7, 435/70.1, 435/70.21, 435/455, 800/13, 800/18
• US Class Current: 435/455
• 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 of producing an antibody-producing cell, said cell comprising a genomically-integrated modified immunoglobulin locus, which method comprises the steps of:

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

(i) a first lox site, and

(ii) a targeting sequence homologous to a first DNA sequence 5′

of a region of a genomically-integrated immunoglobulin locus in said cell, so that said first lox site is inserted 5′

of said region via homologous recombination with the genomically-integrated immunoglobulin locus,

(b) transfecting said cell of step (a) in vitro 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;

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

(d) selecting a transfectant in vitro, which transfectant comprises said modified immunoglobulin locus and which transfectant produces a functional 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 in vitro 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 said region of the immunoglobulin locus, said region being flanked by said first and second DNA sequences, so that said third lox site and said first marker gene are inserted into said immunoglobulin locus via site-specific homologous recombination with genomic DNA in vitro; 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 and said modifying sequence insert into said immunoglobulin locus 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 immunoglobulin locus, said first marker gene and said region 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 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 a constant region or a variable region of the immunoglobulin locus to form an immunoglobulin locus encoding a modified antibody molecule.

7. The method of claim 1 wherein said region of the immunoglobulin locus 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.

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

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

11. The method of claim 10 wherein said constant region gene is a human C_μ

gene and said modifying sequence comprises a human C_γ

constant region gene.

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

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

14. The method of claim 13 wherein said lymphoid cell line is a murine hybridoma cell line.

15. The method of claim 14 wherein said murine hybridoma cell line is producing a human antibody by expression of a human immunoglobulin locus.

16. 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, and wherein said cell comprises a lox site 5′

of said modifying sequence in said modified immunoglobulin locus.

17. A method of producing an antibody from an antibody-producing cell expressing a genomic sequence encoding an antibody, the genomic sequence comprising a modified immunoglobulin locus, which method comprises:

a) incubating the cell according to claim 16 under conditions such that said cell produces said antibody, and

b) recovering said antibody produced by said cell.

18. A method of producing an antibody-producing cell expressing a genomic sequence encoding an antibody, the genomic sequence comprising 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 in vitro with a first homology-targeting vector comprising;

i) a first lox site; and

ii) a targeting sequence homologous to a first DNA sequence 5′

of a region of the immunoglobulin locus 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; and

b) selecting a transfectant with said lox site inserted into said genomic DNA 5′

of said region which is to be converted.

19. A cell expressing an antibody molecule from a genomic sequence of said cell with a lox site 5′

of said region of the immunoglobulin locus encoding said antibody molecule, wherein said cell is obtainable by the method of claim 18.

20. A method of modifying a region of a genomically integrated immunoglobulin locus of an antibody-producing cell wherein said genomic sequence comprises a first lox site 5′

of a region of said immunoglobulin locus which is to be modified using Cre-mediated site-specific recombination, which method comprises;

a) transfecting a cell selected in step (b) of claim 18 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;

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

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

21. An antibody-producing cell comprising a genomically integrated modified immunoglobulin locus wherein said cell is obtainable by the method of claim 20, and wherein said cell comprises a lox site 5′

of a modifying sequence integrated into said modified immunoglobulin locus.

22. A method of producing an antibody from a cell comprising a genomically integrated modified immunoglobulin locus, which method comprises:

a) incubating the cell according to claim 21 under conditions such that said cell produces said antibody, and

b) recovering said antibody produced by said cell.