Production of antibodies using Cre-mediated site-specific recombination

• US 6,091,001 A
• Filed: 03/29/1995
• Issued: 07/18/2000
• Est. Priority Date: 03/29/1995
• Status: Expired due to Term

• Current Assignee: Amgen Fremont Inc. (Amgen Inc.), Japan Tobacco Inc.
• Original Assignee: Abgenix Inc (Amgen Inc.), Japan Tobacco Inc.
• Inventors: Jakobovits, Aya, Zsebo, Krisztina M.
• Primary Examiner(s): HAUDA, KAREN MARIE
• Application Number:
  US08/412,777
• Time in Patent Office: 1,938 Days
• Field of Search: 800/2, 800/18, 800/21, 800/25, 800/4, 435/172.3, 435/354
• US Class Current: 800/18
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• 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: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/902 using homologous recombination
• 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 mouse embryonic stem cell comprising a transgene integrated into the cell'"'"'s genome, wherein the transgene comprises, in order from 5'"'"' to 3'"'"':

i) a human heavy chain immunoglobulin variable region-encoding sequence;

ii) a lox site; and

iii) a human heavy chain immunoglobulin constant region-encoding sequence, wherein the transgene encodes a functional immunoglobulin heavy chain, and wherein the transgene is transcribed and translated in an antibody-producing cell of a mouse produced using the embryonic stem cell.

2. A transgenic mouse comprising a transgene integrated into the genome of the mouse, wherein the transgene comprises, in order from 5'"'"' to 3'"'"':

i) a human heavy chain immunoglobulin variable region-encoding sequence;

ii) a lox site; and

iii) a human heavy chain immunoglobulin constant region-encoding sequence, wherein the transgene encodes a functional immunoglobulin heavy chain, and wherein the transgene is transcribed and translated in an antibody-producing cell of the transgenic mouse.

3. A mouse embryonic stem cell comprising a transgene integrated into the cell'"'"'s genome, wherein the transgene comprises, in order from 5'"'"' to 3'"'"':

i) a human light chain immunoglobulin variable region-encoding sequence;

ii) a lox site; and

iii) a human light chain immunoglobulin constant region-encoding sequence, wherein the transgene encodes a functional immunoglobulin heavy chain, and wherein the transgene is transcribed and translated in an antibody-producing cell of a mouse produced using the embryonic stem cell.

4. A transgenic mouse comprising a transgene integrated into the genome of the mouse, wherein the transgene comprises, in order from 5'"'"' to 3'"'"':

i) a human light chain immunoglobulin variable region-encoding sequence;

ii) a lox site; and

iii) a human light chain immunoglobulin constant region-encoding sequence, wherein the transgene encodes a functional immunoglobulin light chain, and wherein the transgene is transcribed and translated in an antibody-producing cell of the transgenic mouse.