Production of antibodies using Cre-mediated site-specific recombination
First Claim
1. A method to produce an antibody-producing cell comprising a genomically-integrated modified immunoglobulin locus, the method comprising the steps of:
- (a) transfecting a murine embryonic stem (ES) 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,so that said first lox site is inserted 5'"'"' of said region of said immunoglobulin locus via homologous recombination with genomic DNA without disrupting expression of or substantially altering an amino acid sequence of an antibody chain encoded by the immunoglobulin locus;
(b) generating a transgenic mouse from the ES cell;
(c) immunizing the transgenic mouse with an antigen;
(d) isolating an antibody-producing cell from the immunized transgenic mouse;
(e) transfecting said antibody-producing cell 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;
(f) interacting the lox sites with Cre, so that said modifying sequence inserts into said immunoglobulin sequence via Cre-mediated site-specific recombination of said lox sites, thereby forming a modified immunoglobulin locus; and
(g) selecting a transfectant comprising said modified immunoglobulin locus, which transfectant produces a functional antibody molecule.
5 Assignments
0 Petitions
Accused Products
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.
-
Citations
27 Claims
-
1. A method to produce an antibody-producing cell comprising a genomically-integrated modified immunoglobulin locus, the method comprising the steps of:
-
(a) transfecting a murine embryonic stem (ES) 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, so that said first lox site is inserted 5'"'"' of said region of said immunoglobulin locus via homologous recombination with genomic DNA without disrupting expression of or substantially altering an amino acid sequence of an antibody chain encoded by the immunoglobulin locus; (b) generating a transgenic mouse from the ES cell; (c) immunizing the transgenic mouse with an antigen; (d) isolating an antibody-producing cell from the immunized transgenic mouse; (e) transfecting said antibody-producing cell 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; (f) interacting the lox sites with Cre, so that said modifying sequence inserts into said immunoglobulin sequence via Cre-mediated site-specific recombination of said lox sites, thereby forming a modified immunoglobulin locus; and (g) selecting a transfectant comprising said modified immunoglobulin locus, which transfectant produces a functional antibody molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
-
-
16. A method to produce an antibody-producing cell expressing a genomic sequence encoding an antibody, the genomic sequence comprising a lox site adjacent or integrated within a region of an immunoglobulin locus which method comprises:
-
(a) transfecting a murine embryonic stem (ES) 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, wherein said first lox site is inserted 5'"'"' of said region of said immunoglobulin locus via homologous recombination with genomic DNA without disrupting expression of or substantially altering an amino acid sequence of an antibody chain polypeptide encoded by the immunoglobulin locus; and (b) generating a transgenic mouse from the ES cell; (c) immunizing the transgenic mouse with an antigen; (d) isolating an antibody-producing cell from the immunized transgenic mouse, wherein the antibody-producing cell comprises said lox site inserted into said genomic DNA 5'"'"' of said region. - View Dependent Claims (17, 18, 19, 20)
-
-
21. A method to produce an antibody-producing hybridoma, which hybridoma expresses an antibody chain encoded by a modified immunoglobulin locus, the method comprising the steps of:
-
immunizing a transgenic mouse with an antigen, wherein the mouse is transgenic for a sequence encoding a first lox site, which lox site is present within an immunoglobulin sequence selected from; (i) a heavy chain polypeptide encoding sequence, wherein the first lox site is positioned 5'"'"' of a sequence encoding a heavy chain constant region and 3'"'"' of a sequence encoding a heavy chain variable region; and (ii) a light chain polypeptide encoding sequence, wherein the first lox site is positioned 5'"'"' of a sequence encoding a light chain constant region and 3'"'"' of a sequence encoding a light chain variable region; wherein the first lox site is positioned within the immunoglobulin sequence such that the encoded polypeptide retains sufficient functionality for production of a functional antibody; producing hybridomas from antibody-producing cells of the immunized transgenic mouse; identifying a hybridoma producing an antibody having a desired antigen-binding characteristic; transfecting the hybridoma 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; interacting the lox sites with Cre, so that said modifying sequence inserts into the immunoglobulin sequence via Cre-mediated site-specific recombination at the lox sites, wherein a sequence positioned 3'"'"' of the first lox site is displaced by the modifying sequence, producing a modified immunoglobulin locus; and selecting a transfectant having a modified immunoglobulin locus and producing an antibody chain polypeptide from the modified immunoglobulin locus; wherein a hybridoma expressing an antibody chain encoded by a modified immunoglobulin locus is produced. - View Dependent Claims (22, 23)
-
-
24. A method to produce an antibody comprising an antibody heavy chain encoded by a modified heavy chain polypeptide-encoding sequence, the method comprising the steps of:
-
immunizing a transgenic mouse with an antigen, wherein the mouse is transgenic for a sequence encoding a first lox site, which first lox site is present within a heavy chain polypeptide encoding sequence and is positioned 5'"'"' of a sequence encoding a heavy chain constant region and 3'"'"' of a sequence encoding a heavy chain variable region, said positioning being such that the heavy chain polypeptide retains sufficient functionality for production of a functional antibody; producing hybridomas from antibody-producing cells of the immunized transgenic mouse; identifying a hybridoma producing an antibody having a desired antigen-binding characteristic; transfecting the hybridoma 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; interacting the lox sites with Cre, so that said modifying sequence inserts into the heavy chain polypeptide-encoding sequence via Cre-mediated site-specific recombination at the lox sites, wherein all or a portion of the sequence encoding the heavy chain constant region 3'"'"' of the first lox site is displaced by the modifying sequence, producing a modified heavy chain polypeptide-encoding sequence; and selecting a transfectant having a modified heavy chain polypeptide-encoding sequence and expressing a modified heavy chain polypeptide; wherein a hybridoma expressing an antibody comprising a modified heavy chain polypeptide is produced. - View Dependent Claims (25, 27)
-
-
26. A method to produce an antibody comprising an antibody light chain encoded by a modified light chain polypeptide-encoding sequence, the method comprising the steps of:
-
immunizing a transgenic mouse with an antigen, wherein the mouse is transgenic for a sequence encoding a first lox site, which first lox site is present within a light chain polypeptide encoding sequence and is positioned 5'"'"' of a sequence encoding a light chain constant region and 3'"'"' of a sequence encoding a light chain variable region, said positioning being such that the light chain polypeptide retains sufficient functionality for production of a functional antibody; producing hybridomas from antibody-producing cells of the immunized transgenic mouse; identifying a hybridoma producing an antibody having a desired antigen-binding characteristic; transfecting the hybridoma 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 positioned adjacent the second lox site; interacting the lox sites with Cre, so that said modifying sequence inserts into the light chain polypeptide-encoding sequence via Cre-mediated site-specific recombination at the lox sites, wherein all or a portion of the sequence encoding the light chain constant region 3°
of the first lox site is displaced by the modifying sequence, producing a modified light chain polypeptide-encoding sequence; andselecting a transfectant having a modified light chain polypeptide-encoding sequence and expressing a modified light chain polypeptide; wherein a hybridoma expressing an antibody comprising a modified light chain polypeptide is produced.
-
Specification