Generation of Xenogenetic antibodies

• US 6,162,963 A
• Filed: 06/05/1995
• Issued: 12/19/2000
• Est. Priority Date: 01/12/1990
• Status: Expired due to Term

• Current Assignee: Abgenix Inc (Amgen Inc.)
• Original Assignee: Abgenix Inc (Amgen Inc.)
• Inventors: Jakobovits, Aya, Capon, Daniel J., Kucherlapati, Raju, Brenner, Daniel G., Klapholz, Sue
• Primary Examiner(s): Hauda, Karen M.
• Application Number:
  US08/462,513
• Time in Patent Office: 2,024 Days
• Field of Search: 800/2, 800/4, 800/6, 800/8, 800/21, 800/22, 800/25, 800/18, 435/172.3, 536/23.53
• 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 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2217/20 Animal model comprising regulated expression system
• 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: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 16/1282 from Clostridium (G)
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/248 IL-6
• CPC Class Codes: C07K 16/2812 against CD4
• CPC Class Codes: C07K 16/2854 against selectins, e.g. CD62
• CPC Class Codes: C07K 16/2875 : against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 against NGF C07K16/22, against TNF C07K16/241
• CPC Class Codes: C07K 16/462 : Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 2317/21 : from primates, e.g. man
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/87 : Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/90 : Stable introduction of foreign DNA into chromosome

Abstract

The subject invention provides non-human mammalian hosts characterized by inactivated endogenous Ig loci and functional human Ig loci for response to an immunogen to produce human antibodies or analogs thereof. The hosts are produced by multiple genetic modifications of embryonic cells in conjunction with breeding. Different strategies are employed for recombination of the human loci randomly or at analogous host loci. Chimeric and transgenic mammals, particularly mice, are provided, having stably integrated large, xenogeneic DNA segments. The segments are introduced by fusion with yeast spheroplasts comprising yeast artificial chromosomes (YACs) which include the xenogeneic DNA segments and a selective marker such as HPRT, and embryonic stem cells.

Claims

1. A method for producing a transgenic mouse lacking expression of an endogenous immunoglobulin light chain, comprising:

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker; and

(b) producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker,

wherein the transgenic mouse lacks expression of an endogenous immunoglobulin light chain.

5. The method according to any one of claims 1-4, wherein the gene encoding a selectable marker is selected from the group consisting of:

a neomycin resistance gene;

an acyclovir resistance gene and a gancyclovir resistance gene.

13. The method according to any one of claims 1, 2, 6 or 7, further comprising producing progeny of the transgenic mouse by breeding.

2. A method for producing a transgenic mouse lacking expression of an endogenous immunoglobulin light chain, comprising:

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker; and

(b) producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain an immunoglobulin light chain locus in which the Cκ

gene is deleted,

wherein the transgenic mouse lacks expression of an endogenous immunoglobulin light chain.

3. A method for producing a transgenic mouse and its progeny lacking expression of endogenous immunoglobulin light chains, comprising:

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker;

(b) producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker, and

(c) breeding the transgenic mouse as needed to produce a transgenic mouse and its progeny lacking expression of endogenous immunoglobulin light chains.

4. A method for producing a transgenic mouse and its progeny lacking expression of endogenous immunoglobulin light chains, comprising:

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker;

(b) producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain an immunoglobulin light chain locus in which the Cκ

gene is deleted, and (c) breeding the transgenic mouse as needed to produce a transgenic mouse and its progeny lacking expression of endogenous immunoglobulin light chains.

6. A method for preventing the expression of an endogenous immunoglobulin light chain in a transgenic mouse, comprising:

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker; and

(b) producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker,

wherein the transgenic mouse lacks expression of an endogenous immunoglobulin light chain.

10. The method according to any one of claims 6-9, wherein the gene encoding a selectable marker is selected from the group consisting of:

a neomycin resistance gene;

an acyclovir resistance gene and a gancyclovir resistance gene.

7. A method for preventing the expression of an endogenous immunoglobulin light chain in a transgenic mouse, comprising:

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker; and

(b) producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain an endogenous immunoglobulin light chain locus in which the Cκ

gene is deleted,

wherein the transgenic mouse lacks expression of an endogenous immunoglobulin light chain.

8. A method for preventing the expression of endogenous immunoglobulin light chains in a transgenic mouse and its progeny, comprising:

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker;

(b) producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker, and

(c) breeding the transgenic mouse as needed to produce a transgenic mouse and its progeny lacking expression of endogenous immunoglobulin light chains.

9. A method for preventing the expression of endogenous immunoglobulin light chains in a transgenic mouse and its progeny, comprising:

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker;

(b) producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain an immunoglobulin light chain locus in which the Cκ

gene is deleted, and

(c) breeding the transgenic mouse as needed to produce a transgenic mouse and its progeny lacking expression of endogenous immunoglobulin light chains.

11. A method for producing a mouse embryonic stem cell with at least one inactivated endogenous immunoglobulin light chain locus, comprising deleting the Cκ

gene from at least one endogenous immunoglobulin light chain locus in the mouse embryonic stem cell to prevent rearrangement of the locus and to prevent the formation of a transcript of a rearranged immunoglobulin light chain, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker.

12. The method according to claim 11, wherein the gene encoding a selectable marker is selected from the group consisting of:

a neomycin resistance gene;

an acyclovir resistance gene and a gancyclovir resistance gene.

14. A method for producing a transgenic mouse wherein the germ cells comprise at least one inactivated endogenous immunoglobulin light chain locus in which the Cκ

gene is deleted to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged locus and the expression of an endogenous immunoglobulin light chain from the inactivated locus, said method comprising the steps of;

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of said locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker; and

(b) producing from the embryonic stem cell a transgenic mouse whose germ cells comprise at least one immunoglobulin light chain locus in which the Cκ

gene is deleted.

16. The method according to claim 14 or 15, wherein the gene encoding a selectable marker is selected from the group consisting of:

a neomycin resistance gene;

an acyclovir resistance gene and a gancyclovir resistance gene.

15. A method for producing a transgenic mouse and its progeny, wherein the somatic and germ cells comprise at least one inactivated endogenous immunoglobulin light chain locus in which the Cκ

gene is deleted to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged locus and the expression of an endogenous immunoglobulin light chain from the inactivated locus, said method comprising the steps of;

(a) deleting the Cκ

gene from at least one endogenous light chain locus in a mouse embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, the deletion being effected by a targeting vector comprising a gene encoding a selectable marker;

(b) producing from the embryonic stem cell a transgenic mouse whose germ cells comprise at least one immunoglobulin light chain locus in which the Cκ

gene is deleted, and

(c) breeding the transgenic mouse as needed to produce a transgenic mouse and its progeny whose somatic and germ cells comprise at least one inactivated endogenous immunoglobulin light chain locus in which the Cκ

gene is deleted.

17. A mouse embryonic stem cell with at least one inactivated endogenous immunoglobulin light chain locus in which the Cκ

gene is deleted to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin light chain locus, said inactivated endogenous locus comprising a gene encoding a selectable marker.

18. The mouse embryonic stem cell according to claim 17, wherein said gene encoding a selectable marker is selected from the group consisting of:

a neomycin resistance gene;

an acyclovir resistance gene and a gancyclovir resistance gene.

19. A transgenic mouse wherein the germ cells comprise at least one inactivated endogenous immunoglobulin light chain locus in which the Cκ

gene is deleted to prevent rearrangement and to prevent formation of a transcript of a rearranged locus and the expression of an endogenous immunoglobulin light chain from the inactivated locus.

20. A transgenic mouse, wherein the somatic and germ cells comprise at least one inactivated endogenous immunoglobulin light chain locus in which the Cκ

gene is deleted to prevent rearrangement and to prevent formation of a transcript of a rearranged locus and the expression of an endogenous immunoglobulin light chain from the inactivated locus.

21. A transgenic mouse, wherein the somatic and germ cells comprise inactivated endogenous immunoglobulin loci in which the Cκ

gene is deleted to prevent rearrangement and to prevent formation of a transcript of a rearranged locus, wherein the transgenic mouse and progeny lack expression of endogenous immunoglobulin light chains.