COMPOSITIONS AND METHODS FOR INHIBITING ENDOGENOUS IMMUNOGLOBULIN GENES AND PRODUCING TRANSGENIC HUMAN IDIOTYPE ANTIBODIES

• US 20090098134A1
• Filed: 05/30/2008
• Published: 04/16/2009
• Est. Priority Date: 06/01/2007
• Status: Active Grant

• Current Assignee: OmniAb Operations, Inc. (Omniab Incorporated), OMT Inc.
• Original Assignee: OMT Inc.
• Inventors: BUELOW, Roland
• Granted Patent: US 8,703,485 B2
• Time in Patent Office: Days
• US Class Current: 424/141.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• 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 2267/01 Animal expressing industrially exogenous proteins
• 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: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• 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 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• 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: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2015/8518 expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0609 Oocytes, oogonia fertilised oocytes C12N5/0604
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The invention relates to transgenic animals lacking endogenous Ig and capable of producing transgenic antibodies, as well as methods of making the same. The invention further relates to methods for producing transgenic antibodies in such animals, and transgenic antibodies so produced.

Claims

1. A method for producing a viable germ cell having at least one inactivated endogenous Ig locus, comprising expressing at least one meganuclease in a germ cell, fertilized oocyte or embryo, to generate a viable germ cell having at least one inactivated endogenous Ig locus wherein said meganuclease recognizes a meganuclease target sequence present in or proximal to said endogenous Ig locus.

2. A method for producing a transgenic animal comprising at least one germline inactivated endogenous Ig locus, comprising deriving a transgenic animal from a viable germ cell having at least one inactivated endogenous Ig locus produced according to the method of claim 1, or a germ cell descendant thereof, wherein said transgenic animal is selected from the group consisting of birds, rodents, and weasels.

3. The method according to claim 2, wherein said viable germ cell having at least one inactivated endogenous Ig locus further comprises an artificial Ig locus, whereby said transgenic animal comprises an artificial Ig locus.

4. The method according to claim 2, further comprising introducing an artificial Ig locus into said viable germ cell having at least one inactivated endogenous Ig locus, or a germ cell descendant thereof or a fertilized oocyte or embryo derived therefrom, whereby said transgenic animal comprises an artificial Ig locus.

5. The method according to claim 2, wherein said deriving a transgenic animal from a viable germ cell having at least one inactivated endogenous Ig locus comprises combining said viable germ cell, or a germ cell descendant thereof, with a gamete comprising an artificial Ig locus, whereby said transgenic animal comprises an artificial Ig locus.

6. The method according to any one of claims 3-5, wherein said artificial Ig locus comprises (i) a V-region having at least one human V gene segment encoding a germline or hypermutated human V-region amino acid sequence;

(ii) one or more J gene segments; and

(iii) one or more constant region gene segments, wherein said artificial Ig locus is functional and capable of undergoing gene rearrangement and producing a repertoire of artificial immunoglobulins in a transgenic animal derived from said germ cell.

7. The method according claim 5, wherein said gamete has at least one inactivated endogenous Ig locus.

8. The method according to claim 1, wherein said meganuclease target sequence is present in or proximal to a J gene segment within said at least one endogenous Ig locus.

9. The method according to claim 1, wherein said meganuclease target sequence is present in or proximal to an immunoglobulin constant region gene.

10. The method according to claim 1, further comprising expressing a second meganuclease in said germ cell, fertilized oocyte or embryo, wherein said second meganuclease recognizes a second meganuclease target sequence present in or proximal to said endogenous Ig locus.

11. The method according to claim 1, wherein said germ cell, fertilized oocyte or embryo comprises a genomic meganuclease expression construct comprising an inducible expression control region operably linked to a nucleic acid encoding said meganuclease, and wherein said meganuclease is expressed in said germ cell, fertilized oocyte or embryo by inducing expression of said genomic meganuclease expression construct.

12. The method according to claim 11, comprising repeating the step of inducing expression of said genomic meganuclease expression construct.

13. The method according to claim 11, wherein said germ cell, fertilized oocyte or embryo comprises a second genomic meganuclease expression construct comprising a second inducible expression control region operably linked to a second meganuclease-encoding nucleic acid, wherein said second encoded meganuclease recognizes a second meganuclease target sequence present in said endogenous Ig locus, wherein said method further comprises inducing expression of said second meganuclease-encoding nucleic acid in said germ cell, fertilized oocyte or embryo.

14. A viable germ cell having at least one inactivated endogenous Ig locus produced by the method according to claim 1.

15. A transgenic animal produced by the method according to claim 2.

16. A transgenic animal produced by the method according to any one of claims 3-5.

17. The transgenic animal according to claims 15 or 16, wherein said transgenic animal lacks a functional endogenous Ig light chain locus.

18. The transgenic animal according to claims 15 or 16, wherein said transgenic animal lacks a functional endogenous Ig heavy chain locus.

19. The transgenic animal of claim 16, wherein said transgenic animal is capable of producing immunoglobulins having a human idiotype.

20. The transgenic animal of claim 16, wherein said transgenic animal lacks a functional Ig light chain locus and comprises an artificial Ig heavy chain locus, and wherein said transgenic animal is capable of producing heavy chain-only antibodies.

21. The transgenic animal of claim 16, wherein said transgenic animal comprises at least one Ig heavy chain locus with at least one C-region gene lacking sequences encoding a functional CH1 domain, and lacking a functional Ig light chain locus.

24. A method for producing antibodies, comprising immunizing the transgenic animal according to claim 16 with an immunogen.

25. A polyclonal antisera composition produced by the method of claim 24.

26. A method for producing a monoclonal antibody, comprising (i) immunizing the transgenic animal according to claim 16 with an immunogen, (ii) isolating a monoclonal antibody producing cell from said transgenic animal wherein said monoclonal antibody producing cell produces a monoclonal antibody that specifically binds to said immunogen;

and (iii) using said monoclonal antibody producing cell to produce said monoclonal antibody that specifically binds to said immunogen, or using said monoclonal antibody producing cell to produce a hybridoma cell that produces said monoclonal antibody and using said hybridoma cell to produce said monoclonal antibody.

27. A method for producing a monoclonal antibody, comprising (i) immunizing the transgenic animal according to claim 16 with an immunogen, (ii) isolating a monoclonal antibody producing cell from said transgenic animal wherein said monoclonal antibody producing cell produces a monoclonal antibody that specifically binds to said immunogen;

(iii) isolating from said monoclonal antibody producing cell a monoclonal antibody nucleic acid which encodes said monoclonal antibody that specifically binds to said immunogen; and

(iv) using said monoclonal antibody nucleic acid to produce said monoclonal antibody that specifically binds to said immunogen.

28. The method according to claim 26 or 27, wherein said monoclonal antibody has a human idiotype.

29. A method for producing a fully human monoclonal antibody, comprising (i) immunizing the transgenic animal according to claim 16 with an immunogen, (ii) isolating a monoclonal antibody producing cell from said transgenic animal wherein said monoclonal antibody producing cell produces a monoclonal antibody that specifically binds to said immunogen;

(iii) isolating from said monoclonal antibody producing cell a monoclonal antibody nucleic acid which encodes said monoclonal antibody that specifically binds to said immunogen;

(iv) modifying said monoclonal antibody nucleic acid to produce a recombinant nucleic acid encoding a fully human monoclonal antibody; and

(v) using said recombinant nucleic acid encoding a fully human monoclonal antibody to produce the encoded fully human monoclonal antibody.

30. A monoclonal antibody produced by the method according to any one of claims 26, 27, or 29.

31. A method for neutralizing an antigenic entity in a human body component, said method comprising contacting said body component with a polyclonal antisera composition according to claim 25, wherein said polyclonal antisera composition comprises immunoglobulin molecules that specifically bind and neutralize said antigenic entity.

32. A method for neutralizing an antigenic entity in a human body component, said method comprising contacting said body component with the monoclonal antibody according to claim 30, wherein said monoclonal antibody specifically binds to and neutralizes said antigenic entity.

33. A method for producing heavy chain-only antibodies, comprising immunizing a transgenic animal according to claim 20 or 21.

34. A heavy chain-only antibody produced by the method according to claim 33.

22. A transgenic animal comprising a genomic meganuclease expression construct, wherein said construct comprises an inducible expression control region operably linked to a meganuclease-encoding nucleic acid, and wherein said encoded meganuclease recognizes a meganuclease target sequence present in or proximal to an endogenous Ig locus of said transgenic animal, wherein said transgenic animal is selected from the group consisting of birds, rodents, and weasels.

23. The transgenic animal of claim 22, wherein the genome of said transgenic animal further comprises at least one artificial Ig locus.