Humanized Light Chain Mice

• US 20130160153A1
• Filed: 12/17/2012
• Published: 06/20/2013
• Est. Priority Date: 12/20/2011
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Macdonald, Lynn, Gurer, Cagan, Hosiawa, Karolina A., Stevens, Sean, Murphy, Andrew J.
• Granted Patent: US 9,706,759 B2
• Time in Patent Office: Days
• US Class Current: 800/18
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• 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: C12N 2800/204 of bacterial origin, e.g. BAC
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 9/6489 Metalloendopeptidases (3.4.24)

Abstract

Non-human animals, tissues, cells, and genetic material are provided that comprise a modification of an endogenous non-human heavy chain immunoglobulin sequence and that comprise an ADAM6 activity functional in a mouse, wherein the non-human animals express a human immunoglobulin heavy chain variable domain and a cognate human immunoglobulin λ light chain variable domain.

Claims

1. A non-human animal comprising

(a) an insertion of one or more human Vλ

and Jλ

gene segments upstream of an non-human immunoglobulin light chain constant region,

(b) an insertion of one or more human V_H, one or more human D_H and one or more human J_H gene segments upstream of an non-human immunoglobulin heavy chain constant region, and

(c) a nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof, wherein the ADAM6 protein is expressed from an ectopic ADAM6 nucleic acid sequence.

2. The non-human animal of claim 1, wherein the non-human heavy and/or light chain constant regions are rodent constant regions.

3. The non-human animal of claim 2, wherein the light chain constant region is a mouse Cκ

or mouse Cλ

region.

4. The non-human animal of claim 1, wherein the non-human animal comprises at least 12 to at least 40 human Vλ

gene segments and at least one human Jλ

gene segment.

5. The non-human animal of claim 1, wherein the at least one human Jλ

gene segment is selected from Jλ

1, Jλ

2, Jλ

3, Jλ

7 and a combination thereof.

6. The non-human animal of claim 1, wherein the non-human comprises at least four human Jλ

gene segments.

7. The non-human animal of claim 6, wherein the at least four human Jλ

gene segments comprise at least Jλ

1, Jλ

2, Jλ

3 and Jλ

7.

8. The non-human animal of claim 1, wherein the nucleotide sequence that encodes an ADAM6 protein or functional fragment thereof is present the same location as compared to a wild-type type non-human ADAM6 locus.

9. The non-human animal of claim 1, wherein the nucleotide sequence that encodes an ADAM6 protein or functional fragment thereof is present within immunoglobulin gene segments.

10. The non-human animal of claim 9, wherein the immunoglobulin gene segments are heavy chain gene segments.

11. The non-human animal of claim 10, wherein the heavy chain gene segments are human.

12. The non-human animal of claim 10, wherein the heavy chain gene segments are endogenous heavy chain gene segments of the non-human animal.

13. The non-human animal of claim 1, wherein the non-human animal lacks an endogenous immunoglobulin V_L and/or a J_L gene segment at an endogenous immunoglobulin light chain locus.

14. The non-human animal of claim 1, wherein the non-human animal comprises endogenous immunoglobulin V_L and/or J_L gene segments that are incapable of rearranging to form an immunoglobulin V_L domain in the non-human animal.

15. The non-human animal of claim 14, wherein all or substantially all endogenous immunoglobulin Vκ

and Jκ

gene segments are replaced with one or more human Vλ and

Jλ

gene segments.

16. The non-human animal of claim 14, all or substantially all endogenous immunoglobulin Vλ

and Jλ

gene segments are replaced with one or more human Vλ and

Jλ

gene segments.

17. The non-human animal of claim 1, wherein the non-human animal further comprises a human Vκ

-Jκ

intergenic region from a human κ

light chain locus, wherein the human Vκ

-Jκ

intergenic region is contiguous with the one or more human Vλ and

Jλ

gene segments.

18. The non-human animal of claim 17, wherein the human Vκ

-Jκ

intergenic region is placed between a human Vλ

gene segment and a human Jλ

gene segment.

20. A cell derived from the non-human animal of claim 1.

21. The cell of claim 20, wherein the cell is a B cell.

22. A hybridoma made from the B cell of claim 21.

26. A method for making an antibody that binds to an antigen of interest is provided, wherein the method comprises

(a) exposing a non-human animal of claim 1 to an antigen of interest,

(b) isolating one or more B lymphocytes of the non-human animal, wherein the one or more B lymphocytes express an antibody that binds the antigen of interest, and

(c) identifying a nucleic acid sequence that encodes an immunoglobulin light chain of the antibody that binds that antigen of interest, wherein the immunoglobulin light chain comprises a human Vλ

domain and a non-human light chain constant domain, and

(d) employing the nucleic acid sequence of (c) with a human immunoglobulin light chain constant region nucleic acid sequence to make a human antibody that binds the antigen of interest.

27. The method of claim 26, wherein the non-human light chain constant region is a mouse Cκ

or

28. The method of claim 26, wherein the non-human light chain constant region is a mouse Cλ

.

29. The method of claim 26, wherein the antibody comprises an immunoglobulin heavy chain comprising a human V_H domain and a non-human C_H domain.

30. The method of claim 29, wherein the non-human C_H domain is a mouse C_H domain.

31. The method of claim 26, wherein the non-human animal further comprises a human Vκ

-Jκ

intergenic region from a human κ

light chain locus, wherein the human Vκ

-Jκ

intergenic region is contiguous with the one or more human Vλ and

Jλ

gene segments.

32. The method of claim 31, wherein the human Vκ

-Jκ

intergenic region is placed between a human Vλ

gene segment and a human Jλ

gene segment

33. The method of claim 26, wherein the non-human animal is a mouse.

19. A genetically modified non-human animal that comprises

(a) one or more unrearranged human Vλ

gene segments and one or more unrearranged human Jλ

gene segments at an endogenous immunoglobulin light chain locus of the non-human animal,

(b) on or more unrearranged human V_H gene segments, one or more unrearranged human D_H gene segments, and one or more unrearranged human J_H gene segments at an endogenous immunoglobulin heavy chain locus of the non-human animal, wherein the non-human animal is capable of expressing an ADAM6 protein or functional fragment thereof.

23. A cell derived from the non-human animal of claim 19.

24. The cell of claim 23, wherein the cell is a B cell.

25. A hybridoma made from the B cell of claim 24.