Humanized light chain mice

• US 9,622,459 B2
• Filed: 07/26/2013
• Issued: 04/18/2017
• Est. Priority Date: 12/20/2011
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Macdonald, Lynn, Gurer, Cagan, Meagher, Karolina A., Stevens, Sean, Murphy, Andrew J.
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US13/951,996
• Publication Number:
  US 20140017228A1
• Time in Patent Office: 1,362 Days
• Field of Search: 800 4
• 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 method for making an antibody that binds to an antigen of interest, wherein the method comprises:

(a) exposing a mouse to an antigen of interest,

(b) isolating one or more B lymphocytes of the mouse, wherein the one or more B lymphocytes express an antibody that binds the antigen of interest, wherein the expressed antibody that binds the antigen of interest includes an immunoglobulin light chain comprising a human immunoglobulin λ

light chain variable domain and a mouse immunoglobulin light chain constant domain, and comprises an immunoglobulin heavy chain comprising a human immunoglobulin heavy chain variable domain and a mouse immunoglobulin heavy chain constant domain,

(c) identifying a nucleic acid sequence that encodes the human immunoglobulin λ

light chain variable domain of the antibody that binds the antigen of interest, and

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

wherein the mouse comprises

(i) an insertion of one or more human Vλ

gene segments and one or more human Jλ

gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human Vλ

gene segments and one or more human Jλ

gene segments are operably linked to the mouse immunoglobulin light chain constant region gene,

(ii) an insertion of one or more human V_H gene segments, one or more human D_H gene segments and one or more human J_H gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V_H gene segments, one or more human D_H gene segments and one or more human J_H gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene, and

(iii) an ectopic nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof.

2. The method of claim 1, wherein the mouse immunoglobulin light chain constant region gene of (i) is a mouse Cκ

.

3. The method of claim 1, wherein the mouse immunoglobulin light chain constant region gene of (i) is a mouse Cλ

.

4. The method of claim 3, wherein the mouse Cλ

is a mouse Cλ

2.

5. The method of claim 3, wherein the mouse Cλ

is at least 90% identical to mouse Cλ

2.

6. The method of claim 1, wherein the mouse comprises an insertion of at least 12 human Vλ

gene segments and at least one human Jλ

gene segment upstream of the mouse immunoglobulin light chain constant region gene.

7. The method of claim 1, wherein the mouse comprises an insertion of at least 28 human Vλ

gene segments and at least one human Jλ

gene segment upstream of the mouse immunoglobulin light chain constant region gene.

8. The method of claim 1, wherein the mouse comprises an insertion of at least 40 human Vλ

gene segments and at least one human Jλ

gene segment upstream of the mouse immunoglobulin light chain constant region gene.

9. The method of claim 1, wherein the ectopic nucleotide sequence is present at the immunoglobulin heavy chain locus of the mouse.

10. The method of claim 9, wherein the ectopic nucleotide sequence is positioned within the variable region of the human immunoglobulin heavy chain locus.

11. The method of claim 10, wherein the ectopic nucleotide sequence is positioned between two human V_H gene segments.

12. The method of claim 1, wherein the ectopic nucleotide sequence is present at a position other than the immunoglobulin heavy chain locus of the mouse.

13. The method of claim 10, wherein the ectopic nucleotide sequence is positioned between a V_H gene segment and a D_H gene segment.

14. The method of claim 1, wherein the mouse 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 Vκ

gene segments and one or more human Jκ

gene segments.

15. The method of claim 14, wherein the human Vκ

-Jκ

intergenic region is placed between a human Vκ

gene segment and a human Jκ

gene segment.

16. The method of claim 14, wherein the human Vκ

-Jκ

intergenic region comprises SEQ ID NO;

158.

17. The method of claim 1, wherein the nucleic acid sequence of step (c) is isolated from a hybridoma made from the B cell of step (b).

18. The method of claim 3, wherein the mouse Cλ

is at least 95% identical to mouse Cλ

2.

19. The method of claim 3, wherein the mouse Cλ

is at least 97% identical to mouse Cλ

2.

20. The method of claim 3, wherein the mouse Cλ

is at least 98% identical to mouse Cλ

2.