HUMANIZED LIGHT CHAIN MICE

• US 20140017228A1
• Filed: 07/26/2013
• Published: 01/16/2014
• 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.
• Granted Patent: US 9,622,459 B2
• Time in Patent Office: Days
• US Class Current: 424/132.1
• 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 is provided, wherein the method comprises

(a) exposing a non-human animal 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;

wherein the non-human animal comprises

(i) an insertion of one or more human Vλ and

Jλ

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

(ii) 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

(iii) an ectopic nucleotide sequence that encodes an Adam6 protein or a functional fragment thereof.

2. The method of claim 1, wherein the non-human light chain constant region is a mouse Cκ

.

3. The method of claim 1, wherein the non-human light chain constant region 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 60%, at least 70%, at least 80%, at least 90%, at least 95%, 96%, 97%, 98%, or at least 99% identical to mouse Cλ

2.

6. The method of claim 1, wherein the antibody of step (b) comprises an immunoglobulin heavy chain comprising a human V_H domain and a non-human C_H domain.

7. The method of claim 6, wherein the non-human C_H domain is a mouse C_H domain.

8. The method of claim 1, wherein the non-human animal comprises an insertion of at least 12 human Vλ

segments and at least one human Jλ

segment upstream of an non-human immunoglobulin light chain constant region.

9. The method of claim 1, wherein the non-human animal comprises an insertion of at least 28 human Vλ

segments and at least one human Jλ

segment upstream of an non-human immunoglobulin light chain constant region.

10. The method of claim 1, wherein the non-human animal comprises an insertion of at least 40 human Vλ

segments and at least one human Jλ

segment upstream of an non-human immunoglobulin light chain constant region.

11. The method of claim 1, wherein the ectopic nucleotide sequence is present at the immunoglobulin heavy chain locus of the non-human animal.

12. The method of claim 11, wherein the ectopic nucleotide sequence is positioned within a human immunoglobulin heavy chain sequence.

13. The method of claim 12, wherein the ectopic nucleotide sequence is positioned between two human V_H gene segments.

14. The method of claim 1, wherein the ectopic nucleotide sequence is present at a position other than the immunoglobulin heavy chain locus of the non-human animal.

15. The method of claim 14, wherein the ectopic nucleotide sequence is positioned between a V_H and a D_H gene segment.

16. The method 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.

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

-Jκ

intergenic region is placed between a human Vλ

gene segment and a human Jλ

gene segment.

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

-Jκ

intergenic region comprises SEQ ID NO;

158.

19. 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).

20. The method of claim 1, wherein the non-human animal is a mouse or a rat.