Humanized light chain mice
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.
1. A method for generating a human heavy chain variable domain or a human λ light chain variable domain of an antibody that specifically binds an antigen comprising the steps of:
(a) immunizing a genetically modified mouse with an antigen, wherein the mouse:
(i) has a germline genome that comprises:
(1) an insertion of one or more human Vλ gene segments and one or more human Jλ, gene segments upstream of a light chain constant region gene,
(2) 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 heavy chain constant region gene, and
(3) an inserted nucleotide sequence that encodes a mouse ADAM6 protein, wherein the inserted nucleotide sequence is present at the immunoglobulin heavy chain locus of the mouse, and wherein the mouse ADAM6 protein is expressed from the inserted nucleotide sequence;
(ii) generates antibodies when immunized with the antigen, wherein the antibodies each comprise a human heavy chain variable domain operably linked to a mouse heavy chain constant domain and a human λ light chain variable domain operably linked to a light chain constant domain, and
(iii) is fertile; and
(b) identifying a human heavy chain variable domain or a human λ light chain variable domain of an antibody that specifically binds the antigen and that was generated by the genetically modified mouse.
2. The method of claim 1 , wherein identifying the human heavy chain variable domain or the human λ light chain variable domain comprises identifying a nucleotide sequence that encodes the human heavy chain variable domain or the human λ light chain variable domain, respectively.
3. The method of claim 1 , wherein the genome of the mouse comprises endogenous V L gene segments and/or endogenous J L gene segments that are incapable of rearranging to form an immunoglobulin light chain in the mouse.
4. The method of claim 1 , wherein the one or more human Vλ gene segments comprises at least 12 human Vλ gene segments.
5. The method of claim 1 , wherein the one or more human Vλ gene segments comprises at least 28 human Vλ gene segments.
6. The method of claim 1 , wherein the one or more human Vλ gene segments comprises at least 40 human Vλ gene segments.
7. The method of claim 1 , wherein the inserted nucleotide sequence that encodes a mouse ADAM6 protein is positioned between two human V H gene segments.
8. The method of claim 1 , wherein the inserted nucleotide sequence that encodes a mouse ADAM6 protein is positioned between a V H gene segment and a D H gene segment.
9. The method of claim 1 , wherein the one or more human Jλ, gene segments are selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and a combination thereof.
10. The method of claim 1 , wherein the one or more human Jλ, gene segments comprises at least four human Jλ, gene segments.
11. The method of claim 10 , wherein the at least four human Jλ, gene segments comprise at least Jλ1, Jλ2, Jλ3, and Jλ7.
12. The method of claim 1 , wherein the light chain constant region gene is a Cκ gene.
13. The method of claim 12 , wherein the Cκ gene is an endogenous Cκ gene at an endogenous immunoglobulin κ light chain locus.
14. The method of claim 13 , wherein all endogenous Vκ gene segments and endogenous Jκ gene segments are replaced with the one or more human Vλ gene segments and one or more human Jλ gene segments.
15. 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 Vλ, gene segments and one or more human Jλ gene segments.
16. The method of claim 15 , wherein the human Vκ-Jκ intergenic region is placed between a human Vλ gene segment and a human Jλ gene segment.
17. The method of claim 1 , wherein the mouse ADAM6 protein is a mouse ADAM6a or ADAM6b protein.
18. The method of claim 1 , wherein the one or more human Vλ gene segments comprise Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12 or a combination thereof.
19. The method of claim 1 , wherein the one or more human Vλ gene segments comprise human Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27 or a combination thereof.
20. The method of claim 1 , wherein the one or more human Vλ gene segments comprise human Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52 or a combination thereof.
21. The method of claim 1 , wherein at least two λ light chain enhancers are located downstream of both the one or more human Vλ gene segments and the one or more human Jλ gene segments.