Humanized universal light chain mice

Patent metadata

Field	Value
Publication number	US-10130081-B2
Application number	US-201213566765-A
Country	US
Kind code	B2
Filing date	Aug 3, 2012
Priority date	Aug 5, 2011
Publication date	Nov 20, 2018
Grant date	Nov 20, 2018

Abstract

Mice, tissues, cells, and genetic material are provided that comprise a humanized heavy chain immunoglobulin locus, a humanized light chain locus that expresses a universal light chain, and a gene encoding an ADAM6 or ortholog or homolog or functional fragment thereof. Mice are provided that express humanized heavy chains comprising human variable domains, and that express humanized light chains comprising human variable domains wherein the light chains are derived from no more than one, or no more than two, light chain V and J or rearranged V/J sequences. Fertile male mice that express antibodies with universal light chains and humanized heavy chains are provided. Methods and compositions for making bispecific binding proteins are provided.

First claim

We claim:

1. A male mouse whose germline genome comprises:
(a) an insertion comprising at least one unrearranged human V H gene segment, at least one unrearranged human D H gene segment, and at least one unrearranged human J H gene segment, wherein the at least one unrearranged human V H gene segment, at least one human D H gene segment, and at least one unrearranged human J H gene segment are operably linked to a heavy chain constant region gene, wherein the insertion disrupts the function of an endogenous ADAM6 protein, and wherein the disruption of the endogenous ADAM6 function is associated with a reduction in fertility in male mice;
(b) an insertion comprising no more than one, or no more than two, rearranged human light chain V/J sequences, wherein the no more than one, or no more than two, rearranged human light chain V/J sequences are operably linked to a light chain constant region gene; and,
(c) an insertion comprising an ectopic nucleic acid sequence that encodes a functional mouse ADAM6 protein, wherein the functional ADAM6 protein is expressed and the male mouse has wild-type fertility.

2. The mouse according to claim 1 , wherein the ectopic nucleic acid sequence is at a locus other than the immunoglobulin heavy chain variable locus.

3. The mouse according to claim 1 , wherein the heavy chain constant region gene is a mouse gene.

4. The mouse according to claim 1 , wherein the light chain constant region gene is a mouse gene.

5. A mouse cell obtained from the mouse of claim 1 .

6. The mouse cell of claim 5 , wherein the heavy chain constant gene is a non-human heavy chain constant gene.

7. The mouse cell of claim 5 , wherein the light chain constant gene is a non-human light chain constant gene.

8. The mouse cell of claim 5 , wherein the cell is a B cell that expresses:
a chimeric immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable domain that is derived from a rearrangement of one of the at least one unrearranged human V H gene segment, one of the at least one unrearranged human D H gene segment, and one of the at least one unrearranged human J H gene segment, wherein the immunoglobulin heavy chain variable domain is operably linked to a non-human heavy chain constant region; and
a chimeric immunoglobulin light chain comprising an immunoglobulin light chain variable domain that is expressed from one of the no more than one, or no more than two, rearranged human light chain V/J sequences, or a somatically hypermutated variant thereof, and

wherein the immunoglobulin light chain variable domain is fused operably linked to a non-human light chain constant region.

9. The mouse cell of claim 5 , wherein the cell is an embryonic stem (ES) cell.

10. The mouse cell of claim 5 , wherein the immunoglobulin light chain variable domain is expressed from:
(a) a rearranged human Vκ1-39/Jκ sequence, or
(b) a rearranged human Vκ3-20/Jκ sequence.

11. The mouse cell of claim 10 , wherein the rearranged human Vκ1-39/Jκ sequence is a rearranged human Vκ1-39/Jκ5 sequence.

12. The mouse cell of claim 10 , wherein the rearranged human Vκ3-20/Jκ sequence is a rearranged human Vκ3-20/Jκ1 sequence.

13. The mouse of claim 1 , wherein the no more than one, or no more than two, rearranged human light chain V/J sequences are:
(a) a rearranged human Vκ1-39/Jκ sequence, and/or
(b) a rearranged human Vκ3-20/Jκ sequence.

14. The mouse of claim 13 , wherein the rearranged human Vκ1-39/Jκ sequence is a rearranged human Vκ1-39/1κ5 sequence.

15. The mouse of claim 13 , wherein the rearranged human Vκ3-20/Jκ sequence is a rearranged human Vκ3-20/Jκ1 sequence.

16. The mouse of claim 1 , wherein the mouse lacks an endogenous mouse κ immunoglobulin light chain variable region locus that is capable of rearranging and forming a gene that encodes a mouse κ variable region.

17. The mouse of claim 1 , wherein the mouse lacks an endogenous mouse λ immunoglobulin light chain variable region locus that is capable of rearranging and forming a gene that encodes a mouse λ variable region.

18. The mouse of claim 1 , wherein the at least one unrearranged human V H gene segment comprises a gene segment selected from the group consisting of V H 1-2, V H 1-8, V H 1-24, V H 1-69, V H 2-5, V H 3-7, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-20, V H 3-23, V H 3-30, V H 3-33, V H 3-43, V H 3-48, V H 4-31, V H 4-39, V H 4-59, V H 5-51 and V H 6-1.

19. The mouse of claim 1 , wherein the insertion of (b) comprises a single rearranged human light chain V/J sequence, and wherein the single rearranged human light chain V/J sequence is operably linked to a light chain constant region gene.

Assignees

• Mcwhirter John
• Macdonald Lynn
• Stevens Sean
• Murphy Andrew J
• Karow Margaret
• Regeneron Pharma

Inventors

• Mcwhirter John
• Macdonald Lynn
• Stevens Sean
• Murphy Andrew J
• Karow Margaret

Classifications

• C12N9/6489 Primary

  Metalloendopeptidases (3.4.24) · CPC title

• A01K67/0278 Primary

  Knock-in vertebrates, e.g. humanised vertebrates · CPC title

• C07K2317/21

  from primates, e.g. man · CPC title

• A01K2217/072

  maintaining or altering function, i.e. knock in · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title