ADAM6 mice
Mice are provided that comprise a reduction or deletion of ADAM6 activity from an endogenous ADAM6 locus, or that lack an endogenous locus encoding a mouse ADAM6 protein, wherein the mice comprise a sequence encoding an ADAM6 or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the sequence is an ectopic ADAM6 sequence or a sequence that confers upon a male mouse the ability to generate offspring by mating. Mice and cells with genetically modified immunoglobulin heavy chain loci that comprise an ectopic nucleotide sequence encoding a mouse ADAM6 or functional fragment or homolog or ortholog thereof are also provided.
1. A method for generating a fully human antibody specific against an antigen comprising the steps of:
(a) immunizing a mouse with the antigen, wherein the mouse has undergone immunoglobulin gene sequence rearrangement so that it comprises a B cell that comprises:
(i) a rearranged human heavy chain variable region sequence operably linked to a mouse heavy chain constant region sequence, the rearranged human heavy chain variable region sequence comprising a human V H gene segment, a human D H gene segment, and a human J H gene segment; and
(ii) a rearranged human light chain variable region sequence operably linked to a light chain constant region sequence, the rearranged human light chain variable region sequence comprising a human V L gene segment and a human J L gene segment; wherein,
the B cell further comprises in its genome an inserted nucleic acid sequence encoding a mouse ADAM6 protein that is functional in a male mouse;
(b) isolating at least one splenocyte or B cell from the mouse producing an antibody specific against the antigen;
(c) generating at least one cell producing a fully human antibody, which includes a human variable domain encoded by a human variable region sequence found in the at least one splenocyte or B cell of step (b), wherein the fully human antibody is specific against the antigen;
(d) culturing the at least one cell producing the fully human antibody of step (c); and
(e) obtaining the fully human antibody of step (d).
2. A method for generating a fully human antibody specific against an antigen comprising the steps of:
(a) expressing in a mammalian cell said fully human antibody comprising two human light chains and two human heavy chains, wherein each human light chain includes a human light chain domain encoded by a human light chain variable region sequence and each human heavy chain includes a human heavy chain variable domain encoded by a human heavy chain variable region sequence, wherein one or more human variable region sequences were derived by:
(i) immunizing a mouse with the antigen, wherein the mouse has undergone immunoglobulin gene sequence rearrangement so that it comprises a B cell that comprises:
(A) a rearranged human heavy chain variable region sequence operably linked to a mouse heavy chain constant region sequence, the rearranged human heavy chain variable region sequence comprising a human V H gene segment, a human D H gene segment, and a human J H gene segment; and
(B) a rearranged human light chain variable region sequence operably linked to a light chain constant region sequence, the rearranged human light chain variable region sequence comprising a V L gene segment and a human J L gene segment;
wherein the B cell further comprises in its genome an inserted nucleic acid sequence encoding a mouse ADAM6 protein that is functional in a male mouse;
(ii) isolating at least one splenocyte or B cell producing an antibody specific against the antigen from the mouse; and
(iii) determining one or more human variable region sequences of the antibody produced from the at least one splenocyte or B cell;
(b) culturing the mammalian cell so that the fully human antibody is produced; and
(c) obtaining said fully human antibody.
3. The method of claim 1 , wherein the fully human antibody is a monoclonal antibody.
4. The method of claim 1 , wherein immunizing with the antigen of step (a) is carried out with protein, DNA, a combination of DNA and protein, or cells expressing the antigen.
5. The method of claim 1 , wherein the inserted nucleic acid sequence encoding a mouse ADAM6 protein is on a transgene driven by a heterologous promoter.
6. The method of claim 5 , wherein the heterologous promoter is a non-immunoglobulin promoter.
7. The method of claim 1 , wherein the mouse heavy chain constant region sequence comprises a mouse heavy chain sequence selected from the group consisting of a C H 1, a hinge, a C H 2, a C H 3, and a combination thereof.
8. The method of claim 1 , wherein a majority of the B cells of the mouse comprise the inserted nucleic acid sequence encoding a mouse ADAM6 protein.
9. The method of claim 1 , wherein at least 90% of the B cells of the mouse comprise in their genome the inserted nucleic acid sequence encoding a mouse ADAM6 protein.
10. The method of claim 1 , wherein the culturing in step (c) is performed on at least one hybridoma cell generated from the at least one cell obtained in step (b).
11. The method of claim 1 , wherein an inserted nucleic acid sequence encoding a mouse ADAM6 protein
(A) is present on the same chromosome as the rearranged immunoglobulin sequence; and/or
(B) is present at an ectopic position.
12. The method of claim 2 , wherein the fully human antibody is a monoclonal antibody.
13. The method of claim 2 , wherein immunizing with the antigen of step (i) is carried out with protein, DNA, a combination of DNA and protein, or cells expressing the antigen.
14. The method of claim 2 , wherein the inserted nucleic acid sequence encoding a mouse ADAM6 protein is on a transgene driven by a heterologous promoter.
15. The method of claim 14 , wherein the heterologous promoter is a non-immunoglobulin promoter.
16. The method of claim 2 , wherein the mouse heavy chain constant region sequence comprises a mouse heavy chain sequence selected from the group consisting of a C H 1, a hinge, a C H 2, a C H 3, and a combination thereof.
17. The method of claim 2 , wherein a majority of the B cells of the mouse comprise the inserted nucleic acid sequence encoding a mouse ADAM6 protein.
18. The method of claim 2 , wherein at least 90% of the B cells of the mouse comprise in their genome the inserted nucleic acid sequence encoding a mouse ADAM6 protein.
19. The method of claim 2 , wherein an inserted nucleic acid sequence encoding a mouse ADAM6 protein
(a) is present on the same chromosome as the rearranged immunoglobulin sequence; and/or
(b) is present at an ectopic position.
20. The method of claim 1 , wherein the V L gene segment is Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-16, Vκ1-17, Vκ1-27, Vκ1-33, Vκ1-37, Vκ1-39, Vκ2-24, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-40, Vκ3-11, Vκ3-15, Vκ3-20, Vκ4-1, or Vκ6-21.
21. The method of claim 2 , wherein the V L gene segment is Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-16, Vκ1-17, Vκ1-27, Vκ1-33, Vκ1-37, Vκ1-39, Vκ2-24, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-40, Vκ3-11, Vκ3-15, Vκ3-20, Vκ4-1, or Vκ6-21.
22. The method of claim 1 , wherein the V H gene segment is V H 6-1, V H 1-2, V H 1-3, V H 2-5, V H 3-7, V H 1-8, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-16, V H 1-18, V H 3-20, V H 3-21, V H 3-23, V H 1-24, V H 2-26, V H 4-28, V H 3-30, V H 4-31, V H 3-33, V H 4-34, V H 3-35, V H 3-38, V H 4-39, V H 3-43, V H 1-45, V H 1-46, V H 3-48, V H 3-49, V H 5-51, V H 3-53, V H 1-58, V H 4-59, V H 4-61, V H 3-64, V H 3-66, V H 1-69, V H 2-70, V H 3-72, V H 3-73 or V H 3-74.
23. The method of claim 2 , wherein the V H gene segment is V H 6-1, V H 1-2, V H 1-3, V H 2-5, V H 3-7, V H 1-8, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-16, V H 1-18, V H 3-20, V H 3-21, V H 3-23, V H 1-24, V H 2-26, V H 4-28, V H 3-30, V H 4-31, V H 3-33, V H 4-34, V H 3-35, V H 3-38, V H 4-39, V H 3-43, V H 1-45, V H 1-46, V H 3-48, V H 3-49, V H 5-51, V H 3-53, V H 1-58, V H 4-59, V H 4-61, V H 3-64, V H 3-66, V H 1-69, V H 2-70, V H 3-72, V H 3-73 or V H 3-74.