ANTIBODY PRODUCING NON-HUMAN ANIMALS
Described are transgenic, non-human animals comprising a nucleic acid encoding an immunoglobulin light chain, whereby the immunoglobulin light chain is human, human-like, or humanized. The nucleic acid is provided with a means that renders it resistant to DNA rearrangements and/or somatic hypermutations. In one embodiment, the nucleic acid comprises an expression cassette for the expression of a desired molecule in cells during a certain stage of development in cells developing into mature B cells. Further provided is methods for producing an immunoglobulin from the transgenic, non-human animal.
1 . A method to obtain an antibody or fragment thereof that binds to an antigen, the method comprising:
obtaining a population of B cells producing antigen specific antibodies from a non-human transgenic animal that has been immunized with said antigen, wherein said transgenic animal comprises a transgene encoding a rearranged human immunoglobulin variable region and wherein the transgene comprises an immunoglobulin constant region or said rearranged human immunoglobulin variable region is operatively linked to an endogenous immunoglobulin constant region, and wherein the transgenic animal produces a repertoire of antibodies that bind said antigen having the rearranged human immunoglobulin variable region encoded by said transgene and a counterpart immunoglobulin variable region produced by somatic recombination;
isolating nucleic acid comprising a nucleotide sequence encoding at least said counterpart human immunoglobulin variable region from said population of B cells producing antigen specific antibodies;
expressing a nucleotide sequence that encodes said counterpart immunoglobulin variable region and a nucleotide sequence encoding said rearranged human immunoglobulin variable region in a host cell to produce said antibody or fragment; and
recovering the antibody or fragment that binds to the antigen.
2 . The method of claim 1 wherein the transgene encoding a rearranged human immunoglobulin variable region comprises a human germline light chain V gene segment joined to a human germline light chain J gene segment, wherein there is no mutation due to said joining and wherein said immunoglobulin constant region is a light chain constant region,
so that said transgenic animal, in response to said antigen, has produced B cells that secrete antibodies with immunoglobulin light chains comprising said rearranged human light chain variable region and light chain constant region, paired with a diversity of immunoglobulin heavy chains, which antibodies bind said antigen.
3 . The method of claim 1 wherein the transgene encoding a rearranged human immunoglobulin variable region comprises a human germline heavy chain V gene segment joined to a human germline heavy chain D gene segment joined to a human heavy chain J gene segment, wherein there is no mutation due to said joining and wherein said immunoglobulin constant region is a heavy chain constant region,
so that said transgenic animal, in response to said antigen, has produced B cells that secrete antibodies with immunoglobulin heavy chains comprising said rearranged human heavy chain variable region and heavy chain constant region paired with a diversity of immunoglobulin light chains, which antibodies bind said antigen.
4 . A method to obtain an antibody or fragment thereof that binds to an antigen, the method comprising
obtaining a population of B cells producing antigen specific antibodies from a non-human transgenic animal that has been immunized with said antigen, wherein the genome of said transgenic animal comprises a transgene encoding a rearranged human immunoglobulin variable region and wherein the transgene comprises an immunoglobulin constant region or said rearranged human immunoglobulin variable region is operatively linked to an endogenous immunoglobulin constant region, and wherein the transgenic animal produces a repertoire of antibodies that bind said antigen having a rearranged human immunoglobulin variable region encoded by said transgene;
culturing said B cells and allowing secretion of said antibodies; and
recovering the secreted antibodies;
optionally cleaving said antibodies to obtain antigen-binding fragments; thereby obtaining said antibody or fragment that binds to the antigen.
5 . The method of claim 4 wherein the transgene encoding a rearranged human immunoglobulin variable region comprises a human germline light chain V gene segment joined to a human germline light chain J gene segment, wherein there is no mutation due to said joining and wherein said immunoglobulin constant region is a light chain constant region,
so that said transgenic animal, in response to said antigen, has produced B cells that secrete antibodies with immunoglobulin light chains comprising said rearranged human light chain variable region and light chain constant region, paired with a diversity of immunoglobulin heavy chains, which antibodies bind said antigen.
6 . The method of claim 4 wherein the transgene encoding a rearranged human immunoglobulin variable region comprises a human germline heavy chain V gene segment joined to a human germline heavy chain D gene segment joined to a human heavy chain J gene segment, wherein there is no mutation due to said joining and wherein said immunoglobulin constant region is a heavy chain constant region,
so that said transgenic animal, in response to said antigen, has produced B cells that secrete antibodies with immunoglobulin heavy chains comprising said rearranged human heavy chain variable region and heavy chain constant region paired with a diversity of immunoglobulin light chains, which antibodies bind said antigen.
7 . A method to obtain an antibody or fragment thereof that binds to an antigen, the method comprising:
isolating nucleic acid from a B cell from a transgenic non-human animal that has been immunized with the antigen, wherein the genome of the transgenic animal comprises a transgene encoding a rearranged human immunoglobulin variable region and wherein the transgene comprises an immunoglobulin constant region or said rearranged human immunoglobulin variable region is operatively linked to an endogenous immunoglobulin constant region, and wherein the transgenic animal produces a repertoire of antibodies that bind said antigen having the rearranged human immunoglobulin variable region encoded by said transgene and a counterpart immunoglobulin variable region produced by somatic recombination wherein said nucleic acid comprises a nucleotide sequence encoding said counterpart immunoglobulin variable region;
expressing a nucleotide sequence that encodes said counterpart immunoglobulin variable region in a host cell together with a nucleotide sequence encoding said immunoglobulin variable region encoded by the transgene so as to effect production of said antibody or fragment; and
isolating the antibody or fragment produced by said host cell that binds to the antigen.
8 . The method of claim 7 wherein the transgene encoding a rearranged human immunoglobulin variable region comprises a human germline light chain V gene segment joined to a human germline light chain J gene segment, wherein there is no mutation due to said joining and wherein said immunoglobulin constant region is a light chain constant region,
so that said transgenic animal, in response to said antigen, has produced B cells that secrete antibodies with immunoglobulin light chains comprising said rearranged human light chain variable region and light chain constant region, paired with a diversity of immunoglobulin heavy chains, which antibodies bind said antigen.
9 . The method of claim 7 wherein the transgene encoding a rearranged human immunoglobulin variable region comprises a human germline heavy chain V gene segment joined to a human germline heavy chain D gene segment joined to a human heavy chain J gene segment, wherein there is no mutation due to said joining and wherein said immunoglobulin constant region is a heavy chain constant region,
so that said transgenic animal, in response to said antigen, has produced B cells that secrete antibodies with immunoglobulin heavy chains comprising said rearranged human heavy chain variable region and heavy chain constant region paired with a diversity of immunoglobulin light chains, which antibodies bind said antigen.