Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same
A genetically modified non-human animal is provided, wherein the non-human animal expresses an antibody repertoire capable of pH dependent binding to antigens upon immunization. A genetically modified non-human animal is provided that expresses human immunoglobulin light chain variable domains derived from a limited repertoire of human immunoglobulin light chain variable gene segments that comprise histidine modifications in their germline sequence. Methods of making non-human animals that express antibodies comprising histidine residues encoded by histidine codons introduced into immunoglobulin light chain nucleotide sequences are provided.
1 . A genetically modified non-human animal comprising in its germline an immunoglobulin light chain locus comprising at least one human V L gene segment and at least one human J L gene segment operably linked to an immunoglobulin light chain constant region sequence,
wherein each human V L gene segment comprises at least one histidine codon that is not encoded by the corresponding human germline V L gene segment, and
wherein the at least one human V L gene segment and the at least one human J L gene segment are capable of rearranging and encoding a human light chain variable domain of an antibody.
2 .- 22 . (canceled)
23 . The genetically modified non-human animal of claim 1 , comprising in its germline an immunoglobulin light chain locus comprising no more than two human V L gene segments and one or more human J L gene segments operably linked to an immunoglobulin light chain constant region sequence,
wherein each of the no more than two human V L gene segments comprises at least one histidine codon that is not encoded by the corresponding human germline V L gene segment, and
wherein the human V L gene segments and J L gene segments are capable of rearranging and encoding a human light chain variable domain of an antibody.
24 .- 48 . (canceled)
49 . A method of generating an antibody light chain variable domain to an antigen of interest or a nucleic acid sequence encoding same comprising:
immunizing the animal of claim 1 with the antigen of interest,
selecting a B cell that expresses one or more nucleic acids that encodes an antibody that binds to the antigen of interest.
50 .- 52 . (canceled)
53 . A method of the making a non-human animal of claim 1 , the method comprising:
modifying a genome of the non-human animal to delete or render non-functional endogenous immunoglobulin light chain V L and J L gene segments in an immunoglobulin light chain locus, and
placing in the genome of the non-human animal an immunoglobulin light chain variable region comprising at least one human V L gene segment and at least one human J L gene segment, such that the immunoglobulin light chain variable region sequence is operably linked to an immunoglobulin constant region sequence,
wherein each human V L gene segment comprises at least one histidine codon that is not encoded by the corresponding human germline V L gene segment, and
wherein the at least one human V L gene segment and at least one human J L gene segment are capable of rearranging and encoding a human light chain variable domain of an antibody.
54 .- 59 . (canceled)
60 . A B cell selected according to the method of claim 49 .
61 . A nucleic acid expressed by the B cell of claim 60 , wherein the nucleic acid comprises a sequence encoding the heavy chain variable domain of the antibody.
62 . An antibody expressed by the B cell of claim 60 .
63 . A nucleic acid comprising the no more than two human unrearranged immunoglobulin light chain variable (V L ) gene segments and one or more human unrearranged immunoglobulin light chain joining (J L ) gene segments operably linked to an immunoglobulin light chain constant region sequence of claim 23 .
64 . An immunoglobulin light chain locus comprising a human immunoglobulin light chain variable (V L ) gene segment rearranged with an immunoglobulin light chain joining (J L ) gene segment operably linked to a non-human immunoglobulin light chain constant region sequence,
wherein the human V L gene segment comprises a plurality of histidine codons, each of which plurality is not encoded by the corresponding human germline V L gene segment.
65 . A method of generating a binding protein that exhibits pH-dependent binding to an antigen of interest comprising:
co-expressing in a cell:
(i) a first immunoglobulin heavy chain comprising a heavy chain variable domain of a first antibody, wherein the first antibody
(a) binds to the antigen of interest with a desired affinity at a neutral pH,
(b) is generated in a genetically modified non-human animal comprising in its germline a recombinant immunoglobulin light chain nucleic acid sequence comprising at least two unrearranged human V L gene segments and at least one unrearranged human J L gene segment capable of rearranging and encoding a human light chain variable domain of an antibody, and
(c) comprises a light chain component encoded by one of the at least two unrearranged human V L gene segments and the at least one human J L gene segment
(ii) an immunoglobulin light chain comprising a light chain variable domain encoded by an immunoglobulin light chain nucleotide sequence that
(a) is derived from the one of the at least two human V L gene segments and the at least one J L gene segment encoding the light chain component of the first antibody and
(b) modified to comprise a substitution of at least one non histidine codon with a histidine codon, and
selecting a second antibody expressed in the cell that comprises the first immunoglobulin heavy chain and the immunoglobulin light chain, retains the desired affinity for the antigen of interest at neutral pH and displays reduced binding to the antigen of interest at an acidic pH.