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 . The animal of claim 1 , wherein the animal does not comprise an endogenous V L gene segment that is capable of rearranging to form an immunoglobulin light chain.
3 . The animal of claim 1 , wherein the immunoglobulin light chain constant region sequence is a non-human immunoglobulin light chain constant region sequence.
4 . The animal of claim 3 , wherein the non-human immunoglobulin light chain constant region sequence is a mouse or a rat sequence.
5 . The animal of claim 3 , wherein the non-human immunoglobulin light chain constant region sequence is an endogenous immunoglobulin light chain constant region sequence.
6 . The animal of claim 1 , further comprising in its germline an immunoglobulin heavy chain locus that comprises an unrearranged immunoglobulin heavy chain variable region sequence comprising human V H , D H , and J H gene segments operably linked to an immunoglobulin heavy chain constant region sequence.
7 . The animal of claim 6 , wherein the immunoglobulin heavy chain constant region sequence is a non-human immunoglobulin heavy chain constant region sequence.
8 . The animal of claim 7 , wherein the non-human immunoglobulin heavy chain constant region sequence is a mouse or a rat sequence.
9 . The animal of claim 7 , wherein the non-human immunoglobulin heavy chain constant region sequence is an endogenous non-human immunoglobulin heavy chain constant region sequence.
10 . The animal of claim 1 , wherein the at least one human V L gene segment and at least one human J L gene segment are present at the endogenous immunoglobulin light chain locus.
11 . The animal of claim 1 , wherein the immunoglobulin light chain constant region is a Cκ region.
12 . The animal of claim 1 , wherein each human V L gene segment comprises a substitution of at least one non-histidine codon encoded by a corresponding human germline V L gene segment sequence with the histidine codon.
13 . The animal of claim 12 , wherein the substitution is in the CDR3 codon(s).
14 . The animal of claim 13 , wherein the substitution is of three or four non-histidine codons with the histidine codon.
15 . The animal of claim 1 , wherein the at least one human V L gene segment is two human V L gene segments.
16 . The animal of claim 15 , wherein two human V L gene segments are human Vκ1-39 and Vκ3-20 gene segments.
17 . The animal of claim 1 , wherein the animal is a rodent.
18 . The rodent of claim 17 , wherein the rodent is a rat or a mouse.
19 . The rodent of claim 18 , wherein the rodent is a mouse.
20 . The animal of claim 1 , wherein the animal expresses an antibody comprising an amino acid sequence encoded by the at least one human V L gene segment and the antibody retains at least one histidine residue at an amino acid position encoded by the at least one histidine codon of the human V L gene segment.
21 . The animal of claim 1 , wherein the animal comprises a population of B cells in response to an antigen of interest that is enriched for antibodies that exhibit a decrease in dissociative half-life (t 1/2 ) at an acidic pH as compared to neutral pH of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 30-fold.
22 . The animal of claim 21 , wherein the enrichment in antibodies that exhibit a decrease in t 1/2 is at least about 2 fold.
23 . A genetically modified non-human animal 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 . The animal of claim 23 , wherein the animal does not comprise an endogenous V L gene segment that is capable of rearranging to form an immunoglobulin light chain.
25 . The animal of claim 23 , wherein the immunoglobulin light chain constant region sequence is a non-human immunoglobulin light chain constant region sequence.
26 . The animal of claim 25 , wherein the non-human immunoglobulin light chain constant region sequence is a mouse or a rat sequence.
27 . The animal of claim 25 , wherein the non-human immunoglobulin light chain constant region sequence is an endogenous immunoglobulin light chain constant region sequence.
28 . The animal of claim 23 , further comprising in its germline an immunoglobulin heavy chain locus that comprises an unrearranged immunoglobulin heavy chain variable region sequence comprising human V H , D H , and J H gene segments operably linked to an immunoglobulin heavy chain constant region sequence.
29 . The animal of claim 28 , wherein the immunoglobulin heavy chain constant region sequence is a non-human immunoglobulin heavy chain constant region sequence.
30 . The animal of claim 29 , wherein the non-human immunoglobulin heavy chain constant region sequence is a mouse or a rat sequence.
31 . The animal of claim 29 , wherein the non-human immunoglobulin heavy chain constant region sequence is an endogenous non-human immunoglobulin heavy chain constant region sequence.
32 . The animal of claim 23 , wherein the no more than two human V L gene segments and the one or more human J L gene segments are present at the endogenous immunoglobulin light chain locus.
33 . The animal of claim 23 , wherein the immunoglobulin light chain constant region is a Cκ region.
34 . The animal of claim 23 , wherein the animal comprises five human Jκ segments, and the five human Jκ segments are human Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5 segments.
35 . The animal of claim 23 , wherein the no more than two human V L gene segments are human Vκ1-39 and Vκ3-20 gene segments.
36 . The animal of claim 23 , wherein each of the no more than two human V L gene segments comprises a substitution of at least one non-histidine codon encoded by a corresponding human germline V L gene segment sequence with the histidine codon.
37 . The animal of claim 36 , wherein the substitution is in the CDR3 codon(s).
38 . The animal of claim 37 , wherein the substitution is of three or four non-histidine codons with the histidine codon.
39 . The animal of claim 35 , wherein each of the human Vκ1-39 and Vκ3-20 gene segments comprises a substitution of at least one non-histidine codon encoded by a corresponding human germline V L gene segment sequence with the histidine codon.
40 . The animal of claim 39 , wherein each of the human Vκ1-39 and Vκ3-20 gene segments comprises a substitution of three or four non-histidine codons with the histidine codons.
41 . The animal of claim 40 , wherein the substitution is of three non-histidine codons of the human Vκ1-39 gene segment, and the substitution is designed to express histidines at positions 106, 108, and 111 of the human Vκ1-39 gene segment.
42 . The animal of claim 40 , wherein the substitution is of four non-histidine codons of the human Vκ1-39 gene segment, and the substitution is designed to express histidines at positions 105, 106, 108, and 111 of the human Vκ1-39 gene segment.
43 . The animal of claim 40 , wherein the substitution is of three non-histidine codons of the human Vκ3-20 gene segment, and the substitution is designed to express histidines at positions 105, 106, and 109 of the human Vκ3-20 gene segment.
44 . The animal of claim 40 , wherein the substitution is of four non-histidine codons of the human Vκ3-20 gene segment, and the substitution is designed to express histidines at positions 105, 106, 107, and 109 of the human Vκ3-20 gene segment.
45 . The animal of claim 23 , wherein the animal is a rodent.
46 . The rodent of claim 45 , wherein the rodent is a rat or a mouse.
47 . The rodent of claim 46 , wherein the rodent is a mouse.
48 . The animal of claim 23 , wherein the animal expresses an antibody comprising an amino acid sequence encoded by one of the no more than two human V L gene segments and the antibody retains at least one histidine residue at an amino acid position encoded by the at least one histidine codon of the human V L gene segment.
49 . A method of generating an antibody that exhibits pH-dependent binding to an antigen of interest comprising:
generating the animal of claim 1 ,
immunizing the animal with an antigen of interest, and
selecting an antibody that binds to the antigen of interest with a desired affinity at a neutral pH while displaying reduced binding to the antigen of interest at an acidic pH.
50 . The animal of claim 23 , wherein the animal expresses a population of antigen-specific antibodies in response to an antigen wherein all antibodies in the population comprise:
immunoglobulin light chain variable domains derived from a rearrangement of the no more than two V L gene segments and the one or more J L gene segments 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
immunoglobulin heavy chains comprising human heavy chain variable domains derived from a repertoire of human heavy V, D, and J segments.
51 . The animal of claim 23 , wherein the animal comprises a population of B cells in response to an antigen of interest that is enriched for antibodies that exhibit a decrease in dissociative half-life (t 1/2 ) at an acidic pH as compared to neutral pH of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 30-fold.
52 . The animal of claim 51 , wherein the enrichment in antibodies that exhibit a decrease in t 1/2 is at least about 2 fold.
53 . A method of making a non-human animal that comprises a genetically modified immunoglobulin light chain locus in its germline, 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 . The method of claim 53 , wherein the method results in the non-human animal that comprises a population of B cells enriched for antibodies exhibiting pH-dependent binding to an antigen of interest.
55 . The method of claim 53 , wherein the immunoglobulin light chain variable region is at the endogenous non-human immunoglobulin light chain locus.
56 . The method of claim 53 , wherein the animal is a rodent.
57 . The method of claim 56 , wherein the rodent is a mouse or a rat.
58 . The method of claim 53 , wherein the at least one human V L gene segment is two human V L gene segments.
59 . The method of claim 58 , wherein the two human V L gene segments are human Vκ1-39 gene segment and Vκ3-20 gene segment.