Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same

Abstract

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.

What is claimed is: 
     
         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.