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 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.   
     
     
         2 . The method of  claim 1 , wherein the recombinant immunoglobulin light chain nucleic acid sequence comprises no more than two unrearranged human V L  gene segments. 
     
     
         3 . The method of  claim 2 , wherein the two unrearranged human V L  gene segments are human Vκ1-39 and Vκ3-20 gene segments. 
     
     
         4 . The method of  claim 1 , wherein the second antibody comprises a fully human heavy chain and a fully human light chain. 
     
     
         5 . The method of  claim 1 , wherein the immunoglobulin light chain nucleotide sequence further comprises at least one codon that encodes for a somatic mutation not encoded by the germline nucleic acid sequence. 
     
     
         6 . The method of  claim 5 , wherein the second antibody comprises a fully human first immunoglobulin heavy chain and a fully human immunoglobulin light chain. 
     
     
         7 . A binding protein that exhibits pH-dependent binding to an antigen of interest generated according to a method of  claim 1 . 
     
     
         8 . 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 first heavy chain variable domain of a first antibody, wherein the first antibody binds the first antigen of interest with a desired affinity at a neutral pH, 
 (ii) a second immunoglobulin heavy chain comprising a second heavy chain variable domain of a second antibody, wherein the second antibody binds the second antigen of interest with a desired affinity at a neutral pH,
 wherein the first and second antibodies are 
 (a) 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 
 (b) comprise the same 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 
 
 (iii) 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 and second antibodies and 
 (b) modified to comprise a substitution of at least one non histidine codon with a histidine codon, and 
 
   selecting a third 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.   
     
     
         9 . The method of  claim 8 , wherein the recombinant immunoglobulin light chain nucleic acid sequence comprises no more than two unrearranged human V L  gene segments. 
     
     
         10 . The method of  claim 9 , wherein the two unrearranged human V L  gene segments are human Vκ1-39 and Vκ3-20 gene segments. 
     
     
         11 . The method of  claim 8 , wherein the third antibody comprises a fully human heavy chain and a fully human light chain. 
     
     
         12 . The method of  claim 8 , wherein the immunoglobulin light chain nucleotide sequence further comprises at least one codon that encodes for a somatic mutation not encoded by the germline nucleic acid sequence. 
     
     
         13 . The method of  claim 12 , wherein the third antibody comprises a fully human first immunoglobulin heavy chain and a fully human immunoglobulin light chain. 
     
     
         14 . A binding protein that exhibits pH-dependent binding to an antigen of interest generated according to a method of  claim 8 .