Mice That Produce Antigen-Binding Proteins With pH-Dependent Binding Characteristics

Abstract

Genetically modified non-human animals are provided that comprise an immunoglobulin heavy chain locus comprising an unrearranged human heavy chain variable region nucleotide sequence comprising an addition of at least one histidine codon or a substitution of at least one endogenous non-histidine codon with a histidine codon. Compositions and methods for making the genetically modified non-human animals as described herein are provided. Non-human animals capable of expressing an antigen-binding protein characterized by pH-dependent antigen binding, enhanced recyclability and/or enhanced serum half-life are also provided.

What is claimed is: 
     
         1 . A method of producing a nucleic acid encoding a human immunoglobulin heavy chain variable domain with at least one histidine, comprising:
 obtaining from a lymphocyte of a non-human animal, or a hybridoma produced from the lymphocyte, a nucleic acid comprising a rearranged human immunoglobulin heavy chain variable region gene sequence that encodes a human immunoglobulin heavy chain variable domain,   wherein the non-human animal comprises in its germline genome an unrearranged human immunoglobulin heavy chain variable region nucleotide sequence that comprises an addition of at least one histidine codon or a substitution of at least one non-histidine codon with a histidine codon,   wherein the added or substituted histidine codon is not encoded by a corresponding human germline heavy chain variable region gene segment, and   wherein the added or substituted histidine codon is present in a complementary determining region 3 (CDR3) encoding sequence.   
     
     
         2 . The method of  claim 1 , further comprising immunizing the non-human animal with an antigen of interest, and allowing the animal to mount an immune response to the antigen before obtaining the nucleic acid. 
     
     
         3 . The method of  claim 2 , wherein the human immunoglobulin heavy chain variable domain specifically binds the antigen of interest. 
     
     
         4 . The method of  claim 3 , wherein the obtained rearranged human immunoglobulin heavy chain variable region gene sequence comprises at least one somatic hypermutation. 
     
     
         5 . The method of  claim 1 , wherein the unrearranged human immunoglobulin heavy chain variable region nucleotide sequence is operably linked to an endogenous non-human immunoglobulin heavy chain constant region gene sequence at an endogenous non-human immunoglobulin heavy chain locus. 
     
     
         6 . The method of  claim 1 , wherein the non-human animal further comprises in its germline genome an unrearranged human immunoglobulin light chain variable region nucleotide sequence comprising unrearranged human V L  and unrearranged human J L  gene segments; and
 wherein the lymphocyte, or hybridoma produced therefrom, expresses a human immunoglobulin light chain variable domain that is cognate to the human immunoglobulin heavy chain variable domain.   
     
     
         7 . The method of  claim 1 , wherein the unrearranged human immunoglobulin heavy chain variable region nucleotide sequence further comprises an addition of at least one histidine codon or a substitution of at least one non-histidine codon with a histidine codon in a CDR1 encoding sequence, a CDR2 encoding sequence, an N terminal encoding sequence or a loop encoding sequence. 
     
     
         8 . The method of  claim 1 , wherein the CDR3 encoding sequence is selected from a human germline V H  gene segment sequence, a human germline D gene segment sequence, a human germline J H  gene segment sequence, and a combination thereof. 
     
     
         9 . The method of  claim 1 , wherein the non-histidine codon that is substituted with the histidine codon encodes the amino acid selected from the group consisting of Y, N, D, Q, S, W, and R. 
     
     
         10 . The method of  claim 1 , wherein the added or substituted histidine codon is present in at least one reading frame of a human D gene segment. 
     
     
         11 . The method of  claim 10 , wherein the reading frame is a hydrophilic frame of the human D gene segment, and the hydrophilic frame comprises a nucleotide sequence that encodes the amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, and a combination thereof. 
     
     
         12 . The method of  claim 1 , wherein the non-human animal is a rodent selected from a rat, a mouse and a hamster. 
     
     
         13 . The method of  claim 1 , wherein the lymphocyte is a B cell. 
     
     
         14 . A nucleic acid comprising the rearranged human immunoglobulin heavy chain variable region gene sequence produced by the method of  claim 1 . 
     
     
         15 . The nucleic acid of  claim 14 , wherein the nucleic acid further comprises a human constant region gene sequence operably linked to the rearranged human immunoglobulin heavy chain variable region gene sequence. 
     
     
         16 . The nucleic acid of  claim 15 , wherein the human heavy chain constant region gene sequence comprises a modification that increases an affinity of a C H 2-C H 3 region of an IgG heavy chain constant region amino acid sequence to neonatal Fc receptor (FcRn) at a pH ranging from about 5.5 to about 6.0, wherein the modification is a mutation in the IgG heavy chain constant region amino acid sequence selected from the group consisting of M428L, N434S, V259I, V308F, N434A, M252Y, S254T, T256E, T250Q, H433K, N434Y, and a combination thereof. 
     
     
         17 . A cell comprising the nucleic acid of  claim 14 . 
     
     
         18 . A method of obtaining a cell that expresses a human immunoglobulin heavy chain variable domain with at least one histidine comprising:
 isolating a lymphocyte from a non-human animal that comprises in its germline genome an unrearranged human immunoglobulin heavy chain variable region nucleotide sequence that comprises an addition of at least one histidine codon or a substitution of at least one non-histidine codon with a histidine codon,   wherein the added or substituted histidine codon is not encoded by a corresponding human germline heavy chain variable region gene segment, and   wherein the added or substituted histidine codon is present in a complementary determining region 3 (CDR3) encoding sequence.   
     
     
         19 . The method of  claim 18 , further comprising producing a hybridoma from the isolated lymphocyte, wherein the hybridoma expresses a human immunoglobulin heavy chain variable domain with at least one histidine in the CDR3. 
     
     
         20 . The method of  claim 18 , wherein the non-human animal further comprises in its germline genome an unrearranged human immunoglobulin light chain variable region nucleotide sequence comprising unrearranged human V L  and unrearranged human J L  gene segments; and
 wherein the lymphocyte expresses a human immunoglobulin light chain variable domain that is cognate to the human immunoglobulin heavy chain variable domain.   
     
     
         21 . The method of  claim 18 , wherein the unrearranged human immunoglobulin heavy chain variable region nucleotide sequence further comprises an addition of at least one histidine codon or a substitution of at least one non-histidine codon with a histidine codon in a CDR1 encoding sequence, a CDR2 encoding sequence, an N terminal encoding sequence or a loop encoding sequence 
     
     
         22 . The method of  claim 18 , wherein the CDR3 encoding sequence is selected from a human germline V H  gene segment sequence, a human germline D gene segment sequence, a human germline J H  gene segment sequence, and a combination thereof. 
     
     
         23 . A cell obtained according to the method of  claim 18 . 
     
     
         24 . An in vitro method of making a human immunoglobulin heavy chain variable domain comprising:
 expressing in a cell the nucleic acid of  claim 14 .   
     
     
         25 . The method of  claim 24 , wherein the nucleic acid further comprises a human immunoglobulin heavy chain constant region gene sequence operably linked to the rearranged human immunoglobulin heavy chain variable region gene sequence. 
     
     
         26 . The method of  claim 25 , wherein the human immunoglobulin heavy chain constant region gene sequence comprises a modification that increases an affinity of a C H 2-C H 3 region of an IgG heavy chain constant region amino acid sequence to neonatal Fc receptor (FcRn) at a pH ranging from about 5.5 to about 6.0, wherein the modification is a mutation in the IgG heavy chain constant region amino acid sequence selected from the group consisting of M428L, N434S, V259I, V308F, N434A, M252Y, S254T, T256E, T250Q, H433K, N434Y, and a combination thereof. 
     
     
         27 . The method of  claim 24 , further comprising co-expressing in the cell a nucleotide sequence encoding a human immunoglobulin light chain variable domain. 
     
     
         28 . A human immunoglobulin heavy chain variable domain made according to the method of  claim 24 . 
     
     
         29 . A genetically modified immunoglobulin heavy chain locus in a germline of a non-human animal comprising an unrearranged human heavy chain variable region nucleotide sequence, wherein the unrearranged human heavy chain variable region nucleotide sequence comprises an addition of at least one histidine codon or a substitution of at least one endogenous non-histidine codon with a histidine codon.