Non-human animals that make single domain binding proteins

Abstract

Genetically modified non-human animals and methods and compositions for making and using them are provided, wherein the genetic modification comprises (a) a deletion in an immunoglobulin constant region CH1 gene (optionally a deletion in a hinge region) of a heavy chain constant region gene sequence, and (b) replacement of one or all endogenous VH, DH and JH gene segments with at least one unrearranged light chain variable (VL) gene segment and at least one unrearranged light chain joining (JL) gene segment capable of recombining to form a rearranged light chain variable region (VL/JL) nucleotide sequence operably linked to the heavy chain constant region gene sequence comprising a deletion in the CH1 gene and/or insertion of a genetically engineered single rearranged light chain, wherein the mouse is capable of expressing a functional IgM, single domain antigen binding proteins, e.g., VL-single domain binding proteins, and a genetically engineered rearranged light chain.

1 - 100 . (canceled) 
     
     
         101 . A genetically modified rodent comprising in its germline
 (a) a deletion of a nucleotide sequence encoding a C H 1 domain of at least one endogenous immunoglobulin heavy chain constant region gene at an endogenous immunoglobulin heavy chain locus, wherein the at least one endogenous immunoglobulin heavy chain constant region gene is IgG, IgA, IgE, IgD, or a combination thereof, and   (b) an immunoglobulin light chain locus that comprises a single rearranged immunoglobulin light chain variable region V L /J L  sequence comprising V L  and J L  gene segment sequences, wherein the single rearranged immunoglobulin light chain variable region V L /J L  sequence is operably linked to an immunoglobulin light chain constant region sequence,   wherein the rodent further comprises in its serum an antigen-specific single domain antigen binding protein that lacks a functional C H 1 domain,   wherein the rodent further comprises an IgM heavy chain comprising a functional C H 1 domain, and   wherein the IgM heavy chain is associated with a cognate universal light chain encoded by the single rearranged immunoglobulin light chain variable region V L /J L  sequence, comprising V L  and J L  gene segment sequences, operably linked to an immunoglobulin light chain constant region sequence.   
     
     
         102 . The genetically modified rodent of  claim 101 , wherein the single rearranged immunoglobulin light chain variable region V L /J L  sequence is a human sequence. 
     
     
         103 . The genetically modified rodent of  claim 101 , wherein the immunoglobulin light chain constant region sequence is a rodent immunoglobulin light chain constant region sequence. 
     
     
         104 . The genetically modified rodent of  claim 101 , wherein all functional endogenous light chain variable region gene segments are deleted or functionally inactivated. 
     
     
         105 . The genetically modified rodent of  claim 101 , wherein the single rearranged immunoglobulin light chain variable region V L /J L  sequence is a human Vκ1-39/J sequence or a human Vκ3-20/J sequence. 
     
     
         106 . The genetically modified rodent of  claim 105 , wherein the human Vκ1-39/J sequence comprises a human Vκ1-39 gene segment rearranged with a human Jκ5 gene segment or the human Vκ3-20/J sequence comprises a human Vκ3-20 gene segment rearranged with a human Jκ1 gene segment. 
     
     
         107 . The genetically modified rodent of  claim 101 , wherein the endogenous immunoglobulin heavy chain locus comprises human heavy chain variable region gene segments. 
     
     
         108 . The genetically modified rodent of  claim 107 , wherein the human heavy chain variable region gene segments replace all functional endogenous immunoglobulin heavy chain variable region gene segments. 
     
     
         109 . The genetically modified rodent of  claim 101 , wherein the single rearranged immunoglobulin light chain variable V L /J L  sequence replaces all functional endogenous immunoglobulin light chain variable region gene segments of the rodent. 
     
     
         110 . The genetically modified rodent of  claim 101 , wherein the single rearranged immunoglobulin light chain variable region V L /J L  sequence is human and comprises human germline V L  and human germline J L  gene segment sequences. 
     
     
         111 . The genetically modified rodent of  claim 101 , wherein:
 (a) the at least one endogenous immunoglobulin heavy chain constant region gene further comprises an inactivated hinge region and/or   (b) the deletion of a nucleotide sequence encoding a C H 1 domain is in an IgG1 sequence and wherein the endogenous immunoglobulin heavy chain locus further comprises a deletion or inactivating mutation of an immunoglobulin gene selected from the group consisting of IgD, IgG3, IgG2a, IgG2b, IgG2c, IgE, IgA, and a combination thereof.   
     
     
         112 . The genetically modified rodent of  claim 101 , wherein the rodent further comprises an Adam6 gene or portion thereof functional in a male rodent, wherein the Adam6 gene is an Adam6a gene, an Adam6b gene, or both. 
     
     
         113 . The genetically modified rodent of  claim 101 , wherein the rodent is a rat or a mouse. 
     
     
         114 . A genetically modified rodent that is a mouse comprising
 (a) a replacement at a mouse heavy chain locus of all functional endogenous immunoglobulin heavy chain V, D, and J gene segments with at least one unrearranged human immunoglobulin heavy chain V H  gene segment, at least one unrearranged human immunoglobulin heavy chain D H  gene segment, and at least one unrearranged human immunoglobulin heavy chain J H  gene segment,   wherein the human unrearranged immunoglobulin heavy chain V H , D H , and J H  gene segments are operably linked to a mouse heavy chain constant region sequence that comprises a full-length IgM gene and a deletion of a nucleotide sequence encoding a C H 1 domain in an IgG gene selected from the group consisting of an IgG1, IgG2a, IgG2b, IgG2c, IgG3, and a combination thereof, and   (b) a replacement of all functional endogenous immunoglobulin light chain V and J gene segments with a single rearranged human variable Vκ/Jκ sequence, wherein the single rearranged human variable region Vκ/Jκ sequence is operably linked to an immunoglobulin light chain constant region sequence,   wherein the mouse further comprises in its serum an antigen-specific single domain antigen binding protein that lacks a functional C H 1 domain, and   wherein the mouse expresses a B cell receptor that comprises an IgM heavy chain associated with a cognate universal light chain encoded by the single rearranged human variable region Vκ/Jκ sequence, comprising Vκ and Jκ gene segment sequences, operably linked to an immunoglobulin light chain constant region sequence.   
     
     
         115 . A method of making a genetically modified rodent according to  claim 101  comprising
 (a) modifying at least one rodent heavy chain constant region at an endogenous immunoglobulin heavy chain locus of the rodent such that the heavy chain constant region comprises a deletion of a nucleotide sequence encoding a C H 1 domain of IgG, IgA, IgE, IgD, or a combination thereof, and 
 (b) introducing an immunoglobulin light chain locus that comprises a single rearranged immunoglobulin light chain variable region V L /J L  sequence comprising V L  and J L  gene segment sequences, wherein the single rearranged immunoglobulin light chain variable region V L /J L  sequence is operably linked to an immunoglobulin light chain constant region sequence. 
 
     
     
         116 . The method of  claim 115 , wherein:
 (a) the step of modifying further comprises deleting or inactivating a hinge region of the IgG, IgA, IgE, IgD, or a combination thereof, comprising the modified C H 1 domain; and/or   (b) the step of modifying further comprises replacing one or more endogenous immunoglobulin heavy chain variable region gene segments of the heavy chain immunoglobulin locus with human heavy chain variable region gene segments such that expression of the heavy chain immunoglobulin locus results in a heavy chain variable domain that comprises human idiotypes.   
     
     
         117 . The method of  claim 115 , wherein:
 (a) the deletion of a nucleotide sequence encoding a C H 1 domain is in an IgG gene;   (b) the heavy chain immunoglobulin locus comprises variable region gene segments encoding a human variable domain and a constant region gene encoding a rodent constant domain;   (c) the single rearranged immunoglobulin light chain variable region V L /J L  sequence is
 (I) a human V L /J L  sequence, 
 (II) a human Vκ1-39/J sequence, or a human Vκ3-20/J sequence, 
 (III) a human Vκ1-39/J sequence comprising a human Vκ1-39 gene segment rearranged with a human Jκ5 gene segment, or 
 (IV) a human Vκ3-20/J sequence comprising a human Vκ3-20 gene segment rearranged with a human Jκ1 gene segment; and/or 
   (d) the single rearranged immunoglobulin light chain variable region V L /J L  gene sequence is
 (I) operably linked to a rodent immunoglobulin light chain constant region sequence,
 (II) operably linked to a human immunoglobulin light chain constant region sequence, or 
 
 (III) replaces all functional endogenous immunoglobulin light chain V L  and J L  gene segments of the rodent. 
   
     
     
         118 . The method of  claim 115 , wherein:
 (a) the nucleic acid sequence and/or single rearranged immunoglobulin light chain variable region V L /J L  sequence is in the germline of the rodent;   (b) the deletion of a nucleotide sequence encoding a C H 1 domain is in an IgG1 gene and the method further comprises (c) deleting or inactivating an immunoglobulin gene selected from the group consisting of IgD, IgG3, IgG2a, IgG2b, IgG2c, IgE, IgA, and a combination thereof, optionally wherein the IgG2a and IgG2b immunoglobulin genes are deleted or inactivated, the IgG2b and IgG2c immunoglobulin genes are deleted or inactivated, or IgG3, IgD, IgA, and IgE immunoglobulin genes are deleted or inactivated.   
     
     
         119 . The method of  claim 115 , wherein the rodent is a rat. 
     
     
         120 . The method of  claim 115 , wherein the rodent is a mouse.