Non-human animals that select for light chain variable regions that bind antigen

Abstract

Non-human animals, cells, methods and compositions for making and using the same are provided, wherein the non-human animals and cells comprise an immunoglobulin heavy chain locus that includes unrearranged human immunoglobulin light chain gene segments and an immunoglobulin light chain locus that includes a single rearranged human light chain variable region nucleotide sequence. The unrearranged human light chain gene segments may be operably linked to a heavy chain constant region nucleotide sequence and the rearranged human immunoglobulin light chain variable region nucleotide sequence may be operably linked to a light chain constant region nucleotide sequence. Also provided are methods for obtaining nucleic acid sequences that encode immunoglobulin light chain variable domains capable of binding an antigen in the absence of a cognate variable domain, and expressing such nucleic acid sequences in a host cell, e.g., to generate a multispecific antigen-binding protein.

1 . An antigen binding protein comprising a first binding component comprising a first human light chain variable domain that binds a first epitope,
 wherein the first human light chain variable V L/CH×ULC  domain is identical to or derived from a first human light chain variable domain of a first immunoglobulin hybrid chain that has an isotype selected from the group consisting of IgD, IgG, IgE and IgA, comprises a functional C H 1 domain, and is cognate with a first universal light chain variable domain derived from a human universal rearranged V L /J L  gene sequence,   optionally wherein the first V L/CH×ULC  domain is fused to either a heavy chain constant domain or a light chain constant domain, and   optionally wherein the first V L/CH×ULC  domain is associated with a first universal light chain variable domain derived from the human universal rearranged V L /J L  gene sequence.   
     
     
         2 . The antigen binding protein of  claim 1 , further comprising a second binding component comprising a second human variable domain that binds a second epitope,
 wherein the second human variable domain is either
 (i) a second human light chain variable V L/CH×ULC  domain that is identical to or derived from a second human light chain variable domain of a second hybrid immunoglobulin chain that is cognate with a second universal light chain variable domain derived from the human universal rearranged V L /J L  gene sequence, or 
 (ii) a human heavy chain variable V H×ULC  domain that is cognate with a second universal light chain variable domain derived from the human universal rearranged V L /J L  gene sequence; 
   optionally wherein the second human (V L/CH×ULC  or V H×ULC ) variable domain is fused to either a heavy chain constant domain or a light chain constant domain, and   optionally wherein the second human (V L/CH×ULC  or V H×ULC ) variable domain is associated with a second universal light chain variable domain derived from the human universal rearranged V L /J L  gene sequence.   
     
     
         3 . The antigen-binding protein of  claim 2 , wherein the first variable V L/CH×ULC  and second variable domains are linked by a peptide linker such that the first and second binding components pair in an scFv-like format. 
     
     
         4 . The antigen-binding protein of  claim 2 , wherein one of the first variable V L/CH×ULC  or second variable domains is fused to a functional C H 1 domain, wherein the other of first variable V L/CH×ULC  or second variable domains is fused to a light chain constant (C L ) domain, and wherein the C H 1 and C L  domains are linked by a peptide linker or a disulfide bond such that the first and second binding components pair in a Fab-like format or an scFab-like format. 
     
     
         5 . The antigen-binding protein of  claim 2 ,
 wherein the first binding component comprises
 (a) the first variable V l/CH×ULC  domain fused to a first functional C H 1 domain, a first hinge region, a first C H 2 domain, a first C H 3 domain, and optionally a first C H 4 domain, and 
 (b) the first universal light chain variable domain fused to a first light chain constant (C L ) domain, 
 wherein the first functional C H 1 is linked to the first C L  domain by a disulfide bond or a peptide linker such that (a) and (b) pair in a Fab-like structure, 
   wherein the second binding component comprises
 (a′) the second variable domain fused to a second functional C H 1 domain, a second hinge region, a second C H 2 domain, a second C H 3 domain, and optionally a second C H 4 domain, and 
 (b′) the second universal light chain variable domain fused to a second light chain constant (C L ) domain, 
 wherein the second functional C H 1 is linked to the second light chain constant domain by a disulfide bond or a peptide linker such that (a′) and (b′) pair in a Fab-like structure, 
   wherein the first and second binding components pair such that the antigen binding protein forms a tetrameric binding protein, and   optionally wherein the first and second C H 3 domains have different affinities to Protein A.   
     
     
         6 . The antigen-binding protein of  claim 2 , wherein the first and second binding components are identical. 
     
     
         7 . The antigen-binding protein of  claim 2 , wherein the second human variable domain that binds a second epitope is a human heavy chain variable V H×ULC  domain. 
     
     
         8 . The antigen-binding protein of  claim 2 , wherein the second human variable domain that binds a second epitope is a second variable V L/CH×ULC  domain, and wherein the first and second epitopes are not identical. 
     
     
         9 . The antigen-binding protein of any one of the preceding  claim 1 , wherein the human universal rearranged V L /J L  gene sequence is a human universal rearranged Vκ/Jκ gene sequence. 
     
     
         10 . The antigen-binding protein of  claim 9 , wherein the human universal rearranged Vκ/Jκ gene sequence is a human universal rearranged Vκ1-39/Jκ gene sequence or a human universal rearranged human Vκ3-20/Jκ gene sequence. 
     
     
         11 . The antigen-binding protein of  claim 10 , wherein the human rearranged Vκ1-39/Jκ gene sequence is a rearranged human Vκ1-39/R5 gene sequence or wherein the human rearranged Vκ3-20/Jκ gene sequence is a rearranged human Vκ3-20/R1 gene sequence. 
     
     
         12 . The antigen binding protein of  claim 1 , wherein all heavy chain and light chain constant regions are respectively human heavy chain and light chain constant regions. 
     
     
         13 . A non-human animal useful for generating a human V L/CH×ULC  domain comprising in its germline genome:
 (i) a hybrid immunoglobulin locus that encodes an immunoglobulin hybrid chain, wherein the hybrid immunoglobulin locus comprises unrearranged human immunoglobulin light chain variable region gene segments (V L  and J L ) operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes, each of which encodes at least a functional C H 1 domain, wherein the V L  and J L  gene segments are capable of rearranging to form a hybrid sequence comprising a rearranged human V L /J L  gene sequence operably linked to the immunoglobulin heavy chain constant region nucleic acid sequence;   (ii) a light chain locus that encodes a human universal light chain and comprises a human universal rearranged light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence;   wherein the non-human animal is capable of producing an antigen-binding protein that comprises a human immunoglobulin hybrid chain derived from the hybrid locus and a cognate human universal light chain derived from the light chain locus, wherein the human immunoglobulin hybrid chain comprises a human immunoglobulin light chain variable (hV L/CH×ULC ) domain fused to a heavy chain constant IgD, IgG, IgE or IgA region comprising a functional C H 1 domain, and wherein the human universal light chain comprises a human immunoglobulin light chain fused to a light chain constant domain.   
     
     
         14 .- 20 . (canceled) 
     
     
         21 . A nucleic acid encoding a binding component that comprises a human immunoglobulin light chain variable V L/CH×ULC  domain, wherein the nucleic acid comprises a human rearranged V L /J L  gene sequence operably linked with a non-human or human immunoglobulin heavy chain constant gene or a non-human or human immunoglobulin light chain constant gene. 
     
     
         22 . The nucleic acid of  claim 21 , wherein the non-human immunoglobulin heavy chain constant gene is selected from the group consisting of an intact rodent Iv gene, an intact rodent Igδ gene, an intact rodent Igγ gene, an intact rodent Igε gene, an intact rodent Igα gene, and a combination thereof. 
     
     
         23 . The nucleic acid of  claim 21 , wherein the human immunoglobulin heavy chain constant gene is selected from the group consisting of a human Iv gene, a human Igδ gene, a human Igγ gene, a human Igε gene, a human Igα gene, and a combination thereof. 
     
     
         24 . The nucleic acid of  claim 23 , wherein the human immunoglobulin heavy chain constant gene is a human Igγ gene. 
     
     
         25 . The nucleic acid of  claim 24 , wherein the human Igγ gene encodes an IgG subclass selected from the group consisting of IgG1, IgG2 and IgG4. 
     
     
         26 . The nucleic acid of  claim 25 , wherein the human Igγ gene comprises a mutation in the C H 3 encoding sequence that reduces binding affinity of the encoded C H 3 domain to Protein A compared to a wildtype C H 3 domain. 
     
     
         27 . A nucleic acid encoding a binding component that comprises a human immunoglobulin light chain variable V L/CH×ULC  domain fused to a light chain constant region, wherein the nucleic acid comprises a human rearranged V L /J L  gene sequence operably linked with a non-human or human immunoglobulin light chain constant gene. 
     
     
         28 . A cell comprising the nucleic acid of  claim 21 . 
     
     
         29 . The cell of  claim 28 , wherein the cell is a lymphocyte. 
     
     
         30 . The cell of  claim 29 , wherein the cell is a host cell. 
     
     
         31 . A method of making a multi-specific antigen-binding protein comprising co-expressing in a host cell
 (i) a first nucleic acid comprising a nucleic sequence that encodes a human V L/CH×ULC  domain operably linked with a first non-human or human heavy chain constant region gene comprising a functional C H 1 domain encoding sequence, wherein the human V L/CH×ULC  domain binds a first epitope independently of a cognate variable domain,   (ii) a second nucleic acid comprising a nucleic acid sequence that encodes either a human V H×ULC  domain or a second human V L/CH×ULC  domain that is fused with a second non-human or human heavy chain constant region gene comprising a functional C H 1 domain encoding sequence, wherein the V H×ULC  domain or second V L/CH×ULC  domain binds a second epitope independently of a cognate variable domain,   wherein the first and second epitopes are not identical; and   (iii) a third nucleic acid encoding a human universal light chain variable domain fused to a non-human or human light chain constant domain.   
     
     
         32 . The method of  claim 31 , wherein the first and second heavy chain constant region genes are human heavy chain constant region genes, and wherein the light chain constant domain is a human light chain constant domain. 
     
     
         33 . An antigen-binding protein made according to the method of  claim 31 .