Vl antigen binding proteins exhibiting distinct binding characteristics

Abstract

Methods for making, identifying, isolating and/or making binding proteins that contain an immunoglobulin light chain variable domain, including a somatically hypermutated light chain variable domain, fused with a heavy chain constant region, are provided. Exemplary binding proteins specific to small molecules are also provided.

1 . An antigen-binding protein comprising a first and a second immunoglobulin light chain variable domains, wherein the first and the second immunoglobulin light chain variable domains are associated to form a binding pocket that specifically binds a small molecule. 
     
     
         2 . The antigen-binding protein of  claim 1 , wherein the first and/or the second immunoglobulin light chain variable domain is a human immunoglobulin light chain variable domain. 
     
     
         3 . The antigen-binding protein of  claim 1 , wherein the binding pocket of the light chain variable domains binds the small molecule with an affinity of 50 nM or less. 
     
     
         4 . The antigen-binding protein of  claim 1 , wherein the first and/or the second immunoglobulin light chain variable domain is operably linked to a first immunoglobulin heavy chain constant region. 
     
     
         5 . The antigen-binding protein of  claim 4 , wherein the first immunoglobulin heavy chain constant region is from a non-human animal. 
     
     
         6 . The antigen-binding protein of  claim 5 , wherein the non-human animal is a rodent selected from a mouse and a rat. 
     
     
         7 . The antigen-binding protein of  claim 1 , wherein the first immunoglobulin light chain variable domain is encoded by a first rearranged light chain variable region gene derived from (a) a Vκ4-1 gene segment, a Vκ1-5 gene segment, a Vκ3-15 gene segment, a Vκ3-20 gene segment or a Vκ1-33 gene segment and (b) a Jκ1 gene segment, a Jκ3 gene segment, a Jκ4 gene segment or a Jκ5 gene segment 
     
     
         8 . The antigen-binding protein of  claim 7 , wherein the first rearranged light chain variable region gene is derived from a Vκ4-1 gene segment and a Jκ1 gene segment. 
     
     
         9 . The antigen-binding protein of  claim 7 , wherein the first rearranged light chain variable region gene is derived from a 1-5 Vκ gene segment and a Jκ gene segment selected from the group consisting of a Jκ3 gene segment, a Jκ4 gene segment and a Jκ5 gene segment 
     
     
         10 . The antigen binding protein of  claim 7 , wherein the second immunoglobulin light chain variable domain is encoded by a rearranged light chain variable region gene derived from a human 3-20 Vκ gene segment. 
     
     
         11 . A method of producing a V L  antigen binding protein that specifically binds a small molecule comprising the step(s) of:
 (a) immunizing a genetically modified non-human animal with the small molecule, wherein the genetically modified non-human animal comprises
 (i) unrearranged human immunoglobulin light chain variable (V L ) and light chain joining (J L ) gene segments operably linked to a non-human heavy chain constant region nucleic acid sequence, and 
 (ii) unrearranged human immunoglobulin light chain variable (V L ) and light chain joining (J L ) gene segments operably linked to a non-human light chain constant region nucleic acid sequence; and 
   (b) isolating a cell or V L  antigen binding protein from the immunized non-human animal, wherein the cell or V L  antigen binding protein specifically bind the small molecule.   
     
     
         12 . The method of  claim 11 , further comprising
 (c) culturing a cell transfected with a vector in conditions sufficient for expression of the vector, wherein the vector comprises a nucleic acid operably linked to a human heavy chain constant region gene, wherein the nucleic acid is identical to or substantially identical to a nucleotide sequence encoding a variable domain of the V L  antigen binding protein isolated in step (b).   
     
     
         13 . The method of  claim 11 , further comprising the steps of
 (c) collecting a V L  antigen binding protein from supernatant of a hybridoma culture, wherein the hybridoma is produced from the cell isolated in step (b).   
     
     
         14 . The method of  claim 13 , further comprising
 (d) culturing a cell transfected with a vector in conditions sufficient for expression of the vector, wherein the vector comprises a nucleic acid operably linked to a human heavy chain constant region gene, wherein the nucleic acid is identical to or substantially identical to a nucleotide sequence encoding a variable domain of the V L  antigen binding protein collected in step (c).   
     
     
         15 . The method of  claim 11 , wherein the small molecule is a hapten linked to a carrier or an organic compound whose molecular weight is less than 6 kDa. 
     
     
         16 . The method of  claim 11 , wherein the non-human animal is a mammal. 
     
     
         17 . The method of  claim 16 , wherein the non-human animal is a rodent selected from a mouse or a rat. 
     
     
         18 . A hybridoma produced from a cell isolated according to the method of  claim 11 . 
     
     
         19 . A nucleic acid encoding a variable domain of a V L  antigen binding protein isolated according to the method of  claim 11 . 
     
     
         20 . A cell isolated according to the method of  claim 11 . 
     
     
         21 . A genetically modified non-human animal comprising
 (a) in its genome
 (i) unrearranged human immunoglobulin light chain variable (V L ) and light chain joining (J L ) gene segments operably linked to a non-human heavy chain constant region nucleic acid sequence, and 
 (ii) unrearranged human immunoglobulin light chain variable (V L ) and light chain joining (J L ) gene segments operably linked to a non-human light chain constant region nucleic acid sequence; and 
   (b) a V L  antigen binding protein that specifically binds a small molecule.   
     
     
         22 . The genetically modified non-human animal of  claim 21 , wherein the non-human animal exhibits a 2-fold or more increase in small molecule specific binding protein titer as compared to a reference non-human animal. 
     
     
         23 . A method of identifying one or more V L  antigen binding proteins that exhibit a unique binding characteristic to an antigen compared to conventional antibodies comprising:
 (a) profiling one or more binding characteristics of each of a plurality of immunoglobulin proteins that bind an antigen, wherein the plurality of immunoglobulin proteins comprises V L  antigen binding proteins and conventional antibodies,
 wherein each V L  antigen binding protein comprises a hybrid immunoglobulin chain comprising (i) a variable domain derived from one or more light chain variable region gene segments and (ii) a constant domain derived from one or more heavy chain constant region gene segments, and 
 wherein each conventional antibody comprises an immunoglobulin heavy chain variable region derived from one or more heavy chain variable region and an immunoglobulin light chain variable region gene segment derived from one or more light chain variable region gene segments; 
   (b) binning the plurality of immunoglobulin proteins into one or more groups based on at least one binding characteristic of each of the immunoglobulin proteins, wherein V L  antigen binding proteins and conventional antibodies that exhibit a similar binding characteristic are binned into the same group; and   (c) identifying a group comprising all or substantially all V L  antigen binding proteins.   
     
     
         24 . The method of  claim 23 , wherein one or more binding characteristics of each of the plurality of immunoglobulin proteins is profiled by differential antigen disruption 
     
     
         25 . The method of  claim 23 , further comprising mapping one or more epitopes of the antigen bound by each of the plurality of immunoglobulin proteins; wherein immunoglobulin proteins binding the same epitope of the antigen are binned into the same functional group. 
     
     
         26 . The method of  claim 23 , wherein binning each of the immunoglobulin proteins comprises principle component analysis (PCA). 
     
     
         27 . The method of  claim 23 , wherein binning each of the immunoglobulin proteins comprises hierarchical clustering. 
     
     
         28 . The method of  claim 23 , wherein groups are based on a binding profile comprising an association constant, a dissociation constant, epitope specificity, epitope affinity, or a combination thereof. 
     
     
         29 . The method of  claim 23 , further comprising as a last step:
 (d) isolating one or more V L  antigen binding proteins binned in a functional group identified as comprising all or substantially all V L  antigen binding proteins.   
     
     
         30 . The method of  claim 29 , further comprising as a last step
 (e) confirming that the one or more V L  antigen binding proteins isolated binds one or more epitopes of the antigen that are not recognized by conventional antibodies.   
     
     
         31 . The method of  claim 30 , further comprising sequencing the hybrid immunoglobulin chain of the one or more V L  antigen binding proteins isolated.