Methods For Making Fully Human Bispecific Antibodies Using A Common Light Chain

Abstract

A genetically modified mouse is provided, wherein the mouse expresses an immunoglobulin light chain repertoire characterized by a limited number of light chain variable domains. Mice are provided that express just one or a few immunoglobulin light chain variable domains from a limited repertoire in their germline. Methods for making bispecific antibodies having universal light chains using mice as described herein, including human light chain variable regions, are provided. Methods for making human variable regions suitable for use in multispecific binding proteins, e.g., bispecific antibodies, and host cells are provided. Bispecific antibodies capable of binding first and second antigens are provided, wherein the first and second antigens are separate epitopes of a single protein or separate epitopes on two different proteins are provided.

1 . A method for making a bispecific antigen-binding protein, comprising
 exposing a first mouse that expresses a single human immunoglobulin light chain to a first antigen of interest that comprises a first epitope,   exposing a second mouse that expresses a single human immunoglobulin light chain to a second antigen of interest that comprises a second epitope,   allowing the first and the second mouse to each mount immune responses to the antigens of interest,   identifying in the first mouse a first human heavy chain variable region that binds the first epitope of the first antigen of interest,   identifying in the second mouse a second human heavy chain variable region that binds the second epitope of the second antigen of interest,   making a first fully human heavy chain gene that encodes a first heavy chain that binds the first epitope of the first antigen of interest,   making a second fully human heavy chain gene that encodes a second heavy chain that binds the second epitope of the second antigen of interest,   expressing the first heavy chain and the second heavy chain in a cell that expresses a single fully human light chain to form a bispecific antigen-binding protein, and   isolating the bispecific antigen-binding protein.   
     
     
         2 . The method of  claim 1 , wherein the single fully human light chain is derived from a rearranged human Vκ1-39 or a human Vκ3-20 gene segment. 
     
     
         3 . The method of  claim 1 , wherein
 (a) the first antigen and the second antigen are not identical; or   (b) the first antigen and the second antigen are identical, and the first epitope and the second epitope are not identical.   
     
     
         4 . The method of  claim 1 , wherein the human light chain when paired with the first heavy chain specifically binds the first epitope of the first antigen and when paired with the second heavy chain specifically binds the second epitope of the second antigen. 
     
     
         5 . The method of  claim 1 , wherein the first fully human heavy chain gene bears an amino acid modification that reduces its affinity to protein A, and the second fully human heavy chain does not comprise a modification that reduces its affinity to protein A. 
     
     
         6 . The method of  claim 5 , wherein the modification is selected from a 95R (EUR 435R), 96F (EUR 436F), and a combination thereof. 
     
     
         7 . The method of  claim 1 , wherein the human light chain of the cell comprises a germline sequence. 
     
     
         8 . A method for making a human bispecific antibody comprising a step of employing in the bispecific antibody two human heavy chain variable region sequences of two different B cells a mouse that expresses a single human light chain variable domain. 
     
     
         9 . The method of  claim 8 , wherein the single human light chain variable domain comprises a rearranged human Vκ1-39 or human Vκ3-20 gene segment. 
     
     
         10 . The method of  claim 8 , wherein in the single human light chain variable domain further comprises a rearranged human Jκ5 or human Jκ1 gene segment. 
     
     
         11 . The method of  claim 8 , wherein the mouse further comprises a humanized heavy chain locus containing one or more unrearranged human V H  gene segments, one or more unrearranged human D H  gene segments, and one or more unrearranged human J H  gene segments, operably linked to one or more non-human heavy chain constant region genes. 
     
     
         12 . The method of  claim 11 , wherein the humanized heavy chain locus comprises 80 unrearranged human V H  gene segments, 27 unrearranged human D H  gene segments and six unrearranged human J H  gene segments operably linked to one or more mouse heavy chain constant region genes. 
     
     
         13 . The method of  claim 12 , wherein the one or more human V H  gene segments comprises human V H 1-2, V H 1-8, V H 1-24, V H 1-69, V H 2-5, V H 3-7, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-20, V H 3-23, V H 3-30, V H 3-33, V H 3-48, V H 3-53, V H 4-31, V H 4-39, V H 4-59, V H 5-51, V H 6-1, or a combination thereof. 
     
     
         14 . The method of  claim 8 , wherein the mouse does not contain an endogenous light chain variable gene segment that is capable of rearranging to form an immunoglobulin light chain. 
     
     
         15 . A method of selecting two human immunoglobulin heavy chain variable domains for use in a bispecific antibody comprising immunizing a mouse with an antigen of interest, wherein the mouse expresses a single human light chain variable domain, and wherein the two human immunoglobulin heavy chain variable domains independently associate with the single human light chain variable domain to bind the antigen of interest. 
     
     
         16 . The method of  claim 15 , wherein the single human light chain variable domain is derived from a rearranged human Vκ1-39 gene segment. 
     
     
         17 . The method of  claim 16 , wherein the single human light chain variable domain further comprises a rearranged human Jκ5 gene segment. 
     
     
         18 . The method of  claim 15 , wherein the single human light chain variable domain is derived from a rearranged human Vκ3-20 gene segment. 
     
     
         19 . The method of  claim 18 , wherein the single human light chain variable domain further comprises a rearranged human Jκ1 gene segment. 
     
     
         20 . The method of  claim 15 , wherein the mouse does not contain an endogenous light chain variable gene segment that is capable of rearranging to form an immunoglobulin light chain.