METHODS FOR MAKING FULLY HUMAN BISPECIFIC ANTIBODIES USING A COMMON LIGHT CHAIN
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 - 20 . (canceled)
21 . A method of making a bispecific antigen-binding protein comprising:
(a) obtaining a first human immunoglobulin heavy chain variable region sequence and a second human immunoglobulin heavy chain variable region sequence from a first B cell and a second B cell in which they are respectively expressed, wherein:
the first B cell is generated by a first mouse following exposure to a first epitope of a first antigen of interest,
wherein the first mouse is homozygous or heterozygous in its germline genome for a single rearranged human immunoglobulin kappa light chain variable region sequence comprising a human Vκ gene segment and a human Jκ gene segment, wherein all immunoglobulin kappa light chains expressed by B cells of the first mouse comprise immunoglobulin kappa light chain variable domains expressed from the single rearranged human immunoglobulin kappa light chain variable region sequence or a somatically hypermutated version thereof, and wherein the first mouse lacks endogenous immunoglobulin Vκ and/or Jκ gene segments that are capable of rearranging to form an endogenous immunoglobulin light chain variable region sequence, and
wherein the first mouse expresses a first plurality of human immunoglobulin heavy chain variable domains; and
the second B cell is generated by a second mouse following exposure to a second epitope of a second antigen of interest,
wherein the second mouse is homozygous or heterozygous in its germline genome for the single rearranged human immunoglobulin kappa light chain variable region sequence comprising the same human Vκ gene segment and human Jκ gene segment as in the first mouse,
wherein all immunoglobulin kappa light chains expressed by B cells of the second mouse comprise immunoglobulin kappa light chain variable domains expressed from the single rearranged human immunoglobulin kappa light chain variable region sequence or a somatically hypermutated version thereof, and wherein the second mouse lacks endogenous immunoglobulin Vκ and/or Jκ gene segments that are capable of rearranging to form an endogenous immunoglobulin light chain variable region sequence,
wherein the second mouse expresses a second plurality of human immunoglobulin heavy chain variable domains, and
(b) making the bispecific antigen-binding protein, which bispecific antigen-binding protein has:
a first immunoglobulin heavy chain comprising a first human immunoglobulin heavy chain variable domain encoded by the first human immunoglobulin heavy chain variable region sequence;
a second immunoglobulin heavy chain comprising a second human heavy chain variable domain encoded by the second human immunoglobulin heavy variable region sequence; and
an immunoglobulin light chain comprising a human immunoglobulin light chain variable domain encoded by the single rearranged human immunoglobulin kappa light chain variable region sequence or a somatically hypermutated version thereof.
22 . The method of claim 21 , wherein the first mouse and the second mouse are the same mouse, and the first B cell and the second B cell are two different B cells generated by said same mouse.
23 . The method of claim 21 , wherein the single rearranged human immunoglobulin kappa light chain variable region sequence is a Vκ1-39/Jκ sequence.
24 . The method of claim 23 , wherein the single rearranged human immunoglobulin kappa light chain variable region sequence is a Vκ1-39/Jκ5 sequence.
25 . The method of claim 24 , wherein the Vκ1-39/Jκ5 sequence is as set forth in nucleotides 2362 through 2686 of SEQ ID NO: 1.
26 . The method of claim 21 , wherein the single rearranged human immunoglobulin kappa light chain variable region sequence is a Vκ3-20/Jκ sequence.
27 . The method of claim 21 , wherein the single rearranged human immunoglobulin kappa light chain variable region sequence is a Vκ3-20/Jκ1 sequence.
28 . The method of claim 27 , wherein the Vκ3-20/Jκ1 sequence is as set forth in nucleotides 2373 through 2697 of SEQ ID NO: 11.
29 . The method of claim 21 , wherein each of the first mouse and the second mouse comprise in their germline genomes an immunoglobulin 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 mouse immunoglobulin heavy chain constant regions.
30 . The method of claim 29 , wherein the immunoglobulin heavy chain locus in the germline genome of the first mouse and the second mouse comprises 39 unrearranged human V H gene segments operably linked to one or more mouse immunoglobulin heavy chain constant regions.
31 . The method of claim 29 , 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 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 4-31, V H 6-1, or a combination thereof.
32 . The method of claim 31 , wherein each of the first human immunoglobulin heavy chain variable region sequence and the second human immunoglobulin heavy chain variable region sequence comprises a sequence derived from a V H 1-2, V H 1-8, V H 1-24, 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 4- 31, or V H 6-1.
33 . The method of claim 21 , wherein the bispecific antigen-binding protein is a fully human bispecific antigen-binding protein, the method further comprising:
operably linking the first human immunoglobulin heavy chain variable region sequence to a human heavy chain constant region sequence,
operably linking the second human immunoglobulin heavy chain variable region sequence to a human heavy chain constant domain sequence, and/or
operably linking the single rearranged human immunoglobulin light chain variable region sequence or somatically hypermutated version thereof to a human light chain constant region sequence.
34 . The method of claim 21 , wherein the bispecific antigen-binding protein binds:
two different antigens, or
two different epitopes of the same antigen.
35 . A method of making a bispecific antigen-binding protein comprising:
(a) obtaining from a first mouse that has been exposed to a first antigen of interest comprising a first epitope, a first human immunoglobulin heavy chain variable region sequence that encodes a first human immunoglobulin heavy chain variable domain that binds to the first epitope of the first antigen of interest, and
obtaining from a second mouse that has been exposed to a second antigen of interest comprising a second epitope, a second human immunoglobulin heavy chain variable region sequence that encodes a second human immunoglobulin heavy chain variable domain that binds to the second epitope of the second antigen of interest,
wherein each of the first mouse and the second mouse is engineered to comprise in its germline genome a single rearranged human immunoglobulin kappa light chain variable region sequence comprising a human Vκ gene segment and a human Jκ gene segment,
wherein all immunoglobulin kappa light chains expressed by B cells of each of the first mouse and the second mouse comprises immunoglobulin kappa light chain variable domains expressed from the single rearranged human immunoglobulin kappa light chain variable region sequence or a somatically hypermutated version thereof, and wherein each of the first mouse and the second mouse lacks endogenous immunoglobulin Vκ and/or Jκ gene segments that are capable of rearranging to form an endogenous immunoglobulin light chain variable region sequence, and
wherein each of the first mouse and the second mouse expresses a plurality of human immunoglobulin heavy chain variable domains; and
(b) expressing in a cell a first immunoglobulin heavy chain gene comprising the first human immunoglobulin heavy chain variable region sequence, a second immunoglobulin heavy chain gene comprising the second human immunoglobulin heavy chain variable region sequence, and an immunoglobulin light chain gene comprising the single rearranged human immunoglobulin kappa light chain variable region sequence or a somatically hypermutated version thereof, so that the bispecific antigen-binding protein comprising is produced by the cell, said bispecific antigen-binding protein comprising the first immunoglobulin heavy chain, the second immunoglobulin heavy chain and the immunoglobulin light chain.
36 . The method of claim 35 , wherein the first mouse and the second mouse are the same mouse, said same mouse having been exposed to each of said first antigen and said second antigen.
37 . The method of claim 35 , wherein the single rearranged human immunoglobulin kappa light chain variable region sequence is a Vκ1-39/Jκ sequence.
38 . The method of claim 35 , wherein the single rearranged human immunoglobulin kappa light chain variable region sequence is a Vκ1-39/Jκ5 sequence.
39 . The method of claim 38 , wherein the Vκ1-39/Jκ5 sequence is as set forth in nucleotides 2362 through 2686 of SEQ ID NO: 1.
40 . The method of claim 35 , wherein the single rearranged human immunoglobulin kappa light chain variable region sequence is a Vκ3-20/Jκ sequence.
41 . The method of claim 35 , wherein the single rearranged human immunoglobulin kappa light chain variable region sequence is a Vκ3-20/Jκ1 sequence.
42 . The method of claim 41 , wherein the Vκ3-20/Jκ1 sequence is as set forth in nucleotides 2373 through 2697 of SEQ ID NO: 11.
43 . The method of claim 35 , wherein:
the first antigen and the second antigen are not identical; or
the first antigen and the second antigen are identical, and the first epitope and the second epitope are not identical.
44 . The method of claim 35 , wherein the immunoglobulin light chain when paired with the first immunoglobulin heavy chain specifically binds the first epitope of the first antigen and wherein the immunoglobulin light chain when paired with the second immunoglobulin heavy chain specifically binds the second epitope of the second antigen.
45 . The method of claim 35 , wherein the bispecific antigen-binding protein is a fully human bispecific antigen-binding protein, the method further comprising:
operably linking the first human immunoglobulin heavy chain variable region sequence to a human heavy chain constant region sequence,
operably linking the second human immunoglobulin heavy chain variable region sequence to a human heavy chain constant region sequence, and/or
operably linking the single rearranged human immunoglobulin kappa light chain variable region or a somatically hypermutated version thereof to a human light chain constant region sequence.
46 . The method of claim 35 , wherein each of the first mouse and the second mouse comprise in their germline genomes an immunoglobulin 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 mouse immunoglobulin heavy chain constant regions.
47 . The method of claim 46 , wherein the immunoglobulin heavy chain locus in the germline genome of the first mouse and the second mouse comprises 39 unrearranged human V H gene segments operably linked to one or more mouse immunoglobulin heavy chain constant regions.
48 . The method of claim 47 , 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 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 4-31, V H 6-1, or a combination thereof.