NZ617158B2

NZ617158B2 - Non-human animals expressing antibodies having a common light chain

Info

Publication number: NZ617158B2
Application number: NZ617158A
Authority: NZ
Inventors: David R Buckler; Samuel Davis; Lynn Macdonald; John Mcwhirter; Karolina A Meagher; Andrew J Murphy; Sean Stevens
Original assignee: Regeneron Pharmaceuticals Inc
Priority date: 2011-04-25
Filing date: 2012-04-24
Publication date: 2016-08-02

Abstract

Disclosed the use of a genetically modified mouse in making an antibody, wherein the mouse comprises a population of B cells that express antibodies, each of the antibodies comprising a human immunoglobulin light chain variable domain derived from a single rearranged human V?3-20/J? sequence which is present in the germline of the mouse, wherein the single rearranged human V?3-20/J? sequence comprises a human germline V?3-20 gene segment and a human germline J? gene segment, which single rearranged human V?3-20/J? sequence is operably linked to an immunoglobulin light chain constant region sequence, and wherein the human immunoglobulin light chain variable domain is expressed from a sequence that is identical to or a somatically hypermutated variant of the rearranged human V?3-20/J? sequence, and wherein at least one antibody expressed by the population of B cells comprises a human immunoglobulin heavy chain variable domain derived from a human VH3-53 gene segment. s present in the germline of the mouse, wherein the single rearranged human V?3-20/J? sequence comprises a human germline V?3-20 gene segment and a human germline J? gene segment, which single rearranged human V?3-20/J? sequence is operably linked to an immunoglobulin light chain constant region sequence, and wherein the human immunoglobulin light chain variable domain is expressed from a sequence that is identical to or a somatically hypermutated variant of the rearranged human V?3-20/J? sequence, and wherein at least one antibody expressed by the population of B cells comprises a human immunoglobulin heavy chain variable domain derived from a human VH3-53 gene segment.

Description

MAN S EXPRESSING ANTIBODIES HAVING A COMMON LIGHT CHAIN FIELD OF INVENTION A genetically modified mouse is provided that expresses antibodies having a common human variable/mouse nt light chain associated with diverse human variable/mouse constant heavy chains. A method for making a human bispecific dy from human variable region gene sequences of B cells of the mouse is provided.

BACKGROUND Antibodies typically comprise a homodimeric heavy chain component, wherein each heavy chain monomer is associated with an identical light chain. Antibodies having a heterodimeric heavy chain component (e.g., ific antibodies) are desirable as therapeutic antibodies. But making bispecific antibodies having a suitable light chain component that can satisfactorily associate with each of the heavy chains of a bispecific antibody has proved problematic. in one approach, a light chain might be selected by surveying usage statistics for all light chain variable domains, identifying the most ntly ed light chain in human antibodies, and pairing that light chain in vitro with the two heavy chains of ing specificity.

In another approach, a light chain might be selected by observing light chain sequences in a phage display library (9.9., a phage display library sing human light chain variable region sequences, 9.9., a human scFv library) and selecting the most commonly used light chain variable region from the library. The light chain can then be tested on the two different heavy chains of interest.

In another approach, a light chain might be selected by assaying a phage display library of light chain variable sequences using the heavy chain variable sequences of both heavy chains of interest as probes. A light chain that associates with both heavy chain variable sequences might be selected as a light chain for the heavy chains. in another approach, a candidate light chain might be aligned with the heavy chains’ cognate light chains, and cations are made in the light chain to more closely match sequence characteristics common to the cognate light chains of both heavy chains.

If the chances of immunogenicity need to be minimized, the modifications preferably result in sequences that are present in known human light chain sequences, such that proteolytic processing is unlikely to te a T cell epitope based on parameters and methods known in the art for assessing the likelihood of immunogenicity (i.e., in silico as well as wet assays).

All of the above approaches rely on in vitro methods that subsume a number of a priori ints, e.g., sequence identity, ability to associate with specific pre-selected heavy chains, etc. There is a need in the art for compositions and methods that do not rely on manipulating in vitro conditions, but that instead employ more biologically sensible approaches to making human epitope-binding proteins that e a common light chain.

SUMMARY [0007a] In one aspect the present invention provides a genetically modified mouse sing a population of B cells that each express a human globulin light chain variable domain derived from a single rearranged human Vκ1-39/Jκ sequence which is present in the germline of the mouse, wherein the single rearranged human Vκ1-39/Jκ sequence ses a human germline Vκ1-39 gene segment and a human germline Jκ gene segment, which single rearranged human Vκ1-39/Jκ sequence is operably linked to an immunoglobulin light chain constant region sequence, and wherein the human immunoglobulin light chain variable domain is expressed from a sequence that is identical to or a cally hypermutated variant of the rearranged human Vκ1-39/Jκ sequence; and wherein the B cells of the population include at least one B cell that expresses a human immunoglobulin heavy chain variable domain derived from a rearranged human VH/D/JH region selected from the group consisting of 1-69/6-13/4, 1-69/6-6/5, 2-5/3-22/1, 3- 13/6-6/5, 3-23/2-8/4, 3-23/6-6/4, 3-23/7-27/4, 3-30/1-1/4, -3/4, 3-30/5-5/2, and 3-30/7- 27/6.

] In a further aspect the t invention provides a genetically modified mouse comprising a population of B cells that each express a human immunoglobulin light chain variable domain derived from a single rearranged human Vκ3-20/Jκ ce which is present in the germline of the mouse, wherein the single rearranged human Vκ3-20/Jκ sequence ses a human germline Vκ3-20 gene segment and a human ne Jκ gene segment, which single rearranged human Vκ3-20/Jκ sequence is ly linked to an immunoglobulin light chain constant region sequence, and wherein the human immunoglobulin light chain variable domain is expressed from a sequence that is identical to or a somatically hypermutated variant of the rearranged human /Jκ sequence; and wherein the B cells of the population include at least one B cell that expresses a human immunoglobulin heavy chain variable domain d from a rearranged human H region selected from the group consisting of 3-30/3-3/3, 3-33/1-7/4, 3-33/2-15/4, and 3-53/1-1/4. [0007c] In a further aspect the present invention provides use of a genetically modified mouse in making an antibody, wherein the mouse is terized in that it comprises a population of B cells that express dies, each of the antibodies comprising a human immunoglobulin light chain variable domain derived from a single rearranged human Vκ1- 39/Jκ sequence which is t in the germline of the mouse, wherein the single rearranged human Vκ1-39/Jκ sequence comprises a human germline Vκ1-39 gene t and a human germline Jκ gene segment, which single rearranged human Vκ1-39/Jκ ce is ly linked to an immunoglobulin light chain constant region sequence, and wherein the human immunoglobulin light chain variable domain is expressed from a sequence that is identical to or a somatically hypermutated variant of the rearranged human Vκ1-39/Jκ sequence, and wherein at least one antibody expressed by the tion of B cells comprises a human immunoglobulin heavy chain variable domain derived from a human VH1- 69 gene segment. [0007d] In another aspect the present invention provides use of a genetically modified mouse in making an antibody, wherein the mouse is terized in that it ses a population of B cells that express antibodies, each of the antibodies comprising a human immunoglobulin light chain le domain derived from a single rearranged human Vκ3- /Jκ sequence which is t in the germline of the mouse, wherein the single nged human Vκ3-20/Jκ sequence comprises a human germline Vκ3-20 gene segment and a human germline Jκ gene segment, which single rearranged human Vκ3-20/Jκ sequence is operably linked to an immunoglobulin light chain constant region sequence, and wherein the human immunoglobulin light chain variable domain is expressed from a sequence that is identical to or a somatically hypermutated variant of the rearranged human Vκ3-20/Jκ sequence, and wherein at least one antibody expressed by the population of B cells comprises a human immunoglobulin heavy chain variable domain derived from a human VH3- 53 gene segment.

Genetically modified mice that express human immunoglobulin heavy and light chain variable domains, wherein the mice have a limited light chain variable repertoire, are provided. A biological system for generating a human light chain variable domain that associates and expresses with a diverse repertoire of affinity-matured human heavy chain variable domains is provided. Methods for making binding proteins comprising immunoglobulin variable s are provided, comprising immunizing mice that have a limited immunoglobulin light chain repertoire with an antigen of interest, and employing an immunoglobulin variable region gene sequence of the mouse in a binding protein that specifically binds the antigen of interest.

Methods include methods for making human immunoglobulin heavy chain variable s suitable for use in making multi-specific n-binding proteins. cally engineered mice are provided that select suitable affinity-matured human immunoglobulin heavy chain variable domains derived from a repertoire of ranged human heavy chain variable region gene segments, wherein the affinitymatured human heavy chain variable domains associate and express with a single human light chain variable domain derived from one human light chain variable region gene segment.

Genetically engineered mice that present a choice of two human light chain variable region gene segments are also provided.

Genetically engineered mice are provided that express a limited repertoire of human light chain le domains, or a single human light chain variable , from a limited repertoire of human light chain variable region gene segments. The mice are genetically ered to include a single unrearranged human light chain variable region gene segment (or two human light chain variable region gene segments) that rearranges to form a rearranged human light chain variable region gene (or two nged light chain variable region genes) that express a single light chain (or that express either or both of two light chains). The rearranged human light chain variable domains are capable of g with a plurality of affinity-matured human heavy chains selected by the mice, wherein the heavy chain variable regions specifically bind different epitopes. cally engineered mice are provided that express a limited repertoire of human light chain variable domains, or a single human light chain variable domain, from a limited repertoire of human light chain variable region sequences. The mice are genetically engineered to include a single V/J human light chain sequence (or two V/J sequences) that express a le region of a single light chain (or that express either or both of two variable regions). A light chain comprising the le sequence is capable of pairing with a plurality of affinity—matured human heavy chains clonally ed by the mice, wherein the heavy chain variable regions ically bind different epitopes.

In one , a genetically modified mouse is provided that comprises a single human immunoglobulin light chain variable (VL) region gene segment that is capable of rearranging with a human J gene t (selected from one or a plurality of JL segments) and encoding a human VL domain of an immunoglobulin light chain. In another aspect, the mouse comprises no more than two human VL gene segments, each of which is e of rearranging with a human J gene segment (selected from one or a plurality of JL segments) and encoding a human VL domain of an immunoglobulin light chain. in one embodiment, the single human VL gene segment is operably linked to a human JL gene segment selected from JK1, JKZ, JK3, JK4, and JK5, wherein the single human VL gene segment is e of rearranging to form a sequence encoding a light chain variable region gene with any of the one or more human JL gene segments. in one embodiment, the genetically modified mouse comprises an globulin light chain locus that does not comprise an endogenous mouse VL gene segment that is capable of rearranging to form an immunoglobulin light chain gene, wherein the VL locus comprises a single human VL gene segment that is capable of rearranging to encode a VL region of a light chain gene. in a specific embodiment, the human VL gene segment is a human VK1-39JK5 gene segment or a human VK3-20JK1 gene t. In one embodiment, the genetically modified mouse comprises a VL locus that does not comprise an endogenous mouse VL gene segment that is capable of rearranging to form an immunoglobulin light chain gene, n the VL locus comprises no more than two human VL gene segments that are capable of rearranging to encode a VL region of a light chain gene. ln a specific embodiment, the no more than 2 human VL gene segments are a human VK1-39JK5 gene segment and a human VK3—20JK1 gene segment. in one aspect, a genetically modified mouse is provided that comprises a single rearranged (V/J) human globulin light chain variabie (VL) region (Le, a VL/JL region) that encodes a human VL domain of an immunoglobulin light chain. In another aspect, the mouse comprises no more than two rearranged human VL regions that are capable of encoding a human VL domain of an immunoglobulin light chain.

WO 48873 In one embodiment, the VL region is a rearranged human VK1—39/J sequence or a rearranged human VK3-20/J sequence. In one embodiment, the human JL segment of the rearranged VL/JL sequence is selected from JK1, JK2, Jx3, JK4, and JK5. In a specific embodiment, the VL region is a human VK1-39JK5 sequence or a human VK3-20JK1 sequence. In a specific embodiment, the mouse has both a human VK1-39JK5 sequence and a human VK3-20JK1 sequence.

In one embodiment, the human VL gene segment is operably linked to a human or mouse leader sequence. In one embodiment, the leader sequence is a mouse leader sequence. In a specific embodiment, the mouse leader sequence is a mouse VK3-7 leader sequence. In a specific embodiment, the leader sequence is operably linked to an unrearranged human VL gene t. In a specific embodiment, the leader sequence is operably linked to a rearranged human VL/JL sequence.

In one ment, the VL gene segment is operably linked to an globulin promoter sequence. In one embodiment, the promoter sequence is a human er sequence. In a specific embodiment, the human immunoglobulin promoter is a human VK3-15 promoter. In a specific embodiment, the promoter is ly linked to an unrearranged human VL gene segment. In a specific embodiment, the promoter is operably linked to a rearranged human VL/JL sequence.

In one embodiment, the light chain locus comprises a leader sequence flanked 5’ (with respect to riptional direction of a VL gene segment) with a human immunoglobulin promoter and flanked 3’ with a human VL gene segment that nges with a human J segment and encodes a VL domain of a reverse chimeric light chain comprising an nous mouse light chain constant region (CL). In a specific embodiment, the VL gene segment is at the mouse VK locus, and the mouse CL is a mouse In one embodiment, the light chain locus comprises a leader sequence flanked 5’ (with respect to transcriptional direction of a VL gene segment) with a human immunoglobulin promoter and flanked 3’ with a nged human VL region (VL/JL sequence) and encodes a VL domain of a reverse chimeric light chain comprising an nous mouse light chain constant region (CL). In a specific embodiment, the nged human VL/JL sequence is at the mouse kappa (K) locus, and the mouse CL is a mouse CK.

In one embodiment, the VL locus of the modified mouse is a K light chain locus, and the K light chain locus comprises a mouse K intronic enhancer, a mouse K 3’ enhancer, or both an intronic enhancer and a 3’ enhancer.

In one embodiment, the mouse comprises a nonfunctional immunoglobulin lambda 0») light chain locus. In a specific embodiment, the A light chain locus comprises a deletion of one or more sequences of the locus, wherein the one or more deletions renders the it light chain locus incapable of rearranging to form a light chain gene. In another embodiment, all or substantially aII of the VL gene segments of the 7» light chain locus are deleted.

In one embodiment, mouse makes a light chain that comprises a cally mutated VL domain derived from a human VL gene segment. In one embodiment, the light chain ses a somatically mutated VL domain derived from a human VL gene segment, and a mouse CK region. In one embodiment, the mouse does not express a x light chain.

In one ment, the genetically modified mouse is capable of somatically hypermutating the human VL region sequence. In a specific embodiment, the mouse comprises a cell that comprises a nged globulin light chain gene derived from a human VL gene segment that is capabie of rearranging and encoding a VL domain, and the nged immunoglobulin light chain gene comprises a somatically mutated VL In one embodiment, the mouse comprises a cell that expresses a light chain comprising a somatically mutated human VL domain linked to a mouse CK, wherein the light chain associates with a heavy chain comprising a somatically mutated VH domain derived from a human VH gene segment and wherein the heavy chain comprises a mouse heavy chain constant region (CH). In a specific embodiment, the heavy chain comprises a mouse CH1, a mouse hinge, a mouse CH2, and a mouse CH3. In a specific embodiment, the heavy chain comprises a human cm, a hinge, a mouse CH2, and a mouse CH3.

In one embodiment, the mouse comprises a replacement of endogenous mouse VH gene segments with one or more human VH gene ts, wherein the human VH gene segments are operably linked to a mouse CH region gene, such that the mouse nges the human VH gene segments and expresses a reverse chimeric immunoglobuiin heavy chain that comprises a human VH domain and a mouse CH. In one embodiment, 90-100% of unrearranged mouse VH gene ts are replaced with at Ieast one unrearranged human VH gene segment. In a specific embodiment, all or substantially all of the endogenous mouse VH gene segments are replaced with at least one unrearranged human VH gene segment. In one embodiment, the replacement is with at least 19, at least 39, or at least 80 or 81 unrearranged human VH gene segments. In one embodiment, the replacement is with at least 12 functional unrearranged human VH gene segments, at least onal unrearranged human VH gene segments, or at least 43 onal ranged human VH gene segments. In one embodiment, the mouse comprises a 2012/034737 replacement of all mouse DH and JH segments with at least one unrearranged human DH segment and at least one ranged human JH segment. in one embodiment, the at least one unrearranged human DH segment is selected from 1-1, 1—7, 1-26, 2-8, 2—15, 3-3, 3—10, 3—16, 3-22, 5—5, 5-12, 6—6, 6-13, 7-27, and a combination thereof. in one embodiment, the at least one unrearranged human JH t is selected from 1, 2, 3, 4, , 6, and a ation thereof. in a specific embodiment, the one or more human VH gene segment is selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3—15, 3—20, 3-23, 3-30, 3-33, 3—48, 3-53, 4-31, 4—39, 4-59, 5-51, a 6-1 human VH gene segment, and a combination thereof. in one embodiment, the mouse comprises a B cell that expresses a binding protein that specifically binds an antigen of interest, wherein the binding protein comprises a light chain derived from a human VK1~39/JK5 rearrangement or a human VK3-20/JK1 rearrangement, and wherein the cell comprises a rearranged immunoglobuiin heavy chain gene derived from a rearrangement of human VH gene segments selected from a 1—69, 2— , 3-13, 3—23, 3—30, 3-33, 3-53, 4-39, 4-59, and 5-51 gene t. in one embodiment, the one or more human VH gene segments are nged with a human heavy chain JH gene t selected from 1, 2, 3, 4, 5, and 6. in one ment, the one or more human VH and JH gene segments are rearranged with a human DH gene segment selected from 1—1, 1-7, 1—26, 2-8, 2-15, 3-3, 3-10, 3-16, 3-22, 5—5, 5-12, 6—6, 6—13, and 7-27. In a specific embodiment, the iight chain gene has 1, 2, 3, 4, or 5 or more somatic hypermutations. in one embodiment, the mouse comprises a B cell that comprises a nged immunoglobulin heavy chain variable region gene sequence comprising a VH/DH/JH region selected from 2-5/6-6/1, 2-5/3-22/1, 3-13/6-6/5, 3—23/2-8/4, 3-23/3—3/4, 3—23/3-10/4, 3-23/6- 6/4, 3-23/7-27/4, 3—30/1—1/4, 3-30/1-7/4, 3—30/3—3/3, 3—30/3-3/4, 3-30/3~22/5, 3-30/5-5/2, 3- 2/4, 3—30/6—6/1, —6/3, 3-30/6—6/4, 3~30/6—6/5, 3-30/6-13/4, 3-30/7-27/4, - 27/5, 3-30/7-27/6, 3-33/1-7/4, 3-33/2-15/4, 4-39/1-26/3, 4-59/3-16/3, 4-59/3-16/4, 4-59/3- 22/3, 5-51/3-16/6, 5-51/5-5/3, 5—51/6-13/5, 3-53/1-1/4, 1—69/6—6/5, and 1—69/6—13/4. in a specific embodiment, the B cell expresses a binding protein comprising a human immunoglobulin heavy chain variable region fused with a mouse heavy chain constant region, and a human immunoglobulin light chain variable region fused with a mouse light chain constant region. In one embodiment, the rearranged human VL region is a human JK5 sequence, and the mouse expresses a e chimeric light chain comprising (i) a VL domain derived from the human VL/JL sequence and (ii) a mouse CL; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse CH and (ii) a somatically mutated human VH domain derived from a human VH gene segment ed from a 1-2, 1-8, 1-24, 1-69, 2—5, 3-7, 3-9, 3—11, 3-13, 3-15, 3-20, 3—23, 3-30, 3-33, 3-48, 3—53, 4—31, 4-39, 4-59, 5-51, a 6—1 human VH gene segment, and a combination thereof. In one ment, the mouse expresses a light chain that is somatically mutated. ln one ment the CL is a mouse CK. In a specific embodimentthe human VH gene segment is selected from a 2—5, 3—13, 3—23, 3-30, 4-59, 5-51, and 1-69 gene segment. In a specific embodiment, the somatically mutated human VH domain comprises a sequence derived from a DH segment selected from 1-1, 1-7, 2-8, 3-3, 3-10, 3-16, 3-22, -5, 5—12, 6-6, 6—13, and 7-27. ln a specific embodiment, the somatically mutated human VH domain comprises a sequence d from a JH segment ed from t, 2, 3, 4, 5, and 6. in a specific embodiment, the somatically mutated human VH domain is encoded by a rearranged human VH/DH/JH sequence selected from 2—5/6-6/1, 2—5/3—22/1, 3-13/6-6/5, 3- 23/2—8/4, 3—23/3-3/4, 3-23/3-10/4, 3-23/6—6/4, -27/4, 3-30/1-1/4, 3-30/1-7/4, 3-30/3~ 3/4, 3—30/3-22/5, 3—30/5-5/2, 3-30/5—12/4, 3-30/6—6/1, 3-30/6-6/3, 3-30/6-6/4, 3-30/6-6/5, 3- /6—13/4, 3—30/7-27/4, 3-30/7—27/5, —27/6, 4—59/3-16/3, 4—59/3-16/4, 4-59/3-22/3, 5- 51/5—5/3, 1—69/6—6/5, and 1—69/6-13/4.

In one ment, the mouse comprises a B cell that expresses a binding protein that specifically binds an antigen of interest, wherein the binding protein comprises a light chain derived from a human VK1-39/JK5 rearrangement, and wherein the cell comprises a nged immunoglobulin heavy chain variable region gene sequence comprising a VH/DH/JH region selected from 2-5/3—22/1, 3-13/6—6/5, 3—23/2—8/4, 3—23/6-6/4, 3—23/7—27/4, 3—30/1-1/4, 3-30/3—3/4, 3-30/5-5/2, —27/6, —6/5 and 1-69/6—13/4. In a specific ment, the B cell expresses a binding protein comprising a human immunoglobulin heavy chain variable region fused with a mouse heavy chain constant region, and a human immunoglobulin light chain variable region fused with a mouse light chain constant region. in one embodiment, the rearranged human VL region is a human VK3-20JK1 sequence, and the mouse expresses a reverse chimeric light chain comprising (i) a VL domain derived from the rearranged human VL/JL sequence, and (ii) a mouse CL; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse CH, and (ii) a somatically mutated human VH derived from a human VH gene segment selected from a 1-2, 1-8, 1—24, 1-69, 2-5, 3—7, 3—9, 3-11, 3-13, 3-15, 3—20, 3—23, 3-30, 3-33, 3-48, 3-53, 4—31, 4—39, 4—59, 5-51, a 6-1 human VH gene segment, and a combination thereof. in one embodiment, the mouse expresses a light chain that is somatically mutated. in one ment the CL is a mouse CK. ln a specific embodiment, the human VH gene segment is selected from a 3-30, 3-33, 3—53, 4—39, and 5-51 gene t. ln a specific ment, the somatically mutated human VH domain comprises a sequence derived from a DH segment selected from 1-1, 1—7, 1-26, 2—15, 3-3, 3-16, and 6—13. In a specific embodiment, the somatically mutated human VH domain comprises a sequence derived from a JH segment selected from 3, 4, 5, and 6. In a specific embodiment, the somatically d human VH domain is encoded by a rearranged human VH/DH/JH sequence selected from 3-30/1-1/4, 3-30/3—3/3, 3-33/1-7/4, 3-33/2—15/4, 4-39/1-26/3, 5- 6/6, 5-51/6-13/5, and 3-53/1-1/4.

In one embodiment, the mouse comprises a B cell that expresses a binding n that specifically binds an antigen of interest, wherein the binding protein comprises a light chain derived from a human VK3-20/JK1 rearrangement, and wherein the cell comprises a rearranged immunoglobulin heavy chain variable region gene sequence comprising a VH/DH/JH region seIected from 3-30/3-3/3, 3-33/1—7/4, -15/4, and 3-53/1—1/4. In a specific embodiment, the B cell expresses a binding protein comprising a human immunoglobulin heavy chain le region fused with a mouse heavy chain constant region, and a human immunoglobulin light chain le region fused with a mouse light chain constant region.

In one embodiment, the mouse comprises both a nged human VK1-39JK5 sequence and a nged human VK3-20JK1 sequence, and the mouse expresses a reverse chimeric light chain comprising (i) a VL domain derived from the human VK1-39JK5 sequence or the human VK3-20JK1 sequence, and (ii) a mouse CL; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse CH, and (ii) a cally mutated human VH derived from a human VH gene segment selected from a 1— 2, 1-8, 1—24, 1-69, 2—5, 3—7, 3—9, 3-11, 3-13, 3-15, 3-20, 3—23, 3-30, 3—33, 3-48, 3-53, 4-31, 4—39, 4—59, 5-51, a 6~1 human VH gene segment, and a combination thereof. In one embodiment, the mouse expresses a light chain that is somatically mutated. In one ment the CL is a mouse CK.

In various embodiments, the human immunoglobulin heavy chain variable region fused with a mouse heavy chain constant region and human immunoglobulin light chain variable region fused with a mouse light chain constant region expressed by the B cell are cognate in the mouse. In various embodiments, the chimeric light chain and chimeric heavy chain expressed by the mouse are e in the mouse.

In one embodiment, 90-100% of the endogenous unrearranged mouse VH gene segments are replaced with at least one unrearranged human VH gene segment. In a ic embodiment, all or substantially all of the nous unrearranged mouse VH gene segments are replaced with at least one unrearranged human VH gene t. In one embodiment, the replacement is with at least 18, at least 39, at least 80, or 81 unrearranged human VH gene segments. In one embodiment, the replacement is with at least 12 functional unrearranged human VH gene segments, at least 25 functional ranged human VH gene segments, or at least 43 unrearranged human VH gene segments.

In one embodiment, the genetically modified mouse is a CS7BL strain, in a specific embodiment ed from CS7BL/A, CS7BL/An, CS7BL/GrFa, C57BL/KaLwN, C57BL/6, 057BL/6J, GByJ, CS7BL/6NJ, C57BL/10, C57BL/1OScSn, C57BL/10Cr, CS7BL/Ola. In a specific embodiment, the genetically modified mouse is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain. In another specific embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains. In a specific embodiment, the 129 strain of the mix is a 12986 (129/SvaTac) strain.

In one embodiment, the mouse expresses a reverse chimeric antibody comprising a light chain that comprises a mouse CK and a somatically mutated human VL domain derived from a rearranged human VK1—39JK5 sequence or a rearranged human VK3-20JK1 ce, and a heavy chain that comprises a mouse CH and a somatically mutated human VH domain derived from a human VH gene segment selected from a 1-2, 1-8, 1-24, 1-69, 2—5, 3-7, 3-9, 3—11, 3—13, 3—15, 3-20, 3-23, 3-30, 3—33, 3-48, 3—53, 4-31, 4—39, 4-59, 5- 51, and a 6-1 human VH gene segment, wherein the mouse does not express a fully mouse antibody and does not express a fully human antibody. In one embodiment the mouse comprises a K light chain locus that comprises a replacement of nous mouse K light chain gene segments with the nged human VK1—39JK5 sequence or the rearranged human VK3—20JK1 sequence, and comprises a replacement of all or substantially all nous mouse VH gene segments with a complete or substantially complete repertoire of human VH gene segments.

In one aspect, a mouse that expresses an immunoglobulin light chain from a rearranged immunoglobulin light chain sequence in the germline of the mouse is provided, wherein the immunoglobulin light chain comprises a human variable ce.

In one ment, the germline of the mouse lacks a functional unrearranged immunoglobulin light chain V gene segment. In one embodiment, the germline of the mouse lacks a functional unrearranged immunoglobulin light chain J gene segment.

In one ment, the germline of the mouse comprises no more than one, no more than two, or no more than three nged (V/J) light chain sequences.

In one embodiment, the nged V/J sequence comprises a K light chain ce. In a ic embodiment, the K light chain sequence is a human K light chain sequence. In a specific embodiment, the K light chain sequence is selected from a human VK1—39/J sequence, a human VK3—20/J sequence, and a combination thereof. In a specific embodiment, the K light chain sequence is a human VK1-39/JK5 sequence. in a specific embodiment, the K light chain sequence is a human VK3—20/JK1 sequence. in one embodiment, the mouse further comprises in its germline a sequence selected from a mouse K ic enhancer 5’ with respect to the rearranged immunoglobulin light chain sequence, a mouse K 3’ enhancer, and a combination thereof. in one embodiment, the mouse comprises an unrearranged human VH gene segment, an unrearranged human DH gene segment, and an unrearranged human JH gene segment, n said VH, DH, and JH gene segments are e of rearranging to form an immunoglobulin heavy chain variable gene sequence operably linked to a heavy chain constant gene sequence. ln one ment, the mouse comprises a ity of human VH, DH, and JH gene segments. ln a specific embodiment, the human VH, DH, and JH gene segments replace endogenous mouse VH, DH, and JH gene segments at the endogenous mouse immunoglobulin heavy chain locus. in a ic embodiment, the mouse comprises a replacement of all or substantially all functional mouse VH, DH, and JH gene segments with all or substantially all functional human VH, DH, and JH gene segments.

In one embodiment, the mouse expresses an immunoglobulin light chain that comprises a mouse constant sequence. ln one embodiment, the mouse expresses an immunoglobulin light chain that comprises a human constant sequence. in one embodiment, the mouse expresses an globulin heavy chain that comprises a mouse sequence selected from a CH1 sequence, a hinge ce, a CH2 sequence, a CH3 sequence, and a combination thereof.

In one embodiment, themouse expresses an globulin heavy chain that comprises a human sequence selected from a CH1 sequence, a hinge sequence, a CH2 sequence, a CH3 sequence, and a combination thereof.

In one embodiment, the rearranged immunoglobulin light chain sequence in the germline of the mouse is at an nous mouse immunoglobulin light chain locus. In a specific embodiment, the rearranged immunoglobulin light chain sequence in the germline of the mouse replaces all or substantially all mouse light chain V and J sequences at the endogenous mouse immunoglobulin light chain locus.

In one aspect, a mouse cell is provided that is isolated from a mouse as described herein. ln one embodiment, the cell is an ES cell. in one ment, the cell is a lymphocyte. In one embodiment, the lymphocyte is a B cell. in one embodiment, the B cell ses a chimeric heavy chain comprising a variable domain derived from a human gene segment; and a light chain d from a rearranged human VK1-39/J sequence, rearranged human VK3-20/J sequence, or a combination thereof; wherein the heavy chain variable domain is fused to a mouse constant region and the light chain variable domain is fused to a mouse or a human nt region. in one embodiment, a mouse B cell is provided that is isolated from a mouse as described herein, wherein the B cell expresses a chimeric heavy chain derived from a nged human VH/DH/JH sequence selected from 2-5/3-22/1, 3-13/6—6/5, 3-23/2—8/4, 3- 23/6-6/4, 3-23/7—27/4, 3-30/1-1/4, -3/4, 3-30/5-5/2, 3—30/7—27/6, 1—69/6—6/5 and 1~ 69/6-13/4; and a chimeric light chain derived from a rearranged human VK1-39/JK5 sequence; wherein the variable domain is fused to a mouse constant region and the light chain variable domain is fused to a mouse constant region. in one embodiment, a mouse B cell is provided that is ed from a mouse as described herein, wherein the B cell expresses a ic heavy chain derived from a rearranged human VH/DH/JH sequence selected from 3—30/3—3/3, 3-33/1—7/4, 3-33/2-15/4, and 3—53/1-1/4; and a chimeric light chain derived from a nged human VK3-20/JK1 sequence; wherein the variable domain is fused to a mouse constant region and the light chain variable domain is fused to a mouse nt region. in various embodiments, the chimeric heavy and light chains expressed by the B cell isolated from a mouse as described herein are cognate in the mouse. ln one aspect, a hybridoma is provided, n the hybridoma is made with a B cell of a mouse as described herein. in a specific embodiment, the B cell is from a mouse as described herein that has been zed with an immunogen comprising an epitope of interest, and the B cell expresses a binding protein that binds the epitope of interest, the binding protein has a somatically d human VH domain and a mouse CH, and has a human VL domain derived from a nged human VK1-39JK5 or a rearranged human VK3-20JK1 and a mouse CL. in one embodiment, a hybridoma is provided that is made with a B cell of a mouse as described herein, wherein the hybridoma expresses a chimeric heavy chain derived from a rearranged human VH/DH/JH sequence selected from 2-5/3-22/1, 3-13/6-6/5, 3-23/2— 8/4, 3-23/6—6/4, 3—23/7-27/4, 3—30/1—1/4, 3-30/3-3/4, 3-30/5-5/2, —27/6, 1-69/6—6/5 and 1—69/6-13/4; and a chimeric light chain derived from a rearranged human VK1-39/JK5 sequence; wherein the variable domain is fused to a mouse constant region and the light chain variable domain is fused to a mouse constant region.

In one embodiment, a hybridoma is provided that is made with a B cell of a mouse as described herein, wherein the hybridoma expresses a chimeric heavy chain derived from a rearranged human VH/DH/JH sequence selected from -3/3, 3—33/1-7/4, 3-33/2— /4, and -1/4; and a chimeric light chain derived from a rearranged human VK3- /th sequence; wherein the le domain is fused to a mouse constant region and the light chain variable domain is fused to a mouse constant region. in various embodiments, the chimeric heavy and light chains expressed by the oma that is made with a B cell of a mouse as bed herein are cognate in the hybridoma. in various embodiments, the chimeric heavy and light chains expressed by the oma that is made with a B cell of a mouse as described herein are cognate in the B cell of the mouse. in one aspect, a mouse embryo is provided, wherein the embryo comprises a donor ES cell that is derived from a mouse as described herein. ln one aspect, a targeting vector is provided, comprising, from 5’ to 3’ in transcriptional direction with reference to the sequences of the 5’ and 3’ mouse homology arms of the vector, a 5’ mouse homology arm, a human or mouse immunoglobulin promoter, a human or mouse leader sequence, and a human VL region selected from a rearranged human JK5 or a nged human VK3-20JK1, and a 3’ mouse homology arm. in one embodiment, the 5’ and 3’ homology arms target the vector to a sequence 5’ with respect to an enhancer sequence that is present 5’ and proximal to the mouse CK gene. ln one embodiment, the promoter is a human globulin variable region gene segment promoter. In a specific embodiment, the promoter is a human VK3- er. ln one embodiment, the leader sequence is a mouse leader sequence. in a specific embodiment, the mouse leader sequence is a mouse VK3-7 leader ce. in one , a targeting vector is ed as described above, but in place of the ’ mouse homology arm the human or mouse promoter is flanked 5’ with a site-specific recombinase recognition site (SRRS), and in place of the 3’ mouse homology arm the human VL region is flanked 3’ with an SRRS.

In one aspect, a reverse chimeric antibody made by a mouse as described herein, wherein the reverse chimeric antibody comprises a light chain comprising a human VL and a mouse CL, and a heavy chain comprising a human VH and a mouse CH. in one aspect, a method for making an antibody is provided, comprising expressing in a single cell (a) a first VH gene sequence of an immunized mouse as described herein fused with a human CH gene sequence; (b) a VL gene sequence of an immunized mouse as described herein fused with a human CL gene sequence; and, (c) maintaining the cell under conditions sufficient to express a fully human antibody, and isolating the antibody. in one embodiment, the cell comprises a second VH gene sequence of a second immunized mouse as described herein fused with a human CH gene sequence, the first VH gene sequence encodes a VH domain that recognizes a first epitope, and the second VH gene sequence encodes a VH domain that recognizes a second epitope, wherein the first epitope and the second epitope are not identical. in one aspect, a method for making an epitope-binding protein is ed, comprising exposing a mouse as bed herein with an immunogen that comprises an epitope of interest, maintaining the mouse under conditions sufficient for the mouse to generate an immunoglobulin molecule that specifically binds the epitope of interest, and ing the immunoglobulin le that specifically binds the epitope of st; wherein the epitope-binding n comprises a heavy chain that comprises a somatically d human VH and a mouse CH, associated with a light chain comprising a mouse CL and a human VL derived from a rearranged human VK1-39JK5 or a rearranged human VK3- 20JK1. in one aspect, a cell that expresses an epitope-binding protein is provided, wherein the cell comprises: (a) a human nucleotide sequence encoding a human VL domain that is derived from a rearranged human VK1-39JK5 or a rearranged human JK1, wherein the human nucleotide sequence is fused (directly or through a linker) to a human immunoglobulin light chain constant domain cDNA sequence (9.9., a human K constant domain DNA sequence); and, (b) a first human VH nucleotide ce encoding a human VH domain derived from a first human VH nucleotide ce, wherein the first human VH nucleotide sequence is fused (directly or through a linker) to a human immunoglobulin heavy chain constant domain cDNA sequence; wherein the epitope-binding protein recognizes a first epitope. In one embodiment, the e-binding protein binds the first epitope with a dissociation constant of lower than 10‘6 M, lower than 10'8 M, lower than 10'9 M, lower than 1040 M, lower than 10’11 M, or lower than 10'12 M.

In one embodiment, the cell comprises a second human nucleotide sequence ng a second human VH domain, wherein the second human ce is fused (directly or through a linker) to a human immunoglobulin heavy chain constant domain cDNA sequence, and wherein the second human VH domain does not ically recognize the first epitope (e.g., displays a dissociation constant of, 6.9., 10'6 M, 10'5 M, 10' 4 M, or higher), and wherein the epitope—binding protein recognizes the first epitope and the second epitope, and wherein the first and the second immunoglobulin heavy chains each associate with an identical light chain of (a). in one embodiment, the second VH domain binds the second epitope with a dissociation constant that is lower than 10'6 M, lower than 10'7M, lowerthan 10‘8 M, lower than 10'9 M, lower than 1040 M, lower than 10‘11 M, or lower than 10‘12 M. in one embodiment, the epitope-binding protein comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, each associated with an identical light chain derived from a rearranged human VL region selected from a human VK1-39JK5 or a human VK3-20JK1, wherein the first immunoglobulin heavy chain binds a first epitope with a dissociation constant in the nanomolar to picomolar range, the second immunoglobulin heavy chain binds a second epitope with a dissociation constant in the nanomolar to picomolar range, the first epitope and the second epitope are not identical, the first immunoglobulin heavy chain does not bind the second epitope or binds the second epitope with a dissociation constant weaker than the micromolar range (e.g., the millimolar , the second immunoglobulin heavy chain does not bind the first epitope or binds the first epitope with a dissociation nt weaker than the micromolar range (e.g., the millimolar , and one or more of the VL, the VH of the first immunoglobulin heavy chain, and the VH of the second immunoglobulin heavy chain, are cally mutated.

In one embodiment, the first immunoglobulin heavy chain comprises a protein A- binding residue, and the second immunoglobulin heavy chain lacks the protein A-binding ln one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral c acid sequence (9.9., a PERC.6TM cell).

In one aspect, a reverse chimeric antibody is provided, comprising a human VH and a mouse heavy chain constant domain, a human VL and a mouse light chain constant domain, wherein the antibody is made by a s that comprises immunizing a mouse as described herein with an immunogen comprising an epitope, and the antibody specifically binds the epitope of the immunogen with which the mouse was immunized. In one embodiment, the VL domain is somatically mutated. in one embodiment the VH domain is somatically mutated. In one embodiment, both the VL domain and the VH domain are somatically mutated. In one embodiment, the VL is linked to a mouse CK domain.

In one aspect, a mouse is provided, comprising human VH gene segments replacing all or substantially all mouse VH gene segments at the nous mouse heavy chain locus; no more than one or two nged human light chain VL/JL sequences selected from a rearranged VK1-39/J and a rearranged VK3—20/J or a combination thereof, replacing all mouse light chain gene segments; wherein the human heavy chain variable gene segments are linked to a mouse constant gene, and the rearranged human light chain sequences are linked to a human or mouse constant gene.

In one aspect, a mouse ES cell comprising a replacement of all or substantially all mouse heavy chain variable gene ts with human heavy chain le gene segments, and no more than one or two nged human light chain VL/JL ces, wherein the human heavy chain variable gene segments are linked to a mouse globulin heavy chain constant gene, and the rearranged human light chain VL/JL sequences are linked to a mouse or human immunoglobulin light chain constant gene. In a specific embodiment, the light chain constant gene is a mouse constant gene. in one aspect, an antigen-binding protein made by a mouse as described herein is ed. in a specific embodiment, the n-binding n comprises a human immunoglobulin heavy chain variable region fused with a mouse constant region, and a human immunoglobulin light chain variable region derived from a VK’i-39 gene segment or a VK3—20 gene segment, wherein the light chain constant region is a mouse constant region. in one aspect, a fully human antigen-binding protein made from an immunoglobulin variable region gene sequence from a mouse as described herein is provided, wherein the n-binding protein comprises a fully human heavy chain comprising a human variable region derived from a sequence of a mouse as described , and a fully human light chain comprising a VK1—39 or a VK3-20. in one embodiment, the light chain variable region ses one to five c mutations. in one embodiment, the light chain variable region is a cognate light chain variable region that is paired in a B cell of the mouse with the heavy chain variable region.

In one embodiment, the fully human antigen-binding protein comprises a first heavy chain and a second heavy chain, wherein the first heavy chain and the second heavy chain comprise non-identical variable regions ndently derived from a mouse as bed herein, and wherein each of the first and second heavy chains express from a host cell associated with a human light chain derived from a VK1—39 gene segment or a VK3-20 gene segment. in one embodiment, the first heavy chain comprises a first heavy chain variable region that specifically binds a first epitope of a first antigen, and the second heavy chain comprises a second heavy chain variable region that specifically binds a second epitope of a second antigen. in a specific ment, the first antigen and the second antigen are different. in a specific embodiment, the first antigen and the second antigen are the same, and the first epitope and the second epitope are not cal; in a specific embodiment, binding of the first epitope by a first molecule of the binding protein does not block binding of the second epitope by a second molecule of the binding protein. in one aspect, a fully human binding protein derived from a human immunoglobulin sequence of a mouse as described herein comprises a first immunoglobulin heavy chain and a second globulin heavy chain, wherein the first immunoglobulin heavy chain comprises a first variable region that is not identical to a variable region of the second immunoglobulin heavy chain, and wherein the first immunoglobulin heavy chain comprises a wild type protein A binding determinant, and the second heavy chain lacks a wild type WO 48873 protein A binding determinant. In one embodiment, the first immunoglobuiin heavy chain binds protein A under isolation conditions, and the second immunoglobulin heavy chain does not bind protein A or binds n A at Ieast 10-foId, a hundred-fold, or a thousand- foId weaker than the first immunoglobulin heavy chain binds protein A under isolation conditions. In a specific embodiment, the first and the second heavy chains are lth isotypes, wherein the second heavy chain comprises a modification ed from 95R (EU 435R), 96F (EU 436F), and a combination thereof, and wherein the first heavy chain Iacks such modification.

In one aspect, a method for making a bispecific antigen—binding protein is provided, sing exposing a first mouse as described herein to a first antigen of interest that comprises a first epitope, exposing a second mouse as described herein to a second antigen of interest that ses a second epitope, ng 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 n of interest, expressing the first heavy chain and the second heavy chain in a cell that expresses a single fuIly human light chain derived from a human VK1-39 or a human VK3-20 gene segment to form a bispecific antigen—binding protein, and isolating the bispecific antigen— binding n.

In one embodiment, the first antigen and the second n are not identical.

In one ment, the first antigen and the second antigen are identical, and the first epitope and the second epitope are not identical. In one embodiment, binding of the first heavy chain variabIe region to the first epitope does not block binding of the second heavy chain variabIe region to the second epitope.

In one embodiment, the first antigen is selected from a soluble antigen and a cell surface antigen (e.g., a tumor antigen), and the second antigen comprises a cell surface receptor. In a specific embodiment, the cell surface or is an immunoglobuiin receptor. In a specific embodiment, the immunoglobulin receptor is an F0 or. In one embodiment, the first antigen and the second antigen are the same ceII surface receptor, and binding of the first heavy chain to the first epitope does not block binding of the second heavy chain to the second epitope.

In one embodiment, the light chain variabIe domain of the light chain comprises 2 to somatic ons. In one embodiment, the light chain variable domain is a somatically mutated cognate light chain expressed in a B cell of the first or the second immunized mouse with either the first or the second heavy chain variable domain. in one embodiment, the first fully human heavy chain bears an amino acid modification that reduces its affinity to protein A, and he second fully human heavy chain does not comprise a cation that reduces its ty to protein A. ln one aspect, an antibody or a bispecific antibody comprising a human heavy chain variable domain made in accordance with the invention is provided. in another aspect, use of a mouse as described herein to make a fully human antibody or a fully human bispecific antibody is provided. in one aspect, a genetically modified mouse, embryo, or cell described herein comprises a K light chain locus that retains endogenous regulatory or control elements, e.g., a mouse K intronic enhancer, a mouse K 3’ er, or both an ic er and a 3’ enhancer, wherein the regulatory or control elements facilitate somatic mutation and affinity maturation of an expressed sequence of the K light chain locus. in one aspect, a mouse is provided that comprises a B cell population characterized by having immunoglobulin light chains derived from no more than one, or no more than two, rearranged or unrearranged immunoglobulin light chain V and J gene segments, wherein the mouse exhibits a KI)» light chain ratio that is about the same as a mouse that comprises a wild type complement of immunoglobulin light chain V and J gene segments.

In one embodiment, the immunoglobulin light chains are derived from no more than one, or no more than two, rearranged immunoglobulin light chain V and J gene segments.

In a ic embodiment, the light chains are derived from no more than one rearranged immunoglobulin light chain V and J gene segments. in one embodiment, the mouse exhibits a Km light chain ratio that is about from 55:1 to 75:1, 60:1 to 70:1, 63:1 to 68:1, or about from 65:1 to 67:1 as compared to a mouse that comprises a wild type complement of immunoglobulin light chain V and J gene segments. In a specific embodiment, the mouse exhibits a K37» light chain ratio that is 66:1 as compared to a mouse that comprises a wild type complement of immunoglobulin light chain V and J gene segments. in one embodiment, the immunoglobulin light chains derived from no more than one, or no more than two, rearranged or ranged immunoglobulin light chain V and J gene segments include human VK and JK gene ts selected from human , human VK3-20, human JK1 and human JK5. In a specific embodiment, the immunoglobulin light chains are d from a single human light chain sequence comprising a human VK1-39 sequence.

In one embodiment, the mouse exhibits a K37» light chain ratio that is about from 18:1 to 23:1 or about from 19:1 to 22:1 as compared to a mouse that comprises a wild type WO 48873 complement of immunoglobulin light chain V and J gene segments. in one embodiment, the mouse exhibits a KI)» light chain ratio that is 21 :1 as ed to a mouse that comprises a wild type complement of immunoglobulin light chain V and J gene segments.

In one embodiment, the mouse exhibits a KI)» light chain ratio that is about the same or :1 as compared to a mouse that comprises a wild type complement of immunoglobulin light chain V and J gene segments. in one embodiment, the immunoglobulin light chains derived from no more than one, or no more than two, rearranged or unrearranged immunoglobulin light chain V and J gene segments include human VK and JK gene segments selected from human VK1—39, human , human JK1 and human JK5. in a specific embodiment, the immunoglobuiin light chains are derived from a single human light chain ce comprising a human VK3—20 sequence. ln one , a mouse as described herein is provided that expresses an immunoglobulin light chain derived from no more than one, or no more than two, human VK/JK sequences, wherein the mouse comprises a replacement of ali or substantially all endogenous mouse heavy chain variable region gene segments with one or more human heavy chain variable region gene segments, and the mouse exhibits a ratio of (a) CD19+ B cells that express an immunoglobulin having a A light chain, to (b) CD19+ B cells that express an globulin having a K light chain, of about 1 to about 20. in one embodiment, the mouse expresses a single K light chain derived from a human VK1-39JK5 sequence, and the ratio of CD19+ B cells that express an immunoglobulin having a x light chain to CD19+ B cells that express an immunoglobulin having a K light chain is about 1 to about 20; in one embodiment, the ratio is about 1 to at least about 66; in a specific embodiment, the ratio is about 1 to 66. in one embodiment, the mouse expresses a single K light chain derived from a human VK3-20JK5 sequence, and the ratio of CD19“ B cells that express an immunoglobulin having a 7» light chain to CD19+ B cells that express an immunoglobulin having a K light chain is about 1 to about 20; in one embodiment, the ratio is about 1 to about 21. in ic embodiments, the ratio is 1 to 20, or 1 to 21. in one aspect, a genetically modified mouse is provided that ses a single rearranged K light chain, wherein the mouse comprises a functional k light chain locus, and n the mouse expresses a B cell population that ses ng+ cells that express a K light chain derived from the same single rearranged K light chain. in one embodiment, the percent of ngJ'lgk+ B cells in the mouse is about the same as in a wild type mouse. In a ic embodiment, the percent of ng+lgk+ B cells in the mouse is about 2 to about 6 percent. in a specific embodiment, the percent of igK”lg7t+ B cells in a mouse wherein the single rearranged K light chain is derived from a VK1-39JK5 sequence is about 2 to about 3; in a specific embodiment, about 2.6. in a specific embodiment, the percent of ng“lg}»+ B cells in a mouse wherein the single rearranged K light chain is derived from a VK3-20JK1 sequence is about 4 to about 8; in a specific embodiment, about 6.

In one aspect, a genetically modified mouse is provided, wherein the mouse expresses a single rearranged K light chain derived from a human VK and JK gene segment, wherein the mouse expresses a B cell population that comprises a single K light chain derived from the single rearranged K light chain sequence, wherein the genetically modified mouse has not been rendered resistant to somatic hypermutations. in one embodiment, at least 90% of the K light chains expressed on a B cell of the mouse t from at least one to about five somatic hypermutations. in one aspect, a genetically modified mouse is provided that is modified to express a single K light chain d from no more than one, or no more than two, rearranged K light chain sequences, wherein the mouse exhibits a K light chain usage that is about two- fold or more, at least about three-fold or more, or at least about four-fold or more greater than the K light chain usage exhibited by a wild type mouse, or greater than the K light chain usage exhibited by a mouse of the same strain that comprises a wild type repertoire of K light chain gene ts. in a specific embodiment, the mouse expresses the single K light chain from no more than one nged K light chain sequence. in a more ic ment, the rearranged K light chain sequence is selected from a VK1—39JK5 and VK3—20JK1 sequence. in one embodiment, the nged K light chain sequence is a VK1-39JK5 sequence. in one embodiment, the rearranged K light chain sequence is a VK3-20JK1 sequence. ln one aspect, a genetically modified mouse is provided that expresses a single K light chain derived from no more than one, or no more than two, rearranged K light chain sequences, n the mouse exhibits a K light chain usage that is about ld or more, at least about ZOO—fold or more, at least about BOO—fold or more, at least about 400— fold or more, at least about SOD-fold or more, at least about GOO-fold or more, at least about 700~fold or more, at least about 800—fold or more, at least about ld or more, at least about 1000—fold or more greater than the same K light chain usage exhibited by a mouse g a complete or substantially complete human K light chain locus. in a specific embodiment, the mouse bearing a complete or ntially complete human K light chain locus lacks a functional unrearranged mouse K light chain sequence. in a specific embodiment, the mouse expresses the single K light chain from no more than one rearranged K light chain ce. In one embodiment, the mouse comprises one copy of 2012/034737 a rearranged K light chain sequence (9.9., a heterozygote). In one embodiment, the mouse comprises two copies of a rearranged K light chain sequence (tag, a gote).

In a more specific embodiment, the rearranged K light chain sequence is selected from a VK1-39JK5 and VK3-20JK1 sequence. In one embodiment, the nged K light chain ce is a VK1-39JK5 sequence. In one embodiment, the rearranged K light chain sequence is a JK1 sequence.

In one aspect, a genetically modified mouse is ed that expresses a single light chain derived from no more than one, or no more than two, rearranged light chain sequences, wherein the light chain in the genetically modified mouse ts a level of expression that is at least 10-fold to about LOGO—fold, 100~fold to about 1,000-fold, ZOO-fold to about 1,000-fold, BOO-fold to about fold, 400—fold to about 1,000-fold, SOD-fold to about 1,000—fold, (SOD-fold to about 1,000-fold, 700-fold to about 1,000-fold, BOO-fold to about 1,000-fold, or 900-fold to about 1,000-fold higher than expression of the same rearranged light chain exhibited by a mouse bearing a complete or substantially complete light chain locus. In one embodiment, the light chain comprises a human sequence. In a ic embodiment, the human sequence is a K sequence. In one embodiment, the human sequence is a A sequence. In one embodiment, the light chain is a fully human light chain.

In one embodiment, the level of expression is terized by quantitating mRNA of transcribed light chain sequence, and comparing it to transcribed light chain sequence of a mouse bearing a complete or substantially complete light chain locus.

In one aspect, a genetically modified mouse is provided that expresses a single K light chain derived from no more than one, or no more than two, rearranged K light chain sequences, wherein the mouse, upon immunization with antigen, exhibits a serum titer that is comparable to a wild type mouse immunized with the same antigen. In a specific embodiment, the mouse expresses a single K light chain from no more than one rearranged K light chain sequence. In one ment, the serum titer is characterized as total immunoglobulin. In a ic ment, the serum titer is characterized as IgM specific titer. In a specific embodiment, the serum titer is characterized as lgG ic titer. In a more specific embodiment, the rearranged K light chain sequence is selected from a VK1-39JK5 and VK3-20JK1 sequence. In one embodiment, the rearranged K light chain sequence is a VK1-39JK5 sequence. In one embodiment, the rearranged K light chain sequence is a VK3-20JK1 ce.

In one aspect, a genetically modified mouse is provided that expresses a plurality of immunoglobulin heavy chains associated with a single light chain. In one embodiment, the heavy chain comprises a human sequence. In various embodiments, the human sequence is ed from a variable sequence, a CH1, a hinge, a CH2, a CH3, and a combination f. ln one embodiment, the single light chain comprises a human sequence. in various embodiments, the human sequence is selected from a variable ce, a constant sequence, and a combination thereof. In one embodiment, the mouse comprises a disabled endogenous immunoglobulin locus and expresses the heavy chain and/or the light chain from a transgene or extrachromosomal episome. in one embodiment, the mouse comprises a replacement at an endogenous mouse locus of some or all endogenous mouse heavy chain gene ts (i.e., V, D, J), and/or some or all endogenous mouse heavy chain constant sequences (9.9., CH1, hinge, CH2, CH3, or a combination thereof), and/or some or all endogenous mouse light chain sequences (9.9., V, J, constant, or a combination thereof), with one or more human immunoglobulin ces. in one aspect, a mouse suitable for making antibodies that have the same light chain is provided, wherein all or substantially all antibodies made in the mouse are expressed with the same light chain. ln one embodiment, the light chain is sed from an endogenous light chain locus. ln one aspect, a method for making a light chain for a human antibody is provided, sing obtaining from a mouse as described herein a light chain sequence and a heavy chain sequence, and employing the light chain sequence and the heavy chain sequence in making a human antibody. ln one embodiment, the human antibody is a bispecific antibody.

Any of the ments and aspects described herein can be used in conjunction with one another, unless otherwise ted or apparent from the context. Other embodiments will become apparent to those skilled in the art from a review of the ensuing descnpﬁon.

BRIEF DESCRIPTION OF THE FIGURES FlG. 1 illustrates a targeting strategy for replacing endogenous mouse immunoglobulin light chain variable region gene segments with a human VK1-39JK5 gene region. illustrates a targeting strategy for replacing nous mouse globulin light chain variable region gene segments with a human VK3-20JK1 gene region. illustrates a ing strategy for replacing endogenous mouse immunoglobulin light chain variable region gene segments with a human VpreB/JAS gene region. shows the percent of CD19+ B cells (y~axis) from peripheral blood for wild type mice (WT), mice homozygous for an engineered human rearranged VK1-39JK5 light chain region (VK1-39JK5 HO) and mice gous for an engineered human rearranged VK3—20JK1 light chain region (VK3-20JK1 HO). shows the ve mRNA expression (y-axis) of a VK1derived light chain in a quantitative PCR assay using probes specific for the junction of an engineered human rearranged VK1~39JK5 light chain region (VK1-39JK5 Junction Probe) and the human VK1-39 gene segment (VK1-39 Probe) in a mouse homozygous for a replacement of the endogenous VK and JK gene segments with human VK and JK gene ts (HK), a wild type mouse (WT), and a mouse heterozygous for an ered human rearranged VK1—39JK5 light chain region (VK1—39JK5 HET). Signals are normalized to expression of mouse CK. N.D.: not detected. ] shows the relative mRNA expression (y—axis) of a VK1-39—derived light chain in a quantitative PCR assay using probes specific for the junction of an engineered human rearranged VK1-39JK5 light chain region (VK1-39JK5 Junction Probe) and the human VK1-39 gene segment (VK1-39 Probe) in a mouse homozygous for a replacement of the nous VK and JK gene segments with human VK and JK gene segments (HK), a wild type mouse (WT), and a mouse homozygous for an engineered human rearranged VK1—39JK5 light chain region (VK1—39JK5 HO). s are normalized to sion of mouse CK. shows the relative mRNA expression (y—axis) of a VK3derived light chain in a quantitative PCR assay using probes specific for the junction of an engineered human rearranged VK3-20JK1 light chain region (VK3-20JK1 Junction Probe) and the human VK3—20 gene segment 0 Probe) in a mouse homozygous for a replacement of the endogenous VK and JK gene segments with human VK and JK gene segments (HK), a wild type mouse (WT), and a mouse heterozygous (HET) and homozygous (HO) for an engineered human rearranged VK3—20JK1 light chain region, Signals are normalized to sion of mouse CK. shows lgM (left) and lgG (right) titer in wild type (WT; N=2) and mice homozygous for an engineered human rearranged VK1-39JK5 light chain region (VK1-39JK5 HO; N=2) immunized with B-galactosidase.

FIG. GB shows total immunoglobulin (lgM, lgG, lgA) titer in wild type (WT; N=5) and mice homozygous for an engineered human rearranged VK3—20JK1 light chain region (VK3-20JK1 HO; N=5) immunized with [S—galactosiolase.

DETAILED DESCRIPTION This invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. it is also to be understood that the terminology used herein is for the e of describing particular ments only, and is not intended to be limiting, since the scope of the present invention is defined by the claims.

Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or y apparent from the context in which the term or phrase is used.

Although any methods and materials similar or equivalent to those described herein can be used in the ce or testing of the present invention, particular methods and materials are now described. All ations mentioned are hereby incorporated by reference.

The term "antibody", as used , includes immunogiobulin molecuies comprising four polypeptide , two heavy (H) chains and two light (L) chains inter- connected by disuifide bonds. Each heavy chain comprises a heavy chain variable (VH) region and a heavy chain constant region (CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable (VL) region and a tight chain constant region (CL). The VH and VL regions can be further subdivided into regions of ariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework s (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino- us to carboxy-terminus in the following order: FR1, CDR1, FR2, CDRZ, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDRZ and HCDRS; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3. The term “high affinity” antibody refers to an antibody that has a KD with respect to its target epitope about of 10'9 M or tower (e.g., about 1 x 10'9 M, 1 x10'10 M, 1 x 10”11 M, or about 1 x 10'12 M). in one embodiment, KD is measured by e plasmon resonance, e.g., BlACORETM; in another embodiment, KD is measured by ELiSA.

The phrase cific antibody” includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two nonidentical heavy , with each heavy chain icaiiy binding a different epitope-either on two different molecules (e.g., different epitopes on two different immunogens) or on the same molecule (e.g., different epitopes on the same immunogen). if a ific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four or more orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. Epitopes specifically bound by the bispecific dy can be on the same or a different target (e.g., on the same or a different protein).

Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same immunogen. For example, nucleic acid ces encoding heavy chain variable sequences that recognize different epitopes of the same immunogen can be fused to nucleic acid sequences encoding the same or different heavy chain constant s, and such ces can be expressed in a cell that expresses an globulin light chain. A typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer epitope—binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain epitope-binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.

The term “cell” includes any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include those of yotes and eukaryotes (single—cell or multiple—cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc), mycobacteria cells, fungal cells, yeast cells (e.g., S. siae, S. pombe, P. pastoris, P. methanolica, etc), plant cells, insect cells (e.g., SF-Q, SF-21, baculovirus—infected insect cells, plusia ni, etc), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. in some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO K1, DXB—11 CHO, Veggie—CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, Wl38, MRC 5, Col0205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV—1, U937, 3T3, L cell, C127 cell, SP2/0, NS-O, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. in some ments, the cell comprises one or more viral genes, 9.9., a retinal cell that expresses a viral gene (e.g., a PER.C6TM cell).

The phrase “complementarity determining region,” or the term “CDR,” includes an amino acid sequence encoded by a nucleic acid sequence of an sm’s immunoglobulin genes that normally (i.e., in a wild type animal) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). A CDR can be d by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell. A CDR can be cally mutated (e.g., vary from a ce encoded in an animal’s germline), humanized, and/or modified with amino acid substitutions, additions, or deletions. In some circumstances (e.g., for a CDRS), CDRs can be encoded by two or more sequences (e.g., germiine sequences) that are not uous (e.g., in an unrearranged nucleic acid ce) but are uous in a B cell nucleic acid sequence, e.g., as the result of splicing or connecting the sequences (e.g., V—D—J recombination to form a heavy chain CDR3).

The term ”conservative,” when used to describe a conservative amino acid tution, es substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). in general, a vative amino acid substitution will not substantially change the functional properties of st of a protein, for example, the ability of a variable region to specifically bind a target epitope with a desired affinity. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, , leucine, and cine; aliphatic- hydroxyl side chains such as serine and threonine; amide—containing side chains such as gine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, ne, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur—containing side chains such as cysteine and methionine. Conservative amino acids substitution groups e, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, ate/aspartate, and asparagine/glutamine. in some embodiments, a conservative amino acid substitution can be substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis. in some embodiments, a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix sed in Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45, hereby incorporated by reference. In some embodiments, the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 kelihood matrix.

In some embodiments, residue positions in an immunoglobulin light chain or heavy chain differ by one or more conservative amino acid substitutions. In some embodiments, residue positions in an immunoglobulin light chain or functional fragment thereof (e.g., a fragment that allows expression and secretion from, e.g., a B cell) are not identical to a light chain whose amino acid sequence is listed herein, but differs by one or more conservative amino acid substitutions.

The phrase “epitope—binding protein” es a protein having at least one CDR and that is capable of selectively recognizing an epitope, e.g., is capable of binding an epitope with a KD that is at about one micromolar or lower (e.g., a KD that is about 1 x '6 M, 1 x10”7 M, 1 x10’9M, 1 x10‘9 M, 1 x 10-10 M, 1 x 10'11 M, or about1 x 10'12 M).

Therapeutic epitope-binding proteins (e. g., therapeutic antibodies) frequently require a KD that is in the nanomolar or the picomolar range.

The phrase “functional fragment” includes fragments of epitope-binding proteins that can be expressed, secreted, and specifically bind to an epitope with a KD in the micromolar, nanomolar, or picomolar range. Specific recognition includes having a KD that is at least in the olar range, the nanomolar range, or the picomolar range.

The term “germline” includes reference to an immunoglobulin nucleic acid sequence in a non-somatically mutated cell, e.g., a non-somatically mutated B cell or pre—B cell or hematopoietic cell.

The phrase “heavy ” or “immunoglobulin heavy chain” includes an immunoglobulin heavy chain constant region sequence from any organism. Heavy chain variable domains include three heavy chain CDRs and four FR s, unless ise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and ations thereof. A typical heavy chain has, following the variable domain (from N-terminal to C- al), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain es a fragment that is capable of specifically recognizing an epitope (e.g., recognizing the e with a K0 in the micromolar, nanomolar, or picomolar range), that is capable of expressing and ing from a cell, and that comprises at least one CDR.

The term “identity” when used in connection with sequence includes identity as determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments described herein, identities are determined using a lW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap y of 0.1, and using a Gonnet similarity matrix (MACVECTORTM , MacVector lnc., 2008). The length of the sequences compared with respect to identity of sequences will depend upon the particular sequences, but in the case of a light chain nt domain, the length should contain sequence of sufficient length to fold into a light chain constant domain that is capable of self-association to form a canonical light chain constant domain, e.g., capable of forming two beta sheets comprising beta strands and capable of interacting with at least one CH1 domain of a human or a mouse. ln the case of a CH1 domain, the length of ce should contain sequence of sufficient length to fold into a CH1 domain that is capable of forming two beta sheets comprising beta strands and capable of cting with at least one light chain constant domain of a mouse or a human.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavy chains and two immunoglobulin light chains. The heavy chains may be cal or different, and the light chains may be identical or different.

The phrase “light chain” includes an immunoglobulin light chain sequence from any organism, and unless otherwise specified es human K and A light chains and a VpreB, as well as surrogate light chains. Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise ied. Generally, a full—length light chain includes, from amino us to carboxyl terminus, a VL domain that includes R1-FR2—CDR2-FR3-CDR3-FR4, and a light chain constant domain. Light chains include those, 6.9., that do not selectively bind either a first or a second epitope selectively bound by the epitope-binding protein in which they appear. Light chains also include those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more epitopes ively bound by the epitope- binding protein in which they appear. Common light chains are those derived from a rearranged human V1<1~39Ji<5 sequence or a rearranged human VK3-20JK1 sequence, and include somatically mutated (e.g., affinity matured) versions.

The phrase “micromolar range” is intended to mean 1-999 micromolar; the phrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase olar range” is intended to mean 1—999 lar.

The phrase “somatically d” includes reference to a nucleic acid sequence from a B cell that has undergone class—switching, wherein the nucleic acid sequence of an immunoglobulin variable region (9.9., a heavy chain le domain or including a heavy chain CDR or FR sequence) in the class—switched B cell is not identical to the nucleic acid sequence in the B cell prior to class-switching, such as, for example, a difference in a CDR or ork nucleic acid sequence n a B cell that has not undergone class—switching and a B cell that has undergone class-switching. “Somatically mutated” includes reference to nucleic acid sequences from affinity-matured B cells that are not identical to corresponding immunoglobulin variable region sequences in B cells that are not ty—matured (i. e., sequences in the genome of germline cells). The phrase “somatically mutated” also includes reference to an immunoglobulin variable region c acid ce from a B cell after exposure of the B cell to an epitope of interest, wherein the nucleic acid sequence differs from the corresponding nucleic acid sequence prior to exposure of the B cell to the epitope of interest. The phrase “somatically mutated” refers to sequences from antibodies that have been generated in an animal, e.g., a mouse having human immunoglobulin variable region nucleic acid sequences, in response to an immunogen challenge, and that result from the selection processes ntly operative in such an animal.

The term rranged,” with reference to a nucleic acid sequence, includes nucleic acid sequences that exist in the germline of an animal cell.

The phrase “variable domain” includes an amino acid sequence of an immunoglobulin light or heavy chain (modiﬁed as d) that comprises the following amino acid regions, in sequence from N-terminal to C-terminal s otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Common Light Chain Prior efforts to make useful multispecific epitope-binding proteins, e.g., bispecific antibodies, have been hindered by variety of problems that frequently share a common paradigm: in vitro selection or manipulation of sequences to rationally engineer, or to engineer through and-error, a suitable format for pairing a dimeric bispecific human immunoglobulin. Unfortunately, most if not all of the in vitro engineering approaches provide largely ad hoc fixes that are le, if at all, for individual molecules.

On the other hand, in vivo methods for employing x organisms to select appropriate pairings that are capable of leading to human therapeutics have not been realized. ] lly, native mouse ces are frequently not a good source for human therapeutic sequences. For at least that , generating mouse heavy chain immunoglobulin variable regions that pair with a common human light chain is of limited practical utility. More in vitro engineering efforts would be ed in a trial-and-error process to try to humanize the mouse heavy chain variable sequences while hoping to retain epitope specificity and affinity while maintaining the ability to couple with the common human light chain, with uncertain outcome. At the end of such a process, the final product may maintain some of the specificity and affinity, and associate with the common light chain, but ultimately immunogenicity in a human would likely remain a profound risk.

Therefore, a suitable mouse for making human therapeutics would include a suitably large repertoire of human heavy chain variable region gene segments in place of endogenous mouse heavy chain variable region gene segments. The human heavy chain variable region gene segments should be able to rearrange and recombine with an endogenous mouse heavy chain constant domain to form a reverse chimeric heavy chain (i.e., a heavy chain comprising a human variable domain and a mouse constant region).

The heavy chain should be capable of class switching and somatic hypermutation so that a suitably large repertoire of heavy chain variable domains are available for the mouse to select one that can associate with the limited repertoire of human light chain variable regions.

A mouse that selects a common light chain for a plurality of heavy chains has a practical y. in various ments, antibodies that express in a mouse that can only express a common light chain will have heavy chains that can associate and express 2012/034737 with an identical or substantially cal light chain. This is ularly useful in making bispecific dies. For example, such a mouse can be immunized with a first gen to generate a B cell that expresses an antibody that specifically binds a first epitope. The mouse (or a mouse genetically the same) can be immunized with a second immunogen to generate a B cell that expresses an antibody that specifically binds the second epitope. Variable heavy s can be cloned from the B cells and expresses with the same heavy chain constant region, and the same light chain, and expressed in a cell to make a bispecific antibody, wherein the light chain component of the bispecific antibody has been selected by a mouse to associate and s with the light chain component.

The inventors have engineered a mouse for generating immunoglobulin light chains that will suitably pair with a rather diverse family of heavy chains, including heavy chains whose variable regions depart from germline sequences, e.g., affinity matured or somatically mutated variable regions. ln various embodiments, the mouse is devised to pair human light chain variable domains with human heavy chain variable domains that comprise somatic mutations, thus enabling a route to high affinity binding proteins suitable for use as human therapeutics.

The genetically engineered mouse, through the long and complex process of antibody selection within an organism, makes biologically appropriate s in pairing a diverse collection of human heavy chain variable domains with a limited number of human light chain options. ln order to achieve this, the mouse is engineered to present a limited number of human light chain variable domain options in ction with a wide diversity of human heavy chain le domain options. Upon challenge with an immunogen, the mouse maximizes the number of solutions in its repertoire to develop an antibody to the immunogen, limited largely or solely by the number or light chain options in its repertoire. in various embodiments, this includes allowing the mouse to achieve suitable and compatible c mutations of the light chain variable domain that will eless be compatible with a relatively large variety of human heavy chain variable domains, including in particular somatically mutated human heavy chain variable domains.

] To achieve a limited repertoire of light chain options, the mouse is engineered to render ctional or substantially nonfunctional its ability to make, or rearrange, a native mouse light chain variable domain. This can be ed, e.g., by deleting the mouse’s light chain variable region gene segments. The endogenous mouse locus can then be modified by an exogenous le human light chain variable region gene segment of choice, operably linked to the endogenous mouse light chain constant domain, in a manner such that the exogenous human variable region gene segments can combine with the endogenous mouse light chain constant region gene and form a rearranged e chimeric light chain gene (human variable, mouse constant). in various embodiments, the light chain variable region is capable of being somatically mutated. in various ments, to maximize ability of the light chain variable region to acquire somatic mutations, the appropriate enhancer(s) is retained in the mouse. For example, in modifying a mouse K light chain locus to replace endogenous mouse K light chain gene segments with human K light chain gene segments, the mouse K intronic enhancer and mouse K 3’ enhancer are functionally maintained, or undisrupted.

A genetically engineered mouse is ed that expresses a limited repertoire of reverse chimeric (human variable, mouse constant) light chains ated with a diversity of reverse chimeric (human variable, mouse constant) heavy . In various embodiments, the endogenous mouse K light chain gene segments are deleted and ed with a single (or two) rearranged human light chain region, operably linked to the endogenous mouse CK gene. in embodiments for zing somatic hypermutation of the rearranged human light chain region, the mouse K intronic enhancer and the mouse K 3’ enhancer are maintained. in various ments, the mouse also comprises a nonfunctional )V light chain locus, or a deletion thereof or a deletion that renders the locus unable to make a )V light chain.

A genetically engineered mouse is provided that, in various embodiments, comprises a light chain variable region locus g endogenous mouse light chain VL and JL gene segments and comprising a rearranged human light chain variable , in one embodiment a rearranged human VL/JL sequence, operably linked to a mouse constant region, n the locus is capable of undergoing somatic hypermutation, and wherein the locus expresses a light chain comprising the human VL/JL sequence linked to a mouse constant region. Thus, in various embodiments, the locus comprises a mouse K 3’ enhancer, which is correlated with a normal, or wild type, level of somatic hypermutation.

] The genetically ered mouse in various embodiments when immunized with an antigen of interest generates B cells that exhibit a diversity of rearrangements of human immunoglobulin heavy chain variable regions that express and function with one or with two rearranged light , including embodiments where the one or two light chains comprise human light chain variable regions that comprise, e.g., 1 to 5 somatic mutations. in various embodiments, the human light chains so expressed are capable of ating and expressing with any human immunoglobulin heavy chain variable region expressed in the mouse.

Epitope-binding Proteins Binding More Than One Epitope The compositions and methods of described herein can be used to make binding proteins that bind more than one epitope with high affinity, e.g., bispecific dies. Advantages of the invention include the ability to select suitably high binding (e.g., affinity d) heavy chain immunoglobulin chains each of which will associate with a single light chain.

Synthesis and expression of bispecific binding ns has been problematic, in part due to issues associated with fying a le light chain that can associate and express with two different heavy chains, and in part due to isolation issues. The methods and compositions described herein allow for a genetically modified mouse to select, through otherwise natural processes, a suitable light chain that can associate and express with more than one heavy chain, including heavy chains that are somatically mutated (e.g., affinity matured). Human VL and VH sequences from le B cells of immunized mice as bed herein that express affinity d antibodies having e chimeric heavy chains (i.e., human variable and mouse constant) can be identified and cloned in frame in an expression vector with a suitable human constant region gene sequence (e.g., a human lgG1). Two such constructs can be prepared, wherein each construct encodes a human heavy chain variable domain that binds a different epitope. One of the human VLs (e.g., human J5 or human V3-20J1), in germline sequence or from a B cell wherein the sequence has been somatically mutated, can be fused in frame to a suitable human constant region gene (e.g., a human  constant gene). These three fully human heavy and light constructs can be placed in a suitable cell for expression. The cell will express two major species: a homodimeric heavy chain with the identical light chain, and a heterodimeric heavy chain with the cal light chain. To allow for a facile tion of these major species, one of the heavy chains is ed to omit a Protein A-binding determinant, resulting in a differential affinity of a meric binding protein from a heterodimeric binding protein.

Compositions and methods that address this issue are described in USSN 12/823,838, filed June 2010, entitled “Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format,” published as US 2010/0331527A1, hereby incorporated by reference.

In one aspect, an epitope-binding protein as described herein is provided, wherein human VL and VH sequences are derived from mice described herein that have been zed with an antigen comprising an epitope of interest.

In one embodiment, an epitope-binding protein is provided that comprises a first and a second polypeptide, the first polypeptide comprising, from N-terminal to C-terminal, a first epitope-binding region that selectively binds a first epitope, followed by a constant region that ses a first CH3 region of a human IgG selected from lgG1, IgG2, lgG4, and a combination thereof; and, a second polypeptide comprising, from N-terminal to C-terminal, a second epitope-binding region that selectively binds a second epitope, followed by a constant region that comprises a second CH3 region of a human lgG selected from lgG1, lgGZ, lgG4, and a combination thereof, wherein the second CH3 region comprises a modification that reduces or eliminates binding of the second CH3 domain to protein A.

In one embodiment, the second CH3 region comprises an H95R modification (by lMGT exon numbering; H435R by EU numbering). in another embodiment, the second CH3 region r ses a Y96F modification (lMGT; Y436F by EU). in one embodiment, the second CH3 region is from a modified human lth, and further comprises a modification ed from the group consisting of D16E, L18M, N448, K52N, V57M, and V82l (lMGT; D356E, L358M, N384S, K392N, V397M, and V422l by EU).

In one embodiment, the second CH3 region is from a modified human lgGZ, and further comprises a modification selected from the group consisting of N448, K52N, and V82l (IMGT; N384S, K392N, and V422l by EU). in one embodiment, the second CH3 region is from a ed human lgG4, and further comprises a cation selected from the group consisting of 015R, N448, K52N, V57M, R69K, E790, and V82l (IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422l by EU).

One method for making an epitope-binding protein that binds more than one epitope is to immunize a first mouse in accordance with the invention with an antigen that comprises a first epitope of st, wherein the mouse comprises an endogenous immunoglobulin light chain le region locus that does not contain an endogenous mouse VL that is e of rearranging and forming a light chain, n at the endogenous mouse immunoglobulin light chain variable region locus is a single rearranged human VL region operably linked to the mouse nous light chain nt region gene, and the rearranged human VL region is selected from a human VK1-39JK5 and a human VK3—20JK1, and the endogenous mouse VH gene segments have been replaced in whole or in part with human VH gene segments, such that immunoglobulin heavy chains made by the mouse are solely or substantially heavy chains that comprise human variable domains and mouse constant s. When immunized, such a mouse will make a reverse chimeric antibody, comprising only one of two human light chain variable domains (e.g., one of human VK1-39JK5 or human VK3-20JK1). Once a B cell is identified that encodes a VH that binds the epitope of interest, the nucleotide sequence of the VH (and, optionally, the VL) can be retrieved (e.g., by PCR) and cloned into an expression construct in frame with a suitable human immunoglobulin constant domain. This process can be repeated to identify a second VH domain that binds a second epitope, and a second VH gene sequence can be retrieved and cloned into an expression vector in frame to a second suitable immunoglobulin constant domain. The first and the second globulin constant domains can the same or different isotype, and one of the immunoglobulin constant domains (but not the other) can be modified as described herein or in US 2010/0331527A1, and e-binding protein can be expressed in a suitable cell and isolated based on its differential affinity for n A as compared to a homodimeric epitope—binding protein, e.g., as described in US 2010/0331527A1.

In one embodiment, a method for making a bispecific epitope-binding protein is provided, comprising fying a first affinity-matured (e.g., comprising one or more somatic hypermutations) human V... nucleotide ce (VH1) from a mouse as described herein, identifying a second affinity-matured (e.g., comprising one or more somatic hypermutations) human VH nucleotide sequence (VH2) from a mouse as described herein, cloning VH1 in frame with a human heavy chain lacking a Protein A—determinant modification as described in US 2010/0331527A1 for form heavy chain 1 (H01), g VH2 in frame with a human heavy chain comprising a Protein A-determinant as described in US 2010/0331527A1 to form heavy chain 2 (HC2), introducing an expression vector comprising HC1 and the same or a different expression vector comprising H02 into a cell, wherein the cell also ses a human immunoglobulin light chain that comprises a human VK1-39/human JK5 or a human VK3—20/human JK1 fused to a human light chain constant domain, allowing the cell to express a bispecific e—binding protein sing a VH domain encoded by VH1 and a VH domain encoded by VH2, and isolating the bispecific epitope-binding protein based on its differential ability to bind Protein A as compared with a monospecific homodimeric epitope-binding protein. In a specific ment, HC1 is an lgG1, and H02 is an |gG1 that comprises the modification H95R (lMGT; H435R by EU) and further comprises the modification Y96F (lMGT; Y436F by EU). in one embodiment, the VH domain encoded by VH1, the VH domain encoded by VH2, or both, are somatically mutated.

Human VH Genes That Express with a Common Human VL A variety of human variable s from affinity—matured antibodies raised against four different antigens were expressed with either their cognate light chain, or at least one of a human light chain selected from human VK1-39JK5, human JK1, or human VpreBJkS (see Example 1). For antibodies to each of the antigens, somatically mutated high affinity heavy chains from different gene families paired sfully with rearranged human germline VK1-39JK5 and VK3—20JK1 regions and were secreted from cells expressing the heavy and light . For VK1-39JK5 and VK3-20JK1, VH domains d from the ing human VH gene families expressed favorably: 1—2, 1-8, 1-24, 2- , 3-7, 3-9, 3—11, 3-13,3—15,3-20, 3—23, 3-30, 3-33, 3-48, 4-31, 4-39, 4—59, 5-51, and 6—1.

Thus, a mouse that is engineered to express a limited repertoire of human VL s from one or both of VK1-39JK5 and VK3—20JK1 will generate a diverse population of cally mutated human VH domains from a VH locus modified to replace mouse VH gene segments with human VH gene segments.

Mice cally engineered to express reverse chimeric (human variable, mouse nt) immunoglobulin heavy chains associated with a single rearranged light chain (9.9., a /J or a VK3—20/J), when immunized with an antigen of interest, generated B cells that comprised a diversity of human VH rearrangements and sed a ity of high-affinity antigen-specific dies with diverse properties with respect to their ability to block binding of the antigen to its ligand, and with respect to their y to bind variants of the antigen (see Examples 5 through 10).

Thus, the mice and methods bed herein are useful in making and selecting human immunoglobulin heavy chain variable domains, including somatically mutated human heavy chain variable domains, that result from a diversity of rearrangements, that exhibit a wide variety of affinities (including exhibiting a KB of about a nanomolar or less), a wide variety of specificities (including binding to different epitopes of the same antigen), and that associate and s with the same or substantially the same human immunoglobulin light chain variable region.

The following examples are provided so as to describe to those of ordinary skill in the art how to make and use methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc) but some experimental errors and ions should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is indicated in Celsius, and pressure is at or near atmospheric.

EXAMPLES The following examples are provided so as to describe how to make and use methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, temperature is indicated in Celsius, and pressure is at or near atmospheric.

Example 1 identification of Human VH Regions That ate with Selected Human VL Regions ] An in vitro expression system was constructed to determine if a single rearranged human germline light chain could be co-expressed with human heavy chains from n specific human antibodies.

Methods for generating human antibodies in genetically modified mice are known (see e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOClMMUNE®). The VELOClMMUNE® technology involves generation of a genetically ed mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse es an antibody comprising a human variable region and a mouse nt region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibodies produced from a VELOClMMUNE® mouse are fully human. initially, high ty chimeric antibodies are isolated having a human variable region and a mouse constant region. As described below, the antibodies are characterized and selected for ble characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate a fully human antibody containing a non-lgM isotype, for example, wild type or modified lgG1, lgGZ, lgG3 or lgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

A VELOClMMUNE® mouse was immunized with a growth factor that promotes angiogenesis (Antigen C) and antigen-specific human antibodies were isolated and sequenced for V gene usage using standard techniques ized in the art.

Selected antibodies were cloned onto human heavy and light chain constant s and 69 heavy chains were selected for pairing with one of three human light chains: (1) the cognate K light chain linked to a human K constant region, (2) a rearranged human germline VK1—39JK5 linked to a human K nt region, or (3) a rearranged human germline JK1 linked to a human K constant region. Each heavy chain and light chain pair were co-transfected in CHO-K1 cells using standard techniques. Presence of dy in the supernatant was detected by uman lgG in an ELlSA assay. Antibody titer (ng/ml) was determined for each heavy chain/light chain pair and titers with the different rearranged germline light chains were compared to the titers obtained with the parental antibody molecule (i.e., heavy chain paired with cognate light chain) and percent of native titer was calculated (Table 1). VH: Heavy chain variable gene. ND: no expression detected under current experimental conditions. 2012/034737 Table 1 Antibody Titer (ng/mL) Percent of Native Titer Cognate LC VK1-39JK5 VK3—20JK1 VK1-39JK5 VK3-20JK1 .m-—_ ()0 00 157.1 .2 254.3 N(.0 2508.3 225.9 ND .3 81 _.\ 150.7 .4 A 01A 203.0 38 2U 23 212.7 0) (JO (.0 .4 C) CD ._.\ 0) 00 182 3C») “’1‘moo wd 133.8 I mow NM ‘4‘ IN Ax] ooxnco—s NOONN 0'! 253.0 3—23 53 93 151 175.4 3-33 .4 .A L ()0 0) 3-15 \1 (A) U) (.0 AN Ax: 53.7 0)J) .3 07 5600.0 57 192.9 N N5 A(0.4 91 478.4 (A) NU1 U1 (0 U10) 342.7 H0'! A O 19 184.4 (II .b. MO .53 ()0 b)N 83-3 NO .3. 00 24 (JOA Nto 137.3 17 33 105.2 \l \lL 284.6 87 _\ N ND 145.1 — 01 O) 00 K) NZ 00 .1 ‘r’00 Q) _\ O 01 03 ‘r’ ()3 00 NO 0) \l IIII_—\——\ 316.8 —158 4 -m- as A —-m_— m.-—- 3-7 124 --—41 130.0 In a similar experiment, VELOCIMMUNE® mice were immunized with several different antigens and selected heavy chains of antigen specific human antibodies were tested for their y to pair with different rearranged human germline light chains (as described above). The antigens used in this experiment included an enzyme involved in cholesterol homeostasis (Antigen A), a serum hormone ed in regulating glucose homeostasis (Antigen B), a growth factor that promotes angiogenesis (Antigen C) and a cell-surface receptor (Antigen D). Antigen specific antibodies were ed from mice of each immunization group and the heavy chain and light chain le regions were cloned and sequenced. From the sequence of the heavy and light chains, V gene usage was determined and selected heavy chains were paired with either their cognate light chain or a rearranged human ne VK1-39JK5 region. Each heavy/light chain pair was sfected in CHO-K1 cells and the presence of antibody in the supernatant was detected by anti-human lgG in an ELISA assay. Antibody titer (pg/ml) was determined for each heavy chain/light chain pairing and titers with the different rearranged human germline light chains were compared to the titers obtained with the parental antibody molecule (i.e., heavy chain paired with cognate light chain) and percent of native titer was calculated (Table 2). VH1 Heavy chain variable gene. VK: K light chain variable gene. ND: no expression detected under t experimental conditions.

Table 2 Titer (pg/ml) Percent of Antigen Antibody VH VK VH+ VHAlone VH+VK Native Titer VK1-39JK5 320 1—18 2—30 0.3 3.1 2.0 66 321 2—5 2-28 0.4 0.4 1.9 448 334 2—5 2-28 0.4 2.7 2.0 73 A 313 3~13 3-15 0.5 0.7 4.5 670 316 3—23 4-1 0.3 0.2 4.1 2174 315 330 4-1 0.3 0.2 3.2 1327 318 4—59 1-17 0.3 4.6 4.0 86 257 3-13 1-5 0.4 3.1 3.2 104 283 313 1—5 0.4 5.4 3.7 69 637 3-13 1-5 0.4 4.3 3.0 70 638 3-13 1-5 0.4 4.1 3.3 82 B 624 3—23 1-17 0.3 5.0 3.9 79 284 3-30 1-17 0.3 4.6 3.4 75 653 3-33 1—17 0.3 4.3 0.3 7 268 4-34 1—27 0.3 5.5 3.8 69 633 4-34 1-27 0.6 6.9 3.0 44 730 3—7 1—5 0.3 1.1 2.8 249 728 3-7 1—5 0.3 2.0 3.2 157 691 3—9 3-20 0.3 2.8 3.1 109 749 3-33 3~15 0.3 3.8 2.3 62 750 3—33 1-16 0.3 3.0 2.8 92 724 3—33 1-17 0.3 2.3 3.4 151 706 3-33 1-16 0.3 3.6 3.0 84 744 1-18 1-12 0.4 5.1 3.0 59 696 3-11 1-16 0.4 3.0 2.9 97 685 3-13 3-20 0.3 0.5 3.4 734 732 3-15 1-17 0.3 4.5 3.2 72 694 3-15 1-5 0.4 5.2 2.9 55 743 3—23 1-12 0.3 3.2 0.3 10 742 3-23 2-28 0.4 4.2 3.1 74 693 3-23 1-12 0.5 4.2 4.0 94 136 3-23 2-28 0.4 5.0 2.7 55 155 3—30 1—16 0.4 1.0 2.2 221 163 3-30 1-16 0.3 0.6 3.0 506 171 3-30 1—16 0.3 1.0 2.8 295 145 3-43 1-5 0.4 4.4 2.9 65 49 3-48 3-11 0.3 1.7 2.6 155 51 3—48 1-39 0.1 1.9 0.1 4 159 3—7 6-21 0.4 3.9 3.6 92 169 3-7 6-21 0.3 1.3 3.1 235 134 3—9 1-5 0.4 5.0 2.9 58 141 4-31 1-33 2.4 4.2 2.6 63 142 4-31 1-33 0.4 4.2 2.8 67 The results obtained from these ments demonstrate that somatically mutated, high affinity heavy chains from different gene families are able to pair with rearranged human ne VK1—39JK5 and VK3-20JK1 regions and be secreted from the cell as a normal antibody molecule. As shown in Table 1, antibody titer was increased for about 61% (42 of 69) heavy chains when paired with the rearranged human VK1-39JK5 light chain and about 29% (20 of 69) heavy chains when paired with the rearranged human VK3-20JK1 light chain as compared to the cognate light chain of the al antibody. For about 20% (14 of 69) of the heavy chains, both rearranged human ne light chains conferred an increase in expression as compared to the cognate light chain of the parental antibody. As shown in Table 2, the rearranged human germline VK1-39JK5 region conferred an increase in expression of several heavy chains specific for a range of different classes of antigens as compared to the cognate light chain for the parental antibodies.

Antibody titer was increased by more than two—fold for about 35% (15/43) of the heavy chains as ed to the cognate light chain of the parental antibodies. For two heavy chains (315 and 316), the increase was greater than ten-fold as compared to the parental antibody. Within all the heavy chains that showed increase expression ve to the cognate light chain of the parental antibody, family three (VH3) heavy chains are over represented in comparison to other heavy chain variable region gene families. This demonstrates a favorable relationship of human VH3 heavy chains to pair with rearranged human germline VK1-39JK5 and VK3-20JK1 light chains.

Example 2 Generation of a Rearranged Human Germline Light Chain Locus Various rearranged human germline light chain targeting vectors were made using VELOClGENE® technology (see, 9.9., US Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652—659) to modify mouse genomic Bacterial Artificial Chromosome (BAC) clones 302g12 and 254m04 (lnvitrogen). Using these two BAC , c constructs were ered to contain a single rearranged human germline light chain region and inserted into an endogenous K light chain locus that was previously ed to delete the endogenous K variable and joining gene ts.

Construction of Rearranged Human ne Light Chain Targeting Vectors. Three different rearranged human germline light chain regions were made using rd molecular biology techniques ized in the art. The human variable gene ts used for constructing these three regions included rearranged human VK1- 39JK5 sequence, a rearranged human VK3-20JK1 sequence and a rearranged human VpreBJkS sequence.

A DNA segment containing exon 1 (encoding the leader peptide) and intron 1 of the mouse VK3-7 gene was made by de novo DNA sis (integrated DNA Technologies). Part of the 5’ untranslated region up to a naturally occurring Blpl restriction enzyme site was included. Exons of human VK1-39 and VK3-20 genes were PCR amplified from human genomic BAC libraries. The fonivard primers had a 5’ extension containing the splice acceptor site of intron 1 of the mouse VK3-7 gene. The reverse primer used for PCR of the human VK1—39 sequence included an extension encoding human JK5, whereas the reverse primer used for PCR of the human VK3-20 sequence included an extension ng human JK1. The human VpreBJkS sequence was made by de novo DNA synthesis (integrated DNA Technologies). A n of the human JK-CK intron including the splice donor site was PCR amplified from plasmid pBS—296—HA18- PlScel. The forward PCR primer included an extension encoding part of either a human JK5, JK1, or JKS ce. The reverse primer included a Pl-Scel site, which was previously engineered into the intron.

The mouse VK3-7 exon1/intron 1, human variable light chain exons, and human JK-CK intron fragments were sewn together by overlap extension PCR, digested with Blpl and l, and ligated into plasmid pBSHA18-PlScel, which contained the promoter from the human VK3—15 le gene segment. A loxed hygromycin cassette within plasmid pBS—296-HA18—PlScel was replaced with a FRTed hygromycin cassette d by Notl and Ascl sites. The Notl/Pl—Scel fragment of this plasmid was ligated into modified mouse BAC 254m04, which contained part of the mouse JK-CK intron, the mouse CK exon, and about 75 kb of genomic sequence downstream of the mouse K locus, which provided a 3’ homology arm for homologous recombination in mouse ES cells. The Notl/Ascl nt of this BAC was then ligated into modified mouse BAC , which contained a FRTed neomycin cassette and about 23 kb of genomic sequence upstream of the endogenous K locus for homologous recombination in mouse ES cells.

Rearranged Human Germline VK1-39JK5 Targeting Vector (.

Restriction enzyme sites were introduced at the 5’ and 3’ ends of an engineered light chain insert for cloning into a targeting vector: an Ascl site at the 5’ end and a Pl-Scel site at the 3’ end. Within the 5’ Ascl site and the 3’ Pl-Scel site the targeting construct from 5’ to 3’ included a 5’ homology arm containing sequence 5’ to the endogenous mouse K light chain locus obtained from mouse BAC clone 302912, a FRTed neomycin resistance gene, an genomic sequence ing the human VK3-15 promoter, a leader sequence of the mouse VK3-7 variable gene segment, a intron sequence of the mouse VK3-7 variable gene segment, an open reading frame of a rearranged human ne VK1-39JK5 region, a genomic sequence containing a portion of the human JK-CK intron, and a 3’ homology arm containing sequence 3’ of the endogenous mouse JK5 gene t obtained from mouse BAC clone 254m04 e 1, middle), Genes and/or sequences am of the nous mouse K light chain locus and downstream of the most 3’ JK gene segment (e.g., the endogenous 3’ enhancer) were unmodified by the targeting construct (see Figure 1). The sequence of the engineered human VK1-39JK5 locus is shown in SEQ lD N021.

Targeted insertion of the rearranged human germline VK1-39JK5 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located at sequences within the rearranged human germline light chain region. Briefly, the intron sequence 3’ to the mouse VK3-7 leader sequence was confirmed with primers ULC-m1 F (AGGTGAGGGT ACAGATAAGT GTTATGAG; SEQ lD N02) and ULC-mtR (TGACAAATGC CCTAATTATA GTGATCA; SEQ lD N03). The open reading frame of the rearranged human germline VK1-39JK5 region was confirmed with primers 2F AGTCA GAGCATTAGC A; SEQ lD NO:4) and 1633—h2R (TGCAAACTGG ATGCAGCATA G; SEQ ID NO:5). The neomycin cassette was confirmed with primers neoF (GGTGGAGAGG CTATTCGGC; SEQ lD NO:6) and neoR (GAACACGGCG GCATCAG; SEQ lD NO:7). Targeted BAC DNA was then used to oporate mouse ES cells to d modiﬁed ES cells for generating chimeric mice that express a nged human germline VK1-39JK5 .

Positive ES cell clones were confirmed by TAQMAN TM screening and karyotyping using probes specific for the engineered VK1-39JK5 light chain region inserted into the endogenous locus. Briefly, probe neoP (TGGGCACAAC AGACAATCGG CTG; SEQ lD N018) which binds within the neomycin marker gene, probe ULC-m1 P (CCATTATGAT GCTCCATGCC TCTCTGTTC; SEQ lD N029) which binds within the intron sequence 3’ to the mouse VK3—7 leader sequence, and probe 1633h2P (ATCAGCAGAA ACCAGGGAAA GCCCCT; SEQ lD NO:10) which binds within the rearranged human germline VK1-39JK5 open reading frame. Positive ES cell clones were then used to implant female mice to give rise to a litter of pups sing the germline VK1-39JK5 light chain .

Alternatively, ES cells bearing the rearranged human germline JK5 light chain region are ected with a construct that expresses FLP in order to remove the FRTed neomycin cassette introduced by the ing construct. Optionally, the neomycin te is removed by breeding to mice that express FLP recombinase (e.g., US 6,774,279). Optionally, the neomycin te is retained in the mice.

Rearranged Human Germline Vx3-20Jx1 Targeting Vector (. In a similar fashion, an engineered light chain locus expressing a rearranged human germline VK3-20JK1 region was made using a targeting construct including, from 5’ to 3’, a 5’ homology arm containing sequence 5’ to the endogenous mouse K light chain locus obtained from mouse BAC clone 302g12, a FRTed neomycin resistance gene, a genomic sequence including the human VK3-15 promoter, a leader sequence of the mouse VK3-7 variable gene segment, an intron sequence of the mouse VK3—7 le gene segment, an open reading frame of a rearranged human germline VK3—20JK1 region, a genomic sequence containing a portion of the human JK-CK intron, and a 3’ homology arm containing sequence 3’ of the nous mouse JK5 gene segment obtained from mouse BAC clone 254m04 (Figure 2, middle). The sequence of the engineered human VK3- 20JK1 locus is shown in SEQ ID NO:11.

Targeted insertion of the rearranged human germline VK3-20JK1 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located at sequences within the rearranged human germline VK3—20JK1 light chain . Briefly, the intron sequence 3’ to the mouse VK3-7 leader sequence was med with primers ULC-th (SEQ lD N022) and ULC-mtR (SEQ ID NO:3). The open reading frame of the rearranged human ne VK3-20JK1 region was confirmed with primers 1635~h2F (TCCAGGCACC CTGTCTTTG; SEQ lD NO:12) and 1635-h2R (AAGTAGCTGC TGCTAACACT CTGACT; SEQ lD NO:13). The neomycin cassette was confirmed with primers neoF (SEQ lD NO:6) and neoR (SEQ lD N027). Targeted BAC DNA was then used to electroporate mouse ES cells to created modified ES cells for generating chimeric mice that express the rearranged human germline VK3-20JK1 light chain.

Positive ES cell clones were confirmed by TM screening and karyotyping using probes specific for the engineered JK1 light chain region inserted into the endogenous K light chain locus. Briefly, probe neoP (SEQ lD NO:8) which binds within the in marker gene, probe ULC-th (SEQ lD NO:9) which binds within the mouse VK3-7 leader sequence, and probe 1635h2P (AAAGAGCCAC CCTCTCCTGC AGGG; SEQ lD NO:14) which binds within the human VK3-20JK1 open reading frame.

Positive ES cell clones were then used to implant female mice. A litter of pups expressing the human germline VK3-20JK1 light chain region.

Alternatively, ES cells bearing human germline VK3-20JK1 light chain region can be transfected with a construct that expresses FLP in order to remove the FRTed neomycin cassette uced by the targeting construct. Optionally, the neomycin cassette may be removed by breeding to mice that s FLP recombinase (e.g., US 6,774,279). ally, the neomycin cassette is retained in the mice.

Rearranged Human Germline k5 ing Vector (. in a similar fashion, an engineered light chain locus expressing a rearranged human germline VpreBJk5 region was made using a targeting construct including, from 5’ to 3’, a 5’ homology arm containing sequence 5’ to the endogenous mouse K light chain locus obtained from mouse BAC clone 302912, a FRTed neomycin resistance gene, an genomic sequence including the human VK3-15 promoter, a leader sequence of the mouse VK3-7 le gene t, an intron sequence of the mouse VK3-7 variable gene segment, an open reading frame of a rearranged human germline VpreBJAS region, a genomic sequence containing a portion of the human JK-CK intron, and a 3’ homology arm containing sequence 3’ of the endogenous mouse JK5 gene t obtained from mouse BAC clone 254m04 (Figure 3, ). The sequence of the engineered human VpreBJx5 locus is shown in SEQ lD NO:15.

Targeted insertion of the nged human germline VpreBJk5 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located at sequences within the rearranged human germiine VpreBJx5 region light chain region.

Briefly, the intron sequence 3’ to the mouse VK3-7 leader sequence was confirmed with primers ULC—mlF (SEQ lD N022) and R (SEQ lD NO:3). The open reading frame of the nged human germiine VpreBJkS region was confirmed with primers 1616-h1 F (TGTCCTCGGC CCTTGGA; SEQ lD NO:16) and 1616-h1R (CCGATGTCAT GGTCGTTCCT; SEQ lD NO:17). The neomycin te was confirmed with primers neoF (SEQ lD N026) and neoR (SEQ ID N027). Targeted BAC DNA was then used to electroporate mouse ES cells to created modified ES cells for generating chimeric mice that express the rearranged human germiine VpreBJKS light chain.

Positive ES cell clones are confirmed by TAQMAN TM screening and yping using probes specific for the engineered VpreBJk5 light chain region ed into the endogenous K light chain locus. Briefly, probe neoP (SEQ lD N028) which binds within the neomycin marker gene, probe ULC-m1 P (SEQ ID NO:9) which binds within the mouse lgVK3—7 leader sequence, and probe 1616h1P (ACAATCCGCC GCAC CCT; SEQ ID NOziB) which binds within the human VpreBJkS open reading frame.

Positive ES cell clones are then used to implant female mice to give rise to a litter of pups expressing a germiine light chain region.

Alternatively, ES cells bearing the rearranged human germiine VpreBJkS light chain region are transfected with a construct that expresses FLP in order to remove the FRTed neomycin cassette introduced by the targeting construct. ally, the neomycin cassette is d by breeding to mice that express FLP recombinase (e.g., US 6,774,279). Optionally, the neomycin cassette is retained in the mice.

Example 3 Generation of Mice expressing a single rearranged human light chain Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOClMOUSE® method (see, 9.9., US Pat. No. 754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate ypic analyses Nature Biotech. 25(1):91-99. MlCE® independently bearing an engineered human germiine VK1-39JK5 light chain , a VK3-20JK1 light chain region or a VpreBJKS light chain region are identified by genotyping using a modification of allele assay (Valenzuela et a/., supra) that detects the presence of the unique rearranged human germiine light chain region.

Pups are genotyped and a pup heterozygous or homozygous for the unique rearranged human germline light chain region are selected for characterizing expression of the rearranged human germline light chain .

Flow Cytometry. Expression of the rearranged human light chain region in the normal antibody repertoire of common light chain mice was validated by analysis of immunoglobulin K and it expression in splenocytes and peripheral blood of common light chain mice. Cell suspensions from harvested spleens and peripheral blood of wild type (n=5), VK1-39JK5 common light chain zygote (n=3), VK1-39JK5 common light chain homozygote (n=3), VK3-20JK1 common light chain heterozygote (n=2), and VK3-20JK1 common light chain homozygote (n=2) mice were made using standard methods and d with CD191 lg)»+ and ng+ using fluorescently labeled antibodies (BD Pharmigen).

Briefly, 1x106 cells were incubated with anti-mouse CDtS/CD32 (clone 2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling with the following antibody il for 30 minutes on ice: APC conjugated anti-mouse C019 (clone 1D3, BD Pharmigen), PerCP-Cy5.5 conjugated anti-mouse CD3 (clone 17A2, BioLegend), FlTC conjugated ouse ng (clone 187.1, BD Pharmigen), PE ated anti-mouse lg)» (clone , BioLegend). Following staining, cells were washed and fixed in 2% dehyde. Data acquisition was performed on an LSRll flow cytometer and analyzed with FIowJo. Gating: total B cells (CD19+CD3'), lgic+ B cells (ng+lg>»‘CD19+CD3'), lg)»+ B} cells (ng'lg’MCDtyCDB‘). Data gathered from blood and splenocyte samples demonstrated similar results. Table 3 sets forth the percent positive CD19+ B cells from peripheral blood of one representative mouse from each group that are lgx’“, ng‘”, or lgiflgiﬂ. Percent of CD19+ B cells in peripheral blood from wild type (WT) and mice homozygous for either the VK1-39JK5 or JK1 common light chain are shown in FlG.

Table 3 CD19+BcellS Mouse lgk” ng+ ng’lgiC Wild type VK1-39JK5 VK3-20JK1 Common Light Chain Expression. Expression of each common light chain (VK1—39JK5 and VK3-20JK1) was analyzed in zygous and gous mice using a quantitative PCR assay (e.g. TAQMAN"‘“). y, CD19+ B cells were purified from the spleens of wild type, mice gous for a replacement of the mouse heavy chain and K light chain variable region loci with ponding human heavy chain and K light chain le region loci (HK), as well as mice homozygous and zygous for each nged human light chain region (VK1—39JK5 or VK3—20JK1) using mouse CD19 Microbeads (Miltenyi Biotec) according to manufacturer’s specifications. Total RNA was purified from CD19+ B cells using RNeasy Mini kit n) according to manufacturer’s specifications and genomic RNA was removed using a RNase—free DNase on-column treatment (Qiagen). 200 ng mRNA was e-transcribed into cDNA using the First Stand cDNA Synthesis kit (lnvitrogen) and the resulting cDNA was ampliﬁed with the Taqman Universal PCR Master Mix (Applied Biosystems). All reactions were performed using the ABl 7900 Sequence ion System (Applied Biosystems) using primers and Taqman MGB probes spanning (1) the VK-JK junction for both common light chains, (2) the VK gene alone (i.e. VK1—39 and VK3— ), and (3) the mouse CK region. Tabie 4 sets forth the sequences of the primers and probes employed for this assay. Relative expression was normalized to expression of the mouse CK . Results are shown in , SB and 5C.

Table 4 Region Primer/Probe Description (5’—3’) SEQ lD NOs: (sense) AGCAGTCTGC AACCTGAAGA TTT VK1—39JK5 (anti~sense) GTTTAATCTC CAGTCGTGTC CCTT 20 Junction (probe) CCTCCGATCA CCTTC 21 (sense) AAACCAGGGA AAGCCCCTAA 22 VK1-39 (anti—sense) ATGGGACCCC ACTTTGCA 23 (probe) CTCCTGATCT ATGCTGCAT 24 (sense) CAGCAGACTG GAGCCTGAAG A 25 VK3-20JK1 (anti-sense) TGATTTCCAC CTTGGTCCCT T Junction (probe) TAGCTCACCT TGGACGTT (sense) CTCCTGATCT ATGGTGCATC CA Jr" VK3-20 (anti-sense) GACCCACTGC CACTGAACCT (probe) CCACTGGCAT CCC (sense) TGAGCAGCAC CCTCACGTT Mouse CK (anti—sense) GTGGCCTCAC AGGTATAGCT GTT (probe) ACCAAGGACG AGTATGAA 33 ] Antigen Specific Common Light Chain dies. Common light chain mice bearing either a VK1—39JK5 or VK3-20JK1 common light chain at the endogenous mouse K light chain locus were immunized with ﬁ-galactosidase and antibody titer was measured.

Briefly, ﬁ-galactosidase (Sigma) was emulsified in ax adjuvant (Sigma), as per manufacturers directions. Wild type (n=7), VK1-39JK5 common light chain homozygotes (n=2) and VK3-20JK1 common light chain homozygotes (n=5) were immunized by subcutaneous ion with 100 pg (S-galactosidase/Titermax. Mice were boosted by subcutaneous injection two times, 3 weeks apart, with 50 pg [3- galactosidase/Titermax. After the second boost, blood was collected from hetized mice using a retro-orbital bleed into serum separator tubes (BD Biosciences) as per manufacturer’s directions. To measure anti-B-galactosidase lgM or lgG antibodies, ELlSA plates (Nunc) were coated with 1 pg/mL B-galactosidase ght at 4°C. Excess antigen was washed off before blocking with PBS with 1% BSA for one hour at room temperature.

Serial dilutions of serum were added to the plates and incubated for one hour at room temperature before washing. Plates were then incubated with HRP conjugated anti—lgM (Southern Biotech) or anti-lgG (Southern Biotech) for one hour at room temperature.

Following another wash, plates were developed with TMB substrate (BD Biosciences).

Reactions were stopped with 1N sulfuric acid and OD450 was read using a Victor X5 Plate Reader (Perkin Elmer). Data was analyzed with GraphPad Prism and signal was calculated as the dilution of serum that is two times above background. Results are shown in and BB.

As shown in this Example, the ratio of Kl)» B cells in both the splenic and peripheral compartments of VK1-39JK5 and JK1 common light chain mice demonstrated a near wild type pattern (Table 3 and HG. 4). VpreBJx5 common light chain mice, however, demonstrated fewer peripheral B cells, of which about 1—2% express the engineered human light chain region (data not shown). The sion levels of the VK1- 39J1<5 and VK3~20JK1 rearranged human light chain regions from the nous K light chain locus were elevated in comparison to an endogenous K light chain locus containing a complete replacement of mouse VK and JK gene segments with human VK and JK gene segments (, SB and SC). The sion levels of the VpreBJkS nged human light chain region trated similar high expression from the endogenous K light chain locus in both heterozygous and homozygous mice (data not shown). This demonstrates that in direct competition with the mouse A, K, or both endogenous light chain loci, a single rearranged human VL/JL sequence can yield better than wild type levei sion from the endogenous K light chain locus and give rise to normal splenic and blood B cell frequency. Further, the presence of an engineered K light chain locus having either a human VK1-39JK5 or human JK1 sequence was well tolerated by the mice and appear to function in wild type fashion by representing a substantial portion of the light chain oire in the humoral component of the immune response (FlG 6A and GB).

Example 4 ng of Mice Expressing a Single Rearranged Human Germline Light Chain This Example describes several other genetically modified mouse strains that can be bred to any one of the common light chain mice bed herein to create multiple genetically modified mouse strains ing le genetically modified immunoglobulin loci.

Endogenous lg)» Knockout (KO). To optimize the usage of the engineered light chain locus, mice bearing one of the rearranged human germline light chain regions are bred to r mouse containing a deletion in the nous 7» light chain locus. in this manner, the progeny obtained will express, as their only light chain, the rearranged human germline light chain region as bed in e 2. Breeding is performed by standard techniques recognized in the art and, alternatively, by a commercial breeder (6.9., The Jackson tory). Mouse strains bearing an engineered light chain locus and a deletion of the endogenous 7» light chain locus are screened for presence of the unique light chain region and absence of endogenous mouse A light chains.

] Humanized Endogenous Heavy Chain Locus. Mice bearing an engineered human germline light chain locus are bred with mice that contain a replacement of the endogenous mouse heavy chain variable gene locus with the human heavy chain variable gene locus (see US 6,596,541; the VELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc). The VELOCIMMUNE® mouse comprises a genome comprising human heavy chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces antibodies comprising a human heavy chain variable region and a mouse heavy chain constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy chains of the antibodies is isolated and operably linked to DNA encoding the human heavy chain constant regions.

The DNA is then expressed in a cell capable of expressing the fully human heavy chain of the antibody.

Mice bearing a replacement of the endogenous mouse VH locus with the human VH locus and a single rearranged human germline VL region at the endogenous K light chain locus are obtained. Reverse chimeric antibodies containing somatically mutated heavy chains (human VH and mouse CH) with a single human light chain (human VL and mouse CL) are obtained upon immunization with an antigen of interest. VH and VL nucleotide sequences of B cells expressing the antibodies are identified and fully human antibodies are made by fusion the VH and VL tide sequences to human CH and CL nucleotide sequences in a le expression system.

Example 5 Generation of Antibodies from Mice Expressing Human Heavy Chains and a Rearranged Human Germline Light Chain Region After breeding mice that contain the engineered human light chain region to various desired strains containing cations and ons of other endogenous lg loci (as described in Example 4), selected mice can be immunized with an antigen of interest.

Generally, a VELOClMMUNE® mouse containing one of the single rearranged human germline light chain regions is challenged with an antigen, and lymphatic cells (such as B-cells) are recovered from serum of the animals. The lymphatic cells are fused with a myeloma cell line to prepare immortal oma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies containing human heavy chain variables and a rearranged human ne light chains which are ic to the antigen used for zation. DNA encoding the variable regions of the heavy chains and the light chain are ed and linked to desirable isotypic nt regions of the heavy chain and light chain. Due to the presence of the endogenous mouse sequences and any additional cis-acting elements present in the endogenous locus, the single light chain of each antibody may be somatically mutated.

This adds additional diversity to the antigen-specific repertoire comprising a single light chain and diverse heavy chain sequences. The resulting cloned antibody sequences are subsequently expressed in a cell, such as a CHO cell. atively, DNA encoding the antigen-specific chimeric antibodies or the variable s of the light and heavy chains are fied directly from antigen—specific lymphocytes. lly, high affinity chimeric antibodies are isolated having a human le region and a mouse constant region. As described above, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody containing a somatically mutated human heavy chain and a single light chain derived from a rearranged human germline light chain region of the invention. Suitable human constant regions include, for example wild type or modified lth or lgG4.

Separate cohorts of VELOClMMUNE® mice containing a replacement of the endogenous mouse heavy chain locus with human VH, DH, and JH gene segments and a replacement of the endogenous mouse K light chain locus with either the engineered germline VK1—39JK5 human light chain region or the engineered germline VK3-20JK1 human light chain region ibed above) were immunized with a human cell-surface receptor n (Antigen E). Antigen E is administered directly onto the hind footpad of mice with six consecutive injections every 3-4 days. Two to three micrograms of Antigen E are mixed with 10 pg of CpG oligonucleotide (Cat # tlrl—modn — ODN1826 oligonucieotide; anivogen, San Diego, CA) and 25 pg of Adju-Phos (Aluminum phosphate gel adjuvant, Cat# H-71639—250; Brenntag Biosector, Frederikssund, k) prior to injection. A total of six injections are given prior to the final antigen recall, which is given 3-5 days prior to sacrifice. Bleeds after the 4th and 6th injection are collected and the antibody immune response is monitored by a standard antigen-specific immunoassay.

When a desired immune response is achieved cytes are harvested and fused with mouse myeloma cells to preserve their viability and form oma cell lines. The hybridoma cell lines are screened and selected to identify cell lines that produce Antigen E-specific common light chain antibodies. Using this technique several anti- Antigen E-specific common light chain antibodies (i.e., antibodies possessing human heavy chain variable domains, the same human light chain le domain, and mouse constant domains) are obtained.

Alternatively, anti-Antigen E common light chain dies are ed directly from n—positive B cells t fusion to myeloma cells, as described in US. 2007/0280945A1, herein specifically incorporated by nce in its entirety. Using this method, several fully human anti—Antigen E common light chain antibodies (i.e., antibodies possessing human heavy chain variable domains, either an engineered human VK1-39JK5 light chain or an ered human VK3-20JK1 light chain region, and human constant domains) were obtained.

The biological properties of the exemplary ntigen E common light chain antibodies generated in accordance with the methods of this Example are described in detail in the sections set forth below.

Example 6 Heavy Chain Gene Segment Usage in Antigen-Specific Common Light Chain Antibodies To analyze the structure of the human anti-Antigen E common light chain dies produced, nucleic acids encoding heavy chain antibody variable regions were cloned and sequenced. From the nucleic acid sequences and predicted amino acid ces of the antibodies, gene usage was identified for the heavy chain variable region (HCVR) of selected common light chain antibodies obtained from immunized MMUNE® mice containing either the engineered human VK1—39JK5 light chain or engineered human V1<3~20J1<1 light chain region. Results are shown in Tables 5 and 6, which demonstrate that mice according to the invention generate antigen-specific common light chain antibodies from a variety of human heavy chain gene segments, due to a variety of ngements, when employing either a mouse that expresses a light chain from only a human V1<1 or a human VK3derived light chain. Human VH gene segments of the 2, 3, 4, and 5 families rearranged with a variety of human DH segments and human JH segments to yield antigen-specific antibodies.

Table5 VK1-39JK5 Common Light Chain Antibodies HCVR HCVR dy. _ ------------- Antibody VH DH JH VH DH JH 5932 3-30 6-13 4 6002 3-30 6-13 4 6003 3-30 6-13 6012 3-30 6-13 4 6013 3-30 6-13 4 I 6014 3-30 6-13 4 1 4 6015 3304 6-13 4 l 4 6016 3-30 (MET 4 4 6017 3-30 6-13 4 4 6020 3-30 643+ 4 4 6034 3-30 6-13 4 4 2943 3-30 7-27 4 3g 4 2987 3-30 7-27 4 2932 3-30 3-22 5 2996 3-30 7-27 4 6001 3-30 3-22 5] i3005 3-30 7-27 4 {5005 3-30 [3-22 5 3012 | 7-27 4 15035 3-30 5-5 2 :{3-30 4 3013 3301512 4 I 4 3-30 5-12 4 4 13-30 5-5 1 4 -5 3 4 -5 4 4 4 “5— 5 5 3 3.] 3 3 3 5029 3 5035 3 5037 3 -5 i 5 2954 3 -5 5 3027 4 55 st 3045 3 —5 5 5000 4 -5 5 5005 5 55 J 5—1 6008 4:1 TabieS VK3-20JK1 Common Light Chain Antibodies HCVR HCVR Antibody —--- Antibody_ VH DH JH VH DH JH —51 3—16 6 -51 3-16 6 —--n —--n Example 7 Determination of Blocking Ability of Antigen—Specific Common Light Chain Antibodies by XTM Assay Ninety-eight human common light chain antibodies raised against Antigen E were tested for their ability to block g of Antigen E’s l ligand (Ligand Y) to Antigen E in a bead-based assay.

The extracellular domain (ECD) of Antigen E was conjugated to two myc epitope tags and a 6X histidine tag (Antigen E-mmH) and amine—coupled to carboxylated microspheres at a concentration of 20 ug/mL in MES buffer. The mixture was ted for two hours at room temperature followed by bead deactivation with 1M Tris pH 8.0 followed by washing in PBS with 0.05% (v/v) Tween-20. The beads were then blocked with PBS (lrvine ScientificfSanta Ana, CA) containing 2% (w/v) BSA (Sigma—Aldrich Corp, St. Louis, MO). In a 96-well filter plate, supernatants containing Antigen ific common light chain antibodies were diluted 1:15 in buffer. A negative control containing a mock supernatant with the same media components as for the antibody supernatant was prepared. Antigen E-labeled beads were added to the supernatants and incubated overnight at 4°C. Biotinylated-Ligand Y protein was added to a final concentration of 0.06 nM and incubated for two hours at room temperature. Detection of biotinylated-Ligand Y bound to Antigen E-myc—myc-BHis d beads was determined with R—Phycoerythrin conjugated to Streptavidin (Moss lnc, Pasadena, MD) followed by measurement in a XTM flow cytometry-based analyzer. Background Mean Fluorescence intensity (MFl) of a sample t Ligand Y was subtracted from all samples. Percent blocking was calculated by division of the background-subtracted MFI of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100. in a r experiment, the same 98 human common light chain dies raised against Antigen E were tested for their ability to block binding of Antigen E to Ligand Y—labeled beads.

Briefly, Ligand Y was amine-coupled to carboxylated pheres at a concentration of 20 ug/mL diluted in MES . The mixture and incubated two hours at room temperature followed by deactivation of beads with 1M Tris pH 8 then washing in PBS with 0.05% (v/v) Tween—20. The beads were then blocked with PBS (lrvine Scientific, Santa Ana, CA) containing 2% (w/v) BSA -Aldrich Corp, St. Louis, MO). in a 96- well filter plate, supernatants ning Antigen E—specific common light chain antibodies were diluted 1:15 in buffer. A negative control containing a mock supernatant with the same media components as for the antibody supernatant was ed. A biotinylated- Antigen E-mmH was added to a final concentration of 0.42 nM and incubated overnight at 4°C. Ligand Y-labeled beads were then added to the antibody/Antigen E mixture and incubated for two hours at room temperature. Detection of biotinylated-Antigen E-mmH bound to Ligand Y-beads was determined with R-Phycoerythrin conjugated to Streptavidin (Moss lnc, Pasadena, MD) followed by measurement in a XTM flow cytometry- based analyzer. Background Mean Fluorescence lntensity (MFl) of a sample without Antigen E was subtracted from all samples. Percent blocking was calculated by division of the ound—subtracted MFl of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100.

Tables 7 and 8 show the percent blocking for all 98 anti—Antigen E common light chain antibodies tested in both LUMlNEXTM assays. ND: not determined under t experimental conditions.

Table 7 Common Light Chain Antibodies % ng of % Blocking of Antibody Antigen E-Labeled Beads Antigen E in Solution 2949 97.6 78.8 2949G 97.1 73.7 2950 96.2 81.9 2950G 89.8 31.4 2952 96.1 74.3 2952G 93.5 39.9 2954 93.7 70.1 2954G 91.7 30.1 2955 75.8 30.0 2955G 71.8 ND 2964 92.1 J 31.4 2964G 94.6 43.0 2978 98.0 95.1 2978G 13.9 94.1 29826 41.9 52.4 2985 39.5 31.2 2985G 2.0 5.0 2987 81.7 67.8 WO 48873 2987G 26.6 29.3 2996 87.3 55.3 29966 95.9 38.4 \I 01 30046 60.3 40.7 3005 97.4 93.5 3005G 77.5 75.6 3010 82.6 301OG 81.0 3011 42.8 3011G 41.7 3012 60.8 .7 3014G 28.5 3016 97.1 81.6 3016G 93.1 66.4 3017 94.8 70.2 3013s 25.1 12.7 3019 99.3 92.4 30196 99.3 88.1 3020 95.7 90.3 3020c; 35.2 41.5 74.5 25.1 3022 - 30229 3023 3023s 3024 30246 89.0 10.0 3025 70.7 1 15.6 WO 48873 30256 76.7 24.3 962 613 936 753 924 290 813 233 |§ﬁﬁﬂ||l 60 100 30303 3032 30320 3033 30330 3036 327 3041 G 92.4 51.6 3042 88.1 73.3 3042G 60.9 25.2 Table 8 VK3—20JK1 Common Light Chain Antibodies % Blocking of % Blocking of Antibody Antigen E—Labeied Beads Antigen E in Solution In the first XTM experiment bed above, 80 common light chain antibodies containing the VK1-39JK5 engineered light chain were tested for their ability to block Ligand Y binding to n E-labeled beads. Of these 80 common light chain antibodies, 68 demonstrated >50% blocking, while 12 demonstrated <50% blocking (6 at -50% blocking and 6 at <25% blocking). For the 18 common light chain antibodies containing the VK3—20JK1 engineered light chain, 12 demonstrated >50% blocking, while 6 demonstrated <50% blocking (3 at 25-50% blocking and 3 at <25% blocking) of Ligand Y binding to Antigen E—labeled beads. in the second LUMlNEXTM experiment bed above, the same 80 common light chain antibodies containing the VK1-39JK5 engineered light chain were tested for their ability to block g of Antigen E to Ligand Y—labeled beads. Of these 80 common light chain antibodies, 36 trated >50% blocking, while 44 demonstrated <50% blocking (27 at 25-50% blocking and 17 at <25% blocking). For the 18 common light chain dies containing the VK3-20JK1 engineered light chain, 1 demonstrated >50% blocking, while 17 demonstrated <50% blocking (5 at 25-50% blocking and 12 at <25% ng) of Antigen E binding to Ligand Y-labeled beads.

The data of Tables 7 and 8 establish that the rearrangements described in Tables 5 and 6 ted anti—Antigen E—specific common light chain antibodies that d binding of Ligand Y to its cognate receptor Antigen E with varying degrees of efficacy, which is consistent with the anti-Antigen E common light chain antibodies of Tables 5 and 6 comprising antibodies with overlapping and non-overlapping epitope specificity with respect to Antigen E.

Example 8 Determination of Blocking Ability of Antigen-Specific Common Light Chain Antibodies by ELISA Human common light chain antibodies raised against Antigen E were tested for their ability to block Antigen E binding to a Ligand Y—coated surface in an ELISA assay.

Ligand Y was coated onto 96—weil plates at a concentration of 2 ug/mL diluted in PBS and incubated overnight followed by g four times in PBS with 0.05% . The plate was then blocked with PBS (lrvine Scientific, Santa Ana, CA) containing 0.5% (w/v) BSA (Sigma-Aldrich Corp, St. Louis, M0) for one hour at room temperature. in a separate plate, supernatants containing anti-Antigen E common light chain dies were diluted 1:10 in buffer. A mock supernatant with the same components of the antibodies was used as a negative control. Antigen E—mmH (described above) was added to a final concentration of 0.150 nM and incubated for one hour at room temperature. The antibody/Antigen E-mmH mixture was then added to the plate containing Ligand Y and incubated for one hour at room temperature. Detection of Antigen E-mmH bound to Ligand Y was determined with Horse—Radish Peroxidase (HRP) conjugated to anti-Penta—His antibody (Qiagen, Valencia, CA) and developed by standard colorimetric response using tetramethylbenzidine (TMB) substrate (BD ences, San Jose, CA) neutralized by sulfuric acid. Absorbance was read at OD450 for 0.1 sec. Background absorbance of a sample without Antigen E was subtracted from all samples. t blocking was ated by division of the background-subtracted MFl of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100.

Tables 9 and 10 show the t blocking for all 98 ntigen E common light chain antibodies tested in the ELISA assay. ND: not determined under current experimental conditions.

Table 9 VK’l -39JK5 Common Light Chain Antibodies ArmsfeBlockIng ofO ' 0A) Blocking of' Antibody Antibody n E In Solution n E In Solution WO 48873 59.0 3023 9.1 1 29820 20.4 30230 19.2 i 2985 10.5 3024 7.5 29850 ND 30240 15.2 1 31.4 3025 ND 29870 ND 30250 13.9 2995 29.3 3027 51.4 29950 ND 1 30270 82.7 2997 48.7 i 3028 40.3 ND | 30280 12.3 .7 1 3030 ND 3.5 30300 9.5 No i 54.3 30320 13.1 i 74.5 3033 77.1 30100 84.6 L30330 32.9 19.4 3035 17.5 30110 ND 30350 45.0 3041 ”“3040 39.0 3042 30130 9.5 30420 15.1 ___I 3014 5.2 _l {3043 57.4 30140 17.1 30430 45.1 Table 10 VK3-20JK1 Common Light Chain Antibodies % Blocking of % Blocking of Antibody Antibody Antigen E In Solution Antigen E In Solution 2968 29688 2969 2969G 2970 29706 2971 2971 G 2976G As described in this Example, of the 80 common light chain antibodies containing the VK1-39JK5 engineered light chain tested for their ability to block Antigen E binding to a Ligand ed surface, 22 demonstrated >50% blocking, while 58 demonstrated <50% blocking (20 at 25-50% ng and 38 at <25% blocking). For the 18 common light chain antibodies containing the VK3-20JK1 engineered light chain, one demonstrated >50% blocking, while 17 demonstrated <50% ng (5 at 25—50% blocking and 12 at <25% blocking) of Antigen E binding to a Ligand Y—coated surface.

] These results are also consistent with the Antigen E—specific common light chain antibody pool comprising antibodies with overlapping and erlapping epitope icity with respect to Antigen E.

Example 9 BlACORETM Affinity Determination for Antigen-Specific Common Light Chain Antibodies Equilibrium dissociation constants (KD) for selected antibody supernatants were determined by SPR (Surface n Resonance) using a ET'V‘ T100 instrument (GE Healthcare). All data was obtained using HBS-EP (10mM Hepes, 150mM NaCl, 0.3mM EDTA, 0.05% Surfactant P20, pH 7.4) as both the running and sample buffers, at 25°C. Antibodies were captured from crude supernatant samples on a CM5 sensor chip surface previously derivatized with a high density of anti-human Fc antibodies using rd amine coupling chemistry. During the capture step, supernatants were injected across the anti—human Fc surface at a flow rate of 3 pL/min, for a total of 3 minutes. The capture step was followed by an injection of either running buffer or analyte at a concentration of 100 nM for 2 minutes at a flow rate of 35 pL/min. Dissociation of antigen from the captured antibody was monitored for 6 minutes. The captured dy was d by a brief injection of 10 mM glycine, pH 1.5. All sensorgrams were double referenced by subtracting sensorgrams from buffer injections from the analyte sensorgrams, thereby removing artifacts caused by dissociation of the antibody from the capture surface. Binding data for each antibody was fit to a 1:1 binding model with mass transport using e T100 Evaluation software v2.1. Results are shown in Tables 11 and 12.

Table 11 VK1-39JK5 Common Light Chain Antibodies 100 nM Antigen E 100 nM n E Antibody —-———————-————- Antibody KD (HM) T1/2 (min) KD (HM) T1/2 (min) 1 30156 55.9 0 9 4 30176 55.4 7 3018 11.3 35 4 30186 32.5 3 24 3019 59 9 30196 42 29556 4 30206 5 2954 14.8 5 3021 5 1 29546 9 30216 4 i 2978 1.91 49 I302 17 29786 1.80 58 30226 12 2982 19 3023 253 “ 29826 15.3 9 30236 103 2985 54.4 9 3024 58.8 29856 2.44 8 30246 7.09 10 2987 [—21.0 11 3025 35.2 5 E987G 37.5 4 30256 42.5 n 2995 10.8 9 3027 7.15 n 29956 24.0 2 30276 4.24 2997 19 3028 5.89 37 29976 151 1 30286 7.23 22 3004 45.5 14 3030 45.2 30046 1.93 91 30306 128 3 3005 2.35 1M 3032 53.2 9 30056 27 30326 13.0 1 3010 25 3033 4.51 17 30106 2.10 49 3033G 12.0 5 3011 59.1 5 _3035 284 12 Table 12 Common Light Chain Antibodies 100 nM Antigen E 100 nM Antigen E dyw_ Antibody KD(nM) T1,2(min) KD(nM) T1/2 (mm)_ 2970G 12.3 2972 6.02 13 The binding affinities of common light chain antibodies comprising the rearrangements shown in Tables 5 and 6 vary, with nearly all exhibiting a KD in the nanomolar range. The affinity data is consistent with the common light chain antibodies resulting from the combinatorial ation of rearranged variable domains described in Tables 5 and 6 being high-affinity, clonaliy selected, and somatically mutated. Coupled with data usly shown, the common light chain dies described in Tables 5 and 6 comprise a collection of diverse, high-affinity antibodies that exhibit specificity for one or more epitopes on Antigen E.

Example 10 Determination of Binding icities of Antigen-Specific Common Light Chain Antibodies by LUMlNEXTM Assay Selected anti-Antigen E common light chain dies were tested for their ability to bind to the ECD of Antigen E and Antigen E ECD variants, including the cynomolgous monkey ortholog (MfAntigen E), which differs from the human protein in approximately 10% of its amino acid residues; a deletion mutant of Antigen E lacking the last 10 amino acids from the C-terminal end of the ECD (Antigen E-ACT); and two mutants ning an alanine substitution at suspected locations of interaction with Ligand Y (Antigen E-Ala1 and AntigenE—Ala2). The Antigen E proteins were produced in CHO cells and each contained a c-His C-terminal tag.

For the binding studies, n E ECD protein or variant protein (described above) from 1 mL of culture medium was captured by incubation for 2 hr at room temperature with 1 x 106 microsphere (LUMINEXTM) beads covalently coated with an anti- myc onal antibody (MAb 9E10, hybridoma cell line CRL-1729TM; ATCC, as, VA). The beads were then washed with PBS before use. Supernatants containing anti- Antigen E common light chain antibodies were diluted 1:4 in buffer and added to 96-well filter plates. A mock supernatant with no antibody was used as negative l. The beads ning the captured Antigen E proteins were then added to the antibody samples (3000 beads per well) and incubated overnight at 4°C. The following day, the sample beads were washed and the bound common light chain antibody was detected with a R—phycoerythrin-conjugated anti—human lgG antibody. The fluorescence intensity of the beads (approximately 100 beads counted for each antibody sample binding to each Antigen E protein) was measured with a LUMlNEXTM flow cytometry—based analyzer, and the median fluorescence ity (MFI) for at least 100 counted beads per bead/antibody interaction was ed. Results are shown in Tables 13 and 14.

Table 13 VK1-39JK5 Common Light Chain Antibodies Mean Fluorescence intensity (MFI) Antibody Antigen E- Antigen E- Antigen E— Antigen E— .

ECD ACT Ala 1 Ala2 MfAntigen E 3358 244 2808 3396 2643 2955 1310 2955G 1324 4910 3755 1623 WO 48873 1125 4185 346 44 29646 4999 729 4646 534 91 2800 14542 10674 8049 3117 1674 7646 5944 2546 3068 1537 9202 6004 4744 -2996 4666 9046 6459 -2996G 2752 6150 4873 -2997 5164 8361 5922 —2997G 658 2325 1020 -3004 6268 3083 2753 5808 4345 5683 5868 30106 51 3011 3863 30126 968 378 3013 2343 1791 30136 327 144 3015 3202 2068 8262 5554 3796 531 4246 2643 1611 1277 6344 4288 4091 2553 8700 5547 5098 1081 5763 3825 3038 3018 2339 1971 6140 4515 2293 30186 254 118 978 1020 345 3019 5235 1882 7108 4249 1 54 30196 1 4090 1270 4769 3474 l 214 3020 3883 3107 8591 I 6602 I 4420 139206 2165 1209 6489 I 4295 1 2912 WO 48873 1472 6872 4641 2742 1005 6430 3988 2935 3022 2418 793 7523 2679 36 30226 2189 831 6182 3051 132 3023 1692 1411 5788 3898 2054 3023G 1770 825 5702 3677 2648 3024 1819 1467 6179 4557 2450 3024G 100 87 268 433 131 3025 1853 1233 6413 4337 2581 3025G 1782 791 5773 3871 2717 3027 4131 1018 582 2510 22 30276 3492 814 1933 2596 42 3028 4361 2545 9884 5639 975 3028G 2835 1398 7124 3885 597 3030 463 277 1266 1130 391 30306 3420 2570 1186 3032 2083 1496 2405 295 106 292 30336 2499 4210 3036 1755 30366 2313 30416 2519 6468 4274 3320 463 4205 2762 1519 2128 7607 5532 3366 30436 2293 1319 6573 4403 1 3228 Table 14 VK3-20JK1 Common Light Chain Antibodies Mean Fluorescence intensity (MFi) Antibody Antigen E- Antigen E- Antigen E- Antigen E- MfAntigen E ECD ACT Ala1 A|a2 297OG 4683 2971 501 2976 [397% The anti-Antigen E common light chain dy supernatants exhibited high ic binding to the beads linked to n E-ECD. For these beads, the negative control mock supernatant resulted in negligible signal (<10 MFl) when combined with the Antigen E-ECD bead sample, whereas the supernatants containing anti—Antigen E common light chain antibodies exhibited strong binding signal (average MFl of 2627 for 98 antibody supernatants; MFl > 500 for 91/98 antibody samples).

As a measure of the ability of the selected anti—Antigen E common light chain antibodies to identify different epitopes on the E00 of Antigen E, the relative binding of the antibodies to the variants were ined. All four Antigen E ts were captured to the anti-myc XTM beads as described above for the native Antigen E- ECD g studies, and the relative binding ratios (MFlvaﬁam/MFIAmigen 5.500) were determined. For 98 tested common light chain antibody supernatants shown in Tables 12 and 13, the average ratios (MFlvargam/MFlAmigen EECD) differed for each variant, likely reflecting different capture amounts of proteins on the beads (average ratios of 0.61, 2.9, 2.0, and 1.0 for Antigen E-ACT, Antigen E-Ala1, Antigen E-Ala2, and MfAntigen E, respectively). For each protein variant, the binding for a subset of the 98 tested common light chain antibodies showed greatly reduced binding, indicating sensitivity to the on that characterized a given variant. For example, 19 of the common light chain antibody samples bound to the MfAntigen E with iam/MFIAMgen EECD of <8%. Since many in this group include high or moderately high affinity antibodies (5 with KD < 5nM, 15 with KD < 50 nM), it is likely that the lower signal for this group results from sensitivity to the sequence (epitope) differences between native Antigen E-ECD and a given t rather than from lower affinities.

These data establish that the common light chain antibodies described in Tables and 6 represent a diverse group of Antigen-E specific common light chain antibodies that specifically recognize more than one epitope on Antigen E.

Where the terms “comprise”, “comprises”, “comprised” or “comprising” are used in this specification (including the ) they are to be interpreted as specifying the presence of the stated features, rs, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

The discussion of documents, acts, als, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present ion. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common l knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A genetically modified mouse comprising a population of B cells that each express a human immunoglobulin light chain variable domain derived from a single rearranged human /Jκ sequence which is present in the germline of the mouse, wherein the single rearranged human Vκ1-39/Jκ sequence comprises a human germline Vκ1-39 gene segment and a human germline Jκ gene segment, which single rearranged human Vκ1-39/Jκ sequence is operably linked to an immunoglobulin light chain constant region sequence, and wherein the human immunoglobulin light chain variable domain is expressed from a sequence that is cal to or a somatically hypermutated variant of the rearranged human Vκ1-39/Jκ ce; and wherein the B cells of the population include at least one B cell that expresses a human immunoglobulin heavy chain variable domain derived from a rearranged human VH/D/JH region selected from the group consisting of 1-