NZ710301B2

NZ710301B2 - Humanized universal light chain mice

Info

Publication number: NZ710301B2
Application number: NZ710301A
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: 2011-08-05
Filing date: 2012-08-03
Publication date: 2017-08-29

Abstract

Disclosed is a method of making a mouse comprising genetically modifying the mouse to include in its germline: (a) a humanised immunoglobulin heavy chain locus comprising at least one unrearranged human VH gene segment, at least one unrearranged human DH gene segment, and at least one unrearranged human JH gene segment operably linked to a heavy chain constant region gene; (b) a humanised immunoglobulin light chain locus comprising a single rearranged human light chain V/J sequence operably linked to a light chain constant region gene; and (c) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein or ortholog or homolog or functional fragment thereof, wherein the mouse ADAM6 protein or ortholog or homolog or functional fragment thereof is expressed from the ectopic nucleic acid sequence. human JH gene segment operably linked to a heavy chain constant region gene; (b) a humanised immunoglobulin light chain locus comprising a single rearranged human light chain V/J sequence operably linked to a light chain constant region gene; and (c) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein or ortholog or homolog or functional fragment thereof, wherein the mouse ADAM6 protein or ortholog or homolog or functional fragment thereof is expressed from the ectopic nucleic acid sequence.

Description

HUMANIZED UNIVERSAL LIGHT CHAIN MICE RELATED APPLICATION This application is a divisional of New Zealand Patent Application No. 620586, the entire contents of which are herein incorporated by reference.

FIELD OF INVENTION [0001A] Genetically modified mice, cells, embryos, tissues, and isolated nucleic acids for making antibodies and sequences encoding human immunoglobulin heavy chain variable domains, including bispecific antibodies, and including bispecific antibodies that comprise universal light chains. Compositions and methods include genetically modified mice with germline replacements at the endogenous mouse heavy chain variable locus, which comprise modified light chain loci that express light chains derived from no more than one or two different light chain V gene segments, wherein the mice are further genetically modified in their germline such that male mice bearing these modifications are fertile. Genetically modified mice that express universal light chains and humanized heavy chain variable domains are provided, wherein the mice se an ADAM6 activity that is onal in a male mouse.

BACKGROUND The development of antibodies for use as human therapeutics has a long and complex history. One icant e has been the ability to make essentially fully human antibody sequences to use in ing effective human therapeutics with reduced potential for genicity. Mice now exist that are modified in their ne to te human antibody sequences derived from unrearranged gene segments (heavy and light) either as transgenes or as replacements at endogenous mouse immunoglobulin loci. ement of mouse variable sequences with human variable sequences at endogenous loci in mice, as with VELOCIMMUNE® humanized mice, allow for the mouse immune system to function essentially normally. As a result, exposing these mice to an antigen of choice generates a marvelously diverse, rich population of clonally selected B cells that express high affinity somatically mutated human variable s that can be used in making fully human antibodies ed against the antigen of choice.

Human le domains made in humanized mice can be used to design fully human bispecific antibodies, i.e. , binding proteins that are heterodimers of heavy , where the identities and binding specificities of the heavy chain le domains differ. But selecting light chains that can effectively associate and express with the heavy chain heterodimers has no facile solution. Developing human light chain variable domains for use in human therapeutics is nly possible in humanized mice, but there are no easy solutions to selecting which light chains will effectively associate and express with heavy chains having desired binding characteristics, where the light chains are not detrimental to the expression or binding behavior of both heavy chains.

Thus, there remains a need in the art for compositions and methods for developing human globulin variable regions for use in human therapeutics, including human immunoglobulin variable regions ted from nucleic acid sequences at endogenous mouse immunogiobulin loci.

SUMMARY Mice are described that express human immunoglobulin variable domains that are le for use in bispecific binding proteins, including bispecific antibodies, wherein the mice comprise a humanization of an endogenous mouse heavy chain variable locus, wherein male mice comprising the humanization are fertile, and wherein the mice r comprise a humanization of an endogenous immunoglobulin light chain locus that results in the mouse expressing an immunoglobulin light chain repertoire that is d from no more than one, or no more than two, 7» and/or K V gene segments.

Genetically engineered mice are provided that select suitable affinity—matured human immunoglobulin heavy chain variable domains derived from a repertoire of unrearranged human heavy chain V, D, and J segments, wherein the affinity-matured human heavy chain variable s associate and express with a humanized sai light chain. The humanized universal light chain is expressed from a locus that comprises either no more than one or no more than two human light chain V segments and a human J t operably linked to a light chain constant gene, or no more than one or no more than two rearranged (VA/J7», VK/JK, VMJK, or Vic/J7») human nucleic acid ces encoding a light chain variable domain operably linked to a light chain nt gene. in various embodiments the universal humanized light chain domain pairs with a plurality of affinity-matured human heavy chain variable s, wherein the plurality of heavy chain variable domains specifically bind different epitopes or antigens. ln one aspect, nucleic acid ucts, cells, embryos, mice, and methods are provided for making mice that comprise a humanized heavy chain immunoglobulin variable locus and a humanized light chain immunoglobulin variable locus, wherein the mouse expresses one of no more than two universal light chains, and mice that are males exhibit wild-type fertility.

In one aspect, a modified mouse is provided that comprises in its germline a humanized heavy chain immunoglobulin le locus at an endogenous mouse heavy chain locus, and a humanized light chain immunoglobulin variable locus, wherein the mouse expresses a universal light chain, and wherein the mouse comprises a nucleic acid sequence encoding a mouse ADAM6 or ortholog or homolog or onal fragment thereof. in various embodiments the humanized light chain immunoglobulin variable locus is at an nous mouse light chain locus. in one ment, the humanized heavy chain immunoglobulin variable locus ses a ement at the endogenous mouse heavy chain variable locus of all or substantially all functional mouse immunoglobulin heavy chain V, D, and J gene segments with one or more human V, human D, and human J gene segments, wherein the one or more human V, D, and J segments are operably linked and capable of rearranging to form a rearranged V/D/J gene that is operably linked to a heavy chain constant sequence. in one embodiment, the mouse comprises a light chain locus that this engineered to make a universal light chain, wherein the universal light chain is a light chain that is derived from a light chain locus that ses no more than one light chain V segment and no more than one light chain J segment, or a light chain locus that comprises a single rearranged light chain V/J sequence. in one embodiment, the mouse comprises an immunoglobulin light chain locus that comprises single human immunoglobulin light chain V segment that is capable of rearranging with a human light chain J gene segment (selected from one or a ity of J segments) and encoding a human light chain variable domain. in another embodiment, the mouse comprises no more than two human light chain V segments at the light chain locus, each V segment of which is capable of rearranging with a human J gene segment (selected from one or a plurality of light chain J ts) and encoding a rearranged human light chain variable domain. in one embodiment, the single human light chain V segment is operably linked to a human light chain J segment selected from JK1, JK2, JK3, JK4, and JK5, wherein the single human light chain V segment is capable of rearranging to form a sequence encoding a light chain variable region gene with any of the one or more human light chain J segments. in one embodiment, the mouse comprises an nous light chain locus that comprises a ement of all or substantially all mouse V and J gene segments with no more than one, or no more than two, rearranged (V/J) nucleic acid sequences. in one embodiment, the no more than one or no more than two rearranged (V/J) nucleic acid sequences are ed from a human VK1-39JK5, a VK3—20JK1, and a combination In one embodiment, the mouse lacks a functional endogenous light chain locus that is capable of expressing a mouse light chain variable domain. in one embodiment, the mouse comprises a nucleic acid sequence encoding a variable domain of a universal light chain at a K locus. in one embodiment, the mouse comprises a nucleic acid sequence encoding a variable domain of a universal light chain at a it locus. in one embodiment, the human V segment (or rearranged V/J sequence) is operably linked to a human or mouse leader sequence. In one embodiment, the leader sequence is a mouse leader sequence. ln a specific embodiment, the mouse leader sequence is a mouse VK3—7 leader sequence.

In one embodiment, the human V segment (or rearranged V/J sequence) is operably linked to an immunoglobulin promoter ce. 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 one embodiment, the unrearranged V and J segments or the rearranged (V/J) sequence is operably linked to a light chain immunoglobulin constant region gene. in a specific embodiment, the constant region gene is a mouse CK gene. in one embodiment, the unrearranged V and J segments or the rearranged (V/J) sequence are present at 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 a specific embodiment, the K locus is an endogenous K locus. in one embodiment, the mouse comprises a K locus comprising a sequence ng a variable domain of a universal light chain, and the mouse comprises a nonfunctional immunoglobulin lambda (A) 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 A light chain locus incapable of rearranging to form a light chain gene. In another ment, all or substantially all of the V segments of the 9» light chain locus are deleted. In one another embodiment, the mouse comprises a deletion of all, or ntially all, of the endogenous light chain variable locus. in one embodiment, the mouse further comprises in its germline a sequence selected from a mouse K ic er 5’ with respect to rearranged immunoglobulin light chain sequence or the unrearranged gene segments, a mouse K 3’ enhancer, and a combination thereof.

In one embodiment, the universal light chain variable domain sequence of the mouse comprises one or more somatic hypermutations; in one ment, the variable domain ce ses a plurality of somatic utations. in one embodiment, the mouse makes a universal light chain that comprises a cally mutated human variable domain. ln one ment, the light chain comprises a somatically mutated human variable domain derived from a human V segment, a human J segment, and a mouse CK gene. In one embodiment, the mouse does not express a 7» light chain. in one embodiment, the human variable sequence is a rearranged human VK‘l— 39JK5 sequence, and the mouse expresses a reverse chimeric light chain comprising (i) a variable domain d from VK1-39JK5 and (ii) a mouse CL; wherein the light chain is associated with a reverse ic heavy chain comprising (i) a mouse CH and (ii) a somatically mutated human heavy chain variable domain. in one embodiment, the mouse expresses a light chain that is somatically mutated. in one embodiment the CL is a mouse in one embodiment, the human variable sequence is a nged human Vx3- 20JK1 sequence, and the mouse expresses a reverse ic light chain comprising (i) a variable domain d from VK3-20JK1, 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 heavy chain le domain.

In one embodiment, the mouse comprises both a nged human VK1—39JK5 sequence and a rearranged human VK3-20JK1 sequence, and the mouse ses a reverse chimeric light chain comprising (i) a light chain comprising a variable domain derived from the VK1—39JK5 ce or the 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 somatically mutated human heavy chain variable domain. In one embodiment, the mouse expresses a light chain that is somatically mutated. in one embodiment the CL is a mouse CK. in one embodiment, the mouse expresses a reverse chimeric antibody sing a light chain that comprises a mouse CK and a somatically mutated human variable domain derived from a rearranged human VK‘l—39JK5 sequence or a rearranged human JK1 sequence, and a heavy chain that comprises a mouse CH and a somatically mutated human heavy chain variable , 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 endogenous mouse K light chain gene segments with the rearranged human VK1-39JK5 sequence or the rearranged human VK3-20JK1 sequence, and comprises a replacement of all or substantially all nous mouse heavy chain V, D, and J gene segments with a complete or substantially complete repertoire of human heavy chain V, D, and J gene segments.

In one , a genetically modified mouse is provided that expresses a single 1c 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 sequence, wherein the single K light chain sequence is derived from no more than one rearranged K light chain sequence. in one embodiment, the serum titer is characterized as total immunoglobulin. In a specific embodiment, the serum titer is characterized as lgM specific titer. in a specific embodiment, the serum titer is characterized as lgG specific titer. in a more specific embodiment, the rearranged K light chain sequence is selected from a VK1-39JK5 and VK3-20JK1 ce. in one embodiment, the rearranged K light chain sequence is a VK1—39JK5 sequence. in one embodiment, the rearranged K light chain ce is a VK3-20JK1 sequence. in one , a genetically modified mouse is provided that expresses a plurality of immunoglobulin heavy chains ated with a single light chain sequence. in one embodiment, the heavy chain comprises a human sequence. in s embodiments, the human sequence is selected from a variable sequence, a CH1, a hinge, a CH2, a CH3, and a combination thereof. in one embodiment, the single light chain ses a human sequence. in various embodiments, the human sequence is selected from a variable sequence, a constant sequence, and a combination thereof. In one embodiment, the mouse comprises a disabled endogenoLis immunoglobulin locus and ses 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 segments (i.e., V, D, J), and/or some or all nous mouse heavy chain constant sequences (e.g., CH1, hinge, CH2, CH3, or a combination thereof), and/or some or all endogenous mouse light chain ces (6.9., V, J, constant, or a combination thereof), with one or more human immunoglobulin sequences. in one embodiment, the mouse of the one or more V, , following rearrangement D, and J gene segments, or one or more V and J gene segments, the mouse comprises in its genome at least one nucleic acid sequence encoding a mouse ADAM6 gene or homolog or ortholog or functional fragment f. In one ment, following rearrangement the mouse comprises in its genome at least two nucleic acid ces encoding a mouse ADAM6 gene or homolog or ortholog or functional fragment thereof. in one embodiment, following rearrangement the mouse comprises in its genome at least one nucleic acid ce encoding a mouse ADAMS gene or homolog or ortholog or functional fragment thereof. in one embodiment, the mouse comprises the ADAM6 gene or homolog or ortholog or onal fragment thereof in a B cell. in one embodiment, the male mice comprise a single unmodified endogenous ADAM6 allele or ortholog of homolog or functional fragment thereof at an endogenous ADAM6 locus. in one embodiment, the male mice comprise an ADAM6 sequence or homolog or ortholog or functional fragment thereof at a location in the mouse genome that approximates the location of the endogenous mouse ADAM6 allele, 6.9., 3’ of a final V gene segment sequence and 5’ of an initial D gene t. in one embodiment, the male mice comprise an ADAM6 sequence or homolog or ortholog or functional fragment thereof flanked am, downstream, or upstream and downstream (with respect to the direction of transcription of the ADAM6 sequence) of a nucleic acid sequence encoding an immunoglobulin le region gene t. in a specific embodiment, the immunoglobulin variable region gene segment is a human gene t. in one embodiment, the globulin variable region gene segment is a human gene segment, and the sequence encoding the mouse ADAM6 or ortholog or homolog or nt thereof functional in a mouse is between human V gene segments; in one embodiment, the mouse comprises two or more human V gene segments, and the sequence is at a position n the final V gene segment and the penultimate V gene segment; in one embodiment, the sequence is at a on following the final V gene segment and the first D gene segment. in one embodiment, the humanized heavy chain immunoglobulin variable locus lacks an endogenous mouse ADAM6 gene. In one embodiment, the humanized heavy chain immunoglobulin variable locus comprises an ADAM6 gene that is onal in a male mouse. in a specific embodiment, the ADAM6 gene that is functional in the male mouse is a mouse ADAM6 gene, and the mouse ADAM6 gene is placed within or immediately nt to the humanized heavy chain immunoglobulin variable locus. in one ment, the humanized heavy chain immunoglobulin variable locus lacks an endogenous mouse ADAM6 gene, and the mouse comprises an ectopic ADAM6 sequence that is functional in a male mouse. in one embodiment, the ectopic ADAM6 gene that is onal in the male mouse is a mouse ADAM6 gene. in one embodiment, the mouse ADAM6 gene is on the same chromosome as the humanized heavy chain immunoglobulin variable locus. in one embodiment, the mouse ADAM6 gene is on a different chromosome than the humanized heavy chain immunoglobulin le locus. in one embodiment, the mouse ADAM6 gene is on an episome. in one embodiment, the mouse comprises a first endogenous heavy chain allele and a second endogenous heavy chain allele, and the first endogenous heavy chain allele comprises a deletion of a mouse ADAM6 locus, and the first endogenous heavy chain allele comprises a replacement of all or substantially all functional mouse V, D, and J segments with one or more human V, D, and J segments. In one embodiment, the first and the second endogenous heavy chain alleles each comprise a deletion of an endogenous mouse ADAM6 locus, and the first and the second endogenous heavy chain alleles comprise a replacement of all or substantially all functional mouse V, D, and J segments with one or more human V, D, and J segments. in one embodiment, the first and/or the second allele comprises an ectopic nucleic acid sequence that encodes a mouse ADAM6 or ortholog or homoiog or functional fragment thereof. in one embodiment, the ectopic nucleic acid ce is located 3’ (with respect to the transcriptional ionality of the heavy chain variable locust) of a final mouse V gene segment and located 5’ (with respect to the transcriptional ionality of the constant sequence) of a mouse (or chimeric human/mouse) heavy chain constant gene or fragment thereof (e.g., a nucleic acid sequence encoding a human and/or mouse: CH1 and/or hinge and/or CH2 and/or CH3). in one embodiment, the ectopic nucleic acid ce is located downstream (with respect to direction of transcription of the V segment locus) of a V segment and upstream of a D segment. in one embodiment, the ectopic nucleic acid sequence is located between the imate 3’—most V segment and the ultimate t V segment. in a specific embodiment, the ectopic c acid sequence is located between human V segment VHi-Z and human V segment VH6—1. in one embodiment, the nucleotide sequence between the two human V gene ts is placed in opposite transcription ation with respect to the human V gene segments. in a specific embodiment, nucleotide sequence encodes, from 5’ to 3’ with respect to the direction of transcription of ADAM6 genes, and ADAM6a sequence followed by an ADAM6b sequence. in a specific embodiment, the ADAM6 gene(s) is oriented in te transcriptional orientation as compared with the upstream and downstream flanking V segments. in one embodiment, the nucleic acid sequence comprises a sequence encoding mouse ADAM6a or functional fragment thereof and/or a sequence encoding mouse ADAM6b or functional fragment thereof, wherein the ADAM6a and/or ADAM6b or functional fragment(s) thereof is operably linked to a promoter. In one embodiment, the er is a human promoter. in one embodiment, the promoter is the mouse ADAM6 promoter. in a ic embodiment, the ADAM6 promoter ses sequence located between the first codon of the first ADAM6 gene closest to the mouse 5’-most DH gene segment and the recombination signal sequence of the 5’-most DH gene segment, n ’ is indicated with respect to direction of transcription of the mouse immunoglobulin genes. in one embodiment, the promoter is a viral promoter. in a ic embodiment, the viral promoter is a cytomegalovirus (CMV) promoter. in one embodiment, the promoter is a tin promoter.

In one embodiment, the mouse ADAM6a and/or ADAM6b are selected from the ADAM6a of SEQ ID NO:1 and/or ADAM6b of sequence SEQ ID NO:2. In one embodiment, the mouse ADAMS promoter is a promoter of SEQ ID NO:3. In a ic ment, the mouse ADAMS er comprises the nucleic acid sequence of SEQ ID NO:3 directly upstream (with respect to the direction of transcription of ADAM6a) of the first codon of ADAMGa and extending to the end of SEQ ID NO:3 upstream of the ADAM6 coding . In another specific embodiment, the ADAM6 er is a fragment extending from within about 5 to about 20 nucleotides upstream of the start codon of ADAM6a to about 0.5kb, 1kb, 2kb, or 3kb or more am of the start codon of ADAM6a.

In one embodiment, the nucleic acid sequence comprises SEQ ID NO:3 or a fragment thereof that when placed into a mouse that is infertile or that has low fertility due to a lack of ADAMS, improves fertility or restores fertility to about a wild-type fertility. In one ment, SEQ ID NO:3 or a fragment thereof confers upon a male mouse the ability to produce a sperm cell that is capable of traversing a female mouse oviduct in order to fertilize a mouse egg.

In one embodiment, the mice comprise a nucleic acid sequence that encodes an ADAM6 protein, or ortholog or homolog or fragment thereof, that is functional in a male mouse. In a specific embodiment, the nucleic acid sequence is within or adjacent to a human nucleic acid sequence that ses one or more immunoglobulin variable region gene segment. In one embodiment, the one or more immunoglobulin variable region gene segments is at a modified endogenous mouse immunoglobulin heavy chain variable locus.

In one embodiment, the modification comprises a replacement of all or substantially all functional mouse immunoglobulin heavy chain variable gene segments with a plurality of ranged human heavy chain gene segments that are operably linked to an endogenous mouse constant region gene. In a specific embodiment, the nucleic acid sequence is between two human V segments. In a specific embodiment, the nucleic acid ce is between a human V segment and a human D segment. In a specific embodiment, the nucleic acid sequence is n a human D segment and a human J t. In a ic ment, the nucleic acid sequence is upstream of the 5’-most (with respect to ion of transcription of the V segment) human V segment. In a specific embodiment, the nucleic acid sequence is between a human J segment and an endogenous mouse heavy chain constant region gene sequence.

In one embodiment, the male mice are capable of generating offspring by mating, with a frequency that is about the same as a wild-type mouse. In one embodiment, the male mice produce sperm that can transit from a mouse uterus through a mouse oviduct to fertilize a mouse egg; in a specific embodiment, sperm of the mice transit through the oviduct about as efficiently as sperm from a wild-type mouse. In one embodiment, about 50% or more of the sperm produced in the mouse t the ability to enter and/or transit an oviduct to fertilize a mouse egg. in one embodiment, the mouse lacks a functional endogenous ADAM6 locus, wherein the mouse comprises an ectopic nucleotide sequence that complements the loss of mouse ADAM6 function in a male mouse. in one embodiment, the ectopic tide sequence confers upon the male mouse an ability to produce offspring that is comparable to a corresponding ype male mouse that contains a onal endogenous ADAM6 gene. in one embodiment, the sequence s upon the mouse an ability to form a complex of ADAM2 and/or ADAM3 and/or ADAMS on the surface of sperm cell of the mouse. ln one embodiment, the sequence confers upon the mouse an ability to travel from a mouse uterus through a mouse oviduct to a mouse ovum to fertilize the ovum.

In one embodiment, the mouse lacks a functional endogenous ADAM6 locus and comprises an ectopic nucleotide sequence ng an ADAM6 or ortholog or homolog or fragment thereof that is onal in a male mouse and wherein the male mouse produces at least about 50%, 60%, 70%, 80%, or 90% of the number of litters a ype mouse of the same age and strain produces in a six—month time . in one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide ce produces at least about 1.5—fold, about 2-fold, about 2.5-fold, about 3—fold, about 4-fold, about 6—fold, about 7-fold, about 8- fold, or about 10-fold or more progeny when bred over a nth time period than a mouse of the same age and the same or similar strain that lacks the functional endogenous ADAM6 gene and that lacks the ectopic nucleotide sequence that is bred over substantially the same time period and under substantially the same conditions. [0.043] in one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces an average of at least about 2-fold, 3—fold, or 4—fold higher number of pups per litter in a 4- or 6-month breeding period than a mouse that lacks the functional endogenous ADAMB gene and that lacks the ectopic nucleotide sequence, and that is bred for the same period of time. ln one embodiment, the mouse lacking the functional endogenous ADAMS gene and comprising the ectopic nucleotide sequence is a male mouse, and the male mouse produces sperm that when recovered from oviducts at about 5—6 hours post- copulation reflects an oviduct ion that is at least 10—fold, at least 20~fold, at least 30- fold, at least 40-fold, at least 50-fold, at least 60—fold, at least 70-fold, at least 80—fold, at least 90—fold, too-fold, ttO-fold, or iZO-fold or higher than sperm of a mouse that lacks the functional endogenous ADAM6 gene and that lacks the ectopic nucleotide sequence. in one ment, the mouse lacking the onal endogenous ADAM6 gene and comprising the ectopic tide sequence when copulated with a female mouse generates sperm that is capable of traversing the uterus and entering and traversing the oviduct within about 6 hours at an efficiency that is about equal to sperm from a wild-type mouse. in one embodiment, the mouse lacking the functional endogenous ADAMS gene and comprising the ectopic nucleotide sequence produces about 1.5-fold, about 2- fold, about 3-fold, or about 4-fold or more litters in a comparable period of time than a mouse that lacks the functional ADAMS gene and that lacks the ectopic nucleotide sequence. in one aspect, a mouse is provided that ses a humanized nous mouse heavy chain variable immunoglobulin locus and a modification of a mouse light chain immunoglobulin locus, wherein the mouse expresses a B cell that comprises a rearranged human heavy chain immunoglobulin sequence ly linked to a human or mouse heavy chain constant region gene sequence, and the B cell comprises in its genome (e.g., on a B cell chromosome) a gene encoding an ADAMS or ortholog or homolog or nt thereof that is functional in a male mouse (9.9., a mouse ADAMS gene, e.g,, mouse ADAMSa and/or mouse ADAMSb), wherein the variable domains of immunoglobulin A or K light chains of the mice are derived from no more than one or no more than two light chain V gene segments. in one embodiment, the nged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a human heavy chain V, D, and/or J sequence; a mouse heavy chain V, D, and/or J ce; a human or mouse light chain V and/or J sequence. ln one embodiment, the heavy chain constant region gene sequence comprises a human or a mouse heavy chain ce selected from the group consisting of a CH1, a hinge, a CH2, a CH3, and a combination thereof. 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, wherein the light chain comprises a human le domain, and wherein the antibodies comprise a heavy chain that comprises a human variable domain. in one aspect, a mouse is provided that is terized by an inability of the mouse to make a B cell that expresses an immunoglobulin light chain variable domain that is derived from a rearranged light chain sequence that is not a human VK1-39JK5 or a human VK3-20JK1 sequence. ln one embodiment, the mouse exhibits a K2?» light chain ratio that is about the same as a mouse that comprises a wild type complement of globulin light chain V and J gene segments.

In one aspect, a mouse as described herein is provided that ses an immunoglobulin light chain derived from a human VK1-39JK5 or a human JK1 sequence, wherein the mouse comprises a replacement of all or substantially all nous mouse heavy chain variable region gene ts with one or more human heavy chain le 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 s an immunoglobulin having a K light chain, of about 1 to about 20. ln one embodiment, the mouse expresses a single K light chain, wherein the single K light chain is derived from a human VK1-39JK5 ce, 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, wherein the single K light chain is 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 specific embodiments, the ratio is 1 to 20, or 1 to 21.

In one embodiment, the percent of ng‘”lg>t+ B cells in the mouse is about the same as in a wild type mouse. In a specific embodiment, the percent of ng+ng+ B cells in the mouse is about 2 to about 6 percent. in a specific embodiment, the percent of ng+lg>t+ 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 t of ng4’lgk+ 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 ic embodiment, about 6.

In one embodiment, the mouse is does not comprise a modification that reduces or eliminates an ability of the mouse to somatically mutate any functional light chain locus of the mouse. in one embodiment, the only functional light chain locus in the mouse expresses a light chain that ses a human variable domain derived from a nged sequence selected from a human VK1-39JK5 sequence, a human VK3-20JK1 sequence, and a combination thereof.

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 exhibits usage of the K light chain that is about 100—fold 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 600—fold or more, at ieast about 700—fold or more, at least about BOO—fold or more, at least about 900-fold or more, at least about old or more greater than the usage of the same K light chain (i.e., derived from the same V segment and the same J segment, or derived from the same rearranged V/J segment) exhibited by a mouse bearing 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 ranged 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 sequence. in one embodiment, the mouse comprises one copy of a rearranged K light chain sequence (e.g., a heterozygote). in one embodiment, the mouse comprises two copies of a rearranged K light chain sequence (e.g., a homozygote). 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 nged K light chain sequence is a VK3—20JK1 ce. in one aspect, a genetically modified mouse is provided that expresses a single light chain derived from no more than one, or no more than two, rearranged K light chain sequences, wherein the light chain in the genetically modified mouse exhibits a level of expression that is at least 10—fold to about 1,000-fold, 100-fold to about 1,000-fold, ZOO-fold to about 1,000-fold, BOO—fold to about 1,000—fold, 400-fold to about 1,000—fold, 500-fold to about 1,000—fold, GOO—fold to about 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 human K light chain variable locus. in one ment, the light chain ses a human sequence. In one ment, the single light chain is derived from a rearranged K light chain sequence selected from a human VK1-39JK5, a human VK3-20JK1, and a ation thereof. in one embodiment, the level of expression of the light chain, for the purpose of comparing the expression of the light chain with sion in a mouse comprising a substantialiy completely humanized light chain le locus, is characterized by quantitating mRNA of transcribed light chain sequence (from the one or two rearranged ces), and comparing it to transcribed light chain sequence of a mouse bearing a complete or substantially complete light chain locus.

In one aspect, a method for making an dy is provided, comprising expressing in a cell (a) a first human heavy chain variable domain nucleic acid sequence of an immunized mouse as described herein fused with a human CH gene sequence; (b) a human light chain le domain nucieic acid sequence of an immunized mouse as described herein fused with a human CL gene sequence; and, (c) ining the cell under ions sufficient to express a fully human antibody, and isolating the antibody. In one embodiment, the cell comprises a second human heavy chain variable domain c acid sequence of a second immunized mouse as described herein fused with a human CH gene sequence, the first heavy chain nucleic acid sequence encodes a first heavy chain variable domain that recognizes a first epitope, and the second heavy chain nucleic acid sequence encodes a second heavy chain variable domain that recognizes a second epitope, wherein the first e and the second epitope are not identical. in one aspect, a method for making an epitope—binding protein is provided, comprising exposing a mouse as described herein with an antigen that comprises an epitope of interest, maintaining the mouse under conditions sufficient for the mouse to generate an immunoglobulin ie that specifically binds the epitope of interest, and isolating the globuiin molecule that specifically binds the epitope of interest; wherein the epitope-binding protein comprises a heavy chain that comprises a somatically mutated human variable domain and a mouse CH, associated with a light chain comprising a mouse CL and a human variable domain derived from a rearranged human VK1-39JK5 or a rearranged human JK1.

In one aspect, a method for making a bispecific n—binding protein is provided, comprising exposing a first mouse as described herein to a first antigen of interest that comprises a first epitope, exposing a second mouse as bed herein to a second antigen of interest that comprises a second epitope, allowing the first and the second mouse to each mount immune responses to the antigens of st, identifying in the first mouse a first human heavy chain le region that binds the first epitope of the first antigen of interest, fying in the second mouse a second human heavy chain variable region that binds the second epitope of the second antigen of interest, making a first fully human heavy chain gene that encodes a first heavy chain that binds the first epitope of the ﬁrst antigen of interest, making a second fully human heavy chain gene that encodes a second heavy chain that binds the second epitope of the second antigen of interest, expressing the first heavy chain and the second heavy chain in a cell that expresses a single fully human light chain derived from a human VK1-39 or a human VK3- gene segment to form a bispecific antigen-binding protein, and ing the bispecific antigen~binding protein.

In one embodiment, the first antigen and the second antigen are not identical. in one embodiment, 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 variable region to the first epitope does not block binding of the second heavy chain le region to the second epitope. in one embodiment, the first antigen is selected from a soluble antigen and a cell surface antigen (9.9., a tumor antigen), and the second n comprises a cell surface receptor. in a specific embodiment, the cell surface receptor is an globulin receptor. In a specific embodiment, the immunoglobulin receptor is an Fc receptor. in one embodiment, the first n and the second antigen are the same cell 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 variable domain of the light chain comprises 2 to 5 somatic mutations. 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 zed mouse with either the first or the second heavy chain variable domain.

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 light chain variable domain that is derived from a rearranged human VK1-39JK5 or a rearranged human VK3-20JK1, wherein the human nucleic acid sequence is fused (directly or h a linker) to a human immunoglobulin light chain constant domain c acid sequence (9.9., a human K constant domain DNA sequence); and, (b) a first human heavy chain variable domain nucleic acid sequence encoding a human heavy chain variable domain derived from a first human heavy chain variable domain nucleotide sequence, wherein the first human heavy chain variable domain nucleotide sequence is fused (directly or through a linker) to a human immunoglobulin heavy chain constant domain nucleic acid sequence (e.g., a human lth, lgG2, lgG3, lgG4, or lgE sequence); wherein the epitope—binding protein recognizes a first e. 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 10‘10 M, lower than 10’11 M, or lower than 10'12 M. in one embodiment, the cell comprises a second human nucleotide sequence encoding a second human heavy chain variable domain, wherein the second human sequence is fused (directly or through a linker) to a human globulin heavy chain constant domain nucleic acid sequence, and wherein the second human heavy chain variable domain does not specifically recognize the first epitope (e.g., displays a dissociation nt of, e.g., 10‘ 6 M, 10'5 M, 10‘4 M, or ), and wherein the epitope—binding protein binds both the first epitope and the second epitope, and wherein the first and the second globulin heavy chains each associate with a light chain ing to (a). ln one embodiment, the second VH domain binds the second epitope with a dissociation constant that is lower than '6 M, lowerthan 10'7M, lower than 10'8 M, lower than 10'9 M, lower than 10‘10 M, lower than 10'11 M, or lower than 10'12 M. ln one embodiment, the epitope—binding protein comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, each associated with a universal light chain (e.g., a light chain derived from a rearranged human light chain variable sequence selected from a human VK1—39JK5 or a human JK1), wherein the first immunoglobulin heavy chain binds a first epitope with a dissociation constant in the nanomolar (e.g., 1 nM to 100 nM) to picomolar range (e.g., 1 pM to 100 pM), the second immunoglobulin heavy chain binds a second e with a dissociation constant in the nanomolar to lar range (e.g., 1 pM to 100 nM), 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 iation constant weaker than the micromolar range (e.g., the millimolar range), the second immunoglobulin heavy chain does not bind the first epitope or binds the first epitope with a dissociation constant weaker than the micromolar range (e.g., the millimolar , and one or more of the variable domains (i.e., one or more of the light chain variable domain, the heavy chain variable domain of the first immunoglobulin heavy chain, and the heavy chain variable domain) of the second immunoglobulin heavy chain is somatically mutated. In one embodiment, g of the epitope—binding protein to the first epitope does not block binding of the epitope-binding n to the second epitope. ln one embodiment, the first immunoglobulin heavy chain comprises a wild type protein A binding determinant, and the second heavy chain lacks a wild type protein A binding determinant. ln one embodiment, the first immunoglobulin heavy chain binds protein A under isolation conditions, and the second immunoglobulin heavy chain does not bind protein A or binds protein A at least 10-fold, a d-fold, or a thousand-fold 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 lgG1 isotypes, wherein the second heavy chain comprises a modification selected from 95R (EU 435R), 96F (EU 436F), and a combination thereof, and wherein the first heavy chain lacks such modification, In aspect, a mouse, embryo, or cell as described herein comprises a K light chain locus that retains endogenous regulatory or control ts, 6.9., a mouse K ic enhancer, a mouse K 3’ enhancer, or both an intronic enhancer and a 3’ er, wherein the regulatory or control elements facilitate somatic mutation and affinity maturation of an expressed sequence of the K light chain locus. {0071] in one aspect, a mouse cell is provided that is isolated from a mouse as bed herein. in one embodiment, the cell is an ES cell. in one embodiment, the cell is a lymphocyte. In one ment, the lymphocyte is a B cell. ln one embodiment, the B cell expresses a chimeric heavy chain comprising a variable domain derived from a human V gene segment; and a light chain derived from (a) a rearranged human VK1-39/J ce, (b) a rearranged human VK3-20/J sequence, or (c) a combination thereof; wherein the heavy chain le domain is fused to a mouse constant region and the light chain variable domain is fused to a mouse or a human constant region. in one embodiment, the mouse cell comprises at least one gene that encodes a mouse ADAM6 or ortholog or homolog or functional fragment thereof. in one embodiment, the cell is a B cell and the B cell comprises a sequence encoding a rearranged human heavy chain immunoglobulin variable domain and a sequence encoding a universal light chain variable , n the B cell comprises on a chromosome a nucleic acid sequence encoding an ADAM6 protein or og or homolog or fragment thereof that is functional in a male mouse; in one embodiment, the mouse B cell comprises two alleles of the nucleic acid sequence. in one aspect, a mouse cell is ed, comprising a first chromosome that comprises a humanized immunoglobulin heavy chain locus comprising unrearranged human V, D, and J segments; a second chromosome that comprises a humanized globulin light chain locus that encodes or is capable of rearranging to encode a light chain, n the light chain locus comprises no more than one V segment (or no more than two V segments) and no more than one J segment (or no more than two J segments) operably linked to a light chain constant region gene, or no more than one or no more than two rearranged light chain V/J sequences operably linked to a light chain constant gene; and a third chromosome that comprises nucleic acid sequence encoding a mouse ADAM6 or ortholog or homolog or fragment thereof that is functional in a male mouse. in one embodiment, the first and third chromosomes are the same. In one embodiment, the second and third chromosomes are the same. In one embodiment, the first, the second, and the third chromosomes are each different. ln one ment, the nucleic acid sequence encoding the mouse ADAM6 or ortholog or homolog or functional fragment thereof is present in two copies. in one ment, the cell is a somatic cell. in a specific embodiment, the somatic cell is a B cell. in one embodiment, the cell is a germ cell. in one , a hybridoma is provided, wherein the hybridoma is made with a B cell of a mouse as described herein. In a specific ment, the B cell is from a mouse as described herein that has been immunized with an antigen comprising an epitope of interest, and the B cell expresses a binding protein that binds the epitope of interest, the binding protein has a cally mutated human heavy chain variable domain and a mouse heavy chain constant region, and has a human light chain variable domain d from a rearranged human VK1-39JK5 or a rearranged human VK3-20JK1 and a mouse CL.

In one aspect, a cell is provided that comprises a fully human heavy chain gene comprising a nucleic acid sequence encoding a first heavy chain variable domain of a mouse as described herein, and a fully human light chain gene comprising a nucleic acid sequence encoding a universal light chain sequence as described herein. in one ment, the cell further comprises a nucleic acid sequence encoding a second heavy chain variable domain of a mouse as described herein, wherein the first and the second heavy chain variable domains are different. In one embodiment, the cell is selected from CHO, C08, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6TM cell). in one , a mouse embryo is provided, wherein the embryo comprises a donor ES cell that is d from a mouse as described herein.

In one aspect, use of a mouse embryo that comprises a c modification as described herein is provided, wherein the use ses making a cally modified mouse as described herein. ln one aSpect, a human heavy chain variable domain and a human light chain variable domain amino acid sequence of an antibody made in a mouse as described herein are provided.

In one aspect, a human heavy chain variable domain nucleotide sequence and a human light chain variable domain nucleotide sequence of an antibody made in a mouse as described herein is provided. in one aspect, an antibody or antigen—binding protein or antigen—binding fragment thereof (e.g., Fab, F(ab)2, scFv) made in a mouse as described herein is provided. ln one aspect, a mouse made using a targeting vector, nucleotide construct, or cell as described herein is provided. in one aspect, a progeny of a mating of a first mouse as described herein with a second mouse that is a ype mouse or genetically modified is provided.

In one aspect, use of a mouse as described herein to make a fully human antibody, or a fully human antigen-binding protein comprising an immunoglobulin variable domain or functional nt f, is provided. in one aspect, use of a mouse or tissue or cell as described herein to make a fully human bispecific dy is provided.

In one aspect, use of a nucleic acid sequence made by a mouse as described herein is provided, wherein the use comprises expressing the nucleic acid sequence in the manufacture of a human eutic. [0084A] In one aspect, there is provided use of nucleic acid sequence in the manufacture of a medicament for the treatment of a human disease or disorder, wherein the nucleic acid sequence is from a mouse made by a method described herein.

In one aspect, use of a mouse as described herein to make an immortalized cell line is provided.

In one aspect, use of a mouse as described herein to make a hybridoma or quadroma is ed.

In one aspect, use of a mouse as described herein to make a c acid sequence encoding an immunoglobulin variable region or fragment thereof is ed. In one embodiment, the nucleic acid ce is used to make a human antibody or n-binding fragment thereof. In one embodiment, the mouse is used to make an antigen-binding protein selected from an antibody, a multispecific antibody (e.g. , a bispecific antibody), an scFv, a bisscFV , a diabody, a triabody, a tetrabody, a V-NAR, a VHH, a VL, an F(ab), an F(ab)2, a DVD (i.e. , dual variable domain antigen-binding protein), an SVD (i.e. , single variable domain antigen-binding protein), or a bispecific T-cell engager (BiTE).

In one aspect, use of the mouse as described herein for the manufacture of a medicament (e.g. , an antigen-binding n), or for the manufacture of a sequence encoding a variable sequence of a medicament (e.g., an n-binding protein), for the treatment of a human disease or disorder is provided. [0088A] In one aspect, there is provided, a method of making a genetically modified mouse comprising genetically modifying the mouse to include in its germline: (a) a humanized immunoglobulin heavy chain locus comprising at least one unrearranged human VH gene t, at least one unrearranged human DH gene t, and at least one unrearranged human JH gene segment operably linked to a heavy chain constant region gene; (b) a humanized globulin light chain locus comprising a single rearranged human light chain V/J ce operably linked to a light chain constant region gene; and, (c) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein or functional nt thereof, wherein the mouse ADAM6 protein or functional fragment thereof is expressed from the ectopic nucleic acid sequence. [0088B] In one , there is provided a method of making a genetically modified mouse comprising genetically modifying the mouse to include in its ne: (a) a humanized immunoglobulin heavy chain locus comprising at least one unrearranged human VH gene segment, at least one unrearranged human DH gene segment, and at least one unrearranged human JH gene segment ly linked to a heavy chain constant region gene; (b) a zed immunoglobulin light chain locus comprising a single rearranged human light chain V/J sequence operably linked to a light chain constant region gene; and, (c) an inserted c acid sequence encoding a mouse ADAM6 protein or functional fragment thereof, wherein the mouse ADAM6 protein or functional fragment thereof is expressed from the inserted nucleic acid sequence. [0088C] In one aspect, there is provided, a method of making a genetically modified mouse comprising genetically modifying the mouse to express a plurality of different IgG heavy chains each comprising a human heavy chain variable domain, wherein each of the plurality of different IgG heavy chains are associated with an immunoglobulin light chain including a human immunoglobulin light chain le domain that is derived from a single human immunoglobulin VL gene segment, n the mouse ses an ectopic nucleic acid sequence encoding an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse. [0088D] In one aspect, there is ed a cell from a mouse made by a method according to the present disclosure. [0088E] In one aspect, there is provided a human immunoglobulin heavy chain variable region tide sequence of an dy expressed in a mouse made by a method according to the present disclosure. [0088F] In one aspect, there is provided a human immunoglobulin light chain variable region nucleotide sequence of an dy expressed in a mouse made by a method according to the present disclosure. [0088G] In one aspect, there is provided a human immunoglobulin heavy chain variable domain amino acid sequence of an antibody sed in a mouse made by a method according to the present disclosure. [0088H] In one aspect, there is provided a human immunoglobulin light chain variable domain amino acid sequence of an antibody expressed in a mouse made by a method according to the present disclosure.

] In one aspect, there is provided an dy or antigen-binding protein or antigenbinding fragment thereof expressed in a mouse made by a method according to the present disclosure. [0088J] In one aspect, there is provided use of a mouse made by a method according to the present sure, to make a fully human antibody, or a fully human antigen-binding protein, the fully human antibody or fully human antigen-binding protein comprising an immunoglobulin variable domain or functional fragment f. [0088K] In one , there is provided use of a mouse made by a method according to the t sure, to make a fully human bispecific antibody. [0088L] In one aspect, there is provided use of a mouse made by a method according to the present disclosure in the manufacture of an immortalized cell line. [0088M] In one aspect, there is provided use of a mouse made by a method according to the present disclosure in the manufacture of a hybridoma or ma. [0088N] In one aspect, there is provided use of a mouse made by a method according to the present disclosure to make a nucleic acid sequence encoding an immunoglobulin variable region or fragment thereof.

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

BRIEF DESCRIPTION OF THE FIGURES shows a general illustration, not to scale, for direct genomic ement of about three megabases (Mb) of the mouse immunoglobulin heavy chain variable gene locus (closed s) with about one megabase (Mb) of the human immunoglobulin heavy chain variable gene locus (open symbols). shows a general illustration, not to scale, for direct genomic ement of about three megabases (Mb) of the mouse immunoglobulin κ light chain variable gene locus (closed symbols) with about 0.5 megabases (Mb) of the first, or proximal, of two nearly identical repeats of the human immunoglobulin κ light chain variable gene locus (open symbols). shows a ed illustration, not to scale, for three initial steps (A–C) for direct genomic replacement of the mouse immunoglobulin heavy chain variable gene locus that results in deletion of all mouse VH, DH and JH gene segments and replacement with three human VH, all human DH and JH gene segments. A targeting vector for the first insertion of human immunoglobulin heavy chain gene segments is shown (3hVH BACvec) with a 67 kb 5’ mouse homology arm, a selection cassette (open rectangle), a site-specific recombination site (open triangle), a 145 kb human c fragment and an 8 kb 3’ mouse homology arm. Human (open symbols) and mouse (closed symbols) globulin gene segments, onal selection cassettes (open rectangles) and sitespecific recombination sites (open triangles) inserted from subsequent targeting vectors are shown. shows a detailed illustration, not to scale, for six additional steps (D—l) for direct genomic replacement of the mouse immunoglobulin heavy chain variable gene locus that results in the insertion of 77 additional human VH gene segments and removal of the final selection cassette. A targeting vector for insertion of additional human VH gene ts (18hVH BACvec) to the initial insertion of human heavy chain gene segments (3hVH—CRE Hybrid ) is shown with a 20 kb 5’ mouse homology arm, a selection cassette (open rectangle), a 196 kb human genomic fragment and a 62 kb human homology arm that overlaps with the 5’ end of the initial insertion of human heavy chain gene segments which is shown with a site-specific ination site (open triangle) located 5’ to the human gene segments. Human (open symbols) and mouse d symbols) globulin gene segments and additional selection tes (open rectangles) inserted by subsequent ing vectors are shown.

FlG. 2C shows a detailed illustration, not to scale, for three initial steps (A—C) for direct genomic replacement of the mouse immunoglobulin K light chain variable gene locus that results in deletion of all mouse VK, and JK gene segments (ng—CRE Hybrid Allele). Selection cassettes (open rectangles) and site-specific recombination sites (open triangles) inserted from the targeting vectors are shown. shows a ed illustration, not to scale, for 5 additional steps (D—H) for direct genomic replacement of the mouse immunoglobulin K light chain variable gene locus that results in the insertion of all human VK and JK gene ts in the proximal repeat and deletion of the ﬁnal selection cassette (4OhVKdHyg Hybrid Allele). Human (open s) and mouse (closed symbols) immunoglobulin gene ts and additional selection cassettes (open rectangles) inserted by subsequent targeting vectors are shown. shows a general illustration of the locations of quantitative PCR (qPCR) primer/probe sets for screening ES cells for insertion of human heavy chain gene sequences and loss of mouse heavy chain gene sequences. The ing strategy in ES cells and mice for the first human heavy gene insertion is shown with qPCR primer/probe sets for the deleted region (“loss” probes C and D), the region inserted (“hlgH” probes G and H) and flanking regions ntion” probes A, B, E and F) on an fied mouse chromosome (top) and a correctly targeted chromosome (bottom). shows a representative calculation of observed probe copy number in parental and modified ES cells for the first insertion of human immunoglobulin heavy chain gene segments. Observed probe copy number for probes A through F were calculated as 2/2AACt. AACt is calculated as ave[ACt(sample) — medACt(control)] where ACt is the difference in Ct between test and reference probes (between 4 and 6 reference probes depending on the assay). The term medACt(control) is the median ACt of multiple (>60) non-targeted DNA samples from parental ES cells. Each modified ES cell clone was assayed in sextuplicate. To calculate copy numbers of lgH probes G and H in parental ES cells, these probes were assumed to have copy number of 1 in modified ES cells and a maximum Ct of 35 was used even though no amplification was observed. shows a representative calculation of copy numbers for four mice of each genotype were calculated in a r manner using only probes D and H, ype mice: WT Mice; Mice heterozygous for the first insertion of human immunoglobulin gene segments: HET Mice; Mice homozygous for the first insertion of human immunoglobulin gene ts: Homo Mice. shows an illustration of the three steps employed for the construction of the 3hVH BACvec by bacterial homologous ination (BHR). Human (open symbols) and mouse (closed symbols) immunoglobulin gene segments, selection cassettes (open rectangles) and site-specific recombination sites (open triangles) inserted from targeting vectors are shown. shows pulse-field gel electrophoresis (PFGE) of three BAC clones (B1, 82 and B3) after Notl ion. Markers M1, M2 and M3 are low range, mid range and lambda ladder PFG markers, respectively (New England BioLabs, Ipswich, MA). ] shows a schematic illustration, not to scale, of sequential modifications of the mouse immunoglobulin heavy chain locus with increasing s of human immunoglobulin heavy chain gene segments. Homozygous mice were made from each of the three ent stages of heavy chain humanization. Open symbols t human sequence; closed symbols reflect mouse sequence.

FIG. SB shows a schematic illustration, not to scale, of sequential modifications of the mouse immunoglobulin 1c light chain locus with increasing amounts of human globulin K light chain gene segments. Homozygous mice were made from each of the three different stages OfK light chain humanization. Open s reflect human sequence; closed symbols reflect mouse sequence. shows FACS dot plots of B cell populations in wild type and VELOCIMMUNE® humanized mice. Cells from spleen (top row, third row from top and bottom row) or inguinal lymph node d row from top) of wild type (wt) or MMUNE® 1 (V1), VELOCIMMUNE® 2 (V2) or VELOCIMMUNE® 3 (V3) mice were stained for surface IgM expressing B cells (top row, and second row from top), surface immunoglobulin containing either K or I» light chains (third row from top) or surface lgM of specific haplotypes m row), and populations separated by FACS. shows representative heavy chain CDR3 sequences of randomly selected VELOCIMMUNE® antibodies around the JH (CDR3) junction, demonstrating onal diversity and nucleotide additions. Heavy chain CDR3 sequences are grouped according to DH gene segment usage, the germline of which is provided above each group in bold. VH gene segments for each heavy chain CDR3 sequence are noted within parenthesis at the 5’ end of each sequence (e.g. 3-72 is human VH3-72). JH gene segments for each heavy chain CDR3 are noted within hesis at the 3’ end of each sequence (eg. 3 is human JHS). SEQ ID NOs for each ce shown are as follows proceeding from top to bottom: SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID N0234; SEQ ID NO:35; SEQ ID N0236; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:39. shows representative light chain CDR3 sequences of randomly selected VELOCIMMUNE® antibodies around the VK-JK (CDR3) junction, demonstrating junctional diversity and nucleotide additions. VK gene segments for each light chain CDR3 sequence are noted within parenthesis at the 5’ end of each sequence (6.9. 1-6 is human VK1-6). JK gene segments for each light chain CDR3 are noted within parenthesis at the 3’ end of each sequence (9.9. 1 is human JK1). SEQ ID N05 for each sequence shown are as follows proceeding from top to bottom: SEQ ID NO:40; SEQ ID NO:41; SEQ ID NO:42; SEQ ID NO:43; SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58. shows somatic hypermutation frequencies of heavy and light chains of VELOCIMMUNE® antibodies scored (after alignment to matching ne sequences) as percent of sequences changed at each nucleotide (NT; left column) or amino acid (AA; right ) position among sets of 38 (unimmunized IgM), 28 (unimmunized lgG), 32 (unimmunized Igic from lgG), 36 (immunized lgG) or 36 (immunized ng from IgG) sequences. Shaded bars indicate the locations of CDRs. shows levels of serum immunoglobulin for lgM and lgG isotypes in wild type (open bars) or VELOClMMUNE® mice (closed bars).

FIG. SB shows levels of serum immunoglobulin for lgA isotype in wild type (open bars) or VELOCIMMUNE® mice (closed bars). ] shows levels of serum immunoglobulin for lgE isotype in wild type (open bars) or VELOClMMUNE® mice (closed bars).

A shows antigen specific lgG titers against eukin-6 receptor of serum from seven VELOCIMMUNE® (VI) and five wild type (WT) mice after two (bleed 1) or three (bleed 2) rounds of immunization with interleukin—6 receptor ectodomain. 8 shows anti-interleukin-6 receptor-specific lgG isotype—specific titers from seven VELOCIMMUNE® (Vl) and five wild type (WT) mice.

FlG. 11A shows the affinity distribution of nterleukin—6 receptor monoclonal antibodies generated in VELOCIMMUNE® mice. 8 shows the antigen—specific blocking of anti-interleukin—G receptor monoclonal antibodies generated in VELOCIMMUNE® (VI) and wild type (WT) mice. shows a tic illustration, not to scale, of mouse ADAM6a and ADAM6b genes in the mouse immunoglobulin heavy chain locus. A targeting vector (mADAM6 Targeting Vector) used for the insertion of mouse ADAM6a and ADAM6b into a humanized endogenous heavy chain locus is shown with a ion cassette (HYG: hygromycin) d by site-specific recombination sites (Frt) ing engineered restriction sites on the 5’ and 3’ ends. shows a schematic illustration, not to scale, of a human ADAMS pseudogene (hADAM61P) d between human heavy chain variable gene segments 1- 2 (VH1—2) and 6-1 (VHS-1). A ing vector for bacterial homologous recombination (hADAMG‘P Targeting ) to delete a human ADAM6 pseudogene and insert unique restriction sites into a human heavy chain locus is shown with a selection cassette (NEO: neomycin) flanked by site-specific recombination sites (onP) including engineered restriction sites on the 5’ and 3’ ends. An illustration, not to scale, of the resulting targeted humanized heavy chain locus containing a genomic fragment that s for the mouse ADAM6a and ADAM6b genes including a selection cassette flanked by site-speciﬁc recombination sites is shown.

A shows FACS contour plots of lymphocytes gated on singlets for surface expression of lgM and 8220 in the bone marrow for mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozygous for human heavy and human K light chain variable gene loci having an ed mouse genomic fragment comprising mouse ADAMS genes (H/K-AS). Percentage of immature (BZZOintlgM+) and mature (BZZOh‘ghlgMU B cells is noted in each r plot. 3 shows the total number of immature (BZZO‘r‘tlgMU and mature (BZZOhighlgM+) B cells in the bone marrow ed from femurs of mice gous for human heavy and human K light chain variable gene loci (H/K) and mice homozyogous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic nt encoding for mouse ADAMS genes (H/K-AS).

A shows FACS contour plots of CD19+—gated B cells for surface expression of c—kit and CD43 in the bone marrow for mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozyogous for human heavy and human K light chain variable gene loci having an c mouse genomic fragment encoding for mouse ADAMS genes (H/K-AS). Percentage of pro—B (CD19+CD43+ckit+) and pre-B (CD19+CD43‘ckit‘) cells is noted in the upper right and lower left nts, respectively, of each contour plot.

FlG. 158 shows the total number of pro-B cells (CD19+CD43+ckit*) and pre-B cells (CD19+CD43'ckit‘) in the bone marrow isolated from femurs of mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment comprising mouse ADAMS genes (H/K-AS).

A shows FACS contour plots of cytes gated on ts for surface expression of CD19 and CD43 in the bone marrow for mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding for mouse ADAMS genes (H/K-AS). Percentage of immature B (CD19+CD43'), pre-B (CD19+CD43‘“‘) and pro-B (CD19+CD43+) cells is noted in each contour plot.

FlG. 168 shows histograms of immature B (CD19+CD43') and pre-B (CD19+CD43'”‘) cells in the bone marrow of mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic nt encoding for mouse ADAMS genes (H/K-AS).

FlG. 17A shows FACS contour plots of lymphocytes gated on singlets for surface expression of CD19 and CD3 in splenocytes for mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding for mouse ADAM6 genes (H/K-A6). Percentage of B (CD19+CD3‘) and T (CD19'CD3*) cells is noted in each r plot. 8 shows FACs contour plots for CD19+-gated B cells for surface expression of lg)» and ng light chain in the spleen of mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment comprising mouse ADAM6 genes (H/K-AB). Percentage of lg)»+ (upper left quadrant) and ng+ (lower right quadrant) B cells is noted in each contour plot.

C shows the total number of CD19“ B cells in the spleen of mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse c nt comprising mouse ADAMS genes (H/K-A6).

A shows FACs contour plots of gated B cells for surface expression of lgD and lgM in the spleen of mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice gous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment comprising mouse ADAM6 genes (H/K-A6). Percentage of mature B cells (CD19*Igoh‘Q“igM‘“‘) is noted for each contour plot. The arrow on the right contour plot illustrates the process of maturation for B cells in on to lgM and lgD surface expression.

B shows the total number of B cells in the spleen of mice homozygous for human heavy and human K light chain variable gene loci (H/K) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding for mouse ADAMS genes (H/K-A6) during maturation from CD19”lthi9hlgD”‘t to CD19+lgM'"‘lgDhi9“. illustrates a targeting strategy for replacing endogenous mouse immunoglobulin light chain variable region gene segments with a human VK1-39JK5 gene region. illustrates a ing strategy for replacing endogenous mouse immunoglobulin light chain le region gene segments with a human VK3-20JK1 gene region. rates a targeting strategy for replacing endogenous mouse immunoglobulin light chain variable region gene segments with a human Jk5 gene region. ] shows the percent of CD19+ B cells (y—axis) from peripheral blood for wild type mice (WT), mice homozyogous for an ered human rearranged VK1—39JK5 light chain region (VK1-39JK5 HO) and mice homozygous for an engineered human rearranged VKB'ZOJK‘I light chain region (VK3-20JK1 HO).

A shows the relative 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 segments (HK), a wild type mouse (WT), and a mouse zygous for an engineered human rearranged VK1-39JK5 light chain region (VK1-39JK5 HET). Signals are normalized to expression of mouse CK. N.D.: not detected. 8 shows the relative mRNA expression (y-axis) of a VK1-39—derived light chain in a quantitative PCR assay using probes ic for the on of an engineered human rearranged VK1-39JK5 light chain region (VK1-39JK5 Junction Probe) and the human VK1-39 gene t (VK1-39 Probe) in a mouse homozygous for a ement of the endogenous VK and JK gene segments with human VK and JK gene ts (HK), a wild type mouse (WT), and a mouse homozygous for an engineered human rearranged VK1-39JK5 light chain region (VK1—39JK5 HO). Signals are normalized to expression of mouse CK. 0 shows the relative mRNA expression (y-axis) of a VK3derived light chain in a quantitative PCR assay using probes specific for ction of an engineered human rearranged VK3-20JK1 light chain region (VK3-20JK1 Junction Probe) and the human VK3-20 gene segment (VK3-20 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 expression of mouse CK.

A shows IgM (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 ﬁ-galatosidase.

] B shows total immunoglobulin (IgM, lgG, IgA) 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 B—galatosidase.

DETAILED DESCRIPTION ? The term ody', as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable (V H) region and a heavy chain constant region (C H). The heavy chain constant region comprises three domains, CH 1, CH2 and CH3. Each light chain comprises a light chain variable (V L) region and a light chain nt region (CL). The VH and V L regions can be further subdivided into s of ariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; 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 lower (e.g., about 1 x 10-9 M, 1 x 10-10 M, 1 x 1011 M, or about 1 x 10 -12 M). In one embodiment, KD is measured by surface plasmon resonance, e.g., BIACORETM; in another embodiment, KD is measured by ELISA. [00136A]? hout this specification the word "comprise", or variations such as "comprises" or ising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

? The phrase "bispecific antibody" includes an antibody e of selectively binding two or more epitopes. Bispecific antibodies generally comprise two nonidentical heavy chains, with each heavy chain specifically 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 bispecific dy is capable of selectively binding two ent 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 antibody can be on the same or a different target (e.g., on the same or a different protein). ific antibodies can be made, for example, by ing heavy chains that recognize different epitopes of the same immunogen. For example, nucleic acid sequences ng heavy chain variable sequences that recognize different epitopes of the same gen can be fused to nucleic acid sequences encoding the same or different heavy chain constant s, and such sequences can be expressed in a cell that expresses an immunoglobulin 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 e-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 prokaryotes and eukaryotes (single-cell or le-cell), bacterial cells (9.9., strains of E. coli, Bacillus spp., Streptomyces spp., etc), mycobacteria cells, fungal cells, yeast cells (9.9., S. siae, S. pombe, P. pastoris, P. methanolica, etc), plant cells, insect cells (9.9., SF-9, SF—21, baculovirus-infected insect cells, Trichoplusia 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 (9.9., CHO K1, DXB—11 CHO, Veggie-CHO), COS (9.9., COS-7), retinal cell, Vero, CV1, kidney (9.9., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepGZ, Wl38, MRC 5, Col0205, HB 8065, HL-60, (9.9., 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 entioned cell. in some embodiments, the cell comprises one or more viral genes, 9.9., a l cell that expresses a viral gene (9.9., a PER.C6TM cell).

The phrase “complementarity determining region,” or the term “CDR,” includes an amino acid ce encoded by a nucleic acid sequence of an organism’s immunoglobulin genes that normally (i.e., in a wild type ) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin le (9.9., an antibody or a T cell receptor). A CDR can be encoded 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 somatically mutated (9.9., vary from a sequence encoded in an animal’s ne), zed, and/or modified with amino acid substitutions, additions, or deletions. In some circumstances (9.9., for a CDR3), CDRs can be encoded by two or more sequences (9.9., germline sequences) that are not contiguous (9.9., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, 9.9., as the result of ng or connecting the sequences (9.9., V-D-J recombination to form a heavy chain CDR3).

] The term "conservative,” when used to describe a conservative amino acid substitution, includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (9.9., charge or hydrophobicity). in general, a vative amino acid substitution will not substantially change the functional properties of interest of a protein, for e, the ability of a variable region to specifically bind a target epitope with a desired affinity. es of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic- hydroxyl side chains such as serine and threonine; containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur—containing side chains such as cysteine and nine. Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine. In some embodiments, a conservative amino acid tution 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 disclosed in Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, e 256:1443-45, hereby orated by reference. In some embodiments, the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log—likelihood 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 sion and secretion from, 9. 9., 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” includes 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 ,1x10‘7 M,1x10’9 M, 1 x10'9M, 1 x10'1OM, 1 x 10‘11 M, orabout1 M).

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

The phrase ”functional nt” includes fragments of epitope-binding proteins that can be expressed, secreted, and specifically bind to an epitope with a K0 in the micromolar, nanomolar, or lar range. Specific recognition includes having a KD that is at least in the micromolar range, the nanomolar range, or the picomolar range.

The term “germline” includes reference to an immunoglobulin nucleic acid ce in a non-somatically d cell, 9.9., a non-somatically mutated B cell or pre-B » cell or hematopoietic cell.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes an globulin heavy chain constant region sequence from any sm. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise 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- terminal), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an epitope (e.g., izing the epitope with a KD in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.

The term ity” 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 e tide and/or amino acid ce identity. in some embodiments bed herein, identities are determined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MacVectorTM 10.0.2, MacVector Inc, 2008). The length of the ces compared with respect to identity of sequences will depend upon the particular ces, but in the case of a light chain constant 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 g two beta sheets comprising beta strands and capable of interacting with at least one CH1 domain of a human or a mouse. In the case of a CH1 domain, the length of sequence 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 identical or different, and the light chains may be identical or different.

The phrase “light chain” includes an immunoglobulin light chain ce from any organism, and unless otherwise specified includes 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 specified.

Generally, a full-length light chain includes, from amino terminus to carboxyl terminus, a VL domain that includes FR1-CDR1-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 e selectively bound by the epitope-binding protein in which they appear.

Light chains also e those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more epitopes selectively bound by the epitope-binding protein in which they appear.

Universal light chains, or common light chains, refer to light chains made in mice as bed , wherein the mice are highly restricted in the selection of gene segments available for making a light chain variable domain. As a result, such mice make a light chain d from, in one ment, no more than one or two unrearranged light chain V segments and no more than one or two unrearranged light chain J segments (9.9., one V and one J, two V's and one J, one V and two J's, two V's and two J's). ln one embodiment, no more than one or two rearranged light chain V/J sequences, e.g., a rearranged human VK1~39JK5 sequence or a rearranged human VK3-20JK1 sequence. In various embodiments universal light chains include somatically mutated (e.g., affinity matured) versions.

] The phrase “somatically mutated” 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 (e.g., a heavy chain le domain or including a heavy chain CDR or FR sequence) in the 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 framework nucleic acid sequence between a B cell that has not undergone class— switching and a B cell that has undergone class~switching “Somatically mutated” es reference to nucleic acid sequences from affinity-matured B cells that are not identical to ponding immunoglobulin variable region sequences in B cells that are not affinity- matured (i.e., sequences in the genome of germline cells). The phrase “somatically mutated” also includes reference to an immunoglobulin variable region nucleic acid sequence from a B cell after exposure of the B cell to an epitope of interest, n the nucleic acid sequence differs from the corresponding nucleic acid sequence prior to re of the B cell to the epitope of interest. The phrase “somatically d” 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 inherently operative in such an animal.

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

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

Mice with Humanized immunoglobulin Loci The mouse as a genetic model has been greatly enhanced by transgenic and knockout technologies, which have allowed for the study of the effects of the directed over- expression or deletion of specific genes. Despite all of its advantages, the mouse still presents genetic obstacles that render it an imperfect model for human diseases and an imperfect platform to test human therapeutics or make them. First, although about 99% of human genes have a mouse homolog ston, R.H., et. al. (2002). initial sequencing and comparative analysis of the mouse genome. Nature 420, 520-562.), potential eutics often fail to cross-react, or cross-react inadequately, with mouse orthologs of the intended human targets. To obviate this problem, selected target genes can be “humanized,” that is, the mouse gene can be eliminated and replaced by the corresponding human orthologous gene sequence (e.g., US 6,586,251, US 541 and US 7,105,348, incorporated herein by reference). initially, efforts to humanize mouse genes by a “knockout—plus-transgenic humanization” strategy entailed crossing a mouse carrying a deletion (i.e., ut) of the nous gene with a mouse ng a randomly integrated human transgene (see, 9.9., Bril, W.S., et al. (2006). Tolerance to factor Vlll in a transgenic mouse expressing human factor Vlll cDNA carrying an Arg(593) to Cys substitution. Thromb Haemost 95, 341-347; Homanics, G.E., et al. (2006). Production and terization of murine models of classic and intermediate maple syrup urine disease.

BMC Med Genet 7, 33; , D., et al. (2006). A humanized BAC transgenic/knockout mouse model for HbE/beta—thalassemia. Genomics 88(3):309-15; Pan, Q., at al. (2006).

Different role for mouse and human CD3delta/epsilon dimer in preT cell receptor R) function: human CD3delta/epsilon heterodimer restores the defective preTCR on in CD39amma— and madelta-deficient mice. Mol lmmunol 43, 1741-1750).

But those efforts were hampered by size limitations; tional knockout technologies were not sufficient to directly replace large mouse genes with their large human genomic counterparts. A straightforward approach of direct homologous replacement, in which an endogenous mouse gene is directly replaced by the human counterpart gene at the same precise genetic location of the mouse gene (i.e., at the endogenous mouse locus), is rarely attempted because of technical difficulties. Until now, efforts at direct ement involved elaborate and burdensome procedures, thus limiting the length of genetic al that could be handled and the precision with which it could be manipulated.

Exogenously introduced human immunoglobulin transgenes rearrange in precursor B-cells in mice (Alt, F.W., Blackwell, T.K., and Yancopoulos, GD. (1985). immunoglobulin genes in transgenic mice. Trends Genet 1, 6). This finding was exploited by engineering mice using the knockout—plus—transgenic approach to express human antibodies (Green, L.L. et al. (1994). Antigen—specific human monoclonal antibodies from mice engineered with human lg heavy and light chain YACs. Nat Genet 7, 13-21; Lonberg, N. (2005). Human antibodies from transgenic animals. Nat Biotechnol 23, 1117- 1125; Lonberg, N., et al. (1994). Antigen—specific human antibodies from mice comprising four distinct genetic cations. Nature 368, 856-859; Jakobovits, A., etal. (2007). From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice. Nat Biotechnol 25, 1134-1143). The endogenous mouse immunoglobulin heavy chain and K light chain loci were inactivated in these mice by targeted deletion of small but critical portions of each endogenous locus, followed by introducing human immunoglobulin gene loci as randomly integrated large transgenes, as described above, or romosomes (Tomizuka, K., etal. (2000). Double trans-chromosomic mice: maintenance of two individual human some nts containing lg heavy and kappa loci and expression of fully human antibodies. Proc Natl Acad Sci U S A 97, 722- 727). Such mice represented an important advance in genetic engineering; fully human monoclonal dies isolated from them d promising eutic potential for treating a variety of human diseases (Gibson, T.B., et al. (2006). Randomized phase lll trial s of panitumumab, a fully human pidermal growth factor receptor monoclonal antibody, in metastatic colorectal cancer. Clin Colorectal Cancer 6, 29-31; Jakobovits et al., 2007; Kim, Y.H., et al. (2007). Clinical efficacy of zanolimumab (HuMax-CD4): two Phase ll studies in refractory cutaneous T—cell lymphoma. Blood 109(11):4655~62; Lonberg, 2005; Maker, A.V., et al. (2005). Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte—associated antigen 4 blockade and interleukin 2: a phase l/ll study.

Ann Surg Oncol 12, 1005-1016; McClung, M.R., et al. (2006). Denosumab in postmenopausal women with low bone mineral density. N Engl J Med 354, 821-831). But, as discussed above, these mice exhibit compromised B cell development and immune deficiencies when compared to wild type mice. Such problems potentially limit the ability of the mice to support a vigorous humoral response and, consequently, generate fully human antibodies against some antigens. The deficiencies may be due to: (1) inefficient functionality due to the random introduction of the human immunoglobulin transgenes and resulting ect expression due to a lack of upstream and downstream control elements (Garrett, F.E., et al. . Chromatin architecture near a potential 3' end of the igh locus involves modular regulation of histone modifications during B—Cell development and in vivo occupancy at CTCF sites. Mol Cell Biol 25, 525; Manis, J.P., et al. (2003).

Elucidation of a downstream boundary of the 3' lgH regulatory region. Mol lmmunol 39, 753-760; Pawlitzky, l., et al. (2006). identification of a candidate tory element within the 5' flanking region of the mouse lgh locus defined by pro-B cell-specific hypersensitivity ated with g of PU.1, Pax5, and E2A. J lmmunol 176, 6839-6851); (2) cient pecies interactions between human constant domains and mouse ents of the B-cell receptor signaling complex on the cell surface, which may impair signaling processes required for normal maturation, proliferation, and survival of B cells (Hombach, J., et al. (1990). Molecular components of the B-celi antigen receptor complex of the igM class. Nature 343, 760-762); and (3) cient interspecies ctions between soluble human immunoglobulins and mouse Fc receptors that might reduce affinity selection (Rao, S.P., et al. (2002). Differential expression of the inhibitory lgG Fc receptor chammaRiiB on germinal center ceiis: implications for ion of high-affinity B cells. J immunol 169, 1859-1868) and immunoglobulin serum concentrations eil, F.W., et al. . A Theoretical Model of Gamma-Giobulin Catabolism. Nature 203, 1352—1354; Junghans, R.P., and Anderson, C.L. (1996). The protection receptor for IgG catabolism is the beta2— lobulin-containing neonatal intestinal transport receptor. Proc Natl Acad Sci U S A 93, 5512-5516; Rao etal., 2002; Hjeim, F., et al. (2006). Antibody-mediated regulation of the immune response. Scand J Immunol 64, 177-184; Nimmerjahn, F., and Ravetch, J.V. (2007). Fc-receptors as regulators of immunity. Adv immunol 96, 179—204). These deficiencies can be corrected by in situ humanization of only the variable regions of the mouse immunoglobulin loci within their natural ons at the endogenous heavy and light chain loci. This would effectively result in mice that make reverse chimeric (i.e., human V: mouse C) antibodies that would be capable of normal interactions and selection with the mouse environment based on retaining mouse constant regions. r, such e chimeric antibodies are readily reformatted into fully human antibodies for therapeutic purposes.

A method for a large in situ genetic replacement of the mouse ne immunoglobulin variable genes with human germline immunoglobulin variable genes while maintaining the ability of the mice to generate offspring is described. Specifically, the precise replacement of six megabases of both the mouse heavy chain and K light chain immunoglobulin variabie gene ioci with their human counterparts while leaving the mouse nt regions intact is described. As a result, mice have been created that have a precise replacement of their entire germline immunoglobulin variable repertoire with lent human germline immunoglobulin variable sequences, while maintaining mouse constant regions. The human variable regions are linked to mouse constant regions to form chimeric human—mouse immunoglobulin loci that rearrange and express at physiologically appropriate levels. The antibodies expressed are “reverse chimeras,” i. 9., they comprise human variable region ces and mouse constant region ces.

These mice having humanized immunoglobulin variable regions that express antibodies having human variable regions and mouse constant regions are called VELCOIMMUNE® humanized mice.

] VELOCIMMUNE® humanized mice exhibit a fully functional humoral immune system that is essentially indistinguishable from that of wild-type mice. They dispiay normal cell populations at all stages of B cell development. They exhibit normal lymphoid organ morphology. Antibody sequences of VELOClMMUNE® humanized mice exhibit normal variable segment ngement and normal somatic hypermutation. dy populations in these mice reflect isotype distributions that result from normal class switching (9.9., normal isotype cis-switching). lmmunizing VELOClMMUNE® humanized mice results in robust humoral responses that te a large diversity of antibodies having human immunoglobulin variable domains suitable as eutic candidates. This platform provides a plentiful source of affinity-matured human globulin variable region sequences for making pharmaceutically acceptable antibodies and other antigen- binding proteins.

It is the precise replacement of mouse immunoglobulin variable sequences with human immunoglobulin variable sequences that allows for making VELOClMMUNE® humanized mice. Yet even a precise replacement of endogenous mouse immunoglobulin sequences at heavy and light chain loci with equivalent human immunoglobulin sequences, by sequential recombineering of very large spans of human immunoglobulin sequences, may present certain challenges due to ent evolution of the immunoglobulin loci between mouse and man. For example, intergenic sequences interspersed within the immunoglobulin loci are not cal between mice and humans and, in some circumstances, may not be functionally equivalent. Differences between mice and humans in their immunoglobulin loci can still result in abnormalities in humanized mice, particularly when humanizing or manipulating certain portions of endogenous mouse immunoglobulin heavy chain loci. Some modifications at mouse immunoglobulin heavy chain loci are rious. Deleterious modifications can include, for example, loss of the ability of the modified mice to mate and e offspring.

A e, large-scale, in situ replacement of six megabases of the variable regions of the mouse heavy and light chain immunoglobulin loci (VH-DH-JH and VK-JK) with the corresponding 1.4 megabases human genomic sequences was performed, while leaving the flanking mouse ces intact and functional within the hybrid loci, including all mouse constant chain genes and locus riptional control s (Figure 1).

Specifically, the human VH, DH, JH, VK and JK gene sequences were introduced through stepwise insertion of 13 chimeric BAC targeting vectors g overlapping fragments of the human germline variable loci into mouse ES cells using VELOClGENE® genetic engineering technology (see, e.g., US Pat. No. 6,586,251 and Valenzuela, D.M., et al. (2003). hroughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21, 652-659).

Humanization of the mouse immunoglobulin genes represents the largest genetic modification to the mouse genome to date. While previous efforts with randomly integrated human immunoglobulin transgenes have met with some success ssed above), direct replacement of the mouse immunoglobulin genes with their human rparts dramatically increases the efficiency with which fully—human antibodies can be efficiently generated in othenivise normal mice. Further, such mice exhibit a dramatically increased diversity of fully-human antibodies that can be obtained after immunization with virtually any antigen, as compared with mice bearing disabled endogenous loci and fully human antibody enes. Multiple versions of replaced, humanized loci exhibit completely normal levels of mature and immature B cells, in st to mice with ly integrated human transgenes, which exhibit significantly reduced B cell populations at various stages of differentiation. While efforts to increase the number of human gene segments in human transgenic mice have reduced such defects, the expanded immunoglobulin repertoires have not ther ted reductions in B cell populations as compared to wild-type mice.

Notwithstanding the near wild-type humoral immune function observed in mice with replaced immunoglobulin loci, there are other nges encountered when employing a direct ement of the immunoglobulin that is not encountered in some approaches that employ randomly integrated transgenes. Differences in the genetic composition of the immunoglobulin loci between mice and humans has lead to the discovery of sequences cial for the propagation of mice with replaced immunoglobulin gene segments. Specifically, mouse ADAM genes located within the endogenous immunoglobulin locus are optimally present in mice with replaced immunoglobulin loci, due to their role in fertility.

Genomic Location and Function of Mouse ADAM6 Male mice that lack the ability to express any functional ADAM6 protein exhibit a severe defect in the ability of the mice to mate and to generate offspring. The mice lack the ability to express a functional ADAM6 protein by virtue of a replacement of all or substantially all mouse immunoglobulin variable region gene segments with human variable region gene segments. The loss of ADAM6 function s because the ADAM6 locus is located within a region of the endogenous mouse immunoglobulin heavy chain variable region gene locus, proximal to the 3’ end of the VH gene segment locus that is am of the DH gene segments. In order to breed mice that are homozygous for a replacement of all or substantially all endogenous mouse heavy chain variable gene segments with human heavy chain le gene ts, it is generally a cumbersome approach to set up males and s that are each homozygous for the replacement and await a tive mating. Successful litters are relatively rare, and average litter size is very low. Instead, males heterozygous for the replacement have been employed to mate with females homozygous for the replacement to generate progeny that are heterozygous for the replacement, then breed a homozygous mouse therefrom. The inventors have determined that the likely cause of the loss in fertility in the male mice is the absence in gous male mice of a functional ADAM6 protein.

The ADAM6 protein is a member of the ADAM family of proteins, where ADAM is an acronym for A Disintegrln And Metalloprotease. The ADAM family of proteins is large and e, with e functions. Some members of the ADAM family are implicated in spermatogenesis and ization. For example, ADAM2 encodes a subunit of the protein fertilin, which is ated in sperm-egg interactions. ADAM3, or cyritestin, appears necessary for sperm binding to the zona pellucida. The absence of either ADAM2 or ADAM3 results in infertility. It has been postulated that ADAM2, ADAM3, and ADAM6 form a complex on the surface of mouse sperm cells.

The human ADAM6 gene, normally found between human VH gene segments VH1—2 and VH6-1, s to be a pseudogene (Figure 12). in mice, there are two ADAM6 genes—ADAM6a and —that are found in an intergenic region between mouse VH and DH gene segments, and in the mouse the a and b genes are oriented in a transcriptional orientation opposite to that of the transcription orientation of the surrounding immunoglobulin gene segments (Figure 11). In mice, a functional ADAM6 locus is apparently required for normal fertilization. A functional ADAM6 locus or sequence, then, refers to an ADAM6 locus or sequence that can complement, or rescue, the drastically reduced fertilization ted in male mice with missing or damaged endogenous ADAM6 loci.

The position of the intergenic sequence in mice that encodes ADAM6a and ADAM6b renders the intergenic sequence susceptible to cation when modifying an nous mouse heavy chain. When VH gene segments are deleted or replaced, or when DH gene ts are deleted or replaced, there is a high ility that a resulting mouse will exhibit a severe deficit in fertility. in order to compensate for the deficit, the mouse is ed to include a nucleotide sequence that encodes a protein that will complement the loss in ADAM6 activity due to a modification of the endogenous mouse ADAM6 locus. In various embodiments, the complementing nucleotide sequence is one that s a mouse , a mouse ADAMGb, or a g or ortholog or functional fragment thereof that rescues the fertility deficit.

The nucleotide sequence that rescues fertility can be placed at any suitable position. it can be placed in the intergenic region, or in any suitable position in the genome (i.e., ectopically). in one embodiment, the nucleotide sequence can be introduced into a transgene that randomly integrates into the mouse genome. in one embodiment, the sequence can be maintained episomally, that is, on a separate nucleic acid rather than on a mouse chromosome. Suitable positions include positions that are transcriptionaily permissive or , e.g., a ROSA26 locus.

The term “ectopic” is ed to include a displacement, or a placement at a position that is not normally tered in nature (e.g., placement of a nucleic acid ce at a position that is not the same position as the nucleic acid sequence is found in a wild-type mouse). The term in various embodiments is used in the sense of its object being out of its normal, or proper, position. For example, the phrase “an ectopic nucleotide sequence encoding refers to a nucleotide sequence that appears at a position at which it is not normally encountered in the mouse. For example, in the case of an ectopic nucleotide sequence encoding a mouse ADAM6 protein (or an ortholog or homolog or fragment thereof that provides the same or similar ity benefit on male mice), the sequence can be placed at a different position in the mouse’s genome than is normally found in a wild-type mouse. A functional homolog or ortholog of mouse ADAM6 is a sequence that confers a rescue of fertility loss (e.g., loss of the ability of a male mouse to generate offspring by mating) that is observed in an ADAM6" mouse. Functional homologs or orthologs include proteins that have at least about 89% identity or more, e.g., up to 99% identity, to the amino acid sequence of ADAM6a and/or to the amino acid sequence of ADAM6b, and that can complement, or rescue ability to sfully mate, of a mouse that has a genotype that includes a deletion or knockout of ADAM6a and/or ADAM6b.

The ectopic position can be anywhere (e.g., as with random insertion of a transgene ning a mouse ADAM6 sequence), or can be, e.g., at a position that approximates (but is not precisely the same as) its location in a wild—type mouse (e.g., in a ed endogenous mouse immunoglobuiin locus, but either upstream or downstream of its l position, e.g., within a modified immunoglobuiin locus but between different gene segments, or at a different position in a mouse V-D intergenic sequence). One example of an ectopic placement is placement within a humanized immunoglobuiin heavy chain locus.

For example, a mouse comprising a replacement of one or more endogenous VH gene segments with human VH gene segments, wherein the replacement removes an endogenous ADAM6 sequence, can be ered to have a mouse ADAM6 sequence located within sequence that contains the human VH gene segments. The resulting modification would generate an (ectopic) mouse ADAM6 sequence within a human gene sequence, and the (ectopic) placement of the mouse ADAM6 sequence within the human gene sequence can approximate the position of the human ADAM6 pseudogene (i.e., between two V ts) or can approximate the position of the mouse ADAM6 sequence (i.e., within the V-D intergenic ). in various aspects, mice that comprise deletions or repiacements of the endogenous heavy chain variable region locus or portions thereof can be made that contain an ectopic nucleotide sequence that encodes a n that confers similar fertility benefits to mouse ADAM6 (e.g., an ortholog or a g or a fragment thereof that is functional in a male mouse). The ectopic nucleotide sequence can include a nucleotide sequence that encodes a protein that is an ADAMS g or ortholog (or fragment f) of a different mouse strain or a ent species, e.g., a different rodent species, and that confers a benefit in fertility, e.g., increased number of litters over a specified time period, and/or increased number of pups per litter, and/or the ability of a sperm cell of a male mouse to traverse through a mouse oviduct to fertilize a mouse egg. ] in one embodiment, the ADAM6 is a homolog or og that is at least 89% to 99% cal to a mouse ADAMG protein (e.g., at least 89% to 99% identical to mouse ADAM6a or mouse ADAMGb). in one embodiment, the ectopic nucleotide sequence encodes one or more proteins independently selected from a protein at least 89% identical to mouse ADAM6a, a protein at least 89% identical to mouse ADAM6b, and a combination thereof. in one embodiment, the homolog or ortholog is a rat, hamster, mouse, or guine pig protein that is or is ed to be about 89% or more cal to mouse ADAM6a and/or mouse ADAMBb. in one embodiment, the homolog or ortholog is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a mouse ADAM6a and/or mouse ADAMBb.

Ectopic ADAMS in Humanized Heavy Chain Mice Mice that make human antibodies have been available for some time now.

Although they represent an important advance in the development of human therapeutic antibodies, these mice display a number of significant abnormalities that limit their usefulness. For example, they display compromised B cell development. The compromised development may be due to a variety of differences n the transgenic mice and wild~type mice.

Human antibodies might not optimally interact with mouse pre B cell or B cell receptors on the surface of mouse cells that signal for maturation, eration, or survival during clonal selection. Fully human antibodies might not lly interact with a mouse Fc receptor system; mice express Fc receptors that do not display a one-to-one correspondence with human Fc receptors. Finally, various mice that make fully human antibodies do not include all genuine mouse sequences, e.g., downstream enhancer elements and other locus control elements, which may be required for wild-type B cell development.

Mice that make fully human antibodies generally comprise endogenous immunoglobulin loci that are disabled in some way, and human enes that comprise variable and constant immunoglobulin gene segments are introduced into a random location in the mouse genome. As long as the nous locus is sufficiently disabled so as not to rearrange gene segments to form a functional immunoglobulin gene, the goal of making fully human antibodies in such a mouse can be achieved—albeit with compromised B cell development. gh compelled to make fully human antibodies from the human transgene locus, generating human antibodies in a mouse is apparently an unfavored process. ln some mice, the process is so unfavored as to result in formation of chimeric human variable/mouse constant heavy chains (but not light chains) through the mechanism of trans—switching. By this mechanism, transcripts that encode fully human dies undergo isotype switching in trans from the human isotype to a mouse isotype. The process is in trans, because the fully human transgene is located apart from the endogenous locus that retains an undamaged copy of a mouse heavy chain constant region gene. Although in such mice trans-switching is readily apparent the phenomenon is still insufﬁcient to rescue B cell development, which s y impaired. in any event, trans-switched antibodies made in such mice retain fully human light chains, since the phenomenon of trans-switching apparently does not occur with respect to light chains; trans-switching presumably relies on switch sequences in nous loci used t differently) in normal isotype switching in cis. Thus, even when mice engineered to make fully human antibodies select a trans-switching mechanism to make antibodies with mouse nt regions, the strategy is still icient to rescue normal B cell development.

A primary concern in making antibody-based human therapeutics is making a sufficiently large diversity of human immunoglobulin variable region sequences to identify useful variable domains that specifically recognize particular epitopes and bind them with a desirable affinity, usually—but not always—with high affinity. Prior to the pment of VELOClMMUNE® humanized mice, there was no indication that mice expressing human variable regions with mouse nt regions would exhibit any significant differences from mice that made human antibodies from a transgene. That supposition, however, was incorrect.

] VELOClMMUNE® humanized mice, which contain a precise replacement of mouse immunoglobulin variable regions with human immunoglobulin variable regions at the nous mouse loci, display a surprising and able similarity to wild-type mice with respect to B cell development. ln a surprising and ng development, VELOClMMUNE® humanized mice displayed an essentially normal, wild-type response to immunization that differed only in one significant respect from wild-type mice—the variable s generated in response to immunization are fully human.

VELOClMMUNE® humanized mice contain a precise, large-scale replacement of germline variable s of mouse immunoglobulin heavy chain (lgH) and immunoglobulin light chain (e.g., K light chain, ng) with corresponding human immunoglobulin le regions, at the endogenous loci. In total, about six megabases of mouse loci are replaced with about 1.4 megabases of human genomic sequence. This precise replacement results in a mouse with hybrid immunoglobulin loci that make heavy and light chains that have a human le s and a mouse constant region. The precise replacement of mouse VH-DH-JH and VK-JK segments leave flanking mouse sequences intact and functional at the hybrid immunoglobulin loci. The humoral immune system of the mouse functions like that of a wild-type mouse. 8 cell development is ered in any icant respect and a rich diversity of human variable regions is generated in the mouse upon antigen challenge.

VELOClMMUNE® humanized mice are possible because immunoglobulin gene segments for heavy and K light chains rearrange rly in humans and mice, which is not to say that their loci are the same or even nearly so—clearly they are not. r, the loci are r enough that humanization of the heavy chain variable gene locus can be accomplished by replacing about 3 million base pairs of contiguous mouse sequence that contains all the VH, DH, and JH gene segments with about 1 million bases of contiguous human genomic sequence covering basically the equivalent sequence from a human immunoglobulin locus.

] In some embodiments, r replacement of certain mouse constant region gene sequences with human gene sequences (e.g., replacement of mouse CH1 sequence with human CH1 sequence, and replacement of mouse CL ce with human CL sequence) results in mice with hybrid immunoglobulin loci that make antibodies that have human variable regions and partly human constant regions, suitable for, e.g., making fully human antibody fragments, e.g., fully human Fab’s. Mice with hybrid immunoglobulin loci exhibit normal variable gene segment rearrangement, normal somatic hypermutation, and normal class ing. These mice exhibit a humoral immune system that is indistinguishable from wild type mice, and display normal cell populations at all stages of B cell development and normal lymphoid organ structures—even where the mice lack a full repertoire of human variable region gene segments. immunizing these mice results in robust humoral responses that display a wide diversity of variable gene segment usage.

The precise replacement of mouse germline variable region gene ts allows for making mice that have partly human immunoglobulin loci. Because the partly human immunoglobulin loci rearrange, hypermutate, and class switch normally, the partly human immunoglobulin loci generate antibodies in a mouse that se human variable regions. Nucleotide sequences that encode the variable regions can be identified and cloned, then fused (e.g., in an in vitro system) with any sequences of choice, e.g., any immunoglobulin isotype suitable for a ular use, resulting in an antibody or antigen— binding protein derived wholly from human sequences. scale humanization by recombineering methods were used to modify mouse embryonic stem (ES) cells to precisely replace up to 3 megabases of the mouse heavy chain immunoglobulin locus that included essentially all of the mouse VH, DH, and JH gene segments with equivalent human gene segments with up to a 1 megabase human genomic sequence containing some or essentially all human VH, DH, and JH gene segments. Up to a 0.5 se segment of the human genome comprising one of two repeats encoding essentially all human VIC and JK gene segments was used to replace a 3 megabase segment of the mouse immunoglobulin K light chain locus containing essentially all of the mouse VK and JK gene segments.

Mice with such ed immunoglobulin loci can comprise a disruption or deletion of the endogenous mouse ADAM6 locus, which is normally found between the 3’- most VH gene segment and the 5’—most DH gene segment at the mouse immunoglobulin heavy chain locus. Disruption in this region can lead to reduction or elimination of onality of the endogenous mouse ADAMS locus. lf the 3’-most VH gene segments of the human heavy chain oire are used in a replacement, an intergenic region containing a pseudogene that s to be a human ADAMS pseudogene is present n these VH gene segments, i.e., between human VHi—Z and VH1-6. However, male mice that comprise this human intergenic sequence exhibit little or no fertility.

Mice are described that comprise the replaced loci as described above, and that also se an ectopic nucleic acid sequence encoding a mouse ADAM6, where the mice exhibit essentially normal fertility. In one embodiment, the ectopic nucleic acid sequence is SEQ ID NO:3, placed between human VHi—Z and VH1-6 at the modified endogenous mouse heavy chain locus. The ion of transcription of the ADAM6 genes of SEQ lD N023 are opposite with respect to the direction of transcription of the nding human VH gene segments. Although examples herein show rescue of fertility by placing the ectopic sequence between the indicated human VH gene segments, skilled persons will recognize that placement of the ectopic sequence at any suitable transcriptionally-permissive locus in the mouse genome (or even extrachromosomally) will be expected to similarly rescue fertility in a male mouse.

The phenomenon of complementing a mouse that lacks a functional ADAM6 locus with an ectopic sequence that comprises a mouse ADAM6 gene or og or homolog or functional nt f is a general method that is applicable to ng any mice with nonfunctional or lly functional endogenous ADAM6 loci. Thus, a great many mice that comprise an ADAM6-disrupting modification of the immunoglobulin heavy chain locus can be rescued with the compositions and methods of the invention.

Accordingly, the invention comprises mice with a wide variety of modifications of immunoglobulin heavy chain loci that compromise endogenous ADAM6 function. Some (non-limiting) examples are provided in this description. in addition to the VELOClMMUNE® humanized mice described, the itions and methods related to ADAM6 can be used in a great many ations, e.g., when modifying a heavy chain locus in a wide y of ways.

In one aspect, a mouse is provided that comprises an ectopic ADAM6 sequence that encodes a functional ADAM6 protein (or og or homolog or functional fragment thereof), a replacement of all or substantially all mouse VH gene segments with one or more human VH gene segments, a replacement of all or substantially all mouse DH gene segments and JH gene segments with human DH and human JH gene segments; wherein the mouse lacks a CH1 and/or hinge region. In one embodiment, the mouse makes a single variable domain binding protein that is a dimer of immunoglobulin chains selected from: (a) human VH — mouse CH1 — mouse CH2 — mouse CH3; (b) human VH — mouse hinge — mouse CH2 — mouse CH3; and, (0) human VH - mouse CH2 —- mouse CH3, in one aSpect, the nucleotide sequence that rescues fertility is placed within a human immunoglobulin heavy chain variable region sequence (e.g., n human VH1-2 and VH1-6 gene segments) in a mouse that has a replacement of all or substantially all mouse immunoglobulin heavy chain variable gene segments (mVH’s, mDH’s, and mJH’s) with one or more human immunoglobulin heavy chain variable gene segments , hDH’s, and hJH’s), and the mouse further comprises a replacement of all or substantially all mouse immunoglobulin K light chain variable gene segments (mVK’s, mJK’s) with one or more human immunoglobulin K light chain variable gene segments (th’s and hJK’s). in one embodiment, the nucleotide sequence is placed between a human VHt—Z gene segment and a human VHt—S gene t in a VELOClMMUNE® humanized mouse (US 6,596,541 and US 348, incorporated herein by nce). ln one embodiment, the VELOCIMMUNE® humanized mouse so modified comprises a replacement with all or substantially all human immunoglobulin heavy chain variable gene segments (all hVH’s, hDH’s, and hJH’s) and all or substantially all human immunoglobulin K light chain variable gene segments (hVic’s and hJK’s).

In one aspect, a functional mouse ADAMS locus (or ortholog or homolog or functional fragment thereof) can be placed in the midst of human VH gene segments that e endogenous mouse VH gene segments. in one embodiment, all or substantially all mouse VH gene segments are removed and replaced with one or more human VH gene segments, and the mouse ADAMS locus is placed immediately adjacent to the 3’ end of the human VH gene segments, or between two human VH gene segments. in a specific embodiment, the mouse ADAMS locus is placed between two VH gene segments near the 3’ terminus of the inserted human V... gene segments. in a ic embodiment, the replacement includes human VH gene segments Vin-2 and VHS-1, and the mouse ADAMS locus is placed ream of the VHi—Z gene segment and upstream of the VHS-1 gene segment. in a specific embodiment, the arrangement of human VH gene segments is then the following (from upstream to downstream with respect to ion of transcription of the human VH gene segments): human VH1-2 — mouse ADAMS locus — human VHS-1. in a specific ment, the ADAMS pseudogene between human VH1-2 and human VHS-1 is replaced with the mouse ADAMS locus. in one embodiment, the orientation of one or more of mouse ADAMSa and mouse ADAMSb of the mouse ADAMS locus is opposite with respect to direction of transcription as compared with the orientation of the human VH gene segments. Alternatively, the mouse ADAMS locus can be placed in the intergenic region between the t human VH gene segment and the 5’-most DH gene segment. This can be the case whether the 5’-most DH segment is mouse or human.

Similarly, a mouse modified with one or more human VL gene segments (e.g., VK or V)» segments) replacing all or ntially all nous mouse VH gene segments can be modified so as to either maintain the endogenous mouse ADAMS locus, as described above, e.g., by employing a targeting vector having a ream homology arm that includes a mouse ADAM6 locus or functional fragment thereof, or to replace a damaged mouse ADAM6 locus with an ectopic sequence oned between two human VL gene segments or n the human VL gene segments and a DH gene segment (whether human or mouse, 9.9., V)» + m/hDH), or a J gene segment (whether human or mouse, e.g., VK + JH). ln one embodiment, the replacement includes two or more human VL gene segments, and the mouse ADAM6 locus or functional fragment thereof is placed n the two t VL gene segments. ln 3 specific embodiment, the arrangement of human VL gene segments is then the following (from am to downstream with respect to direction of transcription of the human gene segments): human VL3’-1 — mouse ADAM6 locus - human VL3’. ln one embodiment, the ation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is opposite with respect to direction of transcription as compared with the orientation of the human VL gene segments.

Alternatively, the mouse ADAM6 locus can be placed in the intergenic region between the 3’-most human VL gene segment and the 5’-most DH gene segment. This can be the case whether the 5’—most DH segment is mouse or human.

] In one aspect, a mouse is provided with a replacement of one or more endogenous mouse VH gene segments, and that comprises at least one endogenous mouse DH gene segment. In such a mouse, the modiﬁcation of the endogenous mouse VH gene segments can comprise a modification of one or more of the 3’—most VH gene segments, but not the 5’-most DH gene segment, where care is taken so that the modification of the one or more 3’—most VH gene segments does not disrupt or render the endogenous mouse ADAM6 locus ctional. For example, in one embodiment the mouse comprises a replacement of all or ntially all endogenous mouse VH gene ts with one or more human VH gene segments, and the mouse comprises one or more endogenous DH gene segments and a functional endogenous mouse ADAM6 locus. ln another embodiment, the mouse comprises the modification of endogenous mouse 3’-most VH gene segments, and a modification of one or more endogenous mouse DH gene segments, and the modification is carried out so as to in the integrity of the endogenous mouse ADAM6 locus to the extent that the endogenous ADAM6 locus remains functional. ln one example, such a modification is done in two steps: (1) replacing the 3’-most endogenous mouse VH gene segments with one or more human VH gene ts employing a targeting vector with an upstream homology arm and a ream homology arm wherein the downstream homology arm includes all or a portion of a functional mouse ADAM6 locus; (2) then replacing and endogenous mouse DH gene segment with a targeting vector having an upstream gy arm that includes a all or a functional portion of a mouse ADAM6 locus. ln various s, employing mice that contain an ectopic sequence that encodes a mouse ADAMS protein or an orthoiog or homolog or functional homolog thereof are useful where cations disrupt the function of endogenous mouse ADAMS. The probability of disrupting endogenous mouse ADAMS function is high when making cations to mouse immunoglobulin loci, in particular when modifying mouse immunogiobulin heavy chain le regions and surrounding sequences. Therefore, such mice provide particular benefit when making mice with immunoglobulin heavy chain loci that are deleted in whole or in part, are humanized in whole or in part, or are replaced (9.9., with VK or V?» sequences) in whole or in part. s for making the genetic modifications described for the mice described below are known to those skilled in the art.

Mice containing an ectopic sequence encoding a mouse ADAMS protein, or a substantially cal or similar protein that confers the fertility benefits of a mouse ADAMS protein, are particularly useful in conjunction with cations to a mouse immunoglobulin heavy chain variable region gene locus that disrupt or delete the endogenous mouse ADAMS sequence. Although primarily described in connection with mice that express dies with human variable regions and mouse constant s, such mice are useful in connection with any genetic modifications that disrupt the endogenous mouse ADAMS gene. Persons of skill will recognize that this encompasses a wide variety of genetically modified mice that contain modifications of the mouse immunoglobulin heavy chain variable region gene locus. These include, for example, mice with a deletion or a replacement of all or a portion of the mouse immunoglobulin heavy chain gene segments, regardless of other modifications. Non-limiting examples are described below, in some aspects, genetically modified mice are provided that comprise an ectopic mouse, rodent, or other ADAMS gene (or ortholog or homolog or fragment) functional in a mouse, and one or more human immunoglobulin variable and/or constant region gene segments. in one , a mouse is provided that comprises an ectopic ADAMS sequence that encodes a functional ADAMS protein, a ement of all or substantially all mouse VH gene segments with one or more human VH gene segments; a ement of all or substantially all mouse DH gene segments with one or more human DH gene segments; and a replacement of all or substantially all mouse JH gene segments with one or more human JH gene segments.

In one embodiment, the mouse r ses a replacement of a mouse CH1 tide sequence with a human CH1 nucleotide sequence. In one embodiment, the mouse further comprises a replacement of a mouse hinge nucleotide sequence with a human hinge nucleotide sequence. in one embodiment, the mouse further comprises a replacement of an immunoglobulin light chain variable locus (VL and JL) with a human immunoglobulin light chain variable locus. in one embodiment, the mouse further ses a replacement of a mouse immunoglobulin light chain constant region nucleotide ce with a human immunoglobulin light chain constant region nucleotide ce. In a specific embodiment, the VL, JL, and CL are immunoglobulin K light chain sequences. in a specific embodiment, the mouse comprises a mouse CH2 and a mouse CH3 immunoglobulin constant region sequence fused with a human hinge and a human CH1 sequence, such that the mouse immunoglobulin loci rearrange to form a gene that encodes a binding protein comprising (a) a heavy chain that has a human variable region, a human CH1 region, a human hinge region, and a mouse CH2 and a mouse CH3 region; and (b) a gene that encodes an immunoglobulin light chain that comprises a human variable domain and a human constant region. in one aspect, a mouse is ed that ses an ectopic ADAM6 sequence that encodes a functional ADAMG protein, a replacement of all or substantially all mouse VH gene segments with one or more human VL gene segments, and optionally a replacement of all or substantially all DH gene segments and/or JH gene segments with one or more human DH gene segments and/or human JH gene segments, or optionally a ement of all or substantially all DH gene segments and JH gene segments with one or more human JL gene segments. ] in one embodiment, the mouse comprises a replacement of all or substantially all mouse VH, DH, and JH gene segments with one or more VL, one or more DH, and one or more J gene segments (9.9., JK or J)»), wherein the gene segments are ly linked to an nous mouse hinge region, wherein the mouse forms a rearranged immunoglobulin chain gene that contains, from 5’ to 3’ in the direction of transcription, human VL -— human or mouse DH -— human or mouse J - mouse hinge —- mouse CH2 —- mouse CH3. in one embodiment, the J region is a human JK region. In one embodiment, the J region is a human JH region. In one embodiment, the J region is a human J)» region. in one embodiment, the human VL region is selected from a human V)» region and a human VK region. in specific embodiments, the mouse expresses a single variable domain antibody having a mouse or human constant region and a variable region derived from a human VK, a human DH and a human JK; a human VK, a human DH, and a human JH; a human V)», a human DH, and a human J)»; a human V)», a human DH, and a human JH; a human VK, a human DH, and a human J)»; a human V)», a human DH, and a human JK. in ic embodiment, recombination recognition sequences are modified so as to allow for productive rearrangements to occur between recited V, D, and J gene segments or n recited V and J gene segments. ] in one aspect, a mouse is provided that comprises an ectopic ADAM6 sequence that encodes a functional ADAM6 protein (or ortholog or homolog or functional fragment thereof), a replacement of all or substantially all mouse VH gene segments with one or more human VL gene segments, a replacement of all or substantially all mouse DH gene t and JH gene segments with human JL gene segments; wherein the mouse lacks a CH1 and/or hinge region.

In one embodiment, the mouse lacks a sequence ng a CH1 domain. in one embodiment, the mouse lacks a sequence encoding a hinge . in one embodiment, the mouse lacks a sequence encoding a CH1 domain and a hinge region. ] in a ic embodiment, the mouse ses a binding protein that comprises a human immunogiobulin light chain variable domain (A or K) fused to a mouse CH2 domain that is attached to a mouse CH3 domain. in one aspect, a mouse is provided that comprises an ectopic ADAM6 sequence that s a functional ADAM6 protein (or ortholog or homolog or onal fragment thereof), a replacement of all or substantially all mouse VH gene segments with one or more human VL gene segments, a replacement of all or substantially all mouse DH and JH gene segments with human JL gene segments. in one embodiment, the mouse comprises a deletion of an immunogiobulin heavy chain constant region gene sequence encoding a CH1 region, a hinge region, a CH1 and a hinge region, or a CH1 region and a hinge region and a CH2 region. in one ment, the mouse makes a single variable domain binding protein comprising a homodimer selected from the following: (a) human VL — mouse CH1 — mouse CH2 — mouse CH3; (b) human VL —— mouse hinge -— mouse CH2 — mouse CH3; (c) human VL — mouse CH2 —- mouse CH3. in one aspect, a mouse is provided with a disabled endogenous heavy chain immunogiobulin locus, comprising a disabled or deleted nous mouse ADAM6 locus, wherein the mouse comprises a nucleic acid sequence that expresses a human or mouse or human/mouse or other chimeric antibody. in one embodiment, the nucleic acid ce is present on a transgene integrated that is randomly integrated into the mouse genome. In one embodiment, the nucleic acid sequence is on an episome (9.9., a chromosome) not found in a wiid-type mouse.

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

On the other hand, in vivo methods for employing complex organisms to select appropriate pairings that are capable of leading to human therapeutics have not been realized. lly, native mouse sequences are frequently not a good source for human therapeutic ces. For at least that reason, generating mouse heavy chain immunoglobulin variable regions that pair with a common human light chain is of d practical utility. More in vitro engineering efforts would be expended 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 ain 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 ine with an nous mouse heavy chain constant domain to form a reverse chimeric heavy chain (Le, a heavy chain comprising a human variable domain and a mouse nt region).

The heavy chain should be capable of class switching and somatic hypermutation so that a suitably large oire of heavy chain variable s 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 utility. In various embodiments, antibodies that express in a mouse that can only s a common light chain will have heavy chains that can associate and express with an identical or substantially identical light chain. This is particularly useful in making bispecific antibodies. For example, such a mouse can be immunized with a first immunogen 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 regions can be cloned from the B cells and expresses with the same heavy chain constant region, and the same light chain, and sed in a cell to make a bispecific dy, wherein the light chain component of the bispecific dy has been selected by a mouse to associate and express with the light chain component.

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

The cally engineered mouse, through the long and complex process of antibody selection within an organism, makes biologically appropriate choices in pairing a diverse collection of human heavy chain variable domains with a limited number of human light chain options. in order to achieve this, the mouse is engineered to present a limited number of human light chain variable domain options in conjunction with a wide diversity of human heavy chain variable domain options. Upon nge with an antigen, the mouse maximizes the number of solutions in its oire to develop an antibody to the antigen, limited largely or solely by the number or light chain options in its repertoire. in various embodiments, this includes allowing the mouse to e suitable and compatible somatic mutations of the light chain variable domain that will nonetheless 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 nonfunctional or substantially nonfunctional its ability to make, or nge, a native mouse light chain variable domain. This can be achieved, 6.9., by deleting the mouse’s light chain variable region gene segments. The endogenous mouse locus can then be modified by an exogenous suitable human light chain variable region gene segment of , operably linked to the endogenous mouse light chain constant domain, in a manner such that the ous human variable region gene segments can combine with the endogenous mouse light chain constant region gene and form a nged reverse chimeric light chain gene (human variable, mouse constant). in various embodiments, the light chain variable region is capable of being somatically mutated. in various embodiments, to ze ability of the light chain variable region to acquire somatic mutations, the appropriate enhancer(s) is ed in the mouse. For example, in modifying a mouse K light chain locus to e 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 provided that expresses a limited repertoire of reverse chimeric (human variable, mouse constant) light chains associated with a diversity of e chimeric (human variable, mouse constant) heavy chains. in various embodiments, the endogenous mouse K light chain gene segments are d and replaced with a single (or two) rearranged human light chain , operably linked to the endogenous mouse CK gene. ln ments for maximizing somatic hypermutation of the rearranged human light chain region, the mouse K intronic enhancer and the mouse K 3’ enhancer are maintained. in various embodiments, the mouse also comprises a nonfunctional x light chain locus, or a deletion thereof or a deletion that renders the locus unable to make a A light chain.

A genetically engineered mouse is provided that, in various embodiments, comprises a light chain variable region locus lacking 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, wherein 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 engineered mouse in various embodiments when zed with an antigen of interest tes B cells that t a diversity of rearrangements of human globulin heavy chain variable regions that express and function with one or with two rearranged light chains, including embodiments where the one or two light chains comprise human light chain variable regions that comprise, 9.9., 1 to 5 somatic ons. in various embodiments, the human light chains so expressed are e of associating and expressing with any human immunoglobulin heavy chain variable region expressed in the mouse.

Epitope-binding ns That Bind 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 antibodies. Advantages of the invention include the ability to select suitably high binding (e.g., affinity matured) heavy chain immunoglobulin chains each of which will associate with a single light chain.

Synthesis and expression of bispecific binding proteins has been problematic, in part due to issues associated with identifying a suitable light chain that can ate and express with two ent heavy chains, and in part due to isolation . The methods and compositions described herein allow for a genetically ed 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 (6.9., affinity matured). Human VL and VH sequences from suitable B cells of zed mice as bed herein that s affinity matured antibodies having reverse 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 s a human heavy chain variable domain that binds a different epitope.

One of the human VLs (e.g., human VK1-39JK5 or human VK3-20JK1), 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 (6.9., a human K 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 meric heavy chain with the cal light chain, and a heterodimeric heavy chain with the identical light chain. To allow for a facile separation of these major species, one of the heavy chains is modified 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 12/832,838, filed 25 June 2010, entitled “Readily isolated Bispecific Antibodies with Native lmmunoglobulin 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 immunized with an antigen sing an epitope of st. in one embodiment, an epitope-binding protein is provided that comprises a first and a second ptide, 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 comprises a first CH3 region of a human lgG selected from lgG1, lgG2, 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 nt region that comprises a second CH3 region of a human 196 selected from lgG1, lgGZ, lgG4, and a combination thereof, wherein the second CH3 region comprises a modification that reduces or ates g 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 further comprises 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 selected from the group consisting of D16E, L18M, N448, K52N, V57M, and V82i (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 ses a modification selected from the group consisting of N448, K52N, and V82i (lMGT; N3848, K392N, and V422l by EU).

In one embodiment, the second CH3 region is from a modified human lgG4, and further comprises a modification selected from the group consisting of 015R, N448, K52N, V57M, R69K, E79Q, and V82i (lMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422| 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 e of interest, wherein the mouse comprises an endogenous immunoglobulin light chain variable region locus that does not contain an endogenous mouse VL that is capable of rearranging and forming a light chain, wherein at the endogenous mouse immunglobulin light chain variable region locus is a single rearranged human VL region operably linked to the mouse endogenous light chain constant 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 se human variable domains and mouse constant s. When immunized, such a mouse will make a reverse ic dy, comprising only one of two human light chain variable s (9.9., 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 ved (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 immunoglobulin constant domains can the same or different isotype, and one of the immunoglobulin nt domains (but not the other) can be modified as described herein or in US 2010/0331527A1, and epitope-binding protein can be expressed in a suitable cell and isolated based on its differential affinity for Protein A as compared to a homodimeric e-binding protein, tag, as described in US 2010/0331527A1. in one embodiment, a method for making a bispecific epitope-binding protein is provided, comprising identifying a first affinity-matured (e.g., comprising one or more somatic hypermutations) human VH nucleotide sequence (VH1) from a mouse as described herein, identifying a second affinity-matured (6.9., comprising one or more somatic hypermutations) human VH nucleotide sequence (VH2) from a mouse as described herein, g VH1 in frame with a human heavy chain lacking a Protein rminant modification as described in US 2010/0331527A1 for form heavy chain 1 (HC1), cloning VH2 in frame with a human heavy chain sing a Protein A-determinant as described in US 2010/0331527A1 to form heavy chain 2 (HCZ), introducing an expression vector comprising HC1 and the same or a different expression vector comprising HC2 into a cell, wherein the cell also expresses a human immunoglobulin light chain that comprises a human VK1-39/human JK5 or a human VK3-20/human JKl fused to a human light chain constant domain, allowing the cell to express a bispecific epitope—binding protein comprising a VH domain encoded by VH1 and a VH domain d by VH2, and isolating the ific epitope—binding protein based on its differential y to bind Protein A as compared with a ecific homodimeric epitope—binding protein. in a specific embodiment, HC1 is an lgG1, and H02 is an lgG1 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 d by VH2, or both, are somatically mutated.

Human VH Genes That Express with a Common Human VL A variety of human variable regions from affinity-matured antibodies raised against four ent antigens were sed with either their cognate light chain, or at least one of a human light chain selected from human JK5, human VK3-20JK1, or human VpreBJkS (see Example 10). 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 ed from cells expressing the heavy and light chains. For VK1-39JK5 and VK3-20JK1, VH domains derived from the following human VH gene families expressed favorably: 1-2, 1-8, 1~24, 2- , 3-7, 3-9,3—i1,3-13,3—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 domains from one or both of VK1~39JK5 and VK3—20JK1 will generate a diverse population of somatically mutated human VH domains from a VH locus modified to replace mouse VH gene ts with human VH gene segments.

Mice genetically engineered to express reverse chimeric (human variable, mouse nt) globulin heavy chains associated with a single rearranged light chain (e.g., a VK1-39/J or a VK3—20/J), when zed with an antigen of interest, generated B cells that comprised a ity of human VH ngements and expressed a diversity of high—affinity antigen—specific antibodies with diverse properties with respect to their ability to block binding of the antigen to its ligand, and with respect to their ability to bind variants of the antigen (see Examples 14 through 15).

Thus, the mice and methods described herein are useful in making and selecting human immunoglobulin heavy chain le domains, including cally 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 lar or less), a wide variety of specificities (including binding to different epitopes of the same antigen), and that associate and express with the same or ntially the same human immunoglobulin light chain variable region. in one aspect, a first mouse comprising a humanized heavy chain variable region locus is bred with a second mouse comprising a nucleic acid sequence encoding a common, or universal, light chain locus as described . in one embodiment, the first or the second mouse comprises an ectopic nucleic acid sequence encoding a mouse ADAM6 or ortholog or homolog or functional fragment thereof. Progeny are bred to obtain mice homozygous for a humanized heavy chain locus, and homozygous for the universal light chain locus. in one embodiment, the first mouse or the second mouse comprises a modification of an endogenous mouse light chain locus to render the endogenous mouse light chain locus ctional (e.g., a deletion or a knockout of, 9.9., a X and/or K endogenous . in one embodiment, the first mouse ses a replacement of all or substantially all functinoal endogenous mouse V, D, and J gene segments with one or more unrearranged human V, D, and J gene ts (e.g., all or substantially all functional human V, D, and J gene segments); and the mouse comprises a replacement of all or substantially all functional light chain V and J gene segments with no more than one or no more than two nged light chain V/J sequences. In one embodiment the first mouse further comprises an ectopic nucleic acid sequence that encodes a mouse ADAM6 or ortholog or homolog or functional fragment thereof. in one embodiment, the ectopic nucleic acid sequence is at a humanized immunoglobulin heavy chain locus. ] in one embodiment, mice that comprise the ectopic sequence and that are homozygous for the universal light chain locus and for the humanized heavy chain locus are immunized with an antigen of interest to generate antibodies that comprise a plurality of somtatically mutated human variable domains that associate and express with a universal light chain. In one embodiment, human heavy chain variable domain nucleic acid sequences identified in the mouse are employed in an expression system to make a fully human antibody comprising the human heavy chain variable domain and a light chain comprising a universal light chain sequence of the mouse.

The following examples are provided so as to be to those of ry 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 (9.9., amounts, temperature, etc.) but some mental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average lar weight, temperature is indicated in Celsius, and pressure is at or near atmospheric.

EXAMPLES Example l Humanization of Mouse lmmunoglobulin Genes Human and mouse bacterial artificial chromsomes (BACs) were used to engineer 13 different BAC targeting s (BACvecs) for humanization of the mouse immunoglobulin heavy chain and K light chain loci. Tables 1 and 2 set forth detailed ptions of the steps performed for the uction of all BACvecs employed for the zation of the mouse immunoglobulin heavy chain and K light chain loci, respectively. fication of human and mouse BACs.

Mouse BACs that span the 5’ and 3’ ends of the globulin heavy chain and K light chain loci were identified by hybridization of filters spotted with BAC library or by PCR screening mouse BAC y DNA pools. Filters were hybridized under standard conditions using probes that corresponded to the regions of interest. Library pools were screened by PCR using unique primer pairs that flank the targeted region of interest.

Additional PCR using the same primers was performed to deconvolute a given well and isolate the corresponding BAC of interest. Both BAC filters and library pools were generated from 129 SvJ'mouse ES cells (lncyte Genomics/lnvitrogen). Human BACs that cover the entire immunoglobulin heavy chain and K light chain loci were identified either by hybridization of filters spotted with BAC library (Caltech B, C, or D libraries & RPCl—11 library, Research Genetics/lnvitrogen) through screening human BAC library pools (Caltech library, lnvitrogen) by a PCR—based method or by using a BAC end sequence database (Caltech D library, TlGR).

Construction of BACvecs (Tables 1 and 2).

Bacterial homologous recombination (BHR) was performed as described (Valenzuela et al., 2003; Zhang, Y., et al. (1998). A new logic for DNA engineering using ination in Escherichia coli. Nat Genet 20, 123-128). in most cases, linear fragments were generated by ligating PCR-derived homology boxes to cloned cassettes followed by gel isolation of ligation products and oporation into BHR-competent bacteria harboring the target BAC. After selection on appropriate antibiotic petri dishes, correctly recombined BACs were fied by PCR across both novel junctions followed by restriction analysis on pulsed—field gels (Schwartz, DC, and Cantor, CR. (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis.

Cell 37, 67—75) and spot-checking by PCR using primers buted across the human sequences.

A 3hVH BACvec was constructed using three sequential BHR steps for the initial step of humanization of the immunoglobulin heavy chain locus (FlG. 4A and Table 1). ln the first step (Step 1), a cassette was introduced into a human parental BAC upstream from the human VH1-3 gene segment that contains a region of homology to the mouse immunoglobulin heavy chain locus (HB1), a gene that confers kanamycin resistance in bacteria and G418 resistance in animals cells (kanR) and a site-specific recombination site (e.g., loxP). in the second step (Step 2), a second cassette was introduced just downstream from the last JH t that ns a second region of homology to the mouse immunoglobulin heavy chain locus (H82) and a gene that confers resistance in bacteria to spectinomycin (specR). This second step included deleting human immunoglobulin heavy chain locus sequences ream from JH6 and the BAC vector chloramphenicol resistance gene (cmR). in the third step (Step 3), the doubly modified human BAC (B1) was then linearized using l—Ceul sites that had been added during the first two steps and integrated into a mouse BAC (B2) by BHR through the two s of homology (H81 and H82). The drug selections for first (cm/kan), second (spec/kan) and third (cm/kan) steps were designed to be specific for the desired ts. ed BAC clones were analyzed by filed gel electrophoresis (PFG E) after digestion with restriction enzymes to determine appropriate construction (). in a similar n, 12 additional BACvecs were engineered for humanization of the heavy chain and K light chain loci. in some instances, BAC ligation was performed in lieu of BHR to conjoin two large BACs through introduction of rare restriction sites into both parental BACvecs by BHR along with careful placement of selectable s. This allowed for the survival of the desired ligation t upon selection with specific drug marker combinations. Recombinant BACs obtained by ligation after digestion with rare restriction enzymes were identified and screened in a r fashion to those obtained by BHR (as described above).

Table 1 BACvec Step Description Process insert upstream mouse homology box into human proximal BAC OTB-257202 insert downstream mouse homology box into human proximal BAC 7202 insert 3hVH/27hDH/9hJH into mouse proximal BAC CT7-302a07 to create 3hVH BACvec insert cassette at distal end of mouse lgH locus using mouse BAC CT7-253i20 insert specR marker at ream end of :5th insertion 1 BHR using human BAC CTD-257202 insert i-Ceul and Not sites flanking puroR at upstream end of 2 BHR 3:th insertion insert Not site at downstream end of Rel2—408p02 BAC (=10 kb downstream of VH2-5) .1... insert i—Ceu1 site at upstream end of Rel2~408p02 BAC (z23 kb upstream of VH1-18) Ligate 184kb fragment from step 4 into 153kb vector from step Ligation 18hVH Trim human homology from OTB-257202 BAC deleting =85kb and g 65kb homology to 3hVH insert cassette and Not site at distal end of mouse lgH locus in CT7-253i20 BAC Subclone mouse distal homology arm for insertion upstream Ligation from human BACs insert 20 kb mouse arm upstream of Rei2-408p02 BHR Swap selection cassette from hng to neoR to create 18hVH BHR BACvec insert l-Ceui and Pi-Scei sites flanking hng into distal end of 1 BHR human BAC CTD-2534n10 insert CmR at proximal end of CTD-2534n10 BAC to allow for 2 BHR selection for ligation to RP11—72n10 BAG insert Pl-Scel site into RP11-72n10 BAC for on to CTD- 3 BHR 2534n10 BAG insert l—Ceui and Ascl sites flanking puroR at distal end of 4 BHR 2n10 BAG 39hVH Ligate 161kb fragment from construct of step 4 into construct of Ligation step 2 replacing hng __....—i__ insert neoR and Ascl site at proximal end of mouse distal 6 BHR homology arm using 3i20 BAG insert specR and i—Ceui site at distal end of mouse distal 7 BHR homology arm l 8 Ligate mouse distal homology arm onto human insert from step Ligation Swap selection cassette from neo to hyg using UbCp and pA as homolgy boxes to create 39hVH BACvec 2 lnsert Ascl site at distal end of human CTD-3074b5 BAC BHR lnsert hng and Ascl site at proximal end of mouse distal 3 BHR 53hVH gy arm using 3i20 BAG 4 Ligate mouse distal homology arm onto construct from step 2 Ligation Swap selection cassette from hyg to neo using UbCp and pA BHR as homolgy boxes to create 53th BACvec lnsert Pl-Scei and l-Ceul sites flanking spec at distal end of 1 BHR human CTD—2195p5 BAG lnsert l-Ceul site at proximal end of RP11—926p12 BAC for 2 BHR ligation to CTD-2195p5 BAC lnsert Pl-Scel and Ascl sites at distal end of RP11—926p12 7OhVH 3 . . BHR BAC for ligation of mouse arm l—4_ Ligate mouse distal homology arm onto construct from step 3 Ligation Ligate mouse distal homology arm and hlgH fragment from 26p12 BAC onto CTD—2195p5 BAC to create 70 hVH Ligation BACvec lnsert l-Ceul and Ascl sites flanking hng at distal end of CTD- 1 BHR 2313e3 BAG 80hVH Ligate mouse dista homology arm onto human CTD—2313e3 2 Ligation BAC from step 1 to create 80hVH BACvec Table 2 BACvec Step Description Process Insert loxP Site Within mouse J-C intron usmg CT7-254m04 [91(4)C 1 BHR Insert onP site at distal end of mouse ng locus using 0T7— "Insert PI—Scel site z400 bp ream from hJK5 in CTD— BHR 2 BAC Insert l-Ceul and Ascl sites flanking hng at distal end of CTDBHR 2366j12 BAG Insert l-Ceul and Pl—Scel sites flanking puroR zxxbp 3 BHR downstream from mex using CT7—254m04 BAC 6hV1< Insert hlgVK/JK upstream from mouse EnhK/CK usmg uct 4 Ligation from step 3 Replace cmR in construct of step 4 with specR Insert Neo selection cassette at distal end of mouse ng locus using CT7-302912 BAG Ligate mouse distal homology arm upstream of human insert in 7 Li9ation construct of step 6 to create 6hVK BACvec insert NeoR at distal end of RP11—1061b13 BAC' —BHR Replace cmR in construct of step 1 With specR B Insert Hyg selection cassette at distal end of mouse ng locus 16an 3 BHR using CT7-302912 BAG Ligate mouse distal homology arm upstream of human insert . 4 Ligation from construct of step 2 to create 16hV1< BACvec insert Hng at distal end of RP1 1-9996 BAC’ BHR Replace cmR in construct of step 1 With specR -BHR Insert Neo ion cassette at distal end of mouse ng locus' 30an BHR using CT7-302912 BAC Ligate mouse distal homology arm upstream of human insert from construct of step 2 to create 30th( BACvec Insert NeoR at distal end of hlgH locus in CTD—2559d6 BAC Replace cmR in construct of step 1 With specR —BHR 4(3th Ligate mouse distal homology arm upstream of hlgH locus in Ligation construct of step 2 to create 40hVK BACvec Modification of embryonic stem (ES) cells and generation of mice.

ES cell (F1 H4) targeting was med using the VELOClGENE® genetic engineering method as described (Valenzuela et al., 2003). tion of mice from modiﬁed ES cells by either biastocyst (Valenzuela et al., 2003) or 8—celi ion (Poueymirou et al., 2007) was as bed. Targeted ES cells and mice were confirmed by screening DNA from ES cells or mice with unique sets of probes and primers in a PCR based assay (e.g., , 3B and 3C). All mouse studies were overseen and approved by Regeneron’s institutional Animal Care and Use Committee (iACUC).

Karyotype Analysis and Fluorescent in situ Hybridization (FISH).

Karyotype Analysis was performed by Coriell Cell Repositories (Coriell institute for Medical Research, , NJ). FiSH was performed on targeted ES cells as described (Valenzuela et al., 2003). Probes corresponding to either mouse BAC DNA or human BAC DNA were labeled by nick translation (lnvitrogen) with the fiuorescentiy labeled dUTP nucleotides spectrum orange or spectrum green (Vysis). immunoglobulin Heavy Chain Variable Gene Locus.

Humanization of the variable region of the heavy chain locus was achieved in nine sequential steps by the direct replacement of about three million base pairs (Mb) of contiguous mouse genomic sequence ning all VH, DH and JH gene segments with about one Mb of contiguous human genomic ce containing the equivalent human gene segments ( and Table 1) using VELOClGENE® genetic engineering technology (see, e.g., US Pat. No. 251 and uela et al., 2003).

The intron between JH gene segments and constant region genes (the J—C intron) contains a transcriptional enhancer (Neuberger, MS. (1983) Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells. EMBO J 2, 1373-1378) followed by a region of simple repeats required for ination during isotype switching (Kataoka, T. etal. (1980) Rearrangement of immunoglobulin gamma 1— chain gene and mechanism for heavy—chain class switch. Proc Natl Acad Sci U S A 77, 919-923). The junction n human VH-DH-JH region and the mouse CH region (the proximal junction) was chosen to maintain the mouse heavy chain intronic enhancer and switch domain in order preserve both efﬁcient expression and class ing of the humanized heavy chain locus within the mouse. The exact nucleotide position of this and subsequent junctions in all the ements was possible by use of the VELOClGENE® genetic engineering method (supra), which employed bacterial homologous ination driven by synthesized oligonucleotides. Thus, the proximal on was placed about 200 bp downstream from the last JH gene segment and the distal junction was placed several hundred upstream of the most 5’ VH gene segment of the human locus and about 9 kb downstream from the mouse VH1—86 gene segment, also known as J558.55. The mouse VH1-86 (J558.55) gene segment is the most distal heavy chain variable gene segment, ed to be a pseudogene in CS7BL/6 mice, but potentially active, albeit with a poor RSS sequence, in the targeted 129 allele. The distal end of the mouse heavy chain locus reportedly may contain control elements that regulate locus expression and/or rearrangement (Pawlitzky et al., 2006).

A first insertion of human globulin DNA sequence into the mouse was achieved using 144 kb of the proximal end of the human heavy chain locus containing 3 VH, all 27 DH and 9 JH human gene segments inserted into the proximal end of the mouse lgH locus, with a concomitant 16.6 kb deletion of mouse c sequence, using about 75 kb of mouse homology arms (Step A, ; Tables 1 and 3, 3hVH). This large 144kb ion and accompanying 16.6 kb deletion was performed in a single step (Step A) that occurred with a frequency of 0.2% (Table 3). Correctly targeted ES cells were scored by a loss-of—native-allele (LONA) assay (Valenzuela et al., 2003) using probes within and flanking the deleted mouse sequence and within the inserted human sequence, and the ity of the large human insert was verified using multiple probes spanning the entire insertion (, BB and BC). e many rounds of tial ES cell ing were anticipated, targeted ES cell clones at this, and all subsequent, steps were subjected to karyotypic analysis (supra) and only those clones showing normal ypes in at least 17 of 20 spreads were utilized for subsequent steps.

Targeted ES cells from Step A were re—targeted with a BACvec that produced a 19 kb on at the distal end of the heavy chain locus (Step B, FlG. 2A). The Step B BACvec contained a hygromycin resistance gene (hyg) in contrast to the neomycin resistance gene (neo) contained on the BACvec of Step A. The resistance genes from the two BACvecs were designed such that, upon successful targeting to the same some, approximately three Mb of the mouse heavy chain variable gene locus containing all of the mouse VH gene segments other than VH1-86 and all of the DH gene segments other than D052, as well as the two resistance genes, were flanked by loxP sites; D052 and all of the mouse JH chain gene segments were deleted in Step A. ES cell clones doubly targeted on the same chromosome were identified by driving the 3hVH proximal te to homozygosity in high G418 (Mortensen, R.M. et al. (1992) Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol 12:2391- 2395) and following the fate of the distal hyg cassette. Mouse segments up to four Mb in size, having been modified in a manner to be flanked by loxP sites, have been successfully deleted in ES cells by transient expression of CRE recombinase with high efficiencies (up to =11%) even in the absence of drug selection (Zheng, B., et al. (2000). Engineering mouse chromosomes with Cre—loxP: range, efficiency, and c applications. Mol Cell Biol 20:648—655). in a similar manner, the inventors achieved a three Mb deletion in 8% of ES cell clones following transient Cre expression (Step C, ; Table 3). The deletion was scored by the LONA assay using probes at either end of the deleted mouse sequence, as well as the loss of neo and hyg and the appearance of a PCR product across the deletion point containing the sole remaining loxP site. Further, the deletion was confirmed by fluorescence in situ hybridization (data not shown).

The remainder of the human heavy chain variable region was added to the 3hVH allele in a series of 5 steps using the GENE® genetic engineering method (Steps E—H, HS. 28), with each step involving precise insertion of up to 210 kb of human gene ces. For each step, the proximal end of each new BACvec was ed to overlap the most distal human sequences of the us step and the distal end of each new BACvec contained the same distal region of mouse gy as used in Step A. The s of steps D, F and H contained neo selection cassettes, whereas those of steps E and G contained hyg selection cassettes, thus selections were alternated between G418 and hygromycin. Targeting in Step D was assayed by the loss of the unique PCR product across the distal loxP site of 3hVH Hybrid Allele. Targeting for Steps E through l was d by loss of the previous selection cassette. ln the final step (Step l, ), the neo selection cassette, flanked by Frt sites (McLeod, M. et al. (1986) ldentification of the crossover site during FLP-mediated recombination in the Saccharomyces siae plasmid 2 microns circle. Mol Cell Biol 6, 3357—3367), was removed by ent FLPe expression (Buchholz, F. etal. (1998) lmproved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16, 657-662). The human sequences of the BACvecs for Steps D, E and G were derived from two parental human BACs each, whereas those from Steps F and H were from single BACs. Retention of human sequences was confirmed at every step using multiple probes spanning the inserted human sequences (as described above, 9.9. , BB and 30). Only those clones with normal karyotype and germline potential were carried forward in each step. ES ceils from the final step were still able to contribute to the germline after nine sequential manipulations (Table 3). Mice homozygous for each of the heavy chain alleles were viable, appeared healthy and demonstrated an essentially wild—type l immune system (see Example 3).

Table 3 Hybrid Human Targeting Targeting % Total Functional Allele sequence construct efficiency usage VH VH 3hVH 3hVH/DC 80hVHdNeo lmmunoglobulin K Light Chain Variable Gene Locus.

The K light chain variable region was zed in eight sequential steps by the direct replacement of about three Mb of mouse sequence containing all VK and JK gene segments with about 0.5 Mb of human sequence containing the al human VK and JK gene segments in a manner similar to that of the heavy chain (HQ 18; Tables 2 and 4).

The variable region of the human K light chain locus contains two nearly identical 400 kb repeats separated by a 800 kb spacer (Weichhold, G.M. et al. (1993) The human lmmunoglobulin kappa locus consists of two copies that are organized in opposite polarity, Genomics 162503-511). Because the repeats are so similar, nearly all of the locus diversity can be reproduced in mice by using the proximal repeat. Further, a natural human allele of the K light chain locus missing the distal repeat has been reported (Schaible, G. et al. (1993) The immunoglobulin kappa locus: polymorphism and haplotypes of Caucasoid and non-Caucasoid duals, Hum Genet 91 261-2637). About three Mb of mouse K light chain variable gene sequence were replaced with about 0.5 Mb of human K light chain le gene sequence to effectively replace all of the mouse VK and JK gene ts with the proximal human VK and all of the human JK gene segments (FlG. 2C and 2D; Tables 2 and 4). In contrast to the method described in Example 1 for the heavy chain locus, the entire mouse VK gene , containing all VK and JK gene segments, was deleted in a three-step s before any human sequence was added. First, a neo cassette was introduced at the proximal end of the variable region (Step A, HQ 20). Next, a hyg cassette was inserted at the distal end of the K locus (Step B, ). LoxP sites were again situated within each selection cassette such that Cre treatment induced deletion of the remaining 3 Mb of the mouse VK region along with both resistance genes (Step C, ).

A human genomic fragment of about 480 kb in size containing the entire immunoglobulin K light chain variable region was inserted in four sequential steps (; Tables 2 and 4), with up to 150 kb of human immunoglobulin K light chain sequence inserted in a single step, using methods similar to those employed for the heavy chain (see Example 1). The final hygromycin ance gene was d by transient FLPe expression. As with the heavy chain, targeted ES cell clones were evaluated for integrity of the entire human insert, normal karyotype and germ—line potential after every step. Mice homozygous for each of the K light chain chain alleles were generated and found to be healthy and of normal appearance.

Table 4 Hybrid Human Targeting ing % Total Functional Allele sequence construct efficiency usage VK VK ng—PC 0 132 kb 1.1% - — ng-PC/DC o 90 kb 0.4% -j - = ng-CRE 0 - 1% - — — 6an 110 kb 122 kb 0.3% 14_._ 6 4 l 163th 240 kb 203 kb 0.4% 47 16 11 30hVK 390 kb 193 kb 0.1% 70 30 18 40hVK 480 kb 185 kb 0.2% 100 40 25 40hV1<dHyg 480 kb — 0.7% 100 40 25 Example ll Generation of Fully Humanized Mice by Combination of Multiple Humanized Immunoglobulin s At several points, ES cells g a portion of the human immunoglobulin heavy chain or K light chain variable repertoires as described in Example 1 were microinjected and the resulting mice bred to create multiple versions of VELOClMMUNE® humanized mice with ssively larger fractions of the human germline immunoglobulin repertoires (Table 5; FlG. 5A and SB). VELOClMMUNE® 1 (V1) humanized mice possess 18 human VH gene segments and all of the human DH and JH gene ts combined with 16 human VK gene segments and all the human JK gene segments.

VELOClMMUNE® 2 (V2) humanized mice and VELOClMMUNE® (V3) humanized mice have increased variable oires bearing a total of 39 VH and 30 VK, and 80 VH and 40 VK, respectively. Since the genomic regions encoding the mouse VH, DH and JH gene segments, and VK and JK gene segments, have been completely replaced, antibodies produced by any n of VELOClMMUNE® humanized mice contain human variable regions linked to mouse nt regions. The mouse 7» light chain loci remain intact in all versions of the MMUNE® humanized mice and serve as a comparator for efficiency of sion of the various MMUNE® humanized K light chain loci.

Mice doubly homozygous for both immunoglobuiin heavy chain and K light chain humanizations were generated from a subset of the alleles described in e 1. All genotypes observed during the course of breeding to generate the doubly homozygous mice occurred in roughly Mendelian proportions. Male progeny homozygous for each of the human heavy chain alleles showed d fertility. Reduced fertility resulted from loss of mouse ADAMS activity. The mouse heavy chain variable gene locus contains two embedded functional ADAMS genes (ADAMSa and ADAMSb). During humanization of the mouse heavy chain variable gene locus, the inserted human genomic ce contained an ADAMS pseudogene. Mouse ADAMS may be required for fertility, and thus lack of mouse ADAMS genes in humanized heavy chain variable gene loci might lead to reduced fertility in these mice notwithstanding the presence of the human pseudogene. Examples 7—9 describe the e replacement of deleted mouse ADAMS genes back into a humanized heavy chain variable gene locus, and restoration of a wild-type level of fertility in mice with a humanized heavy chain immunoglobulin locus.

Table 5 Version of Heavy Chain 1: Light Chain VELOClMMUNE® Human 5, VH Human "m—s,VK Mouse Allele Allele VH VIC gene -18 ‘18th VH1-18 Example lll Lymphocyte Populations in Mice with Humanized globulin Genes Mature B cell populations in the three different versions of VELOClMMUNE® mice were evaluated by flow cytometry.

Briefly, cell suspensions from bone marrow, spleen and thymus were made using standard methods. Cells were resuspended at 5x105 cells/mL in BD Pharmingen FACS staining buffer, blocked with anti—mouse CD16/32 (BD Pharmingen), stained with the appropriate cocktail of antibodies and fixed with BD XTM all according to the manufacturer’s instructions. Final cell pellets were resuspended in 0.5 mL ng buffer and analyzed using BD FACSCALlBURTM and BD EST F’ROTM software. All antibodies (BD Pharmingen) were prepared in a mass dilution/cocktail and added to a final concentration of 0.5 mg/105 cells. Antibody cocktails for bone marrow (A——D) staining were as follows: A: anti-mouse lgMb-FITC, anti-mouse lgMa-PE, ouse CD45R(8220)-APC; B: anti—mouse CD43(S7)—PE, anti—mouse CD45R(8220)-APC; C: anti-mouse CD24(HSA)— PE; ouse CD45R(8220)—APC; D: anti-mouse BPPE, ouse CD45R(8220)- APC. Antibody cocktails for spleen and inguinal lymph node (E—H) staining were as follows: E: anti-mouse lgMb-FlTC, anti-mouse lgMa-PE, anti-mouse CD45R(8220)-APC; F: anti-mouse lg, M, 72, )3 Light Chain-FlTC, anti mouse ng Light Chain—PE, anti-mouse CD45R(B220)-APC; G: anti-mouse Ly6G/C—FITC, anti-mouse CD49b(DX5)-PE, anti- mouse CD11b-APC; H: anti—mouse CD4(L3T4)-FlTC, anti—mouse CD45R(8220)—PE, anti- mouse CD8a-APC. Results are shown in FlG. 6.

Lymphocytes isolated from spleen or lymph node of homozygous VELOClMMUNE® humanized mice were d for surface expression of the markers 8220 and lgM and analyzed using flow cytometry (. The sizes of the 8220+ lgM+ mature B cell populations in all versions of VELOClMMUNE® humanized mice tested were lly identical to those of wild type mice, regardless of the number of VH gene ts they contained. ln addition, mice containing homozygous hybrid humanized immunoglobulin heavy chain loci, even those with only 3 VH gene ts but normal mouse immunoglobulin K light chain loci or mice containing homozygous hybrid zed K light chain loci with normal mouse globulin heavy chain loci, also had normal numbers of 8220+ lgM+ cells in their peripheral compartments (not shown). These results indicate that chimeric loci with human variable gene segments and mouse constant regions can fully populate the mature B cell compartment. Further, the number of variable gene segments at either the heavy chain or K light chain loci, and thus the theoretical diversity of the antibody repertoire, does not correlate with the ability to generate wild type populations of mature B cells. ln contrast, mice with randomly integrated fully-human immunoglobulin transgenes and inactivated mouse immunoglobulin loci have reduced numbers of B cells in these compartments, with the severity of the deficit depending on the number of variable gene segments included in the transgene (Green, LL, and Jakobovits, A. (1998) tion of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificiai chromosomes, J Exp Med 188:483-495). This demonstrates that the “in situ c humanization” strategy results in a fundamentally different functional outcome than the randomly integrated transgenes achieved in the “knockout-plus—transgenic” approach.

Allelic Exclusion and Locus .

The ability to maintain allelic exlusion was examined in mice heterozygous for different versions of the humanized immunoglobulin heavy chain locus.

The humanization of the immunogiobulin loci was carried out in an F1 ES iine (F1 H4 (Valenzuela et al., , derived from 12986/SvaTac and C57BL/6NTac heterozygous embryos. The human heavy chain germline variable gene sequences are targeted to the 12986 allele, which carries the lgMa haplotype, whereas the unmodified mouse C576BL/6N allele bears the lgMb haplotype. These allelic forms of lgM can be distinguished by flow cytometry using antibodies specific to the poiymorphisms found in the lgMa or lgMb alleles. As shown in (bottom row), the B cells identified in mice heterozygous for each version of the zed heavy chain locus oniy express a single allele, either lgMa (the humanized allele) or igMb (the wild type allele). This trates that the mechanisms involved in allelic exclusion are intact in VELOClMMUNE® zed mice. in addition, the relative number of B ceiis positive for the zed alleie (lgMa) is roughly proportional to the number of VH gene segments present. The humanized immunoglobulin iocus is expressed in approximately 30% of the B ceils in MMUNE® 1 humanized heterozygote mice, which have 18 human VH gene segments, and in 50% of the B cells in MMUNE® 2 and 3 (not shown) humanized heterozygote mice, with 39 and 80 human VH gene ts, tively. Notably, the ratio of cells expressing the humanized versus wild type mouse allele (0.5 for VELOClMMUNE® 1 humanized mice and 0.9 for VELOClMMUNE® 2 humanized mice) is greater than the ratio of the number of variable gene segments ned in the zed versus wild type loci (0.2 for VELOClMMUNE® 1 humanized mice and 0.4 for VELOClMMUNE® 2 humanized mice). This may indicate that the probability of aliele choice is intermediate between a random choice of one or the other chromosome and a random choice of any particular V segment RSS. Further, there may be a fraction of B- cells, but not all, in which one allele becomes accessible for recombination, completes the process and shuts down recombination before the other aiiele becomes accessible. In addition, the even distribution of ceils that have surface lgM (slgM) derived from either the hybrid humanized heavy chain locus or the wild type mouse heavy chain locus is evidence that the hybrid locus is operating at a normal level in st, randomly integrated human immunoglobulin transgenes e poorly with wild type mouse immunoglobulin loci (Bruggemann, M., et al. (1989) A repertoire of monoclonal antibodies with human heavy chains from transgenic mice. PNAS 86, 6709-6713; Green et al. (1994); Tuaillon, N. et al. (1993) Human globulin heavy-chain minilocus recombination in transgenic mice: gene—segment use in mu and gamma transcripts, Proc Natl Acad Sci USA 0—3724).

This further demonstrates the immunoglobulins produced by VELOCIMMUNE® humanized mice are functionally different than those produced by ly integrated enes in mice made by “knockout—plus-transgenic” approaches.

Polymorphisms of the CK regions are not available in 12986 or C57BL/6N to examine allelic exclusion of humanized versus non-humanized K light chain loci. However, VELOCIMMUNE® humanized mice all possess wild type mouse it light chain loci, therefore, it is possible to observe whether rearrangement and expression of humanized K light chain loci can prevent mouse x light chain expression. The ratio of the number of cells expressing the humanized K light chain ve to the number of cells expressing mouse x light chain was relatively unchanged in VELOCIMMUNE® humanized mice compared with wild type mice, regardless of the number of human VK gene segments ed at the K light chain locus (HQ 6, third row from top). in addition there was no increase in the number of double positive (K plus 9») cells, indicating that productive recombination at the hybrid K light chain loci results in riate suppression of recombination of the mouse 7» light chain loci. in contrast, mice containing randomly integrated K light chain transgenes with inactivated mouse K light chain loci—but wild type mouse k light chain loci—exhibit dramatically increased MK ratios (Jakobovits, 1998), implying that the introduced K light chain transgenes do not on well in such mice.

This further demonstrates the different onal outcome observed in immunoglobulins made by VELOCIMMUNE® humanized mice as compared to those made by “knockout- plus-transgenic” mice.

B cell Development. e the mature B cell populations in VELOCIMMUNE® humanized mice le those of wild type mice (described above), it is possible that defects in early B cell differentiation are compensated for by the expansion of mature B cell populations. The various stages of B cell differentiation were examined by analysis of B cell populations using flow cytometry. Table 6 sets forth the ratio of the fraction of cells in each B cell lineage defined by FACs, using specific cell surface markers, in MMUNE® humanized mice compared to wild type littermates.

Early B cell pment occurs in the bone marrow, and different stages of B cell differentiation are characterized by s in the types and amounts of cell e marker expression. These differences in e expression correlate with the molecular changes occurring at the immunoglobulin loci inside the cell. The pro-B to pre-B cell transition requires the successful ngement and expression of onal heavy chain protein, while transition from the pre-B to mature B stage is governed by the correct ngement and expression of a K or 7» light chain. Thus, inefficient transition between stages of B cell differentiation can be detected by changes in the relative populations of B cells at a given stage.

Table6 Bone Marrow Spleen Version of pro-B pre-B Immature Mature Emerging Mature VELOCIMMUNE® . h, 3220'“ Mice CD43” c024” B220'° 3220' B220hi B22o'° 322o'° lgM" 1ch+ 9 lgM+ No major defects were observed in B cell entiation in any of the VELOClMMUNE® humanized mice. The introduction of human heavy chain gene segments does not appear to affect the pro-B to pre-B transition, and introduction of human K light chain gene segments does not affect the pre—B to B transition in VELOCIMMUNE® humanized mice. This demonstrates that “reverse chimeric” immunoglobulin molecules possessing human variable regions and mouse constants function normally in the context of B cell signaling and co-receptor molecules leading to appropriate B cell differentiation in a mouse environment. in contrast, the balance between the different populations during B cell differentiation are perturbed to varying extents in mice that n randomly integrated immunoglobulin transgenes and inactivated endogenous heavy chain or K light chain loci (Green and vits (1998)).

Example IV Variable Gene Repertoire in Humanized Immunogiobulin Mice Usage of human variable gene segments in the humanized antibody repertoire of VELOCIMMUNE® humanized mice was analyzed by reverse transcriptase-polymerase chain reaction (RT—PCR) of human variable regions from multiple sources ing splenocytes and oma cells. Variable region sequence, gene segment usage, somatic hypermutation, and junctional diversity of rearranged variable region gene segments were determined. y, total RNA was extracted from 1 x 107-2 x 107 splenocytes or about 104— 105 hybridoma cells using TRlZOLTM (lnvitrogen) or Qiagen RNEASYTM Mini Kit (Qiagen) and primed with mouse constant region specific primers using the SUPERSCRIPTTM lil One—Step RT-PCR system rogen). Reactions were carried out with 2-5 pL of RNA from each sample using the entioned 3’ constant specific primers paired with pooled leader primers for each family of human variable regions for both the heavy chain and K light chain, separately. Volumes of reagents and primers, and RT-PCR/PCR conditions were performed according to the manufacturer’s instructions. Primers ces were based upon multiple sources (Wang, X. and r, 8D. (2000) Human immunoglobulin variable region gene analysis by single cell RT-PCR, J lmmunol Methods 244217-225; lg- primer sets, Novagen). Where appropriate, nested secondary PCR reactions were carried out with pooled family-specific ork primers and the same mouse 3’ immunoglobulin constant-specific primer used in the primary reaction. Aliquots (5 pL) from each reaction were analyzed by agarose electrophoresis and reaction products were purified from agarose using a MONTAGETM Gel Extraction Kit (Millipore). ed products were cloned using the TOPOTM TA Cloning System (lnvitrogen) and transformed into DH108 Eco/i cells by electroporation. individual clones were selected from each transformation on and grown in 2 mL LB broth cultures with antibiotic selection overnight at 37°C. Plasmid DNA was purified from bacterial cultures by a kit—based approach (Qiagen). lmmunoglobulin Variable Gene Usage. d DNA of both heavy chain and K light chain clones were sequenced with either T7 or M13 reverse primers on the AB! 3100 Genetic er (Applied Biosystems).

Raw sequence data were imported into SEQUENCHERTM (v4.5, Gene Codes). Each sequence was assembled into contigs and aligned to human immunoglobulin ces using lMGT V-Quest (Brochet, X. at al. (2008) lMGTN—QUEST: the highly customized and ated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res 36:W503—508) search function to identify human VH, DH, JH and VK, JK segment usage. Sequences were compared to germline sequences for somatic hypermutation and recombination junction analysis.

Mice were generated from ES cells containing the initial heavy chain modification (3hVH-CRE Hybrid Allele, bottom of ) by RAG complementation (Chen, J. et al. (1993) RAG-2—deficient blastocyst complementation: an assay of gene function in lymphocyte development, Proc Natl Acad Sci USA 90:4528—4532), and cDNA was prepared from splenocyte RNA. The cDNA was amplified using primer sets (described above) speciﬁc for the predicted chimeric heavy chain mRNA that would arise by V(D)J recombination within the ed human gene segments and subsequent ng to either mouse lgM or [96 constant domains. Sequences derived from these cDNA clones (not shown) trated that proper V(D)J recombination had ed within the human variable gene sequences, that the rearranged human V(D)J gene segments were properly spliced in-frame to mouse constant domains and that switch recombination had occurred. Further sequence analysis of mRNA products of subsequent hybrid immunoglobulin loci was performed.

In a similar experiment, B cells from non-immunized wild type and VELOClMMUNE® humanized mice were separated by flow cytometry based upon surface sion of 8220 and lgM or lgG. The 8220+ lgM+ or surface lgG+ ) cells were pooled and VH and VK sequences were obtained ing RT—PCR amplification and cloning (described above). Representative gene usage in a set of RT—PCR amplified cDNAs from unimmunized VELOClMMUNE® 1 humanized mice (Table 7) and VELOClMMUNE® 3 humanized mice (Table 8) was recorded (*defective RSS; Tmissing or pseudogene).

Table 7 VH Observed Observed V1; ed 11 8 .33333132116.nm4449345344P* 6521. 531. 11119336521.* JK Observed «12345 Observed Table 8 Observed DH Observed Vx Observed 2—2 4 3324.13344_/.n7/.__/.66 3 A. 5 5345 96 65 7.. 212 ......... 433332222 097309874 65555444.. 30.09 5331.. 5 2 6 1111 3 1.1141. 765 303 86 1 1—45 3A...4..3 7..7..7._27_.227.. LIL: 4343 33332 3 652 A... 08 0b md Observed 33 22 33 4.I1..

As shown in Tables 7 and 8, nearly all of the functional human VH, DH, JH, VK and JK gene segments are utilized. Of the functional variable gene segments described but not detected in the VELOClMMUNE® humanized mice of this experiment, several have been reported to possess defective recombination signal sequences (RSS) and, thus, would not be ed to be expressed (Feeney, A.J. (2000) Factors that influence formation of B cell oire. lmmunol Res 21 :195~202). Analysis of several other sets of immunoglobulin sequences from various VELOClMMUNE® humanized mice, isolated from both naive and zed repertoires, has shown usage of these gene segments, albeit at lower frequencies (data not shown). Aggregate gene usage data has shown that all functional human VH, DH, JH, VK, and JK gene segments contained in VELOClMMUNE® humanized mice have been observed in various naive and immunized repertoires (data not . Although the human VH7-81 gene segment has been identified in the analysis of human heavy chain locus sequences (Matsuda, F. et al. (1998) The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus, J Exp Med 51-2162), it is not present in the VELOClMMUNE® humanized mice as confirmed by re-sequencing of the entire VELOClMMUNE® 3 zed mouse genome.

Sequences of heavy and light chains of antibodies are known to show exceptional ility, especially in short polypeptide segments within the rearranged variable domain. These regions, known as hypervariable regions or mentary determining regions (CDRs) create the binding site for antigen in the structure of the antibody molecule. The intervening polypeptide sequences are called framework regions (FRs). There are three CDRs (CDRi, CDRZ, CDRB) and 4 FRs (FR1, FR2, FR3, FR4) in both heavy and light chains. One CDR, CDRB, is unique in that this CDR is d by recombination of both the VH, DH and JH and VK and JK gene segments and generates a significant amount of repertoire diversity before antigen is encountered. This joining is imprecise due to both nucleotide deletions via exonuclease activity and non-template d additions via al deoxynucleotidyl erase (TdT) and, thus, allows for novel sequences to result from the recombination process. Although FRs can show substantial somatic mutation due to the high mutability of the variable region as a whole, variability is not, however, distributed evenly across the le region. CDRs are concentrated and localized regions of high variability in the surface of the antibody molecule that allow for antigen binding. Heavy chain and light chain sequences of selected antibodies from VELOClMMUNE® humanized mice around the CDR3 junction demonstrating junctional diversity are shown in and 78, respectively.

As shown in , non-template encoded nucleotide additions (N-additions) are observed at both the VH-DH and DH-JH joint in dies from VELOClMMUNE® humanized mice, indicating proper function of TdT with the human segments. The endpoints of the VH, DH and JH ts relative to their germline counterparts indicate that exonuclease activity has also occurred. Unlike the heavy chain locus, the human K light chain rearrangements exhibit little or no TdT additions at CDR3, which is formed by the recombination of the VK and JK segments (HQ 78). This is expected due to the lack of TdT expression in mice during light chain rearrangements at the pre-B to B cell transition. The ity observed in the CDR3 of rearranged human VK s is introduced predominantly through exonuclease activity during the recombination event.

Somatic hypermutation.

] Additional diversity is added to the variable regions of rearranged immunoglobulin genes during the al center reaction by a process termed somatic hypermutation. B cells expressing somatically d variable regions compete with other B cells for access to antigen presented by the follicular dendritic cells. Those 8 cells with higher ty for the antigen will further expand and undergo class switching before exiting to the periphery. Thus, B cells expressing switched isotypes lly have encountered antigen and undergone germinal center reactions and will have increased numbers of mutations relative to halve B cells. Further, variable region sequences from predominantly naive slgM’r B cells would be expected to have relatively fewer mutations than variable ces from slgG’“ B cells which have undergone antigen ion.

Sequences from random VH or VK clones from slgM+ or sigG+ B cells from non- immunized VELOClMMUNE® humanized mice or slgG+ B cells from immunized mice were compared with their ne variable gene ts and changes relative to the germline sequence ted. The resulting tide sequences were translated in silica and mutations leading to amino acid changes also annotated. The data were collated from all the variable regions and the percent change at a given position was calculated (.

As shown in human heavy chain variable regions derived from slgG+ B cells from non—immunized VELOClMMUNE® humanized mice exhibit many more nucleotides relative to slgM+ B cells from the same splenocyte pools, and heavy chain variable regions derived from immunized mice exhibit even more changes. The number of changes is increased in the complementarity-determining regions (CDRs) relative to the framework regions, indicating antigen selection. The ponding amino acid sequences from the human heavy chain variable regions also exhibit significantly higher numbers of mutations in lgG vs lgM and even more in immunized lgG. These mutations again appear to be more frequent in the CDRs compared with the framework sequences, suggesting that the dies were antigen-selected in vivo. A similar increase in the number the nucleotide and amino acid mutations are seen in the VK sequences derived from lgG+ B cells from immunized mice.

The gene usage and somatic utation observed in VELOClMMUNE® humanized mice demonstrate that essentially all gene segments t are capable of rearrangement to form fully functionally reverse ic antibodies in these mice. Further, VELOClMMUNE® humanized mouse derived antibodies fully participate within the mouse immune system to undergo affinity selection and maturation to create fully mature human antibodies that can effectively neutralize their target antigen. VELOClMMUNE® humanized mice are able to mount robust immune responses to multiple classes of antigens that result in usage of a wide range of human antibodies that are both high ty and suitable for therapeutic use (data not shown).

Example V Analysis of Lymphoid Structure and Serum Isotypes The gross structures of spleen, inguinal lymph nodes, Peyer’s patches and thymus of tissue samples from wild type or VELOClMMUNE® humanized mice stained with H&E were examined by light copy. The levels of immunoglobulin isotypes in serum collected from wild-type and VELOClMMUNE® humanized mice were analyzed using LUMlNEXTM technology.

Lymphoid Organ Structure.

] The structure and function of the lymphoid tissues are in part dependent upon the proper development of hematopoietic cells. A defect in B cell development or function may be exhibited as an alteration in the structure of the lymphoid tissues. Upon analysis of stained tissue ns, no significant difference in appearance of secondary lymphoid organs between wild type and VELOClMMUNE® humanized mice was identified (data not shown).

Serum globulin Levels.

The level of expression of each isotype is r in wild type and VELOClMMUNE® zed mice (, BB and QC). This trates that humanization of the variable gene segments had no apparent adverse effect upon class ing or immunoglobulin expression and secretion and therefore apparently maintain all the endogenous mouse ces ary for these ons.

Example Vl Immunization and Antibody Production in Humanized immunoglobulin Mice Different versions of VELOClMMUNE® humanized mice were immunized with antigen to examine the humoral response to foreign antigen challenge.

Immunization and Hybridoma Development.

VELOClMMUNE® humanized and wild-type mice can be immunized with an antigen in the form of protein, DNA, a ation of DNA and protein, or cells expressing the antigen. Animals are typically boosted every three weeks for a total of two to three times. Following each antigen boost, serum samples from each animal are collected and analyzed for antigen—specific antibody responses by serum titer determination. Prior to fusion, mice received a final pre-fusion boost of 5 pg protein or DNA, as desired, via intra- peritoneal and/or intravenous injections. Splenocytes are harvested and fused to 3 myeloma cells in an electrofusion chamber according to the manufacture’s suggested protocol (Cyto Pulse Sciences lnc., Glen Burnie, MD). Ten days after culture, hybridomas are screened for n icity using an ELISA assay (Harlow, E. and Lane, D. (1988) dies: A Laboratory . Cold Spring Harbor Press, New York). Alternatively, antigen specific B cells are isolated ly from immunized VELOClMMUNE® humanized mice and screened using rd techniques, ing those described here, to obtain human antibodies specific for an antigen of interest.

Serum Titer Determination.

To monitor animal anti-antigen serum response, serum samples are collected about 10 days after each boost and the titers are determined using antigen specific ELISA.

Briefly, Nunc MAXlSORPi’M 96 well plates are coated with 2 pg/mL antigen ght at 4° C and blocked with bovine serum albumin (Sigma, St. Louis, MO). Serum samples in a serial 3 fold dilutions are allowed to bind to the plates for one hour at room temperature.

The plates are then washed with PBS containing 0.05% 20 and the bound lgG are detected using HRP-conjugated goat anti-mouse Fc (Jackson lmmuno Research Laboratories, lnc., West Grove, PA) for total lgG titer, or biotin—labeled isotype specific or light chain specific polyclonal antibodies (SouthernBiotech inc.) for isotype specific titers, respectively. For biotin-labeled antibodies, following plate wash, HRP—conjugated streptavidin (Pierce, Rockford, IL) is added. All plates are developed using colorimetric substrates such as BD OPTElATM (BD Biosciences Pharmingen, San Diego, CA). After the reaction is stopped with 1 M phosphoric acid, optical absorptions at 450 nm are recorded and the data are analyzed using PRISMTM software from Graph Pad. Dilutions required to obtain two-fold of background signal are defined as titer. in one experiment, VELOClMMUNE® humanized mice were immunized with human interleukin-6 receptor (hlL-6R). A representative set of serum titers for VELOCIMMUNE® and wild type mice immunized with hlL-BR is shown in A and 108.

VELOClMMUNE® humanized and wild—type mice d strong responses towards the lL—6R with similar titer ranges (FlG. 10A). Several mice from the VELOCIMMUNE® humanized and wild-type cohorts reached a maximal response after a single n boost. These s te that the immune response strength and kinetics to this antigen were similar in the VELOClMMUNE® humanized and wild type mice. These antigen-specific dy responses were further analyzed to e the particular isotypes of the antigen—specific antibodies found in the sera. Both VELOCIMMUNE® humanized and wild type groups predominantly elicited an lgG1 response (FlG. 108), suggesting that class switching during the humoral response is similar in mice of each type.

Affinity Determination of Antibody Binding to Antigen in Solution.

An ELlSA—based solution competition assay is typically designed to determine antibody-binding affinity to the antigen.

Briefly, antibodies in conditioned medium are premixed with serial dilutions of antigen protein ranging from 0 to 10 mg/mL. The solutions of the antibody and antigen mixture are then incubated for two to four hours at room ature to reach binding equilibria. The amounts of free antibody in the mixtures are then measured using a quantitative sandwich ELISA. Ninety-six well MAXISORBTM plates (VWR, West Chester, PA) are coated with 1 pg/mL antigen protein in PBS solution overnight at 4°C followed by BSA nonspecific blocking. The antibody—antigen mixture solutions are then transferred to these plates followed by one-hour incubation. The plates are then washed with washing buffer and the plate-bound antibodies were detected with an HRP-coniugated goat anti- mouse lgG polyclonal antibody reagent (Jackson lmmuno Research Lab) and ped using colorimetric ates such as BD OPTElATM (BD Biosciences Pharmingen, San Diego, CA). After the reaction is d with 1 M phosphoric acid, optical absorptions at 450 nm are recorded and the data are analyzed using PRISMTM software from Graph Pad.

The dependency of the signals on the trations of antigen in solution are analyzed with a 4 parameter fit analysis and reported as leo, the n concentration required to achieve 50% ion of the signal from the antibody s without the presence of antigen in solution.

In one experiment, VELOCIMMUNE® humanized mice were immunized with hIL—6R (as described above). A and 118 show a representative set of affinity measurements for anti-hILGR antibodies from MMUNE® humanized and wild—type mice.

After immunized mice receive a third antigen boost, serum titers are determined by ELISA. Splenocytes are isolated from selected wild type and VELOCIMMUNE® zed mouse s and fused with Ag8.653 myeloma cells to form hybridomas and grown under selection (as described above). Out of a total of 671 anti-lL-GR hybridomas produced, 236 were found to express antigen-specific antibodies. Media harvested from antigen positive wells was used to determine the antibody affinity of binding to antigen using a solution competition ELISA. Antibodies derived from VELOCIMMUNE® humanized mice t a wide range of affinity in g to antigen in solution (A).

Furthermore, 49 out of 236 anti-lL-6R omas were found to block lL—6 from binding to the receptor in an in vitro bioassay (data not shown). Further, these 49 anti-lL-6R blocking antibodies exhibited a range of high solution affinities similar to that of blocking antibodies derived from the parallel immunization of wild type mice (8).

Example Vll Construction of a Mouse ADAMS ing Vector A targeting vector for ion of mouse ADAM6a and ADAM6b genes into a humanized heavy chain locus was constructed using VELOCIGENE® c engineering technology (supra) to modify a Bacterial Artificial Chromosome (BAC) 929d24 obtained from Dr. Fred Alt d University). 929d24 BAC DNA was engineered to n genomic fragments containing the mouse ADAM6a and ADAMGb genes and a hygromycin cassette for targeted deletion of a human ADAMS pseudogene (hADAMBLlJ) d between human VH1—2 and VH6—1 gene segments of a humanized heavy chain locus ().

First, a genomic fragment containing the mouse ADAM6b gene, ~800 bp of upstream (5’) ce and ~4800 bp of downstream (3’) sequence was ned from the 929d24 BAC clone. A second genomic fragment containing the mouse ADAM6a gene, ~300 bp of upstream (5’) sequence and ~3400 bp of downstream (3’) sequence, was separately subcloned from the 929d24 BAC clone. The two genomic fragments containing the mouse ADAM6b and ADAM6a genes were ligated to a hygromycin cassette flanked by Frt recombination sites to create the targeting vector (Mouse ADAM6 Targeting Vector, Figure 20; SEQ ID N023). Different restriction enzyme sites were engineered onto the 5’ end of the targeting vector following the mouse ADAM6b gene and onto the 3’ end following the mouse ADAM6a gene (bottom of ) for ligation into the humanized heavy chain locus.

A separate modification was made to a BAC clone containing a replacement of the mouse heavy chain locus with the human heavy chain locus, including the human ADAM6 pseudogene located between the human VH1-2 and VH6-1 gene segments of the humanized locus for the subsequent ligation of the mouse ADAM6 targeting vector ().

Briefly, a neomycin cassette flanked by loxP recombination sites was ered to contain homology arms containing human genomic sequence at positions 3’ of the human VH1—2 gene segment (5’ with respect to hADAMGW) and 5’ of human VH6-1 gene segment (3’ with respect to W; see middle of ). The location of the insertion site of this targeting construct was about 1.3 kb 5’ and ~350 bp 3’ of the human ADAM6 pseudogene. The targeting construct also included the same restriction sites as the mouse ADAM6 targeting vector to allow for subsequent BAC ligation n the modified BAC clone containing the deletion of the human ADAM6 pseudogene and the mouse ADAM6 targeting vector.

Following digestion of BAC DNA derived from both constructs, the c fragments were ligated together to construct an ered BAC clone containing a humanized heavy chain locus containing an ectopically placed genomic sequence comprising mouse ADAM6a and ADAM6b nucleotide sequences. The final targeting construct for the on of a human ADAM6 gene within a humanized heavy chain locus and insertion of mouse ADAM6a and ADAM6b sequences in ES cells contained, from 5’ to 3’, a 5’ genomic fragment ning ~13 kb of human genomic sequence 3’ of the human VH1—2 gene segment, ~800 bp of mouse c ce downstream of the mouse ADAM6b gene, the mouse ADAM6b gene, ~4800 bp of genomic sequence upstream of the mouse ADAM6b gene, a 5’ Frt site, a hygromycin cassette, 3 3’ Frt site, ~300 bp of mouse c sequence downstream of the mouse ADAM6a gene, the mouse ADAM6a gene, ~3400 bp of mouse genomic sequence upstream of the mouse ADAM6a gene, and a 3’ genomic fragment containing ~30 kb of human genomic sequence 5’ of the human VH6-1 gene segment (bottom of ).

The engineered BAC clone (described above) was used to electroporate mouse ES cells that contained a humanized heavy chain locus to created modified ES cells sing a mouse genomic ce ectopically placed that comprises mouse ADAM6a and ADAM6b sequences within a humanized heavy chain locus. Positive ES cells containing the ectopic mouse genomic fragment within the humanized heavy chain locus TM probes (Lie, Y8. and were identified by a quantitative PCR assay using TAQMAN Petropoulos, C.J. (1998) Advances in quantitative PCR technology: 5’nuclease assays.

Curr Opin Biotechnol 9(1):43-48). The upstream and downstream regions outside of the modified portion of the humanized heavy chain locus were confirmed by PCR using primers and probes located within the modified region to m the presence of the ectopic mouse genomic sequence within the humanized heavy chain locus as well as the hygromycin cassette. The nucleotide ce across the upstream insertion point ed the following, which indicates human heavy chain genomic sequence upstream of the insertion point and an l—Ceu l restriction site (contained within the parentheses below) linked contiguously to mouse genomic sequence present at the insertion point: (CCAGCTTCAT TAGTAATCGT TCATCTGTGG TAAAAAGGCA GGATTTGAAG CGATGGAAGA TGGGAGTACG GGGCGTTGGA AGACAAAGTG CCACACAGCG CAGCCTTCGT CTAGACCCCC GGGCTAACTA TAACGGTCCT AAGGTAGCGA G) ACAG ATTCTCTGTT CAGTGCACTC AGGGTCTGCC TCCACGAGAA TCACCATGCC CTTTCTCAAG ACTGTGTTCT GTGCAGTGCC CTGTCAGTGG (SEQ lD NO:4). The nucleotide sequence across the downstream insertion point at the 3’ end of the targeted region included the ing, which indicates mouse genomic ce and a Pl-Sce l ction site (contained within the parentheses below) linked contiguously with human heavy chain genomic sequence downstream of the insertion point: (AGGGGTCGAG GGGGAATTTT ACAAAGAACA AAGAAGCGGG CATCTGCTGA CATGAGGGCC GAAGTCAGGC TCCAGGCAGC GGGAGCTCCA CCGCGGTGGC GCCATTTCAT TACCTCTTTC TCCGCACCCG ACATAGATAAAGCTT) ATCCCCCACC AAGCAAATCC CCCTACCTGG GGCCGAGCTT CCCGTATGTG TGAA TCCCTGAGGT CGATTGCTGC ATGCAATGAA ATTCAACTAG (SEQ ID N025).

Targeted ES cells bed above were used as donor ES cells and introduced into an 8—cell stage mouse embryo by the MOUSE® mouse ering method (see, 9.9., US Pat. Nos. 7,6598,442, 7,576,259, 7,294,754). Mice bearing a humanized heavy chain locus containing an c mouse genomic sequence comprising mouse ADAM6a and ADAMGb ces were identified by genotyping using a modification of allele assay (Valenzuela et a/., 2003) that detected the presence of the mouse ADAM6a and ADAM6b genes within the humanized heavy chain locus.

Mice bearing a humanized heavy chain locus that ns mouse ADAM6a and ADAMGb genes are bred to a FLPe deletor mouse strain (see, 9.9., Rodriguez, C.l. et al. (2000) High—efficiency deleter mice show that FLPe is an alternative to Cre-onP.

Nature Genetics 25:139—140) in order to remove any FRTed hygromycin cassette introduced by the targeting vector that is not removed, e.g., at the ES cell stage or in the embryo. Optionally, the hygromycin cassette is ed in the mice.

Pups are genotyped and a pup heterozygous for a humanized heavy chain locus containing an c mouse genomic fragment that comprises mouse ADAM6a and ADAM6b ces is selected for characterizing mouse ADAM6 gene expression and fertility.

Example Vlll Characterization of ADAMG Rescue Mice ] Flow Cytometry.

Three mice at age 25 weeks homozygous for human heavy and human K light chain variable gene loci (H/K) and three mice at age 18-20 weeks homozygous for human heavy and human K light chain having the ectopic mouse genomic fragment encoding the mouse ADAM6a and ADAM6b genes within both alleles of the human heavy chain locus (H/K-AB) were sacrificed for identification and analysis of lymphocyte cell populations by FACs on the BD LSR ll System (BD Bioscience). Lymphocytes were gated for specific cell lineages and analyzed for progression through various stages of B cell development.

Tissues collected from the animals included blood, spleen and bone marrow. Blood was ted into BD microtainer tubes with EDTA (BD Biosciences). Bone marrow was collected from femurs by flushing with complete RPMI medium supplemented with fetal calf serum, sodium pyruvate, HEPES, aptoethanol, non—essential amino acids, and gentamycin. Red blood cells from blood, spleen and bone marrow preparations were lysed with an ammonium chloride-based lysis buffer (e.g., ACK lysis buffer), followed by washing with complete RPMI medium.

For staining of cell populations, 1 x 106 cells from the s tissue sources were incubated with anti-mouse CD16/CD32 (2.4G2, BD Biosciences) on ice for 10 minutes, followed by labeling with one or a combination of the following antibody cocktails for 30 min on ice.

Bone marrow: anti-mouse FlTC-CD43 (1 B1 1, BioLegend), PE—ckit (288, BioLegend), PeCy7-lgM (ll/41, eBioscience), PerCP—Cy5.5-lgD c.2a, BioLegend), APC-eFluor780-BZZO (RAB-682, eBioscience), A700~CD19 (1 D3, BD Biosciences). eral blood and spleen: anti-mouse FlTC—K , BD Biosciences), PE-A (RML-42, BioLegend), PeCy7-lgM (ll/41, eBioscience), PerCP-Cy5.5-lgD 0.2a, BioLegend), APC-CD3 (145—201 1, BD), A700—CD19 (1D3, BD), APC—eFluor780-8220 (RAB—682, eBioscience). Following incubation with the labeled antibodies, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on an LSRll flow ter and analyzed with FlowJo. Results from a representative H/K and H/K-A6 mouse are shown in Fle. 14—18.

The results demonstrate that B cells of H/K-A6 mice progress h the stages of B cell pment in a similar fashion to H/K mice in the bone marrow and peripheral compartments, and show normal patterns of maturation once they enter the periphery. H/K-A6 mice demonstrated an increased CD43‘mCD19+ cell population as compared to H/K mice (FlG. 168). This may indicate an accelerated lgM expression from the humanized heavy chain locus containing an ectopic mouse c fragment comprising the mouse ADAM6a and ADAM6b sequences in H/K-A6 mice. in the periphery, B and T cell populations of H/K-A6 mice appear normal and similar to H/K mice.

Testis Morphology and Sperm Characterization.

To determine if infertility in mice having humanized immunoglobulin heavy chain variable loci is due to testis and/or sperm production defects, testis morphology and sperm content of the epididymis was examined.

Briefly, testes from two groups of five mice per group (Group 1: mice homozygous for human heavy and K light chain variable gene loci, mADAMG"‘; Group 2: mice heterozygous for human heavy chain variable gene loci and gous for K light chain variable gene loci, mADAMB”) were dissected with the ymis intact and d. The specimens were then fixed, embedded in in, sectioned and d with xylin and eosin (HE) stain. Testis sections (2 testes per mouse, for a total of ) were examined for defects in morphology and ce of sperm production, while epididymis sections were ed for presence of sperm. in this experiment, no differences in testis weight or morphology was observed between mADAM6"' mice and mADAMG” mice. Sperm was observed in all pes, both in the testes and the ymis. These results establish that the absence of mouse ADAMBa and ADAMBb genes does not lead to detectable changes in testis morphology, and that sperm is produced in mice in the presence and absence of these two genes.

Defects in fertility of male ADAM6"‘ mice are therefore not likely to be due to low sperm production.

Sperm Motility and Migration.

Mice that lack other ADAM gene family members are infertile due to defects in sperm motility or migration. Sperm migration is defined as the ability of sperm to pass from the uterus into the oviduct, and is normally necessary for fertilization in mice. To determine if the deletion of mouse ADAM6a and ADAMGb affects this process, sperm migration was evaluated in mADAM6'/'mice. Sperm motility was also examined.

Briefly, sperm was obtained from testes of (1) mice heterozygous for human heavy chain variable gene loci and homozygous for human K light chain variable gene locui (ADAM6*"); (2) mice homozyogous for human heavy chain le gene loci and homozygous for human K light chain variable gene loci (ADAMB'l'); (3) mice homozygous for human heavy chain variable gene loci and homozygous for wild-type K light chain (ADAM6'I'mK); and, (4) wild-type C57 BL/G mice (WT). No significant abnormalities were observed in sperm count or l sperm motility by inspection. For all mice, cumulus dispersal was observed, indicating that each sperm sample was able to penetrate the s cells and bind the zona pellucida in vitro. These s establish that ADAM6"‘ mice have sperm that are capable of penetrating the cumulus and binding the zona peliucida.

Fertilization of mouse ova in vitro (IVF) was done using sperm from mice as described above. A slightly lower number of cleaved embryos were present for ADAM6"’ the day following lVF, as well as a reduced number of sperm bound to the eggs. These results establish that sperm from ADAMS" mice, once exposed to an ovum, are capable of penetrating the cumulus and binding the zona pellucida. in another experiment, the abiiity of sperm from ADAMS" mice to migrate from the uterus and through the oviduct was determined in a sperm migration assay.

Briefly, a first group of five superovulated female mice were set up with five ADAM6"' males. A second group of five superovulated female mice were set up with five ADAM6"" males. The mating pairs were ed for copulation, and five to six hours post-copulation the uterus and attached oviduct from all females were removed and flushed for analysis. Flush solutions were checked for eggs to verify ovulation and obtain a sperm count. Sperm migration was evaluated in two different ways. First, both oviducts were removed from the uterus, flushed with , and any sperm identified were counted.

The presence of eggs was also noted as evidence of ovulation. Second, ts were left ed to the uterus and both tissues were ﬁxed, embedded in paraffin, sectioned and stained (as described . Sections were examined for presence of sperm, in both the uterus and in both oviducts.

For the five females mated with the five ADAM6"‘ males, very little sperm was found in the flush solution from the oviduct. Flush solutions from oviducts of the five females mated with the five ADAM6+" males exhibited a sperm level about 25- to d higher (avg, n = 10 ts) than present in flush solutions from the oviducts of the five females mated with the five ADAM6”‘ males.

Histological sections of uterus and oviduct were prepared. The ns were examined for sperm presence in the uterus and the oviduct (the coliicuius us).

Inspection of histological sections of oviduct and uterus revealed that for female mice mated with ADAMB"’ mice, sperm was found in the uterus but not in the oviduct. r, sections from femaies mated with ADAM6"' mice revealed that sperm was not found at the uterotubal junction (UTJ). in ns from females mated with ADAM6+" mice, sperm was identified in the UTJ and in the oviduct.

These results establish that mice lacking ADAM6a and ADAM6b genes make sperm that exhibit an in vivo migration defect. in all cases, sperm was observed within the uterus, indicating that copulation and sperm release apparently occur as normal, but little to no sperm was observed within the oviducts after copulation as ed either by sperm count or histological observation. These results establish that mice lacking ADAM6a and ADAM6b genes produce sperm that t an inability to migrate from the uterus to the t. This defect apparently leads to infertility because sperm are unable to cross the uterine-tubule junction into the oviduct, where eggs are fertilized. Taken together, all of these s converge to the support the esis that mouse ADAM6 genes help direct sperm with normal motility to migrate out of the uterus, through the uterotubal junction and the oviduct, and thus approach an egg to achieve the fertilization event. The mechanism by which ADAM6 achieves this may be directly by action of the ADAM6 proteins, or through coordinate expression with other proteins, e.g., other ADAM proteins, in the sperm cell, as described below.

ADAM Gene Family sion.

A complex of ADAM proteins are known to be present as a complex on the surface of maturing sperm. Mice lacking other ADAM gene family members lose this x as sperm mature, and exhibit a reduction of multiple ADAM proteins in mature sperm. To ine if a lack of ADAM6a and ADAM6b genes affects other ADAM proteins in a similar manner, n blots of protein extracts from testis (immature sperm) and epididymis (maturing sperm) were analyzed to determine the expression levels of other ADAM gene family s. in this experiment, protein extracts were ed from four ADAM6'/‘ and four ADAM6+" mice. The results showed that expression of ADAM2 and ADAM3 were not affected in testis extracts. However, both ADAM2 and ADAM3 were dramatically reduced in epididymis extracts. This demonstrates that the absence of ADAM6a and ADAM6b in sperm of ADAM6"‘ mice may have a direct affect on the expression and perhaps function of other ADAM proteins as sperm matures (e.g., ADAM2 and ADAM3). This suggests that ADAM6a and ADAMGb are part of an ADAM protein complex on the surface of sperm, which might be critical for proper sperm migration.

Example lX Human Heavy Chain Variable Gene Usage in ADAM6 Rescue Mice ed human heavy chain variable gene usage was determined for mice homozygous for human heavy and K light chain le gene loci either lacking mouse ADAM6a and‘ADAMBb genes (mADAM6"') or containing an ectopic genomic fragment encoding for mouse ADAM6a and ADAMBb genes +’+; see Example 1) by a quantitative PCR assay using TAQMAN TM probes (as described above).

Briefly, CD19+ B cells were purified from the spleens of mADAM6"‘ and ADAMS”+ mice using mouse CDtQ Microbeads (Miltenyi Biotec) and total RNA was purified using the RNEASYTM Mini kit (Qiagen). Genomic RNA was removed using a RNase-free DNase on—column treatment (Qiagen). About 200 ng mRNA was reverse- transcribed into cDNA using the First Stand cDNA Synthesis kit rogen) and then amplified with the TAQMANTM Universal PCR Master Mix (Applied Biosystems) using the AB! 7900 Sequence Detection System (Applied Biosystems). Relative expression of each gene was ized to the mouse K Constant (mCK). Table 9 sets forth the antlsense/TAQMANTM MGB probe combinations used in this experiment.

Table 9 Human vH Sequence (5’-3’) SEQ ID NOs: Sense: CAGGTACAGCTGCAGCAGTCA Anti—sense: GGAGATGGCACAGGTGAGTGA Probe: TCCAGGACTGGTGAAGC Sense: TAGTCCCAGTGATGAGAAAGAGAT Anti—sense: GAGAACACAGAAGTGGATGAGATC Probe: TGAGTCCAGTCCAGGGA Sense: AAAAATTGAGTGTGAATGGATAAGAGTG Anti-sense: GGTCAGAAACTGCCA Probe: AGAGAAACAGTGGATACGT -—l- Sense: AACTACGCACAGAAGTTCCAGG Anti-sense: GCTCGTGGATTTGTCCGC Probe: CACGATTACC -—-1 Sense: TGAGCAGCACCCTCACGTT Anti-sense: GTGGCCTCACAGGTATAGCTGTT Probe: ACCAAGGACGAGTATGAA in this experiment, expression of all four human VH genes was observed in the samples analyzed. Further, the sion levels were comparable n mADAM6"’ and ADAMS“+ mice. These results demonstrate that human VH genes that were both distal to the modification site (VHS—23 and VH1—69) and proximal to the modification site (VH1-2 and VH6-1) were all able to recombine to form a functionally sed human heavy chain. These results demonstrate that the ectopic genomic fragment comprising mouse ADAMSa and ADAMBb sequences inserted into a human heavy chain genomic sequence did not affect V(D)J recombination of human heavy chain gene segments within the locus, and these mice are able to recombine human heavy chain gene segments in normal fashion to produce functional heavy chain immunoglobulin proteins.

Example X ldentification of Human Heavy Chain Variable Regions That Associate with Selected Human Light Chain Variable Regions An in vitro expression system was constructed to determine if a single nged human germline light chain could be co-expressed with human heavy chains from antigen-specific human antibodies.

] Methods for ting human antibodies in genetically modified mice are known (see 6.9., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE® humanized mouse). The VELOCIMMUNE® humanized mouse technology involves generation of a genetically modified mouse having a genome comprising human heavy and light chain le regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA ng the variable regions of the heavy and light chains of the antibodies produced from a VELOCIMMUNE® humanized mouse are fully human. lly, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As described below, 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 a fully human antibody containing a non—lgM isotype, for example, wild type or modified lng, lgGZ, lgG3 or lgG4. While the constant region selected may vary according to ic use, high affinity n-binding and target specificity characteristics reside in the variable region.

A VELOCIMMUNE® humanized 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 recognized in the art.

Selected antibodies were cloned onto human heavy and light chain constant regions and 69 heavy chains were selected for pairing with one of three human light : (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 constant region, or (3) a nged human germline VK3—20JK1 linked to a human K constant . Each heavy chain and light chain pair were nsfected in CHO—K1 cells using standard techniques. ce of dy in the supernatant was detected by anti-human 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 10). VH: Heavy chain variable gene. ND: no expression detected under current mental conditions.

Table 10 Antibody Titer (ng/mL) Percent of Native Titer Cognate LC VK1-39JK5 VK3-20JK1 JK5 VK3-20JK1 5600.0 192.9 478.4 342.7 184.4 137.3 3\ICD 195.2 284.6 3—33 145.1 — 0) .13. 3-33 3—33 520.6 In 3—33 3-33 3-23 3-33 3-15 123.1 - 210-9 170.9 — 56550.0 - 1385.5 163.5 164.6 122.7 130.0 in a similar ment, VELOCIMMUNE® humanized mice were immunized with several different antigens and selected heavy chains of antigen specific human antibodies were tested for their ability to pair with different rearranged human germiine light chains (as described above). The antigens used in this experiment included an enzyme invoived in cholesterol homeostasis (Antigen A), a serum hormone involved in ting 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 variable 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 germline VK1-39JK5 region. Each heavy/light chain pair was co-transfected 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 g and titers with the different rearranged human germline light chains were compared to the titers ed with the parental antibody molecule (i.e., heavy chain paired with cognate light chain) and percent of native titer was calculated (Table 11). VH: Heavy chain variable gene. VKI K light chain variable gene.

ND: no expression detected under current mental conditions.

Table 11 Titer (pg/ml) Antigen. Antibody. t of VH VK VH Alone VH + VK VKxgg-JKS Native Titer 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 3—30 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 3-13 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 C 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 L743 323 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 6-214 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 experiments demonstrate that somatically mutated, high affinity heavy chains from different gene families are able to pair with rearranged human germline JK5 and VK3-20JK1 regions and be secreted from the cell as a normal dy molecule. As shown in Table 10, antibody titer was increased for about 61% (42 of 69) heavy chains when paired with the rearranged human JK5 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 parental antibody. For about 20% (14 of 69) of the heavy chains, both rearranged human germline light chains red an increase in sion as compared to the cognate light chain of the parental antibody. As shown in Table 11, the rearranged human germline VK1-39J1c5 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 compared to the cognate light chain of the al 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 sion relative 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 trates a favorable relationship of human VH3 heavy chains to pair with rearranged human germline VK1-39JK5 and VK3-20JK1 light chains.

Example Xl Generation of a Rearranged Human Germline Light Chain Locus Various nged human germline light chain targeting vectors were made using VELOClGENE® genetic engineering technology (see, 9.9., US Pat. No. 6,586,251 and uela et al. (2003) hroughput engineering of the mouse genome d with high-resolution expression analysis, Nature Biotech. 21(6):652-659) to modify mouse c Bacterial Artificial Chromosome (BAC) clones 302912 and 254m04 (lnvitrogen).

Using these two BAC clones, genomic ucts were engineered to contain a single rearranged human germline light chain region and ed into an endogenous K light chain locus that was previously modified to delete the endogenous K variable and joining gene segments.

] Construction of Rearranged Human Germline Light Chain Targeting Vectors.

Three different rearranged human germline light chain regions were made using standard molecular y techniques recognized in the art. The human variable gene segments used for constructing these three regions included rearranged human VK1- 39JK5 sequence, a rearranged human VK3—20JK1 sequence and a rearranged human VpreBJlS 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 synthesis (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 forward primers had a 5’ extension ning 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 encoding human JK1. The human VpreBJkS sequence was made by de novo DNA synthesis rated DNA Technologies). A portion of the human JK-CK intron including the splice donor site was PCR amplified from plasmid pBSHA1 8- PlScel. The forward PCR primer included an extension encoding part of either a human JK5, JK1, or JKS sequence. The reverse primer included a PI—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-Pl8cel, which contained the promoter from the human VK3-15 variable gene segment. A loxed hygromycin cassette within plasmid pBS-296—HA18—PlScel was replaced with a FRTed hygromycin cassette flanked by Notl and Ascl sites. The Notl/Pl-Scel fragment of this plasmid was ligated into modified mouse BAC 254m04, which ned 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 Noti/Ascl fragment of this BAC was then ligated into ed mouse BAC 302g12, 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 (FlG. 19).

Restriction enzyme sites were uced 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 ’ to 3’ included a 5’ homology arm containing ce 5’ to the endogenous mouse K light chain locus obtained from mouse BAC clone 302912, 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, a intron sequence of the mouse VK3-7 variable gene segment, an open reading frame of a rearranged human germline JK5 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 segment obtained from mouse BAC clone 254m04 (Figure 19, middle). Genes and/or sequences am of the endogenous mouse K light chain locus and downstream of the most 3’ JK gene segment (9.9., the endogenous 3’ er) were unmodified by the targeting construct (see Figure 19). The sequence of the engineered human JK5 locus is shown in SEQ lD NO:59. ed insertion of the rearranged human germline VK1-39JK5 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using s located at sequences within the rearranged human ne light chain region. Briefly, the intron ce 3’ to the mouse VK3-7 leader sequence was confirmed with primers ULC-m1 F (AGGTGAGGGT ACAGATAAGT GTTATGAG; SEQ lD NOIBO) and R (TGACAAATGC CCTAATTATA GTGATCA; SEQ lD NO:61). The open reading frame of the rearranged human germline VK1-39JK5 region was confirmed with primers 1633-h2F (GGGCAAGTCA GAGCATTAGC A; SEQ ID NO:62) and 1633-h2R (TGCAAACTGG ATGCAGCATA G; SEQ ID NO:63). The neomycin cassette was confirmed with primers neoF agagg ctattcggc; SEQ ID NO:64) and neoR cggcg gcatcag; SEQ ID NO:65). Targeted BAC DNA was then used to electroporate mouse ES ceIIs to created modified ES cells for generating chimeric mice that express a rearranged human germline VK1-39JK5 region.

Positive ES cell clones were confirmed by TM screening and karyotyping using probes specific for the ered VK1-39JK5 light chain region inserted into the endogenous Iocus. Briefiy, probe neoP (TGGGCACAAC AGACAATCGG CTG; SEQ ID NO:66) which binds within the neomycin marker gene, probe ULC-m1 P (CCATTATGAT GCTCCATGCC TCTCTGTTC; SEQ ID NO:67) which binds within the intron sequence 3’ to the mouse VK3-7 leader sequence, and probe 1633h2P (ATCAGCAGAA ACCAGGGAAA GCCCCT; SEQ ID NO:68) which binds within the rearranged human germiine JK5 open reading frame. ve ES ceII clones were then used to impIant female mice to give rise to a litter of pups expressing the germline VK1-39JK5 light chain region.

] Alternatively, ES cells bearing the rearranged human germline VK1-39JK5 light chain region are transfected with a ct that expresses FLP in order to remove the FRTed neomycin cassette introduced by the targeting construct. Optionally, the neomycin cassette is removed by breeding to mice that express FLP recombinase (9.9., US 6,774,279). Optionally, the neomycin cassette is ed in the mice.

Rearranged Human Germiine Vx3-20JK1 Targeting Vector ().

In a similar fashion, an engineered Iight chain Iocus 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 Iight chain locus ed from mouse BAC clone , a FRTed neomycin resistance gene, a genomic sequence ing the human VK3-15 promoter, a leader sequence of the mouse VK3-7 variable gene segment, an intron sequence of the mouse VK3-7 variable gene segment, an open reading frame of a rearranged human germline VK3-20JK1 , 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 20, middle). The sequence of the engineered human VK3- 20th locus is shown in SEQ ID NO:69.

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 nged human germline VK3—20JK1 light chain region. Briefly, the intron sequence 3’ to the mouse VK3-7 leader sequence was confirmed with s ULC—th (SEQ ID N0260) and ULC-m1 R (SEQ lD . The open reading frame of the rearranged human germline VK3—20JK1 region was confirmed with primers 1635—h2F (TCCAGGCACC CTGTCTTTG; SEQ lD NO:70) and 1635-h2R GCTGC TGCTAACACT CTGACT; SEQ lD . The neomycin cassette was confirmed with s neoF (SEQ lD NOz64) and neoR (SEQ ID NO:65). 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 TaqmanTM screening and karyotyping using probes specific for the engineered VK3-20JK1 light chain region inserted into the endogenous K light chain locus. Briefly, probe neoP (SEQ lD NO:66) which binds within the neomycin marker gene, probe ULC-m1 P (SEQ ID NO:67) which binds within the mouse VK3-7 leader sequence, and probe 1635h2P (AAAGAGCCAC CCTCTCCTGC AGGG; SEQ lD NO:72) 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 .

Alternatively, ES cells bearing human germline VK3-20JK1 light chain region can be transfected with a constuct that expresses FLP in oder to remove the FRTed in cassette introduced by the targeting consruct. Optionally, the neomycin cassette may be removed by ng to mice that express FLP recombinase (e.g., US 6,774,279).

Optionally, the neomycin cassette is retained in the mice.

Rearranged Human Germline VpreBJl5 Targeting Vector (). in a similar fashion, an engineered light chain locus expressing a rearranged human germline VpreBJl5 region was made using a ing construct including, from 5’ to 3’, a 5’ homology arm containing sequence 5’ to the nous mouse K light chain locus obtained from mouse BAC clone 302912, a FRTed neomycin resistance gene, an genomic ce 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 variable gene segment, an open reading frame of a nged human germline VpreBJkS region, a genomic sequence containing a portion of the human JK-CK intron, and a 3’ homology arm ning sequence 3’ of the endogenous mouse JK5 gene segment obtained from mouse BAC clone 254m04 (Figure 21, middle). The ce of the engineered human VpreBJlS locus is shown in SEQ lD NO:73.

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

Briefly, the intron sequence 3’ to the mouse VK3-7 leader sequence was confirmed with primers ULC-miF (SEQ lD N060 and ULC-mlR (SEQ lD NO:61). The open reading frame of the rearranged human germline x5 region was confirmed with primers 1616—h1F (TGTCCTCGGC CCTTGGA; SEQ lD NO:74) and 1616-h1R GTCAT TCCT; SEQ lD NO:75). The in cassette was confirmed with primers neoF (SEQ lD NO:64) and neoR (SEQ lD N0265). Targeted BAC DNA was then used to oporate mouse ES cells to created modified ES cells for generating chimeric mice that express the rearranged human germline VpreBJx5 light chain.

Positive ES cell clones are confirmed by TaqmanTM screening and karyotyping using probes ic for the engineered VpreBJkS light chain region inserted into the endogenous K light chain locus. Briefly, probe neoP (SEQ lD NO:66) which binds within the neomycin marker gene, probe ULC-m‘lP (SEQ lD NO:67) which binds within the mouse lgVK3-7 leader sequence, and probe 1616h1 P (ACAATCCGCC TCACCTGCAC CCT; SEQ lD NO:76) 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 sing a ne light chain region. atively, ES cells bearing the rearranged human germline l5 light chain region are ected with a construct that expresses FLP in order to remove the FRTed neomycin cassette introduced by the targeting consruct. Optionally, the neomycin cassette is removed by breeding to mice that express FLP recombinase (e.g., US 6,774,279). Optionally, the neomycin cassette is retained in the mice.

Example Xll Generation of Mice expressing a single rearranged human light chain ed ES cells described above were used as donor ES cells and introduced into an 8—cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., US Pat.

No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene—targeted ES cells allowing immediate phenotypic analyses, Nature Biotech. 25(1):91-99. VELOClMlCE® independently bearing an engineered human germline VK1-39JK5 light chain region, a VK3—20JK1 light chain region or a it5 light chain region are identified by genotyping using a modification of allele assay (Valenzuela et al.’, supra) that detects the presence of the unique rearranged human germline 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 region.

Flow Cytometry.

Expression of the nged human light chain region in the normal antibody oire of common light chain mice was validated by analysis of immunoglobulin K and 7» sion in cytes 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 heterozygote (n=3), VK1-39JK5 common light chain homozygote (n=3), VK3—20JK1 common light chain heterozygote (n=2), and JK1 common light chain homozygote (n=2) mice were made using standard methods and stained with CD19+, lgl” and lgk+ using fluorescently labeled antibodies (BD Pharmigen).

Briefly, 1x106 cells were incubated with anti-mouse CD16/CD32 (clone 2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling with the following antibody cocktail for 30 minutes on ice: APC conjugated ouse CD19 (clone 1D3, BD Pharmigen), PerCP—Cy5.5 conjugated anti-mouse CD3 (clone 17A2, BioLegend), FlTC conjugated anti-mouse ng (clone 187.1, SD Pharmigen), PE conjugated anti—mouse lg?» (clone , BioLegend). Following ng, cells were washed and fixed in 2% formaldehyde. Data acquisition was med on an LSRll flow cytometer and analyzed with FlowJoT’V'. Gating: total B cells (CD19+CD3”), lgk+ B cells (lgk+lgl'CD19+CD3‘), lgl” B cells (ng'lgl+CD19+CD3‘). Data gathered from blood and splenocyte samples demonstrated similar s. Table 12 sets forth the percent positive CD19+ B cells from peripheral blood of one representative mouse from each group that are lgF, lgk+, or lgl*lgk+. Percent of C019+ B cells in peripheral blood from wild type (WT) and mice homozygous for either the VK1-39JK5 or VK3-20J1<1 common light chain are shown in HS.

Table 12 CD19" B cells Mouse Genotype --I.=l Common Light Chain Expression.

Expression of each common light chain (VK1—39JK5 and JK1) was analyzed in heterozygous and homozygous mice using a quantitative PCR assay (e.g.

Taqman”).

Brieﬂy, CD19” B ceils were purified from the spleens of wild type, mice homozygous for a replacement of the mouse heavy chain and K light chain variable region loci with corresponding human heavy chain and K light chain variable region loci (HK), as well as mice homozygous and heterozygous for each rearranged human light chain region (VK1-39JK5 or VKS-ZOJK1) using mouse CD19 Microbeads (Miltenyi Biotec) according to manufacturer’s specifications. Total RNA was purified from CD19+ B cells using RNeasyTM Mini kit (Qiagen) according to the manufacturer’s specifications and genomic RNA was removed using a RNase-free DNase on-column treatment n). 200 ng mRNA was reverse—transcribed into cDNA using the First Stand cDNA Synthesis kit (lnvitrogen) and the resulting cDNA was ied with the TM sal PCR Master Mix (Applied Biosystems). All reactions were performed using the ABI 7900 Sequence Detection System ed Biosystems) using primers and TaqmanTM MGB probes spanning (1) the VK-JK junction for both common light chains, (2) the VK gene alone (Le. VK1-39 and VK3- ), and (3) the mouse CK . Table 13 sets forth the sequences of the primers and probes employed for this assay. Relative sion was normalized to expression of the mouse CK region. s are shown in A, 238 and 23C.

Table 13 Region Primer/Probe Description (5’-3’) SEQ iD NOs: (sense) CTGC AACCTGAAGA TTT 77 VK1-39JK5 Junction (anti-sense) GTTTAATCTC TGTC CCTT 78 ) CCTCCGATCA CCTTC 79 (sense) AAACCAGGGA AAGCCCCTAA 8O VK1-39 (anti-sense) ATGGGACCCC ACTTTGCA 81 (probe) CTCCTGATCT ATGCTGCAT 82 (sense) CAGCAGACTG GAGCCTGAAG A 83 VK3-20JK1 Junction (anti-sense) TGATTTCCAC CTTGGTCCCT T 84 (probe) TAGCTCACCT TGGACGTT 85 (sense) CTCCTCATCT ATGGTGCATC CA 86 VK3-20 (anti-sense) GACCCACTGC CACTGAACCT 87 (probe) CCACTGGCAT 000 88 (sense) TGAGCAGCAC CCTCACGTT 89 Mouse CK (anti-sense) GTGGCCTCAC AGGTATAGCT GTT 90 (probe) ACCAAGGACG AGTATGAA 91 Antigen Specific Common Light Chain Antibodies.

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 [3- galactosidase and antibody titer was measured.

Briefly, B-galactosidase (Sigma) was emulsified in TITERMAXTM adjuvant (Sigma), as per the manufacturer’s ctions. Wild type (n=7), VK1-39JK5 common light chain homozgyotes (n=2) and VK3-20JK1 common light chain gotes (n=5) were immunized by subcutaneous injection with 100 pg B-galactosidase/TITERMAXTM. Mice were boosted by subcutaneous injection two times, 3 weeks apart, with 50 pg [3— galactosidase/TITERMAXTM. After the second boost, blood was collected from anaesthetized mice using a retro—orbital bleed into serum separator tubes (BD ences) as per the manufacturer’s instructions. To measure anti—B-galactosidase IgM or IgG antibodies, ELISA plates (Nunc) were coated with 1 pg/mL {S-galactosidase overnight 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 g. Plates were then incubated with HRP conjugated anti-lgM (Southern Biotech) or anti-lgG (Southern Biotech) for one hour at room temperature. ing another wash, plates were developed with TMB substrate (BD Biosciences). Reactions were stopped with N sulfuric acid and OD450 was read using a Victor X5 Plate Reader (Perkin Elmer). Data was analyzed with GRAPHPADT'V‘ Prism and signal was ated as the diIution of serum that is two times above background. Results are shown in A and 248.

As shown in this Example, the ratio of Kl?» B cells in both the splenic and peripheral compartments of VK1-39JK5 and VK3-20JK1 common light chain mice demonstrated a near wild type pattern (Table 12 and ). VpreBJkS common light chain mice, however, trated fewer peripheral B cells, of which about 1-2% express the engineered human light chain region (data not shown). The expression levels of the VK1~39JK5 and VK3-20JK1 nged human light chain regions from the endogenous K light chain locus were elevated in comparison to an endogenous K light chain locus ning a complete ement of mouse VK and JK gene segments with human VK and JK gene segments (A, 238 and 23C). The sion levels of the VpreBJkS rearranged human light chain region demonstrated similar high expression from the endogenous K light chain locus in both heterozygous and gous mice (data not shown). This demonstrates that in direct competition with the mouse 7», K, or both endogenous light chain loci, a single rearranged human VL/JL sequence can yield better than wild type level expression from the endogenous k light chain locus and give rise to normal splenic and blood B cell frequency. r, the presence of an engineered K light chain locus having either a human VK1-39JK5 or human VK3-20JK1 sequence was well ted by the mice and appear to function in wild type fashion by representing a substantial portion of the light chain repertoire in the humoral component of the immune se (FIG 24A and 24B).

Example XIII Breeding 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 described herein to create le genetically modified mouse strains harboring multiple 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 another mouse containing a deletion in the nous 9» light chain locus. In this manner, the progeny obtained will express, as their only light chain, the rearranged human germline light chain region as described in Example 11. Breeding is performed by standard techniques recognized in the art and, alternatively, by a cial breeder (e.g., The Jackson Laboratory). Mouse s bearing an engineered light chain locus and a deletion of the endogenous A 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® humanized mouse, Regeneron ceuticals, Inc.). The VELOCIMMUNE® humanized mouse comprises a genome comprising human heavy chain variable regions operably linked to nous 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 nic stimulation. The DNA ng the le regions of the heavy chains of the antibodies is isolated and operably linked to DNA ng 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. e 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 tide sequences of B cells expressing the antibodies are identified and fully human dies are made by fusion the VH and VL nucleotide sequences to human CH and CL nucleotide sequences in a suitable expression system.

Example XlV 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 modifications and ons of other nous lg loci (as described in Example 12), selected mice can be immunized with an n of st.

Generally, a VELOClMMUNE® zed 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 hybridoma 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 germline light chains which are specific to the antigen used for immunization. DNA encoding the variable regions of the heavy chains and the light chain are isolated and linked to desirable isotypic constant 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. Alternatively, DNA ng the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains are identified directly from antigen-specific cytes. lly, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As described above, the antibodies are characterized and ed for desirable characteristics, ing affinity, ivity, epitope, etc. The mouse constant regions are replaced with a d 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. le human constant regions include, for example wild type or modified lgG1 or igG4.

Separate cohorts of VELOCIMMUNE® zed 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 ered germline VK1-39JK5 human light chain region or the ered germline VK3~ 20th human light chain region (described above) were immunized with a human cell surface receptor protein (Antigen E). Antigen E is administered directly onto the hind footpad of mice with six consecutive ions every 3-4 days. Two to three micrograms of n E are mixed with 10 pg of CpG oligonucleotide (Cat # tlrl-modn - ODN1826 oligonucleotide; anivogen, San Diego, CA) and 25 pg of Adju-Phos num phosphate gel adjuvant, Cat# H250; Brenntag Biosector, Frederikssund, Denmark) prior to ion. A total of six injections are given prior to the final antigen recall, which is given 3— 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 assay.

When a desired immune response is achieved splenocytes are harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines.

The oma 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 variable domain, and mouse constant domains) are obtained.

Alternatively, anti-Antigen E common light chain antibodies are isolated directly from antigen—positive B cells without fusion to myeloma cells, as described in US. 280945A1, herein specifically incorporated by reference 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 engineered human VK3-20JK1 light chain region, and human constant domains) were obtained.

] The biological ties of the exemplary anti-Antigen E common light chain antibodies ted in accordance with the methods of this Example are described in detail below.

Example XV Heavy Chain Gene Segment Usage in n-Specific Common Light Chain dies To analyze the structure of the human anti—Antigen E common light chain antibodies produced, nucleic acids encoding heavy chain antibody variable regions were cloned and sequenced. From the nucleic acid sequences and predicted amino acid sequences of the antibodies, gene usage was identified for the heavy chain variable region (HCVR) of selected common light chain antibodies ed from immunized VELOClMMUNE® humanized mice containing either the engineered human VK1-39JK5 light chain or engineered human JK1 light chain region. Results are shown in Tables 14 and 15, 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 rearrangements, when employing either a mouse that expresses a light chain from only a human 9— or a human VKB-ZO-derived light chain.

Human VH gene segments of the 2, 3, 4, and 5 families rearranged with a y of human DH segments and human JH segments to yield antigen—specific antibodies.

Table 14 VK1-39JK5 Common Light Chain Antibodies HCVR HCVR Antibody Antibody VH DH JH VH DH JH 6032 3-30 m- 2997 6-13 3047 3-30 6-13 4 [ 5982 3-30 6-13 4 6002 3-30 6-13 4 6014 3-30 6-13 4 6015 3-30 6017 3-30 6 6020 3-30 6-13 6034 3-30 2948 3-30 7-27 2996 3-30 3005 3-30 7-27 3012 3-30 7-27 4 3020 3-30 727] 4 3021 3-30 7-27 4 3030 3-30 7-27 4 5997 3-30 7-27 4 - 4 _2950-3—30 \I 6004 3—30 (A) Table 15 VK3-20JK1 Common Light Chain dies HCVR HCVR Antibody —-—---- Antibody —-—---——-—- VH DH JH VH DH JH -51 6—13 5 —51 6 4—39 1—26 3 4-39 1—26 3 Example XVI Determination of Blocking Ability of Antigen-Specific Common Light Chain Antibodies by LUMlNEXTM Assay ] Ninety-eight human common light chain antibodies raised against Antigen E were tested for their ability to block binding of Antigen E’s natural 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 pg/mL in MES buffer. The mixture was incubated 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 (lrvlne Scientific, Santa Ana, CA) containing 2% (w/v) BSA (Sigma-Aldrich Corp, St. Louis, MO). in a 96-well filter plate, supernatants containing Antigen E-specific common light chain antibodies, were diluted 1:15 in buffer. A negative control containing a mock supernatant with the same media ents 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 labeled beads was determined with R-Phycoerythrin conjugated to Streptavidin (Moss lnc, Pasadena, MD) followed by measurement in a LUMINEXTM flow cytometry—based analyzer. Background Mean Fluorescence Intensity (MFl) of a sample without Ligand Y was subtracted from all samples. Percent blocking was ated by on of the background-subtracted MFl of each sample by the adjusted negative control value, multiplying by 100 and subtracting the ing value from 100.

In a similar ment, the same 98 human common light chain antibodies raised against Antigen E were tested for their ability to block binding of Antigen E to Ligand led beads.

Briefly, Ligand Y was amine—coupled to ylated microspheres at a concentration of 20 ug/mL diluted in MES buffer. The mixture and incubated two hours at room temperature followed by vation 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 (lrvlne 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 atant was prepared. 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-Phycoeiythrin ated to Streptavidin (Moss lnc, Pasadena, MD) foilowed by measurement in a LUMlNEXTM flow cytometry- based analyzer. Background Mean Fluorescence lntensity (MFl) of a sample without n E was subtracted from all samples. Percent blocking was calculated by division of the background-subtracted MF l of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100.

Tables 16 and 17 show the percent blocking for all 98 anti-Antigen E common light chain antibodies tested in both LUMlNEXTM assays. ND: not ined under current experimental conditions.

Table 16 VK1-39JK5 Common Light Chain Antibodies . % Blocking of % Blocking of Antibody Antigen led Beads Antigen E In Solution 29506 89.8 31.4 2952 96.1 74.3 2952G 93.5 39.9 2964 92.1 31.4 2964G 94.6 43.0 2978 98.0 95.1 2978G 13.9 94.1 78.5 52.4 31.2 No 01 o. 81.7 67.8 26.6 29.3 87.3 55.3 95.9 38.4 2997 93.4 70.6 29% N01 3004 79.0 48.4 3004G 60.3 40.7 30116 83.5 41.7 3012 91.0 60.8 30126 52.4 16.8 3013 80.3 65.8 3014 63.4 20.7 30146 74.4 28.5 30156 58.8 17.3 3017 94.8 70.2 sows 3018 85.4 54.0 30186 26.1 + 12.7 3020 96.7 90.3 30206 85-2 3021 74.5 26.1 30216 81.1 27.4 3022 65.2 17.6 3023 71.4 28.5 3024 73.9 32.6 30246 1 89.0 10.0 3025 70.7 15.6 30306 41.3 14.2 Table 17 Common Light Chain Antibodies . % Blocking of % Blocking of Antibody Antigen E-Labeled Beads Antigen E In Solution In the first LUMlNEXTM experiment described above, 80 common light chain antibodies containing the VK1-39JK5 engineered light chain were tested for their ability to block Ligand Y binding to Antigen 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 trated >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 XTM experiment described above, the same 80 common light chain antibodies containing the VK1~39JK5 engineered light chain were tested for their ability to block binding of n E to Ligand Y—labeled beads. Of these 80 common light chain dies, 36 demonstrated >50% blocking, while 44 demonstrated <50% blocking (27 at 25-50% blocking and 17 at <25% blocking). For the 18 common light chain antibodies 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% blocking) of Antigen E binding to Ligand Y-labeled beads.

The data of Tables 16 and 17 establish that the rearrangements described in Tables 14 and 15 generated anti-Antigen E-speciﬁc common light chain antibodies that blocked binding of Ligand Y to its cognate receptor Antigen E with varying degrees of cy, which is consistent with the anti-Antigen E common light chain antibodies of Tables 14 and 15 comprising antibodies with overlapping and non—overlapping epitope specificity with respect to n E.

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

Ligand Y was coated onto l plates at a concentration of 2 pg/mL diluted in PBS and incubated overnight followed by washing four times in PBS with 0.05% Tween- . 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 antibodies 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 tration 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 Biosciences, San Jose, CA) lized 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. Percent ng was calculated by division of the background-subtracted MFl of each sample by the ed negative control value, multiplying by 100 and subtracting the resulting value from 100.

Tables 18 and 19 show the percent blocking for all 98 anti—Antigen E common light chain antibodies tested in the ELISA assay. ND: not determined under current experimental conditions.

Table 18 VK1-39JK5 Common Light Chain Antibodies o - o .

Antibody Antigeﬁlzcilggigtion dy Antigeﬁlzcllggiiﬁtion some 3016 78.1 r_30166 37.4 3017 61.6 29546 44.7 30196 2955 12.1 L 3020 80.8 29556_‘— 25.6 30206 ND 2964 34.8 3021 29786 90.2 30226 2982 59.0 3023 29826 20.4 30236 2985 10.5 3024 2985L ND 30246 29876 ND 30256 2996 29.3 3027 61.4 29966 ND [#30276 82.7 2997 48.7 3028 40.3 29976 ND 30286 3004 16.7 3030 ND 303OG 9.5 30326 13.1 3033 77.1 30336 32.9 3036 17.6 3036G 24.6 3012 45.0 3041 59.3 Table 19 Common Light Chain Antibodies . % Blocking of % Blocking of Antibody dy Antigen E In Solution Antigen E In Solution 2968 68.9 2968G ’i 5.2 2969G 23.6 2970 34.3 297OG 4i .3 2971 6.3 2972 9.6 As described in this Example, of the 80 common light chain antibodies ning the VK1~39JK5 engineered light chain tested for their ability to block Antigen E binding to a Ligand Y-coated surface, 22 demonstrated >50% blocking, while 58 demonstrated <50% blocking (20 at 25-50% blocking and 38 at <25% blocking). For the 18 common light chain antibodies containing the VK3—2OJK1 engineered light chain, one demonstrated >50% blocking, while 17 demonstrated <50% blocking (5 at 25-50% blocking and 12 at <25% blocking) of Antigen E binding to a Ligand Y—coated e.

These results are also consistent with the Antigen E-specific common light chain antibody pool sing antibodies with pping and non—overlapping epitope specificity with respect to Antigen E.

Example XVlll BiACORE]M Affinity Determination for Antigen-Specific Common Light Chain Antibodies Equilibrium dissociation constants (KD) for selected antibody supernatants were determined by SPR (Surface Plasmon Resonance) using a BlAcoreTM T100 instrument (GE Healthcare). All data was obtained using HBS-EP (1 OmM HEPES, 150mM NaCl, 0.3mM EDTA, 0.05% Surfactant P20, pH 7.4) as both the g and sample buffers, at °C. Antibodies were captured from crude supernatant samples on a CM5 sensor chip surface previously tized with a high density of anti-human Fc antibodies using standard amine coupling chemistry. During the capture step, supernatants were injected across the anti-human Fc surface at a flow rate of 3 uL/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 uL/min. Dissociation of n from the captured antibody was monitored for 6 minutes. The captured antibody was removed 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, y ng 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 ort using BlACORETM T100 Evaluation software v2.1. Results are shown in Tables and 21.

Table 20 VK1-39JK5 Common Light Chain Antibodies 100 nM Antigen E 100 nM Antigen E Antibody ——-—————-—-—j---. Antibody --—--_-———_ K0(“M) T1,2(mln) Ko(nM) T1,2(mln) 2949 3.57 18 3016 4.99 17 sonj 9.83 s 6 3020 5.41 39 4 30206 41.9 6 -2964 14.8 3021 50.1 2964G 13.0 (O 3021 G 26.8 4 2978 1.91 49 3022 25.7 17 2978G 1.80 58 3022G 20.8 12 2982 6.41 19 3023 263 30236 103 5 64.4 3024 7 29876 37.6 30256 -“ 30276 4.24 3028 6.89 37 30286 7.23 22 3030 46.2 7 W306 128 3032 53.2 n 3033 4.61 -17 3033G 12.0 —5 3036 284 .4 N . -2 Table 21 VK3-20JK1 Common Light Chain Antibodies 100 nM Antigen E 100 nM Antigen E AntibodyW dyW KO (HM) T112 (min) KD (nM) T112 (mm) 29696 The binding affinities of common light chain dies comprising the rearrangements shown in Tables 14 and 15 vary, with nearly all exhibiting a K0 in the nanomolar range. The affinity data is consistent with the common light chain antibodies resulting from the combinatorial association of rearranged variable s described in Tables 14 and 15 being high-affinity, clonally selected, and somatically mutated. Coupled with data previously shown, the common light chain antibodies described in Tables 14 and comprise a collection of diverse, high-affinity antibodies that exhibit specificity for one or more epitopes on Antigen E.

Example XlX Determination of Binding Specificities of Antigen—Specific Common Light Chain Antibodies by LUMINEXTM Assay ed ntigen E common light chain antibodies 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 n E lacking the last 10 amino acids from the inal end of the ECD (Antigen E-ACT); and two mutants containing 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 myc—myc—His C-terminal tag.

] For the binding studies, Antigen 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 ntly coated with an anti- myc monoclonal antibody (MAb 9E10, hybridoma cell line CRL-1729TM; ATCC, Manassas, 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 control. The beads containing the captured Antigen E proteins were then added to the antibody s (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 n E protein) was measured with a LuminexT'V' flow cytometry-based er, and the median scence intensity (MFl) for at least 100 counted beads per bead/antibody interaction was recorded. Results are shown in Tables 22 and 23.

Table 22 VK1-39JK5 Common Light Chain Antibodies Mean Fluorescence intensity (MFI) dy Antigen E- Antigen E- Antigen E- Antigen MfAntigen E ECD ACT A|a1 E-Ala2 2948 1503 2746 4953 3579 1648 2948G 537 662 2581 2150 863 2405 7532 5079 3455 1519 1259 155 911 3515 7114 5039 3395 2472 4735 3755 1537 29545 4999 729 2975 5955 2500 29755 5454 3295 29525 3222 2013 5572 5509 4949 29555 43 43 125 2957 3117 1574 7545 5944 2545 29575 3055 1537 9202 5004 4744 2995 4555 1917 12575 2997 5154 2159 12157 5351 5922 29975 555__L 355 2325 1020 3004 2794 1397 5542 5255 3053 30045 2753 1505 5257 5505 301 OG 3685 1097 3011 2859 2015 7855 5513 3863 3011G 2005 1072 6194 4041 3181 3012 3233 2221 8543 5837 30128 988 378 3115 2281 3013 2343 1791 8715 4810 2528 30138 327 144j 1333 j 1225 370 3014 1225 1089 5438 3821 1718 30148 1585 851 5178 3015 3202 2088 8282 5554 30158 1243 531 4248 2843 3018 4220 2543 8920 3017 3545 2553 8700 30178 7 1972 1081 5783 3825 3038 3018 2339 1971 8140 4515 2293 I 30188 254 118 978 1020 345 3019 5235 1882 7108 4249 30198 4090 1270 4789 3474 30208 2185 1209 8489 4295 30218 2091 1005 8430 3988 3022 2418 793 7523 2879 38 30228 2189 831 8182 3051 132 30236 1770 825 1487 8179 4557 2450 30248 100 87 288 433 3025 1853 1233 4337 2581 30258 1782 791 5773 3871 2717 | 3027 4131 1018 582 2510 22 30278 3492 814 1933 2598 3028 4381 '1 2545 9884 975 30288 2835 1398 7124 3885 597 3030 483 277 1288 1130 l: 30308 943 302 3420 2570 3032 2083 I 1498 8594 4402 30328 295 108 814 902 3033 4409 2774 8971 3038 1755 1382 8137 30388 2313 1073 8387 5837 4274 2653 2137 5124 3042s 1117 463 2762 5532 4403 Table 23 JK1 Common Light Chain Antibodies Mean Fluorescence intensity (MFl) Antibod ' Y AntEgCeSE' ' _ AntﬁﬁE AntligsnE' - Anggng- MfAntigenE 4683 599 12321 31 1148 3967 44 84 2974s 2036 228 8172 135 26 2975 1990 1476 8669 2975G 890 1376 2976 147 1079 181 The anti-Antigen E common light chain antibody supernatants exhibited high ic binding to the beads linked to Antigen 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 g 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 es on the ECD of Antigen E, the relative g of the antibodies to the ts were ined. All four Antigen E variants were captured to the anti-myc LUMlNEXTM beads as described above for the native Antigen E-ECD binding studies, and the relative binding ratios (MFlvariam/MFlAmggen 5-509) were determined. For 98 tested common light chain antibody supernatants shown in Tables 21 and 22, the average ratios (MFlvariam/MFIAWgen 5-500) 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 Mf Antigen E, respectively). For each protein variant, the binding for a subset of the 98 tested common light chain antibodies showed y reduced binding, indicating sensitivity to the mutation that characterized a given variant. For example, 19 of the common light chain antibody samples bound to the Mf Antigen E with am/MFIAnﬁgen 5-509 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 variant rather than from lower affinities.

These data establish that the common light chain antibodies described in Tables 14 and 15 represent a diverse group of n—E-specific common light chain dies that specifically recognize more than one epitope on Antigen E.

Claims

The claims defining the ion are as follows:

1. A method of making a cally modified mouse sing genetically modifying the mouse to include in its germline: (a) a humanized immunoglobulin heavy chain locus comprising at least one unrearranged human VH gene segment, at least one unrearranged human DH gene segment, and at least one unrearranged human JH gene segment operably linked to a heavy chain constant region gene; (b) a humanized immunoglobulin light chain locus comprising a single rearranged human light chain V/J sequence ly linked to a light chain constant region gene; and, (c) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein or functional fragment thereof, wherein the mouse ADAM6 protein or functional fragment thereof is sed from the ectopic nucleic acid ce.

2. The method according to claim 1, wherein the ectopic nucleic acid sequence encoding the mouse ADAM6 protein or functional fragment thereof is at a locus other than the endogenous immunoglobulin heavy chain locus.

3. The method according to claim 1 or 2, wherein the heavy chain constant region gene is: (i) a non-human heavy chain constant region gene; or (ii) a mouse heavy chain nt region gene, ably an endogenous mouse heavy chain constant region gene.

4. The method according to any one of claims 1-3, wherein the light chain constant region gene is: (i) a non-human light chain constant region gene; (ii) a human light chain constant region gene; or (iii) a mouse light chain constant region gene.

5. A method of making a genetically modified mouse comprising genetically modifying the mouse to include in its germline: (a) a humanized immunoglobulin heavy chain locus comprising at least one unrearranged human VH gene segment, at least one unrearranged human DH gene segment, and at least one unrearranged human JH gene segment operably linked to a heavy chain constant region gene; (b) a humanized immunoglobulin light chain locus comprising a single rearranged human light chain V/J sequence operably linked to a light chain constant region gene; and, (c) an inserted nucleic acid sequence encoding a mouse ADAM6 protein or onal fragment thereof, wherein the mouse ADAM6 protein or functional fragment f is expressed from the inserted nucleic acid sequence.

6. The method of claim 5, wherein the humanized heavy chain immunoglobulin locus includes: (i) a non-human heavy chain constant region gene; or (ii) a mouse heavy chain constant region gene, preferably an endogenous mouse heavy chain nt region gene.

7. The method of claim 5 or 6, wherein the light chain constant region gene is: (i) a non-human light chain constant region gene; (ii) a human light chain constant region gene; or (iii) a mouse light chain constant region gene.

8. The method of any one of claims 5-7, wherein the ed nucleic acid sequence encodes a mouse ADAM6 protein or fragment thereof that is functional in a male mouse.

9. The method of any one of claims 5-8, wherein the ed nucleic acid sequence is at a position that is not within an endogenous globulin heavy chain locus.

10. A method of making a genetically modified mouse comprising genetically modifying the mouse to express a plurality of different IgG heavy chains each sing a human heavy chain variable domain, wherein each of the plurality of different IgG heavy chains are ated with an immunoglobulin light chain including a human immunoglobulin light chain le domain that is derived from a single human immunoglobulin VL gene segment, wherein the mouse comprises an ectopic nucleic acid sequence encoding an ADAM6 protein or fragment thereof that is functional in a male mouse.

11. The method of claim 10, wherein the ADAM6 protein or fragment thereof is sed from the ectopic nucleic acid sequence.

12. A cell from a mouse made by a method of any one of claims 1-11.

13. The cell of claim 12, wherein the cell is a B cell or splenocyte.

14. Use of a mouse made by the method of any one of claims 1-11, to make a fully human antibody, or a fully human antigen-binding protein, the fully human antibody or fully human antigen-binding protein comprising an immunoglobulin variable domain or functional fragment thereof.

15. Use of a mouse made by the method of any one of claims 1-11, to make a fully human bispecific antibody.

16. Use of the mouse made by the method of any one of claims 1-11 in the manufacture of an immortalized cell line.

17. Use of the mouse made by the method of any one of claims 1-11 in the manufacture of a hybridoma or quadroma.

18. Use of a mouse made by the method of any one of claims 1-11 to make a nucleic acid sequence ng an globulin variable region or fragment thereof.

19. The use of claim 14, wherein the antigen-binding protein is selected from an antibody, a multispecific dy, an scFv, a bis-scFV, a diabody, a triabody, a tetrabody, a V-NAR, a VHH , a VL, an F(ab), an F(ab)2, a dVD, an sVD, or a bispecific T- cell engager.

20. Use of a mouse made by the method of any one of claims 1-11 in the manufacture of an antigen-binding protein for the treatment of a human disease or disorder.

21. The method according to claim 1, 5, or 10, or the cell according to claim 12, or the use according to any one of claims 14-20 substantially as herein described with reference to the figures and/or es, excluding comparative examples.