NZ612643B2

NZ612643B2 - Adam6 mice

Info

Publication number: NZ612643B2
Application number: NZ612643A
Authority: NZ
Inventors: Lynn Macdonald; Andrew J Murphy; Sean Stevens
Original assignee: Regeneron Pharmaceuticals Inc
Priority date: 2011-02-25
Filing date: 2012-02-24
Publication date: 2015-09-29

Abstract

Discloses a genetically modified mouse whose genetic modifications achieve: a) ectopic placement of a nucleotide sequence that expresses an ADAM6 protein or fragment thereof that is functional in a male mouse; and b) a modified immunoglobulin heavy chain variable region locus.

Description

ADAMS MICE FIELD OF INVENTION Genetically modified mice, cells, embryos, and tissues that comprise a nucleic acid sequence encoding a functional ADAMS locus are described. Modifications include human and/or humanized immunoglobuiin loci. Mice that lack a functional endogenous ADAMS gene but that comprise ADAMS function are described, including mice that comprise an c nucleic acid sequence that encodes an ADAMS n. Genetically modified male mice that comprise a cation of an endogenous giobulin VH locus that renders the mouse incapable of making a functional ADAMS protein and results in a loss in fertility, and that further comprise ADAMS function in the male mice are described, including mice that comprise an ectopic nucleic acid sequence that restores ity to the male mouse.

BACKGROUND OF INVENTION Mice that contain human antibody genes are known in the art. ceutical applications for antibodies in the last two decades have fueled a great deal of research into making antibodies that are suitable for use as human therapeutics. Early antibody therapeutics, which were based on mouse antibodies, were not ideal as human therapeutics because repeated administration of mouse dies to humans results in immunogenicity that can confound long-term treatment regimens. Solutions based on humanizing mouse antibodies to make them appear more human and less mouse-like were developed. Methods for expressing human immunoglobulin sequences for use in antibodies followed, mostly based on in vitro expression of human immunoglobulin libraries in phage, bacteria, or yeast. Finally, attempts were made to make useful human antibodies from human iymphocytes in vitro, in mice engrafted with human hematopoietic ceils, and in transchromosomal or transgenic mice with disabled endogenous immunoglobulin loci. In the transgenic mice, it was necessary to disable the nous mouse immunoglobulin genes so that the randomly ated fully human transgenes would function as the source of immunoglobulin sequences expressed in the mouse. Such mice can make human antibodies suitable for use as human therapeutics, but these mice display substantial problems with their immune systems. These ms (1) make the mice impractical for generating a sufficiently diverse antibody repertoire, (2) require the use of extensive re-engineering fixes, (3) provide a suboptimal cional ion s likely due to incompatibility between human and mouse elements, and (4) render these mice an able source of large and diverse populations of human variable sequences needed to be truly useful for making human therapeutics.

There remains a need in the art for making improved genetically ed mice that are useful in generating immunoglobulin sequences, including human antibody ces. There also remains a need for mice that are capable of rearranging immunoglobulin gene segments to form useful rearranged immunoglobulin genes, or capable of making proteins from altered immunoglobulin loci, while at the same time reducing or eliminating deleterious changes that might result from the genetic modifications.

Y OF INVENTION in one , nucleic acid constructs, cells, embryos, mice, and methods are provided for making mice that comprise a modification that results in a nonfunctional endogenous mouse ADAM6 protein or ADAM6 gene (9.9., a knockout of or a deletion in an endogenous ADAM6 gene), wherein the mice se a nucleic acid sequence that encodes an ADAM6 protein or og or homolog or fragment thereof that is functional in a male mouse. in one aspect, nucleic acid constructs, cells, embryos, mice, and methods are provided for making mice that comprise a modification of an endogenous mouse immunoglobulin locus, wherein the mice comprise an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse. in one embodiment, the nous mouse immunoglobulin locus is an immunoglobulin heavy chain locus, and the modification reduces or eliminates ADAM6 activity of a cell or tissue of a male mouse. in one aspect, mice are provided that se an ectopic nucleotide sequence encoding a mouse ADAM6 or ortholog or homolog or functional fragment thereof; mice are also provided that comprise an nous nucleotide sequence encoding a mouse ADAM6 or ortholog or homolog or nt thereof, and at least one genetic modification of a heavy chain globulin locus.

In one aspect, methods are provided for making mice that comprise a cation of an endogenous mouse immunoglobulin locus, wherein the mice comprise an ADAM6 protein or og or homolog or fragment thereof that is functional in a male mouse. Mice according to the invention are obtainable, for example, by the methods described herein.

In one aspect, methods are ed for making mice that se a genetic modification of an immunoglobulin heavy chain locus, wherein application of the methods result in male mice that comprise a modified immunoglobulin heavy chain locus (or a deletion thereof), and the male mice are capable of generating offspring by mating. In one embodiment, the male mice are capable of producing sperm that can transit from a mouse uterus through a mouse oviduct to fertilize a mouse egg.

In one aspect, methods are provided for making mice that comprise a genetic modification of an immunoglobulin heavy chain locus, n application of the methods result in male mice that comprise a modified immunoglobulin heavy chain locus (or a deletion thereof), and the male mice exhibit a reduction in fertility, and the mice comprise a genetic modiﬁcation that restores in whole or in part the reduction in fertility. In various embodiments, the reduction in fertility is terized by an inability of the sperm of the male mice to migrate from a mouse uterus through a mouse oviduct to fertilize a mouse egg. in various ments, the reduction in fertility is characterized by sperm that exhibit an in vivo migration defect. In s embodiments, the c modification that es in whole or in part the reduction in fertility is a nucleic acid sequence encoding a mouse ADAM6 gene or og or homolog or fragment thereof that is functional in a male mouse.

] In one embodiment, the genetic modification ses replacing endogenous immunoglobulin heavy chain variable loci with immunoglobulin heavy chain variable loci of another species (6.9., a non—mouse species). ln one embodiment, the genetic modification comprises insertion of orthologous immunoglobulin heavy chain variable loci into endogenous globulin heavy chain variable loci. in a specific embodiment, the species is human. In one embodiment, the genetic cation comprises deletion of an endogenous immunoglobulin heavy chain variable locus in whole or in part, wherein the deletion results in a loss of nous ADAM6 function. in a specific embodiment, the loss of endogenous ADAM6 function is associated with a ion in fertility in male mice.

In one aspect, mice are provided that comprise a cation that reduces or eliminates mouse ADAM6 expression from an endogenous ADAM6 allele such that a male mouse having the modification exhibits a reduced fertility (e.g., a highly reduced y to generate offspring by mating), or is essentially infertile, due to the reduction or ation of endogenous ADAM6 function, wherein the mice further comprise an ectopic ADAM6 sequence or homolog or ortholog or functional fragment thereof. In one , the modification that reduces or eliminates mouse ADAM6 expression is a modification (9.9., an insertion, a deletion, a replacement, etc.) in a mouse globulin locus. in one embodiment, the reduction or loss of ADAM6 function comprises an inability or substantial inability of the mouse to produce sperm that can travel from a mouse uterus through a mouse oviduct to fertilize a mouse egg. in a specific embodiment, at least about 95%, 96%, 97%, 98%, or 99% of the sperm cells produced in an ejaculate volume of the mouse are incapable of traversing through an oviduct in vivo following copulation and fertilizing a mouse ovum. in one embodiment, the reduction or loss of ADAM6 function comprises an inability to form or substantial inability to form a complex of ADAM2 and/or ADAM3 and/or ADAM6 on a surface of a sperm cell of the mouse. In one embodiment, the loss of ADAM6 function ses a substantial inability to fertilize a mouse egg by copulation with a female mouse.

In one aspect, a mouse is ed that lacks a functional endogenous ADAMS gene, and comprises a protein (or an ectopic nucleotide ce that encodes a n) that confers ADAMS functionality on the mouse. In one embodiment, the mouse is a male mouse and the functionality comprises enhanced fertility as compared with a mouse that lacks a functional endogenous ADAM6 gene.

In one embodiment, the protein is encoded by a genomic sequence d within an immunoglobulin locus in the germline of the mouse. In a specific embodiment, the immunoglobulin locus is a heavy chain locus. In another specific embodiment, the heavy chain locus comprises at least one human VH, at least one human DH and at least one human JH gene segment. In one embodiment, the ectopic protein is enCoded by a genomic sequence located within a non—immunoglobulin locus in the germline of the mouse. In one embodiment, the non-immunoglobulin locus is a transcriptionally active locus. In a specific embodiment, the transcriptionally active locus is the ROSA26 locus. In a specific embodiment, the riptionally active locus is associated with tissue-specific expression. In one embodiment, the tissue-specific expression is present in reproductive tissues. In one embodiment, the protein is encoded by a genomic sequence randomly inserted into the ne of the mouse.

In one embodiment, the mouse comprises a human or chimeric mouse or chimeric human/rat light chain (e.g., human variable, mouse or rat constant) and a chimeric human variable/mouse or rat nt heavy chain. In a specific embodiment, the mouse comprises a transgene that ses a chimeric human le/rat or mouse constant light chain gene operably linked to a transcriptionally active promoter, 9.9., a ROSA26 promoter. In a further specific embodiment, the chimeric human/mouse or rat light chain transgene comprises a rearranged human light chain variable region sequence in the germline of the mouse.

] In one embodiment, the ectopic nucleotide sequence is located within an globulin locus in the germline of the mouse. In a specific embodiment, the immunoglobulin locus is a heavy chain locus. In one embodiment, the heavy chain locus comprises at least one human VH, at least one human DH and at least one human JH gene segment. In one embodiment, the ectopic nucleotide sequence is located within a non- immunoglobulin locus in the germline of the mouse. In one embodiment, the non- immunoglobulin locus is a transcriptionally active locus. In a specific embodiment, the transcriptionally active locus is the ROSA26 locus. In one embodiment, the c nucleotide sequence is positioned ly inserted into the germline of the mouse.

In one aspect, a mouse is provided that lacks a functional endogenous ADAMS gene, wherein the mouse ses an ectopic nucleotide sequence that complements the loss of mouse ADAMS function. In one embodiment, the ectopic tide sequence confers upon the mouse an ability to produce offspring that is comparable to a corresponding ype mouse that contains a functional endogenous ADAMS gene. In one embodiment, the sequence confers upon the mouse an ability to form a complex of ADAMZ and/or ADAM3 and/or ADAMS on the surface of sperm cell of the mouse. In one ment, the sequence confers upon the mouse an ability to travel from a mouse uterus through a mouse oviduct to a mouse ovum to ize the ovum.

In one embodiment, the mouse lacking the functional endogenous ADAMS gene and comprising the ectopic nucleotide sequence produces at least about 50%, 60%, 70%, 80%, or 90% of the number of litters a wild—type mouse of the same age and strain es in a six-month time .

In one embodiment, the mouse lacking the functional endogenous ADAMS gene and comprising the ectopic nucleotide sequence produces at least about 1.5—fold, about 2-fold, about d, about 3—fold, about , about 6—fold, about 7-fold, about 8- fold, or about 10~foId or more progeny when bred over a six-month time period than a mouse of the same age and the same or similar strain that lacks the functional endogenous ADAMS gene and that lacks the ectopic nucleotide sequence that is bred over substantially the same time period and under substantially the same conditions.

In one ment, the mouse lacking the functional endogenous ADAMS 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 ng period than a mouse that lacks the functional endogenous ADAMS gene and that lacks the ectopic nucleotide sequence, and that is bred for the same period of time.

In one ment, 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 migration 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, 100-fold, 110-fold, or 120-fold or higher than a mouse that lacks the functional endogenous ADAMS gene and that lacks the ectopic nucleotide sequence.

In one ment, the mouse g the functional nous ADAMS gene and comprising the ectopic nucleotide 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 ADAM6 gene and sing 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 ADAM6 gene and that lacks the ectopic nucleotide sequence. in one , a mouse comprising in its germline a non-mouse nucleic acid sequence that encodes an immunoglobulin protein is provided, wherein the non-mouse immunoglobulin sequence comprises an insertion of a mouse ADAM6 gene or homolog or ortholog or functional fragment thereof. In one embodiment, the non-mouse immunoglobulin sequence comprises a human immunoglobulin sequence. In one embodiment, the sequence comprises a human globulin heavy chain sequence. In one embodiment, the sequence comprises a human immunoglobulin light chain sequence.

In one embodiment, the sequence comprises one or more V gene segments, one or more D gene segments, and one or more J gene segments; in one embodiment, the sequence comprises one or more V gene ts and one or more J gene segments. in one embodiment, the one or more V, D, and J gene segments, or one or more V and J gene segments, are not rearranged. In one embodiment, the one or more V, D, and J gene segments, or one or more V and J gene segments, are rearranged. ln one embodiment, following rearrangement of the one or more V, D, and J gene segments, or one or more V and J gene segments, the mouse ses in its genome at least one nucleic acid sequence 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 two nucleic acid sequences encoding a mouse ADAM6 gene or homolog or og or functional fragment thereof. in one embodiment, foliowing rearrangement the mouse comprises in its genome at least one c acid sequence ng a mouse ADAM6 gene or homolog or ortholog or functional fragment thereof. In one embodiment, the mouse comprises the ADAM6 gene or homolog or ortholog or functional fragment thereof in a B cell. In one embodiment, the mouse comprises the ADAM6 gene or homolog or ortholog or onal fragment thereof in a non-B cell.

In one aspect, mice are provided that express a human immunoglobulin heavy chain le region or onal fragment thereof from an endogenous mouse immunoglobulin heavy chain locus, wherein the mice comprise an ADAM6 activity that is functional in a male mouse.

In one embodiment, the male mice comprise a single unmodified endogenous ADAM6 ailele or ortholog of homolog or onal fragment thereof at an endogenous ADAM6 locus.

] In one embodiment, the male mice comprise an ectopic mouse ADAM6 sequence or homolog or orthoiog or functional fragment thereof that encodes a protein that confers ADAM6 function.

In one embodiment, the maIe mice comprise an ADAM6 sequence or homoiog or orthoiog or onal 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 segment.

In one embodiment, the maIe mice se an ADAM6 sequence or homolog or orthoiog or functional fragment f flanked upstream, downstream, or upstream and downstream (with respect to the ion of transcription of the ADAM6 sequence) of a nucleic acid sequence encoding an globuiin variable gene segment. In a specific embodiment, the immunoglobuiin le gene segment is a human gene segment. In one embodiment, the immunoglobuiin variabIe gene segment is a human gene segment, and the ce ng the mouse ADAM6 or ortholog or homolog or fragment f functionaI 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 on between the final V gene segment and the imate V gene segment; in one embodiment, the sequence is at a position following the final V gene segment and the first D gene segment.

In one aspect, a male mouse is provided that comprises a ctional endogenous ADAM6 gene, or a deletion of an endogenous ADAM6 gene, in its germline; wherein sperm ceIIs of the mouse are capable of transiting an t of a female mouse and fertilizing an egg. In one embodiment, the mice comprise an extrachromosomal copy of a mouse ADAM6 gene or ortholog or homolog or functional fragment f that is functional in a male mouse. In one embodiment, the mice comprise an ectopic mouse ADAM6 gene or orthoiog or homolog or functional fragment thereof that is functional in a male mouse.

In one aspect, mice are provided that comprise a c modification that reduces endogenous mouse ADAM6 function, wherein the mouse comprises at least some ADAM6 functionality provided either by an endogenous unmodified aIIele that is functional in whole or in part (e.g., a heterozygote), or by expression from an c sequence that encodes an ADAM6 or an orthoiog or homolog or functional fragment thereof that is functionaI in a male mouse.

In one embodiment, the mice comprise ADAM6 function sufficient to confer upon male mice the ability to generate offspring by mating, as compared with male mice that lack a functional ADAM6. In one embodiment, the ADAM6 function is conferred by the presence of an ectopic nucleotide sequence that encodes a mouse ADAM6 or a g or ortholog or functional fragment thereof. ADAM6 homoiogs or orthologs or fragments thereof that are functional in a male mouse include those that restore, in whole or in part, the loss of ability to generate offspring ed in a male mouse that lacks sufficient endogenous mouse ADAM6 activity, e.g., the loss in ability ed in an ADAM6 knockout mouse. In this sense ADAM6 knockout mice include mice that se an endogenous locus or fragment thereof, but that is not functional, i.e., that does not express ADAM6 (ADAM6a and/or ADAM6b) at all, or that ses ADAM6 (ADAM6a and/or ) at a level that is insufficient to support an essentially normal ability to generate ing of a wild-type male mouse. The loss of on can be due, e.g., to a modification in a structural gene of the locus (i.e., in an ADAM6a or ADAM6b coding region) or in a regulatory region of the locus (e.g., in a sequence 5’ to the ADAM6a gene, or 3’ of the ADAM6a or ADAM6b coding , wherein the sequence controls, in whole or in part, transcription of an ADAM6 gene, expression of an ADAM6 RNA, or expression of an ADAM6 protein). in s embodiments, orthologs or homologs or fragments thereof that are functional in a male mouse are those that enable a sperm of a male mouse (or a majority of sperm cells in the ejaculate of a male mouse) to transit a mouse oviduct and fertilize a mouse ovum. ] in one embodiment, male mice that express the human immunoglobulin variable region or functional fragment thereof comprise sufficient ADAM6 activity to confer upon the male mice the ability to generate offspring by mating with female mice and, in one embodiment, the male mice exhibit an ability to generate offspring when mating with female mice that is in one embodiment at least 25%, in one embodiment, at least 30%, in one embodiment at least 40%, in one embodiment at least 50%, in one embodiment at least 60%, in one ment at least 70%, in one embodiment at least 80%, in one embodiment at least 90%, and in one embodiment about the same as, that of mice with one or two endogenous unmodified ADAM6 alleles. in one embodiment male mice express sufficient ADAM6 (or an ortholog or homolog or functional fragment thereof) to enable a sperm cell from the male mice to traverse a female mouse oviduct and fertilize a mouse egg. in one embodiment, the ADAM6 functionality is red by a nucleic acid sequence that is contiguous with a mouse chromosomal sequence (e.g., the nucleic acid is randomly integrated into a mouse some; or placed at a specific location, e.g., by targeting the nucleic acid to a specific location, e.g., by site-specific recombinase-mediated (e.g., Ore-mediated) insertion or homologous recombination). In one embodiment, the ADAM6 sequence is present on a nucleic acid that is ct from a chromosome of the mouse (e.g., the ADAM6 sequence is present on an episome, i.e., extrachromosomally, e.g., in an expression construct, a vector, a YAC, a transchromosome, etc.) In one aspect, genetically modified mice and cells are provided that comprise a modification of an nous immunoglobulin heavy chain locus, wherein the mice express at least a portion of an immunoglobulin heavy chain ce, e.g., at least a portion of a human sequence, wherein the mice comprise an ADAM6 activity that is functional in a male mouse. In one embodiment, the cation reduces or eradicates ADAM6 activity of the mouse. In one embodiment, the mouse is modified such that both alleles that encode ADAM6 activity are either absent or express an ADAM6 that does not substantially function to support normal mating in a male mouse. In one embodiment, the mouse further comprises an ectopic nucleic acid sequence encoding a mouse ADAM6 or ortholog or homolog or functional fragment thereof.

In one aspect, genetically ed mice and cells are provided that comprise a modification of an endogenous immunoglobulin heavy chain locus, wherein the modification reduces or eliminates ADAM6 activity expressed from an ADAM6 sequence of the locus, and wherein the mice comprise an ADAM6 protein or ortholog or homolog or functional fragment thereof. In various embodiments, the ADAM6 protein or fragment thereof is encoded by an ectopic ADAM6 sequence. In various embodiments, the ADAM6 protein or nt thereof is expressed from an endogenous ADAM6 allele. in various embodiments, the mouse comprises a first immunoglobulin heavy chain allele comprises a first modification that reduces or eliminates sion of a functional ADAM6 from the first immunoglobulin heavy chain allele, and the mouse ses a second immunoglobulin heavy chain allele that ses a second modification that does not substantially reduce or does not ate expression of a functional ADAM6 from the second immunoglobulin heavy chain allele. in one embodiment, the second modification is located 3’ (with respect to the transcriptional directionality of the mouse V gene segment) of a final mouse V gene segment and d 5’ (with respect to the transcriptional directionality of the constant sequence) of a mouse (or chimeric human/mouse) immunoglobulin heavy chain constant gene or fragment thereof (9.9., a nucleic acid sequence encoding a human and/or mouse: CH1 and/or hinge and/or CH2 and/or CH3).

In one embodiment, the modification is at a first immunoglobulin heavy chain allele at a first locus that encodes a first ADAM6 allele, and the ADAM6 on results from sion of an endogenous ADAM6 at a second immunoglobulin heavy chain allele at a second locus that encodes a onal ADAM6, wherein the second immunoglobulin heavy chain allele comprises at least one modification of a V, D, and/or J gene segment. in a specific embodiment, the at least one modification of the V, D, and or J gene segment is a deletion, a replacement with a human V, D, and/or J gene segment, a ement with a camelid V, D, and/or J gene segment, a replacement with a humanized or camelized V, D, and/or J gene segment, a replacement of a heavy chain sequence with a tight chain sequence, and a combination thereof. In one embodiment, the at least one modification is the deletion of one or more heavy chain V, D, and/or J gene segments and a ement with one or more light chain V and/or J gene segments (6.9., a human light chain V and/or J gene segment) at the heavy chain locus.

In one embodiment, the modification is at a first immunoglobulin heavy chain allele at a first locus and a second immunoglobulin heavy chain allele at a second locus, and the ADAM6 function results from expression of an ectopic ADAM6 at a non- immunoglobulin locus in the germline of the mouse. in a specific embodiment, the non- immunoglobulin locus is the ROSAZS locus. in a specific embodiment, the non- immunoglobulin locus is transcriptionally active in reproductive tissue. in one aspect, a mouse comprising a heterozygous or a homozygous knockout of ADAM6 is provided. In one embodiment, the mouse further comprises a ed immunoglobulin sequence that is a human or a zed immunoglobulin sequence, or a camelid or zed human or mouse immunoglobulin sequence. ln one embodiment, the modified immunoglobulin ce is present at the nous mouse heavy chain immunoglobulin locus. in one embodiment, the modified immunoglobulin sequence comprises a human heavy chain variable gene sequence at an endogenous mouse immunoglobulin heavy chain locus. in one embodiment, the human heavy chain variable gene ce es an endogenous mouse heavy chain variable gene sequence at the endogenous mouse immunoglobulin heavy chain locus.

In one aspect, a mouse incapable of expressing a functional nous mouse ADAM6 from an endogenous mouse ADAM6 locus is provided. In one ment, the mouse comprises an ectopic nucleic acid sequence that encodes an ADAM6, or functional fragment thereof, that is functional in the mouse. In a specific embodiment, the ectopic nucleic acid sequence encodes a protein that rescues a loss in the ability to generate offspring exhibited by a male mouse that is homozygous for an ADAM6 knockout. in a Specific embodiment, the ectopic nucleic acid sequence encodes a mouse ADAM6 protein. ln one aspect, a mouse is provided that lacks a functional endogenous ADAM6 locus, and that comprises an ectopic nucleic acid sequence that confers upon the mouse ADAM6 function. in one embodiment, the nucleic acid sequence comprises an endogenous mouse ADAM6 sequence or functional fragment thereof. In one embodiment, the endogenous mouse ADAM6 sequence ses — and ADAMBb-encoding sequence located in a wild-type mouse between the 3’—most mouse immunoglobulin heavy chain V gene t (VH) and the 5’-most mouse immunoglobulin heavy chain D gene t (DH). in one embodiment, the nucleic acid sequence comprises a sequence encoding mouse ADAM6a or functional fragment f and/or a sequence encoding mouse ADAMSb or functional fragment thereof, wherein the ADAM6a and/or ADAM6b or functional nt(s) thereof is operably linked to a promoter. In one embodiment, the promoter is a human promoter. In one embodiment, the promoter is the mouse ADAMS promoter. In a specific embodiment, the ADAMS er ses sequence d between the first codon of the first ADAMS gene closest to the mouse 5’—most DH gene segment and the recombination signal ce of the 5’-most DH gene segment, wherein ’ is indicated with t to direction of transcription of the mouse immunoglobulin genes.

In one embodiment, the promoter is a viral promoter. In a specific embodiment, the viral promoter is a galovirus (CMV) promoter. In one embodiment, the promoter is a ubiquitin promoter.

In one embodiment, the promoter is an inducible promoter. In one embodiment, the inducible promoter regulates expression in non-reproductive tissues. In one ment, the inducible promoter regulates expression in reproductive tissues. In a specific ment, the expression of the mouse ADAM6a and/or ADAM6b sequences or functional fragments(s) thereof is developmentally regulated by the inducible promoter in reproductive tissues. in one embodiment, the mouse ADAM6a and/or ADAM6b are selected from the ADAMSa of SEQ lD NO:1 and/or ADAM6b of sequence SEQ lD N012. in one embodiment, the mouse ADAMS promoter is a er of SEQ ID NO:3. in a specific embodiment, the mouse ADAMS promoter 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 ADAM6a and extending to the end of SEQ lD NO:3 upstream of the ADAMS coding region. in r specific embodiment, the ADAMS promoter 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 upstream of the start codon of ADAMSa.

In one embodiment, the nucleic acid ce comprises SEQ ID NO:3 or a fragment thereof that when placed into a mouse that is infertile or that has low fertility clue to a lack of ADAMS, es fertility or restores fertility to about a wild—type fertility. in one embodiment, SEQ lD NO:3 or a fragment thereof confers upon a male mouse the ability to produce a sperm cell that is capable of sing a female mouse t in order to fertilize a mouse egg. in one aspect, a mouse is provided that comprises a deletion of an endogenous nucleotide sequence that encodes an ADAMS protein, a replacement of an endogenous mouse VH gene segment with a human VH gene segment, and an ectopic nucleotide sequence that encodes a mouse ADAMS protein or ortholog or homolog or fragment thereof that is functional in a male mouse.

In one embodiment, the mouse comprises an globulin heavy chain Iocus that comprises a deletion of an endogenous immunogiobulin locus nucleotide sequence that comprises an endogenous ADAM6 gene, comprises a nucleotide sequence encoding one or more human immunoglobulin gene segments, and wherein the ectopic nucleotide sequence encoding the mouse ADAMS protein is within or directly adjacent to the nucleotide sequence encoding the one or more human immunoglobuiin gene In one embodiment, the mouse comprises a replacement of all or substantially all endogenous VH gene segments with a nucleotide sequence encoding one or more human VH gene segments, and the ectopic nucleotide sequence encoding the mouse ADAM6 protein is within, or directly adjacent to, the nucleotide sequence encoding the one or more human VH gene segments. In one embodiment, the mouse further comprises a replacement of one or more endogenous DH gene ts with one or more human DH gene segments at the endogenous DH gene locus. In one embodiment, the mouse further ses a replacement of one or more nous JH gene segments with one or more human JH gene segments at the endogenous JH gene locus. In one embodiment, the mouse ses a replacement of all or substantially aII endogenous VH, DH, and JH gene segments and a replacement at the endogenous VH, DH, and JH gene loci with human VH, DH, and JH gene segments, wherein the mouse comprises an c ce encoding a mouse ADAM6 protein. In a specific embodiment, the ectopic sequence encoding the mouse ADAMS protein is placed between the penuItimate 3’—most VH gene segment of the human VH gene segments present, and the ultimate 3’ VH gene segment of the human VH gene segments present. In a specific embodiment, the mouse ses a deletion of all or ntially all mouse VH gene ts, and a replacement with all or substantially all human VH gene segments, and the ectopic nucleotide sequence encoding the mouse ADAM6 protein is placed downstream of human gene segment VH1-2 and upstream of human gene segment VH6-i.

] In a specific embodiment, the mouse comprises a replacement of aII or substantiaIIy aII endogenous VH gene segments with a nucleotide sequence encoding one or more human VH gene segments, and the ectopic nucleotide sequence encoding the mouse ADAM6 protein is within, or directly adjacent to, the nucleotide sequence encoding the one or more human VH gene ts.

In one embodiment, the ectopic nucleotide sequence that encodes the mouse ADAMS protein is t on a transgene in the genome of the mouse. In one embodiment, the ectopic nucleotide sequence that encodes the mouse ADAMG protein is present extrachromosomally in the mouse.

] In one aspect, a mouse is provided that comprises a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse expresses a B cell that ses a rearranged immunoglobulin sequence operably linked to a 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 ADAM6 or ortholog or g or fragment thereof that is functional in a male mouse. in one embodiment, the rearranged 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 sequence; a human or mouse light chain V and/or J sequence. In one embodiment, the heavy chain constant region gene sequence comprises a human or a mouse heavy chain sequence ed from the group consisting of a CH1, a hinge, a CH2, a CH3, and a combination thereof.

In one aspect, a genetically ed mouse is provided, wherein the mouse comprises a functionally silenced immunoglobulin light chain gene, and further comprises a replacement of one or more endogenous globulin heavy chain variable region gene segments with one or more human globulin heavy chain variable region gene segments, n the mouse lacks a functional endogenous ADAM6 locus, and wherein the mouse comprises an ectopic nucleotide ce that expresses a mouse ADAM6 protein or an ortholog or homolog or fragment thereof that is functional in a male mouse. ] in one aspect, a mouse is provided that lacks a functional endogenous mouse ADAM6 locus or sequence and that comprises an ectopic nucleotide sequence encoding a mouse ADAM6 locus or functional fragment of a mouse ADAM6 locus or sequence, wherein the mouse is capable of mating with a mouse of the opposite sex to produce a progeny that ses the ectopic ADAM6 locus or sequence. in one embodiment, the mouse is male. in one embodiment, the mouse is female. in one aspect, a genetically ed mouse is provided, wherein the mouse comprises a human immunoglobulin heavy chain variable region gene segment at an endogenous mouse immunoglobulin heavy chain variable region gene locus, the mouse lacks an nous functional ADAM6 sequence at the endogenous mouse immunoglobulin heavy chain variable region gene locus, and wherein the mouse comprises an c nucleotide sequence that expresses a mouse ADAM6 protein or an ortholog or homolog or fragment thereof that is functional in a male mouse. in one embodiment, the ectopic nucleotide sequence that expresses the mouse ADAM6 protein is extrachromosomal. in one embodiment, the c nucleotide sequence that expresses the mouse ADAM6 protein is integrated at one or more loci in a genome of the mouse. in a specific embodiment, the one or more loci include an immunoglobulin locus. in one aspect, a mouse is provided that expresses an immunoglobulin heavy chain ce from a modified endogenous mouse immunoglobulin heavy chain locus, wherein the heavy chain is derived from a human V gene segment, a D gene segment, and a J gene segment, wherein the mouse comprises an ADAM6 activity that is functional in the mouse.

In one embodiment, the mouse ses a plurality of human V gene segments, a plurality of D gene segments, and a plurality of J gene segments. In one embodiment, the D gene segments are human D gene segments. In one embodiment, the J gene ts are human J gene segments. In one embodiment, the mouse r comprises a zed heavy chain constant region sequence, wherein the zation comprises replacement of a sequence selected from a CH1, hinge, CH2, CH3, and a combination thereof. in a specific embodiment, the heavy chain is derived from a human V gene segment, a human D gene segment, a human J gene segment, a human CH1 sequence, a human or mouse hinge sequence, a mouse CH2 sequence, and a mouse CH3 sequence. In another specific embodiment, the mouse further ses a human light chain nt sequence.

In one embodiment, the D gene t is flanked 5’ (with t to transcriptional direction of the D gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse. in one embodiment, the ADAM6 activity that is functional in the mouse results from expression of a nucleotide sequence located 5’ of the 5’-most D gene segment and 3’ of the 3’-most V gene segment (with respect to the direction of ription of the V gene segment) of the modified endogenous mouse heavy chain immunoglobulin locus. in one embodiment, the ADAM6 activity that is functional in the mouse results from expression of a nucleotide sequence located between two human V gene segments in the modified endogenous mouse heavy chain immunoglobulin locus. in one embodiment, the two human V gene segments are a human VH1-2 gene segment and a VHS-1 gene segment. ] in one embodiment, the nucleotide sequence ses a sequence selected from a mouse ADAM6b ce or functional fragment thereof, a mouse ADAM6a sequence or functional fragment thereof, and a combination thereof.

In one embodiment, the nucleotide sequence between the two human V gene segments is placed in opposite transcription orientation with respect to the human V gene segments. in a specific embodiment, tide 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 one embodiment, the mouse comprises a replacement of a human ADAM6 pseudogene sequence between human V gene segments VH1—2 and VH6-1 with a mouse ADAM6 sequence or a functional fragment thereof. ] in one embodiment, the sequence encoding the ADAM6 activity that is functional in the mouse is a mouse ADAM6 sequence or functional fragment thereof. in one embodiment, the mouse comprises an endogenous mouse DFL16.1 gene segment (9.9., in a mouse heterozygous for the modified endogenous mouse immunoglobulin heavy chain locus), or a human DH1-1 gene segment. in one embodiment, the D gene segment of the globulin heavy chain expressed by the mouse is derived from an endogenous mouse DFL16.1 gene segment or a human DH1-1 gene segment.

In one aspect, a mouse is provided that comprises a nucleic acid sequence ng a mouse ADAM6 (or homolog or ortholog or functional fragment thereof) in a DNA-bearing cell of non-rearranged B cell lineage, but does not comprise the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment f) in a B cell that comprise rearranged immunoglobulin loci, n the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment thereof) occurs in the genome at a position that is different from a position in which a mouse ADAM6 gene appears in a wild~type mouse. in one embodiment, the nucleic acid sequence ng the mouse ADAM6 (or homolog or ortholog or functional nt thereof) is present in all or substantially all DNA-bearing cells that are not of rearranged B cell lineage; in one ment, the nucleic acid ce is present in germline cells of the mouse, but not in a some of a rearranged B cell. in one aspect, a mouse is provided that comprises a nucleic acid sequence encoding a mouse ADAM6 (or homolog or og or functional fragment f) in all or substantially all DNA—bearing cells, including B cells that comprise rearranged immunoglobulin loci, wherein the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment f) occurs in the genome at a position that is different from a position in which a mouse ADAM6 gene appears in a wild-type mouse. in one embodiment, the nucleic acid sequence encoding the mouse ADAM6 (or homolog or og or functional fragment thereof) is on a nucleic acid that is contiguous with the rearranged immunoglobulin locus. in one ment, the nucleic acid that is contiguous with the rearranged immunoglobulin locus is a chromosome. in one embodiment, the some is a chromosome that is found in a wild-type mouse and the chromosome comprises a modification of a mouse immunoglobulin locus. in one aspect, a genetically modified mouse is provided, wherein the mouse comprises a B cell that comprises in its genome an ADAM6 sequence or ortholog or homolog thereof. in one embodiment, the ADAM6 sequence or ortholog or homolog thereof is at an immunoglobulin heavy chain locus. in one embodiment, the ADAM6 sequence or ortholog or homolog thereof is at a locus that is not an immunoglobulin locus.

In one embodiment, the ADAM6 sequence is on a ene driven by a heterologous promoter. In a specific embodiment, the heterologous promoter is a non-immunoglobulin promoter. In a specific embodiment, B cell expresses an ADAM6 protein or ortholog or homolog thereof.

In one embodiment, 90% or more of the B cells of the mouse comprise a gene encoding an ADAM6 protein or an ortholog thereof or a homolog thereof or a fragment thereof that is functional in the mouse. In a specific embodiment, the mouse is a male mouse.

In one embodiment, the B cell genome comprises a first allele and a second allele comprising the ADAM6 sequence or og or homoIog thereof. In one embodiment, the B cell genome comprises a first allele but not a second allele comprising the ADAM6 sequence or ortholog or homolog f.

In one aspect, a mouse is provided that comprises a modification at one or more endogenous ADAM6 alleles.

In one embodiment, the modification renders the mouse incapable of expressing a functional ADAM6 protein from at least one of the one or more endogenous ADAM6 s. In a ic embodiment, the mouse is incapable of expressing a functional ADAM6 protein from each of the nous ADAM6 alleles.

In one embodiment, the mice are incapable of expressing a functional ADAM6 protein from each endogenous ADAM6 allele, and the mice comprise an ectopic ADAM6 sequence.

In one embodiment, the mice are ble of expressing a functional ADAM6 protein from each endogenous ADAM6 allele, and the mice comprise an ectopic ADAM6 sequence located within 1, 2, 3, 4, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, or 120 or more kb upstream (with respect to the direction of transcription of the mouse heavy chain locus) of a mouse immunoglobulin heavy chain constant region sequence. In a specific embodiment, the ectopic ADAM6 sequence is at the endogenous globulin heavy chain locus (e.g., in an intergenic V-D region, between two V gene ts, between a V and a D gene t, between a D and a J gene segment, etc). In a specific embodiment, the ectopic ADAM6 sequence is located within a 90 to 100 kb intergenic ce between the final mouse V gene segment and the first mouse D gene segment.

In another specific embodiment, the endogenous 90 to 100 kb intergenic V—D sequence is removed, and the c ADAM6 sequence is placed n the final V and the first D gene segment.

In one aspect, an infertile male mouse is provided, wherein the mouse comprises a on of two or more endogenous ADAM6 alleles. In one aspect, a female mouse is provided that is a r of a male infertility trait, wherein the female mouse ses in its germline a nonfunctional ADAM6 allele or a ut of an endogenous ADAM6 allele. in one aspect, a mouse that lacks an endogenous immunoglobulin heavy chain V, D, and J gene segment is provided, wherein a majority of the B cells of the mouse comprise an ADAM6 sequence or ortholog or homolog thereof. in one embodiment, the mouse lacks endogenous immunoglobulin heavy chain gene segments selected from two or more V gene ts, two or more D gene segments, two or more J gene segments, and a combination thereof. In one embodiment, the mouse lacks immunoglobulin heavy chain gene segments selected from at least one and up to 89 V gene segments, at least one and up to 13 D gene segments, at least one and up to four J gene segments, and a combination thereof. in one embodiment, the mouse lacks a genomic DNA fragment from chromosome 12 sing about three megabases of the endogenous immunoglobulin heavy chain locus. In a specific embodiment, the mouse lacks all onal endogenous heavy chain V, D, and J gene segments. In a specific embodiment, the mouse lacks 89 VH gene segments, 13 DH gene segments and four JH gene segments. in one aspect, a mouse is provided, wherein the mouse has a genome in the ne comprising a modification of an immunoglobulin heavy chain locus, wherein the modification to the immunoglobulin heavy chain locus comprises the replacement of one or more mouse immunoglobulin variable region sequences with one or more non-mouse immunoglobulin variable region sequences, and wherein the mouse comprises a nucleic acid sequence ng a mouse ADAMS n. in a preferred embodiment, the DH and JH sequences and at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 VH sequences of the immunoglobulin heavy chain locus are replaced by non-mouse globulin variable region sequences. In a further preferred embodiment, the DH, JH, and all VH sequences of the immunoglobulin heavy chain locus are replaced by non-mouse immunoglobulin variable region sequences. The non-mouse immunoglobulin variable region sequences can be non-rearranged. in a preferred embodiment, the use immunoglobulin variable region sequences se complete non-rearranged DH and JH regions and at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 non— rearranged VH sequences of the non—mouse species. in a further preferred embodiment, the non-mouse immunoglobulin variable region sequences comprise the te le region, including all VH, DH, and JH regions, of the non-mouse species. The non-mouse species can be Homo sapiens and the non-mouse immunoglobulin variable region sequences can be human sequences.

] In one aspect, a mouse that expresses an antibody that comprises at least one human variable domain/non—human constant domain immunoglobulin polypeptide is provided, wherein the mouse expresses a mouse ADAMG protein or ortholog or homolog thereof from a locus other than an immunoglobulin locus.

] In one embodiment, the ADAMS protein or ortholog or homolog thereof is expressed in a B cell of the mouse, wherein the B cell comprises a rearranged immunoglobulin ce that comprises a human variable sequence and a non-human constant sequence.

In one embodiment, the non-human constant sequence is a rodent sequence.

In one ment, the rodent is selected from a mouse, a rat, and a hamster. in one aspect, a method is provided for making an infertile male mouse, sing rendering an endogenous ADAM6 allele of a donor ES cell nonfunctional (or knocking out said allele), introducing the donor ES cell into a host embryo, gestating the host embryo in a ate mother, and ng the ate mother to give birth to progeny derived in whole or in part from the donor ES cell. In one embodiment, the method further comprises breeding progeny to obtain an ile male mouse. in one aspect, a method is provided for making a mouse with a genetic modification of interest, wherein the mouse is infertile, the method comprising the steps of (a) making a genetic modification of interest in a genome; (b) modifying the genome to knockout an endogenous ADAM6 allele, or render an endogenous ADAMS allele nonfunctional; and, (c) employing the genome in making a mouse. in various embodiments, the genome is from an ES cell or used in a r er experiment.

In one aspect, a mouse made using a targeting vector, nucleotide construct, or cell as bed herein is provided.

In one aspect, a progeny of a mating of a mouse as described herein with a second mouse that is a wild—type mouse or cally modified is provided. in one aspect, a method for maintaining a mouse strain is provided, wherein the mouse strain comprises a replacement of a mouse immunoglobulin heavy chain sequence with one or more heterologous immunoglobulin heavy chain sequences. In one ment, the one or more heterologous immunoglobulin heavy chain sequences are human immunoglobulin heavy chain sequences.

In one ment, the mouse strain comprises a deletion of one or more mouse VH, DH, and/or JH gene segments. in one embodiment, the mouse further comprises one or more human VH gene segments, one or more human DH gene segments, and/or one or more human JH gene segments. In one embodiment, the mouse comprises at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 human VH segments, at least 27 human DH gene segments, and at least six JH gene segments. in a specific embodiment, the mouse comprises at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 human VH segments, the at least 27 human DH gene segments, and the at least six JH gene segments are operably linked to a constant region gene. In one embodiment, the constant region gene is a mouse constant region gene. In one embodiment, the constant region gene comprises a mouse constant region gene sequence selected from a CH1, a hinge, a CH2, a CH3, and/or a CH4 or a combination thereof.

In one embodiment, the method comprises generating a male mouse heterozygous for the replacement of the mouse immunoglobulin heavy chain sequence, and breeding the heterozygous male mouse with a wild-type female mouse or a female mouse that is homozygous or heterozygous for the human heavy chain sequence. In one ment, the method comprises ining the strain by repeatedly ng heterozygous males with females that are wild type or homozygous or heterozygous for the human heavy chain sequence.

In one embodiment, the method ses obtaining cells from male or female mice homozygous or heterozygous for the human heavy chain sequence, and employing those cells as donor cells or nuclei therefrom as donor , and using the cells or nuclei to make genetically modified animals using host cells and/or gestating the cells and/or nuclei in surrogate mothers.

In one embodiment, only male mice that are heterozygous for the replacement at the heavy chain locus are bred to female mice. In a specific embodiment, the female mice are gous, heterozygous, or wild type with t to a replaced heavy chain locus. in one embodiment, the mouse further comprises a replacement of k and/or K light chain variable sequences at an nous immunoglobulin light chain locus with heterologous immunoglobulin light chain sequences. In one embodiment, the heterologous immunoglobulin light chain sequences are human immunoglobulin 7» and/or K light chain variable sequences. in one embodiment, the mouse further comprises a transgene at a locus other than an endogenous immunoglobulin locus, wherein the transgene comprises a sequence encoding a rearranged or unrearranged heterologous 7» or K light chain ce (e.g., unrearranged VL and unrearranged JL, or nged VJ) operably linked (for unrearranged) or fused (for rearranged) to an immunoglobulin light chain constant region sequence. In one embodiment, the heterologous k or K light chain sequence is human. In one ment, the constant region sequence is selected from rodent, human, and non- human primate. In one embodiment, the nt region sequence is selected from mouse, rat, and hamster. In one embodiment, the transgene comprises a non- immunoglobulin promoter that drives expression of the light chain sequences. In a specific embodiment, the promoter is a transcriptionally active promoter. In a specific embodiment, the promoter is a ROSAZB promoter. ] in one aspect, a nucleic acid construct is provided, comprising an am gy arm and a downstream homology arm, wherein the upstream homology arm comprises a sequence that is identical or substantially identical to a human immunoglobulin heavy chain variable region sequence, the downstream homology arm comprises a sequence that is identical or ntially cal to a human or mouse immunoglobulin le region sequence, and disposed between the am and downstream homology arms is a sequence that comprises a nucleotide sequence encoding a mouse ADAM6 protein. in a specific embodiment, the ce encoding the mouse ADAM6 gene is ly linked with a mouse promoter with which the mouse ADAM6 is linked in a wild type mouse. in one aspect, a targeting vector is provided, comprising (a) a nucleotide sequence that is identical or substantially identical to a human variable region gene segment nucleotide sequence; and, (b) a nucleotide sequence encoding a mouse ADAM6 or ortholog or homolog or fragment thereof that is functional in a mouse.

In one embodiment, the targeting vector further comprises a promoter ly linked to the ce encoding the mouse ADAM6. in a specific embodiment, the er is a mouse ADAM6 promoter.

In one , a nucleotide construct for modifying a mouse immunoglobulin heavy chain variable locus is provided, wherein the construct ses at least one site- ic recombinase recognition site and a sequence encoding an ADAM6 protein or ortholog or g or fragment thereof that is functional in a mouse.

In one aspect, mouse cells and mouse embryos are provided, including but not limited to ES cells, pluripotent cells, and induced pluripotent cells, that comprise genetic modifications as described herein, Cells that are XX and cells that are XY are ed.

Cells that comprise a nucleus containing a modification as described herein are also provided, e.g., a modification introduced into a cell by pronuclear injection. Cells, embryos, and mice that comprise a virally introduced ADAM6 gene are also provided, e.g., cells, embryos, and mice comprising a transduction construct comprising an ADAM6 gene that is functional in the mouse. in one aspect, a genetically modified mouse cell is provided, wherein the cell lacks a functional endogenous mouse ADAM6 locus, and the cell comprises an ectopic nucleotide sequence that encodes a mouse ADAM6 protein or functional fragment thereof. in one embodiment, the cell further comprises a modification of an endogenous immunoglobulin heavy chain variable gene sequence. in a specific embodiment, the modification of the endogenous immunoglobulin heavy chain variable gene sequence comprises a deletion selected from a deletion of a mouse VH gene segment, a deletion of a mouse DH gene segment, a deletion of a mouse JH gene t, and a combination thereof. in a specific embodiment, the mouse comprises a replacement of one or more mouse immunoglobulin VH, DH, and/or JH sequences with a human immunoglobulin sequence. In a ic embodiment, the human immunoglobulin sequence is selected from a human VH, a human VL, a human DH, a human JH, a human JL, and a combination thereof.

In one embodiment, the cell is a totipotent cell, a pluripotent cell, or an induced pluripotent cell. In a specific embodiment, the cell is a mouse ES cell.

In one aspect, a mouse B cell is provided, wherein the mouse B cell comprises a rearranged immunoglobulin heavy chain gene, wherein the B cell comprises on a chromosome of the B cell a nucleic acid sequence encoding an ADAM6 n or ortholog 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. 4] In one embodiment, the c acid sequence is on a c acid molecule (e.g., a B cell chromosome) that is contiguous with the rearranged mouse immunoglobulin heavy chain locus.

In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (e.g., a B celt chromosome) that is distinct from the c acid molecule that comprises the rearranged mouse immunoglobulin heavy chain locus.

In one embodiment, the mouse B cell comprises a rearranged non-mouse immunoglobulin variable gene sequence operably linked to a mouse or human immunoglobulin nt region gene, wherein the B cell comprises a nucleic acid sequence that encodes an ADAM6 protein or ortholog or g or fragment thereof that is functional in a male mouse. 7] In one , a somatic mouse cell is provided, comprising a chromosome that comprises a modified immunoglobulin heavy chain locus, and a nucleic acid sequence encoding a mouse ADAM6 or ortholog or g or fragment thereof that is functional in a male mouse. in one embodiment, the nucleic acid sequence is on the same chromosome as the modified globulin heavy chain locus. In one embodiment, the nucleic acid is on a different chromosome than the modified immunoglobulin heavy chain locus. in one embodiment, the somatic cell comprises a single copy of the nucleic acid sequence. In one embodiment, the somatic cell comprises at least two copies of the nucleic acid sequence. ln a specific embodiment, the c cell is a B cell. In a specific embodiment, the cell is a germ cell. ln a specific embodiment, the cell is a stem cell.

In one aspect, a mouse germ cell is provided, comprising a c acid sequence encoding a mouse ADAM6 (or homolog or ortholog or functional fragment f) on a chromosome of the germ cell, wherein the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment thereof) is at a position in the chromosome that is ent from a position in a chromosome of a wild-type mouse germ cell. in one embodiment, the nucleic acid sequence is at a mouse immunoglobulin locus. In one embodiment, the nucleic acid sequence is on the same chromosome of the germ cell as a mouse immunoglobulin locus. In one ment, the nucleic acid sequence is on a different chromosome of the germ cell than the mouse immunoglobulin locus. In one embodiment, the mouse immunoglobulin locus comprises a replacement of at least one mouse immunoglobulin sequence with at least one non—mouse immunoglobulin sequence. In a ic embodiment, the at least one non—mouse immunoglobulin sequence is a human immunoglobulin sequence.

In one aspect, a pluripotent, induced pluripotent, or tent cell derived from a mouse as described herein is ed. ln a specific embodiment, the cell is a mouse embryonic stem (ES) cell.

In one , a tissue derived from a mouse as described herein is provided. in one ment, the tissue is derived from , lymph node or bone marrow of a mouse as described herein. 1] ln one aspect, a nucleus derived from a mouse as described herein is provided.

In one embodiment, the nucleus is from a diploid cell that is not a B cell. in one aspect, a nucleotide sequence encoding an immunoglobulin variable region made in a mouse as described herein is provided. in one aspect, an immunoglobulin heavy chain or immunoglobulin light chain le region amino acid sequence of an antibody made in a mouse as described herein is provided.

In one aspect, an immunoglobulin heavy chain or immunoglobulin light chain variable region nucleotide ce encoding a variable region of an antibody made in a mouse as described herein is provided.

In one aspect, an antibody or antigen-binding fragment thereof (e.g., Fab, F(ab)2, scFv) made in a mouse as bed herein is provided. in one aspect, a method for making a genetically modified mouse is provided, comprising replacing one or more globulin heavy chain gene segments upstream (with respect to transcription of the immunoglobulin heavy chain gene segments) of an endogenous ADAM6 locus of the mouse with one or more human immunoglobulin heavy chain gene segments, and replacing one or more immunoglobulin gene segments downstream (with respect to transcription of the immunoglobulin heavy chain gene segments) of the ADAM6 locus of the mouse with one or more human immunoglobulin heavy chain or light chain gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments upstream of an endogenous ADAMS locus of the mouse include V gene segments. In one embodiment, the human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments upstream of an endogenous ADAMS locus of the mouse include V and D gene segments. In one embodiment, the one or more human globulin gene segments replacing one or more endogenous immunoglobulin gene segments downstream of an nous ADAMS locus of the mouse include J gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments ream of an endogenous ADAMS locus of the mouse include D and J gene segments. In one embodiment, the one or more human globulin gene segments replacing one or more endogenous globulin gene segments downstream of an endogenous ADAMS locus of the mouse include V, D and J gene segments.

In one ment, the one or more immunoglobulin heavy chain gene segments upstream and/or downstream of the ADAMS gene are ed in a pluripotent, induced pluripotent, or totipotent cell to form a genetically modified progenitor cell; the genetically modified progenitor cell is uced into a host; and, the host comprising the genetically modified itor cell is gestated to form a mouse comprising a genome d from the genetically modified progenitor cell. In one embodiment, the host is an embryo. In a specific embodiment, the host is selected from a mouse pre—morula (e. g., 8- or 4-cell stage), a tetraploid , an aggregate of embryonic cells, or a cyst.

In one aspect, a method for making a genetically modified mouse is provided, comprising replacing a mouse nucleotide sequence that comprises a mouse immunoglobulin gene t and a mouse ADAMS (or ortholog or homolog or fragment thereof functional in a male mouse) nucleotide sequence with a sequence comprising a human immunoglobulin gene segment to form a first chimeric locus, then inserting a sequence comprising a mouse ADAMS-encoding sequence (or a sequence encoding an ortholog or homolog or functional fragment thereof) into the sequence comprising the human immunoglobulin gene segment to form a second chimeric locus.

In one embodiment, the second chimeric locus comprises a human immunoglobulin heavy chain variable (VH) gene segment. In one embodiment, the second chimeric locus comprises a human immunoglobulin light chain variable (VL) gene segment.

In a specific embodiment, the second chimeric locus comprises a human VH gene segment or a human VL gene segment operably linked to a human DH gene segment and a human JH gene t. In a further specific embodiment, the second chimeric locus is operably linked to a third chimeric locus that comprises a human CH1 sequence, or a human CH1 and human hinge sequence, fused with a mouse CH2 + CH3 sequence. in one aspect, use of a mouse that comprises an ectopic tide sequence comprising a mouse ADAMS locus or sequence to make a fertile male mouse is provided, wherein the use comprises mating the mouse comprising the ectopic nucleotide sequence that comprises the mouse ADAM6 locus or sequence to a mouse that lacks a functional endogenous mouse ADAM6 locus or sequence, and obtaining a progeny that is a female capable of producing progeny having the ectopic ADAM6 locus or sequence or that is a male that comprises the ectopic ADAMS locus or sequence, and the male exhibits a fertility that is approximately the same as a fertility exhibited by a wild-type male mouse. in one aspect, use of a mouse as described herein to make an immunoglobulin variable region nucleotide sequence is provided.

In one aspect, use of a mouse as described herein to make a fully human Fab or a fully human F(ab)2 is provided.

In one , use of a mouse as described herein to make an immortalized cell line is ed. in one aspect, use of a mouse as described herein to make a hybridoma or quadroma is provided. 4] in one aspect, use of a mouse as described herein to make a phage library containing human heavy chain variable regions and human light chain variable regions is provided. in one aspect, use of a mouse as described herein to generate a le region sequence for making a human antibody is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) isolating a lymphocyte from the immunized mouse of (a), (c) exposing the lymphocyte to one or more labeled antibodies, (d) identifying a lymphocyte that is capable of binding to the antigen of interest, and (e) amplifying one or more variable region c acid ce from the cyte thereby generating a variable region sequence. in one embodiment, the cyte is derived from the spleen of the mouse. in one embodiment, the lymphocyte is derived from a lymph node of the mouse. In one embodiment, the lymphocyte is derived from the bone marrow of the mouse. 7] in one embodiment, the labeled antibody is a ﬂuorophore-conjugated antibody. in one embodiment, the one or more phore—conjugated antibodies are selected from an lgM, an lgG, and/or a combination thereof. in one embodiment, the lymphocyte is a B cell. ln one embodiment, the one or more variable region nucleic acid ce ses a heavy chain le region sequence. In one ment, the one or more variable region nucleic acid sequence comprises a light chain variable region sequence. in a specific embodiment, the light chain variable region sequence is an immunoglobulin K light chain variable region sequence. in one embodiment, the one or more variable region nucleic acid sequence comprises a heavy chain and a K light chain variable region sequence.

In one embodiment, use of a mouse as described herein to generate a heavy and a K light chain variable region ce for making a human antibody is provided, sing (a) zing a mouse as described herein with an antigen of interest, (b) isolating the spleen from the immunized mouse of (a), (c) exposing B lymphocytes from the spleen to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the n of interest, and (e) amplifying a heavy chain variable region nucleic acid sequence and a K light chain variable region nucleic acid sequence from the B lymphocyte thereby generating the heavy chain and K light chain variable region sequences.

In one ment, use of a mouse as described herein to generate a heavy and a K light chain variable region sequence for making a human antibody is provided, sing (a) zing a mouse as described herein with an antigen of st, (b) ing one or more lymph nodes from the immunized mouse of (a), (c) exposing B lymphocytes from the one or more lymph nodes to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of g to the antigen of interest, and (e) amplifying a heavy chain variable region nucleic acid sequence and a K light chain variable region nucleic acid sequence from the B lymphocyte thereby generating the heavy chain and K light chain variable region sequences.

In one embodiment, use of a mouse as described herein to generate a heavy and a K light chain variable region sequence for making a human antibody is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) isolating bone marrow from the immunized mouse of (a), (c) exposing B lymphocytes from the bone marrow to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest, and (e) amplifying a heavy chain variable region nucleic acid sequence and a K light chain variable region nucleic acid sequence from the B lymphocyte thereby ting the heavy chain and K light chain variable region sequences. in various embodiments, the one or more labeled antibodies are selected from an lgM, an lgG, and/or a ation thereof. in various embodiments, use of a mouse as bed herein to generate a heavy and K light chain variable region sequence for making a human antibody is provided, further sing fusing the amplified heavy and light chain variable region sequences to human heavy and light chain constant region sequences, expressing the fused heavy and light chain sequences in a cell, and recovering the expressed heavy and light chain sequences thereby generating a human antibody.

In various embodiments, the human heavy chain nt regions are selected from lgM, lgD, lgA, IgE and IgG. In various ic embodiments, the IgG is selected from an IgG1, an IgGZ, an lgG3 and an IgG4. In various embodiments, the human heavy chain constant region comprises a CH1, a hinge, a CH2, a CH3, a CH4, or a combination thereof.

In various embodiments, the light chain constant region is an immunoglobulin K nt region. In various embodiments, the cell is selected from a HeLa cell, a DU145 cell, a anap cell, a MCF-7 cell, a MDA—MB-438 cell, a PC3 cell, a T47D cell, a THP-1 cell, a U87 cell, a SHSY5Y (human neuroblastoma) cell, a Saos-2 cell, a Vero cell, a CHO cell, a GH3 cell, a P012 cell, a human retinal cell (e.g., a PERCGT'V‘ cell), and a MC3T3 cell. In a specific ment, the cell is a CHO cell.

In one aspect, a method for generating a reverse-chimeric rodent-human antibody specific against an antigen of interest Is provided, comprising the steps of zing a mouse as described herein with the antigen, ing at least one cell from the mouse producing a reverse—chimeric mouse-human antibody specific against the antigen, culturing at least one cell producing the reverse—chimeric mouse-human dy specific t the antigen, and obtaining said antibody.

In one embodiment, the reverse-chimeric mouse—human antibody comprises a human heavy chain variable domain fused with a mouse or rat heavy chain constant gene, and a human light chain variable domain fused with a mouse or rat or human light chain constant gene.

In one embodiment, culturing at least one cell producing the e-chimeric rodent-human antibody specific against the n is performed on at least one hybridoma cell generated from the at least one cell isolated from the mouse.

In one aspect, a method for generating a fully human antibody specific against an antigen of interest is provided, comprising the steps of immunizing a mouse as described herein with the antigen, isolating at least one cell from the mouse producing a reverse-chimeric rodent—human antibody specific against the antigen, generating at least one cell producing a fully human dy derived from the reverse—chimeric rodent-human antibody specific against the antigen, and culturing at least one cell producing the fully human antibody, and obtaining said fully human antibody.

In s embodiments, the at least one cell isolated from the mouse producing a reverse-chimeric rodent—human antibody specific against the n is a splenocyte or a B cell.

In various embodiments, the antibody is a monoclonal antibody.

In various embodiments, immunization with the antigen of interest is d out with protein, DNA, a combination of DNA and protein, or cells expressing the antigen.

In one aspect, use of a mouse as described herein to make a nucleic acid sequence encoding an immunoglobulin variable region or fragment f is provided. in one embodiment, the nucleic acid sequence is used to make a human antibody or antigen— binding fragment thereof. In one embodiment, the mouse is used to make an n- g protein selected from an antibody, at multi-specific antibody (e.g., a bi-specific antibody), an scFv, a bi-specific scFv, a diabody, a triabody, a tetrabody, a V-NAR, a VHH, a VL, a F(ab), a F(ab)2, a DVD (i.e., dual variable domain antigen-binding protein), a an SVD (i.e., single variable domain antigen—binding protein), or a bispecific T-cell engager (BiTE). in one aspect, use of a mouse as described herein to introduce an ectopic ADAMS sequence into a mouse that lacks a functional endogenous mouse ADAMS sequence is provided, wherein the use comprises mating a mouse as bed herein with the mouse that lacks the functional endogenous mouse ADAMS sequence.

In one aspect, use of c material from a mouse as bed herein to make a mouse having an ectopic ADAMS sequence is provided. in one embodiment, the use comprises nuclear transfer using a nucleus of a cell of a mouse as described herein.

In one embodiment, the use ses cloning a cell of a mouse as described herein to produce an animal d from the cell. in one embodiment, the use comprises ing a sperm or an egg of a mouse as described herein in a process for making a mouse comprising the ectopic ADAMS sequence. in one aspect, a method for making a fertile male mouse comprising a modified immunoglobulin heavy chain locus is provided, comprising fertilizing a first mouse germ cell that comprises a modification of an endogenous immunoglobulin heavy chain locus with a second mouse germ cell that comprises an ADAMS gene or ortholog or homolog or fragment f that is onal in a male mouse; forming a fertilized cell; ng the ized cell to develop into an embryo; and, gestating the embryo in a surrogate to obtain a mouse.

In one embodiment, the fertilization is achieved by mating a male mouse and a female mouse. in one embodiment, the female mouse comprises the ADAMS gene or ortholog or homolog or fragment thereof. in one embodiment, the male mouse comprises the ADAMS gene or ortholog or homolog or fragment thereof. ln one aspect, use of a nucleic acid sequence encoding a mouse ADAMS protein or an ortholog or homolog thereof or a functional fragment of the corresponding ADAMS protein for restoring or enhancing the fertility of a mouse having a genome comprising a modification of an immunoglobulin heavy chain locus is provided, wherein the cation reduces or eliminates endogenous ADAMS function. in one ment, the nucleic acid sequence is integrated into the genome of the mouse at an ectopic position. In one embodiment, the c acid sequence is integrated into the genome of the mouse at an endogenous immunoglobulin locus. In a specific embodiment, the endogenous immunoglobulin locus is a heavy chain locus. In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at a position other than an endogenous immunoglobulin locus.

In one aspect, use of the mouse as bed herein for the manufacture of a medicament (e.g., an antigen—binding protein), or for the manufacture of a sequence ng a variable sequence of a medicament (e.g., an antigen—binding protein), for the treatment of a human disease or disorder is provided.

BRIEF DESCRIPTION OF FIGURES shows a general illustration, not to scale, of direct genomic ement of about three megabases (Mb) of a mouse immunoglobulin heavy chain variable gene locus (closed symbols) with about one megabase (Mb) of the human immunoglobulin heavy chain variable gene locus (open symbols).

FiG. 1B shows a general ration, not to scale, of direct c replacement of about three megabases (Mb) of a mouse immunoglobulin K light chain variable gene locus (closed symbols) with about 0.5 megabases (Mb) of the first, or proximal, of two nearly identical repeats of a human immunoglobulin K light chain variable gene locus (open symbols). shows a detailed illustration, not to scale, of three initial steps (A—C) for direct genomic replacement of a mouse immunoglobulin heavy chain variable gene locus that resuits in deletion of all mouse VH, DH and JH gene segments and ement with three human VH, all human DH and JH gene segments. A ing vector for a first insertion of human immunoglobulin heavy chain gene segments is shown (23th BACvec) with a 67 kb 5’ mouse gy arm, a selection cassette (open rectangle), a site—specific recombination site (open triangle), a 145 kb human genomic fragment and an 8 kb 3’ mouse homology arm. Human (open symbols) and mouse (closed s) immunoglobulin gene segments, additional selection cassettes (open rectangles) and site— specific recombination sites (open triangles) inserted from subsequent targeting vectors are shown. shows a detailed illustration, not to scale, of six additional steps (D—l) for direct genomic replacement of a mouse immunoglobulin heavy chain variable gene locus that s in the insertion of 77 additional human VH gene segments and l of a final ion cassette. A targeting vector for insertion of additional human VH gene segments (18hVH BACvec) to the initial insertion of human heavy chain gene segments (3hVH-CRE Hybrid Allele) 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 ts which is shown with a site-specific recombination site (open triangle) d 5’ to the human gene segments. Human (open s) and mouse (closed symbols) immunoglobulin gene segments and additional selection cassettes (open rectangles) inserted by subsequent targeting vectors are shown. shows a detailed illustration, not to scale, of three initial steps (A—C) for direct genomic ement of a mouse immunoglobulin K light chain variable gene locus that results in deletion of all mouse VK, and JK gene ts (ng-CRE Hybrid Allele).

Selection cassettes (open rectangles) and site-specific recombination sites (open triangles) ed from the targeting vectors are shown. shows a detailed illustration, not to scale, of five additional steps (D—H) for direct genomic replacement of a mouse immunoglobulin K light chain variable gene locus that results in the insertion of all human VK and JK gene segments of the proximal repeat and deletion of a final selection cassette (40hVKdHyg Hybrid Allele). Human (open symbols) and mouse (closed symbols) immunoglobulin gene segments and onal selection cassettes (open rectangles) inserted by subsequent targeting vectors are shown. 6] shows a general illustration, not to scale, of a screening strategy including the locations of quantitative PCR (qPCR) primer/probe sets to detect ion of human heavy chain gene sequences and loss of mouse heavy chain gene sequences in targeted embryonic stem (ES) cells. The screening strategy in ES cells and mice for a first human heavy gene insertion is shown with qPCR primer/probe sets for the d region (“loss” probes C and D), the region ed (“hlgH” probes G and H) and flanking regions (“retention” probes A, B, E and F) on an unmodified mouse chromosome (top) and a correctly targeted some (bottom). shows a representative calculation of observed probe copy number in parental and modified ES cells for a 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 al ES cells, these probes were assumed to have copy number of 1 in ed ES cells and a maximum Ct of 35 was used even though no amplification was observed. shows a representative calculation of copy s for four mice of each genotype calculated using only probes D and H. Wild-type mice: WT Mice; Mice zygous for a first insertion of human immunoglobulin gene segments: HET Mice; Mice homozygous for a first insertion of human immunoglobulin gene segments: Homo Mice. shows a detailed ration, not to scale, of the three steps employed for construction of a 3hVH BACvec by bacterial gous recombination (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 s are shown. shows pulse-field gel eiectrophoresis (PFGE) of three BAC clones (B1, 82 and B3) after Noti digestion. 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 sequentiaI modifications of a mouse immunoglobulin heavy chain locus with increasing amounts of human immunoglobulin heavy chain gene segments. Homozygous mice were made from each of the three different stages of heavy chain humanization. Open symbols indicate human ce; closed symbols indicate mouse sequence.

FIG. SB shows a schematic illustration, not to scale, of sequential modifications of a mouse globulin K light chain locus with increasing amounts of human immunoglobulin K light chain gene segments. Homozygous mice were made from each of the three different stages OfK Iight chain zation. Open symbols indicate human sequence; closed symbols indicate 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 (second row from top) of wild type (wt), VELOCIMMUNE® 1 (V1), VELOCIMMUNE® 2 (V2) or VELOCIMMUNE® 3 (V3) mice were stained for surface lgM expressing B cells (top row, and second row from top), surface immunoglobulin containing either K or A light chains (third row from top) or surface lgM of specific haplotypes (bottom row), and pOpulations separated by FACS. shows representative heavy chain CDR3 sequences of randomly ed VELOCIMMUNE® dies around the VH-DH-JH (CDR3) junction, demonstrating junctional diversity and tide additions. Heavy chain CDR3 sequences are grouped ing 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 (9.9., 3—72 is human VH3-72). JH gene segments for each heavy chain CDR3 are noted within parenthesis at the 3’ end of each sequence (e.g., 3 is human JH3). SEQ ID N05 for each sequence shown are as follows proceeding from top to bottom: SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID N024; SEQ ID NO:25; SEQ ID N026; SEQ ID NO:27; SEQ ID N028; SEQ ID N0229; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:39. shows entative 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 (9.9., 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 (e.g., 1 is human JK1). SEQ ID N05 for each sequence shown are as follows ding 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 ncies 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 column) position among sets of 38 (unimmunized lgM), 28 (unimmunized lgG), 32 (unimmunized ng from lgG), 36 (immunized IgG) or 36 (immunized IgK 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 VELOCIMMUNE® mice (closed bars). 8] shows levels of serum immunoglobulin for lgA e in wild type (open bars) or VELOCIMMUNE® mice (closed bars). shows levels of serum immunoglobulin for lgE isotype in wild type (open bars) or VELOCIMMUNE® mice (closed bars).

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

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

A shows FACS r plots of lymphocytes gated on singlets for surface expression of lgM and 3220 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 mouse ADAMS genes (H+’+AS’esK+/+). Percentage of immature (BZZOm‘lgM+) and mature (BZZOhighlgM+) B cells is noted in each contour plot. 8 shows the total number of immature (82205“tlgM") and mature (BZZOhighlgM+) B cells in the bone marrow ed from femurs of mice homozygous for human heavy and human K light chain variable gene loci (H+/+ +l+ K ) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding mouse ADAMS genes (H+/+A6resK+/+).

A shows FACS r 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 +l+ +/+ K ) and mice gous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment ng mouse ADAMS genes S’eSK+’+). Percentage of pro-B (CD19+CD43*ckit+) and pre-B (CD19+CD43'ckit') cells is noted in the upper right and lower left quadrants, respectively, of each contour plot. 8 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 encoding mouse ADAM6 genes (H+/+A6resK+/+).

A shows FACS contour plots of lymphocytes gated on singlets for surface expression of CD19 and CD43 in the bone marrow for mice homozygous for +/+ +/+ human heavy and human K light chain le 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 mouse ADAM6 genes (H+’+A6’esK+’+). Percentage of immature B (CD19+CD43'), pre—B (CD19+CD43"“) and pro-B (CD19+CD43+) cells is noted in each contour plot. 3 shows histograms of immature B (CD19+CD43') and pre-B (CD19*CD43"“) cells in the bone marrow of mice homozygous for human heavy and 4-H» + human K light chain le gene loci (H K H) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding mouse ADAM6 genes (H+’+A6”53K+’+).

A 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 le gene loci (H K ) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment ng mouse ADAM6 genes 6“*SK+’+). tage of B (CD19+CD3') and T (CD19'CD3+) cells is noted in each contour plot. 3] B shows FACs contour plots for CD19”-gated B cells for surface expression of lg)» and IgK light chain 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 ng mouse ADAM6 genes (H+/+A6resl( ). Percentage of ng+ (upper left quadrant) and IgK+ (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+l+) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding mouse ADAM6 genes (H+’+A6'ESK*’*), A shows FACs contour plots of ated 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 homozygous for human heavy and human K Iight chain variable gene loci having an ectopic mouse genomic fragment encoding mouse ADAM6 genes (H+’+A6'eSK*’+). Percentage of mature B cells (CD19+lgDhi9“|gM‘"t) is noted for each contour pIot. The arrow on the right contour pIot illustrates the process of maturation for B cells in relation to IgM and IgD e expression. 8 shows the totaI number of B cells in the spleen of mice gous for human heavy and human K light chain variable gene loci (H+/+K+l+) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic nt encoding mouse ADAM6 genes (H+’+A6’°SK+’+) during maturation from CD19+lngghlgDint to CD19+IgMi”tlgDmgh. shows the antibody titer for first and second bleeds from mice homozygous for human heavy and human K light chain variable gene loci (H+"‘K+’+; n=5) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding mouse ADAM6 genes (H+’+A6’eSK+’+; n=5) that were immunized with a human cell e receptor (Antigen A). shows the antibody titer for first and second bleeds from mice homozygous for human heavy and human K light chain variabIe gene loci (H*’*K+’+; n=5) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding mouse ADAM6 genes (H“’*A6'e5K*/+; n=10) that were immunized with a human dy specific for a human receptor tyrosine-protein kinase (Antigen B). 9] shows the antibody titer for first and second bleeds from mice homozygous for human heavy and human K light chain variable gene loci (H+l+K+/+; n=12) and mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic nt encoding mouse ADAM6 genes (H+’+A6'93K”*; n=12) that were immunized with a secreted human protein that functions in regulation of the TGF- (3 ing pathway (Antigen C). shows the antibody titer for first and second bleeds from mice homozygous for human heavy and human K light chain variable gene loci having an ectopic mouse genomic fragment encoding mouse ADAM6 genes (H+’+A6'eSK*’+; n=12) that were immunized with a human reCeptor tyrosine kinase (Antigen D).

DETAILED DESCRIPTION OF ION This ion is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is aIso to be understood that the terminoiogy used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is defined by the claims.

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

Although any methods and materials similar or lent to those described herein can be used in the practice or g of the t ion, particular methods and materials are now described. All publications mentioned are hereby incorporated by reference.

The phrase “substantial” or “substantially” when used to refer to an amount of gene segments (e.g., “substantially all” V gene segments) includes both onal and non functional gene segments and include, in various embodiments, 9.9., 80% or more, 85% or more, 90% or more, 95% or more 96% or more, 97% or more, 98% or more, or 99% or more of all gene segments; in various embodiments, “substantially all” gene ts includes, 6.9., at least 95%, 96%, 97%, 98%, or 99% of functional (i.e., eudogene) gene segments.

The term “replacement” includes wherein a DNA sequence is placed into a genome of a cell in such a way as to replace a sequence within the genome with a logous sequence (e.g., a human sequence in a mouse), at the locus of the genomic sequence. The DNA sequence so placed may include one or more regulatory sequences that are part of source DNA used to obtain the sequence so placed (e.g., ers, enhancers, 5’— or 3’-untranslated regions, appropriate recombination signal ces, etc). For example, in various embodiments, the replacement is a tution of an nous sequence for a heterologous sequence that results in the production of a gene product from the DNA sequence so placed (comprising the heterologous sequence), but not expression of the endogenous sequence; the replacement is of an endogenous genomic sequence with a DNA sequence that encodes a protein that has a similar function as a protein encoded by the endogenous genomic sequence (e.g., the nous genomic sequence encodes an immunoglobulin gene or domain, and the DNA fragment encodes one or more human immunogiobulin genes or domains). In s embodiments, an endogenous gene or fragment thereof is replaced with a ponding human gene or fragment thereof. A corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of, a homolog of, or is substantially identical or the same in structure and/or function, as the endogenous gene or fragment thereof that is replaced.

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. e all of its advantages, the mouse still presents genetic obstacles that render it an imperfect model for human diseases and an ect platform to test human therapeutics or make them. First, although about 99% of human genes have a mouse homolog (Waterston et al. 2002, lnitial sequencing and comparative analysis of the mouse genome, Nature 420:520—562), potential therapeutics 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 6,596,541 and US 7,105,348, orated herein by reference). lly, efforts to humanize mouse genes by a “knockout-plus—transgenic humanization” strategy entailed crossing a mouse carrying a deletion (i.e., knockout) of the endogenous gene with a mouse carrying a randomly ated human transgene (see, 6.9., Bril et al., 2006, Tolerance to factor Vlll in a transgenic mouse expressing human factor Vlll cDNA carrying an 3) to Cys substitution, Thromb Haemost 95:341-347; Homanics et al., 2006, Production and characterization of murine models of classic and intermediate maple syrup urine disease, BMC Med Genet 7:33; Jamsai et al., 2006, A humanized BAC transgenic/knockout mouse model for HbE/beta—thalassemia, Genomics 88(3):309-15; Pan et al., 2006, Different role for mouse and human ta/epsilon heterodimer in preT cell receptor (preTCR) function:human CD3deIta/epsilon heterodimer restores the defective preTCR on in CDSQamma- and CD39ammadelta-deficient mice, Mol Immunol 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 ly ed 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 replacement involved elaborate and burdensome procedures, thus limiting the length of genetic material that could be handled and the precision with which it could be lated. ously introduced human immunoglobulin transgenes rearrange in precursor B cells in mice (Alt et al., 1985, lmmunoglobulin genes in transgenic mice, Trends Genet 1:231—236). This finding was ted by engineering mice using the knockout—plus—transgenic approach to express human dies (Green et al., 1994, n-specific human monoclonal antibodies from mice engineered with human lg heavy and light chain YACs, Nat Genet 7:13-21; Lonberg et al., 1994, Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 368:856-859; Jakobovits et al., 2007, From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice, Nat hnol25:1134-1143). The mouse globulin heavy chain and K light chain loci were inactivated in these mice by targeted deletion of small but critical portions of each endogenous locus, ed by introducing human globulin gene loci as randomly integrated large transgenes, as described above, or minichromosomes (Tomizuka et al., 2000, Double trans-chromosomic mice: maintenance of two dual human chromosome fragments containing lg heavy and kappa loci and expression of fully human antibodies, PNAS USA 972722-727). Such mice represented an important advance in genetic engineering; fully human monoclonal antibodies isolated from them yielded promising therapeutic potential for treating a variety of human diseases (Gibson et al., 2006, Randomized phase Ill trial results of panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody, in metastatic colorectal cancer, Clin Colorectal Cancer 6:29—31; Jakobovits et al., 2007; Kim et al., 2007, Clinical efficacy of zanolimumab -CD4): two Phase II studies in refractory cutaneous T-cell lymphoma, Blood 109(11):4655—62; Lonberg, 2005, Human antibodies from transgenic s, Nat Biotechnol23z11 17-1125; Maker et al., 2005, Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte- associated antigen 4 blockade and interleukin 2: a phase II” study, Ann Surg Oncol 12:1005-1016; McClung et al., 2006, Denosumab in postmenopausal women with low bone mineral density, New 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 clue to the random uction of the human immunoglobulin transgenes and resulting incorrect expression due to a lack of upstream and downstream control elements (Garrett et al., 2005, Chromatin architecture near a potential 3' end of the lgH locus involves modular regulation of histone cations during B-Cell development and in vivo occupancy at CTCF sites, MOI Cell Biol 252151 1-1525; Manis et al., 2003, Elucidation of a downstream boundary of the 3' lgH regulatory region, Mol Immunol 392753-760; Pawlitzky et al., 2006, identification of a candidate regulatory element within the 5' ng region of the mouse lgH locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A, J Immunol 176:6839—6851); (2) inefficient interspecies interactions between human constant domains and mouse components of the B—cell receptor signaling x on the cell surface, which may impair ing processes required for normal maturation, proliferation, and survival of B cells ch et al., 1990, Molecular components of the B- cell antigen or complex of the lgM class, Nature 343:760-762); and (3) inefficient pecies ctions between soluble human immunoglobulins and mouse PC receptors that might reduce affinity selection (Rao et al., 2002, Differential expression of the inhibitory lgG Fc receptor RllB on germinal center cells: implications for selection of high-affinity B cells, J Immunol 59-1868) and globulin serum concentrations (Brambell et a/., 1964, A Theoretical Model of Gamma-Globulin Catabolism, Nature 203:1352-1354; Junghans and Anderson, 1996, The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor, PNAS USA 93:5512-5516; Rao et 3]., 2002; Hjelm et a/., 2006, Antibody-mediated tion of the immune response, Scand J Immunol 64:177-184; Nimmerjahn and Ravetch, 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 locations at the endogenous heavy and light chain loci. This would effectively result in mice that make “reverse chimeric” (i.e., human Vzmouse C) antibodies which would be capable of normal interactions and selection with the mouse environment based on ing mouse constant regions. Further such e chimeric antibodies may be readily reformatted into fully human antibodies for therapeutic purposes. 7] cally modified animals that se a replacement at the endogenous immunogtobulin heavy chain locus with heterologous (e.g., from another species) immunoglobulin sequences can be made in conjunction with replacements at endogenous immunoglobulin light chain loci or in conjunction with immunoglobulin tight chain transgenes (e.g., chimeric immunoglobulin light chain transgenes or fully human fully mouse, etc.) The species from which the heterologous immunoglobulin heavy chain sequences are derived can vary widely; as with immunoglobulin light chain sequences employed in immunoglobulin light chain sequence replacements or immunoglobulin light chain transgenes. lmmunoglobulin variable region nucleic acid sequences, 9.9., V, D, and/or J segments, are in various embodiments obtained from a human or a non—human animal.

Non—human animals suitable for providing V, D, and/or J segments include, for example bony fish, cartilaginous fish such as sharks and rays, amphibians, reptiles, mammals, birds (e.g., chickens). Non-human animals e, for e, mammals. Mammals include, for exampie, man primates, goats, sheep, pigs, dogs, bovine (e.g., cow, bull, buffalo), deer, , ferrets and rodents and non-human primates (e.g., chimpanzees, orangutans, gorillas, marmosets, rhesus monkeys baboons). Suitable non-human animals are selected from the rodent family including rats, mice, and hamsters. in one embodiment, the non-human animals are mice. As clear from the context, various non- human animais can be used as s of variable domains or variable region gene segments (e.g., , rays, mammals (e.g., camels, rodents such as mice and rats). 9] ing to the context, non-human s are also used as sources of constant region sequences to be used in connection with variable sequences or segments, for example, rodent constant sequences can be used in transgenes operably linked to human or non-human variable sequences (e.g., human or non-human primate variable sequences operably linked to, e.g., rodent, e.g., mouse or rat or hamster, constant sequences). Thus, in various ments, human V, D, and/or J segments are operably linked to rodent (e.g., mouse or rat or r) constant region gene sequences. In some ments, the human V, D, and/or J segments (or one or more rearranged VDJ or VJ genes) are operably linked or fused to a mouse, rat, or hamster nt region gene sequence in, 9.9., a transgene integrated at a locus that is not an endogenous immunoglobulin locus. in a specific embodiment, a mouse is provided that comprises a replacement of VH, DH, and JH segments at an endogenous immunoglobulin heavy chain locus with one or more human VH, DH, and JH segments, wherein the one or more human VH, DH, and JH segments are operably linked to an endogenous immunoglobulin heavy chain gene; n the mouse comprises a transgene at a locus other than an endogenous immunoglobulin locus, wherein the transgene comprises an unrearranged or rearranged human VL and human JL segment operably linked to a mouse or rat or human constant region.

A method for a large in situ genetic replacement of the mouse germline immunoglobulin variable gene loci with human ne immunoglobulin variable gene loci while maintaining the ability of the mice to generate ing is described. ically, the precise replacement of six megabases of both the mouse heavy chain and K light chain immunoglobulin variable gene loci with their human counterparts while leaving the mouse constant regions intact is described. As a result, mice have been d 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.e., they comprise human variable region sequences and mouse constant region sequences.

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

MMUNE® humanized mice exhibit a fully functional humoral immune system that is essentially indistinguishable from that of wild-type mice. They display normal cell tions at all stages of B cell development. They exhibit normal lymphoid organ morphology. Antibody sequences of VELOCIMMUNE® mice exhibit normal V(D)J rearrangement and normal somatic hypermutation frequencies. Antibody populations in these mice reflect e distributions that result from normal class switching (6.9., normal isotype itching). immunizing VELOCIMMUNE® mice s in robust humoral immune responses that te a large, diverse antibody repertoires having human immunoglobulin variable domains suitable for use as therapeutic candidates. This platform provides a plentiful source of naturally affinity-matured human immunoglobulin variable region sequences for making pharmaceutically able 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 VELOCIMMUNE® 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 divergent evolution of the immunoglobulin loci between mouse and man. For example, intergenic sequences interspersed within the immunoglobulin loci are not identical 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 alities 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 deleterious.

Deleterious modifications can include, for e, loss of the ability of the modified mice to mate and produce offspring.

A precise, 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 ces was performed, while g the flanking mouse sequences intact and functional within the hybrid loci, including all mouse constant chain genes and locus transcriptional control regions ( and ). Specifically, the human VH, DH, JH, VK and JK gene ces were introduced through stepwise insertion of 13 ic BAC ing vectors bearing overlapping fragments of the human ne variable loci into mouse ES cells using VELOClGENE® genetic engineering technology (see, 9.9., US Pat. No. 251 and Valenzuela et al., 2003, High-throughput ering of the mouse genome coupled with high-resolution expression analysis, Nat Biotechnol 21:652-659). zation 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 (discussed above), direct replacement of the mouse immunoglobulin genes with their human counterparts dramatically increases the efficiency with which fully-human dies can be efficiently generated in otherwise normal mice. r, such mice exhibit a dramatically sed 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 transgenes. Multiple versions of replaced, humanized loci exhibit completely normal levels of mature and immature B cells, in contrast to mice with randomly integrated human transgenes, which exhibit significantly reduced B cell populations at various stages of differentiation. While s to increase the number of human gene segments in human transgenic mice have reduced such defects, the expanded immunoglobulin repertoires have not altogether corrected reductions in B cell populations as compared to wild-type mice.

Notwithstanding the near wild-type l immune function observed in mice with replaced immunoglobulin loci (i.e., VELOCIMMUNE® mice), there are other challenges encountered when employing a direct replacement of the immunoglobulin that is not encountered in some approaches that employ randomly integrated transgenes.

Differences in the genetic composition of the globulin loci between mice and humans has lead to the ery of sequences beneficial for the propagation of mice with ed immunoglobulin gene segments. Specifically, mouse ADAM genes d within the endogenous immunoglobulin locus are optimally present in mice with replaced immunoglobulin loci, due to their role in fertility. c Location and Function of Mouse ADAM6 Male mice that lack the ability to express any functional ADAM6 protein surprisingly exhibit a defect in the ability of the mice to mate and to generate offspring.

The mice lack the ability to s 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 results 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 upstream 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 variable gene segments, it is generally a cumbersome approach to set up males and females that are each homozygous for the replacement and await a productive mating. Successful litters are low in ncy and size. Instead, males heterozygous for the replacement have been employed to mate with females homozygous for the replacement to generate progeny that are zygous for the replacement, then breed a homozygous mouse therefrom. The inventors have determined that the likely cause of the toss in fertility in the male mice is the absence in homozygous male mice of a functional ADAMS protein. 8] In various aspects, male mice that comprise a damaged (is, nonfunctional or marginally functional) ADAMS gene exhibit a reduction or elimination of fertility. Because in mice (and other rodents) the ADAMS gene is located in the immunoglobulin heavy chain locus, the inventors have ined that in order to propagate mice, or create and maintain a strain of mice, that comprise a replaced immunoglobulin heavy chain locus, various modified breeding or propagation schemes are employed. The low fertility, or infertility, of male mice homozygous for a replacement of the endogenous immunoglobulin heavy chain variable gene locus renders ining such a cation in a mouse strain difficult. In various embodiments, maintaining the strain comprises avoiding infertility problems exhibited by male mice homozygous for the replacement.

In one , a method for maintaining a strain of mouse as described herein is provided. The strain of mouse need not se an ectopic ADAMS sequence, and in various embodiments the strain of mouse is homozygous or heterozygous for a knockout (6.9., a functional knockout) of ADAMS. 0] The mouse strain comprises a modification of an endogenous immunoglobulin heavy chain locus that s in a reduction or loss in fertility in a male mouse. In one embodiment, the modification comprises a deletion of a regulatory region and/or a coding region of an ADAMS gene. In a specific embodiment, the modification comprises a modification of an endogenous ADAMS gene (regulatory and/or coding region) that reduces or eliminates fertility of a male mouse that comprises the modification; in a specific embodiment, the modification s or eliminates fertility of a male mouse that is homozygous for the modification.

In one embodiment, the mouse strain is homozygous or heterozygous for a ut (e.g., a functional knockout) or a deIetion of an ADAMS gene.

In one embodiment, the mouse strain is maintained by isolating from a mouse that is homozygous or heterozygous for the cation a cell, and employing the donor cell in host embryo, and gestating the host embryo and donor cell in a surrogate mother, and obtaining from the surrogate mother a progeny that comprises the genetic modification. In one embodiment, the donor cell is an ES cell. In one embodiment, the donor cell is a pluripotent cell, 6.9., an induced pluripotent cell.

In one ment, the mouse strain is maintained by isolating from a mouse that is gous or heterozygous for the modification a nucIeic acid sequence comprising the modification, and introducing the nucleic acid sequence into a host nucleus, and ing a cell comprising the nucleic acid sequence and the host nucleus in a suitable animal. in one embodiment, the nucleic acid sequence is introduced into a host oocyte embryo.

In one embodiment, the mouse strain is maintained by isolating from a mouse that is homozygous or heterozygous for the modification a s, and introducing the nucleus into a host cell, and gestating the nucleus and host cell in a le animal to obtain a progeny that is homozygous or heterozygous for the modification. in one embodiment, the mouse strain is maintained by employing in vitro ization (IVF) of a female mouse (wild-type, homozygous for the modification, or heterozygous for the cation) employing a sperm from a male mouse comprising the genetic modification. In one embodiment, the male mouse is heterozygous for the genetic modification. In one embodiment, the male mouse is homozygous for the genetic modification.

In one embodiment, the mouse strain is maintained by breeding a male mouse that is heterozygous for the c modiﬁcation with a female mouse to obtain progeny that comprises the genetic modification, identifying a male and a female progeny sing the genetic modification, and employing a male that is zygous for the genetic modification in a breeding with a female that is wild-type, homozygous, or heterozygous for the genetic modification to obtain progeny comprising the genetic modification. ln one embodiment, the step of breeding a male heterozygous for the genetic modification with a wild-type female, a female heterozygous for the c modification, or a female homozygous for the genetic modification is repeated in order to maintain the genetic modification in the mouse strain.

In one aspect, a method is provided for maintaining a mouse strain that comprises a replacement of an endogenous immunoglobulin heavy chain variable gene locus with one or more human immunoglobulin heavy chain sequences, comprising breeding the mouse strain so as to generate zygous male mice, wherein the heterozygous male mice are bred to maintain the genetic modification in the strain. In a specific embodiment, the strain is not maintained by any ng of a homozygous male with a wild-type female, or a female gous or heterozygous for the c modification.

The ADAMB protein is a member of the ADAM family of proteins, where ADAM is an m for A Disintegrin And Metalloprotease. The ADAM family of proteins is large and diverse, with diverse functions including cell adhesion. Some members of the ADAM family are implicated in spermatogenesis and ization. For example, ADAMZ 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 ADAMZ, ADAM3, and ADAM6 form a complex on the surface of mouse sperm cells. The human ADAM6 gene, normally found between human VH gene ts VH1-2 and VH6-1, appears to be a pseudogene e 12). In mice, there are two ADAM6 genes—ADAM6a and ADAM6b—that are found in an intergenic region between mouse VH and DH gene segments, and in the mouse the ADAMGa and ADAM6b genes are ed in opposite transcriptional orientation to that of the surrounding immunoglobulin gene segments (). In mice, a functional ADAM6 locus is apparently required for normal fertilization. A onal ADAM6 locus or sequence, then, refers to an ADAM6 locus or sequence that can complement, or rescue, the drastically reduced fertilization exhibited in male mice with missing or nonfunctional endogenous ADAM6 loci.

The on of the intergenic sequence in mice that encodes ADAM6a and ADAM6b renders the intergenic sequence susceptible to cation when ing an endogenous mouse heavy chain. When VH gene segments are deleted or replaced, or when DH gene ts are deleted or replaced, there is a high probability that a resulting mouse will exhibit a severe deficit in fertility. In order to compensate for the deficit, the mouse is modified to include a tide sequence that encodes a protein that will ment the loss in ADAM6 activity due to a modification of the endogenous mouse ADAM6 locus. In various embodiments, the complementing tide sequence is one that encodes a mouse ADAMBa, a mouse ADAM6b, or a homolog 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 ly integrates into the mouse genome. in one embodiment, the sequence can be ined episomally, that is, on a te nucleic acid rather than on a mouse chromosome. Suitable positions include positions that are transcriptionally permissive or active, e.g., a ROSA26 locus (Zambrowicz et al., 1997, PNAS USA 94:3789- 3794), a BT-5 locus (Michael et al., 1999, Mech. Dev. 85:35-47), or an Oct4 locus (Wallace et al., 2000, Nucleic Acids Res. 28:1455-1464). Targeting nucleotide sequences to transcriptionally active loci are described, 6.9., in US 7,473,557, herein incorporated by reference.

Alternatively, the nucleotide sequence that rescues fertility can be coupled with an inducible promoter so as to facilitate optimal expression in the appropriate cells and/or tissues, e.g., reproductive tissues. Exemplary inducible promoters include ers activated by physical (9.9., heat shock promoter) and/or chemical means (e.g,, lPTG or Tetracycline).

Further, expression of the nucleotide sequence can be linked to other genes so as to achieve expression at specific stages of development or within speciﬁc tissues. Such expression can be achieved by placing the nucleotide sequence in operable linkage with the promoter of a gene expressed at a specific stage of development. For example, immunoglobulin sequences from one species engineered into the genome of a host s are place in operable linkage with a promoter sequence of a CD19 gene (a B cell ic gene) from the host species. B cell-specific expression at precise developmental stages when immunoglobulins are expressed is achieved.

Yet another method to achieve robust expression of an inserted nucleotide sequence is to employ a constitutive promoter. Exemplary constitutive promoters include SV40, CMV, UBC, EFlA, PGK and CAGG. In a similar fashion, the desired nucleotide sequence is placed in operable linkage with a selected constitutive promoter, which provides high level of expression of the protein(s) encoded by the tide sequence.

The term “ectOpic” is intended to include a displacement, or a placement at a position that is not normally encountered in nature (e.g., placement of a nucleic acid sequence 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 c 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 nt thereof that es the same or similar fertility benefit on male mice), the sequence can be placed at a different position in the s genome than is normally found in a wild-type mouse. ln such cases, novel sequence junctions of mouse sequence will be created by placing the sequence at a different position in the mouse’s genome than in a wild-type mouse. A functional homolog or ortholog of mouse ADAM6 is a ce that confers a rescue of fertility loss (e.g., loss of the ability of a male mouse to te 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, 6.9., up to 99% ty, to the amino acid sequence of ADAM6a and/or to the amino acid sequence of ADAM6b, and that can ment, or rescue ability to successfully mate, of a mouse that has a genotype that includes a on or ut of ADAM6a and/or ADAMBb.

The ectopic position can be anywhere (9.9., as with random insertion of a transgene containing a mouse ADAM6 sequence), or can be, 6.9., at a position that approximates (but is not precisely the same as) its location in a wild-type mouse (9.9., in a modified endogenous mouse immunoglobulin locus, but either upstream or ream of its l position, 9.9., within a modified immunoglobulin locus but n 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 immunoglobulin heavy chain locus.

For e, 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 engineered to have a mouse ADAM6 sequence located within a sequence that contains the human VH gene segments. The ing modification would generate a (ectopic) mouse ADAM6 sequence within a human gene sequence, and the (ectopic) placement of the mouse ADAM6 ce within the human gene sequence can approximate the position of the human ADAM6 pseudogene (ie., between two V ts) or can approximate the position of the mouse ADAM6 sequence (i.e., within the V-D intergenic region). The resulting sequence junctions created by the joining of a (ectopic) mouse ADAM6 sequence within or adjacent to a human gene sequence (9.9., an immunoglobulin gene sequence) within the germline of the mouse would be novel as compared to the same or similar position in the genome of a wild-type mouse.

In various embodiments, non-human animals are provided that lack an ADAM6 or ortholog or homolog thereof, wherein the lack renders the man animal infertile, or substantially s fertility of the non-human animal. in various embodiments, the lack of ADAM6 or ortholog or homolog thereof is due to a modification of an endogenous immunoglobulin heavy chain locus. A substantial ion in fertility is, e.g., a reduction in fertility (e.g., breeding frequency, pups per litter, litters per year, etc.) of about 50%, 60%, 70%, 80%, 90%, or 95% or more. in various ments, the man animals are supplemented with a mouse ADAM6 gene or ortholog or homolog or functional fragment thereof that is functional in a male of the non—human animal, wherein the supplemented ADAM6 gene or ortholog or homolog or onal fragment thereof rescues the reduction in ity in whole or in substantial part. A rescue of fertility in substantial part is, 9.9., a restoration of fertility such that the non-human animal exhibits a fertility that is at least 70%, 80%, or 90% or more as compared with an unmodified (i.e., an animal without a modification to the ADAM6 gene or ortholog or homolog f) heavy chain locus.

The sequence that s upon the genetically modified animal (i.e., the animal that lacks a functional ADAM6 or ortholog or homolog f, due to, 9.9., a modification of a immunoglobulin heavy chain locus) is, in s embodiments, selected from an ADAM6 gene or ortholog or homolog thereof. For example, in a mouse, the loss of ADAM6 function is d by adding, in one embodiment, a mouse ADAM6 gene. In one embodiment, the loss of ADAM6 function in the mouse is rescued by adding an ortholog or homolog of a closely related specie with respect to the mouse, 69., a rodent, 9.9., a mouse of a different strain or species, a rat of any species, a rodent; wherein the addition of the ortholog or homolog to the mouse rescues the loss of fertility due to loss of ADAMS function or loss of an ADAMS gene. Orthologs and homologs from other species, in s embodiments, are selected from a phylogenetically related species and, in various embodiments, exhibit a percent identity with the nous ADAMS (or ortholog) that is about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, or 97% or more; and that rescue ADAMS-related or (in a non-mouse) ADAMS og-related loss of ity. For example, in a genetically ed male rat that lacks ADAMS function (9.9., a rat with an endogenous immunoglobulin heavy chain variable region replaced with a human immunoglobulin heavy chain variable region, or a ut in the rat globulin heavy chain region), loss of fertility in the rat is rescued by addition of a rat ADAMS or, in some embodiments, an ortholog of a rat ADAMS (e.g., an ADAMS ortholog from another rat strain or species, or, in one embodiment, from a mouse).

Thus, in various embodiments, genetically modified animals that t no fertility or a reduction in fertility due to modification of a nucleic acid sequence ng an ADAMS protein (or ortholog or homolog thereof) or a regulatory region operably linked with the nucleic acid sequence, comprise a nucleic acid sequence that complements, or restores, the loss in ity where the nucleic acid sequence that complements or restores the loss in fertitity is from a different strain of the same species or from a phylogenetically related species. In various embodiments, the complementing nucleic acid sequence is an ADAMS ortholog or homolog or functional fragment thereof. In various embodiments, the complementing ADAMS ortholog or homolog or functional fragment thereof is from a non- human animal that is closely related to the genetically modified animal having the fertility defect. For example, where the genetically modified animal is a mouse of a particular strain, an ADAMS ortholog or homolog or functional fragment thereof can be obtained from a mouse of another strain, or a mouse of a related species. In one embodiment, where the genetically ed animal comprising the fertility defect is of the order ia, the ADAMS ortholog or g or functional fragment thereof is from another animal of the order Rodentia. In one embodiment, the genetically modiﬁed animal comprising the fertility defect is of a suborder Myomoropha (e.g., jerboas, jumping mice, mouse-like hamsters, hamsters, New World rats and mice, voles, true mice and rats, gerbils, spiny mice, crested rats, climbing mice, rock mice, white-tailed rats, malagasy rats and mice, spiny dormice, mole rats, bamboo rats, ), and the ADAMS ortholog or homolog or functional nt thereof is selected from an animal of order Rodentia, or of the suborder Myomorpha. in one embodiment, the genetically ed animal is from the superfamily Dipodoidea, and the ADAMS ortholog or homolog or functional fragment thereof is from the amily Muroidea. in one embodiment, the genetically modified animal is from the superfamily Muroidea, and the ADAMS ortholog or homolog or functional nt thereof is from the superfamily Dipodoidea. in one embodiment, the genetically modified animal is a . In one embodiment, the rodent is selected from the superfamily Muroidea, and the ADAMS ortholog or homolog is from a different species within the superfamily ea. in one embodiment, the genetically modified animal is from a family selected from Calomyscidae (e.9., mouse-like hamsters), Cricetidae (e.9., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.9., spiny dormice), and Spalacidae (e.9., mole rates, bamboo rats, and zokors); and the ADAMS og or g is selected from a different species of the same family. In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), and the ADAMS ortholog or homolog is from a species selected from a gerbil, spiny mouse, or crested rat. in one embodiment, the genetically modified mouse is from a member of the family Muridae, and the ADAMS og or homolog is from a different species of the family Muridae. in a specific embodiment, the genetically modified rodent is a mouse of the family Muridae, and the ADAMS ortholog or homolog is from a rat, gerbil, spiny mouse, or crested rat of the family Muridae.

In various embodiments, one or more rodent ADAMS ogs or homologs or onal fragments thereof of a rodent in a family restores fertility to a genetically modified rodent of the same family that lacks an ADAMS og or g (e.9., Cricetidae (e.9., rs, New World rats and mice, voles); Muridae (e.9., true mice and rats, gerbils, spiny mice, crested rats)).

In various embodiments, ADAMS ogs, homologs, and nts thereof are assessed for functionality by ascertaining whether the ortholog, homolog, or fragment restores fertility to a genetically modiﬁed male non—human animal that lacks ADAMS activity (9.9., a rodent, e.9., a mouse or rat, that comprises a ut of ADAMS or its ortholog). in various embodiments, functionality is defined as the ability of a sperm of a cally modified animal lacking an endogenous ADAMS or ortholog or homolog thereof to migrate an oviduct and fertilize an ovum of the same specie of genetically modified animal. in various aspects, mice that comprise deletions or replacements of the endogenous heavy chain variable region locus or portions thereof can be made that contain an ectopic nucleotide ce that encodes a protein that confers similar fertility benefits to mouse ADAMS (9.9., an ortholog or a homolog or a fragment thereof that is onal in a male mouse). The ectopic nucleotide sequence can include a nucleotide sequence that encodes a protein that is an ADAMS homolog or ortholog (or fragment thereof) of a different mouse strain or a different species, 9.9., a different rodent species, and that confers a benefit in fertility, e.g., increased number of litters over a specified time period, and/or sed 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. 4] in one embodiment, the ADAM6 is a homolog or ortholog that is at least 89% to 99% identical to a mouse ADAM6 protein (e.g., at least 89% to 99% identical to mouse ADAM6a or mouse ADAMBb). 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 , and a combination thereof. In one embodiment, the g or ortholog is a rat, hamster, mouse, or guinea pig protein that is or is modified to be about 89% or more identical to mouse ADAM6a and/or mouse ADAM6b. in one embodiment, the homolog or ortholog is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% cal to a mouse ADAM6a and/or mouse ADAM6b.

Ectopic ADAM6 in Humanized Heavy Chain Mice Developments in gene targeting, e.g., the development of bacterial artificial chromosomes (BACs), now enable the recombination of relatively large genomic fragments. BAC engineering has allowed for the ability to make large deletions, and large insertions, into mouse ES cells.

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 icant abnormalities that limit their usefulness. For e, they display compromised B cell development. The compromised development may be due to a y of ences between the transgenic mice and wild—type mice. 7] Human antibodies might not optimally ct with mouse pre B cell or B cell receptors on the e of mouse cells that signal for maturation, proliferation, or survival during clonal selection. Fully human antibodies might not optimally interact with a mouse Fc receptor system; mice s Fc receptors that do not display a -one correspondence with human Fc receptors. Finally, various mice that make fully human dies 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 transgenes that comprise variable and constant immunoglobulin gene segments are introduced into a random location in the mouse genome. As long as the endogenous locus is sufficiently disabled so as not to rearrange gene ts to form a functional immunoglobulin gene, the goal of making fully human antibodies in such a mouse can be ed—albeit with compromised B cell development.

Although compelled to make fully human antibodies from the human transgene locus, generating human antibodies in a mouse is apparently an unfavored process. in 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 antibodies undergo isotype switching in trans from the human isotype to a mouse isotype. The process is in trans, e the fully human ene is d apart from the endogenous locus that retains an undamaged copy of a mouse heavy chain nt region gene. Although in such mice switching is readily apparent the enon is still insufficient to rescue B cell development, which remains frankly impaired. In any event, trans-switched antibodies made in such mice retain fully human light chains, since the phenomenon of trans—switching ntly does not occur with respect to light chains; trans-switching presumably relies on switch sequences in endogenous loci used (albeit differently) in normal e switching in cis. Thus, even when mice engineered to make fully human antibodies select a trans-switching mechanism to make antibodies with mouse constant regions, the gy is still insufficient to rescue normal B cell development.

A primary concern in making dy-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 development of VELOClMMUNE® mice (described herein), there was no indication that mice expressing human variable regions with mouse constant regions would exhibit any significant differences from mice that made human antibodies from a ene. That supposition, however, was incorrect.

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

VELOClMMUNE® mice contain a precise, large—scale replacement of germline variable regions of mouse globulin heavy chain (lgH) and immunoglobulin light chain (e.g., K light chain, ng) with ponding human immunoglobulin variable regions, at the endogenous loci. in total, about six megabases of mouse loci are replaced with about 1.5 megabases of human c sequence. This precise replacement results in a mouse with hybrid immunoglobulin loci that make heavy and light chains that have a human le regions and a mouse constant region. The precise replacement of mouse VH-DH-JH and VK-JK ts 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. B cell development is unhindered in any significant respect and a rich ity of human le regions is generated in the mouse upon antigen challenge.

VELOClMMUNE® mice are le because immunoglobulin gene segments for heavy and K light chains rearrange similarly in humans and mice, which is not to say that their loci are the same or even nearly so—~—clearly they are not. However, the loci are similar enough that humanization of the heavy chain variable gene locus can be accomplished by replacing about three million base pairs of contiguous mouse sequence that contains all the VH, DH, and JH gene segments with about one million bases of contiguous human genomic sequence covering basically the equivalent sequence from a human immunoglobulin locus. in some embodiments, further replacement of n 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 sequence with human CL sequence) results in mice with hybrid immunoglobulin loci that make dies that have human variabie regions and partly human constant regions, suitable for, 9.9., making fully human antibody fragments, e.g., fully human Fab’s. Mice with hybrid immunoglobulin loci exhibit normal le gene segment rearrangement, normal somatic hypermutation frequencies, and normal class switching. These mice exhibit a humoral immune system that is indistinguishable from wild type mice, and y normal cell populations at all stages of B cell development and normal id organ structures—even where the mice lack a full repertoire of human variable region gene segments. lmmunizing 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 segments 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 comprise human variable regions. Nucleotide sequences that encode the variable regions can be identified and cloned, then fused (9.9., in an in vitro system) with any ces of choice, e.g., any immunoglobulin e suitable for a particular use, resulting in an antibody or antigenbinding protein derived wholly from human sequences.

Large-scale humanization by recombineering s were used to modify mouse embryonic stem (ES) cells to precisely replace up to three 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 one megabase human genomic sequence containing some or essentially all human VH, DH, and JH gene segments, Up to a one-half se segment of the human genome comprising one of two repeats encoding ially all human VK and JK gene ts was used to replace a three megabase segment of the mouse globulin K light chain locus containing essentially all of the mouse VK and JK gene segments.

Mice with such replaced 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 globulin heavy chain locus. Disruption in this region can lead to reduction or elimination of functionality of the endogenous mouse ADAM6 locus. if the 3’-most VH gene segments of the human heavy chain repertoire are used in a replacement, an enic region containing a pseudogene that appears to be a human ADAM6 pseudogene is present between these VH gene segments, i.e., between human VH’l-Z and VHt-G. However, male mice that comprise this human enic sequence exhibit a reduction in fertility.

Mice are described that comprise the ed loci as described above, and that also comprise an ectopic nucleic acid sequence encoding a mouse ADAM6, where the mice exhibit essentially normal fertility. in one embodiment, the c nucleic acid ce ses a mouse ADAM6a and/or a mouse ADAM6b sequence or functional fragments thereof placed between a human VH1-2 and a human VH6-1 at a modiﬁed endogenous heavy chain locus. In one embodiment, the ectopic nucleic acid sequence is SEQ lD NO:3, placed between a human VH1-2 and a human VHS-1 at a modified endogenous heavy chain locus. The direction of transcription of the ADAM6 genes of SEQ ID N013 are opposite with respect to the direction of transcription of the surrounding human VH gene segments. Although examples herein show rescue of fertility by placing the ectopic sequence between the ted human VH gene segments, skilled persons will recognize that placement of the ectopic sequence at any suitable riptionallypermissive 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 ortholog or homolog or functional fragment thereof is a general method that is applicable to rescuing any mice with nonfunctional or minimally functional endogenous ADAMS loci. Thus, a great many mice that comprise an ADAMS-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 ADAMS function, Some (non-limiting) examples are provided in this description. In addition to the MMUNE® mice described, the itions and methods related to ADAMS can be used in a great many applications, 6.9., when modifying a heavy chain locus in a wide variety of ways.

In one , a mouse is ed that comprises an ectopic ADAMS sequence that encodes a functional ADAMS 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 VH gene segments, a replacement of all or substantially all mouse DH gene segments and JH gene ts with human DH and human JH gene ts; wherein the mouse lacks a CH1 and/or hinge region. in one ment, 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 (6.9., between human VH1-2 and VHt-S gene segments) in a mouse that has a replacement of one or more mouse immunoglobulin heavy chain variable gene segments (mVH’s, mDH’s, and/or mJH’s) with one or more human immunoglobulin heavy chain le gene segments , hDH’s, and/or hJH’s), and the mouse further ses a replacement of one or more mouse immunoglobulin K light chain variable gene segments (mVK’s and/or mJK’s) with one or more human immunoglobulin 1c light chain variable gene segments (hVK’s and/or hJK’s). ln one embodiment, the one or more mouse immunoglobulin heavy chain variable gene segments comprises about three megabases of the mouse immunoglobulin heavy chain locus. In one embodiment, the one or more mouse immunoglobulin heavy chain variable gene segments comprises at least 89 VH gene segments, at least 13 DH gene segments, at least four JH gene ts or a combination thereof of the mouse immunoglobulin heavy chain locus. in one embodiment, the one or more human globulin heavy chain variable gene segments comprises about one megabase of a human immunoglobulin heavy chain locus. in one embodiment, the one or more human immunoglobulin heavy chain variable gene segments comprises at least 80 VH gene segments, at least 27 DH gene segments, at least six JH gene segments or a combination thereof of a human immunoglobulin heavy chain locus.

In one ment, the one or more mouse immunoglobulin K light chain variable gene segments comprises about three megabases of the mouse immunoglobulin K light chain locus. In one embodiment, the one or more mouse immunoglobulin K light chain variable gene segments comprises at least 137 VK gene segments, at least five JK gene segments or a ation thereof of the mouse immunoglobulin K light chain locus.

In one ment, the one or more human globulin K light chain le gene segments comprises about one-half megabase of a human immunoglobulin K light chain locus. In a specific embodiment, the one or more human globulin K light chain variable gene segments comprises the proximal repeat (with respect to the immunoglobulin K constant region) of a human immunoglobulin K light chain locus. In one embodiment, the one or more human immunoglobulin K light chain variable gene segments comprises at least 4OVK gene segments, at least five JK gene segments or a combination thereof of a human immunoglobulin K light chain locus.

In one embodiment, the nucleotide sequence is place between two human globulin gene segments. In a specific embodiment, the two human immunoglobulin gene segments are heavy chain gene segments. In one embodiment, the nucleotide sequence is placed between a human VH1-2 gene segment and a human VH1-6 gene segment in a VELOCIMMUNE® mouse (US 6,596,541 and US 7,105,348, orated herein by reference). In one embodiment, the VELOCIMMUNE® mouse so modified comprises a replacement of mouse immunoglobulin heavy chain variable gene segments with at least 80 human VH gene segments, 27 human DH gene segments and six human JH gene segments, and a replacement of mouse immunoglobulin K light chain variable gene segments with at least 40 human VK gene segments and five human JK gene segments.

In one aspect, a functional mouse ADAM6 locus (or ortholog or homolog or functional fragment thereof) is present in the midst of human VH gene segments that replace nous mouse VH gene segments. In one embodiment, at least 89 mouse VH gene segments are removed and replaced with one or more human VH gene segments, and the mouse ADAM6 locus is present immediately adjacent to the 3’ end of the human VH gene segments, or between two human VH gene ts. In a specific embodiment, the mouse ADAM6 locus is t n two VH gene ts within about 20 kilo bases (kb) to about 40 kilo bases (kb) of the 3’ terminus of the inserted human VH gene segments. In a specific embodiment, the mouse ADAM6 locus is present between two VH gene segments within about 29 kb to about 31 kb of the 3’ terminus of the inserted human VH gene segments. In a specific embodiment, the mouse ADAM6 locus is present within about 30 kb of the 3’ terminus of the inserted human VH gene segments. In a specific embodiment, the mouse ADAM6 locus is t within about 30,184 bp of the 3’ terminus of the inserted human VH gene segments. In a specific embodiment, the ement includes human VH gene segments VHi-Z and VHS-1, and the mouse ADAM6 locus is present downstream of the VH1—2 gene segment and am of the VHS-1 gene segment.

In a specific embodiment, the mouse ADAM6 locus is present between a human VH1-2 gene t and a human VH6—1 gene segment, wherein the 5’ end of the mouse ADAM6 locus is about 13,848 bp from the 3’ terminus of the human VH1-2 gene segment and the 3’ end of the ADAM6 locus is about 29,737 bp 5’ of the human VH6-1 gene t. In a specific embodiment, the mouse ADAM6 locus comprises SEQ ID NO:3 or a fragment thereof that confers ADAM6 function within cells of the mouse. In a specific embodiment, the arrangement of human VH gene segments is then the following (from upstream to downstream with respect to direction of transcription of the human VH gene segments): human VH1-2 -— mouse ADAM6 locus —- human VHS-1. In a specific embodiment, the ADAM6 pseudogene between human VH1-2 and human VH6-1 is replaced with the mouse ADAM6 locus. In one embodiment, the orientation 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 VH gene ts. Alternatively, the mouse ADAM6 locus is present in the intergenic region n the 3’-most 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 endogenous mouse VH gene segments can be ed so as to either maintain the endogenous mouse ADAM6 locus, as described above, e.g., by employing a targeting vector having a downstream gy arm that includes a mouse ADAM6 locus or functional nt thereof, or to replace a damaged mouse ADAM6 locus with an ectopic sequence positioned between two human VL gene segments or between the human VL gene segments and a DH gene segment (whether human or mouse, e.g., VA + m/hDH), or a J gene segment (whether human or mouse, e.g., VK + JH). In one embodiment, the replacement includes two or more human VL gene segments, and the mouse ADAM6 locus or functional fragment thereof is present between the two 3’-most VL gene ts. In a specific embodiment, the arrangement of human VL gene segments is then the following (from upstream to downstream with respect to direction of transcription of the human gene segments): human VL3’-1 — mouse ADAM6 locus -— human VL3’. In one ment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is opposite with t to direction of transcription as compared with the orientation of the human VL gene segments. atively, the mouse ADAM6 locus is present 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 t or render the endogenous mouse ADAM6 locus nonfunctional. For example, in one embodiment the mouse comprises a replacement of all or substantially all endogenous mouse VH gene segments with one or more human VH gene segments, and the mouse ses one or more endogenous DH gene segments and a functional endogenous mouse ADAM6 locus. in another embodiment, the mouse comprises the modification of endogenous mouse t VH gene segments, and a modification of one or more endogenous mouse DH gene ts, and the modification is carried out so as to maintain the integrity of the endogenous mouse ADAM6 locus to the extent that the endogenous ADAM6 locus remains functional. In 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 segments employing a ing vector with an upstream homology arm and a downstream 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 homology arm that includes a all or a functional portion of a mouse ADAM6 locus. in various aspects, employing mice that contain an ectopic sequence that encodes a mouse ADAM6 protein or an ortholog or homolog or functional homolog thereof are useful where modifications disrupt the function of endogenous mouse ADAM6. The probability of disrupting endogenous mouse ADAM6 function is high when making cations to mouse globulin loci, in ular when modifying mouse globulin heavy chain variable s 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 (e.g., with VK or V)» sequences) in whole or in part. Methods for making the genetic modifications described for the mice described below are known to those skilled in the art.

Mice containing an ectopic sequence ng a mouse ADAM6 protein, or a substantially identical or similar n that confers the fertility benefits of a mouse ADAM6 protein, are particularly useful in conjunction with modifications to a mouse immunoglobulin heavy chain variable gene locus that disrupt or delete the endogenous mouse ADAM6 sequence. Although primarily described in connection with mice that express antibodies with human variable regions and mouse constant regions, such mice are useful in connection with any genetic modifications that t endogenous mouse ADAM6 genes.

Persons of skill will recognize that this encompasses a wide variety of genetically modified mice that contain modifications of mouse immunoglobulin heavy chain variable gene loci.

These include, for example, mice with a deletion or a replacement of all or a portion of mouse globulin heavy chain gene segments, regardless of other modifications.

Non-limiting examples are bed below. 1] In some aspects, genetically modified mice are ed that comprise an ectopic mouse, , or other ADAM6 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 various embodiments, other ADAM6 gene orthologs or homologs or fragments functional in a mouse may include ces from bovine, canine, primate, rabbit or other man sequences.

In one aspect, a mouse is provided that comprises an ectopic ADAM6 sequence that encodes a functional ADAM6 protein, a replacement of all or ntially all mouse VH gene segments with one or more human VH gene segments; a replacement of all or substantially all mouse DH gene ts 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 ment, the mouse further ses a replacement of a mouse CH1 nucleotide sequence with a human CH1 nucleotide sequence. In one embodiment, the mouse r comprises a ement of a mouse hinge nucleotide ce 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 comprises a replacement of a mouse immunoglobulin light chain constant region nucleotide sequence with a human immunoglobulin light chain constant region nucleotide sequence. 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 comprises an ectopic ADAMS sequence that encodes a functional ADAM6 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 ally 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 ts (e.g., JK or J7»), wherein the gene segments are operabiy linked to an endogenous mouse hinge region, wherein the mouse forms a nged immunoglobulin chain gene that contains, from 5’ to 3’ in the direction of ription, 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 . in one embodiment, the J region is a human JH region. ln one embodiment, the J region is a human J)» region.

In one embodiment, the human VL region is selected from a human V7t 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 VIC, 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 J7»; 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 speciﬁc embodiment, recombination recognition sequences are modified so as to allow for productive rearrangements to occur between recited V, D, and J gene segments or between d V and J gene segments.

In one aspect, a mouse is provided that comprises an ectopic ADAMS sequence that encodes a onai ADAMS 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 gene segment and JH gene segments with human JL gene ts; wherein the mouse lacks a CH1 and/or hinge region. in one embodiment, the mouse lacks a sequence encoding a CH1 domain. in one embodiment, the mouse lacks a sequence encoding a hinge region. In one embodiment, the mouse lacks a ce encoding a CH1 domain and a hinge region.

In a specific embodiment, the mouse expresses a g protein that comprises a human immunogiobulin light chain variable domain 0» 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 encodes a functional ADAMS protein (or ortholog or homolog or functional fragment f), a replacement of all or substantially all mouse VH gene ts 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 nt region gene ce encoding a CH1 region, a hinge region, 3 CH1 and a hinge , or a CH1 region and a hinge region and a CH2 region.

In one embodiment, the mouse makes a single le domain binding n comprising a homodimer selected from the ing: (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 endogenous 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 ce is on an episome (e.g., a chromosome) not found in a wild-type mouse. in one embodiment, the mouse further comprises a disabled endogenous immunogiobulin light chain locus. in a specific embodiment, the endogenous immunogiobulin light chain locus is selected from a kappa (K) and a lambda (A) light chain locus. in a specific embodiment, the mouse comprises a disabled endogenous K light chain locus and a disabled A light chain locus, wherein the mouse expresses an antibody that comprises a human immunogiobulin heavy chain variable domain and a human giobulin light chain domain. In one embodiment, the human immunogiobulin light chain domain is selected from a human K light chain domain and a human h light chain domain.

In one aspect, a genetically modified animal is provided that expresses a chimeric antibody and expresses an ADAM6 protein or og or homolog thereof that is functional in the cally modified animal. in one embodiment, the genetically modified animal is selected from a mouse and a rat. In one embodiment, the genetically modified animal is a mouse, and the ADAMS protein or ortholog or homolog f is from a mouse strain that is a different strain than the genetically ed animal. In one embodiment, the genetically modified animal is a rodent of family Cricetidae (e.g., a hamster, a New World rat or mouse, a vole), and the ADAMS protein ortholog or homolog is from a rodent of family Muridae (e.g., true mouse or rat, gerbil, spiny mouse, crested rat). In one embodiment, the genetically modified animal is a rodent of the family Muridae, and the ADAMS protein ortholog or homolog is from a rodent of family Cricetidae. in one ment, the chimeric dy comprises a human variable domain and a constant region sequence of a rodent. ln one embodiment, the rodent is selected from a rodent of the family Cricetidae and a rodent of family Muridae, in a specific embodiment, the rodent of the family Cricetidae and of the family Muridae is a mouse. In a specific embodiment, the rodent of the family Cricetidae and of the family Muridae is a rat. in one embodiment, the chimeric antibody comprises a human variable domain and a constant domain from an animal selected from a mouse or rat; in a specific embodiment, the mouse or rat is selected from the family Cricetidae and the family Muridae. in one embodiment, the chimeric antibody comprises a human heavy chain variable domain, a human light chain variable domain and a constant region sequence derived from a rodent selected from mouse and rat, wherein the human heavy chain variable domain and the human light chain are cognate. In a specific embodiment, cognate includes that the human heavy chain and the human light chain variable domains are from a single B cell that expresses the human light chain variable domain and the human heavy chain variable domain together and present the variable domains together on the e of an individual B cell. 8] In one embodiment, the chimeric antibody is expressed from an immunoglobulin locus. in one embodiment, the heavy chain variable domain of the chimeric dy is expressed from a rearranged endogenous immunoglobulin heavy chain locus. In one embodiment, the light chain variable domain of the chimeric dy is expressed from a rearranged endogenous immunoglobulin light chain locus. in one embodiment, the heavy chain variable domain of the chimeric antibody and/or the light chain variable domain of the chimeric antibody is sed from a rearranged transgene (e.g., a rearranged nucleic acid ce derived from an unrearranged nucleic acid sequence integrated into the ’s genome at a locus other than an endogenous immunoglobulin locus). in one embodiment, the light chain variable domain of the chimeric antibody is expressed from a rearranged transgene (e.g., a rearranged nucleic acid sequence derived from an unrearranged nucleic acid sequence integrated into the animal’s genome at a locus other than an nous immunoglobulin locus).

In a specific embodiment, the ene is expressed from a transcriptionally active locus, e.g., a ROSA26 locus, 6.9., a murine (e.g., mouse) ROSA26 locus.

In one aspect, a non—human animal is ed, comprising a humanized immunoglobulin heavy chain locus, wherein the zed immunoglobulin heavy chain locus comprises a non-human ADAMS sequence or ortholog or homolog thereof.

In one embodiment, the man animal is a rodent selected from a mouse, a rat, and a hamster.

In one embodiment, the non-human ADAMS ortholog or homolog is a sequence that is orthologous and/or homologous to a mouse ADAMS sequence, wherein the ortholog or homolog is onal in the non-human animal.

In one embodiment, the non-human animal is selected from a mouse, a rat, and a hamster and the ADAMS ortholog or homolog is from a non-human animal selected from a mouse, a rat, and a hamster. In a specific embodiment, the non—human animal is a mouse and the ADAMS ortholog or homolog is from an animal that is ed from a different mouse species, a rat, and a hamster. In specific embodiment, the non-human animal is a rat, and the ADAMS ortholog or homolog is from a rodent that is selected from a different rat species, a mouse, and a hamster. In a specific embodiment, the non-human animal is a hamster, and the ADAMS og or homolog is form a rodent that is selected from a different hamster species, a mouse, and a rat.

In a specific embodiment, the non—human animal is from the suborder Myomorpha, and the ADAMS sequence is from an animal selected from a rodent of superfamily Dipodoidea and a rodent of the superfamily Muroidea. In a specific embodiment, the rodent is a mouse of amily Muroidea, and the ADAMS ortholog or homolog is from a mouse or a rat or a hamster of superfamily Muroidea.

In one embodiment, the humanized heavy chain locus comprises one or more human VH gene ts, one or more human DH gene segments and one or more human JH gene segments. In a specific embodiment, the one or more human VH gene ts, one or more human DH gene segments and one or more human JH gene segments are ly linked to one or more human, ic and/or rodent (e.g., mouse or rat) constant region genes. In one embodiment, the constant region genes are mouse.

In one embodiment, the nt region genes are rat. In one embodiment, the constant region genes are hamster. In one embodiment, the constant region genes comprise a ce selected from a hinge, a CH2, a CH3, and a combination thereof. In ic embodiment, the constant region genes comprise a hinge, a CH2, and a CH3 sequence.

In one embodiment, the non-human ADAMS sequence is contiguous with a human immunoglobulin heavy chain sequence. In one embodiment, the non—human ADAMS sequence is positioned within a human immunoglobulin heavy chain sequence. In a specific embodiment, the human immunoglobulin heavy chain sequence comprises a V, D and/or J gene segment.

In one embodiment, the non-human ADAM6 sequence is positioned between two V gene segments. In one embodiment, the non—human ADAM6 sequence is juxtaposed between a V and a D gene segment. In one embodiment, the mouse ADAM6 sequence is positioned between a V and a J gene segment. In one ment, the mouse ADAM6 ce is juxtaposed between a D and a J gene segment.

In one aspect, a genetically modified non-human animal is provided, comprising a B cell that expresses a human VH domain e with a human VL domain from an immunoglobulin locus, wherein the non-human animal expresses a non-immunoglobulin man protein from the immunoglobulin locus. In one embodiment, the non- immunoglobulin non-human protein is an ADAM protein. In a specific embodiment, the ADAM protein is an ADAM6 protein or homolog or ortholog or functional fragment thereof.

In one embodiment the non—human animal is a rodent (e.g., mouse or rat). In one embodiment, the rodent is of family Muridae. In one embodiment, the rodent is of subfamily Murinae. In a ic embodiment, the rodent of subfamily Murinae is selected from a mouse and a rat.

In one ment, the non-immunoglobulin non-human protein is a rodent protein. In one embodiment, the rodent is of family e. In one embodiment, the rodent is of subfamily Murinae. In a specific embodiment, the rodent is selected from a mouse, a rat, and a hamster.

In one embodiment, the human VH and VL domains are attached directly or through a linker to an immunoglobulin constant domain sequence. In a speciﬁc embodiment, the constant domain sequence comprises a sequence selected from a hinge, a CH2 a CH3, and a combination thereof. In a specific embodiment, the human VL domain is selected from a VK or a V?» .

In one aspect, a genetically modified non-human animal is provided, comprising in its germline a human immunoglobulin sequence, wherein the sperm of a male non- human animal is characterized by an in vivo migration defect. In one embodiment, the in vivo ion defect comprises an ity of the sperm of the male non—human animal to e from a uterus through an t of a female non-human animal of the same s. In one embodiment, the man animal lacks a nucleotide sequence that s and ADAM6 protein or functional fragment thereof. In a specific embodiment, the ADAM6 protein or functional fragment thereof includes an ADAM6a andi’or an ADAMBb protein or functional fragments thereof. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from a mouse, a rat, and a hamster.

In one aspect, a non—human animal is provided, comprising a human immunoglobulin sequence contiguous with a man sequence that s an ADAMS protein or ortholog or homolog or functional nt thereof. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from a mouse, a rat, and a hamster.

In one embodiment, the human immunoglobulin sequence is an immunogtobulin heavy chain sequence. In one embodiment, the immunoglobulin sequence comprises one or more VH gene segments. In one embodiment, the human immunoglobulin sequence ses one or more DH gene segments. In one embodiment, the human immunoglobutin sequence comprises one or more JH gene segments. In one embodiment, the human immunogIobuIin sequence comprises one or more VH gene ts, one or more DH gene segments and one or more JH gene segments.

In one embodiment, the gIobuIin sequence comprises one or more VH gene segments have a high frequency in natural human repertoires. In a specific ment, the one or more VH gene ts se no more than two VH gene ts, no more than three VH gene segments, no more than four VH gene segments, no more than five VH gene segments, no more than six VH gene segments, no more than seven VH gene segments, no more than eight VH gene segments, no more than nine VH gene segments, no more than 10 VH gene segments, no more than 11 VH gene segments, no more than 12 VH gene segments, no more than 13 VH gene segments, no more than 14 VH gene segments, no more than 15 VH gene segments, no more than 16, VH gene segments, no more than 17 VH gene segments, no more than 18 VH gene segments, no more than 19 VH gene segments, no more than 20 VH gene segments, no more than 21 VH gene segments, no more than 22 VH gene segments or no more than 23 VH gene segments.

In a specific embodiment, the one or more VH gene segments comprise five VH gene ts. In a specific embodiment, the one or more VH gene segments comprise VH gene segments. In a specific embodiment, the one or more VH gene segments comprise 15 VH gene segments. In a specific embodiment, the one or more VH gene segments comprise 20 VH gene segments.

In various embodiments, the VH gene segments are selected from VH6-1, VH1~2, VH1-3, VH2-5, VHS-7, VH1-8, VH3-9, VH3-11, , VH3—15, VH3-16, VH1-18, VH3-20, VH3— 21, VH3-23, VH1-24, VH2-26, VH4-28, , VH4—31, VH3-33, VH4-34, VH3—35, VH3-38, VH4-39, VH3-43, VH1-45, , VH3-48, VH3—49, VHS-51, VH3-53, , VH4-59, VH4—61, VHS-64, VHS-68, VH1-69, VH2-70, VH3—72, VHS—73 and VH3—74.

In various embodiments, the VH gene segments are selected from VH1-2, VH1—8, VH1-18, VH1-46, VH1-69, VHS—7, VHS-9, VH3-11, VH3—13, VH3-15, VHS-21, VHS-23, VH3—30, VHS-33, VHS-43, VH3-48, VH4-31, VH4-34, VH4-39, VH4—59, VHS—51 and VH6-1.

In various embodiments, the VH gene segments are selected from VH1-18, VH1- 46, VH1-69, VH3-7, VH3-11, VH3-15, VHS-21, VH3-23, VH3-30, VH3-33, VH3—48, VH4-34, VH4- 39, VH4-59 and VHS-51.

In various embodiments, the VH gene segments are selected from , VH1- 69, VH3-7, VHS-11, , VHS-21, VH3—23, VH3-30, VH3-43, VH3-48, VH4-39, VH4-59 and VHS—51.

In various embodiments, the VH gene ts are selected from VH1-18, VH3— 11, VH3-21, VH3—23, VH3—30, VH4-39 and VH4-59.

In various embodiments, the VH gene segments are selected from VH1-18, VH3- 21, VHS-23, VH3—30 and .

In various embodiments, the VH gene segments are selected from VH1—18, VH3- 23 and VH4-39.

In various embodiments, the VH gene segments are ed from VHS-21, VH3- 23 and VH3-30.

In various embodiments, the VH gene segments are ed from , VH3— and VH4-39.

In a specific embodiment, human immunoglobuIin sequence comprises at least 18 VH gene ts, 27 DH gene segments and six JH gene segments. In a ic embodiment, the human immunoglobuIin sequence comprises at Ieast 39 VH gene segments, 27 DH gene segments and six JH gene segments. In a specific embodiment, the human immunoglobuIin ce comprises at least 80 VH gene segments, 27 DH gene segments and six JH gene segments.

In one embodiment, the non-human animal is a mouse, and the mouse comprises a replacement of endogenous mouse VH gene ts with one or more human VH gene ts, wherein the human VH gene segments are operably Iinked to a mouse CH region gene, such that the mouse rearranges the human VH gene segments and expresses a reverse chimeric immunoglobuIin heavy chain that comprises a human VH domain and a mouse CH. In one embodiment, 90—100% of unrearranged mouse VH gene segments are replaced with at least one ranged human VH gene segment. In a specific embodiment, all or substantially all of the endogenous mouse VH gene segments are replaced with at Ieast one unrearranged human VH gene segment. In one embodiment, the replacement is with at Ieast 19, at least 39, or at least 80 or 81 unrearranged human VH gene segments. In one embodiment, the replacement is with at least 12 functionai unrearranged human VH gene segments, at least 25 functional unrearranged human VH gene segments, or at least 43 functional ranged human VH gene segments. In one embodiment, the mouse comprises a replacement of all mouse DH and JH segments with at least one unrearranged human DH segment and at least one unrearranged human JH segment. In one ment, the at least one unrearranged human DH segment is selected from 1-1, 1—7, 1-26, 2-8, 2-15, 3-3, 3-10, 3-16, 3-22, 5-5, 5- 12, 6-6, 6—13, 7-27, and a ation thereof. In one embodiment, the at least one unrearranged human JH segment is selected from 1, 2, 3, 4, 5, 6, and a combination thereof. In a specific embodiment, the one or more human VH gene segment is selected from a 1—2, 1—8, 1-24, 1-69, 2-5, 3—7, 3—9, 3—1 1, 3-13, 3-15, 3-20, 3—23, 3-30, 3-33, 3-48, 3- 53, 4—31, 4-39, 4—59, 5-51, a 6-1 human VH gene segment, and a combination thereof.

In s embodiments, the human immunoglobulin ce is in operable linkage with a constant region in the germline of the non-human animal (e.g., the , e.g., the mouse, rat, or hamster). In one embodiment, the constant region is a human, chimeric human/mouse or chimeric human/rat or chimeric human/hamster, a mouse, a rat, or a hamster constant region. In one embodiment, the constant region is a rodent (e.g., mouse or rat or hamster) constant . In a specific embodiment, the rodent is a mouse or rat. In various ments, the constant region comprises at least a CH2 domain and a CH3 domain.

In one embodiment, the human immunoglobulin heavy chain sequence is located at an immunoglobulin heavy chain locus in the germline of the non-human animal (e.g., the rodent, e.g., the mouse or rat or hamster). In one ment, the human immunoglobulin heavy chain sequence is located at a non-immunoglobulin heavy chain locus in the germline of the non—human animal, wherein the non-heavy chain locus is a transcriptionally active locus. In a specific ment, the non-heavy chain locus is a ROSA26 locus.

In various aspects, the non-human animal further comprises a human immunoglobulin light chain sequence (e.g., one or more unrearranged light chain V and J sequences, or one or more rearranged VJ sequences) in the ne of the non-human animal. In a specific embodiment, the immunoglobulin light chain sequence is an immunoglobulin x light chain sequence. In one embodiment, the human immunoglobulin light chain sequence comprises one or more VL gene ts. In one embodiment, the human immunoglobulin light chain sequence comprises one or more JL gene segments. In one embodiment, the human immunoglobulin light chain sequence comprises one or more VL gene segments and one or more JL gene segments. In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 16 VK gene segments and five JK gene segments. In a ic embodiment, the human immunoglobulin light chain sequence comprises at least 30 VK gene segments and five JK gene segments. In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 4O VK gene segments and five JK gene segments. in various embodiments, the human globulin light chain sequence is in operable linkage with a nt region in the germline of the non-human animal (e.g., rodent, e.g., mouse or rat or hamster). In one embodiment, the constant region is a human, chimeric human/rodent, mouse, rat, or hamster nt region. In a specific embodiment, the nt region is a mouse or rat constant region. in a specific embodiment, the constant region is a mouse K constant (mCK) region or a rat K constant (rCK) region. ln one embodiment, the non-human animal is a mouse and the mouse ses a replacement of all or substantially all VK and JK gene segments with at least six human VK gene segments and at least one JK gene segment. in one embodiment, all or substantially all VK and JK gene segments are replaced with at least 16 human VK gene segments (human VK) and at least one JK gene segment. ln one embodiment, all or substantially all VK and JK gene segments are replaced with at least 30 human VK gene segments and at least one JK gene t. in one embodiment, all or substantially all VK and JK gene segments are replaced with at least 40 human VK gene segments and at least one JK gene segment. in one embodiment, the at least one Jic gene segment comprises two, three, four, or five human JK gene segments. in one embodiment, the human VK gene segments comprise VK4—1,Vi<5-2, VK7-3, VK2-4, VK1-5, and VK1-6. in one embodiment, the VK gene segments comprise VK3-7,VK1-8,VK1-9,VK2-10,VK3-11,VK1-12,VK1-13,VK2-14,VK3-15 and . in one embodiment, the human VK gene segments comprise VK1-17, VK2-18, VK2-19, VIC?)- , VK6-21, VK1-22, VK1-23, VK2-24, VK3-25, VK2-26, VK1—27, VK2-28, VK2-29, and VK2- . in one embodiment, the human VK gene segments comprise VK3-31, VK1-32, VK1-33, VK3-34, VK1-35, , VK1-37, VK2-38, VK1-39, and .

In a specific embodiment, the VK gene segments comprise contiguous human immunoglobulin K gene segments spanning the human immunoglobulin K light chain locus from VK4-1 through VK2-40, and the JK gene segments comprise contiguous gene segments spanning the human immunoglobulin K light chain locus from JK1 through JK5.

In one embodiment, the human globulin light chain ce is located at an immunoglobulin light chain locus in the germline of the non-human animal. in a specific embodiment, the immunoglobulin light chain locus in the germline of the non- human animal is an immunoglobulin K light chain locus. in one embodiment, the human immunoglobulin light chain sequence is located at a non-immunoglobulin light chain locus in the germline of the non-human animal that is riptionally active. In a specific embodiment, the non-immunoglobulin locus is a ROSA26 locus.

In one aspect, a method of making a human antibody is ed, wherein the human antibody comprises variable domains derived from one or more le region nucleic acid sequences encoded in a cell of a non-human animal as described herein. 6] in one aspect, a pharmaceutical composition is provided, comprising a ptide that ses dy or antibody fragment that is derived from one or more variable region nucleic acid ces isolated from a non-human animal as described herein. In one embodiment, the polypeptide is an antibody. In one embodiment, the polypeptide is a heavy chain only antibody. In one embodiment, the polypeptide is a single chain variable fragment (e.g., an scFv).

In one aspect, use of a non-human animal as described herein to make an antibody is provided. In various embodiments, the antibody comprises one or more variable domains that are derived from one or more variable region nucleic acid sequences isolated from the non—human animal. in a specific embodiment, the variable region nucleic acid sequences comprise globulin heavy chain gene segments. In a ic ment, the variable region nucleic acid sequences comprise immunoglobulin light chain gene segments.

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

Example 1 Humanization of Mouse lmmunoglobulin Genes 9] Human and mouse bacterial artificial chromosomes (BACs) were used to engineer 13 different BAC ing s (BACvecs) for humanization of the mouse immunoglobulin heavy chain and K light chain loci. Tables 1 and 2 set forth descriptions of the steps performed for construction of all BACvecs employed for the humanization of mouse immunoglobulin heavy chain and K light chain loci, respectively.

Identification of human and mouse BACs. Mouse BACs that span the 5’ and 3’ ends of the immunoglobulin heavy chain and K light chain loci were identified by hybridization of filters spotted with BAC library or by PCR screening mouse BAC library 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 s 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 ch D library, TIGR).

Construction of BACvecs by bacterial homologous recombination and ligation. Bacterial homologous recombination (BHR) was performed as described (Valenzuela et al., 2003; Zhang et al., 1998, A new logic for DNA engineering using recombination in Escherichia coli, Nat Genet 20:123-128). in most cases, linear fragments were generated by ligating PCR-generated gy boxes to cloned cassettes followed by gel isolation of ligation products and electroporation into BHR-competent bacteria harboring the target BAC. After selection on appropriate antibiotic petri dishes, correctly recombined BACs were identified by PCR across both novel junctions followed by restriction anaiysis on pulsed-field gels (Schwartz and Cantor, 1984, Separation of yeast chromosome-sized DNAs by pulsed field nt gel electrophoresis, Cell 37:67-75) and spot-checking by PCR using s distributed across the human sequences. [0003221 A 3hVH BACvec was constructed using three sequential BHR steps for the l step of humanization of the immunoglobulin heavy chain locus ( and Table 1). In 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 (H81), a gene that confers kanamycin resistance in bacteria and G418 resistance in animals cells (kanR) and a site-specific ination site (9.9., onP). In the second step (Step 2), a second te was introduced just downstream from the last JH segment that contains a second region of homology to the mouse immunoglobulin heavy chain locus (H82) and a gene that s ance in bacteria to nomycin (specR). This second step included deleting human immunoglobulin heavy chain locus ces downstream 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 ated into a mouse BAC (82) by BHR h the two regions of homology (H81 and H82). The drug selections forfirst (cm/ken), second (spec/kan) and third (cm/ken) steps were designed to be specific for the d products. Modified BAC clones were analyzed by pulse-filed gel electrophoresis (PFGE) after digestion with restriction enzymes to determine appropriate construction (FlG. 4B).

In a similar fashion, 12 additional BACvecs were engineered for humanization of the heavy chain and K light chain loci. In some instances, BAC ligation was med in lieu of BHR to conjoin two large BACs h introduction of rare ction sites into both parental BACvecs by BHR along with careful placement of selectable markers. This allowed for the survival of the desired ligation product upon selection with specific drug marker combinations. Recombinant BACs obtained by ligation after ion with rare restriction enzymes were identified and screened in a similar fashion to those obtained by BHR (as described above).

Table 1 BACvec Step Description Process Insert upstream mouse homology box Into human 1 BHR proximal BAC OTB—257202 Insert downstream mouse homology box Into human proximal BAC OTB-257202 Insert 7hDH/9hJH into mouse proximal BAC CT7— 3 BHR 302a07 to create .3th BACvec Insert cassette at distal end of mouse IgH locus using Insert specR marker at downstream end of 3hVH insertion using human BAC OTB-257202 Insert l-Ceul and Not sites flanking puroR at am and of 3hVH insertion Insert Not site at downstream end of Rel2-408p02 BAC (JO BHR (=10 kb downstream of VH2-5) Insert l—Ceul site at am end of Rel2-408p02 BAC (=23 kb upstream of VH1-18) gigate 1842kb fragment from step 4 into 153kb vector Ligation 18th r°.m Step n—.=85kb and g 65kb homology to 3hVH Insert cassette and Not site at distal end of mouse IgH locus in CT7—253i20 BAC nSubclone mouse distal homology arm for insertion Ligation upstream from human BACs a Insert 20 kb mouse arm upstream of Rel2-408p02 Swap selection cassette from hng to neoR to create 18th BACvec Insert I-Ceul and Pl-Scel sites flanking hng into distal end of human BAC CTD-2534n10 Insert CmR at proxrmal end of CTD—2534n10 BAC to 39th allow for selection for ligation to RP11-72n10 BAC Insert l site into RP11—72n10 BAC for ligation to CTD—2534n10 BAC of RP11—72n10 BAC uct of step 2 repIacmg hng —-distalhomologyarm usrng CT7-253i20 BAC Insert specR and l-Ceul srte at distal end of mouse distal homology arm Ligate mouse distal homology arm onto human insert L'iga iont' from step 5 Swap selection cassette from neo to hyg usrng UbCp and pA as homology boxes to create 39hVH BACvec Insert specR at proximal end of human CTD—3074b5 1 BHR Insert Ascl site at distal end of human CTD-3074b5 2 BHR Insert hng and Ascl site at proximal end of mouse 53ml” BHR distal homology arm using CT7-253i20 BAC Ligate mouse distal homology arm onto construct from A L''gat'onl step 2 Swap ion cassette from hyg to neo using UbCp BHR and pA as homology boxes to create 53hVH BACvec Insert Pl-Scel and l-Ceul sites flanking spec at distaI 1 BHR end of human CTD-2195p5 BAC insert l—Ceul site at proximal end of RP11-926p12 BAC for ligation to CTD-2195p5 BAC 70hVH-—-926p12 BAC for ligation of mouse arm -Ligate mouse distal gy arm onto construct from A Ligation step 3 Ligate mouse distal gy arm and hIgH fragment from RP11—926p12 BAC onto CTD-2195p5 BAC to Ligation create 70 hVH BACvec of CTD-Z313e3 BAC 8()th Ligate mouse distal homology arm onto human CTD— 2 Li9ation 2313e3 BAC from step 1 to create 80th BACvec Table 2 BACvec Step Description Process Insert loxP site within mouse J-C intron usrng CT7- I 9K'PC 1 BHR 254m04 BAC Insert onP site at distal end of mouse IgK locus usmg | 9“-DC 1 BHR . CT7-302912 BAG Insert PI—Scel site R400 bp downstream from hJK5 in 1 BHR 66j12 BAC Insert I Ceul and Ascl Sites ng hng at distal- - BHR end of CTD-2366112 BAG - Insert I—Ceul and PI Scel sites flanking puroR 3 BHR downstream from mJK using CT7 254m04 BAC Insert hlgVK/JK upstream from mouse EHhK/CK using 6hVK 4 Ligation uct 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 BAC Ligate mouse distal homology arm upstream of human insert in construct of step 6 to create 6th( BACvec 1 Insert NeoR at distal end of RP11-1061b13 BAC Replace cmR in construct of step 1 with SpecR Insert Hyg selection cassette at distal end of mouse 16an ng locus using CT7—302912 BAG Ligate mouse distal homology arm upstream of human insert from construct of step 2 to create 1(5th 1 insert Hng at distal end of RP1 1-9996 BAG HR 2 e cmR in construct of step 1 with specR HR Insert Neo selection cassette at distal end of mouse 30hVK ng locus using CT7-302g12 BAG Ligate mouse distal homology arm upstream of human insert from construct of step 2 to create 30hVK Insert NeoR at distal end of hIgH locus in CTD— 1 BHR 2559d6 BAC Replace cmR in construct of step 1 with specR HR 40hVK Ligate mouse distal homology arm upstream of 3 human Insert in construct of step 2 to create 4Oth( Ligation Modification of embryonic stem (ES) cells and generation of mice. ES cell (F1 H4) targeting was performed using the VELOCIGENE® genetic engineering method as described (Valenzuela et al., 2003). Derivation of mice from modified ES cells by either blastocyst zuela et al., 2003) or 8-cell injection (Poueymirou et at, 2007, F0 generation mice fully derived from gene-targeted embryonic stem cells ng immediate phenotypic analyses, Nat hnol 25:91-99) was as described. Targeted ES cells and mice were confirmed by screening DNA from ES cells or mice with unique sets of probes and s in a PCR based assay (e.g., , SB and 30). 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 l Cell Repositories ll Institute for Medical Research, Camden, NJ). FISH was performed on targeted ES cells as described (Valenzuela etal., 2003). Probes corresponding to either mouse BAC DNA or human BAC DNA were d by nick translation (Invitrogen) with the fluorescently labeled dUTP tides spectrum orange or spectrum green (Vysis). lmmunoglobulin 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 containing all VH, DH and JH gene segments with about one Mb of contiguous human genomic ce containing the equivalent human gene segments ( and Table i) using VELOCIGENE® c engineering technology (see, 6.9., US Pat. No. 6,586,251 and Valenzuela et al., 2003), The intron between JH gene segments and constant region genes (the J—C intron) contains a transcriptional enhancer (Neuberger, 1983, Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells, EMBO J -1378) followed by a region of simple repeats required for recombination during isotype switching (Kataoka et al., 1980, Rearrangement of immunoglobulin gamma 1-chain gene and mechanism for heavy-chain class switch, PNAS USA 77:919-923). The junction between 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 ve both efficient expression and class switching of the humanized heavy chain locus within the mouse. The exact nucleotide position of this and subsequentjunctions in all the replacements was possible by use of the VELOCIGENE® genetic engineering method (supra), which employed ial homologous recombination driven by synthesized oligonucleotides. Thus, the proximal junction was placed about 200 bp downstream from the last JH gene t and the distal on 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 5. The mouse VH1-86 55) gene segment is the most distal heavy chain variable gene segment, reported to be a gene in C57BL/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). 8] A first ion of human immunoglobulin 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 ts inserted into the proximal end of the mouse lgH locus, with a concomitant 16.6 kb deletion of mouse genomic sequence, using about 75 kb of mouse homology arms (Step A, FiG. 2A; Tables 1 and 3, 3hVH). This large 144kb insertion 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 integrity of the large human insert was ed using multiple probes spanning the entire insertion (FlG. 3A, 3B and 30). Because many rounds of sequential ES cell ing were anticipated, targeted ES cell clones at this, and all subsequent, steps were subjected to ypic analysis ) and only those clones showing normal karyotypes in at least 17 of 20 s were utilized for subsequent steps.

Targeted ES cells from Step A were re—targeted with a BACvec that produced a 19 kb deletion 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 chromosome, 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 DQ52, as well as the two resistance genes, were flanked by loxP sites; DQ52 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 cassette to homozygosity in high G418 (Mortensen et al., 1992, Production of homozygous mutant ES cells with a single targeting construct, Mo/ Celt 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 z11%) even in the absence of drug selection (Zheng et al., 2000, Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications, Mol Cell Biol :648—655). ln 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 on 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 ance of a PCR product across the deletion point containing the sole ing loxP site. Further, the deletion was confirmed by fluorescence in situ hybridization (data not shown). 0] The remainder of the human heavy chain variable region was added to the 3hVH allele in a series of 5 steps using the VELOClGENE® genetic engineering method (Steps E-H, ), with each step involving precise insertion of up to 210 kb of human gene sequences. For each step, the proximal end of each new BACvec was designed to overlap the most distal human sequences of the previous step and the distal end of each new BACvec contained the same distal region of mouse homology as used in Step A. The BACvecs 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 23th Hybrid Allele. ing for Steps E through i was assayed by loss of the previous selection cassette. in the final step (Step I, ), the neo selection cassette, flanked by Frt sites (McLeod et a/., 1986, Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 s circle, Mol Cell Biol 6:3357-3367), was removed by transient FLPe expression (Buchholz et al, 1998, Improved properties of FLP recombinase evolved by cycling mutagenesis, Nat hnol 162657—662). The human sequences of the s 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 med at every step using multiple probes spanning the inserted human sequences (as described above, 9.9., FlG. 3A, 3B and 3C). Only those clones with normal karyotype and germline potential were carried forward in each step. ES cells from the final step were still able to contribute to the germline after nine tial manipulations (Table 3). Mice homozygous for each of the heavy chain alleles were viable, appeared y and demonstrated an essentially ype humoral immune system (see Example 3).

Table 3 Hybrid Human ing Targeting % Total Functional Allele sequence construct efficiency usage VH VH 0) L0 53th 655 kb 186 kb 0.4% 65 70th 238 kb 05% 80th 940 kb 124 kb 0.2% “-100 Immunoglobulin K Light Chain le Gene Locus. The K light chain variable region was humanized 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 proximal human VK and JK gene segments in a manner similar to that of the heavy chain (HS. 13; Tables 2 and 4).

The variable region of the human K light chain locus contains two nearly identical 400 kb repeats separated by an 800 kb spacer (Weichhold et at, 1993, The human immunoglobulin kappa locus consists of two copies that are zed in te polarity, Genomics 161503-511). Because the repeats are so similar, nearly all of the locus diversity can be uced 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 et al., 1993, The immunoglobulin kappa locus: polymorphism and haplotypes of Caucasoid and non-Caucasoid individuals, Hum Genet 91:261-267). The inventors replaced about three Mb of mouse K light chain variable gene sequence with about 0.5 Mb of human K light chain variable gene sequence to effectively replace all of the mouse VK and JK gene segments 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 region, containing all VK and JK gene segments, was deleted in a step process before any human sequence was added. First, a neo cassette was introduced at the al end of the le region (Step A, FlG. 20). Next, a hyg cassette was inserted at the distal end of the K locus (Step B, ). Recombinase recognition sites (e.g., loxP) 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, FlG. 2C). 3] 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 globulin K light chain sequence inserted in a single step, using methods similar to those ed for the heavy chain (see Example 1). The final hygromycin resistance gene was removed by transient FLPe expression. As with the heavy chain, ed ES cell clones were evaluated for integrity of the entire human insert, normal ype and germ-line potential after every step. Mice homozygous for each of the K light chain s were generated and found to be healthy and of normal ance.

Table 4 Hybrid Human Targeting Targeting % Total Functional Allele sequence construct efficiency usage V1< VK --.--—-— —-_——--_ “n—--— 6th 0.3% 14 16th 0.4% 47 16 11 Example 2 Generation of Fully Humanized Mice by Combination of Multiple Humanized immunoglobulin Alleles At several points, ES cells bearing a portion of the human globulin heavy chain or K light chain variable repertoires as described in Example 1 were microinjected and the resulting mice bred to create le versions of VELOCIMMUNE® mice with progressively larger fractions of the human germline immunoglobulin repertoires (Table 5; and 5B). VELOCIMMUNE® 1 (V1) mice possess eighteen human VH gene segments and all of the human DH and JH gene segments combined with sixteen human VK gene segments and all the human JK gene segments. VELOClMMUNE® 2 (V2) and VELOCIMMUNE® (V3) mice have increased variable repertoires bearing a total of thirty—nine VH and thirty VK, and eighty VH and forty 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 all versions of VELOCIMMUNE® mice contain human variable regions linked to mouse constant regions.

The mouse k light chain loci remain intact in various ments of the VELOCIMMUNE® mice and serve as a comparator for efficiency of sion of the s VELOCIMMUNE® K light chain loci.

Mice doubly homozygous for both immunoglobulin heavy chain and K light chain humanizations were generated from a subset of the alleles described in Example 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 demonstrated reduced fertility, which resulted from loss of mouse ADAM6 activity. The mouse heavy chain le gene locus contains two embedded functional ADAM6 genes (ADAMBa and ). During humanization of the mouse heavy chain variable gene locus, the inserted human genomic sequence contained an ADAMS pseudogene. Mouse ADAM6 may be ed for fertility, and thus lack of mouse ADAM6 genes in zed heavy chain variable gene loci might lead to a reduction in fertility notwithstanding the presence of the human pseudogene. Examples 7- 11 describe the neering of mouse ADAM6 genes into a humanized heavy chain variable gene locus, and restoration of wild-type level fertility in mice with a humanized heavy chain immunoglobulin locus.

Table 5 Version of Heavy Chain K Light Chain VELOCIMMUNE® Human 5 VH, Human 5 VK.

Allele Allele Mouse VH (D :5e VK gene Example 3 cyte Populations in Mice with Humanized globulin Genes Mature B cell populations in the three different versions of VELOCIMMUNE® 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 , blocked with anti—mouse CD16/32 (BD Pharmingen), stained with the appropriate cocktail of antibodies and fixed with BD CYTOFIXTM all ing to the manufacturer’s instructions. Final cell pellets were resuspended in 0.5 mL staining buffer and analyzed using a BD FACSCALIBURTM and BD CELLQUEST PROTM software. All antibodies (BD Pharmingen) were prepared in a mass dilution/cocktail and added to a final concentration of 0.5 mg/1O5 cells.

Antibody cocktails for bone marrow (A—D) staining were as follows: A: anti— mouse lgMb—FlTC, anti—mouse lgMa—PE, anti—mouse CD45R(8220)-APC; B: anti-mouse CD43(S7)-PE, anti-mouse CD45R(8220)-APC; C: anti-mouse CDZ4(HSA)-PE; anti—mouse CD45R(BZZO)—APC; D: anti-mouse BP—1-PE, anti—mouse B220)-APC.

Antibody cocktails for spleen and inguinal lymph node (E—H) staining were as follows: E: anti-mouse lgMb-FITC, ouse lgMa—PE, anti-mouse B220)—APC; F: anti-mouse lg, M, x2, A3 Light Chain-FlTC, anti mouse lgx Light Chain-PE, ouse CD45R(8220)-APC; G: anti-mouse Ly6G/C—FlTC, anti-mouse DX5)-PE, anti- mouse CDHb-APC; H: anti-mouse CD4(L3T4)—FlTC, anti-mouse CD45R(BZZO)-PE, anti— mouse CDBa-APC. Results are shown in FlG. 6.

Lymphocytes isolated from spleen or lymph node of gous VELOClMMUNE® mice were stained for surface expression of the markers 8220 and lgM and analyzed using flow cytometry (. The sizes of the 8220* IgM“ mature B cell populations in all versions of VELOClMMUNE® mice tested were virtually identical to those of wild type mice, regardless of the number of VH gene segments they ned. In 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 humanized K light chain loci with normal mouse immunoglobulin 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 ate with the ability to generate wild type populations of mature B cells. In st, mice with randomly integrated fully—human immunoglobulin transgenes and inactivated mouse immunoglobulin loci have reduced s of B cells in these compartments, with the ty of the deficit depending on the number of variable gene segments included in the transgene (Green and Jakobovits, 1998, Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial 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 exclusion was examined in mice heterozygous for different versions of the humanized immunoglobulin heavy chain locus.

The humanization of the immunoglobulin loci was carried out in an F1 ES line (F1 H4, Valenzuela et al., 2003), derived from 129$6/SvaTac and C57BL/6NTac heterozygous embryos. The human heavy chain germline variable gene sequences are targeted to the 12936 allele, which carries the lgMa haplotype, whereas the unmodified mouse C57SBL/6N allele bears the lgMb haplotype. These allelic forms of lgM can be guished by flow cytometry using antibodies specific to the polymorphisms found in the lgMa or lgMb s. As shown in (bottom row), the B cells identified in mice zygous for each version of the humanized heavy chain locus only express a single allele, either lgMa (the humanized allele) or lgMb (the wild type alleie). This demonstrates that the mechanisms involved in c exclusion are intact in VELOCIMMUNE® mice. in addition, the relative number of B cells positive for the humanized allele (lgMa) is y proportional to the number of VH gene segments present. The humanized immunoglobulin locus is expressed in approximately 30% of the B cells in VELOCIMMUNE® 1 heterozygote mice, which have 18 human VH gene segments, and in 50% of the B cells in VELOCIMMUNE® 2 and 3 (not shown) heterozygote mice, with 39 and 80 human VH gene ts, respectively. Notably, the ratio of cells expressing the humanized versus wild type mouse allele (0.5 for MMUNE® 1 mice and 0.9 for VELOCIMMUNE® 2 mice) is greater than the ratio of the number of le gene segments contained in the zed versus wild type loci (0.2 for VELOCIMMUNE® 1 mice and 0.4 for MMUNE® 2 mice). This may indicate that the probability of allele 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 allele becomes accessible. In addition, the even distribution of cells 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 contrast, randomly integrated human immunoglobulin transgenes compete poorly with wild type mouse immunoglobulin loci (Bruggemann et al., 1989, A repertoire of monoclonal antibodies with human heavy chains from transgenic mice, PNAS 86:6709-6713; Green etal., 1994; Tuaillon etal., 1993, Human immunoglobulin heavy-chain minilocus recombination in enic mice: gene— segment use in mu and gamma transcripts, PNAS USA 90:3720-3724). This further demonstrates the immunoglobulins produced by VELOCIMMUNE® mice are functionally different than those produced by randomly ated transgenes in mice made by “knockout—plus—transgenic” ches. rphisms of the CK regions are not available in 12986 or CSYBL/GN to e allelic exclusion of humanized versus non-humanized K light chain loci. However, VELOClMMUNE® mice all s 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 relative to the number of cells expressing mouse A light chain was vely unchanged in VELOClMMUNE® mice compared with wild type mice, regardless of the number of human VK gene segments inserted at the K light chain locus ( third row from top). in addition there was no increase in the number of double positive (K plus A) cells, ting that productive recombination at the hybrid K light chain loci results in appropriate suppression of recombination of the mouse A light chain loci. in contrast, mice containing randomly integrated K light chain transgenes with inactivated mouse K light chain loci—but wild type mouse x light chain loci—exhibit dramatically sed MK ratios (Jakobovits, 1998), implying that the introduced K light chain transgenes do not function well in such mice. This further demonstrates the different functional outcome observed in immunoglobulins made by VELOClMMUNE® mice as compared to those made by “knockout-plus-transgenic” mice.

B cell Development. e the mature B cell populations in VELOClMMUNE® mice resemble those of wild type mice (described above), it is le 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 VELOClMMUNE® mice compared to wild type littermates.

Early B cell pment occurs in the bone marrow, and different stages of B cell differentiation are terized by changes in the types and amounts of cell surface marker expression. These differences in surface 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 rearrangement and sion of functional heavy chain protein, while transition from the pre-B to mature B stage is governed by the correct rearrangement and expression of a K or k 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.

Table 6 Bone Marrow Spleen Version of pro-B pre-B Immature Mature Emerging Mature MMUNE® , . . Bzzom Mice CD43” 0024‘“ 3220'° 8220'" BZZOhi 3220“) 3220'° g lgM” lgM" igivr No major defects were observed in B cell differentiation in any of the VELOClMMUNE® 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® mice. This demonstrates that ”reverse chimeric” immunoglobulin molecules possessing human le 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 contain randomly ated immunoglobulin transgenes and inactivated endogenous heavy chain or K light chain loci (Green and Jakobovits, 1998).

Example 4 Variable Gene Repertoire in Humanized globulin Mice Usage of human variable gene segments in the humanized antibody repertoire of VELOCIMMUNE® mice was ed by e transcriptase—polymerase chain reaction (RT—PCR) of human variable regions from multiple sources including splenocytes and hybridoma cells. Variable region sequence, gene t usage, somatic hypermutation, and junctional diversity of rearranged variable region gene segments were determined.

Briefly, total RNA was ted from 1x107 — 2x107 splenocytes or about 104 -— 105 hybridoma cells using TM (lnvitrogen) or Qiagen RNEASYTM Mini Kit n) and primed with mouse constant region specific primers using the SUPERSCRIPTTM l|| One-Step RT-PCR system (lnvitrogen). Reactions were d out with 2-5 pL of RNA from each sample using the aforementioned 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 /PCR conditions were med according to the manufacturer’s instructions. Primers sequences were based upon le sources (Wang and Stollar, 2000, Human immunogiobulin variable region gene analysis by single cell RT-PCR, J Immunol Methods 244:217-225; Ig-primer sets, Novagen). Where appropriate, nested secondary PCR reactions were carried out with pooled family-specific framework primers and the same mouse 3’ immunoglobulin constant—specific primer used in the y reaction. Aliquots (5 uL) from each reaction were analyzed by agarose ophoresis and reaction products were purified from agarose using a MONTAGETM Gel Extraction Kit (Millipore). Purified products were cloned using the TOPOTM TA Cloning System (lnvitrogen) and transformed into DH1OB E. coli cells by electroporation. individual clones were selected from each transformation on and grown in 2 mL LB broth cultures with otic selection ght at 37°C. Plasmid DNA was purified from bacterial es by a kit—based approach (Qiagen). immunogiobulin Variable Gene Usage. Plasmid DNA of both heavy chain and K light chain clones were sequenced with either T7 or M13 reverse primers on the ABl 3100 Genetic Analyzer (Applied Biosystems). Raw sequence data were imported into SEQUENCHERTM (v4.5, Gene Codes). Each sequence was assembled into contigs and aligned to human immunogiobulin ces using IMGT V-Quest (Brochet et al., 2008, IMGTN-QUEST: the highly customized and integrated system for IG and TR standardized V—J and V-D-J sequence analysis, Nucleic Acids Res 362W503-5O8) 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 is.

Mice were generated from ES cells containing the initial heavy chain cation (3hVH-CRE Hybrid Allele, bottom of HG. 2A) by RAG compiementation (Chen et al., 1993, RAG-Z-deficient cyst complementation: an assay of gene function in cyte development, PNAS USA 90:4528-4532), and cDNA was prepared from spienocyte 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 inserted human gene segments and subsequent splicing to either mouse lgM or lgG constant domains. Sequences derived from these cDNA clones (not shown) demonstrated that proper V(D)J recombination had occurred within the human variable gene sequences, that the rearranged human V(D)J gene segments were properly spliced in-frame to mouse constant s, and that class-switch recombination had occurred. Further sequence is of mRNA products of subsequent hybrid immunogiobulin loci was performed. {000351} in a similar experiment, B cells from munized wild type and VELOCIMMUNE® mice were separated by flow cytometry based upon surface expression of 8220 and IgM or IgG. The 8220* lgM‘” or surface lgG+ (slgG+) cells were pooled and VH and VK sequences were obtained following RT—PCR amplification and cloning (described above). Representative gene usage in a set of RT-PCR amplified cDNAs from unimmunized VELOCIMMUNE® 1 mice (Table 7) and VELOCIMMUNE® 3 mice (Table 8) was recorded (*defective RSS; ng or pseudogene). Asterisk: gene ts with ive RSS. T: gene segment is missing or pseudogene.

Table 7 VH Observed DH Observed VK Observed 'P00 lt.”co 9" ...\ O 9“? 4.4 NA JK Observed _.4. N .A L A -_ —.4 O ‘F‘P'?’ .44.; \IQU‘I .- -._\ O-_ u 09 .A CO ‘3"? mmAm 40: ll NN mmu T'N\l__\ o Table 8 Observed Observed VK ed ..3321334418747.7.20 4 90 _ .an. 7.8 98 3 O 0 21112221263464664444240047309874.?0 O 3. 4r 3. 4| 3. 1. 4| 4.. 4.! 76521 1 . 4... 303331 3. 47| 11111.11 493. 9 ..n 8 O Q...5 7 4M. 22 3Qw 23 L13131131154111114436644 2 1 4m0 5 2 6 2 Jx ObmWd 6-1 .- 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® mice of this experiment, several have been reported to possess defective recombination signal sequences (RSS) and, thus, would not be expected to be expressed (Feeney, 2000, Factors that nce formation of B cell repertoire, Immunol Res —202). Analysis of several other sets of immunoglobulin sequences from various VELOClMMUNE® mice, isolated from both naive and immunized 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® mice have been observed in various naive and immunized repertoires (data not shown). Although the human VH7—81 gene segment has been identified in the analysis of human heavy chain locus sequences (Matsuda 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® mice as confirmed by re-sequencing of the entire VELOClMMUNE® 3 mouse genome.

Sequences of heavy and light chains of antibodies are known to show exceptional variability, especially in short ptide segments within the rearranged variable domain. These regions, known as hypervariable regions or complementary determining regions (CDRs), create the binding site for antigen in the structure of the antibody molecule. The intervening ptide sequences are called framework regions (FRs). There are three CDRs (CDR1, CDR2, CDR3) and 4 FRs (FR1, FR2, FR3, FR4) in both heavy and light chains. One CDR, CDR3, is unique in that this CDR is created 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 g is imprecise due to both nucleotide deletions via exonuclease ty and non—template encoded ons 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 variable region. CDRs are trated 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® mice around the CDR3 on demonstrating junctional diversity are shown in HS. 7A 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 antibodies from VELOCIMMUNE® mice, indicating proper on of TdT with the human segments. The endpoints of the VH, DH and JH segments relative to their germline counterparts indicate that exonuclease activity has also occurred. Unlike the heavy chain locus, the human K light chain rearrangements t little or no TdT additions at CDR3, which is formed by the recombination of the VK and JK segments (). 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 diversity observed in the CDR3 of rearranged human VK regions 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 germinal center reaction by a s termed somatic hypermutation. B cells expressing somatically mutated variable regions compete with other B cells for access to antigen presented by the follicular dendritic cells.

Those B cells with higher affinity for the antigen will r expand and undergo class switching before exiting to the periphery. Thus, B cells expressing switched isotypes typically have encountered antigen and one germinal center reactions and will have sed numbers of mutations relative to naive B cells. Further, variable region ces from predominantly naive slgM" B cells would be expected to have relatively fewer mutations than variable sequences from slgG+ B cells which have undergone antigen selection.

Sequences from random VH or VK clones from slgM+ or slgG+ B cells from non- immunized VELOCIMMUNE® mice or slgG+ B cells from immunized mice were compared with their germline variable gene segments and changes relative to the germline sequence annotated. The ing nucleotide sequences were translated in silica and mutations leading to amino acid s also ted. 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 le regions derived from slgG" B cells from non-immunized VELOCIMMUNE® mice t 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 s (CDRs) relative to the framework regions, indicating antigen selection. The corresponding amino acid sequences from the human heavy chain variable regions also t significantly higher numbers of mutations in lgG versus lgM and even more in immunized lgG. These mutations again appear to be more frequent in the CDRs compared with the framework ces, suggesting that the antibodies 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 c hypermutation frequency ed in VELOClMMUNE® mice demonstrate that essentially all gene segments present are capable of rearrangement to form fully functionally reverse chimeric antibodies in these mice. Further, VELOClMMUNE® antibodies fully ipate 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® mice are able to mount robust immune responses to le classes of antigens that result in usage of a wide range of human antibodies that are both high affinity and suitable for therapeutic use (data not shown).

Example 5 Analysis of Lymphoid Structure and Serum es The gross structures of spleen, inguinal lymph nodes, Peyer’s patches and thymus of tissue samples from wild type or VELOClMMUNE® mice stained with H&E were examined by light microscopy. The levels of immunoglobulin isotypes in serum collected from wild type and VELOClMMUNE® mice were analyzed using LUMlNEXTM technology.

Lymphoid Organ Structure. The structure and function of the lymphoid tissues are in part ent 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 sections, no signiﬁcant difference in ance of secondary lymphoid organs n wild type and VELOClMMUNE® mice was identified (data not shown). 1] Serum lmmunoglobulin Levels. The level of expression of each isotype is similar in wild type and VELOClMMUNE® mice (, QB and QC). This demonstrates that humanization of the variable gene segments had no apparent adverse effect upon class switching or immunoglobulin expression and secretion and therefore apparently maintain all the endogenous mouse sequences necessary for these functions.

Example 6 Immunization and Antibody Production in Humanized Immunoglobulin Mice Different versions of VELOClMMUNE® mice were immunized with antigen to examine the humoral response to foreign antigen nge. zation and oma Development. MMUNE® and wild- type mice can be immunized with an antigen in the form of protein, DNA, a combination of DNA and protein, or cells expressing the antigen. Animals are typically boosted every three weeks for a total of two to three times. ing 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 A98.653 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 antigen specificity using an ELISA assay (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York). atively, antigen specific B cells are isolated directly from immunized VELOClMMUNE® mice and screened using rd techniques, including those described here, to obtain human antibodies specific for an antigen of interest (9.9., see US 2007/0280945A1, herein incorporated by reference in its ty).

Serum Titer Determination. To monitor animal anti-antigen serum se, serum samples are collected about 10 days after each boost and the titers are determined using antigen specific ELlSA. Briefly, Nunc MAXISORPTM 96 well plates are coated with 2 pg/mL antigen overnight 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% Tween-20 and the bound lgG are detected using HRP-conjugated goat anti-mouse Fc (Jackson lmmuno Research Laboratories, lnc., West Grove, PA) fortotal lgG titer, or biotin-labeled isotype specific or light chain specific polyclonal antibodies (Southern Biotech lnc.) for e specific titers, respectively. For biotin—labeled antibodies, ing 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 on 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 ld of background signal are d as titer.

] In one experiment, MMUNE® mice were immunized with human eukin-6 receptor (hlL-6R). A representative set of serum titers for VELOClMMUNE® and wild type mice immunized with hlL-6R is shown in FlG. 10A and 108.

VELOClMMUNE® and wild-type mice mounted strong responses towards lL-6R with similar titer ranges (A). Several mice from the VELOClMMUNE® and wild- type cohorts reached a maximal se after a single antigen boost. These results te that the immune response strength and kinetics to this antigen were similar in the VELOClMMUNE® and wild type mice. These antigen-specific antibody responses were further analyzed to examine the particular isotypes of the antigen—specific antibodies found in the sera. Both VELOClMMUNE® and wild type groups predominantly elicited an IgG1 se (8), ting that class switching during the humoral response is similar in mice of each type.

Affinity Determination of Antibody g to Antigen in Solution. An based solution competition assay is typically ed to determine antibody- g afﬁnity to the antigen.

Briefly, antibodies in ioned medium are premixed with serial dilutions of antigen protein ranging from O to 10 mg/mL. The solutions of the antibody and antigen e are then incubated for two to four hours at room temperature to reach binding equilibria. The amounts of free antibody in the mixtures are then measured using a quantitative sandwich ELISA. Ninety-six well RBTM plates (VWR, West Chester, PA) are coated with 1 ug/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—conjugated goat anti- mouse IgG polyclonal antibody reagent (Jackson lmmuno Research Lab) and developed using colorimetric substrates such as BD OPTEIATM (BD ences 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.

The dependency of the signals on the concentrations of antigen in solution are analyzed with a 4 parameter fit analysis and ed as |Cso, the antigen concentration required to achieve 50% reduction of the signal from the antibody samples without the presence of antigen in solution.

In one experiment, VELOClMMUNE® mice were immunized with hIL—6R (as described above). A and 113 show a representative set of affinity measurements for anti-hlL6R antibodies from VELOClMMUNE® and wild-type mice.

After immunized mice receive a third antigen boost, serum titers are ined using an ELISA assay. Splenocytes are isolated from selected wild type and MMUNE® mouse cohorts and fused with Ag8.653 a cells to form omas and grown under selection (as described above). Out of a total of 671 anti—IL- 6R hybridomas produced, 236 were found to express antigen-specific antibodies. Media harvested from antigen positive wells was used to ine the antibody ty of binding to antigen using a solution competition ELISA. Antibodies derived from VELOClMMUNE® mice exhibit a wide range of affinity in binding to antigen in solution (A).

Furthermore, 49 out of 236 anti-IL—6R hybridomas were found to block lL-6 from binding to the receptor in an in vitro bioassay (data not shown). Further, these 49 anti-IL-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 7 Construction of a Mouse ADAM6 Targeting Vector Due to replacement of mouse immunoglobulin heavy chain variable gene loci with human globulin heavy chain variable gene loci, early versions of VELOCIMMUNE® mice lack expression of mouse ADAM6 genes. in particular, male MMUNE® mice demonstrate a reduction in fertility. Thus, the ability to express ADAM6 was neered into VELOClMMUNE® mice to rescue the fertility defect. 2] A targeting vector for insertion of mouse ADAMGa and ADAM6b genes into a humanized heavy chain locus was constructed using VELOClGENE® genetic engineering technology (supra) to modify a Bacterial Artificial Chromosome (BAC) 929d24, which was obtained from Dr. Frederick Alt (Harvard University). 929d24 BAC DNA was ered to contain genomic fragments containing the mouse ADAM6a and ADAM6b genes and a hygromycin cassette for targeted deletion of a human ADAM6 pseudogene (hADAMGW) located between human VH1—2 and VHS—1 gene segments of a humanized heavy chain locus ().

First, a genomic fragment containing the mouse ADAM6b gene, ~800 bp of upstream (5’) sequence and ~4800 bp of downstream (3’) sequence was subcloned from the 929d24 BAC clone. A second genomic nt ning the mouse ADAM6a gene, ~300 bp of upstream (5’) ce 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 te flanked by Frt ination sites to create the targeting vector (Mouse ADAM6 Targeting , Figure 12; SEQ lD 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 zed heavy chain locus.

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

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

Following digestion of BAC DNA d from both ucts, the genomic fragments were ligated together to construct an engineered BAC clone containing a humanized heavy chain locus containing an ectopically placed genomic sequence comprising mouse ADAMSa and ADAMSb nucleotide ces. The final targeting uct for the deletion of a human ADAMS gene within a humanized heavy chain locus and insertion of mouse ADAMSa and ADAMSb sequences in ES cells contained, from 5’ to 3’, a 5’ genomic fragment containing ~13 kb of human genomic sequence 3’ of the human VH1—2 gene segment, ~800 bp of mouse genomic sequence downstream of the mouse ADAMSb gene, the mouse ADAMSb gene, ~4800 bp of genomic sequence upstream of the mouse ADAMSb gene, a 5’ Frt site, a hygromycin cassette, a 3’ Frt site, ~300 bp of mouse genomic sequence downstream of the mouse ADAMSa gene, the mouse ADAMSa gene, ~3400 bp of mouse genomic sequence upstream of the mouse ADAMSa gene, and a 3’ genomic fragment containing ~30 kb of human genomic sequence 5’ of the human VHS-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 d modified ES cells comprising a mouse genomic sequence ectopically placed that comprises mouse ADAMSa and ADAMSb sequences within a humanized heavy chain locus. Positive ES cells containing the ectopic mouse genomic fragment within the humanized heavy chain locus were identified by a quantitative PCR assay using TAQMAN TM probes (Lie and Petropoulos, 1998, Advances in quantitative PCR technology: 5’nuclease assays, Curr Opin Biotechno/ 9(1):43-48). The upstream and downstream s outside of the modified portion of the humanized heavy chain locus were confirmed by PCR using primers and probes d within the modified region to confirm the presence of the ectopic mouse genomic sequence within the humanized heavy chain locus as well as the hygromycin cassette. The nucleotide sequence across the upstream ion point included the ing, which indicates human heavy chain genomic sequence upstream of the insertion point and an l-Ceul restriction site (contained within the heses below) linked contiguously to mouse genomic sequence present at the insertion point: (CCAGCTTCAT TAGTAATCGT TCATCTGTGG TAAAAAGGCA GGATTTGAAG CGATGGAAGA TACG GGGCGTTGGA AGACAAAGTG CCACACAGCG CAGCCTTCGT CTAGACCCCC GGGCTAACTA TAACGGTCCT AAGGTAGCGA G) GGGATGACAG ATTCTCTGTT ACTC AGGGTCTGCC TCCACGAGAA TCACCATGCC CAAG 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 tes mouse genomic sequence and a Pl-Scel restriction site (contained within the heses below) linked contiguously with human heavy chain c sequence downstream of the ion point: (AGGGGTCGAG GGGGAATTTT ACAAAGAACA AAGAAGCGGG CATCTGCTGA CATGAGGGCC GAAGTCAGGC TCCAGGCAGC GGGAGCTCCA CCGCGGTGGC GCCATTTCAT TACCTCTTTC TCCGCACCCG ACATAGATAAAGCTT) ATCCCCCACC AAGCAAATCC CCCTACCTGG GGCCGAGCTT CCCGTATGTG GGAAAATGAA TCCCTGAGGT CGATTGCTGC ATGCAATGAA ATTCAACTAG (SEQ ID NO:5).

Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOClMOUSE® mouse engineering method (see, eg., US Pat Nos. 7,6598,442, 7,576,259, 7,294,754). Mice bearing a humanized heavy chain locus containing an ectopic mouse genomic sequence comprising mouse ADAM6a and ADAM6b sequences were identified by genotyping using a cation of allele assay (Valenzuela et al., 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 contains mouse ADAM6a and ADAM6b genes are bred to a FLPe r mouse strain (see, 9.9., Rodriguez et al., 2000, High-efficiency deleter mice show that FLPe is an alternative to Cre-onP. Nature Genetics 252139-140) in order to remove any Frt’ed hygromycin cassette uced by the targeting vector that is not removed, e.g., at the ES cell stage or in the embryo. Optionally, the hygromycin cassette is retained in the mice.

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

Example 8 Characterization of ADAM6 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 gous 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+/+A6resK+/+) 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 es and analyzed for progression h various stages of B cell development. s 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, 2—mercaptoethanol, sential amino acids, and ycin. 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 RPMl medium.

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

Bone marrow: anti-mouse FlTC-CD43 (1 B1 1, BioLegend), PE-ckit (288, BioLegend), PeCy7-lgM (ii/41, eBioscience), Cy5.5-lgD (11—26c.2a, BioLegend), APC-eFluor780-8220 (RA3-GBZ, eBioscience), A700-CD19 (1 DB, BD Biosciences).

Peripheral blood and spleen: anti-mouse FlTC-K (187.1, BD Biosciences), PE-A (RML-42, BioLegend), PeCy7-IgM (ll/41, eBioscience), PerCP-Cy5.5-lgD (11-260.2a, BioLegend), APC-CD3 (145-2011, BD), A700—CD19 (1D3, BD), APC—eFluor780-8220 (RA3-682, eBioscience), Following tion with the labeled antibodies, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on an LSRll flow cytometer and analyzed with FlowJo (Treestar, lnc.). Results from a representative H+’*x*’*and H*’+A6‘esi<+’* mouse are shown in Fle. 14 -— 18.

The results demonstrate that B cells of H""‘A6“§SK"’+ mice progress through the stages of B cell development in a similar fashion to H*’+K+’+mice in the bone marrow and eral compartments, and show normal patterns of maturation once they enter the periphery. H+I+A6reSK+/+ mice demonstrated an increased CD43intCD19+ cell population as compared to H+14» +l+ K mice (HS. 168). This may indicate an accelerated igM expression from the humanized heavy chain locus containing an ectopic mouse genomic fragment comprising the mouse ADAM6a and ADAMGb sequences in H+’+A6resi<+’+mice. in the periphery, B and T cell populations of H+/+A6resi<+’+mice appear normal and similar to H+’+K+’+mice.

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

Briefly, testes from two groups (n=5 per group; group 1: mice gous for human heavy and K light chain variable gene loci, H+/+K+/+ , group 2: mice heterozygous for human heavy chain variable gene loci and homozygous for K light chain le gene loci H”K”) were dissected with the epididymis intact and weighed The ens were then fixed, embedded in paraffin, sectioned and d with hematoxylin and eosin (HE) stain.

Testis sections (2 testes per mouse, for a total of 20) were examined for defects in morphology and evidence of sperm production, while epididymis sections were examined for presence of sperm.

In this experiment, no ences in testis weight or morphology was observed between H+l+Kmmice and H+"K+’+mice. Sperm was observed in both the testes and the epididymis of all genotypes. These results establish that the absence of mouse ADAM6a and ADAM6b genes does not lead to detectable changes in testis morphology, and that sperm is ed in mice in the presence and absence of these two genes. Defects in ity of male HHKmmice are therefore not likely to be due to low sperm production.

Sperm Motility and ion. 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 ADAM6b affects this s, sperm migration and motility was evaluated in H+/+K+/+ mice.

Briefly, sperm was obtained from testes of (1) mice heterozygous for human heavy chain le gene loci and homozygous for human K light chain variable gene loci (H+"K+’+); (2) mice homozygous for human heavy chain variable gene loci and homozygous for human K light chain variable gene loci (H+l+K+l+); (3) mice homozygous for human heavy chain variable gene loci and homozygous for wild-type K light chain (H+’+mK); and, (4) wild- type 057 BL/6 mice (WT). No significant abnormalities were observed in sperm count or overall sperm motility by inspection. For all mice, cumulus dispersal was observed, indicating that each sperm sample was able to penetrate the cumulus cells and bind the zona ida in vitro. These results establish that H+I+K+/+ mice have sperm that are capable of penetrating the cumulus and binding the zona pellucida.

Fertilization of mouse ova in vitro (lVF) was done using sperm from mice as described above A slightly lower number of cleaved embryos were observed for H+/+K+/+ mice the day following lVF as well as a reduced number of sperm bound to the eggs.

These results establish that sperm from H +/+ mice, once exposed'to an ovum, are capable of penetrating the cumulus and g the zona pellucida in r experiment, the ability of sperm from H+/+K+/+ mice to migrate from the uterus and through the t was determined in a sperm migration assay.

Briefly, a first group of super-ovulated female mice (n=5) were set up with H“’+1<+’+ males (n=5) and a second group of super-ovulated female mice (n=5) were set up with H*"t<+f+ males (n=5). The mating pairs were observed for copulation, and five to six hours post-copulation the uterus and ed oviduct from all females were removed and flushed for is. Flush solutions were checked for eggs to verify ovulation and obtain a sperm count. Sperm migration was ted in two different ways. First, both oviducts were removed from the uterus, flushed with saline, and any sperm identified were counted.

The presence of eggs was also noted as ce of ovulation. Second, oviducts were left attached to the uterus and both tissues were fixed, embedded in paraffin, ned and stained (as bed above). Sections were examined for presence of sperm, in both the uterus and in both oviducts.

For the females mated with the five H“"‘i<“’+ males, very little sperm was found in the flush solution from the oviduct. Flush solutions from oviducts of the females mated with the W5?” males ted a sperm level about 25— to 30—fold higher (avg, n = 10 oviducts) than present in flush solutions from the oviducts of the females mated with the H+I+K+’+males. A representative breeding comparison of mef’+ and H+I+A6resxw mice is shown in Table 9.

Histological sections of uterus and oviduct were ed. The sections were ed for sperm presence in the uterus and the oviduct (the colliculus tubarius). inspection of histological sections of oviduct and uterus revealed that for female mice mated with H+i+K+i+ mice, sperm was found in the uterus but not in the oviduct. Further, sections from females mated with H+/+ +H mice revealed K that sperm was not found at the uterotubal junction (UTJ). in sections from females mated with H+"1<”’+ mice, sperm was identified in the UTJ and in the oviduct.

These results ish 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 measured either by sperm count or histological observation. These results establish that mice lacking ADAM6a and ADAMBb genes produce sperm that exhibit an inability to migrate from the uterus to the oviduct. 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 results converge to the support the hypothesis that mouse ADAMS genes help direct sperm with normal motility to migrate out of the , 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 directed by one or both of the ADAM6 proteins, or through nate expression with other proteins, e.g., other ADAM proteins, in the sperm cell, as described below.

Table 9 Male ng .

. Duration of An'ma's “tiers. Oﬁsprlng_ Genotype ng (Male/Female) Mares ——— ADAM Gene Family Expression. 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 complex as sperm mature, and t a reduction of multiple ADAM proteins in mature sperm. To determine if a lack of ADAM6a and ADAM6b genes affects other ADAM proteins in a similar manner, Western blots of protein extracts from testis (immature sperm) and epididymis (maturing sperm) were ed to determine the expression levels of other ADAM gene family members.

In this experiment, protein extracts were analyzed from groups (n=4 per group) of HW“and Hf“):“mice. The results showed that expression of ADAM2 and ADAM3 were not affected in testis extracts. However, both ADAM2 and ADAMS were dramatically reduced in ymis extracts. This demonstrates that the absence of ADAM6a and ADAM6b in sperm of H+I+K+l+ mice may have a direct affect on the expression and s function of other ADAM proteins as sperm matures (e.g., ADAM2 and . This suggests that ADAM6a and ADAM6b are part of an ADAM protein complex on the surface of sperm, which might be critical for proper sperm ion.

Example 9 Human Heavy Chain Variable Gene Utilization in ADAM6 Rescue Mice Selected human heavy chain variable gene usage was determined for mice gous for human heavy and K light chain variable gene loci either lacking mouse ADAM6a and ADAM6b genes (H+/+K+/+) or containing an ectopic genomic fragment ng for mouse ADAM6a and ADAM6b genes (H+’+A6’esi<+”) by a quantitative PCR assay using TAQMANTM probes (as described above).

Briefly, CD19 B cells were purified from the spleens of H+/+K*” and H+’”‘Al8’esi<+’+ mice using mouse CD19 Microbeads (Miltenyi Biotec) and total RNA was purified using the RNEASYTM Mini kit (Qiagen). Genomic RNA was removed using an RNase-free DNase umn treatment (Qiagen). About 200 ng mRNA was reverse-transcribed into cDNA using the First Stand cDNA Synthesis kit (lnvitrogen) and then amplified with the TAQMANTM Universal PCR Master Mix ed Biosystems) using the ABI 7900 Sequence Detection System (Applied Biosystems). Relative expression of each gene was normalized to the expression of mouse K light chain constant region (mCK). Table 10 sets forth the sense/antisensefl'AQMANTM MGB probe combinations used in this ment.

Table 10 Human VH Sequence (5’—3’) SEQ ID NO: Sense: CAGGTACAGCTGCAGCAGTCA Anti-sense: GGAGATGGCACAGGTGAGTGA Probe: TCCAGGACTGGTGAAGC Sense: CAGTGATGAGAAAGAGAT ense: GAGAACACAGAAGTGGATGAGATC Probe: TGAGTCCAGTCCAGGGA Sense: AAAAATTGAGTGTGAATGGATAAGAGTG Anti—sense: AACCCTGGTCAGAAACTGCCA Probe: AGAGAAACAGTGGATACGT Sense: AACTACGCACAGAAGTTCCAGG Anti—sense: GCTCGTGGATTTGTCCGC Probe: CAGAGTCACGATTACC Sense: TGAGCAGCACCCTCACGTT Anti-sense: GTGGCCTCACAGGTATAGCTGTT Probe: ACCAAGGACGAGTATGAA In this ment, expression of all four human VH genes was observed in the samples analyzed. Further, the expression levels were comparable between H+I+K+l+ and H+I+A6resxm mice. These results trate that human VH genes that were both distal to the modification site (VH3-23 and VH1—69) and proximal to the modification site (VH1-2 and VHS-1) were all able to recombine to form a functionally expressed human heavy chain.

These results demonstrate that the ectopic genomic fragment comprising mouse ADAMGa and ADAM6b ces ed 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 10 Humoral Immune Response in ADAMS Rescue Mice The humoral immune response was determined for mice homozygous for human heavy and K light chain variable gene loci either lacking mouse ADAM6a and ADAM6b genes (H+/+K+’*) or containing an c genomic fragment ng for mouse ADAM6a and ADAM6b genes (H+’+A6resx+’+) by a multi-antigen immunization scheme followed by antibody ion and characterization. Results were compared for determination of any effect on V(D)J recombination involving the human immunoglobulin gene segments, assessment of serum titer progression, production of antibodies by hybridomas and affinity for antigen.

Immunization protocol. A human cell surface receptor (Antigen A), a human antibody specific for a human receptor tyrosine—protein kinase (Antigen B), a secreted human protein that functions in regulation of the TGF-B signaling pathway (Antigen C), and a human receptor tyrosine kinase (Antigen D) were employed for comparative immunizations in groups of mice. Serum was ted from groups of mice prior to immunization with the above antigens. Each antigen (2.3 pg each) was administered in an initial priming immunization mixed with 10 pg of CpG oligonucleotide as adjuvant (lnvivogen). The immunogen was administered via footpad (f.p.) in a volume of 25 pl per mouse. Subsequently, mice were boosted via f.p. with 2.3 pg of antigen along with 10 pg CpG and 25 pg Adju-Phos tag) as adjuvants on days 3, 6, 11, 13, 17, and 20 for a totai of six boosts. Mice were bled on days 15 and 22 after the fourth and sixth boosts, respectively, and antisera were assayed for antibody titer to each specific antigen.

Antibody titers were determined in sera of immunized mice using an ELISA assay. Ninety six-well microtiter plates (Thermo Scientific) were coated with the tive antigen (2 pg/ml) in phosphate—buffered saline (PBS, lrvine Scientific) overnight at 4°C.

The following day, plates were washed with phosphate—buffered saline containing 0.05% Tween 20 , Sigma—Aldrich) four times using a piate washer ular Devices).

Plates were then blocked with 250 pi of 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS and ted for one hour at room temperature. The plates were then washed four times with PBS—T. Sera from immunized mice and pre-immune sera were serially diluted three-fold in 0.5% BSA—PBS starting at 1:300 or 1:1000 and added to the blocked plates in duplicate and incubated for one hour at room ature. The last two wells were left blank to be used as secondary antibody control. The plates were again washed four times with PBS-T in a plate washer. A 1:5000/1:10,000 dilution of goat anti—mouse lgG-Fc-Horse Radish dase (HRP, Jackson lmmunoresearch) or goat anti—mouse IgG-kappa-HRP (Southern Biotech) conjugated secondary dy was added to the plates and incubated for one hour at room temperature. Plates were again washed eight times with PBS-T and developed using TMB/H202 as ate. The substrate was incubated for twenty minutes and the reaction stopped with 2 N HZSO4(VWR) or 1 N H3PO4 (JT Baker). Plates were read on a spectrophotometer (Victor, Perkin Elmer) at 450 nm. Antibody titers were ated using Graphpad PRlSM re.

] Serum titer was calculated as serum dilution within experimental titration range at the signal of antigen binding equivalent to two times above background. Results for the l immune response are shown in (Antigen A), FIG, 20 (Antigen B), (Antigen C), and HG. 22 (Antigen D). Antigen positive score of hybridomas made using two spleens isolated from mice from each group of selected immunizations is shown in Table 11 (Antigen score is equal to 2X/background).

As shown in this Example, antibody titers generated in Adam6 rescue mice (H+/+A6resK+/+) were comparable to those generated in mice lacking ADMA6a and ADAM6b and having humanized heavy chain (H+/+ +/+ K ). Further, spleens from H+I+A6'esx+’+ mice d antigen-positive hybridomas for all antigens tested, including antibodies of high affinity, at levels comparable to H+I+K+l+ mice. Thus, no impairment of V(D)J recombination of human immunoglobulin gene segments in Adam6 rescue mice is ed to exist given the tion of dies with high affinity containing human immunoglobulin genes.

Table 11 Antigen Mouse Strain Antigen Score waxes?“ Example 11 Antigen Binding Affinity Determination Binding affinities of antibodies showing specific binding to Antigen B were ed using a real—time surface plasmon resonance sor (BlAcore 2000).

Conditioned media from hybridomas isolated from two strains of mice immunized with Antigen B (H*’+K*’+ and H+I+A6resK+/+) were used during BlAcore screening. BlAcore sensor surface was ﬁrst derivatized with polyclonal rabbit anti—mouse antibody (GE) to capture anti—Antigen B antibodies from conditioned media. During the entire ing method, HBST (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v/v tant P20) was used as the running buffer. Fab fragment of Antigen B was injected over the anti-Antigen B antibody captured surface at a flow rate of 50 pl/minute at 100nM concentration.

Antibody-antigen association was monitored for three minutes while the dissociation of antigen from the ed antibody was monitored for five minutes in HBST running buffer.

The experiment was performed at 25°C. c association (ka) and dissociation (kd) rate nts were determined by processing and fitting the data to a 1:1 binding model using Scrubber 2.0 curve fitting software. Binding dissociation equilibrium constants (KD) and dissociative half-lives (Tyz) were calculated from the kinetic rate constants as: KD (M) = kd / ka; and T72 (min) = (In2/(60*kd). Results for selected anti-Antigen B antibodies are shown in Table 12.

Table 12 Antibody Mouse strain KD (M) Ty, (min) 5D6 H+/+ +/+ 1 .62E—08 00 8G10 H+/+ +/+ 1 .20E-08 5 1OF10 H+/+ +/+ 1.09E—08 3 1F5 H+/+ +/+ 1.00E—07 0.3 1OG8V H+/+ +/+ 1.47E-07 0.3 1811 reSK+/+ 1.98E-08 _D11 H" +A6resi<+ + -5.6OE:E 704 H+/+A6resK+l+ 06 0.05 H*’*A6"~‘SK*’* 2.31E—09 resx*’+ 3.47E-09 1034 H+I+A6reSK+I+ 3.60E-09 -23 H+/+A6’esr<+’+ 3.06E-08 H*’*A6resl<+’+ 2.705-07 H*"‘Ac3“’3$t<+’+ 7.00E-10 H+’+A6'esi<””' 5.80E-10 In a similar experiment, kinetics of different monoclonal antibodies present in hybridoma-conditioned media binding to Antigen A was determined using a real-time e plasmon resonance biosensor (BlAcore 4000) assay. All hybridoma clones used in this assay were produced in H+’+A6""si<+’+ mice. y, to capture the Antigen A-specific antibodies, polyclonal rabbit anti- mouse antibody (GE Catalog# BR38) was first immobilized on the sensor chip.

BlAcore screening was med in two different buffers —- PBSP, pH7.2 and PBSP, pH6.0. Both the buffers were supplemented with 0.1 mg/ml BSA. Following the capture of anti-Antigen A antibodies from the conditioned media, 1 uM of Antigen A monomer (prepared in respective running buffer) was ed over the captured antibody e for 1.5 minutes at 30 ul/minute and the dissociation of bound Antigen A r was monitored for 1.5 minutes in the respective running buffer at 25°C. Kinetic association (ka) and dissociation (kd) rate constants were determined by processing and fitting the data to a 1:1 binding model using Scrubber 2.0 curve fitting software. Binding iation equilibrium constants (KD) and dissociative half-lives (Tyz) were ated from the kinetic rate nts as: KD (M) = kd / ka; and T1/2 (min) = (ln2/(60*kd). Table 13 sets forth the binding kinetics parameters for selected anti-Antigen A antibody binding to Antigen A monomer at pH7.2 and pH6.0. NB: no binding detected under current experimental conditions.

Table 13 pH7.2 pH6.0 Antibody Kn (M) Ty; (mm) Kn (M) Ty: (min) — 2.98EB-09 2.06E-11 _Im- 6.-79E1 7,-42E11 .11:- -05E1 -197E0 _ 1. 58E—08 2.59E-09 _702E 11 14281 As shown above, high affinity antibodies were ed from both H*’*A6re‘°‘x*’+ and H+I+K+l+ mice in a comparable manner. Among the twenty~five antibodies represented in Table 12, twenty produced in H‘WAES’eSK”+ mice demonstrated an affinity range of 0.5 nM to 1 µM, while the five generated in H+/+ κ+/+ mice demonstrated an affinity range of 10 nM to 150 nM. Further, the fifty-five antibodies shown in Table 13 demonstrated an affinity range of 20 pM to 350 nM for g to Antigen A monomer.

As demonstrated in this Example, the reinsertion of mouse Adam6 genes into a humanized immunoglobulin heavy chain locus does not impair the ability of the mouse to mount a robust immunize response to multiple antigens characterized by repertoires of human antibodies having diverse affinities in the subnanomolar range, which are derived from human gene segments rearranged from a engineered germline.

Any discussion of nts, acts, materials, s, es or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general dge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

Throughout 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.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A genetically modified mouse whose c modifications achieve: a) ectopic placement of a nucleotide sequence that ses an ADAM6 protein or fragment thereof that is functional in a male mouse; and b) a modified immunoglobulin heavy chain variable region locus.

2. The mouse of claim 1, wherein the modification of the immunoglobulin heavy chain variable region locus reduces or eliminates endogenous ADAM6 activity of a cell or tissue of a male mouse.

3. The mouse of claim 1 or 2, wherein the nucleotide sequence that expresses an ADAM6 protein or fragment thereof is t at the modified immunoglobulin heavy chain variable region locus.

4. The mouse of claim 1 or 2, wherein the nucleotide sequence that expresses an ADAM6 protein or fragment thereof is present at a position other than the ed immunoglobulin heavy chain le gene locus.

5. The mouse of any one of claims 1 to 4, wherein the nucleotide sequence that expresses an ADAM6 protein or fragment thereof includes a mouse ADAM6a and/or mouse ADAM6b.

6. The mouse of any one of claims 1 to 5, n the modified immunoglobulin heavy chain variable region locus comprises an insertion of one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments.

7. The mouse of claim 6, wherein the modified immunoglobulin heavy chain variable region locus comprises at least 18 human VH gene segments, at least one and up to 27 human DH gene segments, and at least one and up to six human JH gene segments.

8. The mouse of claim 6, wherein the modified immunoglobulin heavy chain variable region locus comprises at least 39 human VH gene segments, at least one and up to 27 human DH gene ts and at least one and up to six human JH gene segments.

9. The mouse of claim 6, wherein the modified immunoglobulin heavy chain variable region locus comprises 80 human VH gene segments, at least one and up to 27 human DH gene segments, and at least one and up to six human JH gene segments.

10. The mouse of claim 6, wherein the one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments are ly linked to an immunoglobulin constant region gene.

11. The mouse of claim 10, wherein the immunoglobulin nt region gene is a mouse immunoglobulin constant gene.

12. The mouse of claim 11, wherein the immunoglobulin mouse constant region gene comprises an immunoglobulin constant region selected from a CH1, a hinge, a CH2, a CH3, a CH4, and a combination thereof.

13. The mouse of claim 6, the mouse r comprising one or more human VK gene segments and one or more human JK gene segments.

14. The mouse of claim 13, wherein the one or more human VK gene segments and one or more human JK gene segments are present at an endogenous immunoglobulin K light chain locus.

15. The genetically modified mouse of any one of claims 1 to 14, wherein the placement is c in that it is inserted or extra-chromosomal.

16. An isolated cell of the mouse of any one of claims 1 to 15.

17. The isolated cell of claim 16, wherein the cell is an nic stem cell.

18. A mouse embryo comprising the embryonic stem cell of claim 17.

19. The isolated cell of claim 16, wherein the cell is a B cell.

20. A hybridoma made from the B cell of claim 19.

21. An immortalized cell made from the isolated cell of claim 16.

22. A method of providing an antibody specific for an antigen of interest, the method sing the steps of: a) immunizing a genetically modified mouse of any one of claims 1-15 with the antigen of interest; b) isolating at least one cell from the mouse producing an antibody ic against the antigen; and c) culturing at least one cell producing an antibody of step b) and obtaining said antibody.

23. The method of claim 22, n the genetically modified mouse is a genetically modified mouse whose genetic modifications achieve an inserted nucleotide sequence that expresses an ADAM6 protein or fragment thereof.

24. The method of claim 22 or 23, wherein the culturing in step c) is performed on at least one hybridoma cell isolated from the at least one cell obtained in step b).

25. The method of claim 22 or 23, wherein the at least one cell isolated in step b) is derived from the spleen, a lymph node, or bone marrow of the mouse from step a).

26. The method of claim 22 or 23, wherein immunizing with the antigen of st in step a) is carried out with protein, DNA, a combination of DNA and protein, or cells expressing the antigen.

27. An antibody produced by a method of any one of claims 22 to 26.

28. A mouse of any one of claims 1 to 15, a cell of any one of claims 16, 17, 19 or 21, an embryo of claim 18, a hybridoma of claim 20, a method of any one of claims 22 to 26, an antibody of claim 27, substantially as herein described with nce to the