Adam6 mice

Abstract

To provide ADAM6 mice.SOLUTION: Mice are provided that comprise a reduction or deletion of ADAM6 activity from an endogenous ADAM6 locus, or that lack an endogenous locus encoding a mouse ADAM6 protein, wherein the mice comprise a sequence encoding an ADAM6 or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the sequence is an ectopic ADAM6 sequence or a sequence that confers, upon a male mouse, ability to generate offspring by mating. Mice and cells with genetically modified immunoglobulin heavy chain loci that comprise an ectopic nucleotide sequence encoding a mouse ADAM6 or functional fragment or homolog or ortholog thereof are also provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] Genetically modified mice, cells, embryos and tissues are described that contain a nucleic acid sequence encoding a functional ADAM6 locus. The modification includes a human and / or humanized immunoglobulin locus. Mice that lack a functional endogenous ADAM6 gene but contain ADAM6 function are described, including mice that contain an ectopic nucleic acid sequence encoding the ADAM6 protein. Endogenous immunoglobulin V H We describe a genetically modified male mouse that comprises a modification of a genetic locus that renders the mouse unable to produce functional ADAM6 protein, thereby rendering it infertile, and further comprises ADAM6 function in the male mouse, including a mouse that comprises an ectopic nucleic acid sequence that restores fertility to the male mouse. [Background technology]

[0002] Mice containing human antibody genes are known in the art. The pharmaceutical application of antibodies during the past two decades has inspired extensive research into the generation of antibodies suitable for use as human therapeutics. Early antibody therapeutics based on mouse antibodies were not ideal as human therapeutics, since repeated administration of mouse antibodies to humans results in immunogenicity that can disrupt long-term treatment regimens. Solutions based on humanizing mouse antibodies to make them more human and less mouse-like were developed. Methods for expressing human immunoglobulin sequences for use in antibodies followed, and these were primarily based on the in vitro expression of human immunoglobulin libraries in phage, bacteria, or yeast. Finally, attempts were made to generate useful human antibodies from human lymphocytes in vitro in mice engrafted with human hematopoietic cells, and in transchromosomic or transgenic mice in which the endogenous immunoglobulin loci have been disabled. In the case of transgenic mice, it was necessary to disable the endogenous mouse immunoglobulin genes so that randomly integrated, fully human transgenes could serve as the source of immunoglobulin sequences expressed in the mice. Although such mice are capable of generating human antibodies suitable for use as human therapeutics, they present significant problems with the mouse immune system. These problems (1) render the mice infeasible for the production of sufficiently diverse antibody repertoires, (2) require the use of extensive re-engineering fixes, (3) result in a suboptimal clonal selection process, likely due to incompatibilities between human and mouse elements, and (4) make these mice an unreliable source of the large and diverse population of human variable sequences required to be truly useful in the generation of human therapeutics. Summary of the Invention [Problem to be solved by the invention]

[0003] There remains a need in the art for improved genetically modified mice that are useful for generating immunoglobulin sequences, including human antibody sequences. There also remains a need for mice that can rearrange immunoglobulin gene segments to form useful rearranged immunoglobulin genes, or that can produce proteins from altered immunoglobulin loci while reducing or eliminating deleterious changes that may result from genetic modifications. [Means for solving the problem]

[0004] In one embodiment, a mouse comprising a modification resulting in a non-functional endogenous mouse ADAM6 protein or ADAM6 gene (e.g., a knockout of the endogenous ADAM6 gene or a deletion in the endogenous ADAM6 gene) is functional in male mice. The present invention provides nucleic acid constructs, cells, embryos, mice and methods for generating mice comprising a nucleic acid sequence encoding an ADAM6 protein or an ortholog or homolog or fragment thereof.

[0005] In one aspect, the present invention provides a nucleic acid construct, a cell, an embryo, a mouse and a method for producing a mouse comprising a modification of an endogenous mouse immunoglobulin locus, the mouse comprising an ADAM6 protein or its ortholog or homolog or fragment that is functional in male mice.In one embodiment, the endogenous mouse immunoglobulin locus is an immunoglobulin heavy chain locus, and the modification reduces or eliminates the ADAM6 activity in cells or tissues of the male mouse.

[0006] In one embodiment, a mouse is provided that comprises an ectopic nucleotide sequence encoding mouse ADAM6 or its ortholog or homolog or functional fragment; a mouse is also provided that comprises an endogenous nucleotide sequence encoding mouse ADAM6 or its ortholog or homolog or functional fragment and that comprises at least one genetic modification of the heavy chain immunoglobulin locus.

[0007] In one embodiment, a method is provided for producing a mouse comprising a modification of endogenous mouse immunoglobulin locus, comprising an ADAM6 protein or its ortholog or homolog or fragment that is functional in male mice.The mouse according to the present invention can be obtained, for example, by the method described herein.

[0008] In one aspect, methods are provided for producing mice comprising genetic modifications of the immunoglobulin heavy chain locus, and application of these methods results in male mice comprising modified immunoglobulin heavy chain loci (or deletions thereof) that can produce offspring upon mating. In one embodiment, the male mice are capable of producing sperm capable of migrating from the mouse uterus through the mouse oviduct to fertilize mouse eggs.

[0009] In one aspect, methods are provided for producing mice comprising genetic modifications of immunoglobulin heavy chain loci, and application of these methods results in male mice comprising modified immunoglobulin heavy chain loci (or deletions thereof), the male mice exhibiting reduced fertility, and the mice comprising genetic modifications that restore the reduced fertility in whole or in part. In various embodiments, the reduced fertility is characterized by the inability of male mouse sperm to migrate from the mouse uterus through the mouse oviduct to fertilize mouse eggs. In various embodiments, the reduced fertility is characterized by sperm exhibiting in vivo migration defects. In various embodiments, the genetic modification that restores the reduced fertility in whole or in part is a nucleic acid sequence encoding a mouse ADAM6 gene or its ortholog or homolog or fragment that is functional in male mice.

[0010] In one embodiment, the genetic modification comprises the replacement of endogenous immunoglobulin heavy chain variable locus with the immunoglobulin heavy chain variable locus of another species (e.g., non-mouse species). In one embodiment, the genetic modification comprises the insertion of an orthologous immunoglobulin heavy chain variable locus into the endogenous immunoglobulin heavy chain variable locus. In a particular embodiment, the species is human. In one embodiment, the genetic modification comprises the deletion of all or part of the endogenous immunoglobulin heavy chain variable locus, which deletion results in the loss of endogenous ADAM6 function. In a particular embodiment, the loss of endogenous ADAM6 function is associated with reduced fertility in male mice.

[0011] In one embodiment, male mice having a modification that reduces or eliminates mouse ADAM6 expression from an endogenous ADAM6 allele are characterized in that they exhibit the same phenotype as mice that exhibit reduced or eliminated endogenous ADAM6 function. Thus, the mouse is provided that comprises the modification and further comprises ectopic ADAM6 sequence or its homolog or ortholog or functional fragment, so that it shows reduced fertility (e.g., highly reduced offspring production ability by mating) or is essentially infertile.In one embodiment, the modification that reduces or eliminates mouse ADAM6 expression is the modification (e.g., insertion, deletion, substitution, etc.) of mouse immunoglobulin locus.

[0012] In one embodiment, the reduction or loss of ADAM6 function comprises the inability or substantial inability of the mouse to produce sperm that can travel from the mouse uterus through the mouse oviduct to fertilize a mouse egg. In certain embodiments, at least about 95%, 96%, 97%, 98% or 99% of the sperm cells produced in the ejaculate volume of the mouse are unable to travel through the oviduct in vivo after mating and are unable to fertilize a mouse egg.

[0013] In one embodiment, the reduction or loss of ADAM6 function comprises a failure or substantial failure to form ADAM2 and / or ADAM3 and / or ADAM6 complexes on the surface of sperm cells of said mouse. In one embodiment, the loss of ADAM6 function comprises a substantial failure to fertilize mouse eggs by mating with female mice.

[0014] In one aspect, provide a mouse that lacks functional endogenous ADAM6 gene, and the mouse comprises a protein (or an ectopic nucleotide sequence encoding the protein) that confers ADAM6 functionality to the mouse.In one embodiment, the mouse is a male mouse, and the functionality comprises enhanced fertility compared to the mouse that lacks functional endogenous ADAM6 gene.

[0015] In one embodiment, the protein is encoded by a genomic sequence located within an immunoglobulin locus in the germline of the mouse. In a particular embodiment, the immunoglobulin locus is a heavy chain locus. In another particular embodiment, the heavy chain locus is encoded by at least one human V H , at least one human D H and at least one human J H 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 particular embodiment, the transcriptionally active locus is the ROSA26 locus. In a particular embodiment, the transcriptionally active locus is associated with tissue-specific expression. In one embodiment, the tissue-specific expression is present in reproductive tissue. In one embodiment, the protein is encoded by a genomic sequence that is randomly inserted into the germline of the mouse.

[0016] In one embodiment, the mouse comprises a human or chimeric human / mouse or chimeric human / rat light chain (e.g., human variable, mouse or rat constant) and a chimeric human variable / mouse or rat constant heavy chain. In a specific embodiment, the mouse comprises a transgene comprising a chimeric human variable / rat or mouse constant light chain gene operably linked to a transcriptionally active promoter, e.g., the 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.

[0017] In one embodiment, the ectopic nucleotide sequence is within an immunoglobulin locus in the germline of the mouse. In a particular embodiment, the immunoglobulin locus is a heavy chain locus. In one embodiment, the heavy chain locus is a human V H , at least one human D H and at least one human J H Genetics In one embodiment, the ectopic nucleotide sequence is located in a non-immunoglobulin locus in the germline of the mouse. In one embodiment, the non-immunoglobulin locus is a transcriptionally active locus. In a particular embodiment, the transcriptionally active locus is the ROSA26 locus. In one embodiment, the ectopic nucleotide sequence is located at a randomly inserted position in the germline of the mouse.

[0018] In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 gene, and the mouse comprises an ectopic nucleotide sequence that compensates for the loss of mouse ADAM6 function. In one embodiment, the ectopic nucleotide sequence confers on the mouse the ability to produce offspring comparable to the corresponding wild-type mouse that contains a functional endogenous ADAM6 gene. In one embodiment, the sequence confers on the mouse the ability to form ADAM2 and / or ADAM3 and / or ADAM6 complexes on the surface of the mouse sperm cells. In one embodiment, the sequence confers on the mouse the ability to travel from the mouse uterus through the mouse oviduct to the mouse egg and fertilize the egg.

[0019] In one embodiment, the mouse lacking a functional endogenous ADAM6 gene and containing the ectopic nucleotide sequence produces litters that are at least about 50%, 60%, 70%, 80% or 90% of the number of litters produced by a wild-type mouse of the same age and strain in a six month period.

[0020] In one embodiment, the mouse lacking a functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence, when bred for a period of 6 months, produces at least about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 4 times, about 6 times, about 7 times, about 8 times, or about 10 or more progeny compared to a mouse of the same age and same or similar strain that lacks a functional endogenous ADAM6 gene and lacks the ectopic nucleotide sequence bred under substantially the same conditions for substantially the same period of time.

[0021] In one embodiment, the mice lacking a functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produce, on average, at least about two, three, or four times more offspring per litter during a four or six month mating period than mice mated for the same period that lack a functional endogenous ADAM6 gene and do not contain the ectopic nucleotide sequence.

[0022] In one embodiment, the mouse lacking a functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence is a male mouse, which, when recovered from the oviduct at about 5 to 6 hours after mating, produces sperm that exhibit oviduct migration that is at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, 100 times, 110 times, or 120 times more than a mouse lacking a functional endogenous ADAM6 gene and not comprising the ectopic nucleotide sequence.

[0023] In one embodiment, the mouse lacking a functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence, when mated with a female mouse, produces sperm that are able to traverse the uterus and enter and traverse the fallopian tubes within about six hours with an efficiency nearly equal to that of sperm from a wild-type mouse.

[0024] In one embodiment, the mouse lacking a functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces about 1.5 times, about 2 times, about 3 times or about 4 times more litters in a comparable period of time than a mouse lacking a functional ADAM6 gene and not comprising the ectopic nucleotide sequence.

[0025] In one aspect, a mouse is provided that comprises in its germline a non-mouse nucleic acid sequence encoding an immunoglobulin protein, wherein the non-mouse immunoglobulin sequence comprises an insertion of a mouse ADAM6 gene or a 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 immunoglobulin 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 segments and one or more J gene segments. In one embodiment, the one or more V, D and J gene segments, or the 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 the one or more V and J gene segments, are rearranged. In one embodiment, after rearrangement of the one or more V, D and J gene segments, or one or more V and J gene segments, the mouse comprises in its genome at least one nucleic acid sequence encoding the mouse ADAM6 gene or a homolog or ortholog or functional fragment thereof. In one embodiment, after rearrangement, the mouse comprises in its genome at least two nucleic acid sequences encoding the mouse ADAM6 gene or a homolog or ortholog or functional fragment thereof. In one embodiment, after rearrangement, the mouse comprises in its genome at least one nucleic acid sequence encoding the mouse ADAM6 gene or a homolog or ortholog or functional fragment thereof. In one embodiment, the mouse comprises the ADAM6 gene or a homolog or ortholog or functional fragment thereof in a B cell. In one embodiment, the mouse comprises the ADAM6 gene or a homolog or ortholog or functional fragment thereof in a non-B cell.

[0026] In one embodiment, a mouse is provided that expresses a human immunoglobulin heavy chain variable region, or a functional fragment thereof, from an endogenous mouse immunoglobulin heavy chain locus, wherein the mouse comprises ADAM6 activity that is functional in male mice.

[0027] In one embodiment, the male mouse comprises a single unmodified endogenous ADAM6 allele or an ortholog or of a homolog or functional fragment thereof at the endogenous ADAM6 locus.

[0028] In one embodiment, the male mouse comprises an ectopic mouse ADAM6 sequence, or a homolog or ortholog or functional fragment thereof, that encodes a protein that confers ADAM6 function.

[0029] In one embodiment, the male mouse comprises an ADAM6 sequence or a homolog or ortholog or functional fragment thereof at the location of an endogenous mouse ADAM6 allele, e.g., at a location in the mouse genome proximal to the last V gene segment sequence and 5' to the first D gene segment.

[0030] In one embodiment, the male mouse comprises an ADAM6 sequence or a homolog or ortholog or functional fragment thereof adjacent to, upstream, downstream, or both upstream and downstream (relative to the transcriptional direction of the ADAM6 sequence) of a nucleic acid sequence encoding an immunoglobulin variable gene segment. In a particular embodiment, the immunoglobulin variable gene segment is a human gene segment. In one embodiment, the immunoglobulin variable gene segment is a human gene segment, and the sequence encoding a mouse ADAM6 or an ortholog or homolog or fragment thereof functional in the mouse is a human V gene segment. in one embodiment, the mouse comprises two or more V gene segments and the sequence is located between the last V gene segment and the penultimate V gene segment, in one embodiment, the sequence is located following the last V gene segment and the first D gene segment.

[0031] In one aspect, a male mouse is provided that comprises a non-functional endogenous ADAM6 gene or a deletion of endogenous ADAM6 gene in its germline, and the sperm cells of the mouse can pass through the oviduct of a female mouse and fertilize an egg.In one embodiment, the mouse comprises an extrachromosomal copy of the mouse ADAM6 gene or its orthologue or homologue or functional fragment that is functional in male mice.In one embodiment, the mouse comprises an ectopic mouse ADAM6 gene or its orthologue or homologue or functional fragment that is functional in male mice.

[0032] In one embodiment, a mouse is provided that contains a genetic modification that reduces endogenous mouse ADAM6 function, and that contains at least some ADAM6 functionality provided by an endogenous unmodified allele (e.g., heterozygote) that is fully or partially functional, or by expression from an ectopic sequence encoding ADAM6 or an ortholog or homolog or functional fragment thereof that is functional in male mice.

[0033] In one embodiment, the mouse comprises sufficient ADAM6 function to confer male mice the ability to produce offspring by mating, compared to male mice lacking functional ADAM6. In one embodiment, the ADAM6 function is conferred by the presence of an ectopic nucleotide sequence encoding mouse ADAM6 or its homologue or orthologue or functional fragment. An ADAM6 homologue or orthologue or fragment that is functional in male mice includes those that fully or partially restore the loss of offspring production observed in male mice that do not have sufficient endogenous mouse ADAM6 activity, for example, the loss of ability observed in ADAM6 knockout mice. In this sense, ADAM6 knockout mice include mice that contain endogenous locus or fragments thereof but are not functional, i.e., do not express ADAM6 (ADAM6a and / or ADAM6b) at all, or express ADAM6 (ADAM6a and / or ADAM6b) at a level that is insufficient to support the essentially normal offspring production ability of wild-type male mice. Loss of function can result, for example, from modification of the structural gene of the locus (i.e., the ADAM6a or ADAM6b coding region) or modification of a regulatory region of the locus (e.g., a sequence 5' to the ADAM6a gene or 3' to the ADAM6a or ADAM6b coding region that controls, in whole or in part, transcription of the ADAM6 gene, expression of ADAM6 RNA, or expression of the ADAM6 protein). In various embodiments, the ortholog or homolog or fragment thereof that is functional in a male mouse is one that allows male mouse sperm (or the majority of sperm cells in a male mouse ejaculate) to pass through the mouse oviduct and fertilize a mouse egg.

[0034] In one embodiment, the male mouse expressing the human immunoglobulin variable region or functional fragment thereof comprises sufficient ADAM6 activity to confer to the male mouse the ability to produce offspring upon mating with a female mouse, and in one embodiment, when mated with a female mouse, the male mouse exhibits an offspring production capacity 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 embodiment, at least 70%, in one embodiment, at least 80%, in one embodiment, at least 90%, and in one embodiment approximately the same as the offspring production capacity of a mouse having one or two endogenous unmodified ADAM6 alleles.

[0035] In one embodiment, a male mouse expresses sufficient ADAM6 (or an ortholog or homolog or functional fragment thereof) to enable sperm cells from the male mouse to pass through a female mouse ovary and fertilize a mouse egg.

[0036] In one embodiment, the ADAM6 functionality is conferred by a nucleic acid sequence adjacent to a mouse chromosomal sequence (e.g., the nucleic acid is randomly integrated into the mouse chromosome; or is 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., Cre-mediated) insertion or homologous recombination). In one embodiment, the ADAM6 sequence is present on a nucleic acid distinct from the mouse chromosome (e.g., the ADAM6 sequence is present on an episome, i.e., extrachromosomally, e.g., in an expression construct, vector, YAC, transchromosome, etc.).

[0037] In one aspect, a genetically modified mouse and cell are provided that comprises a modification of endogenous immunoglobulin heavy chain locus, the mouse expresses at least a portion of an immunoglobulin heavy chain sequence, for example, at least a portion of a human sequence, and the mouse comprises functional ADAM6 activity in male mice.In one embodiment, the modification reduces or eradicates the ADAM6 activity in the mouse.In one embodiment, the mouse is modified such that both alleles encoding ADAM6 activity are absent or express a substantially non-functional ADAM6 in male mice to support normal mating.In one embodiment, the mouse further comprises an ectopic nucleic acid sequence encoding mouse ADAM6 or its ortholog or homolog or functional fragment.

[0038] In one aspect, a genetically modified mouse and cell are provided that comprises a modification of an endogenous immunoglobulin heavy chain locus, the modification reducing or eliminating the ADAM6 activity expressed from the ADAM6 sequence of the locus, and the mouse comprises an ADAM6 protein or an orthologue or homologue or functional fragment thereof. In various embodiments, the ADAM6 protein or a fragment thereof is encoded by an ectopic ADAM6 sequence. In various embodiments, the ADAM6 protein or a fragment thereof is expressed from an endogenous ADAM6 allele. In various embodiments, the mouse comprises a first immunoglobulin heavy chain allele that comprises a first modification that reduces or eliminates the expression of functional ADAM6 from the first immunoglobulin heavy chain allele, and a second immunoglobulin heavy chain allele that comprises a second modification that does not substantially reduce or eliminate the expression of functional ADAM6 from the second immunoglobulin heavy chain allele.

[0039] In one embodiment, the second modification is 3' (relative to the transcriptional orientation of the mouse V gene segment) of the last mouse V gene segment and is located in a mouse (or chimeric human / mouse) immunoglobulin heavy chain constant gene or fragment thereof (e.g., human and / or mouse: H1 and / or hinge and / or C H 2 and / or C H 3) (based on the transcription direction of the constant sequence).

[0040] In one embodiment, the modification is in a first immunoglobulin heavy chain allele at a first locus encoding a first ADAM6 allele, and the ADAM6 function results from expression of endogenous ADAM6 at a second immunoglobulin heavy chain allele at a second locus encoding a functional ADAM6, the second immunoglobulin heavy chain allele comprising at least one modification of a V, D and / or J gene segment. In a particular embodiment, the at least one modification of the V, D and / or J gene segment is a deletion, a substitution with a human V, D and / or J gene segment, a substitution with a camelid V, D and / or J gene segment, a substitution with a humanized or camelized V, D and / or J gene segment, a substitution of a heavy chain sequence with a light chain sequence, and or a combination thereof. In one embodiment, the at least one modification is a deletion of one or more heavy chain V, D and / or J gene segments at the heavy chain locus and replacement with one or more light chain V and / or J gene segments (e.g., human light chain V and / or J gene segments).

[0041] In one embodiment, the modification is in 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 ectopic ADAM6 expression at a non-immunoglobulin locus in the germline of the mouse. In a particular embodiment, the non-immunoglobulin locus is the ROSA26 locus. In a particular embodiment, the non-immunoglobulin locus is transcriptionally active in reproductive tissue.

[0042] In one aspect, a mouse is provided that comprises a heterozygous or homozygous knockout of ADAM6. In one embodiment, the mouse further comprises a modified immunoglobulin sequence that is a human or humanized immunoglobulin sequence or a camelid or camelized human or mouse immunoglobulin sequence. In one embodiment, the modified immunoglobulin sequence is present at an endogenous 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 sequence replaces an endogenous mouse heavy chain variable gene sequence at the endogenous mouse immunoglobulin heavy chain locus.

[0043] In one aspect, a mouse is provided that cannot express functional endogenous mouse ADAM6 from endogenous mouse ADAM6 locus.In one embodiment, the mouse comprises an ectopic nucleic acid sequence that encodes ADAM6 or a functional fragment thereof that is functional in the mouse.In a particular embodiment, the ectopic nucleic acid sequence encodes a protein that rescues the loss of offspring production exhibited by male mice that are homozygous for ADAM6 knockout.In a particular embodiment, the ectopic nucleic acid sequence encodes mouse ADAM6 protein.

[0044] In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 locus, the mouse comprising an ectopic nucleic acid sequence that confers ADAM6 function to the mouse. In one embodiment, the nucleic acid sequence comprises an endogenous mouse ADAM6 sequence or a functional fragment thereof. In one embodiment, the endogenous mouse ADAM6 sequence is located within the 3'-most mouse immunoglobulin heavy chain V gene segment (V H ) and the 5'-most mouse immunoglobulin heavy chain D gene segment (D H ) containing the ADAM6a and ADAM6b coding sequences.

[0045] In one embodiment, the nucleic acid sequence comprises a sequence encoding mouse ADAM6a or a functional fragment thereof, and / or a sequence encoding mouse ADAM6b or a functional fragment thereof, wherein the ADAM6a and / or ADAM6b or functional fragment(s) thereof are operably linked to a promoter. In one embodiment, the promoter is a human promoter. In one embodiment, the promoter is a mouse ADAM6 promoter. In a particular embodiment, the ADAM6 promoter is operably linked to the 5'-most mouse D H The first codon of the first ADAM6 gene closest to the gene segment and the 5'-most D H The promoter comprises a sequence between the recombination signal sequence of the gene segment, where 5' is indicated relative to the transcriptional direction of mouse immunoglobulin genes. In one embodiment, the promoter is a viral promoter. In a particular embodiment, the viral promoter is a cytomegalovirus (CMV) promoter. In one embodiment, the promoter is , the ubiquitin promoter.

[0046] In one embodiment, the promoter is an inducible promoter. In one embodiment, the inducible promoter controls expression in non-reproductive tissue. In one embodiment, the inducible promoter controls expression in reproductive tissue. In a particular embodiment, the expression of the mouse ADAM6a and / or ADAM6b sequence or functional fragment(s) thereof is developmentally regulated in reproductive tissue by an inducible promoter.

[0047] In one embodiment, the mouse ADAM6a and / or ADAM6b is selected from ADAM6a of SEQ ID NO:1 and / or ADAM6b of SEQ ID NO:2. In one embodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO:3. In a particular embodiment, the ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO:3 immediately upstream (relative to the transcriptional direction of ADAM6a) of the first codon of ADAM6a, and extending to the end of SEQ ID NO:3 upstream of the ADAM6 coding region. In another particular embodiment, the ADAM6 promoter is a fragment that extends from within about 5 to about 20 nucleotides upstream of the start codon of ADAM6a to about 0.5 kb, 1 kb, 2 kb, or 3 kb or more upstream of the start codon of ADAM6a.

[0048] In one embodiment, the nucleic acid sequence comprises SEQ ID NO:3 or a fragment thereof that, when placed into a mouse that is infertile or has low fertility due to lack of ADAM6, improves fertility or restores fertility to near wild-type fertility. In one embodiment, SEQ ID NO:3 or a fragment thereof confers on male mice the ability to produce sperm cells that can traverse female mouse oviducts and fertilize mouse eggs.

[0049] In one embodiment, a deletion of the endogenous nucleotide sequence encoding the ADAM6 protein and an endogenous mouse V H Gene segment, human V H A mouse is provided that comprises a replacement with a gene segment and an ectopic nucleotide sequence encoding a mouse ADAM6 protein, or an ortholog or homolog or fragment thereof, that is functional in male mice.

[0050] In one embodiment, the mouse comprises an immunoglobulin heavy chain locus comprising a deletion of an endogenous immunoglobulin locus nucleotide sequence that comprises the endogenous ADAM6 gene, and comprises a nucleotide sequence encoding one or more human immunoglobulin gene segments, and an ectopic nucleotide sequence encoding the mouse ADAM6 protein is within or directly adjacent to the nucleotide sequence encoding the one or more human immunoglobulin gene segments.

[0051] In one embodiment, the mouse is mutated with one or more human V H All or substantially all endogenous V in the nucleotide sequence encoding the gene segment H The ectopic nucleotide sequence encoding the mouse ADAM6 protein, comprising a replacement of a gene segment, is H Within the nucleotide sequence encoding the gene segment or H In one embodiment, the mouse is directly adjacent to a nucleotide sequence encoding an endogenous D H One or more endogenous D at a gene locus H One or more human D H In one embodiment, the mouse further comprises a replacement with an endogenous J gene segment. H One or more endogenous J at a gene locus H One or more human J of a gene segment H In one embodiment, the mouse further comprises a replacement of all or substantially all endogenous V H , D H and J. H Gene segment replacements and endogenous V H , D H and J H At the gene locus, human V H , D H and J. HIn a specific embodiment, the mouse comprises an ectopic sequence encoding a mouse ADAM6 protein, the ectopic sequence encoding the mouse ADAM6 protein being a replacement of a gene segment present in a human V H The second V from the 3'-most end of the gene segment H Gene segments and the human V H The ultimate 3'V of the gene segment H In certain embodiments, the mouse is a mouse having all or substantially all of the mouse V H Deletions of gene segments and all or substantially all human V H The ectopic nucleotide sequence encoding the mouse ADAM6 protein, comprising a substitution in a gene segment, is H Downstream of 1-2 and human gene segment V H It is located upstream of 6-1.

[0052] In certain embodiments, the mouse comprises one or more human V H All or substantially all endogenous V in the nucleotide sequence encoding the gene segment H The ectopic nucleotide sequence encoding the mouse ADAM6 protein, comprising a replacement of a gene segment, is H Within the nucleotides encoding the gene segment or H It immediately flanks the nucleotides encoding the gene segment.

[0053] In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is present on a transgene in the genome of the mouse. In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is present extrachromosomally in the mouse.

[0054] In one aspect, a mouse is provided that comprises an alteration of an endogenous immunoglobulin heavy chain locus, the mouse expressing B cells comprising a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence, the B cells comprising a gene in their genome (e.g., on a B cell chromosome) encoding ADAM6 or an orthologue or homologue 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 is selected from the group consisting of C, D, J and / or J sequences. H 1, hinge, C H 2. C H 3 and combinations thereof.

[0055] In one aspect, a genetically modified mouse is provided, the genetically modified mouse comprising a functionally silenced immunoglobulin light chain gene and further comprising a replacement of one or more endogenous immunoglobulin heavy chain variable region gene segments with one or more human immunoglobulin heavy chain variable region gene segments; lacking a functional endogenous ADAM6 locus; and comprising an ectopic nucleotide sequence that expresses a mouse ADAM6 protein, or an ortholog or homolog or fragment thereof, that is functional in a male mouse.

[0056] In one embodiment, the mouse is provided that lacks functional endogenous mouse ADAM6 locus or sequence and comprises ectopic nucleotide sequence encoding mouse ADAM6 locus or functional fragment of mouse ADAM6 locus or sequence, and can be crossed with mouse of opposite sex to produce offspring that comprises said ectopic ADAM6 locus or sequence.In one embodiment, the mouse is male.In one embodiment, the mouse is female.

[0057] In one embodiment, a genetically modified mouse comprises a human immunoglobulin heavy chain variable region gene segment at an endogenous mouse immunoglobulin heavy chain variable region gene locus; and lacks an endogenous functional ADAM6 sequence at the endogenous mouse immunoglobulin heavy chain variable region gene locus. and an ectopic nucleotide sequence expressing a mouse ADAM6 protein, or an ortholog or homolog or fragment thereof, that is functional in a male mouse.

[0058] In one embodiment, the ectopic nucleotide sequence expressing the mouse ADAM6 protein is extrachromosomal.In one embodiment, the ectopic nucleotide sequence expressing the mouse ADAM6 protein is integrated into one or more loci in the genome of the mouse.In a particular embodiment, the one or more loci include immunoglobulin loci.

[0059] In one embodiment, a mouse is provided that expresses immunoglobulin heavy chain sequences derived from human V, D and J gene segments from a modified endogenous mouse immunoglobulin heavy chain locus, the mouse comprising ADAM6 activity that is functional in the mouse.

[0060] In one embodiment, the mouse comprises 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 segment is a human D gene segment. In one embodiment, the J gene segment is a human J gene segment. In one embodiment, the mouse further comprises a humanized heavy chain constant region sequence, the humanization being a C H 1, hinge, C H 2. C H In a specific embodiment, the heavy chain comprises a substitution of a sequence selected from a human V gene segment, a human D gene segment, a human J gene segment, a human C gene segment, a human D gene segment, a human H ... H 1 sequence, human or mouse hinge sequence, mouse CH 2 sequence, and mouse C H In another specific embodiment, the mouse further comprises a human light chain constant sequence.

[0061] In one embodiment, the D gene segment is adjacent 5' (relative to the transcriptional orientation of the D gene segment) to a sequence encoding an ADAM6 activity that is functional in the mouse.

[0062] In one embodiment, ADAM6 activity that is functional in the mouse results from expression of a nucleotide sequence that is 5' of the 5'-most D gene segment and 3' of the 3'-most V gene segment (relative to the direction of transcription of the V gene segments) of the modified endogenous mouse heavy chain immunoglobulin locus.

[0063] 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. H 1-2 gene segment and V H 6-1 gene segment.

[0064] In one embodiment, the nucleotide sequence comprises a sequence selected from a mouse ADAM6b sequence or a functional fragment thereof, a mouse ADAM6a sequence or a functional fragment thereof, and a combination thereof.

[0065] In one embodiment, the nucleotide sequence between the two human V gene segments is arranged in opposite transcriptional orientation relative to the human V gene segments. In a specific embodiment, the nucleotide sequence encodes, from 5' to 3' relative to the transcriptional direction of the ADAM6 gene, an ADAM6a sequence followed by an ADAM6b sequence.

[0066] In one embodiment, the mouse comprises a human V gene segment V H 1-2 and V H It involves the replacement of the human ADAM6 pseudogene sequence between cDNA 6-1 with the mouse ADAM6 sequence or a functional fragment thereof.

[0067] In one embodiment, the sequence encoding ADAM6 activity that is functional in the mouse is a mouse ADAM6 sequence or a functional fragment thereof.

[0068] In one embodiment, the mouse is modified with an endogenous mouse DFL16.1 gene segment (e.g., in the case of a mouse heterozygous for the modified endogenous mouse immunoglobulin heavy chain locus) or a human DFL16.1 gene segment. H In one embodiment, the immunoglobulin heavy chain D gene segment expressed by the mouse comprises an endogenous mouse DFL16.1 gene segment or a human DFL16.1 gene segment. H Derived from the 1-1 gene segment.

[0069] In one aspect, a mouse is provided that comprises a nucleic acid sequence encoding mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) in DNA-bearing cells of a non-rearranged B cell lineage, but does not comprise a nucleic acid sequence encoding said mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) in a B cell comprising a rearranged immunoglobulin locus, wherein the nucleic acid sequence encoding said mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) is present in a location in the genome that is different from the location where the mouse ADAM6 gene occurs in a wild-type mouse. In one embodiment, the nucleic acid sequence encoding said mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) is present in all or substantially all DNA-bearing cells that are not of a rearranged B cell lineage; in one embodiment, the nucleic acid sequence is present in germline cells of said mouse, but not in the chromosomes of rearranged B cells.

[0070] In one aspect, a mouse is provided that comprises a nucleic acid sequence encoding mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) in all or substantially all DNA-bearing cells, including B cells, that comprise a rearranged immunoglobulin locus, and the nucleic acid sequence encoding mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) is present in a location in the genome that is different from the location where the mouse ADAM6 gene appears in a wild-type mouse. In one embodiment, the nucleic acid sequence encoding mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) is on a nucleic acid adjacent to the rearranged immunoglobulin locus. In one embodiment, the nucleic acid adjacent to the rearranged immunoglobulin locus is a chromosome. In one embodiment, the chromosome is a chromosome found in a wild-type mouse, and the chromosome comprises an alteration of the mouse immunoglobulin locus.

[0071] In one aspect, a genetically modified mouse is provided, comprising a B cell comprising an ADAM6 sequence or its orthologue or homologue in its genome. In one embodiment, the ADAM6 sequence or its orthologue or homologue is in an immunoglobulin heavy chain locus. In one embodiment, the ADAM6 sequence or its orthologue or homologue is in a locus that is not an immunoglobulin locus. In one embodiment, the ADAM6 sequence is on a transgene driven by a heterologous promoter. In certain embodiments, the heterologous promoter is a non-immunoglobulin promoter. In certain embodiments, the B cell expresses an ADAM6 protein or its orthologue or homologue.

[0072] In one embodiment, greater than 90% of the B cells of the mouse express an ADAM6 protein, or an orthologue thereof, or a homolog thereof, or a fragment thereof, that is functional in the mouse. In a particular embodiment, the mouse is a male mouse.

[0073] In one embodiment, the B cell genome comprises a first allele and a second allele comprising the ADAM6 sequence or an orthologue or homolog thereof. In one embodiment, the B cell genome comprises a first allele but not a second allele comprising the ADAM6 sequence or an orthologue or homolog thereof.

[0074] In one embodiment, a mouse is provided that comprises an alteration in one or more endogenous ADAM6 alleles.

[0075] In one embodiment, the modification renders the mouse incapable of expressing functional ADAM6 protein from at least one of the one or more endogenous ADAM6 alleles. In a specific embodiment, the mouse is incapable of expressing functional ADAM6 protein from each of the endogenous ADAM6 alleles.

[0076] In one embodiment, the mouse is incapable of expressing functional ADAM6 protein from each endogenous ADAM6 allele, and the mouse comprises an ectopic ADAM6 sequence.

[0077] In one embodiment, the mouse is unable to express functional ADAM6 protein from each endogenous ADAM6 allele, and the mouse comprises an ectopic ADAM6 sequence within 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 kb or more upstream (relative to the direction of transcription of the mouse heavy chain locus) of the mouse immunoglobulin heavy chain constant region sequence. In a specific embodiment, the ectopic ADAM6 sequence is in the endogenous immunoglobulin heavy chain locus (e.g., within an intergenic VD region, between two V gene segments, between a V gene segment and a D gene segment, between a D gene segment and a J gene segment, etc.). In a specific embodiment, the ectopic ADAM6 sequence is within a 90-100 kb intergenic sequence between the last mouse V gene segment and the first mouse D gene segment. In another specific embodiment, the endogenous 90 to 100 kb intergenic VD sequence is deleted and the ectopic ADAM6 sequence is placed between the last V gene segment and the first D gene segment.

[0078] In one embodiment, an infertile male mouse is provided, comprising a deletion of two or more endogenous ADAM6 alleles.In one embodiment, a female mouse is provided that is a carrier of the male infertility trait, comprising a non-functional ADAM6 allele or an endogenous ADAM6 allele knockout in its germline.

[0079] In one embodiment, a mouse is provided that lacks endogenous immunoglobulin heavy chain V, D and J gene segments, wherein a majority of the B cells of the mouse comprise an ADAM6 sequence or an ortholog or homolog thereof.

[0080] In one embodiment, the mouse lacks endogenous immunoglobulin heavy chain gene segments selected from two or more V gene segments, two or more D gene segments, two or more J gene segments, and combinations thereof. In one embodiment, the mouse lacks immunoglobulin heavy chain gene segments selected from at least one and no more than 89 V gene segments, at least one and no more than 13 D gene segments, at least one and no more than 4 J gene segments, and combinations thereof. In one embodiment, the mouse lacks a genomic DNA fragment from chromosome 12 that comprises about 3 megabases of the endogenous immunoglobulin heavy chain locus. In a particular embodiment, the mouse lacks all functional endogenous heavy chain V, D, and J gene segments. In a particular embodiment, the mouse lacks an immunoglobulin heavy chain gene segment selected from at least one and no more than 89 V gene segments, at least one and no more than 13 D gene segments, at least one and no more than 4 J gene segments, and combinations thereof. In one embodiment, the mouse lacks a genomic DNA fragment from chromosome 12 that comprises about 3 megabases of the endogenous immunoglobulin heavy chain locus. In a particular embodiment, the mouse lacks all functional endogenous heavy chain V, D, and J gene segments. In an embodiment, the mouse has a V H Gene segment, 13 D H Gene segments and four J H There is no gene segment.

[0081] In one aspect, a mouse is provided having in its germline a genome comprising an alteration at the immunoglobulin heavy chain locus, the alteration at the immunoglobulin heavy chain locus comprising replacement of one or more mouse immunoglobulin variable region sequences with one or more non-mouse immunoglobulin variable region sequences, the mouse comprising a nucleic acid sequence encoding a mouse ADAM6 protein. H and J. H Sequence and at least 3, at least 10, at least 20, at least 40, at least 60 or at least 80 V H In a further preferred embodiment, the D of the immunoglobulin heavy chain locus is replaced by a non-mouse immunoglobulin variable region sequence. H , J H and all V HThe sequence is replaced by a non-mouse immunoglobulin variable region sequence. The non-mouse immunoglobulin variable region sequence may be unrearranged. In a preferred embodiment, the non-mouse immunoglobulin variable region sequence is a complete non-rearranged D of a non-mouse species. H and J. H region and at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 unrearranged V H In a further preferred embodiment, the non-mouse immunoglobulin variable region sequence comprises the V H , D H and J. H The non-mouse species may be Homo sapiens, and the non-mouse immunoglobulin variable region sequences may be human sequences.

[0082] In one aspect, a mouse is provided that expresses an antibody comprising at least one human variable domain / non-human constant domain immunoglobulin polypeptide, wherein the mouse expresses a mouse ADAM6 protein or an ortholog or homolog thereof from a locus other than the immunoglobulin locus.

[0083] In one embodiment, the ADAM6 protein, or an ortholog or homolog thereof, is expressed in a B cell of the mouse, and the B cell comprises a rearranged immunoglobulin sequence comprising a human variable sequence and a non-human constant sequence.

[0084] In one embodiment, the non-human constant sequences are rodent sequences, hi one embodiment, the rodent is selected from a mouse, a rat, and a hamster.

[0085] In one aspect, a method for producing an infertile male mouse is provided, the method comprising: rendering the endogenous ADAM6 allele of a donor ES cell non-functional (or knocking out the allele), introducing the donor ES cell into a host embryo, gestating the host embryo in a surrogate mother, and allowing the surrogate mother to give birth to offspring derived in whole or in part from the donor ES cell. In one embodiment, the method further comprises mating the offspring to obtain an infertile male mouse.

[0086] In one aspect, a method is provided for producing mice with targeted genetic modification, the mice being infertile, the method comprises: (a) making targeted genetic modification in genome; (b) modifying said genome to knock out endogenous ADAM6 allele or make endogenous ADAM6 allele non-functional; and (c) using said genome to produce mice.In various embodiments, said genome is from ES cell or used in nuclear transfer experiments.

[0087] In one embodiment, there is provided a mouse generated using the targeting vector, nucleotide construct or cell described herein.

[0088] In one embodiment, progeny of a cross between a mouse described herein and a second mouse that is a wild-type mouse or that has been genetically modified are provided.

[0089] In one aspect, a method is provided for maintaining a mouse strain comprising replacement of a mouse immunoglobulin heavy chain sequence with one or more xenogenous immunoglobulin heavy chain sequences, in one embodiment, the one or more xenogenous immunoglobulin heavy chain sequences are human immunoglobulin heavy chain sequences.

[0090] In one embodiment, the mouse strain comprises one or more mouse V H , D H and / or J. H In one embodiment, the mouse comprises a deletion of one or more human V gene segments.H Gene segment, one or more human D H A gene segment and / or one or more human J H In one embodiment, the mouse further comprises at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 human V gene segments. H Segment, at least 27 human D H A gene segment, and at least six J H In certain embodiments, the mouse comprises at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 human V gene segments operably linked to constant region genes. H segment, said at least 27 human D H A gene segment and said at least six J H In one embodiment, the constant region gene is a mouse constant region gene. In one embodiment, the constant region gene is a C H 1, hinge, C H 2. C H 3 and / or C H 4, or a combination thereof.

[0091] In one embodiment, the method comprises generating male mice heterozygous for the mouse immunoglobulin heavy chain sequence substitution and mating the heterozygous male mice with wild-type female mice or with female mice homozygous or heterozygous for the human heavy chain sequence, hi one embodiment, the method comprises maintaining the line by repeatedly mating heterozygous males with females that are wild-type or homozygous or heterozygous for the human heavy chain sequence.

[0092] In one embodiment, the method comprises obtaining cells from a male or female mouse homozygous or heterozygous for a human heavy chain sequence, utilizing these cells as donor cells or nuclei from these cells as donor nuclei, and using the cells or nuclei to generate a genetically modified animal using a host cell and / or by gestation of the cells and / or nuclei in a surrogate mother.

[0093] In one embodiment, only male mice heterozygous for the replacement at the heavy chain locus are bred to female mice, in certain embodiments, the female mice are homozygous, heterozygous or wild type for the replaced heavy chain locus.

[0094] In one embodiment, the mouse further comprises a replacement of a lambda and / or kappa light chain variable sequence at an endogenous immunoglobulin light chain locus with a heterologous immunoglobulin light chain sequence, in one embodiment, the heterologous immunoglobulin light chain sequence is a human immunoglobulin lambda and / or kappa light chain variable sequence.

[0095] In one embodiment, the mouse further comprises a transgene at a locus other than an endogenous immunoglobulin locus, wherein the transgene comprises a rearranged or unrearranged heterologous λ or κ light chain sequence (e.g., an unrearranged V L and non-reconstructed J L In one embodiment, the VJ comprises a sequence encoding the VJ. In one embodiment, the heterologous λ or κ light chain sequence is human. In one embodiment, the constant region sequence is selected from rodent, human and non-human primate. In one embodiment, the constant region sequence is selected from mouse, rat and hamster. In one embodiment, the transgene comprises a non-immunoglobulin promoter driving expression of the light chain sequence. In a particular embodiment, the promoter is a transcriptionally active promoter. In a particular embodiment, the promoter is a ROSA26 promoter.

[0096] In one embodiment, a nucleic acid construct is provided that includes an upstream homology arm and a downstream homology arm, the upstream homology arm including a sequence identical or substantially identical to a human immunoglobulin heavy chain variable region sequence, the downstream homology arm including a sequence identical or substantially identical to a human or mouse immunoglobulin variable region sequence, and a sequence including a nucleotide sequence encoding a mouse ADAM6 protein is disposed between the upstream homology arm and the downstream homology arm. In a particular embodiment, the sequence encoding the mouse ADAM6 gene is operably linked to a mouse promoter to which the mouse ADAM6 is linked in a wild-type mouse.

[0097] In one aspect, a targeting vector is provided comprising (a) a nucleotide sequence identical or substantially identical to a human variable region gene segment nucleotide sequence, and (b) a nucleotide sequence encoding a mouse ADAM6 or an ortholog or homolog or fragment thereof that is functional in a mouse.

[0098] In one embodiment, the targeting vector further comprises a promoter operably linked to the sequence encoding the mouse ADAM6. In a specific embodiment, the promoter is a mouse ADAM6 promoter.

[0099] In one aspect, a nucleotide construct for modifying a mouse immunoglobulin heavy chain variable locus is provided, the construct comprising at least one site-specific recombinase recognition site and a sequence encoding an ADAM6 protein or an ortholog, homolog, or fragment thereof that is functional in a mouse.

[0100] In one embodiment, mouse cells and mouse embryos are provided, including but not limited to ES cells, pluripotent cells and induced pluripotent cells, that comprise the genetic modifications described herein. XX cells and XY cells are provided. Cells that comprise nuclei that contain modifications described herein, for example, modifications that are introduced into cells by pronuclear injection, are also provided. Cells, embryos and mice that comprise a virus-introduced ADAM6 gene, for example, cells, embryos and mice that comprise a transduction construct that comprises an ADAM6 gene that is functional in the mouse, are also provided.

[0101] In one aspect, a genetically modified mouse cell is provided that lacks a functional endogenous mouse ADAM6 locus, the genetically modified mouse cell comprising an ectopic nucleotide sequence encoding a mouse ADAM6 protein or a functional fragment thereof. In one embodiment, the cell further comprises a modification of an endogenous immunoglobulin heavy chain variable gene sequence. In a particular embodiment, the modification of the endogenous immunoglobulin heavy chain variable gene sequence comprises a modification of a mouse V H Gene segment deletion, mouse D H Gene segment deletion, mouse J H In a specific embodiment, the mouse comprises a deletion selected from a deletion of one or more mouse immunoglobulin V gene segments and combinations thereof. H , D H and / or J. H In certain embodiments, the human immunoglobulin sequence comprises a substitution of a human V H , Human V L , Human D H , Human J H , Human J Land combinations thereof.

[0102] In one embodiment, the cell is a totipotent cell, a pluripotent cell, or an induced pluripotent cell. In a particular embodiment, the cell is a mouse ES cell.

[0103] In one aspect, a mouse B cell is provided that comprises a rearranged immunoglobulin heavy chain gene, the B cell comprising a nucleic acid sequence on a chromosome of the B cell that encodes an ADAM6 protein or an 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.

[0104] In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (eg, a B cell chromosome) that is adjacent to the rearranged mouse immunoglobulin heavy chain locus.

[0105] In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (eg, a B cell chromosome) that is separate from the nucleic acid molecule that comprises the rearranged mouse immunoglobulin heavy chain locus.

[0106] In one embodiment, the mouse B cell comprises a rearranged non-mouse immunoglobulin variable gene sequence operably linked to a mouse or human immunoglobulin constant region gene, wherein the B cell comprises a nucleic acid sequence encoding an ADAM6 protein, or an ortholog or homolog or fragment thereof, that is functional in a male mouse.

[0107] In one aspect, a mouse somatic cell is provided, comprising a chromosome comprising a modified immunoglobulin heavy chain locus and a nucleic acid sequence encoding mouse ADAM6 or an orthologue or homologue 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 immunoglobulin 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. In a particular embodiment, the somatic cell is a B cell. In a particular embodiment, the cell is a germ cell. In a particular embodiment, the cell is a stem cell.

[0108] In one aspect, a mouse germ cell is provided, the mouse germ cell comprising a nucleic acid sequence encoding mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) on a chromosome of the germ cell, the nucleic acid sequence encoding mouse ADAM6 (or a homolog or ortholog or functional fragment thereof) being at a chromosomal location that is different from the chromosomal location 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 embodiment, 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 substitution of at least one mouse immunoglobulin sequence with at least one non-mouse immunoglobulin sequence. In a particular embodiment, the at least one non-mouse immunoglobulin sequence is a human immunoglobulin sequence.

[0109] In one aspect, there is provided a pluripotent, induced pluripotent or totipotent cell derived from a mouse as described herein. In a particular embodiment, the cell is a mouse embryonic stem (ES) cell.

[0110] In one aspect, a tissue is provided from a mouse as described herein. In one embodiment, the tissue is from the spleen, lymph node, or bone marrow of a mouse as described herein.

[0111] In one aspect, there is provided a nucleus from a mouse as described herein, hi one embodiment, the nucleus is from a diploid cell that is not a B cell.

[0112] In one embodiment, a nucleotide sequence encoding an immunoglobulin variable region produced in a mouse as described herein is provided.

[0113] In one embodiment, the immunoglobulin heavy chain variable region amino acid sequence or the immunoglobulin light chain variable region amino acid sequence of an antibody made in a mouse described herein is provided.

[0114] In one embodiment, an immunoglobulin heavy chain variable region nucleotide sequence or an immunoglobulin light chain variable region nucleotide sequence encoding the variable region of an antibody made in a mouse as described herein is provided.

[0115] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof (e.g., Fab, F(ab)2, scFv) produced in the mouse described herein. In one aspect, the present invention provides a method for producing a genetically modified mouse, comprising replacing one or more immunoglobulin heavy chain gene segments upstream (based on the transcription of immunoglobulin heavy chain gene segments) of the 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 (based on the transcription of immunoglobulin heavy chain gene segments) of the ADAM6 locus of the mouse with one or more human immunoglobulin heavy or light chain gene segments. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments upstream of the endogenous ADAM6 locus of the mouse comprise a V gene segment. In one embodiment, the human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments upstream of the mouse's endogenous ADAM6 locus comprise V and D gene segments. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments downstream of the mouse's endogenous ADAM6 locus comprise J gene segments. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments downstream of the mouse's endogenous ADAM6 locus comprise D and J gene segments. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments downstream of the mouse's endogenous ADAM6 locus comprise V, D and J gene segments.

[0116] In one embodiment, the one or more immunoglobulin heavy chain gene segments upstream and / or downstream of the ADAM6 gene are replaced in a pluripotent cell, an induced pluripotent cell, or a totipotent cell to form a genetically modified progenitor cell; the genetically modified progenitor cell is introduced into a host; and the host containing the genetically modified progenitor cell is allowed to become pregnant to form a mouse containing a genome derived from the genetically modified progenitor cell.In one embodiment, the host is an embryo.In a particular embodiment, the host is selected from a mouse pre-morula (e.g., 8- or 4-cell stage), a tetraploid embryo, an aggregate of embryonic cells, or a blastocyst.

[0117] In one embodiment, a method of making a genetically modified mouse is provided, the method comprising: replacing a mouse nucleotide sequence comprising a mouse immunoglobulin gene segment and a mouse ADAM6 (or an 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; thereafter, replacing the mouse ADAM6 coding sequence (or a sequence encoding an ortholog or homolog or functional fragment thereof) with the human immunoglobulin gene segment. The method includes inserting the progeny segment into a sequence comprising the progeny segment to form a second chimeric locus.

[0118] In one embodiment, the second chimeric locus comprises a human immunoglobulin heavy chain variable (V H In one embodiment, the second chimeric locus comprises a human immunoglobulin light chain variable (V L In certain embodiments, the second chimeric locus comprises a human D H Gene segments and human J H Human V operably linked to a gene segment H Gene segment or human V L In a further specific embodiment, the second chimeric locus comprises a gene segment of mouse C H 2+C H 3. Fusion with human CH 1 sequence, or human C H 1 and a third chimeric locus comprising a human hinge sequence.

[0119] In one embodiment, there is provided a use of a mouse comprising an ectopic nucleotide sequence comprising a mouse ADAM6 locus or sequence for producing a fertile male mouse, the use comprising mating a mouse comprising the ectopic nucleotide sequence comprising the mouse ADAM6 locus or sequence with a mouse lacking a functional endogenous mouse ADAM6 locus or sequence, and obtaining female progeny capable of producing progeny having the ectopic ADAM6 locus or sequence, or obtaining male progeny comprising the ectopic ADAM6 locus or sequence and exhibiting fertility approximately the same as that exhibited by a wild-type male mouse.

[0120] In one embodiment, there is provided use of a mouse as described herein for generating immunoglobulin variable region nucleotide sequences.

[0121] In one embodiment, there is provided the use of a mouse as described herein to generate fully human Fab or fully human F(ab)2.

[0122] In one embodiment, there is provided the use of a mouse as described herein to generate an immortalized cell line.

[0123] In one embodiment, there is provided a use of a mouse as described herein for generating hybridomas or quadromas.

[0124] In one embodiment, there is provided the use of a mouse as described herein to generate a phage library containing human heavy chain variable regions and human light chain variable regions.

[0125] In one aspect, there is provided a use of a mouse described herein for generating variable region sequences for making human antibodies, the use comprising: (a) immunizing a mouse described herein with an antigen of interest; (b) isolating lymphocytes from the immunized mouse of (a); (c) exposing the lymphocytes to one or more labeled antibodies; (d) identifying lymphocytes capable of binding to the antigen of interest; and (e) amplifying one or more variable region nucleic acid sequences from the lymphocytes, thereby generating variable region sequences.

[0126] In one embodiment, the lymphocytes are derived from the spleen of the mouse. In one embodiment, the lymphocytes are derived from the lymph nodes of the mouse. In one embodiment, the lymphocytes are derived from the bone marrow of the mouse.

[0127] In one embodiment, the labeled antibody is a fluorophore-conjugated antibody. In one embodiment, the one or more fluorophore-conjugated antibodies are selected from IgM, IgG and / or a combination thereof.

[0128] In one embodiment, the lymphocytes are B cells.

[0129] In one embodiment, the one or more variable region nucleic acid sequences comprise a heavy chain variable region sequence. In one embodiment, the one or more variable region nucleic acid sequences comprise a light chain variable region sequence. In one particular embodiment, the light chain variable region sequence is an immunoglobulin kappa light chain variable region sequence. In one embodiment, the one or more variable region nucleic acid sequences comprise a heavy chain variable region sequence and a kappa light chain variable region sequence.

[0130] In one embodiment, there is provided a use of a mouse described herein for generating heavy chain variable region sequences and κ light chain variable region sequences for making human antibodies, the use comprising: (a) immunizing a mouse described herein with an antigen of interest; (b) isolating a spleen from the immunized mouse of (a); (c) exposing B lymphocytes from the spleen to one or more labeled antibodies; (d) identifying B lymphocytes of (c) capable of binding to the antigen of interest; and (e) amplifying heavy chain variable region nucleic acid sequences and κ light chain variable region nucleic acid sequences from the B lymphocytes, thereby generating the heavy chain variable region sequences and κ light chain variable region sequences.

[0131] In one embodiment, there is provided a use of a mouse described herein for generating heavy chain variable region sequences and κ light chain variable region sequences for making human antibodies, the use comprising: (a) immunizing a mouse described herein with an antigen of interest; (b) isolating 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 B lymphocytes of (c) capable of binding to the antigen of interest; and (e) amplifying heavy chain variable region nucleic acid sequences and κ light chain variable region nucleic acid sequences from the B lymphocytes, thereby generating the heavy chain variable region sequences and κ light chain variable region sequences.

[0132] In one embodiment, there is provided a use of a mouse as described herein for generating heavy and kappa light chain variable region sequences for making human antibodies, 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 the B lymphocytes of (c) capable of binding to the antigen of interest, and (e) amplifying the heavy and kappa light chain variable region nucleic acid sequences from the B lymphocytes, thereby generating the heavy and kappa light chain variable region sequences. In various embodiments, the one or more labeled antibodies are selected from IgM, IgG, and / or a combination thereof.

[0133] In various embodiments, there is provided a use of a mouse as described herein for generating heavy and kappa light chain variable region sequences for making human antibodies, further comprising 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 producing a human antibody.

[0134] In various embodiments, the human heavy chain constant region is selected from IgM, IgD, IgA, IgE, and IgG. In various specific embodiments, the IgG is selected from IgG1, IgG2, IgG3, and IgG4. In various embodiments, the human heavy chain constant region is selected from C H 1, hinge, C H 2. C H 3. C H In various embodiments, the light chain constant region is an immunoglobulin kappa constant region. In various embodiments, the cells are HeLa cells, DU145 cells, Lncap cells, MCF-7 cells, MDA-MB-438 cells, PC3 cells, T47D cells, THP-1 cells, U87 cells, SHSY5Y (human neuroblastoma) cells, Saos-2 cells, Vero cells, or a combination thereof. cell, C H In certain embodiments, the cells are selected from C0 cells, GH3 cells, PC12 cells, human retinal cells (e.g., PER.C6™ cells), and MC3T3 cells. H O cells.

[0135] In one embodiment, a method is provided for producing a reverse chimeric rodent-human antibody specific to an antigen of interest, the method comprising immunizing a mouse as described herein with the antigen, isolating at least one cell from the mouse that produces a reverse chimeric mouse-human antibody specific to the antigen, culturing at least one cell that produces the reverse chimeric mouse-human antibody specific to the antigen, and obtaining the antibody. In one embodiment, the reverse chimeric mouse-human antibody comprises a human heavy chain variable domain fused to a mouse or rat heavy chain constant gene and a human light chain variable domain fused to a mouse or rat or human light chain constant gene.

[0136] In one embodiment, the culturing of at least one cell producing the reverse chimeric rodent-human antibody specific to the antigen is carried out with at least one hybridoma cell produced from at least one cell isolated from the mouse.

[0137] In one embodiment, a method for producing a fully human antibody specific to an antigen of interest is provided, the method comprising immunizing a mouse as described herein with the antigen, isolating at least one cell from the mouse that produces a reverse chimeric rodent-human antibody specific to the antigen, producing at least one cell that produces a fully human antibody derived from the reverse chimeric rodent-human antibody specific to the antigen, and culturing the at least one cell that produces the fully human antibody, and obtaining the fully human antibody.

[0138] In various embodiments, the at least one cell isolated from the mouse that produces reverse chimeric rodent-human antibodies specific for said antigen is a splenocyte or a B cell.

[0139] In various embodiments, the antibody is a monoclonal antibody.

[0140] In various embodiments, immunization with the antigen of interest is carried out using protein, DNA, a combination of DNA and protein, or cells expressing the antigen.

[0141] In one aspect, there is provided a use of a mouse as described herein to generate a nucleic acid sequence encoding an immunoglobulin variable region or fragment thereof. In one embodiment, the nucleic acid sequence is used to generate a human antibody or antigen-binding fragment thereof. In one embodiment, the mouse is used to generate an antibody, a multispecific antibody (e.g., a bispecific antibody), a scFv, a bispecific scFv, a diabody, a triabody, a tetrabody, a V-NAR ... H H, V L , F(ab), F(ab)2, DVD (i.e., dual variable domain antigen binding protein), SVD (i.e., single variable domain antigen binding protein) or bispecific T-cell engager (BiTE).

[0142] In one aspect, there is provided a use of a mouse described herein for introducing an ectopic ADAM6 sequence into a mouse lacking a functional endogenous mouse ADAM6 sequence, the use comprising breeding the mouse described herein with a mouse lacking the functional endogenous mouse ADAM6 sequence.

[0143] In one aspect, there is provided a use of genetic material from a mouse described herein to generate a mouse having an ectopic ADAM6 sequence. In one embodiment, the use comprises In one embodiment, the use includes nuclear transfer using a nucleus of a cell of a mouse described herein. In one embodiment, the use includes cloning a cell of a mouse described herein to produce an animal derived from the cell. In one embodiment, the use includes utilizing sperm or eggs of a mouse described herein in a process to generate a mouse comprising the ectopic ADAM6 sequence.

[0144] In one embodiment, a method is provided for producing a fertile male mouse comprising a modified immunoglobulin heavy chain locus, the method comprising the steps of: fertilizing a first mouse germ cell comprising a modification of an endogenous immunoglobulin heavy chain locus with a second mouse germ cell comprising an ADAM6 gene or an ortholog or homolog or fragment thereof that is functional in a male mouse; forming a fertilized cell; allowing the fertilized cell to develop into an embryo; and gestating the embryo in a surrogate mother to obtain a mouse.

[0145] In one embodiment, the fertilization is achieved by mating a male mouse with a female mouse.In one embodiment, the female mouse comprises the ADAM6 gene or its ortholog or homolog or fragment.In one embodiment, the male mouse comprises the ADAM6 gene or its ortholog or homolog or fragment.

[0146] In one aspect, there is provided the use of a nucleic acid sequence encoding a mouse ADAM6 protein or its ortholog or homolog or a functional fragment of the corresponding ADAM6 protein to restore or enhance fertility in a mouse having a genome comprising a modification in the immunoglobulin heavy chain locus, where the modification reduces or eliminates endogenous ADAM6 function.

[0147] In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at an ectopic location. In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at an endogenous immunoglobulin locus. In a particular 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 location other than the endogenous immunoglobulin locus.

[0148] In one embodiment, the present invention provides a use of a mouse described herein to produce a drug (e.g., an antigen binding protein), or to produce a sequence encoding a variable sequence of a drug (e.g., an antigen binding protein), for the treatment of a human disease or disorder. The present invention provides, for example, the following items. (Item 1) A mouse, wherein the mouse has a genome comprising a modification of an immunoglobulin heavy chain locus, the modification comprising a reduction or elimination of endogenous ADAM6 function, and the mouse further comprises a nucleic acid sequence encoding a mouse ADAM6 protein or its ortholog or homolog or a functional fragment of the corresponding ADAM6 protein. (Item 2) The mouse of item 1, wherein the nucleic acid sequence is located in an ectopic location. (Item 3) 3. The mouse of item 1 or 2, wherein the nucleic acid sequence is located in an endogenous immunoglobulin locus. (Item 4) 3. The mouse of item 1 or 2, wherein the nucleic acid sequence is integrated into the mouse genome at a location other than an endogenous immunoglobulin locus. (Item 5) 5. The mouse of any one of items 1 to 4, wherein the modification of the immunoglobulin heavy chain locus comprises the arrangement of one or more human immunoglobulin gene sequences. (Item 6) 5. The mouse of any one of items 1 to 4, wherein the modification of the immunoglobulin heavy chain locus comprises replacement of one or more sequences within the mouse immunoglobulin heavy chain locus with one or more human immunoglobulin gene sequences. (Item 7) The modification of the immunoglobulin heavy chain locus comprises modifying one or more endogenous heavy chain V(V H ) gene segments, H 7. The mouse of item 6, comprising a replacement with a .ALPHA.) gene segment. (Item 8) 2. The mouse of item 1, wherein the modification of the immunoglobulin heavy chain locus comprises replacement of an endogenous heavy chain variable gene sequence with a human heavy chain variable gene sequence. (Item 9) The mouse of any one of items 1 to 8, wherein the nucleic acid sequence encodes mouse ADAM6a protein and / or ADAM6b protein, or an ortholog, homolog or functional fragment thereof. (Item 10) 1. A method for modifying an immunoglobulin heavy chain locus in a mouse, comprising: (a) making a first modification of the mouse immunoglobulin heavy chain locus that results in a reduction or elimination of endogenous mouse ADAM6 activity in a male mouse; and (b) making a second modification to the mouse to add a nucleic acid sequence that confers to the mouse an ADAM6 activity that is functional in male mice. The method includes: (Item 11) 11. The method according to item 10, wherein the nucleic acid sequence of step (b) is added to an ectopic position. (Item 12) 12. The method of claim 10 or 11, wherein the first modification comprises the arrangement of one or more human immunoglobulin gene sequences. (Item 13) 12. The method of claim 10 or 11, wherein the first modification comprises replacement of one or more sequences within the mouse immunoglobulin heavy chain locus with one or more human immunoglobulin gene sequences. (Item 14) The first modification is selected from one or more endogenous V H One or more human V of a gene segment H The method of claim 13, comprising a substitution in a gene segment. (Item 15) 11. The method of claim 10, wherein the first modification comprises replacing the endogenous heavy chain variable gene sequence with a human heavy chain variable gene sequence. (Item 16) 16. The method according to any one of items 10 to 15, wherein the first and second modifications are carried out simultaneously. (Item 17) A mouse obtainable by the method according to any one of items 10 to 16. (Item 18) 18. The isolated cell from the mouse according to any one of items 1 to 9 and 17. (Item 19) 18. An isolated tissue from a mouse according to any one of items 1 to 9 and 17. (Item 20) 18. Use of the mouse according to any one of items 1 to 9 and 17 for the production of human-mouse reverse chimeric antibodies, fully human antibodies, fully human Fab fragments and / or fully human F(ab)2 fragments. (Item 21) A method for producing a reverse chimeric mouse-human antibody specific to an antigen, comprising the steps of: a) immunizing a mouse according to any one of items 1 to 9 and 17 with the antigen; b) isolating at least one cell from the mouse that produces a reverse-chimeric mouse-human antibody specific for the antigen; and c) culturing at least one cell that produces the antibody of step b) and obtaining said antibody. (Item 22) 22. The method according to item 21, wherein the culturing in step c) is carried out on at least one hybridoma cell produced from the at least one cell obtained in step b). (Item 23) A method for producing a fully human antibody specific to an antigen, comprising the steps of: a) immunizing a mouse according to any one of items 1 to 9 and 17 with the antigen; b) isolating at least one cell from the mouse that produces a reverse-chimeric mouse-human antibody specific for said antigen; c) producing at least one cell that produces a fully human antibody specific for said antigen derived from said antibody of step b); and d) culturing at least one cell producing the antibody of step c) and obtaining said antibody. The method includes: (Item 24) 24. The method according to any one of items 21 and 23, wherein the at least one cell obtained in step b) is a splenocyte or a B cell. (Item 25) 25. The method according to any one of items 21 to 24, wherein the antibody is a monoclonal antibody. (Item 26) 26. The method according to any one of items 21 to 25, wherein the immunization with said antigen in step a) is performed with a protein, DNA, a combination of DNA and protein, or with cells expressing said antigen. (Item 27) Use of a nucleic acid sequence encoding a mouse ADAM6 protein or its ortholog or homolog, or a functional fragment of the corresponding ADAM6 protein, to restore or improve fertility in mice having a genome containing a modification of the immunoglobulin heavy chain locus that reduces or eliminates endogenous ADAM6 function. (Item 28) 28. The use of item 27, wherein the nucleic acid sequence is integrated at an ectopic location in the genome of the mouse. (Item 29) 29. The use of item 27 or 28, wherein the nucleic acid sequence is integrated into an endogenous immunoglobulin locus of the genome of the mouse. (Item 30) 29. The use of item 27 or 28, wherein the nucleic acid sequence is integrated into the genome of the mouse at a location other than an endogenous immunoglobulin locus. [Brief description of the drawings]

[0149] [Figure 1A] FIG. 1A shows a general illustration (not to scale) of the direct genomic replacement of an approximately 3 megabase (Mb) mouse immunoglobulin heavy chain variable gene locus (filled symbols) with an approximately 1 megabase (Mb) human immunoglobulin heavy chain variable gene locus (open symbols). [Figure 1B]FIG. 1B shows a general illustration (not to scale) of the direct genomic replacement of the approximately 3 megabase (Mb) mouse immunoglobulin kappa light chain variable gene locus (filled symbols) with the approximately 0.5 megabase (Mb) first, or proximal, repeat of two nearly identical repeats of the human immunoglobulin kappa light chain variable gene locus. [Figure 2A] Figure 2A shows a detailed schematic (not to scale) of the first three steps (A-C) for direct genomic replacement of the mouse immunoglobulin heavy chain variable gene locus resulting in the deletion of all mouse VH, DH and JH gene segments and replacement with three human VH, all human DH and JH gene segments. The targeting vector (3hVHBACvec) for the first insertion of a human immunoglobulin heavy chain gene segment is shown, which has a 67 kb 5' mouse homology 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. The human (open symbols) and mouse (filled symbols) immunoglobulin gene segments to be inserted from the subsequent targeting vector, an additional selection cassette (open rectangle) and a site-specific recombination site (open triangle) are shown. [Figure 2B]2B shows a detailed schematic (not to scale) of six additional steps (D-I) for direct genomic replacement of a mouse immunoglobulin heavy chain variable gene locus resulting in the insertion of 77 additional human VH gene segments and the final removal of a selection cassette. A targeting vector (18hVHBACvec) for the insertion of additional human VH gene segments into the first human heavy chain gene segment insert (3hVH-CRE hybrid allele) is shown, which has 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 the 5' end of the first human heavy chain gene segment insert shown, with a site-specific recombination site (open triangle) 5' to the human gene segment. The human (open symbols) and mouse (filled symbols) immunoglobulin gene segments inserted by the subsequent targeting vectors as well as an additional selection cassette (open rectangle) are indicated. [Figure 2C] Figure 2C shows a detailed schematic (not to scale) of the first three steps (A-C) for direct genomic replacement of the mouse immunoglobulin κ light chain variable gene locus resulting in a deletion of all mouse Vκ and Jκ gene segments (Igκ-CRE hybrid allele). The selection cassette (open rectangle) and site-specific recombination sites (open triangles) inserted from the targeting vector are shown. [Figure 2D] Figure 2D shows a detailed schematic (not to scale) of five additional steps (D-H) of direct genomic replacement of the mouse immunoglobulin κ light chain variable gene locus resulting in the insertion of all human Vκ and Jκ gene segments of the proximal repeat and the final deletion of the selection cassette (40hVκdHyg hybrid allele). The human (open symbols) and mouse (filled symbols) immunoglobulin gene segments inserted by the subsequent targeting vector as well as the additional selection cassette (open rectangle) are shown. [Figure 3A]Figure 3A shows a general illustration (not to scale) of the screening strategy, including the location of quantitative PCR (qPCR) primer / probe sets for detecting the insertion of human heavy chain gene sequences and the loss of mouse heavy chain gene sequences in targeted embryonic stem (ES) cells. The screening strategy for the first human heavy chain gene insertion in ES cells and mice is shown using qPCR primer / probe sets for the deleted region ("Loss" probes C and D), the inserted region ("hIgH" probes G and H) and the flanking regions ("Keep" probes A, B, E and F) on the unmodified mouse chromosome (top) and the correctly targeted chromosome (bottom). [Figure 3B] FIG. 3B shows a representative calculation of the observed probe copy number in parental and modified ES cells for the first insertion of a human immunoglobulin heavy chain gene segment. The observed probe copy number for probes A through F was calculated as 2 / 2ΔΔCt. ΔΔCt was calculated as ave[ΔCt(sample)-medΔCt(control)], where ΔCt is the difference in Ct between the test probe and the reference probe (reference probes between 4 and 6 depending on the assay). The term medΔCt(control) is the median ΔCt of multiple (>60) non-target DNA samples from parental ES cells. Each modified ES cell clone was assayed in sextuplicate. To calculate the copy number of IgH probes G and H in parental ES cells, we assumed that these probes have a copy number of 1 in modified ES cells and used a maximum Ct of 35 even if no amplification was observed. [Figure 3C] Figure 3C shows a representative calculation of copy numbers for four mice of each genotype, calculated using only probes D and H. Wild-type mice: WT mice; mice heterozygous for the first insertion of a human immunoglobulin gene segment: HET mice; mice homozygous for the first insertion of a human immunoglobulin gene segment: Homo mice. [Figure 4A]Figure 4A shows a detailed schematic (not to scale) of the three steps used to construct 3hVHBACvec by bacterial homologous recombination (BHR). Shown are the human (open symbols) and mouse (filled symbols) immunoglobulin gene segments inserted from the targeting vector, the selection cassette (open rectangle) and the site-specific recombination sites (open triangles). [Figure 4B] Figure 4B shows pulsed-field gel electrophoresis (PFGE) of three BAC clones (B1, B2, and B3) after Notl digestion. Markers M1, M2, and M3 are low-range, mid-range, and lambda ladder PFG markers, respectively (New England BioLabs, Ipswich, Massachusetts). [Figure 5A] Figure 5A shows a schematic illustration (not to scale) of the sequential engineering of mouse immunoglobulin heavy chain loci with increasing amounts of human immunoglobulin heavy chain gene segments. Homozygous mice were generated from each of three different stages of heavy chain humanization. Open symbols indicate human sequences and filled symbols indicate mouse sequences. [Figure 5B] FIG 5B shows a schematic illustration (not to scale) of the sequential engineering of the mouse immunoglobulin kappa light chain locus with increasing amounts of human immunoglobulin kappa light chain gene segments. Homozygous mice were generated from each of the three different stages of kappa light chain humanization. Open symbols indicate human sequences and filled symbols indicate mouse sequences. [Figure 6]Figure 6 shows FACS dot plots of B cell populations in wild type and VELOCIMMUNE® humanized mice. Cells from spleens (top, third and bottom rows) or inguinal lymph nodes (second row) of wild type (wt), VELOCIMMUNE® 1 (V1), VELOCIMMUNE® 2 (V2) or VELOCIMMUNE® 3 (V3) mice were stained for B cells expressing surface IgM (top and second rows), surface immunoglobulins containing either kappa or lambda light chains (third row from the top) or surface IgM of specific haplotypes (bottom row) and populations were separated by FACS. [Figure 7A] Figure 7A shows representative heavy chain CDR3 sequences of randomly selected VELOCIMMUNE® antibodies around the VH-DH-JH (CDR3) junction, which evidences junctional diversity and nucleotide addition. The heavy chain CDR3 sequences are grouped according to DH gene segment usage, and the germline is provided in bold above each group. The VH gene segment for each heavy chain CDR3 sequence is listed in brackets at the 5' end of each sequence (e.g., 3-72 is human VH3-72). The JH gene segment for each heavy chain CDR3 is listed in brackets at the 3' end of each sequence (e.g., 3 is human JH3). The sequence numbers for each sequence shown, proceeding from top to bottom, are as follows: SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:39. [Figure 7B]FIG. 7B shows representative light chain CDR3 sequences of randomly selected VELOCIMMUNE® antibodies around the Vκ-Jκ (CDR3) junction, which evidences junctional diversity and nucleotide addition. The Vκ gene segment for each light chain CDR3 sequence is listed in brackets at the 5′ end of each sequence (e.g., 1-6 is human Vκ1-6). The Jκ gene segment for each light chain CDR3 is listed in brackets at the 3′ end of each sequence (e.g., 1 is human Jκ1). The SEQ ID NOs for each sequence shown, proceeding from top to bottom, are as follows: 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. [Figure 8] Figure 8 shows somatic hypermutation frequencies for the heavy and light chains of VELOCIMMUNE® antibodies scored (after alignment to matching germline sequences) as the percentage of sequences that are altered at each nucleotide (NT; left column) and amino acid (AA; right column) position in a set of 38 (non-immune IgM), 28 (non-immune IgG), 32 (non-immune Igκ from IgG), 36 (immune IgG) or 36 (immune Igκ from IgG) sequences. Shaded bars indicate the locations of the CDRs. [Figure 9A] FIG. 9A shows serum immunoglobulin levels for IgM and IgG isotypes in wild-type (open bars) or VELOCIMMUNE® mice (filled bars). [Figure 9B] FIG. 9B shows serum immunoglobulin levels for the IgA isotype in wild-type (open bars) or VELOCIMMUNE® mice (filled bars). [Figure 9C] FIG. 9C shows serum immunoglobulin levels for IgE isotypes in wild-type (open bars) or VELOCIMMUNE® mice (filled bars). [Figure 10A] FIG. 10A shows antigen-specific IgG titers against the interleukin-6 receptor (IL-6R) in serum from seven VELOCIMMUNE® (VI) and five wild-type (WT) mice after two rounds of immunization (bleed 1) or three rounds of immunization (bleed 2) with the ectodomain of IL-6R. [Figure 10B] FIG. 10B shows anti-IL-6R specific IgG isotype specific titers from seven VELOCIMMUNE® (VI) and five wild-type (WT) mice. [Figure 11A] FIG. 11A shows the affinity distribution of anti-Interleukin-6 receptor monoclonal antibodies generated in VELOCIMMUNE® mice. [Figure 11B] FIG. 11B shows antigen-specific blocking of anti-Interleukin-6 receptor monoclonal antibodies generated in VELOCIMMUNE® (VI) and wild-type (WT) mice. [Figure 12] 12 shows a schematic diagram (not to scale) of the mouse ADAM6a and ADAM6b genes in the mouse immunoglobulin heavy chain locus. The targeting vector used for insertion of mouse ADAM6a and ADAM6b into the humanized endogenous heavy chain locus (mADAM6 targeting vector) is shown, which has a selection cassette (HYG: hygromycin) flanked by site-specific recombination sites (Frt) containing engineered restriction enzyme recognition sites at the 5' and 3' ends. [Figure 13]Figure 13 shows a schematic illustration (not to scale) of the human ADAM6 pseudogene (hADAM6Ψ) between human heavy chain variable gene segments 1-2 (VH1-2) and 6-1 (VH6-1). A targeting vector (hADAM6Ψ targeting vector) for bacterial homologous recombination to delete the human ADAM6 pseudogene and insert a unique restriction site into the human heavy chain locus is shown, which has a selection cassette (NEO: neomycin) flanked by site-specific recombination sites (loxP) containing engineered restriction sites at the 5' and 3' ends. A diagram (not to scale) of the resulting targeted humanized heavy chain locus containing a genomic fragment encoding the mouse ADAM6a and ADAM6b genes, including a selection cassette flanked by site-specific recombination sites, is shown. [Figure 14A] Figure 14A shows FACS contour plots of lymphocytes gated on singlets for IgM and B220 surface expression in bone marrow of mice homozygous for the human heavy and human kappa light chain variable gene loci (H+ / +K+ / +) and mice homozygous for the human heavy and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resK+ / +). The percentages of immature (B220intIgM+) and mature (B220highIgM+) B cells are plotted on each contour plot. [Figure 14B] FIG. 14B shows the total numbers of immature (B220intIgM+) and mature (B220highIgM+) B cells in bone marrow isolated from femurs of mice homozygous for the human heavy chain and human kappa light chain variable gene loci (H+ / +κ+ / +) and mice homozygous for the human heavy chain and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resκ+ / +). [Figure 15A]Figure 15A shows FACS contour plots of CD19+ gated B cells for surface expression of c-kit and CD43 in bone marrow of mice homozygous for the human heavy and human kappa light chain variable gene loci (H+ / +K+ / +) and mice homozygous for the human heavy and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resK+ / +). The percentages of pro-B (CD19+CD43+ckit+) and pre-B (CD19+CD43-ckit-) cells are plotted in the upper right and lower left quadrants of each contour plot, respectively. [Figure 15B] FIG. 15B shows the total numbers of pro-B (CD19+CD43+ckit+) and pre-B (CD19+CD43-ckit-) cells in bone marrow isolated from femurs of mice homozygous for the human heavy chain and human kappa light chain variable gene loci (H+ / +K+ / +) and mice homozygous for the human heavy chain and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resK+ / +). [Figure 16A] Figure 16A shows FACS contour plots of singlet-gated lymphocytes for surface expression of CD19 and CD43 in the bone marrow of a mouse homozygous for the human heavy and human kappa light chain variable gene loci (H+ / +K+ / +) and a mouse homozygous for the human heavy and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resK+ / +). The percentages of immature B (CD19+CD43-), pre-B (CD19+CD43int) and pro-B (CD19+CD43+) cells are plotted in each contour plot. [Figure 16B]FIG. 16B shows histograms of immature B (CD19+CD43−) and pre-B (CD19+CD43int) cells in the bone marrow of mice homozygous for the human heavy chain and human kappa light chain variable gene loci (H+ / +κ+ / +) and mice homozygous for the human heavy chain and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resκ+ / +). [Figure 17A] Figure 17A shows singlet-gated lymphocyte FACS contour plots for CD19 and CD3 surface expression in splenocytes from mice homozygous for the human heavy and human kappa light chain variable gene loci (H+ / +K+ / +) and mice homozygous for the human heavy and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resK+ / +). The percentages of B (CD19+CD3-) and T (CD19-CD3+) cells are plotted on each contour plot. [Figure 17B] Figure 17B shows contour plots of CD19+ gated B cells for surface expression of Igλ and Igκ light chains in the spleen of mice homozygous for the human heavy and human kappa light chain variable gene loci (H+ / +K+ / +) and mice homozygous for the human heavy and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resK+ / +). The percentages of Igλ+ (upper left quadrant) and Igκ+ (lower right quadrant) B cells are plotted in each contour plot. [Figure 17C] Figure 17C shows the total number of CD19+ B cells in the spleens of mice homozygous for the human heavy chain and human kappa light chain variable gene loci (H+ / +κ+ / +) and mice homozygous for the human heavy chain and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resκ+ / +). [Figure 18A]Figure 18A shows FACs contour plots of CD19+ gated B cells for surface expression of IgD and IgM in the spleen of mice homozygous for the human heavy and human kappa light chain variable gene loci (H+ / +K+ / +) and mice homozygous for the human heavy and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resK+ / +). The percentage of mature B cells (CD19+IgDhighIgMint) is plotted on each contour plot. Arrows on the right contour plot illustrate the maturation process of B cells in relation to IgM and IgD surface expression. [Figure 18B] FIG. 18B shows the total numbers of B cells in the spleens of mice homozygous for the human heavy and human kappa light chain variable gene loci (H+ / +κ+ / +) and mice homozygous for the human heavy and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resκ+ / +) during maturation from CD19+IgMhighIgDint to CD19+IgMintIgDhigh. [Figure 19] Figure 19 shows antibody titers for the first and second bleed samples from mice homozygous for the human heavy chain and human kappa light chain variable gene loci (H+ / +κ+ / +; n=5) immunized with a human cell surface receptor (antigen A) and mice homozygous for the human heavy chain and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resκ+ / +; n=5). [Figure 20] Figure 20 shows antibody titers for the first and second bleed samples from mice homozygous for the human heavy chain and human kappa light chain variable gene loci (H+ / +κ+ / +; n=5) immunized with a human antibody specific for human receptor tyrosine-protein kinase (antigen B) and from mice homozygous for the human heavy chain and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resκ+ / +; n=10). [Figure 21]Figure 21 shows antibody titers for first and second bleed samples from mice homozygous for the human heavy chain and human kappa light chain variable gene loci (H+ / +κ+ / +; n=12) immunized with a secreted human protein (antigen C) that functions to regulate the TGF-β signaling pathway, and from mice homozygous for the human heavy chain and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resκ+ / +; n=12). [Figure 22] Figure 22 shows antibody titers for the first and second bleed samples from mice homozygous for the human heavy chain and human kappa light chain variable gene loci carrying an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+ / +A6resκ+ / +; n=12) immunized with human receptor tyrosine kinase (antigen D). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0150] The present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. Since the scope of the present invention is defined by the claims, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0151] Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have acquired in the art, unless the contrary is clearly indicated or clearly evident from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are now described. All publications mentioned are incorporated herein by reference.

[0152] The phrases "substantial" or "substantially" when used to refer to an amount of gene segments (e.g., "substantially all" V gene segments) include both functional and non-functional gene segments, and in various embodiments includes, e.g., 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 segments includes, e.g., at least 95%, 96%, 97%, 98% or 99% of functional (i.e., non-pseudogene) gene segments.

[0153] The term "replacement" includes when a DNA sequence is placed in the genome of a cell in such a way that a sequence in the genome is replaced with a heterologous sequence (e.g., a human sequence in a mouse) at the locus of the genomic sequence. The DNA so placed may include one or more regulatory sequences (e.g., promoters, enhancers, 5' or 3' untranslated regions, appropriate recombination signal sequences, etc.) that are part of the source DNA used to obtain the sequence so placed. For example, in various embodiments, the replacement is the replacement of an endogenous sequence for a heterologous sequence, but not the expression of the endogenous sequence, resulting in the production of a gene product from the DNA sequence (including the heterologous sequence) so placed; the replacement is the replacement of an endogenous genomic sequence with a DNA sequence that encodes a protein having a similar function as the protein encoded by the endogenous genomic sequence (e.g., the endogenous genomic sequence encodes an immunoglobulin gene or domain, and the DNA fragment encodes one or more human immunoglobulin genes or domains). In various embodiments, an endogenous gene or fragment thereof is replaced with a corresponding human gene or fragment thereof. The corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of the endogenous gene or fragment being replaced, a homolog of the endogenous gene or fragment being replaced, or that is substantially identical or similar in structure and / or function to the endogenous gene or fragment being replaced.

[0154] Mice as genetic models have been greatly improved by transgenic and knockout techniques, which allow the study of the effects of directed overexpression or deletion of specific genes. Despite all their advantages, the mice have limitations that make them imperfect models for human diseases and the inability to test or generate human therapeutics. However, human targeting also presents genetic obstacles that make it an imperfect platform for gene targeting. First, although about 99% of human genes have mouse homologs (Waterston et al., 2002, Initial sequencing and comparative analysis of the mouse genome, Nature 420:520-562), potential therapeutics often fail to cross-react, or cross-react inappropriately, with the mouse orthologs of the intended human target. To obviate this problem, selected target genes can be "humanized," i.e., the mouse gene can be erased and replaced with the corresponding orthologous human gene sequence (e.g., U.S. Patent Nos. 6,586,251, 6,596,541, and 7,105,348; which are incorporated herein by reference). Initial efforts to humanize mouse genes by a "knockout-plus-transgenic humanization" strategy involved crossing mice carrying a deletion (i.e., knockout) of the endogenous gene with mice carrying a randomly integrated human transgene (see, e.g., Bril et al., 2006, Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg(593) to Cys substitution, Thromb Haemost 95:341-347; Homanics et al., 2006, Production and characterization of murine factor VIII cDNA carrying an Arg(593) to Cys substitution, Thromb Haemost 95:341-347). (See 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 CD3delta / epsilon heterodimer in preT cell receptor(preTCR) function:human CD3delta / epsilon heterodimer restores the defective preTCR function in CD3gamma- and CD3gammadelta-deficient mice, Mol Immunol 43:1741-1750). However, these efforts were hampered by size limitations; traditional knockout techniques were not sufficient to directly replace large mouse genes with their large human genomic counterparts. The straightforward approach of direct homologous replacement, in which an endogenous mouse gene is directly replaced by a human counterpart gene at the same exact genetic location of the mouse gene (i.e., at the endogenous mouse locus), is rarely attempted due to technical difficulties. To date, efforts at direct replacement have required elaborate and cumbersome procedures, thus limiting the length of genetic material that can be handled and the precision with which it can be manipulated.

[0155] Exogenously introduced human immunoglobulin transgenes rearrange in precursor B cells of mice (Alt et al., 1985, Immunoglobulin genes in transgenic mice, Trends Genet 1:231-236). This discovery was exploited to engineer mice using a knockout-plus-transgenic approach to express human antibodies (Green et al., 1994, Antigen-specific human monoclonal antibodies from mice engineered with human Ig 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 368:856-8 59;Jakobovits et al., 2007, From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice, Nat Biotechnol 25:1134-1143). The mouse immunoglobulin heavy and kappa light chain loci were inactivated in these mice by targeted deletion of small but significant portions of each endogenous locus, followed by introduction of the human immunoglobulin gene loci either as randomly integrated large transgenes as described above or as minichromosomes (Tomizuka et al., 2000, Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies, PNAS USA 97:722-727). Such mice represented an important advance in genetic engineering; fully human monoclonal antibodies isolated from them offered promising therapeutic potential for the treatment of a variety of human diseases (Gibson et al., 2006, Randomized phase III 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(HuMax-CD4):two Phase II studies in refractory cutaneous T-cell lymphoma, Blood 109(11):4655-62; Lonberg, 2005, Human antibodies from transgenic animals, Nat Biotechnol 23:1117-1125;Maker et al., 2005, Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2:a phase I / 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). However, as discussed above, these mice exhibit impaired B cell development and immunodeficiency compared to wild-type mice. Such problems potentially limit the ability of the mice to maintain a vigorous humoral response and thus produce fully human antibodies against some antigens. The deficiencies include (1) insufficient functionality due to random introduction of human immunoglobulin transgenes and resulting incorrect expression due to the absence of upstream and downstream regulatory elements (Garrett et al., 2005, Chromatin architecture near a potential 3'end of the IgH locus involves modular regulation of histone modifications during B-Cell development and in vivo occupancy at CTCF sites, Mol Cell Biol 25:1511-1525; Manis et al., 2003, Elucidation of a downstream boundary of the 3'IgH regulatory region, Mol Immunol 39:753-760;Pawlitzky et al., 2006, Identification of a candidate regulatory element within the 5'flanking region of the mouse IgH 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 cell surface B cell receptor signaling complex that may impair signaling processes required for normal maturation, proliferation, and survival of B cells (Hombach et al., 1990, Molecular components of the B-cell antigen receptor complex of the IgM class, Nature 343:760-762); and (3) affinity selection (Rao et al., 2002, Differential expression of the inhibitory IgG Fc receptor FcgammaRIIB on germinal center cells: implications for selection of high-affinity B cells, J Immunol 169:1859-1868) and immunoglobulin serum concentrations (Brambell et al., 1964, A Theoretical Model of Gamma-Globulin Catabolism, Nature 203:1352-1354; Junghans and Anderson, 1996, The protective receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor, PNAS USA 93:5512-5516; Rao et al., 2002; Hjelm et al., 2006, Antibody-mediated regulation of the immune response, Scand J Immunol 64:177-184;These deficiencies may be due to inefficient inter-species interactions between soluble human immunoglobulins and mouse Fc receptors, which may reduce the ability of mouse Fc receptors to bind to human immunoglobulins (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 in their natural locations at the endogenous heavy and light chain loci. This would effectively result in mice making "reverse chimeric" (i.e., human V:mouse C) antibodies that would be capable of normal interaction and selection in the mouse environment based on the retention of the mouse constant regions. Moreover, such reverse chimeric antibodies can be easily rearranged into fully human antibodies for therapeutic purposes;

[0156] Genetically modified animals can be produced that contain a replacement at the endogenous immunoglobulin heavy chain locus with a heterologous (e.g., from another species) immunoglobulin sequence, either in conjunction with a replacement at the endogenous immunoglobulin light chain locus, or in conjunction with an immunoglobulin light chain transgene (e.g., a chimeric immunoglobulin light chain transgene or a fully human full mouse, etc.). The species from which the heterologous immunoglobulin heavy chain sequence originates can be widely varied, as can the immunoglobulin light chain sequence utilized for the immunoglobulin light chain sequence replacement, or the immunoglobulin light chain transgene.

[0157] In various embodiments, immunoglobulin variable region nucleic acid sequences, such as V, D and / or J segments, are obtained from humans or non-human animals. Suitable non-human animals 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). Suitable non-human animals include, for example, mammals. Suitable mammals include, for example, non-human primates, goats, sheep, pigs, dogs, cattle (e.g., cows, bulls, buffalo), deer, camels, ferrets, and rodents and non-human primates (e.g., chimpanzees, orangutans, gorillas, marmosets, rhesus monkeys, baboons). Suitable non-human animals are selected from the family of rodents, including rats, mice and hamsters. In one embodiment, the non-human animal is a mouse. As will be apparent from the context, various non-human animals (e.g., sharks, rays, mammals (e.g., camels, rodents, e.g., mice, Human and rat models can be used as a source of variable domains or variable region gene segments.

[0158] According to the above context, non-human animals are also used as a source of constant region sequences to be used with variable sequences or segments. For example, rodent constant sequences can be used in transgenes (e.g., rodent, e.g., mouse or rat or hamster, human or non-human primate variable sequences operably linked to constant sequences) that are operably linked to human or non-human variable sequences. Thus, in various embodiments, human V, D and / or J segments are operably linked to rodent (e.g., mouse or rat or hamster) constant region gene sequences. In some embodiments, the human V, D and / or J segments (or one or more rearranged VDJ or VJ genes) are operably linked or fused to mouse, rat or hamster constant region gene sequences, for example, in transgenes that are integrated into loci that are not endogenous immunoglobulin loci.

[0159] In certain embodiments, the V at the endogenous immunoglobulin heavy chain locus H , D H and J. H Segment, one or more human V H , D H and J. H and wherein the one or more human V segments include a substitution at H , D H and J. H and a mouse in which the mouse comprises a transgene at a locus other than an endogenous immunoglobulin locus, the transgene comprising an unrearranged or rearranged human V segment operably linked to a mouse, rat or human constant region. L and human J L Includes segments.

[0160] A method is described for large-scale in situ gene replacement of mouse germline immunoglobulin variable gene loci with human germline immunoglobulin variable gene loci while maintaining the ability of the mouse to produce offspring. Specifically, the precise replacement of 6 megabases of both mouse heavy and kappa light chain immunoglobulin variable gene loci with their human counterparts while leaving the mouse constant regions intact is described. As a result, mice are generated in which the entire mouse germline immunoglobulin variable repertoire is precisely replaced with the equivalent human germline immunoglobulin variable sequences while maintaining the mouse constant regions. The human variable regions are linked to mouse constant regions to form chimeric human-mouse immunoglobulin loci that are rearranged and expressed at physiologically relevant levels. The antibodies expressed are "reverse chimeras", i.e., they contain human variable region sequences and mouse constant region sequences. These mice with humanized immunoglobulin variable regions that express antibodies with human variable regions and mouse constant regions are called VELCOIMMUNE® mice.

[0161] VELOCIMMUNE® humanized mice exhibit a fully functional humoral immune system essentially indistinguishable from that of wild-type mice. The humanized mice display normal cell populations at all stages of B cell development. The humanized mice exhibit normal lymphoid organ morphology. VELOCIMMUNE® mouse antibody sequences exhibit normal V(D)J rearrangements and normal somatic hypermutation frequencies. The antibody populations in these mice display an isotype distribution resulting from normal class switching (e.g., normal isotype switching). Immunization of VELOCIMMUNE® mice results in a robust humoral immune response that produces a large and diverse repertoire of antibodies with human immunoglobulin variable domains suitable for use as therapeutic candidates. This platform provides an abundant source of natural affinity matured human immunoglobulin variable region sequences for generating pharma- ceutically acceptable antibodies and other antigen binding proteins.

[0162] The VELOCIMMUNE® mouse can be generated by precise replacement of mouse immunoglobulin variable sequences with human immunoglobulin variable sequences. However, precise replacement of endogenous mouse immunoglobulin sequences at heavy and light chain loci with equivalent human immunoglobulin sequences by sequential recombineering of very large lengths of human immunoglobulin sequences may pose certain challenges due to the divergent evolution of the immunoglobulin loci between mice and humans. For example, intergenic sequences scattered within the immunoglobulin loci are not identical between mice and humans, and in some circumstances may not be functionally equivalent. The differences in the immunoglobulin loci between mice and humans may also result in abnormalities in the humanized mice, especially when certain parts of the endogenous mouse immunoglobulin heavy chain loci are humanized or engineered. Some modifications in the mouse immunoglobulin heavy chain loci are deleterious. Detrimental modifications may include, for example, loss of ability of modified mice to mate and produce offspring. The mouse heavy and light chain immunoglobulin loci (VLCs) were cloned into a hybrid locus containing all of the mouse constant chain genes and adjacent mouse sequences intact and functional, including the transcriptional regulatory regions of the locus. H -D H -J H We performed precise large-scale in situ replacement of the 6 megabase variable regions of the Vκ-Jκ subunits (Vκ-Jκ and Vκ-Jκ) with the corresponding 1.4 megabase human genomic sequence (Figures 1A and 1B). Specifically, we used VELOCIGENE® genetic engineering technology (see, for example, U.S. Pat. No. 6,586,251 and Valenzuela et al., 2003, High-throughput engineering of the mouse genome coupled with high-resolution Using the expression analysis, see Nat Biotechnol 21:652-659, human V H , D H , J H, Vκ and Jκ gene sequences were introduced into mouse ES cells by stepwise insertion of 13 chimeric BAC targeting vectors carrying overlapping fragments of human germline variable loci.

[0163] Humanization of 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 antibodies can be efficiently produced in otherwise normal mice. Furthermore, such mice exhibit a dramatic increase in the diversity of fully human antibodies that can be obtained after immunization with virtually any antigen, compared to mice carrying disabled endogenous loci and fully human antibody transgenes. In contrast to mice with randomly integrated human transgenes that exhibit significantly reduced B cell populations at various stages of differentiation, the diverse versions of the replaced and humanized loci exhibit completely normal mature and immature B cell levels. Efforts to increase the number of human gene segments in human transgenic mice have reduced such defects, but their expanded immunoglobulin repertoires have not entirely corrected the reduced B cell populations compared to wild-type mice.

[0164] Despite the near-wild-type humoral immune function observed in mice with replaced immunoglobulin loci (i.e., VELOCIMMUNE® mice), there are other challenges encountered using direct replacement of immunoglobulins that are not encountered with some approaches that utilize randomly integrated transgenes. Differences in the genetic composition of the immunoglobulin loci between mice and humans have led to the discovery of sequences beneficial for breeding mice with replaced immunoglobulin gene segments. Specifically, mouse ADAM genes located within the endogenous immunoglobulin loci are optimally present in mice with replaced immunoglobulin loci due to their role in fertility.

[0165] Genomic location and function of mouse ADAM6 Male mice that are incapable of expressing any functional ADAM6 protein surprisingly show a defect in the ability of the mice to mate and produce offspring. These mice are incapable of expressing functional ADAM6 protein by replacing all or substantially all mouse immunoglobulin variable region gene segments with human variable region gene segments. The ADAM6 locus is a D H V upstream of the gene segment H This loss of ADAM6 function results because it is located in the region of the endogenous mouse immunoglobulin heavy chain variable region gene locus proximal to the 3' end of the gene segment locus. To breed mice homozygous for the 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 provide males and females, each homozygous for the replacement, waiting for productive mating. Successful litters are infrequent and small in size. Instead, males heterozygous for the replacement are used to breed with females homozygous for the replacement to produce offspring heterozygous for the replacement, from which homozygous mice are then bred. The inventors have determined that the possible cause of the loss of fertility in the male mice is the absence of functional ADAM6 protein in homozygous male mice.

[0166] In various embodiments, male mice containing damaged (i.e. non-functional or slightly functional) ADAM6 gene show reduced or eliminated fertility.Because ADAM6 gene is located in immunoglobulin heavy chain locus in mice (and other rodents), the inventors have decided to utilize various modified mating or breeding schemes to breed mice containing replaced immunoglobulin heavy chain locus or to create and maintain a line of said mice.The low fertility or sterility of male mice homozygous for replacement of endogenous immunoglobulin heavy chain variable gene locus makes it difficult to maintain such modified mouse line.In various embodiments, maintaining said line includes avoiding the sterility problem exhibited by male mice homozygous for said replacement.

[0167] In one aspect, the present invention provides a method for maintaining the mouse line described herein.The mouse line does not need to contain an ectopic ADAM6 sequence, and in various embodiments, the mouse line is homozygous or heterozygous for ADAM6 knockout (e.g., functional knockout).

[0168] The mouse line comprises a modification of endogenous immunoglobulin heavy chain locus that results in reduced or lost fertility in male mice. In one embodiment, the modification comprises a deletion of the regulatory and / or coding region of the ADAM6 gene. In a particular embodiment, the modification comprises a modification of the endogenous ADAM6 gene (regulatory and / or coding region) that reduces or eliminates fertility in male mice that comprise the modification; in a particular embodiment, the modification reduces or eliminates fertility in male mice that are homozygous for the modification.

[0169] In one embodiment, the mouse strain is homozygous or heterozygous for a knockout (eg, functional knockout) or deletion of the ADAM6 gene.

[0170] In one embodiment, the mouse line is maintained by isolating cells from the mouse that is homozygous or heterozygous for the modification, using the donor cells in a host embryo, and carrying the host embryo and the donor cells in a surrogate mother, and obtaining progeny from the surrogate mother that contain the genetic modification.In one embodiment, the donor cell is an ES cell.In one embodiment, the donor cell is a pluripotent cell, for example an induced pluripotent cell.

[0171] In one embodiment, the mouse line is maintained by isolating a nucleic acid sequence comprising the modification from a mouse homozygous or heterozygous for the modification, and introducing the nucleic acid sequence into a host nucleus and gestating 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 an embryo of a host oocyte.

[0172] In one embodiment, the mouse line is maintained by isolating a nucleus from a mouse that is homozygous or heterozygous for the modification, and introducing the nucleus into a host cell, and gestation of the nucleus and host cell in a suitable animal to obtain progeny that are homozygous or heterozygous for the modification.

[0173] In one embodiment, the mouse line is maintained by using in vitro fertilization (IVF) of female mice (wild type, homozygous for the modification, or heterozygous for the modification) with semen from male mice containing the genetic modification. In one embodiment, the male mice are heterozygous for the genetic modification. In one embodiment, the male mice are homozygous for the genetic modification.

[0174] In one embodiment, the mouse line is maintained by mating male mice heterozygous for the genetic modification with female mice to obtain offspring containing the genetic modification, identifying male and female offspring containing the genetic modification, and using males heterozygous for the genetic modification to breed with wild-type, homozygous or heterozygous females for the genetic modification to obtain offspring containing the genetic modification.In one embodiment, the process of mating males heterozygous for the genetic modification with wild-type females, females heterozygous for the genetic modification, or females homozygous for the genetic modification is repeated to maintain the genetic modification in the mouse line.

[0175] In one aspect, a method is provided for maintaining a mouse strain comprising a replacement of an endogenous immunoglobulin heavy chain variable gene locus with one or more human immunoglobulin heavy chain sequences, comprising breeding said mouse strain to produce heterozygous male mice, and breeding said heterozygous male mice to maintain said genetic modification in said strain. In certain embodiments, said strain is not maintained by breeding homozygous males with wild type females, or females homozygous or heterozygous for said genetic modification.

[0176] The ADAM6 protein is a member of the ADAM family of proteins, where ADAM is an acronym 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 involved in spermatogenesis and fertilization. For example, ADAM2 encodes a subunit of the protein fertilin, which is involved in sperm-egg interaction. ADAM3, or cyritestin, appears to be required for sperm binding to the zona pellucida. Absence of either ADAM2 or ADAM3 results in infertility. ADAM2, ADAM3 and ADAM6 are hypothesized to form a complex on the surface of mouse sperm cells. Human VH Gene segment V H 1-2 and V H The human ADAM6 gene, which is usually found among the 6-1 subtypes, appears to be a pseudogene (Figure 12). There are two ADAM6 genes in mice - ADAM6a and ADAM6b - which are expressed in mouse V H Gene Segment and D H Found in the intergenic region between gene segments, in said mouse, ADAM6a gene and ADAM6b gene are oriented in the opposite transcriptional orientation to that of surrounding immunoglobulin gene segments (Figure 12). In mouse, functional ADAM6 locus is obviously required for normal fertilization. Thus, functional ADAM6 locus or sequence refers to ADAM6 locus or sequence that can compensate or rescue the drastic reduction in fertility shown in male mice that lack endogenous ADAM6 locus or have non-functional endogenous ADAM6 locus.

[0177] The location of the intergenic sequence in mice, which encodes ADAM6a and ADAM6b, renders the intergenic sequence susceptible to modification when modifying endogenous mouse heavy chains. H When deleting or replacing gene segments, or H When gene segment is deleted or replaced, the resulting mouse is likely to show severe fertility defects.To compensate for this defect, the mouse is modified to include the nucleotide sequence that encodes the protein that compensates for the loss of ADAM6 activity caused by the modification of endogenous mouse ADAM6 locus.In various embodiments, the complementary nucleotide sequence encodes mouse ADAM6a, mouse ADAM6b, or its homolog or ortholog or functional fragment that rescues fertility defects.

[0178] The nucleotide sequence that rescues fertility can be placed in any suitable location. The sequence can be placed in the intergenic region or can be placed in any suitable location in the genome (i.e., ectopically). In one embodiment, the nucleotide sequence can be introduced into a transgene that randomly integrates into the mouse genome. In one embodiment, the sequence can be maintained episomally, i.e., on a separate nucleic acid, not on a mouse chromosome. Suitable locations include transcriptionally permissive or active locations, such as the ROSA26 locus (Zambrowicz et al., 1997, PNAS USA 94:3789-3794), the BT-5 locus (Michael et al., 1999, Mech.Dev.85:35-47) or the Oct4 locus (Wallace et al., 2000, Nucleic Acids Res.28:1455-1464). Targeting of nucleotide sequences to transcriptionally active loci is described, for example, in US Pat. No. 7,473,557, which is incorporated herein by reference.

[0179] Alternatively, the fertility-rescuing nucleotide sequence can be coupled to an inducible promoter to facilitate optimal expression in appropriate cells and / or tissues, such as reproductive tissues. Exemplary inducible promoters include promoters that are activated by physical means (e.g., heat shock promoters) and / or promoters that are activated by chemical means (e.g., IPTG or tetracycline).

[0180] Furthermore, expression of the nucleotide sequence can be related to other genes to achieve expression at a particular developmental stage or in a particular tissue. Such expression can be achieved by operably linking the nucleotide sequence to the promoter of a gene that is expressed at a particular developmental stage. For example, an immunoglobulin sequence from one species engineered into the genome of a host species is operably linked to the promoter sequence of the CD19 gene (a B-cell specific gene) from that host species. B-cell specific expression at the exact developmental stage at which the immunoglobulin is expressed is achieved.

[0181] Yet another method for realizing robust expression of inserted nucleotide sequence is to utilize constitutive promoter.Exemplary constitutive promoters include SV40, CMV, UBC, EF1A, PGK and CAGG.Similarly, desired nucleotide sequence is operably linked with selected constitutive promoter, thereby obtaining high expression level of protein(s) encoded by said nucleotide sequence.

[0182] The term "ectopic" is intended to include transposition or placement in a position not normally encountered in nature (e.g., placement of a nucleic acid sequence in a position that is not the same position as the nucleic acid sequence is found in a wild-type mouse). In various embodiments, the term is used to mean that the subject is outside its normal or correct position. For example, the phrase "ectopic nucleotide sequence encoding ..." refers to a nucleotide sequence that appears in a position not normally encountered in mice. For example, in the case of an ectopic nucleotide sequence encoding mouse ADAM6 protein (or its ortholog or homolog or fragment that provides the same or similar fertility benefit to male mice), the sequence may be placed in a position in the mouse genome that is different from the position that is normally found in wild-type mice. In such a case, placing the sequence in a position in the mouse genome that is different from the position in wild-type mice will create a novel sequence junction of the mouse sequence. A functional homolog or ortholog of mouse ADAM6 is a sequence that provides rescue of the fertility loss observed in ADAM6- / - mice (e.g., loss of ability of male mice to produce offspring by mating). Functional homologs or orthologs include proteins having at least about 89% or more identity, for example 99% or less identity, to the amino acid sequence of ADAM6a and / or to the amino acid sequence of ADAM6b, as well as proteins that can complement or rescue the ability of mice having a genotype that includes a deletion or knockout of ADAM6a and / or ADAM6b to mate successfully.

[0183] The ectopic location may be anywhere (e.g., as in the case of random insertion of a transgene containing a mouse ADAM6 sequence) or may be near (but not exactly the same as) its location in, for example, a wild-type mouse (e.g., within a modified endogenous mouse immunoglobulin locus but either upstream or downstream of its natural location, e.g., within a modified immunoglobulin locus but between different gene segments, or at a different location within the mouse VD intergenic sequence). One example of an ectopic location is within a humanized immunoglobulin heavy chain locus. For example, one or more endogenous VDs may be inserted into the humanized immunoglobulin heavy chain locus. H Gene segment, human V H Mice containing a gene segment replacement that eliminates the endogenous ADAM6 sequence are cultured to identify mice in which the mouse ADAM6 sequence aligns with the human V H The ADAM6 sequence can be engineered to be located within the sequence containing gene segments. The resulting modification will result in the (ectopic) mouse ADAM6 sequence being located within the human gene sequence, and the (ectopic) placement of the mouse ADAM6 sequence within the human gene sequence may be close to the position of the human ADAM6 pseudogene (i.e., between two V segments) or close to the position of the mouse ADAM6 sequence (i.e., within the VD intergenic region). The resulting sequence junction created by joining the (ectopic) mouse ADAM6 sequence within or adjacent to the human gene sequence (e.g., immunoglobulin gene sequence) in the germline of mouse will be new compared to the same or similar position in the genome of wild-type mouse.

[0184] In various embodiments, a non-human animal is provided that does not have ADAM6 or its orthologue or homologue, and this lack renders the non-human animal infertile or substantially reduces the fertility of the non-human animal. In various embodiments, the lack of ADAM6 or its orthologue or homologue is due to modification of endogenous immunoglobulin heavy chain locus. The substantially reduced fertility is, for example, about 50%, 60%, 70%, 80%, 90% or 95% or more reduced fertility (e.g., mating frequency, pups per litter, litters per year, etc.). In various embodiments, the non-human animal is supplemented with a mouse ADAM6 gene or its orthologue or homologue or functional fragment that is functional in males of the non-human animal, and the supplemented ADAM6 gene or its orthologue or homologue or functional fragment rescues all or a substantial part of the reduced fertility. Rescue of a substantial portion of fertility is, for example, restoration of fertility such that the non-human animal exhibits at least 70%, 80% or 90% or more fertility compared to an unmodified (i.e., an animal not having a modification to the ADAM6 gene or its ortholog or homolog) heavy chain locus.

[0185] In various embodiments, the sequence provided to the genetically modified animal (i.e., an animal that lacks a functional ADAM6 or its ortholog or homologue, e.g., due to modification of the immunoglobulin heavy chain locus) is a sequence that encodes an ADAM6 gene or its ortholog or homologue. For example, the loss of ADAM6 function in mice is rescued in one embodiment by adding mouse ADAM6 gene. In one embodiment, the loss of ADAM6 function in mice is rescued by adding orthologs or homologs of closely related species, such as mice, e.g. rodents, of different strains or species of mice, any species of rats, rodents, in which case the addition of said mouse orthologs or homologs rescues the loss of fertility caused by loss of ADAM6 function or loss of ADAM6 gene. Orthologs and homologs from other species are selected from phylogenetically related species in various embodiments, and in various embodiments, show a percent identity with endogenous ADAM6 (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 rescue the fertility loss associated with ADAM6 or (in the case of non-mouse) ADAM6 orthologs. For example, in the case of genetically modified male rats lacking ADAM6 function (e.g., rats having their endogenous immunoglobulin heavy chain variable region replaced with a human immunoglobulin heavy chain variable region, or rats having a knockout of the rat immunoglobulin heavy chain region), the loss of fertility of the rats is rescued by the addition of rat ADAM6, or, in some embodiments, by the addition of an ortholog of rat ADAM6 (e.g., an ADAM6 ortholog from another rat strain or species, or in one embodiment, from mouse).

[0186] Thus, in various embodiments, the genetically modified animal that does not exhibit fertility or exhibits reduced fertility due to modification of the nucleic acid sequence encoding ADAM6 protein (or its ortholog or homolog) or the regulatory region operably linked thereto comprises a nucleic acid sequence that compensates for or restores the loss of fertility, where the nucleic acid sequence that compensates for or restores the loss of fertility is from a different lineage of the same species or from a phylogenetically related species. In various embodiments, the complementary nucleic acid sequence is an ADAM6 ortholog or homolog or a functional fragment thereof. In various embodiments, the complementary ADAM6 ortholog or homolog or a functional fragment thereof is from a non-human animal that is closely related to the genetically modified animal that has fertility defects. For example, when the genetically modified animal is a certain lineage of mouse, the ADAM6 ortholog or homolog or a functional fragment thereof can be obtained from another lineage of mouse, or from a related species of mouse. In one embodiment, when the genetically modified animal with fertility defect is from the Rodentia order, the ADAM6 ortholog or homolog or its functional fragment is from another animal of the Rodentia order.In one embodiment, the genetically modified animal with fertility defect is from the suborder Myomoropha (e.g., jerboas, jumping mice, kangaroo hamsters, hamsters, New World rats and mice, voles, true mice and rats, gerbils, spiny mice, maned mice, arboretum mice, rock mice, white-tailed rats, Madagascar rats and mice, spiny dormice, mole rats, bamboo rats, and mole rats), and the ADAM6 ortholog or homolog or its functional fragment is selected from rodents or rodents.

[0187] In one embodiment, the genetically modified animal is from the superfamily Dipodoidea and the ADAM6 ortholog or homolog or functional fragment thereof is from the superfamily Muroidea. In one embodiment, the genetically modified animal is from the superfamily Muroidea and the ADAM6 ortholog or homolog or functional fragment thereof is from the superfamily Dipodoidea.

[0188] In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from the superfamily Murinea and the ADAM6 orthologs or homologs are from different species within the superfamily Murinea. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (e.g., kangaroo hamsters), Cricetidae (e.g., hamsters, New World rats and mice, bank voles), Muridae (pure-breed mice and rats, gerbils, spiny mice, maned mice), Nesomyidae (tree mice, rock mice, white-tailed crested mice, Madagascar rats and mice), Platacanthomyidae (e.g., spiny dormice), and Platacanthomyidae (e.g., mole rats, bamboo rats, and mole rats); and the ADAM6 ortholog or homolog is selected from a different species of the same family. In a particular embodiment, the genetically modified rodent is selected from a pure breed of mouse or rat (Muridae), and the ADAM6 ortholog or homolog is from a species selected from gerbil, spiny mouse, or maned rat. In one embodiment, the genetically modified mouse is from a member of the Muridae family, and the ADAM6 ortholog or homolog is from a different species of the Muridae family. In a particular embodiment, the genetically modified rodent is a Murine mouse, and the ADAM6 ortholog or homolog is from a Murine rat, gerbil, spiny mouse, or maned rat.

[0189] In various embodiments, one or more rodent ADAM6 orthologs or homologs or functional fragments thereof of rodents in a family restore fertility to genetically modified rodents of the same family (e.g., Cricetidae (e.g., hamsters, New World rats and mice, bank voles); Muridae (e.g., purebred mice and rats, gerbils, spiny mice, maned mice)) that lack an ADAM6 ortholog or homolog.

[0190] In various embodiments, ADAM6 orthologs, homologs, and fragments thereof are evaluated for functionality by determining whether the orthologs, homologs, or fragments restore fertility to genetically modified male non-human animals lacking ADAM6 activity (e.g., rodents, such as mice or rats, that contain a knockout of ADAM6 or its orthologs). In various embodiments, functionality is defined as the ability of sperm from a genetically modified animal lacking endogenous ADAM6 or its orthologs or homologs to travel through the oviduct of the genetically modified animal of the same species to fertilize an egg.

[0191] In various embodiments, mice can be produced that contain deletions or replacements of endogenous heavy chain variable region loci or parts thereof, containing ectopic nucleotide sequences that code for proteins that provide fertility benefits similar to mouse ADAM6 (e.g., its orthologs or homologs or fragments that are functional in male mice).The ectopic nucleotide sequences include nucleotide sequences that code for proteins that are ADAM6 homologs or orthologs (or fragments) of different mouse strains or different species, such as different rodent species, and that provide fertility benefits, such as increased litter size over a specified period of time, and / or increased number of pups per litter, and / or the ability of male mouse sperm cells to pass through mouse oviducts and fertilize mouse eggs.

[0192] In one embodiment, the ADAM6 is a homolog or ortholog that is at least 89% to 99% identical to mouse ADAM6 protein (e.g., at least 89% to 99% identical to mouse ADAM6a or mouse ADAM6b). In one embodiment, the ectopic nucleotide sequence encodes one or more proteins independently selected from a protein that is at least 89% identical to mouse ADAM6a, a protein that is at least 89% identical to mouse ADAM6b, and combinations thereof. In one embodiment, the homolog or ortholog is a rat, hamster, mouse, or guinea pig protein that is identical to mouse ADAM6a and / or mouse ADAM6b or has been modified to be about 89% or more identical to mouse ADAM6a and / or mouse ADAM6b. In one embodiment, the homolog or ortholog is identical to mouse ADAM6a and / or mouse ADAM6b, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to mouse ADAM6a and / or mouse ADAM6b.

[0193] Ectopic ADAM6 in humanized heavy chain mice Advances in gene targeting, such as the development of bacterial artificial chromosomes (BACs), now allow the recombination of relatively large genomic fragments. BAC engineering has made it possible to create large deletions and large insertions in mouse ES cells.

[0194] Mice that make human antibodies have been available for some time. Although these mice represent an important advance in the development of human therapeutic antibodies, they exhibit a number of significant abnormalities that limit their usefulness. For example, these mice exhibit impaired B cell development. This impaired development can be attributed to a variety of differences between the transgenic and wild-type mice.

[0195] Human antibodies may not interact optimally with mouse pre-B cell receptors or B cell receptors on the surface of mouse cells that transmit signals for survival during maturation, proliferation, or clonal selection. Fully human antibodies may not interact optimally with the mouse Fc receptor system, and mice express Fc receptors that do not display a one-to-one correspondence with human Fc receptors. Ultimately, the various mice that make fully human antibodies do not contain all the authentic mouse sequences that may be required for wild-type B cell development, e.g., downstream enhancer elements and other locus control elements.

[0196] Mice that make fully human antibodies generally contain endogenous immunoglobulin loci that have been disabled in some way, and human transgenes containing variable and constant immunoglobulin gene segments are introduced into random locations within the mouse genome. As long as the endogenous loci are disabled such that they cannot rearrange the gene segments to form functional immunoglobulin genes, the goal of making fully human antibodies in such mice can be achieved even if B-cell development is impaired.

[0197] Although one is forced to generate fully human antibodies from a human transgene locus, the production of human antibodies in mice appears to be an unfavorable process. In some mice, this process is unfavorable enough to result in the formation of chimeric human variable / mouse constant heavy chains (but not light chains) by a mechanism of trans-switching, whereby transcripts encoding fully human antibodies are isotype-switched in trans from a human to a mouse isotype. This process is in trans because the fully human transgene is located away from the endogenous locus carrying an intact copy of the mouse heavy chain constant region gene. Although trans-switching is readily evident in such mice, this event is still insufficient to rescue B-cell development, which remains clearly impaired. In any case, the trans-switching event does not occur with respect to the light chain, so that trans-switched antibodies in such mice carry fully human light chains; presumably, trans-switching depends on switch sequences in the endogenous locus that are used (albeit differently) for normal isotype switching in cis. Thus, even if mice engineered to make fully human antibodies choose a trans-switching mechanism to generate antibodies with mouse constant regions, the strategy is still insufficient to rescue normal B cell development.

[0198] A major concern in generating antibody-based human therapeutics is to generate a sufficient diversity of human immunoglobulin variable region sequences to identify useful variable domains that specifically recognize a particular epitope and bind that epitope with a desired affinity, usually, but not always, high affinity. Prior to the development of the VELOCIMMUNE® mouse (described herein), there was no indication that mice expressing human variable regions along with mouse constant regions would exhibit any significant differences from mice generating human antibodies from transgenes. However, that assumption was incorrect.

[0199] VELOCIMMUNE® mice, which contain precise replacements of mouse immunoglobulin variable regions 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 indeed impressive developmental context, VELOCIMMUNE® mice displayed an essentially normal wild-type response to immunization, differing from wild-type mice in only one important respect: the variable regions produced in response to immunization are fully human.

[0200] VELOCIMMUNE® mice contain precise large-scale replacement of the germline variable regions of mouse immunoglobulin heavy chains (IgH) and immunoglobulin light chains (e.g., kappa light chain, Igκ) at the endogenous loci with the corresponding human immunoglobulin variable regions. A total of approximately 6 megabases of the mouse locus are replaced with approximately 1.5 megabases of human genomic sequence. This precise replacement results in mice with hybrid immunoglobulin loci that create heavy and light chains with human variable regions and mouse constant regions. The mouse V H -D H -J H The precise replacement of Vκ-Jκ segments leaves the adjacent mouse sequences intact and functional in the hybrid immunoglobulin locus. The humoral immune system of the mouse functions similarly to that of a wild-type mouse. B cell development is not disrupted in any significant way, and diverse human variable regions are produced in the mouse upon antigen challenge.

[0201] VELOCIMMUNE® mice are feasible because the immunoglobulin gene segments for heavy and kappa light chains rearrange similarly in humans and mice, but the loci are not the same or nearly the same - apparently they are not. However, the loci are distinct from all V H , D H and J. HThe similarity is such that humanization of the heavy chain variable gene locus can be achieved by replacing approximately 3 million base pairs of contiguous murine sequence containing the gene segments with approximately 1 million bases of contiguous human genomic sequence that essentially covers the equivalent sequences from the human immunoglobulin loci.

[0202] In some embodiments, certain mouse constant region gene sequences are further replaced with human gene sequences (e.g., mouse C H One sequence of human C H 1, and mouse C L Sequence of human C L Substitution of the human variable region with a partial human constant region (e.g., substitution with a human variable region with a partial human constant region) results in mice with hybrid immunoglobulin loci that generate antibodies with human variable regions and partial human constant regions, e.g., suitable for generating fully human antibody fragments, e.g., fully human Fabs. Mice with hybrid immunoglobulin loci exhibit normal variable gene segment rearrangements, normal somatic hypermutation frequencies, and normal class switching. These mice exhibit humoral immune systems indistinguishable from wild-type mice, and display normal cell populations at all stages of B cell development and normal lymphoid organ structure, even when the mice lack a full repertoire of human variable region gene segments. Immunization of these mice results in a robust humoral response that displays a wide diversity of variable gene segment usage.

[0203] Precise replacement of mouse germline variable region gene segments allows the generation of mice with partial human immunoglobulin loci, which rearrange, hypermutate, and class switch normally, resulting in the production of antibodies in mice containing human variable regions. Nucleotide sequences encoding the variable regions can be identified, cloned, and fused (e.g., in an in vitro system) to any sequence of choice, e.g., any immunoglobulin isotype suitable for a particular application, resulting in an antibody or antigen-binding protein derived entirely from human sequences.

[0204] Large-scale humanization using recombineering techniques to modify mouse embryonic stem (ES) cells to produce mouse V H , D H and J. H A mouse heavy chain immunoglobulin locus of 3 megabases or less that contains essentially all of the gene segments is synthesized by combining some or essentially all of the human V H , D H and J. H The equivalent human gene segment was precisely replaced with a one megabase or less human genomic sequence containing the gene segment. A one-half megabase or less segment of the human genome containing one of two repeats encoding essentially all of the human Vκ and Jκ gene segments was used to replace a three megabase segment of the mouse immunoglobulin kappa light chain locus containing essentially all of the mouse Vκ and Jκ gene segments.

[0205] Mice with such a replaced immunoglobulin locus have a 3'-most V H Gene segment and 5'-most D H This may include a disruption or deletion of the endogenous mouse ADAM6 locus that is normally found between the gene segments. Disruption of this region may result in reduced or eliminated functionality of the endogenous mouse ADAM6 locus. H Using gene segments for replacement, it was found that an intergenic region containing a pseudogene that appears to be the human ADAM6 pseudogene was located between these V H Between gene segments, i.e., human V H 1-2 and V H 1-6. However, male mice containing this human intergenic sequence exhibit reduced fertility.

[0206] The present invention describes a mouse that contains the above-described replacement locus and also contains an ectopic nucleic acid sequence encoding mouse ADAM6, and that exhibits essentially normal fertility. In one embodiment, the ectopic nucleic acid sequence is a human V-type chromosome 1 (V-type chromosome 2) of a modified endogenous heavy chain locus. H 1-2 and Human V HIn one embodiment, the ectopic nucleic acid sequence comprises a mouse ADAM6a sequence and / or a mouse ADAM6b sequence, or a functional fragment thereof, located between human V ... H 1-2 and Human V H 6-1. The transcription direction of the ADAM6 gene in SEQ ID NO:3 is the same as that of the human V H The direction of transcription of the gene segment is reversed. H Although fertility rescue by placing an ectopic sequence between gene segments is shown, one of skill in the art will recognize that placement of an ectopic sequence into any suitable transcription-permissive locus within the mouse genome (or even extrachromosomally) would be expected to similarly rescue fertility in male mice.

[0207] The phenomenon of complementing mice lacking functional ADAM6 locus with ectopic sequences comprising mouse ADAM6 gene or its orthologue or homologue or functional fragment is a general method that can be applied to rescue any mouse with non-functional or minimally functional endogenous ADAM6 locus. Therefore, a large number of mice with ADAM6-disruptive modifications of immunoglobulin heavy chain locus can be rescued with the compositions and methods of the present invention. Thus, the present invention includes mice with a wide variety of modifications of immunoglobulin heavy chain locus including endogenous ADAM6 function. Some (non-limiting) examples are provided herein. In addition to the VELOCIMMUNE® mice described, the compositions and methods related to ADAM6 can be used in a wide variety of applications, for example, when modifying heavy chain locus in a wide variety of ways.

[0208] In one embodiment, an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or an ortholog or homolog or functional fragment thereof) is combined with all or substantially all mouse V H One or more human V of a gene segment H Substitutions in gene segments and all or substantially all mouse D HGene segments and J H Gene segment, human D H Gene segments and human J H and a replacement with a gene segment, H In one embodiment, the mouse is provided with: (a) a mouse lacking a human V1 region and / or a hinge region; H - Mouse C H 1-Mouse C H 2-Mouse C H 3;(b) Human V H -Mouse Hinge-Mouse C H 2-Mouse C H 3; and (c) human V H - Mouse C H 2-Mouse C H A single variable domain binding protein is produced which is a dimer of an immunoglobulin chain selected from three.

[0209] In one embodiment, one or more mouse immunoglobulin heavy chain variable gene segments (mV H , mD H and / or mJ H ) one or more human immunoglobulin heavy chain variable gene segments (hV H , hD H and / or hJ H In a mouse having a substitution of one or more mouse immunoglobulin κ light chain variable gene segments (mVκ and / or mJκ) with one or more human immunoglobulin κ light chain variable gene segments (hVκ and / or hJκ), a fertility-rescuing nucleotide sequence is inserted within a human immunoglobulin heavy chain variable region sequence (e.g., a human V H 1-2 gene segment and V H 1-6 gene segments).

[0210] In one embodiment, the one or more mouse immunoglobulin heavy chain variable gene segments comprise about 3 megabases of the mouse immunoglobulin heavy chain locus. In one embodiment, the one or more mouse immunoglobulin heavy chain variable gene segments comprise at least 89 V of the mouse immunoglobulin heavy chain locus. H Gene segments, at least 13 D H Gene segment, at least 4 J H In one embodiment, the one or more human immunoglobulin heavy chain variable gene segments comprise at least 80 V of the human immunoglobulin heavy chain locus. H Gene segments with at least 27 D H Gene segments, at least 6 J H It includes gene segments or combinations thereof.

[0211] In one embodiment, the one or more mouse immunoglobulin κ light chain variable gene segments comprise about 3 megabases of the mouse immunoglobulin κ light chain locus. In one embodiment, the one or more mouse immunoglobulin κ light chain variable gene segments comprise at least 137 Vκ gene segments, at least 5 Jκ gene segments, or a combination thereof, of the mouse immunoglobulin κ light chain locus. In one embodiment, the one or more human immunoglobulin κ light chain variable gene segments comprise about one-half megabase of the human immunoglobulin κ light chain locus. In a particular embodiment, the one or more human immunoglobulin κ light chain variable gene segments comprise the proximal repeat (with respect to the immunoglobulin κ constant region) of the human immunoglobulin κ light chain locus. In one embodiment, the one or more human immunoglobulin κ light chain variable gene segments comprise at least 40 Vκ gene segments, at least 5 Jκ gene segments, or a combination thereof, of the human immunoglobulin κ light chain locus.

[0212] In one embodiment, the nucleotide sequence is placed between two human immunoglobulin gene segments. In a particular embodiment, the two human immunoglobulin gene segments are heavy chain gene segments. In one embodiment, the nucleotide sequence is placed between two human immunoglobulin gene segments. In a particular embodiment, the two human immunoglobulin gene segments are heavy chain gene segments. In one embodiment, the nucleotide sequence is placed between two human immunoglobulin gene segments. H 1-2 gene segment and human V H 1-6 gene segment (see U.S. Patent Nos. 6,596,541 and 7,105,348, which are incorporated herein by reference). In one embodiment, such modified VELOCIMMUNE® mice contain at least 80 human V H Gene segment, 27 human D H Gene segments and human J H Mouse immunoglobulin heavy chain variable gene segment in gene segment The present invention includes replacement of mouse immunoglobulin κ light chain variable gene segments with at least 40 human Vκ gene segments and 5 human Jκ gene segments.

[0213] In one embodiment, the functional mouse ADAM6 locus (or an ortholog or homolog or functional fragment thereof) is H Human V gene segment replacement H In one embodiment, at least 89 mouse V H Remove gene segments to generate one or more human V H The mouse ADAM6 locus was replaced with a gene segment of human V H Immediately adjacent to the 3' end of the gene segment are two human V H In a specific embodiment, the mouse ADAM6 locus is located between the inserted human V H Two V within about 20 kilobases (kb) to about 40 kilobases (kb) of the 3' end of the gene segment HIn a specific embodiment, the mouse ADAM6 locus is located between the inserted human V H Two V within approximately 29 kb and 31 kb of the 3' end of the gene segment H In a specific embodiment, the mouse ADAM6 locus is located between the inserted human V H In a specific embodiment, the mouse ADAM6 locus is located within about 30 kb of the 3' end of the gene segment. H In certain embodiments, the substitution is within about 30,184 bp of the 3' end of the gene segment. H Gene segment V H 1-2 and V H 6-1, and the mouse ADAM6 locus comprises the V H 1-2 gene segment downstream and the V H In a specific embodiment, the mouse ADAM6 locus is located upstream of the human V6-1 gene segment. H 1-2 gene segment and human V H 6-1 gene segment, in which the 5' end of the mouse ADAM6 locus is located between the human V H The 3' end of the ADAM6 locus is about 13,848 bp from the 3' end of the 1-2 gene segment, and the 3' end of the ADAM6 locus is about 13,848 bp from the 3' end of the 1-2 gene segment. H In a particular embodiment, the mouse ADAM6 locus comprises SEQ ID NO:3, or a fragment thereof, that confers ADAM6 function in cells of the mouse. H The gene segments are organized as follows (human V H From upstream to downstream in terms of the transcription direction of the gene segment): Human V H 1-2-Mouse ADAM6 locus-Human V H 6-1. In certain embodiments, human V H 1-2 and Human V HIn one embodiment, the ADAM6 pseudogene between human ADAM6 locus 6-1 is replaced with the mouse ADAM6 locus. In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b in the mouse ADAM6 locus with respect to the transcription direction is the same as that of human ADAM6 locus 6-1. H Alternatively, the mouse ADAM6 locus is oriented in the 3′-most human V H Gene segment and 5'-most D H Located in the intergenic region between gene segments. H This should be the case whether the segment is mouse or human.

[0214] Similarly, all or substantially all endogenous mouse V H the one or more human V gene segments L Mice modified with gene segments (e.g., Vκ or Vλ segments) can be modified to maintain the endogenous mouse ADAM6 locus, for example, by utilizing a targeting vector with downstream homology arms that include the mouse ADAM6 locus or a functional fragment thereof, as described above, or can be modified to incorporate two human V L Between gene segments, or in human V L Gene Segment and D H Between gene segments (whether human or mouse, e.g., Vλ+m / hD H ) or between J gene segments (e.g., Vκ+J, whether in human or mouse). H In one embodiment, the replacement can be performed by substituting two or more human V L and the mouse ADAM6 locus or functional fragment thereof comprises the two 3'-most V L Thus, in certain embodiments, the human V L The organization of the gene segments is as follows (from upstream to downstream with respect to the transcriptional direction of the human gene segments): human V L3'-1-Mouse ADAM6 locus-Human V L In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b in the mouse ADAM6 locus with respect to the transcription direction is the same as that of the human V L Alternatively, the mouse ADAM6 locus is inverted relative to the 3'-most human V L Gene segment and 5'-most D H Located in the intergenic region between gene segments. H This should be the case whether the segment is mouse or human.

[0215] In one embodiment, one or more endogenous mouse V H A gene segment replacement and at least one endogenous mouse D H The present invention provides a mouse comprising the endogenous mouse V gene segment. H The modification of the gene segment is H The 3'-most V, not the gene segment H The one or more gene segments may include a modification of the 3′-most V H Care is taken to ensure that modifications of the gene segments do not disrupt or render non-functional the endogenous mouse ADAM6 locus. For example, in one embodiment, the mouse has all or substantially all of the endogenous mouse ADAM6 locus. H One or more human V of a gene segment H and the mouse comprises a replacement with one or more endogenous D H Includes gene segments and a functional endogenous mouse ADAM6 locus.

[0216] In another embodiment, the mouse has a 3′-most endogenous mouse V H Modification of a gene segment and one or more endogenous mouse D HThe present invention includes modification of gene segments such that the integrity of the endogenous mouse ADAM6 locus is maintained to the extent that the endogenous ADAM6 locus remains functional. In one example, such modification is performed in two steps: (1) targeting the 3'-most endogenous mouse V gene using a targeting vector having an upstream homology arm and a downstream homology arm that includes all or a portion of the functional mouse ADAM6 locus; H Gene segments can be divided into one or more human V H (2) replacing the endogenous mouse ADAM6 gene segment with a targeting vector having upstream homology arms containing all or a functional portion of the mouse ADAM6 locus. H Replacing gene segments.

[0217] In various embodiments, the use of mice containing ectopic sequences encoding mouse ADAM6 protein or its orthologue or homologue or functional homologue is useful when modification destroys the function of endogenous mouse ADAM6.The probability of destroying endogenous mouse ADAM6 function is high when modification is made to mouse immunoglobulin locus, especially when modifying mouse immunoglobulin heavy chain variable region and surrounding sequence.Therefore, such mice are particularly useful when making mice with immunoglobulin heavy chain locus that is completely or partially deleted, completely or partially humanized, or completely or partially replaced (for example, with Vκ or Vλ sequence).The methods for carrying out the described genetic modifications to mice described below are known to those skilled in the art.

[0218] Mice containing ectopic sequences encoding mouse ADAM6 protein or ectopic sequences encoding substantially the same or similar proteins that provide the fertility benefits of mouse ADAM6 protein are particularly useful in conjunction with modifications to mouse immunoglobulin heavy chain variable gene locus that disrupt or delete endogenous mouse ADAM6 sequence.Although described primarily in conjunction with mice expressing antibodies with human variable regions and mouse constant regions, such mice are useful in conjunction with any genetic modification that disrupts endogenous mouse ADAM6 gene.Those skilled in the art will recognize that this encompasses a wide variety of genetically modified mice that contain modifications to mouse immunoglobulin heavy chain variable gene locus.These include, for example, mice that have deletions or replacements of all or part of mouse immunoglobulin heavy chain gene segments, regardless of other modifications.Non-limiting examples are described below.

[0219] In some aspects, a genetically modified mouse is provided that comprises an ectopic mouse, rodent or other ADAM6 gene (or ortholog or homolog or fragment) functional in mouse and one or more human immunoglobulin variable and / or constant region gene segments. In various embodiments, the other ADAM6 gene ortholog or homolog or fragment functional in mouse can comprise bovine, canine, primate, rabbit or other non-human sequences.

[0220] In one embodiment, an ectopic ADAM6 sequence encoding a functional ADAM6 protein and all or substantially all of the mouse V H One or more human V of a gene segment H Substitutions in gene segments and all or substantially all mouse D H One or more human D H Replacement of gene segments with all or substantially all mouse J H One or more human J of a gene segment H and a replacement with a gene segment.

[0221] In one embodiment, the mouse is mouse C H 1 nucleotide sequence of human C H In one embodiment, the mouse further comprises a substitution of a mouse hinge nucleotide sequence with a human hinge nucleotide sequence. In one embodiment, the mouse further comprises a substitution of a mouse hinge nucleotide sequence with a human hinge nucleotide sequence. L and J. L ) 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 V L , J L and C L is an immunoglobulin κ light chain sequence. In a specific embodiment, the mouse has a human hinge and a human C H 1 sequence fused to mouse C H 2 and mouse C H Since the mouse immunoglobulin locus contains three immunoglobulin constant region sequences, the mouse immunoglobulin locus rearranges to contain (a) a human variable region, a human C H 1 region, human hinge region and mouse C H 2 and mouse C H A gene encoding a binding protein comprising a heavy chain having three regions and (b) a gene encoding an immunoglobulin light chain comprising a human variable domain and a human constant region is formed.

[0222] In one embodiment, an ectopic ADAM6 sequence encoding a functional ADAM6 protein and all or substantially all of the mouse V H One or more human V of a gene segment L Substitution of a gene segment and, optionally, all or substantially all of the D H Gene segment and / or J H One or more human D H Gene segments and / or human J Hand optionally, all or substantially all of the D H Gene segments and J H One or more human J of a gene segment L and a replacement with a gene segment.

[0223] In one embodiment, the mouse is a mouse that is H , D H and J. H One or more Vs in a gene segment L , one or more D H and one or more J gene segments (e.g., Jκ or Jλ), which are operably linked to an endogenous mouse hinge region, and the mouse has, in the transcriptional direction 5' to 3', a human V L -Human or mouse D H -human or mouse J -mouse hinge -mouse C H 2-Mouse C H In one embodiment, the J region is a human Jκ region. In one embodiment, the J region is a human J H In one embodiment, the J region is a human Jλ region. In one embodiment, the human V L The regions are selected from human Vλ and human Vκ regions.

[0224] In a specific embodiment, the mouse comprises a mouse or human constant region and a human Vκ, a human D H and human Jκ; human Vκ, human D H and human J H ; human Vλ, human D H and human Jλ; human Vλ, human D H and human J H ; Human Vκ, human D H and human Jλ; human Vλ, human D Hand a variable region derived from a human Jκ. In certain embodiments, the recombination recognition sequences are engineered to allow productive rearrangements to occur between the recited V, D and J gene segments or between the recited V and J gene segments.

[0225] In one embodiment, an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or an ortholog or homolog or functional fragment thereof) is combined with all or substantially all mouse V H One or more human V of a gene segment L Substitutions in gene segments and all or substantially all D H Gene segments and J H Gene segment, human J L and a replacement with a gene segment, H 1 and / or a mouse lacking the hinge region.

[0226] In one embodiment, the mouse is H In one embodiment, the mouse lacks a sequence encoding the C1 domain. In one embodiment, the mouse lacks a sequence encoding a hinge region. In one embodiment, the mouse lacks a sequence encoding the C2 domain. H 1 domain and the sequence encoding the hinge region is missing.

[0227] In certain embodiments, the mouse is a mouse C H Mouse C binding to the 3 domain H A binding protein containing a human immunoglobulin light chain variable domain (lambda or kappa) fused to the two domains is expressed.

[0228] In one embodiment, an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or an ortholog or homolog or functional fragment thereof) is combined with all or substantially all mouse V H One or more human V of a gene segment L Replacement of gene segments with all or substantially all mouse DH and J. H Gene segment, human J L and a replacement with a gene segment.

[0229] In one embodiment, the mouse is H 1 region, hinge region, C H 1 and the hinge region, or C H 1 region and hinge region and C H It contains a deletion of the immunoglobulin heavy chain constant region gene sequence that encodes two regions.

[0230] In one embodiment, the mouse makes a single variable domain binding protein that comprises a homodimer selected from: (a) a human V L - Mouse C H 1-Mouse C H 2-Mouse C H 3;(b) Human V L -Mouse Hinge-Mouse C H 2-Mouse C H 3;(c) Human V L - Mouse C H 2-Mouse C H 3.

[0231] In one aspect, a mouse is provided that has a disabled endogenous heavy chain immunoglobulin locus, including a disabled or deleted endogenous mouse ADAM6 locus, and 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 sequence is present on an integrated transgene, which is randomly integrated into the mouse genome. In one embodiment, the nucleic acid sequence is present on an episome (e.g., chromosome) that is not found in wild-type mice.

[0232] In one embodiment, the mouse further comprises a disabled endogenous immunoglobulin light chain locus. In a particular embodiment, the endogenous immunoglobulin light chain locus is selected from a kappa (κ) and a lambda (λ) light chain locus. In a particular embodiment, the mouse comprises a disabled endogenous κ light chain locus and a disabled λ light chain locus, wherein the mouse expresses an antibody comprising a human immunoglobulin heavy chain variable domain and a human immunoglobulin light chain domain. In one embodiment, the human immunoglobulin light chain domain is selected from a human κ light chain domain and a human λ light chain domain.

[0233] In one embodiment, a genetically modified animal is provided that expresses a chimeric antibody, wherein the genetically modified animal expresses an ADAM6 protein or an ortholog or homolog thereof that is functional in the genetically modified animal.

[0234] In one embodiment, the genetically modified animal is selected from mouse and rat. In one embodiment, the genetically modified animal is a mouse, and the ADAM6 protein or its ortholog or homolog is from a mouse lineage that is a different lineage from the genetically modified animal. In one embodiment, the genetically modified animal is a rodent of the Cricetidae family (e.g., hamster, New World rat or mouse, bank vole), and the ADAM6 protein ortholog or homolog is from a rodent of the Muridae family (e.g., purebred mouse and rat, gerbil, spiny mouse, maned mouse). In one embodiment, the genetically modified animal is a rodent of the Muridae family, and the ADAM6 protein ortholog or homolog is from a rodent of the Cricetidae family.

[0235] In one embodiment, the chimeric antibody comprises a human variable domain and a rodent constant region sequence. In one embodiment, the rodent is selected from a Cricetidae rodent and a Murine rodent. In a particular embodiment, the Cricetidae and Murine rodent is a mouse. In a particular embodiment, the Cricetidae and Murine rodent 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 a rat; in a particular embodiment, the mouse or rat is selected from the Cricetidae and Murine families. In one embodiment, the chimeric antibody comprises a human heavy chain variable domain, a human light chain variable domain, and a constant region sequence from a rodent selected from a mouse and a rat, where the human heavy chain variable domain and the human light chain are of the same species. In certain embodiments, allogeneic includes those in which the human heavy chain and 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 presents the variable domains together on the surface of the individual B cell.

[0236] In one embodiment, the chimeric antibody is expressed from an immunoglobulin locus. In one embodiment, the heavy chain variable domain of the chimeric antibody is expressed from a rearranged endogenous immunoglobulin heavy chain locus. In one embodiment, the light chain variable domain of the chimeric antibody 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 expressed from a rearranged transgene (e.g., a rearranged nucleic acid sequence derived from a non-rearranged nucleic acid sequence integrated into the genome of the animal at a locus other than the 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 a non-rearranged nucleic acid sequence integrated into the genome of the animal at a locus other than the endogenous immunoglobulin locus).

[0237] In certain embodiments, the transgene is expressed from a transcriptionally active locus, e.g., the ROSA26 locus, e.g., the murine (e.g., mouse) ROSA26 locus.

[0238] In one aspect, a non-human animal is provided, comprising a humanized immunoglobulin heavy chain locus, wherein the humanized immunoglobulin heavy chain locus comprises a non-human ADAM6 sequence, or an orthologue or homologue thereof.

[0239] In one embodiment, the non-human animal is a rodent selected from a mouse, a rat, and a hamster.

[0240] In one embodiment, the non-human ADAM6 ortholog or homolog is a sequence that is orthologous and / or homologous to a mouse ADAM6 sequence, which ortholog or homolog is functional in the non-human animal.

[0241] In one embodiment, the non-human animal is selected from mouse, rat and hamster, and the ADAM6 ortholog or homolog is from a non-human animal selected from mouse, rat and hamster. In a specific embodiment, the non-human animal is a mouse, and the ADAM6 ortholog or homolog is from an animal selected from different mouse species, rat and hamster. In a specific embodiment, the non-human animal is a rat, and the ADAM6 ortholog or homolog is from a rodent selected from different rat species, mouse and hamster. In a specific embodiment, the non-human animal is a hamster, and the ADAM6 ortholog or homolog is from a rodent selected from different hamster species, mouse and rat.

[0242] In certain embodiments, the non-human animal is from the suborder Murine and the ADAM6 sequence is from an animal selected from Jerboidea rodents and Murine rodents. In certain embodiments, the rodent is a Murine mouse and the ADAM6 ortholog or homolog is from a Murine mouse or rat or hamster.

[0243] In one embodiment, the humanized heavy chain locus comprises one or more human V H Gene segment, one or more human D H A gene segment and one or more human J H In certain embodiments, the one or more human V gene segments. H Gene segment, one or more human D H A gene segment and one or more human J H The gene segments are operably linked to one or more human, chimeric and / or rodent (e.g., mouse or rat) constant region genes. In one embodiment, the constant region gene is mouse. In one embodiment, the constant region gene is rat. In one embodiment, the constant region gene is hamster. In one embodiment, the constant region gene is a hinge, C H 2. C H In certain embodiments, the constant region gene comprises a sequence selected from the group consisting of hinge, C, C-terminal, and combinations thereof. H 2 and C H Contains 3 sequences.

[0244] In one embodiment, the non-human ADAM6 sequence is adjacent to a human immunoglobulin heavy chain sequence. In one embodiment, the non-human ADAM6 sequence is located 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.

[0245] In one embodiment, the non-human ADAM6 sequence is located between two V gene segments. In one embodiment, the non-human ADAM6 sequence is juxtaposed between a V gene segment and a D gene segment. In one embodiment, the mouse ADAM6 sequence is located between a V gene segment and a J gene segment. In one embodiment, the mouse ADAM6 sequence is juxtaposed between a D gene segment and a J gene segment.

[0246] In one embodiment, a human V L Domain homologous to human V H The present invention provides a genetically modified non-human animal, comprising a B cell expressing a domain, and expressing a non-immunoglobulin non-human protein from the immunoglobulin locus. In one embodiment, the non-immunoglobulin non-human protein is an ADAM protein. In a particular embodiment, the ADAM protein is an ADAM6 protein or a homologue or ortholog or a functional fragment thereof.

[0247] In one embodiment, the non-human animal is a rodent (e.g., a mouse or a rat). In one embodiment, the rodent is of the family Muridae. In one embodiment, the rodent is of the family Murinoidea. In a particular embodiment, the Murinoidea rodent is selected from a mouse and a rat.

[0248] In one embodiment, the non-immunoglobulin non-human protein is a rodent protein. In one embodiment, the rodent is of the family Muridae. In one embodiment, the rodent is of the family Murinoidea. In a particular embodiment, the rodent is selected from a mouse, a rat, and a hamster.

[0249] In one embodiment, the human V H and V LThe domain is linked to an immunoglobulin constant domain sequence directly or via a linker. In certain embodiments, the constant domain sequence includes a hinge, C H 2. C H 3 and combinations thereof. L The domain is selected from a Vκ or Vλ domain.

[0250] In one aspect, a genetically modified non-human animal is provided, the genetically modified non-human animal comprising a human immunoglobulin sequence in its germline, the sperm of the male non-human animal being characterized by an in vivo migration defect. In one embodiment, the in vivo migration defect comprises the sperm of the male non-human animal being unable to migrate from the uterus through the oviduct of a female non-human animal of the same species. In one embodiment, the non-human animal lacks a nucleotide sequence encoding an ADAM6 protein or a functional fragment thereof. In a particular embodiment, the ADAM6 protein or a functional fragment thereof comprises an ADAM6a and / or an ADAM6b protein or a functional fragment thereof. In one embodiment, the non-human animal is a rodent. In a particular embodiment, the rodent is selected from a mouse, a rat, and a hamster.

[0251] In one aspect, a non-human animal is provided that comprises a human immunoglobulin sequence adjacent to the non-human sequence that encodes ADAM6 protein or its ortholog or homolog or functional fragment.In one embodiment, said non-human animal is a rodent.In a particular embodiment, said rodent is selected from mouse, rat and hamster.

[0252] In one embodiment, the human immunoglobulin sequence is an immunoglobulin heavy chain sequence. In one embodiment, the immunoglobulin sequence includes one or more V H In one embodiment, the human immunoglobulin sequence comprises one or more D HIn one embodiment, the human immunoglobulin sequence comprises one or more J H In one embodiment, the human immunoglobulin sequence comprises one or more V H Gene segment, one or more D H A gene segment and one or more J H Contains gene segments.

[0253] In one embodiment, the immunoglobulin sequence comprises one or more V H In certain embodiments, the one or more V H A gene segment may contain no more than two V H Gene segment, 3 or fewer Vs H Gene segment, 4 or fewer Vs H Gene segment, 5 or fewer Vs H Gene segment, 6 or fewer Vs H Gene segment, 7 or fewer Vs H Gene segment, 8 or fewer Vs H Gene segment, 9 or fewer Vs H Gene segment, 10 or fewer Vs H Gene segment, 11 or fewer V H Gene segment, 12 or fewer V H Gene segment, 13 or fewer V H Gene segment, 14 or fewer Vs H Gene segment, 15 or fewer Vs H Gene segment, 16 or fewer V H Gene segment, 17 or fewer V H Gene segment, V less than 18 H Gene segment, 19 or fewer V H Gene segment, 20 or fewer Vs H Gene segment, V less than 21 H Gene segment, V less than 22 H Gene segments or Vs up to 23 H Contains gene segments.

[0254] In certain embodiments, the one or more V H The gene segment consists of five V H In certain embodiments, the one or more V H The gene segment is 10 V H In certain embodiments, the one or more V H The gene segment consists of 15 V H In certain embodiments, the one or more V H The gene segment is composed of 20 V H Contains gene segments.

[0255] In various embodiments, the V H The gene segment is V H 6-1, V H 1-2, V H 1-3, V H 2-5, V H 3-7, V H 1-8, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-16, V H 1-18, V H 3-20, V H 3-21, V H 3-23, V H 1-24, V H 2-26, V H 4-28, V H 3-30, V H 4-31, V H 3-33, V H 4-34, V H 3-35, V H 3-38, V H 4-39, V H 3-43, V H 1-45, V H 1-46, V H 3-48, V H 3-49, V H 5-51, V H 3-53, V H 1-58, VH 4-59, V H 4-61, V H 3-64, V H 3-66, V H 1-69, V H 2-70, V H 3-72, V H 3-73 and V H Selected from 3-74.

[0256] In various embodiments, the V H The gene segment is V H 1-2, V H 1-8, V H 1-18, V H 1-46, V H 1-69, V H 3-7, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-21, V H 3-23, V H 3-30, V H 3-33, V H 3-43, V H 3-48, V H 4-31, V H 4-34, V H 4-39, V H 4-59, V H 5-51 and V H It is selected from 6-1.

[0257] In various embodiments, the V H The gene segment is V H 1-18, V H 1-46, V H 1-69, V H 3-7, V H 3-11, V H 3-15, V H 3-21, V H 3-23, V H 3-30, V H 3-33, V H 3-48, V H 4-34, V H 4-39, VH 4-59 and V H Selected from 5-51.

[0258] In various embodiments, the V H The gene segment is V H 1-18, V H 1-69, V H 3-7, V H 3-11, V H 3-15, V H 3-21, V H 3-23, V H 3-30, V H 3-43, V H 3-48, V H 4-39, V H 4-59 and V H Selected from 5-51.

[0259] In various embodiments, the V H The gene segment is V H 1-18, V H 3-11, V H 3-21, V H 3-23, V H 3-30, V H 4-39 and V H Selected from 4-59.

[0260] In various embodiments, the V H The gene segment is V H 1-18, V H 3-21, V H 3-23, V H 3-30 and V H Selected from 4-39.

[0261] In various embodiments, the V H The gene segment is V H 1-18, V H 3-23 and V H Selected from 4-39.

[0262] In various embodiments, the V H The gene segment is VH 3-21, V H 3-23 and V H Selected from 3-30.

[0263] In various embodiments, the V H The gene segment is V H 3-23, V H 3-30 and V H Selected from 4-39.

[0264] In certain embodiments, the human immunoglobulin sequence comprises at least 18 V H Gene segment, 27D H Gene segment and 6 J H In certain embodiments, the human immunoglobulin sequence comprises at least 39 V gene segments. H Gene segment, 27D H Gene segment and 6 J H In certain embodiments, the human immunoglobulin sequence comprises at least 80 V gene segments. H Gene segment, 27D H Gene segment and 6 J H Contains gene segments.

[0265] In one embodiment, the non-human animal is a mouse, and the mouse is H One or more human V of a gene segment H the human V gene segment H The gene segment is mouse C H The mouse has a human V region gene operably linked thereto. H Gene segment rearrangements and human V H Domain and mouse C H In one embodiment, the recombinant human IgG1 expresses a reverse chimeric immunoglobulin heavy chain comprising a non-rearranged mouse V H 90–100% of gene segments contain at least one nonconstitutive human V H In certain embodiments, the endogenous mouse VH All or substantially all of the gene segments are at least one unrearranged human V H In one embodiment, the replacement is at least 19, at least 39, or at least 80 or 81 unrearranged human V gene segments. H In one embodiment, the replacement is in at least 12 functional unrearranged human V gene segments. H Gene segments, at least 25 functionally unrearranged human V H Gene segments or at least 43 functionally unrearranged human V H In one embodiment, the mouse is a mouse D H and J. H At least one unrearranged human D segment H segment and at least one unrearranged human J H In one embodiment, the at least one unrearranged human D H In one embodiment, the at least one unrearranged human J segment is selected from the group consisting of 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 combinations thereof. H In certain embodiments, the one or more human V segments are selected from 1, 2, 3, 4, 5, 6, and combinations thereof. H The gene segments are 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, 6-1 Human V H The gene segments and combinations thereof are selected.

[0266] In various embodiments, the human immunoglobulin sequence is in operably linkage with a constant region in the germline of a non-human animal (e.g., a rodent, such as a mouse, rat or hamster). In one embodiment, the constant region is a human, chimeric human / mouse or chimeric human / rat or chimeric human / hamster, mouse, rat or hamster constant region. In one embodiment, the constant region is a rodent (e.g., a mouse or rat or hamster) constant region. In a particular embodiment, the rodent is a mouse or rat. In various embodiments, the constant region is comprised of at least C H 2 domain and C H Contains 3 domains.

[0267] In one embodiment, the human immunoglobulin heavy chain sequence is at an immunoglobulin heavy chain locus in the germline of a non-human animal (e.g., a rodent, such as a mouse, rat or hamster). In one embodiment, the human immunoglobulin heavy chain sequence is at a non-immunoglobulin heavy chain locus in the germline of the non-human animal, and the non-heavy chain locus is a transcriptionally active locus. In a particular embodiment, the non-heavy chain locus is the ROSA26 locus.

[0268] In various embodiments, the non-human animal further comprises a human immunoglobulin light chain sequence in the germline of the non-human animal (e.g., one or more unrearranged light chain V and J sequences, or one or more rearranged VJ sequences). In a particular embodiment, the immunoglobulin light chain sequence is an immunoglobulin κ light chain sequence. In one embodiment, the human immunoglobulin light chain sequence is one or more V L In one embodiment, the human immunoglobulin light chain sequence comprises one or more J L In one embodiment, the human immunoglobulin light chain sequence comprises one or more V L A gene segment and one or more J LIn certain embodiments, the human immunoglobulin light chain sequence comprises at least 16 Vκ gene segments and 5 Jκ gene segments. In certain embodiments, the human immunoglobulin light chain sequence comprises at least 30 Vκ gene segments and 5 Jκ gene segments. In certain embodiments, the human immunoglobulin light chain sequence comprises at least 40 Vκ gene segments and 5 Jκ gene segments. In various embodiments, the human immunoglobulin light chain sequence is in operable linkage with a constant region in the germline of a non-human animal (e.g., a rodent, such as a mouse, rat or hamster). In one embodiment, the constant region is a human, chimeric human / rodent, mouse, rat or hamster constant region. In certain embodiments, the constant region is a mouse or rat constant region. In certain embodiments, the constant region is a mouse kappa constant (mCκ) region or a rat kappa constant (rCκ) region.

[0269] In one embodiment, the non-human animal is a mouse, and the mouse comprises a replacement of all or substantially all Vκ and Jκ gene segments with at least 6 human Vκ gene segments and at least one Jκ gene segment. In one embodiment, all or substantially all Vκ and Jκ gene segments are replaced with at least 16 human Vκ gene segments (human Vκ) and at least one Jκ gene segment. In one embodiment, all or substantially all Vκ and Jκ gene segments are replaced with at least 30 human Vκ gene segments and at least one Jκ gene segment. In one embodiment, all or substantially all Vκ and Jκ gene segments are replaced with at least 40 human Vκ gene segments and at least one Jκ gene segment. In one embodiment, the at least one Jκ gene segment comprises 2, 3, 4 or 5 human Jκ gene segments.

[0270] In one embodiment, the human Vκ gene segments include Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, and Vκ1-6. In one embodiment, the Vκ gene segments include Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, and Vκ1-16. In one embodiment, the human Vκ gene segments include Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, and Vκ2-30. In one embodiment, the human Vκ gene segments include Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, and Vκ2-40.

[0271] In a specific embodiment, the Vκ gene segments comprise contiguous human immunoglobulin κ gene segments spanning the human immunoglobulin κ light chain locus from Vκ4-1 to Vκ2-40, and the Jκ gene segments comprise contiguous gene segments spanning the human immunoglobulin κ light chain locus from Jκ1 to Jκ5.

[0272] In one embodiment, the human immunoglobulin light chain sequence is at an immunoglobulin light chain locus in the germline of the non-human animal. In a particular embodiment, the immunoglobulin light chain locus in the germline of the non-human animal is an immunoglobulin kappa light chain locus. In one embodiment, the human immunoglobulin light chain sequence is at a non-immunoglobulin light chain locus in the germline of the non-human animal that is transcriptionally active. In a particular embodiment, the non-immunoglobulin locus is the ROSA26 locus.

[0273] In one embodiment, a method is provided for making a human antibody comprising variable domains derived from one or more variable region nucleic acid sequences encoded by a cell of a non-human animal as described herein.

[0274] In one aspect, a pharmaceutical composition is provided comprising a polypeptide comprising an antibody or antibody fragment derived from one or more variable region nucleic acid sequences 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., scFv).

[0275] In one aspect, the present invention provides a use of a non-human animal described herein for producing an antibody. In various embodiments, the antibody comprises one or more variable domains derived from one or more variable region nucleic acid sequences isolated from the non-human animal. In certain embodiments, the variable region nucleic acid sequence comprises an immunoglobulin heavy chain gene segment. In certain embodiments, the variable region nucleic acid sequence comprises an immunoglobulin light chain gene segment. EXAMPLES

[0276] The following examples are provided to illustrate how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise indicated, temperatures are given in degrees Celsius and pressures are at or near atmospheric.

[0277] Example 1 Humanization of mouse immunoglobulin genes Using human and mouse bacterial artificial chromosomes (BACs), 13 different BAC targeting vectors (BACvecs) were engineered for the humanization of mouse immunoglobulin heavy and kappa light chain loci. Tables 1 and 2 provide a description of the steps taken to construct all BACvecs utilized for humanization of mouse immunoglobulin heavy and kappa light chain loci, respectively.

[0278] Identification of human and mouse BACs. Mouse BACs spanning the 5' and 3' ends of the immunoglobulin heavy and kappa light chain loci were identified by hybridization of filters spotted with the BAC libraries or by PCR screening of mouse BAC library DNA pools. Filters were hybridized under standard conditions using probes corresponding to the regions of interest. Library pools were screened by PCR using unique primer pairs flanking the target regions of interest. Additional PCRs using the same primers were performed to deconvolute a given well and isolate the corresponding BAC of interest. Both BAC filters and library pools were generated from 129SvJ mouse ES cells (Incyte Genomics / Invtrogen). Human BACs covering the entire immunoglobulin heavy and kappa light chain loci were identified either by hybridization of spotted filters with BAC libraries (Caltech B, C or D libraries and RPCI-11 library, Research Genetics / Invitrogen) and screening of human BAC library pools (Caltech libraries, Invitrogen) by PCR-based methods or by using a BAC end sequence database (Caltech D libraty, TIGR). BACvec construction 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 ligation of PCR-generated homology boxes into the cloned cassette, followed by gel isolation of the ligation product 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 enzyme analysis on pulsed-field gels (Schwartz and Cantor, 1984, Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis, Cell 37:67-75), and spot checking by PCR using primers spanning the entire human sequence.

[0279] 3hV for the first humanization step of the immunoglobulin heavy chain locus using three sequential BHR steps H A BACvec was constructed (Figure 4A and Table 1). In the first step (step 1), the human V H A cassette containing a region of homology to the mouse immunoglobulin heavy chain locus (HB1), a gene conferring kanamycin resistance in bacteria and G418 resistance in animal cells (kanR), and a site-specific recombination site (e.g., loxP) was introduced upstream from the 1-3 gene segment. H Immediately downstream from the segment, a second cassette was introduced containing a second region of homology to the mouse immunoglobulin heavy chain locus (HB2) and a gene that confers resistance to spectinomycin in bacteria (specR). This second step was carried out by inserting the H The third step (step 3) involved deleting the human immunoglobulin heavy chain locus sequence downstream from 6 and the BAC vector chloramphenicol resistance gene (cmR). Then, in the third step (step 3), the doubly modified human BAC (B1) was linearized using the I-CeuI site added during the first two steps and integrated into the mouse BAC (B2) by BHR through two homology regions (HB1 and HB2). Drug selection for the first (cm / kan), second (spec / kan) and third (cm / kan) steps was designed to be specific for the desired product. The modified BAC clones were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with restriction enzymes to determine the appropriate construct (Figure 4B).

[0280] Similarly, 12 additional BACvecs were engineered for humanization of the heavy and kappa light chain loci. In some cases, BAC ligation was performed in place of BHR to join two large BACs by introducing rare restriction sites into both parental BACvecs by BHR, along with careful placement of selectable markers. This allowed survival of the desired ligation product upon selection with a particular drug marker combination. Recombinant BACs obtained by ligation after rare restriction digestion were identified and screened (as described above) in a similar manner to those obtained by BHR.

[0281] [Table 1-1]

[0282] [Table 1-2]

[0283] [Table 1-3]

[0284] [Table 2] Embryonic stem (ES) cell modification and mouse production. ES cell (F1H4) targeting was performed using the VELOCIGENE® genetic engineering method as described (Valenzuela et al., 2003). Derivation of mice from modified ES cells by either blastocysts (Valenzuela et al., 2003) or 8-cell injection (Poueymirou et al., 2007, F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses, Nat Biotechnol 25:91-99) was as described. Targeted ES cells and mice were confirmed by screening DNA from ES cells or mice with a unique set of probes and primers in a PCR-based assay (e.g., Figures 3A, 3B, and 3C). All mouse studies were overseen and approved by Regeneron's Institutional Animal Care and Use Committee (IACUC). Karyotyping and fluorescent in situ hybridization (FISH). Karyotyping was performed by the Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ). FISH was performed using targeted ES cells as described (Valenzuela et al., 2003). Probes corresponding to either mouse or human BAC DNA were labeled with fluorescently labeled dUTP nucleotides Spectral Orange or Spectral Green (Vysis) by nick translation (Invitrogen).

[0285] Immunoglobulin heavy chain variable gene locus. Humanization of the variable regions of the heavy chain locus was performed in nine sequential steps using VELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., 2003). H , D Hand J. H This was achieved by directly replacing approximately 3 million base pairs (Mb) of contiguous mouse genomic sequence containing the gene segments with approximately 1 Mb of contiguous human genomic sequence containing the equivalent human gene segments (Figure 1A and Table 1).

[0286] J H The intron between the gene segment and the constant region gene (JC intron) contains a transcriptional enhancer (Neuberger, 1983, Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells, EMBO J 2:1373-1378), followed by a simple repeat region 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). To preserve both efficient expression of the humanized heavy chain locus in mice and class switching, the human V gene was modified to maintain the mouse heavy chain intron enhancer and switch domains. H -D H -J H Region and Mouse C H The junction between the regions (proximal junction) was selected. Using the VELOCIGENE® genetic engineering method (described above) using bacterial homologous recombination driven by synthetic oligonucleotides, the exact nucleotide position of this and subsequent junctions in all substitutions was feasible. In this way, the proximal junction was selected as the final J H The gene segment was placed approximately 200 bp downstream, with the distal junction located at the 5'-most V of the human locus. H A few hundred genes upstream of the mouse V gene, also known as J558.55 H The mouse V HThe 1-86 (J558.55) gene segment is the most distal heavy chain variable gene segment, which is reported to be a pseudogene in C57BL / 6 mice but potentially active despite poor RSS sequences in the targeted 129 allele. The distal end of the mouse heavy chain locus may reportedly contain regulatory elements that regulate locus expression and / or rearrangements (Pawlitzky et al., 2006).

[0287] The first insertion of human immunoglobulin DNA sequences into mice was performed using approximately 75 kb of mouse homology arms to create three V H , 27 all D H and 9 J's H The 144 kb proximal end of the human heavy chain locus, containing human gene segments, is located proximal to the mouse IgH locus. This was achieved by inserting the 3hV H ). This large 144 kb insertion and accompanying 16.6 kb deletion were performed in a single step (Step A), which occurred at a frequency of 0.2% (Table 3). Correctly targeted ES cells were scored by loss-of-native-allele (LONA) assay (Valenzuela et al., 2003) using probes within and adjacent to the deleted mouse sequence as well as probes within the inserted human sequence, and the integrity of the large human insert was verified using multiple probes across the entire insert (Figures 3A, 3B and 3C). Because multiple rounds of sequential ES cell targeting were expected, at this and all subsequent steps targeted ES cell clones were subjected to karyotype analysis (described above) and only clones showing normal karyotype in at least 17 of 20 spreads were used for subsequent steps.

[0288] The targeted ES cells from step A were retargeted with a BACvec, thereby generating a 19 kb deletion at the distal end of the heavy chain locus (step B, FIG. 2A). The step B BACvec contained a hygromycin resistance gene (hyg) in contrast to the neomycin resistance gene (neo) contained in the step A BACvec. The resistance genes from these two BACvecs were successfully targeted to the same chromosome, resulting in the deletion of not only the two resistance genes but also the V H All Mouse V except 1-86 H Gene segments and all D except DQ52 H Approximately 3 Mb of the mouse heavy chain variable gene locus containing gene segments is flanked by loxP sites, and DQ52 and all mouse J H The 3hV gene segment was designed to be deleted in step A. Doubly targeted ES cell clones on the same chromosome were HThe proximal cassette was made homozygous at high G418 (Mortensen et al., 1992, Production of homozygous mutant ES cells with a single targeting construct, Mol Cell Biol 12:2391-2395) and the fate of the distal hyg cassette was identified by tracing. Mouse segments of 4 Mb or less in size modified in such a way that they were flanked by loxP sites were successfully deleted in ES cells by transient expression of CRE recombinase with high efficiency (~11%) 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 20:648-655). Using a similar approach, we achieved a 3 Mb deletion in 8% of ES cell clones after transient CRE expression (Step C, Figure 2A, Table 3). The deletion was scored by LONA assay using probes at either end of the deleted mouse sequence and the loss of neo and hyg, and the appearance of a PCR product spanning the deletion point containing the only remaining loxP site. The deletion was further confirmed by fluorescent in situ hybridization (data not shown).

[0289] The remainder of the human heavy chain variable region was engineered into 3hV in a series of five steps (steps EH, FIG. 2B) using VELOCIGENE® genetic engineering techniques, each step involving precise insertion of up to 210 kb of human gene sequence. H Alleles were added. For each step, the proximal end of each new BACvec was designed to overlap with the distal most human sequence of the previous step, and the distal end of each new BACvec contained the same distal mouse homology region used in step A. The BACvecs of steps D, F and H contained a neo selection cassette, whereas those of steps E and G contained a hyg selection cassette, and therefore alternated between G418 and hygromycin selection. Targeting in step D was performed using 3hV HTargeting for steps E through I was assayed by loss of a unique PCR product spanning the distal loxP site of the hybrid allele. Targeting for steps E through I was assayed by loss of the previous selection cassette. In the last step (step I, FIG. 2B), the neo selection cassette flanked by Frt sites (McLeod et al., 1986, Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns 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 Biotechnol 16:657-662). Human sequences in BACvec for steps D, E, and G were derived from each of the two parental human BACs, whereas human sequences from steps F and H were from a single BAC. Retention of human sequences was confirmed at every step using multiple probes across the inserted human sequences (as described above, e.g., Figures 3A, 3B and 3C). At each step, only clones with normal karyotype and germline potential were carried forward to the next step. ES cells from the last step were still able to contribute to the germline after nine sequential manipulations (Table 3). Mice homozygous for each of the heavy chain alleles were viable, appeared healthy, and demonstrated an essentially wild-type humoral immune system (see Example 3).

[0290] [Table 3] Immunoglobulin κ light chain variable gene locus. In an approach similar to that of the heavy chain, the κ light chain variable region was humanized in eight sequential steps by directly replacing ∼3 Mb of mouse sequence containing all the Vκ and Jκ gene segments with ∼0.5 Mb of human sequence containing the proximal human Vκ and Jκ gene segments ( FIG. 1B ; Tables 2 and 4 ).

[0291] The variable region of the human kappa light chain locus contains two nearly identical 400 kb repeats separated by an 800 kb spacer (Weichhold et al., 1993, The human immunoglobulin kappa locus consists of two copies that are organized in opposite polarity, Genomics 16:503-511). These repeats are so similar that nearly all locus diversity can be reproduced in mice by using the proximal repeats. In addition, natural human alleles of the kappa light chain locus that lack the distal repeats have been reported (Schaible et al., 1993, The immunoglobulin kappa locus: polymorphism and haplotypes of Caucasoid and non-Caucasoid individuals, Hum Genet 91:261-267). We replaced approximately 3 Mb of mouse kappa light chain variable gene sequence with approximately 0.5 Mb of human kappa light chain variable gene sequence, effectively replacing all mouse VK and JK gene segments with the proximal human VK and all human JK gene segments (Figures 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, including all VK and JK gene segments, was deleted in a three-step process before adding any human sequence. First, a neo cassette was introduced at the proximal end of the variable region (Step A, Figure 2C). Then, a hyg cassette was inserted at the distal end of the kappa locus (Step B, Figure 2C). Recombinase recognition sites (e.g., loxP) were placed again in each selection cassette so that deletion of both resistance genes together with the remaining 3 Mb of mouse VK region could be induced by CRE treatment (Step C, Figure 2C).

[0292] Using a method similar to that used for the heavy chain (see Example 1), ~150 kb of human immunoglobulin kappa light chain sequence was inserted in one step, and ~480 kb sized human genomic fragment containing the immunoglobulin kappa light chain variable region was inserted in four sequential steps (Figure 2D; Tables 2 and 4). The final hygromycin resistance gene was removed by transient FLPe expression. As with the heavy chain, after each step, targeted ES cell clones were evaluated for complete human insert, normal karyotype and germline potential. Mice homozygous for each of the kappa light chain alleles were generated and found to be healthy and of normal appearance.

[0293] [Table 4] Example 2 Generation of fully humanized mice by combining multiple humanized immunoglobulin alleles Several sites were microinjected with ES cells carrying portions of the human immunoglobulin heavy or kappa light chain variable repertoire as described in Example 1, and the resulting mice were bred to generate multiple versions of VELOCIMMUNE® mice carrying progressively larger fractions of the human germline immunoglobulin repertoire (Table 5; Figures 5A and 5B). VELOCIMMUNE® 1 (V1) mice contain 18 human V H Gene segments and all human D H and J. H VELOCIMMUNE® 2 (V2) and VELOCIMMUNE® (V3) mice contain a total of 39 Vκ gene segments, together with 16 human Vκ gene segments and all human κ gene segments. H and 30 Vκ and 80 V H and 40 Vκ, respectively. H , D H and J. HThe gene segments, as well as the genomic regions encoding the Vκ and Jκ gene segments, were completely replaced so that antibodies produced by all versions of VELOCIMMUNE® mice contain human variable regions linked to mouse constant regions. The mouse λ light chain locus remains intact in various embodiments of VELOCIMMUNE® mice and serves as a comparator for the expression efficiency of the various VELOCIMMUNE® κ light chain loci.

[0294] Mice doubly homozygous for humanization of both immunoglobulin heavy and kappa light chains were generated from a subset of alleles described in Example 1. All genotypes observed during the breeding course to generate the doubly homozygous mice occurred in approximately Mendelian ratios. Male progeny homozygous for each of the human heavy chain alleles demonstrated reduced fertility, which was attributed to the loss of mouse ADAM6 activity. The mouse heavy chain variable gene locus contains two nested functional ADAM6 genes (ADAM6a and ADAM6b). During humanization of the mouse heavy chain variable gene locus, the inserted human genomic sequence contained an ADAM6 pseudogene. Mouse ADAM6 may be required for fertility, and therefore it is possible that the absence of the mouse ADAM6 gene in the humanized heavy chain variable gene locus resulted in reduced fertility despite the presence of the human pseudogene. Examples 7-11 describe the re-engineering of the mouse ADAM6 gene into a humanized heavy chain variable gene locus and the restoration of wild-type levels of fertility in mice bearing the humanized heavy chain immunoglobulin locus.

[0295] [Table 5] Example 3 Lymphocyte populations in mice carrying humanized immunoglobulin genes Mature B cell populations in the three different versions of VELOCIMMUNE® mice were assessed by flow cytometry.

[0296] Briefly, cell suspensions from bone marrow, spleen and thymus were generated using standard methods. Cells were resuspended at 5x105 cells / mL in BD Pharmingen FACS staining buffer, blocked with anti-mouse CD16 / 32 (BD Pharmingen), stained with the appropriate antibody cocktail and fixed with BD CYTOFIX™, all according to the manufacturer's instructions. The final cell pellet was resuspended in 0.5 mL staining buffer and analyzed using BD FACSCALIBUR™ and BD CELLQUEST PRO™ software. All antibodies (BD Pharmingen) were prepared in mass dilutions / cocktails and added to a final concentration of 0.5mg / 105 cells.

[0297] Antibody cocktails for bone marrow (A-D) staining were as follows: A: anti-mouse IgMb-FITC, anti-mouse IgMa-PE, anti-mouse CD45R(B220)-APC; B: anti-mouse CD43(S7)-PE, anti-mouse CD45R(B220)-APC; C: anti-mouse CD24(HSA)-PE; anti-mouse CD45R(B220)-APC; D: anti-mouse BP-1-PE, anti-mouse CD45R(B220)-APC.

[0298] The antibody cocktails for spleen and inguinal lymph node (E-H) staining were as follows: E: anti-mouse IgMb-FITC, anti-mouse IgMa-PE, anti-mouse CD45R(B220)-APC; F: anti-mouse Ig, λ1, λ2, λ3 light chain-FITC, anti-mouse Igκ light chain-PE, anti-mouse CD45R(B220)-APC; G: anti-mouse Ly6G / C-FITC, anti-mouse CD49b(DX5)-PE, anti-mouse CD11b-APC; H: anti-mouse CD4(L3T4)-FITC, anti-mouse CD45R(B220)-PE, anti-mouse CD8a-APC. The results are shown in Figure 6.

[0299] Lymphocytes isolated from the spleens or lymph nodes of homozygous VELOCIMMUNE® mice were stained for surface expression of the markers B220 and IgM and analyzed using flow cytometry (Figure 6). B220+IgM in all versions of VELOCIMMUNE® mice tested + The size of the mature B cell population depends on the number of V H Regardless of the number of gene segments, the gene sequences were virtually identical to those of wild-type mice. In addition, mice containing homozygous hybrid humanized immunoglobulin heavy chain loci also possessed a normal mouse immunoglobulin kappa light chain locus, but only three V H Even mice lacking the gene segment or containing homozygous hybrid humanized kappa light chain and normal mouse immunoglobulin heavy chain loci have normal numbers of B220+IgM in their peripheral compartments. + cells (not shown). These results indicate that chimeric loci with human variable gene segments and mouse constant regions can fully assemble into mature B cell compartments. Furthermore, the number of variable gene segments in either the heavy or kappa light chain loci, and therefore the theoretical diversity of the antibody repertoire, does not correlate with the ability to generate a wild-type population of mature B cells. In contrast, mice with randomly integrated complete human immunoglobulin transgenes and inactivated mouse immunoglobulin loci have reduced numbers of B cells in these compartments, with the severity of the defect 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 provides evidence that the "in situ gene humanization" strategy results in fundamentally different functional outcomes than the randomly integrated transgenes achieved in the "knockout plus transgenic" approach.

[0300] Allelic Exclusion and Locus Selection The ability to maintain allelic exclusion was investigated in mice heterozygous for different versions of the humanized immunoglobulin heavy chain locus.

[0301] Humanization of the immunoglobulin locus was performed in an F1 ES line (F1H4, Valenzuela et al., 2003) derived from 129S6 / SvEvTac and C57BL / 6NTac heterozygous embryos. Human heavy chain germline variable gene sequences are targeted to the 129S6 allele. This allele carries the IgMa haplotype, whereas the unmodified mouse C576BL / 6N allele has the IgMb haplotype. These allelic forms of IgM can be distinguished by flow cytometry using antibodies specific for polymorphisms found in the IgMa or IgMb alleles. As shown in Figure 6 (bottom row), B cells identified in mice heterozygous for each version of the humanized heavy chain locus express only a single allele, either IgMa (humanized allele) or IgMb (wild type allele). This provides evidence that the mechanisms involved in allelic exclusion are intact in VELOCIMMUNE® mice. In addition, the relative number of B cells positive for the humanized allele (IgMa) was significantly higher than that of the VELOCIMMUNE® mice. H The number of gene segments is roughly proportional to the number of gene segments. The humanized immunoglobulin locus consists of 18 human V H VELOCIMMUNE®1 heterozygous mice carrying gene segments expressed in approximately 30% of B cells and 39 and 80 human V HIn VELOCIMMUNE® 2 and 3 (not shown) heterozygous mice carrying each gene segment, it is expressed in 50% of B cells. Notably, the ratio of cells expressing humanized mouse alleles to cells expressing wild-type mouse alleles (0.5 for VELOCIMMUNE® 1 mice and 0.9 for VELOCIMMUNE® 2 mice) is greater than the ratio of the number of variable gene segments contained at the humanized locus to the number of variable gene segments contained at the wild-type locus (0.2 for VELOCIMMUNE® 1 mice and 0.4 for VELOCIMMUNE® 2 mice). This indicates that the probability of allele selection is intermediate between random selection of one or the other chromosome and random selection of any particular V-segment RSS. Furthermore, there may be some, but not all, B cells in which one allele becomes available for recombination, completes the process, shuts off recombination, and then the other allele becomes available. In addition, the uniform distribution of cells with surface IgM (sIgM) 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 normal levels. 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 et al., 1994; Tuaillon et al., 1993, Human immunoglobulin heavy-chain minilocus recombination in transgenic mice: gene-segment use in mu and gamma transcripts, PNAS USA 90:3720-3724).This provides further evidence that the immunoglobulins produced by VELOCIMMUNE® mice are functionally distinct from those produced by randomly integrated transgenes in mice generated by a "knockout plus transgenic" approach.

[0302] In 129S6 or C57BL / 6N, polymorphisms in the Cκ region are not available to investigate allelic exclusion of the humanized κ light chain locus relative to the non-humanized κ light chain locus. However, because all VELOCIMMUNE® mice have a wild-type mouse λ light chain locus, it is possible to observe whether rearrangement and expression of the humanized κ light chain locus can prevent mouse λ light chain expression. The ratio of the number of cells expressing humanized κ light chain to the number of cells expressing mouse λ light chain was relatively unchanged in VELOCIMMUNE® mice compared to wild-type mice, regardless of the number of human Vκ gene segments inserted into the κ light chain locus (Figure 6, third row from the top). In addition, there was no increase in the number of double-positive (κ+λ) cells, indicating that productive recombination at the hybrid κ light chain locus results in appropriate suppression of recombination at the mouse λ light chain locus. In contrast, mice containing a randomly integrated kappa light chain transgene and an inactivated mouse kappa light chain locus - but not the wild-type mouse lambda light chain locus - show a dramatic increase in the lambda / kappa ratio (Jakobovits, 1998). This means that the introduced kappa light chain transgene is not fully functional in such mice. This further supports the different functional outcomes observed in immunoglobulins produced by VELOCIMMUNE® mice compared to those produced by "knockout plus transgenic" mice.

[0303] B cell development. Because the mature B cell populations in VELOCIMMUNE® mice resemble those in wild-type mice (as described above), it is possible that defects in early B cell differentiation could be compensated for by expansion of the mature B cell population. The various stages of B cell differentiation were investigated by analyzing the B cell populations using flow cytometry. Table 6 shows the ratio of the fraction of cells of each B cell lineage defined by FAC in VELOCIMMUNE® mice compared to wild-type littermates using specific cell surface markers.

[0304] Early B cell development occurs in the bone marrow, and different B cell differentiation stages are characterized by changes in the type and amount of cell surface marker expression. These differences in surface expression correlate with molecular changes that occur at the immunoglobulin locus inside the cell. While the transition from pro-B to pre-B cells requires successful rearrangement and expression of functional heavy chain proteins, the transition from pre-B to mature B stage is governed by the correct rearrangement and expression of kappa or lambda light chains. Thus, inefficient transitions between B cell differentiation stages can be detected by changes in the associated population of B cells at a given stage.

[0305] [Table 6] There was a significant defect in B cell differentiation in both VELOCIMMUNE® mice. No defects were observed. In VELOCIMMUNE® mice, introduction of human heavy chain gene segments does not appear to affect the pro-B to pre-B transition, and introduction of human kappa light chain gene segments does not affect the pre-B to B transition. This demonstrates that "reverse chimeric" immunoglobulin molecules carrying human variable regions and mouse constant regions (constants) function normally in conjunction with B cell signaling and co-receptor molecules to result in proper B cell differentiation in the mouse environment. In contrast, in mice containing randomly integrated immunoglobulin transgenes and inactivated endogenous heavy or kappa light chain loci, the balance between different populations during B cell differentiation is perturbed to various degrees (Green and Jakobovits, 1998).

[0306] Example 4 Variable gene repertoires in humanized immunoglobulin mice. Human variable gene segment usage in the humanized antibody repertoire of VELOCIMMUNE® mice was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) of human variable regions from multiple sources, including splenocytes and hybridoma cells. Variable region sequences, gene segment usage, somatic hypermutation, and junction diversity of rearranged variable region gene segments were determined.

[0307] Briefly, total RNA was extracted from 1 × 107–2 × 107 splenocytes or approximately 104–105 hybridoma cells using TRIZOL™ (Invitrogen) or Qiagen RNEASY™ Mini Kit (Qiagen) and primed with mouse constant region-specific primers using the SUPERSCRIPT™ III One-Step RT-PCR System (Invitrogen). Reactions were performed with 2–5 μL of RNA from each sample using the 3′ constant region-specific primers described above individually paired with pooled leader primers for each family of human variable regions for both heavy and kappa light chains. Reagent and primer volumes, and RT-PCR / PCR conditions were performed according to the manufacturer's instructions. Primer sequences were based on multiple sources (Wang and Stollar, 2000, Human immunoglobulin variable region gene analysis by single cell RT-PCR, J Immunol Methods 244:217-225; Ig-primer sets, Novagen). Where appropriate, a second nested PCR reaction was performed using the pooled family-specific framework primers and the same mouse 3' immunoglobulin constant-specific primer used in the first reaction. An aliquot (5 μL) from each reaction was analyzed by agarose electrophoresis, and reaction products were purified from the agarose using the MONTAGE™ Gel Extraction Kit (Millipore). Purified products were cloned using the TOPO™ TA Cloning System (Invitrogen) and isolated by electroporation. H The plasmids were transformed into 10β E. coli cells. Individual clones were selected from each transformation reaction and grown overnight at 37°C in 2 mL LB broth cultures with antibiotic selection. Plasmid DNA was purified from the bacterial cultures by a kit-based approach (Qiagen).

[0308] Immunoglobulin variable gene usage. Plasmid DNA from both heavy and kappa light chain clones was sequenced with either T7 or M13 reverse primers using an ABI 3100 Genetic Analyzer (Applied Biosystems). Raw sequence data was analyzed using SEQUENC H The sequences were imported into ER™ (v4.5, Gene Codes). Each sequence was assembled into a contig and aligned to human immunoglobulin sequences using the IMGT V-Quest (Brochet et al., 2008, IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized VJ and VDJ sequence analysis, Nucleic Acids Res 36:W503-508) search function to identify human VDJ sequences. H , D H , J H and Vκ, Jκ segment usage were identified. Sequences were compared to germline sequences for somatic hypermutation and recombination junction analysis.

[0309] The first heavy chain modification (3hV) was achieved by RAG complementation (Chen et al., 1993, RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development, PNAS USA 90:4528-4532). HMice were generated from ES cells containing the -CRE hybrid allele (bottom of FIG. 2A) and cDNA was prepared from splenocyte RNA. The cDNA was amplified using a primer set (above) specific for the predicted chimeric heavy chain mRNA that would result from V(D)J recombination within the inserted human gene segments and subsequent splicing into either the mouse IgM or IgG constant domains. Sequences derived from these cDNA clones (not shown) demonstrated that correct V(D)J recombination had occurred within the human variable gene sequences, that the rearranged human V(D)J gene segments had correctly spliced ​​in frame into the mouse constant domains, and that class switch recombination had occurred. Further sequence analysis of the mRNA products of the subsequent hybrid immunoglobulin loci was performed.

[0310] In a similar experiment, B cells from non-immunized wild-type and VELOCIMMUNE® mice were separated by flow cytometry based on surface expression of B220 and IgM or IgG. + Alternatively, surface IgG+ (sIgG+) cells were pooled and subjected to RT-PCR amplification and cloning (as above) followed by V H and Vκ sequences were obtained. Representative gene usage in a set of RT-PCR amplified cDNA from non-immune VELOCIMMUNE® 1 mice (Table 7) and VELOCIMMUNE® 3 mice (Table 8) was recorded (*defective RSS; †missing or pseudogene). Asterisk: gene segment with defective RSS. †: gene segment missing or pseudogene.

[0311] [Table 7-1]

[0312] [Table 7-2]

[0313] [Table 8-1]

[0314] [Table 8-2] As shown in Tables 7 and 8, functional human V H , D H , J H Nearly all of the V, Vk, and Jk gene segments are utilized. Several of the functional variable gene segments described that were not detected in the VELOCIMMUNE® mice in this study have been reported to possess defective recombination signal sequences (RSSs) and would therefore not be expected to be expressed (Feeney, 2000, Factors that influence formation of B cell repertoire, Immunol Res 21:195-202). Analysis of several other sets of immunoglobulin sequences from various VELOCIMMUNE® mice, isolated from both naive and immune repertoires, showed usage of these gene segments, albeit at lower frequencies (data not shown). The aggregate gene usage data indicates that VELOCIMMUNE® mice contain all functional human Vk and Jk gene segments. H , D H , J H , Vκ and Jκ gene segments were observed in a variety of naive and immune repertoires (data not shown). HThe 7-81 gene segment has been identified in analysis of the human heavy chain locus sequence (Matsuda et al., 1998, The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus, J Exp Med 188:2151-2162), but is absent in VELOCIMMUNE® mice, as confirmed by resequencing of the entire VELOCIMMUNE® 3 mouse genome.

[0315] It is known that antibody heavy and light chain sequences exhibit exceptional variability, especially in short polypeptide segments within the rearranged variable domains. These regions are known as hypervariable regions or complementarity determining regions (CDRs) and create the antigen binding site within the structure of the antibody molecule. The intervening polypeptide sequences are called framework regions (FRs). There are three CDRs (CDR1, CDR2, CDR3) and four FRs (FR1, FR2, FR3, FR4) in both the heavy and light chains. One CDR, CDR3, is a CDR that is specific to the V H , D H and J. H They are unique in that they are created by recombination of both gene segments and Vκ and Jκ gene segments, generating a significant amount of repertoire diversity before antigen encounter. This junction is not precise due to both nucleotide deletions by exonuclease activity and non-template encoded addition by terminal deoxynucleotidyl transferase (TdT), thus allowing novel sequences as a result of the recombination process. FRs can exhibit substantial somatic mutations due to the high variability of the variable region as a whole, however, variability is not evenly distributed throughout the variable region. CDRs are highly variable regions concentrated and localized on the surface of the antibody molecule that allow antigen binding. Heavy and light chain sequences around the CDR3 junctions of selected antibodies from VELOCIMMUNE® mice, demonstrating junctional diversity, are shown in Figures 7A and 7B, respectively.

[0316] As shown in FIG. 7A, non-template encoded nucleotide additions (N-additions) were found to mediate the V H -D H Junction and D H -J H Both of these junctions were observed, indicating the correct function of TdT on the human segment. H , D H and J. H The end points of the segments compared to their germline counterparts indicate that endonuclease activity has also occurred. Unlike the heavy chain locus, human kappa light chain rearrangements show little or no TdT addition at the CDR3 formed by recombination of Vkappa and Jkappa segments (Figure 7B). This is expected to be due to the lack of TdT expression in mice during light chain rearrangement at the pre-B to B cell transition. The diversity observed at the CDR3 of rearranged human Vkappa regions is primarily introduced by exonuclease activity during recombination events.

[0317] Somatic hypermutation. Further diversity is added to the variable regions of rearranged immunoglobulin genes during the germinal center reaction by a process called somatic hypermutation. B cells expressing somatically mutated variable regions compete with other B cells for access to antigens presented by follicular dendritic cells. Those B cells with higher affinity for antigen further proliferate and are class switched before exiting to the periphery. Hence, B cells expressing switched isotypes have typically encountered antigen and undergone the germinal center reaction and have an increased number of mutations compared to naive B cells. Furthermore, mainly naive sIgM + Variable region sequences from B cells would be expected to have relatively fewer mutations than variable sequences from sIgG+ B cells that have been subjected to antigen selection.

[0318] sIgM from non-immunized VELOCIMMUNE® mice + or random V from sIgG+ B cells or sIgG+ B cells from immunized mice HSequences from VK or VK clones were compared to their germline variable gene segments and changes relative to the germline sequence were annotated. The resulting nucleotide sequences were computer translated and mutations resulting in amino acid changes were also annotated. Data was collated from all variable regions and the percent change at a given position was calculated (Figure 8).

[0319] As shown in FIG. 8, human heavy chain variable regions derived from sIgG+ B cells from non-immunized VELOCIMMUNE® mice were significantly higher than sIgM from the same splenocyte pool. + Heavy chain variable regions from immunized mice show more nucleotide and even more changes compared to B cells. The number of changes is increased in the complementarity determining regions (CDRs) compared to the framework regions, indicating antigen selection. The corresponding amino acid sequences from human heavy chain variable regions also show a significantly higher number of mutations in IgG compared to IgM, and even higher in immunized IgG. These mutations also appear to be more frequent within the CDRs compared to the framework sequences, suggesting that the antibodies are antigen selected in vivo. A similar increase in the number of nucleotide and amino acid mutations is found in the Vκ sequences derived from IgG+ B cells from immunized mice.

[0320] The gene usage and somatic hypermutation frequency observed in VELOCIMMUNE® mice demonstrate that in these mice, essentially all gene segments present can rearrange to form fully functional reverse chimeric antibodies. Furthermore, VELOCIMMUNE® antibodies are sufficiently committed within the mouse immune system to undergo affinity selection and maturation to generate fully mature human antibodies capable of effectively neutralizing their target antigens. VELOCIMMUNE® mice are able to mount robust immune responses to multiple classes of antigens, resulting in the availability of a broad range of human antibodies that are both high affinity and suitable for therapeutic use (data not shown).

[0321] Example 5 Analysis of lymphatic structure and serum isotypes The gross structure of spleen, inguinal lymph nodes, Peyer's patches and thymus of tissue samples from wild-type or VELOCIMMUNE® mice stained with H&E was examined by light microscopy. Levels of immunoglobulin isotypes in serum collected from wild-type and VELOCIMMUNE® mice were analyzed using LUMINEX™ technology.

[0322] Lymphoid Organ Structure. The structure and function of lymphoid tissues depend in part on the correct development of hematopoietic cells. Defects in B cell development or function may manifest as alterations in the structure of lymphoid tissues. Analysis of stained tissue sections identified no significant differences in the appearance of secondary lymphoid organs between wild-type and VELOCIMMUNE® mice (data not shown).

[0323] Serum immunoglobulin levels. Expression levels of each isotype are similar in wild-type and VELOCIMMUNE® mice (Figures 9A, 9B and 9C). This demonstrates that humanization of variable gene segments has no apparent adverse effects on class switching or immunoglobulin expression and secretion, thus apparently maintaining all endogenous mouse sequences required for these functions.

[0324] Example 6 Immunity and antibody production in humanized immunoglobulin mice Different versions of VELOCIMMUNE® mice were immunized with antigens to investigate the humoral response to foreign antigen challenge.

[0325] Immunization and Hybridoma Development. VELOCIMMUNE® and wild-type mice can be immunized with antigen in the form of protein, DNA, a combination of DNA and protein, or cells expressing the antigen. Typically, animals are boosted every 3 weeks for a total of 2 to 3 times. After each antigen boost, serum samples are collected from each animal and analyzed for antigen-specific antibody responses by serum titration. Prior to fusion, mice were given a final pre-fusion boost of 5 μg protein or DNA by intraperitoneal and / or intravenous injection as appropriate. Splenocytes are harvested and fused to Ag8.653 myeloma cells in an electrofusion chamber according to the manufacturer's suggested protocol (Cyto Pulse Sciences Inc., Glen Burnie, MD). After 10 days of 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). Alternatively, antigen-specific B cells can be directly isolated from immunized VELOCIMMUNE® mice and screened using standard techniques, including those described herein, to obtain human antibodies specific to the antigen of interest (see, e.g., U.S. Patent Application Publication No. 2007 / 0280945 A1, which reference is incorporated herein by reference in its entirety).

[0326] Serum titer determination. To monitor the animal anti-antigen serum response, serum samples are collected approximately 10 days after each boost and the titers are determined using antigen-specific ELISA. Briefly, Nunc MAXISORP™ 96-well plates are coated with 2 μg / mL of antigen overnight at 4° C. and blocked with bovine serum albumin (Sigma, St. Louis, MO). Three-fold serial dilutions of serum samples are allowed to bind to the plates for 1 hour at room temperature. The plates are then washed with PBS containing 0.05% Tween-20 and bound IgG is detected using HRP-conjugated goat anti-mouse Fc (Jackson Immuno Research Laboratories, Inc., West Grove, PA) for total IgG titers or biotin-labeled isotype-specific or light chain-specific polyclonal antibodies (Southern Biotech Inc.) for isotype-specific titers, respectively. For biotin-labeled antibodies, HRP-conjugated streptavidin (Pierce, Rockford, IL) is added after plate washing. All plates are developed using a colorimetric substrate such as BD OPTEIA™ (BD Biosciences Pharmingen, San Diego, CA). After stopping the reaction with 1 M phosphoric acid, the light absorbance at 450 nm is recorded and the data is analyzed using PRISM™ software from Graph Pad. The dilution required to obtain a signal twice the background signal is defined as the titer.

[0327] In one experiment, VELOCIMMUNE® mice were immunized with human interleukin-6 receptor (hIL-6R). A representative set of serum titers for VELOCIMMUNE® and wild-type mice immunized with hIL-6R are shown in Figures 10A and 10B.

[0328] VELOCIMMUNE® and wild-type mice mounted robust responses to IL-6R with similar titer ranges (Figure 10A). Several mice from the VELOCIMMUNE® and wild-type cohorts reached maximum responses after a single antigen boost. These results indicate that the immune response strength and kinetics to this antigen were similar in VELOCIMMUNE® and wild-type mice. These antigen-specific antibody responses were further analyzed to investigate the specific isotypes of antigen-specific antibodies found in the serum. Both the VELOCIMMUNE® and wild-type groups mounted primarily IgG1 responses (Figure 10B). This suggests that class switching during the humoral response was similar in each type of mouse.

[0329] Affinity Determination of Antibody Binding to Antigen in Solution To determine antibody binding affinity to an antigen, an ELISA-based solution competition assay is typically designed.

[0330] Briefly, antibodies in conditioned medium are premixed with a dilution series of antigen protein ranging from 0 to 10 mg / mL. The antibody-antigen mixture solution is then incubated for 2 to 4 hours at room temperature until binding equilibrium is reached. The amount of free antibody in the mixture is then measured using a quantitative sandwich ELISA. 96-well MAXISORB™ plates (VWR, West Chester, PA) are coated overnight at 4°C with 1 μg / mL of antigen protein in PBS solution, followed by non-specific blocking with BSA. The antibody-antigen mixture solution is then transferred to these plates, followed by incubation for 1 hour. The plates are then washed with wash buffer, and plate-bound antibodies are detected with HRP-conjugated goat anti-mouse IgG polyclonal antibody reagent (Jackson Immuno Research Lab) and developed using a colorimetric substrate such as BD OPTEIA™ (BD Biosciences Pharmingen, San Diego, CA). After stopping the reaction with 1M phosphoric acid, the optical absorbance at 450 nm is recorded and the data is analyzed using PRISM™ software from Graph Pad. The dependence of the signal on the concentration of antigen in solution is analyzed by a four-parameter fit analysis and reported as IC50 (the antigen concentration required to achieve a 50% reduction in the signal from the antibody sample without the antigen in solution).

[0331] In one experiment, VELOCIMMUNE® mice were immunized with hIL-6R (as described above). Figures 11A and 11B show a representative set of affinity measurements for anti-hIL6R antibodies from VELOCIMMUNE® and wild-type mice.

[0332] After the immunized mice were given a third antigen boost, serum titers were determined using an ELISA assay. Splenocytes were isolated from selected wild-type and VELOCIMMUNE® mouse cohorts, fused with Ag8.653 myeloma cells to form hybridomas, and grown under selection (as described above). Of the total 671 anti-IL-6R hybridomas produced, 236 were found to express antigen-specific antibodies. Media collected from antigen-positive wells was used to determine antibody affinity for binding to antigen using a solution competition ELISA. Antibodies from VELOCIMMUNE® mice show a wide range of affinities for binding to antigen in solution (Figure 11A). Furthermore, 49 of the 236 anti-IL-6R hybridomas were found to block IL-6 binding to the receptor in an in vitro bioassay (data not shown). Furthermore, these 49 anti-IL-6R blocking antibodies showed a range of high solution affinities similar to those of blocking antibodies derived from parallel immunization of wild-type mice (Figure 11B).

[0333] Example 7 Construction of mouse ADAM6 targeting vector Due to the replacement of the mouse immunoglobulin heavy chain variable gene locus with the human immunoglobulin heavy chain variable gene locus, the initial version of the VELOCIMMUNE® mouse lacks expression of the mouse ADAM6 gene. In particular, male VELOCIMMUNE® mice show a marked reduction in fertility. Therefore, to rescue the fertility defect, the ability to express ADAM6 was re-engineered into the VELOCIMMUNE® mouse.

[0334] Using VELOCIGENE® genetic engineering technology (described above), a targeting vector for insertion of the mouse ADAM6a and ADAM6b genes into the humanized heavy chain locus was constructed to modify bacterial artificial chromosome (BAC) 929d24 obtained from Dr. Frederick Alt (Harvard University). A genomic fragment containing the mouse ADAM6a and ADAM6b genes and the human V at the humanized heavy chain locus were inserted into the bacterial artificial chromosome (BAC) 929d24 obtained from Dr. Frederick Alt (Harvard University). H1-2 gene segment and human V H The 929d24 BAC DNA was engineered to contain a hygromycin cassette for targeted deletion of the human ADAM6 pseudogene (hADAM6Ψ) between the 6-1 gene segments (Figure 12).

[0335] First, a genomic fragment containing the mouse ADAM6b gene and about 800 bp of upstream (5') and about 4800 bp of downstream (3') sequences was subcloned from the 929d24 BAC clone. A second genomic fragment containing the mouse ADAM6a gene and about 300 bp of upstream (5') and about 3400 bp of downstream (3') sequences was subcloned separately from the 929d24 BAC clone. The two genomic fragments containing the mouse ADAM6b and ADAM6a genes were ligated to a hygromycin cassette flanked by Frt recombination sites to generate a targeting vector (mouse ADAM6 targeting vector, FIG. 12; SEQ ID NO: 3). Different restriction enzyme recognition sites were engineered on the 5' end of the targeting vector following the mouse ADAM6b gene and on the 3' end of the targeting vector following the mouse ADAM6a gene for ligation into the humanized heavy chain locus (bottom of FIG. 12).

[0336] The BAC clones containing the replacement of the mouse heavy chain variable gene locus with the human heavy chain variable gene locus contain the human V of the humanized locus for subsequent ligation of the mouse ADAM6 targeting vector. H 1-2 gene segment and human V H Another modification was made that includes a human ADAM6 pseudogene (hADAM6Ψ) located between the 6-1 gene segments (Figure 13).

[0337] Briefly, a neomycin cassette flanked by loxP recombination sites was inserted into human V H 3' of the 1-2 gene segment (5' to hADAM6Ψ) and human V HThe 6-1 gene segment was engineered to contain a homology arm containing human genomic sequences at the 5' (3' to hADAM6Ψ; see FIG. 13, center) position. The insertion site of this targeting construct was located approximately 1.3 kb 5' and approximately 350 bp 3' to the human ADAM6 pseudogene. This targeting construct also contained the same restriction enzyme recognition sites as the mouse ADAM6 targeting vector to allow for subsequent BAC ligation between the modified BAC clone containing the deletion of the human ADAM6 pseudogene and the mouse ADAM6 targeting vector.

[0338] After digestion of the BAC DNA from both constructs, the genomic fragments were ligated together to construct an engineered BAC clone containing a humanized heavy chain locus containing an ectopically placed genomic sequence containing mouse ADAM6a and ADAM6b nucleotide sequences. The final targeting construct for deletion of the human ADAM6 gene within the humanized heavy chain locus and insertion of mouse ADAM6a and ADAM6b sequences into ES cells contained, 5' to 3', the human V H a 5' genomic fragment containing approximately 13 kb of human genomic sequence 3' of the 1-2 gene segment; approximately 800 bp of mouse genomic sequence downstream of the mouse ADAM6b gene; the mouse ADAM6b gene; approximately 4800 bp of genomic sequence upstream of the mouse ADAM6b gene; a 5' Frt site; a hygromycin cassette; a 3' Frt site; approximately 300 bp of mouse genomic sequence downstream of the mouse ADAM6a gene; the mouse ADAM6a gene; approximately 3400 bp of mouse genomic sequence upstream of the mouse ADAM6a gene; and a human V H The 3' genomic fragment contained approximately 30 kb of human genomic sequence 5' of the 6-1 gene segment (Figure 13, bottom).

[0339] The engineered BAC clone (above) was used to electroporate mouse ES cells containing humanized heavy chain locus to generate modified ES cells containing ectopic mouse genomic sequences, including mouse ADAM6a and ADAM6b sequences, within the humanized heavy chain locus. Positive ES cells containing ectopic mouse genomic fragments within the humanized heavy chain locus were identified by quantitative PCR assay using TAQMAN™ probes (Lie and Petropoulos, 1998, Advances in quantitative PCR technology: 5'nuclease assays, Curr Opin Biotechnol 9(1):43-48). The upstream and downstream regions outside the modified portion of the humanized heavy chain locus were confirmed by PCR using primers and probes located within the modified region to confirm the presence of ectopic mouse genomic sequences within the humanized heavy chain locus and the presence of the hygromycin cassette. The nucleotide sequence spanning the upstream insertion point includes the following, which shows the human heavy chain genomic sequence upstream of the insertion point and an I-CeuI restriction enzyme recognition site (below in brackets) adjacent and linked to the mouse genomic sequence present at the insertion point: (CCAGCTTCAT TAGTAATCGT TCATCTGTGG TAAAAAGGCA GGATTTGAAG CGATGGAAGA TGGGAGTACG GGGCGTTGGA AGACAAAGTG CCACACAGCG CAGCCTTCGT CTAGACCCCC GGGCTAACTA TAACGGTCCT AAGGTAGCGA G)GGGATGACAG ATTCTCTGTT CAGTGCACTC AGGGTCTGCC TCCACGAGAA TCACCATGCC CTTTCTCAAG ACTGTGTTCT GTGCAGTGCC CTGTCAGTGG (SEQ ID NO: 4).The nucleotide sequence spanning the 3' end of the target region downstream of the insertion point includes the following, which shows mouse genomic sequence with a PI-SceI restriction enzyme recognition site (below in brackets) adjacently linked to human heavy chain genomic sequence downstream of the insertion point: (AGGGGTCGAG GGGGAATTTT ACAAAGAACA AAGAAGCGGG CATCTGCTGA CATGAGGGCC GAAGTCAGGC TCCAGGCAGC GGGAGCTCCA CCGCGGTGGC GCCATTTCAT TACCTCTTTC TCCGCACCCG ACATAGATAAAGCTT)ATCCCCCACC AAGCAAATCC CCCTACCTGG GGCCGAGCTT CCCGTATGTG GGAAAATGAA TCCCTGAGGT CGATTGCTGC ATGCAATGAA ATTCAACTAG (SEQ ID NO:5).

[0340] The above-described target ES cells were used as donor ES cells and introduced into 8-cell stage mouse embryos by VELOCIMOUSE® mouse engineering method (see, for example, U.S. Patent Nos. 7,6598,442, 7,576,259 and 7,294,754). Mice carrying humanized heavy chain loci containing ectopic mouse genomic sequences including mouse ADAM6a and ADAM6b sequences were identified by genotyping using a modified method of allele assay (Valenzuela et al., 2003) that detects the presence of mouse ADAM6a and ADAM6b genes in the humanized heavy chain locus.

[0341] Mice carrying a humanized heavy chain locus containing mouse ADAM6a and ADAM6b genes were bred to an FLPe deleter mouse line (see, e.g., Rodriguez et al., 2000, High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nature Genetics 25:139-140) to remove any Frt'ed hygromycin cuts introduced by the targeting vector that are not removed at the ES cell stage or in the embryo. Optionally, the hygromycin cassette was retained in the mice.

[0342] The pups were genotyped and pups heterozygous for the humanized heavy chain locus containing an ectopic mouse genomic fragment including mouse ADAM6a and ADAM6b sequences were selected for characterization of mouse ADAM6 gene expression and fertility.

[0343] Example 8 Characterization of ADAM6 rescue mice Flow cytometry. Homozygous (H) for the human heavy and human kappa light chain variable gene loci. + / + κ + / + Three 25-week-old mice homozygous for human heavy and human kappa light chains (H) harboring ectopic mouse genomic fragments encoding the mouse ADAM6a and ADAM6b genes within both alleles of the human heavy chain locus. + / + A6 res κ + / + ) three mice aged 18–20 weeks were sacrificed for identification and analysis of lymphocyte cell populations by FACs on a BD LSR II System (BD Bioscience). Lymphocytes were gated for specific cell lineages and analyzed for development through various stages of B cell development. Tissues collected from animals included blood, spleen and bone marrow. Blood was collected into BD microtainer tubes (BD Biosciences) containing EDTA. Bone marrow was collected from femurs by flushing with complete RPMI medium supplemented with fetal bovine serum, sodium pyruvate, HEPES, 2-mercaptoethanol, non-essential amino acids and gentamicin. Red blood cells from blood, spleen and bone marrow preparations were lysed with an ammonium chloride-based lysis buffer (e.g., ACK lysis buffer) and then washed with complete RPMI medium.

[0344] To stain cell populations, 1x106 cells from various tissue sources were incubated with anti-mouse CD16 / CD32 (2.4G2, BD Biosciences) for 10 min on ice and then labeled with one or a combination of the following antibody cocktails for 30 min on ice:

[0345] Bone marrow: anti-mouse FITC-CD43 (1B11, BioLegend), PE-ckit (2B8, BioLegend), PeCy7-IgM (II / 41, eBioscience), PerC P-Cy5.5-IgD (11-26c.2a, BioLegend), APC-eFluor780-B220 (RA3-6B2, eBioscience), A700-CD19 (1D3, BD Biosciences).

[0346] Peripheral blood and spleen: anti-mouse FITC-κ (187.1, BD Biosciences), PE-λ (RML-42, BioLegend), PeCy7-IgM (II / 41, eBioscience), PerCP-Cy5.5-IgD (11-26c.2a, BioLegend), APC-CD3 (145-2C11, BD), A700-CD19 (1D3, BD), APC-eFluor780-B220 (RA3-6B2, eBioscience). After incubation with labeled antibodies, cells were washed and fixed with 2% formaldehyde. Data collection was performed on an LSRII flow cytometer and analyzed with FlowJo (Treestar, Inc.). Representative H + / + κ + / + and H + / + A6 res κ + / + The results from mice are shown in Figures 14-18.

[0347] The result is H. + / + A6 res κ + / + Mouse B cells express H in the bone marrow and peripheral compartments. + / + κ + / + We demonstrate that they progress through the stages of B cell development in a manner similar to that in mice and that they display normal maturation patterns once in the periphery. + / + A6 res κ + / + The mouse is H + / + κ + / + Increased CD43 compared to mice int CD19 + The cell population was clearly shown (Figure 16B). + / + A6 res κ + / + Enhancement of IgM expression in mice can be shown from a humanized heavy chain locus containing an ectopic mouse genomic fragment containing mouse ADAM6a and ADAM6b sequences. + / + A6 res κ + / + The B and T cell populations of the mice were normal and + / + κ + / + It appears to be similar to mice.

[0348] Characterization of testicular morphology and sperm. To determine whether infertility in mice with a humanized immunoglobulin heavy chain variable locus was due to defects in testicular and / or sperm production, testicular morphology and epididymal sperm content were investigated.

[0349] Briefly, two groups (n=5 per group; group 1: mice homozygous for human heavy and kappa light chain variable gene loci, H + / + κ + / + Group 2: mice heterozygous for the human heavy chain variable gene locus and homozygous for the kappa light chain variable gene locus, H + / - κ + / + ) were dissected along with intact epididymides and weighed. The specimens were then fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) stain. Testis sections (two testes per mouse, 20 in total) were examined for defects in morphology and evidence of sperm production, while epididymal sections were examined for the presence of sperm.

[0350] In this experiment, H + / + κ + / + Mouse and H + / - κ + / + No differences in testis weight or morphology were observed between the mice. Sperm were observed in both the testes and epididymis of all genotypes. These results confirm that the absence of mouse ADAM6a and ADAM6b genes does not result in detectable changes in testis morphology, and that sperm are produced in the presence and absence of these two genes in mice. Thus, male H + / + κ + / + Fertility defects in mice are unlikely to be due to low sperm production.

[0351] Sperm motility and migration. Mice lacking other ADAM gene family members are infertile due to defects in sperm motility or migration. Sperm migration, defined as the ability of sperm to progress from the uterus to the oviduct, is normally required for fertilization in mice. To determine whether deletion of mouse ADAM6a and ADAM6b affects this process, we performed a multivariate analysis of mouse ADAM6a and ADAM6b-dependent chromosomes. + / + κ + / + Sperm migration and motility were assessed in mice.

[0352] Briefly, (1) mice heterozygous for the human heavy chain variable gene locus and homozygous for the human kappa light chain variable gene locus (H + / - κ + / + (2) mice homozygous for the human heavy chain variable gene locus and homozygous for the human kappa light chain variable gene locus (H + / + κ + / + (3) mice homozygous for the human heavy chain variable gene locus and homozygous for wild-type kappa light chains (H + / + mκ); and (4) sperm were obtained from the testes of wild-type C57BL / 6 mice (WT). Examination revealed no significant abnormalities in sperm count or overall sperm motility. Cumulus dispersal was observed in all mice, indicating that each sperm sample was able to penetrate cumulus cells and bind the zona pellucida in vitro. These results are consistent with the H + / + κ + / + It is established that the mice have sperm that are able to penetrate the cumulus cumulus and bind the zona pellucida.

[0353] In vitro fertilization (IVF) of mouse eggs was performed using sperm from the above mice. + / + κ + / + A slightly lower number of cleaved embryos was observed in mice, and a reduced number of sperm bound to the eggs was also observed. + / + κ + / + It is established that sperm from mice, when exposed to eggs, are able to penetrate the cumulus cumulus and bind the zona pellucida.

[0354] In another experiment, H + / + κ + / + The performance of sperm from the mice was determined in a sperm migration assay.

[0355] Briefly, the first group of superovulated female mice (n=5) was cultured in H + / + κ + / + A second group of superovulated female mice (n = 5) were cultured at 4 °C for 12 h. + / - κ + / + Males (n=5) were prepared. These mating pairs were observed for mating, and 5-6 hours after mating, the uterus and associated oviducts were removed and flushed from all females for analysis. The flush solution was checked for eggs to verify ovulation and sperm counts were obtained. Sperm migration was assessed 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 evidence of ovulation. Second, the oviducts were left attached to the uterus, and both tissues were fixed, embedded in paraffin, sectioned, and stained (as above). Sections were examined for the presence of sperm in the uterus as well as in both oviducts.

[0356] Five H + / + κ + / + In females mated with males, very few sperm were found in the flush solution from the oviduct. + / - κ + / + Flushing fluid from the oviducts of females mated with males was H + / + κ + / + The sperm levels (mean, n=10 oviducts) were approximately 25-30 times higher than those present in flush solutions from the oviducts of females mated with males. + / + κ + / + and H + / + A6 res κ + / + A representative mating comparison of mice is shown in Table 9.

[0357] Histological sections of the uterus and oviduct were prepared. The sections were examined for the presence of spermatozoa in the uterus and oviduct (colliculus tubarius). Examination of the histological sections of the oviduct and uterus revealed that H + / + κ + / + In female mice mated with H mice, sperm were found in the uterus but not in the oviducts. + / + κ + / + Sections from females mated with mice revealed that sperm were not observed at the uterotubal junction (UTJ). + / - κ + / + In sections from females mated with mice, sperm were identified in the UTJ and oviducts.

[0358] These results confirm that mice lacking the ADAM6a and ADAM6b genes produce sperm that exhibit in vivo migration defects. In all cases, sperm were observed in the uterus, indicating that mating and sperm release appeared to occur normally, but little or no sperm were observed in the oviducts after mating, as measured by either sperm count or histological observation. These results confirm that mice lacking the ADAM6a and ADAM6b genes produce sperm that exhibit an inability to migrate from the uterus to the oviduct. This defect appears to result in infertility, as the sperm are unable to migrate across the uterine-tubule junction to the oviduct where the egg is fertilized. Taken together, all of these results lend support to the hypothesis that the mouse ADAM6 gene directs sperm with normal migration ability to migrate out of the uterus through the uterine-tubule junction and the oviduct, thereby gaining access to the egg and achieving the fertilization event. The mechanism by which ADAM6 achieves this may be mediated by one or both of the ADAM6 proteins, or by coordinated expression with other proteins in sperm cells, such as other ADAM proteins, as described below.

[0359] [Table 9] ADAM gene family expression. It is known that a complex of ADAM proteins is present as a complex on the surface of maturing sperm. Mice lacking other ADAM gene family members lose this complex as sperm mature and show a reduction in multiple ADAM proteins in mature sperm. To determine whether the absence of ADAM6a and ADAM6b genes similarly affects other ADAM proteins, Western blots of protein extracts from testis (immature sperm) and epididymis (maturing sperm) were analyzed to determine the expression levels of other ADAM gene family members.

[0360] In this experiment, H + / + κ + / + and H + / - κ + / + Protein extracts from groups of mice (n=4 per group) were analyzed. The results showed that the expression of ADAM2 and ADAM3 was unaffected in testis extracts. However, both ADAM2 and ADAM3 were dramatically reduced in epididymal extracts. This suggests that H + / + κ + / + We demonstrate that the absence of ADAM6a and ADAM6b in mouse semen may have a direct effect on the expression and possibly function of other ADAM proteins (e.g., ADAM2 and ADAM3) as sperm mature, suggesting that ADAM6a and ADAM6b are present on the sperm surface as part of an ADAM protein complex that may be crucial for correct sperm migration.

[0361] Example 9 Human heavy chain variable gene usage in ADAM6 rescue mice Quantitative PCR assays using TAQMAN™ probes (as described above) revealed that the mouse ADAM6a and ADAM6b genes were deleted (H + / + κ + / + ) or containing ectopic genomic fragments encoding the mouse ADAM6a and ADAM6b genes (H + / + A6 res κ+ / + Selected human heavy chain variable gene usage was determined for mice homozygous for the human heavy and kappa light chain variable gene loci, either 1) or 2) .

[0362] Briefly, mouse CD19 Microbeads (Miltenyi Biotec) were used to detect H + / + κ + / + and H + / + A6 res κ + / + CD19 from mouse spleens + B cells were purified and total RNA was purified using the RNEASY™ Mini kit (Qiagen). Genomic RNA was removed using RNase-free DNase on-column treatment (Qigaen). Approximately 200 ng of mRNA was reverse transcribed into cDNA using the First Stand cDNA Synthesis kit (Invitrogen) and then amplified with TAQMAN™ Universal PCR Master Mix (Applied Biosystems) using an ABI 7900 Sequence Detection System (Applied Biosystems). The relative expression of each gene was normalized to the expression of mouse kappa light chain constant region (mCκ). Table 10 shows the sense / antisense / TAQMAN™ MGB probe combinations used in this experiment.

[0363] [Table 10] In this study, all four human V H Expression of the gene was observed. Furthermore, the expression level was + / + κ + / + Mouse and H + / + A6 res κ + / + These results were similar between the human V H Gene (V H 3-23 and V H 1-69) and human V proximal to the modification siteH Gene (V H 1-2 and V H Both of the ADAM6a and ADAM6b sequences were able to recombine to form functionally expressed human heavy chains. These results demonstrate that the ectopic genomic fragments containing the mouse ADAM6a and ADAM6b sequences inserted into the human heavy chain genomic sequence did not affect V(D)J recombination of human heavy chain gene segments within the locus, and that these mice are able to recombine human heavy chain gene segments in the normal manner to produce functional heavy chain immunoglobulin proteins.

[0364] Example 10 Humoral immune responses in ADAM6 rescue mice Lacking the mouse ADAM6a and ADAM6b genes (H + / + κ + / + ) or containing ectopic genomic fragments encoding the mouse ADAM6a and ADAM6b genes (H + / + A6 res κ + / + Humoral immune responses were determined in mice homozygous for the human heavy and kappa light chain variable gene loci, either immunized with the human IgG1 gene or immunized with the human IgG1 gene, by a multivalent antigen immunization scheme, followed by antibody isolation and characterization. Results were compared to determine any effects on V(D)J recombination involving human immunoglobulin gene segments, the evolution of serum titers, and evaluation of antibody production by hybridomas and affinity to antigen.

[0365] 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 to regulate the TGF-β signaling pathway (antigen C), and a human receptor tyrosine kinase (antigen D) were utilized for comparative immunization in groups of mice. Serum was collected from groups of mice prior to immunization with the above antigens. For the priming immunization, each antigen (2.3 μg each) was administered mixed with 10 μg of CpG oligonucleotide (Invivogen) as an adjuvant. Immunogens were administered via the footpad (fp) in a volume of 25 μL per mouse. Mice were then boosted via fp on days 3, 6, 11, 13, 17, and 20 with 2.3 μg of antigen along with 10 μg of CpG and 25 μg of Adju-Phos (Brenntag) as an adjuvant for a total of six boosts. After the fourth and sixth booster immunizations, on days 15 and 22, respectively, mice were bred and antisera were assayed for antibody titers against each specific antigen.

[0366] Antibody titers in the sera of immunized mice were determined using an ELISA assay. 96-well microtiter plates (Thermo Scientific) were coated overnight at 4°C with the respective antigens (2 μg / mL) in phosphate-buffered saline (PBS, Irvine Scientific). The next day, the plates were washed four times with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T, Sigma-Aldrich) using a plate washer (Molecular Devices). The plates were then blocked with 250 μL of 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS and incubated for 1 h 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 in duplicate to the blocked plates and incubated for 1 h at room temperature. The last two wells were left empty and used as secondary antibody controls. Plates were washed again 4 times with PBS-T in a plate washer. A 1:5000 / 1:10,000 dilution of goat anti-mouse IgG-Fc-horseradish peroxidase (HRP, Jackson Immunoresearch) or goat anti-mouse IgG-kappa-HRP (Southern Biotech) conjugated secondary antibody was added to the plate and incubated for 1 hour at room temperature. Plates were washed again 8 times with PBS-T and developed using TMB / H2O2 as substrate. The substrate was incubated for 20 minutes and the reaction was stopped with 2N H2SO4 (VWR) or 1N H3PO4 (JT Baker). Plates were read at 450 nm using a spectrophotometer (Victor, Perkin Elmer). Antibody titers were calculated using Graphpad PRISM software.

[0367] Serum titers were calculated as the serum dilution within the experimental titration range at which the signal for antigen binding corresponded to 2-fold above background. The results for humoral immune responses are shown in Figure 19 (antigen A), Figure 20 (antigen B), Figure 21 (antigen C) and Figure 22 (antigen D). The antigen positive scores of hybridomas generated using two spleens isolated from mice from each of the selected immunization groups are shown in Figure 11 (antigen score is equal to 2X / background).

[0368] As shown in this example, Adam6 rescue mice (H + / + A6 res κ + / + The titers of antibodies produced in mice lacking ADAM6a and ADAM6b and with humanized heavy chains (H + / + κ + / + ) was comparable to H + / + A6 res κ + / + Spleens from mice were cultured to yield antigen-positive hybridomas for all antigens tested, including high affinity antibodies. + / + κ + / + Thus, given the production of high affinity antibodies containing human immunoglobulin genes, there appears to be no defect in V(D)J recombination of human immunoglobulin gene segments in Adam6 rescue mice.

[0369] [Table 11] Example 11 Determination of antigen-binding affinity The binding affinity of the antibodies that showed specific binding to antigen B was screened using a real-time surface plasmon resonance biosensor (BIAcore 2000). Two strains of mice (H + / + κ + / + and H + / + A6 res κ + / +Conditioned medium from hybridomas isolated from GE was used during the BIAcore screening. The BIAcore sensor surface was first derivatized with a polyclonal rabbit anti-mouse antibody (GE) to capture anti-antigen B antibodies from the conditioned medium. HBST (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20) was used as the running buffer during the entire screening procedure. Fab fragments of antigen B were injected at 100 nM concentration at a flow rate of 50 μL / min over the anti-antigen B antibody capture surface. Antibody-antigen association was monitored for 3 min, while dissociation of antigen from the captured antibody was monitored for 5 min in HBST running buffer. The experiment was performed at 25° C. Association (ka) and dissociation (kd) kinetic constants were determined by processing and fitting of the data to a 1:1 binding model using Scrubber 2.0 curve fitting software. The binding dissociation equilibrium constant (KD) and dissociation half-life (T1 / 2) were calculated from the reaction rate constants as follows: KD(M)=kd / ka; and T1 / 2(min)=(ln2 / (60*kd). The results for selected anti-antigen B antibodies are shown in Table 12.

[0370] [Table 12] In a similar experiment, the kinetics of different monoclonal antibodies present in hybridoma-conditioned medium binding to antigen A was determined using a real-time surface plasmon resonance biosensor (BIAcore 4000) assay. All hybridoma clones used in this assay were H + / + A6 res κ + / + Produced in mice.

[0371] Briefly, to capture antigen A specific antibodies, polyclonal rabbit anti-mouse antibodies (GE catalog #BR-1008-38) were first immobilized on a sensor chip. BIAcore screening was performed in two different buffers - PBSP, pH 7.2 and PBSP, pH 6.0. Both buffers were supplemented with 0.1 mg / mL BSA. After capture of anti-antigen A antibodies from conditioned media, 1 μM antigen A monomer (prepared in the respective running buffer) was injected over the capture antibody surface at 30 μL / min for 1.5 min, and dissociation of bound antigen A monomer was monitored for 1.5 min in the respective running buffer at 25°C. Association (ka) and dissociation (kd) kinetic constants were determined by processing and fitting of data to a 1:1 binding model using Scrubber 2.0 curve fitting software. The binding dissociation equilibrium constants (KD) and dissociation half-lives (T1 / 2) were calculated from their kinetic constants as follows: KD(M)=kd / ka; and T1 / 2(min)=(ln2 / (60*kd). The binding kinetic parameters for selected anti-Antigen A antibodies that bind to Antigen A monomer at pH 7.2 and pH 6.0 are shown in Table 13. NB: No binding was detected under the experimental conditions.

[0372] [Table 13-1]

[0373] [Table 13-2] As shown above, H + / + A6 res κ + / + Mouse and H + / + κ + / + High affinity antibodies with similar characteristics were obtained from both mice. Among the 25 antibodies presented in Table 12, H + / + A6 res κ + / + Twenty antibodies produced in mice clearly exhibited affinities ranging from 0.5 nM to 1 μM, while H + / + κ + / +Five antibodies raised in mice demonstrated affinities ranging from 10 nM to 150 nM, and 55 antibodies shown in Figure 13 demonstrated affinities ranging from 20 pM to 350 nM for binding to antigen A monomer.

[0374] As demonstrated in this example, reinsertion of the mouse Adam6 gene into a humanized immunoglobulin heavy chain locus does not impair the ability of the mice to mount a robust immune response to multiple antigens characterized by a repertoire of human antibodies with diverse affinities in the subnanomolar range derived from engineered germline rearranged human gene segments.

[0375] As another embodiment, the present invention includes the following items. [Item 101] 1. A method for producing a fully human antibody specific to an antigen, comprising: (a) immunizing a genetically modified mouse with the antigen, (i) the genome contained in the mouse is (A) One or more human V operably linked to a mouse heavy chain constant region at an endogenous mouse immunoglobulin heavy chain locus. H Gene segment, one or more human D H A gene segment and one or more J H Gene segments, (B) one or more human V operably linked to a light chain constant region. L A gene segment and one or more human J L gene segments, and (C) a nucleotide sequence integrated into the genome of the mouse encoding the mouse ADAM6a and mouse ADAM6b proteins expressed in the genetically modified mouse; Includes; (ii) upon immunization with an antigen, produce antibodies that each comprise a human heavy chain variable domain operably linked to a mouse heavy chain constant region; and (iii) is fertile); (b) isolating from the mouse at least one splenocyte or B cell that produces an antibody that is specific for the antigen; (c) generating at least one cell that produces a fully human antibody specific for the antigen, the cell comprising a human variable domain encoded by a human variable region sequence found in at least one splenocyte or B cell of step (b); (d) culturing at least one cell that produces the fully human antibody of step (c); and (e) obtaining the fully human antibody of step (d); The method includes: [Item 102] One or more human V H The gene segment is V H 6-1, V H 1-2, V H 1-3, V H 2-5, V H 3-7, V H 1-8, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-16, V H 1-18, V H 3-20, V H 3-21, V H 3-23, V H 1-24, V H 2-26, V H 4-28, V H 3-30, V H 4-31, V H 3-33, V H 4-34, V H 3-35, V H 3-38, V H 4-39, V H 3-43, V H 1-45, V H 1-46, V H 3-48, V H 3-49, V H 5-51, V H 3-53, V H 1-58, V H4-59, V H 4-61, V H 3-64, V H 3-66, V H 1-69, V H 2-70, V H 3-72, V H 3-73, V H The method according to [Item 101], wherein the compound is selected from the group consisting of 3-74 and combinations thereof. [Item 103] One or more human V L A gene segment and one or more human J L The method of [Item 101], wherein the gene segment is operably linked to a mouse light chain constant region at an endogenous mouse light chain locus. [Item 104] One or more human V L The method of [Item 101], wherein the gene segment is selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-16, Vκ1-17, Vκ1-27, Vκ1-33, Vκ1-37, Vκ1-39, Vκ2-24, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-40, Vκ3-11, Vκ3-15, Vκ3-20, Vκ4-1, Vκ6-21, and combinations thereof. [Item 105] Nucleic acid sequences encoding mouse ADAM6a and mouse ADAM6b proteins are (a) is present on the same chromosome as an endogenous mouse immunoglobulin heavy chain locus; and / or (b) present in an ectopic configuration; The method described in [Item 101]. [Item 106] The nucleotide sequences encoding the mouse ADAM6a and mouse ADAM6b proteins are H Gene segment, one or more human D H A gene segment, or one or more J H The method described in [Item 101], wherein the gene segment is flanked. [Item 107] The method described in [Item 101], wherein the fully human antibody is a monoclonal antibody. [Item 108] The method according to [Item 101], wherein the immunization with the antigen in step (a) is carried out using a protein, DNA, a combination of DNA and protein, or a cell expressing the antigen. [Item 109] The method described in [Item 101], wherein the inserted nucleic acid sequences encoding mouse ADAM6a and mouse ADAM6b proteins are located on a transgene driven by a heterologous promoter. [Item 110] The method described in [Item 109], wherein the heterologous promoter is a non-immunoglobulin promoter. [Item 111] The mouse heavy chain constant region sequence is H 1, hinge, C H 2. C H The method of [Item 101], comprising a mouse heavy chain sequence selected from the group consisting of 3 and combinations thereof. [Item 112] The method described in [Item 101], wherein a majority of the mouse's B cells contain inserted nucleic acid sequences encoding mouse ADAM6a and mouse ADAM6b proteins. [Item 113] The method described in [Item 101], wherein at least 90% of the B cells of the mouse contain within their genome an inserted nucleic acid sequence encoding mouse ADAM6a and mouse ADAM6b proteins. [Item 114] The method according to [Item 101], wherein the culture in step (c) is carried out on at least one hybridoma cell prepared from at least one cell obtained in step (b). [Item 115] The genome of the genetically modified mouse comprises one or more human V operably linked to a mouse heavy chain constant region sequence at an endogenous mouse immunoglobulin heavy chain locus. H Gene segment, one or more human D H A gene segment and one or more J HThe method of [Item 101], wherein the gene segment is homozygous. [Item 116] The genome of the genetically modified mouse comprises one or more human VLK vectors operably linked to a light chain constant region sequence. L A gene segment and one or more human J L The method of [Item 101], wherein the gene segment is homozygous.

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Claims

[Claim 1] The method described in the specification.

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